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    Indian journal of research in pharmacy and biotechnology  4 Indian journal of research in pharmacy and biotechnology 4 Document Transcript

    • Volume 1 Issue 4 www.ijrpb.com July - August 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
    • Volume 1 Issue 4 www.ijrpb.com July - August 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
    • Indian Journal of Research in Pharmacy and Biotechnology ISSN: 2320-3471 (Online) ISSN: 2321-5674 (Print) Volume 1 Issue 4 www.ijrpb.com July – August 2013 S.No. Contents Page No. 1. Controversial role of antipsychotics in the treatment of Alzheimer’s disease Mahesh G, G Praveen Kumar 469-471 2. Formulation and evaluation of oro dispersible tablets of Amlodipine besylate Shobha Krushnan G, Ravi M Britto, Perianayagam J, Rajendra Prasad R 472-477 3. Comparision of potency of anti bacterial activity and anti inflammatory activity of 10 years and 100 years old bark extracts of Azadirachta indica Vijaya Kumar G, Srinivas N, P Sravanthi, Sravani B 478-483 4. Development and evaluation of carisoprodol tablets with improved dissolution efficiency using solid dispersion technique Mogili Daya Sagar, Mohammed Shahidullah, Shaik Rabbani Basha, Shaik Shahnaz, Harish.G 484-487 5. Transdermal drug delivery systems R.sowjanya, Salman Khan, D.Bhowmik, Harish.G, S.Duraivel 488-495 6. Synthesis of new thiazolidine-2,4-dione derivatives and their antimicrobial and antitubercular activity Faiyazalam M Shaikh, Navin B Patel and Dhanji Rajani 496-503 7. Effects of permeability characteristics of different polymethacrylates on the pharmaceutical characteristics of diltiazem hcl-loaded microspheres V. Kamalakkannan, K.S.G.Arul Kumaran, C. Kannan, S.Bhama, R. Sambath Kumar 504-511 8. Importance of safety health environment in preventing occupational health hazards in indian industries Murty TN, Md Aasif Siddique Ahmed Khan, Abhinov T, Abhilash T 512-516 9. Optimization of Thiocolchicoside tablet with permeation enhancers using 32 factorial design Devendra Singh, Pankaj Kumar Sharma, Udai Vir Singh Sara 517-524 10. Method development and validation for the simultaneous estimation of Desvenlafaxine and Clonazepam in bulk & tablet formulation by RP-HPLC method Regalagadda Mallikarjuna, Nanda Kishore Agarwal, Prem Kumar Bichala, Sukhen Som 525-532 11. Plant seeds used for anthelmintic activity: A review Shambaditya Goswami, Sanjeev Nishad, Mayank Rai, Sarvesh Madhesiya, Ankita Malviya, Pawan Pandey, Vikram Gautam, Sujeet Yadav 533-536 12. Development and validation of pemetrexed by RP-HPLC method in bulk drug and pharmaceutical dosage forms Suresh Kumar Agrawal, Devendra Singh Rathore 537-542 13. Stability indicating RP-HPLC method for the estimation of Ceftazidime pentahydrate and Tazobactam sodium in bulk and dosage forms S. Amareswari, Nandakishore Agarwal, Md Aasif Siddique Ahmed Khan 543-548 14. Effect of hydrotropic solute on in-vitro charecterization of Valsartan fast disintegrating tablets Madhu Sudhan Reddy A, Kishore Babu G, Srinivasa Babu P, Bhardwaj G 549-553 15. A review on Gloriosa superba l as a medicinal plant Kavithamani D, Umadevi M, Geetha S 554-557 16. Formulation and evaluation of floating drug delivery system of Clarithromycin tablets Priyanka Shukla, Ajay Yadav 558-561 17. Antifungal activity of ethanolic extract of Eupatorium adenophorum leaves Dharmendra Kumar Singh, Ranjeet Singh 562-564
    • Indian Journal of Research in Pharmacy and Biotechnology ISSN: 2320-3471 (Online) ISSN: 2321-5674 (Print) Volume 1 Issue 4 www.ijrpb.com July – August 2013 18. Formulation of mouth dissolving tablets of Naproxen Rajesh Reddy K, Nagamahesh Nandru, Desam Asha Latha, Srinivasa Rao Chekuri 565-569 19. Preparation of immediate release Atorvastatin and sustained release matrix tablets of Gliclazide using retardant hydroxypropyl methyl cellulose Vinod Raghuvanshi, Jayakar B, Debjit Bhowmik, Harish G, Dureivel S 570-574 20. Phytochemical sreening and antidiabetic antioxidant effect of Ecbolium ligustrinum flowers extracts Ranjitsingh B Rathor, Rama Rao D, Prasad Rao 575-580
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Mahesh and Praveen Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 469 CONTROVERSIAL ROLE OF ANTIPSYCHOTICS IN THE TREATMENT OF ALZHEIMER’S DISEASE Mahesh G*1 , G Praveen kumar2 1. School of Pharmaceutical sciences, Vels University, Chennai. 2. C.L. Baid Metha College of Pharmacy, Chennai, Tamil Nadu. *Corresponding author: Mail Id: udaynagamahesh@hotmail.com ABSTRACT Antipsychotics are the commonly prescribed drugs in the treatment of Alzheimer’s disease, which is the most common form of dementia. Atypical antipsychotics are an effective short-term (6-12 weeks) treatment in relieving the depression, psychotic symptoms (hallucinations and delusions) and behavioral disturbances (physical and verbal aggression, motor hyperactivity, repetitive mannerisms and activities, and combativeness). But several placebo studies & clinical based evidences which recorded the deaths of the patients concluded that this medication nearly doubles the risk of death in patients over two to three years by developing cerebrovascular adverse events, upper respiratory infections, oedema or extra pyramidal symptoms. The use of selective serotonin reuptake inhibitors (SSRI’s), Nor- epinephrine reuptake inhibitors (NERI’s) and Tricyclic antidepressants (TCA’S) may relieve depression but still they are associated with serious adverse effects such as insomnia, agitation, confusion and GI adverse effects. So there is a need for applying non-pharmacological treatment i.e. Psychotherapy rather than the Pharmacotherapy in minimizing the symptoms & anticipates further research in developing the appropriate medication, alternative to the antipsychotics which minimizes the suffering of the patient. Typical antipsychotics were the first generation of the drugs aimed to treat psychosis by antagonizing D2 receptors. As a result, they reduce dopaminergic neurotransmission in the four dopamine pathways. Typical Antipsychotics include Chlorpromazine, Chlorprothixene, and Haloperidol etc. Atypical Antipsychotics are the drugs which not only block dopamine receptors but also serotonin receptors.Risperidone, Olanzapine, Quetiapine, Aripiprazole, Clozapine, Ziprasidone include Atypical Antipsychotics. Key words: Antipsychotics, Alzheimer’s disease, Atypical Antipsychotics, Typical antipsychotics INTRODUCTION Atypical antipsychotics are not the FDA approved drugs for the treatment of behavioral & psychotic symptoms in dementia (BPSD). Placebo-controlled trials revealed increased mortality rate in patients those treated with Atypical Antipsychotics. The mostly prescribed Antipsychotics include Risperidone, olanzapine, quetiapine & Haloperidol (Typical Antipsychotic). Alzheimer’s disease majorly affects the Hippocampus & Cerebral cortex of the brain with the formation of Neurofibrillary tangles & Neuritic plaques which leads to the degeneration of cortex, cholinergic & other neurons (Amresh Shrivastava, 1999). 15 out of 17 Placebos controlled trials showed increased mortality in the drug treated group compared to the Placebo treated patients (Monasterio E, 2011). It involves Risperidone (7trials), Olanzapine (5trials), Quetiapine (2 trials) & Aripiprazole (3 trials). 1.6-1.7 fold (i.e almost 2 times) increase in mortality is observed in active treatment over placebo (Forbes DA, 2005). Rate of death in drug treated patients was about 4.5%, compared to rate of about 2.6% in placebo group (Ballard, 2009). Specific causes of these deaths are cerebrovascular adverse events (heart failure with sudden death) or infections (mostly pneumonia). In 2005, FDA approved the Black box warning that “Atypical Antipsychotics increase the risk of death in dementia patients” (Cummings JL, 2002). The only FDA approved drugs for the treatment of Alzheimer’s disease is for improving the cognition i.e. for cognitive symptoms (memory loss, disorientation, impaired executive functions such as poor problem solving, planning, and attention, thinking, remembering & reasoning). Examples of drugs used for improving cognitive symptoms are Donepezil, Rivastigmine, Galantamine (Cholinesterase Inhibitors) and Memantine (N-methyl D-aspartate receptor antagonist).
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Mahesh and Praveen Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 470 The U.S. Food and Drug Administration (FDA) have approved only five medications till now to treat the symptoms of Alzheimer's disease (Rayner AV, 2006). Slow titration of drugs with continuous monitoring of patient is essential to minimize the risk of adverse effects. The common adverse effects of AD medications include depression, insomnia, confusion, decreased weight, and diarrhea. So the Cholinesterase inhibitors, NMDA receptor antagonist & Atypical antipsychotics which are used in treating cognitive & non-cognitive symptoms (BPSD & depression) have wide side effects & high risk of adverse effects (Steffens, 2008) CONCLUSION The serious adverse effects due to the use of Atypical Antipsychotics in treating Behavioral & Psychotic symptoms in Dementia (BPSD) of Alzheimer’s disease concludes the limitation for the use of atypical-antipsychotics and their controversial role in the current existing treatment. Despite the FDA black box warning, antipsychotic use in dementia has remained remarkably frequent; a recent study of 16,586 nursing home patients reported that 29% receive at least one antipsychotic medication. As the warnings initially slowed the rate of increase in new prescriptions for atypical antipsychotics in patients with dementia, but there is no decrease in the overall prescription rate. (Devanand, 2011) Non pharmacological interventions which include Psychotherapy should be the primary intervention in treatment. The care giver should simplify the tasks to the patient by providing 3 R’s-Repeat, Reassure & Redirect. This improves the activities of daily living. The current existing medication only slows down the worsening of cognition & minimizes the BPSD but cant arrest the progression of Alzheimer’s disease. So there is an immediate need for developing new drugs which curbs & reverses the neuro degeneration with a cost effective treatment for Alzheimer’s disease (Treloar, 2010). Table.1.FDA approved medications for treating Alzheimer’s disease. Drug name Approved For FDA Approved Memantine Moderate to severe 2003 Galantamine Mild to moderate 2001 Rivastigmine Mild to moderate 2000 Donepezil All stages 1996 Tacrine Mild to moderate 1993 Figure 1: showing the presence of neurofibrillary tangles & neuritic plaques Figure 2: Comparison of Normal brain, early & late Alzheimer brain by Positron emission tomography (PET SCAN)
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Mahesh and Praveen Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 471 Figure 3: Risk Perception In Typical & Atypical Antipsychotics Table 2: Cardiovascular risk factors associated with Atypical Antipsychotics REFERENCES Amresh Shrivastava, Megan Johnston, Kristen Terpstra, Larry Stitt, and Nilesh Shah, Atypical antipsychotics usage in long-term follow-up of first episode schizophrenia, Indian J Psychiatry, 54(3), 2012, 248–252. Ballard CG, Gauthier S, Cummings JL, Brodaty H, Grossberg GT, Robert P, Cyketsos CG, Management of agitation and aggression associated with Alzheimer’s disease, Nature Reviews, 5, 2009, 245-255. Cummings JL, Frank JC, Cherry D, Guidelines for managing Alzheimer's disease: part I. Assessment, Am Fam Physician, 65, 2002, 2263-2272 Devanand D P, Susan M D, Schultz K, Consequences of Antipsychotic Medications for the Dementia Patient, Am J Psychiatry, 168, 2011, 767-769. Forbes DA, Peacock S, Morgan D, Nonpharmacological management of agitated behaviors associated with dementia, Geriatrics and Aging, 8, 2005, 26-30. Monasterio E, McKean A, Off-label use of atypical antipsychotic medications in Canterbury, New Zealand, N Z Med J, 124, 2011, 1336. Rayner AV, O'Brien JG, Schoenbachler B, Behavior disorders of dementia: recognition and treatment, Am Fam Physician, 73, 2006, 647-652. Steinberg, M., Shao, H., Zandi, P., Lyketsos, C.G., Welsh-Bohmer, K.A., Norton, M.C.,Breitner, Steffens JC, Tschanz DC, Point and 5-year period prevalence of neuropsychiatric symptoms in dementia: the Cache County study, International Journal of Geriatric Psychiatry, 23(2), 2008, 170-177. Treloar A, Crugel M, Prasanna A, Solomons L, Fox C, Paton C, Katona C, Ethical dilemmas: should anti-psychotics ever be prescribed for people with dementia? British Journal of Psychiatry, 197(2), 2010, 88-90.
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Shoba Krushnan et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 472 FORMULATION AND EVALUATION OF ORO DISPERSIBLE TABLETS OF AMLODIPINE BESYLATE Shobha Krushnan G*, Ravi M Britto, Perianayagam J, Rajendra Prasad R Department of pharmaceutics, Aurobindo College of Pharmaceutical Sciences, Gangadevipally, Geesugonda, Warangal, Andhra Pradesh, India *Corresponding author: E.Mail: avinashjipz@gmail.com ABSTRACT Recent advances in technology have presented viable dosage forms alternative for patients who may have difficulty in swallowing tablets or capsules. Oro-dispersible tablet is one such approach in which the tablets were dispersed in the mouth rapidly. Amlodipine is a calcium channel blocker used in the treatment of hypertension and angina pectoris, where ultra-rapid action is required. In the present study Amlodipine Oro-dispersible tablets are formulated using sodium starch glycolate, croscarmellose sodium, crospovidone superdisintegrants. The tablets were prepared by direct compression technique and were evaluated for weight variation, friability, hardness, drug content, in-vitro disintegration time, wetting time, in-vitro dissolution studies. All the formulations follow compendia specifications. Formulations containing higher concentrations of sodium starch glycolate and cross povidone as superdisintegrant showed better dissolution profile and disintegration time. The bioavailability of amlodipine was increased by formulating amlodipine as ODT. Differential Scanning calorimetric study (DSC) and Fourier transform infrared spectroscopy (FTIR) were conducted for drug excipient compatibility study. Key words: Orodispersible tablets, Amlodipine, hypertension INTRODUCTION United States of America food and drug administration (FDA) defines oral dispersible tablet (ODT) as “A solid dosage form containing medicinal substances (or) active ingredient which disintegrates rapidly usually within a matter of seconds when placed upon a tongue”. Oral route of drug administration have widely accepted up to 50-60% of total dosage forms. Solid dosage forms are popular because of ease of administration, accurate dosage, self-medication, pain avoidance and most importantly the patient compliance. The most popular solid dosage forms are tablets and capsules having the drawback of these dosage forms for some patients, is the difficulty to swallow. Drinking water plays an important role in the swallowing of oral dosage forms. Often people experience inconvenience in swallowing conventional dosage forms such as tablet when water is not available, in the case of the motion sickness and sudden episodes of coughing during common cold, allergic condition and bronchitis. For these reasons, tablets that can rapidly dissolve or disintegrate in the oral cavity have attracted a great deal of attention. Oro- dispersible tablets are not only indicated for people who have swallowing difficulties, but also are ideal for active people (Valleri M, 2004). Fast dissolving tablets are also called as mouth-dissolving tablets, melt-in mouth tablets, oro- dispersible tablets, rapimelts, porous tablets, quick dissolving etc. Fast dissolving tablets are those when put on tongue disintegrate instantaneously releasing the drug, which dissolve or disperses in the saliva (Fu Y, 2004).The faster the drug into solution, quicker the absorption and onset of clinical effect. Some drugs are absorbed from the mouth, pharynx and esophagus as the saliva passes down into the stomach. In such cases, bio-availability of drug is significantly greater than those observed from conventional tablets dosage form (Ghosh TK, 2005, Deepak K, 2004). The basic approach in development of ODT is the use of superdisintegrants like cross linked carboxymethyl cellulose (croscarmellose), sodium starch glycolate (primogel, explotab), polyvinyl pyrollidone (Crosspovidone) etc, which provide instantaneous disintegration of tablet after placing on tongue, there by release the drug in saliva. The bioavailability of some drugs may be increased due to absorption of drug in oral cavity and also due to pre-gastric absorption of saliva containing dispersed drugs that pass down into the stomach. Moreover, the amount of drug that is subjected to first pass metabolism is reduced as compared to conventional tablet. The advantage of mouth dissolving dosage forms are increasingly being recognized in both, industry and academics. Their growing importance was underlined recently when European pharmacopoeia adopted the term “Oro-dispersible tablet” as a tablet that to be placed in the mouth where it disperses rapidly before swallowing. According to
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Shoba Krushnan et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 473 European pharmacopoeia, the ODT should disperse/disintegrate in less than three minutes. MATERIALS AND METHODS Amlodipine Besylate was received as gift sample from Micro labs, Hosur, Tamilnadu, India. Crospovidone(CP),Croscarmellosesodium(CCS),Sodiu mstarchglycolate(SSG),Lactose, Magnesium stearate and talc were used. And all other chemicals/solvents used were of analytical grade. Formulation of Amlodipine oro-dispersible tablets by direct compression method: The drug and all other excipients were accurately weighed and sifted through #40 sieves and mixed thoroughly. The above blend was lubricated with magnesium stearate. The formulation development of Amlodipine ODT was initially developed with different super-disintegrants, SSG, CCS and CP in the concentration range of 5%, 7.5% and 10%. The tablets were prepared by direct compression method. The tablets were compressed on 8 station rotary tablet punching machine (Rimek manufacturers, Gujarat, India) using 6mm round punch and the individual tablet weight was100mg. The prepared tablets were evaluated for different parameters like weight variation, friability, hardness, thickness, disintegration time, wetting time, assay and in vitro dissolution studies. Weight variation: Twenty tablets were randomly selected from each batch and individually weighed. The average weight of these selected tablets was calculated (Indian Pharmacopoeia, Vol ‐ I, 1996). Tablet thickness: Tablet thickness is an important characteristic in reproducing appearance and also in counting by using filling equipment. Thickness was recorded using vernier calliper. Friability: Friability is a measure of mechanical strength of the tablet. If a tablet has more friability it may not remain intact during packaging,transport or handling. Roche friabilator is used to determine the friability by following procedure. Pre weighed tablets are placed in the friabilator. Friabilator consist of a plastic chamber that revolves at 25 rpm, dropping those tablets at a distance of 6 inches with each revolution (Lachman L, 1987). The tablets are rotated in the friabilator for at least 4 minutes. At the end of test tablets are dusted and reweighed; the loss in the weight of tablet is the measure of friability. Crushing strength: Tablet crushing strength, which is the force required to break the tablet, was measured with a Pfizer tablet hardness tester. The hardness (crushing strength) of three tablets per batch was determined and mean taken. Drug content: Drug content was determined by taking randomly ten tablets per batch. An amount equivalent to 10 mg amlodipine was dissolved in methanol, suitably diluted with PH 7.2 Phosphate buffer and filtered (British pharmacopoeia commission 2007). The absorbance of the solution was measured spectrophotometrically against the blank (PH 7.2 Phosphate buffer) at 239 nm using a U.V.spectrophotometer (Shimazdu-1800, Japan). Wetting time: The wetting time of the tablet was measured by placing five circular tissue papers (10 cm in diameter) in a Petri dish of 10 cm diameter. Water (10 ml) containing methylene blue (0.1% w/v) was added to the Petri dish. A tablet was carefully placed on the surface of the tissue paper and the time required for the dye to reach the upper surface of the tablet was recorded as wetting time (Radke RS et al, 2009). The measurements were carried out in triplicate. Disintegration time: One tablet each was placed in each of the six tubes of the apparatus and time in seconds taken for complete disintegration of the tablet with no palatable mass remaining in the apparatus was measured. The tablet was considered disintegrated completely when all the particles passed through the screen. The disintegration time of 6 individual tablets were recorded and the average was reported. The disintegration time set by U.S. Food and Drug Administration (FDA) for all the ODT formulations (60 s) were considered as a specification limit (Bi Y, 1999). In-vitro drug release: In vitro drug release studies were carried out using USP type II apparatus at 50 rpm. Phosphate buffer (500 ml) at 7.2 was used as the dissolution medium. The temperature of the dissolution medium was maintained at 37±0.50 C (Bhagwati ST, 2000). An aliqout (5 ml) of dissolution medium was withdrawn at specific time intervals, filtered and suitably diluted prior to spectrophotometric analysis. Sink condition were maintained by replenishing the medium with an equal amount (5 ml) of dissolution fluid. Absorption of the solution was measured by UV spectroscopy (Shimadzu-1800, Japan) at 239 nm. Drug-Excipient Compatibility Study: Drug-excipient compatibility was performed by FTIR and DSC studies,
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Shoba Krushnan et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 474 a) Fourier Transform Infrared Spectroscopy (FT- IR): The FT-IR spectrums of pure drug and physical mixtures of drug with SSG, CCS and CP. FT-IR (Thermo Nicolet 670 spectrometer) was used for the analysis in the frequency range between 4000 and 400 cm-1 resolution. A quantity equivalent to 2 mg of pure drug was used for the study. b) Differential scanning calorimetric study (DSC study): Thermal properties of pure drug and physical mixtures of drug with SSG and CP were evaluated by Differential scanning calorimetry (DSC) using a Diamond DSC (Mettler Star SW 8.10). The analysis was performed at a rate 50 C min-1 from 500 C to 2000 C temperature range under nitrogen flow of 25 ml min-1. RESULTS AND DISCUSSION Weight variation and Thickness: The weight variation of all the formulations was within the range and the Thickness of the tablets found to be 2.7mm to 2.92mm. Hardness and Friability: The hardness was constantly maintained between 3-3.5 kg / cm2 during compression and Friability for all the formulation shown less than 1% which is in the acceptable limits which indicates formulations have good mechanical strength. Drug content and Wetting time: The drug content of Amlodipine from all the formulations was found in the range of 98% to 99% and Wetting time in above formulations found to be between 41-56 seconds Disintegration time: Disintegration time of formulations containing 5% SSG (F1),5% CCS (F2),5% CP (F3), found to be between 36-39 seconds. Disintegration time of formulations containing 7.5%SSG (F4), 7.5%CCS (F5), 7.5% CP (F6), found to 18-28 seconds. And disintegration time of formulations containing 10% SSG (F7), 10% CCS (F8), 10% CP (F9) found to be 9-13 seconds. Based on the above results it was clearly observed that the above formulations improved the disintegration with increased concentration of superdisintegrants. In-vitro dissolution: In this work the table No 5 shows dissolution profile of different formulation in which F7 and F9shows maximum % released and increases bioavailability hypothetically.As per USFDA guidelines ODT tablets, the tablets should disintegrate in less than 60 seconds, it should directly reflect on the mouth disintegration. Based on these considerations it was decided to increase the concentration of super- disintegrants in the further study. Fourier Transform Infrared Spectroscopy (FT-IR): The FTIR spectrum peak points of pure drug Amlodipine at 561.52, 613.36, 667.66, 753.76, 996.54, 1031.94, 1090.98, 1202.19, 1263.25, 1300.93, 1364.91, 1432.65, 1468.64, 1614.45, 1672.20, 1696.53, 2979.01 and 3154.55. Similar spectrum peak points were observed in all the formulations. This clearly indicates that there is no drug excipient interaction. Table 2 shows the spectrum peak points of the pure drug and the formulations of Amlodipine. Differential scanning calorimetric study (DSC): DSC study was conducted on the selected formulations. The DSC results shows sharp endothermic peak for pure Amlodipine at 209.98 °C. Similar sharp endothermic peaks were observed in the formulations at almost similar temperatures. This clearly indicates that there is no drug excipient interaction. CONCLUSION This present research work demonstrates that orodispersible tablet with higher percentage of superdisintegrant by direct compression technique yields a good pharmaceutically accepted dosage forms and show increased dissolution profiles which reflects enhanced bioavailability. ACKNOWLEDGEMENT The authors are thankful to Micro labs, Hour, Tamilnadu, India for providing gift sample of Amlodipine besylate and thankful to Principal of Aurobindo college of Pharmaceutical sciences, Gangadevipally, Warangal, Andhra Pradesh.
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Shoba Krushnan et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 475 Table 1: Formulation of Amlodipine Oro-dispersible tablet Ingredients (mg) F1 F2 F3 F4 F5 F6 F7 F8 F9 AMLODIPINE 10 10 10 10 10 10 10 10 10 SSG 5 - 7.5 - 10 - CCS - 5 - - 7.5 - - 10 - CP - - 5 - - 7.5 - - 10 LACTOSE 81 81 81 78.5 78.5 78.5 76 76 76 TALC 3 3 3 3 3 3 3 3 3 MG. STEARATE 1 1 1 1 1 1 1 1 1 Total Weight 100mg 100mg 100mg 100mg 100mg 100mg 100mg 100mg 100mg Table 2: FTIR spectrum peak points of pure drug and the formulation of Amlodipine Table 3: DSC melting points of the selected formulations Formulations DSC melting point in °C PURE AMLODIPINE 209.98 AMD -SSG 208.19 AMD -CP 206.12 Pure AMD WITH SSG WITH CCS WITH CP 561.52 561.09 561.58 559.55 613.36 613.38 613.20 609.53 667.66 666.54 667.22 665.63 727.94 727.22 726.36 726.37 753.76 753.48 752.92 752.59 996.54 998.12 997.24 997.02 1031.94 1032.81 1029.36 1029.22 1090.98 1089.94 1089.86 1089.70 1202.19 1202.44 1201.99 1202.13 1263.25 1263.89 1262.43 1262.77 1300.93 1301.42 1301.39 1299.89 1364.91 1365.00 1365.27 1365.22 1432.65 1431.33 1431.76 1431.61 1468.64 1470.12 1469.56 1469.56 1614.45 1613.36 1613.28 1613.12 1672.20 1672.98 1672.99 1672.18 1696.53 1695.30 1695.39 1694.80 2979.01 2980.52 2980.96 2980.34 3154.55 3155.03 3155.64 3155.42
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Shoba Krushnan et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 476 Table 4: Physicochemical Parameters of Amlodipine ODT Parameter F1 F2 F3 F4 F5 F6 F7 F8 F9 Weight Variation 100.1 101 102 100.3 102 102 100.2 100 101 Friability (%) 0.47 0.58 0.78 0.56 0.64 0.87 0.68 0.72 0.92 Hardness(Kg/Cm2 ) 3.0 3.1 3.0 3.0 3.5 3.0 3.5 3.2 3.5 Thickness (mm) 2.78 2.84 2.81 2.78 2.81 2.92 2.8 2.8 2.7 Disintegration time(Sec) 37.6 39.49 36.04 25.9 28.5 18.81 9.3 12.57 9.26 Wetting time(sec) 52 56 50 41 46 43 42 45 42 Drug content (%) 99 98 99 99 98 99 99 99 99 Table 5: In-vitro dissolution data Amlodipine ODT Time (min) F1 F2 F3 F4 F5 F6 F7 F8 F9 5 66.70 64.90 65.15 75.26 72.67 74.63 83.24 80.28 82.56 10 76.51 75.15 75.00 81.82 79.82 80.68 89.90 87.57 88.98 15 81.90 79.40 80.20 88.81 85.85 87.94 93.3 92.79 93.1 20 87.65 85.28 86.1 93.96 90.78 92.96 95.4 94.5 95.1 30 96.90 93.67 95.98 97.96 96.72 97.86 98.82 96.5 98.9 Fig 1: FTIR of pure Amlodipine Fig 2: FTIR of AMD + SSG Fig 3: FTIR of AMD +CCS Fig 4: FTIR of AMD + CP
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Shoba Krushnan et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 477 Fig 7: DSC of AMD + CP REFERENCES Bhagwati ST, Hiremath SN, Sreenivas SA, Comparative evaluation of disintegrants by formulating cefixime dispersible tablets, Indian J. Pharm.Edu.Res, 39, 2000, 194‐197. Bi Y, Evaluation of rapidly disintegrating tablets prepared by direct compression method, Drug Dev Ind Pharm, 25(5), 1999, 571‐581. Deepak K, Orally disintegrating tablets, Tablets Capsule 7, 2004, 30-35. Fu Y, Yang S, Jeong SH, Kimura S, Park K, Orally fast disintegrating tablets: Developments, technologies, taste masking and clinical studies, Crit Rev Ther Drug Carrier Syst, 21, 2004, 433–76. Ghosh TK, Pfister WR, Quickdissolving oral dosage forms: Scientific and regulatory considerations from a clinical pharmacology and biopharmaceuticals perspective; In: Drug delivery to the oral cavity: Molecules to market. New York, CRC Press, 2005, 337-356. Indian Pharmacopoeia, Vol ‐ I, 4th ed. Controller of publication, Govt. of India, New Delhi, 1996, 736. Lachman L, Liberman H, Kanig J, The theory and practice of industrial pharmacy, Varghese Publishing House, Mumbai, 3rd Edn, 1987, 297. Radke RS., Jadhav JK., Chajeed MR. Formulation and evaluation of orodispersible tablets of baclofen. International Journal of Chemtech Research, 1, 2009, 517‐521. Valleri M, Mura P, Maestrelli F, Cirri M, Ballerini R, Development and evaluation of glyburide fast dissolving tablets using solid dispersion technique, Drug Dev Ind Pharm, 30(5), 2004, 525-534. Fig 5: DSC of Pure Amlodipine Fig 6: DSC of AMD + SSG
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Srinivas et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 478 COMPARISION OF POTENCY OF ANTI BACTERIAL ACTIVITY AND ANTI INFLAMMATORY ACTIVITY OF 10 YEARS AND 100 YEARS OLD BARK EXTRACTS OF AZADIRACHTA INDICA Vijaya Kumar G, Srinivas N*, P Sravanthi, Sravani B Department of Pharmacology, A.K.R.G College of Pharmacy, Nallajerla, W.G. Dist, A.P, India. *Corresponding author: Email: srnvs_87@rediffmail.com, 8019189741 ABSTRACT Azadirachta indica (Meliaceae) commonly known as neem contains many biologically active compounds including alkaloids, flavonoids, triterpenoids, phenolic compounds, Carotenoids, steroids and ketones, azadirachtin. Oil from the leaves, seeds and bark possesses a wide spectrum of antibacterial action against Gram-negative and Gram-positive microorganisms, including M. tuberculosis and streptomycin resistant strains. The present study was undertaken to evaluate the comparision of potency of anti bacterial and anti-inflammatory activities of 10 and 100 years old bark extract of Azadirachta indica. The antibacterial activity was performed by using both gram positive and gram negative organisms viz., Bacillus Subtilis, E. coli and Staphylococcus Aureus. The anti-inflammatory activity was evaluated by using carrageenan induced paw edema method in rats. From the results of anti bacterial activity and anti-inflammatory activity, it has been concluded that 100 years old neem bark extract showed greater activities than the 10 years old neem bark extract. Key words: Azadirachta indica, Anti bacterial activity, Anti-inflammatory activity, 10 years and 100 years old plants. 1. INTRODUCTION Azadirachta indica (Meliaceae) commonly known as neem is native of India and naturalized in most of tropical and subtropical countries is of great medicinal value and distributed widespread in the world. The Chemical constituents contain many biologically active compounds that can be extracted from neem, including alkaloids, flavonoids, triterpenoids, phenolic compounds, Carotenoids, steroids and ketones, Azadirachtin is actually a mixture of seven isomeric compounds labeled as azadirachtin A-G and azadirachtin E is more effective (P Sudhir Kumar, 2010). Other compounds that have a biological activity are salannin, volatile oils, meliantriol and nimbin. Oil from the leaves, seeds and bark possesses a wide spectrum of antibacterial action against Gram- negative and Gram-positive microorganisms, including M. tuberculosis and streptomycin resistant strains. In vitro, it inhibits Vibrio cholerae, Klebsiella pneumoniae, M. tuberculosis and M. pyogenes. Antimicrobial effects of neem extract have been demonstrated against Streptococcus mutans and S. faecalis. NIM-76, a new vaginal contraceptive from neem oil showed inhibitory effect on the growth of various pathogens, including bacteria, fungi and virus. Recently, the antibacterial activity of neem seed oil was assessed in vitro against 14 strains of pathogenic bacteria. (Baswa M, 2001) The present study was undertaken to evaluate the comparision of potency of anti bacterial activity of 10 and 100 years old acetonic bark extract of azadiracta indica by using agar disc diffusion method on Bacillus subtilis, Escherichia coli, Staphylococus aureus and also to evaluate the comparision of potency of anti- inflammatory activity of 10 and 100 years old aqueous bark extract of azadiracta indica on carrageenan induced paw edema in rats. 2. MATERIALS Both the 10 years old and 100 years old neem plants were collected from Bapatla, Guntur district, Andhra Pradesh. Gentamycin (Nicholas piramil ltd, Mumbai), Penicillin (Alembic labs, Ahmedabad) and Diclofenac sodium (Novartis pharma ltd, Ahmedabad) were purchased from local medical stores, Nallajerla. Carrageenan was procured from Ozone internation, Mumbai. 2.1. Animals: Albino Wistar rats weighing 180–200g of either sex were obtained from the animal house of A.K.R.G. College of Pharmacy, Nallajerla, Andhra Pradesh, were used for this study. The animals were housed in separate groups (six rats in each cage) in clean sanitized polypropylene cages containing sterile paddy husk as bedding. The bedding material of the cages was changed every day. They had free accessed to standard pellet diet and water ad libitum. The animals were maintained under day and night 12:12 h cycles and with maintenance of room temperature 25 ±
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Srinivas et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 479 2◦ C. All procedures were performed in accordance with the Institutional Animal Ethics Committee (IAEC) constituted as per the direction of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), under ministry of Animal Welfare Division, government of India, New Delhi IAEC approved the experimental protocol (AKRGCP/IAEC/03/2011-12) dated 11/02/2012. 3. METHODS 3.1. Preparation of neem bark extracts (NBEs): The stem bark of neem plants was peeled with sharp knife and chopped into pieces which was sun dried and ground into powder using a blender. The resulting powder stored at room temperature in clean, air tight and wide mouth container. 3.2. Preparation of acetonic neem bark extract: Twenty grams of neem bark was mixed with 200ml of acetone in a conical flask. The mixture was then magnetically stirred for 24hrs at room temperature. The homogenate was vacuum filtered through filter paper. The clarified filtrate was evaporated using at about 35o C and the residue was collected. 3.3. Preparation of aqueoes neem bark extract: Twenty grams of neem bark was mixed with 200ml of distilled water in a conical flask. The mixture was then magnetically stirred for 60hrs at room temperature. The homogenate was vacuum filtered through filter paper. The clarified filtrate was evaporated using at about 350 C and the residue was collected. 3.4. Antimicrobial Studies (A Kottai Muthu, 2010) 3.4.1. Test solution: Test solution of each extract was prepared by dissolving 100mg of each extract separately in 1ml of sterile dimethyl formamide (DMF) in a specific gravity bottle and stored in refrigerator. The solution was removed from the refrigerator one hour prior to each use and allow warming at room temperature. 3.4.2. Standard solution: The standard drugs Gentamycin (200µg/ml) and Pencillin (750µg/ml) was prepared in sterile water for injection. These were used as standard drugs for Antimicrobial studies. 3.4.3. Preparation of medium: Nutrient broth was used for preparation of inoculum of bacteria. Nutrient agar was used for preparation of medium for Antimicrobial screening. The composition of nutrient agar medium was as follows. Peptone - 5.0g Beef extract - 1.5g Yeast extract - 1.5g Agar - 1.5g Distilled water - 1000 ml pH adjusted -7.2 3.4.4. Preparation of inoculums: Inoculum was prepared by transferring a loopful of stock culture to a 150ml of Erlenmeyer containing 80ml of nutrient broth. The composition of inoculum broth was same as that of stock culture with exception of agar. The inoculum flasks were incubated at 370 C for 24 hrs and used for experiments. 3.4.5. Inoculation: The nutrient agar medium was sterilized by autoclaving at 121o C for 15 min. The petridishes and pipette were sterilized in an oven at 150o C for one hour. About 25ml melted nutrient agar medium (40o -50o C) was poured in each sterilized petridishes and 0.5ml of inoculum broth of bacteria was added to the respective petridishes. The content petridishes were thoroughly maintained at rotary motion. The medium containing inoculum was allowed to solidify at room temperature. After solidification of the medium, fine whattman filter paper disc were made it equal distance. The whattman filter paper discs were dipped in test and standard solution and kept in the petridish and the petridish undisturbed for one hour at room temperature. The petridish were incubated at 37o C for 24 hours and the zone of inhibition was recorded in mm. The experiment was performed in triplicate and the average readings are recorded. 3.5. Anti inflammatory activity (A M Mujumdar, 2000) 3.5.1. Experimental design: Male Wistar rats weighing 180-200 g were divided into four groups of six animals each. The treatment groups are designated as follows Group Treatment Group I Control (Solvent) Group II 100 yrs old NBE (200mg/kg) Group III 10 yrs old NBE (200mg/kg) Group IV Standard (Diclofenac 100mg/kg) 3.5.2. Experimental procedure: Male Wistar rats weighing 200 g are starved for 48 h. having access to drinking water ad libitum. The test compounds and standard drugs are administered by oral route. Thirty min later the rat are challenged by a sub-contentious injection of 0.05ml of 1% solution of carrageenan on the plantar surface of the left hind paw. The paw is
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Srinivas et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 480 marked with ink at the level of lateral malleolus and immersed in the mercury column of a plethysmometer for measuring the paw volume after carrageenin injection and then at 0.5, 1, 2, 3 and 4 hrs. The increase in paw volume at each time interval is calculated as percentage compared with the volume measured immediately after the injection of carrageenan for each animal. The percentage edema inhibition was calculated by sing the following formula Table 1: Percentage yield data of 100 yrs and 10 yrs old plants with different solvents Solvent Percentage Yield 100 Yrs 10yrs Acetone 1.5 1.9 Water 1.2 1.6 Figure 1: Percentage yield profile of 100 yrs and 10 yrs old plants with different solvents Table 2: Comparison of inhibition zones of acetonic neem bark extracts of 100 yrs and 10 yrs old plants against different standard organisms Organism Zone of inhibition (mm) 100 Yrs 10yrs Penicillin Gentamicin DMF Bacillus subtilis 31 14 25 22 - E. coli 35 15 19 30 - Staphylococcus aureus 27 10 21 18 - (-) No zone of inhibition DMF – dimethyl Formamide Figure 2: Comparison of inhibition zones of acetonic neem bark extracts of 100 yrs and 10 yrs old plants against different standard organisms 0 0.5 1 1.5 2 Acetone Water PecentageYieldof NeemBarkExtract 100 Yrs 10yrs 0 10 20 30 40 100 Yrs 10yrs Gentamicin Penicillin Zoneofinhibition (mm) Bacillus Subtilis E. coli
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Srinivas et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 481 Table 3: Paw volume data of test and standard drugs on carrageenan induced rat paw edema Group Treatment Change in paw volume (ml) measured by mercury displacement at different time intervals (hrs) (mean±S.D) 0 0.5 1 2 3 4 Group I Control 0 0.13±0.001 0.3±0.002 0.4±0.001 0.5±0.002 0.5±0.001 Group II 100 yrs old NBE 0 0.11±0.002 0.2±0.001 0.2±0.001 0.11±0.002 0.1±0.001 Group III 10 yrs old NBE 0 0.11±0.001 0.23±0.002 0.3±0.001 0.27±0.001 0.2±0.002 Group IV Standard 0 0.1±0.001 0.2±0.001 0.19±0.002 0.1±0.001 0.1±0.001 Figure 3: Paw volume profiles of test and standard drugs on carrageenin induced rat paw edema Table 4: Percentage oedema inhibition of test and standard drugs on carrageenan induced rat paw edema Group Treatment Percentage of edema inhibition measured by mercury displacement at different time intervals (hrs) 0 0.5 1 2 3 4 Group I Control 0 0 0 0 0 0 Group II 100 yrs old NBE 0 15.38 33.3 50 78 80 Group III 10 yrs old NBE 0 15.38 23.33 25 46 60 Group IV Standard 0 23 33.33 52.5 80 80 Figure 4: Percentage oedema inhibition of test and standard drugs on carrageenin induced rat paw edema 0 0.1 0.2 0.3 0.4 0.5 0.6 0 1 2 3 4 5 changeinpawvolume (ml) Time (hrs) Control 100 yrs 10 yrs Standard 0 20 40 60 80 100 0 1 2 3 4 5 Percentageinhibition ofoedema Time (hrs) 100 yrs 10 yrs Standard
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Srinivas et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 482 4. DISCUSSION The present study has been undertaken to compare the potency of anti microbial and anti-inflammatory activities of 100 years and 10 years old neem bark extracts. In this study acetone, and aqueous extracts were used for anti microbial and anti-inflammatory activities respectively. The percentage yield of different extracts were calculated and tabulated in table 1. The inhibition zones of acetonic neem bark extracts of 100 years and 10 years old plants against different standard organisms (Bacillus subtilis, E. coli and Staphylococcus aureus) were measured. Similarly the inhibition zones of standard drugs that are Gentamicin and Penicillin against the same organisms were also measured and data shown in table 2. The data was treated statistically and the statistical interaction implies that the difference in zone of inhibition was statistically significant between 100 years and 10 years old neem plants. It is clear that the both test drugs (100 years and 10 years old) are showed anti microbial activity against gram positive micro organisms (Bacillus Subtilis and Staphylococcus Aureus) and gram negative micro organisms (E. coli). The solvent (DMF) used as vehicle did not showed anti microbial activity and confirmed there is no solvent action on the micro organisms. Anti inflammatory activity was evaluated by using carrageenan induced rat paw oedema method. A single subcutaneous injection of 0.1 ml of 2% formalin in rats produced inflammation significantly (p<0.001). Paw volume was measured by mercury displacement at different time intervals and right leg considered as control for left leg which is received carrageenan on plantar region. The change in paw volume (L-R) was measured and data shown in table 3. From this data percentage oedema inhibition of test and standard drugs was calculated and tabulated in table 4. The data was treated statistically and the statistical interaction implies that the difference in paw volume was statistically significant between 100 years and 10 years old neem plants. At the time of 3 hours the percentages of oedema inhibition were 0, 78, 46 and 80 for control, 100 years old plant extract, 10 years old plant extract and standard drugs respectively. 5. CONCLUSION The bark extracts were extracted by different solvents. All these activities are evaluated and observed that the age of plant is influenced the index of activity. It is may be due to the age of plant influence the chemical cinstients or their potency. The young plant (10 years age neem plant) showed high percentage yield when compared with old plant (100 years age neem plant). The crude extracts are sparingly soluble in water; hence DMF (dimethyl formamide) used as solvent for the test dose preparations. From the results of anti microbial activity, it has been concluded that 100 years old neem bark acetonic extract showed greater anti microbial activity than the 10 years old neem bark acetonic extract and both the drugs showed broad spectrum anti bacterial activity. From the results of anti inflammatory activity, it has been concluded that 100 years old neem bark aqueous extract showed greater anti inflammatory activity than the 10 years old neem bark aqueous extract and the results met the standard NSAID drug that is diclofenac sodium. From this investigation it was concluded that the selection of age of plant is important to their significant pharmacological action. 6. AKNOWLEDGEMENTS The authors are thankful to Management, A.K.R.G College of Pharmacy, Nallajerla, Andhra Pradesh, India for permitting and providing necessary facilities for carrying out to do the project work. REFERENCES A Kottai Muthu, Penugonda Sravanthi, D Sathesh Kumar, A Anton Smith and R Manavalan, International Journal of Pharma Sciences and Research, 1(2), 2010, 127-130. A M Mujumdar, D G Naik, C N Dandge, H M Puntambekar, Anti inflammatory activity of curcuma amada Roxb in albino rats. Indian Journal of Pharmacology, 32, 2000, 375-377. Ara I, Siddiqui B S, Faizi S, Siddiqui S, Diterpenoids from stem bark of Azadirachta indica, Phytochemistry, 28, 1989, 1177-1180. Baswa M, Rath CC, Dash SK, Mishra RK, Antibacterial activity of Karanj (Pngamia pinnata) and neem (Azadirachta indica) seed oil: a preliminary report, Microbios, 105, 2001, 183-189. Biswas, Kausik, Ishita Chattopadhyay, Ranajit K, Banerjee and Uday Bandyopadhyay, Biological activities and medicinal properties of Neem (Azadirachta indica), Current Science, 82(11), 2002, 1336-1345. Naqvi S N H, Pharmacological importance of neem Azadiracta indica A Juss (Meliacae), J. Baqai Med. Uni, 1(2), 1998, 39-50.
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Srinivas et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 483 P Sudhir Kumar Debasis Mishra, Goutam Ghosh and Chandra S Panda, Biological action and medicinal properties of various constituent of Azadirachta indica (Meliaceae) an Overview, Annals of Biological Research, 1 (3), 2010, 24-34. Siddiqui S, Siddiqui B S, Faizi S, Mahmood T, Isolation of a tetranortriepenoid from Azadirachta indica. Phytochemistry, 23, 1984, 2899-2901. Thaker A M and Anjaria JV, Antimicrobial and infected wound healing response of some traditional drugs, Indian Journal of Pharmacology, 18, 1986, 171-174.
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Harish et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 484 DEVELOPMENT AND EVALUATION OF CARISOPRODOL TABLETS WITH IMPROVED DISSOLUTION EFFICIENCY USING SOLID DISPERSION TECHNIQUE Mogili Daya Sagar, Mohammed Shahidullah, Shaik Rabbani Basha, Shaik Shahnaz, Harish.G* Department of Pharmaceutics, Nimra college of Pharmacy, Vijayawada, AP, India *Corresponding author: E.Mail: harishgopinath4u@gmail.com ABSTRACT Carisoprodol is indicated in patients with acute muscular pain. Carisoprodol is typically prescribed as 350 mg tablets. The aim of the present study is to design and development Carisoprodol tablets with improved dissolution efficiency using solid dispersion technique. The present work is planned to prepare solid dispersion system consisting of Carisoprodol with hydrophilic carriers by employing different methods, to study the physicochemical properties of Carisoprodol solid dispersions, develop fast dissolving tablets of Carisoprodol solid dispersions by using super- disintegrant such as starch, Croscarmelose sodium, sodium starch glycolate and to study the effect of the preparation methods of solid dispersions on dissolution characteristics. Key words: Carisoprodol, Solid dispersion, super-disintegrant. INTRODUCTION The potential drug candidates are characterized by a low oral bioavailability. Often poor drug dissolution/solubility rather than limited permeation through the epithelia of the gastrointestinal tract are responsible for low oral bioavailability (Vasconcelos TF, 2007). Thus aqueous solubility of any therapeutically active substance is a key property as it governs dissolution, absorption and thus the in-vivo efficacy (Vemula VR, 2010). Drugs with low aqueous solubility have low dissolution rates and hence suffer from oral bioavailability problems. The rate and extent of dissolution of the active ingredient from any dosage form often determines the rate of extent of absorption of the drug. When an active agent is given orally, it must first dissolve in gastric acid and/or intestinal fluids before it can then permeate the membranes of the GI tract to reach systemic circulation. Therefore, a drug with poor aqueous solubility will typically exhibit dissolution rate limited absorption, and a drug with poor membrane permeability will typically exhibit permeation rate limited absorption. Hence, two areas focus on improving the oral bioavailability of active agents include:  Enhancing solubility and dissolution rate of poorly water-soluble drugs  Enhancing permeability of poorly permeable drugs There are various techniques available to improve the solubility of poorly soluble drugs, such Micronization, Nanosuspension, Modification of the crystal habits, Eutectic mixtures, Solid dispersions, Microemulsions, Self micro emulsifying drug delivery systems, cyclodextrin inclusion and lipid based delivery systems etc (Sharma D, 2010). Solid dispersion is one of the most promising approaches for solubility enhancement. In the biopharmaceutical classification system (BCS) drugs with low aqueous solubility and high membrane permeability are categorized as Class II drugs. Therefore, solid dispersion technologies are particularly promising for improving the oral absorption and bioavailability of BCS Class II drugs. In case of solid dispersion drug disperse in the matrix generally a hydrophilic matrix and a hydrophobic drug, thereby forming a solid dispersion. When the solid dispersion is exposed to aqueous media, the carrier dissolves and the drug releases as fine colloidal particles. The resulting enhanced surface area produces higher dissolution rate and bioavailability of poorly water-soluble drugs. Solid dispersion: Solid dispersion technology is the science of dispersing one or more active ingredients in an inert matrix in the solid stage in order to achieve increased dissolution rate, sustained release of drugs, altered solid state properties, and enhanced release of drugs from ointment and suppository bases, and improved solubility and stability (Mohanachandran PS, 2010). MATERIALS AND METHODS Materials: Carisoprodol was obtained as gift sample from SYNED LABS LIMITED, Medak, AP, Starch, SSG, Cross carmelose sodium, Crospovidone, MCC, Lactose was obtained as a gift sample from ICPAHealthcare, Ankaleshwar. PVP, Talc and Magnesium Stearate were obtained from Signet Mumbai. All other chemicals and Solvents used in this study are of analytical grade.
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Harish et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 485 Pre-formulation Studies: Pre-formulation study relates to pharmaceutical and analytical investigation carried out proceeding and supporting formulation development effort of the dosage forms of the drug substance. Pre-formulation studies yield basic knowledge necessary to develop suitable formulation. It gives information needed to define the nature of the drug substance and provide frame work for the drug combination with pharmaceutical excipients in the dosage forms. Hence the following pre-formulation studies were performed on the obtained sample of drug such as Solubility, bulk density, tapped density, Percentage compressibility, Identification of drug sample, Drug excipient compatibility studies (Patidar Kalpana, 2010). Formulation of Carisoprodol Solid dispersion: The accurately weighed quantity of the drug and polymer in various ratios has been formulated by melting the polymer and dispersing the drug in it. The formulated SD has been dried and grounded by passing through mesh #22. Formulation of Carisoprodol Tablet: Preparation of the Fast dissolving tablet of Carisoprodol: Fast dissolving tablets of Carisoprodol had been formulated by direct Compression method using Super-disintegrants such as SSG, CP, Starch, CCS etc. in various ratios. These ingredients were weighed and mixed stoichometrically to obtain the final formulation. The weight of the tablet in all formulations was kept constant to 130mg. All the batches were prepared by direct compression method using the 16-station rotary punch tablet compression machine using 7 mm biconvex plain on both side die-punches set. The variables maintained in the formulation were the different types of super-disintegrant and their concentration (in mg) in the formulation. Completely dried complex used for the preparation of fast dissolving tablet. Tablets were prepared from blends by direct compression method. All the ingredients including drug were passed through mesh no. 60 excepting lubricants. Lubricants were passed through mesh no.80. Lubricants were added at the time of compression. Blend is mixed uniformly by manually for 30 minutes. Tablets of convex faced weighing 130mg each with 3.3mm thickness and 7mm in diameter. Evaluation of Post-Compression Characteristics: The formulated Carisoprodol SD has been compressed in to tablet and the following evaluation has been performed as per BP pharmacopoeia. The following evaluation of tablets was performed such as Drug content, Weight variation, Hardness, Friability, Content uniformity, Thickness, In-Vitro Dissolution. Table.1. Formulation of Carisoprodol solid dispersion Drug:Polymer (Urea) Drug:Polymer (Mannitol) 1:1 1:1 1:2 1:2 1:3 1:3 Table.2. Formulation of Fast dissolving tablet of Carisoprodol SD INGREDIENTS F1 F2 F3 X4 X5 X6 Z7 Z8 Z9 C10 C11 Carisoprodol SD (mg) 10 10 10 10 10 10 10 10 10 10 10 Starch 62.5 62.5 62.5 62.5 62.5 62.5 62.5 62.5 62.5 62.5 SSG 2 4 6 - - - - - - - - CCS - - - 2 4 6 - - - - CP - - - - - - 2 4 6 - - MCC - - - - - - - - - - 41 PVP 16 16 16 16 16 16 16 16 16 16 16 Lactose 11.5 10.5 8.5 12.5 10.5 8.5 12.5 10.5 8.5 14.5 36 Talc 10 10 10 10 10 10 10 10 10 10 10 Magnesium Stearate 18 17 17 17 17 17 17 17 17 17 17 Total Weight 130 130 130 130 130 130 130 130 130 130 130
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Harish et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 486 RESULTS AND DISCUSSION Evaluation of Blend: Table.3. Pre-compression parameters of Carisoprodol SD Formulation Series Bulk Density(gm/ml) Tapped Density(gm/ml) Compressibility Index Hausner’s Ratio Angle of Repose F1 0.510 0.598 15.81 1.17 26o 28’ F2 0.512 0.597 15.38 1.18 26 o 85’ F3 0.512 0.60 14.87 1.17 27 o 14’ X4 0.505 0.591 14.64 1.17 27 o 75’ X5 0.507 0.595 14.72 1.17 28 o 07’ X6 0.507 0.597 14.97 1.17 28 o 07’ Z7 0.512 0.595 13.84 1.16 29 o 39’ Z8 0.515 0.598 13.91 1.16 29 o 74’ Z9 0.515 0.602 14.43 1.16 29 o 02’ C10 0.510 0.641 20.40 1.22 32 o 82’ C11 0.534 0.714 25.13 1.33 34 o 59’ Table.4. Evaluation of Formulation Series Batch no. Weight variation Hardnes kg/cm2 Thickness (mm) Friability (%) Disintegration time (sec) Wetting time (sec) Water absorption ratio Drug Content (%) F-1 Passes 3.1 2.1 0.41 42 63 75 99.78 F-2 Passes 3.2 2.1 o.37 31 55 88.72 99.62 F-3 Passes 3.1 2.1 0.37 25 49 96.29 100.8 X-4 Passes 2.9 2.1 0.38 48 69 67.40 100.2 X-5 Passes 3.1 2.1 0.4 35 59 85.82 100.4 X-6 Passes 3 2.1 0.41 29 50 94.77 100.3 Z-7 Passes 2.8 2.1 0.41 55 71 64.70 99.9 Z-8 Passes 2.9 2.1 0.41 41 65 82.82 99.7 Z-9 Passes 2.9 2.1 0.43 34 56 93.28 100.1 C-10 Passes 3.5 2.1 0.41 74 79 58.33 99.6 C-11 Passes 4.1 2.1 0.32 161 93 42.69 99.5 M-1 - 5.3 - - 257 429 68.33 101.1 M-2 - 5.6 - - 291 486 63.01 99.7 M1:- Marketed Tablet of Carisoprodol; M2:- Marketed Tablet of Carisoprodol Fig.1.Percentage Drug release profile of Carisoprodol formulations CONCLUSION The Mannitol and Urea is used as polymer for the enhancement of the solubility of Carisoprodol solid dispersion and improve the rate of dissolution by fast dissolving tablet using various super disintegrates which shows rapid onset of action and faster rate of drug delivery. The formulation F3 and X6 showed faster disintegration time a faster rate of in-vitro dissolution above 99% at the end of 8min. hence formulation of Carisoprodol SD using the SSG (6%) and CCS (6%) showed a rapid onset of drug release. Hence, formulation of the poorly soluble drug with improved solubility using solid dispersion
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Harish et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 487 and faster rate of action can be developed by following the method discussed so far in this study. REFERENCES Aggarwal S, Gupta GD and Chaudhary S, Solid dispersion as an eminent strategic approach in solubility enhancement of poorly soluble drugs. International Journal of Pharmaceutical Sciences and Research, 1, 2010, 1-13. Batra V, Shirolkar VS, Mahaparale PR, Kasture PV, Deshpande AD, Solubility and Dissolution Enhancement of Glipizide by Solid Dispersion Technique, Indian J Pharm Educ Res, 42(4), 2008, 373-378. Chaulang G, Patil K, Ghodke D, Khan S, Yeole P, Preparation and Characterization of Solid Dispersion Tablet of Furosemidewith Crospovidone, Research J Pharm andTech, 1(4), 2008, 386-389. Kumar DS, Solubility improvement using solid dispersion; strategy, mechanism and characteristics: responsiveness and prospect way outs. International Research Journal of Pharmacy, 2, 2011, 55-60. Mohanachandran PS, Sindhumo PG and Kiran TS, Enhancement of solubility anddissolution rate: an overview, International Journal of Comprehensive Pharmacy, 4, 2010, 1-10. Patidar Kalpana, Solid Dispersion: Approaches, Technology involved, Unmet need & Challenges in Drug Invention Today, 2(7), 2010, 349-357. Sharma D, Soni M, Kumar S and Gupta GD, Solubility Enhancement –Eminent Role in Poorly Soluble Drugs. Research Journal of Pharmacy and Technology, 2, 2009, 220-224. Vanshiv SD, Rao MRP, Sonar GS, Gogad VK, Borate SG, Physicochemical Characterization and In Vitro Dissolution of Domperidone by Solid Dispersion Technique, Indian J Pharm Educ Res, 43 (1), 2009, 86-90. Vemula VR, LagishettyV and Lingala S, Solubility enhancement techniques, International Journal of Pharmaceutical Sciences Review and Research, 5, 2010, 41-51.
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Sowjanya et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 489 TRANSDERMAL DRUG DELIVERY SYSTEMS R.Sowjanya*, Salman Khan, D.Bhowmik, Harish.G, S.Duraivel Department of pharmaceutics, Nimra college of pharmacy, Nimranagar, Ibhrahimpatnam, Vijayawada, Andhra Pradesh. *Coreesponding author: E. Mail id:debjit_cr@yahoo.com ABSTRACT Transdermal therapeutic systems have been designed to provide controlled continuous delivery of drugs via the skin to the systemic circulation. The relative impermeability of skin is well known, and this is associated with its functions as a dual protective barrier against invasion by microorganism and the prevention of the loss of physiologically essential substances such as water. Elucidation of factors that contribute to this impermeability has made the use of skin as a route for controlled systemic drug delivery possible. The market for Transdermal devices is currently estimated at US$ 1.2 billion, approximately 10% of the entire US $ 28 billion drug delivery market. In addition, Transdermal drug delivery market is currently based on only 10 drugs. Hence, Pharmaceutical scientists are striving to add new deliverables to the short list of approved Transdermal products. Keywords Therapeutic activity, Bioavailability, First pass metabolism, Ionophoresis. 1. INTRODUCTION For many decades, medication of an acute disease or a chronic illness has been accomplished by delivering drugs to the patients via various pharmaceutical dosage forms like tablets, capsules, pills, creams, ointments, liquid aerosols, injectable and suppositories, as carriers. Recently, several technical advancements have been made. They have resulted in the development of new techniques of drug delivery. These techniques are capable of controlling the rate of drug delivery, sustaining the duration of therapeutic activity, and/or targeting the delivery of drug to a tissue. In responses to these advances, several transdermal drug delivery systems have recently been developed, aiming to achieve the objective of systemic medication through topical application on the intact skin surface. The principal of transdermal drug delivery systems is that they could provide sustained drug delivery (and hence constant drug concentrations in plasma) over a prolonged period of time. For these attributes, it is often extrapolated that sustained therapeutic activity will also be obtained with transdermal drug delivery systems. Thus, it is anticipated that transdermal drug delivery systems can be designed to input drugs at appropriate rates to maintain suitable plasma-drug levels for therapeutic efficacy, without the periodic sojourns into plasma concentrations that would accompany toxicity or lack of efficacy. Today, four drugs have been successfully incorporated into transdermal drug delivery systems for clinical use (Scopolamine, Nitroglycerine, Clonidine and Estradiol), which establishes the dermal route for systemic drug delivery. Ultimately, the success of all transdermal systems depends on the ability of the drug to permeate skin in sufficient quantities to achieve its desired therapeutic effect. (Roberts MS, 1997) 1.1. Advantages of TDDS: 1. Avoids the risk and inconveniences of intravenous therapy 2. Bypass the variation in the absorption and metabolism associated with oral administration 3. Permit continuous drug administration and the use of drugs with a short biological half-life. 4. Increase the bioavailability and efficacy of drugs and bypass of hepatic first pass metabolism. 5. Treatment can be continued or discontinued according to the desire of the physician. 6. Greater patient compliance due to the elimination of multiple dosing schedules. 1.2. Selection of drug candidate for transdermal delivery: The transdermal route of administration cannot be employed for a large number of drugs. Judicious choice of the drug substance is the most important decision in the successful development of a transdermal system. The drug candidate should have following ideas characteristics: 1.2.1. Adequate skin permeability:  Drugs with low molecular weight  Drugs with low melting point  Drugs with moderate oil and water solubility 1.2.2. Adequate skin acceptability:
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Sowjanya et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 490  Non-irritating drugs  Non-irritating drugs  Non-metabolizing drugs 1.2.3. Adequate clinical need:  Need to prolong administration  Need to reduce side effects on target tissues  Need to increase patient compliance 1.3. Factors affecting transdermal permeation: The principle transport mechanism across mammalian skin is by passive diffusion through primarily the transepidermal route at steady state or through trans- appendageal route ay initial non-steady state. The factors controlling transdermal permeability can be broadly placed in the following cases 1.3.1. Physico-chemical properties of the penetrant molecules: Partition co-efficient: Drugs possessing both lipid and water solubility are favorably absorbed through the skin. Transdermal permeability co-efficient shows a linear dependency on partition co-efficient. A lipid/water partition co-efficient of one or greater is generally required. 1.3.2. pH conditions: The pH value of very high or very low can be destructive to the skin. With moderate pH values, the flux of ionisable drugs can be affected by changes in pH that alter the ratio of charged and uncharged species and their transdermal permeability. 1.3.3. Penetrant concentration: Increasing concentration of dissolved drug causes a proportional increase in flux. At higher concentrations, excess solid drug functions as a reservoir and prolonged period of time. 1.3.4. Physico-chemical properties of drug molecule: Release characteristics: solubility of the drug in the vehicle determines the release rate. The mechanism of drug release depends on the following factors. Whether the drug molecules are dissolved or suspended in the delivery system. 1.3.5. Enhancement of transdermal permeation: Majority of drugs will not penetrate the skin at the rates sufficiently high for therapeutic efficacy; the permeation can be improved by the addition of permeation enhancer like dimethyl sulfoxide, dimethyl formamide, propylene glycol, etc into the system 1.4. Physiological and pathological conditions of skin: 1.4.1. Reservoir effect of horny layer: The horny layer is deeper layer, can sometimes act as depot and modify the transdermal permeation of drugs. The reservoir effect is due to irreversible binding of a part of the applied drug with the skin. 1.4.2. Lipid film: The lipid film on the skin surface acts as a protective layer to prevent the removal of moisture from the skin and helps in maintaining the barrier function of stratum corneum. 1.4.3. Skin hydration: Hydration of stratum corneum can enhance permeability. Skin hydration can be achieved simply by covering or occluding the skin with plastic sheeting, leading to accumulation of sweat. Increased hydration appears to open up the dense, closely packed cells of the skin and increase its porosity. 1.4.3. Skin temperature: Raising the skin temperature results in an increase in the rate of skin permeation; this may be due to availability of energy required for diffusivity. 1.4.4. Regional variation: Differences in nature and thickness of the barrier of skin causes variation in permeability. 1.4.5. Pathological injuries to the skin: Injuries that disrupt the continuity of the stratum corneum, increases permeability due to increased vasodilatation caused by removal of the barrier layer. 1.4.6. Cutaneous self-metabolism: catabolic enzymes present in the epidermis may render the drug inactive by metabolism and thus the topical bioavailability of the drug. 1.4.7. Penetration enhancers and their use in transdermal therapeutic system: The transdermal route for drug administration is limited by the barrier properties of the skin. Only the most potent drugs with low daily dose and appropriate physicochemical characteristics are candidates for transdermal delivery. To circumvent the low permeability nature of human skin, pharmaceutical scientists are searching for safe and effective skin penetration enhancers. Development of penetration enhancer is important to improve the low permeability of drug across the skin. Although many penetration enhancers are known, their mode of action is still not fully understood. The penetration enhancers are agents that increase the permeability of the skin or substances that reduce the impermeability of the skin. According to Chien et.al., penetration enhancers or promoters or promoters are agents that
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Sowjanya et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 491 have no therapeutic properties of their own but can transport the sorption of drugs from drug delivery systems onto the skin and/or their subsequent transdermal permeation through skin. The accelerant causes the keratin to swell and leaches out essential structural material from the stratum corneum, thus reducing the diffusional resistance and increasing the permeability of drugs through skin. 1.5. Mechanisms of transdermal permeation: For a systemically active drug to reach a target tissue, it has to possess some physicochemical properties which facilitate the sorption of the drug through the skin and enter the microcirculation. The rate of permeation, dq/dt, across various layers of skin tissues can be expressed as: dq/dt = Ps (Cd—Cr) .......... (1) Where Cd and Cr are respectively, the concentrations of a skin penetrant in the donor phase (stratum corneum) and in the receptor phase (systemic circulation), and Ps is the overall permeability coefficient of the skin and is defined by Ps = Ks Dss/ hs ...........(2) Where, Ks = partition coefficient of the penetrant. Dss = apparent diffusivity of penetrant, hs = thickness of skin Thus, permeability coefficient (Ps) may be a constant, if Ks, Dss and hs terms in equation (2) are constant under a given set of conditions. A constant rate of drug permeation is achieved if Cd >> Cr, then the equation (1) may be reduced to dq / dt = Ps Cd Molecular penetration through the various regions of the skin is limited by the diffusional resistances encountered. The total diffusional resistance (Rskin) to permeation through the skin has been described by Chien as: R skin = Rsc + Re + Rpd .............. (4) Where R is the diffusional resistance and subscripts sc, e , pd refer to stratum corneum, epidermis and papillary layer of the dermis respectively. Of these layers, the greatest resistance is put up by the stratum corneum and tends to be the rate –limiting step in percutaneous absorption. When more than one phase of the membrane is capable of supporting separate diffusional currents through each transdermal patch, then the pathways are configured in parallel to one another and the total fluxes of matter across the membrane is the sum of the fluxes of each route and is expressed by: J = A (f1 p1 + f2 p2 + ..........+ fn pn) C Where J = diffusional flux and the term f1p1 + f2p2 + ..........fnpn, defines the overall permeability coefficient, C being the concentration drop. 1.6. Components of transdermal devices: Transdermal drug delivery devices have come of age. It is 24 years since the first US patents were issued to these systems; today more than 100 patents describing transdermal devices have been issued. Transdermal devices are of 3 types, they are adhesive device, monolithic matrix device and the reservoir system. These devices basically contain: 1. Backing layer 2. Drug reservoir 3. Release control layer (polymer matrix) 4. Adhesive and peel strip 5. Enhancers and excipients. The backing layer/membrane is flexible and they provide a good bond to the drug reservoir, prevent drug from leaving the dosage form through the top, and accept printing. It is impermeable substance that protects the product during use on the skin. Eg., metallic plastic laminate, plastic backing with absorbent pad and Occlusive base plate (aluminium foil), adhesive foam pad (flexible polyurethane) with occlusive base plate (aluminium foil disc) etc. The drug reservoir is generally made up of adhesives and allow for the transport of drug at a desired rate. The drug should be selected depending upon clinical need and its physicochemical properties. The following are some of the desirable properties of a drug for transdermal delivery. 1.7. Physicochemical properties: 1. The drug should have a molecular weight less than approximately 1000 daltons. 2. The drug should have affinity for both lipophilic and hydrophilic phases. 3. The drug should have a low melting point. 1.8. Biological properties: 1. The drug should be potent with a daily dose of the order of a few mg/day. 2. The half life (t1/2) of the drug should be short. 3. The drug must not induce a cutaneous irritant or allergic response.
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Sowjanya et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 492 4. Drugs which degrade in the GI tract or/are inactivated by hepatic first-pass effect are suitable candidates for transdermal delivery. 5. Tolerance to the drug must not develop under the near zero-order release profile of transdermal delivery. 6. Drugs which have to administer for a long period of time or which cause adverse effects to non-target tissues can also be formulated for transdermal delivery. 1.9. Polymer Matrix: The polymer controls the release of the drug from the device. The following criteria should be satisfied for a polymer to be used in a transdermal system. 1. Molecular weight, glass transition temperature and chemical functionality of the polymer should be such that the specific drug diffuses properly and gets released through it. 2. The polymer should be stable, non-reactive with the drug, easily manufactured and fabricated into the desired product; and inexpensive. 3. The polymer and its degradation products must be non-toxic or non-antagonistic to the host. 4. The mechanical properties of the polymer should not deteriorate excessively when large amounts of active agent are incorporated into it. 1.10. Possible useful polymers for Transdermal devices are: 1.10.1. Natural Polymers: Cellulose derivatives, Zein, Gelatin, Shellac, Waxes, Proteins, Gums and their derivatives, Natural rubber, Starch etc. 1.10.2. Synthetic elastomers: Polybutadiene, Hydrin rubber, Polysiloxane, Silicone rubber, Nitrile, Acrylonitrile, Butyl rubber, Neoprene etc. 1.10.3. Synthetic Polymers: Polyvinyl alcohol, Polyvinyl chloride, Polyethylene, Polypropylene, Polyacrylate, Polyamide, Polyurea, Polyvinylpyrrolidine, Polymethylmethacrylate, Epoxy etc. 1.10.4. Adhesives: The fastening of all transdermal devices to the skin has so far been done by using a pressure sensitive adhesive. The pressure sensitive adhesive can be positioned on the face of the device or in the back of the device and extending peripherally. Both adhesive systems should fulfil the following criteria. Should not irritate or sensitize the skin or cause an imbalance in the normal skin flora during its contact time with the skin. It should adhere to the skin aggressively during the dosing interval without its position being disturbed by activities such as bathing, exercise etc. It should be removed easily from the skin. It should not leave a un washable residue on the skin. It should have excellent (intimate) contact with the skin at macroscopic and microscopic level. 1.10.4.1. The face adhesive system should also fulfill the following criteria: 1. Physical and chemical compatibility with the drug, excipients and enhancers of the device of which it is a part. 2. Permeation of drug should not be affected. 3. The delivery of simple or blended permeation enhancers should not be affected. 4. Some widely used pressure sensitive adhesives include polyisobutylenes, acrylics and silicones. 1.11. Permeation Enhancers: These are compounds which promote skin permeability by altering the skin as a barrier to the flux of a desired penetrant. Permeation enhancers are hypothesized to affect one or more of these layers to achieve skin penetration enhancement. A large number of compounds have been investigated for their ability to enhance stratum corneum permeability. These may be conveniently be classified under the following main headings 1.11.1. Solvents: These compounds increase penetration possibly by swelling the polar pathway and/or by fluidizing lipids. Eg.,water alcohols- methanol and ethanol ; alkyl methyl sulfoxides- dimethyl sulfoxide, dimethyl acetamide and dimethyl formamide, miscellaneous solvents-propylene glycol, glycerol, isopropyl palmitate. 1.11.2. Surfactants: These compounds are proposed to enhance polar pathway transport, especially of hydrophilic drugs. Anionic surfactants can penetrate and interact strongly with the skin. Cationic surfactants are reportedly more irritant than the anionic surfactants and they have not been widely studied as skin permeation enhancers. Of the 3 major classes of surfactants, the nonionics have long been recognised as those with the least potential for irritation and have been widely studied. Egs., of commonly used surfactants are :
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Sowjanya et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 493 1.11.3. Anionic surfactants: Dioctyl sulphosuccinate, Sodium lauryl sulphate, Decodecylmethyl sulphoxide etc. 1.11.4. Nonionic surfactants: Pluronic F127, Pluronic F68, etc. 1.11.5. Bile salts: Sodium taurocholate, Sodium deoxycholate, Sodium tauroglycocholate. Binary systems systems apparently open up the heterogeneous multilaminate pathway as well as the continuous pathways. Eg. Propylene glycol-oleic acid and 1,4-butane diol-linoleic acid. 1.11.6. Miscellaneous chemicals: Urea, N,N- dimethyl-m-toluamide, Calcium thioglycolate, 1.11.7. Anticholinergic agents: The enhancers used should be pharmacologically inert, non-toxic, non- allergenic and non-irritating. They should show a quick onset of action, reduction of barrier function of the skin only in one direction. On removal from skin, the tissues should quickly and fully recover normal barrier function. It should be compatible with all the formulation components and should be an excellent solvent for drugs. 2. TECHNOLOGIES OF DIFFERENT TYPES OF TRANSDERMAL DRUG DELIVERY SYSTEM Several technologies have been successfully developed to provide a rate-control over the release and skin permeation of drugs. These technologies can be classified into the following approaches. 2.1. Membrane permeation controlled TDDS: In this system, the drug reservoir is sandwiched between a backing membrane and a rate-controlling membrane, through which the drug is released. In the drug reservoir, drugs are either dispersed uniformly in the solid adhesive matrix (polyisobutylene) or suspended in a viscous, leachable liquid (silicone fluid) or dissolved in a releasable solvent (alkyl alcohol). The rate controlling membrane can be either microporous or non-porous membrane (Ethylene vinyl acetate copolymers) 2.2. Adhesive type TDDS: In this system, the drug resrvoir is formulated by directly dispersing the drug in an adhesive polymer (polyisobutylene or polyacrylate), then spreading the medicated adhesive by solvent casting or hot melt, onto a backing support to form a single layer or multiple layers of drug reservoir 2.3. Matrix type TDDS: The drug reservoir here is formed by homogeneously dispersing the drug in a hydrophilic or lipophilic polymer matrix and the medicated polymer formed is then moulded into medicated discs with a defined surface area and controlled thickness. This is then mounted onto a backing membrane and the adhesive is applied outside the disc along the circumference to form a strip of adhesive rim. 2.4. Microreservoir TDDS: This type of drug delivery system is formed by first suspending the drug in the aqueous solution of a water-soluble polymer (eg.PEG) and then dispersing homogeneously, the drug suspension in a lipophilic polymer, by high shear force, to form unleachable microscopic drug reservoirs. These are also known as ‘Microsealed Delivery Devices. 2.5. Poroplastic or Moleculon Type Devices: These systems, developed at Moleculon, (Cambridge, Massachusetts) utilise poroplastic films. The film is made utilizing the concept of water coagulation of cellulose triacetate solution in organic acids at low temperature. The coagulation is performed under controlled conditions and the extent of water content may be varied to a great condition and degree. 2.6. Penetration enhancement: The permeation of drugs across the skin is enhanced by physical means like pulsed DC iotophorosis or effect of ultrasounds may have synergistic effect depending upon the current density of pulse current applied and ultrasound intensity time (Chien YW, 1992). 2.6.1. Iontophoresis: It is a process that utilizes bipolar electric fields to propel ionic drug molecules across the intact skin into the underlying tissues. Positively charged drug ions in solution are transferred from a positive polarity chamber and vice versa. Delivery of positively charged compounds is easier than negatively charged compounds as the skin itself possesses a net negative charge. Iontophoresis can enhance transport across skin by a number of ways including an electrophoretic driving force and an electro-osmotic driving force and thus transiently increasing skin permeability. The transdermal transport can be increased by orders of magnitude relative to passive diffusion-based methods and can be modulated by controlling electrical parameters. Food and Drug Adminstration (FDA) has approved a number of products based on this technique like pilocarpine and lidocaine patches. The delivery of
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Sowjanya et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 494 proteins and peptides and other small macromolecules has been demonstrated in various articles. An iontophoretic electrode, Trans-Q has been developed such that the charge is delivered to a hydrogel pad loaded with the drug solution. Most of the work is going on to develop novel bioadhesive drug containing electrodes for use in iontophoretic drug delivery. Iontopatch SP transdermal drug delivery system is a self-constrained ultra-thin technology that eliminates the need of wires or batteries. It has an active area of 15.5 cm2 containing 40 mcg of the medicament. Mostly this technology has been introduced as an alternative to traditional treatment with injections. Non-steroidal anti-inflammatory drugs and corticosteroids are delivered by this mechanism. Alza Corporation Ltd., has developed electro transport system (E-Trans) for delivering fentanyl to treat acute and post operative pain. The patient has to push the button on the device which causes current to flow between two electrodes and a predetermined amount of drug is released through the skin. Also, a disposable kind of iontophoretic patch called Power Patch for delivering calcitonin to treat osteoporosis is under clinical trial. 2.6.2. Sonophoresis: It involves the introduction of substance into the body by ultrasound energy. Ultrasound energy vibrates molecules and creates tiny holes in the skin surface through ultrasound technology. The pores remain open for 12 hrs only.SonoPrep transdermal system from Sontra Medical uses low frequency ultrasound for skin permeation of lidocaine. It involves exposing the skin to a coat of lipids and then applying ultrasound at a frequency of 55,000 cycles per second causing creation of tiny bubbles which expands both in the liquid layer applied and the lipids of the skin. Thus, the skin of that area becomes leaky and remains as such. However, the pores get changed once the sound is turned off. Similarly, ImaRx Therapeutics has developed ultrasound assisted transdermal system utilizing ultrasound transducers to activate a drug and to open the skin pores for enhanced transdermal delivery. This technique has been employed for large molecular weight drugs such as peptides or proteins having molecular weight between 6000 to 48000 Daltons. 2.6.3. Electroporation: It is known that the mammalian skin is having intercellular lipids arranged in bilayers, which do not allow the transport of the drug transdermally. Electroporation is the technique by which aqueous pores are created by electric pulse of milliseconds causing transient permeability in the outer membrane which facilitates transport of drug. Flux increase upto four orders of magnitude was observed with human skin in vitro for three polar molecules having charges between –1 and –4 and molecular weights up to slightly more than 1000 daltons. Similar increase in flux was observed in- vivo with animal skin. The commercial product MedPulser (Genetronics Biomedical) is used on electroporation therapy system for use in delivering pharmaceuticals and genes. This electroporation system takes about 30 minutes and uses very small dose of the drug. The flux values of the model drugs increases exponentially and reaches the steady state flux. The examples are heparin and leutinizing hormone releasing hormone (LHRH), which show increased transdermal absorption with this technique. 2.6.4. Heat and Microneedles: Heat is also now expected to enhance the transdermal delivery of various drugs by increasing skin permeability, body circulation, blood vessel wall permeability, rate limiting membrane permeability and drug solubility.Heating prior to or during topical application of a drug will dilate penetration pathways in the skin, increase kinetic energy and the movement of particles in the treated area and facilitate drug absorption. Heating the skin after topical application of a drug will increase the drug absorption into vascular network, enhancing the systemic delivery but decreasing the local delivery as drug molecule is carried away from local site. Tempera are necessary to cause measurable changes in cell permeability. Recently, some researchers have reported the use of pressure driven jets for the intradermal delivery of a variety of drugs. The pressure and velocity of the jet were measured using calibrated pressure transducers and high-speed photography and showed the dependence on the drug delivery. Another innovation in this field is controlled heat aided drug delivery system (CHADD), which uses a thin heating device, attached to the top of the transdermal patch. The heat and temperature are controlled to deliver the drug either as bolus or to match circadian rhythms. S- Caine, a pediatric formulation of lidocaine and tetracaine uses CHADD technology for attaining a dense anesthetic effect in 15 to 20 minutes. Another product-Titragesia, uses the same technology to deliver fentanyl for treating pain.
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Sowjanya et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 495 3. CONCLUSION The novel drug delivery system has brought renaissance into the pharmaceutical industry for controlled drug delivery. The novel drug delivery systems include transdermal drug delivery system, mucoadhesive drug delivery system, nasal drug delivery system etc. The transdermal route of drug delivery is gaining accolade with the demonstration of percutaneous absorption of a large number of drugs. This type of drug delivery with the intention of maintaining constant plasma levels, zero order drug input and serves as a constant I.V. infusion. Several transdermal drug delivery systems (TDDS) have recently been developed aiming to achieve the objective of systemic medication through application to the intact skin. The intensity of interest in the pontential bio-medical application of transdermal controlled drug administration is demonstrated in the increasing research activities in a number of health care institutions in the development of various types of transdermal therapeutic systems(TTS) for long term continuous infusion of therapeutic agents including antihypertensives, antianginal, anti-histamine, anti- inflammatory , analgesic drugs etc. REFERNCES Brahmankar DM, Jaiswal SB, Biopharmaceutics and pharmacokinetics A Teatise, Vallabh Prakashan, Delhi, 1995, 335-371. Chien YW, Novel drug delivery systems, Drugs and the Pharmaceutical Sciences, Vol.50, Marcel Dekker, New York, 1992, 797. Roberts MS, Targeted drug delivery to the skin and deeper tissues: role of physiology, solute structure and disease, Clin Exp Pharmacol Physiol, 24(11), 1997, 874-9. Amgaokar MY, Chikhale RV, Lade UB, Biyani DM, Umekar MJ, Design formulation and evaluation of transdermal drug delivery system of budesonide, Dig J Nanomater and Biostruct, 6(2), 2011, 475-97. Patel MP, Patel KN, Patel DR, Patel UL, Formulation and evaluation of transdermal patches of glibenclamide, Int J Pharm Res, 1(2), 2009, 34-42 Jamakandi VG, Mulla JS, Vinay BL, Shivakumar HN, Formulation, characterization and evaluation of matrix- type transdermal patches of a model antihypertensive drug, Asian J Pharm, 3(1), 2009, 59-65. Irfani G, Raj R S, Tondare A, Noola, Design and Evaluation of transdermal drug delivery system of valsartan using glycerine as plasticizer, IJPRD, 3(2), 2011, 185-92 Shivaraj A, Selvam RP, Mani TT, T Sivakumar, Design and evaluation of transdermal drug delivery of ketotifen fumarate Int J Pharm Biomed Res, 1(2), 2010, 42-47 Ashok KJ, Pullakanda N, Prabu SL, V Gopal, Transdermal drug delivery system an overview, IJPSRR, 3(2), 2010, 49-54
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Navin et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 496 SYNTHESIS OF NEW THIAZOLIDINE-2,4-DIONE DERIVATIVES AND THEIR ANTIMICROBIAL AND ANTITUBERCULAR ACTIVITY Faiyazalam M Shaikh1 , Navin B. Patel1* and Dhanji Rajani2 1. Veer Narmad South Gujarat University, Udhana-Magdalla Road, Surat-395 007, Gujarat, India. 2. Microcare Laboratory and Tuberculosis diagnosis & Research Centre, Surat. * Corresponding author: E-mail: faiyaz_online007@yahoo.co.in; drnavin@satyam.net.in, Mobile: +919825350484 ABSTRACT New 1,3-thiazolidine-2,4-dione (TZD) derivatives 16-29 have been prepared by Knoevenagel condensation reaction between TZD and aromatic aldehydes followed by condensation with 3,4- dichloro benzoyl chloride. The structures of the newly synthesized compounds were assigned on the basis of elemental analysis, IR, 1 H NMR and 13 C NMR spectral data. All the synthesized compounds were tested for antibacterial activity against Gram-positive cocci and Gram-negative rods, antifungal activity and antitubercular activity. Moderate to good activity results were found for the newly synthesized compounds. Key Words: 1,3-thiazolidine-2,4-dione, Knoevenagel condensation, antibacterial, antifungal, antitubercular activity 1. INTRODUCTION One of the main objectives of organic and medicinal chemistry is to design, synthesize and produce molecules possessing value as human therapeutic agents. Compounds containing heterocyclic ring systems are of great importance receiving special attention as they belong to a class of compounds with proven utility in medicinal chemistry. Thiazolidine-2,4-dione (TZD) is a heterocyclic ring system with multiple applications. Thiazolidine-2,4-dione inhibits corrosion of mild steels in acidic solution. These are also used in analytical chemistry as highly sensitive reagents for heavy metals and as a brighter in electroplating industry. In 1982 a number of TZDs were intensively studied for their anti-hyperglycaemic property. The first representative of this class was ciglitazone, whereas other derivatives like englitazone, pioglitazone and troglitazone followed soon. The thiazolidine-2,4-dione nucleus has been reported for being responsible for majority of their pharmacological actions. Henceforth, thiazolidine- 2,4-dione derivatives have been studied extensively and found to have diverse chemical reactivities and broad spectrum of biological activities (Jain, 2013). Thiazolidinediones (TZD) are biologically active compounds having five membered rings, with two heteroatoms. Thiazolidinediones displayed a broad spectrum of biological activities including antimicrobial (Gouveia, 2009; Tuncbilek and Altanlar, 2006), antidiabetic (Murugan, 2009; Pattan, 2005), antiobesity (Bhattarai, 2009), anti- inflammatory (Youssef, 2010), antioxidant (Bozdag- Dundar, 2009), antiproliferative (Patil, 2010), antitumor (Shimazaki, 2008), etc. Currently, the antibiotic era is threatened by the convergence of three adverse circumstances: high levels of antibiotic resistance among important pathogens, an uneven supply of novel classes of antibiotics, and a dramatic reduction in the number of pharmaceutical companies engaged in the discovery and development of anti-infective agents (Wenzel, 2004). As a result, multidrug-resistant, and therefore difficult-to-treat, infections continue to occur and are clearly increasing in some areas. New antibiotics can help stave off the catastrophe. But since 1987, no major antibiotic has been discovered. In this regard, it is important to develop new and safe nuclei to combat with multidrug-resistant bacterial and fungal infections. Substantial investment and research in the field of anti-infectives are now desperately needed if a public health crisis is to be averted. Looking towards this turmoil of situation in the field of antibiotics, we are reporting herewith synthesis and antibacterial, antifungal and antitubercular activity of new thiazolidinediones. 2. MATERIALS AND METHODS 2.1. General: Laboratory Chemicals were supplied by Rankem India Ltd. and Ficher Scientific Ltd. Melting points were determined by the open tube capillary method and are uncorrected. The purity of the compounds was determined by thin layer chromatography (TLC) plates (silica gel G) in the solvent system n-hexane: ethyl acetate (7.5:2.5). The spots were observed by exposure to iodine vapors or by UV light. The IR spectra were obtained on Thermo Scientific Nicolet iS10 FT-IR spectrometer (using KBr pellets). The 1 H-NMR & 13 C-NMR spectra were recorded on a Varian Gemini 200 spectrometer using TMS as an internal standard in DMSO-d6. Elemental analyses of the newly
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Navin et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 497 synthesized compounds were carried out on Carlo Erba 1108 analyzer. 2.2. Synthesis of (E)-5-(substitutedbenzylidene) thiazolidine-2,4-diones 2-15: These compounds were prepared according to previously reported procedure (fig.-1) (Scheme-1). Fig.-1. (E)-5-(substitutedbenzylidene)thiazolidine- 2,4-diones 2-15 2.3. Synthesis of (E)-5-substitutedbenzylidene-3- (3,4-dichlorobenzoyl) thiazolidine-2,4-diones 16- 29: To a mixture of appropriate benzylidenes 2-15 (2 mmol) and pyridine (20 mL) was added dropwise 3,4-dichlorobenzoyl chloride (2.4 mmol) with stirring. The reaction was carried out for 3 h at room temperature with stirring and then heated for 4 h at 70 °C with stirring. When the mixture was cooled to room temperature, water (30 mL) was added. Then the mixture was neutralized with aqueous hydrochloric acid. The resultant solid was filtered off and washed three times with water. Product was obtained as a solid to give respective (E)-5- substitutedbenzylidene-3-(3,4- dichlorobenzoyl)thiazolidine-2,4-diones 16-29. (Scheme-2) Fig.-2. Title compounds 16-29 2.3.1. (E) - 5- benzylidene - 3 - (3,4- dichlorobenzoyl) thiazolidine-2,4-dione 16: m.p. 241-242 °C, yield, 71 % ; IR (KBr) ν cm-1 : 1756 and 1658 (>C=O str. of TZD), 1695 (>C=O str. of benzoyl chloride) 792 (C-S-C str.), 714 (C-Cl str.). 1 H NMR (DMSO-d6) δ (ppm): 8.02 (s, 1H, =CH), 7.24-8.11 (m, 8H, aromatic). 13 C NMR (100MHz, DMSO-d6) δ(ppm): 171.6 (C13), (>CO=), 169.5 (C2), (>CO=), 168.6 (C4), (>CO=), 144.7 (C6), (=CH), 137.2 (C17), 136.0 (C7), 134.6 (C14), 133.9 (C16), 130.8 (C18), 130.2 (C15), 129.1 (C9,11), 128.7 (C8,12), 128.4 (C19), 127.5 (C10), 116.8 (C5). Anal. calcd for C17H9O3NSCl2: C 53.98, H 2.40, N 3.70; found C 53.95, H 2.37, N 3.66. 2.3.2. (E) – 5 - (2-chlorobenzylidene) – 3 - (3,4- dichlorobenzoyl)thiazolidine-2,4-dione 17: m.p. 221-222 °C, yield, 60 % ; IR (KBr) ν cm-1 : 1758 and 1655 (>C=O str. of TZD), 1698 (>C=O str. of benzoyl chloride) 790 (C-S-C str.), 719 (C-Cl str.). 1 H NMR (DMSO-d6) δ (ppm): 8.26 (s, 1H, =CH), 7.26-8.13 (m, 7H, aromatic). 13 C NMR (100MHz, DMSO-d6) δ(ppm): 171.7 (C13), (>CO=), 169.4 (C2), (>CO=), 168.5 (C4), (>CO=), 144.4 (C6), (=CH), 137.1 (C17), 134.6 (C8), 134.0 (C14), 133.6 (C16), 133.1 (C7), 130.9 (C18), 130.3 (C15), 129.6 (C9), 128.5 (C19), 127.9 (C10), 127.3 (C12), 126.2 (C11), 116.7 (C5). Anal. calcd for C17H8O3NSCl3: C 49.48, H 1.95, N 3.39; found C 49.45, H 1.93, N 3.38. 2.3.3. (E) – 5 - (4-chlorobenzylidene) – 3 - (3,4- dichlorobenzoyl)thiazolidine-2,4-dione 18: m.p. 250-252 °C, yield, 67 % ; IR (KBr) ν cm-1 : 1757 and 1659 (>C=O str. of TZD), 1697 (>C=O str. of benzoyl chloride) 791 (C-S-C str.), 716 (C-Cl str.). 1 H NMR (DMSO-d6) δ (ppm): 8.03 (s, 1H, =CH), 7.40-8.21 (m, 7H, aromatic). 13 C NMR (100MHz, DMSO-d6) δ(ppm): 171.2 (C13), (>CO=), 169.1 (C2), (>CO=), 168.4 (C4), (>CO=), 144.1 (C6), (=CH), 137.0 (C17), 134.2 (C14), 133.4 (C10,16), 132.6 (C7), 130.7 (C18), 130.2 (C15), 129.1 (C8,12), 128.2 (C9,11), 128.0 (C19), 116.5 (C5). Anal. calcd for C17H8O3NSCl3: C 49.48, H 1.95, N 3.39; found C 49.46, H 1.94, N 3.37. 2.3.4. (E) - 3 - (3,4-dichlorobenzoyl) -5- (4- fluorobenzylidene) thiazolidine-2,4-dione 19: m.p. 230-232 °C, yield, 70 % ; IR (KBr) ν cm-1 : 1752 and 1662 (>C=O str. of TZD), 1702 (>C=O str. of benzoyl chloride) 798 (C-S-C str.), 715 (C-Cl str.), 678 (C-F str.). 1 H NMR (DMSO-d6) δ (ppm): 8.00 (s, 1H, =CH), 7.14-8.10 (m, 7H, aromatic). 13 C NMR (100MHz, DMSO-d6) δ(ppm): 171.1 (C13), (>CO=), 169.8 (C2), (>CO=), 167.8 (C4), (>CO=), 163.6 (C10), 143.7 (C6), (=CH), 137.3 (C17), 134.4 (C14), 133.2 (C16), 132.4 (C8,12), 132.0 (C7), 131.1 (C18), 130.2 (C15), 128.1 (C19), 116.2 (C5), 112.6 (C9,11). Anal. calcd for C17H8O3NSCl2F: C 51.53, H 2.04, N 3.54; found C 51.51, H 2.02, N 3.51. 2.3.5. (E) – 5 - (3-bromobenzylidene) – 3 - (3,4- dichlorobenzoyl) thiazolidine-2,4-dione 20: m.p. 258-259 °C, yield, 65 % ; IR (KBr) ν cm-1 : 1750 and 1654 (>C=O str. of TZD), 1692 (>C=O str. of benzoyl chloride) 795 (C-S-C str.), 714 (C-Cl str.), 618 (C-Br str.). 1 H NMR (DMSO-d6) δ (ppm): 8.04 (s, 1H, =CH), 7.25-8.15 (m, 7H, aromatic). 13 C NMR (100MHz, DMSO-d6) δ(ppm): 169.9 (C13), (>CO=), 168.6 (C2), (>CO=), 167.3 (C4), (>CO=), 143.5 (C6), (=CH), 138.3 (C7), 137.3 (C17), 134.2 (C14), 133.5
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Navin et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 498 (C16), 131.5 (C10), 130.8 (C18), 130.1 (C15), 129.7 (C8), 129.5 (C11), 128.4 (C19), 127.7 (C12), 122.6 (C9), 116.2 (C5). Anal. calcd for C17H8O3NSCl2Br: C 44.67, H 1.76, N 3.06; found C 44.65, H 1.75, N 3.03. 2.3.6. (E) – 3 - (3,4-dichlorobenzoyl) – 5 - (4- methylbenzylidene)thiazolidine -2, 4 - dione 21: m.p. 212-214 °C, yield, 75 % ; IR (KBr) ν cm-1 : 2912 and 2825 (-CH3 asym. and sym. str.), 1759 and 1652 (>C=O str. of TZD), 1699 (>C=O str. of benzoyl chloride) 790 (C-S-C str.), 719 (C-Cl str.). 1 H NMR (DMSO-d6) δ (ppm): 8.03 (s, 1H, =CH), 7.14-8.10 (m, 7H, aromatic), 2.26 (s, 3H, -CH3). 13 C NMR (100MHz, DMSO-d6) δ(ppm): 172.3 (C13), (>CO=), 170.4 (C2), (>CO=), 168.6 (C4), (>CO=), 144.0 (C6), (=CH), 138.4 (C10), 137.2 (C17), 134.7 (C14), 133.5 (C16), 132.0 (C7), 131.3 (C18), 130.2 (C15), 129.0 (C9,11), 128.3 (C8,12), 127.7 (C19), 115.6 (C5), 21.6 (C20) (-CH3). Anal. calcd for C18H11O3NSCl2: C 55.12, H 2.83, N 3.57; found C 55.10, H 2.81, N 3.56. 2.3.7. (E) – 3 - (3,4-dichlorobenzoyl) – 5 - (4- methoxybenzylidene) thiazolidine - 2, 4-dione 22: m.p. 224-225 °C, yield, 70 % ; IR (KBr) ν cm-1 : 1761 and 1650 (>C=O str. of TZD), 1705 (>C=O str. of benzoyl chloride) 789 (C-S-C str.), 721 (C-Cl str.), (- OCH3 str.). 1 H NMR (DMSO-d6) δ (ppm): 8.05 (s, 1H, =CH), 6.86-8.12 (m, 7H, aromatic), 3.76 (s, 3H, -OCH3). 13 C NMR (100MHz, DMSO-d6) δ(ppm): 172.0 (C13), (>CO=), 169.3 (C2), (>CO=), 168.2 (C4), (>CO=), 157.7 (C10), 143.8 (C6), (=CH), 137.3 (C17), 134.5 (C14), 133.4 (C16), 131.6 (C18), 130.7 (C15), 129.9 (C8,12), 128.4 (C19), 127.1 (C7), 115.5 (C5), 112.1 (C9,11), 55.3 (C20) (-OCH3). Anal. calcd for C18H11O4NSCl2: C 52.96, H 2.72, N 3.43; found C 52.93, H 2.70, N 3.41. 2.3.8.(E) – 3 - (3,4-dichlorobenzoyl) – 5 - (4- (dimethylamino) benzylidene) thiazolidine-2,4- dione 23: m.p. 268-269 °C, yield, 57 % ; IR (KBr) ν cm-1 : 1751 and 1653 (>C=O str. of TZD), 1703 (>C=O str. of benzoyl chloride) 790 (C-S-C str.), 714 (C-Cl str.). 1 H NMR (DMSO-d6) δ (ppm): 8.04 (s, 1H, =CH), 6.68-8.15 (m, 7H, aromatic), 3.10 (s, 6H, -N(CH3)2). 13 C NMR (100MHz, DMSO-d6) δ(ppm): 171.5 (C13), (>CO=), 169.6 (C2), (>CO=), 168.3 (C4), (>CO=), 147.9 (C10), 144.2 (C6), (=CH), 137.3 (C17), 134.5 (C14), 133.7 (C16), 131.4 (C18), 130.6 (C19), 124.4 (C7), 116.3 (C5), 111.1 (C9,11), 40.7 (C20,21) (- N(CH3)2). Anal. calcd for C19H14O3N2SCl2: C 54.17, H 3.35, N 6.65; found C 54.15, H 3.35, N 6.63. 2.3.9.(E)-3-(3,4-dichlorobenzoyl)-5-(4- hydroxybenzylidene) thiazolidine-2,4-dione 24: m.p. 236-238 °C, yield, 78 % ; IR (KBr) ν cm-1 : 3489 (-OH str.), 1749 and 1658 (>C=O str. of TZD), 1693 (>C=O str. of benzoyl chloride) 791 (C-S-C str.), 715 (C-Cl str.). 1 H NMR (DMSO-d6) δ (ppm): 8.02 (s, 1H, =CH), 6.62-8.14 (m, 7H, aromatic), 5.24 (s, 1H, -OH). 13 C NMR (100MHz, DMSO-d6) δ(ppm): 170.3 (C13), (>CO=), 168.7 (C2), (>CO=), 167.3 (C4), (>CO=), 157.1 (C10), 143.8 (C6), (=CH), 137.3 (C17), 134.7 (C14), 133.5 (C16), 131.6 (C18), 130.7 (C15), 130.1 (C8,12), 128.5 (C19), 127.4 (C7), 116.3 (C5), 115.1 (C9,11). Anal. calcd for C17H9O4NSCl2: C 51.79, H 2.30, N 3.55; found C 51.77, H 2.29, N 3.52. 2.3.10. (E) – 3 - (3,4-dichlorobenzoyl) – 5 - (4- hydroxy-3-methoxybenzylidene)thiazolidine-2,4- dione 25: m.p. 206-208 °C, yield, 61 % ; IR (KBr) ν cm-1 : 3491 (-OH str.), 1751 and 1657 (>C=O str. of TZD), 1696 (>C=O str. of benzoyl chloride) 788 (C- S-C str.), 717 (C-Cl str.). 1 H NMR (DMSO-d6) δ (ppm): 8.08 (s, 1H, =CH), 6.64-8.13 (m, 6H, aromatic), 5.21 (s, 1H, -OH), 3.74 (s, 3H, -OCH3). 13 C NMR (100MHz, DMSO-d6) δ(ppm): 171.4 (C13), (>CO=), 169.6 (C2), (>CO=), 168.2 (C4), (>CO=), 149.7 (C9), 147.2 (C10), 143.9 (C6), (=CH), 137.4 (C17), 134.8 (C14), 133.3 (C16), 131.5 (C18), 130.6 (C15), 129.3 (C7), 128.4 (C19), 122.7 (C12), 117.5 (C11), 116.3 (C5), 111.5 (C8), 55.7 (C20) (-OCH3). Anal. calcd for C18H11O5NSCl2: C 50.96, H 2.61, N 3.30; found C 50.94, H 2.59, N 3.27. 2.3.11. (E)-3-(3,4-dichlorobenzoyl)-5-(thiophen-2- ylmethylene)thiazolidine-2,4-dione 26: m.p. 214- 215 °C, yield, 69 % ; IR (KBr) ν cm-1 : 1755 and 1661 (>C=O str. of TZD), 1700 (>C=O str. of benzoyl chloride) 793 (C-S-C str.), 718 (C-Cl str.). 1 H NMR (DMSO-d6) δ (ppm): 8.22 (s, 1H, =CH), 7.46-8.18 (m, 6H, aromatic & thiophene). 13 C NMR (100MHz, DMSO-d6) δ(ppm): 172.2 (C13), (>CO=), 169.1 (C2), (>CO=), 168.3 (C4), (>CO=), 144.7 (C6), (=CH), 136.9 (C17), 134.5 (C14), 133.8 (C16), 130.8 (C18), 130.1 (C15), 128.0 (C19), 138.3-128.1 (thiophene carbons), 115.9 (C5). Anal. calcd for C15H7O3NS2Cl2: C 46.89, H 1.84, N 3.65; found C 46.86, H 1.82, N 3.62. 2.3.12. (E) – 3 - (3,4-dichlorobenzoyl) – 5 - (3- phenoxybenzylidene) thiazolidine-2,4-dione 27: m.p. 221-223 °C, yield, 62 % ; IR (KBr) ν cm-1 : 1750 and 1654 (>C=O str. of TZD), 1694 (>C=O str. of benzoyl chloride) 794 (C-S-C str.), 721 (C-Cl str.). 1 H NMR (DMSO-d6) δ (ppm): 8.08 (s, 1H, =CH), 6.98-8.12 (m, 12H, aromatic). 13 C NMR (100MHz, DMSO-d6) δ(ppm): 171.6 (C13), (>CO=), 169.3 (C2), (>CO=), 168.6 (C4), (>CO=), 156.5 (C9), 143.9 (C6), (=CH), 137.2 (C17), 134.9 (C7), 134.1 (C14), 133.4 (C16), 130.6 (C18), 130.0 (C15), 128.7 (C11), 128.1 (C19), 121.2 (C12), 118.3 (C10), 116.4 (C5), 113.2 (C8), 157.2-119.3 (C20-25) (Phenoxy carbons). Anal. calcd
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Navin et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 499 for C23H13O4NSCl2: C 58.74, H 2.79, N 2.98; found C 58.72, H 2.76, N 2.95. 2.3.13. (E) – 3 - (3,4-dichlorobenzoyl) – 5 - (3,4,5- trimethoxybenzylidene)thiazolidine-2,4-dione 28: m.p. 242-244 °C, yield, 66 % ; IR (KBr) ν cm-1 : 1752 and 1656 (>C=O str. of TZD), 1692 (>C=O str. of benzoyl chloride) 788 (C-S-C str.), 720 (C-Cl str.). 1 H NMR (DMSO-d6) δ (ppm): 8.05 (s, 1H, =CH), 6.72-8.15 (m, 5H, aromatic), 3.79 (s, 9H, (-OCH3)3). 13 C NMR (100MHz, DMSO-d6) δ(ppm): 171.5 (C13), (>CO=), 169.6 (C2), (>CO=), 168.3 (C4), (>CO=), 152.2 (C9,11), 143.7 (C6), (=CH), 138.9 (C10), 136.9 (C17), 134.5 (C14), 133.4 (C16), 130.8 (C18), 130.2 (C15), 129.2 (C7), 128.1 (C19), 116.5 (C5), 102.7 (C8,12), 60.3 (C21) (-OCH3), 55.7 (C20,22) (-OCH3)2. Anal. calcd for C20H15O6NSCl2: C 51.29, H 3.23, N 2.99; found C 51.28, H 3.20, N 2.97. 2.3.14. (E) – 3 - (3,4-dichlorobenzoyl) – 5 - (4- (dihexylamino) – 2 - methylbenzylidene) thiazolidine-2,4-dione 29: m.p. 246-247 °C, yield, 62 % ; IR (KBr) ν cm-1 : 2910 and 2823 (-CH3 asym. and sym. str.),1762 and 1664 (>C=O str. of TZD), 1704 (>C=O str. of benzoyl chloride) 795 (C-S-C str.), 721 (C-Cl str.). 1 H NMR (DMSO-d6) δ (ppm): 8.32 (s, 1H, =CH), 6.46-8.17 (m, 6H, aromatic), 2.52 (s, 3H, -CH3), 0.89-3.80 (m, 26H, dihexyl). 13 C NMR (100MHz, DMSO-d6) δ(ppm): 171.2 (C13), (>CO=), 169.1 (C2), (>CO=), 168.6 (C4), (>CO=), 150.5 (C10), 143.7 (C6), (=CH), 138.5 (C8), 137.0 (C17), 134.4 (C14), 133.5 (C16), 131.0 (C18), 130.2 (C15), 128.4 (C19), 127.4 (C12), 125.2 (C7), 116.8 (C5), 113.3 (C9), 108.5 (C11), 53.5 (C21,22) (N-CH2-), 31.3-14.2 (C23-32) (methylene & methyl groups attached to N), 19.1 (C20) (-CH3), Anal. calcd for C30H36O3N2SCl2: C 62.60, H 6.30, N 4.87; found C 62.57, H 6.26, N 4.86. 3. RESULTS AND DISCUSSION 3.1. Antimicrobial activity: The Minimum inhibitory concentrations (MICs) of synthesized compounds were carried out by broth micro dilution method as described by Rattan (Rattan, 2000). MICs of the tested compounds are shown in Table-1, 2 and 3. The different compounds 1-29 were tested for in vitro against two Gram positive (Staphylococcus aureus MTCC 96, Streptococcus pyogenes MTCC 442) and two Gram negative (Escherichia coli MTCC 443, Pseudomonas aeruginosa MTCC 741) bacteria for antibacterial, three fungal species (Candida albicans MTCC 227, Aspergillus niger MTCC 282 and Aspergillus clavatus MTCC 1323) for antifungal and. M. tuberculosis H37RV mycobacterium for antitubercular activity. Ampicillin, greseofulvin and rifampicin were used as standard antibacterial, antifungal and antitubercular agents respectively. 3.1.1. Antibacterial activity: The results of antibacterial activity of the synthesized compounds are presented in Table-1. Moderate to good antibacterial activity is observed with most of the tested compounds. Starting scaffold 1,3-thiazolidine- 2,4-dione 1 exhibited good activity (MIC value 200 g/ml) against S. aureus and moderate activity against other bacteria with ampicillin. Compounds 9, 12 and 15 displayed good activity (MIC value 62.5–100 g/ml) against E. coli relative to the reference drug ampicillin while other compounds showed modest to moderate activity. Additionally, compounds 17, 20 and 27 shows good biological activity (MIC value 62.5–125 g/ml). Compounds 6, 9, and 15 showed comparable activity (MIC value 100 g/ml) against P. aeruginosa relative to the reference drug ampicillin while other compounds showed modest to moderate activity. These results confirming the importance of the presence of nitrogen atom of a tertiary amine at position-4 in antibacterial activity, on the other hand title compounds 17, 19, 20, 24, 27 and 28 exhibited excellent to good activity (MIC value 50-125 g/ml). Compounds 4, 5, 6, 7, 8, 11, 13 and 14 exhibited comparable to good activity (MIC value 100–250 g/ml) against S. aureus relative to the reference drug ampicillin while other compounds showed modest to moderate activity. From final compounds; 17, 20, 21, 24, 25, 26, 27, 28 and 29 displayed good to very good activity (MIC value 62.5-250 g/ml). Finally, all the compounds (MIC value 200- 500 g/ml) exhibited modest to moderate activity against S. pyogenus relative to the reference drug ampicillin. Dramatically, from compounds (having 3,4-dichloro benzoyl core) 17, 20, 21 and 24 (MIC value 62.5-100 g/ml) showed remarkable activity against S. pyogenus. 3.1.2. Antifungal activity: The results of antifungal activity of the synthesized compounds are presented in Table-2. Starting scaffold 1,3-thiazolidine-2,4- dione 1 exhibited good activity (MIC value 250 g/ml) against C. albicans and moderate activity against other fungal species with greseofulvin. Compounds 2, 3, 4, 7, 8, 12, 13 and 14 displayed comparable to good activity (MIC value 100–500 g/ml) against C. albicans with reference drug greseofulvin while other compounds showed modest to moderate activity. Interestingly, addition of 3,4-dichloro benzoyl group also showed enhanced
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Navin et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 500 antifungal activity for compounds 16, 17, 20, 21, 23, 24, 25, 26, 27, 28 and 29 (MIC value 100-500 g/ml). All the compounds with exception of 11 and 29 (MIC value 100 g/ml) exhibited modest to moderate activity against A. niger with reference drug greseofulvin. All the compounds (MIC value 250->1000 g/ml) except 29 (MIC value 100 g/ml) showed modest to moderate activity against A. clavatus relative to the reference drug greseofulvin. 3.2. Antitubercular activity: All the synthesized compounds were also screened against M. tuberculosis H37RV with reference drug rifampicin which is summarized in Table-3. Results indicate that compounds showed moderate to modest antitubercular activity, except compound 14, 22 and 23 that showed good to very good activity (MIC value 25-50 g/ml) against M. tuberculosis and compounds 5, 11, 16, 21 and 28 that showed comparable (MIC value 62.5-100 g/ml) activity whereas remaining compounds exhibited modest to moderate activity (MIC value 200-1000 g/ml). 4. CONCLUSION In summary, new compounds of 1,3- thiazolidine-2,4-diones were synthesized, which showed a variety of biological activities in vitro as potential antibacterial, antifungal and antitubercular agents. Particulary, 2,4-thiazolidinedione ring is a scaffold for development of new antibacterial and anti-giardiasis agents, and attachment of 3,4- dichlorobenzoyl chloride is a good strategy for development of new antibacterial, antifungal and antitubercular agents. Present work provides a good outline of structure activity relationships of 1,3- thiazolidine-2,4-diones. ACKNOWLEDGEMENTS The authors are thankful to Department of Chemistry, VNSGU, Surat for providing necessary research facilities and Atul Limited, Atul for providing some free chemical samples. One of the authors Faiyazalam M. Shaikh is also thankful to University Grants Commission (UGC), New Delhi for awarding fellowship F1-17.1/2010/MANF-MUS- GUJ-7007 /(SA-III/Website). Table.1. Antibacterial activity (MICs, g/ml) for the title compounds Compound Gram negative bacteria Gram positive bacteria E. coli P. aeruginosa S. aureus S. pyogenus MTCC-443 MTCC-741 MTCC-96 MTCC-442 1 500 500 200 200 2 250 500 500 500 3 250 200 500 500 4 200 250 250 250 5 200 250 200 250 6 500 100 250 500 7 500 250 250 250 8 250 500 100 200 9 62.5 100 500 250 10 200 250 500 250 11 250 250 250 250 12 100 200 500 250 13 200 200 200 200 14 250 250 200 500 15 62.5 100 500 500 16 500 500 1000 500 17 62.5 50 100 62.5 18 500 250 500 500 19 250 50 500 200 20 125 62.5 200 100 21 500 500 125 100 22 500 250 500 500 23 500 500 1000 500 24 200 125 100 62.5 25 250 200 250 500 26 250 250 250 250 27 125 100 125 200 28 500 100 62.5 200 29 200 500 200 250 Ampicillin 100 100 250 100
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Navin et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 501 Table.2. Antifungal activity (MICs, g/ml) for the title compounds Compound Fungal species C. albicans A. niger A. clavatus MTCC-227 MTCC-282 MTCC-1323 1 250 500 >1000 2 500 1000 1000 3 500 250 >1000 4 250 1000 500 5 1000 >1000 500 6 1000 >1000 >1000 7 250 500 1000 8 500 1000 1000 9 1000 >1000 >1000 10 1000 1000 1000 11 1000 100 500 12 500 1000 1000 13 250 1000 >1000 14 500 1000 1000 15 1000 >1000 >1000 16 500 1000 1000 17 200 500 >1000 18 >1000 500 >1000 19 >1000 >1000 >1000 20 500 500 >1000 21 500 >1000 500 22 >1000 >1000 >1000 23 200 500 >1000 24 500 500 >1000 25 500 500 >1000 26 500 500 500 27 200 250 250 28 250 500 500 29 100 100 100 Greseofulvin 500 100 100 Scheme-1 Synthesis of intermediates 2-15 Scheme-2 Synthesis of title compounds 16-29
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Navin et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 502 Table.3. Minimal tubercular concentrations (MICs, g/ml) for the title compounds Compound MIC values (g/ml) of M. tuberculosis H37Rv % Inhibition 2 250 98% 3 500 97% 4 250 99% 5 100 98% 6 250 99% 7 200 99% 8 250 98% 9 1000 98% 10 250 99% 11 100 99% 12 1000 98% 13 200 98% 14 50 99% 15 500 98% 16 62.5 98% 17 500 98% 18 1000 99% 19 500 98% 20 250 98% 21 100 99% 22 50 99% 23 25 99% 24 500 98% 25 1000 99% 26 1000 98% 27 250 98% 28 100 99% 29 1000 99% Rifampicin 40 99% REFERENCES Bhattarai BR, Kafle B, Hwang J, Khadka D, Lee S, Kang J, Ham SW, Han I, Park H, Cho H, Thiazolidinedione derivatives as PTP1B inhibitors with antihyperglycemic and antiobesity effects, Bioorg. Med. Chem. Lett., 19, 2009, 6161-6165. Bozdag-Dundar O, Coban T, Ceylan-Unlusoy M, Ertan R, Radical scavenging capacities of some thiazolylthiazolidine-2,4-dione derivatives, Med. Chem. Res., 18, 2009, 1-7. Gouveia FL, de Oliveira RMB, de Oliveira TB, da Silva IM, do Nascimento SC, de Sena KXFR, de Albuquerque JFC, Synthesis, antimicrobial and cytotoxic activities of some 5-arylidene-4-thioxo- thiazolidine-2-ones, Eur. J. Med. Chem. 44, 2009, 2038-2043. Jain VS, Vora DK, Ramaa CS, Thiazolidine-2,4- diones: Progress towards multifarious applications, Bioorg. Med. Chem., 21, 2013, 1599-1620. Murugan R, Anbazhagan S, Lingeshwaran, Narayanan SS, Synthesis and in vivo antidiabetic activity of novel dispiropyrrolidines through [3+2] cycloaddition reactions with thiazolidinedione and rhodanine derivatives, Eur. J. Med. Chem., 44, 2009, 3272-3279. Patil V, Tilekar K, Mehendale-Munj S, Mohan R, Ramaa CS, Synthesis and primary cytotoxicity evaluation of new 5-benzylidene-2,4- thiazolidinedione derivatives, Eur. J. Med. Chem., 45, 2010, 4539-4544. Pattan SR, Suresh C, Pujar VD, Reddy VVK, Rasal VP, Koti BC, Synthesis and antidiabetic activity of 2- amino [5′(4-sulphonylbenzylidine)-2,4- thiazolidinedione]-7-chloro-6-fluorobenzothiazole, Ind. J. Chem., 44B, 2005, 2404-2408. Rattan A, Antimicrobials in Laboratory Medicine, Churchill B.I., Livingstone, New Delhi, 2000, 85- 108. Shimazaki N, Togashi N, Hanai M, Isoyama T, Wada K, Fujita T, Fujiwara K, Kurakata S, Anti-tumour activity of CS-7017, a selective peroxisome proliferator-activated receptor gamma agonist of thiazolidinedione class, in human tumour xenografts and a syngeneic tumour implant model, Eur. J. Cancer, 44, 2008, 1734-1743. Tuncbilek M, Altanlar N, Synthesis of New 3- (Substituted Phenacyl)-5-[39-(4H-4-oxo-1- benzopyran-2-yl)-benzylidene]-2,4- thiazolidinediones and their Antimicrobial Activity, Arch. Pharm. Chem. Life Sci., 339, 2006, 213-216.
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Navin et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 503 Wenzel RP, The Antibiotic Pipeline - Challenges, Costs, and Values, N. Engl. J. Med., 351(6), 2004, 523-526. Youssef AM, White MS, Villanueva EB, El- Ashmawy IM, Klegeris A, Synthesis and biological evaluation of novel pyrazolyl-2,4-thiazolidinediones as anti-inflammatory and neuroprotective agents, Bioorg. Med. Chem., 18, 2010, 2019-2028.
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Kamalkannan et. al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 504 EFFECTS OF PERMEABILITY CHARACTERISTICS OF DIFFERENT POLYMETHACRYLATES ON THE PHARMACEUTICAL CHARACTERISTICS OF DILTIAZEM HCL-LOADED MICROSPHERES V. Kamalakkannan*1 , K.S.G.Arul Kumaran 2, C. Kannan3 , S.Bhama3 , R. Sambath Kumar4 1. Department of Biotechnology (Pharmacy), Periyar Maniammai University, Thanjavur (Dt), Tamilnadu, India 2. K.M.C.H. College of Pharmacy, Coimbatore, Tamilnadu, India 3. Department of Pharmaceutics,J. K. K. Nataraja College of Pharmacy, Komarapalayam, Namakkal (Dt), Tamilnadu, India 4. J.K.K.Nataraja College of Pharmacy, Komarapalayam,Namakkal (Dt), Tamilnadu, India * Corresponding Author: kamalpharma79@rediffmail.com, +918973750397 ABSTRACT Microspheres containing Diltiazem hydrochloride (DTZ Hcl) were prepared with various Polymethacrylates, with different permeability characteristics (Eudragit RSPO and Eudragit RLPO) and also with mixtures of these polymers in a 1:1 and 1:2 ratio using the solvent evaporation method. The aim was to investigate the effects of the permeability of the polymers on drug release rates and the characteristics of the microspheres. To achieve these aims, yield, incorporation efficiency, particle size and the distribution of microspheres were determined, and the influence of the inner phase viscosities prepared with different polymer and polymer mixtures on particle size and the distribution of microspheres were evaluated. Surface morphologies of microspheres were observed by scanning electron microscope. Drug release rates from microspheres were determined by the half-change method using a flow-through cell. The results indicate that microspheres with different surface morphologies and statistically different yields and incorporation efficiencies could be prepared and their particle size and distribution variances resulted from the viscosity of the inner phase. Dissolution profiles showed that the drug release rate could be modified depending on the permeability characteristics of Polymethacrylates. Key words: Polymethacrylates, Diltiazem Hcl, Microsphere, Solvent Evaporation Method. INTRODUCTION Diltiazem hydrochloride are easily absorbed from gastrointestinal tract (GIT) and have a short half-life are eliminated quickly from blood circulation. This drug undergoes substantaintly hepatic first pass effect it shows to oral bioavailability 40%. So they require frequently dosing to avoid these drawback, the oral sustained control release formulation have been developed in an attempt to release the drug surely in to the GIT and maintained an effective drug concentration in the serum for longer period of time Diltiazem hydrochloride an effective drug in treatment of hypertension and angina pectoris is a benzothiazepine derivative calcium and turbonist are drug which cases coronary and peripheral vasodilation by reducing calcium influx through the slow channel of vascular smooth muscle and cardiac cell membranes. Administration of conventional tablet of Diltiazem Hydrochloride has been reported to exhibit fluctuations in plasma drug level resulting either in side effect of reduction in drug concentration at receptor side also the maintenance of constant plasma concentration of cardiac vascular drug is important in ensuring the designed therapeutic response, again since the half life of Diltiazem HCl is 3-4 hrs multiple dose of drug need to maintained constant plasma concentration for good therapeutic response and improve patients compliance. Hence The objective of the present work are To prepare diltiazem hydrochloride loaded microspheres by incorporating the drug into the mixture of two types of polymers (Polymethacrylates), one having high permeability characteristics (Eudragit RLPO) and the other having less permeability characteristics (Eudragit RSPO). To prepare Diltiazem hydrochloride loaded microspheres having different drug: polymer ratios and characterize the release pattern of the drug from different microspheres. MATERIALS AND METHODS Diltiazem Hcl was received as a gift from M/s Microlabs, Bangalore, India. Eudragit® RSPO and RLPO were obtained Gift sample from Dr.reddys Lab,Hyderabath,India. All other reagents and solvents used were of pharmaceutical or analytical grade. Preparation of DTZ Hcl M icrospheres: Method used: Emulsification – solvent evaporation method (Mastiholimath, 2007). Diltiazem hydrochloride loaded microspheres were prepared by solvent evaporation method. The formulation of microspheres is given in Table. Diltiazem
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Kamalkannan et. al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 505 hydrochloride and each polymer mixture were dissolved completely in acetone-methanol mixture by stirring at 500 rpm with magnetic stirrer. Magnesium stearate was added and the mixture was stirred with magnetic stirrer at 500 rpm in ice-bath at 10o C for 10 minute. Above mixture was poured into the liquid paraffin previously cooled at 10o C, while it was being stirred by mechanical stirrer at 1000 rpm. Resulting emulsion was stirrred at 35o C for 4 hours using mechanical stirrer and the organic solvent, acetone-methanol were removed completely by evaporation. Solidified microspheres were filtered through Whatmann filter paper (No.1) using vacuum pump, washed six times with 50 ml n-hexane, dried under vacuum at room temperature for 12 h and stored in desiccator containing calcium chloride. Evaluation of DTZ hcl microspheres: Drug polymer interaction (FTIR) study: FTIR spectroscopy 4was performed on Fourier transformed infrared spectrophotometer (IR-Affinity-1, Shimadzu, Japan). The pellets of drug and potassium bromide were prepared by compressing the powders at 20 psi for 10 min on KBr-press and the spectra were scanned in the wave number range of 4000- 500 cm-1. FTIR study was carried on DTZ Hcl, physical mixture of DTZ Hcl and polymer, DTZ Hcl microspheres and blank microspheres. Surface morphology (SEM): Scanning electron microscopy (Giovanni, 2002) has been used to determine particle size distribution, surface topography, texture, and to examine the morphology of fractured or sectioned surface. SEM is probably the most commonly used method for characterizing drug delivery systems, owing in large to simplicity of sample preparation and ease of operation. SEM studies were carried out by using JEOL JSM T-330A scanning microscope (Japan). Dry DTZ Hcl microspheres were placed on an electron microscope brass stub and coated with in an ion sputter. Picture of DTZ Hcl microspheres were taken by random scanning of the stub. Frequency Distribution Analysis: Determination of average particle size (Nagasamy, 2009) of DTZ Hcl microspheres was carried out by optical microscopy in which stage micrometer was employed. A minute quantity of DTZ Hcl microspheres was spread on a clean glass slide and average size of 300 DTZ Hcl microspheres was determined in each batch. In order to be able to define a frequency distribution or compare the characteristics of particles with many different diameters, the frequency distribution can be broken down into different size ranges, which can be presented in the form of a histogram.. Histogram presents an interpretation of the frequency distribution and enables the percentage of particles having a given equivalent diameter to be determined. Percentage yield: Percentage practical yield (Dandagi, 2004) of DTZ Hcl is calculated to know about percentage yield or efficiency of any method, thus it helps in selection of appropriate method of production. Practical yield was calculated as the weight of DTZ Hcl microspheres recovered from each batch in relation to the sum of starting material. The percentage yield of prepared DTZ Hcl microspheres was determined by using the formula: Determination of percentage drug entrapment (PDE): Efficiency of drug entrapment (Shah, 2009)for each batch was calculated in terms of percentage drug entrapment as per the following formula: Theoretical drug loading was determined by calculation assuming that the entire DTZ Hcl present in the polymer solution used gets entrapped in DTZ Hcl microspheres, and no loss occurs at any stage of preparation of DTZ Hcl microspheres. Drug loading: Weighed amount of DTZ Hcl microspheres equivalent to 100 mg of DTZ Hcl was dissolved in 100 ml of distilled water. This solution was kept overnight for the complete dissolution of the DTZ Hcl in water. This solution was filtered and further diluted to make a conc of 10 µ g/ml solution.The absorbance of the solutions was measured at 269 nm using double beam UV-Visible spectrophotometer against distilled water as blank and calculated for the percentage of drug present in the sample. In-Vitro Dissolution Studies: In-vitro dissolution profile (Soppimath, 2001) of each formulation was determined by employing USP XXIII apparatus by rotating basket method in different media like stimulated gastric fluid pH 1.2 buffer for 2 hrs (since the average gastric emptying time is 2 hrs), stimulated intestinal fluid pH 7.2 buffer for 3hrs (average small intestinal transit time is 3 hrs) and colonic fluid pH 6.8 buffer for subsequent hrs. The dissolution media were maintained at a temperature of 37± 5ºC, the speed rotation of basket maintained were 50 rpm. DTZ Hcl Microspheres equivalent to 40 mg DTZ Hcl was loaded into the basket
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Kamalkannan et. al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 506 of the dissolution apparatus. 5 ml of the samples withdrawn from dissolution media at suitable intervals and same amount was replaced with fresh buffer. The absorbance was measured at 269 nm. Data obtained was also subjected to kinetic treatment to understand release mechanism. Kinetics of drug release: To examine the drug release kinetics and mechanism (Soppimath, 2001) the cumulative release data were fitted to models representing zero order (Q v/s t), first order [Log(Q0- Q) v/s t], Higuchi’s square root of time (Q v/s t1/2 ) and Korsemeyer Peppas double log plot (log Q v/s log t) respectively, where Q is the cumulative percentage of drug released at time t and (Q0-Q) is the cumulative percentage of drug remaining after time t. RESULTS AND DISCUSSION The resulting microspheres formulated by solvent evaporation method were found to be spherical and free flowing in nature. The mean particle size of microspheres ranged from 450 -500 µm Table.3. It was noticed that mean particle size increased with increase in polymer concentration and decrease in magnesium stearate concentration. The entrapment efficiencies ranged from 86.11 – 98.90% found to be dependent on nature of polymer used in the formulation. From the in vitro drug dissolution studies it was found that the sustaining effect of microspheres depended on the polymer concentration, amount of dispersant used and the type of polymer used in the formulation. Increase of mean particle size with increase in polymer concentration may have occurred due to the fact that as polymer concentration increases it produces a significant increase in the viscosity in a fixed volume of solvent, thus leading to an increase of the emulsion droplet size and finally a higher microsphere size (Dandagi, 2009). The encapsulation efficiency of the drug depended on the solubility of the drug in the solvent and continuous phase. An increase in the concentration of polymer in a fixed volume of organic solvent resulted in an increase in encapsulation efficiency. Diltiazem hydrochloride loaded microspheres having a fairly high yield (74.65 – 90.45%). The entrapment efficiencies ranged from 86.11 – 98.90%. The incorporation efficiency of formulations, F4, F5, F6 was more than formulations F1, F2, F3. The highest incorporation efficiency of formulation having drug: polymer ratio 1:2 can be explained through the fact that the amount of polymer in per unit drug is greater than that in other formulations. It has been observed that the particle size increases with increasing polymer amount. The increase in the mean size Fig.10, Table.3 with increasing polymer concentration was attributed to the fact that higher concentration of polymer in the sample leads to increase in viscosity of the dispersed phase, which results in formation of bigger droplets and also, fusion of semi-formed particles and producing an overall increase in the size of the microspheres. Eudragit RL-type microspheres and Eudragit RS-type microspheres prepared with the same polymer concentration did not show any significant variation in their mean size. Scanning electron microscopic photographs of microspheres are shown in Fig.4 to 9. All microspheres were almost spherical in shape and No aggregation of microspheres had taken place. Diltiazem Hcl showed prominent peaks at Characteristic peak at 3057.27, 2837.38, 2391.81, 1743.71, 1681.98 cm-1 . Peak of all the functional groups are intact in the intermediate mixtures and final formulations.No shifting, deleting and broadening of the peak observed. No chemical interactions had been occurred. The same peaks were also observed in the formulation indicating the stable nature of the drug during encapsulation. The release of Diltiazem hydrochloride from different formulations depended on the type of polymer and the ratio of the polymer in the formulations. The release of Diltiazem hydrochloride from microspheres of Eudragit RL-type was more as compared to Eudragit RS-type. This was due to the presence of more functional quaternary ammonium groups (10%) in RL-type than RS-type (5%). It is also observed that as the amount of polymer in the formulation increased, the drug release decreased. It can be explained on the basis that as the polymer amount increases, the matrix wall of microspheres become thicker. A burst effect of drug release can be observed on the various formulations. The burst effect can be attributed to the presence of non- encapsulated drug particles on the surface of the microspheres. The burst effect of drug release also depended upon the drug : polymer ratio. From the figure of release profile, it can be observed that burst effect of drug release is more in formulations having drug : polymer ratio 1:1, while in the formulation having drug : polymer ratio 1:2, burst effect is less. It can be concluded that as the particle size of the microspheres increased, the release rate of Diltiazem hydrochloride decreased. Hence, particle size of microsphere is inversely proportional to the release of drug from microspheres. It can be explained on the basis that as the polymer amount increases, the matrix wall of microspheres become thicker. In order to determine the mechanism of drug release the data were fitted to the
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Kamalkannan et. al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 507 Korsemeyer-Peppas model in order to determine the ‘n’ value, which describes the drug release mechanism (Shah, 2009). The ‘n’ value of all the formulations was between 0.5 and 1 indicating that the mechanism of drug release was non-Fickian type diffusion. Table.1 Formulations of Diltiazem hydrochloride Microspheres prepared with different Polymers and Polymer mixtures (Drug: Polymer =1:1) Contents of Formulations F 1 F 2 F 3 Diltiazem hydrochloride (gm) 2.0 2.0 2.0 Eudragit RLPO (gm) 2.0 - 1.0 Eudragit RSPO (gm) - 2.0 1.0 Magnesium Stearate (gm) (Dispersing Agent) 0.300 0.300 0.300 Methanol (ml) 3.0 3.0 3.0 Acetone (ml) 7.0 7.0 7.0 Liquid paraffin (ml) 100 100 100 Drug : Polymer 1:1 1:1 1:1 Magnesium Stearate (%) 3 3 3 Table.2 Formulations of Diltiazem hydrochloride Microspheres prepared with different Polymers and Polymer mixtures ( Drug : Polymer =1:2 ) Contents of Formulations F 4 F 5 F 6 Diltiazem hydrochloride (gm) 2.0 2.0 2.0 Eudragit RLPO (gm) 4.0 - 2.0 Eudragit RSPO (gm) - 4.0 2.0 Magnesium Stearate (gm) (Dispersing Agent) 0.600 0.600 0.600 Methanol (ml) 6.0 6.0 6.0 Acetone (ml) 14.0 14.0 14.0 Liquid paraffin (ml) 200 200 200 Drug : Polymer 1:2 1:2 1:2 Magnesium Stearate (%) 3 3 3 500750100012501500175020002500300035004000 1/cm 30 40 50 60 70 80 90 100 %T 2910.68 2837.38 2575.05 2542.26 2501.76 2459.32 2391.81 2349.38 1743.71 1681.98 1606.76 1508.38 1475.59 1448.59 1413.87 1255.70 1219.05 1060.88 1028.09 781.20 DTZ Figure.1. IR Spectrum of Diltiazem Hydrochloride in 7501000125015001750200022502500275030003250350037504000 1/cm 30 45 60 75 90 105 120 %T PF3 F3 DT Z PF3 Figure. 2. IR spectrum of DTZ. HCl, F1, PF1 (Physical mixture corresponding to F1) DTZ. HCl = Diltiazem hydrochloride, F1= Formulation F1 (ERL PO = 1:1) PF1 (Physical mixture corresponding to F1)
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Kamalkannan et. al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 508 5007501000125015001750200022502500275030003250350037504000 1/cm 30 40 50 60 70 80 90 100 %T DT Z Smooth 1 F4 DTZ Figure.3.IR spectrum of DTZ.HCl, F2, PF2 (Physical mixture corresponding to F2) DTZ.HCl = Diltiazem hydrochloride, F2= Formulation F2 (ERS PO = 1:1) PF2 (Physical mixture corresponding to F2) Surface morphology (SEM) Figure.4. SEM for Formulation= F1 Magnification:10x20x Magnification:10x30x. Surface morphology (SEM) Figure.5. SEM for Formulation= F2 Magnification:10x20x Magnification:10x30x Figure.6. SEM for Formulation= F3 Magnification:10x20x Magnification:10x30x.
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Kamalkannan et. al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 509 Figure.7. SEM for Formulation= F4 Magnification:10x20x Magnification:10x30x. Figure.8. SEM for Formulation= F5 Magnification:10x20x Magnification:10x30x. Surface morphology (SEM) Figure.9. SEM for Formulation= F6 Magnification:10x20x Magnification:10x30x Table.3.Particle size Analysis Figure.10.Particle size Analysis PARTICLE SIZE RANGE F1 F2 F3 F4 F5 F6 0-50 0 0 0 0 0 0 50-100 0 0 0 0 0 0 100-150 22 14 15 3 2 1 150-200 70 51 56 12 12 3 200-250 53 66 65 24 17 9 250-300 20 32 42 37 32 24 300-350 18 18 9 57 41 52 350-400 6 12 7 32 45 61 400-450 7 4 4 13 32 31 450-500 4 3 2 10 10 11 500-550 0 0 0 7 6 5 550-600 0 0 0 5 3 3 600-650 0 0 0 0 0 0
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Kamalkannan et. al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 510 Table.4. Percentage Yield Values Fig 11 Percentage Yield Formulation code Percentage Yield (%) F1 82.91 F2 76.95 F3 79.3 F4 90.22 F5 86.36 F6 90.45 Table.5.Actual drug content Figure.12.Actual Drug content Formulation code Actual Drug Content (%) F1 40.45 ± 0.13 F2 41.05 ± 0.09 F3 42.28 ± 0.09 F4 27.45 ± 0.13 F5 28.35 ± 0.10 F6 29.97 ± 0.12 Table.6. Entrapment Efficiencies Figure.13. Entrapment Efficiencies Formulation code Entrapment Efficiencies (%) F1 86.97 ± 0.28 F2 88.26 ± 0.19 F3 90.90 ± 0.19 F4 90.59 ± 0.43 F5 93.56 ± 0.33 F6 98.90 ± 0.39 Table.7.Cumalative % drug release Time (Hours) Cumulative percentage drug release F1 F2 F3 F4 F5 F6 0 0 0 0 0 0 0 1 41.1 20.02 20.91 24.27 14.6 13.36 2 60.29 30.05 37.93 44.68 20.97 19.39 3 73.42 37.06 51 58.67 24 24.89 4 81.15 40.09 60.96 67.05 27.71 28.34 5 85.11 42.15 69.14 73.21 31.94 32.58 6 87.6 44.21 75.35 77.72 36.77 36.81 7 92.11 47.22 79.74 82.76 41.12 40.92 8 97.99 51 83.99 87 45.25 45.06 9 98.2 54.3 87.93 88.91 48.24 49.23 10 98.56 59.13 91.08 90.65 50.72 55.09 11 98.6 64.14 93.18 92.41 53.9 60.48 12 98.64 67.51 94.98 94.34 56.27 66.23
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Kamalkannan et. al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 511 Figure.14. Dissolution study of Diltiazem Hcl Microsphares CONCLUSION Eudragit® microspheres containing Diltiazem Hcl can be prepared successfully by using an emulsion solvent evaporation technique. The surface structure of the microspheres was spherical and rough. The entrapment efficiencies ranged from 86.11 – 98.90%.and the mean size was in the range of 450 - 500 µm. The release rate of Eudragit® RSPO microspheres was much slower than that than those prepared with Eudragit® RLPO. The release pattern of the microspheres was found to be of the non- Fickian. REFERENCES Dandagi PM, Manvi FV, Gadad AP, Mastiholimath VS, Patil MB, Balamuralidhara V. Microencapsulation of verapamil hydrochloride by ionotropic gelation technique, Ind J Pharm Sci, 66(5), 2004, 631-635. Dandagi PM, Matiholimath VS, Gadad AP, Kulkarni AR, Jain SS, pH Sensitive mebeverin microspheres for colon delivery Indian J Phrm Sci, 71 (4), 2009, 264-268. Formulation and in vitro Evaluation of Eudragit Microspheres of Stavudine, Tro j pharma research, 4 (1), 2005, 369-375. Giovanni FP, Giulia B, Piera DM, Sante M. Microencapsulation of semisolid of semisolid ketoprofen/polymer microspheres, Int J Pharma, 5(3), 2002, 134-144. Mastiholimath VS, Dandagi PM, Jain SS, Gadad AP, Kulkarni AR, Time and pH dependent colon specific pulsatile delivery of theophylline for nocturnal asthma. Int J Pharm, 328, 2007, 49-56. Nagasamy Venkatesh D, Reddy AK, Samanta MK, Suresh B, Development and In Vitro Evaluation of colonic drug systems for tegaserod maleate, asian j pharm, 2009, 50-53. Pandey V. P, Manavalan R, Rajan T.S and Ganesh K.S, Formulat ion and release characteristics of sustained release diltiazem hydrochloride tablet, Ind J. Pharm Sci., 65(1), 2003, 44-8. Shah SH, Patel JK, Patel NV, Stomach specific floating drug delivery system, Int J Pharm Tech Res, 1(3), 2009, 623-633. Soppimath, K.S., Kulkarni, A.R., Aminabhavi, T.M, Encapsulation of antihypertensive drugs in cellulose- based matrix microspheres: characterization and release kinetics of microspheres and tableted microspheres. J. Microencapsul. 18, 2001, 397-409.
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Murthy et. al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 512 IMPORTANCE OF SAFETY HEALTH ENVIRONMENT IN PREVENTING OCCUPATIONAL HEALTH HAZARDS IN INDIAN INDUSTRIES Murty TN1* , Md Aasif Siddique Ahmed Khan2 , Abhinov T2, Abhilash T2 1. Nimra College of Business Management, Vijayawada, India. 2. Shadan Institute of Medical Sciences, Hyderabad, India. *Corresponding author: Email: thamminaina@yahoo.com ABSTRACT Environmental Health & Safety (EH&S) provides expert guidance and timely service to the Community through our commitment to health and safety. Employing best practices and collaboration, and by building long term relationships, we promote a productive and safety conscious work environment. Maintaining a healthy and safe work environment for staff, students, neighbours and surrounding communities through the recognition, evaluation, and control of personal and environmental hazards, the strives to eliminate individual risk and reduce the environmental impact of its activities. These endeavours are realized through programs such as personnel training, chemical hygiene plan, biological safety, environmental safety, fire safety, occupational safety, and asbestos and lead management in compliance with local, state and federal regulations. Occupational health deals with all aspects of health and safety in the workplace and has a strong focus on primary prevention of hazards. The health of the workers has several determinants, including risk factors at the workplace leading to cancers, accidents, musculoskeletal diseases, respiratory diseases, hearing loss, circulatory diseases, stress related disorders and communicable diseases. Present business scenario which requires highly skilled and competent human resources for the transformation of traditional economies into the modern and industrial economies. Key Words: Occupational Health, Work Environment, Motivation, Safety. INTRODUCTION H R Capital plays a crucial role in the growth and prosperity of any industry of any nation. The workers can be motivated and their productivity can be increased only when a conducive and hygienic physical work environment is created an adequate health and medical facility is provided by the management of irrespective of the sector. The occupational health service at work should vary according to the size of the work group, hazards involved, the location of the plant, and many others factors. The object should be to set-up a preventive occupational service tailored to the needs of the factory and its particular hazards. Occupational health covers a very wide field and calls for specialized knowledge from many disciplines such as medicine, engineering, chemistry, toxicology, physiology, statistics, etc., and above all a close inter-disciplinary team work. The maintenance of cleanliness at work spot is the responsibility of every employer (Bhattacharya S.K., 1988). OCCUPATIONAL HEALTH – AN ANALYSIS Health and medical provision is neither philanthropy nor charity. It is a method of creating more satisfactory working conditions for men and women employed in an industry. This welfare measure would greatly reduce turnover and absenteeism among workers and improves workers efficiency to a greater extent and also create a permanent settled labor force by making service attractive to the labor. A large number of well- motivated workforces is essential for an industrial organization of such nature and magnitude to maintain good industrial relations which lead to high morale, commitment and productivity. And the workforce can be motivated by way of undertaking good Safety and Health Environment (SHE) and preventing occupational health hazards to the workforce (Dale S.Beach, 2002). In this regard, general propaganda by means of posters, notices on works and office board, articles and magazines, and lectures can help to make the factory workers health-minded. Generally, the firms issue handbooks on hygiene to new employees, which contain hints on food, exercise, personal hygiene, and the seeking of medical advice. A good example of which opens with the sentence “Health is won by a way of life rather than a bottle of medicine.” Any company setting-up a new occupational health program can profitably utilize qualified hygienist to make a through survey of hazards requiring control or elimination. Human beings have been subjected to some occupational health hazards or the other from the very beginning of human civilization. In fact, no occupation is free from hazards. In the recent past as a result of rapid escalation of industries, the health hazards in industry have greatly increased. The Government of India became
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Murthy et. al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 513 concerned about the occupational health problems in the country after the recommendations of the Royal Commission on Labor in 1931. The occupational health service in an industry should aim at: Protecting the workers against health hazards which may arise out of their work or the conditions in which it is carried on, and Contributing towards the workers physical and mental adjustments and maintenance of the highest possible degree of physical and mental well being The role of occupational health service should be essentially preventive. Its functions should be: a). to identify occupational hazards and suggest measures for their control, b). to detect occupational or other diseases and give initial treatment, c). to render advice about the placement of people in suitable work, d). to provide necessary advice relating to the supervision of conditions at work which may influence health and general sanitation, and e). to undertake the health education. A large number of employers on globe do not provide occupational health care for their employees. A very small percentage of employers have their own occupational specialists to ensure healthy working conditions at the work-place. The situation in the small and medium size industries is deplorable. There is no machinery to take care of the health of the unorganized labor like those engaged in road-building, stone-pressing, quarrying, construction work, private transport, agriculture, etc. There should be a growing realization that occupational health is the responsibility of management and not the Employees State Insurance Corporation, which renders curative but not preventive services. The unions are also indifferent towards the health of their members. The management and the unions must try to keep their employees healthy, free from not only occupational hazards and diseased but also from infections, communicable diseases, psychosomatic diseases, because they have a great influence on their work performance. In this context a study has been made on the provisions of the health in the selected units India. RESULTS AND DISCUSSIONS The prevalence of job satisfaction varies from person to person and from time to time within the same company in any nation. This is mainly due to the presence of various factors relating to both the job and the individual within the organization. The following are the integrated elements of Safety and Heath Environment (SHE) of the workforce. Physical Working environment influences to a greater extent of the health of workers. Hygienic environment is an important requisite for maintenance of good health. The importance of personal hygiene should continually be brought home to the industrial workers. The pre-employment interview with the doctor, nurse or welfare officer gives an opportunity for hints on personal hygiene. The fundamental conditions of a healthy working environment must be maintained in every factory for keeping the workers in good health conditions. In this connection, proper ventilation assumes as important, in keeping the air fresh and free from germs. Fresh air and light are healthier than the air-conditioning and artificial lighting. To reduce the risk of direct infection, workers should be so spaced that they do not have to inhale each other’s breath. Cleanliness in the shop is necessary to prevent the foresting of germs, which tend to settle in dust particles or walls, floors, machines, etc. First Aid Box: According to the Chapter – V under section 45 of the Factories Act, 1948, every factory should be provided and maintained on First-Aid Box for every 150 workers. First Aid should be given to the workers as soon as the accident occurs. In most of the case accident assumes serious proportions as first aid being neglected. First Aid Box provision would be helpful immediately to the injured workers. Sickness and ill-health are the most wide spread causes of absenteeism, lower morale and tardiness. The National Commission on Labor in India felt that the last two should be left to the process of evolution, whereas primary importance should be given to the first. It further observed that protection of health requires provision of both preventive and curative measures. Curative measures consist of pre-employment medical examination, removal of health hazards, and imparting of training in first aid and hygiene, etc., the curative aspects begin when a worker suffers from ill health and disease. The workers may face accidents during the work of loading, unloading, metal breaking, metal handling, metal carrying, welding, weighing, mechanical, metallurgical, electrical and some other operations which are of hazardous nature. For this, keeping first aid box at the place of work is very essential. The Public Sector Units in India had provided sufficient and adequate number of First Aid Boxes, which are containing the necessary items, ate every work spot inside the plants. There are well-equipped hospitals in the vicinity of Public Sector Units in India (Frederick Herzberg, 1959). The Computed Data shows information on the opinion of workers in the selected unit of the study on the
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Murthy et. al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 514 awareness and availability of First Aid Box at their nearest places of work spot. It is evident from the data that almost all the workers are known about the first aid box facility in Public Sector Units in India. It is also clear from the data cent percent of the workers in Public Sector Units in India had expressed positive opinion on the maintenance of first aid box inside the plants. Thus, it can be concluded that majority of the workers on an average in the unit felt happy with First Aid Box facility Public Sector Units in India (Moorthy M V, 1981). Crèche: Crèche is a welfare facility, which is provided for women workers. A crèche is a place where babies of working mothers are taken care of while the mothers are at work. Section 48 of the Factories Act, 1948 requires that a crèche must be maintained in every factory, where more than 50 women workers are ordinarily employed for the use of children under the age of 6 years of such women. Very few female workers are working in the selected public sector unit in India. With forward looking the management of Public Sector Units in India has been maintaining crèches in the adjacent side of the factory premises for the purpose of women employees. Medical Check-Up: Management of Public Sector Units in India conducts medical check-up to all employees in a year free of cost. In case of detection of any serious disease during the check-up such employees will be given proper medical care and specialists’ attention at company’s expenditure. Public Sector Units in India have been providing medical aid to the employees and their dependents. These medical facilities are not only applicable to the residents of township, but also applicable to the employees who are living outside the township. The management of Public Sector Units in India made payment of medical advances to the employees who are getting out- patient medical treatment. The management is giving bank drafts in favor of liaison officers as medical advances to the employees of Public Sector Units in India. The medical reimbursement policy is not applicable to the person who recovers from his/her sick at home (Misra KK, 1971). Opinions of the Workers on Medical Facilities in PSU: The Data reveals the views of the workers on medical facilities available in the selected public sector unit. The data reveals that 43.59 percent of workers had expressed good opinion and the remaining 56.41 percent of them were expressed satisfactory opinion about medical facilities. It is observed that almost all the workers were satisfied with the medical facilities provided by Public Sector Units in India. SAFETY WORKING CONDITIONS According to the section 12, 16 and 17 of the Factories Act, 1948, the management of every industrial organization should provide good working conditions at the work-spot. Assurance of safety working conditions is again the job of Safety Engineer and Welfare Officer. Accidents could be avoided by providing adequate lighting and ventilation to the workers at their workplaces. Extreme heat and cold and successively altering temperature are harmful to human health. Therefore these should be controlled by modern scientific devices and humidity and temperature should be adjusted to suit the working conditions. Illumination: Day light is the best illumination for work. In the absence of day light, artificial light is needed. A situation where illumination covers the total work environment rather than concentrating on a single aspect of the work areas is better. In the case of the latter, whenever the visual field is shifted to a properly illuminated surrounding, it dilutes the workers. Excessive papillary activity causes fatigue and eye-strain. Ventilation: Adequate ventilation is another aspect of working conditions. In every factory, there should be proper arrangement for adequate ventilation by the circulation of fresh air. It is mainly provided by windows and ventilators etc. Lighting: Adequate and suitable lighting at work places protects the eye-sight of the employees and increases their output. Natural lighting may be derived from roof windows. Artificial lighting may be had through electricity, kerosene or petro max lamps. Sanitation: Sanitation means cleanliness inside the factory. There should not be accumulation of dirt and dust. The floor should be cleaned; walls should be painted and varnished. Besides, there should be effective arrangements for the disposal of wastes and effluents. Cleanliness: According to Section 11 of Factories Act, 1948, the management of Company has to be maintained work spot very cleanly. The work spot should be made elegant and attractive, as far as possible. A cheerful and moderately artistic work environment will be conducive to the toning up of morale, spirits and energies of the workers. Noise/Sound: Noise is generally considered a distracter, when the noise level is high, an employee has to concentrate more or exert greater efforts to perform. In fact, the consistency of noise reduces its distracting character and the person adopts himself to it more easily.
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Murthy et. al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 515 Noise will lead to increased muscle tension and metabolic rate. Shift System: The shift system is a common feature of modern industrial organization in all organized industries. It is necessitated by more demand for production, and has become possible through the modern industrial system. The shift system is an advantage if it makes fuller utilization of machinery which reduces standing expenses in terms of output. The shift system provides leisure time to the employees to look after their families’ welfare also. Temperature: Temperature and humidification are also important aspect of the working conditions. The climatic conditions especially in summer make physical work unpleasant due to high temperature. As the nature of the work in a company it requires high temperature, workers should be protected against it by separating the process, which produces such temperature, from the workroom or by some other methods. The recommended comfortable temperature in india is 690 to 800 F. Under Section 13 (B) of the Factories Act, 1948 the effective and suitable provision of temperature shall be made in every factory for securing and maintaining in every work room/spot to the workers (Narayana Murty, 2000). SAFETY TOOLS AND EQUIPMENT The Factories Act, 1948 laid down in Sections from 21 to 41 certain standards of safety, to be adopted by factories covered under it. The management of plant has to be provided several protective measures like safety shoes, supplied twice in a year in addition to the uniform, belts, chest-guards, helmets, goggles, aprons, gloves, rain coats and face shields etc. as per the nature of job. Maintenance of men and machines is an important object of every management. Most of the industrial organizations are Vulnerable for industrial accidents. While accidents are costly to the employer, they are something personal to the employee, resulting in some damage to his body. In India, the accident rate is higher where the workers are drawn from rural areas with a low literacy rate and on an average 547 persons are killed per year in Industrial accidents. Hence, the employer must assume the responsibility for providing safe working conditions to workers (Rizwana Ansari., 2011). Working environment: Regarding Working environment, it is found that the workers display more job satisfaction towards the sub-factor of working conditions prevailing in the Working environment followed by another sub-factor namely comfort and safety. It has been found that the strong cluster of 24.68 % of respondents feel that the working conditions are excellent, moderate cluster of 62.82 % are of the opinion that the working environment gives them moderate satisfaction and the weak cluster of 12.50 % says that they need some improvement in the maintenance of the working environment. SUGGESTIVE INJECTION FOR PREVENTION OF OCCUPATIONAL HEALTH HAZARDS 1. It is suggested that Government should implement the uniform working conditions to benefit the employees of all Public Sector Enterprises through a legislative measure as desired by most of the respondents. 2. It is suggested that the management of Public Sector Units in India should mitigate the dissatisfied workers by improving healthy physical work environment, since the workers and the employers are affected very much physically and psychologically. 3. It is suggested that the management of Public Sector Units in India should provide more protective devices to their employees to save from the sound and noise problem during their shifts as per the nature of job and to relieve their psychological and physical burdens to perform their job in their units effectively. 4. It is suggested that the workers who are working in high temperature conditions in the unit should be protected against it by providing protective devices in the working place. 5. Maintenance of good working conditions has the effect of increasing job satisfaction and hence, the management must try to achieve this goal by improving the safety and health environment system and upgrading the technologies and expertise in various areas of activities involving production operations. 6. It is suggested that the management of Public Sector Units in India should provide more protective devices as there is every need to provide and educate in the use of safety tools and equipment at their work spot. 7. It is suggested that the management of Public Sector Units in India should take all the necessary steps to mitigate the rest of dissatisfied workers in the provision of First Aid Boxes. 8. It is suggested that the management of Public Sector Units in India should be made elegant and attractive as far as possible at their work spot and to mitigate the dissatisfied segment of the workers and should maintain a cheerful and moderately artistic work environment for the
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Murthy et. al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 516 toning up of the morale, spirits and energies of the workers. 9. It is further suggested that the management of Public Sector Units in India may be segregated the safety and training function from HRD department. 10. It is suggested that the management o Public Sector Units in India should maintain the working conditions for their better performance. All sections of employees may be treated alike and clear instructions may be given to the workers regarding the results expected of them on their work. Some drawbacks in the organization of work may be removed and suitable work program and action plan for the work to be performed in each department may be revised. CONCLUSION On the basis of the results obtained in the study, it is observed that the safety and health environment to employees in the units prevailing have deep impact on workers’ psychology and the presence of such factors always motivate the workers towards their job in a laudable manner. It has also been found that the concept of health services facility assumes immense importance in the present business scenario which requires highly skilled and competent human resources for the transformation of traditional economies into the modern and industrial economies. ACKNOWLEDGEMENTS We thank Dr. Mohd Vizarath Rasool Khan, Chief Promoter of Nimra and Shadan Group of Colleges, India, Dr. Mohd Saqib Rasool Khan, Secretary, Nimra College of Business Management, Vijayawada, India, Dr. Mohd Sarib Rasool Khan, Managing Director, Shadan Institute of Medical Sciences, Hyderabad for their support and encouragement during this study. REFERENCES Bhattacharya S.K (1988), Organizational Culture and Indian Perspectives. Dale S.Beach (2002), Human Resource and Personnel Management, Tata Mc Graw Hill, New Delhi. Frederick Herzberg, B.Mausner and B. Synderman (1959), The Motivation to work, John Wiley and Sons, New York. Garry Steiner (1964), Human Behavior, New York Harcourt, Brace & World. Wheinrich H W (1959), Industrial accident Preventio, Mc Graw Hill, New York Maslow A.H (1954), Motivation and Personality, Harper & Row, New York. Malik (1993), Hand Book on Labour & Industrial Laws, Eastern Book Company, Lucnkow. Moorthy M V (1981), Principles of Labour Welfare, Oxford & IRH Publications, New Delhi. Misra K K (1971), Labour Welfare in Indian Industry, Meenakshi Prakasam, Meerut. Narayana Murty T (2000), Labor Welfare Activities in Indian Industrial Organizations, Unpublished Thesis, Nagarjuna University, Nagarjuna Nagar, India. Rizwana Ansari (2011), Physical Work Environment and Industrial Safety of the Employees in Power Sector, Unpublished Thesis, Vinayaka Mission University, Salem, India.
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Devendra Singh et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 517 OPTIMIZATION OF THIOCOLCHICOSIDE TABLET WITH PERMEATION ENHANCERS USING 32 FACTORIAL DESIGN Devendra Singh*, Pankaj Kumar Sharma, Udai Vir Singh Sara Raj kumar goel institute of technology, Delhi-Meerut Road, Ghaziabad, India *Corresponding address:devendrasingh.pisces@gmail.com, devendra.treasure007@gmail.com ABSTRACT Thiocolchicoside has a selective affinity for g-amino-butyric acid (GABA) receptors and acts on the muscular contracture by activating the GABA-nergic inhibitory pathways thereby acting as a potent muscle relaxant Thiocolchicoside (Muscoril, Myoril, Neoflax) is a muscle relaxant with anti-inflammatory and analgesic effects. It is used for the treatment of orthopedic, traumatic and rheumatologic disorders. It is currently administered by the oral, injective and topical routes. The physico-chemical properties of Thiocolchicoside are not favorable for its permeation across the skin. Thiocolchicoside has a relatively high molecular weight (563), relatively high water solubility (16.1mg/ml) and low octanol/water partition coefficient (logP=-0.34). The various Permeation enhancers were used in different ratio and its permeation enhancement is done by using Franz diffusion cell. The Data Expert software is applied for the 32 factorial design and the ANOVA study of the linear regression model, response surface plot as well as contour plot confirms the predicted batch with the desirability of 0.982. Result of R2 and T-test confirmed that predicted and observed responses showed no significant difference. The optimized batch was found to be B2 showing disintegrating agent 40 mg and binder 2.5 mg quantity with the best response. Key words: Optimisation, Full factorial design, Contour plots, Thiocolchicoside. INTRODUCTION Oral administration still dominates drug therapy and more than 60 % of marketed drugs are oral products. This type of drug administration is preferred due to its convenience, high patient compliance, less stringent production conditions and lower costs. Delivering a drug by oral route is also preferred for its convenience. Tablets and capsules can be prepared in large quantity at low price (Kumar, 2011). Therefore in lead optimization step of drug discovery, oral bioavailability of a drug is important. It depends on various factors the most common being intestinal permeability, solubility during gastrointestinal transit, liberation from dosage form, liability to efflux and metabolism. Development in the field of combinatorial chemistry and high throughput screening has made it possible to generate a large number of drug candidates but it has also resulted in a number of poorly soluble and or poorly absorbable drugs. A new trend of drug development based on pharmacogenomics or development of molecular targeted drugs is also encouraging the tendency, and it does not necessarily lead to good output in terms of new drug development. Therefore it is necessary to improve the membrane permeability as well. The pharmacokinetic profile of a drug is dependent on the drugs ability to cross biological membranes. All drugs are now classified according to the biopharmaceutical classification BCS into four categories on the basis of solubility and permeability to rationalize science of drug delivery and simplify complications in the drug registration of newly evolving diverse compounds for regulatory authorities. Among the different classes of BCS the per oral delivery of class 3 and 4 drugs is partially or completely decreased due to their poor intestinal permeability. A great number of currently available drugs fall under the class III of the biopharmaceutical classification system, possess high therapeutic potential but cannot be delivered by oral route because of its poor permeation across the GIT epithelia (Amidon, 1995). According to the BCS, Thiocolchicoside drug is classified in class III:a high solubility, low permeability compound. Thiocolchicoside is an approved drug having very low bioavailability in the solid dosage forms therefore they are also available in parenteral injections which are also not showing much difference in bioavailability. Therefore an oral dosage formulation would offer several advantages such as stability, cost effectiveness and patient compliance (Artursson P, 1990). In this paper we report the selection of optimized batch of with the best drug-permeation enhancer ratio combination using the 32 factorial design. MATERIAL AND METHODS Materials: Thiocolchicoside Drug (Saico Healthcare (P) Ltd, New Delhi) Sodium Glycolate and Sodium Caprylate (Himedia Laboratories (P) Ltd, Mumbai) EDTA (Qualikems Laboratories Reagent, New Delhi ) Lactose Monohydrate (CDH Analytical Reagent, Central Drug House (P) Ltd, New Delhi) were obtained as gift samples.
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Devendra Singh et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 518 Experimental Design: Experimental design is a statistical method that prescribes or advice a set of combination of variables. The number and layout of these design points within the experimental region depends on the number of effects that must be estimated. Depending on the number of factors, their levels, possible interaction and order of the model, various experimental design are chosen. Each experiment can be represented as a point, within the experimental domain, being defined by its coordinate (the value given to the variables) in the space. Factorial design: Factorial design are used in experiments where the effects of different factors or conditions on experimental results to be elucidated. These are the design of choice for simultaneous determination of the effect of several factors and their interactions. The simplest one is the two-factorial design were two factors are considered, each at two levels, leading to four experiments, which are situated in 2-dimensional factor space at the corner of a rectangle. If there are three factors, each at two levels, eight experiments are necessary which are situated at the corners of the orthogonal cube in a 3- dimensional space. The number of experiments is given by 2n where ‘n’ is the number of factors If the number of factors and levels are large, then the number of experiments needed to complete a factorial design is large. To reduce the number of experiments, fractional factorial design can be used (i.e., ½ or ¼ of the original numbers of experiments with full factorial design) .The fitting of an empirical polynomial equation to the experimental results facilitates the optimization procedure. The general polynomial equation is as follows: Y=B0 + B1X1 + B2X2 + B3X3 + B4X4+…+ B12X1X2 + B13X1X3 + B23X2X3 +…+ B123X1X3. Where Y is the response, X1, X2, X3 are the levels (concentration) of the 1,2,3 factors. B0, B1, B2, B3, B12, B13, B23, B123, are the polynomial coefficients. B0 is the intercept (which represents the response when the level of all factors is low). Software for designs and optimization: Many commercial software packages are available which are either dedicated to experimental design alone or are of a more general statistical type. Software’s dedicated to experimental designs: Various softwares are available for the prediction of successful experimental design e.g.: Design Ease and Design Expert (Stat-ease). Full Factorial Design: A 32 randomized full factorial design was used to optimize the variables in the present study. In the design 2 factors were evaluated, each at 3 levels, and experimental trials were performed for all 9 possible combinations. The amount (20, 40, 60 mg) of starch (X1), and (2, 2.5, 3 mg) of gelatin(X2), were selected as independent variables. The disintegration time and the % drug release (t45) were selected as dependent variables. In-vitro drug release: The USP paddle method was adopted in this study. The release medium consisted of 900 ml of pH-7.5 buffer. A known quantity from the batches were placed in appropriate chamber of the release apparatus and agitated at 100 rpm. Timed for 45 minutes, for each 5 minutes time intervals 1ml of the release medium were withdrawn, appropriately diluted and their absorbance determined at a wavelength of 259.0 nm using UV spectrophotometer. The volume of the release medium was kept constant by replacing it with 1 ml of fresh medium after each withdrawal. The release study was repeated using pH 7.4 as a release medium and the absorbance was determined at a wavelength of 259.0 nm. RESULT AND CONCLUSION By the application of the Data Expert software it is confirmed that out of the 9 formulation prepared the batch B2 is showing the best with the drug release and the disintegration time as the dependent variable, starch and gelatin as the independent variable. Thus the ANOVA study of the linear regression model, response surface plot as well as contour plot confirms the predicted batch with the desirability of 0.982. This conclude that the use of starch with 40 mg as disintegrating agent and the gelatin as binder with 2.5 mg in formulation will give the best formula for the Thiocolchicoside tablet with permeation enhancers incorporated. Result of R2 and T-test confirmed that predicted and observed responses showed no significant difference. ACKNOWLEDGEMENT The author is grateful to Saico Healthcare Pvt Ltd Delhi, India, for providing the Thiocolchicoside drug.
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Devendra Singh et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 519 Table 1: showing batches containing permeation enhancers with different concentration of disintegrating agent and binder Ingredients A 1 A 2 A 3 B 1 B 2 B 3 C 1 C 2 C 3 Thiocolchicoside 8 8 8 8 8 8 8 8 8 Sodium Caprylate 2.4 2.4 2.4 2.4 2.4 2.4 2.4 2.4 2.4 EDTA 64 64 64 64 64 64 64 64 64 Lactose monohydrate 115.3 95.3 75.3 114.8 94.8 74.8 114.3 94.3 74.3 Starch 20 40 60 20 40 60 20 40 60 Sucrose 5 5 5 5 5 5 5 5 5 Gelatin 2 2 2 2.5 2.5 2.5 3 3 3 Talc 8 8 8 8 8 8 8 8 8 Magnesium stearate 1.3 1.3 1.3 1.3 1.3 1.3 1.3 1.3 1.3 Table 2: showing in-vitro drug release of 9 batches Times (min) Batch A1 Batch A2 Batch A3 Batch B1 Batch B2 Batch B3 Batch C1 Batch C2 Batch C3 M.F 0 0 0 0 0 0 0 0 0 0 0 5 10.31 11.31 11.13 11.89 10.53 11.11 9.41 8.31 11.51 10.33 10 16.75 19.21 19.13 22.63 24.98 21.61 19.41 20.51 18.41 17.76 15 21.51 29.31 28.43 32.69 36.42 28.43 25.41 27.21 24.32 22.61 20 26.42 37.42 35.63 41.52 45.25 35.21 32.64 33.13 31.41 26.42 25 39.81 45.12 43.31 47.61 51.43 45.32 41.41 42.52 40.51 39.12 30 45.23 51.29 49.82 54.63 59.54 54.61 51.31 51.21 51.91 44.99 35 50.37 60.43 59.39 61.51 67.56 61.43 59.12 62.49 61.43 50.32 40 56.91 65.34 61.93 66.53 74.59 69.56 68.13 71.42 68.90 56.82 45 58.13 66.21 63.23 68.17 75.31 70.23 69.12 73.14 69.31 56.92 M.F= marketed formulation Figure.1. In-vitro drug release of different batches
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Devendra Singh et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 520 Figure.2. Comparison between drug release of marketed formulation and batch B 2 Table.3. Disintegration time for the 9 batches Batch Disintegration time (min) Batch Disintegration time (min) A 1 12.5 B 3 11.0 A 2 11.5 C 1 12.5 A 3 11.0 C 2 12.0 B 1 12.0 C 3 12.0 B 2 11.0 Table.4. Full Factorial Design Layout* Batch Code Variable Levels in Coded Form Disintegration Time Drug release X1 (mg) X2 (mg) Disintegration Time(min) Drug release (%) A1 -1 -1 12.5 58.131 A2 0 -1 11.5 66.211 A3 1 -1 11.0 63.231 B1 -1 0 12.0 68.172 B2 0 0 11.0 75.312 B3 1 0 11.0 70.234 C1 -1 1 12.5 69.127 C2 0 1 12.0 73.141 C3 1 1 12.0 69.311 *Coded value for -1 X1 =20mg and X2=2.0 mg; for 0 X1 =40mg and X2=2.5 mg; 1 X1 =60mg and X2=3.0mg;
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Devendra Singh et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 521 Table 5: Calculations For Testing The Model In Portions* For disintegration time DF SS MS F R2 Regression FM 5 3.03 0.61 65.40 0.9909 Error FM 3 0.028 9.259 For drug release DF SS MS F R2 Regression FM 5 209.34 41.87 68.86 0.9914 Error FM 3 1.82 0.61 *(DF): Degree Of Freedom, (SS): Sum of Squares, (MS) Mean of Squares, (F): Fischer’s Ratio, (R2 ) Regression Coefficient, (FM): Full Model Table.6.Summary of Regression Analysis Results For Disintegration Time RESPONSE B 0 B 1 B2 B3 B4 B5 FM 11.11 -0.50 0.25 0.25 0.33 0.58 For drug release RESPONSE B 0 B 1 B2 B3 B4 B5 FM 74.70 1.22 4.00 -1.23 -5.19 -4.71 Table.7.Optimized formula obtained and their desirability Name Goal Lower limit Upper limit Factor A In range 20 60 Factor B In range 2.0 3.0 Disintegrate time Minimize 11 12.5 Drug release Maximize 58.131 75.312 Table.8.Predicted Solution Factor A Factor B Disintegration time Drug release Desirability Remarks 46.53 2.52 11 74.7007 0.982 Selected Table.9.Optimized batch B2 Starch (mg) Binder (%) Disintegration time(min) Drug release (%) 40 2.5 11 75.312
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Devendra Singh et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 522 Figure.3.Desirability contour graph of optimized batch B2 Figure.4.Desirability 3D graph of optimized batch B2 Figure.5.Disintegration time contour graph of optimized batch B2.
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Devendra Singh et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 523 Figure.6.Disintegration time of 3D graph of optimized batch B2 Figure 7: Drug release contour graph of optimized batch B2 Figure 8: Drug release 3D graph of optimized batch B2
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Devendra Singh et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 524 Figure 9: Desirability graphical optimization of Batch B2 REFERENCES Amidon GL, Lennernas H, Shah VP, and Crison JR, A theoretical basis for a biopharmaceutics drug classification: The correlation of in vitro drug product dissolution and in vivo bioavailability, Pharm. Res, 12, 1995, 413–420. Artursson P, Epithelial transport of drugs in cell culture. I: A model for studying the passive diffusion of drugs over intestinal absorptive (Caco-2) cells, J Pharm Sci, 79, 1990, 476–482. Biopharmaceutics Classification System Guidance Office of Pharmaceutical Science, CDER/FDA, August 2006. Guidance for industry, Waiver of in- vivo bioavailability and bioequivalence studies for immediate release solid oral dosage forms based on biopharmaceutics classification system, CDER/FDA, August 2000. Kumar A.P, Badarinath AV, Naveen N, Prasad K, Reddy B, Hyndhavi M, Nirosha M, A rationalized description on study of intestinal barrier, drug permeability and permeation enhancers, 4, 2011, 431-449.
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Mallikarjuna et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 525 METHOD DEVELOPMENT AND VALIDATION FOR THE SIMULTANEOUS ESTIMATION OF DESVENLAFAXINE AND CLONAZEPAM IN BULK & TABLET FORMULATION BY RP-HPLC METHOD Regalagadda Mallikarjuna*1 , Nanda Kishore Agarwal1 , Prem Kumar Bichala2 , Sukhen som3 1. Department of Pharmaceutical Analysis, Nimra college of Pharmacy , Vijayawada, A.P, India. 2. Faculty of Pharmaceutical Analysis, Chaitanya college of pharmacy, Markapur, A.P, India. 3. Faculty of Pharmaceutical Chemistry, M.M.U College of pharmacy, Ramanagaram, Karnataka. *Corresponding author: Email:mallipharma2011@gmail.com Mob.no+919010560590 ABSTRACT The present work is a simple, precise, accurate, efficient and reproducible RP-HPLC method was developed and validated for the simultaneous estimation of Desvenlafaxine and Clonazepam in combined dosage form. Chromatographic separation was effectively carried out on zodiac c18 (250mm X 4.6mm; 5μ) column and mobile phase contain KH2PO4 buffer, methanol and acetonitrile in a proportion of 30:20:50v/v at a flow rate of 1ml/min with U.V detection at 222nm.Retention times of Desvenlafaxine and Clonazepam was found to be 2.51 min and 4.40 min respectively. As per ICH guidelines the developed method was validated in terms of accuracy, Precission, linearity, LOD, LOQ, specificity and robustness. The linearity can occurs in between concentration range of 60-140mcg/ml and 0.6-1.4mcg/ml and the co relation co-efficients were 0.998 and 0.997 for Desvenlafaxine and Clonazepam respectively. The effectively developed and validated method can be used for estimation of Desvenlafaxine and clonazepam in tablet pharmaceutical dosage form. Key words: RP-HPLC, Zodiac C18 column, Desvenlafaxine and Clonazepam INTRODUCTION Desvenlafaxine (Figure.1)was chemically 4-[2- dimethylamino-1-(1-hydroxycyclohexyl)ethyl]phenol, an antidepressant in a group of drugs called selective serotonin and nor epinephrine reuptake inhibitors (SNRI), mode of action to blocking the transporter reuptake proteins for key neurotransmitters affecting mood, thereby leaving more active neurotransmitters in the synapse. Clonazepam (Figure.2) was chemically5 - (o - chlorophenyl) -1,3-dihydro-7-nitro-2H-1,4-benzodiazepin- 2-one, It is a benzodiazepine derivative having anxiolytic, anti convulcent, muscle relaxcent and hypnotic activity ZyVen -OD Plus is a marketed brand it contain 50mg of Desvenlafaxine and 0.5mg of Clonazepam it has been used to reduce the symptoms of depression and relieve the anxiety symptoms. MATERIALS AND METHOD The various materials and equipments used for the present study are summarized as follows. Figure.1.Chemical structure of Desvenlafaxine Figure.2.Chemical structure of Clonazepam
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Mallikarjuna et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 526 Table.1.List of various materials used Materials Grade Company Desvenlafaxine (DES) N/A Zydus cadilla formulation Pvt. Ltd. Puducherry Clonazepam (CLO) N/A PJ Pharma Ltd, Hyderabad Potassium dihydrogen phosphate pure Std. Laboratory, Hyderabad Methanol HPLC Std. Laboratory, Hyderabad Aectonitrile HPLC Std. Laboratory, Hyderabad Zyven-OD plus (DES-50 mg & CLO-0.5 mg) N/A Zydus cadilla Lab.ltd, Ahmedabad Table.2. List of various equipment used Chromatographic condition Mobile phase: Buffer: Methanol: Aectonitrile 30:20:50 v/v/v Flow rate 1.0 ml/min Column Zodiac C18 (4.6 x 250 mm, 5μ) Detector wavelength 222 nm Injection volume 20 µL Run time 6 min Column back pressure 128-13(kgf) Temperature Ambient temperature(25o C) Preparation of mobile phase: Buffer (pot.dihydrogen phosphate pH 3)(30ml), methanol (20 ml) and acetonitrile (50 ml) were mixed well and sonicated for 30 min, and filtered through 0.45 µ membrane filter under vaccum filteration. The prepared solution was used as mobile phase. Preparation of standard and sample solutions of Desvenlafaxine succinate and Clonazepam: Preparation of standard stock solution: An accurately weighed quantity of Desvenlafaxine succinate (50 mg) and Clonazepam (2.5 mg) were transferred into a separate 50 ml volumetric flasks and dissolved and diluted to the mark with Mobile phase to obtain standard solutions having concentration of Desvenlafaxine succinate (1000μg/ml) and Clonazepam (50μg/ml). Standard Solution: The 100 percent mixed standard solution of Desvenlafaxine succinate and Clonazepam was prepared by transferring 5 ml of Desvenlafaxine succinate (1000 µg/ml) and 1 ml of Clonazepam (10 µg/ml) to the 50 ml volumetric flasks and made up to the mark with mobile phase to get 100 µg/ml of Desvenlafaxine succinate and 1µg/ml Clonazepam. Preparation of sample stock solution: 20 Tablet contents were weighed and triturate to fine powders. Accurately weighed and transferred 130.2 mg of powder equivalent to 50 mg Desvenlafaxine succinate and 0.5 mg Clonazepam into a 50 ml volumetric flask. To that 10 ml mobile phase is added to dissolve and then the volume is making up with mobile phase. Then it is filtered with 0.45 micron filter and it is sonicated. Sample Solution: From this stock solution 5 ml is pipette out and transferred to 50 ml volumetric flask and volume Name of equipment Software Company HPLC 02 EMPAVAR2 Shimadzu UV-Spectrophotometer SPECTRATREATS ELICO pH Meter N/A ADWA Weighing Balance Ax200 Shimadzu Sonicator 3.5L100H Shimadzu
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Mallikarjuna et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 527 is makeup to 50 ml to prepare 100μg/ml and 1μg/ml final mixed concentrations of Desvenlafaxine succinate and Clonazepam respectively. RESULTS AND DISCUSSION The developed method of analysis was validated as per the ICH for the parameters like system suitability, specificity, linearity, precision, accuracy, robustness and system suitability, limit of detection (LOD) and limit of quantitation (LOQ). System suitability: System suitability test was carried out on freshly prepared mixed standard solution of Desvenlafaxine succinate and Clonazepam. 20 µL of the standard solution was injected under optimized chromatographic conditions and retention time, theoretical plates, area and tailing factor parameters were studied to evaluate the suitability of system and results were presented in Table 3. Specificity: Specificity of the HPLC method was demonstrated by the separation of the analytes from other potential components such as impurities, degradants or excipients. A volume of 20 µl of working placebo sample solution was injected and the chromatogram was recorded. No peaks were found at retention time of 2.50 and 4.40 min. Hence, the proposed method was specific for Desvenlafaxine succinate and Clonazepam. The representative chromatogram of placebo was shown in Fig. 3. The specificity results were presented in Table 4&5. Linearity: Calibration curves for Desvenlafaxine succinate and Clonazepam were prepared individually. Aliquots of 0.6, 0.8, 1,1.2, 1.4 ml of sample stock solutions were transferred individually to the 10 ml of volumetric flasks and made up to the mark with mobile phase to get concentration of 60, 80, 100, 120, 140 µg/ ml Desvenlafaxine succinate for 0.6, 0.8, 1, 1.2, 1.4 µg/ml for Clonazepam respectively. An aliquot (20 µl) of each solution was injected under the operating chromatographic condition as described above and responses were recorded. Calibration curves were constructed by plotting the peak areas versus the concentration and the regression equations were calculated is shown in Fig.6&7 and results were presented in Table 6. Precision: To check the intra-day and inter-day variation of the method, standard concentration was subjected to the proposed HPLC method of analysis. The precision of the proposed method i.e. the intra and inter-day variations in the peak area of the drug solutions was calculated in terms of percent RSD. A statistical evaluation revealed that the relative standard deviation of drugs at different concentration levels for 6 injections was less than 2.0 . The results for intra-day and inter-day precision were presented in Table 6 and Table 7 respectively. Accuracy: The recovery studies were carried out for the accuracy parameter. The recovery was determined at three levels, viz. 85%, 105% and 125% of the selected. Concentrations and percentage recovery for Desvenlafaxine succinate and Clonazepam ranges from 98.97-100.28% indicate that developed method are accurate and reliable. Robustness: The Robustness was evaluated by the analysis of Desvenlafaxine succinate and Clonazepam under different experimental conditions such as making small changes in flow rate (±0.2 ml/min) and detection wavelength (±2 nm) . The results were presented in Table 8&9. Ruggedness: Three assay samples of drug product at 100 % of the working sample concentration were prepared and injected into the chromatographic system by different analysts. The value of %RSD was < 2, tailing factor and the theoretical plates found to be well within the system suitability limits. This indicates the ruggedness of the developed analytical method. The results were presented in Table 10. Limit of detection and limit of quantification: LOD and LOQ were determined by using the formula based on the standard deviation of the response and the slope. LOD and LOQ were calculated by using equations, LOD = 3.3 X /S and LOQ =10 X /S, The results were presented in Table 11. where, is the standard deviation, S is the slope of the calibration curve. The new analytical (RP-HPLC) method was established for simultaneous determination Desvenlafaxine succinate and Clonazepam, then optimized and applied on pharmaceutical dosage forms. Various mobile phase systems were prepared and used to provide an appropriate chromatographic separation, but the proposed mobile phase comprising of buffer, methanol and acetonitrile in the ratio 30:20:50 v/v/v having a better resolution and sensitivity. The detection was carried out by using UV detector at 222 nm. Among the several flow rates tested, the flow rate of 1.0 ml/min was found to be the best for Desvenlafaxine succinate and Clonazepam with respect to retention time and theoretical plates. The retention time was found to be 2.51 and 4.40 min, for Desvenlafaxine
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Mallikarjuna et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 528 succinate and Clonazepam respectively. The asymmetry factor or the tailing factor was found to be 1.30 and 1.09 respectively which indicates symmetrical nature of the peak. System suitability parameters such as retention time, tailing factor area and number of theoretical plates were calculated. The number of theoretical plates was found to be 3509 and 7333 which indicates efficient performance of the column. These parameters represent the specificity of the method. Specificity of the RP-HPLC method was validated by the separation of the analytes from other potential components such as impurities, degradants or excipients. A volume of 20 µl of working placebo sample solution was injected and the chromatogram was recorded. No peaks were found at retention time of 2.51and 4.40 min. Hence, the proposed method was specific for Desvenlafaxine succinate and Clonazepam. Linearity range was evaluated by the visual inspection of plot of peak area as a function of analyte concentration. From the linearity studies, the specified concentration range was determined. It was observed that Desvenlafaxine succinate and Clonazepam were linear in the range of 60-140 and 0.6-1.4μg/ml concentrations. The regression equation of Desvenlafaxine succinate and Clonazepam concentration over its peak area ratio was found to be Y= 437.4 X +932.8 and Y = 35.44 X + 85.22, where Y is the peak area ratio and X is the concentration (μg/ml). The linearity range of 60-140 and 0.6-1.4 μg/ml for Desvenlafaxine succinate and Clonazepam was found to obey linearity with the correlation coefficient of 0.998 and 0.997. The validation of the proposed method was verified by method precision. The method precision was conducted and the percent RSD for Desvenlafaxine succinate and Clonazepam is 1.39 and 0.51 (Intraday) and 0.35 and 0.58 (Interday). The validation of proposed method was verified by recovery studies. The percentage recovery range was found between 98.97- 100.26% and 99.06-100.28% for Desvenlafaxine succinate and Clonazepam. All parameters including flow rate, temperature, detection wavelength and sensitivity are maintained constant throughout the procedure. Thus the systemic suitability parameter has been qualified as per the requirements. Robustness studies were made by varying the chromatographic parameters like detector wavelength and flow rate are within a realistic range and the quantitative influence of the variables is determined. Ruggedness of the proposed method was determined by two different analysts. The mean, standard deviation and % RSD values were found to be less than 2 % which shows the ruggedness of the method. LOD values for Desvenlafaxine succinate and Clonazepam were found to be 0.24 µg/ml and 0.03 µg/ml respectively and LOQ values for Desvenlafaxine succinate and Clonazepam were found to be 0.72 µg/ml and 0.09 µg/ml respectively. The results obtained were satisfactory and good agreement as per the ICH guidelines. CONCLUSION The proposed RP-HPLC method for Desvenlafaxine succinate and Clonazepam is simple, precise, accurate, cost effective and less time consuming. The validation of the analysis proved that the method is reproducible and efficient for the determination of Desvenlafaxine as bulk drug and in pharmaceutical formulation without any interference from the excipients. Hence, the method can be easily and conveniently applied for routine analysis in quality control laboratories for the determination and can also be used for bioequivalence studies. ACKNOWLEDGEMENTS The authors thank to Mr. J. V. Rao managing director of Priyanka Industries, Bollaram, Hyderabad for providing necessary facilities to carry out this research work. They also thank Mr. Athaulla khan area bussiness manager of Zydus cadilla for providing Zyven-OD plus as gift sample and Mr. D. Anil, V. R. Koteswara Rao are involved in this work. Figure.3.Chromatogram of blank
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Mallikarjuna et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 529 Table: 3. Data of system suitability Injection No DES CLO Retention Time (min) Peak area Efficiency (Th.pl) Asymmetry Retention Time (min) Peak area Efficiency (Th.pl) Asymmetr y 1 2.51 2306.666 3735 1.375 4.397 213.54 7437 1.156 2 2.52 2310.546 3527 1.333 4.413 217.64 7928 1.125 3 2.52 2333.793 3775 1.375 4.410 211.633 7482 1.161 4 2.52 2327.288 3304 1.308 4.413 213.371 7094 1.088 5 2.50 2328.979 3260 1.308 4.397 213.643 7040 1.121 6 2.49 2333.935 3453 1.269 4.390 211.399 7019 1.121 Mean 2.51 2323.530 3509 1.33 4..40 211.540 7333 1.13 SD 0.0109 11.920 214.158 0.0418 0.010 2.239 0.027 0.027 %RSD 0.43 0.51 6.10 3.15 0.22 1.05 2.37 2.37 Figure.4.Chromatogram of sample Table: 4. Specificity data of sample Name Retention time (min) Area (mV. s) Efficiency (th.pl) Asymmetry Resolution Desvenlafaxine succinate 2.523 2332.682 3304 1.308 9.977Clonazepam 4.417 207.218 7505 1.156 Figure.5. Chromatogram of standard
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Mallikarjuna et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 530 Table: 5. Specificity data of standard Name Retention time (min) Area (mV. s) Efficiency (th.pl) Asymmetry Resolution Desvenlafaxine succinate 2.520 2336.440 3295 1.308 9.854 Figure.6. Calibration curve of Desvenlafaxine succinate Figure.7. Calibration curve for Clonazepam Table: 6. Data of linearity Table:7.Precision data for Des And Clo S.No Desvenlafaxine succinate Clonazepam Working conc. ( µg/ml) Peak area Working conc. ( µg/ml) Peak area 1 60 1345.991 0.6 117.759 2 80 1843.264 0.8 157.325 3 100 2232.682 1 195.218 4 120 2697.195 1.2 227.674 5 140 3106.06 1.4 259.795 Correlation Coefficient (R2 ) 0.998 0.997 Slope 5334 990 Intercept -479 7010 Injection No Desvenlafaxine succinate Clonazepam Retention time (min) Peak area Retention time (min) Peak area 1 2.51 2306.666 4.397 213.54 2 2.523 2310.546 4.413 217.64 3 2.523 2333.793 4.410 211.633 4 2.523 2327.288 4.413 213.371 5 2.507 2328.979 4.397 213.643 6 2.497 2323.53 4.390 211.399 Mean 2.51 2323.53 4.40 213.54 SD 0.0109 11.920 0.010 2.239 % RSD 0.43 0.51 0.22 1.05
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Mallikarjuna et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 531 Table: 8. Robustness data for flow rate variation Table: 9. Robustness data for effect of wavelength variation Table: 10. Ruggedness data for RTC and CPDG Sample area S.No Analyst -1 (set-1) Analyst -2 (set-2) Desvenlafaxine succinate Clonazepam Desvenlafaxine succinate Clonazepam 1 3260 7040 3304 7094 2 3304 7060 3234 7019 3 3267 7040 3306 7084 Mean 3277 7046.67 3281.33 7065.67 SD 23.643 11.547 41.004 40.723 % RSD 0.72 0.16 1.25 0.58 Table: 11. Data table of LOD & LOQ for Desvenlafaxine succinate and Clonazepam REFERENCES Babita K.S. Srivastava D.V. Seema S. Sudhir K., Selective and non-extractive spectrophotometric determination of cefdinir in formulations based on donor-acceptor complex formation, Quím. Nova, 33(7), 2010, 1471-1475. Hamrapurkar, P. Patil, P. Phale, M., Gandhi, M. Pawar, S., A developed and validated stability-indicating reverse- phase high performance liquid chromatographic method for determination of cefdinir in the presence of its degradation products as per International Conference on Harmonization guidelines, Pharmaceutical Methods, 2(1), 2011, 15-20. Hashem, H., Gouda, A. A., Hassan, W., Development and Validation of a rapid Stability Indicating chromatographic Determination of Cefdinir in Bulk Powder and Dosage Form Using Monolithic Stationary Phase, Journal of Liquid Chromatography & Related Technologies, 35(12), 2012, 1638-1648. ICH, Harmonized Tripartite Guideline. Validation of Analytical Procedure: Methodology (Q2B). International Conference on Harmonization, 1997. Jain, R. Radhapyari, K. Jadon, N., Electrochemical evaluation and determination of cefdinir in dosage form and biological fluid at mercury electrode. Journal of the Electrochemical Society, 154(11), 2007, 199-204. Mashelkar, U. C. Renapurkar, S. D., A LC-MS Compatible Stability Indicating HPLC Assay Method for Cefdinir. Int. J. Chem. Tech. Res, 2, 2010, 114-121. Narala, S. R. Saraswathi, K., RP-HPLC methods for the determination of cephalosporins (Cefditoren Pivoxil and Cefdinir) in pharmaceutical dosage forms. J Pharm Sci Res, 3, 2011, 1002-1004. Flow rate (ml/min) Desvenlafaxine succinate Clonazepam RT (min) Efficiency(t h.pl) Asymmetry RT (min) Efficiency(t h.pl) Asymmetry 0.8 3.13 3568 1.258 5.44 7631 1.051 1 2.51 3735 1.375 4.39 7437 1.156 1.2 2.09 3040 1.036 3.66 5462 1.00 Wave length (nm) Desvenlafaxine succinate Clonazepam RT (min) Efficiency (th.pl) Asymmetry RT (min) Efficiency (th.pl) Asymmetry Low 220 2.513 3277 1.222 4.38 6624 1.19 Actual 222 2.51 3735 1.375 4.39 7437 1.156 High 224 2.517 3286 1.138 4.38 6624 1.057 DRUG LOD (µg/ml) LOQ (µg/ml) Desvenlafaxine succinate 0.24 µg/ml 0.72 µg/ml Clonazepam 0.03 µg/ml 0.09 µg/ml
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Mallikarjuna et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 532 Okamoto Y, Itoh K, Namiki Y, Matsushita J, Fujioka M, Yasuda T., Method development for the determination of cefdinir and its related substances by high-Performance Liquid Chromatography J Pharm Biomed Anal., 14(6), 1996, 739-48. Rao KV, Rani A, Reddy AV, Bharathi CH, Dandala R, Naidu A., Isolation, structural elucidation and characterization of impurities in Cefdinir, J Pharm Biomed Anal., 43(4), 2007, 1476-1482.
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Goswami et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 533 PLANT SEEDS USED FOR ANTHELMINTIC ACTIVITY:A REVIEW Shambaditya Goswami*, Sanjeev Nishad, Mayank Rai, Sarvesh Madhesiya, Ankita Malviya, Pawan Pandey, Vikram Gautam, Sujeet Yadav Dapartment of Pharmacy, ITM, GIDA, Gorakhpur, India *Corresponding author: E-Mail: shambampharma@gmail.com ABSTRACT Parasitic infections are most widespread among humans. The present study deals with a review on plant seeds which have been scientifically evaluated for in-vitro anthelmintic activity for supporting their folklore use as traditional medicine. There are 30 plant seeds so far have been reported for the in-vitro anthelmintic activity. The families of the plants have also been reported in which Fabaceae was found as most common plant family. Combretaceae, Meliaceae, Cucurbitaceae, Myrsinaceae and Anacardiaceae families were also found possessing anthelmintic activity. The current review also clears the picture about choosing the parasites which have been used for the activity. Ascaris Sp (35%), such as Ascaris lumbricoides, Ascaris suum etc, Ascardia Galli (29%), Tapeworm (18%), Earthworm i.e. Pheritima posthuma (18%), Nippostrongyllus braziliensis, Haemonchus contours, Roundworms, Trichostrongylus colubrifromis etc have been reported as different targeted parasites for the evaluation of anthelmintic activity. This review on the plant seeds which have been used in antelmintic activity will help in ethnopharmacology by evaluating possible molecular level mechanism and isolation of active constituents by chromatography. Key words: In-vitro Anthelmintic Activity, Plant Seeds, Fabaceae, Parasites, Ascaris Sp INTRODUCTION Helminthiasis is one of the most important animal diseases worldwide, indicting heavy production losses in grazing animals. The disease is especially prevalent in developing countries in association with poor management practices and inadequate control measures (Akhtar, 2000). Parasitic diseases cause severe morbidity; including lymphatic filariasis (a cause of elephantiasis), onchocerciasis (river blindness), and schistosomiasis (Pandey Awanish, 2011).The development of herbal products depended upon the local botanical flora with the result that different remedies tended to develop in different parts of the world. Nevertheless, in some instances, the same or related plants were used over wide geographical areas (David I, 1990). India, Pakistan and China are the greatest users of medicinal plants; their traditions of plant remedies date back at least 7000 years. Between them, they now account for two fifths of humanity (in other words, more than 2 billion people), the bulk of whom rely heavily on medicinal plants (Zafar Iqbal, 2005). From the ancient times, indigenous drugs have been used in the Indian medicinal system to treat different ailments and to provide therapeutic benefits. Our traditional system of medicine has made use of the different parts of plants in different types of diseases, including anthelmintic, anti-inflammatory and antimicrobial activities. Kavirajes and Hakims are still using several medicinal plants to treat helminthiasis (Chopra, 2009). For the treatment of Helminthiasis, an ideal anthelmintic drugs should be administered which should have high efficacy, broad spectrum activity. Along with it should be free from toxic effects and cost effective, but, unfortunately no synthetic drugs have reached the specification for an ideal drug. That is the reason for depending on the herbal and traditional plants for the treatment of Helminthiasis by the large population. In the current study the evaluation of anthelmintic activity of plant seeds have been reported. The reviewed data of anthelmintic activity of the plant seeds, their family, targeted parasites and reference no has been tabulated in Table No1. RESULTS AND DISCUSSION After reviewing the above data it has been found that there are 30 plant seeds so far have been reported for the in-vitro anthelmintic activity. The above tabulated data also reports about the families of plant seeds among them Fabaceae was found as most common plant family used as anthelmintic herb. Combretaceae, Meliaceae, Cucurbitaceae, Myrsinaceae and Anacardiaceae families were also found possessing anthelmintic activity (Fig. no. 1). The choosing of parasites for different model is important for anthelmintic activity. The current review also clears the picture about choosing the parasites which have been used for the activity. Ascaris Sp (35%), such as Ascaris lumbricoides, Ascaris suum etc, Ascardia Galli (29%), Tapeworm (18%), Earthworm i.e. Pheritima Posthuma (18%), Nippostrongyllus braziliensis, Haemonchus contours, Roundworms, Trichostrongylus Colubrifromis etc have been reported as different targeted parasites for the evaluation of anthelmintic activity.
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Goswami et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 534 Table No 1: List of Plant Seeds which have been evaluated for Anthelmintic Activity: Name of plant Family Model/Parasites Reference Acacia albida Fabaceae Worm infestation on Sheep, Goat Nwude, 1980 Anogeissus leiocarpus Combretaceae Nippostrongyllus braziliensis Ibrahim, 1984 Areca catechu Arecaceae Tape worms Pheritima Posthuma Nethravathi, 2010 Azadirachta indica Meliaceae Haemonchus contours Hördegen, 2003 Bixa orellana Bixaceae Ascaridia galli,Ascaris suum Fernandez, 2011 Butea frondosa Fabaceae Anthelmintic Ascaridia galli Kalesaraj, 1962; Joshi, 1970; Joshi, 1970; Narayana, 1976 Butea monosperma Fabaceae GI Nematodes Iqbal, 2006 Caesalpina crista Caesalpiniaceae Toxocara vitulorum, Ascaridia galli, Haemonchus contortus Akhtar, 1985; Javed, 1994; Sharma, 1971 Carica papaya Caricaceae Ascaris lumbricoides Ascaridia galli Dhar, 1965 Carum copticum Apiaceae Ascaris lumbricoides Kalesaraj, 1974 Cassia alata Fabaceae Ascaridia galli Fernandez, 1991 Cucurbita maxima Cucurbitaceae Pheritima posthuma Pandey, 2011 Cucurbita moschata Cucurbitaceae Cestodes, Xiao, 1986 Diospyros scabra Ebenaceae Lungworms ITDG and IIRR, 1996 Embelica ribes Myrsinaceae Tapeworms Qureshi, 1979 Hyoscyamus niger Solanaceae Mixed Nematode Infection Akhtar, 1990 Lagenaria siceraria Cucurbitaceae Cestodes, Moniezia Akhtar, 1987 Lantana camara Verbenaceae Anthelmintic activity Avadhoot, 1980 Lansium domensticum Meliaceae Ascaris suum Fernandez, 1991 Leucaena lucocephala Fabaceae Ascaridia galli Fernandez, 1991 Mangifera indica Anacardiaceae Ascaris lumbricoides Xiao, 1986 Melia azedarach Meliaceae Trichostrongylus colubriformis Hördegen, 2003 Moringa olefera Moringaceae Ascaris suum Fernandez, 1991 Nigella sativa Ranunculaceae Antifasciolic, Mon-iezia in sheep Kailani, 1995; Akhtar, 1991 Peganum harmala Nitrariaceae Cestode infection Akhtar, 1991 Pisitacia integrrima Anacardiaceae Earthworms Mishra, 1979 Quisqualis indica Combretaceae Ascaris spp Xiao, 1986 Randia dumetorum Rubiaceae Tapeworms, earthworms Mishra, 1979 Rapanea Myrsinaceae Roundworms ITDG and IIRR, 1996 Vernonia anthelmintica Asteraceae Trichostrongylus colubrifromis, GI nematodes Hördegen, 2003 Xylopia aethiopica Annonaceae Nippostrongylus braziliensis Mohammed, 2005 Fig. no. 1: Family of plant seeds most found to have anthelmintic activity Fig. no. 2: Percentage of Parasites chosen for evaluation of Anthelmintic Activity CONCLUSION Intestinal parasitosis lie among the many health problems observed in economically disadvantaged populations of developing countries. Those infections reveal clear social and economical determinants, with high prevalence in regions with deficiency in sanitation, potable water supplies, education and adequate dwelling conditions (Stephenson, 2000). This review on the plant seeds which have been used in anthelmintic activity will
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Goswami et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 535 help in ethnopharmacology of these plants, by evaluating possible molecular level mechanism and isolation of active constituents by chromatography. The data collected will also increase the strength of backbone of new drug development from the traditional medicine. REFERENCE Akhtar M.S and I. Ahmad, Anthelmintic and Phytochemistryical studies on Hyoscyamus nigerLinn. (Ajwain Khurasani) seeds and Morringa oleifera,Lam (Sohanjna) roots, J. Pharm. Punjab Univ. Lahore, Pakistan, 3, 1990, 75–81. Akhtar M.S and I. Ahmad, Evaluation of antinematodal efficacy of tetrahydrohaunine in goats. Vet. Arhiv., 6, 1991, 307–311. Akhtar, M.S and S Riffat, Evaluation of anticestodal activity of Lagenaria siceraria (Kaddoo) seeds in sheep, Pakistan Vet J, 7, 1987, 139–141. Akhtar M.S, I. Javed, C. S. Hayat, and B. H. Shah, 1985. Efficacy and safety of Caesalpinia cristaLinn. seeds and its extracts in water and methanol against natural Neoascaris vituloruminfection in buffalo calves. Pakistan Vet. J, 5, 1985, 192–196. Akhtar M.S, Javed, I, Efficacy of Nigella sativa Linn. Seeds against Moniezia infection in sheep. Indian Vet. J. 68, 1991, 726±729. Avadhoot Y, V. K. Dixit, and K. C. Varma, Anthelmintic activity of essential oil of seeds of Lantana camaravar. aculeata L. Indian Drugs Pharm. Indust,15, 1980, 19–20. Chopra RN, The medical and economic aspect of Indian indigenous Drugs, 6-7, 2009, 503, 510, 675 David I Grove, A history of human Helminthology, C.A.B. International, Wallingford, UK, 1, 1990, 75. Dhar, R.N, L.C. Garg and R.D. Pathak, Anthelmintic activity of Carica papya seeds, Indian J. Pharm, 27, 1965, 335–336 Fernandez T.J, Local plantshaving anthelmintic activity, Asean J. Sci. Technol. Develop, 8, 1991, 115–19. Hördegen, P., H. Hertzberg, J. Heilmann, W. Langhans and V. Maurer, The anthelmintic efficacy of five plant productsagainst gastrointestinal trichostrongylids in artificially infected lambs, Vet. Parasitol, 117, 2003, 51– 60. Ibrahim, M.A, N Nwude, RA Ogunsusi, and YO Aliu, Screening West African plants for anthelmintic activity. ILCA Bull., 17, 1984, 19–23 Iqbal, Z., M. Lateef, A. Jabbar, M.N. Ghayur and A.H. Gilani, In vivoanthelmintic activity of Butea monosperma against Trichostrongylid nematodes in sheep. Fitoterapia, 77, 2006, 137–140. ITDG and IIRR, 1996. Ethnoveterinary medicine in Kenya: A field manual of traditional animal health care practices. Nairobi, Kenya,1996, 136–137. Javed, I., M.S. Akhtar, Z.U. Rahman, T.Khaliq and M. Ahmad, Comparative anthelmintic efficacy and safety of Caesalpinia cristaseed and piperazine adipate in chickens with artificially induced Ascaridia galliinfection. Acta Vet. Hungarica, 42, 1994, 103–109. Joshi, H.C. Some pharmacological studies with the seeds of Butea fiondosa, Orissa Vet J, 5, 1970, 5–8. Kailani S.U.R., M.S. Akhtar and M. Ashraf, Antifasciolic efficacy of indigenous plant drugs: Kalonji, Shahterah and Karanjwa in buffaloes, Pakistan J. Pharm. Sci.,8, 1995, 17–27 Kalesaraj R, Screening of some indigenous plants for anthelmintic action against human Ascaris lumbricoides. Indian J. Physiol.Pharmacol, 18, 1974, 129–131. Kalesaraj, R. and P.A. Kurup, Anthelmintic activity, toxicity and other pharmacological properties of palasonin, the active principle of Butea frondosa seeds and its piperazine salt, Indian J. Med. Res., 56, 1968, 1818. Kalesaraj, R. and P.A. Kurup, Investigation on the anthelmintic principle of Butea frondosaseeds, Indian J. Pharm, 24, 1962, 63–65. Lal, J, S. Chandra and M. Sabir, Modified method for isolation of Palasonin-the anthelmintic principle of Butea frondosa seeds. Indian J. Pharmacol. Sci., 40, 1978, 97-98. Lal, J., S. Chandra, V.R. Prakash and M. Sabir, In vitro anthelmintic action of some indigenous medicinal plants on Ascaridia galli worms, Indian J. Physiol. Pharmacol, 20, 1976, 64-68. M.S. Akhtar, Zafar Iqbal, M.N. Khan, Muhammad Lateef , Anthelmintic activity of medicinal plants with particular reference to their use in animals in the Indo- Pakistan subcontinent, Small Ruminant Research, 38, 2000, 99-107 Mishra, S.H., R.S. Gaud, R.A. Sharma and S.C. Chaturvedi, Anthelmintic activity of some essential oils. Indian Perfumer, 23, 1979, 208–209. Mohammed MS, Mohammed M, Yusuf OA, Joseph OA,A nthelmintic activity of the crude methanol extracts of Xylo
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Goswami et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 536 pia aethiopica against Nippostrongylus brasiliensis in rats, Veterinarski Arhiv, 75, 2005, 487‐495. Nadkarni, A.K, Indian Materia Medica, Vol. I, 3rd Ed. Popular Book Depot, Bombay, India, 1954, 142–143. Narayana, K, D.R.L. Setty, H.S. Rao and P.N. Kamalapur, A clinical and experimental study of "Wopell" in canine cestodiasis, Mysore J. Agri. Sci., 10, 1976, 98–100. Nethravathi R, Pallavi SS, Shruti MN, Nandini HR, Prashith Kekuda TR, Mukunda S, Antibacterial and anthelmintic activity of Chogaru (Betel Nut Concentrate), Research and Review in Biomedicine and Biotechnology, 1 (1), 2010, 20-23. Nwude N and MA Ibrahim, Plants used in traditional veterinary medical practice in Nigeria, J Vet. Pharmacol and Therap, 3, 1980, 261–73 Pandey Awanish, Goswami Shambaditya, Tripathi Poonam, Singh Ajay Pratap, An in-vitro evaluation of anthelmintic activity of zingiber zerumbet rhizomes and cucurbita maxima seeds on Pheritima posthuma model: a comparative study, Journal of Pharmacy and Bioallied Science, 3(2), 2011, 317. Qureshi M.A. and M Sabir, Preliminary study on anthelmintic efficacy of Embellia seeds (Babarung) against tapeworms of poultry, Pakistan J. Sci, 31, 1979, 218–220. Sharma, L.D., H.S. Bagha and P.S. Srivastava, In vitro anthelmintic screening of indigenous medicinal plants against Haemonchus contortus (Rudolphi, 1803) Cobbold, 1898 of sheep and goats. Indian J. Anim. Res., 5, 1971, 33–38. Shilaskar, D.V. and G.C. Parashar, Evalution of indigenous anthehnintics: In vitro screening of some indigenous plants for their anthelmintic activity against Ascaridia galli, Indian J. Indg. Med, 6, 1989, 49–53. Stephenson L.S, Latham M.C & Ottesen E.A, Malnutrition and parasitichelminth infections, Parasitology, 121, 2000, 223-238. Xiao, P.G. and F.S. Lin, Traditional antiparasitic drugs in China. Parasitol. Today, 2, 1986, 353–355 Zafar Iqbal, Abdul Jabbar, Muhammad Shoaib Akhtar, Ghulam Muhammad and Muhammad Lateef, Possible Role of Ethnoveterinary Medicine in Poverty Reduction in Pakistan: Use of Botanical Anthelmintics as an Example, J. Agri. Soc. Sci., 1(2), 2005, 187-195.
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Suresh and Devendra Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 537 DEVELOPMENT AND VALIDATION OF PEMETREXED BY RP-HPLC METHOD IN BULK DRUG AND PHARMACEUTICAL DOSAGE FORMS Suresh Kumar Agrawal1 *, Devendra Singh Rathore2 1. Department of Pharmaceutical Sciences, Suresh GyanVihar University, Jaipur, (R.J.) - 302025, India 2. Rajasthan Pharmacy College, Jaipur, (R.J.), India *Corresponding author: E-mail: suresh_pharma2006@yahoo.co.in, Ph. No- 09920171860 ABSTRACT A new, simple, specific, rapid, accurate and precise RP-HPLC method was developed and validated for the estimation of Pemetrexed in bulk and pharmaceutical formulations. Pemetrexed was chromatographed on a Inertsil C18 column (250×4.6 mm, 5µm) in a mobile phase consisting of 35:65 v/v ratio of acetonitrile and buffer (2.76 gm of sodium dihydrogen orthophosphate and 2.0 gmof sulphonic acid sodium salt in 1L of mili-Qwater, adjust pH to 3.0 by Ortho-phosphoric acid and absorbance was measured at 254 nm. Column oven temperature maintained at 25o C, injection volume was 20µL and 10 min runtime was selected for analysis. Method validation was performed as per ICH guidelines, with respect to linearity, accuracy, precision, ruggedness and robustness. Linearity was evaluated and found to be linear in the range of 12.5 µg/mLto 37.5µg/mL, correlation coefficient is 1.0. Recovery studies also checked and found to be within the limit. This method has applicable for regular analysis. Ker words: RP-HPLC, Pemetrexed, Pemetrexed disodium, Validation, Assay 1.0 INTRODUCTION Pemetrexed is a folate antimetabolites and class of chemotherapy drug, used for the treatment of pleural mesothelioma and non-small cell lung cancer. It is used as a single agent or in combination with other chemotherapeutic agents for the treatment of other types of cancer such as breast cancer, bladder cancer, colorectal carcinoma and cervical cancer. The dosage of pemetrexed is depending on a number of factors, including body height, weight and response to this medication. Pemetrexed is available in injection dosage form. It is injected slowly (infused) into a vein (Nandha, 2010; Das, 2011; Reddy, 2010). The serious side effects are pale skin, easy bruising or bleeding, unusual weakness, fever, chills, body aches, chest pain, trouble breathing and swelling. Pemetrexed is a suitable third-line treatment option with good efficacy and tolerable toxicity profile for Non-small Cell Lung Cancer (NSCLC) (Ahmed, 2009; Chowdhury, 2012). Figure-1 represents the chemical structure of Pemetrexed. IUPAC name of the drug is L-Glutamic acid, N-[4-[2-(2-amino-4,7-dihydro-4-oxo-1Hpyrrolo[2,3- d]pyrimidin-5-yl)ethyl]benzoyl]-, disodium salt, heptahydrate 2.0 MATERIALS AND METHODS HPLC grade acetonitrile, Octane sulphonic acid sodium salt, and Sodium dihydrogen orthophosphate purchased from RFCL Ltd and, AR grade orthophosphoric acid,and other reagents were procured from S.D. Fine chemicals Ltd. High pure standards and all market samples were used for this study. GL Science makes Inertsil column was used for this product. Shimadzu LC 2010A HT system was used for chromatographic analysis and equipment operated with Class VP software. 2.1. Standard Preparation (25 ppm): Accurately weigh about 70 mg of Pemetrexed Disodium (equivalent to about 50 mg of Pemetrexed) in 100 ml volumetric flask. Add 70 ml of diluents dissolve and diluted to volume with same and mix well. Dilute 5 ml resulting solution to 100 ml with diluent. 2.2. Sample Preparation: Prepared the all dosage forms equivalent to 100ppm of pemetrexed with diluent. 2.3. System suitability: The USP tailing factor for standard peak should be not more than 2.0; the % relative standard deviation should be not more than 2.0% and USP theoretical plates for standard solution should be not less than 2000. 3.0 RESULTS AND DISCUSTIONS The method was validated according to the ICH guidelines for the validation of analytical procedures. The parameters which were used to validate the method of analysis were linearity range, limit of detection (LOD), limit of quantification (LOQ), specificity, accuracy, precision and ruggedness and robustness. 3.1 Linearity: The linearity of the detector response is established by plotting a graph concentration Vs Area. Linearity was determined by injecting different concentrationof sample solutions (12.5 – 37.5 µg/mL). The calibration curve showed good linearity in the range
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Suresh and Devendra Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 538 of 12.5 – 37.5 µg/mL, with correlation co-efficient (R2) of 1.0 (Figure 2). 3.2 Limit of detection (LOD) and Limit of quantitation (LOQ): The detection limit is determined by the analysis of samples with the known concentrations of analyte and by establishing the minimum level at which the analyte can be reliably detected. The quantitation limit is determined by the analysis of sample of known concentration of analyte and by establishing the minimum level at which the analyte can be quantified with acceptable accuracy and precision. The LOD and LOQ were calculated as follows: LOD = 3.3 (σ /S) and LOQ = 10 (σ/S); where S: Slope of the calibration curve, σ: Average standarddeviation of the response. The minimum concentration level at which the analyte can be reliably detected (LOD) and quantified (LOQ) results showed in table-2. 3.3 Precision: Precision was measured in term of system precision and method precision. For system precision, standard solution (25 µg/mL )and sample solution (100 µg/mL) was injected to six replicates injections to check %RSD (relative standard deviation) and for method precision six time samples were prepared and injected to six replicates injections to check %RSD. The results of system precision (% RSD = 0.171), method precision (% RSD = 0.298) are found within the prescribed limit of ICH guidelines (% RSD < 1%, and % RSD < 2 % respectively in case of system precision and method precision). 3.4 Accuracy: Accuracy was determined based on the recovery (percentage) of known amounts of standard pemetrexed in assay sample. This was performed by analyzing pemetrexed at three concentration levels (20, 25 and 30 µg/mL). Samples were prepared in triplicate. The accuracy of the assay was determined by comparing the found concentration with the added concentration. 3.5 Method Robustness Robustness of method was investigated by varying the chromatographic conditions such as change of flow rate (± 10%), change in column temperature (± 2 0 C), and pH of buffer in mobile phase (± 5 %). Robustness of the developed method was indicated by the overall %RSD between the data at each variable condition. 3.6 Ruggedness: The ruggedness of the method was determined by carrying out the experiment on different instruments by using different columns of similar types and on different days. Ruggedness Matrix-1: day-1, Instrument-1, and Column-1and Ruggedness Matrix-2: day-2, Instrument-2, and Column-2. 3.7 Stability in analytical solution: To ensure the reliability of the results in relation to handling and storage standard solutions were performed at 4, 8, 12 hrstime periods at room temperature.A solution containing 25 µg mL−1 was analyzed. After storage at room temperature for 12 h, more than 98% of the pemetrexed remained unchanged, on the basis of comparison of peak areas with those obtained from a freshly prepared solution of pemetrexed (25 µg mL−1). This suggests pemetrexed standard solution was stable for at least 12 hrs when stored at room temperature. Tabel-1 Chromatographic conditions Buffer 2.76 gm of sodium dihydrogen orthophosphate and 2.0 gm of sulphonic acid sodium salt in 1000 ml of water, adjust pH to 3.0 by orthophosphoric acid. Mobile Phase buffer and Acetonitrile in the ratio 65:35v/v Diluent Mobile phase Flow rate 0.6 mL per minute Wavelength 254 nm Inj. Volume 20 µL Run time 10 minutes Tabel-2: Calibration curve and regression equation parameters for pemetrexed Parameter Concentration of Pemetrexed (µg/mL) LOD in µg/mL 0.47 LOQ in µg/mL 1.42
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Suresh and Devendra Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 539 Tabel-3: LOD and LOQ of pemetrexed Parametres Optimized Condition Linearity range (µg mL-1 ) 12.5 – 37.5 Regression equation (Y = mX + C) Slope (m) 69476 Intercept © 1710 Correlation Coeffient (r2 ) 1.0 Retentions Time (Mins) 4.17 Table-4: System suitability test parameter for the proposed method Parameters Optimized condition Retention Time (t) min 4.433 Theoretical plates 4461.67 Peak asymmetry 1.14 Table-5: Accuracy results of Pemetrexed Sr. No. 80 % Recovery Level 100% Recovery Level 120% Recovery Level 1 98.65 98.50 99.11 2 97.73 99.95 101.73 3 98.94 98.67 99.12 Mean 98.44 99.04 99.98 SD 0.634 0.790 1.509 %RSD 0.644 0.798 1.509 Table-6: Method Robustness results of pemetrexed Parameters % RSD Assay Change in pH of the mobile phase -10% 0.640 Change in pH of the mobile phase +10% 0.221 Change in flow rate to 0.54 mL/min. 1.123 Change in flow rate to 0.66 mL/min. 0.855 Column Temperature varied by – 2ºC 0.567 Column Temperature varied by + 2ºC 0.367 Table-7: Ruggedness results of Pemetrexed Sr. No. % Assay of matrix 1 % Assay of matrix 2 Assay -1 100.60 100.80 Assay -2 101.19 100.00 Assay -3 100.39 101.37 Assay -4 100.64 101.48 Assay -5 100.90 99.51 Assay -6 100.46 100.59 Mean 100.70 100.63 SD 0.300 0.768 %RSD 0.298 0.763
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Suresh and Devendra Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 540 y = 69476x + 1710.6 R² = 1 0 500000 1000000 1500000 2000000 2500000 3000000 3500000 4000000 0.00 10.00 20.00 30.00 40.00 50.00 60.00 Area Concentration µg/mL Linearity of Pemetrexed Std Solution-01 Minutes 0 1 2 3 4 5 6 7 8 9 10 mAU 0 50 100 150 200 mAU 0 50 100 150 200 4.433 Pemetrexed Detector A (254nm) Pemetrexed for Injection Std Solution-1 Retention Time Name Figure-1: Chemical structure of Pemetrexed Figure-2: Linearity graph of Pemetrexed Figure-3: Chromatograph of pemetrexed by RP-HPLC 4.0 CONCLUTION In this method isocratic elution method is selected for the analysis of pemetrexed because it gave better base line separation and peak width, which is suitable for the routine analysis of pemetrexed. The developed method was validated as per ICH guidelines (ICH, 1996) and its updated international convention (ICH, 2002). Stability testing forms an important part of the process of drug product development. The purpose of stability testing is to provide evidence on how the quality of a drug substance varies with time under the influence of a variety of environmental factors such as temperature, humidity
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Suresh and Devendra Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 541 and light, and enables recommendation of storage conditions, retest periods, and shelf life to be established.The assay of pemetrexed in stability test sample needs to be determined using stability indicating method, as recommended by the International Conference on Harmonization (ICH) guidelines. 5.0 ACKNOWLEDGEMENT The authors wish to thanks the management of Shree Danventary pharmaceutical analysis and research center (SDPARC), kim, Surat (Guj.) providing me samples and research facilities, Thanksful to Suresh GyanVihar University to providing necessary facility and to all my research colleagues for their support in the work. REFERENCES Nandha NP, Bandana SG, Vairale AS, Sherikar AV, Nalamothu V, Development and validation of stability indicating HPLC method for the estimation of 5- Fluorouracil and related substances in topical formulation. Int. J. Res. Pharm. Sci., 1 (2), 2010, 78-85 Das A, Jayanti M, Chakrabarty US, Sahoo BK, Dey G, Choudhury H, Development and validation of RP-HPLC method to determine NandrolonePhenylpropionate in different pharmaceutical formulations. ActaPoloniaePharmaceutica and Drug Research, 68 (2), 2011, 155-160. Reddy BP, Reddy KA and Reddy MS, Validation and stability indicating RP-HPLC method for the determination of tadalafil API in pharmaceutical formulations. Research In Pharmaceutical Biotechnology, 2(1), 2010, 001-006. Ahmed A, Gopinath B, Shetty SK, Shidhar BK, Development and validation of RP-HPLC Method for the determination of Adefovirdipivoxil in bulk and in pharmaceutical formulation. E. Journal of chemistry, 6(2), 2009, 469-474. Chowdhury SR, Maleque M, Hoque M, Development and Validation of a Simple RP-HPLC Method for Determination of Caffeine in Pharmaceutical Dosage Forms, Asian J. Pharm. Ana., 2(1), 2012, 01-04. Syed R, Sandhya B, Harika VC, Babu B, Pammi K, RP- HPLC Method development and validation for the analyisis of Sunitinib in pharmaceutical dosage forms. International Journal of Science Innovations and Discoveries, 1(3),2011, 441-450. Sreekanth N, Rao CH, Mukkanti K, RP-HPLC Method development and validation of Ropinirole hydrochloride in bulk and pharmaceutical dosage forms. International Journal of Pharmacy and Pharmaceutical Sciences, 1(1), 2009, 186-192. Santhosam DS, Bhavani VN,Suresh M, Evelopment and validation of RP-HPLC method for the simultaneous estimation of Emtricitabine and Tenofovir from bulk and dosage form. International Journal of Pharmaceutical Chemistry Research. 1(4), 2012, 1-5. Malipatil SM, Jahan K, Patil SK, Development & Validation of RP-HPLC Method for the Determination of Oseltamivir Phosphate in Bulk Drug & in Dosage. Indo- Global Journal of Pharmaceutical Sciences, 1(1), 2011, 57-62. Setti A, Phazna Devi TA, Srikanth S, Nallapeta SC, Rao JV, Method development and validation of paracetamol drug by RP-HPLC. J Med Allied Sci., 3(1), 2013, 08-14. Bujji NB, Raju RR, Simultaneous analysis of RP-HPLC method development and validation of Terbinafine and Bezafibrate drugs in pharmaceutical dosage form, Pharmacophore, 2(4), 2011,232-238. Ahmed M, babu SG, Shetty SK, Development and Validation of Amoxicillin by RP-HPLC Method in Bulk drug and Pharmaceutical dosage forms, International Journal of ChemTech Research, 3(3), 2011, 1037-1041 Garikapati DR, Bhimanadhuni CN, Srinivas C, Development and validation of RP-HPLC method for determination of Duloxetine hydrochloride in bulk and dosage form. International Current Pharmaceutical Journal,1(5), 2012, 98-102. Rao AL, Srinivas PV, RAO NS, A new validated RP- HPLC method for the estimation of MycophenolateMofetil in pure and tablet dosage form, JPRHC,2(3),266-269. Bandari S, Gannu R, Sudke SG, Rao YM, Shankar BP, Development and validation of a stability-indicating RP- HPLC method for analysis of Doxofylline in human serum. Application of the method to a pharmacokinetic study.ActaChromatographica, 19, 2007, 149-160. Sandhya M, Nasare M, Diwan PV, Development and Validation of RP-HPLC method for simultaneous estimation of Lamivudine and Efavirenz in the Pharmaceutical Dosage Form. Journal of Advanced Pharmacy Education & Research, 2(4), 2012, 232-238. Sahoo M, Syal P, Ingale S, Ingale K, Sindhe S, Sali M, Choudhari VP, Development and Validation of a RP- HPLC-PDA method for Simultaneous Determination of Lornoxicam and Thiocolchicoside in Pharmaceutical dosage form and its Application for Dissolution study.
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Suresh and Devendra Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 542 International Journal of Research in Pharmaceutical Sciences, 2(1), 2011, 1-7. (18) Chandrasekhar DS, Homdeo DP, Kamleshkumar SS, SamrutwarSwapnil, HomdeoDP. Development and validation of RP-HPLC method for NSAIDS in combined pharmaceutical tablet dosage form. Int. J. A. PS. BMS, 2(1), 2013, 026‐036.
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Amareswari et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 543 STABILITY INDICATING RP-HPLC METHOD FOR THE ESTIMATION OF CEFTAZIDIME PENTAHYDRATE AND TAZOBACTAM SODIUM IN BULK AND DOSAGE FORMS S. Amareswari1 , Nandakishore Agarwal1 , Md Aasif Siddique Ahmed Khan2 1. Department of Pharmaceutical Analysis, Nimra College of Pharmacy, Vijayawada, India. 2. Shadan Institute of Medical Sciences, Hyderabad, India. Corresponding author: amareswarisa@gmail.com, phone no: 9441678136. ABSTRACT A simple rapid, accurate, precise and reproducible validated reverse phase HPLC method was developed for the determination ofCeftazidimepentahydrate and Tazobactam sodium in bulk and pharmaceutical dosage forms. The quantification was carried out using Hypersil BDS C18(150 X 4.6mm, 5 µm) columnrun in isocratic way usingmobile phase comprising of phosphate buffer pH 3.0, acetonitrile, and tetrahydrofuran in the ratio of 60:30:10 with a detection wavelength of 205nm and injection volume of 20µL, with a flow rate of 1.0ml/min. The retention timesof the drugs were found to be 3.490min and 2.353min. The linearity ranges of the proposed method lies between 60-140mcg/ml and 7.5-17.5mcg/ml for Ceftazidimepentahydrate and Tazobactam sodium with correlation coefficient of r2 =0.999 for both. The assay of the proposed method was found to be 99.38% and 99.26%. The recovery studies were also carried out and mean % Recovery was and found to be 99.91% and 100.84%. The % RSD from reproducibility was found to be <2%.The proposed method was statistically evaluated and can be applied for routine quality control analysis of Ceftazidimepentahydrate and Tazobactam sodium in bulk and in Pharmaceutical dosage form. Key Words: Ceftazidime, Tazobactam sodium, RP-HPLC, Hypersil BDS, Validation, Forced degradation studies. INTRODUCTION CeftazidimePentahydrateis(Z)-(7R)-7-[2-(2- Aminothiazol-4-yl)-2-(1-carboxy-1 methylethoxyimino) acetamido]-3-(1-pyridiniomethyl)-3-cephem-4- carboxylate pentahydrate. The molecular weight is 636.7, molecular formula is C22H22N6O7S2·5H2O.It is a third generation Cephalosporin with enhanced antibacterial activity against gram negative organisms. Ceftazidime is bactericidal in action, exerting its effect on target cell wall proteins and causing inhibition of cell wall synthesis. It is official in IP and BP. It is used in the treatment of biliary tract infections, lower respiratory tract infections, bone and joint infections and urinary tract infections. Tazobactam is a potent and novel β-lactamases inhibitor which belongs to the class of penicillanic acid sulfones. The molecular formula is C10H11N4NaO5S, molecular weight is 322.3and the chemical name is (2S, 3S, 5R) - 3 - methyl -7- oxo - 3 - ( 1 H - 1, 2, 3 - triazol - 1 -ylmethyl) – 4 - thia-1-azabicyclo [3.2.0] heptanes – 2 - carboxylic acid4,4-dioxide. Tazobactam inhibits the action of bacterial beta lactamases. It broadens the spectrum of penicillin by making it effective against organisms that express beta-lactamases degrade piperacillin.Used to reduce the development of drug resistant bacteria. Literature review reveals that several methods are reported for these drugs alone or in combination with other drugs. For combination of these drugs Spectroscopic method, HPTLC method are reported, there is no single work done for this combination by using RP-HPLC. Hence an attempt has been made for the development of HPLC method for the combination of drugs. MATERIALS AND METHODS UV-3000 LABINDIA double beam with UV win 5software UV-VISIBLE spectrophotometer with 1cm matched quartz cells. Schimadzu HPLC equipped with SPD 20A UV-VIS detector and the column used was HYPERSIL BDS C18 (150*4.6mm, 5µ). The data acquisition was performed by using LC solutions software. Ceftazidime and Tazobactam pure sample was taken as a gift sample from local labs and dosage form “Combitaz” marketed by LUPIN LABS was purchased from local pharmacy. Other chemicals all are of HPLC grade. Preparation of mobile phase: A suitable quantity of degassed mixture of pH 3.0 phosphate buffer, Acetonitrile, Tetra hydro furan in the ratio of 60:30:10 was prepared and filtered through 0.45µ filter under vacuum filtration. Preparation of standard stock solution: About 100mg of Ceftazidime and 12.5mg of Tazobactam sodium were weighed and taken into 100ml, 50ml volumetric flasks. To each flask 25ml of diluents were added and sonicated for
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Amareswari et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 544 15min to dissolve the drugs then made up to required volume with the diluents, to get a concentration of 1mg/ml solutions. From this solution 10ml was taken into a 100ml flask and made up to final volume with diluents to get a concentration of 100ppm filtered through 0.45µ filter under vacuum filtration. From this stock solution further dilutions were made for the validation of the method developed. Preparation of the sample solution: The powder equivalent to 100mg of Ceftazidime and 12.5mg of Tazobactam sodium were weighed and taken into a 100ml, 50ml volumetric flask. To this 25ml of diluents was added and sonicated for 15min to dissolve the drugs then made up the volume to required volume with the diluents. From this solution 10ml was taken into a 100ml flask and made upto final volume with diluents to get a concentration of 100ppm filtered through 0.45µ filter under vacuum filtration. From this stock solution further dilutions were made by taking the two drugs in the ratio of 8:1 for the validation of the method developed. NANDA .R.K et al.., 2012. Method development Method optimization: The chromatographic separation was performed using Hypersil BDS C18 (150×4.6mm, 5µm) column. For selection of mobile phase, various mobile phase compositions were observedfor efficient elution and good resolution.The mobile phase consisting of pH 3.0 phosphate buffer, acetonitrile and Tetra hydro furan in the ratio of 60:30:10 was found to be the optimum composition for efficient elution of analyte. The mobile phase was injected to the column at a flow rate of 1.0 ml/min for 8min. The column temperature was maintained at 35 ± 10 C. The analyte was monitored at 205 nm using UV-detector. The retention time of the drugs was found to be 3.490 and 2.353min. Mobile phase was used as diluent during the standard and test samples preparation.The optimized chromatographic conditions are mentioned in Table-1 and chromatogram for standard was shown in the figure no:1. RESULTS Method Validation: Specificity: Specificity is the ability of analytical method to measure accurately and specifically the analyte in the presence of components that may be expected to be present in the sample. The specificity of method was determined by spiking possible impurities at specific level to standard drug solution (100ppm). The diluent and placebo solutions were also injected to observe any interference with the drug peak. The results are tabulated in the table no-2 and the chromatogram was shown in the figure no- 2. Linearity: Linearity is the ability of the method to produce results that is directly proportional to the concentration of the analyte in samples with given range. The linearity of Ceftazidime was in the concentration range of 60-140%, for Tazobactam sodium 7.5-17.5%. From the linearity studies calibration curve was plotted and concentrations were subjected to least square regression analysis to calculate regression equation. The regression coefficient was found to be 0.999 and shows good linearity for both the drugs. The results are tabulated in the table no-3 and the chromatogram was shown in the figure no-.3,4. Precision: Precision is the degree of closeness of agreement among individual test results when the method is applied to multiple sampling of a homogeneous sample. Study was carried out by injecting six replicates of the same sample preparations at a concentration of 100ppm/ml. The results are tabulated in the table no-4 and the chromatogram was shown in the figure no-10. Accuracy: Accuracy is the closeness of results obtained by a method to the true value. It is the measure of exactness of the method. Accuracy of the method was evaluated by standard addition method. Recovery of the method was determined by spiking an amount of the pure drug (80%,100% ,120%) at three different concentration levels in its solution has been added to the pre analyzed working standard solution of the drug. The results are tabulated in the table no:4. LOD & LOQ: LOD is the lowest concentration of analyte in a sample that can be detected but not quantified under experimental conditions. The LOD values were determined by the formulae LOD=3.3σ/s (where σ is the standard deviation of the responses and s is the mean of the slopes of the calibration curves). LOQ is the lowest concentration of analyte in a sample that can be determined with acceptable precision and accuracy under experimental conditions. It is a parameter of the quantitative determination of compounds in the mixtures. The LOQ values were determined by the formulae LOD=10σ/s. The results are tabulated in the table no: 4 FORCED DEGRADATION OF CEFTAZIDIME AND TAZOBACTAM SODIUM Acid and Base degradation: Acid/base degradation was determined by taking 5ml of stock solution in 10ml volumetric flask and to this 2ml of 0.1N HCl/NaoH was
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Amareswari et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 545 added and sonicate for 5min, kept aside for 12hrs at room temperature. After 12hrs the solution was neutralized with 2ml of 0.1N HCL/NaoH then diluted with diluents to get a concentration of 10µg/ml solution. Oxidative degradation: Oxidative degradation was determined by taking 5ml of stock solution in 10ml volumetric flask and diluted up to the mark with 5% H2O2 and kept aside for 12hrs. After 12hrs the solution was diluted with diluents to get a concentration of 10µg/ml solution. Thermal degradation: Sample powder equivalent to 100mg of Ceftazidime and 12.5mg of Tazobactam was taken and kept in a controlled temperature oven at 800 c for 12hrs. After 12hrs the powder was diluted with diluents to get a concentration of10µg/ml solution. Photolytic degradation: The Ceftazidime and Tazobactam powder and solutions of both were prepared and exposed to light to determine the irradiation of light on the stability of solution and powder form of drugs. Approximately 100mg of drug powder and 1mg/ml solution were spread on a glass dish in a layer that was less than 2mm thickness and were placed in a light cabinet and exposed to UV light for 12hrs. After 12hrs the samples are removed and diluted with diluents to get a concentration of10µg/ml solution and then injected. The summary of results is tabulated in table no: 5and figures are shown in figure no: 5,6,7,8,9. Disscussion: Several trials has made until getting good peak resolution, acceptable plate count and tailing factor. Method was optimized and the retention times of Ceftazidime and Tazobactam was reported as 3.480 & 2.353. Specificity: The Chromatograms of Standard and Sample are identical with nearly same Retention time. There is no interference with blank and placebo to the drugs. Hence the proposed method was found to be specific. Linearity: From the Linearity data it was observed that the method was showing linearity in the concentration range of 60-140μg/ml for Ceftazidime and 7.5-17.5μg/ml for Tazobactam. Correlation coefficient was found to be 0.999 for both the compounds. Accuracy: The recoveries of pure drug from the analyzed solution of formulation were 99.91 % for Ceftazidimepentahydrate and 100.84 % for Tazobactam sodium, which shows that the method was accurate. Precision: The %RSD for the sample chromatograms of method precision were found to be 0.39 & 0.81 for Ceftazidime and 0.23 &0.64 for Tazobactam. Hence it passes method precision. Robustness: All the system suitability parameters are within limits for variation in flow rate (±0.2 ml). Hence the allowable flow rate should be within 0.8 ml to 1.2 ml. All the system suitability parameters are within limits for variation (±2nm) in wavelength. Hence the allowable variation in wavelength is ± 2nm. Ruggedness: Comparison of both the results obtained for two different Analysts shows that the method was rugged for Analyst-Analyst variability. The %RSD for intermediate precision for Ceftazidime was found to be 0.45 & 0.002 and for Tazobactam was found to be 0.62 & 0.012. LOD & LOQ of Ceftazidime was found to be 0.03, 0.10and for Tazobactam was found to be 0.57, 1.72 respectively. All the system suitability parameters are within in the limits when the drugs are subjected to stress conditions like acid, base peroxide, thermal and photolysis.The results obtained were satisfactory and good agreement as per the ICH guidelines. Fig No 1 Structure of Ceftazidimepentahydrate Fig No 2 Structure of Tazobactam Sodium
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Amareswari et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 546 Table no: 1 Optimized chromatogram conditions for Ceftazidime and Tazobactam Column Hypersil BDS C18 (150*4.6mm,5µ) Mobile phase Phosphate Buffer pH 3.0:ACN:THF(60:35:05) Flow rate 1.0 ml/ min Wavelength 205 nm Injection volume 20 l Column temperature Ambient Run time 8 min Table No: 2 Specificity Data for Ceftazidime and Tazobactam Standard Injection Retention time Area Theoretical Plates Retention time Area Theoretical Plates 3.46 2910.42 3745 2.353 247.82 2254 3.50 3091.18 3817 2.357 285.2 2137 3.47 2943.91 3410 2.343 277.824 2210 Sample Injection 3.48 2984.39 3226 2.350 264.30 2125 3.48 2996.31 3781 2.352 262.89 2248 3.47 2989.06 3410 2.360 263.40 2143 Blank injection - - - - - - Table.No: 3 Linearity data for Ceftazidime and Tazobactam For Ceftazidime For Tazobactam Mcg/ml Area Rt Mcg/ml Area Rt 60 1847.225 3.470 7.5 163.031 2.337 80 2526.511 3.463 10 223.261 2.330 100 3072.796 3.490 12.5 276.082 2.353 120 3793.229 3.467 15 337.177 2.336 140 4341.245 3.453 17.5 394.041 2.323 Slope 31.27 Slope 23.037 Correlation coefficient 0.99913 Correlation Coefficient 0.999811 Intercept 11.17 Intercept 9.249 Table No: 4 Summary of validation parameters Parameter Ceftazidime Tazobactam Linearity 60-140µg/ml 7.5-17.5µg/ml Precision(% RSD) 0.39 0.81 0.23 0.64 Accuracy 99.91% 100.84% LOD & LOQ 0.03,0.10 0.57,1.72 Assay 99.38% 99.26%
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Amareswari et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 547 Table No: 5 Summary of Forced degradation data for Ceftazidime and Tazobactam: Stress Condition Time(hrs) Retention Time Time(hrs) Retention Time As such 12hrs 3.490 12hrs 2.353 Acid Hydrolysis (0.1 N, at RT) 12hrs 3.457 12hrs 2.337 Base Hydrolysis (0.1N at RT) 12hrs 3.477 12hrs 2.360 Oxidation (5% H2O2 at RT) 12hrs 3.453 12hrs 2.353 Photolysis(UV Light and sunlight) 12hrs 3.457 12hrs 2.337 Thermal (at 800 c) 12hrs 3.490 12hrs 2.353 Fig: 1 Chromatogram of standard drug Fig: 2 Chromatogram for specificity Fig: 3 linearity plot for Ceftazidime Fig: 4 linearity plot for Tazobactam Fig: 5 Acid Degradation Fig: 6 Base Degradation y = 31.27x - 31.17 r² = 0.99901 0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 0 50 100 150 Area Conc Linearity of Ceftazidime y = 23.03x - 9.249 r² = 0.999811 0 50 100 150 200 250 300 350 400 450 0 5 10 15 20 Area Conc Linearity of Tazobactam
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Amareswari et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 548 Fig: 7 Peroxide Degradation Fig: 8 Thermal Degradation Fig: 9 Photolytic Degradation Fig: 10 Precision data ACKNOWLEDGEMENT The authors thankful to all the staff members, Nimra College of Pharmacy, Vijayawada and Shadan Institute of Medical Sciences, Hyderabad, India, for providing necessary facilities to carry out the research work. CONCLUSION Finally it concludes that all the parameters are within the limits and meet the acceptance criteria of ICH guidelines for method validation. The proposed method was simple, accurate, specific, precise, robust, rugged and economical. Hence this method is validated and can be used for routine and stability sample analysis. REFERENCES M.Gandhimathi, M.Saravanakumar and T.K.Ravi, validated ion pair hplc method for simultaneous estimation of ceftriaxone sodium and tazobactum sodium in dosage form. International Journal of Pharma and Bio Sciences, 1, 2010, 17-22. P. Rama Krishna Veni, N. Sharmila, K.J.P. Narayana, B. Hari Babu, P.V.V. Satyanarayana, Simultaneous determination of piperacillin and tazobactam in pharmaceutical formulations by rp-hplc method, Journal of Pharmacy Research, 7, 2012, 127 – 131. R.K. Nanda, Ashwini V.Shelke, Development and validation of HPTLC method for the simultaneous estimation of ceftazidime sodium and tazobactam sodium in marketed formulation, International Journal of ChemTech Research, 4, 2012, 1701-1707. R.K. Nanda, Ashwini V.Shelke, UV Spectrophotometric methods for simultaneous estimation of ceftazidime sodium and tazobactam sodium in dry powder injection. International Journal of ChemTech Research, 4, 2012, 1778-1785.
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Madhu et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 549 EFFECT OF HYDROTROPIC SOLUTE ON In-VitroCHARECTERIZATION OF VALSARTAN FAST DISINTEGRATING TABLETS Madhu Sudhan Reddy A, Kishore Babu G, Srinivasa Babu P, Bhardwaj G Vignan Pharmacy College, Vadlamudi, Guntur *Corresponding author: Email: msreddympharm@gmail.com ABSTRACT Valsartan is an angiotensin receptor antagonist, commonly called as “angiotensin receptor blocker", or angiotensin receptor blocker. Solubility is an important physicochemical factor affecting absorption of drug and its therapeutic effectiveness. The poor solubility of drug substances in water and their low dissolution rate in aqueous gastro intestinal fluid often leads to insufficient bioavailability. Consequences of poor aqueous solubility would lead to failure in formulation of a dosage form. In the present investigation, an attempt was made to improve the solubility and dissolution rate of a poorly soluble drug valsartan by using hydrotropic solute, Sodium acetate. To increase the disintegration of the formulation some super disintegrants are used such as cross povidone, cross caremellose and sodium starch glycolate.This study revealed the influence of sodium acetate in the improvement of solubility of valsartan. Key words: Valsartan, fast disintegrating tablet, aqueous solvents 1. INTRODUCTION The rise in blood pressure is usually slight to moderate and continues to rise slowly over many years (Nazma Inamdar, 2008). Sometimes complications are the first indication of hypertension, e.g. heart failure, cerebrovascular accident, myocardial infarction. Occasionally the rate of progress increases and the hypertension becomes malignant.There are different classification of drugs used to reduce hypertension, in thisAngiotensin II receptor antagonists are more preferred category of drugs to reduce the hypertension, but these drugs are poorly soluble in water. Hydrotropy is a solubilization phenomenon whereby addition of large amount of a second solute results in an increase in the aqueous solubility of another solute. Concentrated aqueous hydrotropic solutions of sodium benzoate, sodium salicylate, urea, nicotinamide, sodium citrate and sodium acetate have been reported to enhance the aqueous solubilities of many poorly water-soluble drugs (Arun Shirwaikar, 2007). Hencein the present investigation, an attempt was made to improve the solubility and dissolution rate of a poorly soluble drug valsartan by using hydrotropic solute, Sodium acetate. This study revealed the influence of sodium acetate in the improvement of solubility of valsartan (Ashutosh Mohapatra, 2008). 2. MATERIALS AND METHODS Microcrystalline cellulose (MCC), Sodium starch glycolate, Cross caramellose,Cross povidone,Sodium acetate,Magnesium stearate,talc and starch were of analytical grade chemicals purchased from Quligen chemicals, Mumbai.The valsartan is of benevolent gift from the Alembic limited, vadodra. Physical properties of excipients: The following pre- formulation parameters such as Bulk density, Tap density, Angle of repose, Carrs index, Hausner ratio has been performed for the prepared granules (Mukesh, 2008). Preparation of Valsartan tablets: Valsartan tablets are prepared by using wet granulation technique. 12% Starch paste is used as binder in the formulation. Depending on the formula accurately weighed amounts of substances are taken and mixed thoroughly in a mortar. Sufficient quantity of starch paste is added and granules are prepared (Balakrishnan Prabagar, 2006). The prepared granules are dried in hot air oven. The dried granules are then taken and formulated to tablets using (Elite 10 station GMP model tablet machine). Prepared tablets are intended for further studies. Evaluation of tablet :The post compressional parameter such as Hardness, Friability, Weight variation, Disintegration, In vitro Dissolution studies has been carried out for the formulated batch of valsartan FDDS (Shishu, 2008). 3. RESULTS & DISCUSSION Physical Properties of the Blend: Micromeritic studies were carried out and the results were shown in the table 2. All the formulas VSG1, VSG2, VSG3, VCS1, VCS2, VCS3, VCP1, VCP2, and VCP3 were evaluated for bulk density, angle of repose and carr’s index. The results indicated the flowability of the tablet blend is good according to carr’s index and angle repose. In the study pre formulation studies were carried out to strengthen the physicochemical properties of all excipients which are intended for the formulation i.e identification and calibration of the
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Madhu et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 550 selected drug candidate for the formulation, micromeritic parameters such as bulk density, tap density, angle of repose, cars index, the results were tabulated in table no.2 the identification and calibration cure of valsartan was performed by serial dilution method, and the max was found to be 250nm (Shailesh Sharma, 2008). Post- compressional evaluation studies: The formulated tablets were evaluated for the tablet evaluation tests such as hardness, friability, weight variation, disintegration and dissolution tests. The results confirmed that the tablets are within the limits as per the monograph. The dissolution data of various formulations are given in the table no.3. All the formulations showed more release than that of the pure drug (P.V.Swami, 2007). Pre formulation and micrometrics properties Evaluation of tablets: The prepared different formulations were evaluated with varies parameters such as hardness, weight variationand friability to ensure their suitability for tablet dosage form. The values are tabulated in table no.3. Disintegration: Disintegration is an impotent parameter to know the breakdown of the particle. The formulations were intended for disintegration test was performed by USP method (Lab India). The study revealed that disintegration time was varied with the various formulation i.e. VSG1,VSG2,VSG3,VCS1,VCS2,VCS3,VCP1,VCP2,VC P3 (14 min 20sec, 12 min8 min 32sec, 15 min, 9 min 20 sec,8 min 22 sec, 4 min 20 sec, 4 min 20 sec, 4 min20sec. res..). all the formulations showed satisfactory disintegration time that are suitable for the tablet dosage form. Dissolution: The dissolution test is carried by using USP dissolution apparatus II (paddle type).The tablets are placed in the basket containing the buffer. The medium employed is 0.067M phosphate buffer of pH 6.8. The dissolution flask is maintained at 37.5±0.20 C. The dissolution is carried for 90 min and samples are drawn at intervals of 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90 min and the absorbance are recorded using UV visible spectrophotometers at 250 nm. The dissolution rate of different formulations were observed and tabulated in table no. 6. From the dissolution study it was found that formulation VCP1 has paved satisfactory drug release (69.84%) till 90min of dissolution. In the present investigation an attempt was made to study the effect of hydrotropic solute (sodium acetate) on invitro characterization of valsartan fast disintegrating tablets formulated using super disintegrants such as sodium starch glycolate, cross caramellose& cross povidone. The formulas VSG1, VSG2, VSG3, VCS1, VCS2, VCS3, VCP1, VCP2, and VCP3 were shown in table 1. The physical properties of the blend and tablet properties of the drug are evaluated. Compatibility studies: The I.R Spectrum of Valsartan along with selected formulations was taken and the characteristic peaks were shown below in following figures. The characteristic peaks were noted down and positions of the peaks were compared with the I.R spectrum of the selected sample. The results of the I.R analysis revealed that no interaction between drug and excipients. Table 1: Formulation table of valsartan fast disintegrating tablets Ingredients (in mg) VSG1 VSG2 VSG3 VCS1 VCS2 VCS3 VCP1 VCP2 VCP3 Drug 80 80 80 80 80 80 80 80 80 MCC(Micro crystalline cellulose) 191.5 191.5 191.5 191.5 191.5 191.5 191.5 191.5 191.5 SSG(Sodium starch glycolate) 6 12 18 CCS(Cross Caramellose) 6 12 18 CP(Cross Povidone) 6 12 18 Sodium Acetate 18 12 6 18 12 6 18 12 6 Talc 3 3 3 3 3 3 3 3 3 Magnesium stearate 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 Starch paste Q.S Q.S Q.S Q.S Q.S Q.S Q.S Q.S Q.S Total weight of tablet(in mg) 300 300 300 300 300 300 300 300 300
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Madhu et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 551 Table 2: Micromertic properties of the formulated tablets Formulation Bulk density gm/mL Tap density gm/mL Angle of repose Θ Carr’s index % VSG1 0.69 0.78 26.9 11.54 VSG2 0.58 0.66 25.6 12.12 VSG3 0.55 0.64 24.7 14.06 VCS1 0.65 0.74 28.3 12.16 VCS2 0.62 0.72 25.9 13.89 VCS3 0.53 0.64 25.8 17.19 VCP1 0.6 0.69 26.7 13.04 VCP2 0.62 0.71 25.5 12.68 VCP3 0.56 0.63 25.2 11.11 Table-3 Evaluated parameters of the formulated tablets Formulation Hardness in Kg/Cm2 Friability % Weight variation (Mg) Disintegration min VSG1 3.2 0.26 300 14 min 20sec VSG2 4.1 0.31 300 12 min VSG3 3.6 0.44 300 8 min 32sec VCS1 3.4 0.42 300 15 min VCS2 4.2 0.33 300 9 min 20 sec VCS3 3.8 0.29 300 8 min 22 sec VCP1 4 0.36 300 4 min 20 sec VCP2 3.8 0.39 300 4 min 20 sec VCP3 3.9 0.37 300 4 min 20 sec Table no 4: Represents the micromertic properties of the formulated tablets Formulation Bulk density gm/mL Tap density gm/mL Angle of repose θ Carr’s index % VSG1 0.69 0.78 26.9 11.54 VSG2 0.58 0.66 25.6 12.12 VSG3 0.55 0.64 24.7 14.06 VCS1 0.65 0.74 28.3 12.16 VCS2 0.62 0.72 25.9 13.89 VCS3 0.53 0.64 25.8 17.19 VCP1 0.6 0.69 26.7 13.04 VCP2 0.62 0.71 25.5 12.68 VCP3 0.56 0.63 25.2 11.11
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Madhu et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 552 Table no. 5. Evaluated parameters of the formulated tablets Formulation Hardness(avg of 4 tabs) in Kg/Cm2 Friability % Weight variation (Mg) Disintegration (minutes) VSG1 3.2 0.26 300 14 min 20sec VSG2 4.1 0.31 300 12 min VSG3 3.6 0.44 300 8 min 32sec VCS1 3.4 0.42 300 15 min VCS2 4.2 0.33 300 9 min 20 sec VCS3 3.8 0.29 300 8 min 22 sec VCP1 4 0.36 300 4 min 20 sec VCP2 3.8 0.39 300 4 min 20 sec VCP3 3.9 0.37 300 4 min 20 sec Table No 6. Dissolution Data of the Formulations Time VSG1 VSG2 VSG3 VCS1 VCS2 VCS3 VCP1 VCP2 VCP3 Pure Drug VCP1(WOA) 5 14.90 10.65 3.59 6.98 9.42 17.88 4.94 5.90 3.91 10.65 21.23 10 33.69 22.11 6.20 10.55 15.96 42.79 9.91 8.34 6.20 12.22 24.39 15 38.11 34.43 8.66 39.34 41.31 48.20 20.41 11.90 7.20 16.24 26.95 20 40.57 39.10 11.73 49.18 53.61 48.69 31.72 15.61 9.42 19.11 28.13 30 41.07 51.89 21.66 58.28 55.57 49.67 40.82 21.49 17.66 20.22 29.31 40 41.31 57.05 49.18 61.72 57.05 52.13 53.61 34.18 23.63 22.12 32.85 50 41.80 58.77 58.28 62.46 58.77 52.13 60.00 42.54 34.67 28.22 36.20 60 41.80 59.26 60.00 62.70 52.38 52.87 63.20 50.16 35.66 29.36 38.56 70 41.80 60.00 60.49 62.70 57.54 52.87 66.64 56.56 47.21 32.32 39.74 80 42.05 60.49 60.49 63.69 58.03 54.34 67.13 59.02 48.20 36.39 41.90 90 44.26 61.48 60.74 64.18 58.77 58.03 69.84 61.72 49.92 41.96 47.02 Fig 6: In-vitro dissolution studies of Valsartan DT tablet 4. SUMMARY AND CONCLUSION The objective of the present study was to improve the solubility and dissolution behavior of the poorly soluble drug, Valsartan by using hydrotropic solute by formulating fast disintegration tablets. The absorbance of the drug is studied in whole wavelength range of UV and the maximum absorbance is found at 250nm. The standard curves are plotted as per the values and optimized. The wet granulation method used for preparing the tablets & found to be satisfactory as it produced good formulation with qualifying standards. Out of the ten formulations prepared formulation VCP1 showed marked increase in the dissolution when compared to pure drug. The IR study, the UV overlay spectra of the formulations showed no signs of interactions of the drug with the excipients. The effect of the hydrotropic solute is clearly observed in the enhancement of dissolution of
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Madhu et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 553 the drug by comparing the dissolution profiles of VCP1 and the same formula without sodium acetate. Thus it can be concluded that there is enhancement of dissolution of the poorly soluble drug, Valsartan can by using hydrotropic solutes without any interaction. REFERENCES Arun Shirwaikar, Novel co-processed Excipients of Mannitol and Microcrystalline cellulose For preparing fast dissolving tablet of Glipizide, Indain Journal of Pharmaceutical Sciences, Sept-Oct 2007, 633-639. Ashutosh Mohapatra Rajesh K Parikh, Formulation development and evaluation of patient friendly dosage forms of metformin, Part-III: Soluble effervescent tablets, Asian Journal of pharmaceutics, July-September 2008, 177-181. Balakrishnan Prabagar, Enhancement of bioavailability of poorly water soluble Clotrimazole by inclusion complex with β- cyclodextrin; Indain Journal of Pharmaceutical Sciences, Jan-Feb 2006, 89-94. Darío Leonardi, María Gabriela Barrera, María Celina Lamas, and Claudio Javier Salomón, Development of Prednisone:Polyethylene Glycol 6000 Fast-Release TabletsFrom Solid Dispersions: Solid-State Characterization, Dissolution Behavior,and Formulation Parameters, AAPS Pharm Sci Tech, 8 (4), 2007, E1-E8. Mukesh C Gohel, Formulation, development and evaluation of patient friendly dosage forms of metformin, Part-I: Orally disintegrating tablets, Asian Journal of pharmaceutics, July-september 2008, 167- 171. Mutasem M. Rawas-Qalaji, F. Estelle R. Simons, and Keith J. Simons, Fast-disintegrating Sublingual Tablets: Effect of Epinephrine Load on Tablet Characteristics, AAPS Pharm Sci Tech, 7 (2), 2006, E1-E7. Na Zhao1 and Larry L. Augsburger1 Functionality Comparison of 3 Classes of Superdisintegrants in PromotingAspirin Tablet Disintegration and Dissolution, Jan 2005, AAPS, E634-E640. Nazma Inamdar, Kiran Bhise, Shakeel Memon, Enhancement of solubility of Meloxicam and Development of Dispersible Tablet; Asian Journal of pharmaceutics, April 2008 ,128-132. Omaima A. Sammour,Mohammed A. Hammad, Nagia A Megrab, and Ahmed S Zidan, Formulation and Optimization of Mouth Dissolve Tablets Containing Rofecoxib Solid Dispersion, AAPS PharmSciTech 2006; 7 (2), 2006, E1-E9. P.V.Swami, Studies to Enhance Dissolution Properties of Carbamazepine, Indain Journal of Pharmaceutical Sciences, May-June 2007, 427-430. Shagufta Khan, Prashant Kataria,Premchand Nakhat, and Pramod Yeole formulation of rapid-disintegrating tablets of Ondansetron Hydrochloride by Polymer Carrier System. AAPS PharmSciTech, 9 (2), 2006, E1- E9. Shailesh Sharma, G.D.Gupta, Formulation and characterization of fast dissolving tablet of Promethzine theoclate, Asian Journal of pharmaceutics, Jan- 2008, 70-74. Shishu and ashima Bhatti, et.al, Optimization of Fast Dissolving Tablet of Etoricoxib Prepared by sublimation technique, Indian Journal of Pharmaceutical Sciences, Jan-Feb 2008,71-76. Shishu and ashima Bhatti, et.al, Preparation of Tablets Rapidly Disintegrating in Saliva containing bitter taste masked granules by compression method, Indain Journal of Pharmaceutical Sciences, Jan-Feb 2008, 80- 84. Tartara., Mitsutoshi. Matsuyama. Koji., and Shimizu., US patent No., US 6316026, 1999. Yogesh Rane, Rajshree Mashru, Mayur Sankalia, and Jolly Sankalia, Effect of Hydrophilic Swellable Polymers on Dissolution Enhancement of Carbamazepine Solid Dispersions Studied Using Response Surface Methodology, AAPS PharmSciTech, 8 (2), 2007, E1-E11. Yunxia Bi, Yorinobu Yonezawa and Hisakazu Sunada, Rapidly disintegrating tablets prepared by the wet compression method, Journal of pharmaceutical science, 88, 1999, 1004-101.
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Kavithamani et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 554 A REVIEW ON GLORIOSA SUPERBA L AS A MEDICINAL PLANT Kavithamani D*1 , Umadevi M1 , Geetha S2 1. Centre for Plant breeding and Genetics, Tamil Nadu Agricultural University, Coimbatore- 641003 2. Centre for Plant Molecular biology and biotechnology, Tamil Nadu Agricultural University, Coimbatore- 641003 *Corresponding author: Email:umadevitnau@gmail.com ABSTRACT This spectacular lily is a native of Africa, where it is the national flower of Zimbabwe, and also of India where it is the state flower of Tamil Nadu. It’s a herbaceous climber with tuberous roots, using its leaf-tip tendrils to cling to support. If it’s left to scramble through other plants in a conservatory it will grow to a couple of metres tall and typically produces 5-6 blooms. The plant grows from an underground tuber and is easy to cultivate in a warm conservatory in summer. All parts of the plant are poisonous and the tubers particularly so, since they contain the toxic alkaloid colchicine. Colchicine is the important alkaloid extracted from the seed and used in modern medicine. Like many poisonous plants it has a long history of use in folk medicine and along with several related genera that contain colchicine it has been used to treat. In Africa and India there has recently been resurgence in interest in extracting a broad spectrum of medicinally useful and antimicrobial compounds from the plant, even to the extent of growing it as a crop, but it’s those flame-like flowers that make it horticulturally interesting. Key words: Gloriosa superba, gloriosa lily, medicinal activity 1.INTRODUCTION Gloriosa superba lilies valued much for their distinctive, showy and vividly colored blooms. While it’s unusual climbing habits makes Gloriosa superba an eye catching addition to any home garden, its extreme toxicity requires the most cautious of handling. Gloriosa superpa is one of the medicinal plant grown as a commercial crop and will give good returns. Among the medicinal crops it gives more returns like cash crops. The generic name Gloriosa means ‘full of glory’ and superb means ‘superb’, alluding to the striking red and yellow flowers. Gloriosa is a genus of ten species in the plant family Colchicaceae and include the formerly recognised genus Littonia. They are native in tropical and southern Africa to Asia and naturalised in Australia and the Pacific as well as being widely cultivated. The most common English names are flame lily, fire lily, gloriosa lily, glory lily, superb lily, climbing lily, and creeping lily. They are tender, tuberous rooted deciduous perennials, adapted to summer rainfall with a dormant dry season. All parts of the plant, but especially the tubers (swollen, underground stems), are extremely poisonous and the ingestion of flame lily has caused many accidental deaths. Contact with the stems and leaves can cause skin irritation. Various preparations of the plant are used in traditional medicines for a variety of complaints in both Africa and India. It has also been used to commit murder, suicide, to induce abortions and to poison dogs. African porcupines and some moles are reputed to be able to consume the roots with no ill effect (Bhushan Pawar, 2010). 1.1. Botany: Gloriosais an annual climbing perennial herb with tuberous roots. It grows between 3.5 to 6 m in length. They have showy flowers, many with distinctive and pronouncedly reflexed petals, like a Turk’s cap lily, ranging in colour from a greenish-yellow through yellow, orange, red and sometimes even a deep pinkish-red. Some synonyms, arising from the many variations, for Gloriosa superba include G. Rothschildiana (or) G. superba (Rothschildiana), G. simplex, G. virescens, G. abyssinica, G. Carsonii and G. lutea. 1.2. Taxonomy: Class :Equisetopsida Subclass :Magnoliidae Super order :Lilianae Order :Liliales Family :Colchicaceae Genus :Gloriosa 1.3.Genus description: Scan dent herbs, the rootstock a horizontal rhizome, the stem leafy, the leaves spirally arranged or sub opposite, the upper ones with cirrhose tips; flowers solitary, large, borne on long, spreading pedicels, actinomorphic, hermaphrodite; perianth segments 6, free, lanceolate, keeled within at base, long- persistent; stamens 6, hypogynous, the anthers extrorse, medifixed and versatile, opening by longitudinal slits; ovary superior, 3-celled, the carpels cohering only by their inner margins, the ovules numerous, the style deflected at base and projecting from the flower more or less horizontally; fruit a loculicidal capsule with many seeds.
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Kavithamani et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 555 1.4. Species information Scientific name :Gloriosa superba L. Common name(s) :Flame lily, glory lily, climbing lily, creeping lily Synonym(s) :Methonicasuperba, Eugonesuperba Habitat :Sparse savanna woodlands, grasslands, sand dunes, in abandoned fields or at the boundaries of cultivated ground and roadsides; in sandy- loam soil. Key uses : Medicinal, ornamental. Known hazards : All parts of the plant are extremely poisonous due to the presence of toxic alkaloids, including colchicine; ingestion can be fatal; contact can result in skin irritation. 1.5. Types of species: There are ten accepted species of Gloriosa, ignoring hybrids, varieties and cultivars. Gloriosa aurea Chiov. Gloriosa baudii (A.Terracc.) Chiov. Gloriosa flavovirens (Dammer) J.C.Manning&Vinn. Gloriosa lindenii (Baker) J.C.Manning & Vinn. Gloriosa littonioides (Welw. ex Baker) J.C.Manning & Vinn. Gloriosa modesta (Hook.) J.C.Manning & Vinn. Gloriosa revoilii (Franch.) J.C.Manning & Vinn. Gloriosa rigidifolia (Bredell) J.C.Manning & Vinn. Gloriosa sessiliflora Nordal & Bingham Gloriosa superba L. 1.6. Habitat/ Ecology: In Australia, scattered naturalized populations exist in the understorey of coastal dry sclerophyll forest and sand dune vegetation throughout south-east Queensland and New South Wales. It is considered a rampant and dangerous invasive weed in Australia,dominating the coastal dunes at the expense of native species and leading to deaths of native animals and birds when ingested.In India,Gloriosa is distributed in the Western Ghats but the density is rapidly decreasing due to excessive uprooting by the Herbal Medicine producers. 1.7. Toxicology: All parts of the Gloriosa contain colchicine, the roots and seeds are especially rich. Tubers and seeds contain colchicine, isoperlolyrine and related tropolane alkaloids, sitosterol and its glucoside, 2-hydroxy 6-methoxy benzoic acid.Flower’s contain Luteolin, its Glucoside, N-Formyl-de-Me-colchicine, its Glucoside and 2-de-Me-colchicine. The colchicine content varies from 0.15 to 0.3% in the tubers and 0.7 % to 0.9% in the seeds. The lethal dose of colchicine is about 6 mg/kg and it has been used as a means of committing suicide. 1.8. Uses: Flame lily has a wide variety of uses, especially within traditional medicine as practised in tropical Africa and Asia (including Ayurvedic medicine in India). It contains the alkaloid colchicine, which has been used effectively to treat acute gout, intestinal worms, infertility, wounds and other skin problems. The roots and leaves used as an antidote for snake bite, as a laxative, and to induce abortion. It has proven useful in the treatment of chronic ulcers, arthritis, cholera, colic, kidney problems and typhus. Colchicine, an alkaloid extracted from the tubers and seeds gives high price in the market and used in scientific research. Glory lily extract is useful against many skin diseases. It is used to rectify the many respiratory disorders. Colchicine is widely used as an experimental tool in the study of cell division, as it can inhibit mitosis (a type of cell division), induce polyploidy (cells containing more than two sets of chromosomes), and has been used in the treatment of cancer. The sap from the leaf tip is used for pimples and skin eruptions. Tribals of Patalkot apply the powder of rhizome with coconut oil in skin eruptions and related diseases. Gloriosa paste can be applied for curing inflammation like wound, lymphadenopathy, piles and skin related problem. It is also effective in poisoning. Their powder helps in easy digestion of food. It is also helpful in relieving from menstrual disturbance. It’s also providing strength to the body. Gloriosa superba is widely cultivated as an ornamental for its stunning flowers. 1.9. Propagation: Propagation generally occurs from seeds, although mature plants can be divided and grown from tubers. The hard seeds can remain dormant for 6-9 months. 1.10. Soil and Land preparation: A well drained red loamy soil is good for cultivation. Water logged soils are not suitable. Planting can be done during the rainy seasons and required the seed rate of nearly 1800 - 2000kg of tubers per ha.Planting is distributed from June – July. The ideal pH should be 6.0 – 7.0. This can be cultivated up to 600 m from mean sea level with an annual rainfall of 70 cm. Land should be ploughed many times and soil should be in fine tilth and incorporate 10 tonnes of Farm Yard Manure (FYM) during last ploughing. Furrows of 20 cm depth and 120 to 140cm between the furrows are formed and tubers are planted at 20 – 35 cm spacing. It is mainly propagated through tubers. Tubers are treated with 0.1% carbendazim for half an hour for controlling tuber rot.
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Kavithamani et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 556 Glory lily is cultivated in Tamil Nadu mainly in the western parts viz., Mulanur, Dharapuram of Tirupur district, Oddanchatram and Ambilikai of Dindigul district, Markampatty and Aravakurichi of Karur District, Attur of Salem district. The plant requires some support. Vines trained over support plants (Commiphora beryii, Dedonea viscose). Permanent structures like iron wires can also be formed for growing the vines. 1.11. Irrigation: Irrigation is given immediately after planting. Subsequent irrigation is given at 5days interval. After flowering there is no need of irrigation. Nowadays drip irrigation is well suited with growers. During early stages frequent weeding is necessary. 1.12. Manuring: NPK (Nitrogen, Phosphorus and Potash) at 120:50:75 kg/ha is applied in two split doses. Half of N and the entire dose of P and K are applied as basal dressing. Remaining quantity of N is applied at one and two months after planting. 1.13After cultivation practice: Commiphora must be trimmed annually. Care must be taken to avoid the damages to growing portions. Artificial pollination can be done between 8 – 11 am for getting higher yield. 1.14. Plant protection: To eradicate leaf eating caterpillar we can Spray 0.2 % Dichlorovas to control the caterpillar infestation. 1.15. Tuber rot: Proper drainage can be given during rainy season and drench 2g of COC/1 lit of water. 1.16. Harvest:Flowering duringSeptember -October and pods can be harvested 170 to 180 days after planting. Seeds can be dried in shade for 10 to 15 days. The tubers are finally harvested after 5 to 6 years. 1.17. Yield: In initial years, the yield is low and gradually increases year by year. First two years about 100 kg per acre but after 3 years 200-300 kg of dried seeds per acre can be achieved in well maintained fields. Artificial pollination is neededtoget more yield. Market price is around Rs. 600 to 800/kg.There are many companies (extractors of colchicine) purchasing seed material from farmers. It is being used as an abortifacient and for the management of 29 disease conditions such as leprosy, lice, rheumatism etc. Its major chemical constituent colchicine, is reported for its use in various clinical conditions leading to it’s over exploitation. Gloriosa superb has been enlisted under endangered category and needs immediate attention to conserve the spices (Subhasini et al., 2000). Fig 1: Chemical structure of Colchicine Fig 2: Chemical structure of Colchicoside Fig 3: Ariel part of Gloriosa superb Fig 4: Field Preparation Fig 5: Sprouted tuber for Planting
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Kavithamani et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 557 Fig 6: Matured pod with seeds Fig 7: Dried Seeds 2.0. CONCLUSION This is an important medicinal plant, used as an antidote for snake poison, is in demand commercially. The tuber is poisonous, when consumed in high quantities. Colchicine is the important alkaloid extracted from the seed and used in modern medicine. It is also the state flower of Tamil Nadu. REFERENCE Alok Jain and Satish Suryavanshi, Gloriosa superba Linn: A pharmacological review, IJPRD, 2(8), 2010, 24- 28. AratiMalpani, UrmilaAswar, Shiv Kushwaha, Zambare GN, Bodhankar SL, Effect of the aqueous extract of Gloriosa superba Linn (Langli) roots on reproductive system and cardiovascular parameters in female rats, Tropical Journal of Pharmaceutical Research, 10(2), 2010, 169-176. BhargavBhide and Rabinarayan Acharya, Uses of Langali (Gloriosasuperba Linn.): An Ethnomedicinal Perspective, AyurpharmInt J AyurAlli Sci., 1(3), 2012, 65 – 72. BhushanPawar, Vishal Wavhal, NayanaPawar, Mohan Agarwal, Prashant Shinde, Anthelmintic Activity of Gloriosa superb Linn (Liliaceae), Int J Pharm Tech Research, 2(2), 2010, 1483-1487. Frame PS, Dolan P, Kohli R, Eberly SW.1998. Use of colchicine to treat severe constipation in developmentally disabled patients; J Am Board Fam Med, 11(5): 341-346. Haroon Khan, Murad Ali Khan, TahiraMahmood and Muhammad IqbalChoudhary, Antimicrobial activities of Gloriosa superb Linn. (Colchicaceae) extracts. Journal of Enzyme Inhibition and Medicinal Chemistry, 23(6), 2008, 855-859. Rajendran K, Balaji P, JothiBasu M. Medicinal plants and their utilization by villagers in southern districts of Tamil Nadu, Indian J Traditional Knowledge, 7(3), 2008, 417-420. Ritesh Jain, Sanmati K. Jain, Traditional medicinal plants as anticancer agents from Chhattishgarh, India: An overview. International Journal of Phytomedicine, 2, 2010, 186-196. Senthilkumar M, Gurumoorthi P, Janardhanan K, Some medicinal plants used by Irular, the tribal people of Marudhamalai hills, Coimbatore, Tamil Nadu. Natural Product Radiance, 5(5), 2006, 382-388. Subhasini R, Manimaran S, Ruckmani K. Antimicrobial activity of aqueous and alcoholic extracts of leaves of Gloriosa superb Linn, Proc Int Congress on Ayurveda, 2000, 216. http://en.wikipedia.org/wiki/Gloriosa_(genus)
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Priyanka and Ajay Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 558 FORMULATION AND EVALUATION OF FLOATING DRUG DELIVERY SYSTEM OF CLARITHROMYCIN TABLETS Priyanka Shukla*, Ajay Yadav Rajiv Gandhi College of Pharmacy, Nautanwa, Maharajganj, Uttar Pradesh, India *Corresponding author: Email:priyankashukla2222@gmail.com ABSTRACT The present study focused on the development of gastroretentive technology which will deliver the antibiotic at predetermined rate to achieve the local concentration enough to act as antibacterial against H.Pylori. This technology ensures the maximum utilization of the drug with minimum side effects and maximum patient compliance and also with reduced antibiotic resistance by the bacterium. The tablet contain 15% and 20% having Floating lag time (FLT) 63 seconds and 64 seconds and the drug release was found to be 62.73% and 53.72 respectively. Increasing the polymer ratio the FLT was increase but the drug release was decreased. Key words: Floating drug delivery system, HPMCK4M, Clarithromycin, bioavailability. INTRODUCTION Gastric emptying of dosage forms is an extremely variable process and ability to prolong and control the emptying time is a valuable asset for dosage forms, which reside in the stomach for a longer period of time than conventional dosage forms. Several difficulties are faced in designing controlled release systems for better absorption and enhanced bioavailability. One of such difficulties is the inability to confine the dosage form in the desired area of the gastrointestinal tract (Hirtz, 1985). Drug absorption from the gastrointestinal tract is a complex procedure and is subject to many variables. It is widely acknowledged that the extent of gastrointestinal tract drug absorption is related to contact time with the small intestinal mucosa. Thus, small intestinal transit time is an important parameter for drugs that are incompletely absorbed. The aim of the present study is to develop gastroretentive drug delivery system of clarithromycin there by improving the bioavailability by improving absorption through stomach (Ponchel, 1998) MATERIALS AND METHODS Clarithromycin procured from Micro advanced Research centre, HPMC K4M gift sample from Colorcon, Mumbai Bangalore, Lactose, PVP K30,Sodium Bicarbonate Procured from S.D.Fine chemicals, Cochin. FORMULATION STUDIES Floating tablets, containing drug and polymer, are one of the simplest approaches for controlled release of a drug. Among the different types of hydrophilic polymers reported, HPMC was used because of its associated advantages. In addition, HPMC is a pH independent material and the drug release rates from HPMC matrix formulations are generally independent of processing variables, such as compaction pressure, drug particle size and incorporation of lubricant. Selection of formulation process was done based on the results obtained from the flow properties of drug and excipients. Due to poor flow properties of the drug and polymer, wet granulation method was selected for tablet manufacture. Selection of Excipients: The polymers hydroxy propyl methyl cellulose (HPMC) K4M were evaluated for floating drug delivery system Formulation of Floating Tablets Preliminary batches: The tablets were formulated using 5%, 10%, 15%, 20% and 25% of HPMC K4M respectively as shown in table 1. The tablets were evaluated for floating behavior and dissolution, and other parameters as shown in table3. The matrix integrity was poor for tablets formulated with 5% and 10% HPMC K4M and not considered for further studies (Groning, 1984). Evaluation of Tablets: The tablets were evaluated for various parameters as follows and reading recorded. Appearance and Shape: The general appearance of the tablet includes the morphological characteristics like size, shape, colour, odour etc. Also tablets may have lines, break-marks and may bear a symbol or other markings. Uniformity of Thickness and Diameter: The uniformity of the diameter and thickness was measured using vernier caliper. The average diameter and thickness of the tablet was calculated. The test passed if none of the individual diameter and thickness value deviated by ± 5% of the average. Hardness: Monsanto hardness tester was used to check the hardness of the tablet. The tablet was placed vertically between the jaws of the tester. The two jaws placed under tension by spring and screw gauge. By turning the screw,
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Priyanka and Ajay Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 559 the load was increased, and at collapse the applied pressure from the spring was measured in kg/cm2 . Friability: Tablets were subjected to tumbling in Roche friability tester. Six tablets were weighed and tumbled at the rate of 25 rpm for 4 min. The tablets were weighed and percent friability was calculated by the following formula. RESULTS AND DISCUSSION All tablets of the factorial design batches were off white coloured with smooth surface, circular flat faced with good texture. Uniform thickness of the tablets throughout the batches ensures good tablet strength and shape and is also important with respect to packaging and handling. The thickness of the tablets should be controlled within 5% or less of an established standard value. Excessive variation in the tablet thickness can result in problems with packaging as well as consumer acceptance. There were no marked variations in the thickness of tablets within each formulation (<5%) indicating uniform behavior of granules throughout the compression process. The thickness of the factorial design batches were found in range of 3.26 and 3.44 mm. After compression, a tablet requires a certain amount of mechanical strength to withstand the shock of handling in the manufacture, packing, shipping and dispensing. The hardness of the tablet was found to be in the range of 6.9 to 8.1 kg/cm2 . This ensures good mechanical strength. This resulted due constant tablet press setting across all batches of factorial design irrespective of weight variation. The tablet density was close to 1 result in good floating characteristics in vitro. The tablet densities of the factorial design batches were found to be between 1.03 to 1.21 gm/cm3 . Friability of the tablet is the measure of the tablets strength. Tablets with friability less than 1% of their weight are acceptable. The friability of the factorial design batches were in the range of 0.13 to 0.40, which was within the specified limits. To check labeled amount of drug, it is necessary to note the tablet weight. The average weight of tablets within each formulation was found to be uniform. This indicates uniform feeling of the die cavity during tablet compression. According to I.P., for tablets weighing 250 mg or more, The drug content of the formulations was found to be between 97 and 103 % (i.e. variation of ±3%). The value ensures good uniformity of the drug content in the tablets. In vitro buoyancy study was carried out using USP dissolution apparatus II in an acidic medium (0.1N Hcl) and floating lag time (FLT) and floating time (FT) was noted. The time interval between the introduction of the tablet into the dissolution medium and its buoyancy to the top of the dissolution medium was taken as floating lag time and the duration of buoyancy was taken as total floating time. The mechanism involved in buoyancy of the tablet is the entrapment of carbon dioxide gas induced by sodium bicarbonate after reaction with acidic dissolution medium (0.1N Hcl). The gas is trapped and protected within the gel formed by hydration of polymer thus decreasing the density of the tablet below1gm/cm3 and the tablet becomes buoyant. These formulations were analyzed for the floating behavior (i.e. FLT and FT). The formulation containing 18% of the sodium bicarbonate showed maximum floating time (>24hrs) and minimum floating lag time (<60 secs). Thus, sodium bicarbonate concentration 18% was essential to achieve optimum in vitro buoyancy and selected for the further studies Table 1 Formulation of preliminary batches Ingredients(mg) H1 H2 H3 H4 H5 Clarithromycin 250 250 250 250 250 HPMC K4M 30 60 90 120 150 Lactose 128 98 68 38 8 Sodium Bicarbonate 108 108 108 108 108 Table 2 Evaluation characteristics of preliminary batches Formulation FLT (sec) FT (hrs) Matrix Integrity Hardness (kg/cm2 ) Thickness (mm) Drug Release after 12hrs (%) H1 - - - 7.1 3.46 - H2 - - - 6.9 3.42 - H3 63 1 + 7.1 3.46 62.73 H4 74 < 12 + 7.2 3.48 53.72 H5 90 3 + 7.1 3.43 51.70
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Priyanka and Ajay Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 560 Table 3: Swelling Index of Clarithromycin tablets Time (min) % Swelling index H1 H2 H3 H4 H5 0 0 0 0 0 0 15 38 38 39.6 32.14 40.38 30 53.38 51.9 49 35.71 51.92 60 67.73 71.15 64.15 55.35 69.23 120 84.61 84.6 84.9 76.8 88.46 180 103 101.9 105.66 91.07 119.2 240 115.38 119.23 128.3 101.78 123.07 300 121.15 126.9 132 108.92 134.61 360 134.61 136.53 137.7 116 150 420 138.46 142.3 143.39 123.21 153.84 480 145.84 146.84 150.05 121.65 160.35 540 153.84 153.84 157.69 115.84 171.15 600 151.19 150 150.94 105.65 170 660 148 148 150.94 104.77 166 720 136 138 140 102.55 160 Table 4: Floating ability of various cefpodoxime tablet formulations CONCLUSION The gastroretentive drug delivery is suitable for the controlled release of the drugs with absorption window in stomach and proximal part of small intestine. As it holds the drug delivery above the absorption window and release the drug at a controlled rate. Clarithromycin is an advanced generation macrolide antibiotic used in treatment of H. pylori and respiratory infection. In controlled release formulation, if the concentration of antibiotic is maintained above MIC, drug resistance can be reduced. To achieve the desired therapeutic profile with maximum drug utilization and improve the patient compliance present study was applied to develop the gastroretentive drug delivery system for Clarithromycin. Commercially it is available as conventional dosage and is only 50% bioavailable orally. Therefore, present investigation was taken up to formulate a suitable gastroretentive floating tablets of Clarithromycin and to evaluate the dosages in current perspective. This study discussed a positive application of a computer optimization technique for the development of a gastroretentive floating drug delivery in which polymer (HPMC) viscosity grade (K4M) and citric acid significantly affected the floating lag time and release profile of the drug. The dosage form can control the release, avoid dose dumping, and extend the duration of action of a drug with prolonged floating time. This dosage form holds promise for further in vivo studies, which can be extrapolated for the development of other delivery systems. Addition of citric acid, to achieve buoyancy under the elevated pH of the stomach, caused an enhancement in drug release that was retarded by incorporation of different polymer concentration in the formulation. The tablet contain 15% and 20% having FLT 63 and 64 and the drug release were found 62.73% and 53.72. Increasing the polymer ratio the FLT was increase but the drug release was decreased. REFERENCES Davis SS, Stockwell AF, Taylor MJ, The effect of density on the gastric emptying of single and multiple unit dosage forms, Pharm Res, 3, 1986, 208-213. Batch Code Floating Lag time (sec) Floating duration (min) Integrity H1 - - Intact H2 - - Broken after 6-8Hrs H3 63 60 Intact H4 74 >720 Intact H5 90 59 Intact
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Priyanka and Ajay Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 561 Deshpande AA, Shah NH, Rhodes CT, Malick W, Development of a novel controlled-release system for gastric retention, Pharm Res. 1997;14:815-819. Fix JA, Cargill R, Engle K, Controlled gastric emptying, Gastric residence time of a non-disintegrating geometric shape in human volunteers. Pharm Res, 10, 1993, 1087- 1089. Groning R, Heun G, Oral dosage forms with controlled gastrointestinal transit, Drug Dev Ind Pharm, 10, 1984, 10:527-539. Hirtz J, The GIT absorption of drugs in man: a review of current concepts and methods of investigation, Br J Clin Pharmacol, 1985, 19, 1985, 77-83. Kedzierewicz F, Thouvenot P, Lemut J, Etienne A, Hoffm an M, Maincent P. Evaluation of peroral silicone dosage forms in humans by gamma-scintigraphy, J Control Release, 58, 1999, 195-205. Mamajek RC, Moyer ES, inventors, Drug dispensing device and method, US Patent 4 207 890, June 17, 1980. Ponchel G, Irache JM, Specific and non-specific bioadhesive particulate system for oral delivery to the gastrointestinal tract, Adv Drug Del Rev, 34, 1998, 191- 219. Rednick AB, Tucker SJ, inventors, Sustained release bolus for animal husbandry, US patent 3 507 952, April 22, 1970. Urguhart J, Theeuwes F, inventors, Drug delivery system comprising a reservoir containing a plurality of tiny pills, US patent 4 434 153, February 28, 1994.
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Dharmendra and Ranjeet Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 562 ANTIFUNGAL ACTIVITY OF ETHANOLIC EXTRACT OF EUPATORIUM ADENOPHORUM LEAVES Dharmendra Kumar Singh*, Ranjeet Singh Rajiv Gandhi College of Pharmacy, Nautanwa, Maharajganj, Uttar Pradesh *Corresponding author: Email-dksingh.singh22@gmail.com ABSTRACT The present study designed for phytochemical screening, anti fungal activity and physic chemical constants of ethanolic extracts of whole plant of Euphorbia thymifolia linn belongs to the family Euphorbiaceae, which is widely used in Ayurveda to cure many remedies. The anti fungal activity was studied against Bacillus Subtilis, Staphylococcus aureus, Proteus species and E. coli were tested by cup‐plate agar diffusion method. Erythromycin and tetracycline was used standard anti fungal agent. Physic chemical evaluation was carried out by ash values, extractive values and phytochemical screenings. The results of the study revealed that, the ethanolic extracts exhibited anti fungal activity against both gram positive and gram negative bacteria’s. Keywords: Eupatorium adenophorum, Ethanolic Extract, Antifungal activity INTRODUCTION Eupatorium adenophorum locally called “Banmara” of “Kalijhar” in Nepal is an exotic plant species growing prolifically in Sikkim Himalaya and Meghalaya plateau. It is the native species of tropical America. It has recently drawn worldwide attention for its ever increasing potential weed mostly colonizing cultivated lands, cardamom based agroforestry systems, open degraded lands, jhoom fallow and natural forest especially within the altitudinal range between 1000 - 8000 ft. It has an interesting history of its own. It is probable that the species was nowhere noted in Sikkim and Meghalaya hills approximately 3 and 4 decades back respectively. In the Himalayan state of Sikkim it probably started invading from southern via Doars and Assam valley. Due to the increasing resistance and side effect of the synthetic antimicrobial agents, there is a great demand of the natural antimicrobial agents with fewer side effects. So we decided to collect the plant of Banmara and evaluated for the antimicrobial activity. MATERIALS AND METHODS Eupatorium adenophorum was collected From Palpa Botanical Research Center Nepal & Authenticated by Sanjeev Dubey M.Sc Soxhlet extraction: Soxhlet extraction is only required where the desired compound has a limited solubility in a solvent, and the impurity is insoluble in that solvent. If the desired compound has a high solubility in a solvent then a simple filtration can be used to separate the compound from the insoluble substance. The advantage of this system is that instead of many portions of warm solvent being passed through the sample, just one batch of solvent is recycled. This method cannot be used for thermolabile compounds as prolonged heating may lead to degradation of compounds Source of crude plant extracts: The leaves of Eupatorium adenophorum used for the extraction. Three gram of crude drug material was sterilized in 0.1% mercuric chloride followed by proper washing in distilled water and was grinded in presterilized mortar and pestle by adding 5ml of distilled water. The extracts were filtered and centrifuged at 500 rpm for 3 min to obtain clear solution. The volume was maintained to 7 ml and different concentrations (10, 20, 30, 40, 50, 60, 70, 80, 90 and 100%) were made for further use in the experiment. Test fungus: Fusarium solani was isolated from the infected potato tuber using standard pathological techniques. The media used was Potato Dextrose Agar (PDA). The pure culture of the test fungus was maintained. The assessment of fungitoxicity was done by poisoned food technique. Inoculum disc: Seven days old culture of the test fungus was used for the preparation of inoculum disc of 4 mm in diameter. Antifungal assay: A volume of 0.5 ml of each concentration was aseptically poured into the petriplate followed by the addition of 9.5 ml of melted PDA and was swirled gently to achieve thorough mixing of the contents. In the control set, no extract was used. After the solidification of the media, one inoculum disc of the test fungus was aseptically inoculated upside down at the centre of the petriplate and incubated at 25 +20*C. The average diameter of the fungal colonies was measured on the 7th day of incubation and percentage of mycelial growth inhibition was calculated. Mycelial growth inhibition (%) = gc-gt/gc*100
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Dharmendra and Ranjeet Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 563 Where, gc = growth of mycelial colony in control set after incubation period subtracting the diameter of inoculum disc. gt = growth of mycelial colony in treatment set after incubation period subtracting the diameter of inoculum disc . Table.1. Percentage inhibition of fungal growth Concentration (%) Eupatorium Adenophorium 10 68.18 20 70.45 30 70.45 40 75.00 50 75.00 60 75.00 70 77.20 80 79.50 90 81.18 100 84.00 Fig. 3.Total inhibition of fungal growth Fig: 1. Flower of Eupatorium adenophorum Fig: 2.Leaves of Eupatorium adenophorum
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Dharmendra and Ranjeet Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 564 CONCLUSION The crude extracts of Eupatorium adenophorum assessed in vitro for activity against fungi Fusarium solani (Mart.) Sacc.,the causal agent of dry potato tuber rot. Pathogenecity test was confirmed by inoculating the pathogen into the healthy potato tuber. The assessment of fungitoxicity was carried out by poisoned food technique using ten different concentrations (10, 20, 30, 40, 50, 60, 70, 80, 90 and 100%) against the test fungus. Assessment was carried out in terms of percentage of mycelial growth inhibition of the test fungus. The extracts of the plant species was found to be effective in inhibiting the mycelial growth. The extracts showed inhibition effects on the growth of the test fungus. Among the tested plant species, markedly inhibited the fungus growth even at a low concentration of 40% followed by Eupatorium adenophorum. The inhibition of the mycelial growth of the test fungus was found to be 68.18 to 84 percent by the extracts of Eupatorium adenophorum. The plant species showed reasonable effects among themselves. Relative variation in the content of active chemicals in the extracts was speculated to be one of the evident reasons. REFERENCE Shrestha I and N Joshi, Medicinal plants of the Lele village of Latipur District, Nepal, International Journal of Pharmacognosy, 31(2), 1993, 130-134. Shrestha Vaidya G and Piya S, Study of antagonistic action of two ectomycorrhizal fungi isolated from pine forest of Dadeldhura.In: Proc. of international seminar on mountains, (Eds. F.P. Neupane & K.M. Bhattacharya) Kathmandu, 2002, 571-577. Shrestha Vaidya G, K Shrestha and H Wallander, Evaluation of antimicrobial activity of Lantana camara Linn. against pathogenic fungi and bacteria, Journal of Ethnopharmacology, 60, 2009, 163–172. Shrestha Vaidya, G., K Shrestha, BR Khadgi, NC Johnson and H Wallander, Organic matter stimulates arbuscular mycorrhizal fungi in Bauhinia purpurea and Leucaena diversifolia plantations on eroded slopes in Nepal, Restoration Ecology, 16 (1), 2008, 79-87. Tian, Yu, Hou Jing, Wu Jian-Ping, He Lan, Cao Ao- Cheng, Study on the volatile components from Eupatorium adenophorum Spreng and its antifungal activity, Chinese Journal of Pesticide Science, 2. (3), 2007, 49-52. Tomoko N, A Takashi, Hiromu, I Yuku, M Hiroko, I Munekazu, A Fujio and W Kazuhito, Antibacterial activity of extracts prepared from tropical and sub-tropical plants on methicillin resistant staphylococcus aureus, Journal of Health Science, 48, 2002, 273-276.
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Rajesh et.al. Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 565 FORMULATION OF MOUTH DISSOLVING TABLETS OF NAPROXEN Rajesh Reddy K, Nagamahesh Nandru, Desam Asha Latha, Srinivasa Rao Chekuri MRR College of Pharmacy, Nandigama, Andhra Pradesh, India Corresponding author: Email: rajbiz.36@gmail.com ABSTRACT The aim of the present work is to investigate the possibility of preparing mouth dissolving tablet containing naproxen by direct compression method. Basic goals in the development of mouth dissolving tablet are to increase patient compliance, ease of administration, safety and appropriate dosing. It has perceived faster onset of action as the dosage form is disintegrated prior to reaching the stomach and is ideal for acute diseases like allergies, nausea, and vomiting and particularly applicable to manage breakthrough systems. Direct compression method was used to compress the tablets as it is the easiest way to manufacture tablets and less time consuming. Conventional equipment, commonly available excipients and limited number of processing steps are involved in the direct compression and so manufacturing cost is low. Keeping in view the advantages of this delivery system, in the present study, attempts were made to formulate mouth dissolving tablet. Key words: Naproxen, Mouth dissolving, Disintegration. INTRODUCTION Mouth Dissolving Tablet 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. MATERIALS AND METHODS Naproxen was obtained as an gift sample form Granules India, Hyderabad, Microcrystalline cellulose, Mannitol, Crosscarmellose sodium, Sodium starch glycollate, Crosspovidone, Aerosil, Aspartame and Magnesium stearate was obtained from Kniss laboratories Pvt limited, Chennai Pre-formulation studies Preparation of mixed blend of drug and excipients: All the Ingredients were passed through mesh 60. Required quantity of each ingredient was taken for each specified formulation and all the ingredients were cog rind in a mortar and pestle. The powder blend was evaluated for flow properties such as Bulk density, Tapped density, Compressibility index, Hausner ratio and angle of repose. Evaluation of Naproxen Mouth Dissolving Tablets: The formulated tablets has been evaluated for the following parameters such as Thickness, Weight variation test, Hardness test, Friability test, Water absorption Ratio Content uniformity test, Wetting time, In-vitro dispersion time and In-vitro dissolution studies RESULTS AND DISCUSSION Surface Morphology: Morphological characteristics such as colour, form, taste etc of naproxen were studied and the results are tabulated in table. As the API is bitter in taste, taste masking with sweetener may prove beneficial for a palatable dosage form. Physical parameters: The pure drug showed angle of repose value is 350 311 indicates the good flow properties. The compressibility index, Hausners ratio values of the drug are 28.40% and 1.397 indicates that the drug has poor compressibility properties. Drug – Excipient compatibility studies Fourier transforms infra-red spectroscopy (FTIR): The FTIR analysis was conducted for the structure characterization. FTIR spectra of the pure drug, pure polymers and mixture of both were recorded. Formulations were taken in a KBr pellet using BOMEN MB SERIES FTIR instrument. Approximately 5mg of samples were mixed with 50mg of spectroscopic grade Kbr; samples were scanned in the IR range from 500 to 3500 cm-1 , with a resolution of 4 cm-1 . Compatibility Studies: Drug excipient compatibility study showed no interactions as principle peaks are retained. Thus all excipients were compatible with drug. Optimized formula: The dissolution profile of formulations (F1-F6) indicates a faster release of drug i.e. 90% or more of the drug released from all formulations with in 60 min. however the maximum
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Rajesh et.al. Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 566 release of drug (99.4%) was observed in F4.This formulation also exhibited better performance in the other evaluation parameters. Hence, formulation (F4) is selected as optimized formula. Table 1: Formula for Naproxen Mouth Dissolving Tablets Ingredients (Mg) F1 F2 F3 F4 F5 F6 Naproxen 150 150 150 150 150 150 Microcrystalline Cellulose 18 10 18 10 18 10 Mannitol 26 14 26 14 26 14 Croscarmellose sodium 40 60 - - - - Table: 2 Identity Parameters of API Test Specifications Results Description: Colour Odour Form Taste white None Crystalline Bitter Complies Solubility Soluble in ethanol and methanol Complies Identification: IR-Spectrum IR-Spectrum of the test sample should match with the IR-Spectrum of the working standard. Complies Assay 99% w/w 99.0% to 101.0% w/w All the Identity parameters of the API are found to be within the limits Table: 3 Physical parameters of API in Pre- formulation studies Table: 4 Ftir Spectrum of the Naproxen parameters Result Tapped density 0.728 gm/ml Bulk density 0.521 gm/ml Compressibility Index 28.40 % Hausner Ratio 1.397 Angle of repose 350 311 Type of Vibrations Range C=O 1788 C-H 3192 C-C 1631 C-O 1028 Table No: 5 Pre-compression properties of the naproxen mouth dissolving tablets. Formulation Bulk density (g/ml) Tapped density (g/ml) Hausner’s Ratio Carr’s index Angle of repose (0o ) F1 0.53±0.005 0.62±0.02 1.16±0.02 13.65±1.06 31' F2 0.52±0.01 0.61±0.014 1.18±0.03 14.27±1.06 30º. F3 0.526±0.005 0.61±0.014 1.16±0.01 14.78±0.18 27º9' F4 0.52±0 0.60 1.15±0 14.58±0 32º.05' F5 0.526±0.01 0.605±0.01 1.163±0.02 12.51±0.02 29º.82' F6 0.526±0.005 0.63±0.08 1.163±0.005 14.89±0 27º.21' The data are expressed as mean±S.D. (n=3) Table: 6 Post compression properties of the naproxen mouth dissolving tablets. Formulation Weight variation (mg) Thickness (mm) Hardness (kg/cm2 ) Friability (%) Disintegration Time (sec) F1 Passes 3.74±0.02 3.63±0.15 0.50 28± 0.54 F2 Passes 3.76±0.02 3.8±0.1 0.45±0.12 26 ± 0.02 F3 Passes 3.26±0.02 4.03±0.05 0.41±0.22 25 ± 0.14 F4 Passes 3.14±0.02 3.33±0.11 0.22±0.08 26 ± 0.25 F5 Passes 3.21±0.03 3.8±0.1 0.8±0.08 27 ± 0.14 F6 Passes 3.66± 0.05 4.03±0.15 0.62±0.16 24 ± 0.01 The data are expressed as mean±S.D. (n=3)
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Rajesh et.al. Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 567 Table: 7 Post compression properties of the naproxen mouth dissolving tablets Formulation Water absorption Ratio Content uniformity Wetting time (sec) In-vitro dispersion time(Sec) F1 78.92 ± 0.14 98.19 ± 0.51 50.66±3.05 114.66±4.16 F2 72.35 ± 0.41 98.42 ± 1.01 43.33±3.05 93.3±4.16 F3 69.32 ± 0.58 97.77 ± 1.26 26±2 74.3±4.04 F4 75.63 ± 0.47 99.82 ± 0.33 18±2 55±1.0 F5 74.21 ± 0.25 99.74±0.44 72.66±3.05 93.6±2.51 F6 74.23 ± 0.14 98.50±0.55 67.33±3.05 84±2 The data are expressed as mean±S.D. (n=3) Table: 8 Cumulative Percentage drug Release of F1 – F6in pH 6.8 Phosphate Buffer Time (min) Cumulative Percentage Of Drug Release in pH 6.8 Phosphate Buffer F1 F2 F3 F4 F5 F6 10 42.8 56.6 68.6 78.6 68.4 76.8 20 70.6 64.8 74.8 79.8 74.8 83.6 30 78.4 80.6 86.4 84.8 76.6 91.8 40 84.6 84.8 90.6 91.4 88.4 95.8 50 88.8 92.8 94.8 97.8 89.6 97.4 60 92.6 94.7 96 99.4 96.6 98.7 Table: 9. Optimized formulas of Naproxen Mouth dissolving tablet Ingredients Quantity (mg) Naproxen 150 Microcrystalline cellulose 10 Mannitol 14 Crospovidine 60 Aerosil 2 Aspartame 10 Magnesium stearate 4 Total 250 Fig 1: FTIR of naproxen Fig 2: FTIR of naproxen with croscarmellose sodium
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Rajesh et.al. Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 568 Fig 3: Ftir Of Naproxen With Crospovidone Fig 4: FTIR of naproxen with sodium starch glycolate Fig 5: Formulated mouth dissolution for naproxen mouth dissolving tablets 15 Sec 30Sec 45 Sec 55 Sec Fig 6: Disintegration time at the end of Various time interval Fig :7 In-vitro drug release of mouth dissolving tablets of Naproxen (F1-F6)
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Rajesh et.al. Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 569 CONCLUSION The demand for orally disintegrating tablets has enormously increased during the last decade, particularly for geriatric and pediatric patients who feel difficulty in swallowing conventional tablets and capsules. Fast dissolving or fast disintegrating dosage form is advantageous for such patients. Fast dissolvable or fast disintegrating dosage forms are meant to disintegrate immediately upon contact with the saliva leading to faster release of drug in the oral cavity. By administrating the fast disintegrating dosage forms, absorption of the drugs occurs through buccal mucosa and it may reduce the first pass metabolism leading to better efficacy of the drug. In my present work an attempt was made to develop mouth dissolving tablets of naproxen by direct compression method and by using cross povidone, cross carmellose sodium and sodium starch glycolate as superdisintegrants. In the preformulation studies it has been proved that there is no interaction between the drug and the excipients. The blends of varying super disintegrants were formulated into 6 formulations ranging from F1 to F6, and the blends were evaluated for the pre and post comparison parameters and In vitro drug release is also studied. All the pre compression parameters angle of repose, Cars index, Hausner’s ratio, tapped and bulk density and is within the limits. The results shown that the formulations containing the Cross povidone have the good flow properties and the good compactability when compared with the other formulations. The post compression parameters include the Weight variation, Hardness, friability, Thickness, wetting time, In vitro disintegration and In vitro dispersion time and the water absorption ratio. The results shown maximum for the formulation (F4) that containing the cross povidone as superdisintegrant. The In vitro drug release of formulation F4 had shown that maximum drug release 99.4 ± 0.54 when compared with the other formulations. So F4 was choosen as the best formulation which contains crosspovidone as a super disintegrant. The naproxen 250 mg mouth dissolving tablet was prepared by using the finalized formula and optimized manufacturing process showed good results in formulation of stable tablet dosage form. BIBLIOGRAPHY Allen LV and Wang B, Process for making a particulate support matrix for making rapidly dissolving tablets, US Patent No. 5, 1996, 587,180, Bhaskaran S and Narmada G V, Orally disintegrating tablets, Indian Pharmacist, 1(2), 2002, 9-12. Brahmankar DM, Jaiswal SB, Biopharmaceutics & Pharmaceutics, First Edition, 1995, 335. Howard C. Ansel, Nicholas G. Popvich, Loyd V, Allen, Pharmaceutical Dosage Forms and Drug Delivery System, First Edition, 1995, 78. Kaushik D, Dureja H, Saini TR, Mouth Dissolving Tablets: A review, Indian Drugs, 2004, 41(4), 187-193. Kuccherkar BS, Badhan AC, Mahajan HS, Mouth dissolving tablets: A novel drug delivery system, Phrma Times, 2003, 35, 3-10. Lachmann L., Liebermann H. A. and Kiang J.L., The theory and practice of Industrial Pharmacy, Third Edition, Varghese Publishing House, Bombay, 1998, 430-440. Lailla J K, Sharma A. H., Freeze-drying and its applications, Indian Drugs, 1993, 31, 503-513. Mishra B., Panigrahi D and Baghel S., Mouth dissolving tablets: an overview of preparation techniques, evaluation and patented technologies, J. Pharm. Res, 4(3), 2005, 33-38. Seager H, Drug delivery products and zydis fast dissolving dosage form, J. Pharm. Phamacol., 50, 1998, 375-382.
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Vinod et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 570 PREPARATION OF IMMEDIATE RELEASE ATORVASTATIN AND SUSTAINED RELEASE MATRIX TABLETS OF GLICLAZIDE USING RETARDANT: HYDROXYPROPYL METHYL CELLULOSE Vinod Raghuvanshi*, Jayakar B, Debjit Bhowmik, Harish G, Dureivel S Vinayaka Missions College of Pharmacy, Salem,Tamilnadu Nimra College of Pharmacy, Nimranagar,Vijayawda,AP *Corresponding author: Email-raghuwanshivinod@gmail.com ABSTRACT In the present work an attempt has been made to formulate tablet containing immediate release Atorvastatin and sustained release Gliclazide using retardant namely hydroxypropyl methyl cellulose. Two different grade of hydroxypropyl methylcellulose in different concentrations were used. In the preformulation study, compatibility evaluation was performed which implies that drugs, excipients and polymers are compatible with each other. The formulation of tablets was done by using wet granulation technique which was found acceptable. All the formulations were subjected to various evaluation studies. The results are in accord with the marketed product. Key words: Atorvastatin, Sustained release, Gliclazide, immediate release, Hydroxypropyl methyl cellulose, Matrix Tablet 1. INTRODUCTION The oral route of drug administration is the most important method of drug administration for systemic effects. The parenteral route of administration is important in treating the medical emergencies in which subject is comatose or cannot swallow and in providing various types of maintenance therapy. Nevertheless, about 90% of all the drugs used to produce systemic effects are administered by the oral route. Tablet formulation and design may be described as the process whereby the formulator ensures that the correct amount of the drug in the right form is delivered at or over the proper time at the proper rate and in the desired location, while having its chemical integrity protected to that point. Latest concepts and regulations focus on bioavailability, bioequivalence and validation etc. impact formulation designing and manufacture. 2. MATERIAL AND METHODS Atorvastatin Calcium was obtained from Biocon India Ltd, Microcrystalline Cellulose, PH101, Calcium Carbonate IP (Heavy), Lactose Monohydrate IP was obtained from Reliance Cellulose (P) Ltd, Cross Caramellose Sodium BP, Polysorbate 80, Sodium Ascorbate IP was obtained formSilvoliacalChem Ltd, HPMC 4000 cps, HPMC 100 cps, Poly Ethylene Glycol - 400 IP was obtained from Signet Chemicals Corporation. 2.1. Preparation of Atorvastatin blend: During the formulation process, granulation was done in two steps. In first part granules of Atorvastatin were prepared and in second part Gliclazide granules were prepared using two different grade of HPMC in different ratio. Atorvastatin Granules were kept same for each and every batch and variation was made in formulation of Gliclazide granules only. 2.2. Granulating Solution Preparation: Purified water was taken in suitable vessel and heated to boiling. Charged H.P.C. in stirring condition and then charged sodium ascorbate and poly-sorbate 80 under stirring condition. Cooled solution to room temperature under stirring condition until a translucent solution was obtained. 2.3. Dry Mixing & Granulation: Atorvastatin Calcium, Microcrystalline Cellulose I.P., Lactose Monohydrate IP, Calcium Carbonate IP (Heavy), Cross Caramellose Sodium BP was blended and pass through mesh 40 ss screen. Then H.P.C. Granulating Solution was added on the blend while the dry blend was being mixed. Container was rinsed with suitable amount of water. To this required amount of purified water was added slowly. Wet mass was passed through mesh 8 ss screen fitted of shifter and semidried at 600 C in tray drier and finally granules were passed through mesh 20 ss screen fitted of shifter and dried at 600 C in tray drier until L.O.D. was observed in between 1.6% ± 0.4% (IR at 1050 C). 2.4. Lubrication of Granules: Magnesium Stearate IP and Talc IP was bag blended. Blend wad passed through mesh 60 ss screen fitted on shifter and then above dried granules were mixed with the blend in a suitable blender.As the granules were prepared, it was taken and blended with the granules of Gliclazide. 2.5. Gliclazide Granulation Part : For formulation of gliclazide granules differentcocentration of H.P.M.C.4000 cps, H.P.M.C100 cps and combination of thesepolymersweretried to have desired release profile. 2.6. Granulating Solution Preparation: Purified water was taken in suitable vessel add Charged H.P.C. in stirring condition and continue Stirring until the H.P.C. was completely hydrated in Purified water.
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Vinod et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 571 2.7. Dry Mixing & Granulation: Gliclazide BP, Microcrystalline Cellulose I.P. and HPMC were bag blended and passed through mesh 40 ss screen, and add H.P.C. Granulating Solution was added on the blend while the dry blend was being mixed. Container was rinsed with suitable amount of purified water. To this required amount of purified water was added slowly.Wet mass was passed through mesh 8 ss screen fitted of shifter and semidried at 600 C in tray drier and finally granules were passed through mesh 16 ss screen fitted of shifter and dried at 600 C in tray drier until L.O.D. was observed in between 2% ± 2.5% (IR at 105o C). 2.8. Lubrication of Gliclazide Granules: Magnesum Stearate IP, Aerosil and Talc IP were bag blended. Blend wad passed through mesh 60 ss screen fitted on shifter and then above dried granules were mixed with the blend in a sutaible blender. 2.9. Evaluation of Granules: Pre-formulation is a branch of pharmaceutical sciences that utilizes biopharmaceutical principles in the determination of physicochemical properties of a drug substance. The goal of pre-formulation studies is to choose the correct form of the drug pre-requisite for formulation. The pre- formulation studies such as angle of repose, Bulk density, Tapped density, Hausner ratio, L.O.D. of granules, Sieve analysis of granules 2.10. Evaluation of Tablets: The followingevaluation paramètres such as Tablet dimensions, Hardness, Friability, Average Weight, Content uniformity of active ingredient, In-vitro Dissolution 2.11. Comparison with Marketed Product: The promising formulations F6 obtained in evaluation studies were compared with marketed formulation. The evaluation parameters tested and compared, were drug content uniformity and in-vitro dissolution profile. Mean value of drug content uniformity observed was 98.47  0.578 for Gliclazide (Glizide MR) and 99.23 ± 0.580 for Atorvastatin (Lipifol). The marketed product (Glizide MR) gave 94.018 % of drug release in 8 hours of dissolution study and Atorvastatin (lipifol) showed 99.69 % release in 2 hours of dissolution study. The above study has shown that the in-vitro dissolution profile of formulation F6 was found to be comparable with that of marketed product, which was justified by the F2 value. F2 value obtained for Atorvastatin and Gliclazide was 51.56% and 94.4% respectively. 3. RESULTS AND DISCUSSION Tablets with sustained release matrix of Gliclazide and immediate release of Atorvastatin were prepared and evaluated with an aim to obtain controlled release of Gliclazide and to prevent its first pass metabolism & increase bioavailability. Formulations F1- F9 contains Hydroxypropyl methylcellulose as release retarding polymer. Formulation F1 contains 40% of polymer by total weight of tablet. Formulation F2 contains 30% of polymer by total weight of tablet. Formulation F3 contains 10% of polymer by total weight of tablet. Formulation F4-F9 contains 20% of polymer by total weight of tablet. For Formulations F1–F9, 8 hours of dissolution study was performed. It was found that drug release in F1 and F2 were much retarded to that of desired level; this may be due to more concentration of retardant polymer. In formulation F3 it was found that drug release was not prolonged to desired level; this may be due to inadequate concentration of retardant polymer; i.e. due to low concentration of retardant polymer level employed. In formulation F4 and F5 also that drug release was not prolonged to desired level. In formulation F6-F9 combination of polymers in reduced amount were used. In formulation F6 the release of drugs were found as that of desired level, where Gliclazide showed 89.314% release in 8 hour and Atorvastatin showed 97.6% of release in 2 hours. Again in formulation F7 to F9 It was found that drugs release was much retarded to that of desired level; this may be due to inappropriate ratio of retardant polymers. The drug release may be due to diffusion controlled and swelling controlled mechanism because of inherent swelling characteristic of hydroxyl propyl methylcellulose. The tablets were found swollen at the end of 8 hours indicating a hydrophilic matrix system. In all the above formulations, it was observed that drug release rate was inversely proportional to the concentration of retardant polymer i.e., increase in concentration of retardant polymer resulted in a reduction in the drug release rate.
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Vinod et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 572 0 20 40 60 80 100 120 0 0.5 1 1.5 2 2.5 %drugrelease Time in hour F6 Lipif ol 0 20 40 60 80 100 0 1 2 3 4 5 6 7 8 9 %drugrelease Time in hour F6 Figure.1. Comparative dissolution profile of Atorvastatin and Lipifol in F6 Figure.2. Comparative dissolution profile of Gliclazide and Glizide MR in F6 Table.1. Formulation of immediate release Atorvastatin and sustained release matrix tablets of Gliclazide Ingredient F1 F2 F3 F4 F5 F6 F7 F8 F9 Atorvastatin Calcium 10.80 10.80 10.80 10.80 10.80 10.80 10.80 10.80 10.80 M.C.C. PH 101 59.00 59.00 59.00 59.00 59.00 59.00 59.00 59.00 59.00 Calcium Carbonate IP(Heavy) 34.50 34.50 34.50 34.50 34.50 34.50 34.50 34.50 34.50 Lactose Monohydrate IP 34.33 34.333 34.333 34.333 34.333 34.333 34.333 34.333 34.333 Cross Caramellose Sodium BP 8.00 8.00 8.00 8.00 8.00 8.00 8.00 8.00 8.00 Hydroxy Propyl Cellulose 75 cps 3.14 3.14 3.14 3.14 3.14 3.14 3.14 3.14 3.14 Polysorbate 80 0.65 0.65 0.65 0.65 0.65 0.650 0.650 0.650 0.650 Sodium Ascorbate IP 0.0267 0.0267 0.0267 0.0267 0.0267 0.0267 0.0267 0.0267 0.0267 Water purified IP q.s. q.s. q.s. q.s. q.s. q.s. q.s. q.s. q.s. Gliclazide 30 30 30 30 30 30 30 30 30 H.P.M.C. 4000 cps 120 90 15 60 ---- 30 36 24 42 H.P.M.C. 100 cps ---- ---- 15 ---- 60 30 24 36 18 Microcrystalline Cellulose PH101 143.74 173.74 233.74 203.74 203.74 203.74 203.74 203.74 203.74 Klucel LF 6.261 6.261 6.261 6.261 6.261 6.261 6.261 6.261 6.261 Aerosil 3 3 3 3 3 3 3 3 3 Talc IP 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 Magnesium Stearate IP 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 % of polymer to the total tablet weight 40 30 10 20 20 20 20 20 20 Table.2. Evaluation of tablets (Diameter, Thickness, Hardness) Formulations Mean Diameter (mm)  S.D. ( n=3) Mean Thickness (mm)  S.D. (n=3) Mean Hardness (kg/cm2 )  S.D. ( n=3) F1 12.66  0.083 4.54  0.090 7.83  0.215 F2 12.48  0.006 4.59  0.070 7.64  0.20 F3 12.50  0.070 4.52  0.040 7.50  0.20 F4 12.50  0.067 4.54  0.070 7.50  0.20 F5 12.48  0.070 4.51  0.028 7.55  0.20 F6 12.51  0.006 4.52  0.040 7.51  0.20 F7 12.52  0.050 4.50  0.049 7.52  0.20 F8 12.51  0.103 4.53  0.070 7.52  0.20 F9 12.53  0.063 4.54  0.070 7.53  0.20
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Vinod et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 573 Table.3. In-Vitro dissolution study for formulation for Gliclazide Table.4. In-Vitro dissolution study for formulation for Atorvastatin Table.5. Comparative dissolution profile of Atorvastatin and Lipifol in Formula 6 Cumulative % Drug release Time in hrs Lipifol Atorvastatin (F6) Glizide MR Gliclazide (F6) 0.5. 85.54 80.11 10.5 29.68 1. 92.1 90.77 23.604 36.62 2. 99.69 97.6 24.497 40.16 3. - - 41.267 52.59 4. - - 50.00 60.19 5. - - 60.491 67.551 6. - - 69.861 78.474 7. - - 78.904 84.544 8. - - 94.018 89.314 4. SUMMARY AND CONCLUSION The results of evaluation studies can be summarized as follows such as Results of evaluation of granules exhibited good flowability and compressibility, Uniformity in tablet dimensions implies that die fill was uniform and compression force was constant, Hardness values reveals that tablets are having good mechanical strength and handling characteristics, Friability values dictate good compactness of the formulations, The weight variation of all formulated tablets were satisfactory, attributed by the acceptable flow properties of granules, Content uniformity of active ingredient of all the formulations are within acceptable limit and ensures dosage uniformity. REFERENCE Bechgaar H., Baggeson S, Propoxyphene and norpro- poxyphene: Influence of type of controlled release formulation on intra and intersubject variations, Journal of Pharmaceutical Sciences, 69 (11), 1980, 1327-1330. George M., Grass IV, Robinson J, Sustained and controlled release drug delivery systems, chapter 6 in "Modern Pharmaceutics, edited by Banker G.S., Rhodes C.T., 2nd edition, Marcel Dekker, 1990: 639-658. Gudsoorkar V.R., Rambhau D. 1993, Sustained release of drugs, The Eastern Pharmacist, 27-35. Hui ho-wah, Design and fabrication of oral controlled release drug delivery systems, chapter 9 in Controlled drug delivery; fundamentals and applications, edited by Robinson J.R., Vincent Lee, 2nd edition, Marcel Dekker Inc, 29, 1978: 391-420. Kumar S., Sharma S.M, Controlled Release Dosage Forms, The Eastern Pharmacist, 1991, 17-21. Li. V.H., Influence of drug properties and routes of drug administration on the design of sustained and controlled release systems, Chapter 1 in Controlled drug delivery: Fundamentals and applications, edited by Robinson J.R., Vincent Lee, 2nd edition, Marcel Dekker Inc., 29, 1978, 5-36. Longer M.A., Robinson J.R, Sustained release drug delivery system, chapter 91 in Remington's pharmaceutical sciences 18th edition, Mack Publishing Company, 1990: 1675-1684. Time F1 F2 F3 F4 F5 F6 F7 F8 F9 0.5 hr 3.45 5.67 40.22 12.02 21.45 29.68 2.66 3.53 7.00 1 hr 5.66 8.25 48.12 13.274 33.54 36.62 7.739 8.74 8.754 2 hr 9.87 12.58 55.625 16.619 46.119 40.16 14.639 10.6 30.70 3 hr 15.45 20.58 75.635 28.629 54.379 52.59 17.439 17.97 49.30 4 hr 19.561 26.48 78.712 36.458 55.623 60.19 18.289 18.89 50.05 5 hr 26.58 32.47 80.718 41.67 56.116 67.551 18.489 19.19 50.854 6 hr 30.475 39.89 84.823 45.701 56.333 78.474 19.199 19.38 51.901 7 hr 33.97 43.59 89.862 50.776 57.253 84.544 19.359 19.50 55.281 8 hr 39.88 53.82 91.420 52.314 63.344 89.314 20.199 19.66 55.84 Time F1 F2 F3 F4 F5 F6 F7 F8 F9 0.5 hr 28.55 33.45 35.99 31.99 99.70 80.11 30.54 50.23 55.81 1 hr 37.65 45.56 56.75 53.62 109.98 90.77 46.173 63.085 71.539 2 hr 50.22 62.89 64.57 63.24 118.896 97.6 57.071 77.291 83.835
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Vinod et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 574 Lordi N.G, Sustained release dosage form chapter 14 in Theory and practice of Industrial Pharmacy, edited by Lachman et al., 3rd edition, Varghese Publishing House, 1991: 430-431. Popli H, Sharma S.H, Evaluation of sustained release formulations, The EasternPharmacist, Jan, 1990, 75-79. Welling P.G., Dobrinska M.R, Dosing considerations and bioavailability assessment of controlled drug delivery systems chapter 6 in Controlled drug delivery: fundamentals and applications, edited by Robinson J.R., Vincent Lee, 2nd edition, Marcel Dekker Inc., 29, 1978: 254-289
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Ranjitsingh et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 575 PHYTOCHEMICAL SREENING AND ANTIDIABETIC ANTIOXIDANT EFFECT OF ECBOLIUM LIGUSTRINUM FLOWERS EXTRACTS Ranjitsingh B Rathor* , Rama Rao D, Prasad Rao Department of Pharmacology, M.A.M College of Pharmacy, Narasaraopeth, Guntur, AP *Corresponding author: Email:rbnrathor@gmail.com ABSTRACT The antidiabetic and antioxidant activity of Ecbolium ligustrinum (family:Acanthaceae) was investigated in alloxan induced diabetic albino rats. A comparison was made between both plant extracts and a known antidiabetic drug Glibenclamide (5mg/kg body weight). The dried flowers of Ecbolium ligustinum was subjected to extraction by continuous hot extraction method using hydroalcohol and chloroform as a solvent and were subjected to standardization using pharmacognostical and phytochemical screening. Phytochemical estimation was done for the presence of vital phytoconstituents by T.L.C method. Dose selection was made on the basis of acute oral toxicity study (H.E.A 300 mg/kg body weight C.E 200 mg/kg body weight) as per OECD and CPCSEA guidelines. Oral administration of extract of Ecbolium Ligustrinum for 21 days resulted in significant reduction in blood glucose level. Alloxan induced diabetic rat model was used for evaluation of antidiabetic activity. Biochemical parameters were analysed in support of antidiabetic activity. Antioxidant activity was assessed by the lipid peroxidation (LPO) in liver. The antioxidant enzymes Catalase (CAT), Reduced Glutathione (GSH), Superoxide Dismutase (SOD) in liver were decreased. With histopatholgical studies on pancreas and liver hydroalcoholic extract given more effect in comparison to chloroform extract in lowering blood glucose and oxidant levels. The plant has hypoglycaemic and antioxidant effect. Key worlds: - E. Ligustrinum, acute toxicity study, alloxan, antioxidant study. INTRODUCTION Ecbolium legustrinum is a shrub belongs to family acanthaceae, commonly called as ‘nakka toka’ in regional language. Till now antidiabetic and antioxidant work has not been evaluated for this species of acanthaceae family. Studies has been revealed that it act as a good hypoglycaemic and antioxidant effect. MATERIALS AND METHODS Chemicals: Alloxan monohydrate, glibleclamide, dextrose, tween-80, auto-analyser, (analytical technology limited) and One-touch (horizon).All other chemicals and reagents used were analytical grade. Plant material: Fresh flowers were collected from surrounding of Nellore district (A.P), India and authenticated by Dr.C.V.S. Baskar, Professor, Department of Botany, Venkategiri Raja’s College, S.V.University, Nellore, Andhra Pradesh, India. Preparation of plant extraction: The collected were shed dried and powdered in mixer grinder to get a coarse powder. The powdered flowers were defatted with petroleum ether and later extracted with hydroalcohol and chloroform. The extract was evaporated to dryness, gave a residue of 38% w/w and 29% w/w respectively. Phytochemical screening: A preliminary Phytochemical screening of hydroalcoholic and chloroform extracts of Ecbolium Ligustrinum was carried by using standard procedures. Acute Oral Toxicity Studies: Acute oral toxicity studies of extracts was carried out as per OECD guidelines, draft guidelines 423 adopted and received from Committee For the Purpose of Supervision and Control of Experiments on Animals(CPCSEA), Ministry of Social justice and Enpowerment, Goverment of India. Administration of stepwise doses of both extracts of E. ligustrinum 40 mg /k.g body weight to 2000mg /k.g body weight caused no considerable signs of toxicity in the tested animals. One tenth of the upper limit doses were selected as the level for examination of antidiabetic activity. Experimental Model: Alloxan monohydrate was weight indivisually for each animal according to their weight and solublized 0.2 ml saline just prior to injection. Diabetes was induced by injecting it at a dose of 120 mg/kg body weight intrapertonially. After 1 hr of alloxan administration, the animal were given ad libitum and 5% dextrose solution was also given in feeding bottle for a day to overcome the early hypoglycaemic phase. The animals were kept under observation and after 72 hrs blood glucose level was measured by One-touch glucometer. The diabetic rat (glucose level 200-300 mg/dl) were separated and divided in five different groups for experimental studies, with each group containing five animals.
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Ranjitsingh et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 576 Experimental Designs: Different group of rats were used to study the effect of H.A.E and C.E. of E.ligustrinum. The rats were divided into five groups each consist of five rats. Significant hyperglycemia was achieved within 48 hrs after alloxan (120 mg/k.g body weight i.p) injection induced diabetic rats with more than 220 mg/dl of blood glucose were identified as to be diabetic and used for the study. In acute study all the surviving diabetic animals were fasted overnight blood samples were collected from the fasted animals prior to the treatment with above schedule and after administration,at each day upto 21 days. Group I Normal/Controll received 1% tween 80, 1ml per orally. Group II Alloxan (120 mg/kg body weight) induced diabetic animals received in 1% Tween 80, 3ml /body weight i.p Group III Alloxan (120 mg/kg body weight) induced diabetic animals received Glibenclamide ( 5mg/kg body weight) i.p Group IV Alloxan (120 mg/kg body weight) induced diabetic animals received H.A.E (300 mg/kg body weight) per orally Group V Alloxan (120 mg/kg body weight) induced diabetic animals received C.E (200mg/kg body weight) per orally Statical Analysis: The result of the study were subjected to one way analysis of variance (ANNOVA) fallowed by Dunnet’s test for multiple comparisions. Values with p<0.05 were consider significant. RESULTS AND DISCUSSION Phytochemical Screening: Phytochemical screening of Hydroalcoholic extracts of Ecbolium Ligustrinum showed the presence of various chemical constituents mainly tannins flavanoids, glycosides, proteins and chloroform extract showed the presence of alkaloids, sterols. Proteins, glycosides and flavanoids isolated from H.A. Extract may be responsible for antidiabetic and antioxidant properties. The result obtained was comparable and satisfied the standard literature. Acute oral toxicity studies: In acute toxicity study,none of the studied hydroalcoholic extracts of flowers showed any toxicity sign when observed for the parameters during the first four hours and fallowed by daily observations for 14 days and mortality was also not observed. One tenth of this dose level was taken as effective dose. Both extracts were experimented at the dose of 300 mg and 200 mg/k.g.b.w.in order to ascertain a scientific base of for the useful of this plant in the treatment of diabetes. It was decided to evaluate experimental design of antidiabetic activity by Alloxan induced model. Effect of extracts on blood glucose level of normal rats: The hydroalcoholic extract exhibited remarkable blood glucose level after 4hrs and chloroform extract exhibited lowering in blood glucose level after 8 hrs. Alloxan induced diabetic model: As expected in the diabetic control,there was severe hyperglycemia as compared to the normal animals. Compared to the diabetic control,the H.A.E and C.E lowered the elevated blood glucose level in chronic treatment Table-2.It was observed that the standard drug glibenclamide lowered the blood glucose level significantally,ranging back nearly back to normal, whereas H.A.E and C.E significantally(p<0.01) decreased fasting blood glucose level in diabetic rats on 14th and 21st day as compared to intial (0 day) blood serum glucose levels. When H.A.E and C.E were compared for their antidiabetic activity in compare to active control, particularly glibleclamide the result showed that their potential was lesser but significant (**p<0.01) than the standard drug at chronic level. Effect of extracts in Lipid peroxidation and Antioxidants Levels on liver of Alloxan Induced rats: The level of antioxidant enzymes : CAT,GPx and SOD and lipid peroxidation are given in Table 4.the antioxidant enzyme activities significantly decreased in liver but the lipid peroxidation level were increased in alloxan induced rats.when treated with both extracts the alloxan induced diabetic rat showed increased in antioxidant activities than the untreated rats with extract. The lipid peroxidation level were reduced in flower etract treated rats.in flower extract administered control rats,the peroxidation enzyme levels were near normal values similar to untreated control rats. In the recent time many traditionally used medicinally important plants were tested for their antihyperglycemic and antioxidant properties by various
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Ranjitsingh et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 577 investigators in experimental animals. I have undertaken a study on Ecboium ligustrinum for their antidiabetic and antioxidant activity. Preliminary phytochemical screening revealed that H.A.E showed positive response to alkaloids, Tannins, Flavanoids, carbohydrates, lignin, and proteins and in C.E showed positive response. DM has a significant impact on the health, quality of life and life expectancy of patients as well as healthcare expenditure. With increasing incidence and mortality from its complications, prompt and adequate glycaemic control in diabetes is paramount if management can meaningfully improve the quality of life and increase the life expectancy. In this study experimental evaluation of the antidiabetic potential of E. ligustrinum has shown that single oral administration of the extract to normal rats reduced fasting blood glucose which suggest in inherent hypoglycaemic effect. The time couse of the events also showed that the extract reduced glucose level to below basal in less than 2 hrs compared to the 2.5 hrs for glibenclamide to achive a return to the basal levels chronic hyperglymia in DM is a risk factor constantly fuel post-prandial elevation of blood glucose. In the antidiabetic activirty studies daily oral administration of the extracts for 21 days produced a gradual but sustained reduction in blood glucose in diabetic rats from jypts. Alloxan causes hyperglycemia and glucose intolerance are syndromes similar either type I and type II DM effective and sustained reduction in blood glucose levels of treated diabetic rats by the extract indicates it may be usefull overt cases of DM/treatment with the extract also reduced mortalty of diabetic rats from hyyperglycemia and prolonged their survival in this study the diabetic nontreated control animals all died on day 10 post induction of diabeties whereas the extract treated survived beyond the period of experiments. Hence chronic administration of the extract may cause a progressively sustained reduction in hyperglycemia known to reduce the risk of complications associated with the disease. Several factors such as oxidative straess chornic hyperggymia and autoimmunue of fibrocalculus types of chronic pancreatitis damage the pancreas impaired insulin secretion and hense glycemic control.result of histological studies on pancreas isolated from treated diabetic rats showed that the extract may have repaired the pancreas damaged by alloxan. Alloxan owes its diabetogenic potential to destruction of cells of the islets which consequentaly impairs insulin secretion and gives rise to hyperglycemia.tretment with the extract may have restored the integrity and perhaps,functions f the damaged pancreatic tissues.Glibenclamide used as refrance hypoglymic agent did not cause such effect. The presice mechanism of tissue repair is not known.however due to large implication of oxidative stress in damage to the pancreas,it seems responsible to suggest that the antioxidant effect of this plant may play a key role in protecting pancreatic tissues from oxidants including that generated by alloxan.alloxan destroys insulin producing pancreatic cells through the formation of reactive oxygen species that cause tissue damage.the hypoglycaemic effect of the extract may further protect the pancreas from the deleterious effect of chronic hyperglycemia. Rather than possessing a direct tissue repair effect,it is likely that the extract, through antioxidant and hypoglymic effect, protected the already compromised pancreas from further assault repair process to peoceed and restore the tissue. However, it is not clear if the repaired tissues also had their functions fully or partially restored since the blood glucose level of the animals did not return to basal or pre-treatment levels as at the end of experiment. A return to basal or pretretment levels would have indicated full restoration of insulin secretion by the repaired pancreatic tissues. While the antidiabetic effect of the extract may derive from its hypoglycaemic effect, the mechanisms of the hypoglycaemic effect are yet to be elucidated. The hypoglymic effect in normal and diabetic rats suggests insulin like effect probably mediated via peripheral glucose consumption. Phytochemical analysis of the extract revealed the presence of flavanoids, alkaloids, carbohydrates, glycosides and tannins which are typical plant constituents. although there are chances that any of these constituents may possesses antidiabetic properties, extract of the whole plant may be more effective since the result of bioactivity-guided studied did not reveal any increase in magnitude of hypoglycaemic effect fallowing solvent guided fractionation this observation precluded further fractionation. This observation precluded further fractionation of the extract in this study. It would thus be difficult at this stage to attribute the anti-diabetic to any single or group of constituents.
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Ranjitsingh et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 578 Table.1.Effect of different extract on glucose level in normal rats Values are Mean ± SEM (n=5) one war ANOVA followed by Dunnett’s test. Where, *** represent very significant P<0.001, **represent significant at P<0.01, * represent significant P<0.05 and ns represents not significant. H.A.E.L.F: Hydroalcoholic extract of Ecbolium ligustrinum flowers C.E.E.L.F: Chloroform extract of Ecbolium ligustrinum flowers Table.2.Blood glucose level of Alloxan induced diabetic model Group Blood Glucose Level Day 0 Day 7 Day 14 Day 21 Normal 87.27 90.49 93.08 92.03 Diabetic 285.5 308.93 325.72 333.02 Standard 211.4 98.25 91.13 87.22 H.A.E.E.L.F 287.3 109.38 104.86 102.08 C.E.E.L.F 298.9 * 156.78** 117.38** 112.25** Values are Mean ± SEM (n=5) one war ANOVA followed by Dunnett’s test. Where, *** represent very significant P<0.001, **represent significant at P<0.01, * represent significant P<0.05 and ns represents not significant. Table.3. Effect of oral administration of the H.A.E and C.E of Ecbolium ligustrinum flowers on serum profile in experiments. Group Serum insulin (micron I.U / ml) Total protein (g/dl) Serum albumin (g/dl) Serum Globulin (g/dl) Total cholesterol (mg/dl) Serum triglycerides (mg/dl) SGOT (I.U/L) SGPT (I.U/L) Serum amylase SCU/100 ml Normal 45.00+ 1.05 7.65+ 0.12 4.51+ 0.08 2.53+ 0.04 84.59+ 1.39 78.54+ 0.68 135.05+ 2.36 77.43+ 1.15 86.15+ 3.21 Diabetic 17.85+ 0.39a*8 7.13+ 0.21a* 2.34+ 0.7a** 1.84+ 0.06a** 103.87+ 3.36 a** 383.32+ 8.96 a** 221.00+ 7.07a** 107.01+ 2.69a** 181.30+ 11.74a** Standard 39.83+ 0.72 b** 7.89+ 0.14b* 4.58+ 0.08 b** 2.31+ 0.04 b** 84.6+ 1.52 b** 85.60+ 1.47b** 150.92+ 2.25b** 82.73+ 1.44b** 101.72+ 4.00b** Hydro- alcoholic 36.23+ 0.82b** 7.12+ 0.22bNS 3.96+ 0.09b** 2.60+ 0.06b** 90.47+ 2.56b** 92.68+ 0.24b** 167.75+ 3.40b** 87.38+ 2.03b** 116.06+ 5.40b** Chloroform 34.51+ 0.71b** 6.96+ 4.14bNS 4.14+ 0.09b** 2.64+ 0.04b** 93.67+ 2.29b** 98.62+ 1.72b** 182.33+ 3.69b** 96.37+ 1.83b** 124.77+ 6.36b** Values are Mean ± SEM (n=5) one way ANOVA followed by Dunnett’s test. Where, *** represent very significant P<0.001, **represent significant at P<0.01, * represent significant P<0.05 and ns represents not significant. Table.4.Levels of SOD, CAT, GPX and TLP in Liver of control and experimental animals Group SOD(U/mg) CATALASE(n moles/mg GPX(n moles/mg) TLP(n moles/mg) Normal 88.9 65.8 468 21.9 Diabetic 69.0* 59.1* 307* 37.3* Standard 86.5** 63.9** 457** 28.6** H.A.E.E.L.F 83.8** 60.2** 448** 34.2** C.E.E.L.F 71.3** 56.0** 426** 31.8** Values are expressed as mean + SEM of six samples; *P<0.01 when compared to control,**P<0.01 when compared to diabetic; Data were analyzed by One-way ANOVA followed by Dunnett’s test Units: SOD-Units/mg protein; CAT-n moles of H2O2 decomposed /min/mg protein, LPO- nmoles of malondialdehyde/mg protein; GPx n moles of GSH oxidized/min/mg protein. Diabetic: Alloxan induced 45mg/kg; Standard: Glibenclamide 5 mg/kg Groups Treatment Blood glucose (mg/dl) 0 1 2 4 8 12 Group –I Saline 88.00 91.66 95.33 85.5 77.50 63.17 Group-II Glibenclamide (5mg/kg) 83.83 64 .50 65.50 49.50 39.17 38.33 Group-III H.A.E.E.L.F (300mg/kg) 84.50*** 72.66* 70.33** 59.50 51.17 45.16 Group IV C.E.E.L.F (200 mg/kg) 84.16** 88.66* 76.83* 72.00** 66.83* 62.67**
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Ranjitsingh et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 579 Histopathological images of Pancreases Group I : Normal(Saline) Grup II:Alloxan induced(120 mg/kg i.p) Group III:Glibenclamide 5mg/kg i.p Group IV: Hydroalcoholic extract 300mg/kg orally Group V : Chloroform extract 200 mg/kg orally Group –I(normal control) b) Group II(diabetic control) islets with fatty infiltration show damaged and atrophic islets with acni c)Group III(Glibeclamide) islets are small d) Group –IV(H.A.E) islets with normal structural intactness with their nucleus e) Group-V(C.E) islets with normal round and elongated. Photomicrograph of Liver stained with Hematoxylin and Eosin (magnification × 400) Histopathological images of Pancreases Group I:Normal liver(saline) Group II: Diabetic Liver (Alloxan 120mg/kg b.w) Group III: Liver treated with Glinenclamide(5mg/kg b.w) Group IV: Liver treated with H.A.E (300mg/kg b.w) Group V: Liver treated with C.E (200mg/kg b.w) Group-I (normal control)-hepatocytes are normal b)Group –II(Diabetic control) with shrunken nuclei, granular cytoplasm and diluted sinusoids c) Group-III (Glibenclamide) Normal hepatocytes, dilated central veins are seen d) Group IV-focal areas of necrosis are seen and hepatocytes are normal. Group V- Perivascular round cell collection (PVRCC) and portal tract infiltration with lymphocytes are normal.Occassional dilated central veins are seen along with occasional focal areas of necrosis.
    • ISSN: 2321-5674(Print) ISSN: 2320 – 3471(Online) Ranjitsingh et.al Indian Journal of Research in Pharmacy and Biotechnology Volume 1(4) July-August 2013 Page 580 CONCLUSION The antioxidant study measured on the parameters of enzyme levels and histopathology of the liver tissues. The results were proved to be that the extracts showed the effect on improved enzyme level and tissue repairing.Thus above all on the basis of results, the plant extracts has worthy effect on both antidiabetic and antioxidant activity. REFERANCES Abu hasanat, antidiabetic and antioxidant effect of Scoparia Dulcis in alloxan induced albino mice.international journal of pharmtech research, 2(4), 2010, 2527-2534. Anonymous, The Ayurvedic Pharmacopoiea of India, part I, vol. III, 1st Edition, Govt.of India,Ministry of Health and Family welfare,Department of Indian system of Medicine and Homeopathy,New Delhi, 2001, 234-237. Bhakuni D.S, Dhar M.L, Dhar M.M, Dhawan B.N and Mehrotra B.N, Screening of Indian Medicinal Plants for biological activity, Part II, Indian Journal of Experimental Biology, 7, 1969, 250. Md Shamsuddin Sultan Khan, the study of analgesic, antidirroheal and antioxidant effect of ethanolic extacts of Ecbolium linneanum in albino-mice.interantional journal of phytopharmacy, 3(1), 2013, 30-36. OECD guidelines for the for the testing of chemicals,revised draft guidelines 423;acute oral toxicity-acute toxic class method,revised documents, CPCSEA, Ministry of social justice and Enpowerment, Govt.of India, 2000.