1160 Journal of Natural Products, 2009, Vol. 72, No. 6
Matsuo et al.
atmosphere. After cooling, the reaction mixture was diluted with H2O
(6 mL) and extracted with CHCl3 (5 mL × 3). TLC analysis of the
CHCl3-soluble fraction showed that the labile aglycone decomposed
under acidic conditions. The H2O-soluble fraction was neutralized by
passing through an Amberlite IRA-96SB (Organo, Tokyo, Japan)
column and then passed through a Sep-Pak C18 cartridge, using 40%
MeOH to give a sugar fraction (2.3 mg). The sugar fraction analyzed
by HPLC under the same conditions as in the case of 3 showed
L-arabinose and D-glucose.
described for 3 to yield 8a (4.4 mg) and a sugar fraction (0.2 mg).
HPLC analysis of this sugar fraction under the same conditions as in
the case of 3 showed the presence of L-arabinose and D-glucose.
Cytotoxic Assay. HL-60 cells were maintained in an RPMI 1640
medium containing 10% FBS supplemented with L-glutamine, 100
units/mL of penicillin G, and 100 µg/mL of streptomycin sulfate. The
leukemia cells were washed and resuspended in this medium to 4 ×
104 cells/mL, and 196 µL of this cell suspension was placed in each
well of a 96-well flat-bottom plate. The cells were incubated in 5%
CO2/air for 24 h at 37 °C. After incubation, 4 µL of an EtOH-H2O
(1:1) solution containing the sample was added to give the final
concentrations of 0.1-20 µg/mL, and 4 µL of EtOH-H2O (1:1) was
added into the control wells. The cells were further incubated for 72 h
in the presence of each agent, and then the cell growth was evaluated
by a modified MTT reduction assay. Briefly, after terminating the cell
culture, 10 µL of 5 mg/mL of MTT in PBS was added to every well,
and the plate was reincubated in 5% CO2/air for 4 h at 37 °C. The
plate was then centrifuged at 1500g for 5 min to precipitate the cells
and MTT formazan. An aliquot of 150 µL of the supernatant was
removed from each well, and 175 µL of DMSO was added to dissolve
the MTT formazan crystals. The plate was mixed on a microshaker
for 10 min and then read on a microplate reader at 550 nm.
21
Compound 7: amorphous solid; [R]D +4.0 (c 0.10, MeOH); IR
(film) νmax 3376 (OH), 2925, 2871, and 2855 (CH), 1744 (CdO) cm-1
;
1H NMR (C5D5N, 500 MHz) δ 5.19 (1H, d, J ) 7.7 Hz, Ara-1), 4.98
(1H, d, J ) 5.7 Hz, Glc-1), 3.23 (1H, dd, J ) 11.8, 4.5 Hz, H-3), 1.26
(3H, s, Me-30), 1.24 (1H, t-like, J ) 12.9 Hz, H-13), 1.22 (3H, s,
Me-23), 1.04 (1H, dd, J ) 11.9, 5.5 Hz, H-18), 1.03 (3H, s, Me-24),
0.89 (3H, s, Me-27), 0.88 (3H, d, J ) 5.9 Hz, Me-29), 0.87 (3H, s,
Me-26), 0.76 (3H, s, Me-25); 13C NMR, see Table 1; HRESITOFMS
m/z 751.4648 [M + H]+ (calcd. for C41H67O12, 751.4633).
Enzymatic Hydrolysis of 7. A solution of 7 (4.1 mg) with
naringinase (35.2 mg) was subjected to enzymatic hydrolysis as
described for 3 to yield 7a (2.6 mg) and a sugar fraction (0.6 mg).
HPLC analysis showed the presence of L-arabinose and D-glucose.
25
Compound 8: amorphous solid; [R]D +15.6 (c 0.10, MeOH); IR
(film) νmax 3385 (OH), 2928 (CH), 1696 (CdO) cm-1 1H NMR
;
Acknowledgment. We are grateful to Dr. Y. Shida and Mr. H.
Fukaya, Tokyo University of Pharmacy and Life Sciences, for the mass
spectra and elemental analyses.
(C5D5N, 500 MHz) δ 5.61 (1H, br s, H-12), 5.21 (1H, br s, H-16),
5.17 (1H, d, J ) 7.8 Hz, Glc-1), 5.16 (1H, d, J ) 5.9 Hz, Ara-1), 4.20
(1H, d, J ) 11.0 Hz, H-23a), 4.15 (1H, dd, J ) 11.7, 4.5 Hz, H-3),
3.78 (1H, d, J ) 11.0 Hz, H-23b), 3.57 (1H, dd, J ) 14.0, 4.0 Hz,
H-18), 1.75 (3H, s, Me-27), 1.14 (3H, s, Me-30), 1.02 (3H, s, Me-26),
1.01 (3H, s, Me-29), 0.99 (3H, s, Me-24), 0.93 (3H, s, Me-25); 13C
NMR, see Table 1; HRESITOFMS m/z 783.4514 [M + H]+ (calcd.
for C41H67O14, 783.4531).
Supporting Information Available: 1H and 13C NMR data for 1-4,
4a, 5, 5a, and 6-10. This material is available free of charge via the
References and Notes
Enzymatic Hydrolysis of 8. A solution of 8 (10.5 mg) with
naringinase (70.0 mg) was subjected to enzymatic hydrolysis as
described for 3 to yield 8a (0.7 mg) and a sugar fraction (0.7 mg).
HPLC analysis of this sugar fraction showed the presence of L-arabinose
and D-glucose.
(1) Chevallier, A. M. The Encyclopedia of Medicinal Plants; Dorling
Kindersley Limited: London, 1996; p 73.
(2) Ali, Z.; Khan, I. A. Phytochemistry 2008, 69, 1037–1042.
(3) Betz, J. M.; Andrzejewski, D.; Troy, A.; Casey, R. E.; Obermeyer,
W. R.; Page, S. W.; Woldemariam, T. Z. Phytochem. Anal. 1998, 9,
232–236.
(4) Ganzera, M.; Dharmaratne, H. R. W.; Nanayakkara, N. P. D.; Khan,
I. A. Phytochem. Anal. 2003, 14, 1–7.
(5) Power, F. B.; Salway, A. H. J. Chem. Soc. 1913, 103, 191–209.
(6) Jhoo, J. W.; Sang, S.; He, K.; Cheng, X.; Zhu, N.; Stark, R. E.; Zheng,
Q. Y.; Rosen, R. T.; Ho, C. T. J. Agric. Food Chem. 2001, 49, 5969–
5974.
(7) Kitanaka, S.; Yasuda, I.; Kashiwada, Y.; Hu, C. Q.; Bastow, K. F.;
Bori, I. D.; Lee, K. H. J. Nat. Prod. 1995, 58, 1647–1654.
(8) Liao, X.; Li, B. G.; Wang, M. K.; Ding, L. S.; Pan, Y. J.; Chen, Y. Z.
Gaodeng Xuexiao Huaxue Xuebao 2001, 22, 1338–1341.
(9) Wu, F.; Zhu, Z. Huaxue Xuebao 1984, 42, 253–258.
(10) Aoki, T.; Suga, T. Phytochemistry 1978, 17, 771–773.
(11) Sahu, N. P.; Mahato, S. B. Phytochemistry 1994, 37, 1425–1427.
(12) Joshi, B. S.; Moore, K. M.; Pelletier, S. W.; Puar, M. S.; Pramanik,
B. N. J. Nat. Prod. 1992, 55, 1468–1476.
(13) Maeda, C.; Ohtani, K.; Kasai, R.; Yamasaki, K.; Duc, N. M.; Nham,
N. T.; Cu, N. K. Q. Phytochemistry 1994, 37, 1131–1137.
(14) Aoki, T.; Shido, K.; Takahashi, Y.; Suga, T. Phytochemistry 1981,
20, 1681–1686.
(15) Miyakoshi, M.; Shirasuna, K.; Hirai, Y.; Shingu, K.; Isoda, S.; Shoji,
J.; Ida, Y.; Shimizu, T. J. Nat. Prod. 1999, 62, 445–448.
(16) Ikuta, A.; Kamiya, K.; Satake, T.; Saiki, Y. Phytochemistry 1995, 38,
1203–1207.
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D.; Yoshikawa, M. Chem. Pharm. Bull. 2008, 56, 1628–1631.
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26
Compound 9: amorphous solid; [R]D +2.0 (c 0.86, MeOH); IR
(film) νmax 3428 (OH), 2942 (CH), 1643 (CdO) cm-1 1H NMR
;
(C5D5N, 500 MHz) δ 5.62 (1H, br s, W1/2 ) 12.2 Hz, H-12), 5.29 (1H,
br s, W1/2 ) 16.2 Hz, H-16), 4.30 (1H, overlapping, H-3), 4.29 (1H, d,
J ) 12.8 Hz, H-23a), 3.69 (1H, d, J ) 12.8 Hz, H-23b), 3.53 (1H, dd,
J ) 14.2, 4.2 Hz, H-18), 1.78 (3H, s, Me-27), 1.17 (3H, s, Me-26),
1.04 (3H, s, Me-30), 0.98 (3H, s, Me-29), 1.01 (3H, s, Me-25), 0.94
(3H, s, Me-24), signals for the sugar moieties, see Table 2; 13C NMR,
see Table 1; HRESITOFMS m/z: 783.4579 [M + H]+ (calcd for
C41H67O14, 783.4531).
Acid Hydrolysis of 9. A solution of 9 (5.0 mg) was subjected to
acid hydrolysis as described for 6 to give 8a (3.6 mg) and a sugar
fraction (2.2 mg). HPLC analysis of the sugar fraction under the same
conditions as those used for 3 showed the presence of L-arabinose and
D-glucose.
26
Compound 10: amorphous solid; [R]D -46.0 (c 0.10, MeOH);
IR (film) νmax 3384 (OH), 2924 and 2857 (CH), 1733 and 1645 (CdO)
cm-1; 1H NMR (C5D5N, 500 MHz) δ 5.58 (1H, br s, H-12), 5.27 (1H,
br s, H-16), 4.26 (1H, d, J ) 11.0 Hz, H-23a), 4.22 (1H, br d, J ) 5.9
Hz, H-3), 3.65 (1H, d, J ) 11.0 Hz, H-23b), 3.47 (1H, dd, J ) 14.3,
4.3 Hz, H-18), 1.74 (3H, s, Me-27), 1.13 (3H, s, Me-26), 1.00 (3H, s,
Me-30), 0.99 (3H, s, Me-25), 0.92 (3H, s, Me-29), 0.91 (3H, s, Me-
24), signals for the sugar moieties, see Table 2; 13C NMR, see Table
1; HRESITOFMS m/z 967.4857 [M + Na]+ (calcd for C47H76O19Na,
967.4879).
Enzymatic Hydrolysis of 10. A solution of 10 (5.0 mg) with
naringinase (20.0 mg) was subjected to enzymatic hydrolysis as
NP900164B