Study On The Therapeutic Effect And Chemical Basis Of Shixiao San For The Treatment Of Hyperlipemia

Shixiao San,a classical Traditional Chinese Medicine formula containing two herbs Typha Pollen and Faeces Trogopterori,was originally recorded in the Complete Collection of Prescriptions(Taiping Huimin Heji Ju Fang),written in Song Dynasty of ancient China by Imperial Medical Service.In modern clinical therapies,Shixiao San plays a vital role in treatment of cardiovascular disease,such as hyperlipemia,coronary heart disease,angina pectoris,digestive system disease and dysmenorrheal,etc.,which has aroused researcher's great interests.In this study,Shixiao San was selected and investigated employing pharmaceutical analysis,natural product chemistry,pharmacology and molecular biology combined with serum biochemistry and histopathology to screen effective parts of Shixiao San and its potential mechanism for the treatment of hyperlipidemia.Moreover,the effective parts were confirmed based on the determination of endogenous substances.Furthermore,the main chemical constituents were identified by ultra high-performance liquid chromatography coupled with mass spectrometry(UPLC/Q-TOF/MS)method.On this basis,five main components from effective part were simultaneous determinated and successfully applied to the pharmacokinetics study in rat plasma.Finally,cell-based screening method identified the active ingredients from Shixiao San as the inhibitors of endothelial dysfunction.1.The screening of effective parts of Shixiao San for the treatment of hyperlipidemiaEffects of Shixiao San with different dosages on serum levels of lipids,lipoprotein and blood antioxidant enzymes in rats induced by hyperlipidemia were investigated.As a result,the dosage of 2.8 g/kg/d was selected as the optimal dosage.Effects of Shixiao San extracted by various polar solvents were investigated in the same way.Ethyl acetate(EtOAc)and n-butyl alcohol(BuOH)extract exhibited a remarkably decrease in total cholesterol(TC),triglyceride(TG),low density lipoprotein-cholesterol(LDL-C),apolipoprotein B(ApoB)and malondialdehyde(MDA)levels compared to the control rats.In contrast,the high density lipoprotein-cholesterol(HDL-C),apolipoprotein A-I(ApoA-I)andsuperoxide dismutase(SOD)levels after exposing to EtOAc and BuOH extract were significantly increased compared to control group.For arcus aortae,no significant atherosclerotic lesions were presented in EtOAc and BuOH extract groups.Finally,the extracts of EtOAc and BuOH were found to be effective fractions.2.Confirmation the effective parts of Shixiao San and identification of the components of effective parts for the treatment of hyperlipidemiaA rapid and simple method for simultaneous determination of alanine(Ala),arginine(Arg),lysine(Lys),methionine(Met),serine(Ser),threonine(Thr),tyrosine(Tyr)and valine(Val)in rat plasma using high performance liquid chromatography mass spectrometry(HPLC-MS)was developed.After addition of the internal standard(IS)tinidazole,plasma samples were extracted by protein precipitation(PPT)with methanol(0.01 formic acid),separated on an BDS HYPERSIL C18 column and analyzed in a single chromatographic run in less than 12.5 min,using gradient elution with the mobile phase consisting of methanol and 0.01%formic acid in water.The detection of the analytes was performed on 2010 EV MS system with positive ion mode.The developed method provided excellent linear calibration curves for the assay of analytes(r?0.9962).Mean extraction recoveries of analytes and IS from rat brain were all more than 77.30%.The improvement of the rats in the treatment of Shixiao San and its effective fractions were evaluated based on the level changes of Ala,Arg,Lys,Met,Ser,Thr,Tyr and Val in rat plasma.The results showed that the effective fractions of EtOAc and BuOH had definitely therapeutic effect on hyperlipidemia.A UPLC/Q-TOF/MS method was employed for screening and identifying the main constituents of EtOAc and BuOH extracts.Nineteen compounds were identified.Among the nineteen compounds,six compounds of them belong to flavonoid aglycones and eleven compounds of them belong to flavone glycoside.The research determined the foundation to the study of Shixiao San-treated hyperlipidemia and it also laid foundation to investigate the therapeutic mechanism.3.Simultaneous determination of five flavonoids in rat plasma by ultra HPLC-MS/MS and its application to a comparative pharmacokinetic study in normal and hyperlipidemic ratsA simple and rapid ultra-high performance liquid chromatography-tandem mass spectrometry(UHPLC-MS/MS)method has been developed for the simultaneous determination of five free flavonoids(amentoflavone,isorhamnetin,naringenin,kaempferol and quercetin)and their total(free and conjugated)forms.Furthermore,we compared the pharmacokinetics of these active ingredients in normal and hyperlipidemic rats.The free and total forms of these flavonoids were extracted by liquid-liquid extraction with ethyl acetate.The conjugated flavonoids were deconjugated by the enzyme ?-Glucuronidase and Sulfatase.Chromatographic separation was accomplished on a ZORBAX Eclipse XDB-C8 USP L7 column using gradient elution.Detection was performed on a 4000Q UHPLC-MS/MS system from AB Sciex using negative ion mode in the multiple reaction monitoring(MRM)mode.The lower limits of quantification were 2.0-5.0 ng/mL for all the analytes.Intra-day and inter-day precision were less than 15.0%and accuracy ranged from-9.3%to 11.0%,and the mean extraction recoveries of analytes from rat plasma were all more than 82.8%.The validated method was successfully applied to a comparative pharmacokinetics study of five free and total analytes in rat plasma.The results indicated that the absorption of five total flavonoids in hyperlipidemia group were significantly higher than those in normal group with similar concentration-time curves.4.Cell-Based Screening Identifies the Active Ingredients from Shixiao San for the treatment of complication of hyperlipidemiaWe performed a phenotypic screening in human endothelial cells exposed to oxidized low density lipoprotein(an in vitro model of endothelial dysfunction)to identify the effective compounds in Shixiao San.After investigating the suitability and reliability of the cell-based screening method using atorvastatin as the positive control drug,this method was applied in screening Shixiao San and its extracts.The treatment of BuOH fraction on endothelial cells exhibited strongest healing effects against oxidized low density lipoprotein-induced insult when compared with other fractions.Cell viability,the level of nitric oxide,endothelial nitric oxide synthase and endothelin-1 were measured,respectively.The assays revealed BuOH fraction significantly elevated the survival ratio of impaired cells in culture.In parallel,n-butanol fraction exhibited the highest inhibition of inflammation.The generation of prostaglandin-2 and adhesion molecule(soluble intercellular adhesion molecule-1)was obviously declined.Furthermore,n-butanol fraction suppressed the production of reactive oxygen species and malondialdehyde,and restored the activity of superoxide dismutase.Compounds identification of the n-butanol fraction was carried out by UPLC/Q-TOF/MS.The active ingredients including quercetin-3-O-(2G-?-1-rhamnosyl)-rutinoside,quercetin-3-O-neohesperidoside,isorhamnetin-3-O-neohesperidoside and isorhamnetin-3-O-rutinoside revealed the ability of anti-atherosclerosis after exposing on endothelial cells.The current work illustrated the pharmacology effect of Shixiao San and clearly indicated the major active components in Shixiao San.