November 2011
mode) m/z: 567.2440 [MꢀH] (Calcd for C H O , 567.2442).
1429
ꢀ
3) Kawasaki T., Okabe H., Nakatsuka I., Chem. Pharm. Bull., 19, 1144—
1149 (1971).
4) Ono M., Noda N., Kawasaki T., Miyahara I., Chem. Pharm. Bull., 38,
1892—1897 (1990).
5) Yokota T., Takahashi N., Murofushi N., Tamura S., Tetrahedron Lett.,
10, 2081—2084 (1969).
6) Yokota T., Murofushi N., Takahashi N., Tamura S., Agric. Biol. Chem.,
35, 573—582 (1971).
2
8
39 12
2
5
Pharbiniloside (3): Colorless gum; [a]D ꢃ30.6 (cꢅ0.15, MeOH); UV
(
3
(
MeOH) lmax (log e) 220 (3.24), 285 (3.98), 314 (3.76) nm; IR (KBr) n
max
ꢃ1
1
360, 2948, 2843, 1720, 1629, 1595, 1452, 1378, 1273, 1033, 669 cm ; H-
13
500 MHz) and C- (125 MHz) NMR data, see Table 2; ESI-MS (positive-
ꢀ
ion mode) m/z: 771 [MꢀH] . HR-ESI-MS (positive-ion mode) m/z:
ꢀ
7
71.2716 [MꢀH] (Calcd for C H O , 771.2712).
3
5
47 19
Acid Hydrolysis of 1—3 and Sugar Analysis Solutions of 1 (3 mg)
and 2 (2 mg) in 2 N HCl were heated (90 °C) for 2 h. After cooling, each re-
7) Saito N., Cheng J., Ichimura M., Yokoi M., Abe Y., Honda T., Phyto-
chemistry, 35, 687—691 (1994).
action mixture was diluted with H O and extracted with CHCl . The CHCl
2
3
3
layer in each case was evaporated to dryness, and the residue was individu-
ally chromatographed by a silica gel Waters Sep-Pak Vac 6 cc (CHCl3/
8) Saito N., Lu T. S., Yokoi M., Shigihara A., Honda T., Phytochemistry,
33, 245—247 (1993).
MeOH, 30 : 1) to give each aglycone part, (ꢃ)-syringaresinol (1a) and rel-
9) Saito N., Tatsuzawa F., Kasahara K., Yokoi M., Iida S., Shigihara A.,
Honda T., Phytochemistry, 41, 1607—1611 (1996).
10) Yokota T., Yamazaki S., Takahashi N., Iitaka Y., Tetrahedron Lett., 15,
2957—2960 (1974).
11) Jung Y., Ha H., Lee H. Y., Kim C., Lee J. H., Bae K. H., Kim J. S.,
Kang S. S., Chem. Pharm. Bull., 56, 203—206 (2008).
12) Ko S. G., Koh S. H., Jun C. Y., Nam C. G., Bae H. S., Shin M. K.,
Biol. Pharm. Bull., 27, 1604—1610 (2004).
13) Koo J. C., Lee S. Y., Chun H. J., Cheong Y. H., Choi J. S., Kawabata
S., Miyagi M., Tsunasawa S., Ha K. S., Bae D. W., Han C. D., Lee B.
L., Cho M. J., Biochim. Biophys. Acta, 1382, 80—90 (1998).
14) Kim K. H., Jin M. R., Choi S. Z., Son M. W., Lee K. R., Heterocycles,
75, 1447—1455 (2008).
15) Kim K. H., Choi S. U., Lee K. R., J. Nat. Prod., 72, 1121—1127
(2009).
16) Shahata A. A., Abdel-Azimb N. S., Pietersa L., Vlietincka A. J., Fi-
toterapia, 75, 771—773 (2004).
17) Cavalcante S. H., Yoshida M., Gottlieb O. R., Phytochemistry, 24,
1051—1055 (1985).
18) Huo C., Liang H., Zhao Y., Wang B., Zhang Q., Phytochemistry, 69,
788—795 (2008).
7
(
7S,8R)-D -4,7-dihydroxy-3,5,3ꢄ,5ꢄ-tetramethoxy-8-O-4ꢄ-neolignan (2a),
1
which was identified by comparison with H-NMR, MS, and [a]D in the
literature.
3
1
6,17)
2a: CD (MeOH) lmax [q]237 ꢃ6120, [q]275 ꢃ670. Compound
(3 mg) was hydrolyzed by 1 N HCl (dioxane/H O, 1 : 1, 5 ml) under reflux
2
conditions for 3 h. After cooling, the reaction mixture was diluted with H O
2
and extracted with CHCl . A sample of the aqueous layer from each (1—3)
3
was neutralized by passage through an Amberlite IRA-67 column and was
repeatedly evaporated under reduced pressure to give each sugar fraction.
The sugars in the fraction were analyzed by silica gel TLC by comparison
with authentic samples. The solvent system was CHCl /MeOH/
3
H O (8 : 5 : 1). Spots were visualized by spraying with 95% EtOH/H SO /
2
2
4
anisaldehyde (9 : 0.5 : 0.5), then heated at 120 °C for 3 min. The Rf of glu-
cose and apiose were 0.30 and 0.45, respectively for sugars of 1—3. For GC
analysis, each sugar fraction was dissolved in anhydrous pyridine (100 ml),
and 0.1 M L-cysteine methyl ester hydrochloride in anhydrous pyridine
2
0,21)
(
1
200 ml) was added.
The mixture was stirred at 60 °C for 1 h. Then
50 ml of HMDS/TMCS (hexamethyldisilazane/trimethychlorosilane/pyri-
dine, 3 : 1 : 10) was added, and the mixture was stirred at 60 °C for another
0 min. The precipitate was centrifuged off, and the supernatant was concen-
3
trated under an N stream. The residue was partitioned between n-hexane
2
and H O (0.1 ml each), and the hexane layer (1 ml) was analyzed by GC ex-
19) Sugiyama M., Kikuchi M., Phytochemistry, 32, 1553—1555 (1993).
20) Bognar R., Somogyi L., Gyorgydeak Z., Liebigs Ann. Chem., 738,
68—78 (1970).
2
periment. D-Glucose and D-apiose were detected by co-injection of the hy-
drolysate with standard silylated samples (D-glucose: 11.38 min; L-glucose:
1
2.62 min; D-apiose: 5.08; L-apiose: 5.65). The retention times of sugars ob-
21) Hara S., Okabe H., Mihashi K., Chem. Pharm. Bull., 35, 501—506
(1987).
tained by acid hydrolysis were D-glucose (11.36 min) for 1, D-glucose
(
11.32 min) for 2, and D-glucose (11.41 min) and D-apiose (5.05 min) for 3.
Cytotoxicity Assay A sulforhodamine B (SRB) bioassay was used to
22) Vermes B., Seligmann O., Wagner H., Phytochemistry, 30, 3087—
3089 (1991).
determine the cytotoxicity of each compound isolated against four cultured
23) Sugiyama M., Kikuchi M., Chem. Pharm. Bull., 39, 483—485 (1991).
34)
human tumor cell lines. The assays were performed at the Korea Research 24) Warashina T., Nagatani Y., Noro T., Phytochemistry, 66, 589—597
Institute of Chemical Technology. The cell lines used were A549 (non-small
(2005).
cell lung carcinoma), SK-OV-3 (ovary malignant ascites), SK-MEL-2 (skin 25) Kisiel W., Zielinska K., Fitoterapia, 71, 86—87 (2000).
melanoma), and HCT-15 (colon adenocarcinoma). Doxorubicin was used as
a positive control. The cytotoxicities of doxorubicin against the A549, SK-
26) Sridevi K. V., Venkatesham U., VijenderReddy A., Venkateswarlu Y.,
Biochem. Syst. Ecol., 31, 335—337 (2003).
OV-3, SK-MEL-2, and HCT-15 cell lines were IC50 0.010, 0.001, 0.001, and 27) Dawn M., Edwin C. W., Burt Z., Phytochemistry, 8, 393—395 (1969).
1
3
1
0
.028 mM, respectively.
Measurement of NO Production and Cell Viability Inhibition of NO
28) Pouchert C. J., Behnke J., “The Aldrich Library of C- and H-FTNMR
Spectra,” Aldrich Chemical Co., St. Louis, 1992.
production was evaluated in lipopolysaccharide (LPS)-activated murine mi-
croglia BV-2 cells. Cells were stimulated with 100 ng/ml of LPS in the pres-
ence or absence of samples for 24 h. Nitrite in the culture media, a soluble
29) Makropoulou M., Christakopoulos P., Tsitsimpikou C., Kekos D.,
Kolisis F. N., Macris B. J., Int. J. Biol. Macromol., 22, 97—101 (1998).
30) Kim K. H., Lee K. H., Choi S. U., Kim Y. H., Lee K. R., Arch. Pharm.
Res., 31, 983—988 (2008).
31) Park H. B., Lee K. H., Kim K. H., Lee I. K., Noh H. J., Choi S. U., Lee
K. R., Nat. Prod. Sci., 15, 17—21 (2009).
35)
oxidation product of NO, was determined using the Griess reaction. Cell
viability was measured using a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenylte-
37)
G
trazolium bromide (MTT) assay. N -monomethyl-L-arginine (L-NMMA,
Sigma), a NOS inhibitor, was tested as a positive control.
32) Ahmad I., Lie Ken Jie M. S. F., Ind. Eng. Chem. Res., 47, 2091—2095
(2008).
Acknowledgments This work was supported by Grant No. PF 2-1 of 33) Sakakibara J., Kaiya T., Fukuda H., Ohki T., Phytochemistry, 22,
the Plant Diversity Research Center from the Ministry of Science and Tech-
nology in Korea. We thank Drs. E. J. Bang, S. G. Kim, and J. J. Seo at the
Korea Basic Science Institute for their assistance with the NMR spectro-
scopic and mass spectrometric measurements.
2553—2555 (1983).
34) Skehan P., Storeng R., Scudiero D., Monks A., McMahon J., Vistica
D., Warren J. T., Bokesch H., Kenney S., Boyd M. R., J. Natl. Cancer
Inst., 82, 1107—1112 (1990).
3
5) Kim K. H., Moon E., Choi S. U., Kim S. Y., Lee K. R., Bioorg. Med.
Chem. Lett., 21, 2270—2273 (2011).
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