an ioxidan s
A icle
P o ec i e E ec o Cocoa Bean Shell agains In es inal Damage:
An Example o Byp oduc Valo iza ion
Daniela Rossin 1,† , Le icia Ba bosa-Pe ei a 2,† , Noemi Iaia 1, Ba ba a So e o 1, Alice Cos anza Danze o 1,
Giuseppe Poli 1, Giuseppe Zeppa 3,‡ and Fio ella Biasi 1,*,‡
Ci a ion: Rossin, D.; Ba bosa-Pe ei a,
L.; Iaia, N.; So e o, B.; Danze o, A.C.;
Poli, G.; Zeppa, G.; Biasi, F. P o ec i e
E ec o Cocoa Bean Shell agains
In es inal Damage: An Example o
Byp oduc Valo iza ion. An ioxidan s
2021,10, 280. h ps://doi.o g/
10.3390/an iox10020280
Academic Edi o s: A ianna Vignini
and Je ey B. Blumbe g
Recei ed: 20 Decembe 2020
Accep ed: 9 Feb ua y 2021
Published: 12 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 Clinical and Biological Sciences, Uni e si y o Tu in, 10043 O bassano, I aly;
[email p o ec ed] (D.R.); [email p o ec ed] (N.I.); ba ba a.so e [email p o ec ed] (B.S.);
[email p o ec ed] (A.C.D.); [email p o ec ed] (G.P.)
2Depa men o Analy ical Chemis y, Nu i ion and Food Science, Facul y o Pha macy, Uni e si y o
San iago de Compos ela, 15782 San iago de Compos ela, Spain; [email p o ec ed]
3Depa men o Ag icul u al, Fo es y, and Food Sciences (DISAFA), Uni e si y o Tu in, 10095 G ugliasco,
I aly; [email p o ec ed]
*Co espondence: io [email p o ec ed]; Tel.: +39-011-670-5420
† Equally con ibu ed o his manusc ip .
‡ These au ho s sha e senio au ho ship.
Abs ac :
Backg ound: Cocoa bean shell (CBS), a main byp oduc o cocoa p ocessing, ep esen s a
sou ce o componen s such as polyphenols and me hylxan hines, which ha e been associa ed wi h
a educed isk o se e al diseases. The e o e, CBS has po en ial applica ion as a ood ing edien .
In es inal mucosa is exposed o immune and in lamma o y esponses igge ed by die a y agen s,
such as oxys e ols, which de i e om choles e ol oxida ion and a e p o-oxidan compounds able
o a ec in es inal unc ion. We aimed a in es iga ing he capabili y o he Fo as e o cul i a CBS,
added o no added o ice c eam, o p o ec agains he in es inal ba ie damage induced by a die a y
oxys e ol mix u e. Me hods: Composi ion and an ioxidan capaci y o
in i o
diges ed CBS and CBS-
en iched ice c eam we e analyzed by high-pe o mance liquid ch oma og aphy and 1,1-diphenyl-2-
pic yl-hyd azyl adical-sca enging assay, espec i ely. CaCo-2 cells di e en ia ed in o en e ocy e-like
monolaye we e incuba ed wi h 60
µ
M oxys e ol mix u e in he p esence o CBS o mula ions.
Resul s: The oxys e ol mix u e induced igh junc ion impai men , in e leukin-8 and monocy e
chemoa ac an p o ein-1 cell elease, and oxida i e s ess- ela ed nuclea ac o e y h oid 2 p45-
ela ed ac o 2 esponse N 2. Bo h CBSs p o ec ed cells om hese ad e se e ec s, p obably hanks
o hei high phenolic con en . CBS-en iched ice c eam showed he highes an ioxidan capaci y.
Theob omine, which is in high concen a ions o CBS, was also es ed. Al hough heob omine exe ed
no e ec on N 2 exp ession, i s an i-in lamma o y coope a ing ac i i y in CBS e ec canno be
excluded. Conclusions: Ou indings sugges ha CBS-en iched ice c eam may be e ec i e in he
p e en ion o gu in eg i y damage associa ed wi h oxida i e/in lamma o y eac ions.
Keywo ds:
polyphenols; die a y oxys e ols; epi helial ba ie ; in es inal in lamma ion; IL-8; MCP-1;
igh junc ions; N 2; heob omine
1. In oduc ion
Chocola e consump ion has ecen ly been on he ise hanks o wide comme cial
a ailabili y a ound he wo ld, pa icula ly in he Eu opean Union and No h Ame ica,
ha ing an annual consump ion g ow h a e o 1.7% and 3.6% annually, espec i ely [
1
].
Besides being uni e sally amous as one o he op com o oods, chocola e is now well
known o i s heal hy e ec s, p ima ily on he ca dio ascula sys em [
2
] and as an an ide-
p essan [
3
]. In addi ion, i s po en ial bene i s may play an impo an ole in he p e en ion
o o he pa hological condi ions, including cance [4–7].
Cocoa beans, he seeds o he opical Theob oma cacao L. ee, a e he sou ce o he
main ing edien used o chocola e and cocoa-de i a i e ood p oduc ion, namely cocoa
An ioxidan s 2021,10, 280. h ps://doi.o g/10.3390/an iox10020280 h ps://www.mdpi.com/jou nal/an ioxidan s
An ioxidan s 2021,10, 280 2 o 18
mass (liquo ). To ob ain he cocoa mass, cocoa beans unde go se e al ea men s, among
which a e oas ing and husking, which a e necessa y o emo e he so-called cocoa bean
shell (CBS), a ine shell co e ing he bean [8].
CBS accoun s o 12–20% o bean weigh [
8
] and ep esen s an impo an economic issue
in he cocoa indus y o i s disposal [
1
,
9
]. To minimize was e p oduc ion and ela i e cos s,
al e na i e inno a i e s a egies o CBS ecycling as possible addi i es (e.g., supplemen s,
in eg a o s) in unc ional ood and be e ages a e now being e alua ed [8,10].
Recen s udies ha e, in ac , ecognized CBS as a sou ce o disease-p e en ing compo-
nen s, including ibe s, mine als, me hylxan hines (ca eine, heob omine), and polyphenols
(ca echins, epica echins, p ocyanidins), whose ac ual con en highly depends on ac o s
such as cocoa gene ic a ie y, cul i a ion, and cocoa bean p ocessing [8–12].
Heal h-p omo ing e ec s o polyphenols and me hylxan hines ha e been widely
shown. Polyphenols, in pa icula , show ee adical sca enging and me al chela ing e ec s,
and a e ecognized as s ong modula o s o edox sensi i e-pa hways which a e capable
o coun e ac ing cell and issue oxida i e damage in se e al ch onic human pa hologies,
including in lamma o y gu diseases and colo ec al cance [13–15].
Me hylxan hines a e mainly known o hei smoo h muscle elaxan , diu e ic, and
co ona y asodila o p ope ies. The main ac ion mechanisms o ca eine and heob omine
a e adenosine ecep o s’ blockade and phosphodies e ase inhibi ion, which seem o accoun
o hei an i-in lamma o y and an i umo al ac i i y [16,17].
Besides ha , heob omine exhibi s o he impo an adenosine ecep o -independen e ec s,
such as he educ ion o cellula oxida i e s ess o egula ion o gene exp ession [16,17].
Polyphenols, and e y ecen ly also heob omine, ha e been p oposed as po en ial
modula o s o in es inal pe meabili y [
18
,
19
], whose pe u ba ion is an impo an ea u e
o gu unc ion [20].
In es inal mucosa is indeed pa icula ly exposed o en i onmen al and die a y agen s
esponsible o sus aining immune and in lamma o y esponses [
20
]. The ac i a ion o
hese p ocesses can igge an o e p oduc ion o oxidan species, pa icipa ing in in es inal
epi helial pe meabili y damage and mucosal igh junc ion (TJ) de angemen [21,22].
A die a y egimen ich in ui and ege ables and/o he in ake o supplemen s is
able o educe in lamma ion and p ese e in es inal ba ie in eg i y [23].
On he con a y, he Wes e n-s yle die ep esen s one o he main isks o al e ed
in es inal in eg i y [
24
]. The die a y animal a s ha can unde go oxida ion du ing ood
p ocessing ha e been sugges ed o exe in lamma o y and oxida i e insul s in gu mu-
cosa [
25
]. The oxida i e choles e ol de i a i es, oxys e ols, a e in ol ed in modula ing
in lamma ion, ib osis, and cell dea h, hus playing a ole in he pa hogenesis o se e al
ch onic diseases, such as in lamma ion- ela ed bowel diseases [26].
We p e iously p o ed ha a mix u e o oxys e ols co esponding o high die a y
consump ion o choles e ol igge ed oxida i e eac ions and in lamma o y esponses
in human en e ocy e-like CaCo-2 cells. Nico inamide adenine dinucleo ide phospha e
(NADPH) oxidase, mi ogen-ac i a ed p o ein (MAP) kinase, and nuclea ac o kappa-ligh -
chain-enhance o ac i a ed B cells (NF-kB) appea ed o be he main signaling pa hway
in ol ed [
27
]. Those e en s we e p e en ed in he same cellula model by ed wine ex ac s
showing high phenolic con en [27].
Oxys e ols ha e also been ound o modula e he exp ession o genes con olled
by s ess-induced nuclea ac o e y h oid 2 p45- ela ed ac o 2 (N 2) depending on
he ype o a ge cells, he en i onmen al condi ions, and he concen a ion and ype o
oxys e ols [
28
]. N 2 is a key ansc ip ion ac o whose ac i a ion ep esen s a p o ec ing
adap i e cell esponse agains edox s esso s by p omo ing codi ica ion o a ious s ess-
ela ed de oxi ying and an ioxidan enzymes [
29
]. A ecen s udy showed ha die a y
oxys e ols we e able o induce in lamma ion in CaCo-2 cells by also ac i a ing he immune
sys em- ela ed Toll-like Recep o (TLR) supe amily membe s TLR2 and TLR4 [
30
]. In hose
expe imen s, CBS ex ac s om Hondu as cocoa beans we e e ec i e in p o ec ing in es inal
mucosa om oxida i e s ess and in lamma ion h ough TLR down egula ion [30].
An ioxidan s 2021,10, 280 3 o 18
The aim o ou cu en in es iga ion is o gi e
in i o
u he e idence o CBS posi i e
e ec agains oxys e ol-induced oxida i e and in lamma o y in es inal ba ie damage.
Al hough inc easing li e a u e sugges se e al heal hy p ope ies o polyphenols and
me hylxan hines signi ican ly p esen in CBS, mos o he e idence abou an ioxidan and
an i-in lamma o y ac i i ies a e based on s udies conduc ed on single compounds, while
he bioac i i y o CBS, and subsequen ly, i s use in unc ional oods has no been in-dep h
in es iga ed ye .
We hink ha ou indings on he bene icial e ec s o CBS-en iched ice c eam gi e
alue o cocoa byp oduc s as heal hy ood ecycled supplemen s o he p e en ion o
in lamma o y gu diseases.
2. Ma e ials and Me hods
2.1. Ma e ials
Chemicals o he analyses o phenolic compounds, me hylxan hines, and an ioxidan
ac i i y: E hanol (
≥
99.9%), o mic acid (98–100%), 6-hyd oxy-2,5,7,8- e ame hylch oman-2-
ca boxylic acid (97%) (T olox), 2,2-diphenyl-1- pic ylhyd azyl (95%) (DPPH), Folin-Ciocal eu’s
phenol eagen , Na2CO3 (
≥
99.5%), NaNO2 (
≥
99%), AlCl3 (
≥
99%), anillin (
≥
99%), HCl,
(+)-ca echin hyd a e (> 98%), que ce in-3-
β
-D-glucoside (
≥
90%), heob omine (
≥
98.5%), and
ca eine (
≥
98.5%), ob ained om Sigma-Ald ich (Milan, I aly). Gallic acid and (
−
)-epica echin
(≥99%) we e om Fluka (Milan, I aly).
Chemicals o he simula ed gas oin es inal diges ion: Na2CO3 (
≥
99.5%) and HCl
we e ob ained om Sigma-Ald ich (Milan, I aly). NaHCO3, NaCl, KCl, K2HPO4, KH2PO4,
MgCl2
·
6H2O, (NH4)2CO3, NaOH, and CaCl2
·
2H2O we e p o ided by Ca lo E ba (Milan,
I aly). The enzyme
α
-amylase om Bacillus sp., pepsin om po cine gas ic mucosa, pan-
c ea in om po cine panc eas, and bile sal s we e supplied by Sigma-Ald ich (Milan, I aly).
Chemicals and ma e ials o cell cul u e and ea men : Cell cul u e glucose-en iched
Dulbecco’s modi ied Eagle’s medium (DMEM), e al bo ine se um (FBS), and ypsin
(5 g/L solu ion) we e om Eu oclone SpA (Milan, I aly). 5
α
,6
α
-Epoxycholes e ol (
α
-epox),
5
β
, 6
β
-epoxycholes e ol (
β
-epox), 7-ke ocholes e ol (7K), 7
α
-hyd oxycholes e ol (7
α
-HC),
and 7β-hyd oxycholes e ol (7β-HC) we e pu chased om A an i Pola Lipids (Alabas e ,
AL, USA). Mul iwell pla es and 13 mm glass slides we e om VWR In e na ional S l
(Milan, I aly).
Ma e ials and ki s o he biomolecula analyses: Enzyme-linked immunoso ben
assay (ELISA) ki s o he e alua ion o human in e leukin (IL)-8 and monocy e chemoa -
ac an p o ein (MCP)-1 we e ob ained om PeP o ech (DBA I alia S l, Seg a e, Milan,
I aly). San a C uz Bio echnology (DBA I alia S l, Seg a e, Milan, I aly) p o ided he poly-
clonal p ima y an ibodies abbi an i-junc ional adhesion molecule-A (JAM-A) (SC-25629),
mouse an i-occludin (SC-133256), and mouse an i-claudin 1 (SC-166338). Cell Signaling
Technology (Eu oclone SpA, Milan, I aly) p o ided he seconda y an ibodies an i- abbi
IgG HRP-conjuga ed (7074S) and an i-mouse IgG HRP-conjuga ed (7076S). The mo Fishe
Scien i ic (Li e Technologies I alia, Monza, I aly) p o ided goa an i- abbi IgG-Alexa Fluo
546 and goa an i-mouse IgG-Alexa Fluo 488, li hium dodecyl sul a e (LDS) Sample Bu e
4X, and di hio ei ol (DTT) Sample Reduce 10X. P o eases inhibi o s cock ail “cOmple e
ULTRA Table s Mini EASYpack” and he nico inamide adenine dinucleo ide (NADH) we e
pu chased by Roche SpA (Monza, I aly).
Bio-Rad p o ein assay dye eagen and ECL
®
Wes e n Blo ing Sys em we e om
Bio-Rad S l (SIAL, Rome, I aly). Hybond ECL ni ocellulose memb ane was om GE
Heal hca e S l (Milan, I aly).
TRIzol eagen was om In i ogen (S. Giuliano Milanese, I aly). High-Capaci y
Complemen a y DNA (cDNA) e e se ansc ip ion ki , TaqMan gene exp ession assay
ki s o human N 2 and
β
-ac in, TaqMan Fas Uni e sal PCR (polyme ase chain eac ion)
mas e mix, and TaqMan A ay 96-well pla es we e om Applied Biosys ems (Monza,
I aly). Milli-Q il e sys em ul apu e wa e was om Millipo e (Milan, I aly). All o he no
speci ied eagen s and chemicals we e ob ained om Sigma-Ald ich (Milan, I aly).
An ioxidan s 2021,10, 280 4 o 18
2.2. Cocoa Bean Shell Sample P epa a ion and Ice C eam Fo mula ion
Pas iglie Leone S l (Tu in, I aly) kindly supplied cocoa bean shell (CBS) samples
yielded om cocoa beans o Fo as e o cul i a om Sao Tomé.
CBS samples we e educed o ine pa icles in a ball mill o ob ain a powde , 95%
o which showed a size lowe han 20
µ
m (measu ed by a Mas e size 3000, Mal e n
Ins umen s Limi ed, Wo ces e shi e, UK).
CBS samples we e hen used as a eplace s in he p epa a ion o ice c eam (IC) a
di e en concen a ions (0, 2, 4, 6 and 8 w .% o CBS) [
31
]. The ice c eam ob ained by
4% o i ied CBS lou (CBS-IC) was selec ed because o i s educed a and inc eased
ibe con en s compa ed wi h plain IC (conside ed as con ol sample). CBS-IC o mula-
ion main ained simila s uc u al cha ac e is ics o he chocola e ice c eam (con ol) and
highe accep ance a e he consume es . The o mula ions used o ice c eam p oduc-
ion a e shown in Table S1 (see Supplemen a y Ma e ials). The chemical and nu i ional
cha ac e iza ion o samples a e shown in Table S2 (see Supplemen a y Ma e ials).
CBS and CBS-IC we e used o expe imen s wi h he in es inal cells a e
in i o
diges ion as he ea e epo ed.
2.3. In Vi o Simula ed Gas oin es inal Diges ion
The simula ed gas oin es inal diges ion ela ed o he o al, gas ic, and small in es inal
ac s was pe o med o mimic human diges ion acco ding o he ou ine s anda dized s a ic
in i o
INFOGEST p o ocol sui able o ood desc ibed by Minekus and colleagues [
32
].
Fo each diges ion phase, diges i e juices we e p epa ed in Milli-Q wa e e e y 2 days o
he mou h (Simula ed Sali a Fluid, SSF), s omach (Simula ed Gas ic Fluid, SGF), and small
in es ine (Simula ed Duodenal Fluid, SDF), ollowing p o ocol indica ions as desc ibed by
he au ho s. Each diges ion luid had a di e en elec oly e composi ion, and he pH was
adjus ed o 7 in SSF, o 3 in SGF, and 7 in SIF using HCl o NaOH 1M.
Fo he o al phase, 5 g o ice c eam we e mixed wi h 5 mL SSF con aining human
sali a y
α
-amylase solu ion (1500 U/mL). Then, he mix u e was adjus ed o pH 7
±
0.2
and incuba ed a 37 ◦C in a shaking wa e ba h o 2 min.
Fo he gas ic phase, 10 mL SGF con aining po cine pepsin (2000 U/mL) we e added
o he ice c eam. The mix u e was hen adjus ed o pH 3
±
0.2 and incuba ed a 37
◦
C o
2 h.
Finally, 20 mL o SDF con aining panc ea in (100 U/mL ypsin ac i i y) and po cine
bile ex ac (10 mM) we e added in he las s ep o diges ion. The inal mix u e was adjus ed
o pH 7
±
0.2 and samples we e incuba ed wi h o bi al agi a ion a 37
◦
C o u he 2 h.
A e he comple e diges ion, pH was adjus ed o 5.4, and he samples we e immedia ely
kep in ice o minimize enzyme ac i i y and hen cen i uged a 12,500
×
ga 4
◦
C o 10 min.
Based on he bioaccessibili y s udies pe o med in ou labo a o y [
33
], he supe na an s
we e il e ed h ough a 0.22
µ
m cellulose ace a e memb ane il e (VWR, Milan, I aly)
and s o ed a
−
20
◦
C o u he analyses. The simula ed gas oin es inal diges ion was
pe o med in iplica e o each sample. CBS lou was also diges ed wi hou IC o e alua e
he e ec o ood ma ix on bioac i e compound bioa ailabili y.
2.4. Re e se Phase-High-Pe o mance Liquid Ch oma og aphy-Pho odiode A ay Analysis o Cocoa
Bean Shells and Ice C eam Compounds
CBS and IC ch oma og aphic analyses be o e and a e comple ed diges ion we e
pe o med wi h e e se phase-high-pe o mance liquid ch oma og aphy-pho odiode a ay
(RP-HPLC-PDA) The mo-Finnigan Spec a Sys em (The mo-Finnigan, Wal ham, USA)
equipped wi h a P2000 bina y g adien pump, SCM 1000 degasse , AS 3000 au oma ic
injec o and Finnigan Su eyo PDA Plus de ec o .
The phenolic compounds and me hylxan hines we e sepa a ed a 35
◦
C on a e e se
phase Kine ex Phenyl-Hexyl C18 column (150
×
4.6 mm in e nal diame e and 5
µ
m pa icle
size) (Phenomenex, Cas el Maggio e, I aly). The mobile phase consis ed o 0.1% o mic
acid (sol en A) and 100% me hanol (sol en B). A linea g adien elu ion me hod was
An ioxidan s 2021,10, 280 5 o 18
applied as ollows: 0–2 min wi h 90% A and 10% B; 2–18 min om 10 o 50% B; 18–40 min,
om 50 o 80% B; 40–42 min, up o 90% B. Column e-equilib a ion was hen pe o med by
90% A and 10% B 42–45 min elu ion. The mobile phase low a e was 1.0 mL/min, and he
sample injec ion olume was 10
µ
L. Ch omQues so wa e ( e sion 5.0) (The mo Fishe
Scien i ic, Li e Technologies I alia, Monza, I aly) was used o ins umen con ol as well as
da a collec ion and p ocessing. Da a we e acqui ed a he ollowing wa eleng hs: 272 nm
o heob omine and ca eine; 278 nm o (
−
)-epica echin, ca echin, ype-B and ype-A
p ocyanidins; 293 nm o p o oca echuic acid; and 350 nm o la onol-3-O-glucosides
(que ce in and kaemp e ol). The quan i ica ion was assessed by he ex e nal s anda d
me hod using 6-poin eg ession cu es cons uc ed a molecule maximum abso bance
wa eleng h (R2= 0.999).
2.5. To al Phenolic, Tannin, and Fla onoid Con en s
The amoun o o al phenolics (TPC), la onoids (TFC), and annins (TTC) in CBS and
CBS-IC was quan i ied as p e iously epo ed [33].
The BioTek Syne gy HT spec opho ome ic mul i-de ec ion 96-well mic opla e eade
(BioTek Ins umen s, Milan, I aly) was used o eco d he abso bance o each ac ion. All
he analyses we e pe o med in iplica e om 3 independen expe imen s. The concen-
a ion o o al phenolic compounds, exp essed in mg o gallic acid equi alen s (GAE)/L,
was de e mined using a s anda d cu e o gallic acid (20–100 mg/L). TFC and TTC we e
exp essed in mg o ca echin equi alen s (CE)/L e e ing o a s anda d cu e o ca echin
(5–500 mg/L).
2.6. An ioxidan Capaci y
CBS ac ion adical sca enging ac i i y (RSA) was e alua ed by DPPH adical-
sca enging assay. RSA was es ima ed by eco ding DPPH abso bance a 517 nm (using a
96-well mic opla e eade as epo ed abo e) and he analyses we e pe o med in iplica e
om 3 independen expe imen s. The inhibi ion pe cen age (IP) o DPPH adical was
calcula ed using he ollowing equa ion: IP (%) = [(A0
−
A30)/A0]
×
100 (whe e A0 is he
abso bance a ime 0, and A30 he abso bance a e 30 min). A s anda d cu e o T olox was
used (12.5–300
µ
M) o assess he adical-sca enging ac i i y alues, which we e exp essed
as µmoles o T olox equi alen s (TE) o mL o sample (µmol TE/mL).
2.7. Cell Cul u e and T ea men s
The Cell Bank In e lab Cell Line Collec ion (Genoa, I aly) p o ided human colo ec al
adenoca cinoma CaCo-2 cells (accession numbe : ICLC HTL97023). As indica ed on he
Cell Bank Websi e (h p://www.iclc.i /de ails/de _lis .php; [
34
]), cells we e pla ed a
1
×
10
6
/mL densi y, cul u ed in DMEM supplemen ed wi h 10% hea inac i a ed FBS, 1%
an ibio ic/an imyco ic solu ion (100 U/mL penicillin, 0.1 mg/mL s ep omycin, 250 ng/mL
ampho e icin B and 0.04 mg/mL gen amicin), and main ained a 37
◦
C in a humidi ied
a mosphe e con aining 5% CO2. To allow hei spon aneous di e en ia ion in o en e ocy e-
like pheno ype, cells we e g own o addi ional 18 days a e eaching con luence [35].
CaCo-2 di e en ia ion g ade was ou inely pe o med by moni o ing he alkaline
phospha ase (ALP) en e ocy e b ush bo de enzyme [
36
]. The cell monolaye was lysed
by incuba ing cells a oom empe a u e (RT) o 1 h wi h 300
µ
L PBS wi h 0.5% T i on
X-100 ( / ). The lysa es we e dispensed in o 96-well pla e (co esponding o 5
×
10
5
cells),
and 60
µ
L o enzyme subs a e (0.83 M 2-amino-2-me hyl-1-p opanol, 3.3 mM MgCl2,
13.3 mM disodium p-ni ophenol phospha e pH 10) was added o each well. A e 30 min
a 37
◦
C, he o ma ion o p-ni ophenol phospha e clea ed p oduc was s opped by
adding 100
µ
L 0.5 M NaOH o he wells. The p oduc abso bance was eco ded wi h a
96-mul iwell pla e eade (Model 680 Mic o-pla e Reade , Bio-Rad labo a o ies S l, Milan,
I aly) using a 405 nm wa eleng h il e . ALP ac i i y was exp essed as nmoles o p-
ni ophenol phospha e p oduced/mg cell p o ein. P o eins we e e alua ed wi h Bio-Rad
p o ein assay dye eagen , ollowing he p o ocol published by B ad o d [
37
]. Table 1
An ioxidan s 2021,10, 280 6 o 18
shows he ime cou se o ALP ac i i y (mic oscopy images o CaCo-2 cells a con luence
and di e en ia ed CaCo-2 cells a e 18 days pos -con luence a e shown in Figu e S1,
Supplemen a y Ma e ials).
Table 1. Time cou se o alkaline phospha ase ac i i y in CaCo-2 cells a e eaching cell con luence.
Pos -Con luence Days Alkaline Phospha ase Ac i i y (nmoles/mg P o ein)
T0 39.18 ±10.2
T3 83.84 ±6.7 *
T6 287.29 ±22.8 ***
T9 329.87 ±17.6 ***
T15 437.80 ±15.9 ***
T18 450.55 ±18.4 ***
Values o alkaline phospha ase ac i i y a e shown as nmoles/mg p o ein and a e e e ed as means
±
SD o 6
independen expe imen s. Cell cul u es we e g own up o 18 days a e con luence (T0). T3, T6, T9, T15, and
T18 e e o pos -con luence days o cell cul u e (3, 6, 9, 15, 18 days, espec i ely). Signi ican ly di e en s. T0:
*p< 0.05, *** p< 0.001.
A e cell di e en ia ion, CaCo-2 cells we e incuba ed in se um- ee medium o e nigh
o make hem quiescen be o e ea men . Cells we e hen ea ed a 37
◦
C o 24 h (o
6 h o gene exp ession expe imen s) wi h an oxys e ol mix u e (Oxy-mix) a 60
µ
M inal
concen a ion, and 5% FBS was added o DMEM o allow oxys e ol up ake by cells.
The pe cen age composi ion o oxys e ols used in he Oxy-mix was 42.96% o 7 K,
32.3% o
α
-epox, 5.76% o
β
-epox, 4.26% o 7
α
-HC, and 14.71% o 7
β
-HC. The mola i y
o each oxys e ol in 60
µ
M Oxy-mix was calcula ed as 25.8
µ
M 7K, 19.4
µ
M
α
-epox, 3.4
µ
M
β
-epox, 2.6
µ
M 7
α
-HC, and 8.8
µ
M 7
β
-HC by conside ing an a e age molecula weigh o
403 g/mol [30].
The 18-day di e en ia ed CaCo-2 cells we e p eincuba ed wi h CBS o CBS-IC ex ac s
o 1 h be o e he Oxy-mix ea men . Based on cell dea h analyses, CBS o CBS-IC we e
used o each inal 5% concen a ion in he cell cul u e.
In some expe imen s, cells we e p e ea ed wi h 13
µ
M heob omine co esponding o
i s concen a ion in CBS-IC, and simila analyses as o CBS/CBS-IC in he p esence o no
o Oxy-mix we e done.
2.8. Cell Dea h E alua ion
The ex acellula elease o lac a e dehyd ogenase (LDH) was conside ed as a pa ame-
e o cell dea h. Cells we e ea ed wi h IC, CBS-IC, o CBS, inc easing concen a ions o
each hei inal pe cen age in cul u e media o 5%, 10%, 30%, and 50% ( / ), and incuba ed
o no wi h 60
µ
M Oxy-mix. Un ea ed cells incuba ed o he same ime pe iod as ea ed
cells we e conside ed as con ol.
LDH was e alua ed spec opho ome ically a 340 nm wa eleng h by eco ding
NADH p oduc ion/min. LDH elease o 3 independen expe imen s was pe o med
in iplica e and exp essed as a pe cen age o he o al enzyme eleased in o cell cul u e
medium by comple e cell lysis (ob ained by 0.5% T i on X-100 addi ion o he pla e con ain-
ing he same cell densi y as he ea ed cells) (Table S3 Supplemen a y Ma e ials).
2.9. E alua ion o In e leukin-8 and Monocy e Chemoa ac an P o ein-1 P o ein Le els by
Enzyme-Linked Immunoso ben Assay
IL-8 and MCP-1 le els eleased by he cells in o he medium we e e alua ed in he
cul u e media om ea ed di e en ia ed CaCo-2 cell samples. Con ols we e e e ed o
un ea ed di e en ia ed cells incuba ed wi h he same cell cul u e medium quan i y used
o ea men s (5% FBS-DMEM) a he same imes as ea ed di e en ia ed cells. Cy okines
we e analyzed and es ima ed using comme cial ELISA ki s. Cap u e an ibody an i-human
MCP-1 o an i-human IL-8 was added o each ELISA pla e well (0.25
µ
g/mL concen a ion
o MCP-1 and 0.125
µ
g/mL concen a ion o IL-8, ollowing manu ac u e ’s ins uc ion)
and incuba ed o e nigh . A e wa d, he pla e wells we e washed h ice wi h he Wash
Bu e (0.05% Tween-20 in phospha e bu e ed saline, PBS), incuba ed 1 h a RT wi h he
An ioxidan s 2021,10, 280 7 o 18
Block Bu e (1% bo ine se um albumin (BSA) in 1X PBS), and washed h ice wi h he
Wash Bu e . The samples we e hen added o he pla e wells, incuba ed o 2 h a RT,
and inally washed 4 imes wi h he Wash Bu e . The de ec ion an ibody was added o
he pla e wells a a 0.25
µ
g/mL concen a ion and incuba ed o 2 h a RT, hen washed
4 imes wi h he Wash Bu e . S ep a idin-HRP conjuga e was added o he pla e wells a
a 0.05
µ
g/mL concen a ion o 30 minu es a RT. A e 4 washes wi h he Wash Bu e , he
pla e wells we e incuba ed wi h 3,3
0
,5,5
0
- e ame hylbenzidine (TMB) Liquid Subs a e o
20 minu es a RT, and he colo de elopmen was blocked wi h he s op solu ion (1 M HCl).
Colo de elopmen was moni o ed wi h a 96-mul iwell pla e eade (Model 680 Mic opla e
Reade , Bio-Rad labo a o ies S l, Milan, I aly) using a 450 nm wa eleng h il e . Abso bance
a 655 nm wa eleng h was conside ed as a alue e e ence o each sample.
The Bio-Rad p o ein assay dye eagen was used o es ima e he o al p o ein con-
cen a ion in each sample cell medium [
37
]. IL-8 and MCP-1 le els we e no malized o
p o eins o he incuba ion media and alues exp essed as pg cy okines/mg cell cul u e
medium p o eins.
The analyses we e pe o med in iplica e om 3 independen expe imen s, and da a
we e calcula ed using SlideW i e Plus so wa e (Ad anced G aphics So wa e, Rancho
San a Fe, CA, USA).
2.10. Tigh -Junc ion P o ein Immunoblo ing
A he end o each ea men , di e en ia ed cells we e sc aped and washed wi h
ice-cold PBS 1X, p epa ed by dilu ing 10X PBS (con aining 1.37 M NaCl, 27 mM KCl,
100 mM Na2HPO4, and 18 mM KH2PO4) in Milli-Q wa e . Then, 150
µ
L lysis bu e
we e added o p o ein ex ac ion (147
µ
L PBS supplemen ed wi h 1.5
µ
L T i on X-100
( / ), 1.5
µ
g sodium dodecyl sul a e (SDS) (w/ ) ( inal olume)). Once lysed, samples we e
incuba ed o 30 min on ice and cen i uged a 12,052
×
ga 4
◦
C o 15 min. To al cell ex ac
p o ein concen a ion was e alua ed wi h Bio-Rad p o ein assay dye eagen ollowing he
p o ocol published by B ad o d [37].
Each sample con aining 50
µ
g o al p o eins was boiled a 100
◦
C in he Sample
Bu e (LDS Sample Bu e 4X and DTT Sample Reduce 10X) o 5 min. Boiled samples
we e subjec ed o elec opho esis sepa a ion using 10% SDS-polyac ylamide gel, and
p o eins we e ans e ed o Hybond ECL ni ocellulose memb anes. The memb anes
we e hen incuba ed a RT o 1 h in 10 mL TBS supplemen ed wi h 5
µ
L Tween 20
(TTBS) blocking bu e plus 0.5 g skimmed milk powde . Blo s we e hen incuba ed a
4
◦
C o e nigh wi h ei he 10 mL mouse an i-claudin 1 (1:800 dilu ion), 10 mL mouse
an i-occludin (1:500 dilu ion), o 10 mL abbi an i-JAM-A (1:200 dilu ion) polyclonal
an ibodies in TBS con aining 10
µ
L Tween-20 and 0.5 g skimmed milk powde . Th ee
subsequen washes in TTBS we e pe o med, and blo s we e incuba ed wi h an i- abbi o
an i-mouse HRP-conjuga ed IgG (1:1000 dilu ions) in 10 mL TBS wi h 10
µ
L Tween-20 and
0.5 g skimmed milk powde o 1 h. Finally, blo s we e washed wice in TTBS o 10 min.
The Cla i y Wes e n ECL ki and ChemiDoc
™
Touch Imaging Sys em machine (Bio-
Rad labo a o ies S l, Seg a e, I aly) we e used o de ec chemiluminescence. P o ein band
densi ies o he 3 pe o med independen expe imen s we e quan i ied using Image J
So wa e (Be hesda, MD, USA).
2.11. Tigh Junc ion P o ein Immuno luo escence
Cell localiza ion o TJ p o eins (claudin 1, occludin, and JAM-A) was isualized
using immuno luo escence echnique. Fo his analysis, ea men s we e pe o med using
di e en ia ed CaCo-2 cell monolaye s g own on 13 mm diame e glass slides. A e
ea men s, slides we e washed wice wi h PBS, ixed wi h 4% pa a o maldehyde o
15 min, and hen pe meabilized wi h 0.2% T i on X-100 in PBS a RT o 5 min. Two
PBS washes and incuba ions a RT o 30 min wi h a blocking bu e (PBS con aining 5%
goa se um, 3% BSA, and 0.3% Tween) we e pe o med o p e en nonspeci ic binding
o he p ima y an ibodies. A e 2 u he washes wi h PBS, CaCo-2 cell slides we e
An ioxidan s 2021,10, 280 8 o 18
incuba ed o e nigh a 4
◦
C wi h mouse an i-occludin (1:50 dilu ion), mouse an i-claudin
1 (1:50 dilu ion), o abbi an i-JAM-A (1:100 dilu ion) an ibodies. Rega ding claudin 1
and occludin isualiza ion, cells we e hen washed and incuba ed wi h goa an i-mouse
IgG-Alexa Fluo 488 (1:500 dilu ions) a RT o 2 h (an ibody dilu ion was pe o med in
PBS con aining 0.5% BSA and 0.1% T i on X-100). Rega ding JAM-A isualiza ion goa
an i- abbi IgG-Alexa Fluo 546 was used o incuba ion as desc ibed abo e.
A e 2 u he washes, slides we e moun ed wi h Fluo oshield moun ing medium and
obse ed using a LSM 800 con ocal lase mic oscope (Zeiss SpA, Obe kochen, Ge many).
Exci a ion alues o 488 nm and emission alues o 505–550 nm we e used o g een
luo escence, while exci a ion alues o 546 nm and emission alues o 573 nm we e used
o ed luo escence. Images we e p ocessed using LSM 800 Image Examine so wa e
(Zeiss SpA, Obe kochen, Ge many).
2.12. Real-Time Quan i a i e Re e se-T ansc ip ion Polyme ase Chain Reac ion
Di e en ia ed cells we e p e ea ed o no wi h CBS, CBS-IC o 1 h and incuba ed
wi h Oxy-mix o 6 h. This incuba ion ime co esponds o he signi ican maximum N 2
exp ession. In a g oup o expe imen s, cells we e p e ea ed wi h 13
µ
M heob omine
co esponding o i s con en in CBS-IC; p e ea men s wi h 1
µ
M (
−
)-epica echin we e also
inse ed as posi i e con ols.
To al mRNA was ex ac ed om ea ed cells using 1 mL TRIzol
™
ollowing man-
u ac u e ins uc ions and p o ocol published by Rio and colleagues [
38
]. mRNA ex ac
concen a ions we e de ec ed by measu ing ibonucleic acid abso bance a 260 nm. Ul-
a iole (UV) abso bance a io a 260/280 nm was used o assess RNA pu i y. Re e se
ansc ip ion was pe o med o syn hesize cDNA om 2
µ
g mRNA using andom p ime s
and ollowing he manu ac u e ’s ins uc ions o a comme cial ki . Real- ime quan i a i e
e e se- ansc ip ion polyme ase chain eac ion (qRT-PCR) was pe o med on 20 ng cDNA
using TaqMan gene exp ession p obes o N 2, and ampli ied cDNAs we e analyzed using
he 7500 Fas eal- ime PCR sys em (Applied Biosys ems, The mo ishe , Monza, I aly).
Oligonucleo ide sequences ha e no been disclosed by he manu ac u e because hey a e
s ic ly con iden ial. PCR’s cycling pa ame e s we e 40 cycles/3 s each a 95
◦
C (mel ing)
and 30 s a 60
◦
C (annealing/ex ension). Resul s we e no malized o he exp ession o
β
-ac in as a housekeeping gene. Ta ge gene exp ession was quan i ied as p oposed by
Li ak and Schmi gen [39].
2.13. S a is ical Analyses
S a is ical di e ences among da a ega ding molecula expe imen s, pe o med as
3 independen expe imen s, we e e alua ed using he 1-way ANOVA es associa ed wi h
Bon e oni’s mul iple compa ison pos - es .
Di e ences among alues ob ained om ch oma og aphic analyses we e e alua ed by
ANOVA associa ed wi h Duncan’s es , being, in his case, he mos sui able es in mul iple
compa ison.
All alues we e exp essed as mean
±
S anda d De ia ion (SD) o 3 independen
expe imen s, and da a we e analyzed wi h G aphPad InS a so wa e (San Diego, CA, USA).
3. Resul s
3.1. Cocoa Bean Shell-En iched Ice C eam Shows High Reco e y Con en o Phenolic and
Me hylxan hines and An ioxidan Capaci y
CBS lou addi ion o plain ice c eam ga e impo an quan i a i e changes in bo h
phenolic compounds and xan hine de i a i es (Tables 2and 3). Ca echin wi h i s glucoside
isome s, (
−
)-epica echin and p ocyanidins, as well as heob omine and ca eine, displayed
he highes concen a ion alues in CBS-IC samples. Me hylxan hines we e he main
compounds in all samples. Theob omine was one o he main compounds iden i ied in
high concen a ions (47.47
µ
g/mL) in he ac ion ob ained a e comple e gas oin es inal
diges ion o CBS-IC (Table 2).
An ioxidan s 2021,10, 280 9 o 18
Table 2.
Main componen s iden i ied and quan i ied by high-pe o mance liquid ch oma og aphy (HPLC) in solu ions
yielded a e gas oin es inal diges ion o plain ice c eam (IC), ice c eam en iched wi h 4% CBS (CBS-IC), and CBS (CBS).
Compound (µg/mL Ex ac ) IC CBS-IC CBS Sig
Theob omine 18.69 ±0.65 c47.47 ±0.59 a36.6 ±2.78 b***
Ca eine 1.11 ±0.26 c6.49 ±0.09 a5.99 ±0.09 b***
P o oca echuic acid 0.06 ±0.01 a0.22 ±0.01 a0.16 ±0.16 an.s.
N-Couma oyl-L-aspa a e_isome 1 0.11 ±0.01 c0.99 ±0.04 a0.65 ±0.12 b***
Ca echin-3-O-glucoside_isome 1 0.64 ±0.02 c1.83 ±0.04 a0.44 ±0.01 b***
Ca echin 0.19 ±0.01 c1.05 ±0.03 a0.58 ±0.03 b***
Ca echin-3-O-glucoside_isome 2 0.35 ±0.08 c1.07 ±0.05 a0.71 ±0.02 b***
Epica echin 0.00 ±0.00 b0.89 ±0.10 a0.87 ±0.10 a***
P ocyanidin (PC)C 0.60 ±0.06 b1.28 ±0.05 a0.47 ±0.02 c***
PCA pen oside_isome 1 0.33 ±0.05 b0.50 ±0.06 a0.00 ±0.00 c***
PCA pen oside_isome 2 0.16 ±0.02 c0.84 ±0.03 a0.53 ±0.02 b***
Kaemp e ol-3- u inoside 0.00 ±0.00 c0.17 ±0.01 a0.12 ±0.02 b***
Que ce in-3-a abinoside 0.00 ±0.00 c0.19 ±0.01 a0.03 ±0.02 b***
Da a a e exp essed as mean alues o h ee independen expe imen s (n= 3) e alua ed in iplica e
±
SD. Sig: Signi icance. ANOVA
associa ed Duncan’s es was used as a mul iple ange es o highligh he signi ican di e ences among all he samples (IC, CBS-IC, and
CBS). ANOVA signi icance: *** p< 0.001. Means showing di e en lowe case le e s wi hin he same line a e signi ican ly di e en a
p< 0.05.
Table 3. To al phenolic, la onoid, and annin con en and an ioxidan capaci y in IC, CBS-IC, and CBS.
IC CBS-IC CBS Sig
TPC (µg GAE/mL) 349.01 ±8.32 b476.69 ±7.09 a188.85 ±5.80 c***
TFC (µg CE/mL) 47.69 ±3.62 b96.06 ±7.73 a44.18 ±7.06 b***
TTC (µg CE/mL) 52.77 ±4.15 b59.48 ±2.47 a34.24 ±1.86 c***
RSA (
µ
mol TE/mL)
0.74 ±0.08 b1.12 ±0.10 a0.70 ±0.03 b***
To al phenolic con en (TPC), o al la onoid con en (TFC), o al annin con en (TTC), and an ioxidan capaci y (RSA) we e e alua ed
in plain ice c eam (IC), 4% CBS-en iched ice c eam (CBS-IC) and CBS powde (CBS) ob ained a e gas oin es inal diges ion. TPC a e
exp essed as gallic acid equi alen (GAE); TFC and TTC as ca echin equi alen (CE); RSA as T olox equi alen (TE)/mL ex ac s. Analyses
we e pe o med in iplica e. Da a a e exp essed as mean alues o h ee independen expe imen s (n= 3) e alua ed in iplica e
±
SD. Sig:
Signi icance. ANOVA associa ed Duncan’s es was used as a mul iple ange es o highligh he signi ican di e ences among all he
sample (IC, CBS-IC, and CBS). ANOVA signi icance: *** p< 0.001. Means showing di e en lowe case le e s wi hin he same line a e
signi ican ly di e en a p< 0.05.
No ably, RP-HPLC-PDA analyses pe o med in he di e en o mula ions o he
de ec ion o o al phenolics, annins, and la onoids showed he highes eco e y in he
CBS-IC sample. Inc eased an ioxidan capaci y was also ound in CBS-IC (Table 3).
3.2. Cocoa Bean Shell and Cocoa Bean Shell-En iched Ice C eam P e en In e leukin-8 and
Monocy e Chemoa ac an P o ein-1 P oduc ion om Di e en ia ed CaCo-2 Cells T ea ed wi h he
P oin lamma o y Oxys e ol Mix u e
Di e en ia ed CaCo-2 cells we e p e ea ed (1 h) wi h CBS-IC o CBS alone and hen
incuba ed wi h 60
µ
M die a y Oxy-mix o 24 h. Based on he cy o oxici y analyzed as
LDH elease, 5% CBS-IC/CBS (0.150 g in 3 mL cell cul u e inal concen a ion) showed no
cy o oxic e ec s bo h in p esence and absence o he Oxy-mix and was he e o e chosen as
he bes concen a ion o cell ea men .
CBS-IC and CBS an i-in lamma o y e icacy was e alua ed in e ms o IL-8 and MCP-
1 p oduc ion.
As expec ed, Oxy-mix induced a s ong inc ease in he p oin lamma o y cy okines
eleased by CaCo-2 cells in he cul u e medium (2.25- and 3.3- old inc ease o IL-8 and
MCP-1, espec i ely) (Figu e 1).
An ioxidan s 2021,10, 280 16 o 18
de ec able in choles e ol ich oods in e ms o educed in lamma ion, TJ p o ein loss,
and edis ibu ion, as well as s ess- ela ed an ioxidan esponse po en ia ion. Va ious
s udies ha e sugges ed cocoa bean shell euse in unc ional ood because o i s ichness in
polyphenols’ po en ial bene i on heal h [8,33].
The analysis o CBS-en iched ice c eam ex ac showing highe la onoid con en
han plain ice c eam s eng hens he ac ual possibili y o apply his byp oduc as a ood
ing edien . Howe e , we would like o poin ou ha heob omine accumula es in CBS in
e y high concen a ion. Al hough u he s udies a e equi ed o cla i y he mechanisms
by which heob omine can p o ec he in es inal laye om die a y a acks, a coope a ing—
e en hough pa ial— ole in CBS biological ac i i y canno be excluded.
Supplemen a y Ma e ials:
The ollowing a e a ailable online a h ps://www.mdpi.com/2076-392
1/10/2/280/s1, Table S1: Fo mula ion o he con ol ice c eam (IC) and ice c eam o i ied wi h CBS
a 4% (CBS-IC). Table S2: Chemical composi ion o CBS, plain ice c eam (IC), and o i ied ice c eam
wi h 4% CBS powde (CBS-IC). Table S3: Cell iabili y e alua ion in di e en pe cen ages o CBS
ex ac s. Figu e S1. CaCo-2 Cell Imaging by Lase Scanning Con ocal Mic oscopy.
Au ho Con ibu ions:
Concep ualiza ion, F.B., D.R. and L.B.-P.; In es iga ion, D.R., L.B.-P. and N.I.,
A.C.D.; Funding acquisi ion, G.Z., F.B. and L.B.-P.; Supe ision, F.B., G.P. and G.Z.; W i ing-o iginal
d a , F.B., L.B.-P. and D.R.; W i ing— e iew & edi ing, F.B., L.B.-P. and B.S. All au ho s ha e ead
and ag eed o he published e sion o he manusc ip .
Funding:
This wo k was unding by he Uni e si y o Tu in, I aly [g an numbe s BIAF_RILO_17_01;
BIAF_RILO_18_01]; he I alian Minis y o Uni e si y and Resea ch [BIAF_FFABR_17_01]; he p ojec
COVALFOOD “Valo isa ion o high added- alue compounds om cocoa indus y by-p oduc s as
ood ing edien s and addi i es” has ecei ed unding om he Eu opean Union’s Se en h F ame-
wo k p og amme o esea ch and inno a ion unde he Ma ie Skłodowska-Cu ie g an ag eemen
No 609402-2020 esea che s: T ain o Mo e (T2M); Spanish Minis y o Science, Inno a ion and
Uni e si ies “Juan de la Cie a—Inco po ación” G an (Ag eemen No. IJCI-2017-31665).
Ins i u ional Re iew Boa d S a emen : No applicable
In o med Consen S a emen : No applicable
Da a A ailabili y S a emen :
The da a o his s udy a e con ained wi hin he a icle o supplemen a y
ma e ials.
Acknowledgmen s:
The au ho s exp ess hei acknowledgmen s o Pas iglie Leone S l (Tu in, I aly),
which kindly supplied cocoa bean shell.
Con lic s o In e es : The au ho s decla e no con lic o in e es .
Re e ences
1.
Vásquez, Z.S.; de Ca alho Ne o, D.P.; Pe ei a, G.V.M.; Vandenbe ghe, L.P.S.; de Oli ei a, P.Z.; Tibu cio, P.B.; Rogez, H.L.G.;
Ne o, A.G.; Soccol, C.R. Bio echnological app oaches o cocoa was e managemen : A e iew. Was e Manag.
2019
,90, 72–83.
[C ossRe ] [PubMed]
2.
Gammone, M.A.; E hymakis, K.; Pluchino a, F.R.; Be gan e, S.; Te aman i, G.; Riccioni, G.; D’O azio, N. Impac o chocola e on
he ca dio ascula heal h. F on . Biosci. 2018,23, 852–864. [C ossRe ] [PubMed]
3. Pa ke , G.; Pa ke , I.; B o chie, H. Mood s a e e ec s o chocola e. J. A ec . Diso d. 2006,92, 149–159. [C ossRe ]
4.
Goya, L.; Ma ín, M.Á.; Sa iá, B.; Ramos, S.; Ma eos, R.; B a o, L. E ec o Cocoa and I s Fla onoids on Bioma ke s o
In lamma ion: S udies o Cell Cul u e, Animals and Humans. Nu ien s 2016,8, 212. [C ossRe ] [PubMed]
5.
Galleano, M.; O eiza, P.I.; F aga, C.G. Cocoa, chocola e, and ca dio ascula disease. J. Ca dio asc. Pha macol.
2009
,54, 483–490.
[C ossRe ] [PubMed]
6.
De A aujo, Q.R.; Ga wa d, J.N.; Almoosawi, S.; Sil a, M.D.; Dan as, P.A.; De A aujo Júnio , Q.R. Cocoa and Human Heal h:
F om Head o Foo —A Re iew. C i . Re . Food Sci. Nu . 2016,56, 1–12. [C ossRe ] [PubMed]
7.
Mag one, T.; Russo, M.A.; Ji illo, E. Cocoa and Da k Chocola e Polyphenols: F om Biology o Clinical Applica ions. F on .
Immunol. 2017,8, 677. [C ossRe ]
8.
Okiyama, D.C.G.; Na a o, S.L.B.; Rod igues, C.E.C. Cocoa shell and i s compounds: Applica ions in he ood indus y. T ends
Food Sci. Technol. 2017,63, 103–112. [C ossRe ]
9.
Di Ma ia, C.D.; Sacche i, G.; Mas ocola, D.; Se a ini, M. F om Cocoa o Chocola e: The Impac o P ocessing on
in i o
An ioxidan Ac i i y and he E ec s o Chocola e on An ioxidan Ma ke s in i o. F on . Immunol. 2017,8, 1207. [C ossRe ]
An ioxidan s 2021,10, 280 17 o 18
10.
Redgwell, R.; T o a o, R.V.; Me ina , S.; Cu i, D.; Hedige , S.; Manez, A. Die a y ib e in cocoa shell: Cha ac e isa ion o
componen polysaccha ides. Food. Chem. 2003,81, 103–112. [C ossRe ]
11.
˙
Zy˙
zelewicz, D.; Bud yn, G.; O acz, J.; An olak, H.; K ˛egiel, D.; Kaczma ska, M. The e ec on bioac i e componen s and
cha ac e is ics o chocola e by unc ionaliza ion wi h aw cocoa beans. Food Res. In . 2018,113, 234–244. [C ossRe ] [PubMed]
12.
Panak Balen i´c, J.; Aˇcka , Ð.; Joki´c, S.; Jozino i´c, A.; Babi´c, J.; Miliˇce i´c, B.; Šuba i´c, D.; Pa lo i´c, N. Cocoa Shell: A By-P oduc
wi h G ea Po en ial o Wide Applica ion. Molecules 2018,23, 1404. [C ossRe ]
13.
Qu, G.; Chen, J.; Guo, X. The bene icial and dele e ious ole o die a y polyphenols on ch onic degene a i e diseases by egula ing
gene exp ession. Biosci. T ends. 2019,12, 526–536. [C ossRe ] [PubMed]
14.
Kaulmann, A.; Bohn, T. Bioac i i y o Polyphenols: P e en i e and Adju an S a egies Towa d Reducing In lamma o y Bowel
Diseases-P omises, Pe spec i es, and Pi alls. Oxid. Med. Cell Longe . 2016,2016, 9346470. [C ossRe ] [PubMed]
15.
B glez Mojze , E.; Knez H nˇciˇc, M.; Ške ge , M.; Knez, Ž.; B en, U. Polyphenols: Ex ac ion Me hods, An ioxida i e Ac ion,
Bioa ailabili y and An ica cinogenic E ec s. Molecules 2016,21, 901. [C ossRe ] [PubMed]
16.
Ma ínez-Pinilla, E.; Oña ibia-As ibia, A.; F anco, R. The ele ance o heob omine o he bene icial e ec s o cocoa consump ion.
F on . Pha macol. 2015,6, 30. [C ossRe ]
17.
Mon ei o, J.P.; Al es, M.G.; Oli ei a, P.F.; Sil a, B.M. S uc u e-Bioac i i y Rela ionships o Me hylxan hines: T ying o Make
Sense o All he P omises and he D awbacks. Molecules 2016,21, 974. [C ossRe ]
18.
Be na di, S.; Del Bo’, C.; Ma ino, M.; Ga ga i, G.; Che ubini, A.; And és-Lacue a, C.; Hidalgo-Libe ona, N.; Pe on, G.; González-
Dominguez, R.; K oon, P.; e al. Polyphenols and In es inal Pe meabili y: Ra ionale and Fu u e Pe spec i es. J. Ag ic. Food Chem.
2020,68, 1816–1829. [C ossRe ]
19.
Iaia, N.; Rossin, D.; So e o, B.; Venezia, I.; Poli, G.; Biasi, F. E icacy o heob omine in p e en ing in es inal CaCo-2 cell damage
induced by oxys e ols. A ch. Biochem. Biophys. 2020,694, 108591. [C ossRe ]
20.
Chelakko , C.; Ghim, J.; Ryu, S.H. Mechanisms egula ing in es inal ba ie in eg i y and i s pa hological implica ions. Exp. Mol.
Med. 2018,50, 103. [C ossRe ]
21.
Rezaie, A.; Pa ke , R.D.; Abdollahi, M. Oxida i e s ess and pa hogenesis o in lamma o y bowel disease: An epiphenomenon o
he cause? Dig. Dis. Sci. 2007,52, 2015–2021. [C ossRe ] [PubMed]
22.
Deiana, M.; Cal apie a, S.; Incani, A.; A ze i, A.; Rossin, D.; Loi, R.; So e o, B.; Iaia, N.; Poli, G.; Biasi, F. De angemen o in es inal
epi helial cell monolaye by die a y choles e ol oxida ion p oduc s. F ee Radic. Biol. Med.
2017
,113, 539–550. [C ossRe ] [PubMed]
23.
Molendijk, I.; an de Ma el, S.; Maljaa s, P.W.J. Towa ds a Food Pha macy: Immunologic Modula ion h ough Die . Nu ien s
2019,11, 1239. [C ossRe ] [PubMed]
24.
Leech, B.; McIn y e, E.; S eel, A.; Sibb i , D. Risk ac o s associa ed wi h in es inal pe meabili y in an adul popula ion: A
sys ema ic e iew. In . J. Clin. P ac . 2019,73, e13385. [C ossRe ] [PubMed]
25.
Roh , M.W.; Na asimhulu, C.A.; Rudeski-Roh , T.A.; Pa hasa a hy, S. Nega i e E ec s o a High-Fa Die on In es inal Pe meabil-
i y: A Re iew. Ad . Nu . 2020,11, 77–91. [C ossRe ]
26.
O aegui-A azola, A.; Menéndez-Ca eño, M.; Anso ena, D.; As iasa án, I. Oxys e ols: A wo ld o explo e. Food Chem. Toxicol.
2010,48, 3289–3303. [C ossRe ]
27.
Guina, T.; Deiana, M.; Cal apie a, S.; Cabboi, B.; Maina, M.; Tube oso, C.I.; Leona duzzi, G.; Gamba, P.; Ga giulo, S.; Tes a, G.;
e al. The ole o p38 MAPK in he induc ion o in es inal in lamma ion by die a y oxys e ols: Modula ion by wine phenolics.
Food Func . 2015,6, 1218–1228. [C ossRe ]
28.
Xu, M.; Li, X.X.; Wang, L.; Wang, M.; Zhang, Y.; Li, P.L. Con ibu ion o N 2 o A he ogenic Pheno ype Swi ching o Co ona y
A e ial Smoo h Muscle Cells Lacking CD38 Gene. Cell Physiol. Biochem. 2015,37, 432–444. [C ossRe ]
29. He, F.; Ru, X.; Wen, T. NRF2, a T ansc ip ion Fac o o S ess Response and Beyond. In . J. Mol. Sci. 2020,21, 4777. [C ossRe ]
30.
Rossin, D.; Ba bosa-Pe ei a, L.; Iaia, N.; Tes a, G.; So e o, B.; Poli, G.; Zeppa, G.; Biasi, F. A Die a y Mix u e o Oxys e ols Induces
in i o
In es inal In lamma ion h ough TLR2/4 Ac i a ion: The P o ec i e E ec o Cocoa Bean Shells. An ioxidan s
2019
,8, 151.
[C ossRe ]
31.
Ba bosa-Pe ei a, L.; Bo o o Dalla Vecchia, S.; Dal Bello, B.; Be olino, M.; Bel iso, S.; Ghi a dello, D.; Be ino, P.; Zeppa, G. Cocoa
bean shell as ood ing edien o enhance he unc ional p ope ies and nu i ional quali y o ice c eam. In P oceedings o he
Fou h In e na ional Cong ess on Cocoa, Co ee and Tea, Tu in, I aly, 25–28 June 2017.
32.
Minekus, M.; Alminge , M.; Al i o, P.; Ballance, S.; Bohn, T.; Bou lieu, C.; Ca iè e, F.; Bou ou, R.; Co edig, M.; Dupon , D.; e al.
A s anda dised s a ic
in i o
diges ion me hod sui able o ood—An in e na ional consensus. Food Func .
2014
,5, 1112–1113.
[C ossRe ]
33.
Rojo-Po eda, O.; Ba bosa-Pe ei a, L.; El Kha abi, C.; Youl, E.N.; Be olino, M.; Delpo e, C.; Poche , S.; S é igny, C. Polyphenolic
and Me hylxan hine Bioaccessibili y o Cocoa Bean Shell Func ional Biscui s: Me abolomics App oach and In es inal Pe meabili y
h ough Caco-2 Cell Models. An ioxidan s 2020,9, 1164. [C ossRe ]
34.
Rousse , M. The human colon ca cinoma cell lines HT-29 and Caco-2: Two
in i o
models o he s udy o in es inal di e en ia ion.
Biochimie 1986,68, 1035–1040. [C ossRe ]
35.
To , L. CaCo-2 Cell Line. In The Impac o Food Bioac i es on Gu Heal h: In Vi o and Ex Vi o Models, 1s ed.; Ve hoeckx, K., Co e , P.,
Lopez-Exposi o, I., Klei eland, C., Lea, T., Mackie, A., Requena, T., Swia ecka, D., Wiche s, H., Eds.; Sp inge : Cham, Swize land,
2015; pp. 103–111.
An ioxidan s 2021,10, 280 18 o 18
36.
Ma sumo o, H.; E ickson, R.H.; Gum, J.R.; Yoshioka, M.; Gum, E.; Kim, Y.S. Biosyn hesis o alkaline phospha ase du ing
di e en ia ion o he human colon cance cell line CaCo-2. Gas oen e ology 1990,98, 1199–1207. [C ossRe ]
37.
B ad o d, M.M. A Rapid and Sensi i e Me hod o he Quan i a ion o Mic og am Quan i ies o P o ein U ilizing he P inciple o
P o ein-Dye Binding. Anal. Biochem. 1976,72, 248–254. [C ossRe ]
38.
Rio, D.C.; A es, M., J .; Hannon, G.J.; Nilsen, T.W. Pu i ica ion o RNA using TRIzol (TRI eagen ). Cold Sp ing Ha bo P o oc.
2010
.
[C ossRe ] [PubMed]
39.
Li ak, K.J.; Schmi gen, T.D. Analysis o ela i e gene exp ession da a using eal- ime quan i a i e PCR and he 2-
∆∆
CT me hod.
Me hods 2001,25, 402–408. [C ossRe ]
40. Tu ne , J.R. In es inal mucosal ba ie unc ion in heal h and disease. Na . Re . Immunol. 2009,9, 799–809. [C ossRe ]
41.
Hausmann, M. How bac e ia-induced apop osis o in es inal epi helial cells con ibu es o mucosal in lamma ion. In . J. In lam.
2010,6, 574568. [C ossRe ] [PubMed]
42.
Camille i, M. Leaky gu : Mechanisms, measu emen , and clinical implica ions in humans. GUT
2019
,68, 1516–1526. [C ossRe ]
43.
Pla , J.; Nichols, J.A.; Mensink, R.P. Plan s e ols and s anols: E ec s on mixed micella composi ion and LXR ( a ge gene)
ac i a ion. J. Lipid Res. 2005,46, 2468–2476. [C ossRe ] [PubMed]
44.
Ma iu i, L.R.; Noguei a, G.C.; B agagnolo, N. Lipid and choles e ol oxida ion in chicken mea a e inhibi ed by sage bu no by
ga lic. J. Food Sci. 2011,76, C909–C915. [C ossRe ] [PubMed]
45.
Khan, M.I.; Min, J.S.; Lee, S.O.; Yim, D.G.; Seol, K.H.; Lee, M.; Jo, C. Cooking, s o age, and ehea ing e ec on he o ma ion o
choles e ol oxida ion p oduc s in p ocessed mea p oduc s. Lipids Heal h Dis. 2015,14, 89. [C ossRe ] [PubMed]
46.
Chalubinski, M.; Wojdan, K.; Go zelak, P.; Bo owiec, M.; B oncel, M. The e ec o oxidized choles e ol on ba ie unc ions and
IL-10 mRNA exp ession in human in es inal epi helium co-cul u ed wi h dend i ic cells in he answell sys em. Food Chem.
Toxicol. 2014,69, 289–293. [C ossRe ]
47.
Cho, H.R.; Kim, K.; An, W.G.; Eo, S.K.; Bae, S.S.; Kim, C.D.; Son, Y. 7-Oxygena ed choles e ol molecules di e en ially a ec
he exp ession o zonula occludens-1 in ascula smoo h muscle cells and monocy e/mac ophage cells. Biochem. Biophys. Res.
Commun. 2018,497, 521–526. [C ossRe ]
48.
Filomenko, R.; Fou geux, C.; B e illon, L.; Gambe -Nico , S. Oxys e ols: In luence on plasma memb ane a s mic odomains and
de elopmen o ocula diseases. S e oids 2015,99, 259–265. [C ossRe ]
49.
And ews, C.; McLean, M.H.; Du um, S.K. Cy okine Tuning o In es inal Epi helial Func ion. F on . Immunol.
2018
,9, 1270.
[C ossRe ]
50.
Luissin , A.C.; Nus a , A.; Pa kos, C.A. JAM- ela ed p o eins in mucosal homeos asis and in lamma ion. Semin. Immunopa hol.
2014,36, 211–226. [C ossRe ]
51.
S ama o ic, S.M.; Sladoje ic, N.; Keep, R.F.; Andjelko ic, A.V. Relocaliza ion o junc ional adhesion molecule A du ing in lamma-
o y s imula ion o b ain endo helial cells. Mol. Cell. Biol. 2012,32, 3414–3427. [C ossRe ]
52.
Con e as, T.C.; Riccia di, E.; C emonini, E.; O eiza, P.I. (-)-Epica echin in he p e en ion o umo nec osis alpha-induced loss o
Caco-2 cell ba ie in eg i y. A ch. Biochem. Biophys. 2015,573, 84–91. [C ossRe ]
53.
C emonini, E.; Wang, Z.; Be aieb, A.; Adamo, A.M.; Da e i, E.; Mills, D.A.; Kalane a, K.M.; Haj, F.G.; Ka akas, S.; O eiza, P.I.
(-)-Epica echin p o ec s he in es inal ba ie om high a die -induced pe meabiliza ion: Implica ions o s ea osis and insulin
esis ance. Redox Biol. 2018,14, 588–599. [C ossRe ]
54.
Rowley, T.J., 4 h; Bi ne , B.F.; Ray, J.D.; La hen, D.R.; Smi hson, A.T.; Dallon, B.W.; Plowman, C.J.; Bikman, B.T.; Hansen, J.M.;
Do enko , M.R.; e al. Monome ic cocoa ca echins enhance
β
-cell unc ion by inc easing mi ochond ial espi a ion. J. Nu .
Biochem. 2017,49, 30–41. [C ossRe ] [PubMed]
55.
F aga, C.G.; O eiza, P.I.; Galleano, M. Plan bioac i es and edox signaling: (-)-Epica echin as a pa adigm. Mol. Aspec s Med.
2018
,
61, 31–40. [C ossRe ] [PubMed]
56.
B une o, M.; Gu ié ez, L.; Delgado, Y.; Gallignani, M.; Zamb ano, A.; Gómez, Á.; Ramos, G.; Rome o, C. De e mina ion o
heob omine, heophylline and ca eine in cocoa samples by a high-pe o mance liquid ch oma og aphic me hod wi h on-line
sample cleanup in a swi ching-column sys em. Food Chem. 2007,100, 459–467. [C ossRe ]
57.
Co a, I.; Le a, V.; Ma iani, C.; Pan oni, L.; Poma i, S. Explo ing cocoa p ope ies: Is heob omine a cogni i e modula o ?
Psychopha macology 2019,236, 561–572. [C ossRe ] [PubMed]
58.
Jang, M.H.; Mukhe jee, S.; Choi, M.J.; Kang, N.H.; Pham, H.G.; Yun, J.W. Theob omine alle ia es die -induced obesi y in mice ia
phosphodies e ase-4 inhibi ion. Eu . J. Nu . 2020. [C ossRe ]
59.
Gu, R.; Shi, Y.; Huang, W.; Lao, C.; Zou, Z.; Pan, S.; Huang, Z. Theob omine mi iga es IL-1
β
-induced oxida i e s ess, in lamma o y
esponse, and deg ada ion o ype II collagen in human chond ocy es. In . Immunopha macol. 2020,5, 106226. [C ossRe ]
