Bupleurum exerts antiarrhythmic effects by inhibiting L-type calcium channels in mouse ventricular myocytes.

BACKGROUND: Bupleurum (Bup), is a traditional effective medicine to treat colds and fevers in clinics. Multiple studies have demonstrated that Bup exhibites various biological activities, including cardioprotective effects, anti-inflammatory, anticancer, antipyretic, antimicrobial, and antiviral effects, etc. Currently, the effects of Bup on cardiac electrophysiology have not been reported yet. METHODS: Electrocardiogram recordings were used to investigate the effects of Bup on aconitine-induced arrhythmias. Patch-clamp techniques were used to explore the effects of Bup on APs and ion currents. RESULTS: Bup reduced the incidence of ventricular fibrillation (VF) and delayed the onset time of ventricular tachycardia (VT) in mice. Additionally, Bup (40 mg/mL) suppressed DADs induced by high-Ca2+ and shortened action potential duration at 50 % completion of repolarization (APD50) and action potential duration at 90 % completion of repolarization (APD90) to 60.89 % ± 8.40 % and 68.94 % ± 3.24 % of the control, respectively. Moreover, Bup inhibited L-type calcium currents (ICa.L) in a dose-dependent manner, with an IC50 value of 25.36 mg/mL. Furthermore, Bup affected the gated kinetics of L-type calcium channels by slowing down steady-state activation, accelerating the steady-state inactivation, and delaying the inactivation-recovery process. However, Bup had no effects on the Transient sodium current (INa.T), ATX II-increased late sodium current (INa.L), transient outward current (Ito), delayed rectifier potassium current (IK), or inward rectifier potassium current (IK1). CONCLUSION: Bup is an antiarrhythmic agent that may exert its antiarrhythmic effects by inhibiting L-type calcium channels.

Two Omics Methods Expose Anti-Depression Mechanism of Raw and Vinegar-Baked Bupleurum Scorzonerifolium Willd.

Bupleurum scorzonerifolium willd. (BS) and its vinegar-baked product (VBS) has been frequently utilized for depression management in clinical Chinese medicine. This paper aims to elucidate the antidepressant mechanism of BS and VBS from the perspectives of metabonomics and gut microbiota. A rat model of depression was established by CUMS combined with feeding alone to evaluate the antidepressant effects of BS and VBS. UPLC-Q-TOF-MS/MS-based metabolomics and 16S rRNA sequencing of rat feces were applied and the correlation of differential metabolic markers and intestinal floras was analyzed. The result revealed that BS and VBS significantly improved depression-like behaviors and the levels of monoamine neurotransmitters in CUMS rats. There were 27 differential endogenous metabolites between CUMS and normal rats, which were involved in 8 metabolic pathways. Whereas, BS and VBS could regulate 18 and 20 metabolites respectively, wherein fifteen of them were shared metabolites. On the genus level, BS and VBS could regulate twenty-five kinds of intestinal floras in CUMS rats, that is, they increased the abundance of beneficial bacteria and decreased the abundance of harmful bacteria. In conclusion, both BS and VBS exert excellent antidepressant effects by regulating various metabolic pathways and ameliorating intestinal microflora dysfunction.

Discovery and verification of anti-inflammatory-related quality markers in the aerial part of Bupleurum scorzonerifolium by UPLC-Q-TOF-MS/MS and in RAW 264.7 cells and a zebrafish model.

INTRODUCTION: The root of Bupleurum scorzonerifolium Willd. (BS) is officially recognized in the Chinese Pharmacopoeia. In contrast, the aerial part of BS (ABS), accounting for 80% of BS, is typically discarded, causing potential waste of medicinal resources. ABS has shown benefits in the treatment of inflammation-related diseases in China and Spain, and the material basis underlying its anti-inflammatory effects must be systematically elucidated for the rational use of ABS. OBJECTIVE: We aimed to screen and validate the anti-inflammatory quality markers (Q-markers) of ABS and to confirm the ideal time for ABS harvesting. METHODS: The chemical components and anti-inflammatory effects of ABS from 10 extracted parts were analyzed by UPLC-Q-TOF-MS/MS and in a lipopolysaccharide (LPS)-induced cell model. Anti-inflammatory substances were screened by Pearson bivariate analysis and gray correlation analysis, and the anti-inflammatory effects were verified in a zebrafish tail-cutting inflammation model. HPLC was applied to measure the Q-marker contents of ABS in different harvesting periods. RESULTS: Ten ABS extracts effectively alleviated the increase in LPS-induced proinflammatory cytokines in RAW 264.7 cells. Forty components were identified from them, among which 27 were common components. Eight components were correlated with anti-inflammatory effects, which were confirmed to reverse the expression of proinflammatory and anti-inflammatory factors in a zebrafish model. Chlorogenic acid, hypericin, rutin, quercetin, and isorhamnetin can be detected by HPLC, and the maximum contents of these five Q-markers were obtained in the sample harvested in August. CONCLUSION: The anti-inflammatory Q-markers of ABS were elucidated by chromatographic-pharmacodynamic-stoichiometric analysis, which served as a crucial basis for ABS quality control.

Triterpenoid saponins from Bupleurum marginatum and their anti-liver fibrotic activities.

A new triterpenoid saponin (1), along with five known compounds (2-6), was isolated from Bupleurum marginatum Wall. ex DC, of which compounds 2-4 were obtained for the first time from this plant. The structures were confirmed by the analysis of 1D, 2D NMR, and HR-ESIMS data, and comparison with previous spectral data. Anti-liver fibrotic activities of the isolates were determined as proliferation inhibition of LPS-induced activation of HSC-T6 in vitro.

Comprehensive Analysis Reveals the Difference in Volatile Oil between Bupleurum marginatum var. stenophyllum (Wolff) Shan et Y. Li and the Other Four Medicinal Bupleurum Species.

Volatile oil serves as a traditional antipyretic component of Bupleuri Radix. Bupleurum marginatum var. stenophyllum (Wolff) Shan et Y. Li belongs to the genus Bupleurum and is distinguished for its high level of saikosaponins and volatile oils; nonetheless, prevailing evidence remains inconclusive regarding its viability as an alternative resource of other official species. This study aims to systematically compare the volatile oil components of both dried and fresh roots of B. marginatum var. stenophyllum and the four legally available Bupleurum species across their chemical, molecular, bionics, and anatomical structures. A total of 962 compounds were determined via GC-MS from the dried roots; B. marginatum var. stenophyllum showed the greatest differences from other species in terms of hydrocarbons, esters, and ketones, which was consistent with the results of fresh roots and the e-nose analysis. A large number of DEGs were identified from the key enzyme family of the monoterpene synthesis pathway in B. marginatum var. stenophyllum via transcriptome analysis. The microscopic observation results, using different staining methods, further showed the distinctive high proportion of phloem in B. marginatum var. stenophyllum, the structure which produces volatile oils. Together, these pieces of evidence hold substantial significance in guiding the judicious development and utilization of Bupleurum genus resources.

Uncovering the Chemical Composition and Biological Potentials of Bupleurum lancifolium Hornem. from Jordan.

The current study was designed to uncover the chemistry and bioactivity potentials of Bupleurum lancifolium growing wild in Jordan. In this context, the fresh aerial parts obtained from the plant material were subjected to hydrodistillation followed by GC/MS analysis. The main components of the HDEO were γ-patchoulene (23.79%), ß-dihydro agarofuran (23.50%), α-guaiene (14.11%), and valencene (13.28%). Moreover, the crude thanolic extract was partitioned to afford two main major fractions, the aqueous methanol (BLM) and butanol (BLB). Phytochemical investigation of both fractions, using conventional chromatographic techniques followed by careful inspection of the spectral data for the isolated compounds (NMR, IR, and UV-Vis), resulted in the characterization of five known compounds, including α-spinasteryl (M1), ethyl arachidate (M2), ethyl myristate (M3), quercetin-3-O-ß-d-glucopyranosyl-(1-4")-α-L-rhamnopyranosyl (B1), and isorhamnetin-3-O-ß-d-glucopyranosyl-(1-4")-α-L-rhamnopyranosyl (B2). The TPC, TFC, and antioxidant activity testing of both fractions and HDEO revealed an interesting ABTS scavenging potential of the BLB fraction compared to the employed positive controls, which is in total agreement with its high TP and TF contents. Cytotoxic evaluation tests revealed that BLM had interesting cytotoxic effects on the normal breast cell line MDA-MB-231 (ATCC-HTB-26) and the normal dermal fibroblast (ATCC® PCS-201-012) and normal African green monkey kidney Vero (ATCC-CCL-81) cell lines. Despite both the BLB and BLM fractions showing interesting AChE inhibition activities (IC50 = 217.9 ± 5.3 µg/mL and 139.1 ± 5.6 µg/mL, respectively), the HDEO revealed an interestingly high AChE inhibition power (43.8 ± 2.7 µg/mL) that far exceeds the one observed for galanthamine (91.4 ± 5.2 µg/mL). The HDEO, BLM, and BLB exhbitied no interesting antimicrobial activity against Bacillus cereus, Bacillus subtilis, Staphylococcus aureus, Escherichia coli, or Pseudomonas aeruginosa.

[Effects of humic acid water-soluble fertilizer on growth and physiological characteristics of Bupleurum chinense seedlings].

The effects of humic acid water-soluble fertilizer on the growth and physiological characteristics of Bupleurum chinense seedlings(Zhongchai No.1) were studied by using a single factor experiment design. When the seedling age was 60 days, the humic acid water-soluble fertilizer was diluted 1 200 times(T1), 1 500 times(T2), 1 800 times(T3), and 2 100 times(T4) for seedling treatment, respectively, and water was used as the control(CK). The effects of different treatments on growth indexes, biomass accumulation, root activity, antioxidant enzyme activity, membrane lipid peroxidation, and photosynthetic characteristics of B. chinense seedlings were analyzed after 30 days. The results showed that compared with CK, stem height, leaf number, root diameter, and root length of the B. chinense seedlings under T3 treatment were significantly increased by 36.82%, 37.03%, 42.78%, and 22.38%, respectively. Root fresh weight, leaf fresh weight, root dry weight, and leaf dry weight under T3 treatment were significantly increased by 90.36%, 98.68%, 123.84%, and 104.38%, respectively. In addition, humic acid water-soluble fertilizer also enhanced TTC reducing activity of the root of B. chinense seedlings, inhibited malonaldehyde(MDA) content, increased superoxide dismutase(SOD), peroxidase(POD), and catalase(CAT) enzyme activities, improved chlorophyll content, and enhanced P_n, G_s, T_r, and other photosynthetic parameters. In conclusion, the application of humic acid water-soluble fertilizer diluted 1 800 times can significantly promote the growth of B. chinense seedlings, enhance root vitality, improve seedling stress resistance, and enhance photosynthesis. The results of this study can provide a theoretical basis for fertilization of B. chinense seedlings.

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.

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.

[Correlations between appearance traits and internal quality of Bupleurum scorzonerifolium roots based on theory of "quality evaluation through morphological identification"].

It is generally believed that high-quality Bupleurum scorzonerifolium roots possess specific morphological characteristics, being red, robust, and long with strong odor. However, the scientific connotation of these characteristics has not been elucidated. According to the theory of "quality evaluation through morphological identification", we studied the correlations between appearance traits(the RGB value of root surface, root length, root diameter, dry weight, and ratio of phloem to xylem) and content of main chemical components(volatile oils, total saponins, total flavonoids, total polysaccharides, and seven saikosaponins) of B. scorzonerifolium roots. Epson Scanner and ImageJ were used to scan the root samples and measure the appearance traits. Ultraviolet spectrophotometry and HPLC were employed to determine the content of chemical components. The correlation, regression, and cluster analyses were performed to study the correlations between the appearance traits and the content of chemical components. The results showed that the content of volatile oils and saikosaponins were significantly correlated with RGB value, root length, and root diameter, indicating that within a certain range, the roots being redder, longer, and thicker had higher content of volatile oils and saikosaponins. According to the appearance traits and chemical component content, the 14 samples from different producing areas were classified into four grades, and the differences in morphological traits and chemical component content were consistent among different grades. The findings in this study demonstrate that appearance traits(RGB value, root length, and root diameter) can be used to evaluate the quality of B. scorzonerifolium roots. Meanwhile, this study lays a foundation for establishing an objective quality evaluation method for B. scorzonerifolium roots.

Assembly and comparative analysis of the complete mitochondrial genome of Bupleurum chinense DC.

BACKGROUND: Bupleurum chinense(B. chinense) is a plant that is widely distributed globally and has strong pharmacological effects. Though the chloroplast(cp) genome of B. chinense has been studied, no reports regarding the mitochondrial(mt) genome of B. chinense have been published yet. RESULTS: The mt genome of B.chinense was assembled and functionally annotated. The circular mt genome of B. chinense was 435,023 bp in length, and 78 genes, including 39 protein-coding genes, 35 tRNA genes, and 4 rRNA genes, were annotated. Repeat sequences were analyzed and sites at which RNA editing would occur were predicted. Gene migration was observed to occur between the mt and cp genomes of B. chinense via the detection of homologous gene fragments. In addition, the sizes of plant mt genomes and their GC content were analyzed and compared. The sizes of mt genomes of plants varied greatly, but their GC content was conserved to a greater extent during evolution. Ka/Ks analysis was based on code substitutions, and the results showed that most of the coding genes were negatively selected. This indicates that mt genes were conserved during evolution. CONCLUSION: In this study, we assembled and annotated the mt genome of the medicinal plant B. chinense. Our findings provide extensive information regarding the mt genome of B. chinense, and help lay the foundation for future studies on the genetic variations, phylogeny, and breeding of B. chinense via an analysis of the mt genome.

Response of soil microecology to different cropping practice under Bupleurum chinense cultivation.

The effects of cropping practices on the rhizosphere soil physical properties and microbial communities of Bupleurum chinense have not been studied in detail. The chemical properties and the microbiome of rhizosphere soil of B. chinense were assessed in the field trial with three cropping practices (continuous monocropping, Bupleurum-corn intercropping and Bupleurum-corn rotation). The results showed cropping practices changed the chemical properties of the rhizosphere soil and composition, structure and diversity of the rhizosphere microbial communities. Continuous monocropping of B. chinense not only decreased soil pH and the contents of NO3--N and available K, but also decreased the alpha diversity of bacteria and beneficial microorganisms. However, Bupleurum-corn rotation improved soil chemical properties and reduced the abundance of harmful microorganisms. Soil chemical properties, especially the contents of NH4+-N, soil organic matter (SOM) and available K, were the key factors affecting the structure and composition of microbial communities in the rhizosphere soil. These findings could provide a new basis for overcoming problems associated with continuous cropping and promote development of B. chinense planting industry by improving soil microbial communities.

UV-guided isolation of enantiomeric polyacetylenes from Bupleurum scorzonerifolium Willd. with inhibitory effects against LPS-induced NO release in BV-2 microglial cells.

UV-guided fractionation led to the isolation of thirteen new polyacetylenes (1-13) from the roots of Bupleurum scorzonerifolium Willd. All polyacetylenes were analyzed as racemates since the lack of optical activity and Cotton effects in the ECD spectra. The sequent chiral-phase HPLC resolution successfully gave twelve pairs of enantiomers 1a/1b and 3a/3b-13a/13b. Their structures were elucidated based on the HRESIMS and NMR data analyses. The absolute configurations were determined by the combination of Snatzke's method, electronic circular dichroism calculations, and single-crystal X-ray diffraction. Using Griess methods and MTT assays, polyacetylenes 1a, 3a, 4a/4b-12a/12b, and 13a displayed inhibitory activities against LPS-induced NO release in BV-2 microglial cells.

The potential mechanism of Bupleurum against anxiety was predicted by network pharmacology study and molecular docking.

Bupleurum chinense DC. (Chaihu) is a traditional Chinese medicine (TCM) used in the treatment of anxiety. But the anxiolytic mechanisms of bupleurum are still unclear. Therefore, this unknown is predicted by network pharmacology study with molecular docking in the present study. The components of bupleurum were obtained from the databases. Genes associated with components and disease were also provided by databases. Overlapping genes between components and disease were analyzed. The network of medicine-components-targets-disease was constructed, visualized, and analyzed. Protein-protein interaction (PPI), gene ontology (GO), pathway enrichment (KEGG) and molecular docking were conducted to predict the potential mechanisms of bupleurum on anxiety. A total of 9 bioactive components derived from bupleurum with 80 target genes were involved in anxiety. Neurotransmitter receptor activity, G protein-coupled amine receptor activity, regulation of blood circulation, neuroactive ligand-receptor interaction, calcium signaling pathway and salivary secretion may play significant roles in the anxiolytic of bupleurum. Molecular docking implicated that ACHE and MAOA showed high affinity for stigmasterol. Based on network pharmacology study with molecular docking, multi-component-multi-target-multi-pathway action mode of bupleurum on anxiety was elaborated. Stigmasterol might be the core bioactive component, while ACHE and MAOA might be the core target genes in the pharmacological profile of bupleurum on anxiety.

Aromatic glycosides from the aerial part of Bupleurum chinense.

A new aromatic glycoside (1) and twelve (2-13) known aromatic glycosides were isolated from the n-butyl alcohol of the 70% EtOH extract of the aerial part of Bupleurum chinense. Among them, compounds 4-7, 9, 11-13 were isolated from Umbelliferae plants for the first time. Their structures were elucidated by NMR and MS spectroscopy, and the absolute configuration of compound 1 was elucidated by single-crystal X-ray diffraction. Compounds 6 and 10 showed moderate cytotoxic activities on MGC-803 cell lines with IC50 values of 8.63 ± 0.08 and 13.65 ± 1.73 µM, respectively.

Production of Prosaikogenin F, Prosaikogenin G, Saikogenin F and Saikogenin G by the Recombinant Enzymatic Hydrolysis of Saikosaponin and their Anti-Cancer Effect.

The saponins of Bupleurum falcatum L., saikosaponins, are the major components responsible for its pharmacological and biological activities. However, the anti-cancer effects of prosaikogenin and saikogenin, which are glycoside hydrolyzed saikosaponins, are still unknown due to its rarity in plants. In this study, we applied two recombinant glycoside hydrolases that exhibit glycoside cleavage activity with saikosaponins. The two enzymes, BglPm and BglLk, were cloned from Paenibacillus mucilaginosus and Lactobacillus koreensis, and exhibited good activity between 30-37 °C and pH 6.5-7.0. Saikosaponin A and D were purified and obtained from the crude B. falcatum L. extract using preparative high performance liquid chromatography technique. Saikosaponin A and D were converted into saikogenin F via prosaikogenin F, and saikogenin G via prosaikogenin G using enzyme transformation with high ß-glycosidase activity. The two saikogenin and two prosaikogenin compounds were purified using a silica column to obtain 78.1, 62.4, 8.3, and 7.5 mg of prosaikogenin F, prosaikogenin G, saikogenin F, and saikogenin G, respectively, each with 98% purity. The anti-cancer effect of the six highly purified saikosaponins was investigated in the human colon cancer cell line HCT 116. The results suggested that saikosaponins and prosaikogenins markedly inhibit the growth of the cancer cell line. Thus, this enzymatic technology could significantly improve the production of saponin metabolites of B. falcatum L.

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.

Screening saikosaponin d (SSd)-producing endophytic fungi from Bupleurum scorzonerifolium Willd.

Saikosaponin d (SSd) is an important bioactive compound of traditional Chinese medicinal plant Bupleurum scorzonerifolium Willd. and exhibits many effects, such as anti-tumor, anti-inflammation and immunomodulatory. Since endophytic fungi possess the natural capacity to produce the similar secondary metabolite to that of their host plants, they are promising as alternative sources of plant bioactive natural products. In this study, in order to search for SSd-producing strains, endophytes were isolated from B. scorzonerifolium and were authenticated by the ITS sequence and the translation elongation factor-1alpha gene (TEF-1α) sequence analysis. The profile of metabolites present in the crude exacts was carried out by ultra performance liquid chromatography time-of-flight mass spectrometry (UPLC/Q-TOF-MS) analysis. The results showed that two strains, CHS2 and CHS3 from B. scorzonerifolium could produce SSd by UPLC/Q-TOF-MS analysis, and the amount of SSd produced by strain CHS2 and CHS3 were about 2.17 and 2.40 µg/mL, respectively. CHS2 and CHS3 showed a close phylogenetic relationship to Fusarium oxysporum and Fusarium acuminatum, respectively. According to our concern, no endophytic fungi capable of producing SSd from B. scorzonerifolium have been found before. Our clear intention was to isolate and identify these endophytic fungi that produce important active secondary metabolites, and then study the strains that produce this compound on a large scale through fermentation or even genetic study, to provide a feasible and more convenient way for the production of SSd.

[Mechanism of Bupleurum scorzonerifolium and Paeonia lactiflora herbal pair against liver cancer: an exploration based on UPLC-Q-TOF-MS combined with network pharmacology].

This study aimed to decipher the pharmacodynamic material basis and mechanism of herbal pair Bupleurum scorzonerifolium-Paeonia lactiflora(BS-PL) against liver cancer based on UPLC-Q-TOF-MS and network pharmacology. MTT assay and human hepatocellular carcinoma HepG2 cells were used to screen the effective part of BS-PL, the active components of which were further analyzed and identified by UPLC-Q-TOF-MS. Next, we applied Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP) to screen the active ingredients with OB≥30%. Then TCMSP and SwissTargetPrediction were used to collect and predict component targets, followed by the search of liver cancer-related targets with GeneCards and DisGeNET. The intersection targets were obtained using Venny 2.1.0. Protein-protein interaction(PPI) network was constructed using STRING to uncover the core targets, which were subjected to Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment analysis based on DAVID. Finally, the effects of active ingredients on the expression of main proteins enriched in the key pathways of HepG2 cells were verified by Western blot. The results indicated that compared with 30%, 50%, and 70% ethanol extracts of BS-PL, the n-butanol extraction part(CSYZ) from 95% ethanol extract of BS-PL exhibited the best anti-tumor effect. UPLC-Q-TOF-MS revealed 31 ingredients, 14 of which showed OB≥30%. A total of 220 intersection targets were obtained, from which 35 were selected as the key targets under the condition of two times the median of degree. Among the 215 items with P<0.05 obtained through GO enrichment analysis, 154 were classified into biological processes, 22 into cell components and 39 into molecular functions. KEGG enrichment analysis revealed 95 significantly affected signaling pathways, and the ones(sorted in a descending order by P value) closely related to the anti-liver cancer effect of herbal pair were PI3 K-AKT signaling pathway, TNF signaling pathway, MAPK signaling pathway, HIF-1 signaling pathway, and ErbB signaling pathway. Finally, the PI3 K/AKT signaling pathway involving the largest number of targets was extrapolated, and it was found that this pathway contained 15 core targets and 8 active components. Experimental verification showed that the effective components of BS-PL significantly inhibited the expression of p-PI3 K and p-AKT, consistent with the prediction results of network pharmacology. In conclusion, the main pharmacodynamic substances of BS-PL against liver cancer are 14 components like saikosaponin a, saikosaponin d, and paeoniflorin, which exert the anti-liver cancer effect by regulating PI3 K/AKT pathway.