doi: 10.1111/j.1472-8206.2005.00332.

ORIGINAL Ferulic acid, a natural protector against

ARTICLE
carbon tetrachloride-induced toxicity
M. Srinivasan, R. Rukkumani, A. Ram Sudheer, Venugopal P. Menon*
Department of Biochemistry, Faculty of Science, Annamalai University, Annamalainagar – 608 002, Tamil Nadu, India

Keywords
antioxidants,
CCl4,
ferulic acid,
lipid peroxidation,
liver fibrosis
Received 26 August 2004;
revised 9 December 2004;
accepted 10 February 2005
*Correspondence and reprints:
biocmr@sify.com; cdl_cmrana@
sancharnet.in
ABSTRACT
The present work is aimed at evaluating the protective effect of ferulic acid (FA), a
naturally occurring phenolic compound on CCl4 induced toxicity. The activities of
liver markers (alanine transaminase, aspartate transaminase, alkaline phosphatase,
c-glutamyl transferase), lipid peroxidative index (thiobarbituric acid-reactive sub-
stances, hydroperoxides, nitric oxide, protein carbonyl content), the antioxidant status
(superoxide dismutase, catalase, glutathione peroxidase and reduced glutathione)
were used as biomarkers to monitor the protective role of FA. The liver marker
enzymes in plasma and lipid peroxidative index in liver and kidney were increased in
CCl4-treated groups, which were decreased significantly on treatment with FA. The
antioxidants, which were depleted in CCl4-treated groups, were improved significantly
by FA treatment. Administration of FA to normal rats did not produce any harmful
effects. Thus our results show that FA is an effective antioxidant without any side-
effects and may be a great gain in the current search for natural therapy.

oxidant potential [8]. Dietary plant phenolic compounds

INTRODUCTION
Fibrosis results from chronic tissue insults and is a
common and difficult clinical challenge. Development of
fibrosis, particularly cirrhosis, is associated with signifi-
cant morbidity and mortality [1]. CCl4 is used to induce
fibrosis in experimental animal models [2]. Administra-
tion of CCl4 generates free radicals that trigger a cascade
of events resulting in fibrosis [3]. Oxidative stress is an
important stimulus for activation of hepatic stellate cells
and renal messangial cells [4] and these activated cells
are the major producers of the fibrotic neomatrix [5]. In
chronic injury induced by CCl4 there is an increase in the
deposition of the extracellular matrix (ECM), resulting in
severe fibrosis [6].
Both experimental and clinical observations suggest
that fibrosis can be resorbed. Although numerous
pharmaceutical agents have been tried, they all lead to
unacceptable side-effects during long-term therapy. In
this context, the use of an effective antioxidant without
side-effects is necessary to reduce the oxidative stress,
which leads to fibrosis [7].
Currently, there is a great deal of interest in the health
benefits of phenolic compounds because of their anti-
have been described to exert a variety of biological
actions such as free-radical scavenging, metal chelation,
modulation of enzymatic activity and more recently to
affect signal transduction, activation of transcription
factors and gene expression. They received particular
attention in the past 10 years because of their putative
role in the prevention of several human diseases [9].
Ferulic acid (FA) is a phenolic compound, formed
during the metabolism of phenylalanine and tyrosine.
It occurs primarily in rice, wheat, barely, oat, roasted
coffee, tomatoes, vegetables and citrus fruits [10]. FA
acts as a strong membrane antioxidant in humans and
is known to be effective against skin disorders, skin
cancer, ageing, fatigue, muscle wasting, cold, flu and
influenza [11]. The health benefits of FA is gaining a
lot of attention nowadays in the research world, but its
influence against fibrosis has not yet been entirely
proven. As little or no research has been carried out
on the in vivo antioxidant potential of FA, this
study was designed to study the protective effect of
FA on CCl4-induced toxicity in female Wistar rats.
A dose-dependent study revealed that FA, at a dose of
20 mg/kg body weight, was effective in preventing

2005 Blackwell Publishing Fundamental & Clinical Pharmacology 19 (2005) 491–496 491
492
hepatotoxicity [12]. Hence we used the same dose for
the present study.
MATERIALS AND METHODS
Animals
Female Albino rats, Wistar strain of body weight ranging
from 140 to 150 g bred in Central Animals House (Rajah
Muthiah Medical College, Annamalai University, Tamil-
nadu, India), fed on standard pellet diet (Agro Corpora-
tion Private Limited, Bangalore, India) were used for the
study and water was given ad libitum. The standard pellet
diet comprised 21% protein, 5% lipids, 4% crude fibre, 8%
ash, 1% calcium, 0.6% phosphorus, 3.4% glucose, 2%
vitamin and 55% nitrogen-free extract (carbohydrates).
It provides metabolizable energy of 3600 kcal/kg.
The animals were housed in plastic cages under
controlled conditions of 12 h light/12 h dark cycle, 50%
relative humidity and at temperature of 30 ± 2 C. They
were maintained in accordance with the guidelines of
the National Institute of Nutrition (Indian Council of
Medical Research, Hyderabad, India) and the study was
approved by the Animal Ethical Committee, Annamalai
University (proposal number: 168).
Materials used
Carbon tetrachloride was obtained from Merck Ltd
(Mumbai, India) and FA from Sigma Chemical Company
(St Louis, MO, USA). All other chemicals used in this
study were of analytical grade.
Experimental design
The animals were divided into four groups of six animals
each.
Group 1 Control rats given physiological saline
(3 mL/kg body weight/week)
by subcutaneous injection
Group 2 Rats given CCl4 (3 mL/kg body weight/week)
by subcutaneous injection [13]
Group 3 Rats given CCl4 subcutaneously +
FA (20 mg/kg body weight) [12]
dissolved in distilled water orally
using an intragastric tube
Group 4 Rats given FA (20 mg/kg body weight) +
physiological saline (3 mL/kg body weight/week)
by subcutaneous injection
CCl4 was administered to the rats once in a week and
FA was given once in a day throughout the experimental
period.

2005 Blackwell Publishing Fundamental & Clinical Pharmacology 19 (2005) 491–496
M. Srinivasan et al.
At the end of the experimental period (90 days), the
rats were anaesthetized using light ether and killed
by cervical decapitation. Blood and tissues (liver and
kidney) were immediately processed and used for various
biochemical estimations.
Preparation of plasma
Blood was collected in heparinized tubes and plasma was
separated by centrifugation at 2000 g for 10 min for
various biochemical estimations.
Preparation of tissue homogenate
Tissues (liver and kidney) were removed, cleared off
blood and immediately transferred to ice-cold containers
containing 0.9% NaCl for various estimations. A known
amount of tissue was weighed and homogenized in
appropriate buffer (10%) for the estimation of various
biochemical parameters.
Biochemical parameters
To assess the membrane damage, the activities of
liver marker enzymes alanine transaminase (ALT) and as-
partate transaminase (AST) by the Reitman and Frankel
method [14], alkaline phosphatase (ALP) by the King
and Armstrong method [15] and c-glutamyl transferase
(GGT) by the method of Fiala et al. [16], were assayed.
The extent of lipid peroxidation was determined by
analysing the levels of thiobarbituric acid-reactive sub-
stances (TBARS) by Niehaus and Samuelsson method
[17], hydroperoxides (HP) by Jiang et al. method [18],
nitric oxide (NO) by Lepovire et al. method [19] and
protein carbonyl content (PCO) by the method of Levine
et al. [20]. The antioxidant status was evaluated by
estimating the activities of superoxide dismutase (SOD)
by the method of Kakkar et al. [21], catalase (CAT)
by the method of Sinha [22], glutathione peroxidase
(GPx) by the method of Rotruck et al. [23] and reduced
glutathione (GSH) by the method of Ellman [24].
Statistical analysis
Statistical analysis was performed using one-way analy-
sis of variance (ANOVA) followed by Duncan’s multiple
range test (DMRT). The values are mean ± SD for six
rats in each group. P-values £0.05 were considered
significant.
RESULTS
Table I presents the changes in the activities of ALT, AST,
ALP and GGT in plasma. The activitiy of ALT, AST, ALP
Ferulic acid and carbon tetrachloride toxicity 493

Table I Changes in the activity of liver

No. Groups ALT (IU/L) AST (IU/L) ALP (IU/L) GGT (IU/L)
marker enzymes in plasma (values are
mean ± SD from six rats in each group). 1 Normal 73.83 ± 6.85 a
72.16 ± 7.22 a
70.94 ± 6.38 a
0.56 ± 0.05a
b b b
2 CCl4 156.68 ± 13.34 138.91 ± 12.72 193.14 ± 16.08 1.63 ± 0.11b
3 CCl4 + FA 91.47 ± 7.32c 94.77 ± 8.75c 101.82 ± 9.52c 0.84 ± 0.08c
4 FA 71.93 ± 6.42a 73.64 ± 5.69a 71.42 ± 6.78a 0.59 ± 0.06a

ANOVA followed by Duncan’s multiple range test.

Values not sharing a common superscript differ significantly at P £ 0.05.
ALT, alanine transaminase; AST, aspartate transaminase; ALP, alkaline phosphatase; GGT, c-glutamyl
transferase; FA, ferulic acid.

Table II Changes in the levels of TBARS in tissues (values are Table III Changes in the levels of hydroperoxides in tissues (values
mean ± SD from six rats in each group). are mean ± SD from six rats in each group).

No. Groups Liver (mM/100 g tissue) Kidney (mM/100 g tissue) No. Groups Liver (mM/100 g tissue) Kidney (mM/100 g tissue)

a a a
1 Normal 1.95 ± 0.18 1.93 ± 0.11 1 Normal 84.43 ± 5.71 133.60 ± 7.72a
b b b
2 CCl4 8.76 ± 0.75 5.87 ± 0.46 2 CCl4 168.86 ± 16.65 189.15 ± 11.26b
c c c
3 CCl4 + FA 5.68 ± 0.41 3.90 ± 0.32 3 CCl4 + FA 115.07 ± 10.25 154.58 ± 15.27c
4 FA 1.99 ± 0.08a 1.85 ± 0.13a 4 FA 85.06 ± 6.52a 133.48 ± 5.63a

ANOVA followed by Duncan’s multiple range test. ANOVA followed by Duncan’s multiple range test.
Values not sharing a common superscript differ significantly at P £ 0.05. Values not sharing a common superscript differ significantly at P £ 0.05.
TBARS, thiobarbituric acid-reactive substances; FA, ferulic acid.

GPx in liver and kidney compared with the CCl4-

and GGT were increased significantly in the CCl4-treated
group when compared with the control group. FA
treatment significantly decreased the activities of ALT,
AST, ALP and GGT compared with the CCl4-treated group.
The changes in the levels of TBARS in tissues are
shown in Table II. There was a significant elevation in the
TBARS levels in liver and kidney in CCl4-treated group
when compared with the control group. On treatment
with FA there was a significant decrease in the levels of
TBARS when compared with the CCl4-treated group.
Table III shows the changes in the levels of HP in
tissues. HP showed a significant increase in liver and
kidney of the CCl4-treated group when compared with
the control group. FA treatment significantly decreased
the levels of HP when compared with the CCl4-treated
group.
The changes in the levels of NO and PCO are shown
in Table IV. There was a significant elevation in the
levels of NO and PCO in the liver and kidney of the
CCl4-treated group compared with the control group.
On treatment with FA, there was significant decrease
in these levels.
The changes in the activities of SOD, CAT and GPx
are given in Table V. The activities of SOD, CAT and
GPx were decreased significantly in CCl4-treated groups
compared with the control group. FA treatment
significantly increased the activities of SOD, CAT and
treated group.
The levels of GSH are shown in Table VI. GSH levels
were significantly decreased in liver and kidney of the
CCl4-treated group compared with the control group. On
treatment with FA there was a siginificant increase in
the levels of GSH.
DISCUSSION
Carbon tetrachloride has been extensively used in
experimental models to elucidate the cellular mecha-
nisms behind oxidative damage [25]. CCl4 is activated by
cytochrome P450 2E1, 2B1 or 2B2 and possibly CYP
3A, to form the trichloromethyl radicals CCl3* and
trichloromethyl peroxy radical CCl3OO* which lead to
lipid peroxidation and subsequent tissue damage [26].
The elevated level of plasma liver markers is a direct
reflection of oxidative injury of liver. In chronic liver
injury, the transport function of the hepatocytes is
disturbed resulting in the leakage of plasma membrane
[27], thereby causing an increase in the activities of liver
marker enzymes in plasma. The elevated activities of
AST, ALT, GGT and ALP in our study is an indicative of
severe hepatic damage by CCl4.
Enhanced lipid peroxidation associated with depletion
of antioxidants in the tissue is a characteristic observa-
tion in CCl4-treated rats. Sipes et al. [28] reported that

2005 Blackwell Publishing Fundamental & Clinical Pharmacology 19 (2005) 491–496

494 M. Srinivasan et al.

Table IV Changes in the levels of nitric

Nitric oxide Protein carbonyl content
oxide and protein carbonyl content in
(·10)3lm of nitrite/mg of protein) (nmol/mg of protein)
tissues (values are mean ± SD from six
S. No Groups Liver Kidney Liver Kidney rats in each group).
a a a
1 Normal 11.60 ± 1.07 4.41 ± 0.43 4.49 ± 0.43 3.50 ± 0.31a
b b b
2 CCl4 21.59 ± 2.02 8.53 ± 0.76 11.91 ± 1.17 9.62 ± 0.89b
3 CCl4 + FA 14.83 ± 0.74c 5.79 ± 0.53c 6.88 ± 0.68c 5.79 ± 0.45c
4 FA 10.32 ± 1.04a 4.49 ± 0.42a 4.89 ± 0.37a 3.82 ± 0.33a

ANOVA followed by Duncan’s multiple range test.

Values not sharing a common superscript differ significantly at P £ 0.05.

Table V Changes in the activity of superoxide dismutase, catalase and glutathione peroxidase in tissues (values are mean ± SD from six rats
in each group).

Glutathione peroxidase
Superoxide dismutase (UA/mg protein) Catalase (UB/mg protein) (UC/mg protein)

No. Groups Liver Kidney Liver Kidney Liver Kidney

1 Normal 15.59 ± 1.44a 14.85 ± 1.29a 56.17 ± 4.53a 64.26 ± 5.32a 10.40 ± 0.87a 7.59 ± 0.70a
2 CCl4 5.77 ± 0.53b 7.54 ± 0.74b 38.92 ± 3.20b 37.49 ± 3.53b 3.28 ± 0.35b 3.95 ± 0.37b
c c c a c
3 CCl4 + FA 10.54 ± 1.02 11.17 ± 1.05 48.91 ± 4.41 57.56 ± 5.15 7.07 ± 0.54 5.83 ± 0.55c
a a a a a
4 FA 16.80 ± 1.22 15.92 ± 1.36 57.25 ± 4.08 64.35 ± 6.16 11.46 ± 1.02 7.91 ± 0.52a

ANOVA followed by Duncan’s multiple range test.

Values not sharing a common superscript differ significantly at P £ 0.05.
UAEnzyme required for 50% inhibition of NBT reduction per minute.
UBlmol of H2O2 utilized per minute.
UClmol of GSH utilized per minute.

Table VI Changes in the levels of reduced glutathione in tissues we observed increased levels of NO in liver and kidney
(values are mean ± SD from six rats in each group). during chronic administration of CCl4.
The level of PCO was significantly increased in the
No. Groups Liver (mg/100 g tissue) Kidney (mg/100 g tissue)
CCl4-treated groups. The protein oxidation products and
1 Normal 109.33 ± 8.84 a
116.00 ± 7.66a carbonyl derivatives of proteins may result from oxida-
b
2 CCl4 62.67 ± 5.12 66.33 ± 5.66b tive modifications of amino acid side chains and reactive
3 CCl4 + FA 88.00 ± 6.53c 93.33 ± 8.84c oxygen-mediated peptide cleavage [31]. Our study sug-
4 FA 113.50 ± 10.18a 125.33 ± 9.98a
gests that oxidative damage to proteins occurs during
ANOVA followed by Duncan’s multiple range test. CCl4 administration, resulting in increased PCO in liver
Values not sharing a common superscript differ significantly at P £ 0.05. and kidney.
Antioxidants and radical scavengers were to study the
the trichloromethyl radical abstracts a hydrogen atom mechanism of CCl4 toxicity as well as to protect liver cells
from a fatty acid to form a lipid radical. These radicals from CCl4-induced damage [26]. The principal enzymatic
may then react with oxygen to initiate lipid peroxidation. antioxidant defense systems against oxygen-free radicals
The excess lipid peroxidation in the CCl4-treated group as are SOD, CAT and GPx [32]. In this study, we observed a
evidenced by TBARS and HP in our study corroborates decrease in the activities of SOD, CAT and GPx in tissues
these findings. during chronic administration of CCl4. This decrease could
Several studies have reported that NO is produced in be due to a feed-back inhibition or oxidative inactivation of
the liver of rats treated with CCl4 [29]. NO plays an enzyme protein caused by excess ROS generation [33].
important role in various kinds of tissue injury either Reduced glutathione is an important cellular reduc-
directly or by interacting with reactive oxygen inter- tant and is involved in protection against free radi-
mediates to form more toxic species [30]. In our study, cals, peroxides and other toxic components [34]. The

2005 Blackwell Publishing Fundamental & Clinical Pharmacology 19 (2005) 491–496

Ferulic acid and carbon tetrachloride toxicity
decreased GSH levels in the CCl4-treated group in our
study indicates increased oxidative damage.
Administration of FA decreased lipid peroxidation,
improved antioxidant status and thereby prevented the
damage to the liver and leakage of enzymes ALT, AST,
ALP and GGT. This is mainly because of the antioxidant-
sparing action of FA.
The phenolic compounds act by scavenging free
radicals and quenching the lipid peroxides. The hydroxy
and phenoxy groups of phenolic compounds donate their
electron to the free radicals and neutralize them, form-
ing phenolic radical and quinone methide intermediate,
which is excreted via bile [35]. As FA is a phenolic
compound, it might have inhibited lipid peroxidation
in our study. Previous reports showed that FA is an
effective scavenger of free radicals and it has been
approved in certain countries as food additive to pre-
vent lipid peroxidation [36]. Toda et al. [37] have also
reported that FA scavenges superoxide anion radical and
inhibits lipid peroxidation induced by superoxide and the
effect of FA is similar to that of SOD.
Previous studies have proved that FA is a good
antioxidant against alcohol and polyunsaturated fatty
acids (PUFA)-induced toxicity in an experimental animal
model [38]. Reports have shown that ethyl ferulate, the
naturally occurring ester of FA is able to induce heme
oxygenase (HO) mRNA and protein expression for the
protection of brain cells against oxidative and neuro-
degenerative conditions [39]. It has also been reported
that FA protects against bleomycin-induced oxidative
stress and mutagenicity in Salmonella typhimurium
TA102 [40]. Studies showed that FA is an effective
preventive agent against iron-induced neuronal disease
associated with oxidative stress [41].
The antioxidant potential of FA can usually be
attributed to its structural characteristics (Figure 1).
FA, because of its phenolic nucleus and unsaturated side
chain can readily form a resonance-stabilized phenoxy
radical, which accounts for its potent antioxidant
activity. Any reactive radical colliding with FA easily
abstracts a hydrogen atom to form a phenoxy radical.
This radical is highly resonance-stabilized as the unpaired
electron may be present not only on the oxygen but it can
COOH COOH COOH COOH
OCH3 OCH3 OCH3 OCH3
O O O O
Figure 1 Resonance stabilization of ferulic acid radical.

2005 Blackwell Publishing Fundamental & Clinical Pharmacology 19 (2005) 491–496
495
also be delocalized across the entire molecule. Additional
stabilization of the phenoxy radical is provided by the
extended conjugation in the unsaturated side chain. This
resonance stabilization accounts for the effective anti-
oxidant potential of FA. Moreover, this phenoxy radical is
unable to initate or propagate a radical chain reaction,
and its most probable fate is a collision and condensation
with another ferulate radical to yield the dimer curcumin.
Such coupling may lead to a host of products, all of which
still contain phenolic hydroxyl groups capable of radical
scavenging. The presence of a second phenolic hydroxyl
group substantially enhances the radical scavenging
activity due to additional resonance stabilization and
o-quinone formation [10]. Moreover FA is known to
inhibit cytochrome P450, the free-radical generator and
thus known to decrease lipid peroxidation [42].
CONCLUSION
Ferulic acid effectively quenches free radicals, inhibits
lipid peroxidation and improves the antioxidant status in
the tissues. It also inhibits the leakage of liver marker
enzymes into circulation by preventing the membrane
damage caused by CCl4 toxicity. Hence, in our study, FA
was found to be effective against CCl4-induced toxicity.
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