Biotransformation Of Stilbenes And Flavonoids By Peroxidase From Momordica Charantia

Plant peroxidases, which are oxidoreductases utilizing hydrogen peroxide or organic oxide to catalyze oxidative reactions, were found to play an important role in physiology such as metabolism and transformation of small complexes. In the present research, peroxidase was extracted from the fruits of Momordica charantia and the biotransformation of stilbenes and flavonoids by this peroxidase was also investigated. Through systematical chemical studies on the oxidation products of resveratrol, ferulic acid, quercetin and puerarin, 16 compounds were isolated and identified. Among them, 5 compounds are new compounds; 2 compounds were isolated from the oxidation products catalyzed by peroxidase in vitro for the first time and 8 compounds were isolated from the oxidation products catalyzed by MCP for the first time. The antioxidative activity of 3 compounds isolated from the oxidation products were also examined in vitro.1. Extraction of MCPCrude MCP was obtained by combining consecutive treatment of tris-HCl buffer extraction and ammonium sulfate fractionation. It was analyzed by non-denaturing vertical polyacrylamide gel electrophoresis (PAGE) with discontinuous buffer system followed by guaiacol staining and three peroxidase isoenzyme bands were found. The protein content detected using Bradford method was 0.86 mg/ml, and the peroxidase activity determined using guaiacol colorimetric method was 3786.05 U/mg.2. Biotransformation studies of stilbenes by crude MCPStilbenes, which are produced by plants in response to biotic or abiotic stress factors, are a kind of polyphenol secondary metabolites. Resveratrol is a stilbenic phytoalexin widely distributing in fruits, Chinese traditional medicines, and grape wine. Its oligomers exhibit wide bioactivities including cancer chemopreventive activity, cardioprotective effects, antimicrobial, anti-inflammatory, anti-HIV and immunoloregulative activity. To test the biotransformation activity of MCP and further develop the resource of stilbenes, we carried out the biotransformation studies of resveratrol. The reaction was conducted with acetate buffer (pH 5.0)/ crude MCP at 25 ℃ in the presence of acetone and the oxidation products were isolated by silica gel column, Sephadex LH-20 column and semi-preparative HPLC chromatography. 6 compounds, including resveratrol trans-dehydrodimer (I), parthenostilbenins A (II) and B (III), restrytisol B (IV), leachianol G (V) and 7-O-acetylated leachianol G (VI) were obtain and identified. Among them, compound VI is a new compound; compounds II, III and IV are isolated from the oxidation products catalyzed by peroxidase in vitro for the first time. In this article, we also studied the biotransformation of resveratrol by MCP under different conditions applying the means of HPLC. We found that the transformation efficiency of resveratrol was highest in acetate buffer (pH 5.0)/acetone system at 25 ℃; while the optimum concentratration of H₂O₂ was 0.11 mol/L; moreover, the extension of the reaction time did not profit the biotransformation.The hetero-coupling of resveratrol and ferulic acid was conducted with acetate buffer (pH 5.0)/crude MCP at 30 ℃ in the presence of acetone. The products was analyzed applying the means of LC-MS. Besides the dimers of resveratrol and ferulic acid respectively, a hetero-dimer of resveratrol and ferulic acid was found. The products were isolated by silica gel column, semi-preparative HPLC chromatography and methanol recrystallization and 2 compounds, compounds VII and VIII were isolated and identified. Compound VII is a dehydrodimer of ferulic acid, and compound VIII is a novel dehydrodimer of resveratrol and ferulic acid.3. Biotransformation studies of flavonoids by crude MCPFlavonoids are a large group of naturally occurring dietary polyphenols with a wide range of pharmacological activities such as antioxidant, anti-inflammatory, antimutagenic and anticarcinogenic activities. They have been subjects of detailed examination with respect to their antioxidant and oxidative metabolism by oxidative enzymes which occur in plant tissues, such as polyphenol oxidase and peroxidase.The biotransformation of quercetin was conducted with acetate buffer (pH 5.0)/crude MCP at 25 ℃ in the presence of ethanol and 6 oxidation products, IX, X, XI, XII, XIII and XIV were isolated by silica gel column, Sephadex LH-20 column and semi-preparative HPLC chromatography. Among them, compounds XII and XIII are new compounds.The biotransformation of puerarin was conducted with acetate buffer (pH 5.0)/crude MCP at 25 ℃ in the presence of 1,4-dioxane and 2 oxidation products XV, XVI were isolated by silica gel column, Sephadex LH-20 column and semi-preparative HPLC chromatography. Compound XVI is a new compound.4. Pharmaceutical ResearchThe antioxidative activity of 3 compounds isolated from the oxidation products, I, VII, VIII were tested in vitro in terms of free radical scavenging activity test and cell viability assay on rat cardiac H9C2(2-1) cells. Compounds VII and VIII showed stronger antioxidative activity than their respective substrates. The structure-activity relationship is that the amount and site of phenolic hydroxyl group is directly related with the free radical scavenging activity of the molecule.