HPLC chromatograms of the EEBP and the fraction containing the pungent ingredient.

<p>(A) The following major compounds were identified in the ethanol extract of Brazilian green propolis (EEBP) : <i>p</i>-coumaric acid, drupanin, artepillin C, and baccharin. (B) One peak was found at the same retention time as artepillin C in the fraction with the most pungent taste.</p

Effects of artepillin C, baccharin, drupanin, and capsaicin on the responses of hTRPV1-expressing cells.

<p>The cellular responses to the test compounds were examined using a plate reader-based assay in the presence or absence of 30 µM capsazepine in hTRPV1-expressing (A) or mock-transfected (B) HEK293T cells. Each column represents the means ± S.E.M. (n = 3). ** indicates <i>p</i><0.01 for each substance (comparison between the absence and presence of capsazepine) (Dunnett’s multiple comparison test; the equality of the variances was tested using the Bartlett’s test).</p

Effects of artepillin C, baccharin, drupanin, and capsaicin on the responses of hTRPV1-expressing cells.

<p>(A–C) Representative ratiometric images of Fura 2-loaded hTRPV1-expressing (A and B) and mock-transfected (C) HEK293T cells. The upper and lower columns indicate the images obtained before and after stimulation, respectively. The applied concentrations are indicated in the upper part of the panels. The responses were recorded in the absence (A and C) or presence (B) of 30 µM capsazepine, a TRPV1-specific inhibitor. (D) The responses to the test solutions were analyzed in 100 randomly selected cells. The values represent the means ± S.E.M. (n = 4). * or ** indicates <i>p</i><0.05 or 0.01 vs. buffer. (E) Sequential F340/F380 ratiometric values were measured for 40 random selected cells. The red line indicates that the value changed by more than 0.3.</p

Chemical structures of <i>p</i>-coumaric acid, drupanin, artepillin C, baccharin, allyl isothiocyanate (AITC), and capsaicin.

<p>Chemical structures of <i>p</i>-coumaric acid, drupanin, artepillin C, baccharin, allyl isothiocyanate (AITC), and capsaicin.</p

Pungency threshold of artepillin C by organoleptic examination.

<p>Pungency threshold of artepillin C by organoleptic examination.</p

Effects of artepillin C, baccharin, drupanin, and AITC on the responses of hTRPA1-expressing cells.

<p>The cellular responses to the test compounds were examined using a plate reader-based assay in the presence or absence of 50 µM HC-030031, a TRPA1-specific inhibitor, in hTPRA1-expressing cells (A) or Flp-in 293 cells (B). Each column represents the means ± S.E.M. (n = 4). ** indicates <i>p</i><0.01 for each substance (comparison between the absence and presence of HC-030031) (Tukey’s multiple comparison test; the equality of variances was tested using the Levene test). (C) Dose-response curves for hTRPA1 activation. The values represent the means ± S.E.M. (n = 3).</p

The responses of human TRPA1-expressing cells to the application of the compounds in EEBP.

<p>(A–C) The compounds identified in the HPLC chromatogram were applied to hTRPA1-expressing (A and B) and Flp-In 293 cells (C). Representative ratiometric images of the cells obtained by the Ca²⁺ imaging analysis are shown in each panel. The upper and lower columns indicate the images obtained at approximately 2 and 22 sec after the stimulation, respectively. The applied concentrations are indicated in the upper part of the panels. The responses were recorded in the absence (A and C) or presence (B) of 50 µM HC-030031, a TRPA1-specific inhibitor. (D) The responses to the test solutions were analyzed in 100 randomly selected cells. The values represent the means ± S.E.M. (n = 4). ** indicates <i>p</i><0.01 vs. buffer. (E) Sequential F340/F380 ratiometric values were measured for 20 randomly selected cells. The red line indicates that the value changed by more than 0.3.</p

Roles of Y103 and P277 for the reception of the sweeteners.

<p>Dose-dependent responses of cells expressing Y103 mutants (Y103A and Y103F) to sucralose and P277 mutants (P277A, P277G, P277Q, and P277S) to aspartame are shown in A and B, respectively. Each point indicates the mean ± S.E.M. from at least 3 independent experiments.</p

Model of the aspartame-bound hT1R2 ATD.

<p>(A) Complex model of aspartame in the closed form of hT1R2 ATD. (B) The model oriented 90° from (A). (C) Aspartame-binding pocket in detail (orange circle in (A)).</p

Complex model of the sucralose-bound hT1R2 ATD.

<p>(A) Complex model of sucralose in the closed form of hT1R2 ATD. Chlorine atoms are colored light green. (B) The model oriented 90° from (A). (C) Sucralose-binding pocket in detail (orange circle in (A)).</p