Antimutagenic activity of flavonoids from Sozuku

Article abstract of DOI:10.1080/14786419.2017.1408104

Pinocembrin (1) and cardamonin (2) from Sozuku showed a suppressive effect on umu gene expression of SOS response in Salmonella typhimurium TA1535/pSK1002 against the mutagen furylfuramide. Compounds 1 and 2 suppressed 52% and 36% of SOS-inducing activity at a concentration of 0.20?μmol/mL. The ID₅₀ value of 1 was 0.18?μmol/mL. These compounds showed the suppression of 2-amino-3,4-dimethylimidazo-[4,5-f]quinolone (MeIQ) and UV irradiation-induced SOS response. Pinostrobin (3) and 5,7-dimethoxyflavanone (4), methyl ethers of 1, showed similar activity to 1 against MeIQ-induced SOS response, but that of furylfuramide and UV irradiation were decreased. On the other hand, compounds 1–4 did not show the suppression of activated MeIQ-induced SOS response. Furthermore, compounds 1–4 showed potent antimutagenic activity against MeIQ mutagenesis in Ames test using the S. typhimurium TA100 and TA98 strains.

Full text of DOI:10.1080/14786419.2017.1408104

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: http://www.tandfonline.com/loi/gnpl20
Antimutagenic activity of flavonoids from Sozuku
Yoshiharu Okuno, Shinsuke Marumoto & Mitsuo Miyazawa
To cite this article: Yoshiharu Okuno, Shinsuke Marumoto & Mitsuo Miyazawa (2017):
Antimutagenic activity of flavonoids from Sozuku, Natural Product Research, DOI:
10.1080/14786419.2017.1408104
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Date: 02 January 2018, At: 13:44

Natural Product research, 2017
https://doi.org/10.1080/14786419.2017.1408104
SHORT COMMUNICATION
Antimutagenic activity of flavonoids from Sozuku
Yoshiharu Okunoa, Shinsuke Marumotob and Mitsuo Miyazawac,d,e
adepartment of applied chemistry and Biochemistry, National Institute of technology, Wakayama collage,
Gobo-shi, Japan; bJoint research centre, Kindai university (Former name: Kinki university), higashiosaka-
c
shi, Japan; Graduate school of Materials science, Nara Institute of science and technology, Ikoma, Japan;
ddepartment of applied chemistry, Kindai university (Former name: Kinki university), higashiosaka-shi,
Japan; eGraduate school of science, osaka city university, osaka-shi, Japan
ABSTRACT
ARTICLE HISTORY
received 15 June 2017
accepted 13 November 2017
Pinocembrin(1)andcardamonin(2)fromSozukushowedasuppressive
effect on umu gene expression of SOS response in Salmonella
typhimurium TA1535/pSK1002 against the mutagen furylfuramide.
Compounds 1 and 2 suppressed 52% and 36% of SOS-inducing
activity at a concentration of 0.20 μmol/mL. The ID50 value of 1 was
0.18 μmol/mL. These compounds showed the suppression of 2-amino-
3,4-dimethylimidazo-[4,5-f]quinolone (MeIQ) and UV irradiation-
induced SOS response. Pinostrobin (3) and 5,7-dimethoxyflavanone
(4), methyl ethers of 1, showed similar activity to 1 against MeIQ-
induced SOS response, but that of furylfuramide and UV irradiation
were decreased. On the other hand, compounds 1–4 did not show the
suppression of activated MeIQ-induced SOS response. Furthermore,
compounds 1–4 showed potent antimutagenic activity against MeIQ
mutagenesis in Ames test using the S. typhimurium TA100 and TA98
strains.
KEYWORDS
sozuku; Alpinia katsumadae
hayata; pinocembrin;
cardamonin; antimutagenic
activity; umu test; ames test
1. Introduction
Human epidemiology and animal studies have indicated that cancer risk may be modified
by changes in dietary habits or dietary components. Human ingest large numbers of naturally
occurring antimutagens and anticarcinogens with food. These antimutagens and
CONTACT Mitsuo Miyazawa
miyazawa@ms.naist.jp
supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2017.1408104.
© 2017 Informa uK limited, trading as taylor & Francis Group

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Y. OKUNO ET AL.
anticarcinogens may inhibit one or more stages of the carcinogenic process and prevent or
delay the formation of cancer. Antimutagenic compounds have been found in several crude
drugs, and some of these structures have been elucidated (Kim et al. 1991; Zheng et al. 1992).
The SOS response is one of the DNA repair response to DNA damage induced by environ-
mental factors and so on. The umu test system was developed to evaluate the genotoxic
activities of a wide variety of environmental carcinogens and mutagens, using the SOS
response to detect DNA damage (Oda et al. 1985). 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 (Reifferscheid and Heil 1996).
Sozuku is Chinese crude drugs base on dried seed of Alpinia katsumadae HAYATA (syno-
nym: Alpinia hainanensis K. Schum) (Zingiberaceae), and use as an aromatic stomachache
and antiemetic medicine in China. This plant is native to Hainan Island in Southern China,
but is widely ranged in shaded woodland in Hong Kong. Previous investigations of this plant
have reported a variety of diarylheptanoids, chalcones and flavonoids, monoterpenes and
sesquiterpenes (Yang et al. 1999; Hahm et al. 2003). Hahm et al. (2003) reported that 7,8-dihy-
droxyflavanone showed inhibitory effect on the Jun-Fos DNA complex formation. However,
the compounds responsible for antimutagenicity from Sozuku has not been reported. In
our search for new naturally occurring antimutagenic compounds in plants which have a
history of safe use as Chinese crude drugs (Miyazawa et al. 2000), we found that the methanol
extract of Sozuku exhibited a suppression of the SOS-inducing activity of furylfuramide. In
this paper, we report on the isolation and identification of the antimutagenic compounds
in Sozuku.
2. Results and discussions
The methanol extract from Sozuku was fractionated to search for the suppressive compound
using the umu test as a guide. The chloroform fraction, partitioned from MeOH extract,
showed a suppressive effect on umu gene expression of the SOS responses in S. typhimurium
TA1535/pSK1002 against furylfuramide. The chloroform fraction was repeatedly fractionated
by SiO2 column chromatography. Active compounds 1 and 2 were isolated and identified
as pinocembrin and cardamonin by comparison of spectra (1H-NMR, 13C-NMR, EI-MS and IR)
with literature data (Jaipetch et al. 1982; Ichino et al. 1988; Fukui et al. 1988; Liu et al. 1992;
Ngo et al. 1998), respectively (Figure 1). The suppressive effects of compounds 1, 2 and
methylated compounds pinostrobin (3) and 5,7-dimethoxyflavanones (4), obtained by
Figure 1. structures of compounds 1–4.

NATURAL PRODUCT RESEARCH
3
methylation of 1, were determined in the umu test. Compounds 1 and 2 suppressed 53%
and 36% of the furylfuramide-induced SOS response at concentrations of 0.2 μmol/mL, but
3 and 4 did not (Figure S1). The ID50 value of 1 was 0.18 μmol/mL. These compounds were
also assayed with MeIQ, which require the activation by liver-metabolising enzyme mixture
(S9). Compounds 1–4 suppressed more than 75% of the MeIQ-induced SOS response at
concentrations of 0.02 μmol/mL, and the ID50 values were 0.006, 0.003, 0.004 and 0.003 μmol/
mL, respectively (Figure S1). However, these compounds did not show suppressive effects
on activated MeIQ-induced SOS response (Figure S1). Whereas, 1 and 2 suppressed 70% and
50% of the UV irradiation-induced SOS response at concentrations of 0.2 μmol/mL, the ID50
values were 0.11 and 0.22 μmol/mol, respectively (Figure S2). The difference in structure
between active compound 1 and negative compounds 3 and 4 is hydroxyl group at C-7
position. Therefore, it is indicated that a hydroxyl group at the C-7 position is important
factor for suppressing furylfuramide and UV-irradiation-induced SOS response. The modes
of action of antimutagens are mainly divided into desmutagen and bioantimutagen. The
former inactivates or destroys mutagens directly or indirectly out of cell, and the latter sup-
presses the process of mutagenesis itself inside cells or act on the DNA repair system. From
these results, 1 and 2 may have potency as bioantimutagens. However, the mechanism of
bioantimutagen of these compounds requires more study.
Antimutagenic activity of 1–4 against furylfuramide, MeIQ and activated MeIQ were
demonstrated by the Ames test using S. typhimurium TA100 and TA98 (Figure S3 and S4).
Compound 1 suppressed only 53% of the mutagenicity of furylfuramide at 2.0 μmol/plate
in TA100 strain, and ID50 value was 0.19 μmol/plate. While, 1–4 suppressed more than 90%
of the mutagenicity of MeIQ at 0.2 μmol/plate in both strains. The ID50 values of 1–4 were
0.051, 0.049, 0.038 and 0.024 μmol/plate in TA100 strain and 0.048, 0.045, 0.034 and
0.026 μmol/plate in TA98 strain. The suppressive effects of flavanones increased with the
methylation of hydroxyl group. However, these compounds did not inhibit the mutagenicity
of activated MeIQ. These results were similar to the umu test. MeIQ is one of the heterocyclic
amines, and requires the metabolic activation by P450 monooxygenase system in rat liver
microsomes (S9) for mutagenicity. Trakoontivakorn et al. (2001) reported the antimutagenic
activity of 1–3, isolated from Boesenbergia pandurate, against heterocyclic amines, Trp-P-1,
Trp-P-2 and PhIP, in S. typhimuriumn TA98, and their activity is due to the inhibition of
N-hydroxylation of heterocyclic amines by S9. In this presence study, 1–4 exhibited a potent
inhibition against MeIQ-induced mutagenesis, whereas they did not inhibit that of activated
MeIQ in the both test. Therefore, it is suggested that antimutagenic activity of 1–4 against
MeIQ-induced mutagenesis due to the inhibition of N-hydroxylation by P450 monooxygen-
ase systems in S9.
3. Conclusion
The antimutagenic compounds in Sozuku were clearly pinocembrin (1) and cardamonin (2).
These compounds showed suppressive effect and antimutagenic activity against chemical
mutagens-induced SOS response and mutagenicity. Especially, these compounds had potent
activity against MeIQ-induced SOS response and mutagenicity, and their activity was thought
to due to inhibit the N-hydroxylation of MeIQ by S9. Further, in the comparison of structure
in compounds 1, 3 and 4, it was suggested that the free hydroxyl group on C-7 is very
important factor for suppressing the furylfuramide and UV irradiation-induced SOS response.

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Y. OKUNO ET AL.
Disclosure statement
No potential conflict of interest was reported by the authors.
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