Study On The Material Basis And Mechanism Of The Anti-diabetic Effect Of Xiaokeyinshui Extracts Combination Based On In Vivo And In Vitro Components Combining With Multi-omics Analyses

Background and ObjectiveXiaokeyinshui formula,recorded in Bencaogangmu(Compendium of Materia Medica)and Zhengleibencao(Collected Classified Materia Medica),is a traditional prescription for the treatment of Xiao Ke Zheng which can be regarded as T2DM.In our previous study,a new formula,named Xiaokeyinshui extracts combination(XEC),has been developed from the original Xiaokeyinshui formula to ameliorate its side effects.Meanwhile,our research group have studied the preparation of each components extract,optimization of components extract compatibility,quality control and quality standard,anti-diabetic and renal protective effects,and the relating mechanisms of XEC.Futhermore,the effectiveness and safety of XEC as a candidate drug was preliminarily clarified.On the basis of the above findings,present study used multidisciplinary methods,such as analytical chemistry,molecular docking,metabolomics,lipidomics,and gut microbiota,to further annote the material basis and mechanism of the anti-diabetic effect of XEC from the perspective of“multi-component,multi-target,multi-pathway”.MethodsQualitative analysis of components in Coptidis Rhizoma extract(CRE)and Cassiae Semen extract(CSE)by UPLC-QE-Orbitrap MS/MS was established.And the same UPLC-MS/MS analysis method was also employed to discover the major prototype compounds and their metabolites in diabetic rat plasma,liver,kidney,colon and colon contents after taken XEC for four weeks,aiming to identify the metabolites and propose metabolic pathways and tissue distribution.A combination of metabolomics and lipidomics profiling analysis,together with biochemical analysis,were performed to elucidate the effect and molecular mechanisms of the hypoglycemic and lipid lowering after treatment with XEC in HSHF diet-fed/STZ injected KM mice.Futher,molecular docking between the active compounds of XEC and the key metabolic pathway targets AR,FASN,PLA2,COX1 and COX2 from metabolomics and lipicomics research was slso conducted.After diabetic rats were taken different dose of XEC for four weeks,a 16S r DNA gene sequencing technology was employed to detect alterations of intestinal flora,and high-resolution untargeted metabolomics analysis of colon contents was utilized to identity differential intestinal metabolites.Finally,colon contents of SCFA were absolutely quantified using GC-MS.ResultsThrough reference substances,cracking of mass spectrum peak rule analysis and reference the related literature,total 55 compounds were identified from CRE and CSE,including 14 alkaloid components,14 anthraquinone components and 27naphthopyrone components.72 prototype constituents and metabolites were detected in biological samples of diabetic rats after oral administration of XEC.Major metabolic pathways of alkaloids in CRE were hydroxylation,dehydrogenation,reduction,demethylenation,demethylation,glucuronidation and methylation.Anthraquinone and naphthopyrone components mostly exist in the form of glycosides,firstly hydrolyzed to generate aglycones,and then further transformed.These components were low in plasma and abundant in colon and its contents.After treatment with different dose of XEC for 5 weeks,the levels of insulin and HDL-C were markedly increased,levels of FBG,Hb A1c,TC,TG,LDL-C were decreased,and islet structure was repaired compared to DC group.The metabolic profiles of plasma were analyzed and 54 potential biomarkers were screened out,mainly including carbohydrates,lipids and amino acids.These potential biomarkers were found to be correlated with the following pathways:galactose metabolism,fructose and mannose metabolism,TCA cycle,AA metabolism,sphingolipid metabolism,amino acid metabolism,glycerolipid metabolism and glycerophospholipid metabolism.The molecular docking results showed that most compounds(alkaloids in Coptidis Rhizoma,triterpenoids in bitter melon,anthraquinones and naphthopyrones in Cassiae Semen)have good docking activity with AR,FASN and PLA2 proteins.Except for some alkaloids,compouds from Cassiae Semen displayed better docking scores with COX1 and COX2.Apart from improved biochemical indicators,gut microbiota structure of diabetic rats were improved by XEC,mainly via augmenting the relative abundance of Firmicutes and the ratio of Firmicutes/Bacteroidetes,reducing the relative abundance of Bacteroidetes and Proteobacteria.The SCFAs-producing bacteria such as Lachnospiraceae NK4A136 group,[Eubacterium]xylanophilum group,Roseburia,Ruminococcaceae UCG-010,Ruminococcaceae UCG-013,Ruminococcaceae UCG-014,Ruminococcus 1,Allobaculum in T2DM rats were notably enriched after treatment with high dose of XEC.In addition,fecal SCFA levels were elevated in XEC-L and XEC-H group,and the contents of acetic acid,butyric acid and propionic acid were significantly rised in XEC-H group.At the same time,beneficial bacterias such as Christensenellaceae R-7 group,Romboutsia,[Eubacterium]coprostanoligenes group were significantly increased,and harmful bacteria Rikenellaceae RC9 gut group and Bilophila were significantly reduced.ConclusionsThis research comprehensively clarifies the material basis and mechanism of the anti-diabetic effect of XEC from the perspective of compositional change in vivo and in vitro,regulation of metabolism and multi-channel,regulaton of intestinal flora and its metabolism,and provides a basis for the drugabillity of XEC.Futher more,it embodies the therapeutic characteristics of"multi-components,multi-targets,multi-pathways",and also could be taken as a beneficial reference for other TCM formula research.