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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.

Chen L, Yu B, Zhang Y, Gao X, Zhu L, Ma T, Yang H - PLoS ONE (2015)

Bottom Line: However, the active ingredients responsible for their therapeutic effectiveness remain unknown.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.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, Liaoning Provincial Key Laboratory of Biotechnology and Drug Discovery, Liaoning Normal University, Dalian, 116029, P. R. China.

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.

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Fractionation of Rhodiola kirilowii (Regel) Maxim.A. HPLC fractionation of Rhodiola kirilowii (Regel) Maxim showing the chromatogram at 220 nm absorbance. Vertical bars show 80 collected fractions. B. Activity-directed fractionation scheme for the isolation CFTR inhibition compounds from Rhodiola kirilowii (Regel) Maxim. C. Analytical HPLC chromatograms of fraction ethyl acetate phase, Fr.7, Fr.7–2, and C2 at 220 nm absorbance. The gradient of analytical HPLC was developed with mobile phase A (acetonitrile) and mobile phase B (0.2% acetic acid) at 0.2 mL/min flow rate (inset)
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pone.0119122.g002: Fractionation of Rhodiola kirilowii (Regel) Maxim.A. HPLC fractionation of Rhodiola kirilowii (Regel) Maxim showing the chromatogram at 220 nm absorbance. Vertical bars show 80 collected fractions. B. Activity-directed fractionation scheme for the isolation CFTR inhibition compounds from Rhodiola kirilowii (Regel) Maxim. C. Analytical HPLC chromatograms of fraction ethyl acetate phase, Fr.7, Fr.7–2, and C2 at 220 nm absorbance. The gradient of analytical HPLC was developed with mobile phase A (acetonitrile) and mobile phase B (0.2% acetic acid) at 0.2 mL/min flow rate (inset)

Mentions: Fig. 2A shows preparative HPLC chromatogram of the Rhodiola kirilowii (Regel) Maxim 95% ethanol extract. The active fractions 21–27 (appeared as a clusters) were eluted at 11–14 min. We next optimized extraction and purification methods for CFTR inhibitor isolation under bioactivity-directed strategy. Rhodiola kirilowii (Regel) Maxim was first extracted with 95% ethanol by using microwave extraction equipment, and then the crude extract was extracted sequentially using a series of solvents including petroleum ether, dichloromethane (DCM), ethyl acetate, and n-butyl alcohol. The plate reader assay indicated that the ethyl acetate fraction was the most effective one, so, the fraction was subjected to further isolation. After two cycles of silica gel chromatography, two active fractions (Fr.7 and Fr.7–2) were obtained, respectively. The active fraction Fr.7–2 was further purified by HPLC to get active fraction C2. Detailed activity-directed fractionation protocol was seen in the Material and Method section and summarized in Fig. 2B. Analytical HPLC chromatograms of ethyl acetate extract, Fr.7, Fr.7–2, and C2 were shown in Fig. 2C.


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.

Chen L, Yu B, Zhang Y, Gao X, Zhu L, Ma T, Yang H - PLoS ONE (2015)

Fractionation of Rhodiola kirilowii (Regel) Maxim.A. HPLC fractionation of Rhodiola kirilowii (Regel) Maxim showing the chromatogram at 220 nm absorbance. Vertical bars show 80 collected fractions. B. Activity-directed fractionation scheme for the isolation CFTR inhibition compounds from Rhodiola kirilowii (Regel) Maxim. C. Analytical HPLC chromatograms of fraction ethyl acetate phase, Fr.7, Fr.7–2, and C2 at 220 nm absorbance. The gradient of analytical HPLC was developed with mobile phase A (acetonitrile) and mobile phase B (0.2% acetic acid) at 0.2 mL/min flow rate (inset)
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4352019&req=5

pone.0119122.g002: Fractionation of Rhodiola kirilowii (Regel) Maxim.A. HPLC fractionation of Rhodiola kirilowii (Regel) Maxim showing the chromatogram at 220 nm absorbance. Vertical bars show 80 collected fractions. B. Activity-directed fractionation scheme for the isolation CFTR inhibition compounds from Rhodiola kirilowii (Regel) Maxim. C. Analytical HPLC chromatograms of fraction ethyl acetate phase, Fr.7, Fr.7–2, and C2 at 220 nm absorbance. The gradient of analytical HPLC was developed with mobile phase A (acetonitrile) and mobile phase B (0.2% acetic acid) at 0.2 mL/min flow rate (inset)
Mentions: Fig. 2A shows preparative HPLC chromatogram of the Rhodiola kirilowii (Regel) Maxim 95% ethanol extract. The active fractions 21–27 (appeared as a clusters) were eluted at 11–14 min. We next optimized extraction and purification methods for CFTR inhibitor isolation under bioactivity-directed strategy. Rhodiola kirilowii (Regel) Maxim was first extracted with 95% ethanol by using microwave extraction equipment, and then the crude extract was extracted sequentially using a series of solvents including petroleum ether, dichloromethane (DCM), ethyl acetate, and n-butyl alcohol. The plate reader assay indicated that the ethyl acetate fraction was the most effective one, so, the fraction was subjected to further isolation. After two cycles of silica gel chromatography, two active fractions (Fr.7 and Fr.7–2) were obtained, respectively. The active fraction Fr.7–2 was further purified by HPLC to get active fraction C2. Detailed activity-directed fractionation protocol was seen in the Material and Method section and summarized in Fig. 2B. Analytical HPLC chromatograms of ethyl acetate extract, Fr.7, Fr.7–2, and C2 were shown in Fig. 2C.

Bottom Line: However, the active ingredients responsible for their therapeutic effectiveness remain unknown.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.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, Liaoning Provincial Key Laboratory of Biotechnology and Drug Discovery, Liaoning Normal University, Dalian, 116029, P. R. China.

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.

Show MeSH
Related in: MedlinePlus