Z. Wang et al. / Food Chemistry 132 (2012) 295–300
297
3.68 (1H, m, H-5b), 4.28 (1H, m, H-5
a
); Qui2 (1?2Xyl1), dH 5.23
Holotoxin G (5): colourless amorphous powder; ½a D20
ꢁ
ꢂ 92 (c
(1H, d, J = 7.4 Hz, H-1), 4.12 (1H, m, H-2), 4.16 (1H, m, H-3), 3.73
(1H, m, H-4), 3.85 (1H, m, H-5), 1.86 (2H, d, J = 5.9 Hz); Xyl3
(1?4Qui2), dH 4.94 (1H, d, J = 7.5 Hz, H-1), 4.07 (1H, m, H-2),
4.19 (1H, m, H-3), 4.11 (1H, m, H-4), 3.70 (1H, m, H-5b), 4.48
0.15, pyridine); 1H-NMR chemical shifts of the sugar moiety
(500 MHz in C5D5N): Xyl1 (1?C-3), dH 4.82 (1H, d, J = 7.3 Hz, H-
1), 4.11 (1H, m, H-2), 4.28 (1H, m, H-3), 4.39 (1H, m, H-4), 3.76
(1H, m, H-5b), 4.48 (1H, m, H-5a
); Qui2 (1?2Xyl1), dH 5.25 (1H,
(1H, m, H-5
a
); MeGlc4 (1?3Xyl3), dH 5.37 (1H, d, J = 7.8 Hz, H-1),
d, J = 7.6 Hz, H-1), 4.19 (1H, m, H-2), 4.18 (1H, m, H-3), 3.73 (1H,
m, H-4), 3.87 (1H, m, H-5), 1.83 (2H, d, J = 5.8 Hz); Xyl3
(1?4Qui2), dH 4.95 (1H, d, J = 7.6 Hz, H-1), 4.08 (1H, m, H-2),
4.18 (1H, m, H-3), 4.12 (1H, m, H-4), 3.71 (1H, m, H-5b), 4.29
4.16 (1H, m, H-2), 3.79 (1H, m, H-3), 4.21 (1H, m, H-4), 4.03 (1H,
m, H-5), 4.34 (1H, m, H-6b), 4.62 (1H, m, H-6a), 3.94 (3H, s,
OMe); Glc5 (1?4Xyl1) dH 5.08 (1H, d, J = 7.8 Hz, H-1), 4.08 (1H,
m, H-2), 4.19 (1H, m, H-3), 4.48 (1H, m, H-4), 4.03 (1H, m, H-5),
(1H, m, H-5
4.19 (1H, m, H-2), 4.29 (1H, m, H-3), 4.27 (1H, m, H-4), 4.07 (1H,
m, H-5), 4.41 (1H, m, H-6b), 4.61 (1H, m, H-6
); Glc5 (1?4Xyl1)
a
); Glc4 (1?3Xyl3), dH 5.41 (1H, d, J = 7.9 Hz, H-1),
4.30 (1H, m, H-6b), 4.53 (1H, m, H-6a
); Glc6 (1?3Glc5), dH 5.33
(1H, d, J = 8.0 Hz, H-1), 4.03 (1H, m, H-2), 4.06 (1H, m, H-3), 4.27
a
(1H, m, H-4), 4.17 (1H, m, H-5), 4.30 (1H, m, H-6b), 4.51 m(1H,
dH 5.08 (1H, overlap, H-1), 4.10 (1H, m, H-2), 4.29 (1H, m, H-3),
m, H-6a
); 13C-NMR chemical shifts of the sugar moiety (100 MHz
4.27 (1H, m, H-4), 4.07 (1H, m, H-5), 4.41 (1H, m, H-6b), 4.61
in C5D5N): Xyl1 (1?C-3), dC 105.5 (CH, C-1), 83.7 (CH, C-2), 76.0
(CH, C-3), 77.7 (CH, C-4), 64.3 (CH2, C-5); Qui2 (1?2Xyl1), dC
105.8 (CH, C-1), 76.7 (CH, C-2), 75.7 (CH, C-3), 86.1 (CH, C-4),
72.0 (CH, C-5), 18.2 (CH2, C-6); Xyl3 (1?4Qui2), dC 105.4 (CH, C-
1), 73.7 (CH, C-2), 87.7 (CH, C-3), 69.3 (CH, C-4), 66.7 (CH2, C-6);
MeGlc4 (1?3Xyl3), dC 105.7 (CH, C-1), 75.7 (CH, C-2), 88.2 (CH,
C-3), 70.8 (CH, C-4), 78.5 (CH, C-5), 62.7 (CH2, C-6), 60.9 (CH3,
OMe); Glc5 (1?4Xyl1) dC 103.1 (CH, C-1), 73.4 (CH, C-2), 88.4
(CH, C-3), 69.9 (CH, C-4), 78.4 (CH, C-5), 62.4 (CH2, C-6); Glc6
(1?3Glc5), dC 106.0 (CH, C-1), 78.5 (CH, C-2), 75.3 (CH, C-3), 71.8
(CH, C-4), 78.8 (CH, C-5), 62.3 (CH2, C-6); 1H- and 13C-NMR chem-
ical shifts of the aglycone moiety see liouvillosides A1 and A2 (An-
tonov et al., 2008), and neothyonidioside and mollisosides B1
(Moraes et al., 2005). ESI-MS (positive-ion mode) m/z 1401
[M + Na]+; ESI-MS (negative-ion mode) m/z 1377 [M–H]+. HR-ESI-
MS (positive-ion mode) m/z 1401.6313 [M + Na]+ (calcd. for
(1H, m, H-6a
); 13C-NMR chemical shifts of the sugar moiety
(125 MHz in C5D5N): Xyl1 (1?C-3), dC 105.5 (CH, C-1), 83.6 (CH,
C-2), 76.1 (CH, C-3), 77.5 (CH, C-4), 64.4 (CH2, C-5); Qui2
(1?2Xyl1), dC 105.8 (CH, C-1), 76.7 (CH, C-2), 75.7 (CH, C-3), 86.2
(CH, C-4), 72.0 (CH, C-5), 18.3 (CH2, C-6); Xyl3 (1?4Qui2), dC
105.4 (CH, C-1), 73.7 (CH, C-2), 87.9 (CH, C-3), 69.3 (CH, C-4),
66.7 (CH2, C-6); Glc4 (1?3Xyl3), dC 105.8 (CH, C-1), 75.7 (CH, C-
2), 78.4 (CH, C-3), 71.8 (CH, C-4), 79.0 (CH, C-5), 62.7 (CH2, C-6);
Glc5 (1?4Xyl1) dC 103.6 (CH, C-1), 74.5 (CH, C-2), 78.4 (CH, C-3),
71.8 (CH, C-4), 78.8 (CH, C-5), 62.7 (CH2, C-6); 1H- and 13C-NMR
chemical shifts of the aglycone moiety see cladoloside B (8) (Avilov
& Stonik, 1988; Xue et al., 2010). ESI-MS (positive ion mode) m/z:
1213 [M + Na]+; ESI-MS (negative ion mode) m/z: 1189 [M–H]+;
HRESI-MS (positive ion mode) m/z: 1213.5988 [M + Na]+ (calcd.
C
58H94O25Na, 1213.5982); ESI-MS-MS (negative ion mode) m/z:
1189 [M–H]+ (16), 1171 [M–H2O]+ (100), 1009 [M–H2O–Glc]+
(15), 877 [M–H2O–Glc–Xyl]+ (23), 715 [M–H2O–Glc–Xyl–Glc]+ (7).
C
66H104O32Na, 1401.6303). ESI-MS-MS (negative-ion mode) m/z
1377 [M–H]+ (94), 1333 [M–H–CO2]+ (100), 1157 [M–H–CO2–MeG-
lc]+ (11), 1069 [M–H–MeGlc–Xyl]+ (26), 1025 [M–H–CO2–MeGlc–
Xyl]+ (22), 863 [M–H–CO2–MeGlc–Xyl–Glc]+ (31), 701[M–H–CO2–
MeGlc–Xyl–Glc–Glc]+ (38), 555 [M–H–CO2–MeGlc–Xyl–Glc–Glc–
Qui]+ (8), 423 [M–H–CO2–MeGlc–Xyl–Glc–Glc–Qui–Xyl]+ (24).
2.5. Acid hydrolysis of 2, 3 and 4
Each glycoside (3 mg) was heated in an ampoule with 5 mL of
aqueous 2 M CF3COOH at 120 °C for 2 h. The aglycone was ex-
tracted with dichloromethane, and the aqueous residue was evap-
orated in vacuo. To this dry residue was added 1 mL of pyridine and
2 mg of NH2OHꢃHCl, and the mixtures were heated at 90 °C for 1 h.
After the reaction mixtures were cooled, 1.5 mL of Ac2O were
added and the mixtures were heated at 90 °C for 1 h. The reaction
mixtures were evaporated in vacuo, and the resulting aldononitrile
peracetates as reference were analysed by GC–MS using the
Holotoxin F (4): colourless amorphous powder; ½a D20
ꢂ 102 (c
ꢁ
0.15, pyridine); 1H-NMR chemical shifts of the aglycone moiety
(500 MHz in C5D5N): dH 1.48 (1H, m, H-1b), 1.83 (1H, m, H-1
2.02 (1H, m, H-2b), 2.28 (1H, m, H-2 ), 3.30 (1H, dd, J = 3.9,
11.7 Hz, H-3), 1.00 (1H, m, H-5), 1.54 (1H, m, H-6b), 1.78 (1H, m,
H-6 ), 1.61 (2H, m, H-7), 2.39 (1H, m, H-8), 5.45 (1H, m, H-11),
2.23 (1H, m, H-12b), 2.49 (1H, m, H-12 ), 2.19 (1H, d J = 17.7 Hz,
H-15b), 2.41 (1H, d J = 17.7 Hz, H-15 ), 2.97 (1H, s, H-17), 1.20
(3H, s, H-18), 1.18 (3H, s, H-19), 1.57 (3H, s, H-21), 2.01 (1H, m,
H-22), 1.81 (1H, m, H-23b), 1.92 (1H, m, H-23 ), 2.19 (2H, m, H-
a),
a
a
a
a
corresponding authentic
O-methyl- -glucose treated by the same procedure. The oligosac-
charides of the glycosides were determined as -xylose, -glucose
and 3-O-methyl- -glycose in a ratio of 1:1:1 for 2, -xylose, -qui-
novose, -glucose and 3-O-methyl- -glucose in a ratio of 2:1:2:1
for 3, and -xylose, -glucose and 3-O-methyl- -glucose in a ratio
D-xylose, D-quinovose, D-glucose and 3-
a
D
24), 4.92 (2H, d, J = 16.1 Hz, H-26), 1.81 (3H, s, H-27), 1.20 (3H, s,
H-30), 1.37 (3H, s, H-31), 1.00 (3H, s, H-32); 13C-NMR chemical
shifts of the aglycone moiety (125 MHz in C5D5N): dC 36.4 (CH2, C-
1), 27.3 (CH2, C-2), 88.9 (CH, C-3), 40.2 (C, C-4), 53.1 (CH, C-5), 21.4
(CH2, C-6), 28.6 (CH2, C-7), 40.8 (CH, C-8), 149.7 (C, C-9), 39.8 (C, C-
10), 115.4 (CH, C-11), 36.8 (CH2, C-12), 44.2 (C, C-13), 41.3 (C, C-
14), 49.8 (CH2, C-15), 221.0 (C, C-16), 64.7 (CH, C-17), 17.2 (C, C-
18), 22.6 (CH3, C-19), 74.5 (C, C-20), 26.6 (CH3, C-21), 41.9 (CH2, C-
22), 22.6 (CH2, C-23), 38.8 (CH2, C-24), 146.5 (C, C-25), 110.6 (CH2,
C-26), 22.2 (CH3, C-27), 16.9 (CH3, C-30), 28.3 (CH3, C-31), 19.0
(CH3, C-32); 1H- and 13C-NMR chemical shifts of the aglycone moiety
see cladoloside B (8) (Avilov & Stonik, 1988; Xue et al., 2010). ESI-MS
(positive ion mode) m/z: 1227 [M + Na]+; ESI-MS (negative ion
mode) m/z: 1203 [M–H]+; HRESI-MS (positive ion mode) m/z:
1227.6147 [M + Na]+ (calcd. C59H96O25Na, 1227.6138); ESI-MS-MS
(negative ion mode) m/z: 1203 [M–H]+ (20), 1185 [M–H–H2O]+
(36), 1009 [M–H–H2O–MeGlc]+ (23), 895 [M–H–MeGlc–Xyl]+
(100), 877 [M–H–H2O–MeGlc–Xyl]+ (68), 769 [M–H–MeGlc–Xyl–
Glc]+ (65), 715 [M–H–H2O–MeGlc–Xyl–Glc]+ (47), 455 [M–H–MeG-
lc–Xyl–Glc–Qui–Xyl]+ (38).
D
D
D
D
D
D
D
D
D
D
of 2:1:1:1 for 4, respectively.
2.6. Fungus strains and positive controls
The fungus strains Candida albicans SC5314 and Cryptococcus
neoformans BLS108 were provided by the Changzheng hospital,
The Second Military Medical University (SMMU), PR China, and
Candida tropicalis, Trichophyton rubrum 0501124, Microsporum gyp-
seum 31388 and Aspergillus fumigatus 0504656 were provided by
the Changhai hospital, SMMU, PR China. Positive controls Itraco-
nazole (ICZ), Terbinafine (TRB), Ketoconazole (KCZ), Amphotericin
B (AMB), Voriconazole (VCZ) and Fluconazole (FCZ) were supplied
by the Department of Pharmacology, School of Pharmacy, Second
Military Medical University, Shanghai.