Qualitative And Quantitative Characterization Of Chemomes In Sini San Using Liquid Chromatography-mass Spectrometry Method

Sinisan(SNS)is a classic formula was firstly recorded in "Shanghanlun".SNS has widerange of therapeutic activities,by mainly targeting the pathological disorders with stomach,liver and intestine.SNS is composed of four Chinese medicinal plants with the same dosage,involving Radix Bupleurichinensis,Radix Paeoniae Alba,Fructus Citri Aurantii and Radix Glycyrrhizae Uralensi.Currently,SNS and associated formulae are used to treat various gastrointestinal diseases,hepatobiliary diseases,and gynecological diseases.However,the complexity to chemical composition of SNS seriously impeded the modern development and translational applications of this medicine in preventing diseases and improving health.Although,some studies reported chemicals of SNS,using various instruments combined with bioinformatics annotation.These studies is often extremely timeconsumable,and cannot precisely identify the key chemicals at last,which severely limited further elucidation of bioactive compounds and associated therapeutic mechanisms of this formula.In the present study,our effort is going to precisely elucidate chemomes(key chemicals)of SNS,by developing a robust and sensitive LC-MS based chemical profiling method,combined with the MS/MS recognition of available reference-compounds.Firstly,we optimized multiple parameters that significantly affects the extraction rate,peak-volume,features of separation/sensitivity/accuracy,and exposure levels of SNS chemomes,including extraction method,chromatography parameters,MS parameters,and the method of data analysis and visualization.Then we employed the developed LC/MS based chemical profiling method to generally characterize chemomes of SNS,by subjecting to literature mining,database retrieving and MS/MS deduction,as our results reveals 93 proposed chemicals detected from both water extract and ethanol extract of SNS.Furthermore,we tried our best to acquire commercially available reference-compounds based on the identities of 93 chemicals,we eventually obtained 46 reference compounds,and harvested their highresolution MS/MS data.To precisely identify the key chemomes of SNS,MS/MS spectra of reference compounds were assigned to MS/MS spectra of sample-chemicals,we found 39 key compounds were observed to exactly match the available reference-compounds.Among these compounds,saikosaponin D,saikosaponin A,saikosaponin F,and isofraxidin comes from Radix Bupleurichinensis;the(+)catechin,paeoniflorin,oxypaeoniflorin,albiflorin,benzoylpaeoniflorin present in Radix Paeoniae Alba.In addtion,naringenin,naringin,limonin,nobiletin,tangeretin,narirutin,homoeriodictyol,meranzin,isosinensetin,sinensetin,5,7,8,4’-tetramethoxy flavone,and 3’,4’,7,8-tetramethoxyflavone vicenin-2 are existed in Fructus Citri Aurantii,while isoliquiritigenin,formononetin,glycyrrhizic acid,liquiritin,liquiritin apioside,neolicuroside,isoliquiritin,ononin,liquiritigenin,glycycoumarin,licoricone,isoschftoside,glabrol,vicenin-2,licorice saponin G2,isoangustone A,glyasperin D are found in Glycyrrhizae Uralensi.Only hesperidin is observed to present in in four Chinese medicinal plants.Importantly,quantitative method is also established using reference compounds to absolute determination of the key chemomes in two solvent-extracts,we found that two solvents exert differential extraction capability to the chemomes of SNS,especially the exposure levels of nobiletin、sinensetin、glycycoumarin、saikosaponin F、(+)catechin、albiflorin、saikosaponin A、saikosaponin D、licorice saponin G2、glyasperin D and isoangustone A in both solvents are markedly different.In short,two extraction methods work together for precisely capturing 39 key chemicals present in SNS.Taken together,we successfully developed a combinational strategy facilitated by highresolution UPLC/MS method,literature mining,database retrieving and reference compound matching,which has been applied for precise identification of the key chemomes in twosolvent extracts of SNS.This finding should provide importantly scientific foundation for further deciphering bioactive compounds and therapeutic mechanisms of SNS,as well as offer a new strategy for the chemome identities of other Chinese medicinal plants and related formula.