Ferulic acid, a natural protector against carbon tetrachloride induced toxicity

doi: 10.1111/j.1472-8206.2005.00332.x

OR IG INAL

ART ICLE

Ferulic acid, a natural protector againstcarbon 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

INTRODUCT ION

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-

oxidant potential [8]. Dietary plant phenolic compounds

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

Keywords

antioxidants,

CCl4,

ferulic acid,

lipid peroxidation,

liver fibrosis

Received 26 August 2004;

revised 9 December 2004;

accepted 10 February 2005

*Correspondence and reprints:

[email protected]; 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.

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

hepatotoxicity [12]. Hence we used the same dose for

the present study.

MATER IALS 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. Theywere 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.

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

492 M. Srinivasan et al.

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

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 andGGT comparedwith 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

GPx in liver and kidney compared with the CCl4-

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.

DISCUSS ION

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

Table I Changes in the activity of liver

marker enzymes in plasma (values are

mean ± SD from six rats in each group).

No. Groups ALT (IU/L) AST (IU/L) ALP (IU/L) GGT (IU/L)

1 Normal 73.83 ± 6.85a 72.16 ± 7.22a 70.94 ± 6.38a 0.56 ± 0.05a

2 CCl4 156.68 ± 13.34b 138.91 ± 12.72b 193.14 ± 16.08b 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

mean ± SD from six rats in each group).

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

1 Normal 1.95 ± 0.18a 1.93 ± 0.11a

2 CCl4 8.76 ± 0.75b 5.87 ± 0.46b

3 CCl4 + FA 5.68 ± 0.41c 3.90 ± 0.32c

4 FA 1.99 ± 0.08a 1.85 ± 0.13a



TBARS, thiobarbituric acid-reactive substances; FA, ferulic acid.

Table III Changes in the levels of hydroperoxides in tissues (values

are mean ± SD from six rats in each group).

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

1 Normal 84.43 ± 5.71a 133.60 ± 7.72a

2 CCl4 168.86 ± 16.65b 189.15 ± 11.26b

3 CCl4 + FA 115.07 ± 10.25c 154.58 ± 15.27c

4 FA 85.06 ± 6.52a 133.48 ± 5.63a



Ferulic acid and carbon tetrachloride toxicity 493


the trichloromethyl radical abstracts a hydrogen atom

from a fatty acid to form a lipid radical. These radicals

may then react with oxygen to initiate lipid peroxidation.

The excess lipid peroxidation in the CCl4-treated group as

evidenced by TBARS and HP in our study corroborates

these findings.

Several studies have reported that NO is produced in

the liver of rats treated with CCl4 [29]. NO plays an

important role in various kinds of tissue injury either

directly or by interacting with reactive oxygen inter-

mediates to form more toxic species [30]. In our study,

we observed increased levels of NO in liver and kidney

during chronic administration of CCl4.

The level of PCO was significantly increased in the

CCl4-treated groups. The protein oxidation products and

carbonyl derivatives of proteins may result from oxida-

tive modifications of amino acid side chains and reactive

oxygen-mediated peptide cleavage [31]. Our study sug-

gests that oxidative damage to proteins occurs during

CCl4 administration, resulting in increased PCO in liver

and kidney.

Antioxidants and radical scavengers were to study the

mechanism of CCl4 toxicity as well as to protect liver cells

from CCl4-induced damage [26]. The principal enzymatic

antioxidant defense systems against oxygen-free radicals

are SOD, CAT and GPx [32]. In this study, we observed a

decrease in the activities of SOD, CAT and GPx in tissues

during chronic administration of CCl4. This decrease could

be due to a feed-back inhibition or oxidative inactivation of

enzyme protein caused by excess ROS generation [33].

Reduced glutathione is an important cellular reduc-

tant and is involved in protection against free radi-

cals, peroxides and other toxic components [34]. The

Table IV Changes in the levels of nitric

oxide and protein carbonyl content in

tissues (values are mean ± SD from six

rats in each group).S. No Groups

Nitric oxide

(·10)3lm of nitrite/mg of protein)

Protein carbonyl content

(nmol/mg of protein)

Liver Kidney Liver Kidney

1 Normal 11.60 ± 1.07a 4.41 ± 0.43a 4.49 ± 0.43a 3.50 ± 0.31a

2 CCl4 21.59 ± 2.02b 8.53 ± 0.76b 11.91 ± 1.17b 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



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).

No. Groups

Superoxide dismutase (UA/mg protein) Catalase (UB/mg protein)

Glutathione peroxidase

(UC/mg protein)

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

3 CCl4 + FA 10.54 ± 1.02c 11.17 ± 1.05c 48.91 ± 4.41c 57.56 ± 5.15a 7.07 ± 0.54c 5.83 ± 0.55c

4 FA 16.80 ± 1.22a 15.92 ± 1.36a 57.25 ± 4.08a 64.35 ± 6.16a 11.46 ± 1.02a 7.91 ± 0.52a



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

(values are mean ± SD from six rats in each group).

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

1 Normal 109.33 ± 8.84a 116.00 ± 7.66a

2 CCl4 62.67 ± 5.12b 66.33 ± 5.66b

3 CCl4 + FA 88.00 ± 6.53c 93.33 ± 8.84c

4 FA 113.50 ± 10.18a 125.33 ± 9.98a





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

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].

CONCLUS ION

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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Ferulic acid, a natural protector against carbon tetrachloride induced toxicity