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Ci e his: DOI: 10.1039/c0xx00000x
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Hyd oxy y osol and y osol sul a e me aboli es p o ec agains oxidized
choles e ol p o-oxidan e ec in Caco-2 human en e ocy e-like cells.
Angela A ze ia, Rica do Lucasb, Alessand a Incania, Pablo Peñal e c, Albe o Za a-Gómezd, M. Paola
Melisa, Robe o Pizzalae, Juan C. Mo alesb,c* and Monica Deianaa*
Recei ed (in XXX, XXX) X h XXXXXXXXX 20XX, Accep ed X h XXXXXXXXX 20XX
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DOI: 10.1039/b000000x
Abs ac
The aim o his s udy was o in es iga e he abili y o sul a e me aboli es o hyd oxy y osol (HT) and
y osol (TYR) o ac as an ioxidan s coun e ac ing he p o-oxidan e ec o oxidized choles e ol in
in es inal cells. Fo his pu pose, we syn hesized sul a e me aboli es o HT and TYR using chemical
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me hodology and examined hei an ioxidan ac i i y in Caco-2 monolaye s in compa ison wi h he pa en
compounds. Exposu e o oxidized choles e ol led o ROS p oduc ion, oxida i e damage, as indica ed by
MDA inc ease, a dec ease o educed glu a hione concen a ion and an enhancemen o glu a hione
pe oxidase ac i i y. All he es ed compounds we e able o coun e ac he oxidizing ac ion o oxidized
choles e ol; HT and TYR sul a e me aboli es showed an e iciency in p o ec ing in es inal cells
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compa able o ha o he pa en compounds, s eng hening he assump ion ha he po en ial bene icial
e ec o he pa en compounds is e ained, al hough ex ensi e me abolisa ion occu s, being he esul ing
me aboli es able o exe a biological ac ion hemsel es.
Keywo ds:
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sul a e me aboli es, oxida i e s ess, oxidized choles e ol, oli e oil phenolic compounds
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In oduc ion
Polyphenols p esen in ex a i gin oli e oil, he p incipal a componen o he Medi e anean die , ha e been demons a ed o exe
many po en ially bene icial biological e ec s due o, a leas in pa , hei abili y o p o ec agains oxida i e s ess media ed damage 1-2.
Hyd oxy y osol (3,4-dihyd oxyphenyle hanol; HT) and y osol (4-hyd oxyphenyle hanol; TYR) (Fig. 1) a e he mos biologically ac i e
phenolic alcohols p esen in he oil, whe e hey exis in simple o ms o as conjuga es (such as oleu opein); hey a e s uc u ally iden ical
5
excep ha HT has an ex a hyd oxyl g oup in he me a posi ion.
10
Fig. 1 S uc u e o y osol (TYR), hyd oxy y osol (HT) and hei sul a e me aboli es 3-5
The li e a u e on phenolic abso p ion and me abolism a e oli e oil inges ion is ex ensi e 3-4; howe e , he e a e s ill se e al issues o be
cla i ied. Some complex oli e oil polyphenols, among he secoi idoids, a e ela i ely s able unde gas ic condi ions and each he
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in es ine whe e hey may be di ec ly abso bed o me abolised unde abso p ion5; mos o he complex oli e oil polyphenols howe e
seems o unde go gas oin es inal bio ans o ma ion (see de Bock o an excellen e iew ) 6, inc easing he ela i e amoun o simple
phenols, mainly TYR and HT, en e ing he small and la ge in es ine 7. A his loca ion he concen a ion o simple phenols may be qui e
high, in he high M ange 7, and hey migh exe di ec p o ec i e e ec s by sca enging eac i e species and/o p e en ing hei
o ma ion 8. Howe e , in he p ocess o c ossing en e ocy es oli e oil phenolic compounds a e subjec ed o classical phase I/II
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bio ans o ma ion and o an impo an i s pass me abolism, esul ing in almos unde ec able concen a ions o ee HT and TYR in body
luids 9.
Sul a ed and glucu onida ed HT and TYR a e he p edominan me aboli es ound in human plasma and u ine 10-11, and hey ha e also
been shown o concen a e in he in es inal epi helium, since glucu onida ion and sul a ion a e he majo pa hways o phase II xenobio ic
me abolism in he human in es ine 12-13. The po en ial heal h bene i s o HT, TYR and hei de i a i es a e likely o be due o bo h
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pa en al compounds and hei phase I and phase II majo me aboli es. Glucu onida ed me aboli es o HT showed a mo e e icien adical
sca enging po ency han HT i sel 11 and he abili y o p o ec enal cells 14 and e y h ocy es om oxida i e inju y was also be e han
he pa en compound 15. Howe e no da a a e a ailable up- o-da e conce ning he an ioxidan ac i i y o sul a e me aboli es o HT and
TYR.
In his s udy we in es iga ed he possible p o ec i e e ec o he sul a e me aboli es o TYR and HT, in compa ison wi h he pa en
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compounds, agains he oxida i e damage o in es inal mucosa due o oxidized choles e ol exposu e in he human colon adenoca cinoma
cell line, Caco-2. A e con luence, hese cells spon aneously unde go ull di e en ia ion in i o wi h en e ocy e-like ea u es 16. Caco-2
cells ha e been ecognized as a sui able model o e alua ing he e ec o nu ien componen s, o bo h no mal die a y cons i uen s and
oxican s, as oxidizing agen s 17. La ge amoun s o lipid oxida ion p oduc s become a ailable, o bo h exogenous and endogenous o igin,
a he le el o he in es inal mucosa. Die a y oxys e ols, de i ed om choles e ol deg ada ion and oxida ion a e p olonged s o age o
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cooking o oods ich in choles e ol, ha e ecen ly been shown o con ibu e o he onse and u he de elopmen o oxida i e s ess and
in lamma ion ela ed in es inal diseases 18-19. The majo oxys e ols ound in ood may con ibu e o oxida i e unbalance o he in es inal
epi helium by inducing he gene a ion o eac i e oxygen species (ROS) 18, 20. Ou pu pose was o in es iga e he abili y o sul a e
me aboli es o HT and TYR o ac as an ioxidan s coun e ac ing he oxys e ols induced changes o he cellula edox s a e and compa e
hem wi h hei pa en compounds. Fo his pu pose, we syn hesized sul a e me aboli es o HT and TYR using chemical me hodology
40
and e alua e hei up ake/s abili y in Caco-2 monolaye s. Cy o oxici y, p oduc ion o MDA and ROS species, le els o glu a hione and
glu a hione pe oxidase ac i i y we e measu ed o assess he p o ec ing e ec o he sul a e me aboli es in compa ison wi h he ( hei )
pa en oli e ( emo e) phenolic compounds.
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Ma e ials and me hods
P epa a ion o oxidized choles e ol
Oxidized choles e ol was p epa ed essen ially as p e iously desc ibed 21. In b ie , samples o 2 ml (2ml aliquo s) o pu e choles e ol
(Sigma Ald ich, S . Louis, MO) solu ion (5 mg/ml in E OH) we e d ied down in o ound-bo omed glass es ubes unde acuum and
5
hea ed in a 140 °C oil ba h in ai o 3 h. The oxidized mix u e was sepa a ed and analysed by GC-MS o he de e mina ion o he
ela i e oxys e ols. B ie ly, 2 L o he mix u e was dilu ed in 2 mL o E OH (1: 1000 dilu ion). A e e apo a ing unde a ni ogen
s eam, 50 L o py idine (Sigma Ald ich) and 50 L o N,O-Bis( ime hylsilyl) i luo oace amide wi h ime hylchlo osilane
(BSTFA:TMCS; 99: 1) (Sigma Ald ich) we e added, mixed and kep a 60 °C o 45 min. The de i a ized sample (1.5 L) was injec ed
in o an Agilen (Waldb onn, DE) GC-MS in spli less mode and he column empe a u e was p og ammed s a ing om 70 °C o 250 °C
10
(10 °C/minu e), a e up o 290 °C (5 °C/min) main ained o 10 min, ending a 300 °C (2 °C/min) o 5 min (du a ion abou 45 min
o al). The helium low was se a 1.3 mL/min. The speci ic pa ame e s o he ins umen we e as published by Calde on-San iago e al.
22 Spec a we e acqui ed in scan mode.
GS-MS analysis demons a ed ha he mix u e con ained 58.9% o choles e ol, 13.5% o 7-ke ocholes e ol, 9.54% o 7-
hyd oxycholes e ol, 5.6% o 6-hyd oxycholes e ol, 5.87% o 7-hyd oxycholes e ol, 4.5% o choles a-4,6-dien-3-ol and 2.1% o 3-
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ke o-4-choles ene.
Syn hesis o sul a e me aboli es
Gene al me hods.
HT and HT ace a e we e ob ained om Sep ox Bio ech (Mad id, Spain), TYR om Sigma Ald ich. All o he chemicals ob ained om
comme cial sou ces we e used wi hou u he pu i ica ion, unless o he wise no ed. All eac ions we e moni o ed by TLC on p ecoa ed
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Silica-Gel 60 pla es F254, and de ec ed by hea ing wi h Mos ain (500 mL o 10% H2SO4, 25g o (NH4)6Mo7O24•4H2O, 1g
Ce(SO4)2•4H2O). P oduc s we e pu i ied by lash ch oma og aphy wi h Me ck Silica gel 60 (200-400 mesh). High esolu ion mass
spec a we e ob ained on an ESI/quad upole Au oSpec-Q mass spec ome e . NMR spec a we e eco ded on 300 o 500 MHz
spec ome e s, a oom empe a u e o solu ions in CDCl3, o D2O. Chemical shi s a e e e ed o he sol en signal. Me aboli es we e
pu i ied by ch oma og aphy wi h Re e se Phase-C18 Silica gel. Da a we e p ocessed using manu ac u e so wa e, aw da a we e
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mul iplied by shi ed exponen ial window unc ion p io o Fou ie ans o m, and he baseline was co ec ed using polynomial i ing.
Gene al p ocedu e o he mic owa e-assis ed O-sul a ion.
Mic owa e based sul a ion eac ions we e pe o med using a mic owa e syn hesize in sealed eac ion essels. Phenolic de i a i es (1.0
equi ), sul u ioxide– ime hylamine complex, SO3.NMe3, (5 equi pe OH; being his complex p e iously washed wi h H2O, MeOH,
and CH2Cl2 and d ied unde high acuum) and a magne ic s i e ba we e placed in a 2-5 mL mic owa e eac ion ial and i ed wi h a
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sep um, which was hen pie ced wi h a needle. The closed ial was hen e acua ed in high acuum o 2 h. The mix u e was dissol ed in
d y CH3CN (2.0 mL) and NE 3 (0.3-1.0 mL) was hen added. Reac ion mix u e was subjec ed o mic owa e adia ion o 20–40 min
(depending on he compound) a 100 ºC (50-60W a e age powe ). MeOH (1 mL) and CH2Cl2 (1 mL) we e added, and he solu ion was
laye ed on he op o a Sephadex LH-20 ch oma og aphy column which was elu ed wi h CH2Cl2/MeOH (1:1) o ob ain he co esponding
ie hylammonium sal as a whi e powde (94–98% yield).
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T ie hylamine, 4-(2-(bu y yloxy)e hyl)phenyl sul a e sal (7). Ty osol bu y a e 6 23-24 (70 mg, 0.33 mmol) and SO3.NMe3 (233 mg,
1.7 mmol) we e submi ed unde sul a ion condi ions o 20 min. TLC (E hyl ace a e: MeOH, 10:1) showed he o ma ion o a majo
p oduc and comple e consump ion o he ini ial ma e ial. Sol en s we e emo ed and he c ude ex ac was pu i ied by sephadex LH-20
(CH2Cl2:MeOH, 1:1) o a o d 7 (92 mg, 98%) as a whi e powde . 1H-NMR (300 MHz, D2O) : 6.72, 7.18 (2d, 4H, J = 8.7 Hz, Ha om),
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4.24 ( , 2H, CH2OAc), 3.10 (q, 6H, -CH2CH3), 2.88 ( , 2H, CH2A ), 2.19 ( , 2H, J = 7.5Hz, CH2), 1.45 (m, 2H, CH2), 1.18 ( , 9H,
CH2CH3), 0.74 ( , 3H, CH3); 13C-NMR (125 MHz, D2O) δ: 177.1 (CO), 150.0, 136.2, 130.3 (2x CHa om), 121.3 (2x CHa om), 65.4
(CH2OAc), 46.5, 36.0 (CH2A ), 34.0, 18.3, 12.6, 8.0 (CH3). ESI-HRMS (ES-) Calcd. o C12H15O6S (M-H) 287.0589, Found: 287.0594.
Po assium 4-(2-hyd oxye hyl)phenyl sul a e (5). Compound 7 (97 mg, 0.32 mmol) was dissol ed in MeOH (10 mL) and K2CO3 (90
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mg, 0.66 mmol) was added. The eac ion mix u e was s i ed a oom empe a u e o 24 h and hen neu alized wi h IR 120 H+ esin.
Sol en was hen emo ed in acuum and he c ude ex ac was pu i ied by an RP-C18 column elu ed wi h H2O: MeOH ( om 100:0 o
70:30). F ac ions con aining he desi ed p oduc we e concen a ed and eeze-d ied a o ding compound 5 as a whi e solid (68 mg,
94%). 1H-NMR (400 MHz, D2O) : 7.18, 6.86 (2d, 4H, J = 8.4 Hz, Ha om), 3.78, 2,78 (2 , 4H, J = 6.7 Hz, CH2OH, CH2A ); 13C-
NMR (75 MHz, D2O) δ: 157 (Cq), 130.3 (2x CHa om), 129.8 (Cq), 117.1 (2x CHa om), 63.0 (CH2OH), 36.9 (CH2A ). ESI-HRMS (ES-)
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Calcd. o C8H9O5S (M-H) 217.0171, Found: 217.0171.
3-(( e -bu yldime hylsilyl)oxy)-4-hyd oxyphene hyl ace a e (9) and 4-(( e -bu yldime hylsilyl)oxy)-3-hyd oxyphene hyl ace a e
(10). To a solu ion o hyd oxy y osol ace a e 8 23-24 (223 mg, 1.13 mmol) in DMF (anhyd ous, 3 mL) cooled in an ice-wa e ba h unde
a gon we e added sequen ially e -bu yldime hylsilyl- i luo ome hanesul ona e (TBDMSOT , 287 L, 1.25 mmol, 1.10 equi ) and
55
diisop opyle hylamine ( i-P 2Ne , 265 L, 1.52 mmol, 1.35 equi ). The mix u e was allowed o s i o 30 min a 0 ºC, and TLC (hexane:
Jou nal Name
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e hyl ace a e 3:1) a ha poin indica ed ha he eac ion was comple e. The pale yellow eac ion mix u e was dilu ed wi h E OAc (100
mL), cas in o a sepa a o y unnel, and washed wi h wa e (2x50 mL), b ine (50 mL), and he o ganic phase was d ied (Na2SO4).
Fil a ion and concen a ion in acuum a o ded he c ude ex ac ha was pu i ied by lash column ch oma og aphy (hexane:e hyl
ace a e om 15:1 o 10:1) o a o d 9 and 10 (314 mg, 90%, powde ) like a egioisome ic mix u e in a io ~ 1:1 . 1H NMR (400 MHz,
CDCl3) δ 6.88 (d, 1H, J = 8.1 Hz, Ha om), 6.83 (s, 1H, Ha om), 6.77 (d, 1H, J = 8.4 Hz, Ha om), 6.73 (d, 1H, J = 8.4 Hz, Ha om),
5
6.71(s, 1H, Ha om), 6.62 (d, 1H, J = 8.1 Hz, Ha om ), 5.52, 5.45 (2s, 2H, 2xOH), 4.26 ( , 2H, J = 6.7 Hz, CH2OAc), 4.24 ( , 2H,
J = 6.4 Hz, CH2OAc), 2.86 ( , 2H, J = 6.7 Hz, CH2A ), 2,84 ( , 2H, J = 6.4 Hz, CH2A ), 2.07-2.05 (2s, 6H, CH3C=O), 1.05, 1.03
(2s, 18H, C(CH3)3 x2), 0.30, 0.29 (2s, 12H, -Si(CH3)2 x2); 13C-NMR (125 MHz, CDCl3) δ: 171.1, 171.0 (C=O), 147.1, 145.9, 142.3,
141.0, 131.8, 129.6 (Cqa om), 122.4, 120.2, 118.6, 117.7, 115.4, 114.8 (CHa om), 65.2, 65.0 (CH2OAc), 34.5 (2x CH2A ), 25.7
(C(CH3)3), 21.0, 20.9 (CH3C=O), 18.2 (C(CH3)3), -4.2 (Si(CH3)2); HRMS (ES+) Calcd. o C16H26O4NaSi (M+Na) 333.1498, Found:
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333.1508.
T ie hylamine, 5-(2-ace oxye hyl)-2-(( e -bu yldime hylsilyl)oxy)phenyl sul a e sal (11) and ie hylamine, 4-(2-ace oxye hyl)-2-
(( e -bu yldime hylsilyl)oxy)phenyl sul a e sal (12). Regioisome ic mix u e o compounds 9 and 10 (157 mg, 0.506 mmol) and
SO3.NMe3 (351 mg, 2.52 mmol) we e submi ed unde sul a ion condi ions o 2x20 min. TLC (E hyl ace a e:MeOH 10:1) showed he
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o ma ion o a majo p oduc and comple e consump ion o he s a ing ma e ial. Sol en s we e emo ed and c ude was pu i ied by
sephadex LH-20 in a sol en mix u e o CH2Cl2: MeOH 1:1 o a o d 11 and 12 (231 mg, 94%, powde ) like a egioisome ic mix u e in
a io ~ 1:1. 1H-NMR (400 MHz, CDCl3) δ: 7.50 (d, 1H, J = 7.9 Hz, Ha om), 7.46 (s, 1H, Ha om), 6.82 (d, 1H, J = 8.2 Hz, Ha om), 6.78
(d, 1H, J = 8.2 Hz, Ha om), 6.73 (d, 1H, J = 7.9 Hz, Ha om), 6.72 (s, 1H, Ha om), 4.20 ( , 4H, J = 7.08 Hz, CH2OAc), 3.10-3.00 (dq,
12H, CH2CH3), 2.83 ( , 2H, J = 7.1 Hz, CH2A ), 2.82 ( , 2H, J = 7.05 Hz, CH2A ), 2.04, 2.03 (2s, 6H, CH3C=O), 1.26 ( , 18H, CH2CH3),
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1.00, 0.99 (2s, 18H, C(CH3)3 x2), 0.21, 0.20 (2s, 12H, -Si(CH3)2 x2); 13C-NMR (125 MHz, CDCl3) δ: 171.0, 170.9 (C=O), 146.8, 145.6,
143.9, 142.8, 134.0, 130.7 (Cqa om), 124.5, 122.4, 121.9, 121.8, 121.6, 121.0 (CHa om), 65.1 (CH2OAc), 46.3 (CH2CH3), 34.5,
(CH2A ), 25.7 (C(CH3)3), 21.0 (CH3C=O), 18.8 (C(CH3)3), 8.8 (CH2CH3), -4.2 (Si(CH3)2); ESI-HRMS (ES-) Calcd. o C16H25O7SiS (M-
H) 389.1090, Found: 389.1092.
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Po assium 2-hyd oxy-4-(2-hyd oxye hyl)phenyl sul a e (3) and po assium 2-hyd oxy-5-(2-hyd oxye hyl)phenyl sul a e (4).
Regioisome ic mix u e o 11 and 12 (231 mg, 0.47 mmol), po assium luo ide (KF,55 mg, 0.94 mmol) and po assium ca bona e (K2CO3,
130 mg, 0.94mmol) we e dissol ed in MeOH (10 mL). The eac ion mix u e was s i ed a oom empe a u e o 18 h and sol en was
hen emo ed in acuum. The c ude ex ac was pu i ied by column ch oma og aphy wi h RP-C18 silica gel elu ing wi h H2O: MeOH
( om 100:0 o 70:30). F ac ions con aining he desi ed p oduc we e concen a ed and eeze-d ied a o ding compounds 3 and 4 (115
30
mg, 90%, whi e powde ) like a egioisome ic mix u e in a io ~ 1:1. 1H-NMR (300 MHz, D2O) : 7.21 (d, 2H, J = 8.0 Hz, Ha om), 7.17
(s, 1H, Ha om), 6.98 (d, 1H, J = 8.5 Hz, Ha om), 6.88 (d, 1H, J = 8.5 Hz, Ha om), 6.80 (s, 1H, Ha om), 6.70 (d, 1H, J = 8.0 Hz, Ha om),
3.76-3.69 (m, 4H, CH2OAc), 2.73-2.70 (m, 4H, CH2A ); 13C-NMR (125 MHz, D2O) δ: 149.4, 147.4, 139.0, 138.4, 137.8, 130.7, 127.6,
123.0, 122.6, 120.0, 118.2, 117.6, 62.6, 62.4 (CH2OAc), 37.4, 36.9 (CH2A ). HRMS-ESI (ES-) Calcd. o C8H9O6S (M-H) 233.0120,
Found: 233.0126.
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Cell cul u e
Cell cul u e ma e ials we e pu chased om In i ogen (Milano, I aly). Caco-2 cells we e ob ained om he Eu opean Collec ion o Cell
Cul u es (ECACC, Salisbu y UK) and g own in Dulbecco’s modi ied Eagle’s medium (DMEM), supplemen ed wi h 2.5% o hea -
inac i a ed bo ine se um and 100U/mL penicillin and 100 μg/mL s ep omycin, a 37 °C unde a humidi ied a mosphe e o 95% ai and
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5% CO2. Fo expe imen al s udies Caco-2 cells, a passage 45–60, we e pla ed a a densi y o abou 1 × 105/mL and used 21 days pos
seeding
Cy o oxic ac i i y
The cy o oxic e ec caused by he exposu e o inc easing concen a ions o oxidized choles e ol and he p o ec i e e ec o HT, TYR,
and sul a e me aboli es was assessed on Caco-2 cells, seeded in 24-well pla es, by he Neu al Red me hod 25. Cells we e exposed o
45
oxidized choles e ol (0–175 μg/mL) in comple e medium and incuba ed o 24 h. In o de o assess he p o ec i e e ec o he phenolic
compounds cells we e p e ea ed wi h he es ed compounds (2,5-10 µM in wa e solu ion, 30 min) p io o oxidized choles e ol exposu e
(100 μg/mL o 24 h). A e incuba ion, he medium was emo ed; a Neu al Red Solu ion (Sigma Ald ich) (0.033% in medium) was
hen added o he wells o de e mine cell iabili y. A e 30 min o incuba ion, he Neu al ed solu ion was ca e ully emo ed and cells
quickly insed wi h PBS. The inco po a ed dye was hen solubilised in Neu al Red Solubilisa ion Solu ion, ace ic acid/e hanol /wa e
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(1/45/50, / / ), and he abso bance was measu ed a 540 nm.
De e mina ion o MDA
MDA le els we e de e mined in he medium o ea ed cells by he TBARS es wi h HPLC quan i ica ion, using he me hod desc ibed
by Templa e al. 26, wi h some modi ica ions. B ie ly, 100 μL o 10% ichlo oace ic acid (TCA, Sigma Ald ich) was added o 400 μL o
5
he medium, samples we e mixed and le a oom empe a u e. A e 20 min, 200 μL o 2- hioba bi u ic acid (TBA, Sigma Ald ich)
(0.6%) we e added; samples we e incuba ed a 90 °C o 45 min and hen cen i uged a 5000 x g o 15 min a 4 °C. Aliquo s o he
supe na an we e injec ed in o an Agilen 1100 HPLC sys em (Agilen Technologies, San a Cla a, CA) equipped wi h a diode-a ay
de ec o (HPLC–DAD) and sepa a ion was achie ed using a Va ian (Middelbu g, The Ne he lands) Ine sil 5 ODS-2, 150 × 4.6 mm
column; he mobile phase was a mix u e o KH2PO4 50 mM pH 7/MeOH (65/35, / ) a a cons an low a e o 1 mL/min. The o ma ion
10
o he MDA–TBA adduc was e ealed measu ing i s abso bance a 532 nm. A s anda d cu e was p epa ed using a 1,1,3,3,-
e ae hoxyp opane (TEP, Sigma Ald ich) solu ion in PBS (0.05–10 μM).
ROS p oduc ion in Caco-2 cells
In acellula ROS p oduc ion was moni o ed by adding he 2',7'-dichlo odihyd o luo escein diace a e (H2-DCF-DA, Sigma Ald ich),
acco ding o Dinicola e al. 27, in Caco-2 cells exposed o oxidized choles e ol (75 μg/mL) in comple e medium and incuba ed o 0-180
15
min and in cells p e ea ed wi h he es ed phenolic compounds (5-25 µM in wa e solu ion, 30 min), p io o exposu e o oxidized
choles e ol (75 μg/mL o 30 min), wi h some modi ica ions. A e incuba ion he cul u e medium was eplaced wi h PBS and cells we e
loaded wi h 10 µM H2-DCF-DA o 30 min. A e incuba ion, PBS and H2-DCF-DA we e emo ed and he cells we e washed wice.
Inc ease in cell luo escence was measu ed a exci a ion and emission wa eleng hs o 490 and 520 nm, espec i ely, using an In ini e 200
au o mic opla e eade (Tecan, Salzbu g, Aus ia), a 25 °C.
20
Glu a hione (GSH) le el and Glu a hione pe oxidase (GPx) ac i i y
To assess he changes in he le els o GSH and GPx ac i i y, cells we e seeded in pla e dishes and exposed o oxidized choles e ol
(75 μg/mL) in comple e medium o 0-24 h. In o de o assess he e ec o HT, TYR, HT-S and TYR-S, cells we e p e ea ed wi h he
es ed compounds (5-25 µM in wa e solu ion, 30 min) p io o exposu e o oxidized choles e ol 75 μg/mL o 30 min o de e mine GSH
le el and o 18 h o measu e GPx ac i i y. A he end o incuba ion ime, cells we e washed wi h PBS, and subsequen ly sc aped in o
25
500 μL o 5% me aphospho ic acid. Samples we e hen sonica ed and cen i uged a 10000 × g o 20 min a 4 ºC; supe na an s we e
collec ed and used o de e mine GSH le el and GPx ac i i y, using Glu a hione Assay Ki and Glu a hione pe oxidase Assay Ki
(Cayman Chemical Company, Ann A bo , USA) acco ding o he manu ac u e ’s ins uc ions.
S a is ical analysis
Da a a e exp essed as means ± S.D (n=12 o each sample/condi ion). The s a is ical e alua ion o he esul s was pe o med by analysis
30
o a iance (ANOVA) ollowed by a Bon e oni pos -hoc es using G aphPad InS a e sion 3.05 (G aphPad So wa e, San Diego, CA,
USA).

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Resul s
Syn hesis o HT and TYR sul a e me aboli es has been ca ied ou using a p o ec ion-dep o ec ion s a egy oge he wi h he use o
mic owa es in he c i ical sul a ion s ep. In he case o TYR, he p ima y alcohol was acyl- p o ec ed using immobilized lipase No ozym
435® and inyl bu y a e in e -bu yl me hyl e he (98 % yield) (Scheme 1). Nex , mic owa e-assis ed sul a ion was pe o med by
ea men wi h SO3.NMe3 complex and ie hylamine in ace oni ile a 100 ºC (98% yield). Final dep o ec ion wi h K2CO3 in MeOH
5
a o ded y osol sul a e 5 in good yield (94%).
10
Scheme 1 P epa a ion o y osol sul a e 5
15
Mono-sul a ed hyd oxy y osol de i a i es 3 and 4 we e p epa ed as a egioisome ic mix u e using a syn he ic s a egy simila o ha o
TYR. In his case, hyd oxy y osol ace a e 8 was mono-silyl p o ec ed o a oid disul a ed p oduc s di icul o sepa a e om he mono-
sul a ed de i a i es (Scheme 2).
20
25
Scheme 2 P epa a ion o hyd oxy y osol sul a e me aboli es 3 and 4
Random TBDMS-p o ec ion o HT ace a e 8 and subsequen ch oma og aphic sepa a ion a o ded a 1:1 egioisome ic mix u e o he wo
possible mono-phenolic compounds 9 and 10. The same sul a ion eac ion condi ions used be o e we e applied o gi e 94% yield o a 1:1
mix u e o isome s 11 and 12. Finally, ace yl and silyl dep o ec ion was ca ied ou in one s ep using KF and K2CO3 in MeOH o ob ain a
30
mix u e o mono-sul a ed hyd oxy y osol de i a i es 3 and 4 (90% yield).
To in es iga e he po en ial p o ec i e e ec o he HT and TYR sul a e me aboli es, in compa ison wi h hei pa en compounds, agains
he p o-oxidan e ec o oxidized choles e ol in in es inal cells, Caco-2 monolaye s we e ea ed o 24 h wi h a mix u e ob ained om
he oxida ion o choles e ol a 140 °C o 3 h. In hese oxidizing condi ions hal o he ini ial choles e ol was u ned in o oxida ion
p oduc s ( epo ed in he me hods sec ion). T ea men wi h g owing concen a ion o he mix u e induced p oduc ion o MDA and cell
35
dea h (Fig. 2), indica ing he p esence o oxida i e cell inju y.
40
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10
15
Fig.2 Values o MDA (2A) and cell iabili y (2B) measu ed in Caco-2 cells a e 24 h incuba ion wi h di e en concen a ions o oxidized choles e ol.
* = p<0.05 e sus con ol
20
P e ea men wi h he phenolic compounds signi ican ly inhibi ed he inc ease o MDA (Fig. 3) in Caco-2 cells ea ed wi h 75 g/mL o
he oxidizing mix u e, he highes amoun able o induce a signi ican p oduc ion o MDA bu no cell dea h. MDA educ ion was
signi ican om 2.5 M o HT-S and om 5 M o HT; TYR and i s sul a e me aboli e exe ed he same e icacy s a ing om 2.5 M.
25
30
35
40
45
50
Fig.3 Values o MDA measu ed in Caco-2 cells a e 24 h incuba ion wi h 75 g/mL oxidized choles e ol and p e ea ed (30 min) wi h TYR, HT, HT
sul a e me aboli es 3-4 (HT-S) and TYR sul a e me aboli e 5 (TYR-S) (2.5-10 M). * = p<0.05 e sus con ol, a = p<0.05 e sus oxidized choles e ol
ea ed (Ox).
A
B
0
50
100
150
200
250
300
Con ol
25
50
75
100
125
150
175
MDA (% o he con ol)
Oxidized choles e ol (µg/ml)
*
*
*
*
*
0
40
80
120
Con ol
25
50
75
100
125
150
175
Cell iabili y (% o he con ol)
Oxidized choles e ol (µg/ml)
*
*
*
*
0
40
80
120
160
Con ol
Ox
Ox + 2,5
Ox + 5
Ox + 10
MDA (% o he con ol)
Compound (M)
HT
HT-S
*
a
aa
aa
0
40
80
120
160
Con ol
Ox
Ox + 2,5
Ox + 5
Ox + 10
MDA (% o he con ol)
Compound (M)
TYR
TYR-S
*
a
a
a
a
a
a
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Nex we examined he abili y o HT, TYR and hei co esponding sul a e me aboli es o p o ec cells agains dea h in Caco-2 cells
ea ed wi h 100 g/mL o oxidized choles e ol, he lowes amoun able o induce a signi ican cell dea h (Fig. 4). We obse ed ha HT
p ese ed cell iabili y om he concen a ion o 5 M whe eas HT sul a es 3-4 showed p o ec ion a he 10 M concen a ion. In he
case o TYR and i s sul a e de i a i e, bo h imp o ed cell iabili y a 10 M concen a ion.
5
10
15
20
25
Fig.4 Cell iabili y measu ed in Caco-2 cells a e 24 h incuba ion wi h 100g/mL oxidized choles e ol and p e ea ed (30 min) wi h TYR, HT o hei
sul a e me aboli es (2.5-10 M). * = p<0.05 e sus con ol, a=p<0. 05 e sus oxidized choles e ol ea ed (Ox).
30
35
40
45
0
20
40
60
80
100
120
Con ol
OX
OX + 2,5
OX + 5
OX + 10
Cell iabili y (% o he con ol)
Compound (M)
HT
HT-S
*
aa
a
***
0
20
40
60
80
100
120
Con ol
OX
OX + 2,5
OX + 5
OX + 10
Cell iabili y (% o he con ol)
Compound (M)
TYR
TYR-S
aa
**
**
*
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5
10
15
20
Fig.5 ROS le el, exp essed as % o he con ol samples, in Caco-2 cells, ea ed wi h 75 g/ml oxidized choles e ol o di e en incuba ion imes (5A) o
p e ea ed (30 min) wi h TYR, HT o hei sul a e me aboli es (5-25 M) and ea ed wi h oxidized choles e ol o 30 min (5B), exposed o 2',7'-
dichlo odihyd o luo escein diace a e H2-DCF-DA (10 mM) o 30 min. * = p<0.05 e sus con ol, a = p<0.05 e sus oxidized choles e ol ea ed (Ox), °=
p<0.05 e sus 0 min
25
The oxidizing ac ion o he mix u e o oxidized choles e ol was hen in es iga ed moni o ing he al e a ion o he cellula edox s a us
wi h ime: a e 30 min o incuba ion a signi ican p oduc ion o ROS was obse ed in he cells ea ed wi h he oxidized choles e ol in
compa ison wi h he con ol (Fig. 5A). Nex , we measu ed ROS p oduc ion a 30 min, a e p e ea men wi h he di e en phenolic
compounds (5, 10, 25 M). We obse ed ha ROS p oduc ion was signi ican ly lowe om he concen a ion o 10M o all he es ed
30
compounds, excep o TYR-S (Fig. 5B).
A e 30 min o incuba ion wi h he oxidized choles e ol, ROS p oduc ion was associa ed wi h a signi ican educ ion o Caco-2 cellula
GSH, a ound 40% o he ini ial alue, as shown in Figu e 6A. The le el o GSH inc eased wi h ime, eaching he con ol alue a 3/6 h,
o dec ease again he ea e . P e ea men wi h bo h HT and TYR p ese ed he con ol le el o GSH a all he es ed concen a ions and
hei sul a e me aboli es exe ed a compa able e icacy (Fig. 6B). In e es ingly, none o he es ed compounds was able o al e GSH le el
35
when incuba ed alone (25M) in he expe imen al condi ions used (p>0.5 e sus con ol).
Wi h espec o he conside ed pa ame e s, we did no obse e any concen a ion dependen p o ec i e e ec o he es ed phenolic
compounds, no signi ican di e ences among pa en compounds and me aboli es.
40
45
B
A
0
50
100
150
200
250
Con ol
Ox
Ox + 5
Ox + 10
Ox + 25
ROS le el (% o he con ol)
Compound (M)
HT
HT-S
*
a,* a
*
*a,* a,*
0
50
100
150
200
Con ol
Ox
Ox + 5
Ox + 10
Ox + 25
ROS le el (% o he con ol)
Compound (M)
TYR
TYR-S
*
aaa
*
**
0
50
100
150
0
30
60
180
ROS le el (%o he con ol)
Incuba ion ime (min)