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FIGURE 1. (A) The chemical structures of pungent and tingling compounds 1–9c in black pepper and its pungentp and tinglingt threshold concentrations, as reported by Dawid et al. (2012): piperine (1a) [3.0 nmol/cm2p], piperyline (1b) [5.1 nmol/cm2p], piperlonguminine (1c) [10.4 nmol/cm2p], piperettine (2a) [5.2 nmol/cm2p], dehydropipernonaline (3a) [152.1 nmol/cm2p], 1-[1-oxo-9(3,4-methylenedioxyphenyl)-2E,4E,8E-nonatrienyl]-pyrrolidine (3b), brachyamide A (4b), guineensine (4c) [810.1 nmol/cm2p], 1-(octadeca-2E,4E,13Z-trienyl)piperidine (5a), 1-(octadeca-2E,4E,13Z-trienyl)pyrrolidine (5b), (2E,4E,13Z)-N-isobutyl-octadeca-2,4,13-trienamide (5c) [540.5 nmol/cm2p; 2162.1 nmol/cm2t], 1-(octadeca-2E,4E,12Z-trienyl)piperidine (6a), 1-(octadeca-2E,4E,12Z-trienyl)pyrrolidine (6b), (2E,4E,12Z)-N-isobutyl-octadeca-2,4,12-trienamide (6c) [540.5 nmol/cm2p; 2162.1 nmol/cm2t], (2E,4E)-N-isobutyl-octadeca-2,4-dienamide (7c) [763.0 nmol/cm2p], (2E,4E,15Z)-N-isobutyl-eicosa-2,4,15-trienamide (8c) [741.2 nmol/cm2p; 1482.3 nmol/cm2t], (2E,4E,14Z)-N-isobutyl-eicosa-2,4,14-trienamide (9c) [741.2 nmol/cm2p; 1482.3 nmol/cm2t]. The compounds 5c/6c and 8c/9c were tasted as binary mixtures. (B) A RP-HPLC chromatogram (λ = 260 nm) of SPE fraction A (A), SPE fraction B (B) SPE fraction C (C), and SPE fraction D (D) showing the pepper amides 1a–9c.
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FIGURE 2. A dose-response relationship for the effect of piperine (1a) on Xenopus oocytes expressing the mouse orthologues for KCNK3, KCNK9, and KCNK18. N = 3–5 oocytes.
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FIGURE 3. (A) Pie chart indicating the percentage of piperine- (100 μM) and/or capsaicin- (10 μM) responsive trigeminal neurons. N = 619 neurons from 23 separate experiments. (B) Pie chart indicating the percentages of piperine-unresponsive neurons (white slice), and piperine-responsive neurons (gray and green slices). The neurons that responded to a first application of piperine (100 μM) are subdivided into those in which a second response to piperine (100 μM) was prevented by preincubation and simultaneous co-application with an antagonist-mix consisting on 20 μM HC-030031 and 5 μM capsazepine (gray slice) and those in which the antagonist-mix failed to prevent a calcium response (green slice). N = 332 neurons from 13 separate experiments. (C) Representative Ca2+ imaging traces of two mouse trigeminal neurons subjected to the application of piperine 100 μM; capsazepine 5 μM and HC-030031 20 μM; piperine 100 μM, capsazepine 5 μM and HC-030031 20 μM; and KCl 150 mM. (D) Histogram of the distribution of mouse trigeminal neurons in their function to cell diameter and their responsiveness to capsaicin and piperine, N = 179 neurons from nine separate experiments.
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FIGURE 4. (A) Representative current-voltage curves from cultured mouse trigeminal neurons subjected to a voltage ramp from –100 to 50 mV. The traces compare the change in background current after the application of 100 μM piperine under physiological extracellular solution. (B)
Left: A representative current-voltage curve of a cultivated mouse trigeminal neuron under Na+/Ca2+-free solution before application (black trace), after the application of 100 μM piperine (dark green trace), the KCNQ selective blocker XE-991 (100 μM, gray trace) and subsequent co-application of 100 μM XE-991 and 100 μM piperine (light green trace). Right: The comparative effect of piperine (100 μM) alone, co-applied with XE-991 (100 μM) and co-applied with XE-991 under acidic pH. N = 11 neurons, mean values are indicated by an open rectangle, box for a range between 25 and 75 percentiles with a line for the median, whiskers for a range between the 5 and 95 percentiles, maximum and minimum value data are indicated with an x. (C) A representative current-clamp recording from a cultivated mouse trigeminal neuron exposed to piperine (100 μM), piperine co-applied with XE-991 and piperine co-applied with XE-991 under acidic pH.
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FIGURE 5. Normalized currents showing the effect of 12 sensory active compounds isolated from black peppercorns (see Figure 1) at 1 mM on Xenopus laevis oocytes expressing hKCNK3 (A), hKCNK 9 (B), and hKCNK18 (C). N = 3–7 oocytes. The currents were normalized to the current registered prior to the application of each substance (dotted black line, see materials and methods section for further details). If a substance was able to induce a change greater than 20% of the basal activity (as indicated by upper and lower dotted lines), the effect was considered relevant. For all graphics, the reported effect of 1 mM piperine is provided as a reference (gray bar).
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FIGURE 6. (A) Representative voltage-clamp recordings from Xenopus oocytes expressing hKCNK3, before, during and after the application of 1 mM of 1-[1-oxo-9(3,4-methylenedioxyphenyl)-2E,4E,8E-nonatrienyl]-pyrrolidine (3b) (top); brachyamide A (4b) (middle) and 1-(octadeca-2E,4E,13/12Z-trienoyl)pyrrolidine (5b/6b) (bottom). The dotted line represents zero current. (B) Dose-response curves for the effect of 1-[1-oxo-9(3,4-methylenedioxyphenyl)-2E,4E,8E-nonatrienyl]-pyrrolidine (3b) (black squares); brachyamide A (4b) (red circles) and 1-(octadeca-2E,4E,13/12Z-trienoyl)-pyrrolidine (5a/6a) (blue triangles) on hKCNK3. See Table 1 for IC50 values. N = 3–5 oocytes.
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FIGURE 7. The dose-response relationship for piperlonguminine (a, 1c), piperyline (b, 1b), piperettine (c, 1c), dehydropipernonaline (d, 3a), guineensine (e, 4c), (2E,4E,13/12Z)-N-isobutyl-octadeca-2,4,12/13-trienamide (f, 5c/6c), 1-(octadeca-2E,4E,13/12Z-trienyl)piperidine (g, 5a/6a) and (2E,4E)-N-isobutyl-octadeca-2,4-dienamide (h, 7c) on Xenopus oocytes expressing hKNCK3. N = 3–6 oocytes. The dose-response curve for piperine on hKCNK3 (gray curve) from Beltrán et al., 2013 is presented for comparison purposes.
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FIGURE 8. (A)
Left: A representative voltage-clamp recording from a Xenopus oocyte expressing hKCNK9, before, during and after the application to 1 mM of 1-(octadeca-2E,4E,13/12Z-trienoyl)pyrrolidine (5b/6b). Right: Dose-response curves for the effect of 1-(octadeca-2E,4E,13/12Z-trienoyl) pyrrolidine (5b/6b) on hKCNK9. The IC50 value is 236 ± 56 μM. N = 3–6 oocytes. (B)
Left: A representative voltage-clamp recording from a Xenopus oocyte expressing hKCNK18, before, during and after the application to 1 mM of piperyline (1b). Right: Dose-response curves for the effect of piperyline (1b) on hKCNK18. The IC50 value is 252 ± 55 μM. N = 3–6 oocytes. The dose-response curve for piperine (1a) (gray diamonds) on each channel is presented for comparison.
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