Running title: Hepatoprotective effect of Vitis thunbergii in rats
Hepatoprotective effect of the ethanol extract of Vitis thunbergii on
carbon tetrachloride-induced acute hepatotoxicity in rats through
anti-oxidative activities
Jeng-Shyan Denga, Yi-Chih Changb, Chi-Luan Wenc, Jung-Chun Liaod, Wen-Chi Houe, Sakae Amagayaf,Shyh-Shyun Huangd, Guan-Jhong Huang g,*
a
Department of Health and Nutrition Biotechnology, Asia University, Taichung 413, Taiwan
b
Department of Medical Laboratory Science and Biotechnology, China Medical University, Taiwan
c
Taiwan Seed Improvement and propagation Station, Council of Agriculture, Propagation Technology Section, Taichung, Taiwan
d
School of Pharmacy, China Medical University, Taichung 404, Taiwan
e
Graduate Institute of Pharmacognosy, Taipei Medical University, Taipei 250, Taiwan
f
Department of kampo Pharmaceutical Sciences, Nihon Pharmaceutical University, Saitama 362-0806, Japan
g
School of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, China Medical University, Taichung 404, Taiwan
* Corresponding author: Dr. Shyh-Shyun Huang
School of Pharmacy, College of Pharmacy, China Medical University, Taichung 404, Taiwan; E-mail address: [email protected]
Dr. Guan-Jhong Huang
School of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, College of Pharmacy, China Medical University, Taichung 404, Taiwan; Tel: +886- 4- 2205-3366. Ext: 5508. Fax: +886- 4-2208-3362, E-mail address:
1. Introduction
The oxidative damage caused by reactive oxygen species (ROS) and reactive
nitrogen species (RNS) may generate various diseases in the human body, such as
aging, arthritis, cancer, inflammation, heart diseases and other human diseases (Poli,
1993). The enhanced production of oxidative stress can be induced by a variety of
factors, such as ionizing radiation, and exposure to drugs or xenobiotics (e.g., carbon
tetrachloride). CCl4, an analogue of human hepatotoxin, has been used extensively in
animal models to induce liver damage. Liver damage caused by CCl4 is characterized
by inflammation in the early stage. In damaged hepatocytes, CCl4 is reductively
bioactivated by cytochrome P450 2E1 into a trichloromethyl radical, a highly reactive
species that triggers lipid peroxidation and leads ultimately to hepatotoxicity (Goeptar
et al., 1995). Antioxidant action plays an important role by which various natural
products protect against CCl4-induced liver damage (Halim et al., 1997).
Vitis thunbergii Sieb. & Zucc. var. taiwaniana Lu (Vitaceae) an endemic plant,
an original medicinal plant in Taiwan, which has long been used as folk medicines for
treatments of hepatitis, jaundice, diarrhea, and arthritis (Lin et al, 2003). The active
components from Vitis thunbergii were reported to be resveratrol derivatives (Huang
et al, 2005) and polyphenols compounds (Dou et al., 2003). Vitis thunbergii has been
2010) and neuroprotective activities (Chung et al., 2011).
The objective of this study was to better understand EVT antioxidant effects in
vitro and in vivo. The hepatoprotective activity of its plant extracts has been
associated with its antioxidant activity.
2. Materials and methods
2.1. Chemicals
CCl4, silymarin, olive oil, and thiobarbituric acid (TBA) were purchased
from Sigma Chemical Co. (St. Louis, MO, USA). Glutathione peroxidase (GPx),
superoxide dismutase (SOD), and glutathione (GSH) were purchased from Randox
Laboratory Ltd. TNF-α and IL-1 concentrations were quantified using a
commercial ELISA kit (Biosource International Inc., Camarillo, CA). The antibody
against iNOS, COX-2, and -actin were purchased from Cell Signaling Technology
(Beverly, MA). Gallic acid, protocatechuic acid, catechin, vanillic acid, caffeic acid,
and syringic acid were purchased from Sigma Chemicals Co.
2.2. Plant material
Plant materials were collected from Taichung County, Taiwan. They were identified
Department of Chinese Medicine Recourses, China Medical University, Taichung,
Taiwan. A voucher specimen had been deposited in the school of Chinese
Pharmaceutical Sciences and Chinese Medicine Resources, China Medical University,
Taichung, Taiwan.
2.3. Preparation of the extracts of plant materials
Dried sample of the aerial part of Vitis thunbergii (1 kg) was macerated with 3 L
ethanol for 24 h at room temperature. Filtration and collection of the extract were
done three times. The filtrates were collected, concentrated with a vacuum evaporator
until the volume was below 10 mL and then freeze-dried. The yield obtained was
6.3% (w/w).
2.4. Compositional analysis of EVT by HPLC
HPLC was performed with a Hitachi Liquid Chromatography (Hitachi Ltd., Tokyo,
Japan), consisting of two model L-5000 pumps, and one model L-7455 photodiode
array detector (254 nm). Samples (10 mg/mL) were filtered through a 0.45 μm
PVDF-filter and injected into the HPLC column. The injection volume was 10 μL and
250 mm × 4.6 mm I.D.). The method involved the use of a binary gradient with
mobile phases containing: (A) phosphoric acid in water (0.6‰, v/v) and (B) MeOH
(v/v). The solvent gradient elution program was as follows: from 88% A to 78% A in
60 min, from 78% A to 68% A in 15 min. The flow-rate was kept constant at
1.0 mL/min. A precolumn of μ-Bondapak™ C18 (Millipore, Milford, MA, USA) was
attached to protect the analytical column. For photodiode array detection, the
wavelengths of phenolic compounds at their respective maximum absorbance
wavelength can monitored at the same time. Identification is based on retention times
and on-line spectral data in comparison with authentic standards. Quantification is
performed by establishing calibration curves for each compound determined, using
the standards. The crude extract was partitioned five times with 20 mL ethyl acetate.
The ethyl ether portions were combined, filtered and then concentrated by a rotary
evaporator and the residue dissolved in 1 mL of LC-grade methanol and filtered through ultra membrane filter (pore size 0.45 μm: Millipore) before HPLC analysis.
2.5. In vitro antioxidant activities of the crude extracts
2.5.1. Determination of antioxidant activity by ABTS·+ scavenging ability
The ABTS·+ scavenging ability was determined according to the method of
peroxodisulfate (2.45 mM) for 16 hours in the dark at room temperature. The ABTS·+
solution was diluted with 95% ethanol to an absorbance of 0.75 ± 0.05 at 734 nm
(Beckman UV-Vis spectrophotometer, Model DU640B). An aliquot (20 μL) of each
sample (125 μg/mL) was mixed with 180 μL ABTS·+ solution and the absorbance was
read at 734 nm after 1 min. Trolox was used as a reference standard.
2.5.2. Determination of antioxidant activity by DPPH radical scavenging ability
The effects of crude extracts and positive controls (GSH and BHT) on DPPH
radicals were estimated according to the method of Huang et al., (2007). Aliquot (20 μL) of crude extracts at various concentrations were each mixed with 100 mM Tris-HCl buffer (80 μL, pH 7.4) and then with 100 μL of DPPH in ethanol to a final concentration of 250 μM. The mixture was shaken vigorously and left to stand at room temperature for 20 min in the dark. The absorbance of the reaction solution was
measured spectrophotometrically at 517 nm. The percentages of DPPH decolorization
of the samples were calculated according to the equation: % decolorization = [1-
(ABS sample /ABS control)] ×100. EC50 value was the effective concentration at which
DPPH radicals were scavenged by 50% and was obtained by interpolation from linear
2.5.3. Determination of total polyphenol content
The total polyphenol contents of the crude extracts were determined according to
the method of Huang (2007). 20 μL of each extract (125 μg/mL) was added to 200 μL distilled water and 40 μL of Folin-Ciocalteu reagent. The mixture was allowed to stand at room temperature for 5 min and then 40 μL of 20 % sodium carbonate was added to the mixture. The resulting blue complex was then measured at 680 nm.
Catechin was used as a standard for the calibration curve. The polyphenol content was
calibrated using the linear equation based on the calibration curve. The total
polyphenol content was expressed as mg catechin equivalence (CE)/g dry weight.
2.5.4. Determination of total flavonoid content
The total flavonoid content of the crude extracts was determined according to the
method of Lamaison and Carnet (1990). Aliquots of 1.5 mL of extracts were added to
equal volumes of a solution of 2 % AlCl3·6H2O. The mixture was vigorously shaken,
and the absorbance at 430 nm was read after 10 min of incubation. Rutin was used as
a standard for the calibration curve. The total flavonoid content was calibrated using
the linear equation based on the calibration curve. The total flavonoid content was
expressed as mg rutin equivalent/g dry weight. The dry weight indicated was the
2.6. Animals
Male SD rats, aged six to eight weeks and weighing 180-200 g, were selected for
the study. They were maintained at a controlled temperature of 25-28°C with 12h
light/dark cycles and fed a standard diet and water ad libitum. Animal studies were
conducted according to the regulations of the Institute Animal Ethics Committee and
the protocol was approved by the Committee for the Purpose of Control and
Supervision of Experiments on Animals.
Rats were divided into six groups of eight animals each (n=8). Rats in the normal
control and negative control were orally administered with distilled water. The
positive control was orally administered with silymarin (200 mg/kg in 1%
carboxymethyl cellulose) once daily for 7 days. In the three experimental groups, the
rats were pretreated orally with EVT (100, 200, and 400 mg/kg) once daily for seven
consecutive days. One hour after the last treatment, all the rats, except for those in the
normal control, were treated with CCl4 (1.5 mL/kg in olive oil, 20%, i.p.). 24h after
the CCl4 treatment, animals were anesthetized with ethyl ether, and blood samples
were collected through their carotid arteries. The mortality rate and body weight were
recorded daily.
Different doses of EVT were orally administered to 5 groups of 10 mice in order
to estimate acute toxicity. Signs of toxicity during the first hour were observed and
recorded. Ten control animals were given a vehicle of 0.5% CMC. After the acute
phase, animals were observed for 14 days, keeping a record of the toxicity and
mortality (Rivera et al., 2004). Food and water were provided throughout the
experiment. If the mice died, they would be checked for autopsy and biochemical
profiles.
2.8. Histopathology
Small pieces of liver, fixed in 10 % buffered formalin were processed for
embedment in paraffin. Sections of 5-6 μm were cut and stained with hematoxylin and
eosin before they were examined for histopathological changes under the microscope
(Nikon, ECLIPSE, TS100, Japan). Images were taken with a digital camera
(NIS-Elements D 2.30, SP4, Build 387) at original magnification of ×200.
2.9. Antioxidant enzyme activities
The following biochemical parameters were analyzed to check the
hepatoprotective activity of EVT by the methods given below. Total superoxide
(Flohe and Otting 1984). The reductionof cytochrome c was mediated by superoxide
anions generatedby the xanthine/xanthine oxidase system and monitored at 550 nm.
One unit of SOD was defined as the amount of enzyme requiredto inhibit the rate of
cytochrome c reduction by 50%. Total catalase (CAT) activity was estimated as
described elsewhere (Aebi 1984). In brief, the reduction of 10 mM H2O2 in 20 mM of
phosphate buffer (pH 7) was monitored by measuring the absorbance at 240 nm. The
activity was calculated by using a molar absorption coefficient, and the enzyme
activity was defined as nanomoles of dissipating hydrogen peroxide per milligram
protein per minute. Total GPx activity was determined as previously reported (Paglia
and Valentine, 1967). Briefly, the enzyme solution was added to a mixture containing
hydrogen peroxide and glutathione in 0.1 mM Tris buffer (pH 7.2) and the absorbance
at 340 nm was measured. Activity was evaluated from a calibration curve, and the
enzyme activity was defined as nanomoles of NADPH oxidized per milligram protein
per minute.
2.9.1. Determination of GSH
Hepatic GSH level was determined as described previously (Ellman, 1959) with
slight modifications. Briefly, 720 μL of liver homogenate in 200 mM Tris buffer, pH
added to it and mixed thoroughly. The samples were then centrifuged at 10,000 × g
for 5 min at 4°C. Ellman’s reagent (DTNB solution) (660 μL) was added to the
supernatant (330 μL). Finally the absorbance was taken at 405 nm.
2.9.2. Determination of hepatic lipid peroxidation
The malondialdehyde (MDA) content, a measure of lipid peroxidation, was
assayed in the form of thiobarbituric acid-reactive substances (TBARS) as previously
described (Uchiyama et al., 1978). Briefly, 1 g of liver was homogenized in 10 mL of
KCl 1.15 % (w/v). 0.5 mL of liver homogenate was mixed with 3 mL of H3PO4 1 %
(v/v) and 1 mL of TBA 0.6 % (w/v), and then heated to and maintained at 100°C for
45 min. The samples were allowed to cool down to room temperature and 3 mL of
n-butanol was added. After shaking vigorously with the vortex, the butanolic phase
was obtained by centrifugation at 4,000 × g for 10 min to determine the absorbance at
535 nm. The standard was 1, 1, 1, 3-tetraethoxypropane.
2.9.3. Determination of nitric oxide (NO)
The production of NO was assessed indirectly by measuring the nitrite levels in
the plasma by a calorimetric method based on the Griess reaction (Green et al., 1982).
adding 1/20 volume of zinc sulfate (300 g/L) to a final concentration of 15 g/L. After
centrifugation at 10,000 × g for 5 min at room temperature, 100 μL supernatant was
applied to a microliter plate well, followed by 100 μL of Greiss reagent (1 %
sulfanilamide and 0.1 % N-1-naphthylethylenediamine dihydro-chloride in 2.5 %
polyphosphoric acid). After 10 min of color development at room temperature, the
absorbance was measured at 540 nm with a Micro Reader (Hyperion, Inc., FL, USA).
Nitrite was quantified by using sodium nitrate as the standard curve.
2.9.4. Measurement of serum TNF-α and IL-1
The serum level of TNF-α and IL-1 were determined using a commercially
available enzyme linked immunosorbent assay (ELISA) kit (Biosource International Inc., Camarillo, CA) according to the manufacturer’s instructions. TNF-α and IL-1 were determined from a standard curve. The concentrations were expressed in pg/mL.
2.9.5. Protein Lysate Preparation and Western blot Analysis of iNOS and COX-2.
Liver tissues were homogenized in lysis buffer (0.6% NP-40, 150 mM NaCl, 10
mM HEPES (pH 7.9), 1 mM EDTA, and 0.5 mM PMSF) at 4°C. We used BSA
(bovine serum albumin) as a protein standard to calculate equal total cellular protein
sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) using standard methods,
and then were transferred to PVDF membranes by electroblotting and blocking with
1% BSA. The membranes were probed with the primary antibodies (iNOS, COX-2,
and -actin) at 4°C overnight, washed three times with PBST, and incubated for 1 h at
37 °C with horseradish peroxidase conjugated secondary antibodies. The membranes
were washed three times and the immunoreactive proteins were detected by enhanced
chemiluminescence (ECL) using hyperfilm and ECL reagent (Amersham
International plc., Buckinghamshire, U.K.). The results of Western blot analysis were
quantified by measuring the relative intensity compared to the control using Kodak
Molecular Imaging Software and represented in the relative intensities.
2.10. Statistical analysis
All experiments were done in triplicate and results were reported as mean ± S.D.
Data were analyzed by one way ANOVA. Statistically significant effects were further
analyzed and means were compared using Duncan’s multiple range test. Statistical
significance was determined at p < 0.05.
3. Results
To establish the fingerprint chromatogram for the quality control of EVT. Gallic
acid, protocatechuic acid, catechin, vanillic acid, caffeic acid, and syringic acid were
used as markers. An optimized HPLC-PAD (photodiode array detector) technique was
employed. Meanwhile, HPLC chromatograms showed five marker components
present in EVT. As shown in Fig. 1, these phenolic components have been identified
as gallic acid, protocatechuic acid, catechin, vanillic acid, caffeic acid, and syringic
acid by their retention time and UV absorbance of purified standards. According to
the plot of peak-area ratio (y) vs. concentration (x, g/mL), the regression equations of
the five constituents and their correlation coefficients (r) were determined as follows:
gallic acid, y = 1.111x + 8.101 (r2 = 0.999); protocatechuic acid, y = 0.622x + 4.301
(r2 = 0.999); catechin, y = 0.443x +3.104 (r2 = 0.999); vanillic acid, y = 0.836x +5.686
(r2 = 0.999); caffeic acid, y = 1.643x+11.65 (r2 = 0.999), syringic acid,
y = 1.410x +10.19 (r2 = 0.999). The relative amounts of the six phenolic compounds
found in EVT were in the order of catechin (65.93mg/g) > vanillic acid (12.08mg/g)
>gallic acid (4.54 mg/g) > protocatechuic acid (4.11 mg/g) > syringic acid (3.65 mg/g)
> caffeic acid (2.47 mg/g), respectively.
3.2. In vitro assays
total antioxidant power of single compounds and complex mixtures of various plants.
ABTS assay was expressed as trolox equivalent antioxidant activity (TEAC) values.
Higher TEAC value represented that the sample had a stronger antioxidant activity.
Our results showed that EVT (109.35± 0.12g/mg extracts) had higher antioxidant
potentials (Table 1).
3.2.1. DPPH radical scavenging activity.
DPPH radical was scavenged by antioxidants through the donation of a proton
forming the reduced DPPH. The color changed from purple to yellow after the
reduction, which could be quantified by its decrease of the absorbance at wavelength
517 nm. Radical scavenging activity increased with increasing percentage of the free
radical inhibition. In DPPH radical scavenging activity assay, our results indicated
that an EC50 value of EVT was 192.69 ± 2.56 μg/mL while BHT had a value of 42.46
± 1.08 g/mL, respectively (Table 1).
3.3. Total phenolic and flavonoid content.
Most antioxidant activities from plant sources are correlated with phenolic-type
compounds. Total phenol content was estimated as 82.13± 0.58 g catechin
equivalent/ mg dry weight.
3.4. Evaluation of Acute Toxicity in Mice.
When EVT was studied by oral administration in mice, no signs of toxicity were
observed. In the LD50 experiment, no mice died under the dose of 5 g/kg.
3.5. Activities of ALT and AST in serum
The serum activities of ALT and AST were used as biochemical markers for the
early acute hepatic damage. The effect of the oral administration of EVT on the serum
AST and ALT levels of hepatic-damaged rats is shown in Table 2. EVT (400 mg/kg)
reduced serum AST and ALT levels of the rats with hepatic damage, significantly
(p<0.001). Silymarin (200 mg/kg) used as the standard drug indicated the similar
effect. It is confirmed that EVT could ameliorate hepatic function in CCl4 induced
liver injury.
3.6. Effect on serum NO, TNF-, and IL-1 levels
CCl4 induced hepatotoxicity was associated with marked increase in the levels of
NO, TNF- and IL-1. As shown in Table 2, the production of NO in the plasma was
(12.26 ± 0.68 M in control vs. 20.19 ± 1.75 M in only CCl4 treatment). However,
pretreatments of EVT decreased NO production in CCl4-treated rats. NO level was
significantly inhibited in the groups pretreated with 100 mg /kg (18.56±1.88 M, p <
0.05), 200 mg/kg (17.43 ± 0.85 M, p < 0.01) and 400 mg/kg (15.63 ± 1.11 M, p <
0.001) of EVT.
The production of TNF- and IL-1in the serum was significantly increased in
CCl4-treated rats (152.47±1.46 pg/mL and 363.46 ± 21.89 pg/mL) as compared to the
normal control group (81.07 ± 2.23 pg/mL and 152.47±1.46 pg/mL, respectively). At
the dose of 100 and 200 mg/kg, EVT produce any change in the TNF-and
IL-1level while significant decreases in the TNF-and IL-1level were observed
(p<0.001 or p<0.001). Like silymarin, treatment with EVT (400 mg/kg) over 7 days
produced a significant (p < 0.001) dose-dependent decrease in the levels of
TNF-and IL-1 (Table 1).
3.7. Effect of EVT on liver histology
The histological features of the livers from the control and experimental groups
are as shown in Fig. 2. Figure 2A shows the cell structure of livers of the control
animals and it is a representation of normal liver lobular architecture, which has no
Increased fatty degeneration, cytoplasmic vacuolization and necrosis provided
histopathological evidence of tissue injury in the CCl4-treated group (Fig. 2B).
Changes were improved in EVT and silymarin pretreated rats, which exhibited areas
of normal liver architecture and patches of necrotic hepatocytes (Fig. 2. C–F).
3.8. Effect on CAT, SOD, and GPx activities in CCl4-induced hepatic injury.
CAT activities in the total liver homogenate were shown in Table 3. CAT
activity of liver homogenate in CCl4 group (3.21 ± 0.06 U/mg protein) was
conspicuously lower than that of the control group (5.32 ± 0.15 U/mg protein). CAT
activities of liver homogenates from the 200 mg/kg (3.59 ± 0.28 U/mg protein, p <
0.05) and 400 mg/kg (4.23 ± 0.29 U/mg protein, P < 0.01) of EVT groups were
significantly higher than those in the CCl4 group.
The effect of EVT on SOD activity in the liver was shown in Table 3. SOD
activity of the liver homogenate in CCl4 group (9.68 ± 0.23 U/mg protein) was lower
than that of the control group (14.56 ± 0.57 U/mg protein). The SOD activities of liver
homogenates of groups treated with 100 mg/kg (11.57 ± 1.04 U/mg protein, p < 0.05),
200 mg/kg and 400 mg/kg (13.33 ± 0.93 U/mg protein; 13.69 ± 1.01 U/mg protein, p
< 0.01) were significantly higher than those in the CCl4 group.
administration with EVT. GPx activity of liver homogenate in the CCl4 group
(3.23±0.15 U/mg protein) was lower than that of the control group (4.56 ± 0.21 U/mg
protein). The GPx activities of liver homogenates of the experimental groups
pretreated with 100 mg/kg (3.67 ± 0.28 U/mg protein, P < 0.05), 200 mg/kg (4.14 ±
0.24 U/mg protein, P < 0.01), and 400 mg/kg (4.43 ± 0.14 U/mg protein, P < 0.001)
EVT were significantly higher than those of the CCl4 group. The observed increase of
antioxidant activities suggested that EVT had an efficient protective mechanism in
response to ROS.
3.9. Effect on the GSH levels in CCl4 treated rats
GSH protects cells against free radicals, peroxides and other toxic compounds.
Tissue levels of GSH often decrease upon elevation of local oxidative stress.
Deficiency of GSH within living organisms can lead to tissue disorder and injury.
Oxidative stress caused by CCl4 significantly reduced liver GSH level (5.31 ± 0.14
U/mg protein in only CCl4 treatment), however, CCl4-treated rats administrated with
200 and 400 mg/kg EVT (7.67 ± 0.81 U/mg protein, P < 0.01; 8.61± 0.26 U/mg
protein, P < 0.001) increased the GSH levels, as compared to the rats treated with
CCl4, significantly (Table 3). The results indicating that EVT (200-400 mg/kg)
3.10. Effect on hepatic TBARS levels
The localization of radical formation resulting in lipid peroxidation, measured as
MDA in rat liver homogenate, is as shown in Table 3. MDA contents in the liver total
homogenate were dramatically increased in CCl4-treated (1.59 ± 0.12 nmole/mg
protein) compared to the control group 0.48 ± 0.05 nmole/mg protein). MDA level
was significantly inhibited in 100 mg/kg (1.03 ± 0.17 nmole/mg protein, p < 0.05),
200 mg/kg (0.82 ± 0.16 nmole/mg protein, p < 0.01) and 400 mg/kg (0.68 ± 0.09
nmole/mg protein, p < 0.001) of EVT treated groups.
3.11. Analysis of iNOS/COX-2 expression following EVT treatment in rats with
CCl4-induced liver injury
We investigated the changes of the activation of iNOS and COX-2 by EVT in
CCl4-treated rats (Fig. 3). The results showed that CCl4 treatment stimulates to
increase activation of iNOS and COX-2. However, the treatment of EVT decreased
the iNOS and COX-2 expression in CCl4-induced rats. Namely, the relative intensities
of bands about iNOS and COX-2 expressions were reduced by 74.5 % and 71.1 % at
4. Discussion
Oxidative stress plays a crucial role in the development of CCl4-induced
hepatotoxicity and a connection between oxidative stress and lipid peroxidation has
been reported (Somasundaram et al., 2010). Studies have noted that CCl4 is widely
used to induce liver damage because it is metabolized in hepatocytes by cytochrome
P450, generating a highly reactive carbon-centered trichloromethy radical, leading to
initiating a chain of lipid peroxidation and thereby causing liver fibrosis (Fang et al.,
2008). In the present study, we evaluated the hepatoprotective effect of EVT against
CCl4 induced acute hepatotoxicity in rats. The consistency of chemical composition in
EVT is important in safe guarding the reliability of the research results. The chemical
profile of EVT was recorded by HPLC analysis. The HPLC chemical profile could be
delineated by the measurement of relative retention times of major characteristic
peaks using gallic acid, protocatechuic acid, catechin, vanillic acid, caffeic acid and
syringic acid as markers. The resulting chromatogram was used as a standard for the
assessment of all extracts used in the present study.
HPLC was used to quantify the components of EVT. The fingerprint
chromatograms demonstrated gallic acid, protocatechuic acid, catechin, vanillic acid,
caffeic acid, and syringic acid as its ingredients. Our results demonstrated that 1 g of
respectively. This result confirms that total polyphenol contents of natural products
are regular indices of their antioxidant activity.
The DPPH or ABTS have been popular radical scavenging tests for natural
products. Free radicals could induce biological damage and pathological events, such
as inflammation, aging, and carcinogenesis (Halliwell, 1999). In this study, EVT
showed significant antioxidant activities. The HPLC chromatogram of EVT
demonstrated six phenolic components identified as gallic acid, protocatechuic acid,
catechin, vanillic acid, caffeic acid, and syringic acid had higher good antioxidant
activities (Table 1). The higherradical scavenging activity of EVT seems to be closely
correlated with its polyphenolic constituents through other active components.
Apparently, these marker compounds in EVT could contribute to its antioxidant
effects. They could account for the high antioxidative of EVT. In general, free radical
scavenging and antioxidant activities of phenolics (e.g., phenolic acids, flavonoids)
depend on the number and position of hydrogen-donating hydroxyl groups on the
aromatic ring of the phenolic molecules, and are also affected by other factors, such as
glycosylation of aglycones, other H-donating groups (–NH, –SH), etc., In addition,
many scientific papers have reported that gallic acid was evaluated for
its hepatoprotective activity against CCl4-induced physiological and biochemical
protocatechuic acid for 28 consecutive days significantly decreases the intensity of
hepatic damage induced by CCl4 in rats (Hung et al., 2006). Catechin can be
considered as a chemotherapeutic against hepatopathies (Abdel-Hamid et al., 2007).
The administration of syringic acid and vanillic acid suppress hepatic fibrosis in
chronic liver injury (Itoh et al., 2010). Caffeic acid exhibits hepatoprotective activity
against paracetamol and CCl4-induced hepatic damage (Janbaz et al., 2004). Therefore,
the in vitro and in vivo antioxidant activities of EVT may be associated with the
phenolic compounds in the extracts.
Hepatic cells contain higher concentrations of AST and ALT in the cytoplasm,
and AST, particularly exists in the mitochondria. Due to damage caused to hepatic
cells, the leakage of cytosol will cause increased levels of hepatospecific enzymes in
the serum. The elevated serum enzyme levels such as AST and ALT are indicative of
cellular leakage and functional integrity of cell membrane in the liver (Zeashan et al.,
2009). The measurement of serum AST and ALT levels serve as a mean for the
indirect assessment of the condition of the liver. In the present study, the capability of
EVT in controlling CCl4 induced toxicity was demonstrated in that EVT pre-treated
animals which had decreased AST and ALT levels when compared with the CCl4
Because antioxidants are reported to exert anti-inflammatory activity (Huang,
2011), we also evaluated the in vivo anti-inflammatory activity of EVT by measuring
release of TNF-α, a pro-inflammatory cytokine that becomes elevated in acute and
chronic diseases. Some phytochemicals have been shown to inhibit inflammation by
blocking inflammatory pathways downstream of cytokine release, and also by
reducing macrophage production of proinflammatory factors (Tripathi et al., 2007).
The pro-inflammatory factors, TNF-α has been reported to play a key role in the
pathogenesis of various liver diseases. Following its release from activated Kupffer
cells, TNF-α aggravates both oxidative stress and inflammatory responses in the liver
(Nagata et al, 2007). The key role of TNF-α in CCl4 induced liver damage has been
substantiated in an earlier study in which treatment with soluble TNF-α receptors
prevented liver injury and decreased mortality in rats (Hsieh, et al., 2011). TNF-α
also stimulates the release of cytokines from macrophages and induces phagocyte
oxidative metabolism and NO production. Although several studies have showed that
NO protects against CCl4-induced liver injury using a NOS inhibitor, certain
evidence indicates that excessive NO production by iNOS can lead to hepatic injury
(Zhu and Fung, 2000). Recent reports also demonstrated that iNOS overproduction
occurs in the liver of rats with CCl4-induced acute liver injury, which suggested that
and Fung, 2000). Increased expression of COX-2, a known inflammatory mediator
has been observed in the present study. Increased expression of COX-2 and iNOS
indicate that there is a rise in the inflammation in CCl4 treated rats. In this study, we
evaluated that EVT not only inhibited the release of inflammatory mediators NO,
TNF-α, and IL-1, the hepatoprotective effect of EVT also could be attributed to its
anti-inflammatory properties.
CCl4-induced lipid peroxidation is highly dependent on the bioactivation of the
trichoromethyl and trichloromethyl peroxy radicals (Shen et al., 2009). MDA, a
product of lipid peroxidation, was increased in rat liver by CCl4 induction. However,
we showed that EVT significantly reduced MDA formation. In other words, the
mechanism of the inhibitory effects, by which the EVT protects against lipid
peroxidation, may involve radical-scavenging capability. CCl4 not only initiates lipid
peroxidation but also reduces tissue GPx, GR, CAT, and SOD activities, and this
depletion may result from oxidative modification of these proteins (Augustyniak et al.,
2005). Our results also showed that CCl4 challenge significantly decreased the
activities of CAT, SOD and GPx in the liver. Cells have a number of mechanisms to
defend themselves from the toxic effect of ROS including free radical scavengers and
chain reaction terminators such as SOD, CAT, and GPx systems. SOD removes
water by CAT and GPx. Cellular injury occurs when ROS generation exceeds the
cellular capacity of removal (Wu et al., 2009).
EVT administration effectively protected against the loss of these antioxidant
activities after CCl4 administration, and it also significantly reduced the loss of
hepatic GSH. GSH, an endogenous reductant, is well known to serve diverse
biological functions, including protection of cells from oxidative damage by ROS and
free radicals (Gabele et al., 2009). Some phytochemicals have also been shown to
stimulate syntheses of antioxidant enzymes and detoxification systems at the
transcriptional level, through antioxidant response elements (Masella et al., 2005).
Overproduction of NO in the liver has been implicated as an important event in
endotoxin shock and in other models of hepatic inflammation and injury. NO is
known to react with superoxide radical, forming peroxynitrite, an even more potent
oxidizing agent. Therefore, this endotoxin shock may alter the balance existing
between NO production and its target proteins and enzymes, leading to GSH depletion,
free radical generation and up-regulation of iNOS (Foresti, et al., 1997). Previous
report demonstrated that CCl4 administration increased NO level in the blood plasma
or CCl4-treated animals (Muriel, 1998). Similarly, under acute CCl4 intoxication we
revealed considerable NO level in blood plasma (Table 2), and increased expression
level in CCl4-treated rats. The treatment with EVT leads to a reduction in the
expression level of both iNOS and COX-2. This may in part be the result of the
regulatory activity and expression of NF-κB, and need to be proved.
These results have provided the evidence for the pharmacological effect of EVT
in CCl4-induced hepatotoxicity. Overall, EVT not only provided maximum
conjugation with injurious free radicals and diminished their toxic properties, but also
suppressed the inflammatory responses in CCl4-induced liver injuries. The possible
mechanisms could be suggested that EVT is able to protect the liver against cellular
oxidative damage and maintenance of intracellular level of antioxidant enzymes as
well as to act immunoregulatory. However, further studies on the active compounds
and their biochemical mechanisms responsible for the hepatoprotective effect of Vitis
thunbergii will be necessary.
Acknowledgement
The authors want to thank the financial supports from the National Science Council
(NSC100-2313-B-039-004- and NSC 100-2320-B-039-033-), China Medical
University (CMU) (CMU100-ASIA-15 and CMU100-TC-11) and Taiwan Department
References
Abdel –Hamid, N.M., Faddah, L.M., Al-Rehany, M.A., Ali, A.H., Bakeet, A.A., 2007.
New role of antinutritional factors, phytic acid and catechin in the treatment
of CCl4 intoxication. Annals of hepatology 6, 262-266.
Aebi, H., 1984. Catalase in vitro. Methods in Enzymology 105, 121-126.
Anand, K.K., Singh B., Saxena, A.K., Chandan, B.K., Gupta, V.N., Bhardwaj, V.,
1997. 3, 4, 5-Trihydroxy benzoic acid (gallic acid),
the hepatoprotective principle in the fruits of Terminalia belerica-bioassay
guided activity. Pharmacological Research 36, 315-321.
Augustyniak, A., Wazkilwicz, E., Skrzydlewaka, 2005. Preventive action of green tea
from changes in the liver antioxidant abilities of different aged rats intoxicated
with ethanol. Nutrition 21, 925-932.
Chung, I.M., Yeo, M.A., Kim, S.J., Moon, H.I., 2011. Neuroprotective effects of
resveratrol derivatives from the roots of Vitis thunbergii var. sinuate against
glutamate-induced neurotoxicity in primary cultured rat cortical cells. Human
and Experimental Toxicology 30, 1404-1408.
Dou, D.Q., Cooper, J.R.M., He, Y.H., Pei, Y.P., Takaya, Y., Niwa, M., Chen ,Y.J., Yao,
X.S., Zhou, R.P., 2003. Polyphenols from Vitis thunbergii Sieb. et Zucc.
Ellman, G.L., 1959. Tissue sulphydryl group. Archives of Biochemistry and
Biophysics 82, 70-77.
Fang, H.L. Lai, J.T., Lin, W.C., 2008. Inhibitory effect of olive oil on fibrosis induced
by carbon tetrachloride in rat liver. Clinical Nutrition 27, 900-907.
Flohe L, Otting F. 1984. Superoxide dismutase assays. Methods in Enzymology 105,
93–104.
Foresti, R., Clark, J. E., Green, C. J., Motterlini, R., 1997. Thiol compounds interact
with nitric oxide in regulating heme oxygenase- 1 induction in endothelial cells:
involvement of superoxide and peroxynitrite anions. The Journal of Biological
Chemistry 29, 18411-18417.
Gabele, E., Fron, M., Aeteel, G.E., Uesugi, T., Hellerbrand, C., Scholmerich, J., 2009.
TNF-alpha is required for cholestasis-induced liver fibrosis in the
mouse. Biochemical and Biophysical Research Communications 378, 348-353.
Goeptar, A.R., Scheerens, H., Vermeulen, N.P., 1995. Oxygen and xenobiotic
reductase activities of cytochrome P450. Critical Reviews in Toxicology 25,
25-65.
Green, L.C., Wagner, D.A., Glogowski, J., Skipper, P.L., Wishnok, J.S., Tannenbaum,
S.R., 1982. Analysis of nitrate, nitrite, and [15N] nitrate in biological fluids.
Halim, A.B., el-Ahmady, O., Hassab-Allah, S., Abdel-Galil, F., Hafez, Y., Darwish, A.,
1997. Biochemical effect of antioxidants on lipids and liver function in
experimentally-induced liver damage. Annals of Clinical Biochemistry 34,
656-663.
Halliwell, B., 1999. Antioxidant defense mechanisms: from the beginning to the end.
Free Radical Research 31, 261-272.
Huang, Y.L., Tsai, W.J., Shen, C.C., Chen, C.C., 2005. Resveratrol derivatives from
the roots of Vitis thunbergii. Journal of Natural Products 68, 217-220.
Huang, G.J., Chen, H.J., Chang, Y.S, Sheu, M.J., Lin, Y.H., 2007. Recombinant
Sporamin and its synthesized peptides with Antioxidant Activities in vitro.
Botanical studies 48, 133-140.
Huang, C.Y., Wen, C.L., Lu, Y.L., Lin, Y.S., Chen, L.G., Hou, W.C., 2010.
Antihypertensive activities of extracts from tissue cultures of Vitis
thunbergii var. taiwaniana. Botanical Studies 51, 317-324.
Huang, M.H., Huang, S.S., Wang, B.S., Wu, C.H., Sheu, M.J., Hou, W.C., Lin, S.S.,
Huang, G.J., 2011. Antioxidant and anti-inflammatory properties of
Cardiospermum halicacabum and its reference compounds ex vivo and in vivo.
Journal of Ethnopharmacology 133, 743-750.
ulmoides Oliv.) leaves inhibits CCl4-induced hepatic damage in rats. Food and
Chemical Toxicology 44, 1424-1431.
Hsieh, P.C., Ho, Y.L., Huang, G.J., Huang, M.H., Chiang, Y.C., Huang, S.S., Hou,
W.C., Chang, Y.S. 2011. Hepatoprotective effect of the aqueous extract of
Flemingia macrophylla on carbon tetrachloride-induced acute hepatotoxicity in
rats through anti-oxidative activities. American Journal of Chinese Medicine 39,
349-365.
Itoh ,A., Isoda, K., Kondoh, M., Kawase, M., Watari, A., Kobayashi, M., Tamesada,
M., Yagi, K., 2010. Hepatoprotective effect of syringic acid and vanillic acid on
CCl4-induced liver injury. Biological & Pharmaceutical Bulletin 33, 983-987.
Janbaz, K.H., Saeed, S.A., Gilani, A.H., 2004. Studies on the protective effects of
caffeic acid and quercetin on chemical-induced hepatotoxicity in rodents.
Phytomedicine 11, 424-430.
Lamaison, J. L. C., Carnet, A., 1990. Teneurs en principaux flavonoids des fleurs de
Crataegeus monogyna Jacq et de Crataegeus laevigata (Poiret D. C) en fonction
de la vegetation. Pharmaceutica Acta Helvetiae 65, 315-320.
Lin, I. H. 2003. The Catalogue of Medicinal Plant Resources in Taiwan, Committee
on Chinese Medicine and Pharmacy, Department of Health, Taipei, Taiwan.
mechanisms of natural antioxidant compounds in biological systems:
Involvement of glutathione and glutathione-related enzymes. The Journal of
Nutritional Biochemistry 16, 577-586.
Muriel, P., 1998. Nitric oxide protection of rat liver from lipid peroxidation, collagen
accumulation, and liver damage induced by carbon tetrachloride. Biochemical
Pharmacology 56, 773-779.
Nagata, K., Suzuki, H., Sakaguchi, S., 2007. Common pathogenic mechanism in
development progression of liver injury caused by non-alcoholic or alcoholic
steatohepatitis. The Journal of Toxicological Sciences 32, 453-468.
Paglia, D.E., Valentine, W.N., 1967. Studies on the quantitive and qualitative
characterization of erythrocyte glutathione peroxidase. Journal of Laboratory
and Clinical Medicine 70, 158-169.
Poli, G., 1993. Liver damage due to free radicals. British Medical Bulletin 49,
604-620.
Rivera, F., Gervaz, E., Sere, C., Dajas, F., 2004. Toxicological studies of the aqueous
extract from Achyrocline satureioides (Lam.) DC (Marcela). Journal of
Ethnopharmacology 95, 359-362.
Shen, X., Tang, Y., Yang, R., Yu, L., Fang, T., Duan, J., 2009. The protective effect of
anti-oxidative activities. Journal of Ethnopharmacology 122, 555-560.
Somasundaram, A., Karthikeyan, R., Velmurugan, V., Dhandapani, B., Raja, Muthu.,
2010. Evaluation of hepatoprotective activity of Kyllinga nemoralis (Hutch &
Dalz) rhizomes. Journal of Ethnopharmacology 127, 555–557.
Tripathi, S., Maier, K.G., Bruch, D., Kittur, D.S., 2007. Effect of 6-gingerol on
pro-inflammatorycytokine production and costimulatory molecule expression in
murine peritoneal macrophages. The Journal of Surgical Research 138, 209-213.
Uchiyama, M., Mihara, M., 1978. Determination of malonaldehyde precursor in
tissues by thiobarbituric acid test. Analytical Biochemistry 86, 271-278.
Wang, C.K., Chen, L.G., Wen, C.L., Hou, W.C., Hung, L.F., Yen, S.J., Shen, Y.J., Lin,
S.Y., Liang, Y.C., 2010. Neuroprotective activity of Vitis thunbergii var.
taiwaniana extracts in vitro and in vivo. Journal of Medicinal Food 13, 170-178.
Wu, Y.H., Zhang, X.M., Hu, M.H., Wu, X.M., Zhao, Y., 2009. Effect of Laggera alata
on hepatocyte damage induced by carbon tetrachloride in vitro and in vivo.
Journal of Ethnopharmacology 126, 50–56.
Zeashan, H., Amresh, G., Singh, S., Rao, C.V., 2009. Hepatoprotective and antioxidant
activity of Amaranthus spinosus against CCl4 induced toxicity. Journal of
Zhu, W., Fung, P. C., 2000. The roles played by crucial free radicals like lipid free
radicals, nitric oxide, and enzymes NOS and NADPH in CCl(4)-induced acute liver
