Gut microbiota-based metabolites of Xiaoyao Pills (a typical Traditional Chinese medicine) ameliorate depression by inhibiting fatty acid amide hydrolase levels in brain.ETHNOPHARMACOLOGICAL RELEVANCE
 Traditional Chinese medicines (TCMs) are often prepared in oral dosage forms, making TCMs interact with gut microbiota after oral administration, which could affect the therapeutic effect of TCM.  Xiaoyao Pills (XYPs) are a commonly used TCM in China to treat depression.  The biological underpinnings, however, are still in its infancy due to its complex chemical composition.


 The study aims to explore XYPs' underlying antidepressant mechanism from both in vivo and in vitro.


 XYPs were composed of 8 herbs, including the root of Bupleurum chinense DC. , the root of Angelica sinensis (Oliv.) Diels, the root of Paeonia lactiflora Pall. , the sclerotia of Poria cocos (Schw.) Wolf, the rhizome of Glycyrrhiza uralensis Fisch. , the leaves of Mentha haplocalyx Briq. , the rhizome of Atractylis lanceavar.  chinensis (Bunge) Kitam. , and the rhizome of Zingiber officinale Roscoe, in a ratio of 5:5:5:5:4:1:5:5.  The chronic unpredictable mild stress (CUMS) rat models were established.  After that, the sucrose preference test (SPT) was carried out to evaluate if the rats were depressed.  After 28 days of treatment, the forced swimming test and SPT were carried out to evaluate the antidepressant efficacy of XYPs.  The feces, brain and plasma were taken out for 16SrRNA gene sequencing analysis, untargeted metabolomics and gut microbiota transformation analysis.


 The results revealed multiple pathways affected by XYPs.  Among them, the hydrolysis of fatty acids amide in brain decreased most significant via XYPs treatment.  Moreover, the XYPs' metabolites which mainly derived from gut microbiota (benzoic acid, liquiritigenin, glycyrrhetinic acid and saikogenin D were found in plasma and brain of CUMS rats and could inhibit the levels of FAAH in brain, which contributed to XYPs' antidepressant effect.


 CONCLUSIONS
 The potential antidepressant mechanism of XYPs by untargeted metabolomics combined with gut microbiota-transformation analysis was revealed, which further support the theory of gut-brain axis and provide valuable evidence of the drug discovery.
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