“ANTIDIABETIC ACTIVITY OF JASAD BHASMA IN
RETINO AND RENAL COMPLICATIONS ASSOCIATED WITH
STREPTOZOTOCIN-INDUCED DIABETIC RATS’’
M. Pharm Dissertation Protocol Submitted to
RajivGandhiUniversity of Health Sciences, Karnataka
Bangalore – 560041
By
SANJEEVKUMAR MEDLI.
B.Pharm
Under the Guidance of
Dr. R. A. SHASTRI. M. Pharm., Ph. D.
Assistant Professor
Post Graduate Department of Pharmacognosy,
SET’S College of Pharmacy,
S.R.Nagar, Dharwad,
Karnataka – 580 002.
RAJIVGANDHIUNIVERSITY OF HEALTH SCIENCES,
KARNATAKA, BANGALORE.
ANNEXURE-II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION
1 / NAME OF THE CANDIDATE AND ADDRESS / SANJEEVKUMAR. I. MEDLISET’S COLLEGE OF PHARMACY
S.R.Nagar, Dharwad,
Karnataka – 580002.
2 / NAME OF THE INSTITUTION / POSTGRADUATE DEPARTMENT OF PHARMACOGNOSY
SET’S COLLEGE OF PHARMACY
S.R.Nagar, Dharwad,
Karnataka – 580 002.
3 / COURSE OF STUDY AND SUBJECT / MASTER OF PHARMACY IN PHARMACOGNOSY
4 / DATE OF THE ADMISSION / JUNE 2010
5 /
TITLE OF THE TOPIC
“ANTIDIABETIC ACTIVITY OF JASAD BHASMA IN RETINO AND RENAL COMPLICATIONS ASSOCIATED WITH STREPTOZOTOCIN-INDUCED DIABETIC RATS”6 / BRIEF RESUME OF THE INTENDED WORK
6.1 / Need for study
Ayurveda is the medicinal system that has been practiced in India for centuries; the system involves the use of medicines synthesized as per the methods quoted in ancient ayurvedic texts. Drugs of both herbal origin and metal preparations are used for oral administration.The drugs which involve preparations from the calcinated metals are known as “Bhasmas”. Bhasmas are inorganic in nature and are prepared by converting a metal into its compounds like carbonates, oxides etc. Bhasmas of Iron, Calcium, Copper, Tin, Silver, Gold, Lead, Zinc are commonly used.
JasadBhasma is a bhasma of Zinc and used for treating various eye ailments of some diabetic forms and in muscle building1. Various synthetic procedures for the preparation of the bhasmas are quoted in ancient Ayurvedic text. The methods are broadly classified on the basis of the use of Kajjali, Hartal (Ayurvedic preparation containing Mercury and Arsenic respectively) and herbal products in the preparation of bhasma according to Ayurveda ,the bhasma involving Kajjali in its preparation is suppose to have greater value as compared to bhasma prepared by other methods2.
Diabetes mellitus is a chronic metabolic disorder characterized by a high blood glucose concentration-hyperglycaemia (fasting plasma glucose > 7.0 mmol/l or plasma glucose > 11.1 mmol/l2 hours after a meal) – caused by insulin deficiency, often combined with insulin resistance. Hyperglycemia occurs because of uncontrolled hepatic glucose output and reduced uptake of glucose spills over into the urine (glycosuria) and causes an osmotic diuresis (polyuria), which in turn results in dehydration, thirst and increased drinking (polydipsia).3The IDF (International Diabetes federation) has subsequently released estimates of the numbers of people with diabetes for 2003 and forecasts for 2025 of 194 million and 334 million, respectively.4 International Diabetes Federation, (2006) published the number of people with diabetes in India currently is around 40.9 million and is expected to rise to 69.9 million by 2025 unless urgent preventive steps are taken.5
Although diet and exercise are the first steps toward achieving treatment goals of diabetics, 90% of patients with diabetes cannot maintain long term glycemic control with diet and exercise alone. Thus, antihyperglycemic drugs are necessary for the treatment of diabetes6. The oral hypoglycemic agents like Sulfonylureas (TolbutamideGlibenclamide, Glipizide), Biguanides (Phenformin, Metformin), Thiazolidinediones(Troglitazone, Rosiglitazone),-Glucosidsase inhibiotrs (Acarbose, Miglitol)have been extensively used in type-2 Diabetes mellitus. But drawback of insulin and oral hypoglycemic agents is, none of them are ideal in correcting blood glucose levels round the clock, with single dose administration7.
In recent years, there has been renewed interest in the treatment against different diseases using herbal drugs as they are generally non-toxic and WHO has also recommended the evaluation of the effectiveness of plants in condition where we lack safe modern drugs. In the Indigenous system of medicine like Ayurveda, many herbal medicines have been less-toxic recommended for the treatment of diabetes or madhumeha and some of them experimentally evaluated8. Recently, many traditional plants like Phyllanthusamaracus,Liriopespicata, Bruceajavanica, Terminaliabelerica, , Buteamonosperma, Azadirachtaindica and Abromaaugusta have been extensively studied for their antihyperglycemic and antihyperlipidemic activity using animal mode.
Diabetes complications are divided into microvascular and macrovascular. Microvascular complications include damage to eyes (retinopathy) leading to blindness, to kidneys (nephropathy) leading to renal failure and to nerves (neuropathy) leading to impotence and diabetic foot disorders (which include severe infections leading to amputation). Macrovascular complications include cardiovascular diseases such as heart attack, stroke and insufficiency in blood flow to legs9
6.2 / Review of literature
Jasadbhasmais a bhasma of zinc and is used for treating various eye ailments,in some diabetic forms and in muscle building10.
In the present work, Jasadbhasma will be prepared using Kajjali. The general procedure for preaparingbhasmas involve treatment of a metal with various herbal products like Aloe-vera gel, lemon juice, garlic paste, some animal products like cow’s milk, ghee and some inorganic mixtures of mercury and sulphur11.
To our best knowledge no scientific data regarding the antidiabetic activity ofJasadbhasma are available in literature. Therefore, we undertake this project with following objectives.
6.3 / Objectives of the study
Formulation of Jasadbhasma according to traditional methodology.
Standardization of Jasadbhasmaas per standard guidelines.
To investigate the antidiabetic activity of Jasadbhasmausing .Streptozotocin induced diabetic rats.
To assess the effect of Jasadbhasma in diabetic retinopathy and nephropathy.
7 / MATERIALS AND METHODS
7.1 / Sources of data
. International Journal of Pharmaceutics
. Science Direct
. Journal of Pharmaceutical sciences
. Ethnopharmacology Journal
. Psychopharmacology
. Indian Journal of Experimental Biology
. Food Chemical Toxicology
. Planta Med
. Pubmed
. Fitoterapia
. Word Wide Web
. J-Gate@ Helinet etc
Method of collection of Data
The data is generated using laboratory experimental techniques. In-vivo pharmacological activities will be carried out on male Wistar rats. The result and data obtained from present study will be analyzed by one way analysis (ANOVA) test followed by Tukey’s test.
Pharmacological Evaluation
Animals:
Albino wistar male rats weighing 150-200g will be used. The animals will be maintained under controlled conditions of temperature (23 ± 2C), humidity (50 ± 5%) and 12-h light-dark cycles. The animals are randomized into experimental and control groups and housed each in two sanitized polypropylene cages containing sterile paddy husk as bedding. They will have free assess to standard pellets as basal diet and water ad libitum.
Acute toxicity studies:
In order to know the safer dose of bhasma, up and down or staircase method will be employed as described12.
OGTT in normal rats:
Different doses of bhasma/glibenclamide (GLB) and vehicle will be given orally to normoglycemic rats fasted for 18 h. Thirty minutes later, glucose (2 g/kg body weight) will be administered orally. Serum glucose will be estimated at 0 min (i.e. immediately after glucose load), 30, 60, 90 and 120 mins after glucose administration. The results are expressed as integrated area under curve for glucose (AUCglucose) as calculated by trapezoid rule.
Standardization of STZ dose to induce diabetic condition in rats:
A single intraperitoneal injection of different doses of STZ (35,45 and 65mg/kg body weight) will be administered to 6 animals per group. After 7 day post STZ administration, serum glucose levels will be estimated and the extent of glucosuria will be estimated using Diastix (Bayer Diagnostics, India). Rats showing glucose level > 200 mg/dl will be considered as diabetic and included in the study. A single oral dose of 10 mg/kg of GLB will be administered to all groups of rats. Glucose levels will be estimated at 0, 2, 4, 6 and 10 h after drug administration (single-dose one-day study).
Effect of extracts on stz-induced diabetes in rats:
Optimized dose of STZ will be used to induce diabetes in rats. These diabetic rats will be randomizing into different groups based on their glucose levels. Single-dose one-day study will be carried out as mentioned earlier. Further these groups of rats will be treated with different doses of bhasma for 15 consecutive days [Multiple-dose fifteen-day study]. Serum glucose levels will be estimated on days 0, 7, 10 and 15thday.
Oral glucose tolerance test (OGTT) in diabetic rats:
At the end of 15 days of treatment, glucose (2 g/kg body weight) will be administered to 12 h-fasted rats and blood samples will be collected at 0 (immediately after glucose load), 30, 60 and 120 min after glucose administration. Glucose and insulin will be estimated and results were expressed as integrated area under curve for glucose (AUCglucose) and insulin (AUC insulin)
Biochemical Parameter
Serum insulin
Serum triglyceride (TG)
Serum total cholesterol (TC)
HDL-c
VLDL-c, LDL-c calculated as described in literature13
TC/HDL-c & LDL-c/HDL.
Diabetic complication models
Administration ofstreptozotocin(60mg/kg dissolved in 3 mM citrate buffer pH 4.5 intraperitoneally)for longer period develop diabetic complications. In our study, we will investigate the effect of Jasadabhasmain diabetic retinopathy and nephropathy in animal models.
Diabetic Retinopathy
Fluoresce in angiography’s is a test used for abnormal blood vessels. To do the test, a fluorescent dye was injected into a vein in the leg 14. The dye travels through the body including the eyes. With special camera meant for this purpose photography of the retina will be taken.
- Observation of Retina for opaqueness
- Laser spots
- Vessels in retina
Serum urea nitrogen
Serum albumin
Serum Protein
Serum creatinine
Urine glucose
Urine ketonebodies
Histopathological Studies:
At the end of study, the kidneys and hearts will be collected from the animals and then fix in 10% neutral buffer formalin and they will be subjected to histopathological examination.
7.3 / Does the study require any investigation or interventions to be Conducted on patients or other humans or animals?If so please describe briefly.
The above study requires investigations to be done on the albino rats of Wister strain for the determination of antidiabetic activity. The study is planned in accordance with the procedure reported in the literature
7.4 / Has ethical clearance been obtained from your institution in case of 7.3?
The copy of the ethical clearance of certificate is attached.
8 / REFERENCES
1.VaidyaNagindasChhaganlal Shah, Bharat Bhaishajy Ratnakar, B.JainPublishers,NewDelhi,India,Vol 4 (Hindhi and Sanskrit),1985,
2.Kulkarni D.A., Rasaratnasamuchaya,Part-1 MahenchandraLaxumandas, New Delhi,India,Part-1 (Hindhi and Sanskrith),1986,
3. [cited 2008 Nov 30th]
4.Ayashi T, Maruyama H, Kasai R, Hattori K, Takasuga S, Hazeki O, et al Ellagitannins from Lagerstroemia speciosa as activators of glucose transport in fat cells. Planta Med 2002;68(2):173–75.
5. Anthony M. The Evaluation of Diabetes Knowledge in Relation to the
Theory of Scientific Revolutions. DiabEdu 2002;28(5): 688-96.
6.Ratner RE, Basciano H, Federico L, Adeli K. American J. Cardiol
2005;88:1-5
7.Position Statements. International Diabetes Federation, Brussels:2005.
8.Roa K, Kirishna MB, Srinivas N. Effect of chronic administration of Boerhavia diffusa Linn. Leafextract on experimentallyinduceddiabetes in rats Trop J Pharm Res 2004;3(1):305-59.
9. World Health Organization. [Online]. 1999 Available from: URL:
10. Vaidya NS, Bharath BR, Jain B. Publishers, New Delhi, India, Vol 4(Hindi and Sanskrit).1985.
11. Vaidya GG, Siddaushaud Part-1, VaidyakGranth Bhandar, Pune, India, Part-1(Marathi and sanskrit), 1992.
12. Ghosh MN. Fundamentals of Experimental Pharmacology. 2nd ed., Calcutta:Scientific Book Agency; 1984.
13. Friedewald WT, Levy RI , Fredrickson DS. Estimation of low-density lipoprotein cholesterol in plasma without use of the preparative centrifuge. ClinChem1972;18:499.
14. Yotsumoto, T., Naitoh, T., Sakuya ,T. and Tanaka,T.(1997) Effects of specific antagonists of angiotensinII receptors and captopril on diabetic nephropathy in mice. J.Pharmacol.75, 59- 64.
9
/SIGNATURE OF THE CANDIDATE
10 / REMARKS OF THE GUIDE / The above mentioned information and literature has been extensively investigated, verified and was found to be correct. The present study will be carried out under my supervision and guidance.11 / NAME AND DESIGNATION OF GUIDE
SIGNATURE
/ Dr. R. A. SHASTRI,M. Pharm., Ph.D.,Assistant Professor
Post Graduate Department of Pharmacognosy,
SET’s College of Pharmacy, S.R.Nagar,
Dharwad. Karnataka – 580 002.
12
/NAME AND DESIGNATION OF CO – GUIDE
SIGNATURE / …………13 / NAME AND DESIGNATION OF HOD
SIGNATURE / Dr. P. V. HABBU,M. Pharm., Ph.D.,
Professor & HOD
Post Graduate Department of Pharmacognosy,
SET’s College of Pharmacy, S.R.Nagar,
Dharwad. Karnataka – 580 002.
Mobile No.: +91 – 9448224894
E-mail:
14
/REMARKS OF PRINCIPAL
/ The above mentioned information is correct and I recommend the same for approval.15 / NAME OF THE PRINCIPAL
SIGNATURE / Dr. V. H. KULKARNI, M. Pharm., Ph.D.,
Principal, SET’s College of Pharmacy,
S.R.Nagar, Dharwad. Karnataka – 580002.
Mobile No.: +91 – 9448357804
E-mail: