Inhibitory effect of Gymnema Montanum leaves on α-glucosidase activity and α-amylase activity and their relationship with polyphenolic content
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- Ramkumar, K.M., Thayumanavan, B., Palvannan, T. et al. Med Chem Res (2010) 19: 948. doi:10.1007/s00044-009-9241-5
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The present study was attempted to investigate the effect of G. montanum leaf extract on inhibition of α-glucosidase and α-amylase activity. The ethanol extract of G. montanum (GLEt) at various concentrations (1–10 μg/ml) was tested for its inhibition pattern against α-glucosidase and α-amylase activity in vitro and compared with the commercially available α-glucosidase inhibitor, acarbose. The GLEt showed competitive inhibition against yeast α-glucosidase and noncompetitive inhibition against salivary α-amylase in a concentration-dependent manner. Further, we investigated the effect of extract on levels of blood glucose and plasma insulin in neonatal streptozotocin-induced type 2 diabetic rats. Long-term administration (12 weeks) of the GLEt effectively reduced the blood glucose level and also increased the insulin level. In a preliminary phytochemical analysis, G. montanum leaves were rich in phenolic composition and found to be positively correlated with the inhibitory effect of α-glucosidase and α-amylase activity. These results suggest that the GLEt might exert its antidiabetic effect by suppressing carbohydrate absorption from the intestine and thereby reducing hyperglycemia.
Keywordsα-Glucosidase inhibitorα-Amylase inhibitorGymnema montanumType 2 diabetesnSTZ ratsMedicinal plants
Hyperglycemia has been a classical risk factor in the development of diabetes and its complications associated with diabetes. Therefore control of blood glucose levels is critical in the early treatment of diabetes mellitus and reduction of macro- and microvascular complications. One therapeutic approach is the prevention of carbohydrate absorption after food intake, which is facilitated by inhibition of the enteric enzymes including α-glucosidase and α-amylase present in the brush borders of intestine (Toeller, 1994; Inzucchi, 2002). The inhibition of both these enzymes has been a strong option in the prevention of diabetes. Nowadays, inhibitors like acarbose, voglibose, and miglitol are widely used in type 2 diabetic patients. However, it is well documented that synthetic inhibitors have undesirable side effects such as diarrhea and abdominal cramping (Chakrabarti and Rajagopalan, 2002). Nowadays several glucosidase inhibitors have been developed from natural sources, especially from plants (Matsui et al., 2006; Bhat et al., 2008; Kumarappan and Mandal 2008).
In the traditional system of Indian medicine, Gymnema sp. is used for diabetes treatment, as a diuretic, and as a digestive stimulant (Chattopadhyay, 1998). Several researchers have reported the antidiabetic properties of a few Gymnema species such as G.sylvestre, G. inodorum, and G. yunnanense (Persaud et al., 1999; Shimizu et al., 2001; Xie et al., 2003). Gymnema montanum Hook, an Asclepiadaceae member, is an endemic plant species found mainly in Western Ghats, India. We have previously demonstrated that the extract from G. montanum leaves improves hyperglycemia in alloxan-induced diabetic rats (Ananthan et al., 2003a; Ramkumar et al., 2007a, b; 2008a). In addition, we have reported the antihyperlipidemic property of G. montanum leaves on alloxan-induced diabetic rats (Ananthan et al., 2003b; Ramkumar et al., 2008b). It has also been proved to prevent the cholinergic neural and retinal complications of hyperglycemia in diabetes (Ramkumar et al., 2005). However, the mechanism of the antidiabetic effect of G. montanum is not yet understood. Our preliminary phytochemical testing of G. montanum leaves indicated the presence of phenolics, flavonoids, and alkaloids in the extract (Ramkumar et al., 2007c).
In this study, we investigated the inhibitory effect of the ethanol extract of G. montanum (GLEt) on α-glucosidase and α-amylase activity and compared it with the commercially available α-glucosidase inhibitor, acarbose. We also investigated the effect of extract on levels of blood glucose and plasma insulin in neonatal streptozotocin (nSTZ)-induced type 2 diabetic rats. Further, we studied the correlation of the inhibitory effect on α-glucosidase and α-amylase with the polyphenolic composition of the extract.
Rationale and design
Salivary α-amylase, α-glucosidase from yeast Saccharomyces cerevisiae, p-nitrophenyl-α-d-glucopyranoside, and other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO, USA).
Plant material and preparation of the extract
Fresh leaves of G. montanum Hook were collected from the Shola forests of the Nilgiri Biosphere Reserve, Western Ghats, Gudalur, at an altitude of 900–1500 MASL. The plant was identified by the Herbarium of Botanical Survey of India, Southern Circle, Coimbatore (Accession No. 32561-65), and was deposited at the Department of Biotechnology, Anna University Tiruchirappalli. Fresh leaves of G. montanum (500 g) were chopped into small pieces and soaked overnight in 1.5 l of 95% ethanol. This suspension was filtered and the residue was resuspended for 48 h in an equal volume of 95% ethanol and filtered again. The two filtrates were pooled and the solvents were evaporated in a rotavapor at 40–50°C under reduced pressure and then lyophilized. The greenish-black powder obtained (20–30 g) was stored at 0–4°C until use. When needed, the extract residue was suspended in distilled water and used in this study.
Inhibition assay for α-amylase activity
α-Amylase was assayed with starch (0.5%) as substrate in 0.1 M sodium phosphate buffer (pH 7.0). The GLEt at various concentrations (1–10 μg/ml) was premixed with the substrate in phosphate buffer to start the reaction. The reaction was carried out at 37°C for 5 min and terminated by the addition of 2 ml of DNS reagent (1% 3,5-dinitrosalicylic acid and 12% sodium potassium tartrate in 0.4 M NaOH). The reaction mixture was heated for 15 min at 100°C and then diluted with 10 ml of distilled water in an ice bath. α-Amylase activity was determined by measuring absorbance at 540 nm (Maeda et al., 1985).
Inhibition assay for α-glucosidase activity
α-Glucosidase was premixed with the GLEt at various concentrations (1–10 μg/ml) and substrate, p-nitrophenyl-α-d-glucopyranoside (3 mM), in phosphate buffer to start the reaction. The reaction mixture was incubated at 37°C for 30 min and the reaction was stopped by adding 2 ml of 0.1 M Na2CO3. α-Glucosidase activity was determined by measuring release of p-nitrophenol from p-nitrophenyl-α-d-glucopyranoside at 410 nm (Bergmeyer and Bernt, 1974).
Kinetics of inhibition against α-amylase and α-glucosidase
Inhibition modes of the GLEt against α-glucosidase and α-amylase activities were measured with increasing concentrations of p-nitrophenyl-α-d-glucopyranoside or starch as a substrate respectively in the absence or presence of the GLEt at different concentrations. Inhibition type was determined by Lineweaver–Burk plot analysis of the data, which were calculated from the result according to Michaelis–Menten kinetics.
Healthy albino Wistar strain rats kept for breeding in the Central Animal House, Rajah Muthiah Medical College, Annamalai University, were used in this study. The rats were fed on pellet diet (Hindustan Lever Ltd., Mumbai, India) and water ad libitum. Rats used in the present study were maintained in accordance with the guidelines of the National Institute of Nutrition, Indian Council of Medical Research, Hyderabad, and approved by the ethical committee at Bharathidasan University, Tiruchirappalli, Tamil Nadu.
Experimental induction of type 2 diabetes (NIDDM) in rats
The model was developed according to the description of Bonner Weir et al. (1981). Male albino Wistar rats, aged 48 ± 2 h, were injected intraperitoneally with streptozotocin in citrate buffer (pH 4.5) at a dose of 100 mg/kg body weight. After 12 weeks, only rats weighing >150 g were selected for screening in the NIDDM model.
A total of 30 rats (20 diabetic rats and 10 normal rats) were divided into three groups of 10 animals each. A group of 10 normal rats was maintained without any treatment as normal control. Two groups of 10 diabetic rats were treated with either saline (vehicle control) or the GLEt at a dose of 200 mg/kg body weight. The plant extract was given in aqueous solution daily using an intragastric tube for 12 weeks. Fasting blood glucose was monitored every 7 days throughout the experiment. No detectable adverse effects were observed in any of the animals after treatment.
At the end of the experimental period, animals were deprived of food overnight and blood was collected in a tube containing potassium oxalate and sodium fluoride. Blood glucose level was determined by the glucose oxidase method (Sundaram and Hill, 1992) and plasma insulin level was assayed with an enzyme-linked immunosorbent assay kit (Boehringer Mannheim, Germany).
Antioxidant assay with 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical
Total Phenolic Assay
The amount of total phenolics was measured using the Folin–Ciocalteu reagent method (Djeridane et al., 2006). To 1 ml of the GLEt, 1 ml of 95% ethanol, 5 ml of water, and 0.5 ml of 1 N Folin–Ciocalteu reagent were added. After 5 min, 1 ml of 5% Na2CO3 was added and the reaction mixture was allowed to stand for 60 min before the absorbance at 725 nm was measured. A standard curve was established for each assay using 50–500 μg of gallic acid in 95% ethanol.
All data are expressed as the mean ± SD of the experiments (n = 10). Statistical significance was evaluated by one-way analysis of variance (ANOVA) using SPSS version 7.5 (SPSS, Cary, NC, USA). Individual comparisons were obtained using Duncan’s multiple-range test (DMRT). Values were considered statistically significant at p < 0.05.
Despite the great strides that have been made in the understanding and management of diabetes, the disease and its complications are increasing unabated. The incidence and prevalence of type 2 diabetes mellitus (T2DM) continue to rise in world populations (King et al., 1998). α-Glucosidase inhibitors are prescribed to manage blood sugar levels in T2DM and these drugs lower blood sugar levels by slowing or decreasing carbohydrate breakdown in the intestine (Scheen, 2003). α-Glucosidase inhibitors including acarbose, miglitol, and voglibose reportedly can cause adverse effects such as hypoglycemia gastrointestinal side effects, flatulence, and diarrhea (Chakrabarti and Rajagopalan, 2002). Hence, there is an urgent need to identify indigenous natural resources without or with fewer side effects. Recently, many traditionally used medicinally important plants have been tested for their antidiabetic potential in various investigations (Sugihara et al.,2000; Youn et al., 2004; Andrade-Cetto et al., 2008). There is increased interest in the screening of phytochemicals with the ability to delay or prevent glucose absorption (Tiwari and Madhusudana Rao 2002).
In our earlier studies, G. montanum was found to have a modulatory effect on rate-limiting enzymes of glycolysis and gluconeogenesis in alloxan-induced diabetic rats (Ananthan et al., 2003a). There are no previous reports of α-glucosidase and α-amylase inhibitory activity of G. montanum extract. Hence the present experimental study was conducted to find out the possible antihyperglycemic mechanism of the GLEt.
Pancreatic and intestinal glucosidases are the key enzymes in dietary carbohydrate digestion and inhibitors of these enzymes are found to be effective in retarding glucose absorption to suppress hyperglycemia. The fundamental mechanism underlying hyperglycemia includes the excessive hepatic glycogenolysis and gluconeogenesis associated with decreased utilization of glucose by tissues. Liver glucosidase inhibitors inhibit α-1,6-glucosidase of glycogen-debranching enzymes in the liver and reduce the glycogenolytic rate, which increases the accumulation of glycogen stores in the liver (Bollen and Stalmans, 1989). Inhibition of these enzyme reportedly decreased blood glucose levels in diabetic patients (van de Laar et al., 2005).
Our in vitro studies demonstrated appreciable α-glucosidase and α-amylase inhibitory activity of the GLEt. The extract showed significant inhibition against salivary α-amylase (IC50, 5 μg/ml) as well as yeast α-glucosidase (IC50, 7 μg/ml), which is comparable with that of acarbose. Acarbose, a commercially available drug that inhibits α-glucosidase present in the epithelium of the small intestine, has been demonstrated to decrease postprandial hyperglycemia (Sima and Chakrabarti, 1992) and to improve impaired glucose metabolism without promoting insulin secretion in T2DM patients (Carrascosa et al., 2001; Breuer, 2003).
In inhibition mode studies, the GLEt showed competitive inhibition against human yeast α-glucosidase and noncompetitive inhibition against salivary α-amylase. The fact that α-amylase and α-glucosidase showed different inhibition kinetics seemed to be due to structural differences related to the origins of the enzymes (Chiba, 1997). Our findings correlate well with those of earlier studies conducted using G. sylvestre, which reported reduced blood glucose and increased plasma insulin levels in both type 1 and type 2 diabetic rats (Shanmugasundaram et al.,1990; Baskaran et al., 1990). Gymnemic acid (GA), a mixture of triterpene glucuronides, was isolated from G. sylvestre and found not only to inhibit glucose absorption in the small intestine (Yoshioka, 1986; Imoto et al., 1991), but also suppress hyperglycemia and hyperinsulinemia in an oral glucose tolerance test (Hirata et al., 1992; Leach, 2007). The efficiency of GA in inhibiting glucose absorption in the small intestine was found to be increased by a combined effect with acarbose and voglibose (Luo et al., 2001a, b). In the present study, we found that the GLEt showed a greater inhibitory effect on α-glucosidase and α-amylase activity. This is the first report demonstrating the antihyperglycemic activity of G. montanum by inhibition of α-glucosidase activity. Previously few plant extracts have been reported to inhibit α-amylase or α-glucosidase activity (Kim et al., 2005; Ortiz-Andrade et al.,2007; Shinde et al., 2008).
To understand the mechanism(s) of action, the total soluble phenolic content and antioxidant activity of the plant extract were measured. The phenolic content was found to be 156 mg of galic acid equivalent per gram of leaf extract. We speculate that the higher polyphenolic index observed for the extract may be related to the inhibition of α-amylase and α-glucosidase activity. There is increasing evidence that individual polyphenols or classes of polyphenols may cause other beneficial effects, independent of their antioxidant capacities, by directly influencing the activities of key enzymes (Matsui et al., 2001). Some reports have strongly suggested a relationship between polyphenolic content of the extract and inhibition of α-glucosidase activity (Yoshikawa et al., 2001; McDougall et al., 2005; Mai et al.,2007). Polyphenolic extracts from sweet potato (Ipomoea batatas L.) roots and morning glory (Pharbitis nil cv. Scarlett O’Hara) flowers were reported to effectively inhibit rat yeast α-glucosidase and human α-amylase (Matsui et al., 2002). There are also reports that tannins and ellagic acid derivatives from banaba (Lagerstroemia speciosa L.) leaves are potent inhibitors of α-amylase (Hosoyama et al., 2003). A positive and significant correlation was found between polyphenolic content and α-glucosidase inhibition (r2 = 0.92) as well as α-amylase inhibition (r2 = 0.97).
Results of this study clearly show that the GLEt has strong antioxidant activity as measured by DPPH assay. The strong radical scavenging activity of the GLEt may be because of the presence of antioxidant compounds. In addition, numerous laboratory and epidemiological studies have shown that phenolic phytochemicals have inhibitory activity against glucosidase (Tadera et al., 2006). Among the numerous polyphenolics, quercetin, genistein, myricetin, gallic acid, and kempferol are widely distributed in the plant kingdom and reported to possess strong α-glucosidase and α-amylase inhibitory properties (Tadera et al., 2006; Gowri et al., 2007). Based on GC-MS analysis, the GLEt was found to contain 4.58% gallic acid, 3.09% quercetin, 3.46% kempferol, and 1.7% genistein (Ramkumar et al., 2009). According to these results, the phenolic composition of the GLEt possibly plays a significant role in the inhibition of α-glucosidase and α-amylase activities.
In an effort to evaluate the in vivo antihyperglycemic activity of this extract, we studied the effect of the GLEt on nSTZ rats. Neonatal induction of diabetes mimics the progressive but not abrupt β-cell destruction seen in type 2 diabetic patients and exhibits various stages of T2DM, such as impaired glucose tolerance and mild, moderate, and severe hyperglycemia (Lee et al., 2003). nSTZ rats exhibit slightly lowered plasma insulin levels, elevated plasma glucose levels, and a decreased pancreatic insulin content. nSTZ is considered to be a very useful model for a better understanding of T2DM pathophysiology (Arulmozhi et al., 2004).
Administration of G. montanum extract for 12 weeks caused a significant reduction in blood glucose and increased plasma insulin levels. Leaves of the related species Gymnema sylvestre were found to contain GA, which was reported to be responsible for antihyperglycemic activity (Kimura, 2006). Because the test plant belongs to the same genus, the presence of such active constituents in G. montanum also may be envisaged.
In conclusion, the findings of this investigation allowed us to establish that the extract of G. montanum is a potent inhibitor of α-glucosidase and α-amylase and improves hyperglycemia in type 2 diabetic rats, possibly due to several polyphenolic compounds present within the extract. Further isolation of the active components may pave the way to the development of new agents for the treatment of diabetes and its complications.
K. M. Ramkumar is grateful to the Council of Scientific and Industrial Research, New Delhi, for awarding him a Senior Research Fellowship.