J Nat Med
quasimolecular ion peaks were observed at m/z 1543
[M ? Na]? and 1519 [M - H]-, and HRFABMS analysis
established that its molecular formula was C73H116O33. The
IR spectrum of 1 showed absorption bands at 1736 and
1656 cm-1, ascribable to ester carbonyl and olefin func-
tions, and broad bands at 3445 and 1051 cm-1, suggestive
of a glycoside structure. Upon alkaline hydrolysis of 1 with
10 % aqueous potassium hydroxide (KOH)–50 % aqueous
1,4-dioxane (1:1, v/v), polygalacic acid 3-O-a-L-
rhamnopyranoside [6] was obtained, together with acetic
acid and 3-hydroxybutyric acid, which were identified by
HPLC analysis to be their p-nitrobenzyl derivatives [7–13].
Treatment of 1 with 0.5 % sodium methoxide (NaOMe)–
MeOH produced the desacyl derivative, bellissaponin BS1
(1a) [14] and methyl (S)-(?)-3-hydroxybutyrate [15],
which was identified by HPLC using an optical rotation
3HB-C-10 (dC 169.7); 3HB-H-30 and 3HB-C-100 (dC 171.4);
and 3HB-H-300 and 3HB-C-100. These results indicated that
the structure of 1 was 3-O-a-L-rhamnopyranosyl poly-
galacic acid {28-O-a-L-rhamnopyranosyl(1 ? 3)-b-D-xy-
lopyranosyl(1 ? 4)-3-acetyl-a-L-rhamnopyranosyl(1 ? 2)
-4-O-[(3S)-3-[(3S)-3-[(3S)-3-hydroxy-1-oxobutoxy]-1-oxo-
buthoxy]-1-oxobutyl]-b-D-fucopyranosyl} ester.
Perennisaponin O (2) was obtained as an amorphous
powder with a negative optical rotation ([a]2D5–24.2 in
MeOH). In positive-ion FABMS, a quasimolecular ion
peak was observed at m/z 1629 [M ? Na]?, and
HRFABMS analysis indicated that the molecular formula
was C77H122O35. Alkaline hydrolysis of 2 with 10 %
aqueous KOH–50 % aqueous 1,4-dioxane (1:1, v/v) liber-
ated polygalacic acid 3-O-a-L-rhamnopyranoside and two
organic acids, including acetic acid and 3-hydroxybutyric
acid, which were identified by HPLC analysis for their p-
nitrobenzyl derivatives. Treatment of 2 with 0.5 %
NaOMe–MeOH gave 1a together with methyl (S)-(?)-3-
hydroxybutyrate, which was identified by HPLC using an
1
detector [2, 3]. The H (Table 1) and 13C NMR (Table 2)
spectra (pyridine-d5) of 1, which were assigned using
DEPT, TOCSY, 1H–1H COSY, HSQC, and HMBC
experiments, showed signals assignable to six methyls [d
0.93, 1.07, 1.16, 1.24, 1.57, 1.75 (3H each, all s, H3-29, 30,
26, 24, 25, 27)], a methylene and three methines bearing an
oxygen function [d 3.70 (2H, br s, H2-23), 4.35 (1H, d,
J = 3.4 Hz, H-3), 4.71 (1H, br d, J = ca. 3 Hz, H-2), 5.14
(1H, br s, H-16)], an olefinic proton [d 5.65 (1H, t-like,
J = ca. 3 Hz, H-12)], a b-D-fucopyranosyl [d 1.25 (3H, d,
J = 6.4 Hz, Fuc-H3-6), 6.03 (1H, d, J = 7.3 Hz, Fuc-H-
1)], three a-L-rhamnopyranosyl [d 1.60 (3H, d, J = 6.2 Hz,
3-O-Rha-H3-6), 1.65 (3H, d, J = 6.2 Hz, terminal-Rha-H3-
6), 1.74 (3H, d, J = 6.5 Hz, inner-Rha-H3-6), 5.72 (1H, br
s, 3-O-Rha-H-1), 6.02 (1H, br s, inner-Rha-H-1), 6.13 (1H,
br s, terminal-Rha-H-1)], and a b-D-xylopyranosyl moieties
[d 4.94 (1H, d, J = 7.4 Hz, Xyl-H-1)], together with four
ester carbonyl carbons (dC 169.7, 170.6, 170.9, 171.4),
suggesting the presence of an acetyl [d 2.05 (3H, s, Ac-H3)]
and three (S)-3-hydroxybutyryl (3HB) moieties {d 1.31
(3H, d, J = 6.4 Hz, 3HB-H3-40), 1.33 (3H, d, J = 6.2 Hz,
3HB-H3-4), 1.37 (3H, d, J = 6.2 Hz, 3HB-H3-400), [2.60
(1H, dd, J = 5.0, 9.8 Hz), 2.65–2.70 (2H, m), 2.74–2.83
(3H, m), 3HB-H2-2, 20, 200], 4.55 (1H, m, 3HB-H-300),
1
optical rotation detector. The H (Table 1) and 13C NMR
(Table 2) spectroscopic properties (pyridine-d5) were quite
similar to those of 1, except for the signals resulting from
an additional 3HB moiety: a polygalacic acid part {six
methyls [d 0.93, 1.07, 1.14, 1.25, 1.55, 1.74 (3H each, all s,
H3-29, 30, 26, 24, 25, 27)], a methylene and three methines
bearing an oxygen function [d 3.70 (2H, br s, H2-23), 4.36
(1H, d, J = 3.4 Hz, H-3), 4.71 (1H, br d, J = ca. 3 Hz,
H-2), 5.14 (1H, br s, H-16)], an olefinic proton [d 5.65 (1H,
t-like, J = ca. 3 Hz, H-12)]}, a b-D-fucopyranosyl [d 1.24
(3H, d, J = 6.8 Hz, Fuc-H3-6), 6.03 (1H, d, J = 8.3 Hz,
Fuc-H-1)], three a-L-rhamnopyranosyl [d 1.60 (3H, d,
J = 6.7 Hz, 3-O-Rha-H3-6), 1.65 (3H, d, J = 6.7 Hz,
terminal-Rha-H3-6), 1.74 (3H, d, J = 6.2 Hz, inner-Rha-
H3-6), 5.72 (1H, br s, 3-O-Rha-H-1), 6.02 (1H, br s, inner-
Rha-H-1), 6.13 (1H, br s, terminal-Rha-H-1)], and a b-D-
xylopyranosyl moieties [d 4.95 (1H, d, J = 8.3 Hz, Xyl-H-
1)], together with five ester carbonyl carbons (dC 169.6,
169.9, 170.6, 170.9, 171.4), suggesting the presence of an
acetyl [d 2.04 (3H, s, Ac-H3)] and four 3HB moieties {d
1.30 (3H, d, J = 6.7 Hz, 3HB-H3-4), 1.33 (3H, d,
J = 6.7 Hz, 3HB-H3-40), 1.33 (3H, d, J = 6.7 Hz, 3HB-
H3-400), 1.38 (3H, d, J = 7.0 Hz, 3HB-H3-4000), [2.59–2.71
(4H, m), 2.73–2.83 (4H, m), 3HB-H2-2, 20, 200, 2000], 4.55
(1H, m, 3HB-H-3000), 5.48–5.56 (3H, m, 3HB-H-3, 30, 300)}.
Finally, the positions of the sugar components were
determined by HMBC, which showed long-range correla-
tions between the following proton and carbon pairs: 3-O-
Rha-1 and C-3 (dC 81.4); Fuc-1 and C-28 (dC 176.3); inner-
Rha-H-1 and Fuc-C-2 (dC 76.7); Xyl-H-1 and inner-Rha-C-
4 (dC 77.9); terminal-Rha-H-1 and Xyl-C-3 (dC 83.6); in-
ner-Rha-H-3 [d 5.83 (1H, dd, J = 3.4, 9.4 Hz)] and the
acetyl carbonyl carbon (dC 170.9); Fuc-H-4 [d 5.46 (1H,
1
5.52–5.56 (2H, m, 3HB-H-3, 30)}. The H and 13C NMR
spectra of 1 resembled that of perennisaponin F (13) [2],
except for the signals resulting from the acetyl group. As
1
shown in Fig. 2, H–1H COSY experiments of 1 indicated
the presence of partial structures, shown in bold. In the
HMBC experiment of 1, long-range correlations were
observed between the following protons and carbons: 3-O-
Rha-1 and C-3 (dC 81.6); Fuc-1 and C-28 (dC 176.3); inner-
Rha-H-1 and Fuc-C-2 (dC 76.7); Xyl-H-1 and inner-Rha-C-
4 (dC 77.9); terminal-Rha-H-1 and Xyl-C-3 (dC 83.6); in-
ner-Rha-H-3 [d 5.83 (1H, dd, J = 2.8, 9.6 Hz)] and the
acetyl carbonyl carbon (dC 170.9); Fuc-H-4 [d 5.47 (1H, br
d, J = ca. 3 Hz)] and 3HB-C-1 (dC 170.6); 3HB-H-3 and
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