New Umami Amides: Structure-Taste Relationship Studies of Cinnamic Acid Derived Amides and the Natural Occurrence of an Intense Umami Amide in Zanthoxylum piperitum

Article abstract of DOI:10.1021/acs.jafc.5b02359

A series of aromatic amides were synthesized from various acids and amines selected from naturally occurring structural frameworks. These synthetic amides were evaluated for umami taste in comparison with monosodium glutamate. The effect of the substituti

Full text of DOI:10.1021/acs.jafc.5b02359

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Journal of Agricultural and Food Chemistry
New Umami Amides: Structure-Taste Relationship Studies of Cinnamic Acid-Derived Amides
and Natural Occurrence of an Intense Umami Amide in Zanthoxylum piperitum
Eric Frerota,*, Nathalie Neyrincka,b, Isabelle Cayeuxa, Yoyo Yi-Yuanc, Yong-Ming Yuanc
aFirmenich SA, Corporate R&D Division, P.O. Box 239, CH-1211 Geneva 8, Switzerland
bPresent address: Schwarzkopf Professional, 161, rue de Silly. 92 642 Boulogne Billancourt,
France
cFirmenich Aromatics (China) Co., Ltd., No 3901 JinDu Road, Xinzhuang Industry Park,
Shanghai 201108, China
*Author to whom correspondence should be addressed. Tel: +41 22 780 35 66,
Fax: +41 22 780 33 34, e-mail eric.frerot@firmenich.com
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ABSTRACT
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A series of aromatic amides was synthesized from various acids and amines selected from
naturally occurring structural frameworks. These synthetic amides were evaluated for
umami taste in comparison with monosodium glutamate. The effect of the substitution
pattern of both the acid and the amine parts on umami taste was investigated. The only
intense umami-tasting amides were those made from 3,4-dimethoxycinnamic acid. The
amine part was more tolerant to structural changes. Amides bearing an alkyl- or alkoxy-
substituted phenylethylamine residue displayed a clean umami taste as 20 ppm solutions in
water. Ultra-performance liquid chromatography coupled with a high quadrupole-Orbitrap
mass spectrometer (UPLC/MS) was subsequently used to show the natural occurrence of
these amides. (E)-3-(3,4-dimethoxyphenyl)-N-(4-methoxyphenethyl)acrylamide was shown
to occur in the roots and stems of Zanthoxylum piperitum, a plant of the family Rutaceae
growing in Korea, Japan, and China.
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KEYWORDS
Aromatic amides, umami taste, UPLC/MS, Zanthoxylum piperitum
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INTRODUCTION
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Amides are ubiquitous compounds in nature, especially in plants of the genus Piperaceae.1
Many of the amides occurring in foods elicit a trigeminal sensation. Thus, capsaicin is
responsible for the hot sensation of red pepper (Capsicum annuum L.)2 and piperine for that
of black pepper (Piper nigrum L.)3. Red Sichuan pepper (Zanthoxylum bungeanum Maxim.)4,
jambu oleoresin (extract from Spilanthes acmella L. flowers)5 and pellitory roots (Anacyclus
pyrethrum L.)6 provide another trigeminal effect, called tingling. This effect is mostly
attributed to the presence of unsaturated amides: hydroxy-α-sanshool in red Sichuan
pepper, spilanthol in jambu oleoresin and pellitorin in pellitory. There is no mention in the
peer-reviewed literature of naturally occurring alkyl- or aromatic amides that provide umami
taste.
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The interest in new umami molecules was reviewed by Winkel and colleagues.7 In addition
to amino acid derivatives, small peptides, and nucleotide derivatives, recent years have seen
the emergence of umami compounds that are active at very low levels. Tachdjian et al. of
Senomyx used receptor-based assays to screen libraries of thousands of compounds, among
which amides such as FEMA 4233 and FEMA 4232 (Figure 1) were found to possess umami
taste at concentrations as low as 1 μM.8,9
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Dewis et al. of IFF (International Flavors and Fragrances) patented many unsaturated
alkamides as umami or more general taste enhancers.10-11 These amides have structural
similarities to naturally occurring amides. One example of a polyunsaturated isobutylamide
similar to spilanthol, is presented in Figure 1.11 In addition, Backes et al. of Symrise proposed
umami amides that are based on neomenthylamine.12
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The work of Adesina showed the occurrence of various cinnamic acid-related amides in
Nigerian plants of the Rutaceae family, in particular Zanthoxylum rubescens Planch. ex Hook.
(Figure 2).13,14 Plants of this genus are generally used as spice or as phytomedicine to cure
various diseases.15,16 At the beginning of the present work, the taste of the amides occurring
in Z. rubescens13 had not been described. When we tasted Rubemamine 1, we noticed its
umami taste and this prompted us to study other cinnamic acid-derived amides for their
potential umami taste. Rubemamine 1 was included in a patent of 2003 from Symrise
related to the pungent taste of ferulic acid amides17 and in 2012 in another patent claiming
the umami taste of cinnamic acid related amides.18
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The subject of this work includes the synthesis of many cinnamic acid-related amides, their
sensory evaluation for umami taste, the necessary structural features for umami taste,19 and
the subsequent analysis of Zanthoxylum piperitum to show the natural occurrence of these
umami amides.
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MATERIALS AND METHODS
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General. Unless otherwise specified, commercially available reagents and solvents were
purchased from Fluka-Sigma-Aldrich, Buchs, Switzerland; Acros Organics, Geel, Belgium; and
Carlo-Erba, Val de Reuil, France.
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NMR Spectra. 1H- and 13C-NMR spectra were recorded in CDCl3 on a DPX 400 spectrometer
(Bruker, Rheinstetten, Germany) at 25 °C operated at 400 MHz (1H) or 100 MHz (13C), with
tetramethylsilane as the internal standard. The 13C signal assignments were obtained from
standard gradient-selected correlated spectroscopy, heteronuclear single quantum
coherence, and heteronuclear multiple bond correlation experiments performed on a Bruker
Avance 500 spectrometer. NMR spectra were processed with Bruker TopSpin 2.0 software
(s, singlet; d, doublet; t, triplet; m, multiplet).
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UPLC-MS Method. Analyses of samples were performed on a Acquity I-Class UPLC system
(Waters, Milford, USA) coupled with a Q-Exactive Plus mass spectrometer (Thermo, San Jose,
CA, USA). The column was an Acquity HSS-T3, 100 × 2.1 mm i.d. (Waters) operated at 30 °C.
Solvent A was UPLC/MS grade water (BioSolve, Valkenswaard, The Netherlands) containing
0.1% MS grade formic acid (Sigma-Aldrich, Steinheim, Germany). Solvent B was acetonitrile
(Biosolve) containing 0.1% formic acid at 0.3 mL/mL. The gradient elution was as follows:
40%, 1 min; 40-80% B for 9 min; 80% B, 1 min; and re-equilibration at 40% B for 1 min.
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The mass spectrometer was equipped with an H-ESI II electrospray source operated in the
positive mode: spray 3500 V, probe heater 400 °C, heated capillary 300 °C, sheath gas 50 arb
units, auxiliary gas 10. The mass calibration was performed each week. The “full
scan/ddMS2” combined with the “tMS2” acquisition modes were used. The full scan/ddMS2
triggered the recording of the MS2 spectra for the three most intense ions. The targeted MS2
mode (tMS2) recorded the MS2 spectra of 15 amides (see Results and Discussion) on the
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basis of an inclusion list containing their molecular formulae. The full-scan spectra were
acquired with a resolution of 70,000 from 100 to 1000 Da and the MS2 spectra with a
resolution of 17,500 and a higher energy collisional dissociation cell energy of 30 V.
Automated gain control was targeted to 3.106 and 1.105 ions with a maximum injection time
of 100 ms for full scan and 50 ms for ddMS2.
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Synthesis of Amides with Ethyl Chloroformate, General Procedure. The acid component
(typically 33 mmol) and diisopropyl ethyl amine (2 equiv) were diluted in 200 mL of EtOAc
and 50 mL of dichloromethane. The solution was cooled to 15 °C, and ethyl chloroformate (1
molar equiv) was added drop wise. The reaction was stirred for 1 h before the primary
amine component (1 molar equiv, diluted 2-3 times in EtOAc) was added. The reaction was
stirred overnight at room temperature. It was washed with aqueous 5% KHSO4, brine,
aqueous 5% NaHCO3, and brine, and then dried over Na2SO4 and evaporated under high
vacuum for 3 h. The crude product was purified by flash chromatography (silica gel;
cyclohexane/EtOAc, e.g. 2:8). Yields were between 50 and 80% of the purified product. MS
and UV data only are given below. The description of 1H and 13C NMR spectra can be found
as supporting information.
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Amide 1, Rubemamine. (E)-N-(3,4-dimethoxyphenethyl)-3-(3,4-dimethoxyphenyl)acrylamide
ESI (+): 372.1798 (C21H26O5N, -2.1 ppm). UV max (nm): 316, 287, 222.
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Amide 2. (E)-N-(3,4-dimethoxyphenethyl)-3-(4-methoxyphenyl)acrylamide
ESI (+): 342.1690 (C20H24O4N, -2.8 ppm). UV max (nm): 310, 288, 224.
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Amide 3. (E)-3-(benzo[d][1,3]dioxol-5-yl)-N-(3,4-dimethoxyphenethyl)acrylamide
ESI (+): 356.1483 (C20H22O5N, -4.2 ppm). UV max (nm): 322, 286, 228, 219.
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Amide 4. (E)-3-(3,4-dimethoxyphenyl)-N-(4-methoxyphenethyl)acrylamide
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ESI (+): 342.1693 (C20H24O4N, -2.0 ppm). UV max (nm): 316, 291, 223.
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Amide 5. (Z)-3-(3,4-dimethoxyphenyl)-N-(4-methoxyphenethyl)acrylamide. Amide 4 (500
mg) in 50 mL of ethanol was irradiated for 16 h by using a water-cooled 125 W Hg lamp.
UPLC analysis showed that the crude product contained 50% of the (Z)-isomer. The product
was chromatographed over a Puriflash 50 C18 cartridge (Interchim¸ Montluçon, France)
eluted with water:ethanol (60:40). A mixture of amide 5 and 4 (90:10) was obtained (96 mg).
ESI (+): 342.1693 (C20H24O4N, -2.1 ppm). UV max (nm): 310, 276, 224.
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Amide 6. 3-(3,4-dimethoxyphenyl)-N-(4-methoxyphenethyl)propanamide
ESI (+): 344.1849 (C20H26O4N, -2.0 ppm). UV max (nm): 277, 225.
Amide 7. N-(4-methoxyphenethyl)cinnamamide
ESI (+): 282.1481 (C18H20O2N, -2.8 ppm). UV max (nm): 275, 222.
Amide 8. N-(3-methoxyphenethyl)cinnamamide
ESI (+): 282.1491 (C18H20O2N, -2.6 ppm). UV max (nm): 274, 223.
Amide 9. (E)-N-(4-methoxyphenethyl)-3-(4-methoxyphenyl)acrylamide
ESI (+): 312.1585 (C19H22O3N, -2.9 ppm). UV max (nm): 310, 290, 224.
Amide 10. (E)-N-(4-methoxyphenethyl)-3-(2-methoxyphenyl)acrylamide
ESI (+): 312.1586 (C19H22O3N, -2.7 ppm). UV max (nm): 317, 274, 225.
Amide 11. (E)-N-(4-methoxyphenethyl)-3-(3-methoxyphenyl)acrylamide
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ESI (+): 312.1587 (C19H22O3N, -2.4 ppm). UV max (nm): 310, 277, 224.
Amide 12. (E)-3-(2,5-dimethoxyphenyl)-N-(4-methoxyphenethyl)acrylamide
ESI (+): 342.1690 (C20H24O4N, -2.8 ppm). UV max (nm): 342, 276, 223.
Amide 13. (E)-N-(4-methoxyphenethyl)-3-(3,4,5-trimethoxyphenyl)acrylamide
ESI (+): 372.1797 (C21H26O5N, -2.4 ppm). UV max (nm): 299, 228.
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Amide 14. (E)-2-methoxy-4-(3-((4-methoxyphenethyl)amino)-3-oxoprop-1-en-1-yl)phenyl
acetate. ESI (+): 370.1639 (C21H24O5N, -2.6 ppm). UV max (nm): 310, 277, 224.
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Amide 15. (E)-3-(4-hydroxy-3-methoxyphenyl)-N-(4-methoxyphenethyl)acrylamide. Amide
14 was deprotected in MeOH/Na2CO3. The reaction was acidified and extracted from EtOAc.
The product was recrystallized from EtOAc (yield 75%). ESI (+): 328.1535 (C19H22O4N, -2.5
ppm). UV max (nm): 317, 293, 222.
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Amide 16. (E)-3-(benzo[d][1,3]dioxol-5-yl)-N-(4-methoxyphenethyl)acrylamide
ESI (+): 326.1379 (C19H20O4N, -2.4 ppm). UV max (nm): 322, 284, 219.
Amide 17. (E)-3-(3,4-dimethylphenyl)-N-(4-methoxyphenethyl)acrylamide
ESI (+): 310.1794 (C20H24O2N, -2.6 ppm). UV max (nm): 285, 224.
Amide 18. (E)-3-(3,4-dimethoxyphenyl)-N-phenethylacrylamide
ESI (+): 312.1588 (C19H22O3N, -2.0 ppm). UV max (nm): 317, 291, 235, 211.
Amide 19. (E)-3-(3,4-dimethoxyphenyl)-N-(3-methoxyphenethyl)acrylamide
ESI (+): 342.1693 (C20H24O4N, -2.1 ppm). UV max (nm): 317, 291, 218.
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Amide 20. (E)-3-(3,4-dimethoxyphenyl)-N-(2-methoxyphenethyl)acrylamide
ESI (+): 342.1691 (C20H24O4N, -2.7 ppm). UV max (nm): 317, 291, 220.
Amide 21. (E)-N-(3,5-dimethoxyphenethyl)-3-(3,4-dimethoxyphenyl)acrylamide
ESI (+): 372.1797 (C21H26O5N, -2.4 ppm). UV max (nm): 317, 292, 219.
Amide 22. (E)-3-(3,4-dimethoxyphenyl)-N-(3-ethoxyphenethyl)acrylamide
ESI (+): 356.1848 (C21H26O4N, -2.4 ppm). UV max (nm): 316, 292, 219.
Amide 23. (E)-3-(3,4-dimethoxyphenyl)-N-(3-propoxyphenethyl)acrylamide
ESI (+): 370.2005 (C22H28O4N, -2.2 ppm). UV max (nm): 316, 290, 226.
Amide 24. (E)-3-(3,4-dimethoxyphenyl)-N-(4-isopropoxyphenethyl)acrylamide
ESI (+): 370.2005 (C21H28O4N, -2.1 ppm). UV max (nm): 316, 290, 226.
Amide 25. (E)-3-(3,4-dimethoxyphenyl)-N-(4-ethylphenethyl)acrylamide
ESI (+): 340.1899 (C21H26O3N, -2.3 ppm). UV max (nm): 316, 292, 220.
Amide 26. (E)-3-(3,4-dimethoxyphenyl)-N-(4-isopropylphenethyl)acrylamide
ESI (+): 354.2055 (C22H28O3N, -2.4 ppm). UV max (nm): 316, 292, 219.
Amide 27. (E)-3-(3,4-dimethoxyphenyl)-N-(3,4-dimethylphenethyl)acrylamide
ESI (+): 340.1899 (C21H26O3N, -2.6 ppm). UV max (nm): 316, 292, 218.
Amide 28. (E)-3-(3,4-dimethoxyphenyl)-N-(4-methoxybenzyl)acrylamide
ESI (+): 328.1537 (C19H22O4N, -2.6 ppm). UV max (nm): 317, 292, 223.
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Amide 29. (E)-N-(benzo[d][1,3]dioxol-5-ylmethyl)-3-(3,4-dimethoxyphenyl)acrylamide
ESI (+): 342.1327 (C19H20O5N, -2.7 ppm). UV max (nm): 317, 292, 235, 218.
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Amide 30: Beecheyamide. (E)-3-(3,4-dimethoxyphenyl)-N-(4-methoxyphenethyl)-N-
methylacrylamide. A solution of amide 4 (478 mg, 1.4 mmol) in 14 mL of tetrahydrofuran
(THF) was added to a suspension of sodium hydride (120 mg, 5.0 mmol) in 9 mL of THF at 0
°C under argon atmosphere. The reaction was stirred for 30 min at 0 °C before 240 mg of
methyl iodide was added. The reaction was stirred at room temperature overnight, the
mixture poured into 50 mL of cold water, and the pH adjusted to 7 with 5% KHSO4.
Extraction from EtOAc, followed by flash chromatography (pure EtOAc), gave 402 mg of 20
(yield 81%). The product was fully in agreement with the NMR data (showing two rotamers
of the amide bond) described by Cheng et al.20 ESI (+): 356.1847 (C21H26O4N, -2.6 ppm). UV
max (nm): 320, 285, 224.
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Amide 31. Podocarpamide. (E)-N-(2-(benzo[d][1,3]dioxol-5-yl)ethyl)-3-(3,4-
dimethoxyphenyl)-N-methylacrylamide. Amide 16 (1.70 g, 5.2 mmol) in 50 mL of THF was
added drop wise to a suspension of sodium hydride (150 mg, 1.2 equiv) in 34 mL of THF at 0
°C under argon atmosphere. The reaction was stirred for 30 min at 0 °C. Iodomethane (390
µL, 1.2 equiv) was added and the reaction was stirred overnight at room temperature. After
workup and flash chromatography (cyclohexane:EtOAc, 60:40), 1.37 g was obtained (77%
yield). NMR data (showing two rotamers of the amide bond) were compliant with those
reported by Delle Monache et al.21 ESI (+): 340.1535 (C20H22O4N, -2.4 ppm). UV max (nm):
321, 285, 232.
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Amide 32. (E)-3-(4-hydroxy-3-methoxyphenyl)-N-(4-methoxyphenethyl)-N-methylacrylamide
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4-Acetoxy-3-methoxycinnamic acid (360 mg, 1.5 mmol, Alfa Aesar) was condensed on [2-(4-
methoxy-phenyl)-ethyl]methylamine (1 equiv, Aldrich), as described above. The product was
purified by flash chromatography (cyclohexane:EtOAc, 50:50) and subsequently deprotected
(MeOH:1M KOH, 1/1 v:v). The reaction was neutralized by addition of 5% KHSO4 and the
product was extracted 3 x with EtOAc, then with CH2Cl2. After drying over Na2SO4 and
evaporation, 132 mg of pale yellow oil was obtained (26%). NMR showed the presence of
two rotamers (as described for amide 3020) in a 55:45 ratio. ESI (+): 342.1691 (C20H24O4N, -
2.6 ppm). UV max (nm): 321, 285, 232.
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Preparation of the Extracts from Zanthoxylum piperitum (L.) DC. The stems and roots were
separately cut from the fresh plants, and 207.15 g stems and 110.44 g of roots were
obtained. The stems were ground into a fine powder, poured into 4 L of dichloromethane,
stirred for 6 h, and left standing over the weekend. The suspension was filtered and the
solvent was evaporated to yield 1.06 g of brownish oil (0.51% w/w). Similarly, the roots gave
1.6 g of extract (1.45% w/w). The botanical identification of the species was confirmed by a
professional botanist (Y.-M. Yuan). Voucher specimens were prepared and kept in the
herbarium of the company.
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Sensory Evaluation.
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All of the compounds were assessed by a toxicologist and many of them classified as Cramer
class III. Therefore a daily intake of 90 µg/day was allowed. Only one compound of the
cinnamic amides family was tasted per session using the “sip and spit” method for 20 ml of
solution at 20 ppm therefore minimizing the amount of ingested product. All panelists had
signed a consent form informing them about the risk associated with such tasting of new
compounds.
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Umami Score (small panel). Most of the amides were evaluated by a panel of four to nine
trained people. Each amide was evaluated at 20 ppm in Henniez water against 0.05%
monosodium glutamate (MSG) in a blind pair test. The panelists were asked to score the
umami taste on a scale of 0-10 for the sample in each cup. The relative umami score was
calculated as follows: Relative umami score = (amide score/MSG score) × 10
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Umami Score (large panel). To assess the sensory performance of the selected amides with
a larger panel, we asked 15 panelists to evaluate them at 20 ppm or 100 ppm in water
(Henniez mineral water) alone and in a 0.05% MSG solution. They were asked to taste the
sample while using nose clips in order to focus on taste and trigeminal sensations in the
mouth, and then without nose clips in order to describe the olfactive notes. The panelists
evaluated the samples (20 mL served in a black cup) blind by sipping and spitting the sample
out after 5 s before answering the questions. For the pure water system, the panelists had
to select the most appropriate attributes from attributes lists (see below) when they were
and were not using nose clips. The attributes were ranked by decreasing order as a function
of attribute number cited by the panelists. In the MSG model system, all solutions tasted
contained 0.05% MSG and the panelists had to evaluate the perceived umami intensity in
the reference solution and in the samples by using a linear scale score from 0 (not intense)
to 10 (very intense). The potential umami-enhancing effect of the amide was then evaluated
by comparing the perceived intensity of the samples “amide in MSG” vs “MSG alone”
(“delta” score corresponding to the intensities of “amide in MSG” minus “MSG alone”). A
student test (paired test, one tailed) was performed to evaluate the significance of the
observed umami difference. The following is the list of attributes: with nose clips: astringent,
bitter, cooling, fatty, hot, licorice taste, metallic, mouthfeel, pungent, salivating, salty, sour,
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sweet, tingling, umami; without nose clips: earthy, fruity, green, herbal, mushroom, nutty,
tea, woody.
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RESULTS AND DISCUSSION
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Structure Taste Relationship Study of Nature-Inspired Amides. Most of the amides
occurring in Z. rubescens consist of amides of substituted cinnamic acid and
phenethylamine.13 Throughout this study, 29 such amides (1-29) bearing different
substituents were synthesized (figure 3 and table 1). A few amides shown in table 1 were
already identified in plants13,22,23 or microorganisms24. We limited the study to amides
formed from acids and amines whose structural scaffolds were close to those already known
to occur in nature. We thought it might increase the chances of showing the natural
occurrence of these amides afterwards by targeted analysis. Thus, for example, 4-
methoxyphenethylamine clearly derives from the amino acid tyrosine, but 3-
methoxyphenethylamine could also form from the uncommon but naturally occurring meta-
tyrosine.25 The amide bond was formed from the mixed carbonic anhydride generated by
reaction of the acid with ethyl chloroformate, followed by addition of the amine. The
synthetic amides were subsequently evaluated for their umami taste by a panel of four to
nine trained people. A relative umami score was calculated as follows: (amide score/MSG
score) × 10. For a umami score of 10, the umami taste intensity of the amide at 20 ppm was
perceived as being equal to that of 0.05% MSG. Thus, rubemamine 1, which was perceived
as umami with a score of 3.3, was used as a lead compound in this study. The structural
optimization was performed step by step by varying the substituents independently.
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The comparison of the relative umami score for these amides shown in Table 1 led to the
following conclusions. The double bond of the (E) configuration in the acid part was needed
because compound 5 of the (Z)-configuration and the saturated compound 6 did not have a
umami taste. Two methoxy groups were the most favorable (4 vs 7, 9-11, and 13) and they
should be in the meta and para position (4 better than 12). With substituents other than
methoxy groups (14-17), the umami taste intensity decreased significantly. Thus, it appeared
that 3,4-dimethoxycinnamic acid as the acid part of the amide was needed. The
phenethylamine part (n = 1) tolerated much more variation on the aromatic ring. Thus,
compounds 18-19 and 20-27, bearing different R5 and R6 groups, were perceived as umami,
either moderately (18, 21, 22) or strongly (19, 22-27). In particular, compounds having one
alkoxy or one alkyl group in meta position were all very strong (19 > 4 > 20). Benzylamides
(n = 0) 29 and 30 were only very weakly umami.
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In the large panel study, all but two of the evaluated amides significantly enhanced the
umami taste (see Table 2). The delta scores represent the average difference between the
perceived umami intensity of the model solution (MSG) and that with the amide added. A
positive value denotes an increase of umami taste intensity when the amide was present.
The student test (paired test, one tailed) indicates whether this difference is significant at
different levels of confidence (*, **, ***, at 95%, 99%, and 99.9%, respectively) between
these two samples. The best-performing amides were 1, 4, 19, and 23-27. One of the most
interesting compounds was 4, which increased the perceived intensity of umami by 1.3, with
a significance level of 99.9% with and without the nose clip. Furthermore, the comments
regarding the water system with the nose clips were quite positive, except for the
astringency; there was no olfactive off-note (no comment without the nose clips).
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284
285
286
287
288
289
290
291
292
293
Targeted UPLC/MS/MS Analysis and Occurrence of Amides 1, 4, 15, 16 and 30-32 in Z.
piperitum. There are more than 500 species in the genus Zanthoxylum (Rutaceae),15 often
used as medicinal plants, as ornamentals, or in culinary applications. Cheng et al. analyzed Z.
beecheyanum K. Koch, which K. Koch purchased in Taiwan, and it contained many aromatic
amides.20 The description of this plant as an evergreen shrub originating from Japan and
introduced as an ornamental plant in Taiwan is very similar to Z. piperitum. The latter plant is
used as ornamental plant in China and Japan as bonsai, whereas the pericarp of the species
is commonly used as a spice in Japan.15 We decided to focus our study on this plant.
Dichloromethane was selected as the solvent for the extraction because the synthetic
amides described earlier were all very soluble in it. The stems and the roots were extracted
separately and analyzed by UPLC/MS operated in positive electrospray ionization mode. For
this study, we used a high-resolution hybrid quadrupole-Orbitrap mass spectrometer in full
scan/data dependent MS2 experiment followed by a targeted MS2 experiment (t-MS2) within
the same run. The t-MS2 used an inclusion list comprising the molecular formulae of the 15
selected amides (see Table 3) to record the corresponding product ion spectra. Figure 4
shows the UPLC/MS profile of the root extract of Z. piperitum. It was not the aim of this
study to fully characterize the extract, but some major products already known to occur in
Zanthoxylum species could be tentatively identified on the basis of their accurate masses.
The major products were amides such as α-sanshool (Figure 4, peak 11), beecheyamide 30
(peak 4), podocarpamide 31 (peak 6), alkaloids such as skimmianine (peak 3) and
zanthoxyline (peak 2); and probably lignan-related products (peaks 9-10). Amides 30, 31 and
also 32 (Figure 5) were synthesized to be used in the study.
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295
296
297
298
299
300
301
302
303
304
305
306
307
According to the International Organization of the Flavor Industry guidelines for the
positive identification of chemicals by high resolution LC/MS/MS,26 an undisputed
identification of new compounds in plants “requires six points of identification when
comparing the reference synthetic material with the analyte in the plant: 1 precursor in low
resolution + 2 product ions in high resolution + 1 ratio → 6 IPs”. A mixture of 15 amides
(Table 3) was injected in concentrations ranging from 0.1 to 1000 ppb. The t-MS2 data were
then compared with those obtained from the injection of the extract at 1000 ppm after
blank runs were performed. The comparison of the chromatographic and mass spectral data
(Table 3 and Supporting Information) between the synthetic compounds and the natural
extract showed the unambiguous occurrence of amides 1, 4, 15, 16, 30, 31, and 32 in
Zanthoxylum piperitum root extract. Quantitative results presented below should be
considered with care since we did not check for ion suppression effects. Also, the most
abundant amides were in concentration higher than the highest concentration of the
calibration curves.
308
309
310
311
312
313
314
315
316
Amides 1 (rubemamine), 16, 20 (beecheyamide), and 31 (podocarpamide) were already
reported to occur in Zanthoxylum species (see Table 1). Amides 30 and 31 were abundant
compounds in our extract, accounting for 0.57 and 1.87% w/w, respectively. Figure 6 shows
the t-MS2 trace of ion 342.17. The peak at 3.816 min in the plant extract (trace A) and the
synthetic amide 4 eluting at 3.820 min in the reference mixture (trace B) injected separately
showed identical MS/MS spectra (C and D, same fragments, same ratio). As amide 4 was well
separated from its isomers 5 and 19, its occurrence is thus unambiguously supported. The
comparisons between the UPLC/MS/MS data of the synthetic products and the natural
products for the other amides are available as Supporting Information.
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318
319
320
321
322
323
324
325
This work illustrates how compounds already known to occur in plants served as a starting
point for umami taste relationship studies to find more intense ingredients. Targeted
UPLC/MS/MS analyses then unambiguously supported the natural occurrence of a few of
these molecules in a plant extract. The combination of plant knowledge, chemical synthesis,
and modern LC/MS techniques thus provided an efficient way to discover new taste-active
and naturally occurring ingredients. A similar approach was used by Süss et al. who
synthesized a series of “kitchen-like” amides by reaction of lactones and amines, evaluated
them for their umami taste and finally showed the natural occurrence of one of them in blue
cheese.27
326
327
ABBREVIATIONS USED
328
329
MSG, monosodium glutamate; THF, tetrahydrofuran; UPLC/MS, ultra-performance liquid
chromatography mass spectrometry; ESI, electrospray ionization.
330
331
ACKNOWLEDGMENT
332
333
334
335
336
We thank Mr. Alain Bagnoud for his help in chemical synthesis, Mrs. Esmeralda Cicchetti for
preliminary LC/MS analyses, Mrs. Aurore Planchais and Christine Saint-Léger for the
organization of the large panel evaluations, Dr. Sylvain Etter for toxicology assessment and
Mrs. Kasia Aeberhardt for stimulating discussions. The reviewers for their constructive
suggestions to improve the manuscript are also acknowledged.
337
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339
Supporting Information
1H and 13C NMR descriptions of products 1-32.
340
341
Chromatograms and mass spectra comparison of synthetic amides vs natural amides in
Z.piperitum. This material is available free of charge via the Internet at http://pubs.acs.org.
342
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REFERENCES
344
345
346
1. Strunz, G. M. Unsaturated amide in Piper species (Piperaceae). In Studies in Natural
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738.
347
348
349
2. Galopin, C. C.; Furrer, S. M.; Goeke, A. Pungent and tingling compounds in Asian cuisine.
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353
3. Dawid, C.; Henze, A.; Frank, O.; Glabasnia, A.; Rupp, M.; Büning, K.; Orlikowski, D.;
Bader, M.; Hofmann, T. Structural and sensory characterization of key pungent and
tingling compounds from black pepper (Piper nigrum L.). J. Agric. Food Chem. 2012, 60,
2884-2895.
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4. Yang, X. Aroma constituents and alkylamides of red and green huajiao (Zanthoxylum
bungeanum and Zanthoxylum schinifolium). J. Agric. Food Chem. 2008, 56, 1689-1696.
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5. Nagashima, M.; Nakatani, N. LC-MS analysis and structure determination of pungent
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6. Gulland, J.M.; Hopton, G..U. Pellitorine, the pungent principle of Anacyclus pyrethrum. J.
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7. Winkel, C.; de Klerk, A.; Jan Visser, J.; de Rijke, E.; Bakker, J.; Koenig, T.; Renes, H. New
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8. Tachdjian, C.; Patron, A. P.; Qi, M.; Adamski-Werner, S. L.; Tang, X.-Q.; Chen, Q.;
Darmohusodo, V.; Lebl-Rinnova, M.; Priest, C. Aromatic amides and ureas and their uses
as sweet and/or umami flavor modifiers, tastants and taste enhancers. U.S. Pat. Appl.
20060045953 A1 20060302, 2006. To Senomyx.
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9. Shigemura, R.; Chen, Q.; Darmohusodo, V.; Dean, A. R. Comestible compositions
comprising high potency savory flavorants, and processes for producing them. PCT Int.
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10. Dewis, M. L.; Conklin, G.; Pei, T. Saturated and unsaturated N-alkamides exhibiting taste
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11. Bombuwala, K.; Janczuk, A. J.; John, T. V.; Kraut, K.; Liu, Z.; Mannava, N.; Wu, H.; Yang, Y.
Production and application of flavor-enhancing amide compounds. Eur. Pat. Appl. EP
2386212 A2 20111116, 2011. To IFF.
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12. Backes, M.; Paetz, S.; Vössing, T.; Ley, J.P. Synthesis and Sensory Studies of Umami-
Active Scaffolds. Chem.Biodiv. 2014, 11, 1782-1797.
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13. Adesina, S. K.; Reisch, J. Amides from Zanthoxylum rubescens. Phytochemistry 1989, 28,
839-842.
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14. Adesina, S. K. Three new amides from Zanthoxylum rubescens. Planta Med. 1989, 55,
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15. Patiño, L. O. J.; Prieto, R. J. A.; Cuca, S. L. E. Zanthoxylum genus as potential source of
bioactive compounds. In Bioactive Compounds in Phytomedicine; Rasooli, I., Ed.; InTech:
Rijeka, Croatia, 2012; pp. 185-218.
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16. Adesina, S. K. The Nigerian Zanthoxylum; chemical and biological values. Afr. J. Trad.
Complem. Alt. Med. 2005, 2, 282-301.
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17. Ley, J.P.; Krammer, G.; Muche, S.; Kindel, G.; Reiss, I. Use of ferulic acid amides as flavor
compounds. U.S. Patent 2003/0152682, 2003. To Symrise.
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18. Ley, J. P.; Reichelt, K.; Paetz, S.; Backes, M.; Obst, K. Cinnamamides as savory flavorings.
U.S. Pat. Appl. Publ. 2012/0308703, 2012. To Symrise.
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19. Frerot, E.; Aeberhardt, K. Taste modifying product to improve or modify the kokumi or
umami taste of a flavored food. PCT Int. Appl. (2013), WO 2013000673 A1 20130103,
2013. To Firmenich.
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20. Cheng, M. J.; Wu, C. C.; Tsai, I. L.; Chen, I. S. Chemical and antiplatelet constituents from
the stem of Zanthoxylum beecheyanum. J. Chinese Chem. Soc. 2004, 51, 1065-1072.
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21. Delle Monache, F.; Di Benedetto, R.; De Moraes E Souza, M. A.; Sandor, P. Esenbeckia
leiocarpa: II. Further components. Gazzetta Chim. Ital. 1990, 120, 387-389.
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22. Wu, M.-C.; Peng, C.-F.; Chen, I.-S.; Tsai, I.-L. Antitubercular Chromones and Flavonoids
from Pisonia aculeata. J.Nat. Prod. 2011, 74, 976-982.
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23. Kalia, N.K.; Singh, B.; Sood, R.P. A New Amide from Zanthoxylum armatum. J.Nat. Prod.
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24. Takemoto, T.; Uchida, M.; Koike, K.; Hoshina, Y.; Kusano, G. Constituents of Chloranthus
species. I. Structures of two new amides from Chloranthus serratus and the isolation of
isofraxidin from C. japonicas. Chem.Pharm.Bull. 1975, 23, 1161-1163.
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25. Huang, T.; Rehak, L.; Janger, G. Meta-tyrosine in Festuca rubra ssp. commutata
(Chewings fescue) is synthesized by hydroxylation of phenylalanine. Phytochemistry
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26. IOFI Working Group on Methods of Analysis. Guidelines for LC-MS identifications of
flavouring substances in nature, made by the Working Group on Methods of Analysis of
the International Organization of the Flavor Industry (IOFI). Flavour Fragrance J. 2010,
25, 2-3.
412
413
414
27. Suess, B.; Brockhoff, A.; Meyerhof, W.; Hofmann, T. Structures, orosensory activity, and
T1R1/T1R3 receptor activation of amides generated by lactone aminolysis using food-
related processing conditions. Eur.Food Res.Technol. 2013, 237, 57-70.
415
416
417
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Figure captions
Figure 1. High-intensity umami ingredients.
Figure 2. Amides from Zanthoxylum rubescens.13
Figure 3. Structure of cinnamic acid-derived amides synthesized for umami taste evaluation.
Figure 4. UPLC/MS chromatogram (Full scan mode) of Zanthoxylum piperitum root extract.
Figure 5. Amides 30-32 synthesized for targeted UPLC/MS analysis if Z.piperitum
Figure 6. UPLC/MS trace in targeted-MS2 mode (see Material & Methods) of ion 342.17 in Z.
piperitum root extract (A) and in a mixture of synthetic products (B). MS/MS spectra of peak
at 3.827 min in Z.piperitum (C) and peak at 3.820 min in the synthetic mixture (D), showing
the natural occurrence of amide 4
419
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Table 1. Cinnamic Acid-Derived Amides and Their Umami Taste Score
product no.
natural
number of rel. umami product no.
natural
number of rel. umami
occurrence
panelists
score
occurrence
panelists
score
1
2
Z. rubescens13
4
3.3
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
3
2.6
3.8
5
3.9
Chloranthus
serratus24
7
9
7
7
6
7
5
5
5
4
6
b
3
Z. rubescens14
Z. piperituma
10.2
3.2
4
6
4
5
4
4
6
5
4
9
4
6
4
9
5.6
1.3
0.9
1.6
1.5
2.4
1.0
2.2
1.8
0.3
2.2
1.1
2.1
5
3.7
6
9.8
7
Pisonia
9.9
aculeata22
8
6.5
9
11.5
5.9
10
11
12
13
14
15
16
13.3
1.4
6
0.8
b
Z.
beecheyanum20
Z. piperituma
Z. armatum23
Z. piperituma
b
c
Z. piperituma
aThis work
bEvaluated in large panel only (table 2)
cNot evaluated
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Table 2. Sensory Evaluation of Selected Amides by a Large Panel
compound conc. deltab deltab
t-test
t-test
comments for amides in
comments for amides in
water evaluated w/o NCc
no.
(ppm)a with without with NC without water evaluated with NCc
NC
NC
NC
1
20
20
20
20
20
20
20
20
20
100
0.9
0.8
***
***
**
**
umami (4), astringent (3)
no
4
1.1
1.7
1.3
1
1.3
1.7
1.3
1.2
0.9
1.1
1.0
0.6
0.0
***
**
**
**
**
**
NS
*
umami (9), mouthfeel (6),
salty (5), astringent (4)
no
19
23
24
25
26
27
30
31
umami (10), mouthfeel (4),
salty (4), sour (3)
green (3), herbal (3)
**
umami (11), astringent (3),
salty (3)
no
**
umami (4), sweet (3)
no
0.9
1
**
umami (12)
no
**
umami (7), salty (4), sweet
(4), metallic (3)
no
0.8
0.7
0.1
NS
NS
NS
umami (11)
green (3), herbal (3)
no
no
no
NS
bitter (10), astringent (4)
aThe amides were diluted to 10% in ethanol. The quantity of ethanol added to the solution was therefore 0.02% for the compounds
tasted at 20 ppm and 0.1% for compound 31 tasted at 100 ppm.
bDifference in perceived intensity between the amide+MSG vs MSG alone
cThe numbers in parentheses indicate the number of panelists among the 15 who made this comment.
*, **, ***: levels of confidence at 95%, 99%, and 99.9%, respectively
Abbreviations: NC, nose clip; NS, not significant; No, no comments or comments given by less than 20% of the panel (fewer than three panelists).
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Table 3. Identification of Selected Amides in Zanthoxylum piperitum Root Extract
no.
1
RT
(min)
accurate mass
MS/MS fragment 1 MS/MS fragment 2 MS/MS fragment 3
quantity
[mg/kg of extract]
41
2.94
3.82
372.1805
C21H26NO5 (-0.2)
342.1699
191.0702
C11H11O3 (-0.2)
191.0702
C11H11O3 (-0.5)
n.d.
163.0753
C10H11O2 (-0.2)
135.0846
C9H11O, (+0.1)
n.d.
4
29
C20H24NO4 (-0.2)
n.d.
5
3.62
2.85
15
n.d.
177.0546
C10H9O3 (-0.1)
175.0389
C10H7O3 (-0.2)
n.d.
145.0284
C9H5O2 (+0.0)
145.0284
C9H5O2 (-0.2)
n.d.
135.0805
C9H11O (+0.4)
135.0847
6
16
4.47
326.1387
C19H20NO4 (-0.0)
n.d.
527
C9H11O (+0.2)
19
22
23
24
25
26
27
30
3.92
4.79
5.92
5.44
5.80
6.63
5.64
4.26
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
356.1854
C21H26NO4 (-0.6)
340.1541
C20H22NO4 (-0.6)
342.1700
C20H24NO4 (+0.2)
191.0703
C11H11O3 (-0.1)
175.0389
C10H7O3 (+0.0)
175.0390
C10H9O3 (-0.2)
135.0805
C9H11O (+0.5)
145.0284
C9H5O2 (+0.0)
145.0284
C9H5O2 (-0.2)
5784a
18723a
845
31
32
5.01
3.18
135.0805
C9H11O (+0.5)
135.0805
C9H11O (+0.1)
aabove the highest point of calibration
420
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Figure 1.
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Figure 2.
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R5
R8
O
R6
R7
O
R1
R2
OH
1) EtOCOCl, TEA, EtOAc
R1
R2
N
H
n
R4
R5
2)
R4
R6
R7
R3
R3
H2N
n
R8
N°
1
n
double bond
R1 R2 R3 R4 R5 R6 R7 R8
OMe OMe H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OMe OMe H
OMe OMe H
OMe OMe H
2
H
OMe H
3
a
a
H
4
OMe OMe H
OMe OMe H
OMe OMe H
OMe H
OMe H
OMe H
OMe H
H
H
H
H
5
(Z)
6
single bond
7
H
H
H
H
H
H
H
H
8
H
OMe H
9
OMe H
OMe H
OMe H
OMe H
OMe H
OMe H
OMe H
OMe H
OMe H
OMe H
H
H
H
H
H
H
H
H
H
H
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
H
H
H
H
OMe H
OMe H
OMe H
H
H
OMe H
OMe OMe OMe H
OMe OAc H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OMe OH
H
H
H
a
a
Me Me
OMe OMe H
OMe OMe H
OMe OMe H
OMe OMe H
OMe OMe H
OMe OMe H
OMe OMe H
OMe OMe H
OMe OMe H
OMe OMe H
OMe OMe H
OMe OMe H
H
H
H
H
OMe H
OMe
H
OMe H
OMe H
OEt H
OnPrH
H
H
H
H
H
H
H
H
H
H
OiPr H
H
H
H
H
H
H
Et
H
H
iPr
Me Me
OMe H
0
0
a
a
double bond of (E)-configuration except otherwise stated
aR1-R2 or R6-R7 = O-CH2-O (methylenedioxy)
n = 1 (phenethylamine series) except 0 stated (benzylamine series)
Figure 3.
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Figure 4.
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