Academic Edi o : Alessand a
Napoli ano
Recei ed: 2 Feb ua y 2025
Re ised: 18 Feb ua y 2025
Accep ed: 9 Ma ch 2025
Published: 13 Ma ch 2025
Ci a ion: Se a, M.; Bo elho, C.;
Sousa, D.; Almeida, H.; Casas, A.;
Teixei a, J.A.; Ba os, A.N. Bioac i e
Po en ial o a G ape S em Blend: A
Sus ainable App oach o Skin
Regene a ion. An ioxidan s 2025,14,
338. h ps://doi.o g/10.3390/
an iox14030338
Copy igh : © 2025 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/).
A icle
Bioac i e Po en ial o a G ape S em Blend: A Sus ainable
App oach o Skin Regene a ion
Mónica Se a 1,2, Claudia Bo elho 1,3,4 , Diana Sousa 1,2 , Hugo Almeida 2,5,6 , Ana Casas 2,
José An ónio Teixei a 1,3,4 and Ana No o Ba os 7,*
1CEB—Cen e o Biological Enginee ing, Uni e si y o Minho, 4710-057 B aga, Po ugal;
[email p o ec ed] (M.S.); [email p o ec ed] (C.B.); [email p o ec ed] (D.S.);
[email p o ec ed] (J.A.T.)
2Mesosys em In es igação & In es imen os by Spinpa k, Ba co, 4805-017 Guima ães, Po ugal;
[email p o ec ed] (H.A.); [email p o ec ed] (A.C.)
3LABBELS—Associa e Labo a o y, 4710-057 B aga, Po ugal
4Cen e o Molecula and En i onmen al Biology (CBMA), Aqua ic Resea ch Ne wo k (ARNET), Associa e
Ins i u e o Science and Inno a ion o Sus ainabili y (IB-S), Uni e si y o Minho, Campus de Gual a ,
4710-057 B aga, Po ugal
5UCIBIO, Labo a o y o Pha maceu ical Technology, Facul y o Pha macy, Uni e si y o Po o,
4051-401 Po o, Po ugal
6Associa e Labo a o y i4HB Ins i u e o Heal h and Bioeconomy, Facul y o Pha macy, Uni e si y o Po o,
4051-401 Po o, Po ugal
7
Cen e o he Resea ch and Technology o A go-En i onmen al and Biological Sciences (CITAB), Ins i u e o
Inno a ion, Capaci y Building and Sus ainabili y o Ag i-Food P oduc ion (Ino 4Ag o), Uni e si y o
T ás-os-Mon es and Al o Dou o (UTAD), Quin a de P ados, 5000-801 Vila Real, Po ugal
*Co espondence: aba [email p o ec ed]
Abs ac : The Eu opean wine indus y is emb acing sus ainabili y h ough ci cula econ-
omy p inciples, pa icula ly by alo izing by-p oduc s, such as g ape s ems. G ape s ems
a e ich in phenolic compounds wi h ecognized heal h bene i s. This s udy in es iga es he
bioac i e po en ial o molecules ex ac ed om a blend o g ape s ems (GS blend ex ac ).
The GS blend ex ac was chemically cha ac e ized in e ms o o al phenolic con en (TPC),
o ho-diphenol con en (ODC), and la onoid con en (FC), wi h key compounds iden i ied
ia HPLC-MS. The ex ac ’s an ioxidan capaci y was assessed using ABTS, FRAP, and
DPPH assays, while i s an i-aging and depigmen ing p ope ies we e e alua ed h ough
elas ase and y osinase inhibi ion assays. Addi ionally,
in i o
assays we e conduc ed o
assess i s e ec s on skin cells, including mo phology, me abolic ac i i y, cell cycle, and
cell mig a ion. The GS blend ex ac was ound o be ich in p oan hocyanidins and ex-
hibi ed no able an ioxidan and depigmen ing p ope ies.
In i o
assays demons a ed
ha he ex ac had no signi ican impac on cellula me abolic ac i i y o cell mo phology,
al hough a eo ganiza ion o he cell monolaye was obse ed. Fu he mo e, de ia ions in
cell mig a ion and cell cycle egula ion sugges ha he GS blend ex ac may aid in sca
o ma ion managemen . No ably, he ex ac a es ed ib oblas s in he Sub G0-G1 phase
and inhibi ed HaCaT cell mig a ion, suppo ing i s po en ial applica ion in cosme ic and
pha maceu ical o mula ions aimed a sca modula ion and skin heal h.
Keywo ds: ci cula economy; sus ainabili y; by-p oduc alo iza ion; g ape s ems;
phenolic compounds; chemical cha ac e iza ion;
in i o
assays; biological ac i i ies; skin
egene a ion; an ioxidan p ope ies
An ioxidan s 2025,14, 338 h ps://doi.o g/10.3390/an iox14030338
An ioxidan s 2025,14, 338 2 o 25
1. In oduc ion
The Eu opean wine sec o holds a p ominen posi ion in he global economy, wi h
coun ies such as F ance, I aly, Spain, Ge many, and Po ugal iden i ied by he In e -
na ional O ganiza ion o Vine and Wine (OIV) as key con ibu o s o wo ldwide wine
p oduc ion [1,2].
The global wine indus y, deeply oo ed in adi ion and economic signi icance, has
inc easingly gained a en ion o i s po en ial o suppo sus ainable p ac ices, pa icula ly
wi hin he amewo k o ci cula economy p inciples [
3
,
4
]. G apes, essen ial o wine
p oduc ion, no only se e as he ounda ion o he winemaking p ocess bu also gene -
a e aluable by-p oduc s, such as g ape s ems (o s alks), seeds, and skins, which hold
signi ican po en ial as bioac i e esou ces [5–8].
In he con ex o he ci cula economy [
9
], which emphasizes minimizing was e and
maximizing esou ce e iciency, he alo isa ion o by-p oduc s becomes c ucial. G ape
s ems, one o he mos abundan by-p oduc s o winemaking (accoun ing o up o 14%
o o al by-p oduc olume), a e o en o e looked despi e hei high con en o bioac i e
molecules, pa icula ly phenolics [10].
Phenolic compounds a e seconda y me aboli es syn hesized by plan s in esponse
o a ious en i onmen al challenges, ac ing as de ence mechanisms agains pa hogens,
pa asi es, UV adia ion, and o he bio ic and abio ic s esso s [
10
,
11
]. G ape s ems a e a
ich sou ce o hese compounds, including la anols (epica echin, ca echin, que ce in-3-O-
u inoside), hyd oxycinnamic acids (ca a ic acid, couma ic acid), hyd oxybenzoic acids
(gallic acid), and s ilbenes ( ini e in), among o he s [
12
–
17
]. While he phenolic p o ile o
g ape s em ex ac s is simila ac oss di e en a ie ies, he p opo ion o each compound
can a y signi ican ly, depending on ac o s such as soil ype, ma u a ion s age, g ape size,
ha es ime [
18
–
21
], and ex ac ion me hod [
22
]. The dis inc p opo ions o phenolics in
each ex ac signi ican ly impac hei biological po en ial, pa icula ly hei an ioxidan ,
an i-in lamma o y, an i-aging, an icance , and an imic obial p ope ies [11,22,23].
In e ms o chemical an ioxidan e icacy, phenolic compounds ex ac ed om g ape
s ems, pa icula ly om Tou iga Nacional [
13
], Sousa [
14
], Cabe ne Mo a ia [
17
], and
Mazuelo [
12
] a ie ies, exhibi no able ee adical sca enging capaci y. Simila ly, ex-
ac s om g ape s ems o he Mandila ia a ie y ha e demons a ed cellula an ioxidan
p ope ies, especially in endo helial and muscle cells [24,25].
Beyond g ape s ems an ioxidan ac i i y, Vi is ini e a L. g ape s em ex ac s ha e
shown an i-aging p ope ies by inhibi ing elas ase and y osinase enzymes, con ibu ing o
he p e en ion o age spo s and he loss o skin elas ici y and s eng h [
26
]. The inhibi ion
o hese enzymes is pa icula ly ele an due o he ole o y osinase in melanin syn hesis,
which is associa ed wi h hype pigmen a ion and age spo s, as well as o he pigmen a ion-
ela ed skin diso de s. Simila ly, elas ase is esponsible o he deg ada ion o elas in, a key
s uc u al p o ein in he skin. Elas in b eakdown leads o educed mechanical s eng h in
connec i e issues, w inkle o ma ion, and o he skin de o ma ions [
26
–
29
]. The e o e, he
inhibi ion o hese enzymes is o pa amoun impo ance in an i-aging skinca e applica ions.
These indings highligh he di e se and p omising bioac i e p ope ies o phenolic
compounds ex ac ed om g ape s ems, emphasizing hei po en ial applica ions in heal h
and wellness.
While p e ious s udies ha e p ima ily ocused on e alua ing isola ed g ape a ie ies,
his s udy akes a di e en app oach by analysing he ex ac ion o phenolic compounds
om a blend o g ape s ems, speci ically om Tin a Ro iz, Tou iga Nacional, Cas elão,
Sy ah, A in o, and Fe não Pi es. Blending ex ac s om mul iple g ape a ie ies may
ha e inc eased bene i s in cosme ic skinca e o mula ions, as di e en g ape a ie ies
con ain dis inc bioac i e compounds. Thei combina ion can esul in syne gis ic e ec s
An ioxidan s 2025,14, 338 3 o 25
ha ampli y skin egene a ion po en ial [
30
]. Fo ins ance, es e a ol, abundan in ed
g ape a ie ies, exhibi s po en an ioxidan and an i-in lamma o y p ope ies [
31
], while
la onoids, such as que ce in and ca echins, ound in bo h ed and whi e g apes, play
c ucial oles in angiogenesis and ex acellula ma ix pa hways [
32
–
35
]. P oan hocyanidins,
commonly p esen in g ape seeds and s ems, con ibu e o collagen c osslinking [36]. and
imp o ed skin elas ici y, which can u he aid in minimizing sca o ma ion.
In addi ion o enhancing bioac i i y, blended g ape ex ac s may o e imp o ed
s abili y and bioa ailabili y. Ce ain polyphenols a e p one o oxida ion and deg ada ion,
limi ing hei e icacy in opical o mula ions. A mul i- a ie y blend can coun e ac hese
limi a ions by p o iding a mo e chemically balanced and s able composi ion, ensu ing
p olonged bioac i i y when applied o he skin. Mo eo e , a combina ion o bioac i e
molecules a ge s mul iple wound healing pa hways, c ea ing a comp ehensi e app oach
o skin egene a ion ha os e s smoo h, esilien , and sca - ee issue epai .
The e o e, his s udy explo es he po en ial o blended g ape s em ex ac s as an
ad anced cosme ic ing edien o skin egene a ion. By in es iga ing hei bioac i e compo-
si ion, s abili y, and e ec s on skin cell unc ion, we aim o p o ide insigh s in o how a mul i-
a ie y ex ac can con ibu e o he de elopmen o nex -gene a ion skinca e p oduc s.
2. Ma e ials and Me hods
2.1. Chemicals and Reagen s
Reagen s
Gallic acid (3,4,5- ihyd oxybenzoic acid), Folin–Ciocal eu eagen , and ace ic acid
(all ex a pu e, >99%) we e ob ained om Pan eac (Pan eac Química S.L.U., Ba celona,
Spain). Sodium hyd oxide (98%), sodium ni i e, sodium ca bona e (pu i y > 99%), alu-
minum chlo ide (pu i y > 99%), and e hanol we e acqui ed om Me ck (Me ck, Da ms ad ,
Ge many). Sodium molybda e (99.5%) was pu chased om Chem-Lab (Chem-Lab N.V.,
Zedelgem, Belgium).
Addi ionally, ca echin (98%), TROLOX (6-hyd oxy-2,5,7,8- e ame hylch oman-2-
ca boxylic acid, pu i y
≥
98.0%), ABTS ((2,2
′
-azino-bis-(3-e hylbenzo hiazoline-6-sul onic)
acid) diammonium sal , pu i y
≥
98.0%), DPPH (2,2-diphenyl-1-pic ylhyd azyl adi-
cal, 100.0%), po assium pe sul a e (pu i y
≥
99.0%), TPTZ (2,4,6- ipy idyl-s- iazine,
pu i y ≥98.0%
), i on (III) chlo ide (pu i y
≥
99.9%), y osinase and elas ase enzymes,
and all eagen s used in enzyma ic ac i i y assays we e sou ced om Sigma-Ald ich (S ein-
heim, Ge many), while hyd ochlo ic acid (
≈
37%) was ob ained om Honeywell Fluka
(Be lin, Ge many).
All eagen s used in he cell assays we e ob ained om PAN-Bio ech. Dis illed wa e
was gene a ed using a Millipo e wa e pu i ica ion sys em (Millipo e, Bed o d, MA, USA).
2.2. P epa a ion o GS Blend Ex ac
Equal amoun s o g ape s ems om a ious a ie ies (Tin a Ro iz, Tou iga Nacional,
Cas elão, Sy ah, A in o, and Fe não Pi es) we e d ied in an o en (Memme , Schwabach,
Ge many) a 40
◦
C o 72 h. A e d ying, he s ems we e g ound, and he esul ing samples
om di e en a ie ies we e combined in equal p opo ions o c ea e he GS blend ex ac .
The ex ac ion o phenolic compounds ollowed he me hodology de ined by Dias
Cos a e al. [
24
], wi h ce ain modi ica ions. A 1 g sample was homogenized and s i ed
in 50 mL o ex ac ion sol en (e hanol/wa e , 70:30) o 30 min in an o bi al shake (GFL
3005, GEMINI, Apeldoo n, The Ne he lands). Following homogeniza ion, he mix u e was
subjec ed o cen i uga ion (Sigma 2-16KL Re ige a ed Cen i uge, Sigma Labo zen i ugen,
Be lin, Ge many) a 10,000
×
g o 15 min a 4
◦
C, a p ocess ha was epea ed h ee imes.
An ioxidan s 2025,14, 338 4 o 25
The esul ing supe na an s om he epea ed cen i uga ions we e pooled and adjus ed o
a inal olume o 50 mL in a olume ic lask using he ex ac ion sol en .
2.3. De e mina ion o Phenolic Con en
The phenolic con en o he GS blend ex ac was de e mined using adap ed spec-
opho ome ic me hodologies designed o 96-well mic opla es (P imeSu ace MS-9096MZ,
F ilabo, Maia, Po ugal), ollowing he p o ocol o Gou inhas e al. [27]. Abso bance mea-
su emen s we e pe o med using a mic opla e eade (Mul iskan GO Mic opla e Pho ome-
e , The mo Fishe Scien i ic, Van aa, Finland).
2.4. De e mina ion o To al Phenolic Con en
The o al phenolic con en (TPC) o he GS blend ex ac was de e mined by mixing 1
mL o he sample wi h 0.5 mL o Folin–Ciocal eu eagen . Nex , 2.0 mL o Na
2
CO
3
(7.5%
w/ ) and 6.5 mL o wa e we e added o he mix u e. The eac ion was incuba ed a 70
◦
C
o 30 min, p o ec ed om ligh , and hen cooled unde unning wa e . Abso bance was
measu ed a 750 nm. An analy ical cu e was cons uc ed using a ious concen a ions o
gallic acid, and he o al phenolic con en was quan i ied and exp essed as millig ams o
gallic acid equi alen pe g am o sample (mg GAE/g sample).
2.5. De e mina ion o O ho-Diphenol Con en
The o ho-diphenol con en (ODC) o he GS blend ex ac was assessed by adding
1 mL
o Na
2
MoO
4
(50 g/L) o 4 mL o he app op ia ely dilu ed samples. A e o exing,
he mix u es we e le o s and a oom empe a u e o 15 min, p o ec ed om ligh .
Abso bance was hen measu ed a 370 nm. Quan i ica ion was pe o med using gallic acid
as he s anda d, and he esul s we e exp essed as millig ams o gallic acid equi alen pe
g am o d y weigh (mg GAE/g dw).
2.6. De e mina ion o Fla onoid Con en
The me hod used o de e mine la onoid con en (FC) in ol ed he o ma ion o a
la onoid–aluminum complex. In his p ocess, 0.5 mL o he dilu ed sample was added o a
es ube, ollowed by 150
µ
L o a 5% sodium ni i e solu ion. A e a 5 min eac ion, 150
µ
L
o a 10% aluminum chlo ide solu ion was in oduced. Following a 6 min eac ion pe iod,
1 mL
o a 1 M sodium hyd oxide solu ion was added o he well. The mic opla e was hen
s i ed o 30 s, and abso bance was measu ed a 510 nm. To cons uc he calib a ion cu e,
a ious concen a ions o ca echin we e used. The esul s we e exp essed as millig ams o
ca echin pe g am o d y weigh o he sample (mg CAT/g dw).
2.7. Iden i ica ion o Phenolic Compounds by HPLC-MS
The quan i a i e (poly)phenolic p o ile o he g ape s em ex ac was de e mined
using he me hodology desc ibed by Cos a-Pé ez e al. [
24
]. Ch oma og aphic sepa a ion
o (poly)phenols was pe o med on a Luna C18 column (150.0
×
4.6 mm, 5.0
µ
m pa icle
size, Phenomenex, Maccles ield, UK) using an Agilen HPLC 1100 se ies sys em (Agilen
Technologies, Waldb onn, Ge many) consis ing o a bina y pump (model G1312A), an
au osample (model G1313A), a degasse (model G1322A), a pho odiode a ay (PDA) de-
ec o (model G1315B), and an ion ap spec ome e (model G2445A) wi h an elec osp ay
ioniza ion in e ace. The sys em was con olled using LCMSD so wa e, . 4.1 (Agilen
Technologies), ollowing he speci ica ions de ailed by Ba os e al. [15].
The mobile phases used we e H
2
O/ o mic acid (99.0:1.0, / ) (sol en A) and ace oni-
ile/ o mic acid (9.0:1.0, / ) (sol en B). Spec al da a om all peaks we e de ec ed in
he 200–600 nm ange, wi h ch oma og ams eco ded a 280 nm o p oan hocyanidins,
330 nm
o phenolic acids and s ilbenes, and 360 nm o la onols. Mass spec ome y da a
An ioxidan s 2025,14, 338 5 o 25
we e acqui ed in nega i e mode. Phenolic compound iden i ica ion was ca ied ou by
examining e en ion ime (min), pa en ions, and MS2 agmen a ion pa e ns.
Phenolic compounds we e quan i ied using PDA ch oma og ams eco ded a 280 nm
o p oan hocyanidins, 330 nm o phenolic acids, and 360 nm o la onols. Daily p e-
pa ed calib a ion cu es we e applied using ca echin ( o p oan hocyanidins and ca echin
de i a i es), chlo ogenic acid ( o phenolic acids), and que ce in glucoside ( o la onols).
The esul s we e exp essed as µg/g dw.
2.8. De e mina ion o An ioxidan Capaci y
The an ioxidan capaci y o he GS blend ex ac was assessed using spec opho ome -
ic me hods, speci ically he ABTS ((2,2
′
-azino-bis-(3-e hylbenzo hiazoline-6-sul onic) acid)
diammonium sal ), DPPH (2,2-diphenyl-1-pic ylhyd azyl), and FRAP (Fe ic-Reducing
An ioxidan Powe ) assays, acco ding o San os e al. [
28
]. All me hods a e based on he
p inciple o an ioxidan s in he sample dona ing elec ons o educe he oxidized o ms o
he eagen s.
In he ABTS and DPPH assays, he eac ion in ol es a adical o med by he oxida ion
o ABTS and DPPH, espec i ely. In con as , he FRAP assay is based on he educ ion o a
e ic complex.
2.8.1. ABTS Assay
The i s s ep o his assay in ol ed oxidizing he ABTS sal by adding 88
µ
L o a
148 mM
po assium pe sul a e solu ion o 5 mL o a 7 mM ABTS solu ion. The mix u e was
le o s and o 12 o 16 h a oom empe a u e and was p o ec ed om ligh o achie e i s
mos s able oxida i e s a e.
Subsequen ly, he ABTS wo king solu ion was p epa ed by dilu ing he ca ionic ABTS
solu ion in a 20 mM sodium ace a e bu e (pH 4.5) un il an abso bance o 0.700
±
0.020 a
734 nm was ob ained.
The an ioxidan capaci y o he samples was e alua ed by adding 188
µ
L o he ABTS
wo king solu ion o each mic opla e well, ollowed by he addi ion o 12
µ
L o he dilu ed
sample and blank. A e a 30 min o incuba ion a oom empe a u e, p o ec ed om ligh ,
abso bance was measu ed a 734 nm [Abs (734 nm)].
Fo his assay, a ious concen a ions o TROLOX we e used o cons uc he analy ical
cu e, wi h wa e se ing as he blank. The ABTS assay measu ed he abili y o he
samples o sca enge ABTS adicals, and he inhibi ion pe cen age was calcula ed using he
ollowing o mula:
% inhibi ionSample =Abs (734 nm)Blank −Abs (734 nm)Sample
Abs (734 nm)Blank
×100
The esul s we e p esen ed as TROLOX Equi alen An ioxidan Capaci y (TEAC)
and exp essed in millimoles o TROLOX pe g am o d y weigh o he sample (mmol
TROLOX/g dw).
2.8.2. FRAP Assay
The e alua ion o an ioxidan ac i i y using he FRAP me hod began wi h he p epa a-
ion o he FRAP wo king solu ion. This solu ion was p epa ed by combining 10 olumes
o 220 mM ace a e bu e (pH 3.6), 1 olume o 40 mM TPTZ (2,4,6-T ipy idyl-s-T iazine)
solu ion dissol ed in 40 mM HCl, and 1 olume o 20 mM e ic chlo ide. The FRAP
wo king solu ion, p epa ed daily, was p ehea ed o 37 ◦C o 10 min be o e use.
Subsequen ly, 20
µ
L o he sample was in oduced in o he mic opla e well, ollowed
by he addi ion o 280 µL o he FRAP wo king solu ion. The mic opla e was hen shaken
An ioxidan s 2025,14, 338 6 o 25
and incuba ed a 37
◦
C in da kness o 30 min, a e which he abso bance was measu ed a
593 nm. To es ablish he calib a ion cu e, a ying concen a ions o TROLOX we e used.
The esul s we e exp essed in millimoles o TROLOX pe g am o d y weigh o he sample
(mmol TROLOX/g dw).
2.8.3. DPPH Assay
In he ini ial phase o his assay, he DPPH s ock solu ion (8.87 mM) was dilu ed in
e hanol/wa e (70:30, / ) un il an abso bance close o 1.000 a 520 nm was achie ed,
c ea ing he DPPH wo king solu ion.
To e alua e he an ioxidan capaci y o he samples, 190
µ
L o he DPPH wo king
solu ion was added o each mic opla e well, ollowed by 10
µ
L o he dilu ed sample and
blank (e hanol/wa e , 70:30, / ). The mix u e was incuba ed a oom empe a u e o
30 min
and was p o ec ed om ligh , and he abso bance was measu ed a 520 nm [Abs
(520 nm)].
TROLOX, a a ious concen a ions, was used as he s anda d o cons uc he analy i-
cal cu e. The inhibi ion pe cen age was calcula ed using he ollowing o mula:
% inhibi ionSample =Abs (520 nm)Blank −Abs (520 nm)Sample
Abs (520 nm)Blank
×100
The esul s we e p esen ed as TROLOX Equi alen An ioxidan Capaci y (TEAC)
and exp essed in millimoles o TROLOX pe g am o d y weigh o he sample (mmol
TROLOX/g dw).
2.9. E alua ion o An i-Aging and Depigmen ing Ac i i ies
The e alua ion o an i-aging ac i i y was conduc ed h ough assays measu ing he
inhibi ion pe cen age o elas ase and y osinase enzymes, ollowing he me hodology
desc ibed by Taghou i e al. [29].
2.9.1. Ty osinase Inhibi ion Assay
The y osinase inhibi ion assay was pe o med o de e mine he pe cen age o y osi-
nase inhibi ion. In his p ocedu e, 20
µ
L o 1000 U/mL y osinase and 170
µ
L o a mix u e
con aining 1 mM L- y osine solu ion, 50 mM phospha e bu e (pH 6.5), and wa e (in a
10:10:9 p opo ion) we e added o he sample.
Subsequen ly, he mic opla e was incuba ed a 37
◦
C o 10 min, and he abso bance
was measu ed a 490 nm. Posi i e and nega i e con ols—1 mg/mL Kojic acid and 10%
DMSO, espec i ely—we e used.
The pe cen age o inhibi ion was calcula ed using he ollowing equa ion:
% inhibi ionSample =Abs (490 nm)Nega i e Con ol −Abs (490 nm)Sample
Abs (490 nm)Nega i e Con ol
×100
2.9.2. Elas ase Inhibi ion Assay
The elas ase inhibi ion assay was conduc ed o de e mine he pe cen age o elas ase
inhibi ion.
A 50
µ
L aliquo o he sample was combined wi h 160
µ
L o 0.20 mM T is-HCl bu e
(pH 8) and 20
µ
L o 0.80 mM N-Succinyl-Ala-Ala-Ala-p-ni oanilide subs a e (p epa ed in
T is-HCl bu e ). The mix u e was incuba ed a oom empe a u e o 10 min.
Subsequen ly, 20
µ
L o 1 U/L elas ase enzyme (in T is-HCl bu e ) was added, and he
mic opla e was incuba ed o a u he 20 min a oom empe a u e. The abso bance was
hen measu ed a 410 nm. T is-HCl bu e was used as he nega i e con ol.
An ioxidan s 2025,14, 338 7 o 25
The pe cen age o inhibi ion was calcula ed using he ollowing equa ion:
% inhibi ionSample =Abs (410 nm)Nega i e Con ol−Abs (410 nm)Sample
Abs (410 nm)Nega i e Con ol
×100
2.10. Cell Main enance and Assays
2.10.1. Cell Mo phology Assay
HaCaT and BJ-5 a cells we e i s pla ed in a 48-well pla e a an op imized densi y o
3.5 ×104cells/mL and incuba ed o 24 h.
Following his incuba ion pe iod, he ex ac s we e added o he wells, and incuba ion
con inued o ano he 24 h. The medium was hen emo ed, and he cells we e washed
once wi h PBS. To ix he cells, 100
µ
L o 4% pa a o maldehyde was added, ollowed by a
40 min incuba ion a oom empe a u e (RT). The cells we e hen washed again wi h PBS.
Fo pe meabiliza ion, 100
µ
L o 0.1% T i on X-100 in PBS was added, and he cells
we e incuba ed o 30 min a RT. The pe meabiliza ion solu ion was p epa ed by mixing
5
µ
L o T i on X-100 wi h 4995
µ
L o PBS. A e pe meabiliza ion, he cells we e washed
again wi h PBS.
Phalloidin s aining was pe o med by dilu ing 0.25
µ
L o Alexa Fluo 568 Phalloidin
(In i ogen, A12380, Wal ham, MA, USA) om a 400x s ock solu ion in o 100
µ
L o PBS.
The s aining solu ion was p epa ed by combining 3.8
µ
L o he Phalloidin s ock solu ion,
1496.2
µ
L o PBS, and 15 mg o BSA. The cells we e hen incuba ed wi h his Phalloidin
solu ion o 60 min a RT and we e p o ec ed om ligh .
A e s aining, he cells we e washed wice wi h PBS. Subsequen ly, 50
µ
L o DAPI
solu ion was added, and he cells we e incuba ed o 15 min. Finally, he cells we e washed
wice wi h PBS and examined using a Leica In e ed Mic oscope (DMI 3000B, We zla ,
Ge many, 2012).
2.10.2. Cell Me abolism Assay
Cells we e moni o ed un il hey eached 80% con luency. Once con luency was
achie ed, he DMEM medium was emo ed, and he cells we e washed wi h il e ed
PBS (1 mL o T25 lasks). To de ach he cells, 500
µ
L o ypsin was added o he T25 lasks,
ollowed by incuba ion o 5–10 min. De achmen was con i med by mic oscopy o gen le
shaking. A e de achmen , double he olume o he medium was added o neu alize
he ypsin.
Fo u he cul u e, 1000
µ
L o he cell suspension was added o 5 mL o medium in a
T25 lask. The lasks we e hen incuba ed unde s anda d condi ions.
Resazu in Reduc ion Assay as a Measu emen o Cellula Me abolic Ac i i y/Viabili y
In his wo k, he esazu in educ ion assay, which is an es ablished and widely used
al e na i e o e alua ing cell iabili y h ough me abolic ac i i y was used. Resazu in is a
non- oxic, cell-pe meable compound ha is educed o he highly luo escen eso u in by
me abolically ac i e cells. This educ ion p ocess is dependen on cellula espi a ion and
mi ochond ial unc ion, making i a eliable indica o o cell iabili y.
A e subcul u ing, he cell suspension was ans e ed o 15 mL Falcon ubes. A
10 µL
sample was used o cell coun ing wi h a Neubaue Chambe . The a e age cell coun om
ou quad an s was used o adjus he cell concen a ion o 4.0 ×104cells/mL.
Fo he assay, 200
µ
L o he cell suspension was pla ed in iplica e in a 96-well
mic opla e and incuba ed o 24 h. Following incuba ion, he medium was emo ed, and
200
µ
L o ex ac a a ious concen a ions (20.84; 41.49; 83.38; 166.75; 333.50; 667.00
µ
g/mL)
was added, alongside con ols (medium alone and medium wi h 0.5% DMSO). Cells we e
incuba ed o ano he 24 h.
An ioxidan s 2025,14, 338 8 o 25
The nex day, he cells we e washed wi h 1
×
PBS and incuba ed wi h a esazu in
solu ion (10 mL esazu in in 1
×
PBS + 40 mL medium) o 2 h in he da k. Resazu in
educ ion was assessed by measu ing he luo escence o i s educed p oduc , eso u in,
wi h an exci a ion wa eleng h o 560 nm and an emission wa eleng h o 590 nm. The
ob ained esul s we e hen used o e alua e cellula iabili y.
2.10.3. In Vi o Cell Mig a ion Assay
The cell lines we e seeded in o a 24-well pla e a a densi y o 8.0
×
10
4
cells/mL and
incuba ed a 37
◦
C in a 5% CO
2
a mosphe e un il con luence was eached. A sc a ch was
in oduced in o he con luen cell laye using a 10
µ
L pipe e ip. The cul u e medium was
hen eplaced wi h he di e en ex ac dissol ed in he cul u e medium a a concen a ion
o 0.5 mg/mL.
Cell mig a ion was moni o ed mic oscopically, and images we e acqui ed a 0 h,
6 h,
and 24 h o HaCaT cells and a 0 h, 24 h, and 48 h o BJ-5 a cells. The sc a ch a ea was
measu ed a di e en ime poin s using ImageJ’s MRI Wound Healing Tool plugin (upda ed
e sion 2020) [36,37].
2.10.4. E alua ion o Cell Cycle by Flow Cy ome y
Fo cell cycle analysis, cells we e cul u ed un il hey eached o e 90% con luency. The
medium was hen emo ed, and cells we e washed wi h PBS. Cells we e de ached wi h
1 mL
ypsin, incuba ed o 10 min a 37
◦
C in a 5% CO
2
a mosphe e, and neu alized wi h
2 mL o DMEM medium.
A e cell coun ing, cells we e pla ed in a 6-well pla e in iplica e a he ollowing
densi ies: HaCaT: 2.64 ×105cells/well and BJ-5 a: 1.40 ×105cells/well.
Cells we e incuba ed o 24 h o allow adhe ence, ollowed by ea men wi h GS
blend ex ac o an addi ional 24 h. The concen a ions es ed we e 20.84, 83.38, and
166.75 µg/mL.
Pos - ea men , he medium was emo ed, and cells we e washed wi h PBS. Cells
we e de ached wi h 500
µ
L ypsin, incuba ed o 10 min, and neu alized wi h 1 mL o
medium. Cells om h ee wells pe condi ion we e ans e ed in o 15 mL Falcon ubes.
A e cell coun ing, cells we e cen i uged a 400
×
g o 7 min. The cell pelle was
esuspended in 2 mL o an ice-cold Binding Bu e and cen i uged again a 400
×
g o
7 min. Cells we e hen ixed by adding 2 mL o ice-cold 70% e hanol d opwise while
o exing, ollowed by incuba ion on ice o 40 min.
Following ixa ion, cells we e cen i uged a 400
×
g o 7 min and esuspended
in a Binding Bu e , ollowed by ano he cen i uga ion s ep. The inal cell pelle was
esuspended in 500
µ
L o a s aining solu ion (100
µ
L Enzyme A, 400
µ
L Nuclea Dye, and
10 mL Binding Bu e ) and incuba ed in he da k o 30 min. S ained cells we e analyzed
o cell cycle dis ibu ion using low cy ome y.
2.11. S a is ical Analysis
The da a we e analyzed using IBM SPSS 29.0 s a is ical so wa e. Analysis o a iance
(ANOVA) was pe o med, ollowed by Tukey’s mul iple ange es , wi h signi icance se a
p< 0.05.
Pea son co ela ion analysis was conduc ed using Mic oso Excel ( e sion 16.87).
3. Resul s and Discussion
3.1. Phenolic Con en
The phenolic (TPC), o ho-diphenol (ODC), and la onoid (FC) con en s o he ex ac
ob ained om he blend o g ape s em a ie ies (GS blend ex ac ) a e p esen ed in Table 1.
An ioxidan s 2025,14, 338 9 o 25
Table 1. To al phenolic, o ho-diphenol, and la onoid con en o he GS blend ex ac de e mined by
colo ime ic chemical assays.
TPC (mg GAE/g) ODC (mg GAE/g) FC (mg CAT/g)
78.55 ±1.67 80.83 ±2.69 66.77 ±1.96
The esul s a e p esen ed as mean ±s anda d de ia ion (n = 3).
Consis en wi h he exis ing li e a u e [
24
,
26
,
38
], he GS blend ex ac exhibi s a high
concen a ion o phenolic compounds.
I is impo an o ake in o conside a ion ha he p opo ions o each g ape a ie y
p esen in he blend a e equal, making i possible o in e he con ibu ion o each a ie y
o he inal e ec .
Leal e al. [
13
] cha ac e ized me hanolic ex ac s om dis inc g ape s em a ie ies,
namely, Tin a Ro iz, Tou iga Nacional, Cas elão, Sy ah, A in o, and Fe não Pi es based
on hei TPC, ODC, and FC con en s. The au ho s epo ed ha each a ie y exhibi ed
dis inc concen a ions, anging om 30.91 ±0.73 o 96.12 ±8.14 mg GAE/g dw o TPC,
32.17 ±1.04
o 77.26
±
5.31 mg GAE/g dw o ODC, and 25.76
±
1.14 o 65.14
±
0.65 mg
CAT/g dw o FC.
Simila ly, Sil a e al. [
16
] de e mined TPC in e hanolic ex ac s o g ape s ems om
Tou iga Nacional and P e o Ma inho, epo ing alues be ween 45.9
±
10.7
µ
g/mg and
226.8
±
6.9
µ
g/mg. As shown in Table 1, he chemical cha ac e iza ion o he GS blend
ex ac alls wi hin he ange o me hanolic ex ac s. Howe e , compa ed o e hanolic
ex ac s, he GS blend ex ac con ains app oxima ely 1.7 imes highe TPC han Tou iga
Nacional, bu i is 2.8 imes lowe han P e o Ma inho.
The combina ion o g ape s ems can induce chemical eac ions ha lead o he o -
ma ion o new compounds, po en ially esul ing in syne gis ic o an agonis ic e ec s [
39
].
El-Belaga e al. [
40
] desc ibed an inc ease in o al phenolic and la onoid con en s when
g ape seed and g een ea ex ac s we e mixed in equal p opo ions. TPC inc eased by
app oxima ely 4% ( om 22% o 26%), while FC inc eased by 7% ( om 53% o 60%). Con-
e sely, Gio anelli-Vicuña e al. [
34
] demons a ed ha mixing me hanolic ex ac s om
di e en ui s (g ape, lemon, and bluebe y) led o a signi ican dec ease o app oxima ely
87% in TPC and 63% in FC, highligh ing he unp edic abili y o compound composi ion in
a blend.
3.2. Iden i ica ion and Quan i ica ion o Phenolic Compounds by HPLC-MS Gi en he Biological
Rele ance o Phenolic Compounds, he De ailed Phenolic Composi ion o he GS Blend Ex ac Was
Analyzed Using HPLC-MS (Table 2)
As shown in Table 2, he GS blend ex ac is ich in la onoids, con aining
1270.47 ±2.88 µg/g
dw o la an-3-ols, including p oan hocyanidins and ca echin de i a-
i es, and 108.36
±
0.49
µ
g/g dw o la onols. These esul s align wi h he la onoid
con en assay, which yielded 66.77
±
1.96 mg CAT/g dw, demons a ing consis ency
be ween indi idual la onoid quan i ica ions and he o al la onoid con en .
The o ho-diphenol con en was con i med h ough he iden i ica ion o ca echin and
p oan hocyanidins. The p edominan compounds in he GS blend ex ac include p oan-
hocyanidin ime s (B- ype) (Isome s 1, 2, and 3), p oan hocyanidin ime monogalla es
(Isome s 1 and 2), and a ious ca echin de i a i es.
Leal e al. [
13
] iden i ied la an-3-ols (ca echin, epica echin), la onols ( u in), and
s ilbenes ( es e a ol, ini e in) in me hanolic ex ac s o g ape s ems om Tin a Ro iz,
Tou iga Nacional, Cas elão, Sy ah, A in o, and Fe não Pi es. Simila ly, Sil a e al. [
16
] iden-
i ied la an-3-ols (ca echin, epica echin, gallo-ca echin galla e, ca echin galla e), la onols
( u in, que ce in 3-O-galac oside, que ce in 3-O-glucoside, que ce in 3-O- hamnoside), and
An ioxidan s 2025,14, 338 16 o 25
a ions in HaCaT cells ea ed wi h 250
µ
M and 500
µ
M o Annu ca Apple polyphenol
ex ac , including dissocia ion om he g ow h subs a e, nuclea disin eg a ion, and a
dec ease in cellula size. These changes we e linked o he ex ac ’s s ong inhibi o y e ec
on ke a inocy e g ow h, highligh ing i s po en ial o ea ing pso iasis.
Fib oblas s a e cha ac e ized by hei plump, spindle-shaped, o s ella e o ms and
cen ally loca ed o al o ound nuclei [
65
]. A simila app oach was used o e alua e he
impac o he GS ex ac blend on a ib oblas cell line, as shown in Figu e 2. The GS
ex ac blend appea ed o ha e a simila e ec on ib oblas mo phology as obse ed
in ke a inocy es.
An ioxidan s 2025, 14, x FOR PEER REVIEW 17 o 27
Figu e 2. In luence o he GS blend ex ac on ib oblas (BJ-5 a cells) mo phology a e 24 h o ex-
posu e. (A) DMSO con ol, showing a uni o m dis ibu ion o spindle-shaped cells wi h in ac cy o-
skele al o ganiza ion, (B) 20.84 µg/mL GS blend ex ac , (C) 83.38 µg/mL GS blend ex ac , and (D)
166.75 µg/mL GS blend ex ac , exhibi ing dec eased cy oskele al o ganiza ion and al e ed spa ial
o ien a ion in a dose-dependen manne . The ac in cy oskele on is s ained in ed (Alexa Fluo 568
Phalloidin), and nuclei a e s ained in blue (DAPI). Scale Ba 100 µm
Czemplik e al. [66] epo ed compa able changes in no mal human de mal ib oblas
(NHDF) mo phology when ea ed wi h Linum usi a issimum ex ac (140.4 µg TPC). The
ea ed cells exhibi ed a ounded shape ins ead o hei ypical elonga ed o m. Howe e ,
he nuclei emained simila in size and shape o hose in he con ol g oup, consis en wi h
ou obse a ions. The au ho s a ibu ed hese mo phological al e a ions o he oxici y o
high concen a ions o phenolic compounds.
Simila ly, Kikowska e al. [67] obse ed ha human skin ib oblas s ea ed wi h a
callus ex ac a lowe concen a ions (12.5 µg/mL) main ained a p ope elonga ed shape
wi h la ge, ound nuclei. In con as , ib oblas s exposed o highe concen a ions (100
µg/mL) exhibi ed abno mal mo phology, lacking he ypical elonga ions. Bo h s udies co -
obo a e he esul s obse ed in his s udy.
In conclusion, al hough he GS blend ex ac does no appea o signi ican ly al e cell
mo phology, i does induce no iceable changes in he o ganiza ion o he cell monolaye .
Figu e 2. In luence o he GS blend ex ac on ib oblas (BJ-5 a cells) mo phology a e 24 h o
exposu e. (A) DMSO con ol, showing a uni o m dis ibu ion o spindle-shaped cells wi h in ac
cy oskele al o ganiza ion, (B) 20.84
µ
g/mL GS blend ex ac , (C) 83.38
µ
g/mL GS blend ex ac ,
and (D) 166.75
µ
g/mL GS blend ex ac , exhibi ing dec eased cy oskele al o ganiza ion and al e ed
spa ial o ien a ion in a dose-dependen manne . The ac in cy oskele on is s ained in ed (Alexa Fluo
568 Phalloidin), and nuclei a e s ained in blue (DAPI). Scale Ba 100 µm.
Czemplik e al. [
66
] epo ed compa able changes in no mal human de mal ib oblas
(NHDF) mo phology when ea ed wi h Linum usi a issimum ex ac (140.4
µ
g TPC). The
ea ed cells exhibi ed a ounded shape ins ead o hei ypical elonga ed o m. Howe e ,
he nuclei emained simila in size and shape o hose in he con ol g oup, consis en wi h
An ioxidan s 2025,14, 338 17 o 25
ou obse a ions. The au ho s a ibu ed hese mo phological al e a ions o he oxici y o
high concen a ions o phenolic compounds.
Simila ly, Kikowska e al. [
67
] obse ed ha human skin ib oblas s ea ed wi h
a callus ex ac a lowe concen a ions (12.5
µ
g/mL) main ained a p ope elonga ed
shape wi h la ge, ound nuclei. In con as , ib oblas s exposed o highe concen a ions
(
100 µg/mL
) exhibi ed abno mal mo phology, lacking he ypical elonga ions. Bo h s udies
co obo a e he esul s obse ed in his s udy.
In conclusion, al hough he GS blend ex ac does no appea o signi ican ly al e cell
mo phology, i does induce no iceable changes in he o ganiza ion o he cell monolaye .
3.6.2. In luence o he GS Blend Ex ac on Cellula Me abolic Ac i i y
To assess he in luence o he GS blend ex ac on cells, he me abolic ac i i y o
ke a inocy es (HaCaT) and ib oblas s (BJ-5 a) was e alua ed ollowing ea men wi h
he ex ac .
The indings depic ed in Figu e 3demons a e ha he me abolic ac i i y o HaCaT
cells inc eases by app oxima ely 15% in he p esence o he GS blend ex ac , wi h a
h eshold concen a ion o 166.75
µ
g/mL. Abo e his concen a ion, me abolic ac i i y
dec eases, eaching a minimum o 30% a he highes concen a ion es ed (667 µg/mL).
An ioxidan s 2025, 14, x FOR PEER REVIEW 18 o 27
3.6.2. In luence o he GS Blend Ex ac on Cellula Me abolic Ac i i y
To assess he in luence o he GS blend ex ac on cells, he me abolic ac i i y o
ke a inocy es (HaCaT) and ib oblas s (BJ-5 a) was e alua ed ollowing ea men wi h
he ex ac .
The indings depic ed in Figu e 3 demons a e ha he me abolic ac i i y o HaCaT
cells inc eases by app oxima ely 15% in he p esence o he GS blend ex ac , wi h a
h eshold concen a ion o 166.75 µg/mL. Abo e his concen a ion, me abolic ac i i y de-
c eases, eaching a minimum o 30% a he highes concen a ion es ed (667 µg/mL).
Figu e 3. E ec o he GS blend ex ac on he me abolic ac i i y o ke a inocy es (HaCaT cell line)
measu ed by he esazu in educ ion assay a e 24 h o exposu e o di e en concen a ions. The
GS blend esul s a e p esen ed as mean ± s anda d e o (n = 3). Signi ican di e ences be ween
samples a e deno ed by di e en lowe case le e s (p < 0.05).
As shown in Figu e 4, he me abolic ac i i y o BJ-5 a cells emains s a is ically un-
changed when ea ed wi h he GS blend ex ac up o 667.00 µg/mL.
Figu e 4. E ec o he GS blend ex ac on he me abolic ac i i y o ib oblas s (BJ-5 a cell line) meas-
u ed by he esazu in educ ion assay a e 24 h o exposu e o di e en concen a ions. The GS
blend esul s a e p esen ed as mean ± s anda d e o (n = 3). Signi ican di e ences be ween samples
a e deno ed by a lowe case le e (p < 0.05).
aab aa
bc
c
0
20
40
60
80
100
120
140
Me abolic ac i i y / Me abolic
ac i i y con ol (%)
20.84 µg/mL
41.49 µg/mL
83.38 µg/mL
166.75 µg/mL
333.50 µg/mL
667.00 µg/mL
GS ex ac concen a ion, µg/mL
aaaa
a
a
0
20
40
60
80
100
120
140
Me abolic ac i i y / Me abolic
ac i i y con ol (%)
20.84 µg/mL
41.49 µg/mL
83.38 µg/mL
166.75 µg/mL
333.50 µg/mL
667.00 µg/mL
GS ex ac concen a ion, µg/mL
Figu e 3. E ec o he GS blend ex ac on he me abolic ac i i y o ke a inocy es (HaCaT cell line)
measu ed by he esazu in educ ion assay a e 24 h o exposu e o di e en concen a ions. The GS
blend esul s a e p esen ed as mean
±
s anda d e o (n = 3). Signi ican di e ences be ween samples
a e deno ed by di e en lowe case le e s (p< 0.05).
As shown in Figu e 4, he me abolic ac i i y o BJ-5 a cells emains s a is ically un-
changed when ea ed wi h he GS blend ex ac up o 667.00 µg/mL.
This obse a ion is suppo ed by Domínguez-Pe les e al. [
63
], who in es iga ed he
cy o oxic e ec s on HaCaT ke a inocy es ea ed wi h ex ac s om a ious g ape s em
a ie ies (Tin o Cão, Tin a Ba oca, Mosca el B anco, Mal asia Fina) ac oss a ange o
concen a ions (0.04–30.60 ng GAE/mL). Thei s udy demons a ed ha he cells’ me abolic
ac i i y exceeded 97.5% compa ed o he con ol (100%).
Simila ly, Sangio anni e al. [
68
] analysed cell me abolic ac i i y upon con ac wi h
lea es o he Tein u ie s a ie y in HaCaT cells. Thei s udy e ealed no signs o cy o oxici y
in cells exposed o he ex ac wi hin a concen a ion ange o 5 o 500 µg/mL.
The me abolic ac i i y esul s sugges ha he GS blend ex ac is compa ible wi h he
e alua ed cell lines. While i does no signi ican ly a ec ib oblas me abolism, i appea s
o p omo e me abolic ac i i y in ke a inocy es a mode a e concen a ions bu educes i a
highe concen a ions.
An ioxidan s 2025,14, 338 18 o 25
An ioxidan s 2025, 14, x FOR PEER REVIEW 18 o 27
3.6.2. In luence o he GS Blend Ex ac on Cellula Me abolic Ac i i y
To assess he in luence o he GS blend ex ac on cells, he me abolic ac i i y o
ke a inocy es (HaCaT) and ib oblas s (BJ-5 a) was e alua ed ollowing ea men wi h
he ex ac .
The indings depic ed in Figu e 3 demons a e ha he me abolic ac i i y o HaCaT
cells inc eases by app oxima ely 15% in he p esence o he GS blend ex ac , wi h a
h eshold concen a ion o 166.75 µg/mL. Abo e his concen a ion, me abolic ac i i y de-
c eases, eaching a minimum o 30% a he highes concen a ion es ed (667 µg/mL).
Figu e 3. E ec o he GS blend ex ac on he me abolic ac i i y o ke a inocy es (HaCaT cell line)
measu ed by he esazu in educ ion assay a e 24 h o exposu e o di e en concen a ions. The
GS blend esul s a e p esen ed as mean ± s anda d e o (n = 3). Signi ican di e ences be ween
samples a e deno ed by di e en lowe case le e s (p < 0.05).
As shown in Figu e 4, he me abolic ac i i y o BJ-5 a cells emains s a is ically un-
changed when ea ed wi h he GS blend ex ac up o 667.00 µg/mL.
Figu e 4. E ec o he GS blend ex ac on he me abolic ac i i y o ib oblas s (BJ-5 a cell line) meas-
u ed by he esazu in educ ion assay a e 24 h o exposu e o di e en concen a ions. The GS
blend esul s a e p esen ed as mean ± s anda d e o (n = 3). Signi ican di e ences be ween samples
a e deno ed by a lowe case le e (p < 0.05).
aab aa
bc
c
0
20
40
60
80
100
120
140
Me abolic ac i i y / Me abolic
ac i i y con ol (%)
20.84 µg/mL
41.49 µg/mL
83.38 µg/mL
166.75 µg/mL
333.50 µg/mL
667.00 µg/mL
GS ex ac concen a ion, µg/mL
aaaa
a
a
0
20
40
60
80
100
120
140
Me abolic ac i i y / Me abolic
ac i i y con ol (%)
20.84 µg/mL
41.49 µg/mL
83.38 µg/mL
166.75 µg/mL
333.50 µg/mL
667.00 µg/mL
GS ex ac concen a ion, µg/mL
Figu e 4. E ec o he GS blend ex ac on he me abolic ac i i y o ib oblas s (BJ-5 a cell line)
measu ed by he esazu in educ ion assay a e 24 h o exposu e o di e en concen a ions. The GS
blend esul s a e p esen ed as mean
±
s anda d e o (n = 3). Signi ican di e ences be ween samples
a e deno ed by a lowe case le e (p< 0.05).
3.6.3. In luence o he GS Blend Ex ac on he Ke a inocy e and Fib oblas (HaCaT and
BJ-5 a, Respec i ely) Cell Cycle
The cell cycle is a undamen al p ocess by which cells g ow, eplica e hei DNA, and
di ide in o wo daugh e cells. This cycle is essen ial o he g ow h, de elopmen , and
epai o mul icellula o ganisms. I consis s o se e al dis inc phases ha ensu e p ecise
eplica ion and seg ega ion o gene ic ma e ial, as well as p ope cell g ow h [69].
The Sub G0-G1 phase ypically indica es he p esence o apop o ic o dead cells,
cha ac e ized by educed DNA con en due o agmen a ion. In con as , he G0-G1 phase
includes cells ha a e ei he es ing o in he ini ial s age o he cell cycle. Du ing he S
phase, cells unde go ac i e DNA eplica ion, e lec ing a heigh ened le el o p oli e a i e
ac i i y as hey p epa e o di ision. Finally, in he G2-M phase, cells comple e DNA
syn hesis and p epa e o mi osis, he inal s ep in cell di ision [70,71].
Unde s anding hese phases is c ucial o e alua ing cell p oli e a ion dynamics and
he cellula esponse o ea men s. Thus, he e ec o he GS blend ex ac on he HaCaT cell
cycle was assessed using low cy ome y. Fluo escence in ensi y, which is p opo ional o
DNA con en , allowed o he dis inc ion o cells in di e en phases o he cycle. Apop o ic
cells exhibi ed educed DNA con en compa ed o iable cells in he G0/G1 phase.
Flow cy ome y analysis demons a ed ha he GS blend ex ac does no signi i-
can ly in luence he HaCaT cell cycle a concen a ions anging om 20.84 o 83.38
µ
g/mL
(
Figu e 5
). Howe e , a a highe concen a ion (166.75
µ
g/mL), an a es in he G0-G1 phase
was obse ed, leading o a educ ion in he numbe o cells in he S phase. This sugges s
ha a highe concen a ions, he GS ex ac can inhibi cell p oli e a ion by p e en ing
cells om p og essing o he S phase, he eby blocking DNA eplica ion and subsequen
cell di ision. Fu he mo e, when cells we e ea ed wi h he GS ex ac a concen a ions
abo e 166.75
µ
g/mL, a 70–100% dec ease in me abolic ac i i y was obse ed, po en ially
indica ing a educ ion in cell numbe due o he inhibi ion o p oli e a ion o induc ion
o apop osis.
The cell cycle o BJ-5 a ib oblas cells was also e alua ed using low cy ome y, wi h
he ib oblas ga e selec ed in he same manne . The esul s a e shown below (Figu e 6).
Rega ding he BJ-5 a cell cycle, he GS blend ex ac does no appea o signi ican ly
al e cell cycle p og ession. Howe e , a highe concen a ions (83.38 and 166.75
µ
g/mL),
cell cycle a es in he Sub G0-G1 phase was obse ed, sugges ing he po en ial induc ion
o apop osis (Figu e 6).
An ioxidan s 2025,14, 338 19 o 25
An ioxidan s 2025, 14, x FOR PEER REVIEW 19 o 27
a ie ies (Tin o Cão, Tin a Ba oca, Mosca el B anco, Mal asia Fina) ac oss a ange o con-
cen a ions (0.04–30.60 ng GAE/mL). Thei s udy demons a ed ha he cells’ me abolic
ac i i y exceeded 97.5% compa ed o he con ol (100%).
Simila ly, Sangio anni e al. [68] analysed cell me abolic ac i i y upon con ac wi h
lea es o he Tein u ie s a ie y in HaCaT cells. Thei s udy e ealed no signs o cy o oxi-
ci y in cells exposed o he ex ac wi hin a concen a ion ange o 5 o 500 µg/mL.
The me abolic ac i i y esul s sugges ha he GS blend ex ac is compa ible wi h
he e alua ed cell lines. While i does no signi ican ly a ec ib oblas me abolism, i ap-
pea s o p omo e me abolic ac i i y in ke a inocy es a mode a e concen a ions bu e-
duces i a highe concen a ions.
3.6.3. In luence o he GS Blend Ex ac on he Ke a inocy e and Fib oblas (HaCaT and
BJ-5 a, Respec i ely) Cell Cycle
The cell cycle is a undamen al p ocess by which cells g ow, eplica e hei DNA, and
di ide in o wo daugh e cells. This cycle is essen ial o he g ow h, de elopmen , and
epai o mul icellula o ganisms. I consis s o se e al dis inc phases ha ensu e p ecise
eplica ion and seg ega ion o gene ic ma e ial, as well as p ope cell g ow h [69].
The Sub G0-G1 phase ypically indica es he p esence o apop o ic o dead cells, cha -
ac e ized by educed DNA con en due o agmen a ion. In con as , he G0-G1 phase
includes cells ha a e ei he es ing o in he ini ial s age o he cell cycle. Du ing he S
phase, cells unde go ac i e DNA eplica ion, e lec ing a heigh ened le el o p oli e a i e
ac i i y as hey p epa e o di ision. Finally, in he G2-M phase, cells comple e DNA syn-
hesis and p epa e o mi osis, he inal s ep in cell di ision [70,71].
Unde s anding hese phases is c ucial o e alua ing cell p oli e a ion dynamics and
he cellula esponse o ea men s. Thus, he e ec o he GS blend ex ac on he HaCaT
cell cycle was assessed using low cy ome y. Fluo escence in ensi y, which is p opo -
ional o DNA con en , allowed o he dis inc ion o cells in di e en phases o he cycle.
Apop o ic cells exhibi ed educed DNA con en compa ed o iable cells in he G0/G1
phase.
Flow cy ome y analysis demons a ed ha he GS blend ex ac does no signi i-
can ly in luence he HaCaT cell cycle a concen a ions anging om 20.84 o 83.38 µg/mL
(Figu e 5). Howe e , a a highe concen a ion (166.75 µg/mL), an a es in he G0-G1
phase was obse ed, leading o a educ ion in he numbe o cells in he S phase. This
sugges s ha a highe concen a ions, he GS ex ac can inhibi cell p oli e a ion by p e-
en ing cells om p og essing o he S phase, he eby blocking DNA eplica ion and sub-
sequen cell di ision. Fu he mo e, when cells we e ea ed wi h he GS ex ac a concen-
a ions abo e 166.75 µg/mL, a 70–100% dec ease in me abolic ac i i y was obse ed, po-
en ially indica ing a educ ion in cell numbe due o he inhibi ion o p oli e a ion o in-
duc ion o apop osis.
b
aab
b
a
a
b
aab
a
bb
0
10
20
30
40
50
60
Sub G0-G1 G0-G1 S G2-M
Cells (%)
Cell cycle phase
Con ol
20.84 µg/mL
83.38 µg/mL
166.75 µg/mL
Figu e 5. Flow cy ome y analysis o he cell cycle in ke a inocy es (HaCaT cell line) ollowing
exposu e o di e en concen a ions o he GS blend ex ac . The GS blend esul s a e p esen ed as
mean
±
s anda d e o (n = 3). Signi ican di e ences be ween samples o each cell cycle phase a e
deno ed by di e en lowe case le e s (p< 0.05).
An ioxidan s 2025, 14, x FOR PEER REVIEW 20 o 27
Figu e 5. Flow cy ome y analysis o he cell cycle in ke a inocy es (HaCaT cell line) ollowing ex-
posu e o di e en concen a ions o he GS blend ex ac . The GS blend esul s a e p esen ed as
mean ± s anda d e o (n = 3). Signi ican di e ences be ween samples o each cell cycle phase a e
deno ed by di e en lowe case le e s (p < 0.05).
The cell cycle o BJ-5 a ib oblas cells was also e alua ed using low cy ome y, wi h
he ib oblas ga e selec ed in he same manne . The esul s a e shown below (Figu e 6).
Rega ding he BJ-5 a cell cycle, he GS blend ex ac does no appea o signi ican ly
al e cell cycle p og ession. Howe e , a highe concen a ions (83.38 and 166.75 µg/mL),
cell cycle a es in he Sub G0-G1 phase was obse ed, sugges ing he po en ial induc ion
o apop osis (Figu e 6).
Figu e 6. Flow cy ome y analysis o he cell cycle in ib oblas s (BJ-5 a cell line) ollowing exposu e
o di e en concen a ions o he GS blend ex ac . The GS blend esul s a e p esen ed as mean ±
s anda d e o (n = 3). Signi ican di e ences be ween samples o each cell cycle phase a e deno ed
by di e en lowe case le e s (p < 0.05).
These indings suppo he po en ial o he GS ex ac in managing sca o ma ion.
The GS ex ac a es s ke a inocy es and ib oblas s in he G0-G1 and Sub G0-G1 phases,
impeding p oli e a ion and p omo ing apop osis. This is c ucial o managing keloids by
educing excessi e ib o ic esponses [71–73]. Simila ly, ea ing hype ophic sca s in-
ol es inhibi ing ib oblas p oli e a ion and inducing apop osis [70].
O e all, hese indings highligh he he apeu ic po en ial o he GS ex ac in man-
aging sca o ma ion by egula ing he cell cycle and p omo ing apop osis.
3.6.4. E ec o he GS Blend Ex ac on Cell Mig a ion o HaCaT Ke a inocy es and BJ-5 a
Fib oblas s
Cell mig a ion is a c ucial p ocess in wound healing, in ol ing he mo emen o
ke a inocy es o co e he wound si e and ib oblas s o deposi collagen and emodel he
ex acellula ma ix [74,75]. To e alua e he in luence o he GS ex ac on wound healing,
he mig a o y abili y o HaCaT ke a inocy es and BJ-5 a ib oblas s was assessed using he
sc a ch me hod. Figu e 7 demons a es he mig a ion pe o mance o HaCaT cells upon
con ac wi h he GS blend ex ac .
The esul s sugges ha he GS blend ex ac does no a ec BJ-5 a ib oblas mig a-
ion bu educes HaCaT cell mig a ion in a concen a ion-dependen manne (Figu e 7).
b
a
aa
b
a
aa
ab
a
aa
a
a
aa
0
10
20
30
40
50
60
70
80
90
Sub G0-G1 G0-G1 S G2-M
Cells (%)
Cell cycle phase
Con ol
20.84 µg/mL GS ex ac
83.38 µg/mL GS ex ac
166.75 µg/mL GS ex ac
Figu e 6. Flow cy ome y analysis o he cell cycle in ib oblas s (BJ-5 a cell line) ollowing expo-
su e o di e en concen a ions o he GS blend ex ac . The GS blend esul s a e p esen ed as
mean ±s anda d
e o (n = 3). Signi ican di e ences be ween samples o each cell cycle phase a e
deno ed by di e en lowe case le e s (p< 0.05).
These indings suppo he po en ial o he GS ex ac in managing sca o ma ion.
The GS ex ac a es s ke a inocy es and ib oblas s in he G0-G1 and Sub G0-G1 phases,
impeding p oli e a ion and p omo ing apop osis. This is c ucial o managing keloids
by educing excessi e ib o ic esponses [
71
–
73
]. Simila ly, ea ing hype ophic sca s
in ol es inhibi ing ib oblas p oli e a ion and inducing apop osis [70].
O e all, hese indings highligh he he apeu ic po en ial o he GS ex ac in manag-
ing sca o ma ion by egula ing he cell cycle and p omo ing apop osis.
3.6.4. E ec o he GS Blend Ex ac on Cell Mig a ion o HaCaT Ke a inocy es and
BJ-5 a Fib oblas s
Cell mig a ion is a c ucial p ocess in wound healing, in ol ing he mo emen o
ke a inocy es o co e he wound si e and ib oblas s o deposi collagen and emodel he
ex acellula ma ix [
74
,
75
]. To e alua e he in luence o he GS ex ac on wound healing,
he mig a o y abili y o HaCaT ke a inocy es and BJ-5 a ib oblas s was assessed using he
sc a ch me hod. Figu e 7demons a es he mig a ion pe o mance o HaCaT cells upon
con ac wi h he GS blend ex ac .
An ioxidan s 2025,14, 338 20 o 25
An ioxidan s 2025, 14, x FOR PEER REVIEW 21 o 27
Figu e 7. In luence o he GS blend ex ac on ke a inocy e (HaCaT cell line) mig a ion measu ed
using he sc a ch assay a 0, 6, and 24 h. The GS blend esul s a e p esen ed as mean ± s anda d e o
(n = 3). Signi ican di e ences be ween samples a 24 h a e deno ed by di e en lowe case le e s (p
< 0.05).
A a lowe concen a ion (20.84 µg/mL), HaCaT cell mig a ion emained compa able
o ha o he con ol g oup, indica ing no ad e se e ec s. Howe e , a highe concen a-
ions (83.38–166.75 µg/mL), he ex ac signi ican ly educed mig a ion, sugges ing po en-
ial applica ions in he con olled inhibi ion o cell mo emen .
As shown in Figu e 8, he GS blend ex ac does no signi ican ly impac BJ-5 a ib o-
blas mig a ion, as mig a ion a es emained simila ac oss ea ed and un ea ed cells.
Figu e 8. In luence o he GS blend ex ac on ke a inocy e (BJ-5 a cell line) mig a ion measu ed
using he sc a ch assay a 0, 6, and 24 h GS blend. The esul s a e p esen ed as mean ± s anda d e o
(n = 3). Signi ican di e ences be ween samples a 24 h and samples a 48 h a e deno ed by a lowe -
case le e (p < 0.05).
Resea ch by Loggenbe g e al. [72] demons a ed ha plan -based ex ac s can sup-
p ess ke a inocy e mig a ion by educing pla ele -de i ed g ow h ac o (PDGF-AA) ex-
p ession, which plays a key ole in ke a inocy e p oli e a ion and angiogenesis. Since an-
giogenesis is a majo ac o in hype ophic sca s and keloids, he inhibi ion o
b
ab
aa
0
20
40
60
80
100
120
Con ol 20.84 µg/mL 83.38 µg/mL 166.75 µg/mL
Open a ea (%)
Phenolic Compounds Concen a ion, µg/mL
0h
6h
24h
aa
a
a
a
a
aa
0
20
40
60
80
100
120
Con ol 20.84 µg/mL 83.38 µg/mL 166.75 µg/mL
Open a ea (%)
GS ex ac concen a ion, µg/mL
0h
24h
48h
Figu e 7. In luence o he GS blend ex ac on ke a inocy e (HaCaT cell line) mig a ion measu ed
using he sc a ch assay a 0, 6, and 24 h. The GS blend esul s a e p esen ed as mean
±
s anda d e o
(n = 3). Signi ican di e ences be ween samples a 24 h a e deno ed by di e en lowe case le e s
(p< 0.05).
The esul s sugges ha he GS blend ex ac does no a ec BJ-5 a ib oblas mig a ion
bu educes HaCaT cell mig a ion in a concen a ion-dependen manne (Figu e 7).
A a lowe concen a ion (20.84
µ
g/mL), HaCaT cell mig a ion emained compa able
o ha o he con ol g oup, indica ing no ad e se e ec s. Howe e , a highe concen a ions
(83.38–166.75
µ
g/mL), he ex ac signi ican ly educed mig a ion, sugges ing po en ial
applica ions in he con olled inhibi ion o cell mo emen .
As shown in Figu e 8, he GS blend ex ac does no signi ican ly impac BJ-5 a
ib oblas mig a ion, as mig a ion a es emained simila ac oss ea ed and un ea ed cells.
Resea ch by Loggenbe g e al. [
72
] demons a ed ha plan -based ex ac s can supp ess
ke a inocy e mig a ion by educing pla ele -de i ed g ow h ac o (PDGF-AA) exp ession,
which plays a key ole in ke a inocy e p oli e a ion and angiogenesis. Since angiogenesis is
a majo ac o in hype ophic sca s and keloids, he inhibi ion o ke a inocy e mig a ion
by he GS ex ac sugges s po en ial an i-angiogenic p ope ies, which could help con ol
excessi e sca o ma ion [73,74].
S udies indica e ha keloids and hype ophic sca s exhibi inc eased angiogenesis,
sugges ing ha a ge ing angiogenic pa hways may con ibu e o educing excessi e sca
o ma ion [
75
–
78
]. Thus, he obse ed educ ion in HaCaT cell mig a ion may be linked o
an i-angiogenic mechanisms, suppo ing GS ex ac ’s po en ial applica ion in sca he apy.
The GS blend ex ac does no p omo e wound healing h ough inc eased cell mig a-
ion; i s abili y o inhibi mig a ion and p oli e a ion aligns wi h indings ela ed o sca
issue egula ion, sugges ing po en ial he apeu ic applica ions in con olling keloids and
hype ophic sca s.
While ou s udy p o ides p elimina y e idence ha he GS blend ex ac in luences
key cellula beha iou s in ol ed in sca modula ion, pa icula ly h ough he inhibi ion o
ke a inocy e mig a ion and p oli e a ion, i is impo an o men ion ha sca o ma ion is
la gely in luenced by ex acellula ma ix (ECM) emodelling. Abno mal ECM deposi ion,
including al e ed collagen ype I and III exp ession, ma ix me allop o einase (MMP)
ac i i y, and issue inhibi o s o me allop o eases (TIMPs), plays a pi o al ole in ib osis
and sca o ma ion. Taking in o conside a ion he p omising esul s ob ained in his
s udy, u he esea ch will be conduc ed o in es iga e GS ex ac ’s di ec e ec s on ECM
An ioxidan s 2025,14, 338 21 o 25
emodelling, namely, by assessing he ex ac ’s impac on collagen syn hesis, MMP/TIMP
balance, and collagenase.
An ioxidan s 2025, 14, x FOR PEER REVIEW 21 o 27
Figu e 7. In luence o he GS blend ex ac on ke a inocy e (HaCaT cell line) mig a ion measu ed
using he sc a ch assay a 0, 6, and 24 h. The GS blend esul s a e p esen ed as mean ± s anda d e o
(n = 3). Signi ican di e ences be ween samples a 24 h a e deno ed by di e en lowe case le e s (p
< 0.05).
A a lowe concen a ion (20.84 µg/mL), HaCaT cell mig a ion emained compa able
o ha o he con ol g oup, indica ing no ad e se e ec s. Howe e , a highe concen a-
ions (83.38–166.75 µg/mL), he ex ac signi ican ly educed mig a ion, sugges ing po en-
ial applica ions in he con olled inhibi ion o cell mo emen .
As shown in Figu e 8, he GS blend ex ac does no signi ican ly impac BJ-5 a ib o-
blas mig a ion, as mig a ion a es emained simila ac oss ea ed and un ea ed cells.
Figu e 8. In luence o he GS blend ex ac on ke a inocy e (BJ-5 a cell line) mig a ion measu ed
using he sc a ch assay a 0, 6, and 24 h GS blend. The esul s a e p esen ed as mean ± s anda d e o
(n = 3). Signi ican di e ences be ween samples a 24 h and samples a 48 h a e deno ed by a lowe -
case le e (p < 0.05).
Resea ch by Loggenbe g e al. [72] demons a ed ha plan -based ex ac s can sup-
p ess ke a inocy e mig a ion by educing pla ele -de i ed g ow h ac o (PDGF-AA) ex-
p ession, which plays a key ole in ke a inocy e p oli e a ion and angiogenesis. Since an-
giogenesis is a majo ac o in hype ophic sca s and keloids, he inhibi ion o
b
ab
aa
0
20
40
60
80
100
120
Con ol 20.84 µg/mL 83.38 µg/mL 166.75 µg/mL
Open a ea (%)
Phenolic Compounds Concen a ion, µg/mL
0h
6h
24h
aa
a
a
a
a
aa
0
20
40
60
80
100
120
Con ol 20.84 µg/mL 83.38 µg/mL 166.75 µg/mL
Open a ea (%)
GS ex ac concen a ion, µg/mL
0h
24h
48h
Figu e 8. In luence o he GS blend ex ac on ke a inocy e (BJ-5 a cell line) mig a ion measu ed
using he sc a ch assay a 0, 6, and 24 h GS blend. The esul s a e p esen ed as mean
±
s anda d
e o (
n=3
). Signi ican di e ences be ween samples a 24 h and samples a 48 h a e deno ed by a
lowe case le e (p< 0.05).
4. Conclusions
This s udy comp ehensi ely analysed he GS blend ex ac , ocusing on i s phenolic
con en , an ioxidan p ope ies, and biological e ec s. The ex ac was ound o be ich
in bioac i e phenolic compounds, pa icula ly p oan hocyanidin ime s, ca echins, u in,
que ce in, and es e a ol e ame s. The GS ex ac exhibi ed s ong an ioxidan ac i i y,
pa icula ly in he ABTS assay, likely due o i s high con en o p oan hocyanidin ime s,
which a e known o hei po en adical sca enging p ope ies.
In
in i o
assays, he GS ex ac was non-cy o oxic a low concen a ions. Howe e , a
highe concen a ions, i inhibi ed cell p oli e a ion and mig a ion, pa icula ly in HaCaT
ke a inocy es, induced cell cycle a es in he G0-G1 and Sub G0-G1 phases, and inc eased
ib oblas apop osis.
These indings sugges ha he GS ex ac may play a ole in egula ing cell p oli e -
a ion. Howe e , he da a do no suppo i s di ec applica ion in wound healing o sca
o ma ion, pa icula ly in keloids, as no e idence o ex acellula ma ix emodelling was
p o ided. Fu he s udies a e needed o cla i y i s mechanisms o ac ion.
Au ho Con ibu ions: M.S.: concep ualiza ion, da a cu a ion, o mal analysis, in es iga ion, me hod-
ology, and w i ing—o iginal d a . C.B.: me hodology, supe ision, w i ing—o iginal d a , and
w i ing— e iew and edi ing. D.S.: da a cu a ion and me hodology. H.A.: da a cu a ion and me hod-
ology. A.C.: da a cu a ion and w i ing— e iew and edi ing. J.A.T.: supe ision, and w i ing— e iew
and edi ing. A.N.B.: unding acquisi ion, p ojec adminis a ion, supe ision, w i ing—o iginal d a ,
and w i ing— e iew and edi ing. All au ho s ha e ead and ag eed o he published e sion o
he manusc ip .
Funding: This wo k was suppo ed by he Na ional Funds by he FCT, he Po uguese Founda ion o
Science and Technology, unde p ojec 2023.03608.BD and was unded by he Cen e o he Resea ch
and Technology o Ag o-En i onmen al and Biological Sciences (CITAB) esea ch uni suppo ed by
he FCT unde p ojec s UID/04033 and LA/P/0126/2020 (h ps://doi.o g/10.54499/LA/P/0126/2
020) and WASTELESS unding om HORIZONRIA (HORIZON-CL6-2022-FARM2FORK-01) unde
G an Ag eemen No. 101084222.
Da a A ailabili y S a emen : Da a is con ained wi hin he a icle.
An ioxidan s 2025,14, 338 22 o 25
Con lic s o In e es : Au ho s Monica Se a, Hugo Almeida, Diana Sousa and Ana Casas a e
employed by he company Mesosys em. The emaining au ho s decla e ha he esea ch was
conduc ed in he absence o any comme cial and inancial ela ionships ha could be cons uc ed as
a po en ial con lic o in e es .
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OIV—In e na ional O ganisa ion o Vine and Wine. Annual Assessmen o he Wo ld Vine and Wine Sec o ; OIV: Dijon, F ance, 2022.
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Fe e -Gallego, R.; Sil a, P. The Wine Indus y By-P oduc s: Applica ions o Food Indus y and Heal h Bene i s. An ioxidan s
2022,11, 2025. [C ossRe ] [PubMed]
4.
Ch is , K.L.; Bu i , R.L. C i ical En i onmen al Conce ns in Wine P oduc ion: An In eg a i e Re iew. J. Clean. P od. 2013,53,
232–242. [C ossRe ]
5.
Sil a, A.; Sil a, V.; Ig ejas, G.; Ai es, A.; Falco, V.; Valen ão, P.; Poe a, P. Phenolic Compounds Classi ica ion and Thei Dis ibu ion
in Winemaking By-P oduc s. Eu . Food Res. Technol. 2023,249, 207–239. [C ossRe ]
6. Maicas, S.; Ma eo, J.J. Sus ainabili y o Wine P oduc ion. Sus ainabili y 2020,12, 559. [C ossRe ]
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G aça, A.; Co be -Milwa d, J.; Schul z, H.R.; Oze , C.; de la Fuen e, M. Managing By-P oduc s o Vi i inicul u al O igin; OIV
COLLECTIVE EXPERTISE; OIV–In e na ional O ganiza ion o Vine and Wine: Pa is, F ance, 2017.
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