Several parameters mostly affecting the precision and accuracy of vanillin assay were reexamined and optimized. The reexamination was performed both by vanillin reaction with catechins and by vanillin reaction with purified proanthocyanidins. In addition to the acid nature and concentration, the reaction time, the temperature, and the vanillin concentration, other factors such as the water content, the presence of interfering substances, and the standard utilized, for both vanillin reaction with catechins and vanillin reaction with proanthocyanidins, were also important. However, the kinetics of the two types of reactions were markedly different. For estimating accurately catechins or proanthocyanidins that exist simultaneously in plant tissues, it is necessary to preliminarily separate them from each other. Keywords: Catechins; proanthocyanidins; vanillin assay

A method was developed for the fractionation of grape and wine proanthocyanidins according to their degree of polymerization. The grape and wine proanthocyanidins were fractionated on C18 Sep-Pak cartridges into three fractions by different organic solvents. The combination of TLC, analytic HPLC, and degradation with toluene-α-thiol confirmed that these three fractions contained, respectively, monomeric flavan-3-ols (catechins), oligomeric proanthocyanidins, and polymeric proanthocyanidins. The mean degrees of polymerization for oligomeric and polymeric proanthocyanidins in red wine were, respectively, 4.8 and 22.1, and those in the seed extract, 9.8 and 31.5. The method proposed is very interesting for the study of grape and wine proanthocyanidins according to their degree of polymerization, and a further quantification is also possible. Keywords: Grape; wine; catechins; proanthocyanidins; C18 Sep-Pak cartridge; fractionation

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.

The influence of different types of winemaking technology on the contents of catechins, proanthocyanidins, and anthocyanins in Tinta Miúda red wines was studied. The Tinta Miúda red wines were made by fermentation with carbonic maceration, fermentation with stem contact, and fermentation without stem contact, respectively. The analysis of individual catechins, procyanidins, and anthocyanins in these wines was performed by HPLC, and quantification of total catechins, total oligomeric proanthocyanidins, total polymeric proanthocyanidins, and total anthocyanins was carried out by spectrophotometric methods. The wine made by carbonic maceration contained the highest amounts of both catechins and oligomeric and polymeric proanthocyanidins, followed by the wine made by fermentation with stem contact, whereas the wine made by fermentation without stem contact contained the lowest of these compounds. On the other hand, the concentrations of total anthocyanins and nearly all individual anthocyanins in the carbonic maceration wine were lower than those in the wines made by fermentation with stem contact and fermentation without stem contact. These results indicated that, although the carbonic maceration technique could retain higher amounts of catechins and proanthocyanidins in wine, it did not favor retaining or stabilizing anthocyanins in wine.

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.