Based On Network Pharmacology And Molecular Docking To Explore The Antidiarrheal Compounds And Mechanisms Of Macleaya Cordata (Willd.) R.Brown.

There are few studies on Macleaya cordata(Willd.)R.Brown.(Macleaya cordata)’s antidiarrheal ingredients and mechanisms,and the toxicity of the antidiarrheal ingredient is also unclear,which limits the clinical application of Macleaya cordata.Therefore,it is of great significance to study the antidiarrheal active ingredients and mechanisms of Macleaya cordata.In this study,on the basis of the antidiarrheal effect of the ethanol extract of Macleaya cordata,the dichloromethane extract was screened as the effective fraction,and the chemical components of this fraction were further identified by UPLC-ESI-MS.We further discussed the potential targets and signaling pathways of Macleaya cordata in the treatment of diarrhea by network pharmacological analysis,and at the same time,molecular docking was used to detect the binding ability of various chemical components to targets to screen out the most predominant component.Finally,from the perspective of inhibition of the calcium ion signaling pathway and the NF-kappa B signaling pathway by active components,we could reveal the mechanism of the antidiarrheal effect of Macleaya cordata.The specific contents of this study are as follows:(1)The results of the castor oil-induced diarrhea model test showed that ethanol extract(50,100,and 200 mg/kg)dose-dependently attenuated the castor oil-induced diarrhea(24.03%,37.50%,and 42.31%)compared to the control.The inhibition of castor oil-induced diarrhea by ethyl acetate and n-butyl alcohol extracts 50,100,and 200 mg/kg was 22.11%,32.69%,36.54% and 10.57%,25.10%,28.85%,respectively.The water residue was not effective at all tested doses.The inhibition rates of dichloromethane extract were 35.57%,46.15% and 67.31% at 50,100 and 200 mg/kg,respectively.(2)UPLC-ESI-MS was used to identify the chemical components of the dichloromethane extraction part of Macleaya cordata.A total of 10 compounds with a content greater than 1% were identified: protopine,allocryptopine,oxysanguinarine,dihydrocoptisine,arnottianamide,6-methoxydihydrosanguinarine,ethoxychelerthrine,6-methoxyldihydrochelerythrine,dihydrochelerythrine and dihydrosanguinarine,respectively.(3)Through the analysis of ten chemical components and diarrhea diseases using network pharmacology,it was found that the core targets are EGFR,ERBB2,and LCK,and the pharmacological pathways are the NF-kappa B signaling pathway and the calcium ion signaling pathway.The binding effect of ten active ingredients and the core target was further evaluated by molecular docking technology.Since dihydrosanguinarine has the best binding effect,it is speculated that it is the predominant chemical ingredient of Macleaya cordata that has an antidiarrheal effect.(4)The castor oil-induced diarrhea and the EGFR-TKI,osimertinib-induced diarrhea models were pretreated with dihydrosanguinarine(25,50,and 100 mg/kg,respectively).The results have shown that dihydrosanguinarine exhibited a significant inhibition in the frequency of wet stools compared to the castor oil control group(36.37%,44.73%,and 66.53%),could decrease the diarrhea incidence after osimertinib treatment,and reduced the water content.The results of q RT-PCR all showed that it upregulated the expression of EGFR and ERBB2,and downregulated the m RNA expression of LCK,CACC,IL-8,IL-1β,and TNF-α.The results of Western blotting all showed that it upregulated the protein expression of P-EGFR and downregulated the expression of P-P65 and CACC.These experimental results demonstrated that in both the castor oil-induced diarrhea model and the osimertinib-induced diarrhea model,dihydrosanguinarine affected the NF-kappa B signaling pathway and the calcium ion signaling pathway by regulating the expression of the key target gene EGFR,thereby inhibiting the occurrence of diarrhea.In conclusion,the ethanol extract of Macleaya cordata has an antidiarrheal effect,and the dichloromethane extraction is the part with the most antidiarrheal effect.Ten chemical components in the dichloromethane extraction fraction were identified by UPLC-ESI-MS analysis.Furthermore,network pharmacology and molecular docking techniques were used to hypothesize that dihydrosanguinarine is the main component that exerts an antidiarrheal effect,and it plays an antidiarrheal role by regulating the expression of the key target gene EGFR and inhibiting the NF-kappa B signaling pathway and calcium ion signaling pathway.This study provides a basis for the clinical application of Macleaya cordata in the treatment of diarrhea-related diseases.