Antimutagenic activity of (+)-β-eudesmol and paeonol from Dioscorea japonica

Article abstract of DOI:10.1021/jf950792u

A methanol extract from Dioscorea japonica showed a suppressive effect on umu gene expression of the SOS response in Salmonella typhimurium TA1535/pSK1002 against the mutagen 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (furylfuramide). The methanol extract from D. japonica was reextracted with dichloromethane, n-butanol, and water, respectively. The suppressive compounds in the dichloromethane extract fraction were isolated by SiO₂ column chromatography and identified as (+)-β-eudesmol and 2-hydroxy-4-methoxyacetophenone (paeonol) by EI-MS and ¹H and ¹³C NMR spectroscopy. (+)-β-Eudesmol suppressed the SOS-inducing activity of furylfuramide in the umu test. Gene expression was suppressed 80% at <0.18 μmol/mL, and the ID₅₀ value was 0.09 μmol/ mL. Paeonol also suppressed the SOS-inducing activity of furylfuramide in the umu test. Gene expression was suppressed 60% at <1.2 μmol/mL, and the ID₅₀ value was 0.99 μmol/mL. (+)-β-Eudesmol and paeonol were assayed with the mutagen 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), which requires liver metabolizing enzymes, and showed suppressive effects on the SOS induction of the mutagen. (+)-β-Eudesmol suppressed gene expression 48% at <0.18 μmol/mL. Paeonol suppressed gene expression 76% at <1.2 μmol/mL, and the ID₅₀ was 0.41 μmol/mL. The antimutagenic activities of these compounds against furylfuramide and Trp-P-1 were tested by an Ames test using S. typhimurium TA100, which indicated that these compounds showed antimutagenic activity.

Full text of DOI:10.1021/jf950792u

J. Agric. Food Chem. 1996, 44, 1647
−1650
1647
An tim u ta gen ic Activity of (+)-â-Eu d esm ol a n d P a eon ol fr om
Dioscor ea ja pon ica
Mitsuo Miyazawa,*,† Hideo Shimamura,† Sei-ichi Nakamura,‡ and Hiromu Kameoka†
Department of Applied Chemistry, Faculty of Science and Engineering, Kinki University, Kowakae,
Higashiosaka-shi, Osaka 577, J apan, and Osaka Prefectural Institute of Public Health, Nakamichi-1,
Higashinari-ku, Osaka 537, J apan
A methanol extract from Dioscorea japonica showed a suppressive effect on umu gene expression of
the SOS response in Salmonella typhimurium TA1535/pSK1002 against the mutagen 2-(2-furyl)-
3-(5-nitro-2-furyl)acrylamide (furylfuramide). The methanol extract from D. japonica was re-
extracted with dichloromethane, n-butanol, and water, respectively. The suppressive compounds
in the dichloromethane extract fraction were isolated by SiO2 column chromatography and identified
as (+)-â-eudesmol and 2-hydroxy-4-methoxyacetophenone (paeonol) by EI-MS and 1H and 13C NMR
spectroscopy. (+)-â-Eudesmol suppressed the SOS-inducing activity of furylfuramide in the umu
test. Gene expression was suppressed 80% at <0.18 µmol/mL, and the ID50 value was 0.09 µmol/
mL. Paeonol also suppressed the SOS-inducing activity of furylfuramide in the umu test. Gene
expression was suppressed 60% at <1.2 µmol/mL, and the ID50 value was 0.99 µmol/mL. (+)-â-
Eudesmol and paeonol were assayed with the mutagen 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole
(Trp-P-1), which requires liver metabolizing enzymes, and showed suppressive effects on the SOS
induction of the mutagen. (+)-â-Eudesmol suppressed gene expression 48% at <0.18 µmol/mL.
Paeonol suppressed gene expression 76% at <1.2 µmol/mL, and the ID50 was 0.41 µmol/mL. The
antimutagenic activities of these compounds against furylfuramide and Trp-P-1 were tested by an
Ames test using S. typhimurium TA100, which indicated that these compounds showed antimu-
tagenic activity.
Keyw or d s: Dioscorea japonica; Dioscoreaceae; Chinese yam; (+)-â-eudesmol; paeonol; antimutagenic
activity; umu test; Ames test
INTRODUCTION
new naturally occurring antimutagenic compounds in
plants that have a history of safe use as Chinese crude
drugs (Miyazawa et al., 1995a-c), we found that the
methanol extract of D. japonica (sanyaku in J apanese)
exhibited suppression of the SOS-inducing activity of
2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (furylfuramide).
In this paper, we report the isolation and identification
of the antimutagenic compounds contained in D. japon-
ica.
Chinese yam (Dioscoreaceae) occurs naturally in
J apan, where it is cultivated and its storage rhizome is
used mainly for treatment of diarrhea, asthma, polyuria,
and diabetes. Several phenanthrenes, dihydrophenan-
threnes, and bibenzyls have been isolated from Dioscorea
species (Takasugi et al., 1987; Sunder et al., 1978), and
antifungal compounds were isolated from Dioscorea
batatas (Takasugi et al., 1987). Hikino et al. (1986)
reported on the isolation of hypoglycemic active com-
pounds (dioscorans A-F) from Dioscorea japonica.
In the evaluation of the carcinogenicity or mutage-
nicity of environmental chemicals, it is quite important
to determine factors present in our environment that
may affect these activities. With the development of
techniques for detecting possible environmental car-
cinogens and mutagens (Ames et al., 1975), it has been
shown that ordinary diets contain many kinds of mu-
tagens and antimutagens, and as one example Kaki-
numa et al. (1984a) reported the identification of an
antimutagen (cinnamaldehyde) from Cinnamomum cas-
sia using Escherichia coli WP2 B/r uvrA- trpE-.
MATERIALS AND METHODS
Gen er a l P r oced u r e. Electron-impact mass spectra (EI-
MS) were obtained on a Hewlett-Packard 5972A mass spec-
trometer. 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 OEL GSX 270 NMR spectrometer. Tetra-
methylsilane (TMS) was used as the internal reference (δ 0.00)
for 1H NMR spectra measured in CDCl3. This solvent was
used for 13C NMR spectra. Specific rotation was determined
with a J asco DIP-140 digital polarimeter.
Ma ter ia ls. Commercially available air-dried rhizome of D.
japonica was purchased from Takasago Yakugiyo Co. (Osaka,
J apan). The rhizomes for use as crude drug were collected in
1994 from plants cultivated in Nagano prefecture in J apan.
Furylfuramide and 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]in-
dole (Trp-P-1) were purchased from Wako Pure Chemical Co.
S9 metabolizing enzyme was purchased from Oriental Yeast
Co.
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 (Oda
et al., 1985; Nakamura et al., 1987). In our search for
Um u Test. The umu test for detecting the SOS-inducing
activity of chemicals was carried out essentially as described
by Oda et al. (1985) using Salmonella typhimurium TA1535/
pSK1002, having the plasmid pSK1002 which carries a umuC′-
lacZ′ fused gene. The SOS-inducing potency is estimated by
the measurement of the level of umu operon expression in
terms of cellular â-galactosidase activity.
* Author to whom correspondence should be ad-
dressed (telephone +81-6-721-2332; fax +81-6-727-
4301).
† Kinki University.
‡ Osaka Prefectural Institute of Public Health.
S0021-8561(95)00792-8 CCC: $12.00
© 1996 American Chemical Society
+
+

1648 J. Agric. Food Chem., Vol. 44, No. 7, 1996
Miyazawa et al.
Ta ble 1. Su p p r essive Effect of D. ja pon ica F r a ction s on
F u r ylfu r a m id ea Usin g S. typh im u r iu m TA1535/p SK1002
dose responsec
200
100
50
0
sample
controlb µg/mL µg/mL µg/mL µg/mL
MeOH extract
173.6
815.9
912.3 956.2 972.5
CH2Cl2 fraction
n-BuOH fraction
water fraction
183.9
183.9
183.9
648.6
763.2
792.7
746.8 785.4 830.3
790.5 850.9 830.3
809.6 824.3 830.3
fraction 1
fraction 2
176.7
176.7
592.3
882.9
634.2 792.2 945.6
960.2 953.1 945.6
fraction 3
fraction 4
fraction 5
fraction 6
203.5
203.5
203.5
203.5
812.2
432.5
923.0 921.7 994.7
587.9 860.7 994.7
867.8 1011.9 962.4 994.7
1025.3 968.6 982.5 994.7
a
Furylfuramide (1 µg/mL in DMSO) was added at 60 µL.
b Control was a treatment without furylfuramide. c â-Galactosidase
activity (units).
Am es Test. The mutation test was carried out according
to the preincubation method (Yahagi et al., 1977), which is a
modification of Ames method (Ames et al., 1975).
P u r ifica tion of th e Su p p r essive Com p ou n d s 1 a n d 2.
The dry powder of rhizome (2 kg) of D. japonica was refluxed
with methanol for 12 h to give a methanol extract (21.7 g).
This extract was suspended in water (1 L) and partitioned
between dichloromethane (1 L) and water and then n-butanol
(1 L) and water, successively. Each soluble fraction was
concentrated under reduced pressure to give dichloromethane
(6.7 g), n-butanol (3.4 g), and water (11.6 g) fractions, and each
fraction was tested for suppression of the SOS-inducing
activity. As shown in Table 1, the dichloromethane extract
fraction had positive activity, whereas the n-butanol and water
extract fractions did not show activity. The suppressive
fraction (dichloromethane extract fraction) was dissolved with
dichloromethane and partitioned with 5% NaHCO3 solution.
The organic layer was given as a neutral fraction (fraction 1).
The aqueous layer was acidified with diluted HCl and then
extracted with dichloromethane to yield the acidic fraction
(fraction 2). The results of the umu test showed that fraction
1 had positive activity, whereas fraction 2 did not show
activity. Fractionation of fraction 1 was then carried out as
described in Figure 1. Finally, suppressive compounds 1 (45
mg) and 2 (7 mg) were isolated.
F igu r e 1. Isolation scheme for the suppressive compounds
from D. japonica.
methoxyacetophenone (paeonol) (Fukuhara et al., 1987). Com-
pound 2 was identified as paeonol from these spectral data
and physical properties.
Acetyla tion of 1. The acetylate of 1 (compound 1A) was
obtained by the reaction with acetic anhydride and pyridine.
Compound 1A was an oil; MS, m/ z (%) 204 (M+ - CH3COOH,
KBr
42.5), 189 (25.8), 161 (50.8), 59 (23.3), 43 (100); IRγmax 3080,
2940, 2873, 1733, 1646, 1456, 1368, 1257, 1210, 1134, 1018,
886 cm-1
.
The 1H NMR spectrum of 1A confirmed the
presence of an acetate methyl group at δ 1.98 and agreed with
that of the monoacetate of â-eudesmol (El-Sayed et al., 1989).
Compound 1A was identified as the monoacetate of â-eudesmol
from these spectral data.
Suppressive Compound 1: crystalline; mp 69.8-72.5 °C;
[R]20 +37.5 (CHCl3; c 1.0); MS, m/ z (%) 222 (M+, 1.7), 204
D
Deh yd r a tion of 1. The dehydrated derivative of 1 (com-
pound 1D) was obtained by the reaction of 1 with thionyl
chloride and pyridine (Iwamoto et al., 1982). Compound 1D
was an oil; MS, m/ z (%) 204 (M+, 61.7), 189 (50.8), 175 (25.8),
161 (57.5), 147 (50.8), 133 (55.0), 121 (63.3), 107 (88.3), 105
(100), 93 (96.7), 91 (84.2), 79 (95.8), 67 (72.5), 55 (50.0), 41
KBr
(5.0), 189 (10.0), 164 (32.5), 149 (70.8), 59 (100%); IRγmax
3500, 2972, 2931, 2866, 1646, 1441, 1378, 886 cm-1. The 1H
NMR spectrum of 1 confirmed the presence of a methyl group
at δ 0.70 (C-15) and 1.20 (C-12 and C-13), and the signals at
δ 4.49 and 4.73 could be assigned as the two exocyclic vinyl
hydrogens (C-14). The 13C NMR agreed with those of (+)-â-
eudesmol (Cordano et al., 1978). Suppressive compound 1 was
identified as (+)-â-eudesmol from these spectral data and
physical properties.
1
(86.7). The H NMR spectrum of 1D confirmed the presence
of a methyl group at δ 0.73 (s, 3H) and 1.75 (s, 3H) and vinyl
hydrogens at δ 4.44 (dd, J ) 2.0, 3.1, 1H) and 4.72 (m, 3H).
Compound 1D was identified as â-selinene (Lionel et al., 1983).
RESULTS
F r a ction a tion of th e Extr a ct fr om D. ja pon ica
a n d Isola tion of 1 a n d 2. The methanol extract was
fractionated to search for suppressive compounds using
the umu test as a guide. To obtain dose-response data,
test samples were evaluated at dose levels of 0.2, 0.1,
and 0.04 mg/mL. As shown in Table 1, the methanol
extract from D. japonica showed a suppressive effect on
umu gene expression of the SOS responses in S. typh-
imurium TA1535/pSK1002 against furylfuramide. To
prepare the suppressive fraction, fractionation of the
methanol extract was carried out as described in Figure
1 and Table 1. Finally, suppressive compounds 1 (45
mg) and 2 (7 mg) were isolated.
Suppressive Compound 2: crystalline; mp 48.8-50.3 °C;
[R]20 -0.71 (CHCl3; c 0.25); MS, m/ z (%) 166 (M+, 3.9), 151
D
KBr
(100), 108 (5.8), 95 (9.2), 43 (6.7); IRγmax 2921, 1620, 1505,
1464, 1369, 1334, 1258, 1209, 1141, 1071, 1022, 978, 950, 859,
816 cm-1. The 1H NMR spectrum of 2 indicated the appear-
ance of a δ 12.75 for the strong intramolecular hydrogen
bonding. The EI-MS of 2 agreed with that of 2-hydroxy-4-
+
+

Antimutagenic Activity of Eudesmol from Dioscorea japonica
J. Agric. Food Chem., Vol. 44, No. 7, 1996 1649
Ta ble 2. Su p p r essive Effect of 1 a n d 2 on F u r ylfu r a m id ea a n d Tr p -P -1b Usin g S. typh im u r iu m TA1535/p SK1002
dose responsed
chemical
furylfuramide
930.1
Trp-P-1
567.6
controlc
0.18 µmol/mL
0.11 µmol/mL
0.07 µmol/mL
0.04 µmol/mL
1
252.9
210.0
382.9
395.9
451.7
411.0
645.5
448.7
787.7
477.8
dose responsed
chemical
furylfuramide
930.1
Trp-P-1
567.6
controlc
1.20 µmol/mL
0.60 µmol/mL
0.24 µmol/mL
2
252.9
210.0
529.2
295.1
668.0
346.6
782.5
420.3
a
b
Furylfuramide (1 µg/mL in DMSO) was added at 60 µL. Trp-P-1 (40 µg/mL in DMSO) was added at 50 µL. c Control was treatment
d
without mutagens. â-Galactosidase activity (units).
F igu r e 3. Suppressive effect of 2 on the mutagenicity of
furylfuramide using S. typhimurium TA100. Furylfuramide
(0.5 µg/mL in DMSO) was added at 20 µL/plate.
F igu r e 2. Effect of 1 on the mutagenicity of furylfuramide
(b) and Trp-P-1 (9) in S. typhimurium TA100. Furylfuramide
(0.5 µg/mL in DMSO) was added at 20 µL/plate. Trp-P-1 (40
µg/mL in DMSO) was added at 50 µL/plate. In the case of the
effect of 1 on the mutagenicity of furylfuramide, 1 was toxic
at under 0.56 µmol/plate.
plate. As shown in Figure 3, compound 2, which was
also an authentic sample, suppressed only 20% of the
mutagenicity of furylfuramide.
Rela tion sh ip of th e Str u ctu r e a n d Activity of 1.
To explore the structure-activity relationship of 1,
compounds 1A and 1D were examined for their ability
to suppress the SOS-inducing activity of furylfuramide.
Neither of them suppressed the SOS-inducing activity
of furylfuramide at all, even at higher concentrations
than those of 1 (data not shown). This result suggests
that a hydroxyl group at C-11 is a necessary feature for
â-eudesmol to show a suppressive effect.
Str u ctu r e Deter m in a tion of 1 a n d 2. The identi-
ties of compounds 1 and 2 as â-eudesmol and paeonol
were established by comparison of spectra (1H NMR,
13C NMR, MS) and physical constants (mp, [R]D) with
literature data.
In h ibition of th e SOS-In d u cin g Activity of 1 a n d
2. The suppressive effects of 1 and 2 were determined
in the umu test. As shown in Table 2, 1 and 2 exhibited
inhibition of the SOS induction of furylfuramide. Com-
pound 1 suppressed 80% of the SOS-inducing activity
at concentrations <0.18 µmol/mL, and the ID50 value
was 0.09 µmol/mL. Compound 2 suppressed 60% of the
SOS-inducing activity at concentrations <1.2 µmol/mL,
and the ID50 value was 0.99 µmol/mL. Compounds 1
and 2 also showed suppression of the SOS-inducing
activity of Trp-P-1, which requires metabolic activation.
The former suppressed 48% of the SOS-inducing activity
at concentrations <0.18 µmol/mL. The latter sup-
pressed 76% of the SOS-inducing activity at a concen-
tration <1.2 µmol/mL, and the ID50 value was 0.41 µmol/
mL.
An tim u ta gen ic Activity of 1 a n d 2. The antimu-
tagenic activities of these compounds against furylfura-
mide and Trp-P-1 were also demonstrated by the Ames
test using S. typhimurium TA100. As shown in Figure
2, compound 1, which was an authentic sample, sup-
pressed 93% of the mutagenicity of furylfuramide at
0.45 µmol/plate, and the ID50 value was 0.13 µmol/plate.
Compound 1 also suppressed 70% of the mutagenicity
of Trp-P-1 at 0.56 µmol/plate, with an ID50 of 0.39 µmol/
plate. 1 showed toxicity at a concentration of 0.56 µmol/
DISCUSSION
The antimutagenic compounds in D. japonica were
clearly identified as 1 and 2. These compounds had a
suppressive effect on umu gene expression of the SOS
response in S. typhimurium TA1535/pSK1002 against
furylfuramide and Trp-P-1. As shown in Table 2, 1
suppressed the SOS-inducing activity of both mutagens
at lower concentrations than did 2. In the Ames test
using S. typhimurium TA100, 1 similarly inhibited the
mutagenicity of furylfuramide and Trp-P-1 and 2 showed
a low suppressive effect of the mutagenicity against
furylfuramide (Figures 2 and 3).
Compounds 1A and 1D did not show any suppressive
effects on the SOS-inducing activity of furylfuramide,
which indicated that a hydroxyl group at C-11 is
important for the suppressive effect. Compound 1 has
other biological activities; for example, it has a preven-
tive activity against experimental ulcerations (Nogami
et al., 1986) and antianoxic activity (Yamahara et al.,
1990). Further, 1 has an inhibitory activity on Epstein-
+
+

1650 J. Agric. Food Chem., Vol. 44, No. 7, 1996
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cortex. Agric. Biol. Chem. 1984a , 48 (7), 1905-1906.
Received for review December 4, 1995. Revised manuscript
received April 18, 1996. Accepted April 25, 1996.X
J F950792U
X Abstract published in Advance ACS Abstracts, J une
1, 1996.
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