Prokinetic effect of herbomineral unani formulation (dolabi) in diabetic rats

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Prokinetic effect of herbomineral unani formulation (dolabi) in diabetic rats

  1. 1. ISSN 0970-7700 THE JOURNAL OF RESEARCH AND EDUCATION IN INDIAN MEDICINE An International Quarterly Volume XVIII : 1 e-Version Jan. - March, 2012 Prokinetic Effect of Herbomineral Unani Formulation ( Dolabi ) in Diabetic Rats Rahul Somani, Abu Shaikh, Dilpesh Jain and Rajkumar Shete Energy Dispersive X-Ray Spectroscopy in Quality Control of Powdered Herbal Formulation - Avipattikar Churna K. Jayaram Kumar and Mouli Nandi Clinical Trial of Garbhpal Ras in Pregnancy Outcome Deepa Mishra, Mukta Sinha and Vikas Kumar Inhibitory Effect of the Root of Sida acuta Burm. F. on Calcium Oxalate Crystal Growth T. Vimala and S. Gopalakrishnan Spectroscopic Investigations of Palakarai (Cowrie shell) Parpam S. Joseph Vedhagiri, K. Ganesan and P.C. Jobe Parabakar Hypoglycaemic Activity of Indian Medicinal Plants in Streptozotocin Diabetic Rats E.N. Sundaram, K.P. Singh and P. Umamaheswara Reddy Pharmacological Screening of Cassine albens (Retz.) Kosterm (Celastraceae) for Antidepressant and Anxiolytic Activity in Rodents P.H. Patil, M.B. Gagarani, K.R. Patil and S.J. Surana Screening of Ximenia americana L. for it’s Anti-inflammatory Activity M. Siddaiah, K.N. Jaya Veera, P. Mallikarjuna Rao, K. Yogananda Reddy and C. Madhusudhana Chetty In-vitro Antioxidant Capacity of Graded Doses of Methanolic Extract from Luffa cylindrica (L) Seeds K. Nagarajan, Satyajit Dutta, Sumit Das, Surabhi Singhal, Pallavi Saxena, Avijit Mazumder and L.K. Ghosh Antibacterial Activity of Alcoholic Extract of Aloe vera L. by Disc Diffusion Method G.S. Niture, M.K. Patil, A.G. Karpe and A.V. Bhonsle
  2. 2. e-Version J. Res. Educ. Indian Med., Vol. XVIII (1) : 2012 Conten EDITOR - IN - CHIEF Prof. Em. R.H. SINGH CONTENTS ABMS, Ph.D, D.Sc. Professor Emeritus Banaras Hindu University Formerly Vice Chancellor, Rajasthan Ayurveda University; Dean, Faculty of Ayurveda, IMS, BHU, Varanasi - 221005 UP (India) FOUNDING Editor Prof. (Dr.) Suresh Kumar M.D.(Ay.), Ph.D. Kayachikitsa (BHU) Formerly Dean, Faculty of Ayurveda, Himachal Pradesh University, Shimla Director, Indian Institute of Panchakarma, CCRAS (AYUSH,MoH&FW,GoI) (Kerala) CONSULTANT SUBJECT EDITORS Prof. Ranjana Patnaik Ph.D. (BHU) Professor, School of Bio Medical Engg, Institute of Technology, BHU, Varanasi - 221005 (India) Dr. Sanjeev Rastogi E-mail: rastogisanjeev@rediffmail.com Ravindra G. Mali Assistant Professor, Pharmacognosy & Phytochemistry L.B.Rao Institute of Pharmaceutical Educ. & Research, B.D.Rao College Campus, Khambhat - 388620 (Gujarat) ADVISORY-EDITORS (INTERNATIONAL) M.Sc., Ph.D. Associate Professor, Immunology & Cell Biology Institute for Technology & Research Innovation (ITRI), Deakin University, Victoria - 3217 (Australia) E-mail: jagat.kanwar@deakin.edu.au Dr. Marc Halpern President, California College of Ayurveda E-mail: drh@ayurvedacollege.com Clinical Trial of Garbhpal Ras in Pregnancy Outcome Deepa Mishra, Mukta Sinha and Vikas Kumar ... 13-19 Inhibitory Effect of the Root of Sida acuta Burm. F. on Calcium Oxalate Crystal Growth T. Vimala and S. Gopalakrishnan ... 21-26 Spectroscopic Investigations of Palakarai (Cowrie shell) Parpam S. Joseph Vedhagiri, K. Ganesan and P.C. Jobe Parabakar ... 27-32 Hypoglycaemic Activity of Indian Medicinal Plants in Streptozotocin Diabetic Rats E.N. Sundaram, K.P. Singh and P. Umamaheswara Reddy ... 33-43 Pharmacological Screening of Cassine albens (Retz.) Kosterm (Celastraceae) for Antidepressant and Anxiolytic Activity in Rodents P. H. Patil, M.B. Gagarani, K.R. Patil and S.J. Surana ... 45-50 Screening of Ximenia americana L. for it’s Anti-inflammatory Activity M. Siddaiah, K.N. Jaya Veera, P. Mallikarjuna Rao, K. Yogananda Reddy and C. Madhusudhana Chetty ... 51-54 Girish Mishra M.D.(Ay) Kayachikitsa (BHU) Samita Puri M.S.(Ay) Shalakaya (HPU) Aditya Puri BAMS (JU), Dip.Yoga (BHU), Davinder Singh M.D.AncientAyurveda P. Ltd. JREIM ADMIN. OFFICE: Dr. (Mrs.) Laxmi Bhargava Ph.D. (BHU) Executive Editor In-vitro Antioxidant Capacity of Graded Doses of Methanolic Extract from Luffa cylindrica (L) Seeds K. Nagarajan, Satyajit Dutta, Sumit Das, Surabhi Singhal, Pallavi Saxena, Avijit Mazumder and L.K. Ghosh Antibacterial Activity of Alcoholic Extract of Aloe vera L. by Disc Diffusion Method G.S. Niture, M. K. Patil, A.G. Karpe and A.V. Bhonsle ... 55-59 Conferences and Forthcoming Events ASSISTANT EDITORS Dr. Dr. Dr. Dr. Energy Dispersive X-Ray Spectroscopy in Quality Control of Powdered Herbal Formulation - Avipattikar Churna K. Jayaram Kumar and Mouli Nandi ... 7-11 M.S.(Ay.) (BHU) Assistant Professor-Shalakya Tantra Institute of Medical Sciences, BHU, Varanasi - 221005 (India) Dr. Jagat Kanwar Prokinetic Effect of Herbomineral Unani Formulation ( Dolabi ) in Diabetic Rats Rahul Somani, Abu Shaikh, Dilpesh Jain and Rajkumar Shete ... 1-6 M.D.(Ay.) (BHU) Head, Dept. of Pancha Karma State Ayurvedic College & Hospital Lucknow - 226004 (India) Dr. Manoj Kumar ISSN 0970-7700 (Linking) ... ... 61-63 E-mail: drlaxmi_ayush_journal@yahoo.com TYPE SETTING Arvind Kumar 64 Dip. Computer Engg. E-mail: managerjreimhp@gmail.com The articles published in this Journal are selectively abstracted / indexed in M EDICINAL & A ROMATIC P LANTS A BSTRACTS (MAPA) CSIR publication Reporting Current World Literature (Bimonthly), Standard International ISO Abbreviation of JREIM is: J. Res. Educ. Indian. Med.
  3. 3. NLM ID : 8602438 (NlmId) ISSN : 0970-7700 (Print) 0970-7700 (Linking) THE JOURNAL OF RESEARCH & EDUCATION IN INDIAN MEDICINE Journal of Research and Education in Ayurveda, Yoga, Naturopathy, Unani, Siddha, Homeopathy, Complementary and Alternative Medicine, Integrative Medicine, Medicinal and Aromatic Plants, Pharmaceutical Sciences …… An International Quarterly Call for Authors The Editorial Board of The Journal of Research and Education in Indian Medicine (JREIM) (Website: www.jreim.com/aboutus.htm ) is interested in receiving and reviewing manuscripts in all areas of Research and Education in Ayurveda, Yoga, Naturopathy, Unani, Siddha, Homeopathy, CAM, Integrative Medicine, Medicinal plants and Pharmaceutical Sciences in general and on education of AYUSH sector in particular. Papers submitted for consideration for publication in The Journal of Research and Education in Indian Medicine (JREIM) will be reviewed by at least two expert members of the Editorial Review Board with appropriate credentials and expertise in the specific topic covered. On our web site click on the links under the “Information for Authors” section for complete guidelines for authors and submission requirements. Manuscripts can be electronically submitted by e-mail to editor@jreim.com or any Subject Editor The Journal of Research and Education in Indian Medicine (JREIM) invites submission of Review articles and research papers in all areas of Research & Education in Ayurveda, Yoga, Naturopathy, Unani, Siddha, Homeopathy and other Traditional/Alternative/Oriental Systems of Medicine. The sole criterion for publishing is academic and scientific accuracy. We invite anyone who is involved in research in any relevant area to submit his or her manuscript for consideration of publication. The Journal of Research and Education in Indian Medicine (JREIM) is committed to rapid review and publication. Your manuscript will be published, once accepted, within 90 days of acceptance. An Invitation to Subscribe the Journal I hope that after examination of our Journal and other JREIM publications, you will decide that some or all of these publication merit being recommended to your main or departmental library for current subscription and for acquisition of Special Issues of some of the back volumes. If you have any questions, please submit them through our website: www.jreim.com/aboutus.htm or e-mail to editor@jreim.com Prof. Suresh Kumar Founder Editor Prof. Em. R.H. Singh Editor-in-Chief * The manuscripts submitted in proper JREIM format/typesetting/style are only Registered for publication and assigned Manuscript Number and Reviewers
  4. 4. J. Res. Educ. Indian Med., Jan. - March, 2012 Vol. XVIII (1) : 1-6 ISSN 0970-7700 PROKINETIC EFFECT OF HERBOMINERAL UNANI FORMULATION (DOLABI) IN DIABETIC RATS RAHUL SOMANI,1* ABU SHAIKH,1 DILPESH JAIN1 AND RAJKUMAR SHETE2 Sinhgad College of Pharmacy,1 Pune - 411041 Maharashtra (India) RD’s College of Pharmacy,2 Bhor - 412206 Maharashtra (India) Abstract: This study was undertaken to investigate the prokinetic activity of Unani herbomineral formulation (Dolabi) in streptozotocin induced diabetic rats and its in vitro antioxidant activity. Rat model of diabetes was established by intraperitoneal injection of streptozotocin (55 mg/kg, i.p). Rats were divided into three groups: Normal control, diabetic control and treatment groups. After two weeks of treatment, rats were administered with phenol red meal followed by last dose of Dolabi and they were screened for gastric emptying (GE), intestinal transit (IT) and in vitro study of distal colonic smooth muscle. In vitro antioxidant activity of Dolabi was assessed on the basis of radical scavenging activity of the stable Diphenyl-2-picryl-hydrazyl (DPPH) free radical. Percentage of GE and IT was significantly (P < 0.05) decreased in diabetic rat as compared to normal control groups. In streptozotocin induced diabetic rats, Dolabi significantly (P < 0.05) accelerated both GE and IT as compared to diabetic control rats. Significant (P<0.01) increase in EC50 of ACh in rat distal colon was observed in diabetic rats as compared to normal rats. Where as diabetic rats treated with Dolabi showed significant (P<0.01) decrease in EC50 of ACh in distal colon as compared to diabetic control group. In in vitro study, Dolabi showed potent radical scavenging activity to stable DPPH-free radical with IC50 value of 231.09. Dolabi may exert its prokinetic effect by reducing oxidative stress and therefore can be used as drug for treating diabetic patients with gastrointestinal impairments. Keywords: Dolabi, Unani medicine, Herbal medicine, Diabetes, Oxidative stress, Medicinal plants, Diabetic neuropathy. Introduction Oxidative stress play an important role in the pathogenesis of chronic complications of diabetes mellitus (Ziegler and Gries, 1997) including gastroparesis (James et al., 2008). The conditions that lead to the over production of the precursors of ROS and/or reduce the efficiency of scavenging system are shown to be responsible for the development of oxidative stress. Hyperglycemia play an important role in generation of reactive oxygen species (ROS) and that lead to chronic diabetic complications including diabetic autonomic neuropathy. Gastroparesis is the most common symptom of diabetic autonomic neuropathy (Kong et al., 1999) which is reported to cause considerable morbidity in patients. Gastroparesis leads to abnormal gastric motility, characterized by delayed gastric emptying (GE) and intestinal 1* Corresponding Author transit (IT) (Shamaila et al., 2009). The abnormal gastrointestinal motility among diabetic patients seemes to be a clinical manifestation of diabetic autonomic neuropathy (Punkkinen et al., 2008) and some reports concluded that these gastrointestinal disturbances may be due to the damage of peripheral cholinergic neurons as a result of oxidative stress (Bijender et al., 2003; De Winter et al., 2005). Herbomineral formulation (Dolabi) is used for treatment of diabetes and its complications in an Unani system of medicine. It contains Gymnema sylvestre, Eugenia jambolana, Bambusa arundinacea, Rumex vescarricus, Acacia arabica, oxide of egg shell, oxide of iron rust, zinc oxide (Table 1). Some of these ingredients have been reported to possess both anti-diabetic as well as antioxidant activity such
  5. 5. 2 Somani et al. IAEC/Approval/2008-09/05) by the Institutional Animal Ethics Committee. Animals were housed under standard laboratory conditions at controlled temperature 25 ± 1ºC with 50-60% relative humidity in a normal 12 h light and dark cycle with free access to water and standard laboratory feed ad libitum. as E. jambolana (Sagrawat et al., 2006), A. arabica (Wadood et al., 1989; Sundaram and Mitra, 2007), zinc oxide (Robert and Silvestro, 2000) where as Gymnema sylvestre known for its antidiabetic effect (Gholap and Kar, 2003). So far there is no scientific evidence about efficacy of this formulation in preclinical models of impaired gastrointestinal motility and in vitro antioxidant activity. The present investigation was under taken to evaluate the effect of Dolabi on impaired gastrointestinal motility, colonic smooth muscle response to exogenous acetylcholine (ACh) in streptozotocin (STZ) induced diabetic rats and in vitro antioxidant activity. Overnight fasted rats were injected with streptozotocin (55 mg/kg, i.p.) dissolve in 0.1 M cold citrate buffer (pH 4.45). The blood was withdrawn 48 h later by retro orbital method, serum was separated and fasting serum glucose level was determined using the glucose oxidase -peroxidase method (Miskiewicz et al., 1973). Rats with a serum glucose level > 250 mg/dl were considered as diabetic and used for further study. Age matched five non-diabetic rats were used as normal control group and received 0.5ml of 0.1 M cold citrate buffer (pH 4.45). Materials and Methods Drug and Chemicals Dolabi (Hamdard) a herbomineral formulation, purchased from local market. Streptozotocin, phenol red and DPPH were purchased from Sigma (U.S.A.). Study design for gastrointestinal transit and in vitro study on rat distal colon After persistent hyperglycemia for two weeks, diabetic rats were divided into two groups. Group one was diabetic control, received distilled water (10 ml/kg) and second group served as treatment, received suspension of Dolabi in distilled water (140 mg/kg, p.o) for next two Rat model of diabetes Wistar rats of either sex weighing 180230g were purchased from Haffkine Bio-Pharma Corporation Ltd., Mumbai (India). All the experimental procedures and protocols used in this study were reviewed and approved (SCOP/ Table 1. Contents of Unani herbomineral formulation (Dolabi) Sr. No Name of content Unani Name 1 Aqaqiya 2 Banslochen 3 Tukhm Hammaz 4 Gurmar Booti 5 Maghz Jamun Labba Buz 6 Kushta Baiz Murgh 7 8 9 10 Kushata Khabsul Hadeed Kushta Jast Gond Safaid Labba Buz Botanical Name (Family) Acacia arabica (Fabaceae) Bambusa arundinaceae (Bambusaceae) Rumex vesicarius (Polygonaceae) Gymnema sylvestre (Asclepiadaceae) Syzygium cumini (Myrtaceae) ------ Quantity English Name Gum Arabic Tree 166.6 mg Thorny bamboo 132.0 mg Rosy Dock, Dock Sorrel, Bladder Dock Gymnema 83.3 mg 27.7 mg Black Plum, Java Plum 27.7 mg Egg gallius domestics (oxide of egg shell) Iron (oxide of iron rust) Zinc oxide --- 13.8 mg 13.8 mg 41.6 mg 41.6 mg 200.0 mg
  6. 6. Prokinetic Effect of Herbomineral Formulation in Diabetic Rats weeks. Age matched five non-diabetic rats were used as normal control group and received distilled water (10 ml/kg). Gastrointestinal transit and in vitro study on rat distal colon were performed. Gastric emptying and intestinal transit After administration of last dose of Dolabi to the overnight fasted rats, 1.5 ml of a phenol red meal, consisting of phenol red (0.05%, w/w) in 1.5% methylcellulose, was given through gavage feeding. Twenty minutes later, the rats were sacrificed by cervical dislocation. Their stomachs were clamped with a string above the lower oesophageal sphincter and a string beneath the pylorus to prevent leakage of phenol red. Gastric emptying was determined spectrophotometrically. The stomach of each rat resected just above the lower oesophageal sphincter and pyloric sphincter. Phenol red remained partly in the lumen of the stomach. The stomach and its contents were put into 5 ml of 0.1 mol/l NaOH. The stomach was minced. The samples containing the total amount of phenol red present in the stomach were further diluted to 25 ml with 0.1 mol/l NaOH and left at room temperature for 1 h. The supernatant (5 ml) was then centrifuged at 800 g for 20 min. The absorbance was read at a wavelength of 546 nm on a spectrophotometer (Shimadzu, Japan) and the phenol red content in the stomach was calculated. Percentage of gastric emptying of the phenol red was calculated as [(infusion amount-remains) / infusion amount] × 100. The intestinal transit (IT) of phenol red meal was determined by modified Janseen method (Janseen and Jagenerous, 1957). The small intestine was removed from the pyloric sphincter to the ileocecal junction and the distance travelled by the phenol red meal was noted and expressed as percentage of intestinal transit calculated as [distance traveled by phenol red meal/ total length of small intestine] × 100 In vitro study on rat distal colon Immediately after cleaning and measuring the length of large intestine, 2 cm distal colon 3 was cut and used for in vitro study. The distal colon was dissected out and mounted under resting tension of 0.5 g in an organ bath containing continuously aerated tyrode’s solution. Dose response curves were obtained with ascending doses of ACh (100μg/ml). EC50 values were calculated from graph plotted using percent responses against log dose. In vitro antioxidant activity In vitro antioxidant activity of Dolabi was assessed on the basis of radical scavenging effect of the stable DPPH-free radical. Free radical scavenging activity of different concentration of Dolabi and ascorbic acid were measured using DPPH, employing method of Blois (Blois, 1958). Solutions of different concentration (50, 100, 150, 200, 250 μg/ml) of Dolabi and ascorbic acid were added to 0.01mM, solution of DPPH in methanol. After 30 min, absorbance was measured at 517 nm, using spectrophotometer (Shimadzu, Japan). 0.01mM solution of DPPH in methanol was used as control. All tests were performed in triplicate. IC50 value was calculated. The DPPH radical scavenging activity was calculated according to the following equation, DPPH radical scavenging activity (%) = A0 – A1/ A0 x 100 Where, A0 is the absorbance of DPPH, A1 is the absorbance of DPPH solution in presence of the extract. Statistical analysis Statistical analysis of data was conducted using one-way ANOVA followed by Dunnett’s test. Data were expressed as mean ± SEM, P<0.05 was considered statistically significant. Results Effect of Dolabi on delayed gastric emptying in diabetic rats Gastric emptying (GE) was significantly (P < 0.01) decreased in diabetic rats as compared to normal rats (46.24 ± 3.64 vs 57.57 ± 1.96 %,
  7. 7. 4 Somani et al. Figure 1). In STZ induced diabetic rats, Dolabi significantly (P<0.05) accelerated gastric emptying (GE) as compared to diabetic control rats (59.05 ± 2.09 vs 46.24 ± 3.64 %, Figure 1). Effect of Dolabi on delayed small intestinal transit in diabetic rats Intestinal transit (IT) was significantly (P < 0.05) decreased in diabetic rats compared to normal rats (45.43 ± 1.94 vs 60.93 ± 5.00 %, Figure 2). In STZ induced diabetic rats, Dolabi significantly (P<0.05) accelerated intestinal transit (IT) as compared to diabetic control rats (64.32 ± 4.16 vs 45.43 ± 1.942 %, Figure 2). as compared to normal rats (41.52±3.94 vs 20.89±3.25 μg, Figure 3). In STZ induced diabetic rats, Dolabi show significant (P<0.01) decrease in EC50 value of ACh in distal colon as compared to diabetic control rats (14.23±2.59 vs 41.52±3.94 μg, Figure 3). Effect of Dolabi on in vitro antioxidant activity Dolabi exhibited in vitro antioxidant activity with IC 50 value of 232.11 μg/ml (Figure 4). Effect of Dolabi on EC50 of ACh in distal colon of diabetic rats EC50 value of Ach in rat distal colon was significantly (P<0.01) increased in diabetic rats Discussion Propulsive motility is termed as peristalsis and it is subserved by a complex pattern of neural reflexes that aim to relax intestinal muscle downstream (descending inhibitory reflex) and Figure 1. Effect of two weeks repeated dose treatment of Dolabi on % of gastric emptying (GE %) in STZ induced diabetic rats Figure 3. Effect of two weeks repeated dose treatment of Dolabi on EC50 of ACh in rat distal colon in STZ induced diabetic rats Data represented as mean ± SEM. ## P< 0.01, compared to normal control group; **P< 0.05, compared to diabetic control group (ANOVA followed by Dunnett’ test) Data represented as mean ± SEM. ## P< 0.01, compared to normal control group; **P< 0.01, compared to diabetic control group (significance by one way ANOVA followed by Dunnett’ test) Figure 2. Effect of two weeks repeated dose treatment of Dolabi on % of intestinal transit (IT %) in STZ induced diabetic rats Figure 4. Effect of Dolabi on DPPH free radical scavenging activity Data represented as mean ± SEM # P< 0.05, compared to normal control group; * P< 0.05, compared to diabetic control group (significance by one way ANOVA followed by Dunnett’ test) Data expressed as mean ± SEM from three observations
  8. 8. Prokinetic Effect of Herbomineral Formulation in Diabetic Rats contract the muscle upstream (ascending excitatory reflex) of the intestinal bolus. Intestinal transit is controlled by both neural and myogenic mechanisms (Huizinga et al., 1998). An increase of the contractile activity of the smooth muscle layers is in general responsible for acceleration of intestinal propulsion. Several mediators and neurotransmitters are responsible for these motor patterns. Acetylcholine is the main excitatory neurotransmitter in the enteric nervous system, whereas NO is the major transmitter of the inhibitory motor neurons (Waterman and Costa, 1994). Disorder of autonomic functions (Punkkinen et al., 2008 ) and extrinsic nerve supply to the gut are known to be responsible for the disturbed gastric motility associated with diabetes. In the present study, STZ induced diabetic rats had mild or moderate gastroparesis which is characterized by slow gastric emptying and intestinal transit as compared with normal controls. Similar delayed gastric emptying, intestinal transit were seen in the STZ diabetic rats which is in agreement with previous studies (Bijender et al., 2003; Young et al., 2006; ElSalhy, 2002a; Anjaneyulu and Ramarao, 2002; El-Salhy, 2002b). Thus, rat with STZ induced diabeties could be used as an animal model of diabetic gastroparesis. Disturbed motility of gastrointestinal tract was also reported in the human with diabetes mellitus (Russo et al., 1997). The exact cause of slow gastrointestinal transit in diabetic patients is not known, but several mechanisms have been proposed. Most important among them, is damage of peripheral cholinergic nerve as a result of oxidative stress (Bijender et al., 2003). A significant reduction in the contractile response of distal colonic smooth muscle to exogenous Ach was reported in STZ induced diabetic rats. Treatment with antioxidant vitamin E, significantly increase contractile response of distal colonic smooth muscle to exogenous Ach as well as accelerate small intestinal transit in diabetic rats which confirm role of oxidative stress in damage of peripheral cholinergic neuron 5 associated with diabetic autonomic neuropathy (Bijender et al., 2003). Acute change in blood glucose concentration has also major effect on gastrointestinal motor function in healthy subjects (Russo et al., 1997). In particular, acute hyperglycemia inhibits both the gastrointestinal and ascending components of peristaltic reflex. Poor glycemic control has the potential to cause delayed gastrointestinal transit in diabetic patients (Jung et al., 2003). Therefore drug which control both blood glucose as well as having antioxidant activity will be the best one, for treating diabetic associated gastrointestinal trouble. To the best of our knowledge, this is the first report on the effect of herbomineral formulation (Dolabi) on the gastrointestinal dysmotility in streptozotocin induced diabetic rats and its in vitro antioxidant activity using DPPH. Dolabi significantly accelerate gastric emptying, intestinal transit and increase contractile response of distal colonic smooth muscle to exogenous ACh in STZ induced diabetic rats. It also exhibit in vitro antioxidant activity to DPPH. Mechanism underlying the action of Dolabi on impaired gastric motility may be neuronal dependent and its antioxidant activity may play an important role. Our data strongly reveals that Dolabi exert a prokinetic action on gastric emptying and intestinal transit in diabetic rats. The present study also suggests that antioxidant property of Dolabi may be responsible for halting progressive changes of chronic diabetes leading to gastric impairment. However, further detailed studies like estimation of endogenous antioxidant enzyme levels are needed. Acknowledgments The authors wish to thank Prof. M.N. Navale, Founder President, STES and Dr. K.S. Jain, Principal, SCOP for providing facilities to carryout this work. 1. References Anjaneyulu, M. and Ramarao, P: Studies on gastrointestinal tract functional changes in diabetic
  9. 9. 6 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. Somani et al. animals. Methods Find. Exp. Clin. Pharmacol. 24: 71-75 (2002). Bijender, S., Harish, D., Rishi, S. and Patil B.M: Effect of vitamin E on the impaired gastrointestinal activity of streptozotocin induced diabetic rats. Ind. J. Pharmacol. 35: 186-187 (2003). Blois, M.S: Antioxidant determination by the use of stable free radical. Nature. 29: 1199-1200 (1958). De Winter, B.Y., Van Nassauw, L., De Man, J.G., De Jonge, F., Bredenoord, A.J., Seerden, T.C., Herman, A.G., Timmermans J.P. and Pelckmans P.A: Role of oxidative stress in the pathogenesis of septic ileus in mice. Neurogastroenterol. Motility. 17(2): 251-61(2005). El-Salhy, M: Gastrointestinal transit in an animal model of human diabetes type 2: relationship to gut neuroendocrine peptide contents. Ups. J. Med. Sci. 107: 101-110 (2002a). El-Salhy, M: Gastrointestinal transit in relation to gut endocrine cells in animal models of human diabetes. Ups. J. Med. Sci. 107: 23-33 (2002b). Gholap, S. and Kar A: Effect of Inula racemosa root and Gymnema sylvestre leaf extract in the regulation of corticosteroid induced diabetes mellitus. involvement of thyroid hormones. Pharmazie. 58: 413-415 (2003). Huizinga, J.D., Ambrous, K. and Der-Silaphet, T: Co-operation between neural and myogenic mechanisms in the control of distension-induced peristalsis in the mouse small intestine. J. Physiol. 506: 843–856 (1998). James, L.E., Andrea, M.V., Hsinlin, T.C. and Eva, L.F: Diabetic neuropathy: Mechanisms to management. Pharmacol. Therapeut. 120: 1–34 (2008). Janseen, P.A. and Jagenerous, A.H: New series of potent analysis. J. Pharm. Pharmacol. 6: 38-40 (1957). Jung, H.K., Kim, D.Y., Moon, I.H. and Hong, Y.S: Colonic transit time in diabetic patients comparison with healthy subjects and the effect of autonomic neuropathy. Yonsei. Med. J. 44: 265-272 (2003). Kong, M.F., Horowitz, M., Jones, K.L., Wishart, J.M. and Harding, P.E: Natural history of diabetic gastroparesis. Diab. Care. 22(3): 503-507 (1999). 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. Miskiewicz, S.J., Arnett, B.B., and Simon, G.E: Evaluation of a glucose oxidase-peroxidase method adapted to the single channel autoanalyzer and SMA 12-60. Clin. Chem. 19: 253-257 (1973). Punkkinen, J., Farkkila, M., Matzke, S., Korppi-Tommola, T., Sane, T., Piirila, P. and Koskenpato: Upper abdominal symptoms in patients with Type 1 diabetes: unrelated to impairment in gastric emptying caused by autonomic neuropathy. Diab. Med. 25(5): 570-577 (2008). Robert, A. and Silvestro D: Zinc in relation to diabetes and oxidative disease. J. Nutr. 130: 1509S-1511S (2000). Russo, A., Sun, W.M., Sattawatthamrong, Y., Fraser, R., Horowitz, M., Andrews, J.M. and Read, N.W: Acute hyperglycaemia affects anorectal motor and sensory function in normal subjects. Gut. 41: 494-499 (1997). Sagrawat, H., Mann, A.S. and Kharya, M.D: Pharmacological potential of Eugenia jambolana: A review. Pharmacog. Mag. 2(6): 96-105 (2006). Shamaila, W., Baharak, M., and Peter V.D: Gastroparesis: Current diagnostic challenges and management considerations. World. J. Gastroenterol. 15(1): 25-37 (2009). Sundaram, R. and Mitra S.K: Antioxidant activity of ethyl acetate soluble fraction of Acacia arabica bark in rats. Ind. J. Pharmacol. 39: 33-38 (2007). Wadood, A., Wadood, N. and Shah, S: Effect of Acacia arabica and Caralluma edulis blood glucose levels of normal and alloxan diabetic rabbits. J. Pak. Med. Assoc. 39: 208-212 (1989). Waterman, S.A. and Costa, M: The role of enteric inhibitory motor neurons in peristalsis in the isolated guinea-pig small intestine. J. Physiol. 477: 459–468 (1994). Young, C., Shou-Dong, L., Guey-Hwa Y. and Paulus S.W: Influence of blood glucose levels on rat liquid gastric emptying. Dige. Dise. Sci. 41(3): 528-532 (2006). Ziegler, D., and Gries F.A: Alpha-lipoic acid in the treatment of diabetic peripheral and cardiac autonomic neuropathy. Diabetes. 46(2): 62-66 (1997). Address for correspondence: Prof. Rahul Somani, Sinhgad College of Pharmacy, Pune - 411041 Maharashtra (India). E-mail: rahulsomani2001@yahoo.com 011_2010
  10. 10. J. Res. Educ. Indian Med., Jan. - March, 2012 Vol. XVIII (1) : 7-11 ISSN 0970-7700 ENERGY DISPERSIVE X-RAY SPECTROSCOPY IN QUALITY CONTROL OF POWDERED HERBAL FORMULATION - AVIPATTIKAR CHURNA K.JAYARAM KUMAR* AND MOULI NANDI Department of Pharmaceutical Sciences, Birla Institute of Technology, Mesra, Ranchi - 835215 Bihar (India) Abstract: Energy dispersive X-ray spectroscopy (EDX) is an analytical technique used for the elemental analysis and chemical characterization of a sample. Many of the Ayurvedic formulations contain salt and trace elements, which help to optimize the therapeutic efficacy of the main drugs. The elements generally present are sodium, potassium, chlorine, mercury, sulphur, silver etc. EDX not only helps to quantify the minerals or salts added as one of the ingredients but also helps to quantify the presence of the elements of a formulation as well. It can be used as an important measure for quality control of Ayurvedic formulation. Thus, it can be used for their quantification even in minor amounts. In the present study quantification of different trace elements of two different marketed formulations and in house formulation and a formulation without salt of Avipattikar churna has been done by flame photometry and EDX. The result shows the presence of sodium, potassium, chlorine, calcium, copper and zinc. Quantification of sodium, potassium has been done by flame photometry but the results obtained by EDX showed better sensitivity, precision and accuracy. Keywords: Energy dispersive X-ray spectroscopy, Flame photometry, Trace elements, Quality control, Avipattikar churna, Ayurvedic drugs. Introduction According to an estimate of World Health Organization (W.H.O.) nearly 80% of population of developing countries relies on traditional medicines, mostly on plant drugs for their primary health care needs. Most of the traditional systems of medicines are effective but the need is just to validate them. Validation of herbal medicines is one of the toughest challenges for the scientist. There is a need to develop standards to bring this system of medicine in the main stream of health sciences (Mukherjee, 2003). In India Ayurveda, Unani and Siddha have been used for many centuries. Central Council for Research in Ayurveda and Siddha (C.C.R.A.S) has given preliminary guidelines for standardization of these formulations but for the uniformity of batches in production of Ayurvedic formulations. It is necessary to develop methods using phytochemical markers (Sane, 2005). However, the analysis and the structure of the * Reader in Pharmacognosy finished product, safety and side effects of the formulations, correct doses and duration of the treatment, mode of action remains unanswered (Prakash, 1999). Energy dispersive x-ray spectroscopy (EDX) is a technique used for the elemental analysis and chemical characterization of a sample. Different sample types (solid materials, liquids, powders, metals, minerals, etc.) can be analyzed with simple sample preparation over a wide range of concentrations (from traces to main components) is possible without dilution (Juillet, 1996). It is a new technique used to identify and quantify the trace elements present in a sample. Many of the Ayurvedic formulations contain salt and trace elements, which help to optimize the therapeutic efficacy of the main drugs. So EDX can be used as an important tool in standardization of Ayurvedic formulation. In classical Ayurvedic books, Avipattikar churna is mentioned to be used in digestive and irritable bowel disorders. It is composed of twelve
  11. 11. 8 Kumar and Nandi Table 1. Sodium and potassium content by flame photometer. Sample Sodium content (% weight) Potassium content (% weight) A 0.2508 0.322 B 0.3812 0.417 In house 0.325 0.473 In house without salt 0.1142 chart_ back_ bw_final.pdf]. accessed on date 13.11.2010. Vida lavana contains a small amount of natural amounts of trace elements such as iron and magnesium. It is carminative and tonic to the digestive system. It replenishes salt lost in exercise and adding to trace elements essential for normal health and fitness. Potassium, sodium, chloride helps in regulation of Na/K/CA ATPase, glucose transport, transport of some amino acids including alanine, proline, tryptophan and tyrosine. 0.3554 Table 2. Trace element obtained by EDX (in % weight). Sample Sodium Potassium Calcium Copper Zinc Chloride A 0.50 0.46 0.55 1.39 1.24 0.72 B 0.58 0.82 0.46 3.15 2.71 1.04 In house - 0.60 1.42 2.58 2.24 0.65 In house without salt - 0.34 0.40 1.94 1.89 - Table 3. Comparison of sodium and potassium by flame photometry and EDX (in % weight). Flame photometry EDX Table 4. Constituents of herbal formulation Avipattikar churna. Sodium Potassium Sodium Potassium Sl. No. A 0.2508 0.322 0.50 0.46 B 0.3812 0.417 0.58 0.82 In house 0.325 0.473 - 0.60 Sample In house without salt Sanskrit / Hindi Name Botanical Name 1 Shunthi (Zingiber officinale) 2 Maricha (Piper nigrum) 3 0.3554 - 0.34 (Piper longum) Amalaki (Embelica officinalis) 5 0.1142 Pippali 4 Vibhitaki (Terminalia bellirica) 6 Haritaki (Terminalia chebula) 7 Vidang (Embelia ribes) 8 different herbs and spices along with sugar and salt (Table 4). Herbs include Emblic myrobalan, Ginger, Belliric myrobalan, Nutgrass, Myrobalan, Long pepper, Black pepper, Vidang, Cardamom, Indian cinnamon, Clove, Trivrt (Indian jalap), sugar and Vida lavana (black salt). Though Indian jalap being the main active ingredient for therapeutic efficacy but the presence of sugar and salt is also important as sugar reduces pitta. People with a predominantly pitta constitution are thought to be susceptible to hypertension, heart disease, infectious diseases, and digestive conditions[http://www.amritaveda. c o m / l e a r n i n g / a r t i c l e s / Ta s t e Nagarmotha (Cyperus rotundus) 9 Ela (Elettaria cardamomum) 10 Tejapatara (Cinnamomum zeylanicum) 11 Launga (Syzygium aromaticum) 12 Nishotha (Operculina turpethum) 13 Candy sugar Potassium ions are involved in a number of essential physiological processes, such as the maintenance of intracellular acid-base balance and tonicity. Copper aid enzymes, specifically in oxidation of iron. Zinc is essential in reactions involving synthesis such as carbohydrates, lipids, proteins and nucleic acids. [http://web.uct.ac.za/ depts/git/ibd/vits.htm# Minerals2] accessed on date 13.11.2010.
  12. 12. EDX in Quality Control of Herbal Formulations 9 Figure 2 Figure 1 Figure 4 Figure 3 Figure 6 Figure 5 Figure 1,3,5,7 shows the surface electron image of formulation A, B, in house and in house without salt respectively Figure 2,4,6,8 shows the graph showing the quantity of the present trace element of formulation A,B, in house and in house without salt respectively.
  13. 13. 10 Kumar and Nandi Figure 8 Figure 7 Materials and Methods Instruments and Chemicals Systronics flame photometer 128 was used for flame photometry and energy dispersive X-ray spectroscopy was done in JEOL-JSM 6390LV–SEM analyzer. Millipore water was used for flame photometry. Methods for Flame Photometry 1000 ppm standard solution of analytical grade sodium chloride and potassium chloride was prepared. From that stock solution 20,40,60,80,100 ppm solution of sodium chloride and potassium chloride was prepared. The sample was prepared by taking 1 g of four different formulations in 100 ml of water. It is then filtered and 10 ml of the filtrate was taken and volume was made up to 100 ml. Their sodium content and potassium content was measured by Systronics flame photometer 128 (McNaught and Wilkinson, 1997). They were converted to percentage weight for the convenience of comparison as the results obtained from EDX were in percentage weight. Methods for Energy Dispersive X-ray Spectroscopy EDX analysis was done in JEOL- JSM 6390LV –SEM analyzer. At first the atmospheric air was poured inside the chamber and vacuum was released. It is called Vent mode. Then the chamber was opened and the sample was placed. Again vacuum was regenerated with pumps. After creation of vacuum, the sample was moved towards the beam. Accelerating voltage was 20 kv, working distance was 10 m.m. and spot size was 40-70. Few milligrams of the samples were used in for the analysis. Comparison of results obtained from flame photometry and EDX was done (Seiichi and Hiroyuki, 1986; Kazuo et al., 1996). The results obtained from flame photometry and EDX are given in Table 1 and Table 2. Comparison of the amount of sodium and potassium by flame photometry and EDX are shown in Table 3. Results In house formulation without salt also shows the presence of sodium, potassium, chlorine and other trace elements. The results of flame photometry show presence of sodium ranges from 0.1142% to 0.3812% weight and potassium ranges from 0.322% to 0.473%. In EDX the presence of sodium ranges from 0.50% to 0.58% weight and potassium ranges from 0.34% to 0.82% weight. In EDX presence of other trace elements was also detected and quantified. Discussion The results obtained in EDX and flame photometry shows quite a difference. However, in EDX presence of other trace elements was
  14. 14. EDX in Quality Control of Herbal Formulations found like chlorine, copper, zinc calcium, etc. In house formulation without salt also shows the presence of sodium, potassium, chlorine and other trace elements. It indicates the presence of trace elements in the ingredients other than the salt. Thus EDX gives a better comprehension of trace elements. Acknowledgements Financial help rendered by AICTE to carry out this research work is gratefully acknowledged. We are thankful to the Head, Department of Pharmaceutical Sciences, BIT, Mesra, Ranchi (India) for permission to carry out this experimental work. 1. 2. 3. 4. References McNaught AD and Wilkinson A: IUPAC. Compendium of Chemical Terminology. 2nd edition (the Gold Book). Blackwell Scientific Publications, Oxford (1997). Ayurveda’s Six Tastes and their Effects on the Doshas http://www.amritaveda.com/learning/ articles Taste_chart_back_bw_final.pdf(13.11.10) Backgrounder Ayurvedic Medicine: An Introduction http://nccam.nih.gov/health/ayurveda/ D287_BKG.pdf(13.11.10) The Ayurvedic Formulary: Government of India Ministry of Health and Family Planning, Department of Health, Part-1, 1 st edition. 5. 6. 7. 8. 9. 10. 11. 12. Government of India Press, Faridabad, Controller of Publication, New Delhi, India. pp 87 (1978). Juillet HM: Spectroscopy for the determination of trace and main elements. SPECTRO Analytical Instruments, Kleve. Journal de physique IV. Colloque, supplkment au Journal de Physique III,C4 619-625 (1996). Kazuo F, Mitsuaki O, Mitsuhiro A and Tetsuya S: Practical performance of energy dispersive xray spectroscopy with a high- voltage TEM up to 1,000 kv. Journal of Electron Microscopy. 45(4): 285-290 (1996). Lee. R and Kligler B: Interactive Medicine: Principles for Practice. McGraw-Hill Companies (2004). Mukherjee PK: Exploring botanicals in Indian system of medicine-regulatory perspectives. Clinical Research and Regulatory Affairs. 20: 249– 264 (2003). Prakash VB: In Standardisation of Ayurvedic products including GMP of Ayurvedic industry, Kerala. Ayurvedic Medicine Manufacturer Association, Palakkad, Kerala, India (1999). Sane VC: Past, present and future of Ayurvedic medicines in India. The Pharma Review. 3: 29-33 (2005). Seiichi S and Hiroyuki K: Energy dispersive X-ray spectroscopy in medium voltage electron microscope. Journal of Electron Microscopy. 35(4): 335-342 (1986). Vitamins, Minerals and Trace Elements http:// web.uct.ac.za/depts/git/ibd/vits.htm# Minerals2(13.11.10) Address for correspondence: K. Jayaram Kumar, Department of Pharmaceutical Sciences, Birla Institute of Technology, Mesra, Ranchi - 835215 Bihar (India). E-mail: jayaram_res@yahoo.com 036_2009 11
  15. 15. J. Res. Educ. Indian Med., Jan. - March, 2012 Vol. XVIII (1) : 13-19 ISSN 0970-7700 CLINICAL TRIAL OF GARBHPAL RAS IN PREGNANCY OUTCOME DEEPA MISHRA,1* MUKTA SINHA2 AND VIKAS KUMAR3 Department of Prasuti Tantra,1,2 Institute of Medical Sciences, Pharmacology Laboratory, Department of Pharmaceutics,3 Institute of Technology, Banaras Hindu University, Varanasi – 221005 (India) Abstract: Introduction: Ayurvedic sages suggest that a wholesome diet along with sheetvirya, balya, rasayan and madhur drugs during the period of pregnancy is must. This ultimately results in fetal growth, maternal health and post delivery lactation. Garbhpal ras, a drug mentioned in the text of 17th century namely ‘Ras Chandanshu’ has such properties and is being used since then. It was claimed that Garbhpal ras is a panacea for feto-maternal well being. The aim of present study was to assess the efficacy of Garbhpal ras in pregnancy outcome. Method: In present study, 170 pregnant women were registered. Haematological and biochemical parameters were observed. Neonatal parameters like gestational age, weight, height, head circumference, chest circumference, mid arm circumference and Apgar score were assessed after delivery. Result and conclusion: Normal haematological picture, maintained renal and liver function tests show non-toxic nature of Garbhpal ras. Thus, it can be recommended safely in pregnancy at therapeutic doses. Keywords: Garbhpal ras, Rasayan, Balya, Garbhsrava, Anupan, Antioxidant, Immunomodulatory, Pregnancy, Prevention of abortion, Ayurvedic medicine, Lactation, Meternal health. Introduction Ayurveda, the oldest existing medical system, recognized by World Health Organization, is widely practiced. Obstetric care in ayurveda is an unique feature. Ayurveda ensures a safe and natural delivery of a healthy baby, which is achieved by a regulated diet and regimen during pregnancy and administration of herbal preparations needed in each month. A special unusual glow, beauty, serenity is seen on a pregnant woman’s face. This glow is the indicator of health of baby inside her body. Garbhpal ras, a drug mentioned in the text of 17th century namely ‘Ras Chandanshu’ has occupied a respectable place in ayurveda, for preventing miscarriage and ensures better nourishment to fetus (Rasyog sagar, 1907-10; Chhangadi, 1999; Goyal and Mahajan, 1988; Indian Medical Science Series, 1994). Garbhpal ras contains minerals like Hingula (Cinnabar/Hgs), Nag (lead/Pb), Vang (Tin/Sn) and Loh bhasma (Iron/Fe). Herbal contents of Garbhpal ras include Dalchini (Cinnamomum zeylanicum), Ela (Elettaria cardamomum), Tejpatra (Cinnamomum tamala), Shunthi (Zingiber officinale), Marich (Piper nigrum), Dhanyak (Coriandrum sativum), Chavya (Piper retrofractum), Krishna jeerak (Carum bulbocastanum), Draksha (Vitis vinifera) and Devdaru (Cedrus deodara). All ingredients in same quantity (1 karsh each) except Loh bhasma (1/2 karsh) were triturated in extract of Vishnukranta (Clitoria ternatea) for seven days. (1 karsh = 12 grams) Prior to this clinical study of Garbhpal ras in pregnant women, this drug has been evaluated for acute, sub-chronic and chronic toxicity. It was also evaluated for teratogenic effects, if any, in dams and was found safe at therapeutic doses (Mishra et al., 2008; Mishra et al., 2009a; Mishra et al., 2009b; Mishra et al., 2009c). Garbhpal ras was also found helpful to reduce symptoms like nausea, vomiting, leg cramps, heart *1. Incharge and Lecturer, Department of Prasuti Tantra, A & U Tibbia College and Hospital, New Delhi (India). 2. Professor and Head of Department, Prasuti Tantra, IMS, BHU, Varanasi (India). 3. Associate Professor, Department of Pharmaceutics, IT, BHU, Varanasi (India).
  16. 16. 14 Mishra, Sinha and Kumar burn, low back pain, abdominal pain, oedema, constipation and flatulence during pregnancy (Mishra et al., 2009d; Mishra et al., 2009e). The aim of this study was to assess the efficacy of Garbhpal ras in pregnancy outcome, if any. Materials and Methods Inclusion criteria Women belonging to age group, 18 to 38 years, having amenorrhoea due to pregnancy (as early as pregnancy was detected and up to five month of pregnancy), history of abortion, history of intra uterine death/still birth, history of preterm delivery or any other obstetric problem during previous pregnancies. Exclusion criteria Women having essential hypertension, severe aneamia, diabetes mellitus, hypo/ hyperthyroidism and severe degree of pregnancy induced hypertension. Patients having tuberculosis (except women already on ATT for suspected genital tuberculosis), liver or renal disorder were also excluded. Initially 178 pregnant women were randomly selected, out of which 170 cases registered from out patient department (during period of Aug. 2006 to Sept. 2007), Prasuti Tantra, Sir Sunderlal Hospital, BHU, Varanasi (India). At the time of registration written consent for becoming a volunteer of this study was taken from each pregnant woman. All the registered cases have been categorized in 4 groups as follows: Group 1. Patients registered from first trimester having normal course of pregnancy with no specific obstetric or medical history. Group 2. Patients registered from first trimester with high risk factor like history of abortion, pregnancy induced hypertension in previous pregnancy, preterm delivery and previous caesarean section within two years, elderly primigravida, TORCH infection, on anti tubercular treatment. Group 3. Patients registered from second trimester having normal course of pregnancy with no specific obstetric or medical history. Group 4. Patients registered from second trimester with high risk factor like history of abortion, pregnancy induced hypertension in previous pregnancy, preterm delivery and previous caesarean section within two years, elderly primigravida, TORCH infection, on anti-tubercular treatment. Each group was further subdivided into control and trial: Control group (C1 ,C2,C3 ,C4): Patients received iron (Livogen), calcium (Calcimax forte) after the first trimester, and folic acid from the very beginning till last month. Natural progesterone 200 μg once or twice a day advised in cases having history of spontaneous abortion. Trial group (T 1 ,T 2 ,T 3 ,T 4 ): Patients additionally received Garbhpal ras (120 mg bid with milk and munakka or draksha). As the women registered for the study, they were advised Garbhpal ras to be taken with anupan after meals. Maximum duration for drug administration was six months. In all the groups folic acid was advised as soon as pregnancy was detected till delivery and iron (Livogen), calcium (Calcimax forte) were advised after the first trimester. Blood samples were taken before initiation of treatment and in the last month of gestation for analysis. Pregnant women were reviewed for routine antenatal checkup ( Fortnightly up to 12 weeks, monthly till 32 weeks, fortnightly up to 36 weeks and weekly afterwards). Parameters studied 1. Neonatal parameters (Anthropometry like weight, height, head circumference, chest circumference, mid arm circumference, gestational age and Apgar score at the time of delivery). 2. Outcome in cases of previous history of spontaneous abortion(s) (Table 1). 3. Haematological and bio-chemical blood parameters (Table 2 and 3). Statistical analysis All data were expressed as Mean±SD. Treated and control groups were compared with
  17. 17. Clinical Trial of Garbhpal ras in Pregnancy Outcome Table 1. History of spontaneous abortion(s). 15 independent sample ‘t’ test, intragroup comparison was done with paired ‘t’ test. A 95% confidence level was used to determine statistically significant differences between Garbhpal ras treated and control groups. Effect of Garbhpal ras on heamatological parameters Haemoglobin was improved overall, but statistically significant difference was observed in pregnant women registered in first trimester treated with Garbhpal ras in comparison to control group. It shows that Garbhpal ras causes no interference/ impairment in absorption of iron rather it might have synergistic effect. It was observed that average mean value of total leukocytes count was increased significantly in last month, though it was within normal limits. It shows that Garbhpal ras does not produce any haemopoitic toxicity (Table 2). Results and Discussion Results were assessed on the basis of differences in neonatal parameters (like gestational age at the time of delivery, weight, height, head circumference, chest circumference, mid arm circumference and Apgar score), successful outcome in cases of history of spontaneous abortion(s) and untoward effect on haematological and bio-chemical blood parameters, if any. Effect of Garbhpal ras on biochemical parameters of blood Level of blood sugar was found within normal range in all groups. No incidence of hypo/ hyperglycemia was observed in any of the case. Liver function (serum bilirubin, SGOT, SGPT and alkaline phosphatase) and renal function (blood urea, serum creatinine) are maintained during antenatal period in trial group as well as Group Number of cases Percentage (%) 1 2 3 11 57.89 10 1 0 1 C2(n=19) Cases having 1, 2 or 3 no. of abortion(s) T2(n=25) 15 60.00 9 5 C4(n=14) 08 57.14 6 2 0 T4(n=20) 13 65.00 9 3 1 Table 2. Effect of Garbhpal ras on haematological and biochemical parameters. Group Haemoglobin (gm%) TLC (mm3) FBS (mg/dl) Bl Urea (mgdl) Serum Creat (mg/dl) BT AT BT AT BT AT BT AT BT AT T1 (n=30) 10.25 ±1.22 12.14 ±1.10†† 8347.22 ±1103.37 11244.44 ±1162.87†† 72.04 ±12.98 73.25 ±10.51* 16.29 ±3.08 16.76 ±2.12 0.65 ±0.08 0.70 ±0.08 C1 (n=20) 10.23 ±1.23 11.58 ±1.10†† 8652.15 ±1192.18 11808.71 ±1240.20†† 78.76 ±11.98 79.42 ±10.58 17.20 ±3.20 17.9 ±2.32† 0.66 ±0.13 0.67 ±0.09 T2 (n=25) 10.32 ±1.63 12.20 ±0.92††* 9373.33 ±1038.03 11295.65 ±1157.38†† 76.06 ±11.97 76.29 ±9.38 16.36 ±2.25 16.33 ±1.90 0.68 ±0.12 0.72 ±0.09 C2 (n=19) 10.50 ±1.39 11.81 ±0.88† 9121.08 ±1003.10 11989.47 ±1191.12†† 75.05 ±11.29 77.93 ±10.64 17.98 ±3.32 16.77 ±3.50 0.69 ±0.13 0.71 ±0.10 T3 (n=25) 9.77 ±1.04 11.85 ±0.73†† 9733.33 ±829.52 12013.33 ±1046.00†† 71.64 ±10.71 73.74 ±10.82 17.20 ±4.99 16.95 ±3.82 0.65 ±0.05 0.66 ±0.06 C3 (n=17) 10.32 ±1.44 11.88 ±1.00†† 9494.12 ±872.12 12276.47 ±1056.84†† 77.54 ±10.62 76.65 ±10.66 17.34 ±4.19 17.91 ±3.69 0.65 ±0.09 0.68 ±0.09 T4 (n=20) 10.16 ±1.57 11.85 ±1.22†† 9552.00 ±1037.87 12468.75 ±1189.8†† 73.43 ±13.06 71.57 ±6.32* 15.08 ±0.93* 15.04 ±0.83 0.68 ±0.07 0.70 ±0.08 C4 (n=14) 10.24 ±1.26 11.78 ±1.15†† 9186.67 ±1010.56 12320 ±1166.96†† 75.09 ±11.01 80.36 ±12.44† 17.49 ±4.10 15.85 ±2.62† 0.67 ±0.09 0.69 ±0.08 BT= Before treatment ; AT= At last month of pregnancy ; * p<0.01, compared to control ; † p<0.01, compared to BT ; †† p<0.001, compared to BT.
  18. 18. 16 Mishra, Sinha and Kumar Table 3. Effect of Garbhpal ras on biochemical parameters. Group Serum Bilirubin (mg/dl) SGOT (U/ml) AT SGPT (U/ml) BT AT BT BT T1 (N=30) 0.64 ±0.12 0.60 ±0.11 28.03 ±4.67 27.56 ±4.40 35.12 ±4.78 C1 (N=20) 0.68 ±0.08 0.56 ±0.11 29.01 ±5.88 29.10 ±4.94 T2 (N=25) 0.62 ±0.13 0.57 ±0.12 27.78 ±6.28 C2 (N=19) 0.63 ±0.12 0.60 ±0.13 T3 (N=25) 0.62 ±0.11 C3 (N=17) Alk Phosp (U) AT Serum Protien (gm/dl) Serum Alb (gm/dl) BT AT BT AT AT BT 35.32 ±4.65 142.77 ±24.34 149.80 ±23.50† 6.48 ±0.58 6.44 ±0.50 3.59 ±0.40 3.56 ±0.41 36.05 ±6.39 33.90 ±5.06 154.45 ±25.03 158.90 ±25.68 6.67 ±0.60 6.62 ±0.58 3.51 ±0.43 3.60 ±0.40 27.53 ±4.19 35.40 ±4.37 35.23 ±4.16 142.44 ±37.12 147.22 ±32.28 6.47 ±0.49 6.58 ±0.43 3.36 ±0.41 3.40 ±0.40 27.32 ±6.57 27.21 ±4.21† 34.69 ±6.65 35.79 ±5.80 144.21 ±31.16 140.63 ±34.16 6.38 ±0.49 6.60± 0.36 3.44 ±0.37 3.55 ±0.36 0.57 ±0.10 24.91 ±5.55 26.02 ±4.17 32.26 ±6.15 31.7 ±6.01 147.64 ±29.14 157.21 ±30.41† 6.66 ±0.64 6.70 ±0.50 3.47 ±0.56 3.66 ±0.41 0.63 ±0.11 0.61 ±0.11 27.52 ±6.89 28.28 ±4.63 35.61 ±4.55 36.38 ±5.50 143.29 ±27.85 156.29 ±27.43† 6.25 ±0.52 6.50 ±0.41 3.45 ±0.46 3.56 ±0.41 T4 (N=20) 0.59 ±0.12 0.55 ±0.11 26.66 ±6.28 27.95 ±6.39 36.16 ±4.06 37.29 ±4.30 137.21 ±42.33 140.50 ±46.09 6.24 6.41 3.28 ±0.31* ±0.26* ±0.32* 3.37 ±0.36 C4 (N=14) 0.59 ±0.10 0.58 ±0.09† 25.14 ±7.35 26.07 ±4.92 32.21 ±8.28 32.64 ±8.12 163.29 ±24.36 167.86 ±41.65 6.99 ±0.67 6.98 ±0.45 3.74 ±0.40 3.76 ±0.40 * p<0.01, compared to control ; † p<0.01, compared to BT Table 4. Effect of Garbhpal ras on neonatal parameter. Gestational age (weeks) Weight (gm) Height (cm) CC (cm) HC (cm) MAC (cm) APGAR score (1stmin) APGAR score (5thmin) APGAR score (10thmin) C1 (n=20) 38.40 ±1.60 2.66 ±0.31 48.13 ±1.70 31.89 ±1.25 33.50 ±1.14 9.23 ±0.75 6.42 ±0.61 7.52 ±0.51 8.63 ±0.50 T1 (n=29) 39.17 ±0.93 2.99 ±0.33** 49.74 ±1.27** 32.24 ±1.24 34.10 ±0.91 9.61 ±0.51* 6.69 ±0.89 7.79 ±0.68 8.79 ±0.68 C2 (n=15) 38.40 ±1.12 2.63 ±0.32 48.00 ±1.80 32.50 ±1.10 33.68 ±1.12 9.04 ±0.70 6.50 ±0.76 7.82 ±0.50 8.78 ±0.43 T2 (n=22) 38.91 ±1.15 2.94 ±0.29** 49.45 ±1.41* 32.75 ±1.32 33.81 ±0.72 9.69 ±0.81* 6.68 ±0.65 7.64 ±0.63 8.82 ±0.40 C3 (n=14) 38.43 ±1.02 2.65 ±0.31 48.23 ±1.79 32.92 ±1.71 33.42 ±1.32 9.19 ±0.50 6.61 ±0.65 7.75 ±0.45 8.77 ±0.44 T3 (n=16) 39 ±1.21 2.92 ±0.40 49.10 ±1.12 32.34 ±1.29 33.97 ±0.85 9.78 ±1.10 6.81 ±0.54 7.88 ±0.50 8.88 ±0.50 C4 (n=13) 38.23 ±1.24 2.54 ±0.46 47.75 ±1.91 31.71 ±1.71 32.96 ±1.57 9.25 ±0.69 6.42 ±1.00 7.41 ±1.00 8.42 ±1.00 T4 (n=12) 38.67 ±0.94 2.76 ±0.36 48.83 ±1.47 31.46 ±0.87 33.50 ±0.80 9.33 ±0.89 6.62 ±0.52 7.61 ±0.51 8.69 ±0.48 Group * p<0.05, compared to control ; ** p<0.01, compared to control.
  19. 19. Clinical Trial of Garbhpal ras in Pregnancy Outcome control group (Table 2 and 3). Normal levels of bio-chemical parameters till the end of treatment, show non-toxic nature of Garbhpal ras. Effect of Garbhpal ras on outcome of pregnancy We are able to ascertain here the outcome of 141 pregnancies. There were 143 live births including 2 twins in Garbhpal ras group. No stillbirth, spontaneous abortion or major malformation was observed in any group during study. However there were 3 cases of intrauterine growth restriction, one case of intrauterine death (due to pregnancy induced hypertension) and 2 preterm deliveries (of gestational age 34-35 weeks) came across during study in control group (Table 6). Two patients of preterm delivery (of gestational age 35-36 weeks) were also observed in Garbhpal ras group (T2), though they were positive for TB IgM and VDRL respectively. Total 62.5% and 57.52% women out of total trial and total control group respectively had history of spontaneous abortion(s) in their previous pregnancies (Table 1). Women belong to control group, having previous history of spontaneous abortion advised progesterone therapy during present study, while women of trial group did not received such therapy. It showed that Garbhpal ras might have some role in successful outcome of pregnancy. But its efficacy and mode 17 of action yet to be determined on larger population. Ayurvedic sages have suggested several precautions to minimize the risk of abortions. If the uterus has vitiated doshas, then it expels the fetus, termed garbhsrava / garbhpata (S.S.Ni.8/10; M.Ni.64/2; B.P.Chi.70/72; Y.R.Stri Garbh Roga Ni.). Such uterus does not cause proper upbringing of fetus and sometimes baby may die soon after birth. Amongst all doshas, vata is predominant. By its dynamic and randomness nature, it expels the fetus. Garbhpal ras contains balya and vata-shamak herbs. They Table 5. Incidence of spontaneous vaginal delivery/ caesarean section. Group SVD LSCS C1 (n=20) 15 5 T1 (n=29) 18 (1LPV) 11 C2 (n=15) 7 8 T2 (n=22) 12 (1-Br, 1-LPV, 2-PT) 10 C3 (n=14) 9 (1-IUGR) 5 T3 (n=16) 11 5 C4 (n=13) 3 (1-IUD, 2PT) 10 T4 (n=12) 5 7 Control (n=62) 34 (54.84%) Trial (n=79) 46 (58.23%) 28 (45.16%) 33 (41.77%) Table 6. Indication for caesarean section. FD IUGR PIH Post Dated FPOL + LPV Abn. Presen. CPD Twins Previous C1 (n=20) 1 - - - - 1 2 - 1 T1 (n=29) 1 - - - 1 1 4 - 4 C2 (n=15) 2 - 1 - 2 - - - 3 T2 (n=22) 1 - - - - 1 3 1 4 C3 (n=14) 1 1 1 - - - - - 2 T3 (n=16) 1 - - 1 - - 1 - 2 C4 (n=13) 3 - - - 1 1 2 - 3 T4 (n=12) 1 - - 1 - 1 - 1 3 Group (SVD= Spontaneous vaginal delivery; LSCS=Lower segment caesarean section; PIH=Pregnancy induced hypertension; FPOL=Failed progress of labour; Br=Breech presentation; PT=preterm delivery ( 36 weeks gestational age); FD=Fetal distress; LPV= Leaking per vaginum; IUGR=Intra uterine growth restriction; CPD=Cephalo-pelvic disproportion; IUD=Intrauterine death of fetus) CS
  20. 20. 18 Mishra, Sinha and Kumar may probably help in strengthening the ligaments which hold the uterus in place, supporting the weight of the fetus and prevent garbhsrava or garbhpata. The incidence of spontaneous vaginal delivery was observed little more in Garbhpal ras treated group (58.23%) in comparison to control (54.84%) (Table 5). Effect of Garbhpal ras on neonatal parameters Average mean of anthropometry of neonate (weight, height, head circumference, chest circumference, mid arm circumference) were found improved in Garbhpal ras group in comparison to control group. It was also observed that pregnant women registered during first trimester of pregnancy, treated with Garbhpal ras have statistically significant changes in weight of baby at birth (2.99 kg in T1 and 2.94 kg in T2) with respective control (2.66 kg in C1 and 2.63 kg in C2). Length (crown heel length) and mid arm circumference in T1 and T2 groups had also shown statistically significant changes with respective control (Table 4). Apgar score and gestational age at the time of delivery had not shown significant difference in comparison to control (Table 4). It means Garbhpal ras has not any effect on these parameters. Health status of women along with good nutrition is prime key to the programming of the proper fetal growth in the uterus (C.S.Sh.2/6). Good maternal body composition due to good nutrition ensures good immune adaptations and it also provides plenty of antioxidants and micronutrients for benefit of fetal growth. Ginger (Asnani and Verma, 2007), Cinnamon (Jayaprakash et al., 2006), Black pepper (Gulcin, 2005), Long pepper (Singh et al., 2007) and Munakka (Koga et al., 1999) have antioxidant activity. In a state of oxidative stress i.e. pregnancy, antioxidants help to protect the body from the formation of free radicals which impair the immune system (Palep, 2007). Piperine has immunomodulatory and cytoprotective activity, thus pregnant women * Devdaru (Devdar) = Cedrus deodara taking Garbhpal ras have better immunity to overcome several diseases (Pathak and Khandelwal, 2007; Choi et al., 2007). Herbs like ginger, black pepper, long pepper and coriander helps in digestion, thus nourish mother as well as fetus. Most of the contents of the Garbhpal ras (Cinnamon, long pepper, cardamom coriander, ginger, Devdaru*) have madhur vipak, hence being anabolic, it improves weight of mother as well as fetus. Small doses of mercury to human beings cause an increase in the number of red blood corpuscles, while body weight and general nutrition are also improved (Nadkarni, 1982). In such condition, fetus gets better nutrition. Ginger also plays a role in increased growth of fetus (Wilkinson, 2000). Anupan of Garbhpal ras i.e. milk and draksha are madhur in ras, add glucose in diet and consequently improve general nutrition of fetus. These may be the possible reasons of increase in weight of fetus of trial group in comparison to control group. No gross congenital anomaly was observed in any of the neonates in all the groups. Conclusion Normal haematological picture, maintained renal and liver function tests show non-toxic nature of drug. Improvement in hemoglobin percentage and normal bio-chemical parameters verify safe use of Garbhpal ras. Successful outcome in cases of previous spontaneous abortion with improved neonatal parameters justify the efficacy of Garbhpal ras. It gave a crystal clear impression that Garbhpal ras can be recommended safely at therapeutic dose in pregnancy. Acknowledgements Authors are thankful to Dabur India Ltd., Sahibabad, U.P. (India) for providing gift sample of Garbhpal ras. 1. References Asnani, V. and Verma, R.J: Antioxidative effect of rhizome of Zinziber officinale on paraben induced lipid peroxidation: an in vitro study. Acta Pol Pharm. 64(1): 35-7 (2007).
  21. 21. Clinical Trial of Garbhpal ras in Pregnancy Outcome 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. Bhav Prakash: Vidyotini Hindi commentary and notes by Pt. Brahm Shankar Mishra. Chaukhambha Sanskrit Sansthan, Varanasi. Ed. VIII. Vikram samvat (2050). Charak Samhita: Hindi Commentry by Kashinath Shastri and Dr. Gorakh Nath. Chaukhambha Sanskrit Sansthan, Varanasi. Ed. XVII (1991). Chhangadi, G.S: In Ras Tantra Sar Va Siddhi Prayog Sangrah. Krishna Gopal Ayurveda Bhavan, Ajmer. Ed. IX. Vol. I. pp 554-55 (1999). Choi, B.M., Kim, S.M., Park, T.K., Li, G., Hong, S.J., Park, R., Chung, H.T. and Kim, B.R.: Piperine protects cisplatin-induced apoptosis via heme oxygenase-1 induction in auditory cells. J Nutr Biochem. 18(9): 615-22 (2007). Goyal, R.K. and Mahajan, R: In Adhyatan Ras Shastra. Chaukhambha Surbharati Prakashan, Varanasi. Ed. I. pp 309 (1988). Gulcin, I: The antioxidant and radical scavenging activities of black pepper (Piper nigrum) seeds. Int J Food Sci Nutr. 56(7): 491-9 (2005). Indian Medical Science Series: No. 3. Clinical Application of Ayurvedic Research and a list of Ayurvedic preparations by a panel of Vaidyas. Sri Satguru Publication, Delhi. Ed. III. (ISBN 81-7030101-07) pp160-61 (1994). Jayaprakasha, G.K., Ohnishi-Kameyama, M., Ono, H., Yoshida, M. and Jaganmohan Rao, L: Phenolic constituents in the fruits of Cinnamomum zeylanicum and their antioxidant activity. J Agric Food Chem. 8; 54(5): 1672-9 (2006). Koga, T., Moro, K., Nakamori, K., Yamakoshi, J., Hosoyama, H., Kataoka, S. and Arigo, T: Increase of antioxidative potential of rat plasma by oral administration of proanthocyanidin-rich extract from grape seeds. J Agric Food Chem. 47(5): 1892-1897 (1999). Madhav Nidan: Vidyotini Hindi commentary and notes by Sri Sudarshan Shastri. Chaukhambha Sanskrit Sansthan, Varanasi. Ed. XVIII (1989). Mishra, D., Sinha, M., Singh, P.N. and Kumar, V: Acute and sub-chronic toxicity study of Garbhpal ras. Electronic J Pharmacol Therapy. 1: 31-34 (2008). Mishra, D., Sinha, M., Kumar, M. and Kumar, V: Chronic toxicity study of ‘Garbhpal ras’, An Ayurvedic medicine. J Herb Med Toxicol. 3(1): 13-17 (2009a). 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. Mishra, D. , Sinha, M. and Kumar, V: Teratological study of ‘Garbhpal ras’, An Ayurvedic formulation. J Herb Med Toxicol. 3(1): 37-40 (2009b). Mishra, D. , Sinha, M. and Kumar, V: Safety evaluation of Garbhpal ras, an Ayurvedic formulation. In National Scientific Seminar on Reproductive Health of Women through Ayurveda. Rashtriya Ayurveda Vidyapeeth, Research papers, New Delhi. pp 251-261 (2009c). Mishra, D., Sinha, M. and Kumar,V: Effect of ‘Garbhpal ras’ on pregnancy induced nausea and vomiting (NVP). Ayurvedic Rennaince. (2009d). Accepted for publication. Mishra, D., Sinha, M. and Kumar, V: Clinical evaluation of ‘Garbhpal ras’ in pregnancy related gastrointestinal disorders. Ayurvedic Rennaince. 7(2): 13-16 (2009e). Nadkarni, A.K: Indian Materia Medica. Vol 2. Bombay Popular Prakashan. pp 72-73 (1982). Palep, H.S. Beyond safe motherhood to programming for a super baby II (2007). available on www. Pharmabiz.com/article/ detnews.asp?Arch=&articleid=11331&sectioned=46. Pathak, N. and Khandelwal, S: Cytoprotective and immunomodulating properties of piperine on murine splenocytes: an in-vitro study. Eur J Pharmacol (2007). In press. Rasyog Sagar: By Hari Prapanna Sharma. Krishna Das Academy, Varanasi. Garbhpal ras II 1907-10/ Ras Chandanshu. Vol. I. pp 374-75 (1983). Singh, S.K., Shanmugavel, M., Kampasi, H., Singh, R., Mondhe, D.M., Rao, J.M., Adwankar, M.K., Saxena, A.K. and Qazi, G.N: Chemically standardized isolates from Cedrus deodara stem wood having anticancer activity. Planta Med. 73(6): 519-26 (2007). Sushrut Samhita: Translation of Ayurveda Tatva Sandeepika by Kaviraj Ambika Dutta Shastri. Chaukhambha Sanskrit Series, Varanasi. Ed. VII (1990). Wilkinson, J. M: Effect of Ginger tea on the fetal development of Sprague- Dawley rats. Reprod Toxicol. 507-512 (2000). Yog Ratnakar: Vidyotini Hindi commentary by Vaidya Sri Lakshmi Pati Shastri edited by Sri Brahma Shankar Shastri. Chaukhambha Sanskrit Sansthan, Varanasi. Ed. II (1973). Address for correspondence: Dr. Deepa Mishra, Incharge and Lecturer, Department of Prasuti Tantra, A & U Tibbia College and Hospital, New Delhi - 110005 (India). E-mail: deepamishra219@gmail.com 009_2009 19
  22. 22. J. Res. Educ. Indian Med., Jan. - March, 2012 Vol. XVIII (1) : 21-26 ISSN 0970-7700 INHIBITORY EFFECT OF THE ROOT OF SIDA ACUTA BURM. F. ON CALCIUM OXALATE CRYSTAL GROWTH T. VIMALA AND S. GOPALAKRISHNAN* Department of Chemistry, Manonmaniam Sundaranar University, Abishekappatti, Tirunelveli - 627012 Tamil Nadu (India) Abstract: The root of Sida acuta Burm. F. is useful in nervous and urinary diseases, disorders of blood and bile and in chronic bowel complaints. Calcium oxalate is the most important constituent of urinary stone crystals and several research workers have been attracted to carry out in-vitro studies on urinary stone crystals. However, the inhibitory effect of the root of S. acuta on calcium oxalate crystal growth in-vitro has not been carried out. In the present work an attempt has been made to grow crystals of calcium oxalate and to study the inhibitory effect of the methanolic and aqueous extracts of the root of S. aucta on calcium oxalate crystal growth invitro. Calcium oxalate crystals were grown in silica gel media in Hane’s tubes by single diffusion method. The crystal growth after the addition of the methanolic and aqueous extracts in 20 mg/ 5 ml and 10 mg/5 ml doses was studied. In both the extracts there were reductions in sizes of calcium oxalate crystal columns and also the size of the individual calcium oxalate crystals when compared to that of the controls. The results of in-vitro experimental models, IR and SEM together attribute an inhibitory capacity with respect to calcium oxalate crystals for the methanolic and aqueous extracts of Sida acuta. Keywords: Urinary disorders, Calcium oxalate, Crystal growth, Sida acuta. Introduction Sida acuta Burm.F. (syn. S. carpinifolia Mast; family – Malvaceae) is an erect, perennial undershrub or shrub, distributed throughout the hotter parts of India and Nepal. It is commonly known as Vattatiruppi in Tamil and Bala in Sanskrit. This species is not only important as a medicine, but also yields a good fibre. The root is commonly used for nervous and urinary disorders and also for curing various ailments (Anonymous, 1952). The major alkaloid of S. acuta was cryptolepine and this exhibited antimicrobial activity against Proteus vulgaris (Leslie et al., 1980). The aqueous extract of the whole plant showed significant hepato protective activity against carbontetrachloride, paracetamol and rifampicin induced hepato toxicities in experimental albino rats (Rao, 1998). Chemical analysis of the whole plant has led to the isolation of many compounds (Saraswathy et al., 1998; Prakash et al., 1981). Ethanolic * Professor and Head, Pharmaceutical Chemistry extract of the whole plant of S. acuta showed partial neutralization effect against venom lethal effect (Otero et al., 2000). Deposition of calcium oxalate microcrystal in human body can be a significant problem and it is recognised that 70 - 80 % of kidney stones contain calcium oxalate (Seftel and Resnick, 1990). Barring D- penicillamine and allopurinol for cystine and uric acid stones, no effective drug therapy is available in Allopathy for the treatment or prevention of other types of stones. However, a large number of indigenous drugs have been used for this purpose in our country since ancient times (Chopra et al., 1956). Cystone is claimed to have properties of crushing stones in-situ and expelling them without surgery (Dandia et al., 1976). Saxifraga liqulata and Tribulus terrestris are two common herbs of this herbo - mineral formulation (Handa and Kapoor, 2000). The chances of another stone formation can be minimised and dissolution of existing
  23. 23. 22 Vimala and Gopalakrishnan stones enhanced if the composition of urine is modified. The rationale behind the use of herbal remedies is not well established except for a few plants. Studies on the growth of urinary crystals in the presence of some substances which act as inhibitors or promoters of crystal growth may help to pick out potential substances for use by recurrent stone formers (Natarajan et al., 1997). In the present work an attempt has been made to study the inhibitory effect of the methanolic and aqueous extracts of the root of Sida acuta on calcium oxalate crystal growth in-vitro. Materials and Methods The roots of S. acuta were collected from Tirunelveli, Tirunelveli District, Tamil Nadu, India during the month of September. The botanical identity was confirmed by comparing the sample with the Herbarium specimens preserved in the Department of Botany, by Dr. V. Chelladurai, Govt. Siddha Medical College, Palayamkottai, Tirunelveli District, Tamil Nadu, India. A voucher specimen of the plant had been deposited at the Herbarium, Department of Chemistry, Manonmaniam Sundaranar University, Tirunelveli, Tamil Nadu, India [Voucher specimen number MSU 054]. The plant material was thoroughly washed with water, cut into small pieces, dried under shade and powdered. The powdered root was successively extracted with petroleum ether (40º- 60ºC), benzene, chloroform, methanol and water. The methanol and aqueous extracts were condensed and evaporated to dryness under vacuum. There are previous reports on the growth of calcium oxalate crystals from aqueous solution and gel methods (Sindhu et al., 1992; Srinivasan and Natarajan, 1996; Aravindakshan and Jayanthi Bai, 1996). In the present study calcium oxalate crystals were grown in -vitro in silica gel media in Hane’s tubes by single diffusion method. The gel was prepared by treating sodium metasilicate solution of density 1.03 g/ml with 3M acetic acid and the pH was adjusted to 6.3. One of the reactants, calcium chloride (1M) was incorporated inside the gel. After gellation, 1M oxalic acid was slowly added over the gel along with ethanol/water as the supernatant solution for control group and dried methanolic extract dissolved in ethanol / aqueous extract of the test plant for other groups. The crystals appeared as cloudy precipitate and in due course, the depth of the column was found to increase. The thickness (length) of crystal column was measured in centimeter on days 1, 3, 7, 10, 15, 20, 25 and 30. The sizes of the crystals formed were noted in microns (μm) on days 1, 3, 7, 14, 21 and 28 using an optical microscope and the values thus obtained were compared with each other. The whole experiment was carried out at room temperature (27o + 3oC). The inhibitory effect of methanolic and aqueous extracts were studied at 20 mg/5 ml and 10 mg/5 ml doses. The experiments were repeated six times and the average value was taken. All the results were expressed as mean + SE. The test of significance was statistically analyzed using Student’s t-test (Fisher, 1950). After the crystal growth, the crystals were cleared off the gel by repeated washing with distilled water, filtered and air dried. The purity of the crystals thus obtained was assessed by infra-red spectroscopy using JASCO FT-IR-410 by KBr pellet method. For SEM studies a JEOL JSM 35 - C scanning electron microscope was used. Results and Discussion The inhibitory effect of the methanolic and aqueous extracts of the root of S. acuta on calcium oxalate crystal growth have been studied in-vitro at 20 mg/5 ml and 10 mg/5 ml doses and the results are presented in Tables 1 and 2. Concentration dependent inhibitory effect was observed in methanolic extract of S. acuta where the degree of inhibition was more with 20 mg/ 5 ml concentration. In the case of aqueous extract, the size of the crystal columns were almost equal for higher and lower concentrations but slightly less than the size of crystal columns in the control set up. Reductions in the lengths
  24. 24. Effect of Sida acuta on Calcium Oxalate Crystal Growth of crystal columns were observed even on the first day of the experiment for methanolic extract and the reduction in growth rate was more significant from fifth day onwards. Aqueous extract of S. acuta did not produce significant reductions in the lengths of crystal columns. The mean lengths of the crystal columns in the control set up were 3.48 cm and 3.97 cm respectively on the 30th day when ethanol and distilled water were used. The mean lengths of crystal columns in the extract set up were 2.88 cm (20 mg/5 ml) and 2.93 cm (10 mg/5 ml) for methanolic extract and 3.83 cm (20 mg/5 ml) and 3.83 cm (10 mg/5 ml) for aqueous extract. Calcium oxalate crystals from the extract treated tubes when viewed under microscope showed considerable reduction in size when compared to that of the control. The mean lengths of the grown crystals on 28th day of the experiment in the control set up were 180 μm and 294 μm 23 respectively when ethanol and distilled water were used. The mean lengths of the crystals in the extract set up were 96 μm (20 mg/5 ml) and 162 μm (10 mg/5 ml) for methanolic extracts and 246 μm (20 mg/5 ml) and 262 μm (10 mg/ 5 ml) for aqueous extracts on 28th day. The results are presented in Fig. 1 and Fig. 2. The drug treated crystals are found to exhibit IR spectra similar to that of the control suggesting no possibility of any type of chemical bonding between the drug and the oxalate crystals. These IR spectra are shown in Fig. 3-6. The calcium oxalate crystals were also subjected to SEM analysis for understanding the morphological changes if any. SEM of in-vitro grown calcium oxalate crystals by single diffusion method in silica gel media are presented in Table 1. Effect of methanolic extract of Sida acuta root on the lengths of calcium oxalate crystal columns in cm. Days Control Sida acuta 20 mg/ 5 ml 1 3 7 10 15 20 25 30 1.783 + 0.08 2.200 + 0.05 2.716 + 0.01 3.016 + 0.03 3.266 + 0.03 3.483 + 0.04 3.533 + 0.04 3.483 + 0.04 10 mg/ 5ml 1.700 + 0.00 2.150 + 0.00 2.383 + 0.01 ** 2.533 + 0.03 ** 2.783 + 0.01 ** 2.883 + 0.01 ** 2.883 + 0.01 ** 2.883 + 0.01 ** 1.633 + 0.03 2.133 + 0.03 2.383 + 0.03 * 2.550 + 0.05 * 2.783 + 0.08 * 2.933 + 0.08 * 2.933 + 0.08 * 2.933 + 0.08 * All values are mean + SEM of six experiments in each set * P< 0.05; ** P< 0.005 Table 2. Effect of aqueous extract of Sida acuta root on the lengths of calcium oxalate crystal columns in cm. Days Control Sida acuta 20 mg/5 ml 1 3 7 10 15 20 25 30 1.733 + 2.566 + 3.133 + 3.616 + 4.016 + 4.683 + 3.966 + 3.966 + 0.02 0.02 0.02 0.02 0.02 0.04 0.07 0.07 All values are mean + SEM of six experiments in each set * P< 0.05; ** P< 0.005 10 mg/5 ml 1.700 + 2.533 + 3.083 + 3.566 + 3.950 + 4.566 + 3.833 + 3.833 + 1.750 +0.03 2.583 +0.03 3.166 +0.03 3.650 +0.03 4.050 +0.04 4.566 +0.04 3.766 +0.03 3.833 +0.04 0.02 0.03 0.04 0.03 0.03 0.02 0.02 0.03
  25. 25. 24 Vimala and Gopalakrishnan Fig. 7-10. The crystals from control experiments contained individual prismatic crystals, twinned crystals and some aggregates. Crystals from methanolic extract set up contained some deformed and smaller crystal aggregates in addition to single and twinned crystals. Addition of the extract resulted in partial dissolution of the crystals with cracks and crevices and a tendency for chipping away of the parts as compared to control. Similar results were reported by Aravindakshan and Jayanthi Bai (1996). Aqueous extract showed a marked reduction in the tendency to form crystal aggregates as is evident from Fig. 10. Aqueous extract when included in the crystal growth set up also resulted in corrosion, crevices in the crystals which could indicate a dissolution effect for drug. Agglomeration of calcium oxalate crystals plays a major role in urinary stone formation and has the potential to produce very large crystals in a short period of time with no Fig. 3. IR spectra of calcium oxalate crystals treated with ethanol. Fig. 4. IR spectra of calcium oxalate crystals treated with water Fig. 1. Effect of methanolic extract of S. acuta on the size of the crystals. Fig. 2. Effect of aqueous extract of S. acuta on the size of the crystals. Fig. 5. IR spectra of calcium oxalate crystals treated with methanolic extract of S. acuta Fig. 6. IR spectra of calcium oxalate crystals treated with aqueous extract of S. acuta
  26. 26. Effect of Sida acuta on Calcium Oxalate Crystal Growth 7a 7b Fig. 7. Scanning electron micrographs of calcium oxalate crystals (a) On adding ethanol (b) On adding ethanol showing sharp edges. 8a 8b Fig. 8. Scanning electron micrographs of calcium oxalate crystals (a) On adding methanolic extract of S. acuta (b) On adding methanolic extract of S. acuta showing corrosion. dissolution of the crystals. Natarajan et al. (1997) have reported that no nucleation, reduction in the number and size of the crystals, reduction in total mass of the crystals formed, the change in the morphology of crystals and change in the crystalline quality may be considered as inhibitory effects in crystal growth experiments. In the present investigation the morphology of the crystals have changed with uneven surfaces and less defined edges. Platy crystals were obtained (Fig. 8b) when methanolic extract was used. Change in the lengths of crystal columns, shape, size, transparency were also observed indicating the inhibitory effect of the two extracts of Sida acuta. Acknowledgements One of the authors (T. Vimala) wishes to thank the University Grants Commission, New Delhi and the Principal and Management of St. Jude’s College, Thoothoor, Kanyakumari District (India) for selecting her under FIP Programme. The authors also thank Dr. Y.M. Fazil Marickar, Govt. Medical College, Trivandrum for technical assistance and Dr. Peter Koshy, R. R. L. Trivandrum (India) for SEM analysis. 1. 9a 9b 2. Fig. 9. Scanning electron micrographs of calcium oxalate crystals (a) On adding water showing twinning (b) On adding water showing aggregation. 3. 4. 10a 10b Fig. 10. Scanning electron micrographs of calcium oxalate crystals (a) On adding aqueous extract of S. acuta showing minimal aggregation. (b) On adding aqueous extract of S. acuta showing less defined edges. reduction of supersaturation (Hounslow et al., 1988). The crystals also showed less defined edges with dots of corrosion which could facilitate 25 5. 6. 7. References Anonymous: Wealth of India, Raw Materials. CSIR, New Delhi. pp322-323 (1952). Aravindakshan, C. and Jayanthi Bai, N: Effect of Premna latifolia Roxb and Imperata arundinacea Cyril on in-vitro oxalate crystal growth. Ind. J. Clinical. Biochem. 11: 42-45 (1996). Chopra, R.N., Nayar, S.L. and Chopra, I.C: Glossary of Indian Medicinal Plants. Council of Scientific and Industrial Research, New Delhi (1956). Dandia, D.S., Bhatia, S., Narula, I.M.S. and Pendse, A.K: Role of cystone in prevention of urolithiasis : An experimental study in dogs. Ind. J. Surg. 3: 122 (1976). Fisher, R.A: Statistical Methods for Research Workers. Oliver and Boyd, Edinburgh (1950). Handa, S.S. and Kapoor, V.V: Pharmacognosy. 2nd ed. Vallabh Prakashan, Delhi. pp215 (2000). Hounslow, M.J., Ryall, R.L. and Marshall, V.R: A discretized population balance for nucleation, growth and aggregation. AICHE Journal. 34: 1821-32 (1988).
  27. 27. 26 8. 9. 10. 11. Vimala and Gopalakrishnan Leslie Gunatilaka, A.A., Sotheeswaran, S., Balasubramaniam, S., Chandrasekara, A.I. and Badra Sriyani, H.T: Studies on medicinal plants of Sri Lanka III : Pharmacologically important alkaloids of some Sida species. Planta Med. 39: 66-72 (1980). Natarajan, S., Ramachandran, E. and Blisin Suja, D: Growth of some urinary crystals and studies on inhibitors and promoters. II. X–ray studies and inhibitory or promotery role of some substances. Cryst. Res. Technol. 32: 553-559 (1997). Otero, R., Nunez, V., Jimenez, S.L., Fonnegra, R., Osorio, R.G., Garcia, M.E. and Diaz, A: Neutralization of lethal and enzymatic effects of Bothropsatrox venom. J. Ethnopharmacol. 71: 505-511 (2000). Prakash Anand, Varma, R.K. and Ghosal, S: Chemical constituents of Malvaceae Part III. Alkaloidal constituents of Sida acuta, S. humilis, S. rhombifolia and S. spinosa. Planta Med. 43: 384-388 (1981). 12. 13. 14. 15. 16. Rao, K.S. and Mishra, S.H: Anti–inflammatory and hepato protective activities of Sida acuta. Indian Drugs. 35: 92-97 (1998). Saraswathy, A., Susan, T., Gnana Ravi, R., Govindarajan, S. and Kundu, A.B: Chemical investigation of Sida acuta Burm. F. Bulletin of Medico – Ethnobotanical Research. 19: 176-180 (1998). Seftel, A. and Resnick, M.I: Metabolic evaluation of urolithiasis. Urol. Clin. North Am. 17: 159-69 (1990). Sindhu, S., Krishnamoorthy, H., Thomas, N.E., Roshni, S.V., Vathsala, K.R., Aravindakshan, C. and Marickar, Y.M.F: Diffusion controlled growth of calcium oxalate. Indian J. Pure Appl. Phys. 30: 82-83 (1992). Srinivasan, N. and Natarajan, S: Growth of some urinary crystals and studies on inhibitors and promoters. I standardisation of parameters for crystal growth and characterization of crystals. Indian J. Phys. 70A: 563-568 (1996). Address for correspondence: S. Gopalakrishnan, Department of Chemistry, Manonmaniam Sundaranar University, Abishekappatti, Tirunelveli - 627012 Tamil Nadu (India). E-mail: sgkmsu@yahoo.com 020_2009
  28. 28. J. Res. Educ. Indian Med., Jan. - March, 2012 Vol. XVIII (1) : 27-31 ISSN 0970-7700 SPECTROSCOPIC INVESTIGATIONS OF PALAKARAI (COWRIE SHELL) PARPAM S. JOSEPH VEDHAGIRI,* K.GANESAN AND P.C. JOBE PRABAKAR Research and PG Department of Physics, T.B.M.L. College, Porayar - 609307 Nagai District, Tamil Nadu (India) Abstract: Parpam (Bhasma) is one form of internal medicine widely and effectively used in Siddha system of medicine. Shells of Cypraea moneta Linn. is commonly known as cowrie or cowry. In Siddha, it is known as Palakarai and in Ayurveda it is used as Varatika bhasma. The ingredients that are being used for the preparation of a parpam have to be subjected to several stages of pudam, known as calcination process. The three intermediate stages during the preparation of Palakarai parpam were taken for the study. All the three stages were subjected to particle size analysis, atomic and weight percentage of elemental variations using Scanning Electron Microscopy and Energy Dispersive Spectroscopy (SEM/EDS). Also the vibrational frequencies and their related functional groups were assigned using Fourier Transform Infrared Spectroscopy (FTIR) and finally the crystallographic phase changes by X-ray diffraction (XRD) were analyzed. The results of SEM/ EDS, FTIR Spectra and XRD are reported and discussed. Keywords: Cypraea moneta shells, Palakarai parpam, SEM – EDS analysis, FTIR and XRD analysis, Siddha medicine, Varatika bhasma. Introduction Shells of Cypraea moneta Linn. commonly known as cowries are called chozhi (or) palakarai in Tamil. It belongs to the phylum mollusca and the class gastropoda. Similar to the shells of oyster, palakarai also is the shell for protecting the sea mollusc Cypraea moneta. The shell, palakarai is obtained abundantly along the Indian coast. In ancient days palakarai were valued as currency equal to gold, and used as ornamental materials. In traditional system of medicine the shells of Cypraea moneta, have been used as medicine to cure various ailments mainly related with stomach. Palakarai parpam was found to be effective in anti-pyretic and anti-inflammatory, in experimentally induced albino rats (Devanathan et al., 2002). Also used in treating stomach poisoning and all toxaemic states, colic, retention of urine, gonorrhoea and inflammation of urino-genital tract (Anonymous, 1993). Materials and Methods The samples of initial, intermediate and final stages during the preparation of palakarai parpam * Associate Professor (Physics) were collected and labeled as JVP1, JVP2, and JVP3. Scanning electron microscope (SEM-Model: JEOL JSM 5610 Series) was used to examine the morphology and the particle size of the three stages and SEM images of four hundred magnifications of three stages are considered. Probable elements present in atomic and weight percentage at each stage are analyzed using EDS. The fundamental vibration band frequencies are also assigned for all the three stages by recording FTIR spectra over the region 400-4000 cm-1 at 4 cm-1 resolution using KBr wafer on a Nicolet Avatar-360 Series. The XRD technique is employed to assess the phase purity and crystallographic changes during calcinations process of parpam. The XRD patterns of initial and final stages of parpam were recorded with high-resolution powder X-ray diffractometer, PANalytical–Philip–Netherlands / Model XPert Pro, with Cu-Kα 1 radiation (λ = 1.54056 A°) at 40 kev and 30 mA at the scanning rate of 2° per minute and 2θ was varied from 5 to 60°.
  29. 29. 28 Vedhagiri, Ganesan and Prabakar Results and Discussions SEM Particle size Analysis Figure 1a is representing SEM images of 400 magnifications of the three stages. It is observed that the morphology of the particles is of granular amorphous nature, varies from a minimum to maximum size. In comparing all the three stages most significant changes of particle size have been observed due to calcinations process. The range of particle size is varied from stage one to stage two from 41.67-16.66 μm to 33.67-8.34 μm due to first time calcinations. The maximum size of the particle at the second stage is further reduced to 12.82 μm from 33.97 μm and the minimum size has been retained to 8.34 μm at the final stage which clearly indicates the particle size reducing due to the repeated calcinations. The size of the particles is decreasing in the order of JVP1 > JVP2 > JVP3. The FTIR spectra of the samples could reveal the significant vibrational bands (Petsch et al., 1989). The presence of hydroxyl O-H stretching and bending bands are observed around ~3660 to 3800 cm-1 and ~1650cm-1 (Ana et al., 1996) in all the stages and are pronounced less in the final stage which could be attributed to the reduction in the hydrogen bonded structure due to repeated high temperature treatment of the sample. The presence of calcium carbonate is confirmed in all the three stages due to strong vibrational band frequencies prevailed around ~1420 to 1500 cm-1 and 860 to 875 cm -1 (Prabakaran et al., 2005). The vibrational band observation around ~1080 cm-1 is attributed to the presence of calcium carbonate in the form of aragonite. At the final stage due to calcinations this band gets diminished, which clearly indicates that the form of calcium carbonate is not completely getting changed, from aragonite to calcite. It is noticeable that calcite has no vibrational band assignment at ~1080 cm-1 (Narasimhulu and Lakshmanan, 2000). SEM-EDS Analysis The Electron Density Spectrum of 400 magnifications of the three stages is given in Figure 1b. The elemental compositions of palakarai parpam found in the three stages are given in Table 1.The weight percentage of calcium for all the stages are found to be prominent. First stage JVP1 is found to contain 75.07 wt% of calcium and rose to 93.56 wt% at the second stage after calcinations. Finally the weight percentage of calcium is maintained to 80.02% at the last stage. Magnesium, zinc, strontium and barium were the other elements present as listed in Table 1. After three repeated calcinations, palakarai parpam is found to contain calcium as the major constituent to the value of 80.02 wt%. It is concluded that palakarai parpam is consisting mainly of calcium and magnesium, barium and a trace amount of strontium as reported. The presence of calcium carbonate in all the three stages is further confirmed by the existence of vibrational frequencies at ~875 cm-1 and ~712 cm-1 (Konwar et al., 2004). In the final stage, calcium carbonate has been further confirmed by the strong vibrational band frequencies exhibited at ~1420, ~874 and ~712 cm-1 (Gosh, 1978). In comparing the FTIR spectra of JVP1 and JVP2, it is observed that the vibrational band frequencies due to methyl amide and hydroxyl compounds related C-H, NH and O-H stretching have been shifted. At the final stage all these bands are found weak indicating that due to calcinations, the organic substances have been progressively reduced. FTIR Analyses The recorded FTIR spectrum of the three stages is given in Figure 2. Each compound has its own unique absorption pattern in the finger print region ~1500 to 650 cm-1. (Kalsi, 1995). XRD Analyses The XRD patterns are analyzed and the peaks are indexed. XRD patterns of JVP1 and JVP3 are presented in Figure 3. Powdered XRD patterns of initial stage showed the characteristic peaks of
  30. 30. Spectroscopic Investigations of Palakarai (Cowrie Shell) Parpam Figure 1a: Stage I Figure 1b: Stage I Figure 1a: Stage II 29 Figure 1b: Stage II Figure 1b: Stage III Figure 1a: Stage III Figure 1a: Three stages of SEM images of Palakarai Parpam (400 magnification) Figure 1b: Three stages of EDS of Palakarai Parpam (400 magnification)
  31. 31. Vedhagiri, Ganesan and Prabakar %T 30 %T Wave numbers (cm-1) Wave numbers (cm-1) %T Figure 3. XRD Palakarai Parpam Wave numbers (cm-1) Figure 2. FTIR – Spectrum of Palakarai Parpam poorly crystalline calcium carbonate. All the peaks are indexed from their observed d-values. The structure is found to be orthorhombic with lattice parameters a =3.8218 A°, b = 4.9827 A°, c = 24.2633 A° and α = β = γ = 90° with unit cell volume 462.04 (A°). 3 The lattice parameters of JVP3 (a =3.8502 A°, b = 10.8675 A°, c = 23.3284 A° and α = β = γ = 90° with unit cell volume 976.19 A°)3 are slightly changed and crystal system prevails the same as orthorhombic. It is a known fact that calcite form of calcium carbonate is always of hexagonal structure. If it changes from aragonite to calcite, the crystal system would be of hexagonal.
  32. 32. Spectroscopic Investigations of Palakarai (Cowrie Shell) Parpam 31 Table 1. Elemental composition of Palakarai Parpam. Sl. No. Element and Symbol Weight % Atomic % JVP1 JVP2 JVP3 JVP1 JVP2 JVP3 75.07 93.56 80.02 83.03 96.68 78.38 - 00.20 11.70 - 00.34 18.90 1 Calcium, C 2 Magnesium, Mg 3 Iron, Fe 11.50 00.10 - 9.13 00.07 - 4 Zinc, Zn 06.06 - - 04.11 - - 5 Strontium, Sr 07.36 06.14 02.19 03.73 02.90 00.98 6 Barium, Ba - - 06.08 - - 01.74 No such change of crystal structure from initial to final stage is observed, except the variations in sides and volume of unit cell. Moreover the peak around 2θ of 29° at the initial stage has not been shifted and remains almost the same in the final stage, confirming no phase transition of crystal system. The increase in the intensity of the peaks is indicating the improvement of crystallinity due to calcinations process. The increase in unit cell volume is attributed to the increase of surface area which is a vital factor in drug interaction. The peaks around 2θ of 29.2° to 29.6° are attributed to the presence of calcium carbonate (Davis et al., 1997). The sharp peaks observed at JVP3 clearly imply that the repeated calcinations promote crystallinity which is accountable for bioavailability and dissolution rate. Conclusion The particle size of parpam is reduced due to repeated heat treatment. SEM-EDS analysis reveals that palakarai parpam is having calcium as prominent element and magnesium, barium and strontium as trace elements. The organic substances are progressively reduced from initial to the final stage. The presence of calcium carbonate in aragonite form is observed. The XRD study reveals that the crystallinty has been improved due to calcinations. The orthorhombic crystal structure has not been changed and remains as orthogonal up to the final stage. Acknowledgements Authors are grateful to Kaviraj Pharmaceuticals, Erode, Tamil Nadu (India) for providing the samples. Research grant received under a minor research project of UGC by one of the authors (SJV) is gratefully acknowledged. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. References Ana H Delgado, Ralph M, Paroli and James J Beaudock: Applied Spectroscopy. 50: 970 (1996). Anonymous: Formulary of Siddha Medicines. IVth ed. IMPCOPS, Chennai. pp 228 (1993). Davis EE, Fisher AT and Firth JV Procee: Ocean Drilling Program - Initial Reports. Vol. 168: 35-45 (1997). Devanathan Deva R, Prema S and Saraswathy Jeyaendra: Proce. of Fifth International Congress on Traditional Asian Medicine. pp 10 (2002). Gosh SN: Jour. Mat. Sci. Vol. 13: 1877-1886 (1978). Konwar R, Changmai R and Baruah GD: Indian Jour. of Pure and Applied Phy. Vol. 42: 812-815 (2004). Kalsi PS: Spectroscopy of Organic Compounds. IInd ed. New Age International Ltd., New Delhi. pp 74 (1995). Narasimhalu, KV and Lakshmana RJ: Spectrochim. Acta- A. Vol. 56: 1345-1353 (2000). Petsch, Clerc, Seibel and Simon: Tables of Spectral Data for Structure Determination of Organic Compounds, IInd ed. Springer – Verlag, Hong Kong (1989). Prabakaran K, Balamurugan A and Rajeswari S: Bull. Mater. Sci. Vol. 28(2): 115-119 (2005). Address for correspondence: S. Joseph Vedhagiri, Research & PG Department of Physics, T.B.M.L. College, Porayar - 609307 Nagai District, Tamil Nadu (India). E-mail: jvedha@rediffmail.com 008_2009
  33. 33. J. Res. Educ. Indian Med., Jan. - March, 2012 Vol. XVIII (1) : 33-43 ISSN 0970-7700 HYPOGLYCAEMIC ACTIVITY OF INDIAN MEDICINAL PLANTS IN STREPTOZOTOCIN DIABETIC RATS E. N. SUNDARAM,1 K. P. SINGH2 AND P. UMAMAHESWARA REDDY3 Drug Standardisation Unit,1,2 Department of Zoology,3 Osmania University, Hyderabad – 500007 Andhra Pradesh (India) Abstract: In the present study, the hypoglycaemic effect of alcoholic extracts of M. charantia, A. marmelos and E. jambolana was studied in streptozotocin (STZ) induced diabetes. Rats were made diabetic by intraperitoneal injection of STZ (30 mg/kg) in citrate buffer. On confirmation of diabetes after 48 hrs of injection, alcoholic extract of medicinal plant (250 or 500 mg/kg) or glibenclamide (300 μg/kg) administered orally to rats for 30 days. These three plants produced dose and duration dependent hypoglycaemia very similar to that of glibenclamide. At the end of one month, serum glucose levels of STZ diabetic rats with daily doses of 500 mg/kg of any one of the alcoholic extract were ‘more or less’ comparable to that of normal rats. The anti-diabetic effect of these plants might be due to enhanced insulin secretion from the viable β-cells of islets of Langerhans as evidenced by presence of more viable β-cells and less necrotic changes in the pancreas of diabetic rats as compared to that of control diabetic rats. Thus, these plants appear to be better alternative for the diabetic patients who are prone to develop side effects with the regular use of synthetic hypoglycaemic drugs as these are also plants devoid of any untoward/toxic effects. Keywords: STZ diabetic rats, Hypoglycaemic effect, Histopathology, Momordica charantia, Aegle marmelos, Eugenia jambolana. Introduction In the present era, diabetes mellitus is a global problem. There are an estimated 143 million people worldwide, suffering from diabetes.1 This number may probably double by the year 2030.2 The diabetes enters in man’s life unnoticed as its symptoms remain silent for many years and is considered to be the mother of many diseases. Reports from the World Health Organization (WHO) indicate that diabetes mellitus is one of the major killers of our time, with people in South - East Asia and Western Pacific being most at risk. 3 Prolong and continuous use of currently available oral hypoglycaemic drugs in modern medicine not only associated with side effects like anorexia, nausea, vomiting, flatulence, abdominal discomfort but their use is also contraindicated in patients suffering from renal, hepatic, cardiorespiratory insufficiency, alcoholism and in advanced age. Likewise, repeated injections of insulin may lead complications like insulin allergy, insulin resistance and localized lipoatrophy at the site of injections.4, 5 On the contrary, long before the use of insulin, medicinal plants have been used since ancient times by the physicians and laymen to treat not only the diabetes but also a great variety of human diseases such as heart diseases, cancer etc.6, 7 The text of Charaka, Sushruta, Vagbhatta and Ayurveda also elaborated in details the clinical features of diabetes mellitus, its complications and treatment with medicinal plants. The therapeutic approach of traditional medicines is more holistic because multifactorial pathogenicity of diabetes demands multi - model therapeutic approach.8 Considering the economic resource constraints, easy availability and cheapness and day to day use of M. charantia, A. marmelos and E. jambolana in diabetes mellitus by laymen since ancient time. The present study was undertaken to elucidate the effectiveness of these three plants as 1. Research Officer (Endocrinology) 2. Research Officer (Pharmacology) 3. Professor

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