INHIBITORY EFFECT OF GYMNEMA SYLVESTRE, STEVIA REBAUDIANA, PHYLLANTHUS EMBLICA AND SYZYGIUM CUMINI ON PORCINE PANCREATIC AMYLASE

Abstract

Diabetes has become a common global health problem that affects >170 million people worldwide. It is one of the leading causes of death and disability. It is estimated that by 2030, the number will rise to 366 million. The majority of diabetes is type 2 diabetes (T2D) caused by a combination of impaired insulin secretion from pancreatic beta cells and insulin resistance of the peripheral target tissues, especially muscle and liver. The 'top' three countries in terms of the number of T2D individuals with diabetes are India, China; and the US. Clearly, T2D has become an epidemic in the 21st century where India leads the world with the largest number of diabetic subjects. In this study, the inhibitory effect of Syzygium cumini, Gymnema sylvestre, Stevia rebaudiana and Phyllanthus emblica extract on porcine pancreatic amylase. The most significant inhibitory activity was obtained with the aqueous and ethanolic extracts of Gymnema sylvestre and Syzygium cumini.

Headnote

ABSTRACT

Diabetes has become a common global health problem that affects >170 million people worldwide. It is one of the leading causes of death and disability. It is estimated that by 2030, the number will rise to 366 million (www.who.int). The majority of diabetes (~90%) is type 2 diabetes (T2D) caused by a combination of impaired insulin secretion from pancreatic beta cells and insulin resistance of the peripheral target tissues, especially muscle and liver. The 'top' three countries in terms of the number of T2D individuals with diabetes are India (31.7 million in 2000; 79.4 million in 2030), China (20.8 million in 2000; 42.3 million in 2030); and the US (17.7 million in 2000; 30.3 million in 2030). Clearly, T2D has become an epidemic in the 21st century where India leads the world with the largest number of diabetic subjects. Traditional Medicines obtained from medicinal plants are used by about 40-60% of the world's population. Though there are many approaches to control diabetes and its secondary complications, herbal formulations are preferred due to lesser side effects and low cost. In this study,the inhibitory effect of Syzygium cumini, Gymnema sylvestre, Stevia rebaudiana and Phyllanthus emblica extract on porcine pancreatic amylase.The most significant inhibitory activity was obtained with the aqueous and ethanolic extracts of G. sylvestre and S. cumini.

KEY WORDS: Amylase, Diabetes, Inhibition, Syzygium cumini, Gymnema sylvestre, Stevia rebaudiana, Phyllanthus emblica, Porcine Pancreatic amylase

INTRODUCTION

Type 2 diabetes is a major public health issue in India and South Asians, when compared to Caucasians (IDF) and are more likely to develop Diabetes at younger ages (Banerji, 1999; McKeigue et al., 1991) and at lower body mass indices (McKeigue, 1991; Ramachandran et al., 2001). India has the highest number of diabetes cases worldwide (40 million) (Mohan V et al., 2006). Another 30 million Indians have pre-diabetes and are at high risk of developing T2DM.5 T2DM is an economically costly disease (Yusuf, et al., 2004) and a major cause of mortality and morbidity. Indians and other South Asians with diabetes have worse glycemic control, (chandelier, 2007; Ramachandran, et al., 2006) a higher prevalence of microalbuminuria, (Pan et al., 1997) hypertension, retinopathy, and cardiovascular disease, (Lindstrom et al., 2003) and a higher incidence and faster progression of renal disease than most other diabetic populations. (Ramachandran, et al., 2007) unexplained weight loss. The prevalence of T2DM risk factors, such as insulin resistance, increased fat mass, and central obesity are higher in South Asian populations. (Knowler, 2002; Eriksson, 1991; Narayan KM, et al., 2004).T2DM prevention is a priority for South Asian populations.

Diabetes is a chronic disorder of carbohydrate, fat and protein metabolism characterized by increased fasting and postprandial blood sugar levels. The global prevalence of diabetes is estimated to increase by 5.4% by the year 2025. WHO has predicted that the major burden will occur in developing countries. Studies conducted in India in the last decade have highlighted that not only is the prevalence of diabetes high but also that it is increasing rapidly in the urban population (Alberti KG, et al., 2007). Type I diabetes (insulin dependent) is caused due to insulin insufficiency because of lack of functional beta cells. Though patho physiology of diabetes remains to be fully understood, experimental evidences suggest the involvement of free radicals in the pathogenesis of diabetes and more importantly in the development of diabetic complications (Mather, 1998; Mukhopadhyay,2006; Snehalatha et al., 2003). Free radicals are capable of damaging cellular molecules, DNA, proteins and lipids leading to altered cellular functions. Many recent studies reveal that antioxidants capable of neutralizing free radicals are effective in preventing experimentally induced diabetes in animal models (WHO, Bjork, et al., 2003) as well as reducing the severity of diabetic complications (Mather, et al., 1998). For the development of diabetic complications, the abnormalities produced in lipids and proteins are the major etiologic factors. In diabetic patients, extra- cellular and long lived proteins, such as elastin, laminin, collagen are the major targets of free radicals. These proteins are modified to form glycoproteins due to hyperglycemia. (Dixon, et al., 2006). During diabetes, lipoproteins are oxidized by free radicals. There are also multiple abnormalities of lipoprotein metabolism in very low density lipoprotein (VLDL), low density lipoprotein (LDL), and high density lipoprotein (HDL) in diabetes. Lipid peroxidation is enhanced due to increased oxidative stress in diabetic condition. Apart from this, advanced glycation end products (AGEs) are formed by non-enzymatic glycosylation of proteins. AGEs tend to accumulate on long-lived molecules in tissues and generate abnormalities in cell and tissue functions (Chandie, 2006; Retnakaran, et al., 2006). To manage post-prandial hyper- glycaemia at digestive level, glucosidase inhibitors such as Acarbose, miglitol and voglibose are used. Although several therapies are in use for treatment, there are certain limitations due to high cost and side effects such as development of hypoglycemia, weight gain, gastrointestinal disturbances, liver toxicity etc (Raji, et al., 2001). Based on recent advances and the involvement of oxidative stress in complicating diabetes mellitus, efforts are on to find suitable antidiabetic and antioxidant therapy.

To date, over 400 traditional plant treatments for diabetes have been reported, although only a small number of these have received scientific and medical evaluation to assess their efficacy. The hypoglycemic effect of some herbal extracts has been confirmed in human and animal models of type 2 diabetes. The World Health Organization Expert Committee on diabetes has recommended that traditional medicinal herbs be further investigated. (Lucy Dey et al., 2002). Major hindrance in amalgamation of herbal medicine in modern medical practices is the lack of scientific and clinical data proving their efficacy and safety. There is a need for conducting clinical research in herbal drugs, developing simple bioassays for biological standardization, pharmacological and toxicological evaluation, and developing various animal models for toxicity and safety evaluation. It is also important to establish the active component/s from these plant extracts.

It has been shown that the activity of the HPA (human pancreatic a-amylase) in the small intestine correlates to an increase in post- prandial glucose levels, the control of which is therefore an important aspect in the treatment of type 2 diabetes. Herbal medicines are getting more importance in the treatment of diabetes as they are free from side effects and less expensive when compared to synthetic hypoglycemic agents.

In India, indigenous herbal remedies such as Ayurveda and other Indian traditional medicine have, since ancient times used plants in treatment of diabetes Ethnobotanical studies of traditional herbal remedies used for diabetes have identified more than 1,200 species of plants with hypoglycemic activity. A number of medicinal plants and their formulations are used for treating diabetes in the traditional Indian Ayurvedic system as well as in ethnomedicinal practices. WHO (World Health Organization) (1980) has recommended the evaluation and mechanistic properties of the plants effective in such systems. Pharmacological properties a-glucosidase inhibitors such as acarbose that can also inhibit pancreatic a-amylase revealed that the complications of DM such as onset of renal, retinal, lens and neurological changes and the development of ischaemic myocardial lesions are prevented or delayed. Long-term day-to- day management of diabetes, with acarbose is well tolerated and can improve glycaemic control as monotherapy, as well as in combination therapy Gymnema sylvestre, Stevia rebaudiana, Phyllanthus emblica and syzygium cumini are well known in Ayurveda to possess anti-diabetic properties. Structurally as well as mechanistically, PPA (Porcine pancreatic a-amylase) is closely related to the HPA (Human pancreatic a-amylase). Hence, sequential solvent extracts of the above mentioned plants were screened for the presence of PPA inhibitors, Pancreatic a- amylase (E.C. 3.2.1.1), is a key enzyme in the digestive system and catalyses the initial step in hydrolysis of starch to maltose and finally to glucose. Degradation of this dietary starch proceeds rapidly and leads to elevated post prandial hyperglycemia (PPHG). It has been shown that activity of Human Pancreatic a- amylase (HPA) in the small intestine correlates to an increase in post-prandial glucose levels, the control of which is therefore an important aspect of treatment of diabetes .Hence retardation of starch digestion by inhibition of enzymes such as a-amylase would play a key role in the control of diabetes. However, the discovery of specific high-affinity inhibitors of pancreatic a-amylase for the development of therapeutics has remained elusive. Inhibitors currently in clinical, as for example, acarbose, miglitol, and voglibose, are known to inhibit a wide range of glycosidases such as a- glucosidase and a-amylase. Because of their nonspecificity in targeting different glycosidases, these hypoglycemic agents have their limitations and are known to produce serious side effects (Sebedio, et al., 1989). An effort has been made to study the inhibitory effect of the above said medicinal plants extract on PPA.

MATERIALS AND METHODS

Starch, porcine pancreatic a-amylase (PPA), methanol, isopropanol, acetone, methyl- butyl-tertiary ether, cyclohexane, and dimethylsulfoxide (DMSO) were purchased from SRL Pvt. Ltd, Mumbai, India. 3,5- dinitrosalicylicacid (DNSA) was obtained from HiMedia Laboratories, Mumbai, India. Human pancreatic a-amylase (HPA) and acarbose were purchased from Sigma Aldrich, USA. All other chemicals procured were of AR grade.

Preparation of Plant Extracts

The air-dried plant material (60-100 g) was crushed with liquid nitrogen, powdered, and successively extracted in polar to nonpolar solvent on an increasing degree of non- polarity.The different extracts obtained sequentially were with cold water, hot water, methanol, isopropanol, acetone, methyl-butyl- tertiary ether, and cyclohexane. This kind of sequential extraction was performed taking into consideration the fact that traditional methods of preparing herbal formulations are mainly aqueous. Also, aqueous extracts contain peptides, proteins, or glycans, which would otherwise be denatured by organic solvents and high-temperature extraction. Distilled water was added to the crushed material in a ratio of 1: 4 (w/v) and kept at 30°C (24 h) and 55°C (2 h) at 130RPM for cold-and hot-water extracts, respectively. For each solvent, the extract was filtered, centrifuged, and the residue collected for subsequent solvent extraction. The organic solvents were added in a ratio of 1:3 (w/v) and refluxed with the residue for 3 h at their respective boiling temperatures. Each extract was filtered and stored at -20°C

«-Amylase Inhibition

PPA was used for preliminary screening of a-amylase inhibitors from the extracts. The inhibition assay was performed using the chromogenic DNSA method (28). The total assay mixture composed of 500//L of 0.02 M sodium phosphate buffer (pH 6.9 containing 6 mM sodium chloride), 0.04 units of PPA solution, and extracts at concentration from 0.1-1.5 mgmL 1 (w/v) were incubated at 37°C for 10 min. After pre-incubation, 500//L of 1% (v/v) starch solution in the above buffer was added to each tube and incubated at 37°C for 15 min. The reaction was terminated with 1.0 mL DNSA reagent, placed in boiling water bath for 5 min, cooled to room temperature, diluted, and the absorbance measured at 540 nm. The control reaction representing 100% enzyme activity did not contain any plant extract. To eliminate the absorbance produced by plant extract, appropriate extract controls were also included. One unit of enzyme activity is defined as the amount of enzyme required to release one micromole of maltose from starch per min under the assay conditions.

For the determination of the inhibitor concentration at which 50% inhibition of enzyme activity occurs (IC50), the assay was performed as above except that the inhibitor/plant extract concentrations were varied from 0.1-150//g. Acarbose was used as a positive control in a concentration range of 6.5/^-32.8/^. The IC50 values were determined from plots of percent inhibition versus log inhibitor concentration and calculated by logarithmic regression analysis from the mean inhibitory values. The IC50 values were defined as the concentration of the extract, containing the a-amylase inhibitor that inhibited 50% of the PPA.

% Relative enzyme activity = (enzyme activity of test/enzyme activity of control) *100;

% inhibition in the a-amylase activity = (100 - % relative enzyme activity).

RESULTS

Screening of Plant Extracts for «-Amylase Inhibition

The plants from the Indian subcontinent exhibiting potential hypoglycemic properties were sequentially extracted with polar to non- polar solvents. It should be noted that while generally cold-and hot-water extracts are most commonly used in the traditional method of preparing medicines in Ayurveda, the chances of missing out on bioactive principles with better amylase inhibitory potential from less- polar solvents are high. Hence the rationale for performing extractions from polar to non-polar solvents is not only to confirm and validate the inhibitory activity, if found, in any of the aqueous extractions performed in the traditional manner but also to search for newer and higher specific affinity inhibitors in less-polar solvents. PPA was used as a target enzyme for screening of inhibitory activity from the above mentioned plant extracts. The control reaction representing 100% enzyme activity was 0.20 U/mL for PPA. Extract samples dissolved in DMSO contained as a final yield of 1.5 mg mL 1 of the dried extract and the enzyme activity of PPA was not affected by DMSO at the concentration used. The appropriately diluted plant extract was used for enzyme inhibition assay and the activity obtained with each extract was normalized to percent relative activity (figure-1) from which the percent inhibition was calculated.. The most significant inhibitory activity was obtained with the aqueous and ethanolic extracts of, Gymnema sylvestre and S. cumini the known PPA inhibitor, acarbose, taken as a positive control exhibited an IC50 value of 82.78/zgml (Table- !)*

DISCUSSION

The aim of the present study was to investigate the PPA inhibitory activity of medicinal plants known in the Indian Ayurvedic system for their anti-diabetic properties. To date no reports of compounds responsible for PPA inhibition from these plants exist in the literature and we report here for the first time their inhibitory activity on PPA. These plants chosen in this study are used by the Indian population not only for food purposes but also form a part of the local pharmacopoeia for the treatment of diabetes. Our results indicate that retardation of starch hydrolysis by inhibition of PPA activity of some of these extracts leads to a reduction in glucose concentrations,Of the 4 plants and their extracts tested, Gymnema exhibited significant inhibition of PPA, suggesting that they contain compounds capable of PPA inhibition. Preliminary phytochemical analysis to indicate the kind of compounds present in these extracts suggests the occurrence of proteins/peptides and polyphenols in cold-and hot-water extracts while the tannins, alkaloids, flavonoids, and saponins are found in non-polar extracts. Flavonoids and polyphenolics may be responsible for hypoglycemic activity.

CONCLUSION

Plants chosen in this study are used by the Indian population not only for food purposes but also form a part of the local pharmacopoeia for the treatment of diabetes. Our results indicate that retardation of starch hydrolysis by inhibition of PPA activity of some of these extracts leads to a reduction in glucose concentrations. Of the 4 plants and their extracts tested, Gymnema exhibited significant inhibition of PPA, suggesting that they contain compounds capable of PPA inhibition. Preliminary phytochemical analysis to indicate the kind of compounds present in these extracts suggests the occurrence of proteins/peptides and polyphenols in cold-and hot-water extracts while the tannins, alkaloids, flavonoids, and saponins are found in non-polar extracts. Flavonoids and polyphenolics may be responsible for hypoglycemic activity.

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Cite this article:

Dayananda. K. S., Gopinath. S. M., Murthy. R. K., (2013), INHIBITORY EFFECT OF GYMNEMA SYLVESTRE, STEVIA REBAUDIANA, PHYLLANTHUS EMBLICA AND SYZYGIUM CUMINI ON PORCINE PANCREATIC AMYLASE, Global J Res. Med. Plants & Indieen. Med., Volume 2(8): 554-561

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