Table_5_Metabolomics Analysis Reveals the Differences Between Bupleurum chinense DC. and Bupleurum scorzonerifolium Willd..XLSX

Bupleurum chinense DC. and Bupleurum scorzonerifolium Willd. are two varieties of Bupleuri Radix in Chinese Pharmacopoeia 2020. The clinical efficacy of the two bupleurum species is different. The difference in clinical efficacy is closely related to the composition of plant metabolites. In order to analyze the difference in metabolites, we used liquid chromatography coupled with mass spectrometry (LC-MS) for untargeted metabolome and gas chromatography coupled with mass spectrometry (GC-MS) for widely targeted metabolome to detect the roots (R), stems (S), leaves (L), and flowers (F) of two varieties, and detected 1,818 metabolites in 25 classes. We performed a statistical analysis of metabolites. Differential metabolites were screened by fold-change and variable importance in the projection values of the OPLS-DA model, and significant differences were found among different groups. The content of active components (triterpenoid saponins) was found to be high in the BcR group than in the BsR group. Other pharmacological metabolites were significantly different. By Kyoto Encyclopedia of Genes and Genomes annotation and enrichment analysis, we found that differential metabolites of the aboveground parts mainly concentrated in monoterpenoid biosynthesis, while the differential metabolites of the root mainly concentrated in sesquiterpenoid and triterpenoid biosynthesis. Differences in metabolic networks may indirectly affect the metabolic profile of Bc and Bs, leading to differences in clinical efficacy. Our study provides a scientific basis for subsequent biosynthesis pathway and related bioactivity research, and provides a reference for developing non-medicinal parts and guiding the clinical application of Bupleuri Radix.北柴胡（Bupleurum chinense DC.）与狭叶柴胡（Bupleurum scorzonerifolium Willd.）是《中华人民共和国药典2020年版》中柴胡药材（Bupleuri Radix）的两个基原品种，二者临床疗效存在显著差异，且该差异与植物代谢物组成密切相关。为解析二者代谢物组成差异，本研究采用液相色谱-质谱联用（liquid chromatography coupled with mass spectrometry, LC-MS）进行非靶向代谢组学（untargeted metabolome）检测、气相色谱-质谱联用（gas chromatography coupled with mass spectrometry, GC-MS）进行广泛靶向代谢组学（widely targeted metabolome）检测，对两个品种的根（R）、茎（S）、叶（L）及花（F）进行取样分析，共鉴定得到25类共计1818种代谢物。随后对代谢物进行统计分析：通过倍数变化（fold-change, FC）值与正交偏最小二乘判别分析（orthogonal partial least squares discriminant analysis, OPLS-DA）模型的投影变量重要性（variable importance in the projection, VIP）值筛选差异代谢物，发现不同分组间代谢物存在显著差异。活性成分三萜皂苷（triterpenoid saponins）在北柴胡根（BcR）中的含量显著高于狭叶柴胡根（BsR），其余药理活性代谢物也存在显著差异。通过京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）注释与富集分析发现，地上部分差异代谢物主要富集于单萜类生物合成通路，而根部差异代谢物则主要富集于倍半萜与三萜类生物合成通路。代谢网络差异可能间接影响北柴胡与狭叶柴胡的代谢谱，进而导致其临床疗效出现差异。本研究为后续柴胡的生物合成通路及相关生物活性研究提供了科学依据，同时也为柴胡非药用部位的开发利用及临床合理用药提供了参考。