Structure elucidation of a pungent compound in black cardamom: Amomum tsao-ko Crevost et Lemarie (Zingiberaceae)

Article abstract of DOI:10.1021/jf072707b

Natural plant extracts containing taste modifier compounds will gain more commercial interest in the future. Black cardamom, Amomum tsao-ko Crevost et Lemarie, used as a spice in Asia, produces a nice refreshing effect in the mouth. Therefore, an ethyl acetate extract was prepared, and constituents were separated by liquid chromatography. Guided by the tasting of each fraction (LC tasting), a new pungent compound was discovered, (±)-trans-2,3,3a,7a- tetrahydro-1H-indene-4-carbaldehyde. To confirm this new structure, a synthesis was performed starting from cyclopentene-1-carbaldehyde. The Wittig conditions were determined to control the stereochemistry of the ring fusion to prepare (±)-trans-(2,3,3a,7a-tetrahydro-1H-inden-4-yl) methanol and (±)-cis-(2,3,3a,7a-tetrahydro-1H-inden-4-yl) methanol. After oxidation, (±)-trans-2,3,3a,7a-tetrahydro-1H-indene-4-carbaldehyde and (±)-cis-2,3,3a,7a-tetrahydro-1H-indene-4-carbaldehyde were tasted in water and only the trans-2,3,3a,7a-tetrahydro-1H-indene-4-carbaldehyde, present in black cardamom, produced a trigeminal effect in the mouth.

Full text of DOI:10.1021/jf072707b

10902 J. Agric. Food Chem. 2007, 55, 10902–10907
Structure Elucidation of a Pungent Compound in
Black Cardamom: Amomum tsao-ko Crevost et
Lemarié (Zingiberaceae)
CHRISTIAN STARKENMANN,* FABIENNE MAYENZET, ROBERT BRAUCHLI,
LAURENT WUNSCHE, AND CHRISTIAN VIAL
Firmenich SA, Corporate R&D Division, Post Office Box 239, CH-1211 Geneva 8, Switzerland
Natural plant extracts containing taste modifier compounds will gain more commercial interest in the
future. Black cardamom, Amomum tsao-ko Crevost et Lemarié, used as a spice in Asia, produces a
nice refreshing effect in the mouth. Therefore, an ethyl acetate extract was prepared, and constituents
were separated by liquid chromatography. Guided by the tasting of each fraction (LC tasting), a new
pungent compound was discovered, (()-trans-2,3,3a,7a-tetrahydro-1H-indene-4-carbaldehyde. To
confirm this new structure, a synthesis was performed starting from cyclopentene-1-carbaldehyde.
The Wittig conditions were determined to control the stereochemistry of the ring fusion to prepare
(()-trans-(2,3,3a,7a-tetrahydro-1H-inden-4-yl) methanol and (()-cis-(2,3,3a,7a-tetrahydro-1H-inden-
4-yl) methanol. After oxidation, (()-trans-2,3,3a,7a-tetrahydro-1H-indene-4-carbaldehyde and (()-
cis-2,3,3a,7a-tetrahydro-1H-indene-4-carbaldehyde were tasted in water and only the trans-2,3,3a,7a-
tetrahydro-1H-indene-4-carbaldehyde, present in black cardamom, produced a trigeminal effect in
the mouth.
KEYWORDS: (()-trans-2,3,3a,7a-Tetrahydro-1H-indene-4-carbaldehyde; (()-trans-(2,3,3a,7a-tetrahydro-
1H-inden-4-yl); trigeminal effect; Amomum tsao-ko Crevost et Lemarié
INTRODUCTION
ethanol and carbon dioxide can also generate a trigeminal
sensation (12).
Chemosensation is mediated by taste and smell receptor
organs and transmitted to the brain along their cognate nerves.
However, the trigeminal nerve also carries sensory information
(1). Free nerve endings in the facial mucosa are responsible for
the detection of sharp, pungent, cold, or hot stimuli, such as
capsaicin (2). Recent research has shown that the behavior of
transient receptor potential (TRP) ion channels involved in the
detection of hot or cold temperatures can be modulated by
chemical stimuli (3–5). Thus, menthol elicits a cold sensation
by raising the opening of the TRPM8 channel (6, 7). The
compounds present in foodstuffs, which interact with TRP
receptors, are often derivatives of vanilloid-like capsaicin,
gingerol, and eugenol (8) or originate from closely related
structures, such as piperine and galangal acetate. Polyunsaturated
amides, widely distributed in plants, such as Szechuan pepper,
Spilantes acmella, and Echinacea, also generate trigeminal
effects (9, 10). In the family of terpene derivatives, menthol,
cubebol, and eucalyptol generate a cooling, refreshing sensation
and polygodial is described as pungent (11). A few other
examples, such as phorbol esters, isothiocyanate derivatives, and
allicin, are also known (3, 8). Very simple molecules, such as
Spices are a good source for natural compounds with oral
sensory qualities distinct from olfaction and taste. The ginger
family (Zingiberaceae) contains several important plants used
as spices in food and compounds producing trigeminal effects,
such as gingerols in Aframomum melegueta (grains of paradise)
or Zingiber officinale (ginger) (13, 14) or galangal acetate in
Alpinia galanga (greater galanga) (15), which were extensively
studied. The sensory properties of black cardamom (Amomum
tsao-ko Crevost et Lemarié), native in the Eastern Himalayas,
are smoky, phenolic, with a fresh citrus, camphor odor
reminiscent of green cardamom (Elettaria cardamomum) and
Amomum subulatum Roxb. The smoky flavor comes from a
traditional drying procedure over an open fire. Although there
are many distinct varieties of black cardamom from China and
Vietnam, ranging in pod size from 2 to more than 5 cm, their
tastes do not differ much. In central and southern China, the
seeds are optional ingredients of the five spice powders. A. tsao-
ko produces a refreshing and pungent effect, which cannot be
solely explained by the presence of 1,8-cineol and other known
constituents (16–21). This observation led us to a careful analysis
of black cardamom, to understand why this spice produces a
trigeminal effect. This paper describes the identification of a
new compound guided by tasting, and the structure elucidated
was confirmed by its synthesis.
* To whom correspondence should be addressed. Telephone: +41-
22-780-34-77. Fax: +41-22-780-33-34. E-mail christian.starkenmann@
firmenich.com.
10.1021/jf072707b CCC: $37.00
2007 American Chemical Society
Published on Web 11/20/2007

Analysis of Black Cardamom
J. Agric. Food Chem., Vol. 55, No. 26, 2007 10903
Figure 1. GC/MS (total ion current) on a SPB-1 column of A. tsao-ko (Rui Li) ethyl acetate extract.
MATERIALS AND METHODS
nuclear Overhauser effect spectrometry (NOESY) experiments were
performed with standard Bruker software (Topspin 1.3) (Bruker Biospin,
Fallanden, Switzerland).
General Procedures. Commercially available reagents and solvents
of adequate quality were used without further purification. Reactions
were performed in standard glassware. Yields were not optimized.
Gas Chromatography/Electron Impact-Mass Spectrometry (GC/
EI-MS). An Agilent-GC-6890 system connected to an Agilent-MSD-
5973 quadrupole mass spectrometer was operated at an electron energy
of ca. 70 eV. Helium was the carrier gas set at a constant flow rate of
0.7 mL/min. Separations were performed on 30 m × 0.25 mm i.d.,
0.25 µm fused-silica capillary columns, either coated with SPB-1 or
coated with Supelcowax 10 (Supelco, Buchs, Switzerland). The standard
oven program was the following: 50 °C for 5 min, then 50-240 °C at
5°/min, and held at 240 °C. Retention indices (I) were calculated by
linear extrapolation from the retention times (tR in minutes) of the
analytes and the two closest alkanes. The chiral column was a 25 m ×
0.25 mm i.d., 0.25 µm fused-silica capillary column CP-Chirasil-Dex
CB (Varian, Zug, Switzerland). The oven temperature was programmed
from 100 to 145 °C at 2°/min. The mass spectra are listed as follows:
fragment ions m/z (relative intensity).
Analysis of A. tsao-ko Crevost et Lemarié The seeds (1031 g),
purchased from ZhaoYang Perfume Trading Shop, number 10-13, JunQi
dry vegetable market, XiaoBanQiao town, Kunming City, 650000,
Kunming of Yunnan Province, were manually separated from the pods,
ground into a fine powder, and macerated at room temperature in ethyl
acetate (2000 mL) for 90 min under mechanical stirring. The suspension
was decanted, and the solvent was separated, dried (Na2SO4), filtered, and
concentrated in a Vigreux apparatus at 45 °C under reduced pressure (6
mbar). The resulting oleoresin (41 g) was tasted in plain water at 50 ppm.
The oleoresin was fractionated by two consecutive chromatographic
columns using SiO2 [silica gel 60, 35–70 µm (Toulouse, France) and
Lobar LiChroprep size B SiO2 60, 40–63 µm (Merck, VWR, Nyon,
Switzerland)] with 9:1 pentane/ethyl acetate, as a mobile phase. Each
fraction was tasted by dipping a smelling strip into it. The solvent was
removed by shaking the smelling strip in the air, and then it was placed
onto the tongue. We isolated a pungent fraction of 211 mg, which
contained mainly peak 7 (68%) and peak 8 (18%), plus minor impurities
(Figure 1).
Time-of-flight GC/MS analyses were performed on a GCT Premier
(Waters, Milford, MA). Separations were performed on 30 m × 0.25
mm i.d., 1.0 µm film thickness fused silica capillary columns, coated
with a SPB-1 (Supelco). The oven program started at 100 °C with a
temperature gradient of 10°/min to 240 °C, at a constant helium flow
of 1.0 mL/min. The injection volume was 1.0 µL, and the injector
temperature was 250 °C with a split 1:50. The acquisition time was
set to 0.5 s with an interscan delay of 0.01 s over a mass range of
1–300 Da. Spectra were recorded using an electron energy of 70 eV,
emission current of 600 µA, trap current of 200 µA, and source
temperature of 150 °C. Calibration was performed using heptacosa
(perfluorotributylamine, mass spectrometry grade, Apollo Scientific
LTD, Bradbury, U.K.). Calibration data were collected for 3 min in
continuum mode. A total of 180 spectra were summed to generate a
16 point calibration curve from m/z 69 to 614 Da. The curve was fitted
to a second-order polynomial such that the standard deviation of the
residuals was 0.001 amu or lower. Heptacosa was continuously
introduced into the ion source, and the ion m/z 218.9856 was used as
a lock mass. Mass spectra and molecular formula were obtained using
MassLynx software (Waters). The difference δ, between the exact mass
calculated from the molecular formula and that measured, is calculated
by the sofware and expressed in ppm (δ ) (Mmeasured - Mcalculated)/
Mcalculated × 106).
GC retention indices: peak 7, ISPB-1 1254 and ISPWAX 1848; peak 8,
ISPB-1 1270 and ISPWAX 1876. GC/MSCP-ChirasilDex CB: the major peak (peak
7) gave two peaks of the same intensity at RT ) 12.7 and 12.9 min
(ratio of 1:1); the minor peak (peak 8) also gave two peaks at RT
)
14.5 and 14.9 min. 1H, 13C NMR showed a mixture having signals
corresponding to synthetic 1 and 2. The MS of peak 7 was the same as
1, and the MS of peak 8 was the same as 2. Elemental analysis of 1
gave 148.0881 Da (C10H12O, 4.8 ppm).
Quatitative Analysis of (()-trans-2,3,3a,7a-Tetrahydro-1H-in-
dene-4-carbaldehyde, 1. The powder prepared as described above (3
g) was extracted with 15 mL of ethyl acetate containing methyl
octanoate (3.3 mg) as an internal standard, during 15 min in an
ultrasonic bath. After filtration on a paper filter, the sample was injected
in triplicate on GCSPB-1/MS and 1 (peak 7) was quantified by a
comparison to the internal standard without applying any response
factor. Quantifications were performed on two different lots of A. tsao-
ko from Vietnam and China, qualities A. rui li (three different lots)
and A. he ko (two different lots).
Reduction of the Fraction Having a Pungent Effect. An aliquot
of the fraction obtained above (20 mg) was diluted in ethanol (1 mL)
and treated with NaBH4 (∼20 mg). The crude mixture was diluted in
0.1% H2SO4 (1 mL) and extracted with ethyl acetate. The organic phase
was dried over MgSO4, concentrated (45 °C, 12 mbar), and purified
on a LiChroprep column with a mixture of 1:1 hexane/diethylether,
leading to 4.2 mg of 10.
1H and 13C Nuclear Magnetic Resonance (NMR) Spectra. The
NMR spectra were recorded on a Bruker-Avance-500 spectrometer at
500.13 and 125.76 MHz, respectively. The solvent was CDCl3. δ Values
are in ppm downfield from (CH3)4Si ()0 ppm). The assignments by
correlation spectroscopy (COSY), heteronuclear single-quantum coher-
ence (HSQC), heteronuclear multiple-bond correlation (HMBC), and
GC retention indices of the main peak (reduced peak 7): ISPB-1 1299
and ISPWAX 2115.

10904 J. Agric. Food Chem., Vol. 55, No. 26, 2007
Starkenmann et al.
1
Synthesis of 1-Cyclopentene-1-carbaldehyde, 6. Methyl-1-cyclo-
pentene-1-carboxylate (Fluka, Buchs, Switzerland) (20 g, 0.16 mol)
was diluted in toluene (200 mL). Vitride (sodium bis(2-methoxy-
ethoxy)aluminum hydride, 3.5 M in toluene (Acros, Chemie Brunschwig
A.G., Basel, Switzerland) (100 mL, 0.35 mol) was added at 0 °C
dropwise. The reaction was stirred for 90 min, and then the organic
phase was washed with brine. The crude product was filtered through
a column of 15 × 7 cm SiO2 with 100% pentane and then eluted with
diethyl ether, to give the corresponding alcohol, 14.9 g (yield 95%)
after concentration.
GC retention indices: ISPB-1 1299 and ISPWAX 2115. H NMR: 1.39,
(2H, m, H-1 and H-3), 1.80 (2H, m, H-2), 1.81 (1H, m, H-1′), 1.87
(1H, m, H-3′), 2.11 (2H, m, H-7a and H-3a), 4.22 (1H, d, J ) 13.5
Hz, H-10), 4.26 (1H, d, J ) 13.5 Hz, H-10′), 5.91 (1H, m, H-5), 5.97
(1H, m, H-6), 6.06 (1H, m, H-7). 13C NMR: 143.7 (C-4), 131.7 (C-7),
125.4 (C-6), 120.3 (C-5), 64.2 (C-10), 44.6 (C-9), 44.4 (C-8), 26.5
(C-1), 25.0 (C-3), 22.8 (C-2). MS: 150 (20, M+·), 132 (14), 119 (42),
117 (38), 91 (100), 79 (18), 77 (20).
Synthesis of (()-cis-2,3,3a,7a-Tetrahydro-1H-indene-4-carbal-
dehyde, 2. The pure alcohol 11 (32 mg, 0.21 mmol) was oxidized with
MnO2 (185 mg, 2.1 mmol) in CH2Cl2 (0.5 mL). After 2 h of stirring at
22 °C, the reaction mixture was filtered through Celite and then through
SiO2 using dichloromethane. The filtration was performed using a glass
pipet. Compound 2 was obtained with a GC purity of ∼80%; the major
impurity was the aromatic compound 4 (GC < 20%).
This crude product (14 g, 0.143 mol) was diluted in pentane and
treated with MnO2 (118 g, 1.36 mol) overnight. The reaction mixture
was filtered through Celite; the solvent was removed by distillation;
and the crude product 6 (yield 84%) was used without further
purification for the following Wittig reaction.
Compound 2. GC retention indices: ISPB-1 1270 and ISPWAX 1876.
Synthesis of (()-Methyl cis-2,3,3a,7a-Tetrahydro-1H-indene-4-
carboxylate, 8 (22). To methyl 4-(triphenylphosphonio)crotonate
bromide 7 (Alfa Aesar, Karlsruhe, Germany) (50 g, 0.113 mol) in
methanol (50 mL) was added a freshly prepared sodium methylate
solution (2.26 M in methanol). After 15 min, the aldehyde 6 (10 g,
0.104 mol) in tetrahydrofuran (THF, 280 mL) was added and the
reaction was stirred overnight at 22 °C. The crude product was extracted
with diethylether and washed with 0.5 M H2SO4, followed with brine,
and then dried over Na2SO4, filtered, and concentrated. The crude extract
was filtered through a column of 15 × 7 cm SiO2 with a mixture of
9:1 pentane/diethylether. Compound 8, 2.1 g (yield 11.7%), was
obtained with a GC purity of 80%. The major compounds formed were
methyl (2E,4E)-5-(1-cyclopentene-1-yl)-2,4-pentadienoate and methyl
(2E,4Z)-5-(1-cyclopentene-1-yl)-2,4-pentadienoate.
1
GC/MSCP-ChirasilDex RT ) 14.5 and 14.9 min. H NMR: 1.26 (1H, m,
CB
H-3), 1.42 (1H, m, H-2), 1.50 (1H, m, H-2′), 1.59 (1H, m, H-1), 2.16
(1H, m, H-1′), 2.22 (1H, m, H-3′), 2.88 (1H, m, H-3a), 2.95 (1H, m,
H-7a), 6.03 (1H, m, H-7), 6.04 (1H, m, H-6), 6.63 (1H, m, H-5), 9.50
(1H, s, H-10). 13C NMR: 193.7 (C-10), 141.3 (C-4 and C-7), 140.1
(C-5), 120.4 (C-6), 38.4 (C-8), 34.6 (C-1 and C-3), 33.8 (C-9), 22.9
(C-2). MS: 148 (58, M+·), 119 (50), 105 (38), 92 (25), 91 (100), 77
(27).
Compound 4. GC retention indices: ISPB-1 1307 and ISPWAX 1991.
Significant signals from the mixture: 1H NMR, 2.15 (2H, m, H-2), 2.94
(2H, m, H-1), 3.29 (2H, m, H-3), 7.32 (1H, m, H-6), 7.47 (1H, m,
H-7), 7.62 (1H, m, H-5), 10.15 (1H, s, H-10); 13C NMR, 192.8 (C-
10), 146.3 (C-8 and C-9), 132.3 (C-4), 129.9 (C-7), 129.2 (C-5), 126.7
(C-6), 32.1 (C-1), 31.8 (C-3), 25.2 (C-2); MS, 146 (85, M+·), 145 (28),
117 (100), 116 (29), 115 (60), 91 (18).
1
GC retention indices: ISPB-1 1390 and ISPWAX 1933. H NMR: 1.36
(1H, m, H-3), 1.44 (1H, m, H-2), 1.49 (1H, m, H-2′), 1.63 (1H, m,
H-1), 2.11 (1H, m, H-1′), 2.16 (1H, m, H-3′), 2.85 (1H, ddd, J ) 8,
10, 12 Hz, H-3a), 2.96 (1H, m, H-7a), 3.75 (3H, s, MeO-), 5.79 (1H,
dd, J ) 3, 9 Hz, H-7), 5.89 (1H, ddd, J ) 3, 6, 9 Hz, H-6), 6.88 (1H,
d, J ) 6 Hz, H-5). 13C NMR: 168.4 (C-10), 137.8 (C-7), 131.0 (C-4),
130.3 (C-5), 120.4 (C-6), 51.5 (MeO-), 38.4 (C-8), 36.4 (C-9), 34.6
(C-3), 34.4 (C-1), 22.7 (C-2). NOESY observed between H-7a and H-3a
was in agreement with a cis configuration. MS: 178 (50, M+·), 149
(35), 147 (30), 119 (90), 105 (45), 91 (100), 77 (28).
Synthesis of (()-trans-2,3,3a,7a-Tetrahydro-1H-indene-4-carbal-
dehyde, 1. The pure alcohol 10 (275 mg, 1.8 mmol) was oxidized with
MnO2 (770 mg, 8.6 mmol) in CH2Cl2 (30 mL). After 2 h of stirring at
22 °C, the reaction mixture was filtered through Celite and then on
SiO2 using dichloromethane. The filtration was performed using a glass
pipet. Compound 1 (220 mg, yield 83%) was obtained with a GC purity
of ∼98%.
GC retention indices: ISPB-1 1254 and ISPWAX 1848. GC/MSCP-ChirasilDex CB
:
enantiomers 1a RT ) 12.7 min and 1b RT ) 12.9 min, at a ratio of 1:1.
1H NMR: 1.37 (1H, m, H-1), 1.51 (1H, m, H-3), 1.83 (3H, m, H-1′
and H-2), 2.17 (2H, H-7a, H-3a), 2.36 (1H, m, H-3′), 6.24 (1H, ddd, J
) 2.2, 4.7, 9.0 Hz, H-6), 6.56 (1H, d, J ) 9.0 Hz, H-7), 6.77 (1H, J
) 4.7 Hz, H-5), 9.56 (1H, s, H-10). 13C NMR: 193.0 (C-10), 144.2
(C-5), 142.8 (C-4), 141.9 (C-7), 125.3 (C-6), 44.6 (C-8), 41.6 (C-9),
25.8 (C-3), 25.7 (C-1), 23.0 (C-2). MS: 148 (40, M+·), 119 (38), 117
(23), 115 (20), 105 (18), 92 (21), 91 (100), 77 (15).
Energy Calculations for 1 and 2. Each molecule was first
minimized using the standard Monte-Carlo procedure as implemented
in MacroModel using the OPLS_2005 Molecular Force Field (23). For
each molecule, the lower energy conformer was then minimized at the
density functional theory (DFT) level (B3LYP/6.31G**) using the
Jaguar software (24). The DFT energies were then used to calculate
the energy difference. Compound 1, 463.491484HT; compound 2,
463.499707HT (corresponding to an energy difference of 5.16 kcal or
21.60 kJ).
Synthesis of (()-Methyl trans-2,3,3a,7a-Tetrahydro-1H-indene-
4-carboxylate, 9. To methyl 4-(triphenylphosphonio)crotonate bromide
7 (22 g, 0.05 mol) in 1,4-dioxane (100 mL) was added potassium
carbonate (8.62 g, 0.062 mol) and the aldehyde 6 (4 g, 0.042 mol).
The reaction mixture was heated at 70 °C during 5 h. The same workup
was described as above. Compound 9, 1.86 g (yield 24.9%), was
obtained with a GC purity of 70%.
1
GC retention indices: ISPB-1 1368 and ISPWAX 1910. H NMR: 1.39
(1H, m, H-1), 1.46 (1H, m, H-3), 1.79 (2H, m, H-2), 1.84 (1H, m,
H-1′), 2.21 (2H, m, H-7a and H-3a), 2.26 (1H, m, H-3′), 3.75 (3H, s,
MeO-), 6.08 (1H, m, H-6), 6.40 (1H, m, H-7), 6.97 (1H, m, H-5).
13C NMR: 168.0 (C-10), 139.2 (C-7), 134.9 (C-5), 133.6 (C-4), 125.2
(C-6), 51.3 (MeO-), 44.5 (C-8), 42.8 (C-9), 27.2 (C-3), 26.4 (C-1),
22.7 (C-2). MS: 178 (45, M+·), 149 (28), 147 (28), 119 (90), 117 (35),
105 (20), 91 (100), 77 (15).
Synthesis of (()-cis-(2,3,3a,7a-Tetrahydro-1H-inden-4-yl) Metha-
nol, 11. Compound 8 (130 mg, 0.73 mmol) was reduced with Vitride
with the same procedure described above. Compound 11, 102 mg (yield
93.1%), was obtained with a GC purity of 88%.
Synthesis of (3aRS,4SR,7aRS)-(Octahydro-1H-inden-4-yl) Metha-
nol, 15. The alcohol 11 (200 mg, 1.3 mmol) was treated with hydrogen
in the presence of methanol (6 mL) and PtO2 (30 mg). In 1 h at 22 °C,
60 mL of hydrogen was absorbed. The reaction mixture was filtered
through Celite, and the solvent was evaporated to give 200 mg of 15
(yield 97%).
1
GC retention indices: ISPB-1 1311 and ISPWAX 2134. H NMR: 1.42
(2H, m, H-2 and H-3), 1.56 (2H, m, H-1 and H-2′), 2.05 (1H, m, H-3′),
2.09 (1H, m, H-1′), 2.51 (1H, m, H-3a), 2.88 (1H, m, H-7a), 4.12 (1H,
d, J ) 14 Hz, H-10), 4.15 (1H, d, J ) 14 Hz, H-10′), 5.48 (1H, m,
H-7), 5.74 (1H, m, H-6), 5.75 (1H, m, H-5). 13C NMR: 140.9 (C-4),
130.3 (C-7), 120.5 (C-6), 116.5 (C-5), 66.0 (C-10), 38.4 (C-9), 38.1
(C-8), 34.8 (C-1), 34.0 (C-3), 23.2 (C-2). MS: 150 (40, M+·), 132 (39),
119 (58), 117 (60), 91 (100), 79 (28), 77 (22).
1
GC retention indices: ISPB-1 1316 and ISPWAX 2006. H NMR: 1.00
(2H, m, H-5, H-7), 1.21 (1H, m, H-6), 1.34 (1H, m, H-1), 1.39 (1H,
m, H-7′), 1.42 (2H, m, H-2), 1.52 (1H, m, H-5′), 1.59 (1H, m, H-3),
1.64 (1H, m, H-1′), 1.70 (1H, m, H-6′), 1.71 (1H, m, H-3′), 1.84 (1H,
m, H-7a), 1.88 (1H, m, H-4), 2.01 (1H, dddd, J ) 10.0, 10.0, 5.1, 5.1
Hz, H-3a), 3.45 (1H, dd, J ) 7.1, 10.5 Hz, H-10), 3.49 (1H, dd, J )
Synthesis of (()-trans-(2,3,3a,7a-Tetrahydro-1H-inden-4-yl) Metha-
nol, 10. Compound 9 (150 mg, 0.84 mmol) was reduced with Vitride
using the same procedure described above. Compound 10, 112 mg
(yield 89%), was obtained with a GC purity of 90%.
7.1, 10.5 Hz, H-10′). 13C NMR: see Figure 6. MS: 136 (33, M+·
H2O), 123 (60), 121 (41), 94 (28), 81 (100), 79 (28), 67 (52), 41
-
(22).

Analysis of Black Cardamom
J. Agric. Food Chem., Vol. 55, No. 26, 2007 10905
Figure 3. Preparation of compounds 1 and 2.
Figure 2. Structures of (()-cis/trans-2,3,3a,7a-tetrahydro-1H-indene-4-
carbaldehyde 1 and 2 and related structures 3–5.
Synthesis of (3aRS,4RS,7aSR)-(Octahydro-1H-inden-4-yl) Metha-
nol, 13, and (3aRS,4SR,7aSR)-(Octahydro-1H-inden-4-yl) Methanol,
14. The alcohol 10 (200 mg, 1.3 mmol) was treated with hydrogen in
the presence of methanol (6 mL) and PtO2 (30 mg). In 3 h at 22 °C, 50
mL of hydrogen was absorbed. The reaction mixture was filtered
through Celite, and the solvent was evaporated. After filtration on SiO2
(55:45 pentane/diethylether), we obtained 189 mg (yield of 90%) of a
Figure 4. Chemical structures of 10 and 11 obtained either by the
reduction of esters 9 and 8 or by the reduction of natural aldehydes 1
and 2. Compound 12 corresponds to the chemical structure of tsaokoin
(11).
1
mixture of 13 and 14, in a 77:23 ratio by H NMR.
Compound 13. GC retention indices: ISPB-1 1275 and ISPWAX 1920.
1H NMR: 0.83 (1H, dddd, J ) 11.3, 10.8, 10.8, 6.6 Hz, H-3a), 0.95
(1H, m, H-7), 0.98 (1H, m, H-5), 1.09 (1H, m, H-1), 1.10 (1H, m,
H-7a), 1.15 (1H, m, H-3), 1.28 (1H, m, H-6), 1.30 (1H, m, H-4), 1.60
(2H, m, H-2), 1.74 (1H, m, H-1′), 1.82 (1H, m, H-6′), 1.85 (2H, m,
H-3′ and H-5′), 1.88 (1H, m, H-7′), 3.44 (1H, dd, J ) 7.0, 10.6 Hz,
H-10), 3.67 (1H, dd, J ) 4.3, 10.6 Hz, H-10′). 13C NMR: see Figure
6. MS: 154 (3, M+·), 136 (23), 123 (95), 121 (28), 81 (100), 79 (25),
67 (45).
Compound 14. GC retention indices: ISPB-1 1310 and ISPWAX 1989.
1H NMR: 0.94 (1H, m, H-7), 1.04 (1H, m, H-1), 1.23 (1H, m, H-7a),
1.33 (4H, m, H-3, H-5, H-6 and H-3a), 1.55 (3H, m, H-2, H-2′ and
H-6′), 1.60 (1H, m, H-3′), 1.73 (1H, m, H-1′), 1.90 (1H, m, H-7′),
1.92 (1H, m, H-5′), 2.07 (1H, m, H-4), 3.63 (1H, dd, J ) 8.8, 10.6 Hz,
H-10), 3.78 (1H, dd, J ) 5.2, 10.6 Hz, H-10′). 13C NMR: see Figure
6. MS: 136 (38, M+· - H2O), 123 (25), 121 (98), 94 (41), 93 (45), 81
(100), 79 (48), 67 (62).
Figure 5. Catalytic hydrogenation of 10 and 11. Relative stereochemistry
of three of the eight possible diastereoisomers.
then fractionated by medium-pressure chromatography. This
allowed for a partial structure elucidation of peak 7 by H and
RESULTS AND DISCUSSION
1
13C NMR. The HMBC and COSY NMR 2D correlations
showed the backbone of the 8,9-dihydroindane, but the cor-
relations between H-1′, H-2, H-7a, and H-3a were not clear
because of overlapping signals. The presence of both 4-indan-
ecarbaldehyde (4) and 5-indanecarbaldehyde (5) (peaks 9 and
10) (Figure 1) in the oleoresin, suggested a possible position
for the carbaldehyde on C-4 (1 or 2) as well as on C-5 (3). The
presence of both compounds 4 and 5 has already been reported
in A. tsao-ko (25). It was difficult to obtain peak 7 in high purity,
because it was not stable on preparative GC or on reverse-phase
preparative high-performance liquid chromatography (HPLC).
Therefore, an enriched fraction containing peaks 7 and 8 was
reduced with sodium borohydride to transform the aldehyde
functionality into a hydroxyl group. The reaction mixture was
purified by flash chromatography on SiO2. The NMR spectrum
of the major stereoisomer (reduced peak 7) based on HMBC
correlations between C-10 and C-5 and C-9 confirms the
presence of the functional group on C-4. It was still not possible
to determine the cis or trans configuration of reduced peak 7
because H-7a and H-3a both have the same chemical shift at
2.11 ppm. The only possible way to determine whether reduced
peak 7 corresponds to structure 10 or 11 was by synthesis.
Synthesis of the Authentic Samples (Figure 3). The Wittig
reaction with cyclopentene-1-carbaldehyde 6 and methyl 4-
(triphenylphosphonio)crotonate bromide 7, deprotonated with
Isolation of the Fraction Producing a Trigeminal Effect.
The oleoresin prepared from the seeds was analyzed by gas
chromatography and mass spectrometry. The major volatiles
detected were 1-phellandrene, 1,8-cineol, R-terpineol, neral, (E)-
2-decenal (co-eluting with geraniol), and geranial. They cor-
respond respectively to peaks 1-6 (Figure 1). Peaks 7 (1) and
8 (2) (Figure 2) were unknown, and none of the published data
helped to attribute structures to these compounds. Peak 9 had
the same mass spectrum as peak 10, and it was attributed to
4-indanecarbaldehyde (4); peak 10 was 5-indanecarbaldehyde
(5); peaks 11-13 were geranyl acetate, (Z)-2-decenyl acetate,
and (E)-2-dodecenal, respectively; peak 14 was unknown; and
peak 15 corresponded to nerolidol. These attributions were based
on a comparison to the mass spectra of the authentic sample
and to the retention indices on both polar and apolar columns.
The oleoresin was tasted at 50 ppm in water, and an irritating,
pungent, biting effect was perceived.
This extract was fractionated by flash chromatography, and
each fraction was tasted using smelling strips. The fractions
presenting some pungency were pooled. The major compound
was the unknown peak 7, which has a molecular ion of m/z
148, and the elemental analysis suggested a raw molecular
formula of C10H12O. The fragmentation pattern (m/z 105, 91,
and 77) is typical of compounds having a benzene ring; the ion
at m/z 119 corresponds to a loss of -CHO. This fraction was

10906 J. Agric. Food Chem., Vol. 55, No. 26, 2007
Starkenmann et al.
Figure 6. Display of key elements of 13C NMR chemical shifts of indan-4-yl methanol 13–15.
sodium methylate in methanol, gave mainly (()-methyl cis-
2,3,3a,7a-tetrahydo-1H-indene-4-carboxylate 8 (22). The NOESY
experiment suggests an interaction between H-7a and H-3a,
indicating a cis stereochemistry, but this evidence alone was
insufficient to prove the cis stereochemistry. Compound 8 was
thus reduced to give 11. When the Wittig reaction conditions
were modified, using K2CO3 as a base, it was possible to prepare
9. The chemical pathway to the 8,9-dihydroindane implies the
formation of two double bonds by the Wittig reaction and then
cyclization via an intramolecular electrocyclic reaction. Chang-
ing the conditions of the Wittig reaction influences the stereo-
chemistry of the pericyclic reaction that leads to the formation
of the 8,9-dihydroindane. Using potassium carbonate and
dioxane instead of sodium methylate, the reaction gave two
products corresponding to noncyclic structures and compound
9 as a side product, which was latter purified, fully analyzed
by 1H and 13C NMR, and then reduced to give 10. The retention
indices of reduced peak 7 (ISPB-1 1299 and ISPWAX 2115) as well
Compound 12 (tsaokoin), which is compound 2 hydroxylated
on the C-7 position (Figure 4), has a closely related structure
and was previously isolated by bioassay-guided purification of
the A. tsoa ko extract (26). Peak 14 (Figure 1) had a mass
spectrum with m/z 166 corresponding to the molecular weight
of 12 and m/z 148, 119, 105, and 91, which are exactly the
same fragments as those of 1 and 2. Therefore, we isolated peak
14 from our samples of A. tsoa ko. The 1H and 13C NMR spectra
confirm the structure 12, in full agreement with Moon et al.
(26). Tsaokoin 12 was tasted at 50 ppm in plain water, and no
trigeminal effect was detected. Tsaokoin 12 has a cis
configuration. Isotsaokoin, the other cis diastereoisomer
(3aRS,7SR,7aSR)-7-hydroxy-2,3,3a,6,7,7a-hexahydro-1H-in-
dene-4-carbaldehyde was also detected in black cardamom.
A very recent paper (27) reported for the first time the
1
preparation of 2, but their H and 13C NMR data corresponds
to 1, according to our interpretation. We decided to confirm
our results concerning the trans stereochemistry of 1 by catalytic
hydrogenation of 10 and 11 (Figure 5). Platinum oxide was
preferred to palladium to avoid any double-bond migration.
From 10, two isomers were obtained in a 4:1 ratio and were
fully characterized as compounds 13 and 14 (Figure 6). This
result supports the trans configuration of 10, because of the
lack of a good facial differentiation for the hydrogenation
reaction. Conversely, we expected to obtain only one diastere-
oisomer from the reduction of 11 because of the good facial
discrimination presented by the cis configuration. This was
indeed the case, and 15 was isolated and fully characterized.
The 13C NMR of 15 shows C-8 and C-9 at 40.1 and 41.0 ppm,
respectively, whereas there is a clear downfield shift for C-8
and C-9 at 46.4 and 48.2 ppm for 13 (Figure 6), in agreement
with a trans-indanyl stereochemistry (28). The 1H NMR of 13
shows a signal at 0.83 ppm for H-3a, with four coupling
constants (ddd, J ) 11.3, 10.8, 10.8, and 6.6 Hz), which also
confirm a trans configuration. All of these results clearly
differentiate the stereochemistries of 13, 14, and 15 and prove
that Hong et al. (27) synthesized 1 instead of 2.
1
as the H and 13C NMR spectra were compared to those of
synthetic 10 and 11. All data clearly indicate that reduced peak
7 corresponds to the trans compound 10 (Figure 4).
Synthetic compounds 10 and 11 were oxidized in ethanol
with MnO2 to give 1 and 2. Again, the retention indices of the
natural peak 7 corresponded perfectly to those of the synthetic
compound 1 (trans) and not compound 2 (cis). Compound 2 is
more stable than 1 (∆E ) 5.16 kcal) (24, 25), which explains
the interconversion of 1 into 2, as observed in extracts of A.
tsao ko stored 6 months at 4 °C. The oxidation of 11 produced
4 as a side product.
A total of 10 trained panelists tasted both 1 and 2 in plain
water at 10 and 50 ppm. At 10 ppm, a strong, short lasting
pungency was perceived for compound 1 but not for compound
2. The tasting was repeated at 50 ppm, and compound 2 was
surprisingly still not pungent, except for one panelist, who
perceived a faint throat-burning effect. The odor was described
as fatty, fishy, fresh almond for compound 1. Compound 2 is
also almond but with a more phenolic, coumarin type of odor.
The hydroxyl derivatives 10 and 11 were tasted under the same
conditions and were neither hot nor pungent. Compound 10 had
a green, fishy, oyster odor, and compound 11 was terpenic,
earthy, and citrus. Compounds 10 and 11 were not detected in
the natural extract. Compound 1and 2 obtained by synthesis,
as well as natural 1 and 2, were injected onto a chiral capillary
column. This analysis showed that both enantiomers of 1 and 2
are present in A. tsoa ko as a racemic mixture.
To the best of our knowledge, the pungent active compound
1 found in A. tsao-ko has never been described before in
Amomum sp. or in any other natural source.
ACKNOWLEDGMENT
We thank Dr. Horst Sommer for careful discussions, Dr. Jean-
Yves de Saint Laumer for energy calculations, Dr. Estelle Delort
for elemental analysis, and Mrs. Sandy Frank, apprentice, and
Dr. Didier Roguet, curator of the botanical garden of Geneva,
Switzerland, for botanical discussions.
We received different qualities of Amomums: the A. tsoa ko
from China and Vietnam and A. rui li and A. he ko from China.
Compound 1 was present at a slightly higher concentration
(approximately 0.12–0.09%) in the A. tsao-ko qualities compared
to the A. he ko and A. rui li qualities (approximately 0.08–0.05%).

Analysis of Black Cardamom
J. Agric. Food Chem., Vol. 55, No. 26, 2007 10907
LITERATURE CITED
(16) Lawrence, B. M. Terpenes in two Amomum species. Phytochem-
istry 1970, 9, 665.
(1) Purves, D.; Augustine, G. J.; Fitzpatrick, D.; Katz Lawrence, C.;
LaMantia, A. S.; McNamara, J. O.; Williams, M. S. The chemical
senses. Neuroscience, 2nd ed.; Sinauer Associates, Inc.: Sunder-
land, MA, 2001; Chapter 15, pp 317–344.
(2) Rentmeister-Bryant, H.; Green Barry, G. Percieved irritation during
ingestion of capsaicin or piperine: Comparison of trigeminal and
nontrigeminal areas. Chem. Senses 1997, 22, 257–266.
(3) Dhaka, A.; Viswanath, V.; Patapoutian, A. TRP ion channels and
temperature sensation. Annu. ReV. Neurosci. 2006, 29, 135–161.
(4) Peterlin, Z.; Chesler, A.; Firestein, S. A. Painful TRP can be a
bonding experience. Neuron 2007, 53, 635–638.
(5) Clapham, D. E. TRP channels as cellular sensors. Nature 2003,
426, 517–524.
(6) McKermy, D. D.; Neuhausser, W. M. Julius David identification
of a cold receptor reveals a general role for TRP chanels in thermo
sensation. Nature 2002, 416, 52–58.
(7) Zucker Charles, S. A cool ion channel. Nature 2002, 416, 27–28.
(8) Szallasi, A.; Blumberg, P. M. Vanilloid (capsaicin) receptors and
mechanisms. Pharmacol. ReV. 1999, 51, 199–211.
(9) Bryant, B.; Mezine, I.; Epple, G. M. Trigeminal sensory stimuli
and animal repellents from plants. Patent WO 99/21425, 1999.
(10) Galopin, C. C.; Furrer, S. M.; Goeke, A. Pungent and tingling
compounds in Asian cuisine. ACS Symposium Series 867,
American Chemical Society, Washington, D.C., 2003; pp 139–
152.
(11) Barnes, C. S.; Loder, J. W. The structure of polygodial: A new
sesquiterpene dialdehyde from Persicaria hydropiper L. Aust.
J. Chem. 1962, 15, 322–327.
(12) Trevisani, M.; Smart, D.; Gunthorpe, M. J.; Tognetto, M.; Barbieri,
M.; Campi, B.; Amadesi, S.; Gray, J.; Jerman, J. C.; Brough, S. J.;
Owen, D.; Smith, G. D.; Randall, A. D.; Harrison, S.; Bianchi,
A.; Davis, J. B.; Geppetti, P. Ethanol elicits and potentiates
nociceptor responses via the vanilloid receptor-1. Nat. Neurosci.
2002, 5, 546–551.
(17) Volatile Compounds in Food (VCF): database; Nijssen, L. M.,
van Ingen-Visscher, C. A., Donders, J. J. H., Eds.; version 9.2.,
TNO Quality of Life, Zeist, The Netherlands, 2007; p 1963.
(18) Dung, N. X.; Bien, L. K.; Leclercq, P. A. The essential oil of
Amomum tsao-ko Crevost et Lemarie from Vietnam. J. Essent.
Oil Res. 1992, 4, 91–92.
(19) Yi, Z.; Qin, Q.; Guoying, Z.; Zhonghua, X.; Tingli, L. Study on
chemical constituents of volatile oils from Amomum tsao-ko
Crevost et Lemaire. Zhongcaoyao 2004, 35, 1225–1227.
(20) Zhuqiang, L.; Lei, L.; Wanghua, D.; Rong, H.; Yuanqing, Z.
Chemical constituents of essential oil on Amomum tsao-ko from
Yunnan Province. Yunnan Zhiwu Yanjiu 1998, 20, 119–122.
(21) Essential oil from seeds of Amomum tsao-ko Crevost et Lemarie
(Zingiberaceae). II. Oxygen-containing compounds. Sofiya 1991,
31, 203–211.
(22) Bohlmann, F.; Zdero, C. Ein neue Synthese von Cyclohexadien-
derivaten. Chem. Ber. 1973, 106, 3779–3787.
(23) MacroModel, 9.1 ed.; 2007, Schrödinger, LLC: New York, Mo-
hamadi, F.; Richards, N. G. J.; Guida, W. C.; Liskamp, R.;
Lipton, M.; Caufield, C.; Chang, G.; Hendrickson, T.; Still,
W. C. MacroModel. An integrated software system for model-
ing organic and bioorganic molecules using molecular mechan-
ics. J. Comput. Chem. 1990, 11, 440–467.
(24) Jaguar, 6.5 ed.; Schrödinger, LLC: New York, 2005.
(25) Takido, M.; Yoshikawa, Y.; Yamanouchi, S.; Kimura, Y. 1H-
Indene-2,3-dihydro-4-carboxaldehyde and 1H-indene-2,3-dihydro-
5-carboxyaldehyde from the seeds of Amomum medium. Phy-
tochemistry 1978, 17, 327–328.
(26) Moon, S. S.; Lee, J. Y.; Cho, S. C. Isotsaokin, an antifungal agent
from A. tsao-ko. J. Nat. Prod. 2004, 67, 889–891.
(27) Hong, B.-C.; Tseng, H.-C.; Chen, S.-H. Synthesis of aromatic
aldehydes by organocatalytic [4 + 2] and [3 + 3] cycloaddition
of R,ꢀ-unsaturated aldehydes. Tetrahedron 2007, 63, 2840–2850.
(28) Metzger, P.; Cabestaing, C.; Casadevall, E.; Casadevall, A. Ring
distortion effect on δ carbon-13 values of bicycle[4.n.0]alkanes.
Org. Magn. Reson. 1982, 19, 144–147.
(13) Connell, D. W.; Sutherland, M. D. A re-examination of gingerol,
shogaol and zingerone, the pungent principle of ginger (Zingiber
officinale Roscoe). Aust. J. Chem. 1969, 22, 1033–1043.
(14) Connell, D. W. Natural pungent compounds III. The paradols and
associated compounds. Aust. J. Chem. 1970, 23, 369–376.
(15) Yang, X.; Eilerman, R. G. Pungent principal of Alpinia galangal
(L.) Swartz and its applications. J. Agric. Food Chem. 1999, 47,
1657–1662.
Received for review September 12, 2007. Revised manuscript received
November 1, 2007. Accepted November 1, 2007.
JF072707B