* Author to whom correspondence should be addressed; Tel.:

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1 Moleules 212, 17, ; doi:1.339/moleules Artile OPEN ACCESS moleules ISSN Protetive Effets of Extrts nd Flvonoids Isolted from Suti uxifoli Reissek ginst Chromosome Dmge in Humn Lymphoytes Exposed to Hydrogen Peroxide Aline Augusti Boligon 1, *, Mihele Rorto Sgrillo 2,3, Luiz Filipe Mhdo 3, Olmiro de Souz Filho 3, Mihel Mnsur Mhdo 4, Ivn Betrie Mni d Cruz 3 nd Mrgreth Linde Athyde Progrm of Post-Grdution in Phrmeutil Sienes, Federl University of Snt Mri, Cmpus Cmoi, Snt Mri, RS, , Brzil Frnisn University Center UNIFRA, Andrds Street, 1614, Snt Mri, RS , Brzil Progrm of Post-Grdution in Phrmology, Federl University of Snt Mri, Cmpus Cmoi, Snt Mri, RS, , Brzil Federl University of Pmp UNIPAMPA, Uruguin, RS , Brzil * Author to whom orrespondene should e ddressed; E-Mil: lineoligon@hotmil.om; Tel.: Reeived: 26 Mrh 212; in revised form: 25 April 212 / Aepted: 26 April 212 / Pulished: 14 My 212 Astrt: Flvonoids re limed to protet ginst rdiovsulr disese, ertin forms of ner nd geing, possily y preventing initil DNA dmge. Therefore, we investigted the protetive effets of rude extrt, ethyl ette frtion nd flvonoids (queretin, queritrin, isoqueritrin nd rutin) isolted from the leves from Suti uxifoli ginst hromosome dmge indued y H 2 O 2 in humn lymphoytes y nlyzing ellulr growth rte, ell viility, mitoti index nd hromosoml instility. We found differentil response mong the ompounds tested, with the ethyl ette frtion eing more effetive thn the rude extrt, differene perhps relted to the presene of the ntioxidnts identified nd quntified y HPLC/DAD. In generl, queretin, isoqueritrin nd rutin reovered the mitoti index nd hromosoml instility more thn queritrin fter tretment with hydrogen peroxide. Keywords: Suti uxifoli; flvonols; lymphoyte ulture; hromosomes dmge; HPLC/DAD

2 Moleules 212, Introdution In reent yers, fous on plnt reserh hs inresed ll over the World [1]. The onsumption of plnt produts is ssoited with lowering risk of numer of hroni diseses, inluding theroslerosis nd ner [2]. These enefiil effets hve een prtly ttriuted to ntioxidnts, whih my ply importnt roles in inhiition of free rdils nd oxidtive hin-retions within tissues nd memrnes [3]. Phenoli ompounds, suh s flvonoids, phenoli ids, oumrins nd tnnins re seondry plnt metolites, nd n importnt prt of oth humn nd niml diets [4,5]. They exert wide vriety of iologil effets, inluding ntirinogeni, ntimutgeni nd ntioxidtive tivities [6 9]. The ntioxidnt tivity of these ompounds is minly due to their redox properties, whih llow them to t s reduing gents or hydrogen-tom donors [1]. Additionlly, phenolis nd flvonoids re known to possess ntimutgeni tivity, mong other importnt iologil properties [11]. Suti uxifoli Reissek elongs to the Rhmnee fmily nd is populrly known s oronilh. It is ntive tree from South Ameri, with dispersion re tht omprises Rio Grnde do Sul Stte in Brzil, nd the ountries of Argentin nd Uruguy. The root rk infusion is populrly used s rdiotoni, ntihypertensive nd diureti [12]. Antimiroil tivities of some ylopeptide lkloids isolted from the root rk of this speies were reported y Morel et l. [13] who used the ioutogrphy method. Extrts from leves, twigs nd stem rk of S. uxifoli were onsidered toxi fter n Artemi slin ssy, nd in ddition showed in vitro ntimiroil nd ntimyoteril tivity ginst pnel of miroorgnism strins [14,15]. Extrts from the leves nd stem rk of S. uxifoli were effetive inhiitors of thiorituri id retive speies (TBARS) prodution nd lso presented 1,1-diphenyl-2-pirylhydrzyl (DPPH) rdil svenger tivity. Queretin, queritrin, isoqueritrin nd rutin were isolted from S. uxifoli, inditing tht this plnt ontins promising ompounds to e tested s potentil drugs for the tretment of diseses resulting from oxidtive stress [16]. In line of these findings, this study ompres the ility of the rude extrt, ethyl ette frtion nd four flvonoids (queretin, rutin, isoqueritrin nd queritrin) isolted of the leves from S. uxifoli to modulte the DNA dmge indued in lymphoytes y hydrogen peroxide. 2. Results 2.1. HPLC Anlysis The HPLC profile of rude extrt nd ethyl ette from the leves of S. uxifoli ws lso quired (Figure 1). Leves of S. uxifoli ontins other minor ompounds in ddition to glli id (retention time-t R 12.4 min, pek 1), hlorogeni id (t R = 23.1 min, pek 2), ffei id (t R = 27.6 min, pek 3), rutin (t R = 37.5 min, pek 4), isoqueritrin (t R = 4.1 min, pek 5), queritrin (t R = 43.4 min, pek 6), queretin (t R = 48.2 min, pek 7) nd kempferol (t R = 54.9 min, pek 8). Sine extrts of nturl origin usully ontin rnge of hemilly diverse onstituents ourring in vrying onentrtions, it is importnt to use hromtogrphi methods to nlyze these inherently omplex mixtures. The HPLC profile of rude extrt nd ethyl ette frtion ws quired, s well the quntifition of queretin, rutin, kempferol, nd glli, hlorogeni nd ffei ids y HPLC-DAD sed on referenes stndrds lirtion urves (Tle 1).

3 Moleules 212, Figure 1. High performne liquid hromtogrphy profile of rude extrt () nd ethyl ette frtion () from the leves of S. uxifoli. Glli id (pek 1), hlorogeni id (pek 2), ffei id (pek 3), rutin (pek 4), isoqueritrin (pek 5), queritrin (pek 6), queretin (pek 7) nd kempferol (pek 8). Chromtogrphi onditions re desried in the mteril nd methods setion. Tle 1. Phenolis nd flvonoids omposition of Suti uxifoli leves; LOD nd LOQ vritions for ompounds. Compounds Crude extrt Ethyl ette frtion LOD Quntities (mg/g) Quntities (mg/g) (μg/ml) LOQ (μg/ml) Glli id 18.1 ± ± Chlorogeni id 4.9 ± ± Cffei id 7.3 ± ± Rutin 14.2 ± ± Isoqueritrin * 1.5 ± ± Queritrin * 26.5 ± ± Queretin 42.1 ± ± Kempferol 5.6 ± ± Results re expressed s men ± stndrd devition (SD) of three determintions. Different letters in eh olumn represent signifint differenes using nlysis of vrine followed y Tukey test (p vlues <.5 were onsidered s signifint). * Quntified s queretin. LOD: limit of detetion; LOQ: limit of quntifition Cell Viility After one hour the ell viility deresed signifintly in hydrogen peroxide tretment (78 ± 2.2%) when ompred to ontrol tretment (1 ± 2.9%) (p =.1). Tretments with S. uxifoli rude extrt, ethyl ette frtion nd isolted ompounds (rutin, queretin, isoqueritrin nd queritrin) inresed the ell viility, despite the presene of hydrogen peroxide (p =.1). Rutin tretments t 5 µg/ml nd 1 µg/ml onentrtions reverted 1% of mortlity ssoited to hydrogen peroxide (Figure 2).

4 Moleules 212, Figure 2. Effet of queretin (Q), rutin (R), isoqueritrin (Iso), queitrin (Qr), ethyl ette frtion (EA) nd rude extrt (CE) of S. uxifoli on humn lymphoyte ell viility inution with H 2 O 2. Conentrtion rnge of queretin (A), rutin (B), isoqueritrin (C), queritrin (D), ethyl ette frtion (E) nd rude extrt (F). Results re men ± SD for n = 3 (p =.1). Different letters in eh tretment represent signifint differenes using nlysis of vrine followed y Tukey test, p =.1. Men ell viility (%) µg/mL Q + 1µg/mL Q + 5µg/mL Q + 1µg/mL Q + (A) Men ell viility (%) µg/mL R + 1µg/mL R + 5µg/mL R + 1µg/mL R + (B) (C) (D) Men ell viility (%) µg/mL Iso + 1µg/mL Iso + 5µg/mL Iso + 1µg/mL Iso + Men ell viility (%) µg/mL Qr + 1µg/mL Qr + 5µg/mL Qr + 1µg/mL Qr + Men ell viility (%) µg/mL EA + 1µg/mL EA + 5µg/mL EA + 1µg/mL EA + (E) Men ell viility (%) µg/mL CE + 1µg/mL CE + 5µg/mL CE + 1µg/mL CE + (F)

5 Moleules 212, Mitoti Index Although the tretment period with hydrogen peroxide nd S. uxifoli ompounds ws short (one hour) we oserved tht the tretments ffeted the mitoti index. The men numer of metphses ws higher in ontrol group when ompred with ll tretments (p =.1). On the other hnd, the hydrogen peroxide nd ll tretments with queritrin presented lower numer of metphses; the other tretments only t low onentrtions hd no effet on numer of ells in mitosis (Figure 3). Figure 3. Effet of queretin (Q), rutin (R), isoqueritrin (Iso), queitrin (Qr), ethyl ette frtion (EA) nd rude extrt (CE) of S. uxifoli on humn lymphoyte numer of ells in mitosis inution with H 2 O 2. Conentrtion rnge of queretin (A), rutin (B), isoqueritrin (C), queritrin (D), ethyl ette frtion (E) nd rude extrt (F). Results re men ± SD for n = 3 (p =.1). Different letters in eh tretment represent signifint differenes using nlysis of vrine followed y Tukey test, p =.1. Men numer of ells in mitosis d e (A) Men numer of ells in mitosis d d (B) Men numer of ells in mitosis d d (C) Men numer of ells in mitosis (D) Men numer of ells in mitosis (E) Men numer of ells in mitosis (F)

6 Moleules 212, Chromosoml Instility The hromosoml instility is shown in Figure 4. After one hour the hromosoml instility inresed signifintly in hydrogen peroxide tretment (18 ± 1.5) when ompred to ontrol tretment (.66 ±.35) (p =.1). Figure 4. Effet of queretin (Q), rutin (R), isoqueritrin (Iso), queitrin (Qr), ethyl ette frtion (EA) nd rude extrt (CE) of S. uxifoli on hromosoml instility inution with H 2 O 2. Conentrtion rnge of queretin (A), rutin (B), isoqueritrin (C), queritrin (D), ethyl ette frtion (E) nd rude extrt (F). Results re men ± SD for n = 3 (p =.1). Different letters in eh tretment represent signifint differenes using nlysis of vrine followed y Tukey test, p =.1. Chromosoml instility (A) 1µg/mL Q + 1µg/mL Q + 1µg/mLM Q + 5µg/mL Q + Chromosoml instility d 1µg/mL R + 1µg/mL R + 5µg/mL R + 1µg/mL R + (B) Chromosoml instility (C) 1µg/mL Iso + 1µg/mL Iso + 5µg/mL Iso + 1µg/mL Iso + Chromosoml instility µg/mL Qr + 1µg/mL Qr + 5µg/mL Qr + 1µg/mL Qr + d (D) Chromosoml instility (E) d 1µg/mL EA + 1µg/mL EA + 5µg/mL EA + 1µg/mL EA + Chromosoml instility µg/mL CE + 1µg/mL CE + 5µg/mL CE + 1µg/mL CE + (F)

7 Moleules 212, The CE tretment in the onentrtions 1, 1 nd 5 µg/ml did not present signifint influene on hromosoml instility used y hydrogen peroxide (Figure 4F). However, tretments with rude extrt (1 µg/ml), ethyl ette frtion nd isolted ompounds (1, 1, 5 nd 1 µg/ml) reverted the hromosoml instility despite the presene of hydrogen peroxide (p =.1). Queretin (1, 1, 5 nd 1 µg/ml), rutin nd isoqueritrin (5 nd 1 µg/ml) nd ethyl ette (1 µg/ml) gve the est results, reversing the dmge signifintly ompred with ontrol group (p =.1) Genotoxiity Anlyzed y Alkline Comet Assy DNA dmge indued y hydrogen peroxide exposure ws evluted using the omet ssy under lkline onditions. Tle 2 shows the omet lss nd dmge index in the lymphoyte ell ultures from rude extrt, ethyl ette frtion, queretin, rutin, isoqueritrin nd queritrin (onentrtion the 5 µg/ml) otined of the S. uxifoli fter 1 h of exposure to H 2 O 2. Queretin reverted in signifint mnner the dmge used y hydrogen peroxide (p =.5) when ompred with ontrol. The ethyl ette, isoqueritrin nd rutin ell ultures presented similr dmge indexes etween tretment groups, DNA dmge ws signifintly deresed when ompred with the hydrogen peroxide group (p =.1) nd when ompred to lymphoytes from the isolted queritrin (p =.1). Conentrtion (5 µg/ml) Tle 2. Evlution of DNA dmge y the omet test in leukoytes Comet Clss ( 4) Index of DNA dmge 88 ± 3 5 ± 1 3 ± 1 2 ± 1 2 ± 1.12 H 2 O 2 42 ± 5 1 ± 2 5 ± 1 12 ± 2 29 ± 1.56 Crude extrt 53 ± 3 17 ± 2 11 ± 2 13 ± 3 6 ± 1.47 Ethyl ette frtion 62 ± 6 12 ± 2 8 ± 1 14 ± 2 4 ± 1.38 Queretin 77 ± 4 13 ± 1 4 ± 1 3 ± 1 3 ± 1.23 Queritrin 46 ± 3 11 ± 2 8 ± 1 16 ± 3 2 ± 2.55 Isoqueritrin 62 ± 2 1 ± 3 9 ±1 1 ± 2 9 ± 1.38 Rutin 66 ± 5 16 ± 2 8 ± 1 5 ± 1 8 ± 1.37 Index of DNA dmge: ( omet lss)/1. = nuleus without DNA dmge. Results re men ± SD for n Disussion The present investigtion reported the onentrtion-dependent protetive effets of ethyl ette frtion, queretin, rutin nd isoqueritrin isolted from Suti uxifoli ginst oxidtive DNA dmge indued y hydrogen peroxide in humn lymphoytes. Crude extrt nd queritrin hd less effet on hromosoml instility nd DNA dmge. These results re onsistent with severl previous studies tht reported the effets of mediinl plnts nd their extrts rih in polyphenoli/flvonoid ompounds s protetive ompounds ginst dmge medited y ROS, due to enhnement of ntioxidnt defenses [6,16,17]. In this study the presene of phenoli nd flvonoid ompounds in rude extrt nd ethyl ette frtion (12.2% nd 35.68%, respetively, Tle 1) is diretly relted to their protetive effets. Even low onentrtions suh s 1 µg/ml queretin, rutin nd isoqueritrin lredy provide lrge effet. The in vitro

8 Moleules 212, protetion ginst oxidtive DNA dmge in humn lymphoytes y queretin tretment hs een desried erlier [17 2]. Hydrogen peroxide is retive ompound tht in elevte onentrtions n generte hydroxyl rdils (OH. ) vi the Fenton retion. The OH hs highly ffinity to DNA using strnd rekge. This proess n result in DNA instility, mutgenesis nd ultimtely rinogenesis [18,2]. Therefore, the enefiil effets of flvonoids hve een reported. The ntioxidnt properties inlude their ility to inhiit xnthine oxidse tivity, to svenge superoxide nions nd hydroxyl rdils, nd to inhiit lipid peroxidtion s showed from lrge vriety of experimentl systems [21]. Free rdil svenging flvonoids effiieny is ssoited with the presene nd numer of hydroxyl groups in the B-ring of the flvonoid [22]. Experimentl prmeters, suh s growth, density nd viility, n ffet the ell response to speifi toxin. Hydrogen peroxide (25 µm) ffeted memrne integrity, the tretment with flvonoids nd extrts of the Suti uxifoli inresed the ell viility despite the presene of peroxide hydrogen. Duthie et l. [17] emphsized tht queretin deresed speifi oxidtive DNA se dmge nd the ytoprotetion ws mintined even fter the ells were wshed free of flvonoid, thus voiding its diret retion with hydrogen peroxide, queretin must t within the ell or ell memrnes nd is metolized, possily y the ytohrome P 45 mixed funtion oxidse system [23]. In this present study, we used the omet ssy, s fst nd relile method tht is le to detet genotoxiity in virtully ny mmmlin ell type [17,18], nd only queritrin hd no protetive effet in humn lymphoytes exposed to hydrogen peroxide ording to the omet ssy. 4. Experimentl 4.1. Chemils, Apprtus nd Generl Proedures Methnol, hydrogen peroxide, eti id, glli id, hlorogeni id nd ffei id purhsed from Merk (Drmstdt, Germny). Queretin, rutin, kempferol, trypn lue, peniillin G nd streptomyin sulphte were quired from Sigm Chemil Co. (St. Louis, MO, USA). Fetl ovine serum (FBS) nd RPMI 164 medium were purhsed from GIBCO Co. (Grnd Islnd, NY, USA). Milli-Q ultr-purified wter ws used in prepring the smples. High performne liquid hromtogrphy (HPLC-DAD) ws performed with Shimdzu Prominene Auto Smpler (SIL-2A) HPLC system (Shimdzu, Kyoto, Jpn), equipped with Shimdzu LC-2AT reiproting pumps onneted to DGU 2A5 degsser with CBM 2A integrtor, SPD-M2A diode rry detetor (DAD) UV-VIS detetor nd LC solution 1.22 SP1 softwre (Shimdzu, Kyoto, Jpn) Plnt Colletion nd Extrtion Leves of S. uxifoli were olleted in Dom Pedrito (Rio Grnde do Sul, Brzil) in Otoer of 27 (oordintes S nd W). Exsite ws rhived s vouher speimen in the herrium of Deprtment of Biology t Federl University of Snt Mri y register numer SMBD The leves were dried t room temperture nd powdered in knife mill (.86 µm), resulting in mss of grms of plnt mteril, whih ws sumitted to mertion t room temperture with ethnol 7% for week with dily shke. After filtrtion, the extrt ws evported under

9 Moleules 212, redued pressure to remove the ethnol nd fter this step the queous extrt ws prtitioned suessively with dihloromethne, ethyl ette nd n-utnol (3 2 ml for eh solvent). Detiled isoltion of the flvonoids used in this experiment is pulished elsewhere [16] Quntifition of Phenolis nd Flvonoids Compounds y HPLC-DAD Reverse phse hromtogrphi nlyses were rried out under grdient onditions using C 18 olumn (4.6 mm 25 mm) pked with 5 μm dimeter prtiles; the moile phse ws wter ontining 2% eti id (A) nd methnol (B), nd the omposition grdient ws: 5% (B) for 2 min; 25% (B) until 1 min; 4, 5, 6, 7 nd 8% (B) every 1 min; following the method desried y Lghri et l. [24] with slight modifitions. The rude extrt, ethyl ette frtion nd moile phse were filtered through.45 µm memrne filter (Millipore) nd then degssed y ultrsoni th prior to use, the smples of the plnt were nlyzed dissolved in methnol t onentrtion of 8 mg/ml. Stok solutions of stndrds referenes were prepred in methnol t onentrtion rnge of mg/ml for kempferol, queretin nd rutin, nd.6.25 mg/ml for glli, hlorogeni nd ffei ids. Quntifition ws rried out y integrtion of the peks using the externl stndrd method, t 254 nm for glli id, 325 nm for ffei nd hlorogeni ids, nd 365 nm for queretin, rutin nd kempferol. The flow rte ws.8 ml/min nd the injetion volume ws 4 µl. Chromtogrphi peks were onfirmed y ompring its retention time with those of referene stndrds nd y UV spetr (2 6 nm). Clirtion urve for glli id: Y = 53985x (r =.9998); hlorogeni id: Y = 52548x (r =.9985); ffei id: Y = 87846x (r =.9996); rutin: Y = 13861x (r =.9991); queretin: Y = 15833x (r =.9999) nd kempferol: Y = 13745x (r =.9989). All hromtogrphy opertions were rried out t mient temperture nd in triplite. The limit of detetion (LOD) nd limit of quntifition (LOQ) were lulted sed on the stndrd devition of the responses nd the slope using three independent nlytil urves, s defined y ICH [25]. LOD nd LOQ were lulted s 3.3 nd 1 σ/s, respetively, where σ is the stndrd devition of the response nd S is the slope of the lirtion urve Tretments To test the protetive effets of Suti uxifoli on ell viility nd protetive effet ginst hromosome dmge in humn lymphoytes we performed protool similr to tht desried y Wilms et l. [16]. Briefly, we preinuted the ell ultures with rude extrt (CE), ethyl ette frtion (EA) nd four flvonoids (queretin, queritrin, isoqueritrin nd rutin) isolted from Suti uxifoli (1 h, 37 C), four onentrtions (1, 1, 5 1 µg/ml) of these ompounds were tested here, fter the ells were wshed with PBS, ph 7.4, nd then exposed n effetive dose of hydrogen peroxide (25 µm, 1 h, 37 C) [16]. The medium ulture ws used s negtive ontrol nd the medium dded 25 µm hydrogen peroxide ws used s positive ontrol group Blood Colletion nd Lymphoyte Culture Peripherl lood smples otined from femle helthy volunteer (28 yers of ge), tht did not smoke, drink, or use hroni medition ws olleted fter 12-h overnight fsting y venipunture

10 Moleules 212, using top Vutiner (BD Dignostis, Plymouth, UK). Tues with heprin nd 5 ml ws used for glss to ultured lymphoytes in ulture medi ontining 1 ml RPMI 164 with 1% foetl lf serum (FCS) nd 1% peniillin/streptomyin nd phytohemtoglutin (PHA). The ells were mintined in suspension ulture t 37 C in humidified 5% CO 2 tmosphere in RPMI 164 growth medium (RPMI medium) for 72 h. Further we exposed the ells to tretments during 1 hour with smples, nd fter for 1 h with hydrogen peroxide. Cell viility, omet ssy, mitoti index nd hromosoml instility were nlyzed. All tretments were done in triplite Cell Viility The ell viility ws evluted efore nd fter tretment exposure using the Trypn lue dye exlusion method [26]. To perform the test we used 1 µl of eh ell suspension, whih were mixed with 1 µl of.4% Trypn lue solution nd heked for viility 3 min lter. At lest 3 ells were ounted for eh survivl determintion. The ells were nlyzed through mirosopi oservtion (Neuuer hmer) nd the perentge of vile ells ws determined. Cell viility ws expressed s perentge of the ontrol vlue Single Cell Gel Eletrophoresis (Comet Assy) The lkline omet ssy ws performed s desried y Singh et l. [27] in ordne with the generl guidelines for use of the omet ssy [28,29]. One hundred ells (5 ells from eh of the two replite slides) were seleted nd nlysed. Cells were visully sored ording to til length nd lssified s follows: lss (sene of til); lss 1 (til of up to 1 the dimeter of the nuleus of negtive ontrol); lss 2 (til of up to 2 the dimeter of the nuleus); lss 3 (til of up to 3 the dimeter of the nuleus); lss 4 (til of more thn 3 the dimeter of the nuleus). Apoptoti ells were not ounted [3]. Reeived sores from (no migrtion) to 4 (mximl migrtion). Therefore, the dmge index for ells rnged from (ll ells with no migrtion) to 4 (ll ells with mximl migrtion). The slides were nlysed under lind onditions y t lest two different individuls Mitoti Index nd Chromosoml Instility in Humn Lymphoytes After tretment exposure, one replite of eh tretment ws used to investigte the mitoti index nd hromosoml instility using G-nd ytogeneti nlysis [31]. At lest 5 mitoses were nlysed in eh smple Sttistil Anlysis All nlyses were rried out using the sttistil pkge for soil studies SPSS version 12. (SPSS In., Chigo, IL). To ompre the tretments in ll tests we performed nlysis of vrine (One-wy) followed y post ho Tukey test. We hose these sttistil tests euse previous sttistil nlysis performed using the Kolmogorov-Smirnov test showed norml distriution of the vriles investigted here. All p vlues were two-tiled. The lph vlue onsidered to e sttistilly signifint ws p =.1.

11 Moleules 212, Conlusions In onlusion, the rude extrt, ethyl ette frtion nd flvonoids isolted otined from Suti uxifoli show protetive effets ginst dmge used y hydrogen peroxide in humn lymphoytes, possily y deresing oxidtive stress due to their ntioxidnt nture [9,32]. Thus, diet ontining flvonoids ould e effetive in reduing seline nd exogenously indued oxidtive DNA dmge, however, depending on the type nd mount of flvonoids, in whih se the speifi flvonoids supplied s with supplements re more effetive. Aknowledgments The uthors wish to express grtitude to V. Btist for the ollet of S. uxifoli in this property nd to Prof. N.C.B.Záhi for the identifition of the plnt. The uthors re grteful to CNPq, CAPES nd FAPERGS/Brzil for their reserh fellowships. Referenes 1. Nsim, R.; Kikuzki, H.; Konishi, Y. Antioxidnt tivity of vrious extrts nd frtions of Chenopodium quino nd Amrnthus spp. seeds. Food Chem. 28, 16, Prk, E.J.; Pezzuto, J.M. Botnils in ner hemoprevention. Cner Metstsis Rev. 22, 21, Vlko, M.; Leifritz, D.; Monol, J.; Cronin, M.T.; Telser, J. Free rdils nd ntioxidnts in norml physiologil funtions nd humn disese. Int. J. Biohem. Cell Biol. 27, 39, Nzk, M.; Shhidi, F. Phenolis in erels, fruits nd vegetles: ourrene, extrtion nd nlysis. J. Phrm. Biomed. Anl. 26, 41, Mošovsk, S.; Mikulšov, M.; Brindzov, L.; Vlik, L.; Mikušov, L. Genotoxi nd ntimutgeni tivities of extrts from pseudoerels in the Slmonell mutgeniity ssy. Food Chem. Toxiol. 21, 48, Hyder, N.; Adelwhed, A.; Kilni, S.; Ben Ammr, R.; Mhmoud, A.; Ghedir, K.; Chekir-Ghedir, L. Antimutgeni nd free rdil svenging tivities of extrts from (Tunisin) Myrtus ommunis. Mutt. Res. 24, 564, Kilni, S.; Ben Ammr, R.; Adelwhed, A.; Hyder, N.; Mhmoud, A.; Ben Chini, J.; Chekir-Ghedir, L.; Ghedir, K. Evlution of the ntimutgeni nd ntirdil potentils of extrts from the tuers of (Tunisin) Cyperus rotundus. Environ. Toxiol. Chem. 25, 87, Yilmz, O.; Keser, S.; Tuzu, M.; Cetints, B. Resvertrol (trns-3,4,5- trihydroxystilene) dereses lipid peroxidtion level nd protets ntioxidnt pity in ser nd erythroytes of old femle Wistr rts indued y the kidney rinogen potssium romte. Environ. Toxiol. Phrm. 27, 24, Bhouri, W.; Derel, S.; Skndrni, I.; Bouker, J.; Bouhlel, I.; Sghier, M.B.; Kilni, S.; Mriotte, A.M.; Dijoux-Frn, M.G.; Ghedir, K.; Chekir-Ghedir, L. Study of genotoxi, ntigenotoxi nd ntioxidnt tivities of the diglli id isolted from Pisti lentisus fruits. Toxiol. In Vitro 21, 24,

12 Moleules 212, Amrowiz, R.; Pegg, R.B.; Rhimi-Moghddm, P.; Brl, B.; Weil, J.A. Free rdil svenging pity nd ntioxidnt tivity of seleted plnt speies from the Cndin priries. Food Chem. 21, 84, Bhtthry, S. Nturl ntimutgens: A review. Res. J. Med. Plnt 211, 5, Wsiky, R.; Wsiky, M.; Johimovits, R. Erstuntersuhungen n Coronilh Suti uxifoli Reissek. Plnt Med. 1964, 12, Morel, A.F.; Mldner, G.; Ilh, V.; Bissu, F.; Silv, U.F.; Dlol, II. Cylopeptide lkloids from Suti uxifoli Reiss nd their ntimiroil tivity. Phytohemistry 25, 66, Boligon, A.A.; Jnovik, V.; Frohlih, J.K.; Spder, T.B.; Froeder, A.L.F.; Alves, S.H.; Athyde, M.L. Antimiroil nd ytotoxi tivities of leves, twigs nd stem rk of Suti uxifoli Reissek. Nt. Prod. Res. 211, doi:1.18/ Boligon, A.A.; Agertt, V.; Jnovik, V.; Cruz, R.C.; Cmpos, M.M.A.; Guillume, D.; Athyde, M.L.; dos Sntos, A.R.S. Antimyoteril tivity of the frtions nd ompounds from Suti uxifoli. Br. J. Phrm. 212, 22, Boligon, A.A.; Pereir, R.P.; Feltrin, A.C.; Mhdo, M.M.; Jnovik, V.; Roh, J.B.T.; Athyde, M.L. Antioxidnt tivities of flvonol derivtives from the leves nd stem rk of Suti uxifoli Reiss. Bioresour. Tehnol. 2, 1, Wilms, L.C.; Hollmn, P.C.H.; Boots, A.W.; Kleinjns, J.C.S. Protetion y queretin nd queretin-rih fruit juie ginst indution of oxidtive DNA dmge nd formtion of BPDE- DNA dduts in humn lymphoytes. Mutt. Res. 25, 582, Duthie, S.J.; Collins, A.R.; Duthie, G.G.; Doson, V.L. Queretin nd myrietin protet ginst hydrogen peroxide-indued DNA dmge (strnd reks nd oxidized pyrimidines) in humn lymphoytes. Mutt. Res. 27, 393, Duthie, S.J.; Johnson, W.; Doson, V.L. The effet of dietry flvonoids on DNA dmge (strnd reks nd oxidised pyrimdines) nd growth in humn ells. Mutt. Res. 27, 39, Min, K.; Eeler, S.E. Queretin inhiits hydrogen peroxide-indued DNA dmge nd enhnes DNA repir in Co-2 ells. Food Chem. Toxiol. 29, 47, Afns ev, I.B.; Dorozhko, A.I.; Brodskii, A.V.; Kostyuk, V.A.; Potpovith, A.I. Chelting nd free rdil svenging mehnisms of inhiitory tion of rutin nd queretin in lipid peroxidtion. Biohem. Phrmol. 1989, 38, Cotelle, N.; Bernier, J.-L.; Ctteu, J.-P.; Pommery, J.; Wllet, J.-C.; Gydou, E.M. Antioxidnt properties of hydroxy- flvones. Free Rdi. Biol. Med. 1996, 2, Oermeier, M.T.; White, R.E.; Yng, C.S. Effets of ioflvonoids on hepti P 45 tivities. Xenoioti 1995, 25, Lghri, A.H.; Memon, S.; Nelofr, A.; Khn, K.M.; Ysmin, A. Determintion of free phenoli ids nd ntioxidnt tivity of methnoli extrts otined from fruits nd leves of Chenopodium lum. Food Chem. 211, 126, ICH (25). Vlidtion of Anlytil Proedures: Text nd Methodology Q2 (R1). Aville online: essed (essed on 24 April 212). 26. Burow, M.E.; Weldon, C.B.; Tng, Y.; Nvr, G.L.; Krjewski, S.; Reed, J.C.; Hmmond, T.G.; Clejn, S.; Bkmn, B.S. Differenes in suseptiility to tumour nerosis ftor lph-indued poptosis mong MCF-7 rest ner ell vrints. Cner Res. 1998, 58,

13 Moleules 212, Singh, N.; MCoy, M.; Tie, R.; Shneider, E. A simple tehnique for quntifition of low levels of DNA dmge in individuls ells. Exp. Cell Res. 1995, 175, Tie, R.R.; Agurell, D.; Anderson, D.; Burlinson, B.; Hrtmnn, A.; Koyshi, H.; Miyme, Y.; Rojs, E.; Ryu, J.C.; Sski, Y.F. Single ell gel/omet ssy: Guidelines for in vitro nd in vivo geneti toxiology testing. Environ. Mol. Mutgen. 2, 35, Hrtmnn, A.; Agurell, E.; Beevers, C.; Brendler-Shw, S.; Burlinson, B.; Cly, P.; Collins, A.; Smith, G.; Speit, G.; Thyud, V.; Tie, R.R. Reommendtions for onduting the in vivo lkline omet ssy. Mutgenesis 23, 18, Speit, G.; Hnelt, S.; Helig, R.; Seidel, A.; Hrtmnn, A. Detetion of DNA effets in humn ells with the omet ssy nd their relevne formutgenesis. Toxiol. Lett. 1996, 88, Yunis, J.J. High resolution of humn hromosomes. Siene 26, 1976, Rusk, G.; Pintnid, I.; Msi, L.; Kpurlin, K.; Durgo, K.; Kopjr, N. Spetrophotometri nlysis of flvonoid-dna intertions nd DNA dmging/proteting nd ytotoxi potentil of flvonoids in humn peripherl lood lymphoytes. Chem.-Biol. Int. 211, 188, Smple Avilility: Smples of the ompounds nd extrts re ville from the uthors. 212 y the uthors; liensee MDPI, Bsel, Switzerlnd. This rtile is n open ess rtile distriuted under the terms nd onditions of the Cretive Commons Attriution liense (