988
Vol. 53, No. 8
7.5 mg),7) protosappanin B (5, 19.7 mg),18) and protosappanin C dimethyl ac-
etal (6, 8.6 mg).9)
for International Scientific Research (No. 16406002) from The Ministry of
Education, Culture, Sports, Science and Technology, Japan.
Fraction 6 (IC50, 19.6 mg/ml) was separated by silica gel comlumn chro-
matography with MeOH–CHCl3, followed by reversed-phase preparative
TLC with CH3CN : MeOH : H2Oꢃ1 : 1 : 3, to yield neoprotosappanin (1,
2.8 mg) and 5 (12.2 mg).
References
1) Oettl K., Reibnegger G., Biochim. Biophys. Acta, 1430, 387—395
(1999).
2) Ishibuchi S., Morimoto H., Oe T., Ikebe T., Inoue H., Fukunari A.,
Kamezawa M., Yamada T., Naka Y., Bioorg. Med. Chem. Lett., 11,
879—882 (2001).
3) Cos P., Ying L., Calomme M., Hu J. P., Cimanga K., Van Poel B.,
Pieters L., Vlietinck A. J., Berghe D. V., J. Nat. Prod., 61, 71—76
(1998).
4) Do T. L., “Vietnamese Medicinal Plants,” Medicine Publisher, Hanoi,
2001, p. 50.
Neoprotosappanin (1): Yellow amorphous solid; [a]D25 ꢂ239.0° (cꢃ0.30,
MeOH). IR (KBr) cmꢂ1: 3300, 1610, 1450, 1350. 1H- and 13C-NMR
(CD3OD, 400 MHz), see Table 1. HR-FAB-MS m/z: 571.1586 [MꢁH]ꢁ
(Calcd for C32H27O10: 571.1604).
Protosappanin E-2 (2): Yellow amorphous solid; [a]D20 ꢂ16.9° (cꢃ0.175,
MeOH). IR (KBr) cmꢂ1: 3300, 1640, 1600, 1450, 1380, 1270, 1180, 1070.
1H- and 13C-NMR (CD3OD, 400 MHz), see Table 2. HR-FAB-MS m/z:
587.1558 [MꢁH]ꢁ (Calcd for C32H27O11: 587.1553).
5) Nguyen M. T. T., Awale S., Tezuka Y., Tran Q. L., Watanabe H.,
Kadota S., Biol. Pharm. Bull., 27, 1414—1421 (2004).
6) Nguyen M. T. T., Awale S., Tezuka Y., Tran Q. L., Kadota S., Tetrahe-
dron Lett., 45, 8519—8522 (2004).
Protosappanin A Dimethyl Acetal (3): Light yellow amorphous solid; IR
(KBr) cmꢂ1
:
3300, 1600, 1445, 1350. 1H- and 13C-NMR (CD3OD,
400 MHz), see Table 2. HR-FAB-MS m/z: 341.1001 [MꢁNa]ꢁ (Calcd for
C17H18O6Na: 341.0996).
7) Nagai M., Nagumo S., Chem. Pharm. Bull., 38, 1490—1494 (1990).
8) Nagai M., Nagumo S., Heterocycles, 24, 601—605 (1986).
9) Nagai M., Nagumo S., Chem. Pharm. Bull., 35, 3002—3005 (1987).
10) Nagai M., Nagumo S., Lee S., Eguchi I., Kawai K., Chem. Pharm.
Bull., 34, 1—6 (1986).
11) Kim D. S., Baek N., Oh S. R., Jung K. Y., Lee I. S., Lee H., Phyto-
chemistry, 46, 177—178 (1997).
12) Namikoshi M., Saitoh T., Chem. Pharm. Bull., 35, 3597—3602
(1987).
13) Namikoshi M., Nakata H., Saitoh T., Phytochemistry, 26, 1831—1833
(1987).
14) Namikoshi M., Nakata H., Yamada H., Nagai M., Saitoh T., Chem.
Pharm. Bull., 35, 2761—2773 (1987).
15) Namikoshi M., Nakata H., Nuno M., Ozawa T., Saitoh T., Chem.
Pharm. Bull., 35, 3568—3575 (1987).
16) Saitoh T., Sakashita S., Nakata H., Shimokawa T., Kinjo J., Yamahara
J., Yamasaki M., Nohara T., Chem. Pharm. Bull., 34, 2506—2511
(1986).
Acid Treatment of 3 Compound 3 (3.0 mg) was treated with tri-
flouroacetic acid (50 ml) in THF (2 ml) at room temperature for 3 h, followed
by neutralization with sodium bicarbonate. The reaction mixture was ex-
tracted with ether and dried over anhydrous MgSO4. The crude product was
purified by preparative TLC with MeOH–CHCl3 (1 : 9) to give 7.
XO Inhibitory Assay The XO inhibitory activity was assayed spec-
trophotometrically at 290 nm under aerobic conditions by using 96-well
plates as described previously.6) XO inhibitory activity was expressed as the
percentage inhibition of XO in the above assay system, calculated as
(1ꢂB/A)ꢄ100, where A and B are the activities of the enzyme without and
with test material. IC50 values were calculated from the mean values of data
from four determinations.
Lineweaver–Burk Plots To determine the mode of inhibition by active
compounds from the plant, Lineweaver–Burk plot analysis was performed.
This kinetics study was carried out in the absence and presence of active
compounds with varying concentrations of xanthine as substrate. The initial
rates were determined on the basis of the rate of increase in absorbance at
290 nm between 0.5 and 3 min. The data represent the meanꢆS.D. of four
determinations. The inhibition constants (Ki) were determined from the
slopes of the Lineweaver–Burk plot for competitive inhibition, and intercept
on vertical axis for noncompetitive inhibition.
17) Nagai M., Nagumo S., Eguchi I., Lee S., Suzuki T., Yakugaku Zasshi,
104, 935—938 (1984).
18) Craig J. C., Naik A. R., Pratt R., Johnson E., J. Org. Chem., 30,
1573—1576 (1965).
Acknowledgement This work was supported in part by a Grant-in-Aid