Individual and Joint Effect of Alpha-Tocopherol and Hydroxytyrosol Acetate on the Oxidation of Sunflower Oil Submitted to Oxidative Conditions: A Study by Proton Nuclear Magnetic Resonance

Ci a ion: Caño-Ochoa, S.d.;
Ruiz-A acama, A.; Guillén, M.D.
Indi idual and Join E ec o
Alpha-Tocophe ol and
Hyd oxy y osol Ace a e on he
Oxida ion o Sun lowe Oil
Submi ed o Oxida i e Condi ions:
A S udy by P o on Nuclea Magne ic
Resonance. An ioxidan s 2022,11,
1156. h ps://doi.o g/10.3390/
an iox11061156
Academic Edi o : Daniel F anco Ruiz
Recei ed: 27 May 2022
Accep ed: 10 June 2022
Published: 13 June 2022
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Licensee MDPI, Basel, Swi ze land.
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A ibu ion (CC BY) license (h ps://
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an ioxidan s
A icle
Indi idual and Join E ec o Alpha-Tocophe ol and
Hyd oxy y osol Ace a e on he Oxida ion o Sun lowe Oil
Submi ed o Oxida i e Condi ions: A S udy by P o on Nuclea
Magne ic Resonance
So ía del Caño-Ochoa, Ainhoa Ruiz-A acama and Ma ía D. Guillén *
Food Technology, Facul y o Pha macy, Lasca ay Resea ch Cen e , Uni e si y o he Basque Coun y (UPV-EHU),
Paseo de la Uni e sidad n 7, 01006 Vi o ia-Gas eiz, Spain; [email p o ec ed] (S.d.C.-O.);
[email p o ec ed] (A.R.-A.)
*Co espondence: ma iadolo [email p o ec ed]; Tel.: +34-945-013081; Fax: +34-945-013014
Abs ac :
This s udy ackles he indi idual and join e ec o alpha- ocophe ol and hyd oxy y osol
ace a e on he oxida ion o sun lowe oil submi ed o accele a ed s o age condi ions a in e media e
empe a u e, in o de o deepen he unde s anding o an ioxidan –p ooxidan beha iou . This was
accomplished by
1
H Nuclea Magne ic Resonance. Fo his pu pose, he e olu ion o he deg ada ion
o bo h he main componen s o he oil and he a o emen ioned added compounds was moni o ed
by his echnique h oughou he s o age ime. Fu he mo e, he o ma ion o a e y la ge numbe
o oxylipins and he e olu ion o hei concen a ion up o a e y ad anced s age o oil oxida ion,
as well as he occu ence o lipolysis, we e also simul aneously s udied. The esul s ob ained show
e y clea ly and ho oughly ha in he oxida ion p ocess o he oil en iched in bina y mix u es,
in e ac ions occu be ween alpha- ocophe ol and hyd oxy y osol ace a e ha no ably educe he
an ioxidan e ec o he la e compound wi h he co esponding nega i e consequences ha his
en ails. The me hodology used he e has p o ed o be e y e icien o e alua e he an ioxidan powe
o mix u es o compounds.
Keywo ds:
polyunsa u a ed edible oil; alpha- ocophe ol; hyd oxy y osol ace a e; bina y mix u es
and in e ac ions; accele a ed s o age condi ions; p o on nuclea magne ic esonance; linoleic acyl
g oup deg ada ion a e; oxylipins o ma ion; an ioxidan –p ooxidan e ec s
1. In oduc ion
An ioxidan capaci y is a e y impo an a ibu e o ce ain ood componen s. I
p e en s o delays he oxida ion o ood lipids which, as is well known, causes hei
deg ada ion wi h he consequen loss o nu i ional alue and he gene a ion o oxic
compounds [
1
–
3
]. In addi ion, ollowing ood in ake, compounds wi h an ioxidan capaci y
can also ac endogenously, p e en ing o delaying biological oxida ion p ocesses wi h
impo an epe cussions on human heal h [4].
Fo hese easons, much e o has been de o ed o he sea ch o sou ces o compounds
capable o ac ing as an ioxidan s [
5
–
7
], and also o assessing he le el o his capabili y. This
abili y has been ma ched o di e en capaci ies, such as ha o sca enging ee adicals,
es ima ed by he DPPH (2,2-diphenyl-1-pic ylhyd azyl) [
8
], and ABTS (2,2
0
-azino-bis(3-
e hylbenzo hiazoline-6-sul onic acid) assays [
9
], o ha o educing e ic ions, FRAP [
10
],
all o which a e based on elec on ans e (ET). Likewise, he an ioxidan abili y has also
been ma ched wi h he capaci y o abso b oxygen adicals (ORAC) [
11
,
12
] which is based
on hyd ogen a om ans e (HAT) [
13
]. Howe e , he esul s p o ided by hese me hods a e
no always in ag eemen wi h each o he , no wi h he an ioxidan ac i i y demons a ed.
This may be because hese assays only co e a pa ial, albei impo an , aspec o an ioxidan
capaci y, and do no ake in o accoun o he essen ial aspec s ha in luence his abili y.
An ioxidan s 2022,11, 1156. h ps://doi.o g/10.3390/an iox11061156 h ps://www.mdpi.com/jou nal/an ioxidan s
An ioxidan s 2022,11, 1156 2 o 30
I should be emembe ed ha he an ioxidan capaci y o a compound depends no
only on i s na u e and on he dose used, bu also on o he ac o s ou side he compound
ha canno be o e looked. Among hese ac o s, he sys em in which he compound will
p e en o delay he oxida ion and he oxida i e condi ions o which i is subjec ed a e
essen ial, since i is known ha , depending on hem, a compound can p esen di e en
le els o an ioxidan powe o e en p ooxidan capaci y. The e o e, in o de o a ain
eliable da a on he an ioxidan powe o a compound in a gi en sys em when submi ed
o ce ain condi ions, i is necessa y o ca y ou a de ailed and exhaus i e moni o ing o
he e olu ion o he oxida ion p ocess ha , unde hese condi ions, unde goes he sys em
no en iched and en iched in ha compound a de e mined concen a ions.
In ag eemen wi h he abo emen ioned, some s udies aimed a es ima ing he an ioxi-
dan abili y o di e en compounds ha e been made using, as sys ems able o be oxidized,
pu e compounds, such as me hyl linolea e o iglyce ides o di e en edible oils, submi ed
o ce ain oxida i e condi ions [
14
–
19
]. The esul s ob ained wi h hese lipid sys ems e e
o hem and canno be ex apola ed o o he lipid sys ems. In hese s udies, he moni o ing
o he oxida ion p ocess was pe o med by es ima ing one o wo pa ame e s, conside ed
o be oxida ion ma ke s. Among hese a e pe oxide alue, conjuga ed dienes [
15
,
19
–
22
],
p-anisidine alue [
15
,
19
], o he concen a ion o some oxida ion compounds, measu ed
a ce ain poin s o he p ocess [
14
,
16
,
17
,
23
,
24
]. Howe e , in some cases, hese pa ame e s
ha e p o en o be insu icien o adequa ely con ol he e olu ion o he oxida ion p ocess,
leading, in some s udies, o e oneous conclusions abou he an ioxidan capaci y o he
es ed compound [22,25].
Al hough he a o emen ioned me hods could p o ide some in o ma ion on he an-
ioxidan capaci y o he compounds es ed, his does no allow us o analyse he ull
e ec o hei p esence on he oxida ion p ocess o he lipid sys em o o unde s and hei
beha iou . Recen ly, howe e , he de elopmen o a new me hodology based on P o on
Nuclea Magne ic Resonance,
1
H NMR, allows, in a e y simple way, he moni o ing
and s udy o he oxida ion p ocesses o oils, and o lipids in gene al, in an exhaus i e
and comple e way. This makes i e y sui able o he in-dep h s udy o he an ioxidan
o p ooxidan capaci y [
25
–
31
]. The in o ma ion p o ided by his me hodology allows
us o know he p og ession o he oxida ion p ocess in a global way om he poin o
iew o he compounds deg aded, and om he poin o iew o he oxylipins o med
and hei e olu ion. Thus, knowing o wha ex en and in wha way he an ioxidan o
p ooxidan compound in luences he oxida ion p ocess and he o ma ion o oxylipins
can con ibu e no only o unde s anding i s cu en beha iou , bu also o an icipa ing i s
u u e beha iou .
I is no only in e es ing o know he e ec o indi idual compounds wi h po en ial
an ioxidan capaci y in di e en lipid sys ems, bu also he e ec o hei mix u es. In
ac , he beha iou o compounds in mix u es con ibu es o b oadening he knowledge
o he an ioxidan o p ooxidan capaci y o each o he componen s. Mo eo e , in oods
such as edible oils, ege ables and ui s, hey a e always o ming mix u es, and i is o be
expec ed ha he an ioxidan capaci y o he mix u e is condi ioned and de e mined by he
po en ial es ablishmen o in e ac ions be ween i s componen s. In o de o ad ance he
knowledge o he capaci y o bina y mix u es o p e en o delay he oxida ion o di e en
lipid sys ems subjec ed o oxida i e condi ions, some s udies ha e been ca ied ou o
in es iga e he possible in e ac ions be ween hei componen s and hei e ec s on he
o e all an ioxidan capaci y o he mix u e. In a gene al, summa ized and simplis ic way, i
has been desc ibed ha hese in e ac ions can lead o syne gis ic, addi i e o an agonis ic
e ec s [
32
,
33
]. Howe e , mos o he published s udies on his opic we e pe o med
using elec on ans e (ET) o hyd ogen a om ans e (HAT) based assays [
34
], which as
explained abo e ha e impo an limi a ions. Conside ing all o he abo e, and also he
sca ce co ela ions ound be ween he p esence o liposoluble compounds, supposedly wi h
an ioxidan powe , and he ac ual an ioxidan e ec ha hey cause on edible oils [
33
,
35
,
36
],
An ioxidan s 2022,11, 1156 3 o 30
i seems e iden ha u he s udies a e needed on he e ec p oduced by mix u es o his
kind o compound in lipid sys ems, in o de o shed ligh on his complex issue.
In his con ex , he aim o his s udy is o deepen he unde s anding o he e ec ha
he o i ica ion o sun lowe oil in alpha- ocophe ol,
α
T, in hyd oxy y osol ace a e, HTy-Ac,
o in bina y mix u es o hem has on he e olu ion o all o hem unde accele a ed s o age
condi ions. The s udy was ca ied ou using
1
H NMR spec oscopy. Alpha- ocophe ol
has shown, in p e ious s udies, o ha e an ioxidan o p ooxidan abili y, depending on
he lipid sys em and he expe imen condi ions [
14
,
17
,
21
,
22
,
26
,
27
,
30
,
37
–
39
]. Hyd oxy y-
osol ace a e is a mino componen o i gin oli e oil wi h a well- ecognized an ioxidan
abili y [31,40–44]
. The s o age condi ions used a e oxida i e and sui able o es ing he
an ioxidan o p ooxidan capaci ies o hese compounds indi idually, and hei bina y mix-
u es, hus making i possible o analyse in-dep h he po en ial in e ac ions be ween hese
compounds and he e ec o hese in e ac ions on he esul ing an ioxidan o p ooxidan
capaci y. The s udy ocuses on he e olu ion o he deg ada ion o he main componen s
o he oil, and o he added compounds h oughou he s o age p ocess in oil samples
en iched wi h hese compounds indi idually and join ly. Simul aneously, in he same oil
samples men ioned abo e, he o ma ion o a la ge numbe o oxylipins, as well as he
e olu ion o hei concen a ion h oughou he s o age p ocess is add essed. I is expec ed
ha he esul s ob ained will p o ide aluable in o ma ion ha will con ibu e o a be e
unde s anding o he in e ac ions be ween hese compounds wi h po en ial an ioxidan
abili y, as well as hei e ec , which may ha e an impac on ood echnology, nu i ion and
human heal h.
2. Ma e ials and Me hods
2.1. Samples o S udy
The samples o be s udied a e sun lowe oil, S, pu chased om a local supe ma -
ke , and se e al samples o he same oil en iched wi h alpha- ocophe ol,
α
T, wi h hy-
d oxy y osol ace a e, HTy-Ac, o wi h bina y mix u es o hese compounds. The mola
pe cen ages o he di e en kinds o acyl g oups o he sun lowe oil, es ima ed om
1
H NMR spec al
da a [45–47]
, a e: 57.6
±
0.3% o linoleic, 32.0
±
0.7% o oleic and
10.4
±
0.5% o sa u a ed acyl g oups. Al hough sun lowe oil is one o he edible oils
iches in
alpha- ocophe ol [48,49]
he concen a ion o his ocophe ol in his sun lowe oil
is no high enough o be de ec ed by 1H NMR spec oscopy in he sample S.
The
α
T (98.2% pu i y) and HTy-Ac (99.54% pu i y) used o en ich he oil we e pu -
chased om Sigma-Ald ich (S . Louis, MO, USA) and Sep ox Bio ech (Mad id, Spain),
espec i ely. The sun lowe oil was en iched wi h
α
T a wo di e en concen a ions. The
esul ing samples we e named S
5T
and S
10T,
in which he en ichmen le els we e 5 mmol
and 10 mmol o
α
T, espec i ely, pe mol o iglyce ide (TG). The sun lowe oil was also
en iched wi h HTy-Ac a a concen a ion o 10 mmol HTy-Ac/mol TG and his sample was
named S10H.
In addi ion, he sun lowe oil was also en iched wi h wo di e en bina y mix u es
o
α
T and HTy-Ac. These samples we e named S
5T10H
and S
10T10H
. In he o me , he
sun lowe oil was en iched wi h (5 mmol
α
T + 10 mmol HTy-Ac)/mol TG and in he la e
wi h (10 mmol αT + 10 mmol HTy-Ac)/mol TG.
2.2. Accele a ed S o age Expe imen s and S udy o he Samples E olu ion by 1H
NMR Spec oscopy
Aliquo s o 10 g o he samples S, S
5T
, S
10T
, S
10H
, S
5T10H
and S
10T10H
, we e subjec ed
o accele a ed s o age condi ions a 70
◦
C, as in p e ious s udies [
50
,
51
], un il he samples
we e almos comple ely polyme ized. The expe imen s we e ca ied ou in duplica e.
The e olu ion o each sample unde he a o emen ioned condi ions was moni o ed
using
1
H NMR spec oscopy. The equipmen used was a B uke A ance 400 spec ome e
ope a ing a 400 MHz. The ope a ing condi ions, he acquisi ion pa ame e s, and he
An ioxidan s 2022,11, 1156 4 o 30
so wa e employed we e he same used in p e ious s udies [
50
,
51
]. The spec um o each
sample was acqui ed in duplica e.
The iden i ica ion o he compounds p esen in he di e en samples was ca ied
ou on he basis o he assignmen o he
1
H NMR signals o he hyd ogen a oms o he
di e en s uc u es, like in p e ious s udies [
26
–
31
,
52
], wi h he suppo o he s anda d
compounds, indica ed in Table S1 (Supplemen a y Ma e ial). These signals, hei chemical
shi s and hei assignmen s o he a ious hyd ogen a oms a e gi en in Tables S2–S15
(Supplemen a y Ma e ial).
The es ima ion o he concen a ion o linoleic acyl g oup, o
α
T, and HTy-Ac, as well
as o he se e al oxida ion compounds p esen in he se e al samples o e he s o age ime,
in ela ion o ha o iglyce ides (TG) was ca ied ou as in p e ious s udies [29–31].
2.3. S a is ical Analysis
The s a is ical analysis and g aphical ep esen a ions we e pe o med using Mic oso
O ice Excel 2016, as in p e ious s udies [
30
,
31
]. The s a is ical analysis was used o wo
pu poses. Fi s ly, o calcula e he s anda d de ia ions o he nume ous de e mina ions o he
concen a ion o a e y la ge numbe o compounds p esen in he oil samples h oughou
he accele a ed s o age. Secondly, o s udy he deg ada ion pa hways h oughou he
accele a ed s o age o he sun lowe oil main componen , o he alpha- ocophe ol and o he
hyd oxy y osol ace a e added in he di e en samples.
3. Resul s and Discussion
The objec i e o his s udy, as men ioned abo e, is o elucida e he join an ioxidan o
p ooxidan e ec o alpha- ocophe ol,
α
T, and hyd oxy y osol ace a e, HTy-Ac, in sun lowe
oil and, a he same ime, o analyse whe he his join e ec can be simply desc ibed
as syne gis ic, addi i e o an agonis ic [
32
–
34
] in ela ion o he e ec s caused by hese
compounds indi idually. The e o e, he esul s e e ing o he e olu ion o he oxida ion
p ocess unde accele a ed s o age condi ions a 70
◦
C, o sun lowe oil wi hou en ichmen
in any o he men ioned compounds (S), o en iched in each o hem (samples S
5T
, S
10T
,
and S
10H
), o in any o hei mix u es (samples S
5T10H
and S
10T10H
), will be p esen ed and
discussed sequen ially. These esul s will i s conce n he e olu ion o he deg ada ion o
bo h he main componen o he oil, ha is o he linoleic acyl g oup, and o he added com-
pounds, ha is o
α
T and HTy-Ac, du ing accele a ed s o age. They can p o ide impo an
in o ma ion on he o e all e olu ion o he oxida ion p ocess in each sample and on he
ole played by he added compounds. A e his, he o ma ion o oxylipins in all samples
will also be add essed, in o de o analyse in-dep h he join e ec o he added compounds
bo h in he onse o hei o ma ion and in he e olu ion o hei concen a ion h oughou
he s o age ime. Finally, he occu ence o lipolysis and 1,2-diglyce ide o ma ion du ing
accele a ed s o age in all sun lowe oil samples will also be add essed, in o de o analyse
he join e ec o bina y mix u es on his issue.
3.1. E olu ion o he Concen a ion o he Linoleic Acyl G oup in he Di e en Sun lowe Oil
Samples o e he S o age Time: E ec o Sun lowe Oil En ichmen in
α
T, in HTy-Ac and in Bina y
Mix u es o αT and HTy-Ac
The es ima ion o he concen a ion o he linoleic acyl g oup in he di e en oil
samples du ing s o age was pe o med as indica ed in he expe imen al sec ion om he
1
H NMR spec al signal in ensi y, cen ed a 2.77 ppm, o he bis-allylic p o ons, shown in
Table S2 (Supplemen a y Ma e ial), as desc ibed in p e ious s udies [
30
,
31
,
51
]. The esul s
ob ained a e depic ed in Figu e 1. The e olu ion o he concen a ion o he linoleic acyl
g oup e sus ime i s qui e well, in mos cases, o wo linea pa hs wi h di e en leng h
and slope in each sample, shown in Table 1, and will be discussed below.
An ioxidan s 2022,11, 1156 5 o 30
An ioxidan s 2022, 11, x FOR PEER REVIEW 5 o 31
NMR spec al signal in ensi y, cen ed a 2.77 ppm, o he bis-allylic p o ons, shown in
Table S2 (Supplemen a y Ma e ial), as desc ibed in p e ious s udies [30,31,51]. The esul s
ob ained a e depic ed in Figu e 1. The e olu ion o he concen a ion o he linoleic acyl
g oup e sus ime i s qui e well, in mos cases, o wo linea pa hs wi h di e en leng h
and slope in each sample, shown in Table 1, and will be discussed below.
Figu e 1. E olu ion o he concen a ion, exp essed as mmol o linoleic acyl g oup pe mol o i-
glyce ide (mmol/mol TG) o e he s o age ime a 70 °C, up o a e y ad anced oxida ion s age in:
(a) samples S, S
5T
, S
10T
, and S
10H
; (b) samples S
5T10H
and S
10T10H
.
Table 1. Deg ada ion a es (D
R1L
and D
R2L
), exp essed in mmol o linoleic acyl g oup pe mol o
iglyce ide and day (mmol mol
−1
TG day
−1
), in he wo linea s ages o i s deg ada ion pa h depic ed
in Figu e 1, o each one o he samples, S, S
5T
, S
10T
, S
10H
, S
5T10H
and S
10T10H
, o e he s o age ime a 70
°C, which coincide wi h he slopes o hese linea s ages. The co ela ion coe icien s o he co e-
sponding equa ions a e gi en in b acke s.
Samples
Fi s s age Second s age
Time (days) D
R1L
(mmol/mol TG day)
Time (days) D
R2L
(mmol/mol TG day)
S 0–5 −23.5 (0.95) 5–12 −185.8 (0.97)
S
5T
0–5 −44.1 (1.00) 5–14 −137.2 (0.95)
S
10T
0–7 −61.7 (0.96) 7–14 −133.5 (0.95)
S
10H
0–26 −12.0 (0.99) 26–34 −140.8 (0.99)
S
5T10H
0–14 −29.4 (0.98) 14–24 −110.8 (0.91)
S
10T10H
0–12 −38.9 (0.99) 12–24 −102.8 (0.99)
(i) In sun lowe oil sample S. Sun lowe oil subjec ed o accele a ed s o age condi ions
unde goes oxida ion, esul ing in he deg ada ion o i s componen s, he main one being
he linoleic acyl g oup. As can be seen in Figu e 1a and in he da a o Table 1, he i s
s age o i s deg ada ion co e s he i s 5 days o s o age, in which abou 118 mmol o
linoleic acyl g oup pe mol o iglyce ide (TG) a e los . A e his s age, he e is an accel-
e a ion in he deg ada ion o his acyl g oup, so ha in he second s age, which co e s he
ollowing 7 days, he e is an addi ional deg ada ion o abou 1300 mmol o linoleic acyl
g oup pe mol o TG, p ac ically comple ing he en i e oxida ion p ocess in a o al o 12
days.
(ii) In sun lowe oil samples S
5T
and S
10T
en iched in αT. As expec ed, he e olu ion o
sun lowe oil oxida ion unde accele a ed s o age condi ions a 70 °C is a ec ed by he
en ichmen o he oil in αT. As shown in Figu e 1a and he da a in Table 1, his causes an
Figu e 1.
E olu ion o he concen a ion, exp essed as mmol o linoleic acyl g oup pe mol o
iglyce ide (mmol/mol TG) o e he s o age ime a 70
◦
C, up o a e y ad anced oxida ion s age in:
(a) samples S, S5T, S10T, and S10H; (b) samples S5T10H and S10T10H.
Table 1.
Deg ada ion a es (D
R1L
and D
R2L
), exp essed in mmol o linoleic acyl g oup pe mol o
iglyce ide and day (mmol mol
−1
TG day
−1
), in he wo linea s ages o i s deg ada ion pa h depic ed
in Figu e 1, o each one o he samples, S, S
5T
, S
10T
, S
10H
, S
5T10H
and S
10T10H
, o e he s o age ime
a 70
◦
C, which coincide wi h he slopes o hese linea s ages. The co ela ion coe icien s o he
co esponding equa ions a e gi en in b acke s.
Samples
Fi s S age Second S age
Time (Days) DR1L
(mmol/mol TG Day) Time (Days) DR2L
(mmol/mol TG Day)
S0–5 −23.5 (0.95) 5–12 −185.8 (0.97)
S5T 0–5 −44.1 (1.00) 5–14 −137.2 (0.95)
S10T 0–7 −61.7 (0.96) 7–14 −133.5 (0.95)
S10H 0–26 −12.0 (0.99) 26–34 −140.8 (0.99)
S5T10H 0–14 −29.4 (0.98) 14–24 −110.8 (0.91)
S10T10H 0–12 −38.9 (0.99) 12–24 −102.8 (0.99)
(i)In sun lowe oil sample S. Sun lowe oil subjec ed o accele a ed s o age condi ions
unde goes oxida ion, esul ing in he deg ada ion o i s componen s, he main one being
he linoleic acyl g oup. As can be seen in Figu e 1a and in he da a o Table 1, he i s s age
o i s deg ada ion co e s he i s 5 days o s o age, in which abou 118 mmol o linoleic
acyl g oup pe mol o iglyce ide (TG) a e los . A e his s age, he e is an accele a ion in
he deg ada ion o his acyl g oup, so ha in he second s age, which co e s he ollowing
7 days, he e is an addi ional deg ada ion o abou 1300 mmol o linoleic acyl g oup pe
mol o TG, p ac ically comple ing he en i e oxida ion p ocess in a o al o 12 days.
(ii)In sun lowe oil samples S
5T
and S
10T
en iched in
α
T. As expec ed, he e olu ion o
sun lowe oil oxida ion unde accele a ed s o age condi ions a 70
◦
C is a ec ed by he
en ichmen o he oil in
α
T. As shown in Figu e 1a and he da a in Table 1, his causes an
accele a ion in he i s s age o i s deg ada ion, compa ed o ha which occu ed in he
non-en iched sample. Consequen ly, abou 220 and 430 mmol o linoleic acyl g oup pe
mol o TG a e los in he S
5T
and S
10T
samples, espec i ely, in his i s s age. Likewise, he
second s age o he deg ada ion o he acyl linoleic g oup is also a ec ed by he en ichmen
o he oil in
α
T. This causes a slowing down o he deg ada ion a e o his main componen
o sun lowe oil (see Table 1), compa ed o i s deg ada ion a e in sample S, esul ing in
a leng hening o he whole oxida ion p ocess up o 14 days. These esul s a e consis en

An ioxidan s 2022,11, 1156 6 o 30
wi h and ein o ce p e ious esul s on he e ec o he
α
T en ichmen o o he edible oils
subjec ed o accele a ed s o age condi ions [26,27,30].
(iii)In sun lowe oil in sample S
10H
en iched in HTy-Ac. Acco ding o he da a ep esen ed
in Figu e 1a and gi en in Table 1, he en ichmen o sun lowe oil in HTy-Ac, in ag eemen
wi h ou p e ious esul s [
32
], causes a slowing down o he deg ada ion a e o he linoleic
acyl g oup in bo h s ages o i s deg ada ion pa hway, wi h espec o hose o sample S. Due
o his, he o al oxida ion p ocess o sun lowe oil, o in o he wo ds he o al deg ada ion
o he linoleic acyl g oup in he S
10H
sample, akes abou 34 days, 26 o hem co esponding
o he i s s age o deg ada ion o he linoleic acyl g oup, in which only abou 312 mmol
a e deg aded, and he es is deg aded in he ollowing 8 days, e idencing he delay caused
by HTy-Ac in he oxida ion o sun lowe oil subjec ed o accele a ed s o age unde he
condi ions o his s udy.
(i )In sun lowe oil samples S
5T10H
and S
10T10H
en iched in bina y mix u es o
α
T and HTy-
Ac. The e olu ion o he concen a ion o he linoleic acyl g oup in each o hese sun lowe
oil samples also i s well o wo linea pa hs whose da a a e ep esen ed in Figu e 1b and
shown in Table 1.
In bo h samples, he deg ada ion a e in he i s s age o he deg ada ion o he linoleic
acyl g oup is somewha highe han ha o he S sample. This shows ha he p esence
o
α
T cancels he e ec o HTy-Ac o ice e sa, since each o hese compounds has an
opposi e e ec on he a e o deg ada ion o he linoleic acyl g oup in his i s s age o i s
deg ada ion, as has been no ed in poin s (ii) and (iii). Ano he a iable also a ec ed in his
i s s age o deg ada ion is he du a ion o he s age, which is much longe in samples
S
5T10H
and S
10T10H
han in samples S, S
5T
o S
10T
, bu sho e han in S
10H
. Consequen ly,
in he i s 12–14 days o accele a ed s o age, in which he i s s age o linoleic deg ada ion
in samples S
5T10H
and S
10T10H
akes place, abou 400–460 mmol o linoleic acyl g oup pe
mol o TG a e deg aded in hese samples; howe e , in his same pe iod o ime in samples
S, S
5T
and S
10T
he linoleic acyl g oup is almos comple ely deg aded. I should be no ed
ha al hough he e is no g ea di e ence be ween he deg ada ion a e and he du a ion o
his i s s age o deg ada ion o he linoleic acyl g oup in samples S5T10H and S10T10H, he
e ec o a highe en ichmen in HTy-Ac han in
α
T in sample S
5T10H
is sligh ly e lec ed in
he a iables men ioned abo e.
Fu he mo e, he e ec o sun lowe oil en ichmen in bina y mix u es o
α
T and
HTy-Ac also a ec s he second s age o he linoleic acyl g oup deg ada ion. The du a ion o
his s age in samples S
5T10H
and S
10T10H
is longe han in he es o he samples. This could
be because he en ichmen o sun lowe oil in
α
T and HTy-Ac indi idually (samples S
5T
,
S
10T
and S
10H
) also causes, in bo h cases (see da a in Table 1), a lowe deg ada ion a e o
he linoleic acyl g oup in his second s age han ha obse ed in sample S, and in his case
he e ec s o bo h
α
T and HTy-Ac go in he same di ec ion. Fo his eason, al hough in
samples S
5T10H
and S
10T10H
mos o he linoleic acyl g oup is deg aded du ing his second
s age, his occu s mo e slowly han in he es o he samples.
As a summa y, i can be said ha , conside ing ha he linoleic acyl g oup is no
only he main componen o sun lowe oil, bu also he mos p one o be oxidized, he
e olu ion o i s deg ada ion can be conside ed a e y good ep esen a ion o he e olu ion
o he oxida ion o sun lowe oil. This s udy shows he complexi y o he e ec s ha he
es ed bina y mix u e p o okes in he e olu ion o he oxida ion p ocess o sun lowe oil
submi ed o accele a ed s o age. Taking as a e e ence he oxida ion p ocess in sample
S, he
α
T en ichmen o he oil accele a es he p ocess in he i s s age, while he HTy-Ac
en ichmen slows i down. Howe e , in he second s age o he p ocess, he en ichmen in
ei he o hem slows i down. The en ichmen o he oil in bo h bina y mix u es (samples
S
5T10H
and S
10T10H
) causes, in he i s s age o he oxida ion p ocess, an oxida ion a e
lowe han ha caused by he en ichmen in
α
T (samples S
5T
and S
10T
), bu highe han ha
obse ed in sample S. This e idences he p edominan e ec o
α
T e sus ha o HTy-Ac,
a bo h concen a ions in he bina y mix u es, in his i s s age o he p ocess. Howe e , in
he second s age o he oxida ion p ocess, as he indi idual en ichmen o he oil in
α
T o
An ioxidan s 2022,11, 1156 7 o 30
in HTy-Ac slows down he p ocess in ela ion o ha o sample S, he join e ec o bo h
compounds in he bina y mix u es causes a g ea e slowing down o he p ocess han ha
p oduced by hese compounds indi idually. As a esul , his second s age o he p ocess is
longe in samples S5T10H and S10T10H han in he es o he samples.
The se o a iables, oxida ion a e a each s age, du a ion o each s age and amoun
o linoleic acyl g oup deg aded a each s age, p o ides an o e all pic u e o he oxida ion
p og ess o sun lowe oil du ing accele a ed s o age. All hese a iables a e go e ned by
he p esence o
α
T and HTy-Ac in he oil sample and he e olu ion o he concen a ion o
he la e in he oxida ion p ocess can also shed complemen a y ligh o ob ain a deepe
unde s anding o he global e olu ion o he sun lowe oil oxida ion p ocess unde he
condi ions o his s udy.
3.2. E olu ion o he Concen a ion o αT and HTy-Ac in he Di e en Sun lowe Oil Samples
En iched in These Compounds o e Accele a ed S o age Time
I is known ha when edible oils a e subjec ed o oxida i e condi ions, deg ada ion
o bo h i s majo and mino componen s occu s, whe he he la e a e na u al o added,
and ega dless o whe he hey beha e as an ioxidan s o p ooxidan s [
25
–
27
,
30
,
31
,
49
,
53
].
The e o e, unde accele a ed s o age condi ions, simul aneously wi h he deg ada ion o he
main componen s o he oil, he deg ada ion o he added
α
T and HTy-Ac will also occu .
The e olu ion o hese deg ada ions can be ollowed om he da a p o ided by he
1
H NMR
spec a o he a o emen ioned samples, since hese compounds ha e some p o ons gi ing
speci ic spec al signals, shown in Table S3 (Supplemen a y Ma e ial) no o e lapping wi h
o he s. The e o e, om he in ensi y o hese signals in he di e en spec a, he e olu ion
o
α
T and HTy-Ac concen a ion in he di e en sun lowe oil samples can be es ima ed
o e he s o age ime. The esul s ob ained a e depic ed in Figu e 2. I can be obse ed ha
he e olu ion o he concen a ion o hese compounds qui e adequa ely ma ches one o wo
linea s ages whose slopes a e shown in Table 2, oge he wi h he co ela ion coe icien s o
hese linea s ages gi en in b acke s. The slopes o hese lines coincide wi h he deg ada ion
a es o hese compounds a each deg ada ion s age o he di e en samples. The esul s
ob ained in each sample will be discussed below.
An ioxidan s 2022, 11, x FOR PEER REVIEW 7 o 31
αT en ichmen o he oil accele a es he p ocess in he i s s age, while he HTy-Ac en-
ichmen slows i down. Howe e , in he second s age o he p ocess, he en ichmen in
ei he o hem slows i down. The en ichmen o he oil in bo h bina y mix u es (samples
S
5T10H
and S
10T10H
) causes, in he i s s age o he oxida ion p ocess, an oxida ion a e lowe
han ha caused by he en ichmen in αT (samples S
5T
and S
10T
), bu highe han ha ob-
se ed in sample S. This e idences he p edominan e ec o αT e sus ha o HTy-Ac, a
bo h concen a ions in he bina y mix u es, in his i s s age o he p ocess. Howe e , in
he second s age o he oxida ion p ocess, as he indi idual en ichmen o he oil in αT o
in HTy-Ac slows down he p ocess in ela ion o ha o sample S, he join e ec o bo h
compounds in he bina y mix u es causes a g ea e slowing down o he p ocess han ha
p oduced by hese compounds indi idually. As a esul , his second s age o he p ocess
is longe in samples S
5T10H
and S
10T10H
han in he es o he samples.
The se o a iables, oxida ion a e a each s age, du a ion o each s age and amoun
o linoleic acyl g oup deg aded a each s age, p o ides an o e all pic u e o he oxida ion
p og ess o sun lowe oil du ing accele a ed s o age. All hese a iables a e go e ned by
he p esence o αT and HTy-Ac in he oil sample and he e olu ion o he concen a ion
o he la e in he oxida ion p ocess can also shed complemen a y ligh o ob ain a deepe
unde s anding o he global e olu ion o he sun lowe oil oxida ion p ocess unde he
condi ions o his s udy.
3.2. E olu ion o he Concen a ion o αT and HTy-Ac in he Di e en Sun lowe Oil Samples
En iched in These Compounds o e Accele a ed S o age Time
I is known ha when edible oils a e subjec ed o oxida i e condi ions, deg ada ion
o bo h i s majo and mino componen s occu s, whe he he la e a e na u al o added,
and ega dless o whe he hey beha e as an ioxidan s o p ooxidan s [25–27,30,31,49,53].
The e o e, unde accele a ed s o age condi ions, simul aneously wi h he deg ada ion o
he main componen s o he oil, he deg ada ion o he added αT and HTy-Ac will also
occu . The e olu ion o hese deg ada ions can be ollowed om he da a p o ided by he
1
H NMR spec a o he a o emen ioned samples, since hese compounds ha e some p o-
ons gi ing speci ic spec al signals, shown in Table S3 (Supplemen a y Ma e ial) no
o e lapping wi h o he s. The e o e, om he in ensi y o hese signals in he di e en
spec a, he e olu ion o αT and HTy-Ac concen a ion in he di e en sun lowe oil sam-
ples can be es ima ed o e he s o age ime. The esul s ob ained a e depic ed in Figu e 2.
I can be obse ed ha he e olu ion o he concen a ion o hese compounds qui e ade-
qua ely ma ches one o wo linea s ages whose slopes a e shown in Table 2, oge he wi h
he co ela ion coe icien s o hese linea s ages gi en in b acke s. The slopes o hese lines
coincide wi h he deg ada ion a es o hese compounds a each deg ada ion s age o he
di e en samples. The esul s ob ained in each sample will be discussed below.
Figu e 2.
E olu ion o he concen a ion, exp essed as mmol/mol TG, o alpha- ocophe ol,
α
T, and o
hyd oxy y osol ace a e, HTy-Ac, in sun lowe oil en iched in hese compounds indi idually (samples
S
5T
, S
10T
and S
10H
), o in bina y mix u es o hem (samples S
5T10H
and S
10T10H
), h oughou he
accele a ed s o age a 70 ◦C up o a e y ad anced oxida ion s age.
An ioxidan s 2022,11, 1156 8 o 30
Table 2.
Deg ada ion a es o alpha- ocophe ol (D
RT
) and o hyd oxy y osol ace a e (D
RH
) exp essed
in mmol mol
−1
TG day
−1
in he linea s ages o i s deg ada ion pa h, depic ed in Figu e 2, in each
one o he samples, S
5T
, S
10T
, S
10H
, S
5T10H
and S
10T10H
o e he s o age ime a 70
◦
C. The co ela ion
coe icien s o hese lines a e gi en in b acke s.
Samples Deg ada ion
S age
Alpha-Tocophe ol (αT) Hyd oxy y osol Ace a e
(HTy-Ac)
Time
(Days)
DRT
(mmol/mol
TG Day)
Time
(Days)
DRH
(mmol/mol
TG Day)
S5T - 0–5 1.05 (0.99) - -
S10T - 0–7 1.30 (0.99) - -
S10H 1s - - 0–12 0.20 (0.95)
2nd - - 12–28 0.52 (0.99)
S5T10H 1s 0–8 0.58 (0.96) 0–7 0.54 (0.98)
2nd - - 7–14 0.92 (0.98)
S10T10H 1s 0–10 0.99 (0.99) 0–10 0.56 (0.97)
2nd - - 10–14 1.11 (0.94)
(i)E olu ion o he concen a ion o
α
T in sun lowe oil samples S
5T
and S
10T
.As shown in
Figu e 2and Table 2, he deg ada ion o his compound s a s om he beginning o s o age,
and he s o age ime a which i is o ally deple ed depends on i s ini ial concen a ion
in he oil, occu ing la e he highe he en ichmen o he oil in his compound (day 5 a
S
5T
and day 7 a S
10T
). Mo eo e , he o al deple ion o
α
T coincides app oxima ely wi h
he change in he deg ada ion a e o he linoleic acyl g oup (see Table 1), e idencing he
decisi e ole o his compound in he deg ada ion o he oil main componen and he e o e
in he oxida ion o sun lowe oil.
Fu he mo e, he highe he ini ial concen a ion o
α
T in he oil sample, he highe i s
deg ada ion a e (see Table 2). This compound accele a es he deg ada ion o he linoleic
acyl g oup in i s i s s age o deg ada ion in ela ion o he non-en iched sample S, ha is,
i beha es as a p ooxidan , as demons a ed in p e ious s udies [
26
,
27
,
30
,
54
]. In addi ion,
and al hough his compound is absen in he second deg ada ion s age o he linoleic acyl
g oup, he ini ial en ichmen o he oil in i slows down he a e o deg ada ion o he
linoleic acyl g oup in his second s age, compa ed o ha o he non-en iched sample S
(see Table 1).
(ii)E olu ion o he concen a ion o HTy-Ac in sun lowe oil sample S
10H
. This compound
beha es as an an ioxidan in he S
10H
sample subjec ed o accele a ed s o age condi ions,
as expec ed [
31
,
40
,
41
,
43
,
44
,
55
], and deg ades du ing he p ocess. I s deg ada ion pa h i s
well o wo linea s ages. As Table 2shows, du ing he i s s age, las ing abou 12 days,
he deg ada ion a e o HTy-Ac is e y small. In he second s age, las ing abou 16 days,
he deg ada ion a e o his compound is highe han ha o he i s s age, bu bo h a e
much lowe han ha o
α
T in he S
10T
sample. The di e ences in he deg ada ion a es o
α
T and HTy-Ac a e e y no iceable, as shown in Figu e 2and Table 2. The consequence
o hese di e ences in deg ada ion a e is ha HTy-Ac does no ully deg ade in he S
10H
sample un il a e 28 days unde accele a ed s o age condi ions, while
α
T disappea s
in he S
10T
sample a e 7 days unde he same condi ions. I is obse ed ha as in he
samples en iched in
α
T, he e is a coincidence be ween he ime a which HTy-Ac is o ally
deple ed and he ime a which he deg ada ion a e o he linoleic acyl g oup unde goes
an impo an change (see Figu es 1and 2and Tables 1and 2), e idencing he impo ance
o his compound in he e olu ion o he deg ada ion o he linoleic acyl g oup, and in
u n in he e olu ion o he oxida ion p ocess o he sun lowe oil. I is also wo h no ing
ha , e en once HTy-Ac is ully deg aded, in he second s age o he oxida ion p ocess o
he S
10H
sample, i s in luence is e idenced by slowing down he deg ada ion a e o he
linoleic acyl g oup, compa ed o ha o he second s age o deg ada ion o his same g oup
in he S sample.
An ioxidan s 2022,11, 1156 9 o 30
(iii)E olu ion o he concen a ion o
α
T and o HTy-Ac in sun lowe oil samples S
5T10H
and
S
10T10H
. When hese samples a e subjec ed o accele a ed s o age, as shown in Figu e 2
and Table 2, he deg ada ion a e o
α
T is lowe in bo h samples S
5T10H
and S
10T10H
han
ha obse ed in samples S
5T
and S
10T
, and as a consequence,
α
T is p esen in he o me
samples o up o abou 8–10 days unde accele a ed s o age condi ions, e sus 5 and 7
days in samples S
5T
and S
10T
, espec i ely. Howe e , he deg ada ion a e o HTy-Ac in he
wo s ages o i s deg ada ion p ocess in samples S
5T10H
and S
10T10H
is conside ably highe
han ha obse ed in sample S
10H
, and as a consequence HTy-Ac is p esen in he o me
samples o app oxima ely hal as long as in he las sample. In addi ion, he ime a which
HTy-Ac is comple ely deple ed coincides wi h he change in he a e o he deg ada ion o
he linoleic acyl g oup (see Figu es 1and 2and Tables 1and 2).
In sho , in he samples en iched exclusi ely in
α
T, his deg ades e y apidly a bo h
concen a ions, while he deg ada ion a e o HTy-Ac in he samples en iched exclusi ely
in his compound is almos ou imes lowe han ha o
α
T. In he sun lowe oil samples
en iched in bina y mix u es, he esul s ob ained demons a e he exis ence o in e ac ions
be ween HTy-Ac and
α
T, since he deg ada ion a e o
α
T dec eases somewha , while ha
o HTy-Ac inc eases conside ably, compa ed o hose obse ed in he samples en iched
in each o hese compounds indi idually. This sugges s ha he an ioxidan capaci y o
HTy-Ac inhibi s, up o a ce ain le el, he deg ada ion o
α
T by delaying i , and as a
consequence he deg ada ion o HTy-Ac is accele a ed so ha he an ioxidan capaci y
exhibi ed by his compound in he oxida ion o sun lowe oil is educed in ela ion o ha
shown by his compound when he oil is en iched exclusi ely in i . In o he wo ds, i could
be said ha HTy-Ac, he s ong an ioxidan , egene a es o some ex en
α
T, he p ooxidan ,
and as a consequence he an ioxidan capaci y o he o me is educed due o i s oxida ion.
Mo eo e , i has been obse ed in all samples ha he ime a which he deg ada ion a e
o he linoleic acyl g oup changes coincides wi h he o al disappea ance o
α
T and HTy-Ac
in he samples en iched indi idually in hese compounds, and wi h he disappea ance o
HTy-Ac in he samples en iched in he bina y mix u es o
α
T and HTy-Ac, e idencing he
impo an ole o hese compounds in he e olu ion o oil oxida ion.
In any oxida ion p ocess, he o ma ion o new compounds occu s simul aneously
wi h he deg ada ion o o he ones. In o de o ha e a comple e iew o he e ec ha
he en ichmen o sun lowe oil in he a o emen ioned compounds and hei mix u es has
on i s oxida ion p ocess, when i is subjec ed o accele a ed s o age condi ions, i is also
necessa y o s udy he o ma ion o oxylipins and hei e olu ion in his p ocess.
3.3. E olu ion o he Concen a ion o he Di e en Oxylipins Fo med h oughou he Accele a ed
S o age in he Di e en Sun lowe Oil Samples: E ec o he En ichmen o Sun lowe Oil in αT
and HTy-Ac and in Bina y Mix u es o These Compounds
As is well known, he deg ada ion o he main componen s o edible oil leads o he
o ma ion o oxida ion compounds, some o which a e well known, such as hyd ope oxides,
also called p ima y oxida ion compounds, and seconda y o u he oxida ion compounds,
such as hyd oxy-, ke o- and epoxy-de i a i es, as well as di e en ypes o aldehydes and
o he de i ed compounds. The o ma ion o many o hese compounds and he e olu ion
o hei concen a ion h oughou he accele a ed s o age was moni o ed by
1
H NMR
spec oscopy simul aneously wi h he e olu ion o linoleic acyl g oup and ha o he added
compounds
α
T and HTy-Ac discussed abo e. Using he a o emen ioned analy ical ool and
he me hodology indica ed in he expe imen al sec ion, a la ge numbe o oxylipins we e
iden i ied and quan i ied in he di e en sun lowe oil samples. Thei iden i ica ion, as in
p e ious s udies [
26
–
31
,
52
] was possible by he appea ance in he spec a o speci ic signals
o some o hei p o ons, indica ed in Tables S4–S15 (Supplemen a y Ma e ial). Using
he in ensi y o hese signals, he concen a ion o he oxylipins de ec ed in he di e en
sun lowe oil samples o e he accele a ed s o age ime was de e mined and ep esen ed
in he igu es ha will be shown la e on.
The oxylipins ound ha e been g ouped in o h ee g oups. And he e ec o sun lowe
oil en ichmen in
α
T, HTy-Ac and bina y mix u es o
α
T and HTy-Ac on he onse o
An ioxidan s 2022,11, 1156 16 o 30
1
Figu e 7.
E olu ion o he concen a ion, exp essed in mmol pe mol o iglyce ide (mmol/mol
TG), in samples S, S
5T
, S
10T
, S
10H
, S
5T10H
and S
10T10H
, o e he s o age ime a 70
◦
C, up o a e y
ad anced oxida ion s age, o : (a) mKO-c(Z,E)-dEs; (b) mKO-c(E,E)-dEs.
The e olu ion o he concen a ion o mKO-c(Z,E)-dEs has some simila i y wi h ha o
mHO-c(Z,E)-dEs, as shown in Figu es 6a and 7a. In ac , some au ho s ha e desc ibed ha
he la e can be p ecu so s o he o me [
100
]. In he samples en iched in
α
T, he onse o
he o ma ion o hese oxylipins is ea lie han in he S sample and hei a e o o ma ion is
g ea e he highe he en ichmen deg ee in
α
T. Howe e , in he sample en iched in HTy-Ac,
he onse o he o ma ion o hese oxylipins is la e han in he S, S
5T
and S
10T
samples and
hey each he maximum concen a ion a e app oxima ely 32 days o s o age. In he oil
samples en iched in he bina y mix u es, he o ma ion o hese oxylipins is a ec ed by he
in e ac ions be ween
α
T and HTy-Ac. I is due o hem ha he o ma ion o hese oxylipins
occu s ea lie in sample S
10T10H
han in sample S, and only sligh ly la e in sample S
5T10H
.
The maximum concen a ion is eached a e 15 days o s o age, a e which i begins o
dec ease, coinciding wi h he o al deple ion o HTy-Ac.
The e olu ion o he concen a ion o mKO-c(E,E)-dEs in he di e en samples o e
he s o age ime also shows he exis ence o h ee pa h g oups, namely, he one including
samples S, S
5T
and S
10T
, he one including he sample en iched exclusi ely in HTy-Ac,
S
10H
, and he one co esponding o samples S
5T10H
and S
10T10H
in which he e ec o he
in e ac ions be ween
α
T and HTy-Ac is e idenced. Fu he mo e, he e olu ion o he
concen a ion o hese oxylipins in all he samples esembles ha o mHPO-c(E,E)-dEs,
dHPO-nc(E,E)-dEs, non- icinal mHPO-mEPO-mEs and non- icinal mHO-mEPO-E-mEs, as
shown in Figu es 4,5,6b and 7b.
(F) E olu ion o he concen a ion o o he long chain oxylipins bea ing ke o g oup. This
subg oup includes non- icinal monoke o Z-monoepoxy E-monoenes (non- icinal mKO-Z-
mEPO-E-mEs), and non- icinal monoke o E-monoepoxy E-monoenes (non- icinal mKO-
E-mEPO-E-mEs). The o ma ion o bo h ypes o compounds has ecen ly been epo ed
in he oxida ion p ocesses o edible oils subjec ed o accele a ed s o age [
28
–
31
] and hey
de i e om mHPO-c(Z,E)-dEs [
82
,
83
,
85
,
100
].These oxylipins a e also o med du ing he
accele a ed s o age o he sun lowe oil samples s udied he e. The e olu ion o hei
concen a ion in he di e en samples o e he s o age ime is shown in Figu e 8.

An ioxidan s 2022,11, 1156 17 o 30
An ioxidan s 2022, 11, x FOR PEER REVIEW 17 o 31
mEPO-E-mEs). The o ma ion o bo h ypes o compounds has ecen ly been epo ed in
he oxida ion p ocesses o edible oils subjec ed o accele a ed s o age [28–31] and hey
de i e om mHPO-c(Z,E)-dEs [82,83,85,100].These oxylipins a e also o med du ing he
accele a ed s o age o he sun lowe oil samples s udied he e. The e olu ion o hei con-
cen a ion in he di e en samples o e he s o age ime is shown in Figu e 8.
Figu e 8. E olu ion o he concen a ion, exp essed in mmol pe mol o iglyce ide (mmol/mol TG),
in samples S, S
5T
, S
10T
, S
10H
, S
5T10H
and S
10T10H
o e he s o age ime a 70 °C, up o a e y ad anced
oxida ion s age, o : (a) non- icinal mKO-Z-mEPO-E-mEs; (b) non- icinal mKO-E-mEPO-E-mEs.
The o ma ion o bo h ypes o compounds is clea ly di e en ia ed in he h ee
g oups men ioned abo e, again showing he indi idual e ec s o αT, HTy-Ac and hei
mix u es in he cou se o sun lowe oil oxida ion and also in he o ma ion o bo h kinds
o oxylipins.
Thei o ma ion, in samples S
5T
and S
10T
occu s when αT is o ally deple ed o i s con-
cen a ion is e y low, in samples S
5T10H
, S
10T10H
and S
10H
when HTy-Ac is o ally deple ed
o i s concen a ion is e y low, and in all cases when he concen a ion o hei p ecu so ,
mHPO-c(Z,E)-dEs, is diminishing. Ne e heless, he o ma ion o non- icinal mKO-Z-
mEPO-E-mEs occu s somewha ea lie han ha o non- icinal mKO-E-mEPO-E-mEs,
p obably due o hei di e en isome ism. I is no ewo hy ha , as Figu e 8 indica es, hese
compounds in all samples can be conside ed end oxida ion p oduc s.
3.3.2. Oxylipins O igina ing om he Clea age o Long Chain Oxylipins
The numbe o oxylipins o med by he clea age o long chain oxida ion compounds
is e y la ge and a conside ablenumbe o hem a e de ec able by
1
H NMR, as has been
demons a ed in p e ious s udies on he oxida ion o di e en edible oils unde e y a -
ied condi ions [25–27,29–31,45,50,51,101–104]. They can be small molecules o hey can be
unca ed acyl g oups. Mos o hose de ec ed he e con ain he aldehyde g oup and u -
he mo e, in some cases, hey also con ain a second oxygena ed unc ional g oup. Thei
o ma ion and he e olu ion o hei concen a ion, as could no be o he wise, is also a -
ec ed by he en ichmen o he oil in αT and HTy-Ac o in bina y mix u es o hem. Ac-
co ding o hei unc ional g oups, hey ha e been g ouped in o wo subg oups, which
will be discussed below.
(A) E olu ion o he concen a ion o oxylipins bea ing aldehyde unc ional g oup. These
oxylipins a e conside ed o be de i ed di ec ly om mHPO-c(Z,E)-dEs o om in e me-
dia e compounds de i ed om he la e [92,94,95,98,105]. I is also known ha some o
hem a e in u n p ecu so s o o he oxylipins o his same subg oup. This is he case o 4-
hyd ope oxy-2E-alkenals ha ha e been desc ibed as p ecu so s o 4-hyd oxy-2E-
alkenals and 4-oxo-2E-alkenals [98,106,107]. Likewise, i has also been desc ibed ha
Figu e 8.
E olu ion o he concen a ion, exp essed in mmol pe mol o iglyce ide (mmol/mol TG),
in samples S, S
5T
, S
10T
, S
10H
, S
5T10H
and S
10T10H
o e he s o age ime a 70
◦
C, up o a e y ad anced
oxida ion s age, o : (a)non- icinal mKO-Z-mEPO-E-mEs; (b)non- icinal mKO-E-mEPO-E-mEs.
The o ma ion o bo h ypes o compounds is clea ly di e en ia ed in he h ee g oups
men ioned abo e, again showing he indi idual e ec s o
α
T, HTy-Ac and hei mix u es in
he cou se o sun lowe oil oxida ion and also in he o ma ion o bo h kinds o oxylipins.
Thei o ma ion, in samples S
5T
and S
10T
occu s when
α
T is o ally deple ed o i s
concen a ion is e y low, in samples S
5T10H
, S
10T10H
and S
10H
when HTy-Ac is o ally
deple ed o i s concen a ion is e y low, and in all cases when he concen a ion o hei
p ecu so , mHPO-c(Z,E)-dEs, is diminishing. Ne e heless, he o ma ion o non- icinal
mKO-Z-mEPO-E-mEs occu s somewha ea lie han ha o non- icinal mKO-E-mEPO-E-
mEs, p obably due o hei di e en isome ism. I is no ewo hy ha , as Figu e 8indica es,
hese compounds in all samples can be conside ed end oxida ion p oduc s.
3.3.2. Oxylipins O igina ing om he Clea age o Long Chain Oxylipins
The numbe o oxylipins o med by he clea age o long chain oxida ion compounds
is e y la ge and a conside able numbe o hem a e de ec able by
1
H NMR, as has been
demons a ed in p e ious s udies on he oxida ion o di e en edible oils unde e y
a ied condi ions [
25
–
27
,
29
–
31
,
45
,
50
,
51
,
101
–
104
]. They can be small molecules o hey
can be unca ed acyl g oups. Mos o hose de ec ed he e con ain he aldehyde g oup
and u he mo e, in some cases, hey also con ain a second oxygena ed unc ional g oup.
Thei o ma ion and he e olu ion o hei concen a ion, as could no be o he wise, is
also a ec ed by he en ichmen o he oil in
α
T and HTy-Ac o in bina y mix u es o hem.
Acco ding o hei unc ional g oups, hey ha e been g ouped in o wo subg oups, which
will be discussed below.
(A) E olu ion o he concen a ion o oxylipins bea ing aldehyde unc ional g oup. These
oxylipins a e conside ed o be de i ed di ec ly om mHPO-c(Z,E)-dEs o om in e media e
compounds de i ed om he la e [
92
,
94
,
95
,
98
,
105
]. I is also known ha some o hem
a e in u n p ecu so s o o he oxylipins o his same subg oup. This is he case o 4-
hyd ope oxy-2E-alkenals ha ha e been desc ibed as p ecu so s o 4-hyd oxy-2E-alkenals
and 4-oxo-2E-alkenals [
98
,
106
,
107
]. Likewise, i has also been desc ibed ha 2E,4E-alkenals
a e p ecu so s o 4,5-epoxy-2E-alkenals [
97
,
108
]. The e olu ion o he concen a ion o
each o hose ound in he di e en samples h oughou he s o age ime is ep esen ed in
Figu e 9, and he e ec o oil en ichmen on hei o ma ion will be discussed below.
An ioxidan s 2022,11, 1156 18 o 30
An ioxidan s 2022, 11, x FOR PEER REVIEW 18 o 31
2E,4E-alkenals a e p ecu so s o 4,5-epoxy-2E-alkenals [97,108]. The e olu ion o he con-
cen a ion o each o hose ound in he di e en samples h oughou he s o age ime is
ep esen ed in Figu e 9, and he e ec o oil en ichmen on hei o ma ion will be dis-
cussed below.
Figu e 9. E olu ion o he concen a ion, exp essed in mmol pe mol o iglyce ide (mmol/mol TG),
in samples S, S
5T
, S
10T
, S
10H
, S
5T10H
and S
10T10H
, o e he s o age ime a 70 °C, up o a e y ad anced
oxida ion s age, o : (a) 4-hyd ope oxy-2E-alkenals, 4-hyd oxy-2E-alkenals, n-alkanals and 2E-
alkenals; (b) 2E,4E-alkadienals, 4,5-epoxy-2E-alkenals, 4-oxo-2E-alkenals and 2Z-alkenals.
(i) E ec caused by he en ichmen in αT. The onse o o ma ion o hese oxylipins in
samples S, S
5T
and S
10T
is e y close, as shown in Figu e 9, al hough i occu s sligh ly la e
and, in gene al, hei concen a ion inc eases a a somewha slowe a e in he samples
Figu e 9.
E olu ion o he concen a ion, exp essed in mmol pe mol o iglyce ide (mmol/mol
TG), in samples S, S
5T
, S
10T
, S
10H
, S
5T10H
and S
10T10H
, o e he s o age ime a 70
◦
C, up o a e y
ad anced oxida ion s age, o : (
a
) 4-hyd ope oxy-2E-alkenals, 4-hyd oxy-2E-alkenals, n-alkanals and
2E-alkenals; (b) 2E,4E-alkadienals, 4,5-epoxy-2E-alkenals, 4-oxo-2E-alkenals and 2Z-alkenals.
(i)E ec caused by he en ichmen in
α
T. The onse o o ma ion o hese oxylipins in sam-
ples S, S
5T
and S
10T
is e y close, as shown in Figu e 9, al hough i occu s sligh ly la e and,
in gene al, hei concen a ion inc eases a a somewha slowe a e in he samples en iched
in
α
T han in sample S. Consequen ly, in gene al, he maximum a ained concen a ion o
hese oxylipins is also somewha lowe in he samples en iched in
α
T han in S, excep in
he case o 4-hyd ope oxy-2E-alkenals and 4-hyd oxy-2E-alkenals. This ac is impo an
An ioxidan s 2022,11, 1156 19 o 30
because he la e oxylipins a e conside ed o be esponsible o di e en degene a i e
diseases [78,102,109–111].
(ii)The e ec caused by he en ichmen in HTy-Ac. Two di e en pa hs can be dis in-
guished ega ding he o ma ion and e olu ion o he concen a ion o hese oxylipins
in he sample en iched in HTy-Ac. In one o hem (see Figu e 9a), he o ma ion onse
o some o hese oxylipins occu s a e 18–20 days o s o age, when he concen a ion o
mHPO-c(Z,E)-dEs is s ill in he maximum, and o e a long pe iod o ime hei concen-
a ion inc eases e y slowly un il he concen a ion o HTy-Ac is e y low, close o i s
o al deple ion, a e which he concen a ion o hese oxylipins inc eases e y apidly. In
he o he pa h (see Figu e 9b), he o ma ion onse o some o hese oxylipins occu s la e ,
a e 26 days o s o age, coinciding in ime wi h a e y low concen a ion o HTy-Ac, and
wi h he s a o he dec ease o he concen a ion o mHPO-c(Z,E)-dEs. The maximum
concen a ion eached by all hese oxylipins in sample S
10H
is e y simila o ha eached
in samples S, S5T and S10T as Figu e 9shows.
(iii)E ec caused by he en ichmen in bina y mix u es o
α
T and HTy-Ac. The mos
impo an di e ences ound in he o ma ion o aldehydes be ween he samples en iched
in
α
T and in HTy-Ac a e hose ela ed o he onse o hei o ma ion. The e o e, i is
o be expec ed ha he e ec o he in e ac ions be ween
α
T and HTy-Ac in he samples
en iched in he bina y mix u es o hese compounds will also be obse ed in he onse o
he o ma ion o hese oxylipins. As Figu e 9shows, he onse o aldehydes o ma ion in
he S
5T10H
and S
10T10H
samples is delayed wi h espec o ha obse ed in he samples
en iched in
α
T, wi h his e ec being mo e no iceable he highe he a io be ween he
concen a ions o HTy-Ac and
α
T. Howe e , his onse is ad anced wi h espec o ha
obse ed in he sample en iched in HTy-Ac, his e ec being mo e no iceable he highe
he a io be ween he concen a ions o
α
T and HTy-Ac. These same e ec s ha e been
obse ed in he o ma ion o some long chain oxylipins which o igina e in he pe oxida ion
o linoleic g oup, such as mHPO-c(E,E)dEs, dHPO-nc(E,E)-dEs, non- icinal mHPO-mEPO-
E-mEs, non- icinal mHO-mEPO-E-mEs, non- icinal mKO-Z-mEPO-E-mEs, and non- icinal
mKO-E-mEPO-E-mEs, some o which ha e been p e iously desc ibed as p ecu so s o
oxylipins bea ing an aldehyde g oup [
71
,
93
–
98
,
105
] as men ioned abo e. This sugges s
ha he empo al miles ones occu ed in he o ma ion o hese long chain oxylipins by
he e ec o bina y mix u es allows one o an icipa e he e ec on he o ma ion o hose
bea ing an aldehyde g oup.
I only emains o add ha , as Figu e 9clea ly shows, some aldehydes, such as
4-hyd ope oxy-2E-alkenals and 2E,4E-alkenals a e in e media e oxida ion compounds.
(B) E olu ion o he concen a ion o oxylipins no bea ing he aldehyde unc ional g oup. This
subg oup includes oxida ion compounds wi h e y di e en unc ional g oups, such as
5-alkyl-(5H)- u an-2-ones, 5-alkyl- u ans and o mic acid. The o ma ion o hese h ee
ypes o oxylipins in he oxida ion p ocesses o edible oils has been demons a ed in
p e ious s udies bo h by solid-phase mic oex ac ion ollowed by gas ch oma og aphy-
mass spec ome y [
47
,
112
–
114
], and by
1
H NMR spec oscopy [
29
–
31
]. Figu e 10 shows
he e olu ion o he concen a ion o each o hese oxylipins in he di e en samples o e
he s o age ime.
This igu e shows ha he onse o hei o ma ion ollows he same pa e ns obse ed
in he onse o he o ma ion o many o he oxylipins men ioned abo e. This akes place
a ound h ee s o age imes (days: 7–8; 14–16; and 26–28) in he h ee g oups o samples (S,
S
5T
and S
10T
; S
5T10H
and S
10T10H
; and S
10H
). In sun lowe oil samples en iched in bina y
mix u es, he onse o he o ma ion o all hese oxylipins occu s a an in e media e s o age
ime, compa ed o ha obse ed in sun lowe oil samples en iched in
α
T o HTy-Ac, due
o he in e ac ions be ween hem discussed abo e.
An ioxidan s 2022,11, 1156 20 o 30
An ioxidan s 2022, 11, x FOR PEER REVIEW 20 o 31
Figu e 10. E olu ion o he concen a ion, exp essed in mmol pe mol o iglyce ide (mmol/mol
TG), in samples S, S
5T
, S
10T
, S
10H
, S
5T10H
and S
10T10H
, o e he s o age ime a 70 °C, up o a e y ad-
anced oxida ion s age, o : (a) 5-alkyl-(5H)- u an-2-one; (b) alkyl u ans; (c) o mic acid.
This igu e shows ha he onse o hei o ma ion ollows he same pa e ns ob-
se ed in he onse o he o ma ion o many o he oxylipins men ioned abo e. This akes
place a ound h ee s o age imes (days: 7–8; 14–16; and 26–28) in he h ee g oups o sam-
ples (S, S
5T
and S
10T
; S
5T10H
and S
10T10H
; and S
10H
). In sun lowe oil samples en iched in bina y
mix u es, he onse o he o ma ion o all hese oxylipins occu s a an in e media e s o age
ime, compa ed o ha obse ed in sun lowe oil samples en iched in αT o HTy-Ac, due
o he in e ac ions be ween hem discussed abo e.
3.3.3. Long Chain Oxylipins wi h O igin in Epoxida ion o he Linoleic Acyl G oup
The o ma ion o epoxy g oups on he unmodi ied linoleic acyl g oup has been e-
po ed in p e ious s udies on lipid oxida ion and se e al o ma ion mechanisms ha e
been desc ibed as possible [67,115–121]. In ac , he p esence o long chains bea ing mo-
noepoxy monoene g oups in edible oils subjec ed o accele a ed s o age condi ions,
among which he well-known leuco oxin and isoleuco oxin s uc u es can be ound, has
been p o ed [26,27,29–31].
Fu he mo e, he opening o he oxi ane ing o he a o emen ioned monoepoxy mo-
noene long chains p o ides di e en pa hways, leading o he o ma ion o o he ox-
ylipins, some o which inco po a e b anches o he long chains. This is possible because
in he oxida ion p ocess o he sun lowe oil unde accele a ed s o age condi ions, a la ge
numbe o acids and p ima y and seconda y alcohols a e o med [112,113,122] ha a e
capable o opening he oxi ane ing by hyd olysis [60,123–125]. Due o his, he o ma ion
o oxylipins bea ing icinal dihyd oxy g oups can be p oduced, including he well-known
leuko oxin and isoleuko oxin diols [117,123,124,126–131]. Likewise, he opening o he
oxi ane ing caused by acids leads o he o ma ion o es e g oups [124,126–129,132]. In
ac , in edible oils submi ed o accele a ed s o age, he o ma ion o o ma e g oups has
been p o ed, which may be ei he o ming icinal di o ma e s uc u es o icinal mono o -
ma e monohyd oxy s uc u es [29–31,126]. Finally, when he oxi ane ing opening is
caused by p ima y o seconda y alcohols, e he g oups a e o med, gi ing ise o icinal
monoe he monohyd oxy s uc u es [29–31,124,127,130,131,133–135]. As men ioned
abo e, he o ma ion o he la e wo ypes o oxylipins bea ing es e o e he g oups
in ol es he in oduc ion o b anching in he long chains o he acyl g oups, which leads
o an inc ease in he iscosi y o he oil and e en o i s polyme iza ion when he eac ions
occu be ween acyl g oups. Some o hese oxylipins could also be o med endogenously
a e he ac ion o cy och ome P450 epoxygenases [136,137]. The e ec o he en ichmen
o sun lowe oil in αT, in HTy-Ac and in hei bina y mix u es on he o ma ion and e o-
lu ion o he concen a ion o he a o emen ioned oxylipins will be discussed below.
(A) E olu ion o he concen a ion o long chains bea ing monoepoxy monoene g oups. This
subg oup includes wo ypes o long chains bea ing monoepoxy monoene g oups (mEPO-
mE) de ec ed in sun lowe oil and o he edible oils subjec ed o accele a ed s o age con-
di ions [25–27,29–31]. They a e di e en ia ed by hei isome ism, some o hem exhibi Z-
Figu e 10.
E olu ion o he concen a ion, exp essed in mmol pe mol o iglyce ide (mmol/mol
TG), in samples S, S
5T
, S
10T
, S
10H
, S
5T10H
and S
10T10H
, o e he s o age ime a 70
◦
C, up o a e y
ad anced oxida ion s age, o : (a) 5-alkyl-(5H)- u an-2-one; (b) alkyl u ans; (c) o mic acid.
3.3.3. Long Chain Oxylipins wi h O igin in Epoxida ion o he Linoleic Acyl G oup
The o ma ion o epoxy g oups on he unmodi ied linoleic acyl g oup has been e-
po ed in p e ious s udies on lipid oxida ion and se e al o ma ion mechanisms ha e been
desc ibed as possible [
67
,
115
–
121
]. In ac , he p esence o long chains bea ing monoe-
poxy monoene g oups in edible oils subjec ed o accele a ed s o age condi ions, among
which he well-known leuco oxin and isoleuco oxin s uc u es can be ound, has been
p o ed [26,27,29–31].
Fu he mo e, he opening o he oxi ane ing o he a o emen ioned monoepoxy
monoene long chains p o ides di e en pa hways, leading o he o ma ion o o he
oxylipins, some o which inco po a e b anches o he long chains. This is possible because
in he oxida ion p ocess o he sun lowe oil unde accele a ed s o age condi ions, a la ge
numbe o acids and p ima y and seconda y alcohols a e o med [
112
,
113
,
122
] ha a e
capable o opening he oxi ane ing by hyd olysis [
60
,
123
–
125
]. Due o his, he o ma ion
o oxylipins bea ing icinal dihyd oxy g oups can be p oduced, including he well-known
leuko oxin and isoleuko oxin diols [
117
,
123
,
124
,
126
–
131
]. Likewise, he opening o he
oxi ane ing caused by acids leads o he o ma ion o es e g oups [
124
,
126
–
129
,
132
].
In ac , in edible oils submi ed o accele a ed s o age, he o ma ion o o ma e g oups
has been p o ed, which may be ei he o ming icinal di o ma e s uc u es o icinal
mono o ma e monohyd oxy s uc u es [
29
–
31
,
126
]. Finally, when he oxi ane ing opening
is caused by p ima y o seconda y alcohols, e he g oups a e o med, gi ing ise o icinal
monoe he monohyd oxy s uc u es [
29
–
31
,
124
,
127
,
130
,
131
,
133
–
135
]. As men ioned abo e,
he o ma ion o he la e wo ypes o oxylipins bea ing es e o e he g oups in ol es he
in oduc ion o b anching in he long chains o he acyl g oups, which leads o an inc ease
in he iscosi y o he oil and e en o i s polyme iza ion when he eac ions occu be ween
acyl g oups. Some o hese oxylipins could also be o med endogenously a e he ac ion
o cy och ome P450 epoxygenases [
136
,
137
]. The e ec o he en ichmen o sun lowe
oil in
α
T, in HTy-Ac and in hei bina y mix u es on he o ma ion and e olu ion o he
concen a ion o he a o emen ioned oxylipins will be discussed below.
(A) E olu ion o he concen a ion o long chains bea ing monoepoxy monoene g oups. This
subg oup includes wo ypes o long chains bea ing monoepoxy monoene g oups (mEPO-
mE) de ec ed in sun lowe oil and o he edible oils subjec ed o accele a ed s o age con-
di ions [
25
–
27
,
29
–
31
]. They a e di e en ia ed by hei isome ism, some o hem exhibi
Z-monoepoxy-Z-monoene g oups (Z-mEPO-Z-mEs) and o he s E-monoepoxy-Z-monoene
g oups (E-mEPO-Z-mEs) [
29
–
31
,
117
]. Figu e 11 shows he e olu ion o hei concen a ion
in he di e en sun lowe oil samples h oughou he s o age ime.
An ioxidan s 2022,11, 1156 21 o 30
An ioxidan s 2022, 11, x FOR PEER REVIEW 21 o 31
monoepoxy-Z-monoene g oups (Z-mEPO-Z-mEs) and o he s E-monoepoxy-Z-monoene
g oups (E-mEPO-Z-mEs) [29–31,117]. Figu e 11 shows he e olu ion o hei concen a ion
in he di e en sun lowe oil samples h oughou he s o age ime.
Figu e 11. E olu ion o he concen a ion, exp essed in mmol pe mol o iglyce ide (mmol/mol
TG), in samples S, S
5T
, S
10T
, S
10H
, S
5T10H
and S
10T10H
, o e he s o age ime a 70 °C, up o a e y ad-
anced oxida ion s age, o : (a) Z-mEPO-Z-mEs; (b) E-mEPO-Z-mEs.
I can be obse ed in his igu e ha he s o age ime a which he o ma ion o he
long chains bea ing Z-monoepoxy-Z-monoene g oups begins is simila o ha o he 4-
hyd ope oxy-2E-alkenals in he di e en samples, and he e is also a ce ain pa allelism
in he e olu ion o hei concen a ion, al hough he o me each a much highe concen-
a ion han he la e , which is only sligh ly lowe han ha eached by mHPO-c(E,E)-dEs
in all samples, as Figu es 4 and 11a show.
The o ma ion o long chains bea ing E-monoepoxy-Z-monoene g oups, as shown in
Figu e 11b, s a s somewha la e han ha o hei homologous isome s men ioned abo e
and coincides in ime wi h ha o oxylipins ha o igina e om he clea age o long chains
bu do no bea aldehyde g oups (see Figu e 10). The concen a ion eached by hese ox-
ylipins, al hough much lowe han ha o hei isome s, is also impo an , and he e ec
ha he en ichmen o he oil in αT, in HTy-Ac, o in bina y mix u es o hem, p oduces
on hei o ma ion, is he same as explained abo e in he o ma ion o some oxylipins such
as 5-alkyl-(5H)- u an-2-ones o o mic acid.
(B) E olu ion o he concen a ion o oxylipins wi h o igin in he oxi ane ing opening. As
men ioned p e iously, his subg oup includes long chains bea ing icinal dihyd oxy mo-
noene g oups ( icinal dHO-mEs), icinal di o ma e monoene g oups ( icinal dF-mEs), ic-
inal mono o ma e monohyd oxy monoene g oups ( icinal mF-mHO-mEs) o icinal mo-
noe he monohyd oxy monoene g oups ( icinal mE -mHO-mEs). The e olu ion o he
concen a ion in he di e en samples o e he s o age ime o some o he a o emen ioned
g oups such as icinal dHO, o ma e and icinal mE -mHO g oups, is shown in Figu e 12.
Figu e 11.
E olu ion o he concen a ion, exp essed in mmol pe mol o iglyce ide (mmol/mol
TG), in samples S, S
5T
, S
10T
, S
10H
, S
5T10H
and S
10T10H
, o e he s o age ime a 70
◦
C, up o a e y
ad anced oxida ion s age, o : (a)Z-mEPO-Z-mEs; (b)E-mEPO-Z-mEs.
I can be obse ed in his igu e ha he s o age ime a which he o ma ion o he
long chains bea ing Z-monoepoxy-Z-monoene g oups begins is simila o ha o he 4-
hyd ope oxy-2E-alkenals in he di e en samples, and he e is also a ce ain pa allelism in
he e olu ion o hei concen a ion, al hough he o me each a much highe concen a ion
han he la e , which is only sligh ly lowe han ha eached by mHPO-c(E,E)-dEs in all
samples, as Figu es 4and 11a show.
The o ma ion o long chains bea ing E-monoepoxy-Z-monoene g oups, as shown
in Figu e 11b, s a s somewha la e han ha o hei homologous isome s men ioned
abo e and coincides in ime wi h ha o oxylipins ha o igina e om he clea age o long
chains bu do no bea aldehyde g oups (see Figu e 10). The concen a ion eached by hese
oxylipins, al hough much lowe han ha o hei isome s, is also impo an , and he e ec
ha he en ichmen o he oil in
α
T, in HTy-Ac, o in bina y mix u es o hem, p oduces on
hei o ma ion, is he same as explained abo e in he o ma ion o some oxylipins such as
5-alkyl-(5H)- u an-2-ones o o mic acid.
(B) E olu ion o he concen a ion o oxylipins wi h o igin in he oxi ane ing opening.
As men ioned p e iously, his subg oup includes long chains bea ing icinal dihyd oxy
monoene g oups ( icinal dHO-mEs), icinal di o ma e monoene g oups ( icinal dF-mEs),
icinal mono o ma e monohyd oxy monoene g oups ( icinal mF-mHO-mEs) o icinal
monoe he monohyd oxy monoene g oups ( icinal mE -mHO-mEs). The e olu ion o he
concen a ion in he di e en samples o e he s o age ime o some o he a o emen ioned
g oups such as icinal dHO, o ma e and icinal mE -mHO g oups, is shown in Figu e 12.
An ioxidan s 2022, 11, x FOR PEER REVIEW 21 o 31
monoepoxy-Z-monoene g oups (Z-mEPO-Z-mEs) and o he s E-monoepoxy-Z-monoene
g oups (E-mEPO-Z-mEs) [29–31,117]. Figu e 11 shows he e olu ion o hei concen a ion
in he di e en sun lowe oil samples h oughou he s o age ime.
Figu e 11. E olu ion o he concen a ion, exp essed in mmol pe mol o iglyce ide (mmol/mol
TG), in samples S, S
5T
, S
10T
, S
10H
, S
5T10H
and S
10T10H
, o e he s o age ime a 70 °C, up o a e y ad-
anced oxida ion s age, o : (a) Z-mEPO-Z-mEs; (b) E-mEPO-Z-mEs.
I can be obse ed in his igu e ha he s o age ime a which he o ma ion o he
long chains bea ing Z-monoepoxy-Z-monoene g oups begins is simila o ha o he 4-
hyd ope oxy-2E-alkenals in he di e en samples, and he e is also a ce ain pa allelism
in he e olu ion o hei concen a ion, al hough he o me each a much highe concen-
a ion han he la e , which is only sligh ly lowe han ha eached by mHPO-c(E,E)-dEs
in all samples, as Figu es 4 and 11a show.
The o ma ion o long chains bea ing E-monoepoxy-Z-monoene g oups, as shown in
Figu e 11b, s a s somewha la e han ha o hei homologous isome s men ioned abo e
and coincides in ime wi h ha o oxylipins ha o igina e om he clea age o long chains
bu do no bea aldehyde g oups (see Figu e 10). The concen a ion eached by hese ox-
ylipins, al hough much lowe han ha o hei isome s, is also impo an , and he e ec
ha he en ichmen o he oil in αT, in HTy-Ac, o in bina y mix u es o hem, p oduces
on hei o ma ion, is he same as explained abo e in he o ma ion o some oxylipins such
as 5-alkyl-(5H)- u an-2-ones o o mic acid.
(B) E olu ion o he concen a ion o oxylipins wi h o igin in he oxi ane ing opening. As
men ioned p e iously, his subg oup includes long chains bea ing icinal dihyd oxy mo-
noene g oups ( icinal dHO-mEs), icinal di o ma e monoene g oups ( icinal dF-mEs), ic-
inal mono o ma e monohyd oxy monoene g oups ( icinal mF-mHO-mEs) o icinal mo-
noe he monohyd oxy monoene g oups ( icinal mE -mHO-mEs). The e olu ion o he
concen a ion in he di e en samples o e he s o age ime o some o he a o emen ioned
g oups such as icinal dHO, o ma e and icinal mE -mHO g oups, is shown in Figu e 12.
Figu e 12.
E olu ion o he concen a ion, exp essed in mmol pe mol o iglyce ide (mmol/mol
TG), in samples S, S
5T
, S
10T
, S
10H
, S
5T10H
and S
10T10H
, o e he s o age ime a 70
◦
C, up o a e y
ad anced oxida ion s age, o : (a) icinal dHO; (b) o ma e g oup; (c) icinal mE -mHO.

An ioxidan s 2022,11, 1156 22 o 30
The e ec o sun lowe oil en ichmen in HTy-Ac, in
α
T and in bina y mix u es o
HTy-Ac and
α
T on he onse o he o ma ion o long chains wi h icinal dihyd oxy g oups
( icinal dHO) o wi h icinal monoe he monohyd oxy g oups ( icinal mE -mHO) is simila ,
o some ex en o ha obse ed in he onse o he o ma ion o 4-hyd oxy-2E-alkenals,
as shown in Figu es 9a and 12. Howe e , he e ec o di e en ypes o oil en ichmen
on he onse o o ma e g oup o ma ion is e y simila o ha obse ed on he onse o
o ma ion o i s p ecu so , o mic acid, and on he onse o he o ma ion o long chains wi h
E-monoepoxy-Z-monoene g oups, which could also be p ecu so s o o ma e g oups (see
Figu es 10,11b and 12). The concen a ion eached by o ma e g oups can be conside ed
ema kable, which indica es he ele ance o he o ma ion o es e g oups du ing oil
oxida ion unde accele a ed condi ions and, as a consequence, he gene a ion o b anchings
in he long chain acyl g oups wi h epe cussions on oil iscosi y o e en i s polyme iza ion.
3.4. Lipolysis Ex en and 1,2-diglyce ides Fo ma ion in he Di e en Sun lowe Samples
h oughou he Accele a ed S o age Time: In luence o he En ichmen in
α
T, HTy-Ac and in Bina y
Mix u es o These
This sun lowe oil has a e y small concen a ion o 1,2-diglyce ides as usual. When
his oil is subjec ed o accele a ed s o age, lipolysis occu s o a e y low deg ee, p oducing
1,2-diglyce ides [
31
]. Figu e 13 shows he e olu ion o he concen a ion o hese glyce ides
in he di e en oil samples.
An ioxidan s 2022, 11, x FOR PEER REVIEW 22 o 31
Figu e 12. E olu ion o he concen a ion, exp essed in mmol pe mol o iglyce ide (mmol/mol
TG), in samples S, S5T, S10T, S10H, S5T10H and S10T10H, o e he s o age ime a 70 °C, up o a e y ad-
anced oxida ion s age, o : (a) icinal dHO; (b) o ma e g oup; (c) icinal mE -mHO.
The e ec o sun lowe oil en ichmen in HTy-Ac, in αT and in bina y mix u es o
HTy-Ac and αT on he onse o he o ma ion o long chains wi h icinal dihyd oxy g oups
( icinal dHO) o wi h icinal monoe he monohyd oxy g oups ( icinal mE -mHO) is simi-
la , o some ex en o ha obse ed in he onse o he o ma ion o 4-hyd oxy-2E-alkenals,
as shown in Figu es 9a and 12. Howe e , he e ec o di e en ypes o oil en ichmen on
he onse o o ma e g oup o ma ion is e y simila o ha obse ed on he onse o o -
ma ion o i s p ecu so , o mic acid, and on he onse o he o ma ion o long chains wi h
E-monoepoxy-Z-monoene g oups, which could also be p ecu so s o o ma e g oups (see
Figu es 10, 11b and 12). The concen a ion eached by o ma e g oups can be conside ed
ema kable, which indica es he ele ance o he o ma ion o es e g oups du ing oil ox-
ida ion unde accele a ed condi ions and, as a consequence, he gene a ion o b anchings
in he long chain acyl g oups wi h epe cussions on oil iscosi y o e en i s polyme iza-
ion.
3.4. Lipolysis Ex en and 1,2-diglyce ides Fo ma ion in he Di e en Sun lowe Samples
h oughou he Accele a ed S o age Time: In luence o he En ichmen in αT, HTy-Ac and in
Bina y Mix u es o These
This sun lowe oil has a e y small concen a ion o 1,2-diglyce ides as usual. When
his oil is subjec ed o accele a ed s o age, lipolysis occu s o a e y low deg ee, p oducing
1,2-diglyce ides [31]. Figu e 13 shows he e olu ion o he concen a ion o hese glyce -
ides in he di e en oil samples.
Figu e 13. E olu ion o he concen a ion o 1,2-diglyce ides, exp essed in mmol pe mol o iglyc-
e ide (mmol/mol TG), in samples S, S
5T
, S
10T
, S
10H
, S
5T10H
and S
10T10H
, o e he s o age ime a 70 °C,
up o a e y ad anced oxida ion s age.
I can be obse ed in Figu e 13 ha he s o age ime a which hese glyce ides a e
o med is also a ec ed by he ype o oil en ichmen . Thus, in samples S, S
5T
and S
10T
he
o ma ion o 1,2-diglyce ides begins almos a he same ime o s o age in he h ee sam-
ples, al hough in he samples en iched in αT he concen a ion eached is highe han in
sample S. In he sample en iched in HTy-Ac he o ma ion o his glyce ide s a s much
la e han in he samples men ioned abo e, and i occu s when he HTy-Ac is almos com-
ple ely deple ed. In his sample, 1,2-diglyce ides each a highe concen a ion han in he
o he samples. Finally, in samples S
5T10H
and S
10T10H
, as a consequence o he in e ac ions
be ween αT and HTy-Ac, he o ma ion o hese glyce ides occu s ea lie han in S
10H
, bu
also when HTy-Ac is o ally deple ed and he concen a ion hey each is only sligh ly
Figu e 13.
E olu ion o he concen a ion o 1,2-diglyce ides, exp essed in mmol pe mol o iglyc-
e ide (mmol/mol TG), in samples S, S
5T
, S
10T
, S
10H
, S
5T10H
and S
10T10H
, o e he s o age ime a
70 ◦C, up o a e y ad anced oxida ion s age.
I can be obse ed in Figu e 13 ha he s o age ime a which hese glyce ides a e
o med is also a ec ed by he ype o oil en ichmen . Thus, in samples S, S
5T
and S
10T
he o ma ion o 1,2-diglyce ides begins almos a he same ime o s o age in he h ee
samples, al hough in he samples en iched in αT he concen a ion eached is highe han
in sample S. In he sample en iched in HTy-Ac he o ma ion o his glyce ide s a s much
la e han in he samples men ioned abo e, and i occu s when he HTy-Ac is almos
comple ely deple ed. In his sample, 1,2-diglyce ides each a highe concen a ion han
in he o he samples. Finally, in samples S
5T10H
and S
10T10H
, as a consequence o he
in e ac ions be ween
α
T and HTy-Ac, he o ma ion o hese glyce ides occu s ea lie han
in S
10H
, bu also when HTy-Ac is o ally deple ed and he concen a ion hey each is only
sligh ly highe han ha eached in samples S
5T
and S
10T
. I is e iden ha , in sun lowe
oil subjec ed o accele a ed s o age condi ions, no only does oxida ion occu , bu so does
hyd olysis. Al hough he la e eac ion occu s o a e y small ex en , i is a ec ed by he
en ichmen o he oil in αT, in H y-Ac o in hei bina y mix u es, as has been shown.
An ioxidan s 2022,11, 1156 23 o 30
4. Conclusions
The me hodology based on
1
H NMR spec oscopy used he e o s udy he indi idual
and join e ec o alpha- ocophe ol,
α
T, and hyd oxy y osol ace a e, HTy-Ac, on he e olu-
ion o an edible oil ich in omega-6 acyl g oups subjec ed o oxida i e condi ions, such as
accele a ed s o age, p o ides simul aneously a weal h o in o ma ion impossible o ob ain
by any o he me hod o da e, and wi hou subjec ing he sample o chemical modi ica ions
o p io sepa a ion s eps. This in o ma ion conce ns no only he deg ada ion o he main
componen o he oil and he added
α
T and HTy-Ac, bu also he o ma ion and e olu ion
o he concen a ion o a la ge numbe o oxylipins, as well as he occu ence o some
lipolysis du ing he accele a ed s o age o he oil.
The main componen o he oil, ha is o say he linoleic acyl g oup, deg ades du ing
accele a ed s o age, and wo s ages wi h di e en deg ada ion a es can be dis inguished
in i s deg ada ion pa h. Taking he deg ada ion p ocess o he non-en iched sample as a
e e ence, he en ichmen o he oil in
α
T accele a es he deg ada ion o he linoleic acyl
g oup in he i s s age o his p ocess and slows down i s deg ada ion in he second s age,
whe eas he en ichmen o he oil in HTy-Ac slows down he deg ada ion o he linoleic
acyl g oup in bo h s ages o he p ocess. I ollows ha en ichmen o he oil in
α
T leads
o a p ooxidan e ec in he i s s age o he oxida ion p ocess, bu an an ioxidan e ec
in he second s age, whe eas en ichmen o he oil in HTy-Ac causes an an ioxidan e ec
in bo h s ages o he oxida ion p ocess. The join en ichmen o he oil in
α
T and HTy-Ac
accele a es he deg ada ion o he linoleic acyl g oup in he i s s age, compa ed o ha
obse ed in he non-en iched S sample, al hough o a lesse ex en han ha caused by he
en ichmen in
α
T, and slows down he deg ada ion in he second s age o a g ea e ex en
han ha p oduced by he en ichmen wi h ei he o hese compounds indi idually, so ha
he du a ion o his second s age in he samples en iched in bina y mix u es is he longes
o all. I ollows ha he en ichmen o he oil in bo h bina y mix u es leads o a p ooxidan
e ec in he i s s age o oil deg ada ion, and an an ioxidan e ec in he second s age,
and a o al du a ion o he oxida ion p ocess in e media e be ween ha o he oil en iched
indi idually in αT and in HTy-Ac.
Unde he a o emen ioned oxida i e condi ions,
α
T and HTy-Ac a e also deg aded,
he o me being ully deple ed much ea lie han he la e . The s o age ime a which
he o al deple ion o hese compounds occu s is e y impo an in he e olu ion o he oil
oxida ion p ocess. Thus, he end o he i s s age o he deg ada ion o he linoleic g oup
and he beginning o he second s age coincides wi h he s o age ime a which he o al
deple ion o
α
T occu s in he samples en iched in his compound, o wi h he s o age ime
a which he o al deple ion o HTy-Ac occu s in he samples en iched in HTy-Ac, and
also in he bina y mix u es o
α
T and HTy-Ac. I should be highligh ed ha , consequen ly,
he second s age o deg ada ion o he linoleic acyl g oups in all samples akes place in
he absence o
α
T and HTy-Ac, howe e , i can be said ha he e olu ion o he oil a
his s age is also a ec ed by he ini ial en ichmen in hem. I is also e y no ewo hy
ha in bo h samples en iched in bina y mix u es he o al deple ion o
α
T occu s la e
han in he samples en iched exclusi ely in his compound, while he opposi e happens o
HTy-Ac. Bo h ac s a e due o he in e ac ions be ween
α
T and HTy-Ac which de e mine
he e olu ion o oil oxida ion in he samples en iched in bina y mix u es.
Among he oxylipins o med, a la ge numbe o igina e om he pe oxida ion o
he main oil componen ; ano he la ge g oup o igina es om he clea age o long-chain
oxylipins; and inally, ano he g oup o igina es om he epoxida ion o he main componen
o he oil. The la e , in u n, a e opening hei oxi ane ing wi h acids o alcohols, gi e
ise o o he s ha ha e b anches in he chains o may e en cause polyme iza ion o he oil.
The i s oxylipins o med ca y monohyd ope oxy-conjuga ed dienes, mHPO-c-dEs,
and can be ei he mHPO-c(Z,E)-dEs o mHPO-c(E,E)-dEs. In sample S, he o ma ion
and deg ada ion o bo h ypes o oxylipins uns in pa allel, he la e always in a highe
concen a ion han he o me .
An ioxidan s 2022,11, 1156 24 o 30
En ichmen in
α
T ad ances and accele a es he o ma ion o mHPO-c(Z,E)-dEs, bu
delays and slows down he o ma ion o mHPO-c(E,E)-dEs in ela ion o wha occu s in S.
The e o e, he concen a ion eached by he o me compounds in he samples en iched in
α
T is highe han in he S sample, and ha eached by he la e is lowe o a g ea e ex en
he highe he le el o en ichmen . This is in line wi h he a o emen ioned dual e ec o
α
T en ichmen , a p ooxidan e ec in he i s s age o oil deg ada ion and an an ioxidan
e ec in he second s age o oil deg ada ion.
The en ichmen o he oil in HTy-Ac delays and slows down he o ma ion o bo h
mHPO-c-dEs in ela ion o wha occu s in he S sample, and as a consequence, he maximum
concen a ion eached by hem is lowe han in he S sample and is also eached much la e .
Fu he mo e, his en ichmen leads o la ge di e ences in he e olu ion o he concen a ion
o mHPO-c(Z,E)-dEs and mHPO-c(E,E)-dEs. Thus, mHPO-c(Z,E)-dEs each hei highes
concen a ion when HTy-Ac exhibi s he highes deg ada ion a e, and his concen a ion
emains s abilized o a long pe iod o ime, un il he o al deple ion o HTy-Ac, a e which
i dec eases, coinciding wi h he sudden inc ease in he concen a ion o mHPO-c(E,E)-dEs
and wi h he beginning o he second s age o linoleic deg ada ion.
The en ichmen o he oil in bina y mix u es o
α
T and HTy-Ac ad ances and accele -
a es he o ma ion o mHPO-c(Z,E)-dEs o a lesse ex en han in he samples en iched in
α
T, bu o a g ea e ex en han in he S sample, and hei concen a ion inc eases du ing a
longe pe iod o ime han in he samples en iched in
α
T, achie ing a highe concen a ion
han in hese la e samples. This is because in he samples en iched in bina y mix u es
he o al deple ion o
α
T occu s la e han in he oil samples en iched only in
α
T, which
could be a ibu ed o he ac ha HTy-Ac egene a es i o some ex en and, he e o e,
HTy-Ac deg ades ea lie in hese samples han in he sample en iched only in HTy-Ac.
These ac s de e mine no only he concen a ion o mHPO-c(Z,E)-dEs, bu also hei subse-
quen e olu ion and ha o mHPO-c(E,E)-dEs, as well as he main miles ones o he o al
oxida ion p ocess. Among hem, in addi ion o he abo emen ioned, he du a ion o he
s abiliza ion o mHPO-c(Z,E)-dEs concen a ion, he beginning o hei deg ada ion, he
o al deple ion o HTy-Ac, he change in he a e o linoleic acyl g oup deg ada ion, he
sudden inc ease in mHPO-c(E,E)-dEs concen a ion, he ime a which hese la e oxylipins
each hei maximum concen a ion, and he o al du a ion o he oxida ion p ocess, as well
as he o ma ion and e olu ion o he concen a ion o he o he oxylipins and e en he
occu ence o ce ain lipolysis can be ci ed.
I only emains o be added ha he o ma ion and e olu ion o he concen a ion
o he o he oxylipins men ioned abo e a e also go e ned by
α
T o HTy-Ac in samples
en iched in hese compounds and by in e ac ions be ween hem in samples en iched in
bina y mix u es. To he bes o ou knowledge, his is he i s ime ha he abo e indica ed
miles ones conce ning he main aspec s in ol ed in he oxida ion p ocess o eal sys ems,
such as hose s udied he e, ha e been shown o i oge he like he pieces o a puzzle.
Supplemen a y Ma e ials:
The ollowing suppo ing in o ma ion can be downloaded a : h ps:
//www.mdpi.com/a icle/10.3390/an iox11061156/s1, Table S1. S anda d compounds used o
iden i ica ion pu poses; Table S2.
1
H NMR signals ob ained in CDCl
3
o p o ons o main sun lowe
oil componen s, hei chemical shi s, mul iplici ies and assignmen s o p o ons o di e en unc ional
g oups p esen in edible oils; Table S3. Chemical shi assignmen s and mul iplici ies o
1
H NMR
signals in CDCl
3
o p o ons o hyd oxy y osol ace a e and alpha- ocophe ol; Table S4. Chemical
shi assignmen s and mul iplici ies o
1
H NMR signals in CDCl
3
o p o ons o monohyd ope oxy-
conjuga ed oc adecadienes (mHPO-c-dEs) de i ed om linoleic g oups; Table S5. Chemical shi
assignmen s and mul iplici ies o
1
H NMR signals in CDCl
3
o p o ons o dihyd ope oxy non-
conjuga ed E,E-oc adecadienes (dHPO-nc(E,E)-dEs); Table S6. Chemical shi assignmen s and
mul iplici ies o
1
H NMR signals in CDCl
3
o p o ons o non icinal monohyd ope oxy monoepoxy
E-oc adecamonoenes (mHPO-mEPO-E-mEs); Table S7. Chemical shi assignmen s and mul iplici ies
o
1
H NMR signals in CDCl
3
o p o ons o monohyd oxy-conjuga ed Z,E-oc adecadienes (mHO-
c(Z,E)-dEs); Table S8. Chemical shi assignmen s and mul iplici ies o
1
H NMR signals in CDCl
3
o
p o ons o monohyd oxy monoepoxy E-oc adecamonoenes (mHO-mEPO-E-mEs); Table S9. Chemical
An ioxidan s 2022,11, 1156 25 o 30
shi assignmen s and mul iplici ies o
1
H NMR signals in CDCl
3
o p o ons o monoke o-conjuga ed
oc adecadienes (mKO-c-dEs); Table S10. Chemical shi assignmen s and mul iplici ies o
1
H NMR
signals in CDCl
3
o p o ons o monoke o monoepoxy E-oc adecamonoenes (mKO-mEPO-E-mEs);
Table S11. Chemical shi assignmen s and mul iplici ies o
1
H NMR signals in CDCl
3
o p o ons o
di e en ypes o aldehydes; Table S12. Chemical shi assignmen s and mul iplici ies o
1
H NMR
signals in CDCl
3
o p o ons o u an g oups; Table S13. Chemical shi assignmen s and mul iplici ies
o
1
H NMR signals in CDCl
3
o p o ons o epoxy de i a i es de i ed om linoleic acyl g oups;
Table S14. Chemical shi assignmen s and mul iplici ies o
1
H NMR signals in CDCl
3
o p o ons o
di e en ypes o dihyd oxy g oups (dHO); Table S15. Chemical shi assignmen s and mul iplici ies
o 1H NMR signals in CDCl3o p o ons o o mic acid and o ma es.
Au ho Con ibu ions:
Concep ualiza ion, M.D.G.; Da a cu a ion, S.d.C.-O. and M.D.G.; Fo mal
analysis, S.d.C.-O., A.R.-A. and M.D.G.; Funding acquisi ion, M.D.G.; In es iga ion, S.d.C.-O. and
M.D.G.; Me hodology, S.d.C.-O. and M.D.G.; P ojec adminis a ion, M.D.G.; Supe ision, M.D.G.;
W i ing—o iginal d a , S.d.C.-O. and M.D.G.; W i ing— e iew & edi ing, S.d.C.-O. and M.D.G. All
au ho s ha e ead and ag eed o he published e sion o he manusc ip .
Funding:
This wo k has been unded by he Spanish Minis y o Science and Inno a ion (MINECO,
AGL2015-65450-R, AEI/FEDER-EU) and by he Basque Go e nmen and i s Depa men s o Uni e -
si ies and Resea ch (EJ-GV, IT-916-16).
Ins i u ional Re iew Boa d S a emen : No applicable.
In o med Consen S a emen : No applicable.
Da a A ailabili y S a emen : Da a is con ained wi hin he a icle and supplemen a y ma e ial.
Acknowledgmen s:
So ia del Caño-Ochoa hanks he Spanish Minis y o Science and Inno a ion o
a p edoc o al g an (BES-2016-079037).
Con lic s o In e es : The au ho s decla e no con lic o in e es .
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