Phenolic Cha ac e iza ion o a Pu ple Maize (Zea mays c .
“Mo ag o”) by HPLC−QTOF-MS and S udy o I s Bioaccessibili y
Using a Simula ed In Vi o Diges ion/Caco‑2 Cul u e Model
Ma ianela Desi eéRod iguez, Luisina Monsie a, Pablo Sebas ián Mansilla, Gab iela Te esa Pé ez,
and Sonia de Pascual-Te esa*
Ci e This: J. Ag ic. Food Chem. 2024, 72, 6327−6338
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ABSTRACT: The p esen wo k aimed o cha ac e ize he phenolic and an ioxidan con en o he A gen inian pu ple maize
“Mo ag o” cul i a . Addi ionally, he INFOGEST simula ed in i o diges ion model was used o es ablish he e ec o diges ion on
bioac i e compounds. Finally, diges ion samples we e used o ea Caco-2 cells in he answell model o be e unde s and hei
bioa ailabili y. Twen y-six phenolic compounds we e ound in pu ple maize c . “Mo ag o”, 15 nonan hocyanins and 11
an hocyanins. Se e al compounds we e iden i ied in maize o he i s ime, such as py ogallol, ci ic acid, gallic acid, kaemp e ol 3-
(6″- e ulylglucoside), and kaemp e ol 3-glucu onide. An hocyanins accoun ed o 24.9% o o al polyphenols, wi h he p edominan
an hocyanin being cyanidin-3-(6″malonylglucoside). Ca echin-(4,8)-cyanidin-3,5-diglucoside and ca echin-(4,8)-cyanidin-3-
malonylglucoside-5-glucoside we e de ec ed as cha ac e is ics o his Ame ican maize a ie y. To al polyphenol con en (TPC;
by he Folin−Ciocal eu me hod), HPLC-DAD/MSMS, and an ioxidan ac i i y [by DPPH and e ic- educing an ioxidan powe
(FRAP)] we e e alua ed h oughou in i o diges ion. TPC, DPPH, and FRAP esul s we e 2.71 mg gallic acid equi alen s (GAE)/
g, 24 μmol T olox equi /g, and 22 μmol T olox eq/g, espec i ely. The in i o diges ion p ocess did no cause signi ican
di e ences in TPC. Howe e , he an ioxidan ac i i y was signi ican ly dec eased. Mo eo e , he bioa ailabili y o an hocyanins was
s udied, showing ha a small ac ion o polyphenols in hei in ac o m was conse ed a he end o diges ion. Finally, a p o ec i e
e ec o diges ed maize polyphenols was obse ed in he Caco-2 cell iabili y. The esul s sugges ha “Mo ag o” pu ple maize is a
good sou ce o bioa ailable an hocyanins in he die and an in e es ing sou ce o his g oup o compounds o he ood indus y.
KEYWORDS: an hocyanins, Zea mays L, pu ple maize lou , an ioxidan ac i i y
1. INTRODUCTION
Maize (Zea mays L.) is a aluable c op wi h nu i ional,
cul u al, en i onmen al, and economic impac s in mos
coun ies in he wo ld.
1
Rega ding i s nu i ional impo ance,
maize is an excellen sou ce o ca bohyd a es; i is na u ally
glu en- ee, sui able o people su e ing om celiac disease,
and has special phy ochemical componen s ha can be
bene icial o human heal h.
2
This c op has g ea ag onomical
di e si y, wi h di e en shapes and colo s o g ains anging
om whi e o yellow, ed, blue, and pu ple. Besides, he e a e
di e en maize a ie ies cha ac e ized acco ding o he inal use
o which hey a e in ended and hei quali y o he s uc u e
and composi ion o he g ains. In his sense, he de elopmen
o new ge mplasm is a g ea oppo uni y o he di e si ica ion
and di e en ia ion o his c op in he ma ke wi h po en ial use
o he ood indus y.
3
Pu ple maize has been widely cul i a ed and consumed in
he Andean and Sou h Ame ican egions, mainly in Pe u,
Boli ia, Ecuado , and some egions o no he n A gen ina.
2
In
A gen ina, he p edominan ly semia id clima e in he cen al
a ea o he coun y limi s maize p oduc ion; he e o e, one o
he main gene ic imp o emen e o s has been based on he
de elopmen o adap ed cul i a s o hese speci ic condi ions.
4
The Special Maizes P og am a he Uni e sidad Nacional de
Co doba ocuses on he in oduc ion, adap a ion, and
cha ac e iza ion o pigmen ed maize ge mplasm in he cen al
semia id egion o A gen ina. A new cul i a o pu ple maize
(“Mo ag o”) has been ob ained wi hin his b eeding p og am,
which was egis e ed o he i s ime in he coun y a he
Ins i u o Nacional de Semillas (INASE). The comme cial maize
ypes g own in A gen ina a e adi ional hyb id a ie ies;
howe e , “Mo ag o” is an open-pollina ed a ie y, which is
cha ac e ized by minimizing dependence on ex e nal seed
sou ces wi hou causing yield losses and educing he cos o
p oduc ion in ag icul u al sys ems.
5
Mo eo e , i is a
non ansgenic a ie y, adap ed o la e sowing da es o he
cen al egion o A gen ina (la e Decembe /ea ly Janua y).
Tole ance o semia id clima es is a dis inc i e ea u e o his
cul i a , which pe o ms well unde ain ed condi ions.
4
Recei ed: No embe 30, 2023
Re ised: Feb ua y 26, 2024
Accep ed: Feb ua y 29, 2024
Published: Ma ch 14, 2024
A iclepubs.acs.o g/JAFC
© 2024 The Au ho s. Published by
Ame ican Chemical Socie y 6327
h ps://doi.o g/10.1021/acs.ja c.3c08960
J. Ag ic. Food Chem. 2024, 72, 6327−6338
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Mo eo e , ecen s udies ha e shown ha “Mo ag o” maize
lou can be used as a unc ional ing edien in glu en- ee
b ead, inc easing i s o al polyphenol con en (TPC), o al
an hocyanins, and an ioxidan capaci y. The lou also has a
highe con en o slowly diges ible and esis an s a ch in
compa ison o adi ional whi e maize lou . These indings
sugges ha “Mo ag o” maize has he po en ial o be a aluable
c op o bo h human heal h and sus ainable ag icul u e.
6
Pu ple maize is a ich sou ce o phenolic compounds, mainly
an hocyanins, ha gi e a da k pu ple- ed colo o he g ains.
The an hocyanin composi ion o some pu ple maize has been
well s udied. The 6 majo an hocyanins include cyanidin-3-
glucoside, pela gonidin-3-glucoside, peonidin-3-glucoside, and
hei malonic acid de i a i es.
7
Among mino i y compounds,
condensed la anol-an hocyanin pigmen s ha e been de ec ed
in pu ple maize om Pe u and Mexico and can in luence colo ,
p oduce a da ke ed colo , and migh ha e s abili y
ad an ages.
8
An hocyanins ha e a ole in human heal h. Some bene i s
ha e been shown in diabe es, obesi y, and ca dio ascula
disease.
9
The heal h bene i s o pu ple maize an hocyanins and
o he phenolic compounds depend on hei bioa ailabili y.
Du ing diges ion, phenolics can unde go enzyma ic and
chemical modi ica ions due o he di e en pH alues o he
medium.
10
Mo eo e , some an hocyanins such as cyanidin-3-
glucoside and pela gonidin-3-glucoside could be abso bed in
hei in ac o m in he gas oin es inal ac .
10
O he
an hocyanins and phenolics can each he la ge in es ine in
signi ican amoun s and unde go me abolism by he gu
mic obio a.
11
In his sense, in es iga ing he bioa ailabili y o
non-nu ien s is a challenge o ood echnology due o he
di e en mechanisms o hei abso p ion and he o en-
complex na u e o bioac i e compounds.
12
Howe e , ew
s udies ha e ocused on he heal h e ec s o pu ple maize
phenolic compounds.
Nowadays, ou in e es is in s udying he nu i ional and
echnological quali y o g ains and lou o he “Mo ag o”
cul i a o p oduce heal hy oods based on hei nu aceu ical
p ope ies. The aim o he p esen wo k was o in es iga e he
phenolic composi ion o whole-g ain pu ple maize “Mo ag o”
om A gen ina using high-pe o mance liquid ch oma og a-
phy−quad upole ime-o - ligh andem mass spec ome y
(HPLC−QTOF-MSMS), as well as he TPC and i s
an ioxidan capaci y. Addi ionally, hei accessibili y o
abso p ion and hei con en a e in i o diges ion we e
s udied. Finally, he bioac i i y o he phenolic compounds was
de e mined h ough a Caco-2 model. The esul s o his s udy
may con ibu e o a be e unde s anding o he composi ion o
he bioac i e compounds o “Mo ag o” and hei bioa ail-
abili y.
2. MATERIALS AND METHODS
2.1. Gene ic Ma e ial and he Adap a ion P ocess. The
“Mo ag o” cul i a was ob ained by c ossing in oduced gene ic
ma e ial om di e en o igins: no he n A gen ina, Pe u, Boli ia, and
In e na ional Maize and Whea Imp o emen Cen e (CIMMYT)
seeds. The o iginal popula ion ob ained was plan ed and assessed o
i e cycles (2011/12, 2013/14, 2014/15, 2015/16, and 2016/17) and
he a ie y was s abilized in 2019. The adap a ion p ocess was ca ied
ou in each cycle h ough adap i e mass selec ion a he expe imen al
s a ion o he Facul ad de Ciencias Ag opecua ias, Uni e sidad Nacional
de Co doba, A gen ina (geog aphical loca ion: 31°28′49.42″S,
64°00′36.04″W). The ield is loca ed in he cen al semia id egion
o he coun y in he p o ince o Co doba, wi h an al i ude o 425
m.a.s.l. The soil is En ic Haplus oll and p esen s a sil -loam ex u e on
he supe icial ho izon. I is sligh ly acidic o neu al and well supplied
wi h o ganic ma e (Minis e io de Ag icul u a y Ganade ia 2019).
The ield zone has a his o ical a e age ange o medium, minimum,
and maximum empe a u es o 15−20, 8−13.7, and 21.8−25.1 °C,
espec i ely, and an annual p ecipi a ion o 300 o 1000 mm (Bolsa de
Ce eales de Co doba 2016). The g ains used in he p esen wo k we e
ob ained om he 2020/21 cycle.
2.2. Flou Ob en ion. The g ains o pu ple maize (Z. mays L.),
he “Mo ag o” cul i a , we e milled on a cyclonic mill (Cyclo ec
CT193, Foss, Suzhou) o a ine powde (pa icle size ange less han
500 μm). The whole-g ain maize lou ob ained was s o ed in
da kness a −20 °C un il chemical analysis.
2.3. “Mo ag o” Flou Cha ac e iza ion (P oxima e Compo-
si ion). The mois u e, p o ein, lipid, and ash con en s o he
“Mo ag o” whole-g ain lou we e measu ed acco ding o he AACC
me hods (AACC In e na ional 2010)
13
and exp essed as g/100 g o
lou o d y weigh (DW). All analyses we e pe o med in duplica e.
2.4. Cha ac e iza ion o Phenolic Compounds o “Mo ag o”
Flou . 2.4.1. Phenol Ex ac ion. Sample ex ac ion o iden i y he
ini ial compounds p esen in he “Mo ag o” lou was pe o med using
he me hod o Chamo o e al.
14
wi h modi ica ions. In b ie , 50 mg o
sample was suspended in 1 mL o me hanol/wa e (50:50 /
acidi ied wi h o mic acid 0.1%). The mix u e was o exed and
sonica ed o 15 min and hen cen i uged a 10000 pm o 15 min a
4°C. The supe na an was collec ed, and he esidue was e-ex ac ed
wice wi h 0.5 mL o acidi ied MeOH/H2O (1:1, 0.1% o mic acid)
ollowing he same me hod and e-ex ac ed ollowing he same
p ocedu e wo imes. Supe na an s we e combined, il e ed (0.45
μm), and s o ed a −20 °C un il analysis.
2.4.2. To al Polyphenol Con en . TPC o he pu ple maize lou
was ca ied ou by he Folin−Ciocal eu eagen me hod, in he
ex ac s ob ained in he p e ious sec ion, acco ding o Sil an e al.
15
Gallic acid was used as he s anda d o he calib a ion cu e. The
abso bance was eco ded a 725 nm in a BioTek Syne gy HT
mul imode mic opla e eade (BioTek Ins umen s Inc., Winooski,
VT, USA). Resul s we e exp essed as millig ams o gallic acid
equi alen s pe g am o sample on a d y basis (mg o GAE/g o DW).
2.4.3. An ioxidan Ac i i y. An ioxidan ac i i y was assessed as
an i adical ac i i y and e ous- educing powe . Radical-sca enging
capaci y was measu ed using he DPPH me hod, as epo ed by Puell
and de Pascual-Te esa.
16
The e ic- educing an ioxidan powe
(FRAP) assay was pe o med using he p o ocol o So iano-
Maldonado e al.
17
All samples we e measu ed in iplica e using
T olox (Sigma-Ald ich) as he s anda d. Resul s we e exp essed as
μmol o T olox equi alen s pe g ams o sample (μmol o T olox eq/g
DW).
2.4.4. Iden i ica ion and Quan i ica ion o Phenolic Compounds
Using HPLC−QTOF-MS. The iden i ica ion and quan i ica ion o
“Mo ag o” pu ple maize phenolic compounds, including an hocyanin
compounds, we e pe o med using HPLC wi h mass spec ome y
de ec ion (Agilen 1200, Agilen Technologies) comp ising a
qua e na y pump (G1311A), a diode a ay de ec o (Agilen
G1315B), and a C18 analy ical column (Phenomenex Luna, 3 μm,
4.6 mm ×150 mm) se he mos a ically a 25 °C. The mobile phase
consis ed o wa e / o mic acid, 99.9:0.1 / (sol en A), and
ace oni ile/ o mic acid, 99.9:0.1 / (sol en B). The low a e was
kep a 0.5 mL/min. The g adien p og am was as ollows: 90% A/
10% B, 0−30 min; 70% A/30% B, 30−35 min; 65% A/35% B, 35−45
min; 60% A/40% B, 45−50 min; and 90% A/10% B, 50−60 min
18
The injec ion olume was 5 μL o all samples and s anda ds. Peaks
we e iden i ied by compa ing hei e en ion ime wi h he
co esponding s anda ds. Fo mass spec ome ic analysis, an Agilen
6530 Accu a e-Mass QTOF LC/MS wi h elec osp ay ioniza ion
(ESI) and Je S eam echnology (Agilen Technologies) ope a ed a
325 °C was used. The capilla y ol age and nebulize gas low we e se
o 4000 kV and 45 psi, espec i ely. Ni ogen was used as he d ying
gas a a low a e o 8 L/min. The agmen ed ions o he analy es
we e de ec ed in posi i e and nega i e modes o p o ide ex a
ce ain y in he de e mina ion o he molecula masses. Fo he
Jou nal o Ag icul u al and Food Chemis y pubs.acs.o g/JAFC A icle
h ps://doi.o g/10.1021/acs.ja c.3c08960
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iden i ica ion and quan i ica ion o compounds, MS and MSMS
agmen a ion spec a expe imen s we e pe o med, and spec al
signal da a we e also acqui ed a 280, 320, and 520 nm. Fo MSMS
expe imen s, a qui e gene ic collision ene gy o 20 V was used, as a
comp omise, o simpli y he de elopmen o he me hod and ensu e
good agmen a ion o he majo i y o a ge ed compounds. Da a
acquisi ion and p ocessing we e pe o med wi h Masshun e Da a
Acquisi ion (B.05.01) and Masshun e Quali a i e Analysis (B.07.00
SP2) so wa e. Compounds we e iden i ied by compa ing mass
spec a and e en ion ime wi h he co esponding s anda d, i
a ailable. In he case o compounds o which s anda ds we e no
a ailable, iden i ica ion was based on a p edic ion o chemical o mula
om accu a e ion mass measu emen and con i med by compa ing
MSMS wi h da a p o ided by ele an li e a u e e e ences (see Table
1). The analy ical me hod was alida ed o all quan i ied compounds,
wi h a minimum eco e y o 85%, a minimum de ec ion limi o 0.01
μg/mL, and a minimum quan i ica ion limi o 0.05 μg/mL o each
quan i ied compound. The quan i ica ion was pe o med by
in e pola ion in o he calib a ion cu e o an iden ical s anda d o a
s uc u ally ela ed compound used o quan i y i (equi alen ) and
exp essed as μg pe g o DW sample as ollows: cyanidin-3-O-
glucoside was used o he quan i ica ion o cyanidin de i a i es,
pela gonidin de i a i es, and an hocyanin condensed o ms; peonidin-
3-glucoside was used as s anda d o peonidin de i a i es; ca eic acid
o cinnamoyl-quinic acids, gallic, e ulic, and ci ic acids; 3
ca eoylquinic acid o chlo ogenic acid; que ce in-3-O-glucoside o
que ce in-3-O-glucoside; que ce in o que ce in de i a i es and
mo in; kaemp e ol o kaemp e ol de i a i es; epica echin o
epica echin and na ingenin; apigenin o i exin; and phlo oglucinol
o py ogallol.
2.5. Bioaccessibili y o Phenolic Compounds in “Mo ag o”
Flou . 2.5.1. S a ic In Vi o Diges ion. The pu ple maize “Mo ag o”
lou was in i o diges ed using he INFOGEST p o ocol
19
wi h o al
(pH 7), gas ic (pH 3), and in es inal (pH 7) phases. The enzymes
used o each gas oin es inal phase we e sali a y amylase (75 U/mL),
pepsin (2000 U/mL), panc ea in (100 U ypsin/mL), and po cine
bile ex ac (10 mM). A con ol ube lacking he lou se ed as a
diges ion blank. Following his, o al and gas ic aliquo s (0.5 mL)
we e collec ed. The in es inal diges was cen i uged (5000 pm, 10
min), and i s supe na an was collec ed in 2 mL Eppendo ubes. All
samples we e s o ed a −20 °C un il analysis.
2.5.2. Cha ac e iza ion o Diges ed Aliquo s. 2.5.2.1. Phenol
Ex ac ion, TPC, and An ioxidan Ac i i y. Polyphenol ex ac ion
om diges ed samples was pe o med as men ioned abo e in Sec ion
2.4.1 wi h one modi ica ion. Fo he i s s ep, 0.5 mL aliquo s we e
aken a each diges ion phase, placed in an Eppendo , and added wi h
0.5 mL o me hanol (acidi ied wi h o mic acid, 0.1%). Then, he
p ocedu e con inued as desc ibed be o e. TPC was de e mined as
desc ibed in Sec ion 2.4.2, and an ioxidan ac i i y was de e mined
acco ding o Sec ion 2.4.3.
2.5.2.2. Quan i a i e Analysis o An hocyanins. Quan i a i e
analysis o an hocyanins be o e (in he undiges ed “Mo ag o” lou ),
du ing, and a e diges ion was ca ied ou using an Agilen 1200
se ies liquid ch oma og aph wi h a qua e na y pump and a
pho odiode a ay de ec o equipped wi h a Phenomenex Luna C18
column (3 μm; 4.6 ×150 mm) se a 25 °C. Aqueous 0.1% o mic
acid (sol en A) and 0.1% o mic acid ace oni ile (sol en B) we e
used a a low a e o 0.5 mL/min. We s a ed wi h 90% A/10% B, 0−
30 min o 68% A/32% B, 30−35 min o 62% A/38% B, and 35−40
min o 53% A/47% B, ollowed by an addi ional 5 min isoc a ically a
47% B and 10 min column s abiliza ion a 10% B p io o he nex
Table 1. Cha ac e iza ion o he Indi idual Phenolic Compounds in Mo ag o Flou Ex ac s Using HPLC−QTOF-MS
peak compound assignmen
b
,
c
R
a
(min) [M]−iden i ied MS/MS−[M]+iden i ied MS/MS+
1 py ogallol 2.9 127.0396 81, 53
2 ci ic acid 4.1 191.0198 111
3 ca echin-(4,8)-cy-3,5-diGlu 4.9 899.2250 737, 575, 423, 329, 287
4 gallic acid 5.3 169.0453 125
5 ca echin-(4,8)-Cy-3-MalGlu-5Glu 8.2 985.2241 823, 737, 575, 423, 329
6 Cy-3-Glu 9.9 449.1099 287
7 Pg-3-Glu 11.7 431.1025 269 433.1112 287
8 Cy-3-MalGlu 12.5 535.1075 449, 287
9 chlo ogenic acid 12.5 353.0873 191 287
10 Pn-3-Glu 12.7 463.1240 301
8 Cy-3-MalGlu 13.6 535.1075 449, 287
8 Cy-3-(6′MalGlu) 15.2 535.1070 449, 287
11 ca eic acid 15.6 179.0358 135
12 Pg-3,6-MalGlu 17.2 519.1123 433, 271
13 Pn-3,6-MalGlu 17.9 549.1229 463, 301
14 Cy-3-(diMalGlu) 18.0 621.1084 535, 449, 287
14 Cy-3-(3,6-diMalGlu) 18.8 621.1092 535, 449, 287
15 Pg-3,6-diMalGlu 21.2 605.1136 519, 433, 271
16 p-couma ic acid 21.4 165.0581 147, 45
17 Pn-3,6-diMalGlu 21.9 635.1136 549, 463, 301
18 que ce in-3- u inoside 22.3 609.1507 301 611.1606
19 e ulic acid 23.6 195.0636 176, 144
20 que ce in-3-Glu 23.8 463.1002 301 465.1019
21 kaemp e ol 3-(6″- e uloylglu) 24 625.1549 287
22 kaemp e ol-3-Glu 27.2 449.1082 287
23 i exin 35.2 433.1723 283
24 kaemp e ol-3-glucu onide 36.0 463.1826 287
25 na ingenin 43.1 273.0763 189, 153
26 mo in 43.6 301.0718 149
a
RT, e en ion ime.
b
Iden i ica ion was con i med acco ding o he s anda d (S d) abo e ci ed and/o he MS agmen a ion pa e n p e iously
desc ibed by o he s udies.
c
Cy, cyanidin; Glu, glucoside; Mal, malonyl; Pg, pela gonidin; Pn, peonidin.
Jou nal o Ag icul u al and Food Chemis y pubs.acs.o g/JAFC A icle
h ps://doi.o g/10.1021/acs.ja c.3c08960
J. Ag ic. Food Chem. 2024, 72, 6327−6338
6329
analysis. An hocyanins we e de ec ed a 520 nm, and hei peak a eas
we e e e ed o a calib a ion cu e ob ained wi h cyanidin-3-
glucoside. Limi s o de ec ion and quan i ica ion we e calcula ed
and we e in e e y case below 0.1 μg/mL.
2.5.3. Bioac i i y Analysis. 2.5.3.1. Cell Cul u e and Di e -
en ia ion. Cell cul u e and di e en ia ion we e pe o med ollowing
he p o ocol epo ed by Huba sch e al.
20
In b ie , be o e hei use in
his assay, Caco-2 cells we e cul u ed in cul u e lasks con aining
Dulbecco’s modi ied minimal essen ial medium (DMEM) supple-
men ed wi h 10% hea -inac i a ed e al bo ine se um (FBS),
nonessen ial amino acids (1%), and 1% an ibio ic (s ep omycin/
penicillin) solu ion a 37 °C and in a humidi ied a mosphe e wi h 5%
CO2. The medium was eplaced e e y 2 days. Cells we e subcul u ed
weekly upon 85−95% con luence by ypsiniza ion. Caco-2 cells we e
used in a maximum passage o 60 and seeded in o 24-well ans-wells
a a concen a ion o 6 ×105cells/mL in DMEM wi h 10% FBS,
nonessen ial amino acids (1%), and 1% an ibio ic (s ep omycin/
penicillin). The medium was changed in he apical (150 μL) and
basola e al (700 μL) chambe s e e y 2 days. The ans-epi helial
elec ical esis ance alues we e measu ed o con i m monolaye
o ma ion and cell di e en ia ion.
2.5.3.2. Cell Viabili y. The (3-[4,5-dime hyl hiazol-2-yl]-2,5
diphenyl e azolium b omide) (MTT) assay was used o de e mine
he cell iabili y. Caco-2 cells we e pla ed in 96-well pla es (1.6 ×106
cells/mL) and cul u ed o 7 days a 37 °C in 5% CO2 o
di e en ia ion. The di e en ia ed Caco-2 cells we e ea ed wi h
dilu ed in es inal diges ion aliquo s in he ollowing p opo ions: 1:1,
1:10, 1:100, 1:250, 1:500, and 1:1000, all o hem suspended in
se um- ee DMEM. The medium was emo ed a e 18 h. The cells
we e sequen ially washed wi h phospha e bu e ed saline (PBS),
which was hen emo ed, 200 μL se um- ee DMEM was added, and
20 μL o an MTT solu ion (5 mg/mL in PBS) was added o each well
and incuba ed o an addi ional 2 h a 37 °C in 5% CO2. Fo mazan
c ys als o med in he wells we e solubilized in 200 μL o DMSO
(dime hyl sul oxide). The measu emen was pe o med wi h an
abso bance a a 570 nm wa eleng h employing a mic opla e eade
(Powe Wa eTM XS) in a UV spec opho ome e (BioTek Ins u-
men s, Inc., Winooski, VT, USA). The assay was epea ed in wo
independen expe imen s. The iabili y was calcula ed in compa ison
o con ol expe imen s in which a sol en con ol was added in place
o polyphenols, and ha was used as a 100% iable e e ence.
18
Dilu ion was pe o med wi h one-pa in es inal aliquo and one-pa
DMEM (sample: DMEM). The con ol sample consis ed o an
in es inal aliquo o he diges ion blank (pu ple maize lou was
eplaced wi h wa e , and in i o diges ion was pe o med).
2.6. S a is ical Analysis. The esul s we e exp essed as he mean
o wo eplica es ± he s anda d de ia ion. Analysis o a iance was
pe o med, and da a we e compa ed by he DGC means-compa ison
es
21
wi h a signi ican le el a 0.05. These analyses we e pe o med
using In os a S a is ical So wa e (Facul ad de Ciencias Ag o-
pecua ias, UNC, A gen ina).
3. RESULTS AND DISCUSSION
3.1. “Mo ag o” Flou Cha ac e iza ion. 3.1.1. P oxima e
Composi ion. The mac onu ien composi ion o “Mo ag o”
lou ob ained was 1.85 ±0.03% ash, 6.1 ±0.1% lipids, and
10.2 ±0.2% p o ein. In compa ison wi h o he maize a ie al
ypes, he “Mo ag o” cul i a p esen ed highe p o ein, lipid,
and ash con en s han hose o blue and whi e maize lou om
Mexico.
4,22
“Mo ag o” lou p esen ed highe p o ein and lipid
con en s and lowe ash con en han ha o se e al pu ple
maize geno ypes om India.
23
As maize lou is gene ally ich
in an ioxidan compounds and s a ch, i is ideal o he
de elopmen o unc ional oods.
24
3.1.2. TPC and An ioxidan Ac i i y. The ex ac able TPC
o “Mo ag o” lou was 2.71 ±0.04 mg GAE/g DW. The
an ioxidan po en ial o “Mo ag o” lou was 24 ±1μmol o
T olox eq/g DW assessed by analyzing i s an i adical ac i i y
(DPPH) and 22 ±1μmol o T olox eq/g DW by FRAP.
Polyphenols exhibi an ioxidan capaci y and ac as ee adical
inhibi o s; we es ablished signi ican co ela ions ( ) be ween
TPC and DPPH ( = 0.76, p< 0.05) and DPPH and FRAP (
= 0.80, p< 0.01), sugges ing a di ec ela ionship be ween
polyphenol con en and an ioxidan ac i i y.
“Mo ag o” maize p esen ed highe TPC in compa ison o
ha o whi e and yellow maize om India, wi h a alue o 1.6
mg GAE/g and 1.3 mg GAE/g o TPC, espec i ely.
23
This
highe polyphenolic con en o pu ple maize could be expec ed
because i con ains an hocyanins in addi ion o e ulic and p-
couma ic acids ha ha e been de ec ed in whi e maize.
25
In compa ison wi h he blue maize lou om Mexico,
“Mo ag o” lou showed lowe TPC bu highe an ioxidan
ac i i y (DPPH, FRAP) han i .
22
In addi ion, “Mo ag o” lou
p esen ed lowe an i adical ac i i y han ha epo ed by
Ranilla e al.
26
o a Pe u ian a ie y o pu ple maize
″Can eno″. On he o he hand, in a a ie y o pu ple waxy
maize ( a . “Ce a ina”) om Thailand, he alue o TPC and
e ic- educing powe we e simila o ou esul s ob ained.
27
These esul s indica ed ha “Mo ag o” lou is an impo an
sou ce o phenolic compounds wi h an ioxidan ac i i y.
3.1.3. Cha ac e iza ion o he Composi ion by HPLC−
QTOF-MS. The phenolic compounds iden i ied by HPLC−
QTOF-MS analysis in “Mo ag o” lou a e shown in Table 1. A
o al o 26 compounds we e iden i ied: 15 nonan hocyanin and
11 an hocyanin pigmen s. Fo p ac ical easons, we ha e
classi ied he compounds in o an hocyanins and nonan hocya-
nins o u he analysis and desc ip ion. Also, he phenolic
compounds iden i ied we e quan i ied, and he esul s a e
shown in Tables 2 and 3.
3.1.3.1. Nonan hocyanin Compounds. The 15 non-
an hocyanin compounds iden i ied in he “Mo ag o” cul i a
(Table 1) we e classi ied acco ding o hei s uc u e as
benzoic acids, cinnamoyl-quinic acids, la onols, and o he
phenolic compounds (Table 2).
Table 2. Nonan hocyanin Con en o Mo ag o Flou
compound assignmen concen a ion
a
(μg/g)
benzoic acids
ci ic acid 755.7 ±60.4
gallic acid 1.3 ±0.1
chlo ogenic acid 6.6 ±0.5
cinnamoyl-quinic acids
ca eic acid 1296.8 ±103.7
p-couma ic acid 6.5 ±0.2
e ulic acid 6.0 ±0.4
la onols
que ce in-3- u inoside 588.0 ±29.4
que ce in-3-Glu 14.7 ±0.6
kaemp e ol 3-Glu 25.8 ±1.5
kaemp e ol 3-glucu onide 1201.0 ±9.2
mo in 11.5 ±0.7
kaemp e ol 3-(6″- e uloylGlu) nq
o he compounds
na ingenin 2.0 ±0.1
i exin 254.5 ±12.7
py ogallol 229.4 ±13.8
o al nonan hocyanin compounds 4399.9 ±395.9
a
A e age alue ± he s anda d de ia ion (n= 3).
Jou nal o Ag icul u al and Food Chemis y pubs.acs.o g/JAFC A icle
h ps://doi.o g/10.1021/acs.ja c.3c08960
J. Ag ic. Food Chem. 2024, 72, 6327−6338
6330
Benzoic acid de i a i es we e ecognized as ci ic, gallic, and
chlo ogenic acids. Peak 2 o be assigned ci ic acid was
iden i ied om hei p ecu so ion [M −H]−a m/z191.
Gallic acid was p esen ed by peak 4 wi h a molecula ion o [M
−H]−a m/z= 169. Chlo ogenic acid (peak 9) was iden i ied
by compa ison o i s molecula mass ion o [M −H]−a m/z
353 and e en ion imes o 12 and 5 min wi h he da a
ob ained wi h he comme cial s anda d.
Peaks 11, 16, and 19 we e iden i ied as cinnamoyl-quinic
acids based on da a om a p e ious su ey
14
and hei
p ecu so ion agmen s a [M −H]−179 and [M + H]+165
and 195, co esponding o ca eic, p-couma ic, and e ulic
acids, espec i ely. Ca eic acid iden i ica ion was u he
con i med using a comme cial s anda d.
14
O he compounds o peaks 18, 20, 21, 22, 23, and 26 we e
iden i ied as la onol de i a i es om kaemp e ol and
que ce in. Compound 20 was nega i ely iden i ied as
que ce in-3-glucoside a m/z452 and posi i ely a m/z465
by compa ison wi h he co esponding comme cial s anda d.
Compounds 18 and 26 we e nega i ely iden i ied a m/z609
and 301 as que ce in-3- u inoside and mo in acco ding o a
p e ious in es iga ion.
18,28
Peak 22 was iden i ied as
kaemp e ol-3-glucoside wi h a molecula ion a m/z449.
Peaks 21 and 24 we e named kaemp e ol 3-(6″- e uloylgluco-
side) and kaemp e ol-3-glucu onide wi h a molecula ion a m/
z625 and 463 wi h a ypical agmen o 287 co esponding o
kaemp e ol, he ee aglycone moie y, esul ing om he loss o
he glucose and e uloylglucose, espec i ely. Kaemp e ol 3-O-
glucu onide has been iden i ied in di e en ui s such as
Sa cand a glab a,
29
be ies o he Rosaceae amily,
30
and
s awbe y,
31
bu i has no been iden i ied o quan i ied in
pu ple maize un il now. The same happens wi h kaemp e ol 3-
(6″- e uloylglucoside), which has only been epo ed so a in
Polylepis incana.
32
Fu he mo e, o he compounds we e iden i ied as py ogallol,
i exin ( la one), and na ingenin ( la anone) wi h molecula
ions wi h m/z alues o 127, 433, and 273, espec i ely.
Py ogallol was iden i ied as epo ed by Hidalgo e al.
33
Na ingenin and i exin we e con i med wi h da a om
Cha ham e al.
34
The nonan hocyanin compounds ound in his wo k a e
cha ac e is ic o pu ple maize, and HPLC−MSMS agmen a-
ion o hei main compounds has been desc ibed by Pauca -
Menacho e al.
35
Likewise, hese compounds we e iden i ied,
cha ac e ized, and quan i ied in o he ma ixes in p e ious
s udies by he g oup.
14,18,33
Addi ionally, o he epo s by
Gal ez Ranilla e al.,
36
Del Pozo-Ins an e al.,
37
and Ramos-
Escude o e al.
28
p o ide da a on he quan i ica ion o some
phenolic compounds in pu ple maize. All o hese epo s ha e
been consul ed o con i m he iden i y o he nonan hocyanin
compounds in his esea ch. Howe e , he p esence o
py ogallol, ci ic acid, gallic acid, kaemp e ol 3-(6″- e uloyl-
glucoside), and kaemp e ol 3-glucu onide has no been
p e iously epo ed in pu ple maize, esul ing in compounds
dis inc i e o he “Mo ag o” cul i a .
28,35−38
Rega ding quan i ica ion, he esul s showed di e ences
be ween he amoun o nonan hocyanin and an hocyanin
compounds (Tables 2 and 3). “Mo ag o” lou ound a o al o
4399.9 μg/g DW o nonan hocyanin compounds, p esen ing
75.1% o o al phenol compounds quan i ied, and he majo i y
main compound was ee ca eic acid wi h 22.1%, and he
second majo compound was kaemp e ol 3-O-glucu onide ha
accoun ed o 20.5%. In con as wi h ou esul s, a p e ious
s udy by Pauca -Menacho e al.
35
p esen ed a lowe amoun
(323.9 μg/g DW) o nonan hocyanin compounds, and e ulic
acid de i a i es we e he mos dominan nonan hocyanin
compound ound in pu ple maize (Z. mays L. a . PMV-581).
Fu he mo e, Ramos-Escude o e al.
28
epo ed a lowe
concen a ion o ca eic acid on pu ple maize (INIA-601),
and Gal ez Ranilla e al.
36
epo ed a lowe concen a ion o
ee ca eic acid and e ulic acid, which was no ably highe han
in ou s udy. On he o he hand, a mo e ecen s udy showed
ha e ulic acid is he mos abundan phenolic acid in pu ple
maize om Mexico.
25
Cinnamoyl-quinic acids ep esen ed a pe cen age o 22.3%,
la onols accoun ed o 31.4%, and benzoic acids accoun ed o
13.1% o o al polyphenol compounds. The emaining
pe cen age o o al polyphenol compounds (8.3%) consis ed
o na ingenin, i exin, and py ogallol. The esul s ob ained
showed he p esence o que ce in and kaemp e ol de i a i es
wi hin he la onol class in ele an quan i ies. Some o hese
compounds we e iden i ied by Pauca -Menacho e al.,
35
and an
in e es ing di e ence was he lowe concen a ion o que ce in-
3- u inoside (35.91 μg/g) han ha in “Mo ag o” lou (588.0
μg/g). Que ce in and kaemp e ol ha e been iden i ied and
quan i ied in se e al s udies, bu hei de i a i es ha e no been
de ailed ye , and in compa ison, “Mo ag o” lou showed a
highe concen a ion o que ce in de i a i es han ha
epo ed.
2,28,39
In addi ion, among benzoic acids, ci ic acid
was p esen in “Mo ag o” lou wi h a 12.9% phenol o al
quan i ied, while in o he pu ple maize, i has no been
de ec ed.
The “Mo ag o” cul i a was dis inguished om he o he
pu ple maize a ie ies by he p esence o py ogallol, ci ic acid,
gallic acid, kaemp e ol 3-(6″- e uloylglucoside), and kaemp e -
ol 3-glucu onide, as men ioned abo e. Fu he mo e, in he
quan i ica ion, he “Mo ag o” cul i a was dis inc i e by
showing ele an amoun s o ca eic acid, kaemp e ol 3-O-
glucu onide, ci ic acid, and que ce in-3- u inoside as he ou
majo cons i uen s among he o al phenols iden i ied and
quan i ied. On he o he hand, p e ious s udies showed ha
Table 3. An hocyanin Con en o Mo ag o Flou
a
peak compound assignmen concen a ion (μg/g)
ca echin-(4,8)-Cy-3,5diGlu 15.9
ca echin-(4,8)-Cy-3-MalGlu-5Glu 9.0
1 Cy-3-Glu 314.3
2 Pg-3-Glu 115.7
3 Cy-3-(MalGlu) 4.2
4Pn3-Glu 142.6
5 Cy-3-(MalGlu) 68.1
6 Cy-3-(6′MalGlu) 356.5
7 Pg-3-(6′MalGlu) 108.3
8Pn3-(6′MalGlu) 123.4
9 Cy-3-(diMalGlu) 8.9
9 Cy-3-(3″,6″, diMalGlu) 107.3
10 Pg 3-O-3′,6′-O-diMalGlu 41.3
11 Pn 3-O-3′,6′-O-diMalGlu 44.8
o al an hocyanin concen a ion 1460.4
cyanidin de i a i es 859.3
peonidin de i a i es 310.8
pela gonidin de i a i es 265.3
condensed o ms 24.9
a
Cy, cyanidin; Glu, glucoside; Mal, malonyl; Pg, pela gonidin; Pn,
peonidin.
Jou nal o Ag icul u al and Food Chemis y pubs.acs.o g/JAFC A icle
h ps://doi.o g/10.1021/acs.ja c.3c08960
J. Ag ic. Food Chem. 2024, 72, 6327−6338
6331

e ulic acid and i s de i a i es we e he mos dominan
nonan hocyanin compounds in di e en pu ple maize a ie ies,
while in ou s udy, e ulic acid only ep esen ed 0.1% o he
o al phenols quan i ied, being ano he dis inc i e ea u e o
his cul i a .
3.1.3.2. An hocyanins. Up o 11 an hocyanin pigmen s
we e iden i ied in “Mo ag o” lou , pa icula ly cyanidin (Cy),
pela gonidin (Pg), and peonidin (Pn) de i a i es (Table 1).
The iden i y o each compound was elucida ed by compa ison
o he comme cial s anda d used. In his ega d, peaks 6 a 9.9
min and 10 a 12.7 min we e iden i ied as cyanidin-3-glucoside
and peonidin-3-glucoside, espec i ely, and peak 7 was
iden i ied as pela gonidin-3-glucoside by compa ison o hei
e en ion ime and spec um mass wi h da a in ou lib a y and
p e ious s udies. Acyl de i a i es o cyanidin, peonidin, and
pela gonidin we e obse ed in peaks 8, 12−15, and 17,
espec i ely. In addi ion, peak 8 ma ches a molecula ion a m/
z535, eleasing he MS/MS+ agmen a m/z449 ([M-86]+,
loss o a malonyl esidue) and a m/z287 ([M-248]+, loss o
malonyl glycoside moie y), co esponded wi h cyanidin-3-
malonylglucoside. Fo peak 12, he molecula ion was a m/z
519 ha eleased wo agmen s MS/MS+a m/z433 ([M-86]
+, loss o a malonyl esidue) and a m/z271 (pela gonidin),
co esponded wi h pela gonidin-3,6-malonylglucoside. Also,
peak 13 wi h a molecula ion 549, which eleases wo
agmen s MS/MS+a m/z463 ([M-86] +, loss o a malonyl
esidue) and m/za 301 (peonidin), co esponded wi h
peonidin-3,6-malonylglucoside. Peaks 14, 15, and 17 we e [M
+86] +g ea e han ha o peaks 8, 1, 2, and 13, espec i ely,
and showed a simila agmen a ion pa e n, so hey can be
assigned, espec i ely, o cyanidin-3-(6′malonylglucoside),
pela gonidin-3,6-malonylglucoside, and peonidin-3,6-malonyl-
glucoside. Fu he con i ma ion o he iden i ica ion pe o med
was p o ided by a compa ison o he same compounds wi h
o he s p e iously iden i ied in pu ple maize.
7,40,41
In addi ion, wo compounds in ol ing he condensa ion o
an an hocyanin uni (Cy) and ca echin esidues we e also
ound. These compounds we e iden i ied as ca echin-(4,8)-
cyanidin-3,5 diglucoside (peak 3) and i s acyla ed condensed
o m, ca echin-(4,8)-cyanidin-3-malonylglucoside-5 glucoside
(peak 5) by compa ison wi h pigmen s wi h simila spec um
mass, ac ions, and s uc u al cha ac e is ics o Apache Red
Pu ple Co n (Siskiyou Seeds, Williams, OR).
34
An exhaus i e iden i ica ion and quan i ica ion we e
pe o med o he an hocyanin p o ile, he HPLC-DAD
ch oma og am o “Mo ag o” lou ex ac is shown in Figu e
1, and he compounds we e classi ied acco ding o hei
an hocyanidin and quan i ied in Table 2. An hocyanin
compounds ep esen 24.9% (1460 μg/g) o o al phenolic
compounds wi h a high molecula di e si y. The main
an hocyanin compounds p esen in he “Mo ag o” cul i a
we e cyanidin de i a i es, ep esen ing mo e han 58% o he
o al an hocyanin con en . The p e alen an hocyanins we e
cyanidin-3-(6″malonylglucoside), which accoun ed o 29%,
and i s espec i e nonmalonyl cons i uen , cyanidin-3-gluco-
side wi h 21% o he o al an hocyanin con en . The
an hocyanin p o ile was in ag eemen wi h o he s udies wi h
di e ences in he concen a ion and dominan compounds.
The o al concen a ion o an hocyanin con en was highe
in pu ple maize om Pe u (Z. mays L. a . PMV-581),
accoun ing o 92% o he o al phenolic compound wi h
3636.41 μg/g, han ha in he “Mo ag o” cul i a , and in
pu ple maize om Pe u, Cy-3-Glu was he majo an hocya-
nin.
35
Also, Ped eschi and Cisne os-Ze allos
39
epo ed ha
Cy-3-Glu was he majo an hocyanin, cons i u ing ∼38%,
ollowed by he acyla ed cyanidin-3-glucoside wi h ∼26% o
he o al an hocyanin con en . Al hough, in Apache ed pu ple
maize om he USA, he mos abundan we e pela gonidin
de i a i es (1400 mg/g).
42
In ag eemen wi h ou dominan
an hocyanin, Camelo-Mendez e al.
43
epo ed cyanidin-3-(6″
malonylglucoside) as he majo an hocyanin. Fu he mo e, in
black swee maize om China, despi e being a qui e di e en
a ie y, he o al an hocyanin con en was simila , bu he main
compounds we e pela gonidin de i a i es, ollowed by
cyanidin and hen peonidin de i a i es.
44
The di e ences in
he majo an hocyanins among he men ioned s udies sugges
ha each pu ple maize a ie y had i s own dominan
an hocyanin ype. Mo eo e , he dominan an hocyanins
we e ela ed o plan pigmen ; in a s udy, he au ho s showed
ha while he p edominan an hocyanins in blue-aleu one and
pu ple-pe ica p maize we e cyanidin-based glucosides, pela go-
nidin-based glucosides we e he dominan in eddish-pu ple-
pe ica p and che y-aleu one accessions.
45
The second place was o peonidin de i a i es, accoun ing
o 21% o he o al an hocyanins. Among hem, he majo
compound was peonidin-3-O-glucoside. Simila ly, in Andean
pu ple maize, peonidin de i a i es we e he second mos
abundan compound o o al an hocyanins,
39
whe eas in o he
s udies, peonidin de i a i es we e he mino i y compound
an hocyanins.
8,44,45
Thi d among o al an hocyanins, pela gonidin de i a i es
we e obse ed o each 18%. Pela gonidin-3-glucoside was he
mos concen a ed wi h 115.7 μg/g DW. The same
compounds we e iden i ied in Gonzalez-Manzano e al.,
8
bu
in his s udy, he p edominan compound was pela gonidin-3-
(6″malonylglucoside).
As was men ioned, in “Mo ag o” lou we e de ec ed
ca echin-(4,8)-cyanidin-3,5 diglucoside and ca echin-(4,8)-
Figu e 1. An hocyanin compounds iden i ied in Mo ag o maize lou ex ac a 520 nm.
Jou nal o Ag icul u al and Food Chemis y pubs.acs.o g/JAFC A icle
h ps://doi.o g/10.1021/acs.ja c.3c08960
J. Ag ic. Food Chem. 2024, 72, 6327−6338
6332
cyanidin-3-malonylglucoside-5 glucoside. These condensed
pigmen s we e p esen as mino i y compounds o he o al
an hocyanin de i a i es wi h a 1.7% alue. Fla anol an hocya-
nin condensed o ms we e also iden i ied in many s udies,
being be ween 0.3 and 3.2% o condensed pigmen s, acco ding
o Gonzalez-Manzano e al.
8
In “Mo ag o” lou , only
condensed e sions con aining cyanidin we e iden i ied, bu
i has been epo ed in Apache Red pu ple maize ha o he
condensed o ms o pela gonidin, o peonidin, and (epi)-
a zelechin ha e been iden i ied by Cha ham e al.
34
The iden i ica ion o la anol-an hocyanin condensed
compounds in ou pu ple maize ( a . “Mo ag o”) and a ious
Ame ican a ie ies, including wo dis inc Mexican pu ple
maize a ie ies (c . A ocillo and c . Pe uano),
8
Pe u ian
pu ple maize cul i a s ( a . PMV-581),
35
and Apache Red
pu ple maize om he USA,
42
unde sco es a po en ially
dis inc i e gene ic ai sha ed among Ame ican ge mplasms o
pu ple maize. This inding may se e as a gene ic ma ke ha
di e en ia es hem om hei Eu opean coun e pa s. In
con as , Eu opean pigmen ed a ie ies o pu ple maize, such
as “Millo Co o”
46
om Spain and “Mo adyn”
38
om I aly,
ha e no been epo ed o exhibi he p esence o iden i ica ion
o hese condensed compounds, as a as cu en knowledge
ex ends. This dispa i y in he occu ence o la anol-
an hocyanin compounds could po en ially se e as a
dis inguishing ea u e be ween Ame ican and Eu opean
ge mplasms o pu ple maize.
3.2. Su i al o Polyphenols du ing In Vi o Diges ion
and Bioaccessibili y. 3.2.1. E ec o In Vi o Diges ion on
TPC and An ioxidan Ac i i y. The inc ease in esea ch aimed
a he s udy o polyphenols and hei heal hy p ope ies has
been a u ning poin in he ield o ood science. Nume ous
in es iga ions ha e been ca ied ou o de e mine he o al
con en , an ioxidan ac i i y, and composi ion o polyphenols
in a wide a ie y o oods. Howe e , i is impo an o s udy
how polyphenols ge h ough he diges i e p ocess as his is
undamen al o e alua ing hei heal h bene i s. The diges ion
p ocess induces changes in he ood composi ion, including
polyphenol con en and an ioxidan ac i i y, and dese es o be
in es iga ed, so a s udy o he aw ma e ial ( lou ) is a
undamen al s ep o p oducing ood p oduc s.
The impac o in i o diges ion o “Mo ag o” pu ple maize
lou on i s TPC and an ioxidan ac i i y is shown in Table 4.
The o al phase induces a signi ican modi ica ion o he
polyphenolic con en , as a es ed by eco e y a es o 57% by
he Folin−Ciocal eu me hod compa ed o hose o he
undiges ed ma ix (con ol). In he gas ic and in es inal
phases, he e a e no signi ican di e ences om he con ol.
The an ioxidan ac i i y showed a signi ican dec ease a e o al
phase diges ion conside ing bo h mechanisms wi h a emaining
ac i i y o 34% o DPPH and 53% o FRAP acco ding o a
dec ease in TPC du ing his in i o diges ion s ep. In con as
wi h TPC esul s, gas ic condi ions p oduced a signi ican
dec ease in an ioxidan p ope ies, which was obse ed
compa ed o hose o he undiges ed ma ix (Table 4). The
lowes an ioxidan ac i i y was ob ained in he gas ic phase,
wi h a eco e y a e o 38.8% o an i adical ac i i y and 6.1%
o educing powe . Finally, in he in es inal phase, 61.8% and
64.6% o he an i adical ac i i y and educing powe ,
espec i ely, we e e ained compa ed o hose o he con ol.
In he o al phase, TPC and an ioxidan ac i i y we e lowe
han hose in he undiges ed ma ix, al hough sali a helps wi h
polyphenol solubiliza ion, which subs an ially inc eases hei
a ailabili y.
47
O he ac o s such as he a ia ion o pH,
phenolic composi ion, and p esence o enzymes (in his case,
α-amylase)
48
a ec an ioxidan ac i i y. Du ing he gas ic
phase, he pH was he lowes in he in i o diges ion p ocess,
which could p o ec some polyphenols agains deg ada ion,
such as phenolic acids, la onols, and an hocyanins, while o he
polyphenols can be des oyed as he la onoids oligome s ha
deg ade o smalle uni s.
49
TPC did no show signi ican
di e ences du ing he gas ic phase compa ed wi h he con ol,
bu he an ioxidan ac i i y dec eased. This can be explained by
he ac ha p o ona ion o dep o ona ion eac ions can occu
when pH a ies, and his a ec s he oxida i e s a e and
p ope ies o he polyphenol’s compounds.
50
The high
eco e y a e o he TPC a e in i o diges ion ends o
indica e ha he e y la ge majo i y o he p oduc s a e s ill
p esen , maybe indica ing a libe a ion o he s uc u e o
polyphenols and, consequen ly, an inc ease in po en ially
bioaccessible compounds h ough in i o diges ion phases.
51
Howe e , he an ioxidan ac i i y was lowe a he end o
diges ion compa ed wi h ha o he undiges ed ma ix. This
ac could be mainly a ibu ed o he high pH in he in es inal
phase and he eac ions ha could occu such as
deglycosyla ion, glucu onida ion, me hyla ion, sulphona ion,
and hyd oxyla ion.
49
The esul s o ou s udy showed ha in
i o diges ion o pu ple maize lou a ec s an ioxidan ac i i y
bu e ains abou 60%, while he TPC emained wi hou
signi ican changes in compa ison o ha a he end o
diges ion wi h he undiges ed ma ix, so “Mo ag o” lou will
be an impo an aw ma e ial o elabo a ed p oduc s.
Despi e ex ensi e cha ac e iza ion, iden i ica ion, and
quan i ica ion o phenolic compounds o pu ple maize, only
a ew s udies ha e examined he e ec on compounds o pu ple
maize a e in i o diges ion.
38,43
Compa ed wi h Fe on e
al.
38
TPC, mainly an hocyanins and la onols, we e de ec ed
by RP-HPLC-UV, and a he end o he diges ion, all ma ke
compounds educed no ably, while in ou esul s, he TPC
be ween he undiges ed and diges ed pu ple maize ma ixes
did no di e signi ican ly.
An in e es ing s udy was conduc ed by Seczyk e al.
52
whe e
he e ec o in i o diges ion on he bioaccessibili y o TPC o
ce eal lou s (whea , du um whea , whole whea , yellow maize,
and whi e ice lou ) has been well s udied. In con as o ou
esul s, hey epo ed ha in i o diges ion and combina ion
wi h he ood ma ix had a nega i e e ec on he TPC
compa ed o hei ini ial amoun . Also, ou esul s con as
wi h hose epo ed by Mendez Lagunas e al.,
53
who ound a
signi ican inc emen o TPC in he in es inal phase o blue
Table 4. In luence o In Vi o Diges ion on TPC and An ioxidan Ac i i y
assay undiges ed ma ix o al phase gas ic phase in es inal phase
TPC (mg GAE/g DW) 2.71 ±0.04b1.54 ±0.03a2.30 ±0.10b2.60 ±0.30b
an isca enging ac i i y (μmol o T olox eq/g DW) 24.0 ±1.0c8.3 ±0.2a9.0 ±1.0a15.1 ±0.8b
educing powe (μmol o T olox eq/g DW) 22.0 ±1.0c11.7 ±0.6b1.4 ±0.4a14.0 ±2.0b
Di e en le e s wi hin a line indica e s a is ically signi ican di e ences in he DGS es (p< 0.05). DW: d y weigh ; GAE: gallic acid equi alen .
Jou nal o Ag icul u al and Food Chemis y pubs.acs.o g/JAFC A icle
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maize o illas, and he di e ence is due o he ac ha hei
p oduc was p e iously p ocessed and cooked, while ou s was
aw lou . Conce ning an ioxidan ac i i y, in pu ple lou ,
FRAP and ORAC alues inc eased a e in i o diges ion.
54
Fu he mo e, a simila beha io was epo ed in cooked whole-
whea pas a when he phenolic con en was highe and he
an ioxidan capaci y was lowe han hose in i s con ol.
55
The con adic o y esul s ound in he di e en epo s
con i m ha bioaccessibili y is in luenced by diges ion
condi ions, pH, empe a u e, enzymes used, as well as he
cha ac e is ics and composi ion o he ood ma ix, ex u e, and
he syne gis ic o an agonis ic e ec o he mac omolecules
wi h he bioac i e compounds.
53
Ano he pa ame e in luen is
he ype o polyphenol cons i uen s; some amilies o
polyphenols a e mo e esis an han o he s. Fo example,
p oan hocyanidins (ca echin-(4,8)-Cy-3,5-diGlu and ca echin-
(4,8)-Cy-3-MalGlu-5-Glu) ha e shown signi ican esis ance o
diges ion due o hei chemical s uc u e, which allows hem o
esis enzyma ic hyd olysis in he gas oin es inal ac .
Speci ically, he p esence o C−C and C−O−C la onoid
bonds in hei s uc u e allows hem o each he la ge in es ine
in ac , whe e hey can be e men ed by he gu mic obio a and
gene a e heal h-bene icial me aboli es. This esis ance o
diges ion may ha e implica ions o he bioa ailabili y and
physiological e ec s o hese compounds in he human body.
These condensed compounds ha e demons a ed g ea e
e en ion in he human gas oin es inal ac and, he e o e,
g ea e abso p ion in he human body compa ed o ha o
o he phenolic compounds
56
o an hocyanins ha ha e mo e
esis ance o in i o diges ion han ha o o he phenolic
compounds due o he p esence o glycosidic bonds in hei
chemical s uc u e. These bonds a e mo e di icul o hyd olyze
by diges i e enzymes compa ed o o he es e o ca bona e
bonds p esen in o he phenolic compounds. In addi ion, he
posi ion o hyd oxyl g oups in he molecula s uc u e o
an hocyanins may also con ibu e o hei esis ance o
enzyma ic diges ion.
57
3.2.2. E ec o In Vi o Diges ion on An hocyanin
Con en . As an hocyanins can be abso bed in ac despi e
hei di e en molecula sizes and ypes o suga o acyla ed
g oups a ached, he s a ic in i o diges ion me hod can
p o ide in e es ing in o ma ion abou hei bioaccessibili y.
An hocyanin ex ac s om each in i o diges ion phase
(o al, gas ic, and in es inal) we e analyzed by HPLC-DAD o
e alua e changes in he an hocyanin con en and p o ile.
The e ec o diges ion a each phase and on he di e en
an hocyanins can be seen in Figu e 2. Based on he HPLC−
QTOF esul s (Table 1), h ee di e en ma ke compounds
we e selec ed in he sample and moni o ed du ing diges ion.
Cyanidins we e he main compounds among an hocyanins,
and cyanidin-3-glucoside (peak 1), cyanidin-3-(6′malonylglu-
coside) (peak 6), and cyanidin-3-(3″,6″, dimalonylglucoside)
(peak 9) we e chosen because hey we e p esen a he end o
he diges ion and can be di e en ia ed om o he signals. In
gene al, a he end o he diges ion, i can be seen which
an hocyanins su i ed and which did no . The e is a clea
dec ease in he peaks o each compound owa d he end o he
diges ion, pa icula ly in peaks ha appea du ing he i s 17
min ha seem o disappea a he in es inal phase; Cy-3-Glu,
Cy-3-(6″MalGlu), and Cy-3-(3″,6″, diMalGlu) we e he majo
compounds a he end o he diges ion, wi h he Cy-3-
(6″MalGlu) being dominan .
The quan i ica ion o h ee cyanidin de i a i es is shown in
Figu e 3. The pe cen age eco e y o he ma ke an hocyanins
a each phase o in i o diges ion was calcula ed by compa ing
hei concen a ion a a pa icula diges ion phase wi h ha o
he undiges ed (con ol). In he o al phase, a dec ease in he
o al con en o he h ee cyanidin de i a i es was obse ed,
wi h he emaining 54% o cyanidin-3-glucoside (144.7 μg/g
DW), 71% o cyanidin-3-(6″malonylglucoside) (345.5 μg/g
Figu e 2. An hocyanin p o ile o Mo ag o lou a 520 nm in he o al phase (blue), gas ic phase ( ed), and in es inal phase (g een). Peak 1,
cyanidin-3-glucoside; Peak 6, cyanidin-3-(6′malonylglucoside); and Peak 9, cyanidin-3-(3″,6″, dimalonylglucoside).
Figu e 3. Ma ke an hocyanin con en o Mo ag o lou a 520 nm in
undiges ed ma ix-con ol (whi e), o al phase (g een), gas ic phase
(blue), and in es inal phase ( ed). Cy-3-Glu, Cy-3-(6″diMalGlu), and
Cy-3-(3″,6″, diMalGlu) ma ke s. Cy, cyanidin; Glu, glucoside; Mal,
malonyl; Pg, pela gonidin; Pn, peonidin.
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DW), and 65% o cyanidin-3-(3″,6″, dimalonylglucoside)
(155.5 μg/g DW). In con as , du ing he gas ic phase, an
inc ease o cyanidin-3-(6″malonylglucoside) compa ed o ha
o he con ol was ound, wi h a concen a ion o 436.6 μg/g
DW and p ese a ion o 90%, while cyanidin-3-(3″,6″,
dimalonylglucoside) p esen ed 219.8 μg/g DW, which
co esponds o 92%. Finally, he bioaccessible ac ion
ob ained a e he in es inal phase was 44.4 μg/g DW o
Cy-3-Glu, 130.7 μg/g DW o Cy-3-(6″MalGlu), and 57.1 μg/
g DW o Cy-3-(3″,6″, diMalGlu) ha co esponds o he
emaining pe cen age conce ning he con ol o 17, 27, and
24%, espec i ely. The h ee di e en ma ke an hocyanin
losses accoun ed o a ound 80%; despi e he deg ada ion o
an hocyanins, cyanidin-3-(6″malonylglucoside) emained he
mos p edominan compound among hose a ailable o
up ake. Impo an changes in he p o ile a e he in es inal
phase we e eco ded o he ma ke compounds; in pa icula ,
he concen a ion o each an hocyanin dec eased, con i ming
i s high ins abili y du ing he diges ion p ocess.
The esul s desc ibed a e in ag eemen wi h he da a
epo ed by Da id e al.,
58
who e alua ed he bioaccessibili y o
Co nelia che y an hocyanin. Indeed, his wo k epo ed ha
Co nelian che ies’ an hocyanins we e s able in he s omach,
and he duodenal diges ion d ama ically dec eased he o al
an hocyanin con en and an ioxidan capaci y le els in he ui
ex ac , as in ou s udy. Fe on e al.
38
ha e shown esul s ha
a e in line wi h ou s in a new I alian pu ple maize a ie y,
“Mo adyn” lou , inding an inc ease in he concen a ion o
an hocyanins a e he gas ic phase and a s ong educ ion
he ea e a he end o he in es inal phase.
The e ec o diges ion on he con en o pu ple ice
an hocyanins was explo ed by Sun e al.
59
In con as wi h ou
esul s, a he end o in i o gas oin es inal diges ion,
peonidin-3-glucoside emained he mos p edominan com-
pound.
As he esul s and epo ed da a show, an hocyanins a e
highly uns able and e y suscep ible o deg ada ion by oxygen,
empe a u e, enzymes, and pH, which a e some o he many
ac o s ha may a ec he chemis y o an hocyanins and,
consequen ly, hei s abili y, colo , and molecula s uc u e. As
known, an hocyanins a e s able in acidic solu ions (pH 1−3),
and his is impo an because hey a e exposed o di e en pH
condi ions h ough he gas oin es inal ac , which a ec s hei
bioa ailabili y and hence hei bioac i i y.
60
In e es ingly,
an hocyanins had he highes concen a ion in he gas ic
phase o in i o diges ion, which is posi i e because, in human
diges ion, an hocyanins could be abso bed in he s omach in
hei in ac o m. Mo eo e , in he in es inal phase, a sligh
ac ion o an hocyanins was ound despi e hei ins abili y a
his pH, and his is pa icula ly impo an because, in he
human diges i e sys em, an hocyanins could also be adso bed
in ac o could be b oken down in he la ge in es ine by he
ac ion o he mic obio a.
10
3.3. Bioac i i y Analysis. 3.3.1. Cell Viabili y Assay. The
iabili y o Caco-2 cells was e alua ed o de e mine he oxici y
o he diges ed ex ac s in cell cul u e and o de e mine he
bioac i i y o “Mo ag o” polyphenols. Figu e 4 shows he e ec
o bioaccessible po en ial a di e en dilu ions. A signi ican
di e ence was obse ed a 1:100 dilu ion be ween he
in es inal aliquo s o “Mo ag o” lou and he con ol, wi h
an inc ease in cell iabili y o 55.2% in he in es inal aliquo o
“Mo ag o” lou compa ed o ha o he con ol. A 1:250 and
1:500 dilu ions, signi ican di e ences we e obse ed wi h 43.9
and 29.3% o cell iabili y, espec i ely. The e o e, he e ec s
ound we e due o a p o ec i e e ec p o ided by he
compounds p esen in he “Mo ag o” lou since he cy o oxic
e ec o he con ol sample emains cons an and could be
explained due o he eagen s in ol ed in he in i o diges ion.
Despi e he low an ioxidan ac i i y a he end o in i o
diges ion, he esul s sugges ha he polyphenol compounds
emain bioac i e and p o ec he cells.
To ou knowledge, he e a e ew s udies wi h he cell model
a e in i o diges ion o pu ple maize.
25,61
Mo eo e , some o
hem used an an hocyanin ex ac , which esul ed in much
in o ma ion los , such as he in e ac ion o polyphenols wi h
ma ix compounds and he changes ha occu du ing
diges ion, and he bioa ailabili y o he compounds has no
been well s udied. U ias-Lugo e al.
25
analyzed he an i-
p oli e a i e ac i i y in di e en cells o an hocyanins and
phenolic acids om blue maize ex ac . Cell iabili y o cance
cells (Caco-2) epo ed iabili ies below 30%. Ano he s udy
was pe o med on blue maize ex ac s and e alua ed hei
an ip oli e a i e ac i i y in se e al cell lines, bu in i o
diges ion was no pe o med.
61
Fu u e pe spec i es could ocus on s udying he bioa ail-
abili y o phenolic compounds and hei heal h e ec s,
conside ing whole oods.
O e all, “Mo ag o” maize a ie y phenols ha e been
cha ac e ized in de ail. Showing ha his pu ple maize
cons i u es an excellen sou ce o phenolic compounds, some
o hem speci ic o his a ie y. A o al o wen y-six phenols,
15 nonan hocyanins and 11 an hocyanins we e ound. We
showed o he i s ime he p esence o py ogallol, ca eic
acid, kaemp e ol 3-O-glucu onide, kaemp e ol 3-(6″- e uloyl-
glucoside), ci ic acid, and gallic acid among nonan hocyanin
compounds in pu ple maize. The mos abundan phenol was
ca eic acid, ep esen ing 22.1% o he o al phenols iden i ied.
The pe cen age o an hocyanin compounds was 24.9%, and
cyanidin-3-(6″malonylglucoside) was he mos abundan
an hocyanin. These cha ac e is ic composi ions o “Mo ag o”
ep esen a bene icial ea u e due o he ac ha me hoxyla ed
de i a i es and condensed compounds p esen highe s abili y
and po en ial as colo an s. In addi ion, he “Mo ag o” cul i a
showed simila i ies in p oximal composi ion, an hocyanin
p o ile, and con en wi h o he pu ple maize cul i a s, as well
as in many nonan hocyanin cons i uen s. This may be due o
i s di ec p ogeni o a ie ies as he A gen inean pu ple maize
“Mo ag o” is de i ed om a ie ies descended om Mexico,
Pe u, Boli ia, and no he n A gen ina. This is suppo ed by he
p esence o condensed o ms o an hocyanin- la anol ha ha e
Figu e 4. Cell iabili y assays in Caco-2 o he con ol (whi e ba s)
and in es inal aliquo s (blue ba s) o Mo ag o lou . Di e en le e s
indica e s a is ically signi ican di e ences in he DGS es (p< 0.05).
Jou nal o Ag icul u al and Food Chemis y pubs.acs.o g/JAFC A icle
h ps://doi.o g/10.1021/acs.ja c.3c08960
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6335