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An Insight into Antidiabetic Mechanisms of Polyphenol Rich (Catechin, Epicatechin) Tamarind Seeds

Sushant Shivdas Sole, et al., BAOJ Pharm Sci 2015, 1: 3 1: 014

BAOJ Pharm Sci, an open access journal Volume1; Issue 3; 014

Sushant Shivdas Sole1* and Bhartur Parthsarthy Srinivasan2

1College of Veterinary and Animal Sciences (Maharashtra Animal and Fishery Sciences University), Udgir-413517, Maharashtra, India2Delhi Institute of Pharmaceutical Sciences and Research, (University of Delhi), Pushp Vihar, Sect-3, M. B.Road, New Delhi-110017, India

BAOJ Pharmaceutical Sciences

*Corresponding author: Sushant Shivdas Sole, College of Veterinary and Animal Sciences (Maharashtra Animal and Fishery Sciences University), Udgir-413517, Maharashtra, India, Tel: +91-11-29554327; Fax: +91-11-29554503; E-mail: [email protected]

Sub Date: November 11, 2015, Acc Date: December 17, 2015, Pub Date: December 19, 2015.

Citation: Sushant Shivdas Sole and Bhartur Parthsarthy Srinivasan (2015) An Insight into Antidiabetic Mechanisms of Polyphenol Rich (Catechin, Epicatechin) Tamarind Seeds. BAOJ Pharm Sci 1: 014.

Copyright: © 2015 Sushant Shivdas Sole, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Research Article

AbstractTamarindus indica L. is widely used as a traditional medicine for the management of diabetes mellitus beside its antihepatotoxic and anti-inflammatory activity. However, the influence of Tamarind seed on hyperglycemia and gastrointestinal dysfunctions of diabetes has not been explored so far at molecular level. The effect of 4 weeks oral treatment (120 and 240 mg/kg) of HPLC standardized Tamarind Seed aqueous extract (TSE) was studied in streptozotocin (STZ) model of diabetes in Wistar rats. The GI functions assessed in terms of gastric emptying and small intestinal transit rate by phenol red and activated charcoal method exhibited significant increase in emptying without any effect on intestinal transit rate after TSE treatment. The insulin mimetic effect of TSE was found to reduce fasting and postprandial hyperglycemia with increase in glucose uptake through improvement in expression of glucose transporters (GLUT) 2 and 4 proteins in the liver and skeletal muscle. The proadiponectin action of TSE was devoid of any effect on pancreatic GLP-1 mRNA concentration. In addition, TSE as a non competitive inhibitor inhibited α-glucosidase activity (in vitro) dose dependently. These findings revealed correlation of antihyperglycemic action of TSE with moderate α-glucosidase inhibition, proadiponectin effect beside its pronounced GLUT-2 and GLUT-4 expression in liver and skeletal muscle.Keywords: Type 2 Diabetes Mellitus; α- glucosidase; GLUT-2 GLUT-4; Adiponectin; Glucagon like peptide-1 receptor; Tamarind Seed Extract; Gastrointestinal Functions.

IntroductionDiabetic patients often experience gastrointestinal (GI) dysfunctions such as flatulence, early satiety, esophageal reflux, nausea, and vomiting, which exist even after the ant diabetic therapy [1]. Increasing gut motility with prokinetic agents helps in attenuating these dysfunctions. However, change in gut motility affects the absorption of oral antidiabetic agents. Therefore, an antidiabetic agent is needed to effectively target hyperglycaemia and the GI dysfunctions associated with it. GI disturbances in diabetes can be attributed to oxidative stress-mediated autonomic neuropathy [2]. A gut hormone, glucagon like peptide 1 (GLP-1) that is secreted in response to the absorbed nutrients, contributes to the GI functions by reducing gastric emptying and stimulating insulin release [3]. Furthermore, findings reported by Nogueiras et al. (2009) [4], provided the connecting link between GLP-1, hyperlipidemia, and adiponectin. Previous studies have reported that exogenous GLP-1 administration can control hyperlipidemia via direct modulation of adiponectin as the adipose tissue, involved

in the regulation of both glucose and lipid homeostasis, confer a gatekeeper role on adipocytes in the regulation of lipid metabolism through adiponectin [5]. Type 2 diabetes is characterized by insulin resistance in peripheral tissues such as liver, muscle and fat, with low serum adiponectin level [6]. Delayed gastric emptying affects the postprandial glucose profile, as it impairs the delivery of nutrients to the small intestine. On the other hand it has been observed that hyperglycemia tends to slow gastric emptying. Thus, there is the need to focus on suppression of postprandial hyperglycemia, not only in diabetic patients but also in individuals with impaired glucose tolerance. The prevention of absorption of carbohydrates after food uptake, is one of the therapeutic approaches for reducing postprandial hyperglycemia in patients with DM. Mammalian α-glucosidase, located in the brush-border surface membrane of intestinal cells, is the key enzyme that catalyzes the final step in the digestive process of carbohydrates thereby retarding the liberation of D-glucose from oligosaccharides and disaccharides and delaying glucose absorption, resulting in reduced postprandial hyperglycemia [7].In addition to the drugs in modern medicine, several plant species having hypoglycemic activity are also prescribed widely even though their biological markers have not been assessed [8]. These are used because of their perceived effectiveness and minimal side effects in clinical experience. Tamarindus indica L. [(Leguminosae (Caesalpiniaceae)], commonly known as Tamarind, occurs in the tropical regions of the world, and can be found in more than 50 countries. It is indigenous to tropical Africa but has become naturalized in North and South America and is also cultivated in subtropical China, India, Pakistan, Indochina, Philippines, Java and Spain. T. indica pulp fruit and seed are used for seasoning, as a food component and in juices. Its fruit is regarded as a

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digestive, carminative, laxative, expectorant and blood tonic [9]. Other parts of the plant, present antioxidant [10], antihepatotoxic [11] and antiinflammatory [12] potential. T. indica is used in traditional medicine for the management of diabetes mellitus [13]. Furthermore, the aqueous extract of Tamarind seeds has been found to have potent antidiabetic and antihyperlipidemic activity in streptozotocin (STZ) induced diabetic rats [14].The antihyperglycemic potential of Tamarind seeds in diabetic gastrointestinal complications and its effect on the glucose transporter proteins and α-glucosidase has not been investigated. In view of the multidimensional activity of this plant and putting the above explained observations together, the present study was undertaken to assess the molecular mechanisms involved in the antihyperglycemic action of Tamarind seeds. Thus, this study investigated the effect of HPLC standardized aqueous extract of Tamarind seeds (TSE) on following (1) gastric emptying and small intestinal transit rate (2) α-glucosidase inhibitory effect (3) serum adiponectin level and lipid profile (4) GLUT-2, GLUT-4 protein expression in the liver and skeletal muscle, respectively and (5) pancreatic GLP-1R mRNA concentration in an STZ model of type 2 diabetes in male rats.

Material and MethodsDrugs and Chemicals

The biochemical kits used in the experiment were α-glucosidase (Bioassay, USA), adiponectin (Mediagnost, Germany), HDL and LDL/VLDL Cholesterol quantification Kit (Catalog #K613-100; Biovision, USA), STZ (Sigma Chemicals U.S.A), metformin (Ranbaxy Ltd, Gudgaon, India), Taq DNA polymerase (Bioline Ltd, Catalog-A5209, 0200), horse radish peroxidase conjugated IgG (Jackson Immunoresearch Laboratories, USA) and anti-GLUT antibodies (Abcam, UK).Preparation of Aqueous Extract of T. Indica Seeds: Seeds of T. indica were collected from Kharibauli, New Delhi in the month of May 2011 and its authentication was done by Dr. Roshini Nayar, Scientist at the National Bureau of Plant Genetic Resources, New Delhi, India (Voucher No. NHCP/NBPGR/2010-52). Aqueous extract of the seeds of T. indica was prepared using the method described by National Institute of Health and Family Welfare, India [15]. Briefly, after incubation for 2 days at 40°C, the seeds of T. indica were powdered in a grinder. 100 g powder suspended in 500 mL redistilled water and the extraction was performed in Soxhlet apparatus for 18 h. A deep brown aqueous extract was obtained which was filtered using a coarse sieve filter paper. The filtrate was then dried under reduced pressure and finally lyophilized. Phytochemical screening and standardization of the extract was done before commencement of the in vivo study [14]. The aqueous extract yielded 2.8 g lyophilized powder (0.28%) from 1 kg of Tamarind seeds.Analytical High Performance Liquid Chromatography (HPLC)

Standard Solutions and Sample Preparation: Standard stock solutions were prepared by accurately weighing 4.8 mg of catechin and epicatechin each into separate 100 mL volumetric flasks and dissolved in 100 mL of 0.2% aqueous acetic acid with the aid of sonication. Working standard solutions, 2.4-19.2 μg/mL, were

prepared by dilution with water from the stock standard solutions. The calibration curve of Concentration vs. Area was plotted. The solutions were stored at 4°C. In this study, freeze-dried material (TSE 5 g) was extracted with petroleum ether in a Soxhlet apparatus (3 h) to remove the lipid content. After drying, the solids were extracted with methanol (3 × 3 h). The dry powder (0.1 g) of TSE was sonicated with 10 ml methanol for 30 min. The extracted solution was then centrifuged at 2500 rpm at room temperature for 10 min. The supernatant was filtered through a 0.45 µm membrane filter prior to HPLC analysis [16].HPLC Analysis: HPLC was carried out on a Hitachi liquid chromatograph model 665-II equipped with an Autosampler (Model 655A-40, Hitachi Ltd., Tokyo, Japan) fitted with a C-18 (250 × 4, 6.5 µ) column. For the separation of individual compounds, the mobile phase consisted of phosphoric acid dissolved in double distilled water (A) and acetonitrile (B) utilizing the following gradient: 95% A for 0.01 min, 70% A for 35 min, 70% A for 36 min, and 95% A for 40 min. The flow rate of the mobile phase in both cases (A and B) was 1.0 ml/min. The column temperature and injection volume was maintained at 30°C and 10 µl, respectively. The chromatogram was scanned up to 20 min, which was detected at 280 nm, followed by washing and reconditioning of the column. The analysis was done in triplicate.Animals

Male Wistar rats weighing between 150-200 g used in the study were obtained from the Animal House at the Delhi Institute of Pharmaceutical Sciences and Research, New Delhi, India. Rats were housed in colony cages (4 rats per cage), at an ambient temperature of 25°C with 12 h light: 12 h dark cycle. Rats had free access to standard food and water repetitive. The Principles of Laboratory Animal Care (NIH, 1985) were followed throughout the duration of experiment. All experimental procedures were conducted according to the Institutional Animal Ethical Committee (Protocol No. 2/DIPSAR/IAEC/2010) and CPCSEA guidelines.Experimental Design/ Animal Group

Induction of type 2 Diabetes: STZ, at a dose of 90 mg/kg body weight in 0.1M freshly prepared citrate buffer pH 4.5, was injected intraperitonially to 2 day old neonatal rats. The control rats were injected with equivalent volume of citrate buffer. After 6 weeks of injection, animals were evaluated for fasting blood glucose level. The fasting glucose level above 140 mg/dl was considered for the selection of diabetic rats [17]. A total of 40 male rats were selected and divided into five groups with eight rats per group: Group 1, normal untreated rats; Group 2, diabetic control rats; Group 3, diabetic rats treated with TSE extract (120 mg/kg); Group 4, diabetic rats treated with TSE (240 mg/kg); and Group 5, diabetic rats treated with metformin (100 mg/kg). After the treatment period of 4 weeks, blood samples were collected under fasting condition and processed for further analysis. Gastrointestinal Functions in Diabetic Rats: Either the vehicle or the test compound was administered to the diabetic rats that had been fasted overnight. 30 min later (compound treatment examination), glucose solution (0.2 g/mL glucose, 0.25% methylcellulose, 1 mg/mL phenol red and 10 mg/mL charcoal) was orally administered



at a volume of 15 mL/kg. Under ether anesthesia, the stomach was ligated and removed, after which it was transferred to a tube and cryopreserved. The entire length of the small intestine (between the pylorus of the stomach and the end of the ileum) and the distance to the charcoal front was measured. The rats in the control group were given vehicle solution in order to measure the total amount of glucose solution injected into the stomach. 15 min after administration, the pylorus of the stomach was ligated under ether anesthesia; after which the stomach was immediately removed, and small intestinal transit was checked. To measure the gastric emptying rate, 0.1 mol/L NaOH solution (5 mL) was added to the stomach sample and the mixture was homogenized. After centrifugation (3000 rpm, 10 min), 20% TCA solution (50 mL) was added to a 500-mL aliquot of the supernatant. The mixture was then stirred and centrifuged (15,000 rpm, 10 min). A 100 mL aliquot of the supernatant was then dispensed into a 96-well assay plate and to it, 0.5 mol/L NaOH solution (50 mL) was added. After stirring, phenol red concentration in the sample was determined using the phenol red (0-1000 mg/mL) calibration curve. The gastric emptying rate (%) was calculated using the following equation: [(Mean value of the control group) – (Mean value of the sample value)]/ (Mean value of the control group). The small intestinal transit rate (%) was calculated using the following equation: (Distance travelled by the charcoal front)/ (Entire length of the small intestine) [18].Measurement of α-glucosidase Inhibitory Activity (in vitro): The α-glucosidase inhibitory activity was determined according to Earnst et al. (2005)[19], by measuring the release of 4-nitrophenol from 4-nitrophenyl α-dglucopyranoside (4-NPGP). The assay procedure was according to the protocol of a micro-well kit. TSE samples were prepared at dilutions of 1, 10, 100, 1000, 10000 µg/mL concentrations in 50 mM phosphate buffer (pH 7). The assay media contained 200 µL (1 mM) α-NPG substrate, 20 µL samples and 200 µL of calibrator. Absorbance of the reactants was measured at 405 nm using a microplate reader (Model 550, BIO-RAD Lab, Japan). The rate of reaction was directly proportional to the enzyme activity.Determination of Biochemical Metabolic Parameters: The postprandial and fasting blood glucose was measured on day 0, 14, 21, and 28. After four weeks of administration of drugs (TSE, metformin), blood was collected from the hearts of animals by cardiac puncture prior to killing, placed in EDTA vacutainer tubes, and centrifuged at 4000 × g at 4°C for 15 min and stored at -80°C until analysis. The level of plasma insulin, serum HDL, LDL, and cholesterol were determined using enzyme linked immunosorbent assay (ELISA kit) on Day 28. The assay procedures for assessing the biochemical parameters were according to the Manufacturer’s protocol. The estimation of lipid profile was based on the principle that cholesterol oxidase specifically recognizes free cholesterol and produces products which react with probe to generate color (wavelength=570 nm). Whereas, in cholesterol assay cholesterol esterase hydrolyzes cholesteryl ester into free cholesterol, therefore, cholesterol ester and free cholesterol are detected separately in the presence and absence of cholesterol esterase in the reactions.The adiponectin determination was based on the use of specific high affinity monoclonal antibodies (Sandwich Assay) wherein

adiponectin, in the sample, binds to the first antibody coated on microwell plate. In subsequent steps, the second specific anti-adiponectin antibody binds in turn to the immobilized adiponectin. The color reaction of biotinylated conjugate was measured at 570 nm after 30 min of incubation.

Real-Time PCR Analysis of Pancreatic GLP-1: Real-time PCR amplifications for GLP-1(Gene ID: 25051) was conducted using Light-Cycler® 480 SYBR Green I Master (Roche) according to the manufacturer’s instructions. Reaction conditions were 10X PCR buffer [20 mM Tris (pH 8.4), 50 mM KCl and 2.5 mM MgCl2], 20 pM oligonucleotide, 300 μM dNTPs, 1:1000 SYBR Green I nucleic acid stain, 0.5 U Taq DNA polymerase (recombinant), and 50 ng templates cDNA in a 25 ml reaction volume. The forward primer was 5’-CAACCGGACCTTTGATGACTA-3’; the reverse primer was 5’- GCTGTGCAGAACCGGTACAC-3’. Thermal cycling conditions were 95ºC for a 3 min denaturation step, followed by 40 PCR cycles (94ºC for 30 s, 56ºC for 30 s and 72ºC for 1 min) and reactions were performed in a Light Cycler 480 (Roche) instrument. Fluorescence was detected at the end of the 56°C segment in the PCR step. PCR products of β-actin-F 5’-TCACCCACACTGTGCCCCATCTACGA-3’ and β-actin-R 5’ CAG CGGAACCGCTCATTGCCAATGG-3’ primers gene, were used as internal standards. All assays were carried out in triplicate. Real-time PCR analysis and subsequent calculations were performed on Light- Cycler® 480 software (Roche, version LCS480 1.2.0.169). GLUT-2 Expressions in Liver and GLUT-4 Expressions in Soleus Muscle: For determination of GLUT-2 protein expression in liver and GLUT-4 protein expression in skeletal muscle; each sample was mixed with 1% sodium dodecyl sulfate and 50 mM dithiothreitol, and the mixture was then subjected to electrophoresis using 10% polyacrylamide gel. The separated proteins on the gel were electrotransferred to a polyvinylidene difluoride membrane. After blocking overnight at 4°C with 5% skim milk solution including 0.05% poly (Oxyethylene) sorbitan monolaurate (Tween 20) the membrane was reacted with anti-GLUT-2 antibody and anti-GLUT-4 antibody for 2 h. Subsequently, it was incubated with horseradish peroxidase conjugated IgG (diluted 1: 2000) for 2 h at room temperature. The blots were detected using chemiluminescence reagents (Western Blot) [20].Statistical Analysis

All values are means ± S.E.M. Data analysis was done with one way analysis of variance (ANOVA) followed by Dunnet’s multiple test where diabetic group was considered as positive control (Graphpad Prism Software Inc, Version 5, San Diego, CA). Group means were considered to significantly differ at p<0 .05, as determined by Dunnet’s multiple range analysis.

ResultsAnalysis Of Catechin and Epicatechin In Tamarind Seeds (RP-HPLC)

The analytical reversed-phase HPLC of the extract of Tamarind seeds (Fig.1) revealed the presence of epicatechin and catechin.



The peaks for epicatechin and catechin, at retention time 20.58 and 16.35, were observed in the chromatogram of the extract along with other components. These peaks were corroborated with the peaks of epicatechin and catechin standards at 20.48 and 16.33, respectively. There was no interference in analysis from the other components present in the extract.Influence on Insulin, Postprandial and Fasting Hyperglycemia

The oral administration of TSE, for 4 weeks, to the diabetic rats resulted in a significant decrease (p<0.05) in postprandial and fasting blood glucose when compared with diabetic control group (Table 1). The plasma insulin level decreased significantly in the

diabetic rats in comparison to non-diabetic control. After 28 days of TSE supplementation to the diabetic rats, there was a significant increase in the insulin level in respect to the diabetic control group, in a dose dependent manner at both the doses of TSE (p<0.05, Table1).Kinetic Analysis of α-glucosidase Inhibition

The in vitro α-glucosidase inhibitory studies demonstrated that TSE had α-glucosidase inhibitory activity. The type of inhibition by TSE was determined using the Lineweaver-Burk plot analysis of the data which was calculated (Fig. 2) by Michaelis-Menten kinetics [21]. The percentage inhibition at 1, 10, 100, 1000, 10000 µg/ml concentrations of TSE showed a concentration-dependent reduction in α-glucosidase activity since percentage inhibition increased. The highest concentration, 10,000 µg/ml, showed maximum inhibition at nearly 42%. Apparent kinetic parameters (Km and Vmax) for α-glucosidase inhibitory activity were estimated by fitting apparent formation rates of the metabolite vs. TSE concentration in a single or two-site Michaelis-Menten enzyme

kinetic equation using nonlinear regression analysis software (Graphpad Prism Software Inc, Version 5, San Diego, CA).Influence on Gastrointestinal Functions

TSE showed a dose dependent prokinetic action on the gastric smooth muscles, as a significant increase was noted in the emptying rate (35.63%) at the dose of 240 mg/kg when compared to the non treated diabetic control (31.33%). Metformin showed inhibition of

Reversed phase high performance liquid chromatogram (RP-HPLC) of a) catechin b) epicatechin and c) TSE. TSE-Tamarind seed extract. The peaks for epicatechin and catechin, at retention time 20.58 and 16.35, were observed in the chromatogram of the extract along with other components.

Table 1: Effect of Tamarind seed extract on blood glucose and insulin

Fasting Blood Glucose (mg/dL) Postprandial Blood glucose (mg/dL) Insulin (µg/L)

Before treatment After treatment Before treatment After treatment

Control 87.62±2.94* 89.25±11.31* 115±8.70* 114±7.10* 2.33±0.14*

Diabetic 173.50±6.59 174.87±9.74 319±9.70 306±14.30 0.36±0.03

TSE 120 mg/kg 167.37±4.13* 140.12±10.90* 295±9.10* 235±12.70* 1.08±0.06

TSE 240 mg/kg 166.87±4.80* 130.00±8.70* 290±11.76* 215±16.70* 1.38±0.10*

Metformin 171.25±12.03* 113.50±7.10* 288±12.56* 189±7.50* 1.46±0.22*

a The values are the means ± S.E.M. from eight animals in each group One way ANOVA followed by Dunnnetts test *p < 0.05 vs. diabetic group. TSE- Tamarind seed extract.


In vitro dose dependent inhibition of α-glucosidase activity of Tamarind seeds. The inhibition activity of TSE were measured at concentration of 0, 1, 10, 100, 1000, 10,000 µg/mL. Fig. 1 (a) represent: time dependent % activity of α-glucosidase, where, positive values indicate enzyme-inhibitor (TSE) complex formation which leads to enzyme inhibition (negative values); (B) dose dependent % inhibition of α-glucosidase activity; (c) Lineaweaver burk plot-uncompetitive inhibition of α-glucosidase. TSE- Tamarind seed extract.

gastric emptying but these values were not statistically significant. TSE did not significantly influence the small intestinal transit rates at either dose levels considered under study (Table 2).

Influence on Plasma Adiponectin in Relation to Serum Lipid Profile Plasma adiponectin concentration was significantly higher in TSE treated group in a dose dependent manner compared to untreated diabetic group (p<0.05). The aqueous extract of Tamarind seeds at both the dose levels 120 and 240 mg/kg body weight decreased the elevated levels of serum LDL and cholesterol, significantly (p<0.05). Significant improvement was also noted in serum HDL level (Fig. 3).Influence on Pancreatic GLP-1 mRNA Concentrations, Liver GLUT-2 and Muscle GLUT-4 Expression

As shown in Fig. 4, amplification efficiency (E) for gene was determined by linear regression analysis of the fluorescent data from the exponential phase of PCR [22]. Quantitative RT-PCR results showed no significant changes in GLP-1 mRNA concentration after TSE treatment; however, metformin treatment resulted in moderate rise in concentration. Because of the physiological importance of insulin dependent GLUT-2 and GLUT-4 translocation to the cell membrane, the effect of TSE treatment on skeletal muscle tissue

Table 2: Effect of Tamarind seed extract on gastrointestinal functions in diabetic rats

Gastric emptying rate (%)

Intestinal transit rate (%)

Control 48.23±7.04* 52.27±6.91*

Diabetic 31.33±6.95 37.13±8.86

TSE 120 mg/kg 33.78±5.34 38.77±3.56

TSE 240 mg/kg 35.63±4.52* 41.02±5.35

Metformin 29.11±3.29 37.71±4.66a The values are the means ± S.E.M. from eight animals in each group. One way ANOVA followed by Dunnnetts test *p < 0.05 vs. diabetic group. TSE- Tamarind seed extract.




membrane GLUT-4 and liver GLUT-2 levels were observed. In the liver and muscle membrane fractions of diabetic rats, the translocation of GLUT-2 and GLUT-4 were very much reduced when compared with the band density of healthy controls.

DiscussionHPLC chromatogram of the aqueous extract of TSE was recorded in gradient conditions to identify the marker components in the extract. Herbal extracts are a mixture of many compounds and produce complex chromatograms. However, under the experimental conditions used in this work, no interference was observed from these constituents. The presence of catechin and epicatechin along with other chemical constituents has been reported in Tamarind seed pericarp [23] which is further validated in the present findings (Fig. 1). In general, the antihyperglycemic nature of TSE is supported by the fact that the polyphenols considered for their insulin mimetic action [24] present in the extract, exhibit antioxidant activity and radical scavenging ability.It has been reported that acute marked hyperglycemia caused by glucose-injection induced delayed gastric emptying in diabetic rats, healthy volunteers, and type 1 diabetic patient [25]. Therefore, the diabetic rats considered in the present study for assessing the gastrointestinal functions showed blood glucose levels of more than 300 mg/dL. Delayed gastric emptying affects the

postprandial glucose profile as it impairs the delivery of nutrients to the small intestine. Conversely, hyperglycemia tends to slow gastric emptying. In diabetic patients, there is evidence for both hyperglycemia and delayed gastric emptying and these two causes are linked by the likelihood that prolonged hyperglycemia causes autonomic neuropathy [26]. The STZ model of diabetes exhibit various stages of type 2 diabetes such as impaired glucose tolerance, mild to severe (based on dose) hyperglycemia, lowered plasma and pancreatic insulin. Also the β-cells in STZ rats bear resemblance to the insulin secretory characteristics found in type 2 diabetic human patients [27,28]. In the present study, STZ induced diabetic rats (mortality 30%) had mild gastroparesis with slow gastric emptying and intestinal transit rate in comparison to normal control rats, thus indicating that STZ induced diabetic rats could be used as the animal model for diabetic gastroparesis. These dysfunctions of gastrointestinal transit in diabetic animals were significantly improved by the administration of TSE whereas metformin reduced the gastric emptying rate. Metformin results are in accordance with the findings of Maida et al. (2011) [29], where it reduced the gastric emptying with improvement in GLP-1. Studies have revealed that, exogenous GLP-1 or GLP-1 derivatives cause a delay in gastric emptying and intestinal transit rates, which was considered to be partially responsible for the inhibition of postprandial hyperglycemia [30]. However, TSE was unable to

Effect of Tamarind seeds on serum lipid profile and adiponectin in diabetic rats. One way ANOVA followed by Dennett’s test *p<0.05 vs. Diabetic group. TSE-Tamarind seed extract. HDL-high density lipoproteins, LDL-low density lipoproteins.



ameliorate GLP-1 mRNA when compared to metformin and normal rats thus excluding the possibility of its antidiabetic action through GLP-1. The improvement in gastric emptying rate and small intestinal transit time may be a secondary response to the insulin mimetic action of TSE, as it has been reported that insulin increases the gastric emptying directly by its hypoglycaemic effect [31] and indirectly on stomach through the vagus nerve [32].Several clinical studies have demonstrated an association between hypoadiponectinemia and the development of insulin resistance and type 2 diabetes. Plasma adiponectin levels correlate positively with HDL cholesterol and negatively with triglyceride levels [33]. A decrease in adiponectin concentration may therefore result in an increased blood lipid concentration, as shown in our study (Fig. 2). TSE exhibited a significant hypolipidemic activity, decreasing total cholesterol, triglycerides and LDL cholesterol in serum with improved adiponectin level in the diabetic rats. The decrease in serum triglycerides and cholesterol may be associated with the epicatechin content of TSE [34]. The results obtained in this study are in accordance with those obtained by Martinello et al. (2006)[35], showing the hypolipidemic and antioxidant action of T. indica. In the current investigation, the improved level of adiponectin by

Effect of Tamarind seeds on (a) mRNA concentration of glucagon like peptide-1 in pancreas; (b) GLUT-4 expression in the muscles and (c) GLUT-2 expression in the liver of diabetic rats. (d) glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as a control and is shown on the bottom of each lane. Amplification efficiency (e) for gene was determined by linear regression analysis of the fluorescent data from the exponential phase of PCR. TSE- Tamarind seed extract.

TSE treatment in diabetes may focus light on the connecting link between TSE and adiponectin induced AMP activated protein kinase. Since, activation of protein kinase results in stimulation of glucose uptake in muscle and inhibition of hepatic glucose production, cholesterol and triglyceride synthesis [36]. Therefore, besides its profound insulin-sensitizing effects, this study support adiponectin’s role as a hypolipidemic agent.Maiti et al. (2004) [37] reported the antidiabetic potential of Tamarind seeds by targeting the liver and kidney as the site of action. Supplementation of TSE for 4 weeks restored the level of transaminases and liver glucose-6-phosphatase activity in conjunction with insulin. Furthermore, the antihyperglycemic activity of TSE was correlated with restoration of pancreatic β-cell mass in STZ diabetic rats for 8 weeks of treatment [38]. As the dysregulation of GLUT-2 and GLUT-4 controlling mechanism can result in the pathophysiologic states associated with diabetes and insulin resistance [39], we investigated this aspect of diabetes for antihyperglycemic action of T. indica. TSE treated diabetic rats improved liver and muscle glucose transporter expression thereby resulting in decrease in insulin resistance and increased the glucose uptake in muscle and liver.



In present study, the hypo-adiponectinemia in diabetic rats may partly explain the decreased phosphorylation of the 5’ adenosine monophosphate-activated protein kinase (AMPK-α Thr172) signalling pathway [40]. AMPK has been reported to be an important mediator of glucose metabolism and increases glucose transport by stimulating the translocation of GLUT-4. Our study shows that the protein expression of membrane GLUT-4 is decreased, indicating that glucose metabolism would be reduced in diabetic rat. Thus, it can be hypothesized that the decreased GLUT-4 may be due to the reduced phosphorylation of AMPK in the diabetes, which was ultimately improved by TSE treatment to diabetic rats.Mammalian intestinal α-glucosidase inhibitors have become exciting candidates to slow down the digestion of carbohydrates and in turn mitigate postprandial hyperglycaemia. It catalyzes the final step in the digestive process of carbohydrates. Studies have suggested that polyphenols such as flavonoid [41] and tannin [42] have potent α-glucosidase inhibitory activities contributing to the suppression of postprandial hyperglycaemia. These findings led us to substantiate the anti-hyperglycaemic action of Tamarind seeds, which have been reported to possess polyphenols and tannins [43] in significant amount, via glucosidase inhibition Its aqueous extract showed α-glucosidase inhibitory activity in vitro (Fig. 2). The present results revealed that TSE possessed α-glucosidase inhibition (42%) at the highest concentration (10,000 µg/mL) in dose dependent manner. These findings are complementary to the reports by Funk and Melzig (2005) [44], where different plant materials including Tamarind leaf extract showed strong α-amylase inhibitory activity. The in vivo anti-hyperglycaemic effect of TSE

therefore, may include its α-glucosidase inhibitory action in conjunction with other mechanisms described above.

ConclusionThe proposed mechanism of the anti-hyperglycaemic action of TSE has been diagrammatically represented in Fig. 5. Tamarind seeds exhibited strong anti-hyperglycaemic action which might comprise of an array of mechanisms like α-glucosidase inhibition, pro-adiponectin effect along with improved GLUT-2 and GLUT-4 expression in liver and skeletal muscle. Together, these effects resulted in an increase in the gastric emptying time. Thus, we can conclude that TSE along with its anti-diabetic action may be further be explored for the treatment of GI dysfunctions associated with diabetes. The findings reported in this study are new and are not related to known function of Tamarind seeds (e.g. antioxidant action). These findings show the scope for formulating a new herbal drug for diabetes therapy.

Conflict of InterestThe authors have declared that there is no conflict of interest.

AcknowledgmentsThe author acknowledges Ranbaxy, India for providing metformin and Shweta Sharma and Bhavna Dora for editing support. The funding of the study supported by Govt. of NCT, Delhi [MH-2203-O.E.1.1 (1)(1)(1)(4)], India.

Fig. 5: Digramatic representation of proposed mechanism of action of Tamarind seeds for antihyperglycemic activity.



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http://www.sciencedirect.com/science/article/pii/S0023643806001988






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