Activity-directed isolation of active compounds from Chinese wild grapevine.

<p>A. Preparative HPLC chromatogram of 80 fractions from the ethanol extract of Chinese wild grapevine. Compound absorbance (220 nm) was continuously recorded using a Photodiode Array (PDA) Detector. B. Analytical HPLC chromatogram of fraction 51 in positive cluster 1 (left) and fraction 55 in positive cluster 2 (right). C. Activity-directed fractionation scheme for the isolation of CFTR inhibitory single compounds from Chinese wild grapevine.</p

Structural determination of purified CFTR inhibitors.

<p>A. Analytical HPLC of compound C2 (left) and C3 (right). B. Mass spectra of purified compound C2 (upper) and C3 (down). C. Chemical structures of C2 (trans-ε-viniferin, left) and C3 (r-2-viniferin, right).</p

Construction of natural compounds fraction library from Chinese medicinal herbs.

<p>A. Ethanol extracts prepared from 500 Chinese herbs. B. Natural compounds fraction library constructed by preparative HPLC with a linear gradient of 0–90% methanol. Eighty fractions were collected from the ethanol extract of each herb, which line up with hydrophobicity gradient. C. Determination of a randomly selected fraction by analytical HPLC. D. Working library. Each fraction was dissolved in DMSO to make 5 mg/mL working solution. Each 96-well plate contains 80 fractions from one herb.</p

Inhibition of CFTR activity by fractions from Chinese wild grapevine.

<p>A. A representative plate with positive fractions in screening by cell-based fluorescent assay. Data from the 96-well plate of Chinese wild grapevine showing 14-s time courses (I⁻ added at 2 s) of EYFP-H148Q fluorescence. Controls (C: saline; N: cocktail; P: cocktail plus 20 μM CFTR_inh-172) are shown on the right. Asterisks indicate positive wells with decreased I⁻ influx (representing CFTR inhibition). B. Percentage inhibition rate of the positive fractions indicating Gaussian distributions of the positive clusters. C. Activity of positive fractions. Dose-response results showing inhibition of cocktail-stimulated (5 μM forskolin, 100 μM IBMX and 25 μM genistein) I⁻ influx by fraction 51 (left) and fraction 55 (right) at indicated concentrations. Mean ± SE is shown from three sets of experiments.</p

Inhibition of intestinal fluid secretion by TV and RV.

<p>A. Inhibition of CFTR-mediated short-circuit Cl⁻ currents by TV and RV in isolated rat colonic mucosa. TV and RV were added to serosal and then mucosal surfaces at indicated time after stimulation by forskolin (20 μM). Indomethacin (10 μM) and amiloride (10 μM) were present in the solution to inhibit Na⁺ current and prostaglandin generation. One experiment typical of four or five is shown. B. Photograph of isolated mouse ileal loops at 6 hours after luminal injection of saline, 0.5 μg cholera toxin, 0.5 μg cholera toxin plus 1 μg CFTR_inh-172, 0.5 μg cholera toxin plus 2.5 μg TV or 0.5 μg cholera toxin plus 4.5 μg RV. C. Summarized data of weight/length ratio (g/cm). Mean ± SE; four mice per group; *<i>P</i><0.001).</p

Inhibition of intestinal fluid secretion by EGCG and ECG.

<p><b>A.</b> Photograph of isolated mouse ileal loops at 6 hours after lumenal injection of saline, 0.5 μg cholera toxin, 0.5 μg cholera toxin plus 10 μg EGCG (or 10 μg ECG, or 2 μg CFTR_inh-172). (B) Ratio of loop weight/length (g/cm) at 6 hours before versus after luminal fluid removal (SE; six mice per group;*<i>p</i> < 0.001).</p

Structure-activity analysis of catechin analogs.

<p>IC₅₀ was determined from concentration-inhibition data from cell-based fluorescence measurements.</p

Inhibition of CFTR Cl^¯ channels by fractions from <i>Rhodiola kirilowii</i> (Regel) Maxim.

<p>A. Principle of cell-based fluorescence assay. FRT cells stably co-transfected with human CFTR and YFP-H148Q were prestimulated with a cocktail (5 μM FSK, 100 μM IBMX and 25 μM GEN) for 10 min before addition of a test sample to final concentration of 100 μg/ml. CFTR-mediated I^¯ influx was measured from the kinetics of decreasing YFP-H148Q fluorescence in response to addition of I^¯ solution. B. Time-course fluorescence data from a 96-well microplate of CFTR-expressing FRT cells. Controls (C: PBS; N: cocktail; P: cocktail plus 20 μM CFTR_inh-172) are shown on the right. Asterisk show wells with decreased I^¯ influx (representing CFTR inhibition). C. % inhibition rate of the active wells indicated Gaussian distributions of the active sites.</p

Identification of purified active fraction as EGCG.

<p>Mass spectra of purified fraction C2. Inset: Structure of EGCG.</p

CFTR inhibition by EGCG and ECG.

<p><b>A.</b> Dose—response relationship of EGCG and ECG determined in the iodide influx assay. Data were expressed as mean±SE, n = 3). B. EGCG and ECG inhibition short-circuit current after amiloride and indomethacin addition and stimulation by FSK (20 μM) in isolated rat colonic mucosa. EGCG and ECG were added to mucosal surfaces as indicated. One experiment typical of four or five is shown.</p