114
J. Chai et al. / Phytochemistry Letters 10 (2014) 113–117
Fig. 1. Structures of compounds 1 and 2.
carbons including six olefinic carbons (dC 113.4, 116.1, 123.4,
136.0, 139.2, 146.3), one oxygenated methine carbon ( C 89.9), one
was defined as (2,3-S-trans,10R,6E)-7,11-dimethyl-3-methylene-
1,6-dodecadien-10,11-diol 10-O-
-glucopyranosyl-(1!4)-O-
-glucopyranosyl-(1!4)-O- -glucopyranoside (Fig. 2).
Compound 2 was obtained as an amorphous powder. Positive
coloration reactions for Molish and Liebermann–Buchard tests
indicated 2 as a steroidal saponin. Its molecular formula was
determined as C43H68O19 from the HR-ESI–MS at m/z
911.4243[M + Na]+ (calcd for C43H68O19Na 911.4252). The 1H
NMR spectrum of 2 showed four methyl groups including two
singlet methyl [dH 1.09 (3H, s, H-18) and 1.38 (3H, s, H-19)], one
doublet methyl [dH 0.96 (3H, J = 6.9 Hz, H-27)], and one olefinic
d
b
-D
b-
oxygenated quaternary carbon (dC 72.8), four sp3 methylene
carbons (dC 26.6, 30.8, 31.5, 36.1), and three tertiary methyl
carbons (dC 15.9, 25.1, 26.8), while the remaining carbon signals
were characteristic to three glucosyl moieties. These data
suggested 1 was a farnesane-type sesquiterpene glycoside with
the aglycone similar to 7,11-dimethyl-3-methylene-1,6-dodeca-
dien-10,11-diol (Nakano et al., 1983), the glucosyl moieties was
attached to C-10 due to the resonance shifted to dC 89.9 after
glycosidation (Seo et al., 1978).
D
b-D
The above conclusion and the sugar linkages of 1 were
determined by 1H–1H COSY, HSQC and HSBC experiments.
1H–1H COSY correlation of H-1/H-2, H-5/H-4 and H-6, H-9/H-8
and H-10, and HSBC correlation of H-2/C-3, C-4 and C-15, H-6/C-8
and C-14, H-10/C-11 and C-12, H-12/C-10, C-11 and C-13, verified
the presence of the farnesane-type sesquiterpene aglycone as 7,11-
dimethyl-3-methylene-1,6-dodecadien-10,11-diol. Three glucosyl
proton [dH 5.55 (1H, d, J = 5.9 Hz, H-6)], ascribable to the aglycone
proton signals of this steroidal saponin. The 13C NMR spectrum
(Table 2) exhibited 45 carbon signals, 27 of which belonged to the
aglycone carbons, including three methyl (13.5, 15.4, 17.4), eight
methylene (24.5, 32.4, 32.9, 37.9, 40.8, 44.3, 61.2, 62.8), 20 methine
(33.6, 36.5, 46.1, 50.8, 57.4, 58.2, 68.7, 70.9, 73.6, 84.0, 84.3, 125.2),
and four quaternary carbons (43.3, 57.4, 113.1, 140.0). Among
moiety was identified as
D
-glucose by acid hydrolysis of 1 followed
them, the quaternary carbon signal at dC 113.1 was identified as an
by TLC comparison with reference compound and optical rotation
determination (Fukuda et al., 1981; Hudson and Dale, 1917), and
judged as b-configuration (Zhang et al., 2012) from their coupling
acetal carbon (C-22), a characteristic signal of spirostanol or
norspirostanol saponin (Yan et al., 1996). In addition, three
anomeric signals were observed in the 1H NMR spectrum at [dH
4.72 (1H, d, J = 7.5 Hz, Ara-10), 4.99 (1H, d, J = 7.5 Hz, Xyl-1000), and
constant of anomeric protons (7.9, 7.8 and 7.8 Hz respectively).
Besides, H-Glc-10/C-10, H-Glc-100/C-Glc-40, and H-Glc-1000/C-Glc-400
in HSBC spectrum, indicated that three glucosyl groups were
connected as (Glc-1000-O-Glc-400), (Glc-100-O-Glc-40) and (Glc-10-O-
C-10). In the NOESY spectrum of 1, NOE correlations of H-1a/H-4/
H-6, and H3-14/H2-5 were observed, indicating the geometry of
1,3-conjugated diene to be S-trans configuration, and the double
bond between C-6 and C-7 to be E configuration. The absolute
configuration at C-10 of 1 was confirmed as R by the values of
6.33 (1H, br s, Rha-100)] and in the 13C NMR spectrum at
102.3 and 107.0. Compound 2 was hydrolyzed with 2.0 M HCl, the
products were separated and identified as -rhamnose, -xylose,
and -arabinose by the same way as described in the case of 1.
Moreover, the -anomeric configurations of the -arabinose unit
dH 4.72 (d, J = 7.5 Hz)], the -anomeric configurations of the
rhamnose unit [dH 6.33 (br s)] and -anomeric configurations of
the -xylose unit [dH 4.99 (d, J = 7.5 Hz)] were confirmed by their
dC 101.0,
L
D
L
b
L
[
a
L-
a
D
glycosylation shift of
a
-,
b
-(pro-S side), and
b-(pro-R side) carbons
coupling constants and chemical shifts.
of secondary alcohols to which glucosyl moieties was attached
(Seo et al., 1978), and the 13C chemical shifts at C-8–C-13 of 1 were
quite similar to those of a natural product (10R,6E)-7,11-dimethyl-
In comparison of the proton and carbon resonances of 2 with
those of trikamsteroside E (Ono et al., 2007a), most NMR data of
them were almost consistent, the difference was that the presence
3-mehylene-6-dodecaene-1,2,10,11-tetraol 10-O-
b
-D
-glucopyra-
of D-apiofuranose in trikamsteroside E was not observed in
nosyl-(1!4)-O-
b
-
D
-glucopyranoside (Ono et al., 2007b) and a
compound 2. The above inference was supported by 2D NMR
data analysis. The proton and protonated carbon resonances in the
NMR spectra of 2 were unambiguously assigned by the HSQC
experiment. HSBC correlations of H-19/C-1, C-5, C-9 and C-10,
H-6/C-4, C-7 and C-10, H-3/C-2 and C-4, H-8/C-9, C-11 and C-6;
synthesis compound icariside C4 (10R) (Miyase et al., 1987), and
different from those of icariside C1 (10S) (Miyase et al., 1987),
although the assignments at C-8 and C-9 of icariside C4 and
icariside C1 in the literature should be interchanged. Therefore, 1
Fig. 2. Key HSBC, 1H–1H COSY and NOESY correlations of the compound 1.