Bioactivity-guided fractionation of an antidiarrheal Chinese herb Rhodiola kirilowii (Regel) Maxim reveals (-)-epicatechin-3-gallate and (-)-epigallocatechin-3-gallate as inhibitors of cystic fibrosis transmembrane conductance regulator.

Lei Chen,Liang Zhu,Xin Gao,Bo Yu,Hong Yang,Yaofang Zhang,Tonghui Ma,Ranji Cui

doi:10.1371/journal.pone.0119122

Abstract

Cystic fibrosis transmembrane conductance regulator (CFTR) is the principal apical route for transepithelial fluid transport induced by enterotoxin. Inhibition of CFTR has been confirmed as a pharmaceutical approach for the treatment of secretory diarrhea. Many traditional Chinese herbal medicines, like Rhodiola kirilowii (Regel) Maxim, have long been used for the treatment of secretory diarrhea. However, the active ingredients responsible for their therapeutic effectiveness remain unknown. The purpose of this study is to identify CFTR inhibitors from Rhodiola kirilowii (Regel) Maxim via bioactivity-directed isolation strategy. We first identified fractions of Rhodiola kirilowii (Regel) Maxim that inhibited CFTR Cl- channel activity. Further bioactivity-directed fractionation led to the identification of (-)–epigallocatechin-3-gallate (EGCG) as CFTR Cl- channel inhibitor. Analysis of 5 commercially available EGCG analogs including (+)–catechins (C), (-)–epicatechin (EC), (-)–epigallocatechin (EGC), (-)–epicatechin-3-gallate (ECG) and EGCG revealed that ECG also had CFTR inhibitory activity. EGCG dose-dependently and reversibly inhibited CFTR Cl- channel activity in transfected FRT cells with an IC50 value around 100 μM. In ex vivo studies, EGCG and ECG inhibited CFTR-mediated short-circuit currents in isolated rat colonic mucosa in a dose-dependent manner. In an intestinal closed-loop model in mice, intraluminal application of EGCG (10 μg) and ECG (10 μg) significantly reduced cholera toxin-induced intestinal fluid secretion. CFTR Cl- channel is a molecular target of natural compounds EGCG and ECG. CFTR inhibition may account, at least in part, for the antidiarrheal activity of Rhodiola kirilowii (Regel) Maxim. EGCG and ECG could be new lead compounds for development of CFTR-related diseases such as secretory diarrhea.

Highlights

Maintenance of an appropriate amount of intestinal fluid is vital for digestion and clearance of the luminal contents
Rhodiola kirilowii (Regel) Maxim was extracted using 95% ethanol on Soxhlet reflux apparatus, and the extract was fractionated into 80 fractions by preparative HPLC with a linear gradient of 0–90% methanol (MeOH)
Rhodiola kirilowii (Regel) Maxim was first extracted with 95% ethanol by using microwave extraction equipment, and the crude extract was extracted sequentially using a series of solvents including petroleum ether, dichloromethane (DCM), ethyl acetate, and n-butyl alcohol

Summary

Introduction

Maintenance of an appropriate amount of intestinal fluid is vital for digestion and clearance of the luminal contents. It is a passive process driven by the active anion, predominantly Cl-, transport from blood to the intestinal lumen [1, 2]. The major components in fluid secretion involve Cl- intake via Na+/K+/2Cl- cotransporter (NKCC1) through the basolateral membrane and Cl- exit to the lumen via cystic fibrosis transmembrane conductance regulator (CFTR) and Ca2+-activated Cl- channels (CaCCs) in apical membrane of secretory epithelial cells [1, 3, 4]. Activity of CFTR is regulated by binding and hydrolysis of ATP at NBDs and by phosphorylation of the R region [5, 6]. CFTR is a well-validated target for development of inhibitors for therapy of secretory diarrheas [10,11,12]

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