Chemical Constituents With Antiosteoporosis Effects In The Bioactive Fraction Of Sambucus Willamsii Hance

Sambucus williamsii Hance(SWH)is one species of Sambucus genera in Caprifoliaceae.It has the potence of expelling wind-dampness,rejoining fracture and restoring soft tissue,invigorating blood circulation and stopping pains.It has been usually used in treatment of fracture,traumatic injury,rheumatoid arthritis,gout,kashi-beck disease and traumatic hemorrhage.In this thesis,the fractions separated from 60%ethanol crude extract of SWH using D101 macro adsorptive resin column were administrated to OVX mice and their effects on bone properties were studied.The 50%ethanol eluate(SWC)of SWH subjected to macro adsoptive resin chromatography significantly restored bone mineral density(BMD)and improved bone microarchitecture at femur and tibia as well as increased biomechanical strength at tibia diaphysis in OVX mice.Similarly,SWC was the most potent fraction in stimulating cell proliferation and differentiation(concentration from 0.1 to 100 ?g/ml)in UMR 106 cells.Besides,SWC did not alter uterus weight in OVX mice,therefore SWC was determined as the bioactive fraction of SWH.Then the HPLC characteristic spectrum of SWC was established and 14 major peaks(occupied 82.3%total peak areas)were isolated,identified and characterized.The proliferation effects of the 14 compounds on UMR 106 cells and MCF 7 cells were evaluated.The results showed that 8-0-4'lignans,tetrafuran lignans and phenolic acids promoted the proliferation of UMR 106 cells,while benzofuran lignans without such effects.Amoung 14 compounds,only one compound increased the number of MCF 7 cells.For further isolation of the micro and semimicro compounds in bioactive fraction,more amount of SWC was applied to systemic separation.48 compounds were isolated from SWC through various chromatographic techniques.The structures were elucidated on the basis of theirs physicochemical properties and spectroscopic characters as 1*:(7R,8R)-3-methoxy-4',8-epoxy-7,3'-oxyneolignan-4,9,1'-triol;2*:(7R,8S)-7,8-dihydro-7-(4-hydroxy-3-methoxyphenyl)-1'-hydroxy-3'-methoxy-8-benzofuranmethanol;3:(7R,8S)-ceplignan;4:(7R,8S)-Ficusal;5:8R-Evofolin B;6:3,9-dimethoxy-11-hydroxypropyl-6H-benzo-furo-[3,2-c]-[1]benzopyran;7:(7S,8R)-dihydrode-hydrodiconiferyl alcohol-9-O-?-D-apiofuranosyl-(1?6)-O-?-D-glucopyranoside;8:(7S,8R)-hierochin D;9:(7S,8R)-dihydro-3'-hydroxy-8-hydroxy-methyl-7-(4-hydroxy-3-methoxy-phenyl)-1'-benzofuranpropanol;10:(7R,8S)-dihydrodehydrodi-coniferyl alcohol;11:(7R,8S)-dehydrodi-coniferyl alcohol;12:(7S,8R)-dihydrodehydrodiconiferyl alcohol-9'-O-?-glucosdie;13:(7R,8S)-2,3-dihydro-2-(4-hydroxyl-3-methoxyphenyl)-7-methoxy-5-(3-methoxy-1-(propenyl)-3-be nzofuranmethanol;14:(7R,8S)-1-(4-hydroxy-3-methoxy-phenyl)-2-[4-(3-hydroxypropyl)-2-methoxyphenoxy]-1.3-propanediol;15:(7R,8R)-1-(4-hydroxy-3-methoxyphenyl)-2-[4-(3-hydroxyl-propyl)-2-methoxyphenoxy]-1,3-propanediol;16:(7S,8R)-guaiacylglycerol-?-o-4'-conifery ether;17:(7R,8R)-guaiacylglycerol-?-o-4'-conifery ether;18:(7R,8R)-3-methoxy-8,4'-oxyneolignan-3',4,7,9,9'-pentol;19:(7S,8R)-guaiacylglycerol-?-O-4'-sinapyl ether;20:(1S,2R)-1-(4'-hydroxy-3'-methoxyphenyl)-2-[4"-(3-hydroxypropyl)-2",6"-dimethoxyphenoxy]propane-1,3-diol;21:(7S,8R)-guaiacylglycerol-?-coniferyl aldehyde ether;22:(7S,8R)-syingylglycerol-?-O-4'-coniferyl ether;23:(8S,7'R,8'S)-olivil;24:(8S,7'R,8's)-lariciresinol;25:(8S,7'R,8'S)-5'-methoxylariciresinol;26:(8S,7'R,8'S)-5,5'-dimethoxylariciresinol;27:(8S,7'S,8'R)-ciwujiatone;28:(77R,8R,7'R,8'S)-4,4',9,7'-tetrahydroxy-3,3'-dimethoxy-7.9'-epoxylignan;29:(7R,8S,7'R,8'S)-syringaresinol;30:(7R,8S,-7'R,8'S)-tortoside A;31:(7R,8S,7'R,8'S)-medioresinol;32:gmelinol;33:(7S,8S,8'R)-Lyoniresinol;34:(7S,8R,8'S)-isolariciresino;35:(7S,8S,8'S)-Burselignan;36:(7R,7"R,8"R)-4'-guaiacyl-glycerol-Evofolin B;37:(7R,8S,7'R,8'S,7"S,8"R)4'-guaiacylglycerol-medioresinol-4-O-?-d-gluco-pyranoside;38:(7R,8S,8'S,7"R,8"S)-2,3-dihydro-2-(4-hydroxy-3-methoxyphenyl)-7-methoxy-5-[[tetrahydro-5-(4-hydroxy-3-methoxyphenyl)-4-(hydroxymethyl)-3-furanyl]methyl]-3-Benzofuran-methanol;39:(7R,8S,7'R,8'S,8"S)-2,3-dihydro-2-(4-hydroxy-3-methoxyphenyl)-7-methoxy-5-[tetrahydro-4-[(4-hydro-xyl-3-methoxyphenyl)methyl]-3-(hydroxylmethyl)-2-furanyl]-3-Benzofura nmethanol;40:(7R,8S,7'R,8'S)-Vitrifol A;41:(7R,8S,7'R,8'S,8"S)-Seslignanoccidentaliol A;42:2?,4?,10?-trihydroxyl-1?H,5?H-guaia-6-ene;43:Coniferol;44:Ferulic acid;45:Methyl caffeate;46:Vanilic acid;47:Umbelliferon;48:Tiashic acid.Among them,7 compounds(1,2,6,7,36,37,42)were new compounds and 20 compounds(3-5,8,11,13,17,18,21,22,25-28,32,35,38-41)were isolated from the Sambucus genus for the first time.The mechanism of two compounds,(7R,8S)-4,7,9,9'-tetrahydroxy-3,3'-dimethoxy-8-O-4'-neolignan and Vanilic acid with proliferation effects on UMR 106 cells and without such effects on MCF 7 cells,was preliminary studied.The results suggested that the two compounds might inhibit bone resorption by suppressing osteocalstogenesis via modulation of OPG/RANKL/RANK system.The blocking effects of ICI 182780 on the proliferation effects of the two compounds on UMR 106 cells indicated that the action of both compounds was involved in ER pathway.The results of ER binding assay showed that the two compounds did not bind to ER? or ER ?,which was different from E2 and isoflavonoid.Meanwhile,the two compounds did not activate ERE-dependent transcription and AP-1-dependent transctiption.The concrete pathway of bioactive compounds on bone protection need be further studied in future work.Our present investigation provided the scientific evidences for research and developement of SWH on osteoporosis in the future.