Determination of the phytochemical profile , in vitro the antioxidant and antimicrobial activities of essential oil from Arbutus andrachne L . wood growing in Turkey

This study aimed to determine the chemical composition, antioxidant and antibacterial activities of the essential oil of Arbutus andrachne L. wood, collected from Köyceğiz region of Muğla. According to GC-MS results 25 compounds were identified, accounting for 80.5 % of the wood oil. The predominant compounds were cinnamyl alcohol (21.97 %), 4-tertbutylcyclohexyl acetate (16.59 %) and isobornyl acetate (15.37 %). The investigated wood oil showed significant bioactivities. Furthermore, this study showed that A. andrachne L. essential oil with preferential constituents can be used as potential antioxidant and antibacterial agents for food, perfume and pharmaceutical industries.


Introduction
Arbutus andrachne L. (Greek or Eastern strawberry tree) is among the two species of Arbutus genus which belongs to the family Ericaceae (Serçe et al., 2010).It is an evergreen small tree which is native to the Mediterranean region and southwestern Asia (Markovski, 2017;Bertsouklis and Papafotiou, 2013;Dönmez et al., 2016).The red coloured edible berries of strawberry tree have traditionally been used for human consumption in many countries (Molina et al., 2011;Tardío et al., 2006;Çavuşoğlu et al., 2015).The Arbutus andrachne L. is called as "Sandal" tree (Dönmez, 2018) and the berries of Arbutus species are called as "Davulga" or "Kocayemis" in Turkey (Şeker and Toplu, 2010).Many food products can be prepared from berries in a wide range including alcoholic beverages (liqueur spirits and wine), jam, fruit jelly and marmalades (Ayaz et al., 2000;Pallauf et al., 2008;Oliveira et al., 2009).
Screening natural plants based essential oils and extracts for biological activity has been a historically significant research field that has resulted in the development of several phytopharmaceuticals, perfumes and natural antioxidant&antibacterial agents in food industry (Abu-rish et al., 2016;Djouahri et al., 2015;Sıcak et al., 2017).In this context, there is an increasing demand on the medicinal plant studies with the essential oils composition and their bioactivity potentials. A. andrachne L. is also a special plant used in folk medicine due to many medicinal properties of its fruits and leaves (e.g.astringent, antidiarrheal, depurative, laxative and urinary antiseptic etc.) (Mostafa et al., 2010;Oliveira et al., 2009).Indeed, the composition and potent antioxidant activity of the extracts of A. andrachne L. fruits and leaves has been verified in the preliminary studies (Serçe et al., 2010;Şeker and Toplu, 2010).
To the best of our knowledge there is no comparative work has been published on the evaluation of chemical composition and bioactivity properties of A. andrachne L. wood essential oil from Köyceğiz.The aim of the present study was to evaluate chemical composition, antioxidant and antibacterial activity of A. andrachne L. wood branch oil obtained by hydrodistillation method.For this purpose, in vitro antioxidant activities were evalauted by using four complementary assays, namely, β-carotene/linoleic acid assay, cation radical scavenging activity (ABTS assay), free radical scavenging activity (DPPH assay) and cuprac reducing power (CUPRAC assay).In vitro antimicrobial activities were determined by using broth microdilution method againsts Escherichia coli (ATCC 25293), Bacillus subtilis (ATCC 6633), Staphylococcus aureus (ATCC 25925), Pseudomonas aureginosa, Candida albicans and Candida parapsilosis.

Plant material and Essential oil extraction
A. andrachne L. wood were collected from Köyceğiz region of Muğla, Turkey in 2017.It was identified at the Herbarium of Biology, Faculty of Science, Muğla Sıtkı Koçman University, Turkey.The plant sample was confirmed by comparing it with the specimen located at the stated herbarium.
The essential oil extraction was performed via hydrodistillation which is generally known to be one of the most extensively used techniques to extract volatile compounds from several matrix (Golmakani and Rezaei, 2008).The essential oil was obtained from approximately 100 g of the dried A. andrachne L. wood by hydrodistillation method for 2 hours.The resulting mixture was taken to the separation funnel and liquid-liquid extraction was performed by adding hexane to the water-oil mixture.Anhydrous magnesium sulphate was added to the organic phase and the water in the mixture was filtered to remove the salt.Finally, the solvent of the organic phase was removed using the rotary evaporator under vacuum.

Chemical composition
The qualitative and quantitative composition of essential oil analysis were conducted at Giresun University central Research Laboratories Application and Research Center by GC-MS 7890A-(5975C inert MSD) instrument equipped with an Agilent 19091S-433 column (30m X 250 μm film X 0.25 μm thickness).Helium was used as a carrier gas.The temperature was raised from 50°C to 270°C by an increase of 5°C / minutes and then 25 minutes of waiting time were implemented during the analysis.Injection port and detector temperatures were 250°C and 260°C, respectively.Characterization of essential oil components was based on the library (Wiley and NIST) comparison with the mass spectra of the injected essential oil samples.

Antioxidant activity
Solutions of essential oil of A. andrachne L. were prepared at four different concentrations as 400-200-100-50 ppm in EtOH.EtOH was used as a control, while BHA and α-tocopherol (α-TOC) were used as antioxidant standards for comparison of the activity tests.The results were given as 50% concentration (IC 50 ) for ABTS .+scavenging activity, β-carotene-linoleic acid and DPPH˙ assay while in the CUPRAC assay are expressed as A 0.5 .
The 50 µL (415 µg/mL) of A. andrachne oil were dissolved at 1 mL with dimethyl sulfoxide (10 % DMSO).The experiment were performed on 96-well microtiter plates and firstly 50 µL of Mueller Hinton Broth (MHB) medium were added into all wells.Two-fold serial dilutions of 50 µL oil was made on all x-axis along of elisa plate.Columns 11 and 12 were used as negative and positive controls.Finally, 10 μL culture of microorganisms was inoculated on all wells except medium control wells.All of the plates were incubated at 37°C for 24 hours, the growth (turbidity) was measured at 600 nm for bacteria, 415 nm for yeasts.For MIC analysis, the optical density was read both before, T0 and after 24 hours-incubation, T24.For each plate, MIC were calculated using the following formula: The OD for each replicate at T0 was subtracted from the OD for each replicate at T24.The Percent growth = (ODtest /OD control)x100.Percent Inhibition = 1-(OD test well/OD of corresponding control well)x100 for each row of the 96-well plate.
The dose-response curves obtained from plotting the linear of the concentration of the oils against the resulting percent inhibition of microbial growth were obtained with the regression analysis, giving an R 2 value.MIC (the lowest concentration of test material which results in 99.9% or 50% inhibition of growth) were calculated using the R 2 formula on inhibition curve (Patton et al., 2006;Erdoğan et al., 2017).

Statistical analysis
All data on biological activity assay studies were the averages of triplicate analysis.All the results are presented as 50% concentration (IC 50 ) (%).Data were recorded as mean ± SEM (standard error of the mean).Significant differences between means were determined by Student's-t test and p values < 0.05 were regarded as significant.
The SPSS-one way ANOVA, Tukey test were performed for between MICs and % Cell viability values.The experiment was repeated at least 3 times.Differences were considered significant at p ≤ 0.05.

Antioxidant activity
The in vitro antioxidant activity of essential oil obtained from the wood of A. andrachne L. collected from Köyceğiz-Turkey was reported in this study for the first time.The antioxidant activity results of A. andrachne L. essential oil given Table 2.According to the β-carotene/ linoleic acid assay results, the oil exhibited better lipid peroxidation inhibitory activity value of (IC 50 ) 3.22±0.18µg/mL than standard α-TOC (IC 50 =4.48±0.17µg/mL).In the ABTS ˙+ assay, essential oil (IC 50 : 3.47±0.22µg/mL) showed better cation radical scavenging activity than standard α-TOC (IC 50 =54.97±0.99µg/mL).The essential oil demonstrated activity IC 50 value of 48.31±0.13µg/mL in DPPH free scavenging activity, than standard BHT (IC 50 =54.80±0.78µg/mL).The essential oil indicated better the CUPRAC activity with an A 0.5 value of 27.25±0.01µg/mL, than α-TOC (A 0.5 =40.55±0.04µg/mL) using as a pharmaceutical standard.
It is prominent to point out that the antioxidant activity of the essential oils depends on the species, harvest time and geographical attitude (Mechergui et al., 2016) which can be explained by fact that the oils chemical composition varies because of extrinsic and intrinsic factors (Dutra et al., 2019).The determined antioxidant activities were related to compounds, such as cinnamyl alcohol, isobornyl acetate and 4-tert-butylcyclohexyl acetate which are commonly presented in the essential oils.It was shown that cinnamyl alcohol have significant DPPH scavenning potential (Suryanti et al., 2018).
The 4-tert-butylcyclohexyl acetate extracted from Decalepis hamiltoni was also found to be an effective antioxidant agent (Rayar and Manivannan, 2015).

Antimicrobial activity
The 24 hours incubation of A. andrachne L. oil with microorganisms was found to be statistically significant in terms of the resultant cell viability (p˂0.05)(Table 3).Accordingly, the lowest cell viability value 24-time incubation with A. andrachne L. oil were obtained in C. parapsilosis culture (48.28%), while the highest cell viability was in P. aureginosa (90.2%).
Generally, all tested microorganisms were sensitive to A. andrachne L. oil at MIC 99.9 range of 8.3-18.9µg/mL.The MIC 99.9 and MIC 50 of A. andrachne L. oil results for E. coli was 9.7 and 5.8 µg/mL, B. subtilis 12.1 and 7.03 µg/mL, S. aureus 18.9 and 11.7 µg/mL, C. albicans 13.4 and 7.2 µg/L, C. parapsilosis 8.3 and 5.3 µg/mL and P. aureginosa 10.1 and 7.5 µg/mL (Figure 1).Therefore, the maximum antimicrobial activity were determined against C. parapsilosis (8.3 µg/mL) while the minimum activity were determined against S. aureus (18.9 µg/mL).Although there is a few information in literature about A. andrachne L. oil's antimicrobial performance, extracts prepared from A. unedo, another type of Arbutus genus, leaves were showed antimicrobial effeciency against many microorganisms B. cereus, B. subtilis, S. aureus and S. epidermis, E. coli and P. aeruginosa and C. albicans, C. krusei, C. parapsilosis and C. glabrata at range of 0.1-20 mg/mL (Malheiro et al., 2012).In the study of Orak et al. (2011), antimicrobial and antioxidant activities of ethanol and methanol extracts of Arbutus unedo leaves were investigated to detemine a correlation between the extract and activity.The extracts showed no inhibitory effect against Escherichia coli, while the extracts exhibited antibacterial activity against S. aureus at concentration of 250 mg/mL.In the antioxidant assay, the EC 50 values of extracts were found between 0.42 mg/mL and 0.65 mg/mL (Orak et al., 2011).In another study, the methanolic, ethanolic, ethyl acetate and n-hexanic extracts from the leaves of Arbutus unedo exhibited an antibacterial activity against Escherichia coli, Staphylococus aureus, Listeria monocytogenes and Pseudomonas aeruginosa between the MIC of 0.2 mg/mL and 8 mg/mL (Bouyahya et al., 2016).In Kahraman and coworkers's study, the major component of essential oil extracted from Arbutus unedo flower and fruit were reported to be terpineol (16.3%) and hexadecanoic acid, respectively.In addition, they were showed moderate antibacterial activity against Listeria monocitogenes and Enterococcus faecalis (Kahraman et al., 2010).
These findings indicate that the antimicrobial and antioxidant activity of A. andrachne could be mainly attributed to its major compounds as given in previous studies.For example, Cinnamomum zeylanicum (cinnamon), rich in cinnamyl alcohol (8.21%), was reported to have strong antimicrobial and antioxidant activity (El-Baroty et al., 2010;Chang et al., 2001).The essential oil obtained from the leaves of Chamaecyparis obtusa, rich in isobornyl acetate, were found to have strong antibacterial activities against Gram (+) bacteria such as S. aureus, Bacillus cereus and some fungi such as C. albicans, C. tropicalis (Yang et al., 2007).

Conclusion
The wood essential oils have shown to have significant bioactivities including antibacterial, antioxidant, antiviral, antifungal and insecticide.The essential oil of A. andrachne after successive extraction were analysed by GC-MS.The cinnamyl alcohol, 4-tert-butylcyclohexyl acetate and isobornyl acetate was determined in the chemical characterization of the essential oil of A. andrachne as major compounds.The antimicrobial activity of essential oil was effective in the control of B. subtilis, C. albicans, C. parapsilosis, E. coli, P. aureginosa and S. aureus, which may be related to the well antioxidant activity of the components present in oil, such as cinnamyl alcohol, isobornyl acetate and 4-tert-butylcyclohexyl acetate.This study showed that A. andrachne L. essential oil with preferential constituents can also be used as potential antioxidant and antibacterial agents for food, perfume and pharmaceutical industries.Moreover, the findings obtained from biological activity assays showed that A. andrachne L. essential oil have been a promising candidate for the discovery of new drugs and the preparation of new natural products for aromatherapy and phytotherapy applications.However, furture in vivo studies should be carried out to verify such actions in different matrices.

Table 1 .
Chemical composition of A. andrachne L. essential oil

Table 2 .
Antioxidant activity of A. andrachne L. essential oil a