Inhibition of Intestinal Î±-Glucosidases and Anti-Postprandial

Chapter 25

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Inhibition of Intestinal α-Glucosidases and Anti-Postprandial Hyperglycemic Effect of Grape Seed Extract Kequan Zhou,*,1,2 Shelly Hogan,1,3 Corene Canning,2 and Shi Sun2 1Department

of Food Science and Technology, Virginia Tech, Blacksburg 24061 2Current address: Montana State University, Bozeman, Montana 59717 3Current address: Department of Nutrition and Food Science, Wayne State University, Detroit, MI 48202 *E-mail: [email protected].

Because intestinal α-glucosidase plays a key role in carbohydrate digestion, its inhibition provides a therapeutic option for diabetes by suppressing postprandial blood glucose. We recently identified a grape seed extract (GSE) significantly inhibiting α-glucosidases. The inhibitory activity of GSE on yeast α-glucosidase was significantly stronger than that of acarbose. GSE also inhibited rat α-glucosidases in vitro in a dose- and time-dependent manner. The potential anti-diabetic effect of GSE was further evaluated in an animal model. Male 6 week-old C57BLK/6NCr mice were treated by streptozocin to induce diabetes. The results showed the oral intake of GSE (400mg/kg, body weight) suppressed postprandial blood glucose in STZ-induced diabetic mice. Oral administration of GSE reduced postprandial blood glucose in the diabetic mice by 11.5% and 16.6% at 30 and 60 min after the starch meal. Overall, GSE intake significantly reduced the incremental AUC0-120min (area under postprandial glycemic curve) by 27.3% as compared to the control. Our results strongly suggest the potential of developing GSE, as a novel inhibitor of α-glucosidases, for diabetes prevention and treatment.

© 2012 American Chemical Society In Emerging Trends in Dietary Components for Preventing and Combating Disease; Patil, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Introduction Diabetes has been one of the major public health problems in the United States. Diabetes is at least in part related to the amount of carbohydrates in the diet. The digestion of dietary carbohydrates such as starch primarily occurs in the small intestine by α-amylase to yield both linear maltose and branched isomaltose oligosaccharides, neither of which can be absorbed into the bloodstream without further hydrolysis by α-glucosidases to release glucose (1). Therefore, intestinal α-glucosidase is critical in carbohydrate digestion and glucose release, and its inhibition provides an important anti-diabetic option by reducing postprandial hyperglycemia. Postprandial hyperglycemia is an early symptom of type 2 diabetes (2), which occurs when pancreatic β cells fail to secrete a sufficient amount of insulin (3). Postprandial hyperglycemia induces glucose toxicity and further deteriorates β cell function (4, 5). Treatment of postprandial hyperglycemia has been proven to improve overall glycemic control (6–9). On the other hand, inhibition of α-glucosidases has been demonstrated to be effective in both preventing and treating diabetes through improvement of postprandial hyperglycemia (1, 10–14). Commercial inhibitors, in particular acarbose, have been used for diabetes treatment. However, acarbose has been associated with significant adverse gastrointestinal (GI) side effects which is partly attributable to its non-specific inhibition of α-amylase, causing excessive accumulation of undigested carbohydrates in the large intestine (8, 15). Therefore, specific α-glucosidase inhibitors may provide a novel antidiabetic effect but with fewer GI side effects than currently available inhibitors. We recently found that a grape seed extract (GSE) significantly inhibits the activity of α-glucosidase and the inhibition appears to be selective because GSE does not inhibit structure-comparable α-amylase, suggesting that GSE may be a specific α-glucosidase inhibitor. This study aimed to determine time- and doseresponses of GSE on α-glucosidases and its inhibition mode, and to further assess whether acute intake of GSE can suppress postprandial blood glucose in diabetic animals.

Materials and Methods Materials Yeast type I α-glucosidase (EC 3.2.1.20, G5003), rat intestinal acetone powder (N1377-5G), p-nitrophenyl α-D-glucopyranoside (pNPG), acarbose, porcine pancreatic α-amylase, type VI-B (A3176), porcine pancreatic lipase, Type II (L3216), and streptozotocin (STZ) were purchased from the Sigma Chemical Co. (St. Louis, MO, USA). The organic solvents used in this study were HPLC grade (Fisher Scientific Co.).

432 In Emerging Trends in Dietary Components for Preventing and Combating Disease; Patil, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

GSE Preparation Grape seeds were obtained from a local Virginia vineyard (Blackstone, VA, USA). The seeds were then ground to fine powder by a Thomas Wiley mini-mill (Swedesboro, NJ). The samples were extracted with 80% ethanol at 1:10 ratio (m/v) under overnight shaking. The extracts were filtered through Whatman No. 4 filter paper to remove unwanted residues. After evaporating off the organic solvent, the filtrates were frozen and lyophilized to obtain GSE.


Yeast and Mammalian α-Glucosidase Inhibition Assays Both the yeast and mammalian α-glucosidase activity was assayed using the substrate pNPG, which is hydrolyzed by α-glucosidase to release the product pnitrophenol, a color agent that can be monitored at 405 nm (16). The yeast αglucosidase (EC 3.2.1.20) is categorized as type I α-glucosidase and the inhibition assay was conducted according to the previous assays with slight modification (16, 17). In brief, 80 µl of each sample solution (1mg/ml) was mixed with 20 µl of the enzyme solution (1 U/ml) and incubated in a 96-well plate at 37ºC for 3 min. After incubation, 100 µl of 4 mM pNPG solution in 0.1 M phosphate buffer (pH 6.8) was added and the reaction was conducted at 37ºC. The release of p-nitrophenol was monitored at 405 nm every minute for 75 min spectrophotometrically (Victor, PerkinElmer, USA). The α-glucosidase activity was determined by measuring area under the curve (0-75 min) for each sample and compared with that of the control where GSE was replaced by the dissolving solvent. The mammalian α-glucosidases were prepared from 1g of rat intestinal acetone powder suspended in 20 mL of 0.1 M potassium phosphate buffer (pH 7.0) containing 5 mM EDTA at ambient temperature. The suspension was sonicated for 15 min and after vigorous stirring for 1 h, the suspension was centrifuged (at 12000xg for 15 minutes). The supernatant was dialyzed against 0.01 M potassium phosphate buffer (pH 7.0) for 24 hours. The activity of rat α-glucosidase extract was verified using pNPG as the substrate by comparing with the pure yeast α-glucosidase. The assays were conducted as described above. Acarbose was used as a positive control.

Pancreatic α-Amylase Inhibition Assay The α-amylase inhibitory activity was determined using Type VI-B porcine pancreatic α-amylase (18). In brief, 500 µl of GSE dilutions and 500 µl of 0.02 M sodium phosphate buffer (pH 6.9 with 0.006 M NaCl) containing α-amylase solution (0.5 mg/ml) were incubated at 25 °C for 10 min. After preincubation, 500 µl of a 0.5% starch solution in 0.02 M sodium phosphate buffer (pH 6.9 with 0.006 M NaCl) was added to each tube at timed intervals. The reaction was stopped with 1.0 ml of dinitrosalicylic acid color reagent. The test tubes were then incubated at 90 °C in a water bath for 10 min and cooled to room temperature. The reaction mixture was then diluted 1:15 with distilled water, and absorbance was measured 433 In Emerging Trends in Dietary Components for Preventing and Combating Disease; Patil, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

at 540 nm with the PerkinElmer plate reader. The readings were compared with the controls, containing buffer instead of sample extract. The results were expressed as percent α-amylase inhibition.


Pancreatic Lipase Inhibition Assay The inhibition of pancreatic lipase by GSE was determined by measuring the amount of 4-methylumbelliferone product released by lipase spectrofluorometrically using the plate reader (18). Pancreatic lipase (Type II, from porcine pancreas) and 4-methylumbelliferyl oleate (4-MU oleate) served as the reaction enzyme and substrate, respectively. The reaction mixture was prepared with 25 μl of the GSE dilutions and 25 μl of 16.7 U/ml lipase in Tris-HCl, pH 8.0 buffer solution. The reaction was initiated by adding 50 µl of 0.1M 4-MU oleate in Tris-HCl, pH 8.0 buffer solution. After incubation at 37°C for 30 min, the rate of release of the 4-methylumbelliferone product was measured at an excitation wavelength of 355 nm and emission wavelength of 460 nm. Commercial lipase inhibitor, Orlistat, was used as the positive control. The readings were compared with the controls, containing buffer instead of sample extract. The results were expressed as percent α-lipase inhibition.

Time and Dose-Responses of GSE on Yeast α-Glucosidases The enzyme reactions were performed on various GSE concentrations (2.9285.7 µg/ml) within 75 min. The assays were conducted under the same conditions as described above and the release of p-nitrophenol was monitored every 5 min.

IC50 of GSE against Mammalian Intestinal α-Glucosidases To determine IC50 of GSE on rat intestinal α-glucosidases, we conducted the enzymatic reactions with various GSE concentrations (0.2-1mg/ml) incubated with pNPG ranging from 0.4-2 mM. Animal Experiments Animals Male 6-week old mice (C57BLKS/6NCr, National Cancer Institute, Frederick, MD, USA) were housed in groups of four mice per cage and maintained on a 12-hour light-dark cycle at 20 °C to 22 °C. Mice were acclimatized for a 2-week period before starting the experiment and had ad libitum access to food and water. Mice were maintained on rodent feed (Harlan Tekland Gobal Diets 2018 rodent diet containing 60% of calories from carbohydrate, 23% of calories from protein, and 17% of calories from fat; digestible energy of 3.4 Kcal/g, Madison WI, USA) for the duration of the experiment. Animal husbandry, care, 434 In Emerging Trends in Dietary Components for Preventing and Combating Disease; Patil, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

and experimental procedures were conducted in compliance with the “Principles of Laboratory Animal Care” NIH guidelines, as approved by the Institutional Animal Care and Use Committee (IACUC) at Virginia Tech.


STZ Induction of Diabetes in Mice Diabetes was induced by intraperitoneal injection of STZ dissolved in 10 mM sodium citrate buffer (pH 4.5) at a dose of 50 mg/kg body weight (bw). The STZ was dissolved in ice-cold citrate buffer protected from light and injected immediately to avoid STZ degradation. Five to seven days after STZ injection, mice with non-fasting blood glucose ≥250 mg/dl were considered to have diabetes (19).

Oral GSE Treatment and Starch Challenge The experiment was designed to determine the effect of acute intake of GSE on postprandial glycemic response in STZ-induced diabetic mice following a potato starch challenge. Mice were fasted for 14 hours in freshly cleaned cages with free access to water before the experiment (4/group). Mice in the control group were given 0.2 mL of water by oral gavage. The treatment group were administered 0.2 mL of GSE suspension (400 mg/kg bw) by oral gavage immediately after vortexing the suspension. After approximately 30 minutes post water or GSE administration, 0.2 mL of potato starch suspension (2 g/kg bw) was administered to each mouse by gavage. Approximately 5 µL of whole blood samples were collected from the tail vein of each mouse. The blood samples were acquired at 0, 30, 60, and 120 minutes after the oral starch challenge. Blood glucose levels were measured with a blood glucometer and accompanying test strips (ACCUCHEK Meter®, Roche Diagnostics, Kalamazoo, MI). The area under the glucose tolerance curve (AUC0–120 min) was calculated using a trapezoidal method (20). The total antihyperglycemic response (AUC0–120 min) was expressed as mean ± standard deviation.

Statistical Analysis The statistical significance comparing data between groups was assessed by compared by a two-sample t-test or one-way analysis of variance (ANOVA) followed by Duncan’s multiple range post-hoc tests. Statistical analysis was performed using SPSS (Windows, Version Rel. 10.0.5, 1999, SPSS Inc., Chicago, IL). Statistical significance was declared when P < 0.05.


Results


Time and Dose-Responses of GSE against Yeast α-Glucosidase Figure 1A shows time-responses of GSE at different concentrations against yeast α-glucosidase. The inhibition of yeast α-glucosidase activity by GSE was sustainable during 75 min of the reaction period. The significant inhibition was seen when GSE concentration was as low as 1.4 µg/ml. A dose-dependent inhibition of yeast α-glucosidase by GSE was also observed (Figure 1B). The inhibitory activity of GSE was compared with acarbose. GSE at 1.4 µg/ml exerted significantly stronger inhibition than acarbose at 285.7 µg/ml. GSE inhibited more than 90% of yeast α-glucosidase activity at a concentration of 28.6 µg/ml or higher. We further determined the IC50 of GSE against yeast α-glucosidase which was 1.5 µg/ml. Furthermore, we examined whether GSE also inhibits other digestive enzymes including pancreatic α-amylase and lipase. However, GSE at comparable or even higher concentrations showed no significant inhibition on both enzymes with GSE concentrations up to 1 mg/ml.

Figure 1. Kinetics of yeast α-glucosidase inhibition by GSE at different concentrations (A); Dose-dependent inhibition of GSE on yeast α-glucosidase (B). Enzyme activity was determined by measuring p-nitrophenol released from pNPG at 405 nm. Acarbose is the positive control and denoted as AC. Bars with the different letters are significantly different (p < 0.05).


Dose-Dependent Inhibition of Mammalian Intestinal α-Glucosidase by GSE


GSE also significantly inhibited the activity of rat intestinal α-glucosidases. As shown in Figure 2, the enzymatic inhibition was highly dose-dependent. GSE inhibited the activity of rat α-glucosidases by 25.4%, 69.8%, and 78.6% at the doses of 0.4, 1, and 2 mg/ml, respectively. For comparison, acarbose at 0.08 mg/ml showed 47.2% inhibition of rat α-glucosidases. The IC50 of GSE was determined to be 0.73 mg/ml on rat α-glucosidases.

Figure 2. Dose-dependent Inhibition of GSE on rat α-glucosidases. Rat intestinal α-glucosidase activity was determined by measuring p-nitrophenol released from pNPG at 405 nm. The reaction was conducted at 37ºC for 75 min. Bars marked by different letters are significantly different (p < 0.05).

Inhibition of Postprandial Blood Glucose by the Acute Intake of GSE in STZ-Treated Mice Figure 3 shows the effect of GSE on postprandial blood glucose in STZ-induced mice after oral loads of potato starch (Fig 3A & 3B). Oral administration of GSE (400mg/kg, bw) reduced postprandial blood glucose by 11.5% and 16.6% at 30 and 60 min after the starch meal, respectively (Fig 3A, P > 0.05). Overall, GSE intake reduced the incremental AUC0-120min (area under postprandial glycemic curve) by 27.3% as compared to the control (P < 0.05).



Figure 3. Postprandial glycemic response after acute intake ofGSE in STZ-treated mice after a starch meal. The glycemic response curve in diabetic mice after starch challenge (A).The incremental AUC0-120min in diabetic mice after starch administration (B); The fasted diabetic mice were administered with 100µl of either vehicle or GSE suspension (400mg/kg, bw) by gavage. After 30 min, 100µl of potato starch suspension (2g/kg, bw) was administered and blood was collected from tail vein at 0, 30, 60, and 120 min to determine glucose levels.*, P

Inhibition of Intestinal Î±-Glucosidases and Anti-Postprandial

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