Baoshan Sun

CHEMICAL CHARACTERIZATION AND ANTIOXIDANT ACTIVITIES OF OLIGOMERIC AND POLYMERIC PROCYANIDIN FRACTIONS FROM GRAPE SEEDS

Two procyanidin fractions, namely oligomers and polymers isolated from grape seed methanolic extract were characterized. Phenolic composition and procyanidin purity of these fractions were determined by normal-phase and reverse-phase HPLC, thioacidolysis-HPLC, ESI-MS analyses, formaldehyde-HCl precipitation and elemental analysis. Antioxidant activities of these fractions and other well-known antioxidants were measured using xanthine-xanthine oxidase system for generating superoxide radical ({O2(-)}), the DPPH (1,1-diphenyl-2-picrylhydrazyl) radical method and the Fenton system for generating hydroxyl radical (HO). The results showed that both oligomeric and polymeric procyanidin fractions were highly pure, with the degree of polymerization ranging from 2 to 17-18 and 12 to 32-37, respectively. On the basis of molar concentration, polymeric procyanidins appeared the highest antioxidant activities, followed by oligomeric procyanidins, whereas catechins presented a lower antioxidant activity than its oligomers and polymers. These results indicate that the antioxidant activities of grape seed procyanidins are positively related to their degree of polymerization. Moreover, grape seed procyanidins presented higher antioxidant activities than other well-known antioxidants such as vitamin C, suggesting that grape seed procyanidins might be of interest to be used as alternative antioxidants.

Isolation and purification of dimeric and trimeric procyanidins from grape seeds

An improved method used for the isolation and purification of low-molecular-mass procyanidins (i.e., dimeric B1, B2, B3, B4, B1-3-O-gallate, B2-3-O-gallate, B2-3′-O-gallate, trimer C1 and trimer T2) from grape seeds, is described. Pre-fractionation in oligomers and polymers was performed by chromatography on an open column (LiChroprep RP-18). The oligomer fraction, rich in dimeric and trimeric procyanidins, was then chromatographed on a Toyopearl TSK HW-40 (F) column. The major fractions obtained from Toyopearl TSK HW 40 (F) chromatography were further separated by semi-preparative HPLC, to obtain pure procyanidins. The main advantages of this method are that it is easy to use, less time-consuming and gives very high yield of dimeric and trimeric procyanidins by each manipulation.

Reactivity of polymeric proanthocyanidins toward salivary proteins and their contribution to young red wine astringency.

Recent studies have indicated the presence of significant amount of highly polymerized and soluble proanthocyanidins in red wine and such compounds interacted readily with proteins, suggesting that they might be particularly astringent. Thus, the objective of this work was to verify the astringency of polymeric proanthocyanidins and their contribution to red wine astringency. The precipitation reactions of the purified oligomeric procyanidins (degree of polymerization ranging from 2 to 12-15) and polymeric procyanidins (degree of polymerization ranging from 12-15 to 32-34) with human salivary proteins were studied; salivary proteins composition changes before and after the reaction was verified by SDS-PAGE and procyanidins composition changes by spectrometric, direct HPLC and thiolysis-HPLC methods. The astringency intensity of these two procyanidin fractions was evaluated by a sensory analysis panel. For verifying the correlation between polymeric proanthocyanidins and young red wine astringency, the levels of total oligomeric and total polymeric proanthocyanidins and other phenolic composition in various young red wines were quantified and the astringency intensities of these wines were evaluated by a sensory panel. The results showed that polymeric proanthocyanidins had much higher reactivity toward human salivary proteins and higher astringency intensity than the oligomeric ones. Furthermore, young red wine astringency intensities were highly correlated to levels of polymeric proanthocyanidins, particularly at low concentration range (correlation coefficient r = 0.9840) but not significant correlated to total polyphenols (r = 0.2343) or other individual phenolic compounds (generally r < 0.3). These results indicate the important contribution of polymeric proanthocyanidins to red wine astringency and the levels of polymeric polyphenols in red wines may be used as an indicator for its astringency.

Red Wine Phenolic Complexes and Their in Vitro Antioxidant Activity

Phenolic complexes are a major group of polyphenols in aged red wine. The objective of this work was to evaluate the in vitro antioxidant activity of the phenolic complexes. Thus, red wine polyphenols were fractionated into various fractions including monomers, oligomers, polymers, anthocyanins, and complexes. The in vitro antioxidant activities of these fractions and other phenolic standards (catechin, epicatechin, quercetin, and malvidin 3-glucoside) as well as ascorbic acid were verified by DPPH* test. On the other hand, the variation of antioxidant activities during the reaction between epicatechin and malvidin 3-glucoside mediated by acetaldehyde in a model wine solution was also monitored. The results showed that both the phenolic complex fraction and newly formed condensation products between epicatechin and malvidin 3-glucoside maintain antioxidant activities as strong as those of their compositional phenolics. This work provides, for the first time, direct evidence about the in vitro antioxidant activities of red wine phenolic complexes.

Effect of Addition of Commercial Grape Seed Tannins on Phenolic Composition, Chromatic Characteristics, and Antioxidant Activity of Red Wine

The effect of addition of grape seed tannins on the phenolic composition, chromatic characteristics, and antioxidant activity of red wine was studied. Two highly pure commercial grape seed tannins (GSE100 and GSE300) were selected, and their phenolic compositions were determined. Two types of red wines were made with Castelão/Tinta Miúda (3/2, w/w) grapevine varieties by fermentation on skin using two different maceration times, which correspond to the wines rich and poor in polyphenols, respectively. Each of these wines was used for experimentation with the addition of GSE100 and GSE300 before and immediately after alcoholic fermentation. Phenolic composition, chromatic characteristics, and antioxidant activity of the finished red wines were analyzed by HPLC-DAD, CIElab 76 convention, and DPPH radical test, respectively. The results showed that the addition of grape seed tannins had obvious effects of increasing color intensity and antioxidant activity only in the wines poor in polyphenols. Although GSE300 contained much higher amounts of di- and trimer procyanidins and a lower amount of polymeric proanthocyanidins, it provided effects of increasing the color intensity and antioxidant activity of the wines poor in polyphenols similar to those of GSE100. Furthermore, GSE100 released more gallic acid to wines than GSE300, although no gallic acid was detected in GSE100. Tannins added after alcoholic fermentation had a better effect on phenolic composition of red wine than tannins added before alcoholic fermentation.

Formation of new stable pigments from condensation reaction between malvidin 3-glucoside and (−)-epicatechin mediated by acetaldehyde: Effect of tartaric acid concentration

The objective of this work was to study the effect of tartaric acid concentration on the condensation reaction between malvidin 3-glucoside (Mv-glc) and flavanols mediated by acetaldehyde in the model solution. The model wine solutions were prepared by 12% ethanol in water (v/v) with two different l-tartaric acid concentrations (5g/l and 25g/l, respectively) and at two different pH values (3.2 and 1.7, respectively). Four new pigments were detected in model wine solutions containing Mv-glc, (-)-epicatechin and acetaldehyde. By reverse-phase HPLC-DAD, ESI-MS and MS(n) fragmentation analysis, the four new pigments were tentatively identified as four isomers of hydroxyethyl malvidin-3-glucoside-ethyl-flavanol. The decrease in the concentration of Mv-glc and (-)-epicatechin and the increase in the concentration of the new identified pigments were more pronounced at higher tartaric acid concentration. At pH 1.7, although the two well-recognized ethyl-linked Mv-glc-flavanol isomers were quantitatively the major pigmented products in the reaction solution throughout the assay period, they appeared less stable than the four new pigments. At pH 3.2, the rate of formation of ethyl-linked Mv-glc-flavanol pigments was much slower than at pH 1.7, whereas the four new pigments were quantitatively the predominant pigmented products at the latter stage of the reaction.

A new class of anthocyanin‐procyanidin condensation products detected in red wine by electrospray ionization multi‐stage mass spectrometry analysis

In our previous work, we have identified, in a model wine solution containing malvidin 3-glucoside, epicatechin and acetaldehyde, a new condensation product--hydroxylethyl-malvidin-3-glucoside-ethyl-epicatechin. The objective of this work was to verify the presence of such new condensation products in red wine. For this purpose, red wine was fractionated into various fractions by column chromatography on LiChroprep RP 18 and on Toyopearl 40 (F). The phenolic composition of each fraction was verified by HPLC-DAD and direct-infusion ESI-MS(n) analysis. In addition to the well-known anthocyanins and their acetyl and coumaroyl derivatives, and several direct and indirect anthocyanin-(epi)catechin condensation products, a new class of pigmented products, namely hydroxyethyl-anthocyanin-ethyl-flavanol compounds, have been detected in red wine. The new class of pigmented products would be expected to be the major pigments responsible for the color of aged red wine.