The Study Of Er-Xian Decoction On Anti-osteoporotic Pharmacological Effects And Proteomics

Er-xian Decoction(EXD),an effective Chinese herbal formula developed for the treatment of menopause syndrome,menopause hypertension with Shen-essence deficiency,infertility and osteoporosis,was designed and named with clinical validation by professor Bo-Na Zhang in the 1950’s.The formula consists of six Chinese herbs:Curculi Rhizoma,Epimedii Folium,Morindae Officinalis Radix,Anemarrhenae Rhizoma,Phellodendri Cortex,Angelicae sinensis Radix,which have dual regulation efficacy of Warm Shen and Tonify Yang,Nourish Yin and Reduce Pathogenic Fire.This thesis include four parts:I.Quantitative analysis of EXD extracts and HPLC-DAD-MS/MS spectra qualitative analysis of EXD.Ⅱ.The study of anti-osteoporotic effect on ovariectomized(OVX)and retenoic acid-induced osteoporosis rat,and analgesic effect on ICR mice of EXD extracts.Ⅲ.The study of EXD and its active ingredients on osteoblasts and osteoclasts.Ⅳ.Set up the proteomic protocol involved of 2-DE,image analysis and spectrometry detection,on osteoblasts and osteoclasts treat with EXD and active ingredients.Aim to clarify the multi-component,multi-target,multi-channel mechanism of EXD mechanism action.Ⅰ.Quantitative and qualitative analysis results shows that:1.EXD extracts mainly contains flavonoids,alkaloids,saponins.Total flavonoids should not less than 24%in content,of icariin,total saponins should not less than 16.0%in content of timosaponin B,total alkaloids should not less than 2.0%in content of berberine.2.Attribution of 21 peaks and structures of 10 peaks in EXD HPLC-DAD-MS/MS spectra are identified.Peak 1 and peak 6 belongs to Anemarrhenae Rhizoma,peak 2 and peak 11 belongs to Curculi Rhizoma,,peak 3,peak 4,peak 5,peak 7,peak 8,peak 9 and peak 20 belongs to Phellodendri Cortex,peak 10 and 21 peak belongs to Angelicae sinensis Radix,peak 12、peak 14、peak 15、peak 16、peak 17、peak 18 and peak 19 belongs to Epimedii Folium.Peak 2:curculigoside,peak 5:phellodendrine,peak 6:mangiferin,peak 12:thalphenine,peak 13:berberine,peak 14:baohuoside,peak16:icariin,peak 19:anhydroicaritin,peak 20:kihadalactone B and peak 21:decursin.Ⅱ.The study of anti-osteoporotic effect in vivo shows that:1.EXD extracts have significantly anti-osteoporotic effect on OVX rats whether preventive medicine treatment or medicine therapeutic administration in the dose of 27-108mg/kg.EXD extracts increase bone mineral density,maximum deflection,maximum stress of femur,trabecular bone area,trabecular bone thickness of tibia,calcitonin in serum,decrease trabecular bone gap of tibia,TRAP,but no significant effect on the body and uterine weight of OVX rats.2.EXD extracts have significant anti-osteoporosis effect on retenoic acid-induced osteoporosis rats,especially postmenopausal osteoporosis.3.EXD extracts have significantly analgesic effect on female ICR mice in the dose of 55-220mg/kg.Ⅲ.The study of anti-osteoporotic effect in vitro shows that:1.EXD,icariin and timosaponin B significantly promote the proliferation,increase alkaline phosphatase(ALP)activity and bone nodules formation of osteobalstic cells UMR-106.High-dose of EXD group increase bone nodules the most,which is 147.3%of the control group.2.EXD and icariin significantly inhibit the preosteoclasts RAW264.7 proliferation,and the inhibition rate on RAW264.7 of EXD is 31.39%.High-dose of berberine inhibit preosteoclasts RAW264.7 proliferation,but medium and low-dose of berberine has no effect on the proliferation of RAW264.7.3.High,medium and low-dose of EXD,icariin and berberine significantly inhibit proliferation,activity of TRAP and bone resorption pits of osteoclasts induced by RAW264.7.The inhibition rate of EXD on TRAP activity and bone resorption pits is 23.86%and 52.28%,better than icariin and berberine.The effect of EXD on promotion of osteoblast formation and inhibition of bone resorption are better than that of monomer composition,probably due to the receptor of each active monomer composition is different.The roles of multi-component of Chinese formula are cumulative and synergy.Ⅳ.Proteomic protocol involved of 2-DE,image analysis and spectrometry detection is set up.Proteomics study of EXD and its components shows that:proteins separation in two-dimensional gel electrophoresis of EXD and active compositions on osteoblasts and osteoclasts meet the requirement of PDQuestTM 2-D Analysis Software.54 proteins of 88 differentially expressed proteins were successfully identified,the successful identification rates are 61.2%,more than the 50%identification successful rates of silver nitrate staining proteins method in generally,means results reliable.Almost all peptides ion score indexed in MASCOT is more than 100,means mass spectrometry reports credible.Bioinformatics and literature review showed that differentially expressed proteins functions related to apoptosis,cell proliferation,cell differentiation,proteins aggregation,proteins folding,molecular chaperones,cytoskeleton formation,cell proliferation signals transduction,oxidative stress and mineral crystal fiber et.,reveals the multi-component,multi-target,multi-channel mechanism of Chinese formula EXD,provides the new technological platform for the study of Chinese formula pharmacology mechanism.