Effect of saikosaponin A regulating MLCK/MLC2 signaling pathway on intestinal injury in SAP rats / 国际检验医学杂志

Objective To investigate the effect of saikosaponin A regulating myosin light chain kinase(MLCK)/myosin light chain 2(MLC2)signaling pathway on intestinal injury in rats with severe acute pan-creatitis(SAP).Methods A total of 10 rats were randomly selected as sham operation group,and the other rats were injected with sodium taurine cholate solution to construct SAP rat model.SAP rat models were ran-domly divided into SAP group,saikosaponin A group(10.0 mg/kg intraperitoneal injection of saikosaponin A)and iE-DAP group(3.5 mg/kg intraperitoneal injection of MLCK/MLC2 pathway activator iE-DAP),sai-kosaponin A+iE-DAP group(intraperitoneally injected with 10.0 mg/kg saikosaponin A+3.5 mg/kg iE-DAP),10 rats in each group were injected once a day for 1 week,sham operation group and SAP group were injected with the same amount of normal saline.The serum levels of amylase(AMY),lipase(LIP),diamine oxidase(DAO),interleukin(IL)-1β,IL-6 and tumor necrosis factor-α(TNF-α)were detected by enzyme-linked immunosorbent assay(ELISA).HE staining was used to detect the pathological morphology of ileum tis-sue in each group.The levels of oxidative stress indexes in ileum tissue were detected by ELISA.Intestinal barrier re-lated proteins and MLCK/MLC2 pathway related proteins were detected by western blot.Results Compared with SAP group,the levels of AMY,LIP,DAO,IL-1β,IL-6 and TNF-α in saikosonin A group were significantly de-creased,while the levels of AMY,LIP,DAO,IL-1β,IL-6 and TNF-α in iE-DAP group were significantly in-creased,with statistical significance(P＜0.05).Compared with SAP group,the structure of ileum tissue was improved and the pathological score of ileum tissue was significantly decreased in SA group(P＜0.05).Com-pared with SAP group,the levels of glutathione and superoxide dismutase in SA group were significantly in-creased,and the levels of malondialdehyde were significantly decreased in SA group,with statistical signifi-cance(P＜0.05).Compared with SAP group,the protein levels of MLCK,p-MLC2/MLC2 in SA group were significantly decreased,and the difference was statistically significant(P＜0.05).Conclusion Saikosaponin A may improve intestinal injury in SAP rats by down-regulating the MLCK/MLC2 signaling pathway.

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.

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.

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.

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.

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.

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.

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.

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.

Determination of five saponins in Ginseng Radix et Rhizoma-Bupleuri Radix herb pair and its preparations by QAMS method / 中草药

Objective To study the effects of ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, saikosaponin a, and saikosaponin d in Ginseng Radix et Rhizoma-Bupleuri Radix (GRRBR) herb pair and its preparations by using quantitative analysis of multi-components by single-marker (QAMS). Methods On the basis of ginsenoside Rg1, the relative correction factors between the ginsenoside Rg1 and the other four saponins were established, and then the contents of the other four saponins were calculated. At the same time, the contents of the five components were determined by external standard method and compared with those evaluated by QAMS. The relative retention time was determined by different chromatographic columns. It could be considered that QAMS was feasible and accurate in the determination of saponins in GRRBR herb pair. Results A quantitative control method of five kinds of saponins was established, and the methodological results were good. The results showed that there was no significant difference in the contents of five kinds of saponins in GRRBR herb pair, Xiaochaihu Decoction, and Kaixin Jieyu Prescription between QAMS group and external standard method group. Conclusion QAMS is suitable for the determination of saponin in GRRBR herb pair, which can be used as a reference for the determination of its effective components and the establishment of compound quality control method.

Changes of chemical composition of Bupleuri Radix-Paeoniae Radix Alba herb pair before and after compatibility by UPLC-MS background subtraction and metabonomics / 中草药

Objective To explore the overall change of chemical components of Bupleuri Radix-Paeoniae Radix Alba herb pair before and after compatibility. Methods The change of chemical constituents of Bupleuri Radix and Paeoniae Radix Alba before and after compatibility were analyzed based on UPLC-MS background subtraction and metabonomics. Chemical constituents of eight batches in Bupleuri Radix, Paeoniae Radix Alba, and Bupleuri Radix-Paeoniae Radix Alba were analyzed by UPLC-MS. Mass spectrometry data of the extracts of Paeoniae Radix Alba and Bupleuri Radix were respectively subtracted from mass spectrum data of the extracts of Bupleuri Radix-Paeoniae Radix Alba herb pair by mass spectrometry background subtraction method, finally mass spectrum data of chemical constituents of Bupleuri Radix and Paeoniae Radix Alba after compatibility were obtained. The chemical components of Bupleuri Radix, Paeoniae Radix Alba before and after compatibility were analyzed by PCA and OPLS-DA separately. Results A total of 57 constituents of Bupleuri Radix-Paeoniae Radix Alba herb pair, Bupleuri Radix, and Paeoniae Radix Alba were detected by UPLC-MS, and 32 of them were identified by the literature. Multivariate statistics showed that there were six ingredients which had significant changes in Bupleuri Radix before and after compatibility and three of them were identified. There were three components which were significantly changed in Paeoniae Radix Alba and two of them were identified. The saikosaponin a, 3’-O-acetylation of saikosaponin a, and 4’’-O-acetylation of saikosaponin A decreased after compatibility in Bupleuri Radix, and paeoniflorin, gallic acidpaeoniflorin or their isomers increased after compatibility in Paeoniae Radix Alba. Conclusion The overall differences of chemical composition in Bupleuri Radix-Paeoniae Radix Alba before and after compatibility were compared, which laid the foundation for the explanation of its compatibility mechanism.

Optimization of Fried with Wheat Bran Processing Technology of Bupleurum Chinense DC.by Orthogonal Test / 中国药师

Objective:To research the best processing method for Bupleurum chinense DC.by orthogonal tests.Methods:With the contents of saikosaponin a and saikosaponin d as the indices,the L9(34) orthogonal table was used to study three factors including the amount of wheat bran,pot temperature before heating and processing time.The orthogonal design was applied to study the processing technology of Bupleurum chinense DC.fried with wheat bran.Results:The best processing method was as follows:100 g Bupleurum chinense DC.was mixed with 10 g wheat bran and fried at 290 ℃ for 80 seconds.Conclusion:The optimized processing technology is reasonable,reliable and highly reproducible,which provide reference for the processing of Bupleurum chinense DC.with wheat bran.

Research on biotransformation of saikosaponin A in vitro based on HPLC-DAD-MSn / 中草药

Objective: To study the biotransformation of saikosaponin A in vitro and analyze its metabolites. Methods: Saikosaponin A was incubated in artificial gastric juice and intestinal contents of rats in anaerobic conditions, respectively, and the metabolites were analyzed and identified by HPLC-DAD-MSn. Results: By incubation of saikosaponin A in artificial gastric juice, saikosaponin b1 and g were detected in the reaction mixture. By the anaerobic incubation of saikosaponin A with intestinal flora, prosaikogenin F was soon detected, but it was further converted to saikogenin F. Conclusion: Saikosaponin A can be transformed to secondary glycosides and glycosides in artificial gastrointestinal environment. And the products can be identified by HPLC-DAD-MSn. The distribution and concentration of saikosaponin A in vivo can not reflect its fate fully, and the metabolites should be considered simutaneously.

Simultaneous determination of four saikosaponins in Xiaozheng Pellets by QAMS / 中成药

AIM To establish a quantitative analysis of multi-components by single-marker (QAMS) method for the simultaneous content determination of four saikosaponins in Xiaozheng Pellets (Bupleuri Radix,Cyperi Rhi-zoma,Rhei Radix et Rhizoma,etc.).METHODS The analysis of 5％ ammonia-methanol extract of this drug was performed on a Waters Xbridge C18column (250 mm ×4.6 mm,5 μm),with the mobile phase comprising of acetonitrile-water flowing at 1.0 mL/min in a gradient elution manner,and the detection wavelengths were set at 210 nm and 254 nm.With saikosaponin a as a internal standard,the relative correction factors of saikosaponins b1,b2 and c were calculated,followed by the determination of their contents.RESULTS Four saikosaponins showed good linear relationships within their own ranges (r ≥ 0.999 5),whose average recoveries were 98.08％-102.94％ with the RSDs of 0.85％-1.82％.The results obtained by QAMS approximated those obtained by external standard method.CONCLUSION This simple,precise and feasible method can be used for the quality control of Xiaozheng Pellets.

Content Comparison of Saikosaponin a, d and Total Flavonoids in Different Parts of Hollow Bupleurumin / 中国药师

Objective: To compare the contents of saikosaponin a, d and total flavonoids in different parts of Hollow bupleurum to provide reference for the clinical use of medicinal parts.Methods: The determination was performed on a SHIMADZU Inertsil C18(250 mm×4.6 mm,5 μm)column.The mobile phase consisted of acetonitrile and water with the ratio of 40∶60, and the flow rate was 1.0 ml·min-1.The detection wavelength was 210nm, the column temperature was 40℃, and the injection volume was 10 μl.A colorimetric method was used to detect total flavonoids with the detection wavelength of 500 nm.Results: The calibration curve of Saikosaponin a, d and total flavonoids showed a good linear relationship respectively over the range of 0.21-1.26 mg·ml-1(r=0.999 6),0.25-1.51 mg·ml-1(r=0.9997) and 4.00-25.00 μg·ml-1(r=0.999 6).The average recovery was 99.27%(RSD=2.15%, n=6),99.4%(RSD=2.14%,n=6)and 99.03%(RSD=1.34%,n=6), respectively.The content of saikosaponin a and d respectively was 0.31% and 0.50% in the root, while that in the other parts was low.The content of total flavonoids was as high as 8.48% in flowers, and that in leaves, stem and root reduced in turn.Conclusion: The aerial parts of Hollow bupleurum are rich in flavonoids, and the content of saikosaponin in root is higher, therefore, the whole plant with root is more reasonable in the clinical use.

Quality Evaluation of Radix Bupleuri Fructus from Different Habitats by Grey Correlation Analysis and Principal Components Analysis / 中国药师

Objective: To evaluate the quality of Radix bupleuri from different producing areas and make a comparison by using grey correlation analysis(GRA)and principal components analysis(PCA).Methods: Fifteen samples of Radix bupleuri were collected from different producing areas.The percentage of total ash, acid-insoluble ash, extract, saikosaponin a and saikosaponin d of Radix bupleuri from different habitats were determined.The grey correlation quality evaluation model was built for Radix bupleuri from different producing areas and the results were compared with those of PCA.Results: According to the grey correlation analysis on components in the different batches of Radix bupleuri , the samples from Shaanxi province were the most highly correlated with the optimal reference sequence (0.79), which suggested the best quality.Radix bupleuri Fructus from Hebei province was the most poorly correlated with the optimal reference sequence (0.23), which showed the worst quality.The results were basically identical with those of PCA, indicating good repeatability and stability.Conclusion: Grey incidence degree method and the established Radix bupleuri quality evaluation models can be used to evaluate the quality of Radix bupleuri .

Saikosaponin A attenuates cognitive function via cAMP/CREB signaling pathway in mice after traumatic brain inj ury / 临床麻醉学杂志

Objective To investigate the effects of Saikosaponin A (SSA)on cognitive function and cAMP/CREB signaling pathway and expression of BDNF in mice after traumatic brain injury. Methods Sixty SD male mice were randomized into three groups:shame operation group (group S, n =20),trauma group (group T,n =20)and SSA treatment group (group A,n =20).Mice received an administration of SSA 5 mg/kg (group A)or equal volume saline (group S,group T)immediately and once daily for 5 consecutive days after trauma.The cognitive function was detected by Morris wa-ter maze test on day 1,3,7 and 14 after trauma.The hippocampal tissues were harvested after be-havioral tests and homogenized for measuring the levels of brain derived neurophic factor (BDNF)and cyclic AMP (cAMP)by ELISA as well as the levels of cAMP-response element binding protein (CREB)and phosphorylation-cAMP-response element binding protein (pCREB)by western bolt. Results Compared with group S,the escape latency and swimming distance were significantly pro-longed in group T on day 1,3,7 and 14 and group A on day 1,3 after trauma (P <0.05 );while compared with group T,they were significantly shorter in group A on day 7,14 after trauma (P <0.05).Compared with group S,the levels of BDNF,cAMP,CREB and pCREB were significantly de-creased in group T(P < 0.05 ).Compared with group T,the levels of BDNF,cAMP,CREB and pCREB were significantly increased in group A (P <0.05).Conclusion SSA can significantly improve cognitive dysfunction in mice after traumatic brain injury,and the mechanism may be related to the activation of cAMP/CREB signaling pathway and up-regulation of BDNF.

Effect of different stem branches of Bupleurum chinense on saikosaponins content in its roots and root yield / 中草药

Objective: To evaluate the effect of different stem branches of Bupleurum chinense on saikosaponins content in roots and its root yield. Methods: The contents of saikosaponin a, saikosaponin d, and total saikosaponins in the roots of B. chinense were determined by HPLC and UV-vis spectrophotometry. And parameters of growth characteristics were analyzed. Results: The contents of saikosaponin a, saikosaponin d, and total saikosaponins in the roots of B. chinense and its root yield were the highest when the B. chinense stem had no branch or two branches. And with the increase of stem branch's numbers, the content of saikosaponins in the roots of B. chinense and its root yield decreased. Conclusion: Different stem branches have a significant effect on the content of saikosaponins in the roots of B. chinense and its root yield.

HPLC-DAD conbined with TOF/MS technique in determination of saikosaponin a, baicalin and glycyrrhizic acid in Xiaochaihu Decoction / 第二军医大学学报

Objective: To establish a method for determination of saikosaponin a, baicalin and glycyrrhizic acid in Xiaochaihu Decoction. Methods: A combined method of HPLC-DAD and time of flight mass spectrometry (TOF/MS) was used; the chromatography condition was as following: Agilent Zorbax XDB C18 column (25°C, 2.1 mm × 50 mm, 1.8 μm); mobile phase: methanol (0.025%, V/V, formic acid)-water 0.025%, V/ V, formic acid) with gradient elution at a flow rate of 0.15 ml · min-1. The injection volume was 0.25 μl. Atomospheric pressure electronic spray ionization was used to quantify saikosaponin a at the capillary voltage of 4 000 V, the flow and temperature of drying gas were 10.0 L · min-1 and 350°C, respectively; nebulizer pressure was 40 psi (275 792 Pa), and the fragment voltage was 200 V; the m/z of saikosaponin a was 803.420 0-803.490 0 for quantitative analysis. The wavelengths for detection of baicalin and glycyrrhizic acid were 275 nm and 250 nm, respectively. Results: The calibration curves were Y=461 182X + 2 000 000(r=0.999 0),Y=0.949 9X-1.046 4(r=0.999 5) and Y=5.978 9X-27.418(r=0.999 5) for saikosaponin a (0.211 6-127.3 μg · ml-1), baicalin (0.758-455 μg · ml-1) and ammonium glycyrrhizinate (2.268-136.8 μg · ml-1), respectively. The recovery rates of the low, medium and high concentrations were (95.54 ± 1.60)%, (99.39 ± 3.97)% and (103.8 ± 1.97)% for saikosaponin a, (102.3 ± 0.47)%, (100.9 ± 1.32)% and (97.15 ± 2.10)% for baicalin, and (102.6 ± 1.96)%, (100.3 ± 3.12)% and (97.75 ± 1.25)% for ammonium glycyrrhizinate, respectively. The intra-day and inter-day RSD was both less than 5% and the RSD was less than 5% for 21 days. Conclusion: The method in the present study is simple, sensitive, rapid, and accurate for quality control of Xiaochaihu Decoction.

Determination of four effective components in Chaihu Shugan Powder by HPLC/DAD / 中成药

AIM:To establish a method for determining saikosaponin A,paeoniflorin,hesperidin and ferulic acid in Chaihu Shugan Powder(Pericarpium Citri Reticulatae,Radix Bupleuri,Rhizoma Chuanxiong,Fructus Aurantii,Radix Paeoniae alba,etc.).METHODS:The chromatographic saparation was performed on a Hypersil C_(18) column(250 mm×4.6 mm,5 μm).The mobile phase was acetonitril-water(43:57)for saikosaponin A,and the mobile phase for rest components was acetonitrile-0.5% acetic acid(40:60).All of flow rates were 0.8 mL/min and column temperature maintained at 30℃.The detection wavelength was set at 200-400 nm.RESULTS:The four constituents were separated within 15 min.The linear ranges of saikosaponin A,paeoniflorin,hesperidin and ferulic acid were 38.5-166.7 μg/mL(r = 0.999 6),15.9～254.5 μg/mL(r = 0.999 9),22.1-353 μg/mL(r =0.999 3),6.30-201.5 μg/mL(r =0.999 9),respectively.The average recoveries were 97.57%,97.40%,98.86%,96.37%,respectively.The RSD were 2.1%,1.1%,0.70%,1.3%,respectively.CONCLUSION:The method is rapid,simple and accurate,and can be used for quality control of Chaihu Shugan Powder.