1Department of Biochemistry, Hamadan University of Medical Sciences, Iran
2Student research committee, Hamadan University of Medical Sciences, Iran
3Neurophysiology Research Center, Hamadan University of Medical Sciences, Iran
4Department of Pathology, Hamadan University of Medical Sciences, Iran
5Department of Medical Laboratory Sciences, Hamadan University of Medical Sciences, Iran
Submission: April 11, 2019; Published: May 02, 2019
*Corresponding author: Ebrahim Abbasi Oshaghi, Department of Clinical Biochemistry, School of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran
How to cite this article: Roohollah M, Ebrahim AO, Hamid RGB, Amin M, Hamid RZ, et al. Amelioration of Acetaminophen-Induced Hepatotoxicity in Rat by Co-Administration of Quercetin and Resveratrol in Rats. Dairy and Vet Sci J. 2019; 11(4): 555817. DOI: 10.19080/JDVS.2019.11.555817
Monotherapy of resveratrol (RES) and quercetin (QE) showed potential useful effects. While, the combination properties are remain unknown. Therefore, in the current study we aimed to investigate the hepatoprotective effects of oral co-administration of RES and QE against acetaminophen (AA) induced hepatotoxicity in rats. Acute hepatotoxicity was induced by a single dose of 640 mg/kg AA. Then RES and QE were administrated orally for one week. The male Wistar rats were randomly divided into 6 groups including; normal, hepatotoxic group (AA received rats), N-acetylcysteine (NAC) group: AA + 150mg/kg/day NAC, QE group: AA + 20mg/kg/day QE, RES group: AA + 30 mg/kg/day RES and combination group: AA + RES (30mg/kg/day) + QE (20mg/kg/day). At the end of experiment, serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and gamma-glutamyl transferase (GGT) were measured. Also, serum level of triglyceride, total cholesterol, high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C) and very low-density lipoprotein were measured (VLDL-C). Then the liver structure was assessed by histopathological examination. Serum and liver total antioxidant capacity (TAC) and malondialdehyde (MDA) were measured by photometry and fluorescence spectroscopy method, respectively in serum and tissue homogenate.
The combination treatment significantly alleviated activity of specific liver enzymes and modulated lipid profiles parameters more than RES and QE treatment alone. Histopathological analysis indicated combination treatment remarkably led to improve liver injury more than RES and QE treatment separately. Also, co-administration of RES and QE normalized TAC and MDA levels in serum and tissue more significantly than single treatment. Oral co-administration of RES and QE normalized AA-induced hepatotoxicity by reducing lipid levels, MDA, liver enzymes and histopathological changes.
Acetaminophen (AA) widely utilized in clinical interventions for the management of fever and mild-to-moderate pain . Cheap price and well availability of AA is mainly responsible for its abuse that can be led to liver injury . In the United States, approximately 50% of patients with liver injury were due to drug induced liver toxicity . Malnutrition, fasting, abdominal pain, nausea symptom is the most common clinical sign of the drug induced hepatotoxicity . N-acetylcysteine (NAC) was used
commonly in AA overdose patients to reduce acute liver failure, and in some cases, accompanied by nausea, vomiting and skin allergy. In this context, herbal medicine could be considered as an alternative therapeutic approach for improving AA-induced liver damage.
From ancient times until now natural polyphenols have become widely used for prevention and treatment of many diseases . Their consumption accommodated with fewer side effects than . Natural polyphenols have known as a free radical scavenger where relived oxidative stress and inflammation . Resveratrol
(RES) is one of the most popular natural polyphenols with a huge
number of beneficial effects in clinical interventions . Many
reports introduced RES as an effective protective supplement
against drug hepatotoxicity by relieving tissue oxidative stress
[9-11]. Recent study demonstrated the hepatoprotective effect
of RES against alcohol consumption and AA toxicity . On the
other hand, quercetin (QE) is one of the most prominent dietary
antioxidants that widely found in herbal sources. Previous studies
showed the hepatoprotective effect of QE via decreasing oxidative
stress [13,14]. QE ameliorated diethylnitrosamine induced
hepatotoxicity in rats’ model through reducing the serum level of
alanine aminotransferase (ALT) and aspartate aminotransferase
(AST) and improving hepatic lipid peroxidation and glutathione
content (GSH) . However low bioavailability of RES and QE
that is mainly due to poor stability and intestinal absorption limit
their clinical applications when administrated orally.
Previous experimental researches identified intraduodenal
and intraperitoneally co-administration of natural RES and QE
enhance their bioavailability [16,17]. Over the last years, various
experiment has been done to elevate the bioavailability of natural
polyphenols. Here combination therapy may be considered as an
attractive choice. Recent in vitro study showed co-administration
of QE, RES and curcumin enhances intestinal absorption of RES
and curcumin across epithelial cells . On the other hand, recent
studies reported that co-administration of RES and QE suppressed
cell proliferation, cell cycle progression and primary mammary
tumor growth more effective than single administration [18,19].
Mikstacka showed that co-administration of RES and QE effectively
decreased lipid peroxidation in human erythrocytes . Moreover,
the previous attempt demonstrated RES plus QE prevented liver
steatosis through increasing fatty acid oxidation and inhibiting de
novo lipogenesis . Considering previous reports that mention
above, it could be concluded that the co-administration of RES
plus QE may lead to enhance its hepatoprotective effect against
AA induced hepatotoxicity. Therefore, in the current study, we
investigated the hepatoprotective effect of RES and QE treatment
against AA hepatotoxicity when administrated together and
The 6-week-old male Wistar rats weighing 220-230g were
purchased from Hamadan University of Medical Sciences. Animals
were housed in a temperature-controlled room (23±2 °C) and
humidity environment (60±5%) with 12-hour light/dark cycles
and standard chow diet. After acclimatization for one week in the
animal house, the rats were randomly divided into 6 groups (7 for
each group) as following:
a. group 1: normal healthy controls which received normal
b. group 2: received 640 mg/kg AA (hepatotoxic group).
c. Group 3: received 640 mg/kg AA + 150 mg/kg/day NAC
d. Group 4: received 640 mg/kg AA + 20mg/kg/day QE.
e. group 5: received 640 mg/kg AA + 30mg/kg/day RES
f. group 6: received 640 mg/kg AA + 20mg/kg/day QE +
30mg/kg/day RES (combination group).
AA hepatotoxicity was induced by single dose of AA 640 mg/kg
orally. Then the single and combined administration of QE and RES
were performed orally for one week. In the present study low dose
of RES and QE was selected according to previous studies [22-24].
All procedures of this study were carried out under supervision
of Ethics Committee of Hamadan University of Medical Sciences
(Ethics code: IR.UMSHA.REC.1395.383).
At the end of study, after overnight fasting, all animals were
anesthetized with ketamine 100mg/kg and sacrificed. Blood
samples were collected from the heart and centrifuged at 3000×g
for 10 min at 4 °C then serum was separated and stored at -20 °C
for further biochemical analysis. Liver tissues also were excised
and small portion was fixed in 10% formalin for histopathological
examination. Another section immediately was frozen at the liquid
nitrogen and stored at -70 °C for the determination of oxidative
Small pieces of liver tissues were weighed and homogenized
using lysis buffer and then centrifuged for 15 minutes at 10000g
at 4 °C. The supernatant was separated and maintained at -20 °C
for further analysis.
Specific liver enzymes including; AST, ALT, gamma-glutamyl
transferase (GGT) and serum lipid profile includes; triglyceride
(TG), total cholesterol (Cho) and high-density lipoprotein
cholesterol (HDL-C) were measured by colorimetric assay kit
(Pars Azmun, Tehran, Iran). The levels of low-density lipoprotein
cholesterol (LDL-C) and very low-density lipoprotein (VLDL) was
calculated according to the Friedewald formula.
The formalin treated samples were processed using standard
method. Briefly, paraffin-embedded tissue samples were cut
into 5-mm thickness, after rehydration, slides stained with
haematoxylin and eosin (H & E). Liver structure characteristics
including; tissue inflammation, hepatocytes necrosis and foam
cells was semi-quantitatively evaluated in 10 different fields and
compared with control and treated-groups by an experienced
The reducing potential of biological fluid was determined by
ferric reducing antioxidant potential assay (FRAP) . Briefly,
Fe III was reduced to Fe II by reducing potential of the sample.
Reaction between Fe II and tripyridyltriazine (Fe II-TPTZ)
produce a colored complex with maximum absorbance at 593nm.
In this study, total antioxidant capacity (TAC) in serum and liver
homogenates were measured.
Quantitative measurement of malondialdehyde (MDA), as the
end product of lipid peroxidation, represents lipid damage cause
by free radicals in the biological samples. The reaction between
MDA and thiobarbituric acid produce a colored complex. The
concentrations of peroxidized lipids were expressed as nmol/mg
protein. 1,1,3,3- tetraethoxypropane was used as standard .
All data were analyzed with SPSS 16.0 software (IBM, Armonk,
NY, USA). Statistical significances were determined using one-way
analysis of variance (ANOVA) following by Tukey post-hoc test. All
data were expressed as mean ± standard deviation (SD). P<0.05
was considered significant.
As shown in Table 1, acute hepatotoxicity significantly led to
increase specific liver enzymes compared to the control group.
Our results revealed that administration of RES and QE alone,
inhibited elevation of serum biochemical parameters as compared
with hepatotoxic control group. NAC treatment decreased these
markers more than RES and QE. Interestingly, combination
treatment decreased specific liver enzymes dramatically in a
similar manner to NAC treatment and restored these parameters
to the normal level. As shown in Table 2 AA treatments led to
serum lipid profile disturbance. RES and QE treatment reduced
TG level slightly but didn’t impact on the other parameters. NAC
treatment significantly led to decrease in TG and VLDL-C levels
but other parameters didn’t change. Interestingly combination
treatment decreased TG, VLDL-C, LDL-C more than NAC. Also,
combination treatment increased serum content of HDL-C.
H&E examination was depicted in the Figure 1. As expected
the architecture of hepatic lobule and portal tract in the healthy
control group were normal, while in the hepatotoxic group
the lobular necrosis and portal chronic inflammation are
noted. Complete protection from necrosis and inflammation
were observed in NAC treatment compared to the hepatotoxic
group. Moreover, QE and RES treatment revealed mild portal inflammation without necrosis, that showed partial protection
and then in the combination treatment group was demonstrated
trivial portal inflammation only, that showed a supportive effect
from partial protection more than QE and RES individually.
As illustrated in the Figure 2, TAC levels were significantly
reduced in serum and tissue following AA treatment in the
hepatotoxic group compared to the control group. Administration
of RES and QE avoided reduction of TAC in serum and liver samples.
The NAC treatment decreased this factor in serum and tissue more
than RES and QE treatment. Interestingly co-administration of
RES and QE normalized TAC level in serum and liver.
Animal treatment by RES inhibited elevation of MDA level
cause by AA toxicity partially compared to hepatotoxic group.
QE intake led to decrease MDA level just in liver. NAC treatment
reduced hepatic and serum level of MDA more significantly than
RES and QE. Surprisingly, MDA level in combination treatment was
significantly lower than RES and QE groups and showed similar
pattern like the NAC (Figure 3).
Over the last years, poor pharmacokinetic properties of
natural polyphenols limit their usage in clinical interventions.
A huge amount of data has been proved the beneficial effect of
natural polyphenols when used together [16,20]. Therefore, in the
current study, we investigated the hepatoprotective effect of RES
and QE against AA-induced hepatotoxicity when administrated
together in male Wistar rats.
The overdose of AA in induce experimental hepatotoxicity
model and elevate liver enzymes, which in turn represented
successful induction of acute hepatic injury. Single treatment
partially inhibited elevation of serum level of ALT, AST and GGT.
But oral co-administration of RES and QE restored serum levels
of liver specific markers near to the normal level similar to NAC.
Consistent with earlier reports by Gupta et al, intraperitoneally
administration of 100mg/kg QE normalized ALT and AST level
in diethylnitrosamine (DEN) induced hepatotoxicity model
. Also, Wang et al., showed the high dose of RES 100mg/kg
treatment inhibited elevation of ALT and AST level significantly in
AA induced liver injury in mice .
These results indicated orally co-administration of low
dose of RES and QE have showed protective effect similar to
intraperitoneally administration of the high dose of RES and QE.
In can be concluded co-administration of RES and QE lead to
improve their bioavailability through increase gastric absorption
cooperatively when administrated orally. The serum level of
TG, Cho, VLDL and LDL-C were remarkably elevated during AA
hepatotoxicity and HDL-C content was depleted. Compatible
with the previous studies RES and QE treatment separately
didn’t significant effect on the lipid profile parameters. Heebøll
showed that administration of high dose of RES (408 mg/kg)
prevent hepatic lipid accumulation partially in non-alcoholic
fatty liver disease (NAFLD) mice model . Also, previous
attempt showed administration of high dose of QE (100mg/
kg) had the hypolipidemic and hepatoprotective effect against
high cholesterol diet induced hepatotoxicity in Swiss albino
mice. Likely, low intestinal absorption and rapid metabolism of
polyphenol was due to weak its hypolipidemic effect. Increase
amount of oral intake and combination therapy could be consider
as an alternative approach for this problem. In agreement to our
propose lipid profile was significantly improved by combination
therapy even though better than NAC treatment.
Histopathological examination validated earlier serum biochemical
results. Similar to NAC treatment, combination treatment
compensated tissue necrosis and inflammatory cell infiltration
near to the normal liver. Also, hepatocyte lipid accumulation
was markedly reduced by polyphenol treatment. According to the
Arias et al. finding the antilipidemic effect of RES and QE supplement
may be due to reducing fatty acid oxidation and suppression
of hepatic lipogenesis . Histological observation was in line
with serum lipid profile changes, whereas combination treatment
had a more constructive effect on the vacuolated hepatocytes compared
to the single treatment. Consistent with our results, Arias et
al. reported that RES and QE have a synergistic effect against lipid
accumulation in adipocytes when they are consumed together . Biochemical and histopathological alteration declared that
combination treatment revealed the protective effect of REQ and
QE cooperatively when administrated together. The co-administration
of RES and QE decreased TAC level better than single treatment.
Regarding the previous attempt by Atmaca et al. it could
be concluded oral co-administration of RES and QE represented
anti-oxidant properties similar to RES treatment intraperitoneal
. They showed intraperitoneally injection of RES was accompanied
with a profound modulatory effect on the serum and hepatic
oxidative stress status in sodium fluoride-induced hepatotoxicity
in rats .
It seems to increase intestinal absorption of polyphenols
when combined to etch other may lead to increase circulation
time of RES and QE in blood and improve their protective effect.
Compatible to our propose Lund et al. investigated the effect of
co-administration of RES, QE and curcumin on their intestinal
absorption . They showed that permeability of RES was
elevated across epithelial cells about 3.1 folds when combined
Similar to the TAC results, single treatment approach reduced
serum and tissue MDA level slightly. Although Sebai reported
interaprituneal RES treatment, reduced hepatic oxidative stress
cause by lipopolysaccharide in rat . Also, post-treatment of
rats with DEN induced hepatotoxicity during interaprituneal
injection of QE at dose of 10 and 30 mg/kg retuned hepatic MDA
level. Interestingly combination treatment orally declared similar
effect to interaprituneal administration of RES and QE. NAC and
combination treatment returned serum and tissue level of MDA
content near to the normal level. Mikstacka found similar results.
They reported combination treatment suppressed oxidative
injury of membrane lipids in human erythrocyte synergistically
more than RES and QE treatment alone . This useful finding
presents additional evidence to a cooperative effect of RES and
QE treatment in attenuation of acute hepatotoxicity. Indeed,
combination treatment can be considered as an appropriate
alternative treatment for NAC treatment.
Co-administration of RES plus QE showed potential hepatoprotective
effects against AA induced toxicity by normalizing lipid
profile, liver enzymes and oxidative stress. It can be concluded
that combination treatment could serve as an interesting strategy
to clinical use of natural polyphenols against liver damage cause
by oxidative stress. Further in-depth investigations will be needed
to understand the molecular mechanisms of our findings.