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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.

Background MYB transcription factors play critical roles in secondary metabolite biosynthesis in medicinal plants and are crucial for abiotic stress responses. However, no studies have identified BcMYB genes in Bupleurum chinense DC. or analyzed their expression profiling under temperature stress. The role of BcMYB in ​ B. chinense ’s response to temperature stress and its regulation of saikosaponins synthesis remains unclear. Results This study performed a genome-wide identification of the BcMYB gene family in B. chinense using its genomic data, and analyzed the changes in saikosaponins content, physiological indicators, and BcMYB expression patterns under temperature stress, along with their correlations with saikosaponin biosynthesis. A total of 85 BcMYBs genes were identified, classified into four subfamilies, with amino acid lengths ranging from 306 to 482 aa and relative molecular masses between 23,710.84 and 53,062.36. Except for BcMYB10 and BcMYB50 , all other BcMYB genes were localized in the nucleus. Most BcMYB promoter regions contained low-temperature and drought-responsive elements. BcMYB genes underwent strong purifying selection after duplication. Short-term temperature stress (6 days) significantly stimulated saikosaponins synthesis: Ss-a content increased by 94% under low temperature (15 °C) and 34% under high temperature (35 °C), accompanied by elevated antioxidant enzyme activities (SOD: 58%, POD: 41%) and MDA accumulation. Temperature stress predominantly upregulated upstream genes in the saikosaponins biosynthesis pathway (1-Deoxy-D-xylulose-5-phosphate synthase (DXS) , 1-Deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) , Isopentenyl pyrophosphate isomerase (IDI) , 3-Hydroxy-3-methylglutaryl-CoA reductase (HMGR) ), while mid- and downstream genes (Farnesyl pyrophosphate synthase (FPS) , β-Amyrin synthase (β-AS) , Cytochrome P450 monooxygenase (P450-1 P450-8) ) were more responsive to high temperature. BcMYB28 , 41 , 44 , 46 , 47 , 68 , and 79 were significantly upregulated under both low and high temperatures ( p  < 0.05), whereas BcMYB25 and 56 were specifically induced by low temperature. BcMYB25 , 28 , 41 , 44 , 46 , 47 , 56 , 68 , and 79 demonstrated strong connectivity and correlations with saikosaponins biosynthesis genes and saikosaponins content. Conclusions The physiological responses of B. chinense to temperature stress and its secondary metabolic regulatory network exhibit dynamic synergistic characteristics. BcMYB , a critical transcription factor in B. chinense , plays a pivotal role in adapting to temperature fluctuations under temperature stress. This transcription factor may participate in establishing a “temperature signal-regulatory gene-metabolite” cascade regulatory network, thereby modulating the synthesis of saikosaponins in B. chinense . In agricultural practices, harvesting B. chinense after gradual cooling and exposure to low-temperature conditions for 6–12 days significantly enhances medicinal material quality.

Background Radix Bupleuri, derived from the dried roots of Bupleurum chinense DC., is a well-documented phytomedicine in global pharmacopoeias and a common constituent in herbal formulations. While previous studies have hinted at regional variations in the chemical composition of B. chinense , a comprehensive understanding of its morphological, genetic, and chemical diversity across China remains incomplete. Objective This study aims to investigate the infraspecific variation of B. chinense by analyzing its morphological, genetic, and chemical phenotypes. Methods Wild B. chinense specimens were collected from 31 locations spanning nine Chinese provinces/municipalities, representing a wide range of its natural distribution. A multi-faceted approach combining 21 morphological traits, plastid genome sequencing, and chemical analysis was employed to explore infraspecific variation and clustering patterns. Results Distinct infraspecific variation was revealed through integrated morphological and molecular data. Morphological clustering analysis identified two geographically associated clusters, roughly corresponding to coastal and inland regions. Although plastid genome sequencing of 40 specimens showed high sequence identity, population structure analysis detected variable hotspots. Both maximum likelihood (ML) tree and population structure results consistently identified three distinct clades, which mirrored the patterns observed in morphological clustering. Quantitative analysis of saikosaponins content in 10 representative specimens across the three clades demonstrated significant chemotype variation. Notably, samples from Anhui Province exhibited the highest saikosaponins content, while those from Shanxi Province showed the lowest levels. This chemotype variation, coupled with observed genetic diversity, suggests that B. chinense germplasm from Clade I (particularly from Anhui Province) represents a promising wild resource for further development. Graphical Abstract

Key messageDrastic changes in soil water content can activate the short-term high expression of key enzyme-encoding genes involved in secondary metabolite synthesis thereby increasing the content of secondary metabolites.Bupleurum chinense DC. is a traditional medicinal herb that is famous for its abundant saikosaponins. In the current study, the effects of drought–re-watering–drought on the photosynthesis physiology and biosynthesis of saikosaponins were investigated in 1-year-old B. chinense. The results showed that alterations in soil moisture altered the photosynthesis physiological process of B. chinense. The dry weight and fresh weight of the roots, photosynthesis capacity, chlorophyll fluorescence parameters, and SOD, POD and CAT activities were significantly reduced, and the contents of SP, soluble sugars, PRO and MDA increased. There were strong correlations between different physiological stress indices. All indices promoted and restricted each other, responded to soil moisture changes synergistically, maintained plant homeostasis and guaranteed normal biological activities. It was found that RW and RD_1 were the key stages of the water-control experiment affecting the expression of saikosaponin-related genes. At these two stages, the expression of multiple genes was affected by changes in soil moisture, with their expression levels reaching several-fold higher than those at the previous stage. We noticed that the expression of saikosaponin synthesis genes (which were rapidly upregulated at the RW and RD_1 stages) did not coincide with the rapid accumulation of saikosaponins (at the RD-2 stage), which were found to correspond to each other at the later stages of the water-control experiment. This finding indicates that there is a time lag between gene expression and the final product synthesis. Rapid changes in the external environment (RW to RD_1) have a short-term promoting effect on gene expression. This study reveals that short-term stress regulation may be an effective way to improve the quality of medicinal materials.

Bupleurum chinense DC. and Bupleurum scorzonerifolium Willd. have different clinical efficacies, with the former typically used to treat typhoid fever and the latter mainly used to clear liver heat. The differences in their clinical efficacy are closely related to their complex chemical composition, especially the active components. In this study, the saponins and volatile oils in two varieties of Radix Bupleuri grown in different regions were extracted and analyzed using high-performance liquid chromatography (HPLC) and gas chromatography coupled with mass spectrometry (MS), and the absolute contents of five saikosaponins were accurately quantified using an established HPLC-MS method in the multiple reaction monitoring mode. Multivariate statistical analysis was performed to reveal the difference in the active components between the two varieties. The saikosaponin content was significantly affected by variety and growing region, with all five saikosaponins being significantly higher in Bupleurum chinense DC. than in Bupleurum scorzonerifolium Willd. The results of principal component analysis and hierarchical cluster analysis show a clear distinction between the two varieties in terms of both saponins and volatile oils. Twenty-one saponins, including saikosaponin b2 and b1, and fifty-two volatile oils, including 2-tetradecyloxirane and chloromethyl cyanide, were screened and identified as differential compounds contributing to the significant difference between the two varieties. These compounds may also be responsible for the difference in clinical efficacy between Bupleurum chinense DC. and Bupleurum scorzonerifolium Willd. All the results suggest that the accumulation and diversity of active components in Radix Bupleuri are significantly affected by the variety. In contrast to previous reports, this study provides the absolute contents of five saikosaponins in Radix Bupleuri of different varieties and reduces the influence of the growing region on the analytical results by collecting samples from different regions. The results of this study may provide a reference for the identification and quality evaluation of different varieties of Radix Bupleuri.

Background Radix Bupleuri is a popular traditional Chinese medicinal plant. Its root contains saikosaponin and volatile oil compounds with antipyretic, anti-inflammatory, and hepatoprotective pharmacological effects. However, there are differences in the content and type of main chemical components in the roots of three Bupleurum species: Bupleurum chinense DC. (Bchi), Bupleurum scorzonerifolium Willd. (Bsco), and Bupleurum marginatum var. stenophyllum (Wolff) Shan et Y.Li (Bmar). The molecular mechanism behind these differences is still unclear. The present study used integrated metabolome and transcriptome analyses to uncover the differences in metabolites and expressed genes among the three Bupleurum species. Results Metabolomics results revealed that Bmar contained more saikosaponins than Bchi and Bsco. Conversely, Bsco had the highest content of volatile oil monoterpenes but a lower sesquiterpene content than Bchi and Bmar. Transcriptome analysis showed that several genes were highly expressed in Bchi, Bsco, or Bmar, demonstrating the molecular mechanism responsible for the differences in their metabolic components. We combined the metabolomics and transcriptomics data to investigate the relationship between metabolites and genes. The results showed a high correlation between CYP450, UGT, and β-AS genes and 6’’-acetyl-saikosaponins A, saikosaponins B1, C, and D. The subcellular localization of the two P450 genes ( Bc087391 and Bc036879 ) in the endoplasmic reticulum suggests that they may be involved in saikosaponin biosynthesis. Conclusion We performed an integrated transcriptome and metabolome analysis to investigate the diversity of the terpenoid biosynthetic pathway in three Bupleurum species. The study provides new insights into the molecular basis of the metabolic differences between the three Bupleurum species. It also serves as a theoretical basis for the clinical application and breeding of Bupleurum resources.

The saikosaponins comprise oleanane- and ursane-type triterpene saponins that are abundantly present in the roots of the genus Bupleurum widely used in Asian traditional medicine. Here we identified a gene, designated CYP716Y1 , encoding a cytochrome P450 monooxygenase from Bupleurum falcatum that catalyzes the C-16α hydroxylation of oleanane- and ursane-type triterpenes. Exploiting this hitherto unavailable enzymatic activity, we launched a combinatorial synthetic biology program in which we combined CYP716Y1 with oxidosqualene cyclase, P450, and glycosyltransferase genes available from other plant species and reconstituted the synthesis of monoglycosylated saponins in yeast. Additionally, we established a culturing strategy in which applying methylated β-cyclodextrin to the culture medium allows the sequestration of heterologous nonvolatile hydrophobic terpenes, such as triterpene sapogenins, from engineered yeast cells into the growth medium, thereby greatly enhancing productivity. Together, our findings provide a sound base for the development of a synthetic biology platform for the production of bioactive triterpene sapo(ge)nins.

Background As an important medicinal plant, the synthesis of active ingredient saikosaponin in Bupleurum chinense DC. is significantly influenced by environmental factor stress. However, there is limited understanding of how secondary metabolites in medicinal plants change under the multiple combined stresses. Results This study investigated the changes in saikosaponins content in Bupleurum chinense under drought combined with temperature stress, as well as the potential molecular and ecological mechanisms involved. The experimental plants were divided into four groups: control (CK), drought with low temperature (DL), drought with medium temperature (DM), and drought with high temperature (DH). DL, DM, and DH inhibited plant height and root length, with DH exhibiting the strongest inhibitory effect. The degree of stress followed the order DH > DM > DL > CK. B. chinense adapted to the combined stresses by modulating osmotic regulatory substances and protective enzyme activities. Under combined stress, five enzymes in the terpenoid synthesis pathway showed positive changes. The content of saikosaponins, specifically saikosaponin a and saikosaponin d, increased significantly under DH treatment in the short term. On day 4 of treatment, their levels reached 4.52, 2.07, and 1.54 mg/g, representing increases of 40.82%, 32.76%, and 36.97%, respectively, compared to CK. Abscisic acid levels under DL, DM, and DH were 12.90-, 6.38-, and 16.27-fold higher, respectively, than those under CK. High-quality transcriptome sequencing revealed active changes in gene expression profiles. Weighted gene co-expression network analysis identified the turquoise module genes (12,753 target genes), which were strongly correlated with physiological indices, plant hormones, functional enzymes, and saikosaponins. Saikosaponins synthesis was found to be regulated by multiple transcription factors and functional genes, with distinct regulatory networks governing abscisic acid and saikosaponins. Conclusions Our findings demonstrate that the synthesis and accumulation of saikosaponins exhibit a “short-term promotional effect” under combined stress, regulated by multiple factors. This study elucidates the molecular and ecological mechanisms underlying the rapid accumulation of saikosaponins and provides technical insights for ecological strategies to support B. chinense high-quality cultivation.

Bupleurum , a plant of the genus Bupleurum L. in the family Umbelliferae, is prevalent and extensively applied in traditional medicine systems across East and Southeast Asian countries for the treatment of colds, malaria, hepatitis and other diseases. In the current Chinese herbal medicine market, only the dried roots of two species, Bupleurum chinense DC. and Bupleurum scorzonerifolium Willd., are authentic herbs, and their cultivars dominate as the commercial source, contributing to about 80% of the market share. Shanxi Province, known as the suitable habitat for Bupleurum in China, has a diverse ecosystem and geographical areas with diverse environmental conditions. These diversity ecosystem and environmental conditions cause prominent variations in the content of active ingredients of Bupleurum L. across different sites. Therefore, analyze the ecological, geographical, and soil factors that influence the quality of Bupleurum and to recommend the best suitable sites for cultivation of Bupleurum . This study demonstrates a close correlation between the quality of Shanxi Bupleurum sp. and different ecological factors. A total of 70 sets of Bupleurum and soil samples were collected from 25 counties across 6 cities in Shanxi Province. Consequently, the saponin contents of Datong, Shuozhou and Yizhou were generally higher than those of Linfen, Jincheng and Yuncheng. Bupleurum from northern Shanxi exhibits higher saikosaponin content compared to that from southern Shanxi; The total content of the five saponins shows a significant positive correlation with longitude (* P  < 0.05) and a highly significant positive correlation with latitude and altitude (** P  < 0.01). Saikosaponin levels positively correlate with latitude, longitude, and altitude; Meanwhile, the significance ranking of these ecological factors is: monthly average temperature is equal to monthly average surface temperature is greater than monthly sunshine hours. Low temperatures, arid conditions, and longer sunlight exposure are optimal conditions for the accumulation of saponin components; Besides, high-saponin Bupleurum is typically cultivated in low alkaline soils with low nitrogen, while the habitat of Shanxi Bupleurum is differentiated into four regions. Overall, the current study presents a foundation for selecting the best cultivation sites for Bupleurum and provides a valuable reference for evaluating other medicinal herb production regions.

Background/Objectives: Bupleuri Radix is a plant in the Apiaceae family Bupleurum Chinense DC. or Bupleurum scorzonerifolium Willd. root. The dissimilarities in the metabolite profiles of plants directly correlate with the disparities in their clinical efficacy. Methods: Therefore, the wild Bupleurum Chinense DC. (YBC) and wild Bupleurum scorzonerifolium Willd. (YNC) were used as research materials. They were analyzed using the UPLC-MS/MS and the similarities and differences were uncovered based on differential metabolites. Results: Our results proved that the differences in clinical efficacy between YBC and YNC may be attributed to their distinct metabolite profiles, as follows: (1) a total of 12 classes of 2059 metabolites were identified in the roots, with phenolic acids, terpenoids, and flavonoids being the most abundant metabolic products, with 2026 shared components between the two, 2045 in YBC, and 2040 in YNC; (2) a total of 718 differential metabolites were identified, accounting for 35.44% of the shared metabolites. Among them, YBC had 452 metabolites with higher content relative to YNC, representing 62.95%, and 266 components with lower content, representing 37.05%; (3) the KEEG enrichment analysis results show that the differential metabolic pathways are flavone and flavonol biosynthesis, linoleic acid metabolism, arachidonic acid metabolism, sesquiterpenoid and triterpenoid biosynthesis, and linolenic acid metabolism. Conclusions: These new findings will serve as a foundation for further study of the BR biosynthetic pathway and offer insights into the practical use of traditional Chinese medicine in clinical settings.