Bioactivity-guided Fractionation Of An Herbal Antidiarrheal Rhodiola Kirilowwi Reveals EGCG And ECG As Inhibitors Of Cystic Fibrosis Transmembrane Conductance Regulator

Maintenance of an appropriate amount of intestinal fluid is vital for digestion andclearance of the luminal contents. The process is a passive process driven by the active anion,predominantly Clˉ, transport from blood to the intestinal lumen. CFTR belongs to thesuperfamily of ATP-binding cassette (ABC) proteins, it is the predominant pathway for Clˉtransport in active fluid secretion. Cystic fibrosis transmembrane conductance regulator(CFTR) is the principal apical route for transepithelial fluid transport induced by enterotoxin.Inhibition of CFTR has therefore been proposed as a potential pharmaceutical approach forthe treatment of secretory diarrhea. Many traditional Chinese herbal medicines, like Rhodiolakirilowii (Regel) Maxim, have long been used for the treatment of secretory diarrhea.However, molecular mechanisms responsible for their therapeutic effectiveness are remainedlargely under investigated. Systematic investigation on the pharmacology of activeingredients and mechanisms are crucial for transforming traditional herbal practices intoevidence-based medicine. The purpose of this study is to identify CFTR inhibitors fromRhodiola kirilowii (Regel) Maxim under bioactivity-directed isolation strategy.We identified fractions of Rhodiola kirilowii (Regel) Maxim that inhibited CFTR Cl-channel activity. Bioactivity-directed fractionation of the active fractions led to theidentification of (-)–epigallocatechin-3-gallate (EGCG) as CFTR Clˉchannel inhibitor.Further studies of5commercial available analogs, including (+)–catechins (C),(-)–epicatechin (EC),(-)–epigallocatechin (EGC),(-)–epicatechin-3-gallate (ECG) and (-)–epigallocatechin-3-gallate (EGCG) revealed that ECG also had CFTR Clˉchannelinhibition activity. EGCG reversibly inhibited CFTR Clˉchannel activities in transfectedFRT cells with IC50value around100mM and~100%inhibition at higher concentrations. Inex vivo studies, EGCG and ECG inhibited short-circuit current in isolated rat colonic mucosa.In a closed-loop mice model, intraluminal EGCGand ECG significantly reduced choleratoxin-induced intestinal fluid secretion in mice. CFTR Clˉchannel inhibition adds to the listof EGCG and ECG molecular targets and may account for some of their biological activities;EGCG and ECG could be new candidates for blockers of CFTR-mediated intestinal fluidsecretion.