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OBJECTIVE To explore the intestinal absorption characteristics of saikosaponins. METHODS Based on everted intestinal sac model， using accumulative absorption amount （Q） and absorption rate constant （Ka） as indexes， UHPLC-MS/MS technique as a method， the absorption of saikosaponin A， B2， C， D and F from total saponins of Bupleurum chinense （8 g/mL， by crude drug） in the duodenum， jejunum and ileum was detected. RESULTS The correlation coefficients （r） of the regression equations for the absorption of saikosaponins A， B2， C and F in the duodenum， jejunum and ileum were all higher than 0.95， while the r of saikosaponin D in the above intestinal segments was lower than 0.95； compared with the absorption of the same composition in the duodenum， the Q and Ka of saikosaponin A and C circulating in jejunum and ileum for 120 min， as well as the Q and Ka of saikosaponin F circulating in the ileum for 120 min were significantly decreased （P＜0.05）. CONCLUSIONS Saikosaponin A and the other 4 saikosaponins are all absorbed in the duodenum， jejunum and ileum； among them， saikosaponin A， B2， C and F are linearly absorbed， which conforms to the zero-order absorption characteristics， but saikosaponin D shows non- linear absorption.
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Radix Bupleuri(RB)is commonly used to treat depression,but it can also lead to hepatotoxicity after long-term use.In many anti-depression prescriptions,RB is often used in combination with Radix Paeoniae Alba(RPA)as an herb pair.However,whether RPA can alleviate RB-induced hepatotoxicity remain unclear.In this work,the results confirmed that RB had a dose-dependent antidepressant effect,but the optimal antidepressant dose caused hepatotoxicity.Notably,RPA effectively reversed RB-induced hepatotoxicity.Afterward,the mechanism of RB-induced hepatotoxicity was confirmed.The results showed that saiko-saponin A and saikosaponin D could inhibit GSH synthase(GSS)activity in the liver,and further cause liver injury through oxidative stress and nuclear factor kappa B(NF-KB)/NOD-like receptor thermal protein domain associated protein 3(NLRP3)pathway.Furthermore,the mechanisms by which RPA attenuates RB-induced hepatotoxicity were investigated.The results demonstrated that RPA increased the abundance of intestinal bacteria with glycosidase activity,thereby promoting the conversion of saikosaponins to sai-kogenins in vivo.Different from saikosaponin A and saikosaponin D,which are directly combined with GSS as an inhibitor,their deglycosylation conversion products saikogenin F and saikogenin G exhibited no GSS binding activity.Based on this,RPA can alleviate the inhibitory effect of saikosaponins on GSS activity to reshape the liver redox balance and further reverse the RB-induced liver inflammatory response by the NF-κB/NLRP3 pathway.In conclusion,the present study suggests that promoting the conversion of saikosa-ponins by modulating gut microbiota to attenuate the inhibition of GSS is the potential mechanism by which RPA prevents RB-induced hepatotoxicity.
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OBJECTIVE: To investigate the effect of saikosaponins-b2 (SS-b2) on the migration of HepG2 cells, and to explore its potential molecular mechanism. METHODS: The effect of SS-b2 on the viability of HepG2 cells was detected by MTT method before the cells were divided into normal control group, SS-b2 (15, 30 and 60 mg·L-1) groups and positive control [doxorubicin (DOX) 2 mg·L-1] group according to the results of MTT. Wound-healing assay was performed to observe the influence of SS-b2 on the migration of HepG2 cells. The protein expressions of vascular endothelial growth factor (VEGF), matrix metalloproteinase 2 (MMP-2), MMP-9, hypoxia inducible factor-lot (HIF-1α) and the phosphorylation of extracellular regulated protein kinase 1/2 (ERK1/2) in HepG2 cells were measured by Western blotting. RESULTS: The wound-healing rate was 74.71%, 72.50% and 66.82% after treatment with SS-b2 15, 30 and 60 mg·L-1for 48 h, respectively. The wound-healing rate decreased significantly with the increae of SS-b2 doses. Compared with the normal control group, the wound-healing rate of SS-b2 30 and 60 mg·L-1was significantly reduced (P<0.05). The expressions of VEGF and HIF-1α in HepG2 cells were significantly decreased in SS-b2 groups (P<0.05, P<0.01). SS-b2 60 mg·L-1could reduce the protein expressions of MMP-2 and MMP-9 and the phosphorylation of ERK1/2 significantly (P< 0.05, P<0.01). The positive control group could significantly reduce the expressions of the above four proteins and the phosphorylation of ERK1/2 (P<0.05, P<0.01). Compared with the positive control group, the protein expressions of VEGF and MMP-2 were significantly down-regulated in 60 mg·L-1SS-b2 group (PO.05, P<0.01), but there was no significant difference in the expression of HIF-1α between SS-b2 groups and the positive control group. However, the down-regulation effect of SS-b2 on MMP-9 protein expression was not so significant as in the positive control group (P<0.01). The effect of SS-b2 60 mg·L-1on the phosphorylation of ERK1/2 was similar to that of the positive control group. CONCLUSION: SS-b2 can inhibit the migration of HepG2 cells, which may be related to the inhibition of angiogenesis-related proteins.
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Synthetic biology is an emerging discipline that analyzes the biosynthesis pathways of active constituents in traditional Chinese medicine and explores genes involved in biosynthesis. Bupleuri Radix is one of the most commonly used Chinese medicinal materials with remarkable medicinal value, its index component is saikosaponins, which has significant anti-inflammatory, anti-viral and anti-tumor activities. However, the current wild resources of Bupleuri Radix have been destroyed, and there were some problems in the process of artificial cultivation. The application of biological culture technology and synthetic biology can expand the sources of saikosaponins and protect resources of Bupleuri Radix. The culture conditions of different plants can be followed without a fixed pattern, and the biosynthetic pathways of different medicinal active ingredients are also inconsistent. At present, there is no review report on the culture technology of Bupleuri Radix and the research on the biosynthesis pathway of saikosaponins. This paper introduces the research progress of biological culture techniques, such as callus culture, adventitious root culture, hairy root culture and suspension cell culture used in synthetic biology, and the biosynthesis pathway of saikosaponins and its key enzyme functional genes. It is suggested to optimize the biological culture technology of Bupleuri Radix by referring to the tissue culture technology of other traditional root medicinal materials, so as to provide a reference for the in-depth study on the biosynthesis pathway and metabolic regulation of saikosaponins.
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OBJECTIVE To study saikosaponins (SSs) and their metabolites (a total of 30 compounds) in order to predict and verify partially potential target proteins. METHODS ①PharmMapper and Sybyl X were used to predict potential target proteins of SSs and their metabolites,and the binding model of saikosaponin a (SSa) and glutathione s-transferase A2 (GST A2) was investigated. ② In vitro experi?ments:HepRG cells were treated with SSa 0,0.03,0.06,0.13,0.25 and 0.50 mmol·L-1for 24 h,and cell viability was detected by CCK-8 analysis. HepaRG cells were treated with SSa 0, 0.03, 0.13 and 0.50 mmol·L-1for 24 h, and GST activity was detected. RESULTS ① PharmMapper and Sybyl X predicted the potential target proteins of SSs and their metabolites,including GST A2,phosphatidylcholine transfer protein, adenosine kinase, thymidylate synthase, estrogen receptor β, estradiol 17-beta-dehy?drogenase 1,proto-oncogene serine/threonine-protein kinase Pim1,bile acid receptor and cellular reti?noic acid-binding protein 2. The prediction results showed that SSa and GST A2 had the highest total score and a well-matched binding model, so they were most likely to interact. ② In vitro experiments:SSa could inhibit HepaRG cell viability in a concentration-dependent manner (R2=0.8848,P<0.05).Significant increases in activity of GST were observed after treatment with SSa. CONCLUSION The potential target proteins of SSs and their metabolites are GST, phosphatidylcholine transfer protein etc. GST may be one of the target proteins of SSa.
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Objective To investigate the effect and related mechanism of saikosaponin-d (SS-d) on the proliferation and migration of breast cancer MDA-MB-231 cells. Methods The MDA-MB-231 cells were treated with varying concentrations of SS-d.CCK-8 and colony formation analysis were used to evaluate the proliferation ability of MDA-MB-231 cells;3D cell culture, wound healing assay and Transwell migration and invasion assays were used to analyze the ability of cell migration and invasion;flow cytometry was utilized to analyze the cell cycle and apoptosis;semi-quantitative RT (qRT)-PCR and Western blot were used to explore the action mechanism of SS-d on the cell migration and invation. Results SS-d inhibited the proliferation and colony formation ability of MDA-MB-231 cells, both dose-and time-dependently;migration and invasion abilities of the MB-231 cells were also significantly inhibited by the treatment with SS-d (P＜0.01);the treatment with SS-d caused the significant cell cycle arrest at G1 phase and also significant apoptosis-induction in MDA-MB-231 cells;SS-d could up-regulate the expression of P21, P27 E-cadherin (P＜0.01), but down-regulate the expression of Bcl-2, vimentin and N-cadherin (P＜0.01), all detected in both the m RNA and protein levels. Conclusion SS-d was likely to be a potential antitumor drug for the breast cancer therapy to inhibit the proliferation and metastasis of the cancer cells.
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OBJECTIVE Angiogenesis therapy has attracted interest as a potential treatment for hepatocellular carcinoma (HCC). In this study, we investigated the anti-proliferative activities and anti-angiogenesis effects of saikosaponins (SS)-b on hepatocellular carcinoma (HCC) and its regulation on VEGF/ERK/HIF-1α signal pathway. METHODS H22 hepatoma-bearing mice model and HepG-2 cells were used to study the anti-tumor and anti-angiogenesis effects of SS-b in vivo and in vitro. Pathological change of tumor tissue was observed by HE staining, the microvascular changes were detected by immunohistochemical method. The effects of SS-b on angiogenesis were examined by using the chick embryo chorioallantoic membrane (CAM) model. The effects of SS- b on proliferation, migration and invasion were investigated by MTT assay, scratch wound healing assay and transwell assay inhuman umbilical vein endothelial cell (HUVEC) and HepG2 cells in vitro. Vascular endothelial growth factor (VEGF), matrix metalloproteinase-2/9(MMP-2/9), hypoxia-inducible factor-1α (HIF-1α) expression and the phosphorylation of extracellular regulated kinase(ERK) were analyzed using RT-PCR and Western-blot. RESULTS SS-b effectively inhibited the tumor growth of H22 mice in vivo. The inhibitory rate of tumor was 49.1%, 50.7%, 66.1% in SS-b 5, 10 and 20 mg·kg-1 group respectively. HE staining results showed that SS-b induced tumor necrosis and nuclear dissolution in H22 mice. Moreover, SS-b also reduced the number of microvessels of tumor tissue in H22 mice significantly and suppressed the angiogenesis of CAM induced by b-FGF. SS-b had an obvious inhibitory effect on cell proliferation, migration and invasion of HUVEC cells and HepG-2 cells. These effects were associated with down-regulation of the expression of MMP2/9 and suppression of VEGF/ERK/HIF-1α signaling in H22 mice and Hep-G2 cells. CONCLUSION Our findings showed that SS-b exerts anti-tumor effects by inhibit?ing tumor angiogenesis via regulating VEGF/ERK/HIF-1α signal pathway in vivo and in vitro.
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Objective To identify the genes of WRKY transcription factors (TFs) from roots of Bupleurum chinense and genes that potentially regulate saikosaponin (SS) biosynthesis. Methods Firstly, the subfamily cluster analysis was mainly based on Arabidopsis thaliana WRKYs for 27 putative WRKY TFs selected from previous transcriptome sequencing data. Secondly, qPCR was used to screen such genes of WRKY TFs that could be induced by NaCl and PEG6000 in adventitious roots of B. chinense. Meanwhile, saikosaponins (SSs) in treated adventitious roots were determined by HPLC. The roots were collected at 0, 2, 4, 8, 12, 24, 48, and 72 h after treatments, and 120 h only for PEG. Finally, the tissue-specific expression was analyzed on screened genes by qPCR. Results The 27 genes were grouped into three categories: There were nine in Group I, 15 in Group II, and two in Group III. Four genes of WRKYTFs, BCWRKY6, BCWRKY16, BCWRKY32, and BCWRKY35 were obviously induced by NaCl in adventitious roots of B. chinense, while only BCWRKY32 was induced by PEG. The content of SSs increased at different levels in NaCl and PEG6000 treatment. Three genes including BCWRKY6, BCWRKY32, and BCWRKY35, expressed most in roots, were similar to the accumulation pattern of SS. Conclusion The three WRKY genes, BCWRKY6, BCWRKY32, and BCWRKY35, may be involved in the biosynthesis of SS.
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5-Hydroxytryptamine 2C (5-HT) receptor is one of the major targets of anti-obesity agents, due to its role in regulation of appetite. In the present study, the 70% EtOH extract of the roots of Bupleurum chinense was revealed to have agonistic activity on 5-HT receptor, and the subsequent bioassay-guided isolation led to identification of several saikosaponins as the active constituents with 5-HT receptor agonistic activity in vitro and anti-obesity activity in vivo. The new compound, 22-oxosaikosaponin d (1), was determined by extensive spectroscopic analyses (HR-ESI-MS, IR, and 1D and 2D NMR). The primary structure-activity relationship study suggested that the intramolecular ether bond between C-13 and C-28 and the number of sugars at C-3 position were closely related to the 5-HT receptor agonistic activity. Saikosaponin a (3), the main saponin in B. chinense, showed obviously agonistic activity on 5-HT receptor with an EC value of 21.08 ± 0.33 μmol·Lin vitro and could reduce food intake by 39.1% and 69.2%, and weight gain by 13.6% and 16.4%, respectively, at 3.0 and 6.0 mg·kgin vivo. This investigation provided valuable information for the potential use of B. chinense as anti-obesity agent.
Subject(s)
Animals , Male , Rats , Anti-Obesity Agents , Chemistry , Pharmacology , Biological Assay , Bupleurum , Chemistry , Oleanolic Acid , Chemistry , Pharmacology , Rats, Sprague-Dawley , Saponins , Chemistry , Pharmacology , Serotonin 5-HT2 Receptor Agonists , Chemistry , Pharmacology , Structure-Activity RelationshipABSTRACT
Bupleuri Radix is one of the most frequently used herbal medicines in China with a 2000-year medicinal history. However, the use of Bupleuri Radix is very confused. Twenty-five species and eight varieties of Bupleurum have been used as Bupleuri Radix in different regions of China. It is very difficult to identify these Bupleurum species using traditional morphological method. In order to establish a fast and effective method to identify these Bupleurum species, we collected 168 Bupleurum medicinal plants from 14 populations of 9 provinces, and amplified their ITS sequences. 168 ITS sequences with a full length of 600-606 bp were obtained. DNAMAN analyzing results showed that 86 variable sites were present in these sequences and 19 haplotypes (TH1-TH19) were determined. After calculating K2P distance and analyzing an NJ tree, we established a molecular identification method based on ITS sequence. Using this method,52 samples of Bupleuri Radix were identified successfully. Furthermore, we tested saikosaponin a, c, d contents in these Bupleuri Radix by HPLC and analyzed the results by ANOVA and LSD T test to evaluate the quality of Bupleuri Radix. This method is significant for effective identification of Bupleurum medicinal plants, and quality control of Bupleuri Radix in the market.
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Objective To study the effect of anti-hepatitis B virus of Lamivudine combined with Saikosaponins in vivo. Methods Chongqing duck hepatitis B model was selected and given to Lamivudine combined with Saikosaponins(combined group) oral treatment 28d, then the ducks were observed 7d after drug withdrawal. The levels of serum HBV DNA, HBsAg and transaminase(ALT, AST) were detected before and after treatment, and compared with that in Lamivudine group and Saikosaponins group as control. Results In combined group,the levels of serum HBV DNA and HbsAg were significantly decreased(P ＜ 0.05), and 7d after drug withdrawal the levels of serum HBV DNA and HBsAg showed no" anti-jump" compared with that at drug treatment 28d(P ＞ 0.05). In Lamivudine group and Saikosaponins group the levels of those increased again 7d after drug withdrawal (P ＜ 0. 05). In each group the levels of serum ALT and AST did not change within the same phase during drug treatment 28d and 7d after drug withdrawal(P ＞ 0. 05). Conclusion Lamivudine combined with Saikosaponins in vivo had the effect of anti-hepatitis Bvirus, and the effect was stable and lasting.
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OBJECTIVE:To determine the content of volatile oil and saikosaponins in Radix Bupleuri collected in different harvesting seasons.METHODS:Volatile oil was extracted from Radix Bupleuri by using volatile oil extractor; the content of sai-kosaponins in Radix Bupleuri harvested in different seasons were determined by visible spectrophotometry taking saikosaponin a as index.RESULTS:The content of volatile oil was the highest in Radix Bupleuri harvested in July but the lowest in November;the content of saikosaponins was the highest when harvested in May and the lowest in September. CONCLUSION:The method is simple and rapid,and it provides scientific basis for the establishment of the best harvesting season of Radix Bupleuri.
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Objective To set up a simple and accurate method for the determination of saikosaponins in Bupleurum chinense DC. by UV-Visible Spectrophotometry. Methods The content of saikosaponins was determined by UV-Visible Spectrophotometry. The absorbency was measured at 536nm. Results The calibration curves was linear within 194~1940mg/L(r=0.9996). The average recovery was 97.35%,and the RSD was 1.02%(n=9). Conclusion The method was proved to be simple, precise and reproduciable. It can be used for the quantitative determination of B upleurum chinense DC.