JKAU: Sci., Vol. 22 No. 1, pp: 239-248 (2010 A.D. / 1431 A.H.); DOI: 10.4197 / Sci. 22-1.16

239

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)

alalmalki@kau.edu.sa

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.

Abdulrahman L. Al-Malki 240

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 G-250. 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

Normal Control

group CCI4 group

Thymol treated

group

BHT

Treated group

SOD

MU/mg protein

Mean+SD

226.8+ 13.8

117.5+ 34.0

224.7+ 25.8

185.3+ 23.2

P1 value

P2 value

P3-value

---

---

<0.001

---

N.S

<0.001

<0.001

0.01

0.01

GSHPx

mol/min/mg

protein)

Mean+SD

9772.4+ 2667.7

2192.0+ 146.6

8582.6+ 1482.5

4281.3+ 935.3

P1 value

P2 value

P3-value

---

---

<0.001

----

N.S

<0.001

<0.001

0.01

0.05

Malandialdhyde

(Mmol/mg/protein)

Mean+SD

1.31+ 0.14

4.14+ 0.57

1.84+ 0.27

2.23+ 0.32

P1 value

P2 value

P3-value

---

---

<0.001

---

<0.001

<0.001

<0.001

0.01

0.05

Serum ALT

IU/ml

28.4+4.56

64.0+7.86

39.9+7.14

46.6+5.08

P1 value

P2 value

P3-value

---

---

P<001

---

0.001

0.001

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.

Abdulrahman L. Al-Malki 244

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.

References

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Antioxidant Properties of Thymol and Butylated Hydroxytoluene In Carbon … 247

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