Study On The Formula Optimization Of Xiaokeyinshui Extract Combination,and Its Effects And Mechanisms For The Treatment Of Type 2 Diabetes Mellitus

Background:Diabetes mellitus(DM)is a severe disease,causing great harm to the health of people.In China,about 116.4 million people suffered from DM in 2019,among which,65.2 million people remained undiagnosed,according to a survey conducted by International Diabetes Federation.DM has become a major concern of public health in China.DM is categorized as“Xiaoke”symptom in traditional Chinese medicine(TCM),resulting from several factors,including physical or mental factors,excessive intake of high fat diet,lacking exercises,etc.“Xiaokeyinshui”is an ancient TCM formula treating“Xiaoke”,and could be traced to Tang dynasty,in Haishang Collection of Effective Formula.It was also recorded in famous ancient medical books from different dynasty,for example,Zhengleibencao(Collected Classified Materia Medica)and Bencaogangmu(Compendium of Materia Medica).The focus of the current study,Xiaokeyinshui extract combination(XEC),originated from this formula,consisting extracts of four herbal drugs,namely,Coptidis Rhizoma,Liriopes Radix,bitter melon,and Cassiae Semen.Aim:The aim of the current study was to form well quality-controlled herb extracts with clearly verified active components,optimize the formula of XEC,assess the anti-diabetic and renoprotective effects,explore the mechanisms,and evaluate the safety of XEC.The above-mentioned aim may lay the foundation for the assessment of the druggability of XEC.Methods:According to both traditional medical experience and bioactive-guided fractionation strategy,extracts of four herbal drugs were prepared,namely,Coptidis Rhizoma extract(CRE),Liriopes Radix polysaccharides(LRP),bitter melon extract(BME),and Cassiae Semen extract(CSE).Quality control methods,both quantitative and qualitative,were established mainly with high performance liquid chromatography on different batches of XEC.Hereby,standards of herb extracts were established.Main components determined in quality control were then subjected to in silico screening and subsequently,network pharmacology analysis,using various online bioinformatic resources.Uniform experimental design was applied in the optimization of XEC formula,in diabetic mice model induced with high-sucrose-high-fat(HSHF)diet and injection of streptozotocin.Anti-diabetic and renal protective effects were assessed in diabetic rats,with exploration of mechanisms as well.Maximum toleration dose(MTD)method was applied to assess the acute toxicity of XEC.Results:Ten batches of samples were prepared with same methods.Research showed that preparation of herb extracts was consistent and stable.Standards of quality control were set as followed.(1)CRE,the content of epiberberine,coptisine,palmatine and berberine should be not less than 3%10%,5%,28%,respectively.In addition,the total content of four alkaloids should be not less than 50%.(2)LRP,the range of average molecular weight was 3000-5000 Da.Total carbohydrate content should be not less than 90%.The ratio of fructose and glucose should be 17-22:1.(3)BME,the content of momordicoside L,7?,25-dihydrocucurbita-5,23(E)-dien-19-al-3-O-?-D-allopyranoside,momordicoside F₂should be not less than 0.1%,0.05%,0.1%,respectively.The total content of three compounds should be not less than 0.3%.(4)CSE,the content of cassiaside,rubrofusarin-6-O-?-D-gentiobioside,glucoaurantio-obtusin,cassiaside C and aurantio-obtusin should be not less than 3%,2%,1.5%,1%and 1%,respectively.The total content of five compounds should be not less than 10%.According to network pharmacology analysis,76 targets were found and could be defined as the key targets regulated by XEC in the treatment of T2DM as well as its complications.The key nodes of the targets are protein kinase B(Akt),phosphatidylinositol 3-kinas(PI3K),insulin receptor substrate(IRS),tumor necrosis factor(TNF),etc.XEC could regulate the glucose and lipid metabolism,and alleviate insulin resistance mainly though insulin,PI3K/Akt,adenosine 5'-monophosphate activated protein kinase(AMPK),adipocytokine and forkhead box protein O(Fox O)pathways.In addition,XEC could also be used in the treatment of diabetic complications due to its effect on PI3K/Akt,TNF,hypoxia-inducible factor 1(HIF-1)and vascular endothelial growth factor(VEGF)pathways.The optimized formula of XEC was determined as followed,CRE:LRP:BME:CSE 1.81:4.63:2.63:0.93,with a combined consideration on experimental results,TCM theory,and regression-fitting analysis.Experimental results showed that XEC could ameliorate both glucose and lipid metabolisms,and exerts renopretective effects.XEC could also reduce inflammatory cytokines and oxidative stress in serum.XEC achieved anti-diabetic effects through up-regulation of Ins R?/IRS-1/PI3K/Akt/GLUT4 signaling pathway and phosphorylation of AMPK.In addition,renal protective effects were also achieved with down-regulation of expression of both receptor for advanced glycation end products(RAGE)and VEGF in kidney.Results showed that the MTD of XEC in KM mice was 25200 mg/kg,approximately 434-fold to the daily dose of human.Conclusion:According to TCM theory,XEC could be applied in the treatment of“heat within lung and stomach”,“fire within bowel and stomach”,in other words,early and middle stage of T2DM combined with lipid dysfunctions.In this research,stable preparation technique of herb extracts and quality control methods for extracts had been established.An optimized formula of XEC was determined.Both network analysis and in vivo experimental results demonstrated that XEC exerts promising anti-diabetic and renal protective effects in multi-components-multi-target mechanisms,without obvious side effects.In brief,this research provided basis for further development of new TCM drugs.