| The leaves of Crataegus pinnatifida Bge, var. major N. E. Br. is a traditional Chinese medicine (TCM), which is collected in Chinese Pharmacopoeia, 2005 as one species of hawthorn leaves. The anti-fatty liver effect and active components of the extract of the leaves of C. pinnatifida Bge, var. major N. E. Br. were investigated with the modern pharmacological methods and analytical technologies in this study.Firstly, a composite model of hyperlipemia of rat was established to study the pharmacological function of the extract of the leaves of C. pinnatifida Bge. var. major N. E. Br. The results of experiment were analyzed using one-way ANOVA, which indicated that the part of 70 ethanol extract was the active part of anti-fatty liver. It remarkably affected the results of TC, TC, ALT, ALT, HDL, LDL, the ratio of liver-body weight and pathological examination. The active part of anti-fatty liver was further fractionated into three parts. The extract of 70% ethanol was loaded on porous polymer resin column, and the n-Butanol fraction of the elute was established to be the optimal active part.The preparation technique of active extract of C. pinnatifid Bge, var. major N. E. Br. was optimized: The drug material was extracted with 24 times of 70% ethanol twice, the extract was further isolated by using a porous polymer resin(AB-8)column, the eluate of 70% ethanol was fractionated with chloroform and n-Butanol successively. The n-Butanol layer was found to be the most active part with the model of fatty liver of rat induced by hyperlipermia.The components in n-Butanol of C. pinnatifid Bge, var. major N. E. Br. were isolated by using column and thin layer chromatography. Nine compounds were isolated and identified by ESI-MS and NMR. Among them vitexin-4′-O-glucoside and vitexin-4′-rhamnoside were for the first time isolated in the leaves of C. pinnatifida Bge. var. major N. E. Br. An HPLC method, using the internal standard (I. S.) baicalin, was developed for the first time to determinate the 8 active components in the leaves of C. pinnatifida Bge, var. major N. E. Br. The method is reliable, accurate and was applied to investigate the features of dynamic change of 8 components in the leaves of C. pinnatifida Bge, var. major N. E. Br. and to establish the optimal collection time of the leaves of C. pinnatifida Bge var. major N. E. Br.Oxidation was related with the fatty liver. Therefore, the antioxidation effect of vitexin-2″-O-rhamnoside was investigated via the human umbilical cord endothelial cell culture technology. VOR significantly inhibited 0.5 mol·L-1 TBHP induced ECV304 cells oxidization at dose range from 15.6-125μmol·L-1 for 24 h. The inhibition was significantly enhanced with dose increasing up to showing an antioxidation effect in dose dependent manner.An HPLC method for the determination of vitexin-2″-O-rhamnoside in rat plasma was developed, and applied to an in vivo kinetic study in rats after intravenous administration of four doses. This investigation contributes not only to the determination vitexin-2″-O-rhamnoside in rat plasma by HPLC but also to our understanding of the linear pharmacokinetic characteristics of vitexin-2″-O-rhamnoside over the dose range studied in rats after intravenous administration.A sensitive and selective UPLC-ESI-MS/MS method was developed and validated for the determination of vitexin-2″-O-rhamnoside in rat plasma, which showed great advantages of not only simple sample preparation but also high selectivity and short run time of 3.0 min. The method was applied to the pharmacokinetic study of vitexin-2″-O-rhamnoside in rat plasma after oral administration of standardized extract of leaves of C. pinnatifida Bge, var. major N. E. Br. and vitexin-2″-O-rhamnoside at a single dosage for the first time. The dynamic process of absorption and elimination in vivo of vitexin-2″-O-rhamnoside from the leaves of C. pinnatifida Bge. var. major N. E. Br. was elucidated.In conclusion, this study investigated the anti-fatty liver action and the preparation of the extract of leaves of C. pinnatifida Bge, var. major N. E. Br., and the isolation of its active components based on the combination of interdisciplinary subjects. HPLC combined with the technology of cell culture was employed to investigate the antioxidation of vitexin-2″-O-rhamnoside in vitro. The pharmacokinetics of vitexin-2″-O-rhamnoside was investigated in vivo administered by either intravenous or oral route. The results would be useful for new drug development from the leaves of Crataegus pinnatifida Bge, var. major N. E. Br. |
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