New Triterpenoid Saponins from the Rhizomes of Anemone amurensis
Results and Discussion
ment S in 2 was confirmed by a wavelength dispersive
X-ray fluorescence spectrum.[14] Acid hydrolysis of 2
also afforded D-glucuronic acid, D-glucose, and
Compound 1 was obtained as an amorphous powder.
The molecular formula was established as C60H96O28 by
its HRESIMS (m/z 1263.6019 [M-H]- , calcd for
C60H95O28: 1263.6015). The IR spectrum showed the
1
L-rhamnose. The H NMR and 13C NMR data indicated
2 possessed the same aglycon (oleanolic acid) and sugar
chains as 1. In the 1H-1H COSY spectrum, the spin sys-
tem of D-glucuronic acid was observed as the correla-
tions between H-1' (δH 4.89) and H-2' (δH 3.95), be-
tween H-2' and H-3' (δH 5.12), between H-3' and H-4'
(δH 4.60), as well as between H-4' and H-5' (δH 4.40).
The downfield shifts of the H-3' (Δδ +0.78) and C-3'
(Δδ +5.7) signals of 2 indicated the sulfate group was
attached to C-3' position. Based on the above results,
compound 2 was identified to be 3-O-[3-O-sulfonato]-
β-D-glucuronopyranosyl olean-12-en-28-oic acid 28-O-
β-D-glucopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→
4)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester
monosodium salt.
-
1
presence of hydroxyl (3374 cm ) and carboxyl (1734
-
1
1
cm ) groups. The H NMR spectrum of 1 showed the
signals for seven angular methyl groups (δH 0.82, 0.86,
0.89, 0.99, 1.07, 1.25, and 1.29, each 3H, s), one oxy-
genated methine [δH 3.37 (dd, J=11.5, 4.4 Hz)], as well
as one olefinic proton (δH 5.39, br s). In the 13C NMR
and DEPT spectra of 1, 60 carbon signals were observed
including seven methyl carbons (δC 15.5, 16.9, 17.5,
23.6, 26.1, 28.2, and 33.1), one oxygenated methine
carbon (δC 89.0), two olefinic carbons (δC 122.7 and
144.1), and one carbonyl carbon (δC 176.5). Inter-
pretation of the 2D NMR (1H-1H COSY, HSQC and
HMBC) spectra allowed the assignment of all proton
and carbon resonances of 1 as shown in Tables 1 and 2.
The aglycon of compound 1 was identified to be oleano-
lic acid by comparison of the NMR data with that pub-
lished in literature.[5]
Compound 3 was also obtained as amorphous pow-
der. The molecular formula was established as
C54H85NaO26S by the combination of its HRESIMS (m/z
=1181.5048 [M-Na]-, calcd for C54H85O26S:
1181.5055), and ESI-MS (1182.0 [M-Na]-, 1205.5
[M+H]+, and 1227.6 [M+Na]+), which was 146 mass
units less than that of 2. Acid hydrolysis of 3 afforded
D-glucose, D-glucuronic acid, and L-rhamnose. The 2D
NMR (1H-1H COSY, HSQC, HMBC, TOCSY and
ROESY) spectra of 3 allowed the assignment of all pro-
ton and carbon resonances (Tables 1 and 2). Further
1
The H NMR and 13C NMR spectra of 1 exhibited
five sugar anomeric proton signals at δH 4.92 (d, J=7.7
Hz), 5.02 (d, J=7.8 Hz), 5.36 (d, J=7.8 Hz), 5.86 (br s)
and 6.23 (d, J=8.1 Hz), corresponding to five anomeric
carbon signals at δC 105.0, 107.2, 106.7, 102.4 and 95.6,
respectively (Tables 1 and 2). Acid hydrolysis of 1 af-
forded D-glucuronic acid, D-glucose, and L-rhamnose,
which were identified by HPLC analysis.[11] The relative
anomeric configurations of the sugar moieties were de-
termined to be β for the D-glucuronic acid and
1
comparison of the H NMR and 13C NMR spectra of 3
with those of 2 indicated that they possessed the same
oleanolic acid as its aglycon, and two sugar chains at-
tached at C-3 and C-28 position. The only difference
between them was the absence of the anomeric proton
and carbon signals of the glucose moiety linked to Rha'''',
and the chemical shifts of C-3'''' in 3 was shifted upfield
from δ 82.8 to 72.6. The sequences and linkages of the
four sugars were similarly deduced by the distinct
HMBC correlations, which were also identical to those
of 2. Based on the above results, compound 3 was iden-
tified as 3-O-[3-O-sulfonato]-β-D-glucuronopyranosyl
olean-12-en-28-oic acid 28-O-α-L-rhamnopyranosyl-
(1→4)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl
ester monosodium salt.
3
D-glucose on the basis of the JH1-H2 coupling constants
of the anomeric protons. The α configuration for the
L-rhamnose could be established by comparison of the
NMR data with those reported in the literature.[12,13] The
sugar sequence and linkage positions could be deduced
by an HMBC experiment. In the HMBC spectrum, the
correlations (Figure 1) between H-1 (δH 5.02) of glu-
curonic acid and C-3 (δC 89.0) of the aglycon, between
H-1 (δH 5.36) of terminal glucose and C-3 (δC 83.7) of
the rhamnose, between H-1 (δH 5.86) of rhamnose and
C-4 (δC 77.2) of the centre glucose, between H-1 (δH
4.92) of the centre glucose and C-6 (δC 69.2) of the in-
ner glucose, as well as between H-1 (δH 6.23) of the in-
ner glucose and C-28 (δC 176.5) of the aglycon were
observed. Based on the above results, the structure of 1
was established as 3-O-β-D-glucuronopyranosyl
olean-12-en-28-oic acid 28-O-β-D-glucopyranosyl-(1→
3)-α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranosyl-
(1→6)-β-D-glucopyranoside.
Compound 4 was also obtained as amorphous pow-
der. The molecular formula was established as
C56H87NaO27S by the combination of its HRESIMS (m/z
=1223.5144 [M-Na]-, calcd for C56H87O27S:
1223.5161), and ESI-MS (1224.1 [M-Na]-, 1247.5
[M+H]+, and 1269.6 [M+Na]+), which is 42 mass
1
units more than that of 3. The H NMR spectrum of 4
showed the presence of seven tertiary methyl signals at
δH 0.75, 0.80, 0.82, 0.86, 0.92, 1.18 and 1.23 (each s,
3H), an olefinic protons at δH 5.29 (br s). The 13C NMR
data of 4 were similar to those of 3, except for the ap-
pearance of additional acetyl group signal [δH 2.00 (3H,
s); δC 170.6, 20.8]. Comparison of the NMR data of 4
Compound 2 was purified as amorphous powder.
The molecular formula of 2 was displayed as
C60H95NaO31S by the combination of its HRESIMS (m/z
=1343.5573 [M-Na]-, calcd for C60H95O31S:
1343.5584), and the ESI-MS (1344.2 [M-Na]-, 1366.9
[M-H]-, and 1389.5 [M+Na]+). The presence of ele-
Chin. J. Chem. 2012, 30, 1249—1254
© 2012 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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