antioxidant and antiradical properties of selected flavonoids and phenolic...

Research ArticleAntioxidant and Antiradical Properties ofSelected Flavonoids and Phenolic Compounds

Zuumlbeyir Huyut1 Fuumlkruuml Beydemir2 and Elhami Guumllccedilin3

1Department of Biochemistry Medical Faculty Yuzuncu Yıl University 65080 Van Turkey2Department of Biochemistry Faculty of Pharmacy Anadolu University 26210 Eskisehir Turkey3Department of Chemistry Faculty of Sciences Ataturk University 25240 Erzurum Turkey

Correspondence should be addressed to Zubeyir Huyut zubeyirhuyutgmailcom

Received 13 June 2017 Accepted 7 September 2017 Published 12 October 2017

Academic Editor Paul W Doetsch

Copyright copy 2017 Zubeyir Huyut et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Phenolic compounds and flavonoids are known by their antioxidant properties and one of the most important sources forhumans is the diet Due to the harmful effects of synthetic antioxidants such as BHA and BHT natural novel antioxidants havebecome the focus of attention for protecting foods and beverages and reducing oxidative stress in vivo In the current study weinvestigated the total antioxidant metal chelating Fe3+ and Cu2+ reduction and free radical scavenging activities of some phenolicand flavonoid compounds including malvin oenin ID-8 silychristin callistephin pelargonin 34-dihydroxy-5-methoxybenzoicacid 246-trihydroxybenzaldehyde and arachidonoyl dopamine The antioxidant properties of these compounds at differentconcentrations (10ndash30120583gmL) were compared with those of reference antioxidants such as BHA BHT 120572-tocopherol and troloxEach substance showed dose-dependent antioxidant activity Furthermore oenin malvin arachidonoyl dopamine callistephinsilychristin and 34-dihydroxy-5-methoxybenzoic acid exhibited more effective antioxidant activity than that observed for thereference antioxidants These results suggest that these novel compounds may function to protect foods and medicines and toreduce oxidative stress in vivo

1 Introduction

Reactive oxygen species (ROS) are continuously formed bynormal cellular processes endogenously and environmentalfactors exogenously [1] ROS include nonradical speciessuch as hydrogen peroxide (H2O2) hypochlorous acid(HOCl) singlet oxygen (1O) and free radicals such assuperoxide anion radical (O2

∙minus) hydroxyl radical (OH∙) andhydroperoxide (ROO∙) [2ndash4] Free radicals at physiologicalconcentrations have a series of useful biological functionssuch as acting as a cell signaling molecule functioningagainst cellular responses controlling cell viabilitymigrationand differentiation protecting cells against pathological andinfectious agents and inactivating them [5ndash7] Howeverlevels of ROS higher than physiological concentrations causeoxidative-antioxidant imbalance and oxidative stress [8]Oxidative stress is a factor that induces a number of diseasessuch as atherosclerosis cardiovascular diseases diabetesinflammation aging skin lesions rheumatoid arthritis and

neurological diseases [9ndash11] When enzymatic or nonenzy-matic endogenous antioxidants are inadequate to removeROS from the body it becomes important for the bodyto receive exogenous natural antioxidants such as phenoliccompounds

Phenolic compounds are secondary plant metabolitesthat are found naturally in all plantmaterials including plant-based food products [12] These compounds are thoughtto be an integral part of human and animal diets Theyrepresent the most important group of natural antioxidants[13] The most common phenolic compounds in plants canbe classified into phenolic acids tocopherols and flavonoids[14] It has been reported that phenolic and flavonoidcompounds act as antioxidants to exert antiallergic anti-inflammatory antidiabetic antimicrobial antipathogenicantiviral antithrombotic and vasodilatory effects and pre-vent diseases such as cancer heart problems cataracts eyedisorders and Alzheimerrsquos [15ndash17] In addition the mostimportant features of flavonoids include their ability to

HindawiBiochemistry Research InternationalVolume 2017 Article ID 7616791 10 pageshttpsdoiorg10115520177616791

2 Biochemistry Research International

protect against oxidative diseases activate or inhibit variousenzymes bind specific receptors and protect against cardio-vascular diseases by reducing the oxidation of low-densitylipoproteins [18]

Various methods have been developed to investigate theantioxidant properties of a substance These include assaysfor total antioxidant capacity (TAC) NO∙ H2O2 O2

∙minus andOH∙ radical scavenging capacity oxygen radical scavengingactivity (ORAC) Fe3+ and Cu2+ reducing activity (FRAPand CUPRAC assay resp) metal chelating activity ABTS∙+DMPD∙+ and DPPH∙ free radical scavenging activity andlipid peroxidation inhibition capacity [19ndash21] Among thesethe most commonly used methods are TAC determinationand assays for determining Fe+3 and Cu+2 reduction activitymetal chelating ability and free radical (ABTS∙+ DPPH∙DMPD∙+ OH∙ and O2

∙minus) scavenging activity [21]In the current study we investigated the antioxidant

capacities of malvin oenin ID-8 silychristin callistephinand pelargonin with flavonoid structures and 34-dihydroxy-5-methoxybenzoic acid arachidonoyl dopamine and 246-trihydroxybenzaldehyde with phenolic structures (Figure 1)by assaying Fe3+ and Cu2+ reduction activity metal chelatingactivity and O2

∙minus ABTS∙+ DPPH∙ and DMPD∙+ radicalscavenging capacity

2 Materials and Methods

21 Chemicals Sodium dihydrogen phosphate (NaH2PO4)potassium ferrocyanide (K3Fe(CN)6) trichloroacetic acid(TCA) iron-III-chloride (FeCl3) potassium peroxydisulfate(K2O8S2) copper-II-chloride (CuCl2) sodium acetate(NaCH3COO) hydrochloric acid (HCl) tris iron-II-sul-fate (FeSO4) iron-II-chloride (FeCl2) disodium hydro-gen phosphate (Na2HPO4) methionine ethanol ethylene-diaminetetraacetic acid (EDTA) ammonium thiocyanate(NH4SCN) sodium bicarbonate (NaHCO3) sodium hy-droxide (NaOH) disodium sulfate (Na2SO4) sodium per-chlorate (NaClO4) and disodium carbonate (Na2CO3) wereobtained from Merck (Merck made in Germany) Cis-9cis-12-octadecanoic acid (linoleic acid) 22-diphenyl-1-pi-crylhydrazyl (DPPH) polyoxyethylene sorbitanmonolaurate(Tween 20) nitroblue tetrazolium (NBT) 3-(2-pyridyl)-56-diphenyl-124-triazine-41015840410158401015840-disulfonic acid sodium salt(ferrozine) riboflavin (vitamin B2) N-N-Dimethyl-p-phe-nyl-enediamine dihydrochloride (DMPD) neocuproine hy-drate (C4H12N2sdotXH2O) 221015840-azino-bis(3-ethylbenzothia-zoline-6-sulfonic acid) diammonium sulfate (ABTS) ID-8 (C6H14N2O4) 34-dihydroxy-5-methoxybenzoic acid(C8H8O5) silychristin (C25H22O10) malvin (C29H28O17)pelargonin (C27H31O15) oenin (C23H25O2) arachidonoyldopamine (C28H41NO3) callistephin (C21H21O10) and246-trihydroxy-benzaldehyde (C6H2CHO) were purchasedfrom Sigma Aldrich Company (St Louis MO USA)

22 Determination of TAC TAC was determined by thethiocyanate method [22] Different concentrations of thestock solutions (10 20 30 120583gmL) of phenolic and flavonoidcompounds were added to tubes and volume was broughtto 25mL using phosphate buffer (001M and pH 74)

Subsequently 25mL of linoleic acid emulsion was added toeach tube and the mixture was incubated at 37∘C in the darkSamples (100 120583L) were taken every 12 h during incubationto which 47mL of ethanol 100 120583L of SCNminus solution and100 120583L of Fe2+ solution (20mM) prepared in HCl (35)were added The absorbance of the samples at 500 nm wasmeasured and compared to that of blank solution Alcoholwas used instead of the sample for the blank while buffersolution was used instead of the sample for the control Theincubation and absorbance measurements were continueduntil the maximum absorbance values of the control samplewere reached (about 15 d)

23 Determination of Fe3+-Fe2+ Reduction Activity by FRAPReagent (FRAP Assay) The reduction power as FRAP reac-tivity was determined using the method of Oyaizu with slightmodification [23] Different solutions (10 20 30 120583gmL) wereprepared from the 1mgmL stock solutions of the phenolicand flavonoid compounds The sample volume in the tubeswas to 05mL using acetate buffer (pH 36) Subsequently225mL of FeCl3 solution and 225mL of FRAP reagent wereadded to each tube (total volume 5mL) After incubation for10min the absorbance of the mixture was read at 593 nmagainst the blank Acetate buffer was used as a blank controlsample

24 Cu2+-Cu+ Reduction Activity (CUPRAC Assay) TheCu2+ reduction activity was determined using the methodpreviously described byApak et al with aminormodification[24] Different concentrations (10 20 and 30 120583gmL) ofphenolic and flavonoid compounds were mixed with 125 120583Lof CuCl2 solution (001M) 125120583L of ethanolic neocuprinsolution (75mM) and 125 120583L of ammonium acetate buffersolution (1M) After incubation in the dark for 30minabsorbance was measured at 450 nm against a distilled waterblank

25 Superoxide Anion Radical (O2∙minus) Scavenging Activity

Superoxide anion radical scavenging activity was determinedusing the method described by Zhishen et al with slightmodification [25]Thismethod is based on the spectrophoto-metric measurement of nitroblue tetrazolium (NBT) Differ-ent concentrations of samples and standards were preparedin phosphate buffer (005M and pH 78) To the samplesolutions riboflavin methionine and NBT were added atconcentrations of 133 446 and 815times 10minus2 120583M respectivelyThe reactionmixture was stimulated with 20Wof fluorescentlight at room temperature for 2 h Absorbance was measuredat 560 nm against a distilled water blank

26 DPPH Radical Scavenging Activity The DPPH radicalscavenging activity was analyzed according to the method ofBlois [26] DPPH solution (1mM)was used as the free radicalThe previously prepared 1mgmL antioxidant stock solutionswere used Samples were added to test tubes at concentrationsof 10 20 and 30 120583gmL and the total volume was broughtto 25mL using pure ethanol Subsequently 05mL of thestock DPPH solution was added to each sample tube After

Biochemistry Research International 3

O

OH

OHOH

OH

OH

OH

OH

H

OHOH

OH

OH

OH HO

HO

HO

HON

HO

HO

CallistephinPelargoninOenin

Silychristin ID-8 Malvin

246-TrihydroxybenzaldehydeArachidonoyl Dopamine34-Dihydroxy-5-methoxybenzoic acid

HO

HO

O

OOH

OH

O

OHOH

HO

O

O

OH

OHOH

OH

OH

OH

OH

HO

HO

OH

OH

OH

HO

HO

HO

OH

O

（3

（3

（3

（3

（3

（3

O

O

O

O

OO

NO2

CH3

CH3

O

O

O

O

O

HO

HO NH

O

CH3

Figure 1 Molecular structures of flavonoid and phenolic substances used as antioxidant in this study

incubation at room temperature in the dark for 30minthe absorbance values were measured at 517 nm against theethanol blank A solution of 2mL of ethanol and 05mL ofDPPH solution was used as a control Decreasing absorbancevalues indicated higher free radical scavenging activity

27 ABTS Radical Scavenging Activity ABTS radical elimi-nation activity was measured using the method of Re et al[27] First ABTS solution (2mM) was prepared in phosphatebuffer (1M and pH 74) ABTS radicals were produced byadding 245 nM persulfate solution to the mixture Next theabsorbance of the control solution at 734 nM was adjustedto 700 plusmn 0025 nm using phosphate buffer (01M and pH74) ABTS radical solution (05mL) was added to different

concentrations (10ndash30 120583gmL) of the antioxidants used inthis study and incubated for 30min The absorbance wasmeasured against an ethanol blank at 734 nm

28 DMPD Radical Scavenging Activity For this assay acolored radical cation (DMPD∙+) was first obtained For thispurpose 1mL of DMPD solution and 02mL of 005M FeCl3were added to 100mL of acetate buffer (pH 53 100mM) thusforming the DPPH radical solutionThe optical density of thecontrol solution at 505 nm was adjusted to 0900 plusmn 0100 nmusing phosphate buffer (01M and pH 53) The absorbanceof freshly prepared DMPD∙+ solution is stable for 12 hDifferent concentrations (10ndash30 120583gmL) of some phenolicand flavonoid compounds and reference antioxidants were


transferred to the test tubes and the total volume was broughtto 05mL using distilled water One milliliter of DMPD∙+solution was added to the solution and absorbance valueswere measured at 505 nm after incubation for 50min Buffersolution was used as a blank [28]

29 Fe2+ Chelating Activity Metal chelating activities of thephenolic and flavonoid compounds and positive control sub-stances were assay using the method previously described byDinis et al [29]The phenolic and flavonoid compounds wereadded at different concentrations (10 20 and 30 120583gmL) to asolution containing 50120583L of FeCl2sdot4H2O (2M) and 350 120583L ofpurified water The final volume was brought to 4mL usingdistilled waterThe reaction was initiated by adding 02mL offerrozine solution (5mM) After the solution was thoroughlymixed by vortexing it was incubated at room temperature for10min Subsequently the absorbance values were measuredat 562 nm against an ethanol blank As a control a solutionlacking any phenolic or flavonoid compounds was used

3 Results and Discussion

Antioxidant compounds exert their effects through differentmechanisms such as inhibiting hydrogen abstraction bindingtransition metal ions radical scavenging and disintegratingperoxides [30 31] One of the most important factors influ-encing antioxidant capacity is the ability of the antioxidantto donate electrons Due to the harmful effects of syntheticantioxidants such as BHA and BHT antioxidant capacitiesof flavonoids and phenolic compounds in plant-derived ornatural origin have garnered substantial research interest andare being investigated extensively [32] Many methods havebeen developed to determine the antioxidant capacities ofsynthetic or naturally sourced compounds plant extracts andother samples Among these methods TAC reducing powerDPPH DMPD ABTS∙+ and O2

∙minus scavenging ability andmetal chelating activities are the most frequently used [21]

TAC determination is a method that encompasses manyfactors which are captured individually by other methodsSince TAC is affected by metal chelating capacity reducingpower and free radical scavenging activity of compounds(eg by the number of -OH groups bound to aromatic ringsand conjugate diene structure of antioxidant molecules) it isobvious that each method should be applied and evaluatedseparately [33]

TAC determination is widely used for clinically usedbioactive substances and compounds that are food ingre-dients TAC can also be defined as the capacity to inhibitlipid peroxidation of compounds [34] The ability to inhibitlinoleic acid emulsion is tested to determine possible totalantioxidant effects of a bioactive compound [35] Linoleicacid emulsion ultimately produces hydroperoxides and theresulting hydroperoxides decompose to form secondaryproducts In this method the amount of hydroperoxide fromthe linoleic acid resulting fromautoxidation ismeasured indi-rectly during the test period Hydroperoxides react with Fe2+to form Fe3+ These secondary ions (Fe3+) form complexeswith thiocyanate (SCNminus) The resulting Fe(CN)2+ complexexhibits a maximum absorbance at 500 nm The oxidation

of linoleic acid is slow in the presence of antioxidants [36]Therefore the greater the ability to inhibit the oxidationof Fe2+ to Fe3+ of the antioxidant substance the lower theabsorbance will be In this study the thiocyanate method wasused to determine the TAC of a reference antioxidant andvarious phenolic and flavonoid compounds their ability toinhibit linoleic acid emulsion at a 20120583gmL concentrationwas determined ID-8 callistephin malvin and oenin hadhigher inhibitory effects than all reference antioxidants usedwith 9798 9890 9675 and 967 inhibition valuesrespectively at 36th h (Table 1)

In additionmalvin pelargonin and silychristin exhibitedinhibition values of 9516 9393 and 9545 respectivelyshowing better lipid peroxidation inhibitory activity than thereference antioxidants BHT 120572-tocopherol and trolox Whenthe TACs of the reference antioxidants and the phenolicand flavonoid compounds were compared the antioxidantactivity observed from highest to lowest was as follows ID-8 gt callistephin gt oenin gt BHA gt silychristin gt malvin gtpelargoningt troloxgtBHTgt arachidonoyl dopaminegt 246-trihydroxybenzaldehydegt 34-dihydroxy-5-methoxybenzoicacid gt 120572-tocopherol

Elemental species such as Fe2+ accelerate ROS productionin the bodyTherefore the Fe chelating activity of a substancemay be related to its antioxidant activity Among transitionmetals Fe is known as the most important prooxidant thatcauses lipid peroxidation due to its high reactivity EffectiveFe2+ ion chelators prevent oxidative damage and oxidativestress-based diseases by binding Fe2+ ions which can produceOH∙ radicals and are very reactive in Fenton-type reactions[37]

Similarly this method is also performed using bipyridylreactives With this method 34-dihydroxy-5-methoxyben-zoic acid with 92 metal chelating capacity at 10120583gmLconcentration was more effective than the reference antiox-idants and other phenolic and flavonoid compounds didwith the exception of EDTA (9580) In addition ID-8and arachidonoyl dopamine with 8806 and 7386 metalchelating activity respectively demonstrated higher metalchelating activity than the other phenolic and flavonoidcompounds and reference antioxidants did with exceptionof EDTA and 120572-tocopherol Reference antioxidants and somephenolic and flavonoid compounds exhibitedmetal chelatingactivity to varying degrees (Table 1)

In the presence of chelating agents the red color of theFe2+-ferrozin complex which exhibits maximum absorbanceat 562 nm as a result of the reduction decreases Measuringthe color decrease provides an estimate of the metal chelatingactivity of the chelating agent Low absorbance indicates highmetal chelating activity [38] Kazazica et al reported thatflavonoids such as campherol exhibit Cu2+ and Fe2+ chelatingactivity via their functional groups [39] Similarly Fiorucci etal showed that quercetin exhibits metal ion binding activity[40] In another study it was determined that L-carnitinechelates Fe2+ ions via its carbonyl and hydroxyl functionalgroups Likewise it has been proposed that curcumin chelatesferrous ions via its carbonyl and hydroxyl functional groups[41] Similarly L-adrenaline binds iron ions via its amine and


Table 1The comparison of lipid peroxidation inhibition percentages and ferrous ion (Fe2+) chelating activities of reference antioxidants andsome phenolic and flavonoid compounds (10120583gmL for chelating activity and 20120583gmL for lipid peroxidation inhibition)

AntioxidantFe2+ ions chelating activity with

ferrozine reagentFe2+ ions chelating activity with

bipyridyl reagentTotal antioxidant

activityIC50 (120583gmL) activity IC50 (120583gmL) activity inhibition

BHA 3247 2734 4203 2446 9639BHT 3007 3489 3198 2886 6363120572-Tocopherol 2573 3841 1266 7253 910Trolox 4944 2135 1856 3086 9357EDTA mdash mdash 732 9580 mdashMalvin 1854 5221 1434 6573 9516ID-8 1833 5416 880 8806 9798Pelargonin 2572 3958 1430 5146 9393Silychristin 3678 2578 2483 1420 9545Callistephin 3451 2273 2080 3306 9758Oenin 2895 2200 2647 1640 9675Arachidonoyl Dopamine 2037 5065 1108 7386 511434-Dihydroxy-5-methoxybenzoicacid 2693 3632 5237 9200 1723

246-Trihydroxy benzaldehyde 3216 3216 1793 4800 2824The IC50 values were calculated by means of metal chelating and total antioxidant activity graphs from values measured at different concentrations(10ndash30 120583gmL) of reference antioxidants and the phenolic and flavonoid compounds

hydroxyl groups [42] We tested metal chelating activities ofreference antioxidants and selected phenolic and flavonoidcompounds at different concentrations (10ndash30 120583gmL) usingferrozine and bipyridyl reagents In our study ID-8 malvinarachidonoyl dopamine and pelargonin exhibited higherFe2+ chelating activity than reference antioxidants and otherphenolic and flavonoid compounds did at 10 120583gmL bychelating metal ions at levels of 5416 5221 5065and 3958 respectively (Table 1) In addition ID-8 arachi-donoyl dopamine malvin and pelargonin with IC50 valuesof 1833 1854 2572 and 2037 120583gmL respectively exhibitedmore effective Fe2+ ion chelating activity than referenceantioxidants and the other phenolic and flavonoid com-pounds tested did (Table 1) Additionally we hypothesizedthat Fe2+ chelating activities of the compounds in this studymay be due to their -OH groups

Determining metal chelating activity using the bipyri-dyl reagent was performed at different concentrations(10ndash30 120583gmL) of reference antioxidants and selected phe-nolic and flavonoid compounds In the absorbance-quantityplot drawn according to the results obtained using bipyridylreagent (Table 1) IC50 values of each substance were calcu-lated from the curve corresponding to the 10120583gmL concen-tration ID-8 and arachidonoyl dopamine exhibited bettermetal chelating activity than other phenolic and flavonoidcompounds tested and reference antioxidants did exceptEDTA with IC50 values of 880 and 1108120583gmL respectively(Table 1)

Free radical scavenging activity is very important becauseof the harmful effects of free radicals in biological systems andfoods Radical scavengers can react with free radicals directlyto clear peroxide radicals enhance the stability and quality

of food products and drugs and terminate peroxidationchain reactions [43] This test is one of the standard tests inantioxidant activity studies and provides rapid results for theradical scavenging activity of specific compounds [44] Freeradicals scavenging assays based on the scavenging ofDPPH∙DMPD∙+ ABTS∙+ and O2

∙minus radicals are the most popularspectrophotometric methods used to determine the antiox-idant capacities of foods beverages and plant extracts Inaddition these have advantages such as inexpensive reagentsless labor requirements ease of use high sensitivity andability to rapidly analyze antioxidant properties of numeroussampleswithout complicated instruments [45]When antiox-idants are added to a medium containing radicals DPPH∙DMPD∙+ and ABTS∙+ radicals are converted into theirreduced forms resulting in decolorization of the solution

The DPPH radical scavenging assay is one of the oldestmethods for determination of antioxidant activity [34] TheDPPH radical is an unstable organic nitrogen radical witha dark blue color In this method antioxidants reduce thestable DPPH radicals to yellow diphenyl-picrylhydrazineThis method is based on the fact that these radicals areconverted to DPPH-H the nonradical reduced form of theDPPH radicals upon hydrogen donation by antioxidantsin the alcohol solution [46] The purple-colored stablefree DPPH radical exhibits maximum absorbance at 517 nmWhen DPPH radicals contact a proton donor substrate theyare cleared and the absorbance decreases [47]

Resveratrol is one of the main phenolic compoundsfound in grapes Gulcin showed that resveratrol is an effec-tive DPPH radical scavenger [48] The DPPH∙ scaveng-ing activity of the reference antioxidants and pheno-lic and flavonoid compounds at different concentrations


Table 2 Free radical scavenging activities () of reference antioxidants and selected phenolic and flavonoid compounds at 10120583gmLconcentration for ABTS and DMPD and at 20120583gmL for O2

∙minus and DPPH∙

AntioxidantDPPH∙ DMPD∙+ ABTS∙+ O2

∙minus

IC50(120583gmL) activity IC50

(120583gmL) activity IC50(120583gmL) activity IC50

(120583gmL) activity

BHA 809 9864 1534 1534 360 9980 2337 4106BHT 1189 9628 1526 1526 604 9980 1502 6460120572-Tocopherol 1725 9385 1514 6414 847 7063 2321 4200Trolox 1413 9521 1390 6457 739 8372 2321 4400Malvin 2136 9221 1647 5985 720 8172 3097 3306ID-8 53641 9385 1756 6028 680 8263 3473 3066Pelargonin 6773 9150 1586 6585 671 9354 1413 6340Silychristin 8616 9107 1762 6171 671 8918 1819 5186Callistephin 2064 9557 1280 7271 554 9972 1970 4960Oenin 1672 9535 1546 6585 660 9972 1670 5640Arachidonoyl dopamine 8410 9850 1802 5971 1254 4200 2395 394034-Dihydroxy-5-methoxybenzoicacid 1069 9864 1780 6442 605 9980 1147 7340

246-Trihydroxy benzaldehyde 2886 9678 1793 7000 528 9972 1654 5680The IC50 values were calculated bymeans of radical scavenging activity graphs from the values measured at different concentrations (10ndash30120583gmL) of referenceantioxidants and some phenolic and flavonoid compounds

(10ndash30 120583gmL) wasmeasured at 517 nm As the concentrationof the substance increased the amount of free radicals inthe mixture decreased proportionally for almost all phenolicand flavonoid compounds In our study 34-dihydroxy-5-methoxybenzoic acid with an IC50 value of 1069 120583gmLshowed more DPPH radical scavenging activity than thereference antioxidants BHT 120572-tocopherol and trolox ID-8 with an IC50 value of 53641 120583gmL exhibited the lowestDPPH radical scavenging activity of the compounds exam-ined However all the test materials showed dose-dependentDPPH radical scavenging activity (Table 2)

Superoxide anion radicals are biologically highly toxicand are produced by the immune system to kill microor-ganisms In vivo superoxide can be produced as a result ofan electron being transferred to oxygen because of variousmetabolic processes or activation of oxygen by a radical [49]Although superoxide radicals have relatively limited chemicalreactivity and are a weak oxidant they can produce verydangerous reactive components such as singlet oxygen andhydroxyl radicals that cause lipid peroxidation [50] It has alsobeen observed that superoxide radical directly initiates lipidperoxidation [51] When the riboflavin used in this methodis photochemically activated it reacts with NBT to produceNBTH∙ The NBTH radical leads to formazan formation Inthe presence of oxygen radical species are controlled by asemiequilibrium reaction With the presence of antioxidantsthat donate electrons to NBT the degradation of the typicalpurple color of formazan can bemonitored spectrophotomet-rically at 560 nm Antioxidants have the ability to inhibit theconversion of NBT Decreased absorbance at 560 nm in thepresence of antioxidants indicates that the superoxide anionradicals are scavenged [9] The results obtained with thismethod showed that 34-dihydroxy-5-methoxybenzoic acid

and pelargonin with IC50 values of 1147 and 1413 120583gmLrespectively possessed better O2

∙minus anion radical scavengingactivity than the other phenolic and flavonoid compoundsand the reference antioxidants BHA BHT 120572-tocopherol andtrolox did (Table 2) Additionally oenin callistephin sily-christin and 246-trihydroxybenzaldehyde showed betterO2∙minus anion radical scavenging properties than the reference

antioxidants BHA 120572-tocopherol and trolox did and allother substances exhibited dose-dependent O2

∙minus scavengingactivity

ABTS is oxidized by oxidants into the intensely coloredABTS∙+ cation In this method antioxidant capacity wasmeasured by the decolorization ability of some phenolic andflavonoid compounds from reaction of ABTS radicals and theantioxidants added to the medium The ABTS assay can beapplied to both lipophilic and hydrophilic compounds [52]This method is based on the principle that the ABTS radicalcation shows maximum absorbance at 734 nm Reactionwith the ABTS radical occurs in a time as short as 025 to05min The radical scavenging performance of free radicalscavengers can be determined by monitoring the decrease inabsorbance at 734 nm [47]

The ABTS∙+ radical scavenging activity was determinedfor concentrations of 5 10 and 20 120583gmL of reference antiox-idants and the phenolic andflavonoid compoundsAccordingto the results 246-trihydroxybenzaldehyde oenin callis-tephin and ID-8 at 5120583gmL concentration exhibited higherABTS∙+ radical scavenging activities than the other phenolicand flavonoid compounds and the reference antioxidants(BHT trolox and 120572-tocopherol) (Table 2) In addition cal-listephin and 246-trihydroxybenzaldehyde with IC50 val-ues of 528 and 554 120583gmL respectively showed higherABTS∙+ radical scavenging activity than the phenolic and


flavonoid compounds and the reference antioxidants BHT120572-tocopherol and trolox did In addition 34-dihydroxy-5-methoxybenzoic acid oenin silychristin pelargonin ID-8and malvin exhibited IC50 values of 605 660 671 671680 and 720 (120583gmL) respectively They showed betterABTS radical scavenging activity than the examined phenolicand flavonoid compounds and the reference antioxidants 120572-tocopherol and trolox In addition all compounds examinedshowed dose-dependent ABTS∙+ radical scavenging activity

Another method that is similar to the ABTS radicalscavenging assay is the DMPD radical scavenging methodTohma and Gulcin proposed this new version of the ABTStest [35] In this method the ABTS radical is substitutedwith the stable DMPD∙+ radical cation formed from NN-dimethylphenylenediamine [28 53] They reported thatDMPD∙+ radical scavenging activity was more efficient andthe test was less expensive than the ABTS∙+ radical scaveng-ing method DMPD is converted to colored stable DMPD∙+radical cation in the presence of oxidants and an acidicmedium The visible spectrum of DMPD∙+ radical exhibitsmaximum absorbance at 505 nm However DMPD cannotbe used with hydrophobic antioxidants because it dissolvesin water only [28] When hydrophobic antioxidants areused the sensitivity and reproducibility of the assay dropdramatically [54] Antioxidant compounds decolorize thesolution by donating a hydrogen atom to DMPD radicals [2841] DMPD∙ radical scavenging activity was assayed for differ-ent concentrations (10ndash30 120583gmL) of reference antioxidantsand some phenolic and flavonoid compounds The resultsshowed that theDMPD∙ scavenging activities of the referenceantioxidants and some phenolic and flavonoid compoundswere very similar (Table 2) At 30 120583gmL concentration 246-trihydroxy-benzaldehyde exhibited better DMPD scavengingactivity than the other phenolic and flavonoid compoundsand the reference antioxidants BHA BHT and 120572-tocopherolIn addition callistephin and 246-trihydroxybenzaldehydewith IC50 values of 1327 and 1280 120583gmL respectivelyexhibited better DMPD∙+ radicals removal activity than theother phenolic and flavonoid compounds and the referenceantioxidants BHA BHT 120572-tocopherol and trolox did Inaddition all the compounds tested here exhibited dose-dependent DMPD∙+ radical scavenging activity

Antioxidants which can effectively reduce prooxidantscan also effectively reduce Fe3+ to Fe2+ [55] Therefore thereducing power of a compound provides important informa-tion about its antioxidant activity Reduction ability is oneof the most important antioxidant properties of a compound[56]The three methods used to determine reduction activityin this study measured the reduction of Cu2+ Fe3+ (usingferrozine reagent) and Fe3+ (using the FRAP reagent)

The Fe3+-Fe2 reduction activity of the reference antiox-idants and some phenolic and flavonoid compounds usingtripyridyltriazine (TPTZ) was determined by measuring theformation of the blue Fe2+-TPTZ complex at a wavelength of593 nm (FRAP assay)

Fe3+-Fe2 reduction activities of almost all of the refer-ence antioxidants and phenolic and flavonoid compoundsincreased proportionally with their concentration (Table 3)

Table 3 Fe3+ and Cu2+ reducing activities of reference antioxidantsand selected phenolic and flavonoid compounds at 30 120583gmL con-centration

Antioxidant FRAP assay(593 nm)

CUPRAC assay(450 nm)

BHA 2344 0489BHT 2430 0476120572-Tocopherol 2259 0403Trolox 2086 0330Malvin 2189 0431ID-8 0615 0145Pelargonin 2064 0385Silychristin 1181 0259Callistephin 2328 0456Oenin 2351 0464Arachidonoyl Dopamine 1392 030834-Dihydroxy-5-methoxybenzoicacid

2458 0474

246-Trihydroxybenzaldehyde 1839 0466

34-dihydroxy-5-methoxybenzoic acid exhibited better Fe3+-Fe2 reduction capacity than the reference antioxidants andthe other phenolic and flavonoid compounds examined Inaddition callistephin and oenin showed more effective Fe3+-Fe2 reduction activity than the reference antioxidants troloxand 120572-tocopherol did When Fe3+-Fe2 reduction activitiesof the reference antioxidants and phenolic and flavonoidcompounds were compared at 30120583gmL using the FRAPreagent the antioxidant activities observed from highestto lowest were as follows 34-dihydroxy-5-methoxybenzoicacidgtBHTgt oeningtBHAgt callistephin gt troloxgtmalvingt120572-tocopherolgtpelargoningt 246-trihydroxybenzaldehydegtarachidonoyl dopamine gt silychristin gt ID-8

The CUPRAC assay measuring the reduction of Cu2+to Cu+ was described by Gulcin et al [57] This method isbased on the reduction of Cu2+ to Cu+ at pH 7 in aqueousethanol with the combined effect of antioxidants in thepresence of neocuproine (29-dimethyl-110-phenanthrene)The Cu+ complex formed by the phenols shows maximumabsorbance at 450 nm [58]This method is suitable for a widevariety of antioxidants both hydrophilic and hydrophobicsubstances because it is low-cost fast stable and selectiveFurthermore the chromogenic CUPRAC redox reactionoccurs at physiological pH and is commonly used to comparenonprotein thiol-type antioxidants such as glutathione asopposed to the FRAP method which does not respond toantioxidants containing SH groups [59]

The results obtained with this method showed dose-dependent Cu2+ reduction activity for all phenolic and fla-vonoid compounds In addition 30120583gmL 246-trihy-droxybenzaldehyde 34-dihydroxy-5-methoxybenzoic acidmalvin oenin and callistephin exhibited absorbance valuesof 0431 0464 0456 0466 and 0474 respectively in theCu2+-Cu+ reduction assay which were higher than the values


obtained for other phenolic and flavonoid compounds andtrolox and 120572-tocopherol the reference antioxidants (Table 3)

4 Conclusion

Our data demonstrate the difference in antioxidant activitiesof the reference antioxidants and selected phenolic andflavonoid compounds in different assays This may be due tothe fact that the different antioxidant capacity determiningmethods have different specificities for different solventsreagents pH conditions or hydrophilic and hydrophobicsubstances Furthermore molecular size and the numberand type of functional groups of the phenolic and flavonoidcompounds may be important Oenin malvin arachidonoyldopamine callistephin silychristin and 34-dihydroxy-5-methoxybenzoic acid exhibited better antioxidant activitiesthan the reference antioxidants did Therefore these com-pounds may have the potential to protect and maintainfood and medicines and reduce oxidative stress or increaseantioxidant capacity in vivo this conclusion should be furthervalidated by future studies

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this article

Authorsrsquo Contributions

ZubeyirHuyut and Sukru Beydemir researched literature andconceived the study Zubeyir Huyut was involved in pro-tocol development experimental studies and data analysisZubeyir Huyut Sukru Beydemir and Ilhami Gulcin wrotethe first draft of the manuscript All authors reviewed andedited the manuscript and approved the final version of themanuscript

Acknowledgments

This study was performed with opportunities from Depart-ments of Biochemistry Faculty of Science Ataturk Univer-sity and Faculty of Medicine Yuzuncu Yıl University

References

[1] K Aksu B Ozgeris P Taslimi A Naderi I Gulcin and SGoksu ldquoAntioxidant Activity Acetylcholinesterase and Car-bonic Anhydrase Inhibitory Properties of Novel Ureas Derivedfrom Phenethylaminesrdquo Archiv der Pharmazie vol 349 no 12pp 944ndash954 2016

[2] I Gulcin V Mshvildadze A Gepdiremen and R EliasldquoScreening of antiradical and antioxidant activity of mon-odesmosides and crude extract from Leontice smirnowii tuberrdquoPhytomedicine vol 13 no 5 pp 343ndash351 2006

[3] M M Ibrahim G A M Mersal A-M M Ramadan S YShabanM AMohamed and S Al-Juaid ldquoSynthesis character-ization and antioxidantcytotoxic activity of oxovanadium(IV)complexes ofmethyliminodiacetic acid and ethylenediaminete-tracetic acidrdquo Journal of Molecular Structure vol 1137 pp 742ndash755 2017

[4] C A Prauchner ldquoOxidative stress in sepsis Pathophysiologicalimplications justifying antioxidant co-therapyrdquo Burns vol 43no 3 pp 471ndash485 2017

[5] J Li O Wuliji W Li Z-G Jiang and H A GhanbarildquoOxidative stress and neurodegenerative disordersrdquo Interna-tional Journal of Molecular Sciences vol 14 no 12 pp 24438ndash24475 2013

[6] J-M Lu P H Lin Q Yao and C Chen ldquoChemical and molec-ular mechanisms of antioxidants experimental approaches andmodel systemsrdquo Journal of Cellular andMolecularMedicine vol14 no 4 pp 840ndash860 2010

[7] S Catarzi C Romagnoli G Marcucci F Favilli T Iantomasiand M T Vincenzini ldquoRedox regulation of ERK12 activationinduced by sphingosine 1-phosphate in fibroblasts involvementofNADPHoxidase andplatelet-derived growth factor receptorrdquoBiochimica et Biophysica Acta vol 1810 no 4 pp 446ndash456 2011

[8] A Rahal A Kumar and V Singh ldquoOxidative stress prooxi-dants and antioxidants the interplayrdquo BioMed Research Inter-national vol 2014 Article ID 761264 19 pages 2014

[9] L A Pham-Huy H He and C Pham-Huy ldquoFree radicalsantioxidants in disease and healthrdquo International Journal ofBiomedical Science vol 4 pp 89ndash96 2008

[10] E Bursal and I Gulcin ldquoPolyphenol contents and in vitroantioxidant activities of lyophilised aqueous extract of kiwifruit(Actinidia deliciosa)rdquo Food Research International vol 44 no5 pp 1482ndash1489 2011

[11] Y Cetinkaya H Gocer A Menzek and I Gulcin ldquoSynthe-sis and antioxidant properties of (34-dihydroxyphenyl)(234-trihydroxyphenyl)methanone and its derivativesrdquo Archiv derPharmazie vol 345 no 4 pp 323ndash334 2012

[12] D Giordano M Locatelli F Travaglia et al ldquoBioactive com-pound and antioxidant activity distribution in roller-milledand pearled fractions of conventional and pigmented wheatvarietiesrdquo Food Chemistry vol 233 pp 483ndash491 2017

[13] S Goncalves E Moreira C Grosso P B Andrade P Valentaoand A Romano ldquoPhenolic profile antioxidant activity andenzyme inhibitory activities of extracts from aromatic plantsused in Mediterranean dietrdquo Journal of Food Science andTechnology vol 54 no 1 pp 219ndash227 2017

[14] P A Hannan J A Khan I Ullah and S Ullah ldquoSynergisticcombinatorial antihyperlipidemic study of selected naturalantioxidants Modulatory effects on lipid profile and endoge-nous antioxidantsrdquo Lipids in Health and Disease vol 15 no 1article no 151 2016

[15] T A Comunian R Ravanfar I A de Castro R Dando C SFavaro-Trindade and A Abbaspourrad ldquoImproving oxidativestability of echium oil emulsions fabricated by Microfluidicseffect of ionic gelation and phenolic compoundsrdquo Food Chem-istry vol 233 pp 125ndash134 2017

[16] F Shahidi and P Ambigaipalan ldquoPhenolics and polyphenolicsin foods beverages and spices Antioxidant activity and healtheffects - A reviewrdquo Journal of Functional Foods vol 18 pp 820ndash897 2015

[17] D C Vodnar L F Calinoiu F V Dulf B E Stefanescu GCrisan and C Socaciu ldquoIdentification of the bioactive com-pounds and antioxidant antimutagenic and antimicrobialactivities of thermally processed agro-industrial wasterdquo FoodChemistry vol 231 pp 131ndash140 2017

[18] P G Pietta ldquoFlavonoids as antioxidantsrdquo Journal of NaturalProducts vol 63 no 7 pp 1035ndash1042 2000

[19] J Anissi M El Hassouni A Ouardaoui and K Sendide ldquoAcomparative study of the antioxidant scavenging activity of


green tea black tea and coffee extracts A kinetic approachrdquoFood Chemistry vol 150 pp 438ndash447 2014

[20] H NThatoi J K Patra and S K Das ldquoFree radical scavengingand antioxidant potential of mangrove plants A reviewrdquo ActaPhysiologiae Plantarum vol 36 no 3 pp 561ndash579 2014

[21] I Gulcin ldquoAntioxidant activity of food constituents an over-viewrdquo Archives of Toxicology vol 86 no 3 pp 345ndash391 2012

[22] HMitsuda K Yuasumoto andK Iwami ldquoAntioxidation actionof indole compounds during the autoxidation of linoleic acidrdquoEiyo to Shokuryo vol 19 pp 210ndash214 1996

[23] M Oyaizu ldquoStudies on product of browning reaction preparedfrom glucoseaminerdquo Japanese Journal of Nutrition and Dietete-ics vol 44 pp 307ndash315 1986

[24] RApakKGucluM Ozyurek S EsinKarademir andE ErcagldquoThe cupric ion reducing antioxidant capacity and polyphenoliccontent of some herbal teasrdquo International Journal of FoodSciences and Nutrition vol 57 no 5-6 pp 292ndash304 2006

[25] J Zhishen TMengcheng andW Jianming ldquoThedeterminationof flavonoid contents in mulberry and their scavenging effectson superoxide radicalsrdquo Food Chemistry vol 64 no 4 pp 555ndash559 1999

[26] M S Blois ldquoAntioxidant determinations by the use of a stablefree radicalrdquo Nature vol 181 no 4617 pp 1199-1200 1958

[27] R Re N Pellegrini A Proteggente A PannalaM Yang andCRice-Evans ldquoAntioxidant activity applying an improved ABTSradical cation decolorization assayrdquo Free Radical Biology ampMedicine vol 26 no 9-10 pp 1231ndash1237 1999

[28] V Fogliano V Verde G Randazzo and A Ritieni ldquoMethod formeasuring antioxidant activity and its application to monitor-ing the antioxidant capacity of winesrdquo Journal of Agriculturaland Food Chemistry vol 47 no 3 pp 1035ndash1040 1999

[29] T C P Dinis V M C Madeira and L M Almeida ldquoActionof phenolic derivatives (acetaminophen salicylate and 5-aminosalicylate) as inhibitors of membrane lipid peroxidationand as peroxyl radical scavengersrdquo Archives of Biochemistry andBiophysics vol 315 no 1 pp 161ndash169 1994

[30] A T Diplock ldquoErratum Will the rsquogood fairiesrsquo please prove tous that vitamin E lessens human degenerative disease (FreeRadical Research 26 (565-583))rdquo Free Radical Research vol 27no 5 pp 511ndash532 1997

[31] R L Prior X Wu and K Schaich ldquoStandardized methodsfor the determination of antioxidant capacity and phenolicsin foods and dietary supplementsrdquo Journal of Agricultural andFood Chemistry vol 53 no 10 pp 4290ndash4302 2005

[32] M Qasim Z Abideen M Y Adnan et al ldquoAntioxidant proper-ties phenolic composition bioactive compounds and nutritivevalue of medicinal halophytes commonly used as herbal teasrdquoSouth African Journal of Botany vol 110 pp 240ndash250 2017

[33] I Gulcin M Elmastas and H Y Aboul-Enein ldquoAntioxidantactivity of clove oil - A powerful antioxidant sourcerdquo ArabianJournal of Chemistry vol 5 no 4 pp 489ndash499 2012

[34] V Roginsky and E A Lissi ldquoReview of methods to determinechain-breaking antioxidant activity in foodrdquo Food Chemistryvol 92 no 2 pp 235ndash254 2005

[35] H S Tohma and I Gulcin ldquoAntioxidant and radical scavengingactivity of aerial parts and roots of Turkish liquorice (Gly-cyrrhiza glabra L)rdquo International Journal of Food Properties vol13 no 4 pp 657ndash671 2010

[36] I Gulcin O I Kufrevioglu M Oktay and M E Buyuk-okuroglu ldquoAntioxidant antimicrobial antiulcer and analgesicactivities of nettle (Urtica dioica L)rdquo Journal of Ethnopharma-cology vol 90 no 2-3 pp 205ndash215 2004

[37] S Hippeli and E F Elstner ldquoTransition metal ion-catalysedoxygen activation during pathogenic processesrdquo FEBS Lettersvol 443 no 1 pp 1ndash7 1999

[38] I GulcinMOktay E Kirecci and O I Kufrevıoglu ldquoScreeningof antioxidant and antimicrobial activities of anise (Pimpinellaanisum L) seed extractsrdquo Food Chemistry vol 83 no 3 pp 371ndash382 2003

[39] S P Kazazic V Butkovic D Srzic and L Klasinc ldquoGas-phaseligation of Fe2+ and Cu+ ions with some flavonoidsrdquo Journal ofAgricultural and Food Chemistry vol 54 pp 8391ndash8396 2006

[40] S Fiorucci J Golebiowski D Cabrol-Bass and S AntonczakldquoDFT study of quercetin activated forms involved in antiradicalantioxidant and prooxidant biological processesrdquo Journal ofAgricultural and Food Chemistry vol 55 no 3 pp 903ndash9112007

[41] T Ak and I Gulcin ldquoAntioxidant and radical scavenging prop-erties of curcuminrdquoChemico-Biological Interactions vol 174 no1 pp 27ndash37 2008

[42] I Gulcin ldquoAntioxidant activity of l-adrenaline A structure-activity insightrdquo Chemico-Biological Interactions vol 179 no 2-3 pp 71ndash80 2009

[43] E Koksal I Gulcin S Beyza O Sarikaya and E BursalldquoIn vitro antioxidant activity of silymarinrdquo Journal of EnzymeInhibition and Medicinal Chemistry vol 24 no 2 pp 395ndash4052009

[44] B Ozcelik J H Lee andD BMin ldquoEffects of light oxygen andpHon the absorbance of 22-diphenyl-1-picrylhydrazylrdquo Journalof Food Science vol 68 no 2 pp 487ndash490 2003

[45] I Gulcin D Berashvili and A Gepdiremen ldquoAntiradicaland antioxidant activity of total anthocyanins from Perillapankinensis decnerdquo Journal of Ethnopharmacology vol 101 no1ndash3 pp 287ndash293 2005

[46] M Elmastas I Turkekul L Ozturk I Gulcin O Isildak andH Y Aboul-Enein ldquoThe antioxidant activity of two wild ediblemushrooms (Morchella vulgaris and Morchella esculanta)rdquo inCombinatorial Chemistry amp High Throughput Screening vol 9pp 443ndash448 2006

[47] I Gulcin R Elias A Gepdiremen K Taoubi and E KoksalldquoAntioxidant secoiridoids from fringe tree (Chionanthus vir-ginicus L)rdquo Wood Science and Technology vol 43 no 3-4 pp195ndash212 2009

[48] I Gulcin ldquoAntioxidant properties of resveratrol a structure-activity insightrdquo Innovative Food Science and Emerging Tech-nologies vol 11 no 1 pp 210ndash218 2010

[49] B Halliwell ldquoPhagocyte-derived reactive species salvation orsuiciderdquo Trends in Biochemical Sciences vol 31 no 9 pp 509ndash515 2006

[50] B Halliwell and S Chirico ldquoLipid peroxidation its mechanismmeasurement and significancerdquoThe American Journal of Clini-cal Nutrition vol 57 pp 715ndash725 1993

[51] A P Wickens ldquoAgeing and the free radical theoryrdquo RespirationPhysiology vol 128 no 3 pp 379ndash391 2001

[52] L M Magalhaes M A Segundo S Reis and J L F CLima ldquoMethodological aspects about in vitro evaluation ofantioxidant propertiesrdquo Analytica Chimica Acta vol 613 no 1pp 1ndash19 2008

[53] K Schlesier M Harwat V Bohm and R Bitsch ldquoAssessmentof antioxidant activity by using different in vitromethodsrdquo FreeRadical Research vol 36 no 2 pp 177ndash187 2002

[54] C Sanchez-Moreno ldquoReview methods used to evaluate thefree radical scavenging activity in foods and biological systemsrdquo


Food Science and Technology International vol 8 no 3 pp 121ndash137 2002

[55] N Oztaskin Y Cetinkaya P Taslimi S Goksu and I GulcinldquoAntioxidant and acetylcholinesterase inhibition properties ofnovel bromophenol derivativesrdquo Bioorganic Chemistry vol 60pp 49ndash57 2015

[56] S Meir J Kanner B Akiri and S Philosoph-Hadas ldquoDeter-mination and involvement of aqueous reducing compounds inoxidative defense systems of various senescing leavesrdquo Journalof Agricultural and Food Chemistry vol 43 no 7 pp 1813ndash18191995

[57] I Gulcin R Elias A Gepdiremen A Chea and F TopalldquoAntioxidant activity of bisbenzylisoquinoline alkaloids fromStephania rotunda Cepharanthine and fangchinolinerdquo Journalof Enzyme Inhibition andMedicinal Chemistry vol 25 no 1 pp44ndash53 2010

[58] G LeeM V Rossi N Coichev andHDMoya ldquoThe reductionof Cu(II)neocuproine complexes by some polyphenols Totalpolyphenols determination in wine samplesrdquo Food Chemistryvol 126 no 2 pp 679ndash686 2011

[59] R Apak K Guclu M Ozyurek and S E Celik ldquoMechanismof antioxidant capacity assays and the CUPRAC (cupric ionreducing antioxidant capacity) assayrdquo Microchimica Acta vol160 no 4 pp 413ndash419 2008

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 201


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


protect against oxidative diseases activate or inhibit variousenzymes bind specific receptors and protect against cardio-vascular diseases by reducing the oxidation of low-densitylipoproteins [18]

Various methods have been developed to investigate theantioxidant properties of a substance These include assaysfor total antioxidant capacity (TAC) NO∙ H2O2 O2

∙minus andOH∙ radical scavenging capacity oxygen radical scavengingactivity (ORAC) Fe3+ and Cu2+ reducing activity (FRAPand CUPRAC assay resp) metal chelating activity ABTS∙+DMPD∙+ and DPPH∙ free radical scavenging activity andlipid peroxidation inhibition capacity [19ndash21] Among thesethe most commonly used methods are TAC determinationand assays for determining Fe+3 and Cu+2 reduction activitymetal chelating ability and free radical (ABTS∙+ DPPH∙DMPD∙+ OH∙ and O2

∙minus) scavenging activity [21]In the current study we investigated the antioxidant

capacities of malvin oenin ID-8 silychristin callistephinand pelargonin with flavonoid structures and 34-dihydroxy-5-methoxybenzoic acid arachidonoyl dopamine and 246-trihydroxybenzaldehyde with phenolic structures (Figure 1)by assaying Fe3+ and Cu2+ reduction activity metal chelatingactivity and O2

∙minus ABTS∙+ DPPH∙ and DMPD∙+ radicalscavenging capacity

2 Materials and Methods

21 Chemicals Sodium dihydrogen phosphate (NaH2PO4)potassium ferrocyanide (K3Fe(CN)6) trichloroacetic acid(TCA) iron-III-chloride (FeCl3) potassium peroxydisulfate(K2O8S2) copper-II-chloride (CuCl2) sodium acetate(NaCH3COO) hydrochloric acid (HCl) tris iron-II-sul-fate (FeSO4) iron-II-chloride (FeCl2) disodium hydro-gen phosphate (Na2HPO4) methionine ethanol ethylene-diaminetetraacetic acid (EDTA) ammonium thiocyanate(NH4SCN) sodium bicarbonate (NaHCO3) sodium hy-droxide (NaOH) disodium sulfate (Na2SO4) sodium per-chlorate (NaClO4) and disodium carbonate (Na2CO3) wereobtained from Merck (Merck made in Germany) Cis-9cis-12-octadecanoic acid (linoleic acid) 22-diphenyl-1-pi-crylhydrazyl (DPPH) polyoxyethylene sorbitanmonolaurate(Tween 20) nitroblue tetrazolium (NBT) 3-(2-pyridyl)-56-diphenyl-124-triazine-41015840410158401015840-disulfonic acid sodium salt(ferrozine) riboflavin (vitamin B2) N-N-Dimethyl-p-phe-nyl-enediamine dihydrochloride (DMPD) neocuproine hy-drate (C4H12N2sdotXH2O) 221015840-azino-bis(3-ethylbenzothia-zoline-6-sulfonic acid) diammonium sulfate (ABTS) ID-8 (C6H14N2O4) 34-dihydroxy-5-methoxybenzoic acid(C8H8O5) silychristin (C25H22O10) malvin (C29H28O17)pelargonin (C27H31O15) oenin (C23H25O2) arachidonoyldopamine (C28H41NO3) callistephin (C21H21O10) and246-trihydroxy-benzaldehyde (C6H2CHO) were purchasedfrom Sigma Aldrich Company (St Louis MO USA)

22 Determination of TAC TAC was determined by thethiocyanate method [22] Different concentrations of thestock solutions (10 20 30 120583gmL) of phenolic and flavonoidcompounds were added to tubes and volume was broughtto 25mL using phosphate buffer (001M and pH 74)

Subsequently 25mL of linoleic acid emulsion was added toeach tube and the mixture was incubated at 37∘C in the darkSamples (100 120583L) were taken every 12 h during incubationto which 47mL of ethanol 100 120583L of SCNminus solution and100 120583L of Fe2+ solution (20mM) prepared in HCl (35)were added The absorbance of the samples at 500 nm wasmeasured and compared to that of blank solution Alcoholwas used instead of the sample for the blank while buffersolution was used instead of the sample for the control Theincubation and absorbance measurements were continueduntil the maximum absorbance values of the control samplewere reached (about 15 d)

23 Determination of Fe3+-Fe2+ Reduction Activity by FRAPReagent (FRAP Assay) The reduction power as FRAP reac-tivity was determined using the method of Oyaizu with slightmodification [23] Different solutions (10 20 30 120583gmL) wereprepared from the 1mgmL stock solutions of the phenolicand flavonoid compounds The sample volume in the tubeswas to 05mL using acetate buffer (pH 36) Subsequently225mL of FeCl3 solution and 225mL of FRAP reagent wereadded to each tube (total volume 5mL) After incubation for10min the absorbance of the mixture was read at 593 nmagainst the blank Acetate buffer was used as a blank controlsample

24 Cu2+-Cu+ Reduction Activity (CUPRAC Assay) TheCu2+ reduction activity was determined using the methodpreviously described byApak et al with aminormodification[24] Different concentrations (10 20 and 30 120583gmL) ofphenolic and flavonoid compounds were mixed with 125 120583Lof CuCl2 solution (001M) 125120583L of ethanolic neocuprinsolution (75mM) and 125 120583L of ammonium acetate buffersolution (1M) After incubation in the dark for 30minabsorbance was measured at 450 nm against a distilled waterblank

25 Superoxide Anion Radical (O2∙minus) Scavenging Activity

Superoxide anion radical scavenging activity was determinedusing the method described by Zhishen et al with slightmodification [25]Thismethod is based on the spectrophoto-metric measurement of nitroblue tetrazolium (NBT) Differ-ent concentrations of samples and standards were preparedin phosphate buffer (005M and pH 78) To the samplesolutions riboflavin methionine and NBT were added atconcentrations of 133 446 and 815times 10minus2 120583M respectivelyThe reactionmixture was stimulated with 20Wof fluorescentlight at room temperature for 2 h Absorbance was measuredat 560 nm against a distilled water blank

26 DPPH Radical Scavenging Activity The DPPH radicalscavenging activity was analyzed according to the method ofBlois [26] DPPH solution (1mM)was used as the free radicalThe previously prepared 1mgmL antioxidant stock solutionswere used Samples were added to test tubes at concentrationsof 10 20 and 30 120583gmL and the total volume was broughtto 25mL using pure ethanol Subsequently 05mL of thestock DPPH solution was added to each sample tube After


O

OH

OHOH

OH

OH

OH

OH

H

OHOH

OH

OH

OH HO

HO

HO

HON

HO

HO




HO

HO

O

OOH

OH

O

OHOH

HO

O

O

OH

OHOH

OH

OH

OH

OH

HO

HO

OH

OH

OH

HO

HO

HO

OH

O

（3

（3

（3

（3

（3

（3

O

O

O

O

OO

NO2

CH3

CH3

O

O

O

O

O

HO

HO NH

O

CH3






































∙minus
























2458 0474







4 Conclusion






Acknowledgments


References







































































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


O

OH

OHOH

OH

OH

OH

OH

H

OHOH

OH

OH

OH HO

HO

HO

HON

HO

HO




HO

HO

O

OOH

OH

O

OHOH

HO

O

O

OH

OHOH

OH

OH

OH

OH

HO

HO

OH

OH

OH

HO

HO

HO

OH

O

（3

（3

（3

（3

（3

（3

O

O

O

O

OO

NO2

CH3

CH3

O

O

O

O

O

HO

HO NH

O

CH3






































∙minus
























2458 0474







4 Conclusion






Acknowledgments


References







































































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

































∙minus
























2458 0474







4 Conclusion






Acknowledgments


References







































































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology











2458 0474







4 Conclusion






Acknowledgments


References







































































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



4 Conclusion






Acknowledgments


References







































































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




















































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

antioxidant and antiradical properties of selected flavonoids and phenolic...

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