Antimutagenic Activity of Phenylpropanoids from Clove (Syzygium aromaticum)

Article abstract of DOI:10.1021/jf030247q

Phenylpropanoids that possess antimutagenic activity were isolated from the buds of clove (Syzygium aromaticum). The isolated compounds suppressed the expression of the umu gene following the induction of SOS response in the Salmonella typhimurium TA1535/pSK1002 that have been treated with various mutagens. The suppressive compounds were mainly localized in the ethyl acetate extract fraction of the processed clove. This ethyl acetate fraction was further fractionated by silica gel column chromatography, which resulted in the purification and subsequent identification of the suppressive compounds. Electron impact mass spectrometry, IR, and ¹H and ¹³6 NMR spectroscopy were then used to delineate the structures of the compounds that confer the observed antimutagenic activity. The secondary suppressive compounds were identified as dehydrodieugenol (1) and trans-coniferyl aldehyde (2). When using 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (furylfuramide) as the mutagen, compound 1 suppressed 58% of the umu gene expression as compared to the controls at a concentration of 0.60 μmol/mL, with an ID₅₀ (50% inhibitory dose) value of 0.48 μmol/mL, and compound 2 suppressed 63% of the umu gene expression as compared to the controls at a concentration of 1.20 μmol/mL, with an ID₅₀ value of 0.76 μmol/mL. Additionally, compounds 1 and 2 were tested for their ability to suppress the mutagenic activity of other well-known mutagens such as 4-nitroquinolin 1-oxide (4NQO) and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), which do not require liver metabolizing enzymes, and aflatoxin B1 (AfB₁) and 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), which require liver metabolizing enzymes and activated Trp-P-1 and UV irradiation. Compounds 1 and 2 showed dramatic reductions in their mutagenic potential of all of the aforementioned chemicals or treatment. For the search of the structure-activity relationship, the derivatives of 1 and 2 (1a and 2a-c) were also assayed with all mutagens. Finally, the antimutagenic activities of compounds 1, 1a, 2, and 2a-c against furylfuramide, Trp-P-1, and activated Trp-P-1 were assayed by the Ames test using the S. typhimurium TA100 strain.

Full text of DOI:10.1021/jf030247q

J. Agric. Food Chem. 2003, 51, 6413−6422 6413
Antimutagenic Activity of Phenylpropanoids from Clove
Syzygium aromaticum)
(
MITSUO MIYAZAWA* AND MASAYOSHI HISAMA
Department of Applied Chemistry, Faculty of Science and Engineering, Kinki University,
Kowakae, Higashiosaka-shi, Osaka 577-8502, Japan
Phenylpropanoids that possess antimutagenic activity were isolated from the buds of clove (Syzygium
aromaticum). The isolated compounds suppressed the expression of the umu gene following the
induction of SOS response in the Salmonella typhimurium TA1535/pSK1002 that have been treated
with various mutagens. The suppressive compounds were mainly localized in the ethyl acetate extract
fraction of the processed clove. This ethyl acetate fraction was further fractionated by silica gel column
chromatography, which resulted in the purification and subsequent identification of the suppressive
compounds. Electron impact mass spectrometry, IR, and H and 13C NMR spectroscopy were then
used to delineate the structures of the compounds that confer the observed antimutagenic activity.
The secondary suppressive compounds were identified as dehydrodieugenol (1) and trans-coniferyl
aldehyde (2). When using 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (furylfuramide) as the mutagen,
compound 1 suppressed 58% of the umu gene expression as compared to the controls at a
concentration of 0.60 µmol/mL, with an ID50 (50% inhibitory dose) value of 0.48 µmol/mL, and
compound 2 suppressed 63% of the umu gene expression as compared to the controls at a
concentration of 1.20 µmol/mL, with an ID50 value of 0.76 µmol/mL. Additionally, compounds 1 and
1
2
were tested for their ability to suppress the mutagenic activity of other well-known mutagens such
as 4-nitroquinolin 1-oxide (4NQO) and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), which do not
require liver metabolizing enzymes, and aflatoxin B (AfB ) and 3-amino-1,4-dimethyl-5H-pyrido[4,3-
1
1
b]indole (Trp-P-1), which require liver metabolizing enzymes and activated Trp-P-1 and UV irradiation.
Compounds 1 and 2 showed dramatic reductions in their mutagenic potential of all of the
aforementioned chemicals or treatment. For the search of the structure-activity relationship, the
derivatives of 1 and 2 (1a and 2a-c) were also assayed with all mutagens. Finally, the antimutagenic
activities of compounds 1, 1a, 2, and 2a-c against furylfuramide, Trp-P-1, and activated Trp-P-1
were assayed by the Ames test using the S. typhimurium TA100 strain.
KEYWORDS: Clove; Syzygium aromaticum; dehydrodieugenol; trans-coniferyl aldehyde; antimutagenic
activity
INTRODUCTION
changes in dietary habits or dietary supplements. Humans ingest
large numbers of naturally occurring antimutagens, and anti-
carcinogens may inhibit one or more stages of the carcinogenic
process and prevent or delay the formation of cancer. Recent
studies indicate that compounds with antioxidant or antiinflam-
matory properties, as well as certain phytochemicals, can inhibit
tumor initiation, promotion, and progression in experimental
animal models. Epidemiological studies indicate that dietary
factors play an important role in the development of human
cancer. Attempts to identify naturally occurring dietary anti-
mutagens and anticarcinogens may lead to new strategies for
cancer prevention.
Cancer is currently one of the most dreaded diseases, although
methods for its detection at early stages and therapeutic remedies
have greatly advanced. In Japan, cancer has been the leading
cause of mortality since 1981 (1). Kee (2) reported that the
causes of cancer exist in the environment, especially in the diet
and tobacco. Cancer can be initiated by DNA damage, which
is caused by natural and man-made chemical substances
(mutagens) in the environment (3).
Efforts in cancer chemotherapy have intensified over the past
several decades, but many cancers still remain difficult to cure;
cancer prevention could become an increasingly useful strategy
in our fight against cancer. Human epidemiology and animal
studies have indicated that cancer risk may be modified by
In the evaluation of the carcinogencity or mutagenicity of
environmental chemicals, it is quite important to determine
factors present in the environment that may affect these
activities. With the development of laboratory techniques for
*
To whom correspondence should be addressed. Tel: +81-6-6721-2332.
Fax: +81-6-6727-4301. E-mail: miyazawa@apch.kindai.ac.jp.
1
0.1021/jf030247q CCC: $25.00 © 2003 American Chemical Society
Published on Web 09/17/2003

6414 J. Agric. Food Chem., Vol. 51, No. 22, 2003
Miyazawa and Hisama
the detection of possible environmental carcinogens and mu-
tagens (4), it has been shown that ordinary human diets contain
several mutagens and antimutagens. In particular, the umu test
system was developed as a simple but sensitive tool to evaluate
the genotoxic activities of a wide variety of environmental
carcinogens and mutagens, using the expression of one of the
SOS genes to detect DNA-damaging agents (5, 6). The results
of this test are in agreement with the results of the Ames test
and may be more useful with respect to simplicity and rapidity
toward the furylfuramide-induced SOS response. We thought
that this plant might be useful as a cancer chemopreventive
agent, and we were interested in the yet unidentified antimu-
tagenic compounds in clove. In this paper, we report the isolation
by chemical extraction and silica gel column chromatography
and the identification by mass spectrometry, IR spectrometry,
and NMR of these secondary antimutagenic compounds con-
tained in clove.
(7). The umu test detects the induction of the SOS response
MATERIAL AND METHODS
following treatment of Salmonella typhimurium TA1535 with
test compounds. This strain carries the plasmid pSK1002 in
which the umuC′ gene is fused inframe to the lacZ′ gene. The
SOS-inducing potency of test compounds would therefore be
estimated by the measurement of induction of the level of umu
operon in terms of intracellular â-galactosidase activity.
Furylfuramide is one of the nitrofuran derivatives that has
been widely used as a food preservative in Japan. Its genetic
effects were reported by many researchers, which led to its
classification as a potent mutagen (8, 9). Likewise, 4-nitro-
quinolin 1-oxide (4NQO), a nitroheterocyclic compound, and
N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), a direct-acting
alkylating agent, were classified as potent mutagens (10-13).
Alternatively, aflatoxin B1 (AfB1), a difurofuran ring fused to
a substituted coumarin moiety, and 3-amino-1,4-dimethyl-5H-
pyrido[4,3-b]indole (Trp-P-1), a heterocyclic amine derived from
protein pyrolysis, were shown to be highly mutagenic following
their activation by the enzymes contained in the liver S9 fraction
General Procedure. Electron impact mass spectra (EI-MS) were
obtained on a Hewlett-Packard 5972A mass spectrometer. IR spectra
were determined with a FT/IR-470 Plus Fourier Transform Infrared
Spectrometer. NMR spectra (δ, J in Hertz) were recorded on a JEOL
FX-500 NMR spectrometer. Tetramethylsilane (TMS) was used as the
1
internal reference (δ 0.00) for H NMR spectra measured in CDCl
3
.
This solvent was also used for 13C NMR spectra.
Materials. Commercially available air-dried tips of clove (Tyouji)
were obtained from Yamada Yakken Co., Ltd. Furylfuramide, 4NQO,
MNNG, Trp-P-1, and AfB were purchased from Wako Pure Chemical
1
Co. S9 (supernatant of 9000g) and coenzyme, NADPH, NADH, and
G-6-P were purchased from Oriental Yeast Co.
umu Test. The umu test for detecting the chemical-induced SOS
response was carried out according to the method of Oda et al. (5)
using S. typhimurium TA1535/pSK1002, in which a plasmid (pSK1002)
carrying a fused gene (umuC′-lacZ) had been introduced. The
overnight culture of bacterial strain was diluted 50-fold into TGA
medium (1% Bactotryptone, 0.5% NaCl, and 0.2% glucose; supple-
mented with 20 mg/L of ampicillin) and incubated at 37 °C until the
bacterial density reached 0.25-0.30 in OD600. The bacterial culture
was subdivided into 2.1 mL portions in test tubes, and the test compound
(14-16).
Antimutagenic effects of some naturally occurring compounds
(
(
50 µL, diluted in dimethyl sulfoxide (DMSO)), 0.1 M phosphate buffer
300 µL, pH 7.4), and mutagens furylfuramide (50 µL, 2 µg/mL in
against mutagens have been investigated, including S-(N,N-
diethyldithiocarbamoyl)-N-acetyl-L-cysteine (17), R-pinene-7â-
O-â-D-2,6-diacetylglucopyranoside (18), cinnamic acid (19), and
palmitic acid (20). The resveratrol, a stilbene derivative, was
shown to suppress Trp-P-1-induced SOS as measured by the
umu test and also to suppress its mutagenic potential as measured
by the Ames test (21); Jang et al. (22) have also shown its ability
to act as a potent cancer chemopreventive agent.
DMSO), 4NQO (50 µL, 20 µg/mL in DMSO), MNNG (50 µL, 200
µg/mL in DMSO), and activated Trp-P-1 (50 µL, 10 µg/mL in DMSO)
were added to each tube. In the case of AfB (50 µL, 20 µg/mL in
1
DMSO) and Trp-P-1 (50 µL, 40 µg/mL in DMSO), 300 µL of S9-
metablizing enzyme mixture including the cofactors was added instead
of the phosphate buffer. As a positive control, an equivalent volume
of DMSO was added instead of the test compound, whereas with
negative control an equivalent volume of DMSO was added instead of
both the test compound and the mutagen. After 2 h of incubation at 37
Clove (Syzygium aromaticum, Myrtaceae) is a plant that is
cultivated as a spice in many tropical countries. The most
important clove-exporting countries are Tanzania, Indonesia, Sri
Lanka, and the Malagasy Republic. For oil production, the clove
buds are brought to European and American distilleries. Clove
oil is frequently used in perfumery and medicine, but the largest
part by far is used in flavorings. The dried flower buds of clove
°C with shaking, the culture was centrifuged (3000 rpm) to collect cells,
which were centrifuged in 2.5 mL of phosphate-buffered saline (PBS).
The level of â-galactosidase activity was measured according to a slight
modification of Miller’s method (33). Fractions (0.25 mL) of the culture
were diluted with 2.25 mL of Z buffer, and 0.1% sodium dodecyl sulfate
solution (50 µL) and chloroform (10 µL) were added to each fraction.
The enzyme reaction was initiated by the addition of 0.25 mL of 2-nitro-
phenyl-â-D-galactopyranoside solution (ONPG; 4 mg/mL in 0.1 M
phosphate buffer, pH 7.4) at 28 °C. After 15 min, the reaction was
(“Tyouji” in Japanese) are an oriental drug, which has been used
as vermifuge, an antibacterial agent, and to treat toothaches (23).
The clove species have been demonstrated to produce a wide
variety of potentially useful chemical compounds that include
sesquiterpenes (24), tannins (25), and triterpenoids (26). It is
well-known that clove possesses a phenolic compound, 4-allyl-
2 3
stopped by 0.1 M Na CO , and the absorbance at OD420 and OD550
was measured. Using the remainder of culture, the bacterial density
was measured at OD600. The unit of â-galactosidase activity was
calculated according to the method of Miller (33).
Preparation of Activated Trp-P-1. Preparation of activated Trp-
P-1 was carried out according to the method of Arimoto et al. (34).
UV Irradiation. An overnight culture of the tester bacterial strain
2
-methoxyphenol, commonly called eugenol. Eugenol acts as
an antioxidant on oleogenous foods, as an anticarminative,
antispasmodic, antiseptic in pharmacy, and also as an anti-
microbial agent (27-29).
(S. typhimurium TA1535/pSK1002) incubated at 37 °C in Luria broth
In our search for novel naturally occurring antimutagenic
compounds derived from plants that have a history of safe use
as Chinese crude drugs (30, 31), we reported the suppression
of chemical mutagen-induced SOS response by alkylphenols
from the hexane extract fraction of clove (S. aromaticum) (32).
These compounds suppressed furylfuramide, 4NQO, AfB1, and
Trp-P-1-induced SOS responses in the umu test.
was diluted 50-fold with fresh TGA medium and incubated at 37 °C
until the optical density at 600 nm of 0.25-0.30 was reached. The
cultures were then collected by centrifugation and suspended in 5 mL
of 0.1 M phosphate buffer. The cell suspensions were then poured into
Petri dishes and exposed to UV light (2.0 J/m ) for 10 s using a
germicidical lamp at room temperature.
2
Effect of Suppressive Compounds on mRNA Synthesis Induced
+
by IPTG. The test strain Escherichia coli CSH 26T/Flac , which was
Additional investigations demonstrated that the ethyl acetate
extract fraction of clove had secondary suppressive properties
kindly supplied by Dr. Yoshimitsu Oda (Osaka Prefectural Institute of
Public Health, Japan), was used to investigate the effect on mRNA

Antimutagenic Activity of Phenylpropanoids
J. Agric. Food Chem., Vol. 51, No. 22, 2003 6415
Finally, suppressive compound 1 (81 mg) was isolated from fraction
8, and compound 2 (270 mg) was isolated from fraction 9. Compounds
1
and 2 were identified as dehydrodieugenol (1) and trans-coniferyl
1
13
aldehyde (2) by EI-MS, IR, and H and C NMR, respectively.
Figure 1. Isolation scheme for the suppressive compounds from Clove
(S. aromaticum).
synthesis in the umu test; this strain induces â-galactosidase activity
by addition of IPTG instead of mutagens. The affect on â-galactosidase
synthesis by suppressive compound was assayed as follows: an
+
overnight culture of the tester bacterial strain (E. coli CSH 26T/Flac )
incubated at 37 °C and diluted 50-fold with TGA medium (1%
Bactotryptone, 0.5% NaCl, and 0.2% glucose) was incubated at 37 °C
until the bacterial density at 600 nm reached 0.25-0.30. The culture
was divided into 1.9 mL portions in the test tubes. The test compound
Compound 1. Compound 1 was a yellow crystalline solid; mp 106-
+
108 °C. MS: m/z 326 (M , 100%), 297 (18.6%), 284 (7.2%), 253
-
1
(50 µL, diluted in DMSO), 0.1 M phosphate buffer (300 µL, pH 7.4),
(16.0%), 244 (9.3%), 229 (4.1%), 221 (5.2%). IR γmax KBr (cm ):
1
and 0.01 M IPTG (200 µL) were added to each tube. After 60 min of
incubation at 37 °C with shaking, the culture was centrifuged to collect
cells, which were resuspended in 2.5 mL of PBS; the cell density was
read at 600 nm with one portion (1.0 mL) of the suspension. Using the
other portion (50 µL), the level of â-galactosidase activity was assayed
according to the method of Miller (33).
3242, 1599, 1490, 1454, 1257. H NMR (CDCl
6.6, CH -R,R′), 3.91 (6H, s, OCH ), 5.06 (2H, dd, J ) 1.5, 10.0, CH
-γ,γ′), 5.11 (2H, dd, J ) 1.5, 17.2, CH -H -γ,γ′), 5.98 (2H, ddt, J
) 6.6, 10.0, 17.2, CH-â,â′), 6.72 (2H, d, J ) 1.8, H-6,6′), 6.75 (2H,
d, J ) 1.8, H-2,2′). 13C NMR (CDCl
): δ 147.2 (C- 3,3′), 140.9 (C-
4,4′), 137.6 (C-â,â′), 131.9 (C-1,1′), 124.4 (C-5,5′), 123.1 (C-6,6′),
115.7 (C-γ,γ′), 110.7 (C-2,2′), 56.1 (OCH ), 39.9 (C-R,R′). Compound
3
): δ 3.36 (4H, d, J )
2
3
2
-
H
a
2
b
3
Ames Test. The mutation test was carried out according to the
preincubation method (35), which is a modification of the Ames method
3
1 was identified as dehydrodieugenol 1,1′-di-2-propenyl-4,4′-dihydroxy-
3,3′-dimethoxy-5,5′-biphenyl from these spectral data.
(4). The test compound (50 µL), 0.1 M phosphate buffer (500 µL),
and mutagens frylfuramide (50 µL, 0.5 µg/mL in DMSO), activated
Trp-P-1 (50 µL, 10 µg/mL in DMSO) were added to each test tube. In
the case of Trp-P-1 (50 µL, 20 µg/mL in DMSO), 500 µL of
S9-metabolizing enzyme mixture instead of 0.1 M phosphate buffer
was added. The culture of the tester bacterial strain (S. typhimurium
TA100) was divided into 100 µL portions into the test tube. The mixture
was preincubationed at 37 °C for 20 min, mixed with 2.0 mL of top
agar at 45 °C, and poured onto a minimal glucose agar plate. After
incubation for 2 days at 37 °C, the colonies on the plate were counted.
Purification and Identification of the Suppressive Compounds
Compound 2. Compound 2 was a yellowish crystalline solid; mp
+
75-76 °C. MS: m/z 178 (M , 100%), 147 (48.5%), 135 (72.1%), 107
-
1
(81.5%), 77 (85.0%), 51 (70.0%), 40 (48.3%). IR γmax KBr (cm ):
1
3239, 1661, 1587, 1514, 1134. H NMR (CDCl
OCH ), 6.59 (1H, dd, J ) 7.7, 15.8, CH-â), 6.95 (1H, d, J ) 8.2,
H-5), 7.06 (1H, d, J ) 2.1, H-2), 7.11 (1H, dd, J ) 2.1, 8.2, H-6), 7.40
3
): δ 3.93 (3H, s,
3
1
3
(1H, d, J ) 15.8, CH-R), 9.64 (1H, d, J ) 7.7, CHO-γ). C NMR
(CDCl ): δ 193.4 (C-γ), 153.0 (C- R), 148.9 (C-3), 146.9 (C-4), 126.5
(C-1), 126.2 (C- â), 123.9 (C-6), 114.9 (C-5), 109.5 (C-2), 56.0 (OCH ).
3
3
Compound 2 was identified as trans-coniferyl aldehyde [trans-3-(4-
hydroxy-3-methoxy phenyl)-2-propenaldehyde] from these spectral data.
Methyl Ethers of Compounds 1 and 2. Methyl ethers of compounds
1 and 2 (1a and 2a) were obtained by reaction with diazomethane.
These structures were identified by EI-MS and IR.
1
and 2. As shown in Figure 1, the dry powder (5 kg) of clove was
refluxed with methanol for 12 h to give a methanol extract (1076.6 g).
This extract was suspended in water and reextracted with hexane,
dichloromethane, ethyl acetate, butanol, and water, respectively. Each
soluble fraction was concentrated under reduced pressure to give hexane
+
Methyl Ether of Compound 1 (1a). MS: m/z 354 (M , 100%),
(
(
170. 8 g), dichloromethane (40.1 g), ethyl acetate (165.6 g), butanol
190.1 g), and water (510.0 g) fractions. To purify the compound
353 (8.1%), 298 (36.7%), 283 (24.1%), 282 (21.2%), 115 (10.0%), 41
-
1
(28.3%). IR υmax KBr (cm ): 2944, 2838, 1645, 1588, 1488. The
methyl ether of compound 1 (1a) was identified as di-O-methyldehy-
drodieugenol [1,1′-di-2-propenyl-3,4,3′,4′-tetramethoxy-5,5′-biphenyl]
from these spectral data.
responsible for suppression of the furylfuramide-induced SOS response,
these fractions were evaluted to the umu test. The ethyl acetate fraction
showed a secondary suppressive effect and was fractionated to fractions
+
1
-6 by silica gel column chromatography with hexane and ethyl acetate
Methyl Ether of Compound 2 (2a). MS: m/z 192 (M , 100%),
as eluents. Fraction 2 showed a suppression of the furylfuramide-
induced SOS response in the umu test, and this fraction was repeatedly
fractionated to fractions 7-11 by silica gel column chromatography
with hexane and ethyl acetate as eluents. Fractions 8 and 9 showed a
suppression of the furylfurmaide-induced SOS response in the umu test.
177 (18.8%), 161 (66.7%), 149 (19.8%), 121 (16.7%), 91 (17.7%), 77
-
1
(25.0%). IR υmax KBr (cm ): 1671, 1620, 1597, 1514, 1270. The
methyl ether of compound 2 (2a) was identified as trans-3,4-dimethoxy
cinnamaldehyde [trans-3-(3,4-dimethoxy phenyl)-2-propenaldehyde]
from these spectral data.

6416 J. Agric. Food Chem., Vol. 51, No. 22, 2003
Miyazawa and Hisama
a
Table 1. Suppression of Furylfuramide-Induced SOS Response by Clove Fractions in S. typhimurium TA1535/pSK1002
c
dose response (g/mL)
sample
controlb
200
100
40
0
MeOH extractd
141.8 (±4.6)
341.3 (±3.6)
364.4 (±2.8)
389.9 (±4.4)
407.4 (±6.6)
hexane fractiond
CHCl3 fraction
EtOAc fraction
BuOH fraction
water fraction
141.8 (±4.6)
141.8 (±4.6)
141.8 (±4.6)
141.8 (±4.6)
141.8 (±4.6)
260.6 (±4.5)
386.9 (±7.1)
303.1 (±3.8)
379.4 (±6.8)
366.2 (±9.9)
338.7 (±1.9)
381.0 (±2.9)
365.9 (±5.8)
393.8 (±5.7)
384.1 (±5.2)
374.2 (±5.6)
393.4 (±8.9)
388.7 (±1.5)
398.5 (±8.9)
394.6 (±11.4)
407.4 (±6.6)
407.4 (±6.6)
407.4 (±6.6)
407.4 (±6.6)
407.4 (±6.6)
d
fraction 1
fraction 2
fraction 3
fraction 4
fraction 5
fraction 6
107.2 (±3.9)
107.2 (±3.9)
107.2 (±3.9)
107.2 (±3.9)
107.2 (±3.9)
107.2 (±3.9)
651.2 (±4.2)
449.0 (±2.8)
586.3 (±7.3)
542.0 (±3.0)
668.2 (±6.7)
727.3 (±9.3)
723.6 (±6.1)
532.8 (±4.0)
664.3 (±8.1)
639.9 (±11.8)
713.4 (±3.3)
740.0 (±2.7)
754.3 (±2.2)
641.4 (±3.9)
723.1 (±2.0)
709.0 (±4.8)
760.7 (±9.7)
759.0 (±5.5)
794.1 (±5.8)
794.1 (±5.8)
794.1 (±5.8)
794.1 (±5.8)
794.1 (±5.8)
794.1 (±5.8)
d
fraction 7
94.4 (±5.5)
94.4 (±5.5)
94.4 (±5.5)
94.4 (±5.5)
94.4 (±5.5)
612.9 (±4.6)
344.7 (±1.9)
397.9 (±4.2)
552.2 (±6.0)
659.0 (±9.2)
678.8 (±10.6)
459.2 (±4.8)
497.4 (±5.8)
618.1 (±8.3)
694.2 (±7.3)
704.2 (±2.3)
600.0 (±6.3)
616.3 (±5.0)
679.7 (±1.8)
715.2 (±3.5)
774.7 (±7.6)
774.7 (±7.6)
774.7 (±7.6)
774.7 (±7.6)
774.7 (±7.6)
d
fraction 8
d
fraction 9
fraction 10
fraction 11
a
Furylfuramide (2 µg/mL in DMSO) was added at 50 µL. b Control was exposed to DMSO. c â-Galactosidase activity (units). d Suppressive fraction.
a
b
c
Table 2. Suppressive Effect of Compounds 1 and 1a on Furylfuramide, 4NQO, and MNNG Using S. typhimurium TA1535/pSK1002
d
dose response (µmol/mL)
mutagen
compd
control
0.60
0.48
0.36
0.24
0
ID50e
0.48
furylfuramide
1
1a
97.2 (±1.8)
97.2 (±1.8)
370.2 (±2.4)
590.8 (±5.0)
422.7 (±5.3)
607.7 (±6.9)
490.9 (±4.1)
636.2 (±9.9)
585.3 (±7.2)
670.0 (±6.7)
748.8 (±9.3)
748.8 (±9.3)
4NQO
1
1a
130.7 (±4.2)
130.7 (±4.2)
288.2 (±1.7)
472.8 (±4.9)
345.7 (±3.3)
500.9 (±2.6)
426.6 (±5.9)
517.0 (±6.9)
486.0 (±1.1)
517.2 (±7.8)
639.2 (±5.8)
639.2 (±5.8)
0.42
MNNG
1
1a
106.6 (±5.1)
106.6 (±5.1)
458.9 (±0.4)
273.9 (±8.3)
491.8 (±3.2)
318.8 (±1.9)
528.8 (±5.0)
382.0 (±4.0)
529.2 (±6.3)
465.2 (±8.2)
687.0 (±5.7)
687.0 (±5.7)
0.33
a
Furylfuramide (2 µg/mL in DMSO) was added at 50 µL. b 4NQO (40 µg/mL in DMSO) was added at 50 µL. c MNNG (200 µg/mL in DMSO) was added at 50 µL.
d
e
â-Galactocidase activity (units). 50% inhibition dose.
Reduction of Compounds 2 and 2a (2b,c). Alcohols of compounds
and 2a were obtained by reaction with sodium borohydride. These
hexane and ethyl acetate as eluents. Fraction 8 eluted with 88:
2
1
8
2 hexane/ethyl acetate as eluents and fraction 9 eluted with
5:15 hexane/ethyl acetate as eluents showed a suppression of
structures were identified by EI-MS and IR.
+
Alcohol of Compound 2 (2b). MS: m/z 180 (M , 54.9%), 137
100%), 124 (57.0%), 119 (30.5%), 91 (42.3%), 77 (42.9%). IR υmax
the furylfuramide-induced SOS response in the umu test. Finally,
suppressive compound 1 (81 mg) was isolated from fraction 8,
and suppressive compound 2 (270 mg) was isolated from
fraction 9.
(
-1
KBr (cm ): 3595, 3525, 3000, 1600, 1510. The alcohol of compound
2
3
(2b) was identified as trans-coniferyl alcohol [trans-3-(4-hydroxy-
-methoxy phenyl)-2-propenol] from these spectral data.
+
Alcohol of Compound 2a (2c). MS: m/z 194 (M , 100%), 165
Suppression of Chemical Mutagen-Induced Responses by
Compounds 1 and 1a. Compounds 1 and 1a were examined
for their ability to suppress the SOS-inducing activity on
furylfuramide, 4NQO, and MNNG, which do not require a liver-
metabolizing enzyme mixture (Table 2). Compounds 1 and 1a
suppressed 58 and 24% of the SOS-inducing activity on
furylfuramide at a concentration of 0.60 µmol/mL, and the ID50
value of 1 was 0.48 µmol/mL, respectively. Compounds 1 and
-
1
(
2
52.0%), 151 (38.8%), 139 (29.6%), 124 (6.1%). IR υmax KBr (cm ):
925, 1516, 1263, 1139, 1026. The alcohol of compound 2a (2c) was
identified as trans-3,4-dimethoxy cinnamyl alcohol [trans-3-(3,4-
dimethoxy phenyl)-2-propenol] from these spectral data.
RESULTS
Fractionation of the Extract from Clove and Isolation of
Suppressive Compounds 1 and 2. The initial methanol extract
of clove was further fractionated to identify a suppressive
compound using the umu test as a guide (Figure 1). To obtain
dose-response data for purification of the suppressive com-
pound, test samples were evaluated at dose levels of 0.2, 0.1,
and 0.04 mg/mL. As shown in Table 1, the ethyl acetate fraction
exhibited a secondary suppressive effect of the furylfuramide-
induced SOS response in S. typhimurium TA1535/pSK1002.
After fractionating the ethyl acetate fraction, only the suppressive
fraction 2 eluted with 80:20 hexane/ethyl acetate as eluents had
a clear-cut dose-response effect in the first fractionation
1
4
a suppressed 69 and 33% of the SOS-inducing activity on
NQO at a concentration of 0.60 µmol/mL, and the ID50 value
of 1 was 0.42 µmol/mL, respectively. Compounds 1 and 1a
suppressed 39 and 71% of the SOS-inducing activity on MNNG
at a concentration of 0.60 µmol/mL, and the ID50 value of 1a
was 0.33 µmol/mL, respectively. Compounds 1 and 1a were
also assayed with AfB1 and Trp-P-1, which require liver
metabolic activation (Table 3). Compounds 1 and 1a suppressed
93 and 50% of the SOS-inducing activity on AfB1 at a
concentration of 0.60 µmol/mL, and the ID50 value of 1 was
0.26 µmol/mL. Compounds 1 and 1a suppressed 86 and 41%
of the SOS-inducing activity on Trp-P-1 at a concentration of
0.60 µmol/mL, and the ID50 value of 1 was 0.31 µmol/mL. As
(
fractions 1-6). The fraction 2 was repeatedly fractionated to
fractions 7-11 by silica gel column chromatography with

Antimutagenic Activity of Phenylpropanoids
Table 3. Suppressive Effect of Compounds 1 and 1a on AfB a
J. Agric. Food Chem., Vol. 51, No. 22, 2003 6417
b
c
1
, Trp-P-1, and Activated Trp-P-1 Using S. typhimurium TA1535/pSK1002
d
dose response (mol/mL)
mutagen
AfB1
compd
control
0.60
0.48
0.36
0.24
0
ID50e
1
1a
92.3 (±3.1)
92.3 (±3.1)
127.3 (±0.9)
349.9 (±4.3)
176.6 (±3.0)
398.7 (±6.8)
261.1 (±1.7)
428.0 (±2.9)
363.9 (±5.8)
504.2 (±8.4)
605.9 (±10.5)
605.9 (±10.5)
0.26
0.60
Trp-P-1
1
1a
109.7 (±5.5)
109.7 (±5.5)
179.9 (±1.9)
390.9 (±0.7)
222.8 (±5.3)
429.5 (±3.9)
316.0 (±3.2)
470.4 (±8.3)
392.5 (±7.9)
523.7 (±4.1)
590.0 (±7.3)
590.0 (±7.3)
0.31
activated Trp-P-1
1
1a
94.9 (±4.8)
94.9 (±4.8)
365.5 (±6.6)
510.6 (±8.5)
391.1 (±2.3)
539.0 (±5.2)
422.5 (±7.4)
561.8 (±6.9)
477.0 (±1.5)
590.0 (±2.0)
633.3 (±5.8)
633.3 (±5.8)
0.60
a
AfB1 (20 µg/mL in DMSO) was added at 50 µL. b Trp-P-1 (40 µg/mL in DMSO) was added at 50 µL. c Activated Trp-P-1 (10 µg/mL in DMSO) was added at 50 µL.
d
e
â-Galactocidase activity (units). 50% inhibition dose.
a
b
c
Table 4. Suppressive Effect of Compounds 2 and 2a−c on Furylfuramide, 4NQO, and MNNG Using S. typhimurium TA1535/pSK1002
d
dose response (mol/mL)
ID50e
(mol/mL)
0.76
mutagen
compd
control
1.20
0.84
0.60
0.36
0
furylfuramide
2
102.8 (±4.1)
102.8 (±4.1)
102.8 (±4.1)
102.8 (±4.1)
333.9 (±3.1)
457.4 (±0.9)
470.0 (±4.7)
530.8 (±3.3)
397.4 (±2.7)
507.7 (±4.2)
522.5 (±2.5)
567.0 (±10.1)
454.2 (±5.1)
552.0 (±3.6)
571.7 (±7.2)
621.9 (±2.3)
493.8 (±4.2)
604.1 (±8.3)
633.4 (±3.8)
658.7 (±7.0)
727.3 (±6.9)
727.3 (±6.9)
727.3 (±6.9)
727.3 (±6.9)
2
a
2
b
2c
4
NQO
2
112.1 (±3.4)
112.1 (±3.4)
112.1 (±3.4)
112.1 (±3.4)
258.6 (±3.7)
366.5 (±6.6)
417.7 (±3.7)
472.0 (±4.7)
303.5 (±1.6)
408.3 (±7.2)
455.9 (±3.8)
497.8 (±6.7)
354.0 (±7.2)
454.4 (±2.0)
473.2 (±8.3)
533.1 (±2.2)
420.5 (±4.3)
509.0 (±6.2)
521.1 (±10.0)
549.3 (±5.5)
605.8 (±9.1)
605.8 (±9.1)
605.8 (±9.1)
605.8 (±9.1)
0.58
0.84
2
a
2
b
2c
MNNG
2
133.0 (±1.9)
133.0 (±1.9)
133.0 (±1.9)
133.0 (±1.9)
447.7 (±1.6)
373.9 (±3.6)
534.1 (±5.2)
483.0 (±7.9)
480.9 (±7.3)
422.2 (±4.9)
564.8 (±10.3)
514.7 (±4.7)
536.3 (±5.0)
484.0 (±5.9)
606.2 (±3.5)
573.0 (±6.3)
604.0 (±8.7)
538.7 (±2.8)
651.9 (±4.9)
628.2 (±3.2)
711.4 (±10.3)
711.4 (±10.3)
711.4 (±10.3)
711.4 (±10.3)
2
a
2
b
2c
a
Furylfuramide (2 µg/mL in DMSO) was added at 50 µL. b 4NQO (40 µg/mL in DMSO) was added at 50 µL. c MNNG (200 µg/mL in DMSO) was added at 50 µL.
d
e
â-Galactocidase activity (units). 50% inhibition dose.
Table 5. Suppressive Effect of Compounds 2 and 2a−c on AfB a
, Trp-P-1, and Activated Trp-P-1 Using S. typhimurium TA1535/pSK1002
b
c
1
d
dose response (mol/mL)
ID50e
mutagen
AfB1
compd
control
1.20
0.84
0.60
0.36
0
(mol/mL)
2
80.8 (±4.5)
80.8 (±4.5)
80.8 (±4.5)
80.8 (±4.5)
122.0 (±4.1)
210.7 (±3.9)
185.6 (±4.3)
243.1 (±1.9)
160.8 (±2.2)
249.0 (±4.1)
214.4 (±7.5)
277.0 (±3.8)
220.3 (±6.3)
310.3 (±5.4)
282.0 (±11.2)
359.9 (±4.1)
309.8 (±4.8)
382.5 (±6.6)
363.7 (±4.5)
431.2 (±8.6)
524.4 (±9.9)
524.4 (±9.9)
524.4 (±9.9)
524.4 (±9.9)
0.38
0.63
0.54
0.76
2
a
2
b
2c
Trp-P-1
2
139.9 (±5.1)
139.9 (±5.1)
139.9 (±5.1)
139.9 (±5.1)
219.5 (±4.3)
304.6 (±6.2)
279.7 (±4.0)
362.0 (±5.0)
268.3 (±7.2)
362.5 (±3.7)
330.0 (±8.3)
420.6 (±3.5)
338.1 (±6.1)
423.8 (±10.0)
393.6 (±3.1)
497.2 (±7.5)
427.6 (±9.0)
496.8 (±5.1)
394.2 (±6.9)
551.3 (±6.8)
625.7 (±11.4)
625.7 (±11.4)
625.7 (±11.4)
625.7 (±11.4)
0.48
0.76
0.64
1.07
2
a
2
b
2c
activated Trp-P-1
2
107.3 (±3.6)
107.3 (±3.6)
107.3 (±3.6)
107.3 (±3.6)
365.7 (±2.8)
421.2 (±1.9)
485.5 (±2.9)
540.5 (±4.5)
398.0 (±4.3)
448.8 (±3.3)
510.1 (±1.4)
561.2 (±4.7)
452.6 (±5.1)
486.5 (±2.2)
551.7 (±5.6)
579.2 (±5.7)
520.7 (±4.2)
487.3 (±6.9)
590.0 (±3.3)
602.0 (±3.0)
625.0 (±5.7)
625.0 (±5.7)
625.0 (±5.7)
625.0 (±5.7)
1.20
2
a
2
b
2c
a
AfB1 (20 µg/mL in DMSO) was added at 50 µL. b Trp-P-1 (40 µg/mL in DMSO) was added at 50 µL. c Activated Trp-P-1 (10 µg/mL in DMSO) was added at 50 µL.
d
e
â-Galactocidase activity (units). 50% inhibition dose.
these results of the umu test, the suppressive effects of 1 and
a on 4NQO are similar to the suppressive effects observed in
the case of furylfuramide, and the suppressive effects of 1 and
a on AfB1 are similar to the suppressive effects observed in
the case of Trp-P-1. Compound 1 had stronger suppressive
effects on chemical mutagens except MNNG than compound
and the ID50 value of 2 was 0.76 µmol/mL, respectively.
Compounds 2 and 2a-c suppressed 70, 48, 38, and 27% of the
SOS-inducing activity on 4NQO at a concentration of 1.20
µmol/mL, and the ID50 value of 2 was 0.58 µmol/mL,
respectively. Compounds 2 and 2a-c suppressed 46, 58, 31,
and 39% of the SOS-inducing activity on MNNG at a
concentration of 1.20 µmol/mL, and the ID50 value of 2a was
0.84 µmol/mL, respectively. Compounds 2 and 2a-c were also
assayed with AfB1 and Trp-P-1, which require liver metabolic
activation (Table 5). Compounds 2 and 2a-c suppressed 91,
71, 76, and 63% of the SOS-inducing activity on AfB1 at a
concentration of 1.20 µmol/mL, and the ID50 values of 2 and
2a-c were 0.38, 0.63, 0.54, and 0.76 µmol/mL. Compounds 2
and 2a-c suppressed 84, 66, 71, and 54% of the SOS-inducing
1
1
1
a did.
Suppression of Chemical Mutagen-Induced Responses by
Compounds 2 and 2a-c. Compounds 2 and 2a-c were
examined for their ability to suppress the SOS-inducing activity
on furylfuramide, 4NQO, and MNNG, which do not require a
liver-metabolizing enzyme mixture (Table 4). Compounds 2
and 2a-c suppressed 63, 43, 41, and 31% of the SOS-inducing
activity on furylfuramide at a concentration of 1.20 µmol/mL,

6418 J. Agric. Food Chem., Vol. 51, No. 22, 2003
Miyazawa and Hisama
a
Table 6. Suppressive Effect of Compounds 1, 2, 1a, and 2a−c on UV Irradiation Using S. typhimurium TA1535/pSK1002
b
dose response (µmol/mL)
compd
control
0.60
0.48
0.36
0.24
0
1
1a
103.3 (±5.8)
103.3 (±5.8)
662.6 (±4.4)
431.8 (±6.4)
463.0 (±5.2)
677.2 (±5.8)
512.2 (±4.2)
693.5 (±4.9)
585.7 (±7.9)
715.3 (±11.2)
731.2 (±10.9)
731.2 (±10.9)
compd
control
1.20
0.84
0.60
0.36
0
2
90.9 (±3.6)
90.9 (±3.6)
90.9 (±3.6)
90.9 (±3.6)
412.6 (±3.3)
494.2 (±5.1)
636.3 (±8.4)
650.0 (±6.3)
454.0 (±2.9)
536.8 (±3.0)
656.9 (±6.4)
663.3 (±5.7)
524.8 (±1.4)
577.4 (±8.8)
684.0 (±9.0)
686.6 (±7.9)
643.2 (±6.4)
653.5 (±7.6)
704.7 (±3.8)
706.2 (±3.4)
712.9 (±7.8)
712.9 (±7.8)
712.9 (±7.8)
712.9 (±7.8)
2
a
2
b
2c
a
The cells were exposed to UV light (2.0 J/m ) with a germicidal lamp at room temperature. b â-Galactocidase activity (units).
2
activity on Trp-P-1 at a concentration of 1.20 µmol/mL, and
the ID50 values of 2 and 2a-c were 0.48, 0.76, 0.64, and 1.07
µmol/mL. As these results of the umu test, the suppressive
effects of 2 and 2a-c on 4NQO are similar to the suppressive
effects observed in the case of furylfuramide, and the suppres-
sive effects of 2 and 2a-c on AfB1 are similar to the suppressive
effects observed in the case of Trp-P-1. Compound 2 had
stronger suppressive effects on chemical mutagens except
MNNG than compounds 2a-c did.
Suppressive Effect of Compounds 1, 2, 1a, and 2a-c on
Metabolic Activation of Trp-P-1. The suppressive effect of
compounds 1, 2, 1a, and 2a-c on metabolic activation of Trp-
P-1 was determined by the umu test. The value of â-galacto-
sidase activity observed in the absence of compounds 1, 2, 1a,
and 2a-c was for activated Trp-P-1. As shown in Table 3,
compounds 1 and 1a suppressed 50 and 23% of the SOS-
inducing activity on activated Trp-P-1 at a concentration of 0.60
µmol/mL. As shown in Table 5, compounds 2 and 2a-c
suppressed 50, 39, 27, and 16% of the SOS-inducing activity
on activated Trp-P-1 at a concentration of 1.20 µmol/mL. The
suppressive effect of compounds 1, 2, 1a, and 2a-c on activated
Trp-P-1 was decreased as compared with that on Trp-P-1.
Suppressive Effects of Compounds 1, 2, 1a, and 2a-c on
UV Irradiation. The suppressive effects of compounds 1, 2,
1
a, and 2a-c on UV irradiation-induced SOS response were
determined using the umu test. Compounds 1 and 1a were
evaluated at dose levels of 0.60, 0.48, 0.36, and 0.24 µmol/mL
to obtain dose-response data, and compounds 2 and 2a-c were
evaluated at dose levels of 1.20, 0.84, 0.60, and 0.36 µmol/mL
to obtain dose-response data. Compounds 1, 2, and 2a exhibited
inhibition on the UV irradiation-induced SOS response, and
compounds 1a and 2b,c did not exhibit any inhibition (Table
Figure 2. Effect of compounds 1, 2, 1a, 2a−c on mRNA synthesis induced
by IPTG in E. coli CSH26T/Flac : effect of 1 (0), effect of 2 (]), effect
+
of 1a (1), effect of 2a (b), effect of 2b (2), and effect of 2c (right pointing
-
2
solid triangle). IPTG (10 M) was added at 200 µL.
that compounds 1, 2, 1a, and 2a-c inhibit the lexA-recA
regulation of the umu operon.
Antimutagenic Activity of Compounds 1, 2, 1a, and 2a-c
in the Ames Assay. The antimutagenic activity of compounds
6
). Compound 1 suppressed 48% of the SOS-inducing activity
due to UV irradiation at a concentration of 0.60 µmol/mL,
respectively. Compounds 2 and 2a suppressed 48 and 35% of
the SOS-inducing activity due to UV irradiation at a concentra-
tion of 1.20 µmol/mL, respectively.
1, 2, 1a, and 2a-c against furylfuramide, Trp-P-1, and activated
Trp-P-1 were also demonstrated by the Ames test using S.
typhimurium TA100. Compounds 1 and 1a suppressed 52 and
36% of the mutagenicity of furylfuramide at a concentration of
0.60 µmol/plate, and the ID50 value of 1 was 0.57 µmol/plate,
respectively (Figure 3). Compounds 2 and 2a-c suppressed
62, 54, 47, and 38% of the mutagenicity of furylfuramide at a
concentration of 1.20 µmol/plate, and the ID50 values of 2 and
2a were 0.64 and 1.05 µmol/plate, respectively (Figure 4). As
shown in Figure 3, compounds 1 and 1a suppressed 91 and
78% of the mutagenicity of Trp-P-1 at a concentration of 0.60
µmol/plate, and the ID50 values of 1 and 1a were 0.14 and 0.27
µmol/plate. As shown in Figure 4, compounds 2 and 2a-c
suppressed 57, 47, 79, and 68% of the mutagenicity of Trp-
Effects of Compounds 1, 2, 1a, and 2a-c on mRNA
Synthesis. In the mechanism for inhibition of mutagen-induced
SOS response by compounds 1, 2, 1a, and 2a-c, there are some
possibilities that compounds 1, 2, 1a, and 2a-c have an effect
on the lexA-recA regulation (mRNA synthesis) of the umu
operon. Compounds 1, 2, 1a, and 2a-c were assayed for the
+
effect on mRNA synthesis using E. coli CSH26T/Flac , which
produces â-galactosidase without mutagens. This strain has a
good characteristic function, which produces â-galactosidase
activity, after treated with IPTG. As shown in Figure 2,
compounds 1, 2, 1a, and 2a-c did not affect â-galactosidase
activity caused by IPTG. The result can exclude the possibility

Antimutagenic Activity of Phenylpropanoids
J. Agric. Food Chem., Vol. 51, No. 22, 2003 6419
Figure 3. Effect of compounds 1 and 1a on the mutagenicity of
furylfuramide, Trp-p-1, and activated Trp-p-1 in S. typhimurium TA100:
effect of 1 (0) and effect of 1a (1). Furylfuramide (0.5 µg/mL in DMSO)
was added at 50 µL/plate. Trp-p-1 (20 µg/mL in DMSO) was added at
Figure 4. Effect of compounds 2 and 2a−c on the mutagenicity of
furylfuramide, Trp-p-1, and activated Trp-p-1 in S. typhimurium TA100:
effect of 2 (]), effect of 2a (b), effect of 2b (2), and effect of 2c (right
pointing solid triangle). Furylfuramide (0.5 µg/mL in DMSO) was added
at 50 µL/plate. Trp-p-1 (20 µg/mL in DMSO) was added at 50 µL/plate.
Activated Trp-P-1 (10 µg/mL in DMSO) was added at 50 µL/plate.
50 µL/plate. Activated Trp-P-1 (10 µg/mL in DMSO) was added at 50
µL/plate.
P-1 at a concentration of 1.20 µmol/plate, and the ID50 values
of 2 and 2b,c were 0.83, 0.36, and 0.66 µmol/plate. On the
other hand, these antimutagenic activities against activated
Trp-P-1 were remarkably decreased as compared with those on
Trp-P-1. From this result, it is suggested that antimutagenic
activity of these compounds on Trp-P-1 is due to the inhibition
of metabolic activation of Trp-P-1 by S9.
effects on umu gene expression of the SOS response in S.
typhimurium TA1535/pSK1002 against furylfuramide, 4NQO,
and MNNG, which do not require liver-metabolizing enzymes,
and AfB1 and Trp-P-1, which require liver-metabolizing en-
zymes and UV irradiation.
As shown in Tables 2, 3, and 6, compound 1 had stronger
suppressive potencies than 1a against mutagens all except
MNNG. The differences in structure between 1 and 1a are the
presence of hydroxy groups at the C-4 and C-4′ positions in 1,
as opposed to methoxy groups in 1a. These results indicated
that hydroxy groups at the C-4 and C-4′ positions are important
DISCUSSION
The antimutagenic compounds in clove (S. aromaticum) were
clearly identified as due to dehydrodieugenol (1) and trans-
coniferyl aldehyde (2). These compounds showed suppressive

6420 J. Agric. Food Chem., Vol. 51, No. 22, 2003
Miyazawa and Hisama
factors for suppressing the SOS-inducing activity on mutagens
except MNNG. On the other hand, compound 1a had stronger
suppressive potencies against MNNG than 1 did. These results
indicated that methoxy groups at the C-4 and C-4′ positions
are important factors for suppressing the SOS-inducing activity
on MNNG.
COOH > CH3 on the functional group at the C-γ position in
trans-3,4-dimethoxy cinnamic derivatives. The suppressive
effects of MNNG-induced SOS response decreased in the order
trans-methyl isoeugenol > trans-3,4-dimethoxy methyl cin-
namate > trans-3,4-dimethoxy cinnamaldehyde > trans-3,4-
dimethoxy cinnamyl alcohol > trans-3,4-dimethoxy cinnamic
acid in trans-3,4-dimethoxy cinnamic derivatives. These results
indicated that the importance for suppressing the SOS-inducing
activity on MNNG decreased in the order CH3 > COOCH3 >
CHO > CH2OH > COOH on the functional group at the C-γ
position in trans-3,4-dimethoxy cinnamic derivatives. The
suppressive effects of AfB1- and Trp-P-1-induced SOS response
decreased in the order trans-3,4-dimethoxy methyl cinnamate
As shown in Tables 4-6, compound 2 had stronger sup-
pressive potencies against mutagens except MNNG than 2a did,
and compound 2b had stronger suppressive potencies against
mutagens except MNNG than 2c did. The difference in structure
between 2 and 2a and between 2b and 2c is the presence of a
hydroxy group at the C-4 position in 2 and 2b, as opposed to
a methoxy group in 2a and 2c. These results indicated that a
hydroxy group at the C-4 position is an important factor for
suppressing the SOS-inducing activity on mutagens except
MNNG. On the other hand, compound 2a had stronger
suppressive potencies against MNNG than 2 did, and compound
>
trans-methyl isoeugenol = trans-3,4-dimethoxy cinnamal-
dehyde > trans-3,4-dimethoxy cinnamyl alcohol > trans-3,4-
dimethoxy cinnamic acid in trans-3,4-dimethoxy cinnamic
derivatives. These results indicated that the importance for
suppressing the SOS-inducing activity on AfB1 and Trp-P-1
decreased in the order COOCH3 > CH3 = CHO > CH2OH >
COOH on the functional group at the C-γ position in trans-
2
c had stronger suppressive potencies against MNNG than 2b
did. These results indicated that a methoxy group at the C-4
position is an important factor for suppressing the SOS-inducing
activity on MNNG.
3
,4-dimethoxy cinnamic derivatives.
Compounds 1, 2, and 2a had a suppressive effect on umu
gene expression of SOS response in S. typhimurium TA1535/
pSK1002 against UV irradiation, which is a physical mutagen
36). The antimutagenic factors are divided into two main
Previously, we reported the suppression of chemical mutagen-
induced SOS response by cinnamic acid derivatives from
Scrophulia ningpoensis (30) and by alkylphenols from clove
(
(S. aromaticum) (31). These compounds suppressed furylfura-
classes: one type, desmutagen, inactivates or destroys mutagens
directly or indirectly out of the cell, and the other type of factor
is called bioantimutagen, which suppresses the process of
mutagenesis itself in the cells. From this result, the mechanism
for inhibition of the SOS-inducing activity by compounds 1, 2,
and 2a may involve not only acted action on the mutagens but
also involvement with cellular repair systems, and so, com-
pounds 1, 2, and 2a might have the ability to be potent
bioantimutagens.
mide, 4NQO, MNNG, AfB1, and Trp-P-1-induced SOS re-
sponses in the umu test. These results indicated the importance
of a functional group at the C-γ position in trans-cinnamic
derivatives on suppressive effects, changed by chemical mu-
tagens and functional groups at C-3 and C-4 positions. The
suppressive effects of furylfuramimde- and 4NQO-induced SOS
response decreased in the order trans-isoeugenol = trans-
coniferyl aldehyde > trans-methyl ferulate > trans-coniferyl
alcohol > trans-ferulic acid in trans-4-hydroxy-3-methoxy
cinnamic derivatives. These results indicated that the importance
for suppressing the SOS-inducing activity on furylfuramide and
Compounds 1, 2, 1a, and 2a-c were examined for the ability
to suppress the metabolic activation of Trp-P-1 by S9. As shown
in Figure 3, compounds 1, 2, 1a, and 2a-c suppressed the
weaker SOS induction on activated Trp-P-1 than on Trp-P-1.
This result suggested the possibility that the inhibition of the
SOS-inducing activity on Trp-P-1, which was caused by
compounds 1, 2, 1a, and 2a-c, was due to the inhibition of
metabolic activation by S9.
4NQO decreased in the order CH3 = CHO > COOCH3 > CH2-
OH > COOH on the functional group at the C-γ position in
trans-4-hydroxy-3-methoxy cinnamic derivatives. The suppres-
sive effects of MNNG-induced SOS response decreased in the
order trans-coniferyl aldehyde > trans-methyl ferulate > trans-
coniferyl alcohol > trans-isoeugenol = trans-ferulic acid in
trans-4-hydroxy-3-methoxy cinnamic derivatives. These results
indicated that the importance for suppressing the SOS-inducing
activity on MNNG decreased in the order CHO > COOCH3 >
CH2OH > CH3 = COOH on the functional group at the C-γ
position in trans-4-hydroxy-3-methoxy cinnamic derivatives.
The suppressive effects of AfB1- and Trp-P-1-induced SOS
response decreased in the order trans-isoeugenol > trans-methyl
ferulate > trans-coniferyl aldehyde > trans-coniferyl alcohol
The SOS regulatory system involves the action of two
proteins: the LexA protein, which represses a set of unlinked
genes, and the RecA protein, which is activated as a protease
by an inducing signal and specifically inactivates the repressor.
The SOS response is activation of the protease, and the later
manifestations of the SOS response are a secondary consequence
of these events (37). The mechanism for inhibition of mutagen-
induced SOS response by compounds 1, 2, 1a, and 2a-c
includes the following possibilities: (i) suppression of inactiva-
tion of the LexA repressor by the RecA protease, (ii) suppression
of the transcription of the recA gene, and (iii) suppression of
RecA protein synthesis. Because the expression of the umuC
gene is known to be regulated by the recA and lecA gene
products (38, 39), the present data with E. coli may exclude
the possibility that the lexA-recA regulation of the umu operon
is suppressed. Compounds 1, 2, 1a, and 2a-c were assayed
>
trans-ferulic acid in trans-4-hydroxy-3-methoxy cinnamic
derivatives. These results indicated that the importance for
suppressing the SOS-inducing activity on AfB1 and Trp-P-1
decreased in the order CH3 > COOCH3 > CHO > CH2OH >
COOH on the functional group at the C-γ position in trans-4-
hydroxy-3-methoxy cinnamic derivatives. On the other hand,
the suppressive effects of furylfuramimde- and 4NQO-induced
SOS response decreased in the order trans-3,4-dimethoxy
cinnamaldehyde > trans-3,4-dimethoxy methyl cinnamate =
trans-3,4-dimethoxy cinnamyl alcohol > trans-3,4-dimethoxy
cinnamic acid > trans-methyl isoeugenol in trans-3,4-dimethoxy
cinnamic derivatives. These results indicated that the importance
for suppressing the SOS-inducing activity on furylfuramide and
+
for its effect on mRNA synthesis using E. coli CSH26T/Flac ,
which produces â-galactosidase without mutagens. Compounds
1, 2, 1a, and 2a-c did not affect â-galactosidase activity.
Therefore, it suggested that compounds 1, 2, 1a, and 2a-c had
a potent suppressive effect on the mutagen-induced SOS
response.
4
NQO decreased in the order CHO > COOCH3 = CH2OH >

Antimutagenic Activity of Phenylpropanoids
J. Agric. Food Chem., Vol. 51, No. 22, 2003 6421
Recently, the antimutagenic activity of cinnamaldehyde was
reported frequently. Cinnamaldehyde reduced 4NQO- and UV-
induced mutagenesis, as well as mutagenesis induced by
furylfuramide (AF-2) in E. coli WP2s, and it might act by
interfering with an inducible error prone DNA repair pathway
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(
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95 (3), 221-224.
(
(40). Cinnamaldehyde was shown to suppress Hprt mtations in
UV- and X-ray-exposed V79 cells (41). In human-derived
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micronuclei induced by a variety of heterocyclic amines (42).
Dehydrodieugenol has been isolated from Litsea turfosa (43),
Ocotea cymbarum (44), Virola carinata (45), and Nectandra
polita (46). Coniferyl aldehyde also has been isolated from some
natural sources including SalVia plebeia (47) and Balanophora
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coniferyl aldehyde had hydroxyl radical scavenging abilities
(
(
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(49). However, inhibition of mutagen-induced SOS response
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reported. In summary, this research suggests that suppressive
compounds on SOS response against chemical and physical
mutagens in clove (S. aromaticum) were primarily dehydro-
dieugenol (1) and trans-coniferyl aldehyde (2) and that com-
pounds 1 and 2 and their derivatives showed potent suppressive
effects of the SOS-inducing activity by chemical and physical
mutagens and potent inhibition of the mutagenicity against
chemical mutagens.
We expect that the antimutagenic compounds isolated from
clove (S. aromaticum) will be useful cancer chemopreventive
agents. However, these compounds may not exhibit their
expected effects in vivo if they are adversely affected by factors
such as absorption, biodisposition, and metabolism after they
are incorporated into the human body. Further studies with
mammalian cells in vitro or in vivo are needed to determine
the efficacy of these compounds for the prevention of human
cancer.
(
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(
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ACKNOWLEDGMENT
We thank Dr. Yoshimitsu Oda for the generous gift of the test
+
strain E. coli CSH 26T/Flac .
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Received for review April 3, 2003. Revised manuscript received July
23, 2003. Accepted July 24, 2003.
JF030247Q