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Curs Ginkgo Biloba
Curs Ginkgo Biloba
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
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
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 to yield a product as brown oil, which was further purified by
serum ALT, AST, ALP and antioxidant parameters were molecular distillation at feed temperature of 60 °C, distillation
investigated on carbon tetrachloride (CCl4) induced hepatic temperature of 280 °C, feed flow rate of 180 mL/h, scraper
damages in Sprague–Dawley rats. rate of 300 rpm, and operating pressure of 0.1–0.5 Pa to give
crude GBP product with purity of 38.5%.
2. Materials and methods Crude GBP sample (3 g) was further purified by flash column
chromatography (200 mesh silica gel, Φ2.5 cm × 40 cm), using
2.1. Drugs and chemicals petroleum ether (400 mL) and 1%, 2%, 3% ethyl ether–
petroleum ether (v/v, 300 mL respectively) as eluents. Thin-
The standard GBP (C75–C105) was purchased from Larodan layer chromatography (TLC) was used to analyze the separation
Fine Chemical Co., Ltd, Sweden. Silica gel plate (GF254) was effect of the column chromatography (Fig. 1). Enough GBP
obtained from Qingdao Haiyang Chemical Co., Ltd, China. sample with purity over 90% used for the following pharmaco-
Essentiale as a commercial available hepatoprotective drug logical experiment was prepared according to the method
was produced by A. Nattermann & Cie. GmbH (Germany). All mentioned above.
biochemicals were purchased from Beijing Zhongshan Bio-
technology Co., Ltd, China. Other chemical reagents of 2.5. External standard method for the determination of GBP
analytical grade were provided by Nanjing Chemical Reagent purity performed by HPLC
Co., Ltd, China.
The GBP contents in the samples were determined by High
2.2. Plant material Performance Liquid Chromatography (HPLC) using external
standard method. HPLC was performed using a Shimadzu
G. biloba L. leaves were collected from ginkgo nursery of SPD-10A, consisting of a vacuum degasser, binary pump, and
Taixing, Jiangsu province, China, on October 10th 2009. photodiode array detection, controlled by class-up station
Botanical authentication was done by Dr. Zhaobang Shen of (Shimadzu Co. Ltd., Japan). The HPLC column was a Kromasil
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.
2.3. Animals
ODS-1 C18 column (150 mm × 4.6 mm, 5 μm). The isocratic 2.8. Histopathological examination
mobile phase consisted of isopropanol–methanol–hexane–
water (50:25:10:2, v/v) was used to elute for 65 min, and the The left hepatic lobule in each group was sliced and
flow rate was kept at 1.0 mL/min. Column temperature was washed in saline. Small pieces were preserved in 10%
kept constantly at 25 °C. UV detection was at 210 nm. formasal (formalin diluted to 10% with normal saline) and
The HPLC analysis of GBP standard and all the GBP samples were sent for histopathology examination. After paraffin
were carried out under the established experimental condi- embedding and block making, serial sections of 4–6 μm in
tion as mentioned above. The concentration of the GBP thickness were made, stained with haematoxylin and eosin
standard was 1.168 mg/mL, and the injection volumes were and examined under the microscope.
4, 8, 12, 16, and 20 μL, respectively. The standard curve was
obtained with the content of GBP as the abscissa and the 2.9. Statistical analysis
relevant five main peaks area as the ordinate. All the GBP
samples were filtered with 0.25 μm filter membrane and The results of hepatoprotective effect were recorded for
dissolved in hexane (chromatographically pure) before HPLC individuals within all groups. All data were presented as
measurement. The injection volume was 20 μL. 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.
2.6. Animal grouping and experimentation
3. Results
SD rats after acclimatization (a week) in the animal
quarters were randomly divided into 6 groups, GroupI 3.1. Determination of the purity of GBP
(Normal), GroupII (CCl4), GroupIII (CCl4 + 360 mg/kg Essen-
tiale), GroupIV (CCl4 + 10 mg/kg GBP), GroupV (CCl4 + The purity of GBP was determined by TLC and HPLC. Ethyl
40 mg/kg GBP), and GroupVI (CCl4 + 160 mg/kg GBP), each acetate–petroleum ether (1:9, v/v) mixture as the developing
group consisting of 10 rats (5 males and 5 females). solvent was used for TLC. The spots were visualized by iodine
The model of liver injury induced by CCl4 is commonly staining. The Rf value of GBP was 0.35 (Fig. 1). The regression
used for the evaluation of drug efficacy in liver injury. CCl4 equation obtained from the standard curve of GBP was
was previously dissolved in olive oil to make a final y = 1.5751x + 1.1546 (R 2 = 0.9991), and the purity of GBP
concentration of 40%. The method for feeding CCl4 to the was calculated according to the equation. The linear relation
experimental animals was according to Lin et al. [25]. between injection volume (4.6–23.3 μg) and peak area was
GroupII–VI received subcutaneous injection of 40% CCl4 at a good.
dose of 5 mL/kg for the first time and later at a dose of After purification, the final purity of GBP was 91.3%. GBP
3 mL/kg every 4 days, while GroupI subcutaneously received composed of 15 to 21 unsaturated isoprene units, and the
equal volume of olive oil. Simultaneously, GroupI–II was results showed that the relative content of C75, C80, C85, C90,
given 0.9% of physiological saline, and the treatment groups C95, C100, and C105 was 0.17%, 8.39%, 30.47%, 35.61%, 16.86%,
were given corresponding drugs once daily for a period of 6.48%, and 1.99%, respectively, indicating that C85 and C90
6 weeks. prenols were dominated (Fig. 2).
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
TP were significantly (P b 0.01) diminished. GBP (40, 160 mg/kg) injury. Enzyme activity values for GBP at a dose of 160 mg/kg
caused significant (P b 0.05, Pb 0.01) dose-dependent reductions were mostly comparable and not significantly different from
in CCl4-elevated levels of ALT, AST and ALP. GBP also dose- values obtained from the normal group. Essentiale markedly
dependently increased the reduced serum levels of ALB and TP (P b 0.01) reversed CCl4 suppressed antioxidant enzyme
with effects being significant (P b 0.05, P b 0.01) at 40 mg/kg and activity. However, enzyme activity value for Essentiale
160 mg/kg. GBP (10 mg/kg) had slight effect compared to the group was higher than normal for GSH but lower for SOD.
CCl4 group, and the levels of ALP, ALB, and TP changed little. The effects of GBP at the highest dose of 160 mg/kg were
Essentiale only produced significant (P b 0.05) reduction in CCl4- comparable and not markedly different from the effects of
elevated ALP level and significantly (P b 0.05) increased CCl4- Essentiale.
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 3.4. Effect of GBP on the contents of hydroxyproline and collagen
by reducing CCl4-elevated levels of serum ALT, AST, ALP and in liver injury tissues
increasing CCl4-reduced ALB and TP. The effects of GBP were
more prominent than Essentiale and peak effects were produced One of the main pathological changes of chronic liver
at the highest dose of 160 mg/kg, though there was a little injury induced by CCl4 is liver fibrosis. The liver collagen
difference between the normal and GBP160 group. content, an indication of fibrosis, can be calculated by the
content of hydroxyproline. Hydroxyproline is a unique amino
3.3. Effect of GBP on the in vivo antioxidative parameters acid in collagen, and its content in liver tissues was detected
by spectrophotometer.
As shown in Table 2, the MDA level was significantly As can be seen from Table 3, the content of collagen in the
(P b 0.01) increased due to CCl4 intoxification. GBP signifi- CCl4-treated animals was significantly (P b 0.01) increased
cantly and dose-dependently caused diminution of CCl4- when compared with those of the normal group. GBP caused
elevated MDA level with the value being slightly lower significant (P b 0.05) dose-dependent reductions in CCl4-
produced at the highest dose of 160 mg/kg. Essentiale elevated levels of hydroxyproline and collagen and peak
significantly (P b 0.01) reversed CCl4-induced MDA level effects were generally produced at the highest dose of
elevation with value being a little lower than the normal 160 mg/kg. However, the level of hydroxyproline still didn't
group. The effect of Essentiale was not significantly different restore to normal at the dose of 160 mg/kg. It is important to
from that of GBP at a dose of 160 mg/kg. note that the level of hydroxyproline was greatly (P b 0.05)
CCl4 significantly (P b 0.01) lowered the activity of reduced at the dose of 40 mg/kg, while the collagen level was
hepatic in vivo antioxidant enzymes GSH and SOD. GBP (40, not significantly affected. Essentiale effectively (P b 0.05)
160 mg/kg) caused significant (P b 0.01) increase in CCl4 decreased CCl4-induced hydroxyproline and collagen levels
suppressed activity of the antioxidant enzymes. Both GBP40 elevation. Again, GBP showed excellent protecting effect as
and GBP160 showed promising effects on such a hepatic 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 4. Discussion
injury induced by CCl4
Results indicated that GBP were able to suppress the
The degree of liver fibrosis can be reflected by the levels of hepatotoxicity of CCl4 and the hepatoprotective effect was
serum HA and LN, and the blood lipid level can be reflected by comparable to the conventionally reputed Essentiale. Levels
the levels of serum TG and CHO. Compared to the normal of some important biochemical parameters in serum are
group, CCl4 caused significant (P b 0.01) increase in serum HA used as diagnostic markers of hepatic injury. Increased
and LN levels. Meanwhile, serum TG and CHO levels were also levels of ALT, AST and ALP in serum of the CCl4-treated
increased (P b 0.05). GBP (10, 40, and 160 mg/kg) caused animals indicate liver damage as these enzymes leak out
significant (P b 0.05, P b 0.01) dose-dependent reductions in from liver into the blood at the instance of tissue damage,
CCl4-elevated levels of HA, LN, TG, and CHO. The effects of which is always associated with hepatonecrosis [29,30].
GBP40 were not significantly different from the Essentiale With the treatment of GBP or Essentiale, the levels of these
group and peak effects were produced at the highest dose of marker enzymes were near normal or only slightly elevated,
160 mg/kg. However, GBP160 significantly reduced CCl4- indicating protection against liver damage. ALP activity is
induced HA and LN levels elevation with values being related to the functioning of hepatocytes. Suppression of
slightly higher than the normal group. In addition, GBP (40, increased ALP activity suggests the stability of biliary
160 mg/kg) exhibited better efficiency than Essentiale dysfunction in rat liver during chronic hepatic injury with
(Table 4). These results suggest that GBP (40, 160 mg/kg) CCl4 [31]. Diminution of TP and ALB induced by CCl4 is a
possibly protect the structural integrity of the cell membrane further indication of liver damage [32]. GBP has increased
of liver cells or enhance regeneration of damaged liver cells. 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
3.6. Effect of GBP on liver tissues of CCl4-induced hepatotoxicity contributory hepatoprotective mechanism which acceler-
in rats ates the regeneration process and the production of liver
cells [33,34].
In rats of the normal group (Fig. 3A), the structure of liver Lipid peroxidation is the main mechanism of liver injury.
lobule and portal areas was normal. There was no obvious CCl4 can induce hepatotoxicity and can be converted into
degeneration or necrosis, no congestion, no proliferation of trichloromethyl free radical by cytochrome P-450. Trichlor-
sinusoids, no pathological changes, and no formation of omethyl free radical can cause peroxidate degradation in the
fibrous intervals. While in rats of CCl 4 -treated group adipose tissue [35]. In the present study, free radicals seem to
(Fig. 3B), there was severe steatosis, diffuse degeneration initiate peroxidate degradation of membrane lipids. This leads
and necrosis, streak proliferation of fibrous tissue, and to formation of lipid peroxides which in turn give products
infiltration of a small amount of inflammatory cells in portal like MDA that cause loss of integrity of cell membrane and
areas. These symptoms are very similar to the clinical damage to hepatic tissue [27]. Antioxidant enzymes can
pathological changes of viral hepatitis. Such phenomenon detoxify free radicals by converting them back to more stable
was greatly alleviated by GBP (Fig. 3D–F) as evidenced with molecules within the cell [36]. Decreased GSH level in CCl4-
slighter extent of fibrosis and less deformation occurred in administered rats may be due to its increased utilization for
liver lobules. Infiltration of inflammatory cells in portal areas enhancing the activities of GSH related enzymes GPX and GR
was especially alleviated significantly (P b 0.01). Different [37]. GBP and Essentiale prevented significantly lipid perox-
doses of GBP had obvious different protective effects on idation either directly or through GSH by scavenging the free
chronic liver injury damaged by CCl4. Especially GBP40 had radicals thus protecting the histostructural integrity of the
equivalent protective effects on steatosis compared to liver cells. Liver injury has been observed when GSH storage is
Essentiale (Fig. 3C). The structure of liver lobule and portal markedly depleted, and the level is restored to normal with
areas was restored to normal at the highest dose of 160 mg/kg, GBP treatment. It may be understood that the effect of GBP
which was in accordance with the reduced contents of TG and may be due to an initial reduction in hepatic peroxidative
CHO in serum. activities followed by inhibition of the activities of GSH related
Table 4
Effect of GBP on serum HA, LN, TG, and CHO levels of liver injury induced by CCl4.
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. ity of hydroxyl group of GBP is responsible for this free radical
SOD decreased significantly in CCl4-treated animals due to an scavenging activity. However, further investigation is needed
excessive formation of superoxide anions. Administration of to ascertain the precise mechanism of hepatoprotective
GBP effectively prevented the decrease in SOD activity, which action of GBP.
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 Acknowledgment
effectively recovered or improved.
As well known, the major component in extracellular The authors gratefully acknowledge the financial support
matrix is collagen. The collagen in CCl4-damaged liver by the National Natural Science Foundation of China (No.
apparently increased and severely accumulated (P b 0.01). 30871986) and professor Ying Lu from Nanjing University of
The liver lobules were hampered with huge amount of Chinese Medicine, who provided the histopathology exami-
collagen leading to extensive liver fibrosis. Moreover, liver nation results.
fibrosis was found to be significantly suppressed by GBP due
to the down-regulation of collagen synthesis, which demon-
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