Simplified Methods for Microtiter Based Analysis of In Vitro Antioxidant Activity. Somanjana Khatua, Sandipta Ghosh, Krishnendu Acharya*

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1 ORIGINAL ARTICLE Simplified Methods for Mirotiter Bsed Anlysis of In Vitro Antioxidnt Ativity Somnjn Khtu, Sndipt Ghosh, Krishnendu Ahry* Moleulr nd Applied Myology nd Plnt Pthology Lortory, Deprtment of Botny, Centre of Advned Study, University of Clutt, Kolkt, West Bengl, Indi Astrt Context: The reserh in exploring the ntioxidnt tivity of phrmeutils hs reently een inresed onsiderly nd to determine suh property vriety of testing methods re ville. However, the mjor drwk of these onventionl tests is their lrge retion volumes vrying from 2 to 6 ml tht in turn demnd high quntity of regents nd iologil resoures. Aims: Thus, this work ws foused for optimiztion of routinely used five ntioxidnt experiments suh s superoxide rdil (O 2 ) inhiition, 2, 2-diphenyl-1-pirylhydrzyl (DPPH) quenhing, 2 -zinois (3-ethylenzothizoline-6-sulfoni id) (ABTS.+ ) svenging, helting ility of ferrous ion nd reduing power y mens of 96-well pltes in miniml retion volume rnging from 60 to 200 µl. Sujets nd Methods: To uthentite the proesses some renowned stndrds suh s sori id, utylted hydroxynisole, Trolox s well s ethylenediminetetreti id were used, nd their lirtion urves were prepred. Results: Anlysis depited similr tivities of ll referenes s reported y trditionl protools suggesting vlidtion of stndrdized proedures. Conlusion: Thus, the reommended systems lerly improve originl one in numer of wys. First, the methods provide onurrent multi-smple investigtion with utomti dt storge. Seond, the pproh is time-sving with the use of multihnnel pipette. Third, ssys re inexpensive s the use of hemils is redued y 10 times. Fourth, the investigting pure ompounds or extrts re required in low quntity. Fifth, exept O 2 quenhing ssy, ll the methods re pplile to lipophili nd queous omponents oth. Key words: 2, 2 -zinois (3-ethylenzothizoline-6-sulfoni id) rdil, helting ility, 2, 2-diphenyl-1- pirylhydrzyl rdil, reduing power, stter plots, superoxide rdil, 96-well pltes INTRODUCTION Antioxidnt tivity is rodly used term to hrterize sustnes with the ility of svenging or neutrlizing free rdils. [1] These rdils n e produed inside humn ody y oth exogenous nd endogenous soures suh s inflmmtion, immune ell tivtion, exessive exerise, mentl stress, infetion, ishemi, ner, geing, wter pollution, lohol, igrette smoke, hevy metls, industril solvents, ertin drugs, rdition, nd ooking. [2] The presene of these retive omponents in exess mount n generte phenomenon known s oxidtive stress, deleterious proess tht n lter the struture of proteins, lipids, lipoproteins, nd DNA. [3] Consequently, numer of disorders my propgte tht n led to degenertive, rdiovsulr, renl, neurologil, liver, nd utoimmune diseses. [4] In this kdrop, ntioxidnt omponents externlly supplied through diets re required to neutrlize exess rdils, protet ells ginst toxi effets nd prevent ilments. Nowdys, severl syntheti ntioxidnts suh s utylted hydroxynisole (BHA), utylted hydroxytoluene (BHT), nd tert-utylhydroquinone (TBHQ) re routinely used in foods nd mediines. [5] However, rinogeni nture, strit legisltion on the use of syntheti food dditives nd onsumer preferenes hve shifted the ttention of industrilists from syntheti to nturl ntioxidnts. [6] Thus, there hs een Address for orrespondene: Krishnendu Ahry, Moleulr nd Applied Myology nd Plnt Pthology Lortory, Deprtment of Botny, Centre of Advned Study, University of Clutt, 35, Bllygunge Cirulr Rod, Kolkt , West Bengl, Indi. E-mil: krish_pper@yhoo.om Reeived: Revised: Aepted: Asin Journl of Phrmeutis Apr-Jun 2017 (Suppl) 11 (2) S327

2 worldwide trend towrd the use of nturl sustnes s therpeuti ntioxidnts to redue risk ftors of humn from dedly diseses. [7] To dte, gret numer of in vitro methodologies hve een estlished to mesure the effiy of nturl ntioxidnts either s pure ompound or extrt. However, there re severl impediments tht limited the ppliility of these tehniques nd hndling smples with different onentrtions. [8] For instne, n enormous volume of retion mixture rnging from 3 to 6 ml is required for onduting onventionl superoxide rdil (O 2 ) svenging tivity nd reduing power ssy. As onsequene, not only regents re utilized in high quntity ut the iologil mterils re lso needed in signifint mount. This eomes of mjor onern in the se of pure omponents or omplex smples tht hve een isolted in low extent. Besides, oth these tehniques offer roust nd time-onsuming proesses demnding their modifition in simplified wy. While, methods of 2, 2 -zinois (3-ethylenzothizoline-6-sulfoni id) (ABTS.+ ) s well s 2, 2-diphenyl-1-pirylhydrzyl (DPPH. ) rdil quenhing tivity re opertionlly simple nd hve een used in mny reserh lortories; however high prie of these hemils is the foremost nd unvoidle limittion from the routine nlysis. [9] Therefore, the development of new, esy to perform, rpid nd inexpensive methods re n urgent ll to determine rdil svenging potentility of phrmeutils, nutreutils, nd foods. In this ontext, miroplte tehnology n e onsidered s n effetive step towrd more sensitive nd fst mesurement of spetrl sorption vlues. [10] Eventully, some ntioxidnt experiments suh s DPPH, ABTS, nd reduing power tests hve een modified y dopting 96 s well s 48-well pltes in retion volume of µl. [10-14] However, to the est of our knowledge, no previous study hs een performed to simplify O 2 inhiition nd helting ility of ferrous ion proesses. In this kdrop, here we desrie rpid, smllsle, high-throughput miroplte-sed methodologies for those forementioned ntioxidnt ssy using µl retion mixture. The modified pproh is more preise, smple-sving, rpid, nd eo-friendly s well whih would e more suitle for sustituting the trditionl lorious tehniques. Overll, the proposed ssys re esy to exeute for mnul sreening of ntioxidnt pity of lrge numer of smples in dy. Chemils SUBJECTS AND METHODS Sodium dihydrogen phosphte, disodium hydrogen phosphte, L-methionine, nitrolue tetrzolium (NBT), rioflvin, ferrous hloride, ferrozine, trihloroeti id (TCA), potssium ferriynide, ferri hloride, DPPH, ABTS, sodium persulfte, sori id, ethylenediminetetreti id (EDTA), BHA, nd Trolox were purhsed from Sigm Chemils Co. (St. Louis, MO, USA). All hemils were of nlytil regent grde. Instrument In this study, Bio-Rd imrk TM Miroplte Reder (USA) ws used for sorne reding nd spetr reording. The mhine is omprised n eight-hnnel, vertil pth length photometer tht mesures the sorne of ontents in 96-well mirotitrtion plte t speifi wvelengths suh s 415, 450, 490, 595, 655, nd 750 nm. Before every red, the plte n e shken t low, medium, or high speed for s to mix the retion mixture. Superoxide rdil svenging ssy Eh 3 ml retion mixture sequentilly ontined 750 µl sodium phosphte uffer (50 mm, ph 7.8), 390 µl methionine (13 mm), 300 µl EDTA (100 nm), 1300 µl of stndrds or ntioxidnt ompounds, 250 µl NBT (75 mm), nd 6 µl rioflvin (2 mm). The retion ws strted y turning on fluoresent lmp (15 W), nd illumintion ws run for 10 min. The prodution of violet olor ws monitored y mesuring sorne t 560 nm. Tues with identil retion mixture were kept in the drk nd served s lnks. [15] Methionine solution ws prepred y dissolving g in 5 ml wter (2 nm) g EDTA ws dissolved in 10 ml wter to mke 0.1 nm solution. NBT nd rioflvin were prepred dily y dissolving g in 5 ml (0.1 nm) nd 100 ml (1 nm) wter, respetively. In 200 µl retion mixture, x µl of smple ws dded followed y seril inlusion of wter (83-x µl), 50 µl uffer, 26 µl methionine, 20 µl EDTA, 17 µl NBT, nd 4 µl rioflvin. The plte ws shken for 10 seonds in medium speed y miroplte reder instrument, nd initil sorne ws mesured t 595 nm. Further, the 96-well pltes were pled under 15 W lights nd inuted for 10 min t room temperture to initite the retion. Finlly, hnge in sorne ws reorded t the sme wvelength, nd initil sorne ws deduted to nullify kground olor. Asori id t different onentrtions ( µg/ml) ws onsidered s referene in this tehnique. The degree of svenging ws lulted y the following eqution: Svenging effet (%) = {(Asorne of ontrol Asorne of smple)/asorne of ontrol} 100 Asin Journl of Phrmeutis Apr-Jun 2017 (Suppl) 11 (2) S328

3 DPPH rdil svenging ssy Aout 5 ml of stndrd or ntioxidnt ompound solution ws mixed with 5 ml of 0.008% DPPH prepred in 50% ethnol. The retion ws inuted for 30 min t room temperture in the drk. Deoloriztion of DPPH solution ws mesured t 528 nm. [16] Aout g DPPH regent ws dissolved in 100 ml methnol or 4:1 queous methnol solution depending on nture of investigting omponent. In 96-well pltes, x µl smple nd 200-x µl DPPH solution were dded. The plte ws inuted for 30 min t room temperture in the drk, nd finlly, sorne ws reorded t 595 nm wvelength. To uthentite the proess, three syntheti ntioxidnts inluding sori id, BHA, nd Trolox t different onentrtions rnging from 1 to 50 µg/ml were used. The degree of svenging ws lulted y the following eqution: Svenging effet (%) = {(Asorne of ontrol Asorne of smple)/asorne of ontrol} 100 ABTS rdil svenging ssy At first, ABTS ws dissolved in wter to prepre 7 mm onentrted solutions. Further, ABTS rdil tion (ABTS + ) ws generted y reting the stok solution with 2.45 mm of potssium persulfte followed y inution in the drk t room temperture efore use. After h, the ABTS + solution ws diluted with ethnol or PBS (ph 7.4) to n sorne of 0.7 t 734 nm. 1 ml of diluted ABTS + solution ws mixed with 10 ml of stndrds or ntioxidnt ompounds nd sorne ws mesured ginst the pproprite solvent lnk t 734 nm. [17] ABTS rdils were prepred freshly in eh time ording to the originl method. After h, ABTS + solution ws diluted to n sorne of 0.7 ± 0.02 t 750 nm. In 200 µl retion mixture, x µl stndrd ws dded followed y ddition of 200-x µl regent solution. The plte ws shken for 10 seonds t medium speed, nd sorne ws mesured t 750 nm following 5 min inution in the drk. Herein, three referenes suh s sori id, BHA, nd Trolox were used t different onentrtions (1, 3, 5, nd 10 µg/ml). The degree of svenging ws lulted y the following eqution: Svenging effet (%) = {(Asorne of ontrol Asorne of smple)/asorne of ontrol} 100 Chelting ility of ferrous ion Aout 1850 µl of stndrds or ntioxidnt ompounds were inuted with 50 µl Fe 2+ (20 µm) (mmonium ferrous sulfte) in 5% mmonium ette, ph 6.9. Retion ws initited y ddition of 100 µl of ferrozine (100 µm) nd fter 10 min inution sorne ws red t 562 nm. [18] Aout 10 mg ferrous hloride ws dissolved in 40 ml wter to prepre 3 nm solutions. While g ferrozine ws dissolved in 5 ml wter (0.12 nm). In 200 µl retion mixture, x µl smple ws dded to 96-well pltes long with 5 µl of ferrous hloride nd mixed well. Then, 10 µl ferrozine ws inluded followed y ddition of 185-x µl of wter or methnol depending on investigting omponent. The system ws inuted t room temperture for 10 min, nd sorne ws mesured t 595 nm. EDTA t onentrtions of 5, 10, 15, nd 20 µg/ml ws onsidered s referene. The degree of svenging ws lulted y the following eqution: Svenging effet (%) = {(Asorne of ontrol Asorne of smple)/asorne of ontrol} 100 Reduing power Aout 2.5 ml of 0.2 M phosphte uffer (ph 6.6) nd 2.5 ml of K 3 [Fe(CN) 6 ] (1% w/v) were dded to 1 ml of smple or ntioxidnt ompounds dissolved in distilled wter. The resulting mixture ws inuted t 50 C for 20 min, followed y ddition of 2.5 ml of TCA (10% w/v). The mixture ws entrifuged t 3000 rpm for 10 min to ollet n upper lyer of solution (2.5 ml), mixed with distilled wter (2.5 ml) nd 0.5 ml of FeCl 3 (0.1%, w/v). The sorne ws then mesured t 700 nm ginst lnk smple. [19] Regents were prepred t per the originl method. In 96-well pltes, 10 µl of smple solution, 25 µl of uffer nd 25 µl of K 3 [Fe(CN) 6 ] were dded sequentilly. The mixture ws inuted for 20 min t room temperture, nd retion ws stopped y dding 25 µl of TCA solution. Further, Asin Journl of Phrmeutis Apr-Jun 2017 (Suppl) 11 (2) S329

4 85 µl of wter nd 8.5 µl of FeCl 3 were dded to eh well. The ontents were mixed, inuted for nother 15 min t room temperture nd sorne ws mesured t 750 nm. Asori id, BHA, nd Trolox t onentrtions of 10, 30, 50, nd 70 µg/ml were onsidered s stndrds in this tehnique. Dt nlysis All dt were expressed s men ± stndrd devition of three independent experiments. The smple onentrtions providing 0.5 of sorne or 50% of ntioxidnt tivity were lulted from grphs of ntioxidnt tivity perentges nd regrded s EC 50 vlue. Liner regression nd orreltion nlyses were performed using Mirosoft Exel (USA). RESULTS AND DISCUSSION Superoxide rdil svenging ssy Superoxide nion (O 2 ) is generted y one-eletron redution of oxygen nd ts s the preursor of most retive oxygen speies. Dismuttion of this primry rdil n produe hydrogen peroxide tht in turn my e prtilly redued to one of the strongest oxidnts in nture, hydroxyl rdil. In ddition, the nion my ret with other rdils suh s nitri id resulting more powerful oxidnt, peroxynitrite. [20] These hrmful omponents n initite ellulr dmge tht my led to different pthophysiologil onditions. [3] Therefore, evlution of superoxide nion svenging is extremely importnt for determining ntioxidnt tivities. To ssess suh potentility of phrmeutils, Mrtinez et l. proposed n in vitro method sed on the genertion of O 2 y utooxidtion of rioflvin in the presene of light. The rdil in turn redued NBT to lue olored formzn tht n e mesured photometrilly. However, derese in sorne indited onsumption of O 2 y ntioxidnts present in resulting mixture. [15] This originl method hs een revised herein y dopting 96-well pltes to redue the retion volume from 3000 to 200 µl t miniml expense of smples. In ddition, less onentrtion of ll reting regents ws used s ompred to the trditionl one. Besides, the mximum sorption wvelength (λmx) ws hnged from 560 nm to 595 nm due to the limittion of the utomted nlyzer. The proposed tehnique ws stndrdized y using sori id s referene whih showed exellent rdil svenging tivity [Figure 1]. At the onentrtion of 10, 50, nd 100 µg/ml, the referene ws le to inhiit ± 2.732%, ± 1.838%, nd ± 0.285% rdils, respetively, whih rehed to the level of 59.6 ± 3.085% t the dose of 150 µg/ml. Figure 1: Superoxide rdil svenging tivity () Photogrph of 96-well pltes presenting dose-dependent rdil quenhing potentility of sori id with respet to ontrol (Lne A1-3: Blnk, B1-3: 10 µg/ml, B4-6: 50 µg/ml, B7-9: 100 µg/ml, B10-12: 150 µg/ml), () grphil presenttion of the effet of sori id on rdils in terms of perent inhiition, () stter plot of the stndrd showing Person s orreltion oeffiient (R 2 ). Experiments were triplited, nd vlues re presented s men ± stndrd devition DPPH rdil svenging ssy Use of DPPH. to nlysis ntioxidnt potentility dtes k to 1950s, when Brnd-Willims et l. pulished the first tehnique to quntify svenging property of omponents using spetrophotometer. [21] Presently, this ommerilly ville stle nitrogen-entered free rdil is the most extensively used regent to determine rdil svenging tivity of nutreutils. The violet olored rdil possesses hrteristi sorption nd redily epts eletron from ntioxidnt ompounds. Consequently, it is redued to yellow olored diphenylpirylhydrzine tht n e mesured through olorimeter. [22] However, lrge volume of freshly prepred DPPH solution (1-5 ml) is required eh time to ondut the onventionl method. To overome the limittion, protool hs een lter modified y dopting mirowell-sed tehnique to mke the ssy simpler s well s fster. [23] So fr, few reserhers hve vlidted nd desried the ssy y pplying 96-well pltes lthough retion mixture ws in the rnge of µl. [12] For further simplifition, minituriztion of volume nd redution in expenditure of rdils, the originl method hs een improved herein. In this ontext, the stok solution ws prepred with the low onentrtion of DPPH. nd retion system ws stndrdized to only 200 µl signifying less use of the ostly regent. Besides, λmx ws hnged from 528 nm to 595 nm due to limittion of the utomted nlyzer. To uthentite the tehnique, three stndrds were used, nd results hve een summrized in Tle 1. Asori id exhiited dose-dependent response y inhiiting ± 4.255%, ± 1.766%, ± 1.138%, nd Asin Journl of Phrmeutis Apr-Jun 2017 (Suppl) 11 (2) S330

5 Tle 1: Antioxidnt tivity of stndrds Antioxidnt ssys Asori id BHA Trolox EDTA Superoxide rdil svenging ssy ± NT NT NT DPPH rdil svenging ssy 7.695± ± ±0.022 NT ABTS rdil svenging ssy 3.18± ± ±0.226 NT Chelting ility of ferrous ion NT NT NT ±0.585 Reduing power ± ± ±0.001 NT The results re presented in EC 50 vlues (µg/ml) orresponding to effetive onentrtion t whih 50% rdils re svenged or sorne is 0.5. Experiments were triplited, nd vlues re presented s men±sd. SD: Stndrd devition, NT: Not tested, DPPH: 2, 2 diphenyl 1 pirylhydrzyl, ABTS: 2, 2 zinois (3 ethylenzothizoline 6 sulfoni id), BHA: Butylted hydroxynisole, EDTA: Ethylenediminetetreti id, EC 50 : Effetive onentrtion 50% ± 0.189% of rdils t the level of 5, 10, 25, nd 50 µg/ml, respetively. While BHA showed ± 0.576%, ± 3.029%, ± 0.383%, nd ± 0.197% svenging ility of DPPH t those ove-mentioned onentrtions. However, Trolox presented lower potentility y quenhing ± 1.267%, ± 3.168%, ± 0.198%, nd ± 0.31% rdils t the level of 5, 10, 25, nd 50 µg/ml, respetively [Figure 2]. Nevertheless, our findings were found to e in greement with the previous reports. [24-26] ABTS rdil svenging ssy Originl ABTS + svenging ssy ws proposed y Wolfenden nd Wilson where the rdil tion ws generted y tivtion of metmyogloin with hydrogen peroxide in the presene of ABTS. [27] Lter the method ws modified y diret prodution of lue-green ABTS + hromophore through retion etween ABTS slt nd strong oxidizing gent suh s potssium persulfte or potssium permngnte. [17] However, the lue olored ABTS + is onverted k to its olorless neutrl form in the presene of hydrogen-donting ntioxidnt ompounds mesured y derese of its hrteristi sorption spetrum. [28] As onsequene, the ssy eomes opertionlly simple, nd nowdys it hs een routinely used in mny reserh lortories to determine rdil svenging tivity of omponents. However, with sophistition nd dvnement in instrumentl tehniques, the protool hs experiened severl modifitions, while si priniple remined unique. Reently, miroplte dpttion of ABTS rdil quenhing method hs een desried y some investigtors in retion volume of not <255 µl. [10] In n im to redue the quntity of retion mixture nd use of expensive regent, the ssy ws revised herein. The system ws optimized in resulting solution of only 200 µl, nd three referenes were onsidered for vlidtion [Figure 3]. Anlysis showed tht ll stndrds possessed strong rdil svenging pity tht inremented in dose-dependent mnner. At the onentrtion of 1, 3, 5, nd 10 µg/ml, rdil svenging ilities of sori id were ± 0.647%, ± 3.38%, ± 5.586%, nd ± 2.387%, respetively, depiting strong ntioxidnt potentil. Wheres, BHA presented ± 7.246%, ± 1.923% of quenhing ility t the levels of 1 nd 3 µg/ml tht grdully elevted to ± 7.333% nd ± 1.931% t the dose of 5 nd 10 µg/ml. Conversely, inhiition tivities of Trolox were 8.21 ± 7.451%, ± 8.542%, ± 7.597%, nd ± 3.661% t those onentrtions rnges demonstrting moderte tivity. Thus, it n e sid tht sori id nd BHA presented extremely powerful potentil while Trolox exhiited lower effets [Tle 1]. However, properties of Trolox were deteted to e in ordne with the previously pulished reports. [26,29] Chelting ility of ferrous ion Some trnsition metls inluding Fe 2+, Cu 2+, nd P 2+ s well s Co 2+ n trigger oxidtive stress nd mgnify ellulr dmge. Among them, Fe 2+ hs ility to ret with H 2 O 2 nd produes highly retive hydroxyl rdil vi Fenton s retion. Thus, ntioxidnts with metl helting ility my e of enefiil use in the tretment of severl pthophysiologil disorders. [30] To determine suh tivity, Dinis et l. proposed method where ferrozine ompetitively rets with Fe 2+ forming red omplexes. When other heltors re present in retion mixture, this omplex formtion is disrupted, nd red olor of solution dereses. Thus redution in sorne signifies Fe 2+ trpping potentility of investigting omponent. [18] The ssy is quite populr tehnique for determintion of ntioxidnt property of smples, though the method hs not eing modified so fr for higher smple throughput nd etter reproduiility. In this kground, this study ws onduted in n effort to improve the trditionl method. As shown in Figure 4, EDTA, the referene, reveled exeptionl dose-dependent helting pttern t the tested onentrtions. The helting tivity of ± 3.04% nd ± 0.961% ws hieved t the onentrtion of 5 nd 10 µg/ml of EDTA, respetively. While it inresed stedily to ± 2.333% nd ± 0.209% t the level of 15 nd 20 µg/ml of stndrd. In reent study, Chi et l. reported ferrous ion helting potentility of EDTA following lssil method whih ws found to e in greement with our dt. [31] Asin Journl of Phrmeutis Apr-Jun 2017 (Suppl) 11 (2) S331

6 d Figure 2: 2, 2-diphenyl-1-pirylhydrzyl (DPPH) rdil svenging tivity () Photogrph of 96-well pltes where Lne B represents negtive ontrol; Lne C, E, nd G depits DPPH. quenhing potentilities of sori id, utylted hydroxynisole (BHA), nd Trolox, respetively. The stndrds were investigted t different onentrtions suh s 5, 10, 25, nd 50 µg/ml in triplites, () grphil presenttion of the effet of three referenes on rdils in terms of perent inhiition. Stter plot of sori id (), BHA (d) nd Trolox (e) ws prepred to lulte Person s orreltion oeffiient (R 2 ). Experiments were triplited, nd vlues re presented s men ± stndrd devition e d Figure 3: 2, 2 -zinois (3-ethylenzothizoline-6-sulfoni id) (ABTS) rdil svenging tivity () Photogrph of 96-well pltes where Lne B represents negtive ontrol; Lne C, E, nd G depits ABTS + quenhing potentilities of sori id, utylted hydroxynisole (BHA), nd Trolox, respetively. The stndrds were investigted t vrile onentrtions of 1, 3, 5, nd 10 µg/ml in triplites, () grphil presenttion of the effet of three referenes on rdils in terms of perent inhiition. Stter plot of sori id (), BHA (d), nd Trolox (e) ws prepred to lulte Person s orreltion oeffiient (R 2 ). Experiments were triplited, nd vlues re presented s men ± stndrd devition e Reduing power It is well-known tht free rdils re moleules, toms or ions tht ontin unpired eletrons; s result, they re highly retive in nture. Antioxidnts re sustnes tht re stle enough to donte their own eletrons to stilize rdils nd inhiit further dmges. [5] Thus, determintion of reduing the power of sustne my serve s signifint indition for its ntioxidnt potentility. In tht view, Oyizu desried proess sed on redution of Fe 3+ to Fe 2+ depending on the tivity of investigting ompounds whih in turn genertes KFe [Fe(CN) 6 ] (Prussin lue) pproprite for spetrophotometri mesurement. [19,32] Thus inrese in sorne denotes n inrement in reduing Asin Journl of Phrmeutis Apr-Jun 2017 (Suppl) 11 (2) S332

7 power s well s ntioxidnt effets. However, originlly the ssy ws designed for plsm, ut fter relizing its ppliility, it is now used in n enormous numer of mtrixes. [4] Susequently, the methodology hs een modified using 48 well mirotitrtion pltes in retion mixture of 1500 µl. [13] Figure 4: Chelting ility of ferrous ion () Photogrph of 96-well pltes presenting dose-dependent tivity of ethylenediminetetreti id (EDTA) in respet to negtive ontrol (Lne B1-3: Blnk, C1-3: 5 µg/ml, B4-6: 10 µg/ml, B7-9: 15 µg/ml, nd B10-12: 20 µg/ml), () grphil presenttion of the effet of EDTA in terms of perent heltion, () stter plot of the stndrd showing Person s orreltion oeffiient (R 2 ). Experiments were triplited, nd vlues re presented s men ± stndrd devition To perform the test in 96-well pltes, the ssy needs to e re-designed. In this ontext, the protool hs een modified herein nd to fulfill requirement resultnt solution ws deduted to only 60 µl. Moreover, the entrifugtion step s desried in originl tehnique hs een removed to simplify the method. Besides, λmx ws hnged from 700 to 750 nm due to limittion of utomted nlyzer. To uthentite the revised tehnique, three stndrds were used, nd their tivities hve een presented in Figure 5. All the referenes were ple of reduing Fe 3+ to Fe 2+ in liner dose-dependent mnner. Asori id exhiited dose-dependent response y inresing sorne ± 0.011, ± 0.025, 1.2 ± , nd 1.2 ± t the level of 10, 30, 50, nd 70 µg/ml, respetively. While BHA showed ± 0.015, ± 0.074, ± 0.018, nd ± reduing power t those ove-mentioned onentrtions. However, Trolox presented lower potentility y inresing sorne of 0.07 ± 0.011, ± 0.041, ± 0.08, nd ± t the level of 10, 30, 50, nd 70 µg/ml, respetively. Thus, it n e oserved tht sori id possesses the strongest reduing effet followed y BHA nd Trolox [Tle 1]. CONCLUSION The work presents improved version of routinely used ntioxidnt ssys sed on miroplte reder tehnique. Mjor dvntge of the proposed methods is redution in retion volume rnging from 200 µl for DPPH nd d Figure 5: Reduing power () Photogrph of 96-well pltes where Lne B represents negtive ontrol; Lne C, E, nd G depits reduing power tivity of sori id, utylted hydroxynisole (BHA), nd Trolox, respetively. The stndrds were investigted t vrile onentrtions of 10, 30, 50, nd 70 µg/ml in triplites, () Grphil presenttion of the reduing ility of three referenes in terms of inrese in sorne. Stter plot of sori id (), BHA (d) nd Trolox (e) ws prepred to determine Person s orreltion oeffiient (R 2 ). Experiments were triplited, nd vlues re presented s men ± stndrd devition e Asin Journl of Phrmeutis Apr-Jun 2017 (Suppl) 11 (2) S333

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