Effects Of Fresh Allium Sativa Extract On Lipid Peroxidation, Glutathione Depletion, And Oxidative Stress Induced By Acetaminophen In Mice
Abstract
Oxidative stress and lipid peroxidation reactions are some of the mechanisms through which many diseases produce their effects. Allium sativa (garlic) is widely used as spice or eaten raw in many cultures, and has it been reported to exert several health benefits. This study was designed to evaluate the antioxidant and anti-lipid peroxidative effects of fresh extract of Ugandan cultivars of garlic in acetaminophen induced toxicity in mice. The local Ugandan varieties of the garlic were obtained from a local market in Ishaka Town in Western Uganda, ground to paste and extracted at room temperature with 80 % ethyl alcohol. Graded doses of the extract were administered intraperitonially (i.p.) to Swiss mice for 5 days before a single i.p. dose of 250 mg/kg acetaminophen. Levels of thiobarbituric acid reactive substances (TBARS) and glutathione (GSH) concentrations, and superoxide dismutase (SOD) and catalase (CAT) activities in liver homogenates were determined and compared to controls. Results showed that fresh extract of the local garlic prevented lipid peroxidation, preserved liver GSH stores, and up regulated SOD and CAT activities in the liver in a dose dependent manner. These results suggest that regular consumption of local Ugandan garlic could protect the body from oxidative stress and lipid peroxidation reactions induced by several diseases.
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 (Allium sativum L.) were obtained from Ishaka Town in Western Uganda, and identified by a qualified taxonomist. Cold extraction of the garlic was carried out at room temperature (18-22 o C) as follows: Fresh garlic bulbs were ground to a fine paste using a mechanical grinder and 50 g of the paste was put in a 250 ml conical flask and covered with 100 ml of 80 % ethanol, stoppered with cotton wool, and allowed to stand in the dark at room temperature for 48 hours. The ethanolic extract was filtered off with a Whatman no. paper into pre-weighed evaporating dishes, while the residue in the flask was washed with a further 100 ml of 80 % ethanol and added to the extracts in the evaporating dishes. The filtrates were then evaporated to a syrupy residue using a rotary extractor at 40 O C. The dishes were then weighed again on a triple beam balance and the percentage yield was calculated as follows:
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 LD50
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 Allium sativa extract and silymarin protected against these changes in a dose dependent manner and brought the values to levels comparable to those of the negative controls (p >0.01) as shown in table 1 and in figure 1.
Table 1: Liver TBARS, GSH, SOD, and CAT of mice in the six groups
Figure 1: Changes in liver TBARS, GSH, SOD, and CAT of mice in the six groups caused by the administration of acetaminophen
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 Allium sativa extract exerted significant protection against oxidative stress and lipid peroxidation induced by acetaminophen overdose. It also showed that fresh Allium sativa preserved liver GSH, and up-regulated superoxide dismutase and catalase activities in the liver. These observations are consistent with the observed effects of extracts from other plants in preserving liver GSH 26, and more so agrees with the report of Sabaya and others 27 in the relation to the action of Allium sativa extract on valproic acid induced hepatotoxicity. In this respect, Allium sativa mimics the activities of cysteine prodrugs such as N-acetyl cysteine (NAC) and S-adenosyl methionine (SAM), which are known to preserve liver GSH levels in acetaminophen hepatotoxicity 28, 29.
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 Allium sativa inhibited cytochrome P450 enzymes such as CYP2E1 that act in phase I metabolism of acetaminophen 33, 34 Also, several other studies have reported that Allium sativa and Allium cepa (onion) organic sulphides are capable of enhancing glutathione - S transferase activity in the liver,35 and isothiocyanate is a very potent inducer of phase II metabolising enzymes such as quinone reductase and glutathione –S transferase.36, 37 Allium sativa may also accelerate NAPQI excretion by providing substrates that are required for its conjugation. Such substrates may include thiol (organosulphure) compounds, amino acids, and sulphate ions. It may also achieve this by increasing NAPQI binding to glucuronic acid.38 Investigation of these possibilities requires studies of the pharmacokinetics of NAPQI in animals receiving Allium sativa extract, and the effects of Allium sativa extract on cytochrome P450 enzymes responsible for NAPQI metabolism.
GSH preservation could result from the supply of substrates for GSH biosynthesis by the Allium sativa extract. Allium sativa is known to contain organic sulphides such as S-allyl cysteine (SAC) and S-allyl mercaptocysteine (SAMC) which could be utilized for GSH biosynthesis (221,222). Allium sativa extract also contains dially sulphide (DAS) and diallyl disulphide (DADS, known to have strong reducing properties), and allixin, antioxidant minerals (e.g. selenium), and fructosyl amino acids such as fructosyl glutamic acid and fructosyl arginine.39, 40
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.
