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Dose dependent hypoglycemic effect of aqueous extract of Enicostemma littorale Blume in alloxan induced diabetic rats.

Summary

Previous studies in our lab had confirmed the blood glucose lowering effect of E. littorale Blume in alloxan induced diabetic rats with no change in normoglycemic control rats. Present paper deals with dose dependent (0.5, 1.0, 1.5,2.5,3.5 g dry plant equivalent extract /100 g body wt., p.o.) blood glucose lowering effect of aqueous extract of E. littorale Blume in alloxan induced diabetic rats. The effective dose was found to be 1.5 g dry plant equivalent extract/100 g body wt.. The above dose caused significant decrease in glycosylated haemoglobin, liver glucose-6-phosphatase activity and significant increase in serum insulin levels of the diabetic rats. No significant changes were observed in the toxicity parameters of extract treated diabetic rats as compared to diabetic control rats. The above results suggest that E. littorale is a potent antidiabetic agent without any toxic effect at this particular dose (1.5 g dry plant equivalent extract/100 g body wt.).

Key words: Enicostemma littorale, alloxan induced diabetic rat model, antidiabetic activity

Introduction

Diabetes mellitus is an endocrinological metabolic disorder. The prevalence of diabetes mellitus is rapidly increasing worldwide and India is estimated to have 19.5 million diabetics, the largest number for any country in the world (King et al., 1998).Various remedies used for the treatment of diabetes are not able to achieve glycemic control effectively. Also, well established antidiabetic drugs are known to have adverse side effects (Sameul et al., 1997). Herbal preparations are preferred therapeutic agents to manage the glycemic control in diabetics because of their lesser side effects. Antidiabetic efficacy of various plants and herbs have been proved scientifically (Atta-Ur-Rahman et al., 1989). Enicostemma littorale Blume or chhota chirayata (in Hindi) used in various ayurvedic formulations, is also used as antidiabetic agent by the rural folks of Gujarat.

Previous studies in our lab had confirmed the blood glucose lowering effect of E. littorale Blume in alloxan induced diabetic rats with no change in normoglycemic control rats (Vijayvargia et al., 2000). We had also reported that it can enhance glucose-induced insulin release from isolated rat pancreatic islets (Maroo et al., 2002). But no report is available on the safety and efficacy of this herb. Hence, in the present study different doses of aqueous extract of E. littorale was administered and safety and efficacy was evaluated in alloxan induced diabetic rats.

Materials and Methods

Animals

Male Charles foster rats weighing 200-250 g were provided food and water ad libitum. Diabetes was induced chemically by alloxan. Animals received 120 mg/kg body wt. alloxan intra-peritonially after overnight fasting. These animals were kept for 15 days to stabilize the diabetic condition. Animals showing fasting blood glucose levels more than 200 mg/dl were considered as diabetic.

Extract Preparation

Whole dried plant was used for extract preparation as described earlier (Viyavargia et al., 2000). In short, dried plant material was cut into small pieces and boiled twice in water for 30 minutes. Filtrates were combined and boiled to get desired concentration (1 g dry plant equivalent/ml extract).

All chemicals used were of AR grade and purchased from SRL, Mumbai (India). Alloxan was purchased from Sigma Chemicals, USA and RIA kit for insulin from BRIT, Mumbai (India).

Experimental Protocol

Animals were divided into six groups. Group I as untreated diabetic control rats and Group II-VI were extract treated diabetic rats which received 0.5, 1.0, 1.5, 2.5 and 3.5 g dry plant equivalent extract/100 g body wt./day respectively for 20 days. Extract was administered by tube feeding. Diabetic control animals were given equivalent amount of saline for 20 days. Blood samples were collected at 0 and 20th day from orbital sinus and animals were sacrificed on 20th day.

Parameters

The parameters studied were blood glucose by GOD-POD kit method (Trinder 1969) (Monozyme, India), Glycosylated Haemoglobin by calorimetric method (Parker et al., 1981), liver glucose-6-phosphatase activity (Baingsky et al., 1967) and serum insulin levels estimated by RIA method. The insulin kit was purchased from BRIT, Mumbai (India). The intra-assay coefficient of variation was 2.49% and all samples were measured in the same assay to avoid inter-assay variations. Liver function tests [serum glutamate pyruvate transaminase-SGPT (Reitman et al., 1957), alkaline phosphatase-ALP (Bowers et al., 1975)] and kidney function test-serum creatinine (Bonsnes, 1945) were also estimated.

Statistical Analyses

Statistical analyses were performed by using Student's t-test to test differences between untreated and treated groups and within individual treated groups before and after treatment. The results were also subjected to Multiple comparison test of variance (MANCOVA by Tukey's test) followed by ANOVA to test the differences among various treated groups.

Results

Diabetic rats treated with the extract showed dose dependent decrease in the blood glucose levels which was significant when compared with day 20 of diabetic control rats or compared within the group between 0 and 20 days, whereas Multiple comparison following ANOVA (MANCOVA) did not show any significant difference between the treated groups. The dose of 1.5 g dry plant equivalent extract/l00 g body wt. was found to be effective (Fig. 1). Above dose caused 50% decrease in the blood glucose levels along with significant increase in serum insulin levels of the diabetic rats without any mortality at the 20th day (Fig. 2) where as 40-60% mortality was found at higher doses of 2.5 and 3.5 g dry plant equivalent extract/100 g body wt. (Data of higher doses not shown). Extract treatment also caused significant decrease in the elevated glycosylated haemoglobin (Gly Hb) levels and liver glucose-6-phosphatase activity of the diabetic rats (Fig. 3 and 4).

Extract treatment to the diabetic rats caused slight GIT disturbances initially along with weight loss which was not statistically significant. Liver function tests like SGPT and ALP and kidney function test like serum creatinine were done to check any toxic effect of the extract and it was found that this extract was non-toxic at this particular dose (1.5 g dry plant equivalent extract/100 g body wt.) with no significant change in the above toxicity parameters (Table 1).

Discussion

We had already reported that the blood glucose lowering effect of E. littorale Blume is glucose dependent as the aqueous extract of E. littorale Blume showed blood glucose lowering effect only in hyperglycemic i.e. alloxan induced diabetic rats and not in normoglycemic i.e. control rats (Vijayvargia et al., 2000). Similarly in this study different doses of aqueous extract of E. littorale did not show any change in blood glucose levels of normoglycemic rats (Data not shown). In the present study, there was a gradual decrease in blood glucose levels of extract treated diabetic rats in a dose dependent manner and 1.5 g dose showed almost 50% decrease (p < 0.01) in blood glucose levels at 20th day without any mortality (Fig. 1). But at higher doses (2.5-3.5 g dry plant equivalent/100 g body wt.) 40-60% mortality was observed. There was mild GIT disturbances after the extract treatment which was more profound in rats treated with higher doses of the extract and might be the reason for the mortality rate.

Significant increase in the serum insulin levels of the extract treated diabetic rats was observed at the dose of 1.5 g dry plant equivalent extract/100 g body wt. (Fig. 2). Aqueous extract of E. littorale Blume may be potentiating the glucose dependent insulin release from the pancreatic [beta]-cells as demonstrated by us in in-vitro experiments by incubating isolated rat pancreatic islets with aqueous extract of E. littorale (Maroo et al., 2002). This possibly reflects one of the factors responsible for the decrease in blood glucose levels after extract treatment in diabetic rats and thereby the decreased Gly Hb levels (Fig. 3).

In case of diabetes there is increased hepatic glucose production (Defronzo et al., 1992). The extract treatment caused significant increase in serum insulin levels and decrease in blood glucose levels as well as a decrease in the liver glucose-6- phosphatase activity (p < 0.05, Fig. 4). This suggests a possible role of the extract in lowering hyperglycemia by decreasing the liver glucose-6- phosphatase activity resulting in decreased hepatic glucose production.

SGPT and serum creatinine levels in extract treated diabetic rats were similar to that of diabetic control rats. ALP levels have been reported to be increased in alloxan induced diabetic rats (Gonzalez et al., 1992). Extract treatment to the diabetic rats did not cause any significant change in ALP levels as compared to the diabetic control rats thus showing no toxicity.

It can be concluded from the above results that aqueous extract of E. littorale has hypoglycemic potential with 1.5 g dry plant equivalent extract as [ED.sub.50] without any toxic effect and thus could be a candidate for therapeutic intervention against diabetes mellitus.

Studies are going on in this laboratory to purify and characterize the compounds responsible for glucose lowering effect and would be reported in future.

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Table 1

Effect of aqueous extract of E. littorale on Serum GPT, ALP and
creatinine levels at 20th day in diabetic rats.

 Untreated Diabetic Treated Diabetic rats
 (n=6 in each group)
 rats (n=6) 0.5 g/100 g body wt.

SGPT (IU/L) 73.62 [+ or -] 8.61 59.37 [+ or -] 5.92
ALP (IU/L) 402.7 [+ or -] 72.53 421.5 [+ or -] 78.32
Creatinine (mg/dl) 1.15 [+ or -] 0.04 0.92 [+ or -] 0.22

 Treated Diabetic rats (n=6 in each group)
 1.0 g/100 g body wt. 1.5 g/100 g body wt.

SGPT (IU/L) 78.57 [+ or -] 9.47 72.25 [+ or -] 7.05
ALP (IU/L) 357.8 [+ or -] 45.66 398.6 [+ or -] 77.10
Creatinine (mg/dl) 1.29 [+ or -] 0.30 1.25 [+ or -] 0.30

(IU - [micro]M product formed/mm), Values expressed as mean [+ or -]
SEM.


Acknowledgement

The financial support from CSIR [2-14/1996(1)-E.U. II], New Delhi, India to Jyoti Maroo is gratefully acknowledged.

References

Atta-Ur-Rahman, Zaman K (1989) Medicinal plants with hypoglycemic activity. J Ethnopharmacology 26: 1-55

Baingsky ES, Foa PP, Zak B (1967) Determination of rat liver microsomal glucose-6-phosphatase activity: study of citrate and G-6-P inhibition. Anal Biochem 21: 20 1-207

Bonsnes RM, Taussky HH (1945) Colorimetric determination of creatinine by Jaffe reaction. J Biol Chem 158: 581-591

Bowers GN Jr., McComb RB (1975) Measurement of total alkaline phosphatase activity in human serum. Clin Chem 21: 1988-1995

Defronzo RA, Bonadonna RC, Ferrannini E (1992) Pathogenesis of NIDDM: a balanced overview. Diabetologia 35: 389-397

Gonzalez J, Fevery J (1992) Spontaneously diabetic biobreeding rats and impairment of bile acid independent bile flow and increased biliary bilirubin, calcium and lipid secretion. Hepatology 16: 426-432

King H, Aubert RE, Herman WH (1998) Global burden of diabetes, 1995-2025: Prevalence, numerical estimates and projections. Diabetes Care 21: 1414-1431

Maroo J, Vasu VT, Aalinkeel R, Gupta S (2002) Glucose lowering effect of aqueous extract of Enicostemma littorale Blume in diabetes: A possible mechanism of action. J Ethnopharmacology 81: 317-320

Parker KM. England JD, Da Costa J (1981) Improved colorimetric assay for glycosylated haemoglobin. Clin Chem 27: 669-672

Reitman S, Frankel S (1957) Colorimetric method for determination of serum transaminase activity. Am J Clin Pathol 28: 56-58

Sameul DJ, Julio VS (1997) Pathophysiology of type 2 diabetes and modes of action of therapeutic interventions. Arch Int Med 157: 1802-1817

Tinder, P (1969) Determination of blood glucose using 4-amino phenazone as oxygen acceptor. J Clin Pathol 22: 246

Vijayvargia R, Kumar M, Gupta S (2000) Hypoglycemic effect of aqueous extract of Enicostemma littorale Blume (chhota chirayata) on alloxan induced diabetes mellitus in rats. Ind I Exp Biol 38: 781-784

Address

Sarita Gupta, Department of Biochemistry, Faculty of Science, M.S. University of Baroda, Vadodara, Gujarat, India-390 002

Tel: ++91-265-2795594; e-mail: saritagupta9@yahoo.com
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Article Details
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Author:Maroo, J.; Vasu, V.T.; Gupta, S.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Geographic Code:1USA
Date:Mar 1, 2003
Words:1986
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