Study On The Quality Evaluation Of Bupleuri Radix Based On LC-MS Metabolomics

Rationales:Bupleuri radix has been a common traditional Chinese medicine in the clinic since ancient times.It has the effect of evacuating fever,soothing liver and relieving depression,and lifting yang qi.The main varieties of Bupleuri radix include Bupleurum chinense DC.and Bupleurum scorzonerifolium Willd.The main production areas of Bupleurum chinense DC.are Shanxi,Shaanxi,Gansu,Hebei,Inner Mongolia,and other regions.Its planting areas in Shanxi Province are mainly concentrated in Yuncheng,Linfen,Changzhi,Datong,Lvliang,Xinzhou,and other regions.The chemical composition is the material basis for fully exerting the therapeutic effect of Bupleuri radix.Therefore,determining the chemical composition differences between different production areas and harvesting seasons of Bupleuri radix is of great significance for the quality evaluation of Bupleuri radix.Objective: The purpose of this study was to characterize the chemical components of Bupleuri radix by UHPLC-Q-TOF-MS,and use non targeted metabolomics techniques to conduct overall analysis of all chemical components.A relative quantitative method using UHPLC-Q-Trap-MS s MRM and HPLC-DAD was established for the saponins and polyynes,which are important pharmacological active components in Bupleuri radix,respectively,to further analyze their content changes.Methods:(1)The mass spectrometric data of Bupleuri radix from different places of origin were collected by UHPLC-Q-TOF-MS,and the chemical components of Bupleuri Radix were identified by using the self-built local library of Bupleuri radix,the library contained in SCIEX OS software,and the GNPS molecular network.Multivariate statistical analysis was used to evaluate the differences of chemical components between different habitats and different harvest seasons of Bupleuri radix.(2)A method for the determination of saponins by UHPLC-Q-Trap-MS s MRM was established,and its stability,repeatability,intra-day precision and intra-day precision were investigated.The method was applied to 37 batches of Bupleuri radix samples for pseudotargeted metabonomic analysis to compare the differences of saponins among different Bupleuri radix samples.(3)The single factor method was used to screen the optimal extraction method by examining different extraction times and solvents,using the extraction rate of petroleum ether fraction and the peak area of UHPLC as indicators.A comparative quantitative analysis was conducted on the content of polyacetylenes in 35 batches of samples of Bupleuri radix from inside and outside Shanxi Province,and the content differences between samples from different habitats and different harvest seasons were compared.Result:(1)A total of 79 chemical components were identified by the self-built local library,including 46 saponins,7 flavonoids,7 coumarins,6 alkynes,5 fatty acids,2 caffeic quinic acids,1 chromogen ketone and 1 saccharol;The GNPS molecular network was used to infer 7 possible saponins;the above two methods identified a total of 86 chemical compositions.Using multivariate statistical analysis techniques to analyze the differences in chemical composition of Bupleuri radix under different classification methods,PCA results showed that there was no significant clustering of Bupleuri radix from different regions,only slight differences were observed in the production of Bupleuri radix from Datong and Yuncheng;However,there is a clear clustering trend among the samples of Bupleuri radix collected in different seasons.Further utilizing OPLS-DA analysis,14 differential metabolites of Datong and Yuncheng Bupleuri radix were identified;There are 12 differential metabolites between harvesting Bupleuri radix in spring and autumn and harvesting in summer.(2)The established UHPLC-Q-Trap-MS s MRM method can simultaneously detect 25 saponins in Bupleuri radix,and the method has good repeatability,stability,intra-day and intra-day precision.A pseudotargeted metabolomics analysis was conducted on 37 batches of Bupleuri radix samples,and the results showed that based on the 25 saponin components measured,Bupleuri radix samples from different regions did not form a clear cluster;There are certain differences among the samples of Bupleuri radix collected in different seasons,and a total of 9 different metabolites were found.Correlation analysis showed that 25 saponin components were divided into two groups based on whether they were acetylated or malonylated.The content of acetylated and malonylated saikosaponins in summer samples was significantly higher than that in spring and autumn samples.Four volatile saponin components,including Clinoposaponin XII,were identified through coefficient of variation analysis.(3)The 95% ethanol ultrasound method was selected to extract and separate the petroleum ether fractions of 35 batches of Bupleuri radix in Shanxi Province,and the HPLC-DAD method was used for relative quantitative analysis of the polyacetylene components.The results show that the total content of polyynes in Bupleuri radix in Shanxi Province is higher than that outside Shanxi Province,and the total content of polyynes of Bupleuri radix in Yuncheng production area of Shanxi Province is higher than that in Datong and Linfen;The content of polyacetylene components in the Bupleuri radix samples collected in spring and autumn is higher than the sample collected in summer.Conclusion: This study found that different origins have little overall impact on the chemical composition of Bupleuri radix,indicating that Bupleuri radix has strong growth adaptability,high chemical consistency,and good quality uniformity of medicinal materials,making it suitable for widespread cultivation.Different harvesting seasons have a certain impact on the chemical composition of Bupleuri radix.The content of polar components such as saponins and flavonoids is higher in non traditional harvesting seasons(summer)of Bupleuri radix;However,low polarity components such as polyynes and fatty acids are more abundant in the samples collected during the traditional harvesting season(spring and autumn).The key rate limiting enzymes for acetylation and malonylation of saikosaponin sugar chains may have higher activity in summer.This study has laid a certain foundation for the quality evaluation of Bupleuri radix.