Inhibitory effect of leaf extract of Newbouldia laevis on the metabolic activities of alpha-glucosidase and alpha-amylase
Abstract
In this study, the effect of ethanol extract of the leaves of Newbouldia laevis on the activity of alpha-amylase and alpha-glucosidase was investigated. Inhibitory effect of N. laevis extract on alpha-glucosidase was tested in vitro using baker's yeast alpha-glucosidase and rat intestinal alpha-glucosidase while alpha-amylase inhibitory effect was assayed using rat pancreatic alpha-amylase. alpha-Glucosidase inhibitory effect of the extract was also tested in vivo in diabetic and non-diabetic rats. N. laevis extract exhibited good alpha-glucosidase inhibitory activity in vitro with IC50 values of 2.2 µg/mL and 43.5 µg/mL for baker's yeast and rat intestinal alpha-glucosidase respectively. The extract also inhibited rat pancreatic alpha-amylase activity with IC50 value of 58.7 µg/mL. In both diabetic and non-diabetic rats, N. laevis extract caused a significant reduction in postprandial blood glucose level after oral sucrose load. The results of this study indicate that N. laevis extract exerts its glucose-lowering effect through inhibition of alpha-glucosidase and alphaα-amylase.
Introduction
Diabetes mellitus is a metabolic disorder which affects millions of people around the world. It is one of the leading causes of blindness, cardiovascular complications and end-stage renal failure that results in dialysis and kidney transplantation. Diabetes is characterized by disturbance in glucose homeostasis which results in chronic hyperglycemia. This occurs when there is defect in insulin secretion and/or insulin action (Kumar et al., 2012). Autoimmunity, obesity, high carbohydrate diet and sedentary life style are the major causes of diabetes (Patel et al., 2008). Postprandial hyperglycemia has been widely reported to be the key feature of impaired glucose tolerance and early diabetes (Tentolouris et al., 2007). Therefore, one of the important therapeutic interventions employed in the management of diabetes mellitus is targeted at decreasing postprandial blood glucose level (Alexander-Lindo et al., 2007). This can be achieved by reducing the digestion of carbohydrate and absorption of glucose using inhibitors of enzymes that are involved in the process of digestion. Inhibitors of alpha-amylase and alpha-glucosidase have been effectively used to achieve good glycemic control in diabetic patients (Lelono and Tachibana, 2013).
α-Glucosidase inhibitors are also beneficial in decreasing postprandial plasma insulin level and improving insulin sensitivity (Shinozaki et al., 1996; Breuer, 2003). Examples of alpha-glucosidase and alpha-amylase inhibitors that are currently in clinical use are acarbose, voglibose and miglitol. In spite of their therapeutic benefits, these drugs induce some adverse effects such as abdominal discomfort, bloating, flatulence and diarrhea. These effects have been attributed to excessive enzyme inhibition which results in abnormal fermentation of undigested carbohydrate by intestinal bacteria (Bischoff, 1994). Researchers are therefore interested in discovering more enzyme inhibitors with less side effect. In addition, many antidiabetic drugs are not readily available to diabetic patients in developing nations and rural areas due to economic hardship and other hindrances. Majority of these people depend on herbal remedy (Kolawole et al., 2012). Therefore it is necessary to search for new inhibitors of alpha-amylase and α-glucosidase especially from natural sources such as medicinal plants. This will be of great assistance to those who have little or no access to synthetic drugs to care for their health needs. The search may also result in the discovery of lead molecules for the development of new antidiabetic drugs.
One of the medicinal plants that are widely used in the management of diabetes mellitus across African countries is Newbouldia laevis (P. Beauv). It is an angiosperm which belongs to the Bignoniaceae family. Its common names are "Fertility tree" and "African border tree". In Nigeria, it is known by various names among different ethnic groups. It is called "Akoko" in Yoruba, "Aduruku" in Hausa and "Ogirisi" in Igbo. The extract of the leaves has been reported to lower blood glucose level in diabetic rats (Owolabi et al., 2011). In the present study, we investigated the effects of ethanol extract of N. laevis leaves on alpha-amylase and alpha-glucosidase in diabetic rats.
Materials and Methods
Drugs and chemicals: Streptozotocin, rat intestinal acetone powder, baker's yeast, rat pancreatic amylase, p-nitrophenyl alpha-D-glucopyranoside (pNPG), acarbose and sucrose were obtained from Sigma Chemical Co. (St. Louis, MO, USA). All other reagents were of analytical grade.
Preparation of plant extract: Leaves of Newbouldia laevis were collected from the premises of College of Health Sciences, Ladoke Akintola University of Technology, Mercyland, Osogbo Campus, Nigeria. The leaves were identified and authenticated by a taxonomist in Forest Research Institute of Nigeria (FRIN) and a voucher specimen was deposited in the herbarium of the institute (voucher specimen no: FHI 107753). The leaves were thoroughly washed with distilled water to remove soil and other debris that may contaminate the plant sample. The washed sample was then air-dried under shade in the laboratory for 5 days and then pulverized using an electric grinding machine. The powder sample (500 g) was extracted with 80% ethanol at 70°C by continuous hot percolation using a Soxhlet apparatus. The extraction was carried out for 24 hours and the resulting ethanol extract (NLet) was concentrated at 40°C in a rotary evaporator. The solid sample obtained weighed 47.5 g (yield = 9.5%). The crude ethanol extract was kept in airtight container and stored in a refrigerator at 4°C until the time of use.
Experimental animal: Male Wistar rats weighing 180-200 g were obtained from the Animal Holding Unit of the Department of Pharmacology and Therapeutics, Ladoke Akintola University of Technology (LAUTECH), Nigeria. The animals were housed in polypropylene cages inside a well-ventilated room. The animals were maintained under standard laboratory conditions of temperature (22 ± 2°C), relative humidity (55-65%) and 12 hours light/dark cycle. They were allowed to acclimatize for 2 weeks before the experiment. During the experimental period, animals were fed with a standard balanced commercial pellet diet (Ladokun Feeds Ltd. Ibadan, Nigeria) and potable tap water ad libitum.
Ethical consideration: All experimental procedures were conducted in accordance with National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH, 1985) as well as Ethical Guidelines for the Use of Laboratory Animals in LAUTECH, Nigeria.
Induction of diabetes in experimental animal: Experimental diabetes was induced in rats which had fasted for 12 hours by a single intravenous injection of a freshly prepared solution of streptozotocin (STZ) (60 mg/kg body weight) dissolved in 0.1 M cold citrate buffer, pH 4.5 (Chen et al., 2005). The rats were allowed to drink 5% glucose solution overnight to overcome drug-induced hypoglycemia. Estimation of fasting blood glucose (FBG) was done 72 hours after injection of STZ to confirm induction of diabetes and then on the 7th day to investigate the stability of diabetic condition. Fasting blood glucose was estimated by One Touch® glucometer (Lifescan, Inc. 1995 Milpas, California, USA). Blood sample for the FBG determination was obtained from the tail vein of the rats and those with blood glucose value ≥ 200 mg/dL were selected for the study.
Bakers yeast alpha-glucosidase inhibition assay: The method of Shibano et al. (1997) was adapted for the assay of alpha-glucosidase of baker's yeast (Saccharomyces cerevisiae). The reaction mixture consisted of 50 uL of 0.1 M phosphate buffer (pH 7.0), 25 uL of 0.5 mM pNPG dissolved in 0.1 M phosphate buffer, pH 7.0), 10 uL of extract (20 µg/mL) and 25 uL of alpha-glucosidase solution. This reaction mixture was then incubated at 37°C for 30 min. The reaction was terminated by the addition of 100 uL of 0.2 M sodium carbonate solution. The enzymatic hydrolysis of the substrate was monitored spectrophotometrically at 400 nm based on the amount of p-nitrophenol released in the reaction mixture. The inhibition percentage of α-glucosidase was calculated by the following formula:
% Inhibition = 100 %reaction (where %reaction = mean enzyme activity in sample/mean enzyme activity in control x 100).
IC50 values were determined from dose-response curve of percentage inhibition versus extract concentration and compared with the IC50 of acarbose (standard drug) determined under similar conditions. All experiments were carried out in triplicate.
Rat intestinal alpha-glucosidase inhibition assay: The method of Kim et al. (2005) was adapted for the assay of rat intestinal alpha-glucosidase. Rat intestinal acetone powder (0.5 g) was suspended in 10 mL of 0.9%saline, and the suspension was sonicated for 30 sec at 4°C. After centrifugation at 10,000x g, 4°C and for 30 min, the resulting supernatant was used for the assay. The reaction mixture consisted of 50 uL of 0.1 M phosphate buffer (pH 7.0), 30 uL of 0.5 mM pNPG (dissolved in 0.1 M phosphate buffer, pH 7.0), 10 uL of plant extract (20 µg/mL) and 15 uL of rat intestinal alpha-glucosidase solution. This reaction mixture was then incubated at 37°C for 30 min. The reaction was terminated by the addition of 100 uL of 0.2 M sodium carbonate solution. The a-glucosidase activity was determined by measuring the yellow colored p-nitrophenol released from pNPG at 400 nm. Percentage inhibition was calculated by the following formula:
%Inhibition = 100 %reaction (where %reaction = mean enzyme activity in sample/mean enzyme activity in control x 100).
IC50 values were determined from dose-response curve of percentage inhibition versus extract concentration and compared with the IC50 of acarbose determined under similar conditions. All experiments were carried out in triplicate.
Effect of N. laevis extract on blood glucose level after oral sucrose load: This experiment was performed in normal and diabetic rats to confirm alpha-glucosidase inhibitory activity of N. laevis extract. Prior to the experiment, all the rats were made to fast for 12 hours. Distilled water (control), a reference drug, acarbose (50 mg/kg body weight) or NLet (500 mg/kg body weight) were orally administered to different groups of 6 rats each. Thirty minutes later, sucrose (2 g/kg body weight) was orally administered to each rat with a feeding syringe (Sancheti et al., 2011). Blood samples were collected from the tail vein by tail milking at -30 min (just before the administration of extracts and acarbose), 0 (just before the oral administration of sucrose), 60, 120, and 180 min after sucrose load. Blood glucose level was determined by glucose oxidase/peroxidase method.
alpha-Amylase inhibition assay: The method of Giancarlo et al. (2006) was adapted in the assay of rat pancreatic α- amylase activity. The starch substrate was prepared by dissolving 1 g of starch in 100 mL of 0.1M phosphate buffer (pH 7.0). Concentrated HCl and 0.5N NaOH were used to adjust the pH. The dinitrosalicylic acid (DNS) reagent was prepared by dissolving 5 g of 3, 5-dinitrosalicylic acid and 150 g of sodium potassium tartarate in 150 mL of distilled water and 200 mL of 1N sodium hydroxide. The mixture was refluxed in a water bath at 60°C until all components were totally dissolved and then made up to 500 mL with distilled water. Rat pancreatic amylase was dissolved in ice-cold 20 mM phosphate buffer (pH 6.7) containing 6.7 mM sodium chloride to give a concentration of 5 units/mL. Test tubes were prepared in duplicates including blank and control. In each test tube, 1 mL of the plant extract (20 µg/mL) and 1 mL enzyme solution were mixed and incubated at 25°C for 30 min. After incubation, 250 uL of the starch preparation was transferred into each test tube to start the reaction. The mixture was vortexed and then incubated at 37°C for 15 min. Two millilitres of DNS (color reagent) was added and the mixture was stirred in a vortex and boiled in a water bath at 100°C for 10 min. Thereafter the mixture was cooled down in a running tap water and the absorbance was read at 540 nm using a spectrophotometer. The percentage inhibition of α-amylase was calculated by the following formula:
%Inhibition = 100 %reaction (where %reaction = mean enzyme activity in sample/mean enzyme activity in control x 100).
The percentage inhibition of alpha-amylase activity was plotted against the sample concentration and a linear regression curve was established in order to calculate the IC50 value.
Statistical analysis: Data were recorded as mean ± standard error of mean (SEM) and analyzed using one-way ANOVA followed by Student's t-test. Results were considered significant at p<0.05.
Results
NLet exerted good inhibitory effect on baker's yeast alpha-glucosidase (Figure 1). The concentration of the extract required to cause 50%inhibition (IC50) against alpha-glucosidase was 2.2 µg/mL. Acarbose which was used as positive control, exerted lower alpha-glucosidase inhibitory potential with IC50 value of 3.8 µg/mL. NLet also showed potent inhibitory effect against rat intestinal alpha-glucosidase (Figure 2). The IC50 value of the extract against rat intestinal alpha-glucosidase was 43.5 µg/mL while that of acarbose was 62.7 µg/mL. Figure 3 shows the effect of NLet and acarbose on blood glucose level in normal rats after sucrose administration. Thirty minutes after sucrose load, blood glucose in all the groups increased rapidly and reached a peak at 60 min. Thereafter, it gradually decreased. NLet caused a significant reduction (p<0.05) in blood glucose level at 60, 120 and 180 min compared to normal control. In diabetic rats, NLet also caused significant decrease in blood glucose at 60, 120 and 180 min (p<0.05) compared to the diabetic control (Figure 4). The reduction in blood glucose in NLet-treated diabetic rats was significantly different (p<0.05) compared to the acarbose-treated group. In vitro alpha-amylase inhibitory study demonstrated that NLet inhibited rat pancreatic alpha-amylase activities (Figure 5). The IC50 value for NLet was 58.7 µg/mL and that of acarbose was 92.3 µg/mL.
Figure 1: Inhibitory effect of N. laevis extract on baker's yeast alpha-glucosidase Values represent mean ± SEM of three replicates
Figure 2: Inhibitory effect of N. laevis extract on rat intestinal α-glucosidase Values represent mean ± SEM of three replicates
Discussion
One of the important goals in the treatment of diabetes is to maintain fasting and postprandial blood glucose near normal levels. The suppression of production or absorption of glucose from the gastrointestinal tract through inhibition of alpha-amylase and alpha-glucosidase enzymes is one therapeutic approach that has been successfully employed to achieve this goal (Cheng and Fantus, 2005; Bhandari et al., 2008). Polysaccharides and disaccharides are digested to produce monosaccharides by these carbohydrate-hydrolyzing enzymes. alpha-Amylase is secreted by the salivary glands in the mouth where digestion of carbohydrate begins. However, the acidic medium of the stomach halts this process. In the upper part of the small intestine, digestion of carbohydrate continues with alpha-amylase secreted by the pancreas. Oligosaccharides and disaccharides which are the products of alpha-amylase metabolic activity are then broken down to monosaccharides such as glucose and galactose by alpha-glucosidase enzymes which include maltase, isomaltase, glucoamylase and sucrase (Akintunde and Oboh, 2012). These enzymes are located in the brush border of intestinal mucosa and their digestive activity results in a sharp rise in the level of blood glucose after the consumption of a carbohydrate-rich diet (Tentolouris et al., 2007).
Figure 3: Effect of N. laevis extract on blood glucose level in normal rats after oral sucrose load Values are expressed as the mean ± SEM (n = 6). *p<0.05 compared to control. NLet = ethanol extract of N. laevis; NC = normal control.
Figure 4: Effect of N. laevis extract on blood glucose level in diabetic rats after oral sucrose load Values are expressed as the mean ± SEM (n = 6). *p<0.05 compared to diabetic control. NLet = ethanolic extract of N. laevis; DC = diabetic control
Figure 5: Inhibitory effect of N. laevis extract on rat pancreatic alpha-amylase. Values represent mean ± SEM of three replicates
Preclinical as well as clinical studies have shown that alpha-glucosidase and alpha-amylase inhibitors can suppress the production and absorption of glucose from the small intestine without causing hypoglycemia (Matsui et al., 2007). They are also beneficial in decreasing postprandial plasma insulin level and improving insulin sensitivity (Breuer, 2003). Acarbose and miglitol are examples of enzyme inhibitors that are already in clinical use. Some medicinal plants have also been reported to inhibit the activity of alpha-amylase and alpha-glucosidase (Dimo et al., 2007). Examples of such plants are Musa paradisiacal (Shodehinde and Oboh, 2012), Tourneforia hartwegiana (Oritz et al., 2007) and Allium spp. (Nickavar and Yousefian, 2009). Secondary metabolites such as flavonoids, tannins, saponins and alkaloids present in these plants are generally believed to be responsible for their enzyme inhibition activity (Mahesh and Menon, 2004).
In the current study, the results of in vitro assays showed that extract of N. laevis inhibited the activities of alpha-amylase and alpha-glucosidase but the inhibitory effect against alpha-glucosidase was more pronounced. The results also showed that NLet produced better inhibitory effects than acarbose. Attempt was made in this study to confirm the observed in vitro inhibitory effect of N. laevis leaf extract on alpha-glucosidase through in vivo study by determining postprandial glucose levels in normal and streptozotocin-induced diabetic rats after oral administration of sucrose. Sucrose needs to be digested to glucose before it is absorbed into the systemic circulation. Administration of N. laevis leaf extract 30 min before oral sucrose load significantly suppressed postprandial glucose level in both normal and diabetic rats. This indicates that the extract inhibited the catabolic activity of alpha-amylase and alpha-glucosidase. Therefore the enzyme inhibitor present in the leaves of N. laevis is a promising therapeutic remedy for the management of postprandial hyperglycemia in diabetic patients. As far as we know, this is the first time the inhibitory activity of the leaves of N. laevis against alpha-amylase and alpha-glucosidase is reported.
Conclusion
This study indicates that ethanol extract of the leaves of N. laevis exerts its glucose-lowering effect through inhibition of alpha-amylase and intestinal brush border alpha-glucosidase. Further study is necessary to isolate the active compound of the plant that is responsible for its enzyme inhibition activity and determine its structure and toxicity profile.
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