Hepatoprotective iridoid glucosides from Callicarpa nudiflora

Article abstract of DOI:10.1080/10286020.2015.1074572

Two new iridoid glucosides, callicoside A (1) and callicoside B (2), were isolated from the leaves of Callicarpa nudiflora. Their structures were elucidated by means of spectroscopic methods and chemical evidences. 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.

Full text of DOI:10.1080/10286020.2015.1074572

Journal of Asian Natural Products Research
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Hepatoprotective iridoid glucosides from
Callicarpa nudiflora
Yue-Hua Luo, Hui-Zheng Fu, Bo Huang, Wei-Kang Chen & Shuang-Cheng Ma
To cite this article: Yue-Hua Luo, Hui-Zheng Fu, Bo Huang, Wei-Kang Chen & Shuang-Cheng Ma
(2015): Hepatoprotective iridoid glucosides from Callicarpa nudiflora, Journal of Asian Natural
Products Research, DOI: 10.1080/10286020.2015.1074572
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Date: 12 November 2015, At: 06:54

Journal of Asian Natural Products Research, 2015
http://dx.doi.org/10.1080/10286020.2015.1074572
Hepatoprotective iridoid glucosides from Callicarpa nudiflora
Yue-Hua Luoa, Hui-Zheng Fua*, Bo Huanga, Wei-Kang Chena and Shuang-Cheng Mab*
aJiangxi Provincial Institute for Drug Control, Jiangxi Provincial Engineering Research Center for
Drug and Medical Device Quality, Nanchang 330029, China; National Institutes for Food and
Drug Control, Beijing 100050, China
b
(Received 4 April 2015; final version received 15 July 2015)
Two new iridoid glucosides, callicoside A (1) and callicoside B (2), were isolated from
the leaves of Callicarpa nudiflora. Their structures were elucidated by means of
spectroscopic methods and chemical evidences. 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.
Keywords: Verbenacase; Callicarpa nudiflora; iridoid glucosides; hepatoprotective
activity
1. Introduction
ether, EtOAc, and n-BuOH, successively.
Callicarpa nudiflora Hook, belonging to the
family Verbenacase, is distributed widely in
Guangdong, Guangxi, and Hainan Pro-
vinces in China. The plant is used as
traditional Chinese herbal medicines for the
treatment of inflammation and bleeding [1].
Previous investigation on C. nudiflora has
led to the isolation of iridoids, flavonoids,
triterpenoids, and phenylpropanoid glyco-
sides [2–5]. Some of them exhibit anti-
inflammatory, antibacterial, cytotoxic, and
hemostatic activities [6]. In our previous
papers, phytochemical studies of
C. nudiflora yielded six triterpenoid glyco-
sides, three flavones, and three furofuran
lignans [6–8]. Aspartofa continuingsearch
for bioactive constituents from C. nudiflora,
an 80% EtOH extract of C. nudiflora has
been investigated and two new compounds
(1 and 2) obtained. Reported herein are the
isolation, structure elucidation, and biologi-
cal activity of these compounds.
The n-BuOH-soluble portion was separ-
ated by a combination of silica gel, ODS
column chromatography, and preparative
HPLC and afforded two new compounds
(1 and 2) (Figure 1). Their structures were
elucidated by extensive NMR techniques
including 1D NMR (1H and 13C NMR),
2D NMR (COSY, NOESY, HSQC and
HMBC), and HRESIMS, as well as
chemical evidences.
Compound 1 was obtained as a white
amorphous powder. Its molecular formula,
C24H30O13, was determined from HRE-
SIMS at m/z 549.1575 [M þ Na]þ and
supported by the NMR spectroscopic data.
1
The H NMR spectrum of 1 in CD3OD
exhibited two trans olefinic protons at dH
7.57 (1H, d, J ¼ 15.6Hz) and 6.29 (1H, d,
J ¼ 15.6Hz), a set of ABX-type coupled
aromatic protons at dH 7.05 (1H, d,
J ¼ 1.8 Hz), 6.96 (1H, dd, J ¼ 8.4,1.8 Hz),
and 6.78 (1H, d, J ¼ 8.4 Hz), and an
anomeric proton at dH 4.70 (1H, d,
J ¼ 7.8 Hz) correlated with an anomeric
carbon at dC 99.4 in the HSQC spectrum
(Table 1). In addition, a group of character-
2. Results and discussion
The 80% EtOH extract of Callicarpa
nudiflora was partitioned with petroleum
*Corresponding authors. Email: fhzfhz620@sohu.com; masc@nifdc.org.cn
q 2015 Taylor & Francis

2
Y.-H. Luo et al.
O
9'
7'
3'
H
O
H
O
H
O
H
1'
H
O
3
7
5
9
7
5
9
H
O
3
H
O
O
5
'
O
O
O
9'
H
O
1
O
7'
H
H
O
1
O
H
H
O
10
3'
H
O
10
O
O
O
O
H
1'
H
O
H
H
O
H
O
1''
O
1''
H
O
5'
H
O
H
O
2
1
Figure 1. Chemical structures of compounds 1 and 2.
istic signals for catalpol skeleton [9] were
observedatdH 4.60(1H,d,J ¼ 9.0 Hz),4.07
(1H, d, J ¼ 13.8 Hz), 4.01 (1H, d,
J ¼ 9.0 Hz), 3.83~3.85 (1H, m), 3.64 (1H,
d, J ¼ 13.8Hz), 3.39~3.41 (1H, m), 2.28
(1H, dd, J ¼ 9.0, 7.8 Hz), 2.00 (1H, dd,
Table 1. 1H (600 MHz) and 13C NMR (150 MHz) spectral data of compounds 1 and 2 in CD3OD
(d in ppm, J in Hz).
1
2
Position
dH
dC
dH
dC
Catalpol
1
4.60 (1H, d, 9.0)
97.9
63.0
5.67 (1H, d, 1.8)
5.32 (1H, d, 3.0)
93.1
95.9
3a
3b
4a
4b
5
6
7
8
9
3.83-3.85 (1H, m)
3.39-3.41 (1H, m)
1.73-1.75 (1H, m)
1.56 (1H, d, 13.8)
2.00 (1H, dd, 13.8, 7.8)
4.01 (1H, d, 9.0)
23.9
2.44-2.46 (1H, m)
2.36-2.38 (1H, m)
2.09 (1H, dd, 13.8, 3.0)
5.01 (1H, dd, 8.4, 3.0)
4.44 (1H, d, 8.4)
34.4
38.3
73.3
62.0
66.0
43.3
61.5
34.7
87.7
70.4
79.9
48.3
62.1
3.39-3.41 (1H, m)
2.28 (1H, dd, 9.0, 7.8)
4.07 (1H, d, 13.8)
3.64 (1H, d, 13.8)
2.62 (1H, br d, 9.6)
4.04 (1H, d, 13.8, 1.2)
3.68 (1H, d, 13.8, 1.2)
10a
10b
caffeoyl
10
127.6
115.1
146.9
149.7
116.6
123.1
147.2
114.9
169.0
127.6
115.2
146.8
149.8
116.5
123.1
147.6
114.5
168.9
20
7.05 (1H, d, 1.8)
7.07 (1H, d, 1.8)
30
40
50
6.78 (1H, d, 8.4)
6.80 (1H, d, 8.4)
60
6.96 (1H, dd, 8.4, 1.8)
7.57 (1H, d, 15.6)
6.29 (1H, d, 15.6)
6.97 (1H, dd, 8.4, 1.8)
7.58 (1H, d, 15.6)
6.32 (1H, d, 15.6)
70
80
90
Glc
100
200
300
400
500
600
4.70 (1H, d, 7.8)
3.25-3.27 (1H, m)
3.38-3.40 (1H, m)
3.38-3.40 (1H, m)
3.50-3.52 (1H, m)
4.46 (1H, dd, 11.4, 3.0)
3.40 (1H, m)
99.4
74.8
77.6
71.7
75.8
64.1
4.70 (1H, d, 7.8)
98.9
73.8
78.1
71.6
74.7
62.7
3.65-3.67 (1H, m)
3.29-3.31 (1H, m)
3.28-3.30 (1H, m)
3.15-3.17 (1H, m)
3.88 (1H, dd, 12.0, 1.2)
3.67 (1H, dd, 12.0, 1.2)

Journal of Asian Natural Products Research
3
J ¼ 13.8, 7.8 Hz), 1.73~1.75 (1H, m), and
vicinally coupled protons (H-1 and H-9)
[14]. H-9 gave a double doublet (dH 4.60,
J ¼ 9.0, 7.8 Hz) due to coupling with H-1
(dH 4.60) and H-5 (dH 2.00). Appreciable
coupling with H-5 indicated that the fused
ringwas cis [14]. Inthe NOESY spectrum of
1, the NOESY cross peaks of H-4a/H-6, H-
7/H-10, and H-1/H-10 indicated that the
hydroxy group at C-6 and the oxo-bridge
from C-7 to C-8 should be b-oriented. Full
assignments of the proton and carbon
resonances of 1 could be achieved by
1.56 (1H, d, J ¼ 13.8Hz) (Table 1). The 13
C
NMR spectrum of 1 in combination with the
HSQC spectrum displayed 25 carbon
signals, including those for a carbonyl
carbon at dC 169.0, a (E)ZCvC bond at
dC 147.2 and 114.9, for an aromatic ring at
dC 149.7, 146.9, 127.6, 123.1, 116.6, 115.1,
and for a sugar moiety at dC 99.4, 77.6, 75.8,
74.8, 71.7, and 64.1. These data suggested
thepresenceofacaffeoylmoietyandasugar
unit in the structure of 1. Comparison of the
NMR spectroscopic data of 1 (Table 1) with
those of piscroside B [10] demonstrated that
two compounds were almost identical
except that the p-coumaroyl residue in
piscroside B was replaced by a caffeoyl
group. Acid hydrolysis of 1 with 2 M HCl
afforded D-glucose, which was identified by
GC analysis of their trimethylsilyl L-
cysteine derivatives [11–12]. In the
HMBC spectrum of 1, the HMBC corre-
lation from H-600 (dH 4.46) to C-90 (dC 169.0)
confirmed that the caffeoyl ester group is
attached to C-600 of the glucose (Figure 2).
In addition, the HMBC correlation from
Glc-H-100 (dH 4.70) to C-1 (dC 97.9)
indicated the b-D-glucopyranosyl unit is
located at C-1. The relative configuration of
1was establishedbya combinationofthe1H
NMR spectral data and the NOESY
experiment, as well as a comparison with
thespectroscopicdataofthose reportedones
[13–15]. The large coupling of H-1
(J ¼ 9.0 Hz) with H-9 (dH 2.28) demon-
strated the dihedralangle tobe 1808 between
1
comprehensive analysis of H NMR, 13C
1
NMR, H-1H COSY, HSQC, HMBC, and
NOESY spectra (Table 1). Thus, compound
1 was determined as 60-O-caffeoyl-dihydro-
catalpol, named callicoside A.
Compound 2 was obtained as a white
amorphous powder. The HRTOFMS of 1
showed a quasi-molecular ion peak at m/z
559.1656 [M–H]–, indicating a molecular
formula of C24H32O15. Acid hydrolysis of
2 with 2 M HCl afforded D-glucose, which
was identified by GC analysis of their
trimethylsilyl L-cysteine derivatives [10,
11]. On the basis of the coupling constant
of anomeric proton and the chemical shift
of the anomeric carbon, the anomeric
configuration of the glucose moiety was
determined as b [16]. Comparison of the
NMR data of 2 with those of 1 indicated
2 consisted of the following structural
fragments: a catalpol skeleton, a caffeoyl
group, and a glucose moiety. The differ-
ences between 1 and 2 were in the catalpol
moiety and the connection position of
O
H
O
H
O
H
H
H
O
O
H
H
H
O
H
O
H
O
H
O
O
O
O
O
H
O
H
O
O
O
O
O
O
H
O
H
O
H
O
H
O
H
O
H
O
H
O
H
O
2
1
Figure 2. Key HMBC (H ! C) correlations of compounds 1 and 2.

4
Y.-H. Luo et al.
caffeoyl residue. Meanwhile, detailed
NMR analysis showed an additional
hydroxy group in 2. In the 13C NMR
spectrum of 2, C-3 (dC 95.9) was
deshielded by DdC 32.9 as compared to
1, indicating that the hydroxyl group is
located at C-3 in 2. In the HMBC spectrum
of 2, HMBC correlations from Glc-H-100
(dH 4.70) to C-1 (dC 93.1) and H-6 (dH
5.01) to C-90 (dC 168.9) indicated b-D-
glucopyranosyl unit and the caffeoyl ester
group are attached to C-1 and C-6 of
aglycone, respectively. The relative con-
figuration of 2 was established by a
combination of the NOESY experiment
and the biogenetic ground. The small
coupling of H-1 (J ¼ 1.8 Hz) with H-9 (dH
2.62) confirmed the b-orientation of H-9,
since, naturally occurring iridoids display
an a-orientation for H-1 [17–18]. The
large coupling of H-9 (J ¼ 9.6 Hz) with H-
5 (dH 2.09) confirmed the b-orientation of
H-5, demonstrating that the stereochem-
istry of the catalpol ring fusion was cis.
In the NOESY spectrum of 2, the presence
of NOE interactions of H-3/H-5 and H-7/
H-9 indicated that the hydroxy groups at
C-3 and C-7 are in a-positions. Mean-
while, NOE interactions were observed
among H-1, H-6, and H-10, which
indicated the hydroxy groups at C-6 and
C-8 should be b-oriented. Thus, com-
pound 2 was elucidated as 7-O-caffeoyl-3,
6, 8-trihydroxy-8-(hydroxymethyl)cyclo-
penta[c ]pyran-1-O-b-D-glucopyranoside,
named callicoside B.
Table 2. Hepatoprotective effects of com-
pounds 1–2 against D-galactosamine-induced
toxicity in WB-F344 cells.a
Cell survival
rate (% of normal)
Compound
X ^ s
Normal
Control
1.026 ^ 0.037
0.325 ^ 0.076
100
28##
50*
50*
26
Bicyclolb 0.537 ^ 0.049
1
2
0.558 ^ 0.085
0.323 ^ 0.058
a Results are expressed as means ^ SD (n ¼ 3);
##p , 0.001, significantly different from control by
Student’s t-test; *p , 0.05, significantly different
from normal by Student’s t-test.
b Positive control compound.
point apparatus with a microscope and are
uncorrected (Shanghai Eastern Analytical
Instrument Co., Ltd., Shanghai, China).
UV spectra were recorded in MeOH on a
Jasco V650 spectrophotometer (JASCO,
Inc., Easton, MD, USA). The 1H
(600 MHz), 13C (150 MHz), and 2D
NMR spectra were recorded on a Bruker
AVANCE III 600 instrument using TMS
(Tetramethylsilane) as an internal refer-
ence (Bruker Company, Boston, MA,
USA). HRTOFMS data were obtained on
an Agilent 7890-7000A mass spectrometer
(Agilent Technologies, Santa Clara, CA,
USA). Preparative HPLC (high perform-
ance liquid chromatography) was con-
ducted with an Agilent Technologies 1200
series instrument with a MWD (multiple
wavelength detector) using a YMC-pack
ODS (Octadecylsilyl)-A column (5 mm,
250 mm £ 20 mm). Column chromatog-
raphy was performed with silica gel
(200–300 mesh, Qingdao Marine Chemi-
cal Ltd., Qingdao, China), Develosil ODS
(50 mm, Nomura Chemical Co. Ltd.,
Osaka, Japan), Sephadex LH-20 (GE
Healthcare Bio-Sciences AB, Uppsala,
Sweden). TLC (thin layer chromatog-
raphy) was carried out with glass pre-
coated with silica gel GF254. Spots were
visualized under UV light or by spraying
with 10% sulfuric acid in EtOH followed
by heating.
In an in vitro bioassay, compound 1 at
1025 M showed pronounced hepatopro-
tective activities against D-galactosamine-
induced toxicity in WB-F344 rat hepatic
epithelial stem-like cells (Table 2).
3. Experimental
3.1. General experimental procedures
Optical rotations were measured on an
Autopol IV-T/V (Rudolph Research Ana-
lytical, New Jersey, USA). Melting points
were determined on an XT digital melting-

Journal of Asian Natural Products Research
5
3.2. Plant material
3.3.1. Callicoside A (1)
The leaves of Callicarpa nudiflora Hook
were collected from Wuzhishan, Hainan,
China, in August 2011 and identified by
Prof. Guiping Yuan at Jiangxi Provincial
Institute for Drug and Food Control,
White amorphous powder; mp 179–
20
1808C; ½aꢀD 258.8 (c 0.08, MeOH); UV
(MeOH) lmax (log 1): 205 (4.72) and 329
1
(4.38) nm; H NMR (600 MHz, CD3OD)
and 13C NMR (150 MHz, CD3OD) spec-
tral data see Table 1; HRESIMS: m/z
549.1575 [M þ Na]þ (calculated for
C24H30O13Na, 549.1579).
China.
A
voucher specimen (No.
20110817) has been deposited in the
Herbarium of Jiangxi Provincial Institute
for Drug and Food Control.
3.3.2. Callicoside B (2)
Yellow amorphous powder; mp 117–
20
3.3. Extraction and isolation
1198C; ½aꢀD 210 (c 0.12, MeOH); UV
The powdered dried leaves of Callicarpa
nudiflora (9.6 kg) were extracted three
times with 80% EtOH under reflux (2 h
each). The extracting solution was evap-
orated under reduced pressure to yield a
dark brown residue (1.8 kg). The residue
was suspended in water (15 L) and then
successively partitioned with petroleum
ether (3 L £ 15 L), EtOAc (3 L £ 15 L),
and n-BuOH (3 L £ 15 L). After removing
the solvent, the n-BuOH extract (545 g)
was passed through a XAD-7 macroporous
resin column eluted with H2O and H2O-
EtOH (5:95, v/v), respectively. The H2O-
EtOH (5:95, v/v) fraction (236 g) was
separated by silica gel column chromatog-
raphy (CC) using CHCl3-MeOH gradient
mixtures (99:1 2 60:40, v/v) to give 13
fractions (E1–E13). Fraction E6 (23.9 g)
was subjected to silica gel CC and eluted
with CHCl3-MeOH (85:15 2 75:25, v/v)
to afford five fractions (E6-1 2 E6-5).
Fraction E6-4 (7.18 g) was again subjected
to silica gel CC and eluted with CHCl3-
MeOH (95:5 2 80:20, v/v) to afford 12
fractions (E6-4-1 2 E6-4-12). Fraction
E6-4-11 (1.53 g) was separated by ODS
CC (10 2 70%, MeOH 2 H2O) to give 21
subfractions. Subfraction 4 (145 mg) was
further separated by preparative HPLC
(YMC-ODS-A 5 mm, 250 mm £ 20 mm,
detection at 210 nm) using 15% CH3CN-
H2O (5 ml/min) containing 0.01% TFA
(trifluoroacetic acid) as mobile phase to
yield compounds 1 (25.0 mg, tR 87 min)
and 2 (2.1 mg, tR 105 min).
(MeOH) lmax (log 1): 204 (4.46), 244
(3.65), and 330 (4.23) nm; 1H NMR
(600 MHz, CD3OD) and 13C NMR
(150 MHz, CD3OD) spectral data see
Table 1; HRTOFMS: m/z 559.1656
[M 2 H]2 (calculated for C24H31O15,
559.1663).
3.4. Determination of absolute
configurations of the sugar moieties in 1–2
The determination of the absolute con-
figuration of the sugars in compounds 1–2
was conducted as described previously [7].
3.5. Protective effect on cytotoxicity
induced by D-galactosamine in
WB-F344 cells
The hepatoprotective effects of com-
pounds 1–2 was conducted as described
previously [8].
Acknowledgment
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
Financial support was provided by the National
Natural Science Foundation of China (NSFC,
[grant number 81373955]), Natural Science

6
Y.-H. Luo et al.
Foundation of Jiangxi, China [grant number
20142BAB205085], and Fund for the Devel-
opment of Youth in Inspection Institute of
Research (No. 2013WA9).
[10] S.X. Huang, Y. Zhou, Q.J. Nie, L.S. Ding,
and L. Peng, J. Asian Nat. Prod. Res 8,
259 (2006). doi:10.1080/102860205000
34543.
[11] 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). doi:10.
1248/cpb.40.3269.
[12] H.Z. Fu, R.J. Zhong, D.M. Zhang, and
D. Wang, J. Asian Nat. Prod. Res 15,
1139 (2013). doi:10.1080/10286020.
2013.817393.
[13] E. Helfrich and H. Rimpler, Phytochem-
istry 50, 619627 (1999). doi:10.1016/
S0031-9422(98)00559-7.
[14] N. Tiwari, A.K. Yadav, P. Srivastava,
K. Shanker, R.K. Verma, and M. Gupta,
Phytochemistry 69, 2387 (2008). doi:10.
1016/j.phytochem.2008.06.016.
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 F. Dai, Nat. Prod. Res 24,
899 (2010). doi:10.1080/14786410802
267544.
[3] J. Fu, S.T. Kuang, S.X. Wang, and
J. Hainan, Univ. Nat. Sci. Ed 20, 154
(2002).
[4] F.P. Gao, H. Wang, W.C. Ye, and
X. Zhao, J. China Pharm. Univ 41, 120
(2010).
[15] R.M. Taskova, T. Kokubun, P.J. Garnock-
Jones, and R. Jensen, Phytochemistry 77,
209 (2012). doi:10.1016/j.phytochem.
2012.02.001.
[16] T. Miyase, H. Noguchi, and X.M Chen,
J. Nat. Prod 62, 993 (1999). doi:10.1021/
np990084t.
[5] Z.N. Wang, Z. Han, H.B. Cui, and F. Dai,
J. Trop. Subtrop. Bot 15, 359 (2007).
[6] Y.H. Luo, Z.Q. Zhou, S.C. Ma, and Z. Fu,
Phytochem. Lett 7, 194 (2014). doi:10.
1016/j.phytol.2013.12.001.
[7] Z.Q. Zhou, X.Y. Wei, H.Z. Fu, and
H. Luo, Fitoterapia 88, 91 (2013). doi:10.
1016/j.fitote.2013.05.007.
[8] B. Huang, H.Z. Fu, W.K. Chen, Y.H. Luo,
and C. Ma, Chem. Pharm. Bull. 62, 695
(2014). doi:10.1248/cpb.c14-00159.
[9] R.K. Chaudhuri, O. Sticher, and
R.K. Chaudhuri, O. Sticher, Helv. Chim.
Acta 62, 644 (1979). doi:10.1002/hlca.
19790620232.
[17] L.F. Tietze, U. Niemeyer, P. Marx, and
H. Glu¨senkamp, Tetrahedron 36, 1231
(1980).
87024-4.
doi:10.1016/0040-4020(80)
[18] F.W. Dong, L. Yang, Z.K. Wu, W. Gao,
C.T. Zi, D. Yang, H.R. Luo, J. Zhou, and
M. Hu, Fitoterapia 102, 27 (2015).
doi:10.1016/j.fitote.2015.01.021.