Study On Synthesis Pathway Started By P-coumaric Acid Of Polygonum Multiflorum Stilbene Glucoside By Biotransformation

Stilbene glycoside, or stilbene polyphenol, an active ingredient of traditional Chinese medicine Polygonum Multiflorum, has much physiological activity and medicinal value. It has been proved that stilbene glycoside has the effects of reducing blood press, anti-aging, tumor inhibiting and so on, which shows its significant research and development value in this aging society nowadays. Previous studies have made us comprehensively understand in stilbene glycoside’s physical and chemical properties, such as stability, activity etc. These studies are based on extraction, separation, purification and detection, but there is no study about stilbene glycoside’s biosynthesis metabolism in plant.Stilbene glycoside differs to resveratrol by only a phenolic hydroxyl in molecular structure, and resveratrol is the stilbene compound being thoroughly studied. In this paper, we presumed synthesis pathway of stilbene glycoside according to synthesis pathways of resveratrol in the plant, combined with our preliminary work, and verified a series of synthesis pathways under p-coumaric acid acted as substrate by the method of biotransformation. Finally, we confirmed that in the synthesis pathway of stilbene glycoside, p-coumaric acid turn into p-coumaroyl-Co A by the catalysis of polygonum multiflorum callus crude enzyme, and p-coumaroyl-Co A and malonyl Co A turn into resveratrol by stilbene synthase. Specific research contents and results are as follows:(1) We induced and cultured polygonum multiflorum callus, extracted callus crude enzyme, setted up enzymatic system, and detected target product by HPLC and LC-MS method. The results of HPLC and LC-MS showed that the product of p-coumaric acid was p-coumaroyl-Co A, the product of p-coumaroyl-Co A and malonyl Co A was resveratrol.(2) In the essay, we studied the influencing factor of synthetizing p-coumaroyl-Co A in different reaction time, different content of crude enzyme and different component of crude enzyme. The results showed that the optimal reaction time was 1 hour. In a certain concentration range of crude enzyme, the output of p-coumaroyl-Co A was positive correlation with the content of crude enzyme. Ammonium sulfate(AS) precipitation method showed AS of 20% ~ 60% had catalytic activity, and 40 %~50 % was the optimum.(3) In the essay, we studied the influencing factor of synthetizing resveratrol in different content of substrate and different component of crude enzyme.The results showed that the output of resveratrol was positive correlation with p-coumaroyl-Co A. AS precipitation method showed AS of 40% ~ 70% had catalytic activity, and 50 %~60 % was the optimum. The enzyme involved in the catalysis is mostly to be stilbene synthase, and the molecular weight was nearly 42 KDa.In this paper, we come to a conclusion that the synthesis from p-coumaric acid to p-coumaroyl-Co A and from p-coumaroyl-Co A and malonyl Co A to resveratrol exist in Polygonum Multiflorum callus. This conclusion is helpful to the further study of synthesis pathway of stilbene glycoside.