Free radical scavenging and hepatoprotective effects of salidroside analogs on CCl4-induced cytotoxicity in LO2 cells

Article abstract of DOI:10.1007/s00044-012-0247-z

Salidroside, a phenylpropanoid glycoside isolated from a traditional Chinese medicinal plant Rhodiola rosea L. displays a broad spectrum of pharmacological properties. It has been found to play a hepatoprotective role in liver diseases through inhibiting apoptosis of hepatocytes and proliferation of hepatic stellate cells, decreasing serum aminotransferase, reversing hepatic fibrosis, and improving liver function. In this study as an ongoing study on the discovery and development of new hepatoprotective agents, about 12 novel glycosides were synthesized, and 2,2-diphenyl-1-picrylhydrazyl radical scavenge activity of each glycoside was evaluated. 2-(3,4,5-trihydroxyphenyl)ethyl β-d-glucopyranoside (4g) and 2-(3,4,5-trihydroxyphenyl)ethyl β-d-galactopyranoside (4h) exhibited significant activity prior to salidroside with an IC₅₀ value of 38.05 and 35.85 μM, respectively. The hepatoprotective effect of compounds 4g and 4h on CCl ₄-induced cytotoxicity in LO2 cells was assessed for further research. Graphical abstract: 2-(3,4,5-Trihydroxyphenyl)ethyl β-d-glucopyranoside (4g) and 2-(3,4,5-trihydroxyphenyl)ethyl β-d-galactopyranoside (4h) exhibited significant hepatoprotective activity prior to salidroside.[Figure not available: see fulltext.]

Full text of DOI:10.1007/s00044-012-0247-z

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:2524–2530
DOI 10.1007/s00044-012-0247-z
ORIGINAL RESEARCH
Free radical scavenging and hepatoprotective effects of salidroside
analogs on CCl4-induced cytotoxicity in LO2 cells
•
Yibing Guo Cheng Zheng Wen Xu Yongxing Si
•
•
•
•
Sufeng Dou Yumin Yang
Received: 29 January 2012 / Accepted: 14 September 2012 / Published online: 26 September 2012
Ó Springer Science+Business Media New York 2012
Abstract Salidroside, a phenylpropanoid glycoside iso-
lated from a traditional Chinese medicinal plant Rhodiola
rosea L. displays a broad spectrum of pharmacological
properties. It has been found to play a hepatoprotective role
in liver diseases through inhibiting apoptosis of hepato-
cytes and proliferation of hepatic stellate cells, decreasing
serum aminotransferase, reversing hepatic fibrosis, and
improving liver function. In this study as an ongoing study
on the discovery and development of new hepatoprotective
agents, about 12 novel glycosides were synthesized, and
2,2-diphenyl-1-picrylhydrazyl radical scavenge activity of
each glycoside was evaluated. 2-(3,4,5-trihydroxyphenyl)
ethyl b-D-glucopyranoside (4g) and 2-(3,4,5-trihydrox-
yphenyl)ethyl b-D-galactopyranoside (4h) exhibited sig-
nificant activity prior to salidroside with an IC50 value of
38.05 and 35.85 lM, respectively. The hepatoprotective
effect of compounds 4g and 4h on CCl4-induced cyto-
toxicity in LO2 cells was assessed for further research.
Introduction
Salidroside, a traditional Chinese medicine, can be syn-
thesized by glucosylation of 2-(4-hydroxyphenyl)ethanol
(Fig. 1). It has been shown to have a wide range of phar-
macological properties, including resisting anoxia (Zhang
et al., 2009), eliminating fatigue (Darbinyan et al., 2000),
preventing cardiovascular disease (Wu et al., 2009a),
neuroprotection (Hoi et al., 2010; Li et al., 2010; Zhang
et al., 2011), and anti-tumor activities (Hu et al., 2010a, b).
Knowing that salidroside has a moderate radical scav-
enging activity, its hepatoprotective activity has become a
focus of research in recent years. The hepatoprotective
activity against tacrine-induced cytotoxicity in human
liver-derived Hep G2 cells was prior to silybin, which was
known for its hepatoprotective efficiency (Song et al.,
2003). Salidroside can also protect liver tissue against
paracetamol-induced oxidative damage by suppressing the
depletion of glutathione (GSH) (Wu et al., 2008). The
effect of salidroside on oxidative stress in rat hepatic
stellate cells may be related to inhibition of lipid peroxi-
dation (Zhang and Liu, 2005). There are also studies
reporting that salidroside could protect liver tissue against
oxidative stress elicited by D-galactosamine and lipopoly-
saccharide, which were reported to relate to antioxidant
activity and inhibition of hypoxia-inducible factor-1a
(HIF-1a) (Wu et al., 2009b). Salidroside has been shown to
ameliorate the effects of experimental liver fibrosis and
partially inhibit experimental liver fibrosis. Findings in
recent studies suggest that salidroside could differentiate
rMSCs toward hepatocytes. Treatment with rMSCs trans-
plantation and salidroside may exert synergistic effects on
the experimental hepatic fibrosis via suppressing the
expression of transforming growth factor-b1 (TGF-b1)
(Ouyang et al., 2010).
Keywords Salidroside Á Analogs Á
Hepatoprotective effects Á DPPH
Y. Guo
Surgical Comprehensive Laboratory of Affiliated Hospital,
Nantong University, No. 20, Xisi Road, Nantong 226001,
Jiangsu, People’s Republic of China
C. Zheng Á W. Xu Á Y. Si Á S. Dou Á Y. Yang (&)
Jiangsu Key Laboratory of Neuroregeneration, Nantong
University, No. 19, Qixiu Road, Nantong 226001,
Jiangsu, People’s Republic of China
e-mail: yangym@ntu.edu.cn
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Med Chem Res (2013) 22:2524–2530
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Synthesis
General procedures for preparation of compounds
3a–l (Schemes 1, 2)
Fig. 1 The chemical structure of salidroside
A mixture of corresponding alcohol (4 mmol) and Ag2CO3
(1.1 g, 4 mmol) was dissolved in dry ether–dichlorometh-
ane (1:1, 25 ml) with addition of powdered molecular
Carbon tetrachloride (CCl4) is a potent hepatotoxin
producing centrilobular hepatic necrosis. It is biotrans-
formed to the trichloromethyl radical (ÁCCl3) by the cyto-
chrome P450 system in liver microsomes, often leading to
liver injury (Azri et al., 1992). It is important to use hep-
atotoxic agents that are more relevant to human liver tox-
icosis in the search for hepatoprotective agents (Perez
Tamayo, 1983). In this study, we selected CCl4-induced
LO2 cells insult as a cell model. Although previous
research has reported that salidroside possesses a hepato-
protective activity, no study in the literature has been
reported about the protective effect against CCl4-induced
hepatotoxicity of salidroside analogs. The aim of this study
was to find substances from salidroside analogs that could
prevent the hepatotoxic effect of CCl4 for clinical use.
About 12 novel glycosides were synthesized with the
acetobromo-glucose and acetobromo-galactose as glycosyl
donor, and 4-hydroxyphenyl ethanol differ in the substit-
uents as glycosyl acceptor. The antioxidant properties
of these compounds on 2,2-diphenyl-1-picrylhydrazyl
(DPPH) radical scavenging activity were investigated. The
hepatoprotective effect of compounds prior to salidroside
on CCl4-induced cytotoxicity in LO2 cells was preliminary
assessed.
˚
sieves (4 A, 2.0 g), and then stirred at room temperature
under N2 atmosphere. After 1 h, acetobromo-glucose 1a or
acetobromo-galactose 1b (2.5 g, 6 mmol) and another
Ag2CO3 (550 mg, 2 mmol) were added rapidly to the
mixture and stirred continuously for 14 h to avoid light.
The reaction mixture was filtered and evaporated under
reduced pressure. The residue was purified by short column
chromatography on silica gel eluting with 1: 2 AcOEt–
petroleum to afford a colorless gummy mass.
The colorless gummy mass was dissolved in MeOH with
addition of NaOCH3 (10.8 mg, 0.2 mmol), and stirred for
2 h at room temperature. After neutralization of the mix-
ture by adding Amberlite IRA-120, the resin was filtered
and washed with MeOH. The filtrate was concentrated in
vacuo and recrystallized from ether to afford the desired
product in yield 55–75 % as solid.
2-(4-Benzyloxy-3-methoxyphenyl)ethyl b-D-glucopyrano-
side (3a) m.p. 122–124 °C. 1H NMR (CD3OD,
500 MHz) d (ppm): 2.86 (2H, t, J = 7.2 Hz), 3.17 (1H, t,
J = 7.8 Hz), 3.25–3.27 (2H, m), 3.35–3.37 (1H, m),
3.62–3.67 (1H, m), 3.70–3.77 (1H, m), 3.83 (3H, s),
3.84–3.87 (1H, m), 4.03–4.11 (1H, m), 4.29 (1H, d,
J = 7.7 Hz), 5.05 (2H, s), 6.73 (1H, dd, J = 2.1 and
8.1 Hz), 6.87 (1H, d, J = 8.1 Hz), 6.92 (1H, d,
J = 2.1 Hz), 7.27–7.36 (3H, m), 7.40–7.43 (2H, m).
Materials and methods
Melting points were determined on an X-6 melting point
apparatus and were uncorrected. 1H NMR spectra were
recorded using TMS as an internal standard on a Bruker
AC 400 instrument at 500 MHz with D2O or CD3OD as
solvent. IR was recorded using AVATAR-370 FT-IR
Thermo Nicolet with KBr disk. Elemental analysis (C and
H) was performed using Elementar VarioEL III analyzer
(German). Flash chromatography was performed on Silica
gel (200–300 mesh). Commercial reagents were used
without further purification unless otherwise stated. The
normal human hepatocyte cell line LO2 was purchased
from the Type Culture Collection of the Chinese Academy
of Sciences, (Shanghai, China). Salidroside and Vitamin C
were purchased from Aladdin. Detection kits for mal-
ondialdehyde (MDA), superoxide dismutase (SOD), glu-
tathione peroxidase (GSH-Px) were purchased from
Nanjing Jiancheng Bioengineering.
2-(4-Benzyloxy-3-methoxyphenyl)ethyl b-D-galactopyrano-
side (3b) m.p. 139–142 °C. 1H NMR (CD3OD,
500 MHz) d (ppm): 2.87 (2H, t, J = 7.0 Hz), 3.45 (1H, dd,
J = 3.3 and 9.7 Hz), 3.48–3.53 (2H, m), 3.72–3.77 (3H,
m), 3.82 (1H, d, J = 2.9 Hz), 3.84 (3H, s), 4.03–4.10 (1H,
m), 4.25 (1H, d, J = 7.6 Hz), 5.05 (2H, s), 6.74 (1H, d,
J = 8.1 Hz), 6.87 (1H, d, J = 8.2 Hz), 6.93 (1H, s), 7.28
(1H, t, J = 7.2 Hz), 7.34 (2H, t, J = 7.4 Hz), 7.42 (2H, d,
J = 7.3 Hz).
2-(4-Benzyloxy-3,5-dimethoxyphenyl)ethyl b-D-glucopyran-
1
oside (3c) m.p. 46–48 °C. H NMR (CD3OD, 500 MHz)
d (ppm): 2.89 (2H, t, J = 7.1 Hz), 3.19 (1H t, J = 8.3 Hz),
3.26–3.28 (2H, m), 3.34–3.39 (1H, m), 3.62–3.68 (1H, m),
3.79 (6H, s), 3.84–3.89 (2H, m), 4.06–4.11 (1H, m), 4.31
(1H, d, J = 7.7 Hz), 4.90 (2H, s), 6.59 (2H, s), 7.27–7.46
(5H, m).
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Med Chem Res (2013) 22:2524–2530
Scheme 1 General method for preparation of compounds 4a–h. Reagents: (a) Ag2CO3, CH2Cl2, Et2O; (b) NaOMe, MeOH; (c) HCOONH4, 5 %
Pd/C, CH3OH, reflux
Scheme 2 General method for preparation of compounds 3i–l. Reagents: (a) Ag2CO3, CH2Cl2, Et2O; (b) NaOMe, MeOH
2-(4-Benzyloxy-3,5-dimethoxyphenyl)ethyl b-D-galactopy-
ranoside (3d) m.p. 125–127 °C. 1H NMR (CD3OD,
500 MHz) d (ppm): 2.89 (2H, t, J = 7.2 Hz), 3.46 (1H, dd,
J = 3.4 and 9.7 Hz), 3.49–3.54 (2H, m), 3.73–3.79 (3H,
m), 3.80 (6H, s), 3.83 (1H), 4.04–4.12 (1H, m), 4.27 (1H, d,
J = 7.6 Hz), 4.90 (2H, s), 6.59 (2H, s), 7.28–7.34 (3H, m),
7.45 (2H, d, J = 6.8 Hz).
3.29–3.31 (2H, m), 3.39 (1H, t, J = 8.8 Hz), 3.70 (1H, dt,
J = 3.1 and 2.6 Hz), 3.78 (1H, dt, J = 9.8 and 7.0 Hz),
3.90 (1H, d, J = 10.8 Hz), 4.10 (1H, dt, J = 9.8 and
7.1 Hz), 4.33 (1H, d, J = 7.8 Hz), 4.96 (2H, s), 5.12 (4H,
s), 6.73 (2H, s), 7.24–7.47 (15H, m).
2-(3,4,5-Tribenzyloxyphenyl)ethyl b-D-galactopyranoside
1
(3h) m.p. 144–146 °C. H NMR (CD3OD, 500 MHz) d
2-(4-Benzyloxy-3-ethoxyphenyl)ethyl b-D-glucopyranoside
(ppm): 2.87 (2H, t, J = 6.9 Hz), 3.44–3.57 (3H, m),
3.74–3.77 (3H, m), 3.84 (1H, d, J = 2.5 Hz), 4.04–4.10
(1H, m), 4.26 (1H, d, J = 7.4 Hz), 4.93 (2H, s), 5.09 (4H,
s), 6.70 (2H, s), 7.24–7.47 (15H, m).
1
(3e) m.p. 128–130 °C. H NMR (CD3OD, 500 MHz) d
(ppm): 1.39 (3H, t, J = 7.0 Hz), 2.86 (2H, t, J = 7.1 Hz),
3.18 (1H, t, J = 8.4 Hz), 3.26–3.29 (2H, m), 3.30–3.35
(1H, m), 3.66 (1H, dd, J = 5.2 and 11.8 Hz), 3.72–3.74
(1H, m), 3.86 (1H, dd, J = 1.9 and 12.1 Hz), 4.05–4.08
(3H, m), 4.29 (1H, d, J = 7.8 Hz), 5.06 (2H, s), 6.73–6.91
(3H, m), 7.26–7.48 (5H, m).
2-(3-Bromo-4-hydroxyphenyl)ethyl b-D-glucopyranoside
(3i) Solid, MS: m/z 379 [M?H]?; 1H NMR (D2O,
500 MHz) d (ppm): 2.90 (2H, t, J = 6.6 Hz), 3.26 (1H, t,
J = 8.5 Hz), 3.38–3.53 (3H, m), 3.72 (1H, dd, J = 5.4 and
12.3 Hz), 3.87–3.94 (2H, m), 4.06–4.13 (1H, m), 4.46 (1H,
d, J = 7.8 Hz), 6.98 (1H, d, J = 8.1 Hz), 7.21 (1H, d,
J = 8.3 Hz), 7.55 (1H, s). IR (KBr) cm–1: 3421, 2927,
1613, 1509, 1421, 1368, 1283, 1224, 1077. Anal. Calcd for
C14H19BrO7: C, 44.34; H, 5.05; Found: C, 44.30; H, 5.06.
2-(4-Benzyloxy-3-ethoxyphenyl)ethyl b-D-galactopyrano-
side (3f) 1H NMR (CD3OD, 500 MHz) d (ppm): 1.39
(3H, t, J = 7.0 Hz), 2.86 (2H, t, J = 7.1 Hz), 3.45 (1H, dd,
J = 3.3 and 9.7 Hz), 3.48–3.49 (2H, m), 3.73–3.75 (3H,
m), 3.81 (1H, d, J = 2.3 Hz), 4.06–4.08 (3H, m), 4.25 (1H,
d, J = 7.6 Hz), 5.06 (2H, s), 6.73–6.91 (3H, m), 7.27–7.47
(5H, m).
2-(3-Bromo-4-hydroxyphenyl)ethyl b-D-galactopyranoside
(3j) Solid, MS: m/z 379 [M?H]?; 1H NMR (D2O,
500 MHz) d (ppm): 2.88 (2H, t, J = 6.5 Hz), 3.48 (1H, t,
J = 8.9 Hz), 3.60–3.66 (2H, m), 3.75 (2H, s), 3.86–3.91
(2H, m), 4.09–4.13 (1H, m), 4.39 (1H, d, J = 7.7 Hz), 6.97
2-(3,4,5-Tribenzyloxyphenyl)ethyl
(3g) m.p. 78–80 °C. 1H NMR (CD3OD, 500 MHz) d
(ppm): 2.89 (2H, t, J = 7.0 Hz), 3.22 (1H, t, J = 8.0 Hz),
b-D-glucopyranoside
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Med Chem Res (2013) 22:2524–2530
2527
(1H, d, J = 8.0 Hz), 7.19 (1H, d, J = 8.3 Hz), 7.54 (1H,
s). IR (KBr) cm–1: 3395, 2930, 1609, 1508, 1418, 1372,
1284, 1222, 1079. Anal. Calcd for C14H19BrO7: C, 44.34;
H, 5.05; Found: C, 44.37; H, 5.08.
(2H, m), 7.00 (1H, s). IR (KBr) cm–1: 3520, 3396, 2949,
1602, 1520, 1465, 1379, 1278, 1128, 1061. Anal. Calcd for
C15H22O8: C, 54.54; H, 6.71; Found: C, 54.58; H, 6.73.
2-(4-Hydroxy-3,5-dimethoxyphenyl)ethyl b-D-glucopyran-
oside (4c) White solid, m.p. 124–127 °C. MS: m/z 361
[M?H]?; 1H NMR (D2O, 500 MHz) d (ppm): 2.89 (2H, t,
J = 6.9 Hz), 3.26 (1H, t, J = 8.5 Hz), 3.34–3.51 (3H, m),
3.72 (1H, dd, J = 5.6 and 12.2 Hz), 3.86 (6H, s), 3.89–3.93
(2H, m), 4.08–4.16 (1H, m), 4.46 (1H, d, J = 7.8 Hz), 6.70
(2H, s). IR (KBr) cm–1: 3510, 3404, 2944, 1617, 1523,
1471, 1337, 1240, 1130, 1079, 1025. Anal. Calcd for
C16H24O9: C, 53.33; H, 6.71; Found: C, 53.32; H, 6.70.
2-(3,5-Dibromo-4-hydroxyphenyl)ethyl b-D-glucopyrano-
1
side (3k) Solid, MS: m/z 459 [M?H]?; H NMR (D2O,
500 MHz) d (ppm): 2.82 (2H, t, J = 6.8 Hz), 3.18 (1H, t,
J = 8.4 Hz), 3.26–3.39 (3H, m), 3.61–3.73 (2H, m), 3.87
(1H, d, J = 11.7 Hz), 4.00–4.07 (1H, m), 4.29 (1H, d,
J = 7.7 Hz), 7.40 (2H, s). IR (KBr) cm–1: 3411, 2929,
1717, 1561, 1478, 1407, 1240, 1158, 1028. Anal. Calcd for
C14H18Br2O7: C, 36.71; H, 3.96; Found: C, 36.67; H, 3.98.
2-(3,5-Dibromo-4-hydroxyphenyl)ethyl b-D-galactopyran-
2-(4-Hydroxy-3,5-dimethoxyphenyl)ethyl b-D-galactopy-
ranoside (4d) White solid, m.p. 170–173 °C. MS: m/
1
oside (3l) Solid, MS: m/z 459 [M?H]?; H NMR (D2O,
1
500 MHz) d (ppm): 2.87 (2H, t, J = 6.0 Hz), 3.48 (1H, t,
J = 8.6 Hz), 3.60–3.64 (2H, m), 3.75 (2H, s), 3.87–3.91
(2H, m), 4.07–4.11 (1H, m), 4.39 (1H, d, J = 7.4 Hz), 7.53
(2H, s). IR (KBr) cm–1: 3383, 2887, 1711, 1555, 1479,
1407, 1240, 1159, 1065. Anal. Calcd for C14H18Br2O7: C,
36.71; H, 3.96; Found: C, 36.74; H, 3.92.
z 361 [M?H]?; H NMR (D2O, 500 MHz) d (ppm): 2.89
(2H, t, J = 6.9 Hz), 3.49 (1H, dd, J = 7.9 and 9.9 Hz),
3.62 (1H, dd, J = 3.4 and 9.9 Hz), 3.65–3.67 (1H, m),
3.74–3.77 (2H, m), 3.85 (6H, s), 3.89–3.91 (2H, m), 4.12
(1H, m), 4.39 (1H, d, 7.9 Hz), 6.70 (2H, s). IR (KBr) cm–1:
3512, 3410, 2937, 1612, 1522, 1463, 1335, 1225, 1126,
1086, 1047. Anal. Calcd for C16H24O9: C, 53.33; H, 6.71;
Found: C, 53.35; H, 6.70.
General procedures for preparation of compounds
4a–h (Scheme 1)
2-(4-Hydroxy-3-ethoxyphenyl)ethyl b-D-glucopyranoside
(4e) White solid, m.p. 155–157 °C. MS: m/z 345
[M?H]?; 1H NMR (D2O, 500 MHz) d (ppm): 1.39 (3H, t,
J = 7.0 Hz), 2.88 (2H, t, J = 6.9 Hz), 3.25 (1H, t,
J = 8.6 Hz), 3.36–3.49 (3H, m), 3.71 (1H, dd, J = 5.7 and
12.3 Hz), 3.87–3.92 (2H, m), 4.11–4.16 (3H, m), 4.46 (1H,
d, J = 8.0 Hz), 6.84 (1H, d, J = 8.0 Hz), 6.90 (1H, d,
J = 8.0 Hz), 7.01 (1H, s). IR (KBr) cm–1: 3404, 2985,
1614, 1519, 1437, 1361, 1272, 1160, 1124, 1079, 1043.
Anal. Calcd for C16H24O8: C, 55.81; H, 7.02; Found: C,
55.78; H, 6.99.
A suspension of 3a–h (6 mmol) and 5 % Pd/C (280 mg) in
methanol (50 ml) was stirred under hydrogen atmosphere
at room temperature overnight. The mixture was filtered
through Celite, and the filtrate was concentrated under
reduced pressure. The crude product was purified by flash
chromatography on silica gel eluting with a mixture of
CHCl3 and MeOH (6:1, v/v) to afford the desired product
4a–h.
2-(4-Hydroxy-3-methoxyphenyl)ethyl b-D-glucopyranoside
(4a) White solid, m.p. 153–155 °C. MS: m/z 331
[M?H]?; 1H NMR (D2O, 500 MHz) d (ppm): 2.90 (2H, t,
J = 6.9 Hz), 3.26 (1H, t, J = 8.5 Hz), 3.34–3.50 (3H, m),
3.71 (1H, dd, J = 5.3 and 12.2 Hz), 3.87 (3H, s), 3.89–3.93
(2H, m), 4.12 (1H, dd, J = 7.0 and 16.7 Hz), 4.46 (1H, d,
J = 7.9 Hz), 6.81–6.90 (2H, m), 7.00 (1H, s). IR (KBr)
cm–1: 3514, 3434, 3147, 2967, 1603, 1523, 1454, 1374,
1253, 1091, 1050. Anal. Calcd for C15H22O8: C, 54.54; H,
6.71; Found: C, 54.48; H, 6.69.
2-(4-Hydroxy-3-ethoxyphenyl)ethyl b-D-galactopyranoside
(4f) White solid, m.p. 168–170 °C. MS: m/z 345
1
[M?H]?; H NMR (D2O, 500 MHz) d (ppm) 1.39 (3H, t,
J = 7.0 Hz), 2.89 (2H, t, J = 6.9 Hz), 3.49 (1H, t,
J = 8.9 Hz), 3.62–3.67 (2H, m), 3.72–3.78 (2H, m),
3.87–3.92 (2H, m), 4.11–4.17 (3H, m), 4.40 (1H, d,
J = 7.9 Hz), 6.85 (1H, d, J = 8.1 Hz), 6.89 (1H, d,
J = 8.1 Hz), 7.02 (1H, s). IR (KBr) cm–1: 3411, 2935,
1614, 1520, 1437, 1377, 1270, 1163, 1124, 1084, 1048.
Anal. Calcd for C16H24O8: C, 55.81; H, 7.02; Found: C,
55.79; H, 7.01.
2-(4-Hydroxy-3-methoxyphenyl)ethyl b-D-galactopyrano-
side (4b) White solid, m.p. 153–156 °C. MS: m/z 331
[M?H]?; 1H NMR (D2O, 500 MHz) d (ppm): 2.90 (2H, t,
J = 6.7 Hz), 3.50 (1H, t, J = 8.8 Hz), 3.61–3.67 (2H, m),
3.74–3.79 (2H, m), 3.87 (3H, s), 3.89–3.92 (2H, m),
4.09–4.16 (1H, m), 4.39 (1H, d, J = 7.8 Hz), 6.82–6.89
2-(3,4,5-Trihydroxyphenyl)ethyl b-D-glucopyranoside
(4g) Greenish solid, MS: m/z 333 [M?H]?; 1H NMR
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Med Chem Res (2013) 22:2524–2530
(D2O, 500 MHz) d (ppm): 2.71 (2H, t, J = 6.9 Hz), 3.18
(1H, t, J = 8.5 Hz), 3.28 (2H, t, J = 4.7 Hz), 3.40 (1H, t,
J = 8.9 Hz), 3.65 (1H, dd, J = 12.4 and 5.4 Hz),
3.75–3.85 (2H, m), 4.0 (1H, dt, J = 10.2 and 6.8 Hz), 4.38
(1H, d, J = 7.9 Hz), 6.40 (2H, s). IR (KBr) cm–1: 3384,
2935, 1614, 1537, 1453, 1328, 1198, 1018. Anal. Calcd for
C14H20O9: C, 50.60; H, 6.07; Found: C, 50.59; H, 6.09.
followed by addition of DMSO to dissolve the resulting for-
mazan. OD was measured at 570 nm with an EIX-800 Mic-
roelisa Reader (Bio-Tek Inc., USA). The results were
expressed as the percentage of viability versus control (with-
out treatment). Salidroside was used as a positive control.
Assay for SOD activities and the content of MDA
and GSH-Px
2-(3,4,5-Trihydroxyphenyl)ethyl
b-D-galactopyranoside
(4h) Greenish solid, MS: m/z 333 [M?H]?; H NMR
(D2O, 500 MHz) d (ppm): 2.79 (2H, t, J = 7.0 Hz), 3.49
(1H, t, J = 8.8 Hz), 3.61–3.67 (2H, m), 3.76–3.78 (2H, m),
3.82–3.92 (2H, m), 4.02–4.12 (1H, m), 4.38 (1H, d,
J = 7.7 Hz), 6.47 (2H, s). IR (KBr) cm–1: 3396, 2931,
1615, 1539, 1452, 1328, 1200, 1035. Anal. Calcd for
C14H20O9: C, 50.60; H, 6.07; Found: C, 50.64; H, 6.02.
SOD activity was measured by a modified method of
xanthine/xanthine oxidase assay (McCord and Fridovich,
1969). One unit of SOD activity was defined as the amount
that reduced OD at 550 nm by 50 %. The activities of
GSH-Px and the content of MDA, a compound produced
during lipid peroxidation, were analyzed as previously
described (Xiao et al., 1999).
1
DPPH radical scavenging capacity assay
Results and discussion
DPPH radical scavenging capacity assay was performed
according to the literature with minor modifications (Su
et al., 2010). In brief, 100 ll of synthetic glycoside, sali-
droside or Vitamin C (positive control) at varying con-
centration (10, 50, 100, 150, and 250 lM) in methanol was
added to 100 ll 250 lM DPPH in ethanol solution in a
96-well plate. The plate was shaken for 2 min and stored in
the dark for an additional 30 min at 37 °C. The optical
density (OD) was measured at 517 nm with an EIX-800
Microelisa Reader (Bio-Tek Inc., USA). Samples were
prepared in quintuplicate, and the concentration required
for a 50 % reduction (IC50) of 250 lM DPPH was deter-
mined graphically.
DPPH radical scavenging capacity of salidroside
analogs
Although a large number of antioxidant assays are avail-
able, the DPPH free radical is very stable and thus allows
for easy handling and manipulation. When this compound
accepts an electron or a hydrogen radical to become a more
stable compound, its absorption vanishes. Reduced OD of
the reaction mixture indicates a higher free radical scav-
enging activity. The IC50 value, defined as the amount of
antioxidant necessary to decrease the initial DPPH con-
centration by 50 %, was calculated from the results. As
shown in Table 1, the IC50 of compounds 4a–h and Vita-
min C was below 100 lM. Compounds 4g and 4h showed
Induction of LO2 cell apoptosis assay
LO2 cells were seeded into a 96-well plate (5 9 103 cells/
well), incubated for 24 h to allow attachment, treated with
CCl4 at 2.5, 5.0, 10.0, 20.0, and 30.0 mM, and cultured for
additional 24 h.
Table 1 Scavenging activity of compounds for DPPH radical
Compounds
IC50 (lM)
4a
59.84
40.76
61.65
70.14
66.77
87.50
38.05
35.85
–
4b
MTT cell viability assay
4c
4d
Exponentially growing LO2 cells were seeded at 5 9 103
cells/well (96-well plate) in complete medium containing
RPMI 1640 supplemented with 10 % heat-inactivated FBS,
penicillin G (100 IU/ml), and streptomycin (100 lg/mL), and
incubatedat37 °Cinahumidifiedatmospherecontaining5 %
CO2 and 95 % air. On the following day, the medium was
replaced with 200 ll fresh medium containing different
concentrations (1, 10, 50, and 100 mM) of the test compound
(4g, 4h, or salidroside) prior to 10 mM CCl4 addition. Cyto-
toxicity was assessed after 24 h incubation, and the MTT
solution was added and further incubated at 37 °C for 4 h,
4e
4f
4g
4h
3i
3j
–
3k
–
3l
–
Vitamin C
54.61
‘‘–’’ represent IC50 [ 100 lM
123

Med Chem Res (2013) 22:2524–2530
2529
excellent scavenging capacity, prior to Vitamin C and
salidroside, which was 38.05 and 35.85 lM, respectively.
For further research, the hepatoprotective effect of com-
pounds 4g and 4h on CCl4-induced cytotoxicity in LO2
cells was also assessed.
CCl4 treatment inhibits the growth of LO2 cells
LO2 cells were treated with different concentrations of
CCl4. Cell survival was determined after 24 h using the
control group (none CCl4 treated) as 100 %. The growth of
CCl4-treated cells was inhibited in a dose-dependent
manner (Table 2). Survival of LO2 cells decreased as the
concentrations of CCl4 increased. Treatment with 10 mM
CCl4 was selected in the subsequent experiments.
Fig. 2 Cell viability of compounds 4g, 4h, and salidroside on CCl4-
induced cytotoxicity in LO2 cells The dose-dependent effects of 4g,
4h, and salidroside on CCl4-induced cytotoxicity in LO2 cells. The
cells were exposed to 10 mM CCl4. Indicated doses of compounds 4g,
4h, and salidroside were added prior to CCl4 addition and the viability
of LO2 cells was measured 24 h later by MTT assay. Data were the
mean SEM expressed as percentages of the untreated control
(n = 3). *P \ 0.01 versus control; **P \ 0.01 versus CCl4
Cell viability of compounds 4g, 4h, and salidroside
on CCl4-induced cytotoxicity in LO2 cells
activity by 51 % and SOD activity by 53 %. Treatment
with 4g, 4h, and salidroside attenuated the changes in
GSH-Px and SOD activities in LO2 cells. The toxicity
caused by CCl4 was accompanied by increased lipid per-
oxides. As shown in Table 3, intracellular MDA, a product
of lipid peroxidation, was raised by 70 % 24 h after
exposure of the cultures to CCl4. However, the increased
MDA level induced by CCl4 was significantly attenuated
after treatment of the cells with 4g, 4h, and salidroside.
The hepatoprotective effect of compounds 4g, 4h, and sali-
droside on CCl4-induced cytotoxicity in LO2 cells was
examined. MTT assay showed that cell viability decreased
significantly after 24 h treatment of LO2 cells with 10 mM
CCl4, as compared to control group (none CCl4 treated). The
results of further experiments showed that pretreatment with
compounds 4g, 4h, or salidroside at different concentrations
(1, 10, 50, and 100 mM), displayed a protective effect in a
dose-dependent pattern (Fig. 2). Compound 4g showed
significant protective effects against CCl4-induced hepato-
cytotoxicity at the concentration of 50 and 100 mM, while
4h at concentration of 10, 50, and 100 mM, and salidroside
only at a high concentration of 100 mM.
Table 3 Effects of compounds 4g, 4h, and salidroside on CCl4-
induced antioxidant enzyme activities and level of lipid peroxidation
Percentage of control
SOD
MDA
GSH-Px
Determination of SOD activity and content of MDA
and GSH-Px
Control
100 4.2
47 4.4*
50 5.1
55 3.3
100 5.1
170 4.3*
167 3.6
151 5.2**
100 3.9
49 3.8*
54 4.5
CCl4
GSH-Px and SOD are important antioxidant enzymes in
cells. It was found in our study that CCl4 reduced GSH-Px
CCl4 ? 4g 1 mM
CCl4 ? 4g 10 mM
CCl4 ? 4g 50 mM
CCl4 ? 4g 100 mM
CCl4 ? 4h 1 mM
CCl4 ? 4h 10 mM
CCl4 ? 4h 50 mM
CCl4 ? 4h 100 mM
CCl4 ? Sal 1 mM
CCl4 ? Sal 10 mM
CCl4 ? Sal 50 mM
CCl4 ? Sal 100 mM
57 3.6
70 4.6** 128 6.7**
83 5.4** 117 5.1**
71 5.1**
80 6.4**
55 2.8
Table 2 The dose-dependent effects of LO2 cells on CCl4-induced
apoptosis
54 2.9
164 4.1
65 3.8** 141 5.1**
76 4.7** 119 4.9**
89 4.6** 110 6.9**
64 3.6**
78 5.9**
86 5.2**
51 4.3
Dose (mM)
Viability ( % of control)
Control
CCl4
–
100 0.56
88.42 0.78
74.85 0.59
54.23 1.03
32.76 0.35
13.58 0.66
2.5
5
49 3.4
52 4.6
56 3.1
168 3.3
165 4.0
162 5.7
53 5.0
10
20
30
56 4.7
61 6.1** 157 4.3**
61 3.9**
LO2 cells were exposed to 10 mM CCl4. Compounds 4g, 4h, and
salidroside were added prior to CCl4 addition, and the levels of
antioxidant and MDA were measured 24 h later. Data were the
mean SEM expressed as percentages of the untreated control
(n = 5). * P \ 0.01 versus control; ** P \ 0.01 versus CCl4
LO2 cells were exposed to CCl4 (2.5, 5, 10, 20, and 30 mM), and cell
viability was measured 24 h later by MTT assay. Data were the
mean SEM expressed as percentages of the untreated control
(n = 3)
123

2530
Med Chem Res (2013) 22:2524–2530
Hu X, Zhang X, Qiu S, Yu D, Lin S (2010b) Salidroside induces cell-
cycle arrest and apoptosis in human breast cancer cells. Biochem
Biophys Res Commun 398:62–67
Li QY, Wang HM, Wang ZQ, Ma JF, Ding JQ, Chen SD (2010)
Salidroside attenuates hypoxia-induced abnormal processing of
amyloid precursor protein by decreasing BACE1 expression in
SH-SY5Y cells. Neurosci Lett 481:154–158
Compound 4h exhibited the best hepatoprotective effects,
and 4g was the next, and both of them were prior to sali-
droside. The results are in accordance with the DPPH
radical scavenging capacity, suggesting that the hepato-
protective effects of compounds 4g and 4h on CCl4-
induced LO2 cells injury may be associated with the
anti-oxidative property of the phenolic hydroxy group, and
differ in the glycosyl donor may also play an important part
in it.
McCord JM, Fridovich I (1969) Superoxide dismutase. An enzymic
function for erythrocuprein (hemocuprein).
244:6049–6055
J Biol Chem
Ouyang J, Gao Z, Ren Z, Hong D, Qiao H, Chen Y (2010) Synergistic
effects of rMSCs and salidroside on the experimental hepatic
fibrosis. Pharmazie 65:607–613
Perez Tamayo R (1983) Is cirrhosis of the liver experimentally
produced by CCl4 and adequate model of human cirrhosis?
Hepatology 3:112–120
Song EK, Kim JH, Kim JS, Cho H, Nan JX, Sohn DH, Ko GI, Oh H,
Kim YC (2003) Hepatoprotective phenolic constituents of
Rhodiola sachalinensis on tacrine-induced cytotoxicity in Hep
G2 cells. Phytother Res 17:563–565
In summary, our results suggest that salidroside analog 4g
and4h mayprovetobeleadingcompoundsforreducingCCl4-
induced cytotoxicity in LO2cellsvia the antioxidant pathway.
This finding is interesting, and our future research will focus
on deep mechanisms underlying the two compounds.
Statistical analysis
Su ZH, Zou GA, Preiss A, Zhang HW, Zou ZM (2010) Online
identification of the antioxidant constituents of traditional
Chinese medicine formula Chaihu-Shu-Gan-San by LC-LTQ-
Orbitrap mass spectrometry and microplate spectrophotometer.
J Pharm Biomed Anal 53:454–461
All measurements were repeated in triplicate. Data were
expressed as mean SEM and analyzed by Student’s t test
followed by Dunnett’s multiple comparison.
Wu YL, Piao DM, Han XH, Nan JX (2008) Protective effects of
salidroside against acetaminophen-induced toxicity in mice. Biol
Pharm Bull 31:1523–1529
Wu T, Zhou H, Jin Z, Bi S, Yang X, Yi D, Liu W (2009a)
Cardioprotection of salidroside from ischemia/reperfusion injury
by increasing N-acetylglucosamine linkage to cellular proteins.
Eur J Pharmacol 613:93–99
Acknowledgments Financial supports from the Natural Science
Foundation of Nantong University, Nantong Municipality (Grant No.
11Z011; AS2011016) and the National Natural Science Foundation of
China (Grant No. 81171457) are gratefully acknowledged. This is
also a project funded by the Priority Academic Program Development
of Jiangsu Higher Education Institution.
Wu YL, Lian LH, Jiang YZ, Nan JX (2009b) Hepatoprotective
effects of salidroside on fulminant hepatic failure induced by
D-galactosamine and lipopolysaccharide in mice. J Pharm Phar-
macol 61:1375–1382
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