Chemical composition and some biological activities of the essential oils from basil Ocimum different cultivars

The three basil cultivars (O. basilicum var. purpureum, O. basilicum var. thyrsiflora, and O. x citriodorum) were grown from the seeds sown in the greenhouse, with subsequent transplantation of the seedlings to the same field, in the Kotayk Region of Armenia, where they have been growing side by side, at an elevation of 1600 m above the sea level. Plant materials were collected during blossoming period (July–August, 2014). The plant materials were identified at the Institute of Botany, National Academy of Sciences of Armenia, Yerevan (Armenia). The plants were not included in the herbarium as there were cultivated species and not typical for the flora of Armenia. The samples of basil cultivars are available at the Department of Microbiology & Plants and Microbes Biotechnology, Biology Faculty, Yerevan State University, Yerevan, Armenia.

Essential oils were extracted from air dried plant material (aerial parts only) by hydro-distillation, using a Clevenger-type apparatus and lasted 3 h. The distilled essential oils had been dehydrated with anhydrous sodium sulphate and stored at 4 °C in dark airtight bottles until further analysis [12].

The antibacterial and antifungal activity of the essential oils was determined by the agar diffusion method [13]. This method was preferred over the dilution method because of low solubility of essential oils in water and in meat peptone broth. The following concentrations of essential oils were used: 150; 100; 50; 25; 12.5; 6.25 μL/mL; dimethyl sulfoxide (DMSO) was used as the solvent. The 100 μL of each oil solution was introduced to the wells in the agar with test microorganisms. Different Gram-positive (Bacillus subtilis WT-A, isolated from metal polluted soils of Kajaran, Armenia; Staphylococcus aureus MDC 5233 (Microbial Depository Center, Armbiotechnology Scientific and Production Center, Armenia; laboratory control strain) and Gram-negative (E. coli VKPM-M17 (Russian National Collection of Industrial Microorganisms at the Institute of Genetics and Selection of Industrial Microorganisms, Russia; laboratory control strain), Pseudomonas aeruginosa GRP3 (Soil and Water Research Institute, Iran) bacteria and ampicillin-resistant E. coli dhpα-pUC18 were used. Bacterial cultures were grown on Mueller-Hinton agar. Ampicillin (25 μg/mL) as a positive control and DMSO as a negative control were used. The yeasts (Candida albicans WT-174 isolated from infected vaginal microbiota of hospitalized patients (clinical strain) and Debariomyces hansenii WT (French National Institute for Agricultural Research, France; laboratory control strain) were grown and maintained on Sabouraud-dextrose agar for 24 h at room temperature. As the positive control fluconazole (25 μg/mL) was used. Data were expressed in minimal inhibitory concentrations (MIC) values.

The selected pieces of nutrient medium from the zones of microorganism growth absence were transferred to the nutrient medium corresponding to each microorganism and then they were incubated for 2–3 days at appropriate temperature to determine the bacteriostatic or bactericidal action of the oils. The action of oils is evaluated as bacteriostatic in case of renewed growth of test-microorganisms after the re-cultivation.

Free radical scavenging ability of the essential oils was tested using ethanol solution of 1,1-diphenyl-2-picrylhydrazyl (DPPH) [14]. Catechin was used as a positive reference. Sample solution contained 125 μL (1 mM) DPPH, 375 μL ethanol and 500 μL of test-solution (essential oils or catechin with different concentrations). In the control solution the test-solution was replaced by ethanol. The absorbance was measured at the wavelength of 514 nm.

The radical scavenging activity was calculated using the following formula: Radical scavenging activity (%) = Ac–As/Ac × 100, where Ac is absorbance of control (DPPH without the addition of test solution), and As-the absorbance of the sample.

IC₅₀ calculated denote the concentration of investigated samples required to decrease the DPPH absorbance at 514 nm by 50%.

Tyrosinase inhibition colorimetric assay was carried out according to the method, as described [15, 16]. Each essential oil was dissolved in DMSO to obtain concentration of 20 mg/mL. These stock solutions were diluted to 600 μg/mL concentration in 50 mM potassium phosphate buffer (pH 6.5). Arbutin acid was prepared in similar way and used as positive control. 700 μL of each sample solution or positive control were combined with 300 μL of mushroom tyrosinase (333 Unit per mL in phosphate buffer, pH 6.5). After incubation at 20–22 °C for 5 min, 1100 μL tyrosine (2 mM) were added to each well. Plates were incubated at room temperature for 30 min and the absorbance was measured at the wavelength of 492 nm using the spectrophotometer Genesys 10S UV–vis (Thermo Scientific, USA). Percent inhibition of tyrosinase activity was calculated according to the formula: inhibition (%) = 100-(W_sample/W_blank) × 100, where W is absorbance at 492 nm. W_blank is absorbance of control reaction (containing all reagents without test compound).

Experimental data (n = 4) were expressed as means with standard errors. The latter did not exceed 3% (if not indicated). The validity of differences between experimental and appropriate control data were evaluated by Student’s criteria (P) using Microsoft Excel 2010 with the help of T test function; P < 0.05 (if not indicated).

The results from the quantitative and qualitative analysis of essential oils constituents are presented in Table 1: the average yield of the essential oils was 0.2%. More than 40 compounds were isolated, detected and most of them identified for each essential oil sample. The dominant components were identified to be linalool, methyl chavicol, citral and nerol.

According to the data obtained, O. basilicum var. purpureum contains 57.3% methyl chavicol, with the second largest component being linalool (18%). This places the given variety of O. basilicum into methyl chavicol-rich chemotype. O. basilicum var. thyrsiflora belongs to linalool-rich chemotype, with concentrations of linalool and methyl chavicol being 68 and 20% respectively. These data are in a good accordance with the results reported by Sishu et al. [17]. For the essential oil from O. x citriodorum species the predominant constituents were identified to be citral (21%) and nerol (23%), therefore it could not be classified as belonging to any of the chemotypes mentioned above, but will rather form its own, nerol-rich chemotype. The data on O. x citriodorum are somewhat consistent with the similar results published by Carović-Stanko et al. [18] on essential oil distilled from the plant of the same species, except for the fact that there were more than 45 constituents of O. x citriodorum essential oil identified in the present study, as opposed to 20 components identified by Carović-Stanko et al. [18].

The present investigation revealed that Gram-positive bacteria tested were more sensitive to all three essential oils than Gram-negative bacteria (Fig. 1). Such tendency is also observed by other authors [19]. The essential oil of O. x citriodorum was quite active against B. subtilis and St. aureus, with the MIC of 3.125 μL/mL. The same MIC was recorded for the essential oil of O. basilicum var. thyrsiflora against St. aureus, and O. basilicum var. purpureum essential oil against B. subtilis. The MIC of O. basilicum var. thyrsiflora essential oil against B. subtilis and MIC of O. basilicum var. purpureum against St. aureus were twice as high, 6.25 μL/mL. The ampicillin-resistant E. coli bacteria also displayed sensitivity against the essential oils tested: thus the MIC values of O. x citriodorum and O. basilicum var. purpureum against those bacteria were 6.25 μL/mL, while O. basilicum var. thyrsiflora displayed MIC of 12.5 μL/mL. The action of the essential oils on the all bacteria in this study was evaluated as bactericidal.

All three essential oils have also displayed high antifungal activity, with O. x citriodorum being the strongest antifungal amongst them: MIC of O. x citriodorum against D. hansenii and C. guillermondii were 1.56 and 3.125 μL/mL, respectively (see Fig. 1).

The results of radical DPPH assay for of O. x citriodorum, O. basilicum var.purpureum, O. basilicum var. thyrsiflora essential oils are shown on Fig. 2. The highest antioxidant activity was demonstrated by O. basilicum var. thyrsiflora essential oil: IC₅₀ value for it was equal to the standardized Grapefruit Seed Extract which was used as a control sample (2.5 μL/mL). The antiradical activity for the other two basil species was lower: IC₅₀ value for O. x citriodorum essential oil was 20 μL/mL and for O. basilicum var. purpureum was 22 μL/mL. These results were somewhat unexpected, since usually the oils with higher phenolic content are the ones exhibiting higher radical scavenging abilities, whereas in our case the highest antioxidant properties were displayed by the cultivar with the highest linalool (terpene alcohol) content.

The enzyme tyrosinase inhibition abilities of all three oils were also assessed as a part of our efforts to find a natural treatment for hyper-pigmentation skin disorder. The values for tyrosinase inhibitory activity of O. basilicum var. thyrsiflora, O. basilicum var. purpureum and O. x citriodorum essential oils and arbutin acid (positive control) were calculated to be 20.1 ± 1.4%; 11.5 ± 0.3%; 17.4 ± 0.9% and 81.5 ± 2.6%, respectively (Fig. 3).