Fitoterapia 82 (2011) 834–840

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Fitoterapia
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f i t o t e

Hepatoprotective effects of polyprenols from Ginkgo biloba L. leaves on

CCl4-induced hepatotoxicity in rats
Lan Yang a, Cheng-zhang Wang a,⁎, Jian-zhong Ye a, Hai-tao Li b
a
Institute of Chemical Industry of Forest Products, CAF, Nanjing 210042, People's Republic of China
b
Nanjing University of Chinese Medicine, Nanjing 210046, People's Republic of China

a r t i c l e i n f o a b s t r a c t

Article history: The hepatoprotective effects of polyprenols from Ginkgo biloba L. leaves were evaluated against
Received 22 February 2011 carbon tetrachloride induced hepatic damage in Sprague–Dawley rats. The elevated levels of
Accepted in revised form 8 April 2011 serum ALT, AST, ALP, ALB, TP, HA, LN, TG, and CHO were restored towards normalization
Available online 10 May 2011
significantly by GBP in a dose dependent manner. The biochemical observations were
supplemented with histopathological examination of rat liver sections. Meanwhile, GBP also
Keywords: produced a significant and dose-dependent reversal of CCl4-diminished activity of the
Ginkgo biloba antioxidant enzymes and reduced CCl4-elevated level of MDA. In general, the effects of GBP
Polyprenols
were not significantly different from those of the standard drug Essentiale.
Essentiale
© 2011 Elsevier B.V. All rights reserved.
Hepatoprotective
Antioxidant

1. Introduction [6]. Modern medicines have little to offer for alleviation of

The liver as a vital organ has a wide range of functions in the
body, including detoxification, plasma protein synthesis, and
glycogen storage. The liver is also prone to many diseases
mostly included: infections such as hepatitis A, B, C, E, alcohol
damage, fatty liver, cirrhosis, cancer, and drug damage
(especially acetaminophen, cancer drugs) [1,2]. Liver ailments
remain one of the serious health problems [3]. Pathologically,
chronic hepatitis or long term intoxification can severely injure
hepatocytes. Initially, the damaged cells are denatured, but
subsequently transformed to hypertrophic fibrosis and necrosis
[4,5], and may eventually progress to hepatoma. Clinically, the
hepatofibrotic stage is a very crucial pathogenetic point. To
terminate the serial consequences at the fibrotic stage currently
has become the target strategy in the treatment of liver diseases
Abbreviation: ALT, alanine aminotransferase; AST, aspartate aminotrans-
ferase; ALP, alkaline phosphatase; ALB, albumin; TP, total protein; HA,
hyaluronic acid; LN, laminin; TG, triglyceride; CHO, total cholesterol; MDA,
malondialdehyde.
⁎ Corresponding author. Tel./fax: + 86 2585482471.
E-mail addresses: ylperfect@163.com (L. Yang), wangczlhs@sina.com
(C. Wang).
hepatic diseases and it is chiefly the plant based preparations
which are employed for the treatment of liver disorders [7].
There is a great demand for the development of an efficient
hepatoprotective drug from the natural resource [8].
Polyprenols from G. biloba L. leaves (GBP) as novel natural
active lipids is discovered after ginkgo flavonoids and terpene
lactones, which have already been widely used for their
efficiency in treating cardiovascular and cerebrovascular
diseases [9]. GBP generally composes of 15 to 21 unsaturated
isoprene units and is one type of betulaprenol with an E,E-
farnesyl residue at the ω-end of the prenyl chain and
terminated by an isoprene unit bearing a primary hydroxyl
group. The structure and bioactivity of GBP are similar with
that of dolichols which widely existed in human and
mammalian organs [10,11]. Polyprenols can be converted to
dolichols and dolichyl phosphates in vivo, which are the key
carrier in the biosynthesis of glycoprotein [12]. Previous
studies reported that polyprenols (n = 10–24) from needles
had many biological functions such as antivirus, antitumor,
antioxidant activities and hepatoprotective effects as well as
treating Alzheimer, and enhancing immunity [13–21]. How-
ever, literature reviews indicate that the hepatoprotective
effect of GBP (n = 15–21) has not been reported so far.

0367-326X/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.fitote.2011.04.009
 L. Yang et al. / Fitoterapia 82 (2011) 834–840 835

Therefore, in the present study, effects of GBP on the levels of
serum ALT, AST, ALP and antioxidant parameters were
investigated on carbon tetrachloride (CCl4) induced hepatic
damages in Sprague–Dawley rats.
2. Materials and methods
2.1. Drugs and chemicals
The standard GBP (C75–C105) was purchased from Larodan
Fine Chemical Co., Ltd, Sweden. Silica gel plate (GF254) was
obtained from Qingdao Haiyang Chemical Co., Ltd, China.
Essentiale as a commercial available hepatoprotective drug
was produced by A. Nattermann & Cie. GmbH (Germany). All
biochemicals were purchased from Beijing Zhongshan Bio-
technology Co., Ltd, China. Other chemical reagents of
analytical grade were provided by Nanjing Chemical Reagent
Co., Ltd, China.
2.2. Plant material
G. biloba L. leaves were collected from ginkgo nursery of
Taixing, Jiangsu province, China, on October 10th 2009.
Botanical authentication was done by Dr. Zhaobang Shen of
the Institute of Chemical Industry of Forest Products, Nanjing,
China. The leaves were dried for 12 h at 60 °C in drying oven,
and then the dried leaves with less than 8.0% of water were
powdered and stored in dry, ventilated and dark place.
to yield a product as brown oil, which was further purified by
molecular distillation at feed temperature of 60 °C, distillation
temperature of 280 °C, feed flow rate of 180 mL/h, scraper
rate of 300 rpm, and operating pressure of 0.1–0.5 Pa to give
crude GBP product with purity of 38.5%.
Crude GBP sample (3 g) was further purified by flash column
chromatography (200 mesh silica gel, Φ2.5 cm × 40 cm), using
petroleum ether (400 mL) and 1%, 2%, 3% ethyl ether–
petroleum ether (v/v, 300 mL respectively) as eluents. Thin-
layer chromatography (TLC) was used to analyze the separation
effect of the column chromatography (Fig. 1). Enough GBP
sample with purity over 90% used for the following pharmaco-
logical experiment was prepared according to the method
mentioned above.
2.5. External standard method for the determination of GBP
purity performed by HPLC
The GBP contents in the samples were determined by High
Performance Liquid Chromatography (HPLC) using external
standard method. HPLC was performed using a Shimadzu
SPD-10A, consisting of a vacuum degasser, binary pump, and
photodiode array detection, controlled by class-up station
(Shimadzu Co. Ltd., Japan). The HPLC column was a Kromasil

2.3. Animals

SD rats (180–220 g) of both sexes used in this study were

purchased from Shanghai Slac Laboratory Animal Co., Ltd,
China. They were housed in clean polypropylene cages under
standard laboratory conditions of humidity (55 ± 10% RH),
temperature (25 ± 2 °C) and light (12 h light/12 h dark cycle)
and fed with standard diet (Fu-Sou Brand, specific for rats)
and water (the reverse osmotic water, ROW) ad libitum.
Animal welfare and experimental procedures were strictly in
accordance with the Guide for the Care and Use of Laboratory
Animals published by the US National Institutes of Health
(NIH publication no. 85–23, revised 1996) and the related
ethics regulations of Nanjing University of Chinese Medicine.
All animals were handled with humane care.

2.4. Preparation of GBP

GBP was separated and purified according to the report

[22–24]. Powdered G. biloba L. leaves were extracted 2–3
times with petroleum ether (5–8 mL/g) at room temperature
for 48 h to obtain the crude lipid extract. The extracted lipid
mixture was saponified at 70 °C for 1 h with 95% ethanol
containing 15% (w/v) of NaOH, in order to fully hydrolyze the
lipid fraction and eliminate any phenolic compounds
extracted by the petroleum ether. The fat-soluble unsaponifi-
able matter was extracted 4 times with petroleum ether and
the organic phases were collected. After solvent evaporation, Fig. 1. TLC of fractions obtained by silica gel column chromatography. 1: GBP
a crude product was obtained that was dissolved in acetone– sample; 2–7: fractions obtained by silica gel column chromatography; 2,3:
fractions obtained by petroleum ether as eluent; 4,5: fractions obtained by
methanol (85:15, v/v, 40–50 mL/g) and refrigerated for 2 1% ethyl acetate–petroleum ether as eluent; 6: fraction obtained by 2% ethyl
times at −10 °C for 4 h, and some impurities (i.e., plant sterol, acetate–petroleum ether as eluent; 7: GBP obtained by 3% ethyl acetate–
waxiness) can be precipitated. The filtrate was concentrated petroleum ether as eluent, the purity was over 90%.
836 L. Yang et al. / Fitoterapia 82 (2011) 834–840

ODS-1 C18 column (150 mm × 4.6 mm, 5 μm). The isocratic
mobile phase consisted of isopropanol–methanol–hexane–
water (50:25:10:2, v/v) was used to elute for 65 min, and the
flow rate was kept at 1.0 mL/min. Column temperature was
kept constantly at 25 °C. UV detection was at 210 nm.
The HPLC analysis of GBP standard and all the GBP samples
were carried out under the established experimental condi-
tion as mentioned above. The concentration of the GBP
standard was 1.168 mg/mL, and the injection volumes were
4, 8, 12, 16, and 20 μL, respectively. The standard curve was
obtained with the content of GBP as the abscissa and the
relevant five main peaks area as the ordinate. All the GBP
samples were filtered with 0.25 μm filter membrane and
dissolved in hexane (chromatographically pure) before HPLC
measurement. The injection volume was 20 μL.
2.6. Animal grouping and experimentation
SD rats after acclimatization (a week) in the animal
quarters were randomly divided into 6 groups, GroupI
(Normal), GroupII (CCl4), GroupIII (CCl4 + 360 mg/kg Essen-
tiale), GroupIV (CCl4 + 10 mg/kg GBP), GroupV (CCl4 +
40 mg/kg GBP), and GroupVI (CCl4 + 160 mg/kg GBP), each
group consisting of 10 rats (5 males and 5 females).
The model of liver injury induced by CCl4 is commonly
used for the evaluation of drug efficacy in liver injury. CCl4
was previously dissolved in olive oil to make a final
concentration of 40%. The method for feeding CCl4 to the
experimental animals was according to Lin et al. [25].
GroupII–VI received subcutaneous injection of 40% CCl4 at a
dose of 5 mL/kg for the first time and later at a dose of
3 mL/kg every 4 days, while GroupI subcutaneously received
equal volume of olive oil. Simultaneously, GroupI–II was
given 0.9% of physiological saline, and the treatment groups
were given corresponding drugs once daily for a period of
6 weeks.
2.8. Histopathological examination
The left hepatic lobule in each group was sliced and
washed in saline. Small pieces were preserved in 10%
formasal (formalin diluted to 10% with normal saline) and
were sent for histopathology examination. After paraffin
embedding and block making, serial sections of 4–6 μm in
thickness were made, stained with haematoxylin and eosin
and examined under the microscope.
2.9. Statistical analysis
The results of hepatoprotective effect were recorded for
individuals within all groups. All data were presented as
mean ± SD. Results were statistically analyzed by one-way
ANOVA using SPSS10.0 statistical software. A value of P b 0.05
was considered significant.
3. Results
3.1. Determination of the purity of GBP
The purity of GBP was determined by TLC and HPLC. Ethyl
acetate–petroleum ether (1:9, v/v) mixture as the developing
solvent was used for TLC. The spots were visualized by iodine
staining. The Rf value of GBP was 0.35 (Fig. 1). The regression
equation obtained from the standard curve of GBP was
y = 1.5751x + 1.1546 (R 2 = 0.9991), and the purity of GBP
was calculated according to the equation. The linear relation
between injection volume (4.6–23.3 μg) and peak area was
good.
After purification, the final purity of GBP was 91.3%. GBP
composed of 15 to 21 unsaturated isoprene units, and the
results showed that the relative content of C75, C80, C85, C90,
C95, C100, and C105 was 0.17%, 8.39%, 30.47%, 35.61%, 16.86%,
6.48%, and 1.99%, respectively, indicating that C85 and C90
prenols were dominated (Fig. 2).

3.2. Effect of GBP on serum biochemical parameters

2.7. Sample collection and analysis
From the results obtained in this study (Table 1), CCl4
After the administration, all the animals were sacrificed by significantly (P b 0.01) raised the levels of serum ALT, AST and
cervical dislocation and livers were dissected out immedi- ALP relative to the normal group, while the levels of ALB and
ately, blood was collected from postorbital plexus. The serum
was separated by centrifugation at 3000 rpm for 10 min and
70
used for the determination of ALT, AST, ALP, HA, LN, TG, and 65
CHO by routine medical laboratory assay methods [26]. The 60
55
levels of TP and ALB in serum were determined according to 50
the reported method [27] and bromocresol green method, 45
Voltage (mV)

40
respectively. The same part of the right liver lobule was 35
further chopped into masses, added ten-fold (w/v) buffer 30
solution containing 8 mM of KH2PO4, 12 mM of K2HPO4, and 25
20
1.5% of KCl (pH 7.40) and homogenized. And the homoge- 15
nates were used for the assays of superoxide dismutase 10
5
(SOD), glutathione (GSH), and MDA by following the 0
manufacturer's instructions with the experimental kits -5
supplied by Calbio-chem-Novbiochem Corp. The amount of -10
MDA formed was quantified by reaction with TBA. Other parts 0 5 10 15 20 25 30 35 40 45 50 55 60 65
of the liver were made into powder, the liver fibrosis was Time (min)
measured by the contents of hydroxyproline and collagen
[28]. Fig. 2. Chromatogram of the polyprenols from G. biloba L. leaves.
 L. Yang et al. / Fitoterapia 82 (2011) 834–840 837

Table 1
Effect of GBP on serum biochemical parameters.

Treatment group ALT (U/L) AST (U/L) ALP (U/L) ALB (g/L) TP (g/L)

Normal 58.1 ± 7.31 130.6 ± 23.89 298.65 ± 86.22 32.63 ± 3.62 73.23 ± 9.34
CCl4 89.9 ± 20.35⁎⁎ 246.8 ± 46.36⁎⁎ 644.26 ± 258.89⁎⁎ 25.40 ± 2.97⁎⁎ 61.47 ± 8.74⁎⁎
CCl4 + Essentiale 101.9 ± 23.49 212.4 ± 48.19 428.32 ± 96.22Δ 28.74 ± 4.32Δ 64.52 ± 5.21
CCl4 + GBP10 118.3 ± 25.05 289.4 ± 93.57 537.47 ± 107.35 26.20 ± 3.14 62.86 ± 6.33
CCl4 + GBP40 73.1 ± 17.88Δ 197.2 ± 54.46Δ 359.56 ± 73.48ΔΔ 29.65 ± 3.38ΔΔ 70.71 ± 4.30Δ
CCl4 + GBP160 68.6 ± 15.28ΔΔ 175.6 ± 30.17ΔΔ 312.63 ± 28.44ΔΔ 30.10 ± 3.47ΔΔ 72.13 ± 5.28Δ

Values are mean ± SD (n = 10). ⁎⁎P b 0.01 vs. Normal; ΔP b 0.05, ΔΔ

P b 0.01 vs. CCl4-treated group.

TP were significantly (P b 0.01) diminished. GBP (40, 160 mg/kg)
caused significant (P b 0.05, Pb 0.01) dose-dependent reductions
in CCl4-elevated levels of ALT, AST and ALP. GBP also dose-
dependently increased the reduced serum levels of ALB and TP
with effects being significant (P b 0.05, P b 0.01) at 40 mg/kg and
160 mg/kg. GBP (10 mg/kg) had slight effect compared to the
CCl4 group, and the levels of ALP, ALB, and TP changed little.
Essentiale only produced significant (P b 0.05) reduction in CCl4-
elevated ALP level and significantly (P b 0.05) increased CCl4-
diminished serum ALB level. However, there were not apparent
changes in serum levels of ALT, AST and TP compared to the
CCl4-treated group. GBP demonstrated hepatoprotective effects
by reducing CCl4-elevated levels of serum ALT, AST, ALP and
increasing CCl4-reduced ALB and TP. The effects of GBP were
more prominent than Essentiale and peak effects were produced
at the highest dose of 160 mg/kg, though there was a little
difference between the normal and GBP160 group.
3.3. Effect of GBP on the in vivo antioxidative parameters
As shown in Table 2, the MDA level was significantly
(P b 0.01) increased due to CCl4 intoxification. GBP signifi-
cantly and dose-dependently caused diminution of CCl4-
elevated MDA level with the value being slightly lower
produced at the highest dose of 160 mg/kg. Essentiale
significantly (P b 0.01) reversed CCl4-induced MDA level
elevation with value being a little lower than the normal
group. The effect of Essentiale was not significantly different
from that of GBP at a dose of 160 mg/kg.
CCl4 significantly (P b 0.01) lowered the activity of
hepatic in vivo antioxidant enzymes GSH and SOD. GBP (40,
160 mg/kg) caused significant (P b 0.01) increase in CCl4
suppressed activity of the antioxidant enzymes. Both GBP40
and GBP160 showed promising effects on such a hepatic
injury. Enzyme activity values for GBP at a dose of 160 mg/kg
were mostly comparable and not significantly different from
values obtained from the normal group. Essentiale markedly
(P b 0.01) reversed CCl4 suppressed antioxidant enzyme
activity. However, enzyme activity value for Essentiale
group was higher than normal for GSH but lower for SOD.
The effects of GBP at the highest dose of 160 mg/kg were
comparable and not markedly different from the effects of
Essentiale.
3.4. Effect of GBP on the contents of hydroxyproline and collagen
in liver injury tissues
One of the main pathological changes of chronic liver
injury induced by CCl4 is liver fibrosis. The liver collagen
content, an indication of fibrosis, can be calculated by the
content of hydroxyproline. Hydroxyproline is a unique amino
acid in collagen, and its content in liver tissues was detected
by spectrophotometer.
As can be seen from Table 3, the content of collagen in the
CCl4-treated animals was significantly (P b 0.01) increased
when compared with those of the normal group. GBP caused
significant (P b 0.05) dose-dependent reductions in CCl4-
elevated levels of hydroxyproline and collagen and peak
effects were generally produced at the highest dose of
160 mg/kg. However, the level of hydroxyproline still didn't
restore to normal at the dose of 160 mg/kg. It is important to
note that the level of hydroxyproline was greatly (P b 0.05)
reduced at the dose of 40 mg/kg, while the collagen level was
not significantly affected. Essentiale effectively (P b 0.05)
decreased CCl4-induced hydroxyproline and collagen levels
elevation. Again, GBP showed excellent protecting effect as
Essentiale with values nearly restored to normal.

Table 2 Table 3
Effect of GBP on the in vivo antioxidative parameters. Effect of GBP on the contents of hydroxyproline and collagen in chronic liver
injury tissues in rats.
Treatment group MDA GSH SOD
(nmol/mg protein) (U/mg protein) (U/mg protein) Treatment group Hydroxyproline Collagen
(μg/mg liver powder) (μg/mg liver powder)
Normal 27.5 ± 4.57 93.1 ± 5.44 42.1 ± 4.76
CCl4 38.1 ± 4.56 a 84.4 ± 6.9 a 17.6 ± 4.14 a Normal 1.247 ± 0.458 4.01 ± 0.83
CCl4 +Essentiale 22.6 ± 4.21 b 100.51 ± 3.06 b 39.9 ± 6.31 b CCl4 2.646 ± 0.925 a 10.29 ± 2.40 a
CCl4 + GBP10 30.8 ± 6.32 c 85.6 ± 6.52 25.6.4 ± 6.87 c CCl4 + Essentiale 1.677 ± 0.470 b 4.93 ± 1.57 b
CCl4 + GBP40 27.2 ± 4.56 b 91.0 ± 5.07 b 35.9 ± 6.06 b CCl4 + GBP10 2.077 ± 0.816 8.96 ± 2.61
CCl4 + GBP160 24.7 ± 6.76 b 92.7 ± 6.72 b 41.4 ± 7.83 b CCl4 + GBP40 1.773 ± 0.554 b 7.25 ± 2.56
CCl4 + GBP160 1.546 ± 0.707 b 3.75 ± 1.75 b
Values are mean ± SD (n = 10).
a
P b 0.01 vs. Normal. Values are mean ± SD (n = 10).
b a
P b 0.01 vs. CCl4-treated group. P b 0.01 vs. Normal.
c b
P b 0.05 vs. CCl4-treated group. P b 0.05 vs. CCl4-treated group.
838 L. Yang et al. / Fitoterapia 82 (2011) 834–840
3.5. Effect of GBP on lowering blood lipid level in serum of liver
injury induced by CCl4
The degree of liver fibrosis can be reflected by the levels of
serum HA and LN, and the blood lipid level can be reflected by
the levels of serum TG and CHO. Compared to the normal
group, CCl4 caused significant (P b 0.01) increase in serum HA
and LN levels. Meanwhile, serum TG and CHO levels were also
increased (P b 0.05). GBP (10, 40, and 160 mg/kg) caused
significant (P b 0.05, P b 0.01) dose-dependent reductions in
CCl4-elevated levels of HA, LN, TG, and CHO. The effects of
GBP40 were not significantly different from the Essentiale
group and peak effects were produced at the highest dose of
160 mg/kg. However, GBP160 significantly reduced CCl4-
induced HA and LN levels elevation with values being
slightly higher than the normal group. In addition, GBP (40,
160 mg/kg) exhibited better efficiency than Essentiale
(Table 4). These results suggest that GBP (40, 160 mg/kg)
possibly protect the structural integrity of the cell membrane
of liver cells or enhance regeneration of damaged liver cells.
3.6. Effect of GBP on liver tissues of CCl4-induced hepatotoxicity
in rats
In rats of the normal group (Fig. 3A), the structure of liver
lobule and portal areas was normal. There was no obvious
degeneration or necrosis, no congestion, no proliferation of
sinusoids, no pathological changes, and no formation of
fibrous intervals. While in rats of CCl 4 -treated group
(Fig. 3B), there was severe steatosis, diffuse degeneration
and necrosis, streak proliferation of fibrous tissue, and
infiltration of a small amount of inflammatory cells in portal
areas. These symptoms are very similar to the clinical
pathological changes of viral hepatitis. Such phenomenon
was greatly alleviated by GBP (Fig. 3D–F) as evidenced with
slighter extent of fibrosis and less deformation occurred in
liver lobules. Infiltration of inflammatory cells in portal areas
was especially alleviated significantly (P b 0.01). Different
doses of GBP had obvious different protective effects on
chronic liver injury damaged by CCl4. Especially GBP40 had
equivalent protective effects on steatosis compared to
Essentiale (Fig. 3C). The structure of liver lobule and portal
areas was restored to normal at the highest dose of 160 mg/kg,
which was in accordance with the reduced contents of TG and
CHO in serum.
Table 4
Effect of GBP on serum HA, LN, TG, and CHO levels of liver injury induced by CCl4.
Treatment group HA (μg/L)
Normal 165.56 ± 20.48
CCl4 202.52 ± 22.26 a
CCl4 + Essentiale 179.62 ± 16.23 c
CCl4 + GBP10 180.93 ± 25.55 d
CCl4 + GBP40 178.54 ± 14.31 c
CCl4 + GBP160 172.25 ± 19.31 c
Values are mean ± SD (n = 10).
a
P b 0.01 vs. Normal.
b
P b 0.05 vs. Normal.
c
P b 0.01 vs. CCl4-treated group.
d
P b 0.05 vs. CCl4-treated group.
4. Discussion
Results indicated that GBP were able to suppress the
hepatotoxicity of CCl4 and the hepatoprotective effect was
comparable to the conventionally reputed Essentiale. Levels
of some important biochemical parameters in serum are
used as diagnostic markers of hepatic injury. Increased
levels of ALT, AST and ALP in serum of the CCl4-treated
animals indicate liver damage as these enzymes leak out
from liver into the blood at the instance of tissue damage,
which is always associated with hepatonecrosis [29,30].
With the treatment of GBP or Essentiale, the levels of these
marker enzymes were near normal or only slightly elevated,
indicating protection against liver damage. ALP activity is
related to the functioning of hepatocytes. Suppression of
increased ALP activity suggests the stability of biliary
dysfunction in rat liver during chronic hepatic injury with
CCl4 [31]. Diminution of TP and ALB induced by CCl4 is a
further indication of liver damage [32]. GBP has increased
the levels of serum TP and ALB towards the respective
normal value, which indicates hepatoprotective activity.
Stimulation of protein synthesis has been advanced as a
contributory hepatoprotective mechanism which acceler-
ates the regeneration process and the production of liver
cells [33,34].
Lipid peroxidation is the main mechanism of liver injury.
CCl4 can induce hepatotoxicity and can be converted into
trichloromethyl free radical by cytochrome P-450. Trichlor-
omethyl free radical can cause peroxidate degradation in the
adipose tissue [35]. In the present study, free radicals seem to
initiate peroxidate degradation of membrane lipids. This leads
to formation of lipid peroxides which in turn give products
like MDA that cause loss of integrity of cell membrane and
damage to hepatic tissue [27]. Antioxidant enzymes can
detoxify free radicals by converting them back to more stable
molecules within the cell [36]. Decreased GSH level in CCl4-
administered rats may be due to its increased utilization for
enhancing the activities of GSH related enzymes GPX and GR
[37]. GBP and Essentiale prevented significantly lipid perox-
idation either directly or through GSH by scavenging the free
radicals thus protecting the histostructural integrity of the
liver cells. Liver injury has been observed when GSH storage is
markedly depleted, and the level is restored to normal with
GBP treatment. It may be understood that the effect of GBP
may be due to an initial reduction in hepatic peroxidative
activities followed by inhibition of the activities of GSH related
LN (μg/L) TG (mmol/L) CHO (mmol/L)
80.15 ± 9.28 1.95 ± 0.53 1.95 ± 0.48
116.74 ± 10.84 a 2.21 ± 0.47 b 2.12 ± 0.47 b
103.25 ± 9.62 c 1.37 ± 0.48 d 1.59 ± 0.33 d
107.95 ± 9.27 d 1.59 ± 0.46 1.66 ± 0.24
101.76 ± 12.84 c 1.35 ± 0.59 d 1.60 ± 0.30 d
96.95 ± 12.81 c 1.36 ± 0.36 d 1.58 ± 0.0.32 d
 L. Yang et al. / Fitoterapia 82 (2011) 834–840 839

A B

C D

E F

Fig. 3. Photomicrographs of histopathological studies of liver sections of experimental groups (HE × 200) (A) normal architecture of rat liver (B) CCl4 treated.
Extensive macrovesicular and microvesicular steatosis, large areas of necrosis, and moderate inflammation (C) CCl4 + Essentiale treated. Macrovesicular and
microvesicular steatosis, normal hepatocytes with moderate inflammation (D) CCl4 + GBP10 treated. Microvesicular steatosis (E) CCl4 + GBP40 treated. Balloon
degeneration (F) CCl4 + GBP160 treated. A significant recovery but with mild steatosis.

enzymes, thereby leading to restoration of the GSH content.
SOD decreased significantly in CCl4-treated animals due to an
excessive formation of superoxide anions. Administration of
GBP effectively prevented the decrease in SOD activity, which
may be attributed to the scavenging of radicals by GBP
resulting in protection of this enzyme. The detrimental effects
exerted by CCl4 toxicity on SOD, GSH and MDA were
effectively recovered or improved.
As well known, the major component in extracellular
matrix is collagen. The collagen in CCl4-damaged liver
apparently increased and severely accumulated (P b 0.01).
The liver lobules were hampered with huge amount of
collagen leading to extensive liver fibrosis. Moreover, liver
fibrosis was found to be significantly suppressed by GBP due
to the down-regulation of collagen synthesis, which demon-
strated hepatoprotective effect of GBP.
Furthermore, the histopathological findings of liver sam-
ples are in agreement with the results of biochemical studies.
The results obtained in this study indicate that GBP is able
to inhibit CCl4-induced hepatotoxicity in rats. By scavenging
free radicals, GBP inhibits lipid peroxidation and augments
cellular antioxidant defense system comprising GSH and SOD.
GBP possesses rather potent peroxide eliminating and free
radical scavenging capabilities. Presumably, the high reactiv-
ity of hydroxyl group of GBP is responsible for this free radical
scavenging activity. However, further investigation is needed
to ascertain the precise mechanism of hepatoprotective
action of GBP.
Acknowledgment
The authors gratefully acknowledge the financial support
by the National Natural Science Foundation of China (No.
30871986) and professor Ying Lu from Nanjing University of
Chinese Medicine, who provided the histopathology exami-
nation results.
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