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1 Food Chemistry 125 (211) Contents lists vilble t ScienceDirect Food Chemistry journl homepge: Effect of enzymtic debittering on ntioxidnt cpcity nd protective role ginst oxidtive stress of grpefruit juice in comprison with dsorption on exchnge resin M. Cvi-Siz, P. Muñiz, N. Orteg, M.D. Busto Deprtment of Biotechnology nd Food Science, Are of Biochemistry nd Moleculr Biology, University of Burgos, Plz Misel Bñuelos, s/n, E-91 Burgos, Spin rticle info bstrct Article history: Received 27 April 21 Received in revised form 27 June 21 Accepted 24 August 21 Keywords: Antioxidnt ctivity Debittering DNA dmge Exchnge resin Grpefruit juice Lipoperoxidtion Nringinse Rdicl scvenger Antioxidnt cpcity, rdicl scvenging ctivity, s well s protective effect on lipoperoxidtion, glutthione oxidtion nd DNA dmge, were evluted in grpefruit juice subjected to bitterness removl by nringinse or by physicl dsorption with Amberlite Ò IRA-4. The results showed reduction in nringin content for the nringinse-treted juice (N-PJ) nd those processed with the exchnge resin (R-PJ), which mde both juices cceptble to consumers. Totl ntioxidnt cpcity, mesured by ABTS nd FRAP ssys, ws lower in R-PJ smples. The highest superoxide nd hydroxyl rdicl scvenger ctivity ws observed in N-PJ. With regrd to inhibitory effect of juice smples on lipoperoxidtion, N-PJ lso provided the gretest effectiveness. In ddition, R-PJ showed the lowest levels of GSH. The results showed protective effect on DNA oxidtive dmge for ll juice smples tested. In summry, enzymtic technology ws more effective thn physicl dsorption in preserving the ntioxidnt nd biomolecule protection cpcity of fresh grpefruit juice. Ó 21 Elsevier Ltd. All rights reserved. 1. Introduction The helth benefits of citrus fruit juices hve minly been ttributed to the presence of bioctive compounds, such s scorbic cid, crotenoids, flvonoids, limonoids nd coumrins (Yu et l., 25). Beneficil ctivities of the components of citrus juices for humn helth re primrily scribed to its ntioxidnt cpcity (Xu et l., 28). Indeed, nringin (4,5,7-thrihydroxyflvnone-7- rhmnoglucoside) is the mjor flvonoid present in grpefruit juice nd hs been described to hve ntioxidnt, nti-ulcer, nd ntiinflmmtory ctivity (Chen, Shen, & Lin, 23). However, this flvnone is lso the most dominnt bitter principle in grpefruit. The studies to overcome the bitterness in citrus juices hve been reviewed by Singh, Gupt, Jin, nd Dhtt (23), nd more recently by Puri, Kur, Knwr, nd Singh (28). To reduce/remove bitterness in citrus juices below the threshold level for consumer cceptbility, number of chemicl tretments, physicl seprtion processes, blending with non-bitter citrus juice nd sugrs nd enzyme tretments hve been described. Although mny different methods hve been used since the erly 197s the current debittering technology uses dsorption onto cellulose Corresponding uthor. Tel.: ; fx: E-mil ddress: dbusto@ubu.es (M.D. Busto). cette or mcroporous resin beds or cross-linked styrene divinylbenzene resins (Shw, Bines, Mitnes, & Agmon, 2). Nowdys, consumers demnd the mximum preservtion of the endogenous sensory, nutritionl nd helth relted qulities of fruit products. The serch for new technologies tht cuse minimum dmge to the nutritionl chrcteristics nd helth-promoting effects of grpefruit my be considered s n lterntive to conventionl debittering process by physicl dsorption. Debittering using the enzyme nringinse hs been reported by severl uthors (Puri, Kur, & Kennedy, 25; Sekeroglu, Sibel, & Fhrettin, 26). Commercil enzyme preprtions lbelled s nringinse contin -rhmnosidse nd b-glucosidse. Nringin is first hydrolysed to prunin nd rhmnose by the -rhmnosidse. Orlly dministered nringin cn be metbolised into nringenin nd nringenin glucoronide. Nringenin is metbolite of nringin with bioctive effect on humn helth. In the second step, prunin is broken down into nringenin nd glucose by the b-glucosidse. Due to its substrte specificity, the enzyme tretment seems to cuse minimum chnges in the fruit juice qulity ttributes nd mkes it n interesting option ginst trditionl procedures. To our knowledge, up to now, no direct comprison is published for the ntioxidnt cpcities of grpefruit juice debittered with nringinse nd by physicl dsorption with exchnge resin. Furthermore, reserch bout the effects of enzymtic tretment on /$ - see front mtter Ó 21 Elsevier Ltd. All rights reserved. doi:1.116/j.foodchem

2 M. Cvi-Siz et l. / Food Chemistry 125 (211) functionl vlue of grpefruit juice is very scrce (Ribeiro, Roch, Sepodes, Mot-Filipe, & Ribeiro, 28). In this context, the im of this study ws to evlute the impct of the emerging enzyme technology in comprison with trditionl dsorption technology (exchnge resin) on the protective role of grpefruit juice on DNA oxidtive dmge, glutthione (GSH) oxidtion nd lipid peroxidtion. In ddition, totl ntioxidnt cpcity nd superoxide nd hydroxyl scvenging of debittered grpefruit juice by both procedures were lso studied. 2. Mterils nd methods 2.1. Chemicls 2,2 -Azinobis(3-ethylbenzothizoline-6-sulphonic cid) dimmonium slt (ABTS), 2,2 -dizobis(2-minodinopropne) dihydrochloride (ABAP), 6-hydroxyl-2,5,7,8-tetrmethyl-2-crboxylic cid (Trolox), gllic cid, nringin, 2,4,6-tris(2-pyridyl)-S-trizine (TPTZ), 2-deoxy-D-ribose, tert-butyl hydroperoxide (t-booh), phenzin methosulphte (PMS), nuclese P1 nd clf thymus DNA were from Sigm Aldrich Co. (St. Louis, MO, USA). Sodium hydroxide (NOH), luminium chloride 6-hydrte (AlCl 3 6H 2 O), sodium nitrite (NNO 2 ), diethylene glycol, Folin Cioclteu s regent, potssium persulphte (K 2 O 8 S 2 ), ferric chloride (FeCl 3 ), ferrous sulphte (FeSO 4 ), copper sulphte (CuSO 4 ), L-scorbic cid, nd trichlorocetic cid (TCA) were obtined from Pnrec (Brcelon, Spin). Thiobrbituric cid (TBA) nd cetonitrile ws purchsed from Merck (Drmstdt, Germny). Bovine serum lbumin, NADH disodium slt, DNse I, phosphtse lkline, nd 4-nitroblue tetrzolium chloride (NBT) were from Roche (Mdrid, Spin). Tris, ethidium bromide, nd ethylenediminetetrcetic cid (EDTA) tretrsodium slt were from Amresco (Solon, OH, USA). Amberlite Ò IRA-4 ion exchnge resin ws from Sigm Aldrich Chemie GmbH (Steinheim, Germny). Nringinse from Penicillium decumbens, contining -rhmnosidse nd b-glucosidse ctivity ws purchsed from Sigm Aldrich Co. (St. Louis, MO, USA) Preprtion of grpefruit smples Grpefruits were purchsed from locl supermrket nd keep t 4 C before being processed. Fresh grpefruit juice (FJ) ws obtined by extrction to through domestic squeezer (Brun Citromtic Pulp Control MPZ6) nd filtered using sieve (light of mesh dimeter 1 mm). Smples of fresh juice (FJ) were psteurised (P) ccording to Ttum nd Berry (1973) just before debittering process. Thus, tretment of juice by Amberlite Ò IRA-4, ws crried out in 25 ml beker contining 25 ml of psteurised juice nd 5 g of exchnge resin with constnt stirring for 3 min t room temperture (Mishr & Kr, 23). Resin ws removed by centrifugtion nd by filtering the juice. Tretment of juice by nringinse ws crried out ccording to Busto, Mez, Orteg, nd Perez-Mteos (27). Smples of 25 ml of psteurised juice were treted with nringinse (.4 U/ml) in 25 ml beker nd incubted t 2 C for 24 h Determintion of totl phenols nd nringin Totl phenol content ws evluted by the Folin Cioclteu s rection (Singleton & Rossi, 1965) using gllic cid (GAE) s the stndrd. The results re expressed in milligrmmes of gllic cid equivlent per litre of juice smple. Nringin ws quntified ccording to Dvis (1947) by dding 2 ll of grpefruit juice nd 2 ll of 4 M NOH to 1 ml of 9% diethylene glycol. The rection smple ws mixed well nd llowed to stnd for 15 min to develop the yellow colour. The opticl density ws then red t 42 nm ginst blnk without juice in Hitchi U-2 UV visible spectrophotometer (Tokyo, Jpn). The nringin concentrtion (lg/ml) ws determined from stndrd grph of nringin drwn in the sme mnner. The totl phenol content nd nringin vlues were expressed s the men ± SD of six different experiments repeted three times Assessment of totl ntioxidnt cpcity The ntioxidnt cpcity of the grpefruit juices ws evluted by ABTS method nd ferric reducing ntioxidnt power (FRAP) ssy. The ABTS ssy ws crried out ccording to the procedure of Pellegrini, Proteggente, Pnnl, nd Rice-Evns (1999). The rection mixture contined 3.36 mm ABTS, different liquots of juice (5, 1, 2 or 3 ll t dilution of 1:4), nd phosphte buffered sline (PBS) t ph 7.4 in totl volume of 1 ml. The decrese in the bsorbnce ws mesured t 734 nm. The results were expressed s the millimolr concentrtion of Trolox equivlents, using liner clibrtion obtined with different concentrtions of Trolox. The FRAP ssy is bsed on increse in the bsorbnce t 593 nm due to formtion of TPTZ-Fe(II) complexes ccording to the method described by Benzie nd Strin (1999). The rection mixture ws prepred by mixing 25 ml of.3 M sodium cette buffer solution (ph 3.6), 2.5 ml of 1 mm TPTZ, 2.5 ml of 2 mm FeCl 3, nd 3 ml of wter. Aliquots (5 3 ll) of the juice smples were dded to the rection mixture in totl volume of 1 ml nd incubted t 37 C for 3 min. The results were expressed s mm Fe(II), using liner clibrtion obtined with different concentrtions of FeSO Determintion of superoxide nd hydroxyl rdicl scvenger ctivity The superoxide rdicl scvenging ctivity of grpefruit juice smples ws determined by monitoring the reduction of nitroblue tetrzolium (NBT). The rection mixture contined 2 ll of the juice smple, 78 lm NADH (nicotinmide denine dinucleotide, reduced form), 5 lm NBT nd 1 lm PMS in Tris HCl buffer (16 lm, ph 8.) in totl volume of 2.5 ml. The smples were incubted t room temperture for 5 min nd the bsorbnce ws red t 56 nm. The hydroxyl scvenger ctivity ws mesured by the method described by Hlliwell, Gutteridge, nd Aruom (1987). The rection mixture contining 1 mm deoxyribose, 5 mm phosphte buffer, ph 7.4, 1 mm scorbic cid, 1 mm ferric chloride, 1 mm hydrogen peroxide, 1 mm EDTA nd 2 ll of the juice smples ws incubted for 6 min t 37 C. Afterwrds, 1 ml of the incubted smple ws mixed with.5 ml of trichlorocetic cid (2.8%, w/v) nd.5 ml of thiobrbituric cid regent (1% w/v, in.5 M NOH), followed by heting t 1 C for 15 min nd subsequent cooling to room temperture. The bsorbnce ws recorded t 532 nm in Hitchi U-2 UV visible spectrophotometer (Tokyo, Jpn). The results were expressed s inhibition percentge referred to control test, in the bsence of juice smple Reduced glutthione concentrtion Homogentes from liver rt were obtined using tissue homogentor Ultrturrx Polytron t 4 C. The homogente (1/2, w/v) for GSH determintion ws prepred by using 1 mm sodium phosphte buffer (ph 7) contining 5 mm EDTA. The liver homogente ws incubted with 5 mm t-booh t 37 C for 12 min in the presence or bsence of different liquots of the

3 16 M. Cvi-Siz et l. / Food Chemistry 125 (211) grpefruit juice (1 4 ll). After incubtion, 25% (w/v) HPO 3 ws dded to the superntnt (1/4, v/v) nd the resulting mixture centrifuged t 1,g t 4 C for 3 min. The GSH concentrtion (nmol/mg protein) in the smple ws mesured by the method of Brigelius, Muckel, nd Akerboom (1983) Lipid peroxidtion inhibition The experiments were crried out in rt liver microsoml preprtions (Ubeud, Schmitt, Jeck, Lve, & Cossolo, 1995) using peroxyl rdicls s oxidnt. The totl microsoml protein content ws determined using the Lowry method. The microsoml frction (1 mg/ml of protein) ws incubted in solution of ABAP (1 mm), prepred in 5 mm Tris HCl buffer solution t ph 7 immeditely prior to use, in the presence of juice t different volumes, 1 4 ll. The incubtion temperture ws set t 37 C for period of 9 min. Lipid peroxidtion ws evluted in the incubted smples by the TBARs ssy (Mitsuru & Midori, 1978). Absorbnce mesured t 532 nm (e = 156 mm 1 cm 1 ) ws proportionl to the quntity of peroxyl rdicls generted, nd the results were expressed s nmol/mg protein referred to control test, in the bsence of juice smple DNA oxidtive dmge The DNA oxidtive dmge ws mesured by the method described by Sez et l. (1993). The rection mixture contined clf thymus DNA (2 lg), scorbic cid (1 mm), cupric sulphte (1 lm) nd the smples to be tested t different volumes (25, 5, 1 nd 2 ll). The mixture ws incubted in shking wter bth t 37 C for 1 h. Electrophoresis ws performed fter incubtion of DNA smples in 1% grose gels prepred in electrophoresis buffer (45 mm Tris boric cid with 1 mm EDTA, ph 8.) using Bio-Rd power-pc 1 (Hercules, CA) electrophoresis system. Aliquots (5 ll) of DNA test solutions nd moleculr weight mrkers were premixed with 1 ll of loding buffer solution contining 2% glycerol,.1% bromophenol blue nd 8% buffer solution t ph 7.4 (.1 M sodium phosphte). The resulting mixtures were run t 4 ma. The DNA bnds were viewed under UV light nd photogrphed with digitl cmer Enzymtic DNA hydrolysis nd 8-OH-dG determintion DNA digestion ws performed s described by Muñiz et l. (1995). In brief, 2 lg/ml DNA fter lowering the ph to 5.1 with sodium cette (.5 M), 5 units of nuclese P1 followed by the ddition of 1 ll lkline phosphte (3 units). The DNA hydrolystes were dissolved in HPLC-grde wter nd filtered through.22-lm syringe filter before their nlysis by HPLC. The mount of 8-OH-dG in the DNA digest ws mesured by electrochemicl detection. The results were expressed s inhibition percentge referred to control test, in the bsence of juice smple. Elution conditions were those described by Ritcher, Prk, nd Ames (1988), 5 mm phosphte buffer solution, ph 5.5, contining 1% cetonitrite nd Wters ODS HPLC column (25 cm.46 cm i.d. 5 lm prticle size) Sttisticl procedure Sttisticl nlysis of the dt ws crried out using one-wy nlysis of vrince (ANOVA). Furthermore, the lest significnt difference (LSD) test ws pplied in order to determine the sttisticl significnce between vrious groups. A minimum significnce level of 95% (p <.5) ws considered. Liner regression ws used in order to study the possible correltions between studied prmeters. 3. Results nd discussion Trditionl het psteuristion of citrus juices is necessry in order to preserve their qulity, especilly by minimising pectin methylesterse (PME) enzymtic ctivity nd microbil growth. PME inctivtion is importnt becuse this enzyme ctlyses pectin degrdtion nd lters the colloid stbilizing power of the pectin, which imprts the fvourble ppernce. In this work, the fresh grpefruit juice (FJ) ws first psteurised nd then processed with the exchnge resin or the enzyme nringinse for debittering. Tble 1 shows the content of nringin nd totl phenols of freshly squeezed nd debittered grpefruit juice smples. The reduction in bitterness ws evluted bsed on the mount of residul nringin (Ribeiro et l., 28). From the mount of residul nringin the percentge reduction in bitterness ws evluted. In comprison with fresh juice, reduction of 64.5% nd 46.8% in nringin ws observed in R-PJ nd N-PJ, respectively, which mkes both juices cceptble to the consumers. The totl phenol content of grpefruit juice smples is shown in Tble 1. The highest totl phenol content ws detected in the juice subjected to nringinse tretment. By contrst, the exchnge resin process led to decrese in the totl concentrtion of phenols (21%) compred with smples of fresh juice. The high increse in totl phenols (78%) detected in juice smples treted with nringinse cn be due to the hydroxyl groups of nringenin, metbolite from nringin. The product of enzymtic ctlysis (nringenin) hs hydroxyl group rther thn nringin. Moreover, there is evidence tht the spectrophotometric method overestimtes the polyphenol content by the lck of selectivity of Folin Cioclteu s regent (Escrp & González, 21), which rects not only with phenols but lso with other reducing compounds such s sugrs (e.g. glucose) (Singleton et l., 1965). However, this method hs been shown to be useful nlyticl tool for the routine nlysis of polyphenols nd it is widely used in mny lbortories for the determintion of differences mong fruits nd vegetbles nd their products. On the other hnd, the effect of resin tretment on the totl phenols ws in greement with literture dt for red grpefruit juice concentrte (Lee & Kim, 23). These uthors reported tht the debittering process, utilizing XAD-16 dsorption column, significntly reduced totl phenol content nd completely removed some of the non-bitter flvonoids, such s nrirutin nd hesperidin. In our study the totl ntioxidnt cpcity of grpefruit juice ws evluted using ABTS nd FRAP ssys. The ntioxidnt ctivity mesured by ABTS method is bsed on the cpcity of substnce to stbilize the rdicl ABTS +, while the FRAP ssy mesures directly its reducing cpcity. Both methods re used nd recommended by mny uthors s esy nd ccurte ssys of mesuring the ntioxidnt ctivity of fruit juices (Abeysinghe et l., 27; Rpisrd et l., 1999). Fig. 1 illustrtes the results of the ntioxidnt ctivity obtined for grpefruit juices tested in the present study. All grpefruit juice smples (FJ, N-PJ nd R-PJ) stbilized the rdicl ABTS + nd reduced ferric ion in dose-dependent mnner. Tble 1 Contents of totl phenols nd nringin in grpefruit juice smples. FJ, fresh juice; N-PJ, psteurised nd nringinse-treted juice; R-PJ, psteurised nd resin-treted juice. Grpe juice smple Phenols (mg GAE/l) Nringin (lg/ml) FJ 689 ± 5.2 b 676 ± 26.4 c N-PJ 1227 ± 44.1 c 359 ± 5.9 b R-PJ 545 ± ± 16.4 Results re the men ± SD of eighteen experiments. Different letters for ech column correspond to sttisticlly different vlues (p <.5).

4 M. Cvi-Siz et l. / Food Chemistry 125 (211) A B TEAC (mm Trolox) mm FeSO Fig. 1. Totl ntioxidnt cpcity of grpefruit juice smples by ABTS (A) nd FRAP (B) methods. FJ, fresh juice; N-PJ, psteurised nd nringinse-treted juice; R-PJ, psteurised nd resin-treted juice. Results re the men ± SD of 1 experiments. Different letters for ech volume correspond to sttisticlly different vlues (p <.5). In the ABTS ssy, N-PJ showed the highest TEAC vlues wheres R-PJ showed the lowest for ll volumes of studied smple (Fig. 1A). These results re consistent with those reported in our previous study (Cvi-Siz et l., 21) in which nringin exhibited lower cpcity to inhibit 5% of ABTS rdicl ction thn nringenin. Heim, Tgliferro, nd Bobily (22) lso indicted tht the hydroxyl group in position 7 of nringenin significntly incresed the ntioxidnt cpcity of the flvnone towrds the stble rdicl ABTS +. The ntioxidnt ctivity of the juice smples determined using FRAP ssy re represented in Fig. 1B. All juices nlysed in this study hd bility to reduce Fe 3+ to Fe 2+ but with significnt differences. The FRAP vlues of juice smples were mm FeSO 4 for FJ, mm for N-PJ nd mm for R-PJ. In contrst to the ABTS method, smples of fresh juice hd the highest reducing cpcity. The grpefruit juice treted with the exchnge resin lso showed lower levels of ntioxidnt ctivity with the FRAP method. These results cn be explined bering in mind tht vitmin C is typiclly het-sensitive compound (Yeom, Streker, Zhng, & Min, 2) so it cn produce losses during psteuristion in N-PJ nd R-PJ smples. Furthermore, vitmin C is lso one of the components of the citrus juices with ntioxidnt ctivity tht most contributes to reducing cpcity (Abeysinghe et l., 27). Xu et l. (28) found tht scorbic cid contribution to totl ntioxidnt cpcity of citrus juices ws more thn 5% (by FRAP ssy). These results were in greement with previous reports (Aren, Fllico, & Mccrone, 21), which suggested scorbic cid, not phenolic compounds, ws the mjor contributor of totl ntioxidnt cpcity of citrus juices. However, some studies suggested phenolic compounds dominted totl ntioxidnt cpcity of citrus fruits (Rpisrd et l., 1999). It seemed tht some fctors such s the mturity, mteril preprtion nd nlysing methods might cuse the divergence (Xu et l., 28). Most free rdicl rections involve the reduction of moleculr oxygen, leding to the formtion of rective oxygen species (ROS), including superoxide nion nd hydroxyl rdicl, these ROS re of physiologicl nd technologicl relevnce nd cover brod rnge of rectivity. Nturl ntioxidnts present in citrus juice cn neutrlise free rdicls, due to their bility to ct s free rdicl scvengers nd/or s metl cheltors (Frnke et l., 24). As shown in Fig. 2B, ll juice smples inhibited the hydroxyl rdicl-cused degrdtion of desoxyribose in dose-dependent mnner. The N-PJ gve the highest protection ( % inhibition) ginst oxidtive dmge to desoxyribose when compred to R- PJ ( %) nd FJ ( %). For the NBT reduction ssy, which evlutes the O 2 scvenging bility, our results show tht ll tested juices inhibited NBT reduction (Fig. 2A). The juice processed with nringinse exhibited the highest superoxide scvenging ctivity ( %) in dose-dependent mnner, while juices treted with the exchnge resin showed the lowest inhibition percentge (25 34%). No significnt differences were observed between fresh nd enzyme treted juice t the highest volume tested. In generl, the phenolic compounds re ble to scvenge rdicls nd to chelte metls. However, it depends on the concentrtion nd the type of the phenolic compounds (Glto et l., 21). In this sense, quntittive nd qulittive vritions of these compounds (e.g. ltertion of one or more hydroxyls or oxidtion) my hve occurred during the psteuristion nd debittering tretment, thus leding to differences in the scvenger ctivity of the juice smples (Frnke et l., 24). Previous results (Cvi-Siz et l., 21) indicted, in greement with other uthors (Yu et l., 25), tht the presence of both 7- nd 5-hydroxyl groups in the glycone (nringenin) mkes it n excellent cndidte for superoxide scvenging rection nd lso showed tht nringenin exhibited higher quenching efficiency for hydroxyl rdicls tht nringin. GSH levels in rt liver homogente were studied, using t-booh s inductor of oxidtive stress, s n indictor of the intrcellulr A % Inhibition B %Inhibition Fig. 2. Scvenger effect of grpefruit juice smples on superoxide (A) nd hydroxyl rdicl (B). FJ, fresh juice; N-PJ, psteurised nd nringinse-treted juice; R-PJ, psteurised nd resin-treted juice. Results re the men ± SD of 1 experiments. Different letters for ech volume correspond to sttisticlly different vlues (p <.5).

5 162 M. Cvi-Siz et l. / Food Chemistry 125 (211) GSH (nmol/mg protein) b C t-booh FJ N-PJ R-PJ redox stte. Results in Fig. 3 show tht GSH levels in liver homogente treted with t-booh decresed significntly compred with the control (C). The enzyme tretment slightly reduced thiol group; compred with the fresh juice, no significnt differences were detected t higher doses. In contrst, juice treted with the exchnge resin showed the lowest levels of GSH. Previous reports (Cvi, 21) indicted tht pre-tretment with nringin or nringenin did not prevent the reduction in GSH levels, nd potentil pro-oxidnt effect of both flvonoids in vitro ws lso suggested (Jgeti, Reddy, & Kedly, 24). Nevertheless, severl reserches hve demonstrted the bility of other phenolic compounds nd vitmins present in citrus juices to restore GSH (Weisel et l., 26). Another prmeter evluted ws the inhibitory effect of juice smples tested on lipid peroxidtion in rt liver microsomes exposed to oxidtive stress in the presence of peroxyl rdicl. Free rdicls initite nd cuse lipid peroxidtion prticulrly those tht mke up cell membrnes, so it is importnt tht the inhibitory ctivity of lipid dmge chrcteristic of citrus is not ffected during the tretment of debittering. Our results showed (Fig. 4) tht ll juice smples studied provided protective effect towrds lipid oxidtive dmge in dose-dependent mnner. It is noticeble, tht the nringinse-treted juice provided the gretest effectiveness in the inhibition of lipoperoxidtion. Some uthors hve determined the nti-lipoperoxidtion ctivities of the polyphenols, the flvonoids, nthocynins, hydroxycinmic cids nd scorbic cid contined in citrus juices. In prticulr, Rpisrd et l. (1999) rgued tht the ntioxidnt cpcity of ll exmined ornge b Fig. 3. Effect of grpefruit juice smples on GSH in rt liver homogente exposed to oxidtive stress in the presence of t-booh. FJ, fresh juice; N-PJ, psteurised nringinse-treted juice; R-PJ, psteurised nd resin-treted juice. C: Heptocytes in the bsence of t-booh; t-booh: heptocytes in the presence of t-booh; (1 4 ll): heptocytes preincubted with grpefruit juice smples before dding t- BOOH to induce depletion of GSH. Results re the men ± SD of 1 experiments. Different letters for ech volume correspond to sttisticlly different vlues (p <.5). juices ws due to totl phenol mounts nd to their bility to interct with the biomembrne. Consequently, the highest totl phenol content of fresh juice in comprison with the juice treted with resin, my explin the differences in TBARS vlues obtined in both juices. In ddition, it hs been showed tht the O-glycosyltion (nringin) decresed the efficiency in the inhibition of lipoperoxidtion (Cvi et l., 21). In fct, the concentrtion of nringenin required to rech 5% inhibition using s inductor peroxyl rdicl ws 2.3 lower s compred to the nringin. In contrst, Yu et l. (25) reported similr ntioxidnt ctivity for both flvnones but in hmster LDL model. The oxidtive dmge to the DNA genertes series of injuries s brekge of connections phosphte, or oxidtion of puric nd pirimidinic bses. Some of these modifictions hve been considered potentilly hrmful nd responsible for muttions in the integrity of the genome. In this work the DNA brekge induced by Cu(II)-scorbic cid ws nlysed by grose gel electrophoresis nd bse oxidtion mesurement by the levels of 8-OH-dG. Fig. 5 shows the electrophoretic pttern of DNA fter incubtion with hydroxyl rdicls in the bsence or presence of the grpefruit juice smples. The results showed protective effect for ll juice smples tested t 1 ll of volume, nd it ws clerly observble protection of the integrity of DNA t 2 ll. A slight protection ginst DNA frgmenttion ws lso observed t the lower volumes (25 ll nd 5 ll). The suggestion tht grpefruit juice plys protective effect ginst DNA dmge under the reported experimentl conditions is consistent with number of previous observtions in which the preventive effect by nringin nd nringenin hs been shown (Jgeti et l., 24). Another method used to test the cpcity of the grpefruit juice smples to protect DNA from oxidtion consisted in quntifying their cpcity to inhibit the level of modified bse 8-OH-dG formed during ttck of the OH on the DNA. It is known tht the oxygen free rdicl ttcks DNA, resulting in production of oxidised bses such s 8-OH-dG which contribute to muttions nd tumour promotion (Shen, Deininger, Hunt, & Zho, 27). DNA ws treted in solution with scorbic cid nd copper in the bsence nd presence of different volumes of grpefruit juice smples (Tble 2). 8- OH-dG ws not detected fter incubtion of DNA lone or in the presence of 25, 5, 1 ll of grpefruit (dt not shown), so the juices did not induce ny DNA dmge. The presence of grpefruit juice in the co-incubted DNA system significntly inhibited oxid- 4 TBARS (nmol/mg protein) C ABAP FJ N-PJ R-PJ Fig. 4. Effect of grpefruit juice smples on lipoperoxidtion in rt liver microsomes exposed to oxidtive stress in the presence of peroxyl rdicl. FJ, fresh juice; N-PJ, psteurised nringinse-treted juice; R-PJ, psteurised nd resin-treted juice. C: Microsomes in the bsence of ABAP; microsomes in the presence of ABAP; (1 4 ll): microsomes preincubted with grpefruit juice smples before dding ABAP to induce lipid peroxidtion. Results re the men ± SD of 1 independent experiments. * Vlues significntly different (p <.5) versus C. Different letters for ech volume correspond to sttisticlly different vlues (p <.5). Fig. 5. Effects of grpefruit juice smples t different concentrtions on the protection on DNA ginst Cu(II)-scorbic cid. The numbered lnes represent: DNA lone (C); DNA exposed to Cu(II) (1 lm) nd scorbic cid (1 mm) (OH ); kdna/ Hind III frgments moleculr weight stndrd (1); 1 bp DNA ldder moleculr weight stndrd (2); DNA nd Cu(II)-scorbic cid + fresh juice t 25 ll (3), 5 ll (4), 1 ll (5) nd 2 ll (6); DNA nd Cu(II)-scorbic cid + nringinse processed juice t 25 ll (7), 5 ll (8), 1 ll (9) nd 2 ll (1); DNA nd Cu(II)-scorbic cid + resin-treted juice t 25 ll (11), 5 ll (12), 1 ll (13) nd 2 ll (14).

6 M. Cvi-Siz et l. / Food Chemistry 125 (211) Tble 2 Effect of grpefruit juice smples on the formtion of 8-OH-dG in clf thymus DNA induced by scorbic cid nd copper(ii) t different doses. FJ, fresh juice; N-PJ, psteurised nd nringinse-treted juice; R-PJ, psteurised nd resin-treted juice. Smple Inhibition (%) 25 ll 5ll 1 ll FJ 56.8 ± 7.8 b 64. ± ±.1 N-PJ 59.4 ± 11.2,b 53.6 ± ± 6.4 R-PJ 39. ± ± ± 1.5 Results re the men ± SD of six experiments. Different letters for ech column correspond to sttisticlly different vlues (p <.5). tive DNA dmge induced by copper(ii)-scorbic cid, nd the strongest inhibition effect ws shown by the fresh juice. Nevertheless, no significnt differences were observed between fresh nd debittered juices t 1 ll of smple. Finlly, mong ll grpefruit juice tested, correltion ws found between the percentge of inhibition of modified bse nd totl phenol content (r =.545, p =.37), wheres ny correltion ws found for nringin content. Bsed on these results, the totl phenol content in the grpefruit juice seem to ply role in the protection of DNA ginst oxidtive stress. In summry, the grpefruit juice debittered with nringinse showed significntly higher totl ntioxidnt cpcity, rdicl scvenging bility nd effectiveness in the protection to GSH oxidtion nd lipid peroxidtion thn the juice processed with the exchnge resin. Moreover, both juices were eqully effective in reducing DNA dmge induced by hydroxyl rdicl in dose-dependent mnner. Therefore, enzymtic debittering with nringinse ws more effective thn physicl dsorption with Amberlite Ò IRA-4 in order to preserve the ntioxidnt nd biomolecule protection cpcity of freshly squeezed grpefruit juice. References Abeysinghe, D. C., Li, X., Sun, C. D., Zhng, W. S., Zhou, C. H., & Chen, K. S. (27). Bioctive compounds nd ntioxidnt cpcities in different edible tissues of citrus fruit of four species. Food Chemistry, 14(4), Aren, E., Fllico, B., & Mccrone, E. (21). 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