Antioxidant and hepatoprotective activity of milk thistle (Sylibum marianum L. Gaertn.) seed oil

Open Li e Sci. 2015; 10: 147–158 DOI 10.1515/biol-2015-0017 Recei ed Ma ch 4, 2014; accep ed Augus 20, 2014 1 In oduc ion Se e al xenobio ics a e capable o causing li e inju y and associa ed mo bidi y due o he cen al ole o his o gan in xenobio ic me abolism, i s po al loca ion wi hin ci cula ion, and i s ana omical and physiological s uc u e [1]. Due o he bioac i a ion o xenobio ics du ing he de oxi ica ion eac ions, se e al wa e soluble me aboli es can become mo e ha m ul han he pa en al compounds [2], causing hepa ic degene a ion and cellula dea h [3]. Ca bon e achlo ide (CCl4) has been used as an expe imen al hepa o oxican o in es iga e he induc ion o a y li e , ib osis, ci hosis and nec osis, as a esul o eac i e ichlo ome hyl ee adicals o ma ion [4,5]. Oxida i e s ess, which appea s when he p o-oxidan e ec s o xenobio ics a e no coun e ac ed by he cellula an ioxidan de ense sys em, is impo an in he pa hogenesis o di e en li e diseases. Cu en ly, he e is an inc easing in e es in iden i ying he po en ial o a ious plan p oduc s as hepa op o ec i e/ an ioxidan and as die a y modula o s o oxici y [6,7]. Many seed oil ex ac s a e commonly used as componen s o adi ional heal hy ood in di e en coun ies. To be mo e speci ic, i has been shown ha seed oil ex ac s a e success ul in p e en ing oxida i e s ess, as well as es o ing he no mal le el o choles e ols, iglyce ides, low densi y-lipop o eins and hepa ic ma ke s ela ed wi h li e pa hologies. Such p ope ies ha e been associa ed wi h ea [8], sesame [9], seabuck ho n [10], o g ape [11] seeds. As a as we know, un il now, no expe imen conce ning he an ioxidan and hepa op o ec i e e ec o milk his le (Silybum ma ianum) seed oil has been pe o med. Abs ac : This s udy has assessed he p o ec i e e icacy o Silybum ma ianum seed oil (SMSO) in he con ex o CCl4-induced inju y and oxida i e s ess in mu ine li e . Based on he GC-MS analysis, linoleic and s ea ic acids, ocophe ol, asco bic acid 2,6 dihexadecanoa e and o he cons i uen s we e iden i ied in SMSO. Swiss mice ecei ed o al doses o SMSO daily o 21 days (10 g/kg b.w.) and subsequen ly injec ed i.p. wi h CCl4 (50% / in oli e oil; 1 ml/kg) on he 22nd day. CCl4 adminis a ion induced an ele a ion o se um amino- and glu amyl ans e ases ac i i ies and an inc eased pe oxida ion, as well as a dec ease o SOD, CAT, GPx, GR and GST ac i i ies in li e . SMSO success ully p e en ed oxida i e s ess and es o ed he biochemical pa ame e s, hepa ic a chi ec u e and exp ession o TNF-alpha. These indings sugges ha SMSO was e ec i e in coun e ac ing he damaging e ec s o CCl4-induced inju y in hepa ocy es, p obably due o i s inhe en an ioxidan p ope ies. Keywo ds: Silybum ma ianum, seed oil, CCl4, oxida i e s ess, an ioxidan , hepa op o ec ion Resea ch A icle Open Access © 2015 Anca He menean e al., licensee De G uy e Open. This wo k is licensed unde he C ea i e Commons A ibu ion-NonComme cial-NoDe i s 3.0 License. Anca He menean*, Mi una S an, Au el A delean, Luminița Pila , Cip ian Valen in Mihali, C is ina Popescu, Lajos Nagy, Gyö gy Deák, Miklós Zsuga, Sándo Kéki, Ildikó Bácskay, Fe enc Feny esi, Ma ie a Cos ache, Anca Dinischio u, Miklós Vecse nyés An ioxidan and hepa op o ec i e ac i i y o milk his le (Silybum ma ianum L. Gae n.) seed oil *Co esponding au ho : Anca He menean: Depa men o His ology, Facul y o Medicine, Pha macy and Den is y, Vasile Goldis Wes e n Uni e si y o A ad, Romania, E-mail: anc[email p o ec ed]om Anca He menean, Au el A delean, Cip ian Valen in Mihali, C is ina Popescu: Ins i u e o Li e Sciences, Vasile Goldis Wes e n Uni e si y o A ad, Romania Mi una S an, Ma ie a Cos ache, Anca Dinischio u: Depa men o Biochemis y and Molecula Biology, Uni e si y o Bucha es , Romania Luminița Pila : Depa men o Biochemis y, Facul y o Medicine, Pha macy and Den is y, Vasile Goldis Wes e n Uni e si y o A ad, Romania C is ina Popescu: Depa men o Pha maceu ical Sciences, Facul y o Medicine, Pha macy and Den is y, Vasile Goldis Wes e n Uni e - si y o A ad, Romania Lajos Nagy, Gyö gy Deák, Miklós Zsuga, Sándo Kéki: Depa men o Applied Chemis y, Uni e si y o Deb ecen, Hunga y Ildikó Bácskay, Fe enc Feny esi, Miklós Vecse nyés: Depa men o Pha maceu ical Technology, Uni e si y o Deb ecen, Hunga y 148   A. He menean e al. Silybum ma ianum has been used medicinally o he ea men o li e diseases in Eu ope since he i s cen u y. Silyma in, he p ima y ac i e ing edien in he seeds, has been ex ensi ely s udied o hepa op o ec i e e ec s and se e al pu a i e hepa op o ec i e mechanisms, including an ioxida ion and inhibi ion o lipid pe oxida ion [12,13], as well as an i-in lamma o y [14] and an i- ib o ic e ec s [15], ha e been eco ded. Al hough silyma in is o ally abso bed, i has e y poo bioa ailabili y due o i s lowe wa e solubili y [16]. Consequen ly, esea ch e o s ha e been aimed a de eloping no el d ug deli e y sys ems ha can imp o e he pe o mance o silyma in ac i i y [17-19]. The hepa op o ec i e e ec and an ioxidan ac i i y o Silybum ma ianum seed oil (SMSO) ha e no been p e iously in es iga ed. The aim o his s udy was o e alua e he po en ial o he milk his le na i e seed oil o enhance he an ioxidan de ense sys em and hus p o ide p o ec ion agains CCl4 -induced hepa o oxici y in mice. 2 Expe imen al P ocedu es 2.1 Sylibum ma ianum seed oil ma e ial The sample o milk his le seeds (Silybum ma ianum L. Gae n.) was supplied by a a me om A ad Coun y (Cu ici, Romania) and ouche specimens al eady exis a he Facul y o Na u al Sciences om he Vasile Goldis Wes e n Uni e si y o A ad. 2.2 P epa a ion and GC-MS s udy o Silybum ma ianum seed oil ex ac The d ied Silybum ma ianum seeds we e powde ed, homogenized and de a ed by hexane in a Soxhle ex ac o o 6 h. A e ex ac ion, wo phases we e ob ained: a solid and a liquid one. The solid phase was he de a ed powde (which was d ied be o e u he p ocessing) and he liquid phase was he hexane ex ac . The hexane was e apo a ed om he ex ac unde acuum in a o a y e apo a o and he esidue was a yellow oil (SMSO) ha was cha ac e ized using a Shimadzu QP-2010 GC-MS ins umen . Fo GC-MS measu emen s, one mg oil was dissol ed in one mL hexane and one μL o oil solu ion was injec ed in o he gas ch oma og aph. Compounds we e sepa a ed on a Zeb on ZB-5MS column (30 m × 0.25 mm × 0.25 μm). The ini ial empe a u e o he o en (60°C) was main ained o 3 minu es and subsequen ly hea ed o 300°C a a hea ing a e o 10°C/min and main ained a his empe a u e o 8 minu es. The empe a u es o he elec on ioniza ion (EI) ion sou ce and he in e ace we e 300°C. Spli injec ion was conduc ed wi h a spli a io o 10:1, helium was used as he ca ie gas and he injec o empe a u e was 200°C. 2.3 Animal ea men Male Swiss mice (25±3 g), supplied by he Animal House o he Vasile Goldis Wes e n Uni e si y o A ad, we e used. They we e housed unde con olled condi ions (22– 25°C) on a 12 h ligh /12 h da k cycle. All expe imen al p ocedu es we e done acco ding o he e hics and egula ions o animal expe imen s o Vasile Goldis Wes e n Uni e si y o A ad and app o ed by Ins i u ional Animal E hical Commi ee. The SMSO dose o 10 g/kg b.w. was chosen acco ding o p e ious expe imen s wi h di e en seed oils [8, 9, 20], as well as he oxican (CCl4) dose o 1.0 ml/kg b.w. in 50% oli e oil [21]. Thi y- wo animals we e used o he expe imen and hese we e di ided in o 4 g oups, as ollows: G oup 1: Con ol animals ecei ed iso onic saline solu ion (ISS) e e y day o 21 days and on he 22nd day we e dep i ed o ood o 24 h. G oup 2: CCl4 g oup ecei ed he ISS e e y day o 21 days and we e subsequen ly i.p. injec ed wi h CCl4 a a dose o 1.0 ml/kg b.w. in 50% oli e oil (1:1) on he 22nd day. G oup 3: SMSO p e- ea ed g oup o ally ecei ed Silybum ma ianum seed oil (SMSO) in a dose o 10 g/kg b.w. o 21 days and we e subsequen ly i.p. injec ed wi h CCl4 (1.0 ml/kg b.w.) on he 22nd day. G oup 4: SMSO g oup o ally ecei ed SMSO alone (10 g/kg b.w.) daily o 21 days and on he 22nd day we e dep i ed o ood o 24 h. A e 22 days om he s a o he ea men o g oups 1 and 4, and a 24 hou s a e CCl4 i.p. injec ion o g oup 2 and 3, blood was collec ed om enae ca ae; he mice we e subsequen ly sac i iced by ce ical disloca ion. Li e samples we e used o his opa hology, elec on mic oscopy and biochemical analyses. 2.4 Assay o se um hepa ic ma ke s The collec ed blood was placed in hepa inized ubes and cen i uged o 15 minu es a 1,500 × g in o de o ob ain plasma samples which we e used immedia ely o de e mine alanine amino ans e ase (ALT), aspa a e amino ans e ase (AST) and gamma glu amyl ans e ase (GGT) ac i i ies; hese we e measu ed by spec opho ome ic me hods using comme cially a ailable ki s (Roche Reagen s, F ance) acco ding o he manu ac u e ’s ins uc ions. An ioxidan and hepa op o ec i e ac i i y o milk his le (Silybum ma ianum L. Gae n.) seed oil   149 2.5 Tissue homogena e p epa a ion Mouse li e s (0.1 g o issue) we e homogenized in 10 olumes o ice-cold bu e (0.1M TRIS-HCl, 5mM EDTA bu e , pH 7.4) con aining a p o ease inhibi o cock ail (1:100 dilu ion; Sigma-Ald ich, USA) o 2 min a 16 Hz using a ball mill ( ype MM 301, Re sch GmbH & Co, Haan, Ge many). The homogena es we e cen i uged a 8000 × g o 30 min a 4°C o emo e he cell deb is. The supe na an s we e collec ed and used o biochemical assays. 2.6 An ioxidan enzymes assays Ca alase (CAT) ac i i y was measu ed by he me hod o Aebi [22]. Changes in abso bance a 240 nm co esponded o H2O2 decomposi ion. One uni o CAT ac i i y was calcula ed as one µmole H2O2/min/mL. The de e mina ion o li e supe oxide dismu ase (SOD) ac i i y was pe o med acco ding o he me hod desc ibed by Paole i and Mocali [23]. The amoun o enzyme ha inhibi ed he oxida ion o NADH by 50% a 37°C co esponded o one uni o SOD ac i i y. Glu a hione pe oxidase (GPx) ac i i y was assessed by a coupled eac ion wi h glu a hione educ ase ha ca alyzed NADPH oxida ion as desc ibed by Beu le [24]. Glu a hione educ ase (GR) ac i i y was measu ed by he dec ease o NADPH abso bance a 340 nm [25]. One uni o GR ac i i y was equal o one mic omole o NADPH oxidized pe minu e. The o al glu a hione S- ans e ase (GST) ac i i y was assayed by measu ing he a e o 1-chlo o-2,4- dini obenzene (CDNB) conjuga ion wi h GSH a 340 nm [26]. All enzyma ic ac i i ies we e exp essed as speci ic ac i i ies (uni s/ mg o p o ein). 2.7 Assessmen o lipid pe oxida ion The hepa ic malondialdehyde (MDA) con en was de e mined as a ma ke o lipid pe oxida ion using a luo ime ic echnique desc ibed by Del Rio e  al. [27]. This me hod was based on he eac ion o MDA wi h hioba bi u ic acid. Rela i e luo escence uni s (RFU) eco ded a Spec o luo ome e FP-6300 JASCO (λex= 520 nm; λem= 549 nm) we e con e ed o nmoles malondialdehyde (MDA) using 1,1,3,3- e ame hoxyp opane as s anda d. 2.8 P o ein concen a ion measu emen The p o ein con en was de e mined acco ding o Low y’s me hod using bo ine se um albumin as s anda d [28]. 2.9 His opa hology F eshly p ele a ed agmen s o mice li e s we e ixed in Bouin solu ion, dehyd a ed in e hanol, clea ed in oluene and embedded in pa a in. Fi e mm hick li e sec ions we e depa a inized and p ocessed ou inely o hema oxylin–eosin (H&E) acco ding o Bio Op ica s aining ki . F ozen sec ions we e cu a 8 mm wi h he SLEE MNT c yo ome, ixed in 10% bu e ed o maldehyde and s ained wi h Oil Red O ki acco ding o he me hods o Bio-Op ica s aining ki s. Moun ed slides we e examined unde a ligh mic oscope (Olympus BX43 mic oscope) and pho og aphed using a digi al came a Olympus XC30. 2.10 Immunohis ochemis y Immunohis ochemical s udies we e pe o med on pa a in embedded li e issues using mouse monoclonal an i-TNF-a an ibody dilu ed 1:100 (San a C uz Bio echnology, Cali o nia, USA), incuba ed o e nigh a 4°C, ollowed by HRP seconda y an ibodies incuba ion. The immuno eac ion p oduc was isualized by adding DAB solu ion and coun e s ained wi h hema oxylin, dehyd a ed in a g adien o alcohol and moun ed. The speci ici y o he eac ion was con i med by subs i u ing he p ima y an ibodies wi h i ele an immunoglobulins o ma ched iso ype, used in he same condi ions and dilu ions as he p ima y an ibodies. S ained slides we e analyzed by ligh mic oscopy (Olympus BX43, Tokyo, Japan). 2.11 Elec on mic oscopy Li e specimens we e p e ixed in 2.7% glu a aldehyde solu ion in 0.1 M phospha e bu e o 1.5 hou s, a 4°C. Following his, hey we e washed in 0.15 M phospha e bu e (pH 7.2) and pos - ixed in 2% osmic acid solu ion in 0.15 M phospha e bu e o 1h a 4°C. Dehyd a ion was pe o med in ace one, and inclusion was done in he epoxy embedding esin Epon 812. The blocks we e cu wi h an ul amic o ome ype LKB, a 70 nm hickness. The sec ions we e double con as ed wi h solu ions o u anyl ace a e and lead ci a e and we e analyzed wi h a TEM Tecnai 12 Bio win elec on mic oscope. 2.12 S a is ical analysis All esul s ha e been analyzed o s a is ical signi icance using G aphPad P ism so wa e (Ve sion 5; G aphPad So wa e, Inc., La Jolla, CA) and exp essed as mean alues ± SD (n = 8). Compa isons be ween g oups we e e alua ed 150   A. He menean e al. by one-way ANOVA ollowed by a pos hoc Bon e oni es . A alue o p < 0.05 was conside ed o be s a is ically signi ican . 3 Resul s 3.1 GC-MS esul s o he SMSO Based on he GC-MS measu emen s, sa u a ed and unsa u a ed a y acids, ocophe ol and asco bic acid 2,6 dihexadecanoa e we e iden i ied. In addi ion o hese compounds, lignoce ic acid me hyl es e , squalene and choles e ol could also be de ec ed in he ch oma og am. The linoleic acid and he s ea ic acid appea ed wi h he mos p ominen peaks in he GC-MS ch oma og am. A ep esen a i e GC-MS ch oma og am o SMSO can be seen in Figu e 1A. The iden i ied componen s a e indica ed on he ch oma og am. The spec um lib a y (NIST05) was used o he iden i ica ion o he componen s. The EI mass spec um o ocophe ol (RT 34.5 min) is shown in Figu e 1B. The simila i y pe cen ages we e highe han 90% in all cases conce ning ocophe ol (75%) and choles e ol (71%). 3.2 E ec s o SMSO on he se um hepa ic ma ke s The ea men wi h CCl4 inc eased he se um ac i i y o AST by 67.8 old, ALT by 67.5 olds and GGT by 2.7 old, espec i ely, compa ed o un ea ed indi iduals. The p e ea men o 21 days wi h SMSO oil be o e CCl4 adminis a ion signi ican ly dec eased he ele a ed le els o he AST, ALT and GGT ac i i ies by 42.45%, 50% and 32%, espec i ely, by compa ison o he CCl4- ea ed g oup. In addi ion, he e we e no signi ican changes o hese enzyma ic ac i i ies in he plasma o indi iduals ea ed wi h SMSO only (Figu e 2). 3.3 E ec s o SMSO on hepa ic lipid pe oxi- da ion and an ioxidan enzymes The le el o MDA was signi ican ly inc eased by 2.2 old (p <0.001) in he li e s o indi iduals ea ed wi h CCl4 compa ed o con ol. By con as , he p e ea men wi h SMSO signi ican ly diminished he MDA concen a ion, lowe ing i o con ol le els (Figu e 3). The exposu e o CCl4 induced a educ ion in CAT and SOD speci ic ac i i ies in mice li e (p < 0.001) Figu e 1. (A) GC-MS ch oma og am o SMSO whe e he iden i ied componen s a e indica ed; (B) Elec on-ioniza ion mass spec um o oco- phe ol (RT 34.5 min). An ioxidan and hepa op o ec i e ac i i y o milk his le (Silybum ma ianum L. Gae n.) seed oil   151 by 44% and 47.5%, espec i ely, compa ed o con ol (Figu e4). Ne e heless, in he SMSO p e ea ed g oup, hese enzyma ic ac i i ies we e es o ed o con ol le els (Figu e4). As a as hepa ic enzymes in ol ed in educed glu a hione me abolism a e conce ned, he dec ease o GPX, GST and GR ac i i ies by app oxima ely 44%, obse ed in he CCl4 exposed g oup, was abolished in he g oup p e ea ed wi h SMSO (Figu e 4). 3.4 His opa hologic and immunohis ochemi- cal examina ion Ligh mic oscopic e alua ion o li e issues om he con ol g oup e ealed no mal cellula a chi ec u e (Figu e 5 A1). Li e samples om 24 hou s CCl4 exposed g oup showed nec o ic changes o hepa ocy es including acuola degene a ion, especially in he cen ilobula a ea wi h in il a ion o in lamma o y cells (Figu e 5 B1). Sinusoid conges ion, mac o and mic o esicula s ea osis o hepa ocy es we e obse ed (Figu e 5 B2). P e en i e SMSO ea men gene a ed a ma ked educ ion o hepa ocellula nec osis, acuoliza ion, s ea osis and in lamma o y in il a e, in compa ison wi h he in oxica ed g oup (Figu e 5 C1-2). The p o ec i e e ec s o SMSO on he TNF-a exp ession, ele a ed by CCl4 p o-in lamma o y ac i i y, a e shown in Figu e 5. The li e slides o he con ol mice did no show subs an ial TNFa immunoposi i i y (Figu e 5 A3). The TNF-a exp ession in he SMSO only g oup was simila o he con ols (Figu e 5 D3). By con as , s ong TNF-a exp ession was obse ed o he CCl4 g oup. TNF-a immuno eac i i y was mainly de ec ed in he icini y o cen ilobula eins, wi h he b idges o ma ion be ween neighbo ing eins appea ing as a p edominan ly b own s aining (Figu e 5 B3). This e ec was in he li e o CCl4- in oxica ed mice p e ea ed wi h SMSO (Figu e 5 C3). 3.5 Elec on mic oscopic examina ion The hepa ocy e ul as uc u e was no mal in he con ol g oup. In he CCl4 ea ed g oup, mos o he hepa ocy es showed wo popula ions o lipid d ople s (la ge and small), degene a ed o ganelles and smoo h e iculum Figu e 2. P o ec i e e ec o na i e SMSO (dose o 10 g/kg b.w.) on se um AST, ALT and GGT ac i i ies. Values a e exp essed as means (± SD) (n = 8). *, **, *** S a is ical signi icance a p < 0.05, p < 0.01 and p < 0.001, espec i ely, compa ed o con ol. ### S a is ical signi icance a p < 0.001 compa ed o CCl4- ea ed g oup. Figu e 3. E ec o na i e SMSO ( dose o 10 g/kg) on MDA le els induced by CCl4 exposu e in mu ine li e . Values a e exp essed as means (± SD) (n = 8). *** S a is ical signi icance a p < 0.001 as com- pa ed o con ol. ### S a is ical signi icance a p < 0.001 as compa ed o CCl4- ea ed g oup. 152   A. He menean e al. esicles p oli e a ion (Figu e 6 B1). We also obse ed la ge dila a ions wi h ocal b eaks in he ERs o hepa ocy es in many a eas, associa ed wi h dila ed pe inuclea space and enla ged po es (Figu e 6 B2). In he p e- ea ed g oup, SMSO adminis a ion educed he olume and numbe o he lipid d ople s in he hepa ocy es and he o ganelles and cy oplasm s uc u e we e widely p o ec ed om he e ec s o CCl4 (Figu e 6 C1-2). Figu e 4. P o ec i e e ec o na i e SMSO (10 g/kg b.w.) on speci ic ac i i ies o CAT, SOD, GPx, GR and GST in mu ine li e s. Values a e exp essed as means (± SD) (n = 8). *,**, *** S a is ical signi icance a p < 0.05, p < 0.01 and p < 0.001, espec i ely, compa ed o con ol. #, ### S a is ical signi icance a p < 0.05 and p < 0.001, compa ed o CCl4- ea ed g oup. An ioxidan and hepa op o ec i e ac i i y o milk his le (Silybum ma ianum L. Gae n.) seed oil   153 p o ein oxida ion in he li e , esul ing in memb ane damage and li e inju y. Se e al phy ochemicals a e an ioxidan s and ha e he capaci y o p o ec cells and biomolecules, neu alizing ee adicals and p e en ing oxida i e deg ada ion and ce ain human diseases [31]. Silybum ma ianum seed oil is ich in phy ochemicals such as ocophe ol and shows an ioxidan e ec s [32]. The chemical composi ion o he oil ob ained by us was s udied by se e al me hods. In mo e de ail, he oleic and linoleic acids we e iden i ied as he p edominan componen s o he oil based on gas ch oma og aphy 4 Discussion The hepa o oxici y ollowing CCl4 adminis a ion in oden cells, as well as he gene a ion o oxida i e s ess, is well documen ed by p e ious s udies [29,30]. In CCl4 me abolism, he ichlo ome hyl ee adical o ma ion (CCl3 ·) unde cy och ome P450 ac ion is one o he i s s eps. This ee adical eac s e y apidly wi h an oxygen molecule o yield a highly eac i e ichlo ome hyl pe oxy adical (CCl3OO·). Bo h adicals a e capable o binding o p o eins o lipids, hus ini ia ing lipid pe oxida ion and Figu e 5. The e ec o SMSO on his ological and immunohis ochemical changes in he li e o CCl4 – ea ed mice; (A) Con ol g oup; (B) CCl4 g oup (C) SMSO+CCl4 g oup (D) SMSO g oup; 1.H&E s ain (N-cen ilobula nec osis); 2 Oil Red O s ain (lipid d ops – ed); 3.TNF-a imuno- his ochemical s ain (E) The pe cen age o nec o ic a ea in he li e s in he li e s; (F) TNF-a immunoposi i e hepa ocy es/ ield (uni s abo e con ol). Values a e exp essed as means (± SD) (n = 8). *** p < 0.001 signi ican ly di e en om he con ol g oup; ### p < 0.001 signi ican ly di e en om he CCl4- ea ed g oup. 154   A. He menean e al. Figu e 6. P o ec i e e ec o SMSO (dose o 10 g/kg b.w.) on he ul as uc u e o hepa ocy es agains li e inju ies induced by CCl4. (A) Con ol g oup; no mal aspec o nucleus (N) and nuclea en elope (a owhead), and ew lipid d ops (L); (B) CCl4 g oup; oedema ous cy o- plasm ma ix wi h sER p oli e a ion (as e ix); dila ed ER p o iles (a ow) and enla ged nuclea space (a owhead); inc eased numbe and size o lipid d ops (L); (C) SMSO + CCl4 g oup; no mal aspec o nuclea shape (a owhead) and ER (a ow); Reduc ion o numbe and size o lipid d ops (L); (D) SMSO g oup; no mal aspec o nucleus (N) and nuclea en elope (a owhead) and ER (a ow), ew lipid d ops (L). An ioxidan and hepa op o ec i e ac i i y o milk his le (Silybum ma ianum L. Gae n.) seed oil   155 measu emen s [33]. Beside he sa u a ed and unsa u a ed a y acids, o he compounds such as ocophe ol, s e ols and s e ylglycosides we e also iden i ied by HPLC and capilla y GLC [32]. These esul s highly co obo a e wi h ou GC-MS esul s. In addi ion, he p esence o asco bic acid 2,6 dihexadecanoa e in SMSO was also iden i ied, o he i s ime. Besides ocophe ol and asco bic acid 2,6 dihexadecanoa e, he s e ols o SMSO may be po en an ioxidan s due o hei possibili y o o m esonance-s abilized s uc u es [34]. On he o he hand, s e ylglycosides dissol e be e in pola sol en s because he glucose moie y is pola and hyd ophilic; while hese migh play a ole as an ioxidan s in he cellula hyd ophilic en i onmen [35], squalen, which is an isop enoid compound could unc ion as a highly e ec i e oxygen sca enging agen [36]. I is well known ha chemical agen s inducing hepa ic inju y can lead o signi ican inc eases in ALT and AST ac i i y [5]. One o he mos sensi i e indica o s o li e inju y a e CCl4 adminis a ion is he elease o in acellula enzymes in he ci cula ion, such as amino ans e ases. Becke e al. [37] s a ed ha ele a ed ac i i ies o hese enzymes a e indica i e o cellula leakage and loss o he unc ional in eg i y o li e cell memb ane. The ele a ion o plasma enzymes concen a ion is gene ally ega ded as one o he sensi i e ma ke s o hepa ic damage [38]. In ou s udy, CCl4 was ound o cause signi ican inc eases in plasma AST, ALT and GGT le els compa ed o con ol (p < 0.001). P e- ea men wi h 10 g/kg b.w. o SMSO e ec i ely p o ec ed he animals agains CCl4-induced hepa ic inju y, as e idenced by dec eased plasma AST (by 42.45%), ALT (by 50%) and GGT (by 32%) compa ed o CCl4 g oup. The an ioxidan e ec o all hese na u al p oduc s ( ocophe ol and asco bic acid 6 hexadecanoa e, s e ols, s e ylglycosides and squalene), which a e p esen in SMSO, could diminish he memb ane pe oxida ion as well as he AST, ALT and GGT leakage in plasma [39]. In he case o CCl4-induced hepa opa hy, a 2.5- old highe p oduc ion o supe oxide a he le el o complex I o he mi ochond ial espi a o y chain compa ed o he con ol was egis e ed [40]. This adical is ans o med in hyd ogen pe oxide in he eac ion ca alyzed by SOD. Beside dismu a ion, he Cu/Zn SOD izoenzyme ca alyzes su oga e eac ions, such as he p oduc ion o hyd oxyl adicals, using anionic sca enge s and hyd ogen pe oxide [41]. The e y eac i e hyd oxyl adicals eac apidly wi h a wide ange o biological mac omolecules, causing oxida i e damage [42] and a dec ease in ac i i y/le el o basic cellula enzyma ic and non-enzyma ic an ioxidan s [43]. Recen s udies p o ed ha in CCl4 exposed mice he le el o SOD, CAT and GPX mRNAs dec eased signi ican ly [44]. In addi ion, glu a hione educ ase may be ulne able o oxida i e damage i sel [45]. Also, some isoenzymes o GST, such as pi class, can be deac i a ed by oxida ion o a cys eine esidue nea he ac i e cen e [46]. These could be he easons o which o al SOD and CAT ac i i ies, as well as hose o he enzymes in ol ed in glu a hione me abolism, i.e. GPX, GST and GR, dec eased in he CCl4 ea ed g oup. The i s a ge s o eac i e oxygen species a e polyunsa u a ed a y acids om phospholipids and lipid pe oxida ion, quan i ied as MDA concen a ion, an end p oduc o his p ocess. I seems ha he signi ican dec ease o his biochemical pa ame e in he case o p e ea men wi h SMSO was also due o ocophe ol, asco bic acid 6-hexadecanoa e, s e ols, s e ylglycosides, squalene and linoleic acid p esence. The linoleic acid could be ans o med in he mice gas oin es inal ac in ce ain isome s o conjuga ed linoleic acids (CLA) ha could induce educed glu a hione syn hesis h ough a mechanism independen o ROS p oduc ion. CLA supp ess lipope oxida ion [47] and as a esul , p obably, MDA dec eased. Tocophe ol [39,48], s e ols, s e ylglucosides, and squalene could p o ec cell and o ganelle memb ane polyunsa u a ed a y acids and lipop o eins om oxida ion by eac i e ee adicals and migh inhibi lipid pe oxida ion. Being a chain b eaking an ioxidan , ocophe ol can di ec ly in e ac wi h ee adicals and i is ans o med in ocophe yl adical which can be educed o ocophe ol by asco bic acid 6-hexadecanoa e. Subsequen ly, he dehyd oasco bic acid o med in his eac ion [49] can be ans o med in asco bic acid by educed glu a hione [50]. The ea men o mice wi h SMSO only induced an insigni ican inc ease o li e CAT and SOD ac i i ies. In he indi iduals p e ea ed o 21 days wi h SMSO and hen ea ed wi h CCl4, a eco e y o hese enzyma ic ac i i ies, as well as o GPX, GST and GR le els, was no iced; his could be due o he an ioxidan e ec s o ocophe ol om SMSO [51], bu also o CLA [52], asco bic acid 6-hexadecanoa e [53], s e ols [54] and s e ylglucosides [55]. Memb ane damage p obably esul ed in changes in lipop o ein eleasing and subsequen ly induced lipid d ople s accumula ion in o hepa ocy es [56]. Consequen ly, hepa ocy e acuoliza ion could be due o lipid accumula ion ha was de ec ed ul as uc u ally as la ge lipid d ople s buildup in hese cells. Se e al s udies ha e epo ed hepa ic cen ilobula s ea osis and nec osis a e exposu e o CCl4 [21,57]. In lamma o y cells we e also obse ed, being highligh ed by a signi ican elease o TNF-α om Kup e cells especially, localized a ound he cen al ein and