Effects Of Fresh Allium Sativa Extract On Lipid Peroxidation, Glutathione Depletion, And Oxidative Stress Induced By Acetaminophen In Mice
C Ezeala, I Nweke, P Unekwe
Keywords
acetaminophen, antioxidant enzymes., garlic, glutathione, lipid peroxidation, oxidative stress
Citation
C Ezeala, I Nweke, P Unekwe. Effects Of Fresh Allium Sativa Extract On Lipid Peroxidation, Glutathione Depletion, And Oxidative Stress Induced By Acetaminophen In Mice. The Internet Journal of Pharmacology. 2009 Volume 8 Number 2.
Abstract
Oxidative stress and lipid peroxidation reactions are some of the mechanisms through which many diseases produce their effects.
Introduction
Oxidative stress and lipid peroxidation play central roles in the pathogenesis and progression of several disorders. Cancer, ageing, atherosclerosis, and inflammatory processes have all been linked to the generation of reactive oxygen species and toxic metabolites of lipid peroxidation reactions. 1, 2, 3 In many models, depletion of liver glutathione stores and other antioxidant molecules constitute an important mechanism for the induction of oxidative stress and the concomitant damage to biological molecules such as proteins and nucleic acids, and the activation of nuclear transcription factors that may be important in the generation of pro-inflammatory cytokines 4, 5, 6, 7, 8.
Several anti-oxidants have been used in the treatment of oxidative stress-mediated diseases, including vitamins (C and E), carotenoids, and minerals such as selenium. 9, 10, 11,12 Also, ethnomedical practices have relied on the use of plant products which are now known to contain antioxidant secondary metabolites.13 Garlic and garlic products have been employed in medical practice since antiquity. Various pharmacological studies have also reported on the benefits of its extracts and products on vital physiological functions including their antioxidant, 14 cardioprotective, 15 hepatoprotective, 16 anticancer 17 and anti-inflammatory effects. 18 However, most of these studies focused on the use of aged garlic extract (AGE) or other commercial products. Here we report on the anti-oxidant and anti-lipid peroxidative properties of fresh ethanolic extract of local Ugandan cultivars of garlic in mice models of acetaminophen induced lipid peroxidation and oxidative stress. We hypothesize that regular consumption of fresh garlic could prevent oxidative stress and protect against diseases associated with oxidative stress and lipid peroxidation reactions.
Materials And Methods
Collection, Identification, and Processing of Garlic Bulbs
Bulbs of a local variety of garlic (
Weight of extract = weight of evaporating dish after evaporation – weight of dish before addition of extract;
Percentage yield = total weight of extract ÷ weight of paste used (50 g) × 100.
The extracts were pooled together into an air-tight container and stored refrigerated (at -4 oC) until required for use. For use, a portion of the extract was weighed and dissolved in normal saline solution. Fresh preparations were made on each day of the experiment. The resulting solutions were injected intraperitonially into the mice.
Laboratory Animals
Swiss mice 6-8 weeks old weighing 18-32 g were obtained from the Pharmacology Department of the Mbarara University of Science and Technology in Uganda. They were maintained and habituated in plastic cages in the animal house of the School of Health Sciences, Kampala International University, Western Campus for one week, and then after used for the studies. The mice had free access to water and were fed standard rodent pellets (purchased from a local commercial supplier) ad libitum. Habituation conditions were 12 hr dark/light cycles, and average environmental temperature of 20 o C.
Acute Toxicity Test and Determination of LD
The LD50 of the extract was determined in the mice by the procedure described by Bernas et al. (2004).19 The confidence interval of the LD50 was estimated by the Litchfield – Wilcoxon method using a computer software.20
Experimental Design
Thirty Swiss mice of both sexes were used for the experimental study. The animals were grouped randomly into 6 groups of 5 each and administered with the drugs/extracts as follows: Group I received physiological saline i.p. only; group II received acetaminophen 250 mg/kg i.p. single dose only; group III was given garlic extract 250 mg/kg for 5 days before a single i.p. dose of acetaminophen 250 mg/kg; group IV received 500 mg/kg garlic extract for 5 days before 250 mg/kg acetaminophen; group V were given 750 mg/kg garlic extract for 5 days before 250 mg/kg acetaminophen; group VI received 25 mg/kg silymarin for 5 days before a single i.p dose of acetaminophen 250 mg/kg. The extract was administered as a single once daily dose, while acetaminophen was administered after 12 hours fast.
Sample Collection
The mice were sacrificed under ether anaesthesia, and their livers were obtained from the mice washed with ice cold normal saline, followed by 0.15 M Tris-buffer (pH 7.4), blotted and weighed. The liver was then homogenized in 0.15 M Tris buffer to a concentration of 10 g per 100ml of homogenate and used for TBARS, glutathione, catalase, and SOD assays.
Biochemical Assays
Thiobarbituric acid reactive substances (TBARS) in the liver homogenates were estimated by the method of Ohkawa et al 21 as a measure of lipid peroxidation reactions. Catalase activities in the homogenates were estimated by the method of Johansson and Borg, 22 (which depended on the reaction between methanol and catalase in the presence of hydrogen peroxide) with kits obtained from Calbiochem USA.
Superoxide dismutase assay was estimated by the method of Kakkar et al, 23 using kits obtained from Calbiochem. The NWLSS GSH spectrophotometric assay kit was used for the estimation of glutathione in the homogenates (Northwest Life Sciences Specialties LLC, USA). In this method, 5-5’ – dithiobis (2-Nitrobenzoic acid) DTNB, reacts with glutathione to form 5-thionitrobenzoic acid (TNB) which has optimal absorption at a wavelength of 412 nm. The manufacturer’s protocol was strictly followed.
Data Analysis
Data were presented as mean ± standard error of the mean. Statistical analysis was by the one way analysis of variance (ANOVA) using the SPSS version 10 software, and a p value ≤ 0.05 was considered significant.
Results
Administration of toxic doses of acetaminophen produced marked depletion of the liver glutathione stores and the antioxidant enzymes, superoxide dismutase, and catalase, and significant elevation of lipid peroxidation products estimated as thiobarbituric acid reactive substances (TBARS). Liver glutathione level in group II was significantly lower than in the negative control (p <0.005) as are SOD (p <0.001) and catalase (p <0.05). The liver TBARS level in group II was significantly higher than in group I (p <0.005). The administration of fresh
Figure 1
Figure 2
Discussion
Natural antioxidants play significant roles in the prevention and treatment of many organic and inflammatory diseases associated with oxidative stress.24 Polyphenols and flavonoids that are present in plant-derived products are widely reported to exert significant influences on the removal of reactive oxygen and nitrogen species and have been useful in such diseases as diabetes mellitus and artherosclerosis.25 This study demonstrated that fresh
It is also possible that the extract prevented GSH depletion by preventing NAPQI formation in acetaminophen overdose. The mechanism here could be inhibition of enzymes of phase I metabolism, notably CYP2E1 and CYP3A, which are the primary enzymes responsible for acetaminophen biotransformation into NAPQI. Greenbaltt et al30 have shown that certain water soluble constituents of aged garlic can inhibit CYP3A in normal human liver microsomes. It has been suggested that drugs which can reduce cytochrome P450 mediated NAPQI formation such as cobalt chloride, cimetidine, and piperonyl butoxide could protect the liver against acetaminophen hepatotoxicity 31,
32. Several reports have also shown that isothiocyanate and allyl sulphide compounds of
GSH preservation could result from the supply of substrates for GSH biosynthesis by the
Administration of fresh Ugandan garlic extract prevented lipid peroxidation and depletion of liver glutathione stores and antioxidant enzymes in mice. Regular consumption of Ugandan garlic would therefore protect the body against the toxic effects of oxidative stress and protect from various diseases which are known to be associated with oxidative stress.