Antimutagenic Activity of Gigantol from Dendrobium nobile

Article abstract of DOI:10.1021/jf9603902

A methanol extract from Dendrobium nobile 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 f

Full text of DOI:10.1021/jf9603902

J. Agric. Food Chem. 1997, 45, 2849
−2853
2849
An tim u ta gen ic Activity of Giga n tol fr om Den d r obiu m n obile
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 Dendrobium nobile 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. nobile was re-extracted
with n-hexane, dichloromethane, 1-butanol, and water, respectively. A suppressive compound in
the n-hexane extract fraction was isolated by SiO2 column chromatography and chemical
1
fractionation and was identified as gigantol by EI-MS and H and 13C NMR spectroscopy. Gigantol
suppressed the SOS-inducing activity of furylfuramide in the umu test. Gene expression was
suppressed 90% at <0.73 µmol/mL, and the ID50 value was 0.35 µmol/mL. Gigantol was also assayed
with the mutagen 3-amino-1,4-dimethyl-5H-pyrido[4,3b]indole (Trp-P-1), which requires liver
metabolizing enzymes, and it suppressed the SOS-inducing activity of Trp-P-1 in the umu test.
Gene expression was suppressed 91% at <0.73 µmol/mL, and the ID50 value was 0.32 µmol/mL. In
addition, gigantol was assayed for suppressive effect on UV irradiation in the umu test, where it
showed suppression of the SOS-inducing activity caused by UV irradiation. Gene expression was
suppressed 84% at <0.36 µmol/mL, and the ID50 value was 0.17 µmol/mL. The antimutagenic
activities of gigantol against furylfuramide and Trp-P-1 were assayed by an Ames test using S.
typhimurium TA100, which indicated that gigantol suppressed each of the mutagens.
Keyw or d s: Dendrobium nobile; gigantol; Orchidaceae; antimutagenic activity; umu test; Ames test
INTRODUCTION
exhibited suppression of the SOS-inducing activity of
furylfuramide. In this paper, we report the isolation
and identification of the antimutagenic compounds
contained in D. nobile.
Dendrobium nobile (Orchidaceae) is widely distrib-
uted in China. The cultivated storage stalk is used for
treatment as a roborant and for anorexia and gas-
trointestinal disorders. Dendrobium species are known
to produce alkaloids (Suzuki et al., 1973), fluorenones
and sesquiterpenoids (Talapatra et al., 1985, 1992), and
bibenzyls and phenanthrenes (Majumder et al., 1989,
1992).
With the development of techniques for detecting
possible environmental carcinogens and mutagens (Ames
et al., 1975), it has been shown that ordinary diets
contain many kinds of mutagens and antimutagens.
Ishii et al. (1984) reported on the screening of the
bioantimutagenic capacities of plant extracts with cri-
teria of suppressing UV-induced mutations in Escheri-
chia coli B/r WP2 (trp) and the mutator activity in
Bacillus subtilis NIG 1125 (his met). Protoanemonin
was identified as the factor responsible for the antimu-
tagenicity of ranunculus and anemone plants against
UV- and N-methyl-N′-nitro-N-nitrosoguanidine-induced
E. coli B/r WP2 trp (Minakata et al., 1983).
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
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. nobile (sekkoku in J apanese)
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 EOL GSX 270 NMR spectrometer. Tetrameth-
ylsilane (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.
nobile was purchased from Takasago Yakugiyo Co. (Osaka,
J apan). The rhizomes for use as a crude drug were collected
in 1994 from plants cultivated in Nagano prefecture in J apan.
Furylfuramide [2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide], Trp-
P-1 (3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole), and iso-
propyl â-D-galactopyranoside (IPTG) were purchased from
Wako Pure Chemical Ltd. S9 microsomal preparation was
purchased from Oriental Yeast Co.
u m 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 whose plasmid pSK1002 carries a umuC′-lacZ′ fused
gene. The SOS-inducing potency is estimated by the measure-
ment of the level of umu operon expression in terms of cellular
â-galactosidase activity (Miller, 1972).
Effect of Su p p r essive Com p ou n d s on m RNA Syn th esis
In d u ced by IP TG. The test strain E. coli CSH 26T/Flac+,
which was kindly supplied by Dr. Yoshimitsu Oda (Osaka
Prefectural Institute of Public Health, J apan), was used to
investigate the effect on mRNA synthesis in the umu test; this
strain induces â-galactosidase activity by addition of IPTG
instead of mutagens. The affect on â-galactosidase synthesis
* 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(96)00390-1 CCC: $14.00
© 1997 American Chemical Society

2850 J. Agric. Food Chem., Vol. 45, No. 8, 1997
Miyazawa et al.
Ta ble 1. Su p p r ession of F u r ylfu r a m id ea -In d u ced SOS
Resp on se by D. n obile F r a ction s in 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 extractd,e
240.3
510.2
635.2
793.5
716.8
921.1
n-hexane fractione
CH2Cl2 fraction
1-BuOH fraction
water fraction
226.4
226.4
226.4
226.4
764.1
884.9
800.1 1037.2
947.8 1053.7 1037.2
1067.9 1017.3 1005.2 1037.2
1099.8 1028.6 1052.4 1037.2
fraction 1
fraction 2e
fraction 3
244.7
244.7
244.7
579.8
477.8
589.3
632.0
597.6
652.1
670.6
734.9
717.8
720.9
720.9
720.9
fraction 4
fraction 5
fraction 6e
fraction 7e
166.3
155.3
166.3
166.3
529.2
557.4
278.2
388.8
596.5
604.9
489.6
539.0
708.2
735.3
791.4
740.7
787.5
787.5
787.5
787.5
fraction 8e
fraction 9
254.4
254.4
296.3
782.3
542.7
821.1
732.5
975.6
956.8
956.8
a
Furylfuramide (1 µg/mL in DMSO) was added at 50 µL.
Control was exposed to DMSO. c â-galactosidese activity (units).
MeOH extract was assayed at concentrations of 1000, 500, and
200 µg/mL. Suppressive fraction.
b
d
e
by suppressive compound was assayed as follows: An over-
night culture of the tester bacterial strain (E. coli CSH 26T/
Flac+) diluted 50-fold with TG 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 test tubes. The
test compound (50 µL, diluted with DMSO), 0.1 M phosphate
buffer (350 µL, pH 7.4), 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
in the cell was assayed according to the method of Miller
(1972).
F igu r e 1. Isolation scheme for the suppressive compound
from D. nobile.
Com p ou n d 1. Compound 1 was an oil; [R]20
c 1.0); MS, m/ z 274 [M]+, 137 (base peak); IRγmax
2930, 1700, 1600, 1510, 1450, 1270, 1230, 1200, 1150 cm-1
D -4.19 (CHCl3,
KBr
3480,
.
The 1H NMR spectrum of 1 confirmed the presence of methoxy
groups at δ 3.75 and 3.84, and the signals at δ 5.05 and 5.50
could be assigned to two hydroxy groups. The signal at δ 2.80
could be assigned to four ethane hydrogens. The 13C NMR of
1 was identical to that of gigantol (J uneja et al., 1985, 1987).
Compound 1 was identified as gigantol [5-[2-(3-hydroxy-5-
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. (1980).
UV Ir r a d ia tion . Briefly, an overnight culture of the tester
bacterial strain in Luria broth (1% Bactotryptone, 0.5% NaCl,
and 0.5% yeast extract) was diluted 50-fold with fresh TGA
medium (1% Bactotryptone, 0.5% NaCl, and 0.2% glucose;
supplemented with 20 mg/L ampicillin) and incubated at 37
°C until the bacterial density at 600 nm reached 0.25-0.30.
The cultured cells were centrifuged for collection and then
suspended with 5 mL of 0.1 M phosphate buffer. They were
removed into a Petri dish (4 cm) and UV irradiated for 20 s
(4.0 J /m2) with a germical lamp at room temperature.
methoxyphenyl)ethyl]-2-methoxyphenol] from these spectral
data and physical properties.
Am es Test. The mutation test was carried out according
to the preincubation method (Yahagi et al., 1977), which is a
modification of the Ames method (Ames et al., 1975).
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 358 [M]+, 316, 274, 137
(base peak). The 1H NMR spectrum of 1A confirmed the
presence of the acetate methyl group at δ 2.28 and 2.30, and
the signals at δ 3.75 and 3.78 could be assigned to two methoxy
groups. Compound 1A was identified with that of the diac-
etate of gigantol (J uneja et al., 1985; Majumder et al., 1993).
Compound 1A was identified as diacetoxygigantol from these
spectral data.
Meth yla tion of 1. The methylated derivative of 1 (com-
pound 1M) was obtained by reaction with CH2N2. Compound
1M was an oil; MS, m/ z 302 [M]+, 151 (base peak). The 1H
NMR spectrum of 1M confirmed the presence of the methoxy
groups at δ 3.76 (s, 6H), 3.85 (s, 3H), and 3.86 (s, 3H).
Compound 1M was identical with 3,5,4′,5′-tetramethoxydihy-
drostilbene (J uneja et al., 1985).
P u r ifica tion of th e Su p p r essive Com p ou n d 1. The dry
powder (9 kg) of D. nobile was refluxed with methanol for 12
h to give a methanol extract (545 g). This extract was
suspended in water (2 L) and partitioned between n-hexane
(1 L), dichloromethane (1 L), 1-butanol (1 L), and water
successively. Each soluble fraction was concentrated under
reduced pressure to give n-hexane (83 g), dichloromethane (182
g), 1-butanol (153 g), and water (127 g) fractions. To identify
the compound responsible for the suppression of the SOS-
inducing activity, the fractions were tested. As shown in Table
1, the n-hexane fraction had positive activity, whereas the
dichloromethane, 1-butanol, and water fractions did not show
activity. To prepare the suppressive fraction, fractionation of
the n-hexane fraction was carried out as described in Figure
1. Finally, suppressive compound 1 (240 mg) was isolated.

Antimutagenic Activity of Gigantol
J. Agric. Food Chem., Vol. 45, No. 8, 1997 2851
Ta ble 2. Su p p r ession of F u r ylfu r a m id ea a n d
Tr p -P -1b-In d u ced SOS Resp on se by 1, 1A, a n d 1M in S.
typh im u r iu m TA1535/p SK1002
dose responsed
chem- furylfur- Trp-
0.73
0.37
0.15
ical
amide
P-1 controlc µmol/mL µmol/mL µmol/mL
1
1A
1M
469.0
469.0
469.0
113.5
113.5
113.5
150.1
365.9
344.6
280.6
394.3
362.4
369.5
419.2
390.8
1
1A
1M
468.8 173.4
468.8 173.4
468.8 173.4
200.6
315.2
194.1
176.4
389.0
244.3
208.8
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
exposed to DMSO. â-galactosidase activity (units).
d
RESULTS
F r a ction a tion of th e Extr a ct fr om D. n obile a n d
Isola tion of Com p ou n d 1. 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 200, 100,
and 40 µg/mL. As shown in Table 1, the methanol
extract from D. nobile showed a suppressive effect on
umu gene expression of the SOS responses in S. typhi-
murium 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 compound 1 (240
mg) was isolated.
F igu r e 2. Effect of 1 on â-galactosidase activity in S.
typhimurium TA1535/pSK1002. Compound 1 was added into
each tube at 20 min intervals from the start of the incubation
of 2 h. The concentration of 1 was 0.73 µmol/mL.
Str u ctu r e Deter m in a tion of 1. The identity of
compound 1 as gigantol was established by comparison
of spectral data and physical constants with literature
data.
Su p p r ession of 1, 1A, a n d 1M on th e SOS-
In d u cin g Activity. The suppressive effects of 1, 1A,
and 1M were determined in the umu test. As shown
in Table 2, 1, 1A, and 1M inhibited the SOS induction
of furylfuramide. Compound 1 suppressed 90% of the
SOS-inducing activity at concentrations <0.73 µmol/mL,
and the ID50 value was 0.33 µmol/mL. This result
includes the possibilities that 1 suppressed the furyl-
furamide-induced â-galactosidase activity. In the case
of addition of 1 into the test strain, which was added
with furylfuramide, at 20 min intervals after incubation
of 2 h, the furylfuramide-induced SOS responses were
measured, and the concentration of test compounds was
0.73 µmol/mL (Figure 2). These results suggested that
mutagen-induced SOS response was effected by sup-
pressive compounds, although suppressive compounds
did not affect â-galactosidase activity. Mechanisms for
the inhibition of mutagen-induced SOS response by 1
include the possibilities that suppressive compounds
affect the lexA-recA regulation of the umu operon.
Compound 1 was assayed for its effects on mRNA
synthesis using E. coli CSH26T/Flac+, which produces
â-galactosidase without mutagens. From the assay
using E. coli with suppressive compound, 1 did not affect
â-galactosidase activity (Figure 3). Therefore, it sug-
gested that 1 had the potency of suppressive effect on
the mutagen-induced SOS response. Compound 1 also
showed suppression of the SOS-inducing activity of Trp-
P-1, which requires metabolic activation. Compound 1
suppressed 91% of the SOS-inducing activity at concen-
trations <0.35 µmol/mL, and the ID50 value was 0.32
µmol/mL. Compound 1 similarly suppressed both mu-
tagens. In addition, 1 was assayed for the ability of
F igu r e 3. Effect of 1 on mRNA synthesis induced by IPTG
in E. coli CSH26T/Flac+. IPTG (10-2 M) was added at 200
µL.
suppressive effect on activated Trp-P-1-induced SOS
response (Figure 4). From this result, 1 suppressed the
activated Trp-P-1-induced SOS response; it did not
suggest that suppression of activated Trp-P-1-induced
SOS response by 1 was caused by prevention of meta-
bolic activation. Compound 1A suppressed 29% of the
SOS-inducing activity of furylfuramide at concentra-
tions <0.73 µmol/mL, and 1M also suppressed 35% of
the SOS-inducing activity of furylfuramide at concen-
trations <0.73 µmol/mL. In addition, compound 1A
suppressed 93% of the SOS-inducing activity of Trp-P-1
at concentrations <0.37 µmol/mL, and the ID50 value
was 0.30 µmol/mL. Compound 1M almost suppressed
the SOS-inducing activity of Trp-P-1 completely at
concentrations <0.37 µmol/mL, and the ID50 value was
0.26 µmol/mL.
Su p p r essive E ffect of 1, 1A, a n d 1M on UV
Ir r a d ia tion . Compounds 1, 1A, and 1M showed sup-
pressive effects on umu gene expression of the SOS
responses against both furylfuramide and Trp-P-1 (Table
2). In addition, these compounds were tested for sup-
pressive effects on UV-irradiation-induced SOS response

2852 J. Agric. Food Chem., Vol. 45, No. 8, 1997
Miyazawa et al.
F igu r e 4. Suppression of activated Trp-P-1-induced SOS
response by 1 in S. typhimurium TA1535/pSK1002.
F igu r e 6. Suppression of furylfuramide and Trp-P-1-induced
SOS response by 1, 1A, and 1M in S. typhimurium TA100:
(b) effect of 1 on furylfuramide; (O) effect of 1 on Trp-P-1; (9)
effect of 1A on furylfuramide; (0) effect of 1A on Trp-P-1; (2)
effect of 1M on furylfuramide; (4) effect of 1-OMe on Trp-P-1.
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.
DISCUSSION
The antimutagenic compound in D. nobile was clearly
identified as 1. This compound had a suppressive effect
on umu gene expression of the SOS response in S.
typhimurium TA1535/pSK1002 against furylfuramide,
Trp-P-1, and UV irradiation. As shown in Table 2, 1
suppressed the SOS-inducing activity of furylfuramide
and also of Trp-P-1 with an activity nearly equal to that
of furylfuramide. However, 1 showed inhibition of the
SOS induction due to UV irradiation at a lower concen-
tration than those of chemical mutagens (Figure 4). The
SOS regulatory system involves the action of two
proteins: the LexA protein, which respresses 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 activa-
tion of the protease, and the later manifestations of the
SOS response are a secondary consequence of these
events (Little et al., 1982). Mechanisms for the inhibi-
tion of mutagen-induced SOS response by 1 include the
following possibilities: (i) suppression of inactivation of
the LexA repressor by the RecA protease, (ii) suppres-
sion of the transcription of the recA gene, and (iii)
suppression of RecA protein synthesis. Since the ex-
pression of the umuC gene is known to be regulated by
the recA and lexA gene products (Shimagawa et al.,
1983; Walker, 1984), the present data with E. coli may
exclude the possibility that 1 suppresses the lexA-recA
regulation of the umu operon.
F igu r e 5. Suppression of UV-induced SOS response by 1, 1A,
and 1M in S. typhimurium TA1535/pSK1002: (1) effect of 1
on UV irradiation; (crossed box) effect of 1A on UV irradiation;
(]) effect of 1M on UV irradiation.
using S. typhimurium TA1535/pSK1002. Compounds
1, 1A, and 1M were tested for their suppressive effects
on UV irradiation. As shown in Figure 5, compounds
1, 1A, and 1M suppressed the SOS-inducing activity of
UV irradiation. Compound 1 suppressed 84% of the
SOS-inducing activity at concentrations <0.37 µmol/mL,
with an ID50 of 0.19 µmol/mL. On the other hand,
compounds 1A and 1M suppressed 35 and 40% of the
SOS-inducing activity at <0.37 µmol/mL, respectively.
An tim u ta gen ic Activity of 1, 1A, a n d 1M. The
antimutagenic activity of these compounds against
furylfuramide and Trp-P-1 was also demonstrated by
Ames test using S. typhimurium TA100. As shown in
Figure 6, compound 1 suppressed 38% of the mutage-
nicity of furylfuramide at <0.91 µmol/plate. Compound
1 also suppressed 56% of the mutagenicity of Trp-P-1
at <0.91 µmol/plate, within an ID50 of 0.79 µmol/plate.
Compounds 1A and 1M suppressed 42 and 44% of the
mutagenicity of furylfuramide at <0.91 µmol/plate,
respectively. Compound 1A suppressed 83% of the
mutagenicity of Trp-P-1 at <0.91 µmol/plate, within an
ID50 of 0.30 µmol/plate. Compound 1M suppressed 83%
of the mutagenicity of Trp-P-1 at <0.91 µmol/plate, with
an ID50 of 0.26 µmol/plate.
From the results of the Ames test, 1 did not suppress
the mutagen-induced SOS responses but showed sup-
pression of mutagenicity caused by mutagens. This
result clearly indicated that 1 had potency as an
antimutagenic for suppression of DNA damage.
Recently, it was reported that curucumin suppressed
UV-induced SOS response (Oda, 1995). Shimoi et al.

Antimutagenic Activity of Gigantol
J. Agric. Food Chem., Vol. 45, No. 8, 1997 2853
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(1985) also reported that tannic acid suppressed mu-
tagenesis in E. coli B/r WP2 (trp-) induced by UV or
4-nitroquinoline 1-oxide (4NQO). Paeonol suppressed
the mutagenesis in E. coli WP2s (trp-, uvrA-) induced
by 4NQO (Fukuhara et al., 1987). In our previous
paper, we reported that paeonol also suppressed the
SOS-inducing activity of furylfuramide, using S. typhi-
murium TA1535/pSK1002 in the umu test, and mu-
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Received for review J une 3, 1996. Accepted April 29, 1997.X
We are grateful to the Environmental Science Institute of
Kinki University for financial support. This study was also
supported by a Grant-in-Aid for Science Research from J apan
Private School Promotion Foundation.
J F9603902
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X Abstract published in Advance ACS Abstracts, J uly
1, 1997.