Study On The Anti-osteoporosis Material Basis Of TCM Prescription "Xian-Ling-Gu-Bao"

The traditional Chinese medicine prescription "Xian-ling-gu-bao" is consisted of six common used Chinese materia medica,including Herba Epimedii(Yinyanghuo),Radix Dipsaci(Xuduan),Fructus Psoraleae(Buguzhi),Rhizoma Anemarrhenae(Zhimu),Radix et Rhizoma Salviae(Danshen)and Radix Rehmanniae(Dihuang).It has been proved to be effective in the prevention and treatment of osteoporosis in clinical.In this thesis,the ovariectomized C57BL/6J mice osteoporotic model was adopted,using the bone microstructure of lumbar vertebrae as evalulation index.The results showed that the 60%ethanol extract of "Xian-Ling-Gu-Bao" capsule powder(XLGB)possessed osteoprotective effects on ovariectomized mice(OVX)osteoporotic model.Moreover,among the fractions of XLGB over Daion HP-20 column,XLGB-B(30%ethanol elute)and XLGB-C(95%ethanol elute)were proved to be the anti-osteoporotic active fractions of XLGB in vivo.The further investigation on the chemical constituents of XLGB-B and XLGB-C led to the isolation of 61 compounds through various chromatographic techniques.Their structures were established on the basis of their physic-chemical properties and spectroscopic analysis,including thirty-five flavones,five saponins,five coumarins,four lignans,three iridoids,five phenolic components and other four compounds.Among them,five compounds were new flavonoid glycosides,namely 2"-0-rhamnosylcaohuoside C.(35),4"-O-acetyl-3"-O-xylopyranosylikarisosi de A(37),3"-O-xylopyranosylepimedoside A(38),hydroxylepimedin C(40),4'-methoxylkaempf erol 3-0-[a-L-rhamnopyranosyl(1?2)-?-L-rhamnopyranoside]-7-0-?-D-glucopyranoside(41).The anti-osteoporotic activities of thirty-eight isolated compounds(compounds 1-28,37,42,47-49,52-54,57,59)were evaluated in vitro.Flavonoids(compounds 17-28,37,42,47-49)could promote bone formation,suppress bone resorption and inhibit adipogenic differentiation of preadipocyte.Hederagenin saponins(compounds 57,59)could promote bone formation and suppress bone resorption.Other compounds(compounds 1-16,52-54),including phenolic components,iridoids,lignans and coumarins,showed effect on promoting bone formation.Furthermore,a rapid identification method based on high performance liquid chromatography coupled with diode-array detection and high resolution electrospray ionization tandem mass spectrometry(HPLC-DAD-HR-ESI-MSn)was established.Based on UV spectrum,high-resoluti-on mass spectrum and multi-stage mass spectrum information,combined with the chemical components reported in the literatures of the original plant of "Xian-ling-gu-bao" and the mass cleavage pattern of various compounds,the structures of 117 chemical components were elucidated.And 16 of them might be new compounds.The structure elucidation was confirmed by comparing with the sixty-one compounds isolated from XLGB,and further enriched the chemical composition characterization of "Xian-ling-gu-bao"This work partly revealed the anti-osteoporosis material basis of "Xian-ling-gu-bao",and provided evidence for developing the scientific and rational methods for quality control.This investigation also provided a possible research method for the study of bioactive and effective material basis of traditional Chinese medicine prescription.