ANTIDIABETIC ACTIVITY OF METHANOLIC EXTRACT OF BARK OF BAUHINIA PURPUREA

ANTIDIABETIC ACTIVITY OF METHANOLIC EXTRACT OF BARK OF BAUHINIA PURPUREA

S. Pahwa*¹, R. Mazumder ¹ and S. Bhattacharya ²

Department of Pharmaceutical Technology, Noida Institute of Engineering and Technology, 19, Knowledge Park-II, Greater Noida- 201 306, Uttar Pradesh, India

Shambhunath Institute of Pharmacy ², Jhalwa, Peepal Gaon, Allahabad- 211 012, Uttar Pradesh, India

ABSTRACT

The present investigation was carried out to study the antidiabetic effect of the methanolic extract of bark of Bauhinia purpurea in Streptozotocin induced diabetic model. The antidiabetic activitywas evaluated in normal & Streptozotocin induced diabetic rats. Decreased blood glucose level of the test animals showed that the extract exhibited significant antidiabetic activity when compared to diabetic control group. The results also indicated dose dependent effect. The antidiabetic activity produced by the extract might be due to the increased uptake of glucose at the tissue level or due to an increase in pancreatic beta cell function or due to inhibition of intestinal absorption of glucose.

Streptozotocin

INTRODUCTION:Diabetes mellitus (DM) is a chronic disease caused by inherited and/or acquired deficiency in production of insulin by the pancreas, or by the ineffectiveness of insulin produced.

Such deficiency results in increased concentrations of glucose in the blood, which in turn damage many of the body’s systems, in particular the blood vessels and nerves. As the number of people with diabetes multiply worldwide, the disease takes an ever-increasing proportion of national and international health care budgets. It is projected to become one of the world’s main disablers and killers within the next 25 years.

Regions with greatest potential are Asia and Africa, where DM rates could rise to two to three-folds than the present rates. Apart from currently available therapeutic options, many herbal medicines have been recommended for the treatment of diabetes. Traditional plant medicines are used throughout the world for a range of diabetic presentations ¹.

Bauhinia purpurea (family: Caesalpiniaceae) is a medium sized, evergreen, ornamental tree found throughout India. The leaves are rigidly sub-coriaceous, glabrous and shallowly cordate. The purple colored flowers of the species distinguishes it specifically from other species of Bauhinia. It is presently being used for ailments such as sores, wounds, diarrhea, dropsy, pain, rheumatism, convulsions, delirium, septicemia and so on ². Its decoctions are recommended for ulcers as a useful wash ³. The aerial parts of the plant are reported to contain flavanone glycosides, foliar flavonoids, 6-butyl-3-hydroxy flavanone, amino acids, phenyl fatty ester, lutine and β- sitosterol ^4-9.

Flavonoids are polyphenolic compounds are widely distributed in the plant kingdom. They are reported to exhibit various pharmacological activities such as CNS activity, cardiotonic activity, lipid-lowering activity, antioxidant activity, hepatoprotective activity, and hypoglycemic activity ¹⁰. These active constituents and the above mentioned activities in turn appear to correlate with some other biological activities.

In view of alleged antidiabetic potential of Bauhinia purpurea, we have investigated effect of methanolic extract of its bark onfasting blood sugar levelsin Streptozotocin (STZ) induced diabetic rats. STZ [2-deoxy- 2- {3- (methyl- 3- nitrosoureido)- D-gluco-pyranose}] is synthesized by Streptomycetes achromogenes and is used to induce both  insulin-dependent and non-insulin dependent DM. STZ is also efficacious after intraperitoneal administration of a dose between 40-60 mg/kg body weight or even a higher dose, but single dose below 40 mg/kg body weight may be ineffective ^{11, 12}.

MATERIALS AND METHODS:

Plant Material: The bark of B. purpurea was collected from the surrounding areas of Greater Noida in June, 2010. The plant was identified and authenticated (Voucher No NHCP/ NBPGR/2010-20 dated 10th May 2010) by Dr. Anjula Pandey, Principal Scientist, National Bureau of Plant and Genomic Resources (NBPGR), New Delhi, India. A copy of the herbarium has been preserved in the Department of Pharmaceutical Technology, NIET, Greater Noida, India, for future reference. The part was sun dried after washing and then ground to a coarse powder in a mechanical grinder.

Extraction Procedure: The powdered bark was macerated with different polar and non polar solvents for seven days. After which each extract was subjected to phytochemical screening using standard methods ¹³.

Since the methanolic extract had the maximum constituents henceforth, methanol was chosen as the solvent for extraction.

The coarse powder of the bark (32.81 g) was extracted in a soxhlet apparatus with methanol and the solvent was removed by controlled evaporation under reduced pressure on a heating mantle at temperature below 60^oC. The crude extract thus obtained was tested for its antidiabetic potentiality.

The  extract was used to prepare suspensions of 50 mg/kg and 100 mg/kg concentrations using water and  Tween 80 : Tween 20  (1:1) for treatment.

Drugs: STZ was obtained from SRL Lab, Delhi, India. All other chemicals used for the study were of analytical grade.

Animals: Male Wistar rats weighing between 150 and 200 g were used for antidiabetic activity. Toxicity study was carried out on Albino mice (25-30 g) as per the OECD guidelines and the study was approved by the Institutional Animals Ethics Committee (CPCSEA) (Approval code no. 1121/ac/CPCSEA/07/NIET/IAEC /2010/32P/16). Animals were fed a standard pellet (Lipton India Ltd., Mumbai, India) and water ad-libitum. After randomization into various groups and before initiation of experiment, the rats were acclimatized for a period of 7 days under 24-28^oC temperature, 60-70% relative humidity and 12 h day and night cycle. Animals described as fasted were deprived of food for 16 h but had free access to water.

Sample collection: Blood samples were collected by retro-orbital plexus puncture method and blood glucose levels were estimated using an electronic glucometer (Morepen, Baddi, HP, India).

Effect of Bauhinia purpurea bark Extract on STZ Induced DM ¹⁴: Diabetes was induced in rats by tail vein injection of STZ (50 mg/kg), dissolved in citrate buffer. One group of 6 identical rats was kept without STZ administration as normal control, group I. Forty eight hours after STZ administration, blood samples were drawn by retro-orbital puncture method and glucose levels were determined to confirm diabetes. The diabetic rats exhibiting blood glucose levels in the range of 140 mg/dl or more were selected for the studies. These diabetic rats were sub-divided into 3 groups as follows:

Group II rats, served as diabetic control (STZ induced) were given 0.5 ml of 5% Tween 80 in place of the extract;

Group III diabetic rats were given 50 mg/kg B. purpurea bark extract in 0.5 ml 5% Tween 80;

Group IV diabetic rats were given 100 mg/kg B. purpurea bark extract in 0.5 ml 5% Tween 80;

Group V diabetic rats were given 0.5 ml of 5% Tween 80 containing Glibenclamide (500 μg/kg).

The dose (500 μg/kg) of Glibenclamide was selected based on previous reports.¹⁵ The normal control group of rats (group I) were given 0.5 ml of 5% Tween 80 only.

Each of the control and the test groups consisted of 6 animals. The treatments were continued daily for 15 days. Blood was collected by retro-orbital puncture method for glucose estimation just before drug administration on the 1^st day and 1 h after drug administration on days 4, 7 and 10.

Statistical Analysis: The results were expressed as Mean ± SEM. Comparison between the groups was made by analysis of variance (ANOVA), followed by Dunnett's test. A value of P < 0.001 was considered significant ¹⁶.

RESULTS AND DISCUSSION:

Preliminary Phytochemical Screening: The results of the preliminary phytochemical screening of the various extracts of B. purpurea bark were depicted in Table 1, which showed the presence of alkaloids, glycosides, saponins, phytosterols, phenolic compounds and tannins in the methanolic extract of the part used.

TABLE 1: PRELIMINARY PHYTOCHEMICAL SCREENING OF VARIOUS BARK EXTRACTS OF BAUHINIA  PURPUREA

 Yield: After soxhleting the bark powder (32.81 g) with methanol, a semisolid, dark, viscous crude extract with 16.39% w/w yield was obtained, which was subjected to further antidiabetic studies.

The Effect of Methanolic Extract of Bauhinia purpurea Bark on STZ induced Diabetic Rats: Administration of STZ (50 mg/kg, i.p.) led to many folds elevation of fasting blood glucose levels, which was maintained over a period of 2 weeks. Two weeks of daily treatment of methanolic extract of B. purpurea led to a dose-dependent fall in blood sugar levels. Vehicle control animals were found to be stable in their body weight but diabetic rats showed significant reduction in body weight during 15 days (Table 2).The effect of methanolic bark extract of Bauhinia purpurea on glucose level in STZ induced diabetic ratsare summarized in Table 3 , where a significant reduction (P<0.001) in blood glucose level was observed in the drug treated animals, when compared to the diabetic control group.

TABLE 2: EFFECT OF METHANOLIC EXTRACT OF BARK OF BAUHINIA PURPUREA ON BODY WEIGHT (G) ON STZ (50 MG/KG, I.P.) INDUCED DIABETES IN RATS

Values are mean ± SEM for groups of six animals each; ^* P < 0.01 as compared to normal control on corresponding day

   TABLE 3: EFFECT OF METHANOLIC EXTRACT OF BARK OF  BAUHINIA PURPUREA ON GLUCOSE LEVEL IN STZ INDUCED DIABETIC RATS

Values are mean ± SEM, 6 rats in each group; STZ (50 mg/kg) was injected to control and all other drug treated groups; ^a STZ induced diabetic group vs normal group, #p>0.05; ^b extract treated group vs STZ induced diabetic group, *p<0.01; **p<0.001

CONCLUSION: The extract has exhibited antidiabetic property in streptozotocin induced diabetic rats, as evident from the glucose levels. The hypoglycemic activity may be ascribed to the presence of flavonoids, which have been shown to inhibit cyclooxygenases and promote β-cell regeneration besides having insulin secretary property ^17-19. The results of the present study suggests that the methanolic extract of the bark of B. purpurea  illustrates significant hypoglycemic activity, which may be due to the presence of flavonoids in it, as claimed by earlier reports.

REFERENCES:

1. Nagappa  AN, Thakurdesai  PA, Venkat Rao  N and Singh J:  Antidiabetic activity of Terminalia catappa Linn. fruits. Journal of Ethnopharmacology  2003; 88:45-50.
2. Asolker LV, Kakkar KK and Chakre OJ: 2nd Supplement to Glossary of Indian Medicinal Plants, Part-I (A-K). National Institute of Science Communication, New Delhi, 2000.
3. Kirthikar KR and Basu DB: Indian Medicinal Plants. Oriental Enterprises, Dehradun,  Edition 3, Vol. 4, 2001.
4. Salantino A and Blatt TT: Foliar flavanoids of nine species of Bauhinia. Revista Brsileira de Botanica 1999; 22:17-20.
5. Yadava RN and Tripathi P: A novel flavones glycoside from the stem of Bauhinia purpurea. Fitotherapia 2000; 71:88-90.
6. Panda S and Kar K: Withania somnifera and Bauhinia purpurea in the regulation of circulating thyroid hormone concentrations in female mice. J Ethnopharmacol 1999; 67:233-239.
7. Vijayakumari KP and Siddhuraru: Chemical composition, amino acid content and quality of protein in the legume of Bauhinia purpurea. J Sci. Food Agr 1997; 73:279-286.
8. Ramachandran, Reena and Joshi BC: Chemical examination of Bauhinia purpurea flowers. Current Sci 1967; 36:5774-5775.
9. Pettit GR, Numata A, Iwamota C, Usami Y and Yamada T: Isolation and structures of bauhiniastatins 1-4 from Bauhinia purpurea . J Nat Prod 2006; 69:323-327.
10. Rajanarayana K, Reddy MS, Chaluvadi MR and Krishna DR: Bioflavonoids classification, pharmacological, biochemical effects and therapeutic potential. Indian J Pharmacol 2001; 33:2-16.
11. Szkudelski T: The mechanism of Alloxan and Streptozotocinaction in B cells of the rat pancreas. Physiol Res 2001; 50:536-546.
12. Katsumata K, Katsumata K Jr. and Katsumata Y: Protective effects of diltiazem hydrochloride  on the occurrence of alloxan- or streptozotocin induced diabetes in rats. Horm Metab  1992; 24:508-510.
13. Khandelwal KR: Practical Pharmacognosy and Experiments. Nirali Prakashan, Pune, 2005.
14. Babu V, Gangadevi T and Subramonium A: Antidiabetic activity of ethanol extract of Cassia kleinii leaf in Streptozotocin –induced diabetic rats and isolation of an active extract and toxicity evaluation of the extract. Indian Journal of Pharmacology 2003; 35:290-296.
15. Augusti KT and Sheela CG: Antiperoxide effect of S-allyl cysteine sulfoxide, an insulin secretagogue, in diabetic rats. Experientia1996; 52:115-119.
16. Kulkarni SK: Handbook of Experimental Pharmacology. Vallabh Prakasan, Delhi, 2009.
17. Singh KN, Mittal RK and Barthwal KC: Hypoglycemic activity of Acacia catechu, Acacia suma, Albizzia odoratissima seed diets in normal albino rats. Indian J Med Res 1976; 64:754-757.
18. Geeta BS, Mathew BC and Augusti KC: Hypoglycemic effects of Leucodelphinidin derivative isolated from Licus bengalenses (Linn.). Indian J Physiol Pharmacol 1994; 38:220-222.
19. Gupta SS: Prospects and perspectives of natural plant products in medicine. Indian J Pharmacol 1994; 26:1-12.
    

    ---