Essential oils of basil cultivars selectively affect the activity of antioxidant enzymes in glial cells


 The qualitative and quantitative composition of essential oils (EO) of Ocimum species, cultivated in high altitude Armenian landscape was quite different and the main components of O. basilicum var. purpureum; O. basilicum var. thyrsiflora and O. x citriodorum oils belong to the class of oxygenated monoterpenes. Investigated EOs possess remarkable antioxidant activity. They inhibit the tyrosinase activity, the enzyme responsible not only for the melanin production, but also for various aging-related metabolic processes. Investigated EOs had no any significant effect on catalase at the protein levels, but alter its activity in neuroglial BV-2 different cell lines. Treatment of the neuroglial cell lines with the sub-cytotoxic concentrations of three mentioned EOs influence also the activity of acetyl-CoA oxidase type 1. Practical Applications The microglial cells play a pivotal role as the neuroprotective agents against neuroinflammation. Different data included in the present article are stating that plant origin substances can play a role of regulators of enzymatic antioxidant capacity of cells. EOs extracted from the Ocimum different cultivars are able to trigger the activity of acetyl-CoA oxidase type 1 (or palmytoil-CoA oxidase type 1), which can serve as a basis of regulation of redox deviation in WT cells. So, it can be suggested them to be applied for the prevention of some processes, which can influence on the aging, as the process of ageing is commonly associated with mitochondrial dysfunction, oxidative stress caused by the increased level of free radical production, dysfunction of the microglia, high blood pressure and so on.


Introduction
. Focusing on these data, we aimed to reveal some mechanisms of 61 influence of all three basil EO on the microglial cells, as recent-years research data state that 62 these oils have anti-aging and neuroprotective action, and as it is supposed, against also some 63 forms of neurodegenerations [12; 13]. The process of ageing is commonly associated with 64 complex changes in the organism such as mitochondrial dysfunction, oxidative stress caused by activity of any metabolite is the ability to inhibit the tyrosinase or tyrosine hydroxylase activity, 69 which can prevent the high-blood pressure [19]. These enzymes catalyze the synthesis of 70 dopamine in melanocytes which transports to the neuroglial and adrenal cells with blood flow, 71 where a copper-dependant enzyme dopamine β-oxidase, in the presence of vitamin C, causes its 72 hydroxilation to catecholamine transmitters (norepinephrine and epinephrine), which are able to 73 increase the blood pressure [19; 20]. Some researchers mention, that various models of 74 neurodegenerative disorders are useful for studying the neuroprotective activity of biologically 75 active metabolites. The oxidative neuronal damage in the ageing brain is also associated with the 76 decline in the production and functional activity of antioxidant enzymes [21].

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Essential oil extraction 98 Essential oils were extracted from air dried plant material (aerial parts only) by hydro-99 distillation, using a Clevenger-type apparatus, and the process lasted 3 h as described before [3]. 101 The gas chromatography (GC) mass selective (MS) analysis of the essential oils was 102 performed using a Hewlett-Packard 5890 Series II gas chromatograph, fitted with a fused silica 103 HP -5MS capillary column (30 m × 0.25 mm, in thickness 0.25 μm). Determination of chemical 104 composition was carried out as described previously [3]. The oven temperature varied from 40-105 250 °C with the scanning rate of 3 °C/min. Helium (purity 5.6) was used as a carrier gas at a 106 flow rate of 1 mL/min. The GC was equipped with Hewlett-Packard 5972 Series MS detector.

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The MS operating parameters were ionization voltage 70 eV and ion source temperature 250 °C.

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The diluted samples of essential oils (1/100, v/v in HPLC methanol) of 1 μL had been injected 109 manually. To avoid overloading the GC column, the essential oils were diluted 1:100 (v/v) in  116 Cells, plated in 96-wells plates, were treated for 24-72 h with different concentrations of EO 117 (50 -5.10 -4 µLmL -1 in dimethylsulfoxide). Cells were incubated for 2 h with MTT dye, followed 118 by the absorbance (Abs) measurement at the 570 nm with a microplate reader and the sub-

141
According to the data, obtained from the quantitative and qualitative analysis of the 142 investigated essential oils more than 40 compounds were isolated and most of them identified for 143 each essential oil sample. All dominant components were determined as oxygenated 144 monoterpenes (linalool, methyl chavicol, nerol and geranial) ( Table 1).

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Regarding to our investigations, the sub-cytotoxic concentrations of essential oils were 146 identified which was 0.5 µL/mL for O. basilicum var. purpureum and O. basilicum var. 147 thyrsiflora essential oils, but for the O. x citriodorum essential oil this parameter was higher 148 twice (1 µL/mL) for both microglial cell lines (p < 0.05) (Fig 1 a- ˃ 0.05). Treatment of Acox1 -/cells with essential oils did not affect the activity of catalase (Fig. 3).  (Fig. 2, Fig. 4). The treatment with O. basilicum var. purpureum brings to the significant 169 changes in the SOD activity of BV-2 WT cells (p < 0.05), but there were not any statistically significant 170 data in other cases (Fig. 5).

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As described above, the oxygenated monoterpenes are the main substances in the composition of 173 EO of all three Ocimum cultivars. There is not any direct evidence, which will be able to state whether 174 the described biological activity is due to these substances or not. But one thing is clear, that even small 175 deviations in the chemical composition of any EO can alter their activity [3; 28]. So, the aim of this 176 work was not only the indication of the activity of these EOs, but also to find a common mechanism or 177 feature of influence of these substances which can be considered in their practical application in the 178 future. There is a huge number of literature data stating that EOs generally express the antibacterial and activity of acetyl-CoA oxidase type 1 (or palmytoil-CoA oxidase type 1), which can serve as a basis of 207 regulation of redox deviation in WT cells. So, it can be suggested them to be applied for the prevention 208 of some processes, which can influence on the aging, as the process of ageing is commonly associated 209 with mitochondrial dysfunction, oxidative stress caused by the increased level of free radical production, 210 dysfunction of the microglia, high blood pressure and so on.  The significance is presented with the Student-t test: p < 0.05 for BV-2 both cell lines.