A. SERÇE et al.: Antioxidant Properties of S. marianum Seed Extract, Food Technol. Biotechnol. 54 (4) (2016) 455

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1 A. SERÇE et l.: Antioxidnt Properties of S. mrinum Seed Extrct, Food Technol. Biotechnol. 54 (4) (216) 455 ISSN doi: /ftb originl scientific pper Assessment of the Antioxidnt Activity of Silybum mrinum Seed Extrct nd Its Protective Effect ginst DNA Oxidtion, Protein Dmge nd Lipid Peroxidtion Aynur Serçe 1, Bircn Çeken Toptncı 1, Sevil Emen Tnrıkut 1, Sevcn Altş 1, Göksel Kızıl 1, Süleymn Kızıl 2 nd Murt Kızıl 1 * 1 Chemistry Deprtment, Science Fculty, Dicle University, TR 2128 Diyrbkır, Turkey 2 Deprtment of Field Crops, Fculty of Agriculture, Dicle University, TR 2128 Diyrbkır, Turkey Received: June 2, 215 Accepted: June 16, 216 Summry Antioxidnt properties of ethnol extrct of Silybum mrinum (milk thistle) seeds ws investigted. We hve lso investigted the protein dmge ctivted by oxidtive Fenton rection nd its prevention by Silybum mrinum seed extrct. Antioxidnt potentil of Silybum mrinum seed ethnol extrct ws mesured using different in vitro methods, such s lipid peroxidtion, 1,1 diphenyl 2 picrylhydrzyl (DPPH) nd ferric reducing power ssys. The extrct significntly decresed DNA dmge cused by hydroxyl rdicls. Protein dmge induced by hydroxyl rdicls ws lso efficiently inhibited, which ws confirmed by the presence of protein dmge mrkers, such s protein crbonyl formtion nd by sodium dodecyl sulfte polycrylmide gel electrophoresis (SDS PAGE). The present study shows tht milk thistle seeds hve good DPPH free rdicl scvenging ctivity nd cn prevent lipid peroxidtion. Therefore, Silybum mrinum cn be used s potentilly rich source of ntioxidnts nd food preservtives. The results suggest tht the seeds my hve potentil beneficil helth effects providing opportunities to develop vlue-dded products. Key words: Silybum mrinum (milk thistle) seeds, ntioxidnt ctivity, rdicl scvenging ctivity, oxidtive DNA dmge, protein oxidtion, lipid peroxidtion, crbonyl formtion, SDS PAGE Introduction Silybum mrinum (L.) Gertn. (Astercee), lso known s milk thistle, is n importnt herbl medicine. It is grown primrily for the extrction of its ctive component (silymrin) from edible seeds, which hs been used to tret liver disorders. The liver-protecting bility of silymrin is due to the ntioxidnt nd free rdicl scvenging properties. Silybum mrinum seeds contin more thn seven flvonolignns. Mny studies hve reported the biologicl evlution of silymrin, such s in cncer chemoprevention nd heptoprotection (1,2). Silymrin hs the bility to scvenge free rdicls, it hs been found to increse the production of glutthione in heptocytes nd the ctivity of superoxide dismutse in erythrocytes (3). In one niml study it hs been shown to increse intrcel lulr glutthione level by s much s 5 % (3). Flvonolignns showed rdicl scvenging properties nd protective effects ginst the dmge of lipid membrnes (4) nd low-density lipoproteins (5). The ntioxidnt effect is due to the modultion of pthwys such s cell growth, poptosis nd differentition (6). Rective oxygen species (superoxide rdicl, hydro x- yl rdicl ( OH) nd lipid peroxide (LOO ) rdicls) my cuse mny diseses such s cncer nd Alzheimer s (7). *Corresponding uthor: Phone/Fx: ; E-mil: murtk@dicle.edu.tr

2 456 A. SERÇE et l.: Antioxidnt Properties of S. mrinum Seed Extrct, Food Technol. Biotechnol. 54 (4) (216) They re generted in humn body s by-product of the norml biochemicl rections nd incresed exposure to the environmentl stress (8). Oxidtion of ftty cids in the cell membrnes cn disrupt their fluidity nd permebility nd dmge mcromolecules, such s DNA, RNA, protein nd other cellulr components (9). Silymrin supports the norml fluidity of cell membrne by intercting with its components (1). Mny publictions show the inhibitory effects of plnt extrcts or their components ginst protein oxidtion nd DNA dmge cused by rective oxygen species (11 13). To our knowledge, this is the first study tht hs investigted the inhibition bility of DNA dmge, protein oxidtion nd lipid peroxidtion by ethnol extrct of S. mrinum seeds in vitro. Mterils nd Methods Seed smple Cultivted Silybum mrinum seeds were hrvested in the utumn of 212 from the field of the Deprtment of Field Crops, Fculty of Agriculture, University of Dicle, Diyrbkır, Turkey. The extrct ws prepred from dried S. mrinum seeds, which were ground into fine powder using blender (model 711S; Wring, Atlnt, GA, USA). Chemicls The 1,1 diphenyl 2 picryl hydrzyl (DPPH), 3 (2 pyridyl) 5,6 bis(4 phenyl sulfonic cid) 1,2,4 trizine (Fer- ro zine), butylted hydroxynisole (BHA), trichlorocetic cid (TCA), cetone, grose, Bromophenol Blue, sodium chloride, ethylenediminetetrcetic cid (EDTA), glcil cetic cid, Coomssie Brillnt Blue R 25 nd Trizm bse were purchsed from Sigm-Aldrich GmbH (Steinheim, Germny). Potssium cette nd butylted hydroxytoluene () were purchsed from Merck (Drmstdt, Germny). Ethidium bromide ws obtined from Amresco LLC (Solon, OH, USA). Extrction procedure Silybum mrinum seeds were ground into fine powder with blender to dimeter of.4 mm (14). Silymrin ws extrcted in two step process in which powdered seeds were first deftted. In order to deft the seeds, bout 1 g of finely powdered smples were weighed nd extrcted with petroleum ether (37 ml) for 4 h nd then with ethnol (35 ml) for 8 h in Soxhlet pprtus (Sigm-Aldrich). Ethnol solution ws evported t temperture not exceeding 4 C nd ethnol extrct of seeds (1.8 g) ws obtined s soft yellow powder. Determintion of totl phenolic content The phenolic content of milk thistle seed extrct ws mesured with the Folin-Cioclteu method (15). A mss concentrtion of.5 mg/ml of stock solution of the extrct ws prepred in ethnol. Then, 4 μl of the smple nd the control were diluted with 116 μl of wter nd 2 μl of 2 M Folin-Cioclteu gent. The bsorbnce ws determined t λ=765 nm. A stndrd gllic cid solution (5 4 μg/ml) ws used to construct the clibrtion curve. The phenolic content ws expressed in μg of gllic cid equivlents (GAE). The eqution below ws used to clculte the concentrtion of phenolic compounds: A 765 nm =.24 m(gae)/μg R 2 =.9967 /1/ Determintion of totl flvonoid content The flvonoid content ws determined by the previously described method (16). The stock solution of.5 mg/ml of milk thistle seeds ws prepred in ethnol. In 1 ml test tube, 1 ml of extrct,.1 ml of 1 % Al(NO 3 ) 3,.1 ml of 1 M CH 3 COOK nd 3.8 ml of methnol were mixed. The rection mixture ws incubted t 25 o C for 4 min. The bsorbnce ws mesured t λ=415 nm. The stndrd curve for totl flvonoids ws mde using quercetin stndrd solution (1 25 μg/ml). The eqution below ws used to mesure the concentrtion of flvonoid compounds expressed s quercetin equivlents (QE): A 415 nm =.28 m(qe)/μg R 2 =.9999 /2/ DPPH rdicl scvenging ctivity ssy The 1,1 diphenyl 2 picryl hydrzil (DPPH) rdicl scvenging ctivity of S. mrinum seeds ws studied using previously reported procedure (17). A volume of 3 ml of S. mrinum seed ethnol extrct solutions of different concentrtions (5, 1, 25, 5, 1 nd 15 μg/ml) ws mixed with 1 ml of.1 mm DPPH. The mixture ws kept t room temperture for 3 min. Then the bsorbnce ws mesured t λ=517 nm with Cry 1 Bio UV/Vis spectrophotometer (Vrin Inc., Plo Alto, CA, USA). The following eqution ws used to mesure the rdicl scvenging ctivity: Scvenging effect= =[(A 517 nm control A 517nm smple )/A 517nm control ] 1 Reducing power ssy The reducing power of the S. mrinum seeds ws ev luted using the procedure described previously (18), with some modifictions. Butylted hydroxynisole (BHA) nd butylted hydroxytoluene () were used s positive controls. In test tube, 1 ml of smple (5 15 μg/ ml), 2.5 ml of.2 M phosphte buffer (PBS; ph=6.6) nd 2.5 ml of 1 % K 3 Fe(CN) 6 solution were mixed. After 2 min of incubtion t 5 o C, 2.5 ml of 1 % trichlorocetic cid were dded to ech mixture, nd then centrifuged t 15 g for 1 min. A superntnt volume of 2.5 ml ws tken nd mixed with 2.5 ml of deionized wter contining.5 ml of.1 % iron(iii) chloride. After 1 min of incubtion t room temperture the bsorbnce A ws mesured t λ=7 nm. Lipid peroxidtion ssy Rt liver homogente preprtion The rt liver homogente (1 % by mss per volume) ws prepred ccording to previously reported method (19). For this study, lbino Wistr rts (mss of (15±25) g) were used. Permission ws obtined from Dicle Universi- /3/

3 A. SERÇE et l.: Antioxidnt Properties of S. mrinum Seed Extrct, Food Technol. Biotechnol. 54 (4) (216) 457 ty Ethicl Committee (IAEC; Diyrbkır, Turkey). The nimls were housed in polypropylene cges t controlled temperture ((22±3) o C). Stndrd formul nd wter were given to the nimls d libitum. The liver ws removed nd perfused with 12 mm potssium chloride nd 5 mm phosphte buffer, ph=7.4. The rtio of liver mss per volume of solution ws 1:1. In order to obtin the pellet, the smples were centrifugted t 7 g t 4 C. Thiobrbituric cid ssy The previously described thiobrbituric cid (TBA) ssy ws used to mesure lipid peroxidtion (2). Superntnt (1 μl) ws mixed with ethnol extrct (2 μl) of S. mrinum seeds (5 25 μg/ml). To the rection mixture, 1 μl of 1 mm iron(iii) chloride nd 1 μl of.1 mm scorbic cid were dded nd incubted t 37 C for 1 h. Superntnt bsorbnce ws mesured t λ=532 nm. The following eqution ws used to clculte the percentge of lipid peroxidtion inhibition: Inhibition=[(A c A s )/A c ] 1 /4/ DNA dmge protection cpcity DNA protection cpcity of milk thistle seed extrct ginst oxidtion ws determined ccording to Kızıl (21) using plsmid DNA. The bnd intensity for the forms I nd II of plsmid DNA ws quntified using Quntity One v softwre (Bio Rd, Hercules, CA, USA). Genejet miniprep kit (Thermo Fisher Scientific, Inc, Wlthm, MA, USA) ws employed for the isoltion of plsmid DNA. The following eqution ws used to clculte the inhibition of the DNA clevge: Inhibition=1 [(S m+ S c )/(S m S c )] /5/ where S m+ is the percentge of the remining supercoiled DNA fter tretment with mix consisting of buffer, DNA, H 2 O 2 nd UV, nd the gent (Silybum mrinum extrct), S c is the percentge of the remining supercoiled DNA in the control (untreted plsmid), nd S m is the percentge of the remining supercoiled DNA with the mix, but without the gent. Protein dmge protection Inhibition of protein crbonyl formtion The effect of milk thistle seed extrct on protein oxidtion ws determined using modified method of Wng et l. (22). Bovine serum lbumin (BSA; 4 mg/ml) ws treted with iron(iii) chloride (5 μm), hydrogen peroxide (1 mm) nd scorbic cid (1 μm) in potssium phosphte buffer (2 mm, ph=7.4) in the presence of seed extrct (1 1 μg/ml) t 37 C for 3 min nd then the bsorbnce ws mesured t λ=37 nm. The inhibition of protein crbonyl formtion ws clculted s follows (21): Inhibition=[(A c A s )/A c ] 1 /6/ SDS PAGE nlysis The inhibitory effect of the ethnol extrct of S. mrinum seeds on protein dmge ws lso determined by SDS PAGE (12). The gel ws run t mximum voltge in running buffer contining 25 mm Tris, ph=8.3, 19 mm glycine nd.1 % sodium dodecyl sulphte (SDS). The current of 25 ma ws used per gel. Coomssie Brillnt Blue R 25 (.15 %) ws used for stining of the gel, which ws scnned with Gel Doc XR System (Bio Rd). Sttisticl nlysis In order to determine the differences between the groups, one wy ANOVA ws used. Results re presented s the men vlue±stndrd devition of three replictes. Results nd Discussion Phenolic nd flvonoid content of S. mrinum seed extrct Totl phenolic content (expressed s GAE) of ethnol extrct of milk thistle seeds ws determined to be (62.±4.93) μg/g. In ddition to this, the flvonoid content (expressed s QE) ws found to be (39.32±.11) μg/g. These vlues show tht the ethnol extrct of milk thistle seed hs good ntioxidnt ctivity. Besides tht, hydroxyl groups present in the compounds isolted from the milk thistle extrct hve good rdicl scvenging ctivity. Pereir et l. (23) described the content of phenolics nd flvonoids in milk thistle. In their study, milk thistle plnt ws used s dry mteril for infusion nd pill preprtion. The phenolic nd flvonoid contents (expressed s GAE nd ctechin equivlent, respectively) were found to be (23.26±.22) mg/g nd (6.95±.23) mg/g in infusions, nd (2.92±.45) nd (3.88±.13) mg/g in dietry supplements, respectively (23). On the other hnd, Tupe et l. (24) reported phenolic content (s GAE) of methnolic plnt extrct of milk thistle of (18.33±.16) mg/g. The high contents of phenolics nd flvonoids obtined in infusions, dietry supplements nd methnolic milk thistle plnt extrct my depend on the prt of the plnt used. Phenolics nd flvonoids re very importnt plnt components due to their rdicl scvenging properties (25,26). It is well known tht flvonoids hve nticrcinogenic ctivity, ntillergenic, ntivirl, ntigeing nd nti-inflmmtory properties. DPPH rdicl scvenging ctivity The S. mrinum extrct showed strong DPPH rdicl scvenging bility in concentrtion-dependent mnner (Fig. 1). The order of rdicl scvenging ctivities ws found to be s follows: S. mrinum>>bha nd ws 92.±5.2, 88.±5.7 nd 87.8±2.1 t 15 μg/ml. The ntioxidnt (DPPH scvenging) ctivity of S. mrinum essentil components nd different tissues ws lso reported by Hdrug nd Hdrug (27). There ws no significnt difference between the DPPH rdicl scvenging ctivity of S. mrinum seed extrct nd both stndrds. Reducing power ssy It ws found tht S. mrinum hs beneficl effect ginst cute nd chronic liver filure. Silibinin, mjor ctive constituent of silymrin, ws found to hve ntitumour properties in heptocrcinom cell nd niml models (28). The tested smples t the concentrtions of 5, 1,

4 458 A. SERÇE et l.: Antioxidnt Properties of S. mrinum Seed Extrct, Food Technol. Biotechnol. 54 (4) (216) Inhibition/% ΒHA S. mrinum g/( μg/ml) Fig. 1. Scvenging effect of ethnol extrct of Silybum mrinum seeds on 1,1 diphenyl 2 picrylhydrzyl rdicls. Ech vlue is expressed s men vlue±stndrd devition (S.D.) (N=3). = butylted hydroxytoluene, BHA=butylted hydroxynisole 25, 5, 1, nd 15 μg/ml exhibited dose-dependent reducing power ctivity. Reducing power of 1 μg/ml of milk thistle seed extrct mesured s A 7 nm ws determined to be.11±.. On the other hnd, reducing powers of BHA nd t 1 μg/ml mesured s A 7 nm were determined to be.61±.3 nd.34±.8, respectively. The reducing power of S. mrinum seed ethnol extrct is shown in Fig. 2 with nd BHA s positive controls. There re reports on ntioxidnt ctivity nd reducing power of milk thistle infusions, dietry supplements nd syrups (23,28). Reducing power of infusions, dietry suplements nd syrups (mesured s A 7 nm ) ws found to be 1.73±.3, 1.1±.2, nd.5±., respectively (23,28). Tupe et l. (24) hve investigted 19 frequently used medicinl plnts nd concluded tht the strongest ntioxidnt ctivity ws observed in Terminli chebul since it hd high phenolic content, outstnding reducing power nd highest rdicl scvenging ctivity. Lipid peroxidtion The compound ntioxidnt ctivity depends on their cpcity to dely the oxidtion by inhibiting rective oxygen species (29). As shown in Fig. 3, thiobrbituric cid rective substnces (TBARS), which re formed during lipid oxidtion, were mesured in rt liver homogente nd it ws found tht the S. mrinum extrct modertely repressed TBARS formtion. At the concentrtion of 25 μg/ml of S. mrinum seed extrct, % of TBARS formtion ws inhibited. inhibited 31.4 % t the sme concentrtion. There were no significnt differences between the ctivity of S. mrinum seed extrct nd. The inhibitory ctivity of Mngifer indic frctions ginst lipid peroxidtion ws studied by Bdmus et l. (3), who found tht the ethyl cette frction hd the highest inhibitory ctivity ginst lipid peroxidtion in brin homogente (66.3 %), followed by methnol (61.6 %), chloroform (46.5 %) nd queous (39.5 %) frctions. High percentge of lipid peroxidtion inhibition of 89.1 % ws observed in queous frction of liver homogente, followed by ethyl cette (71.6 %), methnol (59. %) nd chloroform (48.5 %) frctions. 3 S. mrinum 1..8 ΒHA S. mrinum Inhibition/% 2 A 7 nm g(extrct)/ (mg/ml) g(extrct)/( mg/ml) Fig. 2. Reducing power of ethnol extrct of Silybum mrinum seeds, nd BHA mesured by spectrophotometric detection t 7 nm of Fe 3+ Fe 2+ trnsformtion. Ech vlue is expressed s men vlue±s.d. (N=3). Higher bsorbnce t 7 nm indicted greter reducing power. =butylted hydroxy to luene, BHA=buty lted hydroxynisole Fig. 3. Inhibition of Fe 2+ -induced lipid peroxidtion in rt liver homogente by ethnol extrct of Silybum mrinum seeds. Ech vlue is expressed s men vlue±s.d. (N=3). =butylted hydroxytoluene DNA dmge protection The bnd intensity for the forms I nd II of plsmid DNA ws quntified (Fig. 4). Fig. 4b shows plsmid DNA fter UV-induced photolysis of H 2 O 2 (2.5 mm) in the presence or bsence of the S. mrinum etnol extrct (in the concentrtion of 1, 5, 75 or 1 μg/ml). The supercoiled DNA ws converted to form I DNA by UV light (Fig. 4b, lne 4). Formtion of form I DNA ws suppresed in the presence of the extrct (Fig. 4b, lnes 6 9) nd the mount of form II of DNA ws incresed. The inhibitory ctivity of the ethnol extrct of cultivted milk thistle seed ginst oxidtive DNA dmge ws found to be 4.72, 41.8, nd 87.8 % t 1, 5, 75 nd 1 μg/ml,

5 A. SERÇE et l.: Antioxidnt Properties of S. mrinum Seed Extrct, Food Technol. Biotechnol. 54 (4) (216) 459 ) DNA percentge per lne b) form I form II Inhibition/% S. mrinum form II form I 2 Fig. 4. The quntified bnd intensity of the scdna (form I) nd ocdna (form II) (), nd (b) electrophoretic pttern of pbluescript M13+DNA fter UV photolysis of H 2 O 2 in the presence or bsence of ethnol extrct of Silybum mrinum seeds. Rection vils contined 2 ng of supercoiled DNA (31.53 nm) in distilled wter, ph=7 respectively. DNA dmge ws suppressed with the incresed concentrtion of the extrct. The ethnol extrct of milk thistle seeds showed concentrtion dependent free rdicl scvenging ctivity (Fig. 4). The protective effect of the extrct ginst DNA dmge my be ttributed to the presence of flvonoid nd phenolic compounds, such s silymrin nd silibinin, which cn prevent the production of rective oxygen species (ROS) by mking complexes with ctions such s copper nd iron tht prticipte in hydroxyl rdicl formtion (5). Inhibition of protein crbonyl formtion by S. mrinum seed extrct The effect of milk thistle seed extrct on BSA hydroxyl rdicl-medited oxidtion is shown in Fig. 5. nd S. mrinum seed extrct t 5, 1, 25, 5 nd 1 μg/ml decresed the free rdicl-induced protein dmge cus ed by protein crbonyl group formtion, induced by Fenton rection system. The inhibitory effect of t 5, 1, 25, 5 nd 1 μg/ml ws found to be 12.56, 37.33, 38.16, 4.43 nd 45.4 %, respectively, nd of S. mrinum seed extrct t 5, 1, 25, 5 nd 1 μg/ml ws found to be 9.16, 9.46, 11.36, 21.1, nd %, respectively. The inhibition of protein oxidtion by S. mri num seed extrct my be due to its high rdicl scvenging ctivity. Inhibitory effects of methnolic extrct of Feroni limoni pericrp on in vitro protein glycoxidtion ws studied by Kilri et l. (31). The study demonstrted tht F. limoni extrct ws efficient in preventing oxidtive protein dmge, including protein crbonyl formtion during glycoxidtion. They proposed tht the ntioxidnt effect of F. limoni might be due to the presence of voltile flvours nd free ftty cids in the pericrp, which re involved in the inhibitory mechnisms of protein crbonyl formtion. SDS PAGE The different concentrtions of nd S. mrinum seed extrct showed inhibitory ctivity on BSA oxidtion induced by Fenton rection system s mesured with SDS PAGE. Figs. 6 nd b nd Figs. 7 nd b show the bnd pttern of bovine serum lbumin nd densitometry dt of the corresponding bnds. Protein dmge induced by Fenton rection system ws inhibited with buty lted hydroxytoluene (Figs. 6 nd b) nd milk thistle ethnol extrct (Figs. 7 nd b) in concentrtion between 5 nd 1 μg/ml. The inhibitory ctivity of on BSA ws found to be 24.23, 37.54, 61.45, nd %, nd of ) b) Bnd intensity/% 1 5 Control g(extrct)/( mg/ml) Fig. 5. Inhibitory effect of butylted hydroxytoluene () nd ethnol extrct of Silybum mrinum seeds on protein (bovine serum lbumin, BSA) oxidtion, expressed s protein crbonyl inhibition induced by Fenton rection system (Fe 3+ /H 2 O 2 /scorbic cid). Ech vlue is expressed s men vlue±s.d. (N=3). All the vlues were sttisticlly significntly different from the control (p<.5) b Fenton rection b c g(extrct)/(mg/ml) Fig. 6. Protection ginst bovine serum lbumin (BSA) oxidtive dmge by butylted hydroxytoluene (): ) the electrophoretic pttern of BSA, nd b) densitometric nlysis of the corresponding bnd intensity. Ech br represents the men vlue±s.d. (N=3). Men vlues with different letters differ significntly (p<.5), while those with the sme letter re not significntly different (p>.5)

6 46 A. SERÇE et l.: Antioxidnt Properties of S. mrinum Seed Extrct, Food Technol. Biotechnol. 54 (4) (216) ) b) 1 S. mrinum Bnd intensity/% 5 Control S. mrinum seed extrct on BSA ws found to be 65.7, 77.27, 81.96, nd % t the concentrtions of 5 1 μg/ml, respectively. Conclusions b Fenton rection c g(extrct)/(mg/ml) The present work investigted the in vitro ntioxidtive nd oxidtive DNA, protein nd lipid dmge protective effects of milk thistle (Silybum mrinum) seed ethnol extrct. The extrct ws found to hve protective effect ginst DNA, protein nd lipid oxidtion induced by hydroxyl rdicl. The results obtined in this study show tht ethnol extrct of seeds cn be used s dietry supplement nd it cn protect DNA from dmge. The inhibitory ctivity of seed extrct ginst hydroxyl rdicl-induced DNA, protein nd lipid dmge my be minly responsible for the cncer chemoprevention nd heptoprotection effects. The presence of silymrin could ccount for the incresed ntioxidnt ctivity nd enhnced DNA, protein nd lipid dmge protection cpcity of the extrct. Protection ginst the DNA, protein nd lipid oxidtion is due to phenolic nd flvonoid compounds tht scvenge the free rdicls. These findings lso imply tht the presence of ntioxidnts cn decrese the occurrence of degenertive disorders in which free-rdicl dmge my be involed. Conflict of interest Authors stte tht they hve no conflict of interest regrding this submission. d d, e Fig. 7. Protection ginst bovine serum lbumin (BSA) oxidtive dmge by ethnol extrct of Silybum mrinum seeds: ) the electrophoretic pttern of BSA, nd b) densitometric nlysis of the corresponding bnd intensity. BSA ws oxidised by Fenton rection system (Fe 3+ /H 2 O 2 /scorbic cid). Ech br represents the men vlue±s.d. (N=3). Men vlues with different letters differ significntly (p<.5), while those with the sme letters re not significntly different t p>.5 compred with Fenton rection system e e Acknowledgements This reserch (Project no. 12 FF 2) received finncil support from the Dicle University Coordintion Committee of Scientific Reserch Projects (DUBAP), Diyrbkır, Turkey. The uthors thnk DUBAP for finncil support. References 1. Al Anti L, Essid E, Reinehr R, Petzinger E. Silibinin protects OTA medited TNF α relese from perfused rt livers nd isolted rt Kupffer cells. Mol Nutr Food Res. 29;53: Jyrj R, Deb U, Bhskr ASB, Prsd GBKS, Lkishmn Ro PV. Heptoprotective efficcy of certin flvonoids ginst microcystin induced toxicity in mice. Environ Toxicol. 27; 22: Fehér J, Láng I, Deák G, Cornides A, Nékám K, Gergely P. Free rdicls in tissue dmge in liver diseses nd therpeutic pproch. Toki J Exp Clin Med. 1986;1: Křen V, Kubisch J, Sedmer P, Hld P, Prikřylov V, Jegorov A, et l. Glycosyltion of silybin. J Chem Soc Perkin Trns ;17: Mir L, Silv M, Mnso CF. Scvenging of rective oxygen species by silibinin dihemisuccinte. Biochem Phrmcol. 1994;48: (94) Škottová N, Krečmn V, Simánek V. Activities of silymrin nd its flvonolignns upon low density lipoprotein oxidbility in vitro. Phytother Res. 1999;13: (19999)13:6<535: :AID PTR526>3..CO;2 W 7. Miguez MP, Anundi I, Sinz Prdo LA, Lindros KO. Heptoprotective mechnism of silymrin: no evidence for involvement of cytochrome P45 2E1. Chem Biol Interct. 1994;91: (94)96 X 8. Miller AL. Antioxidnt flvonoids: structure, function nd clinicl usge. Altern Med Rev. 1996;1: Wisemn H. Dietry influences on membrne function: importnce in protection ginst oxidtive dmge nd disese. J Nutr Biochem.1996;7: /1.116/ (95) Muriel P, Mourelle M. Prevention by silymrin of membrne ltertions in cute CCl 4 liver dmge. J Appl Toxicol. 199; 1: /jt Emen Tnrıkut S, Çeken B, Altş S, Pirinççioğlu M, Kızıl G, Kızıl M. DNA clevge protecting ctivity nd in vitro ntioxidnt potentil of queous extrct from fresh stems of Rheum ribes. Act Aliment. 213;42: Kızıl G, Kızıl, M, Çeken B, Yvuz M, Demir H. Protective bility of ethnol extrcts of Hypericum scbrum L. nd Hypericum retusum Aucher ginst the protein oxidtion nd DNA dmge. Int J Food Prop. 211;14: Kızıl M, Kızıl G, Yvuz M, Çeken B. Protective ctivity of ethnol extrct of three Achille species ginst lipid peroxidtion, protein oxidtion nd DNA dmge in vitro. Act Aliment. 21;39:

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