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4.2. Cultivation of X. polymorpha NBRC 33288, extraction and isolation
buffer (1.0 ml, 100 ll, pH 5.0). After stirring at 37 °C for 24 h,
of 1–3
the reaction mixture was extracted with EtOAc, and then evapo-
rated to dryness to afford the aglycone 1a (6.1 mg). The aqueous
solubles were also purified by silica gel CC to give mannose
(5.4 mg in 1), whose absolute configuration was determined to
be D by optical rotation analysis. The specific rotation values of
the mannose isolated in this study and an authentic sample were
The producing strain X. polymorpha NBRC 33288 was purchased
from the biological resource center, National Institute of Technology
and Evaluation, Chiba, Japan, and was cultivated on sterilized unpol-
ished rice (20 g/Petri dish ꢁ 55: total 1100 g) at 25 °C for 4 weeks.
The mouldy unpolished rice was extracted with MeOH (2 l) at room
temperaturefor1weekandtheMeOHextractwasconcentrated.The
resulting concentrate (20.4 g) was then partitioned into n-hexane
and EtOAc (1 l of each solvents) layers. Purification of the EtOAc sol-
ubles (5.2 g) was guided by the intense blue characteristic coloration
generated with vanillin-sulfuric acid solution on TLC plates of the
fraction separated by silica gel CC using a gradient of n-hexane–
EtOAc (100:0–0:100) and EtOAc–MeOH (50:50 and 0:100) to give
thirteen fractions (Fr. 1-1 to 1-13). Fr. 13 (EtOAc:MeOH = 0:100,
220 mg) was subjected to ODS CC by eluting with H2O and an
increasing ratio of MeOH (H2O–MeOH, 100:0, v/v to 0:100, v/v) to af-
fordeleven fractions(Fr. 2-1to2-11). Fr. 7 (19 mg) wasapplied to sil-
ica gel flash CC using a mixture of CHCl3–MeOH (80:20) as eluant to
afford compound 2 (3.3 mg) and compound 3 (11.0 mg). Fr. 9
(67 mg) was also subjected to silica gel flash CC CHCl3–MeOH
(80:20) as eluant to afford compound 1 (29 mg).
as follows: ½a 2D0
ꢂ
+ 13.0ꢀ(c 0.1, H2O, measured after 24 h dissolution
in H2O) of 1, indicating the presence of
D
-mannose (½a 2D0
ꢂ
+ 12.4ꢀ(c
4.0, H2O, measured after 24 h dissolving in H2O) in authentic
sample).
1a: FABMS m/z: 321 [M+H+]. 1H NMR dH (400 MHz, C5D5N):
1.07–1.16 (overlapping signals, H-1, 3), 1.89 (1H, d, J = 12.7 Hz,
1H), 1.51–1.58 (overlapping resonances, H-2, 11, 12), 2.27–2.39
(overlapping signals, H-2, 6), 2.47 (1H, m, H-3), 1.40–1.44 (1H, m,
H-5), 2.90 (1H, m, H-6), 5.49 (1H, d, J = 5.8 Hz, H-7), 1.65–1.75
(1H, m, H-9), 1.29–1.33 (overlapping resonances, H-11, 12), 2.07
(2H, s, H-14), 1.80 (2H, t, J = 7.5 Hz, H-15), 4.03 (2H, t, J = 7.5 Hz,
H-16), 0.90 (3H, s, Me-17), 1.38 (3H, s, Me-18), 1.02 (3H, s, Me-
20). 13C NMR dH (100 MHz, C5D5N): 14.9 (C-20), 20.3 (C-2), 21.2
(C-11), 22.0 (C-17), 25.0 (C-6), 29.5 (C-18), 33.3 (C-13), 36.1 (C-
10), 37.5 (C-12), 38.9 (C-3), 40.2 (C-1), 43.9 (C-4), 47.8 (C-14),
48.6 (C-15), 51.6 (C-5), 51.7 (C-9), 58.2 (C-16), 121.6 (C-7), 135.4
(C-8), 180.1 (C-19).
4.3. Compound 1 (16-a-D-mannopyranosyloxyisopimar-7-en-19-oic
acid)
Compound 3 (1.0 mg) was treated with
a-glucosidase in the
same manner to afford aglycone 3a and -glucose. Aglycone 3a
D
was identical to 1a by NMR spectroscopic analysis. The optical
Colorless crystals; m.p. 220–221 °C; ½a D20
ꢂ
+ 40 (c 0.10, MeOH); IR
rotation values of the glucose unit isolated in this study and
(KBr) mmax cmꢀ1; 3646, 2935. 1687, 1241, 1085, 1062; HRFABMS
m/z [MꢀHꢀ]: 481.2799, calcd. for C26H41O8, 481.2801; FABMS
m/z: 481 [MꢀH]ꢀ. For 1H and 13C NMR spectroscopic analyses, see
Tables 1 and 2.
authentic sample were as follows; ½a D20
ꢂ
+ 53.0ꢀ(c 0.10, H2O, mea-
sured after 24 h dissolving in H2O) in 1, ½a D20
ꢂ
+ 52.0ꢀ(c 10.0, H2O,
measured after 24 h dissolving in H2O) for authentic sample).
4.8. Cell viability
4.4. Compound 2 (15-hydroxy-16-a-D-mannopyranosyloxyisopimar-
7-en-19-oic acid)
Human promyelocytic leukemia HL60 (RCB-0041, 4 ꢁ 104 cells/
ml) cells, human chronic myelogenous leukemia K562 (ATCC CCL-
243, 5 ꢁ 104 cells/ml), human cervical carcinoma HeLa (ATCC CCL-
2, 1 ꢁ 105 cells/ml) and human prostate carcinoma LNCaP (ATCC
CRL-1740, 1 ꢁ 105 cells/ml) were treated with the compounds on
various concentrations at 37 °C under a humidified, 5% CO2 atmo-
sphere for 4 days in RPMI 1640 medium supplemented with 10%
White amorphous solid; ½a D20
ꢂ
+ 73 (c 0.10, MeOH); IR (KBr) mmax
cmꢀ1; 3423, 2935, 1691, 1062; HRFABMS m/z [MꢀHꢀ]: 497.2749,
calcd. for C26H41O9, 497.2751; FABMS m/z: 497 [MꢀH]ꢀ. For 1H
and 13C NMR spectroscopic analyses, see Tables 1 and 2.
4.5. Compound 3 (16-a-D-glucopyranosyloxyisopimar-7-en-19-oic
acid)
fetal bovine serum, 50 U/ml of penicillin and 50 lg/ml of strepto-
mycin and cytotoxicity was determined using a 96-well plate with
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bro-
mide) assay. Percentages of viable cells were calculated as ratio
of the A570 values of treated and control cells (treated with 2%
MeOH for HL60 and 5% MeOH for K562, HeLa and LNCaP cells). Val-
ues are the average of two independent experiments.
White amorphous solid; ½a D20
ꢂ
+ 79 (c 0.10, MeOH); IR (KBr) mmax
cmꢀ1; 3363, 2933, 1687, 1060; HRFABMS m/z [MꢀHꢀ]: 481.2799,
calcd. for C26H41O8, 481.2801; FABMS m/z: 481 [MꢀH]ꢀ. For 1H
and 13C NMR spectroscopic analyses, see Tables 1 and 2.
4.6. X-ray structure determination of 1
4.9. Observation of DNA ladder
Crystal data for 1, C26H42O8ꢃCH3OH; MW = 3024.8, monoclinic,
For the internucleosomal DNA laddering, HL60 cells
(5 ꢁ 105 cells) were suspended in 1 ml of the culture medium.
DNA was extracted from the cells using Isoplant according the
manual. The resultant DNA was loaded onto 2% agarose gel (Dojin-
do) with TBE buffer. The DNA was stained with ethidium bromide
and visualized by UV light.
space group P21 (#4); a = 11.380 (1),0 b = 6.8700 (7), c = 17.490 (2)
0
ÅA, b = 101.390 (1)°, V = 1340.4 (2) ÅA3; Z = 2, Dc = 1.275 g/cm3, F
(000) = 560,
l (Mo Ka
) = 0.094 cmꢀ1, Rigaku/MSC CCD diffractom-
eter, T = 173 K, 19398 reflection measured, 6105 unique
(Rint = 0.042), final R1 = 0.0397 and wR (all data) = 0.023,
GOF = 1.084. Complete crystallographic data, as a CIF file, have
been deposited with Cambridge Crystallographic Data Center
(CCDC No. 675598). Copies can be obtained free of charge from:
The Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK
(e-mail: deposit@ccdc.cam.ac.uk).
References
Afiyatullov, S.S.H., Kuznetsova, T.A., Isakov, V.V., Pivkin, M.V., Prokof’eva, N.G.,
Elyakov, G.B., 2000. New diterpenic altrosides of the fungus Acremonium
striatisporum isolated from a sea cucumber. J. Nat. Prod. 63, 848–850 (Erratum
in: 2005, 68, 1308).
4.7. Enzymatic hydrolysis of 1 and 3
Afiyatullov, S.S.H., Kuznetsova, T.A., Isakov, V.V., Pivkin, M.V., Dmitrenok, P.S.,
Elyakov, G.B., 2002. New diterpene glycosides of the fungus Acremonium
striatisporum isolated from a sea cucumber. J. Nat. Prod. 65, 641–644.
Compound 1 (13.9 mg) was hydrolyzed with
a-mannosidase
(100 l; Sigma Chemicals, M-7257 from jack beans) in acetate
l