E. Selenge et al. / Phytochemistry 101 (2014) 91–100
95
moiety. The proton resonances of oxymethylene in the glucose of 3
(dH 3.67, 1H, dd, J = 12.0, 5.5 Hz, H-60; 3.86, 1H, dd, J = 12.0, 2.0 Hz,
H-60) were shifted to a higher field than those of 1. In the differen-
tial HOHAHA spectra of 3, the anomeric proton of the first glucose
moiety (dH 4.43, 1H, d, J = 7.0 Hz, H-10) was correlated with the
oxymethylene protons (H-60). In the HMBC spectrum, the H-600
protons (dH 4.23, 1H, dd, J = 12.0, 5.5 Hz; 4.41, 1H, br d,
J = 12.0 Hz) were long-range coupled with the malonyl carbonyl
carbon (dC 168.7, C-700). These data suggested that 3 has a malonyl
moiety at the C-6 position of the second glucose moiety, not at
the first glucose moiety. Thus, 3 was identified as limonene-10-ol
with 11, corresponded to an additional malonyl unit, C3H2O3, and
supported this conclusion. The sugar analysis using HPLC (Tanaka
et al., 2007) and the coupling constant of the anomeric proton
(dH 4.85, d, J = 7.5 Hz) suggested that the glucosyl moiety was
b-D-glucopyranosyl. The Glc-6 protons (dH 4.32, dd, J = 12.0,
7.0 Hz; 4.56, dd, J = 12.0, 1.5 Hz) and carbon (dC 65.6) of 8 were
downfield shifted relative to those of 11. These shifts suggested
that the malonyl group connected to C-6 of the b-D-glucopyranosyl
unit. Consequently, compound 8 was identified as 3-(6-malonyl-b-
D-glucopyranosyl)-rosmarinic acid.
The molecular formula C34H34O16 for 9 was determined by
10-O-6-malonyl-b-
as shown in Fig. 1.
D
-glucopyranosyl-(1?2)-b-
D
-glucopyranoside,
HRFABMS (m/z 721.1727, calcd for C34H34O16Na, 721.1743). In
the 1H-NMR spectrum, the ABX system proton resonances (dH
7.00, 1H, d, J = 2.0 Hz, H-200, 6.76, 1H, d, J = 8.0 Hz, H-500, 6.90, 1H,
dd, J = 8.0, 2.0 Hz, H-600) and trans olefinic proton resonances (dH
7.53, 1H, d, J = 16.0 Hz, H-700, 6.27, 1H, d, J = 16.0 Hz, H-800) were
observed. The singlet methoxy proton (dH 3.83, 3H) was correlated
with the H-200 in the NOE spectrum. These data suggested that 9
possesses a feruloyl moiety instead of a malonyl moiety for 8.
The absolute configuration at C-4 of 1–3 was not determined.
Compound 4 was isolated from D. kotschyi (Saeidnia et al., 2004),
and d-limonene derivatives were also determined. The expected
aglycones of 1–4 from D. foetidum are likely to be a d-limonene
derivatives.
The molecular formula of 5 (C28H25O12, m/z 553.1358 [M+H]+,
calcd for C28H25O12, 553.1346) was established using HRFABMS.
The 1H- and 13C-NMR spectroscopic data (measured in acetone-
d6 at 30 °C) are shown in Table 2. Three sets of coupling system
protons (dH 6.85, 1H, d, J = 2.0 Hz, H-2, 6.75, 1H, d, J = 8.0 Hz, H-5,
6.67, 1H, dd, J = 8.0, 2.0 Hz, H-6; 7.29, 1H, d, J = 2.0 Hz, H-20, 6.82,
1H, d, J = 8.0 Hz, H-50, 7.07, 1H, dd, J = 8.0, 2.0 Hz, H-60; 7.38, 1H,
d, J = 2.0 Hz, H-200, 6.97, 1H, d, J = 8.0 Hz, H-500, 7.26, 1H, dd,
J = 8.0, 2.0 Hz, H-600) and 27 carbon resonances, except for the pres-
ence of a methoxy resonance at dC 56.2, suggested that 5 was a
phenylpropanoid trimer. These resonances were very similar to
those of melitric acid A (Agata et al., 1993). The methoxy proton
resonance at d 3.87 (3H, s) was long-range coupled with the carbon
resonance at d 150.2 (C-400) in the HMBC spectrum and was corre-
lated with the H-500 in the differential NOE spectrum (Fig. 3). From
Hence,
9 was identified as 3-(6-feruloyl-b-D-glucopyranosyl)-
rosmarinic acid.
The molecular formula C35H36O17 for 10 was determined by
HRFABMS (m/z 751.1843, calcd for C35H36O17Na, 751.1849). In
the NOE spectrum, H-200 and 600 singlet proton resonances (dH
6.73, 2H) were correlated with the methoxy proton resonances
(dH 3.85, 6H) and the trans olefinic proton resonances (dH 7.53,
1H, d, J = 16.0 Hz, H-700, 6.30, 1H, d, J = 16.0 Hz, H-800). These data
suggested that 10 possesses a synapoyl moiety, instead of the
malonyl moiety of 8. Hence, 10 was identified as 3-(6-synapoyl-
b-
D-glucopyranosyl)-rosmarinic acid.
The molecular formula of 12 (HRFABMS, C24H27O13
,
m/z
523.1459 [M+H]+, calcd for C24H27O13, 523.1451) was the same
as that of 11. The 1H- and 13C-NMR resonances of 12 were similar
to 11, except for a caffeoyl moiety on the rosmarinic acid moiety
(Table 2). The coupling constant of the olefinic protons (dH 6.88,
1H, d, J = 13.0 Hz, H-70, 5.82, 1H, d, J = 13.0 Hz, H-80) suggested that
12 has a cis-oriented caffeoyl moiety (Wei et al., 2004). The NOE
correlation between H-70 and H-80 supported this conclusion.
these data, 5 was identified as (aR)-a-[[(2E)-3-[4-[[(1Z)-1-carboxy-
2-(3-hydroxy-4-methoxyphenyl)ethenyl]oxy]-3-hydroxyphenyl]-
1-oxo-2-propen-1-yl]oxy]-3,4-dihydroxy-benzenepropanoic acid,
which is the 400-methoxy derivative of melitric acid A, as shown
in Fig. 1.
The 1H- and 13C-NMR spectroscopic data (measured in CD3OD
at 30 °C) of compounds 8–10 are shown in Table 2. The resonances
corresponding to rosmarinic acid and glucopyranosyl moieties
were observed in the NMR spectra, which were similar to those
of 11. In addition, the resonances of acyl moieties of 8–10 were
observed in each respective spectrum. Their Glc-6 protons and
carbons were downfield shifted relative to that of 11, suggesting
an acyl moiety bond to C-6 of each glucopyranosyl of 8–10.
For 8, typical malonyl carbonyl carbons (dC 168.9 and 170.3),
such as 1–3, were observed in the 13C-NMR spectrum, which
suggested that the acyl moiety of 8 was malonyl. The molecular
formula C27H28O16 established using HRFABMS (m/z 609.1428
[M+H]+, calcd for C27H29O16, 609.1455), which, when compared
Therefore, 12 was identified as the cis-isomer of 3-O-b-D-glucopyr-
anosyl-rosmarinic acid, as shown in Fig. 1.
The absolute stereochemistries of the C-80 of 8–10 and 12 were
determined to be R from the retention time of the amide derivative
of the (S)-2-phenylglycine methyl ester and 3-(3,4-dihydroxy-
phenyl)-2-hydroxypropanoic acid after acidic hydrolysis of the
compounds (Murata et al., 2010b).
Compounds 13–17 were obtained as colorless powders with UV
(measured in MeOH) and NMR (measured in DMSO-d6 at 30 °C,
Table 3) data. Their UV spectra were very similar to that of acacetin
(Greenham et al., 2003). A carbonyl, a methoxy, and 14 phenolic or
olefinic carbons were observed in the 13C-NMR spectrum of each
compound, which suggested the presence of acacetin as the
aglycone of 13–17 (Selenge et al., 2013a). The fragment ion peak
(m/z 285, [C16H12O5+H]+) in the FABMS spectra of 13–17 supported
this conclusion.
The 1H- and 13C-NMR resonances of 13 and 14 showed that they
had glycosyl and malonyl moieties. Sugar analysis of 13 and 14
using HPLC suggested the presence of D-glucopyranosyl moieties.
The coupling constants of the anomeric proton resonances (13:
J = 8.0 Hz; 14: J = 7.5 Hz) showed the b-configuration for the
O
HO
OH
O
MeO
O
O
OH
OH
HO
D-glucopyranosyl component.
O
The molecular formula of 13 was determined as C25H24O13 on
OH
the basis of HRFABMS (m/z 533.1290, calcd for
C25H25O13,
533.1295), which, when compared with 18, corresponded to an
additional malonyl unit of the formula C3H2O3. The methylene
proton and carbon resonances (dH 3.41, 2H, s; dC 41.6) and two
carbonyl carbon resonances (dC 166.4, 167.8) indicated a malonyl
C : key HMBC correlations
: key NOE correlations
H
Fig. 3. Key HMBC and NOE correlations of 5.