α-Glucosidase inhibiting activity and bioactive compounds of six red wine grape pomace extracts

Abstract

Grape pomace contains considerable amounts of polyphenols and it has been reported to exhibit specific inhibitory activity against mammalian intestinal α-glucosidases. This study aims to investigate the anti-diabetes potential of Chambourcin, Merlot, Norton, Petit Verdot, Syrah and Tinta Cão red wine grape pomaces by assessing their rat intestinal α-glucosidase inhibitory activity in relation to their total phenolic content and individual identified phenolic compounds by HPLC. Among the selected pomaces, Tinta Cão, Syrah and Merlot extracts showed higher potency in inhibiting α-glucosidase, and appeared to have higher respective total phenolic contents. Fifteen phenolic compounds were identified in the pomace samples, however, none of them showed significant inhibition of intestinal α-glucosidases. Red grape pomace, namely Tinta Cão, appears to be a promising functional food for the potential future development of a food-derived α-glucosidase inhibitor for preventing and treating diabetes.

1. Introduction

The prevalence of obesity in the US has magnified in the last 20 years (Ogden, Carroll, Kit, & Flegal, 2012). The state of chronic inflammation and oxidative stress that obesity has been associated with is believed to play a role in promoting obesity-related complications such as insulin resistance and type-2 diabetes (Hardy, Czech, & Corvera, 2012; Hotamisligil, 2006). Another common metabolic attribute linked to obesity is hyperglycaemia (Ferguson, Gallagher, Scheinman, Damouni, & Leroith, 2013; Hardy et al., 2012), which in turn has been associated with the precipitation of oxidative stress and inflammation (Colak et al., 2013; Dey & Lakshmanan, 2013), thus further increasing diabetic risks and complications (Giacco & Brownlee, 2010; Henriksen, Diamond-Stanic, & Marchionne, 2011; Zatalia & Sanusi, 2013). It is hence of no surprise that diabetes currently affects 29.1 million people in the U.S. and the number of Americans with prediabetes is on the rise. The costs associated with diabetes management and treatment have become a significant burden in the American society (CDC, 2014).

Type-2 diabetes is a chronic condition characterised by insulin resistance and β-cell failure, resulting from poor lifestyle habits that interact with an underlying genetic susceptibility (Stumvoll, Goldstein, & van Haeften, 2005). Given the overwhelming rise in this disease, it is imperative to explore novel approaches to prevent and control it, particularly in the light of the side effects and limited long-term durability associated with conventional anti-hyperglycaemic agents (Majumdar & Inzucchi, 2013). Inhibition of α-glucosidases has been shown to be effective in both preventing and treating type-2 diabetes through reducing postprandial hyperglycaemia (van de Laar et al., 2005a, 2005b). However, commercial inhibitors are often associated with gastrointestinal side effects due to their non-specific inhibitory activity (Martin & Montgomery, 1996; Santeusanio & Compagnucci, 1994), which necessitates the search for alternatives. Meanwhile, plant sources continue to serve as an inexhaustible reservoir of bioactive compounds (Cowan, 1999). In a screening for natural, food-derived α-glucosidase inhibitors, we identified a red grape pomace extract possessing specific α-glucosidase inhibitory activity (Hogan et al., 2010). However, comparison of a wider range of grape pomaces and obtaining inference on the components responsible for the inhibitory activity have not been achieved, to our knowledge.

Grape pomace, the solid remains of grape after pressing, is commonly considered a waste byproduct generated in the winemaking industry (Lafka, Sinanoglou, & Lazos, 2007). On the other hand, grapes and wines are widely acknowledged as an important source of antioxidants, namely polyphenolic compounds such as flavanols, catechins, anthocyanins, and proanthocyanidins (Frankel, 1999; Frankel, Kanner, German, Parks, & Kinsella, 1993; Katiyar, 2008; Lorrain, Ky, Pechamat, & Teissedre, 2013). Since grape pomace is chiefly comprised of the skins and seeds, it is surmised that this biomass is a rich source of antioxidants (Chidambara Murthy, Singh, & Jayaprakasha, 2002; Kammerer, Claus, Carle, & Schieber, 2004; Ruberto et al., 2007). While the literature associates dietary antioxidants with a reduced risk of type 2 diabetes (Montonen, Knekt, Jarvinen, & Reunanen, 2004; Willcox, Willcox, Todoriki, & Suzuki, 2009), it provides very limited information on the potential of grape pomace as an alternative bioresource for diabetes management. Studies showed that different grapes exhibit varying phenolic contents based on the cultivars and growing environments. The aim of this study is to evaluate the anti-diabetic potential of a selection of six red wine grape pomaces by determining their α-glucosidase inhibitory activity in relation to their total phenolic content. This research may lay the foundation for the future development of a food-derived α-glucosidase inhibitor from grape pomace for preventing and treating type-2 diabetes.

2. Materials and methods

2.1. General

The organic solvents for grape pomace extraction and HPLC analysis were HPLC grade (Fisher Scientific, Atlanta, GA). Intestinal acetone powders from rat, 4-nitrophenyl-α-d-glucopyranoside (pNPG), Folin–Ciocalteu reagent, and phenolic standards including caffeic acid, delphinidin chloride, gallic acid, malvin chloride, malvidin chloride, quercetin hydrate and quercetin 3-O-glucoside were purchased from Sigma-Aldrich (St. Louis, MO). Acarbose and other phenolic standards including catechin, epicatechin gallate, kaempferol, myricetin and resveratrol were obtained from LKT Laboratories, Inc. (St. Paul, MN). Phenolic standards including cyanidin chloride and p-coumaric acid were purchased from Fluka Analytical (Buchs, Switzerland). Rutin was purchased from ACROS (Geel, Belgium).

2.2. Grape pomace

Six red wine grape varieties: Chambourcin (hybrid), Merlot (Vitis vinifera), Norton (Vitis aestivalis), Petit Verdot (V. vinifera), Syrah (V. vinifera) and Tinta Cão (V. vinifera) were kindly provided by Chrysalis Vineyards (Middleburg, VA). The pomaces were shipped immediately after pressing. Upon receipt of the samples, they were immediately dried in a food dehydrator at 95 °F for 28 h.

2.3. Sample extraction

The pomaces were separated from stems and ground to a powder consistency followed by sifting and manual removal of visible solid impurities. Grape pomace powder was soaked and stirred overnight (12 hours) at 450 rpm in aqueous acetone (50%) at a concentration of 0.1 g/mL and collected supernatants were centrifuged at 1000 rpm for 5minutes. Supernatants were retained and filtered using a 20 µm Whatman filter paper via suction filtration with pump-generated vacuum. The filtered extract was then transferred to a Rotavapor (Büchi Labortechnik AG, Flawii, Switzerland) where the solvent was isolated via evaporation at 50–180 rpm and 40–60 °C, in gradual increments, and condensation at 4–8 °C to obtain a solvent-free grape pomace extract in pure water. The extract was frozen at −80 °C, lyophilised and stored in powder form at 4° C for use in screening. The prepared grape pomace extract (GPE) powders were reconstituted with 50% acetone and diluted with ddH₂O to a concentration of 0.5 mg/mL.

3. α-Glucosidase inhibiting potential and bioactive compounds in GPEs

3.1. Preparation of rat intestinal α-glucosidases

Intestinal acetone powders from rat were extracted with 0.05 M phosphate buffer (PB) pH 6.8 at a concentration of 25 mg/mL. The solution was soaked and stirred overnight at 450 rpm and supernatants were isolated and centrifuged at 1000 rpm for 5 minutes. Supernatants were retained and filtered via vacuum filtration using a 20 µM Whatman filter paper. The filtered solution was frozen at −80 °C, lyophilised and reconstituted with 0.05 M PB pH 6.8 to a concentration of 25 mg/mL. Ready-to-use aliquots of this concentration were stored at −20 °C.

3.2. α-Glucosidase inhibition assay

α-Glucosidase enzyme at 25 mg/mL was used from prepared aliquots. 4-Nitrophenyl-α-d-glucopyranoside (pNPG) was used as a substrate at a 4 mM concentration. Briefly, α-glucosidase enzyme complex hydrolyses pNPG and releases p-nitrophenol (pNP). Absorbance reading at 405 nm quantitates the release of pNP thus representing enzymatic activity (Zhang et al., 2011). Acarbose, known to inhibit α-glucosidase enzyme complex and used as an oral blood glucose lowering drug in diabetes (Martin & Montgomery, 1996), served as a positive control at 50 µg/mL. Enzyme, substrate and Acarbose/sample solutions were all prepared in 0.05 M PB pH 6.8, and the PB solution was used as a negative control during experiment. Ninety six-well bioassay microplates were prepared to contain 115 µL of GPE samples or the individual compounds (commercial standards) or control, 90 µL of enzyme solution and 45 µL of substrate solution per well, mixed thoroughly. Absorbance at a wavelength of 405 nm was obtained at start of the reaction using a Perkin Elmer HTS 7000 Bio Assay Reader and software (Perkin Elmer, Norwalk, CT). The microplate was then incubated at 37 °C and absorbance readings were obtained again at 30 and 90 minutes with intense shaking between cycles. The absorbance readings, representing the concentration of pNP, were then used to compare the activity of the tested samples: the lower the value, the less active the enzyme, thus representing greater inhibition by the GPE sample. Percent inhibition by all samples was calculated and compared to controls to determine potency, using the following formula:

$$\%\text{ Inhibition}=100-\{({\text{Abs}}_{\text{sample}}/{\text{Abs}}_{\text{control}})\times 100\}.$$

4. Estimation of total phenolic content (TPC) in the GPE samples

TPC was evaluated with Folin–Ciocalteu’s phenol reagent. Samples were diluted to 2 mg/mL with aqueous acetone. Gallic acid was used as a standard for preparing the standard curve. All the samples and standards were run in triplicate. Each test tube contained 25 µL of a sample or standard and 250 µL distilled water. 750 µL Folin–Ciocalteu’s phenol reagent was then added to each tube and mixed using a vortex mixer. Then, 500 µL of 200 mg/mL sodium carbonate was added to each tube and mixed thoroughly. Samples and standards were incubated for 2 h at room temperature in the dark. Absorbance was detected at 765 nm and the TPC of each sample was expressed as milligrams of gallic acid equivalents (GAE) per mg GPE.

5. High performance liquid chromatography analysis of phenolic compounds in GPEs

Fifteen phenolic compounds, typically reported in grape and wine, were used as standards to identify and quantify our GPE samples. The extracts were first cleaned using solid phase extraction (Oasis HLB 6 cc extraction cartridge, Waters Corporation, Milford, MA) to remove sugar and other contaminants. After drying with nitrogen gas, each sample/standard was dissolved in methanol and filtered using a 0.45 micron, 3 mm syringe filter. Reversed-phase HPLC was employed to profile individual phenolic compounds in the cleaned extracts against known phenolic standards, using a Hitachi HPLC system (Model L-2455 Diode Array Detector, Model L-2200 Autosampler, Model L-2100/2130 Pump) from Hitachi High-Tech Technologies (Tokyo, Japan). A Phenomenex Aqua 5 µm C₁₈ 250 × 4.6 mm analytical column (Phenomenex, Torrance, CA) represents the stationary phase while 0.5% acetic acid in 50% acetonitrile and 2% acetic acid were utilised as mobile phase solvents A and B, respectively. Twenty µL of each sample was injected via the autosampler at a 0–5689 psi pressure range, under room temperature. Gradient systems were used as follows: 10–26% A, 0–8 min; 26% A, 8–15 min; 26–30% A, 15–20 min; 30–55% A, 20–42 min; 55–87% A, 42–75 min; 87–100% A, 75–78 min; 100% A, 78–83 min; 100-10% A, 83–85 min; 10% A, 85–90 min. Flow rate was set at 1 mL/min. Samples and standards were monitored by UV detection at 280 nm and profiled at a wide range of wavelengths (200–700 nm), selecting the optimal wavelength for comparison. Profiles of standards and samples were compared, detected, and quantified on the basis of their retention time and UV spectrum.

5.1. Statistical analysis

Results were analysed via IBM SPSS 22.0 for Windows (IBM Corp., Armonk, NY) using one-way analysis of variance (ANOVA). Tukey’s HSD post-hoc analyses were employed to compare outcomes using P < 0.05 as a cutoff point for statistical significance. Pearson’s correlation was conducted to study the relationship between variables. Data for each dependent variable are reported as mean ± SEM.

6. Results

6.1. Inhibition of mammalian α-glucosidases

Percent enzyme inhibition by GPEs is presented in Fig. 1. With the exception of Petit Verdot, the selected GPEs showed potent inhibition against rat intestinal α-glucosidases. At a concentration of 0.5 mg/mL of dry extract, Tinta Cão exerted the strongest inhibition of intestinal α-glucosidases, measured as 95% (P < 0.05). Chambourcin, Norton, Merlot and Syrah also exhibited significant activity, ranging from 72% to 88% inhibition. The inhibitory effect of these samples surpassed that of Acarbose, a commercial α-glucosidase inhibitor which exerted ~50% inhibition at 50 µg/mL under the described assay conditions. Petit Verdot, on the other hand, demonstrated a poor inhibitory activity of 7%. Fifteen individual phenolic compounds were identified and quantified in the GPEs by HPLC. However, none of these compounds (commercial standards tested) showed a significant inhibition of rat intestinal α-glucosidases at a concentration range of 0.5–1 mg/mL.

Fig. 1.

Percent α-Glucosidase Inhibition per GPE Sample. Enzyme activity was determined by measuring p-nitrophenol release from pNPG at 405 nm. Acarbose (50 µg/mL) is the standard and denoted as Std. C, Chambourcin. M, Merlot. N, Norton. P, Petit Verdot. S, Syrah. T, Tinta Cão. Bars marked with different superscripts are significantly different (P < 0.05).

6.2. Total phenolic content (TPC)

As shown in Fig. 2, all the tested pomace samples contained noticeable amounts of phenolic compounds at the tested concentration of 2 mg/mL, with the exception of PetitVerdot. Merlot GPE contained the highest TPC (0.29 mg GAE/mg) followed by Syrah GPE (0.28 mg GAE/mg), Tinta Cão GPE (0.26 mg GAE/mg), Chambourcin GPE (0.19 mg GAE/mg) and Norton GPE (0.14 mg GAE/mg), while Petit Verdot GPE contained the least TPC (0.06 mg GPE/mg, P < 0.05).

Fig. 2.

Total Phenolic Content (TPC) per GPE Sample. TPC was determined using Folin–Ciocalteu reagent. Data are presented as mg gallic acid equivalents (GAE) per mg dry GPE weight. C, Chambourcin. M, Merlot. N, Norton. P, Petit Verdot. S, Syrah. T, Tinta Cão. Bars marked with different superscripts are significantly different (P < 0.05).

6.3. Correlation

Pearson’s Correlation analysis was used to compare the trends observed in α-glucosidase inhibition and phenolic content. Results indicated strong correlation between the two, with a correlation coefficient of 0.882 (P < 0.01).

6.4. Phenolic composition

HPLC chromatograms of standards and samples are displayed in Fig. 3. All profiled phenolics were detected in the 6 GPE samples, in varying concentrations. The highest and lowest concentrations of most phenolics were observed in the Chambourcin and Petit Verdot varieties, respectively. The sum of concentrations of detected compounds was highest by far in Tinta Cão GPE (169.06 mg/g), most attributable to the anthocyanin malvidin chloride (149.31 mg/g), and lowest in Petit Verdot (9.69 mg/g), with consistently low concentrations of most compounds, except for caffeic acid (2.00 mg/g), which was most concentrated in Petit Verdot GPE among the tested varieties. Sum of concentrations of the profiled compounds ranged from 9.69 to 169.06 mg/g in the remaining varieties. Table 1 summarises the computed data.

Fig. 3.

HPLC Chromatograms of the 15 Selected Antioxidant Standards and 6 Selected GPE Samples. Standards were profiled in duplicate (one shown) to determine the anticipated retention time range for each compound. UV spectrum is shown at 280 nm. 1, gallic acid. 2, malvin chloride. 3, catechin. 4, delphinidin chloride. 5, caffeic acid. 6, cyanidin chloride. 7, p-coumaric acid. 8, epicatechin gallate. 9, rutin. 10, quercetin 3-O-glucoside. 11, malvidin chloride. 12, myricetin. 13, resveratrol. 14, quercetin hydrate. 15, kaempferol. Antioxidant-rich GPE concentrates isolated by solid phase extraction were profiled in triplicate. Each sample is a complex mixture of compounds, including the profiled antioxidants. Peak numbers represent detected antioxidant standards. Spectra are also displayed at 280 nm.

Table 1.

Concentrations of the Detected Antioxidants in the GPE Samples. Following antioxidant detection based on retention time (RT), concentration was determined by measuring and comparing peak area of each detected compound in the sample and the standard chromatogram. Data are presented as milligrams of detected antioxidant per gram of crude GPE, and numbers in bold represent highest and lowest concentrations per row. C, Chambourcin. M, Merlot. N, Norton. P, Petit Verdot. S, Syrah. T, Tinta Cão.

Peak	Phenolic compounds	GPE samples (mg/g crude extract)
		C	M	N	P	S	T
1	Gallic acid	2.42 ± 0.42	0.72 ± 0.10	0.41 ± 0.03	0.52 ± 0.03	0.87 ± 0.06	1.16 ± 0.12
2	Malvin chloride	3.65 ± 0.22	0.29 ± 0.05	0.85 ± 0.18	0.23 ± 0.01	0.35 ± 0.01	1.02 ± 0.16
3	Catechin	6.45 ± 0.93	1.47 ± 0.23	0.92 ± 0.13	0.12 ± 0.01	4.05 ± 0.26	3.40 ± 0.39
4	Delphinidin chloride	0.68 ± 0.17	0.52 ± 0.18	0.43 ± 0.09	0.41 ± 0.00	0.64 ± 0.12	4.36 ± 0.59
5	Caffeic acid	1.14 ± 0.37	0.50 ± 0.02	0.81 ± 0.07	2.00 ± 0.10	1.05 ± 0.09	1.17 ± 0.31
6	Cyanidin chloride	1.65 ± 0.41	0.24 ± 0.06	0.41 ± 0.05	0.19 ± 0.02	0.23 ± 0.02	0.41 ± 0.04
7	p-Coumaric acid	0.35 ± 0.07	0.11 ± 0.03	0.13 ± 0.00	0.02 ± 0.01	0.11 ± 0.01	0.17 ± 0.04
8	Epicatechin gallate	1.72 ± 0.56	0.22 ± 0.04	0.43 ± 0.05	0.04 ± 0.01	0.49 ± 0.06	0.43 ± 0.01
9	Rutin	3.85 ± 1.30	0.42 ± 0.03	1.00 ± 0.11	1.14 ± 0.13	0.83 ± 0.07	2.41 ± 0.16
10	Quercetin 3-O-glucoside	3.90 ± 0.15	0.36 ± 0.01	1.43 ± 0.16	0.09 ± 0.04	0.77 ± 0.04	2.85 ± 0.44
11	Malvidin chloride	22.49 ± 15.16	9.92 ± 8.02	12.38 ± 3.00	3.45 ± 1.13	46.39 ± 3.87	149.31 ± 15.20
12	Myricetin	0.78 ± 0.08	0.32 ± 0.01	0.44 ± 0.04	0.17 ± 0.01	0.31 ± 0.01	0.56 ± 0.09
13	Resveratrol	0.53 ± 0.12	0.20 ± 0.01	0.22 ± 0.05	0.07 ± 0.01	0.38 ± 0.00	0.81 ± 0.07
14	Quercetin hydrate	1.14 ± 0.11	1.03 ± 0.07	0.42 ± 0.13	1.11 ± 0.06	0.35 ± 0.02	0.64 ± 0.20
15	Kaempferol	0.25 ± 0.05	0.22 ± 0.02	0.15 ± 0.08	0.12 ± 0.05	0.23 ± 0.04	0.36 ± 0.03
	Total	51.02	16.53	20.42	9.69	57.06	169.06

7. Discussion

α-Glucosidases play a significant role in carbohydrate digestion and absorption and therefore postprandial blood glucose, a target for diabetes management (Kumar, Narwal, Kumar, & Prakash, 2011). The comparison of the α-glucosidase inhibitory potential of several crude grape pomace extracts allows the identification of the grape variety that is potentially rich in the inhibiting compounds. Although yeast α-glucosidase is readily available in pure form and widely used for nutraceutical investigations (Tan et al., 2013; Zhao et al., 2013), α-glucosidase from mammalian source is more biologically relevant. The mammalian enzyme complex was hence extracted and purified from rat intestinal powder. The presented α-glucosidase inhibition data were consistent with our previous findings indicating that red wine grapes are strong inhibitors of the enzyme (Hogan et al., 2010), with exception to Petit Verdot variety. Having obtained the grape pomaces from the same vineyard and followed a consistent sample preparation protocol, our findings suggest that Tinta Cão exceeds other tested varieties in inhibitory activity due to varietal differences rather than differences in growth and preparation conditions.

The richness of grapes and their pomaces in phenolics (Kammerer et al., 2004; Kanner, Frankel, Granit, German, & Kinsella, 1994; Ruberto et al., 2007; Speisky, López-Alarcón, Gómez, Fuentes, & Sandoval-Acuña, 2012; Torres et al., 2002), and the fact that numerous health protective functions have been attributed to antioxidants over the last few decades (de Camargo, Regitano-d’Arce, Biasoto, & Shahidi, 2014; Frankel, 1999; Rice-Evans, Miller, & Paganga, 1997; Simic Michael & Jovanovic Slobodan, 1994), together suggest that a bioactivity exhibited by a grape extract may be related to its antioxidant content. A review of literature on plant-derived α-glucosidase enzyme inhibitors indicates that known antioxidant compounds such as polyphenols, flavonoids and others have exhibited inhibitory activity in vitro (Benalla, Bellahcen, & Bnouham, 2010; Kumar et al., 2011). This brought about the need to investigate and compare the antioxidant makeup of our 6 grape varieties. Hence, a universal antioxidant assay (TPC) was employed to quantify the phenolic content while HPLC profile comparison allowed the detection of major differences as well as specific phenolic compounds.

According to our results, the tested grape pomace varieties were rich in phenolic compounds, with the exception of Petit Verdot which had the lowest TPC value. Merlot, Syrah and Tinta Cão pomace extracts appeared to contain the highest amounts of phenolic compounds, with these compounds accounting for 29%, 28% and 27% of the dried weight extract of these varieties, respectively. Although higher TPC has been previously reported in red grape pomace extracts, like for example Norton (48%, 80% ethanol extract) (Hogan, Canning, Sun, Sun, & Zhou, 2010) and Bangalore (36%, methanol extract) (Chidambara Murthy et al., 2002), differences may be attributed to different sources and extraction methods/solvents. Interestingly, our observed trend appeared to be consistent with our aforementioned α-glucosidase inhibition results. Our results hence not only indicated that these three varieties were particularly rich in antioxidants, but also suggested that the phenolic content might have contributed to the observed enzyme inhibition potency. α-Glucosidase inhibition data correlated strongly with TPC data suggesting that the varieties with a stronger enzyme inhibition capacity may also exhibit a stronger antioxidant capacity due to their richness in phenolic compounds, which coincide with the conclusion reported recently (de Camargo, Regitano-d’Arce, Biasoto, & Shahidi, 2016).

Information on individual antioxidants in our samples was obtained via HPLC profiling, to detect major differences that may explain the observed trends. The anthocyanins cyanidin chloride, delphinidin chloride, malvidin chloride and malvin chloride, the flavanols catechin and epicatechin gallate, the flavonols kaempferol, myricetin, quercetin hydrate and quercetin 3-O-glucoside, the flavone rutin, the hydroxycinnamates caffeic acid and p-coumaric acid, the stilbenoid resveratrol and the non-flavonoid phenolic compound gallic acid, have been abundantly reported in grapes, particularly red grapes and their extracts and wines (Castillo-Muñoz, Gómez-Alonso, García-Romero, & Hermosín-Gutiérrez, 2007; de Camargo et al., 2014; Iacopini, Baldi, Storchi, & Sebastiani, 2008; Jara-Palacios et al., 2013; Rice-Evans et al., 1997). They were hence selected as standards for phenolic profiling of the GPE samples. As expected, the profiled antioxidant compounds were all detected in the tested samples. Also, the total concentration of detected phenolics was highest with Tinta Cão and lowest with Petit Verdot, in line with the aforementioned assay results indicating that the former possesses strong antioxidant ability (surmised from total phenolic content) while the latter exhibited the weakest antioxidant capacity among the tested varieties. Of interest was the search for phenolic compounds that are particularly deficient in the poor α-glucosidase inhibiting variety, Petit Verdot, and phenolic compounds that are particularly highly concentrated in the most potent α-glucosidase inhibiting variety, Tinta Cão. Catechin, p-coumaric acid, epicatechin gallate, quercetin 3-O-glucoside, malvidin chloride and resveratrol were particularly very low in Petit Verdot GPE. All tested anthocyanins were especially concentrated in Chambourcin and Tinta Cao varieties. The concentration of malvidin chloride in Tinta Cão was 3.22 times higher than the next most concentrated variety. This prompted the evaluation of the α-glucosidase inhibiting capacity of these compounds, to identify the compound(s) that may be responsible for the observed differences between the GPE varieties. We examined a number of known phenolic compounds in grape skin extract including catechin, resveratrol, delphinidin chloride, cyanidin chloride, malvidin-diglucoside, malvin chloride, malvidin chloride, cyanidin-diglucoside, procyanidins B1 and B2, epicatechin gallate, kaempferol, myricetin, quercetin hydrate, quercetin 3-O-glucoside, and phenolic acids (gallic, caffeic, p-coumaric, and ferulic acids). To our surprise, none of the compounds exhibited this bioactivity. Previous studies reported that oligomers of proanthocyanidins from persimmon peels (Lee, Cho, Tanaka, & Yokozawa, 2007) and proanthocyanidins from pine bark extract (Kim, Jeong, Wang, Lee, & Rhee, 2005) exerted strong inhibition on α-glucosidase. However, these studies used the enzymes from yeast (non mammalian species) instead of mammalian derived α-glucosidases (rat intestinal α-glucosidases used in our study). Our finding that procyanidins B1 and B2 had no inhibition on mammalian intestinal α-glucosidases suggests that proanthocyanidins may exert significant different inhibitory activities against non mammalian versus mammalian α-glucosidases. Collectively, our result indicates that the tested GPEs likely contained an unidentified bioactive component that strongly inhibited α-glucosidases and possibly contributed to high phenolic content in our experiment. Of particular concern was the Tinta Cão variety which ranks on the top of the list in terms of α-glucosidase inhibition along with higher content of phenolics, especially malvidin. Interestingly, a recent experiment showed that 6-O-p-trans-coumaroyl-d-glucopyranoside from Tinto Cão is a potential α-glucosidase inhibitor (Sun, Kadouh, Zhu, & Zhou, 2016).

8. Conclusion

Red wine grape extracts, namely Tinta Cão GPE, appear to be novel food-derived extracts that potently inhibited mammalian α-glucosidases. This reported activity is new and likely specific to the grape variety and possibly related to its phenolic content and profile. Although comparing antioxidant activity and content of a sample to those in the literature can be difficult at times due to the absence of one universal method and reporting fashion, the current results did reveal high antioxidant content that strongly correlates with α-glucosidase inhibition. These promising findings may provide a foundation for the future development of natural α-glucosidase inhibitors from Tinta Cão GPE to be potentially used for diabetes management and prevention. Further investigation is required to validate and optimise this property.