Antidiabetic effect of Gymnema montanum leaves: effect on lipid peroxidation induced oxidative stress in experimental diabetes

Pharmacological Research 48 (2003) 551–556

Antidiabetic effect of Gymnema montanum leaves: effect on lipid peroxidation induced oxidative stress in experimental diabetes R. Ananthan a,∗ , C. Baskar a , V. NarmathaBai a , L. Pari b , M. Latha b , K.M. Ramkumar b a

b

Department of Botany, Bharathiar University, Coimbatore 641046, Tamil Nadu, India Department of Biochemistry, Faculty of Science, Annamalai University, Annamalainagar 608002, Tamil Nadu, India Accepted 23 June 2003

Abstract Gymnema montanum is widely used in ancient medicine for the ailment of various diseases. Oral administration of 200 mg kg−1 (body weight) BW of the alcoholic extract of the leaf for 3 weeks resulted in a significant reduction in blood glucose and an increase in plasma insulin, whereas the effect of 50 and 100 mg kg−1 BW was not significant. The alcoholic extract also resulted in decreased free radical formation in plasma of diabetic rats. Thus, this study shows that Gymnema montanum leaf extract (GLEt) possess antihyperglycemic and antiperoxidative effect. The decrease in lipid peroxides and increase in reduced glutathione (GSH), ascorbic acid (Vitamin C) and ␣-tocopherol (Vitamin E) clearly show the antioxidant properties of GLEt. The effect of GLEt was most prominently seen in the case of animals given 200 mg kg−1 BW. In addition, the results suggest that GLEt was highly effective than the reference drug glibenclamide. © 2003 Elsevier Ltd. All rights reserved. Keywords: Alloxan diabetes; Gymnema montanum; Lipid peroxidation; Antioxidants

1. Introduction It is thought that the many stresses inherent in the modern life style may cause an increased incidence of diseases such as cancer, heart diseases and hypertension. The rising incidence of such diseases is alarming and becoming a serious public health problem. Diabetes mellitus is one such disease and it is estimated that the number of diabetic patients will continue to increase in the future [1]. It has been presumed that oxidative stress in the body is a serious contributor to the appearance of this disease. Sato et al. [2] reported that plasma TBARS levels increased in diabetic patients due to vascular lesions induced by hyperglycemia. On the other hand, our bodies possess some defences against oxidative stress through endogenous or exogenous antioxidant substances. Moreover, disturbances of antioxidant defense systems in diabetes were shown: alteration in antioxidant enzymes [3], impaired glutathione metabolism [4] and decreased ascorbic acid levels [5]. Recently an intensive search for novel type of antioxidant has been carried out from numerous plant materials.

∗ Corresponding

author. Fax: +91-422-2422387/2425706. E-mail address: [email protected] (R. Ananthan).

1043-6618/$ – see front matter © 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S1043-6618(03)00219-6

Gymnema montanum Hook.—a rare, endangered and endemic plant species of the family Asclepiadaceae is distributed in India mainly in Western Ghats [6,7]. So for studies pertaining to the antiperoxidative effect of G. montanum has not been carried out. In an effort to find the antiperoxidative effect of this plant, the present study was attempted to investigate the effect of G. montanum leaves on lipid peroxidation induced oxidative stress in experimental diabetes. The effects produced were compared with glibenclamide, a reference drug.

2. Materials and methods 2.1. Animals Male albino Wistar rats 170–200 g BW bred in the Central Animal House, Rajah Muthiah Medical College, were used in this experiment. The animals were fed ad libitum with normal laboratory pellet diet (Hindustan Lever Ltd., India) and water. Animals were maintained under a constant 12-h-light/12-h-dark cycle and an environmental temperature of 21–23 ◦ C [8]. The animals were maintained in accordance with the guidelines of the National Institute of Nutrition, Indian Council of Medical Research, Hyderabad,

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India, and approved by the ethical committee, Annamalai University, Tamil Nadu. 2.2. Drugs and chemicals All the drugs and biochemicals used in this experiment were obtained from Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals used were of analytical grade. 2.3. Plant material Gymnema montanum leaves were collected freshly from the Shola forests of Western Ghats, Gudalur, The Nilgiri Biosphere Reserve at an altitude of 900–1500 m.s.l. The plant was identified at the Herbarium of Botanical Survey of India, Southern Circle, Coimbatore, India (accession no. 32561–65) and was deposited in the Department of Botany, Bharathiar University. 2.4. Preparation of plant extract To prepare the Gymnema montanum leaf extract (GLEt), 500 g of fresh leaves of G. montanum were chopped into small pieces and soaked overnight in 1.5 l of 95% ethanol. This suspension was filtered and the residue was resuspended in an equal volume of 95% ethanol for 48 h and filtered again. The two filtrates were pooled and the solvents were evaporated in a rotavapor at 40–50 ◦ C under reduced pressure and lyophilised. A greenish-black powdered material was obtained (20–30 g). It was stored at 0–4 ◦ C until used. When needed, the residual extract was suspended in distilled water and used in the study [9].

given GLEt (200 mg kg−1 BW); group 6, diabetic rats given glibenclamide (600 ␮g kg−1 BW) [12]. The plant extract and the drug glibenclamide were given in aqueous solution daily using an intragastic tube for 3 weeks. At the end of the experimental period, the animals were deprived of food overnight and then sacrificed by decapitation. Blood was collected in tubes containing potassium oxalate and sodium fluoride solution for the estimation of blood glucose. Plasma was separated for the estimation of insulin and various biochemical parameters. 2.7. Analytical procedure Blood glucose was estimated by the o-toluidine method [13]. Plasma insulin level was estimated with an enzyme linked immunosorbent assay (ELISA) kit using human insulin as standard [14]. Thiobarbituric acid reactive substances (TBARS) and hydroperoxides in plasma was estimated according to the methods of Fraga et al. [15] and Jiang et al. [16]. Glutathione (GSH) was determined by the method of Ellman [17]. Vitamins C and E were estimated by the method of Omaye et al. [18] and Baker et al. [19], respectively. 2.8. Statistical analysis All data were expressed as mean ± S.D. for six experiments. The statistical significance was evaluated by one-way analysis of variance (ANOVA) using SPSS version 7.5 (SPSS, Cary, NC, USA) and the individual comparison were obtained by Duncan’s multiple range test (DMRT) [20].

3. Results 2.5. Experimental induction of diabetes Rats were injected intraperitoneally with freshly prepared solution of alloxan monohydrate in normal saline at a dose of 150 mg kg−1 BW [10]. Because alloxan is capable of producing fatal hypoglycemia as a result of massive pancreatic insulin release, rats were treated with 20% glucose solution (5–10 ml) orally after 6 h. The rats were then kept for the next 24 h on 5% glucose solution bottles in their cages to prevent hypoglycemia [11]. After 2 weeks, rats with moderate diabetes that exhibited glycosuria and hyperglycemia (i.e. blood glucose concentration 200–300 mg dl−1 ) were taken for the experiment. 2.6. Experimental procedure A total of 36 rats (30 diabetic surviving rats, 6 normal rats) were used and they were divided into 6 groups of 6 rats each. The treatment groups can be summarised as follows: group 1, normal untreated rats; group 2, diabetic control; group 3, diabetic rats given GLEt (50 mg kg−1 BW); group 4, diabetic rats given GLEt (100 mg kg−1 BW); group 5, diabetic rats

The changes in the body weight, water and food intake in normal and experimental diabetic rats are represented in Fig. 1. The body weights in the GLEt treated and glibenclamide treated groups increased significantly (P < 0.001) at the end of third week when compared with the diabetic control group. The food and water intake was significantly reduced in GLEt and glibenclamide treated group than diabetic control rats. In all the groups prior to alloxan administration, the basal levels of blood glucose of the rats were not significantly different. However, 15 days after alloxan administration, blood glucose levels were significantly higher in the rats selected for the study. Table 1 shows the effect of GLEt and glibenclamide on blood glucose levels. Although a significant antihyperglycemic effect was evident from the first week onwards, the decrease in blood glucose was maximum on third week with significant increase in plasma insulin in group receiving 200 mg kg−1 BW of GLEt. The effect of GLEt on TBARS and hydroperoxides of normal and experimental rats is summarised in Table 2. A marked increase in the level (TBARS, 0.36 ± 0.024;

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intial

A

Body weight (gm)

250 200

final

*

*

*

*

553

#

150 100 50 0 1

2

3

4

5

6

Groups

Food intake in g -1day -1

250

Before the treatment

B

b

200

After the treatment

150

c 100

e

d e

a

50 0 1

2

3

4

5

6

Groups Fluid intake ml rat -1 day -1

80

Before the treatment After the treatment

C

b 60

c d

40

e

e 20

a

0 1

2

3

4

5

6

Groups Fig. 1. Body weight (A), food (B) and fluid (C) intake in alloxan diabetic rats before and after oral treatment with Gymnema montanum leaf extract for 3 weeks. Groups: 1, normal; 2, diabetic control; 3 diabetic + GLEt (50 mg kg−1 ); 4, diabetic + GLEt (100 mg kg−1 ); 5, diabetic + GLEt (200 mg kg−1 ); 6, diabetic + glibenclamide (600 ␮g kg−1 ). Values are given as mean ± S.D. from six rats in each group. Values not sharing a common superscript letter differ significantly at P < 0.05 (DMRT). Duncan procedure, ranges for the level: 2.95, 3.09, 3.20 and 3.22. Diabetic control was compared with normal, # P < 0.001. Experimental groups were compared with diabetic control ∗ P < 0.001.

hydroperoxides, 14.68 ± 1.99) of plasma lipid peroxidation products was observed in diabetic control rats. Treatment with GLEt and glibenclamide significantly reduced the levels of TBARS and hydroperoxides (TBARS, 0.20 ± 0.014 (GLEt), 0.24 ± 0.022 (glibenclamide); hy-

droperoxides, 10.75 ± 1.30 (GLEt), 12.96 ± 1.76 (glibenclamide)). For studying the effect of GLEt on free radical production, the activities of Vitamin C, Vitamin E and reduced glutathione were measured in plasma (Table 3). Diabetic rats

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Blood glucose (mg dl−1 )

Groups

0 day Normal Diabetic Diabetic Diabetic Diabetic Diabetic

control + GLEt (50 mg kg−1 ) + GLEt (100 mg kg−1 ) + GLEt (200 mg kg−1 ) + glibenclamide (600 ␮g kg−1 )

79.66 81.66 80.00 83.66 79.50 77.48

15 days after alloxan injection ± ± ± ± ± ±

3.03 4.94 5.71 6.59 5.09 4.48

84.16 265.00 255.50 248.00 258.00 245.58

± ± ± ± ± ±

5.14 19.45∗∗ 16.36 14.02 18.61 13.99

First week (after treatment) 82.16 279.66 231.66 215.83 200.00 219.29

± ± ± ± ± ±

5.92 12.93∗∗ 15.45∗ (9.30) 13.04∗ (12.97) 12.58∗∗ (22.48) 7.05∗ (10.70)

Second week 80.66 285.50 207.54 180.40 120.21 191.58

± ± ± ± ± ±

6.04 12.95∗∗ 8.73∗∗ (18.78) 9.83∗∗ (27.25) 6.39∗∗ (53.40) 10.8∗∗ (21.98)

Third week 81.45 298.00 163.75 161.83 86.35 118.22

± ± ± ± ± ±

5.98 15.77∗∗ 8.08∗∗ (35.90) 13.25∗∗ (34.52) 9.25∗∗ (66.53) 4.48∗∗ (51.86)

Plasma insulin (␮U/ml) after 3 weeks 13.62 5.50 6.70 9.16 12.06 10.08

± ± ± ± ± ±

5.52a 2.75b 0.94b 0.62c 4.32d 3.13e

Values are given as mean ± S.D. for six rats in each group. Values in parentheses indicated the percentage lowering of blood glucose in comparison to basal reading after alloxan administration (15 days). Diabetic control was compared with normal. Experimental groups were compared with corresponding values after alloxan injection (15 days). ∗ P < 0.01, ∗∗ P < 0.001. Values not sharing a common superscript letter differ significantly at P < 0.05 (DMRT). Duncan procedure, range for the level: 2.89, 3.03, 3.13, 3.20 and 3.25.

R. Ananthan et al. / Pharmacological Research 48 (2003) 551–556

Table 1 Changes in levels of blood glucose and plasma insulin of normal and experimental animals

R. Ananthan et al. / Pharmacological Research 48 (2003) 551–556 Table 2 Change in the levels of plasma TBARS and hydroperoxides in normal and experimental animals Groups

TBARS (mM dl−1 )

Hydroperoxides (10−5 mM dl−1 )

Normal Diabetic control Diabetic + GLEt (200 mg kg−1 ) Diabetic + glibenclamide (600 ␮g kg−1 )

0.15 ± 0.011a 0.36 ± 0.024b 0.20 ± 0.014c

9.95 ± 0.65a 14.68 ± 1.99b 10.75 ± 1.30a

0.24 ± 0.022d

12.96 ± 1.76d

Values are given as mean ± S.D. for six rats in each group. Values not sharing a common superscript letter differ significantly at P < 0.05 (DMRT). Duncan procedure, range for the level: 2.95, 3.09 and 3.20.

showed a reduced level of antioxidants whereas a significant increase in the levels were observed in GLEt treatment as well as in glibenclamide—the reference drug, when compared with diabetic control rats.

4. Discussion The use of traditional medicine and medicinal plants in most developing countries, as a normative basis for the maintenance of good health, has been widely observed [21]. Free radical induced lipid peroxidation has been associated with a number of disease processes including diabetes mellitus [22]. The increase in oxygen free radicals (OFR) in diabetes could be due to increase in blood glucose levels, which upon autoxidation generate free radicals [23]. This study was therefore undertaken to assess antiperoxidative properties of G. montanum, which is an endemic and endangered plant. The capacity of GLEt to decrease the elevated blood glucose to normal level is an essential trigger for the liver to revert to its normal homeostasis during experimental diabetes. The possible mechanism by which GLEt exerts its hypoglycemic action in diabetic rats may be due to potentiating the insulin release, since the percentage fall in blood glucose levels was very significant at 200 mg kg−1 it implies that antihyperglycemic effect of this plant is dependent upon the dose of diabetogenic agent and therefore, on the degree of ␤-cell destruction. This is probably indicative of efficacy of the plants. Moreover, it indirectly indicates that part of the antihyperglycemic activity of this plant is through release of insulin from the pancreas. Our findings coincide

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with those of earlier studies, which reported that, the extract of Gymnema sylvestrae reduced the blood glucose and increased the plasma insulin levels in diabetic rats. G. sylvestrae is reported to be rich in gymnemagenin and gymnemic acids that are responsible for the antihyperglycemic effect [24]. The presence of such active constituents in G. montanum may be responsible for the blood glucose lowering and insulin stimulatory effect observed in our study. In this context, a number of other plants have also been reported to have antihyperglycemia and insulin-release stimulatory effects [25,26]. The daily administration of GLEt and glibenclamide to alloxan diabetic rats for 3 weeks caused a satistically significant reduction in food and fluid intakes and an increase in the body weight. This could be the result of improved glycemic control produced by G. montanum and glibenclamide. It has been generally reported that diabetic patients with vascular lesions have higher TBARS levels that their healthy counterpart. TBARS and hydroperoxides significantly increased in diabetic rats. Previous studies have reported that there was an increased lipidperoxidation in plasma of diabetic rats [27,28]. Our study shows that administration of GLEt to diabetic rats tends to bring the plasma peroxides to near normal levels. Ascorbic acid is a potent antioxidant, which widely acts on OFR as well as interaction with Vitamin E [29]. Both the Vitamin C and E significantly decreased in plasma of diabetic rats. Administration of GLEt increased the Vitamin C and E levels. This indicates that Vitamin E is used in combating free radicals and if Vitamin C is present, Vitamin E levels are preserved. Frei [30] has previously shown the ability of Vitamin C to preserve the levels of other antioxidants in human plasma. Also Vitamin C regenerates Vitamin E from its oxidised form. GSH is the first line of defense against proxidant status [31] and GSH was evaluated after GLEt administration. GSH systems may have the ability to manage oxidative stress with adaptional changes in enzymes regulating GSH metabolism. In the present study, treatment with GLEt significantly increased the GSH levels. Increase in GSH level may in turn activates the GSH dependent enzymes such as glutathione peroxidase and glutathione-S-transferase. Hence, in addition to antidiabetic effect, G. montanum possess antioxidant potential that may be used for therapeutic purposes. The results of the present study indicate that the preventive effects of G. montanum may be due to inhibition

Table 3 Changes in levels of plasma Vitamin C, Vitamin E and reduced glutathione in normal and experimental animals Groups

Vitamin C (mg dl−1 )

Normal Diabetic control Diabetic + GLEt (200 mg kg−1 ) Diabetic + glibenclamide (600 ␮g kg−1 )

1.68 0.75 1.59 1.40

± ± ± ±

0.07a 0.04b 0.06c 0.04d

Vitamin E (mg dl−1 ) 1.52 0.75 1.4 1.21

± ± ± ±

0.07a 0.04b 0.06c 0.05d

Reduced glutathione (mg dl−1 ) 25.23 12.00 21.25 18.05

± ± ± ±

2.50a 0.46b 2.0c 1.26d

Values are given as mean ± S.D. for six rats in each group. Values not sharing a common superscript letter differ significantly at P < 0.05 (DMRT). Duncan procedure, range for the level 2.95, 3.09 and 3.20.

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