Antioxidant Properties of Urtica pilulifera Root, Seed, Flower, and Leaf Extract

Tevfik Ozen,1 Zeynep Collu,2 and Halil Korkmaz1

1Department of Chemistry, Faculty of Arts and Sciences, Ondokuz Mayis University, Samsun;2Directorates of Provincial Control Laboratory, Ministry of Agriculture and Rural Affairs, Amasya, Turkey

ABSTRACT This study was conducted to evaluate the antioxidative properties of hydroalcoholic (80%) extracts from

different parts of Urtica pilulifera L. (Family Urticaceae), including leaf (UPL), flower (UPF), seed (UPS), and root (UPR).

Antioxidative activity of the extracts was measured using the ferric thiocyanate method, thiobarbituric acid method,

reductive potential, metal chelating, free radical, superoxide anion radical, and hydrogen peroxide scavenging activity. In

addition, the results were compared with antioxidants such as tert-butylated hydroxyanisole (BHA), tert-butylated hy-

droxytoluene (BHT), and a-tocopherol. Total antioxidant activities of UPS, UPF, UPL, UPR, BHA, BHT, and a-tocopherol

were 88.79%, 85.13%, 86.72%, 78.46%, 81.31%, 76.12%, and 46.28%, respectively. Like the antioxidant activity, the

reducing power and the superoxide anion radical and free radical scavenging activities of UPL, UPF, UPS, and UPR are

concentration dependent. A correlation between higher antioxidant activity and the amount of total phenolics was found in

the extracts.

KEY WORDS: � antioxidant activity � hydroalcoholic extract � phenolic compounds � radical scavenging activity� Urtica pilulifera

INTRODUCTION

Plants are potential sources of natural antioxidants.They absorb the sun’s radiation and generate high levels

of oxygen as a product of photosynthesis. Oxygen is easilyactivated by ultraviolet radiation, and heat from sunlightproduces toxic reactive oxygen species. Plants producevarious antioxidative compounds to counteract these reac-tive oxygen species in order to survive.1 Many antioxidantcompounds naturally occurring in plant sources have beenidentified as free radical or active oxygen scavengers.2 Thesecompounds are used to preserve food quality, mainly by theprevention of oxidative deterioration of constituent lipids.The peroxidation of unsaturated lipids in living organismshas recently received increasing attention in relation to thepossible association between lipid oxidation and a widerange of degenerative diseases, including aging, cancer,diabetes, and cardiovascular diseases.3 Thus, antioxidantsare important inhibitors of lipid peroxidation not only forfood protection but also as a defense mechanism of livingcells against oxidative damage.4

Urtica L. stinging nettle (Family Urticaceae) is a group ofcommon annual and perennial herbs that are well known for

their stinging hairs. Leaves are borne opposite, flowers aregreen with yellow stamens, and male and female flowers areon separate plants. Fruits are achene. The main varieties ofthe Urtica genus are Urtica dioica L., Urtica urens L., Urticapilulifera L., Urtica cannabina L., Urtica membranaceaPoiret, and Urtica kiovensis Rogoff. They have a long historyof use as a herbal remedies.5 Tea and a meal made from theleaves, seeds, roots, and flowers have traditionally been usedas tonics and blood purifiers.6 Urticaceae species are beingconsumed without any report of adverse effect.7 U. piluliferaL. has been used for wound healing,7 as a healer of liverdisease,8 and as a hypoglycemic,6,9 anti-inflammation,10

antitumor,11 antifungal,12 and antimicrobial13 agent.U. pilulifera (Roman nettle), locally named ‘‘Kara Isırgan,’’

is one of the most important traditional natural drugs in Tur-key.6 All parts of the plant bristle with stinging hairs, and itflowers from May to August.14 In Turkish traditional folkmedicine, this plant is commonly used as a remedy for dia-betes mellitis and is quite prominent in the Black Sea region ofTurkey.6,15 Despite the existence of several studies on U. pi-lulifera, there is no adequate information about the individualantioxidant properties of its leaves, flowers, roots, and seeds.16

The purpose of the present study is to evaluate the possibleantioxidant properties of the different parts of U. pilulifera byusing the ferric thiocyanate (FTC) method, thiobarbituric acid(TBA) test, reductive potential, and free radical scavenging,superoxide anion radical scavenging, hydrogen peroxide(H2O2) scavenging, and metal chelating activities.

Manuscript received 9 February 2010. Revision accepted 22 April 2010.

Address Correspondence to: Tevfik Ozen, Department of Chemistry, Faculty of Arts andScience, Ondokuz Mayis University, 55139 Samsun, Turkey, Tel: +90 362 312 1919;Fax: +90 362 457 6081; E-mail: ozentevfik@hotmail.com

JOURNAL OF MEDICINAL FOODJ Med Food 13 (5) 2010, 1224–1231# Mary Ann Liebert, Inc. and Korean Society of Food Science and NutritionDOI: 10.1089=jmf.2009.1303

1224

MATERIALS AND METHODS

Chemicals

Ammonium thiocyanate, ferrous chloride, and 1,1-diphenyl-2-picrylhydrazyl radical (DPPH�) were purchased fromE. Merck (Darmstadt, Germany). NADH, tert-butylatedhydroxyanisole (BHA), tert-butylated hydroxytoluene (BHT),a-tocopherol, trichloracetic acid, Tween-20, nitro blue tetra-zolium (NBT), phenazine methosulfate (PMS), potassiumferricyanide, and linoleic acid were purchased from SigmaChemical Co. (St. Louis, MO, USA). All other chemicals andreagents were of analytical grade or obtained from othercommercial sources in Samsun, Turkey.

Plant material and extraction

U. pilulifera was collected in the Samsun center ofbetween May and June 2006 and authenticated by Prof. Dr.Hamdi G. Kutbay, Department of Biology, Faculty of Artsand Sciences, Ondokuz Mayis University, Samsun. U. pi-lulifera seeds (UPS), flowers (UPF), leaves (UPL), and roots(UPR) were separated from the other parts and dried at roomtemperature. Voucher specimens are deposited in the her-barium of the Faculty of Arts and Sciences, Ondokuz MayisUniversity, with the herbarium code number OMUB 2504.

All samples (UPS, UPF, UPL and UPR; 20 g) werepowdered in a mill and were extracted in a Soxhlet apparatuswith hydroalcoholic solvent (80% ethanol in water) until theextracting solution became colorless. All extracts were fil-tered through Whatman (Maidstone, UK) No. 1 paper. Thefiltrate was collected. The filtrates were frozen and lyophi-lized. All the extracts were placed in a plastic bottle and thenstored at �208C until used.

All antioxidant tests and analyses were performed intriplicate, and results were averaged.

Determination of total phenolic contents

The total phenolic compounds presented in extract ofUPS, UPF, UPL, and UPR were determined with Folin-Ciocalteu reagent using pyrocatechol equivalent as thestandard phenolic compound.17 In brief, 0.1 mL of extract(containing 0.1 mg of extract) was mixed with water(46 mL). Folin-Ciocalteu reagent (1 mL) was added andmixed, thoroughly. A solution of Na2CO3 (3 mL, 2%) wasadded, and the mixture was incubated in a water bath for 2hours. The absorbance was measured at 760 nm. The stan-dard curve was prepared from 0–100mg=mL solutions ofpyrocatechol in ethanol. The concentration of total phenolicin all the extracts was determined as 1.0mg of pyrocatecholequivalent using an equation obtained from the standardpyrocatechol graph, given as:

Absorbance¼ 0:001 · lg of pyroctechol

þ 0:0033 (r2¼ 0:9988)

The results were calculated as mg of pyrocatechol equiva-lents=g of dried plant materials.

Determination of total antioxidant activitywith the FTC method

The antioxidant activities of UPS, UPF, UPL, and UPRwere determined according to the FTC method in a linoleicacid emulsion system.17 A solution of 100mg=mL preparedwith dried extract (1 mL) was added to the reaction mixturecontaining linoleic acid in potassium phosphate buffer(2.5 mL, 0.04 M, pH 7.0). The mixed solution was incubatedat 378C in a glass flask in the dark. After that, 0.1 mL ofsolutions was diluted in ethanol (9.7 mL, 99%), NH4SCN(0.1 mL, 10%), and FeCl2 (0.1 mL, 0.02 M). The peroxidevalue was measured at 500 nm, and the percentage of inhi-bition was determined. High inhibition evidenced by lowabsorbance value shows high antioxidant activity. Thepercentage inhibition of lipid peroxidation was calculatedby the following equation:

Inhibition of lipid peroxidation (%)¼100�([A1=A0] · 100)

where A0 is the absorbance of the control reaction at 500 nmafter a 96-hour incubation and A1 is the absorbance in thepresence of the extracts of U. pilulifera or standard com-pounds.

Determination of total antioxidant activitywith the TBA test

The TBA test was conducted according to the combina-tion of the methods of Kikizaki and Nakatani.18 The samplesfrom the FTC method were also used for the TBA test. Tothe 1 mL of sample solution, trichloracetic acid (2 mL) andTBA (2 mL) were added. The mixture was placed in aboiling water bath at 1008C for 20 minutes. After cooling, itwas centrifuged at 3,000 rpm (Mistral 2000 centrifuge,MSE, Loughborough, UK) for 25 minutes. The absorbanceof supernatant was then measured at 532 nm. A high inhi-bition value, evidenced by lower absorbance, indicatesbetter antioxidant activity. The percentage inhibition of theTBA-malonaldehyde complex was calculated by the fol-lowing equation:

Inhibition of the final peroxide value (%)

¼ 100� ([A1=A0] · 100)

where A0 is the absorbance of the control reaction and A1 isthe absorbance in the presence of the extracts of U. piluliferaor standard compounds.

Determination of total reducing capability

The reducing powers of UPS, UPF, UPL, and UPR weredetermined through the method of Oyaizu.19 The extracts ofUPS, UPF, UPL, and UPR (25–250mg=mL) were mixedwith 2.5 mL of phosphate buffer (0.2 M, pH 6.6) andpotassium ferricyanide (2.5 mL; 1%). The mixture wasincubated at 508C for 20 minutes. Then, a portion of tri-chloracetic acid (10%; 2.5 mL) was added to each mixtureand centrifuged at 2,000 g for 20 minutes. Finally, the

ANTIOXIDANT ACTIVITY OF U. PILULIFERA 1225

supernatants (2.5 mL) were mixed with distilled water(2.5 mL) and FeCl3 (0.5 mL; 0.1%). The absorbance of themixture was measured at 700 nm. Reducing power wasdetermined according to the increasing absorbance values.20

A higher absorbance value indicates a higher reducingcapacity.

Determination of superoxide anion scavenging activity

The measurement of superoxide anion radical scavengingactivity of extracts was based on the method described byNishimiki et al.21 Superoxide radicals are generated inPMS-NADH systems by oxidation of NADH and assayedby the reduction of NBT. One milliliter of extracts andstandards, 1.0 mL of NBT solution (156 mM NBT in 100 mMphosphate buffer, pH 7.4), and 1.0 mL of NADH solution(468 mM in 100 mM phosphate buffer, pH 7.4) were mixed.The reaction was started by adding 100mL of PMS solution(60 mM PMS in 100 mM phosphate buffer, pH 7.4) to themixture. The absorbance of the mixture was measured at560 nm. The decreased absorbance value of the reactionmixture shows the results of superoxide anion scavengingactivity. The percentage inhibition of superoxide anionformation was calculated using the following formula:

Inhibition (%)¼ [(A0�A1)=A0] · 100

where A0 is the absorbance of the control and A1 is theabsorbance when the extracts or standards are used.20

Determination of free radical scavenging activity

The stable free radical scavenging activity was deter-mined by the DPPH� assay, which was performed accordingto the method of Blois,22 wherein the bleaching rate of thestable free radical DPPH� is monitored at a characteristicwavelength in the presence of the sample. In its radical form,DPPH� absorbs at 517 nm, but upon reduction by an anti-oxidant or a radical species, its absorption decreases. TheDPPH� concentration (in mM) in the reaction medium wascalculated from the following calibration curve determinedby linear regression (r2¼ 0.9998):

Absorbance¼ 6:5781 · [DPPH�(mM)]þ 0:058

The capability to scavenge free radical was calculatedusing the following equation:

DPPH� scavenging effect (%)¼ ([A0�A1]=A0) · 100

where A0 is the absorbance of the control reaction and A1 isthe absorbance in the presence of UPS, UPF, UPL, or UPRof extracts.20 The results are also expressed as EC50 values,the effective concentrations of the test samples providing50% scavenging of DPPH� radicals.

Determination of ferrous metal ion chelating activity

The chelating of ferrous ions with UPS, UPF, UPL,UPR, and standards was estimated with the method of

Dinis et al.23 In brief, the samples (extracts or standardantioxidants; 100 mg=mL) were added to a solution of2 mM FeCl2 (0.05 mL). The reaction was initiated withaddition of 5 mM ferrozine (0.2 mL), and the mixture wasshaken vigorously. The inhibition of ferrozine-Fe2þ com-plex formation was monitored by measuring the absor-bance values at 562 nm and calculated using the formulagiven below:

Metal chelating effect (%)¼ ([A0�A1]=A0) · 100

where A0 is the absorbance of the control and A1 is theabsorbance in the presence of the sample of UPS, UPF,UPL, UPR, or standards. A lower absorbance indicatesbetter ferrous ion chelating activity. The results are alsoexpressed as EC50 values, the effective concentrations of thetest samples required to chelate 50% of the ferrous ions.

Determination of H2O2 scavenging activity

The H2O2 scavenging ability of UPS, UPF, UPL, andUPR was determined according to the method of Ruchet al.24 A solution of H2O2 (40 mM) was prepared in phos-phate buffer (100 mM, pH 7.4). The samples (extracts orstandard, 100 mg=mL, 3.4 mL) were added to the H2O2

solution (0.6 mL). The absorbance of the mixture was de-termined at 230 nm. The percentages of H2O2 scavengingof UPS, UPF, UPL, UPR. and standard compounds werecalculated as follows:

Scavenging activity of H2O2 (%)¼ ([A0�A1]=A0) · 100

where A0 is the absorbance of the control at 230 nm and A1 isthe absorbance in the presence of the sample of UPS, UPF,UPL, UPR, or standards.

Statistical analysis

Experimental results were given as mean� SD values ofthe three parallel measurements. The experimental valueswere evaluated by using one-way analysis of variance(Tukey’s test). P values of <.05 were regarded as signifi-cant, and P values of <.01 as very significant.

RESULTS AND DISCUSSION

Yield and total phenolic content of extracts

Urticaceae species have secondary metabolites that aremainly phenolic compounds.6 Phenolic compounds haveantioxidant properties because of their ability to scavengefree radicals and active oxygen species such as singlet ox-ygen, free radicals, and hydroxyl radicals.25 Furthermore,phenolic compounds are known to inhibit mutagenesis andcarcinogenesis in humans when ingested up to 1.0 g from adiet rich in fruits and vegetables.26

The yield percentage and total extractable phenolic con-tent of extracts from different parts of U. pilulifera are givenin Table 1. The amounts of phenolics (in mg) per 100 g of drysamples were calculated for comparison. The extracts from

1226 OZEN ET AL.

all four parts of the plant contained high phenolic contents,but UPR and UPF had the highest amounts, which wasconsistent with previous investigations.12,13,17

The antioxidant activities of extracts were assessed by thefollowing seven assays.

Total antioxidant activity determinationby the FTC method

The antioxidant activities of the hydroalcoholic extracts ofUPS, UPF, UPL, and UPR on the peroxidation of linoleic acidwere investigated. The absorbance values after a 96-hourincubation with linoleic acid emulsion are given in Figure 1.The oxidation of linoleic acid was significantly inhibited bythe UPS, UPF, UPL, and UPR extracts (100mg=mL) incomparison with the control (P< .01). The extracts of UPS,UPF, UPL, and UPR exhibited potent antioxidant activitieswith 88.79%, 85.13%, 86.72%, and 78.46% inhibition oflinoleic acid peroxidation, respectively. UPR was the leastactive in the total antioxidant test. However, the same con-centration of BHA, BHT, and a-tocopherol inhibited lipidperoxidation up to 81.31%, 76.12%, and 46.28%, respec-tively. The results suggested that all parts of U. pilulifera havelipid peroxidation inhibition activity.

Total antioxidant activity determinationby the TBA method

In the TBA method, the formation of malonaldehyde isthe basis for evaluating the extent of lipid peroxidation. Atlow pH and high temperature (1008C), malonaldehyde bindsTBA to form a red complex that can be measured at 532 nm.The increase in the amount of red pigment formed correlateswith the oxidative rancidity of the lipid. Figure 2 shows theantioxidative activities of the hydroalcoholic extracts ofUPS, UPF, UPL, and UPR, measured by using TBA. Allextracts had high antioxidant activities. However, no sig-nificant difference was found among the antioxidant activ-ities of any of the extracts and controls. Results indicatedthat the TBA method supported the results of the FTCmethod. The differences in antioxidative activities observedhere could be ascribed to several factors, including the dif-ferent mechanisms involved in the two determinationmethods, structures of the different phenolic compounds, theantioxidative mechanisms exhibited by the compounds, andpossibly the synergistic effects of different compounds. Theantioxidants present in the samples, which include volatileand fixed oils, may have different functional properties, such

as reactive oxygen species scavenging and inhibition of thegeneration of free radicals.27

Determination of reducing power

The reducing capacity of a compound may serve as asignificant indicator of its potential antioxidant activity.28

For the measurements of reducing power, we investigatedFe3þ–Fe2þ transformation in the presence of BHA, BHT, a-tocopherol, and the hydroalcoholic extracts of UPS, UPF,UPL, and UPR (Fig. 3). The reducing power of UPS, UPF,

Table 1. Percentage Yield of Solvent Extracts and Total

Phenolic Constituents of Different Parts from U. pilulifera

Plant part Yield (%)Total phenolics

of extract (mg=100 g)

U. pilulifera L. seed (UPS) 10.96 87.70� 6.97U. pilulifera L. leaf (UPL) 11.49 85.40� 7.77U. pilulifera L. flower (UPF) 14.11 102.64� 9.80U. pilulifera L. root (UPR) 16.52 125.06� 5.54

FIG. 1. Total antioxidant activities of UPS, UPF, UPL, UPR, tert-butylated hydroxyanisole (BHA), tert-butylated hydroxytoluene (BHT),and a-tocopherol (TOC) at the same concentration (100mg=mL) in thelinoleic acid emulsion system by the ferric thiocyanate method.

FIG. 2. Total antioxidant activities of UPS, UPF, UPL, UPR, BHA,BHT, and TOC at the same concentration (100mg=mL) in the thio-barbituric acid test.

ANTIOXIDANT ACTIVITY OF U. PILULIFERA 1227

UPL, and UPR increased with increasing concentrations ofthe sample. Significant changes in reducing power (P< .01)were observed only after the concentration of the extracts,BHA or BHT, or a-tocopherol increased from 25 to250mg=mL. UPR was the least active in the reducing powertest. The results suggested that the reducing power was de-pendent on concentration. The antioxidant activity of putativeantioxidants are attributed to various mechanisms, amongwhich are the prevention of chain initiation, the binding oftransition metal ion catalysts, the decomposition of perox-ides, the prevention of continued hydrogen abstraction,and radical scavenging.17,29 The order of reducing power isBHA> BHT> a-tocopherol> UPL> UPF> UPS> UPR.

Superoxide anion scavenging activity

The production of highly reactive oxygen species such assuperoxide anion radicals, H2O2, and hydroxyl radicals isalso catalyzed by free iron through the Haber-Weiss reac-tion.30 Superoxide anion, which is a reduced form of mo-lecular oxygen, was implicated in the initiation of oxidationreactions associated with aging.31 It was also implicated inseveral pathophysiological processes, because of its trans-formation into more reactive species, such as hydroxyl rad-ical, which initiate lipid peroxidation. Superoxide was alsoobserved to directly initiate lipid peroxidation.32 In thePMS=NADH-NBT system, superoxide anion derived fromdissolved oxygen by the PMS=NADH coupling reaction re-duces NBT. Antioxidants are able to inhibit formation of blueNBT.33 The decrease in absorbance at 560 nm with antioxi-dants thus indicates the consumption of superoxide anion inthe reaction mixture. Figure 4 presents the superoxide radicalscavenging activity of UPS, UPF, UPL, and UPR extractsat 100mg=mL in comparison with the same dose of theknown antioxidants BHA, BHT, and a-tocopherol. UPR,

UPS, and UPL extracts had strong superoxide radical scav-enging activity, higher than BHA, BHT, and a-tocopherolat 100mg=mL. The results of comparisons were found tobe statistically significant (P< 0.01). Superoxide radicalscavenging activity of the samples followed the order UPR>UPS>UPL> BHA> BHT>UPF> a-tocopherol. UPRis the most active in superoxide radical scavenging activitybecause of its high phenolic content.

H2O2 scavenging activity

H2O2 can be formed in vivo by many oxidizing enzymessuch as superoxide dismutase, cross membranes, and slowlyoxidize a number of compounds. The ability of UPS, UPF,UPL, and UPR to scavenge H2O2 is shown in Figure 4. It wasalso compared with that of BHA, BHT, and a-tocopherol ascontrols. UPS, UPF, UPL, and UPR extracts were capable ofscavenging H2O2 in a concentration-dependent manner. At100mg=mL concentration, hydroalcoholic extracts of UPS,UPF, UPL, and UPR exhibited 63.73%, 68.32%, 57.83%, and70.50% scavenging activity, respectively. The differenceswere not found to be statistically significant. On the otherhand, BHA, BHT, and a-tocopherol showed 21.17%, 30.48%,and 54.23% of H2O2 scavenging activity at the same con-centration, respectively. These results showed that the hy-droalcoholic extracts of UPS, UPF, UPL, and UPR exhibitedmore effective H2O2 scavenging activity than BHT. At aconcentration of 100mg=mL, the H2O2 scavenging effect ofthe extracts of UPS, UPF, UPL, UPR, and standards followedthe order UPR> UPF> UPS>UPL> a-tocopherol> BHT> BHA. UPR is the most active in H2O2 scavenging activitybecause of its high phenolic content. H2O2 itself is not veryreactive; however, it can sometimes be toxic to cells becauseit may produce �OH. Addition of H2O2 to cells in culturecondition can lead to transition metal ion-dependent �OH-mediated oxidative DNA damage.4 Thus, removing H2O2 aswell as O2

.- is very important for the protection of pharma-ceutical and food systems.

FIG. 3. Reducing power of different concentrations (25–250mg=mL)of UPS, UPF, UPL, UPR, BHA, BHT, and TOC measured by themethod of Oyaizu.19

FIG. 4. Comparison of hydrogen peroxide and superoxide anionradical scavenging activities of UPS, UPF, UPL, UPR, BHA, BHT,and TOC at the same concentration (100mg=mL).

1228 OZEN ET AL.

DPPH free radical scavenging activity

DPPH� is a useful reagent for investigating the free radicalscavenging activities of phenolic compounds and a substrateto evaluate the antioxidative activity of antioxidants.34 De-creased DPPH� absorption is an indication of the capacity ofthe extracts to scavenge free radicals, independent of anyenzymatic activity. In this method, it was possible to de-termine the antiradical power of an antioxidant by measur-ing the decrease in the absorbance of DPPH� at 517 nm. Inthe radical form (DPPH�), this molecule had an absorbanceat 517 nm that disappears after the acceptance of an electronor hydrogen radical from an antioxidant compound to be-come a stable diamagnetic molecule.35 Therefore, weevaluated the antioxidant potency through free radicalscavenging with the UPS, UPF, UPL, and UPR extracts orthe known antioxidants a-tocopherol, BHA, and BHT.Figure 5 illustrates a significant decrease (P< .01) in theconcentration of DPPH� radical due to the scavenging abilityof UPS, UPF, UPL, and UPR extracts and standards. BHA,BHT, and a-tocopherol were used as reference radicalscavengers. The scavenging effect of UPS, UPF, UPL, UPR,and controls on the DPPH� radical were in the followingorder: BHA > a-tocopherol > BHT > UPR > UPF > UPL> UPS, with percentage scavenging values of 89.99%,80.00%, 67.46%, 58.52%, 51.38%, 38.62%, and 28.41%,respectively, at the concentration of 250mg=mL. UPR is themost active in DPPH� radical scavenging. However, UPRdoes not have more DPPH� radical scavenging ability thanthat of controls. The EC50 values of UPS, UPF, UPL, UPR,BHA, BHT, and a-tocopherol are 907.1, 495.4, 427.7, 281.0,192.8, 178.1, and 106.2mg=mL, respectively. Free radicalscavenging activity of these samples was also enhanced withincreasing concentration.

Metal ion chelating activity

The production of highly reactive oxygen species is alsocatalyzed by free iron through the Haber-Weiss reaction:O2

� þH2O2 ! O2þOH� þOH�.33 Iron is the most im-portant pro-oxidant of lipids. It is known that the Fe2þ ac-celerates lipid peroxidation by breaking down hydrogen andlipid peroxides formed by the Fenton free radical reac-tion: Fe2þþH2O2?Fe3þþOH�þOH�. Fe2þ ion canform complexes with ferrozine. In the presence of chelatingagents, the complex formation is prevented, resulting in adecrease in the red color of the complex. Measurement ofcolor reduction allows determination of metal chelatingactivity. Measurement of the rate of color decrease allowsthe estimation of the chelating activity of the coexistingchelator.36 In this assay, the extracts of UPS, UPF, UPL,UPR, and control antioxidants interfered with the formationof the ferrous–ferrozine complex, suggesting that it haschelating activity and captures ferrous ion before ferrozine.As shown in Figure 6, formation of the ferrozine–Fe2þ

complex is completed in the presence of extracts, indicatingthat UPS, UPF, UPL, and UPR chelate iron. The results werefound to be statistically significant. At a concentration of250mg=mL, the chelating activity of hydroalcoholic extractsof UPS, UPF, UPL, UPR, and standards were the followingorder: BHA > BHT > a-tocopherol > UPF > UPL > UPR> UPS, with percentage chelations of 88.82%, 88.07%,81.81%, 59.46%, 50.81%, 32.33%, and 22.05%, respec-tively. The EC50 values of UPS, UPF, UPL, UPR, BHA,BHT, and a-tocopherol were 325.7, 630.2, 439.1, 819.2,352.2, 499.5, and 169.2mg=mL, respectively. UPR is themost active in chelating activity because of its high phenoliccontent. It was noted that chelating agents that form s-bondswith a metal are secondary antioxidants due to the reductionof the redox potential. Iron can stimulate lipid peroxidationby the Fenton reaction and also accelerate peroxidation by

FIG. 5. Free radical scavenging activity of different concentrations(25–250 mg=mL) of UPS, UPF, UPL, UPR, BHA, BHT, and TOC for1,1-diphenyl-2-picrylhydrazyl (DPPH).

FIG. 6. Metal chelating activity of different concentrations (25–250 mg=mL) of UPS, UPF, UPL, UPR, BHA, BHT, and TOC againstferrous ions.

ANTIOXIDANT ACTIVITY OF U. PILULIFERA 1229

decomposing lipid hydroperoxides into peroxyl and alkoxylradicals that can themselves abstract hydrogen and perpet-uate the chain reaction of lipid peroxidation.4

CONCLUSIONS

In conclusion, the antioxidant activity of the different partof U. pilulifera was concentration dependent, with strongerinhibition of lipid oxidation occurring at higher concentra-tions of the extracts. According to the results, the possiblemechanisms of the antioxidant activity of UPS, UPF, UPL,and UPR include reducing power, superoxide anion andH2O2 scavenging, and metal chelation. In general, a corre-lation between higher antioxidant activity in the extracts andthe amount of total phenolics was found in the extracts.Phenolic compounds could make a significant contribution tothe antioxidant activity in extracts. The antioxidant activitiesof UPS, UPF, UPL, and UPR extracts were established, and,thus, the chemical characteristics of the antioxidative com-ponents in these extracts will be further investigated.

AUTHOR DISCLOSURE STATEMENT

No competing financial interests exist.

REFERENCES

1. Lu F, Foo LY: Phenolic antioxidant component of evening

primrose. In: Nutrition, Lipids, Health and Disease (Ong ASH,

Niki E, Packer L, eds.). American Oil Chemists Society Press,

Champaign, IL, 1995, pp. 86–95.

2. Duh PD: Antioxidant activity of burdock (Arctium lapa Linne):

its scavenging effect on free radical and active oxygen. J Am Oil

Chem Soc 1998;75:455–465.

3. Yagi K: Lipid peroxides and human disease. Chem Phys Lipids

1997;45:337–341.

4. Halliwell B: Reactive oxygen species in living systems: source,

biochemistry, and role in human disease. Am J Med 1991;

91(Suppl 3C):14–22.

5. Launert E: Edible and Medicinal Plants: Covers Plants in Eur-

ope. Hamlyn Publishing Group Ltd., London, 1981, p. 28.

6. Kavalali GM: Urtica: therapeutic and nutritional aspects of

stinging nettles. In: The Chemical and Pharmacological Aspects

of Urtica (Kavalali GM, ed.). Taylor & Francis, New York, 2003,

pp. 25–39.

7. Ali-Shtayeh MS, Yaniv Z, Mahajna J: Ethnobotanical survey in

the Palestinian area: a classification of the healing potential of

medicinal plants. J Ethnopharmacol 2000;73:221–232.

8. Seeff LB, Lindsay KL, Bacon BR, Kresina TF, Hoofnagle JH:

Complementary and alternative medicine in chronic liver dis-

ease. Hepatology 2001;34:595–603.

9. Kavalali G, Tuncel H, Goksel S, Hatemi H: A study on hypo-

glycemic activity of Urtica pilulifera. In: Proceedings of XIIth

International Symposium on Plant Originated Crude Drugs

(Calıs I, Ersoz T, Basaran AA, eds.). Ankara, 1998, 90pp.

10. Kavalali G, Tuncel H: Anti-inflammatory activities of Urtica

pilulifera. Int J Pharm 1997;35:138–140.

11. Abdel-Kader M, Mahmoud AH, Motawa HM, Wahba HE,

Ebrahim AY: Antitumor activity of Urtica pilulifera on Ehrlich

ascites carcinoma in mice. Asian J Biochem 2007;2:375–385.

12. Ali-Shtayeh MS, Yaghmour RMR, Faidi YR, Salem K, Al-Nuri

MA: Antimicrobial activity of 20 plants used in folkloric

medicine in the Palestinian area. J Ethnopharmacol 1998;

60:265–271.

13. Hussain AIA: Isolation, identification, and determination of anti-

fungal constituents from some medicinal plants in Palestine [M.Sc.

Thesis]. An-Najah National University, Nablus, 1995, p. 75.

14. Davis PH: Flora of Turkey and the East Aegean Islands, Vol. 7.

University Press, Edinburgh, 1982, pp. 633–636.

15. Baytop T: Turkiye‘de Tibbi Bitkiler ile Tedavi (Gecmiste ve

Bugun). Nobel Tip Kitapevleri, Istanbul, 1999, pp. 232–238.

16. Alpinar K, Oezyuerek M, Kolak U, Guclu K, Aras C, Altun M,

Celik SE, Berker KI, Bektasoglu B, Apak R: Antioxidant capa-

cities of some food plants wildly grown in Ayvalik of Turkey.

Food Sci Technol Res 2009;15:59–64.

17. Gulcin I, Kufrevioglu OI, Oktay M, Buyukokuroglu ME: Anti-

oxidant, antimicrobial, antiulcer and analgesic activities of nettle

(Urtica dioica L.). J Ethnopharmacol 2004:90:2005–2015.

18. Kikizaki H, Nakatani N: Antioxidant effects of some ginger

constituents. J Food Sci 1993;58:1407–1410.

19. Oyaizu M: Studies on products of browning reaction: anti-

oxidative activity of product of browning reaction prepared from

glucosamine. Jpn J Nutr 1986;44:307–315.

20. Ozen T, Kınalioglu K: Determination of antioxidant activity of

various extracts of Parmelia saxatilis. Biologia (Section Botany)

2008;63:1–6.

21. Nishimiki M, Rao NA, Yagi K: The occurrence of superoxide

anion in the reaction of reduced phenazine methosulfate and

molecular oxygen. Biochem Biophys Res Commun 1972;46:849–

853.

22. Blois MS: Antioxidant determinations by the use of a stable free

radical. Nature 1985;26:1199–1200.

23. Dinis TCP, Madeira VMC, Almeida LM: Action of phenolic

derivatives (acetaminophen, salicylate and 5-aminosalicylate) as

inhibitors of membrane lipid peroxidation as peroxyl radi-

cal scavenging effects. Chem Pharm Bull 1994;36:2090–2097.

24. Ruch RJ, Cheng SJ, Klaunig JE: Prevention of cytotoxicity and

inhibition of intracellular communication by antioxidant cate-

chins isolated from Chinese green tea. Carcinogenesis 1989;10:

1003–1008.

25. Hall CA, Cuppett SL: Structure-activities of natural antioxidants.

In: Antioxidant Methodology: In Vivo and In Vitro Concepts

(Auroma OI, Cuppett SL, eds.). AOCS Press, Champaign, IL,

1997, pp. 141–170.

26. Tanaka M, Kuei CW, Nagashima Y, Taguchi T: Application of

antioxidative maillrad reaction products from histidine and glu-

cose to sardine products. Nippon Suisan Gakk 1998;54:1409–

1414.

27. Ozbek H, Ozturk M, Ozturk A, Ceylan E, Zabit Y: Determination

of lethal doses of volatile and fixed oils of several plants. East J

Med (Health & Medical Complete) 2004;9:4–6.

28. Meir S, Kanner J, Akiri B, Hadas SP: Determination and in-

volvement of aqueous reducing compounds in oxidative defense

systems of various senescing leaves. J Agric Food Chem 1995;

43:1813–1817.

29. Diplock AT: Will the ‘good fairies’ please proves to us that

vitamin E lessens human degenerative of disease? Free Radic

Res 1997;27:511–532.

30. Haber F, Weiss J: The catalytic decomposition of hydrogen

peroxide by iron salts. Proc R Soc Lond 1934;147:332–351.

1230 OZEN ET AL.

31. Cotelle N, Bemier JL, Catteau JP, Pommery J, Wallet JC, Gay-

dou EM: Antioxidant properties of hydroxylflavones. Free Radic

Biol Med 1996;20:35–43.

32. Wickens AP: Aging and the free radical theory. Respir Physiol

2001;128:379–391.

33. Cos P, Ying LY, Calomme M, Hu JH, Cimanga K, Van Poel B,

Pieters L, Vlietinck AJ, Berghe DV: Structure activity relation-

ships and classification of flavonoids as inhibitors of xanthine

oxidase and superoxide scavengers. J Nat Prod 1998;61:71–76.

34. Duh PD, Tu YY, Yen GC: Antioxidant activity of aqueous ex-

tract of harnjyur (Chrysanthemum morifolium Ramat). LWT Food

Sci Technol 1999;32:269–277.

35. Matthaus B: Antioxidant activity of extracts obtained from res-

idues of different oilseeds. J Agric Food Chem 2002;50:3444–

3452.

36. Yamaguchi F, Ariga T, Yoshimira Y, Nakazawa H: Antioxidant

and antilycation of carcinol from Garcinia indica fruit rind.

J Agric Food Chem 2000;48:180–185.

ANTIOXIDANT ACTIVITY OF U. PILULIFERA 1231