| ObjectiveTo clarify the improvement effect of RB before and after vinegar-baked on depression model and liver depression type of depression model,and to initially clarify the anti-depressant substance basis and target network,with a view to preliminary comparison and reveal the effective ingredients and mechanism of antidepressant effect before and after vinegar-baked of RB.This study will provide a theoretical and experimental foundation for reasonable utilization of RB and pharmaceutical research and development used for depression.Methods1.Summarized and analyzed the processing methods,chemical components and pharmacological effects of RB through literature mining and herbal research.2.The active ingredients in RB and vinegar-baked RB were extracted by reflux extraction method with70%alkaline ethanol,and eluted with water,35%ethanol and70%ethanol in D101 macroporous resin column.Different PC12 cell models induced by glutamate(GLU)and corticosterone(CORT)were used to screen the three eluted sites for antidepressant effects in vitro.According to the screening results,the effective parts were prepared by liquid phase knockout.The chemical components(groups)of the antidepressant effects of raw and vinegar-baked RB were initially separated and enriched.At the same time,in vitro antidepressant effects were screened and using LC-MS analyzes the active ingredient(group)by mass spectrometry,and initially confirms the structure of the effective ingredient(group).3.Used TCMSP,Pharmmapper and Swiss Target Prediction databases to explore compound-target network,disease-target research was according to OMIM,TTD,CTD and Drug Bank four databases.Cytoscape 3.6.0 was used to construct candidate compound-target networks,and use the molecular interaction database in Bisogenet to obtain protein protein interaction network(PPI network).We used Clue GO to carry out enrichment analysis of candidate targets in the PPI network,including annotation analysis of Biological Process(BP)and Molecular Function(MF);enrichment analysis of KEGG pathway,and the above analysis results were graphically displayed and combined with network pharmacology technology to predict the target of the effective component(group)of the drug effect.4.Rats were used to establish chronic unpredictable mild stress model(CUMS)of depression.The CUMS model was established to excavate the antidepressant effect of RB.The sucrose preference,open field and forced swimming tests were conducted to behavioral evaluation,detect changes in the hypothalamus-pituitary-adrenal axis(HPA axis)related transmitters and indicators,and observe the brain and hippocampal tissue pathological changes were verified by Western blot to predict the expression of relevant target proteins.In the disease model,the anti-depressant effect of the active ingredient(group)of vinegared was verified,and the network of its mechanism of action was preliminary explored.5.On the basis of CUMS model,the method of tail clamping and restraint at the same time every day to establish the rat model of chronic unpredictable mild stress combined with liver depression syndrome.The rat model of chronic unpredictable mild stress combined with liver depression syndrome were conducted to excavate the antidepressant effect of RB.We conducted syndrome integration,sucrose preference,open field and forced swimming tests.The changes of transmitters and indicators related to the HPA axis,while observing the pathological changes of the brain,hippocampus and liver tissues,on the combination of disease and syndrome models,the antidepressant effect of the active ingredients(group)of RB.Results1.Literature research found that vinegar production of RB was an important medicine for relieving liver and relieving depression,but not all depressions are applicable.RB was only suitable for people with depression in the liver.2.After the macroporous resin eluted,the 70%ethanol elution site had protective effects on the two cell models,and the yields(%)of the RB raw product and the vinegar product RB eluted product are 1.70±0.09 and 1.78±0.03,respectively.Separation and enrichment of the preparative liquid phase of 70%ethanol eluate to obtain the saikosaponin(raw SS and vinegar SS,the same below).The yields(%)were 1.42±0.07 and 1.45±0.05;high-performance liquid phase and mass spectrometry analysis of the enriched saponins showed 18 kinds of saikosaponins,and the structure of 16 kinds of saikosaponins were identified,mainly saikosaponins and acetylation Saikosaponin.The content of saponins increased after vinegar production;After screening the drug efficacy of the cell model,the anti-depressant effect of raw SS and vinegar SS at 5,10 and 15 mg·L-1in vitro was significant,and the effect of vinegar SS was superior to raw SS at the same concentration;The antidepressant effect was significant.3.Using the network pharmacology method,the compound-disease-target network,the PPI protein interaction network and the enrichment analysis were established respectively.Anti-depressant components of SS were constructed,and the release of neurotrophic factor(BDNF)and other closely related signal pathways PI3K/AKT and MAPK-ERK were obtained.4.Both raw and vinegar products can increase the sucrose preference,increase their exploration ability and exercise ability of rats with chronic unpredictable mild stress depression at a dose of 0.54 g·kg-1,and significantly improved the depression-like behavior of rats.5.Both raw and vinegar products at 0.54 g·kg-1dose can improve the appearance and behavior of liver depression rats,through improving the syndrome score,increasing the sucrose preference of rats,and their exploration ability and exercise ability significantly improves depression-like behavior in rats;and the effect of vinegar-made RB preparation was better that Raw.6.The antidepressant effect of saikosaponin a(SSa)was clear.SSa can increase the sucrose preference,increase its exploration ability and exercise ability of rats with chronic unpredictable mild stress depression at a dose of 20 mg·kg-1,and significantly improve the depression-like behavior of rats at 10?mol·L-1.Conclusion1.For the material basis research,used the basic ethanol extraction at p H 9-D101macroporous resin elution-preparation liquid phase separation and enrichment-mass spectrometry analysis strategy,the character basis of raw and vinegar-baked RB was established during the characterization process.A relatively comprehensive,rapid and efficient method for the extraction,separation and identification of saikosaponins.The enriched samples are mainly saikosaponins and acetylated saikosaponins.2.The preparation could regulate the HPA axis,reduce hippocampal neuroinflammation and mediate neuroendocrine recovery,activate PI3K/AKT and MAPK-ERK pathways,thereby promoting the release of BDNF and inhibiting the apoptosis of hippocampal neurons.Antidepressant effect.Moreover,the effect after vinegar preparation is better.This experimental result is consistent with the theory of TCM that the medical doctors of the past generations‘RB,the effect of vinegar system on improving liver and depressing depression'.In the meantime,it was the first time discovered for that SSa could activate the PI3K/AKT and MAPK-ERK pathways by adjusting the HPA axis,adjust the cell cycle and inhibit apoptosis,and produce antidepressant effects. |
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