Study On In-vitro Antioxidant Activities Of Ethanol Extracts Of The Leaves Of Lindera Aggregate (Sims) Kosterm And Purification Technology Of Quercitrin

Lindera aggregate (Sims) Kosterm belongs to the Lauraceae family. In the Tiantai district of the Zhejiang Province in China, the leaves of Lindera aggregate (Sims) Kosterm is known as the native "Tiantai Lindera aggregate" or "Tai Lindera aggregate" and it is a popular tea drink in that region to prevent and treat cardiovascular or cerebrovascular diseases.In order to explore the medicinal value of the leaves of Lindera aggregate (Sims) Kosterm as a new drug to prevent and treat cardiovascular or cerebrovascular diseases, we aimed the quercitrin as target and established a method using HPLC-PDA to detect the content of it. By analyzing the content of quercitrin, the extraction method was optimized, In-vitro antioxidant capability of the ethanol extract of the leaves of Lindera aggregate (Sims) Kosterm was determined by the DPPH free radical scavenging assay and the FRAP assay. Quercitrin was purified by using the macroporous resin and reversed-phase chromatography. This study laid foundation for the development of the new anti-coronary heart disease.By comparing the thin-layer chromatography of 10 batches of the leaves of Lindera aggregate (Sims) Kosterm collected in different months, an identification method of medical material was consummated. The moisture content was determined by the torrefaction method and was limited to 8%. Ethanol extract content was measured by the hot-dip method and was limited to 18%.The extraction method was optimized by single factor experiments and uniform design experiment using quercitrin content as the index. Accordingly, the optimal extracting process was established as follows:the leaves of Lindera aggregate (Sims) Kosterm was extracted for 2 h with70% ethanol (1:22, g/ml) under reflux and repeated three times. Verification tests showed the optimal extracting process was stable and feasible.Macroporous resin and reversed-phase chromatography were used for purification of quercitrin from the leaves of Lindera aggregate (Sims) Kosterm. AB-8 macroporous resin was selected to be the suitable resin by static adsorption and desorption test. The final purification process was optimized by single factor experiments using the combination of the quercitrin yield and the ethanol washing content as composite index. The final purification process was as follows:after concentrating to 0.25 g·mL-1 with pH level 4, the extract was loaded to macroporous resin column. One hour later the column was eluted with sodium hytlroxide solution with pH level 9, water,50% ethanol respectively in the amount of 2 BV,3 BV and 3 BV. The uniform design method was used to optimize the reversed-phase chromatography condition, the final purification process was as follows:the loading amount was 1:40 (g intermediate:ml column volume) and the column was washed by 14.25% ethanol in the speed of 3 BV·h-1. Finally, quercitrin was recrystallized using the ethanol-water system.The total polyphenol content of 8 samples were measured by the Felin-Ciocalteu method and the in-vitro antioxidant capabilities were determined by the 2,2-diphenyl-1-picrylhydrazyl free radical scavenging assay and the ferric reducing/antioxidant power assay. Correlation analysis was used to study the relationship between the total polyphenol content and the in-vitro antioxidant capability. Polyphenol constituents widely exist in the leaves of Lindera aggregate (Sims) Kosterm and they had more or less correlation with the in-vitro antioxidant capabilities.