Study of the Hypoglycemic Effect of Tamarindus indica Linn. Seeds on Non-Diabetic and Diabetic Model Rats

Aim: The present study was undertaken to evaluate the antidiabetic effects of the Tamarindus indica Linn seed in normal (non-diabetic), type-I and type-II model rats and to investigate their effect on gastrointestinal motility and intestinal glucose absorption. Methodology: T. indica seed powder was used at a dose of 1.25g/kg bw/10 ml water. Male Long-Evans rats (160-210g body weight) were used for the experiment. Experiments were done in non-diabetic and streptozotocin-induced diabetic model rats with a single feeding in different prandial states and blood was collected. An intestinal perfusion technique was used to study the effects of T. indica seed powder on intestinal glucose absorption in normal and type-II model rats. Gut motility was evaluated using barium Research Article British Journal of Pharmaceutical Research, 3(4): 1094-1105, 2013 1095 sulfate milk. Glucose was measured by Glucose oxidase-peroxidase (GOD-POD) method. Result: The screening results showed that T. indica seed powder had no effect on fasting or postprandial serum glucose level of normal and type-I diabetic rat. The seed powder also showed no hypoglycemic effect in the fasting state and no antihyperglycemic effect in type-II model rats when fed simultaneously with oral glucose load, but it exhibited significant antihyperglycemic effect when the seed powder was fed 30 minutes prior to the glucose load at 105 minutes (p<0.03). Glibenclamide significantly lowered postprandial serum glucose levels of non-diabetic and type-II diabetic model rats (p<0.02-0.001). T. indica exerted inhibition on glucose absorption in type-II rats during the whole perfusion period when compared with control. On the other hand, T. indica seed powder significantly inhibited the gastrointestinal motility in type-II rats. Conclusion: The present data suggest that T. indica possesses antihyperglycemic properties in type-II rats which are at least partly due to its inhibitory effect on intestinal glucose absorption. This effect cannot be attributed to the acceleration of intestinal transit.


INTRODUCTION
Diabetes mellitus is a disease due to abnormality of carbohydrate metabolism and it is mainly linked with low blood insulin level or insensitivity of target organs to insulin. It is the most prevalent chronic disease in the world affecting a large population and its prevalence is about 6.8% [1].
Hyperglycemia and hyperlipidemia are two important characteristic of diabetes mellitus, an endocrine disorder based disease. In modern medicine, no satisfactory effective therapy is still available to cure diabetes mellitus [2]. Though pharmaceutical drugs like sulfonylureas and biguanides are used for the treatment of diabetes but these are either too expensive or have undesirable side effects or contraindications [3,4]. In recent years, there has been renewed interest in plant medicine [5,6,7] for the treatment against different diseases as herbal drugs are generally out of toxic effect [8,9] reported from research work conducted on experimental model animal. Although in human, whether there is any toxic effect are not investigated but anti-diabetic effect of various plants having "folk medicine reputation" have been screened [10].
Tamarindus indica Linn. (family-Caesalpiniaceae [Fabaceae]), locally known as Tetul tree, is found throughout the South Asian region and some portions of Africa. It is a large ample, evergreen tree, 12-18 m high with round bushy crown and comparatively smaller bole. Phytochemical investigation revealed the presence of many active constituents, such as phenolic compounds, cardiac glycosides, L-(-)mallic acid, tartaric acid, the mucilage and pectin, arabinose, xylose, galactose, glucose, and uronic acid [11,12]. The fruit pulp contains large quantities (16-18%) of tartaric, citric, malic and acetic acids, potassium tartrate, invert sugar, gum and pectin. It also contains traces of oxalic acid. Seed testa contains a fixed oil. Seeds cotyledons contain albuminoids, fat and carbohydrates [13,14]. Traditional healers claim that the seed of the plant possess antidiabetic properties. Scientific reports also support the hypoglycemic activity of this plant [15,16,17,18]. However, no published report supports the underlying mechanism of the hypoglycemic effect of T. indica seed powder in normal, STZ induced type-I and type-II diabetic model rats.
The purpose of this work was to explore the possible hypoglycemic / anti-hyperglycemic activity of T. indica seed extract in type-I and type-II diabetic rats as well as to investigate the possible mode of action beyond this activity.

Plant Materials and Preparation of Test Sample
Tamarindus indica Linn. seeds were collected from Kushtia, a district of Bangladesh. The plant was identified by the Bangladesh National Herbarium (voucher specimen no. DACB-38203). The seeds were collected from the fresh fruit pulp and were washed carefully and dried for two days at 40ºC in an oven. Then the seeds were crushed in an electric grinder to make fine powder. Afterwards the powder was stored immediately in the refrigerator at -20ºC and kept in the same temperature up to end of the experiment.

Experimental Animals
The study was conducted on adult male Long-Evans rats (weighing 160-210g) bred at the BIRDEM animal house maintained at a constant room temperature of 22±5 0 C, 40-70% humidity conditions and the natural day-night cycle with an ad libitum access to food except the day of experimental procedure when animals were used after 12hrs fasting. The rats had no access to food during the whole period of blood sampling. The influence of circadian rhythms was avoided by starting all experiments at 8.30 a.m.

Induction of Diabetes in Rats
Diabetes stimulating type-I was induced by a single intra-peritoneal injection of streptozotocin (STZ, Upjohn Company, Kalamazoo, MI USA) dissolved in 0.1 M citrate buffer, pH 4.5, at a dose of 65mg/kg body weight to adult rats [19]. On the 7 th day rats (fasting blood glucose ≥18mmol/l) were taken for carrying out the experiments.
Type-II diabetes was induced by a single intra-peritoneal injection of STZ at a dose of 90mg/ kg body weight, in citrate buffer to the 48 hours old pups as described by Bonner et al. [19]. Experiments were carried out 3 months after STZ injection an oral glucose (2.5g/kg bw) tolerance test was performed to check the blood glucose levels of the rats. Rats having blood glucose level 8-11 mmol/l at fasting condition were taken to carry out the experiments.
A total number of 250 rats were used to carry out the experiments, which include normal, type-I and type-II diabetic model rats. The animals were divided into 3 groups: Gr 1-water control group; Gr 2-glibenclamide treated positive control group in case of type-II diabetic model rats and insulin treated in case of type-I; Gr-3 T. indica seed powder treated group. Number of rats were 6-8 in each group.

Fasting condition
The powder (1.25g/kg bw/10ml) was fed to overnight fasting (12hrs) rats and blood samples were drawn at 0, 60 and 120 minutes [20]. The positive control group received glibenclamide (5mg/kgbw) for normal and type-II rats and insulin (10µl/rat) for type-I rats whereas the control group received only water (10ml/kg bw) [20]. Blood samples were collected by amputation of the tail tip under mild ethar anesthesia. The rats were kept unfed throughout the experimental period.

Postprandial condition
T. indica seed powder (1.25 g/kg bw), with or without glucose (2.5g/10ml/kg bw), were fed to overnight fasted (12hrs) rats and blood samples were drawn at 0, 30, and 75 min when fed simultaneously with glucose and at 0, 60 and 105 min when fed 30 min prior to glucose load. Both positive control and water control rats were fed with glucose solution (2.5g/10ml/kg bw) and water (10ml/kg bw) following glucose load [21].

Effect of T. indica Seed Powder on Intestinal Glucose Absorption
An intestinal perfusion technique [22] was used to study the effects of T. indica seed powder on intestinal absorption of glucose in nondiabetic and type 2 diabetic rats fasted for 36 hours and anesthetized with sodium pentobarbital (50 mg/kg). The seed powder were added to a kreb's solution (g/L 1.02 CaCl 2 , 7.37 NaCl, 0.20 KCl, 0.065NaH 2 PO 4 .6H 2 O, 0.6 NaHCO 3 , pH 7.4), supplemented with glucose (54.0 g/L) and perfused at a perfusion rate of 0.5 mL/min for 30 min through the duodenum. The perfusate was collected from a catheter set at 40 cm. T. indica seed powder were added to kreb's solution to a final conc. of 25 mg/mL so that the amount of seed powder solution in the perfused intestine was equivalent to the dose of 1.25 g/kg. The control group was perfused only with kreb's buffer supplemented with glucose. The results were expressed as percentage of absorbed glucose, calculated from the amount of glucose in solution before and after the perfusion.

Gastrointestinal (GI) Motility Test
Gastrointestinal motility was evaluated by using barium sulfate (BaSO 4 ) milk method [23]. The experiment was carried out by the method previously described by Chatterjee (1993). Distilled water at a dose of 10ml/kg bw was fed with smooth metallic tube to control rats and T. indica powder (1.25g/kg bw/ 10ml) was fed to treated group. Baso4 milk was prepared by adding Baso 4 as 10% w/v in 0.5% CMC suspension. The milk was given to rats after 1 hour of adminstration of the test material. The rats were sacrificed 15 minutes after the adminstration of the milk. Before sacrificing the rats were anesthetized with di-ethyl ether. Then the abdominal part was opened and the intestinal part from pylorous to the ileoceccal junction as a total length of GI was taken into a petridish filled with distilled water for washing. It was then soaked by tissue paper to make it dry and taken into a white paper marked with 100 cm scale for measurement. The total gastrointestinal tract was measured first. Then the length traversed by Baso 4 was measured (white color). This length traversed by Baso 4 was expressed as a percentage of the total length of small intestine and the result of the test group was compared with that of control group (Chatterjee1993).

Biochemical Analysis
Serum glucose was measured on the same day by glucose-oxidase-peroxidase method (GOD-POD) using a commercial kit (Boehringer-Mannhein, GmbH, Germany) (Sera Pak, USA).

Statistical Analysis
Data from the experiments were analyzed using the Statistical Package for Social Sciences (SPSS) software for Windows version 12 (SPSS Inc, Chicago Illinois, USA). Values were expressed as mean±SD (Standard Deviation), Analysis of variance (ANOVA, Bonferroni Post Hock Test) and independent sample 't'-test were done as the test of significance. p≤0.05 was considered as the minimal level of statistical significance.

Acute effect of Tamarindus Indica Seed Powder on Blood Glucose Level of Normal (Non-Diabetic), Type-I and Type-II Diabetic Model Rats
Blood glucose level was analyzed at the fasting level and the results showed that the seed powder had no significant effect on the fasting state of normal rats but lowered the serum glucose level 2.43% at 60 min and 7.68% at 120 min. Glibenclamide, lowered fasting serum glucose level significantly both at 60 minutes (p=0.000) and at 120 minutes (p=0.000) compared with water control and powder treated groups (Table 1) in normal rats. Glibenclamide also lowered serum glucose level significantly at 120 min in type-II rats (p=0.001). The oral glucose tolerance test (OGTT) was performed and the results showed that powder had glucose lowering effect but non-significantly and the glibenclamide treated group showed a significant fall in serum glucose level at 75 minutes in normal and type-II rats (p=0.000 and p=0.02) respectively (Table 2). When fed 30 min before to glucose load, it was found that glibenclamide treated group showed significant blood glucose lowering effect at 60 min (p = 0.001) and 105 min (p = 0.000) in normal rats compared to water control and powder treated group (Table 3). T. indica seed power showed significant (p=0.03) blood glucose lowering effect at 105 min in type-II diabetic model rats in comparison to water control and glibenclamide treated group (Table 3; Fig. 1). In type-II rats sum of the increments over the basal value for powder treated group was also found to be decreased significantly (p=0.03).

Effect of Tamarindus Indica Seed Powder on Gastrointestinal Motility
The length of GI traversed by BaSO 4 milk in powder treated group was lower than the water treated group. The inhibition in motility was not statistically significant in treated group of normal rats (Table 4).There was decreased percentage of length traversed by BaSO4 with seed powder on type-II model rats in comparison to water control group. T. indica seed powder showed significant inhibitory effect on gastrointestinal motility (p=0.02).

Effect of T. indica Seed Powder on Upper Intestinal Glucose Absorption
It is evident that the upper intestinal glucose absorption was almost constant during 30 minutes of perfusion with glucose. When the seed powder solution was supplemented with the glucose solution, it showed no effect on intestinal glucose absorption in normal rats (Fig. 2).

Fig. 2. Effect of the T. indica seed powder on upper intestinal glucose absorption on normal rats
Results are presented as mean± SD (n=5). Rats were fasted for 36 hours and intestine was perfused with glucose solution (54 g/l) with or without T. Indica seed powder (25mg/ml); glu= glucose; K-g= Kreb's solution + glucose; K-g-ext= Kreb's solution + glucose + seed powder solution.
In case of type-II model rats, intestinal glucose absorption was nearly constant during the 30 min of perfusion with glucose. There was a decrease in glucose absorption with glucose solution when supplemented with seed powder solution (Fig. 3).

Fig. 3. Effect of T. indica seed powder on upper intestinal glucose absorption on type II diabetic rats
Results are presented as mean± SD (n=6). Rats were fasted for 36 hours and intestine was perfused with glucose solution (54 g/l) with or without T. Indica seed powder (25mg/ml); glu= glucose; K-g= Kreb's solution +glucose; K-g-ext= Kreb's solution + glucose + seed powder solution It denotes that T. indica seed powder strongly affected the amount of absorbed glucose throughout the notable period of experiment in type-II rats. Fig. 3 depicts the gradual fall in glucose absorption during the whole perfusion period in type-II rats compared to Krebs solution. Therefore, the obtained results suggest that seed powder of the T. indica delays glucose absorption in the upper part of the gastrointestinal tract.

DISCUSSION
Synthetic drugs such as Sulphonylureas and Biguanides are valuable in the treatment of DM, but their uses are restricted by their limited action, pharmacokinetic properties, secondary failure rates and accompanying side effects [24]. Moreover, these therapies only partially compensate for metabolic derangements seen in diabetes and do not necessarily correct the fundamental biochemical lesion [25]. As the incidence of diabetes is rising relatively around the world, there is an urgent need to expand the range of effective palliatives available to patients.
The present study has been undertaken to screen the hypoglycemic and anti-hyperglycemic activity of T. indica seed powder in nondiabetic, type-I and type-II diabetic model rats. The experimental approach that has been followed, in addition to screening the hypo-/antihyperglycemic activity, gives an approximate idea about the mechanism of action of the plant by analyzing the model, prandial states and timing of hypoglycemic activity. Moreover, the study was also extended to explore the possible mechanism of action by elucidating the effect of the plant on gut motility and intestinal glucose absorption.
Our results demonstrate that T. indica seed powder had no effect in the fasting state of nondiabetic, type-I or type-II rats. At the post prandial state when the seed powder was administered simultaneously with oral glucose load, no significant reduction was noticed with a single feeding in any group of rats. On the contrary, when T. indica seed powder was administered half an hour before oral glucose load in type-II rats, the seed powder caused a significant attenuation in the rise of blood glucose at 105 minutes compared to the control groups (glucose M±SD, mmol/l. 14.65±2.82 in the treated group Vs 19.30±4.27 in the control group, p<0.03). The antihyperglycemic effect of T. indica seed in STZ induced diabetic rats have been found by other investigators [17].
T. indica seed powder was effective in type-II diabetic model rats when fed 30 minutes before glucose load. This effect might be due to a systemic action, i.e. as a result of the stimulation of Beta cells and improving the insulin secretory capacity or enhancement of insulin action by the extract. This effect could not be confirmed by our study since serum insulin level after a single feeding was not determined. It has been claimed that the chronic aqueous extract of T indica improves glycemic status [17,26].
The inhibition of intestinal glucose absorption may contribute to the reduction of postprandial glucose level. One of the objectives of the present study was to investigate whether the hypoglycemic effect is related to the inhibition of glucose absorption in the gut. Since the antihyperglycemic effect of T. indica was found in type II rats, therefore, this gut perfusion experiment was investigated in normal and type-II rats, where T. indica seed powder showed strong inhibition of glucose absorption. This result strongly suggests that the antihyperglycemic effect of T. indica as previously reported [26,27] may be due to, at least in part to the retardation of glucose absorption in the small intestine. T. indica is rich in pectin's and dietary fibers such as cellulose, xylose and mucilage [16] and the presence of such substances in the powder of T. indica may be responsible for the observed effect. Similar results have been reported by some other scientists [16,27]. Moreover, T. indica seed powder also inhibited the BaSO 4 induced gastrointestinal motility in Type-II rats. This result suggests that the decrease of glucose absorption by T. indica seed powder is not achieved by an enhanced intestinal motility. It is now well established that diabetes mellitus is not a single desease entity, but a heterogenous group of disorders with a striking diversity of etiopathogenetic mechanisms as well as clinical manifestations [28]. It is also established that the basic pathophysiology of Type-I and Type-II diabetes is quite different. In type-II diabetes, there are multiple abnormalities in diverse tissues. So, a plant material can show glucose lowering effect in diverse way. It may not be active in type-I diabetes, but may be active in type-II diabetes, which we found in our study.

CONCLUSION
Based on the results of this study, it may be concluded that T. indica seed powder possesses significant antihyperglycemic activity in type II diabetic model rats but not in type I and this is partly due to inhibition of intestinal glucose absorption.

CONSENT
Not applicable.

ETHICAL APPROVAL
All authors hereby declare that all experiments have been examined and approved by the appropriate ethics committee and have therefore been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki.