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Antioxidantandradicalscavengingpropertiesofcurcumin

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Chemico-Biological Interactions 174 (2008) 27–37

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Antioxidant and radical scavenging properties of curcumin

Tuba Ak, Ilhami Gulcin ∗

Faculty of Arts and Sciences, Department of Chemistry, Ataturk University, TR-25240 Erzurum, Turkey

a r t i c l e i n f o

Article history:Received 21 January 2008Received in revised form 30 April 2008Accepted 1 May 2008Available online 7 May 2008

Keywords:Antioxidant activityCurcuminMetal chelatingReducing powerRadical scavenging

a b s t r a c t

Curcumin (diferuoyl methane) is a phenolic compound and a major component of Cur-cuma longa L. In the present paper, we determined the antioxidant activity of curcumin byemploying various in vitro antioxidant assays such as 1,1-diphenyl-2-picryl-hydrazyl freeradical (DPPH•) scavenging, 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS)radical scavenging activity, N,N-dimethyl-p-phenylenediamine dihydrochloride (DMPD)radical scavenging activity, total antioxidant activity determination by ferric thiocyanate,total reducing ability determination by the Fe3+–Fe2+ transformation method, superoxideanion radical scavenging by the riboflavin/methionine/illuminate system, hydrogen perox-ide scavenging and ferrous ions (Fe2+) chelating activities. Curcumin inhibited 97.3% lipidperoxidation of linoleic acid emulsion at 15 �g/mL concentration (20 mM). On the otherhand, butylated hydroxyanisole (BHA, 123 mM), butylated hydroxytoluene (BHT, 102 mM),�-tocopherol (51 mM) and trolox (90 mM) as standard antioxidants indicated inhibitionof 95.4, 99.7, 84.6 and 95.6% on peroxidation of linoleic acid emulsion at 45 �g/mL con-centration, respectively. In addition, curcumin had an effective DPPH• scavenging, ABTS•+

scavenging, DMPD•+ scavenging, superoxide anion radical scavenging, hydrogen peroxidescavenging, ferric ions (Fe3+) reducing power and ferrous ions (Fe2+) chelating activities.Also, BHA, BHT, �-tocopherol and trolox, were used as the reference antioxidant and rad-ical scavenger compounds. According to the present study, curcumin can be used in thepharmacological and food industry because of these properties.

© 2008 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Oxygen consumption inherent in cell growth leads tothe generation of a series of reactive oxygen species (ROS)[1]. They are continuously produced by the body’s normaluse of oxygen such as respiration and some cell-mediatedimmune functions. ROS include free radicals such as super-oxide anion radicals (O2

•−), hydroxyl radicals (OH•) andnon-free radical species such as hydrogen peroxide (H2O2)and singlet oxygen (1O2) [2]. ROS are continuously pro-duced during normal physiologic events and can easilyinitiate the peroxidation of membrane lipids, leading to

∗ Corresponding author. Tel.: +90 442 2314444; fax: +90 442 2360948.E-mail addresses: igulcin@atauni.edu.tr,

igulcin@yahoo.com (I. Gulcin).

the accumulation of lipid peroxides. ROS are also capableof damaging crucial biomolecules such as nucleic acids,lipids, proteins and carbohydrates and may cause DNAdamage that can lead to mutations. If ROS are not effectivelyscavenged by cellular constituents, they lead to diseaseconditions. ROS have been implicated in more than 100diseases [3].

All aerobic organisms have antioxidant defences, includ-ing antioxidant enzymes and antioxidant food constituents,to remove or repair the damaged molecules. Antioxidantcompounds can scavenge free radicals and increase shelflife by retarding the process of lipid peroxidation, whichis one of the major reasons for deterioration of food andpharmaceutical products during processing and storage [4].Antioxidants can protect the human body from free rad-icals and ROS effects. They retard the progress of manychronic diseases as well as lipid peroxidation [5,6]. Hence,

0009-2797/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved.doi:10.1016/j.cbi.2008.05.003

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28 T. Ak, I. Gulcin / Chemico-Biological Interactions 174 (2008) 27–37

a need for identifying alternative natural and safe sourcesof food antioxidants has been created, and the search fornatural antioxidants, especially of plant origin, has notablyincreased in recent years. Antioxidants have been widelyused as food additives to provide protection against oxida-tive degradation of foods. At the present time, the mostcommonly used antioxidants are butylated hydroxyanisole(BHA), butylated hydroxytoluene (BHT), propylgallate andtert-butyl hydroquinone. However, BHA and BHT have beensuspected of being responsible for liver damage and car-cinogenesis [7,8]. Therefore, there is a growing interest innatural and safer antioxidants [9,10].

Curcuma longa L. has been used for hundreds of years as aflavor, color, and preservative. Commercially, it is traded asa dye, spice, and source of industrial starch [11]. Curcumin isa nutriceutical compound reported to possess therapeuticproperties against a variety of diseases ranging from cancerto cystic fibrosis [12]. Recently, it has attracted much atten-tion due to its significant medicinal potential. The aim ofthis study was to investigate the inhibition of lipid peroxi-dation, ferric ions (Fe3+) reducing antioxidant power assay,1,1-diphenyl-2-picryl-hydrazyl (DPPH•) radical scaveng-ing, 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid(ABTS•+) radical scavenging, superoxide anion radical scav-enging in the riboflavin/methionine/illuminate system,hydrogen peroxide scavenging and ferrous ions (Fe3+)chelating activities of curcumin. In addition, an importantmain goal of this study was to clarify the antioxidant andradical scavenging and metal chelating mechanisms of cur-cumin.

2. Materials and methods

2.1. Chemicals

N,N-Dimethyl-p-phenylenediamine dihydrochloride(DMPD), riboflavin, methionine, ABTS, BHA, BHT, nitrobluetetrazolium (NBT), DPPH•, 3-(2-pyridyl)-5,6-bis(4-phenyl-sulfonic acid)-1,2,4-triazine (ferrozine), linoleic acid,�-tocopherol, polyoxyethylenesorbitan monolaurate(Tween-20) and trichloroacetic acid (TCA) were obtainedfrom Sigma (Sigma–Aldrich GmbH, Sternheim, Germany).Curcumin and ammonium thiocyanate were purchasedfrom Merck. All other chemicals used were analytical gradeand obtained from either Sigma–Aldrich or Merck.

2.2. Total antioxidant activity determination by ferricthiocyanate method

The antioxidant activity of curcumin and standards wasdetermined according to the ferric thiocyanate method asdescribed by Gulcin [2]. A stock solution contained 10 mgof curcumin dissolved in 10 mL ethanol. Different concen-trations of curcumin (from 15 to 45 �g/mL) were preparedby diluting the stock solution in 2.5 mL of sodium phos-phate buffer (0.04 M, pH 7.0) and these were added to2.5 mL of linoleic acid emulsion in sodium phosphate buffer(0.04 M, pH 7.0). The linoleic acid emulsion was preparedby homogenising 15.5 �L of linoleic acid, 17.5 mg of Tween-20 as emulsifier, and 5 mL phosphate buffer (pH 7.0). Thecontrol was composed of 2.5 mL of linoleic acid emul-

sion and 2.5 mL, 0.04 M sodium phosphate buffer (pH 7.0).The reaction mixtures (5 mL) were incubated at 37 ◦C inpolyethylene flasks. The peroxide levels were determinedby reading the absorbance at 500 nm. The peroxides formedduring linoleic acid peroxidation will oxidize Fe2+ to Fe3+,which forms a complex with thiocyanate that has a maxi-mum absorbance at 500 nm. The assay step was repeatedevery 5 h until reaching a maximum. The percent inhibi-tion was calculated at this point (30 h). Solutions withoutcurcumin were used as blank samples. The percent inhi-bition of lipid peroxidation in linoleic acid emulsion wascalculated by the following equation:

inhibition of lipid peroxidation (%) =(

100 − AS

AC

)× 100

in which AC is the absorbance of the control reaction, whichcontains only linoleic acid emulsion and sodium phosphatebuffer, and AS is the absorbance of the sample in the pres-ence curcumin or other test compounds [13,14].

2.3. Ferric cyanide (Fe3+) reducing antioxidant powerassay

The ferric reducing antioxidant power method of Oyaizu[15] with slight modification [13] was used to measurethe reducing capacity of curcumin. The FRAP method isbased on the reduction of (Fe3+) ferricyanide in stoichio-metric excess relative to the antioxidants [16]. Differentconcentrations of curcumin (15–45 �g/mL) in 1 mL of dis-tilled water were mixed with 2.5 mL of 0.2 M, pH 6.6 sodiumphosphate buffer and potassium ferricyanide [K3Fe(CN)6].2.5 mL of a 1% mixture was incubated at 50 ◦C for 20 min.After 20 min incubation, the reaction mixture was acidi-fied with 2.5 mL of trichloroacetic acid (10%). Then; 2.5 mLof the acidified sample of this solution was mixed with2.5 mL of distilled water and 0.5 mL of FeCl3 (0.1%) and theabsorbance was measured at 700 nm in a spectrophotome-ter. Increased absorbance of the reaction mixture indicatesgreater reduction capability [17,18].

2.4. Ferrous ion (Fe2+) chelating activity

The chelating of ferrous ion by curcumin was esti-mated by the method of Dinis et al. [19], wherein theFe2+-chelating ability of curcumin was monitored bythe absorbance of the ferrous iron–ferrozine complex at562 nm. Briefly, curcumin (15 �g/mL, 20 mM) in 0.4 mL wasadded to a solution of 2 mM FeCl2 (0.2 mL). The reactionwas initiated by the addition of 5 mM ferrozine (0.4 mL).The total volume was adjusted to 4 mL with ethanol. Then,the mixture was shaken vigorously and left at room tem-perature for 10 min. Absorbance of the solution was thenmeasured spectrophotometrically at 562 nm. The percent-age of inhibition of ferrozine–Fe2+ complex formation wascalculated by using the equation given below:

ferrous ion (Fe2+) chelating effect (%) =(

1 − AS

AC

)× 100

where AC is the absorbance of the control and AS is theabsorbance in the presence of curcumin or standards. Thecontrol contains only FeCl2 and ferrozine [13,20].

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2.5. Hydrogen peroxide scavenging activity

The hydrogen peroxide scavenging assay was carried outfollowing the procedure of Ruch et al. [21]. The principle ofthis method is that there is a decrease in absorbance ofH2O2 upon oxidation of H2O2. A solution of 43 mM H2O2was prepared in 0.1 M phosphate buffer (pH 7.4). Curcuminat 15 �g/mL concentration in 3.4 mL phosphate buffer wasadded to 0.6 mL of H2O2 solution (43 mM) and absorbanceof the reaction mixture was recorded at 230 nm. A blanksolution contained the sodium phosphate buffer withoutH2O2. The concentration of hydrogen peroxide (mM) in theassay medium was determined using a standard curve (r2:0.9895):

absorbance = 0.038[H2O2] + 0.4397

The percentage of H2O2 scavenging by curcumin andstandard compounds was calculated using the followingequation:

H2O2 scavenging effect (%) =(

1 − AS

AC

)× 100

where AC is the absorbance of the control and AS is theabsorbance in the presence of curcumin or other scavengers[14,22].

2.6. DPPH• free radical scavenging activity

The total radical scavenging capacity of the tested com-pounds was determined and compared to that of BHA,BHT, �-tocopherol and trolox by using the DPPH•, ABTS•+,DMPD•+ and superoxide anion radical scavenging meth-ods.

The hydrogen atom or electron donation abilities ofsome pure compounds were measured by the bleachingof a purple colored methanol solution of the stable DPPHradical. This spectrophotometric assay uses the stable rad-ical, 1,1-diphenyl-2-picryl-hydrazyl (DPPH•), as a reagent[23]. The method of Blois [24] previously described byGulcin [25] was used with slight modifications in orderto assess the DPPH• free radical scavenging capacity ofcurcumin. The DPPH radical absorbs at 517 nm, but uponreduction by an antioxidant or a radical species its absorp-tion decreases. When a hydrogen atom or electron wastransferred to the odd electron in DPPH•, the absorbanceat 517 nm decreased proportionally to the increases ofnon-radical forms of DPPH [26]. Briefly, a 0.1 mM solu-tion of DPPH• was prepared in ethanol and 0.5 mL ofthis solution was added to 1.5 mL of curcumin solution inethanol at different concentrations (15–45 �g/mL). Thesesolutions were vortexed thoroughly and incubated in thedark for 30 min. A half hour later, the absorbance was mea-sured at 517 nm against blank samples lacking scavenger.A standard curve was prepared using different concen-trations of DPPH•. The DPPH• scavenging capacity wasexpressed as mM in the reaction medium and calculatedfrom the calibration curve determined by linear regression(r2: 0.9845):

absorbance = 9.692[DPPH•] + 0.215

The capability to scavenge the DPPH• radical was calcu-lated using the following equation:

DPPH• scavenging effect (%) =(

1 − AS

AC

)× 100

where AC is the absorbance of the control (0.5 mL, con-taining DPPH• solution without curcumin), and AS is theabsorbance in the presence of curcumin [27,28]. DPPH•

decreases significantly upon exposure to radical scav-engers.

2.7. ABTS radical cation decolorization assay

ABTS also forms a relatively stable free radical, whichdecolorizes in its non-radical form [29]. The spectropho-tometric analysis of ABTS•+ scavenging activity wasdetermined according to the method of Re et al. [30]. Inthis method, an antioxidant is added to a pre-formed ABTSradical solution and after a fixed time period the remain-ing ABTS•+ is quantified spectrophotometrically at 734 nm[14]. ABTS•+ was produced by reacting 2 mM ABTS in H2Owith 2.45 mM potassium persulfate (K2S2O8), stored in thedark at room temperature for 4 h. The ABTS•+ solution wasdiluted to give an absorbance of 0.750 ± 0.025 at 734 nmin 0.1 M sodium phosphate buffer (pH 7.4). Then, 1 mLof ABTS•+ solution was added to 3 mL of curcumin solu-tion in ethanol at different concentrations (15–45 �g/mL).The absorbance was recorded 30 min after mixing and thepercentage of radical scavenging was calculated for eachconcentration relative to a blank containing no scavenger.The extent of decolorization is calculated as percentagereduction of absorbance. For preparation of a standardcurve, different concentrations of ABTS•+ were used. TheABTS•+ concentration (mM) in the reaction medium wascalculated from the following calibration curve, deter-mined by linear regression (r2: 0.9841):

absorbance (�734 nm) = 4.6788[ABTS•+] + 0.199

The scavenging capability of test compounds was calcu-lated using the following equation:

ABTS•+ scavenging (%) =(

1 − AS

AC

)× 100

where AC is absorbance of a control (blank) lacking any rad-ical scavenger and AS is absorbance of the remaining ABTS•+

in the presence of scavenger [13,31].

2.8. Superoxide anion radical scavenging activity

Superoxide radicals were generated by the method ofBeauchamp and Fridovich [32] described by Zhishen et al.[33] with slight modification. Superoxide radicals are gen-erated in riboflavin/methionine/illuminate and assayed bythe reduction of NBT to form blue formazan. All solutionswere prepared in 0.05 M phosphate buffer (pH 7.8). Thephoto-induced reactions were performed using fluorescentlamps (20 W). The concentration of curcumin in the reac-tion mixture was 15 �g/mL. The total volume of the reactionmixture was 3 mL and the concentrations of the riboflavin,methionine and NBT were 1.33 × 10−5, 4.46 × 10−5 and

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30 T. Ak, I. Gulcin / Chemico-Biological Interactions 174 (2008) 27–37

8.15 × 10−8 M, respectively. The reaction mixture was illu-minated at 25 ◦C for 40 min. The photochemically reducedriboflavin generated O2

•− which reduced NBT to formblue formazan. The unilluminated reaction mixture wasused as a blank. The absorbance was measured at 560 nm.Curcumin was added to the reaction mixture, in whichO2

•− was scavenged, thereby inhibiting the NBT reduction.Decreased absorbance of the reaction mixture indicatesincreased superoxide anion scavenging activity. The per-centage of superoxide anion scavenged was calculated byusing the following equation:

O2•− scavenging (%) =

(1 − AS

AC

)× 100

where AC is the absorbance of the control and AS isthe absorbance in the presence of curcumin or standards[34,35].

2.9. Measurement of DMPD•+ scavenging ability

DMPD radical scavenging ability of curcumin was per-formed according to the method of Fogliano et al. [36].DMPD (100 mM) was prepared by dissolving 209 mg ofDMPD in 10 mL of deionized water and 1 mL of this solu-tion was added to 100 mL of 0.1 M acetate buffer (pH 5.3),and the colored radical cation (DMPD•+) was obtainedby adding 0.2 mL of a solution of 0.05 M ferric chloride(FeCl3). The absorbance of this solution, which is freshlyprepared daily, is constant up to 12 h at room tempera-ture. Different concentrations of standard antioxidants orcurcumin (10–30 �g/mL) were added in test tubes and thetotal volume was adjusted with distilled water to 0.5 mL.Ten minutes later, the absorbance was measured at 505 nm.One millilitre of DMPD•+ solution was directly added tothe reaction mixture and its absorbance at 505 nm wasmeasured. The buffer solution was used as a blank sample.The scavenging capability of ABTS•+ radical was calculatedusing the following equation:

DMPD•+ scavenging (%) =(

1 − AS

AC

)× 100

where AC is the absorbance of the initial concentration ofDMPD•+ and AS is absorbance of the remaining concentra-tion of DMPD•+ in the presence of curcumin [37].

2.10. Energy calculation

All calculations were performed by using SPARTAN04software for Windows, version 1.0.3. The optimizationwas performed at semi-empirical AM1 level for neutralmolecules. No symmetry constraints were imposed duringthe optimization process.

2.11. Statistical analysis

The experiments were performed in triplicate. The datawere recorded as mean ± standard deviation and anal-ysed by SPSS (version 11.5 for Windows 2000, SPSS Inc.).One-way analysis of variance (ANOVA) was performedby standard procedures. Significant differences betweenmeans were determined by Dunnett’s multiple range tests,

and p < 0.05 was regarded as significant and p < 0.01 wasvery significant.

3. Results

The ferric thiocyanate method measures the amountof peroxide, which is the primary product of oxidationproduced during the initial stages of oxidation. Curcuminexhibited effective antioxidant activity in the linoleic acidemulsion system. The effects of different concentrations(15–45 �g/mL) of curcumin on lipid peroxidation of linoleicacid emulsion are shown in Fig. 1A and were found tobe 97.3, 98.8 and 99.2%. This activity was greater than45 �g/mL concentrations of BHA (95.5%), �-tocopherol(84.6%) and trolox (95.6%), but similar to BHT (99.7%). It wasreported that curcumin exhibits strong antioxidant activ-ity in other models, comparable to vitamins C and E [37].The auto-oxidation of linoleic acid emulsion without cur-cumin or standard compounds was accompanied by a rapidincrease of peroxides. Consequently, these results clearlyindicated that curcumin had effective and powerful antiox-idant activity.

As can be seen from Fig. 1B, curcumin had effectivereducing power using the potassium ferricyanide reductionmethod when compared to the standards. For the measure-ments of the reductive ability of curcumin, the Fe3+–Fe2+

transformation was investigated using the method ofOyaizu [15]. At different concentrations (15–45 �g/mL),curcumin demonstrated powerful reducing ability (r2:0.9937) and these differences were statistically very signif-icant (p < 0.01). The reducing power of curcumin, BHA, BHT,�-tocopherol and trolox increased steadily with increasingconcentrations of samples. Reducing power of curcuminand standard compounds exhibited the following order:BHA ≈ BHT > curcumin > �-tocopherol > trolox. The resultsdemonstrate the electron donor properties of curcuminfor neutralizing free radicals by forming stable products.In vivo, the outcome of the reducing reaction is to termi-nate the radical chain reactions that may otherwise be verydamaging.

Curcumin had effective ferrous ions (Fe2+) chelatingcapacity. The difference between the 15 �g/mL concentra-tion of curcumin and the control values was statisticallysignificant (p < 0.01, Table 1). In addition, at 15 �g/mLconcentration, curcumin (20 mM) exhibited 56.7 ± 4.2%chelation of ferrous ion. On the other hand, the ferrousion chelating capacities of the same concentrations of BHA(41 mM), BHT (34 mM), �-tocopherol (17 mM) and trolox(30 mM) were found to be 69.9 ± 7.5, 60.0 ± 9.3, 31.3 ± 5.5and 45.2 ± 6.2%, respectively. These results show that theferrous ion chelating effect of curcumin was statisticallysimilar to BHA (p > 0.05) and BHT (p > 0.05) but higher than�-tocopherol (p < 0.05) and trolox (p < 0.05).

The ability of curcumin to scavenge hydrogen peroxideis shown in Table 1 and compared with that of BHA, BHT�-tocopherol and trolox as reference compounds. Hydro-gen peroxide scavenging activity of curcumin at 15 �g/mL(20 mM) was found to be 28.4 ± 3.9%. On the other hand,BHA, BHT, �-tocopherol and trolox exhibited 13.6 ± 3.5,16.7 ± 4.1, 13.6 ± 2.9 and 25.6 ± 3.3% hydrogen peroxidescavenging activity, respectively, at the same concentration.

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T. Ak, I. Gulcin / Chemico-Biological Interactions 174 (2008) 27–37 31

Fig. 1. (A) Total antioxidant activities of different concentrations (15–45 �g/mL) of curcumin and standard antioxidant compounds such as BHA, BHT,�-tocopherol and trolox at the concentration of 45 �g/mL. The antioxidant activity was determined according to the ferric thiocyanate method in linoleicacid system. Five millilitre of linoleic acid emulsion consists of 15.5 �L of linoleic acid, 17.5 mg of Tween-20 as emulsifier, and 5 mL phosphate buffer (pH7.0). (B) Total reductive potential of different concentrations (15–45 �g/mL) of curcumin (r2: 0.9937) and reference antioxidants: BHA, BHT, �-tocopheroland trolox using spectrophotometric detection of the Fe3+–Fe2+ transformations. In the presence of reductants, Fe3+/ferricyanide complex reduces to theferrous form (BHA: butylated hydroxyanisole, BHT: butylated hydroxytoluene; p < 0.05).

These results show that curcumin has an effective hydrogenperoxide scavenging activity. At the above concentration,the hydrogen peroxide scavenging effect of curcumin andfour standard compounds decreased in the order of cur-cumin > trolox > BHT > BHA ≈ �-tocopherol.

DPPH has been widely used to evaluate the freeradical scavenging effectiveness of various antioxidant sub-stances. In the DPPH assay, the antioxidants were ableto reduce the stable radical DPPH to the yellow-coloreddiphenyl-picrylhydrazine. The method is based on thereduction of DPPH in alcoholic solution in the presence ofa hydrogen-donating antioxidant due to the formation ofthe non-radical form DPPH-H in the reaction. DPPH is usu-ally used as a reagent to evaluate free radical scavengingactivity of antioxidants. DPPH is a stable free radical andaccepts an electron or hydrogen radical to become a stablediamagnetic molecule [15].

Fig. 2A illustrates a significant decrease (p < 0.01) inthe concentration of DPPH radical due to the scavengingability of curcumin and the reference compounds. BHA,BHT, �-tocopherol and trolox were used as references forradical scavenger activity. The scavenging effect of cur-cumin and standards on the DPPH radical decreased inthe order of BHA (123 mM) ≥ �-tocopherol (51 mM) ≥ BHT

(102 mM) ≈ curcumin (60 mM) > trolox (90 mM) (67.8, 64.9,62.5, 62.2 and 29.4%, respectively) at the concentrationof 45 �g/mL. DPPH free radical scavenging activity of cur-cumin also increased with increasing concentrations (r2:0.9947). EC50 for curcumin was 34.86 �g/mL. Lower EC50value indicates a higher DPPH free radical scavenging activ-ity.

All the tested compounds exhibited effective radicalcation scavenging activity. As seen in Fig. 2B, curcumin isan effective ABTS•+ radical scavenger in a concentration-dependent manner (15–45 �g/mL, r2: 0.9250). EC50 forcurcumin in this assay was 18.07 �g/mL. There was asignificant decrease (p < 0.01) in the concentration ofABTS•+ due to the scavenging capacity at all curcuminconcentrations. The scavenging effect of curcumin andstandards on ABTS•+ decreased in the order: BHA > BHT > �-tocopherol > curcumin > trolox (100, 97.8, 96.9, 86.3, 79.6and 4.4%, respectively) at the concentration of 45 �g/mL.No significant differences in ABTS•+ scavenging potentialwere found among curcumin, BHA and BHT.

The inhibition by curcumin of superoxide radical gener-ation is higher than that by for �-tocopherol and trolox butlower than BHA and BHT. As seen in Table 1, the inhibitionof superoxide anion radical generation at the concentra-

Table 1Comparison of hydrogen peroxide (H2O2) scavenging activity, ferrous ion (Fe2+) chelating activity, superoxide anion radical (O2

•−) scavenging activity ofcurcumin and standard antioxidant compounds such as BHA, BHT, �-tocopherol and trolox at the concentration of 15 (g/mL (BHA: butylated hydroxyanisole,BHT: butylated hydroxytoluene)

H2O2 scavenging Ferrous ion chelating Superoxide scavenging

Activity (%) P-value Activity (%) P-value Activity (%) P-value

BHA 13.6 ± 3.5 <0.002 69.9 ± 7.5 <0.0004 75.3 ± 6.5 <0.0004BHT 16.7 ± 4.1 <0.002 60.0 ± 9.3 <0.0026 70.2 ± 7.1 <0.0004Trolox 25.6 ± 3.3 <0.0015 45.2 ± 6.2 <0.0035 16.0 ± 1.9 <0.003�-Tocopherol 13.6 ± 2.9 <0.0001 31.3 ± 5.5 <0.002 22.2 ± 3.3 <0.014Curcumin 28.4 ± 3.9 <0.0031 56.7 ± 4.2 <0.0003 42.7 ± 8.1 <0.013

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32 T. Ak, I. Gulcin / Chemico-Biological Interactions 174 (2008) 27–37

Fig. 2. Radical scavenging activity of different concentrations(15–45 �g/mL) of curcumin and compared with BHA, BHT, �-tocopheroland trolox. *Means in row with different superscripts differ signifi-cantly. (A) DPPH free radical scavenging activity (r2: 0.9947; DPPH•:1,1-diphenyl-2-picryl-hydrazyl free radical). (B) ABTS radical scavengingactivity (r2: 0.9250; ABTS•+: 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid). (C) DMPD radical scavenging activity (r2: 0.9974; DMPD•+:N,N-dimethyl-p-phenylenediamine radical).

tion of curcumin was 42.7 ± 8.1%. On the other hand, at thesame concentration, BHA, BHT and �-tocopherol and troloxexhibited 75.3 ± 6.5, 70.2 ± 7.1, 22.2 ± 3.3 and 16.0 ± 1.9%superoxide anion radical scavenging activity, respectively.According to these results, curcumin had higher superox-

ide anion radical scavenging activity than �-tocopherol andtrolox but lower than BHA and BHT.

As shown in Fig. 2C, curcumin was an effective DMPD•+

radical scavenger in a concentration-dependent man-ner (10–30 �g/mL, r2: 0.9974). EC50 for curcumin was34.5 �g/mL. There was a significant decrease (p < 0.05) inthe concentration of DMPD•+ due to the scavenging capac-ity at all curcumin concentrations. The scavenging effect ofcurcumin and standards on DMPD•+ decreased in the order:trolox > BHA > curcumin, which was at the concentration of30 �g/mL, respectively.

4. Discussion

Many studies have been performed on the in vivo andin vitro properties of curcumin in different systems. Cur-cumin with its proven anti-inflammatory and antioxidantproperties has been shown to have several therapeuticeffects. It was shown to be a potent scavenger of a vari-ety of reactive oxygen species including hydroxyl radicals[38] and nitrogen dioxide radicals [39]. It was also shownto inhibit lipid peroxidation in different animal models[38]. Curcumin is an extremely potent lipid-soluble antiox-idant. It positions itself within the cell membrane, whereit intercepts lipid radicals and becomes a phenoxyl radi-cal. Being more polar than curcumin, the phenoxyl radicalmay move to the surface of the membrane, where it may berepaired by any water-soluble antioxidant such as ascorbicacid [40].

Antioxidant mechanisms of curcumin have been stud-ied by laser flash photolysis and pulse radiolysis [41]. Inthat study, it was found that the keto-enol-enolate equilib-rium of the heptadienone moiety of curcumin determinedits physicochemical and antioxidant properties. In neutraland acidic aqueous solutions, the keto form dominates,and curcumin acts as an extraordinarily potent H-atomdonor. Curcumin reacts with the tert-butoxyl radical inacetonitrile solutions. Phenolic antioxidants usually scav-enge free radicals by an electron-transfer mechanism.The electron-donating ability is determined by the one-electron oxidation potential of the parent antioxidants,expressed by definition as the reduction potential of thecorresponding phenoxyl radicals [41]. In another study,the antioxidant activity of curcumin was determined byinhibition of controlled initiation of styrene oxidation[42].

The reduction of chronic diseases, DNA damage,mutagenesis, carcinogenesis and inhibition of pathogenicbacterial growth is often associated with the termination offree radical propagation in biological systems [43]. Antiox-idant capacity is widely used as a parameter for medicinalbioactive components. In this study, the antioxidant activ-ity of curcumin was compared to BHA, BHT, �-tocopheroland its water-soluble analogue trolox.

Lipid peroxidation consists of a series of free radical-mediated chain reaction processes and is associated withseveral types of biological damage. The ferric thiocyanatemethod measures the amount of peroxide, which is theprimary product of lipid oxidation, produced during theinitial stages of oxidation. In this assay, hydroperoxidesproduced from linoleic acid, which autoxidized during the

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experimental period, added to the reaction mixture wereindirectly measured. Ferrous chloride and thiocyanate reactwith each other to produce ferrous thiocyanate by meansof hydroperoxides [44].

It was suggested that the electron donating capacity,reflecting the reducing power of bioactive compounds, isassociated with antioxidant activity [45]. Antioxidants canbe reductants, and inactivation of oxidants by reductantscan be described as redox reactions in which one reac-tion species is reduced at the expense of the oxidationof the other. The presence of reductants, such as antioxi-dant substances in the samples, causes the reduction of theFe3+/ferricyanide complex to the ferrous form. Fe2+ formedcan be monitored by measuring the formation of Perl’sPrussian blue at 700 nm [46]. There are a number of assaysdesigned to measure overall antioxidant activity, or reduc-ing potential, as an indication of a host’s total capacity towithstand free radical stress [47]. The ferric ion reducingantioxidant power assay takes advantage of an electron-transfer reaction in which a ferric salt is used as an oxidant[16]. In this assay, the yellow color of the test solutionchanges to various shades of green and blue depending onthe reducing power of antioxidant samples. The reducingcapacity of a compound may serve as a significant indicatorof its potential antioxidant activity.

Because elemental species, such as ferrous iron (Fe2+),can facilitate the production of ROS, the ability of sub-stances to chelate iron can be a valuable antioxidantproperty. Iron, in nature, can be found as either ferrous(Fe2+) or ferric ion (Fe3+), with the latter form predomi-nating in foods. Ferrous chelation may render importantantioxidative effects by retarding metal-catalysed oxida-tion [11].

Ferrous ion chelating activities of curcumin, BHA, BHT,�-tocopherol and trolox are shown in Table 1. The chelationof ferrous ions by curcumin and standards was determinedaccording to the method of Dinis et al. [19]. Among the tran-sition metals, iron is known as the most important lipidoxidation pro-oxidant due to its high reactivity. The effec-tive ferrous ion chelators may also afford protection againstoxidative damage by removing iron that may otherwiseparticipate in HO• generating Fenton type reactions.

Fe2+ + H2O2 → Fe3+ + OH− + OH•

Ferric ions also produce radicals from peroxidesalthough the rate is 10-fold less than that of ferrous ion [48]and hence curcumin was assessed for its ability to competewith ferrozine for ferrous ion in the solution.

The data shown in Table 1 reveal that curcumin has amarked capacity for iron binding, suggesting that its mainaction as a peroxidation inhibitor may be related to itsiron binding capacity. In this assay, curcumin interferedwith the formation of the ferrous–ferrozine complex, indi-cating that curcumin has chelating activity and is able tocapture ferrous ion with a higher binding affinity than fer-rozine. As depicted in Fig. 3, curcumin may chelate theferrous ion with its hydroxyl and methoxyl groups. It wasreported that compounds with structures containing C–OHand C O functional groups can chelate metal ions. Kazaz-ica et al. demonstrated that flavonoids, such as kaempferol,

Fig. 3. The proposed reaction for chelating of ferrous ions by curcumin.

chelated Cu2+ and Fe2+ through the functional carbonylgroups [49]. The compounds with structures containingtwo or more of the following functional groups: –OH, –SH,–COOH, –PO3H2, C O, –NR2, –S– and –O– in a favorablestructure-function configuration, can show metal chelationactivity [2,50]. The structure of curcumin and its bindingsites for metal chelation is given in Fig. 3. Recently, Fiorucciet al. demonstrated that quercetin-chelated metal ions inthe same way [51].

Biological systems can produce hydrogen peroxide [52].It also is produced from polyphenol-rich beverages underquasi-physiological conditions and it increases in amountwith the incubation time. Hydrogen peroxide can beformed in vivo by several oxidizing enzymes such as super-oxide dismutase. It can cross-membranes and may slowlyoxidize a number of compounds. It is used in the respi-ratory burst of activated phagocytes [52]. The hydrogenperoxide scavenging capacity of curcumin was determinedaccording to the method of Ruch et al. [21] (Table 1). Cur-cumin has effective hydrogen peroxide scavenging activity.It is known that H2O2 is toxic and induces cell death invitro [53]. Hydrogen peroxide can attack many cellularenergy-producing systems. For instance, it deactivates theglycolytic enzyme glyceraldehyde-3-phosphate dehydro-genase [54].

The free radical chain reaction is widely acceptedas a common mechanism of lipid peroxidation. Radicalscavengers may directly react with and quench peroxideradicals to terminate the peroxidation chain reactions andimprove the quality and stability of food products [55].Assays based upon the use of DPPH• and ABTS•+ radicalsare among the most popular spectrophotometric methodsfor determination of the antioxidant capacity of foods, bev-erages and vegetable extracts. Both chromogens and radicalcompounds can directly react with antioxidants. Addition-ally, DPPH• and ABTS•+ scavenging methods have been usedto evaluate the antioxidant activity of compounds due tothe simple, rapid, sensitive, and reproducible procedures[56].

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34 T. Ak, I. Gulcin / Chemico-Biological Interactions 174 (2008) 27–37

ABTS•+ or DPPH• radical-scavenging methods arecommon spectrophotometric procedures for determiningantioxidant capacities of components. When an antiox-idant is added to the radicals, there is a degree ofdecolorization owing to the presence of the antioxidants,which reverses the formation of the DPPH• radical andABTS•+ cation:

DPPH• + AH → DPPH2 + A•

ABTS•+ + AH → ABTS+ + A•

DPPH and ABTS radical scavenging are easy to use, have ahigh sensitivity, and allow for rapid analysis of the antiox-idant activity of a large number of samples. These assayshave been applied to determine the antioxidant activity ofpure components [57]. In this study, three different assayswere used to assess the radical scavenging activities of cur-cumin.

With the DPPH• method it was possible to determinethe antiradical power of an antioxidant by measuringa decrease in the absorbance of DPPH• at 517 nm. Theabsorbance decreased when DPPH• was scavenged by anantioxidant through donation of hydrogen to form a sta-ble DPPH• molecule. In the radical form, this molecule hadan absorbance at 517 nm, which disappeared after accep-tance of an electron or hydrogen radical from an antioxidantcompound to become a stable diamagnetic molecule [58].

As can be seen in Fig. 4, in the keto form of curcumin,the heptadienone linkage between the two methoxyphenolrings contains a highly activated carbon atom. Curcumincan easily abstract a hydrogen atom from this carbonatom. Hydrogen atom abstraction from phenolic ring isvery difficult since curcumin’s phenolic hydrogen atomsare intramolecularly H-bonded to the adjacent methoxygroups. Based on theoretical calculations shows that Bis the most stable one among the intermediates (A–C)(Fig. 4). While the calculated formation energy (�H)for B is −42.05 kcal/mol, this energy was calculated as

39.45 kcal/mol for A and 54.70 kcal/mol for C. DPPH radicalseasily abstract an H-atom from free hydroxyl group whichwas responsible for the “superb antioxidant” properties ofcurcumin. As a consequence, the reaction of DPPH radicalsdiminishes by curcumin in alcoholic media, and the pheno-lic part of curcumin takes over as (electron donor) reactionsite [41]. The electron donating ability of curcumin isassessed from the measurements of one-electron-transferto DPPH radicals. H-atom transfer reactions of curcuminwere also investigated using the tert-butoxyl [(CH3)3CO•]radicals, with same results. Same mechanism observed inthese radicals scavenging. In addition, the H-atom donationfrom the �-diketone moiety to a lipid alkyl or a lipid per-oxyl radical was described as a potentially more importantantioxidant action of curcumin [41].

ABTS•+ radicals are more reactive than DPPH radicalsand unlike the reactions with DPPH radical, which involveH-atom transfer, the reactions with ABTS•+ radicals involvean electron-transfer process. Generation of the ABTS radi-cal cation forms the basis of one of the spectrophotometricmethods that have been applied to the measurement ofthe total antioxidant activity of pure substances, aqueousmixtures and beverages [59]. A more appropriate formatfor the assay is a decolorization technique, in which theradical is generated directly in a stable form prior to reac-tion with putative antioxidants. The improved techniquefor the generation of ABTS•+ described here involves thedirect production of the blue/green ABTS•+ chromophorethrough the reaction between ABTS and potassium per-sulfate. ABTS•−, the oxidant, was generated by potassiumpersulfate oxidation of ABTS2− and the radical cation ismeasured spectrophotometrically. This is a direct genera-tion of a stable form of radical to create a blue-green ABTS•+

chromophore prior to the reaction with antioxidants [52].Bleaching of a preformed solution of the blue-green rad-

ical cation ABTS•+ has been extensively used to evaluatethe antioxidant capacity of complex mixtures and individ-ual compounds. The reaction of the preformed radical withfree radical scavengers can be easily monitored by follow-

Fig. 4. Proposed reaction of DPPH with curcumin (DPPH: 1,1-diphenyl-2-picryl-hydrazyl, DPPH•: 1,1-diphenyl-2-picryl-hydrazyl free radical).

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T. Ak, I. Gulcin / Chemico-Biological Interactions 174 (2008) 27–37 35

ing the decrease of the sample absorbance at 734 nm. TheABTS radical cation can be prepared employing differentoxidants. Results obtained using K2S2O8 as oxidant showthat the presence of peroxodisulphate increases the rate ofABTS•+. ABTS•+ were generated in the ABTS/K2S2O8 system:

S2O82− + ABTS → SO4

2− + SO4−• + ABTS+•

where the scission of the peroxodisulphate could take placeafter the electron transfer. In the presence of excess ABTS,the sulphate radical will react according to the followingequation:

SO4−• + 2ABTS → SO4

2− + + 2ABTS+•

leading to the overall reaction represented by

S2O82− + 2ABTS → 2SO4

2− + + 2ABTS+•

ABTS•+ radicals are more reactive than DPPH radicals and,unlike the reactions with DPPH radicals, which involve H-atom transfer, the reactions with ABTS•+ radicals involveelectron-transfer [60].

Superoxide is an oxygen-centred radical with selectivereactivity. Although a relatively weak oxidant, superoxideexhibits limited chemical reactivity, but can generate moredangerous species, including singlet oxygen and hydroxylradicals, which cause the peroxidation of lipids [61]. Thesespecies are produced by a number of enzyme systems.Superoxide can also reduce certain iron complexes suchas cytochrome c. Superoxide anions are thus precursorsto active free radicals that have potential for reactingwith biological macromolecules and thereby inducing tis-sue damage [62]. Also, superoxide has been observedto directly initiate lipid peroxidation. It has also beenreported that antioxidant properties of some flavonoids areeffective mainly via scavenging of superoxide anion rad-ical [63]. Superoxide radicals are normally formed first,and their effects can be magnified because they produceother kinds of free radicals and oxidizing agents [12].Superoxide anions derived from dissolved oxygen by theriboflavin/methionine/illuminate system will reduce NBTin this system. In this method, superoxide anion reduces theyellow dye (NBT2+) to produce the blue formazan, whichis measured spectrophotometrically at 560 nm. Antioxi-dants are able to inhibit the blue NBT formation [64]. Thedecrease of absorbance at 560 nm with antioxidants indi-cates the consumption of superoxide anion in the reactionmixture. Table 1 shows the inhibition of superoxide radi-cal generation by 15 �g/mL concentrations of curcumin andstandards.

The principle of the DMPD•+ assay is that at acidic pHand in the presence of a suitable oxidant solution, DMPDcan form a stable and colored radical cation (DMPD•+).The UV–visible spectrum of DMPD•+ shows a maximumabsorbance at 505 nm. Antioxidant compounds which areable to transfer a hydrogen atom to DMPD•+ quench thecolor and produce a decoloration of the solution. This reac-tion is rapid and the end point, which is stable, is takenas a measure of the antioxidative efficiency. Therefore, thisassay reflects the ability of radical hydrogen-donors to scav-enge the single electron from DMPD•+ [36].

In contrast to the ABTS procedure, the DMPD•+ methodguarantees a very stable end point. This is particularlyimportant when a large-scale screening is required. It wasreported that the main drawback of the DMPD•+ methodis the fact that its sensitivity and reproducibility dramat-ically decreased when hydrophobic antioxidants such as�-tocopherol or BHT were used. Hence, these standardantioxidant compounds were not used in this antiradicalassay.

5. Conclusion

Curcumin was found to be an effective antioxidant indifferent in vitro assays including: reducing power, DPPH•,ABTS•+, O2

•− and DMPD•+ radical scavenging, hydrogenperoxide scavenging and metal chelating activities whencompared to standard antioxidant compounds such as BHA,BHT, �-tocopherol, a natural antioxidant, and trolox. Fig. 1shows the total antioxidant activity of curcumin, BHA, BHT,�-tocopherol and trolox as determined by the ferric thio-cyanate method in the linoleic acid system, demonstratingthat curcumin had a marked antioxidant effect in linoleicacid emulsion. Reactive radicals scavenging and antioxi-dant activity of curcumin was interpreted as originatingby H-atom abstraction from the free hydroxyl group. Weconcluded that it was H-atom donation from phenolicgroup which was responsible for the “superb antioxi-dant” properties of curcumin. Based on the discussionabove, it can be used for minimizing or preventing lipidoxidation in pharmaceutical products, retarding the forma-tion of toxic oxidation products, maintaining nutritionalquality and prolonging the shelf life of pharmaceuti-cals.

Acknowledgements

This study partially was supported by the Research Fundof Ataturk University. The author is grateful to the ResearchFund of Ataturk University for financial support (Projectno. 2001/35). The authors thank Prof. Glen Lawrence,Department of Chemistry and Biochemistry, Long IslandUniversity, Brooklyn, NY, USA for language correction ofthis manuscript. Also, the authors thank Dr. Mustafa Arık,Department of Chemistry, Faculty of Science and Arts,Ataturk University for theoretical calculations.

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