In this in vitro model DHTQ was more potent in comparison with TQ and butylated ... hydroxytoluene (BHT) on the non-enzymatic lipid peroxidation of mice liver.
Vol. 47, No. 1, January 1999
BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL pages 153-159
T H Y M O Q U I N O N E PROTECTS A G A I N S T C A R B O N T E T R A C H L O R I D E HEPATOTOXICITY IN MICE VIA AN ANTIOXIDANT MECHANISM Mahmoud N. Nagi, Khurshid Alam, Osama A. Badary, Othman A. A1-Shabanah,Hussein A. A1-Sawaf, and Abdullah M. A1-Bekairi Department of Pharmacology, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh 11451, Saudi Arabia ReceivedJuly 6, 1998. Accepted September24, 1998.
Summary. Thymoquinone (TQ) is the major active component of the volatile oil of Nigella sativa seeds. The effects of TQ on carbon tetrachloride (CC14)-induced hepatotoxicity was investigated in male Swiss albino mice. Carbon tetrachloride (20 gl/Kg, i.p.) injected into mice, induced damage to liver cells and was followed by the increase in serum alanine aminotransferase (ALT) activity after 24 h. Oral administration of TQ in a single dose (100 mg/Kg) resulted in significant (p 99% pure. Animal treatment: Male Swiss albino mice, weighing 22-25 g, were divided into groups of six and allowed ad libitum access to food (Purina chow) and tap water. CC14 (20 gl/Kg) was administered intraperitoneally (i.p.) in corn oil. TQ (100 mg/Kg) was administered by oral intubation in corn oil immediately before CC14 administration~ Control mice received either corn oil (10 ml/Kg o,p.) or TQ (100 mg/Kg). After 24 hr of CCI4 administration, blood was withdrawn from the orbital sinus under light anaesthesia and allowed to clot, this was followed by centrifugation at 1000 g for 5 rain to obtain serum. _Alanine aminotransferase (ALT) assay: Kinetic measurements of serum ALT was carried out spectrophotometrically (9) using a commercially available diagnostic kit (Biosystems, Spain). DT-diaphorase assay: Livers of untreated mice were excised, blotted dry, weighed and a 10% homogenate prepared in ice-cold saline. The DT-diaphorase activity in the liver homogenate was assayed spectrophotometrically by measurement of the rate of oxidation of NADH at 340 nm using TQ as substrate, according to the procedure of (10). Assay of non-enzymatic lipid peroxidaton in liver homogenate: The method of the assay is given in (3) with slight modification. Lipid peroxidation was induced by addition of Fe3+-ascorbate to an incubation mixture of liver homogenate. The 154
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BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL
assay mixture contained varying amounts of antioxidant or test compound (0.2l~tM) in 10 ~tl of ethanol, 0.75 ml phosphate buffered saline (50 mM, pH 7.4), 50~tl liver homogenate (10%), 0.1 ml of lmM Ferric chloride and 0.1 ml o f l m M ascorbic acid. The tubes were incubated at 37~ for 30 min and the extent of peroxidation was measured using the thiobarbituric acid (TBA) test. To the above tubes were added 0.1 ml BHT (2%, w/v) to stop further lipid peroxidation, 1.0 ml TBA(I% w/v in 0.05 M NaOH) and 1.0 ml trichloroacetic acid (2.8% w/v)and placed in water bath at 80~ for 20 min. At the end of incubation, the tubes were cooled and centrifuged for 5 min at 1000 g. The chromogen was extracted with 2.0 ml n-butanol and the absorbance was read at 532 nm. Tubes without antioxidants were subjected to TBA test and served as control. RESULTS The
effect of TQ on CC14-induced hepatotoxicity was evaluated by recording
changes in serum ALT levels. The data of Table 1 demonstrate that serum ALT activity was notably elevated 24 hr after CC14 administration. TQ had no effect on serum ALT activity. Co-treatment with TQ protected the liver against injury produced by CCI4,
Serum
ALT activity was significantly reduced (p
