JKAU: Sci., Vol. 22 No. 1, pp: 239-248 (2010 A.D. / 1431 A.H.); DOI: 10.4197 / Sci. 22-1.16
Antioxidant Properties of Thymol
and Butylated Hydroxytoluene in Carbon
Tetrachloride – Induced Mice Liver Injury
Abdulrahman L. Al-Malki
Department of Biochemistry, Faculty of Science,
King Abdulaziz University, Jeddah, Saudi Arabia (KSA)
[email protected]
Abstract. Thymol and Butylated hydroxytoluene (BHT) were tested
for their ability to inhibit carbon tetrachloride (CCl4)-induced lipid
peroxidation in mice liver. To achieve this purpose, four groups of
mice were tested, GP I: Animals served as normal control, GP II:
Animals injected i.p. with a single dose of CCl4 (0.5 ml/kg B.W), GP
III and IV: Animals pretreated by oral administration of thymol or
BHT for 6 days, then injected with a single dose of CCl4. After the
experimental period, blood was collected, sera separated for ALT
assay. The antioxidant activities of the enzymes; superoxide dismutase
(SOD), glutathione peroxidase (GS-PX) together with malondialdhyde
level were assayed in liver tissues. Results obtained showed that, the
antioxidant enzyme activities were significantly decreased while the
level of Malondialdhyde (MDA), the indicator of lipid peroxidation,
and the activity of alanine aminotransferase (a liver function test) were
significantly increased following i.p injection with single sub-lethal
hepatotoxic dose of CCl4 as compared to control. Pretreatment of
CCl4-intoxicated mice with thymol or BHT for 6 days showed a
significant increase in the activities of antioxidant enzymes with
simultaneous significant decrease in the level of MDA and ALT as
compared to CCI4-treated rats. BHT was a little more potent than
thymol in its action. Histopathological examination revealed a
necrotic lesion in liver of mice injected with CCl4. Pretreatment with
thymol or BHT ameliorates the deleterious effect of CCl4. This is
confirmed by the normal appearance of liver tissue. It was speculated
that thymol and BHT exert their effects by decreasing lipid
peroxidation and enhancing the activities of antioxidant enzymes. For
this reason, thymol could be used as hepatoprotective agent with free
medication side effects.
239
240
Abdulrahman L. Al-Malki
Introduction
Certain toxicants have been characterized to be bioactivated by cytochrome
P450 (CYP450). For example, carbon tertrachloride (CCl4) causes liver damage
following the cleavage by CYP450 to form the trichloromethyl free radical.
This radical quickly adds molecular oxygen to form the trichloromethyl peroxyl
radical[1]. Abstraction of hydrogen atoms from unsaturated lipids by such
radicals creates carbon-centered lipid radicals[2].
These lipid radicals quickly add molecular oxygen to form lipid peroxyl
radicals, thereby initiating the process of lipid peroxidation. Unless scavenged
by vitamin E or other radical scavengers, these lipid peroxyl radicals in turn
abstract hydrogen atoms from other lipid molecules, thereby propagating the
process of lipid peroxidation[3].
Biphenyl dimethyl dicarboxylate (DDB) is a synthetic analogue of
schizandrin C, one of the compounds isolated from Fmctus Schizandrae, which
is a traditional Chinese medicine[4]. DDB was found to markedly improve
impaired liver functions such as the elevated serum alanine aminotransferase
(ALT), bilirubin and ά-fetoprotein in hepatitis patients[5]. Moreover, DDB was
reported to efficiently protect the hepatocytes against CCl4 and D-galactosamine
induced damages[5]. On the other hand, Silymarin is the commercial name for a
flavonoid extract from the seeds of the milk thistle silybum marianum[5].
Butylated hydroxytoluene (BHT) has been shown to protect experimental
animals against various hepatotoxins such as allyl alcohol[6]. With respect to
BHT, it was able to prevent chemically induced tumours or acute toxic effects
of some chemicals.
Thymol (p-methyl-isopropyl-phenol) is the main constituent of the oils of
Thymus vulgaris[7]. It possesses antioxidant properties and has been suggested
as a natural replacement for synthetic antioxidant additives. In the present study,
the aim was to compare between the hepatoprotective effects of thymol or BHT
on CCl4 induced lipid peroxidation in mice.
Materials and Methods
1) Animals
Fourty male Swiss albino mice, weighing 25-27 g, were obtained from the
Animal House, King Fahd Medical Research Center at King Abdul Aziz
University, Jeddah, Saudi Arabia. The animals were kept under standard
conditions throughout the study. Water and food were given ad libitum. The
animals were housed for one week prior to the experiment and were randomly
divided into 4 groups (each contains 10 mice).
Group (I): Animals served as normal control group.
Antioxidant Properties of Thymol and Butylated Hydroxytoluene In Carbon …
241
Group (II): Animals were injected i.p (0.5 ml/kg B.W.) with a single dose
of 0.2% CCl4 in corn oil. After 24 hours, blood was collected from orbital sinus
under light ether anesthesia.
Group III: Animals were orally administrated 300 mg of thymol/kg in corn
oil through gastric tube daily for 6 days then injected i.p with single dose of
0.2% CCl4.
Group (IV): Animals were orally administrated 200 mg of BHT in 1 ml corn
oil/kg B.W through gastric tube daily for 6 days then injected i.p with a single
dose of 0.2% CCl4 as above.
After the above mentioned treatments blood was collected from orbital sinus
under light anaesthesia in centrifuge tubes, the abdomen was excised
immediately and the liver was removed by dissection, washed in ice-cold
isotonic saline, then blotted between two filter papers.
2- Methods
Separated serum was used for immediate determination of alanine
aminotransferase (ALT) using commercial reagent kit from Randox laboratories
Ltd., United Kingdom.
Each liver was homogenized in ice-cold 0.I M potassium phosphate buffer,
pH 7.4. Liver homogenates were used to measure the activities of the
antioxidant enzymes superoxide dismutase and glutathione peroxidase in
addition to malondehydyde (MDA) and protein content .
Glutathione peroxidase was assayed in liver homogenate by the method
described by Letteron et al., (1990)[8]. One unit of glutathione peroxidase
activity is defined as the amount of enzyme which oxidizes 1µmol of NADPH
per min and the specific activity is expressed as µmol/min/mg protein.
Superoxide dismutase was determined by the method of Liu (1989)[9].
One unit SOD is defined as the amount of enzyme which catalyzes a
decrease in the rate of nitrite formation to 50% of its maximal value.
Lipid peroxide level in the liver homogenate was estimated by the
production of thiobarbituric acid reactive substances according to the method
described by McCay et al., (1984)[10]. Protein content was measured by the
method of Meister and Andersen (1983)[11]. using Coomassie brilliant blue G250. Calibration curve was prepared with bovine serum albumin.
Histological Examination
Fragments of liver were immediately removed and immersed in 10%
formalin for histopathological study according to Miguez et al., (1994)[12]. The
formalin was dehydrated with ethyl alcohol, cleared in terpineal, embedded in
Abdulrahman L. Al-Malki
242
paraffin wax and transversely sectioned at thickness of 5 µm then stained with
Mayer's haemtoxylin and eosin.
Statistical Analysis
Results are presented as mean ±SD. Statistical comparison between each
two groups was performed using Student's t-test with P < 0.05 as considered
significant for all analyses. Statistical software system SPSS version 13 was
used for these calculations.
Results
The results of hepatoprotective effect of thymol and BHT on CCI4
intoxicated mice are shown in Table 1. In the CCI4 intoxicated group ALT
activity was significantly increased to 64.0 IU/ml as compared to only
28.4IU/ml in normal control group. The elevated activity of serum ALT was
significantly reduced in groups treated either with BHT or thymol (p<0.001)
compared to CCl4 injected group.
Table 1. The antioxidant liver enzyme activities; superoxide dismutase (SOD), glutathione
peroxidase (GPX), lipid peroxide product (Malendialdlyde and serum alanine
aminotransferase enzyme of all studied groups (Mean+SD).
Animal groups
Parameters
SOD
MU/mg protein
Mean+SD
P1 value
P2 value
P3-value
GSHPx
 mol/min/mg
protein)
Mean+SD
P1 value
P2 value
P3-value
Malandialdhyde
(Mmol/mg/protein)
Mean+SD
P1 value
P2 value
P3-value
Serum ALT
IU/ml
P1 value
P2 value
P3-value
Normal Control
group
CCI4 group
Thymol treated
group
BHT
Treated group
226.8+ 13.8
-----
117.5+ 34.0
<0.001
---
224.7+ 25.8
N.S
<0.001
185.3+ 23.2
<0.001
0.01
0.01
9772.4+ 2667.7
2192.0+ 146.6
8582.6+ 1482.5
4281.3+ 935.3
-----
<0.001
----
N.S
<0.001
<0.001
0.01
0.05
1.31+ 0.14
-----
4.14+ 0.57
<0.001
---
1.84+ 0.27
<0.001
<0.001
2.23+ 0.32
<0.001
0.01
0.05
28.4+4.56
-----
64.0+7.86
P<001
---
39.9+7.14
0.001
0.001
46.6+5.08
0.001
0.05
0.05
P1 – comparison to normal control
P2 compred to CCl4
P3- thymol versus BHT
N.S= non significant
Antioxidant Properties of Thymol and Butylated Hydroxytoluene In Carbon …
243
Results of this study clearly revealed increased levels of lipid peroxidation
in CCI4 intoxicated rats compared to the normal control group, while treatment
with either thymol or BHT significantly elevated the activities of GSPX and
SOD content in treated groups as compared to CCI4 intoxicated group. Thymol
showed maximum protection followed by BHT.
Histopathological studies of liver section of CCl4 intoxication rats showed
massive fatty changes necrosis and broad infiltration of the lymphocytes and
loss of cellular boundaries. Thymol and BHT treated rats showed more normal
lobular pattern with mild degree of fatty changes, necrosis and lymphocyte
infiltration almost comparable to control (Fig. 1).
a) control group
b) CCl4 group
c) CCl4 + Thymol treatment
d) CCl4+ BHT treatment
Fig. 1. Light micrographs of mice liver treated with CC14, without and with treatment of
thymol or BHT compared with normal control. Representative sections from (a)
group 1 normal control liver (b) group 2, mice treated with CC14, showing extensive
hepatocellular necrosis; and (c) group 3, pretreated mice with thymol (d), group 4
pretreated mice with BHT . (magnification × 250).
Discussion
This study shows that pretreatment of rats with Thymol or BHT inhibited
CCl4-induced hepatic damage and lipid peroxidation. Liver damage was
evaluated by measurement of serum level of alanine aminotransferase (ALT)
while the level of Malondialdhyde was used as an indicator of lipid
peroxidation.
244
Abdulrahman L. Al-Malki
Moreover, pretreatment of CCl4-injected rats with Thymol or BHT
improved the activities of antioxidant enzymes (glutathione peroxidase and
superoxide dismutase) compared to CCl4-intoxicated group. Pretreatment of
CCl4-intoxicated group with Thymol exerted the strongest action inducing a
highly significant decrease in MDA and a highly significant increase in the
activities of antioxidant enzymes compared with the CCL4-intoxicated group.
Ip et al.,[7]. have shown that CCl4 treatment increased both plasma ALT and
sorbitol dehydrogenase activities in mice and pretreating mice with thymol
significantly inhibited the CCl4-induced increase in plasma ALT activity.
Previous studies have shown that, ALT activity was increased in mice after the
administration of CC14[13].
Pretreatment of mice with silymarin afforded protection against the
hepatotoxic effects of CCl4. Moreover previous study, have shown that, in dogs,
CCl4 induced a significant increase in the ALT and aspartate aminotransferase
(AST) activity, and histological lesions in the liver. The protective effects of
silymarin were manifested by the significantly lower ALT and AST activities
and by the insignificantly lower extent of lesions in liver parenchyma as
compared to the CCl4-intoxicated group.
Our results also showed that pretreatment of CCl4-intoxicated group with
thymol or BHT improved the activities of antioxidant enzymes (glutathione
peroxidase and SOD but decreased the level of the lipid peroxides indicator,
MDA as compared to CCl4- intoxicated rats.
In a previous study, it has been demonstrated that the suppression of
catalase activity was responsible for the formation of superoxide anion radical
via suppression of superoxide dismutase activity[14]. Glutathione reductase
catalyzes the reduction of GSSG to GSH using NADPH as a source of
electrons[15].
GSH plays an important role in many biological phenomena, including the
protection against toxicities by reactive oxygen compounds and free radicals[16].
Taken together, these observations, therefore, suggest that thyme or BHT may
decrease lipid peroxidation by increasing the activities of antioxidant enzymes
which in turn scavenge free radicals. The protection effects of Thyme was
found to be more effective than BHT effect against CCl4-induced lipid
peroxidation.
In commitment with results, Fu & Liu[12], have reported that thymol
markedly decreased the level of the lipid peroxidation indicator, MDA of rat
hepatocytes. In addition, the damage of the cell surfaces of the hepatocytes was
also reduced as seen under a scanning electron microscope.
Antioxidant Properties of Thymol and Butylated Hydroxytoluene In Carbon …
245
Thymol also inhibited the metabolic activation of CCl4, in vivo as suggested
by a decreased covalent binding of CCl3 metabolites to hepatic lipids in vivo.
Decreased metabolic activation of CCl4 by cytochrome P450 would decrease
the initial formation of the trichloromethyl free radical and therefore decrease
the initiation of lipid peroxidation[17]. A major defense mechanism involves the
antioxidant enzymes including SOD and glutothoine peroxidase which convert
active oxygen molecules into non–toxic compounds hepatoprotective effect of
thymol were compared with BHT where an active antioxidant damage was
assessed by biochemical study of ALT and histopathological examinations.
The toxic metabolite CC13 radical is produced which further reacts with
oxygen to give the chlanomethyl peroxy radical. Cytochrome P450 is the
enzyme responsible for this conversion. This radical binds covalently to
macromolecules and causes peroxidative degradation of lipid membrane of a
dispose tissue in this view the reduction of ALT level by the thyme is an
indication of stabilization of plasma membrane as well as of hepatic tissue
damage caused by CC14. This effect agrees with Thabrew (1987) and Zanetti
(1979)[18,19].
These results indicated that thymol and BHT were of beneficial effects on
both damaged and normal hepatocytes. They prevented partially lipid
peroxidation and improved the activities of antioxidant enzymes of both normal
and CCl4-intoxicated rats. Moreover, this study showed that the protection
effects of thymol was more effective than BHT against CCl4-induced lipid
peroxidation.
The biochemical study was confirmed by histopathological study that
revealed the hepatic damage and necrosis was observed in CCl4 group
compared with normal mice, pretreatment with either thymol or BNT improve
the damage effect of CCl4 but thymol effect is more than BHT.
In summary, this study suggests that administration of thymol or BHT
significantly ameliorates CC14 hepatotoxicity in mice. The compound may be
protecting the liver by preventing peroxidation of lipids of the endoplasmic
reticulum. However, the possibility that thymol might suppress the cytochrome
P-450 mediated metabolic activation of CC14 itself can not be ruled out.
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Antioxidant Properties of Thymol and Butylated Hydroxytoluene In Carbon …
247
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