Effects of Three Different Doses of a Fruit Extract of Terminalia chebula on Metabolic Components of Metabolic Syndrome, in a Rat Model

Inderjeet Singh,1 Pawan Kumar Singh,1 Shobhit Bhansali,1 Nusrat Shafi q,1 Samir Malhotra1*, Promila Pandhi1 and Amrit Pal Singh2

1Postgraduate Institute of Medical Education and Research (PGIMER), Chandigarh City, India2Department of Herbal Medicine, Amity University, Jalandhar, Punjab, India

There is documented evidence of the use of Terminalia chebula for various ailments in the Ayurvedic litera-ture. The extract has been shown to possess glucose lowering activity and to improve insulin sensitivity in animal models of type 2 diabetes mellitus. The present study was carried out to study the dose response rela-tionship of this extract in a rat model of metabolic syndrome. Six groups of rats were fed a high fructose diet (HFD) for a period of 20 days to induce metabolic syndrome. Three doses of fruit extract of T. chebula 50, 100 and 200 mg/kg were administered orally and pioglitazone 2.7 mg/kg was used as a positive control. Blood samples were collected at days 0, 20 and 40 from the tail vein. Systolic blood pressure (SBP) was measured using the tail cuff method and an oral glucose tolerance test (OGTT) was done on the day of blood collection. Administration of HFD for 20 days signifi cantly increased fasting blood glucose (FBG), SBP and the area under the curve of OGTT. On day 40 the FBG in the 50, 100 and 200 mg/kg group was 97.33 ± 5.82 (NS), 86.83 ± 5.08 (p = 0.038) and 85.67 ± 6.74 (p = 0.15), respectively. These results show that the fruit extract of T. chebula exerts a signifi cant and dose-dependent glucose lowering effect in the rat model of metabolic syndrome. Copyright © 2009 John Wiley & Sons, Ltd.

Keywords: insulin resistance; metabolic syndrome; Terminalia chebula.

INTRODUCTION

Metabolic syndrome (MS) refers to the clustering of several cardiometabolic risk factors, including abdomi-nal obesity, hyperglycemia, dyslipidemia and elevated blood pressure, which are linked to insulin resistance (Alberti et al., 2005). Aggressive management of the individual components of the syndrome makes it pos-sible to prevent or delay the onset of diabetes mellitus, hypertension and cardiovascular disease. Pharmaco-logical agents directed towards the individual compo-nents of this syndrome have been the main modality of treatment for metabolic syndrome. These include anti-hyperglycemic agents, antihypertensive agents and lipid lowering therapies (Chang et al., 2007 and Grundy et al., 2005). To date no single pharmacological agent has been developed to correct the metabolic abnormalities associated with MS.

A fruit extract of Terminalia chebula (TC) has been studied for its effectiveness in various animal models of diabetes. It has been shown to possess insulinotropic action. It has been shown to be effective in correcting hyperglycemia associated with streptozotocin induced diabetes in rats (Murali et al., 2004, 2007; Kumar et al., 2006). The present study evaluated the effects of three

different doses of extract of Terminalia chebula in a rat model of metabolic syndrome. This study was based on our preliminary study which showed that this extract has insulin sensitizing action in a high fructose induced animal model of insulin resistance.

MATERIALS AND METHODS

Animals. Animals (Wistar rats weighing 150–180 g) were procured from the central animal house of Post-graduate Institute of Medicala Education and Research (PGIMER), Chandigarh. They were kept individually in polypropylene cages in an environmentally controlled room of the departmental animal house. They were maintained at 25 ± 2 ºC with a 12 h dark/light cycle and 40–70% humidity. The animals had free access to food and water. The rats were fed with a standard rat chow diet or a special high fructose diet according to the protocol. Experiments were carried out after a week of acclimatization according to the guidelines of Animal Ethics Committee of the Institute and the Committee for the Purpose of Control and Supervision of Experi-mentation in Animals (CPCSEA) guidelines for animal care. Approval from the Institute’s Animal Ethics Committee was taken prior to conducting the studies.

Drugs and chemicals. T. chebula fruit extracts and pioglitazone powder were obtained from Ind-Swift Pharmaceuticals, Chandigarh, India.

* Correspondence to: Dr Samir Malhotra, Postgraduate Institute of Medical Education and Research (PGIMER), Chandigarh City, India.E-mail: samirmalhotra345@yahoo.com

Received 05 March 2009Copyright © 2009 John Wiley & Sons, Ltd. Accepted 26 March 2009

PHYTOTHERAPY RESEARCHPhytother. Res. 24: 107–112 (2010)Published online 28 May 2009 in Wiley InterScience(www.interscience.wiley.com) DOI: 10.1002/ptr.2879

Copyright © 2009 John Wiley & Sons, Ltd. Phytother. Res. 24: 107–112 (2010)DOI: 10.1002/ptr

108 SINGH ET AL.

Experimental groups. Six groups of six rats each were taken. Five groups were fed with high fructose diets for 3 weeks ad libitum. The high fructose diet provides 60% of the calories from fructose. T. chebula fruit extracts at 50, 100 and 200 mg/kg were administered orally daily to three groups. As a control, one group received only the normal diet alone, one group received HFD and vehicle solution while the other group received pioglitazone 2.7 mg/kg body weight. The diet was continued for 40 days while the treatment was started on day 20 of feeding. All the drugs were administered once daily by gavage in 1% carboxymethyl cellulose (CMC) suspension.

Blood glucose estimation. Blood samples for plasma glucose were collected from the tail vein of rats. 0.1 mL of blood was collected in heparinized tubes for this purpose, to which sodium fl uoride was added. For fasting blood glucose, the samples were collected after an overnight fast. Glucose level estimation was carried out by the glucose oxidase method described by Trinder (1969). For this purpose, commercial kits purchased from CDR Diagnostics, Hyderabad along with manu-facturer’s instructions were followed.

Plasma insulin measurement. Plasma samples for insulin estimation was stored at −20 ºC. Insulin level was measured by rat insulin ELISA purchased from Mercodia AB, Seminariegatan 29, S-752 28 Uppsala, Sweden and was done according to the manufacturer’s instructions.

Lipid profi le measurement. Blood was collected through the tail vein during the study and by cardiac puncture at the end of the experiment. The plasma was separated by centrifuging at 3000 rpm. Lipid profi le estimation was carried out in the pooled plasma in each group. Plasma total cholesterol and triglycerides were measured enzymatically (Randox Laboratories Ltd, UK) on a digital colorimeter (Digichem economy). HDL cholesterol was measured (AUTOPAK, Bayer Diagnostics India Ltd) on a digital colorimeter (Digichem economy) after precipitation of chylo-microns and very low-density lipoprotein and LDL cholesterol by phosphotungstic acid and magnesium chloride followed by centrifugation. The cholesterol in the HDL fraction, which remained in the supernatant, was assayed with enzymatic cholesterol method, using cholesterol esterase, cholesterol oxidase, peroxidase and the chromogen 4-aminoantipyrine/phenol.

Insulin resistance assessment. Insulin resistance was calculated using homeostasis (HOMA) according to Mathews et al. (1985). This model incorporates in math-ematical expression measures of both fasting plasma concentrations of glucose and insulin. It calculates the index of insulin resistance.

Beta cell function assessment. Beta cell function assess-ment was done according to Mathews et al. (1985).

Area under curve of oral glucose tolerance test (OGTT). The area under the curve of OGTT was cal-culated according to the trapezoidal rule.

Blood pressure measurement. Blood pressure was measured by the tail cuff method [Ugobasile, Biological Research Apparatus, Comerio-(va)-Italy]. After light ether anesthesia the tail of rat was cleaned with xylene. An occluding cuff similar in principle to that used for routine blood pressure measurements in humans was placed over the tail (near the base), with a piezoelectric transducer placed about 0.5–1 cm distal to the cuff. Pressure in the cuff was increased until the tail pulse disappeared and then released slowly. The systolic BP was taken as the average of the value when the tracing disappeared on infl ation and reappeared on defl ation.

The animals were warmed because the tail arteries which are important in thermoregulation are often con-stricted at room temperature. An infrared lamp was placed near the tail to heat the tail to 32 ºC for 10 min before recording the BP.

Statistical analysis. All the data are expressed as mean ± SD. Intra-group comparisons were done by paired t-test. Inter-group comparisons were made using ANOVA followed by post-hoc Bonferoni’s test. A value of p < 0.05 was considered signifi cant.

RESULTS

Feeding of the high fructose diet for 20 days increased signifi cantly the fasting blood glucose levels, fasting plasma insulin levels and systolic blood pressure in rats (Table 1). Similarly the area under curve of the OGTT and the insulin resistance markers, the HOMA index and % beta cell function was increased after 20 days of fructose feeding (Table 1).

Administration of T. chebula extract at a dose of 100 and 200 mg/kg body weight led to a signifi cant reduc-tion in fasting blood glucose levels which was raised by fructose feeding. This effect was signifi cantly different from the vehicle treatment group. There was no differ-ence between the extract treated group and the piogli-tazone treated group, which also reduced the fasting blood glucose levels (Fig. 1).

Similar results were obtained with the fasting plasma insulin levels and the markers of insulin resis-tance. Both 100 and 200 mg/kg dose of the extract led to improvement of insulin resistance, as shown by the reduced insulin levels (Fig. 2), lower HOMA index (Fig. 3) and lower % beta cell function (Fig. 4) in these groups. These were signifi cantly lower when compared with the vehicle treatment group and were similar to the pioglitazone treated group. The administration of 50 mg/kg dose did not have any signifi cant effect on fasting plasma glucose levels and insulin resistance markers.

Treatment with 100 and 200 mg/kg of T. chebula extract for 20 days decreased the mean AUC. Piogli-tazone treatment had a maximum lowering effect on the AUC of the OGTT.

Treatment with T. chebula had no effect on SBP at all doses. Long term administration of the extract for 20 days did not alter the increased systolic blood pres-sure on continuous feeding of fructose.

Total cholesterol, triglycerides and the calculated LDL levels were 110 mg%, 122 mg% and 74 mg%, respectively, on day 40, the HDL levels was 36 mg.

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Table 1. Changes in various parameters after 20 days of fructose feeding in various groups

Group Day Normal diet HFD diet Pioglitazone TC 50 mg/kg TC 100 mg/kg TC 200 mg/kg

Fasting plasma glucose (mg/dL)

0 82 ± 6.93 81.17 ± 7.02 81.83 ± 6.31 82.83 ± 5.15 82.17 ± 8.82 86.67 ± 14.99

20 81.17 ± 6.18 98.33 ± 9.50a 97.17 ± 8.95a 98.67 ± 12.26a 97.33 ± 4.50a 98.33 ± 6.86a

Fasting plasma insulin (μmol/dL)

0 6.67 ± 3.56 6.83 ± 3.56 6 ± 4.09 6.17 ± 3.25 8 ± 4.77 8 ± 3.03

20 5.67 ± 3.78 20.33 ± 9.50a 19.17 ± 8.95a 20.67 ± 12.26a 19.33 ± 4.50a 20.33 ± 6.86a

HOMA IR 0 0.85 ± 0.41 0.83 ± 0.41 0.77 ± 0.48 0.8 ± 0.38 1.03 ± 0.52 1.02 ± 0.3620 0.73 ± 0.45 2.63 ± 1.13a 2.48 ± 1.08a 2.68 ± 1.46a 2.5 ± 0.52a 2.63 ± 0.80a

% Beta cell function 0 99.65 ± 23.94 101 ± 32.48 92.15 ± 27.56 92.27 ± 22.63 114.83 ± 43.49 106.13 ± 23.8520 89.77 ± 23.69 149.2 ± 20.42a 146.45 ± 21.64a 142.68 ± 39.20a 151.4 ± 11.68a 151.92 ± 16.58a

Systolic blood pressure (mm Hg)

0 143.33 ± 10.33 138.33 ± 11.69 141.67 ± 14.72 138.33 ± 16.02 146.67 ± 16.33 141.67 ± 11.69

20 141.67 ± 16.02 188.33 ± 21.37a 193.33 ± 28.71a 195 ± 34.50a 208 ± 27.14a 195 ± 24.29a

Weight (g) 0 157 ± 10.08 162 ± 8.72 161.6 ± 8.42 172.5 ± 9.87 168 ± 5.44 162 ± 7.9220 178.2 ± 11.80 185.8 ± 5.85 181.6 ± 5.55 192.5 ± 10.70 189 ± 7.90 184.33 ± 8.19

a p < 0.05 compared with baseline. TC, Terminalia chebula.

Figure 1. Fasting blood glucose at the end of treatment. Error bars represent standard deviation. @p < 0.05 compared with vehicle group. $p < 0.05 compared with 50 mg/kg group and vehicle group. TC, Terminalia chebula.

Treatment with T. chebula at a dose of 100 mg/kg decreased triglyceride level to 98 mg%. Similarly in the 200 mg/kg group the TGL level decreased to 88 mg% at day 40. Treatment with the extract at any dose level did not alter the total cholesterol levels HDL and LDL levels. However, statistical signifi cance could not be achieved as lipid profi le analysis was done in pooled samples.

Treatment with the extract did not alter the increase in weight after fructose feeding.

DISCUSSION

The fi ndings suggest that long term treatment with the fruit extract of T. chebula lowers both postprandial plasma glucose and fasting glucose levels. The lowering of postprandial glucose levels was consistent with

our previous study. Chebulagic acid, one of the com ponents of the extract has been shown to be a potent inhibitor of α glucosidase enzyme (Gao et al., 2008). This suggests that the glucose lowering activity of the T. chebula extract involves multiple mechanisms.

Similarly, the fasting plasma insulin levels and the % beta cell function decreased signifi cantly after 20 days of treatment with the extract at both doses of 100 and 200 mg/kg body weight. The HOMA IR marker for insulin resistance also decreased on treatment with these two doses. However, the extract has been shown to possess insulin secreting property in various studies. A study done by Murali et al. (2007) showed that the extract at a dose of 200 mg/kg had insulintropic action. They showed that the extract stimulated in vitro beta cells and increased insulin secretion. The present study results show that the glucose lowering effect of the extract may also involve improvement of insulin resis-

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110 SINGH ET AL.

tance as treatment with the extract lowered the beta cell activity and the fasting plasma insulin levels, which were increased by high fructose feeding. These effects were similar to the pioglitazone treated group. Treat-ment with pioglitazone improved insulin resistance thereby lowering the insulin levels which were increased by high fructose feeding. This was consistent with the study done by Sharabi et al. who showed that the PPAR γ agonist rosiglitazone lowered insulin levels in

an animal model of insulin resistance. This effect on insulin levels was associated with an increase in adipo-nectin levels. However, in this study the mechanisms involved in improvement of insulin resistance were not evaluated.

In contrast to the results of insulin sensitivity, after treatment with the extract there was no change in the elevated systolic blood pressure. This was surprising because insulin plays a major role in the development

Figure 2. Fasting plasma insulin levels at the end of treatment. Error bars represent standard deviation. @p < 0.05 compared with vehicle group. $p < 0.05 compared with 50 mg/kg group and vehicle group. TC, Terminalia chebula.

Figure 3. HOMA index at the end of treatment at the end of treatment. Error bars represent standard deviation. @p < 0.05 compared with vehicle group. $p < 0.05 compared with 50 mg/kg group and vehicle group. TC, Terminalia chebula.

TERMINALIA CHEBULA ON METABOLIC SYNDROME 111

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of hypertension in animal models of hypertension. Insulin resistance and hyperinsulinemia has been dem-onstrated in genetic and nongenetic models of hyper-tension such as spontaneously hypertensive (Mondon and Reaven, 1988), Dahl salt-sensitive (Sechi et al., 1997), Milan hypertensive (Dall et al., 1991) and fruc-tose fed rats. This suggests that a similar mechanism is involved in these animal models. In rats which are fed a high fructose diet hypertension develops and this is associated with hyperinsulinemia and insulin resistance (Higashiura et al., 1999). In these rats restoration of normal insulin levels and normalization of insulin sensitivity by somatostatin decreased the raised blood pressure suggesting a pathogenic role of insulin in hypertension (Reaven et al., 1989). Post receptor defects in the molecular pathways of insulin are involved in the pathophysiology. However, the results show that improving insulin resistance did not alter the increased blood pressure. This suggests that there may be other mechanisms involved in the development of hyperten-sion in our animal model of metabolic syndrome.

A similarly descriptive analysis of the lipid profi le showed that the triglyceride levels were lowered after treatment with the extract. There was no effect on the LDL, total cholesterol and HDL levels after treatment. However, no statistical signifi cance could be obtained as the data were from pooled samples.

The fruit extract of T. chebula is a mixture of different tannins mainly chebulinic acid, chebulagic acid and che-bulic acid. The extract has shown to have a variety of pharmacological activities such as antioxidant activity, antiinfl ammatory activity and cytotoxic activity. This may be attributed to the variety of tannins present in this complex extract. The multiple effects of the extract can be due to different constituents in the extract. The results of our study points towards multiple mechanisms involved in glucose lowering and reversing insulin resis-tance in an animal model.

The major limitation of the study is that the mechanism of action of the extract was not evaluated. In addition the individual components of the extract were not studied for their pharmacological properties. The molecular mechanisms leading to these effects need to be evaluated. It is necessary to fi nd which of the above or other constituents are responsible for the antihyperglycemic activity observed in the present studies. Further studies on the exact mechanism of action of this extract and isolation of the active compound in a pure state should be undertaken. This could lead to the development of a drug from this plant which improves insulin resistance thereby help-ful in managing patients with metabolic syndrome and a better understanding of the pathophysiology of metabolic syndrome.

Figure 4. % beta cell function at the end of treatment. Error bars represent standard deviation. @p < 0.05 compared with vehicle group. $p < 0.05 compared with 50 mg/kg group and vehicle group. TC, Terminalia chebula.

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