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Chemical Composition, Antioxidant, andAntibacterial Activities of the EssentialOil and Methanol Extracts of Eucalyptusoleosa LeavesMehdi Rahimi-Nasrabadi a , Seied Mahdi Pourmortazavi b , ShahramNazarian a , Farhad Ahmadi c & Hosein Batooli da Department of Chemistry , Imam Hossein University , Tehran , Iranb Faculty of Material and Manufacturing Technologies , Malek AshtarUniversity of Technology , Tehran , Iranc Department of Medicinal Chemistry, Faculty of Pharmacy ,Kermanshah University of Medical Sciences , Kermanshah , Irand Isfahan Research Center of Natural Sources and Agriculture ,Kashan Station , Kashan , IranAccepted author version posted online: 01 Aug 2011.Publishedonline: 02 Apr 2013.

To cite this article: Mehdi Rahimi-Nasrabadi , Seied Mahdi Pourmortazavi , Shahram Nazarian , FarhadAhmadi & Hosein Batooli (2013) Chemical Composition, Antioxidant, and Antibacterial Activities ofthe Essential Oil and Methanol Extracts of Eucalyptus oleosa Leaves, International Journal of FoodProperties, 16:5, 1080-1091, DOI: 10.1080/10942912.2011.558227

To link to this article: http://dx.doi.org/10.1080/10942912.2011.558227

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International Journal of Food Properties, 16:1080–1091, 2013Copyright © Taylor & Francis Group, LLCISSN: 1094-2912 print / 1532-2386 onlineDOI: 10.1080/10942912.2011.558227

CHEMICAL COMPOSITION, ANTIOXIDANT,AND ANTIBACTERIAL ACTIVITIES OF THE ESSENTIAL OILAND METHANOL EXTRACTS OF EUCALYPTUS OLEOSALEAVES

Mehdi Rahimi-Nasrabadi1, Seied Mahdi Pourmortazavi2,Shahram Nazarian1, Farhad Ahmadi3, and Hosein Batooli41Department of Chemistry, Imam Hossein University, Tehran, Iran2Faculty of Material and Manufacturing Technologies, Malek Ashtar University ofTechnology, Tehran, Iran3Department of Medicinal Chemistry, Faculty of Pharmacy, Kermanshah Universityof Medical Sciences, Kermanshah, Iran4Isfahan Research Center of Natural Sources and Agriculture, Kashan Station,Kashan, Iran

This study was designed to examine the chemical composition of essential oil and in vitroantioxidant and antibacterial activity of the essential oil and methanol extracts of Eucalyptusoleosa F. Muell. The chemical composition of the hydrodistilled essential oil of the leavesof E. oleosa was analyzed by gas chromatography and gas chromatography/mass spec-trometry. The main constituents of the oil were found to be 1,8-cineole (45.1%), α-pinene(14.52%), and α-terpineol (4.35%). The essential oil showed strong antibacterial activityagainst the test microorganisms studied, while polar subfraction of methanol extract hadmoderate antibacterial activity and the nonpolar subfraction of methanol extract did notshow any antibacterial activity. In contrast, the extract showed much better activity thanthe essential oil in antioxidant activity assays employed, e.g., in DPPH systems, the highestradical-scavenging activity was shown by the polar subfraction (15.1 ± 0.7 μg/ml). In thesecond case, the inhibition capacity (%) of the nonpolar subfraction (98.2% ± 1.5) was foundto be the stronger one. In addition, the amounts of total phenol components in the polarsubfraction (186.3 ± 2.1 μg/mg) and the nonpolar subfraction (79.6 ± 1.4) were determined.

Keywords: Eucalyptus oleosa, Essential oil, Antioxidant activity, Antibacterial activity, 1,8-cineole.

INTRODUCTION

Essential oils (EOs) and various extracts from plants are of great interest in industryand scientific research. They can be used as alternative medicine for the treatment of manyinfectious diseases and also they possess antioxidant,[1,2] antibacterial,[1,2] antifungal,[3] andother activities that make them susceptible as natural additives in food and pharmaceuticalindustries.

Received 29 August 2010; accepted 17 January 2011.Address correspondence to Mehdi Rahimi-Nasrabadi, Department of Chemistry, Imam Hossein

University, Tehran, Iran. E-mail: rahiminasrabadi@gmail.com

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ESSENTIAL OIL AND METHANOL EXTRACTS OF EUCALYPTUS OLEOSA 1081

Oxidation of lipid and microbial contamination are the two important causes of fooddeterioration during its processing and storage. In order to prolong the storage stability offoods and to reduce damage to the human body, synthetic antioxidants are mainly used inindustrial processing.[1–3] But side effects of some synthetic antioxidants used in food pro-cessing, such as butylated hydroxyl toluene (BHT) and butylated hydroxyl anisole, havebeen proved. For example, these substances exhibit carcinogenic effects in living organ-isms and are responsible for liver damage.[1,4] Due to these reasons, there is an increasinginterest in extraction of antioxidants from natural sources by various techniques,[5–10] andefforts have been made to identify compounds that can act as appropriate antioxidants andantibacterials to replace synthetic food additives.[11,12]

E. oleasa, which is one of the Myrtaceae families, is native to Australia. Eucalyptus,which includes more than 700 species, is native to Australia with a world-wide distribution.Since the first commercial distillation of Eucalyptus oil, a vast array of Eucalyptus-basedproducts has entered the marketplace, mainly for pharmaceutical, fragrance, and flavoruse. Gums, oils, and resins are some products of Eucalyptus, which are used as flavoringagents, astringents, and aromatics, and formerly to treat diarrhea, asthma, bronchitis, andrespiratory tract infections.[13,14] To the best of knowledge, there is no information on theantioxidant and antibacterial properties of E. oleosa in the literature, except its phytochemi-cal constituents.[15–17] The aim of this work is to evaluate the in vitro antioxidant propertiesof the essential oil and methanol extracts of E. oleosa by DPPH, β-carotene/linoleic acid,and reducing power assays.

MATERIALS AND METHODS

Materials

Plant material and chemicals used. The leaves of E. oleosa were cultivatedin Septembetr 2009 from Kashan (Isfahan province, Iran). The leaves were dried in theshade (at room temperature). A voucher specimen of the plant was deposited at theHerbarium of Kashan botanical garden (KBG 1493). Linoleic acid, 2,6-di-tert-butyl-4-methylphenol (butylated hydroxytoluene, BHT), 2,2-diphenyl-1-picrylhydrazyl (DPPH,95%), gallic acid, and β-carotene were procured from Sigma–Aldrich Chemie (Steinheim,Germany). Analytical grade methanol, ethanol, and dimethyl sulphoxide (DMSO), HPLCgrade chloroform, standard Folin–Ciocalteu’s phenol reagent, anhydrous sodium sulphate,ferric chloride, sodium carbonate, potassium ferricyanide, phosphate buffer solution, andTween 40 were obtained from Merck (Darmstadt, Germany).

Extraction of the Essential Oil

The EOs were extracted by hydrodistillation of dried leaves of plant material for12 h (50 g of sample in 500 mL of distilled water) using a Clevenger-type apparatus asrecommended by British Pharmacopeia.[18] The oils were dried over anhydrous sodiumsulphate and stored in sealed glass vials at 4–5◦C prior to analysis. Yield based on dryweight of the sample was calculated.

Preparation of the Methanol Extract

Thirty grams of the dried and powdered leaves of plant materials were extractedwith methanol by using Soxhlet apparatus at 60◦C for 12 h. The extract was filtered and

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concentrated under vacuum at 40◦C by using a rotary evaporator (Heidolph, Laborota 4000,Schwabach, Germany), yielding a waxy material (2.25 g, 7.5% w/w). This extract was sus-pended in water and extracted with chloroform (4 × 100 mL) to obtain 1.35 g (4.5%) polarand 0.84 (2.8%) nonpolar extracts. The extract was stored in the dark at 4◦C until usedwithin a maximum period of 1 week.

Analysis of the EO

The analytical GC was carried out on a Varian (Walnut Creek, CA, USA) Saturn3400 GC system equipped with flame ionization detectors and a DB-5 capillary fused silicacolumn (30 m × 0.25 mm ID, film thickness of 0.25 μm). The oven temperature was heldat 40◦C for 1 min then programmed at a rate of 3◦C/min to 250◦C and held isothermal for10 min. The carrier gas was nitrogen at a flow rate of 1.1 mL/min; injector temperature:260◦C; detector: 280◦C. Gas chromatography/gas spectrometry analysis of the EO wereperformed using an HP-6890 GC system (Agilent, USA) coupled with a 5973 network massselective detector (Agilent, USA) and was equipped with a HP5-MS capillary fused silicacolumn (60 m, 0.25 mm I.D.; 0.25 μm film thickness [Agilent, USA]). EO solution (1 μL)in hexane (HPLC grade) was injected and analyzed with the column held initially at 40◦Cfor 1 min and then increased to 250◦C with a 3◦C/min heating ramp and subsequently keptat 250◦C for 20 min. Other operating conditions were as follows: carrier gas, He (99.999%);with a flow rate of 1 mL/min; injector temperature, 250◦C; split ratio, 1:50. Mass spectrawere taken at 70 eV. Mass to charge ratio, m/z was in the range of 20–500 amu. Therelative percentage amounts of the separated compounds were calculated from total ionchromatograms by a computerized integrator. Oil constituents were identified by comparinglinear retention indices based on a homologous series of even numbered n-alkanes (C8–C24) (Niles, IL, USA) with those of standard compounds and by comparison with literaturedata and MS data with those of reference compounds (Sigma–Aldrich and Acros Organics)and by MS data obtained from Wiley and NIST libraries.[19,20]

Antioxidant Properties

Scavenging capacity on DPPH radical. The free radical scavenging activitiesof extracts were measured by using 2,2-diphenyl-1-picryl-hydrazyl (DPPH) as describedby Gholivand et al.[21] Various concentrations of the extract or the oil in methanol (3 mL)was added to 1 mL of a 0.5 mM methanol solution of DPPH. The mixture was shakenvigorously and left to stand at room temperature for 60 min in the dark. Then the absorbancewas measured at 517 nm against a blank. Inhibition of free radical, DPPH, in percent (I%)was calculated according to the formula:

I% = [(Ab − As)/Ab) × 100],

where Ab is the absorbance of the control reaction (containing all reagents except the testcompound), and As is the absorbance of the test compound. The sample concentration pro-viding 50% inhibition (IC50) was calculated from the graph of inhibition percentage againstsample concentration. BHT and ascorbic acid were used as positive standards in this test.All experiments were repeated three times.

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ESSENTIAL OIL AND METHANOL EXTRACTS OF EUCALYPTUS OLEOSA 1083

β-Carotene linoleic acid assay. The antioxidant activity was evaluated accordingto the method described by Ahmadi et al.[22] with some modifications. Briefly, 1.5 mL ofβ-carotene solution (1 mg/mL in chloroform), 3 mL of linoleic acid solution (10 mg/mL inchloroform), and 1.0 mL of Tween 40 solution (300 mg/mL in chloroform) were pipettedinto a 250-mL flask. The chloroform was removed by a rotary vacuum evaporator, and150 mL of deionized water was added to the residue and the mixture was shaken to forman emulsion. Three hundred fifty μl of the test sample in methanol (2 mg/mL) was mixedwith 2.5 mL of this reagent, and the emulsion system was incubated for up to 48 h at roomtemperature. The same procedure was repeated with the synthetic antioxidant, BHT, as thepositive control, and a blank containing only 350 μl of methanol. After this incubationperiod, absorbances of the mixtures were measured at 490 nm. Antioxidative capacities ofthe oil and the extract were compared with those of BHT and the blank.

Reducing power. The reducing power was determined according to the method ofGholivand et al.[23] Different concentrations of methanolic extract and oil of the plant inmethanol (1.0 mL) were mixed with 2.5 mL of phosphate buffer (200 mM, pH 6.6) and2.5 mL of 1% potassium ferricyanide, and the mixture was then incubated at 50◦C for20 min. Then, 2.5 mL of trichloroacetic acid (10%) was added to the mixture to stop thereaction; the mixture was then centrifuged at 3000 rpm for 10 min. Next, 2.5 mL from theupper layer was mixed with 2.5 mL of de-ionized water and 0.5 mL of 0.1% ferric chloride,and the absorbance was measured at 670 nm against a blank. The assays were carried out intriplicate and the results were expressed as mean values ± standard deviations. Increasedabsorbance values indicated a higher reducing power.

Determination of total phenolic contents. Total phenolic contents of theextracts were determined using the Folin–Ciocalteu reagent according to the method ofSingleton and Rossi[24] using gallic acid as standard, with some modifications. The extractssolution (0.1 mL) containing 1000 μg of the extracts was mixed with 46 mL of distilledwater in a volumetric flask and 1 mL Folin–Ciocalteu reagent were added, and the flask wasthoroughly shaken. The mixture was allowed to react for 3 min and 3 mL aqueous solutionof 2% Na2CO3 was added. At the end of incubation of 2 h at room temperature, absorbanceof each mixture was measured at 760 nm. The same procedure was also applied to thestandard solutions of gallic acid and a standard curve was obtained. Total phenol contentswere expressed as μg gallic acid equivalents per mg of the extract or the oil. All tests werecarried out in triplicate and gallic acid equivalent values were reported as means ± SD oftriplicates.

Antibacterial Activity

Bacterial strains. The microorganisms used in this study consisted of fourGram-positive (Staphylococcus aureus [ATCC:25923], Staphylococcus epidermidis[ATCC12228], Bacillus cereus [ATCC11778], and Bacillus subtilis [ATCC6633]),and three Gram negative (Escherichia coli [ATCC:25922], Salmonella typhimurium[ATCC19430], and Klebsiella pneumoniae [ATCC13183]) bacteria. The bacterial strainswere grown at 37◦C and maintained on nutrient agar and recovered for testing bysub-culturing in nutrient broth for 24 h.

Disc diffusion assay. The agar disc diffusion method was used as a prelimi-nary assay for testing the antibacterial effect of essential oil and methanol extracts[25]

against seven microorganisms of significant importance. A suspension of the tested bac-teria (0.1 mL of 106 cells per ml) was spread on the solid media plates. Filter paper discs(6 mm in diameter) were individually impregnated with 10 μl of the essential oil or the

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20 mg/mL extracts (200 μg/disc in DMSO) and then placed on the previously inoculatedagar plates. The Petri dishes were kept at 4◦C for 2 h and then incubated at 37◦C for 24 h.Gentamicine (20 μg/disk) was used as the positive control. Negative controls were pre-pared using DMSO. The diameters of the inhibition zones were measured in millimeters.Tests were carried out in triplicate.

Determination of minimum inhibitory concentration (MIC) and mini-

mum bactericidal concentration (MBC). A broth micro dilution method was usedto determine the minimum inhibitory concentration (MIC) and the minimum bactericidalconcentration (MBC).[26] All tests were performed in nutrient broth. The essential oil andextracts were dissolved in 10% dimethylsulphoxide (DMSO). A serial doubling dilutionof oil and extracts was prepared in a 96-well micro titer plate over the range of 7.8–1000μg/ml. In brief, the 96-well plates were prepared by dispensing into each well 95 μl ofnutrient broth and 5 μl of the inoculum (1.5 × 108 cfu mL−1). An aliquot of 100 μl fromthe stock solutions of the essential oil and extracts initially prepared at the concentrationof 1000 μg/ml was added into the first wells. Then, 100 μl from their serial dilutions wastransferred into seven consecutive wells. The last well containing 195 μl of nutrient brothwithout compound and 5 μl of the inocula on each strip was used as the negative con-trol. The final volume in each well was 200 μl. Gentamicine at the concentration range of7.8–1000 μg/ml was prepared in nutrient broth and used as the standard drug for positivecontrol. The plate was covered with a sterile plate sealer. Contents of each well were mixedon a plate shaker at 200 rpm for 40 s and then incubated at appropriate temperatures for24 h. Bacterial growth was determined by absorbance at 600 nm and confirmed by plating5 μl samples from clear wells on nutrient agar medium.

The MIC was defined as the lowest concentration of the essential oil or extracts atwhich the bacteria does not show visible growth. To determine MBC, broth was taken fromeach well and inoculated in nutrient agar for 24 h at 37◦C. The MBC is defined as the lowestconcentration of the essential oil or extract at which inoculated bacteria was totally killed.Gentamicin and 10% DMSO solution served as positive and negative controls, respectively.

RESULTS AND DISCUSSION

Chemical Composition of EO

The hydrodistillation of the leaves of E. oleosa gave light yellowish oil with ayield of 6.7% (w/w). The identified constituents from the leaves of E. oleosa, theirretention indices, and their percentage composition are summarized in Table 1. All thecompounds are arranged in order of their elution from the HP5-MS column. A total of85 compounds have been identified representing around 98.0% of the total oil. Major con-stituents in the volatile oil of E. oleosa were 1,8-cineole (45.1%), α-pinene (14.5%), andα-terpineol (4.3%). Other representative compounds were identified as trans-pinocarveol(3.9%), β-eudesmol (2.2%), and 3-methyl butanal (2.1%). The authors’ results show thatthe oil contains about 18.7% monoterpene hydrocarbons and 68.6% oxygenated monoter-penes. The concentration of sesquiterpene hydrocarbons and oxygenated sesquiterpenes isrelatively low (0.5 and 6.5%, respectively). The oil of E. oleosa consists of 16 monoter-penoids (69.5%) and 5 sesquiterpenoids (7.1%). The essential oil of E. oleosa is rich inmonoterpenoids.

A report from Safaei-Ghomi indicated that the 1,8-cineole (57.89%) was themain constituent of the oil, followed by α-pinene (11.19%), trans-pinocarveol (7.79%),p-cymene (5.86%), and cryptone (2.60%).[27] Also, Safaei-Ghomi et al. identified only

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ESSENTIAL OIL AND METHANOL EXTRACTS OF EUCALYPTUS OLEOSA 1085

Table 1 Essential oil composition of E. oleosa.

Compounda RIb %

3-Methyl butanal 736 2.1Isoamyl acetate 790 trc

NI 890 tralpha-Thujene 930 0.1α-Pinene 939 14.5Camphene 954 0.2Verbenene 968 trNI 971 trSabinine 975 trβ-Pinene 979 1.5β-Myrcene 987 0.3NI 993 tr1-Phellandrene 1003 0.6α-Terpinene 1017 0.11,8-Cineole 1031 45.1γ -Terpinene 1060 0.7α-Terpinolene 1082 0.3Linalool 1097 trIsoamyl valerianate 1106 1.9Amyl isovalerate 1112 0.2Propanoic acid, 2-methyl-, ethyl ester 1115 0.2Fenchol 1117 0.41-Terpineol 1134 0.2α-Campholenal 1126 0.2trans-Pinocarveol 1139 3.9Camphor 1146 0.4exo-Methyl-camphenilol 1150 0.2Neroloxide 1158 tr1,5,7-Octatrien-3-ol, 2,6-dimethyl- 1161 0.3Pinocarvone 1165 1.1Borneol 1169 0.94-Terpineol 1177 1.9Cryptone 1186 1.7α-Terpineol 1189 4.3Myrtenal 1196 0.8NI 1201 0.2trans-Piperitol 1205 trVerbenone 1210 0.1Nd 1215 0.1trans-(+)-Carveol 1217 0.3β-Citronellol 1232 0.82-Methyladamantane 1235 0.3NI 1239 trPropanal, 2-methyl-3-phenyl- 1244 0.6NI 1245 0.1Carvotanacetone 1247 0.3Piperitone 1253 0.5cis-2-Indanone, hexahydro-3a-methyl- 1259 0.42,3-Butanedioldiacetate 1264 trNeral 1268 0.1Phellandral 1270 0.2Thymol 1290 0.1

(Continued)

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Table 1 (Continued).

Compounda RIb %

4-(1-Methylethyl)-benzenemethanol 1294 0.3Carvacrol 1299 trNI 1301 0.1NI 1309 0.5NI 1325 0.11-Methowycarbonyl-2,6-dimethyl-hepta-1,5-diene 1327 0.7Cyclohexyl benzoate 1329 trNI 1331 trNI 1335 trZ-4-Octene-2,7-diol, 2,7-dimethyl 1337 trIsoamyl benzoate 1441 0.2NI 1449 0.3Allo-Aromadendrene 1460 tr2-Methyl-4-(1-methylethyl)-2-cyclohexenone 1463 trNI 1470 0.2Isoamyl phenylacetate 1479 0.2NI 1485 0.1α-Selinene 1498 trNI 1521 trEpiglobulol 1550 0.1NI 1560 0.1(+) Spathulenol 1578 0.4Globulol 1585 1.0Viridiflorol 1593 0.3Nd 1610 0.1Eremophilene 1622 trNd 1625 0.2γ –Eudesmol 1632 0.8NI 1640 0.2β-Eudesmol 1651 2.2α-Eudesmol 1654 1.5Farnesol 1720 0.1NI 1780 trGrouped componentsMonoterpene hydrocarbons 18.7Oxygenated monoterpenes 68.6Sesquiterpene hydrocarbons 0.5Oxygenated sesquiterpenes 6.5Other 3.7Total 98.0

aOrder of elution on HP5-MS.bRetention indices.ctr = trace, less than 0.1%.

22 components in their oil, whereas 85 components were identified in our oil. These differ-ences might have been derived both from harvest time and local, climatic, and seasonalfactors or it may be hypothesized that these samples belong to a different chemotype.However, further investigations are needed to elucidate this hypothesis. According to differ-ent activities of various compounds, these differences between constituents of the essentialoil will be important in nutritional and medicinal uses.

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ESSENTIAL OIL AND METHANOL EXTRACTS OF EUCALYPTUS OLEOSA 1087

Antioxidant Properties

DPPH radical scavenging activity. The effect of antioxidant on DPPH radicalscavenging was thought to be due to their hydrogen donating ability or radical scavengingactivity. When a solution of DPPH is mixed with a substance able to donate a hydrogenatom, it can give rise to the reduced form 1,1-diphenyl-2-picryl hydrazine (non radical)with the loss of violet color. DPPH scavenging activity is usually presented by IC50 value,defined as the concentration of the antioxidant needed to scavenge 50% of DPPH presentin the test solution. Therefore, extract concentrations providing 50% inhibition (IC50) werecalculated and presented in Table 2. Lower IC50 values reflect better DPPH radical scaveng-ing activity. The PS of E. oleosa provided the highest radical scavenging activity with thelowest IC50 value of 15.1 ± 0.7 μg/mL. DPPH scavenging activity of extracts increasedin order of ascorbic acid > BHT > PS > NS > EO. Ascorbic acid and BHT were used asstandards.

β-Carotene linoleic acid assay. The relative anti-oxidative activities (RAAs) ofthe extracts and oil were calculated from the equation, RAA = Asample/ABHT, where ABHT

is the absorbance of the control (BHT) and Asample is the absorbance of the extract or oil.Calculated RAAs of the extract and oil are given in Table 2. Inhibition values of linoleicacid oxidation were estimated as 93.1 ! 0.4%, 98.2 ± 1.5%, and 49.1 ± 0.8% in the presenceof PS, NS, and EO, respectively.

Inhibition of lipid peroxidation by donation of a hydrogen atom is the basis ofβ-carotene/linoleic acid bleaching test. Compounds that contain hydrogen atoms in theallylic/benzylic positions show better activity in this test because of the relatively easyabstraction of atomic hydrogen from these functional groups by peroxy radicals formedunder test circumstances.[28] High bleaching activity of plant extracts in this test may alsobe a consequence of the presence of allylic/benzylic containing compounds.

Reducing power. Different studies have indicated that the electron donationcapacity reflects the reducing power of bioactive compounds in association with antioxidantactivity. Antioxidants can be explained as reducers, and inactivation of oxidants by reducerscan be described as redox reaction in which one reaction species is reduced at the expense ofthe oxidation of the other. Fe3+ reduction is often used as an indicator of electron donatingactivity, which is an important mechanism of phenolic antioxidant action.[29] In the reduc-ing power assay, the presence of antioxidants in the sample would result in the reducingof Fe3+–Fe2+ by donating an electron. An amount of Fe2+ complex can then be monitoredby measuring the formation of Perl’s Prussian blue (Fe4[Fe(CN)6]3) at 670 nm. Increasingabsorbance at 670 nm indicates an increase in reductive ability. At 500 μg/mL, reducingpowers of PS and EO were around 0.941, 0.571, and 0.399, respectively, while a solution

Table 2 Antioxidant activities of E. oleosa essential oil and methanol extract.

SampleDPPH IC20

(μg/mL)β-Carotene bleaching (RAA)

(%)Total phenol contents

(μg/mg)

Essential oil nd 49.1 ± 0.8 —Polar subfraction 15.1 ± 0.7 93.1 ± 0.4 186.3 ± 2.1Nonpolar subfraction 136.0 ± 1.7 98.2 ± 1.5 79.6 ± 1.4Ascorbic acid 5.8 ± 0.3 nd ndBHT 25.4 ± 1.5 100.0 nd

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1088 RAHIMI-NASRABADI ET AL.

0

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0.6

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1

1.2

0 200 400 600 800 1000 1200

Concentration (µg/ml)

Abs

orba

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)

PS

NS

AA

EO

Figure 1 Reducing power of different concentrations of essential oil and methanol extract of E. oleosa comparedto ascorbic acid (spectrophotometric detection of the Fe+3–Fe+2 transformations). PS: Polar subfraction; NS:nonpolar subfraction; AA: ascorbic acid; EO: essential oil.

of 500 μg/mL of ascorbic acid, the positive control used in this test, had a reducing powervalue of 0.942. The reducing power of other concentrations of extracts and essential oils arepresented in Fig. 1. As a result of this study, the methanolic extract and oil were found tobe effective antioxidants in different in vitro assays including β-carotene bleaching, DPPHradical scavenging, and reducing power and can be suggested as a natural additive in foodand pharmaceutical industries.

Total Phenolic Content

The data presented in Table 2 indicates that the total phenolic content in the PS(186.3 ± 2.1 μg/mg as gallic acid equivalents) was higher that non polar ones (79.6 ± 1.4μg/mg). The high phenolic content in PS contributes to its increased antioxidant potentialin comparison to other extracts. Phenolic compounds, biologically active components, arethe main agents that donate hydrogen to free radicals and thus break the chain reactionof lipid oxidation at the first initiation step. This high potential of phenolic compounds toscavenge radicals may be explained by their phenolic hydroxyl groups.[21]

Antibacterial Activity

The in vitro antibacterial activities of E. oleasa EO and extracts were evaluatedagainst the microorganisms employed and their activity potentials were assessed by thepresence or absence of inhibition zones, zone diameters, MIC, and MBC values. Accordingto the results presented in Table 3, the essential oil of E. oleosa had great potential ofantibacterial activity against seven bacteria, and the most activity against Gram-negativeones. When compared to the methanol extracts, the essential oil exhibited stronger andbroader activity.

Based on the results presented in Table 3, the NS did not exhibit antibacterial activ-ity, but the PS showed modest antibacterial activities against E. coli, K. pneumoniae, andS. typhimurium. The EO of E. oleasa exhibited stronger antibacterial activity against themicroorganisms than those of extracts. As can be seen from Table 3, the highest inhibition

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ESSENTIAL OIL AND METHANOL EXTRACTS OF EUCALYPTUS OLEOSA 1089

Table 3 Antibacterial activity of essential oil and methanol extract of E. oleasa.

Methanol sub fractions

Essential oil Polar fraction Non polar fraction Gentamicin

Test bacteria DDa MICb MBCc DD MIC MBC DD MIC MBC DD MIC

S. aureus 11.5 250 250.0 — — — — — — 17.5 10.0S. epidermidis 15 125 250.0 — — — — — — 20.0 5.0E. coli 19 62.5 62.5 14.0 125 250.0 — — — 11.5 17.0K. pneumoniae 15 125 125.0 12.0 250.0 250.0 — — — 8.0 14.0B. subtilis 18 125 125.0 — — — — — — 15.0 20.0B. cereus 10 250 500 — — — — — — 13.0 20.0S. typhimurium 12 250.0 250 10.0 250.0 500.0 — — — 10.5 12.0

aDD: Diameter of inhibition zone including disc diameter of 6 (mm).bMIC: Minimum inhibitory concentration (as μg/mL).cMBC: Minimum bactericidal concentration.A dash (—) indicates no antibacterial activity.

zone (19.0 mm ± 1.1) and lowest MIC value (62.5 μg mL−1) was achieved for E. coli,which shows that this microorganism is the most sensitive to E. oleasa essential oil.

CONCLUSIONS

As a result of the present study, the methanolic extract and the oil were found to beeffective antioxidants and antimicrobials in different in vitro assays and can be suggested asa natural additive in food and pharmaceutical industries. In conclusion, E. oleasa extractsappear to contain compounds with antioxidant and antimicrobial activities. However, thecomponents responsible for the antioxidant and antimicrobial activities of its extracts arecurrently unclear; further works should be performed on the isolation and identificationof the components in the extracts. In addition, the in vivo safety needs to be thoroughlyinvestigated in experimental rodent models prior to its possible application.

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