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1 Elsevier Editoril System(tm) for Food Reserh Interntionl Mnusript Drft Mnusript Numer: Title: Chemo-protetive tivity nd hrteriztion of phenoli extrts from Corem lum. Artile Type: Reserh Artile Keywords: ntioxidnt defenes; iomrkers of oxidtive dmge; hlorogeni id; hydroxyinnmi ids; nturl iotive ompounds; oxidtive stress Corresponding Author: Dr. Luis Goy, Corresponding Author's Institution: First Author: Antonio León-González Order of Authors: Antonio León-González; Rquel Mteos; Soni Rmos; Mri Angeles Mrtín; Betriz Srriá; Crmen Mrtín-Cordero; Miguel López-Lázro; Lur Brvo; Luis Goy Astrt: There is urrently sustntil interest in the yto-protetive effets of nturl ompounds ginst oxidtive stress nd in studying of the defene mehnisms involved. Corem lum fruit is n edile erry onsumed long the Atlnti littorl of the Ierin Peninsul. The im of this study ws to hrterize the phenoli omposition nd evlute the hemo-protetive effets ginst oxidtive stress of three phenoli extrts from this fruit on liver ells. Chrteriztion of phenoli ompounds, hieved y liquid hromtogrphy nd diode-rry, mss spetrometry nd eletrospry ioniztion-time of flight-mss spetrometry detetion, showed min frtion of hydroxyinnmi ids. Liver HepG ells were treted with 1- µg/ml of the extrts nd exposed to oxidtive stress hemilly indued. Cell viility, retive oxygen speies (ROS), redued glutthione (GSH), ntioxidnt enzymes nd iomrkers of oxidtive dmge were evluted. Tretment of HepG ells with the extrts prtilly prevented ROS inrese, GSH depletion, ntioxidnt enzymes over-tivity nd oxidtive dmge to proteins nd lipids indued y stress. The results support the trditionl use of Corem lum s mediinl plnt nd suggest tht inlusion of its erries in the diet would ontriute to the protetion fforded y fruits, vegetles nd plntderived everges ginst oxidtive stress relted diseses.

2 Cover Letter July th, 1 To: Editor of Food Reserh Interntionl Der Editor, Plese find enlosed the mnusript entitled: Chemo-protetive tivity nd hrteriztion of phenoli extrts from Corem lum, y Antonio León- González, Rquel Mteos, Soni Rmos, M. Ángeles Mrtín, Betriz Srriá, Crmen Mrtín-Cordero, Miguel López-Lázro, Lur Brvo nd Luis Goy for sumission to Food Reserh Interntionl. The results reported in this mnusript hve not een sumitted to ny other journl. Corresponding uthor: Dr. Luis Goy (luisgoy@itn.si.es) -Consejo Superior de Investigiones Cientifis, CSIC - ICTAN, José Antonio Novis,, Mdrid, Spin (phone), (fx) Mnusript signifine The results of the present study show tht Corem lum erry phenoli extrts re minly rih in hydroxyinnmi ids nd tretment with the extrts protets ultured liver ells ginst oxidtive stress hemilly indued. Thnking you in dvne. We look forwrd to hering from you. Yours sinerely, Luis Goy Deprtment of Nutrition nd Metolism ICTAN (CSIC)

3 *Highlights (for review) Highlights 1) Corem lum erry phenoli extrts re minly rih in hydroxyinnmi ids ) Tretment with the extrts diretly improves redox ondition of liver ells ) Tretment with the extrts protets liver ells ginst oxidtive stress

4 *Mnusript Clik here to view linked Referenes Chemo-protetive tivity nd hrteriztion of phenoli extrts from Corem lum. Antonio León-González 1, Rquel Mteos, Soni Rmos, M. Ángeles Mrtín, Betriz Srriá, Crmen Mrtín-Cordero 1, Miguel López-Lázro 1, Lur Brvo nd Luis Goy * 1 Deprtmento de Frmologí, Fultd de Frmi, Universidd de Sevill, Prof. Grí González, 1 Sevill (Spin). Deprtmento de Metolismo y Nutriión; Instituto de Cieni y Tenologí de Alimentos y Nutriión ICTAN - CSIC; José Antonio Novis, ; Mdrid (Spin). Running title: Corem lum hydroxyinnmi ids protet HepG ells from oxidtive stress. *Corresponding uthor: Luis Goy, Deprtmento de Metolismo y Nutriión, Instituto de Cieni y Tenologí de Alimentos y Nutriión ICTAN - CSIC; José Antonio Novis, ; Mdrid, Spin Phone: Fx: e-mil: luisgoy@itn.si.es 1

5 Astrt There is urrently sustntil interest in the yto-protetive effets of nturl ompounds ginst oxidtive stress nd in studying of the defene mehnisms involved. Corem lum fruit is n edile erry onsumed long the Atlnti littorl of the Ierin Peninsul. The im of this study ws to hrterize the phenoli omposition nd evlute the hemo-protetive effets ginst oxidtive stress of three phenoli extrts from this fruit on liver ells. Chrteriztion of phenoli ompounds, hieved y liquid hromtogrphy nd diode-rry, mss spetrometry nd eletrospry ioniztion-time of flight-mss spetrometry detetion, showed min frtion of hydroxyinnmi ids. Liver HepG ells were treted with 1- µg/ml of the extrts nd exposed to oxidtive stress hemilly indued. Cell viility, retive oxygen speies (ROS), redued glutthione (GSH), ntioxidnt enzymes nd iomrkers of oxidtive dmge were evluted. Tretment of HepG ells with the extrts prtilly prevented ROS inrese, GSH depletion, ntioxidnt enzymes over-tivity nd oxidtive dmge to proteins nd lipids indued y stress. The results support the trditionl use of Corem lum s mediinl plnt nd suggest tht inlusion of its erries in the diet would ontriute to the protetion fforded y fruits, vegetles nd plnt-derived everges ginst oxidtive stress relted diseses. Keywords: ntioxidnt defenes, iomrkers of oxidtive dmge, hlorogeni id, hydroxyinnmi ids, nturl iotive ompounds, oxidtive stress.

6 Introdution Oxidtive stress is the min use of liver diseses nd plnt extrts with ntioxidnt properties hve reeived extensive ttention s possile therpeuti nd preventive gents whih ountert the prodution of free rdils nd retive oxygen speies (ROS) nd thus omt oxidtive stress. Plnt polyphenols hve gined inresing interest euse of their numerous iologil effets suh s free-rdil svenging, metl heltion, modultion of enzymti tivity, inhiition of ellulr prolifertion nd ltering signl trnsdution pthwys (Slert et l., ). Epidemiologil studies hve lso highlighted the ssoition etween the onsumption of polyphenol-rih foods nd the prevention of degenertive humn diseses suh s rdiovsulr diseses, ner nd other degenertive disorders (Mnh et l., ). Corem lum D. Don (Eriee) fruit is wild edile erry trditionlly onsumed long the Atlnti littorl of the Ierin Peninsul. Berries of Corem lum hve een used in populr mediine s ntipyreti nd re offered in the south of Spin s ppetizers. Unpulished results hve indited tht the min phenoli frtion in Corem lum is omposed y hydroxyinnmi ids. The mjor hydroxyinnmi ids, p-oumri, ffei, feruli nd sinpini ids, re uiquitously found in fruits, vegetles, erels nd lso in high onentrtions in te, mte (Brvo et l., ) nd prtiulrly in offee, verging 1 mg per up (Willimson et l., ). The interest of these ompounds is relted to their ntioxidnt tivity, whih my hve some helth enefiil effets in vivo. The extent of their protetive effet in vivo depends on their iovilility for intestinl sorption, metolism, nd susequent intertion with trget tissues. In this line, different studies hve shown tht hydroxyinnmi ids re extensively sored in ultured ells (Mteos et l., ), rts (Azum et l., ; Lfy et l., ) nd humns (Nrdini et l., ; Olthof et l., 1). The liver is prtiulrly suseptile to toxi nd oxidtive insults sine the portl vein rings lood to this orgn fter intestinl sorption. The sored drugs nd xenoiotis n use ROS- nd free rdil-medited dmge tht my result in inflmmtory nd firoti proesses (Lim et l., ). Therefore, studies deling with the effets of ntioxidnts t ellulr level in ultured hepti ells re essentil. Humn HepG is ell ulture model of humn heptoytes widely used for phrmologil studies sine they retin their morphology nd most of their funtion in ulture (Alí et l., ). Different studies hve demonstrted tht hydroxyinnmi ids (Mteos et l., ), flvonoids (Knzw et l., ) nd olive oil phenols, hydroxytyrosol nd hydroxytyrosyl ette (Mteos et l., ) re sored nd metolized y ultured HepG ells. In this study, the min phenoli ompounds in three different extrts from Corem lum were hrterized nd quntified, nd their heptoprotetive tivity ginst n oxidtive hllenge ws tested in HepG ells.. Mterils nd Methods.1. Regents Formi id nd methnol grde HPLC were otined from Pnre (Brelon, Spin). Chlorogeni id, rutin, hesperetin, resvertrol nd p-hydroxyenzoi id, tert-utylhydroperoxide (t- BOOH), o-phthlldehyde (OPT), glutthione redutse (GR), redued (GSH) nd oxidized glutthione, niotine denine dinuleotide (redued) (NADH), niotine denine dinuleotide phosphte redued slt

7 (NADPH),, -dihlorofluoresin diette (DCFH-DA),,-dinitrophenylhydrzone (DNPH), gentmyin, peniillin G, streptomyin, β-merptoethnol nd EDTA were purhsed from Sigm- Aldrih (Mdrid, Spin). Cynidin -O-gluoside hloride, delphinidin -O-gluoside hloride nd ynidin -O-rinoside hloride were quired from Extrsynthese (Lyon, Frne). The Brdford regent ws from BioRd Lortories (Mdrid, Spin). Other regents were of nlytil or hromtogrphi qulity... Extrtion of Phenoli Compounds from Corem lum Wild Corem lum erries were hrvested in Huelv (Spin) º.1" N - º 1 1." W, in Septemer, nd identified y Dr. Mri Cruz Diz Brrds, from Deprtment of Plnt Biology nd Eology, University of Seville. Corem lum fruits not showing ny physil dmge were seleted, wshed under running tp wter nd lot dried. Ripe fruits were lyophilized nd freeze-dried smples were ground nd stored t C until further nlysis. Three different phenoli ompounds extrtion methods were pplied: Aetone extrt (A) ws otined y homogenizing g of lyophilized Corem lum with ml of etone/formi id/wter (:.:., v/v/v) using ultrsoni equipment for min t room temperture. The finl extrt ws lyophilized nd resulting.% yield with respet to fresh fruit. Ethyl ette extrt (EA) ws otined y homogenizing g of lyophilized fruit with ml of ethyl ette using ultrsoni equipment for min t room temperture. The finl extrt ws evported under vuum produing.% yield with respet to fresh fruit. Wter extrt (W) ws otined y homogenizing g of frozen ripe fruit with ml of wter using ultrsoni equipment for min t room temperture. The finl extrt ws lyophilized nd resulting.% yield with respet to fresh fruit. Finlly, mg/ml stok solutions of A nd W in wter nd EA in ethnol were prepred to hrterize their phenoli omposition y HPLC nd for ell tretment... HPLC Anlysis Phenoli omposition of extrts ws nlyzed using n Agilent 1 liquid hromtogrphi system equipped with n utosmpler, quternry pump nd diode-rry (DAD) detetor. A mm x. mm i.d., - m prtile size Nuleosil 1 RP-1 olumn (Teknokrom) preeded y ODS preolumn ws used. Elution ws performed t flow rte of 1. ml/min, using s moile phse mixture of 1% (v/v) formi id in deionized wter (solvent A) nd methnol (solvent B). The solvent grdient hnged from % A to % A in min, to % A in 1 min, to % A in 1 min, to % A in min, to % A in min mintined for min nd to % A in min. Chromtogrms were quired t nm to register hydroxyenzoi ids, flvnones nd stilenes. Wvelengths, nd nm were seleted to monitor hydroxyinnmi ids, flvonols nd nthoynins, respetively. p-hydroxyenzoi id, hesperetin, resvertrol, hlorogeni id, rutin nd ynidin -O-gluoside were used to quntify hydroxyenzoi ids, flvnones, stilenes, hydroxyinnmi ids, flvonols nd nthoynins, respetively.. LC-MS Anlysis LC-MS mesurements were performed on n Agilent 1 series liquid hromtogrph/mss seletive detetor equipped with DAD detetor nd qudrupole mss spetrometer (Agilent Tehnologies). Chromtogrphi onditions (eluents, olumn, flow rte, grdient, et.) were s desried ove. Eluent flow ws split :1 etween the DAD detetor nd the MS ion soure. The MS ws fitted to

8 n tmospheri pressure eletrospry ioniztion (ESI) soure, operted in negtive ion mode. The eletrospry pillry voltge ws set to V, with neulizing gs (nitrogen) flow rte of 1 L/h nd drying gs temperture of C. Mss spetrometry dt were quired in sn mode (mss rnge m/z -) t sn rte of 1. s... HPLC-ESI-QTOF Anlysis The hromtogrphy ws performed on n Agilent 1 series LC system oupled to n Agilent A Aurte-Mss Qudrupole Time-of-Flight (Q-ToF) with ESI-Jet Strem Tehnology (Agilent Tehnologies). A mm x. mm i.d., μm prtile size Nuleosil 1 RP-1 olumn (Teknokrom) preeded y n ODS preolumn ws used. Eh smple ( μl) ws injeted nd seprted isortilly y using moile phse onsisting of wter nd etonitrile, oth ontining.1% formi id, t flow rte of. ml/min. The Q-ToF quisition onditions were s follows: GHz, mss rnge etween - m/z, negtive polrity, drying gs volume nd temperture L/min nd ºC, sheth gs volume nd temperture L/min nd ºC, neulizer pressure psi, p voltge V, nozzle voltge V, nd frgmentor voltge V. Dt quisition nd qulittive nlysis were performed y using MssHunter Worksttion Softwre... Cell Culture nd tretment Humn hepti HepG ells were mintined in humidified inutor ontining % CO nd % ir t ºC. They were grown in DMEM F-1 medium from Biowhitker (Lonz, Mdrid, Spin), supplemented with. % foetl ovine serum (FBS) nd mg/l eh of gentmiin, peniillin nd streptomyin. The different onentrtions of the three extrts (1,,, nd μg/ml) were dissolved in serum-free ulture medium nd dded to the ell pltes for h exept in the ROS ssy. In the experiments to evlute the protetive role of the ompounds ginst n oxidtive insult, ells were pre-treted with the sme onentrtions of the ompounds for h, then the medium ws disrded nd fresh medium ontining M t-booh ws dded for h, fter whih the ell ultures were proessed for eh ssy... Evlution of ell viility, ROS prodution nd ntioxidnt defenes Cellulr dmge ws evluted y ltte dehydrogense (LDH) lekge (Alí et l., ). Cells were seeded ( x ells per plte) in mm pltes, grown for h with the different tretments nd then the ell ulture medium ws olleted nd the ells were srped off in phosphte uffer sline (PBS). LDH lekge ws estimted from the rtio etween the LDH tivities in the ulture medium nd the totl tivity, ulture medium plus intrellulr. Cellulr ROS were quntified y the dihlorofluoresin ssy using miroplte reder (Alí et l., ). Cells were seeded in -well pltes ( ells per well) Multiwell pltes were seeded s previously referred nd mesured in fluoresent miroplte reder t exittion wvelength of nm nd emission wvelength of nm. GSH ontent ws evluted y fluorometri ssy (Alí et l., ). The method tkes dvntge of the retion of GSH with o-phthlldehyde t ph.. Fluoresene ws mesured t exittion nd emission wvelength of nm nd nm respetively. Determintion of GPx tivity ws sed on the oxidtion of GSH y GPx, using t-booh s sustrte, oupled to the dispperne rte of NADPH y GR (Alí et l., ). GR tivity ws determined y following the derese in sorne due to the oxidtion of

9 NADPH utilized in the redution of oxidized glutthione (Alí et l., ). Protein onentrtion in the smples ws mesured y the Brdford ssy... Determintion of ronyl groups nd mlondildehyde (MDA) Protein oxidtion of ells ws mesured s ronyl groups ontent in superntnts ording to the method of Rihert et l. (). Asorne ws mesured t nm nd ronyl ontent ws expressed s nmol/mg protein using n extintion oeffiient of nmol/l/m. Cellulr MDA ws nlyzed in superntnts y HPLC s its DNPH derivtive (Mteos et l., ). An Agilent 1 Series HPLC-DAD ws used nd MDA vlues were expressed s nmol of MDA/mg protein... Sttistis Sttistil nlysis of dt otined from ell ulture studies ws performed s follows: prior to nlysis the dt were tested for homogeneity of vrines y the test of Levene; for multiple omprisons, one-wy ANOVA ws followed y Bonferroni test when vrines were homogeneous or y Tmhne test when vrines were not homogeneous. The level of signifine ws P <.. A SPSS version 1. progrm ws used.. Results.1. Identifition nd hrteriztion of polyphenols in plnt extrts The phenoli onstituents present in the three extrts of Corem lum were monitored y DAD, MS nd ESI-QTOF detetion. Typil hromtogrms of the three extrts of Corem lum re shown in Figure 1. Tle 1 shows the list of ompounds identified long with their retention time (RT) nd UV hrteristis of the hromtogrphi peks, [M-H] - nd their orresponding frgment ions, urte mss (. mss), moleulr formul (MF) nd md of error etween the mss found nd the urte mss of eh polyphenol. Up to different phenoli ompounds were deteted in the extrts (A, EA nd W) Identifition of phenoli ids Peks 1,,, nd hd similr UV spetr profile, with mximum t 1 nm nd shoulder t - nm, typil of ffei id derivtives. Peks 1, nd presented similr mss spetr, with [M-H] - ion t m/z nd ions t m/z 11, 1 nd, orresponding to deprotonted quini id, ffei id nd dimeri ddut of the ffeoylquini id moleule, respetively. The LC retention time nd UV nd MS spetr of ompound were identil to stndrd hlorogeni id (-Offeoylquini id), while 1 nd would orrespond to hlorogeni id isomers: -O-ffeoylquini id nd -O-ffeoylquini id. Compound showed RT, UV nd MS spetr totlly oinident with p-hydroxyenzoi id stndrd. Compound showed [M-H] - ion t m/z 1 nd frgment with m/z 1. This frgmenttion long with the urte mss nd moleulr formul permitted its identifition s ffei id-ohexoside. Cffei id (pek ) ws identified y ompring its RT, UV nd MS spetr with tht of referene sustne. Finlly, ompound 1 showed n UV spetrum with mx t nd shoulder t 1 nm. The urte mss nd moleulr formul provided y Mss Hunter long with [M-H] - ion t m/z nd frgment t m/z 1 permitted its tenttive identifition s -gingerol, lthough further hrteriztion is neessry to onfirm this point.

10 Identifition of nthoynins Peks, nd hd UV spetr hrteristi of the nthoynin group, with two sorption mxim t nm nd t 1- nm. The RT nd UV spetr were identil to stndrd delphinidin - O-gluoside, ynidin -O-gluoside nd ynidin -O-rinoside, respetively..1.. Identifition of flvonols Six peks (ompounds,, 1, 1, 1 nd 1) hd UV spetr omptile with flvonols, hrterized y two sorption mxim in the rnges - nm nd - nm. Compound hd deprotonted moleulr ion t m/z nd frgment t m/z 1, resulting to the loss of dehydrted hexose moiety to yield myrietin ion nd onfirming the presene of myrietin--o-gluoside. Compound showed MS spetrum with [M-H] - ion t m/z nd frgment t m/z 1, derived from the loss of dehydrted hexose moiety to yield the queretin ion, orresponding to queretin--o-gluoside. Compound 1 showed RT, UV nd MS spetr totlly oinident with rutin stndrd. Regrding ompound 1, the MS spetrum showed [M-H] - ion t m/z nd frgment t m/z 1 orresponding to queretin fter the loss of dehydrted rinose moiety tht enled its identifition s queretin--o-rinose. Chromtogrphi peks 1 nd 1 showed identil MS spetrum, providing deprotonted moleulr ion t m/z nd frgment t m/z inditive of flvonol kmpherol fter the loss of 1 units [M-H-1] -, tht leded to utious identifition s kmpherol -O-gltoside nd -O-gluoside, respetively..1.. Identifition of flvnones Peks 1 nd showed UV spetr inditive of flvnone struture with mx t nm nd shoulder t nm. Prtiulrly, ompound 1 showed n only ion t m/z whih urte mss nd moleulr formul pointed to pinoemrin, s the possile identity of this pek. On the other hnd, ompound showed [M-H] - ion t m/z nd frgment t m/z 1 tht permitted its identifition s -gernylnringenin..1.. Identifition of stilenes Chromtogrphi pek 1 showed n UV spetrum with mx t nd shoulder t nm. Considering the mss spetr, this ompound provided the deprotonted moleulr ion t m/z plus two frgment ions t m/z 1 nd, onsistent with the loss of one nd two methyl groups, respetively. QTOF nlysis ws in greement with these results, onfirming the presene of pterostilene in Corem lum fruit... Quntifition of phenoli ompounds from Corem lum fruit. Contents of phenoli ompounds, exluding unknown hromtogrphi peks 1 nd 1, were determined y HPLC-DAD nd using stndrds nd wvelength speified in mterils nd methods. The totl ontent of phenoli ompounds ws lulted s the sum of eh phenoli group. Results were expressed s mg per ml of eh extrt. The finl omposition determined in the three phenoli extrts (A, EA nd W) otined from Corem lum fruit is summrized in Tle. The results indite different degree of extrtion of the polyphenols groups in ordne with the polrity of the used solvent. Thus, the extrt otined with wter ontined the most polr ompounds suh s phenoli ids nd nthoynins, in detriment of the most lipophyili ones, i.e.

11 flvnones nd stilenes. The extrtion rried out with etone provided the most lned omposition, reovering high mounts of ll lsses of polyphenols. Finlly, when the extrtion ws developed with ethyl ette, the extrt showed high mount of flvonols nd stilenes nd very low mounts of the most polr ompounds. The overll omprison of the three extrtnts pointed out t etone s slightly the est solvent to otin the highest reovery rtes of ll lsses of polyphenols from Corem lum fruit... Antioxidnt effets of Corem lum extrts on HepG in sl onditions Cell viility remined unltered fter h tretment with the three extrts t dose s high s µg/ml (Figure ). Tretment for h with doses up to µg/ml of ny extrt did not ffet ell viility y the rystl violet ssy (dt not shown). Thus, it n e ssumed tht the rnge of onentrtions finlly seleted (1- µg/ml) n e sfely used to study the potentil protetive effet in vitro of Corem lum extrts ginst ondition of oxidtive stress. Additionlly, tretment of ells with 1- µg/ml of ll three extrts signifintly deresed ROS prodution s ompred to those of ontrol ells (Figure ). A dose-response ws oserved; thus, - µg/ml of ny extrt evoked the lrgest derese in ROS (Figure ). Tretment with inresing onentrtions of extrts during h provoked n inrese in GSH onentrtion in ll tretments exept - µg/ml EA (Figure ). No signifint hnges in the tivity of GPx were oserved (Figure ), ut tretment with µg/ml EA nd µg/ml W evoked signifint inrese in the GR tivity (Figure ). Biomrkers of oxidtive dmge to proteins nd lipids were tested with only the intermedite dose of µg/ml from the three extrts. No signifint hnges in the onentrtion of ronyl groups were found (Figure ), ut signifint derese in MDA levels ws oserved fter the tretment with µg/ml of W extrt (Figure )... Protetive effets of Corem lum extrts on HepG in ondition of oxidtive stress Tretment with t-booh for hours evoked prominent ell deth of round % (Figure ). Pre-tretment for h with 1- µg/ml of ll three extrts signifintly redued ell dmge indued y t-booh, limiting ell deth to vlues tht were elow % in A- nd W-treted HepG nd round 1 % in AE-treted ells (Figure ). Cells treted with t-booh showed signifint.-fold inrese in ROS genertion fter h s ompred to non-stressed ontrols (Figure ). Pre-tretment with µg/ml A for h slightly ut signifintly deresed ROS genertion indued y t-booh, yet remrkle ROS reduing effet ws oserved with 1- µg/ml EA nd W (Figure ). Addition of t-booh to ells for h evoked drmti depletion of the GSH onentrtion to % of ontrol vlues (Figure ); this diminution ws prtly overome y pre-tretment with ll doses of EA extrt nd 1- µg/ml of A nd W, nd ompletely surmounted y tretment with µg/ml of A nd W (Figure ). Tretment with t-booh for h indued -fold inrese in the tivity of GPx nd GR (Figure,) s defene response ginst the oxidtive insult. Pre-tretment with ll doses of the three extrts ompletely reversed the hemilly indued inrese in GPx nd GR (Figure,). Tretment with t-booh during h evoked -fold inrese in the onentrtion of ronyl groups, inditing permnent oxidtive dmge to ell proteins (Figure ). Pre-tretment with the three

12 extrts evoked prtil (EA) or omplete (A nd W) reovery of the ronyl onentrtion (Figure ). Tretment with t-booh during h lso evoked -fold inrese in the onentrtion of MDA, inditing permnent oxidtive dmge to ell lipids (Figure ). Pre-tretment with µg/ml of A, EA nd W extrts for h gretly prevented the MDA inrese indued y t-booh. Indeed, omplete redution of the hemilly-indued MDA ws oserved in ells tht hd previously een treted with µg/ml of EA nd W (Figure ).. Disussion In this study, we nlyzed the phenoli omposition of three different extrts otined from the erry of the plnt Corem lum nd investigted the hemo-protetive effet of the extrts ginst indued oxidtive stress nd the different defene mehnisms involved. The results prolim hydroxyinnmi ids s the mjor phenoli frtion in the three plnt extrts nd demonstrte tht pretretment of hepti ells with ny of the extrts grnt signifint protetion ginst n oxidtive hllenge hemilly indued. Chemil hrteriztion of the extrts showed remrkle similrity in the totl mount of phenoli ompounds, g/ g extrt. Around % nd % of the phenoli frtion of extrts A nd W, respetively, is omposed y hydroxyinnmi ids tht lmost doule the mount of these ompounds in EA extrt. However, the ltter frtion ompensted with signifintly higher mount of flvonoids (prtiulrly flvnones), phenoli ids nd stilenes, lthough hydroxyinnmi ids still mounted to % of the totl phenolis. Between 1.-1.% of hydroxyinnmi ids is reltively high mount ompred to other fruits nd vegetles ut remins fr from rosted offee ens, whih omprise higher onentrtion of 1% of hlorogeni ids nd relted ompounds (Frh & Donngelo, ). All three extrts were poor in nthoynins. Therefore, most of the iologil effets of the extrts should e endorsed to the group of hydroxyinnmi ids, in prtiulr, -Offeoylquini id or hlorogeni id. Biologil tivities of nturl hydroxyinnmi ids inlude inhiiting tumour ell prolifertion (Cillet et l., 1; Jnike et l., ) nd nti-inflmmtory tivity (Kim et l., 1; Ngsk et l., ). Aute ingestion of yer mte infusion rih in hydroxyinnmi ids inhiits plsm nd lipoprotein oxidtion in humns (D Silv et l., ). Cffeoylquini id hs shown liverprotetive tivity in experimentl liver injury models (Bsnet et l., 1) nd its dministrtion to rts improved gluose nd lipid homeostsis nd overll ntioxidnt sttus (Jurgoński et l., 1). Besides, hlorogeni id up-regulted ellulr ntioxidnt enzymes in humn denorinomi lveolr ells (Feng et l., ), nd signifint protetive effet of hlorogeni id ginst n indued oxidtive stress hs een reported in PC1 ells (Pvli & Gehrdt, ). Finlly, hlorogeni id enhned the intrinsi ellulr tolerne ginst oxidtive insults in HepG ells oth y tivting survivl/prolifertion pthwys nd inresing ntioxidnt potentil (Grndo-Serrno et l., ). All these properties of hlorogeni id mke Corem lum phenoli extrts interesting ndidtes for ellulr hemo-protetion, nd, to our knowledge, there is no previous dt on ell ulturesed study testing the ntioxidnt effets of Corem lum extrts. Although nturl phenolis my hve potent ntioxidnt effets in vitro nd in vivo, elevted doses of dietry ntioxidnts my lso t s

13 pro-oxidnts in ell ulture systems nd provoke ellulr dmge (Azm et l., ). Consequently, the seletion of the tenttive rnge of doses used ws sed on oth literture serh nd our previous studies with other phenoli extrts. Thus, uthors hve reported humn lood onentrtions up to µm of hlorogeni id fter ingestion of green offee extrt (Frh et l., ), lood levels up to µm in humns fter offee onsumption (Monteiro et l., ) nd up to 1 µm of ll hydroxyinnmi ids nd their metolites in plsm fter ingestion of offee (Stlmh et l., ). In previous work we hve shown hemo-protetive effet on HepG ells with.- µg/ml of oo phenoli extrt (Mrtín et l., ; Mrtín et l., ). Therefore, in order to evlute the effet of Corem lum extrts, the onentrtion rnge seleted, 1- µg/ml, is not fr from relisti. In terms of hlorogeni id, this rnge is equivlent to.- µm. Cell integrity, redox sttus nd oxidtive stress iomrkers were primrily determined under these onditions, then, one ensured tht the rnge of onentrtions is seure, the response of Corem lum extrts-onditioned ells ginst n oxidtive hllenge ws tested. As expeted, tretment of ells with 1- µg/ml of the three extrts for h produed no signifint ell dmge nd evoked dose-response redution in the ellulr ROS prodution, in greement with previous reports inditing tht plnt hydroxyinnmi ompounds re effetive svengers of oxygen rdils in ell ultures (Feng et l., ; Pvli nd Gehrdt, ). The sme tretment with Corem lum extrts evoked sustntil inrese in GSH, refleting redued intrellulr oxidtion whih ould e expeted to prepre the ell ginst potentil oxidtive insult. In line with this result, tretment of HepG ells with hlorogeni id (Grndo-Serrno et l., ) lso resulted in n inrese in stedy-stte GSH onentrtions. The presene of glutthione-dependent enzymes is essentil to prevent the ytotoxiity of ROS. An inrese in the stedy-stte tivity of ntioxidnt enzymes with oo phenoli extrt (Mrtín et l., ) nd olive oil phenoli hydroxytyrosol (Mrtín et l., ) hs een reported. In ordne with those results, the present study shows signifint inrese in GR tivity in HepG ells treted with high onentrtions of EA nd W extrts for h. This outome pointed out tht these treted ells were in etter onditions to fe the inresing genertion of ROS indued y the potent pro-oxidnt t- BOOH. Additionlly, W extrt signifintly redued MDA, three-ron ompound formed y sission of peroxidized polyunsturted ftty ids. Sine MDA hs een found elevted in vrious diseses thought to e relted to free rdil dmge, it hs een widely used s n index of lipid peroxidtion in iologil nd medil sienes (Breusing et l., ). Therefore, ells treted with the extrts seem to e in fvourle onditions to fe n oxidtive hllenge. Tretment of HepG ells with the strong prooxidnt t-booh is n exellent model of oxidtive stress in ell ulture systems (Alí et l., ; Lim et l., ; Mrtín et l., ; Mrtín et l., ). Thus, µm t-booh signifintly enhned ell dmge nd ROS genertion in HepG, nd pre-tretment of HepG ultures with 1- µg/ml of the three Corem lum extrts gretly prevented ell dmge nd slightly ut signifintly redued ROS. These results suggest tht the ROS generted during the period of oxidtive stress were more effiiently quenhed in ells pre-treted with extrts, whih ould e first explntion for the redued ell dmge.

14 Addition of t-booh to ells evoked remrkle depletion of GSH whih ws signifintly overome y pre-tretment of the ells with ny of the three Corem lum extrts. These results implied tht inresed levels of GSH in the extrt-treted ells efore exposure to the oxidtive hllenge gretly helped to prevent the drmti depletion of the intrellulr GSH stok. Mintining GSH onentrtion ove ritil threshold while fing stressful sitution represents deisive dvntge for ell survivl. Indution of GPx nd GR re ritil mehnisms of the ell defene ginst oxidtive insults nd plys mjor role to overome ROS prodution in the presene of t-booh (Alí et l., ; Mrtín et l., ; Mrtín et l.,,). However, rpid return of the ntioxidnt enzyme tivities to sl vlues one the hllenge hs een surmounted will ple the ell in fvorle ondition to del with new insult. In this study, pre-tretment of ells with Corem lum extrts mnged to prevent the longlsting inrese in the tivities of GPx nd GR indued y oxidtive stress. This ensured tht the ells were in optimum onditions to withstnd further oxidtive hllenges. In onert, we hve previously reported tht oo phenoli extrt verted ell dmge y preventing the permnently inresed tivities of GPx nd GR indued y t-booh (Mrtín et l., ; Mrtín et l., ). The signifint inrese in the ellulr onentrtion of ronyl groups nd MDA during oxidtive stress indued y t-booh indited extensive dmge to ellulr proteins nd lipids. Pretretment of HepG with µg/ml of the three extrts signifintly deresed oth iomrkers demonstrting redued degree of protein nd lipid oxidtion in response to the stressful sitution. This hemo-protetive effet on oxidtive mrkers hs lso een reported with other plnt phenoli extrts (Mrtín et l., ; Mrtín et l., ) nd pure nturl iotive ompounds (Alí et l., ; Mrtín et l., ). Therefore, s evidened y the results of ROS prodution, GSH onentrtion nd ntioxidnt enzymes tivity, the rpid reovery of the redox homeostsis evoked y the pre-tretment with Corem lum extrts will limit protein nd lipid degrdtion nd will ensure redued ell dmge. Despite the different omposition of phenoli ompounds in extrts A nd W (round % hydroxyinnmis nd 1% of rest of polyphenols) nd EA (% hydroxyinnmis, % flvonoids nd 1% of stilenes), the hemo-protetive effiieny of the three frtions ws quite similr. Tretment with A nd W redued ell dmge during oxidtive stress to less thn % wheres ells treted with EA remined slightly over 1%; further, only ells treted with A nd W ompletely reovered GSH. However, EA extrt evoked in stressed ells stronger ROS nd MDA redution thn extrt A nd, overll, hd omprle effet on the rest of prmeters. This suggests tht the signifint mount of flvonoids nd stilenes in EA extrt ompenstes for the smller quntity of hydroxyinnmi ids in the hemo-protetive tivity. In onlusion, the results indite tht the three Corem lum extrts tested re rih in hydroxyinnmi ids nd ontin different mounts of flvonoids nd stilenes. Tretment with the tested onentrtions of the three extrts evoked hnges in the sl redox sttus, suh s redued ROS nd inresed GSH, whih ple the ells in fvourle onditions to fe n oxidtive hllenge. Cells pre-treted with the extrts showed n outstnding protetion ginst the hllenge-indued dmge. These results support the trditionl use of Corem lum s mediinl plnt nd its presene in the diet

15 s n ppetizer my ontriute to the protetion fforded y fruits, vegetles nd plnt-derived everges ginst diseses for whih oxidtive stress hs een implited s usl or ontriuting ftor. Aknowledgements: This work ws supported y the grnts AGL-1, AGL-1 nd projet CSD- from Progrm Consolider-Ingenio from the Spnish Ministry of Siene nd Innovtion (CICYT). A. L.-G. reeived pre-dotorl fellowship from Ministerio de Cieni e Innovión (FPU). Referenes Alí, M., Rmos, S., Mteos, R., Brvo, L. & Goy, L. (). Queretin protets humn heptom ell line (HepG) ginst oxidtive stress indued y tertutyl hydroperoxide. Toxiology nd Applied Phrmology, 1, 1-. Azm, S., Hdi, N., Khn, N. U. & Hdi, S. M. (). Prooxidnt property of green te polyphenols epitehin nd epigllotehin--gllte: implitions for ntiner properties. Toxiology In Vitro, 1, -1. Azum, K., Ippoushi, K., Nkym, M., Ito, H., Higshio, H. & Tero, J. (). Asorption of hlorogeni id nd ffei id in rts fter orl dministrtion. Journl of Agriulturl nd Food Chemistry,, -. Bsnet, P., Mtsushige, K., Hse, K., Kdot, S. & Nm, T. (1). Four di-o-ffeoyl quini id derivtives from propolis. Potent heptoprotetive tivity in experimentl liver injury models. Biologil nd Phrmeutil Bulletin, 1, 1 1. Brvo, L., Goy, L. & Leumerri, E. (). LC/MS hrteriztion of phenoli onstituents of mte (Ilex prguriensis, St. Hil.) nd its ntioxidnt tivity ompred to ommonly onsumed everges. Food Reserh Interntionl,, -. Breusing, N., Grune, T., Andrisi, L., Atly, M., Brtosz, G., Bisi, F., Borovi, S., Brvo, L., Csls, I., Csills, R., Dinishiotu, A., Drzewinsk, J., Fer, H., Fuzi, N. M., Gjewsk, A., Gmini, J., Grdinru, D., Kokkol, T., Lojek, A., Luzj, W., Mrgin, D., Msi, C., Mteos, R., Meinitzer, A., Mitjvil, M. T., Mrkovi, L., Muntenu, M. C., Podorsk, M., Poli, G., Siinsk, P., Skrzydlewsk, E., Vin, J., Wiswedel, I., Zrkovi, N., Zelzer, S. & Spikett, C. M. (). An inter-lortory vlidtion of methods of lipid peroxidtion mesurement in UVA-treted humn plsm smples. Free Rdil Reserh,, 1-1. Cillet, S., Lorenzo, G., Cote, J., Doyon, G., Sylvin, J.-F. & Lroix, M. (1). Cner hemopreventive effet of frtions from rnerry produts. Food Reserh Interntionl,, -. 1

16 D Silv, E. L., Neiv, T. J. C., Shiri, M., Tero, J. & Adll, D. S. P. (). Aute ingestion of yer mte infusion (Ilex prguriensis) inhiits plsm nd lipoprotein oxidtion. Food Reserh Interntionl 1, -. Frh, A. & Donngelo, C. M. (). Phenoli ompounds in offee. Brzilin Journl of Plnt Physiology, 1, -. Frh, A., Monteiro, M., Donngelo, C. M. & Lfy, S. (). Chlorogeni ids from green offee extrt re highly ioville in humns. Journl of Nutrition, 1, -1. Feng, R., Lu, Y., Bowmn, L. L., Qin, Y., Cstrnov, V. & Ding, M. (). Inhiition of tivtor protein-1, NF-kppB, nd MAPKs nd indution of phse detoxifying enzyme tivity y hlorogeni id. Journl of Biologil Chemistry, -. Grndo-Serrno, A. B., Mrtín, M. A., Izquierdo-Pulido, M., Goy, L., Brvo, L. & Rmos, S. (). Moleulr mehnisms of (-)-epitehin nd hlorogeni id on the regultion of the poptoti nd survivl/prolifertion pthwys in humn heptom ell line (HepG). Journl of Agriulturl nd Food Chemistry,, -. Jnike, B., Hegrdt, C., Krogh, M., Onning, G., Akesson, B., Cirenjwis, H. M. & Oredsson, S. M. (). The ntiprolifertive effet of dietry fier phenoli ompounds feruli id nd p-oumri id on the ell yle of Co- ells. Nutrition nd Cner,, -. Jurgoński, A., Juśkiewiz, J., Zduńzyk, Z. & Król, B. (1). Cffeoylquini id-rih extrt from hiory seeds improves glyemi, therogeni index, nd ntioxidnt sttus in rts. Nutrition,, -. Knzw, K., Uehr, M., Yngitni, H. & Hshimoto, T. (). Bioville flvonoids to suppress the formtion of -OHdG in HepG ells. Arhives in Biohemistry nd Biophysis,, 1-. Kim, E. O., Min, K. J., Kwon, T. K., Um, B. H., Moreu, R. A. & Choi, S. W. (1). Antiinflmmtory tivity of hydroxyinnmi id derivtives isolted from orn rn in lipopolyshride-stimulted Rw. mropphges. Food nd Chemil Toxiology,, 1-. Lfy, S., Gil-Izquierdo, A., Mnh, C., Mornd, C., Besson, C. & Slert, A. (). Chlorogeni id is sored in its intt form in the stomh of rts. Journl of Nutrition, 1, -. Lim, C. F., Fernndes-Ferreir, M. & Pereir-Wilson, C. (). Phenoli ompounds protet HepG ells from oxidtive dmge: Relevne of glutthione levels. Life Sienes,,. 1

17 Mnh, C., Slert, A., Mornd, C., Remesy, C. & Jiménez, L. (). Polyphenols: food soures nd iovilility. Amerin Journl of Clinil Nutrition,, -. Mrtín, M. A., Rmos, S., Mteos, R., Grndo-Serrno, A. B., Izquierdo-Pulido, M., Brvo, L. & Goy, L. (). Protetion of humn HepG ells ginst oxidtive stress y oo phenoli extrt. Journl of Agriulturl nd Food Chemistry,, -. Mrtín, M. A., Grndo-Serrno, A. B., Rmos, S., Izquierdo-Pulido, M., Brvo, L. & Goy, L. (ª). Coo flvonoids up-regulte ntioxidnt enzymes tivity vi ERK1/ pthwy to protet ginst oxidtive stress-indued poptosis in HepG ells. Journl of Nutritionl Biohemistry, 1, 1-. Mrtín, M. A., Rmos, S., Grndo-Serrno, A. B., Rodríguez-Rmiro, I., Trujillo, M., Brvo, L. & Goy, L. (). Hydroxytyrosol indues ntioxidnt/detoxifint enzymes tivity nd Nrf trnslotion vi ERKs nd PIK/AKT pthwys in HepG ells. Moleulr Nutrition Food Reserh,, -. Mteos, R., Goy, L. & Brvo, L. (). Determintion of mlondildehyde (MDA) y highperformne liquid hromtogrphy s the,-dinitrophenylhydrzine derivtive. A mrker for oxidtive stress in ell ultures of humn heptom HepG. Journl of Chromtogrphy, B,,. Mteos, R., Goy, L. & Brvo, L. (). Metolism of the Olive Oil Phenols Hydroxytyrosol, Tyrosol nd Hydroxytyrosyl Aette y Humn Heptom HepG Cells. Journl of Agriulturl nd Food Chemistry,, -. Mteos, R., Goy, L. & Brvo, L. (). Uptke nd metolism of hydroxyinnmi ids (hlorogeni, ffei nd feruli ids) y HepG ells s model of humn liver. Journl of Agriulturl nd Food Chemistry,, -. Monteiro, M., Frh, A., Perrone, D., Trugo, L. C. & Donngelo, C. (). Chlorogeni id ompounds from offee re differentilly sored nd metolized in humns. Journl of Nutrition, 1, 1 1. Ngsk, R., Chotimrkorn, C., Shfiqul, I. M., Hori, M., Ozki, H. & Ushio, H. (). Antiinflmmtory effets of hydroxyinnmi id derivtives. Biohemil nd Biophysil Reserh Communitions,, 1-. Nrdini, M., Cirillo, E., Ntell, F. & Sini, C. (). Asorption of phenoli ids in humn fter offee onsumption. Journl of Agriulturl nd Food Chemistry,, -1. Olthof, M. R., Hollmn, P. C. H. & Ktn, M. B. (1). Chlorogeni id nd ffei id re sored in humns. Journl of Nutrition,, 1. 1

18 Pvli, S. & Gehrdt, R. (). Protetive effet of ellgi nd hlorogeni ids ginst oxidtive stress in PC1 ells. Free Rdil Reserh,, 1-. Rihert, S., Wehr, N., Stdtmn, E. & Levine, R. (). Assessment of skin ronyl ontent s non invsive mesure of iologil ge. Arhives in Biohemistry nd Biophysis,,. Slert, A., Mnh, C., Mornd, C., Rémésy, C., Jiménez, L. (). Dietry polyphenols nd the prevention of diseses. Critil Reviews in Food Siene nd Nutrition,, -. Stlmh, A., Mullen, W., Brron, D., Uhid, K., Yokot, T., Cvin, C., Steiling, H., Willimson, G. & Crozier, A. (). Metolite profiling of hydroxyinnmte derivtives in plsm nd urine fter the ingestion of offee y humns: Identifition of iomrkers of offee onsumption. Drug Metolism nd Disposition,, 1 1. Willimson, G., Dionisi, F. & Renouf, M. (). Flvnols from green te nd phenoli ids from offee: ritil quntittive evlution of the phrmokineti dt in humns fter onsumption of single doses of everges. Moleulr Nutrition nd Food Reserh,, -. Legends to figures Figure 1.- HPLC hromtogrms of phenoli ompounds extrted from Corem lum erries with etone (A), ethyl ette (EA) nd wter (W) t nm. Figure.- Diret effet of Corem lum extrts on ell viility nd ROS genertion. Pnel, LDH lekge is expressed s perent of LDH tivity in the ulture medium of the totl tivity, ulture medium plus intrellulr. Vlues re mens ± SD (n=-). Pnel, fluoresene units orresponding to intrellulr ROS prodution re expressed s perent of ontrol dt. Vlues re mens (n=-). Mens without ommon letter differ, P<.. Figure.- Diret effet of Corem lum extrts on ntioxidnt defenes. HepG were treted with the noted onentrtions of the extrts for h. Intrellulr onentrtion of GSH () nd enzyme tivity of GPx () nd GR () re expressed s perent of ontrol vlue ± SD of - different smples per ondition. Different letters indite sttistilly signifint differenes (P <.) mong different groups. Figure.- Diret effet of Corem lum extrts on oxidtive stress iomrkers. HepG were treted with g/ml of the extrts for h. Vlues of ronyl groups () nd MDA () re expressed s mens ± SD of different smples per ondition. Different letters indite sttistilly signifint differenes (P <.) mong different groups. Figure.- Protetive effet of Corem lum extrts on ell viility nd ROS genertion. HepG were treted with the noted onentrtions of extrts for h, then the ultures were wshed nd M t- BOOH ws dded to ll the ultures exept ontrols for h (pnel, LDH) or h (pnel, ROS). Results of LDH lekge re expressed s perent of LDH tivity in the ulture medium of the totl tivity, ulture medium plus intrellulr nd intrellulr ROS prodution ws expressed s perent of ontrol 1

19 vlues. Dt re mens ± SD (n=-). Different letters indite sttistilly signifint differenes (P <.) mong different groups. Figure.- Protetive effet of Corem lum extrts on the ntioxidnt defenes. GSH ontent () nd tivity of GPx () nd GR () in HepG ells treted with the noted onentrtions of the extrts for h efore the exposure to M t-booh during h. Vlues re mens ± SD (n=-). Mens without ommon letter differ, P<.. Figure.- Protetive effet of Corem lum extrts on oxidtive stress iomrkers. HepG were treted with the noted onentrtions of the extrts for h, then the ultures were wshed nd M t-booh ws dded to ll the ultures exept ontrols for h. Vlues of ronyl groups () nd MDA () re expressed s mens ± SD of different smples per ondition. Different letters indite sttistilly signifint differenes (P <.) mong different groups. 1

20 Figure Figure 1. mau nm 1 Aetone extrt (A) nm min mau Ethyl ette extrt (EA) min mau Wter extrt (W) min

21 % ROS genertion % LDH in medium Figure ) A EA W µg/ml 1 A EA W ) µg/ml

22 % GR % GPx % GSH Figure ) A EA W ) µg/ml A EA W ) µg/ml A EA W µg/ml

23 nmol MDA/mg protein Protein ronyl ontent (nmol/mg protein) Figure ) 1 A EA W µg/ml ) 1 A EA W µg/ml

24 % ROS genertion % LDH in medium Figure ) A EA W 1 C 1 C 1 C 1 µg/ml t-booh ( µm) t-booh ( µm) t-booh ( µm) ) A EA W 1 e de d d d d d d C 1 C 1 C 1 µg/ml t-booh ( µm) t-booh ( µm) t-booh ( µm)

25 % GR % GPx % GSH Figure ) 1 A EA W C 1 C 1 C 1 µg/ml t-booh ( µm) t-booh ( µm) t-booh ( µm) ) A EA W 1 ) C 1 C 1 C 1 µg/ml t-booh ( µm) t-booh ( µm) t-booh ( µm) A EA W 1 C 1 C 1 C 1 µg/ml t-booh ( µm) t-booh ( µm) t-booh ( µm)

26 nmol MDA/mg prt Protein ronyl ontent (nmol/mg protein) Figure ) 1 C A EA W µg/ml t-booh ( µm) ) C A EA W µg/ml t-booh ( µm)

27 Tle Tle 1. List of ompounds identified in Corem lum fruit. Chr. Pek Compound RT (min) mx [M-H] - Frgment ions A. mss md M.F. 1 -O-Cffeoylquini id.1, sh 11,1,. -1. C 1 H 1 O p-hydroxyenzoi id C H O Cffei id-o-hexoside 1. 1, sh C 1 H 1 O -O-Cffeoylquini id 1., sh 11,1,. -1. C 1 H 1 O Cffei id 1., sh C H O -O-Cffeoylquini id., sh 11,1,. -. C 1 H 1 O Delphinidin -O-gluoside.1, C 1 H O 1 Cynidin -O-gluoside., C 1 H O Cynidin -O-rinoside., 1 C H 1 O Myrietin--O-gluoside 1., C 1 H O 1 Queretin--O-glu oside., C 1 H O 1 1 Rutin., C H O 1 1 Queretin--O-rinoside.1, C H 1 O 1 Kmpherol--O-gltoside.,. -. C 1 H O 1 Kmpherol--O-gluoside.1,. -1. C 1 H O 1 Pinoemrin., sh. -1. C 1 H 1 O 1 Pterostilene., sh 1,. -. C 1 H 1 O 1 Unknown., 1sh C 1 H O 1 Unknown. -Gernylnringenin 1., sh C H O Chr. Pek: hromtogrphi pek; RT (min): retention time (min); A. Mss: urte mss; M.F.: moleulr formul; md: millidlton of error etween the mss found nd the urte mss of eh polyphenol.

28 Tle Tle. Content of phenoli ompounds nd flvonoids in the three extrts (A, EA nd W) otined from Corem lum fruit, expressed in mg per g of extrt. Results re expressed in men ± SD of three determintions. A mg/g extrt EA mg/g extrt W mg/g extrt PHENOLIC ACIDS Hydroxyenzoi ids (nm) p-hydroxyenzoi id. ±.. ±.. ±. Hydroxyinnmi ids (nm) -O-Cffeoylquini id. ±..1 ±.. ±. Cffei id-o-hexoside. ±.. ±.. ±. -O-Cffeoylquini id 1. ±.1. ±.. ±.1 Cffei id. ±.. ±.. ±.1 -O-Cffeoylquini id. ±.. ±.. ±. Totl Phenoli ids 1. ±.. ±. 1. ±. ANTHOCYANINS (nm) Delphinidin -O-gluoside. ±.. ±.. ±. Cynidin -O-gluoside 1.1 ±.. ±. 1.1 ±. Cynidin -O-rinoside. ±.. ±.. ±. Totl Anthoynins 1.1 ±.. ±.. ±. FLAVONOLS (nm) Myrietin--O-gluoside 1. ±.. ±.. ±. Queretin -O-gluoside 1. ±.. ±.. ±. Rutin 1.1 ±. 1. ±. 1.1 ±. Queretin -O-rinoside. ±.. ±.. ±. Kmpherol--O-gltoside. ±.. ±.. ±. Kmpherol -O-gluoside. ±.. ±.. ±. Totl flvonols 11.1 ±. 1. ±.. ±. FLAVANONES (nm) Pinoemrin 1. ±.. ±.. ±. -gernylnringenin. ±.. ±.. ±. Totl flvnones. ±. 1. ±.. ±. STILBENES (nm) Pterostilene 1. ±. 1.±.. ±. Totl stilenes 1. ±. 1. ±.. ±. TOTAL PHENOLIC COMPOUNDS 1. ±. 1. ±. 1.1 ±.