EFFECT OF NENAS HONEY SUPPLEMENTATION ON THE OXIDATIVE STATUS OF UNDERGRADUATE STUDENTS

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1 Act Alimentri, Vol. 43 (1), pp (2014) DOI: /AAlim EFFECT OF NENAS HONEY SUPPLEMENTATION ON THE OXIDATIVE STATUS OF UNDERGRADUATE STUDENTS J.A. GOON*,, C. K. CHOOR, R. NUR AINAA, X.Q. SZE, M. SYAHRIAH, M.S. NURUL SYAMIMI, S. MUHAMAD RASHIDI, M.A. MARDIYANNA nd J. ZAKIAH Deprtment of Biochemistry, Fculty of Medicine, Universiti Kengsn Mlysi, Jln Rj Mud Adul Aziz, Kul Lumpur. Mlysi Deprtment of Medicine, Universiti Kengsn Mlysi Medicl Center, Jln Jco Ltif, Bndr Tun Rzk, Chers, Kul Lumpur. Mlysi (Received: 23 Octoer 2012; ccepted: 23 Novemer 2012) Honey is dietry ntioxidnt s it contins phenolic compounds, such s flvonoids nd phenolic cids. Antioxidnts re non-nutritive, iologiclly ctive ingredients in food tht reduce oxidtive stress. The ntioxidnt content in ech type of honey vries depending on its source. This study ws imed to determine the effect of Nens honey supplementtion on the oxidtive sttus of group of helthy medicl students. They were divided into two groups; control (n=10) nd supplemented (n=13), where 1 tlespoon of Nens honey ws given ech dy. Blood smpling ws done t seline, 1 st nd 2 nd month of the study for determintion of DNA dmge nd ntioxidnt enzyme ctivities, such s superoxide dismutse (SOD), glutthione peroxidise (GPx), nd ctlse (CAT). Results showed tht Nens honey incresed the level of DNA dmge t the 1 st month ut reduced it significntly t the 2 nd month s compred to control. GPx nd CAT ctivities lso decresed significntly with honey supplementtion throughout the study, though no chnges were oserved in SOD ctivity. Fsting glucose levels remined within the norml rnge with honey supplementtion. In conclusion, Nens honey decreses oxidtive stress which leds to reduction of ntioxidnt enzyme ctivities in the ody. Keywords: honey, ntioxidnt, enzymes, DNA Honey is sticky, yellowish-rown, nturl sweet sustnce produced y honey ees through collection of nectr from mny plnts. From the chemicl point of view, honey is highly concentrted solution of complex mixture of sugrs (PYRZYNSKA & BIESAGA 2009). The specific composition of honey depends on the flower ville to the ees producing the honey. The composition nd the source of honey gretly dicttes its iochemicl properties (KISHORE et l., 2011). Honey is known to e rich in oth enzymtic nd non-enzymtic ntioxidnts, including glucose oxidse, ctlse, scoric cid, flvonoids, phenolic cids, crotenoid derivtives, orgnic cids, Millrd rection products, mino cids, nd proteins (BALTRUŠAITYTĖ et l., 2007). Mlysi is well-known for its wide vriety of honeys. Severl types of Mlysin honey re Durin, Gelm, Nens, nd Coconut honey. Nens honey is one of the most common monoflorl Mlysin honeys nd the florl source is from Anns comosus spp. (common nme Nens) trees. The honeys were nmed ccording to their florl sources. Other thn eing nturl sweetener, honey lso serves s dietry ntioxidnt. The mount, composition, nd type of the ntioxidnts in honey depend on the florl source used to collect nectr from, sesonl nd environmentl fctors, s well s processing (GHELDOF et l., 2003). There is growing demnd of nturl products in humn diet due to the possile negtive effects of synthetic food dditives on humn helth (BALTRUŠAITYTĖ et l., 2007). Honey * To whom correspondence should e ddressed. Phone: ; fx: ; e-mil: jon@medic.ukm.my /$ Akdémii Kidó, Budpest

2 GOON et l.: EFFECT OF NENAS HONEY ON OXIDATIVE STATUS 183 is one of the nturl food products tht is well-known for its high nutritionl nd prophylctic medicinl vlue (PYRZYNSKA & BIESAGA, 2009). The ntioxidtive properties in honey hve een proven effective in iding the heling process of wounds nd urns, dietic ulcers, cncer, crdiovsculr nd neurl diseses (TAN et l., 2009; EREJUWA et l., 2010; KHALIL et l., 2010). In ddition, honey lso helps to improve sptil memory nd reduces the nxiety in ging mice, nd hs ntimicroil properties tht ct effectively ginst humn pthogenic microorgnisms (PAUL et l., 2007; CHEPULIS et l., 2009). Rective oxygen species (ROS), which include superoxide nion (O 2 ), hydrogen peroxide (H 2 O 2 ), nd the hydroxyl rdicl (OH ) generted s y-products of cellulr metolism primrily in the mitochondri, hve een implicted s cuses of ging nd disese. With time, the ccumultion of ROS overwhelms the ody s ntioxidnt defence nd results in oxidtive stress. The term oxidtive stress descries the lck of equilirium etween the free rdicls generted nd the ntioxidnt protective ctivity in given orgnism (RATNAM et l., 2006). Among other fctors tht contriute to ROS formtion re smoking, pollutnts, stress, excessive physicl exercise, nd ultrviolet rys (BLOCK et l., 2002). The humn ody uses defence mechnism known s the ntioxidnt network to fight ginst these free rdicls nd ROS. In this network, there re ntioxidnt enzymes, such s SOD, GPx, nd CAT, tht help in scvenging the free rdicls (NAITO et l., 2010). In ddition, there re rdicl-scvenging sustnces, such s coenzyme Q10, vitmins A, C, nd E, tht ct on the free rdicls nd eliminte them directly (KUTLU et l., 2005; KNOWLDEN, 2012). Beside this, the ody lso hs repir nd regenertive ctivities tht ct on lipids, proteins, nd DNAs tht hve een dmged y free rdicls. The primry enzymes tht re involved in these ctivities re phospholipse, protese, trnsferse, nd DNA repir enzymes (NAITO et l., 2010). Despite the existence of well-structured ntioxidnt network, oxidtive stress does occur when there is n imlnce etween free rdicl formtion nd the mentioned defence mechnism. Free rdicls cn trvel through the cell disrupting the structure of other molecules nd resulting in cellulr dmge, which contriutes to ging nd vrious helth prolems. Antioxidnt tht occur nturlly in the ody or re consumed through the diet my lock dmge to cells. However, over time, dmge cells cn ccumulte nd led to ge relted diseses. As such, humns need to protect themselves from these dmging compounds y soring ntioxidnts from high ntioxidnt food (KHALIL et l., 2010). Therefore, it is vitl to cter the vilility of ntioxidnts y mnipulting diet nd life style of individuls in id of the existing network. In n effort to comt the free rdicl ctivities, vrious studies hve een done to determine the effect of diet nd dietry supplementtion in incresing the individul s ntioxidnt level (GHELDOF & ENGESETH, 2002; RATNAM et l., 2006; EREJUWA et l., 2010). Antioxidnt properties of honey hve often een highlighted ecuse they ct s protective elements ginst ging nd other ge relted diseses y scvenging ROS (KNOWLDEN, 2012). Mlysin honeys tht hve een studied re Tulng nd Gelm honey. Studies hve reported the enefits of oth Tulng nd Gelm honey ut reserch on the Nens honey is still scrce (DYRBYE et l., 2005; TAN et l., 2009). Recently, HUSSEIN nd co-workers (2011) reported tht Gelm nd Nens honeys hve comprtively higher ntioxidnt reducing power s compred to honey from Croti, wheres Gelm honey hs higher rdicl scvenging ctivity s compred with commercil Indin honey. Nens honey hs een implicted to contin vrious ntioxidnt components. However, it hs yet to e tested s n ntioxidnt supplement. Therefore, the im of this work ws to evlute the effect of Nens honey in modulting the oxidtive sttus of young dults s Act Alimentri 43, 2014

3 184 GOON et l.: EFFECT OF NENAS HONEY ON OXIDATIVE STATUS preventive mesure to limit oxidtive dmge with ge. We hope tht this preliminry report will promote the use of this honey s n ntioxidnt in the future. 1. Mterils nd methods 1.1. Nens honey The Mlysin Nens honey used in this reserch ws similr to tht studied y HUSSEIN nd co-workers (2011), where flvonoid nd phenolic contents in Nens honey were found to e the mjor components responsile for its ntioxidnt ctivity. The honey ws purchsed from the Deprtment of Agriculture, Btu Pht, Johor, Mlysi, the florl source ws from the Anns comosus spp. tree Sujects The sujects recruited for this study were yers old medicl students of the Universiti Kengsn Mlysi Medicl Centre (UKMMC). They were physiclly helthy, not tking ny form of ntioxidnt supplementtions, nor prcticing ny form of regulr exercise for the lst three months. Students with medicl illness, smoking, or tking lcohol were excluded from this study. Prior to the commencement of the study, informed consents were otined from ll prticipnts nd demogrphic dt, which included ge, weight, height, ody mss index (BMI), gender, nd ethnic group, were lso recorded. The sujects were ssigned into two groups: control group nd supplemented group. The supplemented group ws given 1 tlespoon of Nens honey every dy for 2 months. Fsting lood smples were tken t seline (0 month), 1 st nd 2 nd month from oth groups. Prticipnts were instructed to fst for 10 hours efore lood smple collection Detection of DNA dmge DNA dmge ws determined y using comet ssy (SING et l., 1988). Briefly, 5 μl of fresh whole lood ws mixed with 70 μl 0.6% low melting grose in microcentrifuge tues. The cell suspension ws then emedded onto frosted microscope slides with 0.6% norml melting grose nd lysed with cold lysing solution. After the denturtion of DNA y the lkline solution, neutrliztion uffer ws used to neutrlize the excess lkli, then electrophoresis ws done nd slides were viewed y n epifluorescent microscope Glutthione peroxidse (GPx) ssy The ntioxidnt enzymes ctivities, GPx, SOD, nd CAT were mesured using spectrophotometer. The GPx ctivity ws determined sed on the method y PAGLIA nd VALENTINE (1967). Hemolystes (200 μl) were diluted with 0.8 ml distilled wter nd mixed with 1 ml of cynmethemogloin regent efore dding to sustrte mixture. It consisted of 0.05 M phosphte uffer (ph 7.0), 5 mm ethylenedimine tetrcette (EDTA), M sodium zide (NN 3 ), 8.4 mm reduced nicotine denine dinucleotide phosphte (NADPH), 10 U ml 1 glutthione reductse, nd 0.15 M reduced glutthione (GSH). A volume of 0.1 ml H 2 O 2 (2.2 mm) ws dded to 2.9 ml of the rection mixture nd the rection ws followed y chnge in sornce for 5 min t 340 nm. A control ws lso run y replcing the hemolyste with distilled wter. One unit of GPx ws defined s the mount of enzyme tht would oxidize 1 μm NADPH to NADP + per min t 25 C nd ph 8.0. Act Alimentri 43, 2014

4 GOON et l.: EFFECT OF NENAS HONEY ON OXIDATIVE STATUS Superoxide dismutse (SOD) ssy SOD ctivity ws determined ccording to BEYER nd FRIDOVICH (1987). Blood smples to e nlysed were mixed with n equl volume of distilled wter to prepre hemolystes. The sustrte solution ws mde up of 50 mm phosphte uffer (ph 7.0), 0.2 M L-methionine, 1.72 mm nitro lue tetrzolium (NBT), nd 1% Triton X-100. The hemolyste (20 μl) ws dded to 1 ml sustrte solution nd the mixture ws rected with 10 μl rioflvin (0.117 mm) in rightly illuminted luminium foil-lined ox contining two 20 W Sylvni GroLux fluorescent lmps for 7 min. A control tue in which the smple ws replced y phosphte uffer ws run in prllel nd the sornce ws mesured t 560 nm. One unit of SOD ws defined s the mount of enzyme tht would inhiit the rte of reduction of NBT y 50% t 25 C t ph Ctlse (CAT) ssy CAT ctivity ws determined ccording to AEBI (1984). The hemolyste ws derived y mixing RBC pellet with cold distilled wter. The hemolyste ws mixed with phosphte uffer nd then with 30 mm hydrogen peroxide to initite the rection in which the decrese in sornce ws mesured t 240 nm wvelength. One unit of CAT ws defined s the mount of enzyme tht would decompose 1 μm H 2 O 2 per second t 25 C nd ph Hemogloin content The hemogloin content of ll hemolystes ws determined in duplicte with hemogloin ssy kit (ctlogue numer 6210) y Egle Dignostic (USA). Briefly, hemogloin concentrtion stndrd curve ws prepred with cynmethemogloin stndrd nd regent ccording to the mnufcturer s guide. The hemolystes used in SOD (30 μl), GPx (100 μl), nd CAT (20 μl) nlysis were mixed with cynmethemogloin regent of 4 ml nd the sornces were red t 540 nm Sttisticl nlysis Results otined were expressed s men ± stndrd devition (SD). Sttisticl significnce ws tken t P<0.05. All dt nlysis ws performed using SPSS version Chnges in the mesured prmeters t ech follow-up were nlysed using ANOVA. 2. Results nd discussion A totl of 23 sujects completed the study where 11 of them were mle nd 10 femles. The sujects were from three min ethnic groups; Mly (78.26%), Chinese (13.04%) nd Indin (8.7%). This dt reltively mirrored the popultion of medicl students in the Universiti Kengsn Mlysi Medicl Centre. Severl sujects from oth groups dropped out from the study; however, the men demogrphic chrcteristics remined unltered. Of the 30 sujects enrolled t the eginning, 23 sujects (10 controls nd 13 supplemented) completed the study. Medicl students were selected for this study s they hve een identified s young dults with the highest risk of stress due to exposure to stressful environment, such s exmintions, clinicl works, nd hectic schedules (DYRBYE et l., 2005). The uild-up of these stress fctors my result in oxidtive stress. This corresponds to our result, which Act Alimentri 43, 2014

5 186 GOON et l.: EFFECT OF NENAS HONEY ON OXIDATIVE STATUS showed considerle high level of DNA dmge in oth control nd supplemented group t the eginning of the study. Though the numer of sujects studied ws smll, our results reveled interesting roles of Nens honey, which prelude future reserch Blood glucose Both groups of sujects showed no significnt chnges in the glucose level throughout the study (Tle 1). Despite the high glucose content of honey, this result indicted tht dily consumption of 1 tlespoon of Nens honey hd no effect on the lood glucose level. Tle 1. Fsting glucose levels in control nd supplemented group t 0, 1 st, nd 2 nd month Men±SD (mmol l 1 ) 0 1 st 2 nd Control 5.65± ± ±0.22 Supplemented 5.24± ± ± DNA dmge Interestingly, supplementtion with Nens honey demonstrted non-expected hormesis effect, where the level of DNA dmge ws found to increse t the 1 st month of supplementtion nd decresed t the 2 nd month (Fig. 1). These results suggest novel role of Nens honey, wherey it could stimulte the ntioxidnt system or the DNA repir mechnism y inititing n incresed level of oxidtive stress t the erly stge of supplementtion. The decresed level of DNA dmge therefter is evidence of this hormesis effect. 60 DNA dmge, u Months Fig. 1. DNA dmge in supplemented nd control groups t 0, 1 st, nd 2 nd month. : Control; : supplemented. Significnt chnge (P<0.05) s compred to 0 month; significnt chnge (P<0.05) s compred to 1 month Our findings re similr with the results otined y YAO nd co-workers (2011), where DNA dmge ws found to decrese significntly with Gelm honey supplementtion. Our results lso support previous studies, which climed tht Nens honey hs the ility to scvenge free rdicls (HUSSEIN et l., 2011; KISHORE et l., 2011). Quercetin, which is the Act Alimentri 43, 2014

6 GOON et l.: EFFECT OF NENAS HONEY ON OXIDATIVE STATUS 187 second most undnt flvonoid in Nens honey, prevents DNA dmge y incorporting into the red lood cells structure to provide defence nd promote cell function (ALVAREZ- SUAREZ et l., 2012). Elsewhere, flvonoids in the honey decrese oxidtive stress y trpping rective oxygen species, inhiiting enzymes responsile for producing superoxide nions, chelting trnsition metls involved in processes forming rdicls, nd preventing the peroxidtion process y reducing lkoxyl nd peroxyl rdicls (PYRZYNSKA & BIESAGA, 2009) Antioxidnt enzyme The supplemented group hd decresed GPx ctivity t 1 st nd 2 nd month s compred to the seline (Fig. 2). Conversely, there were no significnt chnges oserved in the GPx ctivity for control group throughout the study. As for CAT enzyme, its ctivity decresed significntly in the supplemented group t the 2 nd month ut showed no significnt chnges in the control group (Fig. 3). Unlike the mentioned enzymes, SOD ctivity did not chnge significntly in either group of sujects GPx ct., mu/mgh Months Fig. 2. GPx ctivities in supplemented nd control groups t 0, 1 st, nd 2 nd month. : Control; : supplemented. Significnt chnge (P<0.05) s compred to 0 month. Significnt chnge (P<0.05) s compred to 1 month 6 5 CAT ct., U/mgH Months Fig. 3. CAT ctivities in supplemented nd control groups t 0, 1 st, nd 2 nd month. : Control; : supplemented. Significnt chnge (P<0.05) s compred to 0 month. Significnt chnge (P<0.05) s compred to 1 month Act Alimentri 43, 2014

7 188 GOON et l.: EFFECT OF NENAS HONEY ON OXIDATIVE STATUS SOD ct., U/mgH Months Fig. 4. SOD ctivities in supplemented nd control groups t 0, 1 st, nd 2 nd month. : Control; : supplemented As GPx, CAT, nd SOD function to scvenge free rdicls, their ctivities depend lrgely on the mount of free rdicls nd ROS present in the humn ody (NAITO et l., 2010). Our findings correspond to the study y EREJUWA nd co-workers (2009), which lso presents reduction in ntioxidnt enzyme ctivities with honey supplementtion. This might e the result of lower mount of free rdicls ville to ctivte the ntioxidnt enzymes s shown y reduction in oxidtive DNA dmge mong the supplemented sujects. This lso indictes tht honey ttenutes the chnges in ntioxidnt enzymes in response to oxidnt genertion (EREJUWA et l., 2010). 3. Conclusions The sustitution of honey in some foods for trditionl sweeteners hs een suggested to result in n enhnced ntioxidnt defence system in helthy dults (SCHRAMM et l., 2003). In line with tht, our dt suggest tht Nens honey in prticulr cn ct s n ntioxidnt gent, either through the direct ntioxidnt effect of flvonoids nd phenolics or stimultion of DNA repir in the humn ody y its residues, which finlly produce eneficil effects to helth. In conclusion, supplementtion of Nens honey improved the oxidtive sttus of medicl students. This study highlights the relevnce of honey supplementtion t n erly stge of life, such s young dults, to prevent further oxidtive dmge with ge. Therefore, more investigtions re highly recommended to elucidte the potentil use of Nens honey s nturl ntioxidnt supplement in dvnced ge nd lso in oxidtive stress ssocited diseses. * We wish to thnk the Fculty of Medicine, Universiti Kengsn Mlysi (UKM) for the UKM short-term grnt FF Act Alimentri 43, 2014

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9 190 GOON et l.: EFFECT OF NENAS HONEY ON OXIDATIVE STATUS T AN, H.T., RAHMAN, R.A., GAN, S.H., HALIM, A.S., HASAN, A.S., SULAIMAN, S.A. & KIRNPAL-KAUR, B. (2009): The nticteril properties of Tulng honey ginst wound nd enteric microorgnisms in comprison to Mnuk honey. BioMed Centrl Complementry Alterntive Med., 15, 34. Y AO, L.K., RAZAK, S.L.A., ISMAIL, N., FAI, N.C., ASGAR, M.H.A.M., SHARIF, N.M., GOON, J.A. & JUBRI, Z. (2011): Mlysin gelm honey reduces oxidtive dmge nd modultes ntioxidnt enzyme ctivities in young nd middle ged rts. J. Medl Pl. Res., 5, Act Alimentri 43, 2014