JOURNAL OF MEDICINAL FOOD J Med Food 14 (9) 2011, 975985 # Mary Ann Liebert, Inc.

and Korean Society of Food Science and Nutrition DOI: 10.1089/jmf.2010.0197

Antioxidant Activity of Eugenol: A StructureActivity Relationship Study

_ Ilhami Gulcin1,2
1

Department of Chemistry, Faculty of Sciences, Ataturk University, Erzurum, Turkey. 2 _ School of Health Services, Ibrahim Cecen University, Agri, Turkey.

ABSTRACT Eugenol (4-allyl-2-methoxyphenol), a major phenolic component from clove oil (Eugenia caryophyllata), has several biological activities. To estimate the capacity of eugenol to act as an antioxidant, the following were studied: 1,1diphenyl-2-picryl-hydrazyl, 2,20 -azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), and N,N-dimethyl-p-phenylenediamine scavenging activity; total antioxidant activity; and ability to reduce ferric ions and cupric ions. Eugenol inhibited 96.7% (r2 = 0.9319) lipid peroxidation of a linoleic acid emulsion at a 15-lg/mL concentration. Butylated hydroxyanisole, butylated hydroxytoluene, a-tocopherol, and Trolox displayed 95.4% (r2 = 0.8482), 99.7% (r2 = 0.7798), 84.6% (r2 = 0.9272), and 95.6% (r2 = 0.8511) inhibition of peroxidation, respectively, at the 15-lg/mL concentration. According to the results of this study, eugenol had the most powerful antioxidant activity and radical-scavenging activity. This study should prompt further studies of the antioxidant properties of eugenol. KEY WORDS:  antioxidant activity  eugenol  radical scavenging  reducing power

INTRODUCTION

ugenol (4-allyl-2-methoxyphenol), a methoxyphenol with a short hydrocarbon chain, is the major component (80%95%) of clove oil.1 Eugenol has been used as a spice because of its strong odor and as a dental antiseptic because of its detergent-like effect.2,3 It has been extensively used as a therapeutic agent in dentistry for sedation in patients with toothache, pulpitis, and dental hyperalgesia.4,5 Pharmacologic studies have demonstrated that eugenol has anticonvulsant,6 local anesthetic7 and antistress,8 bacteriostatic and bactericidal,9 anticandidal,10 and antifungal11 properties. In addition, eugenol has antigenotoxic12 and anticarcinogenic13 potential. Eugenol has induced reactive oxygen speciesmediated apoptosis in HL-60 human promyelocytic leukemia cells. It depresses neuromuscular transmission14 and central nervous system function.6 Eugenol prevented radiation-induced chemical oxidative damage in membranes and modied the membrane-associated signaling process after radiation exposure.15 In view of its nonmutagenic and noncarcinogenic properties, eugenol is generally regarded as safe by the Food and Agricultural Organization of the United Nations, with an acceptable daily intake of up to 2.5 mg/kg body weight in humans.16 Reactive oxygen species (ROS), such as superoxide, hydrogen peroxide, and singlet oxygen, and other free radicals,
Manuscript received 30 July 2010. Revision accepted 7 October 2010. _ lc rk Address correspondence to: Ilhami Gu in, Atatu University, Faculty of Sciences, Department of Chemistry, TR-25240-Erzurum, Turkey, E-mail: igulcin@atauni.edu.tr

such as nitrogen free radicals, appear to be generated in the human body because of various internal or external sources. ROS and other free radicals are byproducts of normal cellular metabolism in aerobic life, where molecular oxygen is ubiquitous. ROS are generated during irradiation by ultraviolet light, x-rays, and gamma rays; are products of metal-catalyzed reactions; are present as pollutants in the atmosphere; are produced by neutrophils and macrophages during inammation; and are byproducts of mitochondriacatalyzed electron transport reactions and other mechanisms.17 ROS may contribute to oxidative damage of lipids, protein, nucleic acids, and polyunsaturated fatty acids in living cells. Free radicals or ROS play important roles in the development of many chronic diseases, such as the aging process, heart disease, and cancer.18 It is signicant for health to look for efcient ways to decrease or depress creation of free radicals in the body. Oxidative stress has been dened as an imbalance between the production of ROS and antioxidant defense. Because of the disturbance in the equilibrium state of prooxidant-antioxidant reaction, ROS are overproduced to induce oxidative stress, which inhibits normal functions of cellular lipids, proteins, DNA, and RNA. Thus, increasing attention has been directed toward nding some natural antioxidant compounds that could be isolated from herbal medicine and efciently clear free radicals. Antioxidants are a defensive factor against free radicals effects in the body. The antioxidants not only eliminate ROS but also adjust the cellular redox state and enable redox signal transduction. They act by inhibiting the initiation and propagation steps, leading to the termination of the reaction and delaying the

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oxidation process. The mechanism of antioxidants may involve the scavenging of free radicals.19,20 At present, a variety of synthetic antioxidants are commonly used. However, the use of these compounds has been restricted by legislation because of doubts over their toxic and carcinogenic effects. Plant foods are potential sources of natural antioxidants, such as vitamin C, tocopherol, carotenoids, avonoid, and phenolic acids, that prevent free radical damage. They can provide phenolic hydroxyl groups to react with free radicals. Consequently, they will inhibit the oxidative mechanisms that degenerative diseases. Hence, there is a growing interest in natural and safer antioxidants for food and pharmaceutical applications and a growing trend in consumer preferences toward natural antioxidants. All of this has given impetus to attempts to explore natural sources of antioxidants.2123 This study investigated the following capacities of eugenol: inhibition of lipid peroxidation in the linoleic acid system, ferric ion (Fe3+)reducing antioxidant power (Fe3+Fe3+ transformation), cupric ion (Cu2+)reducing antioxidant power (CUPRAC method); 1,1-diphenyl-2picryl-hydrazyl (DPPH)scavenging, 2,20 -azino-bis(3ethylbenzthiazoline-6-sulfonic acid) (ABTS+)scavenging, N,N-dimethyl-p-phenylenediamine (DMPD+)scavenging, superoxide anion radicalscavenging, and hydrogen peroxide (H2O2)scavenging; and ferrous ion (Fe2+)chelating activities of eugenol. These multiple methods are recommended to measure antioxidant properties of food or pharmacologic materials that better reect their potential protective effects. MATERIAL AND METHODS Chemicals The following compounds used for antioxidant activities were obtained from Sigma (Sigma-Aldrich GmbH): eugenol (4-allyl-2-methoxyphenol), neocuproine (2,9-dimethyl1,10-phenanthroline), DMPD, ABTS, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), DPPH, 3-(2-pyridyl)-5,6-bis (4-phenyl-sulfonic acid)1,2,4-triazine (Ferrozine), linoleic acid, a-tocopherol, polyoxyethylenesorbitan monolaurate (Tween-20), and trichloroacetic acid. Ammonium thiocyanate was purchased from Merck. All other chemicals used were of analytical grade and obtained from Sigma-Aldrich or Merck. The following experimental procedures were applied to evaluate the performance of the eugenol and to establish, if possible, structureactivity relationships. Determination of total antioxidant activity The ferric thiocyanate method was used to evaluate the effect of eugenol on the prevention of peroxidation of linoleic acid emulsion, as described elsewhere.21,24 A stock solution containing 10 mg eugenol was dissolved in 10 mL ethanol. Eugenol (15 lg/mL) was prepared by diluting the stock solution in 2.5 mL sodium phosphate buffer (0.04 M; pH, 7.0), and these were added to 2.5 mL linoleic acid emulsion in

sodium phosphate buffer (0.04 M; pH, 7.0). The linoleic acid emulsion was prepared by homogenizing 15.5 lL linoleic acid, 17.5 mg Tween-20 as emulsier, and 5 mL phosphate buffer (pH, 7.0). The control was composed of 2.5 mL linoleic acid emulsion and 2.5 mL 0.04 M sodium phosphate buffer (pH, 7.0). The reaction mixtures (5 mL) were incubated at 37C in polyethylene asks. The peroxide levels were determined by reading the absorbance at 500 nm in a spectrophotometer (UV-1208 UV-VIS Spectrophotometer; Shimadzu) after reactions with FeCl2 and thiocyanate at intervals during incubation.2527 The peroxides formed during linoleic acid peroxidation oxidize Fe2+ to Fe3+, and Fe3+ forms a complex with thiocyanate that has a maximum absorbance at 500 nm. The assay steps were repeated every 5 hours until the maximum was reached. The percentage of inhibition was calculated at this point (55 hours). The solution without eugenol was used as the blank sample. Linoleic acid mixture without the addition of sample was used as control. The percentage of inhibition of lipid peroxidation in linoleic acid emulsion was calculated by the following equation:   A500 S 100 Inhibition of lipid peroxidation (%) 1 A500 C where A500-C is the absorbance of the control reaction, which contains only linoleic acid emulsion and sodium phosphate buffer and A500-S is the absorbance of sample in the presence of eugenol or other test compounds.21,28 Fe3+-Fe2+reducing assay Ferric-reducing antioxidant power was measured by the direct reduction of Fe3+(CN-)6 to Fe2+(CN-)6 and was determined by measuring absorbance resulting from the formation of the Perls Prussian blue complex after the addition of excess ferric ions (Fe3+). Thus, the ferric-reducing antioxidant power method of Oyaizu29 with slight modications was used to measure the reducing capacity of eugenol.27 This method is based on the reduction of (Fe3+) ferricyanide in stoichiometric excess relative to the antioxidants.22 Briey, 15-lg/mL concentrations of eugenol in 0.75 mL distilled water were mixed with 1.25 mL 0.2 M sodium phosphate buffer (pH, 6.6) and 1.25 mL potassium ferricyanide [K3Fe(CN)6] (1%). The mixture was incubated at 50C for 20 minutes. The reaction mixture was then acidied with trichloroacetic acid (1.25 mL, 10%). Finally, 0.5 mL of FeCl3 (0.1%) was added to this solution, and the absorbance was measured at 700 nm. Increased absorbance of the reaction mixture indicates grater reduction capability.30,31 Cu2+-Cu + reducing-CUPRAC assay To determine the cupric ion (Cu2+)reducing antioxidant capacity of eugenol, the method proposed by Apak et al.32 and clearly described by Karaman et al.33 was used with slight modications. To this end, 0.25 mL CuCl2 solution (0.01 M), 0.25 mL ethanolic neocuproine solution (7.5 10-3 M), and 0.25 mL CH3COONH4 buffer solution

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(1 M) were added to a test tube, followed by mixing with a 45-lg/mL concentration of eugenol. Total volume was then adjusted to 2 mL with distilled water and thoroughly mixed. The tubes were stoppered and kept at room temperature. Absorbance was measured at 450 nm against a reagent blank 30 minutes later. Increased absorbance of the reaction mixture indicates increased reduction capability.34 DPPH -scavenging activity The hydrogen atom or electron-donation ability of eugenol was measured from the bleaching of the purple ethanol solution of DPPH. When a hydrogen atom or electron was transferred to the odd electron in DPPH, the absorbance at 517 nm decreased proportionally to the increases in nonradical forms of DPPH.35 Free radicalscavenging capacity of eugenol was determined and compared with that of BHA, BHT, a-tocopherol, and Trolox (Hoffman-LaRoche) by using the DPPH, ABTS+, DMPD +, and O2- radical scavenging methods. The DPPH solution is deep violet, and radical-scavenging activity of antioxidant compounds can be measured spectrophotometrically at 517 nm by the loss of absorbance as the pale yellow nonradical form (DPPH-H) is produced. The method of Blois,36 previously described by Gulcin,37 was used with slight modications to assess the DPPH free radicalscavenging capacity of eugenol. The DPPH radical shows absorbance at 517 nm, but its absorption decreases upon reduction by an antioxidant or a radical.27 Briey, 0.1-mM solution of DPPH was prepared in ethanol, and 0.5 mL of this solution was added to a 1.5-mL eugenol solution in ethanol at different concentrations (1030 lg/mL). These solutions were vortexed thoroughly and incubated in the dark for 30 minutes. Thirty minutes later, the absorbance was measured at 517 nm against blank samples lacking scavenger. A standard curve was prepared by using different concentrations of DPPH. The DPPH-scavenging capacity was calculated from the calibration curve determined by linear regression (r2 = 0.9848): Absorbance (A517 ) 0:9692 [DPPH ] 10 4 M 0:215

The experiment was carried out by using an improved ABTS decolorization assay.40 In this method, an antioxidant is added to a preformed ABTS radical solution, and after a xed time period, the remaining ABTS+ is quantied spectrophotometrically at 734 nm.41 The ABTS+ was produced by reacting 2 mM ABTS in H2O with 2.45 mM potassium persulfate (K2S2O8) and was stored in the dark at room temperature for 6 hours. The ABTS+ solution was diluted to give an absorbance of 0.750 0.025 at 734 nm in 0.1 M sodium phosphate buffer (pH, 7.4). Then, 1 mL of ABTS+ solution was added to 3 mL of eugenol solution in ethanol at different concentrations (1030 lg/mL). The absorbance was recorded 30 minutes after mixing, and the percentage of radical scavenging was calculated for each concentration relative to a blank-lacking scavenger. The extent of decolorization is calculated as percentage reduction of absorbance. For preparation of a standard curve, different concentrations of ABTS+ (0.0330.33 mM) were used. The ABTS+ concentration (mM) in the reaction medium was calculated from the following calibration curve, determined by linear regression (r2 = 0.9841): Absorbance (A734 ) 4:6788 [ABTS ] 0:199 ABTS+-scavenging capability of test compounds was calculated by using the following equation:   A734-S 100 ABTS scavenging effect (%) 1 A734-C where A734-C is the absorbance of a control lacking any radical scavenger and A734-S is absorbance of the remaining ABTS+ in the presence of scavenger.42,43 The concentration of eugenol providing 50% inhibition (IC50) was calculated from the graph-plotted inhibition percentage against eugenol concentration (lg/mL).37,44,45 DMPD+-scavenging activity Finally, antiradical capacity was analyzed by DMPD+ assay. DMPD radicalscavenging ability of eugenol was performed by using the method of Fogliano et al.,46 with slight modications.47 In the presence of Fe3+, antioxidant compounds can transfer a hydrogen atom to DMPD+, resulting in a decolorization of the solution, measured by the decrease in absorbance at 505 nm. DMPD (100 mM) was prepared by dissolving 209 mg DMPD in 10 mL deionized water, and 1 mL of this solution was added to 100 mL 0.1 M acetate buffer (pH, 5.3). The colored radical cation (DMPD+) was obtained by adding 0.2 mL of a solution of 0.05 M ferric chloride (FeCl3). The absorbance of this solution, which is freshly prepared daily, is constant up to 12 hours at room temperature. Different concentrations of standard antioxidants or eugenol (1030 lg/mL) were added in test tubes, and the total volumes were adjusted to 0.5 mL with distilled water. Ten minutes later, the absorbance was measured at 505 nm. One milliliter of DMPD+ solution was directly added to the reaction mixture, and its

The capability of scavenging the DPPHradical was calculated by using the following equation:   A517-S 100 Scavenged DPPH (%) 1 A517-C

where A517-C is the absorbance of control reaction (containing all reagents except the test compound) and A517-S is the absorbance at 517 nm containing the test compound. The concentration of eugenol providing 50% inhibition (IC50) was calculated from the graph-plotted inhibition percentage against eugenol concentration (lg/mL).36,37 DPPH signicantly decreases upon exposure to radical scavengers.38,39 ABTS+-scavenging activity The ABTS radical cation decolorization test is widely used to assess the antioxidant activity of various substances.

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Table 1. Total Antioxidant Activity (Thiocyanate Method), Fe3+-Fe2+Reducing Ability (Fe3+-Fe2+ Transformation Method), and Cu2+-Cu +Reducing Ability (by CUPRAC Method) of Eugenol and Standard Compounds Total antioxidant activity Compound BHA BHT Trolox a-Tocopherol Eugenol
a b

Fe3+-Fe2+reducing abilitya,b l700a 1.148 0.905 0.140 0.722 1.180 r2 0.9011 0.9764 0.7506 0.9581 0.9677

Cu2+-Cu +reducing abilitya,b l450a 0.640 0.538 0.604 0.534 0.762 r2 0.9214 0.9622 0.9847 0.8886 0.9957

l500a,b 95.4 99.7 95.6 84.6 96.7

r2 0.8482 0.7798 0.8511 0.9272 0.9319

Values are expressed as absorbance. High absorbance indicates high reducing ability. At the same concentration of 45 lg/mL. BHA, butylated hydroxyanisole; BHT, butylated hydroxytoluene.

absorbance was measured at 505 nm. The buffer solution was used as a blank sample. The DMPD+ concentration (mM) in the reaction medium was calculated from the following calibration curve, determined by linear regression (r2 = 0.9993): Absorbance (A505 ) 0:0088 [DMPD ] The scavenging capability of DMPD+ radical was calculated by using the following equation:   A505-S 100 DMPD scavenging effect (%) 1 A505-C where in A505-C is the initial concentration of the DMPD+ and A505-S is absorbance of the remaining concentration of DMPD+ in the presence of eugenol. The concentration of eugenol providing 50% inhibition (IC50) was calculated from the graph-plotted inhibition percentage against eugenol concentration (lg/mL).48,49 Statistical analysis The experimental analyses were performed in triplicate. The data were recorded as means standard deviations and were analyzed by SPSS software, version 11.5 for Windows 2000 (SPSS Inc.). One-way analysis of variance was performed by following the procedures. Signicant differences between means were determined by using Duncan multiple range tests. A P value less than .05 was considered to represent a signicant difference, and a P value less than .01 represented a very signicant difference. RESULTS Antioxidant capacity is dened as the ability of a compound to inhibit oxidative degradation, such as lipid peroxidation.50 Lipid peroxidation can be dened as the oxidative deterioration of lipids containing several carbon carbon double bonds. To determine the possible effects of eugenol, its afnity to restrict the peroxidation of a linoleic acid emulsion was tested. Oxidation of linoleic acid generates linoleic acid hydroperoxides, which decompose to secondary oxidation products. The ferric thiocyanate

method measures the amount of peroxide produced during the initial stages of oxidation, which are the primary products of linoleic acid oxidation. The oxidized products react with ferrous ions to form ferric ions, then to blood-red ferric thiocyanate. In the presence of antioxidants, oxidation of linoleic acid will be slow. As a result, because of the formation of thiocyonate, the color development will be slow. In the present study, eugenol exhibited effective antioxidant activity in the linoleic acid emulsion system. Table 1 and Figure 1 show the effect of 15 lg/mL eugenol on lipid peroxidation of a linoleic acid emulsion: 96.7% (r2 = 0.9319). Conversely, BHA, BHT, a-tocopherol, and Trolox exhibited 95.4% (r2 = 0.8482), 99.7% (r2 = 0.7798), 84.6% (r2 = 0.9272), and 95.6% (r2 = 0.8511) peroxidation

2,5 2 1,5 1 0,5 0

0

Lipid oxidation (500nm)

Control BHA-45 mg/mL BHT-45 mg/mL a-Tocopherol-45 mg/mL Trolox-45 mg/mL Eugenol-15 mg/mL

10

20 30 Incubation time (h)

40

50

FIG. 1. The total antioxidant activity of eugenol (at a concentration of 15 lg/mL) and standard compounds such as butylated hydroxyanisole, butylated hydroxytoluene, a-tocopherol, and Trolox at the same concentration (45 lg/mL) was determined by using the ferric thiocyanate method in the linoleic acid emulsion system. With this method, peroxide formation occurred during the oxidation of linoleic acid emulsion, and these compounds oxidized Fe2+ to Fe3+; Fe3+ forms a complex with SCN, with a maximum absorbance at 500 nm. Control samples contained only linoleic acid emulsion and sodium phosphate buffer. The linoleic acid emulsion was incubated at 37C until control values reached a plateau. BHA, butylated hydroxyanisole; BHT, butylated hydroxytoluene.

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of linoleic acid emulsion at the same concentration, respectively. Peroxidation of linoleic acid emulsion without eugenol or standard compounds was accompanied by a rapid increase in peroxides. Consequently, these results clearly indicate that eugenol had effective and potent antioxidant activity in the ferric thiocyanate assays. Furthermore, eugenol had effective reducing power, as determined by using the potassium ferricyanide reduction and cupric ions (Cu2+)reducing methods when compared with the standards. To measure the reductive ability of eugenol, Fe3+-Fe2+ transformation was investigated in the presence of eugenol by using the method of Oyaizu.29 As shown in Table 1, eugenol demonstrated powerful, statistically signicant Fe3+-reducing ability (r2 = 0.9677; P < .01). The reducing power of eugenol, BHA, BHT, a-tocopherol, and Trolox increased steadily with increasing concentration of samples. The reducing power of eugenol and the standard compounds was as follows: eugenol > BHA > BHT > atocopherol > Trolox. The results demonstrated that eugenol had marked ferric ion (Fe3+)reducing ability and electrondonor properties for neutralizing free radicals by forming stable products. This reducing power was higher than that of the standard antioxidants used. The outcome of the reducing reaction is to terminate the radical chain reactions that may otherwise be very damaging. The putative CUPRAC method developed by Apak et al.32 was used to determine the antioxidant capacity of eugenol by the Cu2+-neocuproine reagent as the chromogenic oxidizing agent. Table 1 shows a cupric ion (Cu2+) reducing ability of eugenol, and a correlation was observed between the cupric ionreducing ability and eugenol concentrations (r2 = 0.9957). Cu2+-reducing capability of eugenol measured by the CUPRAC method was found to be concentration-dependent (1030 lg/mL). Cu2+ ion reducing power of eugenol and standard compounds at the same concentration (45 lg/mL) was as follows: eugenol Trolox BHT > BHA a-tocopherol. In the DPPH assay, the antioxidants reduced the stable radical DPPH to the yellow diphenyl-picrylhydrazine. This method is based on the reduction of DPPH in alcoholic solution in the presence of a hydrogen-donating antioxidant because of the formation of the nonradical form DPPH-H in the reaction. DPPH is usually used as a reagent to evaluate free radicalscavenging activity of antioxidants.29

DPPH is a stable free radical, showing a maximum absorbance at 517 nm. When DPPH radicals encounter a proton-donor substrate such as an antioxidant, the radicals would be scavenged and the absorbance is reduced.51,52 The decrease in absorbance is taken to evaluate the radical scavenging. Table 2 shows the DPPH radicalscavenging activity of eugenol and eugenol benzoate at different concentrations. The results clearly indicate that the eugenol showed high DPPH free radical activity with the lowest IC50 value (16.06 lg/mL; r2 = 0.9823). The IC50 values for standard compounds (BHA, BHT, a-tocopherol, and Trolox) correspond to 25.51 (r2 = 0.9672), 34.01 (r2 = 0.9857), 33.85 (r2 = 0.9962), and 86.93 lg/mL (r2 = 0.7925), respectively. A lower median effective concentration (EC50) value indicates higher DPPH free radicalscavenging activity. The EC50 value depends on the number of DPPH moles reduced by the eugenol during the reaction. DPPH radicalscavenging capacity of these samples decreased in the following order: eugenol > BHA > a-tocopherol & BHT > Trolox. All the tested compounds exhibited effective radical cationscavenging activity. As seen in Table 2, eugenol is an effective ABTS+ radical scavenger in a concentrationdependent manner (1030 lg/mL, r2 = 0.9995). The EC50 value for eugenol in this assay was 7.84 lg/mL. The concentration of ABTS+ decreased signicantly (P < .01) because of the scavenging capacity at all eugenol concentrations (1030 lg/mL). Conversely, EC50 values for BHA, BHT, a-tocopherol, and Trolox were 4.68 (r2 = 0.9958), 9.12 (r2 = 0.8118), 18.14 (r2 = 0.9910), and 22.80 lg/mL (r2 = 0.9410), respectively. The ABTS+-scavenging effect of eugenol and standards decreased in the following order: BHA > eugenol > BHT > a-tocopherol > Trolox. As shown in Table 2, eugenol was an effective DMPD+ radical scavenger in a concentration-dependent manner (10 30 lg/mL; r2 = 0.9886). EC50 was 10.04 lg/mL for eugenol, 63.25 lg/mL for BHA (r2 = 0.9025), and 14.91 lg/mL for Trolox (r2 = 0.8098). The concentration of DMPD+ decreased signicantly (P < .05) because of the scavenging capacity at all eugenol concentrations. The main drawback of the DMPD+ method is that its sensitivity and reproducibility dramatically decreased when hydrophobic antioxidants, such as a-tocopherol or BHT, were used.53 Lower EC50 values for DMPD+ radicalscavenging correspond to highest DMPD+ radicalscavenging capacity.54

Table 2. Concentration Required for 50% Scavenging (IC50) activities of Eugenol and Standard Compounds DPPH Compounds BHA BHT Trolox a-Tocopherol Eugenol IC50a 25.51 34.01 86.93 33.85 16.06 r2 0.9672 0.9857 0.7925 0.9962 0.9823 IC50a 4.68 9.12 22.8 18.14 7.84 ABTS+ r2 0.9958 0.8118 0.9410 0.9910 0.9995 IC50a 63.25 14.91 10.04 DMPD+ r2 0.9025 0.8098 0.9886

a The values are expressed as lg/mL. Lower IC50 values indicate higher radical-scavenging activity. ABTS+, 2,20 -azino-bis(3-ethylbenzthiazoline-6-sulfonic acid radicals; BHA: butylated hydroxyanisole; BHT, butylated hydroxytoluene; DMPD+: N,Ndimethyl-p-phenylenediamine dihydrochloride radicals; DPPH, 1,1-diphenyl-2-picryl-hydrazyl free radicals.

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DISCUSSION Phenolics share the same general structurethey are composed of an aromatic hydroxyl nucleus and exist in an approximated number of 8000 in nature.33 So far, plant phenolics constitute one of the major groups of compounds acting as primary antioxidants or free radical terminators. Plant polyphenols are multifunctional in the sense that they can act as reducing agents, hydrogen atom donators, and singlet oxygen scavengers. Certain polyphenols are also effective as antioxidants capable of chelating transition metal ions, which may otherwise induce Fenton-type oxidation reactions in their free states.33,55 Eugenol, a major phenolic component from clove oil (Eugenia caryophyllata), has demonstrated several biological activities, such as anti-inammatory activity by inhibiting the enzyme cyclooxygenase-II,56 analgesic activity due to selective binding at the capsaicin receptor, anti-oxidation activity,57 and antibacterial activity against both gram-positive and gram-negative microorganisms.58,59 In addition, it has been widely used as a fragrant and avoring agent in a variety of food and cosmetic products.6062 However, at high concentrations, eugenol has adverse effects, including inammatory and allergic reactions, possibly due to the formation of phenoxyl radicals and, subsequently, of quinine intermediates, via its pro-oxidant activity.63 Eugenol inhibits neuronal excitotoxic or oxidative injury and has protective effects against N-methyl-D-aspartate induced neurotoxicity. The effect of eugenol on lipid peroxidation and oxidation of low-density lipoprotein was studied.6466 It also protected in vivo inhibition of propagation of lipid peroxidation.67 Eugenol reportedly protected nicotine-induced superoxide-mediated oxidative damage in murine peritoneal macrophages in vitro. In a recent study, eugenol inhibited the activity and gene expression of inducible cyclooxygenase in lipopolysaccharide-activated mouse macrophage cells.68 In addition, inhibition of xanthine oxidase by eugenol was reported by Rajakumar and Rao69 and Jeng et al.70 The present study shows the antioxidant and radicalscavenging mechanism of eugenol by using different in vitro bioanalytical methods. As reported in many studies, the activities of natural antioxidants in inuencing diseases are closely related to their ability to reduce DNA damage, mutagenesis, carcinogenesis, and inhibition of pathogenic bacterial growth.71 Antioxidant capacity is widely used as a measure for medicinal bioactive components. In this study, the antioxidant and radical-scavenging activities of eugenol were compared with those of BHA, BHT, a-tocopherol, and its water-soluble analogue Trolox. These comparisons were made by using a series of in vitro tests, including DPPH scavenging, ABTS+ scavenging, DMPD+ scavenging, total antioxidant activity (by the ferric thiocyanate method), and reducing power (by 2 methods). Various antioxidant assay methods have been developed for food and biological samples. Lipid peroxidation in biological systems has long been thought to be a toxicologic phenomenon that can lead to various pathologic conse-

quences.72 The resulting lipid hydroperoxides can affect membrane uidity and the function of membrane proteins. In addition, lipid hydroperoxides can undergo ironmediated, 1-electron reduction, and oxygenation to form epoxyallylic peroxyl radicals. These radicals, in turn, trigger a chain reaction of free radicalmediated lipid peroxidation. The end products of lipid peroxidation are reactive aldehydes, such as 4-hydroxylnonenal and malondialdehyde, many of which are highly toxic to cells.73 In addition, reactive aldehydes generated by lipid peroxidation can attack other cellular targets, such as proteins and DNA, thereby propagate the initial damage in cellular membranes to other macromolecules. Because lipid hydroperoxides formed in membranes are important components of ROS generation in vivo, their detoxication appears to be critical for the survival of an organism in oxidative stress.74 Therefore, antioxidants play a vital role in inhibition of lipid peroxidation or in protection against cellular damage by free radicals. Lipid oxidation consists of a series of free radical mediated chain reaction processes and is associated with several types of biological damage. The ferric thiocyanate method measures the amount of peroxide produced during the initial stages of oxidation, which is the primary product of lipid oxidation. In this assay, the amount of hydroperoxides produced from linoleic acid emulsion by autooxidation was indirectly measured during the experiment period. Ferrous chloride and thiocyanate then react with each other to produce ferrous thiocyanate by means of hydroperoxides.7577 On the other hand, reducing power reects the electrondonating capacity of bioactive compounds and is associated with antioxidant activity. Antioxidants can be reductants and inactivate oxidants. The reducing capacity of a compound can be measured by the direct reduction of Fe[(CN)6]3 to Fe[(CN)6]2. Addition of free Fe3+ to the reduced product leads to the formation of the intense Perls Prussian blue complex, Fe4[Fe(CN-)6]3, which has a strong absorbance at 700 nm. An increase in absorbance of the reaction mixture would indicate an increase in the reducing capacity due to an increase in the formation of the complex. Many assays are designed to measure overall antioxidant activity, or reducing potential, as an indication of a hosts total capacity to withstand free radical stress. The ferric ion reducing antioxidant power assay takes advantage of an electron transfer reaction in which a ferric salt is used as an oxidant.20,47 In this assay, the yellow of the test solution changes to various shades of green and blue depending on the reducing power of antioxidant samples. The reducing capacity of a compound may serve as a signicant indicator of its potential antioxidant activity.23,28 The present study used the CUPRAC assay, which is based on reduction of Cu2+ to Cu + by eugenol. This method is simultaneously cost-effective, rapid, stable, selective, and suitable for a variety of antioxidants, regardless of chemical type or hydrophilicity. Moreover, it was reported that the results obtained from in vitro cupric ion (Cu2+)reducing measurements might be more efciently extended to the

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possible in vivo reactions of antioxidants. CUPRAC chromogenic redox reaction is carried out at a pH (7.0) close to the physiologic pH, and the method is capable of measuring thiol-type antioxidants, such as glutathione, and nonprotein thiols, unlike the widely applied ferric-reducing antioxidant power test, which is nonresponsive to -SH group antioxidants.33,78 It has been reported that the main mechanism of action of phenolic antioxidants was considered to be the scavenging of free radicals, although other mechanisms may be involved.79 The radical-scavenging activity of phenolics depends on structural characteristics that favor phenolic hydrogen donation and the stability of the resulting phenoxyl radicals. Only when at least a second phenol group is attached to this chain (e.g., rosmarinic acid) does its contribution becomes signicant. In addition, extension of the conjugation to the carbon chain is a molecular feature that requires some attention because it could participate by resonance to the stabilization of the phenoxyl radical.79 The free radical chain reaction is widely accepted as a common mechanism of lipid peroxidation. Radical scavengers may directly react with and quench peroxide radicals to terminate the peroxidation chain reactions and improve the quality and stability of food products.80 Assays based on the use of DPPH, ABTS+, and DMPD+ radicals are among the most popular spectrophotometric methods for determination of the antioxidant capacity of foods, beverages, and vegetable extracts. Both chromogens and radical compounds can directly react with antioxidants. Additionally, DPPH- and ABTS+-scavenging methods have been used to evaluate the antioxidant activity of compounds because of the simple, rapid, sensitive, and reproducible procedures.81 In this study, 3 radical-scavenging methods were used to assess the potential radical-scavenging activities of eugenol: DPPH, ABTS+, and DMPD+. With these methods, it is possible to determine the antioxidant power of an antioxidant by measuring a decrease in the absorbance of DPPH at 517 nm. The structure of eugenol provides a chromophoric system that leads to interference in the DPPH method used in this study with the 517 nm wavelength, as described earlier. The absorbance is decreased when DPPH is scavenged by an antioxidant through donation of hydrogen to form a stable DPPH radical molecule. In the radical form, this molecule has an absorbance at 517 nm, which disappears after acceptance of an electron or hydrogen radical from an antioxidant compound to become a stable diamagnetic molecule.82 Free radicalscavenging is one of the known mechanisms by which antioxidants inhibit lipid oxidation. This test is a standard assay in antioxidant activity studies and offers rapid screening of the radical-scavenging activity of specic compounds. The antioxidants are believed to intercept the free radical chain of oxidation and donate hydrogen from the phenolic hydroxyl groups, thereby forming a stable endproduct that does not initiate or propagate further oxidation of lipid.80 Reactions with DPPH radicals have been reported in the literature. Gulcin suggested the best-known mechanism for L-carnitine and the DPPH radical.28 L-carnitine has

a carbonyl group, which stabilized a radical formed on acarbon with conjugation in the L-carnitine molecule. This carboxylate group also had a carbonyl unit. An abstraction of a hydrogen atom from C2 may occur easily. Kawabata et al. reported the formation of dimers from gallic and protocatechuic acids after reaction with DPPH radical.83 Brand-Williams et al.84 and Bondet et al.85 studied the possible mechanism of DPPH with BHT, eugenol, and isoeugenol and proposed the intervention of a more complex reaction mechanism, eventually involving dimeric species. Dehydrodiisoeugenol occurred via oxidative coupling of eugenol.86 To conrm observations made on the role of the side chain, a group of 3 monophenols isoeugenol, dihydroeugenol, and eugenoldiffering in 1 double bond or its position in the chain, was also known. In addition, conjugation seemed to enhance the antioxidant and radical-scavenging activity of eugenol.79 It is accepted that the scavenging mechanism of reaction is abstracting a hydrogen atom from a phenol donor to give DPPH-H and a phenoxy radical.87 It was reported that eugenol reduces 2 or more DPPH radicals, despite the availability of only 1 hydrogen from a hydroxyl group, and different hypotheses have been proposed to explain the antiradical efciencies of the different monophenolic compounds.87,88 In another study, coantioxidant behavior of some relevant phenols, such as eugenol and isoeugenol, was studied. Synergism, implying regeneration of a-tocopherol by the coantioxidant, was observed with combination of a-tocopherol/eugenol with peroxy radicals.89 The dimers with 2 phenolic hydroxyl groups of A, B, and C were present at a very low amount compared with C. All these compounds can be originated from the C8-C8 and C5C5 coupling processes, as hypothesized in Figure 2.86 Eugenol is believed to have an aromatic ring. This phenolic group stabilized a radical formed on a-carbon with conjugation in the eugenol molecule. As can be seen in Figure 2, eugenol scavenged the radical on this aromatic ring. Similarly, the structure of resveratrol provides a chromophoric system that leads to interference in DPPH. It is well known that phenolic groups stabilize a radical formed on phenolic carbon with their resonance structure. Resveratrol has 2 phenolic rings: monophenol and diphenol. An abstraction of a hydrogen atom from monophenolic hydroxyl group may occur easily.23 ABTS+ is applicable for both lipophilic and hydrophilic compounds. ABTS+ radicals are more reactive than DPPH radicals, and unlike the reactions with DPPH radical, which involve H atom transfer, the reactions with ABTS+ radicals involve an electron-transfer process. Generation of the ABTS radical cation forms the basis of one of the spectrophotometric methods that have been applied to measure the total antioxidant activity of pure substances, aqueous mixtures, and beverages.47 A more appropriate format for the assay is a decolorization technique in which the radical is generated directly in a stable form before the reaction with putative antioxidants. ABTS+ was generated by oxidation of ABTS with potassium persulfate. This assay is based on the inhibition of the

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OH
4 3 5

OCH3

2
2 1 7

8 9

Eugenol

DPPH

DPPH-H

O OCH3

O OCH3

O OCH3

O OCH3

Eugenyl intermediate radicals

OH H3CO O HO OCH3 OCH 3

OH OCH3 H3 CO HO OH OCH3

2 Dehydrodieugenol

FIG. 2.

The proposed mechanism between eugenol and DPPH radicals and formation of dehydrodieugenol.

absorbance of the radical cation ABTS+, which has a characteristic long-wavelength absorption spectrum showing absorption at 734 nm. The reaction with ABTS+ was quite fast, and in almost all cases was completed in 0.25 to 0.5 min. Bleaching of a preformed solution of the blue-green radical cation ABTS+ has been extensively used to evaluate the antioxidant capacity of complex mixtures and individual compounds. The reaction of the preformed radical with free radical scavengers can be easily monitored by following the decay of the sample absorbance at 734 nm.20,35

The principle of the DMPD+ assay is that DMPD can form a stable and colored radical cation (DMPD+) at an acidic pH and in the presence of a suitable oxidant solution. The ultraviolet visible-light spectrum of DMPD+ shows a maximum absorbance at 505 nm. Antioxidant compounds, which can transfer a hydrogen atom to , DMPD+ quench the color, and produce a decolorization of the solution. This reaction is rapid, and the end point, which is stable, is taken as a measure of antioxidative efciency. Therefore, this assay reects the ability of radical

ANTIOXIDANT ACTIVITY OF EUGENOL

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hydrogen donors to scavenge the single electron from DMPD+.27,46 Preliminary experiments show that the choice of oxidant solution and the ratio between the concentration of DMPD+ and the concentration of the oxidative compound is crucial for the effectiveness of the method. In fact, formation of radical cation is very slow and results in a continuous increase of the absorbance. The best results were obtained with FeCl3, which gives a stable colored solution up to a nal concentration of 0.1 mM. Moreover, this method ensures low cost and highly reproducible analysis.26 The DMPD assay is particularly suitable for hydrophilic antioxidants but is less sensitive to hydrophobic bioactive compounds; the opposite applies for the other 2 tests. In contrast to the ABTS procedure, the DMPD+ method guarantees a very stable end point. This is particularly important when a large-scale screening is required. It has been reported that the main drawback of the DMPD+ method is that its sensitivity and reproducibility dramatically decreased when hydrophobic antioxidants, such as a-tocopherol or BHT, were used. Hence, these standard antioxidant compounds were not used in this antiradical assay. CONCLUSION The principle of antioxidant activity is based on the availability of electrons to neutralize any free radicals. In addition, antioxidant activity is related to the number and the nature of the hydroxylation pattern on the aromatic ring. It is generally assumed that the ability to act as hydrogen donor and the inhibition of oxidation are enhanced by the increase in the number of hydroxyl groups in the phenol ring. Eugenol has an aromatic ring. In this study, Fe3+-Fe2+ and Cu2+Cu +reducing abilities and DPPH-, ABTS+-, and DMPD+-scavenging activities were adopted to evaluate the antioxidant activities of eugenol at different concentrations. The results were compared with BHA, BHT, a-tocopherol, and Trolox, which were used as standard antioxidants. In addition, the possible mechanisms between antioxidant activity of eugenol and its structure were claried.

3.

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14. 15.

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ACKNOWLEDGMENT
This study was partially supported by the Research Fund of the Ataturk University. The author would like to express gratitude to the Research Fund of the Ataturk University for the funding of Project (BAP-2008/75). AUTHOR DISCLOSURE STATEMENT The author reports no conicts of interest. REFERENCES
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This article has been cited by: 1. Yasin etinkaya, Hlya Ger, Abdullah Menzek, #lhami Glin. 2011. Synthesis and Antioxidant Properties of (3,4Dihydroxyphenyl)(2,3,4-trihydroxyphenyl)methanone and Its Derivatives. Archiv der Pharmazie n/a-n/a. [CrossRef]