EJBR2018v8i3art153 ISSN 2449-8955 European Journal of Biological Research Research Article European Journal of Biological Research 2018; 8 (3): 153-156 Achillea millefolium L. subsp. millefolium essential oil’s antifungal effect Sinem Aydin 1 , Emre Sevindik 2 * 1 Giresun University, Department of Biology, Faculty of Science and Arts, Giresun, Turkey 2 Faculty of Agriculture, Department of Agricultural Biotechnology, Adnan Menderes University, South Campus, Cakmar, Aydin, Turkey *Corresponding author: Emre Sevindik; E-mail: ph.d-emre@hotmail.com ABSTRACT This study was carried out with the aim of determining the antifungal effect of the essential oil isolated from Achillea millefolium subsp. mille- folium plant against pathogenic fungi. In order to test the antifungal effect of the oil, an analysis was conducted on a total of 4 pathogen fungi which included Candida albicans, Candida tropicalis, Candida parapsilosis and Saccharomyces cerevi- siae, and the effect of the essential oil on the growth of these fungi was investigated. The essential oil of A. millefolium ssp. millefolium had varying degrees of effect on the tested fungi. The highest antifungal effect was found against S. cerevisiae; whereas the lowest antifungal effect was found against C. para- psilosis. Nystatin showed a higher activity than the essential oil of A. millefolium subsp. millefolium against the tested fungi. MIC values of the essential oil against the tested fungi ranged from 1.25 μl/ml to 10 μl/ml. The results obtained indicate that essential oil of A. millefolium subsp. millefolium can be used as an alternative to antifungal agents such as amphotericin, ketoconazole, and fluconazole. Keywords: Achillea millefolium subsp. millefolium; Essential oil; Antifungal; Turkey. 1. INTRODUCTION Since ancient times, raw herbal essences of aromatic plants have been used for different purposes, such as food, perfumery and medicines [1]. Primary and secondary metabolites produced by plants have a wide spectrum of functions. Secondary metabolites have been later utilized by humans due to their beneficial roles [2]. Essential oils are secondary metabolites obtained from plants and have been extensively used since the Middle Ages for bactericidal, virucidal, fungicidal, antiparasitic, insecticidal, medical and cosmetic purposes [3, 4]. Achillea L. is a large genus belonging to the family Asteraceae. The genus Achillea L. includes 59 taxa divided into 6 sections. Among them, 31 taxa are endemic to Turkey [5-7]. Achillea mille- folium, known to the public as "milfoil", "common yarrow", "gordaldo", nosebleed plant" is considered to be one of the oldest medical plants [8]. There are many subspecies of A. millefolium species. A. millefolium species is represented by two sub- types in Turkey flora. These are Achillea millefolium subsp. millefolium and Achillea millefolium subsp. pannonica [9]. Achillea species, known as medici- nal plants, are used against fever, colds, digestive complaints, slow-healing wounds and dermatitis. However, A. millefolium plant has been used be- Received: 22 June 2018; Revised submission: 20 July 2018; Accepted: 16 August 2018 Copyright: © The Author(s) 2018. European Journal of Biological Research © T.M.Karpiński 2018. This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial 4.0 International License, which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. DOI: http://dx.doi.org/10.5281/zenodo.1346338 154 | Aydin & Sevindik Achillea millefolium L. subsp. millefolium essential oil’s antifungal effect European Journal of Biological Research 2018; 8 (3): 153-156 cause of its anti-inflammatory, spasmolytic, haemo- static, and cholagogue effects [10]. The herbal tea of A. millefolium has been found to be used against diseases of the gastrointestinal tract, especially in the folk medicine. The aim of the present study was to determine the essential oil A. millefolium subsp. millefolium growing in Ardahan ecological conditions and to investigate their antifungal effect on some strong pathogen fungi. 2. MATERIALS AND METHODS 2.1. Plant material and isolation of essential oils A. millefolium subsp. millefolium samples of the plants were collected as study materials in June 2013 from Ardahan/Turkey surroundings (approxi- mately 2080 m altitude). Extractions were carried out with Clevenger apparatus (Basaran cam, Turkey and Misung Scientific Co., Korea) using water distillation. 2.2. Microorganisms Candida albicans and Candida tropicalis were obtained from Fırat University Department of Biology; Candida parapsilosis were obtained from Giresun University Faculty of Education, Saccharomyces cerevisiae was obtained from Giresun Province Control Laboratory. 2.3. Antifungal activity The antifungal activity of the essential oil was determined by disc diffusion method. The essential oil of A. millefolium subsp. millefolium was steri- lized by filtration through a 0.45 μm membrane filter [13]. The turbidity of fungal suspensions were adjusted with 0.5 Mc Farland standard (107 CFU/ml fungi concentration), then, the fungal suspension spread on Petri dishes [14]. The discs (6 mm diame- ter) were put on the inoculated agar and separately impregnated with 20 µ l of essential oils. Nystatine disc was used as positive control. Plates were kept at 30°C for 48 h. Antifungal activity was assessed by measuring the diameter of the growth-inhibition zone in millimeters [15]. 2.4. Determination of Minimum Inhibition Concentration (MIC) of the essential oils The MIC was defined as the lowest con- centration that completely inhibits the growth of microorganisms. For the determination of values of MIC, a micro-dilution broth assay was utilized. Two-fold serial dilutions (in dimethyl sulphoxide (DMSO)) were prepared from 0.0098 µ l/ml to 20 µ l/ml of the essential oils of A. millefolium subsp. millefolium in a 96-well microplate. Plates were incubated at 30°C for 48 h [16, 17]. 3. RESULTS AND DISCUSSION Medical and aromatic plants are rich and important natural sources of biologically active compounds and have been shown to possess anti- bacterial, antifungal, antiviral, insecticidal and anti- oxidant properties [18]. Table 1 reveals inhibition zones which were created by essential oil of A. millefolium subsp. millefolium against the test fungi. The highest and the lowest activities were found against S. cerevisiae and C. parapsilosis, respectively. Nystatin was more active against the test fungi than the essential oil of A. millefolium subsp. millefolium except for S. cerevisiae. In addi- tion to this, DMSO showed no activity. Table 1. Inhibition zones of essential oil of A. millefolium subsp. millefolium (mm). Fungi A. millefolium subsp. millefolium Nystatin DMSO C. albicans 17 30 - C. tropicalis 20 30 - C. parapsilosis 15 25 - S. cerevisiae 30 17 - 155 | Aydin & Sevindik Achillea millefolium L. subsp. millefolium essential oil’s antifungal effect European Journal of Biological Research 2018; 8 (3): 153-156 Table 2. MIC values of essential oil of A. millefolium subsp. millefolium (µ l/ml). Fungi A. millefolium subsp. millefolium C. albicans 10 C. tropicalis 5 C. parapsilosis 1.25 S. cerevisiae 2.5 Table 2 shows values of MIC. The values range from 1.25 to 10 µl/ml for A. millefolium subsp. millefolium. Essential oils exhibited the lowest MIC value against C. parapsilosis. El-Kalamouni et al. [19] examined antifungal activity of essential oil of A. millefolium collected from France and it was demonstrated that the essential oil were inhibited the growth of Rhizopus stolonifer, Verticillium dahliae, Colletotrichum gloesporoides, Botrytis cinerae and Aspergillus niger. MIC values were found as 1.6 mg/ml, 3.1 mg/ml, 3.4 mg/ml, 3.6 mg/ml and 4.7 mg/ml, respectively. Karamenderes et al. [20] revea- led that essential oil of A. millefolium subsp. millefolium was active against C. albicans. Like- wise, we found that essential oil of A. millefolium possessed affect on the growth of C. albicans. Falconieri et al. [21] studied antifungal activity of the essential oils of flowering aerial parts of wild A. millefolium growing on the Mediterranean coast (Sardina Island, Italy) and on the Atlantic Coast. Both of the essential oils inhibited C. albicans (MIC: 2.5 µ l/ml), C. tropicalis (MIC: 2.5 µ l/ml) and C. parapsilosis (MIC: 2.5 µl/ml). In our study, MIC values were found as 10 µ l/ml, 5 µ l/ml and 1.25 µ l/ml against C. albicans, C. tropicalis and C. parapsilosis, respectively. The difference might be arising from several factors like local, climatic, seasonal, and experimental conditions [22]. Candan et al. [23] reported that essential oil of A. millefolium subsp. millefolium had activity against C. albicans. Similarly, we found activity against C. albicans. 4. 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