Preparation Of New Type Proanthocyanidin Oligomers And Research Of Their Bioactivity

In order to prepare structural different proanthocyanidin dimers, and provide foundation for studying the antioxidant and polyphenol-protein interaction structureactivity relationships of proanthocyanidin oligomers, the conditions of depolymerizating of PT40 using epigallocatechin gallate and epicatechin as chain breakers to produce new proanthocyanidin dimers were optimized. RP-HPLC-MS/MS was used to analyze the structures of the produced proanthocyanidin oligomers. Meanwhile, four typical proanthocyanidin dimers were purified and the antioxidant structure-activity relationships as well as their interactions with snake vernom PLA2 were studied. The main results were shown as follows: 1. Preparation and structural identification of new proanthocyanidin oligomersNew proanthocyanidin dimers by depolymerization of PT40 with epigallocatechin gallate and epicatechin as chain breakers was available. The optimum depolymerized conditions of C with PT40 was as follows: the mass ratio of C and PT40 was 2:1, reacting at 40℃ for 2h in 0.1mol/l HCl-methanol solution. Four new proanthocyanidin dimers, namely B-type EC-EC, B-type ECG-EC, B-type EGCG-EC and B-type(E)AFZ- EC were generated. Similarly, the optimum depolymerized condition of EGCG with PT40 was as follows: the mass ratio of C and PT40 was 8:5, reacting at 40℃ for 2h in 0.1mol/l HClmethanol solution and four proanthocyanidin dimers, namely A-type EGCG-EGCG, Btype EGCG-EGCG, B-type EGCG-ECG and A-type EGCG-EGC-CH3 could be obtained. 2. Antioxidant structure-activity relationships of the proanthocyanidin oligomersAntioxidant activities of the four proanthocyanidin oligomers were assessed in different systems including of the reducing activity, total antioxidant capacity, the capacity of clean up ABTS+·, DPPH· and ORAC. The results showed that all four proanthocyanidin oligomers showed high antioxidant capacities. Structure-activity relationship study revealed that the degree of polymerization, the linkage type and the subunit composition affected the antioxidant activities significantly. The greater polymerization degree, the stronger antioxidant; B-type proanthocyanidins dimers exerted more potent antioxidant activity than A-type ones with the same structure subunits; moreover, proanthocyanidins dimers with pyrogallol and galloyl groups in the structure showed stronger antioxidant activity than dimers without these groups, suggesting pyrogallol and galloyl groups had great contribution to the antioxidant activity of proanthocyanidins dimers. 3. The interaction and structure-activity relationships of proanthocyanidin oligomers with PLA2Incubating of proanthocyanidin dimers with PLA2 in physiological conditions for a long time resulted the formation of covalent complexes. A-type dimers exerted higher ability of forming covalent complex with PLA2 than B-type ones with the same structural units, and proanthocyanidin dimers showed stonger covalent binding ability with PLA2 than the monomer. Incubating of proanthocyanidin dimers with PLA2 in physiological conditionsfor a short time led to significant changes in the secondary structure of PLA2. Proanthocyanidin dimers could bind to a single site of PLA2 by the hydrophobic interaction and quench the fluorescence of PLA2 and induce a red shift in both amide I band and II band of PLA2. Generally, the order of the binding ability of dimers to PLA2 is A-type EGCG dimer>B-type EGCG dimer>B-type EGCG-ECG dimer >EGCG monomer. Structure-activity relationship study suggested that the linkage type of interflavan bonds of proanthocyanidins dimers is the most important factor affecting the interaction of dimers with PLA2, followed by the effect of polymerization degree and the number of phenolic hydroxyl groups.