Analysis Of Ginseng Active Components In Two Places Based On HPLC-Triple TOF/MS Technology

Objective:Since ancient times,ginseng,as a functional food,has been received much attention from local and abroad people.Ginseng has been proved to have anti-cancer,fatigue relief,anti-inflammatory and other effects by modern pharmacological studies.As an important active components in ginseng,ginsenoside determines the efficacy and price of ginseng effectively.The main reason of the differences in the types and contents of ginsenosides in different area are diverse soil conditions,temperature,humidity and altitude conditions in the production areas,which eventually leads to the variety of ginseng effects in different producing areas.In addition,morphological identification is the main methods of verification of ginseng origin,which mainly depends on the experience and knowledge reserve of professional technicians,it is prone to misjudgment and lacks the support of theoretical data.Therefore,it is necessary to figure out the chemical components of ginseng from different area and establish a data spectrum database to provide scientific basis for ginseng quality control,origin identification,quality evaluation.Collectivelly,the main medicinal components of ginseng,ginsenoside,are the research focus.Taking the ginsenosides in two places ginseng as the landmark substances,the main differential marker in two places ginseng were fully discussed.Except that,the origin traceability data database and the qualitative and quantitative methods of ginsenosides were established.Methods:1.To establish a method for the determination of chemical components in ginseng.High Performance Liquid Chromatography Triple Time of Flight Mass Spectrometry,HPLC-Triple TOF/MS.Optimize the liquid chromatography conditions（selection of chromatographic column,mobile phase type and gradient elution procedure）and sample pretreatment conditions（ultrasonic time,extraction solvent,etc.）.The method was applied to extract the mass spectrum information of ginsenosides in ginseng from two producing areas for subsequent analysis.2.Identification of ginsenosides and construction of mass spectrometry database.Based on the mass spectrum information of ginsenosides extracted by the established HPLC-Triple TOF/MS method,ginsenosides were confirmed by SCIEX high resolution secondary database of natural products and ginsenoside standards.Ginsenoside standard samples are scanned and collected by mass spectrometry instrument.Combined with SCIEX OS software,the retention time,accurate quality database and mass spectrum library of ginsenoside are established subsequently.The established library information was matched with the information collected from ginseng samples,and the accurate mass number deviation,retention time,parent ion fragment and daughter ion fragment information were compared to qualitatively analyze ginsenoside.Ginsenosides without standards were confirmed by accurate mass number comparison and data matching of SCIEX high resolution secondary database of natural products。3.Screening differential markers and establishing origin identification data library.Ginsenoside data were combined with metabo Analyst online data analysis platform for Principal Component Analysis（PCA）and Orthogonal Partial Least Squares Discriminant Analysis（OPLS-DA）,According to the degree of separation,VIP value and P value,the difference markers between the two ginseng were judged.After that,the data spectrum database of origin identification was established.4.Quantitative detection of target ginsenosides.Through the prepared ginseng standard working solution,the standard curves of 20 ginsenosides were drawn,and the content of ginsenosides in ginseng from the two places was determined by the established HPLC Triple TOF/MS method,and the content was statistically analyzed.Results:1.After the optimization of the method,the final detection conditions were as follows:ginseng samples were extracted with 70%methanol for 15 min,0.1%formic acid water acetonitrile was used as mobile phase,gradient eluted on C18chromatographic column for 90 min,and detected by HPLC Triple TOF/MS.2.Based on the established HPLC-Triple TOF/MS,ginsenosides in ginseng were determined.According to the obtained mass spectrometry information,combined with SCIEX natural products high-resolution secondary database and ginsenoside standard comparison,24 ginsenosides were identified.3.The obtained ginsenoside data were combined with metaboanalyst online data analysis platform for PCA and OPLS-DA.According to the degree of separation,VIP value and P value,a total of 13 differential markers of ginseng from the two producing areas were finally identified,and the data spectrum database of origin identification was established.4.The established standard curve of 20 ginsenosides was used to accurately quantify the identified ginsenosides,compare the difference of ginsenoside content in the two places of origin,and statistically analyze the content of 20 ginsenosides in the two places by using the quantitative method.It was found that there was a significant difference in the content of ginsenoside Re、Rb₁、Rb₂、Rb₃、Rc、20（S）-Rg₃、Rk₁、F4、F1、Pseudoginsenoside RT₅、Notoginsenoside R1、F3、protopanaxatriol in the two places of ginseng（P<0.05）,but there was no significant difference in the content of Ginsenoside Rg₁、Rf、Rd、Rs₂和20（S）-ginsenoside Rg₂、20（S）-ginsenoside Rh₂、Notoginsenoside R2.Conclusions:1.In this paper,the qualitative and quantitative methods of ginsenosides in ginseng from two places of origin were established by using HPLC-Triple TOF/MS detection technology combined with multivariate statistical analysis.A total of 24ginsenosides were identified in ginseng from two places.2.Combining the data obtained by HPLC-Triple TOF/MS with multivariate statistical analysis,the database of the differences in the types and contents of ginsenosides in ginseng between the two places was successfully constructed,and 13difference markers were identified.3.A quantitative analysis method of 20 ginsenosides in ginseng from two places was established.The contents of 20 ginsenosides in ginseng from the two places were quantitatively detected by standard curve,the contents of ginsenosides Rb₂,Rb₃,Rb₁,Rc,Ro,F3,Re and Notoginsenoside R1 in Russian ginseng were higher than those in Chinese ginseng;The contents of ginsenoside 20（s）-Rg₃,Rk₁,F4,F1,protopanaxatriol and Pseudoginsenoside RT₅ in Chinese ginseng were higher than those in Russian ginseng.The results provide a reference for the scientific and objective evaluation of ginsenoside analysis in ginseng,and also provide data support for combating adulteration and selling.