Quality evaluation of Bupleuri Radix in Shanxi Province by LC-MS based pseudotargeted metabonomics / 药学学报

Bupleuri Radix is commonly used in the traditional Chinese medicine, and saikosaponins are the important active ingredients. In this study, we first established a relative quantitative method for 25 saikosaponins using ultra high performance liquid chromatography-triple quadrupole mass spectrometry (UHPLC-QTrap-MS) in the scheduled multiple reaction monitoring (sMRM) mode. The established method showed good intra-day and intra-day precision, linearity, repeatability and stability. Then the method was applied to compare 37 batches of Bupleuri Radix from different planting areas. The results showed that there was no significant difference in the saikosaponins composition of Bupleuri Radix from different planting areas in Shanxi Province, which indicating that Bupleuri Radix is well adapted to the environment, so it is suitable for widely planting. However, Bupleuri Radix harvested at spring and autumn were differed from those harvested at summer, which indicated that the traditional harvesting experience was reasonable. Correlation analysis showed that saikosaponins a and d were positively correlated with some saponins, and 4 saponins (such as clinoposaponin XII) showed bigger content variation were identified by coefficient of variation analysis. The LC-MS based pseudotargeted metabonomic method established in this study can be applied to the comprehensive detection of saikosaponins, which providing new method for the quality evaluation of Bupleuri Radix.

Intestinal absorption mechanism of saikosaponin d in vitro and in vivo / 中国药科大学学报

@#In order to reveal the intestinal absorption mechanism of saikosaponin d (SSd) in vitro and in vivo, the current research investigated the effects of different experimental conditions (time, concentration, temperature, pH, intestinal segments), transporter inhibitors, paracellular pathway enhancer, metabolic enzyme inhibitors on the intestinal absorption of SSd, in Caco-2 monolayers and a single pass perfusion model in rats.The results showed that the apparent permeability coefficient (Papp) and effective permeability coefficient (Peff) of SSd were 4.75 × 10-7 - 6.38 × 10-7 cm/s and 0.19 × 10-4- 0.27 × 10-4 cm/s, respectively, indicating that it was a low permeability compound, and that the transmembrane transport of SSd was concentration-dependent (0.5-5 μmol/L) and time-dependent (0-180 min).Ileum was the main absorption site for SSd. Experimental results based on Caco-2 monolayers showed that the P-gp inhibitor and paracellular permeability enhancer significantly increased the absorption of SSd (P < 0.05), which was consistent with the results obtained in rats. Inhibitors of OATPs and OCTs showed different results in vitro and in vivo, which may be related to the lower expression of them in jejunum.In summary, the intestinal absorption of SSd occurs through a carrier-mediated and energy-dependent transport, as well as passive diffusion, and P-glycoprotein plays an important role in the active transport of SSd.

Establishment and application of quality evaluation method for Xiaochaihu Granules based on calibrator samples / 中国中药杂志

With reference to the production process documented in Chinese Pharmacopoeia, this paper prepared the calibrator samples of Xiaochaihu Granules from multiple batches and established a method for fingerprint analysis and content determination that could be used to evaluate Xiaochaihu Granules available in market. Multiple batches of Chinese herbal pieces contained in Xiaochaihu Granules were collected for preparing the calibrator samples according to the process in Chinese Pharmacopoeia. Following the establishment of fingerprints for calibrator samples by UHPLC, the method for determining the contents of saikosaponin B2, saikosaponin B1, baicalin, wogonoside, baicalein, liquiritin, glycyrrhizin G2 and glycyrrhizic acid in Xiaochaihu Granules was established. The experimental results showed that the fingerprints of calibrator samples had 26 common peaks, covering the chemical compounds of main herbs Bupleuri Radix, Scutellariae Radix, Changii Radix, Glycyrrhizae Radix et Rhizoma, and Rhizoma Zingiberis Recens. The similarity of fingerprints for 47 batches of Xiaochaihu Granules from 31 companies with the calibrator sample fingerprint ranged from 0.74 to 0.99, indicating good applicability of the established fingerprint. The contents of main components baicalin, saikosaponin B2, and glycyrrhizic acid in Xiaochaihu Granules were within the ranges of 22.917-49.108 mg per bag(RSD 19%), 0.28-2.19 mg per bag(RSD 62%), and 0.897-6.541 mg per bag(RSD 41%), respectively. The quality difference in saikosaponin B2, and glycyrrhizic acid among different manufacturers was significant. The fingerprint analysis and content determination method for calibrator samples of Xiaochaihu Granules prepared according to the production process in Chinese Pharmacopoeia has been proved suitable for evaluating the quality of Xiaochaihu Granules from different manufacturers. Saikosaponin B2, glycyrrhizic acid, and liquiritin should be added as content control indicators for Xiaochaihu Granules, aiming to further improve the product quality.

Comparative permeability of three saikosaponins and corresponding saikogenins in Caco-2 model by a validated UHPLC-MS/MS method / 药物分析学报

Saikosaponins (SSs) are the main active components extracted from Bupleuri Radix (BR) which has been used as an important herbal drug in Asian countries for thousands of years.It has been reported that the intestinal bacteria plays an important role in the in vivo disposal of oral SSs.Although the deglycosylated derivatives (saikogenins,SGs) of SSs metabolized by the intestinal bacteria are speculated to be the main components absorbed into the blood after oral administration of SSs,no studies have been reported on the characteristics of SGs for their intestinal absorption,and those for SSs are also limited.Therefore,a rapid UHPLC-MS/MS method was developed to investigate and compare the apparent permeability of three common SSs (SSa,SSd,SSb2) and their corresponding SGs (SGF,SGG,SGD) through a bidirectional transport experiment on Caco-2 cell monolayer model.The method was validated according to the latest FDA guidelines and applied to quantify the six analytes in transport medium samples extracted via liquid-liquid extraction (LLE).The apparent permeability coefficient (Papp) determined in this study indicated that the permeability of SGs improved to the moderate class compared to the corresponding parent compounds,predicting a higher in vivo absorption.Moreover,the efflux ratio (ER) value demonstrated an active uptake of SSd and the three SGs,while a passive diffusion of SSa and SSb2.

Saikosaponin A Changes Drug Resistance of A549/DDP Cells Through Wnt/β-Catenin Pathway / 中国实验方剂学杂志

Objective:To investigate the effect and mechanism of saikosaponin A （SSA） on the reversal of cisplatin （DDP） resistance in human lung cancer cell line A549/DDP. Methods:The resistance of A549 and A549/DDP cells to DDP and the inhibitory effects of SSA against the proliferation of A549 and A549/DDP cells were detected using cell counting kit-8 （CCK-8）. The apoptosis rates of A549/DDP cells treated with SSA or DDP or SSA combined with DDP and the changes in reactive oxygen species （ROS） were determined by flow cytometry. The mRNA expression levels of C-myc， B-cell lymphoma 2 （Bcl-2） and cysteinyl aspartate-specific protease-3 （Caspase-3） were detected by real-time polymerase chain reaction （Real-time PCR）， followed by the determination of <italic>β</italic>-catenin transcriptional activity using the TopFish dual-luciferase reporter assay system and the measurement of <italic>β</italic>-catenin protein expression in A549/DDP cells by Western blot. Results:The results of CCK-8 assay showed that the DDP resistance of A549/DDP cells was 12.82 times that of A549 cells （<italic>P</italic><0.05）. SSA inhibited the viability of A549 cells with the half maximal inhibitory concentration （IC₅₀） being 34.9 μmol·L^-1， and also suppressed the viability of A549/DDP cells in a concentration-dependent manner. Since the inhibition rate of 20 μmol/L SSA against A549/DDP cells was less than 10%， the reversal concentration was set at 20 μmol/L. Flow cytometry revealed that compared with the control， DDP alone increased the apoptosis rate of A549/DDP cells （<italic>P</italic><0.05）， stimulated the accumulation of intracellular ROS （<italic>P</italic><0.05）， down-regulated the mRNA expression levels of C-myc and Bcl-2 in A549/DDP cells， up-regulated Caspase-3 mRNA expression， and reduced the transcriptional activity of <italic>β</italic>-catenin （<italic>P</italic><0.05）. Compared with the DDP group， the SSA+DDP group exhibited obviously increased apoptosis of A549/DDP cells， enhanced accumulation of intracellular ROS， down-regulated C-myc and Bcl-2 mRNA expression， up-regulated Caspase-3 mRNA expression （<italic>P</italic><0.05）， and weakened <italic>β</italic>-catenin transcription （<italic>P</italic><0.05）. DDP combined with SSA better decreased the <italic>β</italic>-catenin protein expression in contrast to that of control or DDP （<italic>P</italic><0.05）. Conclusions:SSA enhances the sensitivity of A549/DDP cells to DDP possibly by inhibiting the activation of Wnt/<italic>β</italic>-catenin pathway.

Integrative lipidomic and transcriptomic study unravels the therapeutic effects of saikosaponins A and D on non-alcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFLD) has become one of the most prominent causes of chronic liver diseases and malignancies. However, few therapy has been approved. Radix Bupleuri (RB) is the most frequently used herbal medicine for the treatment of liver diseases. In the current study, we aim to systemically evaluate the therapeutic effects of saikosaponin A (SSa) and saikosaponin D (SSd), the major bioactive monomers in RB, against NAFLD and to investigate the underlying mechanisms. Our results demonstrated that both SSa and SSd improved diet-induced NAFLD. Integrative lipidomic and transcriptomic analysis revealed that SSa and SSd modulated glycerolipid metabolism by regulating related genes, like

Saikosaponin-d simultaneously induces apoptotic and paraptosis-like cell death in hepatoma cells / 西安交通大学学报(医学版)

Objective: To investigate the cell death-inducing effect of saikosaponin-d (SSD) on human hepatoma Hep3B cells and its potential mechanism. Methods: Hepatoma Hep3B cells were divided into five groups: blank control group, DMSO (vehicle) group, and three SSD treatment groups treated with various doses of SSD (5, 10, and 15 μmol/L). MTT assay was used to evaluate cell viability. Flow cytometer was employed to quantitatively detect the percentage of dead cells after Annexin V-FITC/PI double staining. Apoptosis was detected morphologically after Hoechst 33258 staining. The activity of caspase-3 apoptotic protease was determined by spectrophotometry. Cell morphologic changes were observed with an inverted microscope. Western blotting and Real-time PCR were employed to evaluate the expression levels of C/EBP homology (CHOP) protein and mRNA, respectively. Results: MTT assay showed that SSD inhibited the viability of human hepatoma Hep3B cells in concentration- and time-dependent manners. Sinomenine hydrochloride induced the death of Hep3B cells in a concentration-dependent manner indicated by flow cytometry. After staining with Hoechst 33258, the nuclei of SSD-treated cells showed nucleosomal agglutination, nucleosomal shrinkage and fragmentation under the fluorescence microscope, which are the characteristics of apoptotic cells. SSD significantly activated the key apoptotic executor caspase-3. The occurrence of paraptosis, characterized by extensive cytoplasmic vacuoles, was observed in SSD-treated cells under an inverted microscope. The pretreatment of a pancaspase inhibitor Z-VAD-FMK completely inhibited caspase-3 activity triggered by SSD, but only partially suppressed cell death and could not reduce the cytoplasmic vacuolation in SSD-treated cells. The protein and mRNA expressions of CHOP, a stress-inducible molecule, were upregulated by SSD, which could not be inhibited by Z-VAD-FMK. Conclusion: SSD can simultaneously induce caspase-dependent apoptosis and caspase-independent paraptosis in human hepatoma Hep3B cells. The upregulated expression of CHOP may be the mechanism involved in SSD-induced paraptosis.

Effects of Saikosaponin d on differentially expressed genes CTLA-4, IL-10 and IL-17 in mice with autoimmune hepatitis / 上海交通大学学报（医学版）

Objective: To explore the effects of Saikosaponin d (SS-d) on autoimmune hepatitis (AIH) by observing the expression changes of some differentially expressed genes screened with the Agilent-085631 gene chip in the liver of AIH mice. Methods: Forty healthy male SPF C57BL/6 mice were divided into chip group (n=8) and SS-d treatment group (n=32). The mice in the chip group were divided into the normal group and the model group [concanavalin A (Con A) was administered to the tail vein at a dose of 15 mg/kg] (both n=4). The mice were sacrificed after 12 h. The differentially expressed genes of liver were screened, some of which were verified by qRT-PCR. The SS-d treatment group was further divided into the normal group, the model group (treatment was the same with the chip group), SS-d low-dose group and SS-d high-dose group [according to the literature and pre-experiment results, 2.5 and 5.0 mg/kg dose of intraperitoneal injection of SS-d were given respectively, and 15 mg/kg of Con A was administered to the tail vein 8 h later] (all n=8). After 12 h, total venous blood, liver total protein and total RNA of mice were collected. The levels of serum glutamic pyruvic transaminase (GPT), glutamic oxaloacetic transaminase (GOT), interleukin (IL)-10 and IL-17 were detected by ELISA, and cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) was detected by Western blotting. qRT-PCR technology was used to detect the mRNA levels of IL-10, IL-17 and CTLA-4. Results: A total of 11 512 differentially expressed genes were screened (up 5 189, down 6 323), which were related to 138 signal pathways. The qRT-PCR results of IL-10, IL-17 and CTLA-4 gene were consistent with the results of chip screening. Compared with the normal group, the serum levels of GPT and GOT in the model group increased, IL-17 mRNA level increased, IL-10 mRNA and CTLA-4 mRNA levels decreased, the content of serum IL-17 increased, the content of serum IL-10 decreased, and the level of CTLA-4 protein expression in the liver tissues decreased. Compared with the model group, the serum GPT and GOT levels of SS-d in the low-dose and high-dose groups decreased, IL-17 mRNA level decreased, the levels of IL-10 mRNA and CTLA-4 mRNA increased, the content of serum IL-17 decreased, the content of serum IL-10 increased, and the level of CTLA-4 protein expression in the liver tissue increased. Conclusion: Multiple signaling pathways are involved in the pathogenesis of AIH, and SS-d can alleviate the liver inflammation in AIH mice by regulating the expression of IL-10, CTLA-4, and IL-17.

Investigating mechanism of Qingfei Dayuan Granules for treatment of COVID-19 based on network pharmacology and molecular docking / 中草药

Objective: To explore the main active components, key targets and signaling pathways of Qingfei Dayuan Granules in treating of COVID-19 based on network pharmacology and molecular docking. Methods: TCMSP, ETCM and YATCM databases were used to search the chemical constituents of Qingfei Dayuan Granules, and the threshold values of OB ≥ 30% and DL ≥0.18 were used to screen the potential active compounds. SIB and STITCH databases were used to query the targets corresponding to the active compounds, and the PPI network and network topology parameters were obtained by using STRING database. Cytoscape 3.6.0 was used to screen the hub targets. The key targets were analyzed by Gene Ontology (GO), the Kyoto Encyclopedia of genes and genomes (KEGG) pathway enrichment and tissue enrichment using DAVID 6.8 software. The molecular docking was performed by AutoDock Tools 1.5.6 software. Results: A total of 251 active compounds and 1 037 targets were obtained, 107 key targets and 185 corresponding compounds were screened. The key targets involved ESR1, AR, EGFR, KDR, MMP2, and 52 genes were coexpressed with ACE2. The results of GO function enrichment analysis showed that Qingfei Dayuan Granules mainly regulated the biological processes of cell surface signaling transduction, molecular function, phosphorylation and transcription. KEGG pathway enrichment mainly involved chemokine signaling pathway, T cell receptor signaling pathway, B cell receptor signaling pathway, natural killer cell mediated cytotoxicity and Toll like receptor signaling pathway. The results of tissue enrichment showed that the key gene expression sites were mainly in lung and epithelial cells, involving a variety of immune cells, such as T cells, B cells, lymphocytes, etc. Molecular docking showed that the compounds with good binding power to SARS-CoV-2-RBD-ACE2 complex in Qingfei Dayuan granules were mainly come from Bupleuri Radix, Codonopsis Radix, Anemarrhenae Rhizoma, and Glycyrrhizae Radix et Rhizoma. Saikosaponin, glycyrrhizic acid, anemarsaponin had good binding power with SARS-CoV-2-S-RBD-ACE2, which may be potential active components against SARS-CoV-2. Conclusion: Qingfei Dayuan Granules has the characteristics of multi-components, multi-targets and multi-pathway regulation. Saikosaponin, glycyrrhizic acid, and anemarsaponin may be the potential active components against SARS-CoV-2. The mechanisms of its treatment against COVID-19 may be related to the regulation of the co-expressed genes with ACE2, inhibition of inflammation and immune related signaling pathways, and the destruction of the complex structure of SARS-CoV-2-S-RBD-ACE2.

Exploring active ingredients and function mechanism of Chaihu Guizhi Ganjiang Decoction against coronavirus disease 2019 based on molecular docking technology / 中草药

Objective: To predict the efficacy components and key targets of Chaihu Guizhi Ganjiang Decoction (CGGD) in the intervention of novel coronavirus pneumonia in the cold-dampness obstructing lungs in early stage, and clarify its mechanism. Methods: The novel coronavirus pneumonia TCM stage, clinical manifestations and the function of CGGD were analyzed by literature mining and clinical reports. TCMSP database was used to screen potential active components and related targets in CGGD. PubMed database was used to screen pneumonia, cough and fever related targets. With the help of Cytoscape software, a “drug-disease-target” visual network diagram and protein interaction network were constructed, and GO and pathway enrichment analysis of key targets was performed through the STRING database. The active ingredients were molecularly docked with SARS-CoV-2 3CL hydrolase protein and ACE2 by AutoDock Vina. Results: The analysis of the relationship between prescriptions and syndromes showed that CGGD could play warm-yang scattered cold, resolve dampness, clear stagnation and heat, and open up membrane’s power to intervene in early cold-dampness lung type COVID-19. Through screening, the therapeutic effects of CGGD were mainly in 156 chemical components acting on 159 related targets. The core 27 genes predicted and analyzed included EGFR, TP53, YWHAZ, HSP90AB1, PIK3R1, GRB2, etc. GO and pathway analysis showed that CGGD was mainly involved in biological processes such as cell regulation and immune system related pathways to play a therapeutic role. The 10 core components were molecularly docked, saikosaponin A, saikosaponin D, and peroxyergosterol in CGGD had good affinity with 3CL hydrolase protein and ACE2. Conclusion: Using network pharmacology and molecular docking technology to predict that CGGD can be used for the treatment of novel coronavirus pneumonia with symptom of cold-dampness obstructing lungs in early stage, potential antiviral ingredients contained in prescription of CGGD, can play a therapeutic role in the treatment of new type of coronavirus pneumonia in the early stage by regulating the immune system. It explains the characteristics of “multi-component-multi-target-multi-disease” of Chinese materia medica, and provides theoretical basis for clinical rational use of medicines.

Effects of soil factors on saikosaponin content of Bupleurum chinense in different habitats / 中草药

Objective: Soil is an important factor affecting the formation and accumulation of active ingredients in Chinese medicinal herbs. Taking 29 sample wild and cultivated Bupleurum chinense of 11 areas as study materails, using mathematical statistical analysis methods to explore the relationship between saikosaponin accumulation and soil factors in order to improve the quality of B. chinense by soil ecological regulation. Methods: HPLC analysis of the contents of saikosaponins a, c, d, e, and f from different habitats; pH, organic matter, conductivity, soil water, total nitrogen, alkaline nitrogen, available phosphorus, available potassium, effective calcium, effective magnesium, effective iron, effective copper, and effective manganese were determined by conventional soil physicochemical property assay methods, cluster analysis method was used to analyze the content of saikosaponin from different habitats, analysis of the relationship between soil factors and the content of saikosaponin by Pearson correlation analysis, analysis of soil factors using principal component analysis. Results: Determination of saikosaponin showed that higher content of saikosaponin in Henan province (habitat 5, habitat 6), total content of saikosaponin a and saikosaponin d in habitat10 was 5.16 times the national standard.Cluster analysis of B. chinense from different origins, according to the content of saikosaponin, the B. chinense from 29 habitats were grouped into three types. Pearson correlation analysis of saikosaponin content and soil factors that the organic matter in the soil was significantly positively correlated with saponin a and total saponins (P < 0.05), there was a significant positive correlation between available zinc and available iron and each saikosaponin (P < 0.05) properly improve the organic matter, effective zinc, effective ironcontent in the soil can promote the accumulation of saikosaponin.Analysis of the principal components of soil from different habitats, the higher score was habitat 9, habitat 10, habitat 11, habitat 14, habitat 19, habitat 28. This is basically consistent with the results of cluster analysis of saikosaponin content. Based on the analysis of the main soil indicators of B. chinense from different habitats that in a certain range, the higher organic and effective zinc content, the more favorable were the accumulation of saikosaponins. This was basically consistent with the results of Pearson correlation analysis. Conclusion: These results indicated enhancement of organic matter, effective zinc, in the soil can improve the saikosaponin content in cultivated B. chinense.

Optimization of Water Extraction Technology of Saikosaponin a and Saikosaponin d from Bupleurum chinense by Electroma- gnetic Cracking / 中国药房

OBJECTIVE： To establish the method for simultaneous determination of saikosaponin a and saikosaponin d in Bupleurum chinense water extract， and to optimize its water extraction technology for electromagnetic cracking. METHODS： HPLC method was used. The determination was performed on SB-C18 column with mobile phase consisted of acetonitrile-water （gradient elution） at the flow rate of 1.0 mL/min. The column temperature was 40 ℃. The detection wavelength was set at 210 nm， and  the sample size was 10 μL. Based on single factor experiment， using extraction time， particle size， solide-liquid ratio as factors， total extraction rate of saikosaponin a to saikosaponin d as indexes， the extraction technology was optimized by using Box-Behnken response surface methdology， and compared with the results of ultrasound method and decoction method. RESULTS： The linear range of saikosaponin a and saikosaponin d were 50.70-202.80 μg/mL （r＝0.999 9） and 50.50-202.00 μg/mL （r＝0.999 9）， respectively. The quantitation limits were 0.16 and 0.13 μg/mL， respectively. The detection limits were 0.05 and 0.04 μg/mL，respectively. RSDs of precision， stability and reproducibility tests were all lower than 2％. The average recoveries were 98.23-102.47％ （RSD＝1.80％， n＝6） and 98.84％-102.06％ （RSD＝1.60％， n＝6）. The optimal extraction technology was as follows： the extraction time of 2.50 min； the particle size of 80 mesh， solid-liquid ratio of 1 ∶ 28 （g/mL）. Results of 3 times of validation tests showed that the optimal technology included the average total extraction rates of saikosaponin a and saikosaponin d were 8.42 mg/g， which was higher than that of ultrasonic method （8.34 mg/g） and decoction method （8.06 mg/g）， and the extration time was shorter. CONCLUSIONS： Established method is simple and accurate， and can be used for simultaneous determination of saikosaponin a and saikosaponin d in B. chinense water extract. The optimized water extraction technology for electromagnetic cracking is stable and feasible.

Simultaneous Determination of 6 Components in Chaihuang Tablets by HPLC-DAD / 中国药房

OBJECTIVE： To establish a method for simultaneous determination of 6 components in Chaihuang tablets， such as baicalin， wogonoside， baicalein， wogonin， saikosaponin a and saikosaponin d in Chaihuang tablets. METHODS： HPLC-DAD method was used to detect 3 batches of Chaihuang tablets from same manufacturers. The determination was performed on Agilent Eclipse XDB-C18 column with mobile phase consisted of acetonitrile-triethylamine phosphate aqueous solution （pH adjusted to 7.0， gradient elution） at flow rate of 1.0 mL/min. The detection wavelengths were set at 210 nm （saikosaponin a， saikosaponin d） and 277 nm （baicalin， wogonoside， baicalein， wogonin）. The column temperature was 30 ℃， and sample size was 5 μL. RESULTS： The linear ranges of baicalin， wogonoside， baicalein， wogonin， saikosaponin a and saikosaponin d were 0.379 5-7.590 4 μg，   0.082 96-1.659 2 μg， 0.039 39-0.787 8 μg， 0.040 72-0.814 4 μg， 0.040 45-0.809 0 μg， 0.038 63-0.772 6 μg （all r≥0.999 3）， respectively. The limits of detection were 0.008， 0.007， 0.005， 0.005， 0.020 and 0.018 μg/mL. The limits of quantitation were 0.025， 0.022， 0.015， 0.015， 0.060， 0.054 μg/mL. RSDs of precision， reproducibility and stability tests （48 h） were all lower than 1.5％ （n＝6）. Average recoveries were 98.46％， 97.06％， 100.90％， 96.13％， 96.91％， 96.57％ （RSD＜2.0％， n＝6）. CONCLUSIONS： Established method is simple， accurate and reproducible for 6 components in Chaihuang tablets， and can be used for quality control of the tablet.

Study on liver protection and hepatotoxicity of saikosaponin a based on zebrafish model / 中国中药杂志

Bupleuri Radix has both liver protection and hepatotoxicity. Saponins are the main pharmacodynamic and toxic components of Bupleuri Radix. Based on zebrafish physical model and the model of alcoholic fatty liver( AFL) pathology,the liver toxic and protective effect of saikosaponin a( SSa) were assessed. The results indicated that 1. 77 μmol·L-1 SSa showed protective effect to AFL zebrafish. 5. 30 μmol·L-1 SSa was hepatotoxic to healthy zebrafish,but it showed protective effect to AFL zebrafish. 5. 62 μmol·L-1 SSa was hepatotoxic to healthy and AFL zebrafish. This study is benefit for clinical safety of saikosaponin a.

Effects of saikosaponin b_2 on inflammation and energy metabolism in mice with acute liver injury induced by LPS/GalN / 中国中药杂志

To study the effects of saikosaponin b2( SS-b2) on inflammatory factors and energy metabolism against lipopolysaccharide/galactosamine( LPS/Gal N) induced acute liver injury in mice. Mice were randomly divided into normal group( equal amount of normal saline),model group( 100 g·kg~(-1) LPS and 400 mg·kg~(-1) Gal N),low,medium,high dose group of SS-b2( SS-b25,10,20 mg·kg~(-1)·d-1) and positive control group( dexamethasone,10 mg·kg~(-1)). All of the groups except for the normal group were treated with LPS/Gal N though intraperitoneally injection to establish the acute liver injury model. The organ indexes were calculated. The levels of serum transaminases( ALT and AST) and the activities of ATPase( Na+-K+-ATPase,Ca2+-Mg2+-ATPase) in liver were detected. The activity of tumor necrosis factor-α( TNF-α),interleukin-1β( IL-1β) and interleukin-6( IL-6) were determined by the enzyme-linked immunosorbent assay( ELISA). The contents of lactate dehydrogenase( LDH) in liver were determined by micro-enzyme method. HE staining was used to observe the histopathological changes of the liver. Histochemical method was used to investigate the protein expression of liver lactate dehydrogenase-A( LDH-A). The protein expressions of Sirt-6 and NF-κB in the liver were detected by Western blot. According to the results,compared with the model group,there were significant changes in organ indexes in the high-dose group of SS-b2( P<0. 05). The level of ALT,AST,TNF-α,IL-1β,IL-6 and the activities of LDH in serum of mice with liver injury were significantly reduced in the medium and high dose groups of SS-b2( P<0. 01). With the increase of the concentration of SS-b2,the range of hepatic lesions and the damage in mice decreased. The activities of Na+-K+-ATPase and Ca2+-Mg2+-ATPase in liver of mice were significantly enhanced in each dose group( P<0. 01). The expression of NF-κB in liver tissues was significantly down-regulated in the medium and high dose group( P<0. 01). Meanwhile,the expression of Sirt-6 protein in the liver of mice with acute liver injury was significantly increased in each dose group( P<0. 01).In summary,SS-b2 has a significant protective effect on LPS/Gal N-induced acute liver injury in mice,which may be related to the down-regulation of NF-κB protein expression and up-regulation of Sirt-6 protein expression to improve inflammatory injury and energy metabolism.

Fingerprint Analysis of Bupleurum chinense Roots from Different Origins by UPLC/Q-TOF-MS / 中国实验方剂学杂志

Objective: The fingerprint of Bupleurum chinense roots was developed with ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry(UPLC/Q-TOF-MS),the main chromatographic peaks were preliminary identified,and combined with principal component analysis(PCA) pattern recognition method to evaluate the quality of this herb from different origins. Method: The chromatographic separation was performed on a ZORBAX Eclipse Plus C18 column(2.1 mm×100 mm,1.8 μm) with a gradient elution of acetonitrile-0.1%formic acid aqueous solution.The mass spectrometer equipped with electrospray ionization(ESI) was used as detector and operated under the negative ion mode.Taking mass spectrometry data processing software of PeakView 1.2 and metabolomics analysis software of MarkerView 1.2.1,the different origins of B. chinense roots were analyzed by PCA. Result: The fingerprint of B. chinense roots was established within 35 min by UPLC/Q-TOF-MS.The samples from different origins were apparently classified by PCA.Eight compounds with significant differences were screened out,and the structures of three of them were identified as 3″-O-acetyl saikosaponin A,3″-O-acetyl saikosaponin D,6″-O-acetyl saikosaponin D. Conclusion: UPLC/Q-TOF-MS can be used for the rapid identification of fingerprint of B. chinense roots from different origins.IBM SPSS Statistics 21.0 and PCA can comprehensively distinguish the differences of chemical components in B. chinense roots from different origins and can be used to evaluate the quality of this herb.

Saikosaponin-b2 alleviates CCL4-induced acute liver injury in mice by inhibiting endoplasmic reticulum stress signal pathway / 中国药理学与毒理学杂志

OBJECTIVE To investigate the protective effect of saikosaponin-b2 (SS-b2) on acute liver injury induced by carbon tetrachloride (CCI4) and its inhibition on endoplasmic reticulum stress pathway in mice. METHODS Sixty male BALB/c mice were randomly divided into normal control, model, SS-b2 (5, 10 and 20 mg·kg-1) and silymarin (10 mg·kg-1) groups. Mice were ig given different drugs or saline for 7 d. Two hours after the last administration, mice were IP given 5% CCI4 to induce acute liver injury, except those in normal control group. The activities of glutamic-pyruvic transaminase (GPT), glutamic-oxaloacetic transaminase (GOT) and superoxide dismutase (SOD) and the content of malondialdehyde (MDA) in mouse serum were detected by colorimetric method. Pathological changes in liver tissue were detected by HE staining. The expression levels of protein kinase R-like ER kinase (PERK), phospho-eukaryotic initiation factory 2a (p-elF2α), activating transcrIPtion factor-4(ATF4), glucose regulated protein 78 (GRP78) and CCAAT/enhancer-binding protein homologous protein (CHOP) were detected by immuno-histochemistry and Western blotting. RESULTS Compared with the normal control group, the activities of GPT and GOT and the content of MDA in mouse serum in model group were significantly increased (P<0.05, P<0.01), but the activity of SOD was significantly decreased (P<0.01). Meanwhile, the expression levels of PERK, p-elF2α, ATF4, GRP78 and CHOP in the liver tissue were significantly increased in the model group (P<0.01). Compared with the model group, SS-b2 groups significantly reduced the activity of GPT, GOT and the content of MDA in the serum of acute liver injury mice (P<0.05, P<0.01), but increased the activity of SOD (P<0.01), and the expression levels of endoplasmic reticulum stress pathway proteins mentioned above were significantly reduced (P<0.05, P<0.01). HE staining results showed that different concentrations of SS-b2 could significantly improve the swelling of hepatocytes, the dissolution of nuclei around hepatic lobules, and the arrangement disorder of hepatic cords induced by CCI4 in mice. The cell morphology was obviously improved. CONCLUSION SS-b2 has a significant protective effect on CCL-induced acute liver injury in mice. The mechanism may be related to the inhibition of oxidative stress and down-requlation of the expressions of ERS related protein.

Correlation between content of saikosaponin and expression of key enzyme genes in different parts of Bupleurum chinense / 中草药

Objective: To select reference genes suitable for the expression analysis of Bupleurum chinense, and analyze the relationship between the content of saikosaponin and the gene expression of key enzymes in different tissues of B. chinense. Methods The roots, stems, leaves and fruits of B. chinense were used as test materials, and five commonly used internal reference genes of Actin, α-tubublin, β-tubulin, Cyclophilin and EF-1α were selected as candidates by real-time quantitative PCR. Based on the selected internal reference gences, tissue expression pattern of ACAT, FPS, HMGR, IPPI, PMD, PMK, SE, SS, β-AS, UGT1, UGT3, UGT6, UGT8, UGT10, P450-7 and P450-12 genes in B. chinense was analyzed. The content of saikosaponin a, saikosaponin c and saikosaponin d were determined by HPLC, and correlation analysis was performed by SPSS. Results:The EF-1α gene with the best stability in the five candidate genes (EF-1α, Cyclophilin, Actin, β-tubulin, α-tubublin) was selected as the internal reference gene. The expression levels of 16 key enzymes in the roots of B. chinense were measured. The results showed that ACAT, PMK, IPPI, SS, SE, UGT1, UGT3, UGT6, and UGT8 were the highest in the aboveground parts, the levels of HMGR, β-AS, P450-7 and P450-12 were higher in the roots than those in the aboveground parts, but PMD, FPS and UGT10 were not significantly different in the tissues. The content of saponins in the root was much higher than that in the aerial parts (stem, leaf and fruit) by HPLC. The results of correlation analysis showed that 16 key enzyme genes in the upstream ACAT, HMGR, PMD, SE and so on were significantly correlated with downstream P450-7, P450-12, UGT3, UGT6 and UGT8 (P < 0.05). It showed that the key enzyme genes were closely related to each other and regulated the synthesis of saikosaponin in common. The correlation analysis between the 16 key enzyme genes and the content of saikosaponin showed: HMGR, P450-7, p450-12 and the total of three saponins were significantly positively correlated (P < 0.01), and β-AS was significantly correlated with the total content of three saponins (P < 0.05), and HMGR, P450-7, P450-12, and β-AS were significantly correlated with the monomer saponins a, c, d (P < 0.05). These four genes jointly regulated the synthesis of saikosaponin and had an important effect on the accumulation of saponin. Conclusion:The HMGR, β-AS, P450-7 and P450-12 genes in the saikosaponin synthesis pathway have a consistent distribution in saikosaponin synthesis and play an important role in the regulation of saponin synthesis.

Network pharmacological analysis of Chaihu Guizhi Decoction for treating different diseases with same method on gastric ulcer and epilepsy / 中草药

Objective: Under the guidance of traditional Chinese medicine theory, this paper used network pharmacology model to explore the effective components and mechanism of “treating different diseases with same method” of Chaihu Guizhi Decoction (CHGZD) in treating gastric ulcer and epilepsy, which provided modern medical evidence for the theory of “treating different diseases with same method” of traditional Chinese medicine. Methods: The chemical constituents and potential targets of CHGZD were collected by TCMSP and TCMID databases. Disease targets of gastric ulcer and epilepsy were obtained in PubMed, CTD, and OMIM databases. Cytoscape 3.6.0 software was used to map CHGZD for gastric ulcer and epilepsy. The pharmacodynamic basis and molecular mechanism of the “treating different diseases with same method” network, and the functional and pathway enrichment analysis of gene targets was analyzed by DAVID 6.8 and KOBAS 3.0. Results: A total of 198 kinds of chemical constituents, 417 target targets, 114 gastric ulcer disease targets, and 461 epileptic disease targets were collected from CHGZD. Network analysis showed that 152 active components such as quercetin, beta-sitosterol, wogonin, kaempferol, and saikosaponin a in CHGZD played a role in the treatment of gastric ulcers and epilepsy through 17 common targets including PTGS2, VEGFA, TP53, IL6, and TNF, and 62 pathways such as pathways in cancer, and proteoglycans in cancer. Conclusion: The similar efficacy network composed of 17 common targets in gastric ulcer and epilepsy was the basis for CHGZD to play the role of “different disease and common treatment”. A total of 152 components work together through 62 pathways and 17 pathways to achieve the effect of “treating different diseases with same method”, which provides modern medical evidence for the traditional Chinese medicine to treat gastric ulcer and epilepsy from the liver and spleen.

Study on efficacy network of Chaihu Guizhi Ganjiang Decoction in treating insomnia / 中草药

Objective: To investigate the mechanisms of effects of Chaihu Guizhi Ganjiang Decoction in the treatment of insomnia by using network pharmacology methods. Methods: TCMSP and TCMID were used to lock the targets of seven herbs in Chaihu Guizhi Ganjiang Decoction. TTD, DrugBank, and PubMed were used to search targets of insomnia and construct a “disease-prescription-target” network. STRING and Cytoscape were used to perform enrichment analysis and clarify the mechanism of core targets in the network. Results: The PPI network of Chaihu Guizhi Ganjiang Decoction contained 640 targets and the PPI network of insomnia included 175 targets. A total of 29 core targets and 80 interactions were found after enrichment analysis between two PPI networks. After GO enrichment analysis and KEGG pathway analysis of 29 key targets, we found that 171 active ingredients in Chaihu Guizhi Ganjiang Decoction such as saikosaponin a, saikosaponin d, quercetin, calcium carbonate, 6-gingerol, kaempferol, and wogonin, which played a role in the treatment of insomnia mainly through 29 core targets such as CACNA1C, GABRA1, GABRA2, GABRB3, GABRA3, with biological processes such as target and synaptic signaling, regulation of membrane potential, G-protein coupled receptor signaling pathway, and molecular functions such as neurotransmitter receptor activity, ion-gated channel activity, GABA-A receptor activity, and functional pathways composed by plasmalemma, synapse, and other cells such as neural active ligand-receptor interaction, retrograde endogenous cannabinoid signal transduction, and serotoninergic synapses. Conclusion: The pharmacological substance basis for the treatment of insomnia was composed of 171 active ingredients such as saikosaponin a and saikosaponin d. The efficacy network of “soothing liver and invigorating spleen, regulating yin and yang” was constituted by several pathways like the neural active ligand-receptor interaction and 29 targets such as CACNA1C. Our results provide network pharmacological evidence for clinical rational use of Chaihu Guizhi Ganjiang Decoction for insomnia.