Hypoglycemic activity of Leaves of Bougainvillea spectabilis extract in Streptozotocin-Induced Diabetic Rats

M. R. Chitra Devi¹*, Dr. B. Ramesh²

¹Assistant Professor in Biochemistry, Navarasam Arts and Science College for women, Arachalur, Erode.

²Associate Professor in Biochemistry, PSG College of Arts and Science, Coimbatore, Tamilnadu, India.

*Corresponding Author E-mail: jvchrsty@yahoo.co.in

ABSTRACT:

Diabetes mellitus is a disease in which blood glucose levels are above normal. The β cells of the pancreas do not make sufficient insulin. Herbal drugs treat diabetes by improving insulin sensitivity, increasing insulin production and/or decreasing the amount of glucose in blood. The present study was aimed to investigate antidaibetic activity of methanolic leaf extract of Bougainvillea spectabilis (B. spectabilis) alone and in combination with glibencliamide in Streptozotocin induced diabetic rats. Primarily acute toxicity study and oral glucose tolerance test were performed. The powdered plant was successfully extracted with methanol by using soxhlet extractor. The Wister strains of male albino rats were used for present study. The methanol extracts of B. spectabilis (200mg/kg and 400mg/kg) were administered to both normal and streptozotocin induced diabetic rats at defined time intervals. Blood glucose levels were measured at 0,30,60,120 minutes and 0, 7, 14, 21 and 28^th day after oral administration of extracts. Of the doses test, highest anti -hyper glycemic effect was observed by the extract of leaves at 400mg/kg after a week treatment. B. spectabilis extract exhibited significant hypoglycemic activity at different doses and intervals. The toxicity study results showed that the medium lethal dose (LD₅₀) of the extract is higher than 2g/kg body weight, and hence, in a single dose administration, the plant extract had no adverse effect. Bougainvillea spectabilis methanol extract exhibited potent hypoglycemic activity.

KEY WORDS: Hypoglycemic activity, leaves, blood glucose level, Bougainvillea spectabilis, streptozotocin.

INTRODUCTION:

Diabetes mellitus is an endocarine disorder in which glucose metabolism is impaired because of total loss of insulin after destruction of pancreatic beta cell or because of inadequate release of insulin from the pancreatic beta cell or insensitivity of target tissue of insulin. The fundamental mechanism underlying hyperglycemia involved over production (excessive hepatic glyconeogenesis and glyconeogenesis) and decrease utilization of glucose by the tissue.

Early symptoms concern hyperglycemia and encompass excessive thirst, excessive eating, production of excessive amount of dilute urine and blurred vision. Later complications encompass an abnormal condition of the blood vessels, peripheral neuropathy, nephropathy and tendency to suffer from infection. Almost every organ system in the body can be affected by diabetes mellitus¹. Diabetes broadly classified in to two categories, type I (Insulin dependent or Juvenil) and type II diabetes (non- insulin dependent or diabetes mellitus). Type II diabetes is much more prevalent which may be due to combination of resistance to insulin action and an inadequate compensatory insulin secretion. The incidence of type 2 diabetes mellitus is increasing worldwide. It may results from the interaction between a genetic predisposition and behavioral and environmental risk factors^2-5. World Health Organization predicted that developing countries like India will bear the brunt of this epidemic in the present century⁶. India has more than 50.8 million people with diabetes and projected to increase to 87 million by year 2030^7-9. Recently, biguanides, thiazolidine- diones, sulfonylureas, D-phenylalanine and α-glucosidase inhibitors along with insulin are extensively used in the treatment of diabetes by a sustained reduction in hyperglycemia. However, due to enormous unwanted side effects and in addition to high cost of allopathic drugs the effectiveness of these compounds are not acceptable and there is an increasing demand for both effective as well as safer drug compounds for the treatment of diabetes Mellitus ^10-13. To avoid side effects of insulin and oral hypoglycemic agents, patient’s interest are expanding to use traditional plants with antidiabetic activity. Therefore, the WHO recommended for continue research for antidiabetic leads from plants and other resources¹⁴. Bougainvillea spectabilis (B. spectabilis) may be a potential candidate for above objective as it is a part of various herbal formulations for diabetes¹⁵. B. spectabilis Wild commonly known as bougainvillea, great bougainvillea (family: Nyctaginaceae) (local Indian names: booganbel, cherei, baganbilas, booganvel, bouganvila, kagithala puvvu). It possess various biological activities like hypoglycemic¹⁶, cholesterol lowering effect¹⁷, thrombolytic activity¹⁸, nematicidal¹⁹, antifertility²⁰, Aflatoxin²¹, antiviral²². antioxidant and antimicrobial²³, antiulcer²⁴ were reported of Bougainvillea spectabilis. Hypoglymic activity of Bougainvillea spectabilis stem bark in normal and alloxan induced diabetic rats also reported²⁵. This research paper reports about the assessment of the hypoglycemic activity of methanolic leaf extracts of Bougainvillea spectabilis from the family of Nyctaginaceae to the standard reference Glibenclamide (Streptozotocin-Induced).

MATERIALS AND METHODS:

Chemicals:

Streptozotocin was purchased from Loba Chemie Indo Austrand Co., Mumbai. Glucose was purchased from Glaxo Smith Kline. Metformin was obtained from Chad well Heath Essex, England. All other chemicals used in the study were of analytical grade. All solutions were prepared on the same day of experiments.

Plant Materials:

The leaves of Bougainvillea spectabilis (Nyctaginaceae) were collected from surrounding area of Kangayam, Tirupur district in May, 2017. The plants were identified by expert of Dr. K. Madhava Chetty, Plant Taxonomist, Department of Botany, Sri Venkateswara University, Tirupathy.

Preparation of Extracts:

The leaves of Bougainvillea spectabilis was shade dried at room temperature individually. The dried plant leaves were subjected to size reduction to a coarse powder (1000 grams) by using dry grinder and passed through sieve. The powder was packed into soxhlet apparatus and extracted successively with Petroleum ether (60 – 80.C) and 90% methanol. The extraction was carried out until the extract becomes colorless. The solvent is removed by distillation under reduced pressure and stored in desiccators for further experiment.

Phytochemical studies:

Phytochemical investigation was carried out for both extracts of leave for the presence of alkaloids with Dragendroff’s reagent, Mayer’s reagent and Wagner’s reagent, Cardiac glycosides with Killer killiani test, Baljet’s test, Raymond’s test and Legal’s test, anthraquinones with Borntrager’s test, modified Borntrager’s test, reducing sugars with Fehling’s reagent, Molisch’s test, Benedicts test, sterols with Liebermann-Burchard’s test, triterpenes with Salkowski test, Phenolic compounds and tannins with Ferric chloride, gelatin test, lead acetate test, Aqueous bromine test, saponins with ability to form foam, Proteins and free amino acids with Millon’s test, Biuret test, Ninhydrin test, flavonoids with metallic magnesium and HCl, alkaline reagent test according to the standard methods reported ^26-27.

Experimental Animals:

Male Wister albino rats (170-230gm) of age 8-12 weeks each were used for this experiment. They were procured from Sri Venkateswara College of Pharmacy, Chittoor, Andhra Pradesh, India. The animals were maintained in a well-ventilated room with at 12:12 h light, dark cycle in polypropylene cages and maintained at 22±1˚C with humidity at 55±5%.They were fed balanced rodent pellet diet and tap water ad libitum throughout the experimental period. Ethical committee clearance was obtained from IAEC (Institutional Animal Ethics Committee) of CPCSEA (Reference No: SVCOP/IAEC/002/2016-17).

Acute Oral Toxicity Studies:

The methanol extract of leaves of B. spectabilis was tested to their acute and short term toxicity (if any) in albino Wistar rats. For determining the acute toxicity of a single oral administration of the herbal drug, the OECD guidelines (OECD/OCDE 2001, 423Annex 2c) were followed²⁸.Dose level of extracts was administered 2000 mg/kg body weight orally. Food was withheld for a further 3 to 4 hr administration of methanol extract of leaves of B. spectabilis (MEBS) observed for signs of toxicity. Rats were kept under observation continuously for the initial 4 h and intermittently for the next 6, 24, 48 hr following drug administration. The body weight of the rats before and after administration was noted. Parameters like grooming, hyperactivity, sedation, loss of righting reflux respiratory rates and convulsion were observed. No considerable signs of toxicity were observed in any of tested albino wistar rats. On the basis of above acute toxicity study 1/10^th (200mg/kg) and 1/5^th (400mg/kg) of this minimum and maximum oral doses were selected for further study.

Diabetes induction:

Diabetes was induced in rats by a single intraperitoneal injection of freshly prepared Streptozotocin (40mg/kg body weight) in 0.1M citrate buffer (pH 4.5) in a volume of 1ml/kg body weight. Diabetes was confirmed by the elevated glucose level in plasma it was determined after 48 hrs of injection. The rats with effective and permanent elevated plasma glucose levels (> 250 mg/ dI) were selected for present study.

Study of extract on normal and glucose loaded rats (OGTT):

Oral glucose tolerance test was performed after overnight fasting (16hr) of normal rats. Vehicle (1% w/v CMC 5ml/kg body weight), methanol extracts of leaves of B. spectabilis (200mg/kg and 400 mg/kg) and standard as glibenclimide (600µg/kg) were administered to five different groups of rats (n=6). Glucose (2g/kg) was fed to 30 min after the extract and glibenclimide. Blood was withdrawn from the tip of tail at 0, 30, 60 and 120 min from normal control and experimental rats. Blood glucose levels were estimated by commercially available glucose strips (Accu – check) using one touch glucometer (Johnson-Johnson, India).

Blood glucose level measurement in diabetic rats up to 28^th day:

Experimental animals were divided randomly in five groups of six rats each. Overnight faster diabetic rats treated orally using gastric gavage needle with vehicle, methanol extract of leaves of B. spectabilis doses of 200mg/kg and 400mg/kg, glibenclimide (600µg/kg) daily once up to 28 days. Blood samples were collected at the defined time intervals from control and experimental animals. Blood glucose was estimated on 0^th, 7^th, 14^th, 21^th and 28^th day by commercially available glucose strips (Accu- check) using one touch glucometer. The effect extract on initial and final body weights were also monitored up to 28 days.

Statistical analysis:

Observed data are represented as means ±SEM statistical significance of dissimilarities of the groups was evaluated by one way and multiple way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test. A difference in the mean value of P< 0.05 was deliberated significant statistically.

RESULTS AND DISCUSSION:

Oral glucose tolerance test:

The experiment showed that oral glucose tolerance test (OGTT) measure the body ability to use glucose, the body’s main source of energy (Arover et al 2002). The test can be diagnosed prediabetes and diabetes. We made an attempt for the first time study the effect of methanol extract of leaves of B. spectabilis in hyperglycemic rats. There was no significant change in the blood glucose level of vehicle treated normal rats. Overnight fasted normal rats loaded with glucose (2g/kg), showed significant increase in blood glucose (p<0.05) after 30 minutes. Leaves extract at the doses of 200mg/kg, 400mg/kg /day, reduced the blood glucose level significantly (P<0.05) from after 30minutes, comparable to glibenclamide (600µg/kg). The reports are shown in table-1 and figure-1. As compare to control, the oral administration of methanol extract of leaves of B. spectabilis (200 mg/kg, 400mg/kg) induced a significant decrease of blood glucose in diabetic rats (P<0.05). Leaves extract at the dose of 400mg/kg was found to be most effective at the doses tested. The potency of the extract was similar to glibenclamide and hypoglycemic effect persists until 120 min.Oral administration of vehicle did not any significant effect on the level of blood glucose level in diabetic rats. In blood glucose level was decreased significantly (p<0.05) after oral administration of extract upto7days. Greater antihyperglymic effect was observed by the methanol extract of leaves at 400mg/kg /day and found to be 97.17±0.044mg/dl more effect than glibenclamide 600µg/kg) table-2.

Table 1. Effect of MEBS on oral glucose tolerance

Groups	Serum Blood Glucose (mg/dl)
Groups	0min	30min	60min	120min
I - Normal control (1% w/v CMC)	89.00±0.7303	137.83±1.222	130.67±0.8028	113.33±0.6667
II - MEBS (200 mg/kg/day p.o)	91.67±0.6146^a	125.83±0.6009^a	114.83±0.7032*^a	98.17±0.8333**^a
III - MEBS (400 mg/kg/day p.o)	91.50±0.5000^a	117.33±1.085*^a	106.00±0.5774*^a	91.17±0.4014**^a
IV - Glibenclamide (600 µg/kg p.o)	90.00±0.6831^a	107.50±0.7638**^a	99.17±0.8724**^a	83.50±0.4282**^a

All values compared with normal control groups. * p < 0.05 ; ** p < 0.001

a – Normal control group I Vs group II, III, IV

Table 2. Effect of MEBS on Serum Blood Glucose in STZ induced diabetic rats

Groups	Serum Blood Glucose (mg/dl)
Groups	day 0	day 7	day 14	day 21	day 28
I - Normal control (1% w/v CMC)	94.50±1.432	98.50±1.765	101.33±0.8028	101.05±0.5774	98.83±0.8724
II - STZ induced Diabetic control	293.50±2.291**^a	295.50±1.565**^a	297.17±1.078**^a	290.33±3.412**^a	287.67±2.565**^a
III - STZ + MEBS (200 mg/kg, p.o)	289.67±3.232^b	265.67±1.308**^b	241.33±0.9888**^b	190.67±1.022**^b	137.33±1.706**^b
IV - STZ + MEBS(400 mg/kg, p.o)	294.83±1.078^b	244.00±1.183**^b	218.50±0.7638**^b	176.17±2.242**^b	104.21±1.7303**^b
V - STZ + Glibenclamide (600 µg/kg, p.o)	296.33±1.229^b	230.12±1.065**^b	197.22±2.805**^b	147.83±1.014**^b	91.33±0.8819**^b

All values expressed in mean ± SEM.

All values compared with diabetic control groups. * p < 0.05 ; ** p < 0.01.

a – Normal control group I Vs diabetic control group II.

b – Groups III, IV & V Vs diabetic control group II.

Figure-1 Effect of MEBS on Oral Glucose Tolerance

The methanol extract of leaves explicitly exhibited significant potent antihyperglycemic activity in diabetic rats, which significant, as compared to the control as well as glibenclamide treated group. The comparable effect of the extract with glibenclamide may suggest the similar mode of action, since streptozotocin permanently destroys the pancreatic β-cells and the extract lowered the blood glucose level in streptozotocin induced diabetic rats, indicating that the extract possesses extrapancreatic effects. The extract may have the properties to stimulate or regenerate the β-cells of islets of langerhans for the secretion of insulin and are most effective for controlling diabetes by various mechanisms which may finally lead to improvement of carbohydrate metabolizing enzyme towards the re-establishment of normal blood glucose level. In our study it is found that extract have hypoglycemic effect in glucose induced hyperglycemic rats. Phytochemical screening showed the presence of alkaloids, terpenoids, tannins, phenolic compound, saponins and flavonoids in the leaves extract of B. spectabilis. Many researchers reported flavonoids having hypoglycemic properties. This may be the reason for the antidiabetic properties of B. spectabilis leaves.

CONCLUSION:

In conclusion, the study showed the antidiabetic activity of the methanol extract of leaves of B. spectabilis on streptozotocin induced diabetic rats. The plant extract as effective as the standard drug (glibenclimide). The ability of B. spectabilis to reduce blood glucose level in the animals may inform the usage of the plant parts by traditional medical practitioners in the management of diabetes. Further studies are required to determine the exact phytochemical component responsible for the action and it mechanism of action in stabilizing blood glucose.

REFERENCES:

1. Kujur RS, Singh V, Ram M, Yadava HN, Singh KK, Kumari S, et al., Antidiabetic Activity and Phytochemical Screening of Crude Extract of Stevia Re-baudianain Alloxan-Induced Diabetic Rats. Pharmacognosy research, 2; 2010: 258-263.

2. American American Diabetes Association. Diagnosis and classification of diabetes mellitus. Diabetes Care. 33(1); 2010: 562-569.

3. Bruno G, Landi A. Epidemiology and costs of diabetes. Transplant Proc. 43(1); 2011: 327-329.

4. Moore K, Dean HJ. Clinical management of type 2 diabetes in indigenous youth. Pediatr Clin North Am 56(6); 2009: 1441-1459.

5. Murugesan N, Snehalatha C, Shobhana R, Roglic G, Ramachandran A. Awarness about diabetes and its complications in the general and diabetic population in a city in southern India. Diabetes Res Clin Pract 77(3); 2007: 433-437.

6. Saxena A, Vikram NK. Role of selected Indian plants in management of type 2 diabetes: a review. J Altern Complement Med 10(2); 2004: 369-378.

7. Lee HS. Rat lens aldose reductase inhibitory activities of Coptis japonica root-derived isoquinoline alkaloids. J Agric Food Chem 50(24); 2002: 7013-7026.

8. Jung M, Park M, Lee HC, Kang YH, Kang ES, Kim SK. Antidiabetic agents from medicinal plants. Curr Med Chem. 13(10); 2006: 1203-1218.

9. U.K Prospective Diabetes study group: Overview of six years therapy of type 2 diabetes: a Progressive disease: Diabetes. 44; 1995: 1249- 1258.

10. Moller, D.E., New drug targets for type 2 diabetes and the metabolic syndrome. Nature.2001; 414: 821-827.

11. Modak M, Dixit P, Londhe J, Ghaskadbi S, Paul A, Devasagayam T. Indian herbs and herbal drugs used for the treatment of diabetes. J Clin Biochem Nutr. 40(3); 2007: 163-173.

1. 12.Mustafa SSS, Eid NI, Jafri SA, El-Latif HAA, Ahmed HMS. Insulinotropic effect of aqueous ginger extract and aqueous garlic extract on the isolated perfused pancreas of streptozotocin induced diabetic rats. Pak J Zool 39(5); 2007: 279-284.

12. Islam MS, Choi H. Dietary red chilli (Capsicum frutescens L.) is insulinotropic rather than hypoglycemic in type 2 diabetes model of rats. Phytother Res 22(8); 2008: 1025- 1029.

13. World Health Organization. WHO Expert Committee on Diabetes Mellitus: second Report. Geneva: World Health Organization; 1980.

14. Soumyanth A. Traditional Medicine for Modern Times: Antidiabetic Plants, 1st ed. London: Taylor and Francis; 2006.

15. Narayanan CR, Joshi DD, Majumdar AM. Hypoglycemic action of Bougainvillea spectabilis leaves. Curr Sci 53; 1984: 579-581.

16. Adebayo JO, Adesokan AA, Olatunji LA, Buoro DO, Soladoye AO. Effect of ethanolic extract of Bougainvillea spectabilis leaves on haematological and serum lipid variables in rats. Biochem 17; 2005: 45-50.

17. Sikandar khan Sherwani. Muhammad Mumtaz Khan., Arif Zubair., Muhammad Ajmal Shah., shahana Urooj Kazmi. Evaluation of Invitro Thrombolytic Activity of Bougainvillea spectabilis Leaf Extract. Int.J. Pharm. Sci. Rev. Res., 21(1); 2013: 6-9.

2. 19.Grewal PS. Nematicidal effects of some plant-extracts to Aphelenchoides comosticola (nematoda) infecting mushroom. Revue-de-Nematologie; 12(3); 1989: 317-322.

3. 20. Mishra N, Joshi S, Tandon VL, Munjal A. Evaluation of antifertility potential of aqueous Extract of B.spectabilis leaves in Swiss Albino Mice. Intnat J Pharma Sci Drug Res. 1(1); 2009: 19-23.

4. 21. Nidhi Mishra, Vijay Lakshmi Tandon, Kuldeep Dhama, Rekha Khandia, Ashok Munjal. Does Bougainvillea spectabilis Protect Swiss Albino Mice from Aflatoxin- induced Hepatotoxicity?. Advances in Animal and Veterinary Sciences. 2016; 4(5): 250-257.

5. 22. Pun KB, Sabitha D, Jeyarajan R. Screening of plant species for the presence of antiviral principles against okra yellow vein mosaic virus. Indian J Phytopathol 1999; 52(3): 221-223.

6. 23. Sandeep Dhinakhar, Mamta Sharma, Sonam Ruhil, Meenakshi Balhara, Manish Kumar, Anil K Chhillar. Evaluation of Antimicrobial and Antioxidant Activities of Bougainvillea Spjectabilis. Intnat J Pharm Pharmaceutical Sci. 2013;5, Suppl 3: 178-182.

7. 24. Malairajan P, Gopalakrishanan G, Narasimhan S, Jessi Kala Veni K. Antiulcer activity of Crude Alcoholic Extracts of Bougainvillea Spectabilis Willd. Jundishapur J Nat Pharma Products. 2007; 2(1):1-6.

8. 25. Sunil Jawla, Y Kumar, MSY Khan. Hypoglycemic activity of Bougainvillea spectabilis stem bark in normal and alloxan-induced diabetic rats. Asian Pacific J Tropical Biomedicine. 2012; S919-S923.

9. 26. Evans WC. Trease and Evans Pharmacognosy, 15^th ed. New Delhi: Sounders-an imprint of Elsevier; 2002.

10. 27. Harborne JB. Phytochemical methods: A Guide to Modern Techniques of Plant Analysis, 3^rd ed., New Delhi: Springer (India); 1998.

11. 28. Organization for Economic Co-operation and Development (OECD). OECD guidelines for the testing of chemicals. Test No.425: Acute Oral Toxicity: Up-and-Down Procedure 2008.

Received on 05.04.2018 Accepted on 30.05.2018

Asian J. Pharm. Res. 2018; 8(2):99-103.

DOI: 10.5958/2231-5691.2018.00017.5