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  • 1. Volume 1 Issue 3 www.ijrpb.com May-June 2013 Indian Journal of Research in Pharmacy and Biotechnology ISSN: 2320-3471 (Online) ISSN: 2321-5674 (Print) Editor B.Pragati Kumar, M.Pharm, Assistant Professor, Nimra College of Pharmacy Consulting editor Dr. S Duraivel, M.Pharm, Ph.D., Principal, Nimra College of Pharmacy Associate Editors Mr. Debjit Bowmick, M.Pharm., (Ph.D) Assistant Professor, Nimra College of Pharmacy Mr. Harish Gopinath, M.Pharm., (Ph.D) Assistant Professor, Nimra College of Pharmacy Dr. M. Janardhan, M.Pharm., Ph.D. Professor, Nimra College of Pharmacy Dr. A. Ravi Kumar, M.Pharm., Ph D. Professor, Bapatla College of Pharmacy Editorial Advisory Board Dr.Y.Narasimaha Reddy, M. Pharm., Ph D. Principal, University college of Pharmaceutical Sciences, Kakatiya University, Warangal. Dr. Biresh Kumar Sarkar, Asstt.Director (Pharmacy), Kerala Dr.V.Gopal, M. Pharm., Ph D. Principal, Mother Theresa Post Graduate & Research Institute of Health Sciences,Pondicherry-6 Dr. M.Umadevi, M.Sc. (Agri), Phd Research Associate, Tamil Nadu Agricultural University, Coimbatore Dr. J.Balasubramanium, M. Pharm., Ph D. General Manager, FR&D R A Chem Pharma Ltd., Hyderabad Dr. V.Prabhakar Reddy, M. Pharm., Ph D. Principal, Chaitanya College of Pharmacy Education & Research, Warangal Dr.P.Ram Reddy, M. Pharm., Ph D. General Manager, Formulation, Dr.Reddy’s Laboratory, Hyderabad Dr. S.D.Rajendran, M. Pharm., Ph D. Director, Pharmacovigilance, Medical Affairs, Sristek Consultancy Pvt. Ltd, Hyderabad
  • 2. Volume 1 Issue 3 www.ijrpb.com May-June 2013 INDIAN JOURNAL OF RESEARCH IN PHARMACY AND BIOTECHNOLOGY Instructions to Authors Manuscripts will be subjected to peer review process to determine their suitability for publication provided they fulfill the requirements of the journal as laid out in the instructions to authors. After the review, manuscripts will be returned for revision along with reviewer’s and/or editor’s comments. Don’t copy and paste the article content from internet or other sources like e-books etc. Authors are the sole responsible persons for the article, article content; results of the research conducted and copy right issues if any. The editor and the editorial board are not entitled to change the article content, results and diagrammatic representations which are given by authors. The article will be published only after getting the approved galley proof from the authors. Kindly follow the below guidelines for preparing the manuscript: 1. Prepare the manuscript in Times New Roman font using a font size of 12. There shall not be any decorative borders anywhere in the text including the title page. 2. Don’t leave any space between the paragraphs. 3. Divide the research article into a. Abstract b. Introduction c. Materials and Methods d. Results e. Discussion f. conclusion g. References 4. References should include the following in the same order given below a) Author name followed by initials b) Title of the book/ if the reference is an article then title of the article c) Edition of the book/ if the reference is an article then Journal name d) Volume followed by issue of the journal e) Year of publication followed by page numbers 5. Download the author declaration form from the web site www.ijrpb.com, fill it and submit it after signing by corresponding and co-authors to IJRPB. You can send the filled in form by post or scanned attachment to ijrpb@yahoo.com. 6. Keep in touch with the editor through mail or through phone for further clarifications as well as for timely publication of your article. Indian Journal of Research in Pharmacy and Biotechnology is a bimonthly journal, developed and published in collaboration with Nimra College of Pharmacy, Ibrahimpatnam, Vijayawada, Krishna District, Andhra Pradesh, India-521456 Printed at: F. No: 501, Parameswari Towers, Ibrahimpatnam, Vijayawada, India -521456 Visit us at www.ijrpb.com Contact us/ send your articles to: Email: ijrpb@yahoo.com Phone no: 9490717845; 9704660406
  • 3. Indian Journal of Research in Pharmacy and Biotechnology ISSN: 2320-3471 (Online) ISSN: 2321-5674 (Print) Volume 1 Issue 3 www.ijrpb.com May-June 2013 S.No. Contents Page No. 1. Development and evaluation of drotoverine taste masked tablets with improved dissolution efficiency using solid dispersion technique Anusha P, Nirajana V.A, Syed Mohammed, Shaik Jilani, Ch. Murali Krishna, Harish.G 275-280 2. Effect of different disintegrants on Ciprofloxacin conventional tablets Harish.G, Ch.Bhargavi, A.Riyajune, Md.Yasmeen, Syed Mohammed, Ch.Murali Krishna, Sk.Jilani, Nirajana.V.A 281-287 3. Chronotherapy for nocturnal asthama Papola Vibhooti , Rajan G, Bisht Seema , Dr. Kothiyal Preeti 288-298 4. Recent trends in scope and opportunity of clinical research in India Hemant Singh, Abhinav Srivastva 299-304 5. Sustained release drug delivery system Navin Dixit, Sheo Dutt Maurya, Bhanu P.S.Sagar 305-310 6. Antibacterial activity of ethanolic extracts of Nyctanthes arbortristis and Nerium oleander A.Ravi Kumar, Ch.S.D.Phani Deepthi Yadav 311-313 7. Food poisoning and its safety precaution M. Umadevi, K.P.Sampath Kumar, Sai Pavan, Sd.Gosia Sultana, D. Bhowmik 314-323 8. Microencapsulation technology K.P.Sampath Kumar,Tejbe.Sk, Shameem Banu, P.Naga Lakshmi , D.Bhowmik 324-328 9. An overview about pharmacy education in India Praneta Desale 329-332 10. Phytochemical evaluation of Nyctanthes arbortristis, Nerium oleander and Catharathnus roseus Ch.S.D. Phani Deepthi Yadav, N.S.P. Bharadwaj, M. Yedukondalu, Ch. Methushala, A. Ravi Kumar 333-338 11. Angina pectoris epidemic in India: a comprehensive review of clinical features, differential diagnosis, and remedies Shravan Paswan, Ranjan Kumar Sharma, Alok Ranjan Gaur, Avinash Sachan, Mahendra Singh Yadav, Preeti Sharma, Mrinmoy Gautam 339-345 12. Stability Indicating HPLC method for the estimation of Cinacalcet hydrochloride API Manikandan Krishnan, Santhana Lakshmi Karunanidhi, Gayathri Sola, Akshitha .Y 346-350 13. Bioavailability: criteria for approving a drug product for marketing Sandhya Singh, Faheem Ajmal Ansari, Shravan Paswan, Rnjan Kumar Sharma, Alok Ranjan Gaur 351-359 14. The effect of superdisintegrants on the dissolution of calcium carbonate fast dissolving tablets Mohammed Farhana, J.Preeti, Md Faizulla, Budda Chellibabu, Harish.G, Rajnesh Kumar Singh 360-364 15. Phytochemical and pharmacological studies on whole plant of Asystasia gangetica T.K.Gopal, Megha.G, D.Chamundeeswari, C.Umamaheswara Reddy 365-370 16. Investigation of in-vitro anti-oxidant, anti-inflammatory and anti-arthritic activity of aerial parts of Securinega leucopyrus (willd.) Muell T.K.Gopal, T.Sheela, D.Chamundeeswari, C.Umamaheswara Reddy 371-378
  • 4. Indian Journal of Research in Pharmacy and Biotechnology ISSN: 2320-3471 (Online) ISSN: 2321-5674 (Print) Volume 1 Issue 3 www.ijrpb.com May-June 2013 17. Transdermal sonophoresis technique- an approach for controlled drug delivery K.P.Sampath Kumar, Debjit Bhowmik, M.Komala 379-381 18. A comprehensive review of Eladi Vati Navin Dixit, Sheo Dutt Maurya , Bhanu P.S.Sagar 382-384 19. Preparation and characterization of some herbal ointment formulations with evaluation of antimicrobial property Pulak Majumder and Susmita Majumder 385-390 20. The effects of air pollution on the environment and human health Shyam Bihari Sharma, Suman Jain, Praveen Khirwadkar, Sunisha Kulkarni 391-396 21. Formulation and evaluation of orodispersible tablets of Cinnarizine by super disintegrants addition method Praveen Khirwadkar, Kamlesh Dashora, Shyam Bihari Sharma 397-400 22. Effective hypoglycemic action of metformin combinations against Dexamethasone induced diabetes mellitus in rats Mohanraghupathy.S, Jayabharath N, Bhuvana Tejay, Hameera Khanam B, Lavanya Lahari B 401-403 23. A review on medicinal plants having antioxidant potential Prof.S.K Sharma, Mr. Lalit Singh, Suruchi Singh 404-409 24. Invitro anti-inflammatory activity of Strychnos potatorum linn seed by HRBC membrane stabilization V.Vijayakumar, Dr C.K.Hindumathy 410-412 25. Synthesis and characterization of 1, 3, 4-oxadiazole and 1,3,4- thiadiazole Ramanji Naik 413-419 26. Preparation, characterization and evaluation of Olmesartan medoxomil β- cyclodextrin complexes V. Prudhvi Raj, Subhashis Debnath, Maleswari, M. Niranjan Babu 420-427 27. Wafers technology – a newer approacah to smart drug dilevery system Papola Vibhooti*, Kothiyal Preeti 428-439 28. Evaluation of anti-ulcer effects of ethanolic extract of Delonix regia flower Samaresh Pal Roy, Kamlesh Prajapati, Ramji Gupta, Dipanwita Bhadra, Nikunj Patel, Archana Batiwala, Gautam Sonara, Neerav Gheewala, T. Kannadasan 440-445 29. A study on medication non-adherence in ambulatory diabetic patients and need for pharmacist intervention for improving patient adherence Dr. Praveen Kumar G 446-447 30. Recent trends in positive and negative aspects of food on bioavalabilty of drugs Gowthami B, Sk Nahida Fazilath, Sanaulla Md, K Prudhvi Raj, Dastagiriah G, Tabassum Sk 448-460 31. A review on collagen based drug delivery systems Sahithi B, Ansari Sk, Hameeda Sk, Sahithya G, Durga Prasad M, Yogitha Lakshmi 461-468
  • 5. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Anusha P et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 275 DEVELOPMENT AND EVALUATION OF DROTOVERINE TASTE MASKED TABLETS WITH IMPROVED DISSOLUTION EFFICIENCY USING SOLID DISPERSION TECHNIQUE Anusha P*1 , Nirajana V.A2 , Syed Mohammed1 , Shaik Jilani1 , Ch. Murali Krishna, Harish.G1 1. Nimra college of Pharmacy, Jupudi, Vijayawada, Andhra Pradesh, India 2. Faculty of Pharmacy, Sri Ramachandra University, Porur, Chennai. *Corresponding author: E.Mail:anupharma88@gmail.com ABSTRACT The purpose of this research is to mask the bitter taste of the drug, Drotoverine using solid dispersion technique. The taste-masked drug is formulated in to a conventional tablet by direct compression method for ease of administration. Taste masking was done by solid dispersion using polymer such as urea and mannitol by melting/fusion method Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM) were performed to identify the physicochemical interaction between drug and carrier, hence its effect on dissolution. Conventional taste masked tablets were evaluated for weight variation, disintegration time, hardness and friability. In vitro drug release studies were performed for conventional tablets of drotoverine. Bitterness score was evaluated on volunteers. FTIR spectroscopy and SEM showed no interaction between drug and carriers. Conventional tablets prepared using solid dispersion, showed faster disintegration and complete bitter taste masking of drotoverine. In addition the prepared tablets exhibited better dissolution profile. Taste evaluation of taste masked tablets in human volunteers rated tasteless with a score of 0. Thus, results conclusively demonstrated successful masking of taste and oral disintegration of the formulated tablets in the oral cavity with improved dissolution. Key words: Drotoverine, conventional tablet, solid dispersion, taste masking INTRODUCTION The oral route is the most convenient, appropriate and acceptable way to administer medications. Several oral active pharmaceuticals ingredients and bulking agents have Unpleasant bitter taste; hence this often times results to non compliance to medications by Patients. Taste masking is a means of masking the bitter taste of drug in order to improve the Palatability of the drug, which in turn improves patience compliance. Drotoverine is a novel non-Anticholinergic smooth muscle antispasmodic drug. Its Chemical name is 1- [(3, 4-diethoxy phenyl) methylene]-6, 7-diethoxy-1, 2, 3, 4-tetrahydroisoquinoline with a molecular formula of C24 H31NO4. HCl. It decreases the influx of active calcium into smooth muscles due to inhibiting of phosphodiesterase and intracellular increase of cAMP level. Its oral bioavailability is about 100% with a biologic half- life of about 7 to 12 h. It adult dose is about 40 to 80mg one to three times a day. Drotoverine has poor aqueous solubility, and thus resulting in incomplete absorption after oral administration. This is due to a large fraction of the dose remaining undissolved for absorption. Under such conditions, the bioavailability can be increased by using, a more water soluble formulation. Solid dispersion is an efficient means of improving the dissolution rate and hence the bioavailability of a range of poorly soluble drug. Further drotoverine has an extremely unpleasant bitter taste. The exact mechanism of bitterness is unknown. Masking of bitter taste of the drotoverine is an extremely important factor in the formulation of tablets to ensure patient compliance. Taster masked tablets were useful in patients, such as pediatric, geriatric, who may face difficulty in swallowing conventional tablets or capsules and liquid orals or syrup. Many reported techniques such as polymer coating, microencapsulation, use of lecithins and related substances, liposomes and various polymeric materials mask the bitterness by controlling drug release at salivary pH. However it is a major challenge to develop taste masked conventional tablets with improved drug release. Thus in the present study an attempt has been made to formulate taste masked conventional tablets of drotoverine with improved dissolution so as to prepare a “patient-friendly dosage form”. Furthermore, the study undertakes to investigate solid-state characterization, and attempts to see the possible mechanism of taste masking and improved dissolution rate. 2. MATERIALS AND METHODS 2.1. Materials: Drotoverine was obtained as a gift sample from Apex laboratory ltd, Chennai; sodium starch glycolate, aspartame, magnesium stearate and micro crystalline cellulose was obtained as a gift sample from Intex chemicals, Chennai.
  • 6. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Anusha P et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 276 2.2. Methods 2.2.1. Preparation of Drotoverine Solid Dispersion Using Mannitol and Urea as Carriers: The solid dispersion of drotoverine with urea and mannitol in 1:1, 1:2 and 1:3 ratios were carried out using melting or fusion method. The preparation of physical mixture of a drug and a water-soluble carrier and heating it directly until it melted. The melted mixture is then solidified orally in an ice-bath under vigorous stirring. The final solid mass is crushed, pulverized and sieved. 2.2.2. Characterization of solid dispersion: The prepared drotoverine and mannitol, drotoverine and urea solid dispersions were characterized for solubility studies, FTIR studies and SEM studies. 2.2.3. Tablet Formulation: Oral conventional tablets containing equivalent of 80 mg of drotoverine were compressed on an eight-station single rotary tabletting press (GMC, using a flat punch with break line by direct compression technique. 3. Characterization of Prepared Tablets: The prepared tablets were evaluated for its physical characteristics like hardness thickness, weight variation, friability, disintegration test. 3.1. In vitro dissolution study: One tablet of F1 or F2 or MKT formulation were placed in a cylindrical basket (aperture size 425μm: diameter 20mm; height 25mm), and immersed in 900ml of leaching fluid (Stimulated gastric fluid maintained at 37 ± 2oC). The fluid was stirred at 100rpm (Model Disso 2000, Lab India). Samples of the leaching fluid (5ml) were withdrawn at selected time intervals with a syringe fitted with a cotton wool plug and replaced with an equal volume of drug-free dissolution fluid. The samples were suitably diluted with blank dissolution fluid and were analyzed for content of drotaverine hydrochloride spectrophotometrically at λmax, 302.8 nm by using an ElicoSL 210 UV-Visible double beam spectrophotometer (Elico, India). The amounts released were expressed as a percentage of the drug content in each dissolution medium. The dissolution test was carried out in quadruplicate and the mean results reported. 3.2. Taste evaluation: Taste evaluation was done on 6 volunteers by using time intensity method. One tablet was held in mouth for 10 seconds bitterness levels were recorded instantly and after 10 seconds, 30seconds, 1 minute and 2 minutes, bitterness levels were recorded. 4. RESULTS Table.1. Formulation table for solid dispersion Table.2. Formulation Table for Tablets Formulation code Polymer Ratio F1 Mannitol 1:1 F2 Mannitol 1:2 F3 Mannitol 1:3 F4 Urea 1:1 F5 Urea 1:2 F6 Urea 1:3 *for all the formulations Name of the ingredient Quantity per single tablet* Drug polymer SD 80mg Sodium starch glycolate 6.6mg Aspartame 1.1mg Microcrystalline cellulose 16.8mg Magnesium stearate 5.5mg Total 110mg Table.3. Pre-compression parameters for drug polymer solid dispersion Formulation code F1 F2 F3 F4 F5 F6 Angle of repose 25°71’ 26°42’ 28°93’ 24°32’ 25°43’ 29°47’ Bulk Density(gm/ml) 0.74 0.72 0.69 0.64 0.75 0.78 Tapped Density(gm/ml) 0.86 0.82 0.87 0.85 0.89 0.89 Compressibility Index (%) 13.95 12.19 20.68 24.70 15.73 12.35 Hausners ratio 1.18 1.14 1.25 1.30 1.16 1.14 Table.4. Post compression studies Formulation code Weight variation (mg) Thickness (mm) Hardness (Kg/cm2) Friability (%) Assay (%) F1 Complies 1.9 4 0.05 99.56 F2 Complies 1.9 4 0.06 98.45 F3 Complies 2.0 5 0.03 99.12 F4 Complies 1.9 4 0.05 98.22 F5 Complies 2.1 5 0.06 97.44 F6 Complies 2.0 4 0.01 99.61
  • 7. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Anusha P et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 277 Table.5. In-vitro dissolution study for Drotoverine solid dispersion Time in min Percent drug release formulation code F1 F2 F3 F4 F5 F6 0 0 0 0 0 0 0 5 14.11 14.75 15.39 16.56 22.25 26.03 10 25.03 26.14 30.01 32.78 39.56 45.35 15 34.22 36.78 40.56 43.06 54.09 69.46 20 41.86 45.67 52.88 55.12 67.03 78.75 25 52.45 55.76 61.75 67.95 73.25 86.95 30 58.56 65.20 67.25 72.15 84.02 92.10 Table.6. In-vitro dissolution study for marketed drotoverine tablets Table.7.Comparison of dissolution profile for formulated and marketed products Time in min Percent drug release 0 0 5 24.36 10 35.12 15 54.32 20 67.75 25 75.55 30 82.36 Time in min %Drug Release Marketed drotoverine tablets Formulated drotoverine tablets 0 0 0 5 24.36 26.03 10 35.12 45.35 15 54.32 69.46 20 67.75 78.75 25 75.55 86.95 30 82.36 92.10 Table.8. Bitterness evaluations of prepared drotoverine tablets: volunteers Formulation code and bitterness F1 F2 F3 F4 F5 F6 1 0 0 0 0 0 0 2 0 0 X 0 0 0 3 0 0 0 X 0 0 4 x 0 0 0 x 0 5 0 0 0 0 0 0 0=No bitterness; x=Threshold bitterness Figure.1. IR spectro of the pure drug, Drotaverine Hcl Figure.2.IR spectro of the formulation F1 Figure.3.IR spectro of the formulation F2 Figure.4. IR spectro of the formulation F3
  • 8. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Anusha P et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 278 Figure.5.IR spectro of the formulation F4 Figure.6.IR spectro of the formulation F5 Figure.6. IR spectro of the formulation F6 Figure.7.SEM image of the pure drug, Drotaverine Hcl Figure.8.SEM image of the formulation F1 Figure.9.SEM image of the formulation F2 Figure.10.SEM image of the formulation F3 Figure.11. SEM image of the formulation F4 Figure.12.SEM image of the formulation F5 Figure.13. SEM image of the formulation F6
  • 9. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Anusha P et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 279 Figure.14. In-Vitro Dissolution studies of the formulated batches Figure.15. In-Vitro Dissolution studies Optimized and marketed 5. DISCUSSION Fourier Transform Infrared Spectroscopy: Infrared spectra matching approach was used for thedetection of any possible chemical reaction between the drug and the polymer. The IR spectrum of the physical mixture was done to detect any appearance or disappearance of peaks. The compatibility between the drug and the polymer were evaluated using FTIR matching method. The IR spectra of pure drug and polymer are shown in (figure 1). Pre-Formulation Studies: The angle of repose of prepared drotaverine tablet mixture was in the range 20°-30°. Normally if the value falls between 20°-30°, it shows good flow property. The bulk density and tapped density were found to be in the range of 0.7 to 0.8 g/cm3. A Hausner’s ratio was within the range of 1.13 to 1.32, lesser than 1.25 is considered to be an indication of good flow property. The compressibility index was within the range of 10-25 hence falls within the good range. Post-Compressional studies: The post compressional characteristic for all the formulated batches was found to be within the limits as per Indian pharmacopeia. The hardness was found to be within 4-5 Kg/cm2 in all the formulations. In all the formulations, the friability value is less than 1% giving an indication that tablets formulated are mechanically stable. All the tablet formulations compile the weight variation test. The weight of all the formulations was found to be within the limits. The assay of all the formulations was found to be within the pharmacopoeial (Table 4). In-vitro dissolution study: All the formulation was subjected to dissolution studies and it was absorbed that the batch F6 showed about 92.10% of release and was found to be maximum when compared to other batches. Formulated batch F1 and F2 showed a slow release pattern with about only 58.56% and 65.20% of drug release at the end of 30mins. (table 5), and the batches F3, F4, F5 showed a release of about 67.25% to 84.02% of drug release. For the formulation f7 the percentage amount of drug release was found to be with the pharmacopeial limits.the comparission of dissolution profiles for formulated drotaverine and marketed drotaverine tablets were shown in the table no 8 and it was concluded that percent drug release for formulated drotaverine tablets was more when comparared to marketed drotaverine tablets. Taste evaluation of all formulations: The time intensity study for taste in human volunteers of the formulated drotaverine hydrochloride with the polymer SD revealed considerable masking of the bitter taste of drotaverine HCl with degree of bitterness below the threshold value within 120seconds (See Table 8). Sensory evaluation of the tablets with both polymers proved good palatability. 6. CONCLUSION This study has established effective taste masking of drotaverine HCl with the use of the solid dispersion techniques using urea and mannitol as carriers. Taste masking and rapid dissolution of drotaverine HCl tablets formulated in this investigation may possibly help in the administration of drotaverine HCl in a more palatable form in the absence of water and more importantly since drotaverine HCl solid dispersion tablet formulations are not presently in the market. Hence, “patient-friendly dosage form” of bitter drugs, especially for pediatric and geriatric patients, can be developed using this technique. 7. REFERENCES A. Rajpoot, Formulation and In-vitro Evaluation of Immediate release tablets of Drotaverine HCl, J. Chem. Pharm. Res, 3(4), 2011, 333-341
  • 10. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Anusha P et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 280 Srikanth MV, Uhumwangho MU, Sunil SA, Design and evaluation of taste masked Drotaverine HCl orodispersible tablets using polymethacrylate polymers, Der Pharmacia Lettre, 2(6), 2010, 223-231. Vijayanand P, Formulation, Development and Evaluation of Novel Dosage Form Containing Silk Fibroin for Elderly Patients, RJPBCS, 3(1), 2010, 524,
  • 11. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Harish G et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 281 EFFECT OF DIFFERENT DISINTEGRANTS ON CIPROFLOXACIN CONVENTIONAL TABLETS Harish.G1 *, Ch.Bhargavi1 , A.Riyajune1 , Md.Yasmeen1 , Syed Mohammed1 , Ch.Murali Krishna1 , Sk.Jilani1 , Nirajana.V.A2 1.Nimra College of Pharmacy, Jupudi, Vijayawada. 2. Faculty of pharmacy, Sri Ramachandra University, Chennai. *Corresponding author: E.Mail: harishgopinath4u@gmail.com ABSTRACT The objective of the present study is to design and evaluate the effect of disintegrating agents such as Starch, Cross Caramellose Sodium and Sodium starch glycolate on ciprofloxacin tablet. The nature of the Ciprofloxacin which is slightly soluble in water which affects the drug disintegration process there by inhibits the drug release from the Conventional Tablet. Hence in the present study the effect of disintegrating agents at different concentrations is carried out on the ciprofloxacin hcl to find out the optimized concentration followed by stability studies for a period of 3 months. INTRODUCTION Despite increasing interest in controlled-release drug delivery systems, the most common tablets are those intended to be swallowed whole and to disintegrate and release their medicaments rapidly in the gastrointestinal tract (GIT). The proper choice of disintegrant and super-disintegrant to improve its consistency of performance is of critical importance to the formulation development of such tablets. Drug release from a solid dosage form can be enhanced by addition of suitable disintegrants. In more recent years, increasing attention has been paid to formulating fast dissolving and/or disintegrating tablets that are swallowed, but also orally disintegrating tablets that are intended to dissolve and/or disintegrate rapidly in the mouth. The present study is an attempt to select best possible combination of drug and disintegrating agent to formulate rapidly disintegrating tablet of ciprofloxacin conventional tablets which disintegrates faster thereby reducing the time of onset of action. Lactose is selected as diluents, Starch, Sodium starch glycolate, CCS and Crospovidone were selected as disintegrants. PVP K 30M paste was used as a binder in all formulations, Magnesium stearate and Talc as a Lubricant, Aerosil as a Glidant. The percentage Drug content of all tablets was found to be between 95% - 105%, which was within the limit. From the data obtained, it is observed that the formulation containing crosprovidone disintegrant disintegrate rapidly when compared to other disintegrating agents such as Starch, SSG, and CCS with ciprofloxacin. MATERIALS AND METHODS Ciprofloxacin obtained as a gift sample from Intex chemicals Pvt ltd, Chennai. Starch, Cross Caramellose Sodium and Sodium Starch Glycolate was obtained from Fischer Ltd, Chennai. All other excipients which are used are of high standard analytical grade. Pre-Formulation Studies: Drug-excipients compatibility studies: Compatibility of drug with excipients was determined by FTIR using kBr pellet technique, in the wavelength region of 4000-400cm-1 . Table 1: Formulation Table of Ciprofloxacin Hcl conventional tablet Formulation FA1 FA2 FA3 FB1 FB2 FB3 FC1 FC2 FC3 FD1 FD2 FE1 FE2 Ciprofloxacin (mg) 500 500 500 500 500 500 500 500 500 500 500 500 500 Starch (%) 50 10 15 - - - - - - - - - - SSG (%) - - - 4 5 6 - - - - - - - CCS (%) - - - - - - 10 20 30 - - - - BCD (%) - - - - - - - - - 40 80 40 80 Aerosil (%) 200 200 200 200 200 200 200 200 200 200 200 200 200 Lactose 220 260 255 246 265 264 260 250 240 230 90 230 90 PVP (%) 50 50 50 50 50 50 50 50 50 50 50 50 50 Talc (%) 50 50 50 50 50 50 50 50 50 50 50 50 50 Magnesium Sterate (%) 30 30 30 30 30 30 30 30 30 30 30 30 30 Total Weight (mg) 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000
  • 12. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Harish G et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 282 Quantity sufficient of Ciprofloxacin for a batch of 50 tablets was separately mixed to ensure complete mixing. A tablet containing 500 mg equivalents of ciprofloxacin was compressed. All ingredients were weighed and passed through 40# sieve, blended in a Poly Bag except Magnesium Stearate for 10 minutes. Mix half the part of the disintegrated with the above mixture after passing through the sieve. The resultant mixture was wet massed using suitable binder (qs) for granulation. This wet mass was passed through 20# sieve in order to form granules. These granules were dried and the dried granules were passed through 30# sieve. These dried granules were lubricated with Magnesium stearate, which was previous, passed through 60# Sieve. The lubricated granules were punched to tablets using single punching machine. Drug content: The estimation of drug content for ciprofloxacin tablets was performed by crushing three tablets and quantity equivalent to 45mg was taken and determined using 0.1M Hcl using UV spectrophotometer at about 276nm Weight Variation: The USP weight variation test was run by weighing 20 tablets individually, calculating the average weight, and comparing the individual tablet weights to the average. The tablets met the USP tests that were not more than 2 tablets were outside the percentage limit and no tablets differed by more than 2 times the percentage limit. Hardness: Hardness of the tablets was determined by breaking it between the second and third fingers with thumb being as a fulcrum. There was a sharp snap the tablet was deemed to have acceptable strength. Hardness of the tablets was determined by Stokes Monsanto Hardness Tester and the hardness should be found within the range of 3.5-5.5 kg/cm². Friability: The friability of tablets was determined by Roche Friablator. 20 tablets were taken and weighed. After weighing the tablets were placed in the Roche Friablator and subjected to the combined effects of abrasion and shock by utilizing a plastic chamber that revolves at 25RPM for minutes dropping the from a distance of six inches with each revolution. After operation the tablets were de-dusted and reweighed. Content Uniformity: In this test, 30 tablets were randomly selected contained for sample, Ciprofloxacin Hydrochloride should contain not less than 98.0 per cent and not more than 102.0 percent. Thickness: The thickness of a tablet was the only dimensional variable related to the process. 10 tablets were measured for their thickness and diameter with a Caliper, Thickness Gauge. Average thickness and diameter were calculated. Disintegration Test: Disintegration time is considered to be one of the important criteria in selecting the best formulation. For most tablets the first important step toward solution is break down of tablet into smaller particles or granules, a process known as disintegration. Place one tablet into each tube and suspend the assembly in to the 1000ml beaker containing water maintained at 37 ± 2o C and operate the apparatus for 30 seconds. Remove the assembly form the liquid. Observe the tablets, if one or two tablets fail to disintegrate completely; repeat the test on 12 additional tablets. The requirement is met if not less than 16 of the total of 18 tablets tested are disintegrated. In-Vitro Drug Release Studies: In our case to study the release kinetics of drug we used USP II apparatus (Paddle type, 2) with 900 ml, pH 6.8 phosphate buffer as the dissolution medium. The paddle was rotated 50 rpm and 5ml of aliquots were withdrawn at pre-determined time intervals and an equal amount of thee medium was replaced to maintain sink conditions. The aliquots were diluted suitably and the amount of drug(s) released was determined spectrophotometrically using U.V. at wavelength 271 nm. Evaluation of Formulated Ciprofloxacin Tablet: Evaluation of blend characteristics: Ciprofloxacin Tablet was prepared by using wet granulation method. Various formulations were made as shown in table: 6. The Formulated Ciprofloxacin tablet were evaluated for Pre-formulation parameters like angle of repose, bulk density, tapped density, Compressibility index and Hausner’s Ratio. The results of disintegration time of all the tablets prepared by wet granulation found to be varied with change in concentration of disintegrating agents from few seconds to several minutes. Formulations FD 1 and FE1 disintegrated within 3min and found to be more effective. The disintegration time of the tablets using different disintegrants decreases in the following order BC > croscarmellose sodium > SSG > Starch. It is observed that, when BC is used as disintegrant, tablets disintegrate rapidly with in less time compared to other tablets prepared using croscarmellose sodium, starch and sodium starch glycolate disintegrants. Though tablets prepared by intra and extra granulation method found to be more effective in comparison with formulation prepared by only extra
  • 13. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Harish G et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 283 granulation. When concentration of Starch, SSG, CCS and BC is increased, the disintegration time was reduced The angle of repose of prepared Ciprofloxacin tablet was in the range of 20°-30°. Normally if the value falls between 20°-30°, it shows good flow property. The bulk density and tapped density were found to be in the range of 0.37 to 0.38 g/cm3 and 0.44 to 0.45g/cm3 respectively. A Hausner ratio was within the range of 1.16 to 1.17, lesser than 1.25 is considered to be an indication of good flow property. The compressibility index was within the range of 5-15 hence falls within the excellent range. The results were tabulated in table 15. The pre-formulation characteristics results for all the formulation of ciprofloxacin tablets using FB as disintegrating agent found to be within the range, compressibility index for FB1 and FB3 was found to be with in good range of 12-16 were as FB2 was in excellent range. The angle of repose of prepared tablet was in the range of 20°-30°. Normally if the value falls between 20°-30°, it shows good flow property. The bulk density and tapped density were found to be in the range of 0.34 to 0.36g/cm3 and 0.39 to 0.40 g/cm3 respectively. A Hausner ratio was within the range of 1.07 to 1.18, lesser than 1.25 is considered to be an indication of good flow property. The compressibility index was within the range of 5- 15 hence falls within the excellent range. The results were tabulated in table 16. The pre-formulation characteristics results for all the formulation of ciprofloxacin tablets using FC as disintegrating agent found to be within the range, angle of repose and compressibility index was found to be within good range. The angle of repose of prepared ciprofloxacin tablet was in the range of 20°-30°. Normally if the value falls between 20°-30°, it shows good flow property. The bulk density and tapped density were found to be in the range of 0.35 to 0.36 g/cm3 and 0.39 to 0.41 g/cm3 respectively. A Hausner ratio was within the range of 1.08 to 1.18, lesser than 1.25 is considered to be an indication of good flow property. The compressibility index was within the range of 5-15 hence falls within the excellent range. The results were tabulated in table 17. The formulation of ciprofloxacin using 4% B.C disintegrant found to be within the limits for both FD1 and FD2 and falls in good range. The angle of repose of prepared ciprofloxacin tablet was in the range of 20°-30°. Normally if the value falls between 20°-30°, it shows good flow property. The bulk density and tapped density were found to be in the range of 0.36 to 0.38g/cm3 and 0.40 to 0.41 g/cm3 respectively. A Hausner ratio was within the range of 1.07 to 1.16, lesser than 1.25 is considered to be an indication of good flow property. The compressibility index was within the range of 5-15 hence falls within the excellent range. The angle of repose of prepared ciprofloxacin using FE as disintegrant was in the range of 20°-30°. Normally if the value falls between 20°-30°, it shows good flow property. The bulk density and tapped density were found to be in the range of 0.36 to 7 g/cm3 and 0.340 to 0.41g/cm3 respectively. A Hausner ratio was within the range of 1.10 to 1.11, lesser than 1.25 is considered to be an indication of good flow property. The compressibility index was within the range of 5-15 hence falls within the excellent range. The post compressional characteristic for all the formulated batches was found to be within the limits as per Indian pharmacopeia 2007. The hardness was found to be within the range of 3.5 to 5.5 Kg/cm2 in all the formulations indicating good mechanical strength with an ability indicating physical and mechanical strength with an ability to withstand physical and mechanical stress conditions while handling. In all the formulations, the friability value is less than 1% giving an indication that tablets formulated are mechanically stable. All the tablet formulations passed the weight variation test. The weight of all the formulations was found to be within the limits. The assay of all the formulations was found to be with in the 90% to 110% acceptable limits. The disintegration time of the entire Formulated batch varies with change in concentration of disintegrating agents from few seconds to several minutes. Formulations FD2 and FE2 disintegrated within 3min and found to be more effective. The disintegration time of the tablets using different disintegrants decreases in the following order Starch > CCS > SSG >CP. It is observed that, when BC is used as disintegrant, tablets disintegrate rapidly with in less time compared to other tablets prepared using croscarmellose sodium, starch and sodium starch glycolate disintegrants. Though tablets prepared by intra and extra granulation method found to be more effective in comparison with formulation prepared by only extra granulation. When concentration of Starch, SSG, CCS and BC is increased, the disintegration time was reduced significantly show in table 2.. In vitro drug release studies were conducted for the formulation using USP dissolution apparatus type-II (paddle), at 50 rpm. The percentage drug release at the end of 30 min was found in the range 48 – 73% using FA as disintegrant and 67-77% using FB as disintegrant. Invitro drug release studies were conducted for the formulation using USP dissolution apparatus type-II (paddle), at 50 rpm. The percentage of drug release was determined at a time interval of 0, 5, 10, 15, 20, 25, 30 min and at the end of 30 min it was found in the range 80-
  • 14. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Harish G et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 284 95% using FC as disintegrant. Invitro drug release studies were conducted for the formulation using USP dissolution apparatus type-II (paddle), at 50 rpm. The percentage of drug release was determined at an time interval of 0, 5, 10, 15, 20, 25, 30 min and at the end of 30 min it was found in the range 81-95% using 4% BC as disintegrant and 78-98% using 8% BC as disintegrant shown in figure 9, 10 and 11. Stability Studies: Drug molecules are of reactive naturally, the additives are considered to be inert substance but this may not be true for all additives in a formulations. Hence, in developing any formulation, when additive are selected the same must be super imposed on to drugs and with other additives present in the formulation, to see how compatible they are with the other formulation ingredients. A lot of literature has got piled up on drug- excipients incompatibilities, which is a handy tool in the hand of a formulation man, to avoid possible drug- excipient interactions. But even with this literature at the back, formulation may be highly individualistic and each formulation may have problems of its own. There are methods called FTIR, differential scanning calorimetric, thin layer chromatography for investigating these interactions in short period of time. On many occasions, even with the sum total of knowledge available, it is not possible to envisage, all the interactions and a formulation while remaining good for a certain period of time, may go bad in the long run. There is not ready made answer for such situation and all that is possible is to “wait and watch”, the method called “Real time study”. As per ICH guide line for stability study, which advice the formulation to store their products at 30 ° c and 65 % RH to find out actual shelf life period or to assure the product quality free from unwanted interactions. Real time study of ICH guidelines involves storage of products at 40° C& 65 % RH for the complete proposed shelf life period, and analyzing the product sample every month in the first 3 months, every 3 months from 4th month onwards up to one year, every 6 month in the second year of storage, afterwards once in a year till shelf life is completed. ICH guidelines also demands for storing samples at 40 ° c and 75 % RH (stress condition or accelerated stability study) for relatively short period of time (3-6 months) which depends on claimed shelf life period as well as the zone (zone 1/2/3/4 of the world) to which the product is meant to be exported. This later study (with stress conditions) is to mine the alternating climates condition during the shelf life of the product. The stability parameters for all the formulation were evaluated after 15, 30, 45, 60, and 90 days for 40 °C at 75% RH. Tab 2: Disintegration Time of the Ciprofloxacin Fast disintegrating tablet Formulation With Disk Without Disk I II III I II III FA1 11min 43 sec 10 min 30 sec 10min 52sec 14min 32 sec 15min 11sec 15min 48 sec FA2 8min 2sec 9min 33 sec 8min 18 sec 11min 14sec 12min 31 sec 11min 56 sec FA3 4min 41 sec 5min 8 sec 4min 55sec 9min 23sec 9min 51 sec 8min 50sec FB1 11min 41 sec 10min 21 sec 10min 54 sec 14min 11sec 14min 56sec 13min 34sec FB2 8min 43sec 9min 21sec 9min 5sec 12min 37sec 14min 12sec 12min 44sec FB3 4min 21sec 5min 32 sec 4min 13sec 7min 23sec 7min 47 sec 6min 43sec FC1 9min 21sec 8min 55 sec 10min 12 sec 11min 15 sec 11min 24 sec 10min 55min FC2 7min 43sec 8min 11 sec 8 min 5sec 9min 22 sec 9min 17 sec 10 min 31 sec FC3 5min 22 sec 5min 42sec 6min 31sec 6 min 4 sec 7min 41sec 7min 18sec FD1 4min 45 sec 4min 52 sec 3min 21sec 7min 19 sec 7min 47 sec 6min 14 sec FD2 2 min 51 sec 2min 11 sec 1min 33sec 3min 46sec 4min 23 sec 4min 11sec FE1 4min 31sec 5min 55sec 4min 14sec 9min 50sec 8min 14sec 8min 19sec FE2 3min 11 sec 2 min 47 sec 3min 17sec 5min 11sec 6min 42sec 5min 4 sec
  • 15. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Harish G et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 285 Fig 1: IR spectra of the pure drug, Ciprofloxacin Hcl. Fig 2: IR spectra of FA Fig 3: IR spectra of FB Fig 4: IR spectra of FC Fig 5: IR spectra of the FD&FE Disintegrant Fig 6: IR spectra of the mixture of the Ciprofloxacin and FA Fig 7: IR spectra of the mixture of the Ciprofloxacin and FB Fig 7: IR spectra of the mixture of the Ciprofloxacin and FC
  • 16. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Harish G et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 286 0 20 40 60 80 100 0 10 20 30 40 %Cumulativeamountofdrug release Time in Minutes FA1 FA2 FA3 FB1 FB2 FB3 0 20 40 60 80 100 0 20 40 %Cumulativeamountofdrug release Time in minutes FC1 FC2 FC3 0 20 40 60 80 100 120 0 20 40 %Cumulativeamountofdrug release Time in minutes FD1 FD2 FE1 FE2 Fig 8: IR spectra of the mixture of the Ciprofloxacin and FD &FE Fig 9: Dissolution profile of Ciprofloxacin tablets Using FA and FB as disintegrant Fig 10: Dissolution profile of Ciprofloxacin tablets Using FC disintegrants Fig 11: Dissolution profile of Ciprofloxacin tablets Using FD disintegrants CONCLUSION Selecting appropriate formulation excipients and manufacturing technology can obtain the design feature of fast disintegrating tablet. The disintegrants have the major function to oppose the efficiency of the tablet binder and the physical forces that act under compression to form the tablet. The stronger the binder, the more effective must be the disintegrating agents in order for the tablet to release its medication. Ideally, it should cause the tablet to disrupt, not only into the granules from which it was compressed, but also into powder particles from which the granulation was prepared. From this study, it is concluded that the disintegrants such as Starch, SSG, CCS was compared with combination of disintegrants and in this study optimized combination of disintegrant prepared by intra and extra granulation method was found to be the most effective as they disintegrate rapidly when compared to other disintegrants, and the percentage drug release also shows a higher dissolution profile. REFERENCES Budavani S O, Neil N J, Smith A, The Merck Index, An Encyclopedia of Chemicals, Drugs and Biologicals, 29th Ed, Published by Merck Research Laboratories, Division of Merck & Co. Inc, 1996, 181
  • 17. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Harish G et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 287 Caramella C, Colombo P, Conte U, La Manna A, Tablet disintegration update: the dynamic approach, Drug. dev.Ind.Pharm, 13 (12), 1987, 2111-2145. Chaudhary K P R, Sujata Rao, Formulation and Evaluation of Dispersible tablets of poorly soluble drugs, Indian J. Pharm. Sci, (2), 1992, 31-32. Cohen Y, Lach JL, Factors affecting the Effect of Disintegrating Agent, J. Pharm. Sci, 52, 1963, 122. Cousin, Rapidly Disintegrable multiparticular Tablets, U S Patent, 5, 464, 632 (1995). E Sallam, Ibrahim H, R Abu Dahab, M Shubair, Enam Khalil, Drug.Dev and Industrial Pharmacy, Vol. 24(6), 1998, 501–507. Garnpimol C, Ritthidej, Parichat Chomto, Sunibhond Pummangura, Piamsak Menasveta, Chitin and Chitosan as Disintegrants in Paracetamol Tablets, Drug Dev. Ind. Pharm, 20(13), 1994, 2109-2134. Grasono Alesandro, US Patent 6,197,336 2001 Gupta G D, Gaud R S, Formulation and Evaluation of Nimesulide Dispersible Tablets Using Natural Disintegrants, Indian.J. Pharm Sci., 62(5), 2000, 339-342. H N Bhargava, D Shah, A Anaebonam, B Oza, An Evaluation of Smecta as a Tablet Disintegrant and Dissolution Aid, Drug. Dev.Ind.Pharm, 17(15), 1991, 2093-2102.
  • 18. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Papola Vibhooti Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 288 CHRONOTHERAPY FOR NOCTURNAL ASTHAMA Papola Vibhooti* Rajan G, Bisht Seema, Dr.Kothiyal Preeti Shri Guru Ram Rai Institute of Technology & Sciences Dehradun, Uttarakhand, India *Corresponding author:papola.vibhooti47@gmail.com ABSTRACT Chronotherapeutic drug delivery system is useful in the treatment of disease, in which drug availability is timed to match rhythm of disease, in order to optimize therapeutic effect and minimize side effect. Nocturnal asthma is defined as any sleep related worsening of reversible airway disease. Approximately 80 percent of severe asthmatic attacks occur between midnight and 8 a.m. Nocturnal asthma is associated with critical symptoms and urgent need for proper medications. The onset of nocturnal asthmatic attacks is rare in the first part of night, 80% of asthmatic attacks occur between midnight and 8 a.m., and deaths from asthma are more common during these hours. In a study of asthma mortality, 79% of the patients who died had a disturbed sleep before the death. In a survey of almost 8000 patients with varying degrees of asthma found that approximately - 75% of asthmatics attacks happened once a week with symptoms, 64 % three times a week, and 39 % every night. Nocturnal asthma is currently controlled by taking either a long-acting β2 agonists like salmetrol inhalers, sustained release theophylline. All the current sustained release formulation has a shortcoming of inability to maintain high blood levels for that long period. This may lead to leaving the patient unprotected against the worse events of nocturnal asthma. Thus, a smart drug delivery that is administrated before sleep and maintains high blood levels for longer period (from midnight to 8 am in the morning) could be very much beneficial for proper management of nocturnal asthma. Key words: Chronotherapy, cardian rhythm, nocturnal, morbidity, diurnal INTRODUCTION In order to increase the effectiveness of drug there are many approaches which have been applied. The pharmaceutical formulations which are for direct ingestion for oral administration and orally administrated drugs are generally absorbed from the gastrointestinal tract. Many functions of the human body show considerable change in a day. These variations cause changes both in disease state and in plasma drug concentrations (Lin, 2011). Coordination of biological rhythms with medical treatment is called chronotherapy. Chronotherapy considers a person’s biological rhythms in determining the timing and amount of medication to optimize a drug’s desired effects and minimize the undesired ones. Study of influence of biological rhythm on the effects of medication is known as chronopharmacology while the science of study of biological rhythms is known as chronobiology. With the understanding of biological time keeping the idea came that these rhythms must affect how the body responds to drugs administered over the course of the day (Hizli, 2009) (Ohdo, 2006). Appropriate timing of administration can improve efficacy and diminish toxicity. Chronotherapy is relevant when the risk or intensity of the symptoms of disease vary with time as in the case of allergic rhinitis, arthritis, asthma, myocardial infarction, congestive heart failure, stroke and peptic ulcer disease (Haus, 2007). The human circadian rhythm is based on sleep activity cycle, is influenced by our genetic makeup and hence, affects the body’s functions day and night (24-hour period) (Devdhawala, 2010). Coordination of biological rhythms with medical treatment is called Chronotherapy. Chronotherapy considers a person’s biological rhythms in determining the timing and amount of medication to optimize a drug’s desired effects and minimize the undesired ones. Study of influence of biological rhythm on the effects of medication is known as chronopharmacology while the science of study of biological rhythms is known as chronobiology. To understanding the biological time keeping the idea came that these rhythms must affect how the body responds to drugs administered over the course of the day (Awasthi, 2010) (Hizli, 2009). raditionally, drug delivery systems have focused on constant/sustained drug output with the objective of minimizing peaks and valleys of drug concentrations in the body to optimize drug efficacy and to reduce adverse effects. A reduced dosing frequency and improved patient compliance can also be expected for the controlled/sustained release drug delivery systems, compared to immediate release preparations (Saigal N, 2009). Some of the rhythms that affect our body are ultradian (cycles shorter than a day like firing of neurons take milliseconds), circadian (cycles lasting 24 h such as sleeping and waking pattern), infradian (cycles longer than a day like menstrual cycles) and seasonal rhythms (such as seasonal affective disorders causing more depression in
  • 19. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Papola Vibhooti Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 289 susceptible individuals in winter). Circadian rhythm governs every process of our body. The term circadian rhythm was first given by Halberg and Stephens in 1959. Chronotherapy: The term "chrono" basically refers to the observation that every metabolic event undergoes rhythmic changes in time. Perhaps the best known and studied chronobiology frequency is the circadian rhythm which approximates the earth's 24-hour rotation around the sun. Researchers have recently concluded that both disease states and drug therapy are affected by a multitude of rhythmic changes that occur within the human body. Chronotherapeutic refers to a treatment method in which in vivo drug availability is timed to match rhythms of disease in order to optimize therapeutic outcomes and minimize side effects. It is based on the observation that there is an interdependent relationship between the peak-thorough rhythmic activity in disease symptoms and risk factors, pharmacologic sensitivity and pharmacokinetics of many drugs. Biological rhythms concern to the control of biological functions including those of the autonomic nerve system, endocrine system, and immune system, are fundamental in homeostasis and in protection against various diseases. Chronotherapeutics: The first chronotherapy to be widely applied in clinical practice was introduced in the 1960s the alternate-day morning schedule of conventional tablet corticosteroid medication. Other chronotherapies have since been widely used in clinical medicine in the U S, Europe, and Asia; These include special evening theophylline systems for chronic obstructive pulmonary disease, conventional evening H2 -receptor ant agonists for peptic ulcer disease, and conventional evening cholesterol medications for hyperlipidemia. Chronotherapeutics refers to a treatment method in which in vivo drug availability is timed tomatch rhythms of disease in order to optimize therapeutic outcomes and minimize side effects. It is based on the observation that there is an interdependent relationship between the peak-to trough rhythmic activity in disease symptoms and risk factors, pharmacologic sensitivity, and pharmacokinetics of many drugs . Figure.1.Human biological lock Circadian Time Structure: Circadian rhythms are the rhythm in the chronotherapeutic and the dysfunction of circadian rhythms can affect the brain functioning and it can be improved by the chronotherapeutic approach. Circadian rhythms are self-sustaining, endogenous oscillations that occur with a periodicity of about 24 hours.Circadian rhythm regulates several body functions such as metabolism, physiology, behavior, sleep patterns, hormone production, and so on. The circadian rhythm not only affects most physiological functions but also influences the absorption, distribution, metabolism, and elimination (ADME) of drugs, leading to changes in drug availability and target cell responsiveness.
  • 20. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Papola Vibhooti Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 290 Figure.2. Human circadian time structure Circadian rhythms can change the sleep-wake cycles, hormone release, body temperature, and other important bodily functions driving the alteration of various physiological, biochemical, and behavioral processes. Circadian Rhythms of Diseases: The biological rhythm studies help in defining the temporal organization of human beings. One means of illustrating the human circadian time structure is to depict the peak time of 24-h rhythms on a clock--like diagram. The 24 h clock pattern of diseases showing prominent day-night patterns when symptoms of disease are most frequent .Variation in the severity of many diseases over a 24-hour period is well known diseases such as bronchial asthma, myocardial infarction, angina pectoris, rheumatic disease, ulcers 43, diabetes, and attention deficit syndrome, hypercholesterolemia and hypertension show symptomatic changes due to circadian rhythm city. Aggravation of asthmatic attacks occur after midnight or in the early morning due to limited lung function promoted by circadian changes at that time. Many common diseases also display a marked circadian variation during onset or exacerbation of symptoms, Figure.3.The circadian pattern of diseases Asthma: Asthma may be the most common disease with the largest circadian variation. It is considered as a chronic condition where airways are hyperreactive to certain irritants which can constrict them, and so making difficulty in breathing. Such a constriction is often called as bronchospasm and is followed by excess production of mucus and inflammation in the membranes lining the walls of the airways. “Breathing through a straw” is a commonly description that can explain the situation. Allergens, fumes, smoke, and/or dry and cold air are common irritants. Laughing or exercise can also cause the constriction of airways. Asthmatics vary considerably in the impact of illness on their life, response to treatment and severity of symptoms. Because asthma has such a striking circadian variation, several types of chronotherapy have been tried. In one study, use of a timed-release formulation of theophylline (Theo- 24) achieved therapeutic drug concentrations during the night and avoided toxic levels during the day. Asthma is well suited for chronotherapy, with beta 2- agonists and oral corticosteroids.
  • 21. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Papola Vibhooti Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 291 Nocturnal asthma: Some biological rhythms come about monthly or even annually, asthma changes fairly predictably on a circadian cycle or 24 hour. Even in normal, lung function differs between day and night. The activity of the lung exhibits a circadian rhythm with a maximum around 4 p.m. and a minimum around 4 a.m. In asthmatic patients, the intensity of variation in lung function is as much as 50% in a day. Bronchial reactivity generally follows the same circadian cycle in Asthmatic patients. It can be defined as any sleep-related Worsening of reversible airway disease. Shortness of breath or wheezing at night is symptoms generally shown. Nocturnal asthma is associated with critical symptoms and urgent need for proper medications. The onset of nocturnal asthmatic attacks is rare in the first part of night 80% of asthmatic attacks occur between midnight and 8 a.m., and deaths from asthma are more common during these 9 hours. In a study of asthma mortality, 79% of the patients who died had a disturbed sleep before the death. In a survey of almost 8000 patients with varying degrees of asthma found that approximately 75% of asthmatics attacks happened once a week with symptoms, 64 % three times a week, and 39 % every night. Causes of Nocturnal Asthma: Nocturnal asthma is probably because of multiple factors than to a single cause. Asthma attacks are aggravated mainly by irritants. Exposure to allergens daytime can be as important as exposure to allergens in the bedroom during sleep. A series of physiological events from three to eight hours are poorest about 4 a.m after the initial exposure called late-asthma response (LAR); it may match to the night time for some people and it can persist over nights. An increase in a susceptible patient's risk of LAR from 40 to 90 % by allergen exposure in the evening. In some people, the inflammation worsens correspondingly with circadian changes in peak expiratory flow rates in night. Another contributing factor to nocturnal asthma may be airway secretions. About 70 % of asthmatics experience postnasal drip and/or chronic sinusitis. Asthma often improves when the sinuses are cleared in daytime. Influence of airway temperature on onset of symptoms was studied. Bronchospasm is produced after a brief exposure to cold and dry air. Breathing warm humidified air can reverse this. Diagnosis of Nocturnal Asthma: If asthma symptoms worsen at night it is important to inform clinician. Monitoring lung function using a peak flow meter is necessary. Peak flow meter is a portable device that measures the lung volume and how time by which air can be expelled from the lungs. Low peak flow meter values indicates that there is a tightening of the airways, and can be an early warning of impending respiratory symptoms, such as shortness of breath and wheezing. Recording peak flow rates at bedtime serves as a document for nocturnal asthma. During any awakening at night and in the morning also serves as a record . Chronotharapy of asthma:Asthma and Sleep: In asthma, the relative role of circadian and sleep systems has been a subject of controversy, and this issue remains unresolved.Initially, it was suspected that sleep systems played the major role. In a study of shift-workers, there appeared to be an immediate phase shift in the circadian rhythm of peak expiratory flow (PEF) when subjects rotated shifts, such that the decline in airway function remained coupled to the sleep period. In asthma, the resistance increases progressively across the night, whether subjects sleep or not, although the increase is much greater during sleep. These results are supported by the observations that the onset of asthmatic attacks is less common in the first part of the night. These data allow us to reach certain conclusions. First, it seems likely that both circadian and sleep factors play a role in asthma. Also, the progressive decline in airway function across the night does not suggest a typical change in a neuronal control process coupled to sleep. Notably, airway function is maximal at the time of increased sleepiness during the afternoon, and declines as sleep pressure dissipates during sleep. Effect of nocturnal asthma on daytime performance, morbidity, and mortality: With the decreased sleep efficiency in asthma, and reports of daytime tiredness/sleepiness, the possibility exists that performance at work or school will be affected. A study of nocturnal asthmatic and control subjects demonstrated that the asthmatic subjects had increased scores for subjective sleepiness. This reflected decreased objective sleep quality. Interestingly, daytime cognitive performance was worse in the nocturnal-asthma group. This area of research needs further investigation. The morbidity of ventilatory failure, and also the mortality in asthma, are linked to the nocturnal worsening of lung function, which may be related to a blunted arousal mechanism caused by fragmented sleep. Interestingly, in one study of asthma mortality, 79% of the 168 patients who died had a sleep disturbance reported prior to the terminal event This contrasts with the usually accepted mortality risk factors of a previous ICU admission (5% in the study cited ), more than two hospitalizations/emergency- room (ER) visits in the preceding year (28%), or psychologic disturbance (13%).
  • 22. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Papola Vibhooti Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 292 Circadian/sleep physiologic and challenge response: Lung function and thoracic blood volume: It has been shown that sleep enhances the nocturnal increases in airway resistance and also leads to marked reduction in the volume of hyperinflated lungs in patients with nocturnal asthma. Such volume changes do not account directly for all of the nocturnal change in airway resistance. However, artificially reducing lung volumes in awake asthmatic individuals to their typical levels during sleep did trigger worsening of airway obstruction, suggesting that the effects of sleep on lung volume could contribute to the nocturnal worsening of asthma. The effects of sleep on lung volume could be mediated by several different mechanisms including:a sleep associated reduction in inspiratory muscle tone, a decrease in pulmonary compliance; and an increase in intrapulmonary blood pooling. In particular, the effects of sleep on intrapulmonary blood volume (IPBV) are intriguing, since there is evidence that such pooling of blood can promote airway narrowing. Using capillary volume (Vc) as a surrogate marker of IPBV, it has been shown that Vc increased overnight in asthmatic individuals with nocturnal worsening of lung function . Gastrointestinal Function and the Lung: There is significant variation in gastrointestinal (GI) function during sleep. The circadian rhythm of human basal gastric-acid secretion is characterized by a peak in the early evening and a nadir between 5:00 and 11:00 in the morning. There are conflicting data as to whether esophageal acid causes a decrease in airway function. In one study of sleeping individuals with nocturnal asthma, no significant acute or sustained change was observed in airflow resistance relative to periods of increased esophageal acid content, suggesting that gastroesophageal reflux (GER) contributed little to the nocturnal worsening of asthma. Although it appears that asthma is more responsive to the ets of GER during the diurnal cycle than during the nocturnal cycle, the exact role of circadian/sleep effects in esophageal acid-induced bronchoconstriction remains unclear. Nasal-Sinus–Lung Interaction: There is evidence that upper-airway disease (i.e., allergic rhinitis, sinusitis, and nasal polyps) influences and may contribute to the intensity of lower-airway disease. Allergic rhinitis, for example, can intensify airway responsiveness and even provoke asthma symptoms. Data indicate that treatment of allergic rhinitis diminishes bronchial responsiveness and asthma. Active sinusitis can also cause an increase in the asthma process as shown in animal models, which appears to involve drainage of nasal mediators into the lower airway. Other processes that link the nasal sinus to the lung have been identified in studies of viral infections of the nose that produce an increase in lower-airway reactivity. Also, there is a day– night cycle in nasal patency and perhaps in inflammation. All of these data suggest an important interaction between the nasal sinus and lower- airway function. Chronotherapy General Principles: Bodily functions have been incorrectly assumed to be relatively constant throughout the 24 h of the day and other periods of time. Numerous studies have shown that the kinetics and dynamics of pharmacotherapies vary significantly according to the biologic time of administration during the 24 h-cycle, menstrual cycle, or annually, owing to the cumulative effect of endogenous rhythms in crucial physiologic and biochemical functions. Chronotherapeutics is the synchronization of medication levels in time with reference to need, taking into account biologic rhythms in the pathophysiology of medical conditions, and/or rhythm- dependencies in patient tolerance for given chemical interventions. Chronotherapeutics can sometimes be achieved by the judicious timing of conventional sustained- release (SR) formulations, although reliance on special drug-delivery systems seems to constitute a more dependable means of matching drug level to biologic need and tolerance. β2-Agonists, Theophylline, and Anticholinergic Therapy: Certain SR formulations of theophylline can be administered so that a rising blood level of the drug occurs when airway obstruction is increasing, while adverse effects are reduced. For this purpose, SR theophylline is administered once daily, in the evening, for the management of nocturnal asthma. Various tablet formulations for the sustained-release of b-agonists have been used in a chronotherapeutic fashion for the management of asthma. As with theophylline, very little information exists about comparing the effects of or adding a long-acting b2-agonist oral preparation to an inhaled corticosteroid using chronotherapeutic techniques. Salmeterol has been shown to control symptoms of nocturnal asthma to a substantial degree, and to improve sleep quality and daytime cognitive performance in patients with chronic asthma. Drugs that antagonize the vagal nervous system should be useful in the management of nocturnal asthma as a means of counteracting the enhanced nocturnal parasympathetic tone that occurs in the disease.
  • 23. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Papola Vibhooti Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 293 Corticosteroids and Leukotriene-active Drugs: Corticosteroids have been used in a chronotherapeutic manner, with the finding that their long-term oral administration at 8:00 A.M. and 3:00 P.M. was more effective in controlling nocturnal asthma than the same doses given at 3:00 P.M. and 8:00 P.M. Other studies have shown that a single 3:00 P.M. dose of prednisone improved lung function and reduced airway inflammation more effectively than the same single dose given at 8:00 A.M. and 8:00 P.M (Beam, 1992). Not only can oral steroids be dosed chronotherapeutically, but inhaled corticosteroids can also be efficacious when used in this manner (Pincus, 1995). Although the leukotriene-active drugs, including zileuton, zafirlukast and montelukast, are new in the treatment of asthma, they have been shown to alleviate the symptoms and the decrement in lung function seen in nocturnal asthma. It has been shown that zileuton in particular decreased nighttime increases in leukotriene B4 (LTB4) and (LTE4) while improving lung function (Wenzel, 1995). Zafirlukast has also been shown to decrease nighttime awakenings and improve morning PEF rates . Although these agents have only been studied at set doses and times regardless of the presence or absence of nocturnalasthma, the improvements observed were significant, and it is likely that these agents will prove very useful in the treatment of nocturnal asthma when used chronotherapeutically. Ideal Characteristics of Chronotherapeutic Drug Delivery System should:  be non-toxic within approved limits of use,  have a real-time and specific triggering biomarker for a given disease state,  have a feed- back control system (e.g. self-regulated and adaptative capability to  circadian rhythm and individual patient to differentiate between awake – sleep status),  be biocompatible and biodegradable, especially for parenteral administration,  be easy to manufacture at economic cost,  be easy to administer in to patients in order to enhance compliance to dosage regimen. Chronotherapeutic drug delivery systems: Controlled release formulations can be divided into subgroups of rate-controlled release,delayed-release and pulsed-release formulations. Delayed-release formulations include time controlled release and site specific dosage forms. When constant drug plasma levels need to be avoided, as in chronotherapy, time-controlled or pulsed-release formulations are preferable, especially in the treatment of early morning symptoms. By timing drug administration, plasma peak is obtained at an optimal time and the number of doses per day can be reduced. Saturable first-pass metabolism and tolerance development can also be avoided. Various technologies to develop timecontrolled peroral drug delivery systems have been extensively studied in recent decades. Some of these systems are discussed in the following subsections. Enteric-coated systems: Enteric coatings have traditionally been used to prevent the release of a drug in the stomach Enteric coatings are pHsensitive and drug is released when pH is raised above 5 in the intestinal fluid. These formulations can be utilised in time-controlled drug administration when a lag time is needed. Because of the unpredictability of gastric residence, such systems cannot be the first choice when a time-controlled release is required. In the treatment of nocturnal asthma, a salbutamol formulation containing a barrier coating which is dissolved in intestinal pH level above about 6, has been successfully used. The system contains a core which is film coated with two polymers, first with HPMC and then with a gastro-resistant polymer (Eudragit® L30D). In this system the duration of the lag phase in absorption can be controlled by the thickness of the HPMC layer. Figure.4.Schematic representation of Enteric coated system Layered systems: These are one or two impermeable or semipermeable polymeric coatings (films or compressed) applied on both sides of the core. To allow biphasic drug release, a three-layer tablet system was developed . The two layers both contain a drug dose. The outer drug layer contains the immediately available dose of drug. An intermediate layer, made of swellable polymers,separates the drug layers. A film of an impermeable polymer
  • 24. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Papola Vibhooti Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 294 coats the layer containing the otherdose of drug. The first layer may also incorporate a drug-free hydrophilic polymer barrier providing delayed (5 h) drug absorption. Conte et al has also studied a multi-layer tablet system (Geomatrix®).It consists of a hydrophilic matrix core containing the drug dose. This kind of three layer device has been used in the treatment of Parkinsonian patients using L-- dopa/benserazide. Nighttime problems and early-morning symptoms of Parkinsonism can be avoided by using a dualrelease Geomatrix@ formulation, which allows daily doses of drug to be reduced and leads to extent of bioavailability 40 % greater than when a traditional controlled release formulation is employed. Figure.5. Geminex TIMERx technology based bilayered dual release tablet Time-controlled explosion systems (TES): These have been developed for both single and multiple unit dosage forms [80],[81] . In Both cases, the core contains the drug, an inert osmotic agent and suitable disintegrants. Individual units can be coated with a protective layer and then with a semipermeable layer, which is the rate controlling membrane for the influx of water into the osmotic core. As water reaches the core, osmotic pressure is built up. The core ultimately explodes, with immediate release of the drug. The explosion of the formulation can also be achieved through the use of swelling agents. Lag time is controllable by varying the thickness of the outer polymer coating. Sigmoidal release systems (SRS): For the pellet-type multiple unit preparations, SRS containing an osmotically active organic acid have been coated with insoluble polymer to achieve different lag-times. By applying different coating thicknesses, lag times in vivo of up to 5 hours can be achieved. Release rates from SRS, beyond the lag time, has been found to be independent of coating thickness. Press-coated systems: Delayed-release and intermittent-release formulations can be achieved by press-- coating. Presscoating, also known as compression coating, is relatively simple and cheap, and may involve direct compression of both the core and the coat, obviating the need for a separate coating process and the use of coating solutions. Materials such as hydrophilic cellulose derivatives can be used and compression is easy on a laboratory scale. On the other hand, for large-scale manufacture, special equipment is needed. The major drawbacks of the technique are that relatively large amounts of coating materials are needed and it is difficult to position the cores correctly for the coating process. Conte et al have developed a press coated device in which the inner core contains the drug and the outer coat is made of different types of polymers. The outer barrier, which controls drug release, can be either swellable or erodible. Lag times can be varied by changing the barrier formulation or the coating thickness (Halsas M, 1998). Hydrophilic polymers such as hydroxypropyl methylcellulose and sodium alginate have been used in the coat to control drug release. In recent years, various controlled release, especially time-controlled release; drug delivery systems based on compression coating technology have been studied. Most such Formulations release drug after a lag phase, followed by a rapid dissolution of a core. Tablets formulated with Penwest's TIMERx ® oral controlled release technology comprise an inner core containing drug and an outer layer compression-coated with TIMERx, a hydrophilic matrix of the heteropolysaccharides xanthan and locust bean gum (Baichwal A, 2002). Figure.6. TIMERx based drug delivery system Examples of chronopharmaceutical technologies: Currently key technologies in chronopharmaceutics includes: CONTINR, physico-chemical modification of the active pharmaceutical ingredient (API), OROSR, CODASR,
  • 25. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Papola Vibhooti Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 295 CEFORMR, DIFFUCAPSR, chronomodulating infusion pumps, TIMERxR, threedimensional printing, controlled-release (CR) erodible polymer and CR microchip strategies. Readers may find advantages and disadvantages of each technology depending on their specific needs on the website of each developer/marketer website before selection. Informations on FDA approval status and dosage formed were compiled from the FDA electronic orange book. For asthma (CONTINR technology): In this technology, molecular coordination complexes are formed between a cellulose polymer and a non-polar solid aliphatic alcohol optionally substituted with an aliphatic group by solvating the polymer with a volatile polar solvent and reacting the solvated cellulose polymer directly with the aliphatic alcohol, preferably as a melt. This constitutes the complex having utility as a matrix in controlled release formulations since it has a uniform porosity (semipermeable matrixes) which may be varied. This technology has concretely enabled the development of tablet forms of sustained-release aminophylline, theophylline, morphine, and other drugs. Research suggested that evening administration of UniphylR (anhydrous theophylline) tablets represented a rational dosing schedule for patients with asthma who often exhibit increased bronchoconstriction in the morning. Patients demonstrated improved pulmonary function in the morning compared with use of twice- daily theophylline when once-daily UniphylR was administered in the evening. Thus, evening administration of once-daily theophylline may block the morning dip in lung function commonly seen. CONTINR technology provides for closer control over the amount of drug released to the bloodstream, and benefits patients in terms of reducing the number of doses they need to take every day, providing more effective control of their disease (particularly at night), and reducing unwanted side effects. Marketed preparation for asthma till now  FDA approval date – Sept.01.1982  API- Theopylline  Proprietary name dosage form - Uniphyl  Chronopharmaceutical technology- CONTIN CONCLUSION Chronopharmaceutics will certainly improve patient outcome and optimize disease management in the future. Research in chronopharmacology has demonstrated the importance of biological rhythms in drug therapy and this has led to a new approach to the development of drug delivery systems. Optimal clinical outcome cannot be achieved if drug plasma concentrations are constant. If symptoms of a disease display circadian variation, drug release should also vary over time. Different technologies have been applied to develop time-controlled, pulsed, triggered and programmed drug delivery devices in recent years. Since it is seems that timing of drug administration in disease therapy has significant impact upon treatment success, chronotherapeutics remains an important area for continuing research. It is concluded that the treatment of asthma with the Chrono-optimized preparation over night is more effective than treatment with a conventional preparation in twice-daily dosage. In addition, lung function showed greater stability, throughout the day, with once-daily evening therapy than with traditional 12 hr dosing. It is well known that human body temperature, blood pressure, and pulse rate reach high values during the day and fall at night. Similarly, all other physiological functions and activities are subject to a daily cyclical variation known as their circadian rhythm. The respiratory function is no exception and is known to experience a trough in activity from late night until early morning. In other words, in application once daily at bedtime could be expected to prevent asthma attacks for practically the entire 24-hour period and, as maximum blood concentration is reached in the early morning, would be particularly effective against attacks caused by morning dip. REFERENCES Alexander A, Dulal KT, Ajazuddin, Mukesh S, Monesh S, Swarna. .Multidose Therapy (MDT) Treatment for Helicobacter Pylori Infection Leading to Gastric Ulcer and Carcinoma: A Review; Res J Pharmacol Pharmacodynamics 3(3): 2011; 140-147. Alexander A, Sharma S, Ajazuddin, Giri TK, Swarna, Shukla P. Various evaluation parameters used for the evaluation of different mucoadhesive dosage forms. A review. Inter. J. Drug Formulation res 2011; 2:1-26. Ali J, Baboota S, Ahuja A, Saigal N. Distinctive features of “chronotherapeutic” and “pulsatile” drug delivery
  • 26. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Papola Vibhooti Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 296 systems negating the practice of their interchangeable terminology. J. Drug Target 2010; 18 (6):413–419. Awasthi R, Kumar P, Pawar VK. Chronotherapy: science and technology of drug scheduling on the basis of biological rhythm. JChrDD 2010; 1: 09-18. Ballard R. D, Pak J,White D.P, Influence of posture and sustained loss of lung volume on pulmonary function in asthmatics. Am. Rev. Respir. Dis. 144, 1991, 499–503 Ballard R. D., Saathoff M. C., Patel D. K., Kelly P. L, Martin RJ, Effect of sleep on nocturnal bronchoconstriction and ventilatory patterns in asthmatics. J. Appl. Physiol. 67, 1989, 243–249. Ballard R. D.,Irvin C. G., Martin R. J., Pak J.,Pandey R., White D.P, Influence of sleep on lung volumes in asthmatic patients and normal subjects, J. Appl. Physiol, 68, 1990, 2034–2041 Ballard, R.D., M. C. Saathoff, D. K. Patel, P. L. Kelly, and R. J. Martin, Effect of sleep on nocturnal bronchoconstriction and ventilator patterns in asthmatics, J. Appl. Physiol. 67, 1989, 243–249. Beam, W. R., D. E. Weiner, and R. J. Martin, Timing of prednisone and alterations of airways inflammation in nocturnal asthma, Am. Rev. Respir. Dis, 146, 1992, 1524–1530. Bellofiore, S., G. U. diMaria, and J. G. Martin, Changes in upper and lower airway resistance after inhalation of antigen in sensitized rats, Am. Rev. Respir. Dis, 136, 1987, 363–368. Brugman S. M.,Larsen G. L.,Henson P.M, Honor J.,Irvin CG, Increased lower airways responsiveness associated with sinusitis in a rabbit model. Am. Rev. Respir. Dis. 147, 1993, 314–320 Catterall, J. R., G. B. Rhind, and I. C. Stewart, Effect of sleep deprivation on overnight bronchial constriction in nocturnal asthma, Thorax 41, 1986, 676–680. Clark, T. J. H., and M. R. Hetzel, Diurnal variation of asthma, Br. J. Dis. Chest 71, 1977, 87–92 Corren J, Adinoff A.D., Irvin CG, Changes in bronchial responsiveness following nasal provocation with allergen, J. Allergy Clin. Immunol, 89, 1992, 611–618. D’Alonzo, G. E, M. H. Smolensky, S. Feldman, Y. Gnosspelius, and K Karlsson, Bambuterol in the treatment of asthma, Chest, 107, 1995, 406– 412. D’Alonzo, G. E., M. H. Smolensky, S. Feldman, L. A. Gianotti, M. B.Emerson, H. Staudinger, and V. W. Steinijans, Twenty-fourhour lung function in adult patients with asthma, Am. Rev. Respir. Dis, 142, 1990, 84–90 Desjardin J. A, Sutarik J. M, Suh B. Y, Ballard R.D, Influence of sleep on pulmonary capillary blood volume in normal and asthmatic subjects. Am. J. Respir. Crit. Care Med. 152, 1995, 193–198 DevdhawalaMehulG.and Seth Avinash K. Current status of chronotherapeutic drug delivery system: An overview. J Chem Pharm Res 2010; 2(3):312-328. Elliott WJ. Timing treatment to the rhythm of disease, A short course in chronotherapeutics. Postgrad Med 2001; 110: 119–212 (1125–126, 129). Fitzpatrick, M. F., H. Engelman, K. F. Whyte, I. J. Deary, C. M. Shapiro, and N. J. Douglas, Morbidity in nocturnal asthma: sleep quality and daytime cognitive performance, Thorax 46, 1991, 569–573. Fitzpatrick, M. F., T. Mackay, H. Driver, and N. J. Douglas, Salmeterol in nocturnal asthma: a double-blind, placebo-controlled trial of a long-acting inhaled b2 agonist, Br. Med. J. 301, 1990, 1365–1368 Halberg F, Haus E, Cardoso SS, Scheving LE, Kuhl JFW, Shiotsuka R, Toward a chronotherapy of neoplasia: tolerance of treatment depends upon host rhythms, Experientia, 29, 1973, 909 - 1044. Halberg F, Stephens AN. Susceptibility to ouabain and physiologic circadian periodicity, Proc Minn Acad Sci, 27, 1959, 139 - 43. Haus E, Chronobiology in the endocrine system, Advanced Drug Delivery Reviews, 59, 2007, 985 - 1014.
  • 27. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Papola Vibhooti Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 297 Hetzel, M. R., and T. J. H. Clark, Comparison of normal and asthmatic circadian rhythms in peak expiratory flow rate, Thorax, 35, 1980, 732– 738. Hizli FG, Agar gun MY. Delayed Sleep Phase Type Sleep Disorder and Chronotherapy. Turkish Journal of Psychiatry 2009:1 - 5. Hrushesky WJM, Von Roemeling R, Lanning RM, Rabatin JT, Circadian-shaped infusion of floxuridine for progressive metastatic renal cell carcinoma, J ClinOncol, 8, 1990, 1504 - 1513. Lemmer B, The clinical relevance of chronopharmacology in therapeutics. Pharmacol Res, 33, 1996, 107–115. Lin SY. Chronotherapeutic approach to design a thermo responsive membrane for transdermal drug delivery. Curr Drug Deliv 2004; 1: 249–263. Litinski M, Scheer FA, Shea SA. Influence of the circadian system on Disease severity. Sleep Med Clin 2009; 4: 143–163. Montplaisir, J., J. Walsh, and J. L. Malo, Nocturnal asthma: features of attacks, sleep and breathing patterns. Am. Rev. Respir. Dis,125, 1982, 18–22. Moore J. G., Englert E, Circadian rhythm of gastric acid secretion in man. Nature 226, 1970, 1261–1262. Morrison, J. F. J., and S. B. Pearson, The effect of the circadian rhythm of vagal activity on bronchomotor tone in asthma, Br. J. Clin. Pharmacol, 28, 1989, 545–549. Ohdo S. Changes in Toxicity and Effectiveness with Timing of Drug administration Implications for Drug Safety Drug Safety. 2003; 26 (14): 999-1010. Ohdo S. Chronopharmaceutics: pharmaceutics focused on biological rhythm. Biol Pharm Bull 2010; 33:159–167. Ohdo S. Chronotherapeutic strategy: rhythm monitoring, manipulation and disruption. Adv Drug Deliv Rev 2010; 62: 859–875. Pincus, D. J., S. J. Szefler, L. M. Ackerson, and R. J. Martin, Chronotherapy of asthma with inhaled steroids: the effect of dosage timing on drug efficacy, J. Allergy Clin. Immunol, 95, 1995, 1172–1178. Reinberg, A., P. Gervais, M. Chaussade, G. Fraboulet, and B. Duburque, Circadian changes in effectiveness of corticosteroids in eight patients with allergic asthma, J. Allergy Clin. Immunol, 71, 1983, 425–433. Ritschel W.A. and Sabouni A, Permeability of [3H] water across a porous polymer material used as rate-limiting shell in compression-coated tablets, J. Controlled Release 12, 1990, 97-102. Robertson, C. E., A. R. Rubinfeld, and G. Bowej, Deaths from asthma in Victoria: a 12-month study. Med. J. Austr. 152, 1990, 511–517 Robertson, C.E., Rubinfeld, A.R., Bowej G, Deaths from asthma in Victoria: a 12-month study. Med. J. Austr. 152, 1990, 511-517 Saigal N, Baboota S, Ahuja A, Ali J. Site Specific Chronotherapeutic Drug Delivery Systems: A Patent Review. Recent Patents on Drug Delivery & Formulation. 2009; 3: 64-70. Selby, C., H. M. Engleman, M. F. Fitzpatrick, P. M. Sime, T. W. MacKay, and N. J. Douglas, Inhaled salmeterol or oral theophylline in nocturnal asthma? Am. J. Respir. Crit. Care Med, 155, 1997, 104–108 Smolensky M.H, Chronobiology and chronotherapeutics: Applications to cardiovascular medicine, Am J Hypertens, 9, 1986, 11S-21 S. Tan W. C.,Martin R. J.,Pandey R.,Ballard R. D, Effects of spontaneous and simulated gastroesophageal reflux on sleeping asthmatics. Am. Rev. Respir. Dis. 141, 1990, 1394–1399. Traynor K, Newton DW, Hrushesky JM, Reiter RJ. A pharmacist's primer on chronotherapeutics. American Pharmacy1992; 3:261-269. Vener KJ, Reddy A. Timed treatment of the arthritic diseases: a review and hypothesis, Arthritis Rheum, 22,
  • 28. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Papola Vibhooti Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 298 1992, 83– 97. Watson W. T. A., Becker A. B., Simons F. E. R, Treatment of allergic rhinitis with intranasal corticosteroids in patients with mild asthma: effect on lower airway responsiveness, J. Allergy Clin. Immunol, 91, 1993, 97–101. Wenzel, S. E., J. B. Trudeau, D. A. Kaminsky, J. Cohn, R. J. Martin, and J. Y. Westcott, Effect of 5-lipoxygenase inhibition on bronchoconstriction and airway inflammation in nocturnal asthma, Am. J. Respir. Crit. Care Med, 152, 1995, 897–905. Youan BIBC. Chronopharmaceutics: Gimmick or clinically relevant approach to drug delivery. J Controlled Release. 2004; 98: 337 - 53. Youan BIBC. Chronopharmaceutics: Gimmick or clinically relevant approach to drug delivery. J Controlled Release 2004; 98: 337 - 53.
  • 29. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Hemant Singh Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 299 RECENT TRENDS IN SCOPE AND OPPORTUNITY OF CLINICAL RESEARCH IN INDIA Hemant Singh* , Abhinav Srivastva I.E.C. College of Eng & Technology, Greater Noida. *Corresponding author:hemanthsingh198611@gmail.com ABSTRACT Clinical Research is one of the most knowledge-intensive industry. It is complete biography of drug from its inception in the lab to its introduction to the consumer market and beyond. Any molecule is identified is subjected to pre-clinical and clinical trials before entering in to market. Though post marketing surveillance is also part of the same. Pre-clinical studies are associated with effect of drug on animals. All the toxicological studies, tests for teratogenicity, carcinogenicity are carried out.After this, the data obtained from the studies are submitted as an IND (Investigational Drug Application) to take permission for human studies. Then, it is enter in to clinical trials. There are 4 phases in it. It is very clear that India has become a very preferred destination for clinical research. The industry is growing exponentially and is expected to reach Rs. 7000 Cr by 2010. Statics shows that if India's clinical trial business grows to 10% of that in US by 2015, the industry will need approximately 50,000 clinical research professionals. Key Words: Clinical Research, healthy volunteers, Clinical Trial Phases. INTRODUCTION Clinical Research is a systematic study to evaluate the effectiveness and safety of medications or medical devices by monitoring their effects on large groups of people. Clinical research has become a multi-billion and multidisciplinary industry. A number of factors favor India as a clinical research hub. There are numerous government-funded medical and pharmaceutical institutions with state-of-the-art facilities, which can serve as ideal centers for multi-centered clinical trials.India boasts of well-trained and qualified manpower, well versed in English. The research and the development process in India can be done at a more affordable price. More importantly, there is vast clinical material, which can be utilised, due to the prevalence of a large variety of diseases, including widespread cases of cancer and diabetes, India is viewed the world over as the ideal location for clinical research trials for the pharmaceutical industry.India is becoming a hub for clinical research; the demand for professionals in this field is growing rapidly. Clinical research business in India will be worth $1 billion by 2010. Thus, there will soon be a massive demand for clinical research professionals, making it an interesting career option with massive growth potential. According to a survey, there are 2,50,000 vacancies available worldwide 50,000 job openings in India by 2010. Highest remuneration packages owing to the shortage of skilled professionals.There are high demand for trained professionals in this field; the pay package is impressive at the entry level. Freshers can expect a pay packet of around three lakhs or more per anum. If you have a master’s degree backing your qualifications, then the amount is almost doubles. Clinical research is an industry where experience counts, thus the longer you are in this field; higher the salary you can expect.So there is really a very good scope for clinical research in India. HUMAN CLINICAL TRIAL PHASES Phase I: Studies assess the safety of a drug or device. This initial phase of testing, which can take several months to complete, usually includes a small number of healthy volunteers (20 to 100), who are generally paid for participating in the study. The study is designed to determine the effects of the drug or device on humans including how it is absorbed, metabolized, and excreted. This phase also investigates the side effects that occur as dosage levels are increased. About 70% of experimental drugs pass this phase of testing. Phase II: Studies test the efficacy of a drug or device. This second phase of testing can last from several months to two years, and involves up to several hundred patients. Most phase II studies are randomized trials where one group of patients receives the experimental drug, while a second "control" group receives a standard treatment or placebo. Often these studies are "blinded" which means that neither the patients nor the researchers know who has received the experimental drug. This allows investigators to provide the pharmaceutical company and the FDA with comparative information about the relative safety and effectiveness of the new drug. About one-third of experimental drugs successfully complete both Phase I and Phase II studies.
  • 30. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Hemant Singh Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 300 Phase III: Studies involve randomized and blind testing in several hundred to several thousand patients. This large-scale testing, which can last several years, provides the pharmaceutical company and the FDA with a more thorough understanding of the effectiveness of the drug or device, the benefits and the range of possible adverse reactions. 70% to 90% of drugs that enter Phase III studies successfully complete this phase of testing. Once Phase III is complete, a pharmaceutical company can request FDA approval for marketing the drug. Phase IV: Studies, often called Post Marketing Surveillance Trials, are conducted after a drug or device has been approved for consumer sale. Pharmaceutical companies have several objectives at this stage to compare a drug with other drugs already in the market; to monitor a drug's long-term effectiveness and impact on a patient's quality of life; and to determine the cost-effectiveness of a drug therapy relative to other traditional and new therapies. Phase IV studies can result in a drug or device being taken off the market or restrictions of use could be placed on the product depending on the findings in the study. SCOPE FOR CLINICAL RESEARCH IN SOUTH ASIAN NATIONS With a very large number of drugs, worth more than $50 billion in annual revenues, coming off patent in the next few years, many western pharma companies are now increasingly relinquishing business activities that are not considered core, such as clinical research, and are moving towards contract research. Since in the western countries, clinical research is characterized by extremely high costs and long gestation periods and the same work can be performed in developing countries at a fraction of the cost and much faster, many global pharma companies are increasingly turning their attention to Asia to benefit from low R&D costs in the region and also to gain access to Asia's drug markets. Clinical trials have thus gone ‘global,’ because CROs find it easier to conduct them in underdeveloped countries, as this is cheaper or has less ethical encumbrances or legal risks. Naïve patient populations, English speaking doctors, low costs and other advantages offered by developing countries have opened up new avenues for the clinical trials market. Hence there is lot of scope for clinical research activities for the South Asian nations if they make use of the upcoming opportunities in this field. India: The future of clinical research in India continues to be hazy. While on the one hand there are predictions that India will be the next hotspot for clinical trials, on the other , industry forums speak of ongoing challenges and stagnant growth for a variety of reasons, such as regulatory delays in approval, escalating costs, inconsistent quality, ethical irregularities etc. While several CROs have started operations in India basing their future on the rosy predictions, it is equally true that existing players are struggling to grow or even sustain their current business. Since registration of clinical trials in Clinical Trial Registry of India (CTRI) was made mandatory from mid-2009 onwards, close to 1800 trials have been registered in this database. Of these, about 69 per cent are sponsored by the pharmaceutical industry (both multinational and Indian companies), the rest being funded by grants from various government and not-for-profit institutions (e.g. DBT, CSIR, AIIMS, ICMR, Ministry of Defence,). Indian CROs can now look forward for more opportunities to conduct clinical studies within the generic space. For drugs that are not absorbed in the GI tract, plasma concentrations are not useful to determine delivery of the drug to the site of activity, and hence bioequivalence of the generic product to the innovator cannot be established by BA/BE studies. In such cases, one needs to demonstrate therapeutic equivalence with clinical or pharmaco-dynamic studies. Assocham had predicted that India would garner about 15 per cent of the global clinical trials opportunity. However, out of over 1,00,000 human trials being conducted in 178 countries, less than 2,000 (two per cent) are being done in India compared to over 9,000 (nine per cent) in China. Some of the advantages that India offers as a clinical trial destination are : availability of diverse patient population across major therapeutic segments such as oncology, metabolics, neurology etc; high degree of compliance to international guidelines such as the ICH GCP and the regulations laid down by the US FDA; availability of well qualified, English speaking research professionals including physicians; lower costs compared to the west; increasing prevalence of illnesses common to both developed and developing countries; availability of good infrastructure and changes in Patent Laws since January 2005. Bangladesh: According to an article in The Pharma World, leading health Journal in Bangladesh, notwithstanding its potential, clinical research is still underdeveloped in Bangladesh. There is a lack of capacity for bioequivalence studies, no analytical capacity (samples have to be shipped to foreign countries such as Singapore for any analytical treatment) and limited bio-banking and documentation of clinical specimens. There
  • 31. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Hemant Singh Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 301 is no CRO activity in the country. This cripples the ability of the health and pharmaceutical industry to move forward, severely stunts the professional growth of health care personnel and limits their ability to become competitive for funding in the global arena. Fortunately, with appropriate input from global scientists and consultants, Bangladesh appears very well placed to fill this void and develop world-class clinical research capacity. There is a significant foundation for such clinical research capacity in the health care sector in Bangladesh. Hospitals in the country have the advantage of access to a large population base presenting a range of clinical conditions. These hospitals have talented physicians who are fluent in English and eager to take advantage of opportunities to expand their expertise and scope of activities into state-of-the-art health care research. Moreover Bangladesh has a significant generic pharma industry, currently marketing its products mainly in the domestic market and in non-regulated international markets. Building Contract Clinical Research capacity in Bangladesh will serve the local pharmaceutical companies and allow Bangladesh to take its deserved position in the rapidly-growing global clinical research products and services market. Bangladesh can be the next frontier in the global CRO industry. Based on GDP and the volume of the pharma industry compared to other Asian economies such as India and China, Bangladesh could potentially capture five per cent of the Asian CRO market by 2020. The country is well poised to launch a CRO for multiple reasons that involve both its own fledgling pharma industry as well as the increasing demands in the global market, the article says. The development of a globally-competitive CRO will have an impact beyond the domestic pharma industry. It will attract foreign companies, seeking high quality research at more affordable prices. It will also have a multiplier effect on other areas of the economy that transcend the pharma industry, and contribute to the transition from low-wage labour-intensive activities into higher-wage knowledge-based industries. Building capacity for upscale knowledge industries, including biotechnology and health science, historically has had a remarkable impact on wealth building and human development in the West and is expected to have a similar positive impact in emerging economies. Global Health Expansion (GHE) LLC is a Washington DC-based company that is currently partnering with Bangladeshi entities to develop a world class CRO in Bangladesh. The goal of GHE is to team international know-how in regulatory issues, clinical pharmacology and contract clinical research with the current capacity in Bangladesh in clinical research and drug manufacturing. This could be done by building synergies between academia and industry in order to develop the capacity (human and physical) for a true knowledge-based industry in Bangladesh. Driving such efforts through an international partnership will create unique opportunities for all partners, the article points out. Pakistan: The clinical research industry is still in its infancy in Pakistan despite having a large pool of patients, a large number of English-speaking physicians, low value of rupee, a network of high volume medical centres and a good know how of this business as many physicians have conducted clinical trials in other countries The country is the sixth most populous country in the world and being a developing country, the patients’ population in Pakistan is also very large. Industry experts are of the view that unless the government encourages research culture and streamlines public-sector universities, the country would continue to lose opportunities in this important arena. Some experts believe that the local private sector is not motivated to conduct research. This is in contrast to China and India, which have emerged as the top destinations for researchers in the global pharmaceutical industry. If Pakistan is interested in attracting more clinical trials to the country, it must take bold steps in these areas, publicize them and inspire the confidence of international sponsors in the measures taken. The speed of current healthcare and regulatory reforms will be crucial in this respect, they point out. Sri Lanka: Sri Lanka is an emerging market for clinical trial business with some contract research organizations already operational in the country. Although Sri Lanka is getting involved more and more in clinical research, it significantly lacks professionals formally trained in conducting trials. The need for clinical research training in Sri Lanka for medical and life science graduates has been stressed upon by various research papers published by Sri Lankan authors. Clinical trials are sets of tests in medical research and drug development that generate safety and efficacy data (or more specifically, information about adverse drug reactions and adverse effects of other treatments) for health interventions (e.g., drugs, diagnostics, devices, therapy protocols). They're conducted only after satisfactory
  • 32. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Hemant Singh Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 302 information has been gathered on the quality of the nonclinical safety, and health authority/ethics committee approval is granted in the country where approval of the drug or device is sought. The U.S. National Institutes of Health (NIH) organizes trials into five different types: Prevention trials look for better ways to prevent disease in people who have never had the disease or to prevent a disease from returning. These approaches may include medicines, vitamins, vaccines, minerals, or lifestyle changes. Screening trials test the best way to detect certain diseases or health conditions. Diagnostic trials are conducted to find better tests or procedures for diagnosing a particular disease or condition. Treatment trials test experimental treatments, new combinations of drugs, or new approaches to surgery or radiation therapy. Quality of life trials (supportive care trials) explore ways to improve comfort and the quality of life for individuals with a chronic illness. Compassionate use trials or expanded access trials provide partially tested, unapproved therapeutics to a small number of patients who have no other realistic options. Usually, this involves a disease for which no effective therapy exists, or a patient who has already attempted and failed all other standard treatments and whose health is so poor, he does not qualify for participation in randomized clinical trials. Usually, case-by-case approval must be granted by both the FDA and the pharmaceutical company for such exceptions. The clinical trial, in its simplest form, involves the application of the experimental variable – treatment to a person or group of persons – and observation during or following application of the treat ment to measure its effect. That measure (outcome measure) may be death, occurrence or recurrence of some morbid condition, or a difference indicativeof change (e.g. difference in blood pressure measured for each person just prior to the start of treatment and again at some point during or after treatment). There is no way to “test” a treatment or to “prove” its effectiveness in the absence of some absolute or relative measure of success. Trials are said to be controlled if the effect of a treatment is measured against a comparison treatment administered over the same time period and under similar conditions. That comparison treatment may be another test treatment or, depending on circumstances, a control treatment consisting of an accepted standard form of therapy, a placebo or sham treatment, or observation only (no treatment). A trial is said to be uncontrolled if it does not have a comparison treatment or if the enrolment to and administration of the test and comparison treatments is not concurrent. History: Prove thy servants, I beseech thee, ten days; and let them give us pulse to eat, and water to drink. Then let our countenances be looked upon before thee, and the countenance of the children that eat of the portion of the King’s meat: and as thou seest, deal with thy servants. So he consented to them in this matter, and proved them ten days. And at the end of ten days their countenances appeared fairer and fatter in flesh than all the children which did eat the portion of the King’s meat. Fortuitous events can produce conditions reminiscent of the features of a trial. One such account is that given by Ambroise Par´e (surgeon, 1510–1590) during the battle in 1537 for the castle of Villaine. The treatment for gunshot wounds in Par´e’s time was boiling oil poured over the wound. Because of the intensity of the battle, Par´e ran out of oil and resorted to using an ointment made of egg yolks, oil of roses, and turpentine. The result of his “trial” is summarized by his observation the morning after the battle: Treatment protocol: The treatment protocol (the general term, study protocolor trial protocol has broader meaning and refers to the constellation of activities involved in conducting a trial) of the trial specifies the treatments being studied, the manner and methodof usage and administration, and conditions under which other treatments are called for when needed for the well-being of those enrolled.Protocols for all research involving human beings are subject to review and approval by institutional review boards (IRBs) or ethics review boards (ERBs) before implementation and at periodic intervals thereafter until the research is finished Only patients judged eligible (as determined by specified may be enrolled, and among those, only those who consent to participate in the trial. Persons are under no obligation to enroll or to continue once enrolled, and must be so informed prior to being enrolled. All trials involve data collection at various time points over the course of enrollment and follow-up of persons. The amount collected per person depends on the nature of the disease or condition being treated and on the nature of the treatment process implied by the study treatments being used. Classes of trials: Most clinical trials involve parallel treatment designs, i.e. designs where an assignment unit (usually a person) is assigned to receive only one of the treatments under study. The word parallel indicates that two or more groups of assignment units are proceeding through the trial side by side, with the only ostensible difference (other than baseline differences in the composition of the groups) being the treatment administered.
  • 33. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Hemant Singh Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 303 The goal in trials with parallel treatment designs is for each person enrolled to receive the assigned treatment and to have no exposure to any of the other treatments under study in the trial (except where the requirements for proper care are overriding and make such exposure necessary). Drug trials: Compounds, no matter how promising or impassioned the pleas for use, have to go through a series of tests in animals before they can be tested in humans. Those considered to lack promise after animal testing do not come to testing in humans. Most drug trials are done under an INDA held by the sponsor of the drug. The “sponsor” in the vernacular of the Food and Drug Administration (FDA) is typically a drug company, but can be a person or agency without “sponsorship” interests in the drug. Regulations require investigators to report adverse events to the FDA. The general guidelines regarding consent are similar, but not identical, to those promulgated by the Office for Protection from Research Risks (OPRR) for IRBs. Randomized trials: A randomized trial is a trial having a parallel treatment design in which treatment assignment for persons (treatment units) enrolled is determined by a randomization process similar to coin flips or tossings of a die. The trialist’s purpose in randomization is to avoid selection bias in the formation of the treatment groups. Randomization does not guarantee comparability of the treatment groups with regard to the various entry characteristics of interest. Indeed, one can, by chance, have differences among the treatment groups. A large difference (one yielding a small P value) can arise by chance and, hence, cannot be taken as prima facie evidence of a “breakdown” (e.g. “peeking” or other purposeful acts aimed at determining assignment before issue) of the randomization process, unless supported by other evidence of a “breakdown”. The randomization may be simple (complete) or restricted. The purpose of restriction is to force the assignments to satisfy the specified assignment ratio at intervals during enrolment. Masking: Masking is the purposeful concealment of some fact or condition and is done to keep knowledge of that fact or condition from influencing the behavior, observation, or reporting of persons so masked. Masking, in the context of trials, is imposed to reduce the likelihood of a treatment-related bias due to knowledge of treatment assignment. Masked treatment administration has been used as a mark of “quality” for trials. There is, therefore, a tendency to view results from masked trials as more reliable than those from unmasked trials. In truth, however, masked treatment administration is rarely 100% effective. All forms of treatment, and especially those involving drugs, produce side-effects and telltale signs that may serve to unmask treatment. Hence, the protection provided by masking can be illusory. As a result, it is better to make assessments of “quality” in terms of the risk of treatment-related bias and the likely effect of such bias, if present, on the results reported. The risk of treatment- related bias is low for “hard” outcome measures and with explicitly defined treatment protocols, even in the absence of masked treatment administration. Analysis: The protection provided against treatment-related bias by the assignment process is futile if the analysis is biased. Treatment comparisons, to be valid, must be based on analyses that are consistent with the design used to generate them. Analyses involving arrangements of data related to treatment administered may be performed, but only as supplements to the primary analyses. They should not and cannot serve as replacements for those analyses. TYPES OF CLINICAL TRIALS We often think of clinical trials as a method of studying new drugs, but many different types of trials are in process to evaluate cancer. Prevention trials:Prevention trials look at substances and lifestyle factors that may raise or lower the risk of developing cancer Screening trials: Screening trials study methods to diagnose cancer in its early stages when it is often more curable Diagnostic trials: Diagnostic trials are done to look for the best methods of finding out if a person has cancer, or to accurately determine the stage of cancer that is present. Treatment trials: Treatment trials evaluate the ability of drugs, radiation, surgery, or other measures to treat cancer. Supportive care trials: Supportive care trials are also called quality-of-life trials. They study the ability of a drug or procedure to lessen the symptoms of cancer or symptoms related to the treatment of cancer. CONCLUSION Clinical trials are conducted to collect data regarding the safety and efficacy of new drug and device development. There are several steps and stages of approval in the clinical trials process before a drug or device
  • 34. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Hemant Singh Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 304 can be sold in the consumer market, if ever.Drug and device testing begins with extensive laboratory research which can involve years of experiments in animals and human cells. If the initial laboratory research is successful, researches send the data to the Food and Drug Administration (FDA) for approval to continue research and testing in humans.Once approved, human testing of experimental drugs and devices can begin and is typically conducted in four phases. Each phase is considered a separate trial and, after completion of a phase, investigators are required to submit their data for approval from the FDA before continuing to the next phase. REFERENCES American Bible Society (1816). The Holy Bible: Old and New Testaments, King James Version (1611). American Bible Society, New York. Applied Clinical Trials (1992–97), Astor, Eugene. Ashby, D, ed. (1993). Conference on methodological and ethical issues in clinical trials, 27–28 June, Statistics in Medicine, 12, 1991, 1373–1534. Beecher H.K, Ethics and clinical research, New England Journal of Medicine , 274, 1966, 1354–1360. Berlin, J.A, Laird, N.M., Sacks, H.S. & Chalmers, T.T, A comparison of statistical methods for combining event rates from clinical trials, Statistics in Medicine, 8, 1989, 141–151. Berry D.A, Interim analysis in clinical trials: classical vs Bayesian approaches, Statistics in Medicine , 4, 1985, 521–526. Blackwelder, W.C, Proving the null hypothesis in clinical trials, Controlled Clinical Trials, 3, 1982, 345–353. Blackwelder, W.C. & Chang, M.A, Sample size graphs for “proving the null hypothesis”, Controlled Clinical Trials, 5, 1984, 97–105. Boissel, J.P, Blanchard J, Panak E, Peyrieux J.C. & Sacks H, Considerations for the meta-analysis of randomized clinical trials: summary of a panel discussion, Controlled Clinical Trials 10, 1989, 254–281. Bristol D, Sample sizes for constructing CI’s and testing hypotheses, Statistics in Medicine 8, 1989, 803–821. Brosgart, C.L, Mitchell, T, Charlebois, E., Coleman, R., Mehalko, S., Young, J. & Abrams, D.I, Off-label drug use in human immunodeficiency virus disease, Journal of Acquired Immune Deficiency Syndrome and Human Retrovirology, 12, 1996, 56–62. Brown, B.W Jr, Designing for cancer clinical trials: Selection of prognostic factors, Cancer Treatment Reports, 64, 1980, 499–502. Bull, J.P, The historical development of clinical therapeutic trials, Journal of Chronic Diseases, 10, 1959, 218– 248. Buyse M. & Ryan L.M, Issues of efficiency in combining proportions of deaths from several clinical trials, Statistics in Medicine, 6, 1987, 565–576. Canner, P.L, Monitoring clinical trial data for evidence of adverse or beneficial treatment effects, INSERM – Essais Controles Multicentres: Principes et Problemes, 76, 1977, 131–149.
  • 35. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Navin Dixit et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 305 SUSTAINED RELEASE DRUG DELIVERY SYSTEM Navin Dixit*, Sheo Dutt Maurya, Bhanu P.S.Sagar I.E.C.College of Eng & Technology, Greater Noida (U.P) *Corresponding author: ndixit515@gmail.com ABSTRACT The oral route is the most popular route used for administration of drugs, which is due in part to the ease of administration and to the fact that gastrointestinal physiology offers more flexibility in dosage form design than most other routes. The terms Sustained release, prolonged release, modified release, extended release or depot formulations are used to identify drug delivery systems that are designed to achieve or extend therapeutic effect by continuously releasing medication over an extended period of time after administration of a single dose. There are several reasons for attractiveness of these dosage forms: provides increased bioavailability of drug product, reduction in the frequency of administration to prolong duration of effective blood levels, reduces the fluctuation of peak trough concentration and side effects and possibly improves the specific distribution of the drug. Key words: Sustained Release, Drug delivery system, Matrix, Drug Release INTRODUCTION Probably the earliest work in the area of sustained drug delivery dosage forms can be traced to the 1938 patent of Israel Lipowski. This work involved coated pallets for prolonged release of drug and was presumably forerunner to the development of the coated particle approach to sustained drug delivery that introduced in the early 1950s. Ideally, a drug should arrive rapidly at the site of action (receptor) in the optimum concentration, remain for the desired time, be excluded from other sites, and be rapidly removed from the site when indicated i.e. the basic goal of the therapy is to achieve a steady state blood level that is therapeutically effective and non-toxic for an extended period of time. Generally, the time course of a dosage form (pharmacokinetics) in man is considered to be controlled by the chemical structure of the drug. Decreasing the rate of absorption and/ or changing the dosage form provide a useful adjunct. When it is feasible or desirable to modify the drug compound on a molecular level, often sought is a product that will requireless frequent administration to obtain the required biologic activity time profile; for example, a tablet that has the same clinical effect when administered every twelve hours. In another instance, it may be desirable to decrease the absorption rate in order to obtain a more acceptable clinical response (Girish K Jani, 2009). Tablets are one of the most stable and commonly administered oral dosage forms. Since the later part of nineteen-century, tablets have been widespread and their popularity continues. Tablets remain popular as dosage form because of the advantages afforded both to the pharmaceutical manufacturers and patients. These includes: simplicity and economy of preparation, stable and convenient in packing, ease of transporting and dispensing, accuracy of single dosage regimen, compactness and portability, and blandness of taste and ease of administration. The goal in designing sustained or controlled delivery systems is to reduce frequency of dosing or to increase the effectiveness of the drug by localization at the site of action, reducing the dose required, providing uniform drug delivery. If one were to imagine the ideal drug delivery system, two prerequisites would be required. First, it would be a single dose for duration of treatment, whether it is for days of weeks, as with infection, or for lifetime of the patient, as in hypertension or diabetes. Second, it should deliver the drug directly to the site of action, thereby minimizing or eliminating side effects. This may necessitate delivery to specific receptors or to localization to cells or to specific areas of the body. Oral ingestion has long been the most convenient and commonly employed route of drug delivery. Indeed, for sustained release systems, oral route of administration has received most of the attention with respect to research on physiological and drug constraints as well as design and testing of products. This is because of the fact that there is more feasibility in dosage form design for oral route than for parenteral or any other route. The design of oral sustained release delivery systems is subject to several intercalated variables of considerable importance. Among these are the types of delivery systems, the disease being treated, the patient and the length of therapy and the properties of the drug. In conventional drug therapy, it can be seen from the Figure 1.1 that the administration of drug by either intravenous injection or an extravascular route e.g. Orally, intramuscularly, or
  • 36. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Navin Dixit et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 306 rectally does not maintain drug blood level within the therapeutic range for an extended period of time. The short action is due to the inability of conventional dosage forms to control temporal delivery (Banks Michael, 1991). DRUG SELECTION FOR ORAL SUSTAINED RELEASE DRUG DELIVERY SYSTEMS The biopharmaceutical evaluation of a drug for potential use in controlled release drug delivery system requires knowledge on the absorption mechanism of the drug form the G. I. tract, the general absorbability, the drug’s molecular weight, pKa, solubility at different pH and apparent partition coefficient. Table.1. Parameter for drug selection Parameter Preferredvalue Molecular weight/ size < 1000 Solubility > 0.1 µg/ml for pH 1 to pH 7.8 Pka Non ionized moiety > 0.1% at pH 1 to pH 7.8 Apparent partition coefficient High Absorption mechanism Diffusion General absorbability From all GI segments Release Should not be influenced by pH and enzymes The pharmacokinetic evaluation requires knowledge on a drug’s elimination half- life, total clearance, absolute bioavailability, possible first- pass effect, and the desired steady concentrations for peak and trough. Table.2. Pharmacokinetic parameter for drug selection Parameter Comment Elimination half life Preferably between 0.5 and 8 h Total clearance Should not be dose dependent Elimination rate constant Required for design Apparent volume of distribution Vd The larger Vd and MEC, the larger will be the required dose size. Absolute bioavailability Should be 75% or more Intrinsic absorption rate Must be greater than release rate Therapeutic concentration Css av The lower Css av and smaller Vd, the loss among of drug required Toxic concentration Apart the values of MTC and MEC, safer the dosage form. Also suitable for drugs with very short half-life. Matrix diffusion controlled drug delivery system: In this type of controlled drug delivery system, the drug reservoir results from the homogeneous dispersion of the drug particles in either a lipophilic or a hydrophilic polymer matrix. Mode of action of hydrophilic matrix dosage form: Hydrophilic matrix dosage forms essentially consist of a compressed blend of hydrophilic polymer and drug. According to the generally accepted mechanism, the drug release from hydrophilic matrix dosage forms starts when the tablet comes in contact with gastrointestinal fluid. The surface of the tablet hydrates to release exposed drug and at the same time form a viscous polymer mucilage or gel. This gel fills the interstices within the tablet, retarding further ingress of liquid. The concentration of polymer within the hydrated layer ranges from dilution at the outer surface to around 90% at the boundary with the drug core. Within this layer, drug in various states of dissolution (undissolved in dilute solution; in saturated solution) is distributed amongst the other ingredients of the tablets.
  • 37. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Navin Dixit et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 307 Fig.1.Matrix diffusion controlled drug delivery system Drug release occurs immediately from the surface (burst effect) followed by diffusion through, and / or erosion of, the hydrated layer. The relative proportions of drug released by diffusion and erosion are determined by the drug’s solubility properties and by the physical and chemical nature of the hydrated polymer. This in turn is influenced by other factors, including drug characteristics, dissolution medium and other, which continue to be investigated. Modified-release drug delivery systems: In order to overcome the drawbacks of conventional drug delivery systems, several technical advancements have led to the development of modified release drug delivery systems. The modified-release delivery systems may be divided conveniently into four categories4 : 1. Delayed release 2. Sustained release 3. Site-specific targeting 4. Receptor targeting Delayed release system: Delayed-release systems are those that use repetitive, intermittent dosing of a drug from one or more immediate-release units incorporated into a single dosage form. Examples of delayed-release systems include repeat-action tablets and capsules, and enteric-coated tablets where timed release is achieved by a barrier coating. Sustained release system: Sustained-release systems include any drug-delivery system that achieves slow release of drug over an extended period of time. If the systems can provide some control, whether this is of a temporal or spatial nature, or both, of drug release in the body, or in other words, the system is successful at maintaining constant drug levels in the target tissue or cells, it is considered a controlled-release system. Site-specific targeting: Site-specific and receptor targeting refer to targeting of a drug directly to a certain biological location. In the case of site-specific release, the target is adjacent to or in the diseased organ or tissue. Receptor targeting: For receptor release, the target is the particular receptor for a drug within an organ or tissue. Both of these systems satisfy the spatial aspect of drug delivery and are also considered to be controlled drug- delivery systems. Sustained release drug delivery systems: During the past few years, conventional dosage forms of drugs are rapidly being replaced by the new and the novel drug delivery systems. Amongst, these the controlled release/sustained release dosage forms have become extremely popular in modern therapeutics. The basic rationale for sustained release drug delivery is to alter the pharmacokinetics and pharmacodynamics of drugs by using novel drug delivery systems or by modifying the molecular structure or physiological parameters inherent in a selected route of administration. It is desirable that the duration of drug action becomes more a design property of a rate controlled dosage form and less or not at all a property of the drug molecule’s inherent kinetic properties. Thus, optimal design of a sustained/ controlled release system necessitates a thorough understanding of the pharmacokinetics and pharmacodynamics of the drug. When the drug is administered in a conventional dosage form, it results in a fluctuation of drug concentration at the site of action (peak and valley pattern) and therefore in systemic circulation and tissue compartment. Figure 1.2 shows the difference between the conventional and sustained release dosage forms. Advantages of sustained release drug delivery: Following are the potential advantages of sustained release products 1. Decreased local and systemic side effects reduced gastrointestinal irritation. 2. Better drug utilization reduction in total amount of drug used.
  • 38. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Navin Dixit et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 308 3. Improved efficiency in treatment, optimized therapy, more uniform blood concentration. 4. Reduction in fluctuation in drug level and hence more uniform pharmacological response, cure of control of condition more promptly, less reduction in drug activity with chronic use. 5. Method by which sustained release is achieved can improve the bioavailability of some drugs e.g. drugs susceptible to enzymatic inactivation can be protected by encapsulation in polymer systems suitable for sustained release. 6. Improved patient compliance, less frequent dosing, reduced night-time dosing, reduced patient care time. The importance of patient compliance in successful drug therapy is well recognized. It has been found that there is an inverse relationship between the number of dosages per day and the compliance rate. 7. Although the initial unit cost of sustained release products is usually greater than that of conventional dosage forms because of the special nature of these products, the average cost of treatment over an extended time period may be less. Economy may also result from a decrease in nursing time and hospitalization time. Disadvantages of sustained release drug delivery: The disadvantages of sustained release drug delivery system are 1. Decreased systemic availability in comparison to immediate release conventional dosage forms, which may be due to incomplete release, increased first-pass metabolism, increased instability, insufficient residence time complete release, site specific absorption, pH dependent stability, etc. 2. Poor in vitro – in vivo correlation. 3. Retrieval of drug is difficult in case of toxicity, poisoning or hypersensitivity reactions. 4. Reduced potential for dose adjustment of drugs normally administered in varying strengths. Classification of oral sustained/controlled release systems Diffusion controlled Systems Reservoir devices: A core of drug (reservoir) surrounded by a polymeric membrane characterizes them. The nature of the membrane determines the rate of drug release. The characteristics of reservoir diffusion systems are 1. Zero order drug release is possible. 2. The release rate is dependent on the type of polymer. 3. High molecular weight compounds are difficult to deliver through the device. Matrix devices: It consists of drug dispersed homogenously in a matrix. The characteristics of matrix diffusion systems are 1. Zero order release cannot be obtained. 2. Easy to produce than reservoir devices. 3. High molecular weight compounds are delivered through the device. Dissolution controlled systems Matrix dissolution controlled systems: Aqueous dispersions, congealing, spherical agglomeration, etc. can be used. Encapsulation dissolution controlled systems: Particles, seeds, granules can be coated by techniques such as microencapsulation. Diffusion and dissolution controlled systems: In a bioerodible matrix, the drug is homogenously dispersed in a matrix and it is released either by swelling controlled mechanism or by hydrolysis or by enzymatic attack. Sustained release matrix tablets: One of the least complicated approaches to the manufacture of sustained release dosage forms is the direct compression of drug, release retardant, and additives to form a tablet in which drug is embedded in a matrix core of retardant. Alternatively drug retardant blend may be granulated prior to compression. Such tablets are called as matrix tablets. Three classes of release retarding materials are used for the formulation of matrix tablets. They include 1. Insoluble or ‘skeleton’ matrices 2. Water insoluble, erodable matrices 3. Hydrophilicmatrices.
  • 39. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Navin Dixit et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 309 Table1.1. Some important materials used for preparing sustained release tablets. Matrix characteristics Release retarding material Insoluble, inert Polyethylene, Polyvinyl Chloride Methyl acrylate-methacrylate copolymer Ethyl Cellulose Insoluble, erodible Carnauba wax, Steryl alcohol Stearic acid, Polyethylene glycol Polyethylene glycol monostearate Triglycerides Hydrophillic Methyl cellulose, Hydroxyethyl cellulose Hydroxypropyl methycellulose Sodium carboxymethylcellulose Carboxypolymethylene Sodium alginate, Galactomannose Tablets prepared from these materials are egested intact and not break apart in GI tract. The rate-liming step in controlling the release of drug from these formulations is liquid penetration into the matrix unless channeling agents are included in the formulation to promote permeation of water into the matrix. This allows drugs dissolution and diffusion from the channel created in the matrix. In these tablets, drug bioavailability is dependent on polymer-ratio. The bioavailability may be modified by addition of diluents such as lactose. These forms of matrix tablets are not useful if dose of drug is high or if the drug is insoluble in water. Waxes, lipids and related materials form matrices that control the release through both pore diffusion and erosion. Release characteristics are more sensitive to digestive fluid composition than the tablets preparation by insoluble material. Total release of drug from the wax-lipid matrices is not possible, since a certain fraction of the dose is coated with impermeable wax films. For dispersion of drug with the base, three methods are used, which include the fusion technique. In absence of additives, the drug release is non-linear from these systems. Additives like polyvinyl pyrrolidone or polyoxyethylene lauryl esters can lead to apparent zero-order release. The third group of matrix formers represents non digestible materials, which form gels in situ. The release of drug from these systems is controlled by penetration of water through a gel layer produced by hydration of polymer and diffusion of drug through the swollen, hydrated matrix, in addition to the erosion of gelled layer. The extent to which the erosion or diffusion controls the release depends on polymer selected as well as on the drug- polymer ratio used in the formulation. High drug polymer ratios results in formulations from which drug release is controlled attrition. Mechanism of drug release: On exposure to aqueous fluid, hydrophilic matrices take up water, and polymer starts hydrating to form a gel layer. An initial burst of soluble drug may occur due to surface leaching when a matrix containing a swellable glassy polymer comes in contact with an aqueous medium, there is an abrupt change from a glassy to a rubbery state which is associated with swelling process with time, water infiltrator deep into the case increasing the thickness by the gel layer. Concomitantly the outer layer becomes fully hydrated and states dissolving or eroding. When water reaches the center of the system and the concentration of drug fells below the solubility value, the release rate of drug begins to reduce. At the same time, an increase in thickness of the barrier layer with time increases the diffusion path length, reducing the rate of drug release. Drug release kinetic associated with these gel – layer dynamic, range initially from Fickian to annomalous (Non – Fickian) and subsequently from quasi – Constant ( near Zero order ) to constant. In general, two major factors control the drug release from swelling controlled matrix system. They include many processes given below. 1. The rate of aqueous medium infiltration into the matrix, followed by a relaxation process (hydration, gelatin or swelling). 2. The rate of matrix erosion As a result of these simultaneous processes, two front are evident, a swelling front, where the polymer get hydrated, and an eroding front. The distance between these two fronts are called diffusion layer thickness. Diffusion layer thickness depends on the selective rate at which the swelling and eroding fronts move in relation to each other. If the polymer gets slowly, solvent can penetrate deep into the glassy matrix the dissolving the drug; there for gel layer thickness and it stability are council in controlling drug release. Swelling of HPMC
  • 40. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Navin Dixit et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 310 matrix tablet was higher for higher a molecular weight. They attributed this to the large hydrodynamic volume occupied by higher molecular weight chain when hydrated. As the polymer chain becomes more hydrated and the gel becomes more dilute, the disentanglement concentration may be reached that is the critical polymer concentration below which the polymer chain disentangle and detached from gelled matrix. CONCLUSION There are several reasons for attractiveness of these dosage forms: provides increased bioavailability of drug product, reduction in the frequency of administration to prolong duration of effective blood levels, reduces the fluctuation of peak trough concentration and side effects and possibly improves the specific distribution of the drug. If one were to develop an ideal drug delivery system, two pre- requisites would be required: Firstly single dose for the duration of treatment whether for days or weeks as with infection, diabetes or hypertension. Second it should deliver the active entity directly to the site of action minimizing the side effects. REFERENCES Anoop Kumar singh et al. Isolation, Characterization and formulation properties of a new plant gum obtained from mangifera indica, Int J Pharm Biomed Res, 1(2), 2010, 35-41. Banks Michael and E.alulton Michael, Fluidized-bed Granulation: A Chronology, DIP, 17(11), 1991, 1437- 1463. Davies WK, Gloor WT, Batch production of pharmaceutical granulations in a fluidized bed: Effects of process variables on physical properties of final granulation.Journal of Pharmaceutical Sciences, 60(12), 1971, 1869- 1874. Davies WL, Gloor WT. Batch production of pharmaceutical granulations in a fluidized bed 2: effect of various binders and their concentrations on granulations and compressed tablets. Journal of Pharmaceutical sciences 1972, 61(4), 1972, 618-622. Georgakopoulos PP, Malamataris S, Dolamidis G, The Effect of using different grades of PVP and gelatin as binder in the fluidized bed granulation and tableting of lactose, Pharmazie, 38(4), 1983, 240-243. Girish K Jani, Dhiren P Shah, Vipul D Prajapati, Vineet C Jain. Gums and Mucilages, AJPS, 4(5), 2009, 309-323. Heng PWS, Roller compaction of crude plant material: Influence of process variables, PVP and Co- milling. Pharmaceutical Development and Technology, 9(2), 2004, 135-144. Leon L, Herbert AL, Joseph LK, The theory and practice of industrial pharmacy, 3rd ed. Bombay, Varghese Publishing House, 1991. Lowenthal W, Disintegration of tablets, Journal of Pharmaceutical Sciences, 61(11), 2006, 1695-1711. Odeku OA, Itiola OA, Evaluation of the effects of khaya gum on the mechanical and release properties of paracetamol tablets, DIP, 29(3); 2003, 311-320. Pereira de Souza T et al. Eudragit E as excipient for production of granules and tablets from phyllanthus niruri l spray-dried extract, AAPS Pharm Scitech, 8(2), 2007, A-34.
  • 41. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Ravi Kumar and Deepthi Yadav et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 311 ANTIBACTERIAL ACTIVITY OF ETHANOLIC EXTRACTS OF NYCTANTHES ARBORTRISTIS AND NERIUM OLEANDER A.Ravi Kumar, CH.S.D.Phani Deepthi Yadav* Department of Pharmacognosy, Bapatla College of Pharmacy, Bapatla, Andhra Pradesh, India *Corresponding author: E-Mail:deepukoushikyadav@gmail.com ABSTRACT In the present study, an attempt was made to investigate the anti-bacterial activity of Nyctanthes arbortristis and Nerium oleander. The crude drug powder extracts of the leaves of the above plants were taken for the study. The antibacterial activity was performed by using both gram positive and gram negative organisms viz., B.subtilis and E.coli respectively. The Phytochemical Screening was done for the selected plants. Phenolic compounds, tannins, flavonoids, cardiac glycosides, saponins and alkaloids were present in Nyctanthes arbortristis. Steroids, alkaloids, flavanoids, carbohydrates, cardiac glycosides and tannins were present in Nerium oleander. Key words: Anti-bacterial activity, Nyctanthes Nerium species plants INTRODUCTION Herbal medicine – It is also called botanical medicine or phytomedicine-refers to using plants seeds, flowers, roots for medicinal purpose. Herbal has a long tradition of use of outside of conventional medicine. It is becoming more main stream as improvements in analysis and quality control along with advances in clinical research show the value of herbal medicine in the treating and preventing disease. The medicinal action of plants is unique to a particular plant species, consistent with the concept that the combination of secondary metabolites in a particular plant is taxonomically distinct. This article discusses the description and uses of two medicinal plants Nyctanthes arbor and Nerium oleander. Nyctanthes arbor-tristis is commonly known as Night-flowering Jasmine, Coral Jasmine and Parijat. It is used for its antibacterial, anthelmintic, anti-inflammatory, hepatoprotective, immunopotential, anti pyretic, antioxidant and anti fungal activity Nerium oleander is an evergreen shrub or small tree in the dogbane family Apocynaceae and it is toxic in all its parts. It is used traditionally in treating dermatitis, abscesses, eczema, psoriasis, sores, warts, corns, ringworm, scabies, herpes, skin cancer, asthma, dysmenorrheal, epilepsy, malaria, abortifacients, emetics, heart tonics, and tumor. MATERIALS AND METHODS Plant Materials: The leaves of plants Nerium and Nyctanthes species were authentified by Prof. V.Satyanarayana, department of plant breeding, Bapatla Agricultural College, Bapatla Andhra Pradesh, India. They were collected from different places of Andhra Pradesh, India. Solvent Extraction: The leaves of Nerium oleander, and Nyctanthes arbortristis were collected, washed, dried and powdered separately. 50g of dried powder of the leaves was weighed and transferred into a conical flask and it was macerated with sufficient amount of ethanol for about a week days. The whole mixture was filtered and filtrate was collected, concentrated in a china dish on a hot plate till the residue was obtained. The extract was collected, labelled and stored for further experimental use. Fig 1: Nyctanthes arbortristis Plant Fig 2: Nerium oleander Plant Microorganisms: The test organisms used were E.coli (ATCC 25922) a Gram –ve strain and B.subtilis (ATCC 21332) a Gram +ve strain which were obtained from PG and Research Department of Biotechnology Bapatla
  • 42. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Ravi Kumar and Deepthi Yadav et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 312 College of Pharmacy Bapatla Andhra Pradesh India The strains were sub-cultured on nutrient agar slants and were incubated for 24 hrs. ANTIBACTERIAL ACTIVITY Agar well diffusion method: Required glass ware was washed and dried in a hot air oven. The sterilized agar medium was transferred into the Petri dishes, was allowed to solidify at room temperature. The selected test organism was spread over the solidified agar with the help of a swab stick. Sterile borer was used to make wells of 8mm diameter .The dilutions of ethanolic extracts of Nyctanthes arbortristis, Nerium oleander solutions of combined ethanolic extracts of Nyctanthes arbortristis with Nerium oleander respectively were poured in the wells with the help of a sterile syringe needle. In each Petri plate a well was prepared for standard i.e., ciprofloxacin 10µg/ml solution. The Petri plates were placed in a refrigerator for 5min to allow diffusion. Later the Petri plates were incubated in inverted position at 370 C for 24 hours in the incubator. After 24hours the zone of inhibition was observed and diameter in mm was measured and recorded. Qualitative analysis: The extracts and crude dried powders of Nyctanthes arbortristis, Nerium oleander were subjected to qualitative analysis for presence of chemical constitutens of Nytanthes arbortristis Nerium oleander Table.1.Anti-bacterial activity of Nyctanthes Arbortristis and Nerium Oleander COMPONENT DOSE Zone of Inhibition (mm) E.COLI B.SUBTILIS CIPROFLOXACIN (STD) 10 µg/ml 20mm 22mm Ethanolic extract of Nyctanthes arbortristis 500µg/ml - - 750µg/ml - - 1000µg/ml 2mm 3mm Ethanolic extract of Nerium oleander 500 µg/ml - - 750µg/ml - - 1000µg/ml 4mm 5mm Combined ethanolic extracts of Nerium oleander and Nyctanthes arbortristis 1000µg/ml - - 1500µg/ml 8mm 10mm 2000µg/ml 12mm 15mm RESULTS AND DISCUSSION The study of the chemical constituents and the active principles of the medicinal plants have acquired a lot of importance all over the world. The present study includes the antibacterial activity of extracts of leaves of Nyctanthes arbortristis in combination with the leaf extracts of Nerium oleander separately were performed. Earlier studies on Nyctanthes arbortristis indicated that the ethylacetate and chloroform extracts showed significant activity on both Gram +ve and Gram –ve strains. But in present study ethanolic leaf extract showed that the activity on bacterial strains was significant. But comparably the activity on B.subtilis was more than that of E.coli. As the activity obtained for the leaf extract was significant the combined leaf extract of Nyctanthes arbortristis and Nerium oleander were used which showed a synergistic effect i.e., Nerium oleander increased the antibacterial activity of the Nyctanthes arbortristis. The results were given in Table 1 CONCLUSION The Screening of Phytochemical constituents of the plants Nyctanthes arbortristis Nerium oleander analysis indicated the presence of carbohydrates, glycosides and alkaloids. The combined ethanolic extract of Nyctanthes arbortristis and Nerium oleander exhibited significant antibacterial activity of 2000µg/ml concentration.
  • 43. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Ravi Kumar and Deepthi Yadav et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 313 AKNOWLEDGEMENTS The authors are thankful to Management Principal, Bapatla College of Pharmacy, Bapatla, Andhra Pradesh, India for permitting and providing necessary facilities for carrying out to do the project work. REFERENCES Aiyer MN, Namboodiri AN and Kolammal M, Pharmacognosy of Ayurvedic Drugs, Trivandrum, 5, 1957, 49-55. Amarite O, Bhuskat P, Patel N and Gadgoli C,Evaluation of antioxidant activity of carotenoid from Nyctanthes arbortristis, Int J Pharmacol Biol Sci, 2, 2007, 57-59. Andrews JM, BSAC Standardised disc susceptibility testing method, J Antimicrob Chemother, 48, 2001, 5– 16. Chattopadhyay RR, Sarkar SK, Ganguli S, Banerjee RN, Basu TK, Antiinflammatory and acute toxicity studieswith leaves of Vinca rosea linn. in experimental animals. Indian Journal of Physiology and Pharmacology, 36, 1992, 291–292. Chattopadhyay RR, Sarkar SK, Ganguli S, Banerjee RN, Basu TK, Hypoglycemic and antihyperglycemic effect ofleaves of Vinca rosea Linn, Indian Journal of Physiology andPharmacology, 35, 1991, 145–151. El-Sayed, A. and G.A. Cordell, Catharanthamine: A new antitumor bisindole alkaloid from Catharanthus roseus, J. Nat. Prod, 44, 1981, 289-293. Evans WC and Trease, Pharmacognosy, Elsevier publication, 19th Edn, 1983, 96-98. Hassan KA, Brenda AT, Patrick V, Patrick OE, In vivo antidiarrheal activity of the ethanolic leaf extract of Catharanthus roseus Linn. (Apocyanaceae) in Wistar rats, Afr J Pharm Pharmacol, 5(15), 2011, 1797-1800. Jaleel CA, R Gopi and R Paneerselvam, Alterationsin non-enzymatic antioxidant componentsof Catharanthus roseus exposed to paclobutrazol gibberellic acid and Pseudomonas fluorescens, Plant Omics J, 2, 2009, 30-40. Kirtikar KR and Basu BD, Indian Medicinal Plants, Vol.VII, (Sri Satguru Publications, New Delhi,) 2000, 2110-2113. Kokate C K, purohit A P, Gokhale S B, A Textbook of Pharmacognosy, Nirali Prakashan, 2009, 27-28 Kokate CK, Purohit AP and Gokhale SB., Pharmacognosy. Nirali prakashan, 2006, 35th Edn: 99-100 Kusum S and Akki, Phytochemical investigation and in vitro evaluation of Nyctanthes arbortristis leaf extract for antioxidant property, J Pharm Res, 2(4), 2009, 752-755 Lokesh R,Leonard Barnalas, Madhuri P,Saurav K,Sundar K, Larvicidal activity of Trigonella, foenumand Nerium oleander Linn, Current research journal of biological sciences, 2(3), 2010, 154-160. Mathuram V and Kundu AB, Occurrence of two new ester of 6-Hydroxyloganin in Nyctanthes arbortristis. J Indian Chem Soc, 68, 1991, 581-584 Muhammad LR, N Muhammad A, Tanveer andS.N. Baqir, 2009. Antimicrobial activity of different extracts of Catharanthas roseus, Clin. Exp. Med J, 3, 2009, 81-85 Ohadoma SC, Micheal HU, Effects of co-administration of methanol leaf extract of Catharanthus roseus on the hypoglycemic activity of metformin and glibenclamide in rats, Asian Pac J Trop Biomed, 4(6), 2011, 475- 477 Omkar A, Jeeja T and Chhaya G, Evaluation of anti-inflammatory activity of Nyctanthes arbortristis and Onosma echiodes, Phrmacog mag, 8, 2006, 258-260.
  • 44. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Sampath Kumar et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 314 FOOD POISONING AND ITS SAFETY PRECAUTION M. Umadevi1 , K.P.Sampath Kumar *2 , Sai Pavan3 , Sd.Gosia Sultana3 , D. Bhowmik3 1. Tamil Nadu Agricultural University, Coimbatore 2. Coimbatore Medical College, Coimbatore 3. Nimra College of Pharmacy,Vijayawada *Corresponding author: kp_sampath@rediffmail.com ABSTRACT Food poisoning occurs when you swallow food or water that contains bacteria, parasites, viruses, or toxins made by these germs. Most cases of food poisoning are from common bacteria such as Staphylococcus or E. coli. Food poisoning, also known as acute gastroenteritis, is an acute inflammation of the lining of the stomach and small bowel. Food poisoning is a common, usually mild, but sometimes deadly illness that occur suddenly (within 48 hours) after consuming a contaminated food or drink. Most of the common contaminants cause nausea, vomiting, diarrhea, and abdominal cramping. Depending on the contaminant, fever and chills, bloody stools, dehydration, and nervous system damage may follow. Food poisoning comes from eating foods that contain germs like bad bacteria or toxins, which are poisonous substances. Bacteria are all around us, so mild cases of food poisoning are common. Key words: Food poisoning, Infectious agents, Food handling, Infectious organisms. INTRODUCTION Food poisoning, also called food-borne illness, is illness caused by eating contaminated food. Infectious organisms including various bacteria, viruses and parasites or their toxins are the most common causes of food poisoning. Infectious organisms or their toxins can contaminate food at any point during its processing or production. Contamination can also occur at home if food is incorrectly handled or cooked. Food poisoning symptoms often include nausea, vomiting or diarrhea, which can start just hours after eating contaminated food. Most often, food poisoning is mild and resolves without treatment. But some cases are severe, requiring hospitalization. Food poisoning is a common, usually mild, but sometimes deadly illness. Typical symptoms include nausea, vomiting, abdominal cramping, and diarrhea that occur. FOOD POISONING CAUSES More than 250 known diseases can be transmitted through food. The estimates unknown or undiscovered agents cause 68% of all food-borne illnesses and related hospitalizations. Many cases of food poisoning are not reported because people suffer mild symptoms and recover quickly. Also, doctors do not test for a cause in every suspected case because it does not change the treatment or the outcome. The known causes of food poisoning can be divided into two categories: Infectious agents and toxic agents. Infectious agents include viruses, bacteria, and parasites. Toxic agents include poisonous mushrooms, improperly prepared exotic foods (such as barracuda - ciguatera toxin), or pesticides on fruits and vegetables.Food usually becomes contaminated from poor sanitation or preparation. Food handlers who do not wash their hands after using the bathroom or have infections themselves often cause contamination. Improperly packaged food stored at the wrong temperature also promotes contamination. Food poisoning can affect one person or a group of people who all ate the same contaminated food. It more commonly occurs after eating at picnics, school cafeterias, large social functions, or restaurants. The germs may get into the food you eat (called contamination) in different ways:  Meat or poultry can come into contact with bacteria from the intestines of an animal that is being processed  Water that is used during growing or shipping can contain animal or human waste  Food handling or preparation in grocery stores, restaurants, or homes  Food poisoning often occurs from eating or drinking:  Any food prepared by someone who does not wash their hands properly  Any food prepared using cooking utensils, cutting boards, and other tools that are not fully cleaned  Dairy products or food containing mayonnaise (such as coleslaw or potato salad) that have been out of the refrigerator too long  Frozen or refrigerated foods that are not stored at the proper temperature or are not reheated properly  Raw fish or oysters
  • 45. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Sampath Kumar et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 315  Raw fruits or vegetables that have not been washed well  Raw vegetables or fruit juices and dairy products (look for the word "pasteurized," which means the food has been treated to prevent contamination)  Undercooked meats or eggs  Water from a well or stream, or city or town water that has not been treated  Many types of germs may cause food poisoning, including:  Campylobacter enteritis  Cholera  E. coli enteritis  Fish poisoning  Staphylococcus aureus  Salmonella  Shigella Infants and elderly people are at the greatest risk for food poisoning. You are also at higher risk if:  You have a serious medical condition, such as kidney disease or diabetes  You have a weakened immune system  You travel outside of the United States to areas where you are exposed to germs that cause food poisoning  Pregnant and breastfeeding women have to be especially careful to avoid food poisoning.More than 250 known diseases can be transmitted through food. Many cases of food poisoning are not reported because people suffer mild symptoms and recover quickly. Also, doctors do not test for a cause in every suspected case because it does not change the treatment or the outcome. The known causes of food poisoning can be divided into two categories: Infectious agents include viruses, bacteria, and parasites. Toxic agents include poisonous mushrooms, improperly prepared exotic foods or pesticides on fruits and vegetables.Food usually becomes contaminated from poor sanitation or preparation. Food handlers who do not wash their hands after using the bathroom or have infections themselves often cause contamination. Improperly packaged food stored at the wrong temperature also promotes contamination. FOOD POISONING SYMPTOMS Symptoms of food poisoning depend on the type of contaminant and the amount eaten. The symptoms can develop rapidly, within 30 minutes, or slowly, worsening over days to weeks. Most of the common contaminants cause:  nausea  vomiting  diarrhea  abdominal cramping  fever Usually food poisoning is not serious, and the illness runs its course in 24-48 hours. Viruses: Viruses account for most food poisoning cases where a specific contaminant is found. Noroviruses are a group of viruses that cause a mild illness (often termed "stomach flu") with nausea, vomiting, diarrhea, abdominal pain, headache, and low-grade fever. These symptoms usually resolve in two to three days. It is the most common viral cause of adult food poisoning and is transmitted from water, shellfish, and vegetables contaminated by feces, as well as from person to person. Outbreaks are more common in densely populated areas such as nursing homes, schools, and cruise ships (hence the viral infection is also known as the "Cruise Ship Illness"). The term Norovirus has been approved as the official name for this group of viruses. Several other names have been used for noroviruses, including Norwalk-like viruses, caliciviruses (because they belong to the virus family Caliciviridae), and small round structured viruses. Rotavirus: Causes moderate to severe illness with vomiting followed by watery diarrhea and fever. It is the most common cause of food poisoning in infants and children and is transmitted from person to person by fecal contamination of food and shared play areas. Hepatitis A: Causes moderate illness with sudden onset of fever, loss of appetite, abdominal pain, and feeling of tiredness followed by jaundice, which is a yellowing of the eyes and skin. Symptoms usually last less than two
  • 46. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Sampath Kumar et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 316 months, but can be prolonged or relapse for up to six months. It is transmitted from person to person by fecal contamination of food. Bacteria: Bacteria can cause food poisoning in two different ways. Some bacteria infect the intestines, causing inflammation and difficulty absorbing nutrients and water, leading to diarrhea. Other bacteria produce chemicals in foods (known as toxins) that are poisonous to the human digestive system. When eaten, these chemicals can lead to nausea and vomiting, kidney failure, and even death. Salmonellae: Salmonellae are bacteria that may cause food poisoning; the illness itself is often referred to as Salmonella or Salmonella infection. TheCDC estimates that each year 1 million people are infected with Salmonella, amounting to $365 million in direct medical costs annually. Salmonellaecause a moderate illness with nausea, vomiting, crampy diarrhea, andheadache, which may come back a few weeks later as arthritis (joint pains). In people with impaired immune systems (such as people with kidney disease,HIV/AIDS, or those receiving chemotherapy for cancer), Salmonellae can cause a life-threatening illness. The illness is transmitted by undercooked foods such as eggs, poultry, dairy products, and seafood. Campylobacter: Causes mild illness with fever, watery diarrhea, headache, and muscle aches. Campylobacter is the most commonly identified food-borne bacterial infection encountered in the world. It is transmitted by raw poultry, raw milk, and water contaminated by animal feces. Staphylococcus aureus: Causes moderate to severe illness with rapid onset of nausea, severe vomiting, dizziness, and abdominal cramping. These bacteria produce a toxin in foods such as cream-filled cakes and pies, salads (most at risk are potato, macaroni, egg, and tuna salads, for example) and dairy products. Contaminated salads at picnics are common if the food is not chilled properly. Bacillus cereus: Causes mild illness with rapid onset of vomiting, with or without diarrhea and abdominal cramping. It is associated with rice (mainly fried rice) and other starchy foods such as pasta or potatoes. It has been speculated that this bacteria may also be used as a potential terrorist weapon. Escherichia coli (E coli): Causes moderate to severe illness that begins as large amounts of watery diarrhea, which then turns into bloody diarrhea. There are many different types of this bacteria. The worst strain can cause kidney failure and death (about 3% to 5% of all cases). It is transmitted by eating raw or undercooked hamburger, unpasteurized milk or juices, or contaminated well water. Outbreaks of food poisoning due to E. coli have also occurred following ingestion of contaminated produce. Shigella (traveler's diarrhea): Causes moderate to severe illness with fever,diarrhea containing blood or mucus or both, and the constant urge to have bowel movements. It is transmitted in water polluted with human wastes. Listeria monocytogenes: Listeriosis is a moderate to severe illness with nausea and vomiting. Some affected individuals can progress to developmeningitis from Listeria. It is transmitted on many tips of uncooked foods such as meats, fruits, vegetables, soft cheeses, unpasteurized milk, and cold cut meats. Pregnant woman and newborns are at increased risk for serious infections. In 2011, in an outbreak caused by tainted cantaloupe, 25 people died and 123 people were infected in 26 states. Clostridium botulinum (botulism): Causes severe illness affecting the nervous system. Symptoms start as blurred vision. The person then develops problems talking and overall weakness. Symptoms then progress to breathing difficulty and the inability to move arms or legs. Infants and young children are particularly at risk. It is transmitted in foods such as home-packed canned goods, honey, sausages, and seafood. Because botulism can be released in the air, it is considered a potential biological weapon for terrorists. Vibrio cholerae: Causes mild to moderate illness with crampy diarrhea, headache, nausea, vomiting, and fever with chills. It strikes mostly in the warmer months of the year and is transmitted by infected, undercooked, or raw seafood. Vibrio parahaemolyticus: Causes moderate to severe abdominal cramping, nausea, vomiting, and fever. In immunocompromised individuals, it can cause severe or deadly disease. It is transmitted by eating raw or undercooked fish, particularly oysters. Parasites rarely cause food poisoning. When they do, they are usually swallowed in contaminated or untreated water and cause long-lasting but mild symptoms. Giardia (beaver fever): Causes mild illness with watery diarrhea often lasting one to two weeks. It is transmitted by drinking contaminated water, often from lakes or streams in cooler mountainous climates. The infection can also be spread from person to person by food or other items contaminated with feces from an infected person.
  • 47. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Sampath Kumar et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 317 Cryptosporidium: Causes moderate illness with large amounts of watery diarrhea lasting two to four days. May become a long-lasting problem in people with poor immune systems (such as people with kidney disease or HIV/AIDS or those on chemotherapy for cancer). It is transmitted by contaminated drinking water. Toxoplasma: The CDC estimates that more than 60 million people in the U.S. carry the Toxoplasma parasite, but few have symptoms because the immune system keeps the parasite from causing illness. When it does cause disease, symptoms include headache, blurred vision, and eye pain. It is transmitted by eating undercooked or raw meat, contaminated water, or contact with contaminated cat feces. Pregnant women and those with compromised immune systems infected with Toxoplasma can have severe health complications. Toxic agents are the least common cause of food poisoning. Illness is often an isolated episode caused by poor food preparation or selection (such as picking wild mushrooms). Mushroom toxins: Illness can range from mild to deadly depending on the type of mushroom eaten. Often there is nausea, vomiting, and diarrhea. Some types of mushrooms produce a nerve toxin, which causes sweating, shaking, hallucinations, and coma. Ciguatera poisoning: Caused by eating fish that contains toxins produced by a marine algae called Gambierdiscus toxicus. It can cause moderate to severe illness with numbness of the area around the mouth and lips that can spread to the arms and legs, nausea, vomiting, muscle pain and weakness, headache, dizziness, and rapid heartbeat. The toxin may cause sensory problems in which hot things feel cold and cold things feel hot. It is transmitted by eating certain large game fish from tropical waters-most specifically barracuda, grouper, snapper, and jacks. According to the CDC, ciguatera has no cure. Symptoms may disappear in days or weeks, but may persist for years. Scombroid: Causes mild to moderate illness with facial flushing, burning around the mouth and lips, peppery- taste sensations, a red rash on the upper body, dizziness, headache, and itchy skin. Severe symptoms may include blurry vision, respiratory distress, and swelling of the tongue and mouth. Symptoms typically last from four to six hours, and rarely more than one or two days. It is transmitted in seafood, mostly mahi-mahi and tuna, but can also be in Swiss cheese. Pesticides: Cause mild to severe illness with weakness, blurred vision, headache, cramps, diarrhea, increased production of saliva, and shaking of the arms and legs. Toxins are transmitted by eating unwashed fruits or vegetables contaminated with pesticides. MEDICAL CARE Contact your doctor if any of the following situations occur:  Nausea, vomiting, or diarrhea lasts for more than two days.  The ill person is a child younger than three years of age.  The abdominal symptoms are associated with a low-grade fever.  Symptoms begin after recent foreign travel.  Other family members or friends who ate the same thing are also sick.  The ill person cannot keep any liquids down.  The ill person does not improve within two days even though they are drinking large amounts of fluids.  The ill person has a disease or illness that weakens their immune system (for example, HIV/AIDS, cancer and undergoing chemotherapy, kidney disease).  The ill person cannot take their normal prescribed medications because of vomiting.  The ill person has any nervous system symptoms such as slurred speech, muscle weakness, double vision, or difficulty swallowing.  The ill person is pregnant. Go to the nearest hospital's emergency department if any of the following situations occur:  The ill person passes out or collapse, become dizzy, lightheaded, or has problems with vision.  A fever higher than 101 F (38.3 C) occurs with the abdominal symptoms.  Sharp or cramping pains do not go away after 10-15 minutes.  The ill person's stomach or abdomen swells.  The skin and/or eyes turn yellow.  The ill person is vomiting blood or having bloody bowel movements.  The ill person stops urinating, have decreased urination, or have urine that is dark in color.
  • 48. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Sampath Kumar et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 318  The ill person develops problems with breathing, speaking, or swallowing.  One or more joints swell or a rash breaks out on the ill person's skin.  The ill person or caretaker considers the situation to be an emergency. FOOD POISONING DIAGNOSIS If the person visits a doctor or a hospital emergency department because they think they may have food poisoning, a thorough examination will be performed, including measurements of blood pressure, pulse, breathing rate, and temperature. The doctor will perform a physical exam, which screens for outward signs and symptoms of the illness. They will assess how dehydrated the patient is and examine the abdominal area to make sure the illness is not serious.The doctor may need to do a rectal examination. The doctor performs this test by inserting a lubricated and gloved finger gently into the rectum. The purpose is to make sure there are no breaks in the rectal wall. A sample of stool is taken and tested for blood and mucus. In some cases, a sample of stool or vomit can be sent to the laboratory for further testing to find out which toxin caused the illness. In a majority of cases, a specific cause is not found.A urine sample helps assess how dehydrated the patient is and may indicate possible kidney damage.Blood tests may be performed to determine the seriousness of the illness. An X-ray of the abdomen or a CT scan may be taken if the doctor suspects the patient's symptoms may be caused by another illness. FOOD POISONING SELF-CARE AT HOME Short episodes of vomiting and small amounts of diarrhea lasting less than 24 hours can usually be cared for at home. Do not eat solid food while nauseous or vomiting but drink plenty of fluids. Small, frequent sips of clear liquids (those you can see through) are the best way to stay hydrated. Avoid alcoholic, caffeinated, or sugary drinks. Over-the-counter rehydration products made for children such as Pedialyte and Rehydralyte are expensive but good to use if available. Sports drinks such as Gatorade and Powerade are fine for adults if they are diluted with water because at full strength they contain too much sugar, which can worsen diarrhea. Home remedies to treat nausea or diarrhea such as tea with lemon and ginger can be used for relief from symptoms. There are no proven herbal food poisoning cures. Consult a health care practitioner before taking any natural food poisoning remedies. After successfully tolerating fluids, eating should begin slowly, when nausea and vomiting have stopped. Plain foods that are easy on the stomach should be started in small amounts. Initially consider eating rice, wheat, breads, potatoes, low-sugar cereals, lean meats, and chicken (not fried). Milk can be given safely, although some people may experience additional stomach upset due to lactose intolerance. Most food poisonings do not require the use of over-the-counter medicines to stop diarrhea, but they are generally safe if used as directed. It is not recommended that these medications be used to treat children. If there is a question or concern, always check with a doctor. SIGN AND TEST Your health care provider will examine you for signs of food poisoning, such as pain in the stomach and signs your body does not have as much water and fluids as it should. This is called dehydration. Tests may be done on your stools or the food you have eaten to find out what type of germ is causing your symptoms. However, tests may not always find the cause of the diarrhea. In more serious cases, your health care provider may order a sigmoidoscopy. A thin, hollow tube with a light on the end is placed in the anus to look for the source of bleeding or infection. MEDICAL TREATMENT SITUATIONS  Medical treatment is necessary if following situations occur:  Nausea, vomiting, or diarrhea lasts for more than two days.  The abdominal symptoms associated with a low-grade fever.  Other family members or friends who ate the same thing are also sick.  The ill person cannot keep any liquids down.  The ill person does not improve within two days even drinking large amounts of fluids.  The ill person has a disease or illness that weakens immune system (for example, HIV/AIDS, cancer and undergoing chemotherapy, kidney disease).  The ill person cannot take normal prescribed medications because of vomiting.  The ill person has any nervous system symptoms such as slurred speech, muscle weakness, double vision, or difficulty swallowing.
  • 49. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Sampath Kumar et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 319 Tests: If the patient visits a doctor or a hospital emergency department because they think they may have food poisoning, a thorough examination will be performed, including  Measurement of blood pressure  Pulse  Breathing rate  Temperature  Examination of the abdominal area  Rectal examination  Sampling of stool for blood and mucus testing  Sample of stool or vomit for testing to find out which toxin caused the illness  A urine sample helps assess how dehydrated the patient is and may indicate possible kidney damage  Blood tests may be performed to determine the seriousness of the illness  An x-ray of the abdomen or a CT scan may be taken RISK Higher risk foods include: 1. Meat, especially undercooked mince and rolled, formed or tenderised meats 2. Raw or undercooked poultry such as chicken, duck and turkey 3. Raw or lightly cooked eggs including foods made from raw egg such as unpasteurised mayonnaise 4. Small goods such as salami and hams 5. Seafood 6. Cooked rice not kept at correct temperatures 7. Cooked pasta not kept at correct temperatures 8. Prepared salads such as coleslaw, pasta salads and rice salads 9. Prepared fruit salads 10. Unpasteurised dairy products. FOOD POISONING TREATMENT Self-Care at Home: Short episodes of vomiting and small amounts of diarrhea lasting less than 24 hours can usually be cared for at home.  Do not eat solid food while nauseous or vomiting but drink plenty of fluids. Small, frequent sips of clear liquids are the best way to stay hydrated. Avoid alcoholic, caffeinated, or sugary drinks. Over-the-counter rehydration products made for children such as Pedialyte and Rehydralyte are expensive but good to use if available. Sports drinks such as Gatorade and Powerade are fine for adults if they are diluted with water because at full strength they contain too much sugar, which can worsen diarrhea.  After successfully tolerating fluids, eating should begin slowly, when nausea and vomiting have stopped. Plain foods that are easy on the stomach should be started in small amounts. Consider eating rice, wheat, breads, potatoes, low-sugar cereals, lean meats, and chicken (not fried) to start. Milk can be given safely, although some people may experience additional stomach upset due to lactose intolerance.  Most food poisonings do not require the use of over-the-counter medicines to stop diarrhea, but they are generally safe if used as directed. Medical Treatment: The main treatment for food poisoning is putting fluids back in the body (rehydration) through an IV and by drinking. The patient may need to be admitted to the hospital. This depends on the severity of the dehydration, response to therapy, and ability to drink fluids without vomiting. Children, in particular, may need close observation.  Anti-vomiting and diarrhea medications may be given.  The doctor may also treat any fever to make the patient more comfortable.  Antibiotics are rarely needed for food poisoning. In some cases, antibiotics worsen the condition. Only a few specific causes of food poisoning are improved by using these medications. The length of illness with traveler's diarrhea can be decreased with antibiotics, but this specific illness usually runs its course and improves without treatment.
  • 50. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Sampath Kumar et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 320  With mushroom poisoning or eating foods contaminated with pesticides, aggressive treatment may include pumping the stomach (lavage) or giving medications as antidotes. These poisonings are very serious and may require intensive care in the hospital. Prevention: Safe steps in food handling, cooking, and storage are essential to avoiding food-borne illness. Bacteria cannot be seen, smelled, or tasted, which may be on any food. Illness can be prevented by: 1) Controlling the initial number of bacteria present. 2) Preventing the small number from growing. 3) Destroying the bacteria by proper cooking. 4) Avoiding re-contamination. Follow the food safety guidelines to keep contaminants away: Safe shopping  Buy cold foods last during shopping trip. Get them home fast.  Never choose torn or leaking packages.  Do not buy foods past their expiration dates.  Keep raw meat and poultry separate from other foods. Safe storage  Keep it safe, refrigerate.  Place raw meat, poultry, or fish in the coldest section of refrigerator.  Check the temperature of appliances. To slow bacterial growth, the refrigerator should be at 40°F, the freezer at 0°F.  Cook or freeze fresh poultry, fish, and meats within 2 days. Safe food preparation  Keep everything clean.  Wash hands before and after handling raw meat and poultry.  Sanitize cutting boards often in a solution of one teaspoon chlorine bleach in one quart of water.  Keep raw meat, poultry, fish, and their juices away from other food. After cutting raw meats, wash hands, cutting board, knife, and counter tops with hot, soapy water. Safe cooking  Keep hot foods hot and cold foods cold.  Use cooked leftovers within four days. Complication: Dehydration is the most common complication. This can occur from any causes of food poisoning. Less common, but much more serious complications depend on the bacteria that are causing the food poisoning. These may include:  Arthritis  Bleeding problems  Damage to the nervous system  Kidney problems  Swelling or irritation in the tissue around the heart Prevention: Here are steps you can take to prevent food poisoning at home: Wash your hands, utensils and food surfaces often. Wash your hands well with warm, soapy water before and after handling or preparing food. Use hot, soapy water to wash the utensils, cutting board and other surfaces you use. Keep raw foods separate from ready-to-eat foods. When shopping, preparing food or storing food, keep raw meat, poultry, fish and shellfish away from other foods. This prevents cross-contamination. Cook foods to a safe temperature. The best way to tell if foods are cooked to a safe temperature is to use a food thermometer. You can kill harmful organisms in most foods by cooking them to the right temperature. Ground beef should be cooked to 160 F (71.1 C), while steaks and roasts should be cooked to at least 145 F (62.8 C). Pork
  • 51. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Sampath Kumar et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 321 needs to be cooked to at least 160 F (71.1C), and chicken and turkey need to be cooked to 165 F (73.9 C). Fish is generally well-cooked at 145 F (62.8 C). Refrigerate or freeze perishable foods promptly. Refrigerate or freeze perishable foods within two hours of purchasing or preparing them. If the room temperature is above 90 F (32.2 C), refrigerate perishable foods within one hour. Defrost food safely. Do not thaw foods at room temperature. The safest way to thaw foods is to defrost foods in the refrigerator or to microwave the food using the "defrost" or "50 percent power" setting. Running cold water over the food also safely thaws the food. Throw it out when in doubt. If you aren't sure if a food has been prepared, served or stored safely, discard it. Food left at room temperature too long may contain bacteria or toxins that can't be destroyed by cooking. Don't taste food that you're unsure about — just throw it out. Even if it looks and smells fine, it may not be safe to eat. Food poisoning is especially serious and potentially life-threatening for young children, pregnant women and their fetuses, older adults, and people with weakened immune systems. These individuals should take extra precautions by avoiding the following foods:  Raw or rare meat and poultry  Raw or undercooked fish or shellfish, including oysters, clams, mussels and scallops  Raw or undercooked eggs or foods that may contain them, such as cookie dough and homemade ice cream  Raw sprouts, such as alfalfa, bean, clover or radish sprouts  Unpasteurized juices and ciders  Unpasteurized milk and milk products  Soft cheeses (such as feta, Brie and Camembert), blue-veined cheese and unpasteurized cheese  Refrigerated pates and meat spreads  Uncooked hot dogs, luncheon meats and deli meats REMEDIES OF FOOD POISONING Here are the top five easy and quick home remedies to get instant relief from food poising: 1. Ginger: Ginger is a well known remedy for various gastrointestinal distresses. Loaded with potent anti- inflammatory compounds ginger helps quell nausea and gastric distress. Chewing a piece of ginger tossed in honey helps relieve the severity of nausea. Ginger tea relieves stomach cramps and upset caused by food poisoning. 2. Cumin: Add a tablespoon of crushed cumin seeds to the soup to soothe the inflammation in your stomach. Crushed cumin with fenugreek powder mixed with a glass of water or half a cup crud helps relive abdominal pain and vomiting. Cumin seeds are of great benefit to the digestive system as they help stimulate the secretion of pancreatic enzymes, compounds necessary for proper digestion and nutrient assimilation. 3. Basil: Basil is another excellent home remedy to cure stomach infection due to its anti-bacterial properties. Taking juice of basil leaves with water empty stomach in the morning enhances digestive power. Basil juice helps stop vomiting immediately. Strain the juice of a few basil leaves and add it to a tablespoon of honey to get instant relief. Mix chopped basil leaves, sea salt and one shake of black pepper to three tablespoon of crud. Take the mixture three times in a day till you are totally cured of food poisoning. It will also cure any cramps or gas problem associated with food poisoning. 4. Lemon: The acidity of the lemon juice kills the micro-organism and toxins in the gastrointestinal tract. Squeeze juice of a lemon and add a pinch of sugar to it and drink, or you can even add lemon to your tea. Since fluid intake is very important as one tends to lose more water through diarrhea taking lemon juice in short intervals helps you keep hydrated. 5. Peppermint tea: Peppermint oil helps relieve symptoms of irritable bowel syndrome, including indigestion, dyspepsia, and colonic muscle spasms. It is extremely beneficial for people suffering from stomach spasms due to food poisoning. Add a few drops to your tea; your cramps will vanish in a couple of hours. TREATMENTS You will usually get better in a couple of days. The goal is to make you feel better and make sure your body has the proper amount of fluids. Getting enough fluids and learning what to eat will help keep you or your child comfortable. You may need to:
  • 52. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Sampath Kumar et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 322  Manage the diarrhea  Control nausea and vomiting  Get plenty of rest If you have diarrhea and are unable to drink or keep down fluids, you may need fluids given through a vein (by IV). This is especially true for young children. If you take diuretics, ask your health care provider if you need to stop taking the diuretic while you have diarrhea. Never stop or change medications without first talking to your health care provider. For the most common causes of food poisoning, your doctor will NOT prescribe antibiotics. You can buy medicines at the drugstore that help slow diarrhea. Do not use these medicines without talking to your health care provider if you have bloody diarrhea, a fever, or the diarrhea is severe. Do not give these medicines to children Treatments and drugs: Treatment for food poisoning typically depends on the source of the illness, if known, and the severity of your symptoms. For most people, the illness resolves without treatment within a few days, though some types of food poisoning may last a week or more.Treatment of food poisoning may include: Replacement of lost fluids: Fluids and electrolytes — minerals such as sodium, potassium and calcium that maintain the balance of fluids in your body — lost to persistent diarrhea need to be replaced. Children and adults who are severely dehydrated need treatment in a hospital, where they can receive salts and fluids through a vein (intravenously), rather than by mouth. Intravenous hydration provides the body with water and essential nutrients much more quickly than oral solutions do. Antibiotics: antibiotics if you have certain kinds of bacterial food poisoning and your symptoms are severe. Food poisoning caused by listeria needs to be treated with intravenous antibiotics in the hospital. And the sooner treatment begins, the better. During pregnancy, prompt antibiotic treatment may help keep the infection from affecting the baby. CONCLUSION Food poisoning is a common infection that affects millions of people in the India each year. Most commonly, patients complain of vomiting, diarrhea, and crampy abdominal pain. People should seek medical care if they have an associated fever, blood in their stool, signs and symptoms of dehydration, or if their symptoms do not resolve after a couple of days. Treatment focuses on keeping the patient well hydrated. Most cases of food poisoning resolve on their own. Prevention is key and depends upon keeping food preparation areas clean, good hand washing, and cooking foods thoroughly. Food poisoning is the name for the range of illnesses caused by eating or drinking contaminated food or drink. It is also sometimes called food borne illness. REFERENCES Food and Agriculture Organization of the United Nations, GASGA Technical Leaflet - 3 Mycotoxins in Grain, Retrieved 12 August 2007. World Health Organization, Chapter 2 Foodborne Hazards in Basic Food Safety for Health Workers Retrieved 12 August 2007. Food and Drug Administration, Sec. 683, 100 Action Levels for Aflatoxins in Animal Feeds (CPG 7126.33), Retrieved 13 August 2007. Henry, Michael H, Mycotoxins in Feeds: CVM’s Perspective , Retrieved 1 January 2012. Webley DJ, Jackson KL, Mullins JD, Hocking AD, Pitt JI, Alternaria toxins in weather-damaged wheat and sorghum in the 1995–1996 Australian harvest , Australian Journal of Agricultural Research, 48 (8), 1997, 1249- 56. Centers for Disease Control and Prevention, Diagnosis and management of foodborne illness, A primer for physicians and other health care professionals, MMWR, 53(RR-4), 2004, 1-32. Centers for Disease Control and Prevention (2008). Toxoplasmosis. www.cdc.gov/toxoplasmosis/factsheet.html. Centers for Disease Control and Prevention (2009). Foodborne infections. www.cdc.gov/nczved/divisions/dfbmd/diseases/foodborne_infections.
  • 53. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Sampath Kumar et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 323 Centers for Disease Control and Prevention (2009). Listeriosis. Available online: www.cdc.gov/nczved/divisions/dfbmd/diseases/listeriosis. Centers for Disease Control and Prevention (2009). Noroviruses and drinking water from private wells. www.cdc.gov/healthywater/drinking/private/wells/disease/norovirus.html. Centers for Disease Control and Prevention (2009).Salmonellosis.: www.cdc.gov/nczved/divisions/dfbmd/diseases/salmonellosis. Centers for Disease Control and Prevention (2009). Shigellosis. www.cdc.gov/nczved/divisions/dfbmd/diseases/shigellosi
  • 54. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Sampath Kumar et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 324 MICROENCAPSULATION TECHNOLOGY K.P.Sampath Kumar1 *,Tejbe.Sk2 , Shameem Banu2 , P.Naga Lakshmi2 , D.Bhowmik3 1. Coimbatore Medical College, Coimbatore 2. Nimra Pharmacy College,Vijayawada 3. Karpagam University,Coimbatore *Corresponding author: kp_sampath@rediffmail.com ABSTRACT Microparticulate drug delivery systems provide tremendous opportunities for designing new controlled and delayed release oral formulations, thus extending the frontier of future pharmaceutical development. The Microparticulate offers a variety of opportunities such as protection and masking, reduced dissolution rate, facilitation of handling, and spatial targeting of the active ingredient. It is the process by which individual particles or droplets of solid or liquid material (the core) are surrounded or coated with a continuous film of polymeric material (the shell) to produce capsules in the micrometer to millimeter range, known as microcapsules. Microencapsulation technology can protect active materials against environment, stabilize them, prevent or suppress volatilization. Microencapsulation technology can provide new forms and features and many polymeric drug delivery systems, biodegradable polymers have been used widely as drug delivery systems because of their biocompatibility and biodegradability. Microencapsulation is a powerful technique to achieve targeted delivery and on-demand release of different active ingredients. Many synthetic and natural biodegradable polymers present exciting opportunities in tailor-making the micro particle formulations for long-term drug release with specific release rates. Hence finally concluded that continuous knowledge up gradation is required in order to make desired drug delivery system and minimization of problems associated with physicomechanical techniques and complete knowledge about selection of raw materials and method for their microencapsulation to get desire goal of study. Key words: Microencapsulation technology, targeted delivery, protection, masking. INTRODUCTION Microencapsulation is a rapidly expanding technology. It is the process of applying relatively thin coatings to small particles of solids or droplets of liquids and dispersions. Microencapsulation provides the means of converting liquids to solids, of altering colloidal and surface properties, of providing environmental protection and of controlling the release characteristics or availability of coated materials. Microencapsulation is receiving considerable attention fundamentally, developmentally and commercially. The term microcapsule is defined as a spherical particle with size varying from 50nm to 2mm, containing a core substance. Microspheres are in strict sense, spherical empty particles. However the terms microcapsule and microsphere are often used synonymously. The microspheres are characteristically free flowing powders consisting of proteins or synthetic polymers, which are biodegradable in nature, and ideally having a particle size less than 200µm. Solid biodegradable microcapsules incorporating a drug dispersed or dissolved throughout the particle matrix have the potential for the controlled release of drug. These carries received much attention not only for prolonged release but also for the targeting of the anticancer drug to the tumour. The concept of miroencapsulation was initially utilized in carbonless copy papers. More recently it has received increasing attention in pharmaceutical and biomedical applications. The first research leading to the development of micro encapsulation procedures for pharmaceuticals was published by Bungenburg de Jong and Kass in 1931 and dealt with the preparation of gelatin spheres and the use of gelatin coacervation process for coating. In the late 1930s, Green and co-workers of National cash register co. Dayton, Ohio, developed the gelatin coacervation process. Since then may other coating materials and processes of application have been developed by the pharmaceutical industry for the microencapsulation of medicines. Over the last 25 years pharmaceutical companies for microencapsulated drugs have taken out numerous patents. Reasons for microencapsulation: The primary reason for microencapsulation is found to be either for sustained or prolonged drug release. This technique has been widely used for masking taste and odor of many drugs to improve patient compliance. This technique can be used for converting liquid drugs in a free flowing powder.The drugs, which are sensitive to oxygen, moisture or light, can be stabilized by microencapsulation. Incompatibility among the drugs can be prevented by microencapsulation. Vaporization of many volatile drugs e.g. methyl salicylate and peppermint oil can be prevented by microencapsulation. Many drugs have been microencapsulated
  • 55. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Sampath Kumar et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 325 to reduce toxicity and GI irritation including ferrous sulphate and KCl. Alteration in site of absorption can also be achieved by microencapsulation. Toxic chemicals such as insecticides may be microencapsulated to reduce the possibility of sensitization of factorial person. Bakan and Anderson reported that microencapsulated vitamin A palmitate had enhanced stability. Microparticles offer various significant advantages as drug delivery systems, including: i. An effective protection of the encapsulated active agent against (e.g. enzymatic) degradation ii. The possibility to accurately control the release rate of the incorporated drug over periods of hours to months. iii. An easy administration (compared to alternative parenteral controlled release dosage forms, such as macro-sized implants). iv. Desired, pre-programmed drug release profiles can be provided which match the therapeutic needs of the patient. Microparticulate drug delivery systems are an interesting and promising option when developing an oral controlled release system.Microcapsules are finally dispersed in various dosage forms, such as hard gelatin capsules, which may be enteric coated, soft gelatin capsules, or suspensions in liquids, all of which allow dispersion of individual microcapsules on release.Microcapsules continue to be of much interest in controlled release because of relative ease in design and formulation and partly on the advantages of microparticulate delivery systems. The latter include sustained release from each individual microcapsule and offer greater uniformity and reproducibility. Additional advantage over monolithic systems containing multiple doses is the greater safety factor in case of a burst or defective individual in subdivided dosage forms. Finally, it has been argued that multiple particle systems are distributed over a great length of gastro-intestinal tract, which should result in, (a) lowered local concentrations and hence reduced toxicity or irritancy, and (b) reduced variability in transit time and absorption rate Basic consideration of microencapsulation technique: Microencapsulation often involves a basic understanding of the general properties of microcapsules, Such as the nature of the core and coating materials, the stability and release characteristics of the coated materials and the microencapsulation methods. The intended physical characters of the encapsulated product and the intended use of the final product must also be considered. a. Core material: The core material, defined as the specific material to be coated, can be liquid or solid in nature. The composition of the core material can be varied as the liquid core can include dispersed and/or dissolved material. The solid core can be a mixture of active constituents, stabilizers, diluents, excipients and release rate retardants or accelerators. b. Coating materials: The coating material should be capable of forming a film that is cohesive with the core materials, be chemically compatible and non reactive with the core material and provide the desired coating properties such as strength, flexibility impermeability, optical properties and stability. The total thickness of the coatings achieved with microencapsulation techniques is microscopic in size. c. Stability, release and other properties: Three important areas of current microencapsulation application are the stabilization of core materials, the control of the release or availability of core materials and separation of chemically reactive ingredients within a tablet or powder mixture. A wide variety of mechanisms is available to release encapsulated core materials; such as disruption of the coating can occur by pressure, shear or abrasion forces, permeability changes brought about enzymatically etc., improved gastro tolerability of drugs can be obtained by microencapsulation. d. Physical character of the final product: Microcapsules should have desirable physical properties like ability to flow, to be compacted or to be suspended and the capsule wall must be capable of resisting the pressure during compression etc. e. coating materials: A number of different substances both biodegradable as well as non-biodegredable have been investigated for the preparation or microcapsules. These materials include the polymers of natural and synthetic origin and also modified natural substances. Some of the polymers used in the preparation of the microcapsules are classified and listed below.
  • 56. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Sampath Kumar et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 326 SYNTHETIC POLYMERS Non-biodegradable 1. PMMA 2. Acrolein 3. Glycidyl methacrylate 4. Epoxy polymers Biodegradable 1. Lactides and glycolides and their copolymers 2. Polyalkyl cyano acrylates 3. Polyanhydrides 4. Corbopol NATURAL MATERIALS A. Proteins B. Albumins C. Gelatin D. Collagen E. Carbohydrates F. Starch , Agarose G. Carrageenan H. Chitosan I. Chemically modified carbohydrates J. DEAE cellulose K. Poly (acryl) dextran L. Poly (acryl) starch METHODS OF MICROENCAPSULATION Preparation of microecapsules as prolonged action dosage form can be achieved by various techniques under following headings. 1. Coacervation phase separation a. By temperature change b. By incompatible polymer addition c. By non-solvent addition d. By salt addition e. By polymer-polymer interaction f. By solvent evaporation 2. Multi orifice centrifugal process. 3. Pan coating 4. Air suspension coating 5. Spray drying and spray congealing 6. Polymerization 7. Melt dispersion technique 1. Coacervation phase separation: Microencapsulation by coacervation phase separation is generally attributed to the national cash register (NCR) corporation and the patents of green et.al. The general outline of the processes consists of three steps carried out under continuous agitation. 1. Formation of three immiscible chemical phases 2. Disposition of the coating, and 3. Rigidization of the coating a. By thermal change: phase separation of the dissolved polymer occurs in the form of immiscible liquid droplets, and if a core material is present in the system, under proper polymer concentration, temperature and agitation conditions, the liquid polymer droplets coalesce around the dispersed core material particles, thus forming the embryonic microcapsules. As the temperature decreases, one phase becomes polymer-poor (the microencapsulation vehicle phase) and the second phase. (The coating material phase) becomes polymer-rich. b. By incompatible polymer addition: it involves liquid phase separation of a polymers coating material and
  • 57. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Sampath Kumar et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 327 microencapsulation can be accomplished by utilizing the incompatibility of dissimilar polymers existing in a common solvent. c. By non-solvent addition: a liquid that is a non-solvent for a given polymer can be added to a solution of the polymer to induce phase separation. The resulting immiscible liquid polymer can be utilized to effect microencapsulation of an immiscible core material. d. By salt addition: there are two types of coacervation: simple and complex. Simple coacervation involves the use of only on colloid, e.g. gelatin in water, and involves removal of the associated water from around the dispersed colloid by agents with a greater affinity for water, such as various alcohols and salts. The dehydrated molecules of polymer tend to aggregate with surrounding molecules to form the coacervate. Complex coacervation involves the use of more than one colloid. Gelatin and acacia in water are most frequently used, and the coacervation is accomplished mainly by charge neutralization of the colloids carrying opposite charges rather than by dehydration. e. By polymer-polymer interaction: the interaction of oppositely charged poly electrolytes can result in the formation of a complex having such reduce solubility that phase separation occurs. f. By solvent evaporation: the processes are carried out in a liquid manufacturing vehicle. The microcapsule coating is dispersed in a volatile solvent, which is dispersed in volatile solvents, which is immiscible with the liquid manufacturing vehicle phase. A core material to be microencapsulated is dissolved or dispersed in the coating polymer solution. With agitation, the core material mixture is dispersed in the liquid manufacturing vehicle phase to obtain the appropriate size microcapsule. The mixture is then heated if necessary to evaporate the solvent for the polymer. In the case in which the core material is dissolved in the coating polymer solution, matrix type microcapsules are formed. The solvent evaporation technique to product microcapsules is applicable to a wide variety of core materials. The core materials may be either water soluble or water insoluble materials. 2. Multiorifice centrifugal process: The South-West research institute (SWRI) has developed a mechanical process for producing microcapsules that utilizes centrifugal forces to hurl, a core material particle through an enveloping microencapsulation membrane therapy effecting mechanical microencapsulation. Processing variables include the rotational speed of the cylinder, the flow rate of the core and coating materials, the concentration and viscosity of the coating material an the viscosity and surface tension of the core material. This method is capable of microencapsulating liquids and solids of varied size ranges, with diverse coating materials. 3. Pan coatings: The microcapsulation of relatively large particles by pan coating method are generally considered essential for effective coating. The coating is applied as a solution or as an automized spray to the desired solid core passed over the coated materials during coatings is being applied in the coating pans. 4. Air suspension coating: The process consists of the dispersing of solid particulate core materials in a supporting air stream and the spray coating of the air suspended particles. Within coating chambers, particles are suspended on an upward moving air stream. The design of the chamber and its operating parameters effect a re-circulating flow of the particles through the coating zone portion of the chamber, where is a coating material, usually a polymer solution is spry-applied to the moving particles. 5. Spray drying and spray congealing: Spray drying and spray congealing processes are similar in that both involve dispersing the core material in liquefied coating substance and spraying or introducing the core coating mixture into some environmental condition, whereby relatively rapid solidification of the coating is effected. The principle difference between the two methods is the means by which coating solidification is accomplished. Coating solidification in the case of spray during is effected by rapid evaporation of solvent in which the coating material is dissolved. Coating solidification in spray congealing method, however, is accomplished by thermally congealing a molten coating material or by solidifying the dissolved coating by introducing the coating core material mixture into a nonsolvent. Removal of the nonsolvent or solvent from the coated product is then accomplished by sorption extraction or evaporation techniques. 6. Polymerization: The method involves the reaction of monomeric unit located at the interface existing between a core material and a continuous phase in which the core material is dispersed. The continuous or core material supporting phase is usually a liquid or gas and therefore the polymerization reaction occurs at a liquid-liquid, liquid-gas, solid-liquid or solid-gas interface e.g., microcapsules containing protein solutions by incorporating the protein in the aqueous diamine phase.
  • 58. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Sampath Kumar et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 328 7. Melt-dispersion technique: In this technique the coating material is melted by heating upto 80o C. The drug is suspended in it and then emulsified in water containing emulsifying agent at 80o C under stirring. Microcapsules are formed as the temperature of the system reaches to room temperature. CONCLUSION Microencapsulation is one of the quality preservation techniques of sensitive substances and a method for production of materials with new valuable properties. Microencapsulation is process of enclosing micron sized particles in a polymeric shell.Significances of microencapsulation For Sustained or prolonged drug release For Masking test and odour of many drugs Converting liquid into free flowing properties Drugs which are sensitive to Light,oxygen, moisture they are easily stabilized. Microencapsulation technologies are applied in any area of the industry. It can be found in: Cell immobilization, Beverage production, Protection of molecules from other compounds, Drug delivery, Quality and safety in food, agricultural & environmental sectors, pharmaceuticals etc. REFERENCES Dziezak JD, Microencapsulation and encapsulated ingredients, Food Technology, 42(4), 1988, 136-51. Fergason JL, Polymer encapsulated nematic liquidcrystals for display and light control applications, SID Int. Symp Digest, 16, 1985, 68-70. Green BK & Schleicher L, The National Cash Register Company, Dayton, Ohio, Oil containing microscopic capsules and method of making them, US Patent 2,800,457.23 July 1957, 11. Green BK, The National Cash Register Company, Dayton, Ohio, Oil containing microscopic capsules and methodof making them, US Patent 2,800,458, 23 July 1957, Jackson LS & Lee K, Microencapsulation and encapsulated ingredients, Lebensmittel WissenschaftTechnol, 24, 1991, 289-97. Mars GJ & Scher HB, Controlled delivery of cropprotecting agents, Wilkens, R.M. (Ed.) Taylor and Francis, London, 1990, 65-90. Scher, H. B. In Proceedings of the 5th InternationalCongress of Pesticides Chemistry, edited by Miyamoto, J.& Kearney, P C Pergamon Press, Oxford. 1982, 295-300. Schnoring H, Dahm M & Pampus G, Fed. Rep. of Germany, Process for the Production of Microcapsules, US Patent 4,379,071, 5 April 1983. 9pp. Shahidi F & Han XQ, Encapsulation of food ingredients, Crit Rev. Food Sci. Nutr, 33, 1993, 501-47. Zhang MQ, Yin T, Rong MZ & Yang GC, Selfhealing epoxy composites–preparation and effect of the healant consisting of microencapsulated epoxy and latent curing agent. Composites Sci. Technol, 67(2), 2007, 201-12.
  • 59. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Praneta Desale Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 329 AN OVERVIEW ABOUT PHARMACY EDUCATION IN INDIA Praneta Desale* ISPOR Asia Consortium Education Committee, Maharashtra-424304, India. * Corresponding author: Email- desalepr@rediffmail.com ABSTRACT If the current trend toward automation continues, there will be severe cuts in the employment of pharmacists in pharmaceutical industries in India. To survive in society as a professional, the pharmacist will have to find suitable alternative avenues. The World Health Organization (WHO) has organized three meetings on the potential role of pharmacists in the health care system: in New Delhi in 1988, in Tokyo in 1993, and in Vancouver in 1997. The first meeting outlined the various activities of pharmacists, namely regulatory control and drug management, legislation, procurement, storage and distribution of drugs, drug information, quality control, community and hospital pharmacy, industrial pharmacy, and academic activities, the second meeting introduced the concept of ph armaceutical care, and the third meeting was on developing curriculums for the education of future pharmacists. The International Pharmaceutical Federation has developed good pharmacy practice standards for the community and hospital settings many pharmacy institutions in India have started offering programs in pharmacy practice and clinical pharmacy. The Pharmacy Council of India (PCI) has initiated steps to raise the minimum qualification of registered pharmacists. This ambitions plan materializes there will be ample opportunity for pharmacists to serve society. Key words: Pharmacy education, Pharmacy council of India, Education Regulations INTRODUCTION The beginning of pharmaceutical education in India was initiated at the Banaras Hindu University (BHU) in 1932 by Professor M. L. Schroff. From there it has been a long journey of almost 80 years for this profession in this country. The enactment of the Pharmacy Act 1948 established the statutory regulation of pharmacy institutions in India. The Pharmacy Council of India (PCI) was established in 1949 under “Ministry of Health” and the first education regulations (ER) framed in 1953, which were subsequently amended in 1972, 1981 and 1991. On the other hand, the pharmacy education has never been part of paramedical team and hence, its development has been quite unique and quite different from rest of the world. Pharmacy Council of India and Pharmacy Act were created to establish minimum qualification required to be a pharmacist. The role of pharmacist in the society was never been given its due place and did not grow due to less paying job compared to job in industry. This would have been the reason for transfer of pharmacy education from PCI to All India Council of Technical Education (AICTE) under the “Ministry of Human Resource Development”. Currently, PCI and AICTE regulate pharmacy profession and education respectively in India. Both of these regulatory bodies have been doing a regulatory function without bothering to create a permanent mechanism of updating curriculum along with development in the field. In short, it can be said that evolution of pharmacy education has been quite confusing and developed like a vagabond. Hence, evolution of pharmacy education has been primarily due to evolution of pharmaceutical industries and has lot of impact under curriculum of “Bachelor and Master in Pharmacy” programmes. Similarly, medical education in India grew with less focus on research and development and hence, India produced medical graduates more with clinical sense acquired through experience and less of a doctors with analytical bent of mind. Due to tight junctions at the entry point, integration of the thoughts of medical sciences, pharmaceutical sciences, nursing, engineering sciences and basic sciences have never taken place. Primarily, this resulted in isolated development of medical education without integration with other sciences including pharmaceutical. It is also true in case of pharmacy education. It may also one of the reason of pharmacy education not to be a part of healthcare system. Today, the global institutes are moving towards excellence in research and capability building in order to better meet the requirements of 21st century. This forces us to evaluate status of pharmacy education in India. There is a rapid transition in pharmacy profession worldwide and in the era of globalization, we cannot be silent spectators. If we have to compete with the rest of the world and become guiding torch for rest of the world, we will have to become proactive. It means, we have to define the goals of pharmacy education for present and future and re-frame our curriculum according to defined goals to meet the global challenges. In the past decade, the technical education in India has spread its roots at an amazing rate. On the other hand, there is sharp decrease in interest and overall admissions to undergraduate programme (B. Pharmacy) in pharmacy during the last three academic years. This decline may be attributed due to changed trends in pharmaceutical industry which has become primarily research and marketing oriented from production oriented. Role of knowledge in giving in increasing employability of the students has become need of the
  • 60. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Praneta Desale Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 330 hour. Simultaneously, other facets of pharmacy profession should be given adequate attention in curriculum development and in creation of knowledge based manpower for service of the society. The products of this form of education lack the much needed professionalism and rational thinking required for problem solving. So, the situation demands a nudge for the system to ensure its revival in order to better meet the needs of 21st century. Pharmacy is related to health sciences. It is the profession responsible for the preparation, dispensing and appropriate use of medication and which provides services to achieve optimal therapeutic outcomes. A Pharmacists job is to prepare, mix, compound or dispense drugs and medicines, ointments, powder, pills, tablets and injections on the prescription of a medical practitioner, dentist or veterinarian. In detail, they are concerned with production of pharmaceutical products, development of the methods or processes of production and quality control. Those in research concern themselves with synthesis of new drugs (what is commonly referred to as molecules), new processes, clinical testing of the effects of such drugs on animals and humans, and obtaining the required License from the drug control authorities. A pharmacist is required to explain the mode and precautions regarding the use of medicines dispensed in a hospital pharmacy, prepare special formulations normally not available in the market, assist the physician in rendering necessary information about various drugs, their contra-indications, incompatibility etc. PHARMACY PROFESSION IN INDIA Currently there are over a million pharmacists in India with around 55% of them in community, 20% in hospital, 10 % in industry & regulatory. And 2 % in academia in India, formal pharmacy education leading to a degree began in 1937, with the introduction of a 3 year industry – oriented Bachelor of Pharmacy course. To meet the varying needs of the profession at different levels the following pharmacy programs are offered in India today: Diploma in Pharmacy (D.Pharm.), Bachelor of Pharmacy (B.Pharm.), Master of Pharmacy (M.Pharm.), practice- based Doctor of Pharmacy (Pharm.D.), and Doctor of Philosophy in Pharmacy (Ph.D.). To practice as a pharmacist in India, one needs at least a diploma in pharmacy, which is awarded after 2 years and 3 months of pharmacy studies & practical training. These diploma-trained pharmacists are currently the mainstay of pharmacy practice in India. Every year nearly 20000 D. Pharm, 30,000 B. Pharm, 6000 M.Pharm and 700 Pharm.D. students graduate in the Country. Pharmacy Council of India (PCI) is the statutory body established in 1949, for regulating pharmacy education and practice of pharmacy profession in India. PCI is actively working towards strengthening and upgrading the curriculum to produce competent workforce that is able to meet the growing demands of the industry & community. In 2003, the Pharma Vision 2020 Charter was released by the then President of India, Dr. A.P.J. Abdul Kalam, at the 55th Indian Pharmaceutical Congress at Chennai. The Vision 2020 is focused on promoting the highest professional ethical standards of pharmacy, focusing the image of pharmacists and competent healthcare professionals, sensitizing the community, government and others on vital professional issues and supporting pharmaceutical education and sciences in all aspects. Indian Pharmaceutical Association once again, with the support of the leaders of the pharmacy profession presented the road map to Pharma Vision 2020 at the 58th Indian Pharmaceutical Congress held in December 2006 at Mumbai. The themes of the subsequent Congresses in the country have been centered on Pharma Vision 2020. PHARMACEUTICAL INDUSTRY IN INDIA It is exciting to be part of the Pharmaceutical industry in India today, In 2007, the turnover of the Indian Pharmaceutical Industry was USD 8.4 bn, with additional USD 5.8 bn generated from exports. If we take a look at the top 10 pharmaceutical companies in India, more than 50% of their annual turnover comes from exports. India exports to over 100 countries & also boasts of having the largest USFDA approved facilities outside USA. The number is rising every year as Indian companies keep on adding facilities to cater to the increasing demand. India is the 3rd largest manufacturer of pharmaceuticals in the global market in terms of volume of sales. In terms of value, India is 14th globally which clearly shows that the prices of the medicines in India are lowest in the world. India has slowly but surely emerged as the global outsourcing hub- be it for manufacturing or R&D, Clinical research or basic drug discovery. The reasons are not hard to fathom. India has a huge pool of qualified & well trained professionals, While maintaining stringent quality standards, it is possible to establish GMP- compliant manufacturing units at around 30% cheaper rates as compared to any of the regulated markets. All these factors make outsourcing various pharmaceutical operations to India a very lucrative option for the big pharma that are trying hard to manage their profit margins.Indian pharmaceutical companies have also been making increasing forays into the global generics market, which is continuously increasing because of many of the blockbuster drugs going off-patent, Identification of molecules going off-patent, well thought out time lines, speed of doing development work and good
  • 61. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Praneta Desale Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 331 regulatory understanding are some of the key factors that define success in the generic industry; attributes that are the forte of Indian companies. TOTAL QUALITY MANAGEMENT IN PHARMACY EDUCATION Applying principles of TQM to pharmacy education in India leads to the development of pharmacy education in India. The concept of Total Quality Management (TQM) although developed by an American was successfully implemented by Japan in their recovery from World War II. The concept of TQM is applicable to academics. Many educators believe that the concept of TQM provides guiding principles for needed educational reform. Education is a fast moving commodity in the market and is mainly business oriented which means it should give some profit to the undertaker. TQM is a philosophy for perfection and continuous improvement in services offered to someone or one's own performance. The TQM principles which are most salient to educational reform are as follows: Synergistic relationship: According to this principle, an organization must focus, first and foremost, on its “suppliers” and “customers”. In other words, teamwork and collaboration are essential. The concept of synergy suggests that performance and production is enhanced by pooling the talent and experience of individuals. In a classroom, teacher-student teams are the equivalent of industry’s front-line workers. The product of their successful work together is the development of the student’s capabilities, interests, and character. Continuous improvement and self evaluation: TQM emphasizes self-evaluation as part of a continuous improvement process. In addition, this principle also laminates to the focusing on students’ strengths, individual learning styles, and different types of intelligences. A system of ongoing process: The recognition of the organization as a system and the work done within the organization must be seen as an ongoing process. Quality speaks to working on the system, which must be examined to identify and eliminate the flawed processes that allow its participants to fail. Leadership: The upper level provides proposes basic way of functioning, provides quality staff, while the lower level are directly linked to the students as lecturers who perform the most important functions of the whole system. The school teachers must establish the context in which students can best achieve their potential. REGULATION BODIES OF PHARMACY EDUCATION There is no doubt that currently there is enormous gap existing between education and practice of pharmacy. Most of the academic institutions providing education in pharmacy are away from practice environment. The overall basis of pharmacy education is still extra biological synthesis, physicochemical studies, analysis, and manufacturing aspects of drug. It is a common feeling that the medical practitioner is better placed for pharmacists' job than the pharmacists themselves. The dispensing services are poor. The syllabus and duration of the two-year diploma course in pharmacy education in India is completely outdated and irrelevant in the present industry context. It is a heterogeneous mixture of clinical and industrial subjects. Since clinical subjects are there PCI comes into the picture and AICTE came in because of industrial orientation of pharmacy syllabus. Pharmacy as a nascent science developed like this in the last century. During 1940s and 50s, hospitals and industries were established in large numbers in India. Consequently, pharmacists and pharmaceutical chemists were required in huge numbers. Hence pharmacy education was developed in such away to satisfy the requirement of industry and hospital. Short- term compounders and or D. Pharm. course to satisfy the needs of hospital and medical shops and B. Pharm. course for the industry were started. This is proved by the fact that in the last few decades D. Pharm. holders are not employed by the industry and B. Pharm. holders are not in many numbers in hospitals or medical shops. In the West, pharmacy education is patient-oriented and is responsible for Healthcare Management, while in India pharmacy education is industry-oriented.Nearly 55 per cent of the jobs are available in the industry sector while 30per cent in education. There are only three per cent jobs in healthcare. There must be revolutionary changes in the healthcare system e.g. making laws for appointing pharmacists at each Primary Health Centre and government hospitals. There should be adequate staff in the state drugs control departments for better control of drug distribution system. It is crystal clear that separation and improvement of clinical and industrial subjects in the pharmacy syllabus is a compulsion of the time. But it is yet to be completed, that is why there is such a situation and a lot of infighting among government authorities. Present. Pharm. syllabus can be divided into 2 major courses like B. Pharm.(Clinical) and B. Pharm. (Industrial) as it has been already decided to abolish D. Pharmacy course. Such an arrangement will increase the confidence and competitive skills of pharmacy graduates among health care team and technocrats and some sort of specialization during under graduation itself. If two B. Pharm.courses are created as above, needless to say clinical course can be controlled by PCI and industrial course by AICTE. Private college managements can opt for
  • 62. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Praneta Desale Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 332 any one of the courses. If any college wants to run both the courses theyshould accept both masters, there is no other go. Existing D. Pharm. colleges who are in the verge of closure can adopt B. Pharm. (Clinical) and continue to serve the profession. This stunted growth of professional pharmacy in our country is the result of misplaced belief that profession is same as vocation. This belief has kept Indian pharmacy academics completely focused on industrial pharmacy at the cost of real - communitypharmacy. While the justification for focusing pharmacy education on Industrial Pharmacy after attaining national freedom was valid, its review to make it relevant in contemporary scenario is already too late. Our present system has produced half a million''qualified'' pharmacists but not many ''trained'' professionals. This has effectively led to a situation where neither there is a need felt by the society nor is there anyone available to fulfill that "professed" need. This situation feeds on itself to such an extent that any attempt to keep one's knowledge updated and work professionally has strong economic disincentives in Indian retail pharmacy practice. Gravity of the situation dawns upon us when we think about petitions filed in High courts that propose scrapping of the Pharmacy Act because the pharmacists - according to petitioners - do not play any role other than selling the drugs like all other commodities. There is virtually a complete lack of any training or incentive to professionalize – as a result of which even the most enthusiastic pharmacists gradually convert into mere traders. The uninspiring implementation of statutory provisions has led to a cancerous proliferation of retail drug shops and the situation now threatens the profession itself. The retail pharmacist shall be relevant to the society ‘only'' if he can make a difference to the patient - by providing him information about drug usage to achieve better outcome than the patient obtains by uninformed usage of drugs. CONCLUSION Each pharmacy institute should operate a model pharmacy; this would not only improve the image of pharmacists in Indian society but provide an opportunity for pharmacy students to train in community practice. The minimum wages established by state governments for pharmacists working in drugstores should be properly implemented and periodically revised. Even though medicines are now dispensed in the manufacturer’s original pack wherever possible, additional labeling should include generic name and strength, dose and frequency, date of dispensing, name of patient, name and address of dispenser and pharmacy, and date after which the product in not to be used. Finally, to improve patient compliance, oral or written instructions should be provided by the pharmacist. Although raising the minimum qualification of registered pharmacists to the B.Pharm, degree is desirable, the economics of employing pharmacists in drug stores, particularly in remote rural areas, need to be considered. Even if standards for good pharmacy practice are set in India, it will take years to meet them fully, until then, pharmacists in hospital and community setting need to take steps on their own to improve their image and protect the health of patients and the public. REFERENCE Good pharmacy practice in community and hospital pharmacy settings, WHO/FARM/ DAPS, 1996, 5-10. Mohanta GP, Manna PK, Valliappan K, Manavalan R, Pharmacy education in India ,Am J Health-Syst Pharm, 2001, 809-810. Seth PD, A responsible pharmacist of the 21st century in India through education and service, Pharma Times, 1999, 31 (Jan), 30. The role of pharmacists in the healthcare system, WHO/FARM, 1994, 30-60.
  • 63. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Phani Deepthi Yadav et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 333 PHYTOCHEMICAL EVALUATION OF NYCTANTHES ARBORTRISTIS, NERIUM OLEANDER AND CATHARATHNUS ROSEUS CH.S.D. Phani Deepthi Yadav*, N.S.P. Bharadwaj, M. Yedukondalu, CH. Methushala, A. Ravi Kumar *PG & Research Department of Pharmacognosy, Bapatla College of Pharmacy, Bapatla-522 101, Andhra Pradesh, India *Corresponding Author: deepukoushikyadav@gmail.com ABSTRACT In the present study, an attempt was made to investigate phytochemical evaluation of Nyctanthes arbortristis, Nerium oleander and Catharathus roseus. The crude drug powder extracts of the leaves of the above plants were taken for the study. The Phytochemical Screening was done for the selected plants. Phenolic compounds, tannins, flavonoids, cardiac glycosides, and alkaloids were present in Nyctanthes arbortristis. Alkaloids, flavanoids, carbohydrates, glycosides and tannins were present in Nerium oleander. Alkaloids, saponins, flavanoids, carbohydrates and anthraquinone glycosides were present in Catharanthus roseus. Key words: Phytochemical screening, Nyctanthes, Nerium and Catharanthus, Plant species. INTRODUCTION Herbal medicine also known as botanical medicine or phytomedicine-refers to using plants seeds, flowers, roots for medicinal purpose. Herbalism has a long tradition of use of outside of conventional medicine. It is becoming more main stream as improvements in analysis and quality control along with advances in clinical research show the value of herbal medicine in the treating and preventing disease. The medicinal action of plants is unique to a particular plant species, consistent with the concept that the combination of secondary metabolites in a particular plant is taxonomically distinct for three medicinal plants and their description and uses respectively. Nyctanthes arbor-tristis is commonly known as Night-flowering Jasmine, Coral Jasmine and Parijat. It is used for its antibacterial, anthelmintic, anti-inflammatory, hepatoprotective, immunopotential, anti pyretic, antioxidant and anti fungal activity Nerium oleander is an evergreen shrub or small tree in the dogbane family Apocynaceae, toxic in all its parts. Used traditionally in treating dermatitis, abscesses, eczema, psoriasis, sores, warts, corns, ringworm, scabies, herpes, skin cancer, asthma, dysmenorrheal, epilepsy, malaria, abortifacients, emetics, heart tonics, and tumor. Catharanthus roseus commonly called Madagascar periwinkle is an evergreen shrub or herbaceous plant which exhibits the anti cancer activity due to the presence of vincristine and vinblastine. Here in the present study the above three plants were taken for Phytochemical screening of plants extracts crude dried powdered drug were taken and evaluated. The phytochemical constituents were studied by qualitative analysis for performing various chemical tests. MATERIALS AND METHODS Plant Materials: The leaves of plants Nerium, Nyctanthes and Catharanthus species were authentified by Prof. V.Satyanarayana Department of Plant Breeding, Bapatla Agricultural College, Bapatla, Andhra Pradesh, India .They were collected from different areas of Guntur, Prakasham and Krishna districts of Andhra Pradesh, India . Solvent Extraction: The leaves of Nerium oleander, Catharanthus roseus and Nyctanthes arbortristis were collected, washed, dried and powdered separately. 50g of dried powder of the leaves was weighed and transferred into a conical flask and it was macerated with sufficient amount of ethanol for about a week days. Process is repeated with water. The whole mixture was filtered and filtrate was collected, concentrated in a china dish on a hot plate till the residue was obtained. The extracts was collected, labeled and stored for further experimental use. Qualitative analysis for detection of Carbohydrates Alkaloids Cardiac Anthraquinone Saponin Glycosides Flavonoids Tannins: The extracts and crude dried powders of Nyctanthes arbortristis, Nerium oleander and Catharanthus roseus were subjected to qualitative analysis for presence of chemical constituents of Nyctanthes arbortristis, Nerium oleander and Catharanthus roseus by performing various chemical tests.
  • 64. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Phani Deepthi Yadav et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 334 TEST FOR CARBOHYDRATES TEST FOR ALKALOIDS To 250 mg of each extracts, 10 ml of dilute HCl was added, mixed and filtered. To the filtrate the following reagents were added and tested. TEST PROCEDURE WAGNER’S TEST 2 ml of Wagner’s reagent was added to the above filtrate solution and observed. HAGER’S TEST To the 2 ml of above filtrate solution, 2 ml of picric acid was added and observed. TEST FOR GLYCOSIDES The extract was tested for the presence of saponin glycosides, cardiac glycosides and anthraquinone glycosides TEST FOR SAPONIN GLYCOSIDES TEST PROCEDURE FOAM TEST To 200 mg of each extracts, 15 ml of distilled water was added, shake it well and observed. TEST FOR CARDIAC GLYCOSIDES TEST PROCEDURE LEGAL’S TEST To 50 mg of each extracts, 1 ml of pyridine, 1 ml of Sodium nitro prusside solution were added and observed. KELLER-KILIANI TEST To 50 mg of each extracts, 2 ml of glacial acetic acid, 1 ml FeCl3 solution were added, heated and then cooled. This was transferred to a test tube containing 2ml conc. H2SO4and observed. TEST FOR ANTHRAQUINONE GLYCOSIDES TEST PROCEDURE BORNTRAGER’S TEST To 200 mg of each extracts, dil. H2SO4was added and boiled. Then it was filtered and cooled. To the cold filtrate, 3 ml of benzene was added and mixed. The benzene layer was separated and to it, ammonia (2 ml) was added and ammonical layer was observed. TEST PROCEDURE MOLISCH’S TEST 200 mg of extracts were dissolved separately in 5ml of water and filtered. 2 ml of the above sample solution is placed in a test tube. Two drops of the Molisch reagent is added. The solution is then poured slowly into a tube containing 2 ml of concentrated sulphuric acid and observed. FEHLING’S TEST 1ml of Fehling’s solution A and 1ml of Fehling’s solution B were added to 100mg of extracts separately. They were heated on a boiling water bath for 5 min and observed. BENEDICT’S TEST To the 150 mg of each extracts, 2ml of Barfoed’s reagent was added. Then the mixture was heated on a boiling water bath for 5 min, cooled and observed.
  • 65. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Phani Deepthi Yadav et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 335 TEST FOR FLAVANOIDS TEST PROCEDURE LEAD ACETATE TEST To the 100 mg of each extracts, lead acetate (5 ml) was added and observed. TEST FOR TANNINS To 100 mg of each extracts, the following reagents were added and observed. 1. 5 ml of 5% w/v FeCl3 solution. 2. 5 ml acetic acid solution. 3. 5 ml dil. KMnO4 solution. TEST FOR STEROIDS TEST PROCEDURE SALKOWSKI TEST To 100 mg of each extracts, 2 ml of CHCl3, 2 ml of conc. H2SO4were added, mixed thoroughly and both the layers were observed for color. LIBERMAN AND BURCHARD TEST To 200 mg of each extracts, 5ml CHCl3, 5 ml acetic anhydride were added. Two drops of H2SO4 was added from the sides of test tube and observed. PHYTOCHEMICAL EVALUATION OF NYCTANTHES ARBORTRISTIS Table 1. Phytochemical evaluation of Nyctanthes arbortristis Chemical tests Result Chemical tests Result Test for carbohydrates A. Molisch's test B. Fehling's test C. Benedict's test D. Barfoed's test Positive Positive Positive Positive Test for alkaloids A. Hager's test B. Wagner's test Positive Positive Test for flavanoids Lead acetate test Positive Test for saponins A. Foam test Negative Test for cardiac glycosides A. Legal test B. Keller-killiani test positive positive Test for steroids A. Lieberman burchard test B. Salkowski test Negative Negative Test for anthraquinone glycosides A. Borntrager's test Negative PHYTOCHEMICAL EVALUATION OF NERIUM OLEANDER Table 2. Phytochemical evaluation of Nerium oleander Chemical tests Result Chemical tests Result Test for carbohydrates A. Molisch's test B. Fehling's test C. Benedict's test Positive Positive Positive Test for steroids A. Lieberman burchard test B. Salkowski test Positive positive Test for alkaloids A. Hager's test B. Wagner's test Positive Test for cardiac glycosides A. Legal test B. Keller-killiani test Positive Positive Test for flavanoids Lead acetate test Positive Test for anthraquinone glycosides Borntragers test Negative Test for saponins Foam test Negative Test for tannins A. Fecl3test B. Acetic acid test C. Kmn04 test C. Kmn04 test Positive Positive Positive
  • 66. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Phani Deepthi Yadav et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 336 PHYTOCHEMCIAL EVALUATION OF CATHARANTHUS ROSEUS Table. 3. Phytochemcial evaluation of Catharanthus roseus Chemical tests Result Chemical tests Result Test for carbohydrates A. Molisch's test B. Fehling's test C. Benedict's test D. Barfoed's test Positive Positive Positive Positive Test for saponins A. Foam test Positive Test for steroids A. Lieberman burchard test B. Salkowski test Negative Negative Test for alkaloids A. Hager's test B. Wagner's test Positive Test for cardiac glycosides A. Legal test B. Keller-killiani test Negative Negative Test for flavanoids Lead acetate test Positive Test for anthraquinone glycosides A. Borntrager's test Positive RESULTS AND DISCUSSION The study of the chemical constituents and the active principles of the medicinal plants have acquired a lot of importance all over the world. The present study includes the phytochemical screening of the plants Nyctanthes arbortristis, Nerium oleander and Catharanthus roseus. The plants were collected and were authentified. Then they were shade dried and powdered and were subjected to phytochemical screening. The dried powdered leaves of Nyctanthes arbortistis, Nerium oleander and Catharanthus roseus were subjected to extraction with ethanol separately. The qualitative chemical tests for the ethanolic extracts were performed. The investigation showed that Nyctanthes arbortristis contains carbohydrates, alkaloids, flavanoids and cardiac glycosides. The screening showed that Nerium oleander possesses carbohydrates, flavanoids, alkaloids, steroids, cardiac glycosides and tannins. The screening showed that Catharanthus roseus possesses carbohydrates, flavanoids, saponins, and alkaloids. The results were given in Table 1 and Table 2 and Table 3 respectively. CONCLUSION The screening of phytochemical constituents of plants Nyctanthes arbortristis, Nerium oleander and Catharanthus roseus indicated the presence of carbohydrates, flavonoids and alkaloids in common. Nerium oleander does not contain saponins and Catharanthus roseus lacks tannins and cardiac glycosides and Nyctanthes arbortristis do not have steroids, saponins and tannins. These plants contain more metabolites and there is a need for further investigations using fractionated extracts and purified chemical components. AKNOWLEDGEMENTS The authors are thankful to Management and Principal of Post Graduate Research Centre Division, Bapatla College of Pharmacy, Bapatla, Andhra Pradesh, India in permitting and providing necessary facilities for carrying out to do the project work. Nyctanthes arbortristis (Aerial Parts of thePlant) Nerium oleander(Aerial Parts of the Plant)
  • 67. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Phani Deepthi Yadav et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 337 Catharanthus roseus (Aerial Parts of the Plant) REFERENCES Mathuram V and Kundu AB, Occurrence of two new ester of 6-Hydroxyloganin in Nyctanthes arbortristis, J Indian Chem Soc, 68, 1991, 581-584 Saxena RS, Gupta B, Saxena KK and Srivastava VK and Prasad DN, Analgesic, antipyretic and ulcerogenic activities of Nyctanthes arbortristis leaf extract, J Ethnopharmacol, 19, 1987, 193-200. Amarite O, Bhuskat P, Patel N and Gadgoli C, Evaluation of antioxidant activity of carotenoid from Nyctanthes arbortristis. Int J Pharmacol Biol Sci, 2, 2007, 57-59. Omkar A, Jeeja T and Chhaya G, Evaluation of anti-inflammatory activity of Nyctanthes arbortristis and Onosma echiodes. Phrmacog.mag, 8, 2006, 258-260 Kirtikar KR and Basu BD. Indian Medicinal Plants, Vol.VII, (Sri Satguru Publications, New Delhi,) 2000; 2110- 2113. Tandon JS, Srivastava V and Guru PY, Iridoids: A new class of leishmanicidal agents from Nyctanthes arbortristis, J Nat Prod. 4, 1991, 1102-1104. Hukkeri VI, Akki KS, SUreban RR, Gopalakrishna B, Byahatti VV and Rajendra SV, Hepatoprtective of the leaves of Nyctanthes arbortristis Linn, Indian J Pharm Sci, 68(4), 2006, 542-543. Vats M, Sharma N and Sardana S, Antimicrobial Activity of stem bark of Nyctanthes arbortristis linn. (Oleaceae), Int. J Pharmacognosy and Photochemical Research, 1(1), 2009, 12-14. Kusum S and Akki, Phytochemical investigation and in vitro evaluation of Nyctanthes arbortristis leaf extract for antioxidant property, J Pharm Res, 2(4), 2009, 752-755 Saxena RS, Gupta B, Saxena KK, Srivstav VKand Prasad DN, Analgesic, Antipyretic and Ulcerogenic activity of Nyctanthes arbortristis Leaf Extract. J. Ethanopharmacol, 19, 1987, 193-200. Andrews JM, BSAC Standardised disc susceptibility testing method. J Antimicrob Chemother, 48, 2001, 5–16. Shanthi R, Lakshmi G, Priyadarshini AM, Anandaraj L, Phytochemical screening of Nerium oleander Linn. Leaves and Momordica charantia leaves, International research journal of pharmacy 2(1), 2011, 131-135. Lokesh R,Leonard Barnalas, Madhuri P,Saurav K,Sundar K, Larvicidal activity of Trigonella, foenumand Nerium oleander Linn. Current research journal of biological sciences, 2(3), 2010, 154-160. El-Sayed A and GA Cordell, Catharanthamine: A new antitumor bisindole alkaloid from Catharanthus roseus, J. Nat. Prod, 44, 1981, 289-293. Jaleel CA, R Gopi and R Paneerselvam, Alterationsin non-enzymatic antioxidant componentsof Catharanthus roseus exposed to paclobutrazol, gibberellic acid and Pseudomonas fluorescens, Plant Omics J, 2, 2009, 30-40. Muhammad LR, N Muhammad, A Tanveer and SN Baqir, Antimicrobial activity of different extracts of Catharanthas roseus, Clin. Exp. Med. J, 3, 2009, 81-85
  • 68. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Phani Deepthi Yadav et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 338 Perez C, M. Pauli and P. Bazerque , An antibioticassay by the agar-well diffusion method. Acta Biol. Med. Exp, 2, 1990, 708-712. Chattopadhyay RR, Sarkar SK, Ganguli S, Banerjee RN Basu, TK, Hypoglycemic and antihyperglycemic effect ofleaves of Vinca rosea Linn. Indian Journal of Physiology andPharmacology 35, 1991, 145–151. Chattopadhyay, R.R., Sarkar, S.K., Ganguli, S., Banerjee, R.N.Basu, T.K., 1992. Antiinflammatory and acute toxicity studieswith leaves of Vinca rosea linn. in experimental animals. IndianJournal of Physiology and Pharmacology 36, 291–292 Ohadoma SC, Micheal HU. (2011) Effects of co-administration of methanol leaf extract of Catharanthus roseus on the hypoglycemic activity of metformin and glibenclamide in rats. Asian Pac J Trop Biomed 4(6): 475-477 Hassan KA, Brenda AT, Patrick V, Patrick OE.(2011) In vivo antidiarrheal activity of the ethanolic leaf extract of Catharanthus roseus Linn. (Apocyanaceae) in Wistar rats, Afr J Pharm Pharmacol, 5(15), 1797-1800. Parekh J Karathia N Chanda S (2006) Evaluation of Antibacterial activity and Phytochemical Analysis of Bauhinia variegate L Bark, African Journal of Biomedical Research, 9:53-56. S Vidyadhar Saidulu M Gopal TK Chamundeeswari D Rao U Banji D, In Vitro Anthelmentic activity of whole plant of Enicostemma littorale by using various extracts International Journal of Applied Biology and Pharmaceutical Technology, 1(3), 2010, 1119-1125.
  • 69. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Shravan Paswan et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 339 ANGINA PECTORIS EPIDEMIC IN INDIA: A COMPREHENSIVE REVIEW OF CLINICAL FEATURES, DIFFERENTIAL DIAGNOSIS, AND REMEDIES Shravan Paswan1 *, Ranjan Kumar Sharma1 , Alok Ranjan Gaur2 , Avinash Sachan1 , Mahendra Singh Yadav1 , Preeti Sharma3 , Mrinmoy Gautam4 1. Advance Institute of Pharmaceutical Education & Research, Kanpur, India 2. Department of Pharmacy, Pranveer Singh Institute of Technology Kanpur, India 3. Department of Pharmaceutical Science, Banasthali University, Rajasthan, India 4. Royal College of Pharmacy and Health Sciences, Berhampur, Odisha, India *Corresponding author: E-mail: paswanshravan@gmail.com ABSTRACT The community pharmacist is the globally accepted professional to cater the pharmaceutical care to the patients at the time of dispensing the medicine it self. This will correct the wrong practices in medicine usage and make the patient a partner in his improvement of health. Angina pectoris is the medical term for chest pain or discomfort due to coronary heart disease. Angina is a symptom of a condition called myocardial ischemia. It occurs when the heart muscle (myocardium) doesn't get as much blood (hence as much oxygen) as it needs. This usually happens because one or more of the heart's arteries (coronary blood vessels that supply blood to the heart muscle) is narrowed or blocked. Insufficient blood supply is called ischemia. Major risk factors for angina include cigarette smoking, diabetes, high cholesterol, high blood pressure, sedentary lifestyle and family history of premature heart disease. The distal part of the heart does not receive any more blood and individuals will develop chest pain. Initially when the coronary disease is mild the angina will occur during exercise, eating heavy meals, extreme heat or stress. As the coronary disease worsens, the angina will come on with minimal work and may even occur at rest. The first approach in the treatment of angina pectoris is to prevent the progression of heart disease. By addressing the known causes of heart disease, such as reducing high cholesterol levels, controlling high blood pressure, stopping smoking, losing weight, exercising and eating a “heart-healthy” diet, the symptoms can be reduced. Most people can live a productive life if they make the necessary lifestyle changes. By following medical advice, taking doctor- prescribed medication, maintaining a good physical condition and eating well, angina can be controlled. There are also natural alternatives to conventional medicine such as herbal and homeopathic remedies useful in controlling angina without the harsh side effects associated with prescription drugs. Herbal and homeopathic remedies are safe and gentle to use, while at the same time addressing the underlying causes of the condition. Key Words: Community pharmacist, Angina pectoris, Coronary Heart Disease, Ischemia, Myocardium. INTRODUCTION Angina pectoris: Angina pectoris is a medical condition that occurs when the heart receives a decreased amount of oxygenated blood. Often, this occurs due to deposits of cholesterol, clogging the blood vessels that carry blood to the heart. Patients who have angina pectoris are at an risk for having a heart attack, chest pain behind the breastbone is the most common sign of angina pectoris. The discomfort may feel like pressure, squeezing, burning or tightness, reports the National Heart Lung and Blood Institute. The chest pain is most common during exercise, physical work or sexual activity. Emotional stress, cold weather and nightmares may also trigger an attack of chest pain, explains Cedars-Sinai Medical Center. Patients with severe angina pectoris may also develop the pain during rest without the presence of any stress. An episode of chest pain caused by angina pectoris may last from 5 to 30 minutes. Angina is a common presenting symptom (typically, chest pain) among patients with coronary artery disease. Statistics reveal that close to 7 million American suffer from angina and countless more do not even know if they have it. Each year close to half a million new cases are diagnosed. The common condition affects nearly ¼ individuals over the age of 55. Each year, there are more than 1 million cases of recurrent acute angina, with a morality rate of close to 40%. In addition, there are a significant number of individual who die suddenly and have no symptoms. No race or ethnic groups are immune from coronary artery disease. Angina pectoris is far more common in women than men. In addition, angina in women can present in an atypical fashion. The pain may not be in the chest area and the pain may have a different quality. To improve the
  • 70. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Shravan Paswan et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 340 prognosis, it is essential that one change the lifestyle. One should adhere to these changes, otherwise angina will recur. The best preventive measures one can take include - start an exercise program, stop smoking, decrease alcohol consumption, avoid stressful situation, avoid heavy and fatty meals. The best treatment of angina is drug therapy. The most common group of drugs used to treat angina is nitroglycerines. Nitroglycerin is available in various formulations. It can be taken by mouth, placed under the tongue and can even be given intravenously. Nitroglycerin has the ability to open up (dilate) blood vessel and allows more blood flow to the heart. Nitroglycerin is usually taken when one feels the chest pain occuring. A tablet is placed underneath the tongue and within a few minutes the pain will disappear. In some cases, two tablets may be required. Nitroglycerin formulations are also available as an oral pill which must be taken 2-3 times a day. Beta blockers have been used to treat coronary disease for decades. They act by decreasing the work of the heart and thus decrease oxygen utilization. Unfortunately they work in the long term and do not work immediately, like nitroglycerin. Beta blockers have to be taken every day and have a few side effects like a decrease in libido. Calcium channel blockers are very effective in the treatment of angina, but like the beta blockers they have to be taken daily. Causes of angina pectoris: In most cases, the cause of angina is coronary atherosclerosis: the thickening of arteries that supply blood, oxygen and nutrients to the heart. This happens when fatty deposits, called plaques or atheroma, narrow the arteries over time and reduce blood flow to the heart. Symptoms may only appear at times when the heart needs more blood supply, such as in stressed condition, exercising or climbing stairs. As the heart tries to pump blood faster to meet the body's increased demands, the narrowed arteries struggle to keep up. The heart then receives less oxygen, which causes pain in the heart that is felt as chest pain.In severe cases this can also happen when the heart is at rest. Types of angina pectoris: Angina is classified as one of the following two types: 1. Stable angina 2. Unstable angina 1. Stable Angina: Stable angina is the most common angina, and the type most people mean when they refer to angina. People with stable angina usually have angina symptoms on a regular basis.The episodes occur in a pattern and are predictable. For most people, angina symptoms occur after short bursts of exertion. Stable angina symptoms usually last less than five minutes. They are usually relieved by rest or medication, such as nitroglycerin under the tongue. 2. Unstable Angina: Unstable angina is less common. Angina symptoms are unpredictable and often occur at rest.This may indicate a worsening of stable angina, but sometimes the first time a person has angina it is already unstable. The symptoms are worse in unstable angina - the pains are more frequent, more severe, last longer, occur at rest, and are not relieved by nitroglycerin under the tongue. Unstable angina is not the same as a heart attack, but it warrants an immediate visit to the healthcare provider or a hospital emergency department. The patient may need to be hospitalized to prevent a heart attack. If the patient has stable angina, any of the following may indicate worsening of the condition. An angina episode that is different from the regular pattern being awakened at night by angina symptoms. Coronary Heart Disease: The most common cause for the heart not getting enough blood is coronary heart disease, also called coronary artery disease. In this disease, the coronary arteries become blocked, narrowed, or otherwise damaged. They can no longer supply the heart with all of the blood it needs. Most cases of coronary heart disease are caused by atherosclerosis (hardening of the arteries). Atherosclerosis is a condition in which a fatty substance/cholesterol builds up inside the blood vessels. These buildups are called plaques, and they can block blood flow through the vessels partially or completely. Multiple risk factors, particularly: diabetes, high blood pressure, smoking, high cholesterol, and genetic predisposition may accelerate this build up. Coronary Artery Spasm: Another cause of unstable angina is coronary artery spasm. Spasm of the muscles surrounding the coronary arteries causes them to narrow or close off temporarily. This blocks the flow of blood to the heart muscle for a brief time, causing angina symptoms. This is called variant angina or Prinzmetal angina. This is not the same as atherosclerosis, although some people have both conditions. The symptoms often come on at rest (or during sleep) and without apparent cause. Cocaine use/abuse can cause significant spasm of the coronary arteries and lead to a heart attack. Risk factors: All of us have fatty deposits in our arteries to some degree. Atherosclerosis can start as early as our 20s and increases with age. But there are risk factors that are known to increase the development of fatty deposits that can cause the arteries to narrow.
  • 71. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Shravan Paswan et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 341  A family history of atherosclerosis  High levels of LDL cholesterol in the blood  High blood pressure  Smoking  Being male  Diabetes  Obesity  Stress  Lack of regular exercise Symptoms of angina pectoris: Symptoms typically start during physical exertion or emotional stress. They are often worse in cold or windy weather and sometimes after big meals.  A squeezing or heavy pressing sensation on the chest.  Increased shortness of breath on exercise.  A sense of heaviness or numbness in the arm, shoulder, elbow or hand, usually on the left side.  A constricting sensation in the throat.  The discomfort can radiate into arms, the jaw, teeth, ears, stomach and in rare cases between the shoulder blades. Unstable angina is associated with the same symptoms at rest. In some cases the fatty deposits that restrict blood flow can rupture. Blood then clots around the rupture, and the clot may be large enough to block the artery and seal off the blood supply. This may cause unstable angina or a heart attack. Pathophysiology: Myocardial ischemia develops when coronary blood flow becomes inadequate to meet myocardial oxygen demand. This causes myocardial cells to switch from aerobic to anaerobic metabolism, with a progressive impairment of metabolic, mechanical, and electrical functions. Angina pectoris is the most common clinical manifestation of myocardial ischemia. It is caused by chemical and mechanical stimulation of sensory afferent nerve endings in the coronary vessels and myocardium. These nerve fibers extend from the first to fourth thoracic spinal nerves, ascending via the spinal cord to the thalamus, and from there to the cerebral cortex. Studies have shown that adenosine may be the main chemical mediator of anginal pain. During ischemia, ATP is degraded to adenosine, which after diffusion to the extracellular space, causes arteriolar dilation and anginal pain. Adenosine induces angina mainly by stimulating the A1 receptors in cardiac afferent nerve endings. Heart rate, myocardial inotropic state, and myocardial wall tension are the major determinants of myocardial metabolic activity and myocardial oxygen demand increases in the heart and myocardial contractile state result in increased myocardial oxygen demand. Increases in both after load (ie, aortic pressure) and preload (ie, ventricular end-diastolic volume) result in a proportional elevation of myocardial wall tension and, therefore, increased myocardial oxygen demand. Oxygen supply to any organ system is determined by blood flow and oxygen extraction. Because the resting coronary venous oxygen saturation is already at a relatively low level (approximately 30%), the myocardium has a limited ability to increase its oxygen extraction during episodes of increased demand. Thus, an increase in myocardial oxygen demand (eg, during exercise) must be met by a proportional increase in coronary blood flow. The ability of the coronary arteries to increase blood flow in response to increased cardiac metabolic demand is referred to as coronary flow reserve (CFR). In healthy people, the maximal coronary blood flow after full dilation of the coronary arteries is roughly 4-6 times the resting coronary blood flow. CFR depends on at least 3 factors: large and small coronary artery resistance, extravascular (ie, myocardial and interstitial) resistance, and blood composition. Myocardial ischemia can result from (1) a reduction of coronary blood flow caused by fixed and/or dynamic epicardial coronary artery (ie, conductive vessel) stenosis, (2) abnormal constriction or deficient relaxation of coronary microcirculation (ie, resistance vessels), or (3) reduced oxygen-carrying capacity of the blood. Atherosclerosis is the most common cause of epicardial coronary artery stenosis and, hence, angina pectoris. Patients with a fixed coronary atheroscerotic lesion of at least 50% show myocardial ischemia during increased myocardial metabolic demand as the result of a significant reduction in CFR. These patients are not able to increase their coronary blood flow during stress to match the increased myocardial metabolic demand, thus they experience angina. Fixed atherosclerotic lesions of at least 90% almost completely abolish the flow reserve;
  • 72. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Shravan Paswan et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 342 patients with these lesions may experience angina at rest.Coronary spasm can also reduce CFR significantly by causing dynamic stenosis of coronary arteries. Prinzmetal angina is defined as resting angina associated with ST-segment elevation caused by focal coronary artery spasm. Although most patients with Prinzmetal angina have underlying fixed coronary lesions, some have angiographically normal coronary arteries. Several mechanisms have been proposed for Prinzmetal angina: focal deficiency of nitric oxide production, hyperinsulinemia, low intracellular magnesium levels, smoking cigarettes, and using cocaine. Approximately 30% of patients with chest pain referred for cardiac catheterization have normal or minimal atherosclerosis of coronary arteries. A subset of these patients demonstrates reduced CFR that is believed to be caused by functional and structural alterations of small coronary arteries and arterioles (ie, resistance vessels). Under normal conditions, resistance vessels are responsible for as much as 95% of coronary artery resistance, with the remaining 5% being from epicardial coronary arteries (ie, conductive vessels). The former is not visualized during regular coronary catheterization. Angina due to dysfunction of small coronary arteries and arterioles is called microvascular angina. Several diseases, such as diabetes mellitus, hypertension, and systemic collagen vascular diseases (eg, systemic lupus erythematosus, polyarteritis nodosa), are believed to cause microvascular abnormalities with subsequent reduction in CFR. The syndrome that includes angina pectoris, ischemia like ST-segment changes and/or myocardial perfusion defects during stress testing, and angiographically normal coronary arteries is referred to as syndrome X. Most patients with this syndrome are postmenopausal women, and they usually have an excellent prognosis Syndrome X is believed to be caused by microvascular angina. Multiple mechanisms may be responsible for this syndrome, including; (1) impaired endothelial dysfunction (2) increased release of local vasoconstrictors, (3) fibrosis and medial hypertrophy of the microcirculation, (4) abnormal cardiac adrenergic nerve function, and/or (5) estrogen deficiency. A number of extravascular forces produced by contraction of adjacent myocardium and intraventricular pressure can influence coronary microcirculation resistance and thus reduce CFR. Extravascular compressive forces are highest in the subendocardium and decrease toward the subepicardium. Left ventricular (LV) hypertrophy together with a higher myocardial oxygen demand (eg, during tachycardia) cause greater susceptibility to ischemia in subendocardial layers. Myocardial ischemia can also be the result of factors affecting blood composition, such as reduced oxygen-carrying capacity of blood, as is observed with severe anemia (hemoglobin, <8 g/dL), or elevated levels of carboxyhemoglobin. The latter may be the result of inhalation of carbon monoxide in a closed area or of long-term smoking. Ambulatory ECG monitoring has shown that silent ischemia is a common phenomenon among patients with established coronary artery disease. In one study, as many as 75% of episodes of ischemia (defined as transient ST depression of >1 mm persisting for at least 1 min) occurring in patients with stable angina were clinically silent. Silent ischemia occurs most frequently in early morning hours and may result in transient myocardial contractile dysfunction (ie, stunning). The exact mechanism(s) for silent ischemia is not known. However, autonomic dysfunction (especially in patients with diabetes), a higher pain threshold in some individuals, and the production of excessive quantities of endorphins are among the more popular hypotheses. Exams and tests: Upon hearing about the patient's symptoms, the primary healthcare provider or the provider in the emergency department will immediately think of angina and other heart problems. Time is of the essence- treatment will probably begin as the evaluation continues. An electrocardiogram (ECG) will be done. This painless test checks for abnormalities in the beating of the heart. Electrodes are attached to the chest and other points on the body. The electrodes read the electrical impulses linked to the beating of the heart. The ECG looks for signs of a heart attack or of impaired blood flow to the heart. For many patients with angina, the ECG result is normal. The patient may have a chest x-ray. This will show any fluid buildup in the lungs. It can also rule out some other causes of chest pain. There is no blood lab test that can tell with certainty that someone is having angina. There are certain blood tests that suggest that a person may be having a heart attack. These tests may be done if a heart attack is suspected.While these tests are going on, the healthcare provider will be asking questions to help with the diagnosis.The questions will be about the symptoms and about the patient's medical history, previous operations, medications, allergies, and habits and lifestyle. The physical exam will include listening to the heart and lungs and feeling the heart through the chest. If, after these tests, the healthcare provider suspects the patient may have coronary heart disease, additional tests will be performed to confirm the possibility.
  • 73. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Shravan Paswan et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 343 Exercise stress test: An ECG is taken before, during, and after exercise (usually walking on a treadmill) to detect inadequate blood flow to the heart muscle indirectly by changes on the ECG. This usually is done only for stable angina. Thallium stress test: This is a more complex and expensive test that injects a radioisotope into the circulation and indirectly detects parts of the heart that may not be getting enough blood during "stress" (usually walking on a treadmill, or after administration of a drug that mimics exercise in those unable to walk on the treadmill). This information indicates more accurately whether any of the coronary arteries may be narrowed, causing inadequate blood flow to the heart muscle or ventricle. Again, this is usually done only for stable angina. Dobutamine echocardiogram stress test: This is done for people who cannot walk on a treadmill. A drug called dobutamine stimulates and speeds up the heart, creating an increased demand or need for blood flow to the left ventricle or muscle. If the muscle shows a slowing of function on the ultrasound image of the heart muscle, then it indirectly indicates inadequate blood flow to the muscle. Coronary angiogram (or arteriogram): This test of the coronary arteries is the most accurate but also the most invasive. It is a type of x-ray. A thin plastic tube called a catheter is threaded through an artery in the arm or groin to one of the main coronary arteries. A contrast or harmless dye is injected into the arteries. The dye depicts the arteries directly and shows any blockage more accurately than the above or more noninvasive procedures. The healthcare provider will make the decision about whether these tests or any treatment need to be done on an urgent basis. If so, the patient will be admitted to the hospital. If not, the tests will be scheduled for the next few days, and the patient may be allowed to go home TREATMENT OF ANGINA PECTORIES The patient may need to take several medicines to control symptoms and improve angina.  Aspirin in low doses reduces the tendency of small blood cells called platelets to stick together, which helps prevent the formation of a blood clots.  Glyceryl trinitrate relaxes the arteries of the heart and relieves angina attacks. GTN comes in sublingual tablet or spray form.  Long-acting nitrates reduce the frequency of angina attacks. These can be in the form of tablets or patches and are very effective. Their main side-effect is headache, but this often disappears once the nitrate has been taken for some weeks.  Beta-blockers block the effect of the hormone adrenaline so that the pulse is slowed and blood pressure lowered. This reduces the heart's need for oxygen and improves the supply of blood to the heart muscle. They are also important in protecting the heart after heart attacks.  Calcium-channel blockers reduce the muscle tension in the coronary arteries, expanding them and creating more room. They also slightly relax the heart muscle, reducing the heart's need for oxygen and reducing blood pressure.  The potassium-channel activator nicorandil (Ikorel) reduces muscle tension in the blood vessel walls, expanding them and improving the flow of blood and the supply of oxygen. Surgery: If the patient have severe angina that is not responding to medication, a cardiologist may decide to need surgery to restore heart function to an adequate level and reduce the likelihood of a heart attack. This can be done by one of the following operations.  Angioplasty: the narrowed coronary artery is dilated (opened up) with a balloon. A small tube called a stent may also be inserted into the artery at this time to help prevent it narrowing down again in the future.  Bypass operation: a superficial blood vessel is taken from another part of the body, usually the leg, and joined to the coronary artery to bypass the obstruction to blood flow. Transmyocardial revascularization: Transmyocardial revascularization is a procedure for patients who cannot undergo angioplasty or surgery. A simple incision is made in the chest, and a laser is used to "drill" small holes through the outside wall of the heart into the left ventricle. About 20-40 holes are made. Bleeding from these holes is minimal and usually stops after a few minutes of pressure. It is not clear why this helps relieve angina. One theory is that it stimulates growth of new blood vessels that improve blood flow to the heart. Other investigators believe it is a placebo effect. Current research is focusing on trying to find growth factors that could be injected into coronary arteries or directly into the left ventricle to encourage growth of new blood vessels. Diet plan for preventing angina pectoris: Obviously, a healthy diet goes a long way in preventing angina pectoris. The following basic guidelines need to be followed with meticulous care.
  • 74. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Shravan Paswan et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 344 Avoid fatty foods to the maximum possible extent. This includes fried food, milk products such as butter and cheese, full cream milk, oils, etc. Fermented milk products are good for people with angina. This includes curds. Use only vegetable oils for cooking. This includes sunflower, olive, groundnut and rapeseed oils. Avoid salt in the diet. Do not consume foods that are too much salty. In meats, red meats such as mutton, beef and pork must be avoided. White meats such as poultry and fish are beneficial. Fishes with high body oil content must be preferred. This includes sardines, tunas, mackerels, salmons, herrings, etc. Canned fish must be strictly avoided. There should be at least two to three fish consumptions per week. Carbohydrates should form the major part of the food. This includes cereals, wheat, rice, bread, potatoes and pasta. It is found that a little bit of alcohol is actually beneficial for angina, but excess is harmful. The safe limit of alcohol is as follows:- For men: 21 units per week, and not more than 4 units on any one day .For women: 14 units per week, and not more than 3 units on any one day. Prevention of angina pectoris: Many of the risk factors for angina can be tackled by lifestyle changes.  Eat a varied and healthy diet with plenty of leafy vegetables. Avoid sugary foods and saturated fats found in meat and full-fat dairy products.  Stop smoking.  Lose weight if you are overweight.  Exercise more: aim for a half-hour walk each day.  If you have diabetes or high blood pressure, maintain treatment for these conditions. Ayurvedic herbs: Ayurveda is a treasure-house of remedies for angina pectoris. There is a long list of herbs that have been used since ancient times for the treatment of the condition. The following is a list of these herbs with their actions on the human body. Table.1. Ayurvedic herbs useful in the treatment of heart diseases Ayurvedic Name of the Herb Biological Name of the Herb Common English Name of the Herb Action on the Human Body Alfalfa Medicago satina Alfalfa Juice of the alfalfa grass is used for people with arterial and heart problems. The benefits of this juice are improved by taking it in a mixture with carrot juice. Amla Emblica officinalis Indian Gooseberry Amla tones up all the organs of the body, and that includes the heart. Thus it betters the pumping action of the heart. Chachinga Trichosanthes anguina Snake Gourd The leaves of the snake gourd have been traditionally used as medicine for treating pain in the heart due to physical exertion. Haldi Curcuma longa Turmeric Curcumin is a chemical compound present in turmeric. This compound is known to lower the amount of serum cholesterol and even the blood sugar level. Kahu Terminalia arjuna Arjuna Arjuna is perhaps the most beneficial herb used by Ayurvedic practitioners in the treatment of angina-related problems. The bark of the arjuna tree is known to have stimulant action on the heart. Lahsoona Allium sativa Garlic Garlic is beneficial for people with angina pectoris as it is a known blood thinner. For this reason, garlic must be included in the diet. Peepal Ficus religiosa Peepal The leaves of the peepal tree are effective in treatment of heart ailments. They are known to strengthen the heart and thus keep angina pectoris at bay. Pyaaza Allium cepa Onion Trials have shown that regular usage of onion for five months is beneficial in decreasing serum cholesterol. It is also beneficial in decreasing thrombosis. Rojmari Achillea millefolium Blood Wort The herb of blood wort is beneficial in the treatment of circulatory problems due to its stimulant action. It can bring down high blood pressure, which is a leading cause of angina pectoris. Tilpushpi Digitalis purpurea Digitalis Treatment of angina pectoris is one of the most elemental purposes digitalis is put to. Digitalis stimulates the muscle activity of the heart and makes it pump better. Thus it forces more blood into the heart and improves nourishment.
  • 75. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Shravan Paswan et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 345 Guggul is an age-old remedy used by Ayurvedic exponents for treating angina pectoris and its complications. Guggul is in fact a mixture of several substances that have been extracted from the Commiphora mukul plant. This medicine is effective in treating atherosclerosis, which is a leading cause of angina. This is because of guggulsterone, which is a compound found in the guggul plant. CONCLUSION Angina Pectoris is a term for chest pain caused by an inadequate supply of blood and oxygen to the heart. More than 9 million people in the United States have angina. With angina, the affected patient’s heart may get sufficient blood for daily activities, but the arteries may not be able to respond appropriately to increased demands for oxygen during exercise, times of emotional or physical stress, and with extremes of temperature. Treatments for angina depend on the type and severity of the angina. For most people with stable angina, symptoms disappear after a period of time. Rest and nitroglycerin may be sufficient to treat the attack. For people with unstable angina, other medications such as antiplatelet medications, beta blockers, calcium channel blockers and blood thinners may be given. In severe cases of angina, or where the risk of a heart attack is high, surgical procedures such as a coronary artery bypass grafting and percutaneous transluminal angioplasty may be recommended. REFERENCES Droste C, Roskamm H, Experimental pain measurement in patients with asymptomatic myocardial ischemia, J Am Coll Cardiol, 1, 1983, 340–345. Falcone C, Sconocchia R, Guasti L, et al. Dental pain threshold and angina pectoris in patients with coronary artery disease. J Am Coll Cardiol,12, 1998, 348–352. Gettes LS, Winternitz SR, Monitoring to detect "silent" ischemia. In: Stern S, ed. Ambulatory ECG Monitoring. Chicago, Ill: Year Book Medical Publishers, Inc; 1978: 93–106. Kannel WB. Unrecognized myocardial infarction.In: Stern S, ed. Silent Myocardial Ischemia. London, UK: Martin Dunitz Ltd; 1998; 47–53. Laukkanen JA, Kurl S, Lakka TA, et al. Exercise-induced silent myocardial ischemia and coronary morbidity and mortality in middle-aged men, J Am Coll Cardiol, 38, 2001, 72–79. Mark DB, Hlatky MA, Califf RM, et al. Painless exercise ST deviation on the treadmill: long term prognosis. J Am Coll Cardiol, 14, 1989, 885–888. Mazzone A, Cusa C, Mazzucchelli I, et al. Increased production of inflammatory cytokines in patients with silent myocardial ischemia, J Am Coll Cardiol, 38, 2001, 1895–1901. Miranda CP, Lehmann KG, Lachterman B, et al. Comparison of silent and symptomatic ischemia during exercise testing in men. Ann Intern Med, 114, 1991, 645–656. Pepine CJ, Sharaf B, Andrews TC, et al. Relation between clinical, angiographic and ischemic findings at baseline and ischemia-related adverse outcomes at 1 year in the Asymptomatic Cardiac Ischemia Pilot study. J Am Coll Cardiol, 29, 1997, 1483–1489. Pierdomenico SD, Bucci A, Costantini F, Circadian blood pressure changes and myocardial ischemia in hypertensive patients with coronary artery disease, J Am Coll Cardiol, 31, 1998, 1627–1634. Schang SJ, Pepine CJ, Transient asymptomatic ST segment depression during daily activity, Am J Cardiol, 39, 1977, 396–402. Stern S, Tzivoni D, Early detection of silent ischemic heart disease by 24-hour electrocardiographic monitoring of active subjects, Br Heart J, 36, 1974, 481–486.
  • 76. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Manikandan K et.al. Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 346 STABILITY INDICATING HPLC METHOD FOR THE ESTIMATION OF CINACALCET HYDROCHLORIDE API Manikandan Krishnan*, Santhana Lakshmi Karunanidhi, Gayathri Sola, Akshitha .Y Department of Pharmaceutical Analysis, SRM College of Pharmacy, SRM University, Kattankulathur, Tamilnadu 603203, India *Corresponding author: E.mail: gurumani12@gmail.com ABSTRACT A stability-indicating liquid chromatography method has been developed and validated for the determination of Cinacalcet hydrochloride in a laboratory mixture as well as in a tablet formulation developed in-house. Efficient chromatographic separation was achieved on phenomenex C18 column (150 mm×4.6 mm, 5.0 μm) with mobile phase containing Methanol: Water (70:30v/v) pH adjusted to 3.6 with dilute Orthophosphoric acid at a flow rate of 1.3 mL/min and the eluent was monitored at 271 nm using Shimadzu LC-10AT-VP & LC-20 AD with Spinotech (Winchrome) software. Linearity range was found to be between 50-300μg/ml and the linear regression coefficient was not more than 0.999. The values of % RSD are less than 2% indicating accuracy and precision of the method. The percentage recovery varies from 97-103%w/w. LOD and LOQ were found to be within limit. The system suitability parameters like tailing factor, number of theoretical plates, Asymmetry were found to be within limit. Forced degradation studies were performed by using this method. There was a significant degradation in the presence of 0.1M HCl, 0.1M NaOH, 30%H2O2, heat and light. The proposed method is simple, accurate and rapid and hence can be employed for routine quality control analysis. Keywords: Cinacalcet Hydrochloride, Stability-indicating, ICH guidelines, HPLC INTRODUCTION Cinacalcet Hydrochloride is N-[(1R)-1(Naphthyl) ethyl] - 3 - [3(trifluoromethyl) phenyl] Propan - 1- amine hydrochloride an oral calcimimetic agent that increases the sensitivity of the calcium-sensing receptor to activation by extracellular calcium. The calcium sensing receptors on the surface of parathyroid gland regulate parathyroid hormone (PTH) secretion. Increased PTH stimulates osteoclastic activity resulting in cortical bone resorption and marrow fibrosis. Increasing the sensitivity of these receptors results in a lowering of PTH which subsequently lowers serum calcium levels. Fig 1: Chemical structure of Cinacalcet hydrochloride EXPERIMENTAL Instrumentation: Quantitative High Performance Liquid Chromatographic analysis was performed on a Shimadzu LC-10AT-VP, LC-20 AD pumps and UV Detector with Spinotech (Winchrome) software. Reagents and Chemicals: Cinacalcet hydrochloride reference standard was obtained as a gift sample from Actavis Pharma Manufacturing Pvt. Ltd. Chennai, India and has been used as reference drug without prior treatment. Synthetic mixture of Cinacalcet Hydrochloride was prepared by mixing Reference standard drug, Magnesium Stearate, Talc, Starch which were procured from local supplier. Analytical / HPLC grade chemicals and solvents used were obtained from Ranbaxy Fine Chemicals Limited (Delhi, India). Chromatography: Chromatography was performed on phenomenex C18 column (150 mm×4.6 mm, 5.0 μm) with mobile phase containing Methanol: Water (70:30v/v) pH adjusted to 3.6 with dilute Orthophosphoric acid at a flow rate of 1.3 mL/min and the eluent was monitored at 271 nm Calibration plot for Cinacalcet hydrochloride: A Stock solution of Cinacalcet hydrochloride (1000µg/ml) was prepared in methanol. The gradient dilutions were prepared by taking 0.5, 1, 1.5, 2, 2.5 and 3ml of the solution and made up to 10 ml with mobile phase. 20μl of each concentration was injected twice. Calibration cure was constructed by plotting mean peak areas against the corresponding drug concentration. From the graphs it was found that the Cinacalcet Hydrochloride obeys Beer’s law and the Range lies between 50- 300μgml.
  • 77. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Manikandan K et.al. Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 347 VALIDATION Precision: Precision of an analytical method is the degree of agreement among individual test results when the procedure is applied repeatedly to multiple samplings of a homogeneous sample. Precision of analytical method is usually expressed as the standard deviation (or) relative standard deviation. Cinacalcet Hydrochloride of concentration 200μg/ml was prepared and injected for six times and analyzed under optimized chromatographic conditions. Limit of detection and limit if quantification: Cinacalcet hydrochloride at concentration in the lower part of the linear range of calibration plot was used to determine limit of detection (LOD) and Limit of quanfication(LOQ). They were determinated from the slope of calibration plot and standard deviation of the blank sample by use of the equations LOD = 3.3×Standard deviation / Slope and LOQ = 10×Standard deviation / Slope Recovery: Recovery studies were carried out by mixing standard quantity of drug with pre analyzed sample formulation and the contents were analyzed by proposed method. From the sample stock solution a dilution of concentration of 50μg/ml was prepared and to this standard solution of series of concentrations from 0, 50, 100, 150, 200, 250 μg/ml was added and scanned. Analysis of Synthetic mixture: Due to non- availability of formulation in the local pharmacies, a synthetic mixture was prepared using drug with suitable excipients. Synthetic mixture of Cinacalcet hydrochloride was used for the assay. Accurately weighed quantity of Cinacalcet Hydrochloride equivalent to 100mg and transferred into a 100ml volumetric flask. To this 30ml of methanol was added and ultrasonicated for half an hour until the drug was extracted. The solution was filtered by using Whatmann filter paper No:45μ and finally made up to the mark with mobile phase to give a concentration of 1mg/ml. Different aliquots of 1.0, 1.5and 2.0ml was taken from the above prepared stock and made up to 10ml with mobile phase to get concentrations of 100, 150, 200μg/ml respectively. 20μl of the aliquots were injected and the chromatograms were recorded. Forced degradation of Cinacalcet hydrochloride Acid and base induced degradation: Cinacalcet hydrochloride (25 mg) of standard was separately taken in a two 50 ml volumetric flask, dissolved in 25 ml methanol and diluted to volume with 0.1M HCl and 0.1M NaOH. The solution was refluxed for 24 hours at 40C to 80C, and sampling was done at different intervals of time and diluted with the mobile phase to the final concentration of 100 µg/ml and are analyzed by proposed HPLC method Hydrogen peroxide induced degradation: Cinacalcet hydrochloride (25 mg) of was taken in a 50 ml volumetric flask, dissolved in methanol and diluted to volume with 1% H2O2, 3%H2O2 and 30 % H2O2.The solution was collected at different time intervals. Then these solutions were diluted with the mobile phase to the final concentration of 100 µg/ml and are analyzed by proposed HPLC method. Thermal Degradation: Cinacalcet hydrochloride (25 mg) was taken in petri dish (10cm in diameter) and spread to a thickness of 1mm and the sample is exposed to a temperature of 105ºC for24 hrs and for wet heat degradation drug was kept in reflux at 80°C, for 24 hrs. Then the samples were taken at different time intervals and diluted with the mobile phase to the final concentration of 100 µg/ml for further analysis by HPLC. Photochemical Degradation: Cinacalcet hydrochloride (25 mg) was taken on Petri dish (10cm in diameter) and spread to a thickness of 1mm and exposed to Sunlight, UV-light and dark place for 24 hrs. Simultaneously the drug solutions (1mg/ml) were taken in different volumetric flasks and exposed to Sunlight, UV-light and dark place for 24 hrs. Then these samples are taken at different time intervals and diluted with the mobile phase to the final concentration of 100 µg/ml for further analysis using HPLC RESULT AND DISCUSSION Chromatographic conditions: The present study of stability indicating high performance liquid chromatographic method of Cinacalcet hydrochloride in bulk and synthetic mixture, different chromatographic condition were applied. Among the different mobile phase employed the mobile phase of Methanol: Water (70:30 v/v) pH adjusted to 3.6 with dilute Orthophosphoric acid at a flow rate of 1.3 mL/min and the eluent was determined in UV detection at 271 nm. Linearity: The calibration curve showed the concentrations ranging from 50-300μg/ml of Cinacalcet Hydrochloride were prepared and 20μl of each concentration was injected two times. The Correlation Coefficient values were found to be within the acceptance criteria for the drug. The Limit of Detection and Quantitation values were calculated. The results were shown in Figure 1, 2 and tabulated in Table-1, 2
  • 78. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Manikandan K et.al. Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 348 Precision: Cinacalcet Hydrochloride of concentration 200μg/ml was prepared and injected for six times and analyzed under optimized chromatographic conditions. The standard deviation, Relative Standard Deviation for Cinacalcet Hydrochloride was found to be within the acceptance criteria. (< 2%) and hence the method was found to be precise. The results are tabulated in Table 2. Limit of detection and limit if quantification: The signal and noise ratios of 3:1 and 10:1 were considered as LOD and LOQ respectively. From the results of linearity the LOD and LOQ were found to be 5.063 μg/ml and 15.20μg/ml respectively. . The results are tabulated in Table 2. Recovery studies: A known amount of the drug was added to formulation and subjected to check the reproducibility of the method. The estimated percentage recovery was found to be within the normal range (97% – 103%), this proves the reproducibility of the method Forced degradation studies: HPLC studies of samples obtained on stress testing of Cinacalcet hydrochloride under different conditions using Methanol: Water: (70:30 v/v) and pH adjusted to 3.6 with O-Phosphoric acid as the mobile solvent system suggested the following degradation behavior. During the study it was observed than upon treatment of Cinacalcet hydrochloride with acid(0.1M HCl), base (0.1M NaOH), hydrogen peroxide (1%, 3%, and 30%), heat(dry and wet heat) and photo degradation(Sunlight, UV light(254nm) and Dark light. Table 3 indicated the extent of Cinacalcet hydrochloride degradation was observed in various Stress condition. Figure 4 to 8 shows the chromatogram of forced degraded sample. Table 1: Linearity data of the HPLC method Cinacalcet hydrochloride Concentration (μg/ml) Peak Area (m V .s) 50 100 150 200 250 300 12171129 21982505 32616363 42370019 53819060 63894844 Table 2: Summary of HPLC Results Parameter Result found Limit Retention time (min) 5,52 - Correlation coefficient (R2 ) 0.999 >0.996 Slope (m) 21097 - Intercept (c) 0.999 - LOD 5.063 - LOQ 15.20 - SD 0.9799 - RSD (%) 0.478 <2% SE 0.4004 - % Purity(w/w) 97-103 97-103 Table 3: Result of force degradation studies of Cinacalcet hydrochloride API Stress Condition / Duration % Degradation Acid Degradation(0.1M HCl, 40˚C, 24h) 88.6 Base Degradation(0.1M NaOH, 80˚C, 12h) 92.2 Oxidative degradation(1%H2O2, 25˚C, 8h) 1.3 Oxidative degradation(3%H2O2, 25˚C, 8h) 3.5 Oxidative degradation(30%H2O2, 25˚C, 8h) 13.2 Thermal degradation(Soild sample, 105˚C, 24h) 28.8 Thermal degradation(Solution, 80˚C, 24h) 92.2 Photo degradation(Soild sample, Sunlight, 24h) 29.5 Photo degradation(Solution, Sunlight, 24h) 65.2 Photo degradation(Soild sample, UV light, 24h) 13.7 Photo degradation(Solution, UV light, 24h) 18.2 Photo degradation(Soild sample, Dark light, 24h) 9.3 Photo degradation(Solution, Dark light, 24h) 11.1
  • 79. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Manikandan K et.al. Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 349 Figure 1: Chromatogram of Cinacalcet Hydrochloride at 271nm Figure 2: Calibration curve of Cinacalcet Hydrochloride using HPLC Figure 3: Chromatogram of Cinacalcet Hydrochloride standard (100 µg/ml) at 271nm. Figure 4: Chromatogram of Cinacalcet Hydrochloride under acid stress condition at 24 hr Figure 5: Chromatogram of Cinacalcet Hydrochloride under alkali stress condition at 12hrs. Figure 7: Chromatogram of Cinacalcet Hydrochloride under wet heat stress condition at 24hrs Figure 8: Chromatogram of Cinacalcet Hydrochloride Solution under UV light at 24 hrs CONCULTION
  • 80. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Manikandan K et.al. Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 350 The proposed HPLC method is precise, specific, accurate and stability indicating. The sample recoveries in Synthetic mixture were in good agreement with their respective amount taken which suggested noninterference of synthetic mixture excipients in the estimation. The method can be used to determine the purity of the drug obtained from different sources by detecting related impurities. Hence, the proposed method stands validated and may be used for routine and stability sample analysis ACKNOWLEDGEMENT The authors are grate full to Actavis Pharma Manufacturing Pvt. Ltd. Chennai, India for providing the pure sample of Cinacalcet hydrochloride REFERENCES Fen Hang, Hangyun wang, Qianzhoo, Hongzong liu, Pei Hu and JiJIang, Determination of Cinacalcet Hydrochloride in Human plasma by Liquid Chromatgraphy - tandem mass spectrometry, Journal of Pharmaceutical & Biomedical Analysis, 10, 2011, 2-18. Ibrahim A Darwish, Mona M Al- Shehri and Manal A El – Gendy, Novel spectrophotometric method for determination of Cinacalcet Hydrochloride in its tablets via derivitization with 1,2 – naphthoquinone 4 – Sulphonate, Chemistry Central Journal, 6,11, 2012, 655-658. ICH Harmonized Tripartite guideline, validation of analytical procedures, Text and Methodology Q2 (R1), 4th version, 27oct 1994, 01-17. ICH Stability testing of new drug substances and products (Q1 A R2), 4th version, 2003, 5. Julia A, Barmon Balfour, Scott and Lesley J, Cinacalcet Hydrochloride, Drugs, 65(2), 2005, 271-281. Martindale, the Complete Drug Reference, 28, 770-772. Narogi R ., Vishvottam K, Prashanth Komarneni, Raghupati Aleti, Naga Surya Prakash Padala and Ilayaraja.K, Quantification of Cinacalcet by LC-MS/MS using Liquid-Liquid Extraction from 50μl of plasma, Journal of Pharmaceutical and Biomedical Analysis, 56(5), 5, 2011, 373-81. Nuin E, Andreu I, Torres MJ, Miranda MA, Miguel A, Jime Nez and Consueloo M, Enhanced Photo safety of Cinacalcet Hydrochloride upon complexation with serum albumin, The Journal of Physical Chemistry, B, 115, 5, 2011, 1158-1164. Ravi Bhushan and Ritual Dubey, Indirect Reversed Phase HPLC and Direct Thin layer Chromatographic enantioresolution of (R,S)- Cinacalcet, Biomedical Chromatography, 25(6), 2011, 674-679. Satinder Ahuja, Michael W Dong, Hand book of pharmaceutical analysis by HPLC, 25, 336-345. Sethi P D, High Performance Liquid Chromatography Quantitative Analysis of Pharmaceutical Formulations, 1, 2001, 02-07. Sigala Ashok, Raghunath Babu CH V, Satish Varma M and BalaSwamy G, A new validated liquid chromatographic method for the determination of impurities in Cinacalcet Hydrochloride, Analytical Chemistry-An Indian Journal, 8(4), 2009, 278-297. Vadde ravinder, Sigala Ashok, Satish varma M, Raghunaath babu C V and Gubba Balaswamy, A validated chiral LC method for the enantiomeric separation of (S)-Cinacalcet from (R) – Cinacalce, Chromatographia, 70, 2009, 229-232.
  • 81. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Shravan Paswan et.al. Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 351 BIOAVAILABILITY: CRITERIA FOR APPROVING A DRUG PRODUCT FOR MARKETING Sandhya Singh1 , Faheem Ajmal Ansari1 , Shravan Paswan2* , Rnjan Kumar Sharma2 , Alok Ranjan Gaur3 1 Azad Institute of Pharmacy and Research Lucknow, India 2 Advance Institute of Pharmaceutical Education & Research, Kanpur, India 3 Department of Pharmacy, Pranveer Singh Institute of Technology, Kanpur, India *Corresponding author: paswanshravan@gmail.com ABSTRACT FDA ensure that the drug product for marketing should be safe, effective and meet all applicable standards, for this FDA requires bioavailability/pharmacokinetic studies and, where necessary, bioequivalence studies for all drug products (FDA Guidance for Industry, 2003). The U.S. Food and Drug Administration (FDA) define bioavailability as "the rate and extent to which the active drug ingredient or therapeutic moiety is absorbed from a drug product and becomes available at the site of drug action". Generally direct and indirect methods use to assess drug bioavailability. For assessing bioavailability or clinical availability of a drug, its rate and extent of absorption and its first-pass metabolism must be evaluated. The clinical response of the patient or the amount of active drug at the target site of action at different time periods should also be assessed. In order to achieve targeted minimum level for therapeutic or clinical effect, the medical practitioner must understand various contributing factors that could affect the bioavailability. For the scientists, they must also be aware of some essential intrinsic factors that influence the formulation. There are basically three factors, which affect bioavailability physiological factors, physicochemical factors and pharmacological factors. Key Words: FDA, Bioavailability, Bioequivalence, Pharmacokinetic, Physicochemical INTRODUCTION In approving a drug product for marketing, the FDA ensures that the drug product is safe and effective for its labeled indications for use. Moreover, the drug product must meet all applicable standards of identity, strength, quality, and purity. To ensure that these standards are met, the FDA requires bioavailability/pharmacokinetic studies and, where necessary, bioequivalence studies for all drug products (FDA Guidance for Industry, 2003). Bioavailability may be considered as one aspect of drug product quality that links in-vivo performance of the drug product used in clinical trials to studies demonstrating evidence of safety and efficacy. For unmarketed drugs that do not have full NDA approval by the FDA, in-vitro and/or in-vivo bioequivalence studies must be performed on the drug formulation proposed for marketing as a generic drug product. Bioavailability: The U.S. Food and Drug Administration (FDA) define bioavailability as "the rate and extent to which the active drug ingredient or therapeutic moiety is absorbed from a drug product and becomes available at the site of drug action". Because in practice it is rare that drug concentrations can be determined at the site of action (e.g., at a receptor site), bioavailability is more commonly defined as "the rate and extent that the active drug is absorbed from a dosage form and becomes available in the systemic circulation." Objectives of bioavailability studies: Bioavailability studies are important as 1. Primary stages of development of a suitable dosage form for a new drug entity. 2. Determination of influence of excipients, patient related factors & possible interaction with other drugs on the efficiency of absorption. 3. Development of new formulations of the existing drugs. 4. Control of quality of a drug product during the early stages of marketing in order to determine the influence of processing factors, storage & stability on drug absorption. Relative and absolute bioavailability: The area under the drug concentration–time curve (AUC) is used as a measure of the total amount of unaltered drug that reaches the systemic circulation. The AUC is dependent on the total quantity of available drug, FD0, divided by the elimination rate constant, k, and the apparent volume of distribution, VD. F is the fraction of the dose absorbed. After IV administration, F is equal to unity, because the entire dose enters the systemic circulation. Therefore, the drug is considered to be completely available after IV administration. After oral administration of a drug, F may vary from a value of 0 (no drug absorption) to 1 (complete drug absorption).
  • 82. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Shravan Paswan et.al. Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 352 Relative availability: Relative (apparent) availability is the availability of the drug from a drug product as compared to a recognized standard. The fraction of dose systemically available from an oral drug product is difficult to ascertain. The availability of drug in the formulation is compared to the availability of drug in a standard dosage formulation, usually a solution of the pure drug evaluated in a crossover study. The relative availability of two drug products given at the same dosage level and by the same route of administration can be obtained using the following equation. (1.1) Where drug product B is the recognized reference standard. This fraction may be multiplied by 100 to give percent relative availability. When different doses are administered, a correction for the size of the dose is made, as in the following equation: (1.2) Urinary drug excretion data may also be used to measure relative availability, as long as the total amount of intact drug excreted in the urine is collected. The percent relative availability using urinary excretion data can be determined as follows: (1.3) Where [D u]∞ is the total amount of drug excreted in the urine. Absolute Availability The absolute availability of drug is the systemic availability of a drug after extravascular administration (eg, oral, rectal, transdermal, subcutaneous) compared to IV dosing. The absolute availability of a drug is generally measured by comparing the respective AUCs after extravascular and IV administration. This measurement may be performed as long as VD and k are independent of the route of administration. Absolute availability after oral drug administration using plasma data can be determined as follows: (1.4) Absolute availability, F, may be expressed as a fraction or as a percent by multiplying F x 100. Absolute availability using urinary drug excretion data can be determined by the following: (1.5) The absolute bioavailability is also equal to F, the fraction of the dose that is bioavailable. Absolute availability is sometimes expressed as a percent, ie, F = 1, or 100%. For drugs given intravascularly, such as by IV bolus injection, F = 1 because the entire drug is completely absorbed. For all extravascular routes of administration, such as the oral route (PO), the absolute bioavailability F may not exceed 100% (F > 1). F is usually determined by Equation 1.4 or 1.5, where PO is the oral route or any other extravascular route of drug administration. Example: The difference between absolute and relative bioavailability is illustrated by the following hypothetical example. Assume that an intravenous injection (Product A) and two oral dosage forms (Product B and Product C), all containing the same dose of the same drug, are given to a group of subjects in a crossover study. Furthermore, suppose each product gave the values for AUC indicated in Table 1. Table.1. Data for Absolute and Relative Bioavailability Drug Product Are Under the Curve (mcg/ml) x hr A. Intravenous injection 100 B. Oral dosage form, brand or reference standard 50 C. Oral dosage form, generic product 40
  • 83. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Shravan Paswan et.al. Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 353 The F for Product B and Product C is 50% (F = 0.5) and 40% (F = 0.4), respectively. However, when the two oral products are compared, the relative bioavailability of Product C as compared to Product B is 80% [3]. FACTOR AFFECTING BIOAVAILABILITY Drug is absorbed from the gastrointestinal (GI) tract after being dissolved according to its intrinsic absorbability. If the test product shows the same pattern of drug dissolution in the GI tract in vivo as the reference one, that product must be equivalent unless other ingredients do not modulate the absorption of active drug. In other words, physicochemical properties of drugs such as water solubility and membrane permeability do not affect the bioequivalency of oral product. A wide range of factors can influence the bioavailability of a drug. Basically, the availability of the drug or its metabolite to the target organ or receptor is controlled by three principal factors: 1. The rate and extent of drug release from its formulation, and its subsequent absorption. 2. The first-pass effect while passing through the liver after absorption. 3. The conjoint effect of plasma protein binding, drug distribution to various body fluids, metabolism and excretion. For assessing bioavailability or clinical availability of a drug, its rate and extent of absorption and its first-pass metabolism must be evaluated. The clinical response of the patient or the amount of active drug at the target site of action at different time periods should also be assessed. In order to achieve targeted minimum level for therapeutic or clinical effect, the medical practitioner must understand various contributing factors that could affect the bioavailability. For the scientists, they must also be aware of some essential intrinsic factors that influence the formulation. In general, the following factors were discussed. Physiological Factors: High variability in oral drug absorption is caused by several factors. Deviations in physiological conditions in the GI tract of volunteers would affect dissolution and permeation of drugs even in the same individual. For example, bile acid secretion into the small intestine promotes the dissolution of poorly soluble drugs to enhance the bioavailability. On the other hand, it was reported that food intake often reduced the oral absorption of BCS class 3 drugs. These facts indicate that low solubility and low permeability of drugs may cause not only the incomplete oral absorption but also the high variability in it. Metabolism in the intestine and liver affects the oral bioavailability as the first-pass effects after absorption. Also deviations of the metabolic activity in pharmacokinetic parameters due to the change in total body clearance of drugs. High clearance of drugs therefore, might be one of the risk factors for high variability in human bioequivalence study. In short, the following physiological factors are known to affect bioavailability: 1. These include the effect of gastrointestinal fluids such as pH, mucus, bile salts, complexing components. 2. Gastric motility such as gastric emptying, presence of food, rest and exercise. 3. Gastrointestinal transit time which can be affected by a large number of drugs. 4. Metabolism of drugs by the gut wall, liver, skin and bronchial mucosa. 5. The pharmacogenetic factors determining the rate of hepatic metabolism. 6. Various disease states such as malabsorption, achlorhydria, thryrotoxicosis and celiac disease. 7. Other factors include the gut flora, age, sex, weight and physical status of the patients. Physicochemical Factors: There are various physicochemical factors that may influence absorption of drugs into the bloodstream. A dissolution testing is essential to establish a profile of each generic product with specific physicochemical characteristic of the solid dosage form. This testing will ensure the permeability and solubility of drugs. At the drug development stage, these factors are essential to be evaluated and should not be the direct causes of failure in bioequivalence study. The physicochemical factors which point to the need for dissolution testing include: 1. Low drug solubility – to establish the evidence that the drug has a low aqueous solubility. 2. Poor product dissolution – to establish from the literature that the dissolution of one or more marketed product to develop is, poor when tested by official compendial test procedure. 3. Drug particle size – to establish the evidence that the particle size may affect bioavailability. 4. The physical form of drug – to establish that certain polymorphs, solvates or complexes have poor dissolution characteristics and hence bioavailability may be affected.
  • 84. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Shravan Paswan et.al. Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 354 5. Presence of specific excipients – to establish evidence that specific excipients may alter dissolution or absorption, hence bioavailability may also be affected. 6. The nature of tablet or capsule coating – to establish evidence that coating may interfere with the disintegration or dissolution of the formulation. Biopharmaceutic Classification System (BCS): The biopharmaceutic classification system was developed primarily in the context of immediate release (IR) solid oral dosage forms. It is the scientific framework for classifying drug substances based on their aqueous solubility and intestinal permeability. It is a drug development tool that allows estimation of the contributions of three major factors namely dissolution, solubility and intestinal permeability that affect oral drug absorption from immediate release solid oral dosage forms. The interest in this classification system is largely because of its application in early drug development and then in the management of product change through its life cycle. Goals of the BCS Guidance: 1. To improve the efficiency of drug development and the review process by recommending a strategy for identifying expendable clinical bioequivalence tests. 2. To recommend a class of immediate-release (IR) solid oral dosage forms for which bioequivalence may be assessed based on in vitro dissolution tests. 3. To recommend methods for classification according to dosage form dissolution, along with the solubility and permeability characteristics of the drug substance. Classification: According to BCS, drug substances are classified as: Class I: High Solubility – High Permeability Class II: Low Solubility – High Permeability Class III: High Solubility – Low Permeability Class IV: Low Solubility – Low Permeability Combined with the dissolution, the BCS takes into account the three major factors governing bioavailability parameters namely, dissolution, solubility and permeability. This classification is associated with drug dissolution and absorption model, which identifies the key parameters controlling drug absorption as a set of dimensionless numbers. Absorption number, defined as the ratio of the mean residence time to mean absorption time. Dissolution number, defined as the ratio of mean residence time to mean dissolution time. METHODS FOR ASSESSING BIOAVAILABILITY  Direct and indirect methods may be used to assess drug bioavailability. The in-vivo bioavailability of a drug product is demonstrated by the rate and extent of drug absorption, as determined by comparison of measured parameters, eg, concentration of the active drug ingredient in the blood, cumulative urinary excretion rates, or pharmacological effects.  For drug products that are not intended to be absorbed into the bloodstream, bioavailability may be assessed by measurements intended to reflect the rate and extent to which the active ingredient or active moiety becomes available at the site of action.  The design of the bioavailability study depends on the objectives of the study, the ability to analyze the drug (and metabolites) in biological fluids, the pharmacodynamics of the drug substance, the route of drug administration, and the nature of the drug product.  Pharmacokinetic and/or pharmacodynamic parameters as well as clinical observations and in-vitro studies may be used to determine drug bioavailability from a drug product. Pharmacokinetic methods: These are indirect methods. Assumption is that pharmacokinetic profile reflects the therapeutic effectiveness of a drug. Advantages include the results are accurate, reliable, reproducible. a) Plasma / blood level time profile: Time for peak plasma (blood) concentration (t max) Peak plasma drug concentration (Cmax) Area under the plasma drug concentration–time curve (AUC). b) Urinary excretion studies: Cumulative amount of drug excreted in the urine (Du) Rate of drug excretion in the urine (dD u/dt). Time for maximum urinary excretion (t). Pharmacodynamic methods: Involves direct measurement.(measurement of pharmacologic or therapeutic end point) Disadvantages: High variability, difficult to measure, limited choices, less reliable, more subjective. Drug response influenced by several physiological & environmental factors  Maximum pharmacodynamic effect (E max).  Time for maximum pharmacodynamic effect.
  • 85. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Shravan Paswan et.al. Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 355  Area under the pharmacodynamic effect–time curve.  Onset time for pharmacodynamic effect. They involve determination of bioavailability from: a) Acute pharmacological response. b) Therapeutic response. In-vitro dissolution studies 1. Closed compartment apparatus 2. Open compartment apparatus 3. Dialysis systems. Plasma drug concentration: Measurement of drug concentrations in blood, plasma, or serum after drug administration is the most direct and objective way to determine systemic drug bioavailability. By appropriate blood sampling, an accurate description of the plasma drug concentration–time profile of the therapeutically active drug substance(s) can be obtained using a validated drug assay. The time of peak plasma concentration, t max, corresponds to the time required to reach maximum drug concentration after drug administration. At t max, peak drug absorption occurs and the rate of drug absorption exactly equals the rate of drug elimination. Drug absorption still continues after t max is reached, but at a slower rate. When comparing drug products, t max can be used as an approximate indication of drug absorption rate. The value for t max will become smaller (indicating less time required to reach peak plasma concentration) as the absorption rate for the drug becomes more rapid. Units for t max are units of time (eg, hours, minutes). The peak plasma drug concentration, C max, represents the maximum plasma drug concentration obtained after oral administration of drug. For many drugs, a relationship is found between the pharmacodynamic drug effect and the plasma drug concentration. C max provides indications that the drug is sufficiently systemically absorbed to provide a therapeutic response. In addition, C max provides warning of possibly toxic levels of drug. The units of C max are concentration units (eg, mg/mL, ng/mL). Although not a unit for rate, C max is often used in bioequivalence studies as a surrogate measure for the rate of drug bioavailability. The area under the plasma level–time curve, AUC, is a measurement of the extent of drug bioavailability. The AUC reflects the total amount of active drug that reaches the systemic circulation. The AUC is the area under the drug plasma level–time curve from t = 0 to t = ∞, and is equal to the amount of unchanged drug reaching the general circulation divided by the clearance. where F = fraction of dose absorbed, D0 = dose, k = elimination rate constant, and VD = volume of distribution.  The AUC is independent of the route of administration and processes of drug elimination as long as the elimination processes do not change. The AUC can be determined by a numerical integration procedure, such as the trapezoidal rule method.  The units for AUC are concentration time (eg, μg hr/mL).  For many drugs, the AUC is directly proportional to dose. For example, if a single dose of a drug is increased from 250 to 1000 mg, the AUC will also show a four fold increase.  In some cases, the AUC is not directly proportional to the administered dose for all dosage levels. For example, as the dosage of drug is increased, one of the pathways for drug elimination may become saturated. Drug elimination includes the processes of metabolism and excretion. Drug metabolism is an enzyme-dependent process.  For drugs such as salicylate and phenytoin, continued increase of the dose causes saturation of one of the enzyme pathways for drug metabolism and consequent prolongation of the elimination half-life.  The AUC thus increases disproportionally to the increase in dose, because a smaller amount of drug is being eliminated (ie, more drug is retained). When the AUC is not directly proportional to the dose, bioavailability of the drug is difficult to evaluate because drug kinetics may be dose dependent.
  • 86. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Shravan Paswan et.al. Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 356 Plasma drug concentration time curve Urinary drug excretion data: Urinary drug excretion data is an indirect method for estimating bioavailability. The drug must be excreted in significant quantities as unchanged drug in the urine. In addition, timely urine samples must be collected and the total amount of urinary drug excretion must be obtained. The cumulative amount of drug excreted in the urine, D∞u is related directly to the total amount of drug absorbed. Experimentally, urine samples are collected periodically after administration of a drug product. Each urine specimen is analyzed for free drug using a specific assay. The relationship between the cumulative amounts of drug excreted in the urine and the plasma level–time curve shows when the drug is almost completely eliminated, the plasma concentration approaches zero and the maximum amount of drug excreted in the urine, D∞u is obtained. The rate of drug excretion, because most drugs are eliminated by a first-order rate process, the rate of drug excretion is dependent on the first-order elimination rate constant k and the concentration of drug in the plasma Cp. In the maximum rate of drug excretion, (dD u/dt) max, is at point B, whereas the minimum rate of drug excretion is at points A and C. Thus, a graph comparing the rate of drug excretion with respect to time should be similar in shape as the plasma level–time curve for that drug. The total time for the drug to be excreted, in and, the slope of the curve segment A–B is related to the rate of drug absorption, whereas point C is related to the total time required after drug administration for the drug to be absorbed and completely excreted t = ∞. The t∞ is a useful parameter in bioequivalence studies that compare several drug products. Rate of excretion Acute pharmacological response  In some cases, the quantitative measurement of a drug in plasma or urine lacks an assay with sufficient accuracy and/or reproducibility. For locally acting, non systemically absorbed drug products, such as topical corticosteroids, plasma drug concentrations may not reflect the bioavailability of the drug at the site of action.  An acute pharmacodynamic effect, such as an effect on forced expiratory volume, FEV1 (inhaled bronchodilators) or skin blanching (topical corticosteroids) can be used as an index of drug bioavailability. In this case, the acute pharmacodynamic effect is measured over a period of time after administration of the drug product.
  • 87. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Shravan Paswan et.al. Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 357  Measurements of the pharmacodynamic effect should be made with sufficient frequency to permit a reasonable estimate for a time period at least three times the half-life of the drug. This approach may be particularly applicable to dosage forms that are not intended to deliver the active moiety to the bloodstream for systemic distribution.  The use of an acute pharmacodynamic effect to determine bioavailability generally requires demonstration of a dose–response curve.  Bioavailability is determined by characterization of the dose–response curve. For bioequivalence determination, pharmacodynamic parameters including the total area under the acute pharmacodynamic effect–time curve, peak pharmacodynamic effect, and time for peak pharmacodynamic effect are obtained from the pharmacodynamic effect–time curve. The onset time and duration of the pharmacokinetic effect may also be included in the analysis of the data.  The use of pharmacodynamic endpoints for the determination of bioavailability and bioequivalence is much more variable than the measurement of plasma or urine drug concentrations.  Effects such as change in ECG or EEG readings, pupil diameter, etc are related to the time course of a given drugs.  Bioavailability can be determined by construction of pharmacologic effect time curve as well as dose response graph.  The drawback of this method is that, the response tends to more variable. Moreover, the observed response may be due to an active metabolite whose concentration is not proportional to concentration of parent drug responsible for the pharmacological effect. Therapeutic response: Theoretically, this method is most definite among all.  It’s based on observing clinical response to a drug formulation given to a patient suffering from disease for which the drug is intended to be used.  A major drawback is that quantification of observed response is unreliable for assessment of bioavailability. In- vitro dissolution study: Drug dissolution studies may under certain conditions give an indication of drug bioavailability. Ideally, the in-vitro drug dissolution rate should correlate with in-vivo drug bioavailability (see and on in-vivo–in-vitro correlation, IVIVC). Dissolution studies are often performed on several test formulations of the same drug. The test formulation that demonstrates the most rapid rate of drug dissolution in vitro will generally have the most rapid rate of drug bioavailability in vivo. Closed compartment apparatus: Non sink condition Open compartment apparatus: Perfect sink condition Dialysis system: This method is useful for very poorly aqueous soluble drugs for which maintenance of sink condition would require large volume of dissolution fluid. Clinical observations: Well-controlled clinical trials in humans establish the safety and effectiveness of drug products and may be used to determine bioavailability. However, the clinical trials approach is the least accurate, least sensitive, and least reproducible of the general approaches for determining in-vivo bioavailability. The FDA considers this approach only when analytical methods and pharmacodynamic methods are not available to permit use of one of the approaches described above. Comparative clinical studies have been used to establish bioequivalence for topical antifungal drug products (eg, ketoconazole) and for topical acne preparations. For dosage forms intended to deliver the active moiety to the bloodstream for systemic distribution, this approach may be considered acceptable only when analytical methods cannot be developed to permit use of one of the other approaches. Analytical methodology: The selected analytical method should be  Sufficiently sensitive to permit detection of low concentration of drug.  Reproducible  Must be specific for unmetabolized drug as well as capable of determining concentration of drug in presence of metabolites, constituents of blood/ urine. Stable Isotope Studies: This approach involves the simultaneous administration of test product and the reference product, using each subject as his own control. The reference contains the drug, which has been synthesized to contain a stable isotope such as such as 2H, 15N, 13C or 18Oin a position in a drug molecule that is not
  • 88. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Shravan Paswan et.al. Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 358 susceptible to metabolism and does not result in kinetic differences due to presence of isotope. The sample is collected and the comparisons are made of quantity of labeled and unlabeled drug in each sample, using sophisticated detection systems involving mass spectroscopy. A method for the calculation of bioavailability in slow release formulations in the presence of within- individual variability: In the present study they propose a model-independent method based on the combination of the area under the curve of serum drug levels and the mean residence time for evaluating the amount of bioavailability when within-individual variability is present in the serum clearance of the drug, administered as a slow release formulation (SRF), and this follows linear pharmacokinetic behavior. The method assumes that the modifications in the area under the curve of the serum levels induced by the within-individual variability in the kinetic behavior of the drug lead to a variation of the same proportions in the mean residence time of the serum levels curve and that this parameter can be used as a correction factor in the ratio of the areas under the curve of serum levels in bioavailability studies. The method allows one to calculate the fraction of dose absorbed from the SRF without having to measure the disposition clearance of the drug either when using the reference formulation or when the drug is administered as a SRF. The method is easy to apply and has a minimum mathematical complexity. The validity of the method was evaluated using simulated data with either no error or containing a random error of 10%. Bioavailability Problems: There are a number of examples of drugs products which have exhibited bioavailability problems in the past. These examples are all pre-1976 [Gibaldi, 1984]. This is an indication that more attention is now being given to formulation development during drug development. Three chlorpropamide formulations were tested and the peak plasma concentration after administration of one brand was less than half the peak concentration after the other two formulations (figure.1.). Figure.1. Plot of Cp versus Time The text reports a number of bioavailability problems with digoxin. One example is particularly interesting. Doctors in Israel noticed 15 cases of digoxin toxicity between Oct/Dec 1975 with almost no reports for the same period the previous year. It was found that the local manufacturer had changed the formulation to improve dissolution without telling the physicians. Urinary data suggested a two-fold increase in availability of the new formulation. Again there are a number of examples in the text. One report described an incidence of phenytoin intoxication in Australia in 1968 and 1969. Apparently the tablet diluent was changed from calcium sulfate to lactose. Later studies showed that the bioavailability was higher from the dosage form containing lactose. Other drugs with problems in the past include Acetazolamide, Aminosalicylate, Ampicillin, Aspirin, Ascorbic Acid, Chloramphenicol, Chlorothiazide, Diazepam, Furosemide, Iron, Levodopa + 10 (Gibaldi, 1984). CONCLUSION Today, various pharmaceutical companies are developing generic drug products. Bioequivalence study is important for generic drug approval process. It is our hope that, this review will provide an easy quick overview for Regulatory consideration required for bioequivalence study. This review covers major aspect of requirement of bioequivalence study along with the regulatory specification.
  • 89. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Shravan Paswan et.al. Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 359 REFERENCES Asif M Tamboli, Pavan Todkar, Priti Zope and FJ Sayyad, An Overview on Bioequivalence: Regulatory Consideration for Generic Drug Products Journal of Bioequivalence & Bioavailability, 2(4), 2010, 086-092. Dalton JT and Yates CR, Bioavailability of Drugs and Bioequivalence, Encyclopedia of pharmaceutical technology, Third Edition, Volume 1, Swarbrick J, Editor, 2007, Informa Healthcare. USA Inc, 164-176. Davit BM, Conner DP, fabian-Firtsch B, Highly Variable Drugs: Observations from Bioequivalence Data Submitted to the FDA for New Generic Drug Applications, AAPS J, 10, 2008, 148-156. Karalis V, Macheras P, Van Peer A, Bioavailability and Bioequivalence: Focus on Physiological Factors and Variability, Pharm Res, 25, 2008, 1956-1962.
  • 90. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Harish G et.al. Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 360 THE EFFECT OF SUPERDISINTEGRANTS ON THE DISSOLUTION OF CALCIUM CARBONATE FAST DISSOLVING TABLETS 1 Mohammed Farhana, 1 J.Preeti, 1 Md Faizulla, 1 Budda Chellibabu, 1 Harish.G*, 2 Rajnesh Kumar Singh 1 Nimra College of Pharmacy, Jupudi,Vijayawada 2 Micro Advance Research Centre, Bangalore *Corresponding author:harishgopinath4u@gmail.com ABSTRACT The objective of the present study is to design and evaluate the effect of disintegrating agents such as Starch, Cross Caramellose Sodium, Sodium starch glycolate and crosprovidone Calcium carbonate conventional tablets simultaneously. The nature of calcium carbonate which forms cake on standing which affects the drug disintegration process there by inhibits the drug release from the Conventional Tablet. Hence in the present study the effect of disintegrating agents at different concentrations is carried out on both the drugs simultaneously and finding out the best concentration followed by stability studies for a period of 3 months. Key words-Calcium Carbonate, Disintegrating agent, fast dissolving Tablets INTRODUCTION Despite increasing interest in controlled-release drug delivery systems, the most common tablets are those intended to be swallowed whole and to disintegrate and release their medicaments rapidly in the gastrointestinal tract (GIT). The proper choice of disintegrant or superdisintegrant and its consistency of performance are of critical importance to the formulation development of such tablets. Drug release from a solid dosage form can be enhanced by addition of suitable disintegrants. In more recent years, increasing attention has been paid to formulating fast dissolving and/or disintegrating tablets that are swallowed, but also orally disintegrating tablets that are intended to dissolve and/or disintegrate rapidly in the mouthThe present study is an attempt to select best possible combination of drug and disintegrating agent to formulate rapidly disintegrating tablet of calcium carbonate conventional tablets which disintegrates faster thereby reducing the time of onset of action. Lactose is selected as diluents, Starch, Sodium starch glycolate, CCS and crospovidone were selected as disintegrants. PVP K 30M paste was used as a binder in all formulations, Magnesium stearate and Talc as a Lubricant, Aerosil as a Glidant. MATERIALS AND METHODS Calcium carbonate Procured from MicroLabs,Bangalore , Cross Caramellose Sodium, Sodium Starch Glycolate Procured from Signet chemicals,Mumbai, Anhydrous lactose Procured from Jain Enterprises,Chennai,Aerosil ,Talc from Nice Chemicals Ltd, Chennai Table 1(a): Formulation Tablet of Calcium carbonate conventional tablet FA- Starch, FB- Croscarmelose Sodium, FC-Sodium starch glycolate, FD & FE- Crospovidone Formulation FA1 FA2 FA3 FB1 FB2 FB3 FC1 FC2 FC3 FD1 FD2 FE1 FE2 Calcium Carbonate (mg) 250 250 250 250 250 250 250 250 250 250 250 500 500 Starch 50 100 150 - - - - - - - - - - CCS - - - 10 20 30 - - - - - - - SSG - - - - - - 40 50 60 - - - - CP - - - - - - - - - 40 80 40 80 Lactose 570 520 470 610 600 590 580 570 560 580 540 340 300 PVP 50 50 50 50 50 50 50 50 50 50 50 20 20 Talc 50 50 50 50 50 50 50 50 50 50 50 50 50 Magnesium Sterate 30 30 30 30 30 30 30 30 30 30 30 50 50 Total Weight 1000 1000 1000 100 0 1000 1000 1000 1000 1000 1000 1000 1000 1000
  • 91. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Harish G et.al. Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 361 EVALUATION OF FORMULATED CALCIUM CARBONATE TABLETS Evaluation of blend characteristics: The Calcium carbonate granules were prepared using wet granulation method. Various formulations were made as shown in table: 1(a). The Formulated calcium carbonate granules were evaluated for Pre-formulation parameters like angle of repose, bulk density, tapped density, Compressibility index and Hausner’s Ratio. Using FA (Starch) as Disintegrant: The angle of repose of formulated calcium carbonate tablet was in the range of 20°-30°. Normally if the value falls between 20°-30°, it shows good flow property. The bulk density and tapped density were found to be in the range of 0.37 to 0.38 g/cm3 and 0.44 to 0.45g/cm3 respectively. A Hausner ratio was within the range of 1.16 to 1.17, lesser than 1.25 is considered to be an indication of good flow property. The compressibility index was within the range of 5-15 hence falls within the excellent range. Using FB (Croscarmelose Sodium) as Disintegrant: The angle of repose of prepared calcium carbonate tablet was in the range of 20°-30°. Normally if the value falls between 20°-30°, it shows good flow property. The bulk density and tapped density were found to be in the range of 0.34 to 0.36 g/cm3 and 0.39 to 0.40g/cm3 respectively. The Hausner’s ratio was within the range of 1.07 to 1.18, lesser than 1.25 is considered to be an indication of good flow property. The compressibility index was within the range of 5-15 hence falls within the excellent range. Using FC (Sodium Starch Glycolate) as Disintegrant: The angle of repose of prepared calcium carbonate tablet was in the range of 20°-30°. Normally if the value falls between 20°-30°, it shows good flow property. The bulk density and tapped density were found to be in the range of 0.35 to 0.36 g/cm3 and 0.39to 0.41g/cm3 respectively. The Hausner’s ratio was within the range of 1.08 to 1.18, lesser than 1.25 is considered to be an indication of good flow property. The compressibility index was within the range of 5-15 hence falls within the excellent range. Using FD (Crospovidone- Internal) as Disintegrant: The angle of repose of prepared calcium carbonate tablet was in the range of 20°-30°. Normally if the value falls between 20°-30°, it shows good flow property. The bulk density and tapped density were found to be in the range of 0.36 to 0.38 g/cm3 and 0.40 to 0.41g/cm3 respectively. The Hausner’s ratio was within the range of 1.07 to 1.15, lesser than 1.25 is considered to be an indication of good flow property. The compressibility index was within the range of 5-15 hence falls within the excellent range. Using FE (Crospovidone- Internal& External)) as Disintegrant: The angle of repose of prepared calcium carbonate tablet was in the range of 20°-30°. Normally if the value falls between 20°-30°, it shows good flow property. The bulk density and tapped density were found to be in the range of 0.36 to 0.37 g/cm3 and 0.40 to 0.41g/cm3 respectively. The Hausner ratio was within the range of 1.10 to 1.11, lesser than 1.25 is considered to be an indication of good flow property. The compressibility index was within the range of 5-15 hence falls within the excellent range. Post- Compressional Characteristic: The post compressional characteristic for all the formulated batches was found to be within the limits as per Indian pharmacopeia 2007. The hardness was found to be within the range of 3.5 to 5.5 Kg/cm2 in all the formulations indicating good mechanical strength with an ability indicating physical and mechanical strength with an ability to withstand physical and mechanical stress conditions while handling. In all the formulations, the friability value is less than 1% giving an indication that tablets formulated are mechanically stable. All the tablet formulations passed the weight variation test. The weight of all the formulations was found to be within the limits. The assay of all the formulations was found to be with in the 98% to 100.5% acceptable limits. The results of disintegration time of all the tablets prepared by wet granulation found to be varied with change in concentration of disintegrating agents from few seconds to several minutes. Formulations FD 1 and FE1 disintegrated within 3min and found to be more effective. The disintegration time of the tablets using different disintegrants decreases in the following order CP > Croscarmellose sodium > SSG > Starch. It is observed that, when CP is used as disintegrant, tablets disintegrate rapidly with in less time compared to other tablets prepared using croscarmellose sodium, starch and sodium starch glycolate disintegrants. Though tablets prepared by intra and extra granulation method found to be more effective in comparison with formulation prepared by only extra granulation. When concentration of Starch, SSG, CCS and BC is increased, the disintegration time was reduced significantly. STABILITY STUDIES Drug molecules are of reactive naturally, the additives are considered to be inert substance but this may not be true for all additives in a formulations. Hence, in developing any formulation, when additive are selected the same must be super imposed on to drugs and with other additives present in the formulation, to see how
  • 92. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Harish G et.al. Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 362 compatible they are with the other formulation ingredients. Real time study of ICH guidelines involves storage of products at 30°C & 65% RH for the complete proposed shelf life period, and analyzing the product sample every month in the first 3 months, every 3 months from 4th month onwards up to one year, every 6 month in the second year of storage, afterwards once in a year till shelf life is completed. ICH guidelines also demands for storing samples at 40 ° c and 75 % RH (stress condition or accelerated stability studys) for relatively short period of time (3-6 months) which depends on claimed shelf life period as well as the zone (zone 1/2/3/4 of the world) to which the product is meant to be exported. This later study (with stress conditions) is to mine the alternating climates condition during the shelf life of the product. The stability parameter for all the formulation were evaluated after 15, 30, 45, 60, and 90 days for 40 °C at 75% RH and the values were been tabulated in table given in Table 11. CONCLUSION Selecting appropriate formulation excipients and manufacturing technology can obtain the design feature of fast disintegrating tablet. The disintegrants have the major function to oppose the efficiency of the tablet binder and the physical forces that act under compression to form the tablet. The stronger the binder, the more effective must be the disintegrating agents in order for the tablet to release its medication. Ideally, it should cause the tablet to disrupt, not only into the granules from which it was compressed, but also into powder particles from which the granulation was prepared. From this study, it is concluded that the disintegrants such as Starch, SSG, CCS was compared with crospovidone disintegrants and in this study calcium carbonate tablet using crospovidone as disintegrant prepared by intra and extra granulation method was found to be the most effective as they disintegrate rapidly when compared to other disintegrants, and the percentage drug release also shows a higher dissolution profile. Table 1: Post-compressional characteristic of Calcium carbonate Tablets Using FA as disintegrant Formulation Weight variation(mg) Thickness (mm) Diameter (mm) Hardness (Kg/cm2 ) Friability (%) Assay (%) FA1 Compiles 7.194 18.99 3.5-5.5 0.291 91.8263 FA2 Compiles 7.29 19.06 3.5-5.5 0.386 93.1792 FA3 Compiles 7.39 18.97 3.5-5.5 0.254 99.9458 Table 2: Post-compressional characteristic of Calcium carbonate Tablets Using FB as disintegrant Formulation Weight variation (mg) Thickness (mm) Diameter (mm) Hardness (Kg/cm2 ) Friability (%) Assay (%) FB1 Compiles 7.31 18.91 3.5-5.5 0.355 99.2762 FB2 Compiles 7.46 19.03 3.5-5.5 0.318 98.4949 FB3 Compiles 7.27 19.11 3.5-5.5 0.347 99.5952 Table 3: Post-compressional characteristic of Calcium carbonate Tablets Using FC as disintegrant Formulation Weight variation (mg) Thickness (mm) Diameter (mm) Hardness (Kg/cm2 ) Friability (%) Assay (%) FC1 Compiles 7.251 17.96 3.5-5.5 0.314 99.6604 FC2 Compiles 7.564 18.42 3.5-5.5 0.389 98.8846 FC3 Compiles 7.387 18.55 3.5-5.5 0.296 98.8863 Table 4: Post-compressional characteristic of Calcium carbonate Tablets Using FD as disintegrant Formulation Weight variation (mg) Thickness (mm) Diameter (mm) Hardness (Kg/cm2 ) Friability (%) Assay (%) FD1 Compiles 7.27 19.11 3.5-5.5 0.214 98.4771 FD2 Compiles 7.39 19.27 3.5-5.5 0.296 99.2737 Table 5: Post-compressional characteristic of Calcium carbonate Tablets Using FE as disintegrant Formulation Weight variation (mg) Thickness (mm) Diameter (mm) Hardness (Kg/cm2 ) Friability (%) Assay (%) FE1 Compiles 6.94 18.72 3.5-5.5 0.184 98.2401 FE2 Compiles 7.05 18.92 3.5-5.5 0.213 99.1880
  • 93. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Harish G et.al. Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 363 Table 6: Disintegration profile of Calcium carbonate tablets using FA as disintegrant Formulation With Disk Without Disk I II I II I II FA1 11min 43 sec 10 min 30 sec 10min 52sec 14min 32 sec 15min 11sec 15min 48 sec FA2 8min 2sec 9min 33 sec 8min 18 sec 11min 14sec 12min 31 sec 11min 56 sec FA3 4min 41 sec 5min 8 sec 4min 55sec 9min 23sec 9min 51 sec 8min 50sec Table 7: Disintegration profile of Calcium carbonate tablets using FB as disintegrant Formulation With Disk Without Disk I II I II I II FB1 9min 21sec 8min 55 sec 10min 05sec 11min 15 sec 11min 24 sec 10min 55min FB2 7min 43sec 8min 11 sec 8 min 5sec 9min 22 sec 9min 17 sec 10 min 31 sec FB3 5min 22 sec 5min 42sec 6min 31sec 6 min 4 sec 7min 41sec 7min 18sec Tab 8: Disintegration profile of Calcium carbonate tablets using FC as disintegrant Formulation With Disk Without Disk I II I II I II FC1 11min 41 sec 10min 21 sec 10min 54 sec 14min 11sec 14min 56sec 13min 34sec FC2 8min 43sec 9min 21sec 9min 5sec 12min 37sec 14min 12sec 12min 44sec FC3 4min 21sec 5min 32 sec 4min 13sec 6min 23sec 6min 47 sec 6min 43sec Tab 9: Disintegration profile of Calcium carbonate tablets using FD as disintegrant Formulation With Disk Without Disk I II I II I II FD1 5 min 51 sec 6min 11 sec 5min 33sec 9min 46sec 8min 23 sec 9min 11sec FD2 3min 11 sec 2 min 47 sec 2min 17sec 5min 25 sec 4min 21 sec 5 min 41 sec Tab 10: Disintegration profile of Calcium carbonate tablets using FE as disintegrant Formulation With Disk Without Disk I II I II I II FE1 6min 19 sec 6min 5sec 5min 54sec 11min 19 sec 10min 47 sec 11min 14 sec FE2 4min 45 sec 3min 52 sec 4min 32sec 9min 11sec 9min 42sec 8 min 4 sec Stability studies: Tab 11: Stability studies of calcium carbonate Optimized batch Characteristics 400 C ± 20 C, 75% ± 5%RH Initial 15days 30days 45days 60days 75days 90days Description White compiles compiles compiles compiles compiles compiles Weight variation (mg) compiles compiles compiles compiles compiles compiles compiles Thickness (mm) 6.31 6.27 6.24 6.28 6.23 6.22 6.24 Diameter (mm) 19.07 19.01 18.89 18.97 18.84 19.84 19.89 Hardness(kg/cm2 ) 3 3 3 3 3 3 3 Friability (%) 0.09 0.04 0.01 0.01 0.01 0.01 0.01 Assay (%) 98.32 98.30 99.74 98.74 98.56 98.25 98.73 Disintegration (With disk) 4min 5sec 5min 19 sec 4min 34sec 4min 55sec 4min 5sec 4min 33sec 4min 56sec Disintegration (Without disk) 8min 15 sec 8min40 sec 9min23 sec 8min 54sec 8min19 sec 7min 55sec 7min 49sec
  • 94. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Harish G et.al. Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 364 REFERENCES Agdish Singh, Current Status of Tablet Disintegrants:A Review, Drug. Dev. Ind. Pharn, 18(3), 1992, 375-383. Alekha K, Charles Gayser, Hector Fausett, Evaluation of Quick Disintegrating Calcium Carbonate Tablets, AAPS Pharm.Sci.Tech, 1(3), 2000, 20. Andries F Marais, Mingna Song, Melgardt M, Effect of compression force, humidity and disintegrant concentration on the disintegration and dissolution of directly compressed furosemide tablets using croscarmellose sodium as disintegrant, Tropical.J .Pharm Res, 2(1), 2003, 125-135. Antony P J, Sanghavi NM, A New Binder for Pharmaceutical Dosage Forms, Drug. Dev. Ind. Pharm, 23(4), 1997, 413-415. Banker SG, Modern pharmaceutics, second edi, Marcel Dekker, newyork, 1990, 372-379. Basak SC, Rao YS, Manavalan R, Rao PR, Development and In vitroevaluation of an oral Floating matrix tablet formulation of ciprofloxacin, Ind.Pharm. Sci, 66, 2004, 313-316. Bi al yen, Rapidly Disintegrable multiparticular Tablets, Chem.Pharma.Bull., 18(9), 1995, 1308-1310. Bi Y, Sunada H, Yonezawa Y, Preparation and evaluation of a compressed tablet rapidly disintegrating in the oral cavity, Chem. Pharm. Bull, 44, 1996, 2121-2127.
  • 95. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) T K Gopal et.al. Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 365 PHYTOCHEMICAL AND PHARMACOLOGICAL STUDIES ON WHOLE PLANT OF ASYSTASIA GANGETICA T.k.Gopal*, Megha.G, D.Chamundeeswari, C.Umamaheswara Reddy, Faculty of Pharmacy, Sri Ramachandra University, Chennai, India *Corresponding author: E.Mail: tkgopal23@gmail.com ABSTRACT Asystasia gangetica (Linn) T.Anderson belonging to the family Acanthaceae is an ornamental plant and used as a vegetable in times of food scarcity. It is having many medicinal properties and is used in folklore medicine for various ailments like asthma, rheumatism, swellings etc. The whole plant of Asystasia gangetica was used for the study. The solvent methanol was used for the extraction as it was reported to extract the maximum phytoconstituents. The preliminary phytochemical analysis indicated the presence of compounds like Flavonoids, Alkaloids, Glycosides, Coumarins, Tannins, Steroids, Terpenoids and Saponins. The nutritive value of the methanolic extract was evaluated by estimating the Carbohydrate, Protein and Lipid content. The results justified its use as a vegetable in times of food scarcity. The secondary metabolites content like Phenols, Tannin and Flavonol content was also estimated. The Methanolic Extract of the whole plant was subjected to fractionation by Column chromatography with solvents of graded polarity. A Flavonoid Mixture Fraction was obtained in the Ethyl acetate: Methanol (50: 50) fraction. This mixture was characterised by chemical tests, Thin Layer Chromatography and High Performance Liquid Chromatography. The HPLC characterisation reported the presence of 4 known Flavonoids and 2 unknown compounds. The four Flavonoids were Luteolin, Quercetin, Kaempferol and Isorhamnetin. The in- vitro pharmacological activities were performed for the methanolic extract of the whole plant of Asystasia gangetica. The methanolic extract was selected for the pharmacological activities because it showed the maximum anti-inflammatory effect as reported in literature. Protein Denaturation produces auto antigens which may be a cause for the development of Rheumatoid arthritis. The study of Anti arthritic activity of the methanolic extract showed a dose dependent inhibition of protein Denaturation and was comparable to that of the standard. The methanolic extract also exhibited a dose dependent inhibition of ADP induced platelet aggregation. The methanolic extract was also evaluated for its Anthelmintic activity. It showed a dose dependent decrease in time taken for paralysis and death of worms. The methanolic extract and Flavonoid Mixture fraction were subjected to tests on blood viscosity. The Flavonoid mixture fraction exhibited a better decrement in blood viscosity than the methanolic extract. KEY WORDS: Asystasia gangetica, Anti-Oxidant, Anti-Arthritic, In-vitro, Anthelmintic INTRODUCTION Plants play a pivotal role in health care. According to World Health Organisation (WHO), 80% of the world’s population relies on traditional medicine, particularly plant drugs for primary health care. The practise of traditional medicine is not new in India since it is the birth place of many traditional practices like Ayurveda, Siddha and Unani. India is particularly well endowed with over 6000 medicinal plants and well recorded practical knowledge of traditional medicine. The scientific mind will not be satisfied by mere claims no matter from whatever source they originate, unless corroborated by experimental and clinical evidences. As it is evident that plants are treasure house for many potent medicines, it is important to scientifically evaluate the traditional practices as well as upgrade the existing knowledge and make it available to the general public. The very important plant molecules like Digitoxin, Morphine, Vincristine, Quinine etc have been used as prototypes for the discovery of newer synthetic molecules. The frequency of life threatening diseases and ailments has increased worldwide and it is becoming an important cause of morbidity and mortality in developing countries. Majority of the diseases like Atheroscelorosis, Arthritis, Diabetes, and Cancer occur due to free radical generation. Plants contain naturally occurring anti-oxidants like Flavonoids, Tannins etc which can be utilised for scavenging free radicals. Many of the plant Alkaloids affect the nervous system and hence have been used as Anaesthetics, Psycho stimulants, Motor end Depressants etc. Asystasia gangetica is an ornamental plant and has been used as a source of nutrition in times of food scarcity. It is also used traditionally for many ailments and diseases. Already the traditional use of the leaves as anti-asthmatic (Akah PA, 2003) was scientifically proven. But no work on its anti-arthritic, Anthelmintic and anti-
  • 96. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) T K Gopal et.al. Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 366 0 20 40 60 80 100 Percentinhibition Concentration µg/ml Metha nolic Extract platelet properties was performed. It was in this context that the multiple pharmacological activities of the whole plant of Asystasia gangetica were investigated in the course of this study. Also an attempt was made to isolate the possible bioactive molecule responsible for the pharmacological actions. Asystasia gangetica (Linn.) T.Anderson is commonly known as Chinese violet. ‘Asystasia’ means inconsistency and it relates to the fact that the corolla is slightly regular which is an unusual characteristic in the Acanthaceae family. The word ‘gangetica’ is derived from Ganges River where it is presumed to occur. It is locally used as a potherb and leafy vegetable in times of food scarcity. It is also promoted as cover plant in orchards as it checks soil erosion and prevents growth of noxious weeds. It also attracts bees to orchards. It has high nutritive value and hence used as forage for cattle, goats and sheep in south East Asia. It is also used as an ornamental plant. MATERIALS AND METHODS a. Plant material: The whole plant was collected from Tirumala hills, Andhra Pradesh, in the month of December 2009. The plant material was authenticated by National Institute of Herbal Science (PARC/2010/542), West Tambaram, Chennai. b.Preparation of the extract: The freshly collected whole plant was dried in shade and coarsely powdered with a blender. 200 grams of the powder was subjected to continuous hot percolation using Soxhlet’s apparatus with the solvent methanol for 48 hrs. The solvent was recovered by distillation in Rotary Vacuum Evaporator at 80o C. The residue was stored in a desiccator and used in further studies. Fig 1: Aerial parts of Asystasia gangetica Fig 2: Asystasia gangetica habit c. Phytochemical Evaluation: Compounds like Flavonoids, Alkaloids, Tannins, and Glycosides etc. are responsible for many pharmacological activities of a plant. Phytochemical evaluation gives the chemical nature of the bioactive molecules responsible for pharmacological activity in a plant. d. Pharmacological studies: The following Pharmacological studies has been carried out on the whole plant of Asystasia gangetic such as determination of Anti-arthritic activity by inhibition of protein denaturation (Mizushima et al., 1966 ), In - vitro determination of anti-platelet activity by inhibition of platelet aggregation induced by ADP, In - vitro anthelmintic activity (Fabiyi, 1986) and Effect of plant extract on blood viscosity. RESULT AND DISCUSSION In- Vitro Pharmacological Activities 1. Anti-arthritic activity by inhibition of protein denaturation method: The percentage of inhibition of protein Denaturation of the methanolic extract and the standard Diclofenac sodium are tabulated in Table.1 & represented in fig 3. Table.1 Percent inhibition of Protein Denaturation by Methanolic Extract & Diclofenac sodium Fig 3: Anti-Arthritic activity of Asystasia gangetica Concentration μg/ml Percentage Inhibition of Methanolic extract Percent Inhibition of Diclofenac sodium 10 17.29 - 50 23.61 - 100 33.34 - 200 42.70 84.47 400 58.11 - 800 65.87 - 1000 78.94 - The methanolic extract exhibited a dose dependent inhibition of Protein Denaturation for Anti Arthritic activity. The maximum percent inhibition (78.94%) was exhibited by 1000 µg/ml of the methanolic extract.
  • 97. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) T K Gopal et.al. Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 367 2. Anti-platelet activity by inhibition of ADP induced platelet aggregation: The percentage transmittance of the methanolic extract and the standard drug aspirin in different time intervals are tabulated in Table.2 & represented in fig 4 Table.2 Percent transmittance of methanolic Extract & Aspirin for Anti-Platelet Activity Concentration (µg/ml) Percentage transmittance 0min 1min 2min 3min 4min 5min 100 2.311 2.433 2.453 2.546 2.743 2.764 200 3.764 3.001 3.437 3.813 3.760 3.941 400 4.111 4.024 4.512 4.589 4.708 4.800 500 5.452 5.675 5.876 5.783 5.863 5.944 ASPIRIN 6.769 6.654 6.540 6.502 6.471 6.411 Fig 4: Percent transmittance of Methanolic Extract & Aspirin for Anti-Platelet Activity The methanolic extract showed a dose dependent inhibition of aggregation for Anti Platelet activity and the maximum inhibition was exhibited by 500 µg/ml of Methanolic extract. 3. In- Vitro Anthelmintic Activity The average values of the time taken for paralysis and death of Pheretima posthuma are tabulated in Table.3 and represented in fig 5 Table.3 Time taken for Paralysis & Death in Albendazole and Methanolic Extract treated groups Group Solution Concentration (mg⁄ml) Time taken for paralysis (min) Time taken for death (min) I Control Solution - - - I Albendazole (Standard) 10 38±0.3 56±0.3 I Test Extract 10 45±0.2 54±0.5 50 37±0.6 43±0.3 100 25±0.4 30±0.8 200 19±0.9 24±0.4 Values are Mean± SEM of 6 observations. 0 2 4 6 8 100 200 400 500 ASPIRIN PercentTransmittance Concentration µg/ml 0 minute 1 minute 2 minutes 3 minutes 4 minutes 5 minutes
  • 98. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) T K Gopal et.al. Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 368 Fig 5: Time taken for Paralysis & Death in Albendazole and Methanolic Extract treated groups The Methanolic extract showed a dose dependent decrease in time taken for paralysis and death of Pheretima posthuma. The maximum dose of 500 mg/ml of methanolic extract showed the least time taken for paralysis and death of the earth worms. 10 mg/ ml of Methanolic Extract 50 mg/ ml of Methanolic Extract 100 mg/ ml of Methanolic Extract 200 mg/ ml of Methanolic Extract 10 mg/ml of Standard solution Control solution Fig.6. In Vitro anthelmintic activity of various concentrations of methanolic extract of whole plant of Asystasia gangetica 0 10 20 30 40 50 60 10 50 100 200 Timeinminutes Concentration mg/ml Methanolic Extract (Paralysis time in min) Methanolic extract (Death time in min) Albendazole (paralysis time) Albendazole (death time)
  • 99. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) T K Gopal et.al. Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 369 4) Effect of methanolic extract and flavanoid mixture fraction on blood viscosity The viscosity of Methanolic extract treated blood is tabulated in Table.4 and represented in fig 7. Table.4 Effect of Methanolic Extract on Blood Viscosity in time span of 90 minutes Concentration (µg/ml) 0 minute 30 Minutes 60 minutes 90 minutes Relative blood viscosity 100 2.2 2.2 2.1 2.1 2.2 200 2.1 2 2 1.9 300 1.9 1.9 1.9 1.8 400 1.8 1.7 1.7 1.6 500 1.6 1.6 1.5 1.5 Fig: 7 Effect of Methanolic extract on Blood viscosity The effect of various concentrations of Flavanoid fraction on blood viscosity at various time intervals is tabulated in Table.5 and represented in fig 8. Table.5 Effect of Flavonoid Mixture Fraction on Blood viscosity in time span of 90 minutes Concentration (µg/ml) 0minute 30minutes 60minutes 90minutes Relative blood viscosity 100 2.2 2.1 1.9 1.9 2.2 200 1.9 1.8 1.8 1.7 300 1.7 1.7 1.6 1.6 400 1.6 1.5 1.5 1.5 500 1.5 1.5 1.4 1.4 Fig 8: Effect of Flavonoid Mixture Fraction on Blood viscosity The methanolic extract showed a dose dependent decrease in Blood viscosity in a span of 90 minutes. The Flavanoid Mixture fraction showed better decrease in viscosity than methanolic extract. 0 0.5 1 1.5 2 2.5 100 200 300 400 500 Viscosity concentration µg/ml 0 minute 30 minutes 60 minutes 90 minutes Relative viscosity 0 0.5 1 1.5 2 2.5 100 200 300 400 500 Viscosity Concentration µg/ml 0 minute 30 minutes 60 minutes 90 minutes Relative viscosity
  • 100. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) T K Gopal et.al. Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 370 CONCLUSION The study justified the use of the plant as a food source by quantifying the nutritive value. The study also justified the use of plant as Anthelmintic and anti-Arthritic in folklore medicine. The methanolic extract also exhibited inhibition of platelet aggregation and decrease in blood viscosity. Also this is the first report of the flavonoids Luteolin, Quercetin, Kaempferol and Isorhamnetin being isolated from the Methanolic Extract of the whole plant of Asystasia gangetica. The study also reports that the decrease in blood viscosity by the methanolic extract was due to the Flavonoid mixture, which was isolated by column chromatography. REFERENCES Akah PA, Ezike AC, Nwafor SV, Okoli CO, Enwerem NM, Evaluation of the anti asthmatic property of Asystasia gangetica leaf extracts. Journal of Ethnopharmacology, 89, 2003, 25-36. Athnasiadau S, Interaction of anti-inflammatory drugs with serum proteins especially with some biologically active proteins. J.pharma.pharmacol, 20, 1968, 69-73. Athnasiadau S, I Kyriazakis, F Jackson, RL Coop, Direct Anthelmintic effects of condensed tannins towards different gastrointestinal nematodes of sheep: in vitro and in vivo studies, Vet.Parasitol, 99, 2001, 205-219. Brown JH, Inhibition of heat-induced denaturation of serum protein by mixtures of non-steroidal anti- inflammatory agents, Proc.soc.exp.biol.med, 128, 1968, 225-228. Chamundeswari D, In vitro antiplatlet activity-guided fractionation of aerial parts of Melthoria maderaspatana. Indian Journal of Pharmaceutical sciences, 68(5), 2006, 668-670. Connar AO, The in vitro anti-denaturation effects induced by natural products and non-steroidal compounds in heat treated (immunogenic) bovine serum albumin is proposed as a screening assay for the detection of anti- inflammatory compounds, without the use of animals, in the early stages of the drug discovery process, West Indian med j, 57, 2008, 327-331. Ezike AC, Akah PA, Okoli CO, Bronchospasmolytic activity of the extract and fractions of Asystasia gangetica leaves. International Journal of Applied Research in Natural Products, 3, 2008, 8-13. Hack B, Analytical method of determination of mineral& metallic elements. Hand book of Analytical practice. Dolphin ands, John(Ed) N.Y.Incorp.press ltd, 2000, 126-149. Harborne, J.B, Phytochemical methods, Chaponann & Hall, London, 1973, 1-271. Iqbal, Z, Lateef M, Ashraf M, Jabbar A, Anthelmintic activity of Artemisia brevifolia in sheep. J.of Ethnopharmacology, 93, 2004, 265-268 John S, Estimation of total Flavonoid content by two complementary colorimetric methods, Journal of food &drug analysis, 10, 2002, 178-182. Kokate, CK Texbook of Pharmacognosy, Nirali prakashnan, Publication, 1985 112-124. Kritikar, K.R and B.D Basu, Indian Medicinal plants 2nd E d., Basu, Allahabad, India, 1933, 231-236. Kumar R, Journal of Experimental Pharmacology, 2, 2010, 29-36. Lancet.S, Screening test for anti-rheumatic drugs, j.nat prod, 2, 1966, 443-445.
  • 101. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) T K Gopal et.al. Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 371 INVESTIGATION OF IN-VITRO ANTI-OXIDANT, ANTI-INFLAMMATORY AND ANTI-ARTHRITIC ACTIVITY OF AERIAL PARTS OF SECURINEGA LEUCOPYRUS (WILLD.) MUELL T.K.Gopal*, T.Sheela, D.Chamundeeswari, C.Umamaheswara Reddy Faculty of Pharmacy, Sri Ramachandra University, Chennai, India Corresponding author: E.Mail: tkgopal23@gmail.com ABSTRACT Securinega leucopyrus (willd.) Muell. (Euphorbiaceae) is a thorny, large shrub or small tree. In Ayurveda it has a lot of potential medicinal uses. The decoction of leaves are used to dress the cancerous wounds. The juice or paste of the leaves along with tobacco used to destroy worms in sores. Its leaf decoction useful externally in the treatment of piles. The present study is designed to carry out Anti-oxidant, Anti-inflammatory, Anti arthritic activity by Invitro methods. The Securinega leucopyrus was authenticated and the anatomical features were done which shows calcium oxalate crystals, fibres, glandular trichomes, sclereids, vascular bundles (secondary xylem and phloem). The microscopical evaluation also performed by determination of stomatal index and vein islet number. The In-vitro anti-oxidant activity revealed that the Alcoholic extract showed more activity as compared to Chloroform, Ethyl acetate, n-Hexane. In DPPH (1,1-diphenyl-2-picrylhydrazyl) radical method Alcoholic extract showed more activity and in nitric oxide scavenging method Hydro alcoholic, Alcoholic extracts showed more activity. Chloroform extract showed more activity in hydroxy radical method. In Total anti-oxidant method Alcoholic extract showed more activity. The Anti-inflammatory activity also performed by Human Red Blood Corpuscle (HRBC) Membrane Stabilization Method. The results were tabulated and the values are represented in the figure. In this Ethyl acetate extract showed more activity. The Anti-arthritic activity is performed by inhibition of protein denaturation method. And the results are given in table and values are represented in graph. This shows Alcoholic extract gave more activity. The above results shows that the presence of phytoconstituents like Glycosides, Flavanoids, Alkaloids, steroids and Tannins may be responsible for Anti-oxidant, Anti-inflammatory, Ant arthritic activities. Key words: Securinega leucopyrus, Anti-oxidant, Anti-inflammatory, Anti-arthritic. INTRODUCTION Plants are used as medicine since time immemorial. Approximately one-third of the top selling drugs in the world are derived from natural products and their derivatives. The traditional approach makes use of material that has been found by trial and error over many years in different systems of medicine (Cotton, 1996). Plants are used for discovery of modern medicine in four basic ways, as a source of direct medicinal agents, serve as a raw material base for elaboration of more complex semi-synthetic chemical compounds, chemical structure derived from phyto constituents can be used as models for new synthetic compound (Anon, 1994), Plants can be used as taxonomic markers for discovery of new therapeutic compounds (Cox.P.A, 1990). According to the world health organization (WHO), (Murry et al.,) 80% of the world’s population has faith in traditional medicine, particularly plant drugs for their primary healthcare. India is a goldmine of well recorded and traditionally well practical knowledge of herbal medicine. India officially recognizes over 3000 plants for their medicinal value. It is estimated generally that over 6000 plants in India are use in traditional, folk and herbal medicine, representing 75% of the medicinal needs of the third world countries. The frequency of life threatening infections has increased worldwide and it is becoming an important cause of morbidity and mortality in immuno-compromised patients in developing countries. Eg: Cancer, Cardio vascular diseases, Rheumatoid arthritis, Cataracts, Alzheimer’s diseases and inflammatory disorders. (Dev,1999). Several anti-oxidants of plants origin are experimentally proved and used as effective protective against oxidative stress. They play an important role in major health problems like cancer. Free radicals are produced in large amount during metabolic disease conditions like diabetes, hypertension, atherosclerosis, urolithiasis, ulcers etc. This free radicals attack DNA, protein molecules, enzymes and cells leading to change in genetic material and cell proliferation (Cancer). Plants which contain carotenoids, flavonoids and Tannins can be utilized to scavenge the excess free radicals from human body. An inflammatory disease like Rheumatoid arthritis is one of the most common immuno inflammatory conditions affecting the population worldwide. About 1% of the worldwide adult population is affected by Rheumatoid arthritis with above twice as many females suffers as male. In recent years, attempts have been made to investigate the indigenous drug against Arthritis. Research in the field of indigenous plant is a significant aspect of developing a safer anti arthritic principle through isolation,
  • 102. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) T K Gopal et.al. Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 372 characterization, identification and biological studies. (Pratt, 1992). Securinega leucopyrus (willd.) Muell, belonging to the family Euphorbiaceae commonly known as Bushweed, Indian snow berry, is a thorny woody shrub or a small tree distributed in different parts of India (Ayensu, 1996). Its leaves and fruits are edible. The berry is sweet. The slender branches are reported to be utilized for preparing wicker-baskets and for thatching (Kirtikar, 1918). The leaves of the plant contain germicidal properties. In Chattisgarh, the decoction of leaves is used to dress the cancerous wounds. It is used in combination with tobacco. The juice or paste of the leaves along with tobacco used to destroy worms in sores. Its leaf decoction useful externally in the treatment of piles and it is used to wash the wounds of cattle. It is used as popular veterinary medicine (Bakshu, 2001). The other uses are sweet, cooling, diuretic, aphrodisiac, tonic and are useful in vitiated conditions of pitta, burning sensation, strangury, seminal weakness, general debility, larvicide, paralysis, paresis, piscicide, insecticide. The anti-microbial properties of securinega leucopyrus is already reported (Bakshu, 2001). The leaves are set as a disinfectant and its paste is used to extract the extraneous materials from body tissues without surgery. Even though Securinega leucopyrus has lot of potential medicinal uses, the study on this plant is very less. Considering the importance of this plant, the present study was undertaken. Plant collection: It consists of dried aerial parts of Securinega leucopyrus (willd.) Muell. (Euphorbiaceae) is a thorny, large shrub or small tree. The plant Securinega leucopyrus were collected from Tribal Women’s Welfare Trust’s Herbal Garden, Thandarai, Chengalpattu (Dist), Tamilnadu in the month of December and authenticated at National Institute of Herbal Science, West Tambaram, Chennai (PARC/2009/289). Fig 1: Morphology of Securinega Leucopyrus (willd.) Muell METHODS AND MATERIALS Collection and processing of plant material: Then the aerial part of plant material was cut into small fragments and dried in shade until the fracture is uniform and smooth. The dried leaves, stem portions were separately powdered by using a blender and sieved in sieve No 60 (Gahan, 1984). The coarse powder 500 gm was subjected to maceration for 72 hours, followed by exhaustive maceration for 48 hours by using various solvents of increasing polarity [n- hexane> Chloroform> Ethyl acetate>Alcohol> Water] by decanting and drying the marc after each extraction. The solvents were recovered by distillation of the extracts at 750 C to 800 C. The extracts were dried under desiccator and the percentage yield was calculated. The residues obtained were used for the screening of the phytochemical analysis. These n-Hexane, Chloroform, Ethyl acetate, Ethanol and Hydroalcoholic extracts were used for the preliminary phytochemical investigation, chromatographic analysis and its pharmacological evaluation (Sundaresan, 1991). In-Vitro Pharmacological Evaluation: The aerial part of the plant of Securinega leucopyrus (willd.) Muell had been stuied for its anti-oxidant activity by DPPH method (Yohozawa, 1998), nitric oxide scavenging activity (Alderson, 2001), determination of total anti-oxidant activity (Prieto, 1999) scavenging of hydroxyl radical deoxyribose method (Elizabeth, 1990), in-vitro anti-inflammatory activity by human red blood corpuscle (HRBC) membrane stabilization method, in-vitro anti-arthritic activity by inhibition of protein denaturation method. RESULTS AND DISCUSSION Anti-oxidant activity by DPPH radical scavenging method: The chloroform and ethyl acetate, alcohol, hydro alcohol extracts showed a dose dependent increase in anti-oxidant activity in DPPH method. About 82.5%, 88.42% were observed in Chloroform and Alcoholic extracts of Securinega leucopyrus. But in Hexane extract the effect was decreased when dose increases. The presence of flavanoids, Alkaloids, Tannins and Steroids in these extracts may be responsible for free radical scavenging activity. From the results it is made clear that Securinega leucopyrus possess free radical scavenging property and the order was, Alcohol > Chloroform > Ethyl acetate > Hydro alcohol >Hexane. DPPH is a relatively stable free radical. The assay is the measurement of scavenging ability of Antioxidants towards the stable radical DPPH. The plant containing
  • 103. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) T K Gopal et.al. Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 373 flavanoids, Alkaloids, Tannins and Steroids reduces the radical to the corresponding hydrazine when it reacts with a hydrogen donors in the anti-oxidant principle. In DPPH method results are highly reproducible. The active constituent present in the plant donates an electron to reduce the DPPH radical to its corresponding hydrazine. The chemical constituents present in this plant like Flavanoids, Tannins, Alkaloids and Steroids may be responsible for this activity. They shows the anti-oxidant activity by inhibition of enzymes involved in oxidation systems. Anti -Oxidant Activity by Nitric Oxide Scavenging Method: All extracts of Securinega leucopyrus shown a dose dependent increase in Nitric oxide scavenging property .About 85% inhibition was observed by Hydroalcoholic extract. But in Hexane and Ethyl acetate extracts the effect was very less compared to standard. The presence of flavonoids Tannins and steroids in these extracts may be responsible for Nitric oxide scavenging activity. Nitric oxide is a radical produced in mammalian cells, involved in the regulation of various physiological processes. In the present study the nitrite produced by the incubation of sodium nitro prusside in standard phosphate buffer at 25◦ c was reduced by plant extract. This may be due to the anti- oxidant principles present in the plant extract which compete with oxygen to react with nitric oxide, there by inhibiting the generation of more deleterious products such as Nitric anhydride (N 2O3 ) and perhydroxy nitrite (ONO --- ) (Chen et al 2001). This activity is due to the presence of flavonaids, Tannins and steroids. They inhibit the free radicals by inhibition of enzymes involved in oxidation systems (5- Lipoxygenase, cycloxygenase, mono oxygenase, xanthine oxidase. Determination of total anti-oxidant activity: The Ethanolic, Hydro alcoholic extracts showed maximum effect due to the presence of flavanoids, Reducing sugars, Alkaloids, Tannins and steroids. The values were expressed as equivalents of Vitamin E. From the results it is made that Securinega leucopyrus possess free radical scavenging activity through anti-oxidant property. Anti-oxidant activity By Hydroxy radical scavenging method: All the extracts of Securinega leucopyrus shown a dose dependant increase in nitric oxide scavenging property. About 87.11% inhibition was observed by Alcoholic extract. The Chloroform and Hydroalcoholic extract showed 78.56%, 78.52% inhibition. But in Ethyl acetate and Hexane extract the effect was very less compared to standard. The presence of flavanoids Tannins and steroids in these extracts may be responsible for Hydroxy radical scavenging activity. Ferrous salts can react with H2O2 and form hydroxyl radical via Fention’s reaction. The iron required for this reaction is obtained either from the pool of iron or the heme containing protein. The hydroxyl radical (OH)- thus produced may attack the sugar of DNA deoxy causing ribose fragmentation , base loss, and DNA strand breakage. The generation of (OH)- in fenton reaction is due to the presence of iron ions. When the Fe2+ /Fe3+ redox couple is bound by certain chelators, the OH● fragmentation is prevented, whereas the increased colour formation in the absence of crude extracts were observed in deoxy rebose assay. In this, the extract act as a chelator of iron ions , binding to them, & preventing the formation of free radicals, though the extracts not directly involved in the OH● scavenging . The results indicates that the extracts of plant plays a major role in the inhibition of ribose fragmentation and hence the decreased color formation in the deoxy rebose assay. The free radical scavenging property of the crude extracts of plant against DPPH, Nitric oxide, Hydroxy radicals and the Total anti-oxidant activity is clearly understood from the results of this chapter. The phytochemical screening of the extract revealed the presence of flavonoids, tannins which is responsible for anti-oxidant property. Anti-inflammatory activity by HRBC membrane stabilization method: The all extracts shows a dose dependent increase in Anti-inflammatory activity in HRBC membrane stabilization method. About 89.66%, 87.42%, 72.32% inhibitions were observed in Ethyl acetate, Chloroform and Alcoholic extracts. But in Hexane and Hydro alcoholic extracts the effect was very less when compared to standard Diclofenac sodium. This anti-inflammatory activity may be due to the presence of flavonoids, Tannins and Alkaloids. Flavonoids may produce their Anti-inflammatory effect by a multitude of ways to inhibit the inflammatory processes. Formation and release of various mediators of inflammation like Histamine and Prostaglandin are affected by Flavanoids. They inhibit the increased capillary permeability during inflammation. The adhesion of leucocyte to endothelial surface and subsequent migration is influenced by flavanoids. They inhibit the prostaglandin and Leucotriene C4 in human platelet. They were investigated for lipoxygenase inhibitory activity. They inhibits cytokine release from cells. Anti-Arthritic Activity by Inhibition of Protein Denaturation Method: The results of anti-arthritic activity of all extracts of Securinega leucopyrus on inhibition of protein denaturation method as shown in table18 and figure 5.6. The Ethanol, Chloroform, Hydro alcoholic extracts showed maximum activity when compared to n-Hexane and Ethyl acetate extracts. The effect is represented as, Ethanol > Water > Chloroform > Ethyl acetate > Hexane extracts. The effect may be due to Alkaloids, Tannins, Flavanoids, Triterpenes and Reducing
  • 104. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) T K Gopal et.al. Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 374 sugars. Denaturation of protein is one of the cause of Rheumatoid arthritis. Production of auto antigen in certain arthritic disease may be due to denaturation of proteins. The mechanism of denaturation probably involves alteration in electrostatic hydrogens and hydrophobic di sulphide bonding. The present study, Securinega leucopyrus capable of controlling the production of auto antigens and thereby it inhibit the denaturation of protein in Rheumatic diseases. Tab 1: The Percent inhibition of different extracts of Securinega leucopyrus on DPPH radical scavenging activity Concentrations in μg/ml Hexane extract CHCL3 extract EtoAc extract Alcoholic extract Hydoalcholic extract Percent Inhibition of Standard (Vit. E) 10 19.50 25.25 23.28 24.45 20.36 - 50 26.51 29.91 26.74 28.43 20.13 32.5 100 47.91 55.23 50.46 51.58 40.01 62.88 200 39.66 68.58 62.85 58.34 52.41 81.02 400 39.51 74.62 71.22 75.66 65.32 95.24 800 37.87 74.56 75.95 76.3 62.45 - 1000 34.77 82.51 78.32 88.42 67.41 - Fig. 2: Effect of securinega leucopyrus on DPPH method Tab 2: Percentage inhibition of different extracts Securinega leucopyrus by Nitric Oxide Scavenging method. Concentrations in μg/ml Hexane extract CHCL3 extract EtoAc extract Alcoholic extract Hydrialcocolic extract Percent Inhibition of Standard (Vit. E) 10 15.43 18.43 18.34 20.35 25.35 - 50 18.34 20.65 21.45 24.17 26.13 28.40 100 22.51 28.47 20.36 35.11 38.53 47.78 200 38.62 46.44 29.4` 62.43 69.42 71.34 400 45.23 55.91 37.22 63.12 67.37 87.28 800 47.54 52.13 37.13 62.56 74,91 - 1000 54.33 71.44 45.78 77.12 85.38 -
  • 105. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) T K Gopal et.al. Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 375 Fig 3: Effect of Securinega Leucopyrus on Nitric Oxide Scavenging activity Determination of Total Antioxidant Activity: In this investigation the absorbance (Y-axis) of different extracts were extrapolated to the concentration (X-axis) of standard Vit – E. Fig 4: Determination of total anti-oxidant activity Tab 3: Absorbance of Test extracts plotted on the above graph is as follows Extracts 200 µg/ml Hexane 0.6215 Chloroform 1.2710 Ethyl acetate 1.0126 Ethanol 1.4205 Hydroalcohol 1.3010 Tab 4: Estimation of Total anti-oxidant activity Concentrations of extracts in μg/ml Equivalent to Standard Vit – E in μg/ml Hexane Chlorofom Ethylacetate Ethanol Hydroalcohol 200 36.5 68 61.5 83.5 72.7 The above table shows the extrapolated values. Hence the effect of 200µg/ml of extracts is equivalent to the effect of µg/ml of Vit – E with respect to the extracts.
  • 106. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) T K Gopal et.al. Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 376 Tab 5: Percent inhibition of Securinega leucopyrus in Hydroxyl Radical Scavenging method Concentrations in μg/ml Hexane extract CHCl3 extract EtoAc extract Alcoholic extract Hydro alcocolic extract Percent Inhibition of Standard (Vit. E) 10 21.91 28.54 29.44 28.22 18.62 - 50 34.56 38.55 28.41 37.45 22.45 40.34 100 33.42 51.91 38.42 35.66 28.64 62.99 200 45.82 66.34 48.54 47.44 34.25 85.59 400 60.44 75.66 66.69 59.23 43.88 95.01 800 65.71 75.52 67.23 61.03 50.52 - 1000 77.30 86.12 71.56 66.45 58.34 - Fig 5: Effect of Securinega Leucopyrus on Hydroxy Radical Scavenging Activity Tab 6: Percent HRBC Membrane Stabilization of different extracts of Securinega leucopyrus Concentrations in μg/ml Hexane extract Chloroform extract Ethyl acetate extract Alcohol extract Hydro alcohol extract Percent stabilization of Diclofenac sodium 10 29.77 34.21 35.67 25.14 20.67 - 50 29.93 34.52 36.73 28’56 29.54 - 100 40.56 42.51 47.43 32.56 37.54 - 200 47.82 55.32 68.23 45.06 36.77 93.52 400 43.18 69.76 71.94 62.45 34.52 - 800 59.43 75.87 82.54 62.44 40.54 - 1000 65.88 87.42 89.66 74.32 56.33 - Fig 6: Effect of Securinega leucopyrus on Anti-inflammatory Activity
  • 107. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) T K Gopal et.al. Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 377 Tab 7: Percent denaturation of protein of different extracts of Securinega leucopyrus Concentrations in μg/ml Hexane extract CHCL3 extract Ethanolic extract Alcohol extract Hydro alcohol extract Percent stabilization of Diclofenac sodium 10 18.34 26.95 27.57 29.45 24.55 - 50 25.67 32.86 32.74 38.56 37.13 - 100 38.33 48.21 30.51 40.54 39.45 - 200 37.34 47.54 38.43 57.82 56.99 94.14 400 45.81 56.90 62.55 67.90 57.82 - 800 45.58 69.44 74.21 73.51 67.95 - 1000 52.80 78.52 76.4 87.11 78.56 - Fig 7: Effect of securinega leucopyrus on Anti Arthritic Activity CONCLUSION From this study we can conclude that Securinega leucopyrus showed the presence of Alkaloids, Sugars, Flavonoids and Tannins. This is identified by phytochemical analysis and confirmed by thin layer chromatography. The present study on Securinega leucopyrus showed that the plant has moderate anti-oxidant activity and it compared with standard vitamin E. The plant also showed anti-inflammatory and anti-arthritic activity and it compared with standard Diclofinac sodium. Chloroform, Ethyl acetate, Ethanol and Hydroalcohol fractions are having significant anti-inflammatory and antiarthritic activity may be due to the presence of alkaloids, Phytostrols, Flavonoids, saponins, Tannins, and Lignans. The future work will be the determination of Anti-oxidant, Anti-inflammatory and Anti arthritic activities by Invivo methods. REFERENCES Alderson WK, Cooper CE, Knowels RG, Nitric oxide systhesis, structure, function & inhibition, Journal of Biochemistry, 2001, 357:593-615. Anon K, Ethnobotany in search for new drugs. Ciba foundation symposium, John Wiley & sons, Newyork, 1994,188. Ayensu ES, World medicinal plant resourses. In conservation for productive agriculture (VL chopra and TN Khoshoo, edds) ICAR, 1996, New Delhi, India-11-42. Bakshu LM, Ram AJ, Raju RBV, Fitoterapia, Volme-72, No-8, Dec 2001, 930-9330 Chen Y, Zhen R, Jia Z, Flavonoids as super oxide scavengers and anti-oxidants, Free radical Bio Med, 9, 1990, 19-26. Cotton, 1996, The traditional approach makes use of material that has been found by trial and error over many years in different countries and systems of medicine Elizabeth K and Rao MNA, 1990, Oxygen radical scavenging activity of curcumin, Indian J Pharm, 58, 1990, 237.
  • 108. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) T K Gopal et.al. Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 378 Gahan, 1984, Plant Histo chemistry and phyto chemistry. An introduction academic press, Florida, U.S.A, 1984, 300. Kirtikar KR, Basu B.D, Indian medicinal plants, volm-5, 1918, 97-98. Murray CJL and Lopez AD, The global burden of Diseases.1996. Pratt DE, natural anti-oxidants from plant material, in phenolic compounds in food & their effects on health II: Anti-oxidants and cancer preservation (AVS symposium series 507) edited by M. Hang, (American chemical society, Washington DC), 1992, 54. Prieto P, Pineda M, Aguilar M. Spectrophotometric quantification of antioxidant capacity through the formation of a phosphomolybdenum complex: specific application of vitamin E. Anal. Biochem, 269, 1999, 337-341. Sundaresan V, De Britto AJ, Preliminary studies on some Phyto medicinal plants of thirunelveli hills. Journal of economic and taxonomic Botany 23, 1991, 377-38. Yohozawa T, Chen CP, Dong E, Tanaka T, Nonaka GT, Nishioka I, Studies on the inhibitory effect of Tannins & flavonoids against radical. Bio chemical pharmacology, 56, 1998, 212-222.
  • 109. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Sampath Kumar et.al. Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 379 TRANSDERMAL SONOPHORESIS TECHNIQUE- AN APPROACH FOR CONTROLLED DRUG DELIVERY K.P.Sampath Kumar1 *, Debjit Bhowmik2 , M.Komala2 1. Department of Pharmaceutical Sciences, Coimbatore medical college, 2. Karpagam University, Coimbatore *Corresponding author: Email:debjit_cr@yahoo.com ABSTRACT Sonophoresis (phonophoresis) has been shown to increase skin permeability to various low and high molecular weight drugs, including insulin and heparin. However, its therapeutic value is still being evaluated. Some obstacles in transdermal sonophoresis can be overcome by combination with other physical and chemical enhancement techniques. Use of ultrasound in therapeutics and drug delivery has gained importance in recent years, evident by the increase in patents filed and new commercial devices launched. The present review discusses new advancements in sonophoretic drug delivery in the last two decades, and highlights important challenges still to be met to make this technology of more use in the alleviation of diseases. new formulations tried in sonophoresis, synergistic effects with techniques such as chemical enhancers, iontophoresis and electroporation, as well as the growing use of ultrasound in areas such as cancer therapy, cardiovascular disorders, temporary modification of the blood-brain barrier for delivery of imaging and therapeutic agents, hormone replacement therapy, sports medicine, gene therapy and nanotechnology. Keywords: Sonophoresis, Permeability, higher frequency, Patient compliances. INTRODUCTION Sonophoresis is a process that exponentially increases the absorption of semisolid topical compounds (transdermal delivery) into the epidermis, dermis and skin appendages. Sonophoresis occurs because ultrasound waves stimulate micro-vibrations within the skin epidermis and increase the overall kinetic energy of molecules making up topical agents. It is widely used in hospitals to deliver drugs through the skin. Pharmacists compound the drugs by mixing them with a coupling agent (gel, cream, ointment) that transfers ultrasonic energy from the ultrasound transducer to the skin. The ultrasound probably enhances drug transport by cavitation, microstreaming and heating. Sonophoresis is also used without drug delivery in physical therapy, and as a complementary modality for iontophoresis. Application of ultrasound to the skin increases its permeability (sonophoresis) and enables the delivery of various substances into and through the skin. Ultrasound has been used extensively for medical diagnostics and to a certain extent in medical therapy.The generation of ultrasound and mechanism of sonophoresis with particular emphasis on the role of cavitation (both inside and outside the skin), thermal effects, convective transport, and mechanical effects also included. Sonophoresis is a localized, non-invasive, convenient and rapid method of delivering low molecular weight drugs as well as macromolecules into the skin. The ultrasound waves generate tiny bubbles of water on skin surface; this causes the skin surface to lightly get worn out. This allows the drug to pass through the skin surface efficiently. Sonophoresis occurs because ultrasound waves stimulate micro- vibrations within the skin epidermis and increase the overall kinetic energy of molecules making up topical agents. Sonophoresis, or ultrasound, creates holes in the skin, and allows fluids to travel into or out of the body. When sound is emitted at a particular frequency, the sound waves disrupt the lipid bilayers. This method can be used for delivery of steroids, systemic drugs such as Insulin and antigens for vaccination. Ultrasound transdermal drug delivery system in noninvasive way is used for Diabetics to control blood sugar level through short term and long term delivery of Insulin. Noninvasive drug delivery (as capsule formulation) is used for acne, psoriasis. These systems enhance activity of transdermal patches. The higher the frequency, the more dispersed the transmission. Advantages of Using Sonophoresis as a Physical Penetration Enhancer  Enhanced drug penetration (selected drugs) over passive transport.  Allows strict control of transdermal penetration rates.  Low risk of introducing infection as the skin remains intact  Reduction of dosing frequency and patient compliance
  • 110. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Sampath Kumar et.al. Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 380  Improved control of the concentrations of drugs with small therapeutic indices.  Reduction of fluctuations in plasma levels of drugs  Avoids hepatic first pass elimination and gastrointestinal irritation.  Substitute’s oral administration when the route is unsuitable as in case of vomiting, diarrhea.  Permit both local and systemic effects and less risk of systemic absorption than injection.  Easy termination of drug delivery in case of toxicity, through termination of ultrasound. Disadvantages of using sonophoresis as a physical penetration enhancer  Stratum corneum must be intact for effective drug penetration.  Can be time consuming to administer Applications of Sonophoresis: 1. Ultrasound helps in treatment of wide varieties of sports injuries such as tennis elbow, tendon problems, repairing damaged ligaments, muscle spasms, stiff joints, fractured bones and cartilage. Also used in healing of wounds, damaged skin, skin rejuvenation, nerve stimulation, and improving the strength and elasticity of scar tissues. 2. Ultrasound with Topical Anesthesia rapidly decreases pain of intravenous cannulation. 3. Low frequency ultrasonic gene delivery. 4. The dolphin therapy arouses a great interest in the whole world, since it causes analgesic effects, removal of depression, and improvement of learning abilities of the children suffering from autism. 5. Sonophoresis is also being used in drug enhancement in granulomas and tumors. 6. Sonophoresis is being investigated as a way of extracting compounds such as glucose. 7. In the treatment of sick fish by University of Maryland‘s Center of Marine Biotechnology. The current method uses intraperitoneal injections which are costly and highly labour intensive. In this experiment, ultrasound was applied to water containing fish and compound of interest. The ultrasound waves increases the permeability of compound into the tissues of the skin and gills. This method is highly cost and labour effective. 8. Sonophoresis also used in treatment of glaucoma, corneal infection and nail delivery, to Increase the permeability of drugs. Future Trends: Vaccination In recent years, the potential for exploiting the skin for purposes of vaccination has received a great deal of attention. One common strategy is to use an adjuvant, which is a compound used to enhance the immune response to vaccine compounds Ultrasound can be used to enhance skin permeability to both the adjuvant and the vaccine, and hence to facilitate their delivery to the target cells. Gene therapy Gene therapy is a technique for correcting defective genes that are responsible for disease development, most commonly by replacing an ‘abnormal’ disease-causing gene with the ‘normal’ gene The most obvious candidate diseases for cutaneous gene therapy are the severe forms of particular geno dermatoses (monogenic skin disorders), such as epidermolysis bullosa. Other applications might be healing of cutaneous wounds such as severe burns and skin wounds of diabetic origin. CONCLUSION: Sonophoresis is the enhancement of migration of drug molecules through the skin by ultrasonic energy Sonophoresis occurs because ultrasound waves stimulate micro-vibrations within the skin epidermis and increase the overall kinetic energy of molecules When sound is emitted at a particular frequency, the sound waves disrupt the lipid bilayers The higher the frequency, the more dispersed the transmission. REFERENCES Allen LV, Popovich NG, Ansel HC, Ansel’s pharmaceutical dosage forms and drug delivery systems. Transdermal drug delivery systems. 8th ed. India: Gopsons papers ltd.; 2006, 298-315. James Swarbrick, Transdermal Delivery: Sonophoresis, Encyclopedia of pharmaceutical technology, 3 rd edition, Volume-6, 2007, 3828-3842. Mitragotri S, Blankschtein D, and Langer R. Transdermal drug delivery using low- frequency sonophoresis. Pharmaceutical research, 13, 1996, 411-420. Mr. Ashish Pahade, Dr. Mrs. V.M.Jadhav, Dr. Mr. V.J.Kadam, Sonophoresis: an overview, International Journal of Pharmaceutical Science, 3(2), 2010, 24-32
  • 111. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Sampath Kumar et.al. Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 381 N.K.Jain, Sonophoresis: Biophysical of Transdermal Drug Delivery, Controlled and Novel Drug Delivery, 1 st edition, 1997, 208-235. Ogura M, Paliwal S, Mitragotri S, Low-frequency sonophoresis: Current status and future prospects. Advanced Drug Delivery Reviews, 60, 2008, 1218–1223. Pahade A, Jadhav VM, Kadam VJ, Sonophoresis: an overview. International Journal of Pharmaceutical Sciences Review and Research, 3, 2010, 24-32. Tang H, Wang CCJ, Blankschtein D, and Langer R, An Investigation of the Role of Cavitation in LowFrequency Ultrasound-Mediated Transdermal Drug Transport, Pharmaceutical Research, 19, 2002, 1160-1169. Terahara T, Mitragotri S, Kost J, Langer R, Dependence of low-frequency sonophoresis on ultrasound parameters; distance of the horn and intensity. International Journal of Pharmaceutics, 235, 2002, 35-42. Tezel A, Sens A, Tuchscherer J, and Mitragotri S. Frequency Dependence of Sonophoresis, Pharmaceutical Research, 18, 2001, 1694-1700.
  • 112. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Navin Dixit et.al. Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 382 A COMPREHENSIVE REVIEW OF ELADI VATI Navin Dixit*, Sheo Dutt Maurya , Bhanu P.S.Sagar I.E.C.College of Eng & Technology, Greater Noida (U.P) *Corresponding author: Email: ndixit515@gmail.com ABSTRACT Herbal medicines are the synthesis of therapeutic experiences of generations of practising physicians of indigenous systems of medicine for over hundreds of years while nutraceuticals are nutritionally or medicinally enhanced foods with health benefits of recent origin and marketed in developed countries. The marketing of the Herbal drugs under the category of the Neutraceutical is unethical. Herbal medicines are also in great demand in the developed world for primary health care because of their efficacy, safety and lesser side effects. They also offer therapeutics for age-related disorders like memory loss, osteoporosis, immune disorders, etc. for which no modern medicine is available. Eladi Vati is a pill containing cardamom, cinnamon, long pepper, dates, raisins and sugar. It treats bronchitis, cough, cold and other respiratory diseases. Keywords: Eladi vati , pill, Herbal medicines, respiratory diseases. INTRODUCTION India despite its rich traditional knowledge, heritage of herbal medicines and large biodiversity has a dismal share of the world market due to export of crude extracts and drugs. WHO too has not systematically evaluated traditional medicines despite the fact that it is used for primary health care by about 80% of the world population? HERBAL medicine is still the mainstay of about 75–80% of the world population, mainly in the developing countries, for primary health care because of better cultural acceptability, better compatibility with the human body and lesser side effects. They have stood the test of time for their safety, efficacy, cultural acceptability and lesser side effects. The chemical constituents present in them are a part of the physiological functions of living flora and hence they are believed to have better compatibility with the human body. A soothing remedy with Ela (Chhoti elaichi / Lesser cardamom), Mulethi, Misri, Munakka and other demulcents that is extremely useful in respiratory and gastric manifestations, such as dry cough, sore throat, nausea, excessive thirst and pittaja disorders. Indications: Eladi Vati is extremely useful in the following conditions  Sore throat, dry cough and cold.  Chronic bronchitis.  Hiccups, nausea and vomiting.  Loss of appetite, hyperacidity and gastritis.  Burning micturition and dysuria. Actions:  Pacifies aggravated pitta.  Soothes the throat.  Relieves excessive thirst. Vati & Gutika: Medicines prepared in the form of tablet or pills are known as Vati and Gutika. These are made of one or more drugs of plant, animal or mineral origin. It has simple composition, no side effect, and contraindication. It has been using since long time. Benefits:  Helps to relieve cough, cold, fever, hiccups, vomiting, dizziness, hematemesis (blood vomiting), and abdominal pain.  It relieves excessive thirst, spleen diseases and gout. Side Effects:  There are no known side effects of this medicine.  Over-dosage may cause slight burning sensation in abdomen.  It is better to avoid this tablet during pregnancy
  • 113. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Navin Dixit et.al. Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 383 MATERIALS USED IN FORMULATION OF ELADI VATI Table 1: Materials used in formulation of eladi vati Ingredients English Name Scientific Name Quantity Ela Cardamom Elettaria cardamomum 6 g Patra Cinnamon Cinnamomum tamala 6 g Twak Long pepper fruit Cinnamomum zeylanicum 6 g Pippali Piper longum 24 g Sita Sugar 48 g Madhuka Licorice Glycyrrhiza glabra 48 g Kharjura Dates Phoenix dactylifera 48 g Draksha Raisin Vitis vinifera 48 g Madhu Honey 48 g EVALUATION PARAMETER OF ELADI VATI Eladi Gutika (EG), a traditional Ayurvedic polyherbal formulation is used as a remedy for Kasa (Cough), Svasa (Asthma), Bhrama (Vertigo),Raktapitta (Bleedingdisorders), Jvara (Fever), Amvata (Rhe umatism) etc. In the present work, an attempt has been made to develop pharmacognostic standards for EG. The raw materials of EG were subjected to proximate analysis prior to preparation of EG. EG was prepared using raw materials of pharmacopoeial quality. Powder microscopy of EG showed the presence of discerning anatomical characters which were present in the raw materials. A simple, rapid, accurate and sensitive HPTLC method was developed and validated for the quantitation of piperine from EG. Method was validated as per ICH guidelines and applied for stability studies of EG stored for different storage periods at room temperature. A comparative evaluation of EG prepared in-house was carried out with three available marketed samples in terms of their respective piperine content. Acute toxicity study of EG in Albino Swiss mice revealed that it is safe at the dose of 2 g/kg body weight in animal. Evaluation of EG by these scientific methods can be used as quality control tool for the manufacturing and processing of EG. Eladi Gutika (EG), is an Ayurvedic polyherbal formulation of seven ingredients. Eladi Vati is an Ayurvedic tablet used in treating cough, cold, fever and vomiting. It is used mainly in respiratory and gastric conditions. Eladi Vati is a soothing remedy, which is prepared using various ingredients like Mulethi, Misri, Ela, Munakka and other demulcents. All these ingredients are useful for sore throat, nausea, dry cough, excessive thirst and pittaja disorders. It has simple composition, no side effect and contraindication. Piperine, a major alkaloid of Pippali (one of the ingredient of EG) is reported as a bioavailability enhancer, anti- inflammatory, anticonvulsant and antiulcer agent. There are reports on extraction of piperine using various extraction techniques and its estimation using analytical tools from single herbs and polyherbal formulations. But, there are no methods reported for its estimation from complex matrix of EG. CONCLUSION Eladi Vati is an Ayurvedic tablet used in treating cough, cold, fever and vomiting. It is used mainly in respiratory and gastric conditions. It helps to relieve cough, cold, fever, hiccups, vomiting, dizziness, haematemesis and abdominal pain. It relieves excessive thirst, spleen diseases and gout. It is a natural aphrodisiac and useful in all bleeding conditions. REFERENCES Ayurvedic Formulary of India (AFI), Part I. Government of India, Ministry of Health and Family Welfare. New Delhi: Department of Health, Controller of Publications Civil Lines; 1989, 181-182. Bajada S, Singla AK, Bedia KL, Liquid chromatographic method for determination of piperine in rat plasma: Application to pharmacokinetics, J Chromatogr B, 776, 2002, 245-9. Capasso R, Izzo AA, Borrelli F, Russo A, Sautebin L, Pinto A, Effect of piperine, the active ingredient of black pepper on intestinal secretion in mice, Life Sci, 71, 2002, 2311-7. Evans WC, Trease and Evans Pharmacognosy, 14 th ed, London: SW Saunders Company Ltd, 1996, 319. Hamrapurkar PD, Jadhav K, Zine S, Quantitative estimation of piperine in Piper nigrum and Piper longum using high performance thin layer chromatography, J Appl Pharm Sci, 1, 2011, 117-20.
  • 114. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Navin Dixit et.al. Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 384 Patel RK, Kanani RJ, Patel VR, Patel MG, Development and validation of HPTLC method for simultaneous quantification of vasicine and piperine in Vasavaleha, Int J Pharm Res, 2, 2010, 14-7 Shailajan S, Singh A, Tiwari B. Quality control and standardization of an ayurvedic Taila formulation. Int J Biomed Res Anal, 1, 2010, 78-81. Shailajan S, Yeragi M, Purohit A, Optimized separation and quantification of eugenol from a traditional Unani medicine Jawarish-e-Bisbasa using HPTLC, Int J Pharm Sci Rev Res, 9, 2011, 146-51. Shialajan S, Menon S, Polymarker based standardization of an ayurvedic formulation Lavangadi Vati using high performance thin layer chromatography, J Pharm Res, 4, 2011, 467-70. Tapadiya G, Metku M, Deokate U, Khadabadi S, Saboo S, Sahu K, Quantitative estimation of piperine from pharmaceutical dosage form by HPTLC, Asian J Pharm Cli Res, 2, 2009, 47-50.
  • 115. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Pulak Majumder and Susmita Majumder Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 385 PREPARATION AND CHARACTERIZATION OF SOME HERBAL OINTMENT FORMULATIONS WITH EVALUATION OF ANTIMICROBIAL PROPERTY *Pulak Majumder1 and Susmita Majumder2 1 Dept. of Pharmacognosy, Rajiv Gandhi Institute of Pharmacy, Trikaripur, Kasaragod Dist, Kerala, India, 2 Dept. of Biotechnology, Heritage Institute of Technology, Anandapur, Kolkata, India. *Corresponding author: Email: pulak2007@gmail.com ABSTRACT The Indian subcontinent is enriched by a variety of flora- both aromatic and medicinal plants. Herbal drugs constitute a major part in all the traditional systems of medicine. There are approximately 1250 Indian medicinal plants, which are used in formulating therapeutic preparation according to Ayurveda and other traditional system of medicine. This work has been an approach to formulate a modern Ayurvedic formulation with single and double drug combination. Both the formulations are found to be very efficacious in all the parameters which was conducted for its characterization and also found enhance antimicrobial property. This study may give a brief importance for modernization of Ayurvedic preparation and also the importance of some herbs which can be distinct within some time period. Key words: Peperomia pellucida, Cymbopogon citrate, Ointment, Antimicrobial property. INTRODUCTION According to survey report by WHO, about 25 per cent of prescribed human medicines are derived from plants and 80 per cent people still depend on traditional system of medicines. The herbal wealth of India and the knowledge of their medicinal properties have a long tradition, as referred in Rig veda and other ancient literature. The topography of India in the tropical belt with its varied climatic zones made it a vast storehouse of medicinal plants. The quality assessment of herbal formulations is of paramount importance in order to justify their acceptability in modern system of medicine. One of the major problems faced by the herbal industry is the unavailability of rigid quality control profiles for herbal materials and their formulations. Regulatory bodies have laid down the standardization procedures and specifications for Ayurvedic preparations. The World Health Organization (WHO) has appreciated the importance of medicinal plants for public health care in developing nations and has evolved guidelines to support the member states in their efforts to formulate national policies on traditional medicine and to study their potential usefulness including evaluation, safety, and efficacy (Organisation Mondiale De La Sante, 1992). Peperomia pellucida (Fig 1) is a commonly known as Shiny Bush or silver bush, fleshy tropical annual, shallow-rooted herb, usually growing to a height of about 15 to 45 cm. It is characterized by fibrous roots, succulent stems, shiny, heart-shaped, fleshy leaves and tiny, dot-like seeds attached to several fruiting spikes. It has a mustard-like odor when crushed. P. pellucida plant possesses various pharmacological properties like Analgesic (Aziba PI et al), Anti-inflammatory (De Fatima Arrigoni, 2004) antipyretic (Alam Khan, 2008), chemotherapeutic, broad spectrum antibiotic, refrigerant, anticancer (Arrigoni-Blank Mde F, 2002) and depressant of Central Nervous System (Khan, 2007) etc. There are popular descriptions of this plant to lower cholesterol levels or used on proteinuria and as diuretic. The Cymbopogon citrates (Fig 2) commonly known as lemongrass or Citroengrass, Fever Grass, a perennial plant with brawny stalks and somewhat broad and scented leaves. This species grows in thick bunches that often develop to a height of six feet (1.8 meters) and approximately four feet (1.2 meters) in breadth. The leaves of the plant are similar to straps and are 0.5 inch to 1 inch (1.3 cm to 2.5 cm) in width and around three feet (0.9 meter) in length, and possess stylish apexes. The plant bears leaves round the year and they are vivid bluish- green and when mashed they emit an aroma akin to lemons. Apart from the herb’s aromatic, ornamental and culinary uses, lemongrass also provides therapeutic benefits to cure grouchy conditions, nervous disorders, colds and weariness. The essential oils extracted from lemongrass have a yellow or yellowish-brown hue and this liquid is known to be antiseptic. Very often the oil is applied externally to treat disorders like athlete’s foot (tinea pedia). Lemongrass is also used as a carminative to emit digestive gas, a digestive tonic, a febrifuge, analgesic as well as an antifungal (Chiori, 1977), anti microbial (Kokate, 1971), astringent , aids in avoiding panic, melancholy, nervousness, rheumatism and sprains (Carbajal, 1989), suppress coughs, and as a diuretic and sedative (Carlini, 1986). The stalks and leaves of the lemongrass are widely used in culinary; In addition, Cymbopogon citratus is extensively used by the cosmetic industry in the manufacture of soaps as well as hair care products. In an earlier
  • 116. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Pulak Majumder and Susmita Majumder Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 386 study, medicinal plants which have been reported individually of its anti microbial activity along with different other pharmacological properties on different experimental studies, but there are no such kind of existing suitable pharmaceutical formulations available for the single or combinational form of those drugs. This attempt has been made to formulate and evaluate the pharmaceutical doses form i.e ointment by first time combining those herbals and also single drug ointment formulation. This approach may provide a suitable and more effective utilization of those pharmaceutically impotent herbs. MATERIALS AND METHODS Collection and Authentication of plant: Plants of Peperomia pellucida and Cymbopogon citrates were collected from the road sides of Trikaripur forest areas, Kasaragod district of Kerala, India, in the month of May 2011 in a quantity sufficient for all the experiments in a single batch and the plant materials were authenticated. One each voucher specimen was submitted and preserved in the Department of Pharmacognosy, Rajiv Gandhi Institute of Pharmacy, Trikaripur (Herbarium no. RGIP/10-12/003 & 004). The plant parts were washed under running tap water, cut into small pieces of 2-3cm and shade dried (300C, 50 ± 5% relative humidity) for 15days. The shade dried plant material was powdered using a dry grinder to get the coarse powder (sieve no. 10/44). The powder was stored in air tight container for further use. Fig 1: Peperomia pellucida Fig 2: Cymbopogon citrate Preparation of plant extract and isolation of oil: The powder of Peperomia pellucida was subjected solvent extraction. The powder material was refluxed with ethanol (90%) in a Soxhlet extractor for 18 hrs in batches of 50g each cycle. The marc was gently pressed and dried before completing the extracting. The extracts obtained by the above techniques were concentrated in vacuum under reduced pressure using a rotary flash evaporator. Fresh leaves of Cymbopogon citrates were subjected to Clevenger apparatus with sufficient quantity of water with glycerin and boiled up to complete extraction of oil from the plant part. Preliminary phytochemical screening: The preliminary phytochemical screening was carried out according to the recommended standard procedures (Kokate, 1997) (Wallis, 1985) (Khandalwal, 2008). Physical Evaluation of formulated ointments: Organoleptic parameters: Organoleptic parameters like color, odor and taste of all the formulations were carried out. Determination of pH: The pH value of a solution was determined by digital pH meter (Mettler Toledo). The pH meter was operated according the manufacturer’s instructions. First the apparatus was calibrated using buffer of 4, 7 and 9 pH. The electrodes were immersed in the solution and the pH was measured. Homogeneity:All the developed ointments were tested for homogeneity by visual inspection. They were tested for their appearance. Evaluation of Herbal Ointment and Polyherbal Ointment Ointment formulations: Two types of drug formulations (ointments) were prepared by using standard ointment base for standardization and evaluation microbial activity. For topical application 5g of each extract was separately incorporated with 100g of simple ointment IP. The formula for simple ointment I.P. is: S.No Ingredients Quantity (gm) 1 White bees wax 20 2 Hard paraffin 30 3 Cetosteryl alcohol 50 4 White soft paraffin 900
  • 117. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Pulak Majumder and Susmita Majumder Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 387 Consistency or hardness of an ointment (Pharmacopeial Forum): The consistency or hardness of an ointment was measured by Penetrometer. Prepare the test samples by one of the following procedures:  Carefully and completely fill three containers, without forming air bubbles. Level if necessary to obtain a flat surface. Store the samples at 25±0.5°C for 24 hrs, unless otherwise prescribed.  Store three samples at 25±0.5°C for 24hrs. Apply a suitable shear to the samples for 5 min. carefully and completely fill three containers, without forming air bubbles, and level if necessary to obtain a flat surface.  Melt three samples and carefully and completely fill three containers, without forming air bubbles. Store the samples at 25±0.5°C for 24hrs, unless otherwise prescribed. Place the test sample on the base of the Penetrometer. Verify that its surface is perpendicular to the vertical axis of the penetrating object. Bring the temperature of the penetrating object to 25±0.5°C and then adjust its position such that its tip just touches the surface of the sample. Release the penetrating object and hold it free for 5sec. Clamp the penetrating object and measured the depth of penetration. Repeat the procedure for remaining two containers. Spreadability studies of ointment (Wood, 1963): Spreadability of the formulation was determined by an apparatus suggested by Muttimer et al., which was suitably modified in the laboratory and used for the study. It consists of a wooden block, which was provided by a pulley at one end. A rectangular ground glass plate was fixed on this block. An excess of ointment (about 3 gm.) under study was placed on this ground plate. The ointment was then sandwiched between this plate and another glass plate having the dimension of fixed ground plate and provided with the hook. A 1 Kg. weight was placed on the top of the two plates for 5 minutes to expel air and to provide a uniform film of the ointment between the plates. Excess of the ointment was scrapped off from the edges. The top plate was then subjected to pull of 80 gms. With the help of string attached to the hook and the time (in seconds) required by the top plate to cover a distance of 10 cm. be noted. A shorter interval indicates better Spreadability. Spreadability is measured as S = M × L /T M= weight tide to upper slide L= length of glass slides T= Time Tube Extrudability studies of ointment: It is a usual empirical test to measure the force required to extrude the material from tube. The method applied for determination of applied shear in the region of the rheogram corresponding to a shear rate exceeding the yield value and exhibiting consequent plug flow one such apparatus is described by Wood et al. In the present study, the method adopted for evaluating ointment formulation for extrudability was based upon the quantity in percentage of ointment and ointment extruded from tube on application of finger pressure. More quantity extruded better was extrudability. The formulation under study was filled in a clean, lacquered aluminum collapsible tube with a nozzle tip of 5 mm opening and applies the pressure on tube by the help of finger. Tube extrudability was then determined by measuring the amount of ointment extruded through the tip when a pressure was applied on tube. PHARMACOLOGICAL STUDIES Topical sensitivity Test: Both two ointments (Herbal and Polyherbal) were tested for their skin sensitivity tests by applying to the elbow of the hand in selected human volunteers and observed the side effects if any, as a set of parameters like skin inflammation, skin irritation reddening of skin (allergic reactions) etc. Microbiological studies (Bauer, 1966): The antibacterial activity of various ointment formulations evaluated against various strain of anaerobic and aerobic microorganism by standard cup plate method and the inhibition zone diameters were measured with the help of zone reader. Mucus fungus, Lactobacillus, Escherichia coli (aerobic organism) were used for testing of antibacterial activity. Nutrient agar media was used for aerobic bacterial culture and incubated at temperature 37°C±2°C for 48 hrs. RESULTS AND DISCUSSION The various physicochemical parameters utilized to evaluate the prepared ointment formulations are shown in table 2.
  • 118. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Pulak Majumder and Susmita Majumder Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 388 Preparation of Ointments: Herbal ointment was prepared by incorporating 5% of Cymbopogon citrates oil in simple ointment base (Fig. 3). Polyherbal ointment was prepared by equal amount of (5%) both the drug Peperomia pellucida plant extract and Cymbopogon citrates, oil incorporated in simple ointment base (Fig.3). Fig.3. Herbal and polyherbal ointment formulation Preliminary Phytochemical Investigation: The isolation, purification and identification of active constituents are chemical methods of evaluation. Qualitative chemical tests are also included under chemical evaluation. Preliminary phytochemical screening which is performed to establish a chemical profile of a crude drug is a part of chemical evaluation. All the three different plant parts extracts were screened for phytochemical investigation by different phytochemical tests to check the presence or absence of a group of phytochemical constituents. These phytochemical tests shown the presence of carbohydrates, alkaloids, tannins, steroids, triterpenoids, flavonoids etc. are present in stem extract whereas in root extract contains all above except flavonoids as mentioned in Table 1. Leaf extract were also found to contain all the constituents including saponins. Table 1: Preliminary phytochemical analysis of various parts of Peperomia pellucida. Chemical Constituents Root Stem leaf Carbohydrates + - - Proteins - - - Alkaloids +++ ++ + Saponins - - + Tannins ++ ++ +++ Flavonoids ++ ++ +++ Steroids + + + Triterpenoids + + + Glycoside +++ ++ ++ + = Present, - = Absent. Table-2 Physical Evaluation of formulated ointments Evaluation parameters Herbal ointment Polyherbal ointment Description Colour yellow Dark brown Odour Aromatic Characteristic Consistency Soft semisolid Soft semisolid Phase separation Nil Nil pH (10℅w/v solution) 6.2 6.5 Hardness or consistency 149mm 198mm Spreadability 47.2g/s 24.6g/s The pH of the formulations lies in the normal pH range of human skin (6.8 ± 1). All the formulations did not produce any skin irritation, i.e , erythema and edema for about a week when applied over the skin. These formulations did not produce any skin irritation for about a week when applied over the skin.
  • 119. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Pulak Majumder and Susmita Majumder Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 389 Fig 4: Spread ability of ointment From the data it is clearly evident all the physicochemical characteristics of both the formulation were found satisfactory and there were no significance changes in evaluation parameters. The formulation (polyherbal ointment) was found to be more satisfactory than Herbal ointment. Spreadability results (graph-1) of both the ointments found satisfactory. The antimicrobial activity of polyherbal ointment and Herbal ointment were determined (Fig. 4). The zones of inhibition of various strains of aerobic and anaerobic organisms are depicted in table 4. The zone of inhibition for both the ointment (after one month) found to be nearer to standard Gentamycin. Table 3. Antimicrobial activity of Herbal Ointment and Polyherbal Ointment Diameter of Zone of inhibition (cm) Ointment E. coli Mucus fungus Lactobacillus Herbal Ointment 1.25± 0.2 1.50±0.12 1.11±0.32 Polyherbal Ointment 1.85±0.42 1.93±0.31 1.71±0.13 Standard(Gentamycin) 2.21±0.23 2.00±0.11 2.11±0.23 Control 0.166±0.11 0.41±0.23 0.08±22 E. coli Mucus fungus Lactobacillus Fig. 5. Antimicrobial activity of Herbal and Polyherbal Ointment formulations CONCLUSION A combinational therapy is the need of hour to treat eczema and pruritis. This can be achieved by Clotrimazole and Ichthammol (an antifungal and antiseptic).In this study, ointment was formulated with different bases like white soft paraffin, cetostearyl alcohol, hard paraffin, and light liquid paraffin. By combining these drugs with appropriate ointment bases (as polyherbal formulation) a better therapy and patient compliance can be achieved. Both the formulations may shows the batter way to use these drugs with more significant way and more over WHO also emphasizes the herbal medicine for treatment. This study showed the convenient preparation and more effective use of both the herbs with modernized formulations with comparatively old form of medicine. This affords may leads a beginning of proper utilization and conservation of medicinally important herbs and cost effective treatment in future.
  • 120. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Pulak Majumder and Susmita Majumder Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 390 ACKNOWLEDGEMENT Authors are thankful to the principal, Rajiv Gandhi Institute of Pharmacy, Trikaripur and Heritage Institute of Technology, Anandapur for their necessary facilities and constant support to successfully completion of this research work. REFERENCES Alam Khan, Moizur Rahman, Shariful Islam Antipyretic Activity of Peperomia pellucida Leaves in Rabbit, Turk J Biol, 32, 2008, 37-41. Arrigoni-Blank Mde F ,Oliveira RL, Mendes SS, et al. Seed germination, phenology, and anti-dematogenic activity of Peperomia pellucida (L.), HBK BMC Pharmacol, 2, 2002, 12-19. Aziba PI, Adedeji A, Ekor M, Analgesic activity of Peperomia pellucida aerial parts in mice, Fitoterapia, 72, 2001, 57-58. Bauer AW, Kirby WM, Sherris JC and Truck M, Antibiotic susceptibility testing by a standardized single disc method, Am J Clin pathol, 45(4), 1966, 493-496. C. K. Kokate, Practical Pharmacognosy, 4th edition, Vallabh prakashan, 53, 1997, 123-124,127. Carbajal D, A Casaco, L Arruzazabala, R Gonzalez, and Z Tolon, Pharmacological study of Cymbopogon citratus leaves, J. Ethnopharmaco, 25, 1989, 103-107. Carlini EA, DP Contar, AR Silva-Filho, DA Silveria-Filho, ML Frochtengarten and FA Bueno, Pharmacology of lemongrass (Cymbopogon citratus stapf) I. Effects of teas prepared from the leaves on laboratory animals, J. Ethnopharmaco, 17, 1986, 37-64 Chiori CO, HN Ezeiruaku, and FA Ogadi, A study of the antiseptic properties of the oils from the fresh leaves of Ocimum viride and Cymbopogon citrates, J. Pharm. Sciences, 1, 1977, 267-270. De Fatima Arrigoni, Blank M, Dmitrieva EG, Franzotti EM, Antoniolli AR , Andrade MR, Marchioro M, Anti- inflammatory and analgesic activity of Peperomia pllucida (L.) HBK (Piperaceae). J Ethnophrmacol, 91, 2004, 215-218. Khan A, Mosaddik MA, Rahman MM, Rahman MM, Haque ME, Jahan SS, Islam MS, Hasan S. Neuropharmacological effects of Laportea crenulata Roots in mice, J Appl Sci Res, 3, 2007, 601-606. Khandalwal KR, Practical Pharmacognosy – Techniques and experiments, Nirali Prakashan, 19th edition, 2008, 149-156. Kokate CK, and KC Verma, A note on the antimicrobial activity of volatile oils of Cymbopogon nardus (Linn.) Rendle and Cymbopogon citrates (Stapf.), Science Culture, 37, 1971, 196-198. Kulyal P, Tiwari, UK, Shukla A, Gaur AK, Indian J. of Chem, 49, 2010, 356-359. Organisation Mondiale De La Sante, Quality control methods for medicinal plant materials, 559, rev.1, Original English, World Health Organisation; 1992, P. 159 Wallis TE, Text book of Pharmacognosy, (CBS Publisher and Distributor, Delhi), 1985, 104-114. Wood J. H., Catacalos G. and Liberman S. V, Adaptation of commercial viscometers for special applications in pharmaceutical rheology-II, J. Pharm. Sci, 1963, 52, 375-378.
  • 121. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Shyam Bihari Sharma et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 391 THE EFFECTS OF AIR POLLUTION ON THE ENVIRONMENT AND HUMAN HEALTH Shyam Bihari Sharma1 *, Suman Jain1 , Praveen Khirwadkar2 , Sunisha Kulkarni1 1. School of Studies in Pharmaceutical Sciences, Jiwaji University, Gwalior, Madhya Pradesh, INDIA 2. Institute of pharmacy, Vikarm University, Ujjain (M.P) *Corresponding Author: shyam_mpharm06@rediffmail.com ABSTRACT Pollution is the introduction of contaminants into the natural environment that cause adverse change. Air pollution includes anything introduced by humans into the atmosphere with a damaging effect. The main cause of air pollution is the burning of fossil fuels in cars, in planes and for the production of electricity. Air pollution occurs when any chemicals or biological matter that can harm humans or other living things is introduced into the atmosphere. Pollutants in the air include carbon dioxide, carbon monoxide, sulphur dioxide and small particles that are a result of burning different materials, especially coal. These pollutants not only harm individuals by causing disease, but also harm the environment by adding to global warming. Pollution affects all humans, children more so than adults because children spend more time outside and take in more air, and air pollution, when breathing, especially when exerting them. The main air pollutants affect the lungs most. Sulphur dioxide irritates eyes, nose and throat. When inhaled, cause severe lung problems, such as asthma, bronchitis, emphysema and lung cancer. Nitrogen dioxide damages lung tissue and can restrict airways and cause emphysema. It also leads to formation of ozone, which can eat holes in lung tissue, aggravate asthma and leave people susceptible to respiratory disease. Carbon monoxide, which is invisible and odourless, can lead to damage of the heart and central nervous system, headaches, dizziness, convulsions and death. Air pollution affects the whole earth ecosystem. Global warming, caused by carbon dioxide building up in the atmosphere, traps heat in the earth's atmosphere, which can cause dramatic climate changes that shift the delicate balance of ecosystems around the world. A number of chemicals that have been released into the air, such as chlorofluorocarbons, have caused depletion of the protective ozone layer, which causes harmful ultraviolet radiation to reach the earth. Air pollution may be prevented only if individuals and businesses stop using toxic substances that cause air pollution in the first place. This would require the cessation of all fossil fuel-burning processes, from industrial manufacturing to home use of air conditioners. Keywords: Pollution, Air pollution, Pollutants, Atmosphere, Carbon dioxide, Global warming. INTRODUCTION Air pollution is the introduction into the atmosphere of chemicals, particulates, or biological materials that cause discomfort, disease, or death to humans, damage other living organisms such as food crops, or damage the natural environment or environment. The atmosphere is a complex dynamic natural gaseous system that is essential to support life on planet Earth. Stratosphericozone depletion due to air pollution has long been recognized as a threat to human health as well as to the Earth's ecosystems. Indoor air pollution and urban air quality are listed as two of the World’s Worst Toxic Pollution Problems in the 2008 Blacksmith Institute World's Worst Polluted Places report. Air pollution, contamination of the air by noxious gases and minute particles of solid and liquid matter (particulates) in concentrations that endanger health. The major sources of air pollution are transportation engines, power and heat generation, industrial processes, and the burning of solid waste. The combustion of gasoline and other hydrocarbon fuels in automobiles, trucks, and jet airplanes produces several primary pollutants: nitrogen oxides, gaseous hydrocarbons, and carbon monoxide, as well as large quantities of particulates, chiefly lead. In the presence of sunlight, nitrogen oxides combine with hydrocarbons to form a secondary class of pollutants, the photochemical oxidants, among them ozone and the eye-stinging peroxyacetylnitrate (PAN). Nitrogen oxides also react with oxygen in the air to form nitrogen dioxide, a foul-smelling brown gas. In urban areas like Los Angeles where transportation is the main cause of air pollution, nitrogen dioxide tints the air, blending with other contaminants and the atmospheric water vapor to produce brown smog. Although the use of catalytic converters has reduced smog-producing compounds in motor vehicle exhaust emissions, studies have shown that in so doing the converters produce nitrous oxide, which contributes substantially to global warming. Pollutants: A substance in the air that can be harmful to humans and the environment is known as an air pollutant. Pollutants can be in the form of solid particles, liquid droplets, or gases. In addition, they may be
  • 122. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Shyam Bihari Sharma et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 392 natural or man-made. Pollutants can be classified as primary or secondary. Usually, primary pollutants are directly emitted from a process, such as ash from a volcanic eruption, the carbon monoxide gas from a motor vehicle exhaust or sulphur dioxide released from factories. Secondary pollutants are not emitted directly. Rather, they form in the air when primary pollutants react or interact. An important example of a secondary pollutant is ground level ozone — one of the many secondary pollutants that make up photochemical smog. Some pollutants may be both primary and secondary: that is, they are both emitted directly and formed from other primary pollutants. Major primary pollutants produced by human activity include Sulphur oxides (SOx) - especially sulphur dioxide, a chemical compound with the formula SO2. SO2 is produced by volcanoes and in various industrial processes. Since coal and petroleum often contain sulphur compounds, their combustion generates sulfur dioxide. Further oxidation of SO2, usually in the presence of a catalyst such as NO2, forms H2SO4, and thus acid rain.This is one of the causes for concern over the environmental impact of the use of these fuels as power sources. Nitrogen oxides (NOx) - especially nitrogen dioxide are emitted from high temperature combustion, and are also produced naturally during thunderstorms by electric discharge. Can be seen as the brown haze dome above or plume downwind of cities. Nitrogen dioxide is the chemical compound with the formula NO2. It is one of the several nitrogen oxides. This reddish-brown toxic gas has a characteristic sharp, biting odor. NO2 is one of the most prominent air pollutants. Carbon monoxide (CO)- is a colourless, odorless, non-irritating but very poisonous gas. It is a product by incomplete combustion of fuel such as natural gas, coal or wood. Vehicular exhaust is a major source of carbon monoxide. Volatile organic compounds - VOCs are an important outdoor air pollutant. In this field they are often divided into the separate categories of methane (CH4) and non-methane (NMVOCs). Methane is an extremely efficient greenhouse gas which contributes to enhancedglobal warming. Other hydrocarbon VOCs are also significant greenhouse gases via their role in creating ozone and in prolonging the life of methane in the atmosphere, although the effect varies depending on local air quality. Within the NMVOCs, the aromatic compounds benzene, toluene and xylene are suspected carcinogens and may lead to leukemia through prolonged exposure. 1,3-butadiene is another dangerous compound which is often associated with industrial uses. Particulates, alternatively referred to as particulate matter (PM), atmospheric particulate matter, or fine particles, are tiny particles of solid or liquid suspended in a gas. In contrast, aerosol refers to particles and the gas together. Sources of particulates can be man made or natural. Some particulates occur naturally, originating from volcanoes, dust storms, forest and grassland fires, living vegetation, and sea spray. Human activities, such as the burning of fossil fuels in vehicles, power plants and various industrial processes also generate significant amounts of aerosols. Averaged over the globe, anthropogenic aerosols—those made by human activities—currently account for about 10 percent of the total amount of aerosols in our atmosphere. Increased levels of fine particles in the air are linked to health hazards such as heart disease,altered lung function and lung cancer. Persistent free radicals connected to airborne fine particles could cause cardiopulmonary disease. Toxic metals, such as lead and quick silver, especially their compounds: Chlorofluorocarbons (CFCs) - harmful to the ozone layer emitted from products currently banned from use. Ammonia (NH3) - emitted from agricultural processes. Ammonia is a compound with the formula NH3. It is normally encountered as a gas with a characteristic pungent odor. Ammonia contributes significantly to the nutritional needs of terrestrial organisms by serving as a precursor to foodstuffs and fertilizers. Ammonia, either directly or indirectly, is also a building block for the synthesis of many pharmaceuticals. Although in wide use, ammonia is both caustic and hazardous. Secondary pollutants include: Particulates created from gaseous primary pollutants and compounds in photochemical smog. Smog is a kind of air pollution; the word "smog" is a portmanteau of smoke and fog. Classic smog results from large amounts of coal burning in an area caused by a mixture of smoke and sulfur dioxide. Modern smog does not usually come from coal but from vehicular and industrial emissions that are acted on in the atmosphere by ultraviolet light from the sun to form secondary pollutants that also combine with the primary emissions to form photochemical smog. Ground level ozone (O3) formed from NOx and VOCs. Ozone (O3) is a key constituent of the troposphere. It is also an important constituent of certain regions of the stratosphere commonly known as the Ozone layer. Photochemical and chemical reactions involving it drive many of the chemical processes that
  • 123. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Shyam Bihari Sharma et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 393 occur in the atmosphere by day and by night. At abnormally high concentrations brought about by human activities (largely the combustion of fossil fuel), it is a pollutant, and a constituent of smog. Peroxyacetyl nitrate (PAN) - similarly formed from NOx and VOCs. Minor air pollutants include: A large number of minor hazardous air pollutants. Some of these are regulated in USA under the Clean Air Act and in Europe under the Air Framework Directive. A variety of persistent organic pollutants, which can attach to particulates: Persistent organic pollutants (POPs) are organic compounds that are resistant to environmental degradation through chemical, biological, and photolytic processes. Because of this, they have been observed to persist in the environment, to be capable of long-range transport, bioaccumulate in human and animal tissue, biomagnify in food chains, and to have potential significant impacts on human health and the environment. Anthropogenic sources (human activity) mostly related to burning different kinds of fuel "Stationary Sources" include smoke stacks of power plants, manufacturing facilities (factories) and waste incinerators, as well as furnaces and other types of fuel-burning heating devices. In developing and poor countries, traditional biomass burning is the major source of air pollutants; traditional biomass includes wood, crop waste and dung. "Mobile Sources" include motor vehicles, marine vessels, aircraft and the effect of sound etc. Chemicals, dust and controlled burn practices in agriculture and forestry management. Controlled or prescribed burning is a technique sometimes used in forest management, farming, prairie restoration or greenhouse gas abatement. Fire is a natural part of both forest and grassland ecology and controlled fire can be a tool for foresters. Controlled burning stimulates the germination of some desirable forest trees, thus renewing the forest. Fumes from paint, hair spray, varnish, aerosol sprays and other solvents Waste deposition in landfills, which generate methane. Methane is highly flammable and may form explosive mixtures with air. Methane is also an asphyxiant and may displace oxygen in an enclosed space. Asphyxia or suffocation may result if the oxygen concentration is reduced to below 19.5% by displacement. Military, such as nuclear weapons, toxic gases, germ warfare and rocketry Natural sources Dust from natural sources, usually large areas of land with little or no vegetation Methane, emitted by the digestion of food by animals, for example cattle Radon gas from radioactive decay within the Earth's crust. Radon is a colorless, odorless, naturally occurring, radioactive noble gas that is formed from the decay of radium. It is considered to be a health hazard. Radon gas from natural sources can accumulate in buildings, especially in confined areas such as the basement and it is the second most frequent cause of lung cancer, after cigarette smoking. 1. Smoke and carbon monoxide from wildfires 2. Vegetation, in some regions, emits environmentally significant amounts of VOCs on warmer days. These VOCs react with primary anthropogenic pollutants—specifically, NOx, SO2, and anthropogenic organic carbon compounds—to produce a seasonal haze of secondary pollutants. 3. Volcanic activity, which produce sulfur, chlorine, and ash particulates Air pollutant emission factors are representative values that people attempt to relate the quantity of a pollutant released to the ambient air with an activity associated with the release of that pollutant. These factors are usually expressed as the weight of pollutant divided by a unit weight, volume, distance, or duration of the activity emitting the pollutant (e.g., kilograms of particulate emitted per tonne of coal burned). Such factors facilitate estimation of emissions from various sources of air pollution. In most cases, these factors are simply averages of all available data of acceptable quality, and are generally assumed to be representative of long- term averages. There are 12 compounds in the list of POPs. Dioxins and furans are two of them and are intentionally created by combustion of organics, like open burning of plastics. The POPs are also endocrine disruptor and can mutate the human genes. The United States Environmental Protection Agency has published a compilation of air pollutant emission factors for a multitude of industrial sources. The United Kingdom, Australia, Canada and many other countries have published similar compilations, as well as the European Environment Agency. Health effects: Air pollution is a significant risk factor for multiple health conditions including respiratory infections, heart disease, and lung cancer, according to the WHO. The health effects caused by air pollution may include difficulty in breathing, wheezing, coughing and aggravation of existing respiratory and cardiac conditions. These effects can result in increased medication use, increased doctor or emergency room visits, more hospital admissions and premature death. The human health effects of poor air quality are far reaching,
  • 124. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Shyam Bihari Sharma et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 394 but principally affect the body's respiratory system and the cardiovascular system. Individual reactions to air pollutants depend on the type of pollutant a person is exposed to, the degree of exposure, the individual's health status and genetics. The most common sources of air pollution include particulates, ozone, nitrogen dioxide, and sulfur dioxide. Both indoor and outdoor air pollution have caused approximately 3.3 million deaths worldwide. Children aged less than five years that live in developing countries are the most vulnerable population in terms of total deaths attributable to indoor and outdoor air pollution. The World Health Organization states that 2.4 million people die each year from causes directly attributable to air pollution, with 1.5 million of these deaths attributable to indoor air pollution. "Epidemiological studies suggest that more than 500,000 Americans die each year from cardiopulmonary disease linked to breathing fine particle air pollution. . ." A study by the University of Birmingham has shown a strong correlation between pneumonia related deaths and air pollution from motor vehicles. Worldwide more deaths per year are linked to air pollution than to automobile accidents. A 2005 study by the European Commission calculated that air pollution reduces life expectancy by an average of almost nine months across the European Union. Causes of deaths include aggravated asthma, emphysema, lung and heart diseases, and respiratory allergies. The US EPA estimates that a proposed set of changes in diesel engine technology could result in 12,000 fewer premature mortalities, 15,000 fewer heart attacks, 6,000 fewer emergency room visits by children with asthma, and 8,900 fewer respiratory-related hospital admissions each year in the United States. The US EPA estimates allowing a ground-level ozone concentration of 65 parts per billion, would avert 1,700 to 5,100 premature deaths nationwide in 2020 compared with the current 75-ppb standard. The agency projects the stricter standard would also prevent an additional 26,000 cases of aggravated asthma and more than a million cases of missed work or school. The worst short term civilian pollution crisis in India was the 1984 Bhopal Disaster. Leaked industrial vapours from the Union Carbide factory, belonging to Union Carbide, Inc., U.S.A., killed more than 25,000 people outright and injured anywhere from 150,000 to 600,000. The United Kingdom suffered its worst air pollution event when the December 4 Great Smog of 1952 formed over London. In six days more than 4,000 died, and 8,000 more died within the following months. An accidental leak of anthrax spores from a biological warfare laboratory in the former USSR in 1979 near Sverdlovsk is believed to have been the cause of hundreds of civilian deaths. The worst single incident of air pollution to occur in the US occurred in Donora, Pennsylvania in late October, 1948, when 20 people died and over 7,000 were injured. A new economic study of the health impacts and associated costs of air pollution in the Los Angeles Basin and San Joaquin Valley of Southern California shows that more than 3800 people die prematurely (approximately 14 years earlier than normal) each year because air pollution levels violate federal standards. The number of annual premature deaths is considerably higher than the fatalities related to auto collisions in the same area, which average fewer than 2,000 per year. Diesel exhaust (DE) is a major contributor to combustion derived particulate matter air pollution. In several human experimental studies, using a well validated exposure chamber setup, DE has been linked to acute vascular dysfunction and increased thrombus formation. This serves as a plausible mechanistic link between the previously described association between particulates air pollution and increased cardiovascular morbidity and mortality. Effects on cardiovascular health: A 2007 review of evidence found ambient air pollution exposure is a risk factor correlating with increased total mortality from cardiovascular events (range: 12% to 14% per a 10 microg/m3 increase). Air pollution is also emerging as a risk factor for stroke, particularly in developing countries where pollutant levels are highest. A 2007 study found that in women air pollution is associated not with hemorrhagic but with ischemic stroke. Air pollution was also found to be associated with increased incidence and mortality from coronary stroke in a cohort study in 2011. Effects on cystic fibrosis Main article: Cystic fibrosis A study from around the years of 1999 to 2000, by the University of Washington, showed that patients near and around particulates air pollution had an increased risk of pulmonary exacerbations and decrease in lung function. Patients were examined before the study for amounts of specific pollutants like Pseudomonas aeruginosa or Burkholderiacenocepacia as well as their socioeconomic standing. Participants involved in the study were located in the United States in close proximity to an Environmental Protection Agency. During the time of the study 117 deaths were associated with air pollution. Many patients in the study lived in or near large metropolitan areas in order to be close to medical help. These same patients had higher level of
  • 125. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Shyam Bihari Sharma et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 395 pollutants found in their system because of more emissions in larger cities. As cystic fibrosis patients already suffer from decreased lung function, everyday pollutants such as smoke, emissions from automobiles, tobacco smoke and improper use of indoor heating devices could further compromise lung function. Effects on COPD and asthma Main article: Chronic obstructive pulmonary disease Chronic obstructive pulmonary disease (COPD) includes diseases such as chronic bronchitis and emphysema. Researches have demonstrated increased risk of developing asthma and COPD from increased exposure to traffic-related air pollution. Additionally, air pollution has been associated with increased hosptializations and mortality from asthma and COPD. A study conducted in 1960-1961 in the wake of the Great Smog of 1952 compared 293 London residents with 477 residents of Gloucester, Peterborough, and Norwich, three towns with low reported death rates from chronic bronchitis. All subjects were male postal truck drivers aged 40 to 59. Compared to the subjects from the outlying towns, the London subjects exhibited more severe respiratory symptoms (including cough, phlegm, and dyspnea), reduced lung function (FEV1 and peak flow rate), and increased sputum production and purulence. The differences were more pronounced for subjects aged 50 to 59. The study controlled for age and smoking habits, so concluded that air pollution was the most likely cause of the observed differences. It is believed that much like cystic fibrosis, by living in a more urban environment serious health hazards become more apparent. Studies have shown that in urban areas patients suffer mucushypersecretion, lower levels of lung function, and more self diagnosis of chronic bronchitis and emphysema. Links to cancer A review of evidence regarding whether ambient air pollution exposure is a risk factor for cancer in 2007 found solid data to conclude that long-term exposure to PM2.5 (fine particulates) increases the overall risk of nonaccidental mortality by 6% per a 10 microg/m3 increase.PMID 19235364 Exposure to PM2.5 was also associated with an increased risk of mortality from lung cancer (range: 15% to 21% per a 10 microg/m3 increase) and total cardiovascular mortality (range: 12% to 14% per a 10 microg/m3 increase). PMID 19235364 The review further noted that living close to busy traffic appears to be associated with elevated risks of these three outcomes (increase in lung cancer deaths, cardiovascular deaths, and overall nonaccidental deaths. PMID 19235364 The reviewers also found suggestive evidence that exposure to PM2.5 is positively associated with mortality from coronary heart diseases and exposure to SO2 increases mortality from lung cancer, but the data was insufficient to provide solid conclusions. In 2011, a large Danish epidemiological study found an increased risk of lung cancer for patients who lived in areas with high nitrogen oxide concentrations. In this study, the association was higher for non- smokers than smokers. An additional Danish study, also in 2011, likewise noted evidence of possible associations between air pollution and other forms of cancer, including cervical cancer and brain cancer. Effects on children Around the world, children living in cities with high exposure to air pollutants are at increased risk of developing asthma, pneumonia and other lower respiratory infections. Because children are outdoors more and have higher minute ventilation they are more susceptible to the dangers of air pollution. Risks of low initial birth weight are also heightened in such cities. The World Health Organization reports that the greatest concentrations of particulates are found in countries with low economic world power and high poverty and population growth rates. Examples of these countries include Egypt, Sudan, Mongolia, and Indonesia. However even in the United States, despite the passage of the Clean Air Act in 1970, in 2002 at least 146 million Americans were living in non-attainment areas—regions in which the concentration of certain air pollutants exceeded federal standards. These dangerous pollutants are known as the criteria pollutants, and include ozone, particulates, sulfur dioxide, nitrogen dioxide, carbon monoxide, and lead. Protective measures to ensure children's health are being taken in cities such as New Delhi, India where buses now use compressed natural gas to help eliminate the "pea-soup" smog. References Davis, Devra. When Smoke Ran Like Water: Tales of Environmental Deception and the Battle Against Pollution. Basic Books. ISBN 0-465-01521-2,2002. Grossni, Mark. "Human cost of valley's dirty air: $6.3 billion". Sacramento Bee. 2008.
  • 126. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Shyam Bihari Sharma et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 396 Sahagun, Louis "Pollution saps state's economy, study says". Los Angeles Times.2008 Kay, Jane. "Bad air costing state's economy billions". San Francisco Chronicle. 2008 Farrah J. Mateen& Robert D. Brook "Air pollution as an emerging global risk factor for stroke" JAMA, 305(12) ,2011,1240-1. Miller K. A., Siscovick D. S., Sheppard L., Shepherd K., Sullivan J. H., Anderson G. L., Kaufman J. D. "Long-term exposure to air pollution and incidence of cardiovascular events in women.".The New England journal of medicine, 356 (5), 2007, 447–458. Christopher H. Goss, Stacey A. Newsom, Jonathan S. Schildcrout, Lianne Sheppard and Joel D. Kaufman "Effect of Ambient Air Pollution on Pulmonary Exacerbations and Lung Function in Cystic Fibrosis". American Journal of Respiratory and Critical Care Medicine, 169 (7), 2004, 816–821. Zoidis, John D. "The Impact of Air Pollution on COPD". RT: for Decision Makers in Respiratory Care, 1999. Gehring, U., Wijga, A. H., Brauer, M., Fischer, P., de Jongste, J. C., Kerkhof, M., Brunekreef, B.Traffic- related air pollution and the development of asthma and allergies during the first 8 years of life.American journal of respiratory and critical care medicine, 181(6), 2010, 596-603. Andersen, Z. J., Hvidberg, M., Jensen, S. S., Ketzel, M., Loft, S., Sorensen, M., Raaschou-Nielsen, O. Chronic obstructive pulmonary disease and long-term exposure to traffic-related air pollution: a cohort study.American journal of respiratory and critical care medicine, 183(4), 2011, 455-461. Committee of the Environmental and Occupational Health Assembly of the American Thoracic Society, Health effects of outdoor air pollution. American journal of respiratory and critical care medicine, 153(1), 1996, 3-50. J. Sunyer, "Urban air pollution and Chronic Obstructive Pulmonary disease: a review". European Respiratory Journal, 17(5), 2001, 1024–1033. Raaschou-Nielsen, O, Andersen ZJ, Hvidberg M, Jensen SS, Ketzel M, Sorensen M, Tjonneland A, Lung cancer incidence and long-term exposure to air pollution from traffic, Environmental health perspectives, 119(6), 2011, 860-865. Raaschou-Nielsen, O., Andersen, Z. J., Hvidberg, M., Jensen, S. S., Ketzel, M., Sorensen, M., Tjonneland, A., Air pollution from traffic and cancer incidence: a Danish cohort study. a global access science source, 10,2011, 67. Committee on Environmental Health, Ambient Air Pollution: Health Hazards to Children, Pediatrics, 114 (6), 2004, 1699–1707.
  • 127. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Shyam Bihari Sharma et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 397 FORMULATION AND EVALUATION OF ORODISPERSIBLE TABLETS OF CINNARIZINE BY SUPER DISINTEGRANTS ADDITION METHOD Praveen Khirwadkar1 , Kamlesh Dashora2 , Shyam Bihari Sharma3 * 1 Institute of Pharmacy, Vikram University, Ujjain, Madhya Pradesh, India 2 Institute of Pharmacy, Vikram University, Ujjain, Madhya Pradesh, India 3 Jiwaji University, Gwalior, Madhya Pradesh, India *Corresponding author: E.Mail: shyam_mpharm06@rediffmail.com Abstract The aim of the present work is to study the Preformulation parameters for Cinnarizine orodipersible tablet. The objective of pre-formulation study is to generate information useful to the formulator in developing stable and bioavailable dosage form. Administration of conventional tablets of Cinnarizine has been reported to exhibit fluctuations in plasma drug levels, either in manifestation of side effects or reduction in drug concentration at the receptor sites. Conventional Cinnarizine tablets available in market are not suitable where quick onset of action is required. Besides, the conventional tablets also show poor patient compliance particularly by the geriatric and pediatric patients who experience difficulty in swallowing, and by those who are bed ridden or who are traveling and do not have an easy access of water. Usage of excipients like low-substituted hydroxypropyl cellulose, crospovidone helps for the faster disintegration and faster release of the drug from the dosage form. The Preformulation studies were carried out in terms of test for identification (physical appearance, melting point, and UV spectrophotometer), solubility profile, and determination of partition coefficient and quantitative estimation of drug. All the observations and results showed that the Cinnarizine could serve as suitable candidate for fast dissolving tablet that may improve the bioavailability. Keywords: Preformulation study, Orodispersible tablet, Patient compliance, Super disintegrants INTRODUCTION Tablets are solid preparations each containing a single dose of one or more active ingredients and are obtained by compressing uniform volumes of particles. The objective of the design and manufacture of the compressed tablet is to deliver orally the correct amount of drug in the desired location and to have its chemical integrity protected to the point. Recent advances in Novel Drug Delivery System (NDDS) aims to enhance safety and efficacy of drug molecule by formulating a convenient dosage form for administration and to achieve better patient compliance. One such approach is “Mouth Dissolving Tablet”. This is an innovative tablet technology where the dosage form containing active pharmaceutical ingredients disintegrates rapidly, usually in a matter of seconds, without the need for water, providing optimal convenience to the patient. Innovators and inventor companies have given these tablets various names such as orally disintegrating tablets (ODT), mouth dissolving (MD), fast melting, fast dissolving or Orodisperse. The European Pharmacopoeia defines Orodisperse as a tablet that can be placed in the mouth where it disperses rapidly before swallowing. Researchers have formulated ODT for various categories of drugs, which are used for therapy in which rapid peak plasma concentration is required to achieve desired pharmacological response. These include neuroleptics, cardiovascular agents, analgesics, anti-allergic and drugs for erectile dysfunction. The concept of Mouth Dissolving Drug Delivery System emerged from the desire to provide patient with conventional mean of taking their medication. Difficulty in swallowing (Dysphasia) is a common problem of all age groups, especially elderly and pediatrics, because of physiological changes associated with these groups of patients. Other categories that experience problems using conventional oral dosage forms includes are the mentally ill, uncooperative and nauseated patients, those with conditions of motion sickness, sudden episodes of allergic attack or coughing. Sometimes it may be difficult to swallow conventional products due to unavailability of water. These problems led to the development of novel type of solid oral dosage form called “Mouth Dissolving Tablets”. This tablet disintegrates instantaneously when placed on tongue, releasing the drug that dissolves or disperses in the saliva. Produce rapid onset of action. In such a cases Bioavailability of drug is significantly greater than those observed from conventional tablet dosage form. MATERIALS AND METHODS Preformulation studies: Preformulation studies are the first step in the rational development of dosage form of a drug substance. The objective of Preformulation studies are to develop a portfolio of information about the drug substance, so that this information useful to develop formulation. Preformulation can be defined as investigation of physical and chemical properties of drug substance alone and when combined with excipients. Preformulation investigations are designed to identify those physicochemical properties and excipients that
  • 128. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Shyam Bihari Sharma et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 398 may influence the formulation design, method of manufacture, and pharmacokinetic-biopharmaceutical properties of the resulting product. Organoleptic Characteristics: The color, odor, and taste of the drug were characterized and recorded using descriptive terminology. Drug Excipients Compatibility Study: IR Study were done for following drug, drug- excipients ratio 1. API alone (Cinnarizine) 2. API: Ac-Di-Sol® → 1: 1 3. API: Kollidone® → 1: 1 Super disintegrants addition method: Specified quantity of Cinnarizine, mannitol, Avicel 102, aspartame, crospovidone, Ac-di-sol, talc and magnesium stearate were weighed accurately and passed through 60 #screen. All the materials were transferred to mortar and triturated till it mixed uniformly. The resulting powdermixture was compressed into tablets using singlepunch tablet machine. Formulation of tablets wasre presented in Table.1. Table.1.Composition of Orodispersible Tablet of Cinnarizine Formulation F1 (mg) F2 (mg) F3 (mg) F4 (mg) F5 (mg) F6 (mg) F7 (mg) F8 (mg) F9 (mg) Cinnarizine 25 25 25 25 25 25 25 25 25 Manitol 30 30 30 30 30 30 30 30 30 crospovidone 8 12 16 8 12 16 8 12 16 Ac-di-Sol 2 2 2 4 4 4 6 6 6 Aerosil 2 2 2 2 2 2 2 2 2 Aspartame 3 3 3 3 3 3 3 3 3 Magnesium Stearate 1 1 1 1 1 1 1 1 1 Avicel 102Up to 200 200 200 200 200 200 200 200 200 RESULTS Evaluation of the blend, evaluation of the tablet for post compression parameters, cumulative percentage of drug release were done and noted in the tables 2 to 4. Stability studies as well as compatibility studies were conducted and presented in table 5 and figures 1 to 3 respectively. Table.2. Evaluation of the Powder Blend Batch Code Bulk Density Tapped Density Angle Of Repose Percentage Compressibility Hausner’s Ratio F1 0.52 0.58 25.32 16.25 1.163 F2 0.53 0.62 26.23 18.23 1.200 F3 0.59 0.60 28.23 20.06 1.164 F4 0.54 0.78 23.56 14.52 1.170 F5 0.57 0.55 25.33 15.25 1.135 F6 0.59 0.77 29.36 17.23 1.152 F7 0.51 0.71 24.56 12.56 1.207 F8 0.53 0.69 30.21 18.31 1.212 F9 0.54 0.74 31.12 19.18 1.237 Table.3. Physical parameters of mouth dissolving Tablet Batch Code Weight Variation Thickne ss (Mm) Hardness (Kg/Cm2 ) Friability (%) Disintegration Time (Sec) Wetting Time (Sec) Assay (%) F1 pass 2.56 3.4 0.73 30.6 ±1.25 64.0 ±1.35 98.14 F2 pass 2.57 2.5 0.76 43.7 ±2.46 65.8 ±0.35 99.02 F3 pass 2.60 2.5 0.79 56.4 ±2.42 79.0 ±0.85 100.51 F4 pass 2.63 3.2 0.74 29.6 ±1.22 32.4 ±1.15 98.91 F5 pass 2.65 3.0 0.78 30.6 ±1.25 65.0 ±1.35 100.04 F6 pass 2.66 2.5 0.80 33.0 ±1.00 35.0 ±0.95 99.86 B7 pass 2.51 3.0 0.69 23.3 ±0.58 29.1 ±1.05 98.92 F8 pass 2.52 2.5 0.65 35.5 ±0.50 41.7 ±1.45 101.05 F9 pass 2.54 2.5 0.66 36.5 ±0.50 34.12 ±1.45 100.3
  • 129. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Shyam Bihari Sharma et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 399 Table.4.The following are the results of the dissolution studies of the formulated Table.5. Stability parameters of formulation B7 stored at temperature 40C/75 % RH 40o C. Parameters Control sample After 15 Days After 30 Days After 60 Days Drug Content (%) 100.34 99.57 99.19 99.00 Disinintegration time (Sec) 25.3 26.2 26.8 25.90 Wetting Time (Sec) 29.1 30.2 30.9 30.25 Hardness(Kg/Cm2 ) 3.00 3.1 3.3 3.4 Fig. No 1: IR Spectrum of Pure Drug (Cinnarizine) Fig. No. 2 IR Spectrum of Cinnarizine + Ac-di-sol Fig. No. 3 IR Spectrum of Cinnarizine + Crospovidone CONCLUSION Cinnarizine is a histamine H1-receptor antagonist is the most frequently prescribed drug in treatment of motion sickness, vomiting, allergic reaction, vertigo and insomnia. Conventional Cinnarizine tablets available in market are not suitable where quick onset of action is required. To overcome these problems, there is a need to develop a rapidly disintegrating dosage form, particularly one that would rapidly disintegrate in saliva and could be administered without water anywhere anytime. No such mouth dissolving tablet of Cinnarizine is available in the market. The present investigation was aimed to evaluate the possibility of using different parameters for the development of fast dissolving of Atenolol. Preformulation studies were done using various parameters such as identification test of Atenolol. The description and appearance, melting point and solubility were also performed for further characterization & it was found that all results are satisfactory. REFERENCES Bhushan SY, Sambhaji SP, Anant RP, and Kakasaheb RM, New drug delivery system for elderly, Indian drugs, 37, 2000, 312-318. Time (Min) Cumulative percentage Drug Release F1 F2 F3 F4 F5 F6 F7 F8 F9 0 0 0 0 0 0 0 0 0 0 2 63.75 60.82 55.00 74.97 71.47 69.06 75.77 73.64 72.65 4 75.09 65.75 62.97 84.39 81.97 79.91 88.09 84.34 80.88 6 81.79 79.04 75.65 92.68 89.75 88.85 93.08 90.85 89.58 8 92.19 86.34 87.78 96.65 95.32 94.90 98.08 97.79 94.46 10 99.3 96.22 97.32 99.6 98.08 97.61 99.89 98.75 97.90
  • 130. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Shyam Bihari Sharma et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 400 British Pharmacopoeia, cinnarizine, the stationery office on behalf of the medicines and healthcare products regulatory agency (mhra), 2001, 398- 400. Chang RK, Guo X, Burniside BA and Couch RA, Fast dissolving tablets, Pharm Tech, 24(6), 2000, 52-58 Cims-93 april-2006 (update-2), 211-212. Cinnarizine, www.redpoll.pharmacy.ualberta.ca/drugbank/index.html, 2006. Cinnarizine, drug information, www.emc.medicines.org.uk, 2006. Cooper J, Gun C, Powder flow and compaction, tutorial pharmacy, New Delhi, Hidix CBS publishers and distributors, 1986, 211-233. Dobetti, Fast melting tablets: developments and technologies, Pharm. Tech, 2001, (suppl.), 44 Herbert a. Liberman and leon lachman, the theory and practice of industrial pharmacy, third edition, verghese publication house, 171, 293. James E F Reynolds: Martindale, The Extra Pharmacopoeia, thirty-first edition, Royal Pharmaceutical Society, London, 1996, 406. Kaushik D, Dureja S and Saini TR, Mouth dissolving tablets- a review, Indian Drugs, April 2003, 41(4), 187- 193 Kuchekar BS, Badhan AC and Mahajan HS, Mouth dissolving tablets: a novel drug delivery system, Pharma times, 35, 2003, 7-9 Kuchekar BS, and Arumugan V, Fast dissolving tablets, Indian journal of pharmaceutical education, 35, 2001, 150 Lindgreen S and Janzon L, Dysphagia: prevalence of swallowing complaints and clinical findings, Medical clinics of North America, 1993, 77, 3-5. Seager H, Drug delivery products and zydus fast dissolving dosage forms, J. Pharm. Pharmacology, 50, 1998, 375-382. The theory and practice of industrial pharmacy, Leon Lachmann, Herbert A Lieberman, Joseph L Kanig, 293- 303. Wilson CG, Washington N, Peach J, Murray GR and Kennerley J, The behavior of fast dissolving dosage, Int. J. Pharm, 40, 1987, 119-123.
  • 131. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Mohanraghupathy S et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 401 EFFECTIVE HYPOGLYCEMIC ACTION OF METFORMIN COMBINATIONS AGAINST DEXAMETHASONE INDUCED DIABETES MELLITUS IN RATS Mohanraghupathy S*, Jayabharath N, Bhuvana tejaY, Hameera khanam B, Lavanya lahari B Department of Pharmacology, Annamacharya College of Pharmacy, Kadapa, Andhra Pradesh *Corresponding author: E.Mail: mohanraghupathy@gmail.com ABSTRACT Diabetes mellitus is a chronic metabolic disorder occurs due to the damage of β-cells of islets of Langerhans in pancreas. In general patients suffering for diabetes mellitus always use the medicines, apart from the medicines they take insulin also. In our study we did the preclinical experimental study of Metformin combinations on Albino Wistar rats such that which combination is effective for the patients who are using purposefully using the insulin. Our studies shown that Metformin + Glipizide shown the effective hypoglycemic results. For inducing diabetes mellitus we introduced Dexamethasone intraperitoneally as it is a steroidal anti-inflammatory agent and it is having hypoglycemic action as adverse effect. Key Words: Diabetes mellitus, metformin, glimepiride, glipizide INTRODUCTION Diabetes Mellitus: It is a metabolic disorder characterized by hyperglycemia, glycosuria, hyperlipidaemia, negative nitrogen balance and sometimes ketonaemia. A widespread pathological change is thickening of capillary basement membrane, increase in blood vessel wall matrix and cellular proliferation resulting in vascular complications like lumen narrowing, early atherosclerosis, and sclerosis of glomerular capillaries, retinopathy, neuropathy and peripheral vascular insufficiency. Insulin-dependent diabetes mellitus (IDDM), juvenile onset diabetes mellitus: There is β cell destruction in pancreatic islets, majority of cases are auto immune (type 1A) antibodies that destroy β cells are detectable in blood, but some are idiopathic (type 1B) no β cell antibody is found. In all type 1 cases circulating insulin levels are low or very low, and patients are more prone to ketosis. This type is less common and has a low degree of genetic predisposition. Noninsulin-dependent diabetes mellitus (NIDDM), maturity onset diabetes mellitus: There is no loss or moderate reduction in β cell mass; insulin in circulation is low, normal or even high, no anti-β-cell antibody is demonstrable; has a high degree of genetic predisposition; generally has a late onset (past middle age). Over 90% cases are type II DM. causes may be: 1) Abnormality in gluco-receptor of β cells so that they respond at higher glucose concentration or relative β cell deficiency. 2) Reduced sensitivity of peripheral tissues to insulin: reduction in number of insulin receptors, ‘down regulation’ of insulin receptors. Many hypertensives are hyperinsuliaemic, but normoglycaemic; exhibit insulin resistance associated with dyslipidaemia (metabolic syndrome). Hyperinsulinaemia has been implicated in causing angiopathy. 3) Excess of hyperglycemic hormones (glucagon, etc.) /obesity: cause relative insulin deficiency-the β cells lag behind. 4) The third main form, gestational diabetes occurs when pregnant women without a previous diagnosis of diabetes develop a high blood glucose level. It may precede development of type 2 DM. MATERIALS AND METHODS Metformin, Glimipride, Glipizide and Dexamethasone were purchased from the near community pharmacy. Kits used for the estimation of albumin, total cholesterol, blood urea nitrogen were purchased from Erba Diagnostics. Blood glucose levels were estimated with the help of Glucometer which was purchased from community pharmacy. Male Wistar albino rats weighing 150-250gms, Manjunatha institute of animal house, Bangalore. Maintenance of animals: Animals were kept for 1 week to acclimatize laboratory conditions before starting the experiment. The animal house temperature was always maintained within the range of approximately 22-290 c. The relative humidity of animal house was maintained within the range of 30-70% throughout the duration of experiment. Sufficient diet and water was supplied every day. All the experimental procedures and protocols used in the study were approved by institutional animal ethical committee (IAEC).
  • 132. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Mohanraghupathy S et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 402 0 50 100 150 200 250 Normal Diabetic Metformin + Glimepride Metformin + Glipizide Body weight Fasting blood glucose levels Post prandial blood glucose level Experimental protocol Group – I: It consists of normal animals which were received no dug and only food was kept. This group consists of four animals. Group – II: It consists of four animals which were received inducing agent Dexamethasone of 0.75mg/kg body weight with normal food. Group – III: It consists of four animals which were received inducing agent Dexamethasone of 0.75mg/kg body weight. And also these animals received the combination of hypoglycemic agents [Metformin (500mg/kg body weight) + Glimepride (1mg/kg body weight)] with normal food. Group – IV: It consists of four animals which were received inducing agent Dexamethasone of 0.75mg/kg body weight. And also these animals received the combination of hypoglycemic agents [Metformin (500mg/kg body weight) + Glipizide (5mg/kg body weight)] with normal food. Biochemical parameters: After intraperitoneal administration of Dexamethasone in rats the Diabetes was induced after thirty days. The dose was calculated by taking the body weight of rat. The blood glucose levels were examined with help of glucometers. When the fasting blood glucose levels shown 150mg/dl in rats those animals were considered as diabetic. And we examined the body weight additionally. The results of Body weight and Blood glucose levels are given below: Table 1: Blood glucose levels of the experimental animals Fig 1: Blood glucose levels of the experimental animals Tab 2: Estimation of Total protein, Cholesterol, Blood Urea Nitrogen, Serum Creatinine Groups Total protein Cholesterol Blood Urea Nitrogen Serum creatinine Control 6.99 82.20 42.66 0.50 Diabetic 3.77 182.2 0.78 0.80 Metformin+Glimipride 5.02 98.8 0.62 0.62 Metformin + Glipizide 6.05 91.9 0.59 0.58 Groups Normal Diabetic Metformin + Glimepride Metformin + Glipizide Body weight (gm) 230 175 185 187 Fasting blood glucose levels (mg/dl) 90 147 150 160 Post prandial blood glucose level (mg/dl) 100 180 114 100
  • 133. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Mohanraghupathy S et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 403 0 20 40 60 80 100 120 140 160 180 200 control diabetic met+gl met+gz Total protein Cholesterol BUN Serum creatinine Fig 2: Estimation of Total protein, Cholesterol, Blood Urea Nitrogen, Serum Creatinine CONCLUSION The Metformin combination therapy on Dexamethasone induced diabetes mellitus on rats proved that instead of administration of insulin to the patients with excess blood glucose levels oral therapy is always safe. It also denotes that the metabolic activity of the organs as well as the different mechanism of action of medicines is going to reduce the blood glucose level perfectly. Glipizide and Metformin combination showed the valuable results on therapy when compared to Glimipride. The both drugs Glimipride and Glipizide are the secretogogues enhancing the insulin levels as well as the Metformin is having the property of reducing the glucose level by glucose reuptake by the cells. Finally we concluded that Metformin and Glipizide combination is the best. DISCUSSION Patients suffering with diabetes mellitus with blood glucose levels above 200mg/dl always have a little pain on taking insulin through intramuscular route. Hypoglycemic agents with different combinations when used through oral route are not only effective but also safe for the patients. The metabolic rate of the medicines should always be examined properly because, in some patients the medicines after prolong use may not work due to improper binding of the drug molecules at the receptor sites. This factor leads to increased blood glucose level. Immediately the patient should not be advised to take insulin because, insulin is having adverse hypoglycemic effect. Patients are advised to have the physical exercise like walking or working like gardening, sweeping, lifting mild luggages etc because whatever the diet we are taking it is not only digested in the stomach but also should be utilized in the form of energy in tissues through work. REFERENCES Akhila shetty J and Divya Choudhary, Effect of insulin plant (Costus igneus) leaves on Dexamethasone induced hyperglycemia, JAR. 1, 2010, 100. Department of health and families, Glucometers, RHA, 2005, 1-4. Good Man and Gilman, Stephen N. Davis and Daryl K. Granner, Insulin and oral hypoglycemic agents and the pharmacology of the endocrine pancreas, The pharmacological basis of therapeutics, 10th edition, 1679. Harsh Mohan, Diabetic Nephropathy, Text book of pathology, 6th editon, 2010, 677. Padmaja Udaykumar, Insulin and oral hypoglycaemics, Text book of Medical pharmacology, 2nd edition, 2009, 486. Vasanth Muthu Swamy, Lal Krishna, Murugan SS, CPCSEA guidelines for laboratory animal facility, 2009, 9-10
  • 134. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Suruchi Singh et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 404 A REVIEW ON MEDICINAL PLANTS HAVING ANTIOXIDANT POTENTIAL S.K Sharma, Lalit Singh, Suruchi Singh* Sunder Deep Pharmacy College, Ghaziabad, U.P, India. *Corresponding author: E.mail: suruchibpharm89@gmail.com ABSTRACT Natural compounds from plants and other life forms (bacteria, fungi, marine organisms) represent a major source of molecules with medicinal properties. Among them, antioxidant substances are of particular interest. The understanding of the central role that oxidative stress holds in the progression of disorders as varied as: cardiovascular diseases, degenerative conditions, rheumatic disorders, metabolic syndrome, and in aging, makes antioxidant capacity to a key-feature of modern, multipotent remedies. A lot of medicinal plants, traditionally used for thousands of years, are present in a group of herbal preparations of the Indian traditional health care system (Ayurveda) named Rasayana proposed for their interesting antioxidant activities. Keywords: Antioxidant, Ayurveda, Rasayana, Oxidative stress. INTRODUCTION Antioxidants are substances that may protect your cells against the effects of free radicals. Free radicals are molecules produced when your body breaks down food, or by environmental exposures like tobacco smoke and radiation. Free radicals can damage cells, and may play a role in heart disease, cancer and other diseases. Studies suggest that a diet high in antioxidants from fruits and vegetables is associated with a lower risk of cancer, cardiovascular disease, Parkinson's disease and Alzheimer's disease. A plant-based diet protects against chronic oxidative stress-related diseases. Dietary plants contain variable chemical families and amounts of antioxidants. It has been hypothesized that plant antioxidants may contribute to the beneficial health effects of dietary plants. Our objective was to develop a comprehensive food database consisting of the total antioxidant content of typical foods as well as other dietary items such as traditional medicine plants, herbs and spices and dietary supplements. Since ancient times, the medicinal properties of the plant materials improve the quality and nutritional value of plants has been investigated in the recent scientific form. While, flavonoids are a group of polyphenolic developments throughout the world, due to their potent compounds with known properties, which include free antioxidant activities. The antioxidants have been reported to have radical scavenging, inhibition of hydrolytic and oxidative to prevent oxidative damage caused by free radical. Antioxidants Potential Plants Free radicals are atoms or molecules with singlet, i.e. unpaired electron which makes them highly reactive. Oxidative free radicals are generated by metabolic reactions create a chain reaction leading to membrane and other lipid peroxidation, DNA damage, etc. This has been implicated in atherosclorosis (oxidated LDL is more atherogenic), cancers, neurodegenerative and inflammatory bowel diseases. Many endogenous and dietary compounds like superoxide dismutase, ferritin, transferrin, reruloplasmin, tocopherol, carotene and ascorbic acid have anti oxidant and free radical scavenging properties. Small amounts of reactive oxygen species are continually formed in the body in the cell membrane and close to the cells organelles. They act where they are generated. Hence, they can damage most cell structures including membrane lipids, proteins, enzymes and nuclic acids. The body has mechanisms to produce the small amounts of oxidants normally formed during metabolic reaction. Reactive species such oxidants are formed in controlled amounts by neutrophil leucocytes on exposure to microbes are beneficial to the body in that they participate in destroying the microbes. Excess of oxidants, however, can be harmful to the body. Liver is also under constant threat of oxidants and some of the free radical especially H2O2. Lipid peroxidation has been demostred as one of the important feature after exposure to hepatotoxic substances and also is a measure of extent of hepatic damage. Several herbs and herbal formulations are available for the scavenging activity. In addition to this there is a global trend to revive the traditional systems of medicines and renewed interest in the natural remedies for treating human ailments. Antioxidants have important preventive roles, not only on undesirable changes in the flavor and nutritional quality of food, but also on tissue damage in various human diseases. Almost all organisms are well protected against free radical damage by either enzymes or compounds, such as ascorbic acid, α- tocopherol and gluthione.
  • 135. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Suruchi Singh et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 405 When the mechanism of antioxidant protection unbalanced by the deterioration of different factors, physiological functions can occur which result in diseases or accelerated aging. Consequently, it is important to find compounds that prevent oxidation. Antioxidants have important preventive roles not only on undesirable changes in the flavor and nutritional quality of food, but also on tissue damage in various human diseases. They are effective in prevention of degenerative illnesses, such as different types of cancers, cardiovascular and neurological diseases, cataracts and oxidative stress dysfunctions. Polyphenols are the most significant compounds for the antioxidant properties of plant raw materials. Then antioxidant activity of polyphenols is mainly due to their redox properties, which allow them to act as reducing agents, hydrogen donors, singlet oxygen quenchers, metal chelators and reductants of ferryl hemoglobin. Medicinal plant parts are commonly rich in phenolic compounds, such as flavonoids, phenolic acids, stilbenes, tannins, coumarins, lignans and lignins. These compounds have multiple biological effects including antioxidant activity. CONCLUSION As antioxidant is a molecule capable of slowing or preventing the oxidation of other molecules. Oxidation is achemical reaction that transfers electron from a substance to an oxidizing agent. Oxidation reactions can produce free radicals, which start chain reactions that damage cells. Antioxidants are the substances that inhibit oxidation and are capable of counteracting the damaging effects of oxidation in body tissue. They prevent damage caused by free radicals. Free radicals are very unstable molecules with an unpaired electron and are important intermediates in natural processes involving control of vascular tone, cytotoxicity and neurotransmission. Free radicals cause many human diseases like cancer, Alzheimer’s disease, cardiac reperfusion abnormalities, kidney disease and fibrosis etc. Antioxidants play many vital functions in a cell and have many beneficial effects when present in foods. Table 1. List of plants exhibit antioxidant characteristics and their chemical constituents PLANT NAME PLANT PART MAIN CHEMICAL CONSTITUENTS Withania somnifera Ocimum sanctum Piper nigrum Arentium lappalo Scutellaria barbata Daucus carrota Coleus ferscoli Salvia sclarea Eugenia caryophylla Allium sativum Zingiber officinalis Ginkgo biloba Vitis vinifera Berries, leaves, roots Leaves, Seeds Fruit Root Leaves, Leaves, Seed, Root Roots Entire plant, seed Inflorescence Leaves, Bud Leaves, Rhizome Plant Fruit, Seed Ascorbic acid,α-tocopherol and reduced glutathione, superoxide dismutase,ascorbate peroxidase, catalase, peroxidase & polyphenol oxidase Ascorbic acid,β-carotene, β-sitosterol, eugenol,Palmitic acid, tannin Ascorbic acid, β carotene,Lauric acid, myristic acid, palmitic acid, piperine Insulin, tannic acid Gallic acid Alanine, α tocopherol, ascorbic acid, camphene,eugenol,γ- terpinene, histidine Antitoxin Ferscolin ˠ-terpinene, linalyl acetate, myrcene, Palmitic acid, rosemarinic acid Acetyl-eugenol, Ascorbic acid, β -carotene, β-sitosterol, caryophyllene oxide, eugenol, isoeugenol Alanine, Ascorbic acid, β-sitosterol,Caffeic acid, Kaemferol, Methionine 6-Gingerol,alanine, Ascorbic acid, Histidine, Lauricacid, Methionine, Myristic acid,Palmitic acid, Tryptophan EGB 761,Ginkgogolide Alanine, α-tocopherol, Ascorbic acid, β -carotene, β- sitosterol, Histidine, OPC, Methionine, Palmitic acid,
  • 136. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Suruchi Singh et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 406 Citrus aurantifolia Cymbopogon citratus Commiphora myrrha Myristica fragrance Olea europaea Mentha piperata Catharanthus roseus Rosemarionus officinalisL Santalum album Curcuma domestica Acorus calamus Alisma plantago- aquatica L. Allium ursinum L. Cotinus coggygria Scop. Angelica sylvestris L. Anthriscus cerefolium Anthriscus sylvestris Carum carvi L. Ery ngium campestre L. Sanicula europaea L. Achillea millefolium s.l. Arctium lappa L. Artemisia absinthium L Artemisia vulgaris L. Bellis perennis L Bidens tripartita L. Carlina acaulis L. Carthamus tinctorius L. Cichorium intybus L. Cirsium arvense (L.) Scop Fruit Leaves Resin, Sap Seed, Leaf Leaf Leaf Leaf Entire Plant Leaf, Oleoresin Fruit, Wood Rhizome Rhizome Flowering aerial parts, roots Leaf Leaf Root, Grains Root, Flowering aerial part Flowering aerial part Fruits Flowering aerial part Flowering aerial part Leaf, root Flowering aerial part Flowering aerial parts Flowering aerial parts Flowering aerial parts Root Flower Flowering aerial part, root Leaf Flowering aerial part Flowering aerial parts selenium Alanine, α –pinene,ascorbic acid,β -Sitosterol, caffeic acid, Eugenol, Linalylacetate, Palmitic acid, Tannin Β-sitosterol,Myrcene,Selenium Β-Sitosterol,campestrol,eugenol Lauric acid,Myrcene, Palmitic acid Α -tocopherol,apigenin, β -carotene, γ - tocopherol,kaempferol, Luteolin Menthol, Limonene Vincristine, Vinblastine Carsonic acid, Rosemaric acid, Β –sitosterol , Caryophyllene oxide, eugenol,isoeugenol Alanine, eugenol, β -sitosterol, Palmitic acid, phenol Curcumin,tannins, phenolic acids Only antioxidative fractions devoid of beta-asarone should be used, Triterpene (alisol B) Flavonoids, sulfur-containing compounds Flavones, aurones, chalcones Flavonoids, coumarins Flavonoids (apiin), lignans Flavonoids (quercetin, apigenin) Flavonoids, volatile oil Flavonoids, triterpenes Rosmarinic acid derivative Flavonoids, tannins, volatile oil Flavonoids Flavonoids Flavonoids Flavonol glycosides Flavonoids Flavonoids Flavonoids Phenolic acids, flavonoids Phenolic acids,acidic polysaccharides with unprecised structure Flavonoids Flavonoids, volatile oils Phenolic acids, flavonoids Flavonoids Phenolic acids, flavonoids Flavonoids, polysaccharides (mucilages) Flavonoids Flavone 6-C-Glycosides
  • 137. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Suruchi Singh et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 407 Conyza canadensis L. Cronq. Hieracium pilosella L Matricaria recutita L. Onopordum acanthium Solidago virgaurea L. Taraxacum officinale agg. Tussilago farfara L Betula pendula Roth Alliaria petiolata Capsella bursa-pastoris Nasturtium officinale Humulus lupulus L. Sambucus nigra L. Sambucus ebulus L. Viburnum lantana L. Viburnum opulus L. Evonymus europaeus L. Cornus mas L. Corylus avellana L. Juniperus communis L. Hippophae rhamnoides Elaeagnus angustifolia . Equisetum arvense L. Calluna vulgaris (L.) Vaccinium myrtillus L. Anthyllis vulneraria L. Genista tinctoria L. Lotus corniculatus L. Melilotus officinalis L. Pallas Ononis spinosa L. Trifolium arvense L. Flowers Flowering aerial parts Flowering aerial parts Root, Flowering aerial parts Leaf Leaf Flowering aerial parts Flowering aerial parts Flowering aerial parts Glandulae Flowers Leaf Branches Branches Grains Fruits Grains, Leaf Fruits Fruits Leaf, Branch Flowering aerial parts Flowering aerial parts Leaf, Fruit Flowering aerial parts Flowering aerial parts Flowering aerial parts Flowering aerial parts Flowering aerial parts Flowering aerial parts Flowering aerial parts Flowering aerial parts Bark, Flowers Bark Flavonoids, glucosinolates Flavonoids, glucosinolates Flavonoids Flavonoids Flavonoids Flavonoids, procyanidins Flavonoids, procyanidins Flavonoids Flavonoids, phenolic acids Phenolic acids Flavonoids Flavonoids, carotenoids Flavonoids Flavonoids Flavonoids Anthocyans Flavonoids,isoflavones (genistein) Flavonoids, triterpenes Flavonoids Triterpenes Isoflavones Isoflavones Isoflavones Tannins, procyanidins, flavonoids Tannins, procyanidins, Flavonoids Xanthones, phenolic acids Tannins, gallic acid Flavonoids, tannins Flavonoids Flavonoids Tannins, flavonoids Flavonoids, phenylpropanoids (verbascoside) Flavonoids, phenolic acids Flavonoids Flavonoids Flavonoids, phenolic acids Flavonoids Flavonoids Flavonoids Flavonoids, phenolic acids Flavonoids
  • 138. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Suruchi Singh et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 408 Trifolium pratense L. Trifolium repens L. Quercus petraea L. Quercus robur L. Centaurium erythraea L. Erodium cicutarium L. Geranium Flowering aerial parts Flowering aerial parts Flowering aerial parts, roots Flowering aerial parts Flowering aerial parts Flowering aerial parts Flavonoids, phenolic acids Flavonoids, phenolic acids Flavonoids Flavonoids, iridoids Phenolic acids,flavonoids, carotenoids Polysacharides, flavanoids Polysaccharides (mucilages), flavonoids Flavonoids, coumarins phenylpropanoids (verbascoside) Tannins, REFERENCE Anchana Chanwitheesuk, Aphiwat Teerawutgulrag, Nuansri Rakariyatham, Screening of antioxidant activity and antioxidant compounds of some edible plants of Thailand, Food Chemistry, 92, 2005, 491–497. ANTAL Diana Simona, Medicinal plants with antioxidant properties from Banat region(Romania): A rich pool for the discovery of multi- target phytochemicals active in free radical related disorders, Analele Universităţii din Oradea - Fascicula Biologie Tom. XVII / 1, 2010, 14-22. Arya Vikrant, Bhardwaj Ankur, Sharma Vinit, Pharmacology of some antioxidant plants from district kangra Himachal Pradesh- A Review, International journal of current npharmaceutical research, 3(2), 26 – 31. Bibi Sedigheh Fazly Bazzaz, Antioxidant and antimicrobial activity of methanol, dichloro methane and, ethyl acetate extracts of Scutellaria litwinowii, Science Asia, 37, 2011, 327–334. HA Ogbunugafor, FU Eneh, AN Ozumba, MN Igwo-Ezikpe, Physico-chemical and Antioxidant Properties of Moringa oleifera Seed Oil, Pakistan Journal of Nutrition, 10 (5), 2011, 409-414. Khanahmadi M, Rezazadeh Sh, Review on Iranian medicinal plants with antioxidant properties, Journal of Medicinal Plants 2010, 9(35), 20-31. Kratchanova Maria, Denev Petko, Ciz Milan, Lojek Antonin, Mihailov Atanas, Evaluation of antioxidant activity medicinal plants containing polyphenol compounds.Comparison of two extraction system, ACTA Biochemia Polonica, 57(2),2012, 229-234. Krishnaiah Duduku, Sarbatly Rosalam, Bono Awang, Phytochemical antioxidants for health and medicine – A move towards nation, Biotechnology and Molecular Biology Review, (4), 2007, 097-104. Luz María Sánchez Perera, Arturo Escobar, Caden Souccar, Antonia Remigio and Betty Mancebo, Pharmacological and toxicological evaluation of Rhizophora mangle L as a potential antiulcerogenic drug: Chemical composition of active extract, Journal of Pharmacognosy and Phytotherapy, 2(4) , 2010, 56-63.
  • 139. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Suruchi Singh et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 409 Mandana B, Russly A R, Farah ST, Noranizan MA, Zaidul I S and Ali G, Antioxidant activity of winter melon (Benincasa Hispida) seeds using conventional soxhlet extraction technique, International Food Research Journal 19(1), 2012, 229-234. P Kanimozhi and J Karthikeyan, A study on antioxidant potential of Glycyrrhiza glabra linn. in 1,4- dichlorobenzene induced liver carcinogenesis, Journal of Chemical and Pharmaceutical Research, 3(6), 2011, 288-292. Pandey Neha, Barve Dushyant, Antioxidant activity of ethanolic extract of Annona squamosa Linn Bark, International Journal of Research in Pharmaceutical and Biomedical Sciences, 2(4), 2011, 1629- 1697. Pratap Sangh.,Pandey Sanjay, A review on herbal antioxidants”, Journal of pharmacognosy and phytocheistry 1(4), 28-38. Rahmat Ali Khan, Evaluation of phenolic contents and antioxidant activity of various solvent extracts of Sonchus asper (L.) Hill, Chemistry Central Journal, 6, 2012,12. Rana Siddhant, Suttee Ashish, Phytochemical investigation and evaluation of free radical scavenging potential of Benincasa hispida peel extracts, International Journal of Current Pharmaceutical Review and Research, 3(3), 43- 46. Sangh Partap, Amit Kumar, Neeraj Kant Sharma, K. K. Jha, Luffa Cylindrica : An important medicinal plant, J. Nat. Prod. Plant Resour, 2 (1), 2012, 27-134. Scartezzini Paolo, Speroni Ester, Review on some plants of indian traditional medicine with antioxidant activity, Journal of Ethnopharmacology, 71, 2000, 23-43. Shyamala BN & Jamuna P, Nutritional Content and Antioxidant Properties of Pulp Waste from Daucus carota and Beta vulgaris, Mal NJ Nutr, 16(3) 2010, 397-408. TK Gopal, Harish G, D Chamundeeswari, C Umamaheswara Reddy, In-vitro Anti-Oxidant Activity of Roots of Boerhaavia diffusa Linn, Research journal of Pharmaceutical, Biological and Chemical Sciences, 4, 2010, 782- 788.
  • 140. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Vijayakumar and Hindumathy Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 410 INVITRO ANTI-INFLAMMATORY ACTIVITY OF STRYCHNOS POTATORUM LINN SEED BY HRBC MEMBRANE STABILIZATION V.Vijayakumar*1 , Dr C.K.Hindumathy 2 1. Department of Pharmaceutical Chemistry, Vinayaka Missions College of Pharmacy. 2. Department of Biotechnology, V.M.K.V. Engineering College. * Corresponding author: E-mail: vijaiy77@gmail.com ABSTRACT In the present study, strychnos potatorum linn belonging to the family Loganiaceae (Strychnaceae) was studied. It is a folkloric medicinal plant used to treat antibacterial, antifungal, anti diabetic, antioxidant and anti-inflammatory. Since many flavonoids have remarkable anti-inflammatory activity the present work aims at evaluating the anti inflammatory activity of seeds strychnos potatorum linn by HRBC membrane stabilization method. The prevention of hypo tonicity induced HRBC membrane lysis was taken a measure of anti inflammatory activity. The anti-inflammatory activity of hydroalcholic extract was comparable to that of the standard drug Hydrocortisone. The percentage protection for the hydroalcholic extract and hydrocortisone were 100 at µg/ml. the hydroalcholic extract of strychnos potatorum linn seed has significant anti-inflammatory activity (54.95±0.74). Key words: Anti-inflammatory, strychnos potatorum linn, HRBC membrane stabilization, Hydroalcholic extract. INTRODUCTION Inflammation is a normal protective response to tissue injury that is caused by physical trauma, noxious chemicals or microbiological agents. Inflammation is the result of concerted participation of more number of proliferative factors (like Vasoactive, chemotatic) at different stages and there are many targets for anti inflammatory activity. (Tripathi, K.D, 2004). The irrespective tissue injury is a type of inflammatory response suppressed by glucocorticoids and this is the basic clinical uses and also it interferes with several steps in the inflammatory response. Numbers of corticoids are only palliative; do not act on the inflammation instead of they favor spread of infections capacity of defensive cells to kill microorganisms is impaired at the same time interfere with healing and scar formation. The alternate use of corticoids is hazardous other than the corticosteroids the NASIDs are also used to treat inflammation .the main mechanism of action of the NASIDs are the inhibition of prostaglandin (PG) synthesis or preferential or selective COX-2 inhibition. Due to the inhibition of prostaglandin (PG) synthesis it may produce toxic effects like bleeding, inhibition of platelet function, gastric mucosal damage, asthma and anaphylactic reactions may cause some individuals. (Tripathi, K.D, 2004).the plant strychnos potatorum linn commonly known as Nirmali and otherwise called clearing nut. The plant is distributed throughout India, Ceylon, Burma, and Deccan. The present work aims at evaluating the anti inflammatory activity of strychnos potatorum linn seed by HRBC membrane stabilization. MATERIALS AND METHODS Plant material: The fresh seeds of strychnospotatorum Linn were collected from shervaroys hills, Salem district, Tamilnadu, India. The collected seeds were identified and authenticated by the Botanist Dr. A. Balasubramanian (consultant central siddha research) Executive Director ABS Botanical Garden, Salem, Tamilnadu. Preparation of extract: The dried seeds are powdered using mixer grinder; 1000gm of the powdered seeds was packed evenly in the soxhelet extractor and subjected to extraction with Hydroalchol (70% ethanol, 30% water).After extraction, the solvent was distilled off and the extracts were concentrated on water bath to a dry residue and kept in a desiccator. Anti inflammatory activity: The HRBC membrane stabilization has been used as a method to study the anti- inflammatory activity. (Gandidasan.R, 1991) Blood was collected from healthy volunteers. The collected blood was mixed with equal volume of sterilized Alsever solution (2% dextrose, 0.8% sodium citrate, 0.05% citric acid and 0.42 % sodium chloride in water).the blood was centrifuged at 3000 rpm and packed cells were washed with isosaline (0.85 % , pH 7.4) and a 10% v/v suspension was made with isosaline. The assay mixture contains the drug (at various concentrations as mentioned in the table), 1 ml phosphate buffer (0.15 M, pH 7.4), and 2 ml of hypo saline (0.36%) and 0.5 ml of HRBC suspension. Hydrocortisone sodium was used as the reference drug. Instead of hypo saline 2 ml of distilled water was used in the control. All the assay mixtures were incubated at 370 C for 30 minutes and centrifuged. The haemoglobin content in the
  • 141. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Vijayakumar and Hindumathy Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 411 supernatant solution was estimated using spectrophotometer at 560 nm. The percentage of hemolysis was calculated by assuming the hemolysis produced in the presence of distilled water as 100%. The percentage of HRBC membrane stabilization or protection was calculated by using the following formula, RESULTS AND DISCUSSION The lysosomal enzymes released during inflammation produce a various disorders. The extra cellular activity of these enzymes is said to acute or chronic inflammation. The non steroidal drugs act either by inhibiting these Lysosomal enzymes or by stabilizing the membrane. (Rajendran Vadivu, 2008) since HRBC membrane are similar to Lysosomal membrane components the prevention of hypo tonicity induced HRBC membrane lysis is taken as a measure of anti inflammatory activity of drugs. The results were reported in table 1. It was observed from the table 1 and figure 1 that the hydroalcholic extract shows significant anti inflammatory activity at the concentration 100 µg/ml, which is comparable to the standard drug hydrocortisone sodium. The anti inflammatory activity of the extract were concentration dependent, with increasing concentration the activity is also increased. The hydroalcholic extract has significant anti inflammatory activity. Table.1. In-vitro anti inflammatory activity strychnos potatorum linn seed Concentration in ( µg/ml ) Activity ( % protection ) Standard Hydrocortisone sodiumHAESP Control - - 20 43.53±0.62 55.48±0.57 40 46.41±0.87 57.71±0.54 60 49.00±0.50 59.21±0.95 80 52.57±0.68 63.56±0.68 100 54.95±0.74 67.50±0.52 (Values are expressed as SEM of 3 readings) Figure1. In-vitro anti inflammatory activity strychnos potatorum linn seed CONCLUSION The extract exhibited membrane stabilization effect by increasing hypo tonicity induced lysis of erythrocyte membrane. The erythrocyte membrane is analogus to the Lysosomal membrane (Chou, 1977) and its stabilization implies that the extract may as well stabilize Lysosomal membrane. Stabilization of Lysosomal membrane is very important in limiting the inflammatory response by preventing the release of Lysosomal substances of activated neutrophil such as bacterial enzymes and proteases which cause further tissue damages. (Murugasan, 1981) From the investigation it was concluded that the hydroalcholic extract of strychnos potatorum linn has significant membrane stabilization property and it was comparable to the standard drug hydrocortisone. 0 20 40 60 80 20 40 60 80 100 Activity%protection concentration in µg /ml Invitro anti inflammatory effect of strychnos potatorum linn HAESP STANDARD DRUG
  • 142. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Vijayakumar and Hindumathy Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 412 REFERENCES Chou CT, The Anti inflammatory effect of Tripterygium Wilfordihook on adjuvant induced paw edema in rats and inflammatory mediator’s release, Phytother Res, 11, 1997, 152-154 Gandidasan R, Thamaraichelvan A, Baburaj S, Anti inflammatory action of Lannea coromandelica by HRBC membrane stabilization. Fitoterapia, 1991, voll LXII, 81-83. Kirtikar K.R, Basu B.D, Indian Medicinal plants. International book distributors, Dehradum.1975; II edn; Vol III; 1971. Murugasan, Vember, S, Damodharan, C, C. Studies on erythrocyte membrane IV, In-vitro hemolytic activities of Oleander extract .Toxicol Lett, 8, 1981, 33-38. Nirmala Devi K, Periyanayagam K, in-vitro Anti inflammatory activity of Plectrnthus Amboinicus (Lour) spring by HRBC membrane stabilization, International journal of pharmaceutical studies and Research, 1(1), 2010; 26- 29. Rajendran Vadivu, Lakshmi K.S.In vitro and in-vivo anti inflammatory activity of Leaves of Smplocos cochinchinenisis (Lour) Moore ssp Laurina. Bangladesh JPharmacol, 3, 2008, 121-124. Ram P Rastogi, Mehrotra B N, Compendium of Indian Medicinal plants, CDRI Lucknow and publication and information Directorate, New Delhi, 2, 1970, 79-201. Tripathi KD, Essentials of Medical Pharmacology, Jaypee brothers medical publishers (P) Ltd, New Delhi, 2004, V edn; 167-181, 257-259. Warrier P.S, Indian Medicinal plants, Arya Vaidhya Sala, Kottakkal, Orient Longmann limited. Hyderabad, Voll IV, 315-317.
  • 143. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Ramanji Naik Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 413 SYNTHESIS AND CHARACTERIZATION OF 1, 3, 4-OXADIAZOLE AND 1,3,4- THIADIAZOLE Ramanji Naik * Vaagdevi College of pharmacacy and research center, Nellore, AP *Corresponding author: ramspharma40@gmail.com ABSTRACT There are vast numbers of pharmacologically active heterocyclic compounds in regular clinical use. The presence of heterocyclic structures in diverse types of compounds is strongly indicative of the profound effects such structure exerts on physiologic activity, and recognition of this is abundantly reflected in efforts to find useful synthetic drugs. The 1, 3, 4-oxadiazole and 1,3,4- thiadiazole nucleus has emerged as one of the potential pharmacophore responsible for diverse pharmacological properties. Literature is flooded with reports of a variety of biological activities of 2, 5-Disubstituted-1,3,4- oxadiazoles and 1,3,4- thiadiazole. The search for better anticonvulsant drug and the importance of 2,5- disubstituted 1,3,4-oxadiazoles and 1,3,4- thiadiazole as anticonvulsant pharmacophores, prompted us to design, synthesize and evaluate a series of differently substituted 1,3,4-oxadiazoles and 1,3,4- thiadiazole for their potential anticonvulsant activity by autodock software. KEY WORDS: 1, 3, 4-oxadiazole and 1, 3, 4- thiadiazole , synthesis INTRODUCTION The search for new effective and safe drugs has led today’s researchers to improve the existing drugs by increasing their potency, duration of action and decreasing their toxic side effects. Structural activity studies shows that a variation in the ring system or incorporation of different biologically active ring systems or minor group modifications extends distinct biologically active ring systems or minor group modifications extends distinct pharmacological effects upon the drug molecules. The past quarter of a century has seen a great increase in the technical applications of heterocyclic compounds, in a Pharmaceutical field due to their various synthetic strategies. This attracted the researchers and enormous approaches were made from their side to evaluate its activities. MATERIAL AND METHODS Isonicotinic Acid, Indole 3-Acetic Acid Procured from SISCO research lab, Quinoline 2-carboxylic acid, Ethanol PROCURED FROM FISCHER LABS, Phenyl Isothiocyanate, Methanol, Potassium Iodide, Sodium Hydroxide PROCURED FROM SISCO research lab SCHEME STEP I STEP II RCOOH RCOOCl RCONHNH2 NH2NH2 .H2O (Ia-e) (IIa-e) PCl5 , CCl4 , 2 hr STEP III RCONHNH2 + N C S RCONHNHC=S NH Conc H2SO4 I2/KI Ethanolic NaOH O N N H N R S N N H N R (IIa-e) (IIIa-e) (IVa-e) (Va-e) STEP IV
  • 144. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Ramanji Naik Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 414 Table.1.Different substitution in compounds IVa-e and Va-e Compound R Compound R Iva N Va N IVb N H H2C Vb N H H2C IVc N Vc N IVd Cl Vd Cl IVe H2N Ve H2N EXPERIMENT Synthesis General method of Synthesis of Acid chlorides (Ia-e): A mixture of substituted acid (0.03 mole) and phosphorus pentachloride (0.05ml) in anhydrous carbon tetra chloride (20ml) was refluxed for 2 hour at 1000 c. Solvent was distilled off and the solid acid chloride thus obtained was used for further reaction. General method of Synthesis of Acid hydrazides (IIa-e): In the acid chloride (0.03 mole), hydrazine hydrate was added (0.1 mole) drop wise at 00 c and the resultant mixture was stirred for 5 hours at room temperature. A solid that separated out was washed with aqueous sodium carbonate (10%) and dried in vaccum. It was recrystalized from methanol to the pure crystalline solid was obtained. General method of Synthesis of substituted thiosemicarbazide (III-a-e): Substituted thiosemicarbazides (IIIa- e)were synthesized by refluxing substituted acid hydrazides (0.03moles) with phenyl isothiocyanates (0.03moles) in 20 mL of ethanol on a boiling water bath for 5-6 h. After completion of reaction, the reaction mixture was concentrated and kept overnight at room temperature. The needle shaped crystals of thiosemicarbazides so obtained were filtered. Synthesis of compound (IVa-e): 2-(phenyl) amino-5-substituted-1,3,4-thiadiazoles (IVa) were synthesized by cyclization of substituted thiosemicarbazides (0.004 mole) with sulfuric acid at 0-5O C. After completion of reaction, the mixture was poured onto crushed ice; the solid so separated was filtered, washed with water and recrystallization from methanol yielded the pure compound. Synthesis of compound (Va): A solution of substituted thiosemicarbazide (0.004 mole) and NaOH (5 M, 2 ml) in 25ml of absolute ethanol was cooled with continuous stirring for half an hour. To this mixture, iodine in KI (5%) was added drop wise, till the color of iodine persisted at room temperature and the mixture was refluxed for 2 h on a water bath. After completion of reaction, the mixture was poured onto crushed ice and the solid so separated was filtered and washed with water. The recrystallization from petroleum ether:diethyl ether mixture (8:2, v/v) gave the pure compounds. RESULTS AND DISCUSSION Synthesis: A series of five membered thiadiazole and oxadiazole heterocyclic compounds was synthesized from substituted thiosemicarbazides were obtained by the reaction between various heterocyclic or aromatic acid hydrazides and phenyl isothiocyanate. The acid hydrazides were prepared by chlorination of the corresponding acids, followed by treatment with hydrazine hydrate in 0-5o C.
  • 145. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Ramanji Naik Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 415 SUMMARY AND CONCLUSION Much attention has been paid to 1, 3, 4-oxadiazole and thiadiazole derivatives in recent years, because of their showing anti-inflammatory, hypoglycemic, anticonvulsant, antimicrobial, anticancer and other activities. A series of five membered thiadiazole and oxadiazole heterocyclic compounds was synthesized from substituted thiosemicarbazides were obtained by the reaction between various heterocyclic or aromatic acid hydrazides and phenyl isothiocyanate. The acid hydrazides were prepared by chlorination of the corresponding acids, followed by treatment with hydrazine hydrate in 0-5o C. Melting points were determined by open capillary tube method and are uncorrected. Purity of the compounds was checked on thin layer chromatography (TLC) plates (Silica gel G) in the solvent system toluene: ethyl acetate: formic acid (5:4:1, v/v/v). The spots were located under iodine vapors and UV light. Docking studies for the synthesized compounds were carried out against androgen receptor for bearing the PDB id- 3PMX by the use of molecular docking software’s such as PYMOLWIN and Auto DOCK and found that all the compounds showed good binding interaction with the receptor. The study also showed that ARG 182 and SER 14 is the target site for binding of the drug to show anti-convulsant activity. Compounds Ve and IVe were found to be more active when compared to other synthesized derivatives. Further, Characterization by IR, 1H NMR and Mass spectra and in vivo studies for the synthesized compounds has to be carried out to confirm the pharmacological activity. Table.2.Structure and IUPAC Name of the synthesised Oxadizoles and thiadiazole derivatives ompound Code Structure of the Compound IUPAC name IVa S N N H N N N-phenyl-5-(pyridin-3-yl)-1,3,4- thiadiazol-2-amine IVb S N N H N H N C H2 5-((1H-indol-3-yl)methyl)-N- phenyl-1,3,4-thiadiazol-2-amine IVd S N N H N Cl 5-(4-chlorophenyl)-N-phenyl-1,3,4- thiadiazol-2-amine IVe S N N H N O2N 5-(4-nitrophenyl)-N-phenyl-1,3,4- thiadiazol-2-amine Va O N N H N N N-phenyl-5-(pyridin-3-yl)-1,3,4- oxadiazol-2-amine Vb O N N H N H N C H2 5-((1H-indol-3-yl)methyl)-N- phenyl-1,3,4-oxadiazol-2-amine Vd O N N H N Cl 5-(4-chlorophenyl)-N-phenyl-1,3,4- oxadiazol-2-amine Ve O N N H N O2N 5-(4-nitrophenyl)-N-phenyl-1,3,4- oxadiazol-2-amine
  • 146. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Ramanji Naik Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 416 Table.3. Physico-chemical data of the synthesised Oxadiazole and thiadiazole Compound code Molecular formula Colour Appearance/ Nature Melting point Solubility TLC Yield Rf value Iva C13H10N4S white crystalline 136-38 Soluble in chloroform 0.72 74.32 IVb C17H14N4S brown powder 142-44 Soluble in chloroform 0.62 77.27 IVc C17H12N4S white powder 168-70 Soluble in chloroform 0.79 73.35 IVd C14H10ClN3S white crystalline 152-54 Soluble in chloroform 0.65 87.56 IVe C14H12N4S brown powder 138-40 Soluble in chloroform 0.69 85.72 Va C13H10N4O white crystalline 266-68 Soluble in chloroform 0.66 76.82 Vb C17H14N4O brown powder 278-80 Soluble in chloroform 0.54 73.56 Vc C17H12N4O white powder 280-82 Soluble in chloroform 0.70 71.67 Vd C14H10ClN3O white crystalline 238-40 Soluble in chloroform 0.58 86.33 Ve C14H12N4O brown powder 236-38 Soluble in chloroform 0.63 84.26 *Solvent used for TLC= Toluene: Ethyl acetate: Formic acid (5:4:1), *Detecting agent – Iodine Table.4. Autodock result of Compound IVa Conformation Binding energy Inhibitory constant Ref RMS Hydrogen bond (Residue Name And Distance) 1 -9.58 94.87 n/a none 2 -4.83 289.23 n/a none 3 -5.36 117.71 n/a SER14:HN1:..N (1.833 A0 ) MET19:HN:..N (2.034 A0 ) 4 -4.45 548.91 n/a none 5 -9.93 52.26 n/a LEU94:O:..S (2.966 A0 ) 6 -9.29 154.41 n/a none 7 -9.72 344.13 n/a THR174:HN:..N (2.029 A0 ) LEU12:O:..S 8 -10.6 17.12 n/a ILE100:O:..N (2.923A0 ) 9 -4.65 392.35 n/a none 10 -4.87 268.09 n/a THR174:HG1:..N (2.056 A0 ) THR174:HN:..N (2.118 A0 ) LEU12:O:..S Table.5. Autodock result of Compound IVb Conformation Binding energy Inhibitory constant Ref RMS Hydrogen bond (Residue Name And Distance) 1 -5.35 120.78 n/a none 2 -4.93 242.14 n/a none 3 -4.55 463.08 n/a none 4 -5.19 157.39 n/a SER14:HN2:..N (2.073 A⁰) 5 -5.35 119.0 n/a none 6 -4.4 594.47 n/a none 7 -4.67 378.96 n/a MET19:HN:..N (2.151 A⁰) 8 -5.23 161.23 n/a none 9 -5.61 122.32 n/a SER14:HN1:..N (1.921 A⁰) 10 -5.47 118.21 n/a none
  • 147. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Ramanji Naik Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 417 Table.6. Autodock result of Compound IVc Conformation Binding energy Inhibitory constant Ref RMS Hydrogen bond (Residue Name And Distance) 1 -5.74 61.84 n/a none 2 -5.07 192.31 n/a none 3 -5.33 123.97 n/a none 4 -5.27 136.93 n/a LEU12:O:..S (2.616 A⁰) 5 -5.33 123.22 n/a none 6 -5.2 154.5 n/a LEU12:O:..N (2.928 A⁰) 7 -5.94 44.42 n/a SER14:HN2:..N (2.19 A⁰) 8 -5.11 131.53 n/a none 9 -5.39 58.89 n/a none 10 -5.31 133.97 n/a LEU12:O:..S (2.527 A⁰) Table.7. Autodock result of Compound IVd Conformation Binding energy Inhibitory constant Ref RMS Hydrogen bond (Residue Name And Distance) 1 -5.35 120.78 n/a none 2 -4.93 242.14 n/a none 3 -4.55 463.08 n/a none 4 -5.19 157.39 n/a SER14:HN2:..N (2.073 A⁰) 5 -5.35 119.0 n/a none 6 -4.4 594.47 n/a none 7 -4.67 378.96 n/a MET19:HN:..N (2.151 A⁰) 8 -5.23 161.23 n/a none 9 -5.61 122.32 n/a SER14:HN1:..N (1.921 A⁰) 10 -5.47 118.21 n/a none Table.8. Autodock result of Compound IVe Conformation Binding energy Inhibitory constant Ref RMS Hydrogen bond (Residue Name And Distance) 1 -5.23 146.38 25.61 LYS104:HZ3:..O 1.775 A⁰ 2 -6.15 31.28 21.67 ARG182:HH12:..O 1.9 A⁰ 3 -5.86 50.41 20.85 ASN214:HD22:..O 2.018 A⁰ LYS251:HZ3:..N 1.983 A⁰ 4 -22.05 68.76 32.44 none 5 -5.87 49.39 22.5 ARG203:HE:..O 2.041 A⁰ 6 -5.25 141.25 21.0 LYS183:HZ3:..O 1.783 A⁰ 7 -5.42 106.12 23.8 ARG182:HH12:..O 1.765 A⁰ 8 -21.18 297.62 30.15 none 9 -6.64 13.55 21.62 ARG182:HH12:..O 2.007A⁰ 10 -6.28 24.95 21.63 SER14:HN2:..N 2.084 A⁰ ARG182:HH12:..O 1.727 A⁰
  • 148. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Ramanji Naik Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 418 Table.9. Autodock result of Compound Va Conformation Binding energy Inhibitory constant Ref RMS Hydrogen bond (Residue Name And Distance) 1 -5.57 82.19 n/a SER14:NH2:..N 2.232 A⁰ THY61:HH:..N 1.916 A⁰ 2 -4.61 419.09 n/a THR174:HG1:..N 1.991 A⁰ THR174:HN:..N 2.139 A⁰ LEU12:O:..O 3 -4.49 513.37 n/a SER14:HN1:..O 2.131 A⁰ 4 -5.12 176.89 n/a SER14:O:..N 2.563 A⁰ 5 -4.39 607.58 n/a PHE102:HN:..N,N 2.158A⁰ ILE100:O:..N 6 -4.52 487.57 n/a none 7 -5.01 212.17 n/a THR174:HN:..N 2.121 A⁰ 8 -4.73 407.29 n/a THR174:HN:..N 2.023 A⁰ 9 -4.47 520.35 n/a SER14:HN1:..O 2.011 A⁰ 10 -5.26 146.87 n/a SER14:NH2:..N 2.123 A⁰ Table.10. Autodock result of Compound Vb Conformation Binding energy Inhibitory constant Ref RMS Hydrogen bond (Residue Name And Distance) 1 -5.55 84.85 n/a LEU12:HN:..N (1.965A0 ) 2 -5.15 168.00 n/a none 3 -4.76 321.79 n/a MET19:HN:..N (1.887A0 ) 4 -5.13 173.75 n/a none 5 -4.73 339.34 n/a ALA175:NH:..N(2.026 A0 ) 6 -5.09 185.26 n/a none 7 -4.69 355.29 n/a ASN83:HD22:..O (2.151 A⁰) 8 -4.87 197.21 n/a MET19:HN:..N (1.927A0 ) 9 -4.63 341.37 n/a none 10 -4.59 183.23 n/a none Table.11. Autodock result of Compound Vd Conformation Binding energy Inhibitory constant Ref RMS Hydrogen bond (Residue Name And Distance) 1 -5.01 211.24 40.2 none 2 -4.89 261.15 39.49 none 3 -4.74 335.84 40.44 none 4 -4.09 998.83 41.13 LEU52:HN:..O (2.035 A⁰) LYS50:O:..N 5 -5.01 211.62 39.76 none 6 -4.98 223.17 39.73 none 7 -3.53 2.58 48.51 ASN83:HD22:..O (2.151 A⁰) 8 -5.01 212.84 39.91 none 9 -5.01 213.61 40.01 none 10 -4.88 265.81 39.36 none
  • 149. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Ramanji Naik Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 419 Table.12. Autodock result of Compound Ve Conformation Binding energy Inhibitory constant Ref RMS Hydrogen bond (Residue Name And Distance) 1 -6.89 8.92 19.06 SER14:HN2:..N(2.08A0 ) ARG182:HH12:..O(2.185A0 ) ARG182:HH22:..O(1.942A0 ) ALA175:NH:..N(2.125 A0 ) 2 -6.99 7.46 19.04 ARG182:HH12:..O(1.806A0 ) ARG182:HH22:..O(1.775A0 ) ALA175:NH:..N(2.184 A0 ) 3 -6.03 37.7 20.26 TYR199:HH:..O(2.249A0 ) ARG203:HH22:..O(1.87A0 ) 4 -6.06 36.43 22.58 MET19:HN:..O(1.997A0 ) 5 -6.9 8.78 18.84 ARG182:HH22:..O(2.045A0 ) 6 -6.92 8.43 19.4 SER14:HN2:..N(2.126A0 ) ARG182:HH12:..O(1.94A0 ) ARG182:HH22:..O(1.865A0 ) 7 -6.49 17.39 19.06 SER14:HN2:..N(1.963A0 ) ARG182:HH12:..O(1.801A0 ) ARG182:HH22:..O(2.022A0 ) 8 -6.93 8.32 19.06 ARG182:HH12:..O(1.805A0 ) ARG182:HH22:..O(1.884A0 ) 9 -6.51 16.87 20.2 LEU94:HN:..N(2.169A0 ) ARG148:HE:..O(1.638A0 ) ARG148:HH22:..O(1.908A0 ) 10 -5.94 44.18 20.2 ARG182:HH12:..O(2.18A0 ) ARG182:HH22:..O(1.855A0 ) REFERENCES Jumat Salimon, Nadia Salih, Ayad Hameed, Hiba Ibraheem, Emad Yousif, Synthesis and Antibacterial Activity of Some New 1,3,4-Oxadiazole and 1,3,4-Thiadiazole Derivatives, Journal of Applied Sciences Research, 6(7), 2010, 866-70. Khare R K and Sing H, Srivastava A K, Synthesis and fungicidal activity of some 3-(5-aryl-1,3,4-thiadiazol- 2-yl)-1-(β-Dglocopyranosyl)-5-alkyl-2-thoio-4-imidazo lidinones, Ind J Chem, 46B, 2007, 875-9. Löscher W, New visions in the pharmacology of anticonvulsion, Eur J Pharm, 342, 1998, 1-13. Mohammad Shahar Yar And Mohammad Wasim Akhter, Synthesis and anticonvulsant activity of substituted Oxadiazole and thiadiazole derivatives, Acta Poloniae Pharmaceutica - Drug Research, 66(4), 2009, 393-7. Srivastava K and Pandeya S N, Synthesis and anticonvulsant activity of 3-arylamino-4- aryl-5(N-4- chlorophenylthiocarbamido)-1,2,4- thiadiazoles. Bioorg and Med Chem, 3, 1993, 547-55. Wei B T and Zhang Y M, Li M N, Synthesis and bioactivity study of 2,5- bismercapto-1,3,4-thiadiazoles heterocyclic derivatives. Ind J Chem, 46, 2007, 544-9. Zarghi A, Hajimahdi Z, Mohebbi S, Rashidi H, Mozaffari S, Sarraf S, Faizi M, Tabatabaee SA, Shafiee A, Design and synthesis of new 2-substituted-5-[2-(2-halobenzyloxy)phenyl]-1,3,4-oxadiazoles as anticonvulsant agents, Chem. Pharm. Bull, 56(4), 2008, 509–12.
  • 150. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Pruthvi Raj et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 420 PREPARATION, CHARACTERIZATION AND EVALUATION OF OLMESARTAN MEDOXOMIL-Β CYCLODEXTRIN COMPLEXES V. Prudhvi Raj*, Subhashis Debnath, Maleswari, M. Niranjan Babu Department of Pharmaceutics, Seven Hills College of Pharmacy, Venkatramapuram, Tirupati – 517 561 * Corresponding author: prudhvikalyan34@gmail.com ABSTRACT Olmesartan medoxomil is a poor water soluble drug. There are numerous approaches available and reported in literature to enhance the solubility of poor water soluble drugs among which cyclodextrin complexation is predominant. β-cyclodextrin was used since this possess a special ability to complex with drugs enabling them to increase solubility, reduce bitterness, enhance stability and decrease tissue irritation upon dosing. Olmesartan medoxomil-β cyclodextrin complexes were prepared and characterized by FT-IR and SEM, studies. The results showed the formation of true inclusion complexes at molar ratio 1:5. In contrast crystalline drug was detectable in all other products. The dissolution of Olmesartan medoxomil from all the prepared complexes has been carried out to determine the most appropriate ratio that can be used for further development like tablet formulation for oral delivery. The complexes prepared by physical mixture method in 1:5 ratio showed superior dissolution profile when compared to complexes prepared in other ratios. From this research work it can be concluded that β CD has potential to increase the solubility of Olmesartan medoxomil and true complexes are formed in 1:5 ratio and because of the higher dissolution profile it can be used for further formulations. KEY WORDS: β-cyclodextrin, complex, inclusion complexes, dissolution INTRODUCTION Orally administered drugs completely absorb only when they show fair solubility in gastric medium and such drugs shows good bioavailability. Recently more than 40% NCEs (new chemical entities) developed in Pharmaceutical Industry are practically insoluble in water. These poorly water soluble drugs are allied with slow drug absorption leading to inadequate and variable bioavailability and gastrointestinal mucosal toxicity. Therefore, the improvement of drug solubility thereby its oral bio-availability remains one of most challenging aspects of drug development process especially for oral drug delivery system. There are numerous approaches available and reported in literature to enhance the solubility of poorly water soluble drug. The techniques are chosen on the basis of certain aspects such as properties of drug under consideration, nature of excipients to be selected and nature of intended dosage form. Among all the solubility enhancement techniques inclusssion complex formation technique has been employed more precisely to improve the aqueous solubility, dissolution rate, and bioavailability of poorly water soluble drugs. Inclusion complexes are formed by the insertion of the nonpolar molecule or the nonpolar region of one molecule (known as guest) into the cavity of another molecule or group of molecules (known as host). The most commonly used host molecules are cyclodextrins. Olmesartan is one of the widely used selective AT1 subtype angiotensin II receptor antagonist which is marketed under the names of Benicar in US, Olmetac in EU and Canada, Golme in India. As many of the drugs even Olmesartan medoxomil is a poor water soluble drug. The present work aims to investigate the potential of β cyclodextrin (β CD) complexes to increase the solubility of Olmesartan Medoxomil. MATERIAL & METHODS Olmesartan medoxomil (MSN Laboratories, Hyderabad), β cyclodextrin (Merck limited, Germany), HCl (Sd fine chemicals Ltd, Mumbai) Phase solubility studies: Phase-solubility studies were carried out with double distilled water according to the method described by Higuchi and Connors (1965). Excess amount of Olmesartan medoxomil (100 mg) was added to 1ml stock solution with varying concentrations (3, 6,9,12, 15 and 18 mM) of β-CD. The suspensions were then shaken at 37.0±0.5 ◦C for at least 2 days. After equilibrium attainment, the samples were filtered and the concentration of Olmesartan medoxomil was determined spectrophotometrically on a UV- spectrophotometer at 260 nm. Amount of drug present in the samples were calculated by using USP-Disso-v3 software. The complexation efficiency, which reflects the solubilizing power of CDs toward the drug, was calculated from the tredline of the phase solubility diagram according to the equation
  • 151. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Pruthvi Raj et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 421 CE=Slope/1-slope=SoK1:1 Where so represents the solubility of the drug in the absence of cyclodextrin K1:1 represents the apparent solubility constant = Slope/ So (1-slope) Synthesis of inclusion complexes: Inclusion complexes of Olmesartan medoxomil with β cyclodextrin was prepared by the Physical mixture method. The Olmesartan medoxomil - β cyclodextrin inclusion complexes in the ratios 1:1, 1:2, 1:3, 1:4, 1:5 and 1:6 (molar ratios) were prepared by mechanical trituration by grinding the accurately weighed amounts in a geometrical dilution method in the mortar and pestle for 30 min. EVALUATION Characterization of prepared complexes: Characterization studies of the Olmesartan medoxomil - β cyclodextrin complexes by FT-IR and SEM were performed. Invitro dissolution studies: Dissolution studies of samples weight equivalent to 20 mg were performed using USP XXII apparatus at the stirring speed of 50 rpm and temperature maintained at 37±0.50C with 0.1N HCl as dissolution medium. The samples of 5ml were withdrawn at regular intervals of 5,15,30,45,60 min. Every time the samples are replaced with 5ml of the fresh dissolution medium. The filtrates of the samples were analyzed at 260 nm. Percentage drug release and other parameters of the samples were calculated by using Disso software PCP Disso V3 software. The dissolution in pH 6.8 phosphate buffer, pH 7.4 phosphate buffer has been performed in the aforementioned procedure. RESULT AND DISCUSSION Phase Solubility Studies: The aqueous solubility of Olmesartan medoxomil at various concentrations of β CD was studied. It was found that the solubility of drug increased with the increasing concentration of β CD. The main reason might be the formation of inclusion complexes however other reasons like formation of aggregations of cyclodextrin which promote the solubility of drugs might also have been involved. The coefficient of determination (r2 ) value of the phase solubility diagram of Olmesartan medoxomil is found to be 0.9878 (<0.990) therefore the diagram can be classified as Ap-type curve. This positive deviation suggests the formation of higher order inclusion complexes with respect to β-cyclodextrin. The complexation efficiency of the curve calculated was 0.04395. Phase solubility curve for the system was found to be of Higuchi’s AP type phase solubility curve was presented in the Figure No. 1, Table No. 1. Table No.1: Phase solubility studies Molar conc. of β-cyclodextrin (*10-3 ) Molar conc. of Olmesartan medoxomil (*10-5 ) 3 0.06 6 0.09 9 0.12 12 0.16 15 0.21 18 0.26 Figure No. 1: Phase solubility studies of olmesartan medoxomil
  • 152. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Pruthvi Raj et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 422 The practical yield obtained and the Percentage yield for drug- β-cyclodextrin complexes prepared in different ratios was given in the table no. 2. Table no.2: percentage yield of the prepared complexes of different ratios Inclusion complexes ratio %yield 1:1 90.6 1:2 89.4 1:3 91.2 1:4 92.4 1:5 91.6 Evaluation of Inclusion Complexes: Fourier Transform Infrared (FT-IR) Spectroscopy of Olmesartan medoxomil- β cyclodextrin complexes: The FT-IR studies showed that the significant peaks of Olmesartan medoxomil are at showed the characteristic peak at 3286.81which can be assigned to the O-H group and a peak at 1737.2 which can be assigned to the presence of Ester carbonyl group. The absence of peaks at 3286.81, 1282.71 and 1552.75 and reduction of intensities of peaks at 1707.06 and 1832.44 to considerable level in the complexes prepared in the ratio 1:5 confirms the formation of true inclusion complex with β-cyclodextrin in 1:5 ratio(Table no 3). Table No.3: comparison of interpretation of IR spectrum of drug, β CD and complexes Olmesartan medoxomil Peak assignment Drug: β CD 1:1 1:2 1:3 1:4 1:5 3286.81 O-H group 3306.10 3317.67 absent absent absent 1282.71 C-N stretching 1282.71 1282.71 absent absent absent 1707.06 Diaryl ketone 1707.06 1707.06 1707.06 1707.06 1707.06 1737.2 Ester carbonyl group 1832.44 1832.44 1832.44 1832.44 1832.44 1552.75 C=C aromatic stretching 1550.82 absent absent absent absent Scanning Electron Microscopy (SEM) Studies: To study about the shape and surface characteristics SEM studies were performed for the individual components and Olmesartan medoxomil- β cyclodextrin complexes. Drug and β-CD inclusion complexes of drug showed significant difference in the microscopic structure. The reduction in the crystallinity was observed in the inclusion complexes. Reduced size of the complexes is due to the method of preparation used, physical mixture method for preparing complexes. Comparative SEM photographs for Olmesartan medoxomil and Complexes were given in Figure No. 2. Except the complexes prepared in 1:5 ratio, the SEM images of remaining complexes showed the presence of minute amounts of drug (irregular shaped particles) which confirms the formation of the true inclusion complexes in 1:5 ratio which supports the results obtained from FT-IR.
  • 153. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Pruthvi Raj et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 423 a. Pure Drug b. β CD c. Drug : β CD (1:1) d. Drug : β CD (1:2) e. Drug : β CD (1:3) f. Drug : β CD (1:4) g. Drug : β CD (1:5) Figure No. 2: Comparative SEM images of Drug-β CD complexes in different ratios In-vitro dissolution studies: The results from dissolution studies carried in 0.1 N HCl and also with pH 6.8 phosphate buffer and pH 7.4 phosphate buffer showed that there is cyclodextrin complexes showed improved dissolution profile with 1:5 complexes the showing the highest. The dissolution studies in 0.1N HCl revealed that drug release for pure drug at 60 min is 33.75% while the complexes prepared in 1:5 ratio exhibited drug release of 65.93% at 60 min. In 6.8 buffer for pure drug release at 60 min is 37.83% whereas the complexes prepared in 1:5 ratio exhibited 76.49% of drug release at the end of 60 min. In 7.4 buffer for pure drug release at 60 min is 38.99% whereas the complexes prepared in 1:5 ratio exhibited 74.83% of drug release at the end of 60 min. The improved release of the drug can be attributed to the formation of the inclusion complex with β CD. Invitro dissolution profile for both pure drug and formulation along with standard deviations in 0.1 N HCl was given in Table No.4 [Figure No.3] for pH 6.8 phosphate buffer was given in Table No.5 [Figure No.4] for pH 7.4 phosphate buffer was given in Table No.6 [Figure No.5] and the comparison of dissolution profile for pure drug and complexes prepared in the ratio 1:5 was given in Table No.7.
  • 154. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Pruthvi Raj et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 424 Table no.4: Dissolution profile for pure drug and complexws in 0.1N HCL Time (min) Average % drug release (n=2) Pure drug 1:1 1:2 1:3 1:4 1:5 0 0 0 0 0 0 0 5 30.35 34.73 39.25 41.11 44.96 45.62 15 34.11 38.11 40.93 44.79 49.06 52.78 30 34.69 40.98 44.74 47.69 52.78 57.32 45 35.55 40.01 45.25 49.28 57.66 60.95 60 33.75 41.56 46.96 51.27 58.55 65.93 Table no.5: Dissolution profile for pure drug and complexws in pH 6.8 phosphate buffer Time (min) Average % drug release (n=2) Pure drug 1:1 1:2 1:3 1:4 1:5 0 0.00 0.00 0.00 0.00 0.00 0.00 5 34.00 39.25 44.65 46.90 51.70 53.50 15 38.24 43.07 46.55 51.06 56.34 61.59 30 38.90 46.31 50.85 54.34 60.55 66.73 45 39.86 45.21 51.43 56.14 62.68 70.85 60 37.83 46.96 53.37 58.40 67.07 76.49 Table no.6: Dissolution profile for pure drug and complex was in pH 7.4 phosphate buffer Time (min) Average % drug release (n=2) Pure drug 1:1 1:2 1:3 1:4 1:5 0 0.00 0.00 0.00 0.00 0.00 0.00 5 34.45 39.10 44.95 47.50 51.55 53.35 15 35.39 42.92 46.40 51.06 56.18 61.44 30 36.64 46.15 50.71 54.34 60.39 66.43 45 37.59 45.06 51.58 56.14 62.38 70.70 60 38.99 46.20 52.62 57.50 66.02 74.83 Table No.7: In vitro dissolution profile for drug and olmesartan medoxomil-β cyclodextrin inclusion complexes (1:5 ratio) Time 0.1N HCL pH 6.8 phosphate buffer pH 7.4 phosphate buffer Pure drug Complexes (1:5 ratio) Pure drug Complexes (1:5 ratio) Pure drug Complexes (1:5 ratio) 0 0.00 0.00 0.00 0.00 0.00 0.00 5 30.35 45.62 34.00 53.50 34.45 53.35 15 34.11 52.78 38.24 61.59 35.39 61.44 30 34.69 57.32 38.90 66.73 36.64 66.43 45 35.55 60.95 39.86 70.85 37.59 70.70 60 33.75 65.93 37.83 76.49 38.99 74.83
  • 155. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Pruthvi Raj et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 425 Figure No. 3: Dissolution profile for pure drug and complex in 0.1 N HCl Figure No. 4: Dissolution profile for pure drug and complex in pH 6.8 phosphate buffer Figure No. 5: Dissolution profile for pure drug and complex in pH 7.4 phosphate buffer SUMMARY AND CONCLUSION Phase solubility curve for the system was found to be of Higuchi’s AP type phase solubility curve. The coefficient of determination (r2 ) value of the phase solubility diagram of Olmesartan medoxomil is found to be 0.9878 (<0.990). This positive deviation suggests the formation of higher order inclusion complexes with respect to β-cyclodextrin. The complexation efficiency of the curve calculated was 0.04395. Results from SEM studies confirmed the formation of true complexes of drug Olmesartan medoxomil with β CD in 1:5 ratio supporting the results from FT-IR. The results from dissolution studies carried in 0.1 N HCl and also with pH 6.8 phosphate buffer and pH 7.4 phosphate buffer showed that cyclodextrin complexes showed
  • 156. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Pruthvi Raj et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 426 improved dissolution profile and complexes prepared in the ratio 1:5 is showing the highest dissolution profile which exhibited drug release of 65.93% in 0.1N HCl and exhibited 76.49% of drug release in pH 6.8 phosphate buffer and 74.83% of drug release in pH 7.4 phosphate buffer at the end of one hour. REFERENCES Adam M Persky and Jeffrey A Hughes, Solutions and Solubility, Referred in the website http://www.cop.ufl.edu/safezone/prokai/ pa5100 /pha5110.htm. Accessed on 2/2/2012 Blagden N, Gavan P T, York P, Crystal engineering of active pharmaceutical ingredients to improve solubility and dissolution rates, Adv. Drug Del. Rev, 59(30), 2007, 617-630. 33. Burt VL, Cutler JA, Higgins M, Trends in the prevalence, awareness, treatment, and control of hypertension in the adult US population, Data from the health examination surveys, 1960 to 1991, Hypertension, 26(1), 1995, 60–9. Carretero OA, Oparil S, Essential hypertension, Part I: definition and etiology, Circulation, 101 (3):2000, 329–35. Chaumeil J C, Micronization: a method of improving the bioavailability of poorly soluble drugs, 20(3), 1998, 211-5. Chen AZ, Pu XM, Kang YQ, Liao L, Yao YD, Yin GF, Preparation of 5 fluorouracil-poly(L-lactide) microparticles using solution-enhanced dispersion by supercritical CO2, Macromol, Rapid Commun, 27, 2006, 1254- 1259. Chingunpituk J, Nanosuspension Technology for Drug Delivery, Walailak J Sci & Tech, 4(2), 2007, 139-153 Chowdary K P R and Madhavi B L R, Novel drug delivery technologies for insoluble drugs, Ind. Drugs, 42(9), 2005, 557-563. Dohrn R, Bertakis E, Behrend O, Voutsas E, Tassios D, Melting point depression by using supercritical CO2 for a novel melt dipersion micronization process, J. Mole. Liq, 2007; 131-132. Kearney PM, Whelton M, Reynolds K, Muntner P, Whelton PK, He J, Global burden of hypertension: analysis of worldwide data, Lancet, 365, 2005, 217–23. Kearney PM, Whelton M, Reynolds K, Whelton PK, He J, Worldwide prevalence of hypertension: a systematic review, J Hypertens, 22 (1), 2004, 11–9. Keck C M and Muller R H, Drug nanocrystals of poorly soluble drugs produced by high pressure homoginisation, Eur. J. Pharm. Biopharm, 62, 2006, 3 16. Kim J H, Paxton T E, Tomasko D L, Microencapsulation of naproxen using rapid expansion of supercritical solutions, Biotechnol Prog, 12(5), 2006, 650-661. Krober H and Teipel U, Materials processing with supercritical antisolvent precipitation: process parameters and morphology of tartaric acid, The Journal of Supercritical Fluids, 22(3), 2002, 229-235. Muhrer G, Meier U, Fusaro F, Albano S and Mazzotti M, Use of compressed gas precipitation to enhance the dissolution behavior of a poorly water-soluble drug: generation of drug microparticles and drug-polymer solid dispersions, Int. J. Pharm, 308, 2006, 69-83. Muller R H, Jacobs C and Kayer O, Nanosuspensions for the formulation of poorly soluble drugs, In: F Nielloud, G Marti-Mestres, Pharmaceutical emulsion and suspension, New York, Marcel Dekker, 2000; 383-407
  • 157. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Pruthvi Raj et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 427 Nash RA, Suspensions, In: J Swarbrick, JC Boylan (ed) Encyclopedia of pharmaceutical technology, Second edition, vol. 3. New York, Marcel dekker, 2002; 2045-3032. Patravale V B, Date A A and Kulkarni R M, Nanosuspensions: a promising drug delivery strategy, J. Pharm. Pharcol, 56, 2004, 827-840. Phillips E M, Stella V J, Rapid expansion from supercritical solutions: application to pharmaceutical processes.” Int. J. Pharm, 94, 1993, 1-10. Reverchon E and Della Porta G, Production of antibiotic micro- and nanoparticles by supercritical antisolvent precipitation, Powder Technol, 106, 1999, 23-29. Rinaki E, Valsami G, and Macheras P, Quantitative Biopharmacuetics Classification System; the central role of dose/solubility ratio, Pharm. Res, 20, 2003, 1917 Sekiguchi K, Obi N, Studies on absorption of eutectic mixtures, I.A. comparison of the behaviour of eutectic mixtures of sulphathiazole and that of ordinary sulphathiazole in man, Chem. Pharm. Bull, 9, 1961, 866-872. Sunkara G, Kampala U B, Drug delivery applications of supercritical fluid technology, Drug. Del. Technol, 2, 2002, 44-50. Williams RQ, Process for Production of Nanoparticles and Microparticles by Spray Freezing into Liquid, US Patent, 20030041602, 2003 Preparation, characterization and evaluation of Olmesartan medoxomil-β cyclodextrin complexes.
  • 158. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Vibhooti and Preethi Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 428 WAFERS TECHNOLOGY – A NEWER APPROACAH TO SMART DRUG DILEVERY SYSTEM Papola Vibhooti* , Kothiyal Preeti Shri Guru Ram Rai Institute of Technology & Sciences Dehradun, Uttarakhand, India *Corresponding author: E-mail: Papola.vibhooti@gmail.com ABSTRACT The lyophilized wafer developed throughout this review is an effective and versatile drug delivery system for oromucosal application. This has been established from the extensive physicochemical and physic mechanical profiling conducted. Through a screening and selection of polymers, HPC had the lowest gelation characteristics and was therefore suitable for the development of the wafer system. Suitable excipient and polymer combinations were established which allowed for the development of rapidly disintegrating and prolonged release wafer systems. The wafer system containing HPC, lactose, mannitol and glycine had the ability to disintegrate within 30 seconds. The modified wafer system, consisting of pectin cross linked with zinc ions serving as the drug reservoir, and mucoadhesive polymer combination of pectin, carmellose and gelatin, provided effective release of model drug diphenhydramine hydrochloride over approximately six hours. The modification of this technology to provide a prolonged release mucoadhesive system seems promising. It is envisaged that this system will be applicable to many drugs requiring the extended release of bioactive material. Therefore, the lyophilized wafer matrices developed in this study are highly effective in the rapid delivery of drugs, using the oral route as a site of administration. KEY WORDS: Glioblastoma, glycospingolipids, acylceramides, matrices and lyophilized. INTRODUCTION Therapeutic value and pharmacoeconomic value have in recent years become major issues in defining health care priorities under the pressure of cost containment. The improvement in drug therapy is a consequence of not only the development of new chemical entities but also the combination of active substances and a suitable Delivery system. The treatment of an acute disease or chronic illness is mostly accomplished by delivery of one or more drugs to the patient using various pharmaceutical dosage forms. Tablets, pills, capsules, suppositories, creams, ointments, liquids, aerosols, and injections are in use as drug carriers for many decades (Shayne, 2008) (Bhalla, 1999). These Conventional types of drug delivery systems are known to provide a prompt release of the drug. Therefore, to achieve as well as to maintain the drug concentration within the therapeutically effective range needed for treatment, it is often necessary to take this type of drug several times a day, resulting in the significant fluctuation in drug levels. For all categories of treatment, a major challenge is to define the optimal dose, time, rate, and site of delivery. Recent developments in drug delivery techniques make it possible to control the rate of drug delivery to sustain the duration of therapeutic activity and/or target the delivery of drug to a special organ or tissue. Many investigations are still going on to apply the concepts of controlled delivery for a wide variety of drugs (Chein, 1992). The basic rationale for controlled drug delivery is to alter the pharmacokinetics and pharmacodynamics of pharmacologically active moieties by using novel drug delivery systems or by modifying the molecular structure and or physiological parameters inherent in a selected route of administration. It is desirable that the duration of drug action become more a design property of a rate - controlled dosage form and less, or not at all, a property of the drug molecules’ inherent kinetic properties (Brannon, 1997). The rationale for development and use of novel drug delivery systems may include one or more of the following arguments  Decrease the toxicity and occurrence of adverse drug reactions by controlling the level of drug and/or metabolites in the blood at the target sites.  Improve drug utilization by applying a smaller drug dose in a controlled – release form to produce the same clinical effect as a larger dose in a conventional dosage form.  Control the rate and site of release of a drug that acts locally so that the drug is released where the activity is needed rather than at other sites where it may cause adverse reactions.
  • 159. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Vibhooti and Preethi Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 429  Provide a uniform blood concentration and/or provide a more predictable drug delivery.  Provide greater patient convenience and better patient compliance by significantly prolonging the interval between administrations. The oral mucosa provides the ideal application site for many active ingredients. Their diffusion into the dense network of capillaries ensures direct access to the blood stream –and excellent patient compliance. Wafer – an innovative oral dosage form: New oral thin films, so-called wafers, thus creating new possibilities for action profiles and patient compliance. Wafers are paper-thin polymer films used as carriers for pharmaceutical agents. The innovative dosage form is taken orally but does not require water or swallowing. Effective absorption of active ingredient: The wafer quickly dissolves in the oral cavity, and the active ingredient can be absorbed into the blood - stream via the oral mucosa. The active ingredient, once absorbed by the oral mucosa, thus bypasses the liver’s first-pass effect, which improves bioavailability. Depending on the selected wafer type, the active ingredient’s release may also be delayed. In this case, it is absorbed after swallowing via the gastrointestinal tract. Positive aspects with wafers (industrial point of view):  Attractive dosage form with new active ingredients.  Improvement of established products.  Access to new indications by means of a new absorption profile even for existing active ingredients.  Optimization of bioavailability.  Increase patient compliance.  Innovative technology for product.  Increase of product appeal through innovative format.  Exclusivity and cutting edge technology position in the market through a step forward. Advantages of wafers:  No first – pass effect*  Conrolled release  Improved bio-availability, translates to lower doses  Reduction of side-effects  Reduced impact on the gastro intestinal tract*  Discrete and easy application (no additional intake of liquids required)  Excellent compliance, especially in children and seniors *Applies to all wafer types except the flash-dispersal wafer whose active ingredient is swallowed and subsequently absorbed into the gastro-intestinal tract. Marketing availability till date: Ranbaxy Laboratories received import permission for marketing the US FDA approved product Gliadel (polifeprosan 20 with carmustine implant) Wafer. The company has signed an exclusive licensing agreement with BioPro Pharmaceutical, USA, to promote and market Gliadel Wafer in India. Gliadel Wafer is for the treatment of newly diagnosed high-grade malignant gliomas and recurrent glioblastoma multiforme. There is very limited data available on the incidence of brain tumours in India, according to unofficial sources, the estimated prevalence of CNS tumours in India is two to five new cases per 1,00,000 per year. Another source estimates the total number of cases to be around 21,000 per year. Glioblastoma multiforme constitutes about 60-65 percent of these primary brain tumours. Type of wafers  Flash dissolved wafers  Melt away wafers  Sustained release wafers  Flash dispersed wafers
  • 160. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Vibhooti and Preethi Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 430 Anatomic and Physiological Considerations: Four sites within the buccal cavity have been used for drug administration. The four regions have varying permeability, which plays a role in the absorption of drugs across the oral mucosa. As seen in the four key areas are the buccal cavity, the lingual area, the palate and gingival region. The most commonly used sites for drug administration of the four mentioned above is the sublingual and buccal route. Using the sublingual route, the medicament is placed under the tongue, usually in the form of a rapidly dissolving tablet. The anatomic site for drug administration between the cheek and gingival is known as the buccal mucosa. The oral mucosa is composed of three layers. The first layer is the stratified squamous epithelium; underneath this layer lies the basement Membrane .The basement membrane overlies the lamina propria and submucosa. Fig 1 : Difference between sustained release and flash-dispersal wafers Fig 2 : Difference between flash dissolve and melt – away wafers The constitution of the epithelium within the different sites of the oral cavity shows dissimilarity. The gingival and hard palate are exposed to mechanical stress during eating, hence the epidermis is keratinized in a similar manner as the skin. The epithelium in the soft palate, buccal and sublingual area is not keratinized, therefore not containing ceramides and acylceramides which are associated with providing a barrier function (Danckwerts, 2003). The mucosa of the buccal and sublingual region have only small amounts of ceramide, and is thus more permeable when compared to other regions of the oral cavity (Squier, 1991). The presence of membrane coating granules (MCGs) accounts for the differences in permeability amongst the various regions of the oral mucosa. When cells go through differentiation from basal to flattened keratinous cells, MCGs are formed. At the apical cell surface, MCGs merge with the plasma membrane and their contents are discharged into the intercellular spaces. This occurs mainly in the upper one-third of the epithelium. MCGs are present in both
  • 161. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Vibhooti and Preethi Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 431 keratinized and nonkeratinised epithelia, however their composition is different. On the other hand, non- keratinised epithelium contains MCGs that are nonlamellar and include cholesterol, cholesterol esters and glycospingolipids. A layer of mucus is present on the surface of the epithelial layer of cells. This plays a major role in cell- to-cell adhesion, oral lubrication, as well as mucoadhesion of mucoadhesive drug delivery systems .A major feature in the environment of the oral cavity is the presence of saliva. The salivary glands produce saliva, responsible for protecting the soft tissues from abrasion during the mastication of food Saliva plays an essential role in facilitating the disintegration of quick-disintegrating drug delivery systems (Shojaei, 1998). The buccal and sublingual regions are different from each other in terms of anatomy, permeability to drug, and their ability to retain a drug delivery system for a desired duration. Although the buccal mucosa is less permeable than the sublingual mucosa and does not yield a rapid onset of action as seen with sublingual delivery, mucosa of the buccal area has an expanse of smooth and relatively immobile surface, which is suitable for placement of a retentive system. For buccal drug delivery, adhesion to the oral mucosa permits not only the intimacy of contact and the possibility of improved drug absorption, but also the ability to achieve an optimum residence time at the site of administration (Rathborne, 1994). These characteristics make the buccal mucosa a more appropriate site for prolonged systemic delivery of drugs. The sublingual route is however more suitable for delivery systems formulated either as rapidly disintegrating matrices or soft gels. These systems create a highly significant drug concentration in the sublingual region prior to systemic absorption across the mucosa. Fig 3. : Mucousal region of mouth [10] Fig 4: Composition of layers of mucosal epithelium (a) Keratinised (b) Non kerantinised Fig 5. : Absorption through oral mucosal Mode of Action: The wafer quickly dissolves in the oral cavity, and the active ingredient can be absorbed into the blood - stream via the oral mucosa. The active ingredient, once absorbed by the oral mucosa, thus bypasses the liver’s first-pass effect, which improves bioavailability. Depending on the selected wafer type, the active ingredient’s release may also be delayed. In this case, it is absorbed after swallowing via the gastrointestinal tract. Manufacturing of wafers: The active ingredient in wafer is integrated into a polymer matrix. The typical size of an oral film is between 2cm2 to 10cm2 , with a thickness of 20 micrometre to 500 micrometre.Oral thin films can be composed of a single-layered system.The active ingredient may be prsented within the wafer matix in either a dissolved an emulsified or a dispersed state.If required, it can also be bound in a complex form,for example,to enable taste masking. Open Matrix-Type Wafers and Tablets: With the introduction of the Zydis ® system in the late 1970s, the concept of quick disintegrating drug delivery systems gained much attention. It was the first of this class of delivery systems to be manufactured on a large scale. It is a freeze-dried wafer made from various standard tablet adjuvants. The wafer essentially works on the principle of forming an open network containing the active ingredient. The Zydis ® manufacturing process. The freeze-dried tablet disintegrates within 2-3 seconds, releasing
  • 162. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Vibhooti and Preethi Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 432 the active ingredient. The drug either forms dispersion or dissolves in the saliva, which is then swallowed and absorbed via the GIT. Fig.6.Production of Zydis ® lyophilized wafer [10] The WOWTab ® (With-Out-Water tablet) has been produce by Yamanouchi Pharmaceutical Co. Ltd. (Tokyo, Japan). This tablet is manufactured using conventional granulating and compression. The rapid disintegration is attributed to the blending of a low and high mold ability saccharide. The unique combination of saccharides provides sufficient mechanical strength as well as quick tablet disintegration.Fuisz Technology Ltd. (Chantily, Virginia, USA) developed the Flash Dose ® tablet, which can dissolve in the patient’s mouth in less than 10 seconds. This has been achieved by the use of Shearform ™ technology. The process involves a unique blend of sugars being placed in a fast spinning machine and subjected to flash heat. By this process, long cotton-like fibres called ‘floss’ are produced. The ‘floss’ is then cured by subjecting it to specific environmental conditions that induce crystallisation, at this stage crystallisation modifiers may also be added. The matrix is then blended with coated or uncoated microspheres containing the active drug. The floss is compressed using standard tabletting equipment (Virely, 1990). Fig.7. Manufacturing process of Flash Dose Of the various open matrix-type wafers on the market, the Zydis ® system remains the most popular; as a result making lyophilisation the most frequently used process for the manufacture of these systems. Preparation of wafers: For the formulation of a rapidly disintegrating wafer, a polymer with low gelation characteristics is desired (Misra, 1999). The gelation potential of polymers is highly dependent on it’s’ solubility. Materials and Methods: Polymers utilised in the study include: sodium alginate, hydroxypropylmethyl cellulose (HPMC), hydroxypropyl cellulose (HPC), hydroxyethyl cellulose (HEC), pectin, polyethylene oxide (PEO), polyvinyl alcohol (PVA) (MW 124,000 - 186,000). Additionally, lactose and polystyrene cylindrical moulds of total volume 60.31mm³ (diameter 16mm and depth of 2.4mm) were utilised. Materials used in the preparation of simulated saliva were: Potassium Phosphate Monobasic (KH2PO4), Disodium Hydrogen Phosphate (Na2HPO4), Sodium Chloride (NaCl). Preparation of wafers: Polymers suitable for oramucosal preparations were identified based on information provided in literature. A polymer (1%w/v) and lactose as a bulking agent (6%w/v) was added to deionised water and mixed for 45 minutes. 1.5mL of the various polymer solutions were pipetted into the cylindrical cavities pre- oiled with mineral oil. The formulation was subjected to a freeze-phase in a freeze-dryer at -60°C for 2 hours. The
  • 163. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Vibhooti and Preethi Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 433 drying-phase was executed at a pressure of 25 mtorr for 24 hours. Wafers were stored in glass jars with 2g of desiccant sachets. ANALYSIS OF WAFERS Weight Uniformity: Weight uniformity was used to assess the reproducibility of wafer production process. Individual wafers were weighed, and standard deviations calculated. All experimentation was conducted in triplicate. The reproducibility of the production process was demonstrated by the low standard deviations (SD) calculated from the mass for each of the various polymer systems. Shows the results obtained from the various polymer wafer systems. Mean weight of wafers manufactured (N=3) Polymer Mean (g) ± SD HPC 0.126 ± 0.0017 HPMC 0.122 ± 0.0002 Pectin 0.134 ± 0.0055 PEO 0.119 ± 0.0045 PVA 0.118 ± 0.0011 Sodium alginate 0.109 ± 0.0007 Although the standard deviation of the samples is low, slightly higher values were observed for polymers such as pectin and PEO. This may be attributed to the high viscosity of the initial solution, and therefore greater variability in the production process. Gelation of Matrices: The main objective of this study was to formulate a rapidly dissolving wafer system. Thus the matrix formation characteristics required assessment and formed the basis for the selection of a suitable polymer. Gelation of the dosage form would delay the disintegration and ultimately the release of active substance. A novel method was developed in order to assess the matrix forming profiles of the wafers. Wafers were weighed before being placed in a Petri dish (diameter 85mm, depth 10mm) containing 20mL of simulated saliva (pH 7.1). The Petri dish was agitated for a period of 30 seconds on a Vortex Genie2 on the slowest setting. The contents of the Petri dish were sieved through a stainless steel mesh (pore size 1mm). The mass of the remaining residue was determined on a balance (and used to calculate the rate of matrix formation.The simulated saliva solution comprised 2.38g Na2HPO4, 0.19g KH2PO4 and 8g NaCl in 1000mL of deionised water (Guo, 1994). Determination Limits for Formulation Variables: The lower and upper limits were determined using a trial and error method. Wafers of varying polymer and diluents concentrations (up to 30%w/v of each) were made and inspected visually. Polymers such as sodium alginate, pectin and PEO tended to form a gel-like substance when hydrated and agitated rather than undergo disintegration. Sodium alginate produced the highest amount of residue, possibly due to its low water solubility. In sharp contrast, the highly hydrophilic polymers such as HPC were completely disintegrated within 30 seconds into small particles which were able to penetrate through the pores on the sieve. The mass of intact material after sieving of the various dissolved wafers tested. Based on the results obtained, HPC was identified as the most suitable polymer for the wafer system, because no residue was produced after 30 seconds of hydration and agitation in simulated saliva. This may be attributed to the fact that HPC is highly soluble in polar solvents and therefore undergoes disintegration rapidly without forming a gel residue, ensuring rapid matrix disintegration. Development of the Manufacturing Process: To establish the suitability of a mould in terms of ease of the system removal, well plates, blister packs and disposable polystyrene trays were assessed. To overcome problems of wafers sticking to the mould, various lubricant systems were considered. Magnesium Stearate, Span 60, Maize oil and mineral oil were evaluated for their anti-adhesive properties. It was also necessary to determine suitable timeframes for the lyophilisation process. Established Parameters of Formulation Variables Concentration of HPC: Lower and upper limits were determined to be 1%w/v and 10%w/v respectively. The upper limit of 10%w/v was set because wafers of higher polymer concentrations were difficult to remove from the
  • 164. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Vibhooti and Preethi Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 434 mould. Some wafers produced with polymer concentrations below 5%w/v collapsed. Less than 1%w/v of HPC was not sufficient to form the wafer matrix. Concentration of Diluent: The concentration of the diluent would affect both the solubility and textural properties of the matrices. Lower and upper limits were determined to be 1%w/v and 5%w/v respectively. Concentrations of lactose higher than 5%w/v caused the wafer to be powdery and extremely fragile. Type of Mouldl: A major problem that was encountered was the removal of the wafers from the moulds without disrupting the delicate structure. Polystyrene trays proved to be the most successful, with minimal deformation of the final product as these moulds could be easily split down the middle to release the wafer. Type of Lubricant: As mentioned above, removal of the wafers from the mould was problematic. Mineral oil produced the greatest ease of removal of the product as compared to the other lubricants analyzed, imparting minimal hydrophobicity and having no effect on the taste of the final product as opposed to other substances such as maize oil. Freeze-Drying Parameters: Although the wafers appeared to be dry after a period of 24 hours, ‘melting’ and discoloration of the matrices occurred on storage. This was attributed to moisture present within the products, indicating that the freeze drying process needed to be conducted for a longer period. In future processes, this was increased to 48 hours. Fig.8. Mass of intact wafer after gelation studies using various polymers (N=3) Concluding Remarks: It was necessary to gain a firm understanding of the key factors involved in the successful production of a lyophilised wafer system. HPC was selected as the most appropriate polymer of the seven that were assessed. It was expected that the type of diluent used in the wafer matrix would affect the disintegration rate of the wafers. Mannitol which is more quickly soluble than lactose will be included in the experimental design to assess its influence of this inclusion on the disintegration rate. The diluents will either be used on their own or in a 1:1 combination. To solve the problem of wafers collapsing, Seager (1998) recommended that glycine be used as a collapse protectant. Therefore, concentrations of up to 0.6%w/v will be included in subsequent formulations.The selection of a suitable polymer, determination of future formulation parameters and creation of problem-free manufacturing techniques formed the basis of this part of the study. General Statistical Approach to Wafer Formulation: A Face Centered Central Composite design was developed with 5 factors and 4 centre points (Table 5.1).The equation for the design was as follows: Response = b0 + b1*s + b2*t + b3*u + b4*v + b5*w + b6*s*s + b7*t*t + b8*u*u + b9*v*v + b10*w*w + b11*s*t + b12*s*u + b13*s*v + b14*s*w + b15*t*u + b16*t*v + b17*t*w + b18*u*v + b19*u*w + b20*v*w Where: s = Polymer Concentration; t = Diluent Type; u = Diluent Amount; v = Glycine Concentration; and w = Fill Volume. The responses that are generally measured are:  Disintegration profiles;  Rate of influx of simulated saliva into the matrix;  Friability;
  • 165. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Vibhooti and Preethi Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 435  Matrix yield value;  Matrix tolerance;  Matrix absorption energy;  Matrix resilience; and  Brinell Hardness Number (BHN). Evaluation of CCF Responses ANOVA Test: An Analysis of Variance (ANOVA) was conducted on the input variables of the wafers to determine which input variables had a significant effect on the recorded output properties of the wafers. The ANOVA was carried out using Essential Regression and Experimental Design V2.207 (Tan, 1994). Only the linear terms were used to regress the data, since we were only interested in the effect that each input variable had on the measured output variables at a 95% confidence interval. Disintegration Profiles: The definition of a fast melting (or disintegrating) tablet appeared in a compendia publication for the first time in 1998. However, neither the US Pharmacopeia north European Pharmacopeia have defined a specific disintegration test (Yong, 2001). As a result, a novel method was developed to assess and compare the disintegration profiles of the 30 samples manufactured according to the CCF. Wafers were weighed before being placed in a petri dish containing 20mL of simulated saliva. The dish was allowed to slowly agitate on a vortex mixer for a period of 20 seconds. The contents of the dish were sieved through a stainless steel mesh (pore size 1mm). Particles that were able to pass through the pores of the sieve were considered to be sufficiently disintegrated, while those captured by the sieve were termed the ‘residue’. The residue represents the portion of the wafer that was not sufficiently disintegrated. The residue was measured in both the hydrated and dry state. For wafers that were eroded very rapidly, the agitation time was reduced to 10 seconds. Tests were conducted in triplicate. Based on the measurements documented, the following information was calculated, providing a comprehensive disintegration profile for each wafer formulation: Normalised Percentage Matrix Disintegrated per second (%/s) Similar to the disintegration profiles, the influx of simulated saliva is calculated as a rate, allowing the various formulations to be compared on the unit percentage per second. Friability: Rapidly disintegrating systems prepared by the process of lyophilisation are known for having the characteristic disadvantage of poor physical resistance (Dobetti, 2001). Problems anticipated as a result of this include: breakage of tablet edges during handling and the inability of the tablet to be ejected and removed from a conventional blister alveolus. These features need to be taken into consideration when determining the packaging of the product. Friabilty was measured using a Roche friabilator (Hoffman la Roche, Basel, Switzerland). The wafers (N=3) were accurately weighed before being placed into the friabilator. A rotation time of 4 minutes at 25 rpm was used. Tablets were removed and loose particles brushed off the surface. Wafers were re-weighed and the percentage weight loss was calculated. Structural Analysis His study focuses on the characterisation of matrix resilience, energy of absorption, matrix yield value and matrix tolerance, using the TAXTplus Texture Analyser fitted with a 5kg load cell. Following method of structural analysis:
  • 166. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Vibhooti and Preethi Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 436 Energy of Absorption: The energy of absorption is an indirect indication of the porosity of the wafers. A highly porous wafer will exhibit a greater value for the energy of absorption because energy is accommodated within the voids in the matrix. The energy of absorption is calculated by determining the area under the curve (AUC) of a profile illustrating force (N) and distance (m) Note that for the AUC, the units of Newton metre (Nm) are equivalent to Joules. Matrix Yield Value: This test is indicative of a surface phenomenon, providing information about the superficial, surface structure of the wafer. The matrix yield value is determined by creating a gradient between anchors 1 and 2. Anchor 2 represents the first point of major inflection on the force-distance profile. This is indicative of primary fracture of the wafer matrix which results in a reduction of force. Matrix Tolerance: On further application of force, the residual intact matrix undergoes complete fracture (Figure 5.3). The matrix tolerance value is indicative of the overall strength of the wafer. The second anchor indicates the point of maximum force. The gradient is between anchors 1 and 2 in the matrix tolerance value. This indicates the point at which total collapse of the matrix occurs. Matrix Resilience: Matrix resilience profiles provide us with an understanding of the deformation characteristics and the ability of the wafer to withstand pressure. The calculation of matrix resilience is provided by the ratio of the AUC between anchors 2 and 3 and between anchors 1 and 2. BHN: The BHN is an indication of the force required to indent the surface of the wafer, and is thus a measure of the hardness of the surface of the wafer. BHN is calculated using the following equation. Where: D = Diameter of ball probe = 3.175mm, d = Depth of indentation = 0.25mm, F = Force CONCLUSION The lyophilized wafer developed throughout this review is an effective and versatile drug delivery system for oramucosal application. This has been established from the extensive physicochemical and physic mechanical profiling conducted. Through a screening and selection of polymers, HPC had the lowest gelation characteristics and was therefore suitable for the development of the wafer system. Suitable excipient and polymer combinations were established which allowed for the development of rapidly disintegrating and prolonged release wafer systems. The wafer system containing HPC, lactose, mannitol and glycine had the ability to disintegrate within 30 seconds. The modified wafer system, consisting of pectin cross linked with zinc ions serving as the drug reservoir, and mucoadhesive polymer combination of pectin, carmellose and gelatin, provided effective release of model drug diphenhydramine hydrochloride over approximately six hours. A successful, reproducible, manufacturing technique was established by the optimization of the lyophilisation cycle, employing mineral oil as a lubricant and polystyrene moulds providing wafers of suitable characteristics. Characteristics that were critical to the mechanistic functioning of the wafer, such as rate of matrix disintegration, rate of simulated saliva influx and friability, were extensively elucidated to determine the effects of the formulation variables using ANOVA technology. A low concentration of polymer was associated with a high Disintegration rate, friability and influx of simulated saliva. As predicted, an increase in the amount of diluents present increased both the disintegration rate and friability. The ANOVA method was used to present a comprehensive profile of the physic mechanical properties such as matrix yield value, matrix tolerance, matrix absorption energy, matrix resilience and Brinell hardness number.A firm understanding of the effects of formulation variables on the responses formed the corner stone of the optimization process. Although the DSC did not form a component of the optimization process, the information provided was integral in the determination of the effect of lyophilisation on the native ingredients. Through this analytical process, it was accepted that lyophilisation did not significantly alter the Tg. The aim of this study, to consider formulation variables in the statistical optimisation of the lyophilised wafer system was achieved.
  • 167. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Vibhooti and Preethi Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 437 Future Prospects and Challenges (Dobetti, 2001): Historically, drug delivery has taken the form of injection, infusion, ingestion, and inhalation, with additional variations of each category. For example ingestion may be in tablet, capsule or liquid form; inhalation may be via use of a dry powder inhaler, an MDI, or a nebulizer. The challenge for both drug and drug delivery companies is to deliver both existing and emerging drug technologies in a manner that improves the benefits to the patients, healthcare workers and the healthcare system. Areas that are being targeted for improvements through device development includes  Improved efficacy  Reduced side effects  Continuous dosing (sustained release)  Reduced pain from administration  Increased ease of use  Increased use compliance  Improved mobility  Decreased involvement of healthcare workers  Improved safety for healthcare workers  Reduced environmental impact (elimination of CFC’s) To provide these benefits, a number of approaches are being (or in some cases have been) developed. The common thread running through the approaches is the concept of self-administered, targeted, sustained release with increased bioavailability. Determining which of the emerging approaches best meets stakeholder needs is a complex, multifaceted problem. Although ingestion is probably the most widely accepted form of delivery it presents difficulties for a number of important classes of drugs. Many drug delivery scientists view oral delivery as the ideal drug delivery method. In the case of proteins and peptides, historical oral delivery mechanisms can only delivery bioavailabities of a few percent. In some cases, dose limiting toxicity levels are caused by lack of selectivity. In addition, due to the well known fragility and hygroscopicity of lyophilized products, an appropriate packaging system for the wafers need to be developed to ensure that the dosage form reaches the patient and is administered intact. The modification of this technology to provide a prolonged release mucoadhesive system seems promising. It is envisaged that this system will be applicable to many drugs requiring the extended release of bioactive material. Therefore, the lyophilized wafer matrices developed in this study are highly effective in the rapid delivery of drugs, using the oral route as a site of administration. The manufacturing process is simple and reproducible. A number of unique opportunities are presented for the formulation of a controlled release drug delivery system. Fig 9: Calculation of energy of absorption (i.e. AUC) Fig.10.Determination of matrix yield
  • 168. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Vibhooti and Preethi Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 438 Fig.11. Determination of matrix tolerance Fig 12 : Determination of matrix resilience Fig.13. Force – Distance profile for the computation of the BHN REFERENCE Shayne COX GAD, Pharmaceuticals Manufacturing Handbook of Production and Process, A John Wiley & Son, INC, Publication, 5(1), 2008, 348. Bhalla HL, Drug delivery Research in India a challenges and opportunity, J Controlled Release, 62, 1999, 65-68. Chein YW, Novel Drug Dilevery Systems , Marcel Dekker , New York 1992, 1-2 Brannon - Peppas L, Polymer in Controlled Drug Delievery, Biometirial, 11, 1997, 1-14. Danckwerts MP, Intraoral drug delivery: A comparative review, Amer. J. Drug Del, 1, 2003, 149-224 Squier CA , The permeability of oral mucosa, Crit. Rev. Oral Biol. Med, 2, 1991, 13-32 Shojaei AH, Buccal mucosa as a route for systemic drug delivery: A review, J. Pharm. Sci, 1(1), 1998, 15-30 Rathborne M, Drummond B and Tucker I, Oral cavity as a site for Systemic drug delivery, Adv. Drug Deliv. Rev, 13, 1994, 1-22. Virely P and Yarwood RJ, Zydis: a novel, fast dissolving dosage form, Manuf. Chem, 61, 1990, 36-37
  • 169. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Vibhooti and Preethi Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 439 Guo J.H. Investigating the surface properties and bioadhesion of buccal patches, J. Pharm. Pharmacol, 46, 1994, 647-650, Tan Y.T.F, Peh KK and Al-Hanbali O, Inveatigation of interpolymer complexation between Carbopol and various grades of polyvinylpyrrolidone and effects on adhesion strength and swelling properties, J. Pharm. Sci, 4(1), 2001, 14, Yong CS., Jung J, Rhee J, Kim C and Choi H, Physicochemical characterization and evaluation of buccal adhesive tablets containing omeprazole, Drug Dev. Ind. Pharm, 27, 2001, 447-455,
  • 170. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Samaresh Pal Roy et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 440 EVALUATION OF ANTI-ULCER EFFECTS OF ETHANOLIC EXTRACT OF DELONIX REGIA FLOWER Samaresh Pal Roy*1 , Kamlesh Prajapati1 , Ramji Gupta2 , Dipanwita Bhadra4 , Nikunj Patel1 , Archana Batiwala1 , Gautam Sonara1 , Neerav Gheewala1 , T. Kannadasan3 1. Department of Pharmacology, Shree Dhanvantary Pharmacy College, Kim, Surat, Gujarat, India 2. Shree Dhanvantary Pharmaceutical Analysis and Research Centre, Kim, Surat 3. Anna University of Technology, Coimbatore, Tamilnadu 4. PDL Dept. Alkem Laboratory Limited, Amulya, Daman *Corresponding author: Email: samareshproy@gmail.com, Mobile: +919377077712 ABSTRACT Ulcer is one of the most common disorder in the world. The available antiulcer drug in the market have different side effect. So the people move to herbal drug for a better result and lesser side effect. Delonix regia is a plant from the family leguminosae is extensively available in the word. The earlier researcher found that the plant contain the chemical constitutent like tannin, saponin, flavonoid, so it have a good antioxident property. So, this study is planned recurred the antiulcer activity by ethanolic extract of the different doses (100mg/kg, 200mg,kg & 400mg/kg) of the flower of Delonix regia (EEDRF) on ethanol induced ulcer model in experimental rat whereas lansoprazole (8mg/kg) was taken as an standard drug. It has been found that the extract shows significant antiulcer activity in a dose dependent manner. The protection of ulcer may due to the presence of antioxidant principles present in the plant. Keywords: Delonix regia, Antiulcer, Ethanol 1. INTRODUCTION Gastric ulcer, the most common disorder of GIT has multifunctional causes in its pathophysiology. The pathophysiology of peptic ulcer has been centralized on an imbalance between aggressive and protective factors in the stomach such as acid-pepsin secretion, mucosal barrier, mucus secretion, blood flow, cellular regeneration, prostaglandins and epidermal growth factors. Although hospital admission for uncomplicated peptic ulcers in developed countries had begun decrease, there was a striking rise in admission for ulcer haemorrhage and perforation among elderly people. This increase has been attributed to the increased use of non-steroidal anti- inflammatory drugs (NSAIDs), alcoholic beverages, cigarettes and Helicobacter pylori infections. It is now considered to be one of the modern age epidemics affecting nearly 10% of world population during last decade have offered new insights in the therapy and prevention of peptic ulceration. Plants provide an alternative strategy in search for new drugs. There is a rich abundance of plants reputed in traditional medicine known to possess antiulcer properties. It is likely that plants will continue to be a valuable source of new molecules which may, after possible chemical manipulation, provide new and improved antiulcer drugs. Borrelli and Izzo reveal the extensive variety of chemical compounds isolated from medicinal plants with antiulcer activity. Literature search revealed that herbs rich in flavonoids show several biological activities including antiulcerogenic activity. This is an important reason to investigate antiulcer effects in medicinal plants with traditional use in gastric diseases. Delonix regia is a plant from the family leguminosae, is extensively cultivated in most regions of the world. The flowers of Delonix regia are commonly used as an antibacterial, analgesic, antiulcer, anti- inflammatory and antimicrobial. Reports indicate that pharmacological activities of Delonix regia flowers include anti-inflammatory and analgesic (Ahmad & Aqil., 2003), antimicrobial, broad spectrum antibacterial and antifungal activities.Wijayasirivardena et al 2009 reported the plant possess antiinflammatory activity. Muruganathan et al. 2011 reported that the bark of Delonix regia showed significant anti-inflammatory and anti-arthritic activity. Pradeepa et al. 2012 reported the leaf extract of Delonix regia showed antinociceptive activity. The plant has been claimed to be useful as antioxidant (Aquil et al., 2006), larvicidal (Chockalingam et al., 1990), antibacterial, antifungal (Ahmed et al., 2003), anti-inflammatory, analgesic (Muruganandam et al., 2000), nutritional (Grant et al., 1991), antimalerial (Ankrah et al., 2003), antiperiodic, febrifuge, emetic, CNS depressant (Rastogi et al., 1993) and antirheumatic (Khare et al., 2007). Its aqueous and alcoholic extracts were active against roundworm. The bark contains leucocyanidin, lupeol, tannin, -sitosterol and free OH-proline as
  • 171. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Samaresh Pal Roy et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 441 major amino acid. Flower anthers are a rich source of zeaxanthin. Leaves contain tannins, lupeol and -sitosterol (Khare et al., 2007). D. regia seeds contain lectins (Pando et al., 2002). However, there is no scientific proof justifying the traditional use of Delonix regia flowers in treatment of ulcer. Hence the present investigation was taken up to evaluate its potential gastroprotective efficacy in ethanol induced experimental models of ulcer in rats. 2. MATERIALS AND METHODS 2.1 Plant material: Flowers of Delonix regia were collected from the field of Surat and the plant was authenticated by Dr. Vinoo Parabia, Department of Bioscience, Veer Narmad South Gujarat University, Surat. A herbarium specimen is deposited in our college museum. 2.2 Preparation of 70% EEDRF: The flowers were shade dried at room temperature and pulverized. The powder obtained was subjected to successive soxhlet extraction with 70% ethanol (hydro-alcoholic extract). The extract were concentrated under reduced pressure and stored in desicator until further use and the percentage yield of corresponding extracts were calculated. 2.3 Animals: Whister albino rats (150-200g) and mice (18-25 g) of either sex were used for the study, obtained from AFC, Shree Dhanvantary Pharmacy College, kim. After one week of acclimatization the animals were used for further experiments. Approval from the Institutional Animal Ethical Committee for usage of animal in the experiment was obtained as per the Indian CPCSEA guidelines. 2.4. Acute Toxicity studies: The acute toxicity was determined on albino mice by fixed dose method of OECD Guide line given by CPCSEA. Groups of 6 mice were administered test drug by oral route in the range of 2000- 3000 mg/kg and mortality was observed after 24 hr. 2.5 Ethanol induced ulcer model (Surana et al., 2007): Albino rats of either sex weighing between 120 – 200 gm were selected and divided into 5 groups of 6 animals each. Table.1.Grouping of animals for the conduction of the trial Group I Control (1 ml/200 g of 99.80% ethanol p.o.) Group II Standard (Lansoprazole 8 mg/kg i.p.) Group III 70% ethanolic extract of DR flowers 100 mg/kg p.o. Group IV 70% ehtanolic extract of DR flowers 200 mg/kg p.o. Group V 70% ethanolic extract of DR flowers 400 mg/kg p.o. The animals were fasted for 24 hours with free access to water. Animals were given different doses of DRF extract and standard drug lansoprazole as mentioned above. Thirty minutes after the treatment 1ml/200 g of 99.80% alcohol was administered p.o. to each animal. Animals were sacrificed 1 hr. after alcohol administration, stomachs were isolated and cut open along the greater curvature and pinned on a soft board. The ulcer index was measured. Table.2.Codes for showing extent of ulcer activity in animals Code Extent of ulcer activity 0 Normal stomach 0.5 Red coloration 1 Spot ulcers 1.5 Hemaorrhagic streaks 2 Ulcer > 3 mm < 5mm 3 Ulcers > 5mm The percentage protection was calculated using the formula Where Ut = Ulcer index of treated group Uc = Ulcer index of control group.
  • 172. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Samaresh Pal Roy et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 442 2.6. Statistical analysis: Results were expressed as mean of # SEM (n=6). Statistical analysis was performed with one way ANOVA followed by Turkey-Kramer multiple comparisons test. P values less than 0.05 was considered to be statistically significant (p<0.05). 3. RESULTS 3.1. Acute toxicity: The acute toxicity study showed No animal died even at 2000mg/kg and hence the extract was treated as non-toxic and 1/25th , 1/10th & 1/5th of the 2000mg/kg was selected for further investigations are mentioned below. I. 100 mg/kg (1/25th of 2000 mg/kg) II. 200 mg/kg (1/10th of 2000 mg/kg) III. 400 mg/kg (1/5th of 2000 mg/kg). 3.2. Antiulcer Activity: The extract (100, 200 and 400 mg/kg) significantly inhibited the ulcerogenic effect of ethanol in rats. The percentage protection of ulcer is increase according to the increase in concentration of extract. The higher doses i.e 200mg & 400 mg/kg of the ethanolic extract of DF flower shows significant ulcer protection whereas 100mg/kg dose does not shows any significant effect. The standard drug lansoprazole (8mg/kg) shows a highly significant result. The ulcer score also reveals the antiulcer protection of the extract. The results are summarized in Table.3. 4. DISCUSSION In the present investigation different doses of ethanolic extract of DR flowers (EEDRF) was screened for gastroprotective properties by employing ethanol induced ulcer models in rats. It has been found that the ethanolic extract of Delonix regia has shown the antiulcer property in a dose dependent manner. Ethanol provoked gastric mucosal lesions are caused by the direct toxic effects of ethanol through the reduction in mucus production, gastric mucosal blood flow and bicarbonate secretion. Endogenous glutathione and prostaglandin levels are also lowered by ethanol while the release of histamine, influx of calcium ions, generation of free radicals and production of leukotriene’s are all increased (somchit et al., 2007). The product of the 5-lipoxygenase pathway may also play a key role in the development of ulcer induced by irritant agents such as ethanol. It has been reported that leukotriene antagonist and 5-lipoxygenase inhibitors are capable of inhibiting alcohol and NSAIDs induced gastric ulceration in rats (surana et al., 2007). In the present study also the significant protection exhibited by the test extract against alcohol induced gastric ulceration may be due to inhibition of 5-lipoxygenase pathway or leukotriene antagonistic activity. 5. CONCLUSION The results of the present study indicate that ethanolic extract of Delonix regia flowers possessed significant antiulcer properties, thus supports the traditional use of DR flowers in treatment of gastrointestinal disorders. 6. ACKNOWLEDGEMENTS The authors express their gratitude to the entire fraternity of Shree Dhanvantary Pharmacy college & Shree Dhanvantary Pharmaceutical Analysis & Research Centre for the completion of this project work. A : Control B : Std. Lansoprazole (8 mg/kg) C : 70% Ethanolic Ext. (100 mg/kg) D : 70% Ethanolic Ext. (200 mg/kg) E : 70% Ethanolic Ext. (400 mg/kg) Figure.1. Antiulcer activity of ethanolic extract of DR flowers
  • 173. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Samaresh Pal Roy et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 443 Table.3.Effect of 70% alcoholic extract of flower of Delonix regia on Ethanol-induced ulcer (Mean ± SEM, n = 6 in each group) in rats ** P < 0.01 and *** P < 0.001 (Vs. Control) respectively Control Standard (lansoprazole) EEDRF 100 mg/kg EEDRF 200 mg/kg EEDRF 400 mg/kg Figure.2. Photograph showing open stomach of alcohol induced gastric ulceration rats Gr. No. Bodyweight Treatment ULCER INDEX Tota l scor e Meanulcer Index±SEM %of protection Normal coloured stomach Red colouration Spotulcer Haemorr h-agic streaks Ulcer ≥3 but ≤5 Ulcer ≥ 5 I 160 Control - 0.5 1.0 1.5 2.0 - 5.0 6.00 ± 0.632 - 160 - 0.5 1.0 1.5 2.0 - 5.0 170 - 0.5 1.0 1.5 2.0 - 5.0 170 - 0.5 1.0 1.5 2.0 - 5.0 150 - 0.5 1.0 1.5 2.0 3.0 8.0 175 - 0.5 1.0 1.5 2.0 3.0 8.0 II 180 Lansoprazol e 8 mg/kg - 0.5 - - - - 0.5 1.00 ± 0.447 *** 83.33 180 - 0.5 - - - - 0.5 175 - 0.5 - - - - 0.5 170 - 0.5 1.0 - - - 1.5 175 - - - - - - - 170 - 0.5 1.0 1.5 - - 3.0 III 160 Alcoholic extract 100 mg/kg - 0.5 1.0 1.5 - - 3.0 3.916 ± 0.757 34.73 170 - 0.5 1.0 1.5 2.0 - 5.0 170 - 0.5 1.0 1.5 2.0 - 5.0 180 - 0.5 1.0 1.5 2.0 - 5.0 180 - 0.5 - - - - 0.5 180 - 0.5 1.0 1.5 2.0 - 5.0 IV 180 Alcoholic extract 200mg/kg - 0.5 1.0 1.5 2.0 - 5.0 2.166 ± 0.909 ** 63.90 180 - 0.5 - - - - 0.5 175 - 0.5 1.0 1.5 2.0 - 5.0 170 - 0.5 1.0 - - - 1.5 170 - 0.5 - - - - 0.5 160 - 0.5 - - - - 0.5 V 160 Alcoholic extract 400mg/kg - 0.5 1.0 1.5 2.0 - 5.0 1.416 ± 0.735 ** 76.40 175 - 0.5 1.0 - - - 1.5 180 - 0.5 - - - - 0.5 170 - 0.5 - - - - 0.5 170 - 0.5 - - - - 0.5 165 - 0.5 - - - - 0.5
  • 174. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Samaresh Pal Roy et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 444 REFERENCES Ahmad I, Aqil F, Broad spectrum antibacterial and antifungal activities and potency of crude alcoholic extract and fractions of Delonix regia flowers, 2nd World Congress on “Biotechnological Development of Herbal Medicine”, NBRI, Lucknow, UP, India, 2003:74. Asolkar LV, Kakkar KK, Chakre OJ, Second Supplement to Glossary of Indian Medicinal Plants with active principles-Part-I, New Delhi: National Institute of science Communication (CSIR), 1992:264. Bhave AL, Bhatt JD, Hemavathi KG, Antiulcer effect of Amlodipine and its interaction with H2 blocker and proton pump inhibitor in pylorus ligated rats, Indian J Pharmacol, 38(6), 2006, 403-07. Biswajit Majumdar, Sursi Guha Ray Chaudhuri, Arun Ray, Sandip K Bandyopadhyay. Effect of ethanol extract of Piper betle Linn leaf on healing of NSAID–induced experimental ulcer–A novel role of free radical scavenging action, Indian J Exp Biol, 41, 2003, 311-15. Borrelli F, Izzo AA, The plant kingdom as a source of anti-ulcer remedies, Phytother Res, 14, 2000, 581-91. Deshpande SS, Shah GB, Parmar NS, Antiulcer activity of Tephrosia purpurea in rats, Indian J Pharmcol 35, 2003, 168-72. Edward F, Gilman and Dennis G, Watson. Delonix regia-Royal Poinciana, Fact Sheet ST-228, a series of Environemtnal Horticulture Departmrnt, Florida Cooperative Extension Service, Institute of Food and Agriculture Sciences, University of Florida, Nov 1993. Etuk EU, Agaie BM, Onyeyill PA, Ottah CU, The effect of Rabeprazole and its isomers on aspirin and histamine- induced ulcers in rats, Indian J Pharmacol, 38(5), 2006, 357-58. Ganachari MS, Shiv kumar. Anti-ulcer properties of Ziziphus jujuba Lam leaves extract in rats, J Nat Rem, 4(2), 2004, 103-08. Goel RK, Sairam K, Dorababu M, Prabha T, Rao V, Effect of standardized extract of Ocimum sanctum Linn. on gastric mucosal offensive and defensive factors, Indian J Exp Bio, 43, 2005, 715-21. Hemamalini K, Vimal kumar Varma M, Antiulcer activity of Indigofera aspalathoids on chemically induced ulcer models in rats and guinea pigs, Adv Pharmacol Tox, 7, 2006, 25-29. Higham J, Kang JY, Majeed A, Recent trends in admissions and mortality due to peptic ulcer in England : increasing frequency of haemorrhage among older subjects, Gut, 50, 2002, 460-64. Jana U, Bhattacharya D, Bandopadhyay S, Pandit S, Debnath PK, Sur TK. Antiulcer activity of Digitrall: A polyherbal drug in rats, Indian J Pharmacol, 37(6), 2005, 406-07. Jungalwala FB, Cama HR. Carotenoids in Delonix regia (Gul Mohr) flower, Biochem J, 1962; 85:1. Karpagam Kumara Sundari S, Murugesh, Nallu M, Antiulcer activity of certain Phenyl tosylates, Adv Pharmacol Toxicol, 7(1), 2006, 7-9. Kumar A, Ram II. Anti-ulcer properties of methanolic extract of Benincasa hispida (Thunb.) Cogn. Indian Drugs, 39(1), 2002, 9-13. Lima ZP, Severi JA, Pellizzon CH, Brito ARMS, Solis PN, Caceres A, Can the aqueous decoction of Mango flowers be used as an antiulcer agent, Ethnopharmacol, 106, 2006, 29-37.
  • 175. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Samaresh Pal Roy et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 445 Muruganathan G, and Mohan S. Anti-inflammatory and Anti-Arthritic activities of Delonix elata bark extracts, Int J Res Ayu Pharm, 2(6), 2011, 1819-1821. Pandit S, Sur TK, Jana U, Bhattacharyya D, Debnath PK. Antiulcer effect of Shankha bhasma: A preliminary study, Indian J Pharmacol, 32, 2000, 378-80. Pradeepa K, Krishna V, Venkatesh, Kumar GK, Kumar SRS, Hoskeri JH, Antinociceptive activity of Delonix elata leaf extract, Asia Pacific J of Tropical biomedicine, 2012, 229-S231. Sairam K, Ch V Rao, Dora Babu M, Vijay Kumar K, Agarwal VK, Goel RK. Anti-ulcerogenic effect of methanolic extract of Emblia officinalis: an experimental study, J Ethnopharmacol, 82, 2002, 1-9. Shah JS, Anand IS, Patel SK, Patel HU, Thakkar VT, A comparative study of antacid and antiulcer activity of two marketed herbal products in North Gujarat region, Indian Drugs, 43(9), 2006, 763-65. Somchit MN, Siti Rahmah S, Zuraini A, Ahmad Bustamam A, Zakaria ZA, Somchit N, Gastroprotective activity of Spirulina platensis in acetic acid and ethanol induced ulcers in rats, J Nat Rem,7(1), 2007, 37-42. Sonowski RA. The changing spectrum of therapy of active peptic ulcer disease, Modern Medicine, 58, 1990, 50- 58. Surana SJ, Tatiya AU, Jain AS and Ushir YV. Antiulcer activity of Eranthemum Roseum (VAHL) R.BR on ethanol induced ulcer in albino rats.Int.J.Pharmacol.Biol.Sci, 1(1), 2007, 65-66 Surana SJ, Tatiya AU, Jain AS, Ushir YV, Antiulcer activity of Eranthemum roseum (VAHL) R.Br on ethanol induced ulcer in albino rats, Int J Pharmacol Biol Sci, 1(1), 2007, 65-69. Wijayasirivardena C, Chauhan NG, Sharma PP, Lahore SK, Shah MB, Anti-inflammatory activity of Delonix elata (L) gamble, J Nat Rem, 9(2), 2009, 209-215.
  • 176. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Dr. Praveen Kumar Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 446 A STUDY ON MEDICATION NON-ADHERENCE IN AMBULATORY DIABETIC PATIENTS AND NEED FOR PHARMACIST INTERVENTION FOR IMPROVING PATIENT ADHERENCE Dr. Praveen Kumar G Assistant Professor, C.L. Baid Metha College of Pharmacy, Chennai, Tamil Nadu. *Corresponding author: Email: praveen.pharmd@gmail.com ABSTRACT Diabetes mellitus is a chronic disease and the prevalence for all age-groups worldwide was estimated to be 2.8% in 2000 and 4.4% in 2030. Medication non adherence is a pervasive medical problem that is common among patients with chronic disease. This study was conducted for a period of 1 month. It includes 154 randomly selected patients who were interviewed by using a questionnaire regarding socio-economic characteristics, adherence rates, barriers that affect adherence to medication use (using Morisky self report scale) and counseled by pharmacist for 20-30 minutes. Of the total, 97(62.9%) were adherent and 57(37.01%) were non adherent to medications. In our study the commonly cited intentional non-adherence was found to be self-decision(35.08%) and ommision of drugs because of experiencing Side effects (22.8%). Confusion in dosage frequency (17.5%), forgetfulness (10.5%) and financial difficulty (8.7%) were the other contributing factors for non-adherence. Efforts were taken to increase the medication adherence and self care through patient education by pharmacists. Patient’s medication compliance is a multifactor behavior in which the role of patient’s attitude is very important. Interventions are needed to increase medication adherence so that patients can realize the full benefit of prescribed therapies. Key words: Diabetes mellitus, non adherent, pharmacist INTRODUCTION Diabetes mellitus is a chronic disease and the prevalence for all age-groups worldwide was estimated to be 2.8% in 2000 and 4.4% in 2030.In India the prevalence of diabetes and pre-diabetes among adults were 5.1% and 13.5%. Medication non adherence is a pervasive medical problem that is common among patients with chronic disease. Factors such as uncontrolled diet, sedentary lifestyle, inappropriate therapeutic regimens as well as medication non adherence have been known to have significant impact on glycemic control and outcome of type 2 diabetes treatment. In unadjusted analyses non adherent patients had higher hospitalization (23.2%) and higher mortality (5.9%). With this background information this study has been proposed to evaluate the probable reasons for patient’s non adherence to prescribed anti diabetic medications in ambulatory care type 2 diabetes patients and to improve the adherence rate through patient education. Objective:  To assess adherence levels and factors affecting adherence in diabetic patients.  To educate patients on self care and adherence to the lifestyle modifications (diet and exercise) as well as pharmacological therapy. Methodology: The study was conducted in the Sri Ramachandra Hospital for a period of 1 month. A total of 154 randomly selected patients who were attending the diabetes outpatient department were interviewed by using a questionnaire regarding socio-economic characteristics, adherence rates,barriers that affect adherence to medication use(using Morisky self report scale) and counseled by pharmacist for 20-30 minutes. RESULTS AND DISCUSSION Among the 154 patients, 42 (27.2%) male and 112(72.7%) female were taking a mean of 5.3 medicines per patient to control diabetes and related comorbidities. Of the total, 97(62.9%) were adherent and 57(37.01%) were non adherent to medications. In our study the commonly cited intentional non-adherence was found to be self-decision(35.08%) and ommision of drugs because of experiencing Side effects (22.8%). Confusion in dosage frequency (17.5%), forgetfulness (10.5%) and financial difficulty (8.7%) were the other contributing factors for non-adherence. All together 128patients (83.12%) were on anti-hypertensive and 73patients (47.4%) were on lipid lowering medicines. Efforts were taken to increase the medication adherence and self care through patient education by pharmacists.
  • 177. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Dr. Praveen Kumar Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 447 Factors for non-adherence Gender No. of patients % Self decision Male 4 7.01 Female 16 28.07 Side effects Male 3 5.26 Female 10 17.54 Confusion Male 2 3.5 Female 8 14.03 Forget Male 2 3.5 Female 4 7.01 Financial difficulty Male 1 1.75 Female 4 7.01 Others Male 1 1.75 Female 2 3.5 GRAPH3 SD1: alter dosing schedule for convenience, SD2: omit drugs if feeling ill, SD3:don’t care to take drugs, SD4: think drugs are not effective. GRAPH.2.SD:selfdecision,SE:sideeffects, C:confusion, F:forget, FD:financial difficutly, O:Others CONCLUSION Patient’s medication compliance is a multifactor behavior in which the role of patient’s attitude is very important. Interventions are needed to increase medication adherence so that patients can realize the full benefit of prescribed therapies. REFERENCES Adisa R, Alutundu MB, Fakeye TO, Factors contributing to nonadherence to oral hypoglycemic medications among ambulatory type 2 diabetes patients in Southwestern Nigeria, Pharmacy Practice, 7(3), 2009, 163-169.
  • 178. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Gowthami B et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 448 RECENT TRENDS IN POSITIVE AND NEGATIVE ASPECTS OF FOOD ON BIOAVALABILTY OF DRUGS Gowthami B1* , Sk Nahida Fazilath2 , Sanaulla Md2 , K Prudhvi Raj2 , Dastagiriah G2 , Tabassum Sk2 1.Vaagdevi College of Pharmacy and Research Center, Nellore 2.Nimra College of Pharmacy Corresponding Author: Email: gowthu.buduru@gmail.com ABSTRACT The therapeutic effectiveness of a drug depends upon the ability of the dosage form to deliver the medicament to its site of action at a rate & amount sufficient to produce the desired pharmacologic response. This attribute of the dosage form is referred to as Physiologic availability or Biologic availability or simply availability. For most of the drugs, the pharmacologic response is directly related to the plasma levels. Thus, the term, Bioavailability is defined as the rate & extent (amount) of absorption of unchanged drug from its dosage form. Bioavailability refers to the difference between the amounts of a substance, such as a drug, herb, or chemical, to which a person is exposed and the actual dose of the substance the body receives. Bioavailability accounts for the difference between exposure and dose. A drug's therapeutic action or a chemical's toxicity is determined by the dose received at the target site in the body. The dose at the target site is determined by the amount of the substance absorbed by the body, which depends on its bioavailability. If a substance is ingested, for example, its bioavailability is determined by the amount that is absorbed by the intestinal tract. If a substance is inhaled, its bioavailability is determined by the amount that is absorbed by the lungs. Understanding bioavailability is critical to determining the amount of a drug to administer or the level of chemical exposure that is likely to produce toxicity. Key words: Bioavailability, Bioavailability studies, Absolute and Relative bioavailability. INTRODUCTION In comparison to drugs, there are significant differences in dietary supplements that impact the evaluation of their bioavailability. These differences include the following: the fact that nutritional supplements provide benefits that are variable and often qualitative in nature; the measurement of nutrient absorption lacks the precision; nutritional supplements are consumed for prevention and well-being; nutritional supplements do not exhibit characteristic dose-response curves; and dosing intervals of nutritional supplements, therefore, are not critical in contrast to drug therapy. In addition, the lack of defined methodology and regulations surrounding the consumption of dietary supplements hinders the application of bioavailability measures in comparison to drugs. In clinical trials with dietary supplements, bioavailability primarily focuses on statistical descriptions of mean or average AUC differences between treatment groups, while often failing to compare or discuss their standard deviations or inter-individual variation. This failure leaves open the question of whether or not an individual in a group is likely to experience the benefits described by the mean-difference comparisons. Further, even if this issue were discussed, it would be difficult to communicate meaning of these inter-subject variances to consumers and/or their physicians. FACTORS INFLUENCING BIOAVAILABILITY Before the therapeutic effect of an orally administered drug can be realized, the drug must be absorbed. The systemic absorption of an orally administered drug in a solid dosage form is comprised of three distinct steps: 1. Disintegration of the drug product 2. Dissolution of the drug in the fluids at the absorption site 3. Transfer of drug molecule across the membrane lining the gastrointestinal tract into the systemic circulation.
  • 179. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Gowthami B et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 449 Bioavailability and oral Dosage Forms Bioavailability Factors related to the dosage form Physicochemical properties of the drug Formulation and manufacturing variables Particle size Crystalline structure Degree of hydration of crystal Salt or ester form Amount of disintegrant Amount of lubricant Special coatings Nature of diluent Compression force Bioavailability Factors related to the patient Physiologic factors Interactions with other substances Variations in absorption power along GI tract Variations in pH of GI fluids Gastric emptying rate Intestinal motility Perfusion of GI tract Presystemic and first-pass metabolism Age, sex, weight Disease states Food Fluid volume Other drugs Factors influencing Gastric Emptying Rate Factor Influence on gastric emptying rate Increased viscosity of stomach contents Decreased Body position lying on left side Decreased Emotional state stress depression anxiety Increased or Decreased Increased Activity, exercise Decreased Type of meal fatty acids, fats carbohydrates amino acids Decreased pH of stomach contents decreased increased Decreased Increased Disease states gastric ulcers Crohn’s disease hypothyroidism hyperthyroidism Decreased Decreased Increased Drugs atropine propantheline narcotic analgesics amitriptyline metoclopramide Decreased Decreased Increased OBJECTIVES OF BIOAVAILABILITY STUDIES 1. Primary stages of development of a suitable dosage form for a new drug entity. 2. Determination of influence of excipients, patient related factors and possible interaction with other drugs on the efficiency of absorption. 3. Development of new formulations of the existing drugs. 4. Control of quality of a drug product during the early stages of marketing in order to determine the influence of processing factors, storage and stability on drug absorption. PURPOSE OF BIOAVAILABILITY STUDIES 1. Bioavailability studies are performed for both approved active drug ingredients and therapeutic moieties not yet approved for marketing by the FDA.
  • 180. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Gowthami B et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 450 2. New formulation of active drug ingredients must be approved by the FDA before marketing to ensure the safe and effective for its labeled indications for use. 3. According to FDA the drug product must meet all applicable standards of identity, strength, quality, and purity. To ensure all these standards are met, the FDA requires bioavailability and pharmacokinetic studies and where necessary, bioequivalence studies for all drug products. 4. Bioavailability links in vivo performance of the drug product used in clinical trials to studies demonstration evidence of safety and efficacy. 5. For unjacketed drug that donor have NDA approval by the FDA, inviter and in vivo bioequivalence studies must be performed on the drug formulation proposed for marketing as a genetic drug product. Essential pharmacokinetic parameters, including the rate and extent of systemic absorption, elimination half life, and rates of excretion and metabolism, should be established after single and multiple dose administration. Data from these in vivo studies are important to establish recommended dosage regimens and to support the drug labeling. 6. Used to define the effect of changes in the physicochemical properties of the drug substance and the effect of the dug product on the pharmacokinetics of the drug. 7. Bioequivalence studies are used to compare the bioavailability of the same drug from various drug products. 8. Bioavailability and bioequivalence can also be considered as the performance measures of the drug product invivo. If the drug products are bioequivalent and therapeutically equivalent then the clinical efficacy and the safety profile of these drug products are assumed to be similar and may be substituted for each other. CONSIDERATION IN BIOAVAILABILITY STUDY DESIGN Bioavailability: Absolute versus Relative Absolute bioavailability: When the systemic availability of a drug administered orally is determined in comparison to its intravenous administration, is called as absolute availability (denoted by F). AUC = Area under the Curve D = Dose of administered drug  Its determination is used to characterize a drug’s inherent absorption properties from extra vascular site.  Intravenous dose is selected as a standard due to its 100% bioavailability.  If the drug is poorly water soluble, intramuscular dose can be taken as standard. Relative bioavailability: - When the systemic availability of a drug after administration is compared with that of standard of the same drug it’s referred to as relative bioavailability (Fr).  In contrast to absolute bioavailability, it is used to characterize absorption of drug from its formulation.  F & Fr are expressed in percentages. Single dose versus multiple dose studies: These are very common.  They are easy, offer less exposure to drugs & are less tedious.  However, it is difficult to predict the steady-state characteristic of a drug & inter-subject variability with such studies.  On the other hand, multiple Dose study is difficult to control (poor subject compliance), exposes the subject to more drugs & is highly tedious & time consuming. Nevertheless, such a study has a several advantages. 1. More accurately reflects the manner in which the drug should be used.
  • 181. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Gowthami B et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 451 2. Easy to predict the peak & valley characteristic of the drug since the bioavailability is determined at steady – state. 3. Requires collection of fewer blood samples. 4. The drug blood levels are higher due to cumulative effect which makes its determination possible even by the less sensitive analytic methods. 5. Can be ethically performed in patients because of the therapeutic benefit to the patient. 6. Better evaluation of the performance of a controlled release formulation is possible. 7. Nonlinearity in pharmacokinetics, if present, can be easily detected. In Multiple Dose study, one must ensure that the steady state has been reached. For this, the drug should be administered for 5-6 elimination half-lives before collecting the blood samples. Human Volunteers- Healthy subjects verses Patients: Ideally, the bioavailability study should be carried out in patients for whom the drug is intended to be used because of the apparent advantages; 1. The patient will be beneficial from the study. 2. Reflects better therapeutic efficacy of a drug. 3. Drug absorption pattern in disease states can be evaluated. 4. Avoids the ethical requirements of administering drugs to the healthy subjects.  In multiple dose study, they prefer patients rather than healthy humans.  The drawbacks of using patients as volunteers are equally large- disease, other drugs, physiologic changes etc. may modify the drug absorption pattern.  Strict study conditions such fasting state required to be followed by the subject is also difficult.  In short, establishing a standard set of conditions necessary for a bioavailability study is difficult with patients as volunteers.  Therefore, such studies are generally carried out in healthy human volunteers (20-40 yrs), preferably male adults with body weight within narrow range +/- 10 %, under restricted dietary & fixed activity conditions.  The consent of volunteers must be obtained and they must be informed about the conditions to be followed during the course of studies – to abstain from any other medication for at least 2 weeks and to fast overnight prior to and for a minimum of 2-4 hours post dosing as well as possible hazards if any. METHODS FOR ASSESSING THE BIOAVAILABILITY 1) Pharmacokinetic methods: These are very widely used and based on the assumption that the pharmacokinetic profile reflects the therapeutic effectiveness of a drug. Thus these are indirect methods. Indirect methods a) Plasma level – time studies b) Urinary excretion studies 2) Pharmacodynamic methods: These methods involve direct measurement of drug effect on a physiologic process as a function of time. Direct methods a) Acute pharmacologic response b) Therapeutics response 1) Pharmacokinetic methods a) Plasma level – time studies: It is the most reliable Method, & based on assumption that two dosage forms exhibit super imposable plasma level-time profiles in a group of subjects should result in identical therapeutic activity. With single dose study: Requires collection of samples for a period of 2 to 3 biological half lives. & make a plot of Conc. Vs time of sample collection. i.v. dose: sampling start within 5 min. and subsequent samples taken at 15 min intervals. The 3 parameters of plasma level-time studies which are considered important for determining bioavailability are: 1. Cmax: The peak plasma concentration that gives an indication whether the drug is sufficiently absorbed systematically to provide a therapeutic response. 2. tmax: The peak time that gives an indication of the rate of absorption.
  • 182. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Gowthami B et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 452 3. AUC: The area under the plasma level-time curve that gives a measure of the extent of absorption or the amount of drug that reaches the systemic circulation. The extent of Bioavailability can be determined from equations: F = [AUC] oral Div / [AUC] iv Doral Fr = [AUC] test Dstd / [AUC] std Dtest Where D stands for dose administered and subscripts i.v and oral indicates the route of administration. Subscripts test and std indicate the test and standard dose of the same drug to determine relative bioavailability. With multiple dose study: The method involves drug administration for at least 5 biological half-lives with a dosing interval equal to or greater than the biological half-life to reach the steady state. The extent of bioavailability for multiple dose study is given as: Fr = [AUC]test Dstd τtest/ [AUC]std Dtest τstd Where [AUC] values are under the plasma level- time curve of one dosing interval in a multiple dosing regimen after reaching the steady state and τ is the dosing interval. The Bioavailability can be determined from peak plasma concentration at steady state Css, max according to following equation: Fr = (Css,max)test Dstd τtest / (Css,max)std Dtest τstd b) Urinary excretion studies: Based on the principle that the urinary excretion of unchanged drug is directly proportional to the plasma concentration of drug. E.g. Certain diuretics and sulfonamides, urinary antiseptics such as nitrofurantion and hexamine. Concentrations of metabolites excreted in urine is never taken into account in calculations since a drug undergo presystemic metabolism before being absorbed. Method involves sampling at regular intervals for a time span equal to7 biological half-lives. Total emptying of the bladder is necessary to avoid errors. The three major parameters examined in urinary excretion studies are: 1) (dxu/dt)max: The maximum urinary excretion rate, obtained from the peak of plot between rate of excretion versus midpoint time of urine collection. It is analogous to Cmax. Its value increases as rate of and/or extent off absorption increases. 2) (tu)max: The time for maximum excretion rate, it is analogous to the tmax. Its value decreases as the absorption increases. 3) Xu: The cumulative amount of drug excreted in the urine, it is related to the AUC and increases as the extent of absorption increases. The extent of bioavailability is calculated from equation: F = (Xu∞)oral Div / (Xu∞)iv Doral Fr = (Xu∞)test Dstd / (Xu∞)std Dtest With the multiple dose study to steady state , the equation is: Fr = (Xu,ss)test Dstd τtest / (Xu,ss)std Dtest τstd Where, (Xu,ss) is the amount of drug excreted unchanged during a single dosing interval at steady state. Bioavailability of few drugs can be also determined by assay of biologic fluids other than plasma and urine. For e.g. Theophyllline  salivary secretion Cephalosporin  CSF and bile 2) Pharmacodynamic methods: a) Acute pharmacologic response: When bioavailability measurement by pharmacokinetic methods is difficult,an acute pharmacological effect such as a change in ECG or EEG readings, pupil diameter,etc.is related to the time course of a given drug. Thus bioavailability can be determined by construction of pharmacological effect-time curve as well as dose-response graphs. The method requires measurement of responses for at least 3 biological half-lives of the drug in order to obtain a good estimate of AUC. Disadvantages:The pharmacological response tends to be more variable and accurate correlation between measured response and drug available from the formulation is difficult. The observed response may be due to an active metabolite whose concentrations not proportional to the concentration of parent drug responsible for the pharm1acological effect. b) Therapeutic response: Based on observing the clinical response to a drug formulation given to patients suffering from disease for which it is to be used.
  • 183. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Gowthami B et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 453 Drawback: Quantitation of observed response is too improper to allow for reasonable assessment of relative bioavailability between two dosage forms of the same drug. EFFECT OF FOOD ON BIOAVAILABILITY Drugs are frequently taken with food, and patients often use mealtimes to remind them to take their medications. However, food can have a significant effect on the bioavailability of drugs. The influence of food on drug absorption has been recognized for some time, and several reviews have been published on the influence of food on drug bioavailability. Food may influence drug absorption indirectly, through physiological changes in the GI tract produced by the food, and/or directly, through physical or chemical interactions between the drug molecules and food components. When food is ingested, stomach emptying is delayed, gastric secretions are increased, stomach pH is altered, and splanchnic blood flow may increase. These may all affect bioavailability of drugs. Food may also interact directly with drugs, either chemically (e.g. chelation) or physically, by adsorbing the drug or acting as a barrier to absorption. In general, gastrointestinal absorption of drugs is favored by an empty stomach, but the nature of drug-food interactions is complex and unpredictable; drug absorption may be reduced, delayed, enhanced or unaffected by the presence of food. Following Table summarizes some of the studies that have indicated the effect of food on the bio- availability of a variety of drugs. FOOD-DRUG INTERACTIONS Medicines can treat and cure many health problems. However, they must be taken properly to ensure that they are safe and effective. Medications should be extremely specific in their effects, have the same predictable effect for all patients, never be affected by concomitant food or other medications, exhibit linear potency, be totally non-toxic in any dosage and require only a single dose to affect a permanent cure. However, this ideal drug is still to be discovered. Many medicines have powerful ingredients that interact with the human body in different ways. Diet and lifestyle can sometimes have a significant impact on drugs. A drug interaction is a situation in which a substance affects the activity of a drug, i.e. the effects are increased or decreased, or they produce a new effect that neither produces on its own. Typically, interactions between drugs come to mind (drug- drug interaction). However, interactions may also exist between drugs and foods (drug-food interactions), as well as drugs and herbs (drug-herb interactions). These may occur out of accidental misuse or due to lack of knowledge about the active ingredients involved in the relevant substances. Interactions between food and drugs may inadvertently reduce or increase the drug effect. Some commonly used herbs, fruits as well as alcohol may cause failure of the therapy up a point of to serious alterations of the patient`s health. The majority of clinically relevant food-drug interactions are caused by food induced changes in the bioavailability of the drug. Major side- effects of some diet (food) on drugs include alteration in absorption by fatty, high protein and fiber diets. Bioavailability is an important pharmacokinetic parameter which is correlated with the clinical effect of most drugs. However, in order to evaluate the clinical relevance of a food-drug interaction the impact of food intake on the clinical effect of the drug has to be quantified as well. The most important interactions are those associated with a high risk of treatment failure arising from a significantly reduced bioavailability in the fed state. Such interactions are frequently caused by chelation with components in food. In addition, the physiological response to food intake, in particular, gastric acid secretion, may reduce or increase the bioavailability of certain drugs. Drug interactions can alter the pharmacokinetics and/or pharmacodynamics of a drug. The pharmacodynamic interaction may be additive, synergistic, or antagonistic effects of a drug. Drug interactions (DIs) represent an important and widely under recognized source of medication errors. The gastrointestinal absorption of drugs may be affected by the concurrent use of other agents that have a large surface area upon which the drug can be absorbed bind or chelate, alter gastric pH, alter gastrointestinal motility, or affect transport proteins such as P-glycoprotein. A reduction only in absorption rate of a drug is seldom clinically important, whereas a reduction in the extent of absorption will be clinically important if it results in sub therapeutic serum levels. Factors such as nonspecific binding, atypical kinetics, poor effector solubility, and varying ratios of accessory proteins may alter the kinetic behavior of an enzyme and subsequently confound the extrapolation of in vitro data to the human situation. Coenzyme Q-10 (CoQ10) is very widely consumed by humans as a food supplement because of its recognition by the public as an important nutrient in supporting human health. It interferes with intestinal efflux transporter P-glycoprotein (P-gp) and as result food-drug interactions arise. The interaction of natural products and drugs is a common hidden problem encountered in clinical practice. The
  • 184. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Gowthami B et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 454 interactions between natural products and drugs are based on the same pharmacokinetic and pharmacodynamic principles as drug-drug interactions. Several fruits and berries have recently been shown to contain agents that affect drug-metabolizing enzymes. Grapefruit is the most well-known example, but also sevillian orange, pomelo and star fruit contain agents that inhibit cytochrome P450 3A4 (CYP3A4), which is the most important enzyme in drug metabolism. The study of drug-drug, food-drug, and herb-drug interactions and of genetic factors affecting pharmacokinetics and pharmacodynamics is expected to improve drug safety and will enable individualized drug therapy. Drugs can show their efficacy only if administered in appropriate quantity with appropriate combination of drugs and foods and at appropriate time. In contrast to the easy access to information on drug-drug interactions, the information about food- drug interaction is not always available conveniently. It is a difficult and complex problem to accurately determine the effects of food and nutrients on a particular drug. This article aims to help the healthcare professionals specially physicians and pharmacists and patients to become more knowledgeable about drug and food interactions. Electronic search of literatures was conducted over a period of two months and all original research and review articles were included in this study. No literature was older than 20 years. The drugs were selected and reviewed on the basis of their general utilization pattern and realizing the need for reporting their interaction with different dietary supplements for better therapeutic use of these drugs within the recommended dose regimen. Fruit Juices: Among all fruit juices, grape fruit juice (GFJ) possesses high interaction with almost all types of drugs. The juice modifies the body`s way of metabolizing the medication, affecting the liver`s ability to work the drug through a person`s system. Taniguchi in 2007 reported a case of purpura associated with concomitant ingestion of cilostazol, aspirin and grapefruit juice in 79 years old man. His purpura disappeared upon cessation of grapefruit juice, although his medication was not altered. The most probable cause of his purpura is an increase in the blood level of cilostazol because of the inhibition of cilostazol metabolism by components of grapefruit juice; Numerous reports have documented drug interactions with GFJ that occur via inhibition of CYP3A enzymes. Furano coumarins present in GFJ inhibit the intestinal CYP 3A4 and have been shown to increase the oral bioavailability of medications that are CYP 3A4 substrates like Felodipine, midazolam, cyclosporine and raise their concentrations above toxic levels. GFJ is generally contraindicated to patients taking psychotropics and it is advised to inform patients about described interaction. The in vitro data suggest that compounds present in grapefruit juice are able to inhibit the P-gp activity modifying the disposition of drugs that are P-gp substrates such as talinolol. The overall exposure of some drugs can be increased by more than fivefold when taken with GFJ and increase the risk of adverse effects. With new anticonvulsants, serum iron and sodium need to be monitored. Additionally, users are advised to avoid drinking grape fruit juice within 1-2 hr(s) of taking these anticonvulsants. Furanocoumarines and active bioflavonoids present in GFJ are also inhibitors of OATP and when ingested concomitantly, can reduce the oral bioavailability of the OATP substrate, fexofenadine. Overall, a series of flavonoids present in GFJ are identified as esterase inhibitors, of which kaempferol and naringenin are shown to mediate pharmacokinetic drug interaction with most of the calcium channel antagonist and the statin groups of drugs such as enalapril and lovastatin due to their capability of esterase inhibition. Cholesterol-lowering agent lovastatin should be taken with food to enhance gastrointestinal absorption and bioavailability. The absorption of rosuvastatin, another anti-hyper lipidemic agent, was significantly decreased in the fed state compared with the fasting state, which suggests that rosuvastatin should be administered on an empty stomach. Simvastatin, ezetimibe, pravastatin and fluvastatin may be taken without regards to food. However, high fiber diets may lower the efficacy of these drugs. Concomitant administration of statins with food may alter statin pharmacokinetics or pharmacodynamics, increasing the risk of adverse reactions such as myopathy or rhabdomyolysis or reducing their pharmacological action. Consumption of pectin or oat bran together with Lovastatin reduces absorption of the drug, while alcohol intake does not appear to affect the efficacy and safety of Fluvastatin treatment. Warfarin: Warfarin is commonly used to treat or prevent thromboembolic events. Patients taking warfarin are at particular risk of interactions with dietary supplements, yet approximately 30% use herbal or natural product supplements on a regular basis. There is a possible interaction between warfarin and a highprotein diet. The
  • 185. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Gowthami B et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 455 potential for increased dietary protein intake to raise serum albumin levels and/or cytochrome P450 activity has been postulated as mechanisms for the resulting decrease in international normalized ratio (INRs). Some vegetables (broccoli, Brussels sprouts, kale, parsley, spinach, and others) are high in vitamin K. Eating large quantities or making sudden changes in the amounts eaten of these vegetables, interferes with the effectiveness and safety of warfarin therapy. Eating charbroiled food may decrease warfarin activity, while eating cooked onions may increase warfarin activity. Soy foods have been reported both to increase and to decrease warfarin activity. The significance of these last three interactions remains unclear. The combination of warfarin administration and cranberry juice ingestion appeared to be associated with an elevated INR without bleeding in elderly patient. A number of studies have been documented on the interaction of warfarin and cranberry juice.26-30 Cranberry juice is a flavonoid, which has been shown to induce, inhibit, or act as a substrate for the biosynthesis of several cytochrome P-450 (CYP) isoenzymes. Specifically, cranberry juice may inhibit the activity of CYP2C9, the primary isoenzyme involved in the metabolism of S-warfarin. It was suggested that cranberry juice increased the International Normalized Ratio (INR) of patients taking warfarin, but neither clearly identified cranberry juice as the sole cause of INR elevation. If warfarin sodium is ingested with leafy green vegetables, the hypoprothrombinemic effect of warfarin may be decreased and thromboembolic complications may develop. Monoamnine oxidases: Antidepressant activity of monoamine oxidase inhibitors (MAOIs) was initially noted in the 1950s. Although older monoamine oxidase inhibitors (MAOIs) are effective in the treatment of depressive disorders, they are under-utilized in clinical practice due to main concerns about interaction with tyramine- containing food (matured cheese, red vine, ripped bananas, yogurt, shrimp paste and salami) or so called cheese reaction, since they are capable of producing hypertensive crisis in patients taking MAOIs. The first-generation MAOIs such as phenelzine and isocarboxazid were largely nonselective inhibitors of both subtypes of MAO, MAO (A) and MAO (B). These medications carried with them dietary restrictions. Tyramine is an indirectly acting sympathomimetic agent, is degraded by MAO but in the presence of MAOIs, it escapes degradation and reaches the systemic circulation where it is taken up by the adrenergic neuron, leading to a hypertensive crisis. However, MAOIs have been well established as an effective intervention for people with treatment resistant depression, and transdermal formulations may provide a valuable therapeutic option and eliminate the drug-food interaction. Antihypertensive Drugs: Patients placed on anti hypertensive drugs will benefit from concomitant moderate sodium restricted diets. Propranolol serum levels may be increased if taken with rich protein food. A change in diet from high carbohydrates/low protein to low carbohydrate/ high protein may result in increased oral clearance. Smoking may decrease its plasma levels of by increasing its metabolism. The intestinal absorption of celiprolol (beta-blocker) is inhibited when it is taken with orange juice. Hesperidin, present in orange juice, is responsible for the decreased absorption of celiprolol. The absorption of ACEs inhibitors is increased when taken on an empty stomach. While GFJ increases the bioavailability of felodipine (Ca2 channel blocker). Licorice extract, a common ingredient of dietary supplement contains glycyrrhizin and glycyrrhetinic acid. It is a potent inhibitor of 11- bet- hydroxyl steroid dehydrogenase, it increases excess of cortisol to mineralocorticoid receptors causing sodium retention and potassium depletion, so it may interfere with various medicines including antihypertensive and antiarrhythmic agents. A high intake of liquorice can cause hypermineralocorticoidism with sodium retention and potassium loss, oedema, increased blood pressure and depression of the renin-angiotensin-aldosterone system. Studies showed that a daily consumption of glycyrrhizic acid of 95 mg or more caused an increase in blood pressure. A practical guideline for an acceptable daily intake of glycyrrhizic acid seems to be 9.5 mg a day. This means no more than 10-30g liquorice and no more than half a cup of liquorice tea a day. Antibiotics: Antibiotics are widely prescribed in medical practice. Many of them induce or are subject to interactions that may diminish their anti-infectious efficiency or elicit toxic effects. Food intake can influence the effectiveness of an antibiotic. Avoid coadministration of antibiotics with milk products which are rich sources of divalent ions, such as calcium and magnesium that complex with some antibiotics and prevent their absorption. The intake of dairy products, however, needs to be monitored and encouraged with appropriate consideration of specific antibiotics involved.
  • 186. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Gowthami B et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 456 A number of studies give evidence that fluoroquinolones forming slightly soluble complex with metal ions of food show reduced bioavailability. Casein and calcium present in milk decrease the absorption of ciprofloxacin. The effect of interaction of five fruit juices on the dissolution and absorption profiles of ciprofloxacin tablets were determined. It was found that the absorption of ciprofloxacin (500 mg) tablets can be reduced by concomitant ingestion of the GFJ. Therefore, to avoid drug therapeutic failures and subsequent bacterial resistance as a result of sub-therapeutic level of the drug in the systemic circulation, ingestion of the juice with ciprofloxacin should be discouraged. Azithromycin absorption is decreased when taken with food, resulting in a 43% reduction in bioavailability. Tetracycline should be taken one hour before or two hours after meals, and not taken with milk because it binds calcium and iron, forming insoluble chelates, and influencing its bioavailability. The effect of milk added to coffee or black tea on the bioavailability of tetracycline was evaluated in healthy individuals. Results showed that even a little quantity of milk containing extremely small amounts of calcium severely impair the absorption of the drug, so that the presence of this metal ion should be carefully controlled in order to avoid decreasing the available tetracycline. Food-drug interactions may reduce the bioavailability of drugs taken after meals (negative food effects). However, enteric-coated tablets that start to disintegrate when they reach the middle-to lower region of the small intestine could reduce negative food effects. Results indicated that food-drug interactions were avoided by separating the main absorption site of drugs from that of food components. Analgesics and Antipyretics: Analgesics and antipyretics are used to treat mild to moderate pain and fever. For rapid relief, acetaminophen should be taken in an empty stomach because food may slow the body absorption of acetaminophen. Co-administration of acetaminophen with pectin delays its absorption and onset. NSAIDs like ibuprofen, naproxen, ketoprofen and others can cause stomach irritation and thus they should be taken with food or milk. Avoid or limit the use of alcohol because chronic alcohol use can increase the risk of liver damage or stomach bleeding. The absorption of ibuprofen and oxycodone when given in the combination tablet was affected by the concomitant ingestion of food. The C max and AUC0-alpha of ibuprofen were significantly increased after single and multiple doses of Coca-Cola, thereby indicating increased extent of absorption of ibuprofen. The daily dosage and frequency of ibuprofen must be reduced when administered with Coca-Cola. Food intake did not appear to affect the extent of absorption (i.e, total exposure) of oral Diclofenac potassium soft gelatin capsule at doses. Bronchiodilators: Bronchodilators like theophylline, albuterol, and epinephrine possess different effects with food. The effect of food on theophylline medications can vary widely. High-fat meals may increase the amount of theophylline in the body, while high carbohydrate meals may decrease it. Avoid alcohol if taking theophylline medications because it can increase the risk of side effects such as nausea, vomiting, headache and irritability. Avoid eating or drinking large amounts of foods and beverages that contain caffeine (e.g., chocolate, colas, coffee, and tea) since theophylline is a xanthine derivative and these substances also contain xanthine. Hence consuming large amounts of these substances while taking theophylline, increases the risk of drug toxicity. Additionally, both oral bronchodilators and caffeine stimulate the central nervous system. Patients may be advised not to consume GFJ when taking theophylline, since it increases the bioavailability and monitoring of plasma theophylline levels in patients consuming GFJ might be helpful in better management of patient care. Antihistamines: Fexofenadine, loratadine, rupatadine, cimetidine cetirizine, are all antihistamines. It is best to take prescription antihistamines on an empty stomach to increase their effectiveness. Rupatadine is commonly used for the management of diseases with allergic inflammatory conditions. A study indicates that concomitant intake of food with a single 20 mg oral dose of rupatadine exhibits a significant increase in rupatadine bioavailability. Cimetidine is given with food to assist the maintenance of a therapeutic blood concentration. A fraction of cimetidine is absorbed in the presence of food, allowing the remaining drug to be dissolved once the gut is cleared. Thus, therapeutic levels are maintained throughout the dosing interval. A study was conducted on a latest molecule esomeprazole (acid-reducer) and it was observe that its bioavailability was reduced when taken within 15 min before eating a high-fat meal vs. that while fasting. Antitubercular Drugs: Anti-tubercular drugs like isoniazid have been associated with tyramine and histamine interactions. Inhibition of monoamine oxidase and histaminase by isoniazid can cause significant drug food interactions. Food greatly decreases isoniazid bioavailability. Oleanolic acid, a triterpenoid exists widely in food,
  • 187. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Gowthami B et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 457 medicinal herbs and other plants, has antimycobacterial activity against the Mycobacterium tuberculosis, when administered with isoniazid, it exerts synergistic effect. High fat meals decrease the serum concentration of cycloserine, a bacteriostatic anti-tubercular drug and results in incomplete eradication of bacteria. Antidiabetics:Glimepiride is an antidiabetic and a new generation sulfonylurea derivative should be administered with breakfast or the first main meal of the day. It has absolute bioavailability and the absence of food interaction guarantee highly reproducible pharmacokinetics. Immediate release glipizide should be taken 30 minutes before meals. However, extended release tablets should be taken with breakfast. The maximum effectiveness of acarbose, an alpha-glucosidase inhibitor is attained when the drug is taken immediately at the start of each meal (not half an hour before or after), because it delays the carbohydrate absorption by inhibiting the enzyme alpha- glucosidase. Thyroxine: Recent evidence pointed out the role of gastric acid secretion on the subsequent intestinal absorption of thyroxine in relation with the timing of food ingestion as well as with pH impairment associated to frequent gastric disorders like Helicobacter pylori infection and gastric atrophy. Levothyroxine is a derivative of thyroxine. Grapefruit juice may slightly delay the absorption of levothyroxine, but it seems to have only a minor effect on its bioavailability. Accordingly, the clinical relevance of the grapefruit juice-levothyroxine interaction is likely to be small. Drug interactions may be theoretical or clinically relevant. A summary table is given to highlight some significant food-drug interactions. Some may be taken advantage of, to the benefit of patients, but more commonly drug interactions result in unnecessary adverse events. Fortunately, undesirable drug interactions can be prevented. Becoming more familiar with potential drug interactions can help clinicians predict and explain a patient`s response to medications. Significant food effects complicate development of new drugs, especially when clinical plans require control and/or monitoring of food intake in relation to dosing. The prediction of whether a drug or drug product will show human food effect is challenging. Antitumor Drugs: Mercaptopurine is a purine analog used for acute lymphoblastic leukemia and chronic myelogenous leukemias. Since it is inactivated by xanthine oxidase (XO), concurrent intake of substances containing XO may potentially reduce bioavailability of mercaptopurine. Cow’s milk is known to contain a high level of XO. This interaction may be clinically significant. Therefore most patients should try to separate the timing of taking mercaptopurine and drinking milk. Tamoxifen is a successful anti-tumor agent. If taken with sesame seeds, it negatively interferes with tamoxifen in inducing regression of established MCF-7 tumor size but beneficially interacts with tamoxifen on bone in ovariectomized athymic mice. Pharmacokinetic Interactions Drug Absorption Interactions: Food may affect drug absorption in the GI tract by altering gastric pH, secretion, gastrointestinal motility and transit time. This may result in a change in the rate of absorption or extent of drug absorption or both. For example, azithromycin absorption is decreased when it is taken with food, resulting in a 43% reduction in bioavailability. Sustained-release theophylline products when taken with high-fat foods may cause a sudden release (dose dumping) of theophylline, resulting in increased theophylline concentrations and possible toxicity. Children are more prone to this interaction than adults. In other cases, the components of the food, such as calcium or iron, may form complexes with the drug that are less easily absorbed. Examples include tetracycline, sodium fluoride and ciprofloxacin .The absorption of alendronate is impaired by food, calcium and almost everything, including orange juice and coffee. It should be taken with plain water and nothing else should be consumed for at least 30 minutes. In many cases, the actual mechanism by which food interferes with absorption is not known. Delayed absorption does not necessarily reduce the total overall exposure to the drug; the area under the curve (AUC) may be equivalent regardless of how the drug is taken. A reduced rate of absorption may sometimes be useful in reducing the side effects of a drug, as in the cases of ibuprofen, without reducing bioavailability. The bioavailability of some drugs may be enhanced by food. For example, an acid environment is necessary for the absorption of ketoconazole. The absorption of griseofulvin is increased by fat in a meal. Fenofibrate, mebendazole, isotretinoin, tamsulosin, carbamazpine and labetalol are examples of drugs that will be better absorbed when taken with food. Improved absorption of a drug may or may not have a significant effect on the drug’s efficacy. Patients taking digoxin should avoid taking bran fiber, pectin-containing foods such as apples or pears, or fibercontaining,
  • 188. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Gowthami B et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 458 bulk-forming laxatives at the same time, since these agents may bind to the digoxin, decreasing its absorption. This interaction could result in decreased serum concentrations of digoxin and therapeutic effectiveness. It is advisable to take some medications with food to reduce gastrointestinal irritation and possible nausea. Examples of these medications include potassium supplements, ferrous sulfate, nonsteroidal anti- inflammatory drugs, estrogen, prednisone, tacrine, terfenadine and nitrofurantoin. Cholesterol-lowering agent lovastatin should be taken with food to enhance gastrointestinal absorption and bioavailability. Simvastatin, pravastatin and fluvastatin may be taken without regard to food. Drug Metabolism Interactions: Food may alter the hepatic metabolism of some drugs. It has been reported that when administered with the antihypertensive drug felodipine, concentrated grapefruit juice caused an increase in the bioavailability of felodipine. The mean felodipine bioavailability with grapefruit juice was 284% (range 164%–469%) that of water. This resulted in lower diastolic blood pressures and increased heart rate in the male study volunteers. Adverse effects such as headaches, facial flushing and lightheadedness were more common after ingestion of 250 ml grapefruit juice (125 ml frozen grapefruit concentrate plus 125 ml of water). The bioavailability of nifedipine with grapefruit juice was 134% (range 108%–169%) of that with water. Orange juice did not have these effects. It is postulated that flavonoid compounds in grapefruit juice concentrate inhibit cytochrome P-450 metabolism of felodipine and nifedipine. This interaction could increase both the efficacy and toxicity of these calcium channel blockers. There is potential clinical significance because citrus juices are frequently consumed at breakfast, when many medications are also taken. Patients should be advised of this possible interaction. First-pass hepatic metabolism of propranolol and metoprolol may be decreased when either medication is taken with food, thereby enhancing bioavailability. Drug levels and therapeutic efficacy may be increased due to this interaction. Monoamine oxidase (MAO) inhibitors are known to interact with foods containing tyramine. Tyramine is normally inactivated by the enzyme monoamine oxidase and this prevents tyramine from accumulating in the body. Monoamine oxidase inhibitors cause increased levels of tyramine which can lead to a hypertensive crisis. Patients taking monoamine oxidase inhibitors should avoid foods high in tyramine such as aged cheeses, pickled fish, yeast extracts, red wine, some types of beer (including nonalcoholic beer), fava beans and fermented products. High protein foods that have been aged, fermented, pickled, smoked or bacterially contaminated are unsafe for patients taking MAO inhibitors. Foods considered safe when used fresh and in moderation include sour cream, yogurt, meat extracts, chopped liver, dry sausage and alcoholic beverages. Drug Excretion Interactions: Foods may alter the urinary pH, which can affect the activity of certain drugs. The half-lives of some medications can be significantly changed by alterations in urinary pH. Therefore, the half-life of acidic drugs will be extended in acidic urine because the drug is in its unionized form. However, the half-life of an acidic drug in alkaline urine is reduced because the drug is in its ionized form. Foods such as milk, vegetables and citrus fruits can alkalinize the urine. Meats, fish, cheese and eggs can acidify the urine. Foods may alter the renal excretion of some medications. Lithium and sodium compete for tubular reabsorption in the kidney. A high-salt diet causes more lithium to be excreted, whereas a low-salt diet causes decreased renal excretion of lithium and an increase in serum lithium levels. Pharmacodynamic Interactions: Foods may interact with medications by altering their pharmacologic actions. Diets high in vitamin K may cause antagonism of warfarin and decreased therapeutic efficacy of the anticoagulant. Foods rich in vitamin K include green leafy vegetables (kale, turnip greens, spinach, broccoli and brussels sprouts), cauliflower, chick peas, green tea, pork liver and beef liver. Alcoholic beverages may increase the central nervous system depressant effects of medications such as benzodiazepines, antihistamines, antidepressants, antipsychotic, muscle relaxants, narcotics or any drug with sedative actions. An example of a food potentiating the effect of a medication is coffee, as caffeine has additive effects on theophylline. It has been reported that caffeine increased serum theophylline levels by 20%–30% and increased the half-life of theophylline by decreasing clearance. Patients may complain of nervousness, tremor or insomnia. Caffeine has some bronchodilatory effects, which may enhance the effects of theophylline. A lower dosage of theophylline may be necessary for those patients who consume excessive quantities of coffee (more than 6 cups daily). ROLE OF PHARMACIST IN PREVENTION OF DRUG-FOOD INTERACTIONS
  • 189. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Gowthami B et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 459 Pharmacists in every practice setting need to be vigilant in monitoring for potential drug-food interactions and advising patients regarding foods or beverages to avoid when taking certain medications. It is imperative for pharmacists to keep upto- date on potential drug-food interactions of medications, especially today’s new drugs, so that they may counsel properly. In providing drug information to patients, pharmacists often discuss potential side effects and how the medication should be taken. It is important to provide information to patients on when to take their medications in relation to food intake. Consequences of drug-food interactions may include delayed, decreased or enhanced absorption of the drug. Food may also affect the bioavailability, metabolism and excretion of certain medications. The patient may experience an adverse side effect or toxicity or may not receive the full therapeutic benefit of the medication. The Joint Commission on the Accreditation of Healthcare Organizations (JCAHO) requires that a patient’s medication profile include potential drug-food interactions, that the pharmacist call the prescriber whenever the potential for a medication-food interaction exists and document the communication and follow-up action on the prescription or order form, and that patients be given instructions and counseling regarding the potential for drug-food interactions before their hospital discharge. Elderly patients may be at a greater risk for drug-food interactions because they typically consume more medications for their chronic medical conditions. A study of drug-nutrient interactions in long-term care facilities found a significant relationship between the number of medications a resident consumed and the number of drug-nutrient interactions for which a resident was at risk. Counseling and Guidance about Drug- Food Interactions: The following information can be given to the patients while dispensing the medicine.  Read the prescription label on the container. If you do not understand something or think you need more information, ask your physician or pharmacist.  Read directions, warnings and interaction precautions printed on all medication labels and package inserts. Even over-the-counter medications can cause problems.  Take medication with a full glass of water.  Do not stir medication into your food or take capsules apart (unless directed by your physician). This may affect the efficacy of medication.  Do not take vitamin pills at the same time you take medication. Vitamins and minerals can interact with some drugs.  Do not mix medication into hot drinks because the heat from the drink may destroy the effectiveness of the drug.  Never take medication with alcoholic drinks.  Be sure to tell your physician and pharmacist about all medications you are taking, both prescription and nonprescription.  Check with the pharmacist on how food can affect specific medications taken with the food. Precautions to be taken  Medications need to be taken at different times relative to meals.  Consult a physician when health problems persist.  During pregnancy and nursing always consult a physician or pharmacist before taking any medication.  Drugs taken by the mother may affect the infant.  Check with a doctor or pharmacist for the proper way and time to take medication. CONCLUSION Interaction between foods and drugs can have profound influence on the success of drug treatment and on the side effect profiles of many drugs. The clinical significance of drug-food interactions can be variable. Some foods greatly affect drug therapy, resulting in serious side effects, toxicity or therapeutic failure. In some instances, the interaction may have a beneficial effect by increasing drug efficacy or diminishing potential side effects. The interactions are not always detrimental to therapy, but can in some cases be used to improve drug absorption or to minimize adverse effects. These interactions have received more attention recently, especially drug interactions with grapefruit juice. As new drug approvals occur with ever increasing speed, there is less information available about their adverse effects and interactions when the drugs reach the market. Pharmacists in every practice setting need to be vigilant in monitoring for potential drug-food interactions and advising patients
  • 190. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Gowthami B et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 460 regarding foods or beverages to avoid when taking certain medications. It is imperative for pharmacists to keep up-to date on potential drug-food interactions of medications, especially today’s new drugs, so that they may counsel properly to the patients. REFERENCES Aihara K, Kajimoto O, Hirata H, Takahashi R, Nakamura Y, Effect of powdered fermented milk with Lactobacillus helveticus on subjects with high-normal blood pressure or mild hypertension, J Am Coll Nutr, 24(4), 2005, 257-65 Central drug standard controller organization, Drug Controller General of India, Guideline for Bioavailability and Bioequivalence studies, Indian bioequivalence studies guidelines, 2005. Essentials of Pharmacotherapeutics by FSK Barar, edition, published by S Chand & Company, 2000, 239-249. Ethical Guidelines for Biomedical Research on Human Subjects Indian Clinical Medical Research, 2000. FitzGerald RJ, Murray BA, Walsh DJ, Hypotensive peptides from milk proteins, J Nutr, 2004, 134: 980-988. Goodman and Gilman, The Pharmacological basis of therapeutics, by Joel Griffith Hardman (Author), Lee E Limbird (Author), Alfred G. Gilman (Author) Published by Mc Graw-hill, 08th international edition, 784-813. International Conferences on Harmonization (ICH) Guidelines, Tripartite Harmonized Guidelines on Good Clinical Practice, Step 4, May 1996. Molinaro G, Cugno M, Perez M, Angiotensin-converting enzyme inhibitor-associated angioedema is characterized by a slower degradation of des-arginine(9)-bradykinin, J Pharmacol Exp Ther, 2002; 303: 232-7. Okumura H, Nishimura E, Kariya S, Angiotensin-converting enzyme (ACE) Yakugaku Zasshi, 121(3), 2001, 253-7. Pharmacology and pharmacotherapeutics by Satoskar RS & Bhandarkar SD, Sixteenth edition, published by popular prakashan, Mumbai, 396-426. Pharmacology, HP Rang, MM Dale, JM Ritter, Fifth Edition, 2003, 299-301. Rockville, Food and Drug Administration, Bioavailability and Bioequivalence studies for orally administered drug products-General considerations, 2003. Rossi S, Editor, Australian Medicines Handbook, Adelaide: Australian Medicines Handbook, 2006, 2-3. Thomas MC, Diuretics, ACE inhibitors and NSAIDs - the triple whammy, Med J Aust, 172(4), 2000, 184,185. Tripathi K.D, Essential of medical Pharmacology, published by Jitendra P Vij, Jaypee Brothers Medical Publishers Pvt. Ltd, New Delhi, fifth edition, 503-518. United States Food and Drug Administration, Guidance for Industry, Computerized Systems Used in Clinical Trials, April 1999.
  • 191. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Sahithi B et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 461 A REVIEW ON COLLAGEN BASED DRUG DELIVERY SYSTEMS Sahithi B* , Ansari Sk, Hameeda Sk, Sahithya G, Durga Prasad M, Yogitha Lakshmi Nimra college of pharmacy, Vijayawada Corresponding author: Email:sahithinaidu60@gmail.com ABSTRACT Due to its biodegradability, biocompatibility, weak antigenicity and well-known safety profile, collagen becomes a very useful carrier for the delivery of various kinds of drugs and agents like growth factors, collagen possess some very unique properties as compare to other drug carriers that’s why a numerous number of researches are in pipeline on this biomaterial. The main application of collagen is collagen shields which are used in ophthalmology. However, fundamental awareness regarding collagen biochemistry and the manufacturing knowledge in combination with understanding of the physico- chemical properties is essential for fruitful application of collagen for drug delivery systems. The purpose of this review article is to summarize information available on collagen dosage forms for drug delivery as well as to communicate an outline regarding current preparation of collagen available in market includes - collagen sponges for burns/wounds, mini-pellets and tablets, gel preparations in combination with liposomes for sustained delivery of drug, formulations for transdermal drug delivery, and Nano spheres for gene delivery, collagen matrices for cell culture. Key words: Collagen, Drug delivery system, Biomaterial, Ophthalmology. INTRODUCTION In the beginning of 1970s and 1980s the research on collagen was initiated by interested scientists and commercial research laboratories expanding medical applications of biomaterials and connective tissue research. In present years biotechnology has given a support to research the collagen material useful for drug delivery. Basically Collagen is a protein which is widely used in medical field. Collagen plays a significant role in the formation of organs and tissues, and is involved in different functional expressions of cells. Many natural polymers and their synthetic analogues are used as a biomaterial, but the characteristics of collagen as a biomaterial are different from those of synthetic polymers mainly in its mode of interaction inside the body. The important features of collagen are its biocompatibility, biodegradability and weak antigenicity [Maeda et al., 1999].In the body as compared with other natural polymers like gelatin and albumin. Collagen possesses good abilityto penetrate a lipid-free interface. The primary reason for the usefulness of collagen in biomedical application is that collagen can form fibers with extra strength and stability through its self-aggregation and cross- linking. Collagen: Basically collagen is a naturally existing protein present in the animal body, fibrous in nature, and especially found in the connective tissue and flesh of mammals. Approximately 25%-35% of total body protein is comprised of collagen, in the form of elongated fibrils; collagen is abundantly present in fibrous tissue like bone, cartilage, tendons, blood vessels, ligament, skin, cornea, inter-vertebral disc and the gut. The synthesis of collagen in the body is made by fibroblast cells. Collagens possess good tensile strength, and found both outside and inside the body cells. In combination with elastic, collagen provides support to body tissues and organs, basically collagen offers firmness and strength and elastic provides flexibility to body tissues. In fact gelatin which is used in food and pharmaceutical industries is collagen that has been hydrolysed irreversibly. Structure of collagen: Basically collagen possesses a triple helix structure, which generally made up of two homologous chains (α-1) and one supplementary chain that varies slightly in its chemical composition (α-2). These chains are polypeptide in nature and coiled around one another in a cable form. Each has a distinct turn in the reverse direction, these chains are connected together chiefly by hydrogen bonds between nearby CO and NH groups. The weight of collagen molecule is 300 kda and its structure is rope shaped and having a length of 300 NM and a width of 1.5 NM. The major content of glycine and amino acid residue is affecting the helix formation; in each of three chains of collagen molecule the amino acids are regularly arranged. The sequence of amino acids follows the pattern glycine-proline-X or glycine-X-hydroxyproline where X is the amino acid other than glycine, proline or hydroxyproline; glycines constitute about 1/3 of total sequence and proline or hydroxyproline accounting for the 1/6 of the sequence. This whole structure is joined with the help of hydrogen bonds and linking peptide bonds. Characteristics possessed by collagen:  Stretch-ability under stress condition collagen stretch rather than break
  • 192. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Sahithi B et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 462  Strength  Biochemical compatibility  These three amino acid monomers are strongly fused and they look like single monomer  Several hydrogen bonds are present in collagen, on applying stress they can be wrecked and re-joined after removal of pressure;  Collagen is biodegradable  Collagen show good absorption in-vivo  Collagen possesses weak antigenicity Isolation and Purification of Collagen: Even though the mammalian body retains a plenty amount of collagen, those tissues rich in fibrous possess collagen such as skin and tendons, are commonly used as preliminary materials to produce collagen for use in transplants, wound dressings, or drug delivery systems. In addition procaine, bovine and sheep collagen varieties derived from many different sources including marine sources, human placenta, and recombinant human collagen from transgenic animals must be labelled. Autologous collagen material deals additional gut alternative mucosa which is consumed in the building of surgical sutures. Collagen is insoluble in organic solvents. Water-soluble collagen denotes only a minor fraction of total collagen and the quantity depends on the age of the animal and kind of tissue extracted. In certain tissues, especially the skin of young animals, cross linking is sufficiently little to extract a few percent in suitable conditions. Still, collagen molecules present inside fibril masses can be separated and brought into aqueous solution. Though, the nature of the crosslink dominant in different tissues decides the particular solvent to be used and the resulting yields. Basically four types of collagen can be isolated and purified for the implementation in pharmaceutical industries for the delivery of drugs are following such as  Natural salt soluble collagen  Alkali and enzyme treated collagen  acid soluble collagen  Insoluble collagen Process to Isolate Neutral Salt Soluble Collagen: Note- Most tissues have minute or no salt-extractable collagen. In demand to increase the yield for research purposes animals can be put on the diet contain b-aminopropionitrile, an inhibitor of peptidyllysyl oxidase, yet this method is in adequate for larger commercial scale.
  • 193. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Sahithi B et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 463 Process to Isolate Acid Soluble Collagen: Why Collagen can be used as a biomaterial for drug delivery:  Collagen is biodegradable and simply absorbed in the body  Collagen is a part of the body that’s why it is non-antigenic  Collagen is a non- toxic biopolymer  Collagen shows better biocompatibility  Collagen can be framed in a number of different forms  Shows synergism with other bioactive compounds  Haemostatic in nature and encourage blood coagulation  Compatible with synthetic polymers  By utilizing its functional group collagen can be easily modified to produce desired materials  Biodegradability of collagen can be controlled by cross-linking  Existing in plenty and simply purified from living organisms (constitutes more than 32% of vertebrate tissues)  Biological plastic due to great ductile strength and negligible express ability. The in vivo absorption of collagen is controlled by the use of cross-linking agents, such as:  Glutaraldehyde  Chromium tanning  Formaldehyde  Poly epoxycompounds  Acylazide  Carbodiimides  Hexamethylenediisocyanate Physical treatment, such as ultra-violet/gamma-ray irradiation and dehydrothermal treatments have been efficiently used for the introduction of cross links to the collagen matrix [10]. The use of collagen as a drug
  • 194. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Sahithi B et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 464 delivery system is very comprehensive and diverse. It can be extracted into an aqueous solution and moulded into various forms of delivery systems. The applications of collagen as drug delivery systems are:  Used in the formation of microspheres for drug delivery  In formulation of nanoparticles for gene delivery  Collagen is used in the manufacturing of collagen Sponges for burns/wounds;  Development of tablets and pellets for the delivery of proteins;  Collagen is used for gel formulation and combined with liposomes for sustained delivery of drugs  In the treatment of cancer collagen is used as aqueous injection  Collagen is used in ophthalmology as collagen shields  Collagen is used as controlling material for the delivery of drugs in transdermal patches  As films for the delivery of human growth hormone, immune-stimulants, tetracycline, growth factors  For the delivery of glucocorticosteroids, microparticles of collagen are used. Collagen delivery systems having a smooth release control can be attained by balancing the configuration of the collagen matrix or attaching other proteins, such as Austin, fibronectin or by fusion of collagen with other polymers, such as collagen/liposome and collagen/silicone. Some specific type of drug delivery systems constructed on Collagen base: Nanoparticles/Nano spheres/Microspheres: In the collagen fold configuration, the crystallites suspended in the gel aggregates seem as a multiple chain system; this property is used to formulate aggregates as colloidal drug delivery carriers. The construction of nanosphereis determined by a mixture of electronic and electrostatic forces with sodium sulphate engaged as a liquefying reagent to facilitate greater charge-charge relations among plasmid DNA and collagen. The stability of the produced collagen nanoparticles is depending on the molecular weight of collagen, and temperature and pH is greatly affecting the molecular weight profile of the collagen solution , and these are also further affect the non-covalent interactions liable for the molecular structure of collagen. The nanoparticles and nanospheres based on biodegradable collagen; are enabled and enhanced uptake of exogenous compounds such as anti-HIV in a number of cells, especially macrophages, that is an advantage of collagen based nanoparticles as systemic delivery carriers; and they are also thermally stable and easily sterilized. Some other drugs like steroids, cytotoxic drugs like Campthocin can be easily delivered in systemic circulation with the help of collagen nanoparticles. Collagen based nanoparticles can be readily used in sustained and delayed release formulation for steroids and antibiotics because of their: –  Large surface area  Smaller size  Great absorptive capability  Capacity to diffusing in water to form a colloidal solution o For example: Dermal delivery of retinol enhanced in collagen nanoparticles. Retinol in collagen nanoparticle was stable shows a quicker transportation of incorporated drug through the skin. Fabrication methods for Collagen nanoparticles: There are basically four type of methods for the manufacturing the protein based nanoparticles namely-  Emulsification  Desolvation  Coacervation  Spray drying o And some additional methods are, Jet milling technique, fluidization and solvent precipitation method, Interfacial polymerization etc. Processes involved: Emulsification: In this process, A collagen aqueous phase containing a hydrophilic surfactant and water, and an organic phase containing a lipophilic surfactant, oil and water miscible solvent is mixed with rapid agitation by a mechanical homogenizer at room temperature to form a homogeneous emulsion. Then the above emulsion will be mixed in preheated oil (120) drop by drop resulting formation of collagen nanoparticles.
  • 195. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Sahithi B et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 465 Aqueous phase (Collagen solution) Organic phase (oil) Emulsification O/w emulsion Heated oil Collagen Nanoparticles Fig.1. Emulsification method for nanoparticles manufacturing Desolvation : The process of Desolvation includes the addition of alcohol or natural salt as desolvation factor to the collagen solution, which alters the tertiary structure of collagen, when the critical level of desolvation attained the formation of collagen mass, starts lastly glutaraldehyde will be added as a cross-linking material, and then nanoparticles is formed. This process was firstly employed by Marty and co-workers. Fig2. Desolvation method for nanoparticles manufacturing Coacervation: This method is similar to desolvation method, the difference is only in various parameters like- temperature, molar ratio of organic solvent and protein, rate of solvent addition, concentration of cross-linker used, pH, speed of homogenizer etc. Spray drying: Basically spherical collagen nanoparticle is fabricated by this process. This process include the spraying of dilute solution of collagen leads to the formation of hollow spheres using elevated temperature;
  • 196. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Sahithi B et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 466 increased temperature can lead denaturation of collagen triple –helical structure. So collagen solution is sprayed into liquid nitrogen to prevent denaturation. After that the fabricated nanospheres are successively frozen, tempered, lyophilized, cross-linked, and sterilized. Collagen based pellets/tablet: Collagen pellets are extensively used in Japan. These pellets are also known as monolithic devices .They are tiny rods of approximately 1mm in diameter and 15 mm in length manufactured from collagen by cutting, moulding, and drying. These devices are cylindrical in structure, and they can be administered via injection using a syringe with a plugar. Pellets are very suitable for the local delivery of lysozyme and minocycline in the treatment of clinical symptoms of periodontitis. These pellet are also proven to be effective in vivo for the delivery of interleukin-2 (half-life =360 min for subcutaneously injected IL-2 mini- pellets as compare to, 15 min and 8 min respectively for subcutaneous and intravenous injections of an aqueous preparation). It is also proved that single subcutaneous injection of a mini-pellet cause a prolonged retention of IL-2 and maximal concentration in the serum. The similar result was also observed for interferon. In 1989 Lucas and his co-workers was designed a collagen pellet type controlled release delivery vehicle which is made up of type-1 collagen for water soluble bone forming proteins. Collagen films: Collagen films are basically sterilized films of collagen having thickness of 0.01-0.5 mm; incorporated with drugs like steroids, antibiotics, hormones (obtained from rDNA technology E.g. human growth hormone, rhBMP-2 etc.) intended for local action. They are simply manufactured by air- drying of casted collagen mainly used as a barrier membrane, the drug incorporation in collagen films done by covalent bonding, hydrogen bonding and by simple entrapment. Bradley used collagen films cross-linked by chromium tanning, formaldehyde to sustain the release of medroxyprogesterone acetate. In this investigation cross-linking was done on films already incorporated with drugs, endangering the Pharmacokinetic and pharmacodynamics activity of the drug. This threat can be overcome with collagen materials which are cross-linked with glutaraldehyde prior to protein incorporation. Collagen films also can be used as a drug carrier for antibiotics. E.g. when collagen films containing tetracycline were implanted in rabbits suffering from periodontal disease; tetracycline was remained in plasma for more than seven days and its activity was sustained for more than ten days. After four and seven weeks the clinical symptoms of disease were decreased significantly. A European patent application was filed which contain the research information regarding sustained release delivery of platelet derived growth factor; was improved by multiple and single layer collagen films and it was found that this specific preparation had got a release factor up to 100 h and thereby it leads to an enhanced wound healing in vivo. In the case of liver cancer or tissue infection collagen film implants are very useful. When collagen film applied to eye it was totally hydrolysed within 5-6 hr in the case of corneal tissue infection. Collagen sheets of micro-fibrous collagen was used as local delivery carrier in the treatment of cancer, locally implanted collagen sheets which incorporated with anticancer agents such as methotrexate and ectopocide needs low plasma concentration management. The formation of bone tissues was enhanced when a collagen matrix and film were used as a carrier for gene delivery. This shows that collagen based gene delivery system is very efficient as implant. Collagen shields: Collagen shields is also known as collagen corneal shield, they are newly developed, potentially versatile ophthalmic lens, which is made up of collagen, since collagen is a natural, commonly available protein involved in the support and protection of vital structures, many researchers have tried to use peripheral collagen to protect the surface of the eye in a variety of diseased states, like traumatic and non- traumatic states after surgery; after corneal transplantation, radial keratomy. Generally collagen shields are manufactured from bovine or procaine collagen, there are three kind of collagen shield available in market having dissolving time of 12, 24, 72 hours. Bausch & Lomb Pharmaceuticals, a division of Bausch & Lomb, Inc. acquired the rights to develop and market these collagen contact lenses, now known as Bio Cora collagen shields. After much research, Bausch & Lomb has been able to produce the shields in a reproducible manner and in a variety of shapes and thickness. Some other marketed preparations are (proshieldo, MediLenso, Fort Worth, Chiron, TX, Irvine). These shields are able to enhance the penetration of corticosteroid, sub-conjunctival antibiotics in eye. They are act as a short term bandage and allow sufficient oxygen transmission for essential metabolism occurring in eye cornea.For the corneal surface lubrication these shields dissolve in collagen solution that minimize lids rubbing. Mainly water soluble antibiotics and steroids are used in combination with collagen shields for example- Vancomycin, Trimethoprim, Amphotericin-B,Gentamycin Polymyxin-B sulfate, Tobramycin, steroids, pilocarpine, Application of collagen shields on cornea is demonstrated in figure-3.
  • 197. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Sahithi B et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 467 Fig 3: Application of collagen shield on cornea Marketed preparation of collagen shields:  Biocora®  ProshieldO®  MediLenso®  Irvine®  Chiron® Collagen sponge: Collagen sponge are manufactured from pure bovine collagen obtained from bovine skin, bovine collagen is firstly put into a solution having pH 3.0 and then stabilize into physical form of a sponge layer. And then this sponge layer is combined with fibronectin, elastin or glycosaminoglycans to achieve a fluid building capacity and elasticity. They are also manufactured by freeze-drying of alkali or acid, swollen collagen containing 0.1- 5% dry matter content. Collagen sponges can be cross-linked with glutaraldehyde and copolymerised with other synthetic as well as natural polymers, for example collagen sponges copolymerized with PHEMA (polyhydroxyethylmethacrylate) are more hydrophilic in nature, retain wetness for longer period and also possesses more tensile strength. Collagen sponges were basically developed as hemostyptics and wound dressings but in present time they are also used for antibiotics, steroids and growth factor delivery for wound healing and for bone forming implants. The application of collagen sponges for delivery of topical agents (table 1) Table.1.Application of collagen sponges for various topical agents Collagen sponges are found very useful in dressing for leg ulcers, decubitus ulcer, donor sites, pressure sores. The major benefits of collagen sponges includes their ability to absorb enormous quantity of tissue exudates and smooth adherence to the wet wounds with preservation of micro climate as well as shielding against secondary bacterial infection and mechanical harm, in addition collagen sponges promote inflammatory cells activity to porous scaffolds and cellular growth. Thus collagen sponges can be considered asactive dressings, which aid in the healing process. Marketed preparations of collagen sponges: These are the some of the marketed formulation of the Collage sponges available in the market such as Collarx®, Collatamp® G, Collatamp® Eg, Sulmycin® Implant, Garamycin® Schwamm, Duracol®, Duracoll®, Gentacol®, Gentacoll®, Garacol®, Garacoll®, And Cronocol® - Gentamicin Surgical Implants Collagen hydrogels/gels: Collagen hydrogels/gels are processed by cross-linking of collagen with chemicals like poly epoxy compounds, carbodiimides, polyphenolic compounds, aldehydes, and acyl azide compounds which leads to the formation of bonds between molecules and fibrils. Collagen hydrogels possess a unique property of soaking and swelling on hydration with biological fluids and they are also capable to maintain their integrity after soaking. Drugs/ agents Uses Gentamycin In septic focus in abdomen Growth factor (rhBMP-2) In bone formation and wound healing
  • 198. ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Sahithi B et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(3) May-June 2013 Page 468 These hydrogels are very patients compliant because ease of application, high bio-adhesion and compatible with large varieties of drugs and agents.Collagen gels are excessively used as injectables the most common form are- 1. Non-fibrillar viscous solution in aqueous medium 2. Fibersinjectables suspensions For ophthalmic purpose these suspensions can be mixed with drugs and administered, these preparations are patented, when inject; initially remains in liquid state and then after some time convert into gel.Shows a great potential for sustained and controlled delivery of medicaments. CONCLUSION AND FUTURE PERSPECTIVES Collagen has various advantages as a biomaterial and is widely used as carrier systems for delivery of drug, protein and gene. The examples described in this paper signify selected applications of collagen in the biomedical field. The effective demonstration of usefulness of human skin substitutes made of collagen has leads to the development of bioengineering tissues, such as blood vessels and ligaments. Although many applications of collagen as a drug vehicle discussed in the paper, it should be noted the information regarding collagen is very less as compare to synthetic polymers in literature because, the pure type-1 collagen is very costly, variability in different forms, complex handling processes, and risk of Bovine spongiform encephalopathy (BSE). Beside them collagen possess some very extraordinary properties which make it a very useful biomaterial for drug delivery includes its biocompatibility, absorbability on biological membranes, no antigenicity, low toxicity, synergism with other bioactive compounds etc. these advantage will carry the future development of this biomaterial. REFERENCES Achim Berthold, Karsten Cremer, JorgKreuter, Collagen microparticles: carriers for glucocorticosteroids, European Journal ofPharmaceutics and Biopharmaceutics, 45, 1998, 23–29 Barbani N, Giusti P, Lazzeri L, Polacco G, Pizzirani G, Bioartificial materials based on collagen: Collagen cross- linking with gaseous glutaraldehyde, J. Biomater, Sci. Polym. Ed. 7, 1995, 461–469 BS Davidson, F Izzo, DM Cromeens, LC Stephens, ZH Siddik, SA Curley. Collagen matrix cisplatin prevents local tumor growth after margin-positive resection, J. Surg. Res., 58, 1995, 618–24 Chavpil M, Medical and Surgical Appliances of Collagen from International Review of Connective Tissue Research , 6, 1993, 1 6, 9 10, 29 30 and 534 F Lefebvre, P Pilet, N Bonzon, G Daculsi, M Rabaud, New preparation and microstructure of the EndoPatch elastin–collagen containing glycosaminoglycans, Biomaterials, 17, 1996, 1813–18. G. L. Wilkes and B.T. Vu, “Superstructure in Films of Bio- and Biorelated Polymers as Noted by Small Angle Light Scattering,” in Structure and Properties of Polymer Films, eds. R. Lenz and R. Stein, Plenum Press (1973) 39-65 Harkness R.D, Biological functions of collagen, Biol. Rev. 36, 1961, 399–463 I Finkelstein, GE Trope, JG Heathcote, DS Rootman, L Spero, IA Menon, Further evaluation of collagen shields as a delivery system for 5-fluoruracil: histopathological observations, Can. J. Ophthalmol, 26, 1991, 129–32