1896
B. T. T. Luyen et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1895–1900
Table 1
Cytokines are soluble, hormone-like protein mediators, and
NMR data of compounds 1 and 2
TNF-a
is one of the major inflammatory cytokines involved in sys-
is a well-characterized,
temic inflammation and immunity. TNF-
a
No.
1
No.
1
pro-inflammatory cytokine released primarily from monocytes
and macrophages upon invasion of the host by a wide variety of
a,b
a,c
d,b
d,c
dH (mult., J in Hz) dC
dH (mult., J in Hz) dC
1
2
3
4
5
6
10
20
30
40
—
152.2
119.7
116.7
153.9
116.7
119.7
103.6
75.0
2
3
4
5
6
7
8
9
—
—
—
—
160.4
117.7
138.3
110.8
113.3
150.2
138.9
110.4
140.0
33.0
pathogens. TNF-
a affects various cell types and is involved in the
6.97 (d, 8.4)
6.69 (d, 8.4)
—
production of cytokines, endothelial gene regulation, chemotaxis,
leukocyte adherence, and fibroblast activation. It also plays a cen-
tral role in the inflammatory response and is involved in the path-
ogenesis of both acute and chronic inflammatory diseases.15
Recently, many compounds isolated from natural sources have
been found to exhibit anti-inflammatory activities by inhibiting
6.69 (d, 8.4)
6.97 (d, 8.4)
4.76 (d, 6.6)
3.44e
7.45 (s)
—
—
—
—
3.44e
77.9
71.2
10
11
3.44e
3.79 (d, 17.4)
3.76 (d, 17.4)
—
TNF-
a
and NO production in macrophages.16–19 Thus, agents that
might be promis-
block excessive production of NO and/or TNF-
a
50
60
3.31 (m)
3.74 (dd, 5.4; 8.4)
4.01e
4.82 (br s)
5.15 (d, 4.2)
4.18 (dd, 4.2; 9.6)
5.09 (t, 9.6)
4.01e
1.09 (d, 6.6)
—
7.90 (d, 8.4)
6.83 (d, 8.4)
—
76.6
67.2
12
13
170.0
75.4
ing candidates for the treatment of inflammatory diseases.
Herein, we report the isolation and structure elucidation of
chemical constituents from E. humifusa, as well as their anti-
inflammatory activity by sEH inhibitory effects, and inhibition of
6.79 (s)
100
98.9
74.0
68.6
75.4
67.8
17.8
122.0
133.0
116.2
163.6
116.2
133.0
167.7
172.2
20.9
14
15
—
—
168.1
161.4
52.5
200
300
12-OMe 3.65 (s)
14-OMe 3.62 (s)
LPS-induced NO and TNF-a production in RAW 264.7 cells.
400
54.2
Dried whole plants of Euphorbia humifusa Willd. were pur-
chased from herbal market in Iksan 2012 and taxonomically iden-
tified by one of authors (Young Ho Kim). A voucher specimen
(CNU-12110) was deposited at the Herbarium of College of Phar-
macy, Chungnam National University, Republic of Korea.
500
600
1000
2000
3000
4000
5000
6000
7000
10000
20000
A methanol extract of E. humifusa was successively separated by
column chromatography (CC) to yield 26 compounds (see Support-
ing data, Fig. 1), including two new compounds (1 and 2). Com-
pound 120 was obtained as a pale yellow powder. The HR-ESI-MS
spectrum of compound 1 contained clusters of quasi-molecular
ion peaks at m/z 579.1713 [MꢀH]ꢀ and 615.1478 [M+Cl]ꢀ, indicat-
ing its molecular formula to be C27H32O14. The 1H NMR spectra of 1
showed signals of two para-disubstituted benzene fragments [dH
7.90 and 6.83 (each 2H, d, J = 8.4 Hz), dH 6.97 and 6.69 (each 2H,
d, J = 8.4 Hz)]. The signals of two anomeric protons at dH 4.76 (d,
J = 6.6 Hz), 4.82 (br s) indicated the presence of two sugar units in
the structure of 1. The 13C NMR spectrum revealed resonant signals
of 27 carbons (see Table 1) which were in good agreement with its
molecular formula, C27H32O14. Among them, two carbonyl groups
were detected at dC 167.7 (C-7000) and 172.2 (C-10000). Twelve aromatic
carbon signals (dC 116.2 through 163.6) belonging to two benzene
rings were also detected. Two anomeric carbons (dC 103.6 and
98.9), nine carbinol carbons (dC 67.2 through 77.9), and a methyl
group (dC 17.8) were assigned as a rhamnose–glucose disaccharide
moiety.
6.83 (d, 8.4)
7.90 (d, 8.4)
—
—
2.15 (s)
a
b
c
Measured in CD3OD.
Measured in 600 MHz.
Measured in 150 MHz.
Measured in DMSO-d6.
d
e
Overlapped signals. Assignments were done by the COSY, HMQC, and HMBC
experiments.
hydrolysis of 1 followed by TLC, GC analysis, as well as comparison
with authentic sugars, which were further confirmed the presence
of glucose and rhamnose moieties in 1 (see Supporting data).
Consequently, the structure of compound 1 was determined
to be 200-O-acetyl-400-O-p-hydroxybenzoyl-p-hydroxyphenyl-
a-L-
rhamnopyranosyl-(1 ? 6)-O-b-D-glucopyranoside, and named
euphorbinoside.
Compound 220 was obtained as a pale yellow powder. Its molec-
ular formula was deduced to be C16H12O10 from a quasi-moleculaꢀr
These NMR data suggests that compound 1 is a phenylglyco-
side. Moreover, the HMBC correlation (see Fig. 2) of a singlet
methyl proton (dH 2.15) with a carbonyl carbon dC 172.2 indicated
the presence of an acetyl group. A pair of aromatic protons (dH 7.90,
H-2000 and H-6000) correlated with C-7000 (dC 167.7) and C-4000 (dC
163.6), suggesting a para-hydroxybenzoic acid moiety. The string
of COSY correlations (H-10/H-20/H-30/H-40/H-50/H-60, and H-100/H-
200/H-300/H-400/H-500/H-600) confirmed main chain assignments of
protons of glucose and rhamnose moieties, respectively (see
Fig. 2). The acetyl group and para-hydroxybenzoic acid moiety lo-
cated at C-200 and C-400 of rhamnose which were supported by
HMBC correlations H-200 (dH 5.15)/C-10000 (dC 172.2) and H-400 (dH
5.09)/C-7000 (dC 167.7). Otherwise, correlations between rhamnose
H-100 (dH 4.82) and glucose C-60 (dC 67.2), and glucose H2-60 (dH
3.74, 4.01) with rhamnose C-100 (dC 98.9) in the HMBC spectra sug-
gested the structure of a (1 ? 6) disaccharide. The glucose linked
with a benzene ring through an ether linkage was confirmed by
HMBC correlation between an anomeric proton H-10 (dH 4.76) with
C-1 (dC 152.2). A hydroxy group substituted at C-4 was suggested
by a downfield shift of resonant signals at C-4 (dC 153.9) in the
13C NMR spectra. It also was in agreement with HMBC correlations
of pair protons H-2, H-6 (dH 6.97) and C-4 (dC 153.9). Finally, acid
ion peak at m/z 363.0360 [MꢀH]ꢀ (calcd for [C16H11O10
]
363.0358) according to the HR-ESI-MS spectra. The 1H NMR spec-
tra of 2 exhibited four sets of signals: aromatic proton H-6 (dH
7.45), oxygenated methine proton H-13 (dH 6.79), methylene group
H2-11 [dH 3.79, 3.76 (each 1H, d, J = 17.4 Hz)], and two methoxy
groups [dH 3.62 and 3.65 (each 3H, s)]. The 13C NMR and DEPT spec-
tra of 2 (see Table 1) revealed signals of 16 carbons including 11
quaternary carbons, two tertiary carbons, a methylene carbon,
and two methoxy groups. Basically, the 1D NMR spectral patterns
of 2 were similar to those of picrorhiza acid (2a),21 with the excep-
tion of the appearance of two methoxy groups and the loss of two
hydrogen signals.
The chemical structure of 2 was further confirmed by the HMQC
and HMBC experiments (see Fig. 2). A singlet of an aromatic proton
signal (dH 7.45, H-6) and its HMBC correlations with C-7 (dC 150.2),
C-8 (dC 138.9), C-10 (dC 140.0), and C-15 (dC 161.4) indicated a
substituted galloyl (3,4,5-trihydroxybenzoyl) moiety. The first lac-
tone linkage between C-13 and carbonyl group (C-15) was con-
firmed by HMBC correlation of H-13 (dH 6.79, s)/C-15 (dC 161.4).
Another lactone linkage between C-2 and C-9 was suggested by
an up-field shift of carbonyl carbon C-2 (dC 160.4), as well as