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.

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