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Effects Of Fresh Allium Sativa Extract On Lipid Peroxidation, Glutathione Depletion, And Oxidative Stress Induced By Acetaminophen In Mice

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
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  The Internet Journal of Pharmacology ISSN: 1531-2976Effects Of Fresh Allium SativaExtract On Lipid Peroxidation,Glutathione Depletion, AndOxidative Stress Induced ByAcetaminophen In Mice Christian Chinyere Ezeala PhD, MICR, CSci (UK),AIMLS Department of Medical Laboratory Sciences,Kampala International University Western Campus Ifeoma Nneka Nweke PhD Department of Pharmacology and Therapeutics, College of Medicine and Health Sciences, Abia State UniversityUturu Nigeria Prince C. Unekw e PhD Department of Pharmacology and Therapeutics, College of Medicine and Health Sciences, Abia State UniversityUturu Nigeria Citation:  C.C. Ezeala, I.N. Nweke, P.C. Unekwe: Effects Of Fresh Allium Sativa Extract On Lipid  Peroxidation, Glutathione Depletion, And OxidativeStress Induced By Acetaminophen In Mice. The Internet Journal of Pharmacology. 2010 Volume 8 Number 2. DOI: 10.5580/1b52 Keywords:  Garlic, oxidative stress, lipid  peroxidation, acetaminophen, glutathione,antioxidant enzymes. Abstract Oxidative stress and lipid peroxidation reactions are some of the mechanisms through which manydiseases 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 healthbenefits. This study was designed to evaluate theantioxidant and anti-lipid peroxidative effects of  fresh extract of Ugandan cultivars of garlic inacetaminophen induced toxicity in mice. The local Ugandan varieties of the garlic were obtained from alocal market in Ishaka Town in Western Uganda, ground to paste and extracted at room temperaturewith 80 % ethyl alcohol. Graded doses of the extract were administered intraperitonially (i.p.) to Swissmice 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 weredetermined 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 inthe liver in a dose dependent manner. These results suggest that regular consumption of local Ugandan garlic could protect the body from oxidative stressand lipid peroxidation reactions induced by several diseases. Introduction Oxidative stress and lipid peroxidation play centralroles in the pathogenesis and progression of severaldisorders. Cancer, ageing, atherosclerosis, andinflammatory processes have all been linked to thegeneration of reactive oxygen species and toxicmetabolites of lipid peroxidation reactions. 1, 2, 3 Inmany models, depletion of liver glutathione storesand other antioxidant molecules constitute animportant mechanism for the induction of oxidativestress and the concomitant damage to biologicalmolecules such as proteins and nucleic acids, and theactivation of nuclear transcription factors that may beimportant in the generation of pro-inflammatorycytokines 4, 5, 6, 7, 8 .Several anti-oxidants have been used in the treatmentof oxidative stress-mediated diseases, includingvitamins (C and E), carotenoids, and minerals such asselenium. 9, 10, 11,12 Also, ethnomedical practices haverelied on the use of plant products which are nowknown to contain antioxidant secondarymetabolites. 13 Garlic and garlic products have beenemployed 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 reporton the anti-oxidant and anti-lipid peroxidative properties of fresh ethanolic extract of local Ugandancultivars of garlic in mice models of acetaminopheninduced lipid peroxidation and oxidative stress. Wehypothesize that regular consumption of fresh garliccould prevent oxidative stress and protect againstdiseases associated with oxidative stress and lipid peroxidation reactions.  Materials And MethodsCollection, Identification, and Processing of GarlicBulbs Bulbs of a local variety of garlic (  Allium sativum L.)were obtained from Ishaka Town in Western Uganda,and identified by a qualified taxonomist. Coldextraction of the garlic was carried out at roomtemperature (18-22 o C) as follows: Fresh garlic bulbswere ground to a fine paste using a mechanicalgrinder and 50 g of the paste was put in a 250 mlconical flask and covered with 100 ml of 80 %ethanol, stoppered with cotton wool, and allowed tostand in the dark at room temperature for 48 hours.The ethanolic extract was filtered off with aWhatman no. paper into pre-weighed evaporatingdishes, while the residue in the flask was washedwith a further 100 ml of 80 % ethanol and added tothe extracts in the evaporating dishes. The filtrateswere then evaporated to a syrupy residue using arotary extractor at 40 O C. The dishes were thenweighed 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-tightcontainer and stored refrigerated (at -4 o C) untilrequired for use. For use, a portion of the extract wasweighed and dissolved in normal saline solution.Fresh preparations were made on each day of theexperiment. The resulting solutions were injectedintraperitonially into the mice. Laboratory Animals Swiss mice 6-8 weeks old weighing 18-32 g wereobtained from the Pharmacology Department of theMbarara University of Science and Technology inUganda. 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 usedfor the studies. The mice had free access to water andwere fed standard rodent pellets (purchased from alocal commercial supplier) ad libitum. Habituationconditions were 12 hr dark/light cycles, and averageenvironmental temperature of 20 o C. Acute Toxicity Test and Determination of LD 50   The LD 50 of the extract was determined in the mice by the procedure described by Bernas et al. (2004). 19  The confidence interval of the LD 50 was estimated bythe Litchfield  –  Wilcoxon method using a computer software. 20   Experimental Design Thirty Swiss mice of both sexes were used for theexperimental study. The animals were groupedrandomly into 6 groups of 5 each and administeredwith the drugs/extracts as follows: Group I received physiological saline i.p. only; group II receivedacetaminophen 250 mg/kg i.p. single dose only;group III was given garlic extract 250 mg/kg for 5days before a single i.p. dose of acetaminophen 250mg/kg; group IV received 500 mg/kg garlic extractfor 5 days before 250 mg/kg acetaminophen; group Vwere given 750 mg/kg garlic extract for 5 days before250 mg/kg acetaminophen; group VI received 25mg/kg silymarin for 5 days before a single i.p dose of acetaminophen 250 mg/kg. The extract wasadministered as a single once daily dose, whileacetaminophen was administered after 12 hours fast. Sample Collection The mice were sacrificed under ether anaesthesia, andtheir livers were obtained from the mice washed withice cold normal saline, followed by 0.15 M Tris- buffer (pH 7.4), blotted and weighed. The liver wasthen homogenized in 0.15 M Tris buffer to aconcentration of 10 g per 100ml of homogenate andused for TBARS, glutathione, catalase, and SODassays. Biochemical Assays Thiobarbituric acid reactive substances (TBARS) inthe liver homogenates were estimated by the methodof Ohkawa et al 21 as a measure of lipid peroxidationreactions. Catalase activities in the homogenates wereestimated by the method of Johansson and Borg, 22  (which depended on the reaction between methanoland catalase in the presence of hydrogen peroxide)with kits obtained from Calbiochem USA.Superoxide dismutase assay was estimated by themethod of Kakkar et al, 23 using kits obtained fromCalbiochem. The NWLSS GSH spectrophotometricassay kit was used for the estimation of glutathione inthe homogenates (Northwest Life Sciences  Specialties LLC, USA). In this method, 5- 5’ –   dithiobis (2-Nitrobenzoic acid) DTNB, reacts withglutathione 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 themean. Statistical analysis was by the one wayanalysis of variance (ANOVA) using the SPSSversion 10 software, and a p value ≤ 0.05 was considered significant. Results Administration of toxic doses of acetaminophen produced marked depletion of the liver glutathionestores and the antioxidant enzymes, superoxidedismutase, and catalase, and significant elevation of lipid peroxidation products estimated asthiobarbituric acid reactive substances (TBARS).Liver glutathione level in group II was significantlylower than in the negative control (p <0.005) as areSOD (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 protectedagainst these changes in a dose dependent manner and brought the values to levels comparable to thoseof the negative controls (p >0.01) as shown in table 1and in figure 1.Table 1: Liver TBARS, GSH, SOD, and CAT of mice in the six groupsFigure 1: Changes in liver TBARS, GSH, SOD, andCAT of mice in the six groups caused by theadministration of acetaminophen   Discussion  Natural antioxidants play significant roles in the prevention and treatment of many organic andinflammatory diseases associated with oxidativestress. 24 Polyphenols and flavonoids that are presentin plant-derived products are widely reported to exertsignificant influences on the removal of reactiveoxygen and nitrogen species and have been useful insuch diseases as diabetes mellitus andartherosclerosis. 25 This study demonstrated that fresh  Allium sativa extract exerted significant protectionagainst oxidative stress and lipid peroxidationinduced by acetaminophen overdose. It also showedthat fresh  Allium sativa preserved liver GSH, and up-regulated superoxide dismutase and catalase activitiesin the liver. These observations are consistent withthe observed effects of extracts from other plants in preserving liver GSH 26 , and more so agrees with thereport of Sabaya and others 27 in the relation to theaction of   Allium sativa extract on valproic acidinduced 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 GSHlevels in acetaminophen hepatotoxicity 28 , 29 .It is also possible that the extract prevented GSHdepletion by preventing NAPQI formation inacetaminophen overdose. The mechanism here could be inhibition of enzymes of phase I metabolism,notably CYP2E1 and CYP3A, which are the primaryenzymes responsible for acetaminophen biotransformation into NAPQI. Greenbaltt et al 30  have shown that certain water soluble constituents of aged garlic can inhibit CYP3A in normal human liver microsomes. It has been suggested that drugs whichcan reduce cytochrome P450 mediated NAPQIformation such as cobalt chloride, cimetidine, and piperonyl butoxide could protect the liver againstacetaminophen hepatotoxicity 31,   32 . Several reportshave also shown that isothiocyanate and allylsulphide compounds of   Allium sativa inhibitedcytochrome P450 enzymes such as CYP2E1 that actin phase I metabolism of acetaminophen 33, 34 Also,several other studies have reported that  Allium sativa  and  Allium cepa (  onion) organic sulphides arecapable of enhancing glutathione - S transferaseactivity in the liver, 35 and isothiocyanate is a very potent inducer of phase II metabolising enzymes suchas quinone reductase and glutathione  –  Stransferase. 36, 37    Allium sativa may also accelerate NAPQI excretion by providing substrates that arerequired for its conjugation. Such substrates mayinclude thiol (organosulphure) compounds, aminoacids, and sulphate ions. It may also achieve this byincreasing 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 organicsulphides such as S-allyl cysteine (SAC) and S-allylmercaptocysteine (SAMC) which could be utilizedfor GSH biosynthesis (221,222).  Allium sativa extractalso contains dially sulphide (DAS) and diallyldisulphide (DADS, known to have strong reducing properties), and allixin, antioxidant minerals (e.g.selenium), and fructosyl amino acids such asfructosyl 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 wouldtherefore protect the body against the toxic effects of oxidative stress and protect from various diseaseswhich are known to be associated with oxidativestress. 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