Gymnemic Acids from Gymnema sylvestre. Potentially Regulates Dexamethasone-Induced Hyperglycemia in Mice

ABSTRACT

Effects of three different doses (6.7, 13.4, and 26.8 mg/kg body wt., i.p.) of gymnemic acids (GA) from the leaves of Gymnema sylvestre. R. Br. in the regulation of dexamethasone induced hyperglycemia have been investigated in mice. Simultaneously, thyroid hormone levels were estimated by radioimmunoassay (RIA) in order to find out whether the effects are mediated through alteration in the thyroid function or not. While the prestandardized dose (1 mg/kg body wt., i.m.) of dexamethasone administration for 22 days increased the serum glucose concentration, it decreased serum concentration of the thyroid hormones thyroxine (T₄) and triiodothyronine (T₃). Although the administration of all three different doses of gymnemic acid decreased the serum glucose concentration in dexamethasone-induced hyperglycemic animals, the decrease was significant only in groups receiving the two higher doses, 13.4 and 26.8 mg/kg. These effects were comparable to a standard corticosteroid-inhibiting drug, ketoconazole. However, the percentage reduction was greater in the 13.4 and 26.8 mg/kg of gymnemic acids–treated groups (28.76% and 21.71% respectively) as compared to that of ketoconazole, where it was only 9.07%. As no significant changes in thyroid hormone concentrations were observed by the administration of any of the doses of GA in dexamethasone-treated animals, it further appears that the effects of the tested material might not have been mediated through alterations in the thyroid function. The changes in hepatic lipid peroxidation (LPO), superoxide dismutase (SOD), and catalase (CAT) activity revealed a toxic effect at the highest dose (26.8 mg/kg body wt.) as evidenced by increased LPO, whereas the medium dose (13.4 mg/kg body wt.) was found to be safe and antiperoxidative. It is suggested that 13.4 mg/kg of gymnemic acids may prove to be potentially effective in the amelioration of corticosteroid-induced diabetes mellitus/hyperglycemia.

Keywords:

• Dexamethasone
• gymnemic acids
• hyperglycaemia
• lipid peroxidation
• T₃
• T₄

Introduction

Diabetes mellitus is basically an endocrine disorder in which carbohydrate metabolism is impaired, leading to excess deposition of glucose in blood. This is broadly of two types, insulin-dependent diabetes mellitus (IDDM) and non-insulin-dependent diabetes mellitus (NIDDM). Under the second category, some hormones such as catecholamine, glucagon, cortisol, and thyroxine, either directly or through their influence on other hormones, affect carbohydrate metabolism so to elevate blood sugar level (hyperglycemia) and cause hormonal diabetes mellitus (Ganong, 1995). Looking to the increasing incidence of NIDDM, there is an increasing drive to develop alternative herbal medicine for its treatment, as plant-based medicines are believed to be safe, economic, and effective. The present investigation is an endeavor in this direction.

The therapeutic potential of Gymnema sylvester. (R. Br. Asclepidaceae) leaves has been known for many years, and it has a key place in Ayurvedic medicine (Asolkar et al., 1992). Its leaf extract was reported for its antihyperglycemic property by many workers in animals and humans (Mhaskar & Caius, 1930; Khare et al., 1983; Shanmugasundaram et al., 1983; 1990; Shrivastava et al., 1985; Baskaran et al., 1990). Recently, we reported that the crude extract of Gymnema sylvestre. leaf ameliorates corticosteroid-induced hyperglycemia (Gholap & Kar, 2003b). In order to determine whether this effect is mediated through its major active constituent, gymnemic acids (GA), the current investigation has been performed in dexamethasone-induced hyperglycemic mice. In fact, almost no scientific work was done to date to evaluate the efficacy of gymnemic acid in regulating corticosteroid-induced diabetes.

Materials and Methods

Plant materials

Processed gymnemic acid in the form of dry powder from the leaves of G. sylvestre. (purity, 75.6%, as compared to the standard GA) was obtained from Amsar Lab. Pvt. Ltd. (Indore, India). As 70% or more gymnemic acids is normally recommended for therapeutic use, we considered this drug in our experiment. The procured GA was dissolved in normal saline and the required doses were made for final administration.

Chemicals

Diethylenetriamine penta-acetic acid, Tris, sodium dodecyl sulfate, and thiobarbituric acid (TBA) were purchased from E. Merck (Mumbai, India), whereas dexamethasone sodium phosphate (Decdan, Wockhard Ltd., Mumbai, India) and ketoconazole (Ranbaxy Lab. Ltd., Mumbai, India), having ketoconazole 200 mg/tablets, were purchased from a standard medical shop of Indore, Mumbai, India. Other chemicals were of reagent grade and were obtained from Loba Chemie (Mumbai, India). Radioimmunoassay (RIA) kits for the estimation of total serum triiodothyronine (T₃) and thyroxine (T₄) were purchased from Bhabha Atomic Research Center (BARC; Mumbai, India).

Animals

Healthy Swiss albino mice of either sex (2 months old), weighing 28 ± 2 g, were maintained in a constant temperature (27 ± 1^○C) and photo-schedule (14 h light and 10 h dark) controlled room. The mice were provided with feed (Golden feeds, New Delhi, India) ad libitum. and free access to drinking water.

Experimental design

Forty mice were rendered hyperglycemic by daily administration of a prestandardized dose of dexamethasone (1 mg/kg body wt., i.m.; Gholap & Kar, 2003a, b) for 7 consecutive days and then divided into five groups of eight each. Group II continued to receive only dexamethasone, whereas the other three groups were treated with equivalent dose of dexamethasone along with three different doses of gymnemic acid, that is, 6.7, 13.4, and 26.8 mg/kg body wt. (G III G IV, and G V, respectively) against a reference dose of 13.4 mg/kg body wt., i.p. (Sugihara et al., 2000), and group VI received ketoconazole (24 mg/kg body wt., p.o.; Marty et al., 2000). Simultaneously, five other groups, each with eight normoglycemic animals, were treated with equivalent amount of vehicle (0.1 ml of normal saline, G I) or three different doses of gymnemic acid, that is, 6.7, 13.4, and 26.8 mg/kg body wt. or equivalent amount of ketoconazole (G VII, G VIII, G IX, and G X, respectively). All the doses of gymnemic acids were administered intraperitoneally between hours 1000 and 1100 of the day to avoid any circadian variation. On the last day, all the animals were weighed, and after an overnight fast were sacrificed by cervical dislocation. Blood samples were collected, allowed to clot, and centrifuged to obtain clear serum, which was then stored at −20°C, until assayed for glucose and thyroid hormones. The liver was removed quickly, cleaned, and washed twice with phosphate-buffered saline (pH 7.4) and immediately processed for the estimation of lipid peroxidation (LPO), superoxide dismutase (SOD), and catalase (CAT) activity.

Biochemical estimations

Serum concentration of tri-iodothyronine (T₃) and thyroxine (T₄) were estimated by radioimmunoassay (RIA) using kits obtained from BARC. The serum glucose was estimated by the modified glucose oxidase method (Hugget & Nixon, 1957) using glucose oxidase and peroxidase (GOD-POD) kits from Qualigens Fine Chemicals (Glaxo India Ltd, Mumbai, India) and the hepatic LPO, SOD, and CAT activities using the routine protocols, followed earlier in our laboratory (Panda & Kar, 2001; Gholap & Kar, 2003ab).

Statistical analysis

Data were analyzed for significance using analysis-of-variance (ANOVA) followed by Student's t.-test and p values of 5% and less were considered as significant (Senedector & Cochran, 1967).

Results and Discussion

Results (Table 1) clearly revealed that administration of dexamethasone significantly increased serum glucose concentration, as observed earlier by us and other workers (Caro & Amatruda, 1982; Gholap & Kar, 2003ab). A concomitant decrease in the level of both thyroid hormones, T₃ and T₄, was also observed. Interestingly, administration of the three different doses of gymnemic acid to dexamethasone-induced hyperglycemic animals reversed the effect produced by dexamethasone. However, the decrease was significant only in groups receiving two higher doses (13.4 and 26.8 mg/kg body wt) of gymnemic acid.

Table 1.. Effect of different doses of gymnemic acid (GA) in serum concentrations of glucose (mg %), T₃ (ng/ml), T₄ (ng/ml), and hepatic LPO (nM MDA formed h⁻¹ mg protein⁻¹), SOD (units/mg protein) and CAT (μM of H₂O₂ decomposed min^-1 mg protein⁻¹) activities in normoglycemic and dexamethasone-induced hyperglycemic mice.

Groups	Glucose	T3	T4	LPO	SOD	CAT
I. Vehicle control	52.47 ± 1.19	0.75 ± 0.05	31.40 ± 1.84	0.79 ± 0.05	5.14 ± 0.24	65.38 ± 0.48
II. Dexamethasone (Dexa)	75.52 ± 2.54***	0.59 ± 0.04	24.06 ± 2.19	0.71 ± 0.04	6.56 ± 0.24***	72.71 ± 1.47***
III. Dexa + GA (6.7 mg/kg)	72.41 ± 2.43	0.63 ± 0.06	25.07 ± 1.75	0.64 ± 0.04	6.72 ± 0.25	71.49 ± 1.39
IV. Dexa + GA (13.4 mg/kg)	53.80 ± 2.44***	0.68 ± 0.06	26.70 ± 2.26	0.55 ± 0.04	7.79 ± 0.34	74.05 ± 1.42
V. Dexa + GA (26.8 mg/kg)	59.12 ± 3.62**	0.65 ± 0.05	28.20 ± 2.41	2.79 ± 0.48**	5.13 ± 0.18***	60.58 ± 2.35**
VI. Dexa + ketoconazole	68.67 ± 1.97	0.66 ± 0.07	25.17 ± 2.76	0.84 ± 0.05	6.11 ± 0.36	71.75 ± 2.12
VII. GA (6.7 mg/kg,)	55.32 ± 1.72	0.65 ± 0.05	26.13 ± 2.75	0.65 ± 0.03	6.05 ± 0.20	67.46 ± 0.98
VIII. GA (13.4 mg/kg)	44.43 ± 1.41†††	0.48 ± 0.04†††	23.31 ± 1.59††	0.77 ± 0.03	6.82 ± 0.23†††	69.21 ± 1.11††
IX. GA (26.8 mg/kg)	45.73 ± 1.29†††	0.49 ± 0.05†††	24.12 ± 1.71	1.20 ± 0.24	4.73 ± 0.22	55.77 ± 2.62†††
X. Ketoconazole	42.50 ± 2.75††	0.61 ± 0.05	28.23 ± 2.17	0.76 ± 0.05	5.25 ± 0.35	66.94 ± 2.34

LPO, lipid peroxidase; SOD, superoxide dismutase; CAT, catalase.

Data are mean ± SEM (n. = 8). †p. < 0.05; ††p. < 0.01 and †††p. < 0.001 as compared to the respective control values.

*p. < 0.05, **p. < 0.01, ***p. < 0.001 as compared to the respective values of the dexamethasone-treated groups.

Although previous studies on the crude extract of Gymnema sylvestre. leaves indicated its hypoglycemic nature (Shanmugasundaram & Panneerselvam, 1981; Shanmugasundaram et al., 1990), the role of gymnemic acids (its principal constituent) in corticosteroid-induced hyperglycemia has not been investigated thus far. The current findings indicate that gymnemic acid is capable of reducing corticosteroid-induced hyperglycemia. In fact the efficacy of the two doses of gymnemic acids was found to be more (28.76% and 21.71%, respectively) than that of the standard antiglucocorticoid drug ketoconazole (9.07%).

Very often, hyperthyroidism is related to hyperglycemia (Ganong, 1995; Kar & Panda, 2003). However, in the current study, when the serum concentrations of thyroid hormones were correlated, it appeared that hypoglycemic effect of the test material is not mediated through the changes in the levels of thyroid hormones, as serum T₃ and T₄ concentrations were not significantly altered in the groups treated with dexamethasone and gymnemic acids. In normoglycemic animals, the two higher doses of gymnemic acids (13.4 and 26.8 mg/kg) decreased both T₃ and T₄ concentration, indicating a thyroid inhibitory nature of GA. This decrease in both T₃ and T₄ concentration by GA could be the result of inhibition in T₄ synthesis at the glandular level and T₃ production by the peripheral conversion of T₄ to T₃, the major source of the generation of the latter hormone, as has been suggested earlier with respect to other plant extracts (Kar & Panda, 2003).

The results on LPO, SOD, and CAT activities in liver, the primary target organ of a drug, revealed no marked alteration in groups receiving 6.7 and 13.4 mg/kg body wt. of gymnemic acids, indicating that the plant material is apparently not toxic when administered in moderate doses. However, a significant increase in LPO with simultaneous decrease in SOD and CAT activity with 26.8 mg/kg body wt. of gymnemic acid indicated its peroxidative nature, suggesting that the drug may be hepatotoxic at the higher dose.

Excess of either endogenous or exogenous glucocorticoids has been shown to increase gluconeogenesis and decrease tissue glucose uptake, thus resulting in hyperglycemia, potentially inducing diabetes (Campbell & Latimer 1984; Peterson et al., 1984; Wolfsheimer, 1989). Therefore, in the current study, dexamethasone-induced increase in serum glucose level is obvious.

Different mechanisms for corticosteroid-induced diabetes mellitus have been postulated from time to time. One of those is the insulin resistance, caused by the alteration in binding of insulin to its receptor (receptor defect) or by the impairment of the intracellular response to insulin (postreceptor defect) (Grunfeld et al., 1981; Caro & Amatruda, 1982; Rizza et al., 1982). Therefore, in the current study, the antihyperglycemic activity of the gymnemic acids in dexamethasone-treated animals appears to be mediated by decreasing the peripheral insulin resistance or by suppressing enzymes involved in hepatic gluconeogenesis as well as by stimulating glucose uptake and use in peripheral tissues as suggested in diabetic rabbit (Shanmugasundaram et al., 1983). Another possible mechanism of decreasing serum glucose through an increase in insulin release from pancreas cannot be ruled out, as has been suggested earlier (Shanmugasundaram et al., 1990; Persaud et al., 1999).

Whatever may be the mode of actions of the gymnemic acid, considering the glucose-lowering efficacy with no hepatotoxic effects, particularly by the first two doses, the current findings certainly suggest that gymnemic acids (in moderate concentration) may be considered as a potential agent for the amelioration of corticosteroid-induced diabetes mellitus and/or hyperglycemia. However, further investigations will be needed before considering it for human therapy.

Acknowledgments

Financial support from University Grant Commission, New Delhi, India, through a research award grant (no. F-30–57/98) to Dr. A. Kar and the gift of gymnemic acid from Amsar Private Limited, Indore, India, is gratefully acknowledged. We also thank Dr. P. Tahiliani for his help in this study.