INTRODUCTION:

Diabetes is prevalent worldwide and is stated to be one of the important causes of death worldwide. The world health organization predicts that this number will rise dramatically by the year 2025, by which time India and China may each face problem of dealing with 50 million affected individuals. Diabetes is a major and growing public health problem throughout the world, with an estimated worldwide prevalence in 2000 of 150millon people², expected to rise 220 million people by 2010¹.

Diabetes mellitus in human and experimental animal models also exhibits high oxidative stress, due to persistent and chronic hyperglycemia and protein glycation³, which thereby depletes the antioxidant defense system⁴ and thus promotes denovo free radicals generation⁵.

Current therapies used for controlling diabetic complications are associated with several side effects⁶. Moreover, as synthetic antioxidants are suspected to be carcinogenic⁷, there is a need for effective, safe and better oral hypoglycemic agents. There is a renewed interest in plant-based drugs in the treatment of diabetes and its complications, due to the efficacy of medicinal plants.

Pterocarpus marsupium Roxb (Indian kino tree) is one such plant for treating diabetes and it is a species of Pterocarpus native to India, Nepal and Srilanka. Parts of the Indian kino have long been used for their medicinal properties in Ayurveda.

The heart wood is used as an astringent and in the treatment of many diseases. The leaves are useful in boils, sores and skin diseases. The flowers are bitter, sweet, cooling, appetizing and febrifuge and are useful in vitiated conditions of anorexia, fever. Kino is powerful astringent and was formerly used widely in the treatment of diarrhea and dysentery. It is locally applied in leucorrhoea and in passive heamorrhages⁸. It is also used for toothache. Kino finds application in dyeing, tanning and printing and is of potential use in the paper industry.

In the present study, hypoglycemic and antimicrobial activity of ethanolic extract of P.marsupium leaves had been evaluated.

MATERIAL AND METHODS:

Animal care and monitoring:

A group of 25 healthy adult Swiss albino rats weighing about 150 – 200g were used for this study. They were housed in polypropylene cages and fed with standard feed. They were maintained at room temperature under standard conditions. They were divided into 4 experimental groups each containing six rats viz. Group I (Normal control), Group II (Alloxan – induced diabetic control), Group III (Alloxan induced diabetic + P. marsupium treated), and Group IV (Alloxan induced diabetic + Glibenclamide treated).

Maintenance and treatment of animals was done in accordance with the principles of Institutional Animal Ethics Committee constituted as per the directions of the committee for the purpose of Control and Supervision of Experiments on Animals (No.265/CPCSEA).

Plant material and Preparation of extract:

The leaves of Pterocarpus marsupium was collected from S.T.E.T women’s college campus, Mannargudi between April and may 2009. The collected plants were shade dried for a period of 7 – 10 days. The powder (100gms) of the given samples were extracted with 500ml of ethanol 95% by continuous hot percolation respectively using soxhelt apparatus until the extraction were completed and it is used for this study.

Biochemical Assays:

Hyperglycemia was induced by a single intraperitoneal injection of freshly prepared aqueous solution of alloxan monohydrate (SD fine chemicals Pvt. Ltd., Biosar) 150 mg / kg, to overnight fasted rats. Control rats received similar volume of vehicle, normal saline (2ml /kg body weight) alone. Rats were classified into six groups of six rats each. Group I received normal saline and served as control. Group II treated with alloxan 150mg / kg served as diabetic control. Group III treated plant extract (200mg/kg) respectively. Group IV treated with Glibenclamide (5mg/ kg). Treatment continued for 14 days. At the end of 14 ^th day plasma levels were estimated⁹.

Blood samples were obtained from tail tip vein of all experimental animals and fasting blood glucose strips¹⁰ at regular time intervals i.e., 0(before alloxan injection), 0,7^th and 14^th day (after alloxan injection in groups II, III and IV) of experiment. Fasting blood glucose level of normal control group (Group I) was also measured simultaneously. Blood Glucose was estimated by using orthotoluidene method¹¹.

For estimating lipid profile, serum was isolated from the blood collected by cardiac puncture under mild ether anaesthesia from overnight fasted rats on day 14 of P.marsupium treatment. Total cholesterol¹², triglycerides¹³, HDL cholesterol¹⁴, LDL cholesterol¹⁵ and total protein¹⁶ were estimated in the serum. From the calculated value of triglycerides, VLDL cholesterol was calculated.

Antimicrobial Assay:

The microorganisms were collected from the National collection of Industrial Microorganism (NICM), Pune and maintained in the laboratory by periodic subculture. Antimicrobial activity was tested by disc diffusion method. The inoculum was prepared by sub culturing the pure culture in Nutrient Broth and incubated at 37ºC for 6 hours. after incubation the inoculum size was standardized by comparing with McFarland standard. The standardized inoculum should contain 1 X 10³organism/ml^17.

All the ingredients were dissolved in distilled water, pH was adjusted to 7.6 then transferred to suitable container and autoclaved at 121°C for 20 minutes. After sterilization, the bacteria was inoculated into the nutrient broth by means of inoculating loop and incubated at 37°C to 6 hours ¹⁸.

All the ingredients were dissolved in distilled water, pH was adjusted to 6.8 then transferred to suitable container and autoclaved at 121°C for 20 minutes ¹⁹.

Whatman number 2 filter paper of 6 mm diameter were cut and sterilized by dry heat sterilization (2 hrs at 160°C). These discs were used for disc diffusion method. The prepared discs were placed over the inoculated agar plate at equal intervals (to avoid merging of zone) at a distance of 15 mm from the periphery to the center of the disc and a 25 mm between each disc.

The prepared discs were placed over the inoculated agar plate at equal intervals. The standard disc (positive control) and solvent control disc were also placed to compare the antimicrobial activity of the solvents²⁰.

Data analysis:

Data were expressed as mean ± S.E. of six values and analysed by student ‘t’ test for differences among controls and treated groups. The values of P < 0.001 and P<0.01 were considered statistically significant ²¹.

RESULTS:

Table -1 depicts that the blood glucose level is higher in diabetic control rats when compared to treated groups. It shows significant hypoglycemic activity.

The triglycerides level is higher in diabetic control rats when compared to treated groups and standard drug. The HDL level is higher in diabetic control rats when compared to treated groups. The VLDL cholesterol level is higher in diabetic control rats when compared to treated groups. The LDL cholesterol level is increased in diabetic control rats when compared to treated groups. The cholesterol level is increased in diabetic rats when compared to treated groups. The total protein level is decreased in diabetic control groups when compared to treated groups

Table -2 displays the antimicrobial activity of ethanolic extract of P. marsupium leaves. The highest value of zone of inhibition was found in E. coli. the lowest value of zone of inhibition was found in C. albicans.

Table-1: Antidiabetic activity of Pterocarpus marsupium

Groups	Glucose mg/dl	TGL mg/dl	HDL mg/dl	VLDL mg/dl	LDL mg/dl	Cholesterol mg/dl	Total Protein mg/dl
Group – I	85.3 ± 3.1	75.15 ± 5.03	38.6 ± 1.83	15.03 ± 1.006	41.9 ± 4.37	82.53 ± 7.21	7.4± 0.56
Group – II	265.6 ± 7.2	124.32 ± 6.5	42.56 ± 5.52	32.14 ± 1.7	171.07 ± 2.27	232.2 ± 9.8	5.2±0.31
Group – III	150.6 ± 2.2*	94.48* ± 1.45	37.21* ± 0.98	21.30 ± 2.32	106.42** ± 2.14	140.0 ± 4.8	6.3± 0.23
Group – IV	72.3 ± 8.2*	82.04* ± 6.7	32.26* ± 1.80	16.92 ± 1.34	33.9** ± 2.66	96.2 ± 4.8	7.6 ±0.61

Values are expressed as Mean ±S.E., n=6 by students ‘t’ test

*P<0.01Vs Control; **P<0.001 Vs Control

Table – 2: Antimicrobial activity of Pterocarpus marsupium

S. no	Name of the microorganism	Diameter zone of inhibition in mm
S. no	Name of the microorganism	Sample	Standard
1.	Staphylococcus aureus	20	38
2.	Escherichia coli	22	35
3.	Aspergillus niger	20	28
4.	Candida albicans	12	25

Standard – standard – Ciprofloxacin 5µg/disc for bacicria Fluconazole 100 units/disc for fungi.

DISCUSSION:

In the present study antidiabetic activity of ethanolic extract of P. marsupium leaves were evaluated in alloxan induced diabetic swiss albino rats. Alloxan was reported to cause a significant reduction of insulin producing β–cells of islets of langerhans, thus inducing hyperglycemia²².The increased blood glucose level in diabetic rats as compared to normal ones might be due to glycogenolysis and /or gluconeogenesis²³.

Liver is the candidate organ for glucose metabolism, glycolysis and gluconeogenesis are two primary complementary events balancing the glucose load in our body. The activity of key glycolytic enzyme hexokinase is lowered during diabetes. Loading to reduced disposal of glucose as glucose-6-phosphatase which inturn causing hyperglycemia²⁴The activities of gluconeogenesic enzyme viz. glucose – 6- phosphatase and fructose 1,6 bisphosphatase are exchanged during diabetes leading to increased synthesis of glucose²⁵. The ethanolic extract of herbs lowers the blood glucose level. The herbal drug act as a good antidiabetic agents.

The elevated triglycerides, total cholesterol, LDL, and VLDL and HDL level in alloxan-induced diabetic rats was in agreement with the previous reports regarding alteration of these parameters under diabetic condition²⁶.

The diabetes-induced hyperlipidemia might be due to excess mobilization of fat from the adipose tissue because of underutilization of glucose²⁷. The P. marsupium treatment for 14days decreased the serum triglycerides, cholesterol, HDL, VLDL, and LDL level significantly. The hypolipidemic action of P. marsupium might be due to inhibition of lipid peroxidation and was consistent with an earlier study, whereas increase the above lipid profile levels in Diabetes Mellitus was indicated²⁸. At the end of the present study, it was observed that treatment with herbs is effective in reducing lipoproteins, TG & TC levels.

Absolute or relative deficiency of insulin leads to decreased protein synthesis in all tissues. The glycogen and protein content of the diabetic rats were significantly lowered when compared to control, but in drug treated diabetic rats, significant changes were observed. The reduced hepatic glycogen content in diabetic condition is replenished after insulin therapy ²⁹.

The ethanolic extract of P. marsupium has potent antimicrobial activity. Both gram positive S.aureus and gram negative E.coli were inhibited. The antifungal activity of P. marsupium is only towards A.niger and not much towards C.albicans. Then it was observed the plate after 4 days, the spores of A. niger were not screened. And few mycelial growth were only seen. Hence, it was concluded that the ethanolic extract of P. marsupium has got sporicidal activity ³⁰.

In conclusion, Pterocarpus marsupium showed significant antidiabetic activity in alloxan induced diabetic rats and also it showed antimicrobial activity. Hence it may find use in the management and/or control of diabetes and act as an antimicrobial agent. The mechanisam of action of Pterocarpus marsupium is to be workout in future.

ACKNOWLEDGEMENT:

The authors are thankful to Dr. V. Dhivakaran, Secretary, S.T.E.T. Women’s College, Mannargudi, for his kind suggestions and Dr. S. Karpagam Kumara Sundari, Head Department of Pharmacology, Periyar College of Pharmaceutical Sciences, Trichy, for providing all facilities to do this research work.

REFERENCES:

1. Cockam C S (2006) The epidemiology of diabetes mellitus in the Asia pacific HK MJ 1, 43-52

2. ceriello A (2000) Metabolism 49, 27-29

3. Wolff P & Dean R T (1987) Biochem J 245, 243-250

4. Halliwell B, Gutteringe J M (1990) Meth Enzymol 186, 1-85

5. Baynes J W, Thorpe S R (1999) Diabetes 48, 1-9

6. Sathyanarayana T, Katyani B M, Hema L E, Mathews A A, & china E M (2006) Phcog Mag 2, 244-253

7. Lavhale M S, Mishra S H (2007) Indian J Exp Biol 45, 376-384

8. Prajapati, Purohit, Sharma, Kumar (2003) A handbook of Medicinal Plants 555

9. Sharma S R, Dwivedi S K, Swarup D (1997)Journal of Ethanopharmacology 58, 39-44

10. Henry J D (1984) Clinical Diagnosis and management by Laboratory methods 17^th edn. Philadelphia P A, WB saunders 1433

11. Trinder P (1969) Ann cain biochem 6, 24

12. Zak (1959) clin chemistry 18, 480

13. Buccolo G, David M (1973) clin chemistry 19, 476

14. Assan G (1979) Intermist 20, 1159

15. Fridewald W T (1937) calculation of LDL cholesterol clin chem 18, 499

16. Lowry O H, Rosebrough N J, Lewisfarr A, Randall R J (1951) Protein measurements with the Folin’s phenol reagent J Bio chemistry 193, 275

17. Jeanne molden hauer (2005) Biopharm International 18(12), 34

18. Jawetz E, Melnick T, Aderlberg S (2000) Medical microbiology 21^st Edn McGraw Hill Medical Pub. Div 112-215

19. Baure A W, Kirby W M M, Sheris J C & Truck M (1996) Ann.J.Clin.Pathol 45, 493-496

20. Cruickshank R, Duguid J O, Marmion B P, swain R H, (1975) Medical microbiology 12^th Edn. Churchill Livingstone publication New york 2, 98

21. Snedeeor G W, Cochran W G in “Statistical methods” (1967) Oxford and IBH publishing co New Delhi 33

22. Wilson G L, Leiter E H (1990) In: Essentials of modern Biochemistry 1^st Edn Jones & wett B publishers Bosten Portala valet 368-369

23. Guyton A C, Hall J E (2000) Textbook of medical physiology 10^th Edn Philadelphia WB saunders 810-818

24. Kochupillai (2001) Diabetes mellitus in India JAMA

25. Muniappan Latha and Leelavinothan Pari (2003) Antihyperglycemic effect of Lassia auriculata in experimental diabetes and its effects on key metabolic enzymes involved in carbohydrate metabolisam Clinical and experimental pharmacology and physiology 30, 38-43

26. Howard B V (1987) J Lipid Res 28, 613-628

27. Krishnakumar K, Augustti K T, vijayammal P L (2000) Med sci 28, 65-67

28. EI-Missiry M A, MI-Gindy A M (2000) Ann Nutr Metab 47, 97-100

29. Church J M,.Hill G L, (1987) Assessing the efficicacy of intravenous nutrition in general surgical patients. Dynamic nutritional assessment with plasma proteins. J.P.E.N 11, 135

30. Buckley R M (1992) Immonodeficiency diseases JAMA 268- 279.

Received on 29.09.2011 Modified on 20.10.2011

Research J. Pharm. and Tech. 4(12): Dec. 2011; Page 1915-1917