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Ci a ion: Mon oya, T.;
Ala cón-de-la-Las a, C.; Cas ejón,
M.L.; O ega-Vidal, J.; Al a ejos, J.;
Sánchez-Hidalgo, M. (−)-Me hyl-
Oleocan hal, a New Oleocan hal
Me aboli e Reduces LPS-Induced
In lamma o y and Oxida i e
Response: Molecula Signaling
Pa hways and His ones Epigene ic
Modula ion. An ioxidan s 2022,11, 56.
h ps://doi.o g/10.3390/an iox
11010056
Academic Edi o : S anley Omaye
Recei ed: 3 Decembe 2021
Accep ed: 24 Decembe 2021
Published: 27 Decembe 2021
Publishe ’s No e: MDPI s ays neu al
wi h ega d o ju isdic ional claims in
published maps and ins i u ional a il-
ia ions.
Copy igh : © 2021 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license (h ps://
c ea i ecommons.o g/licenses/by/
4.0/).
an ioxidan s
A icle
(−)-Me hyl-Oleocan hal, a New Oleocan hal Me aboli e
Reduces LPS-Induced In lamma o y and Oxida i e
Response: Molecula Signaling Pa hways and His ones
Epigene ic Modula ion
Ta iana Mon oya 1, Ca alina Ala cón-de-la-Las a 1, Ma ía Luisa Cas ejón1, Juan O ega-Vidal 2,
Joaquín Al a ejos 2and Ma ina Sánchez-Hidalgo 1,*
1Depa men o Pha macology, Facul y o Pha macy, Uni e sidad de Se illa, 41012 Se illa, Spain;
[email p o ec ed] (T.M.); cala [email p o ec ed] (C.A.-d.-l.-L.); [email p o ec ed] (M.L.C.)
2Depa men o Ino ganic and O ganic Chemis y, Facul y o Expe imen al Sciences, Campus de Excelencia
In e nacional Ag oalimen a io (ceiA3), Uni e si y o Jaén, 23071 Jaén, Spain; jo 00007@ ed.ujaen.es (J.O.-V.);
[email p o ec ed] (J.A.)
*Co espondence: [email p o ec ed]
Abs ac :
The an ioxidan and an i-in lamma o y esponses o (
−
)-me hyl-oleocan hal (me -OLE),
a new me aboli e o he ex a i gin oli e oil (EVOO) phenolic oleocan hal (OLE), we e explo ed
in lipopolysaccha ide (LPS)-induced mu ine pe i oneal mac ophages. Possible signaling pa hways
and epigene ic modula ion o his ones we e s udied. Me -OLE inhibi ed LPS-induced in acellula
eac i e oxygen species (ROS) and ni i e (NO) p oduc ion and dec eased he o e exp ession o he
p o-in lamma o y enzymes COX-2, mPGES-1 and iNOS in mu ine mac ophages. In addi ion, me -
OLE was able o signi ican ly dec ease he ac i a ion o p38, JNK, and ERK mi ogen-ac i a ed p o ein
kinases (MAPKs) and blocked canonical and non-canonical in lammasome signaling pa hways. On
he con a y, me -OLE up egula ed haem oxigenase 1 (HO-1) and nuclea ac o (e y h oid-de i ed
2)-like 2 (N -2) exp ession in ea ed cells. Finally, me -OLE p e ea ed spleen cells coun e ac ed LPS
induc ion, p e en ing H3K18 ace yla ion o H3K9 and H3K27 deme hyla ion. O e all, hese esul s
p o ide no el mechanis ic insigh s in o he bene icial e ec s o me -OLE ega ding he egula ion o
he immune–in lamma o y esponse h ough epigene ic changes in his one ma ke s. This e ealing
e idence sugges s ha he me hyla ed me aboli e o OLE may con ibu e signi ican ly o he bene icial
e ec s ha a e associa ed wi h he secoi idoid- ela ed compound and he usual consump ion o
EVOO.
Keywo ds:
an ioxidan ; his ones; in lamma ion; mac ophages; me aboli e; me hyla ion; oleocan hal;
oli e oil
1. In oduc ion
Mac ophages a e majo componen s o he inna e immune sys em and play a c i i-
cal ole in modula ing in lamma o y and immune esponses [
1
]. Ex acellula bac e ial
lipopolysaccha ide (LPS) ac s as pa hogen-associa ed molecula pa e n and is ecognized
by he Toll-like ecep o (TLR)-4, inducing mac ophages o an ac i a ed s a e, p oducing p o-
in lamma o y cy okines and chemokines and enhancing he exp ession o in lamma o y-
ela ed enzymes, such as inducible ni ic oxide syn hase (iNOS), cyclooxygenase (COX)-2
and mic osomal p os aglandin E syn hase (mPGES)-1, which syn hesize ni ic oxide (NO)
and p os aglandin (PG)E
2
, espec i ely [
2
]. Addi ionally, LPS-s imula ed mac ophages
dis up he balance o he in acellula educ ion–oxida ion s a e, leading o oxida i e s ess,
usually accompanied by damage ha is media ed by eac i e oxygen species (ROS) [
3
].
The p ocess o gene exp ession o hese p o-in lamma o y media o s in ol es mul iple
signal ansduc ion pa hways, which a e mainly h ough mi ogen-ac i a ed p o ein kinases
An ioxidan s 2022,11, 56. h ps://doi.o g/10.3390/an iox11010056 h ps://www.mdpi.com/jou nal/an ioxidan s
An ioxidan s 2022,11, 56 2 o 18
(MAPKs), nuclea ansc ip ion ac o -kappa B (NF-
κ
B), janus kinase/signal ansduce
and ansc ip ion ac i a o o ansc ip ion (JAK/STAT) o in lammasome ac i a ion. Fu -
he mo e, he nuclea ac o (e y h oid-de i ed 2)-like 2 (N -2)/haem oxygenase-1 (HO-1)
an ioxida i e axis, which exe s a egula i e unc ion on he ac i a ion o ROS, MAPKs,
and in lammasome signaling pa hways, is ep essed in he e en o he induc ion o he
ac i a ed mac ophages s a e [2,3].
Eme ging e idence sugges s ha epigene ic p ocesses ha a ec gene exp ession
wi hou causing changes in he nucleo ide sequence occu a e ex e nal s imuli exposu e,
and may con ibu e o he pa hophysiology o in lamma o y p ocesses [
4
]. In pa icula ,
his one H3 me hyla ion a lysine 9 (H3K9), one o he mos conse ed epigene ic ma ke s,
is co ela ed wi h gene silencing and he modula ion o immune cell di e en ia ion and
immune esponses, and he e o e, in luences he ou come o in lamma ion. Simila ly, H3
ace yla ion on lysine 18 (H3K18ac) is a pe missi e ma ke on genes encoding cy okines
ha co ela e o in lamma ion, such as in e leukin (IL)-1
β
, IL-6 o IL-17 [
5
–
7
]. Fu he mo e,
pos - ansla ional his one modi ica ions ha e eme ged as p ospec i e he apeu ic a ge s.
Unde s anding he c oss-link o hese mechanisms could be c ucial in designing new
immune–in lamma o y app oaches ha a e e ec i ely ela ed o se e al in lamma o y
diseases [8].
In his con ex , die a y nu ien s could modi y physiological and pa hological p o-
cesses h ough c i ical epigene ic mechanisms, p omo ing modi ica ions o gene exp ession
wi hou al e a ion o he gene ic code. In pa icula , speci ic, unc ional oods such as ex a
i gin oli e oil (EVOO) ha e displayed an i-in lamma o y ac i i ies in human mac ophages
h ough epigene ic mechanisms [9].
The heal h-p omo ing p ope ies o EVOO ha e been co ela ed wi h i s peculia
chemical composi ion. EVOO polyphenols a e mino seconda y me aboli es ha ha e been
widely s udied due o hei wide unc ional e sa ili y, including hei an i-in lamma o y,
an ioxidan , ca diop o ec i e, chemop e en i e, and neu op o ec i e p ope ies [
10
]. Es-
sen ially, he an i-in lamma o y ac i i ies o oli e oil, especially phenolic compounds, ha e
ecen ly been linked o hei po en ial o induce epigene ic modi ica ions such as gene
exp ession, DNA me hyla ion and his one modi ica ion [10,11].
Examples o he main oli e polyphenols a e y osol, hyd oxy y osol, oleocan hal
(OLE), oleacein, oli e ligus oside and oleu opein. OLE ep esen s up o 10% o he o-
al polyphenol con en in EVOO (0.2–498 mg/kg) [
12
] and has ecei ed mo e scien i ic
a en ion, due o i s in e es ing biological ac i i ies, bo h wi hin
in i o
and
in i o
sys-
ems, including an i-in lamma o y, an ioxidan , ca diop o ec i e, chemop e en i e and
neu op o ec i e p ope ies [
10
,
13
]. In ac , ecen ly, we ha e epo ed he p e en i e ole o
die a y OLE-supplemen ed e ec s in a collagen-induced a h i is (CIA) mu ine model and
he abili y o OLE o diminish he acu e in lamma o y esponse in LPS-induced mu ine
pe i oneal mac ophages [14,15].
Me abolic ans o ma ions o he p esence o me aboli es can a ec he pha macologi-
cal ac i i y o he pa e n compounds. I has been epo ed ha OLE can emain in ac in he
s omach o up o 4 h and en e he small in es ine unhyd olyzed. Then, non-hyd olyzed
OLE ollows u he me abolic eac ions ela ed o phase I and II in he li e , namely,
hyd oxyla ion o hyd a ion and me hyla ion, espec i ely. Consequen ly, López-Ye ena
e al. (2021) p oposed wo plausible me aboli es o OLE, howe e , me -OLE was he main
ci cula ing conjuga e o OLE de ec ed in all issues analyzed om a s a e he acu e in ake
o a e ined oli e oil con aining 0.3 mg/mL o OLE [
16
]. In ela ion o bio ans o ma ion,
he me hyla ed me aboli es a e mainly me abolized by glucu onida ion and sulpha ion, as
hey a e no subs a es o me hyl ans e ases.
Pe haps, he majo disad an age o he EVOO phenols is hei empe a u e ins abili y,
pho olabili y, and inadequa e pha macokine ic p o ile. To educe hese handicaps, me hyla-
ion o he phenolic hyd oxyl g oups (O-me hyla ion) may inc ease he chemical s abili y
o hei s uc u es, while con e ing g ea e lipophilici y, inc easing hei me abolic s abili y
and memb ane anspo and acili a ing abso p ion and g ea e o al bioa ailabili y. Recen
An ioxidan s 2022,11, 56 3 o 18
s udies also indica e ha he me hyla ion p ocess inc eases biological ac i i y wi hou
al e ing he apeu ic indices [17].
Taking his backg ound in o accoun , he aim o he p esen wo k was o in es i-
ga e he po en ial an ioxidan and an i-in lamma o y e ec s o a new OLE me aboli e,
(
−
)-me hyl-oleocan hal (me -OLE), in LPS-induced mu ine pe i oneal mac ophages. Speci -
ically, in acellula ROS, NO, p o-in lamma o y cy okines p oduc ion (IL-1
β
, IL-6, IL-17,
IL-18, umo nec osis ac o (TNF)-
α
and in e e on (IFN)-
γ
), and he p o ein exp essions
o p o-in lamma o y enzymes (COX-2, iNOS, and mPGES-1) we e e alua ed. In addi-
ion, he possible molecula signaling pa hways in ol ed in hei bene icial e ec s, such
as N -2/HO-1, MAPKs, and he canonical and noncanonical in lammasome, we e also
s udied. Finally, o s udy he ole o he epigene ic mechanisms unde lying me -OLE
an i-in lamma o y e ec s, we explo ed me -OLE induced epigene ics changes in his one
ma ke s (H3K9me3, H3K27me3 and H3K18ac) and cy okines-co ela ed p oduc ion com-
pa ed o OLE in spleen cells a e LPS induc ion.
2. Ma e ials and Me hods
2.1. Reagen s
Sol en s used o ex ac ion, analy ical hin-laye ch oma og aphy (TLC), column ch o-
ma og aphy (CC) and as cen i ugal pa i ion ch oma og aphy (FCPC), such as die hyl
e he (E
2
O), e hyl ace a e (E OAc), n-hexane (Hex), dichlo ome hane (DCM) and e hanol
(E OH) we e o analy ical g ade and we e pu chased om VWR Chemicals (P olabo
®
,
Fon enay-sous-Bois, F ance). Wa e (H
2
O), used o ch oma og aphic sepa a ions, was
o ul apu e g ade and was p oduced by Milli-Q wa e (1.8 M
Ω
) equipmen (Me ck
®
,
KGaA, Da ms ad , Ge many). Ace oni ile (ACN) and me hanol (MeOH), used o high-
pe o mance liquid ch oma og aphy (HPLC), and chlo o o m, used o de e mine op ical
o a ion alues, we e o HPLC g ade and we e pu chased om VWR
®
(Mad id, Spain).
Deu e a ed chlo o o m (CDCl
3
) was used o p epa e solu ions o isola ed compounds o
nuclea magne ic esonance (NMR) analysis and we e pu chased om VWR
®
(Mad id,
Spain). Ace ic acid (AcOH), used o HPLC, was pu chased om VWR
®
(Mad id, Spain).
Silica gel 60 F240 p ecoa ed aluminum shee s we e pu chased om Me ck
®
(Da ms ad ,
Ge many). N-me hylu ea, used o p epa e N-me hyl-N-ni osou ea (see Supplemen a y
Ma e ials), was pu chased om Sigma-Ald ich
®
(Mad id, Spain). Sodium hyd oxide was
pu chased om VWR Chemicals®(P olabo, Fon enay-sous-Bois, F ance).
2.2. Ins umen s
HPLC analyses we e pe o med on a Wa e s HPLC ins umen , equipped wi h a C18
e e sed-phase Sphe iso b ODS-2 column, 250
×
3 mm i.d, 5
µ
m (Wa e s Ch oma og a-phy
Di ision®, Mild o d, MA, USA), and a pho odiode a ay de ec o .
Reac ions unde mic owa e i adia ion we e achie ed on a CEM Disco e monomodal
mic owa e eac o wi h empe a u e and p essu e in e nal p obes. A sealed essel was
used o pe o m he eac ion.
Pu i ica ion o syn hesized compounds was ca ied ou on a FCPC-200
®
ins umen
(K oma on Technologies
®
, Ange s, F ance) ha was i ed wi h a o o and wi h a o al
column capaci y o 200 mL. Ro a ion could be adjus ed om 0 o 2000 pm. Sol en was
pumped by an All ech 627 isoc a ic pump (All ech Associa es
®
, Dee ield, IL, USA). The
sample was injec ed in o he FCPC column wi h a 3725i-038 manual injec o (Rheodyne
®
,
Co a i, CA, USA) equipped wi h a 10 mL sample loop. The ins umen was equipped wi h
a UV-Vis Linea UVIS 200 de ec o (Linea Ins umen Co
®
, Reno, NV, USA) se a 280 nm.
Mass spec a (ESIMS) we e eco ded on an Esqui e 6000 ion mass spec ome e (B uke
Dal onics, B emen, Ge many) i ed wi h an elec osp ay ioniza ion (ESI) in e ace, ope a -
ing in posi i e mode. High- esolu ion mass spec a (HRMS) we e pe o med on an Agilen
6520B spec ome e (Agilen echnologies, Waldb onn, Ge many) i ed wi h a quad upole
ime-o - ligh (Q-TOF) mass spec ome e .
An ioxidan s 2022,11, 56 4 o 18
P o on nuclea magne ic esonance (
1
H NMR) and ca bon nuclea magne ic esonance
(
13
C NMR) spec a o he isola ed and syn hesized compounds we e eco ded on a B uke
A ance DPX 400 spec ome e (B uke Dal onic GmbH
®
, Rheins e en, Ge many) a 400 and
100 MHz, espec i ely. Deu e a ed chlo o o m, wi h e ame hylsilane (TMS) as in e nal
e e ence, was used o dissol e he samples. Coupling cons an s (J) a e p o ided in he z
(Hz) and he mul iplici ies o signals a e epo ed using he ollowing abb e ia ions: single
(s), b oad single (b s), double (d), double o double s (dd), double o double o double s
(ddd), iple ( ), double o iple (d ), quad uple (q) and mul iple (m).
Speci ic o a ions ([
α
]
D
) o chi al compounds we e calcula ed by measu ing he co e-
sponding op ical o a ion (
α
) in chlo o o m on a Jasco P-200 au oma ic pola ime e (Jasco
Analy ical Ins umen
®
, Eas on, MD, USA) using cells o qua z wi h a pa h leng h o 1 dm.
2.3. Isola ion o (−)-Oleocan hal (OLE) om Oli e Oil
A sample o OLE was pu i ied om oli e oil phenolic ex ac by a combina ion o
wo echniques ( as cen i ugal pa i ion ch oma og aphy (FCPC) and semi-p epa a i e
high-pe o mance liquid ch oma og aphy (HPLC)), ollowing he p ocedu e desc ibed
in Diez-Bello e al., (2019) [
18
]. In sho , an oli e oil sample (300 g) was ex ac ed wi h a
mix u e o MeOH/H
2
O 8:2, acco ding o a no malized p ocedu e (IOC, 2017), o a o d a
phenolic ex ac (1.50 g), which was ac iona ed wi h a FCPC-200
®
ins umen (K oma on
Technologies, Ange s, F ance) using a qua e na y biphasic sol en sys em composed
o hexane/e hyl ace a e/e hanol/wa e (2:3:2:3, / / / ). A pooling ac ion (180 mg),
mainly con aining OLE, was u he pu i ied by semi-p epa a i e HPLC using he sol en s
ace oni ile/ace ic acid (99.8:0.2, / , sol en A) and wa e /ace ic acid (99.8:0.2, / , sol en
B) unde a linea g adien om 20 o 25% o sol en A. As a esul , pu e OLE (25 mg) was
ob ained, in which nuclea magne ic esonance (NMR) da a (Figu es S1 and S2) ag eed
wi h hose epo ed in he li e a u e [18].
2.4. Syn hesis o (−)-Me hyl-Oleocan hal (Me -OLE) om (−)-Ligus oside
A wood sample o Olea eu opaea L., ob ained om p uning a ee g owing in he
p o ince o Jaén, Spain, was used o isola e (
−
)-ligus oside. A sample o wood chips
(300 g) was ex ac ed wi h E OAc (4 L) o 2 h a e lux. Sol en was e apo a ed unde
educed p essu e a 40
◦
C o gi e he co esponding d y ex ac (15 g), which was column
ch oma og aphed o gi e ligus oside (0.34 g), as epo ed be o e by he au ho s [
19
]. The
pu i y o ligus oside (84%) was de e mined by an ex e nal s anda d me hod using he
HPLC peak a ea a 280 nm. P e iously, a calib a ion cu e (y = 1734.5 x + 1656; R
2
= 0.9981)
(G aphic S1) was cons uc ed wi h six s anda ds (0.1–1 mg/mL in MeOH) (Table S1) o
pu e (
−
)-ligus oside. Fo analy ical HPLC analyses, sepa a ion was ca ied ou by a
s ep g adien wi h mix u es o MeOH/AcOH (99.8:0.2, / , sol en A) and H
2
O/AcOH
(99.8:0.2, / , sol en B). The g adien p og am consis ed o a linea g adien om 20 o
80% A in 55 min, a linea g adien om 80 o 100% A in 5 min and ano he 10 min o e u n
o ini ial condi ions.
Nex , in o de o syn he ize (
−
)-me hyl-ligus oside, a solu ion o diazome hane
in E
2
O (35 mL), eshly p epa ed om N-me hyl-N-ni osou ea (3.5 g) and aq. KOH
(50%), was added d opwise o a solu ion o ligus oside (200 mg) in MeOH (10 mL) and
he eac ion was le o s i o 60–90 min. The eac ion was moni o ed by TLC and by
HPLC using he g adien elu ion desc ibed abo e. Finally, sol en was emo ed unde
educed p essu e o gi e a b own solid (205 mg). The c ude was pu i ied by as cen i ugal
pa i ion ch oma og aphy (FCPC) using a qua e na y biphasic sol en sys em composed o
Hex/E OAc/E OH/H
2
O (2:5:2:5, / / / ) a a low o 7 mL/min and a o a ion speed
o 1200 pm. As a esul , 125 mg (71% yield) o (
−
)-me hyl-ligus oside as a sligh yellow–
whi e solid was ob ained: ESIMS: m/z 561 [M + Na]
+
(Figu e S11);
1
H NMR (400 MHz,
CDCl
3
) (Figu e S9):
δ
(ppm) 7.51 (s, 1H, H-9), 7.15 (m, 2H, H-4
0
, H-8
0
), 6.85 (m, 2H, H-5
0
,
7
0
), 6.07 (m, 1H, H-5), 5.92 (b s, 1H, H-7), 4.81 (d, 1H, J
1”,2”
= 7.8 Hz, H-1”), 4.25 (d , 1H,
J
10a,20
= 6.9 Hz, J
10a,10b
= 10.7 Hz, H-1
0
a), 4.12 (d , 1H, J
10b,20
= 6.9 Hz, J
10b,10a
= 10.7 Hz, H-1
0
b),
An ioxidan s 2022,11, 56 5 o 18
3.96 (dd, 1H, J
3,2a
= 4.5 Hz, J
3,2b
= 9.2 Hz, H-3), 3.89 (dd, 1H, J
6”a,5”
= 1.8 Hz, J
6”a,6”b
= 11.9
Hz, H-6”a), 3.76 (s, 3H, C6
0
-OCH
3
), 3.71 (s, 3H, C11-OCH
3
), 3.67 (dd, 1H, J
6”b,5”
= 5.7
Hz, J
6”b,6”a
= 11.9 Hz, H-6”b), 3.37 (m, 4H, H-2”, H-3”, H-4”, H-5”), 2.85 ( , 2H, J
20,10
= 6.9
Hz, H-2
0
), 2.70 (dd, 1H, J
2a,3
= 4.5 Hz, J
2a,2b
= 14.2 Hz, H-2a), 2.44 (dd, 1H, J
2b,3
= 9.2 Hz,
J
2b,2a
= 14.2 Hz, H-2b), 1.64 (dd, 3H, J
6,7
= 1.5 Hz, J
6,5
= 7.0 Hz, H-6).
13
C NMR (100 MHz,
CDCl
3
) (Figu e S10):
δ
(ppm) 173.1 (C-1), 168.6 (C-11), 159.9 (C-6
0
), 155.1 (C-9), 131.3 (C-4),
131.0 (C-4
0
, C-8
0
), 130.5 (C-3
0
), 124.8 (C-5), 115.0 (C-5
0
, C-7
0
), 109.4 (C-10), 100.8 (C-1”), 95.1
(C-7), 78.5 (C-5”), 77.9 (C-3”), 74.8 (C-2”), 71.5 (C-4”), 66.8 (C-1
0
a,C-1
0
b), 62.8 (C-6”a, C-6”b),
55.7 (C6
0
- O
C
H
3
), 51.9 (C11-O
C
H
3
), 41.2 (C-2a, C-2b), 35.1 (C-2
0
), 31.8 (C-3), 13.5 (C-6).
[α]25
D
:
−
154.0 (c0.7, MeOH). NMR da a o his compound ag ee wi h hose epo ed in
he li e a u e [
20
]. The pu i y o (
−
)-me hyl-ligus oside (90%) was de e mined using he
ligus oside’s calib a ion cu e desc ibed abo e.
Finally, o ob ain me -OLE, a mix u e o me hyl-ligus oside (100 mg), wa e (5 mL)
and DMSO (0.4 mL) was subjec ed o mic owa e i adia ion o 9 min a 180
◦
C. The
eac ion was moni o ed by TLC and HPLC. Then, he c ude was ex ac ed wi h E
2
O
and he sol en was emo ed unde educed p essu e o gi e a b own oil (58 mg). The
pu i ica ion was achie ed by FCPC using Hex:E OAc:E OH:H
2
O (1:1:1:1, / / / ) as a
biphasic sol en sys em. As a esul , 39 mg (64% yield) o me -OLE as a yellow oil was
ob ained: ESIMS: m/z341 [M + Na]
+
(Figu e S7): HRMS (ESI/Q-TOF) m/z319.1540
[M + H]
+
co esponding o C
18
H
22
O
5
(Figu e S8);
1
H NMR (400 MHz, CDCl
3
) (Figu e S3):
δ
(ppm) 9.63 (b s, 1H, H-8), 9.23 (d, 1H, J
9,3
= 2.0 Hz, H-9), 7.10 (m, 2H, H-4
0
,H-8
0
), 6.83 (m,
2H, H-5
0
,H-7
0
), 6.62 (q, 1H, J
5,6
= 7.1 Hz, H-5), 4.19 (m, 2H, H-1
0
), 3.78 (s, 3H, C6
0
-OC
H3
),
3.61 (m, 1H, H-3), 2.95 (ddd, 1H, J
7b,8
= 1.2 Hz, J
7b,3
= 8.7 Hz, J
7b,7a
= 18.3 Hz, H-7b), 2.83 ( ,
2H, J
20,10
= 7.0 Hz, H-2
0
), 2.73 (ddd, 1H, J
7a,8
= 1.0 Hz, J
7a,3
= 5.5 Hz, J
7a,7b
= 18.3 Hz, H-7a),
2.68 (dd, 1H, J
2b,3
= 8.2 Hz, J
2b,2a
= 15.9 Hz, H-2b), 2.61 (dd, 1H, J
2a,3
= 6.7 Hz, J
2a,2b
= 15.9 Hz,
H-2a), 2.07 (d, 3H, J
6,5
= 7.1 Hz, H-6).
13
C NMR (100 MHz, CDCl
3
):
δ
(ppm) 200.5 (C-8),
195.2 (C-9), 172.0 (C-1), 158.5 (C-6
0
), 154.3 (C-5), 143.4 (C-4), 130.0 (C-4
0
,C-8
0
), 129.8 (C-3
0
),
114.1 (C-5
0
,C-7
0
), 65.2 (C-1
0
), 55.4 (C6
0
-O
C
H
3
), 46.3 (C-7), 37.0 (C-2), 34.3 (C-2
0
), 27.4 (C-3),
15.4 (C-6). The ull assignmen o 1H and 13C NMR esonances was suppo ed by DEPT
(Figu e S4), HSQC (Figu e S5) and HMBC (Figu e S6) spec al analyses.
[α]25
D
:
−
1.7 (c1.0,
CHCl
3
). The pu i y o me -OLE (97%) was de e mined by an ex e nal s anda d me hod
using he HPLC peak a ea a 280 nm. P e iously, a calib a ion cu e (y = 3121.8x
−
6706.6;
R
2
= 0.9974) (G aphic S2) was cons uc ed wi h six s anda ds (0.01–0.5 mg/mL in MeOH)
(Table S2) o pu e OLE a ailable in au ho s’ lab.
2.5. Animals
Swiss mice we e p o ided by Ha lan In e auna Ibé ica
®
(Ba celona, Spain) and
main ained in a empe a u e-, humidi y- and ligh -con olled oom, and we e allowed
ee access o wa e and ood. All animal p ocedu es ollowed we e in acco dance wi h
he ecommenda ions o he Eu opean Union on animal expe imen a ion (Di ec i e o he
Eu opean Counsel 2012/707/EU) and we e app o ed by he Animal E hics Commi ee o
he Uni e si y o Se illa (23 July 2018/119).
2.6. Mu ine Mac ophages and Spleen Cells Isola ion and Cul u e
Cells we e collec ed 72 h a e in ape i oneal s e ile hioglycola e injec ion (3.8%
w/ ) as p e iously desc ibed by Mon oya e al. (2019) [
14
]. The collec ed mac ophages
we e cul u ed wi h me -OLE (50, 25, 12.5
µ
M) p e- ea men s o 30 min (min) and hen
s imula ed wi h LPS om Esche ichia coli (5
µ
g/mL) (Sigma-Ald ich
®
, S . Louis, MO,
USA) incuba ing hem o 18 h (h) a 5% CO
2
37
◦
C. The supe na an s and cells samples
we e collec ed and s o ed a
−
80
◦
C un il cy okine measu emen and Wes e n blo ing,
espec i ely.
The mice spleens we e ha es ed and passed h ough a nylon cell s aine (BD
®
Biosciences, F anklin Lakes, NJ, USA) wi h supplemen ed RPMI1640 medium (10% e al
cal se um, 2 mM glu amine, 1 mM sodium py u a e, 50
µ
M 2-me cap oe hanol and 1%

An ioxidan s 2022,11, 56 6 o 18
penicillin and s ep omycin), in o de o ob ain a cell suspension. Pelle ed cells we e
esuspended in ed blood lysis bu e (BD
®
Biosciences, F anklin Lakes, NJ, USA) and
hen washed wi h phospha e bu e solu ion (PBS). Cells (1
×
10
6
cells/mL) we e le
un ea ed o ea ed wi h OLE (50
µ
M) o , me -OLE (50, 25, 12.5
µ
M) o 30 min and we e
LPS-s imula ed (5
µ
g/mL) du ing an 18 h pe iod a 5% CO
2
, a 37
◦
C. A e incuba ion, cell
pelle s and supe na an s we e collec ed and s o ed a
−
80
◦
C un il he quan i ica ion o
cy okine le els and his one ex ac ion, espec i ely.
2.7. Cell Viabili y
To e alua e me -OLE cy o oxici y, a sul o hodamine B (SRB) assay was pe o med [
21
].
An amoun o 1
×
10
5
cells/mL we e cul u ed in he p esence/absence o me -OLE
(200–1.6
µ
M) o 18 h. The abso bance was ead a 510 nm wi h a mic opla e eade
(Bio ad
®
, Mad id, Spain). Abso bance is exp essed as he pe cen age o iabili y when
compa ed o un ea ed con ol cells (100% cell su i al).
2.8. Ni ic Oxide P oduc ion
Ni i es le els, exp essed as he NO gene a ion index, we e quan i ied using G iess
eagen in cul u e supe na an s (Sigma-Ald ich
®
, S . Louis, MO, USA), ollowing he
p o ocol epo ed by Mon oya e al. (2018) [
22
]. A sodium ni a e cu e was used as a
s anda d o ex apola e he ni i e amoun , and he esul s we e exp essed as a pe cen age
compa ed wi h DMSO-LPS ea ed cells (100% ni i es p oduc ion).
2.9. In acellula ROS P oduc ion
The DCDFA assay ki was pe o med acco ding o he manu ac u e ’s ins uc ions
(Abcam
®
, Camb idge, UK). Cells (2.5
×
10
5
cells/mL) we e seeded on a black pla e and
hen DCDFA (25
µ
M) was added o each well ei he p e iously un ea ed o ea ed wi h
me -OLE (50, 25, 12.5
µ
M) and we e LPS-s imula ed du ing 18 h. The esul s we e exp essed
using H
2
O
2
(Sigma-Ald ich
®
, Ba celona, Spain) as a posi i e p o-oxidan con ol (100%
in acellula ROS p oduc ion), compa ing luo escence in ensi y [14].
2.10. His one Ex ac ion
Acid ex ac ion was pe o med as p e iously epo ed by Hajji e al. (2010) [
5
] wi h
b ie modi ica ions. The collec ed spleen cells we e washed wi h PBS, suspended in lysis
bu e (10mM T is pH 6.5, 50 mM sodium bisul a e, 10 nM MgCl
2
, 8.6% suc ose, 1% T i on
X-100) and incuba ed 15 min a 4
◦
C. A e ha , he samples we e cen i uged a 3500 pm
o 10 min a 4
◦
C and ewashed. The supe na an was hen emo ed and disca ded and
T is–EDTA bu e (10 mM T is pH 7.4 and 13 mM EDTA) was added o he samples. The
p ecipi a ed nucleus was esuspended wi h acid sul u ic 0.2M. A e 1 h incuba ion ime,
samples we e cen i uged a 15,000 pm o 1 h a 4C, sal ing supe na an s o incuba ion
wi h ace one o e nigh a
−
20
◦
C. Cen i uged samples we e dilu ed wi h H
2
O and p o ein
con en was measu ed.
2.11. Enzyme-Linked Immunoso ben Assay
IL-6, IFN-
γ
(Diaclone
®
, Besancon Cedex, F ance), IL-1
β
(R&D Sys em
®
, Minneapolis,
Cánada, USA), TNF-
α
and IL-17 (Pep o ech
®
, London, UK) concen a ions in cul u e media
we e measu ed using speci ic enzyme-linked immunoso ben assay (ELISA) ki s.
2.12. Wes e n Blo ing
Whole cell lysa es, p epa ed as desc ibed by Mon oya e al. (2019) [
14
], and ex ac ed
his ones we e p o ided in o 25
µ
g p o ein aliquo s. P o ein samples we e sepa a ed
by SDS-PAGE (15 o 10%) and elec oblo ed on o ni ocellulose memb anes. Speci ic
p ima y an ibodies we e used. The memb anes we e hen incuba ed wi h he co esponding
seconda y an ibody o 2 h. The esul s we e ob ained om a leas six independen
expe imen s. A chemiluminescence ligh de ec ion ki (Pie ce
®
, Rock o d, IL, USA) and
An ioxidan s 2022,11, 56 7 o 18
Ame sham Image 600 equipmen (GE Heal hca e
®
, Chicago, IL, USA) we e used o he
de ec ion o immunosignals. Da a we e no malized wi h a housekeeping con ol and
quan i ied by Image P ocessing and Analysis in Ja a (Image J®, Be hesda, MD, USA).
2.13. S a is ical Analysis
Da a in igu es and ex a e epo ed as a i hme ic means
±
s anda d e o (SEM) om
a leas six independen expe imen s ca ied ou in iplica e. Resul s we e e alua ed using
G aph Pad P ism e sion 5.01 so wa e (San Diego, CA, USA), analyzing he s a is ical
signi icance by a one-way analysis o a iance (ANOVA), ollowed by he Tukey’s mul iple
compa isons es . The p- alues < 0.05 we e conside ed s a is ically signi ican . Figu es
om densi ome y expe imen s a e ep esen a i es o di e en expe imen s pe o med on
a di e en day.
3. Resul s
3.1. Chemis y
The syn hesis o (
−
)-me -OLE was ca ied ou he ein om (
−
)-ligus oside (Figu e 1),
inspi ed by he eac ion epo ed by Skal sounis’ g oup o he con e sion o oleu opein
(same s uc u e as ligus oside bu wi h an addi ional OH a C-5
0
) in o oleacein (same
s uc u e as OLE bu wi h an addi ional OH a C-5
0
) [
23
]. These au ho s achie ed he semi-
syn hesis o oleacein om oleu opein unde K apcho deca bome hoxyla ion condi ions in
one single s ep. They e luxed oleu opein, dissol ed in we DMSO, wi h wo equi alen s
o an ino ganic sal (NaCl) o 10 h o ob ain oleacein wi h a 21% yield, a e pu i ica ion
by silica gel column ch oma og aphy. P ocopio’s g oup la e imp o ed his a ac i e
semi-syn hesis h ough changing DMSO by wa e and hea ing in a mic owa e eac o o
ge oleacein in jus 20 min wi h a 48% yield [
24
]. Ou g oup was also wo king o imp o e
he semi-syn hesis o oleacein and OLE om oleu opein and ligus oside, espec i ely,
ollowing he ecommenda ions o a p e ious wo k aimed a adap ing he classical K apcho
deca boxyla ion expe imen al condi ions o an aqueous mic owa e scena io [
25
], when
P ocopio’s wo k came o ligh (unpublished esul s).
An ioxidan s 2022, 11, x FOR PEER REVIEW 8 o 19
In o de o pu i y he inal compound and aking in o accoun he known sensi i i y
o ela ed OLE and oleacein o decomposi ion when in con ac wi h solid s a iona y
phases, such as silica gel [27], and upon exposu e o oxygen and ligh [12], we used he
FCPC echnique o he pu i ica ion o me -OLE. This allowed us o ob ain pu e me -OLE
wi h he same success as when we p e iously pu i ied OLE om an oli e oil phenolic
ex ac [18]. This silica- ee ch oma og aphic echnique has shown o be a good op ion o
isola e hese compounds [28] and consequen ly imp o e he yield o he eac ion s ep.
Thus, we ha e pe o med he con e sion o me hyl-ligus oside in o me -OLE in a 64%
yield, which is he bes yield epo ed o da e o he K apcho con e sion o ligus o-
side/oleu opein o hei de i a i es in o he co esponding dialdehydes. Me -OLE has
been syn hesized in his wo k o he i s ime.
Figu e 1. Syn hesis o (−)-me hyl-oleocan hal om (−)-ligus oside.
Reagen s and condi ions: (a)
CH
2
N
2
/E
2
O; (b) H
2
O-DMSO, MW, 180 °C, 9 min.
3.2. E ec s o Me -OLE on Cell Viabili y
Fi s , we e alua ed he cell iabili y a e me -OLE ea men s using a SRB assay.
Da a show ha me -OLE was no cy o oxic a e 18 h o ea men wi h concen a ions o
1.6 up o 200 μM and did no comp omise he cell iabili y signi ican ly (≥80%) (Figu e 2).
The e o e, based on ou p e ious wo k using OLE [14], we selec ed 12.5, 25 and 50 μM
concen a ions o me -OLE o be s udied in he ollowing assays.
Figu e 2. E ec o me -OLE on cell iabili y. Mac ophages we e p e ea ed wi h me -OLE (200–1.6
μM) o 18 h. Cell su i als we e exp essed as he pe cen age o iabili y wi h espec o 100% om
con ol, un ea ed cells. Resul s a e p esen ed as mean ± SEM o a leas six independen expe i-
men .
3.3. E ec s o Me -OLE on IL-1β, IL-6, IL-17, IFN-γ and TNF-α P oduc ion
To explo e he e ec s o me -OLE on p o-in lamma o y cy okine p oduc ion, we
e alua ed IL-1β, IL-6, IL-17, IFN-γ and TNF-α le els. As shown in Figu e 3, a e 18 h o
   










μ

 
Figu e 1.
Syn hesis o (
−
)-me hyl-oleocan hal om (
−
)-ligus oside. Reagen s and condi ions:
(a) CH2N2/E 2O; (b) H2O-DMSO, MW, 180 ◦C, 9 min.
Thus, ou syn hesis o me -OLE (Figu e 1) s a ed wi h he isola ion o ligus oside
om oli e wood ollowing a p ocedu e p e iously epo ed by us [
19
]. Then, ligus oside
was me hyla ed using diazome hane, p epa ed in si u om N-me hyl-N-ni osou ea. The
con e sion o ligus oside in o me hyl-ligus oside was quan i a i ely pe o med a oom
empe a u e o a ound 1 h. I he s a ing ma e ial (ligus oside) had been pu e enough,
me hyl-ligus oside would ha e been su icien ly pu e o be used di ec ly in he nex s ep.
Howe e , ligus oside was isola ed wi h a pu i y o 84% om he oli e wood ex ac
and i was con enien o submi c ude me hyl-ligus oside o pu i ica ion, in o de o
elimina e he mino componen s ha accompanied he s a ing ma e ial (ligus oside). On
his occasion, we used as cen i ugal pa i ion ch oma og aphy (FCPC) o ge adequa ely
pu e me hyl-ligus oside in one single un. Then, pu e me hyl-ligus oside was submi ed
o he K apcho eac ion unde mic owa e i adia ion. Se e al a emp s we e ca ied ou ,
wi h a ying empe a u es, eac ions ime and sol en a ios, un il he bes condi ions
An ioxidan s 2022,11, 56 8 o 18
we e ound. Thus, he eac ion o me hyl-ligus oside in wa e , wi h he minimum amoun
o DMSO o dissol e he subs a e, was pe o med in a mic owa e eac o o 9 min a
180
◦
C, eaching he comple e con e sion o me hyl-ligus oside in o me -OLE. I is wo h
no ing ha his K apcho eac ion ook place in he absence o ino ganic sal , as Mu ph ee’s
g oup [
25
] came o obse e in some cases and as Fe nández-Bolaños’ g oup has ecen ly
claimed [26].
In o de o pu i y he inal compound and aking in o accoun he known sensi i i y
o ela ed OLE and oleacein o decomposi ion when in con ac wi h solid s a iona y phases,
such as silica gel [
27
], and upon exposu e o oxygen and ligh [
12
], we used he FCPC
echnique o he pu i ica ion o me -OLE. This allowed us o ob ain pu e me -OLE wi h he
same success as when we p e iously pu i ied OLE om an oli e oil phenolic ex ac [
18
].
This silica- ee ch oma og aphic echnique has shown o be a good op ion o isola e hese
compounds [
28
] and consequen ly imp o e he yield o he eac ion s ep. Thus, we ha e
pe o med he con e sion o me hyl-ligus oside in o me -OLE in a 64% yield, which is
he bes yield epo ed o da e o he K apcho con e sion o ligus oside/oleu opein o
hei de i a i es in o he co esponding dialdehydes. Me -OLE has been syn hesized in
his wo k o he i s ime.
3.2. E ec s o Me -OLE on Cell Viabili y
Fi s , we e alua ed he cell iabili y a e me -OLE ea men s using a SRB assay.
Da a show ha me -OLE was no cy o oxic a e 18 h o ea men wi h concen a ions o
1.6 up o 200
µ
M and did no comp omise he cell iabili y signi ican ly (
≥
80%) (Figu e 2).
The e o e, based on ou p e ious wo k using OLE [
14
], we selec ed 12.5, 25 and 50
µ
M
concen a ions o me -OLE o be s udied in he ollowing assays.
An ioxidan s 2022, 11, x. h ps://doi.o g/10.3390/xxxxx www.mdpi.com/jou nal/an ioxidan s
Figu e 2.
E ec o me -OLE on cell iabili y. Mac ophages we e p e ea ed wi h me -OLE (200–1.6
µ
M) o
18 h. Cell su i als we e exp essed as he pe cen age o iabili y wi h espec o 100% om con ol,
un ea ed cells. Resul s a e p esen ed as mean ±SEM o a leas six independen expe imen .
3.3. E ec s o Me -OLE on IL-1β, IL-6, IL-17, IFN-γand TNF-αP oduc ion
To explo e he e ec s o me -OLE on p o-in lamma o y cy okine p oduc ion, we
e alua ed IL-1
β
, IL-6, IL-17, IFN-
γ
and TNF-
α
le els. As shown in Figu e 3, a e 18 h
o exposu e o LPS, mu ine cells exhibi ed highe le els o p o-in lamma o y cy okines
han uns imula ed con ol cells (++ p< 0.01; +++ p< 0.001 s. uns imula ed cells). On
he con a y, when cells we e ea ed wi h me -OLE, we obse ed a signi ican ly down-
egula ion o IL-1
β
, IL-6, IL-17, IFN-
γ
and TNF-
α
sec e ions when compa ed o he LPS-
DMSO g oup (** p< 0.01; *** p< 0.001 s. LPS-DMSO s imula ed cells).
An ioxidan s 2022,11, 56 9 o 18
3.4. E ec s o Me -OLE on In acellula ROS and NO P oduc ions
In e ms o elucida ing he ole o me -OLE in he oxida i e and in lamma o y esponse
media ed by LPS, we measu ed in acellula ROS and NO le els using DCFDA and G iess
assays, espec i ely, in LPS-induced mu ine pe i oneal mac ophages.
Da a e ealed ema kable o e p oduc ions o ROS and NO induced by LPS in mu ine
mac ophages when compa ed o uns imula ed cells (+ p< 0.05; ++ p<0.01;
+++ p< 0.001 s. uns imula ed con ol cells) (Figu e 4A,B). Meanwhile, le els o bo h
media o s we e signi ican ly educed a e me -OLE ea men s (** p< 0.01; *** p< 0.001 s.
cells s imula ed cells).
An ioxidan s 2022, 11, x FOR PEER REVIEW 9 o 19
exposu e o LPS, mu ine cells exhibi ed highe le els o p o-in lamma o y cy okines han
uns imula ed con ol cells (++ p < 0.01; +++ p < 0.001 s. uns imula ed cells). On he con-
a y, when cells we e ea ed wi h me -OLE, we obse ed a signi ican ly
down- egula ion o IL-1β, IL-6, IL-17, IFN-γ and TNF-α sec e ions when compa ed o he
LPS-DMSO g oup (** p < 0.01; *** p < 0.001 s. LPS-DMSO s imula ed cells).
Figu e 3. P o-in lamma o y cy okine le els we e down- egula ed in me -OLE ea ed cells. Mac-
ophages we e p e ea ed wi h me -OLE (50, 25 o 12.5 μM) o 30 min and hen, LPS-s imula ed
du ing 18 h. (A) IL-1β, (B) IL-6, (C) IL-17, (D) IFN-γ and (E) TNF-α le els we e measu ed by ELISA
on cell supe na an s. Resul s a e p esen ed as he mean ± SEM o a leas six independen expe i-
men s. ++ p < 0.01; +++ p < 0.001 s. uns imula ed con ol cells; ** p < 0.01; *** p < 0.001 s. LPS-DMSO
s imula ed cells.
3.4. E ec s o Me -OLE on In acellula ROS and NO P oduc ions
In e ms o elucida ing he ole o me -OLE in he oxida i e and in lamma o y e-
sponse media ed by LPS, we measu ed in acellula ROS and NO le els using DCFDA
and G iess assays, espec i ely, in LPS-induced mu ine pe i oneal mac ophages.
Da a e ealed ema kable o e p oduc ions o ROS and NO induced by LPS in mu-
ine mac ophages when compa ed o uns imula ed cells (+ p < 0.05; ++ p <0.01; +++ p < 0.001
s. uns imula ed con ol cells) (Figu e 4A,B). Meanwhile, le els o bo h media o s we e
signi ican ly educed a e me -OLE ea men s (** p < 0.01; *** p < 0.001 s. cells s imu-
la ed cells).
B
CD
A
E
Figu e 3.
P o-in lamma o y cy okine le els we e down- egula ed in me -OLE ea ed cells.
Mac ophages we e p e ea ed wi h me -OLE (50, 25 o 12.5
µ
M) o 30 min and hen, LPS-s imula ed
du ing 18 h. (
A
) IL-1
β
, (
B
) IL-6, (
C
) IL-17, (
D
) IFN-
γ
and (
E
) TNF-
α
le els we e measu ed by ELISA
on cell supe na an s. Resul s a e p esen ed as he mean
±
SEM o a leas six independen expe i-
men s. ++ p< 0.01; +++ p< 0.001 s. uns imula ed con ol cells; ** p< 0.01; *** p< 0.001 s. LPS-DMSO
s imula ed cells.
3.5. Me -OLE Down-Regula ed iNOS, COX-2 and mPGES-1 P o ein O e exp ession Induced by
LPS in Mu ine Mac ophages
To in es iga e whe he he e ec s o me -OLE on NO accumula ion we e associa ed
wi h he exp ession o he iNOS p o ein, we pe o med an immunoblo ing assay wi h cell
lysa es. Consis en ly, cells om secoi idoid- ela ed phenolic g oups showed dec eased
iNOS p o ein o e exp ession when compa ed o hose o he LPS-DMSO exposed g oup
(*** p< 0.001 s. LPS-DMSO s imula ed cells) (Figu e 4C).
In addi ion, o gain u he insigh in o he an i-in lamma o y po en ial o me -OLE, we
s udied i s e ec s on COX-2 and PGE
2
ela ed bioma ke s. As expec ed, a e LPS exposu e,
COX-2 and mPGES-1 exp essions inc eased signi ican ly (+++ p< 0.001 s. uns imula ed
con ol cells). On he con a y, me -OLE p e ea men s e ec i ely coun e ac ed induc ion o
An ioxidan s 2022,11, 56 16 o 18
5. Conclusions
Collec i ely, his s udy epo s, o he i s ime, he semi-syn hesis o a new me hy-
la ed OLE me aboli e and i s an ioxidan and an i-in lamma o y e ec s in he esponse
o mu ine mac ophages o LPS exposu e. Gene ally, me -OLE inhibi ed he exp ession o
p o-in lamma o y enzymes, (iNOS, COX-2 and mPGES-1), cy okines (IL-6, IL-17, IL-1
β
,
TNF-
α
, IFN-
γ
and IL-18) and egula ed in acellula ROS and p oduc s ha a e ela ed o
oxida i e damage, such as NO le els, ia MAPKs, N -2/HO-1 and bo h canonical and
non-canonical in lammasome signal pa hway modula ion. Fu he mo e, bo h OLE and
me -OLE we e able o egula e epigene ic mechanisms by modula ing his one me hyla-
ion (H3K9me3 and adnH3K27me) and ace yla ion (H3K18ac) and by down- egula ing
cy okine- ela ed p oduc ion in LPS-exposed spleen immune cells.
This e ealing e idence sugges s ha he me hyla ed me aboli e o OLE may con-
ibu e signi ican ly o he bene icial e ec s ha a e associa ed wi h he secoi idoid- ela ed
compound and he usual consump ion o EVOO. Thus, me -OLE could be a p omising
he apeu ic agen used in a ious immune—in lamma o y pa hologies, and he elucida ion
o hei s uc u e—ac i i y ela ionship migh be a ele an goal o u u e di ec ions.
Supplemen a y Ma e ials:
Supplemen a y ma e ials a e a ailable online h ps://www.mdpi.com/
a icle/10.3390/an iox11010056/s1, syn hesis o diazome hane’s p ecu so ; Wes e n Blo ing; Cali-
b a ion cu es: G aphics S1 and S2, Tables S1 and S2; Spec oscopy da a o OLE (Figu es S1 and S2),
me -OLE (Figu es S3–S8) and me hyl-ligus oside (Figu es S9–S11).
Au ho Con ibu ions:
Concep ualiza ion, M.S.-H. me hodology, T.M.; expe imen a ion con ibu ion,
M.L.C.; p oduc ex ac ions and chemical syn hesis, J.O.-V. and J.A.; so wa e, alida ion and o mal
analysis, T.M.; in es iga ion, T.M.; da a cu a ion, T.M.; w i ing—o iginal d a p epa a ion, T.M.
and M.S.-H.; w i ing— e iew and edi ing, C.A.-d.-l.-L., T.M., J.A. and M.S.-H.; isualiza ion and
supe ision, C.A.-d.-l.-L. and M.S.-H. All au ho s ha e ead and ag eed o he published e sion o
he manusc ip .
Funding:
This esea ch was suppo ed by Minis e io de Economía y Compe i i idad o Spain, g an
numbe AG-2017-89342-P and, Jun a de Andalucía unded by CTS-259, FQM-182.
Ins i u ional Re iew Boa d S a emen :
The s udy was conduc ed acco ding o he guidelines o he
Eu opean Union ega ding animal expe imen a ion (Di ec i e o he Eu opean Counsel 2012/707/EU)
and we e app o ed by he Animal E hics Commi ee o he Uni e si y o Se illa (23 July 2018/119).
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.
Acknowledgmen s:
The au ho s g a e ully acknowledge he assis ance o Cen e o Technology and
Inno a ion Resea ch, Uni e si y o Se ille (CITIUS) and, he Technical Scien i ic Ins umen a ion
Cen e (CICT) o he Uni e si y o Jaen. T. Mon oya hanks suppo om a Pos g adua e P og am o
PIF ellowship and inancial sponso ship om VI Plan P opio de In es igación y T ans e encia a
Uni e sidad de Se illa.
Con lic s o In e es : The au ho s decla e no con lic o in e es .
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