Suppression of the SOS-inducing activity of mutagenic heterocyclic amine, Trp-P-1, by triterpenoid from Uncaria sinensis in the Salmonella typhimurium TA1535/pSK1002 umu test

Article abstract of DOI:10.1021/jf035430y

The methanol extract from Uncaria sinensis showed a suppressive effect on umu gene expression of the SOS response in Salmonella typhimurium TA1535/pSK1002 against the mutagen 3-amino-1,4-dimethyl-5H-pyrido[4,3b]indole (Trp-P-1), which requires liver metabolizing enzymes. The methanol extract from U. sinensis was re-extracted with hexane, CH₂Cl₂, BuOH, and water, respectively. CH₂-Cl₂ extract showed a suppressive effect. A suppressive compound 1 in CH₂Cl₂ extract was isolated by SiO₂ column chromatography. Compound 1 was identified as ursolic acid by IR, electron ionization EI-MS, and NMR spectroscopy. Suppressive effects of ursolic acid (1) and its derivatives, methyl ursolate (1M), acetylursolic acid (1A), and methyl acetylursolate (1MA), were determined in the umu test. These compounds suppressed 61.3, 37.7, 71.5, and 37.8% of the Trp-P-1-induced SOS response at a concentration of 0.4 μmol/mL, respectively. The ID ₅₀ values of compounds 1 and 1A were 0.17 and 0.20 μmol/mL. In addition, these compounds were assayed with the activated Trp-P-1. Suppressive effects on activated Trp-P-1 were decreased as compared with those of Trp-P-1.

Full text of DOI:10.1021/jf035430y

2312 J. Agric. Food Chem. 2005, 53, 2312−2315
Suppression of the SOS-Inducing Activity of Mutagenic
Heterocyclic Amine, Trp-P-1, by Triterpenoid from
Uncaria sinensis in the Salmonella typhimurium TA1535/
pSK1002 Umu Test
MITSUO MIYAZAWA,* YOSHIHARU OKUNO, AND KOJI IMANISHI
Department of Applied Chemistry, Faculty of Science and Engineering, Kinki University Kowakae,
Higashiosaka-shi, Osaka 577-8502, Japan
The methanol extract from Uncaria sinensis showed a suppressive effect on umu gene expression
of the SOS response in Salmonella typhimurium TA1535/pSK1002 against the mutagen 3-amino-
1,4-dimethyl-5H-pyrido[4,3b]indole (Trp-P-1), which requires liver metabolizing enzymes. The methanol
extract from U. sinensis was re-extracted with hexane, CH2Cl2, BuOH, and water, respectively. CH2-
Cl2 extract showed a suppressive effect. A suppressive compound 1 in CH2Cl2 extract was isolated
by SiO2 column chromatography. Compound 1 was identified as ursolic acid by IR, electron ionization
EI-MS, and NMR spectroscopy. Suppressive effects of ursolic acid (1) and its derivatives, methyl
ursolate (1M), acetylursolic acid (1A), and methyl acetylursolate (1MA), were determined in the umu
test. These compounds suppressed 61.3, 37.7, 71.5, and 37.8% of the Trp-P-1-induced SOS response
at a concentration of 0.4 µmol/mL, respectively. The ID50 values of compounds 1 and 1A were 0.17
and 0.20 µmol/mL. In addition, these compounds were assayed with the activated Trp-P-1.
Suppressive effects on activated Trp-P-1 were decreased as compared with those of Trp-P-1.
KEYWORDS: Uncaria sinensis; Rubiaceae; ursolic acid; SOS response; umu test
INTRODUCTION
extract of the bark (8). The plants of the genus Uncaria
(sinensis) are well-known for their rich content of alkaloids and
tannins, triterpenoid saponins. A number of alkaloids displaying
a pronounced enhancement of phagocytosis were isolated as
well as quinovic acid glycosides with antiviral activity (9, 10).
Also, various biological activities have been described for
triterpenoid saponins (11). A strong antitumor activity of
triterpenoid saponins against Ehrich carcinoma ascite was
reported (12) in our search for new naturally occurring sup-
pressive compounds of SOS inducing activity in plants, which
have a history of safe use as Chinese crude drugs (13-15). We
found that the methanol extract of U. sinensis exhibited
suppressive effects of the SOS inducing activity of Trp-P-1. In
this paper, we report the identification of the suppressive
compound in U. sinensis and its structure-activity relationship.
Several sort-term tests for screening environmental mutagens
and carcinogens have been developed and are widely used in
many laboratories (1, 2). The Ames test is a convenient method
for evaluating mutagenic activity (1), and several pieces of
evidence have suggested that the mutagenic activity of a number
of chemicals be correlated well with the carcinogenic activity
so far reported (3, 4).
The SOS response appears to be induced by an alteration in
DNA synthesis, either directly by DNA damage blocking to
the replication fork or indirectly by antibiotic, such as novo-
biocin, that inhibits DNA synthesis. The umu test system was
developed to evaluate the genotoxic activity of a wide variety
of environmental carcinogens and mutagens, using the expres-
sion 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, sensitivity, and rapidity (7).
MATERIALS AND METHODS
General Procedure. Gas chromatography (GC) was performed 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 interfaced 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. NMR spectra (δ, J in hertz) were
recorded on a JEOL GSX 270 NMR spectrometer. Tetramethylsilane
(TMS) was used as the internal reference (δ 0.00) for 1H NMR spectra
measured in CDCl3.
Uncaria sinensis is a plant that belongs to the family
Rubiaceae and is commonly known as Chotoko in Japanese.
The plant is used in traditional Peruvian medicine for the
treatment of cancer, arthrits, gastritis, cytostatic, contraceptive,
antiinflammatory, and certain epidemic diseases, as an aqueous
* To whom correspondence should be addressed. Tel: +81-6-6721-2332.
Fax: +81-6-6727-4301. E-mail: miyazawa@apch.kindai.ac.jp.
10.1021/jf035430y CCC: $30.25
© 2005 American Chemical Society
Published on Web 02/18/2005

Suppression of SOS-Inducing Activity of Triterpenoid
J. Agric. Food Chem., Vol. 53, No. 6, 2005 2313
Materials. The commercially available air-dried rhizome of U.
sinensis was purchased from Yamada Yakken Co. (Osaka, Japan). The
rhizomes for use as a crude drug were cultivated in China. Trp-P-1
was purchased from Wako Pure Chemical Co. (Osaka, Japan). S9
(supernatant of 9000 g) and coenzyme, NADPH, NADH, and G-6-P
were purchased from Oriental Yeast Co., Ltd. (Tokyo, Japan).
Umu Test. The umu test is based upon the abilities of carcinogens
and mutagens to introduce expression of an umu gene in Salmonella
typhimurium TA1535/pSK1002, in which a plasmid (pSK1002) carrying
a fused gene (umuC-lacZ) had been introduced. The SOS response
appears to be induced by an alteration in DNA synthesis, either directly
by DNA damage blocking to the replication fork or indirectly by
antibiotics, such as novobiocin, that inhibits DNA synthesis. The SOS
regulatory system is controlled in part by the interplay of two proteinss
the lexA protein, which represses asset of unlinked genes during normal
cell growth, and the recA protein, which is required in vivo for
inactivation of lexA protein after treatments that derepress the system
by DNA damaging its metabolism (16, 17). The SOS inducing potency
is estimated by the measurement of the level of umu operon expression
in terms of cellular â-galactosidase activity.
Briefly, an overnight culture of the tester bacterial strain was in TGA
broth (1% bactotryptone, 0.5% NaCl, and 0.2% glucose; supplemented
with 50 mg/L ampicillin) and incubated at 37 °C until the bacterial
density at 600 nm reached 0.25-0.30. The culture was divided into
2.1 mL portions in test tubes. The test compound (50 µL, diluted in
DMSO), S9 metabolizing enzyme instead of 0.1 M phosphate buffer
mixture (300 µL, pH 7.4), and Trp-P-1 (50 µL, 40 µg/mL in DMSO)
were added to each tube. After 2 h 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 (0.25 mL), the
level of â-galactosidase activity in the cell was assayed by the method
of Miller (18).
Preparation of Activated Trp-P-1. The preparation of activated
Trp-P-1 was carried out according to the method of Arimoto et al. (19).
Purification and Identification of the Suppressive Compound
from U. sinensis. The dry powder of U. sinensis was refluxed with
methanol for 12 h to give a methanol extract (5 kg). This extract was
suspended in water and partitioned between hexane, dichloromethane,
butanol, and water successively. Each soluble extract was concentrated
under reduced pressure to give hexane (12.8 g), dichloromethane (83.4
g), butanol (112.1 g), and water (122.7 g) extracts. To purify the
compound responsible for suppression of SOS inducing activity, these
extracts were subjected to the umu test. The CH2Cl2 extracts showed a
suppressive effect, whereas the other extracts did not. The CH2Cl2
extract was repeatedly fractionated by SiO2 column chromatography
using the umu test as a guide, and suppressive compound 1 (196 mg)
was isolated (Figure 1). Compound 1 was identified as ursolic acid by
IR, GC-MS, and NMR spectroscopy.
Figure 1. Isolation scheme of suppressive compound from U. sinensis.
and the 13C NMR corresponded with those of methyl ursolate (20-
22). Compound 1M was identified as methyl ursolate (3â-hydroxyurs-
12-en-28-oic acid methyl ester) from these spectra data and physical
properties.
Compound 1A. Compound 1A was a white crystal; mp 242.7-244.1
°C. EI-MS (70 eV): m/z (rel int.) 498 ([M]+, 10), 483 (1), 452 (4),
438 (5), 248 (100), 203 (31), 190 (21), 133 (22), 119 (11), 105 (8). IR
KBr
γmax
(cm-1): 2927, 1736, 1697, 1455, 1371, 1244, 1027; [R]23d
+71.2° (CHCl3; c 1.0). The 1H NMR and the 13C NMR were identified
with those of ursolic acid (20, 21, 23-25). Compound 1A was identified
as acetyl ursolic acid [3â-(acetyloxy)-urs-12-en-28-oic acid] from these
spectra data and physical properties.
Methylation of Compound 1. The methylated derivative of 1
(compound 1M) was obtained by reaction with excess diazomethane
in diethyl ether.
Compound 1MA. Compound 1MA was a white crystal; mp 243.2-
244.8 °C. EI-MS (70 eV): m/z (rel int.) 512 ([M]+, 7), 497 (2), 452
(9), 437 (2), 262 (100), 249 (19), 203 (60), 189 (20), 133 (26), 119
(12), 107 (7). IR γmaxKBr (cm-1): 2940, 2924, 1733, 1730, 1456, 1371,
1243, 1202, 1025; [R]23d +56.4° (CHCl3; c 1.0). The 1H NMR and the
13C NMR were identified with those of ursolic acid (20-22, 25, 26).
Compound 1MA was identified as acetyl ursolic acid [3â-(acetyloxy)-
urs-12-en-28-oic acid methyl ester] from these spectra data and physical
properties.
Acetylation of Compounds 1 and 1M. Pyridine (0.5 mL) was added
to a solution of compound 1 (20 mg) in acetic anhydride (5 mL), and
the solution was refluxed for 3 h. The product was treated with 5%
HCl and 5% NaHCO3 solution and extracted with CH2Cl2, and the
solvent was evaporated. The residue was separated by silica gel column
chromatography, and the acetate 1A (19 mg) was obtained. Similarly,
compound 1M (20 mg) was acetylated to give the acetate 1MA (21
mg).
Suppressive Compound 1 and Derivatives. Compound 1. Com-
pound 1 was obtained as a white crystal; mp 238.4-240.2 °C. EI-MS
(70 eV): m/z (rel int.) 456 ([M]+, 6), 438 (3), 410 (6), 248 (100), 203
RESULT AND DISCUSSION
(25), 189 (10), 133 (20), 119 (11), 105 (9). IR γmax (cm-1): 3432,
KBr
The methanol extract of U. sinensis was fractionated to search
for suppressive compound using the umu test as a guide. To
prepare the suppressive compound, fractionation of the methanol
extract was carried out as described in Figure 1. Suppressive
compound 1 (196 mg) was isolated and identified as ursolic by
spectral comparison and physical constants with literature
data.
1
2926, 1695, 1457, 1387. The specific rotation, the H NMR, and the
13C NMR were measured with methyl ester. Compound 1 was identified
as ursolic acid (3â-hydroxy-urs-12-en-28-oic acid) (20, 21).
Compound 1M. Compound 1M was a white crystal; mp 172.1-
173.0 °C. EI-MS (70 eV): m/z (rel int.) 470 ([M]+, 10), 452 (6), 410
(6), 262 (100), 203 (59), 133 (28), 119 (16), 105 (11). IR γmaxKBr (cm-1):
3525, 2926, 1725, 1456, 1386; [R]23d +49.8° (CHCl3; c 1.0). 1H NMR

2314 J. Agric. Food Chem., Vol. 53, No. 6, 2005
Miyazawa et al.
carboxyl group is an important factor for suppressive effect.
On the other hand, these compounds did not show the suppres-
sive effect against activated Trp-P-1 in the umu test. It may be
expected that the suppressive effect of Trp-P-1 be due to the
inhibition of metabolic activation by S9. Previously, ursolic acid
have been isolated from Eriobotrya japonica and Ligustrum
lucidum as an antimutagenic compound (28, 29). Ursolic acid
exhibited the potent antimutagenic activity against AfB1 and
benzo[a]pyrane using S. typhimurium TA100/TA98 Ames test.
However, structure-activity relationships attend esterification
for exhibition of suppression of SOS inducing activity, and the
metabolic activation of S9 was not investigated. In summary,
this research suggested that the suppressive compound in U.
sinensis was primarily ursolic acid, and free carboxyl group is
an important factor for activity.
Figure 2. Suppressive effects of Trp-P-1- and Act. Trp-P-1-induced SOS
response by compounds 1, 1M, 1A, and 1MA. Key:
Trp-P-1; ( ) effect of 1M on Trp-P-1; ( ) effect of 1A on Trp-P-1; (
effect of 1MA on Trp-P-1; ( ) effect of 1 on Act.Trp-P-1; ( ) effect of
1M on Act.Trp-P-1; ( ) effect of 1A on Act.Trp-P-1; ( ) effect of 1MA
on Act.Trp-P-1. Trp-P-1 (40 g/mL in DMSO) was added at 50 L. Act.Trp-
P-1 (10 g/mL in DMSO) was added at 100 L.
(O) effect of 1 on
0
]
4)
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b
9
[
2
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