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MING-AN OUYANG et al.
extract (100 g) suspended in H2O. The aq. suspen- product was puri®ed by CC (silica gel 3 g,
sion was extracted with CHCl3 and n-BuOH. The CH2Cl2:MeOH, 3:1) to aord hydrolysate
a
n-BuOH layer was evaporated to dryness to give a (15 mg), and a mixture of rhamnose and glucose
residue (50 g). Crude saponin were treated with (1:1) that were detected by HPLC.
Diaion column ®rst eluate 30% MeOH 1000 ml,
Acid hydrolysis of latifolosides F-H
then with 100% MeOH 1000 ml to give two frac-
tions and MeOH fraction was chromatographed on
A soln of each compound (10 mg) was heated at
silica gel (1.5 kg, 200±300 mesh) with 7000 ml, 1008 in 5% H2SO4 and 50% EtOH for 10 hr. The
CHCl3-MeOH-H2O (7:3:0.5) to give twenty frac- reaction mixture was diluted with water, neutralized
tions. No. 16 and No. 20 were separated on HPLC with 2% NaOH and evaporated in vacuo to dry-
[ODS, eluting with MeOH-H2O (8:2±6:4), ¯ow rate: ness. The reaction product was a mixture of arabi-
5 ml min 1, injection: 0.4 ml (10 mg ml 1)] to aord nose, glucose, and rhamnose (1:2:2). The mole ratio
latifolosides F(1, 180 mg), G(2, 80 mg), H(3, of each sugar was determined by using RI detection
75 mg).Latifoloside F(1). m.p. 235±2388, C59H96O26, in HPLC (Shodex RS pak DC-613, 75% MeCN,
1
IR ꢀKmBaxrcm 1: 3430 (OH), 2927 (C-H), 1730 (C.O), 1 ml min
, 708) by comparison with authentic
1640 (C.C). FAB-MS m/z: 1219 [M-H] , 1073 [M- sugars (10 mM each of Ara, Glc and Rha). The
H-146] , 911 [M-H-146±162] , 765 [M-H-146 Â2± retention time of each sugar was as follows: Ara,
162] , 749 [M-H-146±162 Â 2] , 603 [M-H-146 Â2± 6.0 min; Glc, 7.4 min and Rha, 4.8 min.
162 Â2] . 1H NMR: d 0.85, 1.08, 1.10, 1.11, 1.33,
Enzymatic hydrolysis of latifoloside F(1)
1.73 (3H Â6, s, Me-23, Me-24, Me-25, Me-26, Me-
27 and Me-29), 0.87(3H, d, J = 6.5 Hz, Me-30),
Latifoloside F (1) (35 mg) was taken up in 5 ml
3.22 (1H, dd, J = 4.5, 11.5 Hz, H-3), 4.83 (1H, d, each of EtOH-H2O (1:9) and 0.01 M NaH2PO4 buf-
J = 6.0 Hz, H-1 of Ara), 5.19 (1H, d, J = 7.9 Hz, fer (pH 4.0), and incubated with crude cellulase
H-1 of Glc), 6.19 (1H, br.s, H-1 of Rha), 6.21 (1H, (50 mg, Sigma) for two weeks at 378. The soln was
d, J = 7.8 Hz, H-1 of 28-Glc), 6.69 (1H, br.s, H-1 diluted with H2O and then ®ltered. The resulting
of 28-Rha), 13C NMR data see Table 1.Latifoloside residue was subjected to MCI gel (CHP20P).
1
G(2). m.p. 215±2188, C59H96O26, IR ꢀKmaBxrcm
:
Elution with water and MeOH aorded crude
3400±3100 (OH), 2932 (C-H), 1734 (C.O), 1640 genin. The crude genin was chromatographed on a
(C.C). FAB-MS m/z: 1219 [M-H]-, 1073 [M-H- silica gel column with CHCl3-MeOH-H2O
146] , 911 [M-H-146±162] , 765 [M-H-146 Â2± (250:40:1) giving ilexgenin
B
(4, 12 mg).
1
162] , 749 [M-H-146±162 Â 2] , 603 [M-H-146 Â2± Identi®cation was by H and 13C NMR data.
162 Â2] . 1H, 13C NMR: d 0.89, 1.12, 1.16, 1.18,
Enzymatic hydrolysis of latifolosides G(2) and H(3)
1.39, 1.70, (3H Â6, s, Me-23, Me-24, Me-25, Me-
26, Me-27 and Me-29), 1.05 (3H, d, J = 6.4 Hz,
Latifoloside G(2) (25 mg) and H(3) (30 mg) were
Me-30), 3.27 (1H, dd, J = 4.5, 11.5 Hz, H-3), 5.54 hydrolyzed in the same way as for 1 to give pomolic
(1H, br.s, H-12), 4.85 (1H, d, J = 5.4 Hz, H-1 of acid (6, 8 mg), and siaresinolic acid (7, 9 mg), which
Ara), 5.80 (1H, d, J = 7.7 Hz, H-1 of Glc), 6.14 had the same NMR data as an authentic sample.
(1H, br.s, H-1 of Rha), 6.28 (1H, d, J = 8.0 Hz, H-
1 of 28-Glc), 6.65 (1H, br.s, H-1 of 28-Rha). 13C
AcknowledgementsÐThe authors are grateful to the
Laboratory of Kunming Institute of Botany,
Chinese Academy of Sciences for ®nancial support.
NMR data see Table 1.Latifoloside H(3). m.p. 227±
2318, C59H96O26, IR ꢀKmaBxrcm 1: 3430 (OH), 2928
(C-H), 1730 (C.O), 1642 (C.C). FAB-MS m/z:
1219 [M-H] , 1073 [M-H-146] , 911 [M-H-146±
162] , 765 [M-H-146Â 2±162] , 749 [M-H-146±
162 Â2] , 603 [M-H-146 Â2±162Â 2] . 1H NMR: d
0.87, 0.97, 1.10, 1.12, 1.14, 1.19, 1.64 (3H Â7, s),
3.30 (1H, dd, J = 4.3, 11.3 Hz, H-3), 4.86 (1H, d,
J = 5.7 Hz, H-1 of Ara), 5.09 (1H, d, J = 8.1 Hz,
H-1 of Glc), 6.16 (1H, br.s, H-1 of Rha), 6.36 (1H,
d, J = 7.5 Hz, H-1 of 28-Glc), 6.65 (1H, br.s, H-1
of 28-Rha), 13C NMR data see Table 1.
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Alkaline hydrolysis of latifoloside F(1)
LiOH (6 mg) was added to a soln of latifoloside
F(1, 24 mg) in H2O (3.0 ml). The reaction mixture
was heated with stirring at 408 for 10 hr, then
cooled to ambient temperature, and the solvent
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