Anti-α-glucosidase and Anti-dipeptidyl Peptidase-IV Activities of

Jul 2, 2015 - Graduate Institute of Pharmacognosy, Taipei Medical University, Taipei, Taiwan. § Department of Life Science, National Taitung Universi...
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Anti-α-glucosidase and Anti-dipeptidyl Peptidase-IV Activities of Extracts and Purified Compounds from Vitis thunbergii var. taiwaniana Yin-Shiou Lin,† Chiy-Rong Chen,§ Wei-Hau Wu,# Chi-Luan Wen,⊥,‡ Chi-I Chang,*,# and Wen-Chi Hou*,†,‡ †

Graduate Institute of Pharmacognosy, Taipei Medical University, Taipei, Taiwan Department of Life Science, National Taitung University, Taitung, Taiwan # Department of Biological Science and Technology, National Pingtung University of Science and Technology, Pingtung, Taiwan ⊥ Taiwan Seed Improvement and Propagation Station, Council of Agriculture, Taichung, Taiwan ‡ Traditional Herbal Medicine Research Center, Taipei Medical University Hospital, Taipei, Taiwan §

S Supporting Information *

ABSTRACT: Ethanol extracts (Et) from the stem (S) and leaf (L) of Vitis thunbergii var. taiwaniana (VTT) were used to investigate yeast α-glucosidase and porcine kidney dipeptidyl peptidase-IV (DPP-IV) inhibitory activities. Both VTT-Et showed complete α-glucosidase inhibition at 0.1 mg/mL; VTT-S-Et and VTT-L-Et showed 26 and 11% DPP-IV inhibition, respectively, at 0.5 mg/mL. The VTT-Et interventions (20 and 50 mg/kg) resulted in improvements in impaired glucose tolerance of dietinduced obese rats. (+)-Hopeaphenol, (+)-vitisin A, and (−)-vitisin B were isolated from the ethyl acetate fractions of S-Et and showed yeast α-glucosidase inhibition (IC50 = 18.30, 1.22, and 1.02 μM) and porcine kidney DPP-IV inhibition (IC50 = 401, 90.75, and 15.3 μM) compared to acarbose (6.39 mM) and sitagliptin (47.35 nM), respectively. Both (+)-vitisin A and (−)-vitisin B showed mixed noncompetitive inhibition against yeast α-glucosidase and porcine kidney DPP-IV, respectively. These results proposed that VTT extracts might through inhibitions against α-glucosidase and DPP-IV improve the impaired glucose tolerance in diet-induced obese rats. KEYWORDS: blood sugar, dipeptidyl peptidase-IV (DPP-IV), α-glucosidase, impaired glucose tolerance, Vitis thunbergii var. taiwaniana (VTT), (+)-vitisin A, (−)-vitisin B

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inhibitory potency order of acarbose against α-glucosidases is glucoamylase > sucrase > maltase > isomaltase. Therefore, acarbose can slow the intestinal conversion of sucrose to glucose and fructose by inhibiting sucrose. Starch digestion is delayed through the inhibition of glucoamylase, maltase, and isomaltase. 8 DPP-IV (EC 3.4.14.5) inhibitors, such as sitagliptin, can prolong glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) biological activities by inhibiting DPP-IV activity to stimulate glucosedependent insulin secretions for T2DM treatments in vivo.9,10 DPP-IV cleaves preferentially to the N-terminal X-Ala of the active GLP-1(7−37)/GLP-1(7−36)amide or GIP(1−42) and results in inactive GLP-1(9−37)/GLP-1(9−36)amide or GIP(3−42), which blocks the receptor binding for activating signal transduction in the stimulation of insulin releases.10,11 The small-leaf grape (Vitis thunbergii var. taiwaniana, VTT) has leaves smaller than those of the standard grape (Vitis vinifera), and VTT’s small fruit is not as favorable as the standard grape for eating, but the dried leaves of VTT are frequently used as a tea substitute in Taiwan. It has been

INTRODUCTION The World Health Organization has estimated that >1.9 billion adults, 18 years and order, were overweight and >600 million were obese in 2014.1 The is a growing number of patients suffering from type 2 diabetes mellitus (T2DM) worldwide, and the number is expected to reach 380 million in 20 years.2 T2DM and obesity associated with insulin resistance3,4 are involved in a cluster of multiple metabolic risk criteria, a term related to metabolic syndrome observed in cardiovascular diseases.5,6 Moreover, the twin epidemics of obesity and T2DM have illustrated that 80% of newly diagnosed T2DM cases are also overweight.7 Therefore, obesity associated with T2DM is considered not only a clinical problem but also a public health issue in many countries. Several different kinds of oral drugs have been developed for T2DM management, among which inhibitors against two enzymes, α-glucosidase and dipeptidyl peptidase-IV (DPP-IV), which are both associated with insulin secretions, have been developed to improve hyperglycemia. α-Glucosidase (EC 3.2.1.20) inhibitors, such as acarbose obtained from the fermentation processes of Actinoplanes utahensis,8 retard the digestion of starch and sucrose, slow glucose absorption in the gut, and then reduce the insulin and postprandial blood glucose levels. α-Glucosidase hydrolyzes terminal nonreducing α(1−4) glycosidic linkage of starch to release a glucose molecule. The © XXXX American Chemical Society

Received: April 24, 2015 Revised: July 1, 2015 Accepted: July 2, 2015

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DOI: 10.1021/acs.jafc.5b02069 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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and stems (10 kg each) was extracted with ethanol in a solid/solvent ratio of 1:5 at room temperature for 7 days. The combined ethanol extract was filtered through Whatman no. 1 filter paper and evaporated under reduced pressure to obtain the ethanol extracts (Et, 405 and 485 g, respectively), which were suspended in H2O, and then partitioned sequentially using ethyl acetate (EA) and n-butanol (BuOH), to get the ethyl acetate fraction (Et-EA), butanol fraction (Et-BuOH), and water fraction (Et-w). For hot-water extraction, 100 g of leaf and stem powder was extracted with 1 L of distilled water in reflux for 3 h. Once cooled to room temperature, the extract was filtered through Whatman no. 1 filter paper and dried under reduced pressure below 45 °C using a rotary evaporator. It was then freeze-dried to obtain hot-water extracts (Hw 5.3 and 3.6 g, respectively) and was stored at −20 °C until further use. Compounds Isolated from S-Et-EA. The S-Et-EA (39 g) was resolved by silica gel column chromatography (90 × 5 cm) using nhexane−EA gradients as eluent. Twenty-four fractions were collected. Fraction 24 (6.2 g) was in turn passed through an RP-18 reversed phase column (60 × 3 cm) and eluted by using water−methanol gradients (1:1 to 0:1) to obtain seven subfractions (each 200 mL, fractions 24A−24G), among which fraction 24D was further purified by semipreparative HPLC (Thermo Betasil C-18 column; flow rate = 2 mL/min) by water/acetonitrile (9:1 to 1:1) gradients to afford (+)-hopeaphenol (1) (7.2 mg, tR = 39.6 min) and (+)-vitisin A (2) (12.3 mg, tR = 46.8 min), respectively; fraction 24E was finally purified by semipreparative HPLC by water/acetonitrile (9:1 to 5:5) to afford (−)-vitisin B (3) (17.2 mg, tR = 48.7 min). The collections of appropriate sample amounts for studies were carried out by repeated injection under the above HPLC conditions. α-Glucosidase Inhibitory Assays. The α-glucosidase inhibitory activity was assayed according to a previous study with modifications.20 Each test sample was dissolved in DMSO. Ten microliters of VTT extracts and fractions (0.1 mg/mL) and different concentrations of purified compounds [(+)-hopeaphenol, 0.78, 1.56, 3.125, 6.25, 12.5, and 25 μM; (+)-vitisin A and (−)-vitisin B, 0.625, 1.25, 2.5, 5, and 10 μM] or acarbose (positive control, 1.67, 3.33, 5.0, 6.67, 8.3, 10, and 11.67 mM) were premixed with 10 μL of α-glucosidase (1 U/mL) at 37 °C for 10 min. Then, 50 μL of 10 mM pNPG (in 200 mM phosphate buffer, pH 6.8) and 50 μL of 200 mM phosphate buffer (pH 6.8) were added and incubated at 37 °C for 30 min. One hundred microliters of 200 mM sodium carbonate was added to stop reactions. DMSO was used instead of tested samples for the blank. For sample background interference determinations, each sample was added into the phosphate buffer only during reactions. The absorbance at 405 nm was measured by using an ELISA reader (TECAN Sunrise microplate reader; Männedorf, Switzerland). The α-glucosidase inhibition (%) was calculated as follows:

classified by the Endemic Species Research Institute, Council of Agriculture, Taiwan, as an endemic herb in Taiwan, and it has long been used in folk medicine for the treatment of hepatitis, jaundice, diarrhea, and arthritis.12 It is reported that extracts or purified compounds from VTT exhibited several biological activities, including antihypertensive,13,14 antiarthritis,15,16 neuroprotective,17 and hepatoprotective18 activities. The active components were reported to be resveratrol, resveratrol oligomers,14−16 and other polyphenols.17,18 Moreover, the ethanolic extract of VTT has exhibited less lipid accumulation through regulation of the AMPK-ACC pathway in hypercholesterolemic rabbits.19 Therefore, the purpose of this study was to investigate whether VTT could decrease lipid levels to improve glucose tolerances in diet-induced obese rats. In the present study, ethanol extracts (Et) and hot-water extracts (Hw) from the stem (S) and leaf (L) of VTT and their Et partitioned fractions, including ethyl acetate fraction (Et-EA), butanol fraction (Et-BuOH), and water fraction (Et-w), were used to investigate yeast α-glucosidase and porcine DPP-IV inhibitory activities. Later, a high-fat (HF) diet was used to induce Wistar rat obesity for 5 weeks. Then VTT-S-Et and VTT-L-Et (20 and 50 mg/kg) were used daily concurrently with the HF diet (HF + S-Et or HF + L-Et) from day 36 to day 68 and were then used to investigate improvements of the impaired glucose tolerances by OGTT. The isolated compounds from S-Et-EA were tested for yeast α-glucosidase and porcine DPP-IV inhibitory activities and related kinetic properties.

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MATERIALS AND METHODS

Materials. α-Glucosidase (from Saccharomyces cerevisiae, type I, lyophilized powder, ≥10 units/mg protein), DPP-IV (from porcine kidney, lyophilized powder, ≥10 units/mg protein, D-7052), pnitrophenyl α-D-glucopyranoside (pNPG), Gly-Pro-p-nitroanilide, dimethyl sulfoxide (DMSO), acarbose, and sitagliptin phosphate were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The HF diet for diet-induced obesity (fat composition provided 60% of total calories, D12942) was purchased from Research Diets, Inc. (New Brunswick, NJ, USA). The standard mouse/rat chow (fat composition provided 12% of total calories, Prolab RMH2500, 5P14 Diet; PMI Nutrition International, St. Louis, MO, USA). The compositions of the standard chow and high-fat diets for providing total calories are listed in Supplementary Table S1 of the Supporting Information. Other chemicals were from Sigma Chemical Co. (St. Louis, MO, USA). For pure compound identifications, optical rotation measurements were measured with a JASCO DIP-180 digital spectropolarimeter, NMR spectra were analyzed in CDCl3 at room temperature using a Varian Mercury plus 400 NMR spectrometer, and the solvent resonance was used as the internal shift reference [tetramethylsilane (TMS) as standard]; electrospray ionization mass was determined on a VG platform electrospray mass spectrometer. Thin-layer chromatography was performed on silica gel 60 F254 plates (Merck, Darmstadt, Germany). Silica gels (230−400 mesh ASTM, Merck) were used for column chromatography. Semipreparative HPLC was performed on a Hitachi L-7000 chromatograph with a Thermo Betasil C-18 column (5 μm, 250 × 10 mm). Preparation of VTT Extracts and Fractions. VTT leaves and stems were collected in Taichung County, Taiwan, in July 2006. They were cultivated by Dr. Chi-Luan Wen (Taiwan Seed Improvement and Propagation Station, Council of Agriculture, Taichung, Taiwan) and identified by Dr. Tsai-Wen Hsu (Endemic Species Research Institute, Nantou, Taiwan). The specimens were kept in the laboratory of Dr. Chi-I Chang (National Pingtung University of Science and Technology, Pingtung, Taiwan) for reference. All plant materials were oven-dried at 50 °C for 3 days and mechanically ground to a fine powder passing through a 10-mesh sieve. The powder of the leaves

[A 405 blank − (A 405sample − A 405background )] A 405 blank

× 100%

The 50% inhibition (IC50) value was calculated as the concentration of samples that inhibited 50% of α-glucosidase activity under tested conditions. DPP-IV Inhibitory Assays. The DPP-IV inhibitory activity was assayed according to a previous study with modifications.21,22 The enzyme powder (lyophilized powder, ≥10 units/mg protein) was redissolved in 1 mL of 100 mM Tris buffer (pH 8.0) as a stock solution and a 50-fold dilution (∼0.002 unit/mL) as a working solution. Each test sample was dissolved in DMSO. Fifty microliters of VTT extracts and fractions (0.5 mg/mL) and different concentrations of purified compounds [(+)-hopeaphenol, 100, 200, 300, 400, and 500 μM; (+)-vitisin A, 25, 50, 75, 100, 125, 150, and 200 μM; (−)-vitisin B, 2.5, 5, 10, 15, 20, 25, and 30 μM] or sitagliptin phosphate (positive control, 0.01, 0.025, 0.05, 0.1, and 0.2 μM) were premixed with 50 μL of DPP-IV working solution at 37 °C for 10 min. Then, 50 μL of 1 mM Gly-Pro-p-nitroanilide and 50 μL of 100 mM Tris buffer (pH 8.0) were added and incubated at 37 °C for 60 min. Fifty microliters of 3% acetic acid was added to stop reactions. DMSO was used instead of tested samples for the blank. To determine sample background B

DOI: 10.1021/acs.jafc.5b02069 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 1. Effect of 0.5 mg/mL extracts and partitioned fractions on dipeptidyl peptidase-IV (DPP-IV) inhibitory activities from stem of Vitis thunbergii var. taiwaniana (A) and from leaf of V. thunbergii var. taiwaniana (B); effect of 0.1 mg/mL extracts and partitioned fractions on αglucosidase inhibitory activities from stem of V. thunbergii var. taiwaniana (C) and from leaf of V. thunbergii var. taiwaniana (D). S, stem; L, leaf; Et, ethanol extracts; Hw, hot-water extracts; Et-EA, ethyl acetate partitioned fractions from Et; Et-BuOH, butanol partitioned fractions from Et; Et-w, Et partitioned water fractions. Triplicates were determined and expressed as the mean ± SD. Multiple group comparisons under the same treatment time were performed using one-way analysis of variance followed by the post hoc Tukey’s test, and values that have not been marked with the same letter are significantly different (P < 0.05). Impaired Glucose Tolerance in Obese Rat Models. Male 10week-old Wistar rats (N = 48) were purchased from the National Laboratory Animal Center (Taipei, Taiwan) and housed in wirebottomed stainless steel cages in a temperature- and humiditycontrolled room (at 22 °C) with a 12-h light/dark cycle and had free access to feed and water. All animal experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee, Taipei Medical University (LAC-99-0136). After acclimation for 1 week, the rats were randomly divided into three groups (N = 8 for each group), including a normal diet (ND) group given standard mouse/rat chow and five HF diet-induced obese groups (one control as HF group, two VTT-S-Et intervened groups, and two VTT-L-Et intervened groups) for 68 days. The Wistar rats were subjected to diet-induced obesity during days 0−35 and then randomly divided into groups for further interventions at days 36− 68. The HF was changed every 2 days to avoid oxidation. In the VTTS-Et and VTT-L-Et intervened groups, S-Et or L-Et (20 and 50 mg/kg, respectively, Et20 and Et50) was orally administered daily by oral gavage together with HF diets (HF + S-Et20, HF + S-Et50; HF + LEt20, HF + L-Et50) from days 36 to 68. The rat weights were recorded during experiments. The impaired glucose tolerances of HF diet-induced obese rats were measured by OGTT methods following a previous study.22 Rats of each group at day 68 were fasted for 16 h, and glucose (1g/kg) was administered by oral gavage. Blood (0.1 mL) was obtained from the tail vein at 0 (basal level), 5, 30, 60, 90, and 120 min after the glucose loads. Plasma glucose was determined by using a

interference, each sample was added into the Tris buffer only during reactions. The absorbance at 405 nm was measured by using an ELISA reader (TECAN Sunrise microplate reader). The DPP-IV inhibition (%) was calculated as follows:

[A 405 blank − (A 405sample − A 405background )] A 405 blank

× 100%

The 50% inhibition (IC50) value was calculated as the concentration of sample that inhibited 50% of DPP-IV activity under tested conditions. Kinetic Analyses of α-Glucosidase and DPP-IV. The kinetic properties of α-glucosidase (0.083 unit) without or with different concentrations of (+)-vitisin A (0.3125 and 1.25 μM) and (−)-vitisin B (0.3125 and 0.625 μM) were determined using different concentrations of pNPG as substrate (0.625, 1.25, 2.5, and 5 mM). The kinetic properties of DPP-IV (0.005 unit) without or with different concentrations of (−)-vitisin B (5, 15, and 25 μM) were determined using different concentrations of Gly-Pro-p-nitroanilide as substrate (0.1, 0.2, 0.4, 0.6, 0.8, and 1 mM). The Km, K′m, Vmax, and V′max were calculated from Lineweaver−Burk plots. The enzyme− inhibitor dissociation constant (Ki) was the intercept of the x-axis calculated (each line slope in the Lineweaver−Burk plot vs the tested inhibitor concentrations), and the inhibitor-enzyme−substrate dissociation constant (Kis) was the intercept of the x-axis calculated (each line y-axis intercept in the Lineweaver−Burk plot vs the tested inhibitor concentrations). C

DOI: 10.1021/acs.jafc.5b02069 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 2. Effects of (A) stem ethanol extracts (S-Et) interventions (20 and 50 mg/kg) and (B) leaf ethanol extracts (L-Et) interventions (20 and 50 mg/kg) of Vitis thunbergii var. taiwaniana on the changes of increased body weight. Effects of (C) S-Et20 and S-Et50 and (D) L-Et20 and L-Et50 on impaired glucose intolerances of high fat-diet induced obese rats by oral glucose tolerance tests. The HF diet and normal diet (ND) were provided from day 1 to day 68. The arrow indicates the intervened periods from day 36 to day 68. Data are expressed as the mean ± SD. ND, normal diet; HF, high-fat diet; (HF + Et20 or HF + Et50), ethanol extracts (20 mg/kg, Et20; 50 mg/kg, Et50) were intervened daily concurrent with the HF diet. Multiple group comparisons at the end of experiment were performed using one-way analysis of variance (ANOVA) followed by the post hoc Tukey’s test. Values that have not been marked with the same letter are significantly different (P < 0.05).

concentration of 0.1 mg/mL, the order of VTT extracts for αglucosidase inhibitory activity weas S-Et ≥ L-Et ≫ S-Hw ≫ LHw; the order of Et partitioned fractions for α-glucosidase inhibitory activity was S-Et-EA ≅ S-Et-BuOH ≅ S-Et-w ≅ L-EtEA ≫ L-Et-BuOH ≫ L-Et-w, and the maximal α-glucosidase inhibition was close to 100% (Figure 1C,D). Impaired Glucose Tolerance Improvements of S-Et and L-Et in High-Fat (HF) Diet-Induced Obese Rats. HF diets are reported to induce an overweight condition, obesity, dyslipidemia, insulin resistance, and high blood pressure in rodents.23 The α-glucosidase and DPP-IV inhibitions can reduce postprandial blood glucose levels and prolong GLP-1 and GIP biological activities. Therefore, a HF diet was used to preinduce obese rats, and then Et-S/Et-L was used together with HF to investigate the improved possibility of impaired glucose tolerance in obese rats. Figure 2 shows the increased body weights in Wistar rats fed normal diets (ND) or HF without or with S-Et (Figure 2A) or L-Et (Figure 2B) interventions at doses of 20 mg/kg (Et20) and 50 mg/kg (Et50). The increased body weight (g) at day 68 in HF groups was 212.73 ± 21.05, 223.47 ± 7.66, 199.93 ± 10.46, 212.37 ± 19.67, and 193.53 ± 14.19, respectively, for HF + S-Et20, HF + S-Et50, HF + L-Et20, HF + L-Et50, and HF. The rats in the HF groups without or with S-Et or L-Et interventions showed no significant difference (P > 0.05) in weight increases on the day of experiment end. The increased body weight (g) at day 68 in the ND group was only 119.35 ± 9.00. This was significantly

RANDOX glucose kit (Randox Laboratories-US, Ltd., Kearneysville, WV, USA). Statistical Analyses. Data are expressed as the mean ± SD. Multiple group comparisons were performed using one-way analysis of variance (ANOVA) followed by the post hoc Tukey’s test. The animal experiments were compared among groups on the day of the experiment end. Values that have not been marked with the same letter are significantly different (P < 0.05). Statistical analysis was performed using GraphPad Prism 5.0 software (San Diego, CA, USA).

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RESULTS AND DISCUSSION DPP-IV and α-Glucosidase Inhibitory Activities of VTT Extracts and Fractions. VTT was reported to have different biological activities depending on which plant portions (roots, stems, or leaves) were used in combination with different solvent extracts.14−19 Therefore, the Et and Hw from S and L and their Et partitioned fractions, S-Et-EA and L-Et-EA, S-EtBuOH and L-Et-BuOH, and S-Et-w and L-Et-w, were used to investigate inhibitory activities against DPP-IV (Figure 1A,B) and yeast α-glucosidase (Figure 1C,D). Generally, the VTT extracts and Et partitioned fractions exhibited higher yeast αglucosidase inhibitory activities than those of the DPP-IV inhibitory ones. At the same concentration of 0.5 mg/mL, the order of VTT extracts for DPP-IV inhibitory activity was S-Hw ≥ L-Hw > S-Et ≫ L-Et; the order of Et partitioned fractions for DPP-IV inhibitory activity was S-Et-EA > S-Et-BuOH > L-Et-w ≫ L-Et-EA ≅ L-Et-BuOH ≅ S-Et-w, and the maximal DPP-IV inhibition was close to 40% (Figure 1A,B). At the same D

DOI: 10.1021/acs.jafc.5b02069 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 3. Chemical structures of three compounds, (+)-hopeaphenol (1), (+)-vitisin A (2), and (−)-vitisin B (3), isolated from the ethyl acetate fractions of stem ethanol extracts of Vitis thunbergii var. taiwaniana.

tensive rats. The improved OGTT profiles in obese rats of VTT-S-Et or VTT-L-Et interventions together with antihypertensive activities15 provided evidence that VTT extracts could be processed as health-promoting foods. The HF provides 20 and 60% of total calories, respectively, from carbohydrate and fat in the diets. S-Et and L-Et showed excellent yeast α-glucosidase inhibitory and partial DPP-IV inhibitory activities in vitro (Figure 1), which might correlate with the improvement of impaired glucose tolerance in obese rats (Figure 2C,D). The S-Et intervention showed better than L-Et in OGTT results (Figure 2B). The S-Et-EA fraction in vitro screening test showed good anti-α-glucosidase and DPPIV inhibitory activities; therefore, the EA partitioned fraction from S-Et was selected to isolate the active compounds for further studies. Compounds Isolated from S-Et-EA. Stilbenoid compounds are rich in Vitis genus plants and are considered as the active constituents in them. All 24 fractions were analyzed by HPLC method and monitored at the characteristic UV absorption wavelength of stilbenoids, 280 nm. Fraction 24 was found to contain the most abundant stilbenoid compounds and was thus selected for further purification. Three resveratrol tetramers, 1−3 (Figure 3), were isolated from the S-Et-EA and identified by comparing their physical and spectral data (specific rotation, MS, and NMR) with the available values in the literature. The chemical structures of these compounds were identified as (+)-hopeaphenol (1),24 (+)-vitisin A (2),25 and (−)-vitisin B (3),26 and the spectral properties were as follows. (+)-Hopeaphenol (1): brown-yellow powder; [α]25 D +390.0° (c 0.35, MeOH); ESI-MS m/z 907 [M + H]+; 1H NMR (400 MHz, acetone-d6) δ 7.12 (2H, d, J = 8.4 Hz, H-2a, 6a), 6.89 (2H, d, J = 8.4 Hz, H-2b, 6b), 6.77 (2H, d, J = 8.8 Hz, H-3a, 5a), 6.54 (2H, d, J = 8.8 Hz, H-3b, 5b), 6.28 (2H, d, J = 2.4 Hz, H-14a), 5.80 (1H, br s, H-7b), 5.73 (1H, d, J = 12.0 Hz, H-7a), 5.71 (1H, d, J = 2.0 Hz, H-12b), 5.15 (1H, d, J = 2.0 Hz, H14b), 4.22 (1H, d, J = 12.0 Hz, H-8a), 3.93 (1H, d, J = 4.8 Hz, H-7b). (+)-Vitisin A (2): brown solid; [α]D25 +210.5° (c 0.4, MeOH); ESI-MS m/z 907[M + H]+; 1H NMR (400 MHz, acetone-d6) δ 7.19 (2H, d, J = 8.5 Hz, H-2a, 6a), 7.14 (2H, d, J = 8.5 Hz, H-2c, H-6c), 7.02 (2H, d, J = 8.3 Hz, H-2d, H-6d), 6.87 (1H, dd, J = 8.4, 2.2 Hz, H-6b), 6.81 (2H, d, J = 8.5 Hz, H-

different (P < 0.05) compared to the HF group. The standard chow of normal diet (Table S1) in the present experiment was not a real low-fat diet control, and hence the protocol was not easy to compare to the efficiency of the normal diet group and the HF-induced groups in the increased rat body weights. From the present results, S-Et or L-Et interventions at 20 and 50 mg/ kg for around 5 weeks showed no weight reduction in preobese rat models. Meanwhile, rats with similar weights in different HF groups and rats in the ND group were used to evaluate the impaired glucose tolerance by OGTT after fasting for 16 h (Figure 2C,D). Figure 2C shows the basal level (0 min) and plasma glucose concentrations (mg/dL) after glucose loads at 5, 30, 60, 90, and 120 min of HF + S-Et20, HF + S-Et50, HF, and the blank groups. After glucose loads for 120 min, the rats in the HF group still showed high plasma glucose concentrations, a symptom of impaired glucose tolerance. However, the rats in the S-Et intervened groups seemed to improve with respect to impaired glucose tolerance in OGTT profiles. The obese rats in the HF + S-Et20 and HF + S-Et50 groups showed the lowest plasma glucose concentrations at the basal level, and each time the intervals and significant differences (P < 0.05) were compared to those of the HF group and the ND group. Figure 2D shows the basal level (0 min) and plasma glucose concentrations (mg/dL) after glucose loads at 5, 30, 60, 90, and 120 min of HF + L-Et20, HF + LEt50, and HF compared to the ND group. The rats in the L-Et intervened groups seemed to improve with respect to impaired glucose tolerance in OGTT profiles. The plasma glucose concentrations of obese rats in the intervened groups of HF + L-Et20 and HF + L-Et50 showed lower and significant differences (P < 0.05) compared to those of the HF group at the basal level and at each time interval. Rats in the L-Et50 intervened groups showed better and significant difference (P < 0.05) in OGTT profiles at 0, 5, 30, and 60 min after glucose loads and were comparable at 90 and 120 min time intervals compared to rats in the ND group. From the present OGTT data, S-Et and L-Et interventions showed improved activities against impaired glucose tolerance in obese rats, and S-Et interventions seemed to result in greater improvement than LEt interventions. Lin et al.15 reported that VTT-S-Et showed better than VTT-L-Et in angiotensin converting enzyme inhibitory activities, and the oral administration of VTT-S-Et exhibited antihypertensive activities in spontaneously hyperE

DOI: 10.1021/acs.jafc.5b02069 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 4. (A) Effect of different concentrations of (+)-hopeaphenol, (+)-vitisin A, and (−)-vitisin B on α-glucosidase inhibitory activities compared to the positive control of acarbose: Lineweaver−Burk plots of α-glucosidase (0.083 U) (B) without or with (+)-vitisin A (0.3125 and 1.25 μM) or (C) without or with (−)-vitisin B at the different concentrations of p-nitrophenyl α-D-glucopyranoside as substrates (0.625, 1.25, 2.5, and 5 mM). Fifty percent inhibition (IC50) of α-glucosidase activity was calculated as the concentration of sample that inhibited 50% of α-glucosidase activity under tested conditions. Data are expressed as the mean ± SD (n = 3).

H-5d), 6.76 (1H, d, J = 16.8 Hz, H-8b), 6.70−6.62 (5H, m, H5b, H-2b, H-14b, H-2c, H-6c), 6.61 (1H, d, J = 16.8 Hz, H-7b), 6.59 (2H, d, J = 8.6 Hz, H-3c, H-5c), 6.33 (2H, br s, H-12c), 6.24−6.19 (5H, m, H-12b, H-10d, H-14d, H-12d, H-14c, H12a), 6.13 (2H, d, J = 2.2 Hz, H-10a, H-14a), 5.55 (1H, d, J = 4.7 Hz, H-7c), 5.41 (2H, m, H-7d, H-7a), 4.53 (1H, d, J = 4.5 Hz, H-8a), 4.45 (1H, d, J = 5.4 Hz, H-8d), 4.33 (1H, d, J = 4.7 Hz, H-8c). Anti-α-glucosidase Activities of (+)-Hopeaphenol, (+)-Vitisin A, and (−)-Vitisin B. Three isolated compounds were used to determine the α-glucosidase inhibitory activities compared to the positive control of acarbose (Figure 4A). Before the determinations of IC50 inhibitions, the same 10 μM was used for prescreening tests. For hopeaphenol, about 60% inhibition was found, and for vitisin A and vitisin B, almost

3a, 5a), 6.77 (2H, d, J = 8.5 Hz, H-3c, H-5c), 6.68 (1H, d, J = 8.4 Hz, H-5b), 6.65 (2H, d, J = 8.3 Hz, H-3d, H-5d), 6.50 (1H, d, J = 2.2 Hz, H-14b), 6.38 (2H, br s, H-7b, H-8b), 6.25 (1H, d, J = 2.2 Hz, H-12b), 6.23 (1H, d, J = 2.2 Hz, H-14c), 6.21 (1H, d, J = 2.2 Hz, H-12a), 6.16 (2H, d, J = 2.2 Hz, 10a, 14a), 6.08 (1H, d, J = 1.8 Hz, H-12d), 6.07 (1H, d, J = 2.2 Hz, H-2b), 6.04 (1H, d, J = 1.8 Hz, H-14d), 6.02 (1H, d, J = 2.2 Hz, H-12c), 5.87 (1H, d, J = 11.6 Hz, H-7c), 5.47 (1H, d, J = 3.3 Hz, H-8d), 5.38 (1H, d, J = 3.3 Hz, H-7d), 5.35 (1H, d, J = 5.3 Hz, H-7a), 4.40 (1H, d, J = 5.3 Hz, H-8a), 4.23 (1H, d, J = 11.6 Hz, H-8c). (−)-Vitisin B (3): brown solid; [α]25 D −112.4° (c 0.5, MeOH); ESI-MS m/z 907 [M + H]+; 1H NMR (400 MHz, acetone-d6) δ 7.26 (2H, d, J = 8.6 Hz, H-2a, H-6a), 7.20 (2H, d, J = 8.5 Hz, H-2d, H-6d), 7.14 (1H, d, J = 8.3 Hz, H-6b), 6.92 (2H, d, J = 8.6 Hz, H-3a, H-5a), 6.83 (2H, d, J = 8.6 Hz, H-3d, F

DOI: 10.1021/acs.jafc.5b02069 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Table 1. Kinetic Parameters of α-Glucosidase and DPP-IV Inhibition in the Presence of (−)-Vitisin B or (+)-Vitisin A α-glucosidase inhibition (+)-vitisin A control inhibition type K m or K′ma (mM) Vmax or V′maxb Kic (μM) Kisd (μM)

0.719 0.095

(−)-vitisin B

0.3125 μM

1.25 μM

mixed noncompetitive 0.845 0.0871 1.784 3.842

0.981 0.0737

0.3125 μM

control 0.719 0.095

mixed noncompetitive 0.866 0.086 0.633 1.249

0.625 μM 1.058 0.072

DPP-IV inhibition (−)-vitisin B 5 μM

control inhibition type Km or K′ma (mM) Vmax or V′maxb Kic (μM) Kisd (μM)

15 μM

mixed noncompetitive 1.029 0.941 0.0153 0.00878 7.661 11.361

1.25 0.0208

25 μM 0.927 0.00543

a

Km and K′m are Michaelis constants in the absence and presence of inhibitor, respectively. bVmax and V′max are maximal velocities in the absence and presence of inhibitor, respectively. cEnzyme−inhibitor dissociation constant. dEnzyme−substrate dissociation constant.

potency. Tadera et al.27 reported the potent flavonoids against yeast α-glucosidase activities in mixed noncompetitive patterns with Ki and Kis values of 3 and 7.6 μM for epigallocatechin gallate, 3 μM and 7.6 μM for myricetin, 3.4 μM and 14 μM for quercetin, and 52 μM and 4.2 μM for genistein, which were higher than those with (+)-vitisin A (1.784 and 3.842 μM) and (−)-vitisin B (0.633 and 1.249 μM) in the present study. The yeast α-glucosidase has been widely used to evaluate αglucosidase inhibitory ability. However, Oki et al.28 have indicated that the efficacy of α-glucosidase inhibition was greatly affected by different origins. Therefore, α-glucosidase from mammalian will be used for further investigations. Anti-DPP-IV Activities of (+)-Hopeaphenol, (+)-Vitisin A, and (−)-Vitisin B. Three isolated compounds from S-Et-EA were used to determine the DPP-IV inhibitory activities compared to the positive control of sitagliptin (Figure 5A). Before the determinations of IC50 inhibitions, the same 25 μM was used for prescreening tests. For hopeaphenol, about 10% inhibition was found; for vitisin A, about 25% inhibition was found; and for vitisin B, about 80% inhibition was found. Therefore, different dilutions and different concentrations for each purified compound were used to perform DPP-IV inhibitions. These three compounds, (+)-hopeaphenol, (+)-vitisin A, and (−)-vitisin B, showed dose-dependent inhibitions against DPP-IV with IC50 values of 401, 90.75, and 15.3 μM, respectively, compared to sitagliptin (IC50 = 47.35 nM). (−)-Vitisin B showed about 26- and 6-fold better DPP-IV inhibitions than (+)-hopeaphenol and (+)-vitisin A and less than that of sitagliptin. Therefore, the potent DPP-IV inhibitor (−)-vitisin B was selected for further kinetic analyses. (−)-Vitisin B showed mixed noncompetitive type (close to noncompetitive type) inhibition against DPP-IV with respect to Gly-pNA (substrate) and Gly-pNAp−DPP-IV (substrate− enzyme complex) (Figure 5B). In the absence of (−)-vitisin B, the Km and Vmax were 1.25 mM and 0.0208 (ΔA405/min), respectively; with 5, 15, and 25 μM (−)-vitisin B additions, the K′m was decreased to 1.029, 0.941, and 0.927 mM. However, the V′max was also decreased to 0.0153, 0.00878, and 0.00543 (ΔA405/min). The calculated Ki and Kis for (−)-vitisin B, respectively, were 7.661 and 11.361 μM (Table 1). The DPPIV inhibitory activities were frequently screened from protein

100% inhibition was found. Therefore, different dilutions and different concentrations for each purified compound were used to perform α-glucosidase inhibition. These three compounds, (+)-hopeaphenol, (+)-vitisin A, and (−)-vitisin B, showed dose-dependent inhibitions against α-glucosidase with IC50 values of 18.3, 1.22, and 1.02 μM, respectively, compared to the IC50 of acarbose of 6.39 mM. These three resveratrol tetramers with the same molecular mass showed much more significant α-glucosidase inhibitory activities than that of acarbose. (+)-Hopeaphenol, (+)-vitisin A, and (−)-vitisin B showed about 350-, 5340-, and 6265-fold better α-glucosidase inhibitions than acarbose. Therefore, the first two potent αglucosidase inhibitors of (+)-vitisin A and (−)-vitisin B with similar IC50 values were selected for further kinetic analyses. It was found that (+)-vitisin A (Figure 4B) and (−)-vitisin B (Figure 4C) showed mixed-type noncompetitive inhibition against α-glucosidase with respect to pNPG (substrate) and pNPG−α-glucosidase (substrate−enzyme complex). In the absence of (+)-vitisin A, the Km and Vmax were 0.719 mM and 0.095 (ΔA405/min), respectively; whereas with the 0.3125 and 1.25 μM (+)-vitisin A additions, the K′m was increased to 0.845 and 0.981 mM, and V′max was decreased to 0.0871 and 0.0737 (ΔA405/min). The calculated Ki and Kis for (+)-vitisin A, respectively, were 1.784 and 3.842 μM (Table 1). In the absence of (−)-vitisin B, the Km and Vmax were 0.719 mM and 0.095 (ΔA405/min), respectively; with the 0.3125 μM and 0.625 μM (−)-vitisin B additions, the K′m was increased to 0.866 and 1.058 mM and V′max was decreased to 0.086 and 0.072 (ΔA405/min). The calculated Ki and Kis for (−)-vitisin B, respectively, were 0.633 and 1.249 μM (Table 1). Tadera et al.27 tested 16 flavonoids against yeast α-glucosidase activity (IC50, μM), among which epigallocatechin gallate (2 μM), cyanidin (4 μM), myricetin (5 μM), and quercetin and genistein (7 μM) showed excellent yeast α-glucosidase inhibition with IC50 values of