Two new iridoid glycosides from the leaves of Callicarpa nudiflora

Article abstract of DOI:10.1080/10286020.2017.1323884

Two new iridoid glycosides, callicoside C (1) and callicoside D (2), together with three known compounds (3–5), were isolated from the leaves of Callicarpa nudiflora. Their structures were established by 1D and 2D NMR spectroscopy and mass spectrometry. I

Full text of DOI:10.1080/10286020.2017.1323884

Journal of Asian Natural Products Research
ISSN: 1028-6020 (Print) 1477-2213 (Online) Journal homepage: http://www.tandfonline.com/loi/ganp20
Two new iridoid glycosides from the leaves of
Callicarpa nudiflora
Jie Wang, Hui-Zheng Fu, Yue-Hua Luo, Yuan-Yu Ma, Bo Huang & Shuang-
Cheng Ma
To cite this article: Jie Wang, Hui-Zheng Fu, Yue-Hua Luo, Yuan-Yu Ma, Bo Huang & Shuang-
Cheng Ma (2017): Two new iridoid glycosides from the leaves of Callicarpa nudiflora, Journal of
Asian Natural Products Research, DOI: 10.1080/10286020.2017.1323884
To link to this article: http://dx.doi.org/10.1080/10286020.2017.1323884
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Date: 29 May 2017, At: 02:03

Journal of asian natural Products research, 2017
ꢁꢁpꢂ://ꢃꢄꢅ.ꢄꢆg/10.1080/10286020.2017.1323884
ꢀ
Two new iridoid glycosides from the leaves of Callicarpa
nudiflora
Jie Wanga,b#, Hui-Zheng Fu , Yue-Hua Luo , Yuan-Yu Ma , Bo Huang and
Shuang-Cheng Ma
a#
a
a,b
a
c
ꢇ
Jꢅꢇꢈgxꢅ Pꢆꢄvꢅꢈꢉꢅꢇꢊ iꢈꢂꢁꢅꢁꢋꢁꢌ ꢍꢄꢆ dꢆꢋg cꢄꢈꢁꢆꢄꢊ, Jꢅꢇꢈgxꢅ Pꢆꢄvꢅꢈꢉꢅꢇꢊ eꢈgꢅꢈꢌꢌꢆꢅꢈg rꢌꢂꢌꢇꢆꢉꢀ cꢌꢈꢁꢌꢆ ꢍꢄꢆ dꢆꢋg ꢇꢈꢃ
b
Mꢌꢃꢅꢉꢇꢊ dꢌvꢅꢉꢌ Qꢋꢇꢊꢅꢁy, nꢇꢈꢉꢀꢇꢈg 330029, cꢀꢅꢈꢇ; cꢄꢊꢊꢌgꢌ ꢄꢍ Pꢀꢇꢆmꢇꢉy, Pꢀꢇꢆmꢇꢉꢌꢋꢁꢅꢉꢇꢊ dꢌpꢇꢆꢁmꢌꢈꢁ ꢄꢍ
nꢇꢈꢉꢀꢇꢈg uꢈꢅvꢌꢆꢂꢅꢁy, nꢇꢈꢉꢀꢇꢈg 330006, cꢀꢅꢈꢇ; rꢌꢂꢌꢇꢆꢉꢀ ꢇꢈꢃ iꢈꢂpꢌꢉꢁꢅꢄꢈ cꢌꢈꢁꢌꢆ ꢄꢍ tꢆꢇꢃꢅꢁꢅꢄꢈꢇꢊ cꢀꢅꢈꢌꢂꢌ
ꢉ
Mꢌꢃꢅꢉꢅꢈꢌ ꢇꢈꢃ eꢁꢀꢈꢄmꢌꢃꢅꢉꢅꢈꢌ, nꢇꢁꢅꢄꢈꢇꢊ iꢈꢂꢁꢅꢁꢋꢁꢌꢂ ꢍꢄꢆ fꢄꢄꢃ ꢇꢈꢃ dꢆꢋg cꢄꢈꢁꢆꢄꢊ, Bꢌꢅjꢅꢈg 100050, cꢀꢅꢈꢇ
ABSTRACT
ARTICLE HISTORY
Two new iridoid glycosides, callicoside C (1) and callicoside D (2),
together with three known compounds (3–5), were isolated from
the leaves of Callicarpa nudiflora. Their structures were established
by 1D and 2D NMR spectroscopy and mass spectrometry. In an in
vitro bioassay, compound 1 showed pronounced hepatoprotective
activity against d-galactosamine-induced toxicity in WB-F344 rat
hepatic epithelial stem-like cells.
rꢌꢉꢌꢅvꢌꢃ 13 fꢌbꢆꢋꢇꢆy 2017
aꢉꢉꢌpꢁꢌꢃ 23 apꢆꢅꢊ 2017
KEYWORDS
Vꢌꢆbꢌꢈꢇꢉꢇꢂꢌ; Callicarpa
nudiflora; ꢅꢆꢅꢃꢄꢅꢃ gꢊyꢉꢄꢂꢅꢃꢌꢂ;
ꢀꢌpꢇꢁꢄpꢆꢄꢁꢌꢉꢁꢅvꢌ ꢇꢉꢁꢅvꢅꢁy
1
2
1
. Introduction
Callicarpa nudiflora Hook belongs to the family Verbenacase and has been used as traditional
Chinese herbal medicines to treat inflammation and bleeding [1]. Previous investigation
on C. nudiflora has led to the isolation of iridoids, flavonoids, triterpenoides, and phenyl-
propanoid glycosides [2–5]. Some of them have been shown to exhibit anti-inflammatory,
antibacterial, cytotoxic and hemostatic activies [6]. C. nudiflora has been intensively studied
in our previous work and was found containing diverse constituents including triterpenoid
glycosides, flavonoids, furofuran lignans, and iridoid glucosides, among which two iridoid
glucosides, callicoside A and callicoside B, showed pronounced hepatoprotective activity
CONTACT Yꢋꢌ-hꢋꢇ lꢋꢄ
tꢀꢌꢂꢌ ꢇꢋꢁꢀꢄꢆꢂ ꢉꢄꢈꢁꢆꢅbꢋꢁꢌꢃ ꢌqꢋꢇꢊꢊy ꢁꢄ ꢁꢀꢅꢂ wꢄꢆk.
ꢌmꢇꢅꢊꢊꢋꢄ@ꢂꢄꢀꢋ.ꢉꢄm; sꢀꢋꢇꢈg-cꢀꢌꢈg Mꢇ
mꢇꢂꢉ@ꢈꢅꢍꢃꢉ.ꢄꢆg.ꢉꢈ
#
tꢀꢌ ꢂꢋppꢊꢌmꢌꢈꢁꢇꢊ ꢃꢇꢁꢇ ꢍꢄꢆ ꢁꢀꢅꢂ ꢇꢆꢁꢅꢉꢊꢌ ꢉꢇꢈ bꢌ ꢇꢉꢉꢌꢂꢂꢌꢃ ꢇꢁ ꢀꢁꢁp://ꢃx.ꢃꢄꢅ.ꢄꢆg/10.1080/10286020.2017.1323884.
2017 iꢈꢍꢄꢆmꢇ uK lꢅmꢅꢁꢌꢃ, ꢁꢆꢇꢃꢅꢈg ꢇꢂ tꢇyꢊꢄꢆ & fꢆꢇꢈꢉꢅꢂ Gꢆꢄꢋp
©

2
J. WANG ET AL.
against ꢀ-galactosamine-induced toxicity in WB-F344 rat hepatic epithelial stem-like cells
6–9]. In continuation of our investigation on the iridoid composition of C. nudiflora, two
[
further new iridoid glycosides, callicoside C (1), callicoside D (2) and three known com-
pounds 6-O-caffeoyl ajugo (3), nudifloside (4), minecoside (5) were isolated from the 80%
EtOH extract of C. nudiflora. Reported herein are the isolation, structure elucidation and
biological activity of these compounds.
2
. Results and discussion
e 80% EtOH extract of Callicarpa nudiflora was partitioned with petroleum ether, EtOAc,

and n-BuOH, successively. e n-BuOH-soluble portion was separated by a combination
of silica gel, ODS column chromatography and preparative HPLC, and afforded two new
compounds 1 and 2 (Figure 1) together with three known compounds 6-O-caffeoylajugo (3)
[
10], nudifloside (4) [2], minecoside (5) [11]. eir structures were elucidated by extensive
1
13
NMR techniques including 1D NMR ( H and C NMR), 2D NMR (COSY, NOESY, HSQC
and HMBC), and HRESIMS, as well as chemical evidence.
Compound 1 was obtained as a white amorphous powder. Its molecular formula,
–
C H O , was determined from HRESIMS at m/z 521.1656 [M – H] and supported by
2
5
30 12
1
the NMR spectroscopic data. e H NMR spectrum of 1 in CD OD exhibited two trans
3
olefinic protons at δ 7.61 (1H, d, J = 15.6 Hz) and 6.32 (1H, d, J = 15.6 Hz), one disubstituted
H
olefinic protons at δ 7.45 (1H, s), a set of AA′BB′-type coupled aromatic protons at δ 7.46
H
H
(
2H, d, J = 7.8 Hz) and 6.80 (2H, d, J = 7.8 Hz), two hemiacetal protons at δ 5.55 (1H, d,
H
J = 4.2 Hz) and 4.69 (1H, d, J = 7.8 Hz), one secondary methyl at δ 1.12 (3H, d, J = 7.2 Hz)
H
1
3
(
Table 1). e C NMR spectrum of 1 displayed 25 carbon signals including two carboxy
carbonyl carbons at δ 170.6 and 169.0. From the foregoing evidence it was concluded that
C
1
was likely to be a carboxylated iridoid glucoside. Comparison of the NMR spectroscopic
data of 1 (Table 1) with those of linearoside [12–14] demonstrated that two compounds were
almost identical. e only difference between 1 and linearoside is the configurations at C-7
and C-8. Acid hydrolysis of 1 with 2 M HCl afforded ꢀ-glucose, which was identified by GC
analysis of their trimethylsilyl ꢁ-cysteine derivatives [15,16]. In the HMBC spectrum of 1,
the HMBC correlation from H-7 (δ 4.94) to C-9′ (δ 169.0) confirmed that the p-coumaroyl
H
C
ester group is attached to C-7 of the aglycone (Figure 2). In addition, a HMBC correlation
from Glc-H-1″ (δ 4.69) to C-1 (δ 96.0) indicated the β-ꢀ-glucopyranosyl unit is located
H
C
1
1
11
HO
H
O
HO
H
O
O
9'
O
9'
7'
7'
3
3
7
3'
7
3'
5
5
O
O
1'
1'
9
9
O
O
1
HO
1
O
HO
10
HO
10
HO
HO
HO
H
H
5'
5'
O
O
O
HO
HO
1''
1''
OH
OH
1
2
Figure 1. cꢀꢌmꢅꢉꢇꢊ ꢂꢁꢆꢋꢉꢁꢋꢆꢌꢂ ꢄꢍ ꢉꢄmpꢄꢋꢈꢃꢂ 1 ꢇꢈꢃ 2.

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
3
1
13
Table 1. h (600 Mhz) ꢇꢈꢃ c nMr (150 Mhz) ꢂpꢌꢉꢁꢆꢇꢊ ꢃꢇꢁꢇ ꢄꢍ ꢉꢄmpꢄꢋꢈꢃꢂ 1 ꢇꢈꢃ 2 (δ ꢅꢈ ppm, J ꢅꢈ hz).
1a
2b
Position
δH
δC
δH
δC
ꢅꢆꢅꢃꢄꢅꢃ
1
3
4
5
6
5.55 (1h, ꢃ, 4.2)
7.45 (1h, ꢂ)
96.0
152.7
113.7
31.8
5.84 (1h, ꢃ, 5.4)
7.97 (1h, ꢂ)
96.4
152.8
111.9
33.3
3.09–3.11 (1h, m)
2.24–2.26 (1h, m)
3.65 (1h, ꢃ, 7.2)
2.58–2.60 (1h, m)
2.43–2.45 (1h, m)
5.93 (1h, bꢆ ꢁ)
39.1
38.9
1
.98–2.00 (1h, m)
7
8
9
4.94 (1h, ꢄvꢌꢆꢊꢇp)
2.46–2.48 (1h, m)
2.60–2.62 (1h, m)
1.12 (3h, ꢃ, 7.2)
82.6
43.1
43.3
14.3
75.9
149.0
44.8
3.27 (1h, ꢂ)
5.67 (1h, ꢂ)
10
116.4
5.55 (1h, ꢂ)
11
170.6
169.7
p-ꢉꢄꢋmꢇꢆꢄyꢊ
1’
2’
3’
4’
5’
6’
7’
8’
9’
127.2
131.1
116.9
161.2
116.9
131.1
146.5
111.5
169.0
126.6
131.2
117.3
162.0
117.3
131.2
145.8
115.8
167.6
7.46 (1h, ꢃ, 7.8)
6.80 (1h, ꢃ, 7.8)
7.61(1h, ꢃ, 8.4)
7.18 (1h, ꢃ, 8.4)
6.80 (1h, ꢃ, 7.8)
7.46 (1h, ꢃ, 7.8)
7.61 (1h, ꢃ, 15.6)
6.32 (1h, ꢃ, 15.6)
7.18 (1h, ꢃ, 8.4)
7.61 (1h, ꢃ, 8.4)
7.96 (1h, ꢃ, 15.6)
6.59 (1h, ꢃ, 15.6)
Gꢊꢉ
1’’
2’’
3’’
4’’
5’’
6’’
4.69 (1h, ꢃ, 7.8)
99.9
74.8
78.1
71.8
78.4
63.0
5.50 (1h, ꢃ, 8.4)
4.16 (1h, ꢁ, 8.4)
4.32–4.34 (1h, m)
4.30–4.32 (1h, m)
4.05–4.07 (1h, m)
4.59 (1h, ꢃꢃ, 12.0, 2.4)
4.41 (1h, ꢃꢃ, 12.0, 5.4)
101.2
75.4
79.0
72.1
79.4
63.3
3.19–3.21 (1h, m)
3.36–3.38 (1h, m)
3.23–3.25 (1h, m)
3.31–3.33 (1h, m)
3.91 (1h, ꢃ, 11.4)
3
.64–3.66 (1h, m)
ꢇ
rꢌꢉꢄꢆꢃꢌꢃ ꢅꢈ cd od.
rꢌꢉꢄꢆꢃꢌꢃ ꢅꢈ pyꢆꢅꢃꢅꢈꢌ-d5.
3
b
HO
O
HO
O
H
H
H
O
H
H
O
H
H
H
O
O
O
H
O
H
HO
HO
H
H
H
O
O
HO
HO
O
O
HO
HO
HO
HO
OH
OH
1
2
Figure 2. Kꢌy hMBc (h→c) ꢇꢈꢃ noe (h↔h) ꢉꢄꢆꢆꢌꢊꢇꢁꢅꢄꢈꢂ ꢄꢍ ꢉꢄmpꢄꢋꢈꢃꢂ 1 ꢇꢈꢃ 2.
at C-1. In the NOESY spectrum of 1, NOESY correlations were observed between the fol-
lowing proton pairs: H-5/H-6β, H-5/H-9, and H-9/H-8, indicating that methyl group at
C-8 should be α-oriented. However, NOE interactions were observed among H-1, H-7, and
H-10, which indicated the hydroxyl group at C-7 should be β-oriented. Further structure
analysis suggested the configurations at C-7 and C-8 in compound 1 was 7S, 8S, while the

4
J. WANG ET AL.
configurations at C-7 and C-8 in linearoside was 7S, 8R. Full assignments of the proton and
1
13
carbon resonances of 1 could be achieved by comprehensive analysis of H NMR, C NMR,
1
1
H- H COSY, HSQC, HMBC, and NOESY spectra. us, compound 1 was determined as
7
-O-E-p-coumaroyl-8-epi-loganic acid, named callicoside C.
Compound 2 was obtained as a white amorphous powder. e HRESIMS of 2 showed
+
a quasi-molecular ion peak at m/z 543.1479 [M + Na] , indicating a molecular formula
1
of C H O . e H NMR spectrum (Table 1) of 2 was similar to that of 1, including a
2
5
28 12
p-coumaroyl group and a glucose unit, as well as the resonance at δ 7.97 (1H, s, H-3) and
H
δH 5.84 (1H, d, J = 5.4 Hz, H-1). e major differences were in the iridoid moiety. us,
additional resonances [δ 5.67 (1H, s) and 5.55 (1H, s)] were present, while that from a
H
1
3
methyl group was missing. In the C NMR spectrum of 2, the expected 25 carbon signals
were observed. When compared with the spectrum of 1, only the resonances for the iri-
doid aglycone differed. e two resonances at δ 149.0 and 116.4 suggested an iridoid with
C
a 8, 10-double bond, which was further confirmed by HMBC correlations from H2–10 to
C-7, C-8, and C-9. However, comparison of the NMR data of 2 with those of gardoside
[
5
17] indicated that 2 is a derivative of gardoside. HMBC correlations from Glc-H-1″ (δ
.50) to C-1 (δ 96.4) and H-7 (δ 5.93) to C-9′ (δ 167.6) indicated β-ꢀ-glucopyranosyl
C H C
H
unit and p-coumaroyl group are attached to C-1 and C-7 of aglycone, respectively. In the
NOESY spectrum of 2, NOE interactions were observed among H-1, H-7, and H-6α, which
indicated the hydroxyl groups at C-1 and C-7 should be β-oriented. us, compound 2 was
elucidated as 7-O-E-p-coumaroyl-gardoside, named callicoside D.
In an in vitro bioassay, compound 1 at 10 μM showed pronounced hepatoprotective activ-
ities against ꢀ-galactosamine-induced toxicity in WB-F344 rat hepatic epithelial stem-like
cells (Table 2). Structure analysis indicated that compound 1 was very similar to compound
2
except that the methyl group of C-8 in 1 was replaced by the olefinic bond of C-8 in 2.
However, there were significant differences in the activity of the two compounds. It may
be that the methyl group of C-8 is an active group.
3
. Experimental
3
.1. General experimental procedures
Optical rotations were measured on an Autopol IV-T/V (Rudolph Research Analytical,
New Jersey, USA). UV spectra were recorded in MeOH on a Jasco V650 spectropho-
1
13
tometer (JASCO, Inc., Easton, Maryland, USA). e H (600 MHz), C (150 MHz), and
D NMR spectra were recorded on a Bruker AVANCE III 600 instrument using TMS
2
Table 2. hꢌpꢇꢁꢄpꢆꢄꢁꢌꢉꢁꢅvꢌ ꢌffꢌꢉꢁꢂ ꢄꢍ ꢉꢄmpꢄꢋꢈꢃꢂ 1 ꢇꢈꢃ 2 ꢇgꢇꢅꢈꢂꢁ d-gꢇꢊꢇꢉꢁꢄꢂꢇmꢅꢈꢌ-ꢅꢈꢃꢋꢉꢌꢃ ꢁꢄxꢅꢉꢅꢁy ꢅꢈ
ꢇ
WB-f344 cꢌꢊꢊꢂ .
Compound
x ± s
Cell survival rate (% of normal)
nꢄꢆmꢇꢊ
cꢄꢈꢁꢆꢄꢊ
Bꢅꢉyꢉꢊꢄꢊ
1
2
1.026 ± 0.037
0.325 ± 0.076
0.537 ± 0.049
0.572 ± 0.016
0.311 ± 0.067
100
#
#
28
b
*
50
*
*△
52
27
ꢇ
rꢌꢂꢋꢊꢁꢂ ꢇꢆꢌ ꢌxpꢆꢌꢂꢂꢌꢃ ꢇꢂ mꢌꢇꢈꢂ ± sd (n = 3).
b
Pꢄꢂꢅꢁꢅvꢌ ꢉꢄꢈꢁꢆꢄꢊ ꢉꢄmpꢄꢋꢈꢃ.
#
#
*
**
p < 0.001, ꢂꢅgꢈꢅfiꢉꢇꢈꢁꢊy ꢃꢅffꢌꢆꢌꢈꢁ ꢍꢆꢄm ꢉꢄꢈꢁꢆꢄꢊ by sꢁꢋꢃꢌꢈꢁ′ꢂ t-ꢁꢌꢂꢁ; p < 0.05; p < 0.01, ꢂꢅgꢈꢅfiꢉꢇꢈꢁꢊy ꢃꢅffꢌꢆꢌꢈꢁ ꢍꢆꢄm ꢈꢄꢆmꢇꢊ
△
by sꢁꢋꢃꢌꢈꢁ’ꢂ t-ꢁꢌꢂꢁ; p < 0.05, ꢂꢅgꢈꢅfiꢉꢇꢈꢁꢊy ꢃꢅffꢌꢆꢌꢈꢁ ꢍꢆꢄm pꢄꢂꢅꢁꢅvꢌ ꢉꢄꢈꢁꢆꢄꢊ by sꢁꢋꢃꢌꢈꢁ’ꢂ t-ꢁꢌꢂꢁ.

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
5
(
Tetramethylsilane) as an internal reference (Bruker Company, Massachusetts, USA).
HRESIMS data were obtained on an Agilent 7890–7000A mass spectrometer (Agilent
Technologies, Santa Clara, USA). Preparative HPLC (high-performance liquid chroma-
tography) was conducted with an Angilent Technologies 1200 series instrument with a
MWD detector using a YMC-pack ODS (Octadecylsilyl)-A column (5 μm, 250 × 20 mm).
Column chromatography was performed with silica gel (200–300 mesh, Qingdao Haiyang
Chemical Ltd., Qingdao, China), Develosil ODS (50 μm, Nomura Chemical Co. Ltd., Osaka,
Japan), Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). TLC (thin
layer chromatography) was carried out with glass precoated with silica gel GF . Spots
2
54
were visualized under UV light or by spraying with 10% sulfuric acid in EtOH followed by
heating. WB-F344 cells come from the Institute of Materia Medica, Chinese Academy of
Medical Sciences, Beijing.
3
.2. Plant material
e leaves of Callicarpa nudiflora Hook were collected from Wuzhishan, Hainan, China and

identified by Prof. Guiping Yuan at Jiangxi Provincial Institute for Drug and Food Control,
China. A voucher specimen (No. 20110817) has been deposited in the Herbarium of Jiangxi
Provincial Institute for Drug and Food Control.
3
.3. Extraction and isolation
e powdered dried leaves of Callicarpa nudiflora (9.6 kg) were extracted with 80% EtOH

at reflux for 3 × 2 h, and the extract was evaporated under reduced pressure to yield a
dark brown residue (1.8 kg), which was suspended in water (15 L) and then partitioned
with petroleum ether (3 × 15 L), EtOAc (3 × 15 L), and n-BuOH (3 × 15 L), successively.
Af er removing the solvent, the n-BuOH extract (545 g) was passed through a XAD-7
macroporous resin column eluted with H O and H O-EtOH (5:95, v/v), respectively. e
2
2
H O-EtOH (5:95, v/v) fraction (236 g) was separated by silica gel column chromatography
2
(
CC), eluting with CHCl -MeOH gradient mixtures (99:1−60:40, v/v), to afford 13 fractions,
3
E1–E13. Fraction E6 (23.9 g) was fractionated via silica gel CC, eluting with CHCl -MeOH
3
(
85:15−75:25, v/v) to give five fractions (E6-1−E6-5). Fraction E6-4 (7.18 g) was again
subjected to silica gel CC and eluted with CHCl -MeOH (95:5−80:20, v/v) to afford 12
3
fractions (E6-4-1−E6-4-12). Fraction E6–4-10 (4.31 g) was separated by ODS CC (30−100%,
MeOH−H O) to give 17 subfractions. Subfraction 9 (457 mg) was further separated by
2
preparative HPLC (YMC-ODS-A 5 μm, 250 mm × 20 mm, detection at 210 nm) using
1
1
9% CH CN-H O (5 ml/min) containing 0.01%TFA as mobile phase to yield compounds
3 2
(14.0 mg, t 84 min) and 2 (47.4 mg, t 93 min). Subfraction 12 (457 mg) was purified
R R
further by preparative HPLC (YMC-ODS-A 5 μm, 250 mm × 20 mm, detection at 210 nm)
using 16% CH CN-H O (7 ml/min) as mobile phase to yield compounds 3 (7.0 mg, t
3
2
R
1
21 min), 4 (12.6 mg, t 133 min) and 5 (6.7 mg, t 148 min).
R
R
3
.3.1. Callicoside C (1)
2
0
White amorphous powder;[ꢀ] − 58.8(c 0.08, MeOH); UV (MeOH) λ (logε): 228 (4.72)
D
max
1
13
and 312 (4.38) nm; H NMR (600 MHz, CD OD) and C NMR (150 MHz, CD OD) spec-
tral data see Table 1; HRESIMS: m/z 521.1656 [M – H] (calcd for C H O , 521.1659).
3
3
–
2
5
29 12

6
J. WANG ET AL.
3
.3.2. Callicoside D (2)
2
0
White amorphous powder;[ꢀ] − 25.3(c 0.07, MeOH); UV (MeOH) λ (logε): 228 (4.42)
D
max
1
13
and 312 (4.26) nm; H NMR (600 MHz, C D N) and C NMR (150 MHz, C D N) spectral
data see Table 1; HRESIMS: m/z 543.1479 [M + Na] (calcd for C H O Na, 543.1478).
5
5
5
5
+
2
5
28 12
3
.4. Determination of absolute configurations of the sugar moieties in 1 and 2
e determination of the absolute configuration of the sugars in compounds 1 and 2 was
conducted as described previously [7].

3
.5. Protective effect on cytotoxicity induced by d-galactosamine in WB-F344 cells

e hepatoprotective effects of compounds 1 and 5 were determined by a 3-(4,5-dimethylth-
iazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay in WB-F344 cells,
4
with some modification [8]. Each cell suspension of 1 × 10 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 planted in a 96-well microplate and precultured for 24 h at
3
7 °C under a 5% CO atmosphere. Fresh medium (200 μl) containing bicyclol and test
2
samples was added, and the cells were cultured for 1 h. en, the cultured cells were exposed
to 40 mM ꢀ-galactosamine for 24 h. Cytotoxic effects of test samples were measured simul-
taneously in the absence of ꢀ-galactosamine. e medium was changed into a fresh one
containing 0.5 mg/ml MTT. Af er 3.5 h incubation, the medium was removed and 150 μl
of dimethyl sulfoxide was added to dissolve formazan crystals. e optical density (OD) of
the formazan solution was measured on a microplate reader at 492 nm.
Acknowledgments
We thank Prof. Ai-Hong Liu at Center of Analysis And Testing Nanchang University for NMR
measurements.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding

is work was financially supported by the National Natural Science Foundation of China [NSFC,
grant number 81373955 and 81460589], Natural Science Foundation of Jiangxi, China [grant num-
ber 20142BAB205085], and Fund for the Development of Youth in Inspection Institute of Research
[
grant number 2013WA9].
References
[1] Guangdong Institute of Botany, Flora Hainanica, (Science Press, Beijing, 1977), p. 10.
[2] W.L. Mei, Z. Han, H.B. Cui, Y.X. Zhao, Y.Y. Deng, and H.F. Dai, Nat. Prod. Res. 24, 899 (2010).
[3] J. Fu, S.T. Kuang, and S.X. Wang, J. Hainan Univ. Nat. Sci. Ed. 20, 154 (2002).
[4] F.P. Gao, H. Wang, W.C. Ye, and S.X. Zhao, J. China Pharm. Univ. 41, 120 (2010).
[5] Z.N. Wang, Z. Han, H.B. Cui, and H.F. Dai, J. Trop. Subtrop. Bot. 15, 359 (2007).

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
7
[
[
[
[
6] Y.H. Luo, Z.Q. Zhou, S.C. Ma, and H.Z. Fu, Phytochem. Lett. 7, 194 (2014).
7] Z.Q. Zhou, X.Y. Wei, H.Z. Fu, and Y.H. Luo, Fitoterapia. 88, 91 (2013).
8] B. Huang, H.Z. Fu, W.K. Chen, Y.H. Luo, and S.C. Ma, Chem. Pharm. Bull. 62, 695 (2014).
9] Y.H. Luo, H.Z. Fu, B. Huang, W.K. Chen, and S.C. Ma, J. Asian Nat. Prod. Res. 18, 274 (2016).
[
[
[
[
[
[
10] H. Laiva, R. Kasai, M. Rakotovao, and K. Yamasaki, Nat. Med. 55, 187 (2001).
11] J.H. Kwak, H.J. Kim, K.H. Lee, S.C. Kang, and O.P. Zee, Arch. Pharm. Res. 32, 207 (2009).
12] J. Shi, C.J. Li, J.Z. Yang, Y.H. Yuan, N.H. Chen, and D.M. Zhang, Planta Med. 78, 1844 (2012).
13] C. Bergeron, A. Marston, R. Gauthier, and K. Hostettmann, Phytochemistry 44, 633 (1997).
14] S.X. Feng, B. Yi, M. Zhang, J. Xu, H. Lin, and W.T. Xu, Nat. Prod. Res. 31, 181 (2017).
15] J. Kinjo, K. Araki, K. Fukui, H. Higuchi, T. Ikeda, T. Nohara, Y. Ida, N. Takemoto, M. Miyakoshi,
and J. Shoji, Chem. Pharm. Bull. 40, 3269 (1992).
[
[
16] H.Z. Fu, R.J. Zhong, D.M. Zhang, and D. Wang, J. Asian Nat. Prod. Res. 15, 1139 (2013).
17] H. Inouye, Y. Takeda, and H. Nishimura, Phytochemistry 13, 2219 (1974).