International Journal of Biochemistry

Research & Review

1(3): 69-81, 2011

SCIENCEDOMAIN international
www.sciencedomain.org

Evaluation of the Protective Potential of

Chromolaena odorata Linn. Extract on Carbon
Tetrachloride-Induced Oxidative Liver Damage

Chinwe Sylvanus Alisi1*, Geoffrey Okike C. Onyeze1, Okey Alphonsus Ojiako1

and Chidi G. Osuagwu2

1
Department of Biochemistry, Federal University of Technology,PMB 1526,
Owerri, Nigeria.
2
Department of Biomedical Technology, Federal University of Technology, PMB 1526,
Owerri, Nigeria.

Received 1st June 2011

Research Article Accepted 14th June 2011
Online Ready 19th July 2011

ABSTRACT

Carbon tetrachloride and its toxic metabolites consistently produce liver injury in many
species including man. The hepatoprotective potential of Chromolaena odorata Linn. (C.
odorata) was evaluated in male rabbits against carbon tetrachloride-induced liver
damage. Carbon tetrachloride intoxicated control (CCl4) and ethanol extract of C. odorata-
treated rabbits (ETECO TEST) received a single dose of CCl4 (0.2 ml/kg bw in liquid
paraffin 1:1). Pre-treated rabbits received ethanol extract of C. odorata at 400 mg/kg/day
in two divided doses of 200 mg/kg in morning and at night for 6 days prior to CCl4
administration. Sylimarin control received 50 mg/kg bw as a replacement for ETECO prior
to CCl4 intoxication. Normal animals received only extract in the above stated dose and
served as extract controls (ETECO CTRL).
Pre-treatment with C. odorata significantly (p<0.05) prevented the elevation of serum
aspartate aminotransferase (AST), alanineaminotransferase (ALT), lactate
dehydrogenase (LDH), gamma glutamyl transferase (‫ץ‬-GT), total bilirubin and
malondialdehyde (MDA) resulting from carbon tetrachloride intoxication. C. odorata
extract also significantly (p<0.05) prevented a decrease in serum total protein, albumin,
and glutathione (GSH) concentrations. The extract also significantly (p<0.05) prevented a
decrease in superoxide dismutase (SOD), catalase (CAT) and glutathione-s-transferase
(GST) activities. The presence of secondary plant metabolites like alkaloids, saponins,

____________________________________________________________________________________________

*Corresponding author: Email: chinwealisi@yahoo.com;

 International Journal of Biochemistry Research & Review, 1(3): 69-81, 2011

phenolic compouds, flavonoids and tannins found in C. odorata extract could be

responsible for its hepatoprotective action.

Keywords: Herbal drugs; Chromolaena odorata; hepatotoxicity; antioxidants; carbon

tetrachloride; oxidative stress;

1. INTRODUCTION

Liver disease is a worldwide problem. Conventional drugs used in the treatment of liver
diseases are sometimes inadequate and can have some serious adverse effects. It is
therefore, necessary to search for alternative drugs for the treatment of liver disease to
replace currently used drugs of doubtful efficacy and safety (Stickel and Schuppan, 2007).
Chromolaena odorata (L.) R. King and H. Robinson (formerly Eupatorium odoratum L.), a
perennial herb belonging to the plant family Asteraceae (=Compositae), is a diffuse,
scrambling shrub that is mainly a weed of plantation crops and pastures of southern Asia
and western Africa. The common plant is known as Siam weed, ‘Elizabeth’, ‘Independence
leaf’ and ‘Awolowo’ among the Igbos of the South-Eastern Nigeria. Phenolic compounds
from Chromolaena odorata have been reported to protect cultured skin cells from oxidative
damage. Phang et al., (2001) also showed that extracts from the leaves of C. odorata can
protect human dermal fibroblast and epidermal keratinocytes against hydrogen peroxide and
hypoxanthine – xanthine oxidase-induced damage. We already demonstrated a nitric oxide
scavenging ability of ethyl acetate fraction of methanol leaf extracts of Chromolaena odorata
(Alisi and Onyeze, 2008). Biochemical mechanisms of wound healing using ethanol extract
of Chromolaena odorata have also been previously reported (Alisi and Onyeze, 2009).

Carbontetrachloride (CCl4) has been one of the most intensively studied hepatotoxicants to
date and provides a relevant model for other halogenated hydrocarbons that are used widely
(Dahm and Jones, 1996; Weber et al., 2003). It consistently produces liver injury in many
species, (Safaq et al., 2009; Achudume and Ogunyemi, 2007). Carbontetrachloride is well
known to be converted by cytochrome P-450-mixed function oxygenases in smooth
endoplasmic reticulum of liver into toxic metabolite, mainly trichloromethyl radical (CCI3•).
This free radical in the presence of oxygen may cause peroxidation of lipids on target cell
resulting in extensive damage.

Alanine aminotransferase and aspartate aminotransferase are present in the high

concentration in the liver. They are present in both the mitochondria and cytosol of the
hepatocytes. Damage to this tissue may increase plasma ALT and AST activity (Moundipa
et al., 2007, Adinarayana et al., 2011) Since these enzymes are cytoplasmic in nature, upon
liver injury the enzyme enter in to the circulatory system due to altered permeability of
membrane. Increases in plasma ALT, AST and LDH activities have been seen in the
intoxication of liver with carbon tetrachloride (Wills and Asha, 2006). Gamma glutamyl
transferase (‫ץ‬-GT) activity is also known to be elevated in hepatocellular injury.

Protective potential of Chromolaena odorata extract on carbon tetrachloride-induced

oxidative acute liver injury will be investigated in this study in a preventive model.

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2. MATERIALS AND METHODS

2.1 Chemicals/Reagents

2-Thiobarbituric acid (TBA), Bovine Serum Albumin (BSA), trichloroacetic acid (TCA), 1-
chloro-2,4-dinitro benzene (CDNB) (Sigma-Aldrich, MO USA), Sodium dodecyl sulphate
(SDS), Adrenaline, Glutathione (GSH), 5,5-Dithiobis-2-nitro-5-thiobenzoic acid (DTNB)
(Fluka Chemie, Switzerland), All other chemicals and reagents used were from varied
sources and of analytical grade.

2.2 Plant Sample

Fresh aerial parts of Chromolaena odorata were collected from Egbu and Ihiagwa in Owerri
North and Owerri West Local Government Areas of Imo State. The study was conducted in
2008. The plant material was authenticated by a plant taxonomist, at the Department of
Plant Science and Biotechnology, Imo State University, Owerri, Imo State. Voucher
specimen has been retained at the authors’ laboratory in the school of science, federal
university of technology Owerri, Nigeria.

2.3 Preparation of Extract

The aerial part of C. odorata was shed, dried at 300C and dried leaves were reduced to a
coarse powder in a mill (Kenwood BL357). The powder (500g) was extracted with 2 litre
ethanol. Extract were recovered by distillation under reduced pressure at 49 °C in a Buchi
rotavapour (Switzerland). The extract was then dried to solid form in vacuum desiccators,
and stored in a freezer (<4.0 0C) until needed.

2.4 Animals

Thirty rabbits (white New Zealand) acquired from a small animal breeding station belonging
to Engr. E. Anyanwu, an animal breeder in Owerri, Imo State. Animals were maintained
under standard environmental condition (28-30 OC, 60-70 % relative humidity, 12-h dark /
light cycle) in stainless steel cages with free access to standard laboratory animal diet (Vital
finisher) and drinking water.

2.5 Induction of Hepatic Injury

Seven days after acclimatization, animals (rabbits) were separated into five groups of six
animals each. Group I served as normal control (NC) which received food and water only
throughout the treatment period. Group II served as intoxicated controls (CCl4 group) which
received food and water ad libitum and carbon tetrachloride (0.2ml/kgbw in liquid paraffin
1:1) on day 7. Groups III served as intoxicated tests (ETECO test) that received food and
water ad libitum, received ethanol extracts of C. odorata (400mg/kg body weight of animal)
in two divided equal daily doses and carbon tetrachloride (0.2ml/kgbw in liquid paraffin 1:1)
on day 7. Group IV received food and water ad libitum and received ethanol extracts of C.
odorata (400mg/kg body weight of animal) in two divided equal daily doses but did not
receive carbon tetrachloride. Group V received food and water ad libitum and received
Sylimarin (50mg/kg body weight of animal) daily and CCl4 on day 7. At the end of the seven-
day pre-treatment and subsequent intoxication with carbon tetrachloride, animals were
allowed for 48hrs. Animals were anaesthetized and sacrificed by cervical dislocation.

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2.6 Preparation of Liver Homogenate

Liver were obtained, washed in washing buffer (1.15% KCl), and dabbed with filter paper,
weighed in a digital weighing balance (Metler® PT320) and homogenized in KCl [10 mM]
phosphate buffer (1.15%) with ethylene-diamine tetra acetic acid (EDTA; pH 7.4) and
centrifuged at 12,000×g for 60 min.

2.7 Biological Assays

Blood collected from the ear blood vessels under mild chloroform anesthesia was kept for 45
min at 4 °C to cloth. Serum was separated by centrifugation at 600g for 15 min and analyzed
for various biochemical parameters. Serum lactate dehydrogenase (LDH), aspartate
aminotransferase (AST), alanineaminotransferase (ALT), gamma glutamyl transferase (‫ץ‬-
GT) activity, was determined using a chemistry autoanalyser (ciba corning 550 express).
Total bilirubin estimation exploits the use of diazotized sulphanilic acid as described by
Pearlman and Lee (1974) and Zoppi et al., (1976). The Biuret method as described by
Gornall et al. (1949) was employed for the determination of protein concentration in serum
and supernatant. Serum albumin concentration was estimated by the method employing
bromocresol green as described by Doumas et al. (1971).

2.8 Estimatioin of Lipid Peroxides

Lipid peroxidation in the supernatant fractions was determined spectrophotometrically by

assessing the concentration of thiobarbituric acid reactive substances (TBARS) according to
the method of Ohkawa et al. (1979) as described by Liu et al., (1990). The results were
expressed in malondiadehyde (MDA) formed relative to an extinction coefficient of 1.56 x 106
mol/cm.

2.9 Estimation of Reduced Glutathione (GSH)

Reduced glutathione (GSH) was estimated by its reaction with dithio-bis-2-nitrobenzoic acid
(DTNB) that gives a yellow coloured complex with absorption maximum at 412 nm (Raja et
al., 2007).

2.10 Assay of Superoxide Dismutase (SOD) Activity

Superoxide dismutase (SOD) activity was assayed as formerly described by Fridovich

(1989). The ability of the superoxide dismutase to inhibit the autoxidation of adrenalin was
the basis of the SOD assay.

2.11 Assay of Catalase (CAT) Activity

Catalase activity was assayed by the method of Aebi (1974). A 0.1ml portion of supernatant
was added to cuvette containing 1.9mL of 50mM phosphate buffer (pH 7.0). Reaction was
started by addition of 1.0mL of freshly prepared 30mM H2O2. The rate of decomposition of
H2O2 was measured spectrophotometrically at 240nm using the equation for a first-order
reaction.

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2.12 Assay of Glutathione-s-Transferase (GST) Activity

Glutathione-s-transferase activity was assayed by the method of Habig et al. (1974). The
reaction mixture consisted of 2.75mL of sodium phosphate buffer (0.1 M; pH 7.4), 0.1mL
glutathione (reduced) (1 mM), 0.1mL supernatant in a total volume of 3.0 mL. The change in
the absorbance was recorded at 340 nm and enzyme activity was calculated as nanomoles
of 1-chloro-2,4-dinitro benzene (CDNB) conjugate formed/min/mg protein using a molar
extinction coefficient of 9.6×103 M−1 cm−1.

2.13 Statistical analysis

Results of groups are calculated as means ± S.D. and subjected to one-way analysis of
variance (ANOVA). Significant difference between means were determined at alpha = 0.05.
Analysis was done using Excel+Analysi-it v.2.2 (Leeds UK).

3. RESULTS

3.1 Effect of Ethanol Extract of C. odorata on Plasma Alanine Amino

Transferase (ALT) Activity in Carbon Tetrachloride-Induced
Hepatotoxicity

Result (Table 1) showed that ALT activity in carbon tetrachloride-intoxicated animals were
elevated significantly (p < 0.05) (115.93±11.8 U/l) when compared to normal control
(40.86±4.0 U/l), ethanol extract of C. odorata control (62.72±7.2 U/l) and ethanol extract of
C. odorata treated group (77.88±6.8 U/l). Sylimarin (52.6±4.4 U/I) was however more
effective in protecting the hepatocytes against carbon tetrachloride-induced damage.

3.2 Effect of Ethanol Extract of C. odorata on Plasma Aspartate Amino

Transferase (AST) Activity in Carbon Tetrachloride-Induced
Hepatotoxicity

Result (Table 1) showed that AST activity in carbon tetrachloride-intoxicated animals was
elevated significantly (p < 0.05) (194.0±17.8 U/l) when compared to normal control (31.0±3.2
U/l), ethanol extract of C. odorata control (47.0±3.9U/l) and ethanol extract of C. odorata
treated group (143.±15.4 U/l). Sylimarin (80.82±10.5 U/I) was however more effective in
protecting the hepatocytes against carbon tetrachloride-induced damage.

3.3 Effect of Ethanol Extract of C. odorata on Lactate Dehydrogenase (LDH)

Activity in Carbon Tetrachloride-Induced Hepatotoxicity

Result (Table 1) showed that LDH activity in carbon tetrachloride intoxicated animals was
elevated significantly (p < 0.05) (4833±540 IU/l) when compared to normal control
(2740±294 IU/l), ethanol extract of C. odorata control (2992±246 IU/l) and ethanol extract of
C. odorata treated group (3543.±600 IU/l). Ethanol extract of C. odorata was comparable to
sylimarin (3200±85 IU/l) in restoring the ‫ ץ‬-GT activity to normalcy.

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3.4 Effect of Ethanol Extract of C. odorata on Gamma Glutamyl Transferase (‫ץ‬-

GT) Activity in Carbon Tetrachloride-Induced Hepatotoxicity

Result (Table 1) showed that ‫ץ‬-GT activity in carbon tetrachloride intoxicated animals was
elevated significantly (p < 0.05) (26.0±4.5 IU/l) when compared to normal control (8.33±1.12
IU/l), ethanol extract of C. odorata control (11.67±2.5 IU/l) and ethanol extract of C. odorata
treated group (13.0±2.16IU/l). Sylimarin (10.3±2.9 IU/l) was comparable to ethanol extract of
C. odorata in restoring the ‫ ץ‬-GT activity to normalcy.

3.5 Effect of Ethanol Extract of C. odorata on Serum Total Bilirubin

Concentration in Carbon Tetrachloride-Induced Hepatotoxicity

Result (Table 1) showed that total bilirubin concentration in carbon tetrachloride intoxicated
animals was elevated significantly (p < 0.05) (40.34 µmol/l) when compared to normal
control (15.34 µmol/l), ethanol extract of C. odorata control (14.77 µmol/l) and ethanol
extract of C. odorata treated group (19.31 µmol/l). Ethanol extract of C. odorata was
comparable to sylimarin (18.5±2.3 µmol/l) in restoring the total bilirubin concentration to
normalcy.

3.6 Effect of Ethanol Extract of C. odorata on Serum Protein Concentration in

Carbon Tetrachloride Induced Hepatotoxicity

Table 1 showed that total protein concentration in carbon tetrachloride intoxicated animals
was significantly (p < 0.05) decreased (50.20±2.5gl/l) when compared to normal control
(76.6±3.83 g/l), ethanol extract of C.odorata control (68.81±3.5g/l). Pre-treatment with the
extract of C. odorata prevented significantly (p < 0.05) a depletion of protein in the
intoxicated group (57.56±3.4 g/l).

3.7 Effect of Ethanol Extract of C. odorata on Serum Albumin Concentration in

Carbon Tetrachloride Induced Hepatotoxicity

Table 1 showed that total albumin concentration in carbon tetrachloride intoxicated animals
was significantly (p < 0.05) decreased (32.6±1.87gl/l) when compared to normal control
(49.76±1.47g/l), ETECO control (44.73±3.32g/l). Pre-treatment with the extract of C. odorata
prevented significantly (p < 0.05) a depletion of albumin in the intoxicated group (43.92±2.86
g/l).

3.8 Effect of Ethanol Extract of C. odorata on Hepatic Lipid Peroxidation in

Carbon Tetrachloride-Induced Hepatotoxicity

Results obtained from our studies (Figure 1A) showed that intoxication of rabbits with carbon
tetrachloride resulted in elevation of malondialdehyde concentration (25.0 ± 1..2.nmol/g
Liver) which was significantly (p < 0.05) higher than the normal controls (8.0 ± 0.38nmol/g
Liver) and the ETECO controls (10.0 ± 0.48nmol/g Liver). Pre-treatment with ETECO
significantly (p < 0.05) prevented increase in malondialdehyde concentration in the
intoxicated animals (15.0 ± 0.7nmol/g Liver). Sylimarin decreased malondialdehyde
concentration (9.5 ± 0.9 nmol/g Liver) in carbon tetrachloride-induced liver damage.

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Table 1. Effect of Ethanol extract of C. odorata (400mg/kg bw) on liver function parameters

Liver Normal Carbon ETECO ETECO Control Silymarin

function Control tetrachloride Test
parameters (CCl4)
ALT (IU/l) 40.86 ± 4.00 115.93 ± 11.80* 77.88 ± 6.80† 62.72 ± 7.20†* 52.60 ± 4.40†*

AST (IU/l) 31.0 ± 3.2 194.0 ± 17.8* 143.0 ±15.4†* 47.0 ± 3.9†* 80.8 ± 10.5†*

LDH (IU/l) 2740 ± 294 4833 ± 540* 3543 ± 600† 2992 ± 246† 3200 ± 85†

γ-GT (IU/l) 8.33 ± 1.12 26.0 ± 4.50* 13.0 ± 2.16† 11.67 ± 2.5† 10.3 ± 2.90†

Bilirubin-Total 15.34 ±1.33 40.34 ±3.60* 19.31 ±1.57† 14.77 ±1.32† 16.43 ± 1.70†
(mg/dl)
Protein (g/dl) 76.55 ± 3.83 50.20 ± 2.50* 57.56 ± 3.40†* 68.81 ± 3.50† 70.12 ± 3.46†

Albumin (g/dl) 49.76 ±1.47 32.63 ± 1.87* 43.92 ± 2.86†* 44.73 ± 3.32† 48.40 ± 5.50†

Values are mean ± S.D., n=6. * P<0.05 vs. normal control. † P<0.05 vs. CCl4 control. ETECO – Ethanol extract of Chromolaena odorata, CCl4

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30
* 9
25 A C #
MDA (nmol/gTissue)

SOD (U/ g Liver )

20
6 #*
#*
15
#* #*
10
3 *
5

0 0
NC CCL4 ETECO ETECO Sylimarin NC CCL4 ETECO ETECO Silymarin
TEST CTRL CTRL
800
160
# D
GSH (µmol/gTissue)

B # #
600 #

CAT (U/ g Liver )

120
#*
#*
400
* 80

200
40 *

0
NC CCL4 ETECO ETECO Sylimarin 0
TEST CTRL NC CCL4 ETECO ETECO Silymarin
1800 CTRL
GST (µg/min/mgProtein X 104

# #
1400 E Fig. 1 (A-E). Effect of ethanol extract
#* of Chromolaena odorata
(400mg/kgbw) on hepatic (A)
1000 malondialdehyde (MDA), (B)
glutathione (GSH), (C) superoxide
* dismutase (SOD), (D) catalase (CAT)
600 and (E) glutathione-s-transferase
(GST).
Values are mean ± S.D., n=6. * P<0.05 vs.
200 normal control. # P<0.05 vs. CCl4 control
NC CCL4 ETECO ETECO Sylimarin
TEST CTRL

3.9 Effect of Ethanol Extract of C. odorata on Hepatic Glutathione in Carbon

Tetrachloride-Induced Hepatotoxicity

The result (Figure 1B) showed that Carbon tetrachloride intoxication in animals produced a
significant (p < 0.05) decrease in glutathione concentration (300 ± 12µMol/g Liver), when
compared with the normal animals (650 ± 18 µMol/g Liver). This decrease in glutathione
concentration was reversed by pre-treatment with ethanol extract of C. odorata (400 ± 15

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µMol/g Liver). Sylimarin similarly, restored the concentration of glutathione (625 ± 21 µMol/g
Liver) in carbon tetrachloride-induced liver toxicity.

3.10 Effect of Ethanol Extract of C. odorata on Liver Superoxide Dismutase

Activity in Carbon Tetrachloride-Induced Hepatotoxicity

The result (Figure 1C) showed that treatment with carbon tetrachloride substantially
decreased liver superoxide dismutase (SOD) activity (2.3 ± 0.30 U/g Liver) significantly (p <
0.05) when compared to normal control (6.8 ± 1.20 U/g Liver) and ETECO control (6.2 ±
0.80 U/g Liver). Pre-treatment with the extract, significantly (p < 0.05) raised the activity of
liver superoxide dismutase in intoxicated animals (4.9 ± 0.76 U/g Liver). Sylimarin similarly
prevented a drop in SOD activity in intoxicated animals.

3.11 Effect of Ethanol Extract of C. odorata on Liver Catalase Activity in

Carbon Tetrachloride-Induced Hepatotoxicity

The result (Figure 1D) showed that liver catalase (CAT) activity in carbontetrachloride-
intoxicated animals was significantly (p < 0.05) decreased (30.0 ± 3.90 U/g Liver) when
compared to normal control (114.3 ± 15.4 U/g Liver) and ethanol extract of C. odorata
control (106.8 ± 10.6U/g Liver). Ethanol extract of C. odorata significantly (p < 0.05) raised
the activity of liver catalase in the intoxicated group (87.0 ± 9.7 U/gLiver); it however, could
not restored catalase to an activity similar to the normal control activity. Standard drug,
Sylimarin was able to restore catalase to an activity (112±14.65 U/g Liver) similar to normal
control.

3.12 Effect of Ethanol Extract of C. odorata on Hepatic Glutathione-s-

Transferase Activity in Carbon Tetrachloride-Induced Hepatotoxicity

The result (Figure 1E) showed that liver glutathione-s-transferase activity in carbon
tetrachloride treated animals was significantly (p < 0.05) decreased (635 ± 43 µmol/gLiver),
when compared to normal control (1535 ± 120µMol/gLiver) and ETECO control (1420 ± 86
µmol/g Liver). Treatment with ethanol extract of C. odorata increased significantly (p < 0.05)
the activity of glutathione-s-transferase in the intoxicated rabbits (1026 ± 90µmol/g Liver).
Sylimarin treated intoxicated group had a higher GST activity (1420 ± 110 µmol/g Liver) than
C. odorata treated group.

4. DISCUSSION

Results of the study (Table 1) showed that ALT, AST, LDH and ‫ץ‬-GT activities in carbon
tetrachloride intoxicated animals were elevated significantly (p<0.05) when compared to their
normal controls. This indicated that CCl4 intoxication compromised the integrity of the hepatic
cell membranes (Mukherjee, 2003; Drotman and Lawhorn, 1978; Mukherjee, 2002). Pre-
treated with Chromolaena odorata significantly (p<0.05) prevented hepatic damage
manifesting in the non-elevation of serum aspartateaminotransferase (AST),
alanineaminotransferase (ALT), lactate dehydrogenase (LDH), gamma-glutamyltransferase
(‫ץ‬-GT) and total bilirubin. These observations are in agreement with the commonly accepted
view that serum levels of transaminases return to normal with healing of hepatic parenchyma
and the regeneration of hepatocytes (Thabrew et al., 1987). In this study however, there was
a prevention of damage to hepatic parenchyma and hepatocytes rather than healing.

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Pre-treatment with Chromolaena odorata also significantly (p<0.05) prevented a decrease in

serum total protein and albumin concentrations, It is known that increases in ALT, AST,
LDH, and γ-GT activities shows injury to the tissues, while elevations in total bilirubin,
decreases in total protein and albumin concentrations are useful tools in the assessment of
hepatic functions. It is also known that most circulating proteins are synthesized in the liver
and concentrations indicate synthetic ability of the liver since serum albumin accounts for
65% of serum proteins (Deepak et al., 2000). These results indicate that ethanol extract of
C. odorata protected the hepatocytes against injury and prevented loss of functionality.

Many compounds known to be beneficial against carbon tetrachloride-mediated liver injury

exert their protective action via a decreased production of carbon tetrachloride derived free
radicals or through the antioxidant activity of the protective agents themselves (Jayatilaka et
al., 1990; Thabrew et al., 1987). Protection of the liver against antioxidant depletion
observed in this study (Figure 1B-1E), indicated that protective action against carbon
tetrachloride induced damage in the liver could be by anti-oxidation mechanism. During
hepatic injury, superoxide radicals are generated at the site of damage and modulate
superoxide dismutase (SOD) and catalase (CAT) activity, resulting in the loss of activity and
accumulation of superoxide radical, which damages the liver. Decreased CAT activity is
linked up to exhaustion of the enzyme as a result of oxidative stress caused by CCl4. The
SOD and CAT activities were kept at near normal by pre-treatment with ethanol extract of C.
odorata in CCl4-treated rabbits (Figures 1B & 1C), which evidently showed the antioxidant
potentials of the extract against oxygen free radicals. Restoration of SOD and CAT activities
by ethanol extract of C. odorata indicated that the extract scavenged superoxide radicals.
Superoxide and nitric oxide radicals have been found to cause inactivation and nitration of
the human superoxide dismutase enzyme (Demichelli et al., 2007). We had earlier
demonstrated the nitric oxide scavenging ability of C. odorata extract (Alisi and Onyeze,
2008).

Reduction in liver GSH and corresponding increase in lipid peroxidation (Figure 1A&B) and
decrease in GST activity (Figure 1E) in CCl4-treated rabbits as observed in this study
indicated antioxidant depletion resulting in damage to the hepatic cells. Increase in hepatic
GSH concentration in ethanol extract of C. odorata treated group could either be due to an
effect on the de novo synthesis of GSH, its regeneration or both. As a consequence, hepatic
GSH concentration could be sufficiently maintained to counteract the increased formation of
free radicals as in the case of carbon tetrachloride toxicity (Ko et al., 1995).

There seemed to be GST activation in the presence of the extract. GST plays a physiological
role in initiating the detoxification of potential alkylating agents. Chemicals like chloroform
and CCl4 alter the hepatic GST activity (Aniya and Anders, 1985). GST activity was
significantly (p < 0.05) reduced in CCl4-treated group and upward reversal was observed in
the pre-treatment with ethanol extract of C. odorata. This may be attributed to substrate
(glutathione) activation or a direct action of extract on the hepatic GST activation, the
mechanism of which is not clearly understood. Reduction in lipid peroxide concentration
showed that lipid peroxidation in carbon tetrachloride-induced liver damage was prevented
significantly (p<0.05) due to increases in the enzymatic antioxidant activity and non-
enzymatic antioxidant concentration, resulting from pre-treatment with ethanol extract of C.
odorata. Results of this study showed that the extract compared well with sylimarin (a
standard hepatoprotective agent) in normalising hepatic parameters. These observations are
indicative of potentials of the extract in the prevention of oxidative stress induced by toxic
metabolite, mainly trichloromethyl radical (CCI3•). Secondary plant metabolites like
flavonoids, phenolic compounds, saponins, alkaloids, glycosides and tannins have earlier
been shown to be present in this extract (Alisi and Onyeze, 2008; Igboh et al., 2009). The

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hepatoprotective potentials exhibited by the C. odorata extract could be due to the

synergistic effect of these agents.

5. CONCLUSION

In conclusion, our investigation revealed that C. odorata leaf extract has got
hepatoprotective potentials against carbontetrachloride-induced oxidative damage. The
mechanism of hepatoprotection may be multidimensional but not fully understood. However,
the protection conferred on the liver by the extract is thought to be by anti-oxidative action as
inferred from the ability of the extract to keep antioxidant enzyme activities stable at near
normal. Its ability to prevent glutathione depletion and reduce lipid peroxidation is suggestive
of an anti-oxidative mechanism. Intake of C. odorata extract as drug or as supplement in diet
may offer useful benefit in the protection of the liver. Availability of the plant makes the
herbal remedy a reasonable alternative to synthetic drugs that are neither available nor
cheap in the third world.

ACKNOWLEDGEMENT

The authors wish to acknowledge the technical assistance received from Mrs. P. Nc. Alisi
and Mr Emeka Asiwe of Silverpresh Laboratory. We also wish to thank Miss Juliet Opara for
good animal care

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