All-trans-Configuration in Zanthoxylum ... - ACS Publications

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All-trans-Configuration in Zanthoxylum Alkylamides Swaps the Tingling with a Numbing Sensation and Diminishes Salivation Matthias Bader,∥ Timo D. Stark,∥ Corinna Dawid, Sofie Lösch, and Thomas Hofmann* Chair of Food Chemistry and Molecular Sensory Science, Technische Universität München, Lise-Meitner Strasse 34, D-85354 Freising, Germany ABSTRACT: The methanol soluble prepared from a supercritical fluid extract of Szechuan pepper (Zanthoxylum piperitum) was screened for its key tingling and numbing chemosensates by application of taste dilution analysis. Further separation of fractions perceived with the highest sensory impact, followed by LC-TOF-MS, LC-MS, and 1D/2D NMR experiments, led to the structure determination of the known alkylamides hydroxy-γ-sanshool (1), hydroxy-α-sanshool (2), hydroxy-β-sanshool (3), bungeanool (4), isobungeanool (5), and hydroxy-γ-isosanshool (6), as well as hydroxy-ε-sanshool (7), the structure of which has not yet been confirmed by NMR, and hydroxy-ζ-sanshool (8), which has not been previously reported in the literature. Psychophysical half-tongue experiments using filter paper rectangles (1 × 2 cm) as the vehicle revealed amides 1, 2, 4, 5, 7, and 8, showing at least one cis-configured double bond, elicited the well-known tingling and paresthetic orosensation above threshold levels of 3.5−8.3 nmol/cm2. In contrast, the all-trans-configured amides 3 and 6 induced a numbing and anesthetic sensation above thresholds of 3.9 and 7.1 nmol/cm2, respectively. Interestingly, the mono-cis-configured major amide 2 was found to induce massive salivation, whereas the all-trans-configuration of 3 did not. KEYWORDS: Szechuan pepper, tingling, numbing, salivation, hydroxysanshool, bungeanool, half-tongue test, Zanthoxylum piperitum

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INTRODUCTION Due to their delicate orosensory tingling flavor and salivating properties, the fruits of various Zanthoxylum species have been used for decades as a culinary spice, called the Szechuan pepper, and as one of the blended ingredients of the five-spice powder in Chinese and Japanese cuisines, respectively. In traditional folk medicine, Zanthoxylum plants are referred to as “toothache trees” because their anesthetic properties render them useful in pain relief.1 Natural product analysis revealed lipophilic unsaturated aliphatic acid amides such as hydroxy-γ-sanshool, 1 (Figure 1), and hydroxy-α-sanshool, 2, as the sensory active phytochemicals in Zanthoxylum fruits.2 Hydroxy-β-sanshool, 3, was prepared from 2 by irradiation with UV light in the presence of catalytic amounts of iodine.2 Besides 3 and 1, bungeanool (4), isobungeanool (5), and hydroxy-γ-isosanshool (6) were isolated from Huajiao, the pericarps of Zanthoxylum bungeanum Maxim.1 To investigate the activation of tactile and thermal trigeminal neurons, hydroxyalkylamides 2 and 3 were isolated from dried Szechuan pepper.3 Moreover, the authors reported hydroxy-ε-sanshool (7),3 the structure of which was proposed on comparison of spectroscopic data with those allegedly reported by Yasuda et al.1 and Kashiwada et al.,4 who, however, never reported the presence of this amide. In 2008, hydroxy-ε-sanshool, 7, was again mentioned as a phytochemical in Z. bungeanum and Zanthoxylum schinifolium,5,6 respectively, and was reported to be identified by comparison with literature data published by Mizutani et al.1 The latter authors, however, never described the structure elucidation of amide 7. Moreover, Jang et al.5 isolated polyunsaturated fatty acid amides from the seeds of Zanthoxylum piperitum and, again, referenced compounds 2, 3, and 7 to the publications of Yasuda et al.2,7−9 and Kashiwada et al.,4 lacking any data on the isolation © 2014 American Chemical Society

and structure determination of hydroxy-ε-sanshool (7). All together, the literature data on the structure determination of hydroxy-ε-sanshool, 7, from Szechuan pepper are not conclusive. Preliminary sensory analysis revealed hydroxy-α-sanshool, 2, as the tingling principle of Szechuan pepper,6 although any technical details on the sensory evaluation and data on human taste threshold concentrations are lacking. Furthermore, unsaturated amides showing a cis-configured double bond were reported to induce a tingling and pungent orosensation.2 A more systematic structure−activity relationship study performed with a series of synthetic sanshools and bungeanools revealed a minimal structure including a (CHZCH CH2CH2CHECH) motif and an N-(2-methyl-2hydroxypropyl) amide structure to be essential for the tingling activity.10 For all of these sensory studies, neither has the purity of these chemically unstable amides been determined, nor have the sensory protocols been sufficiently described. Others used ternary mixtures of sucrose, ethanol, and propylene glycol as test matrix,11 which, however, cannot be excluded to affect the tingling/pungent sensation of the amides as ethanol is reported to activate the vanilloid receptor TRPV-1.12 Reliable sensory analysis of the polyansaturated amides from Szechuan pepper, therefore, requires an ethanol-free sensory test system as recently reported for the pungent compound in black pepper.13 Besides eliciting a pronounced tingling sensation, some fatty acid amides such as spilanthole were reported to induce salivation.14 Also, Szechuan pepper extract has been reported to Received: Revised: Accepted: Published: 2479

January 23, 2014 March 6, 2014 March 7, 2014 March 7, 2014 dx.doi.org/10.1021/jf500399w | J. Agric. Food Chem. 2014, 62, 2479−2488

Journal of Agricultural and Food Chemistry

Article

Figure 1. Chemical structures of hydroxy-γ-sanshool (1), hydroxy-α-sanshool (2), hydroxy-β-sanshool (3), bungeanool (4), isobungeanool (5), hydroxy-γ-isosanshool (6), hydroxy-ε-sanshool (7), and hydroxy-ζ-sanshool (8).

activate saliva,15 but the key molecule responsible for this activity is not known yet. Therefore, the objectives of the present study were to locate and isolate most sensory active phytochemicals from a highly tingling active supercritical fluid (SCF) extract prepared from dried pods of Z. piperitum by application of the taste dilution analysis (TDA),16,17 to validate their chemical structure by means of LC-TOF-MS, LC-MS/MS, and 1D/2D NMR spectroscopy, and to determine human orosensory recognition thresholds as well as salivation-inducing activity of purified chemosensates using an ethanol-free sensory assay.13

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(Kolenchery, Kerala, India). For sensory analysis, bottled water (Evian, low mineralization = 405 mg/L), sucrose (Sigma-Aldrich, Steinheim, Germany), piperine (Sigma-Aldrich), and capsaicine (Sigma-Aldrich) were used. Filter paper (Rundfilter classic, 94 mm; Melitta, Minden, Germany), cut into a rectangular shape (1 × 2 cm), was used as vehicle for the sensory evaluation of pungent and tingling amides. Sensory Analyses. General Conditions and Panel Training. Twelve volunteers (seven women and five men, ages 25−34 years), who gave their informed consent to participate in the sensory tests of the present investigation and had no history of known taste disorders, were trained in sensory experiments with purified reference compounds at regular intervals for at least two years. The subjects were experienced in evaluating the orosensation of a tingling Szechuan pepper extract (40 μg/1 × 2 cm; Synthite, Kerala, India), isolated from Z. piperitum by means of supercritical carbon dioxide extraction and containing hydroxy-α-sanshool and hydroxy-β-sanshool in amounts of 9.1 and 2.5% (HPLC-UV), respectively, and the pungent compounds capsaicine (3 μg/1 × 2 cm) and piperine (20 μg/1 × 2 cm), respectively, by means of a modified half-tongue test using filter paper rectangles (1 × 2 cm) as the vehicle.13 Panelists were instructed to rinse their mouth after each dilution step with a 3% sucrose solution (15 mL), followed by water (15 mL), and to wait for 2 min between

MATERIALS AND METHODS

Chemicals. The following compounds were obtained commercially: Water for chromatographic separations was purified with an integral 5 system (Millipore, Schwalbach, Germany); n-hexane (Merck, Darmstadt, Germany) and solvents used were of HPLC grade (J. T. Baker, Deventer, The Netherlands). Deuterated solvents containing 0.03% trimethylsilane (TMS) were obtained from EurisoTop (Gif-Sur-Yvette, France). A supercritical fluid (SCF) extract from dried fruit pods of Z. piperitum was obtained from Synthite Industries 2480

dx.doi.org/10.1021/jf500399w | J. Agric. Food Chem. 2014, 62, 2479−2488


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each sample. Sensory analyses were performed at 19−22 °C in a sensory panel room in three different sessions. Precaution Taken for Sensory Analysis of Food Fractions and Taste Compounds. To remove solvent traces and buffer compounds from all fractions and compounds isolated from pepper, the individual fractions were suspended in water and, after removal of the volatiles under high vacuum (98%. N-(2-Methyl-2-hydroxypropyl)-tetradeca-(2E,4E,8Z,10E,12E)-pentaene amide (hydroxy-γ-sanshool), 1, Figure 1: UV−vis (acetonitrile/ 0.1% aqueous formic acid; 6:4, v/v), λmax 240, 272, 292 nm; LC-TOFMS (ESI+), m/z 290.2119 (measured), m/z 290.2120 (calcd for [C18H27NO2 + H]+); MS (ESI+), m/z (%) 290 (100, [M + H]+), 272 (31, [M + H − H2O]+), 312 (28, [M + Na]+), 328 (9, [M + K]+); MS/MS (DP = +40 V), m/z (%) 272 (100), 165 (68), 72 (42), 131 (35), 173 (31), 145 (25), 201 (8); 1H NMR (500 MHz, d3-MeOD, COSY), δ 1.17 [s, 6H, H−C(3′,4′)], 1.76 [d, 3H, J = 6.9 Hz, H− C(14)], 2.26 [m, 2H, J = 6.9, 14.1 Hz, H−C(6)], 2.33 [m, 2H, J = 7.3, 14.5 Hz, H−C(7)], 3.26 [s, 2H, H−C(1′)], 5.37 [dt, 1H, J = 7.5, 10.6, H−C(8)], 5.65−5.76 [m, 1H, H−C(13)], 5.97−6.05 [m, 2H, J = 15.0 Hz, H−C(2, 9)], 6.07−6.19 [m, 3H, H−C(5, 11, 12)], 6.24 [dd, 1H, J = 10.9, 15.1 Hz, H−C(4)], 6.32−6.41 [m, 1H, H−C(10)], 7.12 [dd, 2481



Article

Table 1. 13C NMR Chemical Shifts of Alkylamides 1−8 compound carbon atoma

1b,c,e,f

2b,c,d,f

3b,c,d,g

4b,c,e,f

5b,c,e,f

6b,c,e,f

7b,c,d,f

7b,c,e,f

8b,c,e,f

C(1) C(2) C(3) C(4) C(5) C(6) C(7) C(8) C(9) C(10) C(11) C(12) C(13) C(14) C(1′) C(2′) C(3′) C(4′)

169.6 123.3 142.4 130.3 143.2 34.0 28.1 130.9 130.8 126.7 134.6 133.3 130.6 18.5 51.2 71.7 27.3 27.3

167.0 123.7 144.4 32.1 26.5 129.5 129.7 125.2 133.5 131.8 130.2 18.3

166.9 123.6 144.5 31.9 31.4 132.0 131.6 130.1 131.5 131.6 129.4 18.3

169.6 123.3 142.4 130.3 143.3 33.9 33.2 133.5 133.2 132.7 132.6 131.6 129.8 18.4 51.3 71.5 27.3 27.3

169.4 125.1 145.6 33.2 27.7 131.5 131.0 128.6 129.5 130.9 127.8 13.7

50.4 71.1 27.4 27.4

169.6 123.2 142.5 130.2 143.5 33.9 27.5 129.8 129.9 31.4 128.5 133.5 26.6 14.4 51.2 71.7 27.3 27.3

166.9 123.7 144.4 32.1 26.5 130.3 129.8 127.2 128.4 129.5 127.2 13.5

50.4 71.1 27.3 27.3

169.5 123.3 142.4 130.2 143.4 33.9 27.6 129.7 130.0 26.5 128.3 132.8 21.5 14.7 51.2 71.7 27.3 27.3

50.4 71.0 27.3 27.3

51.1 72.0 27.3 27.3

169.5 123.3 142.5 130.3 131.0 34.0 28.2 131.6 130.9 128.7 129.4 131.0 127.6 13.7 51.3 71.8 27.2 27.2

a

Arbitrary numbering of carbon atoms refers to chemical structures displayed in Figure 1. bChemical shifts are given in relation to solvent signals. Signal assignment was performed by means of gHSQC (1J), gHMBC (2,3J), and DEPT-135 spectroscopy. dCDCl3 was used as solvent. ed3-MeOD was used as solvent. fSpectrum was recorded at 125 MHz. gSpectrum was recorded at 100 MHz. c

NMR (500 MHz, d3-MeOD, COSY), δ 0.96 [t, 3H, J = 7.5 Hz, H− C(14)], 1.17 [s, 6H, H−C(3′,4′)], 2.03−2.11 [m, 2H, H−C(13)], 2.18−2.27 [m, 4H, H−C(6, 7)], 2.78 [dd, 2H, J = 5.8 Hz, H−C(10)], 3.26 [s, 2H, H−C(1′)], 5.25−5.32 [m, 1H, H−C(11)], 5.34−5.41 [m, 3H, H−C(8, 9, 12)], 6.00 [d, 1H, J = 15.1 Hz, H−C(2)], 6.07−6.14 [m, 1H, J = 6.5, 15.1 Hz, H−C(5)], 6.23 [dd, 1H, J = 10.8, 15.1 Hz, H−C(4)], 7.13 [dd, 1H, J = 10.7, 15.1 Hz, H−C(3)]. The 13C NMR data (125 MHz, d3-MeOD, HSQC, HMBC) are shown in Table 1. N-(2-Methyl-2-hydroxypropyl)-tetradeca-(2E,4E,8Z,11E)-tetraene amide (isobungeanool), 5, Figure 1: UV−vis (acetonitrile/0.1% aqueous formic acid; 7:3, v/v), λmax = 260 nm; LC-TOF-MS (ESI+), m/z 292.2274 (measured), m/z 292.2277 (calcd for [C18H29NO2 + H]+); MS (ESI+), m/z 292 (100, [M + H]+), 314 (52, [M + Na]+), 274 (22, [M + H − H2O]+), 330 (18, [M + K]+); MS/MS (DP = +20 V), m/z 274 (100), 72 (29), 131 (26), 119 (26), 95 (26), 203 (9), 175 (8); 1H NMR (500 MHz, d3-MeOD, COSY), δ 0.96 [t, 3H, J = 7.6, H− C(14)], 1.17 [s, 6H, H−C(3′,4′)], 1.95−2.03 [m, 2H, H−C(13)], 2.16−2.26 [m, 4H, H−C(6, 7)], 2.72 [dd, 2H, J=5.4 Hz, H−C(10)], 3.26 [s, 2H, H−C(1′)], 5.33−5.42 [m, 3H, H−C(8, 9, 11)], 5.42− 5.51 [m, 1H, H−C(12)], 5.99 [d, 1H, J = 15.1 Hz, H−C(2)], 6.06− 6.14 [m, 1H, J = 6.6, 15.1 Hz, H−C(5)], 6.22 [dd, 1H, J = 10.8, 15.1 Hz, H−C(4)], 7.12 [dd, 1H, J = 10.7, 15.1 Hz, H−C(3)]. The 13C NMR data (125 MHz, d3-MeOD, HSQC, HMBC) are shown in Table 1. N-(2-Methyl-2-hydroxypropyl)-tetradeca-(2E,4E,8E,10E,12E)-pentaene amide (hydroxy-γ-isosanshool), 6, Figure 1: UV−vis (acetonitrile/ 0.1% aqueous formic acid; 6:4, v/v), λmax = 248, 272 nm; LC-TOF-MS (ESI+), m/z 290.2122 (measured), m/z 290.2120 (calcd for [C18H27NO2 + H]+); MS (ESI+), m/z 290 (100, [M + H]+), 272 (43, [M + H − H2O]+), 312 (53, [M + Na]+), 328 (16, [M + K]+); MS/MS (DP = +40 V), m/z 272 (95), 165 (100), 107 (78), 72 (35), 173 (17), 201 (7); 1H NMR (500 MHz, d3-MeOD, COSY), δ 1.17 [s, 6H, H−C(3′,4′)], 1.74 [d, 3H, J = 7.0 Hz, H−C(14)], 2.22 [m, 2H, J = 6.4, 12.9 Hz, H−C(6)], 2.26 [m, 2H, J = 6.6, 13.0 Hz, H−C(7)], 3.26 [s, 2H, H−C(1′)], 5.60−5.70 [m, 2H, H−C(8, 13)], 5.99 [d, 1H, J = 15.1 Hz, H−C(2)], 6.01−6.10 [m, 4H, H−C(9, 10, 11, 12)], 6.11 [m, 1H, H−C(5)], 6.23 [dd, 1H, J = 10.8, 15.2 Hz, H−C(4)], 7.12 [dd, 1H, J = 10.7, 15.1 Hz, H−C(3)]. The 13C NMR data (125 MHz, d3-MeOD, HSQC, HMBC) are shown in Table 1. N-(2-Methyl-2-hydroxypropyl)-dodeca-(2E,6Z,8E,10Z)-tetraene amide (hydroxy-ε-sanshool), 7, Figure 1: UV−vis (acetonitrile/0.1% aqueous

1H, J = 10.8, 15.1 Hz, H−C(3)]. The 13C NMR data (125 MHz, d3MeOD, HSQC, HMBC) are shown in Table 1. N-(2-Methyl-2-hydroxypropyl)-dodeca-(2E,6Z,8E,10E)-tetraene amide (hydroxy-α-sanshool), 2, Figure 1: UV−vis (acetonitrile/0.1% aqueous formic acid; 1:1, v/v), λmax 244, 272, 220 nm; LC-TOF-MS (ESI+), m/ z 286.1774 (measured), m/z 286.1777 (calcd for [C16H25NO2 + Na]+); MS (ESI+), m/z 264 (100, [M + H]+), 246 (38, [M + H − H2O]+), 286 (15, [M + Na]+), 302 (15, [M + K]+); MS/MS (DP = +40 V), m/z 246 (100), 147 (77), 107 (46), 72 (27), 133 (23), 175 (12); 1H NMR (500 MHz, CDCl3, COSY), δ 1.23 [s, 6H, H−C(3′, 4′)], 1.78 [d, 3H, J = 7.2 Hz, H−C(12)], 2.29 [m, 2H, J = 6.7, 14.44 Hz, H−C(4)], 2.34 [m, 2H, J = 7.6, 14.4 Hz, H−C(5)], 3.33 [d, 2H, J = 6.1 Hz, H−C(1′)], 5.37 [dt, 1H, J = 7.4, 10.6 Hz, H−C(6)], 5.73 [m, 1H, J = 7.0, 14.3, H−C(11)], 5.85 [dt, 1H, J = 1.5, 15.3 Hz, H− C(2)], 5.99 [1H, H−N], 6.02 [pt, 1H, J = 11.0 Hz, H−C(7)], 6.12 [m, 1H, J = 10.5, 14.5 Hz, H−C(10)], 6.18 [dd, 1H, J = 10.5, 14.4 Hz, H− C(9)], 6.33 [dd, 1H, J = 11.4, 14.2 Hz, H−C(8)], 6.86 [dt, 1H, J = 6.7, 15.3 Hz, H−C(3)]. The 13C NMR data (125 MHz, d3-MeOD, HSQC, HMBC) are shown in Table 1. N-(2-Methyl-2-hydroxypropyl)-dodeca-(2E,6E,8E,10E)-tetraene amide (hydroxy-β-sanshool), 3, Figure 1: UV−vis (acetonitrile/0.1% aqueous formic acid; 1:1, v/v), λmax 244, 272, 220 nm; LC-TOF-MS (ESI+), m/ z 286.1774 (measured), m/z 286.1777 (calcd for [C16H25NO2 + Na]+); MS (ESI+), m/z 264 (100%, [M + H]+), 246 (66%, [M + H − H2O]+), 286 (94%, [M + Na]+), 302 (36%, [M + K]+); MS/MS (DP = +40 V): m/z (%) 107 (100), 246 (75), 79 (42), 133 (33), 139 (33), 147 (33), 175 (18); 1H NMR (400 MHz, CDCl3, COSY), δ 1.24 [s, 6H, H−C(3′, 4′)], 1.77 [d, 3H, J = 7.0 Hz, H−C(12)], 2.26 [m, 2H, J = 5.8 Hz, H−C(5)], 2.28 [m, 2H, J = 6.4 Hz, H−C(4)], 3.33 [d, 2H, J = 6.1 Hz, H−C(1′)], 5.63 [m, 1H, J = 7.2, 13.5 Hz, H−C(6)], 5.69 [m, 1H, J = 6.9, 14.3, H−C(11)], 5.82 [d, 1H, J = 15.3 Hz, H−C(2)], 5.84 [1H, H−N], 6.02−6.14 [m, 4H, J = 14.7 Hz, H−C(7−10)], 6.86 [dt, 1H, J = 6.5, 15.2 Hz, H−C(3)]. The 13C NMR data (125 MHz, d3-MeOD, HSQC, HMBC) are shown in Table 1. N-(2-Methyl-2-hydroxypropyl)-tetradeca-(2E,4E,8Z,11Z)-tetraene amide (bungeanool), 4, Figure 1: UV−vis (acetonitrile/0.1% aqueous formic acid; 7:3, v/v), λmax = 272, 248 nm; LC-TOF-MS (ESI+), m/z 292.2278 (measured), m/z 292.2277 (calcd for [C18H29NO2 + H]+); MS (ESI+), m/z 292 (60, [M + H]+), 274 (17, [M + H − H2O]+), 314 (100, [M + Na]+), 330 (70, [M + K]+); MS/MS (DP = +40 V), m/z (%) 274 (100), 119 (20), 131 (19), 72 (17), 203 (5), 175 (4); 1H 2482



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Figure 2. (A) MPLC chromatographic separation of the methanol solubles of Szechuan pepper extract and (B) taste dilution analysis (TDA) recording the tingling orosensation. formic acid; 1:1, v/v), λmax = 208, 276 nm; LC-TOF-MS (ESI+), m/z 286.1774 (measured), m/z 286.1777 (calcd for [C16H25NO2 + Na]+); MS (ESI+), m/z 264 (100, [M + H]+), 246 (81, [M + H − H2O]+), 286 (70, [M + Na]+), 302 (25, [M + K]+); MS/MS (DP = +40 V), m/ z 274 (100), 119 (20), 131 (19), 72 (17), 203 (5), 175 (4); 1H NMR (500 MHz, d3-MeOD, COSY), δ 1.19 [s, 6H, H−C(3′, 4′)], 1.77 [dd, 3H, J = 1.6, 7.2 Hz, H−C(12)], 2.31 [m, 2H, J = 6.7, 14.4 Hz, H− C(4)], 2.39 [m, 2H, J = 7.4, 14.5 Hz, H−C(5)], 3.27 [s, 2H, H− C(1′)], 5.44 [m, 1H, J = 7.6, 10.7 Hz, H−C(6)], 5.52 [m, 1H, J = 7.2, 10.8, H−C(11)], 6.04 [dt, 1H, J = 1.4, 15.4 Hz, H−C(2)], 6.07−6.11 [m, 2H, J = 10.4, 10.9 Hz, H−C(10, 7)], 6.48 [dd, 1H, J = 10.6, 14.9 Hz, H−C(8)], 6.53 [dd, 1H, J = 10.8, 14.9 Hz, H−C(9)], 6.81 [dt, 1H, J = 6.8, 15.3 Hz, H−C(3)]. 1H NMR (500 MHz, CDCl3, COSY): δ/ppm: δ 1.24 [s, 6H, H−C(3′, 4′)], 1.78 [dd, 3H, J=1.6, 7.2 Hz, H− C(12)], 2.29 [m, 2H, J=6.8, 14.4 Hz, H−C(4)], 2.37 [m, 2H, J=7.3, 14.4 Hz, H−C(5)], 3.32 [d, 2H, J=6.1 Hz, H−C(1′)], 5.43 [dt, 1H, J=7.4, 10.8 Hz, H−C(6)], 5.55 [m, 1H, J=7.2, 10.7, H−C(11)], 5.84 [dt, 1H, J = 1.5, 15.3 Hz, H−C(2)], 5.84 [1H, H−N], 6.08 [m, 1H, J = 10.8 Hz, H−C(10)], 6.10 [m, 1H, J = 11.1 Hz, H−C(7)], 6.43 [dd, 1H, J = 11.1, 14.8 Hz, H−C(8)], 6.52 [dd, 1H, J = 11.0, 14.8 Hz, H− C(9)], 6.87 [dt, 1H, J = 6.7, 15.2 Hz, H−C(3)]. The 13C NMR data (125 MHz, d3-MeOD, HSQC, HMBC) are shown in Table 1. N-(2-Methyl-2-hydroxypropyl)-tetradeca-(2E,4E,8Z,10E,12Z)-pentaenwamide (hydroxy-ζ-sanshool), 8, Figure 1: UV−vis (methanol/0.1% aqueous formic acid; 6:4, v/v), λmax = 276, 264 nm; LC-TOF-MS (ESI+), m/z 290.2114 (measured), m/z 290.2120 (calcd for [C18H27NO2 + H]+); MS (ESI+), m/z 290 (100, [M + H]+), 272 (22, [M + H − H2O]+), 312 (70, [M + Na]+), 328 (33, [M + K]+); MS/MS (DP = +40 V), m/z 272 (100), 165 (69), 131 (40), 72 (35), 173 (33), 107 (30), 93 (18), 201 (10); 1H NMR (500 MHz, d3MeOD, COSY), δ 1.18 [s, 6H, H−C(3′,4′)], 1.76 [d, 3H, J = 7.1 Hz, H−C(14)], 2.27 [m, 2H, J = 7.3, 14.5 Hz, H−C(6)], 2.35 [m, 2H, J = 7.4, 14.8 Hz, H−C(7)], 3.26 [s, 2H, H−C(1′)], 5.38−5.46 [m, 1H, H−C(8)], 5.46−5.56 [m, 1H, H−C(13)], 6.00 [d, 1H, J = 15.2 Hz, C−H(2)], 6.03−6.17 [m, 3H, H−C(5, 9, 12)], 6.24 [dd, 1H, J = 10.6, 14.9 Hz, H−C(4)], 6.43−6.49 [dd, 1H, J = 10.2, 14.9 Hz, H−C(10)], 6.49−6.56 [dd, 1H, J = 10.4, 15.5 Hz, H−C(11)], 7.12 [dd, 1H, J = 10.8, 14.9 Hz, H−C(3)]. The 13C NMR data (125 MHz, d3-MeOD, HSQC, HMBC) are shown in Table 1. Ultraperformance Liquid Chromatography−Time-of-Flight Mass Spectrometry (UPLC-TOF-MS). Mass spectra of the compounds were measured on a Waters Synapt G2 HDMS mass spectrometer (Waters, Manchester, UK) coupled to an Acquity UPLC core system (Waters, Milford, MA, USA) consisting of a binary solvent

manager, sample manager, and column oven. Analytes were injected into the UPLC-TOF-MS system equipped with a 2.1 × 150 mm, 1.7 μm, BEH C18 column (Waters, Manchester, UK). Operated with a flow rate of 0.4 mL/min at 45 °C, the following gradient was used for chromatography: starting with a mixture (40:60, v/v) of water and acetonitrile, the acetonitrile content was increased to 100% within 4 min and, then, kept constant for 1 min. Scan time for the MSE method (centroid) was set to 0.1 s. Analyses were performed in the positive ESI and the resolution mode using the following ion source parameters: capillary voltage, +2.0 kV; sampling cone, 30 V; extraction cone, 4.0 V; source temperature, 150 °C; desolvation temperature, 450 °C; cone gas, 30 L/h; and desolvation gas, 850 L/h. Data processing was performed by using MassLynx 4.1 SCN 779 (Waters, Manchester, UK) and the elemental composition tool for determining the accurate mass. All data were lock mass corrected on the pentapeptide leucine enkephaline (Tyr-Gly-Gly-Phe-Leu, m/z 556.2771, [M + H]+) in a solution (2 ng/μL) of acetonitrile/0.1% formic acid (1:1, v/v). Scan time for the lock mass was set to 0.3 s, an interval of 15 and 3 scans to average with a mass window of ±0.3 Da. Calibration of the Synapt G2 in the range from m/z 50 to 1300 was performed using a solution of sodium formate (5 mmol/L) in 2-propanol/water (9:1, v/v). The UPLC and Synapt G2 systems were operated with MassLnyx software (Waters, Manchester, UK). High-Performance Liquid Chromatography−Mass Spectrometry (HPLC-MS/MS). LC-MS/MS analysis was performed using an Dionex Ultimate 3000 HPLC system connected to the API 4000QTrap LC-MS/MS (AB Sciex, Darmstadt, Germany) running in the positive electrospray ionization (ESI+) mode. Zero grade air served as nebulizer gas (45 psi) and as turbo gas (425 °C) for solvent drying (55 psi). Nitrogen served as curtain (20 psi) and collision gas (8.7 × 10−7 psi). Both quadrupoles were set at unit resolution. ESI+ mass and product ion spectra were acquired with direct flow infusion. For ESI+, the ion spray voltage was set at +5500 V. Nuclear Magnetic Resonance Spectroscopy (NMR). 1H, 13C, DEPT-135, homonuclear 1H−1H correlation spectroscopy (1H−1HgCOSY), heteronuclear single-quantum coherence spectroscopy (gHSQC), heteronuclear multiple-bond correlation spectroscopy (gHMBC), and 1H−1H rotating frame nuclear Overhauser enhancement spectroscopy (phase-sensitive ROESY) NMR measurements were performed on an Avance III 500 MHz equipped with a CTCI probe and an Avance III 400 MHz spectrometer with a BBO probe (Bruker, Rheinstetten, Germany), respectively. Chemical shifts were referenced to the solvent signal. Data processing was performed by 2483



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Figure 3. Preparative HPLC separation of MPLC fractions F2 (A) and F3 (B).

Figure 4. MS/MS spectrum (ESI+, +20 V) of (A) hydroxy-ε-sanshool (7) and (B) hydroxy-ζ-sanshool (8), respectively.

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Figure 5. 13C NMR spectra (125 MHz, CDCl3) of alkylamides 2, 3, and 7. using Topspin version 2.1 (Bruker) and MestReNova version 6.2.1 software (Mestrelab Research, Santiago de Compostela, Spain).

Isolation and Identification of Chemosensates in Fractions F2 and F3. Separation of fractions F2 and F3 by means of preparative RP-HPLC revealed nearly baseline separation of their components, which could be isolated or further purified by means of rechromatography (Figure 3). LC-MS analysis using electrospray ionization (ESI+) revealed m/z 264 as the pseudomolecular ion ([M + H]+) and m/z 246 as the main fragment ion for the three main compounds 2, 3, and 7 eluting in fraction F2 (Figure 3A). Exemplified for compound 7, the product ion scan of m/z 264 [M + H]+ revealed the main fragments of m/z 246, 147, 107, and 175 (Figure 4A), indicating the loss of water as well as an alpha and an allyl cleavage, respectively. Moreover, the isobaric compounds 2, 3, and 7 were found by LC-TOF-MS to show identical elemental compositions of C16H25NO2. In comparison, the early-eluting compounds 1, 6, and 8 in fraction F3 (Figure 3B) showed a pseudomolecular ion ([M + H]+) of m/z 290 and a main fragment ion with m/z 272, well in agreement with the molecular formula of C18H27NO2 as indicated by LCTOF-MS analysis. The late-eluting compounds 4 and 5 in fraction F3 differed by a mass increment of 2, consistent with an elemental composition of C18H29NO2 (LC-TOF-MS), and showed m/z 292 and 274 as the [M + H]+ and the main fragment ion, respectively. Unequivocal assignment of protons and carbon atoms could be successfully achieved by means of 1H, 13C, DEPT-135, 1 H−1H-gCOSY, gHMBC optimized for 2JC,H and 3JC,H coupling constants, and gHSQC optimized for 1JC,H coupling constants, respectively (Table 1). Comparison of 1H and 13C NMR data with literature data1,2 led to the identification of the amides hydroxy-γ-sanshool (1), hydroxy-α-sanshool (2), hydroxy-βsanshool (3), bungeanool (4), isobungeanool (5), and hydroxyγ-isosanshool (6), respectively.

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RESULTS AND DISCUSSION Using the recently developed half-mouth test,13 orosensory evaluation of paper vehicles loaded with dilutions of the SCF extract prepared from Z. piperitum revealed a strong and longlasting tingling activity in the oral cavity perceived up to a dilution of 1:16384 (data not shown). To locate the most active chemosensates in Szechuan pepper, the SFC extract was dissolved in methanol and extracted with hexane, both fractions were separated from solvent, and dilutions of the hexane extractables as well as the methanol solubles were judged in natural concentration ratios in their tingling activity by means of a TDA. The methanol solubles induced a strong tingling sensation judged with a TD factor of 8192, whereas the hexane extractables showed comparatively low activity judged with a TD factor of only 128. As a consequence, the following investigation on the primary tingling components in Szechuan pepper was focused on the methanol solubles. Taste Dilution Analysis. To sort out the tingling compounds from the bulk of less taste-active or tasteless substances, the tingling-active methanol solubles of the SFC extract were separated by means of MPLC on RP18 material to give seven fractions, namely F1−F7, which were collected individually, separated from solvent, and used for the TDA using paper disks as vehicles.13 Fractions F2 and F3 showed by far the highest TD factors of 4096 and 2048, respectively, whereas all other fractions showed only low tingling activity judged with TD factors of ≤128 (Figure 2). Aimed at characterizing the molecular structure of the compounds imparting the most intense tingling sensation of Szechuan pepper, further fractionation and LC-MS as well as NMR experiments were focused on MPLC fractions F2 and F3. 2485



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The 1H NMR of compound 7 showed eight olefinic protons of the polyunsaturated fatty acid chain, namely, H−C(2), H− C(3), and H−C(6) to H−C(11), resonating in the range of 5.43−6.87 ppm, and six aliphatic protons, which could be assigned to the 2-methyl-2-hydroxypropyl moiety (H−C(1′), H−C(3′), H−C(4′)), the terminal methyl protons H−C(12), and the two methylene groups H−C(4) and H−C(5) of the fatty acid moiety. Unequivocal assignment of all protons and carbon atoms could be successfully achieved by means of 1D/ 2D NMR spectroscopy, which identified 7 as a geometrical isomer of amides 2 and 3 showing four double bonds at carbons C(2), C(6), C(8), and C(10), respectively. The geometry of the individual double bonds was unambiguously elucidated by comparing the 3JH,H coupling constants of the olefinic protons and the 13C NMR chemical shifts recorded for the individual isomers, as well as by means of ROESY experiments. The protons H−C(2) and H−C(3), both resonating as double triplets, and the two double duplet protons H−C(8) and H−C(9) showed the characteristic transolefinic coupling constants of 15.2 and 14.8 Hz, respectively. In contrast, the olefinic protons H−C(10) and H−C(11) as well as H−C(6) and H−C(7) showed a 3JH,H coupling constant of ∼10.8 Hz, thus indicating a cis-configuration. By comparing the 13 C chemical shifts of compounds 2, 3, and 7, the methylene carbon atom C(5) and the methyl carbon C(12), both adjacent to the cis-configured double bond, showed a clear upfield shift in 7 when compared to the all-trans-isomer 3 (Table 1; Figure 5). The upfield shift of the carbon C(5) from 31.4 to 26.5 ppm in 7 and 2 compared to 3 is well in agreement with a cisshielding effect reported in the literature.2,7−9 Another fingerprint carbon atom, namely C(12), showed an upfield shift of ∼5 ppm in 7 when compared to its geometric isomers 2 and 3, respectively (Table 1; Figure 5). The configurations of the double bonds were further supported by means of a ROESY experiment; for example, a ROESY correlation was detected between the methyl protons H−C(12) and methine proton H−C(9). Taking all spectroscopic data into consideration, LC-TOF-MS, LC-MS/MS, and 1D/2D NMR experiments led to the unequivocal identification of compound 7 as N-(2-methyl-2-hydroxypropyl)dodeca-(2E,6Z,8E,10Z)-tetraene amide (hydroxy-ε-sanshool). which has already been mentioned in the literature,3,5,6,10 although its chemical structure has not yet been confirmed by means of 1D/2D NMR spectroscopy. The MS product ion scan of m/z 290 [M + H]+, the pseudomolecular ion of compound 8, revealed m/z 272, 173, 107, and 202 as the main fragment ions (Figure 4B), thus indicating the cleavage of water (m/z 272), and an alpha cleavage (m/z 173; m/z 202), as well as an allyl cleavage (m/z 107), respectively. The 1H NMR of 8 showed 10 olefinic protons, namely, H−C(2) to H−C(5) and H−C(8) to H− C(13), resonating in the range of 5.38−7.12 ppm, six aliphatic protons assigned to the 2-methyl-2-hydroxypropyl moiety (H− C(1′), H−C(3′), H−C(4′)), the terminal methyl protons H− C(14), and the two methylene groups H−C(6) and H−C(7) of the fatty acid moiety. Assignment of protons and carbon atoms revealed 8 to be a geometrical isomer of the amides 1 and 6, respectively. The 3JH,H coupling constants of 15.5 and 14.9 Hz of the two double duplets detected for H−C(10) and H−C(11) indicated a trans-configuration of this double bond (Figure 6). Moreover, carbon atoms C(7) and C(14) showed an upfield shift of ∼5 ppm in 8 when compared to its geometric isomers 1 and 6 (Table 1; Figure 1), being well in agreement

Figure 6. Excerpt (δ 6.3−6.7) of the 1H NMR spectra (500 MHz, d3MeOD) of alkylamide 8 showing H−C(10) and H−C(11).

with the expected cis-shielding effect.2,7−9 Taking all spectroscopic data into consideration, the structure of compound 8 was determined to be N-(2-methyl-2-hydroxypropyl)tetradeca(2E,4E,8Z,10E,12Z)-pentaene amide, coined hydroxy-ζ-sanshool, which to the best of our knowledge has not been reported before. Sensory Evaluation of Pungent and/or Tingling Chemosensates. Prior to sensory analysis, the purity of all compounds was confirmed by LC-MS as well as 1H NMR spectroscopy to be >98%. To determine the human threshold concentrations for the orosensation induced by the individual chemosensates, paper vehicles loaded with the test compounds in serial dilutions were evaluated by means of a half-tongue test.13 Hydroxy-γ-sanshool (1), hydroxy-α-sanshool (2), bungeanool (4), isobungeanool (5), hydroxy-ε-sanshool (7), and the previously not reported hydroxy-ζ-sanshool (8) were found to elicit an oral tingling and paresthetic sensation above a rather similar threshold concentrations ranging between 3.5 and 8.3 nmol/cm2, respectively (Table 2). It is interesting to note that the position of the cis-configured double bond in 1, 2, 4, 5, 7, and 8 did not have a major impact on the tingling threshold concentration of these amides. Interestingly, the absence of a cis-double bond, as found for hydroxy-β-sanshool (3) and hydroxy-γ-isosanshool (6), resulted in a complete loss of the tingling activity. This is well in agreement with earlier studies Table 2. Orosensory Recognition Thresholds of Alkylamides 1−8 compounda

oral sensation

threshold concnb (nmol/cm2)

1 2 3 4 5 6 7 8

tingling, paresthetic tingling, paresthetic numbing, anesthetic tingling, paresthetic tingling, paresthetic numbing, anesthetic tingling, paresthetic tingling, paresthetic

6.8 8.3 3.9 3.5 3.5 7.1 4.2 7.0

a

Structures of compounds are given in Figure 1. bOrosensory recognition threshold concentrations were determined by means of a modified half-mouth test using paper rectangles (1 × 2 cm) as a vehicle.13

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on synthetic polyunsaturated alkylamides10 and those isolated from black pepper;13 for example, saturation of the double bond in black pepper’s tingling compound (2E,4E,12Z)-Nisobutyloctadeca-2,4,12-trienamide wiped out the tingling activity of (2E,4E)-N-isobutyloctadeca-2,4-dienamide. Although no tingling activity was detectable for the all-trans-configured compounds 3 and 6, most interestingly, the sensory panelists reported an intense and long-lasting numbing and anesthetic oral impression above threshold concentrations of 3.9 nmol/ cm2 (3) and 7.1 nmol/cm2 (6). To answer the question of whether the tingling and numbing amides differ in their salivating activity, the major amides hydroxy-α-sanshool (2, tingling) and hydroxy-β-sanshool (3, numbing) were isolated in suitable amounts and aqueous solutions were used to orally challenge a panel of eight healthy volunteers for 15 s; the saliva flow was measured for 120 s using an assay reported recently.15 A comparative experiment was performed with an aqueous solution of the crude Szechuan pepper extract containing 9.1% hydroxy-α-sanshool and 2.5% hydroxy-β-sanshool. All three stimuli significantly activated a long-lasting saliva flow increase (Figure 7). The tingling

neuron activation through a unique mechanism involving inhibition of the pH- and anesthetic-sensitive two-pore potassium channels TASK-1, TASK-3, and TRESK (previously named KCNK3, KCNK9, and KCNK18) is mediating the tingling effect of hydroxy-α-sanshool.19 The finding that a clear tingling, paresthetic sensation is induced by amides containing at least one cis-configured double bond as found in 1, 2, 4, 5, 7, and 8, whereas an intense numbing and anesthetic impression is caused by all-trans-configured amides (3 and 6), promises the various isomers as valuable molecular probes to test candidate ion channels TASK-1, TASK-3, and TRESK for mediating the tingling effect and to challenge other receptive proteins for mediating the numbing and anesthetic effect. Such experiments will be helpful to deliver a framework for understanding the unique and complex psychophysical sensations associated with the Szechuan pepper experience.

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AUTHOR INFORMATION

Corresponding Author

*(T.H.) Phone: +49-8161-71-2902. Fax: +49-8161-71-2949. Email: [email protected]. Author Contributions ∥

M.B. and T.D.S. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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REFERENCES

(1) Mizutani, K.; Fukunaga, Y.; Tanaka, O.; Takasugi, N.; Saruwatari, Y.-I.; Fuwa, T.; Yamauchi, T.; Wang, J.; Jia, M.-R.; Li, F.-Y.; Ling, Y.-K. Amides from Huajiao, pericarps of Zanthoxylum bungeanum Maxim. Chem. Pharm. Bull. 1988, 36, 2362−2365. (2) Yasuda, I.; Takeya, K.; Itokawa, H. Distribution of unsaturated aliphatic acid amides in Japanese Zanthoxylum species. Phytochemistry 1982, 21, 1295−1298. (3) Bryant, B. P.; Mezine, I. Alkylamides that produce tingling paresthesia activate tactile and thermal trigeminal neurons. Brain Res. 1999, 842, 452−460. (4) Kashiwada, Y.; Ito, C.; Katagiri, H.; Mase, I.; Komatsu, K.; Namba, T.; Ikeshiro, Y. Amides of the fruit of Zanthoxylum. Phytochemistry 1997, 44, 1125−1127. (5) Jang, K. H.; Chang, Y. H.; Kim, D.-D.; Oh, K.-B.; Oh, U.; Shin, J. New polyunsaturated fatty acid amides isolated from the seeds of Zanthoxylum piperitum. Arch. Pharm. Res. 2008, 31, 569−572. (6) Yang, X. Aroma constituents and alkylamides of red and green Huajiao (Zanthoxylum bungeanum and Zanthoxylum schinifolium). J. Agric. Food Chem. 2008, 56, 1689−1696. (7) Yasuda, I.; Takeya, K.; Itokawa, H. Two new pungent principles isolated from the pericarps of Zanthoxylum ailanthoides. Chem. Pharm. Bull. 1981, 29, 1791−1793. (8) Yasuda, I.; Takeya, K.; Itokawa, H. Structures of amides from Asiasarum heterotropoides Maek. var. mandshuricum Maek. Chem. Pharm. Bull. 1981, 29, 564−566. (9) Yasuda, I.; Takeya, K.; Itokawa, H. The geometric structure of spilanthol. Chem. Pharm. Bull. 1980, 28, 2251−2253. (10) Galopin, Ch. C.; Furrer, S. M.; Goeke, A. Pungent and tingling compounds in Asian cuisine. In Challenges in Taste Chemistry and Biology; Hofmann, T., Ho, C. T., Pickenhagen, W., Eds.; ACS Symposium Series 876; American Chemical Sosiety: Washington, DC, USA, 2004; pp 139−152. (11) Sugai, E.; Morimitsu, Y.; Iwasaki, Y.; Morita, A.; Watanabe, T.; Kubota, K. Pungent qualities of sanshool-related compounds evaluated by a sensory test and activation of rat TRPV1. Biosci., Biotechnol., Biochem. 2005, 65, 1951−1957. (12) Trevisani, M.; Smart, D.; Gunthorpe, M. J.; Tognetto, M.; Barbieri, M.; Campi, B.; Amatesi, S.; Gray, J.; Jerman, J. C.; Brough, S. J.; Owen, D.; Smith, G. D.; Randall, A. D.; Harrison, S.; Bianchi, A.;

Figure 7. Time course of saliva flow increase induced by an aqueous stimulus solution (2 mL) containing (●) the Szechuan pepper extract (100 mg/100 mL), (■) hydroxy-β-sanshool (100 mg/100 mL), and (▲) hydroxy-α-sanshool (100 mg/mL). Least significant difference is visualized (LSD = 8.263).

hydroxy-α-sanshool induced a massive increase of saliva flow accounting for a maximum of ∼75% after 45 s when compared to the control. Also, the Szechuan pepper extract showed a strong increase of saliva flow (∼44% after 45 s), whereas the numbing hydroxy-β-sanshool showed only a marginal activity accounting for a saliva flow increase of ∼25%. This demonstrates that at least one cis-configured double bond is needed for strong salivating activity, being well in agreement with literature data on spilanthol,14 whereas the all-transconfiguration diminishes salivation. As the tingling hydroxy-αsanshool is the quantitatively predominant amide in Szechuan pepper, this isomer is concluded to be mainly responsible for the tingling as well as the salivation enhancement activity of Szechuan pepper. The molecular mechanisms by which hydroxy-α-sanshool (2) induces the tingling sensation have been a matter of debate. Although this amide is an agonist at the pain-integrating cation channels TRPV1 and TRPA1,18 newer evidence suggests that 2487



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Davis, J. B.; Geppetti, P. Ethanol elicits and potentiates nociceptor responses via the vanilloid receptor-1. Nat. Neurosci. 2002, 5, 546− 551. (13) Dawid, C.; Henze, A.; Frank, O.; Glabasnia, A.; Rupp, M.; Büning, K.; Orlikowski, D.; Bader, M.; Hofmann, T. Structural and sensory characterisation of key pungent and tingling compounds from pepper (Piper nigrum L.). J. Agric. Food Chem. 2012, 60, 2884−2895. (14) Ley, J. P.; Simchen, U. Quantification of the saliva-inducing properties of pellitorine and spilanthole. Rec. Highl. Flav. Chem. Biol. 2007, 365−368. (15) Lorenz, K.; Bader, M.; Klaus, A.; Weiss, W.; Görg, A.; Hofmann, T. Orosensory stimulation effects on human saliva proteome. J. Agric. Food Chem. 2011, 59, 10219−10231. (16) Frank, O.; Ottinger, H.; Hofmann, T. Characterization of an intense bitter-tasting 1H,4H-quinolizinium-7-olate by application of the taste dilution analysis, a novel bioassay for the screening and identification of taste-active compounds in foods. J. Agric. Food Chem. 2001, 49, 231−238. (17) Stark, T.; Hofmann, T. Application of a molecular sensory science approach on alkalized cocoa (Theobroma cacao): structure determination and sensory activity of non-enzymatically C-glycosylated flavan-3-ols. J. Agric. Food Chem. 2006, 54, 9510−9521. (18) Koo, J. Y.; Jang, Y.; Cho, H.; Lee, C.-H.; Jang, K. H.; Chang, Y. H.; Shin, J.; Oh, U. Hydroxy-α-sanshool activates TRPV1 and TRPA1 in sensory neurons. Eur. J. Neurosci. 2007, 26, 1139−1147. (19) Bautista, D. M.; Sigal, Y. M.; Milstein, A. D.; Garrison, J. L.; Zorn, J. A.; Tsuruda, P. R.; Nicoll, R. A.; Julius, D. Pungent agents from Szechuan peppers excite sensory neurons by inhibiting two-pore potassium channels. Nat. Neurosci. 2008, 11 (7), 772−779.

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