Suppression of chemical mutagen-induced SOS response by alkylphenols from clove (Syzygium aromaticum) in the Salmonella typhimurium TA1535/pSK1002 umu test

Article abstract of DOI:10.1021/jf0103469

A methanol extract from clove (Syzygium aromaticum) showed a suppressive effect of the SOS-inducing activity on the mutagen 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (furylfuramide) in the Salmonella typhimurium TA1535/pSK1002 umu test. The methanol extract was re-extracted with hexane, dichloromethane, ethyl acetate, butanol, and water. The hexane fraction showed a suppressive effect. Suppressive compounds in the hexane fraction were isolated by silica gel column chromatography and identified as trans-isoeugenol (1) and eugenol (2) by GC, GC-MS, IR, and ¹H and ¹³C NMR spectroscopy. Compounds 1 and 2 suppressed the furylfuramide-induced SOS response in the umu test. Compounds 1 and 2 suppressed 42.3 and 29.9% of the SOS-inducing activity at a concentration of 0.60 μmol/mL. These compounds were assayed with other mutagens, 4-nitroquinolin 1-oxide (4NQO) and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). In addition, compounds 1 and 2 were assayed with aflatoxin B₁ (AFB₁) and 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), which require liver metabolizing enzymes. These compounds showed suppressive effects of the SOS-inducing activity against furylfuramide, 4NQO, AFB₁, and Trp-P-1. To research the structure-activity relationship, methyl esters of 1 and 2 (1Me and 2Me) and o-eugenol (3), as compounds similar to 2, were also assayed with all mutagens. Compounds 1Me, 2Me, and 3 showed weak suppressive effects of the SOS-inducing activity against furylfuramide.

Full text of DOI:10.1021/jf0103469

J. Agric. Food Chem. 2001, 49, 4019−4025
4019
Su p p r ession of Ch em ica l Mu ta gen -In d u ced SOS Resp on se by
Alk ylp h en ols fr om Clove (Syzygiu m a r om a ticu m ) in th e Sa lm on ella
typh im u r iu m TA1535/p SK1002 u m u Test
Mitsuo Miyazawa* and Masayoshi Hisama
Department of Applied Chemistry, Faculty of Science and Engineering, Kinki University,
Kowakae, Higashiosaka-shi, Osaka 577-8502, J apan
A methanol extract from clove (Syzygium aromaticum) showed a suppressive effect of the SOS-
inducing activity on the mutagen 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (furylfuramide) in the
Salmonella typhimurium TA1535/pSK1002 umu test. The methanol extract was re-extracted with
hexane, dichloromethane, ethyl acetate, butanol, and water. The hexane fraction showed a
suppressive effect. Suppressive compounds in the hexane fraction were isolated by silica gel column
1
chromatography and identified as trans-isoeugenol (1) and eugenol (2) by GC, GC-MS, IR, and H
and 13C NMR spectroscopy. Compounds 1 and 2 suppressed the furylfuramide-induced SOS response
in the umu test. Compounds 1 and 2 suppressed 42.3 and 29.9% of the SOS-inducing activity at a
concentration of 0.60 µmol/mL. These compounds were assayed with other mutagens, 4-nitroquinolin
1-oxide (4NQO) and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). In addition, compounds 1 and
2 were assayed with aflatoxin B1 (AfB1) and 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-
1), which require liver metabolizing enzymes. These compounds showed suppressive effects of the
SOS-inducing activity against furylfuramide, 4NQO, AfB1, and Trp-P-1. To research the structure-
activity relationship, methyl esters of 1 and 2 (1Me and 2Me) and o-eugenol (3), as compounds
similar to 2, were also assayed with all mutagens. Compounds 1Me, 2Me, and 3 showed weak
suppressive effects of the SOS-inducing activity against furylfuramide.
Keyw or d s: Clove; Syzygium aromaticum; trans-isoeugenol; eugenol; SOS response; umu test;
Salmonella typhimurium TA1535/ pSK1002
INTRODUCTION
bial agent (11-13). In our search for new naturally
occurring antimutagenic compounds in plants, with a
Several short-term tests for screening of environmen-
tal mutagens and carcinogens have been developed and
used widely in many laboratories (1, 2). The Ames test
is a convenient method to evaluate the mutagenic
activities of these chemicals (1), and several lines of
evidence have suggested that the mutagenic activities
of a number of chemicals correlate well with the
history of safe use as Chinese crude drugs (14, 15), we
found that the methanol extract of clove exhibited a
suppression of the furylfuramide-induced SOS response.
In this paper, we report the isolation and identification
of the suppressive compounds on SOS response against
mutagens in clove.
carcinogenic activities reported so far (3, 4).
MATERIALS AND METHODS
The umu test system was developed 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 (7).
The dried flower buds of clove (“tyouji” in J apanese;
Myrtaceae) are used asan oriental drug, which has been
used as a vermifuge, as antibacterial agent, and to treat
toothache (8). Various compounds such as tannins and
triterpenoids were isolated and identified from this
plant (9, 10). It is well-known that clove possesses a
phenolic compound, 4-allyl-2-methoxyphenol, commonly
called eugenol. Eugenol acts as an antioxidant on
oleogenous foods, as an anticarminative, antispasmodic,
and antiseptic in pharmacy, and also as an antimicro-
Gen er a l P r oced u r e. Gas chromatography (GC) was per-
formed on a Hewlett-Packard 5890 gas chromatograph equipped
with a flame ionization detector (FID). GC-MS was performed
on a Hewlett-Packard 5972 series mass spectrometer inter-
faced with a Hewlett-Packard 5890 gas chromatograph fitted
with a column (HP-5MS, 30 m × 0.25 mm i.d.). IR spectra were
determined with a Perkin-Elmer 1760-x infrared Fourier
transform spectrometer. Nuclear magnetic resonance (NMR)
spectra (δ, J in hertz) were recorded on a J EOL GSX 270 NMR
spectrometer. Tetramethylsilane (TMS) was used as the
internal reference (δ 0.00) for 1H NMR spectra measured in
CDCl3. This solvent was also used for 13C NMR spectra.
Ma ter ia ls. Commercially available air-dried tips of clove
(tyouji) were obtained from Yamada Yakken Co., Ltd. Furyl-
furamide, 4-nitroquinoline 1-oxide (4NQO), N-methyl-N′-nitro-
N-nitrosoguanidine (MNNG), 3-amino-1,4-dimethyl-5H-pyrido-
[4,3-b]indole (Trp-P-1), and aflatoxin B1 (AfB1) were purchased
from Wako Pure Chemical Co. S9 (supernatant of 9000g) and
coenzyme, NADPH, NADH, and G-6-P were purchased from
Oriental Yeast Co. o-Eugenol was purchased from Aldrich
Chemical Co.
* Author to whom correspondence should be addressed
(telephone +81-6-6721-2332; fax +81-6-6727-4301; e-mail
miyazawa@apch.kindai.ac.jp).
u m u Test. The umu test for detecting the chemical-induced
SOS response was carried out according to the method of Oda
10.1021/jf0103469 CCC: $20.00 © 2001 American Chemical Society
Published on Web 07/21/2001

4020 J. Agric. Food Chem., Vol. 49, No. 8, 2001
Miyazawa and Hisama
showed a suppressive effect. The hexane fraction was fraction-
ated to fractions 1-6 by silica gel column chromatography with
hexane and ethyl acetate as eluents. Fraction 4 showed a
suppression of the furylfuramide-induced SOS response in the
umu test, and this fraction was repeatedly fractionated by
silica gel column chromatography with hexane and ethyl
acetate as eluents using the umu test as a guide. Finally,
suppressive compounds 1 (20.5 g) and 2 (74.4 g) were isolated
from clove. These compounds were major components in the
hexane fraction. Compounds 1 and 2 were identified as trans-
isoeugenol and eugenol by GC, GC-MS, IR, and 1H and 13C
NMR, respectively.
Com p ou n d 1. Compound 1 was a yellow oil: MS, m/z 164
(M+, 100%), 149 (27%), 133 (15%), 131 (20%), 103 (24%), 77
(19%), 66 (11%); IR 3510.8, 2939.7, 2842.9, 1514.9, 1268.6; 1H
NMR (CDCl3) δ 1.84 (3H, dd, J ) 2, 6, γ-H3), 3.85 (3H, s,
OCH3), 5.64 (1H, s, OH), 6.06 (1H, m, J ) 6, 16, â-H), 6.31
(1H, dd, J ) 2, 16, R-H), 6.81 (1H, dd, H-3), 6.83 (1H, s, H-5),
6.85 (1H, t, H-6); 13C NMR (CDCl3) δ 146.51 (C-1), 144.70 (C-
2), 130.69 (R-C), 130.59 (C-4), 123.31 (C-6), 119.23 (C-3), 114.32
(C-5), 107.87 (â-C), 55.75 (OCH3), 18.24 (γ-C). Compound 1
was identified as trans-isoeugenol [(E)-2-methoxy-4-(1-pro-
penyl)phenol] from these spectral data.
Com p ou n d 2. Compound 2 was a yellow oil: MS, m/z 164
(M+, 100%), 149 (28%), 137 (20%), 131(27%), 103 (30%), 91
(26%), 77(35%); IR 3509.6, 2939.6, 2851.9, 1514.4, 1267.0; 1H
NMR (CDCl3) δ 3.23 (2H, d, R-H2), 3.78 (3H, s, OCH3), 5.00
(2H, dd, γ-H2), 5.45 (1H, s, OH), 5.80-6.00 (1H, m, â-H), 6.60
(2H, m, H-3,5), 6.77 (1H, d, H-6); 13C NMR (CDCl3) δ 146.41
(C-1), 143.87 (C-2), 137.79 (â-C), 131.89 (C-4), 121.15 (C-6),
115.47 (γ-C), 114.23 (C-3), 111.08 (C-5), 55.82 (OCH3), 39.84
(R-C). Compound 2 was identified as eugenol [2-methoxy-4-
(2-propenyl)phenol] from these spectral data.
F igu r e 1. Isolation scheme for suppressive compounds from
clove (S. aromaticum).
et al. (5) using Salmonella typhimurium TA1535/pSK1002, the
pSK1002 plasmid of which carries an umuC′-′lacZ fused gene.
The overnight culture of bacterial strain was diluted 50-fold
into TGA medium (1% Bactotryptone, 0.5% NaCl, and 0.2%
glucose; supplemented with 20 mg/L ampicllin) and incubated
Meth yl Ester s of Com p ou n d s 1 a n d 2 (1Me a n d 2Me).
Methyl esters of 1 and 2 were obtained by reaction with
diazomethane. These structures were identified by GC, GC-
MS, IR, and 1H and 13C NMR.
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 (50 µL), 0.1 M phosphate
buffer (300 µL, pH 7.4), and mutagens, furylfuramide (50 µL,
5 µg/mL in DMSO), 4NQO (50 µL, 20 µg/mL in DMSO), and
MNNG (50 µL, 200 µg/mL in DMSO) were added to each tube.
In the case of AfB1 (50 µL, 40 µg/mL in DMSO) and Trp-P-1
(50 µL, 40 µg/mL in DMSO), 300 µL of S9-metabolizing 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 °C with shaking, the culture was
centrifuged (3000 rpm) to collect cells, which were centrifuged
in 2.5 mL of PBS. The level of â-galactosidase activity was
measured according to a slight modification of Miller’s method
(Miller, 1972). Fractions (0.25 mL) of the culture were diluted
with 2.25 mL of Z buffer, and 0.1% SDS 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 stopped by 0.1 M Na2CO3, and the absorbance
at OD420 and OD550 was measured. Using the remainder of the
culture, the bacterial density was measured at OD600. The
units of â-galactosidase activity was calculated according to
the method of Miller (1972).34
Meth yl Ester 1Me. Methyl ester 1Me was a colorless oil:
MS, m/z 179 (12.2%), 178 (M+, 100%), 163 (31.1%), 147 (10.8%),
135 (8.8%), 107 (36.5%), 103 (23.0%), 89 (5.4%), 77 (14.9%);
IR 2938.9, 2833.6, 1515.6, 1261.8; 1H NMR (CDCl3) δ 1.90 (3H,
dd, J ) 2, 6, γ-H), 3.90 (3H, s, OCH3), 3.92 (3H, s, OCH3), 6.14
(1H, m, J ) 6, 16, â-H), 6.38 (1H, dd, J ) 2, 16, R-H), 6.81(1H,
s, H-3), 6.83 (1H, s, H-5), 6.90 (1H, t, H-6); 13C NMR (CDCl3)
δ 148.86 (C-1), 148.06 (C-2), 131.04 (R-C), 130.50 (C-4), 123.65
(C-6), 118.54 (C-3), 111.08 (C-5), 108.36 (â-C), 55.78 (OCH3),
55.65 (OCH3), 18.26 (γ-C). Methyl ester 1Me was identified
as trans-methyl isoeugenol [(E)-1,2-dimethoxy-4-(1-propenyl)-
benzene] from these spectral data.
Meth yl Ester 2Me. Methyl ester 2Me was a colorless oil:
MS, m/z 179 (12.2%), 178 (M+, 100%), 163 (29.9%), 147 (28.6%),
135 (10.2%), 107 (23.1%), 103 (25.9%), 92 (28.6%), 77 (13.6%);
IR 2932.0, 2834.6, 1515.2, 1264.9; 1H NMR (CDCl3) δ 3.34 (2H,
d, R-H2), 3.85 (3H, s, OCH3), 3.87 (3H, s, OCH3), 5.07 (2H, dd,
γ-H2), 5.97 (1H, m, â-H), 6.73 (2H, m, H-3,5), 6.81 (1H, d, H-6);
13C NMR (CDCl3) δ 148.85 (C-1), 147.33 (C-2), 137.62 (â-C),
132.58 (C-4), 120.34 (C-6), 115.52 (γ-C), 111.83 (C-3), 111.22
(C-5), 55.88 (OCH3), 55.73 (OCH3). Methyl ester 2Me was
identified as methyleugenol [1,2-dimethoxy-4-(2-propenyl)-
benzene] from these spectral data.
P r ep a r a tion of Activa ted Tr p -P -1. Preparation of acti-
vated Trp-P-1 was carried out according to the method of
Arimoto et al. (16).
RESULTS
F r a ction a tion of th e Extr a ct fr om Clove a n d
Isola tion of Su p p r essive Com p ou n d s 1 a n d 2. The
methanol extract of clove was fractionated to search for
suppressive compounds using the umu test as a guide
(Figure 1). To obtain dose-response data, test samples
were evaluated at dose levels of 0.2, 0.1, and 0.04 mg/
mL. If test samples showed toxicity at 0.2 mg/mL, test
samples were evaluated at dose levels of 0.1, 0.05, and
0.02 mg/mL. As shown in Table 1, the hexane fraction
exhibited a suppressive effect of the furylfuramide-
induced SOS response in S. typhimurium TA1535/
P u r ifica tion a n d Id en tifica tion of th e Su p p r essive
Com p ou n d s. 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
re-extracted with hexane, dichloromethane, ethyl acetate,
butanol, and water, respectively. Each soluble fraction was
concentrated under reduced pressure to give hexane (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
responsible for suppression of the SOS-inducing activity, these
fractions were evaluted with the umu test. The hexane fraction

Suppression of Chemical Mutagen-Induced SOS Response
J. Agric. Food Chem., Vol. 49, No. 8, 2001 4021
Ta ble 1. Su p p r ession of F u r ylfu r a m id e-In d u ced a SOS
Resp on se by Clove F r a ction s in S. typh im u r iu m
TA1535/p SK1002
dose responseb
200
100
40
0
sample
controlc µg/mL µg/mL µg/mL µg/mL
MeOH extractd
hexane fractiond
CH2Cl2 fraction
EtOAc fraction
BuOH fraction
water fraction
142
142
142
142
142
142
341
260
387
303
379
366
364
339
381
366
394
384
390
374
373
389
398
395
407
407
407
407
407
407
dose responseb
100
µg/mL
50
µg/mL
20
µg/mL
0
sample
controlc
µg/mL
fraction 1
fraction 2
fraction 3
fraction 4d
fraction 5
fraction 6
261
261
261
261
261
261
687
693
657
592
692
681
707
709
674
627
712
706
716
719
692
661
723
718
736
736
736
736
736
736
F igu r e 2. Suppression of furylfuramide-induced SOS re-
sponse by compounds 1-3, 1Me, and 2Me in S. typhimurium
TA1535/pSK1002: (0) effect of 1 on furylfuramide; (]) effect
of 2 on furylfuramide; (3) effect of 3 on furylfuramide; (O) effect
of 1Me on furylfuramide; (4) effect of 2Me on furylfuramide.
Furylfuramide (5 µg/mL in DMSO) was added at 50 µL.
a
b
Furylfuramide (5 µg/mL in DMSO) was added at 50 µL. â-
Galactosidase acitivity (units). c Control was exposed to DMSO.
d
Suppressive fraction.
(Figure 3). Compounds 1 and 2 showed weak (13.6 and
11.1% 0.60 µmol/mL, respectively) suppressive effects
of the SOS-inducing activity on MNNG (Figure 3). The
suppressive effects of 1 and 2 on 4NQO are similar to
the suppressive effects observed in the case of furyl-
furamide. These compounds were also assayed with
AfB1 and Trp-P-1, which require liver metabolic activa-
tion (Table 3). As shown in Figure 4, compound 1
suppressed 90.4% of the SOS-inducing activity on AfB1
at a concentration of 0.60 µmol/mL, and the ID50 value
was 0.27 µmol/mL. Compound 2 suppressed 61.5% of
the SOS-inducing activity on AfB1 at a concentration
of 0.60 µmol/mL, and the ID50 value was 0.48 µmol/mL.
Compound 1 suppressed 88.1% of the SOS-inducing
activity on Trp-P-1 at a concentration of 0.60 µmol/mL,
and the ID50 value was 0.25 µmol/mL. Compound 2
suppressed 60.8% of the SOS-inducing activity on Trp-
P-1 at a concentration of 0.60 µmol/mL, and the ID50
value was 0.50 µmol/mL. As shown by the umu test,
compounds 1 and 2 had greater suppressive effects on
the SOS genes against mutagens, furylfuramide, 4NQO,
AfB1, and Trp-P-1 than did MNNG.
pSK1002. After the hexane fraction was fractionated,
only the suppressive fraction 4 eluted with 95:5 hexane/
ethyl acetate as eluent had a clear-cut dose-response
effect in the first fractionation (fractions 1-6). Finally,
suppressive compounds 1 (20.5 g) and 2 (74.4 g) were
isolated from the suppressive fraction 4. Compounds 1
and 2 were identified as trans-isoeugenol and eugenol
1
by GC, GC-MS, IR, and H and 13C NMR, respectively.
In h ibition of SOS-In d u cin g Activity by Com -
p ou n d s 1 a n d 2. The suppressive effects of compounds
1 and 2 were evaluted in the umu test. Compounds 1
and 2 exhibited inhibition on the furylfuramide-induced
SOS response (Table 2). Compounds 1 and 2 suppressed
42.3 and 29.9%, respectively, of the SOS-inducing activ-
ity on furylfuramide at a concentration of 0.60 µmol/
mL, although these compounds showed toxicity at 1.2
µmol/mL (Figure 2). Compounds 1 and 2 were also
assayed with other mutagens, 4NQO and MNNG, which
do not require a liver-metabolizing enzymes mixture
(Table 2). Compounds 1 and 2 suppressed 55.4 and
44.0% of the SOS-inducing activity on 4NQO at a
concentration of 0.60 µmol/mL, respectively, and the
ID50 (50% inhibitory dose) value of 1 was 0.53 µmol/mL
Su p p r essive Effect of Meth yl Ester s (1Me a n d
2Me) of Com p ou n d s 1 a n d 2. Methyl esters (1Me and
Ta ble 2. Su p p r essive Effects of Com p ou n d s 1-3, 1Me, a n d 2Me on F u r ylfu r a m id e,a 4NQO,b a n d MNNGc Usin g S.
typh im u r iu m TA1535/p SK1002
dose responsed
compd
furylfuramide
4NQO
MNNG
control
0.60 µmol/mL
0.48 µmol/mL
0.36 µmol/mL
0.24 µmol/mL
1
1074
1074
1074
1074
1074
282
282
282
282
282
740
837
820
905
1034
1022
1024
873
953
1057
1053
1040
958
1032
1060
1062
1064
2
3
1Me
2Me
995
1007
1015
1
759
759
759
759
759
194
194
194
194
194
446
510
701
690
712
499
566
712
697
719
548
606
736
721
727
606
657
740
742
747
2
3
1Me
2Me
1
697
697
697
697
697
304
304
304
304
304
643
653
659
435
486
657
675
672
458
503
673
686
680
496
530
675
688
689
554
573
2
3
1Me
2Me
a
b
Furylfuramide (5 µg/mL in DMSO) was added at 50 µL. 4NQO (20 µg/mL in DMSO) was added at 50 µL. c MNNG (200 µg/mL in
d
DMSO) was added at 50 µL. â-Galactosidase activity (units).

4022 J. Agric. Food Chem., Vol. 49, No. 8, 2001
Miyazawa and Hisama
F igu r e 3. Suppression of 4NQO- and MNNG-induced SOS
response by compounds 1-3, 1Me, and 2Me in S. typhimu-
rium TA1535/pSK1002: (0) effect of 1; (]) effect of 2; (3) effect
of 3; (O) effect of 1Me; (4) effect of 2Me. 4NQO (20 µg/mL in
DMSO) was added at 50 µL. MNNG (200 µg/mL in DMSO)
was added at 50 µL.
2Me) of 1 and 2 were examined for their ability to
suppress the SOS-inducing activity on furylfuramide,
4NQO, and MNNG, which do not require a liver-
metabolizing enzymes mixture (Table 2). Methyl esters
1Me and 2Me showed weaker suppressive effects on
furylfuramide and 4NQO than 1 and 2 (Figures 2 and
3). Methyl esters 1Me and 2Me showed greater sup-
pressive effects (66.7 and 53.8% at 0.60 µmol/mL,
respectively) on MNNG than 1 and 2, and the ID50
values of 1Me and 2Me were 0.35 and 0.49 µmol/mL,
respectively (Figure 3). These methyl esters were also
assayed with AfB1 and Trp-P-1, which require liver
metabolic activation (Table 3). As shown in Figure 4,
methyl esters 1Me and 2Me suppressed 44.0 and 31.4%,
respectively, of the SOS-inducing activity on AfB1 at a
concentration of 0.60 µmol/mL. These methyl esters
suppressed 47.0 and 29.9%, respectively, of the SOS-
inducing activity on Trp-P-1 at a concentration of 0.60
µmol/mL. As shown by the umu test, methyl esters 1Me
and 2Me had greater suppressive effects on the SOS
genes against MNNG than mutagens, furylfuramide,
4NQO, AfB1 and Trp-P-1.
F igu r e 4. Suppression of AfB1-, Trp-P-1-, and activated Trp-
P-1-induced SOS responses by compounds 1-3, 1Me, and 2Me
in S. typhimurium TA1535/pSK1002: (0) effect of 1; (]) effect
of 2; (3) effect of 3; (O) effect of 1Me; (4) effect of 2Me. AfB1
(40 µg/mL in DMSO) was added at 50 µL. Trp-P-1 (40 µg/mL
in DMSO) was added at 50 µL. Activated Trp-P-1 was added
at 50 µL.
In h ibition of SOS-In d u cin g Activity by Com -
p ou n d 3. For confirming the structure-activity rela-
tionship, o-eugenol [1-methoxy-5-(2-propenyl)phenol] (3)
was obtained. Compound 3 was also examined for its

Suppression of Chemical Mutagen-Induced SOS Response
J. Agric. Food Chem., Vol. 49, No. 8, 2001 4023
Ta ble 3. Su p p r essive Effects of Com p ou n d s 1-3, 1Me, a n d 2Me on AfB1,a Tr p -P -1,b a n d Activa ted Tr p -P -1c Usin g
S. typh im u r iu m TA1535/p SK1002
dose responsed
compd
AfB1
Trp-P-1
activated Trp-P-1
control
0.60 µmol/mL
0.48 µmol/mL
0.36 µmol/mL
0.24 µmol/mL
1
2
3
1Me
2Me
1292
1292
1292
1292
1292
296
296
296
296
296
391
679
868
854
979
501
798
920
928
1028
697
877
950
994
1100
829
980
997
1083
1167
1
2
3
1Me
2Me
819
819
819
819
819
387
387
387
387
387
438
556
666
616
690
490
611
702
648
729
548
652
718
690
754
605
688
758
736
777
1
2
3
1Me
2Me
558
558
558
558
558
101
101
101
101
101
370
420
478
464
504
439
480
489
483
510
475
502
493
505
522
496
520
523
519
535
a
b
AfB1 (40 µg/mL in DMSO) was added at 50 µL. Trp-P-1 (40 µg/mL in DMSO) was added at 50 µL. c Activated Trp-P-1 was added at
d
50 µL. â-Galactosidase activity (units).
ability to suppress the SOS-inducing activity on furyl-
furamide, 4NQO, and MNNG, which do not require a
liver-metabolizing enzymes mixture (Table 2). Com-
pound 3 showed a weaker suppressive effect on furyl-
furamide, 4NQO, and MNNG than 2 (Figures 2 and 3).
Compound 3 was also assayed with AfB1 and Trp-P-1,
which require liver metabolic activation (Table 3). As
shown in Figure 4, compound 3 showed a weaker
suppressive effect (42.5% at 0.60 µmol/mL) on AfB1 than
did 2. Compound 3 showed a weaker suppressive effect
(35.5% at 0.60 µmol/mL) on Trp-P-1 than did 2.
Su p p r essive Effects of 1, 2, 1Me, 2Me, a n d 3 on
Meta bolic Activa tion of Tr p -P -1. The suppressive
effects of 1, 2, 1Me, 2Me, and 3 on metabolic activation
of Trp-P-1 were determined by using the umu test. The
value of â-galactosidase activity observed in the absence
of these compounds was for activated Trp-P-1. As shown
in Table 3 and Figure 4, compounds 1, 2, and 3
suppressed 41.0, 30.1, and 17.4%, respectively, of the
SOS-inducing activity on activated Trp-P-1 at a con-
centration of 0.60 µmol/mL. Methyl esters 1Me and 2Me
suppressed 20.6 and 11.8%, respectively, of the SOS-
inducing activity on activated Trp-P-1 at a concentration
of 0.60 µmol/mL. Suppressive effects of 1, 2, 1Me, 2Me,
and 3 on activated-Trp-P-1 were decreased compared
with those of Trp-P-1.
2 and 3 is the C-4 position of a hydroxy group. These
results indicated that the C-4 position of a hydroxy
group is also an important factor for suppressing the
SOS-inducing activity on furylfuramide, 4NQO, AfB1,
and Trp-P-1. As shown in Tables 2 and 3 and Figures
2-4, compound 1 had more suppressive potency against
furylfuramide, 4NQO, AfB1, and Trp-P-1 than did 2. The
difference in structure between 1 and 2 is the position
of an olefinic double bond. These results indicated that
the position of an olefinic double bond is an important
factor for suppressing the SOS-inducing activity on
furylfuramide, 4NQO, AfB1, and Trp-P-1.
These compounds were examined for the ability to
suppress the metabolic activation of Trp-P-1 by S9. As
shown in Figure 4, these compounds suppressed the
SOS induction on activated Trp-P-1 more weakly than
they did on Trp-P-1. These results suggested the pos-
sibility that the inhibition of the SOS-inducing activity
on Trp-P-1, which was caused by compounds 1, 2, 3,
1Me, and 2Me, was due to the inhibition of metabolic
activation by S9.
For mutagenic activation of furylfuramide (cis form),
cis-trans isomerization (17-19) and reduction of the
nitro group of 5-nitrofuran (20, 21) are important in the
metabolic pathway. The cis-trans isomerization is
based on the formation of nitro anion radicals. cis-
Furylfuramide receives a single electron derived from
an enzyme system to form the anion radical. Spin
density on the olefinic double bond results in free
rotation between the olefinic carbons followed by con-
version to its thermodynamically more stable trans
isomer. The nitro group of 5-nitrofuran is activated by
the reductive metabolism associated with nitroreduc-
tases in bacteria. The main pathway for nitrofuran
activation would be via reduction to a hydroxylamine
intermediate, which could react with DNA though a
nitrenium ion. An alternative reactive intermediate, the
ring-opened acrylonitrile derivative, could form through
rearrangement of the hydroxylamine intermediate. It
has been shown that the acrylonitrile derivative readily
forms conjugates with glutathione, mercaptoethanol,
and thiol groups of proteins. These conjugates increase
the mutation frequency in S. typhimurium TA100,
suggesting that the acrylonitryle derivative is also
capable of interacting with DNA. trans-Isoeugenol and
eugenol exhibited inhibition of SOS induction on furyl-
DISCUSSION
The suppressive compounds of SOS-inducing activity
in clove were identified as 1 and 2. These compounds
showed suppressive effects on umu gene expression of
the SOS response in S. typhimurium TA1535/pSK1002
against furylfuramide and 4NQO, which do not require
liver-metabolizing enzymes, and AfB1 and Trp-P-1,
which do require liver-metabolizing enzymes. As shown
in Tables 2 and 3 and Figures 2-4, compounds 1 and 2
had greater suppressive potencies against furylfura-
mide, 4NQO, AfB1, and Trp-P-1 than did 1Me and 2Me.
The difference in structure between 1 and 1Me and
betweem 2 and 2Me is a hydroxy group. These results
indicated that a hydroxy group is an important factor
for suppressing the SOS-inducing activity on furylfura-
mide, 4NQO, AfB1, and Trp-P-1. As shown in Tables 2
and 3 and Figures 2-4, compound 2 had more suppres-
sive potency against furylfuramide, 4NQO, AfB1, and
Trp-P-1 than did 3. The difference in structure between

4024 J. Agric. Food Chem., Vol. 49, No. 8, 2001
Miyazawa and Hisama
furamide. Therefore, trans-isoeugenol and eugenol may
block these reactive metabolic pathways and/or activa-
tion of nitroanion radicals on furylfuramide.
inhibited the mutagenicity of furylfuramide on the Ames
test using S. typhimurium TA100 (30). In addition,
microsomes or S9 prepared from rats that received
eugenol decreased the mutagenic activity of benzo[a]-
pyrene in the Ames test in comparison to microsomes
or S9 from untreated rats (31, 32). Eugenol has the
antimutagenic effect on the mutagenicity of cyclophos-
phamide in the rodent bone marrow micronucleus test
using male Swiss mice (33). However, inhibition of
mutagen-induced SOS response by trans-isoeugenol and
eugenol has not been reported.
In summary, this research suggests that suppressive
compounds on SOS response against mutagens in clove
were primarily trans-isoeugenol (1) and eugenol (2).
Compounds 1 and 2 showed potent suppressive effects
of the SOS-inducing activity by chemical mutagens, and
the characteristic activity of the respective compounds
was dependent upon the hydroxy group at the C-4
position.
With respect to mutagenic activation of 4NQO, meta-
bolic activation of 4NQO by a nitro reduction enzyme
to 4-hydroxyaminoquinoline 1-oxide (4HAQO) (22, 23),
which is believed to be a proximate derivative of the
potent carcinogen 4NQO, reaction of 4HAQO with DNA
to yield three primary adducts (N2-guanine, C8-
guanine, and N6-adenine adducts) (24), and access of
4NQO and/or 4HAQO to the target cell of bacteria are
necessary. trans-Isoeugenol and eugenol exhibited in-
hibition of SOS induction on 4NQO. Therefore, trans-
isoeugenol and eugenol may block this reactive meta-
bolic activation of 4NQO, which is reacted with a nitro
reduction enzyme in cells of bacteria.
Blocking effects of trans-isoeugenol and eugenol may
be caused by the possible involvement of their antioxi-
dant and scavenging properties. Isoeugenol had a strong
antioxidant effect, and eugenol had a weak antioxidant
effect (25). Isoeugenol and eugenol had inhibitory effects
on the peroxidation of lecithin induced by reactive
oxygen derived from the interaction of ferrous salt and
hydrogen peroxide (26). Isoeugenol and eugenol had
hydroxyl radical scavenging abilities (27). Therefore, the
block by trans-isoeugenol and eugenol could arise from
their scavenging ability to trap hydroxyl radicals orig-
inating from metabolites of furylfuramide and 4NQO
and/or activation of nitro anion radicals on furylfur-
amide with a hydroxy group.
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Received for review March 13, 2001. Revised manuscript
received May 30, 2001. Accepted May 30, 2001.
J F0103469