Evaluation of antioxidant properties of a new compound, pyrogallol-phloroglucinol -6,6'-bieckol isolated from brown algae, Ecklonia cava

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1 Nutrition Reserh nd Prtie (Nutr Res Prt) 211;5(6): Evlution of ntioxidnt properties of new ompound, pyrogllol-phlorogluinol -6,6'-iekol isolted from rown lge, Ekloni v Sung-Myung Kng 1*, Seung-Hong Lee 1*, Soo-Jin Heo 2, Kil-Nm Kim 3 nd You-Jin Jeon 1 1 Deprtment of Mrine Life Siene, Jeju Ntionl University, 1 Ar 1-dong, Jejudehk-ro, Jeju , Kore 2 Mrine Living Resoures Reserh Deprtment, Kore en Reserh nd Development Institute, Ansn , Kore 3 Kore Bsi Siene Institute (KBSI), Jeju 69-14, Kore Astrt In this study, ntioxidnt nd free rdil svenging tivities of the nturl ntioxidtive ompound, pyrogllol-phlorogluinol-6,6'-iekol (PPB) isolted from rown lge, Ekloni v ws ssessed in vitro y mesuring the rdil svenging tivities (DPPH, lkyl, hydroxyl, nd superoxide) using eletron spin resonne (ESR) spetrometry, intrellulr retive oxygen speies (RS) svenging tivity, nd DNA dmge ssy. Aording to the results of these experiments, the svenging tivity PPB ginst differene rdils ws in the following order: DPPH, lkyl, hydroxyl, nd superoxide rdils (IC 5;.9, 2.54, nd 19.5 µm). The ntioxidnt tivities of PPB were higher thn tht of the ommeril ntioxidnt, sori id. Furthermore, PPB effetively inhiited DNA dmge indued y H 2 2. These results suggest tht the nturl ntioxidtive ompound, PPB, n e used y the nturl food industry. Key Words: Ekloni v, rown lge, pyrogllol-phlorogluinol-6,6 -iekol, ntioxidnt Introdution 1) Retive oxygen speies (RS) inluding superoxide nion rdil, hydroxyl rdil, lkyl rdil nd hydrogen peroxide, whih re formed nd degrded y ll eroi orgnism, n use oxidtive dmge to ll mjor groups of iomoleules (DNA, protein, lipids nd smll ellulr moleules), nd hs een shown to led to rdiovsulr nd neurodegenertive diseses [1]. Antioxidnts n inhiit or dely the initition or propgtion of the oxidtive hin retion nd thus prevent the repir of ell dmge used y RS. Aordingly, syntheti ntioxidnts, suh s utylted hydroxytoluene (BHT) nd utylted hydroxynisole (BHA), hve een extensively used s dditives for oxidtion suppressnt in foods, osmetis nd drug ompositions. However, the possile toxiity s well s generl onsumer rejetion of syntheti ntioxidnts hs led to derese in the use of these ompounds [2]. Therefore, reserh on nturl ntioxidnts hs reently een on the rise. Mny studies hve een rried out to find new ntioxidnt ompounds from nturl resoures suh s plnts [3], foods [4] nd mrine lge [5,6]. Espeilly, foods phytohemils suh s phenoli ompounds hve potentil protetive effets ginst mny diseses [7]. Therefore, onsumption of vriety of phenoli ompounds with high ntioxidnt effets my redue the risk of serious helth disorders used y RS. Ekloni v, whih is rown lg tht is present in high quntities on Jeju Islnd of Kore, is not ville in Europe. E. v is lso populr in Kore nd Jpn, where these vlule rown lge re utilized in food ingredients, niml feeds, fertilizers nd mediines [8]. The results of reent studies hve shown tht E. v displys strong ntioxidnt tivity. However, further studies will e needed to determine nd isolte the nturl ntioxidtive ompoundss from E. v. The ojetives of the present study were to isolte nd evlute the ntioxidnt properties of ompounds from E. v. Mterils nd Methods Chemils Sili gel 6 ( mm Merk), Celite 545, TLC pltes (Kieselgel 6 F , Merk), 5,5-Dimethyl-1-pyrrolin N- oxide (DMP), 2,2-zois (2-midinopropne) hydrohloride This reserh ws supported y grnt from Mrine Bioproess Reserh Center of the Mrine Bio21 Projet funded y the Ministry of Lnd, Trnsport nd Mritime, Repuli of Kore. * These uthors ontriuted eqully to this mnusript. Corresponding Author: You-Jin Jeon, Tel , Fx , Emil. youjinj@jejunu..kr Reeived: Septemer 8, 211, Revised: Novemer 28, 211, Aepted: Deemer 7, The Koren Nutrition Soiety nd the Koren Soiety of Community Nutrition This is n pen Aess rtile distriuted under the terms of the Cretive Commons Attriution Non-Commeril Liense ( whih permits unrestrited non-ommeril use, distriution, nd reprodution in ny medium, provided the originl work is properly ited.

2 496 Antioxidnt effet of new ompound from E. v (AAPH), -(4-pyridyl-1-oxide)-N-t-utylnitrone (4-PBN), 1,1- diphenyl-2-pirylhydrzyl (DPPH), FeS 4, nd hydrogen peroxide; for the ell ulture experiment regents, 3-(4,5-Dimethylthizol- 2-yl)-2,5-diphenyltetrzolium romide (MTT), 2 7 -dihlorodihydrofluoresein diette (DCFH-DA) were quired from Sigm Chemil Co. (St. Louis, Mo, USA). All other regents were of nlytil grde. Plnt mteril The rown lg, E. v, ws olleted long the ost of Jeju Islnd, Kore, etween Ferury nd My 21. The smples were wshed three times in tp wter to remove ny tthed slt, epiphytes, nd snd, then rinsed refully with fresh distilled wter, nd mintined in medil refrigertor t -2. The frozen smples were then lyophilized nd homogenized using grinder prior to extrtion. Preprtion of the extrts from rown lge, E. v The dried E. v powder (5 g) ws extrted using 5 L of 8% queous methnol three times t room temperture. The liquid lyer ws otined vi filtrtion, nd the filtrte ws onentrted using n evportor under redued pressure. The extrt ws suspended in H 2, nd the queous lyer ws prtitioned with ethyl ette (EtA). The EtA extrt (45.65 g) ws mixed with elite. The mixed elite ws then dried nd pked into glss olumn, nd susequently eluted in the following order: hexne, dihloromethne, diethyl ether, nd Ekloni v ( 5g ) 8% Me extrt Ethyl ette frtion (47.63 g) Celite olumn hromtogphy n-hexne frtion ( 9.4 g) Aqueous frtion Dihloromethne frtion (.45 g) Sili olumn hromtogrphy Aqueous frtion Diethyl ether frtion ( g) Bu frtion ( 9.16 g) Aqueous frtion ECE1 ECE2 ECE3 ECE4 ECE5ECE6 ECE8 ECE7 Ative ompound (15 mg) Aqueous frtion Fig. 1. Shemti of the isoltion nd purifition of the tive ompound from Ekloni v utnol. The diethyl ether frtion (26.69 g) ws sujeted to Sili olumn hromtogrphy using stepwise grdient with hloroform/methnol (2/1 /1) to yield sufrtions (E. vdiethyl ether frtion, ECE) sed on TLC nlysis (Fig. 1). HPLC-MS nlysis nd HPLC seprtion for evlution of mjor tive ompound HPLC-ESI-MS nlysis of ECE ws onduted using n LXQ system (Thermo Fisher Sientifi, USA) equipped with 2.1 mm 1 mm i.d., 1.9 μm prtile size, Hypersil-Gold C18 olumn. The smple ws then seprted for 4 min using grdient moile phse onsisting of 1% to 1% methnol. The flow rte ws set t 2 µl/min nd the injetion volume ws 2 µl. The detetion wvelength ws set to 23 nm. Positive ioniztion mode ws pplied in full sn rnge of 5-1,. The optimized eletrospry opertion onditions were s follows: pillry voltge 5 kv, temperture 275, nd sheth gs 3 ml/min. HPLC seprtion of ECE7 ws onduted using Dionex system (Dionex, USA) equipped with 19 mm ⅹ3 mm i.d., 1 μm prtile size, μbondpk TM C18 olumn. The sme hromtogrphi onditions s desried ove were employed, exept the flow rte ws set to 5 ml/min. Identifition of tive ompound vi Nuler Mgneti Resonne (NMR) NMR spetr were reorded using JNM ECP-4 spetrometer (JNM ECP-4, JEL, Jpn), operted t 5 MHz for 1 H NMR nd 1 MHz for 13 C NMR. The isolted tive ompound ws prepred in deuterted solvents in 5mm NMR tues. The deuterted solvent employed ws DMS-d 6 nd the hemil shifts were mesured reltive to the TMS signl. All experiments were onduted t room temperture. Mesurement of free rdil svenging tivities of the tive ompound PPB y ESR The different rdils tested here were generted ording to previously desried proedures [5], nd the spin dduts were reorded using JES-FA eletron spin resonne (ESR) spetrometer (JES-FA ESR, JEL, Jpn). DPPH rdil svenging ssy The DPPH rdil svenging tivity ws evluted using n ESR spetrometer in ordne with the method desried y Nnjo et l. [9]. A 6 μl smple ws dded to 6 μl of DPPH (6 μm) in ethnol. After 1 seonds of vigorous mixing, the solution ws trnsferred to Teflon pillry tues nd fitted into the vity of the ESR spetrometer extly 2 min lter, under the following mesurement onditions: entrl field 3475 G, modultion frequeny 1 khz, modultion mplitude 2 G,

3 Sung-Myung Kng et l. 497 mirowve power 5 mw, gin , nd temperture 25. All rdil svenging tivities (%) were lulted using the following eqution, where H nd H were the reltive pek heights of the rdil signls with nd without PPB, respetively. % rdil svenging tivity = [1-(H / H)] 1 Alkyl rdil svenging ssy Alkyl rdils were generted vi AAPH. The retion mixture ontining 1 mm AAPH nd 1 mm 4-PBN were mixed with PPB. The solution ws inuted for 3 min t 37 in wter th, nd then trnsferred to Teflon pillry tues [1]. The spin ddut ws reorded using JES-FA ESR spetrometer under the following mesurement onditions: entrl field 3475 G, modultion frequeny 1 khz, modultion mplitude 2 G, mirowve power 1 mw, gin , nd temperture 25. The rdil svenging tivity (%) ws presented s desried in setion 3.1. Hydroxyl rdil svenging ssy Hydroxyl rdils were generted vi Fenton retion, nd rpidly reted with DMP nitrone spin trp. The resultnts DMP- dduts were deteted using n ESR spetrometer [11]. Retion mixtures ontining 1 μl of.3 M DMP, 1 μl of 1 mm FeS 4, nd 1 μl of 1 mm H 2 2 were mixed with PPB, nd trnsferred to Teflon pillry tues. The spin dduts were mesured using n ESR spetrometer extly 2.5 min lter, under the following mesurement onditions: entrl field 3475 G, modultion frequeny 1 khz, modultion mplitude 2 G, mirowve power 1 mw, gin , nd temperture 25. The hydroxyl rdil svenging tivity (%) ws presented s desried in setion 3.1. Superoxide rdil ssy Superoxide rdils were generted vi the UV irrdition of rioflvin/edta system [12]. The retion mixture ontining.3 mm rioflvin, 1.6 mm EDTA, 8 mm DMP, nd the indited onentrtions of PPB were irrdited for 1 min under UV lmp t 365 nm. The retion mixture ws trnsferred to 1 μl Teflon pillry tue in the ESR spetrometer for mesurement. Experimentl onditions were s follows: mgneti field ± 5 mt, power 1 mw, modultion frequeny 9.41 GHz, mplitude 1 1, sweep time 1 min, gin , nd temperture 25. The rdil svenging tivity (%) is shown s desried in setion 3.1. Cell ulture The monkey kidney firolst ell line (Vero) ws mintined t 37 in n inutor with humidified tmosphere of 5% C 2. The ells were ultured in Duleo s modified Egle s medium ontining 1% het-intivted fetl ovine serum, streptomyin (1 µg/ml), nd peniillin (1 unit/ml). Assessment of ell ytotoxiity Cell ytotoxiity ws estimted vi the MTT ssy, whih is test of metoli ompetene predited sed on mitohondril performne. It is olorimetri ssy, whih depends on the onversion of yellow tetrzolium romide to its purple formzn derivtive vi mitohondril suinte dehydrogense in vile ells [13]. Vero ells were seeded in 96-well pltes t onentrtion of ells/ml. After 16 hr, the ells were treted with PPB t different onentrtions for 24 hr t 37. The MTT stok solution (5 µl; 2 mg/ml) ws then pplied to the wells to totl retion volume of 25 μl. After 4 hr of inution, the pltes were entrifuged for 5 min t 8 g, nd the superntnts were spirted. The formzn rystls in eh well were dissolved in 15 µl of dimethylsulfoxide (DMS), nd the sorne ws mesured vi ELISA t wvelength of 54 nm. Reltive ell ytotoxiity ws evluted ording to the quntity of MTT onverted to insolule formzn slt. The optil density of the formzn generted in the ontrol ells ws onsidered to represent 1% viility. The dt were expressed s the men perentge of the vile ells versus the respetive ontrol. Intrellulr retive oxygen speies (RS) mesurement For the detetion of intrellulr RS, Vero ells were seeded in 96-well pltes t onentrtion of ells/ml. After 16 hr, the ells were treted with vrious onentrtions of PPB nd inuted t 37 under humidified tmosphere. After 3 min, H 2 2 ws dded t finl onentrtion of 1 mm, nd the ells were inuted for n dditionl 3 min t 37. Finlly, 2,7 -dihlorodihydrofluoresein diette (DCFH-DA; 5 µg/ml) ws introdued to the wells, nd DCFH fluoresene ws deteted t n exittion wvelength of 485 nm nd n emission wvelength of 535 nm, using Perkin-Elmer LS-5B spetrofluorometer. The perentge of intrellulr RS svenging tivity ws lulted using the following eqution: Intrellulr RS svenging tivity (%) = (1-(C1 / C)) 1 in whih C1 is the fluoresene intensity of ells treted with H 2 2 nd ompound, nd C is the fluoresene intensity of ells treted with only H 2 2. Comet ssy The lkline omet ssy ws onduted ording to the method desried y Ahn et l. [14]. The numer of ultured ells ws djusted to ells/ml, nd the ells were inuted with PPB t onentrtions rnging from 25 to 1 μg/ml, whih

4 498 Antioxidnt effet of new ompound from E. v were seleted sed on the results of the hydrogen peroxide svenging tivity mesurements, for 3 min t 37 in drk inutor. After preinution, the ells were entrifuged t minimum rpm for 5 min nd wshed with phosphte uffer sline (PBS). The ells were then resuspended in PBS with 5 μm H 2 2 for 5 min on ie. The untreted ontrol ells were resuspended in PBS only, without H 2 2. The ells were then wshed with 1 ml PBS nd entrifuged. The ell suspensions were mixed with 1 μl of.7% low melting point grose (LMPA) t 37 nd spred on fully frosted mirosopi slide tht ws pre-oted with 1 μl of 1% norml melting point grose (NMPA). After the solidifition of the grose, the slide ws overed with nother 1 μl of.7% LMPA nd susequently immersed in lysis solution (2.5 M NCl, 1 mm N-EDTA, 1 mm Tris, 1% Trion X-1, nd 1% DMS, ph 1) for 1 hr t 4. The slides were then pled in uffer ontining 3 mm N nd 1 mm N-EDTA (ph 13) for 2 min to llow DNA unwinding nd to mesure the lkli lile dmge. An eletril field ws pplied (3 ma, 25 V) for 2 min t 4 to drw negtively hrged DNA towrd the node. After eletrophoresis, the slides were wshed three times with neutrlizing uffer (.4 M Tris, ph 7.5) for 5 min t 4 nd stined with 5 μl of ethidium romide (2 µg/ml). The slides were oserved using fluoresene mirosope nd the result imges were nlyzed (Kineti Imging, Komet 5.5, UK). The perentge of totl fluoresene in the til nd the til length of the 5 ells/slide were reorded. Sttistil nlysis All the dt were expressed s men ± stndrd devition (SD) of three determintions. Sttistil omprison ws performed vi one-wy nlysis of vrine (ANVA) followed y Dunn s multiple rnge test (DMRT). P-vlues of less thn.5 (P <.5) were onsidered signifint. In te n sity A B Pyrogllol-phlorogluinol-6,6 -iekol (PPB) H H 3" 2"' 3"' 2" 4" 1"' 4" 5" 1" 1" 1"' H 8 7 9" 4"' 1"" 4"' 6"" m/z 9 6 2"" 6" 9" Ative ompound [M-H] - = 973 m/z 9 5 7" 8" H 4"" 5"" 5' 6' 1 4 4' 1' 1 4 2""' 1""' Fig. 2. MS spetrosopi nd hemil struture of the tive ompound isolted from E. v. The spetr were generted in negtive ioniztion mode (A). Chemil struture nd HMBC orreltion of the tive ompound isolted from E. v (B). 3"" 3' 6""' ' ""' 5""' ""' Results Isoltion nd identifition of ntioxidnt tive ompound E. v is present in undne long the ost of Jeju Islnd, nd is generlly regrded s useful mteril for the prevention or tretment of severl humn diseses. Sine there hve een reltively few iologil studies onduted with this lg, here, we ttempted to detet nd isolte novel ntioxidtive ompound from the lg. In this study, we ttempted to ssess the ntioxidnt effets of the Me extrt derived from E. v long with its solvent solule frtions, inluding n-hexne, dihloromethne, diethyl ether, n-uthnol, nd H 2 lyer t onentrtions of 1 μg/ml (dt not shown). Among the prtitioned frtions of the methnol extrt, the diethyl ether frtion (64.1%) displyed notiele DPPH rdil svenging tivity. Therefore, we onduted further experiments nd isolted the tive ompound of the diethyl ether frtion vi repeted olumn hromtogrphy over sili gel nd RP-18 gel. The struture of the tive ompound ws determined y 1D ( 1 H nd 13 C NMR) spetrosopi nlyses nd omprison to pulished dt [15]. The moleulr ion in the negtive ESI-MS of the tive ompound t m/z 973 orresponded to the moleulr formul of C 48H The hemil shifts oserved in the 1 H nd 13 C NMR experiments indited tht the polyphenoli struture ws phlorotnnin, sine resonne signls were in the rnge of δ nd δ The 1 H NMR dt indited the presene of fifteen romti protons nd fifteen phenoli hydroxyl protons. The 13 C NMR signls orresponded to fifteen non-sustituted nd thirty three -ering romti rons. This dt suggested tht the tive ompound ontined eight units of phlorogluinol. The NMR spetrl dt were similr

5 Sung-Myung Kng et l. 499 Tle 1. 1 H NMR dt of pyrogllol-phlorogluinol-6,6'-iekol (PPB) in DMS-d 6 (4 MHz) Position (C # ) δ H (mult, J) Position (C # ) δ H (mult, J) (1H, s) (1H, s) (1H, s) (1H, s) (1H, m) (1H, s) (1H, m) (1H, s) (1H, m) (1H, s) (1H, s) (1H, s,) (1H, s) (1H, s) (1H, m) (1H, s) (1H, m) (1H, s) (1H, m) (1H, s) (1H, d) (1H, s) (1H, d) (1H, s) (1H, m) (1H, s) (1H, m) (1H, s) (1H, m) (1H, s) Tle C NMR dt of PPB in DMS-d 6 (1 MHz) Position (C # ) δ (mult) Position (C # ) δ (mult) (s) (s) (s) (s) (d) (s) (s) (d) (s) (s) (s) (s) (s) (s) (s) (s) (d) (s) (s) (s) (s) (d) (s) (s) (d) (s) (s) (d) (d) (s) (s) (d) (s) (s) (s) (d) (d) (s) (s) (s) (d) (d) (s) (s) (d) (s) (s) (d) to those of 6, 6 -iekol [16]. However, when ompred to 6, 6 -iekol, the hemil shift of C-5 (δ 159.3) ws up field. This suggested tht two dditionl units of phlorogluinol were onneted with C-5. The hemil shifts of C-1 (δ 154.) C-4 (δ 122.5) nd C-1 (δ 159.9) nd HMBC orreltions etween H-2 /H-6 nd C-1 /C-4, H-2 /H-6 nd C- 1 indited the presene of two ether linkges etween three rings. The 1 H nd 13 C NMR dt of the tive ompound re listed in Fig. 2, nd shown in Tle 1 nd 2. Bsed on the nlysis of these spetrl dt, the tive ompound ws dedued nd tenttively reported to e pyrogllol-phlorogluinol-6,6 -iekol (PPB). Rdil svenging tivities of PPB ESR spin trpping is sensitive, diret, nd urte method to monitor retive speies [12]. Therefore, in this study ESR, ws employed to evlute the DPPH, lkyl, hydroxyl, nd superoxide rdil svenging tivities of PPB isolted from E. v, nd ompre these tivities with tht of ommeril ntioxidnt, sori id. DPPH hs een widely used to test the ility of ompounds to t s free rdil svengers or hydrogen donors, nd to evlute the ntioxidnt tivity of foods. Therefore, DPPH ws used to evlute the ntioxidtive tivity of PPB. The ESR spetr of different onentrtions of PPB in the presene of DPPH re shown in Fig. 3A. It ws noted tht the ESR signls of PPB were ttenuted when the onentrtions inresed from.25 to 5 µm. PPB hd higher IC 5 vlue for DPPH rdil svenging tivity (IC 5 =.9 µm) thn tht of the ommeril ntioxidnt, sori id (IC 5 = µm) (Tle 3), nd the rdil svenging tivity ws found to e dose-dependent. The lkyl rdil spin ddut ws oserved when AAPH ws inuted for 3 min with the spin trp 4-PBN t 37, nd these rdils were mesured vi ESR. The ility of PPB to svenge lkyl rdils is presented in Fig. 3B. As shown in this figure, 63.62% of the lkyl rdils were svenged y PPB t onentrtion of 6.25 µm. The ttenution of the ESR signl ws oserved with n inrese in the PPB onentrtion; hene, the tivity ws determined to e dose-dependent. Furthermore, PPB hd higher IC 5 vlue for lkyl rdil svenging (IC 5 = 2.54 µm) thn tht of sori id (IC 5 = µm) (Tle 3). Therefore, this result indites tht PPB possesses remrkle svenging ility for lkyl rdils. Hydroxyl rdils re n extremely retive oxygen speies, ple of modifying lmost every moleule in the living ells. This rdil hs the pity to use strnd dmge in DNA, whih n led to rinogenesis, mutgenesis, nd ytotoxiity. Moreover, hydroxyl rdils re ple of quik initition of the lipid peroxidtion proess y strting hydrogen toms from unsturted ftty ids [17]. The hydroxyl rdil svenging tivities of PPB re shown in Fig. 3C. As shown in this figure, derese in the mount of DMP- dduts ws oserved fter the ddition of PPB, nd rdil svenging tivity ws dose-dependent. The PPB displyed strong hydroxyl rdil svenging tivity nd higher IC 5 vlues (IC 5 = µm) thn sori id (IC 5 = µm) (Tle 3), nd the rdil svenging tivity ws found to e dose-dependent. Superoxide nion rdils ply mjor role in the formtion of other retive oxygen speies, suh s hydroxyl rdil, hydrogen peroxide nd single oxygen in living systems. Superoxide rdils

6 5 Antioxidnt effet of new ompound from E. v DPPH rdil svenging tivity (%) Hydroxyl rdil svenging tivity (%) A Conentrtion (μm) C d Conentrtion (μm) Alkyl rdil svenging tivity (%) Superoxide rdil svenging tivity (%) B Conentrtion (μm) D d Conentrtion (μm) Fig. 3. Free rdil svenging tivities of PPB mesured using n ESR spetrometer. (A) DPPH rdil; (B) Alkyl rdil; (C) Hydroxyl rdil; (D) Superoxide rdil. Experiments were onduted in triplite nd the dt were expressed s the mens ± SE. Vlues with different lphets re signifintly different t P <.5 s nlyzed y Dunn s multiple rnge test (DMRT). Tle 3. Free rdil svenging tivities of PPB sed on the ESR spetr IC 5 (µm) DPPH Alkyl Hydroxyl Superoxide PPB Asoriid n indue ging nd destroy the ell memrne, nd n e generted y oxidtive stress. The ESR signls nd the svenging tivity of PPB ginst superoxide rdils re shown in Fig. 3D. As shown in this figure, PPB showed lmost the sme tivity (IC 5 = 19.5 µm) s the ommeril ntioxidnt (IC 5 = µm) (Tle 3). Bsed on these results, this novel ntioxidnt ompound, PPB, ppers to funtion s n eletron donor tht is ple of neutrlizing free rdils. Consequently, PPB is potentil rdil svenging gent tht suppresses oxidtion in humns nd in food. Cell survivl rte (%) Pyrogllol-phlorogluinol-6,6'-iekol Fig. 4. The ytotoxi effet of the PPB isolted from E. v t different onentrtions. The viility of Vero ells ws determined using the MTT ssy. Experiments were onduted in triplite nd the dt re expressed s the mens ± SE. Vlues with different lphets re signifintly different t P <.5 s nlyzed y Dunn s multiple rnge test (DMRT). Cell viility The survivl rtes of ells were higher thn 95% t ll of onentrtions tested (6.25 to 1 µm), whih indite tht this ompound produed no ytotoxi effets in Vero ells (Fig. 4). Intrellulr retive oxygen (RS) speies mesurement The diret svenging effets of PPB on ellulr rdils were investigted in order to onfirm their ility to svenge free rdils in ellulr environments. To determine whether PPB ould prevent H 2 2-indued RS genertion nd the resultnt oxidtive stresses, levels of RS prodution in the ells were determined using the fluoresene proe DCF. In this study, we ttempted to evlute the ntioxidnt effets of PPB in Vero ells fter H 2 2 tretment. The intrellulr RS svenging tivity of the ompound ws shown in Fig. 5. As shown in this figure, 68.79% of intrellulr RS ws svenged t onentrtion of 5 µm, nd this tivity ws dose-dependent. PPB hd n IC 5

7 Sung-Myung Kng et l. 51 Intrellulr RS svenging tivity (%) Asori id Pyrogllol-phlorogluinol-6,6 -iekol Conentrtion (μm) Fig. 5. Effet of the PPB on svenging intrellulr retive oxygen speies. The intrellulr retive oxygen speies generted were deteted vi the DCFH-DA method. Experiments were onduted in triplite nd the dt were expressed s the mens±se. Vlues with different lphets re signifintly different t P <.5 s nlyzed y Dunn s multiple rnge test (DMRT). % Fluoresene in til A B C D Control μm of PPB + 1 μm H 2 2 Fig. 6. Protetive effet of different onentrtions of the PPB on H 2 2-indued DNA dmge using the omet ssy. Photomirogrphs of DNA dmge nd migrtion oserved under PPB. (A) Control; (B) 1 µm H 2 2 ; (C) 25 µg/ml of PPB + 1 µm H 2 2; (D) 5 µg/ml of PPB + 1 µm H 2 2. The ells dmged y H 2 2-tretment were ssessed vi the omet ssy. Experiments were onduted in triplite nd the dt were expressed s the mens±se. Vlues with different lphets re signifintly different t P <.5 s nlyzed y Dunn s multiple rnge test (DMRT). vlue for intrellulr RS svenging (IC 5 = 3.54 µm) thn the vlues otined for sori id. Protetive effet ginst H 2 2-indued ell dmge The protetive effets of PPB on ell dmge were lso verified vi omet ssys (Fig. 6). Photomirogrphs of different DNA migrtion profiles upon tretment with different PPB onentrtions re provided in Fig. 6. In ells exposed to only H 2 2, the DNA ws ompletely dmged, ut the ddition of PPB during H 2 2 tretment resulted in dose-dependent suppression of DNA dmge. In ft, PPB hd DNA dmge-inhiitory tivity of 54.9% even t onentrtion of 25 µm (IC 5 vlue ws 23.2 µm). d d e e Inhiitory effet of ell dmge (%) Disussion Reent developments in iomedil siene hve emphsized the involvement of free rdils in mny diseses, suh s rin dysfuntion, ner, hert disese nd immune system [1]. Dietry ntioxidnt intke my e n importnt strtegy for inhiiting or delying the oxidtion of suseptile ellulr sustrtes, nd is thus relevnt to disese prevention in mny prdigms. Polyphenoli ompounds, suh s flvonoids, phenoli ids, nd tnnins, hve reeived ttention in this regrd due to their high ntioxidtive tivity. Fruit, vegetle, nd lge, nd other plnt mterils rih in polyphenoli omponents re of inresing interest in the food industry euse they n retrd oxidtive degrdtion of lipids nd improve the qulity nd nutritionl vlue of food [18,19]. Polyphenoli ompounds tht re known to possess high ntioxidtive tivity re ommon phytohemils in fruits nd lefy vegetles. Previous studies hve shown tht there ws diret reltionship etween ntioxidnt tivity nd phenoli ompounds in hers, vegetles nd fruits [2-22]. Sine there re lrge numer of different types of ntioxidnt ompounds tht might ontriute to the totl ntioxidnt pity, it is not ler whih omponents re responsile for the oserved ntioxidnt tivity. To exmine the effet of the phytohemil onstituents of E. v on ntioxidnt pity, we determined the orreltion etween the ntioxidnt pity nd min ntioxidnt sustnes, polyphenoli ompounds. The ntioxidnt pity of E. v ppered to e lrgely ffeted y the levels of polyphenoli ompounds nd the struture-tivity reltionship. Mny studies hve suggested tht the rdil svenging tivities of polyphenols suh s flvonoids re due to the phenoli hydroxyl groups tthed to the enzene ring, nd the struturetivity reltionship of flvonoids hs een exmined using wide rnge of different ntioxidnt ssys [23,24]. Although the struture-tivity reltionship of polyphenol is not ler, it is plusile tht the phenoli hydroxyl groups tthed to the ekol skeleton ply n importnt role in the rdil svenging tivities [25]. In the present study, the rdil svenging tivities of PPB ws muh higher thn tht of phlorogluinol, whih implied tht the mount of hydroxyl groups tthed to the enzene ring indeed plyed n importnt role in the rdil svenging tivities [6]. In onlusion, our results demonstrted tht the novel polyphenol ompound isolted from E. v, PPB, displyed different degrees of poteny in its rdil svenging nd protetive effets ginst H 2 2-indued dmge in Vero ells. Therefore, sed on our results, PPB my prove to e useful nturl rdil svenger nd potentil supplement for use y the food, phrmeutil, nd osmeti industries, due to its profound ntioxidnt properties.

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