Yeast type I α-glucosidase (EC 22.214.171.124, G5003), rat intestinal acetone powder (N1377-5G), p-nitrophenyl α-D-glucoside (pNPG), acarbose, porcine pancreatic α-amylase, type VI-B (A3176), porcine pancreatic lipase, Type ll (L3216), Folin-Ciocalteu reagent, gallic acid, rutin, Trolox™, fluorescein, 2,2'-Azobis(2-amidinopropane) dihydrochloride (AAPH), 1,1-diphenyl-2-picrylhydrazyl (DPPH), and streptozotocin (STZ) were purchased from the Sigma Chemical Co. (St. Louis, MO, USA). The ethanol and acetone solvent was HPLC grade (Fisher Scientific Co.).
Preparation of pomace extracts
Red wine grape pomace (Cabernet Franc) and white wine grape pomace (Chardonnay) were obtained from a local Virginia vineyard (Blackstone, VA, USA). The red apple pomace was obtained from National fruit product company, INC. (Winchester, VA). All the pomace extracts were prepared from a single production lot. A portion of the pomace samples (500 g) were immediately freeze-dried upon receiving. The dried extracts 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 the pomace extracts. The extraction yield was 13.4%, 16.3%, and 13.8% for the dried red grape, white grape, and red apple pomace, respectively. A portion of the lyophilized extracts were freshly reconstituted in dimethyl sulfoxide (DMSO) at 20 mg/mL as the stock solution and stored at -20°C for further investigation.
Total phenolic content (TPC)
The TPC of each pomace extract was determined using Folin-Ciocalteu reagent with gallic acid as the phenolic standard . In brief, appropriate dilutions of extracts were mixed with 3.0 mL of 0.2 N Folin-Ciocalteu reagent and 2.0 mL of 20% sodium carbonate (Na2CO3) at ambient temperature. After incubation for 2 hours, the absorbance of the blue color that developed in each assay mixture was recorded at 760 nm (Thermo Electron Corporation, Genesys 10-UV scanning, Madison, WI, USA). The TPC value of each pomace extract was expressed as micrograms of gallic acid equivalent per gram of pomace (μg GAE/g).
Total flavonoid content (TFC)
The pomace extracts were analyzed for TFC according to an established colormetric method . In brief, 1 mL of the reconstituted pomace extract (500 μg/mL) or rutin standard was mixed with 0.3 mL of 5% sodium nitrite (NaNO2), 0.3 mL of 10% aluminum chloride (AlCl3), and 2 mL of 1 M sodium hydroxide (NaOH). The reaction mixture was incubated at 30°C for 30 min. All samples were measured in duplicate and compared against a blank at an absorbance of 510 nm (Thermo Electron Corporation, Genesys 10-UV scanning, Madison, WI, USA). Results were expressed as micrograms of rutin equivalent per gram of pomace (μg RE/g).
Oxygen radical absorbance capacity (ORAC)
The ORAC assay was conducted to kinetically measure the peroxyl radical scavenging activity of each pomace extract with Trolox™ as the antioxidant standard (Zhou et al., 2007). Fluorescein (FL) was used as the fluorescent probe and the peroxyl radicals were generated from AAPH in 75 mM phosphate buffer (pH 7.4). Specifically, 225 μL of 81.6 nM FL solution was mixed with 30 μL of sample extract, standard, or blank (DMSO) to a black 96-well fat bottom plate and incubate covered plate at 37°C for 20 min. After incubation, 25 μL of 0.36 M APPH solution was added to the mixture and the reaction started. Standards and samples were measured in duplicate. The fluorescence of the reaction mixture was monitored and recorded every minute (λex = 485 nm and λem = 535 nm) and maintained at 37°C until the reading had declined to less than 5% of the initial reading with a Victor3 multilabel plate reader (Perkin-Elmer, Turku, Finland). Results for ORAC were determined by using a regression equation relating Trolox™ concentrations and the net area under the kinetic fluorescein decay curve. The ORAC value was expressed in micromoles of Trolox™ equivalents per gram of pomace (μmol TE/g).
DPPH radical scavenging activity
The DPPH radical scavenging antioxidant activity assay was conducted to obtain the antioxidant activity of pomace extracts . The reaction mixture contained 100 μL of the diluted pomace extracts (1 mg/mL) and 100 μL of 0.208 mM DPPH radical solution. The absorption at 515 nm was determined immediately after the reaction was initiated. Each plate was read once every minute for 30 min with a Victor3 multilabel plate reader. The initial and final absorbance for the control was 0.934 and 0.917, respectively. The percent inhibition of the DPPH radical scavenging activity per milligram of pomace extract was expressed as the inhibition percentage (% DPPH inhibition/mg).
Yeast and mammalian α-glucosidase inhibition assays
Both the yeast and mammalian α-glucosidase activity was assayed using the substrate p-nitrophenyl-α-d-glucopyranoside (pNPG), which is hydrolyzed by α-glucosidase to release the product p-nitrophenol, a color agent that can be monitored at 405 nm . The mammalian α-glucosidases were prepared from 1 g 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. 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 by mixing 80 μL of approximate dilutions of the pomace extracts (10 μg/mL) in 0.1 M phosphate buffer (pH 6.8) with 20 μL of the yeast enzyme solution (1 U/mL) or 20 μL rat intestinal α-glucosidase solution (3 × dilution from the original extract). The acarbose (150 μg/mL) was used as a positive control. The pH of the sample extracts were 6.8 which was the optimal for the enzyme reaction. The blank reagent, 0.1 M phosphate buffer (pH 6.8), was used as the control. The mixture was incubated in a 96-well plate at 37°C for 3 minutes under constant shaking. 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 37°C. The release of p-nitrophenol from pNPG was monitored at 405 nm every minute for 75 minutes with a Victor3 multilabel plate reader. The α-glucosidase activity was determined by measuring the area under the curve (0-75 minutes) for each sample and compared with that of the control (the blank reagent). The results were expressed as the percent of α-glucosidase inhibition.
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. The animals were acclimatized for a 2-week period before starting the experiment and had ad libitum access to food and water. The 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, 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 in 14-hour fasted 8-week old mice (25-27 g) 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 a fasting blood glucose (FBG) level higher than 126 mg/dL were considered to have diabetes and were randomly assigned to one of two groups (n = 8).
Oral RGPE treatment and starch challenge
The experiment was designed to determine the effect of acute RGPE intake on postprandial glycemic response in STZ-induced diabetic mice following a potato starch challenge. Diabetic mice were fasted for 14-hours in freshly cleaned cages with free access to water before the experiment. Ten mg of the lyophilized RGPE was suspended in 0.2 mL water (50 mg/mL) in a small centrifuge tube and vortexed vigorously. The dietary dose was calculated to be 400 mg/kg bw based on mouse weight of 25 g. Mice in the control group were given 0.2 mL of water by oral gavage. The treatment group were administered 0.2 mL of RGPE suspension (400 mg/kg bw) by oral gavage immediately after vortexing the suspension. After approximately 30 minutes post water or RGPE 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 (ACCU-CHEK Meter®, Roche Diagnostics, Kalamazoo, MI). The area under the glucose tolerance curve (AUC0-120 min) was calculated using a trapezoidal method . The total antihyperglycemic response (AUC0-120 min) was expressed as mean ± standard deviation.
The statistical significance comparing data between groups was assessed by 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.