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. Howe e , no
signi ican apop osis was ound in MCF-7 cells. T ea men wi h longe ime (72 h) induced
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 IC50 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.
Figu e 2. Con .
An ioxidan s 2021,10, 243 16 o 17
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