Secondary metabolites of Camellia SinensisL., RP-HPLC analysis and Quantitative Structure Antioxidant Activity Relationships

Abstract

In this study, quantitative analyses by HPLC-DAD of Camellia sinensis (green tea) samples were performed in order to determine the concentrations of four catechins: (-)-Epicatechin (EC), (-)-Epigallocatechin (EGC), (-)-Epicatechingallate (ECg), (-)-Epigallocatechin gallate (EGCg), three methylxantines: Caffeine, Theophylline and Theobromine as well as Gallic acid. Two different extraction methods were performed: an infusion in hot distilled water for 5 min and a multistep extraction process using water/MeOH mixtures. The examination of the obtained chromatograms shows good separation of metabolites, especially for tea infusions. Indeed, all peaks have shown good resolution, symmetry,normal Gaussian shapes and linear baselines. Substantial amounts of polyphenols were found in most tea samples. The catechins contents followed the sequence: EGCg>ECg> EC > EGC in both infusions and water/MeOH extracts. Caffeine is the major methylxantine in all tea samples. Methylxantines level followed the sequence: Caffeine > Theobromine > Theophylline. Levels of methylxantines were higher in infusions than the methanol extracts, whereas catechins levels were found to be higher in methanol extracts than in infusions. In the second part of work, we report, a comparative study of the antioxidant potential of EGCg and EC as compared to resveratrol (RSV). The most favorable mechanism by which each molecule exerts the antioxidant activity is determined. Ascorbic acid (AA) was used as a reference. DPPH and FRAP assays were used for experimental evaluation of antioxidant activity and for theoretical calculations, density functional theory (DFT) method was chosen. Three mechanisms were investigated : Hydrogen Atom Transfer (HAT), Single Electron Transfer Proton Transfer (SET-PT) and Sequential Proton Loss Electron Transfer (SPLET). Calculated thermodynamic parameters correlate well with percentage inhibition (I%) and half maximal inhibitory concentration (IC50) values given by the DPPH test. Both experimental and theoretical approaches showed that EGCg is more potent antioxidant than EC and RSV. The most preferential sites are gallate moiety and 4’-OH in EGCg and OH sites of the B ring in EC. The pKa values confirm this finding. All proposed mechanisms are favored for EGCg, SET-PT is preferred antioxidant mechanism for EC and it is the most suitable in the first step for RSV. Flavanols are more potent antioxidants than the stilbene; RSV.