Determination of the chemical composition and antimicrobial activity of the essential oils of Teucrium polium and Achillea millefolium grown under North Anatolian ecological conditions

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)-β-farnesene from T. polium was with the highest percentage (15.49%), whereas β-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 properties, fruits, grains and spices, which impart flavour 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 damaging 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 phytogeographical regions coincide. It is a bridge between South Europe and south-west Asia floras, and also, Anatolia 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 medicine 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 biological properties such as hypoglycaemic, hypolipidemic, hepatoprotective, antipyretic, anti-inflammatory, antiulcer, antitumour, antibacterial and insect antifeedant activities.[15,17–21] The genus Achillea L. belongs to the family Asteraceae, which consists of about 140 species 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 adaptability.[22] These species have some interesting properties and are used in cosmetics, fragrances and agriculture, for instance, in plant protection.[23] Some Achillea species 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 constitute a scientific basis for future studies and to reveal the scientific basics of the traditional treatment methods used by people.

Materials and methods

Plant materials

T. 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 periods, from Ardahan Çıldır lake (1970 m altitude) and its surroundings, and from Göle 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.

Figure 1. Location of the Ardahan region. Map data: US Dept of State Geographer © 2015 Google © 2015 Basarsoft Image Landsat.

Isolation of essential oils

Approximately 300 g of plant sample was used for essential oil extraction. This was performed with Clevenger 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 chromatography–mass spectrometry (GC–MS, HP 6890 Series Gas Chromatography, 5973 Mass Selective Dedector System) present in Eskişehir Anadolu University Medicinal Plants, Drugs and Scientific Research Center (AÜBİBAM).

GC–MS analysis

The chromatographic procedures were made using Hewlett Packard system, HP 5973 Mass Selective Detector 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 µm) was used. Helium was used as a carrier gas. The injection temperature was 250 °C and the oven temperature programme was as follows: from 60 °C, 4 °C/min up to 220 °C for 10 min, 2 °C/min up to 240 °C for 20 min. The components characterization in the essential oils was made using electronic 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.

Preparation of micro-organism cultures

The antimicrobial activity of the two different plant extracts was detected by using three different gram positive micro-organisms (methicillin-resistant Staphylococcus aureus (MRSA), S. aureus ATCC 6538, Bacillus cereus CCM 99) and two different gram negative micro-organisms (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 37 °C for 24 h to grow the stock bacteria cultures. A 0.5 McFarland standard was prepared with the used micro-organisms and sterile 0.85% physiological 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 µL) was added to each well and an incubation followed at 37 °C for 24 h. The formed inhibition zones were measured in millimetres at the end of the incubation 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. millefolium species are given in Table 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 essential oil yield from T. polium and A. millefolium subsp. millefolium was measured as 0.05 and 0.18 mL, respectively (data not shown).

Table 1. Essential oil composition of T. polium and A. millefolium subsp. millefolium.

T. polium from Çıldır Lake (1970 m)			A. millefolium subsp. millefolium from Göle region (2080 m)
RT (min)	Component	Quantity (%)	RT (min)	Component	Quantity (%)
9.30	α-pinene	0.91	9.38	α-pinene	7.41
9.45	α-thujene	1.35	13.17	β-pinene	5.67
13.07	β-pinene	0.74	13.77	Sabinene	2.38
13.83	β-phellandrene	10.77	18.16	1,8-cineole	22.83
15.88	Myrcene	2.50	19.95	γ-terpinene	1.62
17.68	Limonene	5.86	21.19	p-cymene	0.93
36.61	(Z)-β-farnesene	15.49	28.68	β-thujone	1.31
38.41	Germacrene D	9.69	29.87	Unknown	3.72
38.62	β-bisabolene	2.33	31.75	Camphor	3.38
38.76	δ-cadinene	0.85	33.83	Bornyl acetate	1.17
47.52	α-farnesene	10.71	34.50	β-caryophyllene	2.59
49.18	β-gurjunene	7.50	34.61	Terpinen-4-ol	4.50
52.45	Ledene	6.33	37.69	α-terpineol	3.02
53.12	β-selinene	2.19	37.93	Borneol	1.57
Total		77.22	46.64	Caryophyllene oxide	1.50
			50.57	α-elemene	1.57
			52.70	α-selinene	1.46
			53.00	β-selinene	2.95
			54.77	Unknown	1.88
			56.50	Unknown	1.77
			Total		73.23

Note: Retention time (RT).

The compounds obtained from T. polium were as follows: α-pinene, α-thujene, β-pinene, β-phellandrene, myrcene, limonene, (Z)-β-farnesene, germacrene D, β-bisabolene, δ-cadinene, α-farnesene, β-gurjunene, ledene and β-selinene. The compounds detected with highest percentages were (Z)-β-farnesene (15.49%), β-phellandrene (10.77%) and α-farnesene (10.71%), whereas the β-pinene was with the lowest percentage (0.74%). Belmekki et al. [8] studied the essential oil components 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% α-pinene, 11.69% β-pinene, 0.88% myrcene, 1.77% limonene, 4.31% δ-cadinene and 25.81% germacrene D).[8] Mahmoudi and Nosratpour [24] conducted studies on the phytochemical and antioxidant features of essential oils of T. polium species distributed 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 β-pinene (11.02%) and germacrene D (8.15%) were found to be the most common. Moghtader [25] extracted essential oils from T. polium species collected from Iran. At the end of the study, 29 different components were obtained.[25] These components (12.52% α-pinene, 7.09% β-pinene, 1.89% limonene, 1.46% myrcene and 5.03% germacrene D) were consistent with the components obtained in our study, but with different percentages. Çakır 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 α-pinene (12%), β-pinene (18%), myrcene (6.8%), limonene (3.5%) and germacrene D (5.3%) were common with the components obtained in our study, but had different percentages. 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 components α-pinene (5.5%), α-thujene (0.4%), β-pinene (11%), myrcene (0.5%), limonene (4.2%), germacrene D (6.5%), β-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 α-pinene, β-pinene, sabinene, 1,8-cineole, γ-terpinene, p-cymene, β-thujone, camphor, bornyl acetate, β-caryophyllene, terpinen-4-ol, α-terpineol, borneol, caryophyllene oxide, α-elemene, α-selinene, β-selinene and three unknown compounds were obtained. The detected compounds with the highest percentages were 1,8-cineole (22.83%), α-pinene (7.41%), β-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 cultures 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 compounds were common with ours (6.28% α-pinene, 6.26% β-pinene, 17.58% sabinene, 13.04% 1,8-cineole, 1.11% p-cymene, 1.19% γ-terpinene, 7.98% bornyl acetate, 2.31% β-caryophyllene, 6.17% terpinen-4-ol, 1.04% α-terpineol, 12.41% borneol), but with different percentages. Candan et al. [29] studied the essential oils and antimicrobial activity of A. millefolium subspecies collected from Sivas Kızıldağ. 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 (α-pinene, β-pinene, sabinene, γ-terpinene, camphor, bornyl acetate, terpinen-4-ol, α-terpineol, borneol, 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 α-pinene, β-pinene, sabinene, 1,8-cineole, γ-terpinene, p-cymene, camphor, bornyl acetate, β-caryophyllene, terpinen-4-ol, α-terpineol, borneol and caryophyllene oxide were found to be common with the obtained compounds in our study.

In general, these findings confirmed that the essential oil composition of the plants can have different quality and quantity in different geographical and environmental 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 resistance that micro-organisms have built against antibiotics.[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 determination of the in vitro antimicrobial activity of essential oils. The trials were performed with two repetitions and the average values are given in Table 2.

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).

The essential oil obtained from T. polium plant was effective against MRSA, S. aureus ATCC 6538, P. aeruginosa, 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. aeruginosa and the smallest inhibition zone (11 mm) was generated against E. coli Q157:H7. Toroğlu et al. [39] did not observe an inhibition zone against E. coli and S. aureus Cowan I when they soaked discs with 2 µL T. polium essential oil in their studies, in which they applied the disc diffusion method. They stated that a disc soaked with 4 µL 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. Oğuz et al. [40] investigated the antimicrobial effect of essential oils from some species belonging to Labiatae family growing in Şırnak--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 E. coli was 8 mm and that of B. cereus -- 19 mm.[40] Belmekki 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 essential oils obtained from A. millefolium plant against Staphylococcus 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 Kızıldağ. When the diameters of the antimicrobial 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 generated from the essential oil from A. millefolium subsp. millefolium 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 determined 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 geographical regions and by the type of bacterial strains used and essential oil amounts put into wells. The observation 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 synthetic antibiotics.

Conclusions

In the present work, we assessed the chemical composition 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. aeruginosa and E. coli. These findings may be a valuable resource for further biotechnological, biodiversity, pharmaceutical 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.

Additional information

Funding

This work was supported by Ardahan University [grant number 2013/02].