Hepatoprotective constituents from the roots and stems of erycibe hainanesis

Article abstract of DOI:10.1021/np900593q

Eleven new diglycosides, erycibosides A-K (1-11), four new chlorogenic acid derivatives (14-17), and a new biscoumarin (18), together with 21 known compounds, have been isolated from an EtOH extract of the roots and stems of Erycibe hainanesis. Their structures were elucidated by means of spectroscopic methods and chemical evidence. Inhibitory activities of some of the compounds on D-galactosamine-induced cytotoxicity in WB-F344 rat hepatic epithelial stemlike cells were screened, and compounds 2, 6, 10, 18, and 32 showed potent hepatoprotective activities at concentrations of 1 × 10^-5 to 1 × 10^-4 M.

Full text of DOI:10.1021/np900593q

J. Nat. Prod. 2010, 73, 177–184
177
Hepatoprotective Constituents from the Roots and Stems of Erycibe hainanesis
Shuang Song, Yixiu Li, Ziming Feng, Jianshuang Jiang, and Peicheng Zhang*
Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College (Key Laboratory of BioactiVe
Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Education), Beijing 100050, People’s Republic of China
ReceiVed September 24, 2009
Eleven new diglycosides, erycibosides A-K (1-11), four new chlorogenic acid derivatives (14-17), and a new bis-
coumarin (18), together with 21 known compounds, have been isolated from an EtOH extract of the roots and stems of
Erycibe hainanesis. Their structures were elucidated by means of spectroscopic methods and chemical evidence. Inhibitory
activities of some of the compounds on D-galactosamine-induced cytotoxicity in WB-F344 rat hepatic epithelial stem-
like cells were screened, and compounds 2, 6, 10, 18, and 32 showed potent hepatoprotective activities at concentrations
of 1 × 10-5 to 1 × 10-4 M.
The genus Erycibe roxb. (Convolvulaceae) consists of about 66
species, with 11 species found in China. However, only E.
obtusifolia, E. schmidtii, E. hainanesis, E. expansa, and E.
elliptilimba were chemically investigated previously. Flavonoids,
coumarins, chlorogenic acids, alkaloids, and several other compo-
nents were reported from Erycibe species.1–5 Some of them have
been shown to exhibit anti-inflammatory, muscarinic agonistic, and
cytotoxic activities.3,6–8 Our previous phytochemical study of E.
obtusifolia, used in Chinese folk medicine to relieve symptoms of
rheumatoid arthritis, led to the isolation of two new bis-coumarins,
a new coumarin glucoside, and a new chlorogenic acid derivative,
together with four known coumarins.9 Continuing our study on the
constituents and bioactivities of the plants of the genus Erycibe,
we investigated E. hainanesis Merr., a species growing in Guang-
dong, Hainan, and Guangxi Provinces of the People’s Republic of
China.10 Sixteen new compounds including 11 diglycosides,
erycibosides A-K (1-11), four chlorogenic acid derivatives
(14-17), and a bis-coumarin (18) were isolated, along with 21
known compounds, which were identified by comparison of
experimental and reported spectroscopic data as 1-O-[6-O-(5-O-
syringoyl-ꢀ-D-apiofuranosyl)-ꢀ-D-glucopyranosyl]-3,4,5-trimethoxy-
benzene (12),11 seguinoside E (13),12 caffeic acid (19),13 3,4-
dihydroxybenzoic acid (20),14 trans-N-feruloyltyramine (21),15 7,7′-
dihydroxy-6,6′-dimethoxy-3,3′-bis-coumarin (22),9 trans-N-(p-
coumaroyl)tyramine (23),16 chlorogenic acid (24),17 methyl
nm), typical of a coumarin. The negative HRESIMS data of 1
indicated an [M - H]- ion at m/z 665.1704 corresponding to the
molecular formula C30H34O17 (calcd for C30H33O17, 665.1712). In
chlorogenate (25),18 methyl-3-O-(4′′-hydroxy-3′′,5′′-dimethoxy-
benzoyl)chlorogenate (26),9 (+)-lyoniresinol 3a-O-ꢀ-D-glucopy-
ranoside (27),19 4-hydroxy-3-methoxybenzoic acid (28),20 ethyl
chlorogenate (29),21 3-O-caffeoylquinic acid butyl ester (30),22 ethyl
3,4-dicaffeoylquinate (31),23 7R,8R,8′S-aketrilignoside B (32),24
aketrilignoside B,24 cis-N-feruloyltyramine,25 syringaresinol-di-O-
ꢀ-D-glucopyranoside,26 scopoletin,27 and scopolin.28 Additionally,
the hepatoprotective activities of compounds 1-16 and 18-32
against D-GalN-induced cytotoxicity in the primary cultured mouse
hepatocytes were examined.
1
the H NMR spectrum of 1, a pair of doublets at δ 6.24 (1H, d, J
) 9.5 Hz) and 7.86 (1H, d, J ) 9.5 Hz), and two aromatic singlets
at δ 7.20 (1H, s) and 7.13 (1H, s), indicated the presence of a 6,7-
disubstituted coumarin skeleton. In addition, the characteristic
signals at δ 7.15 (2H, s) and 3.78 (6H, s) suggested the existence
of a syringoyl moiety, while two doublets due to anomeric protons
at δ 5.06 (1H, d, J ) 7.0 Hz, H-1′) and 4.86 (1H, d, J ) 2.5 Hz,
H-1′′), together with the partially overlapped signals between δ
3.08 and 5.28, showed the presence of two glycosyl groups. An
apiofuranose moiety could be assumed from the occurrence of two
pairs of doublets at δ 4.23 (1H, d, J ) 11.0 Hz) and 4.19 (1H, d,
J ) 11.0 Hz) and at δ 4.04 (1H, d, J ) 9.5 Hz) and 3.87 (1H, d,
J ) 9.5 Hz) for the two methylene groups (C-4′′ and C-5′′),
respectively. The 13C NMR spectrum showed 30 signals (see Table
3). Except for 19 carbon signals assigned as a coumarin skeleton
with a methoxy and a syringoyl group, the remaining 11 carbon
signals were attributable to glucosyl and apiosyl moieties. Further-
more, the coupling constant (J ) 7.0 Hz) of the anomeric proton
of the glucosyl moiety as well as the chemical shift (δ 109.4) of
the anomeric carbon of the apiosyl moiety demonstrated that both
sugar moieties had ꢀ-anomeric configurations.29 Comparison of the
Results and Discussion
The EtOH extract of the roots and stems of E. hainanesis was
suspended in H2O and then sequentially partitioned with petroleum
ether, EtOAc, and n-BuOH. The n-BuOH and EtOAc fractions were
subjected to separation using various column chromatographic
techniques to afford 16 new compounds (1-11 and 14-18),
together with the known compounds mentioned above.
Compound 1 was obtained as a white powder, [R]20 -83.6 (c
D
0.05, MeOH), and it showed blue fluorescence under UV light (365
* To whom correspondence should be addressed. Tel: 86-10-63165231.
Fax: 86-10-63017757. E-mail: pczhang@imm.ac.cn.
10.1021/np900593q  2010 American Chemical Society and American Society of Pharmacognosy
Published on Web 01/21/2010

178 Journal of Natural Products, 2010, Vol. 73, No. 2
Song et al.
Table 1. 1H NMR Spectroscopic Data (δ) of Compounds 1-6a
position
1
2
3
4
5
6
3
4
5
6.24, d (9.5)
7.86, d (9.5)
7.20, s
6.21, d (9.5)
7.79, d (9.5)
7.14, s
6.28, d (9.5)
7.83, d (9.5)
7.03, s
6.25, d (9.5)
7.88, d (9.5)
7.23, s
6.28, d (9.5)
7.82, d (9.5)
7.00, s
6.35, d (9.5)
7.88, d (9.5)
7.05, s
8
7.13, s
7.05, s
6.98, s
7.14, s
7.00, s
1′
5.06, d (7.0)
3.11, m
3.80, m
3.30, m
3.67, m
5.06, d (7.0)
3.11, m
3.80, m
3.30, m
3.65, m
5.08, d (8.0)
3.62, m
3.43, m
5.06, d (5.0)
3.12, m
3.61, m
3.25, m
3.25, m
5.10, d (7.5)
3.62, m
3.42, m
3.17, m
3.52, m
5.08, d (7.0)
3.09, m
3.76, m
3.24, m
3.27, m
2′
3′
4′
3.17, m
5′
3.42, m
6′a
3.77, m
3.50, m
3.89, m
3.52, m
3.75, mb
3.86, m
3.49, m
3.72, m
3.45, m
3.77, m
3.43, m
6′b
3.52, m
1′′
4.86, d (2.5)
3.83, d (2.5)
4.04, d (9.5)
3.87, d (9.5)
4.23, d (11.0)
4.19, d (11.0)
7.15, s
5.14, d (2.5)
4.91, d (2.5)
4.02, d (9.5)
3.70, d (9.5)
3.47, s
5.48, br s
3.75, br s
4.26, d (10.0)
3.76, d (10.0)
4.18, d (11.5)
4.06, d (11.5)
7.03, s
4.85, br s
3.79, br s
4.02, d (9.5)
3.81, d (9.5)
4.23, d (11.0)
4.19, d (11.0)
7.37, d (2.0)
6.79, d (7.5)
7.43, dd (7.5, 2.0)
3.79, s
5.48, br s
4.74, d (2.5)
3.66, mb
3.73, d (9.5)
3.67, d (9.5)
4.08, s
2′′
3.75, mb
4′′a
4′′b
5′′a
5′′b
2′′′
4.28, d (10.0)
3.82, d (10.0)
4.14, d (11.0)
4.05, d (11.0)
7.23, br s
6.67, d (7.5)
7.27, d (7.5)
3.66, s
7.08, s
7.19, s
5′′′
6′′′
6-OMe
8-OMe
3′′′-OMe
5′′′-OMe
7.15, s
3.80, s
7.08, s
3.80, s
7.03, s
3.68, s
7.19, s
3.79, s
3.88, s
3.79, s
3.79, s
3.78, s
3.78, s
3.78, s
3.78, s
3.74, s
3.74, s
3.77, s
3.74, s
a
1H NMR data (δ) were measured in DMSO-d6 at 500 MHz. Coupling constants (J) in Hz are given in parentheses. b Overlapping signals.
Table 2. 1H NMR Spectroscopic Data (δ) of Compounds 7-11a
position
7
8
9
10
11
1
2a
2b
3
4a
4b
5
6
1.07, d (6.5)
3.82b
6.99, d (8.5)
6.62, d (8.5)
1.50, t (11.5)
1.20b
1.49, t (11.5)
1.27, dd (11.5, 2.5)
3.82b
1.61, dd (12.5, 4.5)
1.55, t (12.5)
1.50b
1.09, m
3.49, m
1.36b
1.25, q (11.5)
1.66, m
1.04, d (6.5)
3.57, m
1.47b
1.25, q (12.0)
1.75, m
6.62, d (8.5)
6.99, d (8.5)
2.68, m
7
8a
5.44, d (16.5)
5.61, dd (16.5, 6.0)
5.98, d (16.0)
5.71, dd (16.0, 7.0)
1.47b
1.40b
3.82, m
8b
3.55, m
9
10
11
4.24, m
1.15, d (6.5)
0.75, s
4.30, m
1.19, d (6.0)
1.07, s
3.58, m
1.03, d (6.5)
0.84, s
12
0.84, s
0.72, s
0.84, s
13
0.67, d (6.5)
4.17, d (8.0)
2.93, dd (8.5, 7.5)
3.11, dd (9.0, 8.5)
3.02, dd (9.0,9.0)
3.18, m
0.99, s
0.78, d (6.5)
4.14, d (8.0)
2.88, m
3.11, m
2.97, m
1′
4.12, d (7.5)
2.88, m
3.11, m
4.14, d (8.0)
2.94, m
3.13, m
2.98, m
3.30, m
4.22b
2′
2.94, m
3.13, m
3.03, m
3.19, m
3.82b
3.44, m
4.91, d (2.5)
3.84b
3′
4′
2.95, m
5′
3.24, m
3.22, m
6′a
3.82b
3.45, m
3.25, m
3.81b
3.80b
6′b
3.43, m
3.44, dd (11.5, 7.0)
4.90, d (2.5)
3.81b
3.43, m
1′′
4.92, d (2.5)
4.92, d (2.5)
3.82, d (2.5)
3.93, d (9.5)
3.86, d (9.5)
4.23, d (11.0)
4.19, d (11.0)
7.23, s
4.91b
2′′
3.83b
3.80b
4′′a
3.93, d (9.5),
3.77, d (9.5)
4.25, d (11.5)
4.25, d (11.5)
7.23, s
3.93, d (9.0)
3.80, d (9.0)
4.25b
3.94, d (9.5),
3.90, d (9.5)
4′b
3.79b
3.78b
5′′a
4.20b
4.25, d (11.5)
4.22, d (11.5)
7.23, s
5′′b
4.22b
4.19b
2′′′,6′′′
3′′′,5′′′-OMe
7.24, s
3.82, s
7.24, s
3.82, s
3.80, s
3.79, s
3.80, s
1H NMR data (δ) were measured in DMSO-d6 at 500 MHz. Coupling constants (J) in Hz are given in parentheses. b Overlapping signals.
a
NMR data of 1 with those of known compounds 12 and 13
suggested the presence of a 6-O-(5-O-syringoyl-ꢀ-apiofuranosyl)-
ꢀ-glucopyranosyl moiety. This was confirmed by HMBC correla-
tions (see Figure 1) of C-6′ with H-1′′ and C-7′′′ with H-5′′. The
HMBC correlations of C-6 with the methoxy protons at δ 3.80
and of C-7 with H-1′ indicated that the methoxy group and the
sugar chain were located at C-6 and C-7, respectively, of the
coumarin moiety. In addition, the glucose obtained from the
evidence, the structure of 1 was characterized as 7-O-[6-O-(5-O-
syringoyl-ꢀ-D-apiofuranosyl)-ꢀ-D-glucopyranosyl]-6-methoxycou-
marin and named eryciboside A.
Compound 2 was obtained as a white powder, [R]20 -39.3 (c
D
0.05, MeOH). The positive HRESIMS data of 2 showed an [M +
Na]+ ion at m/z 689.1686 corresponding to the same molecular
formula, C30H34O17, as 1. The NMR data of 2 showed close
resemblance to those of 1 (see Tables 1 and 3). Comparison of the
1H and 13C NMR data of 1 and 2 indicated that H-2′′ and C-2′′ of
2 were deshielded by ∆δH 1.08 and ∆δC 2.4 ppm, respectively,
while H-5′′ and C-5′′ were shielded by ∆δH 0.74 and ∆δC 2.1 ppm,
hydrolysis of 1 gave a positive specific rotation, [R]20 +47.4 (c
D
0.2, H2O), suggesting that it was D-glucose. The common D-
configuration for apiose was assumed. According to the above

HepatoprotectiVe Constituents from Erycibe hainanesis
Journal of Natural Products, 2010, Vol. 73, No. 2 179
Table 3. 13C NMR Spectroscopic Data (δ) of Compounds 1-11a
position
1
2
3
4
5
6
7
8
9b
10
11
1
2
3
4
5
6
7
8
23.4
70.1
21.8
128.6
129.7
115.0
155.5
115.0
129.7
34.8
39.2c (40.5)
45.1 (45.9)
40.0c
45.7
62.6
45.2
75.7
40.0c
46.8
160.3
113.3
143.9
109.6
145.8
149.7
102.9
148.8
112.3
160.4
113.3
143.7
109.5
145.8
149.7
102.8
148.9
112.3
160.5
113.1
144.0
109.0
145.5
149.2
102.5
148.7
112.1
160.3
113.3
143.9
109.7
145.9
149.7
103.0
148.8
112.3
160.5
113.1
144.0
109.0
145.5
149.3
102.5
148.7
112.1
159.7
114.7
144.2
105.4
149.4
141.6
140.3
142.3
114.7
64.9 (67.5)
64.9
39.7c (39.9)
33.6 (35.4)
40.1c
33.8
76.0 (78.6)
77.1
73.9
30.9
32.9
74.1
134.4 (135.9)
131.6 (133.4)
74.5 (78.1)
131.7
132.0
74.8
69.9
9
10
20.9 (21.4)
20.8
19.4
11
25.5 (25.3)
25.7
24.5
12
24.5 (26.2)
27.0
25.8
13
16.1 (16.5)
26.9
16.1
1′
99.5
72.9
77.0
69.9
75.4
68.1
109.4
77.0
77.0
73.5
99.6
72.9
76.7
70.0
75.4
67.4
107.3
79.4
78.4
74.3
97.8
77.2
77.1
70.0
74.4
60.5
108.0
76.9
77.4
73.6
99.6
72.9
76.7
69.8
75.4
67.9
109.3
76.9
77.0
73.5
97.8
77.1
77.0
69.9
74.4
60.5
107.9
76.9
77.4
73.5
102.3
73.9
76.3
70.0
75.9
67.4
108.8
77.0
76. 7
73.2
66.4
118.9
107.1
147.6
141.0
147.6
107.1
165.3
56.4
99.5
73.4
76.7
70.3
75.3
67.9
109.0
76.8
77.1
73.3
102.8
73.3
76.9
70.2
75.5
67.7
108.9
76.6
77.1
73.3
100.5 (102.4)
73.7 (75.3)
76.8 (78.2)
70.0 (71.6)
75.5 (77.6)
67.4 (68.7)
100.3
73.6
76.7
70.0
75.3
67.5
109.0
76.9
77.0
73.4
100.4
73.4
76.8
70.3
75.3
67.7
109.0
76.8
77.1
73.3
2′
3′
4′
5′
6′
1′′
109.0 (110.8)
77.1 (77.6)
2′′
3′′
76.8 (79.1)
73.4 (75.1)
66.8 (68.2)
119.5 (121.1)
107.1 (108.5)
147.5 (149.0)
140.2 (142.2)
147.5 (149.0)
107.1 (108.5)
165.4 (167.9)
4′′
5′′
66.5
64.4
66.7
66.4
66.4
66.7
66.6
66.9
66.8
1′′′
2′′′
3′′′
4′′′
5′′′
6′′′
7′′′
6-OMe
8-OMe
3′′′-OMe
5′′′-OMe
a
119.5
107.0
147.4
140.2
147.4
107.0
165.3
56.0
119.0
107.2
147.3
140.7
147.3
107.2
164.7
56.0
119.5
107.0
147.3
140.8
147.3
107.0
165.1
55.6
120.1
112.6
147.3
150.8
115.1
123.6
165.3
55.5
120.1
112.5
147.1
151.4
114.8
123.4
165.1
55.4
119.0
107.1
147.5
141.0
147.5
107.1
165.4
119.5
107.1
147.6
140.0
147.6
107.1
165.4
119.5
107.1
147.5
140.2
147.5
107.1
165.5
119.2
107.1
147.5
140.8
147.5
107.1
165.4
61.3
56.1
56.1
56.0
56.0
56.0
56.0
56.0
56.0
56.0
55.4
55.5
55.5
56.1
56.1
56.1 (57.0)
56.1 (57.0)
56.1
56.1
56.1
56.1
13C NMR data (δ) were measured in DMSO-d6 at 125 MHz. b Chemical shifts in parentheses were measured in MeOH-d4. c Signal overlapped by
solvent peaks.
1H NMR spectrum of 4, suggesting the presence of a vanilloyl
moiety. The 13C NMR spectrum of 4 showed carbon signals
corresponding to the vanilloyl moiety (see Table 3). Furthermore,
the HMBC spectrum displayed long-range correlations of C-7 with
H-1′, C-6′ with H-1′′, and C-7′′′ with H-5′′. Considering these
spectroscopic observations, 4 was determined as 7-O-[6-O-(5-O-
vanilloyl-ꢀ-D-apiofuranosyl)-ꢀ-D-glucopyranosyl]-6-methoxycou-
marin and named eryciboside D.
Figure 1. Selected HMBC correlations of 1.
Compound 5 was obtained as a white powder, [R]20 -32.9 (c
D
respectively. These data showed that the syringoyl group was linked
to C-2′′ of the ꢀ-D-apiofuranosyl moiety, as confirmed by an HMBC
correlation from H-2′′ to C-7′′′. Therefore, 2 was elucidated to be
7-O-[6-O-(2-O-syringoyl-ꢀ-D-apiofuranosyl)-ꢀ-D-glucopyranosyl]-
6-methoxycoumarin and named eryciboside B.
0.05, MeOH). The same molecular formula, C29H32O16, as 4 was
determined by the positive HRESIMS data ([M + Na]+, m/z found
659.1579). Comparison of the NMR data (see Tables 1 and 3) of
5 and 3 showed that the signals of the syringoyl moiety in 3 were
replaced by signals attributed to a vanilloyl moiety in 5. Further
confirmation was derived from HMBC correlations of C-3′′′ with
the methoxy protons at δ 3.74, C-6 with the methoxy protons at δ
3.66, C-7 with H-1′, C-2′ with H-1′′, and C-7′′′ with H-5′′. Thus 5
was assigned as 7-O-[2-O-(5-O-vanilloyl-ꢀ-D-apiofuranosyl)-ꢀ-D-
glucopyranosyl]-6-methoxycoumarin and named eryciboside E.
Compound 3 was obtained as a white powder, [R]20 -33.6 (c
D
0.03, MeOH), and the positive HRESIMS ion at m/z 689.1682 [M
+ Na]+ indicated it had the same molecular formula as 1. The NMR
spectroscopic data of 3 also resembled those of 1 (see Tables 1
and 3). However, the 13C NMR chemical shift differences of C-2′
(∆δC +4.3) and C-6′ (∆δC -7.6) for 3 and 1 suggested that the
apiofuranosyl moiety was located at C-2′ in 3 instead of C-6′ in 1.
This was supported by an HMBC correlation of C-1′′ with H-2′.
From these data, 3 was established as 7-O-[2-O-(5-O-syringoyl-
ꢀ-D-apiofuranosyl)-ꢀ-D-glucopyranosyl]-6-methoxycoumarin and
named eryciboside C.
Compound 6 was obtained as a white powder, [R]20 -3.8 (c
D
0.11, MeOH). Its molecular formula was determined as C31H36O18
by the positive HRESIMS data ([M + Na]+, m/z found 719.1801).
1
The H and 13C NMR data (see Tables 1 and 3) of 6 also showed
characteristic signals for a 6-O-(5-O-syringoyl-ꢀ-apiofuranosyl)-
ꢀ-glucopyranosyl moiety. The 1H NMR spectrum revealed the
presence of an additional methoxy group at δ 3.88 (3H, s) and the
absence of an aromatic proton observed in 1. When compared to
those of 1, C-8 of 6 was deshielded by ∆δC 37.4 ppm, while C-7,
C-9, and C-5 were shielded by ∆δC 8.1, 6.5, and 4.2 ppm,
respectively. These data suggested that the additional methoxy group
was located at C-8. This was confirmed by an HMBC correlation
from this methoxy group to C-8. In addition, the HMBC spectrum
Compound 4 was obtained as a white powder, [R]20 -62.4 (c
D
0.05, MeOH). Its molecular formula was determined to be
C29H32O16 from the positive HRESIMS data ([M + Na]+, m/z found
659.1583). An ABX spin system at δ 7.37 (1H, d, J ) 2.0 Hz),
6.79 (1H, d, J ) 7.5 Hz), and 7.43 (1H, dd, J ) 7.5, 2.0 Hz) and
a singlet for a methoxy group at δ 3.77 (3H, s), instead of the
characteristic signals of the syringoyl moiety, were observed in the

180 Journal of Natural Products, 2010, Vol. 73, No. 2
Song et al.
H-5 (J ) 12.0 Hz), implied that H-3 and H-5 must be in the axial
positions. The 3,6,9-trihydroxymegastigman-7-ene moiety with H-3
and H-5 in the axial positions has been reported,30–33 and except
for C-9, their absolute configurations were determined as 3S, 5R,
and 6S. Thus the ring system of 9 was presumed to have the same
configuration. The absolute configuration of C-9 was further
elucidated by comparing the 13C NMR data of 9 with those of
reported 9-O-glycosides of (3S,5R,6S,9R)-3,6,9-trihydroxymegastig-
man-7-ene and (3S,5R,6S,9S)-3,6,9-trihydroxymegastigman-7-ene.30,33
The 13C NMR data of the aglycone moiety of 9 were consistent
with those of (3S,5R,6S,9R)-3,6,9-trihydroxymegastigman-7-ene
moiety. Therefore, 9 was established as 9-O-[6-O-(5-O-syringoyl-
ꢀ-D-apiofuranosyl)-ꢀ-D-glucopyranosyl]-(3S,5R,6S,9R)-3,6,9-trihy-
droxymegastigman-7-ene and named eryciboside I.
Figure 2. Selected HMBC correlations of 9.
also showed correlations of C-7 with H-1′, C-6′ with H-1′′, and
C-7′′′ with H-5′′. All these data indicated the structure of 6 as 7-O-
[6-O-(5-O-syringoyl-ꢀ-D-apiofuranosyl)-ꢀ-D-glucopyranosyl]-6,8-
dimethoxycoumarin, named eryciboside F.
Compound 7 was obtained as a white powder, [R]20 -50.6 (c
D
0.10, MeOH), and its molecular formula was determined to be
C23H34O14 by the positive HRESIMS data ([M + Na]+, m/z found
557.1841). Signals derived from a 6-O-(5-O-syringoyl-ꢀ-D-api-
ofuranosyl)-ꢀ-D-glucopyranosyl moiety were also observed in the
NMR spectra of 7 (see Tables 2 and 3). However, the signals of
the coumarin unit observed in 1-6 were replaced by signals
attributed to isopropyl at δH 1.07 (3H, d, J ) 6.5 Hz), 3.82 (1H,
overlapped), and 1.04 (3H, d, J ) 6.5 Hz) in the 1H NMR spectrum
and at δC 23.4, 70.1, and 21.8 in the 13C NMR spectrum. HMBC
correlations of C-6′ with H-1′′ and C-7′′′ with H-5′′ confirmed the
sugar chain as 6-O-(5-O-syringoyl-ꢀ-D-apiofuranosyl)-ꢀ-D-glucopy-
ranosyl. Furthermore, the connection between the isopropyl and
sugar moieties was established by HMBC correlations of C-2 with
H-1′ and C-1′ with H-2. These spectroscopic data established 7 as
2-O-[6-O-(5-O-syringoyl-ꢀ-D-apiofuranosyl)-ꢀ-D-glucopyranosyl-
]isopropyl alcohol, named eryciboside G.
Compound 10 was obtained as a white powder, [R]20D -36.9 (c
0.05, MeOH). The positive HRESIMS ion of 10 at m/z 741.2946
([M + Na]+) proved the molecular formula to be C33H50O17. The
NMR spectra (see Tables 2 and 3) indicated that it was also a
megastigmane derivative with a 6-O-(5-O-syringoyl-ꢀ-D-apiofura-
1
nosyl)-ꢀ-D-glucopyranosyl moiety. The H NMR spectrum (see
Table 2) showed the absence of signal of H-5 in 9, and the changes
of coupling patterns of H2-4 [δ 1.61 (1H, dd, J ) 12.5, 4.5 Hz,
H-4eq) and 1.55 (1H, t, J ) 12.5 Hz, H-4ax)] indicated the presence
of a hydroxy group at C-5. This was supported by the deshielded
signal of C-5 at δ 75.7 in the 13C NMR spectrum (see Table 3).
1
The planar structure of 10 was further confirmed by the H-1H
COSY, HSQC, and HMBC spectra. The elucidation of the relative
configuration of the aglycone moiety is based on the NOE difference
experiment and on the observed 1H/1H coupling constants. The large
coupling values of H-3 with H-2ax (J ) 11.5 Hz) and with H-4ax
(J ) 12.5 Hz) indicated H-3 must be in the axial position. The
NOE difference experiment showed enhancements of both H-7 and
H-3 by irradiation of H3-11 and no enhancement of H3-11 or H-3
by irradiation of H3-13. This indicated H3-11, H-3, and H-7 were
on the same side of the six-membered ring, while H3-13 was on
the opposite face. In addition, as 10 differed from 9 only by an
additional hydroxy group at C-5, the absolute configuration of 10
was presumed to be the same as 9. Thus, 10 was assigned as 9-O-
[6-O-(5-O-syringoyl-ꢀ-D-apiofuranosyl)-ꢀ-D-glucopyranosyl]-
(3S,5R,6R,9R)-3,5,6,9-tetrahydroxymegastigman-7-ene and named
eryciboside J.
Compound 8 was obtained as a white powder, [R]20 -41.4 (c
D
0.05, MeOH), and its molecular formula was determined to be
C28H36O15 by the positive HRESIMS data ([M + Na]+, m/z found
1
635.1951). Its H and 13C NMR data (see Tables 2 and 3) also
indicated the presence of a 6-O-(5-O-syringoyl-ꢀ-D-apiofuranosyl)-
ꢀ-D-glucopyranosyl moiety, and this was further confirmed by
HMBC correlations of C-6′ with H-1′′ and C-7′′′ with H-5′′. The
remaining proton signals at δ 6.99 (2H, d, J ) 8.5 Hz, H-2 and
H-6), 6.62 (2H, d, J ) 8.5 Hz, H-3 and H-5), 3.82 (1H, m, H-8a),
3.55 (1H, m, H-8b), and 2.68 (2H, m, H2-7) in the 1H NMR
spectrum were attributable to a 4-substitued phenylethyl alcohol
moiety. HMBC correlations of C-8 with H-1′ and C-1′ with H-8
suggested that the sugar moiety was at C-8. Thus, compound 8
was determined to be 8-O-[6-O-(5-O-syringoyl-ꢀ-D-apiofuranosyl)-
ꢀ-D-glucopyranosyl]-4-hydroxyphenylethyl alcohol and named ery-
ciboside H.
Compound 11 was obtained as a white powder, [R]20D -50.7 (c
0.06, MeOH). The spectroscopic data of 11 indicated that it was
also a megastigmane derivative with a 6-O-(5-O-syringoyl-ꢀ-D-
apiofuranosyl)-ꢀ-D-glucopyranosyl moiety. The molecular formula
was C33H50O17, as indicated by the positive HRESIMS ion ([M +
Compound 9 was obtained as a white powder, [R]20 -38.7 (c
D
1
Na]+, m/z found 727.3148). The H and 13C NMR spectra of 11
0.06, MeOH), and its positive HRESIMS data ([M + Na]+, m/z
(see Tables 2 and 3) also suggested a close structural similarity to
9, with the main difference of the replacement of signals for a
double bond with an additional pair of methylenes [δH 1.47 (2H,
overlapped, H2-7) and 1.40 (2H, overlapped, H2-8), and δC 30.9
(C-7) and 32.7 (C-8)]. This suggestion was confirmed by the 1H-1H
COSY, HSQC, and HMBC spectra. The absolute configuration of
11 was also presumed to be the same as 9. Thus, 11 was elucidated
as 9-O-[6-O-(5-O-syringoyl-ꢀ-D-apiofuranosyl)-ꢀ-D-glucopyrano-
syl]-(3S,5R,6S,9R)-3,6,9-trihydroxymegastigmane and named ery-
ciboside K.
found 725.2983) indicated the molecular formula to be C33H50O16.
1
The H and 13C NMR spectra of 9 (see Tables 2 and 3) displayed
signals assignable to a 6-O-(5-O-syringoyl-ꢀ-D-apiofuranosyl)-ꢀ-
D-glucopyranosyl moiety. The remaining signals in the H NMR
1
spectrum included two methyls at δ 0.75 and 0.84 as singlets, two
methyl doublets at δ 1.15 and 0.67, five aliphatic protons ranging
from δ 1.20 to 1.75, two oxymethine protons as multiplets at δ
3.57 and 4.24, and two olefinic protons at δ 5.44 (d, J ) 16.5 Hz)
and 5.61 (dd, J ) 16.5, 6.0 Hz) for a disubstituted trans double
bond. The 13C NMR spectrum displayed 13 carbon signals due to
the aglycone moiety. The above spectroscopic data suggested the
planar structure of the aglycone moiety was 3,6,9-trihydroxyme-
gastigman-7-ene. This suggestion was further supported by the
vicinal coupling correlations of H-9 with both H3-10 and H-8, H-8
with H-7, H-3 with both H2-2 and H2-4, and H-5 with H3-13 in the
1H-1H COSY spectrum, together with the related HMBC correla-
tions (see Figure 2). In addition, HMBC correlations from the
anomeric proton H-1′ to C-9 and from H-9 to C-1′ indicated the
sugar moiety was attached to C-9. The large couplings of H-2ax
with H-3 (J ) 11.5 Hz), and H-4ax with H-3 (J ) 12.0 Hz) and
Compound 14 was obtained as a white powder, [R]20 -106.7
D
(c 0.05, MeOH), and its molecular formula was determined to be
C25H26O13 by the negative HRESIMS data ([M - H]-, m/z found
533.1287). Signals derived from a syringoyl moiety were observed
in the 1H and 13C NMR spectra (see Table 4). The 1H NMR
spectrum showed an ABX system attributed to a 1,3,4-trisubstituted
aromatic ring at δ 7.00 (1H, d, J ) 1.5 Hz), 6.95 (1H, dd, J ) 8.0,
1.5 Hz), and 6.74 (1H, d, J ) 8.0 Hz) and an AX system assignable
to a trans double bond at δ 7.43 (1H, d, J ) 15.5 Hz) and 6.15
(1H, d, J ) 15.5 Hz), which suggested the presence of a caffeoyl

HepatoprotectiVe Constituents from Erycibe hainanesis
Journal of Natural Products, 2010, Vol. 73, No. 2 181
Table 4. NMR Spectroscopic Data (δ) of Compounds 14-17a
14
15
16
δH (J in Hz)
17
position
δH (J in Hz)
δC
δH (J in Hz)
δC
δC
δH (J in Hz)
δC
1
2
3
74.0
37.8
66.7
72.4
36.4
71.2
73.1
40.0c
68.9
72.8
37.7
65.3
1.97, 2.20, m
4.30, m
2.19, 2.00, m
5.31, m
2.14, 1.99, m
5.53, m
1.91, 2.24, m
4.20, m
4
5
4.97, dd (8.0,2.0)
5.57, m
75.2
67.4
3.84, m
5.28, m
68.3
70.0
4.92, dd (8.0,2.0)
4.16, m
73.1
65.3
5.01, m
5.35, m
73.1
67.5
6
2.08, 2.20, m
37.8
2.00, 1.98, m
34.7
1.99, 1.91, m
36.5
2.00, 2.26, m
36.2
7
175.0
125.6
115.2
145.9
148.8
116.0
121.7
145.9
113.9
166.0
119.6
107.4
147.8
141.1
147.8
107.4
165.5
175.3
125.0
114.3
145.0
147.8
115.2
120.7
144.4
113.8
165.2
119.8
106.8
146.9
139.9
146.9
106.8
164.7
177.4
125.4
115.8
145.6
148.5
113.9
121.4
145.4
114.0
165.9
119.7
106.9
147.4
140.5
147.4
106.9
164.7
173.3
125.2
114.7
145.7
148.6
115.1
121.4
145.6
113.2
165.2
120.4
112.8
147.3
151.6
114.7
123.7
164.9
52.0
1′
2′
7.00, d (1.5)
7.04, br s
7.01, d (1.5)
7.00, br s
3′
4′
5′
6.74, d (8.0)
6.77, d (8.0)
6.99, d (8.0)
7.44, d (16.0)
6.18, d (16.0)
6.74, d (8.0)
6.75, d (8.0)
6.95, d (8.0)
7.40, d (15.6)
6.12, d (15.6)
6′
6.95, dd (8.0, 1.5)
7.43, d (15.5)
6.15, d (15.5)
6.93, dd (8.0, 1.5)
7.43, d (15.5)
6.24, d (15.5)
7′
8′
9′
1′′
2′′
7.21, s
7.21, s
7.31, s
7.31, s
7.20, s
7.20, s
7.44, br s
3′′
4′′
5′′
6.85, d (8.0)
7.49, d (8.0)
6′′
7′′
7-OMe
3′′-OMe
5′′-OMe
3.52, s
3.76, s
3.79, s
3.79, s
56.3
56.3
3.81, s
3.81, s
55.5
55.5
3.72, s
3.72, s
55.9
55.9
55.6
a NMR data (δ) were measured in DMSO-d6 at 500 or 600 MHz for 1H NMR and at 125 MHz for 13C NMR. b Overlapping signals. c Signal
overlapped by solvent peaks.
moiety. The remaining signals of three oxygenated methine protons
at δ 4.30 (1H, m, H-3), 4.97 (1H, dd, J ) 8.0, 2.0, H-4), and 5.57
(1H, m, H-5) and four methylene protons at δ 2.20, 1.97 (2H, m,
H2-2) and 2.20, 2.08 (2H, m, H2-6) indicated the presence of a
quinic acid moiety. This was supported by a set of characteristic
signals in the 13C NMR spectrum at δ 175.0, 75.2, 74.0, 67.4, 66.7,
37.8, and 37.8. Analyses of the HSQC spectrum of 14 led to
unambiguous assignment of proton and corresponding carbon
signals in the NMR spectra. The linkages between the units were
established by HMBC correlations from H-4 to C-7′′ and from H-5
to C-9′. Additional support for the location of the caffeoyl moiety
was obtained from hydrolysis of 14 under alkaline conditions to
afford 5-O-caffeoylquinic acid. Consequently, the structure of 14
was determined to be 5-O-caffeoyl-4-O-syringoylquinic acid.
Compound 15 was obtained as a white powder, [R]20D -96.4 (c
0.05, MeOH), and its negative HRESIMS and NMR data (see
Experimental Section and Table 4) were similar to those of 14.
Comparison of the NMR data of 15 and 14 indicated that C-4 and
H-4 of 15 were shielded by ∆δC 6.9 and ∆δH 1.13 ppm,
respectively, whereas C-3 and H-3 were deshielded by ∆δC 5.2
and ∆δH 1.01 ppm, respectively. These data suggested that the ester
substituents were located at C-3 and C-5 in 15 instead of C-4 and
C-5 in 14. The HMBC spectrum could not confirm the locations
of the ester linkages because of the overlap of the H-3 and H-5
resonance. However, the alkaline hydrolysis of 15 gave 5-O-
caffeoylquinic acid, which suggested the caffeoyl moiety was
located at C-5, while the syringoyl was attached to C-3. This was
corroborated by the comparison of the NMR data of 15 with those
of 5-O-caffeoyl-3-O-syringoylquinic acid methyl ester9 isolated
from E. obtusifolia. Hence, 15 was identified as 5-O-caffeoyl-3-
O-syringoylquinic acid.
the basis of HMBC correlations of C-9′ with H-4 and C-7′′ with
H-3. Thus, 16 was defined as 4-O-caffeoyl-3-O-syringoylquinic
acid.
Compound 17 was obtained as a white powder, [R]20 -108.2
D
(c 0.04, MeOH), and its negative HRESIMS data ([M - H]-, m/z
found 517.1337) indicated the molecular formula to be C25H26O12.
1
Comparison of the H and 13C NMR of 17 and 14-16 revealed
that the signals for the syringoyl unit in 14-16 were replaced by
the signals attributed to the vanilloyl moiety (see Table 4). HMBC
correlation of C-7′′ with H-4 demonstrated that the vanilloyl moiety
was located at C-4. Although the correlation of C-9′ with H-5 was
not observable in the HMBC spectrum, the alkaline hydrolysis of
17 gave 5-O-caffeoylquinic acid, which suggested the location of
the caffeoyl moiety at C-5. An additional methyl ester group in 17
1
was deduced from its H NMR signals at δ 3.52 (3H, s) and the
HMBC correlation between the methoxy protons and the carbonyl
carbon. On the basis of the above results, 17 was elucidated as
5-O-caffeoyl-4-O-vanilloylquinic acid methyl ester.
Compound 18 was obtained as a yellowish powder, and its
molecular formula was determined to be C20H14O8 by negative
HRESIMS data ([M - H]-, m/z found 381.0592). The compound
exhibited blue fluorescence under UV light (365 nm). The 1H NMR
spectrum (see Experimental Section) displayed a typical pair of
doublets at δ 6.41 (1H, d, J ) 9.5 Hz, H-3′) and 8.03 (1H, d, J )
9.5 Hz, H-4′) and five singlets at δ 6.85 (1H, s, H-8), 7.18 (1H, s,
H-5), 7.23 (1H, s, H-8′), 7.48 (1H, s, H-5′), and 7.61 (1H, s, H-4)
for aromatic protons. The 13C NMR spectrum (see Experimental
Section) exhibited 18 carbon signals in the downfield region,
including two conjugated ester carbonyls at δ 160.1 and 156.6,
which indicated that 18 possessed a dimeric coumarin skeleton.
Analysis of the above proton and carbon signals led to the
construction of a 6,7-O-disubstituted coumarin unit and a 3,6,7-
O-trisubstituted coumarin unit, aided by the HMBC spectrum (see
Figure 3). In addition, the substituents at C-6, C-6′, and C-7 were
established by HMBC correlations of C-6 and C-6′ with the
methoxy groups at δ 3.79 (3H, s) and 3.88 (3H, s), respectively,
and C-7 with the OH proton at δ 10.21 (1H, s). Given the fact that
there were only two oxygen-substituted positions remaining, it can
Compound 16 was obtained as a white powder, [R]20D -83.5 (c
0.05, MeOH), and its negative HRESIMS data ([M - H]-, m/z
found 533.1287) indicated that it possessed the same molecular
1
formula as those of 14 and 15. The H and 13C NMR spectra (see
Table 4) of 16 also displayed signals for syringoyl, caffeoyl, and
quinic acid moieties. The locations of the caffeoyl and syringoyl
moieties were determined to be at C-4 and C-3, respectively, on

182 Journal of Natural Products, 2010, Vol. 73, No. 2
Song et al.
mL), EtOAc (3 × 6000 mL), and n-BuOH (3 × 5000 mL), successively.
After evaporation of the solvent under reduced pressure, the n-BuOH
extract (450 g) was subjected to column chromatography over
macroporous resin, eluting successively with H2O, 15% EtOH, 30%
EtOH, 50% EtOH, 70% EtOH, and 95% EtOH (20 L each). After
removing the solvent, the 30% EtOH fraction (30 g) was subjected to
chromatography over Sephadex LH-20 with H2O as the mobile phase
to yield eight fractions (A1-A8) on the basis of HPLC-DAD analysis.
Fraction A3 (1.0 g) was subjected to reversed-phase preparative HPLC,
using MeOH-H2O (33:67) as the mobile phase, to give 1 (300 mg), 2
(9 mg), and 6 (15 mg). Fraction A4 (1.1 g) was chromatographed over
reversed-phase silica gel, eluting with a gradient of increasing MeOH
(0-45%) in H2O, to yield five subfractions (A4-1-A4-5). Subfractions
A4-3 (50 mg) and A4-4 (200 mg) were further separated by reversed-
phase preparative HPLC, using MeOH-H2O (30:70 and 38:62) as the
mobile phase, respectively, to afford 4 (10 mg), and 9 (30 mg), 10 (11
mg), and 11 (50 mg). Fractions A5 (200 mg) and A8 (150 mg) were
separately subjected to reversed-phase preparative HPLC, for fraction
A5 using MeOH-H2O (33:67) as the mobile phase, to afford 3 (10
mg), 5 (10 mg), and 7 (12 mg), for fraction A8 using MeOH-H2O
(35:65) as the mobile phase, to afford 8 (15 mg). After removal of
solvent, the EtOAc extract (100 g) was applied to a normal-phase silica
gel column. Successive elution of the column with a gradient of
increasing acetone (0-100%) in petroleum ether afforded six fractions
(B1-B6) on the basis of HPLC-DAD analysis. Fraction B3 (3.5 g)
was further chromatographed over a normal-phase silica gel column
eluting with a gradient of increasing EtOAc (0-100%) in petroleum
ether, to afford five subfractions (B3-1-B3-5). Subfraction B3-4 (500
mg) was purified by reversed-phase preparative HPLC, using a mobile
phase of MeOH-H2O (50:50), to yield 18 (25 mg). Fraction B5 (20
g) was subjected to chromatography over Sephadex LH-20 with a
gradient of increasing MeOH (0-100%) in H2O as the mobile phase,
to give five subfractions (B5-1-B5-5). Subfractions B5-3 (1000 mg)
and B5-4 (500 mg) were separated by reversed-phase preparative HPLC,
for subfraction B5-3 using MeOH-H2O (34:66) as the mobile phase,
to afford 14 (200 mg), 15 (30 mg), and 16 (20 mg), for subfraction
B5-4 using MeOH-H2O (40:60) as the mobile phase, to afford 17 (10
mg).
Figure 3. Selected HMBC correlations of 18.
Table 5. Hepatoprotective Effects of Compounds 2, 6, 10, 16,
and 32 against D-Galactosamine-Induced Toxicity in WB-F344
Cellsa
cell survival rate
(% of normal)
inhibition
(% of control)
compound
normal
100 ( 8.6
30 ( 1.6
control
bicyclolb
38 ( 2.3**
47 ( 0.7***
44 ( 4.8*
45 ( 2.9**
61 ( 0.7***
45 ( 3.6**
11.2
19.8
15.5
17.4
42.2
17.4
2
6
10
16
32
a Results are expressed as means ( SD (n ) 3; for normal and
control, n ) 6); *p < 0.05, **p < 0.01, ***p < 0.001. 2 was tested at 1
× 10-5 M due to its poor solubility, while other compounds were tested
at 1 × 10-4 M. b Positive control substance.
be inferred that the two coumarin units were linked by an ether
bridge between C-3 and C-7′. Therefore, the structure of 18 was
deduced as 7-hydroxy-6,6′-dimethoxy-3,7′-O-bis-coumarin. This is
the first report of a bis-coumarin with a C-O-C linkage in the
family of Convolvulaceae.
The hepatoprotective activities against D-galactosamine-induced
toxicity of compounds 1-16 and 18-32 were examined in WB-
F344 cells. Compounds 2, 6, 10, 18, and 32 showed potent
hepatoprotective activities, without any obvious cytotoxic effects
(see Table 5), while the other compounds tested were inactive at 1
× 10-4 M.
Eryciboside A (1): white powder; [R]20 -83.6 (c 0.05, MeOH);
D
UV (MeOH) λmax (log ε) 282 (4.16), 341 (3.89) nm; IR νmax 3429,
1734, 1615, 1567, 1516, 1459, 1280, 1222, 1069, 867, 822, 763 cm-1
;
1H NMR (DMSO-d6, 500 MHz) data, see Table 1; 13C NMR (DMSO-
d6, 125 MHz) data, see Table 3; (-)-ESIMS m/z 665 [M - H]-; (-)-
HRESIMS m/z 665.1704 (calcd for C30H33O17, 665.1712).
Experimental Section
General Experimental Procedures. The optical rotations were
measured on a Jasco P-2000 polarimeter. The UV spectra were scanned
by a Jasco V650 spectrophotometer. IR spectra were recorded on an
Eryciboside B (2): white powder; [R]20 -39.3 (c 0.05, MeOH);
D
UV (MeOH) λmax (log ε) 283 (4.16), 340 (3.87) nm; IR νmax 3382,
1
IMPACT 400 (KBr) spectrometer. H NMR (500 or 600 MHz), 13C
1710, 1613, 1565, 1513, 1462, 1279, 1223, 1114, 863, 826, 761 cm-1
;
NMR (125 MHz), and 2D-NMR spectra were run on INOVA 500 and
600 MHz spectrometers. HRESIMS were performed on a Finnigan LTQ
FT mass spectrometer. The ESI mass spectra were recorded on an
Agilent 1100 series LC/MSD TOF from Agilent Technologies. Column
chromatography was performed with macroporous resin (Diaion HP-
20, Mitsubishi Chemical Corp., Tokyo, Japan), Rp-18 (50 µm, YMC,
Kyoto, Japan), Sephadex LH-20 (Pharmacia Fine Chemicals, Uppsala,
Sweden), and silica gel (100-200, 200-300 mesh, Qingdao Marine
Chemical Inc. Qingdao, People’s Republic of China). Preparative HPLC
was carried out on a Shimadzu LC-6AD instrument with an SPD-20A
detector, using a YMC-Pack ODS-A column (250 mm × 20 mm, 5
µm). HPLC-DAD analysis was performed using an Agilent 1200 series
system (Agilent Technologies, Waldbronn, Germany) with an Apollo
C18 column (250 mm × 4.6 mm, 5 µm; Grace Davison). Precoated
silica gel GF-254 plates (Yantai Jiangyou Silica Gel Exploitation
Company) were used for analytical TLC.
Plant Material. The roots and stems of E. hainanesis were collected
in Hainan Province, People’s Republic of China, in March 2008. The
plant material was identified by Mr. Huanqiang Chen (Jianfengling
National Nature Reserve of Hainan Province). A voucher specimen
(ID-21741) was deposited at the Institute of Materia Medica, Chinese
Academy of Medical Sciences, Beijing 100050, People’s Republic of
China.
1H NMR (DMSO-d6, 500 MHz) data, see Table 1; 13C NMR (DMSO-
d6, 125 MHz) data, see Table 3; (+)-ESIMS m/z 689 [M + Na]+; (+)-
HRESIMS m/z 689.1686 (calcd for C30H34O17Na, 689.1688).
Eryciboside C (3): white powder; [R]20 -33.6 (c 0.03, MeOH);
D
UV (MeOH) λmax (log ε) 282 (4.12), 340 (3.86) nm; IR νmax 3368,
1703, 1614, 1568, 1514, 1465, 1279, 1207, 1104, 859, 817, 760 cm-1
;
1H NMR (DMSO-d6, 500 MHz) data, see Table 1; 13C NMR (DMSO-
d6, 125 MHz) data, see Table 3; (-)-ESIMS m/z 665 [M - H]-; (+)-
HRESIMS m/z 689.1682 (calcd for C30H34O17Na, 689.1688).
Eryciboside D (4): white powder; [R]20 -62.4 (c 0.05, MeOH);
D
UV (MeOH) λmax (log ε) 260 (4.06), 290 (4.02), 340 (3.85) nm; IR
1
νmax 3395, 1711, 1611, 1564, 1514, 1281, 1072, 820, 763 cm-1; H
NMR (DMSO-d6, 500 MHz) data, see Table 1; 13C NMR (DMSO-d6,
125 MHz) data, see Table 3; (+)-ESIMS m/z 637 [M + H]+; (+)-
HRESIMS m/z 659.1583 (calcd for C29H32O16Na, 659.1583).
Eryciboside E (5): white powder; [R]20 -32.9 (c 0.05, MeOH);
D
UV (MeOH) λmax (log ε) 261 (4.15), 290 (4.11), 343 (3.95); IR νmax
3407, 1685, 1611, 1565, 1513, 1461, 1284, 1074, 864, 822, 760 cm-1
;
1H NMR (DMSO-d6, 500 MHz) data, see Table 1; 13C NMR (DMSO-
d6, 125 MHz) data, see Table 3; (-)-ESIMS m/z 635 [M - H]-; (+)-
HRESIMS m/z 659.1579 (calcd for C29H32O16Na, 659.1583).
Eryciboside F (6): white powder; [R]20 -3.8 (c 0.11, MeOH); UV
D
(MeOH) λmax (log ε) 285 (4.24), 338 (3.77) nm; IR νmax 3416, 1709,
Extraction and Isolation. The dried roots and stems of E. hainanesis
(22.5 kg) were extracted with 95% EtOH under reflux (3 × 1.5 h).
The EtOH extract was concentrated under reduced pressure to give a
residue (1.3 kg), which was suspended in H2O (7500 mL) with the
suspension sequentially partitioned with petroleum ether (3 × 6000
1
1610, 1568, 1514, 1462, 1337, 1223, 1116, 850, 763 cm-1; H NMR
(DMSO-d6, 500 MHz) data, see Table 1; 13C NMR (DMSO-d6, 125
MHz) data, see Table 3; (+)-ESIMS m/z 719 [M + Na]+; (+)-
HRESIMS m/z 719.1801 (calcd for C31H36O18Na, 719.1794).

HepatoprotectiVe Constituents from Erycibe hainanesis
Journal of Natural Products, 2010, Vol. 73, No. 2 183
Eryciboside G (7): white powder; [R]20 -50.6 (c 0.10, MeOH);
comparing with an authentic standard on HPLC-DAD. The scopolin
dissolved in 1 N HCl (5 mL) was refluxed for 3 h. After cooling, the
reaction mixture was extracted with EtOAc (3 × 5 mL). The aqueous
layer was cryodesiccated to afford D-glucose, which was identified by
D
UV (MeOH) λmax (log ε) 217 (4.46), 277 (4.09) nm; IR νmax 3399,
1702, 1610, 1515, 1462, 1335, 1220, 1112, 763 cm-1 1H NMR
;
(DMSO-d6, 500 MHz) data, see Table 2; 13C NMR (DMSO-d6, 125
MHz) data, see Table 3; (+)-ESIMS m/z 557 [M + Na]+; (+)-
HRESIMS m/z 557.1841 (calcd for C23H34O14Na, 557.1841).
comparison
with
an
authentic
sample
on
TLC
(CH3Cl-MeOH-HOAc-H2O, 14:6:2:1, Rf 0.27) and by its specific
Eryciboside H (8): white powder; [R]20 -41.4 (c 0.05, MeOH);
rotation, [R]20 +47.4 (c 0.2, H2O).
D
D
UV (MeOH) λmax (log ε) 219 (4.60), 268 (4.20), 284 (sh) (4.17); IR
Alkaline Hydrolysis of 14, 15, and 17. To each solution of 14, 15,
and 17 (1.0 mg) in MeOH (1.0 mL) was added one drop of 1 N NaOH,
and the mixture was stirred at room temperature. After 15 min the
reaction mixture was neutralized with 0.1 N HCl and filtrated through
a 0.45 µm filter for injection. HPLC-DAD analysis was performed on
a C18 column using MeOH-0.2% HOAc (35:65) as mobile phase.
5-O-Caffeoylquinic acid was identified by comparing the retention time
and UV spectrum with the authentic standard.
ν
max 3390, 1697, 1613, 1515, 1461, 1336, 1226, 1114, 830, 763 cm-1
;
1H NMR (DMSO-d6, 500 MHz) data, see Table 2; 13C NMR (DMSO-
d6, 125 MHz) data, see Table 3; (+)-ESIMS m/z 635 [M + Na]+; (+)-
HRESIMS m/z 635.1951 (calcd for C28H36O15Na, 635.1946).
Eryciboside I (9): white powder; [R]20 -38.7 (c 0.06, MeOH);
D
UV (MeOH) λmax (log ε) 216 (sh) (4.52), 278 (4.09) nm; IR νmax 3403,
1701, 1610, 1515, 1461, 1335, 1218, 1113, 764 cm-1 1H NMR
;
(DMSO-d6, 500 MHz) data, see Table 2; 13C NMR (DMSO-d6, 125
MHz, and MeOH-d4, 125 MHz) data, see Table 3; (-)-ESIMS m/z
701 [M - H]-; (+)-HRESIMS m/z 725.2983 (calcd for C33H50O16Na,
725.2991).
Protective Effect on Cytotoxicity Induced by D-Galactosamine
in WB-F344 Cells. The hepatoprotective effects of compounds 1-16
and 18-32 were determined by a 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) colorimetric assay in WB-F344
cells.34 Each cell suspension of 1 × 104 cells in 200 µL of Dulbecco’s
modified Eagle’s medium containing fetal calf serum (3%), penicillin
(100 units/mL), and streptomycin (100 µg/mL) was placed in a 96-
well microplate and precultured for 24 h at 37 °C under a 5% CO2
atmosphere. Fresh medium (200 µL) containing bicyclol and test
samples was added, and the cells were cultured for 1 h. The cultured
cells were exposed to 40 mM D-galactosamine for 24 h. The cytotoxic
effects of test samples were measured simultaneously in the absence
of D-galactosamine. The medium was changed into a fresh one
containing 0.5 mg/mL MTT. After 3.5 h incubation, the medium was
removed and 150 µL of DMSO was added to dissolve formazan
crystals. The optical density (OD) of the formazan solution was
measured on a microplate reader at 492 nm. Inhibition (%) was obtained
by the following formula: Inhibition (%) ) [(OD(sample) - OD(control))/
(OD(normal) - OD(control))] × 100.
Eryciboside J (10): white powder; [R]20 -36.9 (c 0.05, MeOH);
D
UV (MeOH) λmax (log ꢀ) 216 (sh) (4.46), 278 (4.06) nm; IR νmax 3401,
1699, 1611, 1515, 1462, 1336, 1223, 1116, 764 cm-1 1H NMR
;
(DMSO-d6, 500 MHz) data, see Table 2; 13C NMR (DMSO-d6, 125
MHz) data, see Table 3; (-)-ESIMS m/z 717 [M - H]-; (+)-HRESIMS
m/z 741.2946 (calcd for C33H50O17Na, 741.2940).
Eryciboside K (11): white powder; [R]20D -50.7 (c 0.06, MeOH);
UV (MeOH) λmax (log ε) 217 (4.49), 278 (4.14) nm; IR νmax 3360,
1701, 1609, 1515, 1461, 1334, 1216, 1111, 763 cm-1 1H NMR
;
(DMSO-d6, 500 MHz) data, see Table 2; 13C NMR (DMSO-d6, 125
MHz) data, see Table 3; (-)-ESIMS m/z 703 [M - H]-; (+)-HRESIMS
m/z 727.3148 (calcd for C33H52O16Na, 727.3170).
5-O-Caffeoyl-4-O-syringoylquinic acid (14): white powder; [R]20
D
-106.7 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 249 (4.10), 289
(4.32), 331 (4.25) nm; IR νmax 3374, 1695, 1605, 1516, 1461, 1346,
1278, 1223, 1114, 764 cm-1; 1H NMR (DMSO-d6, 500 MHz) and 13
C
Statistical Analysis. The Student’s t-test for unpaired observations
between normal and tested samples was carried out to identify statistical
differences; p values less than 0.05 were considered significantly
different.
NMR (DMSO-d6, 125 MHz) data, see Table 4; (-)-ESIMS m/z 533
[M - H]-; (-)-HRESIMS m/z 533.1287 (calcd for C25H25O13,
533.1290).
5-O-Caffeoyl-3-O-syringoylquinic acid (15): white powder; [R]20
D
-96.4 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 249 (4.11), 290 (4.34),
330 (4.26) nm; IR νmax 3421, 1693, 1608, 1517, 1462, 1334, 1278,
1233, 1115, 763 cm-1; 1H NMR (DMSO-d6, 500 MHz) and 13C NMR
(DMSO-d6, 125 MHz) data, see Table 4; (-)-ESIMS m/z 533 [M -
H]-; (-)-HRESIMS m/z 533.1291 (calcd for C25H25O13, 533.1290).
Acknowledgment. We thank Mrs. Y. H. Wang and Mr. H. Y. Liu
for NMR measurements.
Supporting Information Available: NMR spectra of compounds
1-11 and 14-18. This material is available free of charge via the
Internet at http://pubs.acs.org.
4-O-Caffeoyl-3-O-syringoylquinic acid (16): white powder; [R]20
D
-83.5 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 248 (4.10), 290 (4.32),
330 (4.25) nm; IR νmax 3414, 1699, 1608, 1516, 1461, 1334, 1278,
1234, 1116, 762 cm-1; 1H NMR (DMSO-d6, 500 MHz) and 13C NMR
(DMSO-d6, 125 MHz) data, see Table 4; (-)-ESIMS m/z 533 [M -
H]-; (-)-HRESIMS m/z 533.1287 (calcd for C25H25O13, 533.1290).
5-O-Caffeoyl-4-O-vanilloylquinic acid methyl ester (17): white
powder; [R]20D -108.2 (c 0.04, MeOH); UV (MeOH) λmax (log ε) 254
(4.16), 298 (4.28), 330 (4.26) nm; IR νmax 3407, 1694, 1600, 1516,
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NP900593Q