reactive oxygen species ( ros ) are produced in the biological system due to the exposure of various physical and chemical toxins , either directly or indirectly . once they enter into the body , these chemicals undergo the metabolism and detoxification . during the process , intermediate metabolites , ros and hydrogen peroxide are generated . the overload of ros leads to oxidative stress to macromolecules like dna , lipids and proteins ( 1 ) . as a consequence , lipid peroxidation , dna damage , they are implicated in numerous diseases and disorders like cataract , birth defects , reproductive anomalies , heart diseases , neurodegenerative diseases like parkinson and alzheimer , cardiovascular disorder , immune dysfunction , inflammation , ageing and cancer ( 2 ) . the balance between formation and removal of reactive oxygen molecules is required to maintain the normal physiological functions ( 1 ) . the biological system is endowed with an array of antioxidants like superoxide dismutase , catalase , glutathione peroxidase , glutathione s - transferase , reduced glutathione , vitamin c and vitamin e that would neutralize the activity of ross , and other reactive intermediate metabolites ( 3 ) . the in - vivo system of defense may not be adequate to neutralize all the ross produced . there is a need for an external source of antioxidants to neutralize the free radical load in the body ( 4 ) . fruits , vegetables , spices and oilseeds have been increasingly recognized as external sources of antioxidants . they contain vitamins , minerals , soluble / insoluble fibers , carotenoids , phenolic compounds including phenolic acids , flavonoids , isoflavonoids , lignans and tannins ( 5 - 7 ) . these antioxidant compounds may act as free radical scavengers and/or activators of anti - oxidative defense enzyme systems to suppress the radical damages in biological systems ( 8) . therefore , there is a growing interest toward natural antioxidants of herbal resources ( 9 ) . epidemiological and in - vitro studies strongly supported this idea that plant constituents with antioxidant activity are capable of exerting protective effects against ros - induced almighty in biological systems ( 10 ) . mentha spicata l. ( lamiaceae ) is an important aromatic spice which is cultivated throughout the world . its essential oil is widely used in food , cosmetic and pharmaceutical industries ( 11 ) . m. spicata , selected for the present study as it possess large amounts of total antioxidant activity which is better than some well - known antioxidants like quercetin , beta carotene and vitamin c ( 12 ) . it is also known to possess antimicrobial , antimutagenic and anti - inflammatory properties due to the presence of coffeic acid , rosmarinic acid and -tocopherol and anti - histaminic activity due to 5-o - desmethylnobiletin , cirsilineol , thymosin , thymonin and sideritoflavone ( 13 - 16 ) . it was found to be antiallergic , antiplatelet ( 17 ) , cytotoxicity ( 18 ) , chemopreventive ( 19 ) and h2o2 scavenging activities ( 7 ) . therefore , the present study focused on evaluating the in - vivo antioxidant effects of ethyl acetate fraction from ethanol extract of mentha spicata l. on 4-nitroquinoline-1-oxide - induced oxidative stress in mice . swiss albino of either sex ( 10 - 12 weeks old ) weighing 25 - 30 grams were used in this study . animals obtained from the king institute , chennai , india and maintained ( committee for the purpose of control and supervision of experiments on animals -cpcsea ) at the institute s animal house under standard environmental conditions ( temperature : 22  2c and 12/12 h ( light / dark ) period ) . malondialdehyde ( mda ) , 4-nitroquinoline-1-oxide ( 4-nqo ) , giemsa stain and may - grunwald stain were purchased from sigma aldrich ( sigma , usa ) . folin s phenol reagent , 5,5-dithiobis ( 2-nitro benzoic acid ) ( dtnb ) , 1-chloro 2,4-dinitrobenzene ( cdnb ) and reduced glutathione were purchased from sisco research laboratories pvt . extraction from leaves of mentha spicata 
  plant material was commercially obtained from a local market ( chennai ) and identified at the center for advanced studies in botany , university of madras ( voucher number-855 ) . eight hundred grams of shadow dried leaf powder was immersed in 4 l of 95% ethanol and left for 24 h under constant stirring and filtered . concentrated ethanol extract was further fractionated sequentially by hexane , chloroform , ethyl acetate and water . extract method briefly given in the early publication ( 14 ) . 
 experimental group for antioxidant properties of mentha spicata thirty - six mice were divided into six groups of either sex . the concentrated ethyl acetate fraction ( eaf ) was dissolved in 25% dmso and administered by oral gavage to the mice for 5 consecutive days . group 1 , the control , was administered 25% dmso , groups 2 and 3 received doses of 80 and 160 mg / kg bwt eaf , respectively , group 4 was administered 4-nqo alone ( 7.5 mg / kg bwt , intraperitoneally - ip ) , groups 5 and 6 received the doses of 80 and 160 mg / kg bwt of eaf for 5 days , and after 2 h , 4-nqo ( 7 . after 24 h of 4-nqo treatment , all the animals were sacrificed by cervical dislocation . liver homogenate ( 10% ) was made in 0.1 m of tris - hcl buffer ( ph 7.4 ) , using a potter - elvehjem homogenizer with a teflon pestle . the homogenate was used for the following bioassays : total protein ( 20 ) , lipid peroxidation ( lpo ) ( 21 ) , glutathione peroxidase ( gpx ) ( 22 ) , glutathione - s - transferase ( gst ) ( 23 ) , superoxide dismutase ( sod ) ( 24 ) , catalase ( cat ) ( 25 ) , reduced glutathione ( gsh ) ( 26 ) , vitamin e ( 27 ) and vitamin c ( 28 ) were estimated by using conventional procedures . 
 statistical analysis 
  in the present study , results were shown as mean  standard error for each group with six mice . results were performed through one - way anova using spss software version 12.0 with student - neuman - keuls test ( snk test ) to assess the differences among the groups . the enhanced lpo level by 4-nqo was observed to be 1.6 fold higher than the control value , 1.84 nmoles of mda formed / mg protein ( figure 1 ) . ethyl acetate fraction alone tested group showed no effect on the lpo level ( group 1 = group 2 = group 3 , p > 0.05 ; figure 1 ) . pretreatment with eaf significantly decreased the lpo increased by 4-nqo ( group 4 > group 5 > group 6 , p < 0.05 ( figure 1 ) ) . this decreased level was comparable to the control value ( 1.84 nmoles of mda formed / mg protein ) only at the highest dose treated , i.e. , at 160 mg / kg bwt . the enzymatic antioxidants were found to be decreased with 4-nqo in the range of 1.4 - 1.7 fold lesser than that of respective control values ( table 1 ) . pretreatment with eaf , even at the lowest dose ( 80 mg / kg bwt ) , effectively increased all of the four enzymatic antioxidants viz . , gpx , gst , sod and cat to the levels comparable to their control values . in fact , at the highest dose of 160 mg / kg bwt , the levels of gst and sod were significantly greater than their control values ( gst-2.55 nmoles of cdnb conjugated / min / mg protein and sod - 5.36 units / min / mg protein ) . besides , ethyl acetate alone group had no changes in the levels of antioxidants ( table 1 ) . protective effect of ethyl acetate fraction of mentha spicata against 4-nqo altered enzymatic antioxidants in liver of mice eaf : ethyl acetate fraction ; mean  standard error ; lpo : nmoles of mda formed / mg protein ; gpx : moles of reduced glutathione oxidized / min / mg protein ; gst : nmoles of cdnb conjugated / min / mg protein ; sod : units / min / mg protein ; cat :  moles of h2o2 consumed / min / mg protein similarly , 4-nqo - decreased non - enzymatic antioxidant was found to be in the range of 1.6 - 2.5 folds lesser than that of respective control values ( figure 1 ) . pretreatment with eaf , at a dose of 80 mg / kg bwt , the level of gsh was significantly increased but it was still lower than the control value ( group 5 > 4 and < 1 , p < 0.05 ) . at dose , 160 mg / kg bwt the enhanced level of gsh was significantly greater than the control value ( group 6 > 1 , p < 0.05 ) . vitamin e level was effectively enhanced even at the lowest dose , i.e. , 80 mg / kg bwt ( group 5 = 1 , p < 0.05 ) . at the highest dose ( 60 mg / kg bwt ) , its level was found to be higher than the control value ( group 6 > 1 , p < 0.05 ) . vitamin c increased with the increasing dose of eaf and at dose , 160 mg / kg bwt , its level was comparable with the control value ( group 6 = 1 , p > 0.05 ) . protective effect of ethyl acetate fraction from ethanol extract of mentha spicata against 4-nqo altered lipid peroxidation and non - enzymatic antioxidants in liver of mice 4-nqo is an oral synthetic carcinogen and widely used as mutagen ( 29 ) . it is also used as uv - mimetic and s - dependent clastogenic agent ( 30 ) . earlier reports indicate that 4-nqo induced oxidative stress in the biological system by enhancing lpo and suppressing body defense mechanisms during the exposure ( 31 ) . in the present study , the altered activities of enzymatic ( decreased 27 - 41% ) , non - enzymatic ( decreased 38 - 60% ) , antioxidants and promoted ( 60% ) lpo were observed in the 4-nqo alone treated group when compared with respective control values ( table 1 and figure 1 ) . it showed an impaired anti - oxidative capacity and sustained oxidative stress emerged following the exposure of 4-nqo . pretreatment with eaf , at the lowest dose ( 80 mg / kg bwt ) reduced lpo by about 56% . at dose of 160 mg / kg bwt , the reduction of lipid peroxidation reached up to 87% . the decreased lipid peroxidation by the eaf suggests the prevention of free radical formation in the process of 4-nqo toxicity . high phenolic and flavonoid content were observed in eaf which may be responsible for the reduction of lipid peroxidation ( 12 ) . the major phenolics and flavonoids of m. spicata were of caffeic acid , rosmarinic acid , -tocopherol , apigenin , acacetin , luteolin , thymonin , thymosin , sideritoflavone , cirsilineol , sorbitolin , 5-o - desmethylnobiletin and diosmetin ( 32 , 33 , 13 ) . the restoration of 4-nqo enhanced lpo by eaf was observed to be better than aqueous fraction obtained from ethanol extract of m. spicata ( 34 ) . on other hand , the most altered enzymatic antioxidant with 4-nqo was found to be gst and the least was cat . gst is an important enzymatic antioxidant , which is involved in detoxification process of genotoxins by catalyzing the formation of thiol group of glutathione to electrophilic genotoxins . these thiol compounds are also utilized to scavenge the potentially toxic compounds including those produced as a result of oxidative stress . catalase is also one of the most efficient enzymes that protect cells from the overload of hydrogen peroxide generated under the oxidative stress ( 35 ) . pretreatment with eaf ( 160 mg / kg bwt ) , the restoration of gst was found to be better among the enzymatic antioxidants when compared to the control value ( table 1 ) . the restoration of other antioxidants like gpx , sod and cat with eaf , was equal or less than 1.2 fold higher than that of the respective control values ( table 1 ) . similarly , the most affected non - enzymatic antioxidant by 4-nqo was found to be gsh and the least was vitamin c over their control values , respectively ( figure 1 ) . gsh is the major low molecular weight antioxidant in cells and its levels are often decreased due to oxidative stress ( 36 ) . it plays a major role in the protection of biological system against proliferation of lipid peroxidation under the threat of genotoxins . pretreatment with eaf at highest dose enhanced the gsh and vitamin e levels by over 100 percent of the mutagen - induced values which were also found to be 1.2 fold higher than the respective control values ( figure 1 ) . in contrast , the enhanced level of vitamin c with eaf was found near to control level . eaf effectively restored all antioxidant levels when compared to aqueous fraction of ethanol extract of m. spicata ( 34 ) . these results indicate that ethyl acetate fraction plays a major role in protecting the cells from genotoxic insults . in fact , the non - enzymatic antioxidants such as vitamin e , vitamin c and gsh are the major antioxidant involved in the metabolization of genotoxins ( 37 ) . apart from its well - documented anti - oxidant property , m. spicata possessed many other pharmacological activities including cytotoxicity , anti - inflammatory , anti - bacterial and radical scavenging effects ( 14 , 15 , 38 ) . antigenotoxicity of eaf was found to be higher than that of the other fractions such as hexane , chloroform and aqueous obtained from ethanol extract of m. spicata ( 16 , 34 ) . in conclusion , pretreatment with eaf ameliorate 4-nqo altered the antioxidants in which exception of vitamin c , eaf increased all the antioxidants in the range 15 - 34 percent . particularity , increasing gst and vitamin e ( 34% and 23% , respectively ) stood in contrast with the other antioxidants ( 14 - 17% ) . furthermore , ethyl acetate fraction showed better in - vitro and in - vivo antioxidant than that of aqueous fraction of m. spicata ( 12 , 34 ) .
antioxidant effects of ethyl acetate fraction of mentha spicata ( l. ) were evaluated against 4-nitroquinoline-1-oxide injected mice . for this study , experiment setup consisted of 36 albino mice of either sex divided into 6 groups : control ( 25% dmso in water ) , ethyl acetate fraction ( eaf ) alone group ( 80 , 160 mg / kg body weight - bwt ) , 4-nqo ( 7.5 mg / kg bwt - ip ) alone and 4-nqo + eaf . 
 eaf and vehicles were administered orally for five consecutive days . 
 4-nqo ( 7.5 mg / kg bwt ) was injected intraperitoneally on the 6th day . 
 after 24 h , the animals were killed ; liver sample was extracted and used for bio - assay . 
 4-nqo alone treated group decreased ( 27 - 60% ) the antioxidant activities and promoted lipid peroxidation ( lpo-60% ) over their respective control values . 
 pretreatment with eaf , at the maximum dose ( 160 mg / kg bwt ) brought down the lpo up to 87% enhanced by 4-nqo . among the enzymatic antioxidants , glutathione s - transferase ( gst ) was the most affected enzyme with 4-nqo and the least was catalase ( cat ) . 
 pretreatment with eaf ( 160 mg / kg bwt ) , the restoration of antioxidants like glutathione peroxidase , superoxide dismutase , and cat were found equal or less than 1.2 fold higher than that of the respective control values whereas , gst was observed to be the most restored antioxidant . 
 be reduced glutathione ( gsh ) and the least vitamin c over their control values . 
 eaf restored the gsh and vitamin e levels were found to be 1.2 fold higher than the respective control values .