Intercropping with maize and sorghum-induced saikosaponin accumulation in Bupleurum chinense DC. by liquid chromatography-mass spectrometry-based metabolomics.

Bupleuri Radix is an important medicinal plant, which has been used in China and other Asian countries for thousands of years. Cultivated Bupleurum chinense DC. (B. chinense) is the main commodity of Bupleuri Radix. The benefits of intercropping with various crops for B. chinense have been recognized; however, the influence of intercropping on the chemical composition of B. chinense is still unclear yet. In this study, intercropping with sorghum and maize exhibited little effect on the root length, root diameter, and single root mass of B. chinense. Only the intercropping with sorghum increased the root length of B. chinense slightly compared to the monocropping. In addition, 200 compounds were identified by UHPLC-Q-TOF-MS, and metabolomic combined with the Venn diagram and heatmap analysis showed apparent separation between the intercropped and monocropped B. chinense samples. Intercropping with sorghum and maize could both increase the saikosaponins, fatty acyls, and organic acids in B. chinense while decreasing the phospholipids. The influence of intercropping on the saikosaponin biosynthesis was probably related with the light intensity and hormone levels in B. chinense. Moreover, we found intercropping increased the anti-inflammatory activity of B. chinense. This study provides a scientific reference for the beneficial effect of intercropping mode of B. chinense.

Study on characterization of Bupleurum chinense polysaccharides with antioxidant mechanisms focus on ROS relative signaling pathways and anti-aging evaluation in vivo model.

This study explored the structures of three polysaccharides from Bupleurum chinense DC. (BCPRs), and evaluated their antioxidant and anti-aging properties. The HPGPC and ion chromatography analyses revealed that the molecular weights of the BCPRs ranged from 12.05 to 21.20 kDa, and were primarily composed of rhamnose, arabinose, xylose, galactose, glucose and galacturonic acid. Methylation and NMR studies identified 10 PMAAs, establishing the various backbones of BCPRs 1-3. BCPR-3 demonstrated potent antioxidant activities, including DPPH, ABTS, hydroxy, and superoxide radicals scavenging in vitro. At concentrations between 125 and 500 µg/mL, BCPR-3 increased T-AOC, SOD and GSH-Px activities, while decreasing MDA levels in H2O2-induced SH-SY5Y cells. In addition, RNA-seq results indicated that BCPR-3 considerably downregulated the expression of 49 genes and upregulated five genes compared with the control group. KEGG analysis suggested that these differentially expressed genes (DEGs) were predominantly involved in the TNF and PI3K/Akt signaling pathways. Furthermore, in vivo experiment with Drosophila melanogaster showed that BCPR-3 could extend the average lifespan of flies. In conclusion, polysaccharides from B. chinense exhibited potential antioxidant and anti-aging activities, which could be developed as new ingredients to combat oxidative stress damage and slow the aging process.

Homotherapy-for-heteropathy of Bupleurum Chinense DC.-Scutellaria baicalensis Georgi in treating depression and colorectal cancer: A network pharmacology and animal model approach.

ETHNOPHARMACOLOGICAL RELEVANCE: Bupleurum chinense DC.-Scutellaria baicalensis Georgi (BS) is a classic drug pair that has good clinical effects on depression and many tumors. However, the concurrent targeting mechanism of how the aforementioned drug pair is valid in the two distinct diseases, has not been clarified yet. AIM OF THE STUDY: The components of BS were detected by LC-MS, combined with network pharmacology to explore the active ingredients and common targeting mechanism of its multi-pathway regulation of BS in treating depression and CRC, and to validate the dual effects of BS using the CUMS mice model and orthotopic transplantation tumor mice model of CRC. RESULTS: Twenty-nine components were screened, 84 common gene targets were obteined, and the top 5 key targets including STAT3, PIK3R1, PIK3CA, AKT1, IL-6 were identified by PPI network. GO and KEGG analyses revealed that PI3K/AKT and JAK/STAT signaling pathways might play a crucial role of BS in regulating depression and CRC. BS significantly modulated CUMS-induced depressive-like behavior, attenuated neuronal damage, and reduced serum EPI and NE levels in CUMS model mice. BS improved the pathological histological changes of solid tumors and liver tissues and inhibited solid tumors and liver metastases in tumor-bearing mice. BS significantly decreased the proteins' expression of IL-6, p-JAK2, p-STAT3, p-PI3K, p-AKT1 in hippocampal tissues and solid tumors, and regulated the levels of IL-2, IL-6 and IL-10 in serum of two models of mice. CONCLUSION: BS can exert dual antidepressant and anti-CRC effects by inhibiting the expression of IL-6/JAK2/STAT3 and PI3K/AKT pathway proteins and regulating the release of inflammatory cytokines.

Quantitative and Differential Analysis between Bupleurum chinense DC. and Bupleurum scorzonerifolium Willd. Using HPLC-MS and GC-MS Coupled with Multivariate Statistical Analysis.

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.

Bupleurum chinense exerts a mild antipyretic effect on LPS-induced pyrexia rats involving inhibition of peripheral TNF-α production.

ETHNOPHARMACOLOGICAL RELEVANCE: Bupleuri Radix, the dried roots of Bupleurum chinense DC. (BC) or Bupleurum scorzonerifolium Willd., is one of the most frequently used traditional Chinese medicines. As the species in Xiao-Chai-Hu decoction, BC has been used as an antipyretic medicine with a long history. However, its antipyretic characteristics and underlying mechanism(s) remain unclear. AIM OF THE STUDY: To elucidate the antipyretic characteristics and mechanism(s) of BC used in its traditional way. METHODS: The water extract of BC (BCE) was prepared according to the traditional decocting mode. Murine fever and endotoxemia models were induced by intravenous injection of lipopolysaccharide (LPS). In vitro complement activation assay and the levels of TNF-α, IL-6, IL-1ß, and C5a were determined by ELISA. RESULTS: BCE exerted a confirmed but mild antipyretic effect on LPS-induced fever of rat. In vitro, it significantly lowered LPS-elevated TNF-α in the supernatant of rat complete blood cells and THP-1 cells, but failed to decrease IL-6 and IL-1ß. In murine endotoxemia models, BCE markedly decreased serum TNF-α, but had no impact on IL-6 and IL-1ß. BCE also restricted complement activation in vitro and in vivo. Nevertheless, the mixture of saikosaponin A and D could not suppress supernatant TNF-α of monocytes and serum TNF-α of endotoxemia mice. CONCLUSIONS: The present study dissects the peripheral mechanism for the antipyretic effect of BC used in the traditional way. Our findings indicate that BCE directly suppresses monocyte-produced TNF-α, thus decreasing circulating TNF-α, which may be responsible for its mild but confirmed antipyretic action.

Comparative Transcriptome Analysis Provides Insights into the Molecular Mechanism Underlying the Effect of MeJA Treatment on the Biosynthesis of Saikosaponins in Bupleurum chinense DC.

Bupleurum chinense DC. is a well-known traditional Chinese medicinal plant that produces saikosaponins (SSs), which possess hepatoprotective, antipyretic, and anti-inflammatory activities. Methyl jasmonate (MeJA) is a signalling phytohormone that can increase the accumulation of SSs in the root of Bupleurum plants. However, the molecular understanding of MeJA-mediated SS biosynthesis is not clear. Therefore, it is necessary to explore the molecular mechanism underlying the response of B. chinense DC. to MeJA in roots. In this study, we performed comparative transcriptome analysis of B. chinense DC. roots with different MeJA treatment times. In total, 104,057 unigenes were identified, of which 4053 were differentially expressed genes (DEGs). Most of the DEGs were downregulated after MeJA treatment, and GO enrichment analysis showed that they were mainly related to biological processes involved in stress responses and development. A total of 88 DEGs encoding enzymes known to be involved in the SS synthesis pathway were found, and most were significantly downregulated within 24 h. Based on the DEGs, 99 transcription factors (TFs) belonging to the AP2/ERF, WRKY, bZIP, ZFP, and bHLH families with different expression patterns were also identified. Further integrated analysis indicated that 20 DEGs involved in the SS synthesis pathway and 12 DEGs encoding TFs presented strong correlations with the SS contents, and these DEGs may be critical for the biosynthesis and regulation of SSs. These findings will be critical for further study of the response of B. chinense DC. to MeJA for SS biosynthesis.

Saikosaponin A and Its Epimers Alleviate LPS-Induced Acute Lung Injury in Mice.

The purpose of this work was to illustrate the effect of processing with vinegar on saikosaponins of Bupleurum chinense DC. (BC) and the protective effects of saikosaponin A (SSA), saikosaponin b1 (SSb1), saikosaponin b2 (SSb2), and saikosaponin D (SSD) in lipopolysaccharide (LPS)-induced acute lung injury (ALI) mice. We comprehensively evaluated the anti-inflammatory effects and potential mechanisms of SSA, SSb1, SSb2, and SSD through an LPS-induced ALI model using intratracheal injection. The results showed that SSA, SSb1, SSb2, and SSD significantly decreased pulmonary edema; reduced the levels of IL-6, TNF-α, and IL-1ß in serum and lung tissues; alleviated pulmonary pathological damage; and decreased the levels of the IL-6, TNF-α, and IL-1ß genes and the expression of NF-κB/TLR4-related proteins. Interestingly, they were similar in structure, but SSb2 had a better anti-inflammatory effect at the same dose, according to a principal component analysis. These findings indicated that it may not have been comprehensive to only use SSA and SSD as indicators to evaluate the quality of BC, especially as the contents of SSb1 and SSb2 in vinegar-processed BC were significantly increased.

Molecular cloning, functional characterization and expression of the ß-amyrin synthase gene involved in saikosaponin biosynthesis in Bupleurum chinense DC.

Bupleurum chinense DC. is a commonly used plant in traditional Chinese medicine, and saikosaponins(SSs) are the main active oleanane-typetriterpene saponins in B. chinense. ß-Amyrin synthase (ß-AS) is an important enzyme in oleanane-type triterpenoid saponin synthesis, but its role in saikosaponin synthesis has rarely been studied. Here, the putative ß-AS gene BcBAS1(Accession No.ON890382) selected according to metabolomic and transcriptomic analyses was cloned and functionally characterized by heterologous expression in Escherichia coli and Pichia pastoris, and its subcellular localization and expression patterns were examined. The molecular weight of the BcBAS1 recombinant protein was approximately 87 kDa, and this protein could catalyse the production of ß-amyrin, the precursor of SSs. Furthermore, BcBAS1 was located in the cytosol, and relative expression in four tissues of the four genotypes was positively correlated with SSa and SSd contents. Our results indicate that BcBAS1 is a ß-AS gene and may play an important role in saikosaponin biosynthesis and regulation. This study sheds light on the role of ß-AS genes in the synthesis of SSs and provides insights for the metabolic engineering of SSs.

Rapid determination of microbial contamination of Bupleurum chinense DC. decoction slices by ATP bioluminescence technology combined with statistical analysis methods / 药学学报

This study focuses on the microbial quality control of the Chinese herbal decoction pieces. In view of the shortcomings of traditional culture methods such as slow detection speed and inability to detect unculturable microorganisms, a new method based on ATP bioluminescence technology combined with statistical analysis methods was established to rapidly predict and quantitatively detect the total aerobic microbial count (TAMC) and total yeast and mold count (TYMC) contaminated Bupleurum chinense DC. decoction pieces. Based on the optimized ATP bioluminesence detection system, accurate detection of pure bacterial solution of Escherichia coli, Bacillus subtilis and Staphylococcus aureus can be achieved, with detection limits of 47.86, 89.13 and 1 862.09 CFU·mL-1, respectively. The detection time was 6.5 h, and the detection cost was as low as 2 yuan/time. The upper and lower warning limits of TAMC were determined by the misjudgment rates of 10% and 20%, respectively. And the warning limit of TYMC was determined by the misjudgment rate of 20%. The proposed crossing method could quickly predict the amount of microbial contamination in Bupleurum chinense DC. decoction pieces. The constructed partial least squares regression (PLSR) model could accurately quantify the quantity of microbial contamination in Bupleurum chinense DC. decoction pieces. The optimal PLSR prediction model for TAMC had a correction coefficient (R2) of 0.826, a root mean square error of correction set (RMSEE) of 0.468 and a root mean square error of cross-validation set (RMSECV) of 0.465. The R2, RMSEE and RMSECV in the prediction model of TYMC were 0.778, 0.543 and 0.541, respectively. The aim of this study is to establish a kind of rapid detection method and prediction models for the microbial limit of traditional Chinese medicine and Chinese herbal decoction pieces, and to provide a more convenient and sensitive detection technology for the microbial quality process control of traditional Chinese medicine products.

Quantitative analysis of multi-components by single marker method combined with UPLC-PAD fingerprint analysis based on saikosaponin for discrimination of Bupleuri Radix according to geographical origin.

Background: Saikosaponins are regarded as one of the most likely antipyretic constituents of Bupleuri Radix, establishing a comprehensive method that can reflect both the proportion of all constituents and the content of each saikosaponin is critical for its quality evaluation. Methods: In this study, the combination method of quantitative analysis of multiple components with a single marker (QAMS) and fingerprint was firstly established for simultaneous determination of 7 kinds of saikosaponins in Bupleuri Radix by ultra-high performance liquid chromatography (UPLC). Results: The results showed that saikosaponin d was identified as the optimum IR by evaluating the fluctuations and stability of the relative calibration factors (RCFs) under four different conditions. The new QAMS method has been confirmed to accurately quantify the 7 kinds of saikosaponins by comparing the obtained results with those obtained from external standard method and successfully classify the 20 batches of Bupleuri Radix from 8 provinces of China. The experimental time of fingerprint was significantly reduced to approximate 0.5 h through UPLC-PAD method, a total of 17 common peaks were identified. Conclusion: The QAMS-fingerprint method is feasible and reliable for the quality evaluation of Bupleuri Radix. This method could be considered to be spread in the production enterprises of Bupleuri Radix.

Drought Stress Stimulates the Terpenoid Backbone and Triterpenoid Biosynthesis Pathway to Promote the Synthesis of Saikosaponin in Bupleurum chinense DC. Roots.

Bupleurum chinense is an important medicinal plant in China; however, little is known regarding how this plant transcribes and synthesizes saikosaponins under drought stress. Herein, we investigated how drought stress stimulates the transcriptional changes of B. chinense to synthesize saikosaponins. Short-term drought stress induced the accumulation of saikosaponins, especially from the first re-watering stage (RD_1 stage) to the second re-watering stage (RD_2 stage). Saikosaponin-a and saikosaponin-d increased by 84.60% and 75.13%, respectively, from the RD_1 stage to the RD_2 stage. Drought stress also stimulated a rapid increase in the levels of the hormones abscisic acid, salicylic acid, and jasmonic acid. We screened 49 Unigenes regarding the terpenoid backbone and triterpenoid biosynthesis, of which 33 differential genes were significantly up-regulated during drought stress. Moreover, one P450 and two UGTs are possibly involved in the synthesis of saikosaponins, while some transcription factors may be involved in regulating the expression of key enzyme genes. Our study provides a reference for the cultivation of B. chinense and a practical means to ensure the quality (safety and effectiveness) of B. chinense for medicinal use, as well as insights into the modernization of the China Agriculture Research System.

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

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.

Integration of Non-Targeted Metabolomics and Targeted Quantitative Analysis to Elucidate the Synergistic Antidepressant Effect of Bupleurum Chinense DC-Paeonia Lactiflora Pall Herb Pair by Regulating Purine Metabolism.

Bupleurum chinense DC (Chaihu)-Paeonia lactiflora Pall (Baishao) is among the most accepted herb pairs in many classic antidepressant prescriptions. Our previous study has shown that the Chaihu-Baishao herb pair (CBHP) had a better antidepressant effect than Chaihu or Baishao. Nevertheless, the synergistic antidepressant mechanism of this herb pair was not clearly understood. This study aimed to investigate the compatibility mechanism of Chaihu and Baishao for treating depression through a strategy of non-targeted metabolomics combined with targeted quantitative analysis and molecular biology techniques. First, the compatibility effects of CBHP were assessed by the chronic unpredictable mild stress (CUMS) rat model. Next, cortex metabolomics based on ultra-high-performance liquid chromatography combined with quadrupole orbitrap mass spectrometry (UPLC-Q-Orbitrap/MS) was used to discover the metabolic pathway that was synergistically regulated by CBHP. Based on the results of metabolomics analysis, metabolites were quantitatively validated by UPLC-MS/MS combined with the MRM mode in the crucial metabolic pathway. In addition, the signaling pathway associated with this metabolic pathway was detected by molecular biology techniques to further identify the biological meaning of the crucial metabolite on the synergistic antidepressant effect of CBHP. The antidepressant effect of CBHP was significantly better than that of Chaihu or Baishao single administrated in the behavioral test. According to cortex metabolomics, a total of 21 differential metabolites were screened out, and purine metabolism was selected as the crucial metabolic pathway by the enrichment analysis of differential metabolites. Subsequently, purine metabolism was confirmed as disorder in the CUMS group by targeted quantitative analysis, CBHP regulated more purine metabolites (six) than individual administration (two and two). The results showed that purine metabolism was modulated by CBHP through synergistically decreasing xanthine levels and inhibiting the conversion of xanthine dehydrogenase (XDH) to xanthine oxidase (XOD). Finally, the synergistic regulation effect of CBHP on xanthine synthesis was found to be related to inhibition of malondialdehyde (MDA) production, Nod-like receptor protein 3 (NLRP3) inflammasome expression, and interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α secretion. The present study demonstrated that the regulation of purine metabolism, the suppression of oxidative stress, and inflammatory responses in the cortex were involved in the synergistic antidepressant effect of CBHP.

Corrigendum: Systematic Characterization and Identification of Saikosaponins in Extracts From Bupleurum marginatum var. stenophyllum Using UPLC-PDA-Q/TOF-MS.

[This corrects the article DOI: 10.3389/fchem.2021.747987.].

Proprietary Medicines Containing Bupleurum chinense DC. (Chaihu) for Depression: Network Meta-Analysis and Network Pharmacology Prediction.

Background and Aims: The rapid development of society has resulted in great competitive pressures, leading to the increase in suicide rates as well as incidence and recurrence of depression in recent years. Proprietary Chinese medicines containing Bupleurum chinense DC. (Chaihu) are widely used in clinical practice. This study aimed at evaluating the efficacy and safety of oral proprietary Chinese medicines containing Chaihu for treating depression by network meta-analysis (NMA) and exploring the potential pharmacological mechanisms of the optimal drugs obtained based on NMA. Methods: This study searched for clinical randomized controlled trial studies (RCTs) about Chaihu-containing products alone or in combination with selective serotonin reuptake inhibitors (SSRI), serotonin-norepinephrine reuptake inhibitors (SNRI), and cyclic antidepressants (CAS) for depression in eight databases. The search deadline is from data inception to April 2021. For efficacy assessment, the clinical response rate, the Hamilton Depression Scale-17 (HAMD-17), and adverse reactions were calculated. The methodological quality of the included studies was assessed for risk of bias following the Cochrane Handbook for Systematic Reviews of Interventions, and the data were subjected to NMA via the Stata version 16.0 software. Subsequently, the optimal drug obtained from the NMA results, Danzhi Xiaoyao pill (DZXY), was used to conduct network pharmacology analysis. We searched databases to acquire bioactive and potential targets of DZXY and depression-related targets. The protein-protein interaction (PPI) network, component-target network, the Gene Ontology (GO), and the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were performed by the STRING database, Cytoscape 3.9.0 software, and R version 4.1.2, respectively. Results: Thirty-seven RCTs, with a total of 3,263 patients, involving seven oral proprietary Chinese medicines containing Chaihu, were finally included. The results of the NMA demonstrated that the top four interventions with the best efficiency were Jiawei Xiaoyao + SSRI, DZXY + SNRI, Xiaoyao pill + SSRI, and Jieyu pill + SNRI; the top four interventions reducing HAMD score were DZXY + SNRI, Jiawei Xiaoyao, Jieyu pill, and Puyu pill + SNRI; the top four interventions with the least adverse effects were Jieyu pill, Anle pill + SSRI, DZXY + SNRI, and Puyu pill + SNRI. In the aspects above, DZXY + SNRI performed better than other treatments. After network meta-analysis, we conducted a network pharmacology-based strategy on the optimal drugs, DZXY, to provide the pharmacological basis for a conclusion. A total of 147 active compounds and 248 targets in DZXY were identified, of which 175 overlapping targets related to depression. Bioinformatics analysis revealed that MAPK3, JUN, MAPK14, MYC, MAPK1, etc. could become potential therapeutic targets. The MAPK signaling pathway might play an essential role in DZXY against depression. Conclusion: This is the very first systematic review and network meta-analysis evaluating different oral proprietary Chinese medicines containing Chaihu in depressive disorder. This study suggested that the combination of proprietary Chinese medicines containing Chaihu with antidepressants was generally better than antidepressant treatment. The incidence of adverse reactions with antidepressants alone was higher than that with proprietary Chinese medicines containing Chaihu alone or in combination with antidepressants. DZXY + SNRI showed significantly better results in efficacy, HAMD scores, and safety. The antidepressant effect of DZXY may be related to its regulation of neuroinflammation and apoptosis.

The Structural Characteristics of an Acidic Water-Soluble Polysaccharide from Bupleurum chinense DC and Its In Vivo Anti-Tumor Activity on H22 Tumor-Bearing Mice.

This study explored the preliminary structural characteristics and in vivo anti-tumor activity of an acidic water-soluble polysaccharide (BCP) separated purified from Bupleurum chinense DC root. The preliminary structural characterization of BCP was established using UV, HPGPC, FT-IR, IC, NMR, SEM, and Congo red. The results showed BCP as an acidic polysaccharide with an average molecular weight of 2.01 × 103 kDa. Furthermore, we showed that BCP consists of rhamnose, arabinose, galactose, glucose, and galacturonic acid (with a molar ratio of 0.063:0.788:0.841:1:0.196) in both α- and ß-type configurations. Using the H22 tumor-bearing mouse model, we assessed the anti-tumor activity of BCP in vivo. The results revealed the inhibitory effects of BCP on H22 tumor growth and the protective actions against tissue damage of thymus and spleen in mice. In addition, the JC-1 FITC-AnnexinV/PI staining and cell cycle analysis have collectively shown that BCP is sufficient to induce apoptosis and of H22 hepatocarcinoma cells in a dose-dependent manner. The inhibitory effect of BCP on tumor growth was likely attributable to the S phase arrest. Overall, our study presented significant anti-liver cancer profiles of BCP and its promising therapeutic potential as a safe and effective anti-tumor natural agent.

Identification and potential mechanism of different components from the aerial part of Bupleurum chinense DC. for epileptic treatment.

The present study aimed to assess the effects of epileptic treatment of the aerial part of Bupleurum chinense DC. in kainic acid (KA)-induced epilepsy rats and LPS-induced BV2 cells, as well as to identify the active chemical constituents. The in vivo and vitro results showed that 20% ethanol elution fractions of the aerial part of B. chinense DC. (BCE-20) and 70% ethanol elution fractions of the aerial part of B. chinense DC. (BCE-70) could improve the epileptic state of the rats and status epilepticus (SE%). Moreover, ultra-high-performance liquid chromatography (UPLC)-Orbitrap- mass spectrometry (MS) analysis identified BCE-20 and 70 as flavonoids and phenylpropanoids, respectively. The mechanistic analysis also showed that BCE-20 and 70 could regulate neurotransmitter abnormalities and suppresses the expression and secretion of pro-inflammatory cytokines. Notably, BCE-20 and 70 could regulate the Triggering receptor expressed on myeloid cells 2 (TREM2)/nuclear factor-k-gene binding (NF-κB)/inhibitor of NF-κB α (IκBα) pathway to inhibit the neuroinflammation. Our findings support the ethnopharmacological use of the constituent polyphenols and flavonoids from the aerial part of B. chinense DC., as the strong anti-epileptic agents.

Bupleurum chinense DC improves CUMS-induced depressive symptoms in rats through upregulation of the cAMP/PKA/CREB signalling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Bupleurum chinense DC. (B. chinense) is the dried root of B. chinense, belonging to the Umbelliferae family. B. chinense has been reported since ancient times for its effect of soothing the liver and relieving depression. Additionally, its important role in treating depression, depressed mood disorders and anti-inflammation has been proven in previous studies. However, its specific mechanism of action remains unknown. AIM OF THE STUDY: The key targets and metabolites of the antidepressant effect of B. chinense were investigated based on the cAMP signalling pathway. The study examined the mechanism for the antidepressant effect of B. chinense by target prediction, analysis of related metabolites and potential metabolic pathways. MATERIALS AND METHODS: A network pharmacology approach was used to predict the antidepressant targets and pathways of B. chinense. A depression rat model was established through the CUMS (chronic unpredictable mild stress) procedure. The depression model was assessed by body weight, sugar-water preference, water maze and enzyme-linked immunosorbent assay (ELISA) indicators (5hydroxytryptamine, etc.). The key metabolic pathways were screened by correlations between metabolites and key targets. Finally, a quantitative analysis of key targets and metabolites was experimentally validated. RESULTS: B. chinense significantly ameliorated the reduction in body weight, sugar-water preference rate and cognitive performance in the water maze experiment in rats with depression induced by CUMS. ELISA, Western blotting (WB) and reverse transcription-polymerase chain reaction (RT-PCR) assays showed that B. chinense significantly improves the expression of protein kinase cyclic adenylic acid (cAMP)-activated catalytic subunit alpha (PRKACA), cAMP-response element-binding protein (CREB) and cAMP activation in the rat brain induced by CUMS. According to metabolic pathway analysis, B. chinense shows an antidepressant effect primarily by regulating the cAMP metabolic pathway. CONCLUSION: B. chinense upregulated PRKACA and CREB expression and the level of the key metabolite cAMP in the cAMP/PKA/CREB pathway while reducing the inflammatory response to depression treatment. These new findings support future research on the antidepressant effects of B. chinense.

Transcriptome and metabolome analysis to reveal major genes of saikosaponin biosynthesis in Bupleurum chinense.

BACKGROUND: Bupleurum chinense DC. is a widely used traditional Chinese medicinal plant. Saikosaponins are the major bioactive constituents of B. chinense, but relatively little is known about saikosaponin biosynthesis. In the present study, we performed an integrated analysis of metabolic composition and the expressed genes involved in saikosaponin biosynthetic pathways among four organs (the root, flower, stem, and leaf) of B. chinense to discover the genes related to the saikosaponin biosynthetic pathway. RESULTS: Transcript and metabolite profiles were generated through high-throughput RNA-sequencing (RNA-seq) data analysis and liquid chromatography tandem mass spectrometry, respectively. Evaluation of saikosaponin contents and transcriptional changes showed 152 strong correlations (P < 0.05) over 3 compounds and 77 unigenes. These unigenes belonged to eight gene families: the acetoacetyl CoA transferase (AACT) (6), HMG-CoA synthase (HMGS) (2), HMG-CoA reductase (HMGR) (2), mevalonate diphosphate decarboxylase (MVD) (1), 1-deoxy-D-xylulose-5-phosphate synthase (DXS) (3), farnesyl diphosphate synthase (FPPS) (11), ß-amyrin synthase (ß-AS) (13) and cytochrome P450 enzymes (P450s) (39) families. CONCLUSIONS: Our results investigated the diversity of the saikosaponin triterpene biosynthetic pathway in the roots, stems, leaves and flowers of B. chinese by integrated transcriptomic and metabolomic analysis, implying that manipulation of P450s genes such as Bc95697 and Bc35434 might improve saikosaponin biosynthesis. This is a good candidate for the genetic improvement of this important medicinal plant.

Systematic Characterization and Identification of Saikosaponins in Extracts From Bupleurum marginatum var. stenophyllum Using UPLC-PDA-Q/TOF-MS.

Saikosaponins comprise a large group of chemical components present in the Bupleurum species that have attracted attention in the field of medicine because of their significant biological activities. Due to the high polarity, structural similarity, and the presence of several isomers of this class of components, their structural identification is extremely challenging. In this study, the mass spectrometric fragmentation pathways, UV spectral features, and chromatographic behavior of different types of saikosaponins were investigated using 24 standard substances. Saikosaponins containing carbonyl groups (C=O) in the aglycone produced fragment ions by loss of 30 Da, and in addition, type IV saikosaponins could produce [aglycone-CH2OH-OH-H]- and [aglycone-H2O-H]- fragment ions through neutral losses at positions C16 and C17. The above characteristic ions can be used to identify saikosaponins. More notably, the identification process of saikosaponins was systematically summarized, and using this method, 109 saikosaponins were identified or tentatively characterized from the saikosaponins extract of Bupleurum marginatum var. stenophyllum (BMS) using UPLC-PDA-Q/TOF-MS with both data-dependent acquisition (DDA) and data-independent acquisition (DIA) modes, of which 25 were new compounds and 60 were first discovered from BMS. Further studies revealed that the saikosaponins profiles of BMS, Bupleurum chinense DC (BC), and Bupleurum marginatum Wall. ex DC (BMW) were very similar. This work is of great significance for the basic research of the Bupleurum species and provides strong technical support to solve the resource problems associated with Radix Bupleuri.