RESEARCH ARTICLE
Ca o enoids www.mn -jou nal.com
Phy oene and Phy ofluene Isola ed om a Toma o Ex ac
a e Readily Inco po a ed in Mixed Micelles and Abso bed by
Caco-2 Cells, as Compa ed o Lycopene, and SR-BI is
In ol ed in hei Cellula Up ake
Paula Mapelli-B ahm, Cha les Desma chelie , Ma ielle Ma gie , Emmanuelle Reboul,
An onio J. Mel´
endez Ma ´
ınez, and Pa ick Bo el*
Scope: Abso p ion mechanisms o phy oene (PT) and phy ofluene (PTF) a e
poo ly known. The main objec i es o he s udy a e o measu e hei
micelliza ion and in es inal cell up ake efficiencies and o compa e hem o
hose o commonly consumed ca o enoids. O he objec i es a e o assess he
in ol emen o p o ein(s) in hei cellula up ake and whe he hey compe e
wi h o he ca o enoids o micelliza ion and cellula up ake.
Me hods and esul s: Toma o-ex ac -pu ified PT and PTF, mainly p esen as
cis-isome s, a e much be e inco po a ed in syn he ic mixed micelles han
pu e all- ans lycopene. PT impai s lycopene micelliza ion (−56%, P<0.05)
while PT and PTF do no significan ly affec he micelliza ion o o he
ca o enoids, and ice e sa. A low concen a ion, Caco-2 PTF up ake is highe
(P<0.05) han ha o PT and lycopene (29%, 21%, and no de ec able).
SR-BI, bu no CD36 nei he NPC1L1, is in ol ed in PT and PTF up ake. PT
and PTF impai (p<0.05) β-ca o ene up ake (−13 and −22%, espec i ely).
Conclusions: The high bioaccessibili y o PT and PTF can be pa ly explained
by hei high micelliza ion efficiency, which is likely due o hei na u al cis
isome iza ion and/o o hei high molecula flexibili y. SR-BI is in ol ed in
hei cellula up ake, which can explain compe i ions wi h o he ca o enoids.
1. In oduc ion
Phy oene (PT) and phy ofluene (PTF) a e ca o enes, i.e., non-
oxygena ed ca o enoids, which a e ound in a wide a ie y o
ui s and ege ables, e.g., in oma oes, ca o s, and ligh o -
ange ap ico s (a concen a ions o a ound 1.4 and 0.4; 1.4 and
0.6; and 7.2 and 2.4 mg/100 g edible po ion, o PT and PTF,
P. Mapelli-B ahm, D . A. J. Mel´
endez Ma ´
ınez
Food Colou & Quali y Lab.
A ea o Nu i ion & Food Science
Uni e sidad de Se illa
41012, Se ille, Spain
D .C. Desma chelie , M. Ma gie , D . E. Reboul, D . P. Bo el
C2VN, Aix Ma seille Uni , INRA, INSERM
Ma seille, F ance
E-mail: [email p o ec ed]
DOI: 10.1002/mn .201800703
espec i ely).[1,2] These ca o enoids con-
ain h ee (PT) and fi e (PTF) conju-
ga ed double bonds (CDB), while com-
monly consumed ca o enoids con ain a
leas en CDB (Figu e S1, Suppo ing In-
o ma ion). This p o ides hem wi h a
unique ea u e in he ca o enoid king-
dom: hey do no abso b isible ligh and
hus a e colo less o human.[3] In addi-
ion, hei lowe numbe o CDB gi es
hem a mo e wis ed shape compa ed
o commonly consumed ca o enoids,[4,5]
which has been sugges ed o affec hei
bioa ailabili y and biological ac ions.[2]
Mo eo e , i could also be expec ed ha
hei endency o oxida ion would be
lowe .[2]
PT and PTF a e eadily abso bed by
he human body, being ound in blood
and se e al issues.[6,7] They ha e ecen ly
ecei ed inc eased in e es because se -
e al s udies ha e ound posi i e associa-
ions be ween hei consump ion/blood
concen a ion and some heal h benefi s.
The in ake o PT and/o PTF could be
ela ed o an imp o emen o he immune sys em and a educ-
ion in he isk o de elop a ious diseases, including ce ain
cance s.[2,8,9] Mo eo e , se e al s udies ha e indica ed ha hey
could p o ec he skin agains UV-damage and p o ide cosme ic
benefi s.[10–13]
To each he bloods eam and hen a ge issues, ca o enoids
mus fi s be eleased om he ood ma ix in which hey a e
embedded and be inco po a ed in o mixed micelles.[14,15] PT and
PTF ha e been shown o exhibi highe bioaccessibili y han o he
ca o enoids p esen in he same ood ma ices.[16–19] Indeed, a
bioaccessibili y anking o ca o enoid species seems o eme ge
ega dless o he ood ma ix: PT and PTF >lu ein >β-ca o ene
>lycopene. Howe e , a ailable da a do no allow us o conclude
whe he he high bioaccessibili y o PT and PTF o igina es om
a highe ex ac ion efficiency om ood ma ices, due o spe-
cific in acellula localiza ion compa ed o o he ca o enoids,[20]
o om a highe in insic solubili y in mixed micelles,[21] due o
hei peculia chemical and physical p ope ies, o bo h. Once in
mixed micelles, i is assumed ha PT and PTF a e aken up by
Mol. Nu . Food Res. 2018,62, 1800703 C2018 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim1800703 (1 o 9)
www.ad ancedsciencenews.com www.mn -jou nal.com
en e ocy es, anspo ed o hei basola e al side, and inco -
po a ed in o chylomic ons be o e being sec e ed in o he
lymph.[14,15,22] These up ake and anspo p ocesses a e appa -
en ly e y efficien o he colo less ca o enoids because, e.g., PT
is a majo ca o enoid in a ious issues and i s bioa ailabili y
has been shown o be nea ly iple han ha o lycopene.[23] Ye ,
hei in es inal abso p ion mechanisms ha e no been s udied
and compa ed o hose o commonly s udied ca o enoids. Ne -
e heless, s udies in he las decade ha e allowed expe s in his
field o conclude ha en e ocy e up ake o commonly consumed
ca o enoids is no only passi e bu acili a ed by memb ane
p o eins.[24–27] Indeed, i has been shown ha CD36 molecule
(CD36) is in ol ed in cell up ake o p o i amin A ca o enoids[24]
and lu ein,[28] sca enge ecep o class B ype I (SR-BI) is in ol ed
in cell up ake o p o i amin A ca o enoids,[24,29] lycopene,[28] and
lu ein,[26] and NPC1 like in acellula choles e ol anspo e 1
(NPC1L1) is in ol ed in lu ein up ake.[30,31] Ye , i is no known
whe he any o hese p o eins a e in ol ed in cell up ake o he
colo less ca o enoids.
Ou main objec i e was o ob ain undamen al da a on wo
key s eps ha a e assumed o go e n he bioa ailabili y o he
colo less ca o enoids, i.e., micelliza ion and apical up ake by in-
es inal cells. Fo ha , we fi s measu ed he inco po a ion effi-
ciency o oma o ex ac pu ified PT and PTF in syn he ic mixed
micelles and compa ed i o ha o pu e commonly consumed
ca o enoids. We nex assessed whe he p e iously men ioned
p o eins in ol ed in up ake o commonly consumed ca o enoids
we e also in ol ed in ha o hese colo less ca o enoids. Finally,
because hese colo less ca o enoids migh be used as supple-
men s in he u u e, we assessed in all expe imen s whe he hey
compe e wi h he s udied commonly consumed ca o enoids.
2. Expe imen al Sec ion
2.1. Chemicals
PT and PTF (99.6 and 99.8% pu e as checked by HPLC) we e
isola ed om a oma o ex ac as desc ibed p e iously.[19] Pu-
ified PT con ained 96% o 15-cis-isome and 4% o all- ans-
isome and pu ified PTF con ained 94% o cis-isome s and
6% o all- ans-isome . No e ha in mos oods, human is-
sues, and biological fluids, PT and PTF a e expec ed o be
p esen as a mix u e o isome s, he cis isome s assumed o
be p edominan .[5,32] All- ans α-ca o ene, lycopene, and lu ein
(ࣙ95% pu e) we e a gi om DSM L d. (Basel, Swi ze -
land). All- ans β-ca o ene (ࣙ97% pu e), 2-oleoyl-1-palmi oyl-
sn-glyce o-3-phosphocholine (phospha idylcholine), 1-oleoyl- ac-
glyce ol (monoolein), 1-palmi oyl-sn-glyce o-3-phosphocholine
(lysophospha idylcholine), 3β-hyd oxy-5-choles ene ( ee choles-
e ol), oleic acid, and sodium au ochola e we e pu chased om
Sigma–Ald ich (Sain -Quen in-Falla ie , F ance). DMEM con-
aining 4.5 g L−1glucose, ypsin-EDTA (500 and 200 mg L−1,
espec i ely), nonessen ial amino acids, penicillin/s ep omycin,
and PBS we e pu chased om Li e Technologies (Illki ch,
F ance). Fe al bo ine se um (FBS) came om PAA (V´
elizy-
Villacoublay, F ance). Block lipid anspo -1 (BLT1), used as
chemical inhibi o o SR-BI, was pu chased om Sigma–
Ald ich. Eze imibe β-d-glucu onide, used as chemical inhibi o
o NPC1L1, was pu chased om Sequoia-Resea ch (Pangbou ne,
UK). Sul o-N-succin-imidyl olea e (SSO), used as chemical in-
hibi o o CD36, was syn hesized as p e iously published.[33]
2.2. P epa a ion o Ca o enoid-Rich Mixed Micelles
Mixed micelles con aining ca o enoids we e syn hesized as p e-
iously desc ibed,[34] wi h mino modifica ions. In summa y, sol-
en solu ions o ca o enoids we e fi s mixed wi h sol en so-
lu ions o micelle lipids and hen he mix u e was e apo a ed.
Then, DMEM con aining 5 mm sodium au ochola e was added
and mixed micelles we e syn hesized by sonica ion. The mixed
micelle ac ion was op ically clea and s o ed a −20 °C un il
Caco-2 cell expe imen s.
2.3. Micelliza ion Expe imen s
2.3.1. Measu emen o Ca o enoid Micelliza ion
The amoun o ca o enoids ha could be inco po a ed in he
mixed micelle ac ion was measu ed a h ee a ge ca o enoid
concen a ions, 0.5, 2, and 10 µm. These concen a ions a e ex-
pec ed o be ound in he human in es inal lumen a e ei he
low die a y, high die a y, o pha macological in ake o hese
ca o enoids. They we e es ima ed om a p e ious wo k[19] whe e
ca o enoid concen a ions we e measu ed in gas o-in es inal
fluid ollowing in i o diges ions o diffe en doses o hese com-
pounds.
2.3.2. P o ocol o S udy Compe i ions be ween Ca o enoids o
Micelliza ion
To s udy his compe i ion, he amoun o ca o enoid eco e ed
in mixed micelles was compa ed when only one ca o enoid was
added a 0.5 µm du ing mixed micelle syn hesis (con ol) o he
ca o enoid amoun eco e ed in micelles when 0.5 µm o ano he
ca o enoid was added o he p e ious one du ing mixed micelle
syn hesis.
2.4. Caco-2 Cell Expe imen s
2.4.1. Cul u e o Caco-2 Cells
Caco-2 clone TC-7 cells we e a gi om D . M. Rousse (UMR-
S872, Pa is, F ance). Cells we e hawed a passage numbe 70
o highe and we e cul u ed as p e iously desc ibed.[26] Be o e
3 weeks o each expe imen , he cells we e seeded on Millicell
hanging cell cul u e inse s (1 µm po e size polyca bona e mem-
b ane, Millipo e S.A.S., Molsheim, F ance) in six-well pla es a a
densi y o 25 ×104cells pe well o allow o diffe en ia ion. Be-
o e 12 h o each expe imen , media we e changed o FBS- ee
medium a bo h sides. Based on p elimina y esul s (Figu e S2,
Suppo ing In o ma ion) and unless o he wise s a ed, an incuba-
ion ime o 2 h was selec ed o he ollowing Caco-2 cells expe -
imen s.
Mol. Nu . Food Res. 2018,62, 1800703 C2018 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim1800703 (2 o 9)
www.ad ancedsciencenews.com www.mn -jou nal.com
2.4.2. P o ocol o E alua e he Maximal Amoun o Micella PT and
PTF ha could be Theo e ically Taken Up by Caco-2 Cells
The apical side o Caco-2 cell monolaye s ecei ed diffe en con-
cen a ions o micella PT and PTF, mo e p ecisely om 0.3 o
6.9 µm. The amoun o PT and PTF aken up by he cells was mea-
su ed a he end o he incuba ion ime (2 h). Qmax, which is he
maximal amoun o ca o enoid ha could be heo e ically aken
up by he cells, and appa en K, which is he micella ca o enoid
concen a ion a which up ake is hal he Qmax, we e calcula ed.
2.4.3. Compa ison o Up ake o Diffe en Ca o enoid Species by
Caco-2 Cells
Pu ified 0.5 µm PT o PTF, o pu e lu ein, lycopene, o β-ca o ene
was inco po a ed in mixed micelles and added o he apical side
o Caco-2 cells monolaye s. The amoun o ca o enoids aken up
by he cells was measu ed a e 2 h incuba ion.
2.4.4. Compe i ions be ween Micella Ca o enoids o hei Up ake
by Caco-2 Cells
Cells we e incuba ed wi h mixed micelles con aining one
ca o enoid oge he wi h mixed micelles con aining ei he no
ca o enoid (con ol) o ano he ca o enoid. These expe imen s
we e ca ied ou wi h micella ca o enoid concen a ions o abou
1µm.
2.4.5. Apical Efflux o Micella PT and PTF by Caco-2 Cells
Apical efflux was assessed as p e iously desc ibed.[34] Fi s , he
apical side o he cells was incuba ed du ing 4 h wi h ca o enoid-
ich mixed micelles ha con ained a ound 10 µm ca o enoids.
Cells we e hen washed wi h PBS and incuba ed o 15 min wi h
FBS- ee medium. Las ly, cells we e incuba ed du ing 30, 60, o
120 min wi h ca o enoid- ee mixed micelles a he apical side
and he amoun o ca o enoid eco e ed in he apical medium
was measu ed.
2.4.6. Effec o NPC1L1 and SR-BI Chemical Inhibi o s on micella
PT and PTF Up ake by Caco-2 Cells
Cells we e fi s p e-incuba ed wi h ei he 10 µmDMSO(con ol)
o 10 µm o he co esponding chemical inhibi o (eze imibe glu-
cu onide o NPC1L1 o BLT1 o SR-BI) o 1 h. The apical side
hen ecei ed 1 mL o ca o enoid- ich mixed micelles (a 1.4 µm
o PT o 1.2 µm o PTF) supplemen ed wi h ei he 10 µmDMSO
(con ol) o 10 µm o he co esponding chemical inhibi o while
he basola e al side ecei ed FBS- ee medium. The cellula up-
ake o ca o enoids was measu ed a e 2 h incuba ion.
2.5. HEK Cell Cul u e Expe imen s
To confi m p e ious esul s ob ained on he p o ein appa en ly
in ol ed, o no , in PT and PTF up ake by Caco-2 cells, and
o u he assess he po en ial in ol emen o CD36, which is
no exp essed in Caco-2 TC-7 cells,[35] up ake s udies in G ipTi e
cells, i.e., gene ically enginee ed Human Emb yonic Kidney cells
(HEK 293-T cells), we e pe o med.
HEK cells we e cul u ed and ans ec ed as p e iously
desc ibed.[24] Fo ans ec ion, 3 µg o DNA was used, i.e., emp y
pIRES plasmid o human CD36 in pIRES plasmid o s udy
he in ol emen o CD36; and emp y pCDNA3.1 plasmid o
human SCARBI in pCDNA3.1 plasmid o s udy he in ol e-
men o SR-BI. The ans ec ions we e checked by Wes e n blo
analysis.[33]
Ca o enoids in mixed micelles we e no used in hese ex-
pe imen s because bile sal s exe oxic effec s on HEK cells.
The e o e, he ca o enoids ehicles we e p epa ed as ollows:
fi s , ca o enoids in hexane we e inco po a ed in a glass ube
and, a e e apo a ion o he sol en , 6 µL o e hanol we e
added o acili a e he subsequen solubiliza ion o ca o enoids
in FBS. Then, 1.2 mL o FBS and 10.8 mL o DMEM we e
added and he final mix u e was o exed and sonica ed o wo
min.
Be o e each expe imen , ca o enoid concen a ion in he com-
ple e medium was analyzed by HPLC. Th ee condi ions we e
es ed: 1) HEK cells ans ec ed wi h he emp y plasmid (con-
ol condi ion), 2) HEK cells ans ec ed wi h a plasmid con ain-
ing ei he SCARB1, which encodes o SR-BI, o CD36,and3)
HEK cells ans ec ed wi h a plasmid con aining ei he SCARB1
o CD36 oge he wi h an inhibi o o he co esponding p o ein
(BLT1 a 10 µm o SSO a 400 µm,[36] espec i ely). Thus, he cells
ecei ed 1 mL o comple e medium in which 5 µm o ei he PT
o PTF was added, supplemen ed wi h ei he DMSO o he fi s
and second condi ions, o wi h he co esponding inhibi o o
he hi d condi ion. A e 3 h o incuba ion, ca o enoid concen-
a ion was measu ed in he media and he sc aped cells.
2.6. Ca o enoid Ex ac ion and HPLC Analysis
Ca o enoid ex ac ion was ca ied ou as p e iously desc ibed,[33]
using α-ca o ene as an in e nal s anda d. Ca o enoid ex ac s
we e e-dissol ed in 100 µL o e hyl ace a e and 10–80 µL we e
injec ed. HPLC analyses we e ca ied ou on a Dionex sys em,[37]
using a YMC-C30 column (5 µm, 4.6 ×250 mm) kep a 30 °C
wi h a YMC-C30 p e-column (5 µm, 10 ×4 mm). The mobile
phase consis ed o a mix u e o me hanol and me hyl e -bu yl
e he wi h an elu ion g adien ha was desc ibed p e iously.[37]
The quan ifica ion was pe o med by conside ing he da a ex-
ac ed a 286 (PT), 350 (PTF), 450 (β-ca o ene, lu ein, and α-
ca o ene), and 470 nm (lycopene), using Ch omeleon so wa e
( e sion 6.50 SP4 Build 1000, Dionex) and ex e nal calib a ion
cu es.
2.7. Calcula ions and S a is ics
Ca o enoid up ake efficiency by cells was exp essed as he pe -
cen age o ca o enoids eco e ed in he sc aped cells a he end
o he expe imen s ela i e o he sum o ca o enoids eco -
e ed in he apical chambe plus hose eco e ed in he sc aped
cells.
Mol. Nu . Food Res. 2018,62, 1800703 C2018 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim1800703 (3 o 9)
www.ad ancedsciencenews.com www.mn -jou nal.com
Table 1. Pa ame e s o phy oene and phy ofluene up ake by Caco-2 cells.
Ca o enoid Appa en Qmax (nmol) Appa en K(µm) R2
Phy oene 3.17 ±0.05a13.67 ±0.35a1.000
Phy ofluene 0.53 ±0.04b1.44 ±0.25b0.997
Caco-2 clone TC-7 cells we e hawed a passage numbe 67. Th ee weeks be o e each
expe imen , he cells we e seeded on cul u e inse s (1 µm po e size polyca bona e
memb ane) in six-well pla es a a densi y o a ound 25 ×104cells pe well o allow o
diffe en ia ion. Twel e hou s be o e each expe imen , media we e changed o FBS-
ee medium a bo h sides. Cells ecei ed 1 mL o ca o enoid- ich syn he ic mixed
micelles a a ound 0.5 µM on he apical side. Ca o enoid up ake was measu ed a e
2 h incuba ion. Resul s a e shown in Figu e 1B. Bes fi ing cu es we e hype bolic
ones: y=ax/(b +x). Appa en Qmax ep esen s he maximal amoun o ca o enoid
ha could be aken up by cells. Appa en Kis he micella ca o enoid concen a ion
a which he amoun aken up is hal he Qmax. Values ep esen means ±SEM o
h ee eplica es. Mean alues wi h unlike supe sc ip le e s wi hin a column we e
significan ly diffe en (p<0.05).
Table 2. Ca o enoid up ake by Caco-2 cells a a micella concen a ion co -
esponding o a low die a y in ake o ca o enoids.
Ca o enoid Up ake (%)
Phy oene 20.8 ±0.6b
Phy ofluene 28.9 ±1.2a
β-Ca o ene 30.6 ±0.7a
Lu ein 25.8 ±2.1ab
Caco-2 clone TC-7 cells we e hawed a passage numbe 92. Th ee weeks be o e each
expe imen , he cells we e seeded on cul u e inse s (1 µm po e size polyca bona e
memb ane) in six-well pla es a a densi y o abou 25 ×104cells pe well o allow o
diffe en ia ion. Twel e hou s be o e each expe imen , media we e changed o FBS-
ee medium a bo h sides. Cells ecei ed 1 mL o ca o enoid- ich syn he ic mixed
micelles a a ound 0.5 µm on he apical side. Ca o enoid up ake was measu ed a e
2 h incuba ion. Values ep esen mean ±SEM o h ee eplica es. Lycopene up ake
could no be accu a ely measu ed because i was lowe han he HPLC de ec ion limi .
Mean alues wi h unlike supe sc ip le e s we e significan ly diffe en (p<0.05).
Ca o enoid efflux efficiency by cells was calcula ed as he ela-
i e amoun o ca o enoid eco e ed in he apical medium a he
end o he expe imen compa ed o ha measu ed in he cells
a e 4 h incuba ion.
When micelliza ion and up ake expe imen s we e done using
he same mixed micelles han hose used o measu e ca o enoid
micelliza ion, he pe cen age o heo e ical bioa ailabili y o a
ca o enoid was calcula ed as: micelliza ion efficiency (%) ×up-
ake efficiency (%).
All expe imen s we e done in iplica e, excep hose o s udy
he implica ion o SR-BI and NPC1L1 in he up ake o PT and
PTF by Caco-2 cells, which we e pe o med on wo diffe en days
and included ou eplica es pe day. Resul s a e exp essed as
mean ±SEM.
S a is ical analyses we e pe o med using SPSS ( e sion 20,
SPSS Inc., Chicago, IL, USA) s a is ical package. Be o e S u-
den ’s - es o ANOVA, homogenei y o a iances was checked by
Le ene’s es and no mali y o dis ibu ions by Q-Q plo s. When
he F- es in ANOVA was significan , Tukey’s es was used as a
pos hoc es o pai wise compa isons, bu Dunne ’s es was
used when compa ing means om se e al expe imen al g oups
agains a single con ol g oup mean. Fo all es s, he bila e al
alpha isk was α=0.05.
Rela ionships be ween wo con inuous a iables we e exam-
ined by eg ession analysis on KaleidaG aph so wa e ( e sion
3.6, Syne gy so wa e, Reading, PA).
3. Resul s
3.1. Inco po a ion Efficiency o PT and PTF in Syn he ic Mixed
Micelles as Compa ed o ha o Commonly Consumed
Ca o enoids
Ma ked diffe ences in inco po a ion efficiency o he in es iga ed
ca o enoids we e obse ed (Figu e 1A). PT and lu ein displayed
he highes inco po a ion efficiencies, which we e linea o e
he h ee concen a ions es ed. PTF inco po a ion efficiency was
simila o ha o PT and lu ein up o abou 2 µm, i.e., high die a y
concen a ions, bu hen i appa en ly s a ed o pla eau when
he concen a ion inc eased. Lycopene exhibi ed he lowes in-
co po a ion efficiency wi h a maximum micella concen a ion
o 0.06 µm a all h ee concen a ions es ed.
3.2. Compe i ion be ween Colo less Ca o enoids and O he
Ca o enoids o Micelliza ion
Nei he did PT o PTF compe e o hei micelliza ion when hey
we e added concu en ly a 0.5 µm du ing mixed micelle syn he-
sis. The addi ion o 0.5 µm PTF du ing mixed micelle syn he-
sis did no significan ly impai lu ein o lycopene micelliza ion.
Conce ning PT, i s addi ion did no significan ly impai lu ein
micelliza ion whe eas i significan ly (p<0.05) impai ed ha o
lycopene (−55.6%). Finally, he inco po a ion efficiencies o PT
and PTF we e no significan ly affec ed by he simul aneous addi-
ion o lu ein, β-ca o ene, o lycopene du ing mixed micelle syn-
hesis (da a no shown).
3.3. Effec o he Concen a ion o Micella PT and PTF on hei
Up ake Efficiency by Caco-2 Cells
PT and PTF up ake by Caco-2 cells as a unc ion o hei micella
concen a ion ollowed hype bolic cu es (Figu e 1B). Thus, hei
up ake efficiency dec eased when hei micella concen a ion in-
c eased. Mo e p ecisely, PT up ake efficiency dec eased om 23.4
o 14.6% (a 0.3 and 6.9 µm, espec i ely) and ha o PTF om
32.0 o 14.8% (a 0.4 and 2.2 µm, espec i ely). Calcula ed Qmax
and Ko PT we e almos six- and en old highe han ha o PTF,
espec i ely (Table 1).
3.4. Compa ison o Ca o enoid Up ake by Caco-2 Cells
The up ake efficiency o lu ein, β-ca o ene, and PTF was no sig-
nifican ly diffe en (p=0.121). Con e sely, PT up ake efficiency
was significan ly lowe han ha o PTF and β-ca o ene (Table 2).
The up ake o lycopene was oo low o be accu a ely measu ed
and was he e o e ma kedly lowe han ha o he o he s udied
ca o enoids.
Mol. Nu . Food Res. 2018,62, 1800703 C2018 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim1800703 (4 o 9)
www.ad ancedsciencenews.com www.mn -jou nal.com
Figu e 1. Cha ac e iza ion o phy oene and phy ofluene micelliza ion and up ake by Caco-2 cells. A) Inco po a ion o phy oene, lu ein, phy ofluene, and
lycopene in syn he ic mixed micelles. Mixed micelles wi h a ying concen a ions o pu e ca o enoids we e syn hesized and hei ca o enoid concen a ion
was measu ed by HPLC. Linea end lines: Phy oene: y=0.874x(R2=0.999); lu ein: y=0.823x(R2=0.999). Cu ilinea end lines: Phy ofluene:
y=−0.0734x2+0.899x(R2=1.000); lycopene: y=−0.0008x2+0.0137x(R2=0.836). B) Effec o micella phy oene and phy ofluene concen a ions
on hei up ake by Caco-2 cells. The apical side o he cells ecei ed mixed micelles ha con ained diffe en concen a ions o phy oene and phy ofluene.
Basola e al side ecei ed FBS- ee medium. Ca o enoid concen a ions we e measu ed in sc aped cells a e 2 h incuba ion. The bes -fi cu es we e
hype bolic ones: y=ax/(x+b). Values ep esen means o h ee eplica es and e o ba s indica e s anda d e o o he mean.
3.5. Compe i ions be ween Micella Ca o enoids o hei Up ake
by Caco-2 Cells
The effec o he addi ion o ei he micella PT o PTF on he cel-
lula up ake o commonly consumed ca o enoids is shown in Fig-
u e 2A and B. Micella lu ein up ake was no significan ly affec ed
by he addi ion o ei he micella PT o PTF (Figu e 2A). Con-
e sely, micella β-ca o ene up ake was significan ly impai ed by
PT and PTF (–12.9 and –21.6%, espec i ely) (Figu e 2B). The
effec o he addi ion o ei he micella PT o PTF on lycopene
up ake is no shown because i could no be accu a ely measu ed
due o he e y low amoun o lycopene aken up by he cells.
The effec o he addi ion o he o he s udied ca o enoids on
PT and PTF up ake by Caco-2 cells is shown in Figu e 2C and D.
The up ake o micella PT was significan ly impai ed when micel-
la PTF, β-ca o ene, o lu ein we e added in he apical chambe
(−30.8, −52.4, and −27.8%, espec i ely, p<0.001; Figu e 2C).
Con e sely, only micella lu ein significan ly impai ed micella
PTF up ake (–40%, p<0.001) (Figu e 2D).
3.6. Apical Efflux o PT and PTF by Caco-2 Cells
The apical efflux o PT and PTF ollowing hei apical up ake
was no significan ly diffe en , i.e., a ound 14 ±2% o bo h
ca o enoids (p=0.649), and i did no significan ly a y om 30
o 120 min (da a no shown).
3.7. Effec o NPC1L1 and SR-BI Chemical Inhibi o s on Micella
PT and PTF Up ake by Caco-2 Cells
Eze imibe glucu onide, a chemical inhibi o o NPC1L1, did no
significan ly affec PT o PTF up ake (Figu e 3A). Con e sely,
up ake o PT and PTF was significan ly dec eased (−76.9 and
−85.4%, espec i ely, p<0.001) when BLT1, a chemical inhibi o
o SR-BI was added o he apical medium.
3.8. Effec o T ans ec ion o Memb ane P o eins on Micella PT
and PTF Up ake by HEK Cells
PT and PTF up ake was significan ly highe in HEK cells
ans ec ed wi h SCARB1, which encodes o SR-BI, han
in HEK cells ans ec ed wi h an emp y plasmid (p<0.01
and p<0.05 o PT and PTF, espec i ely). Fu he mo e,
he addi ion o BLT1 o he SCARB1 ans ec ed cells led
o abolish he highe up ake obse ed in hese cells (Fig-
u e 3B). In addi ion, ans ec ion o HEK cells wi h CD36 did
no significan ly change hei PT and PTF up ake efficiency
(da a no shown).
4. Discussion
This s udy was based on he hypo hesis ha he high bioa ail-
abili y o PT and PTF ela i e o ha o o he ca o enoids ound
in he same ood ma ices is due, a leas in pa , o hei peculia
molecula p ope ies, which could lead o highe solubili y in
mixed micelles and/o o highe up ake efficiency by in es inal
cells. To e i y his hypo hesis, we fi s pu ified PT and PTF
om oma o ex ac and we compa ed hei micelliza ion
and hei cellula up ake efficiency o ha o pu e commonly
consumed ca o enoids. We obse ed ha PT and PTF possess
a much highe in insic abili y o be inco po a ed in o syn he ic
mixed micelles han lycopene, ano he linea non-oxygena ed
ca o enoid. In ac , a low and high die a y concen a ions, i.e.,
0.5 and 2.0 µm, hei micelliza ion efficiency was simila o ha o
Mol. Nu . Food Res. 2018,62, 1800703 C2018 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim1800703 (5 o 9)

www.ad ancedsciencenews.com www.mn -jou nal.com
Figu e 2. Compe i ions be ween micella ca o enoids o hei up ake by Caco-2 cells. A) Effec o phy oene and phy ofluene on lu ein up ake. B) Effec
o phy oene and phy ofluene on β-ca o ene up ake. C) Effec o phy ofluene and commonly consumed ca o enoids on phy oene up ake. D) Effec o
phy oene and commonly consumed ca o enoids on phy ofluene up ake. The apical side o he cells ecei ed 1 mL o mixed micelles ha con ained
he ca o enoid o in e es plus ei he ca o enoid- ee mixed micelles (con ol) o mixed micelles loaded wi h ano he ca o enoid species. The a ge
micella concen a ion o each ca o enoid in each compe i ion condi ions was 1 µm. Ca o enoid up ake was measu ed a e 2 h incuba ion. The effec
o phy oene and phy ofluene on lycopene up ake could no be accu a ely measu ed because lycopene up ake was oo low o be accu a ely measu ed in
ou expe imen al condi ions. Values ep esen mean o h ee eplica es and e o ba s indica e SEM. As e isks indica e significan diffe ences om he
con ol (abso p ion o he ca o enoid o in e es alone): *p<0.05; ***p<0.001.
Figu e 3. Implica ion o NPC1L1 and SR-BI on phy oene and phy ofluene up ake by cells. A) Effec o chemical inhibi o s o NPC1L1 and SR-BI on
phy oene and phy ofluene up ake by Caco-2 cells. Cell apical sides we e p e-incuba ed o 1 h wi h ei he 10 µmDMSO(con ol)o 10µM chemical
inhibi o (eze imibe glucu onide o NPC1L1 o BLT1 o SR-BI). Apical sides ecei ed he ea e phy oene- o phy ofluene-loaded syn he ic mixed micelles
a 1.4 and 1.2 µm, espec i ely. Ca o enoid up ake was assessed a e 2 h incuba ion. The expe imen was ca ied ou wice, wi h 4 eplica es in each
case. This figu e shows esul s o one expe imen . As e isks indica e significan diffe ences om he con ol (***p<0.001). B) Effec o ans ec ion o
HEK cells wi h SR-BI gene and u he addi ion o SR-BI chemical inhibi o on phy oene and phy ofluene up ake by hese cells. Cells we e fi s ans ec ed
wi h ei he an emp y plasmid (con ol) o a plasmid con aining SCARB1, i.e., he gene encoding he SR-BI p o ein. Then cells ecei ed comple e medium
en iched wi h ei he micella phy oene o phy ofluene a 5 µm, supplemen ed o no wi h 10 µm DMSO o BLT1 ( he chemical inhibi o o SR-BI).
Incuba ion ime was 3 h. Fo each ca o enoid ba s bea ing unlike supe sc ip le e s a e significan ly diffe en (p<0.05). In each figu e, alues ep esen
means o h ee eplica es and e o ba s indica e SEM.
Mol. Nu . Food Res. 2018,62, 1800703 C2018 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim1800703 (6 o 9)
www.ad ancedsciencenews.com www.mn -jou nal.com
lu ein, which is an oxygena ed ca o enoid acknowledged o ha e
a highe micelliza ion efficiency compa ed o ca o enes.[38,39] We
hypo hesize ha his high in insic solubili y in mixed micelles
is due ei he o he ac ha PT and PTF we e mainly p esen in
he o m o cis-isome s, which is simila o hei isome iza ion
s a us in oods, and/o o he ac ha hese ca o enoids ha e a
highe molecula flexibili y han he o he s udied ca o enoids.
Conce ning he fi s hypo hesis, al hough i is no known
whe he he cis-isome s o PT and PTF ha e highe solubili y in
micelles han hei espec i e all- ans isome s, we hypo hesize
ha his is e y likely because his has been shown o ano he
linea ca o ene, i.e., lycopene.[39–41] Conce ning he second
hypo hesis, i has been shown ha , due o hei lowe numbe
o CDB (Figu e S1, Suppo ing In o ma ion), PT and PTF can
old mo e eely and adop less igid shapes han commonly
consumed ca o enoids.[32] Fu he mo e, he highe numbe o
sigma bonds in hese molecules, whe e o a ion is possible,[42]
leads o a mo e p onounced wis in he backbone o hese
molecules.[4,5] In ac , o sional ene gies o he linea ca o enoids
in es iga ed ank as ollows: PT (ࣈ57 kcal mol−1,3CDB)<
PTF (ࣈ61 kcal mol−1,5CDB)<lycopene (ࣈ73 kcal mol−1,11
CDB).[32] This highe flexibili y and wis abili y a e assumed
o ansla e in o be e inse ion o hese ca o enoids be ween
lipid molecules composing mixed micelles. Howe e , we canno
conclude whe he he high bioaccessibili y o PT and PTF is due
o hei cis-isome iza ion, o hei high molecula flexibili y, o
bo h. Second, we s udied he up ake o PT and PTF by in es inal
cells. The fi s key obse a ion was ha hei up ake efficiency
was much highe han ha o lycopene. In ac , he up ake
efficiency o PTF was equi alen o ha o lu ein and β-ca o ene.
The second key obse a ion was ha he sa u able up ake o PT
and PTF s ongly sugges ed a p o ein-media ed up ake. Ano he
in e es ing obse a ion was ha PT up ake was highe han ha
o PTF a high die a y concen a ions, i.e. >2µm(Figu eS2,
Suppo ing In o ma ion), while i was lowe a low die a y
concen a ions (Table 2). This effec o he colo less ca o enoid
concen a ion on hei ela i e cellula up ake efficiency was
unexpec ed bu i was in ag eemen wi h a p e ious s udy.[43] In
his clinical s udy, he bioa ailabili y o PTF was highe han ha
o PT in he g oup o subjec s who inges ed he lowes concen a-
ions o colo less ca o enoids (ࣈ0.9 mg o PT and PTF pe day o
12 weeks), while i was he opposi e in he g oup who inges ed
he highes concen a ions (4.6 mg o PT and 3.2 mg o PTF
pe day o 12 weeks). We hypo hesize ha his concen a ion
effec can be due o diffe ences be ween PT and PTF ega ding
hei ela i e affini y o memb ane anspo e (s). Indeed, he
highe appa en Qmax o PT,ascompa ed o ha o PTF,couldbe
explained by he hypo hesis ha PTF possesses a highe affini y
o he main anspo e o hese colo less ca o enoids han PT.
This las hypo hesis is suppo ed by i s lowe appa en Kand
by he ac ha PTF significan ly inhibi ed PT up ake while
he opposi e was no obse ed. The pe cen ages o heo e ical
bioa ailabili y o PT and PTF a 0.5 µm, i.e., a a low die a y
concen a ion, we e 18.3 and 26.1%, espec i ely, which was in
ag eemen wi h he esul s ob ained in he p e iously men ioned
s udy.[43]
A e ob aining esul s ha sugges ed he colo less ca o enoid
up ake is p o ein media ed, we e alua ed whe he p o eins ha
ha e been shown o pa icipa e in he up ake o commonly con-
sumed ca o enoids, i.e., SR-BI, CD36, and NPC1L1,[27] a e also
in ol ed in PT and PTF up ake. O e all, ou esul s sugges ha
SR-BI is in ol ed in he up ake o PT and PTF while CD36 and
NPC1L1 a e no . The in ol emen o SR-BI is in ag eemen wi h
he esul s ob ained o o he ca o enes, i.e., lycopene and β-
ca o ene.[24,25,44] The lack o in ol emen o CD36 sugges s ha
his p o ein is mo e specifically associa ed wi h he up ake o
p o i amin A ca o enoids.[24] Ano he in e es ing finding was
ha abou 14% o PT and PTF aken up by he cells was appa -
en ly effluxed back o hei apical side. This is consis en wi h p e-
ious da a sugges ing ha o he a -soluble mic onu ien s, such
as ocophe ol, cholecalci e ol, o phylloquinone,[33,45,46] a e pa -
ially effluxed by Caco-2. A e ha ing ob ained key in o ma ion
on he mechanisms implica ed in abso p ion o hese ca o enoids
and because hese phy ochemicals migh be used in he u u e as
die a y supplemen s, whe he hey exhibi demons a ed benefi s
o heal h, we assessed whe he hey compe e wi h commonly
consumed ca o enoids o ei he hei micelliza ion o in es inal
cell up ake. Indeed, significan compe i ions a hese key s eps o
ca o enoid abso p ion could lead o a dec ease in abso p ion o
ca o enoids ha possess well-acknowledged heal h effec s, e.g.,
β-ca o ene and lu ein. Conce ning micelliza ion, only one com-
pe i ion was obse ed, i.e., PT significan ly impai ed lycopene
micelliza ion. I seems logical o obse e ha he ca o enoid ha
has he highes abili y o be inco po a ed in mixed micelles sig-
nifican ly impai ed he micella inco po a ion o he one ha has
he lowes abili y o be inco po a ed in micelles. Conce ning cel-
lula up ake, ou esul s sugges ha PT and PTF can pa ially
impai he in es inal up ake o β-ca o ene, and ice e sa. This
is in ag eemen wi h p e ious esul s showing ha commonly
consumed ca o enoids compe e o hei in es inal up ake,[47]
and his is likely explained by he ac ha all hese ca o enoids
sha e a leas one common memb ane anspo e , in ha case
SR-BI.
In summa y, his s udy has p o ided esul s allowing us o
sugges why he bioaccessibili y o PT and PTF is unexpec -
edly high as compa ed o ha o he o he main linea die a y
ca o ene, i.e., lycopene. Indeed, his is likely because hese colo -
less ca o enoids a e p esen mainly as cis-isome s in oods and/o
because o hei high molecula flexibili y. This s udy has also
p o ided us da a sugges ing ha SR-BI, which is in ol ed in up-
ake o commonly consumed ca o enoids, is also in ol ed in cel-
lula up ake o PT and PTF, which in u n explains compe i ions
o cellula up ake. We ha e also ob ained da a sugges ing ha a
ac ion o abso bed PT and PTF is effluxed back o he in es inal
lumen. We acknowledge some limi a ions o his s udy. Fi s , we
compa ed micelliza ion o mainly cis-isome s o PT and PTF wi h
ha o all- ans isome s o commonly consumed ca o enoids.
Thus, we canno conclude ha all- ans PT and PTF a e be e
inco po a ed in micelles han all- ans common ca o enoids. Ne -
e heless, his would ha e a low in e es o nu i ionis s because
hese colo less ca o enoids a e na u ally p esen in oods as cis-
isome s. Ye , his could be o in e es o people who would like o
chemically syn hesize hese compounds. The second main limi-
a ion is ha we did no use an in i o diges ion model o assess
bioaccessibili y. Ne e heless, his model was used in p e ious
s udies, and ou aim was o go u he by ob aining da a on he
in insic solubili y o hese ca o enoids in micelles o unde s and
why hey a e so bioaccessible.
Mol. Nu . Food Res. 2018,62, 1800703 C2018 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim1800703 (7 o 9)
www.ad ancedsciencenews.com www.mn -jou nal.com
Suppo ing In o ma ion
Suppo ing In o ma ion is a ailable om he Wiley Online Lib a y o om
he au ho .
Acknowledgemen s
P.M.B. and C.D. a e join fi s au ho s. P.B. designed he esea ch p ojec
wi h P.M.B., C.D., and A.M.M.; P.B., C.D., and E.R. designed he p o o-
col; P.M.B. and V.M. conduc ed he micelliza ion s udies; P.M.B., M.M.,
V.M., and M.N. conduc ed he cell s udies; P.M.B. and C.H. measu ed
ca o enoid concen a ions by HPLC; PMB analyzed he esul s wi h C.D.
and E.R.; P.M.B. and C.D. pe o med s a is ical analyses; P.B., P.M.B.,
and C.D. w o e he pape wi h consul a ion om E.R.; P.B. had p i-
ma y esponsibili y o he final con en o he manusc ip . All au-
ho s ha e ead and app o ed he final manusc ip . The au ho s would
like o hank Vincen Migno , Cha lo e Halimi, and Ma ion Nowicki
(C2VN, Ma seille) o aluable echnical assis ance. P.M.B. ecei ed und-
ing o ca y ou a Sho -Te m Scien ific Mission a PB’s human mi-
c onu i ion eam in Ma seille om he Eu opean COST Ac ion EU-
ROCAROTEN (CA15136, Eu opean ne wo k o ad ance ca o enoid e-
sea ch and applica ions in ag o- ood and heal h, www.eu oca o en.eu,
h p://www.cos .eu/COST_Ac ions/ca/CA15136).
The Ca no S a Ins i u e p o ided unding o consumables.
A.M.M. and P.M.B. acknowledged unding om he Andalusian Coun-
cil o Economy, Inno a ion, Science and Employmen (p ojec e .
CAROTINCO-P12-AGR-1287).
Conflic o In e es
A.M.M. is a membe o he ad iso y boa d o IBR-Is aeli Bio echnology
Resea ch, L d. (Ya ne, Is ael).
Keywo ds
bioaccessibili y, bioa ailabili y, ca o enoids, lu ein, β-ca o ene
Recei ed: July 16, 2018
Re ised: Augus 29, 2018
Published online: Sep embe 24, 2018
[1] E. Biehle , A. Alke wi, L. Hoffmann, E. K ause, M. Guillaume, M.-L.
Lai , T. Bohn, J. Food Compos. Anal. 2012,25, 56.
[2] A. J. Mel´
endez-Ma ´
ınez, P. Mapelli-B ahm, A. Ben´
ı ez-Gonz´
alez, C.
M. S inco, A ch. Biochem. Biophys. 2015,572, 188.
[3] D. B. Rod iguez-Amaya, A Guide o Ca o enoid Analysis in Foods,ILSI
P ess, Washing on, D.C. 2001.
[4] I. T. Lima, L. Sousa, R. da S. F ei as, L. A. Ribei o Junio , R. T. de Sousa
Junio , D. A. da Sil a Filho, J. Mol. Model. 2017,23,1.
[5] A. J. Mel´
endez-Ma ´
ınez,C.M.S inco,C.Liu,X.-D.Wang,Food Chem.
2013,138, 1341.
[6] F. Khachik, L. Ca alho, P. S. Be ns ein, G. J. Mui , D.-Y. Zhao, N. B.
Ka z. Exp. Biol. Med. 2002,227, 845.
[7] J. K. Campbell, N. J. Engelmann, M. A. Lila, J. W. E dman. Nu . Res.
2007,27, 794.
[8] N. J. Engelmann, S. K. Clin on, J. W. J. E dman. Ad . Nu . 2011,2, 51.
[9] A.J.Mel
´
endez-Ma ´
ınez, P. Mapelli-B ahm, C. M. S inco, J. Food Com-
pos. Anal. 2018,67, 91.
[10] L. on Oppen-Bezalel, D. Fishbein, F. Ha as, O. Ben-Chi i , A. Khaia ,
Glob. De ma ol. 2015,2, 178.
[11] M. M. Ma hews-Ro h, M. A. Pa hak, Pho ochem. Pho obiol. 1975,21,
261.
[12] M. M. Ma hews-Ro h, Oncol. 1982,39, 33.
[13] L. on Oppen-Bezalel, F. Ha as, O. Ramo , E. Kalo, D. Fishbein, O.
Ben-Chi i , SOFW J. 2014,140,8.
[14] C. Desma chelie , P. Bo el, T ends Food Sci. Technol. 2017,69, 270.
[15] T. Bohn, C. Desma chelie , L. O. D ags ed, C. S. Nielsen, W. S ahl, R.
R¨
uhl, J. Keije , P. Bo el, Mol. Nu . Food Res. 2017,61,1.
[16] D. B. Rod igues, C. Chi chum oonchokchai, L. R. B. Ma iu i,
A. Z. Me cadan e, M. L. Failla, J. Ag ic. Food Chem. 2017,65,
11220.
[17] P. Mapelli-B ahm, C. M. S inco, M. J. Rod igo, L. Zaca ´
ıas, A. J.
Mel´
endez-Ma ´
ınez. J. Func . Foods 2018,46, 38.
[18] J. L. Jeffe y, N. D. Tu ne , S. R. King. J. Sci. Food Ag ic. 2012,92, 2603.
[19] P. Mapelli-B ahm, J. Co e-Real, A. J. Mel´
endez-Ma ´
ınez, T. Bohn,
Food Chem.2017,229, 304.
[20] R. M. Schweigge , R. Ca le. C i .Re .FoodSci.Nu .2017,57, 1807.
[21] C. Sy, B. Gleize, O. Dangles, J. F. Land ie , C. C. Vey a , P. Bo el. Mol.
Nu . Food Res. 2012,56, 1385.
[22] E. Reboul, Nu ien s 2013,5, 3563.
[23] N. E. Mo an, S. K. Clin on, J. W. E dman. J. Nu . 2013,143, 1920.
[24] P. Bo el, G. Lie z, A. Goncal es, F. Szabo de Edelenyi, S. Lecomp e,
P. Cu is, L. Goumidi, M. J. Caslake, E. Miles, C. Packa d, P. Calde , J.
C. Ma he s, A. M. Minihane, F. Tou niai e, E. Kesse-Guyo , P. Galan,
S. He cbe g, C. B eidenassel, M. Gonz´
alez G oss, M. Moussa, A.
Mei haeghe, E. Reboul, J. Nu . 2013,143, 448.
[25] M. Moussa, J.-F. Land ie , E. Reboul, O. Ghi inghelli, C. Com´
e a, X.
Colle , K. F ¨
ohlich, V. B¨
ohm, P. Bo el, J. Nu .2008,138, 1432.
[26] E. Reboul, L. Abou, C. Mikail, O. Ghi inghelli, M. And ´
e, H. Po ugal,
D. Jou dheuil-Rahmani, M.-J. Amio , D. Lai on, P. Bo el, Biochem. J.
2005,387, 455.
[27] E. Reboul, P. Bo el. P og. Lipid Res. 2011,50, 388.
[28] M. Moussa, E. Gou an on, B. Gleize, C. El. Yazidi, I. Nio , M. Moussa,
E. Gou an on, B. Gleize, C. El. Yazidi, P. Besna d, P. Bo el, J.-F. Lan-
d ie , Mol. Nu . Food Res. 2011,55, 578.
[29] A. Van Bennekum, M. We de , S. T. Thuahnai, C. H. Han, P. Duong,
D. L. Williams, P. We s ein, G. Schul hess, M. C. Phillips, H. Hause ,
Biochemis y 2005,44, 4517.
[30] A. Du ing, H. Dawson, E. Ha ison, J. Nu . 2005,135, 2305.
[31] Y. Sa o, R. Suzuki, M. Kobayashi, S. I agaki, T. Hi ano, T. Noda, S.
Mizuno, M. Sugawa a, K. Iseki. J. Pha m. Pha m. Sci. 2012,15, 256.
[32] A. J. Mel´
endez-Ma ´
ınez, M. Paulino, C. M. S inco, P. Mapelli-B ahm,
X.-D. Wang, J. Ag ic. Food Chem. 2014,62, 12399.
[33] E. Reboul, A. Goncal es, C. Come a, R. Bo , M. Nowicki, J. F. Land ie ,
D. Jou dheuil-Rahmani, C. Du ou , X. Colle , P. Bo el, Mol. Nu . Food
Res. 2011,55, 691.
[34] C. Desma chelie , M. Ma gie , D. P. P ´
e ´
e aud, M. Nowicki, V. Rosilio,
P. Bo el, E. Reboul, Nu ien s 2017,9, E1152.
[35] M. We de , C. H. Han, E. Weh li, D. Bimmle , G. Shul hess, H.
Hause , Biochemis y 2001,40, 11643.
[36] S. Coo , J. Willems, W. Coumans, G. Vusse an de , A. Bonen, J. F.
Gla z, J. J. Luiken, Biochem.2002,239, 213.
[37] B. Gleize, M. S eib, M. And ´
e, E. Reboul, Food Chem.2012,134, 2560.
[38] L. Lemmens, I. Colle, S. Van Buggenhou , P. Palme o, A. Van Loey,
M. Hend ickx, T ends Food Sci. Technol. 2014,38, 125.
[39] Q. Sun, C. Yang, J. Li, H. Raza, Lne. Zhang, J. Food Sci. 2016,81,
C2381.
[40] M. L. Failla, C. Chi chum oonchokchai, B. K. Ishida, J. Nu . 2008,138,
482.
[41] J. L. Coope s one, R. A. Rals on, K. M. Riedl, T. C. Hau e, R. M.
Schweigge , S. A. King, C. D. Timme s, D. M. F ancis, G. B. Lesinski,
S. K. Clin on, S. J. Schwa z, Mol. Nu . Food Res. 2015,59, 658.
[42] G. B i on, S. Liaaen-Jensen, H. P ande , Ca o enoids, Vol. 4,
Bi kh¨
ause , Basel, Swi ze land 2008.
Mol. Nu . Food Res. 2018,62, 1800703 C2018 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim1800703 (8 o 9)
www.ad ancedsciencenews.com www.mn -jou nal.com
[43] O. Aus , W. S ahl, H. Sies, H. T onnie , U. Hein ich, In . J. Vi am. Nu .
Res. 2003,75, 54.
[44] A. Du ing, E. Ha ison, J. Lipid Res. 2007,48, 2283.
[45] A. Goncal es, M. Ma gie , S. Roi, X. Colle , I. Nio , P. Goupy, C. Ca is-
Vey a , E. Reboul, J. Biol. Chem. 2014,289, 30743
[46] E. Reboul, A. Klein, F. Bie ix, B. Gleize, C. Maleze -Desmoulins, M.
Schneide , A. Ma go a , L. Lag os , X. Colle , P. Bo el, J. Biol. Chem.
2006,281, 4739.
[47] E. Reboul, S. Thap, F. Tou niai e, M. And ´
e, C. Juhel, S. Mo ange, M.
J. Amio , D. Lai on, P. Bo el, J. Nu . 2007,97, 440.
Mol. Nu . Food Res. 2018,62, 1800703 C2018 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim1800703 (9 o 9)