Separation And Identification Of Three Kinds Of Flavonoid Glycosides From Taxus Chinensis Var Mairei Leaves

Taxus is mainly used for extracting taxol, but a large numbers of flavonoids in Taxus remainder extracts free of toxoids (Treft) are currently not been developed and utilized. In order to solve this problem, this paper used the leaves of Taxus chinensis var mairei as raw material and Treft as experimental subjects, established a spectrophotometric method for quantitative analysis of flavonoids and UPLC method to analyze the flavonoid monomer component, designed an orthogonal experiment to optimize the process parameters of ultrasonic extraction of flavonoids, applied a combination of multiple media chromatographic techniques to separate three flavonoid monomers, and exploited1H NMR,13C NMR, HMBC, ESI spectrums to identify three flavonoid glycosides. The main conclusions are as follows:(1) Employed aluminum nitrate complexation spectrophotometric method for quantitative analysis of flavonoids and the relative standard deviation (RSD) values of the precision, stability, repeatability and recovery tests were0.352%.0.137%、1.33%、1.35%.(2) Ultrasonic extraction parameters influencing the yield of flavonoids showed the following order as:solid-liquid ratio (R=35.03), ultrasonic times (R=28.16), ultrasonic time (R=19.67), ethanol concentration (R=16.30), ultrasonic temperature (R=16.27). The optimum conditions was investigated as solid-liquid ratio of1:20(g/mL), ethanol concentration of70%(v/v), ultrasonic times of3, ultrasonic temperature of45℃, ultrasonic time of30min and the yield of flavonoids was47.99mg/g.(3) Compared preliminary separation processes of flavonoids with polyamide resin and AB-8macro-porous resin respectively. In the preliminary separation process of flavonoids with AB-8macro-porous resin, the recovery of flavonoids was about98.47±1.01%. The fraction of40%ethanol aqueous solution enriched three kinds of target products with the recovery of flavonoids was81.84±1.62%. The silica column separated the fraction of40%ethanol aqueous solution with Ethyl Acetate/Methanol mixtures increasing concentration of methanol:15:1,9:1,0:1. Finally, we obtained compound3(25.lmg) in Ethyl Acetate:Methanol/9:1and the fraction of methanol mainly included compound1and compound2. The ODS column separated the fraction of methanol with Methanol/H2O mixtures increasing concentration of Methanol:0,10%,40%,50%,70%, and got compound1(36.2mg)、2(15.7mg) and3(3.2mg).(4) Three kinds of flavonoids glycosides were identified as quercetin3-O-rutinoside (1), kaempferol3-O-a-L-arabinose (1"’�'6")-β-D-glucoside (2), kaempferol3-O-β-D-rhamnose (1"’�'6")-β-D-glucoside (3) and the compounds (2) and (3) were first isolated from the leaves of Taxus chinensis var. mairei.The research works on the analysis methods, extraction processes and separation technologies of flavonoids were scientific and feasible and could be applied to production practice. This article can provide technical support for the development of flavonoids which belong to the fifth sorts of new drug of Chinese traditional medicine.