1 (Normal) 2.1g ± 0.15 0.42h ± 0.03 0.28f ± 0.02 1.2h ± 0.11 Group 2 (CCl4) 1.3a ± 0.21 0.22a ± 0.04 0.15a ± 0.03 3.8a ± 0.51 Group 3 1.6c ± 0.23 0.28c ± 0.05 0.20c ± 0.04 2.8b ± 0.41 Group 6 (Tert.) 1.8e ± 0.17 0.34f ± 0.06 0.24e ± 0.03

1. © by PSP Volume 33 – No 3. 2011 Advances in Food Sciences
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THE SCAVENGING CAPACITY AND
SYNERGISTIC EFFECT OF VITAMIN C,
GALLIC ACID AND TANNIC ACID ON CCl4-INDUCED
ACUTE HEPATIC INJURY IN EXPERIMENTAL RATS
Fouad A. Ahmed1
, Mohamed M. Rashed1
, Nasser S. A. M. Khalil2
,
Mohamed S. Masoud2
and Mahmoud A. A. M. Hashem2,
*
1 Faculty of Agriculture, Cairo University, Giza, Egypt
2 Regional Center for Food and Feed, Agricultural Research Center, Giza, Egypt
ABSTRACT
The biological activity of the natural antioxidant vi-
tamin C can be enhanced by the presence of some other
active natural antioxidants, such as gallic and tannic acid.
Since many of these natural antioxidants are consumed
together in foods, the potency for synergistic interactions
is high in the human diet. In the current study, the hepato-
protective activity of the natural antioxidants against CCl4-
induced acute hepatic injury by using rats (male albino)
was investigated. The assessment of liver function (AST
and ALT) results indicated that natural antioxidants sig-
nificantly elevated AST and ALT levels, but also creatinine
as kidney marker compared to controls. As liver markers,
significantly elevated levels of MDA as marker of LPO but
lowered levels of GST (p<0.05) were observed following
CCl4 administration. Quantitative and qualitative analysis
of CAT and SOD revealed lower activities of these anti-
oxidant enzymes on the liver of CCl4-administered rats.
An analysis of the isozyme pattern of these enzymes re-
vealed variations in relative concentration presumably due
to hepatotoxicity. Histopathological studies confirmed that
the natural antioxidants act as hepatoprotective substances.
KEYWORDS:
Antioxidant; VC; CCl4; scavenging capacity; synergistic effect
ABBREVIATIONS
Vitamin C (VC); gallic acid (GA); tannic acid (TA);
carbontetrachloride (CCl4); aspartate amino transferase
(AST); alanine amino transferase (ALT); malondialdehyde
(MDA); lipid peroxidation (LPO), glutathione-S-transferase
(GST); catalase (CAT); superoxide dismutase (SOD).
1. INTRODUCTION
Antioxidant compounds like GA, TA, VC and vita-
min E exist naturally in different plants and fruits. These
compounds possess the ability to reduce oxidative dam-
age, which is believed to cause many diseases including
cancer, cardiovascular diseases, cataracts, atherosclerosis,
diabetes, arthritis, immune deficiency diseases and aging
[1-3]. Recently, the ability of phenolic substances in-
cluding flavonoids and phenolic acids to act as antioxi-
dants have been extensively investigated [4]. Natural anti-
oxidants often exist in nature in combination, and such a
combination might act additively, and even synergistically
[5]. Carbon tetrachloride (CCl4), a potent hepatotoxic agent
is biotransformed to a trichloromethyl radical by the cyto-
chrome P450 system in liver microsomes and, consequently,
causes lipid peroxidation of membranes that leads to liver
injury [6-9].
2. MATERIALS AND METHODS
2.1 Materials
All natural antioxidants (GA, TA and VC) and CCl4
were purchased from Sigma-Aldrich (U.S.A) and of ana-
lytical grade.
2.2 Animals
45 Male albino rats weighing 150 ± 20 g were used
for this study. Animals were obtained from the animal
house in Veterinary Medicine, Cairo University, Egypt. The
rats were raised in the animals` house of Regional Center
for Food and Feed, Agriculture Research Center, Giza,
Egypt and kept in wire-bottomed stainless steel cages, main-
tained under standard conditions (12 h light/12 h dark cycle;
20-24 ºC; relative humidity (RH) not less than 55%). The
rats were acclimatized to laboratory conditions for 7 days
before commencement of experiments. During this period,

2. © by PSP Volume 33 – No 3. 2011 Advances in Food Sciences
160
rats were allowed free access of water, and “American
Institute of Nutrition” standard reference diet (AIN-93M;
Table 1) was used as basal diet ad libitum, as well as 1%
AIN-93M vitamin mixture and 3.5% AIN-93M mineral
mixture [10].
TABLE 1 - Formulation of the AIN-93M diets.
Ingredient (g/kg diet)
Corn starch 465.692
Casein (85% protein) 140
Dextrinized corn starch (90–94% tetra-saccharides) 155
Sucrose 100
Soybean oil (no additives) 40
Fiber 50
Mineral mix (AIN-93-MX) 35
Vitamin mix (AIN-93-VX) 10
Choline bitartrate (41.1% choline) 2.5
Values taken from [10]; AIN-93M: American Institute of Nutrition
standard reference diet.
2.3 Experimental design
This study has been designed to evaluate (in vivo) the
hepatoprotective activity of the natural antioxidants against
CCl4-induced acute hepatic injury using male albino rats.
Forty-five rats were divided into 9 groups (5 rats/group)
fed on basal diet, except group 1 (control) which received
daily 1 ml 0.9% saline/200 g B.W. orally and a single dose
of 1 ml corn oil/200 g B.W. intraperitoneally. All groups
received a single dose of 50% CCI4 in corn oil (l ml/200 g
B.W., intraperitoneally). From group 3 to group 9 orally re-
ceived daily doses of natural antioxidants of 1 ml/200g
B.W. as following:
((G3 V.C 6.08); G4 T.A 117.52); (G5 G.A 11.752); (G6
V.C 6.08, T.A 117.52 and G.A 11.752); (G7 V.C 15.2);
(G8 T.A 293.8); (G9 V.C 15.2 and T.A 293.8 mg/kg))
respectively.
2.4 Preparation of serum
After 2-weeks intoxication of CCl4, 45 rats were fasted
for l2 h, anaesthetized with CO2, and the blood samples
were withdrawn from retro-orbital venous plexus through
capillary tubes into a centrifuge tube. Samples were allowed
to coagulate at room temperature and then serum was sepa-
rated at 3000 rpm for 20 min, collected into sterilized Ep-
pendorf tubes and stored at 0 °C until analysis for AST,
ALT and creatinine.
2.5 Preparation of liver samples
Animals were killed by cervical decapitation and
their livers were rapidly removed, washed with ice-cold
0.9% NaCl (w/v) solution to remove the blood, and then
weighed by a digital top balance.
2.6 Preparation of liver homogenate
Liver homogenate was prepared as reported [11].
Dissected livers were excised, washed with ice-cold 0.9%
NaCl (w/v) solution to remove the blood, cut into small
pieces by fine scissors, and then homogenized (10% w/v)
separately in ice-cold 1.15% KCl-0.01M sodium phosphate
buffer, pH 7.4 with a Potter-Elvehjem glass homogenizer.
The homogenate was centrifuged at 5000g and 4 °C for
10 min. Supernatant of the liver homogenate was col-
lected into sterilized Eppendorf tubes and stored at 0 °C
until analyses of GST, CAT, SOD activity, and LPO level.
2.7 Histological technique
Livers were fixed in l0% formalin solution for 2 days,
washed in tap water, dehydrated in ascending grades for
ethyl alcohol and, finally, cleared with xylene and embed-
ded in paraffin wax. The paraffin blocks were cut into 5-
micron sections and stained with haematoxylin and eosin
[12] for histopathological examination by light microscopy.
2.8 Assays
2.8.1 Determination of aspartate amino transferase (AST) and
alanine amino transferase (ALT) as liver markers
AST and ALT were assayed by transaminase activity
proportional to the amount of oxalate or pyruvate formed
over a definite period of time, and measured by a reaction
with 2,4 dinitrophenylhydrazine (DNPH) under alkaline
conditions [13-15].
2.8.2 Determination of creatinine
Creatinine assay based on the reaction of creatinine
with sodium picrate [16]. Creatinine reacts with alkaline
picrate forming a red complex. The time interval chosen
for measurements avoids interferences from other serum
constituents. The intensity of the color formed is propor-
tional to the creatinine concentration in the sample [13-15].
2.8.3 Determination of glutathione-S-transferase (GST)
The reaction mixture consisted of 1.475 ml phosphate
buffer (0.1 M, pH 6.5), 0.2 ml reduced glutathione (l mM),
0.025 ml CDNB (l mM) and 0.3 ml of tissue homogenate
in a total volume of 2.0 ml. Reaction mixture without the
enzyme was used as blank. The changes in absorbance
were recorded at 340 nm within 3 min [17].
2.8.4 Superoxide dismutase (SOD)
SOD was estimated spectrophotometrically in a micro-
cuvette, and 1 ml of tris HCI buffer was added to 10 µl of
pyrogallol solution and 5 µl of sample (standard or liver
homogenate). Absorbance at 420 nm was measured after
1 and 2 min, and difference per min was calculated. Re-
agent blank was prepared by using distilled water instead
of the samples [18].
2.8.5 Determination of catalase (CAT)
The reaction mixture (1.5 ml) contained l.0 ml of
phosphate buffer (0.01 M, PH 7.0), 0.1 ml of the sample
(standard or liver homogenate) and 0.4 ml of H2O2 (0.2 M).
The reaction was stopped by addition of 2.0 ml of dichro-
mate-acetic acid reagent. Addition of reagent to H2O2 in-
stantaneously produced an unstable blue precipitate of per-
chromic acid. All the tubes were heated in a boiling wa-
terbath for exactly l0 min, the color of solution changed to

3. © by PSP Volume 33 – No 3. 2011 Advances in Food Sciences
161
stable green due to the formation of chromic acetate. After
cooling to room temperature, the optical density (O.D.)
was measured at 570 nm in a double-beam spectropho-
tometer (UV-150-02, Shimadzu, Japan). Reagent blank was
prepared by using double-distilled water instead of the
samples [19].
2.8.6 Lipid peroxidation (LPO)
LPO was measured in hepatic tissue homogenates
based on the formation of thiobarbituric acid (TBA) and
expressed as the extent of malondialdehyde (MDA) pro-
duction using a double-beam spectrophotometer (UV-
150-02 Shimadzu, Japan) [20].
2. 9. Statistical analysis
A randomized complete block design was used for
analysis of all data, with three replications for each para-
meter. The treatment means were compared by least sig-
nificant differences (L.S.D.) test as reported [21, 22].
3. RESULTS
3.1 Effect of natural antioxidant on body weight, liver weight
and ratio of hepatotoxic rats
Treatment with binary antioxidant mixture (group 9;
VC 15.2 and TA 293.8 mg/kg B.W.) could significantly
decrease the liver weight when compared with CCl4-
treated group (Table 2).
3.2 Effect of natural antioxidant on serum ALT and AST of
hepatotoxic rats
Treatment of the hepatoprotective rats with binary an-
tioxidant mixture, (group 9; VC 15.2 and TA 293.8 mg/kg
B.W.) and tertiary antioxidant mixture (group 6; VC 6.08,
TA 117.52 and GA 11.752 mg/kg B.W.) significantly de-
creased the levels of ALT and AST enzymes when com-
pared with CCl4 group (Table 3).
TABLE 2 - Effect of natural antioxidant on body weight, liver weight and ratio of hepatotoxic rats.
Groups Rat weight (g) Liver weight (g) Liver/Rat ratio Relative (%)
Group 1(Normal) 163.1d
± 29.81 6.0c
± 0.59 3.68 100.0
Group 2 (CCl4) 190.0a
± 14.36 9.0a
± 0.73 4.74 129
Group 3 184.6b
± 25.03 7.3bc
± 1.00 3.95 107
Group 4 185.0ab
± 20.96 7.5bc
± 1.13 4.05 110
Group 5 194.9a
± 14.55 7.8b
± 1.21 4.00 109
Group 6 (Tert.) 179.0c
± 17.81s 7.0bc
± 1.05 3.91 106
Group 7 186.2ab
± 22.07 7.6b
± 1.48 4.08 111
Group 8 169.2cd
± 10.23 6.8bc
± 0.44 4.02 109
Group 9 (Binary) 183.0bc
± 8.80 7.1bc
± 0.46 3.88 105
LSD (0.05) 25.00 1.31
Each value was obtained by calculating the average of 3 experiments ± S.D. The superscript letters indicated statistically significant differences (P <0.05).
TABLE 3 - Effect of natural antioxidant on serum ALT and AST of hepatotoxic rats.
Group
ALT
(U/I)
AST
(U/I)
Creatinine
(mg/dl)
Group 1 (Normal) 40.0g
± 6.58 80.0i
± 6.93 0.3d
± 0.06
Group 2 (CCl4) 105.0a
± 22.79 250.0a
± 61.30 0.8a
± 0.20
Group 3 75.0b
± 21.83 195.0b
± 58.27 0.4c
± 0.12
Group 4 65.0d
± 11.61 165.0e
± 55.86 0.5b
± 0.12
Group 5 70.0c
± 32.10 180.0c
± 60.73 0.4c
± 0.15
Group 6 (Tert.) 55.0ef
± 22.41 135.3g
± 32.02 0.5b
± 0.00
Group 7 70.0c
± 23.95 170.0d
± 60.54 0.5b
± 0.01
Group 8 59.0e
± 17.69 150.0f
± 24.41 0.4c
± 0.12
Group 9 (Binary) 52.0f
± 26.81 120.0h
± 35.84 0.5b
±0.12
LSD (0.05) 4.09 2.52 0.02
Each value was obtained by calculating the average of 3 experiments ± S.D. The superscript letters indicated statistically significant differences
(P <0.05).

4. © by PSP Volume 33 – No 3. 2011 Advances in Food Sciences
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TABLE 4 - Effect of natural antioxidant on liver homogenate SOD, CAT, GST activity and LPO level of hepatotoxic rats.
Group
SOD (U/mg protein) CAT (µmol/mg protein)
GST (mM/min/mg pro-
tein)
LPO (nmol/mg
protein)
Group 1 (Normal) 45.0a
± 6.97 14.0a
± 0.75 27.8a
± 2.80 3.6f
± 0.12
Group 2 (CCl4) 12.0h
± 6.65 4.0h
± 0.60 11.0f
± 3.12 11.0a
± 0.64
Group 3 16.0g
± 8.35 5.0g
± 0.43 13.5e
± 7.19 6.0b
± 0.46
Group 4 19.0e
± 13.49 5.9e
± 0.12 15.0d
±8.60 5.0c
± 0.12
Group 5 18.0f
± 6.65 5.5f
± 0.00 14.7d
± 0.29 5.2c
± 0.05
Group 6 (Tert.) 23.0c
± 3.37 7.0c
± 0.23 16.8c
± 2.01 4.0e
± 0.23
Group 7 19.0e
± 18.30 5.7ef
± 0.35 15.0d
± 1.85 4.4d
± 0.35
Group 8 21.0d
± 5.00 6.5d
± 0.11 16.5c
± 1.69 4.5d
± 0.12
Group 9 (Binary) 25.0b
±8.40 8.0b
± 0.12 18.5b
± 1.36 4.0e
± 0.12
LSD (0.05) 0.75 0.35 0.51 0.36
Each value was obtained by calculating the average of 3 experiments ± S.D. The superscript letters indicated statistically significant differences (P
<0.05).
3.3 Effect of natural antioxidant on creatinine level
Groups 4, 6, 7 and 9 are not significantly different
from each other with regard creatinine levels in serum
(Table 3) but also groups 3, 5, and 8 are not significantly
different from each other, though they are significantly
different from the previously mentioned groups
3.4 Effect of natural antioxidant on liver homogenate SOD
activity of hepatotoxic rats
Data presented in Table 4 show a significant decrease
in the activity of SOD enzyme of all treatment rat groups
when compared with CCl4 group. On the other hand, the
binary antioxidant mixture treatment (group 9) recorded
the highest significant SOD activity compared to other
groups, and these results are similar with reported data [23].
3.5 Effect of natural antioxidant on liver homogenate CAT
activity of hepatotoxic rats
Data in Table 4 show that group 9 (binary antioxidant
mixture) recorded the highest significant increase in liver
homogenate CAT activity compared to the other treat-
ments.
3.6 Effect of natural antioxidant on liver homogenate GST
activity of hepatotoxic rats
A significant decrease in GST activity of treatment
rats could be observed, and group 9 (binary antioxidant
mixture) evidenced the highest value in GST activity com-
pared to other groups.
3.7 Effect of natural antioxidant on liver homogenate LPO
contents of hepatotoxic rats
Group 6 (tertiary antioxidant mixture) and group 9
(binary antioxidant mixture) showed a significant increase
in liver homogenate MDA level compared to other groups
(Table 4). These results are in agreement with reported
data [24].
3.8 Histopathological examination results of different groups
Sections in the liver tissue from untreated rats group 1
and group 2 (CCl4 group) showed that there is normal
appearing in hepatic lobular architectures, with normal vas-
culature, and portal areas. Microscopical examination of
liver sections from control group 1 revealed a normal histo-
logical structure of hepatic lobule which consists of central
vein and concentrically arranged hepatocytes (Fig. 1).
Meanwhile, examined sections from group 2 showed
Kupffer cells activation, centrilobular hepatic lipidosis, cy-
tomegally of hepatocytes with foamy cytoplasm and pykno-
tic nuclei. Additionally, all examined sections from this
group revealed congestion of hepatoportal blood vessels
and portal infiltration with leucocytic inflammatory cells.
However, liver sections of rats from group 3 revealed a
marked improvement in the histopathological picture.
Most examined sections revealed
Kupffer cells activation and granularity of the cyto-
plasm of hepatocytes. On the other hand, microscopical
examination of group 4 livers showed focal lipidosis,
hepatocellular vacuolization and portal infiltration with
leucocytes. Meanwhile, examined sections from group 5
and some sections from group 6 revealed improvements
in the histopathological picture, as those sections showed
no histopathological alterations, except vacuolizations of
the cytoplasm of some hepatocytes. But other sections from
group 6 showed centrilobular hepatic lipidosis, cytomegally
of hepatocytes with foamy cytoplasm and pyknotic nuclei.
Moreover, liver of rats from groups 7 and 8 revealed cen-
trilobular hepatic lipidosis, cytomegally of hepatocytes
with foamy cytoplasm and pyknotic nuclei as well as
sinusoidal leucocytosis. Conversely, sections from group 9
showed no histopathological alterations, except vacuoliza-
tions of the cytoplasm of hepatocytes (Fig. 1).

5. © by PSP Volume 33 – No 3. 2011 Advances in Food Sciences
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FIGURE 1 - Liver of rats from group 1 (A) showing the normal histological structure of hepatic lobule; group 2 (CCl4) (B) showing centri-
lobular hepatic lipidosis; group 3 (C) showing Kupffer cells activation and granularity of the cytoplasm of hepatocytes; group 4 (D) showing
focal lipidosis, hepatocellular vacuolization and portal infiltration with leucocytes; group 5 (E) showing vacuolizations of the cytoplasm of
hepatocytes; group 6 (F) showing vacuolizations of the cytoplasm of hepatocytes; group 7 (G) showing centrilobular hepatic lipidosis, cy-
tomegally of hepatocytes with foamy cytoplasm and pyknotic nuclei; group 8 (H) showing centrilobular hepatic lipidosis, cytomegally of
hepatocytes with foamy cytoplasm and pyknotic nuclei as well as sinusoidal leucocytosis; group 9 (I) showing vacuolizations of the cytoplasm
of hepatocytes (H & E X 400).
A B
C D
E F
G H
I

6. © by PSP Volume 33 – No 3. 2011 Advances in Food Sciences
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4. DISCUSSION
The liver is the largest organ in the vertebrate body,
and the major site of xenobiotic metabolism and excre-
tion. Liver injury can be caused by toxic chemicals, drugs
and virus infiltration from ingestion or infection. Carbon
tetrachloride (CCl4) has been widely used in animal mod-
els to investigate chemical toxin-induced liver damage.
The most remarkable pathological characteristics of CCl4-
induced hepatotoxicity are fatty liver, cirrhosis and necro-
sis, which have been thought to result from the formation
of reactive intermediates such as trichloromethyl free
radicals (CCl.
3) metabolized by the mixed function cyto-
chrome P450 in the endoplasmic reticulum [25]. Usually,
the extent of hepatic damage is assessed by increased
levels of cytoplasmic enzymes ALT and AST, thus lead-
ing to leakage of large quantities of enzymes into the blood
circulation. This was associated by massive centrilobular
necrosis, ballooning degeneration and cellular infiltration
of the liver [26]. The present study revealed a significant
increase in the levels of serum creatinine too. Antioxidant
activity has been hypothesized that one of the principal
causes of CCl4-induced liver injury is formation of lipid
peroxides by free radical derivatives of CCl4 (CCl3). From
the tables, it is clear that the binary antioxidant mixture
shows dose-dependent activity among concentrations and
combinations of natural antioxidants (VC, GA and TA).
The binary antioxidant mixture as well as the greater syn-
ergistic effect and free radical scavenging activities might
contribute to the hepatoprotective effects. Thus, the anti-
oxidant activity or the inhibition of the generation of free
radicals is important in the protection against CCl4-induced
hepatopathy [27]. The body has an effective defense mecha-
nism to prevent and neutralize the free radical-induced
damage. This is accomplished by a set of endogenous anti-
oxidant enzymes, such as SOD, CAT and GPO. These
enzymes constitute a mutually supportive team of defense
against ROS [28]. The balance between ROS production
and these antioxidant defenses may be lost, and ‘oxidative
stress’ results, which through a series of events deregu-
lates the cellular functions leading to hepatic necrosis. The
increased activity of SOD, CAT and GST observed point
out the hepatic damage in the rats administered with CCl4.
Administration of natural antioxidant, especially binary
antioxidant mixture (group 9), attenuated the increased
levels of the serum enzymes, produced by CCl4 and caused
a subsequent recovery towards normalization when com-
pared with control group. The hepatoprotective effect of the
binary natural antioxidant mixture was further concluded
by the histopathological examinations. The results show
that both VC and TA at different dose levels offer hepato-
protection, but binary antioxidant mixture (group 9) is more
effective than all other groups, and this may be due to its
synergistic effect. The level of lipid peroxide is a measure
of membrane damage and alterations in structure and func-
tion of cellular membranes. The increase in MDA levels in
liver suggests enhanced lipid peroxidation leading to tissue
damage and failure of the antioxidant defense mecha-
nisms to prevent the formation of excessive free radicals.
The results are expected to be a useful guide in the formu-
lation and production of functional food products that
have high antioxidant potency. As a conclusion from the
present work, use of natural antioxidant mixture might be
useful for the hepatoprotective effects against liver oxida-
tive damage.
REFERENCES
[1] Lee, K. G.; Mitchell, A. E. and Shibamoto, T. (2000). Deter-
mination of antioxidant properties of aroma extracts from
various beans. Journal of Agricultural and Food Chemistry,
48: 4817–4820.
[2] Middleton, E.; Kandaswamy, C. and Theoharides, T. C.
(2000). The effects of plant flavonoids on mammalian cells:
Implications for inflammation, heart disease, and cancer.
Pharmacological Reviews, 52: 673–751.
[3] Pietta, P.; Simonetti, P. and Mauri, P. (1998). Antioxidant ac-
tivity of selected medicinal plants. Journal of Agricultural
and Food Chemistry, 46: 4487–4490.
[4] Rice-Evans, C. A.; Miller, N. J. and Paganda, G. (1996). Struc-
ture-antioxidant activity relationships of flavonoids and pheno-
lic acids. Free Radical Biology and Medicine, 20: 933–956.
[5] Fuhrman, B.; Volkova, N.; Rosenblat, M. and Aviram, M.
(2000). Lycopene synergistically inhibits LDL oxidation in
combination with vitamin E, glabridin, rosmarinic acid, car-
nosic acid, or garlic. Antioxidants and Redox Signaling, 2:
491–505.
[6] Recknagel, R. O. (1983). Carbon tetrachloride hepatotoxic-
ity: status quo and future prospects. Trends Pharmacol. Sci.,
4: 129–131.
[7] Slater, T. F. (1984). Free radical mechanism in tissue injury,
Biochem. J., 222: 1–15.
[8] McCay, P. B.; Lai, E. K.; Poyer, J. L.; DuBose, C. M. and
Janzen, E. G. (1984). Oxygen- and carbon-centered free radi-
cal formation during CCL4 metabolism: observation of lipid
radicals in vivo and in vitro. J. Biol. Chem., 259: 2135–2143.
[9] Jayakumar, T.; Ramesh, E. and Geraldine, P. (2006). Antioxi-
dant activity of the oyster mushroom, Pleurotus ostreatus, on
CCl4-induced liver injury in rats. Food and Chemical Toxi-
cology, 44: 1989–1996
[10] Reeves, P. G.; Nielsen, F. H. and Fahey, G. C. (1993). Jr.
AIN-93 purified diets for laboratory rodents: final report of
the American Institute of Nutrition ad hoc writing committee
on the reformulation of the AIN-76A rodent diet. J. Nutr.,
123: 1939-1951.
[11] El-Demerdash, F. M.; Yousef, M. I. and Abou El-Naga, N. I.
(2005). Biochemical study on the hypoglycemic effects of on-
ion garlic in alloxan-inauced diabetic rats. Food Chem. Toxic.,
43(1): 57– 63.
[12] Banchroft, J. D.; Stevens, A. and Turner, D. R. (1996). Theory
Practice of Histological Techniques. Fourth ed. Church, Living
stone, New York, London, San Francisco, Tokyo, 125 p.
[13] Murray, R. and Kaplan A. (1984). Alanine amtnotransferase.
Clin Chern The CV. Masby Co St Louis. Toronto. Princeton;
pp. 1088-1126.

7. © by PSP Volume 33 – No 3. 2011 Advances in Food Sciences
165
[14] Young, D. S. (1995). Effects of drugs on Clinical Lab. Tests,
4th
ed AACC.
[15] Young, D. S. (2001). Effects of disease on Clinical Lab.
Tests, 4th
ed AACC.
[16] Burtis, A. (1999). Textbook of Clinical Chemistry, 3rd ed
AACC.
[17] Habig, W. R.; Pbst, M. J. and Jakpoly, W. B. (1974).
Glutathion-S-transferase. A first enzymatic step in mercaturic
acid form, J. Biol. Chem., 249: 7130.
[18] Marklund, S. and Marklund, G. (1974). Involvement of the
Superoxide anion radical in the autoxidation of pyrogallol
and a convenient assay for superoxide dismutase. Eur. J. Bio-
chem., 47: 469-474.
[19] Sinha, A. K. (1972). Colorimetric assay of catalase. Analyt.
Biochem., 47: 389.
[20] Uchiyama, M. and Mihara, M. (1978). Determination of
Malonaldehyde precursor in tissues by thiobarbituric acid.
Anal. Biochem., 86: 27l-278.
[21] Snedecor, G. A. and Cochran, W. G. (1976). Statistical
Method. Iowa State Univ. Press, Ames. 289 p.
[22] Mstat-c. (1989). Users guide: a microcomputer program for
the design, management and analysis of agronomic research
experiments. Michigan University, East Lansing, MC, USA.
152 p.
[23] Chidambara M. K. N.; Jayaprakasha, G. K. and Singh, R. P.
(2002). Studies on antioxidant activity of pomegranate (Pu-
nica granatum) peel extract using in vivo models. J. Agric.
Food Chem., 50: 4791.
[24] Teselkin, Y.O.; Babenkova, I.V.; Kolhir, V. K.; Baginskaya,
A. I.; Tjukavkina, N. A.; Kolesnik, Y. A.; Selivanova, I. A.
and Eichholz, A. A., (2000). Dihydroquercetin as a means
antioxidative defence in rats with tetrachloromethane hepato-
toxic. Phytother. Res., l4 (3): 160.
[25] Recknagel, R. O.; Glende J. E. A.; Dolak, J. A. and Waller, R.
L. (1989). Mechanisms of carbon tetrachloride toxicity. Phar-
macol. Therapeut., 43: 139–154.
[26] Plaa, G. L. and Charbonneau, M. (1989). Detection and
evaluation of chemically induced liver injury. In: Principles
and Methods of Toxicology. (Hayes, A.W. Ed.) Raven Press,
New York, pp. 399–628.
[27] Castro, J. A.; Ferrya, G. C.; Castro, C. R.; Sasame, H.; Fenos,
O. M. and Gillette, J. R., (1974). Prevention of carbon tetra-
chloride-induced necrosis by inhibitors of drug metabolism.
Further studies on the mechanism of their action. Biochem.
Pharmacol., 23: 295–302.
[28] Venukumar, M. R. and Latha, M. S. (2002). Antioxidant ac-
tivity of Curculigo orchioides in carbon tetrachloride-induced
hepatopathy in rats. Indian J. Clin. Biochem., 17: 80–87.
Received: April 26, 2011
Accepted: June 14, 2011
CORRESPONDING AUTHOR
Mahmoud A. A. M. Hashem
Regional Center for Food and Feed
Agricultural Research Center
Giza
EGYPT
Phone: +002 0105117017
E-mail: mahmoud5000@gmail.com
AFS/ Vol 33/ No 3/ 2011 – pages 159 - 165