Investigation On The Chemical Constituents Of Triterpenoid Saponins For Quality Control In Pulsatilla Chinensis(Bge.) Regel And Identification Of The Metabolites In Vivo And In Vitro Of Betulinic Acid

Pulsatilla chinensis(Bge.)Regel(P.chinensis)is a common traditional Chinese medicine,possessing the effects of heat-clearing and detoxicating,cooling blood and stopping dysentery,which belongs to the genus Pulsatilla(Ranunculaceae).P.chinensis is widely used for the treatment of bacterial dysentery,amoebic diseases,vaginal trichomoniasis bacterial infections and malignant tumor.The distribution of P.chinensis is extensively,including Hebei,Neimeng-gu,Shanxi,Gansu,Anhui,Henan and Hubei provinces,and the contents in P.chinensis with different origins have obvious differences.Hence,it is essential for simultaneous determination and identification of major active chemical constituents for the quality control.Traditional Chinese medicine is a complex component system,and the investigation for quality control focus on “multi-component” and “multi-target” features.Accordingly,in this study,ultra high performance liquid chromatography tandem mass spectrometry(UPLC-ESI-MS/MS)combining with tissue-smashing extraction technique was used for simultaneous determination of 12 major active compounds in P.chinensis,and principle components analysis(PCA)was applied to evaluate and classify P.chinensis samples with different origins.This can provide the scientific basis for the quality control of P.chinensis.In this study,we developed a comprehensive and systematic analytical strategy to discover and identify triterpenoid saponins and isomers from P.chinensis using a simple,rapid and robust method(UPLC-Q-TOF-MS/MS).As a result,a total of 22 triterpenoid saponins(11 pairs of isomers)were tentatively identified or elucidated in crude extracts from P.chinensis.This research provides effective technical methods for the clarification of differences in TCM and theoretical basis for the quality control of TCM,which is abundant with pentacyclic triterpenoid saponins.Thus it can lay a firm foundation for comprehensively and deeply studying the correlation between active chemical composition and the efficacy of TCM.Betulinic Acid(BA)is one of the active compounds in P.chinensis and belongs to pentacyclic organic acid.The pharmacological studies revealed that it had the effects of anti-tumor and inhibitory HIV-1 infection.Based on UPLC-Q-TOF-MS/MS technique,we established a method for identification and characterization the metabolites of betulinic acid in vivo and in vitro.And we used the above method to detect their samples of the rat feces and the biotransformation,and then deduced the metabolic pathways according to the metabolites.The study on the metabolic process of betulinic acid in vivo can provide the theoretical basis for the metabolism of pentacyclic triterpenoid saponins in P.chinensis.Part one Simultaneous determination of twelve active components in theroots of Pulsatilla chinensis using UPLC-ESI-MS/MS andprincipal component analysis Objective: To develop a ultra high performance liquid chromatography tandem mass spectrometry(UPLC-ESI-MS/MS)method for the simultaneous determination of 12 compounds(?-ecdysterone,ajugasterone C,hederasaponin C,cirenshenoside S,pulsatilloside C,betulinic acid glycoside-3-O-?-arabinopyranosyl-(1-3)-?-L-rhamnopyransoyl-28-O-?-glucopyranosyl-23-hydroxy,anemoside B4,hederasaponin B,anemoside A3,?-hederin,23-hydroxybetulinic acid and hederagenin)in P.chinensis.At the same time,principal component analysis(PCA)was used for better evaluate these samples quality,and it can provide the basis for the establishment of quality standard and quality control in P.chinensis.Methods: The chromatographic separation was performed on a Phenomenex Kinetex-C18 column(100×3 mm,2.6 mm)and the column temperature was set at 40°C.The mobile phase,methanol consisted of 0.1% formic acid(A)and 0.1%(v/v)formic acid(B),were run at a flow rate of 0.4 m L/min and the analytes were eluted with a gradient system.Multiple-reaction monitoring(MRM)scanning was employed for determination with positive and negative modes.The conditions of MS/MS detector were set as follows: source temperature 150oC,capillary voltage 2.0 k V,and desolvation temperature 350oC,desolvation and cone gas at a flow rate of 800 and 150 L/h,respectively.Atomization gas pressure 7.0 bar and the interface heater was turned on.Results: The method linearity of twelve analytical response was good with correlation coefficients R2 value greater than 0.9990.And the repeatability of the instrument,intra-day and inter-day precision and solution stability were in accordance with the requirements.The average recoveries were in the range of 97.6-104.2% with RSD values less than 4.7%.The obtained results demonstrated that 33 batches of samples contained cirenshenoside S,although with obvious difference in absolute content.PCA results showed that samples in the main component space were successfully divided into two clusters,which indicated that origins have impact on the quality of P.chinensis.Conclusions: The analytical method established in this study posseses the advantage of simple operation,rapid speed,strong specificity and high sensitivity,and can be employed to the simultaneous quantification of multiple components in P.chinensis.Meanwhile,PCA method can provide the basis for the classification and quality evaluation of Pulsatilla herbs,and it is helpful for the reasonable and normative medication in clinical.Part two Rapid identification and qualitative analysis of triterpenoidsaponins in Pulsatilla chinensis by UPLC-Q-TOF-MS/MSObjective: To identify the triterpenoid saponins and isomers from P.chinensis,the fragmentation pathways of triterpenoid saponins under both positive and negative mode were investigated and summarized.Then a systematic analytical strategy for identification was established.Methods: Firstly,the mass fragmentation patterns of eight major triterpenoid saponins were studied in the positive and negative ion mode by full scan,product ion scan and precursor ion scan.Then the clevage rules were summarized and the analytical strategy was established.The analysis was performed on a UPLC-Q-TOF-MS/MS system.Combined with the relevant reference,a mass spectra library on triterpenoid saponins in Pulsatilla genus was established.According to the accurate mass measurement,fragment ion and retention time,the chemical constituents of pentacyclic triterpenoid saponins in P.chinensis were tentatively identified.The chromatographic separation was performed on a Agilent Poroshell 120 SB-C18 column(2.1 mm×100 mm,2.7 ?m)and the column temperature was set at 40°C.The mobile phase,0.1%(v/v)formic acid(A)and methanol consisted of 0.1% formic acid(B),were run at a flow rate of 0.3 m L/min and the analytes were eluted with a gradient system.The injection volume was 5 ?L.Results: Compared the chromatographic behavior,mass accurate measurement and MS/MS fragment ions with reference standards,eight peaks were assigned.Peaks 3,4,7,14,17,18,21 and 22 were identified as anemoside B4,hederasaponin C,pulsatilloside C,hederasaponin B,anemoside A3,?-hederin,23-hydroxybetulinic acid and hederagenin,respectively.At the same time,22 pentacyclic triterpenoid saponins(11 pairs of isomers)were identified and characterized based on the above strategy.Conclusions: The UPLC-Q-TOF-MS/MS method established in this study has the advantage of high sensitivity and strong specificity.The analytical strategy can provide strong technical support for the qualitative analysis and identification constituents in Pulsatilla genus and other TCM.It is significant for the quality control of TCM.Part three Identification of metabolites of betulinic acid in vivo and vitroby UPLC-Q-TOF-MS/MSObjective: To develop UPLC-Q-TOF-MS/MS method for catching and identifying of betulinic acid metabolites in rat feces and the biotransformation samples,as well as to deduce pathways rule.Methods: At first,liquid-liquid extraction method was used to pretreat biological samples and UPLC-Q-TOF-MS/MS technique was employed to acquire exact mass and MS/MS data.Secondly,the data acquired were analyzed and identified by a combination of data mining methods in Metabolite Pilot 1.5 software(extracted ion chromatography,mass defects filter,product ion filter and neutral loss filter),obtaining the accurate mass measurement,elemental composition and MS/MS spectra of metabolites.Finally,according to the accurate mass measurement,drug biotransformation knowledge and fragmentation regulation of parent drug,the metabolites of betulinic acid were deduced and Clog P values were applied to distinguish isomers.Results: Based on the proposed strategy,a total of 46 metabolites,in intestinal bacteria biotransformation sample and in rat feces,were identified,including 35 metabolites in rat feces,20 metabolites in biotransformation sample and 7 metabolites were both in biotransformation sample and rat feces.Conclusions: In this study,the metabolites of betulinic acid in vivo and vitro were successfully identified using the method of UPLC-Q-TOF-MS/MS,and then the result can be a theoretical foundation for the metabolic processes and pharmacological effects of betulinic acid.The method is rapid,sensitive,and it can be used for the metabolites identification of monomer and TCM in vivo.