| Si-miao-yong-an decoction,a classical Traditional Chinese Medicine formula containing fore herbs,Flos Lonicerae Japonicae(Lonicera japonica Thunb.,Jinyinhua),Radix Scrophulariae Ningpoensis(Scrophularia ningpoensis Hemsl.,Xuanshen),Radix Angelicae Sinensis(Angelica sinensis(Oliv.)Diels,Danggui)and Radix Glycyrrhizae Uralensis(Glycyrrhiza uralensis Fisch.,Gancao).,show effect in treatment of cardiovascular disease,such as hyperlipemia,coronary heart disease,digestive system disease and nonalcoholic fatty liver disease(NAFLD),etc.,which has appeal to attentions.In this study,Si-miao-yong-an decoction was selected and investigated employing pharmaceutical analysis,pharmacology and molecular biology combined with serum biochemistry and histopathology to clarify its antihyperlipidemia effect in rats.Moreover,the main compounds in the decoction and plasma sample obtained after oral administration of Si-miao-yong-an decoction were identified by ultra high-performance liquid chromatography coupled with mass spectrometry(UPLC/Q-TOF/MS)method.Furthermore,fore main components from the decoction were simultaneous determinated and successfully applied to the pharmacokinetics study in rat plasma.Based on the main elimination pathway of cholesterol,a high-performance liquid chromatography coupled with mass spectrometry(HPLC-MS/MS)method was established and validated to investigate the effect of si-miao-yong-an decoction on bile acid synthesis pathway Finally,the remove lipid and protect liver cell effect of si-miao-yong-an decoction was investigated using HL7702 cell model.1.Therapeutic effect of Si-miao-yong-an decoction in hyperlipidemia ratsEffects of Si-miao-yong-an decoction with different dosages on serum levels of lipids,inflammatory factors and blood antioxidant enzymes in rats induced by hyperlipidemia were investigated.The results indicated that si-miao-yong-an decoction exhibited a remarkably decrease in total cholesterol(TC),triglyceride(TG),low density lipoprotein-cholesterol(LDL-C),malondialdehyde(MDA),intercellular cell adhesion molecules-1(s-ICAM),interleukin-6(IL6)compared with model group.In contrast,the high density lipoprotein-cholesterol(HDL-C),reduced glutathione(GSH)and catalase(CAT)levels after oral administration of si-miao-yongan decoction were significantly increased compared with Model group.Liver function test and tissue section examination indicated that si-miao-yong-an decoction improved liver function and reduced fat accumulation in hyperlipidemia rat liver.The results indicated that antihyperlipidemia effect of si-miao-yong-an decoction may be related with its antioxidant,antiinflammatory,and liver protect effect.2.Identification of the components in si-miao-yong-an decoction and compounds from plasma after oral administration of si-miao-yong-an decoctionIn this part,a UPLC-Q-TOF-MS/MS method was employed for screening and identifying the main constituents of si-miao-yong-an decoction.59 compounds were identified from the decoction.Among the 59 compounds,32 compounds were from Flos Lonicerae Japonicae,4 compounds were from Radix Scrophulariae Ningpoensis,13 compounds were from Radix Angelicae Sinensis,and 10 compounds were form Radix Glycyrrhizae Uralensis.11 compounds were identified from rat plasma after oral administration of si-miao-yong-an decoction,6 compouds from Flos Lonicerae Japonicae,1 compound from Radix Scrophulariae Ningpoensis,1 compound from Radix Angelicae Sinensis and 3 compounds were from Radix Glycyrrhizae Uralensis.The research determined the foundation to the study of si-miao-yongan decoction-treated hyperlipidemia and it also laid foundation to investigate the therapeutic mechanism.3.Simultaneous determination of fore compounds in rat plasma by HPLC-MS/MS andits application to a pharmacokinetic study in hyperlipidemic ratsIn this paper,a high performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS)method has been developed and validated for the simultaneous determination of fore analytes(chlorogenic acid,neochlorogenic acid,harpagoside,liquiritin)in rat plasma after oral administration of si-miao-yong-an decoction.Chromatographic separation was accomplished on a CAPCELL CORE ADME(3.0mm×50mm,2.7μm)column using gradient elution.Detection was performed on a LC-MS/MS system from Waters using negative ion mode in the multiple reaction monitoring(MRM)mode.The analytes show good linearity and the lower limits of quantification were 5.0-10.0 ng/mL.Precision of intra-day and inter-day precision were less than 15.0%and the accuracy of the method ranged from-9.9%to 12.2%,and the mean extraction recoveries of analytes from rat plasma were all more than 62.2%.The LC-MS/MS method was successfully applied to a pharmacokinetics study of chlorogenic acid,neochlorogenic acid,harpagoside,liquiritin in hyperlipidemia rat plasma.4.Effect of si-miao-yong-an decoction on bile acid synthesis pathway.A LC-MS/MS method was established and well validated to evaluate major bile acids derived from cholesterol metabolism through the classic neutral pathway and the alternative acidic pathway(cholic acid,chenodeoxycholic acid and their taurine and glycine conjugates)in liver and plasma.Endogenous bile acids were removed by incubation with activated carbon to obtain blank matrix.Chromatographic separation was accomplished on a CAPCELL CORE ADME(3.0mmx 150mm,2.7μm)column using gradient elution.Detection was performed on a HPLC-MS/MS system from Waters using negative ion mode in the multiple reaction monitoring(MRM)mode.The validated method was successfully applied to determination of 6 bile acids in plasma and liver samples.Increased total 6 bile acids concentrations in both liver and plasma were observed after oral administration of 12g/kg/d,24g/kg/d and 36g/kg/d of SMYAD in a dose dependent manner which contributed to eliminate of cholesterol.Cholic acid,taurocholic acid and glycocholic acid act as the main products of bile acid classic neutral synthesis pathway and show sharp increase(p<0.01)after treatment of SMYAD at dosage of 24-36g/kg/d.For liver samples,taurocholic acid level act as the largest growth section,while in plasma samples,cholic acid act as the largest growth section after SMYAD treatment,compared with Model group.By contrast,the main products of alternative acidic pathway(chenodeoxycholic acid and its glycine and taurine conjugates)show no significant increase after treatment of SMYAD.In conclusion,the cholesterol lowing effect of SMYAD may be related with the accelerated transformation of cholesterol into bile acids through the classic neutral pathway.5.Lipid-lowing effect of si-miao-yong-an decoction in HL7702 cell modelA lipid deposition model were successfully established by incubation HL7702 cell with Cell Culture Media contained 900 μM of free fatty acids(OA:PA=2:1),atorvastatin as the positive control drug.Si-miao-yong-an show good lipid lowing effect,cell viability,the level of TC,TG,aspartate transaminase(AST),alanine transaminase(ALT)were determined,respectively.The results indicated that si-miao-yong-an decoction could reduce cell TC,TG levels and show significant lipid lowing effect in oil-red stain.Moreover,si-miao-yong-an decoction decreased AST and ALT levels which show protect effect to liver cells.The results were in accord with animal therapeutical effects. |
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