Journal of Natural Products
Article
structure elucidation of these triterpenes and their inhibitory
effects on VEGF-induced angiogenesis.
confirmed the positions of the three hydroxy groups to be at C-3,
C-6, and C-7, respectively (Figure 1). Furthermore, the HMBC
correlations between H-19/C-1, C-9, C-10, and C-11 and between
Figure 1. Key 1H−1H COSY and HMBC correlations of compounds 1
and 4.
H-31/C-23, C-24, and C-25 indicated that the presence of an
olefinic methylene group at C-24 and a cyclopropane moiety
connected to C-9 and C-10, as found in 7. The β-orientation of the
hydroxy groups at C-3, C-6, and C-7 was deduced from the
NOESY experiment, showing NOE correlations between H-3/
H-5ax and H3-29, H-6/H-7 and H3-29, and H-7/H-6 and H3-28,
respectively (Figure 2). Hence, the structure of 1 was established as
24-methylenecycloartane-3β,6β,7β-triol.
RESULTS AND DISCUSSION
■
An 80% EtOH extract of H. riparia leaves was suspended in H2O
and partitioned successively with n-hexane, EtOAc, and n-BuOH.
The EtOAc-soluble fraction was subjected to a series of
chromatographic techniques, leading to the isolation of six new
triterpenes (1−6), along with one known compound (7), which
was identified as 24-methylenecycloartane-3β,6β,7β,16β-tetraol
3-O-β-D-xylopyranoside by comparing its observed and
published physicochemical data.9
Compound 2 was obtained as a white powder. The molecular
formula of 2 was found to be C31H52O4 on the basis of a molec-
ular ion peak at m/z 511.3750 [M + Na]+ in the HRESIMS. The
1H and 13C NMR chemical shifts of the C-19 methylene group
and C-18, C-28, C-29, and C-30 tertiary methyl groups were very
similar to those of 1, suggesting that 2 has the same cycloartane
nucleus with an exocyclic methylene group. The difference in
Compound 1 was obtained as a white powder with a positive
specific rotation, [α]2D5 +90.4 (c 0.05, pyridine). The HRESIMS
of 1 exhibited a molecular ion peak at m/z 495.3809 [M + Na]+,
corresponding to the molecular formula C31H52O3. The IR
spectrum exhibited absorption bands at 3430 cm−1 (hydroxy
group) and 1640 and 890 cm−1 (1,1-disubstituted double bond).
1
the H NMR spectrum of 2 compared to that of 1 was the
appearance of a signal at δH 4.75, which is assignable to the
hydroxymethine proton instead of the 16-methylene protons in 1.
Inspection of the 13C NMR spectrum when compared to 1 revealed
a marked downfield shift of the C-16 signal (δC 72.3), suggesting
the presence of a hydroxymethine group at C-16 in 2, which was
further supported by the 1H−1H COSY correlation between H-15,
H-16, and H-17, coupled with the HMBC correlation of H-16 with
C-14, C-17, and C-20. The β-configuration of the hydroxy groups
at C-3, C-6, C-7, and C-16 was inferred from the NOE correlations
between H-3/H-5ax and H3-29, H-6/H-7 and H3-29, H-7/H-6 and
H3-28, and H-16/H-17ax and H3-28, respectively, in the NOESY
spectrum. Thus, the structure of 2 was assigned as 24-methylene-
cycloartane-3β,6β,7β,16β-tetraol.
Compound 3 was obtained as a white powder and showed a
molecular ion peak at m/z 495.3803 [M + Na]+ in the HRESIMS,
corresponding to the molecular formula C31H52O3. The 1H NMR
spectrum of 3 was very similar to those of 1 and 2, with the
appearance of two typical high-field doublets at δH 1.61 and 0.60
(each 1H, d, J = 3.5 Hz) as well as two singlet olefinic protons at δH
4.92 and 4.85 (each 1H, s), suggesting that 3 is a cycloartane
triterpene with the same skeleton as 1 and 2. A comparison of the
1H and 13C NMR spectra of 3 with those of 2 revealed them to be
very similar, with the only difference being the appearance of a set
of proton signals at δH 1.70 and 1.46 (each 1H, m) corresponding
to a methylene carbon signal at δC 35.8 in the HMQC experiment.
These methylene proton signals (δH 1.70 and 1.46) revealed
1H−1H COSY correlations with H-6 and H-8, as well as HMBC
correlations with C-6 and C-8, indicating the presence of a methy-
lene group at C-7. The relative configuration of 3 was determined
on the basis of the NOESY experiment and a comparison with that
1
The H NMR spectrum of 1 showed signals for four tertiary
methyls [δH 1.78, 1.50, 1.31, and 1.21 (each s)], three secondary
methyls [δH 1.08 (3H, d, J = 7.0 Hz), 1.07 (3H, d, J = 7.0 Hz), and
1.02 (3H, d, J = 6.5 Hz)], and three hydroxymethine protons [δH
4.51 (1H, br s), 3.85 (1H, dd, J = 11.5, 1.5 Hz), and 3.59 (1H, dd,
J = 11.5, 4.0 Hz)]. The two high-field doublets observed at δH
1.60 (1H, d, J = 3.5 Hz) and 0.60 (1H, d, J = 3.5 Hz) are charac-
teristic of the two germinal protons of a cyclopropane
moiety.10,11 In addition, two singlet olefinic protons at δH 4.97
(1H, s) and 4.87 (1H, s) indicated the presence of an olefinic
methylene moiety. The 13C NMR spectrum, combined with the
DEPT data, showed that 1 has 31 carbons, consisting of seven
methyls, 10 methylenes, eight methines, and six quaternary
carbons. Of these, the signals at δC 79.1, 74.2, and 73.1 indicated
the presence of three oxygenated methines, and two sp2 carbons
at δC 157.6 and 107.0 revealed that 1 contains an exocyclic
(CH2) methylene group. These spectroscopic data suggested
1 to be a cycloartane-type triterpene with an exocyclic methylene
group.12−15 In addition, the 1H and 13C NMR data of 1 were very
similar to those of the aglycone of 7, except for the additional
signals consistent with the presence of a methylene unit [δH 2.06 m,
1.34 m; δC 29.6 (t)] instead of a hydroxymethine group at
C-16 [δH 4.74 (br q); δC 72.3 (d)] in 7. This finding indicates that 1
differs from 7 in the substitution pattern of ring D, which was
supported by analysis of the 2D NMR spectra. The 1H−1H COSY
correlations between H-2/H-3, and H-5, H-6, H-7, and H-8, as well
1
as the long-range H−13C coupling (HMBC) observed between
H-3/C-4 and C-29, H-6/C-5 and C-7, and H-7/C-6 and C-8,
1313
dx.doi.org/10.1021/np300224k | J. Nat. Prod. 2012, 75, 1312−1318