366
Vol. 57, No. 4
ture was neutralized with Amberlite IRA-400 (OHꢂ form) and then the resin
was removed by filtration. After removal of the solvent from the filtrate
under reduced pressure, the residue was separated by Sep-Pak C18 cartridge
column (H2O→MeOH). The H2O-eluted fraction was subjected to HPLC
analysis under the following conditions: HPLC column, Kaseisorb LC NH2-
60-5, 4.6 mm i.d.ꢆ250 mm (Tokyo Kasei Co., Ltd., Tokyo, Japan); detec-
tion, optical rotation [Shodex OR-2 (Showa Denko Co., Ltd., Tokyo, Japan);
mobile phase, CH3CN–H2O (85 : 15, v/v); flow rate 0.8 ml/min]. Identifica-
tion of D-glucose from 1—4 present in the H2O-eluted fraction was carried
out by comparison of its retention time and optical rotation with that of an
authentic sample. tR: 13.9 min (positive optical rotation). On the other hand,
the MeOH-eluted fraction was purified by HPLC [CH3CN–1% aqueous
AcOH (40 : 60, v/v)] to furnish naringenin44) (2.0 mg, 73% from 1, 2.1 mg,
73% from 2, 1.3 mg, 72% from 3, and 1.3 mg, 72% from 4).
18) Yoshikawa M., Sugimoto S., Nakamura S., Matsuda H., Chem. Pharm.
Bull., 56, 1297—1303 (2008).
19) Xie Y., Morikawa T., Ninomiya K., Imura K., Muraoka O., Yuan D.,
Yoshikawa M., Chem. Pharm. Bull., 56, 1628—1631 (2008).
20) Nakamura S., Okazaki Y., Ninomiya K., Morikawa T., Matsuda H.,
Yoshikawa M., Chem. Pharm. Bull., 56, 1704—1709 (2008).
21) Yoshikawa M., Sugimoto S., Nakamura S., Matsuda H., Heterocycles,
in press.
22) Wang Y., Singh R., Lefkowitch J. H., Rigoli R. M., Czaja M. J., J. Biol.
Chem., 281, 15258—15267 (2006).
23) Tilg H., Day C. P., Nature Clincal Practice, Gastroenterology & Hepa-
tology, 4, 24—34 (2007).
24) Seronello S., Sheikh M. Y., Choi J., Free Radic. Biol. Med., 43, 869—
882 (2007).
25) Kouroku Y., Fujita E., Jimbo A., Mukasa T. Tsuru T., Momoi M. Y.,
Momoi T., Biochem. Biophys. Res. Commun., 270, 972—977 (2000).
26) Zapesochnaya G. G., Kurkin V. A., Braslavskii V. B., Filatova N. V.,
Chem. Nat. Comp., 38, 314—318 (2002).
27) Tyukavkina N. A., Demyanovich V. M., Kolesnik Y. A., Ruchkin V. E.,
Rulenko I. A., Litvinenko V. I., Khimiya Prirodnykh Soedinenii, 1989,
185—188 (1989).
28) Rahman W., Ishratullah K., Wagner H., Seligmann O., Chari V. M.,
Österdahl B.-G., Phytochemistry, 17, 1064—1065 (1978).
29) Hashimoto K., Katsuhara T., Niitsu K., Ikeya Y., Okada M., Mitsu-
hashi H., Phytochemistry, 31, 2477—2480 (1992).
30) Morimura K., Gatayama A., Tsukimata R., Matsunami K., Otsuka H.,
Hirata E., Shinzato T., Aramoto M., Takeda Y., Phytochemsitry, 67,
2681—2685 (2006).
Bioassay Method. Inhibitory Effect on TNF-a-Induced Cytotoxicity
in L929 Cells Inhibitory effect on TNF-a-induced cell death in L929 cells
was assayed by the method described in our previous report.19) Briefly, a sus-
pension of 2ꢆ104 cells [obtained from Dainippon Pharmaceutical (Osaka,
Japan)] in 100 ml of minimum essential medium Eagle supplemented with
1% non-essential amino acid solution (Invitrogen), fetal calf serum (FCS,
10%), penicillin G (100 units/ml), and streptomycin (100 mg/ml) was incu-
bated in a 96-well microplate. After 44 h of incubation in the medium con-
taining TNF-a (1 ng/ml) with or without a test sample, the viability of the
cells was assessed by the MTT colorimetric assay. Each test sample was dis-
solved in DMSO, and the solution was added to the medium (final DMSO
concentration was 0.5%).
Statistics Values were expressed as meansꢀS.E.M. One-way analysis of
variance (ANOVA) followed by Dunnett’s test was used for statistical analy-
sis. Probability (p) values less than 0.05 were considered significant.
31) Nørbœk R., Nielsen J. K., Kondo T., Phytochemistry, 51, 1139—1146
(1999).
32) Long C., Wang X., Yang Y., Cai S., Beijing Yike Daxue Xuebao, 32,
229—231, 257 (2000).
33) Redaelli C., Formentini L., Santaniello E., Phytochemistry, 19, 985—
986 (1980).
34) Ahmed A. A., Marby T. J., Matlin S. A., Phytochemistry, 28, 1751—
1753 (1989).
35) Kikuchi M., Matsuda N., J. Nat. Prod., 59, 314—315 (1996).
36) Veitch N. C., Grayer R. J., Irwin J. L., Takeda K., Phytochemistry, 48,
389—393 (1998).
Acknowledgements O. M., T. M., and K. N. were supported by a Grant-
in Aid for Scientific Research from ‘High-tech Research Center’ Project for
Private Universities: matching fund subsidy from The Ministry of Educa-
tion, Culture, Sports, Science and Technology of Japan (MEXT), 2007—
2011 and also supported by a Grant-in Aid for Scientific Research by Japan
Society for the Promotion of Science (JSPS). M. Y., H. M., and S. N. were
supported by the 21st COE Program, Academic Frontier Project, and a
Grant-in-Aid for Scientific Research from MEXT.
37) Markham K. R., Ternai B., Stanley R., Geiger H., Mabry T. J., Tetrahe-
dron, 34, 1389—1397 (1978).
38) Miyaichi Y., Kizu H., Tomimori T., Lin C.-C., Chem. Pharm. Bull., 37,
794—797 (1989).
39) Ulubelen A., Kerr K. M., Mabry T. J., Phytochemistry, 19, 1761—
1766 (1980).
40) Stepanova T. A., Glyzin V. I., Smirnova L. P., Khimiya Prirodnykh
Soedinenii, 1980, 723—724 (1980).
41) Tanaka M., Fujimori T., Uchida I., Yamaguchi S., Takeda K., Phyto-
chemistry, 56, 373—376 (2001).
42) Yoshida T., Saito T., Kadoya S., Chem. Pharm. Bull., 35, 97—107
(1987).
43) Karl C., Pedersen P. A., Müller G., Z. Naturforsch., 35c, 826—828
(1980).
44) Rutin (29), adenosine, and naringenin were identified by comparison
of their physical and spectral data with those of commercially obtained
samples.
References and Notes
1) Part XXVI.: Sugimoto S., Chi G., Kato Y., Nakamura S., Matsuda H.,
Yoshikawa M., Chem. Pharm. Bull., 57, 269—275 (2009).
2) Czinner E., Hagymási K., Blázovics A., Kéry Á, Szöke É, Lem-
berkovics É., J. Ethnopharmacol., 73, 437—443 (2000).
3) Vrkoc J., Dolejs L., Sedmera P., Vasicková S., Sorm F., Tetrahedron
Lett., 3, 247—250 (1971).
4) Mericli A. H., Damadyan B., Cubukcu B., Sci. Pharm., 54, 363—365
(1986).
5) Vrkoc J., Dolejs L., Budesínsky M., Phytochemistry, 14, 1383—1384
(1975).
6) Vrkoc J., Budesínsky M., Dolejs L., Vasicková S., Phytochemistry, 14,
1845—1848 (1975).
7) Yoshikawa M., Sugimoto S., Nakamura S., Matsuda H., Chem. Pharm.
Bull., 55, 571—576 (2007).
8) Yoshikawa M., Xu F., Morikawa T., Pongpiriyadacha Y., Nakamura S.,
Asao Y., Matsuda H., Chem. Pharm. Bull., 55, 308—316 (2007).
9) Nakamura S., Sugimoto S., Matsuda H., Yoshikawa M., Heterocycles,
71, 577—588 (2007).
10) Yoshikawa M., Nakamura S., Kato Y., Matsuhira K., Matsuda H.,
Chem. Pharm. Bull., 55, 598—605 (2007).
45) Barron D., Ibrahim R. K., Phytochemistry, 26, 1181—1184 (1987).
46) Asen S., Plimmer J. R., Phytochemistry, 11, 2601—2603 (1972).
47) Seabra R. M., Andrade P. B., Ferreres F., Moreira M. M., Phytochem-
istry, 45, 839—840 (1997).
48) Matsuda N., Sato H., Yaoita Y., Kikuchi M., Chem. Pharm. Bull., 44,
1122—1123 (1996).
49) Changzeng W., Zhongjian J., Phytochemistry, 45, 159—166 (1997).
50) Satake T., Coskun M., Hori K., Saiki Y., Tanker M., Phytochemistry,
30, 4191—4192 (1991).
51) Kuroyanagi M., Shiotsu M., Ebihara T., Kawai H., Ueno A.,
Fukushima S., Chem. Pharm. Bull., 34, 4012—4017 (1986).
52) Ghosal S., Singh S., Bhagat M. P., Kumar Y., Phytochemsitry, 21,
2943—2946 (1982).
53) Ohmura K., Miyase T., Ueno A., Phytochemistry, 28, 1919—1924
(1989).
11) Yoshikawa M., Morikawa T., Asao Y., Fujiwara E., Nakamura S.,
Matsuda H., Chem. Pharm. Bull., 55, 606—612 (2007).
12) Yoshikawa M., Sugimoto S., Nakmaura S., Sakumae H., Matsuda H.,
Chem. Pharm. Bull., 55, 1034—1038 (2007).
13) Nakamura S., Sugimoto S., Matsuda H., Yoshikawa M., Chem. Pharm.
Bull., 55, 1342—1348 (2007).
14) Yoshikawa M., Wang T., Sugimoto S., Nakamura S., Nagatomo A.,
Matsuda H., Harima S., Yakugaku Zasshi, 128, 141—151 (2008).
15) Yoshikawa M., Sugimoto S., Kato Y., Nakamura S., Wang T., Yama-
shita C., Matsuda H., Chem. Biodiversity, in press.
16) Morikawa T., Li X., Nishida E., Ito Y., Matsuda H., Nakamura S.,
Muraoka O., Yoshikawa M., J. Nat. Prod., 71, 828—835 (2008).
17) Yoshikawa M., Li X., Nishida E., Nakamura S., Matsuda H., Muraoka
O., Morikawa T., Chem. Pharm. Bull., 56, 559—568 (2008).
54) Li H., Nakashima T., Tanaka T., Zhang Y.-J., Yang C.-R., Kouno I., J.
Nat. Prod., 62, 75—78 (2008).
55) Kitajima J., Ishikawa T., Tanaka Y., Ono M., Ito Y., Nohara T., Chem.