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Determination of the chemical composition
and antimicrobial activity of the essential oils of
Teucrium polium
and Achillea millefolium grown
under North Anatolian ecological conditions
Emre Sevindik, Zehra Tugba Abacı, Cigdem Yamaner & Muavviz Ayvaz
To cite this article: Emre Sevindik, Zehra Tugba Abacı, Cigdem Yamaner & Muavviz Ayvaz (2016) Determination of the chemical composition and antimicrobial activity of the essential oils of Teucrium�polium and Achillea�millefolium grown under North Anatolian ecological conditions, Biotechnology & Biotechnological Equipment, 30:2, 375-380, DOI: 10.1080/13102818.2015.1131626
To link to this article: https://doi.org/10.1080/13102818.2015.1131626
© 2016 The Author(s). Published by Taylor &
Francis. Published online: 29 Jan 2016.
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ARTICLE; PHARMACEUTICAL BIOTECHNOLOGY
Determination of the chemical composition and antimicrobial activity of the
essential oils of
Teucrium polium and Achillea millefolium grown under North
Anatolian ecological conditions
Emre Sevindika, Zehra Tugba Abacıb, Cigdem Yamaneraand Muavviz Ayvaz a
aFaculty of Agriculture, Department of Agricultural Biotechnology, Adnan Menderes University, South Campus, Cakmar, Aydin, Turkey;bFaculty of Engineering, Department of Food Engineering, Ardahan University, Ardahan, Turkey
ARTICLE HISTORY
Received 28 September 2015 Accepted 10 December 2015
ABSTRACT
The chemical composition and antimicrobial effect of essential oils derived from Teucrium polium L. and Achillea millefolium subsp. millefolium Afan. in in vitro conditions were investigated in the present study. The results from the gas chromatography mass spectrometry analysis showed that the obtained (Z)-b-farnesene from T. polium was with the highest percentage (15.49%), whereas b-pinene from the same plant was with the lowest percentage (0.74%). The 1,8-cineole in A. millefolium subsp. millefolium was with the highest percentage (22.83%), whereas p-cymene in the same plant was with the lowest percentage (0.93%). In the antimicrobial studies, the essential oils’ effect against methicillin-resistant Staphylococcus aureus (MRSA), S. aureus ATCC 6538, Pseudomonas aeruginosa, Escherichia coli Q157:H7 and Bacillus cereus CCM 99 were investigated using the agar well method. P. aeruginosa and MRSA showed the biggest inhibition zones (15 mm), whereas E. coli Q157:H7 showed the smallest inhibition zone (11 mm), each generated by using T. polium essential oils. MRSA showed the biggest inhibition zone (21 mm), whereas P. aeruginosa showed the smallest inhibition zone (10 mm), both obtained by using A. millefolium subsp. millefolium essential oils. Therefore, it was concluded that the essential oils obtained from the two plant species had an inhibition effect on resistant micro-organisms.
KEYWORDS
Essential oils; GC-MS; antimicrobial activity; Turkey
Introduction
Aromatic and medical plants have been used since ancient times for their preservative and medicinal proper-ties, fruits, grains and spices, which impartflavour to the food.[1,2] They are a rich and significant natural source of
biologically active compounds and have been shown to possess antibacterial, antifungal, antiviral, insecticidal and antioxidant properties.[3 5] With the increase of bacterial resistance to antibiotics, there has been a remarkable interest towards investigation of the antimicrobial effects of different extracts against a range of bacteria and towards developing other classes of antimicrobial agents useful for the infection control or for the preservation of food.[6,7] Therefore, the use of essential oils is less dam-aging to the human health because they are usually less toxic and do not have any side effects.[8]
A variety of plants have been used as tea, spices and for treatment purposes by the people in Turkey and in the whole world. The plant diversity in our country is caused by the fact that it is in a region where three phy-togeographical regions coincide. It is a bridge between
South Europe and south-west Asiafloras, and also, Ana-tolia is possibly the centre of origin of many species and sections. The plant diversity is also caused by the fact that the species endemism, related with ecological and phytogeographical differentiation, is relatively high.[9] The genus Teucrium (L.), which belongs to the family Lamiaceae, is represented by about 300 species in the world and has a cosmopolitan distribution, mainly in Europe, North Africa and in the temperate parts of Asia. [10] Teucrium polium (L.) is widely used in the folk medi-cine for many treatment interventions.[11,12] Teucrium species have been used as medicinal plants for more than 2000 years and some of them are still used in the folk medicine as antispasmodics, tonics, antipyretics and antiseptics.[13 16] Many Teucrium species are known for their medicinal utilization and exhibit interesting bio-logical properties such as hypoglycaemic, hypolipidemic, hepatoprotective, antipyretic, anti-inflammatory, antiul-cer, antitumour, antibacterial and insect antifeedant activities.[15,17 21] The genus Achillea L. belongs to the family Asteraceae, which consists of about 140 species
CONTACT Muavviz Ayvaz muavviz.ayvaz@adu.edu.tr
© 2016 The Author(s). Published by Taylor & Francis.
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License (http://creativecommons.org/licenses/by-nc-nd/ 4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed, or built upon in any way.
VOL. 30, NO. 2, 375 380
that are centred in south-west Asia and south-east Europe, with extensions from Eurasia to North America. The genus exhibits a high level of ecological adaptabil-ity.[22] These species have some interesting properties and are used in cosmetics, fragrances and agriculture, for instance, in plant protection.[23] Some Achillea spe-cies are used generally as teas and household remedies for diarrhoea and stomach gas prevention, as external lotions and creams or as a haemorrhoid and wound treatment.
The aim of the present study was to determine the essential oil contents of T. polium and Achillea millefolium subsp. millefolium plants growing in Ardahan ecological conditions and to investigate their antimicrobial effect on various bacteria. The purpose of this work was to con-stitute a scientific basis for future studies and to reveal the scientific basics of the traditional treatment methods used by people.
Materials and methods
Plant materialsT. polium and A. millefolium subsp. millefolium aerial parts of the plants were collected as study materials in June and August 2013 2014, which are their blooming peri-ods, from Ardahan ¸Cıldır lake (1970 m altitude) and its surroundings, and from G€ole province entrance zone on the Ardahan Oltu road (2080 m altitude), respectively (Figure 1). The collected samples were placed in fabric bags and kept in a room with no sunlight.
Isolation of essential oils
Approximately 300 g of plant sample was used for essential oil extraction. This was performed with Cle-venger apparatus (Basaran cam, Turkey and Misung Scientific Co., Korea) using water distillation. The yield and qualitative and quantitative compositions of the essential oils from T. polium and A. millefolium subsp. millefolium were determined. Chemical analyses of the essential oils were carried out with gas chromatogra-phy mass spectrometry (GC MS, HP 6890 Series Gas Chromatography, 5973 Mass Selective Dedector Sys-tem) present in Eski¸sehir Anadolu University Medicinal Plants, Drugs and Scientific Research Center (A€UB_IBAM).
GC MS analysis
The chromatographic procedures were made using Hewlett Packard system, HP 5973 Mass Selective Detec-tor System and GC MS 6890 GC system. Agilent HP innowax column (60 m in length, inner diameter of 0.25 mm,film thickness of 0.25 mm) was used. Helium was used as a carrier gas. The injection temperature was 250 C and the oven temperature programme was as follows: from 60C, 4 C/min up to 220 C for 10 min, 2C/min up to 240C for 20 min. The components char-acterization in the essential oils was made using elec-tronic libraries (WILEY-NIST Database,http://www.wiley. com/go/databases). The quantity of the characterized components was expressed in percentages and the retention time (RT) was recorded in minutes.
Figure 1.Location of the Ardahan region.
Map data: US Dept of State Geographer © 2015 Google © 2015 Basarsoft Image Landsat.
Preparation of micro-organism cultures
The antimicrobial activity of the two different plant extracts was detected by using three different gram pos-itive micro-organisms (methicillin-resistant Staphylococ-cus aureus (MRSA), S. aureus ATCC 6538, Bacillus cereus CCM 99) and two different gram negative micro-organ-isms (Pseudomonas aeruginosa, Escherichia coli Q157:H7) by using the agar well method. Ampicillin was used as a control in the experiments. The cultures were inoculated into brain heart infusion broth (Merck 1.10493.0500) and were incubated at 37C for 24 h to grow the stock bac-teria cultures. A 0.5 McFarland standard was prepared with the used micro-organisms and sterile 0.85% physio-logical saline water. Then, they were inoculated into Petri dishes containing Mueller Hinton agar (Merck 1.05437.0500) by using the expansion method. The Petri dishes were allowed to dry at room temperature for 20 25 min. Afterwards, a 0.5 mm diameter well was made inside the agar in sterile conditions. Essential oil (5 mL) was added to each well and an incubation fol-lowed at 37 C for 24 h. The formed inhibition zones were measured in millimetres at the end of the incuba-tion period from the lower surface of the Petri dish. The trials were performed with two repetitions.
Results and discussion
Chemical composition of the essential oils
Analysis results of the essential oils from organs above the ground from T. polium and A. millefolium subsp. mille-folium species are given inTable 1. During the analysis,
14 20 components were found from T. polium and A. millefolium, which formed 77.22% and 73.23% of the total essential oil amount, respectively. The total essen-tial oil yield from T. polium and A. millefolium subsp. mil-lefolium was measured as 0.05 and 0.18 mL, respectively (data not shown).
The compounds obtained from T. polium were as fol-lows: a-pinene, a-thujene, b-pinene, b-phellandrene, myr-cene, limonene, (Z)-b-farnesene, germacrene D, b-bisabolene, d-cadinene, a-farnesene, b-gurjunene, ledene and b-selinene. The compounds detected with highest percentages were (Z)-b-farnesene (15.49%), b-phellandrene (10.77%) and a-farnesene (10.71%), whereas the b-pinene was with the lowest percentage (0.74%). Belmekki et al. [8] studied the essential oil com-ponents of T. polium species collected from north-west Algeria. They detected totally 37 components and 6 of them were common with the ones in our study, but had different percentages (3.96% a-pinene, 11.69% b-pinene, 0.88% myrcene, 1.77% limonene, 4.31% d-cadinene and 25.81% germacrene D).[8] Mahmoudi and Nosratpour [24] conducted studies on the phytochemical and antioxi-dant features of essential oils of T. polium species distrib-uted in Kerman Province of Iran. At the end of the study, 21 different components were obtained.[24] The obtained results were different from the results in our study. The components b-pinene (11.02%) and germacrene D (8.15%) were found to be the most common. Moghtader [25] extracted essential oils from T. polium species col-lected from Iran. At the end of the study, 29 different components were obtained.[25] These components (12.52% a-pinene, 7.09% b-pinene, 1.89% limonene,
Table 1.Essential oil composition of T. polium and A. millefolium subsp. millefolium.
T. polium from ¸Cıldır Lake (1970 m) A. millefolium subsp. millefolium from G€ole region (2080 m)
RT (min) Component Quantity (%) RT (min) Component Quantity (%)
9.30 a-pinene 0.91 9.38 a-pinene 7.41 9.45 a-thujene 1.35 13.17 b-pinene 5.67 13.07 b-pinene 0.74 13.77 Sabinene 2.38 13.83 b-phellandrene 10.77 18.16 1,8-cineole 22.83 15.88 Myrcene 2.50 19.95 g-terpinene 1.62 17.68 Limonene 5.86 21.19 p-cymene 0.93 36.61 (Z)-b-farnesene 15.49 28.68 b-thujone 1.31 38.41 Germacrene D 9.69 29.87 Unknown 3.72 38.62 b-bisabolene 2.33 31.75 Camphor 3.38
38.76 d-cadinene 0.85 33.83 Bornyl acetate 1.17
47.52 a-farnesene 10.71 34.50 b-caryophyllene 2.59
49.18 b-gurjunene 7.50 34.61 Terpinen-4-ol 4.50
52.45 Ledene 6.33 37.69 a-terpineol 3.02
53.12 b-selinene 2.19 37.93 Borneol 1.57
Total 77.22 46.64 Caryophyllene oxide 1.50
50.57 a-elemene 1.57 52.70 a-selinene 1.46 53.00 b-selinene 2.95 54.77 Unknown 1.88 56.50 Unknown 1.77 Total 73.23
1.46% myrcene and 5.03% germacrene D) were consis-tent with the components obtained in our study, but with different percentages. ¸Cakır et al. [26] studied the essential oil contents of T. polium plants collected from Gaziantep region and detected totally 30 components. At the end of the study, the components a-pinene (12%), b-pinene (18%), myrcene (6.8%), limonene (3.5%) and germacrene D (5.3%) were common with the compo-nents obtained in our study, but had different percen-tages. Raei et al. [27] studied the essential oil contents of T. polium plants collected from Tahran, Iran and detected totally 20 components. At the end of the study, the com-ponents a-pinene (5.5%), a-thujene (0.4%), b-pinene (11%), myrcene (0.5%), limonene (4.2%), germacrene D (6.5%), b-bisabolene (3.7%) and farnesene (13%) were consistent with the results obtained in our study and only their proportions were different.
The compounds obtained from A. millefolium subsp. millefolium were a-pinene, b-pinene, sabinene, 1,8-cineole, g-terpinene, p-cymene, b-thujone, camphor, bornyl ace-tate, b-caryophyllene, terpinen-4-ol, a-terpineol, borneol, caryophyllene oxide, a-elemene, a-selinene, b-selinene and three unknown compounds were obtained. The detected compounds with the highest percentages were 1,8-cineole (22.83%), a-pinene (7.41%), b-pinene (5.67%) and terpinen-4-ol (4.50%), whereas p-cymene was with the lowest percentage (0.93%). Nadim et al. [28] determined the essential oil contents of A. millefolium cul-tures growing in India under tropical conditions and at the end of the study, 30 different components were obtained. Considering our study, 11 of the obtained com-pounds were common with ours (6.28% a-pinene, 6.26% b-pinene, 17.58% sabinene, 13.04% 1,8-cineole, 1.11% p-cymene, 1.19% g-terpinene, 7.98% bornyl acetate, 2.31% b-caryophyllene, 6.17% terpinen-4-ol, 1.04% a-terpineol, 12.41% borneol), but with different percentages. Candan et al. [29] studied the essential oils and antimicro-bial activity of A. millefolium subspecies collected from Sivas Kızıldag. At the end of the essential oil analysis, 69 components were detected.[29] When compared with the results of the present study, 10 compounds were found to be common (a-pinene, b-pinene, sabinene, g-terpinene, camphor, bornyl acetate, terpinen-4-ol, a-terpineol, bor-neol, caryophyllene oxide). Nenaah [30] determined the essential oil contents of A. millefolium growing in Egypt during the flowering period. At the end of the analysis, the compounds a-pinene, b-pinene, sabinene, 1,8-cineole, g-terpinene, p-cymene, camphor, bornyl acetate, b-phyllene, terpinen-4-ol, a-terpineol, borneol and caryo-phyllene oxide were found to be common with the obtained compounds in our study.
In general, thesefindings confirmed that the essential oil composition of the plants can have different quality
and quantity in different geographical and environmen-tal conditions, and during different periods of the plant growth.[31,32]
Antimicrobial activity
In the last few years, there has been a targeted interest towards biologically active compounds isolated from aromatic plant species for the elimination of pathogenic micro-organisms. The main reason for this is the resis-tance that micro-organisms have built against antibiot-ics.[31,33,34] Because of their hydrophobicity, the essential oils and their components may disintegrate the lipids of the cell membranes. This makes the cells’ and mitochondrias’ membranes more permeable. As a result, leakage of ions and cell contents may happen.[35 38] In our study, the agar well method was used for determi-nation of the in vitro antimicrobial activity of essential oils. The trials were performed with two repetitions and the average values are given inTable 2.
The essential oil obtained from T. polium plant was effective against MRSA, S. aureus ATCC 6538, P. aerugi-nosa, E. coli Q157:H7 and B. cereus CCM 99 in a varying degree. The biggest inhibition zone (15 mm) generated from the essential oil was against MRSA and P. aerugi-nosa and the smallest inhibition zone (11 mm) was gen-erated against E. coli Q157:H7. Toroglu et al. [39] did not observe an inhibition zone against E. coli and S. aureus Cowan I when they soaked discs with 2 mL T. polium essential oil in their studies, in which they applied the disc diffusion method. They stated that a disc soaked with 4 mL essential oil also did not form an inhibition zone against E. coli DM, but it constituted a zone of 12 mm against S. aureus Cowan I. Oguz et al. [40] investi-gated the antimicrobial effect of essential oils from some species belonging to Labiatae family growing in¸Sırnak– Silopi region and in the study, T. polium species were used as well. S. aureus ATCC 25923, E. coli ATCC 29998 and B. cereus ATCC 17778 micro-organisms were used for determination of the antimicrobial effects.[40] In the study, the inhibition zone diameter of S. aureus was found to be 15 mm, the inhibition zone diameter of
Table 2.Antimicrobial activity (inhibition zones) of the essential oils from T. polium and A. millefolium subsp. millefolium.
Micro-organisms Ampicillin T. polium A. millefolium subsp. millefolium Inhibition zone (mm) MRSA 14§ 0 15§ 0 21§ 0.7 S. aureus ATCC 6538 56§ 0 12§ 0.35 14§ 0.7 P. aeruginosa 9§ 0.35 15§ 0.7 10§ 0 E. coli Q157:H7 34§ 0 11§ 0 11§ 0 B. cereus CCM 99 19§ 0 14§ 0.7 12§ 0 Note: Results are expressed as mean§ standard deviation (SD);
methicillin-resistant S. aureus (MRSA). 378 E. SEV_IND_IK ET AL.
E. coli was 8 mm and that of B. cereus– 19 mm.[40] Bel-mekki et al. [8] investigated the antimicrobial effect of essential oils from T. polium species collected from north-west Algeria. In the disc diffusion study, a 15 mm inhibition zone was detected against B. cereus ATCC 11778, a 16 mm inhibition zone was generated against E. coli ATCC 25922, the inhibition zone was 9 mm against P. aeruginosa ATCC 27853 and 16 mm against S. aureus ATCC 25923.
The essential oil obtained from A. millefolium subsp. millefolium plant was effective against MRSA, S. aureus ATCC 6538, P. aeruginosa, E. coli Q157:H7 and B. cereus CCM 99 also in a varying degree. The biggest inhibition zone (21 mm) generated from the essential oil was against MRSA and the smallest generated inhibition zone (10 mm) was against P. aeruginosa. Issabeagloo et al. [41] investigated the antimicrobial effect of essen-tial oils obtained from A. millefolium plant against Staph-ylococcus species. In the disc diffusion study, the inhibition zone against S. aureus ATCC 25923 was detected to be 9.3 mm on the average.[41] Candan et al. [29] studied the essential oils and antimicrobial activity of A. millefolium subsp. millefolium species collected from Sivas Kızıldag. When the diameters of the antimi-crobial inhibition zones from the essential oils were examined, it was seen that the zone generated against S. aureus ATCC 25923 was 8 mm, the one generated against B. cereus ATCC 11778 was 10 mm and there was no inhibition zone generated against E. coli ATCC 25922. [29] In our study, the inhibition zone diameters gener-ated from the essential oil from A. millefolium subsp. mil-lefolium were 14 mm against S. aureus, 12 mm against B. cereus and 11 mm against E. coli.
Essential oil components and antimicrobial effect of T. polium and A. millefolium subsp. millefolium plants grown in Ardahan ecological conditions were deter-mined in this study. The difference of our recent work from the previous studies discussed in the manuscript may be explained by the usage of different methods and different plant collecting periods from different geo-graphical regions and by the type of bacterial strains used and essential oil amounts put into wells. The obser-vation of an antimicrobial effect against micro-organisms shows us that plants containing etheric oils may be used for treatment purposes and may be an alternative to syn-thetic antibiotics.
Conclusions
In the present work, we assessed the chemical composi-tion and antimicrobial effect of the essential oils derived from plant species. Overall, we showed that the essential oils obtained from both plant species had an inhibition
effect on the most resistant and difficult to treat micro-organisms, such as MRSA, B. cereus, S. aureus, P. aerugi-nosa and E. coli. These findings may be a valuable resource for further biotechnological, biodiversity, phar-maceutical and medicinal studies. It will also help to understand the importance of the biological diversity and conservation biology efforts.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was supported by Ardahan University [grant number 2013/02].
ORCID
Muavviz Ayvaz http://orcid.org/0000-0002-1776-0730
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