Grape stem extracts with potential anticancer and antioxidant properties

an ioxidan s A icle G ape S em Ex ac s wi h Po en ial An icance and An ioxidan P ope ies Ja ie Que o 1, Ne ea Jiménez-Mo eno 2, I ene Espa za 2, Jesús Osada 3,4 , Elena Ce ada 5, Ca men Ancín-Azpilicue a 2,* and Ma ía Jesús Rod íguez-Yoldi 1,4,*   Ci a ion: Que o, J.; Jiménez-Mo eno, N.; Espa za, I.; Osada, J.; Ce ada, E.; Ancín-Azpilicue a, C.; Rod íguez-Yoldi, M.J. G ape S em Ex ac s wi h Po en ial An icance and An ioxidan P ope ies. An ioxidan s 2021,10, 243. h ps:// doi.o g/10.3390/an iox10020243 Academic Edi o : Isabel Seique Recei ed: 27 Decembe 2020 Accep ed: 29 Janua y 2021 Published: 5 Feb ua y 2021 Publishe ’s No e: MDPI s ays neu al wi h ega d o ju isdic ional claims in published maps and ins i u ional a il- ia ions. Copy igh : © 2021 by he au ho s. Licensee MDPI, Basel, Swi ze land. This a icle is an open access a icle dis ibu ed unde he e ms and condi ions o he C ea i e Commons A ibu ion (CC BY) license (h ps:// c ea i ecommons.o g/licenses/by/ 4.0/). 1Depa men o Pha macology and Physiology, Ve e ina y Facul y, Uni e si y o Za agoza, 50013 Za agoza, Spain; ja ie que [email p o ec ed] 2Depa men o Science, Public Uni e si y o Na a a, Ins i u e o Ad anced Ma e ials and Ma hema ics (INAMAT2), 31006 Pamplona, Na a a, Spain; [email p o ec ed] (N.J.-M.); [email p o ec ed] (I.E.) 3Depa men o Biochemis y and Molecula Cell Biology, Ve e ina y Facul y, Uni e si y o Za agoza, 50013 Za agoza, Spain; josada@uniza .es 4CIBERobn, ISCIII, IIS A agón, IA2, 50009 Za agoza, Spain 5Depa men o Ino ganic Chemis y, Sciences Facul y, Uni e si y o Za agoza, 50009 Za agoza, Spain; ece ada@uniza .es * Co espondence: [email p o ec ed] (C.A.-A.); mj odyol@uniza .es (M.J.R.-Y.); Tel.: +34-948-169596 (C.A.-A.); +34-976-761649 (M.J.R.-Y.) Abs ac : The applica ion o plan ex ac s o he apeu ic pu poses has been used in adi ional medicine because plan s con ain bioac i e compounds wi h bene icial p ope ies o heal h. Cu en ly, he use o hese compounds ha a e ich in polyphenols o he ea men and p e en ion o diseases such as cance , diabe es, and ca dio ascula diseases, many o hem ela ed o oxida i e s ess, is gaining ce ain ele ance. Polyphenols ha e been shown o ha e an imu agenic, an ioxidan , and an i-in lamma o y p ope ies. The e o e, he objec i e o he p esen wo k was o s udy he po en ial e ec o g ape s em ex ac s (GSE), ich in phenolic compounds, in he ea men o cance , as well as hei ole in he p e en ion o his disease associa ed wi h i s an ioxidan powe . Fo ha pu pose, h ee cance lines (Caco-2, MCF-7, and MDA-MB-231) we e used, and he esul s showed ha g ape s em ex ac s we e capable o showing an an ip oli e a i e e ec in hese cells h ough apop osis cell dea h associa ed wi h a modi ica ion o he mi ochond ial po en ial and eac i e oxygen species (ROS) le els. Addi ionally, g ape s em ex ac s showed an an ioxidan e ec on di e en ia ed in es inal cells ha could p o ec he in es ine om diseases ela ed o oxida i e s ess. The e o e, g ape ex ac s con ain bioac i e p inciples wi h impo an biological p ope ies and could be used as bio- unc ional ood ing edien s o p e en diseases o e en o imp o e ce ain aspec s o human heal h. Keywo ds: cance cells; polyphenols; g ape s em; p o easome; ROS; T xR1 1. In oduc ion G ape s ems a e by-p oduc s gene a ed in g ea quan i y in he winemaking p ocess, and hei elimina ion causes en i onmen al p oblems. The e o e, i is impo an o ind s a egies ha allow he euse o hese p oduc s. This esidue is a ich sou ce o phenolic compounds, celluloses, hemicelluloses, and lignins [ 1 – 6 ]. Among hem, phenolic com- pounds con e an ioxidan p ope ies o he ex ac s ob ained om g ape s ems. Fo his eason, di e en s udies ha e been conduc ed in o de o de e mine he polyphenolic composi ion o g ape s ems, and se e al p oan hocyanidins, an hocyanidins, la onols, hyd oxycinnamic acids, and s ilbenes ha e been ound. Among hem, he mos cha ac- e is ic polyphenolic subs ances e e ed o in mos o he s udies a e ans- es e a ol, ε - ini e in, ca a ic acid, gallic acid, ca echin (one o he mos abundan polyphenols), epica echin, mal idin de i a i es, que ce in, and glycosyla ed de i a i es o que ce in in posi ion 3 [2,3,5,7]. An ioxidan s 2021,10, 243. h ps://doi.o g/10.3390/an iox10020243 h ps://www.mdpi.com/jou nal/an ioxidan s An ioxidan s 2021,10, 243 2 o 17 Acco ding o cu en esea ch esul s, g ape s em ex ac s possess impo an biological ac i i ies wi h mul iple bene i s o human heal h due o an ioxidan and an i-in lamma o y p ope ies [ 5 , 7 – 11 ]. Veskoukis e al. [ 10 ] ound ha his by-p oduc is pa icula ly ich in la onoids and s ilbenes, such as ans- es e a ol and ini e in, which a e ound in conside ably high concen a ions. These au ho s also ound ha such ex ac s exhibi ed signi ican an ioxidan p ope ies, and, e en a low concen a ions, hey showed a s ong abili y o p e en he oxida ion o low-densi y lipop o ein (LDL) and o educe in acellula le els o eac i e oxygen species (ROS). In his way, Gonzalez-Cen eno e al. [ 12 ] and Veskoukis e al. [10] e alua ed he o al phenolic and o al p oan hocyanidin composi ion o di e en g ape s em a ie ies, as well as hei an ioxidan po en ials. G ape s em ex ac s also p e en ROS-induced DNA damage and ha e inhibi o y ac i i y agains li e and ce - ical cance cell g ow h, sugges ing hei po en ial as chemop e en i e agen s [ 13 ]. Using human epide mal ke a inocy es, Domínguez-Pe les e al. [ 14 ] obse ed a p o ec i e e ec o g ape s em ex ac s agains oxida i e s ess. These esea che s ound a close co ela ion be ween he concen a ion o phenolic compounds in he ex ac s and he po en ial o egula e he edox balance in i o , as well as he capaci y o hese ex ac s o e icien ly modula e apop osis in HaCaT ke a inocy es. Cho e al. [ 15 ] s udied he e ec o he opical adminis a ion o g ape s em ex ac s o mice skin be o e subjec ing hem o UVB adia ion o h ee minu es, h ice a week o one mon h. These au ho s demons a ed ha hese ex ac s signi ican ly inhibi ed oxida i e damage induced by UVB adia ion and obse ed dec eased epide mal hype plasia, melanin pigmen a ion, and collagen deg ada ion in he skin o mice. In addi ion, g ape pomace—consis ing o peel, seed, s em, and pulps—is disca ded du ing g ape p ocessing, including juice ex ac ion and winemaking, despi e i s subs an ial phenolic con en [ 8 ]. In his way, Del Pino-Ga cia e al. [ 16 ] s udied he chemop e en i e po en ial o powde ed ed wine seasonings agains colo ec al cance in HT-29 cells. G ape seed ex ac s ha e also been applied as pho ochemop e en i e agen s agains UVB-induced skin cance [ 17 ]. Likewise, Vi is ini e a ex ac s ha e shown an an idiabe ic e ec by inhibi ing he enzyme glycogen phospho ylase [18]. On he o he hand, g ape s em ex ac s ha e signi ican an imic obial ac i i ies ha seem o be in luenced by he s uc u e and unc ion o phenolic compounds, as well as by hei in e speci ic ela ion wi h di e en bac e ial s ains [ 19 , 20 ]. Fo example, hese ypes o ex ac s inhibi ed he g ow h o bo h G am-posi i e (Lis e ia monocy ogenes, S aphylococcus au eus and En e ococcus aecalis) and G am-nega i e (Pseudomonas ae uginosa, Esche ichia coli and Klebsiella pneumoniae) diges i e pa hogens unde in i o condi ions [ 21 ], and hei bioac i e compounds a e used in o al ca e [22]. In addi ion, he ex ac s could be used o con ol he p esence o human pa hogenic bac e ia in esh lea y ege ables [ 6 ]. Leal e al. [ 23 ] s udied he po en ial o g ape s em ex ac s om di e en whi e g ape a ie ies as an imic obial agen s o educe he use o an ibio ics. These au ho s ound ha he bac e icidal ac i i y o he ex ac s was highe , in gene al, agains G am-posi i e han G am-nega i e bac e ia, al hough hey used a di e en me hodology om ha o Dias e al. [21] o e alua e he an imic obial ac i i y (minimum inhibi o y concen a ion s. disc di usion). O he s udies ha e shown ha g ape s em ex ac s a e highly e ec i e agains oo wound ulce s p oduced by G am-posi i e bac e ia, and hey also ha e an i-in lamma o y ac ion, inhibi ing he p oduc ion o ni ic oxide lipopolysaccha ide-s imula ed mac ophages by up o 35.25% [24]. Du ing he las ew yea s, ou esea ch g oup has in es iga ed he chemop e en i e p ope ies o ex ac s ob ained om di e en plan ma ices such as osehips, enug eek, pine ba k, and a ichoke was e on human colon cance [3,25–27]. Though he e ha e only been ew publica ions on his subjec , he an ica cinogenic po en ial o g ape s em ex ac s has also been s udied in di e en cell lines [ 13 , 28 , 29 ]. Addi ionally, g ape s em ex ac s possess impo an bioac i i ies such as an iangiogenic p ope ies [30]. Howe e , he phenolic composi ion—and he e o e he biological ac i i y and e icacy—o a speci ic g ape s em ex ac depends on he p ocedu e used o ob ain he ex ac . In a p e ious s udy, we selec ed an op imized ex ac ion me hod o g ape s ems [ 3 ], and he An ioxidan s 2021,10, 243 3 o 17 ex ac s ob ained by his me hod p esen ed high an ioxidan po en ial and we e demon- s a ed o be good candida es o SO 2 subs i u ion in wines [ 4 ]. Wi h his backg ound, he aim o he p esen esea ch was o comple e he cha ac e iza ion o hose g ape s em ex ac s by s udying hei po en ial o he ea men o human colo ec al adenoca cinoma (Caco-2) and human b eas adenoca cinoma (MCF-7 and MDA-MB-231) cell lines. Thus, we measu ed he possible an ip oli e a i e e ec s o hese ex ac s on cance cells and hei mechanisms o ac ion. Fu he mo e, he p o ec i e e ec s o hese ex ac s, in a model o in es inal ba ie (di e en ia ed Caco-2 cells), we e also es ed h ough he measu emen o he in acellula le els o ROS. 2. Ma e ials and Me hods 2.1. Ex ac s The g ape s em ex ac was ob ained h ough an ex ac ion me hod using GRAS sol en s om Mazuelo- a ie y s ems ha es ed in he 2016 in age [ 3 ]. B ie ly, g ape s ems we e o en-d ied a 25 ◦ C, g ound, and sie ed ( φ < 0.3 mm). The ex ac was ob ained a e mace a ing he g ound and sie ed s ems in 50% e hanol/wa e , wi h a 1:100 (w/ ) a io and a 40 ◦ C o 24 h. Then, he ex ac was cen i uged (8000 pm o 15 min), il e ed h ough il e pape , and lyophilized (Tels a C yodos eeze d ie , Mad id, Spain). 2.2. Chemicals All he used HPLC sol en s we e om Scha lab (Ba celona, Spain). All he used phenolic s anda ds we e om Sigma-Ald ich (Mad id, Spain), wi h he excep ion o mal idin-3-glucoside (enyn-chlo ide, Ex asyn hese, Genay, F ance). Among he chemicals o spec opho ome ic analysis, he Folin–Ciocal eu eagen , T olox (6-hyd oxy-2,5,7,8- e ame hylch oman-2-ca boxylic acid), gallic acid, and que ce in we e supplied by Sigma- Ald ich (Mad id, Spain); glacial ace ic acid, anhyd ous sodium ca bona e, and aluminum chlo ide 6-hyd a e we e supplied by PanReac AppliChem (Ba celona, Spain). 2.3. Iden i ica ion and Quan i ica ion o Phenolic Composi ion o G ape S em Ex ac s by HPLC-DAD The iden i ica ion and quan i ica ion o he phenolic compounds p esen in he g ape s em ex ac s we e pe o med using high-pe o mance liquid ch oma og aphy. The ch o- ma og aph was equipped wi h wo 510 pumps, a 717 Plus au osample , and a 996 pho odi- ode a ay de ec o (Wa e s Di ., Mil o d, MA, USA). A Zo bax Eclipse Plus C18 e e sed phase column (250 × 4.6 mm; pa icle size o 5 µ m) (Agilen , San a Cla a, CA, USA) was used. Fo he analyses o he ex ac , be ween 45.0 ± 0.1 and 70.0 ± 0.1 mg o each sample we e weigh ed and dissol ed in 10 mL o me hanol wi h he aid o an ul asonic ba h (JP Selec a, Ba celona, Spain). Samples we e p epa ed in iplica e and analyzed once. The ch oma og aphic analyses we e ca ied ou acco ding o a modi ied me hod o Ba - os e al. [ 31 ]. Two mobile phases, A (wa e : 85% o mic acid, 99:1 / ) and B (ace oni ile: 85% o mic acid, 99:1 / ) we e used. The low a e was 1 mL/min using he ollowing linea g adien scheme ( in min; % A): 0, 95%; 15, 85%; 22, 80%; 25, 80%; 35, 70%; 45, 50%; 50, 5%; 55, 95%; and 60, 95%. The column empe a u e was 30 ◦ C, and he injec ion olume was 40 µ L. The iden i ica ion o he di e en compounds was pe o med by he double coincidence o he e en ion ime o i s co esponding s anda d and he UV–Vis spec um o each compound. Quan i ica ion was ca ied ou using calib a ion cu es o each analyzed compound. The calib a ion cu es used o es e a ol, gallic acid, que ce in, mal idin-3-glucoside, and ca a ic acid p esen ed linea co ela ion coe icien s highe han 0.999. The calib a ion cu es ob ained o he es o compounds ( ini e in, ca echin, and he de i a i e o que ce in) showed linea co ela ion coe icien s highe han 0.998. In he case o he uniden i ied an hocyanin, i was no possible o iden i y i s s uc u e wi h he me hod used in he labo a o y. Howe e , mos o he an hocyanins desc ibed in he li e a u e ha a e p esen in g ape s ems co espond o de i a i es o mal idin. Fo his eason, and gi en he ac ha all an hocyanins ha e simila gene al s uc u es, we An ioxidan s 2021,10, 243 4 o 17 used he calib a ion cu e o mal idin-3-glucoside o es ima e he concen a ion o he unknown an hocyanin. 2.4. De e mina ion o An ioxidan Capaci y o he G ape S em Ex ac s by DPPH The DPPH (2,2-diphenyl-1-pyc ilhyd acyl) assay was based on he me hod p oposed by B and-Williams e al. [ 32 ]. A s anda d solu ion o 24 mg o DPPH in 100 mL o me hanol was p epa ed, and hen i was dilu ed in me hanol un il we ob ained an abso bance o 0.9 ± 0.1 a 517 nm in a UV–Vis spec opho ome e (Jenway, S a o dshi e, UK). Fo he calib a ion cu e, se en di e en T olox s anda ds we e p epa ed in me hanol in concen a ions om 0.05 o 0.73 mM. Fo sample p epa a ion, be ween 50.0 ± 0.1 and 72.0 ±0.1 mg o ex ac we e dissol ed in 10 mL o me hanol, and he esul ing mix u e was dilu ed 10 imes wi h me hanol. Fo analysis, 150 µ L o he T olox s anda d solu ion o p ocessed sample we e mixed wi h 2.85 mL o he DPPH solu ion. A e 30 min in da kness, he an ioxidan capaci ies o all he s anda ds and samples we e de e mined by measu ing he abso bance a 517 nm. Fo each ba ch o ex ac , h ee di e en p ocessed samples we e p epa ed, and each o hem was analyzed once. The linea co ela ion coe icien ob ained o he calib a ion cu e was R 2 > 0.998. The esul s o an ioxidan capaci y we e exp essed as mmol T olox/g o ex ac . 2.5. Spec opho ome ic De e mina ion o To al Phenolic and Fla onoid Con en o he G ape S em Ex ac s To al phenolic con en was analyzed using he Folin–Ciocal eu me hod, as desc ibed by Single on e al. [ 33 ]. Fo he calib a ion cu e, di e en gallic acid s anda ds we e p epa ed in me hanol in concen a ions om 0.2 o 4.6 mM. Fo sample p epa a ion, be ween 50.0 ±0.1 and 72.0 ± 0.1 mg o ex ac we e dissol ed in 10 mL o me hanol. Fo analysis, 100 µ L o he gallic acid s anda d solu ion o p ocessed sample we e mixed wi h 0.5 L o he Folin–Ciocal eu eagen , 7.9 mL o deionized wa e , and 1.5 mL o Na 2 CO 3 (20% w/w), and he esul ing solu ions we e le o 2 h in da kness. The abso bance was measu ed a 765 nm in a UV–Vis spec opho ome e (Jenway, S a o dshi e, UK). The s anda d used o he calib a ion cu e was gallic acid, anging be ween 0.2 and 5.08 mM. The linea co ela ion coe icien ob ained o he calib a ion cu e was R 2 > 0.999. Fo each ba ch o ex ac , h ee di e en p ocessed samples we e p epa ed, and each o hem was analyzed once. The esul s o o al phenolic con en we e exp essed as mg gallic acid/g ex ac s. The o al la onoid con en was de e mined by he colo ime ic me hod o aluminum chlo ide using a solu ion o 2% AlCl 3 in 5% ace ic acid [ 34 ]. Fo he calib a ion cu e, di e en que ce in s anda d solu ions we e p epa ed in me hanol in concen a ions om 3 o 30 µ g/mL. Fo sample p epa a ion, be ween 50.0 ± 0.1 and 72.0 ± 0.1 mg o ex ac we e dissol ed in 10 mL o me hanol. Fo analysis, 1.5 mL o he que ce in s anda d solu ion o sample we e mixed wi h 1.5 mL o he AlCl 3 solu ion, and he esul ing solu ions we e le o 30 min in da kness. Then, abso bance was measu ed on a Jenway UV–Vis spec opho ome e a 420 nm. The linea co ela ion coe icien ob ained o he calib a ion cu e was R 2 > 0.999. Fo each ba ch o ex ac , h ee di e en p ocessed samples we e p epa ed, and each o hem was analyzed once. The esul s we e exp essed as mg o que ce in/g ex ac s. In all cases, he samples we e analyzed in iplica e. 2.6. Cell Cul u e Human Caco-2 cell line (TC7 clone) was kindly p o ided by D . Edi h B o -La oche (Uni e si éPie e e Ma ie Cu ie-Pa is 6, UMR S 872, Les Co delie s, F ance). Human b eas adenoca cinoma MDA-MB-231 cells we e kindly p o ided by D . Ca los J. Ciudad and D . Ve ònica Noé(Depa amen o de Bioquímica y Fisiología, Facul ad de Fa ma- cia, Uni e sidad de Ba celona, Spain). Human b eas adenoca cinoma MCF-7 cells we e kindly p o ided by C is ina Sanchez-de-Diego (Depa amen o de Fisiología II, Uni e - sidad de Ba celona, Spain). Human ib oblas cells we e kindly p o ided by D . Julio Mon oya (Depa amen o de Bioquimica, Uni e sidad de Za agoza, Spain). All cell lines An ioxidan s 2021,10, 243 5 o 17 we e main ained in a humidi ied a mosphe e o 5% CO 2 a 37 ◦ C. Cells we e g own in Dulbecco’s Modi ied Eagle Medium (DMEM) supplemen ed wi h 20% e al bo ine se um (FBS), 1% non-essen ial amino acids, 1% penicillin (1000 U/mL), 1% s ep omycin (1000 µ g/mL), and 1% ampho e icin (250 U/mL). The cells we e enzyma ically passaged wi h 0.25% ypsin–1 mM EDTA and sub-cul u ed on 25 cm 2 plas ic lasks a a densi y o 5×105cells/cm2 . The cul u e medium was eplaced e e y 2 days. Ex ac ea men s we e added 24 h pos -seeding o assays on undi e en ia ed Caco-2 cells [ 35 ] and 10–15-days pos -seeding on di e en ia ed Caco-2, MCF-7, and MDA cells. Cell con luence (80%) was con i med by op ical mic oscopy obse ance. 2.7. Cell T ea men and An ip oli e a i e P ope y Analysis Ex ac s om g ape s ems we e dilu ed in a cell cul u e medium o a inal concen- a ion 1.5 mg/mL. Fo cy o oxici y sc eening assays, he cells we e seeded in 96-well pla es a a densi y o 4 × 10 3 cells/well. The cul u e medium was eplaced wi h a medium con aining plan ex ac s, and cells we e incuba ed o 48 o 72 h. The an ip oli e a i e e ec was measu ed wi h he sul o hodamine B (SRB) assay, as p e iously desc ibed [ 36 ]. Abso bance a 540/620 nm was measu ed wi h he SPECTROs a Nano (BMG Lab ech, O enbe g, Ge many). The e ec on cell g ow h was exp essed as a pe cen age o he con ol. Finally, he IC 50 alue was calcula ed unde all condi ions es ed. IC 50 ep esen s he concen a ion o compound ha hal es cell p oli e a ion o iabili y. This alue was selec ed o u he analysis o elucida e he ex ac s’ mechanism o ac ion on cance cells. 2.8. Measu emen s o Apop osis The cells we e seeded in 25 cm 2 lasks (5 × 10 5 cells/cm 2 ), exposed o plan ex ac s o 48 h a he IC 50 concen a ion, and hen collec ed and s ained wi h annexin V-FITC and p opidium iodide, as p e iously desc ibed [ 37 ]. A nega i e con ol was p epa ed by un ea ed cells, and i was used o de ine he basal le el o apop o ic and nec o ic o dead cells. A e incuba ion, cells we e ans e ed o low cy ome y ubes and washed wice wi h phospha e-bu e ed saline (PBS), ollowed by a esuspension in 100 µ L o he annexing V binding bu e (100 mM HEPES/NaOH pH 7.4, 140 mM NaCl, and 2.5 mM CaCl 2 ). To each ube, 5 µ L o annexin V-FITC and 5 µ L o p opidium iodide we e added. A e 15 min o incuba ion a oom empe a u e in he da k, 400 µ L o he annexin binding bu e we e added and analyzed by low cy ome y wi hin 1 h. The signal in ensi y was measu ed using a BD FACSA ia TM cell so e (BD Biosciences, San Jose, CA, USA) and analyzed using he BD FASCDi aTM so wa e (BD Biosciences, San Jose, CA, USA). 2.9. Flow Cy ome y Mi ochond ial Memb ane Po en ial Assay Cells we e seeded in 25 cm 2 lasks and hen exposed o plan ex ac s o 48 h. The con ol cells we e incuba ed wi h a new medium wi hou ea men . Then, cells we e washed wice wi h PBS. The pelle was esuspended in PBS a concen a ion o 10 6 cell/mL, and 5 µ L o 10 µ M 1,1 0 ,3,3,3 0 -hexame hylindodica bo-cyanine iodide (DiIC1) we e added o each sample. Tubes we e incuba ed a 37 ◦ C o 15 min, and 400 µ L o PBS we e added p io o analyze luo escence wi h BD FACSa ay TM (BD Biosciences, San Jose, CA, USA) equipped wi h an a gon ion lase . The exci a ion and emission se ings we e 633 and 658 nm, espec i ely [37]. 2.10. De e mina ion o In acellula Le els o Reac i e Oxygen Species (ROS) The cells we e seeded in 96-wells pla e a a densi y o 4 × 10 3 cells/well. The in- acellula le el o ROS was assessed using he dichlo o luo escein assay, as p e iously desc ibed [ 37 ]. Cells we e cul u ed 24 h be o e being incuba ed wi h s em ex ac s and hen unde wen oxida i e s ess induc ion by adding H 2 O 2 (80 mM) o 20 min. A e ha , he medium was emo ed, cells we e washed wice wi h PBS, and cells we e incuba ed o 1 h wi h 20 µ M 2 0 ,7 0 -dichlo o luo escein diace a e (DCFH-DA) in PBS a 37 ◦ C. The o ma ion o he luo escence oxidized de i a i e o DCF was moni o ed a an emission wa eleng h An ioxidan s 2021,10, 243 6 o 17 o 535 nm and an exci a ion o 485 nm in a FLUOs a Omega (BMG Lab ech, O enbe g, Ge many) mul ipla e eade . A measu e a ime “ze o” was pe o med, cells we e incu- ba ed a 37 ◦ C in he mul ipla e eade , and he gene a ion o luo escence was measu ed a e 20 min. ROS le els we e exp essed as a pe cen age o luo escence compa ed o he con ol. The ob ained alues o luo escence in ensi y a e conside ed as a e lec ion o o al in acellula ROS con en . 2.11. De e mina ion o P o easome Ac i i y The cells (5 × 10 5 cells/cm 2 ) we e seeded in a cell cul u e lask (25 cm 2 ). The de e - mina ion o he p o easome ac i i y was ca ied ou wi h a luo ome ic assay using a p o easome 20S ac i i y assay ki (MAK172, Sigma-Ald ich, Mad id, Spain) based on Suc- LLVy-AMC, a luo ogenic subs a e o he p o easome β 5 submi . Caco-2 cells we e ea ed wi h g ape s em ex ac s o 24 h pos -seeding and hen p ocessed ollowing ins uc ions in he ki p o ocol. The luo escence le els co espond o he p o easomal chymo ypsin-like ac i i y (CT-L ac i i y). The ac i i y was measu ed in lysed cells wi h FLUOs a Omega (BMG Lab ech, O enbe g, Ge many), and he alue was ob ained pe mg o p o ein. The da a a e exp essed in % CT-L ac i i y. 2.12. Thio edoxin Reduc ase 1 (T xR1) Ac i i y The cells we e seeded in a 96-well pla e wi h g ape s em ex ac s o 24 h. The cells we e hen lysed and incuba ed wi h a shaking mo ion o 20 min be o e adding 25 µ L/well o he eac ion bu e (500 µ L o PBS pH 7.4, 80 µ L o 100 mM EDTA pH 7.5, 20 µ L o 0.05% BSA, 100 µ L o 20 mM NADPH, and 300 µ L o dis illed H 2 O), and he eac ion was s a ed wi h DTNB (20 mM in pu e e hanol), as p e iously desc ibed by Allaoui e al. [ 26 ]. The abso bance inc ease was ollowed a 405 nm wi h SPECTROs a Nano (BMG Lab ech, O enbe g, Ge many) e e y minu e o 6 min. The alue was ob ained pe mg o p o ein and exp essed in % hio edoxin educ ase (T xR) ac i i y. 2.13. S a is ical Analysis All assays we e pe o med a leas h ee imes. Da a a e p esen ed as mean ± SD. Means we e compa ed using ANOVA. Signi ican di e ences a p< 0.05 we e compa ed using a Bon e oni’s mul iple compa ison es . The s a is ical analyses we e pe o med and he g aphics we e ob ained using he G aphPad P ism Ve sion 5.02 so wa e o Windows (G aphPad So wa e San Diego, CA, USA). 3. Resul s and Discussion Plan polyphenols ep esen a a ie y o bioac i e compounds ha a e capable o p e en ing and con olling cance and diabe es, as well as neu odegene a i e, au oimmune, ca dio ascula , and oph halmic diseases [ 38 ]. The p esence o polyphenol compounds in g ape s ems gi es hem excep ional biological alue [ 39 ]. I has been shown ha ex ac s de i ed om g ape s ems possess po en an ioxidan ac i i y in i o [ 40 ] and in cell lines [7], whe eas hei an ica cinogenic ole has no been widely epo ed. The p esen s udy in es iga ed he biological p ope ies o g ape s em ex ac s on di e en cance cells, as well as hei mechanisms o ac ion. Fu he mo e, he ex ac s’ e ec s on he p e en ion o oxida i e s ess in a model o di e en ia ed in es inal cells was also s udied. 3.1. Phenolic Composi ion and An ioxidan Ac i i y in Mazuelo S em Ex ac s The phenolic composi ion, as well as he o al polyphenol and la onoid con en s, o Mazuelo s em ex ac s a e p esen ed in Table 1. In his ex ac , nine phenolic compounds we e ound, o which he mos abundan we e (+)-ca echin and he que ce in-3-de i a i e. Likewise, Leal e al. [ 24 ] ound ha (+)-ca echin was he mos abundan phenolic compound in Po uguese g ape s em ex ac s om di e en a ie ies (Tin a Ro iz, Tou iga Nacional, Cas elão, Sy ah, A in o, and Fe não Pi es). Anas asiadi e al. [ 11 ] also epo ed he p esence An ioxidan s 2021,10, 243 7 o 17 o se e al phenolic compounds in g ape s em ex ac s om six ed and whi e a ie ies om G eece. In compa ison o hei esul s wi h ou Mazuelo s em ex ac , ans- es e a ol, ε - ini e in, (+)-ca echin, and ca a ic acid coincide, (+)-ca echin was ound o be he mos abundan in bo h s udies. Rega ding he concen a ions o es e a ol and ini e in, hese au ho s obse ed di e ences among a ie ies and in ages. Lambe e al. [ 41 ] analyzed he s ilbene con en o p uning canes o he Ca ignan a ie y, which is he name gi en in F ance o he Mazuelo a ie y. These au ho s ound a highe amoun o es e a ol and ini e in in hei ex ac s (0.88 mg es e a ol/g ex ac and 0.97 mg ini e in/g ex ac ), al hough i mus be conside ed ha g ape ine canes a e p obably iche in s ilbenes han g ape s ems [ 42 , 43 ]. In addi ion o he phenolic compounds ound in he Mazuelo s ems analyzed in his wo k, o he compounds ha e been iden i ied in g ape s em ex ac s o di e en a ie ies. Fo ins ance, in s em ex ac s om Po uguese g apes, kaemp e ol and iso hamne in we e iden i ied [ 2 ], and in g ape s ems om G eek a ie ies, e ulic, couma ic, ca eic, and sy ingic acids we e iden i ied [28]. Table 1. Phenolic composi ion (mg/g ex ac ) and an ioxidan capaci y o he Mazuelo s em ex ac . Phenolic Composi ion & An ioxidan Capaci y G ape S em Ex ac Gallic acid 0.21 ±0.03 Ca a ic acid 0.14 ±0.03 (+)-Ca echin 0.98 ±0.20 Que ce in 0.05 ±0.01 Que ce in-de i a i e 10.91 ±0.08 Mal idin-3-glucoside 0.10 ±0.02 Unknown an hocyanin 20.15 ±0.02 T ans- es e a ol 0.26 ±0.04 T ans-ε- ini e in 0.59 ±0.09 To al phenolic con en 383 ±2 To al la onoid con en 42.6 ±0.1 An ioxidan capaci y by DPPH 50.47 ±0.04 1 Exp essed as que ce in-3-glucoside; 2 exp essed as mal idin-3-glucoside; 3 exp essed as mg gallic acid/g ex ac ; 4exp essed as mg que ce in/g ex ac ; 5exp essed as mmol T olox/g ex ac . Rega ding he an ioxidan capaci y measu ed by he DPPH assay (Table 1), he esul o he Mazuelo s em ex ac was simila o ha o he Sy ah (0.44 ± 0.04 mmol T olox/g) and Fe não Pi es (0.55 ± 0.01 mmol T olox/g) ex ac s and highe han ha o he Cas elão (0.31 ± 0.01 mmol T olox/g) and A in o (0.15 ± 0.01 mmol T olox/g) a ie ies ound by Leal e al. (2020). 3.2. E ec o Ex ac s F om G ape S em on Cance Cells 3.2.1. An ip oli e a i e Ac i i y The oxici y o ex ac s om g ape s ems was e alua ed on undi e en ia ed Caco-2, MCF-7, and MDA-MB-231 cells by an SRB assay. Ini ially, a ange o concen a ions o g ape ex ac s (62.5, 125, 250, 500, and 1000 µ g/mL) was es ed. The concen a ions chosen we e in ela ion o p e ious wo k ca ied ou by ou esea ch g oup wi h o he plan ex ac s [ 25 , 36 ]. The IC 50 was calcula ed in he di e en cell lines a wo ime-poin s o 48 and 72 h. Howe e , in he MDA-MB-231 and MCF-7 cells, when ea ed o 72 h, his ange was le hal in mos concen a ions and he IC 50 could no be calcula ed, so he ange was modi ied o dec eased concen a ions ( ange: 9, 18. 37.5, 75, and 200 µ g/mL). These esul s sugges ha cy o oxic e ec o g ape s em ex ac s (GSE) is concen a ion- and ime- dependen and ha Caco-2 cells a e less sensi i e o GSE a he highes incuba ion ime. A 48 h, simila iabili y cu es we e ob ained in he h ee di e en cell lines ( Figu e 1 , Table 2). An ioxidan s 2021,10, 243 8 o 17 An ioxidan s 2021, 10, x FOR PEER REVIEW 8 o 17 3.2. E ec o Ex ac s F om G ape S em on Cance Cells 3.2.1. An ip oli e a i e Ac i i y The oxici y o ex ac s om g ape s ems was e alua ed on undi e en ia ed Caco-2, MCF-7, and MDA-MB-231 cells by an SRB assay. Ini ially, a ange o concen a ions o g ape ex ac s (62.5, 125, 250, 500, and 1000 µg/mL) was es ed. The concen a ions chosen we e in ela ion o p e ious wo k ca ied ou by ou esea ch g oup wi h o he plan ex ac s [25,36]. The IC50 was calcula ed in he di e en cell lines a wo ime-poin s o 48 and 72 h. Howe e , in he MDA-MB-231 and MCF-7 cells, when ea ed o 72 h, his ange was le hal in mos concen a ions and he IC50 could no be calcula ed, so he ange was modi ied o dec eased concen a ions ( ange: 9, 18. 37.5, 75, and 200 µg/mL). These esul s sugges ha cy o oxic e ec o g ape s em ex ac s (GSE) is concen a ion- and ime- dependen and ha Caco-2 cells a e less sensi i e o GSE a he highes incuba ion ime. A 48 h, simila iabili y cu es we e ob ained in he h ee di e en cell lines (Figu e 1, Table 2). Figu e 1. Measu emen o Caco-2, MCF-7, MDA-MB-231, and ib oblas cell iabili y a 48 and 72 h a e incuba ion wi h g ape s em ex ac s (GSE). The GSE concen a ions es ed in he ou ypes o cells we e 62.5, 125, 250, 500, and 1000 µg/mL, bu a 72 h in he MCF-7 and MDA-MB-232 cells, he chosen concen a ions we e 9, 18, 37.5, 75, and 200 µg/mL. Table 2. IC50 ( he concen a ion o compound ha hal es cell p oli e a ion o iabili y) alues o g ape s em ex ac s on Caco-2, MCF-7, MDA-MB-231, and ib oblas cells a e 72 and 48 h o incuba ion. IC50 (µg/mL) 72 h IC50 (µg/mL) 48 h Selec i i y Index Caco-2 759 ± 51 661 ± 48 2.9 MCF-7 203 ± 53 817 ± 52 * 7.2 MDA-MB-231 85 ± 9 911 ± 10 * 17.0 Fib oblas 1454 ± 6 - - * p < 0.05; incuba ion ime 48 s. 72 h. The esul s showed ha he g ape s em ex ac s we e no selec i e o a single cance line, bu hey p oduced a dec ease in iabili y in he h ee es ed cell lines (Figu e 1). The e ec was as e in Caco-2 cells, al hough hei e ec i eness was g ea e in b eas cells (MCF-7 and MDA-MB-231) a longe imes (72 h). In o de o de e mine he ac ion o hese ex ac s on a noncance ous model, he IC50 was calcula ed on human ib oblas cells, a e 72 h o incuba ion, whe e we obse ed a signi ican ly lowe e ec . These da a could be Figu e 1. Measu emen o Caco-2, MCF-7, MDA-MB-231, and ib oblas cell iabili y a 48 and 72 h a e incuba ion wi h g ape s em ex ac s (GSE). The GSE concen a ions es ed in he ou ypes o cells we e 62.5, 125, 250, 500, and 1000 µ g/mL, bu a 72 h in he MCF-7 and MDA-MB-232 cells, he chosen concen a ions we e 9, 18, 37.5, 75, and 200 µg/mL. Table 2. IC 50 ( he concen a ion o compound ha hal es cell p oli e a ion o iabili y) alues o g ape s em ex ac s on Caco-2, MCF-7, MDA-MB-231, and ib oblas cells a e 72 and 48 h o incuba ion. IC50 (µg/mL) 72 h IC50 (µg/mL) 48 h Selec i i y Index Caco-2 759 ±51 661 ±48 2.9 MCF-7 203 ±53 817 ±52 * 7.2 MDA-MB-231 85 ±9 911 ±10 * 17.0 Fib oblas 1454 ±6 - - *p< 0.05; incuba ion ime 48 s. 72 h. The esul s showed ha he g ape s em ex ac s we e no selec i e o a single cance line, bu hey p oduced a dec ease in iabili y in he h ee es ed cell lines (Figu e 1). The e ec was as e in Caco-2 cells, al hough hei e ec i eness was g ea e in b eas cells (MCF-7 and MDA-MB-231) a longe imes (72 h). In o de o de e mine he ac ion o hese ex ac s on a noncance ous model, he IC 50 was calcula ed on human ib oblas cells, a e 72 h o incuba ion, whe e we obse ed a signi ican ly lowe e ec . These da a could be used o ob ain a selec i i y index (SI), as p e iously desc ibed by Badisa el al. [ 44 ]. The SI esul s a e shown in Table 2, wi h he highes alue being o he MDA-MB-231 cell line, acco ding wi h he highes e ec i e esponse o he ex ac s owa ds hese cells a e 72 h o incuba ion. The obse ed di e ence in he wo b eas cance lines could ha e been due o he ac ha he ac ion o hese ex ac s could be ela ed o he ecep o s’ exp ession o es ogens, which a e only p esen in MCF-7 cells [45]. 3.2.2. Cell Dea h S udies Since he g ape s em ex ac s p oduce a educ ion in cell iabili y, i was decided o de e mine wha ype o cell dea h occu ed. Thus, low cy ome y analyses o e 48 h we e pe o med using bioma ke s o cell dea h. The esul s showed ha ea men o 48 h wi h he IC 50 concen a ion co esponding o each cell line mainly p oduced ea ly apop osis in Caco-2 cells, while la e apop osis was mainly de ec ed in MDA cells. Howe e , no signi ican apop osis was ound in MCF-7 cells. T ea men wi h longe ime (72 h) induced An ioxidan s 2021,10, 243 9 o 17 a signi ican dea h o hese cells by la e apop osis (Figu e 2). The e o e, he ob ained esul s showed ha g ape s em ex ac s a hei IC 50 p oduced apop osis in all es ed cance ous cells by ac i a ing apop o ic pa hways, he eby educing hei abili y o non-selec i ely eac wi h biological a ge s o cause nec osis and i s ela ed side e ec s. Since p e ious s udies on plan ex ac s sugges ed mi ochond ial dys unc ion and in insic apop osis induc ion [ 25 , 26 ], he mi ochond ial memb ane po en ial change was analyzed. Mi ochond ia play a pi o al ole in li e and cell dea h inasmuch as hey p oduce he majo i y o he ene gy equi ed o su i al and egula e he in insic apop osis pa h- way. The in ol emen o mi ochond ia in cell dea h is gene ally measu ed by ollowing mi ochond ial memb ane depola iza ion [ 46 ]. The esul s showed ha g ape s em ex ac s signi ican ly al e ed he mi ochond ial po en ial o he es ed cance cells compa ed o he un ea ed ones (Figu e 3); he e o e, he changes in mi ochond ial po en ial could be ela ed o he obse ed apop osis (Figu e 2). An ioxidan s 2021, 10, x FOR PEER REVIEW 9 o 17 used o ob ain a selec i i y index (SI), as p e iously desc ibed by Badisa el al. [44]. The SI esul s a e shown in Table 2, wi h he highes alue being o he MDA-MB-231 cell line, acco ding wi h he highes e ec i e esponse o he ex ac s owa ds hese cells a e 72 h o incuba ion. The obse ed di e ence in he wo b eas cance lines could ha e been due o he ac ha he ac ion o hese ex ac s could be ela ed o he ecep o s’ exp ession o es ogens, which a e only p esen in MCF-7 cells [45]. 3.2.2. Cell Dea h S udies Since he g ape s em ex ac s p oduce a educ ion in cell iabili y, i was decided o de e mine wha ype o cell dea h occu ed. Thus, low cy ome y analyses o e 48 h we e pe o med using bioma ke s o cell dea h. The esul s showed ha ea men o 48 h wi h he IC50 concen a ion co esponding o each cell line mainly p oduced ea ly apop osis in Caco-2 cells, while la e apop osis was mainly de ec ed in MDA cells. 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