All- trans -Configuration in Zanthoxylum Alkylamides Swaps the Tingling with a Numbing Sensation and Diminishes Salivation

All-trans-Configuration in Zanthoxylum Alkylamides Swaps theTingling with a Numbing Sensation and Diminishes SalivationMatthias Bader,∥ Timo D. Stark,∥ Corinna Dawid, Sofie Losch, and Thomas Hofmann*

Chair of Food Chemistry and Molecular Sensory Science, Technische Universitat Munchen, Lise-Meitner Strasse 34, D-85354Freising, Germany

ABSTRACT: The methanol soluble prepared from a supercritical fluid extract of Szechuan pepper (Zanthoxylum piperitum) wasscreened for its key tingling and numbing chemosensates by application of taste dilution analysis. Further separation of fractionsperceived with the highest sensory impact, followed by LC-TOF-MS, LC-MS, and 1D/2D NMR experiments, led to thestructure 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 hasnot 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 thresholdlevels of 3.5−8.3 nmol/cm2. In contrast, the all-trans-configured amides 3 and 6 induced a numbing and anesthetic sensationabove thresholds of 3.9 and 7.1 nmol/cm2, respectively. Interestingly, the mono-cis-configured major amide 2 was found toinduce massive salivation, whereas the all-trans-configuration of 3 did not.

KEYWORDS: Szechuan pepper, tingling, numbing, salivation, hydroxysanshool, bungeanool, half-tongue test, Zanthoxylum piperitum

■ INTRODUCTION

Due to their delicate orosensory tingling flavor and salivatingproperties, the fruits of various Zanthoxylum species have beenused for decades as a culinary spice, called the Szechuan pepper,and as one of the blended ingredients of the five-spice powderin Chinese and Japanese cuisines, respectively. In traditionalfolk medicine, Zanthoxylum plants are referred to as “toothachetrees” because their anesthetic properties render them useful inpain relief.1

Natural product analysis revealed lipophilic unsaturatedaliphatic acid amides such as hydroxy-γ-sanshool, 1 (Figure1), and hydroxy-α-sanshool, 2, as the sensory activephytochemicals in Zanthoxylum fruits.2 Hydroxy-β-sanshool,3, was prepared from 2 by irradiation with UV light in thepresence 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 Zanthoxylumbungeanum Maxim.1 To investigate the activation of tactile andthermal trigeminal neurons, hydroxyalkylamides 2 and 3 wereisolated from dried Szechuan pepper.3 Moreover, the authorsreported hydroxy-ε-sanshool (7),3 the structure of which wasproposed on comparison of spectroscopic data with thoseallegedly 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 phytochemicalin Z. bungeanum and Zanthoxylum schinifolium,5,6 respectively,and was reported to be identified by comparison with literaturedata 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 theseeds of Zanthoxylum piperitum and, again, referencedcompounds 2, 3, and 7 to the publications of Yasuda etal.2,7−9 and Kashiwada et al.,4 lacking any data on the isolation

and structure determination of hydroxy-ε-sanshool (7). Alltogether, the literature data on the structure determination ofhydroxy-ε-sanshool, 7, from Szechuan pepper are notconclusive.Preliminary sensory analysis revealed hydroxy-α-sanshool, 2,

as the tingling principle of Szechuan pepper,6 although anytechnical details on the sensory evaluation and data on humantaste threshold concentrations are lacking. Furthermore,unsaturated amides showing a cis-configured double bondwere reported to induce a tingling and pungent orosensation.2

A more systematic structure−activity relationship studyperformed with a series of synthetic sanshools and bungeanoolsrevealed a minimal structure including a (CHZCHCH2CH2CHECH) motif and an N-(2-methyl-2-hydroxypropyl) amide structure to be essential for the tinglingactivity.10 For all of these sensory studies, neither has the purityof these chemically unstable amides been determined, nor havethe sensory protocols been sufficiently described. Others usedternary mixtures of sucrose, ethanol, and propylene glycol astest matrix,11 which, however, cannot be excluded to affect thetingling/pungent sensation of the amides as ethanol is reportedto activate the vanilloid receptor TRPV-1.12 Reliable sensoryanalysis of the polyansaturated amides from Szechuan pepper,therefore, requires an ethanol-free sensory test system asrecently reported for the pungent compound in black pepper.13

Besides eliciting a pronounced tingling sensation, some fattyacid amides such as spilanthole were reported to inducesalivation.14 Also, Szechuan pepper extract has been reported to

Received: January 23, 2014Revised: March 6, 2014Accepted: March 7, 2014Published: March 7, 2014

Article

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

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activate saliva,15 but the key molecule responsible for thisactivity is not known yet.Therefore, the objectives of the present study were to locate

and isolate most sensory active phytochemicals from a highlytingling active supercritical fluid (SCF) extract prepared fromdried pods of Z. piperitum by application of the taste dilutionanalysis (TDA),16,17 to validate their chemical structure bymeans of LC-TOF-MS, LC-MS/MS, and 1D/2D NMRspectroscopy, and to determine human orosensory recognitionthresholds as well as salivation-inducing activity of purifiedchemosensates using an ethanol-free sensory assay.13

■ MATERIALS AND METHODSChemicals. The following compounds were obtained commer-

cially: Water for chromatographic separations was purified with anintegral 5 system (Millipore, Schwalbach, Germany); n-hexane(Merck, Darmstadt, Germany) and solvents used were of HPLCgrade (J. T. Baker, Deventer, The Netherlands). Deuterated solventscontaining 0.03% trimethylsilane (TMS) were obtained from Euriso-Top (Gif-Sur-Yvette, France). A supercritical fluid (SCF) extract fromdried fruit pods of Z. piperitum was obtained from Synthite Industries

(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 asvehicle 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 ofthe present investigation and had no history of known taste disorders,were trained in sensory experiments with purified referencecompounds at regular intervals for at least two years. The subjectswere experienced in evaluating the orosensation of a tingling Szechuanpepper extract (40 μg/1 × 2 cm; Synthite, Kerala, India), isolated fromZ. piperitum by means of supercritical carbon dioxide extraction andcontaining hydroxy-α-sanshool and hydroxy-β-sanshool in amounts of9.1 and 2.5% (HPLC-UV), respectively, and the pungent compoundscapsaicine (3 μg/1 × 2 cm) and piperine (20 μg/1 × 2 cm),respectively, by means of a modified half-tongue test using filter paperrectangles (1 × 2 cm) as the vehicle.13 Panelists were instructed torinse 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

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).

Journal of Agricultural and Food Chemistry Article

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each sample. Sensory analyses were performed at 19−22 °C in asensory panel room in three different sessions.Precaution Taken for Sensory Analysis of Food Fractions and

Taste Compounds. To remove solvent traces and buffer compoundsfrom all fractions and compounds isolated from pepper, the individualfractions were suspended in water and, after removal of the volatilesunder high vacuum (<5 mPa), were freeze-dried twice. 1H NMRspectroscopic, UPLC-UV-TOF-MS, and ion chromatographic analysesof an aliquot revealed that food fractions treated by that procedurewere essentially free of the solvents and buffer compounds used.Modified Half-Tongue Test. According to a literature protocol,13

serial 1:1 dilutions of HPLC fractions or purified compounds inethanol were applied on filter paper rectangles (1 × 2 cm) and thesolvent was removed under a stream of nitrogen at 38 °C. As thecontrol vehicle, filter paper rectangles were loaded with ethanol(blank), followed by evaporation at 38 °C. Using a 3% sucrosesolution (15 mL) and water (15 mL) as cleansing solutions and aninterstimulus interval length of 5 min between sample dilutions, thevehicles loaded with the stimulus in order of ascending concentrationwere presented to the sensory panel in three independent sessions andwere randomly placed on the right or the left side of the anteriortongue together with an anonymized control vehicle (blank) on theother side. For each dilution, the subjects were asked to locate the sideof the tongue on which a pungent and/or a tingling sensation wasperceivable.Taste Dilution Analysis (TDA). An aliquot (1.0 g) of the SFC

extract prepared from Szechuan pepper was separated by RP-MPLC togive seven fractions, namely, F1−F7, which were freeze-dried twiceand dissolved in “natural” concentration ratios, which means thefractions were used in concentrations corresponding to their yieldsobtained after fractionation. After each fraction had been sequentiallydiluted 1:1 with ethanol and applied on filter paper rectangles (1 × 2cm), solvent traces were removed with a nitrogen stream at 38 °C, andthe serial dilutions of each of these fractions were presented to thesensory panel in order of ascending concentration. Each dilution wasevaluated for tingling activity by means of the half-tongue test detailedabove. The dilution at which a sensory difference between the paperrectangle loaded with a Szechuan pepper fraction and a blank vehicle(control) could just be detected was defined as taste dilution (TD)factor.13 The TD factors evaluated by six trained assessors in twodifferent sessions were averaged. The TD factors between the trainedindividuals and separate sessions did not differ by more than plus/minus one dilution step.Recognition Threshold Concentration. Threshold concentrations

of the purified compounds were determined by using the half-tonguetest and the filter paper rectangles as detailed above. To preventexcessive fatigue, the trials began at a concentration level two stepsbelow the threshold concentration that had been evaluated inpreliminary sensory experiments. Whenever the panelist selectedincorrectly, the next trial took place at the next higher concentrationstep. Whenever the panelist selected correctly, the same dilution of thestimulus was evaluated again as a proof of the correctness of the data.The threshold value of the sensory group was approximated byaveraging the threshold values of the individuals in three independentsessions. The geometric mean of the two lowest correctly identifiedconcentrations was calculated and taken as the individual recognitionthreshold. Values between individuals and separate sessions differed bynot more than plus or minus two dilution steps; that is, a thresholdvalue of 3.0 nmol/cm2 for the pungent reference compound piperinerepresents a range of 0.75−9.0 nmol/cm2.Salivation Experiments. Following a literature protocol15 with

some modifications, eight healthy volunteers were asked not to eat anddrink for at least 1 h before the experiment started. First, the panelistsrinsed the oral cavity with bottled water (8 mL) for 60 s andswallowed. To measure the nonstimulated saliva flow (control), thepanelists then took bottled water (2 mL) in the mouth, simulatedchewing motions for 30 s, and expectorated into preweighed cups. Tomeasure the stimulated saliva flow, the oral cavity was rinsed againwith water (8 mL) for 60 s; thereafter, the panelists took the stimulussolution (2 mL) containing the Szechuan pepper extract (100 mg/100

mL), hydroxy-α-sanshool (100 mg/100 mL), and hydroxy-β-sanshool(100 mg/100 mL), respectively, in the mouth and simulated chewingfor 15 s and expectorated into preweighed vessels (stimulus sample).Without swallowing between, the subjects were asked to take water (2mL) in the mouth, to simulate chewing for 30 s, and to expectorateagain (poststimulus sample 1). This step was repeated three times(poststimulus samples 2−4). The saliva flow increase was calculatedfrom the weight difference of the expectorated saliva/water mixture,and the aliquot (2 g) of the aqueous stimulus solution used to collectthe stimulus sample and the aliquot (2 g) of water used to obtain theprestimulus sample (control) as well as poststimulus samples 1−4,respectively.

Identification of Chemosensates 1−8. A solution of theSzechuan pepper SCF extract (10 g) in methanol (400 mL) wasdefatted by n-hexane extraction (3 × 400 mL), concentrated undervacuum at 40 °C to about 30 mL, and, after dilution with water (30mL), separated by medium-pressure liquid chromatography (MPLC)using a Sepacore system from Buchi (Flawil, Switzerland) consisting ofa C-620 control unit, three C-605 type pumps, a C-660 fractioncollector, a C-635 UV detector, and an injection valve (20 mL).Chromatography was performed using a 40 × 150 mm polypropylenecartridge (Buchi) filled with 25−40 μm LiChroprep RP-18 material(Merck). Monitoring the effluent (40 mL/min) at 265 nm,chromatography was performed with a mixture of aqueous formicacid (0.1% in water, pH 2.5, A) and methanol (B). A gradient was usedas follows: 0 min, 65% B; 17 min, 90% B; 22 min, 90% B; 23 min,100% B; and 28 min, 100% B. Individual fractions were collected,concentrated under vacuum at 40 °C, freeze-dried twice, and used forTDA to locate those fractions inducing a tingling and/or pungentorosensation. Fractions evaluated with high TD factors were furtherseparated by means of preparative HPLC (Jasco, Groß-Umstadt,Germany) equipped with two PU-2087 Plus pumps, an AS-2055 Plusautosampler, a 7725i type Rheodyne injection valve (Rheodyne,Bensheim, Germany), and a MD-2010 Plus diode array detector.

By monitoring the effluent at 265 nm, isolation of amides 2, 3, and7 was performed using a 21.2 × 250 mm, 5 μm, ODS-Hypersil C18column (ThermoHypersil, Kleinostheim, Germany) using aqueousformic acid (0.1% in water, pH 2.5, A) and acetonitrile (B) as mobilephase (21 mL/min) and the following gradient: 0 min, 40% B; 18 min,65% B; 22 min, 100% B; 24 min, 100% B; 27 min, 40% B; 30 min, 40%B.

Separation and purification of amides 1, 4−6, and 8 were achievedon a 21.2 × 250 mm, 5 μm, Varian Pursuit C18 column (Agilent,Waldbronn, Germany), equipped with a 21.2 × 50 mm guard columnof the same type, and a mixture of aqueous formic acid (0.1% in water,pH 2.5, A) and acetonitrile (B) as mobile phase (18 mL/min) usingthe gradient as follows: 0 min, 50% B; 20 min, 70% B; 21 min, 100%B; 23 min, 100% B; 25 min, 50% B; 30 min, 50% B. After the purity ofeach fraction had been checked by means of analytical RP-HPLC, theisolated phytochemicals were analyzed by LC-MS and NMRspectroscopy, whereas the fractions containing mixtures of individualsubstances were further purified by HPLC rechromatography. Theindividual fractions were collected, separated from solvent undervacuum, and freeze-dried to yield the target amides 1−7 in a purity of>98%.

N-(2-Methyl-2-hydroxypropyl)-tetradeca-(2E,4E,8Z,10E,12E)-pen-taene 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-TOF-MS (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 = 15.0Hz, 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,



1H, J = 10.8, 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,10E)-tetraene amide

(hydroxy-α-sanshool), 2, Figure 1: UV−vis (acetonitrile/0.1% aqueousformic 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, 14.44Hz, 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% aqueousformic 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% aqueousformic acid; 7:3, v/v), λmax = 272, 248 nm; LC-TOF-MS (ESI+), m/z292.2278 (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

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 NMRdata (125 MHz, d3-MeOD, HSQC, HMBC) are shown in Table 1.

N-(2-Methyl-2-hydroxypropyl)-tetradeca-(2E,4E,8Z,11E)-tetraeneamide (isobungeanool), 5, Figure 1: UV−vis (acetonitrile/0.1% aqueousformic acid; 7:3, v/v), λmax = 260 nm; LC-TOF-MS (ESI+), m/z292.2274 (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/z274 (100), 72 (29), 131 (26), 119 (26), 95 (26), 203 (9), 175 (8); 1HNMR (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, 15.1Hz, H−C(4)], 7.12 [dd, 1H, J = 10.7, 15.1 Hz, H−C(3)]. The 13CNMR data (125 MHz, d3-MeOD, HSQC, HMBC) are shown in Table1.

N-(2-Methyl-2-hydroxypropyl)-tetradeca-(2E,4E,8E,10E,12E)-pen-taene 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

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) 169.6 167.0 166.9 169.5 169.6 169.6 166.9 169.4 169.5C(2) 123.3 123.7 123.6 123.3 123.2 123.3 123.7 125.1 123.3C(3) 142.4 144.4 144.5 142.4 142.5 142.4 144.4 145.6 142.5C(4) 130.3 32.1 31.9 130.2 130.2 130.3 32.1 33.2 130.3C(5) 143.2 26.5 31.4 143.4 143.5 143.3 26.5 27.7 131.0C(6) 34.0 129.5 132.0 33.9 33.9 33.9 130.3 131.5 34.0C(7) 28.1 129.7 131.6 27.6 27.5 33.2 129.8 131.0 28.2C(8) 130.9 125.2 130.1 129.7 129.8 133.5 127.2 128.6 131.6C(9) 130.8 133.5 131.5 130.0 129.9 133.2 128.4 129.5 130.9C(10) 126.7 131.8 131.6 26.5 31.4 132.7 129.5 130.9 128.7C(11) 134.6 130.2 129.4 128.3 128.5 132.6 127.2 127.8 129.4C(12) 133.3 18.3 18.3 132.8 133.5 131.6 13.5 13.7 131.0C(13) 130.6 21.5 26.6 129.8 127.6C(14) 18.5 14.7 14.4 18.4 13.7C(1′) 51.2 50.4 50.4 51.2 51.2 51.3 50.4 51.1 51.3C(2′) 71.7 71.1 71.1 71.7 71.7 71.5 71.0 72.0 71.8C(3′) 27.3 27.3 27.4 27.3 27.3 27.3 27.3 27.3 27.2C(4′) 27.3 27.3 27.4 27.3 27.3 27.3 27.3 27.3 27.2

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



formic acid; 1:1, v/v), λmax = 208, 276 nm; LC-TOF-MS (ESI+), m/z286.1774 (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, 14.9Hz, 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)-pen-

taenwamide (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, d3-MeOD, 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 thecompounds were measured on a Waters Synapt G2 HDMS massspectrometer (Waters, Manchester, UK) coupled to an Acquity UPLCcore system (Waters, Milford, MA, USA) consisting of a binary solvent

manager, sample manager, and column oven. Analytes were injectedinto the UPLC-TOF-MS system equipped with a 2.1 × 150 mm, 1.7μm, BEH C18 column (Waters, Manchester, UK). Operated with aflow rate of 0.4 mL/min at 45 °C, the following gradient was used forchromatography: starting with a mixture (40:60, v/v) of water andacetonitrile, the acetonitrile content was increased to 100% within 4min 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 positiveESI and the resolution mode using the following ion sourceparameters: capillary voltage, +2.0 kV; sampling cone, 30 V; extractioncone, 4.0 V; source temperature, 150 °C; desolvation temperature, 450°C; cone gas, 30 L/h; and desolvation gas, 850 L/h. Data processingwas performed by using MassLynx 4.1 SCN 779 (Waters, Manchester,UK) and the elemental composition tool for determining the accuratemass. All data were lock mass corrected on the pentapeptide leucineenkephaline (Tyr-Gly-Gly-Phe-Leu, m/z 556.2771, [M + H]+) in asolution (2 ng/μL) of acetonitrile/0.1% formic acid (1:1, v/v). Scantime for the lock mass was set to 0.3 s, an interval of 15 and 3 scans toaverage with a mass window of ±0.3 Da. Calibration of the Synapt G2in the range from m/z 50 to 1300 was performed using a solution ofsodium formate (5 mmol/L) in 2-propanol/water (9:1, v/v). TheUPLC and Synapt G2 systems were operated with MassLnyx software(Waters, Manchester, UK).

High-Performance Liquid Chromatography−Mass Spec-trometry (HPLC-MS/MS). LC-MS/MS analysis was performedusing an Dionex Ultimate 3000 HPLC system connected to the API4000QTrap LC-MS/MS (AB Sciex, Darmstadt, Germany) running inthe positive electrospray ionization (ESI+) mode. Zero grade air servedas 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 andproduct 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−1H-gCOSY), heteronuclear single-quantum coherence spectroscopy(gHSQC), heteronuclear multiple-bond correlation spectroscopy(gHMBC), and 1H−1H rotating frame nuclear Overhauser enhance-ment spectroscopy (phase-sensitive ROESY) NMR measurementswere performed on an Avance III 500 MHz equipped with a CTCIprobe and an Avance III 400 MHz spectrometer with a BBO probe(Bruker, Rheinstetten, Germany), respectively. Chemical shifts werereferenced to the solvent signal. Data processing was performed by

Figure 2. (A) MPLC chromatographic separation of the methanol solubles of Szechuan pepper extract and (B) taste dilution analysis (TDA)recording the tingling orosensation.



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.



using Topspin version 2.1 (Bruker) and MestReNova version 6.2.1software (Mestrelab Research, Santiago de Compostela, Spain).

■ RESULTS AND DISCUSSIONUsing the recently developed half-mouth test,13 orosensoryevaluation of paper vehicles loaded with dilutions of the SCFextract prepared from Z. piperitum revealed a strong and long-lasting tingling activity in the oral cavity perceived up to adilution of 1:16384 (data not shown). To locate the most activechemosensates in Szechuan pepper, the SFC extract wasdissolved in methanol and extracted with hexane, both fractionswere separated from solvent, and dilutions of the hexaneextractables as well as the methanol solubles were judged innatural concentration ratios in their tingling activity by meansof a TDA. The methanol solubles induced a strong tinglingsensation judged with a TD factor of 8192, whereas the hexaneextractables showed comparatively low activity judged with aTD factor of only 128. As a consequence, the followinginvestigation on the primary tingling components in Szechuanpepper was focused on the methanol solubles.Taste Dilution Analysis. To sort out the tingling

compounds from the bulk of less taste-active or tastelesssubstances, the tingling-active methanol solubles of the SFCextract were separated by means of MPLC on RP18 material togive seven fractions, namely F1−F7, which were collectedindividually, separated from solvent, and used for the TDAusing paper disks as vehicles.13 Fractions F2 and F3 showed byfar the highest TD factors of 4096 and 2048, respectively,whereas all other fractions showed only low tingling activityjudged with TD factors of ≤128 (Figure 2). Aimed atcharacterizing the molecular structure of the compoundsimparting the most intense tingling sensation of Szechuanpepper, further fractionation and LC-MS as well as NMRexperiments were focused on MPLC fractions F2 and F3.

Isolation and Identification of Chemosensates inFractions F2 and F3. Separation of fractions F2 and F3 bymeans of preparative RP-HPLC revealed nearly baselineseparation of their components, which could be isolated orfurther 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 246as the main fragment ion for the three main compounds 2, 3,and 7 eluting in fraction F2 (Figure 3A). Exemplified forcompound 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 andan allyl cleavage, respectively. Moreover, the isobariccompounds 2, 3, and 7 were found by LC-TOF-MS to showidentical elemental compositions of C16H25NO2. In compar-ison, the early-eluting compounds 1, 6, and 8 in fraction F3(Figure 3B) showed a pseudomolecular ion ([M + H]+) of m/z290 and a main fragment ion with m/z 272, well in agreementwith the molecular formula of C18H27NO2 as indicated by LC-TOF-MS analysis. The late-eluting compounds 4 and 5 infraction F3 differed by a mass increment of 2, consistent withan elemental composition of C18H29NO2 (LC-TOF-MS), andshowed m/z 292 and 274 as the [M + H]+ and the mainfragment ion, respectively.Unequivocal assignment of protons and carbon atoms could

be successfully achieved by means of 1H, 13C, DEPT-135,1H−1H-gCOSY, gHMBC optimized for 2JC,H and 3JC,H couplingconstants, and gHSQC optimized for 1JC,H coupling constants,respectively (Table 1). Comparison of 1H and 13C NMR datawith literature data1,2 led to the identification of the amideshydroxy-γ-sanshool (1), hydroxy-α-sanshool (2), hydroxy-β-sanshool (3), bungeanool (4), isobungeanool (5), and hydroxy-γ-isosanshool (6), respectively.

Figure 5. 13C NMR spectra (125 MHz, CDCl3) of alkylamides 2, 3, and 7.



The 1H NMR of compound 7 showed eight olefinic protonsof the polyunsaturated fatty acid chain, namely, H−C(2), H−C(3), and H−C(6) to H−C(11), resonating in the range of5.43−6.87 ppm, and six aliphatic protons, which could beassigned 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 thefatty acid moiety. Unequivocal assignment of all protons andcarbon atoms could be successfully achieved by means of 1D/2D NMR spectroscopy, which identified 7 as a geometricalisomer of amides 2 and 3 showing four double bonds atcarbons C(2), C(6), C(8), and C(10), respectively. Thegeometry of the individual double bonds was unambiguouslyelucidated by comparing the 3JH,H coupling constants of theolefinic protons and the 13C NMR chemical shifts recorded forthe individual isomers, as well as by means of ROESYexperiments. The protons H−C(2) and H−C(3), bothresonating as double triplets, and the two double dupletprotons H−C(8) and H−C(9) showed the characteristic trans-olefinic coupling constants of 15.2 and 14.8 Hz, respectively. Incontrast, the olefinic protons H−C(10) and H−C(11) as wellas H−C(6) and H−C(7) showed a 3JH,H coupling constant of∼10.8 Hz, thus indicating a cis-configuration. By comparing the13C chemical shifts of compounds 2, 3, and 7, the methylenecarbon atom C(5) and the methyl carbon C(12), both adjacentto the cis-configured double bond, showed a clear upfield shiftin 7 when compared to the all-trans-isomer 3 (Table 1; Figure5). The upfield shift of the carbon C(5) from 31.4 to 26.5 ppmin 7 and 2 compared to 3 is well in agreement with a cis-shielding effect reported in the literature.2,7−9 Anotherfingerprint carbon atom, namely C(12), showed an upfieldshift of ∼5 ppm in 7 when compared to its geometric isomers 2and 3, respectively (Table 1; Figure 5). The configurations ofthe double bonds were further supported by means of aROESY experiment; for example, a ROESY correlation wasdetected between the methyl protons H−C(12) and methineproton H−C(9). Taking all spectroscopic data into consid-eration, LC-TOF-MS, LC-MS/MS, and 1D/2D NMR experi-ments led to the unequivocal identification of compound 7 asN-(2-methyl-2-hydroxypropyl)dodeca-(2E,6Z,8E,10Z)-tetraeneamide (hydroxy-ε-sanshool). which has already been men-tioned in the literature,3,5,6,10 although its chemical structurehas not yet been confirmed by means of 1D/2D NMRspectroscopy.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), thusindicating the cleavage of water (m/z 272), and an alphacleavage (m/z 173; m/z 202), as well as an allyl cleavage (m/z107), respectively. The 1H NMR of 8 showed 10 olefinicprotons, 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 aliphaticprotons 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 carbonatoms revealed 8 to be a geometrical isomer of the amides 1and 6, respectively. The 3JH,H coupling constants of 15.5 and14.9 Hz of the two double duplets detected for H−C(10) andH−C(11) indicated a trans-configuration of this double bond(Figure 6). Moreover, carbon atoms C(7) and C(14) showedan upfield shift of ∼5 ppm in 8 when compared to its geometricisomers 1 and 6 (Table 1; Figure 1), being well in agreement

with the expected cis-shielding effect.2,7−9 Taking all spectro-scopic data into consideration, the structure of compound 8was determined to be N-(2-methyl-2-hydroxypropyl)tetradeca-(2E,4E,8Z,10E,12Z)-pentaene amide, coined hydroxy-ζ-san-shool, which to the best of our knowledge has not beenreported before.

Sensory Evaluation of Pungent and/or TinglingChemosensates. Prior to sensory analysis, the purity of allcompounds was confirmed by LC-MS as well as 1H NMRspectroscopy to be >98%. To determine the human thresholdconcentrations for the orosensation induced by the individualchemosensates, paper vehicles loaded with the test compoundsin serial dilutions were evaluated by means of a half-tonguetest.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) werefound to elicit an oral tingling and paresthetic sensation above arather similar threshold concentrations ranging between 3.5 and8.3 nmol/cm2, respectively (Table 2). It is interesting to notethat 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 thresholdconcentration of these amides. Interestingly, the absence of acis-double bond, as found for hydroxy-β-sanshool (3) andhydroxy-γ-isosanshool (6), resulted in a complete loss of thetingling activity. This is well in agreement with earlier studies

Figure 6. Excerpt (δ 6.3−6.7) of the 1H NMR spectra (500 MHz, d3-MeOD) of alkylamide 8 showing H−C(10) and H−C(11).

Table 2. Orosensory Recognition Thresholds of Alkylamides1−8

compounda oral sensation threshold concnb (nmol/cm2)

1 tingling, paresthetic 6.82 tingling, paresthetic 8.33 numbing, anesthetic 3.94 tingling, paresthetic 3.55 tingling, paresthetic 3.56 numbing, anesthetic 7.17 tingling, paresthetic 4.28 tingling, paresthetic 7.0

aStructures of compounds are given in Figure 1. bOrosensoryrecognition threshold concentrations were determined by means of amodified half-mouth test using paper rectangles (1 × 2 cm) as avehicle.13



on synthetic polyunsaturated alkylamides10 and those isolatedfrom black pepper;13 for example, saturation of the doublebond in black pepper’s tingling compound (2E,4E,12Z)-N-isobutyloctadeca-2,4,12-trienamide wiped out the tinglingactivity of (2E,4E)-N-isobutyloctadeca-2,4-dienamide. Althoughno tingling activity was detectable for the all-trans-configuredcompounds 3 and 6, most interestingly, the sensory panelistsreported an intense and long-lasting numbing and anestheticoral 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 amideshydroxy-α-sanshool (2, tingling) and hydroxy-β-sanshool (3,numbing) were isolated in suitable amounts and aqueoussolutions were used to orally challenge a panel of eight healthyvolunteers for 15 s; the saliva flow was measured for 120 s usingan assay reported recently.15 A comparative experiment wasperformed with an aqueous solution of the crude Szechuanpepper extract containing 9.1% hydroxy-α-sanshool and 2.5%hydroxy-β-sanshool. All three stimuli significantly activated along-lasting saliva flow increase (Figure 7). The tingling

hydroxy-α-sanshool induced a massive increase of saliva flowaccounting for a maximum of ∼75% after 45 s when comparedto the control. Also, the Szechuan pepper extract showed astrong increase of saliva flow (∼44% after 45 s), whereas thenumbing hydroxy-β-sanshool showed only a marginal activityaccounting for a saliva flow increase of ∼25%. Thisdemonstrates that at least one cis-configured double bond isneeded for strong salivating activity, being well in agreementwith literature data on spilanthol,14 whereas the all-trans-configuration diminishes salivation. As the tingling hydroxy-α-sanshool is the quantitatively predominant amide in Szechuanpepper, this isomer is concluded to be mainly responsible forthe tingling as well as the salivation enhancement activity ofSzechuan 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 cationchannels TRPV1 and TRPA1,18 newer evidence suggests that

neuron activation through a unique mechanism involvinginhibition of the pH- and anesthetic-sensitive two-porepotassium channels TASK-1, TASK-3, and TRESK (previouslynamed KCNK3, KCNK9, and KCNK18) is mediating thetingling effect of hydroxy-α-sanshool.19 The finding that a cleartingling, paresthetic sensation is induced by amides containingat least one cis-configured double bond as found in 1, 2, 4, 5, 7,and 8, whereas an intense numbing and anesthetic impressionis caused by all-trans-configured amides (3 and 6), promises thevarious isomers as valuable molecular probes to test candidateion channels TASK-1, TASK-3, and TRESK for mediating thetingling effect and to challenge other receptive proteins formediating the numbing and anesthetic effect. Such experimentswill be helpful to deliver a framework for understanding theunique and complex psychophysical sensations associated withthe Szechuan pepper experience.

■ AUTHOR INFORMATIONCorresponding Author*(T.H.) Phone: +49-8161-71-2902. Fax: +49-8161-71-2949. E-mail: [email protected] Contributions∥M.B. and T.D.S. contributed equally to this work.NotesThe authors declare no competing financial interest.

■ 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 unsaturatedaliphatic acid amides in Japanese Zanthoxylum species. Phytochemistry1982, 21, 1295−1298.(3) Bryant, B. P.; Mezine, I. Alkylamides that produce tinglingparesthesia 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 ofZanthoxylum piperitum. Arch. Pharm. Res. 2008, 31, 569−572.(6) Yang, X. Aroma constituents and alkylamides of red and greenHuajiao (Zanthoxylum bungeanum and Zanthoxylum schinifolium). J.Agric. Food Chem. 2008, 56, 1689−1696.(7) Yasuda, I.; Takeya, K.; Itokawa, H. Two new pungent principlesisolated from the pericarps of Zanthoxylum ailanthoides. Chem. Pharm.Bull. 1981, 29, 1791−1793.(8) Yasuda, I.; Takeya, K.; Itokawa, H. Structures of amides fromAsiasarum heterotropoides Maek. var. mandshuricum Maek. Chem.Pharm. Bull. 1981, 29, 564−566.(9) Yasuda, I.; Takeya, K.; Itokawa, H. The geometric structure ofspilanthol. Chem. Pharm. Bull. 1980, 28, 2251−2253.(10) Galopin, Ch. C.; Furrer, S. M.; Goeke, A. Pungent and tinglingcompounds in Asian cuisine. In Challenges in Taste Chemistry andBiology; Hofmann, T., Ho, C. T., Pickenhagen, W., Eds.; ACSSymposium 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 evaluatedby 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 aqueousstimulus 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 isvisualized (LSD = 8.263).



mailto:[email protected]

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