| Bupleurum chinense DC.is one of the Bupleurum species which was recorded in the Chinese Pharmacopoiea.In this study,the antidepressant effects of Bupleuri Radix(BR)and Vingner-baked Bupleuri Radix(VBBR)were evaluated.Moreover,therapeutic constituents basis were identified and relative components were determined in BR and VBBR.In addition,the antidepressant-like effects of BR and VBBR on chronic unpredictable mild stress(CUMS)-induced rats were evaluated.Furthermore,the pharmacokinetics of saikosaponins(SSs)of BR and VBBR in CUMS-induced rats plasma was charaterized and applied to a comparative study.In a word,the studies provided the theoretical foundation for therapeutic basis and antidepressant mechanism of BR and VBBR.1.The isolation and identification of saikosaponins and characterization of chemical constitutents in Bupleuri RadixBy means of the silica gel column chromatography,ODS column chromatography and preparative liquid chromatography,9 components were isolated from Bupleuri Radix,On the basis of NMR spectral data and physicochemical characters,the structures of these compounds were identified as:SSa,SSd,SSc,SSe,SSb1,SSb2,SSb3,SSb4,SSf;An UHPLC-QTOF-MS method was established for the screening and identification of the main chemical components in BR and VBBR.A total of 49 chemical compounds were identified or tentatively characterized by comparing with the reference compounds or matching the molecular weight with that of the published compounds of Bupleuri Radix and other Bupleurum plants.The major types of components including saikosaponins,flavonoids and lignans.The above results preliminarily clarify the therapeutic basis and provide essential data for further study of the material basis and quality control of BR and VBBR.2.The investigation of components variations and determination of relative components in BR and VBBRAn UHPLC-QTOF-MS coupled with multivariate statistical analysis based plant metabonomics method was developed,and it has been successfully find out the difference between raw and vinegar processed samples.The raw data of BR and VBBR samples were processed with principal component analysis(PCA)and orthogonal partial least squares-discriminant analysis(OPLS-DA).The score plot processed through PCA showed good separation of BR and VBBR,and ten components were screened out from the OPLS-DA S-plot which could be considered as chemical markers to discriminate BR from VBBR.Ten of the chemical markers were identified as SSa,SSd,SSb3,SSe,4"-O-acetyl-SSd,SSc,hyperoside,3’,4’-dimethoxy quercetin,SSb2,SSb1.An HPLC-DAD method for the simultaneous determination of eight relative components in BR and VBBR was developed.The contents of the saikosaponins of type Ⅰ(SSa,SSc,SSd)and type Ⅲ(SSb3,SSb4,SSf)were lower in VBBR,and the contents of saikosaponins of type Ⅱ(SSb1,SSb2)were higher in VBBR.However,total saikosaponin had no obvious changing trend after processing.3.Antidepressant effect and the mechanism study of chronic unpredictable mild stress(CUMS)rat model treated with BR and VBBRThe rats used for antidepressant model construction were evaluated in a rat model of chronic unpredictable mild stress(CUMS).Antidepressant effects of BR and VBBR on CUMS-induced rats were evaluated through behavioral tests,neurotransmitter levels,hormone levels as well as 1H-NMR metabolomics coupled with multivariate data analysis.The results demonstrated that VBBR significantly reversed the depressive behaviors,involving increase in sucrose preference test,decrease in the duration of immobility in the forced swimming test(FST),increase the locomotor activity and exploratory activity in the open-field test(OFT)compared with the CUMS group.And the effect of VBBR was better than BR.In addition,5-HT,5-HIAA,DA DOPAC,TPH and TH levels were measured by ELISA kits,the results showed that 5-HT,5-HIAA,DA and DOPAC levels in CUMS group decreased compared with control group.VBBR produced a significant increase in hippocampus and frontal cortex of 5-HT,5-HIAA,DA and DOPAC levels.The relative components in BR and VBBR showed a trend of recovery compared with CUMS group.Especially,there is an obviously increased 5-HT,5-HIAA and DOPAC in hippocampus and prefrontal cortex in VBBR,indicating that VBBR exerted antidepressant effects against CUMS-induced depression better than BR.Moreover,BR and VBBR produced a significant decrease in plasma ACTH and CORT levels compared with the CUMS group.Metabolomics based on 1H-NMR was used to study the therapeutic effect of VBBR/BR on depression rats.Subsequently,metabolomics analysis was conducted using samples of hippocampus,liver and serum from the rats in control group,CUMS group,BR group,VBBR group and FLU group.OPLS-DA combined with PCA were used to detect potential biomarkers.A total of 22 biomarkers were identified involved in the energy metabolism,amino acid metabolism,glycolysis,TCA cycle and lipid metabolism.The results presented here showed that VBBR significantly reversed the pathological process of CUMS-induced depression,partially via regulation of the disturbed metabolic pathways.The study facilitated better understanding of antidepressant mechanisms of BR and VBBR.And depression can disturb the balance of many of these metabolic pathways in vivo.And a number of variations in biomarkers are not only significant for early diagnosis but also for predicting depression.The levels of succinate,creatine,glycine,glycerol,glutamine and citrate in VBBR group were reduced markedly and returned to normal levels,and the levels of leucine,phenylalanine,taurine and 2-oxo-glutarate increased signficantly compared with the CUMS group.In contrast,not many significant changes were observed in the above biomarkers after treatment with BR.All above showed that VBBR exerted antidepressant effects on CUMS-induced rats better than BR.4.Comparative pharmacokinetic study in CUMS-induced rats after oral administration of extracts of BR and VBBRAn UPLC-MS/MS method was developed for the first time to quantitatively detect eight saikosaponins(SSa,SSb1,SSb2,SSb3,SSb4,SSc,SSd and SSf)in CUMS-induced rat plasma.LC separation was performed on an ACQUITY UPLC BEH C18 column(100 × 2.1 mm,1.7μm)with a flow rate at 0.4 mL/min.The mobile phase contained 0.05%formic acid in water and acetonitrile.The electrospray ionization(ESI)source was operated in negative and the detection of the analytes was in the multiple reaction monitoring mode(MRM).Linear calibration curves of eight saikosaponins were obtained as follows:0.6-300 ng/mL for SSa and SSd,0.2-100 ng/mL for SSb1,SSb2,SSb3,SSb4 and SSf,0.4-200 ng/mL for SSc.The lower limits of quantitation(LLOQ)were 0.2-0.62 ng/mL.RSD was found to be less than 11%for both inter-day and intra-day precision assay.RE ranged from-10.6%to 10.3%for the accuracy assay.The extraction recovery of eight analytes was higher than 74%.Eight saikosaponins shown stable when kept at room temperature for 24 h,during three freeze/thaw cycles,and at-80 ℃ for 1 mouth after samples extracted,respectively.The results indicated that the Tmax in SSa and SSd of VBBR were quickly than that in BR.In addition,the Cmax of SSa,SSb3 and SSd in the CUMS group significantly decreased.While,Cmax of only SSb1 in the CUMS group significantly increased.The AUC0-t of SSa in the CUMS group significantly decreased,while,The AUC0-t of SSb1 and SSb2 in the CUMS group significantly increased.However,there were no significant difference for pharmacokinetics parameters of other saikosaponins.These findings indicated that the structure and polarity of the compound and the pathological state of depression affect the pharmacokinetic behavior of SSs and provide a reference for the clinical rational administration of BR and VBBR. |
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