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Antimicrobial and Antioxidant Activity of the EssentialOil of the Turkish Endemic Species Achillea phrygiaBoiss. & Bal.Adnan Akcina, Tülay Aytas Akcinb, Fatih Seyisc, Ahmet Yilmaz Coband & Belma Durupinard

a Department of Biology, Faculty of Arts and Sciences, Amasya University, 05100, Amasya,Turkeyb Department of Biology, Faculty of Arts and Science, Ondokuz Mayis University, 55139,Kurupelit, Samsun, Turkeyc Department of Field Crops, Faculty of Agronomy and Natural Sciences, Recep TayyipErdogan University, 53300, Rize, Turkeyd Department of Microbiology, Faculty of Medicine, Ondokuz Mayis University, 55139Kurupelit, Samsun, TurkeyPublished online: 23 May 2014.

To cite this article: Adnan Akcin, Tülay Aytas Akcin, Fatih Seyis, Ahmet Yilmaz Coban & Belma Durupinar (2014) Antimicrobialand Antioxidant Activity of the Essential Oil of the Turkish Endemic Species Achillea phrygia Boiss. & Bal., Journal of EssentialOil Bearing Plants, 17:2, 219-227, DOI: 10.1080/0972060X.2014.895179

To link to this article: http://dx.doi.org/10.1080/0972060X.2014.895179

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Antimicrobial and Antioxidant Activity of the Essential Oilof the Turkish Endemic Species Achillea phrygia Boiss. & Bal.

Adnan Akcin 1*, Tülay Aytas Akcin 2 , Fatih Seyis 3,Ahmet Yilmaz Coban 4 and Belma Durupinar 4

1Department of Biology, Faculty of Arts and Sciences,Amasya University, 05100, Amasya, Turkey

2 Department of Biology, Faculty of Arts and Science,Ondokuz Mayis University, 55139, Kurupelit, Samsun, Turkey

3 Department of Field Crops, Faculty of Agronomy and Natural Sciences,Recep Tayyip Erdogan University , 53300, Rize, Turkey

4 Department of Microbiology, Faculty of Medicine,Ondokuz Mayis University, 55139 Kurupelit, Samsun, Turkey

Abstract: The essential oil obtained from the dried flowering aerial parts of Achillea phrygia Boiss &Bal. by hydrodistilation was analysed by gas chromotography-mass spectrometry. Camphor (35.55 %), 2-furaldehyde (16.59 %) and 1,8-cineol (eucalyptol) (10.12 %) were detected as the major components of theessential oil. Essential oil of the plant was also tested for antimicrobial activity using the disc-diffusion methodagainst 6 reference bacterial strains and 65 clinical bacterial isolates. Essential oil of A. phrygia showedantimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA), methicillin-sensitive S.aureus (MSSA), Acinetobacter baumannii and bacterial strains as S. aureus ATCC 25923, S. aureus ATCC43300, Acinetobacter baumannii ATCC 19606. In additionally, the essential oil were not effective againstbacterial strains as Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922, Enterococcus faecalisATCC 29212. Morover, the total antioxidant capacity and the scavenging effect on 2,2diphenil-1-picrylhydrazyl(DPPH) radicals of the essential oil were also evaluated. The radical scavenging activity of A. phrygia essentialoil was 40.43 %.

Key words: Achillea phrygia, essential oil, antimicrobial activity, antioxidant activity.

IntroductionHerbs and spices have been used since ancient

times as food flavouring and storing agents. Butonly in the last decade scientific research has beenfocused on their essential oil composition andtheir use as natural extracts of antimicrobial andantioxidant compounds 1,2. Efforts focussedfurther on the use of this naturally occuring anti-oxidants in the prevention of oxidative damage

occurring in the body 3,4,5.The genus Achillea are represented in the list

of the most important indigeneous economicplants of Anatolia 6. The genus Achillea belongsto the Asteraceae family and is represented bytotal 85 species found in the Northern Hemi-sphere, mostly in Europe and Asia 7. The samegenus is represented in Turkey by 46 taxa; 25 ofthem are endemic 8,9. The name of the genus

ISSN Print: 0972-060XISSN Online: 0976-5026

*Corresponding author (A. Akcin)E-mail: < adnanakcin@hotmail.com > © 2014, Har Krishan Bhalla & Sons

TEOP 17 (2) 2014 pp 219 - 227 219

Received 05 November 2012; accepted in revised form 10 May 2013

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originates from the ancient use as a wound-healing remedy by the Trojan hero Achilles 10.Various species of the genus are traditionally usedas diuretic and emmenagog agents in woundhealing for abdominal pain against diarrahea andflatuence in Turkey 11,12,13,14. Particularly, the aerialparts of A. millefolium, which is a well-knownspecies among the members of Achillea, arecommonly used in European traditional medicinefor the treatment of gastrointestinal disorders andhepatobiliary complaints as well as for woundhealing and skin inflammations 15. There are manyreports describing the antinociceptive and anti-inflammatory 16,17, human erythrocyte andleucocyte protective 18, antispazmodic 19, anti-microbial 20,21,22,23 and antioxidant effects 20,21 ofthe constituents of many Achillea species.

Achillea phrygia itself is a perennial herb,grows up to 45 cm and has golden yellow flowers24. This species is endemic to Anatolia and growson steppes and limestone slopes between locatedbetween altitudes of 700 - 1500 m 25.

The essential oil composition of some Achilleaspecies grown in Turkey has been previouslystudied 22,26,27,28. The essential oil compositionsand antimicrobial activity of A. phrygia essentialoil have previously been reported by Baser et al.26 and Karaalp et al. 28 . However, no reports areavailable about the antioxidant activities of A.phrygia essential oil. Therefore, the aim of thepresent study was to compare and investigate thechemical composition, antimicrobial andantioxidant activities of A. phrygia essential oil.

ExperimentalPlant material

The flowering aerial parts of A. phrygia werecollected from Nevsehir, Avanos on limestoneslopes about 930 m above the sea level duringJune 2010. A voucher specimen (Herbarium No:OMUB 5394) has been deposited in theHerbarium of the Faculty of Arts and Science,University of Ondokuz Mayis, Department ofBiology, Turkey. The collected specimens wereair-dried for two weeks at room temperature andprotected from light for later analysis.

Essential oil isolationEssential oils were obtained from flower and

leaf parts by hydrodistillation in a Clevenger-typeapparatus for 3 h. The obtained essential oil wasdried over anhydrous sodium sulphate, filteredand stored in sealed vials protected from light at-4°C until tested and analysed.

Gas chromatography-mass spectrometry (GC-MS) analysis

The GC-MS analysis of the essential oil wasperformed using a Schimadzu 2010 AF gaschromatography apparatus equipped with a flameionization detector (FID). The essential oilcomponents were seperated on a TR-CN 100capillary column (60 m x 0.25 mm i.d.; film thick-ness 0.20 μm). Injector and detector temperatureswere set to 260°C at a rate of 5°C /min. Heliumwas used as carrier gas at a flow rate of 0.12 ml/min. In every sample 1.0 μl was injected usingsplit mode (split ratio: 1.75).

Retention indices were calculated for allcomponents using a homologous series of n-alkanes injected in the same conditions describedby the Van den Dool method 29. The identificationof components was done by comparison of theirmass spectra with those in the Wiley/NIST massspectral library of the GC-MS data system or withmass spectra from literature 30 .

Antimicrobial activitySix reference bacterial strains namely

Pseudomonas aeruginosa ATCC 27853,Escherichia coli ATCC 25922, Enterococcusfaecalis ATCC 29212, Staphylococcus aureusATCC 25923, Staphylococcus aureus ATCC43300, Acinetobacter baumannii ATCC 19606and 65 clinical bacterial isolates including 24 A.baumannii, 27 methicillin resistant S. aureus and14 methicillin susceptible S. aureus were testedin the study. Microorganisms were provided bythe Department of Microbiology, Faculty ofMedicine, Ondokuz Mayis University, Samsun.Antimicrobial activities of the oil was carried outaccording to disc agar diffusion method 31,32 usingfilter paper discs of 6 mm diameter. Bacterialreference strains were cultured onto blood agarand incubated at 37°C for 24 hours. After theincubation, bacterial suspension adjusted toMcFarland 0.5 standard was prepared from grownbacteria. Bacterial suspension was swapped onto

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Mueller-Hinton agar (MHA) plates by sterilecotton swaps. Sterile discs were placed on thecentral part of plates and 30 μl of essential oilwas added onto the disc. All plates were kept atroom temperature for 15 minutes and then theywere incubated at 37°C for 24 hours. Followingincubation period, bacterial growth inhibitionzones around discs were measured. The activitiesare expressed in mm diameter of the inhibitionzones of bacterial growth including the 6 mm discdiameter.

Antioxidant activityAntioxidant activity of hydrodistilled essential

oils of A. phrygia were determined by using:

1. Scavenging effect on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical

Experiments were carried out as previoslydescribed 33. The absorbances were determinedspectrophometrically at 517 nm. The measure-ments were carried out in triplicate and averaged.

2. Total phenolic content assayDetermination of total phenolic content was

performed according to the method of Slinkard& Singleton 34 using Folin-Ciocalteu reagent. Theabsorbances of the mixtures were measured at760 nm. Gallic acid was used as the standard fora calibration curve. Total phenolic content isexpressed as the gallic acid equivalent using anequation based on the calibration curve.

Results and discussionThirty-one compounds representing 87.11 %

of the essential oil were identified by GC-MSanalysis (Table 1). The major components of theessential oil were camphor (35.55 %), 2-furalde-hyde (16.59 %) and 1,8-cineole (eucalyptol)(10.12 %). In addition, A. phrygia essential oilalso contained considerable amounts of variousminor constituents such as borneol (3.62 %),menthone (2.70 %), terpinen-4-ol (2.54 %), α-terpineole (2.32 %), pulegone (2.03 %), caryo-phyllene oxide (1.75 %), carvacrol (1.66 %) and

Table 1. The chemical constituents of the essential oil of A. phrygia

No Compounds Composition (%) RT*

1 α-Pinene 0.30 5.2462 Camphene 0.80 5.5343 1-Butanol 0.39 6.4154 (+)Limonene 0.03 6.4485 Eucalyptol (1,8-cineol) 10.12 7.5536 2-Heptanone 0.65 8.2247 Hexyl acetate 0.14 8.4888 Neryl acetate 0.42 10.0879 2-Methyl-2-hepten-6-on(6-5-2) 0.56 11.472

10 2-Furaldehyde 16.59 15.23611 (-)-Menthone 2.70 15.36512 Terpinen-4-ol 2.54 16.78913 Camphor 35.55 17.37314 (+)-α-Terpineol 2.32 19.01215 Borneol 3.62 19.26516 (+)-Pulegone 2.03 19.66417 Nerol 0.65 20.74018 Geraniol 0.16 21.76319 Humulene 0.82 22.59120 Heneicosane 0.12 24.18621 Benzyl alcohol 0.06 24.819

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tricosane (1.18 %), whose contribution was lessthan 20 %.

With respect to previous investigationsfocussing on the composition of A. phrygiaessential oils from Turkey, our analysis showeda different chemical profile with somesimilarities. Baser et al. 26 reported cis-piperitol(31.2 %), trans-p-ment-2-en-1-ol (14.7 %), cis-p-ment-2-en-1-ol (9.9 %) and 1,8-cineole (9.9 %)as the major compounds. In our study, camphor(35.54 %) and 2-furaldehyde (16.59 %) were themajor components, but these components wereabsent in the mentioned previous study. Further-more, α-terpineole was detected in lower amountin our study. These variations in the chemicalcomposition of essential oil might be attributedto variable climatic, geographical, seasonalgenetic factors and stage of maturity 35. 1,8-cineole, camphor and borneol are frequentlycharacterized components inside the genus 36. 1,8-cineole has been reported as the main componentin about one-third of the species 37. Also camphorand borneol are among the second and third mostfrequently characterized components of yarrow37. Most of these reports show similar patterns ofessential oil compositions compared with thepresent study.

Results of antimicrobial activity (Table 2)showed that most of the bacterial strains weresignificantly inhibited by the essential oil. Thewidest inhibition zone diameters were determined

in A. baumannii 21 and A. baumannii 22 with 40mm. However, no inhibition zone was observedin 2 clinical isolates (MSSA1 and MSSA3). Inadditionally, the essential oil were not effectiveagainst bacterial strains as Pseudomonasaeruginosa ATCC 27853, Escherichia coli ATCC25922, Enterococcus faecalis ATCC 29212.

Previous investigations on the antimicrobialactivity of Achillea essential oils showed inhibi-tory activity against several bacteria. Karaalp etal.28 reported that the hexane extracts of A.phrygia showed low antibacterial activity againstEnterococcus faecalis ATCC 29212, whereas themethanol extract of the oil showed mild anti-bacterial activity against Staphylococcus aureusATCC 6538, S. aureus ATCC 43300 and Bacilluscereus ATCC 7064. The methanol extract of A.millefolium subsp. millefolium showed moderateactivity against Clostridium perfringens and C.albicans 28. Karamenderes et al. 19 also found thatthe essential oils of A. millefolium subsp.pannonica showed activity against the gram-negative bacterias Proteus vulgaris and Salmo-nella thypimurium.

In another study, the hexane-ether-methanolextract of A. millefolium was found to be activeagainst E. coli, P. aeruginosa, S. aureus,Salmonella enteridis, Aspergillus niger and C.albicans 36. Sökmen et al. 21 reported that themethanol extract and water-insoluble part of A.bieberstenii are active against B. cereus, C.

table 1. (continued).

No Compounds Composition (%) RT*

22 2-Phenyletanol 0.12 25.85123 (-)-Linalool 0.30 26.62324 (-)-Caryophyllene oxide 1.75 27.10225 Methyl palmitate 0.41 27.26226 Tricosane 1.18 27.40227 Thymol 0.92 28.72228 Carvacrol 1.66 29.59829 Eugenol 0.12 30.12730 Phytol 0.06 33.37631 Tridecanoic acid 0.03 34.276

Total identified ( %) 87.11

* Retention time (as minutes)

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Table 2. Antimicrobial activity of the essential oil extract from A. phrygia

Bacterial strains Diameter of zone ofinhibition (mm)

Methycillin-resistant Staphylococcus aureus (MRSA)Staphylococcus aureus 1 24Staphylococcus.aureus 2 15Staphylococcus aureus 3 10Staphylococcus aureus 4 18Staphylococcus aureus 5 10Staphylococcus aureus 6 15Staphylococcus aureus 7 20Staphylococcus aureus 8 17Staphylococcus aureus 9 20Staphylococcus aureus 10 20Staphylococcus aureus 11 20Staphylococcus aureus 12 10Staphylococcus aureus 13 24Staphylococcus aureus 14 20Staphylococcus aureus 15 30Staphylococcus aureus 16 18Staphylococcus aureus 17 12Staphylococcus aureus 18 17Staphylococcus aureus 19 16Staphylococcus aureus 20 27Staphylococcus aureus 21 12Staphylococcus aureus 22 10Staphylococcus aureus 23 11Staphylococcus aureus 24 15Staphylococcus aureus 25 14Staphylococcus aureus 26 23Staphylococcus aureus 27 22Methycillin-sensitive Staphylococcus aureus (MSSA)Staphylococcus aureus 1 -Staphylococcus aureus 2 14Staphylococcus aureus 3 -Staphylococcus aureus 4 11Staphylococcus aureus 5 16Staphylococcus aureus 6 11Staphylococcus aureus 7 11Staphylococcus aureus 8 11Staphylococcus aureus 9 25Staphylococcus aureus 10 9Staphylococcus aureus 11 10Staphylococcus aureus 12 16Staphylococcus aureus 13 22Staphylococcus aureus 14 12

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table 2. (continued).

Bacterial strains Diameter of zone ofinhibition (mm)

Acinetobacter baumannii clinical isolatesAcinetobacter baumannii 1 15Acinetobacter baumannii 3 16Acinetobacter baumannii 4 38Acinetobacter baumannii 5 24Acinetobacter baumannii 6 20Acinetobacter baumannii 7 20Acinetobacter baumannii 8 16Acinetobacter baumannii 9 19Acinetobacter baumannii 10 30Acinetobacter baumannii 12 25Acinetobacter baumannii 15 25Acinetobacter baumannii 17 26Acinetobacter baumannii 18 24Acinetobacter baumannii 20 26Acinetobacter baumannii 21 40Acinetobacter baumannii 22 40Acinetobacter baumannii 23 32Acinetobacter baumannii 24 18Acinetobacter baumannii 25 16Acinetobacter baumannii 26 25Acinetobacter baumannii 27 35Acinetobacter baumannii 28 35Acinetobacter baumannii 29 18Other bacterial strainsPseudomonas aeruginosa ATCC 27853 -Escherichia coli ATCC 25922 -Enterococcus faecalis ATCC 29212 -Staphylococcus aureus ATCC 25923 12Staphylococcus aureus ATCC 43300 17Acinetobacter baumannii ATCC 19606 18

- ; no antimicrobial activity, inhibition zones was no greater than 6 mm.

perfringens and C. albicans. The oil of A.clavennae and A. holoserica showed strongactivity against E. coli, Klebsiella pneumoniae,P. aeruginosa, S. aureus, C. albicans and A. niger36. If compared with antimicrobial activity of otherAchillea species, the effectiveness of the essentialoil of A. phrygia exerted a broad antimicrobialspectrum by inhibiting the growth of most of thetested bacteria. Based on these results, it may be

considered as a natural antimicrobial agent andthe observed antimicrobial activity supports themedicinal uses of this plant for traditionalremedies.

The antioxidant assay results of extracts andessential oil of A. phrygia are given in Table 3.In our study the total phenolic content andantioxidant activity values was determinedrespectively as 120.33 and 40.43 (Table 3).

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Table 3. Total phenolic content and in vitro antioxidantactivitiy by using DPPH assay of the extract from A. phrygia

Species Total phenolic content DPPH (mg/l) (mg GAE/g DW)

A. phrygia 120.33±2.52 40.43±2.57

Results are mean ± SD of three replicate analyses

Konyalioglu and Menderes 18 investigated thephenol contents and antioxidant activity ofAchillea species grown in Turkey including A.clypeolata, A. schischkinii, A. teretifolia, A.coarctata, A. phrygia, A. crithmifolia, A. nobilissubsp. neilrechii, A. millefolium subsp.pannonica, A. biebersteinii, A. kotschyi subsp.kotschyi, A. nobilis subsp. sipylea, A. setacea, A.falcata, A. grandifolia and A. multifida. The totalphenol content of this investigated species rangedbetween 100.00 and 181.212 mg/L. A. phrygiashowed a total phenol content of 110.908 mg / Lin the present study. Our material showed highervalues regarding total phenol content.

The DPPH-RSC activity value of collected A.phrygia was 40.43 %. In the mentioned study ofKonyalioglu and Menderes 18 the DPPH % RSCvalues for investigated Achillea species was23.73-43.57 %. The DPPH % RSC value for A.phrygia was 34.81 18. The present materialshowed a value of 40.43 mg/L, which is highercompared with the given literature. Konyaliogluand Karamenderes 38 suggested in their study thatAchillea species having stronger DPPH radicalscavening activity seems to include high contents

of total phenolics.Several investigations revealed that a positive

relationship exists between total phenolic sub-stance content and antioxidant capacity in medi-cinal and aromatic plants 39 . The amounts ofphenol compounds of A. phrygia correlates totheir scavenging effect. These results demonstratethat the antioxidant activities observed can beascribed both to mechanisms exerted by phenoliccompounds and also to synergistic effects ofdifferent phytocompounds. This study reveals thattested plant materials have significant antioxidantactivity and free radical scavenging activity. Theresult of the present study suggests that selectedplant can be used as a source of antioxidants forpharmacological preparations which is very wellevidenced by the present work.

AcknowledgementsThe authors are thankful to Prof. Dr. Sezai

Ercisli for it’s help regarding the antioxidantanalysis. Financial support made by the ResearchFunds of the University of Ondokuz Mayis,Samsun, Turkey is gratefully acknowledged.

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