Bioactive aromatic compounds from leaves and stems of Vanilla fragrans

Article abstract of DOI:10.1021/jf010425k

Alcoholic extracts of leaves and stems of Vanilla fragrans were fractionated with ethyl acetate and aqueous butanol. All three fractions of ethyl acetate, butanol, and water were screened for toxic bioactivity against mosquito larvae. The results of these experiments showed that the fractions from the ethyl acetate and butanol phases were both active in the bioassay. Bioactivity of the ethyl acetate fraction was found to be much greater than that from the butanol fraction in mosquito larvae toxicity. The water phase appeared to contain no substances that impaired mosquito larval growth. Repeated column chromatography of the ethyl acetate fraction on silica gel led to the isolation of 4-ethoxymethylphenol (1), 4-butoxymethylphenol (2), vanillin (3), 4-hydroxy-2-methoxycinnamaldehyde (4), and 3,4-dihydroxyphenylacetic acid (5). Compounds 4 and 5 were isolated from Vanilla species for the first time and 2 has not been reported to have been found in a natural form. 4-Ethoxymethylphenol (1) was the predominant compound, but 4-butoxymethylphenol (2) showed the strongest toxicity to mosquito larvae. The structures of the compounds were determined on the basis of their mass spectra and ¹H or ¹³C NMR data.

Full text of DOI:10.1021/jf010425k

J. Agric. Food Chem. 2001, 49, 5161−5164
5161
Bioa ctive Ar om a tic Com p ou n d s fr om Lea ves a n d Stem s of Va n illa
fr a gr a n s
Rongqi Sun, J ohn N. Sacalis,* Chee-Kok Chin, and Cecil C. Still
Department of Plant Science, Rutgers University, 59 Dudley Road, New Brunswick, New J ersey 08903
Alcoholic extracts of leaves and stems of Vanilla fragrans were fractionated with ethyl acetate and
aqueous butanol. All three fractions of ethyl acetate, butanol, and water were screened for toxic
bioactivity against mosquito larvae. The results of these experiments showed that the fractions
from the ethyl acetate and butanol phases were both active in the bioassay. Bioactivity of the ethyl
acetate fraction was found to be much greater than that from the butanol fraction in mosquito
larvae toxicity. The water phase appeared to contain no substances that impaired mosquito larval
growth. Repeated column chromatography of the ethyl acetate fraction on silica gel led to the isolation
of 4-ethoxymethylphenol (1), 4-butoxymethylphenol (2), vanillin (3), 4-hydroxy-2-methoxycinnamal-
dehyde (4), and 3,4-dihydroxyphenylacetic acid (5). Compounds 4 and 5 were isolated from Vanilla
species for the first time and 2 has not been reported to have been found in a natural form.
4
-Ethoxymethylphenol (1) was the predominant compound, but 4-butoxymethylphenol (2) showed
the strongest toxicity to mosquito larvae. The structures of the compounds were determined on the
1
13
basis of their mass spectra and H or C NMR data.
Keyw or d s: Vanilla fragrans; bioactivity; insecticidal properties; 4-ethoxymethylphenol; 4-butoxy-
methylphenol; vanillin; toxicity
INTRODUCTION
as were some others, and this interesting observation
led us to determine whether the extract of vegetative
portions of vanilla possesses insecticidal activity. The
present paper deals with the isolation, structural iden-
tification, and biological activity against mosquito larvae
of several aromatic compounds from the leaves and
stems of V. fragrans.
Vanilla is a most popular flavoring material that has
been used for hundreds of years. The plant is a climbing
orchid and is said to attain a length of 100 m (1). There
are about 110 wild-growing species of the genus Vanilla
known throughout the tropics and subtropics, of which
only three are cultivated for their economic impor-
tance: V. planifolia Andrews (V. fragrans), V. tahitensis
Moore, and V. pompona Schiede (2). Because of the
specific fragrance of vanilla the plant has become the
only orchid used for commercial purposes other than for
their ornamental value. Vanilla is now widely used for
the production of foods, and in perfumes and pharma-
ceutical preparations. For this reason, the chemical
constituents that are responsible for the flavor, aroma,
and taste of vanilla bean extract have been extensively
investigated. More than 180 volatile constituents from
cured vanilla beans have been identified (3-7), and
among those components over one-third are aromatic
volatiles. Vanillin is the most abundant ingredient, and
the other major volatile constituents of vanilla aroma
are p-hydroxybenzoic acid, p-hydroxybenzaldehyde, van-
illic acid, p-hydroxybenzyl alcohol, and vanillyl alcohol.
It is now well-known that these aromatic compounds
contribute a great deal to the typical vanilla flavor but
little is known about the biological activity of those
aromatic volatiles. Although there is a large amount of
information concerned with the constituents of vanilla
bean extract, little work has been done to characterize
the components of vegetative portions of this plant. It
was observed by us that greenhouse grown V. fragrans
did not appear to be as frequently attacked by insects
MATERIALS AND METHODS
P la n t Ma ter ia l. Vegetative aerial parts of Vanilla fragrans
were collected in J une 1999 from the greenhouse of Cook
College, Rutgers University, New Brunswick, New J ersey.
Ch em ica ls. All reagents used for column chromatography
and thin-layer chromatography were of HPLC grade. Ethyl
alcohol (95%) was used for extraction. Pyridine and acetic
anhydride were obtained from Sigma Chemical Company. All
other solvents were analytical grade or better. Silica gel
(Merck, 230-400 mesh) used for column chromatography was
purchased from Aldrich Chemical Co., Inc.
Extr a ction a n d Isola tion . Extraction. Before extraction,
the freshly collected leaves and stems (3 kg of each) were
washed with distilled water to eliminate impurities and
organic fertilizers. The washed material was then chopped into
fine pieces within an electric blender in the presence of ethyl
alcohol. The chopped tissue was transferred to an extraction
container into which a total volume of 4000 mL of ethyl alcohol
(95%) was added. The container was kept at room temperature
for 4 to 5 days. The alcoholic extraction solution was filtered
and this treatment was repeated twice more with ethyl alcohol
(2 × 3000 mL). The combined aqueous ethyl alcohol phase was
evaporated in a rotary evaporator under reduced pressure until
the final concentration of alcohol was approximately 20%. The
concentrated solution was kept in a freezer to precipitate
chlorophyll and insoluble material, which were than filtered
off. Evaporation of the remaining alcohol in the filtrate left
an orange water phase. This was extracted with ethyl acetate
(3 × 150 mL), followed by aqueous butanol (3 × 300 mL). After
*
Corresponding author. Tel: 732-932-9711, ext. 131. Fax:
7
32-932-9441. E-mail: sacalis@aesop.rutgers.edu.
1
0.1021/jf010425k CCC: $20.00 © 2001 American Chemical Society
Published on Web 10/04/2001

5162 J. Agric. Food Chem., Vol. 49, No. 11, 2001
Sun et al.
(
(
(
CDCl
3
): δ 9.70 (1H, s). 7.40 (2H, m, aromatic protons), 7.01
1H, d, J ) 8.5 Hz, aromatic protons), 6.30 (1H, s, OH), 3.90
3H, s, OCH ).
-Hyd r oxy-2-m eth oxycin n a m a ld eh yd e (4). Yellowish
3
4
+
+
powder. EI-MS (m/z, %): 178 (M , 100), 177 (M -H, 30), 163
(
+
+
+
M -CH
3
, 10) 161 (M -OH, 21), 147 (M -OCH
3
, 38), 135 (44),
1
24 (16), 118 (16), 107 (30), 89 (16), 77 (26), 63 (10), 55 (4), 51
+
+
(12). Acetate: 220 (M , 36), 178 (M -42, 100), 163 (85), 145
(
5), 134 (7), 17 (3), 105 (2), 89 (5), 77 (6), 63 (1), 51 (3), 43 (35).
1
H NMR (acetone-d ): δ 9.65 (1H, d, J ) 6 Hz), 7.50-7.70 (3H,
6
m, aromatic protons), 6.80-6.90 (2H, m, olefinic protons), 3.85
(3H, s, OCH
3
).
3
,4-Dih yd r oxyp h en yla cetic a cid (5). Yellow crystals; mp
+
1
4
(
4
(
26-127 °C (Lit. value: 129 °C, 8). EI-MS (m/z, %): 168 (M ,
0), 151 (M -OH, 1), 139 (5), 123 (M -COOH, 100), 111 (6), 94
10), 77 (14), 65 (12), 51 (16). Acetate: 252 (M , 8), 220 (M -
+
+
+
+
F igu r e 1. Structures of compounds 1-5.
+
2, 35), 168 (M -42 × 2, 100), 151 (4), 139 (5), 123 (55), 111
1
9), 93 (10), 77 (3), 65 (6), 55 (5), 43 (38). H NMR (acetone-
evaporation of solvents, 2 and 22 g of extracts were obtained
from the ethyl acetate and butanol fractions, respectively. The
water phase was also evaporated to dryness under reduced
pressure to provide 43 g of yellowish solid substance.
Each of the above-mentioned extracts was subjected to
toxicity bioassays using mosquito larvae as test organisms for
initial screening. Results of these preliminary bioassays
showed that both the ethyl acetate extract and the butanol
extracts were biologically active, whereas the yellowish solid
substance from the water extract was neither toxic nor
beneficial to mosquito larvae. The lethality of the ethyl acetate
fraction to mosquito larvae was very much stronger than that
of the butanol fraction. Therefore, the ethyl acetate extraction
d
6
): δ 7.0-8.50 (2H, br, OH×2), 6.60-6.70 (2H, m, aromatic
protons), 6.39 (1H, s, aromatic proton), 3.15 (2H, s, Ar-CH
CO).
2
Acetyla tion of Com p ou n d s 1-5. One milligram of each
compound was dissolved in pyridine and then a few drops of
acetic anhydride were added to that solution. The resulting
reaction was kept at room temperature overnight until acet-
ylation was completed (monitored by TLC). Excess acetic
anhydride and pyridine were removed under a stream of
nitrogen. The residue was extracted with methylene chloride
and subjected to mass spectrometry. The mass spectrum of
the acetate was compared with that of each original compound
and molecular weight increase was calculated.
(2 g) was used for the subsequent isolation of insecticidal
compounds.
Gen er a l Sp ectr oscop ic P r oced u r es. H (300 MHz) NMR
spectra were measured on a General Electric QE-300Mz NMR
spectrometer, and the C-13 spectrum of 4-butoxymethylphenol
was obtained on a General Electric Omega-500Mz NMR
spectrometer. Deuterated chloroform or deuterated acetone
was used as solvent, and tetramethylsilane (TMS) was used
as internal standard. Mass spectra were recorded on a
Hewlett-Packard HP-5973 mass selective detector in the EI
mode at 70 eV.
Pretreatment and Isolation. The ethyl acetate fraction (2 g)
was dissolved in a small amount of ethyl acetate and then
transferred onto a chromatography column filled with 75 g of
dry silica gel. Methylene chloride (850 mL) and ethyl alcohol
(300 mL) were used as elution solvents. After removal of the
solvent, the methylene chloride eluant gave 600 mg of residue
that was proved to be the lethal fraction to mosquito larvae.
This portion was repeatedly chromatographed on silica gel
columns with a linear gradient of 20:1 to 2:1 hexane-ethyl
acetate. The eluate was monitored with TLC, and eluates
containing each pure compound were combined. Removal of
the solvents from the combined fractions gave pure compounds
Mosqu ito La r va e Bioa ssa y Scr een in g P r otocol. Mos-
quito eggs, Culex pipiens, were purchased from Carolina
Biological Supply Company, Burlington, North Carolina. After
purchase, the eggs were transferred into a culturing beaker
with distilled water, into which a pinch of powdered milk was
added. Within a short time, larvae were produced from the
eggs. The larvae were then allowed to grow until they were
approximately 7 days old, when the bioassays were conducted.
Flat-bottom multiwell polystyrene cell culture plates with 24
wells were used for performing the bioassay. Bioassay solu-
tions containing predetermined amounts of extracts were
prepared in water for water-soluble samples or prepared in
water with the presence of a trace amount of dimethyl
sulfoxide (DMSO) for the ethyl acetate extract and the pure
compounds isolated. In each case that DMSO was employed
to prepare the solution, the same volume of DMSO was also
added to each water control well. Five 7-day old larvae and a
sample solution were then transferred into each well. The
survivors of mosquito larvae were counted from 0.5 h to 24 h
later at room temperature. The same protocol was used for
controls containing distilled water with or without the presence
of DMSO. All bioassays were performed in triplicate. It was
found that the crude extract was lethal to the larvae and the
extract was partitioned into ethyl acetate, butanol, and water
fractions. Each fraction was then screened for bioactivity
against mosquito larvae. The ethyl acetate fraction at the
concentration of 1.0-2.0 mg/mL displayed a very strong
toxicity to mosquito larvae, resulting in total lethality to larvae
within only a few hours. Larvae exposed to either the butanol
or water fraction showed little or no activity when used at the
same concentration. Tests with the water fraction indicated
that it might even have been slightly beneficial to larvae.
1
(420 mg), 2 (21 mg), 3 (3 mg), 4 (4 mg), and 5 (16 mg).
4
-Eth oxym eth ylp h en ol (1). Yellowish oil. EI-MS (m/z,
+
+
+
%
(
): 152 (M , 40), 135 (M -OH, 1.5), 123 (M - C
2
H
5
, 12), 107
+
M -OC
2 5
H , 100), 95 (13), 77 (24), 65 (4), 51 (8), 41 (11).
+
+
Acetate: 194 (M , 12), 152 (M -42, 85), 135 (5), 123 (18), 107
1
(
100), 94 (14), 77 (17), 65 (4), 51 (4), 43 (15). H NMR(CDCl
δ 7.50 (1H, br, OH), 7.18 (2H, d, J ) 8.5 Hz, aromatic protons),
.34 (2H, d, J ) 8.5 Hz, aromatic protons), 4.45 (2H, s, Ar-
CH OEt), 3.60 (2H, q, J ) 7Hz, OCH Me), 1.25 (3H, t, J ) 7
Hz, CH ).
-Bu toxym eth ylp h en ol (2). Yellowish oil. EI-MS (m/z,
3
):
6
2
2
3
4
+
+
+
%
): 180 (M , 35), 163 (M -OH, 0.5), 123 (15), 107 (M -
CO , 100), 95 (13), 77 (24), 65 (4), 51 (8), 41 (12). Acetate:
22 (M , 8), 180 (M -42, 65), 163 (1), 149 (2), 123 (12), 107
2
2 5
C H
+
+
2
1
(
100), 95 (8), 78 (10), 77 (11), 65 (1), 52 (2), 43 (9), 41 (4). H
NMR(CDCl ): δ 7.18 (2H, d, J ) 8.5 Hz, aromatic protons),
.73 (2H, d, J ) 8.5 Hz, aromatic protons), 6.26 (1H, s, OH),
.43 (2H, s, Ar-CH O), 3.50 (2H, t, J ) 7 Hz, protons italicized
CH CH CH ), 1.59 (2H, quintet, CH ), and 1.39 (2H,
sextet, CH ), 0.90 (3H, t, J ) 7 Hz, CH ). C NMR(CDCl ): δ
56.2, 131.2,130.0, 115.9 (all aromatic carbons), 73.2, 70.7
3
6
4
2
in OCH
2
2
2
3
2
1
3
2
3
3
1
(
carbons italicized in CH
OCH CH CH CH ), 20.0 (CH
).
Va n illin (3). White crystals; mp 80-81 °C. (Lit. value: 82
2
OCH
2
CH
2
2
CH
2
CH
3
), 32.4 (CH
2
-
2
2
2
3
2
OCH
CH
2
CH
2
3
CH ), and 14.6
(CH
3
+
+
°
C, 8). EI-MS (m/z, %): 152 (M , 100), 151 (M -H, 98), 137
+
3
(M -CH , 5), 123 (14), 109 (15), 93 (2), 81(17), 77 (4), 65 (5),
+
+
5
1
3 (7), 41 (1). Acetate: 194 (M , 8), 152 (M -42, 100), 137 (4),
23 (6), 109 (7), 95 (1), 79 (4), 63 (5), 51 (6), 43 (16). H NMR
1

Bioactive Compounds of Vanilla
J. Agric. Food Chem., Vol. 49, No. 11, 2001 5163
Ta ble 1. P er cen t Mor ta lity of Mosqu ito La r va e Exp osed
to Com p ou n d s 1-5 a t Differ en t Tim e In ter va ls
within only 3 h. Vanillin (3) and compound 4 were
aromatic aldehydes, and it was interesting that they
exhibited similar activity toward the larvae. They
resulted in the death of the tested larvae in relatively
concentrated solution, compared with solutions using
compounds 1 or 2. A solution of 1.0 mg/mL of compound
0
.5 h
0
3 h
5 h
10 h
0
24 h
0
distilled H2O
compound 1
0
0
0
0
0
.1 mg/mL
.3 mg/mL
.5 mg/mL
0
0
0
0
7
27
0
47
61
11
53
100
50
73
100
3
or 4 caused death of the larvae during 20 to 24 h.
Unexpectedly, compound 5 did not have an effect on
mosquito larvae within long periods of time, even at the
concentration of 1.0 mg/mL. The mechanism by which
toxicity to mosquito larvae is conferred is not known.
Following exposure to compound 4 or 5 for a few hours,
the inner transparent bodies of the larvae changed to
the yellow color of the corresponding compounds. The
larvae exposed to compounds 1 or 2 for a short time
appeared shrunken after death. These observations,
although open to interpretation, suggest the larvae were
not repelled by those phenolic compounds, some of which
are bitter tasting to human beings (11), indicating that
antifeedant activity was not a factor.
compound 2
0
0
0
0
.05 mg/mL
.1 mg/mL
.2 mg/mL
.4 mg/mL
0
0
10
60
0
67
100
100
0
73
100
100
17
83
100
100
50
100
100
100
compound 3
1
2
.0 mg/mL
.0 mg/mL
0
0
0
15
11
39
50
94
100
100
compound 4
0
1
.6 mg/mL
.0 mg/mL
0
0
0
0
17
33
33
50
67
100
compound 5
0
1
.4 mg/mL
.0 mg/mL
0
0
0
0
0
0
0
0
0
17
RESULTS AND DISCUSSION
Id en tifica tion of Com p ou n d s. Compound 1 (420
mg) was a yellowish oil at room temperature. The mass
spectrum of compound 1 indicated M at m/z 152,
+
P h en olic Com p ou n d s Isola ted a n d Th eir Bio-
logica l Activity a ga in st Mosqu ito La r va e. The
mortality of mosquito larvae exposed to compounds 1-5
is listed in Table 1. In 10 h, compound 1 killed half of
the tested mosquito larvae at the concentration of 0.3
mg/mL and killed all the larvae at the concentration of
suggesting a molecular formula as C9H12O2. The major
fragment ions at m/z 123, 107, 95, 77, 65, and 51
indicated the presence of a substituted benzene skeleton
+
in the molecule. The significant ions at m/z 123 (M -
+
2
9, 12%) and 107 (M -29-16, 100%) suggested the
0
.5 mg/mL. Compound 2 killed 67% of the larvae at a
existence of an ethoxyl group in the molecular structure.
concentration of 0.1 mg/mL and killed all the larvae at
the concentration of 0.2 mg/mL in 3 h. At 1.0 mg/mL of
compound 3 half of the larvae were killed within 10 h
and all the larvae were killed in 24 h. The toxicity of
compound 4 to the larvae was similar to that of
compound 3 when the same concentration was used.
Compound 5 exhibited no toxicity to the larvae when
the concentration was below 1.0 mg /mL.
Phenolic and aromatic compounds were the most
abundant ingredients in the ethyl acetate extract of
Vanilla fragrans leaves and stems. Many naturally
occurring phenolic compounds are known to possess
biological activity in animals, microorganisms, and
plants (9). Compounds 1-5 are phenolic derivatives.
However, the biological activities of these ingredients
have not yet received attention. 4-Ethoxymethylphenol
+
In the mass spectra, the M of its acetate was observed
at m/z 194 (42 mass units higher than that of compound
1
), which showed that there was a free hydroxyl group
1
in this molecule. In the H NMR of compound 1,
characteristic proton signals of p-substituted benzene
were observed at δ 7.18 (2H, d, J ) 8.5 Hz) and 6.34
(2H, d, J ) 8.5 Hz). Other signals at δ 7.50 (1H, broad
s), 4.45 (2H, s) 3.60 (2H, q, J ) 7 Hz), and 1.25 (3H, t,
J ) 7 Hz) reinforced the existence of a free hydroxyl
group and an ethoxymethyl group in the molecule. On
the basis of the evidence mentioned above, the structure
of compound 1 was identified as 4-ethoxymethyl-phenol.
This compound was also reported to be isolated from
fresh Gastrodia elata (12).
Compound 2 (21 mg) was also a yellowish oil. The M+
at m/z 180 in the mass spectrum suggested its molecular
formula as C11H16O2. The presence of major fragment
ions at m/z 123, 107, 95, 77, 65, and 51 was similar to
that of compound 1, which indicated that the compound
was also a substituted benzene. In the mass spectrum
(
1) was the most abundant component, which was
obtained at a 21% yield. But vanillin (3) and 4-hydroxy-
-methoxycinnamaldehyde (4) were found to exist in the
2
extract in only small quantities. It has been reported
that vanillin does not exist in the free form in freshly
harvested vanilla beans (10). The present investigation,
however, revealed that it might exist in fresh leaves and
stems, even if only in trace amounts. It cannot be
discounted that vanillin may be present as a breakdown
product of another compound. Compounds 4 and 5 were
isolated from Vanilla species for the first time. Com-
pound 2 has not been reported as a naturally occurring
compound to this date.
Compound 1 was proven to be a biologically active
component that displayed 100% mortality to mosquito
larvae in 10 h at the tested concentration of 0.5 mg/mL
in water. Compound 2 was the most active of these
compounds, displaying considerable mortality to mos-
quito larvae within 24 h at the concentration between
+
of its acetate, the M was observed at m/z 222, which
confirmed the existence of a free hydroxyl group in
1
molecule. The H NMR of compound 2 was also similar
to that of compound 1 except for two more groups of
proton signals due to two more CH2 groups, which were
observed at δ 1.59 (2H, quintet) and 1.39 (2H, sextet).
The signals of 1,4-disubstituted benzene appeared at δ
7.18 (2H, d, J ) 8.5 Hz) and 6.73 (2H, d, J ) 8.5 Hz).
The signals at δ 6.26 (1H, broad s), 4.43 (2H, s), 3.50
(2H, t, J ) 7Hz), and 0.90 (3H, t, J ) 7 Hz) confirmed
the existence of a butoxymethyl group in the molecule.
Therefore, the structure of compound 2 was determined
to be 4-butoxymethylphenol. The structure was rein-
forced by the observation of carbon signals in its carbon-
13 spectrum at δ 156.2, 131.2, 130.0, 115.9 (all aromatic
carbons), 73.2, 70.7 (two oxygenated carbons), 32.4, 20.0
(carbons of two CH2 groups), and 14.6 (methyl carbon).
0
.05 and 0.1 mg/mL. As an example of the toxicity of
compound 2 to mosquito larvae, a solution of 0.2 mg/
mL of compound 2 killed 100% of all larvae tested

5164 J. Agric. Food Chem., Vol. 49, No. 11, 2001
Sun et al.
This is the first report of 4-butoxymethylphenol as a
naturally occurring compound.
LITERATURE CITED
(
1) Graf, A. B. HORTICA: A Color Cyclopedia of Garden
Flora in All Climates and Indoor Plants, 1st edition;
Roehrs Co.: East Rutherford, NJ , 1992.
Compound 3 (3 mg) was white crystals. The mass
+
spectrum of compound 3 gave M at m/z 152, suggesting
its molecular formula as C8H8O3. In the mass spectrum
of its acetate, M was observed at m/z 194, which
(2) Ehlers, D.; Pfister, M. Compounds of Vanillons. J .
Essent. Oil Res. 1997, 9, 427-431.
+
(
(
(
3) Taylor, S. Improved determination of vanillin and
related phenolic components in vanilla by high-perfor-
mance liquid chromatography. Flavour Fragrance J .
confirmed the existence of a free hydroxyl group in
molecule. The compound was identified as vanillin by
1
comparing its mass spectrum and H NMR spectrum
1
993, 8, 281.
with that of an authentic sample.
4) Schulte-Elte, K. H.; Gautschi, F.; Renold, W.; Hauser,
A.; Frankhauser, P.; Limacher, J .; Ohloff, G. Important
constituents of vanilla aroma. Helv. Chim. Acta 1978,
61, 1125.
Compound 4 (4 mg) was a yellowish powder. In the
+
mass spectrum of compound 4, the M was observed at
m/z 178, and fragment ions of substituted benzene
appeared at m/z 51,55, 63, and 77, suggesting its
molecular formula as C10H10O3. The presence of a
hydroxyl group and a methoxyl group were suggested
in the molecule by the observation of signals at m/z 161
5) Klimes, I.; Lamparsky, D. Vanilla volatiles - A Com-
prehensive Analysis. Int. Flavours Food Addit. 1976, 7
(Nov/Dec), 272.
(
6) Anwar, M. H. Paper chromatography of monohydroxy-
phenols in vanilla extract. Analyt. Chem. 1963, 53, 404.
7) Ranadive, A. S. Vanillin and Related Flavor Compounds
in Vanilla Extracts Made from Beans of Various Global
Origins. J . Agric. Food Chem. 1992, 40, 1922.
(
3
M-OH, 21%), 163 (M-CH3, 10%), and 147 (M-OCH3,
8%). In the mass spectrum of its acetate, M at m/z
(
+
2
20 reinforced the existence of a hydroxyl group. In the
1
(8) Pouchert, C. J .; Behnke, J . The Aldrich Library of 13C
H NMR spectrum of compound 4, the presence of the
methoxyl group and an aldehyde group were confirmed
by the signal at δ 3.85 (3H, s) and 9.60 (1H, d, J ) 6
Hz). The compound was finally identified as 4-hydroxy-
1
and H FT NMR Spectra, 1st ed.; Aldrich Chemical Co.,
Inc.: Milwaukee, WI, 1993; Vol. II.
(
9) Fahey, G. C., J r.; J ung, H. G. Phenolic Compounds in
Forages and Fibrous Feedstuffs. In Toxicants of Plant
Origin; Cheeke, P. R., Ed.; CRC Press: Boca Raton, FL,
2
-methoxycinnamaldehyde by analysis of all the NMR
signals and a comparison with that of related com-
pounds (8, 13). The existence of this compound in
sunflower aroma has been reported (14).
1
989; Vol. IV, p 124.
(
10) Leong, G.; Archavlis, A.; Derbesy, M. Research on the
glucoside fraction of the vanilla bean. J . Essent. Oil Res.
1989, 1, 33.
Compound 5 (16 mg) was yellow crystals. The mass
+
(11) Belitz, H. D.; Wieser, H. Bitter compounds: Occurrence
spectrum of compound 5 gave M at m/z 168, suggesting
and structure-activity relationships. Food Rev. Int.
its molecular formula as C8H8O4. Signals due to a
1
985, 1, 271.
benzene ring were found at m/z 51, 65, and 77. The base
(
12) Zhou, J .; Yang, Y.; Pu, X. Phenolic constituents of fresh
+
peak was observed at m/z 123 (M -COOH), indicating
Gastrodia elata Blume. Yunnan Zhiwu Yanjiu 1980, 2,
the formation of a very stable fragment ion after the
loss of a carboxyl group from the molecule. Two hy-
droxyls were confirmed after acetylation by observation
3
70.
1
(
13) Liptaj, T.; Remko, M.; Polcin, J . Analysis of H NMR
Spectra of Cinnamaldehyde Type Model Substance of
Lignin. Collect. Czecho Chem. Commun. 1980, 45, 330.
+
+
+
of signals at m/z 252 (M ), 210 (M -42), and 168 (M -
4
1
2 × 2) in the mass spectrum. In the H NMR spectrum
(14) Thiery, D.; Bluet, J . M. Pham-Del e` gue; Eti e´ vant, P.;
Masson, C. Sunflower aroma detection by the honeybee:
Study by Coupling Gas Chromatography and Electroan-
tennography. J . Chem. Ecol. 1980, 16, 701.
of compound 5, the presence of a carboxymethyl (CH2-
COOH) attached to a benzene ring was suggested by a
signal at δ 3.14 (2H, s). The above-described evidence
indicated that compound 5 is a trisubstituted benzene.
1
Received for review March 29, 2001. Revised manuscript
received August 7, 2001. Accepted August 9, 2001.
The observation of identical signals of H NMR between
compound 5 and 3,4-dihydroxyphenylacetic acid re-
vealed that they were same compound (12).
J F010425K