Antimicrobial, antioxidant and cytotoxic activity on human breast cancer cells of essential oil from Pinus sylvestris. var mongolica needle

Pinus sylvestris. var mongolica is a major source of timber in Mongolia. The logging process makes many kinds of valuable biomass including bark, cones, and needles, which can be used for obtaining essential oil. The essential oil from the needles of wild growing Pinus sylvestris. var mongolica growing in Mongolia was chemically analyzed and its antibacterial, antifungal and cytotoxic activities were evaluated. The chemical analyses identified 101 compounds in the essential oil with the major compounds of α-pinene (29.87 %), limonene + β-phellandrene (16.15 %), camphene (4.95 %), bornylacetate (4.34 %), and β-pinene (3.88 %). This oil possessed the inhibitory activity against B. subtilis, S. cerevisiae, S. aureus and E. coli, successively with minimum inhibition concentration of 0.125, 0.1, 3.0, and 10.0 μg/mL. Importantly, the oil at 50 μg/mL and 100 μg/mL inhibited the growth of MCF-7 cells by 45.3 % and 99.7 %. The half of inhibition concentration of DPPH (2,2-Diphenyl-1-picrylhydrazyl) radical scavenging was 14.36 ± 0.28 mg/mL. The results, therefore, suggested that the essential oil of a Mongolian Scotch pine could potentially be used as a preservative material in cosmetic and food products, as a bioactive agent in anti-inflammatory and wound healing products in view of its antibacterial activity. Given our findings that this essential oil has such profound activity against MCF-7 cancer cells, a further investigation concerning the full extent of this essential oil’s anticancer activities seems warranted. Furthermore, given the promising antimicrobial effects of this essential oil against various bacterial species, an investigation concerning its effect against drug-resistant bacteria would be of immense interest.

compounds with various pharmacological properties of the Scotch pine [1]. The EO of Scotch pine needles is a colorless and pale yellow liquid that smells like drybalsamic, turpentine and it possesses wide therapeutic effects, including antibacterial [8,9], antifungal [10], antiseptic [11], and anticancer [12]. The EO has been used in the diseases of the respiratory system like cough or catarrh and applied commonly in massages, clinical showers, and packs [1,5] Many studies have currently focused on investigating the new molecules with therapeutic properties in Scotch pine based on its traditional usage. However, there is still limited information on the biological activities of the essential oils from the needles of Mongolian Scotch pine. This study aimed to approve the potential of pharmacological utilization of the needle essential oils from Pinus sylvestris. var mongolica growing in Mongolia. This is the first report on the antimicrobial and anti-oxidant activities and the cytotoxic effect on breast cancer cells of the essential oil. Isolation of essential oil: The pine needles were dried and crushed to 4 -6 cm. The air-dried aerial pine needles (70 -80 g) were further hydro-distilled in the Clevenger type apparatus for 3 h. The essential oil yield was 0.4 %. The essential oil was dried over anhydrous calcium chloride, then put away in fixed vials at 4 °C until examination. Gas chromatography -mass spectrometry analysis (GC-MS): GC analysis of EO has performed on Hewlett Packard HP 5890 II Gas Chromatography with fused silica DB-Wax column (30 m x 0.25 mm; film thickness: 0.25 µm). Nitrogen was carrier gas with a linear velocity of 38 mL/min, the split ratio in 30 : 1. The temperature of the detector and injector were 250 °C, column temperature was programmed from 80 °C to 200 °C at a rate of 2 °C/min. The EO sample (1 %) in dichloromethane was injected with an amount of 0.5 µL. Quantitative data is determined by an electronic integration of the flame ionization detector (FID) peak area. GC/MS analysis was conducted on an instrument HP 5971A with MS detector 5890 II, was operated in EI mode (70 eV). It is using a Supelcowax 10 column (60 m x 0.25 mm; film thickness: 0.25 µm); He was as a carrier gas with linear velocity 10 mL/min, split ratio 30 : 1. The column temperature was programmed from 80 °C to 120 °C at a 3 °C/min rate and the injector and detector temperatures were 250 °C, 280 °C respectively. The identification of the separated components is obtained by matching with the library data of massspectra and comparing Kovat's indices with those genuine components with published data.

Culture medium and inoculum preparation:
Bacillus subtilis MNL-0919, Bacillus cereus MNL-0920, Saccharomyces cerevisiae MNL-0921, Paeonia anomala MNL-09196, Geotrichum candidum MNL-09102, Staphyllacocus aureus, Aspergillus niger, Penicillium sp., and Fusarium sp. were used as test organisms in the study from the microbial collection of the Institute of Biology, Mongolian Academy of Sciences. Yeast extracts were sub-cultured into peptone dextrose (YEPD) agar for 5-7 days at 37 °C, bacteria and fungi were sub-cultured at Potato dextrose agar for 24 h, respectively. Mueller-Hinton broth was used as a suspension for colonies from bacteria plates. Fungi were suspended in Sabouraud dextrose agar, and turbidity was coordinating to 0.5 McFarland standard (10 8 CFU/mL). Then the organisms were incubated properly as specified for each species for a period of 18-24 h [13]. Determination antibacterial activity of the essential oil (Agar well diffusion assay): The antimicrobial activity of EO was tested against Gram-positive bacteria B. cereus, B. subtilis, S. aureus, yeast P. anomala, S. cerevisiae, and G. candidum using the disc-diffusion method [14]. Standardized inoculum of each bacteria and yeasts in the amount of 100 µL were spread onto sterile Muller-Hinton Agar and Sabouraud Dextrose Agar, respectively. A well with 8 mm diameter was cut from the agar; subsequently, 100 µL of the pure and diluted essential oils filled each well, then the plates were incubated as appropriately, as specified for each organism at 28 -40 °C for 2 or 3 days, each sample was tested in triplicates. The antimicrobial activity of essential oils was formed clear zones by inhibiting microbial growth. The clear zone was measured in millimeters at 24 and 48 hours after the incubation. 50 % dimethyl sulfoxide (DMSO) solution was used as a solvent and a control. Determination of the essential oil on growth parameters of micro-organisms: Suitable volumes (40, 20, 10, 5, and 2.5 µL) of the EO were straightforwardly added to 40 mL of ME stock bringing in concentration from 1 to 0.0625 μL/mL. Then, the broths, including the EOs with approximately 10 5 CFU/mL culture suspensions incubated under smooth shaking for 48 h at 28 -37 °C, appropriately to each organism in Erlenmeyer flasks with cotton wool ventpeg and, further sealed with parafilm. Samples were collected every 1 or 2 h. The absorbance of samples was measured at 580 nm; absorbance change revealed the growth rate per time unit (1/h). The growth rate was evaluated by determining the slope of the straight line matched on the growth curves in the exponential phase. The length of the lag phase was calculated by the X value of the straight line at the initial absorbance. All of the calculations were performed by MS Excel. The calibration curve of a cell count versus absorbance at 580 nm gave the maximum total cell count formed in the stationary phase. Measurements were done three times for each sample.

Determination of minimal inhibitory concentrations (MICs) of the essential oil:
Macro dilution assay was used to determine the MICs of the EO, [15]. For the assay, 1 mL of EO in concentrations from 0.0625 µL/ mL to 1 µL/mL were added to 30 mL of ME medium in Erlenmeyer flasks, followed by incubation with approximately 10 5 CFU/mL bacteria and yeast for 24 h at 28 °C or 37 °C respectively. Then, the absorbance of the suspensions was measured at 580 nm. MICs were taken the most reduced concentration at which no noticeable growth happened where any colonies were framed in plate count assay. Negative controls contained EO components in ME medium. The absorbance increments measuring over 5 % at time 0 were served as growth positive samples. Cell culture: Human ER-positive breast cancer MCF -7 cell was bought from Korean Cell Line Research Foundation and was cultured in Dulbecco's Modified Eagle Medium compounded with 10 % fetal bovine serum and 1 % penicillin. The cells were incubated in a humidified atmosphere, including 5 % carbon dioxide at 37 °C. In vitro cytotoxic assay: The effects of EO on the viability of malignant cells were determined by ez-cytox cytotoxic assay [16]. Briefly, human breast cancer cells (MCF -7) were grown in 96 -well microtiter plates, with each well containing 10 4 CFU/mL. After 24 h, 10 μL of test samples with concentrations 50 and 100 μg/mL dissolved in DMSO were added to each well. One plate without a sample is considered as a day 0 control. After cell culturing during 48 h at 37 °C, ez-cytox were fixed, followed by determination of optical densities at 450 nm using a Microplate Reader (Tecan, Switzerland). The percentage of growth inhibition was calculated using the following equation: Where, OD is absorbance values or optical density. Etoposide, a potential anticancer agent, was used as a positive control.

Determination of antioxidant activity (2,2-Diphenyl-1-picrylhydrazyl free radicalscavenging capacity) of the essential oil:
Measurement of the 2,2-diphenyl-1-picrylhydrazyl (DPPH) (Sigma-Aldrich) radical scavenging capacity was performed according to Karamać et al [17]. Briefly, 2 mL methanol solution of 0.5 mmol/L DPPH was mixed with 5, 10, 20, 40 mL of different concentrations of EO of Mongolian Scotch pine. After 20 min incubation, the absorbance was measured at 517 nm with a spectrophotometer. Methanol was served as the positive control. The percentage of free radical-scavenging capacity was calculated by the following equation:

Radical scavenging capacity (%) = (A blank − A sample )/A blank × 100
Where, A sample is the absorbance of DPPH with essential oil, A blank is the absorbance of DPPH with methanol. All measurements were carried out in triplicate and reported as the average value. The results were expressed as half of minimum inhibition concentration-IC 50 (mg/mL) and trolox equivalent per mL of EO or fraction (μg TE/ mg EO). Statistical analysis: Antioxidant data were calculated and expressed as concentrations, at which 50 % of free radical was scavenged (IC 50 values ± standard deviation). All experiments were performed in triplicate and the Excel software was used for the calculation of IC 50 values. P < 0.05 was determined to be significant.

RESULTS AND DISCUSSION
Chemical compositions: The essential oil (EO) of needles from Mongolian Scotch pine obtained by hydrodistillation showed amber color with a mild aromatic odor. The average yield was 0.4 % on a dried weight basis. The chemical composition of the oil is presented in Table 1. A total of 101 constituents, representing 98.36 % of the total EO, were identified by GC/MS. The results showed that monoterpene hydrocarbons (59.20 %) were dominated group and sesquiterpenes (19.29 %) resented the second largest group. The major compounds were the monoterpene α-pinene, followed by limonene + β-phellandrene, camphene, bornyl acetate, ∆-cadinene and β-pinene. In general, the monoterpenes significantly release into the air because of their high enough vapor pressures at normal atmospheric conditions [18]. The published report has previously shown that the chemical compositions of the EO of Scotch pine consist mainly of 50 -90 % monoterpene hydrocarbons, and the other components are sesquiterpene hydrocarbons as well as the oxygenated mono-and sesquiterpenes. The EO is mainly dominated by α-pinene, camphene, β-pinene, 3-carene, β-myrcene, limonene, β-phellandrene, p-cymene, terpinolene, bornyl acetate, and β-caryophyllene, and diterpenes (isopimaral and isoabienol) occurred as mains in few samples [19,20]. Within the cases, the principal components including α-pinene, 3-carene, and β-phellandrene or β-pinene clarified 37.6 %, 23.6 %, and 10.1 % of the whole change, individually, permitting the visualization of more than 70 % of the EO of Scotch pine. For that reason, chemically, Scotch pine can be divided into three groups: α-pinene, 3-carene, and β-phellandrene or β-pinene based on the amount of those compounds accumulated in needle EO [20]. The needles EO of Mongolian Scotch pine belongs to α-pinene chemotype similar to Scotch pine native to Kosovo, Slovakia, Greece, and Russia (Siberia) [1]. They are also rich in α-pinene, camphene, β-pinene, limonene or β-phellandrene which possess high biological activities including antimicrobial, antiinflammatory antimetastatic, and apoptotic effects [21 -25]. Moreover, the compositions of EO of Scotch pine were studied in different geographical areas since the contents of the main compounds and the biological activities of EO are suggested to be varied considerably depending on the geographical areas [3].  Table 3.  Table 3. Effects of the essential oil on the duration of lag phases of bacteria and yeast Notice: Lag phase: h, mean ± SD, Growth rate : 1/h, mean ± SD, SD: Standard deviation*, * Various letters point to significant differences (p<0.05).
The lag phases of the tested bacteria and yeast strain were considerably prolonged by increasing the EO concentrations in all of the cases. The most sensitive species was approved as B. subtilis, the lag phases of this bacterium could lengthen over 48 h at low concentrations of the EO, and, there was no growth at 0.125 μL/mL of it. For S. cerevisiae, the decrease of the lag phase of this strain was observed at 1.0 μL/ mL concentration during 48 h investigation time. The maximum total cell counts were calculated from the growth curves, where indicated the different variance from the control. A moderate change on maximum total cell counts of B. subtilis was shown at a low concentration of EO, whereas 0.5 μL/mL EO decreased to no growth of the maximum total cell counts in this bacterium. In the case of S. aureus, the maximum total cell counts fluctuated at all the concentration of EO. Besides, we also examined the effect of the EO on growth parameters of gram -negative bacteria E. coli and compared the minimum inhibitory concentrations (MIC) against three tested species. As described in the experimental part, the MIC was determined by measuring absorbance accordingly with the growth rate of S. aureus, B. subtilis and S. cerevisiae in ME medium, and the results are shown in Table 4.  [24,28]. β-Pinene and limonene could, therefore, explain the antimicrobial property [27 -29]. Mechanistically, the action of terpenes has been suggested to cause the membrane disturbance through lipophilic compounds and interaction with the intracellular sites. [30,31]. In addition, it is also possible that the components in lower percentage might be involved in some types of synergism with the other active major compounds [32]. As the most interesting observation from the current results, the MIC values were comparatively higher than the obtained results from the agar diffusion method and the previous researches. The main components of the EO might have limited diffusion through the agar medium because of their high volatility during its incubation [18]. In the MIC assay system, the essential oil was tested in an emulsion form, and the less dense oil was partitioned well to the liquid culture system. This may enhance the antibacterial effects in the MIC assay. Antioxidant activity: The antioxidant activity of EO of Mongolian Scotch pine was assessed in a series of in vitro assays. The DPPH assay is a measured form of DPP-H produced by donating hydrogen atoms or electrons of the essential oil for in transformation of DPPH· using a spectrophotometrical method.
In Table 5, the results were expressed as IC 50 and trolox equivalent per mg of EO or fraction (μg TE/mg EO).

DPPH value Concentration
Essential oil IC50 (mg/mL) 14.36 ± 0.28 Trolox IC50 (µg/mL) 9.54 ± 0.14 Essential oil TEAC (µg TE/mg) 0.66 ± 0.02 Notice: ± mean -Standard deviation treatment. Previously, the extracts prepared from the needles of various pine species have been shown to exert some anticancer effects [12,39,40]. Hoai et al. reported that the EO of Estonian Scotch pine needle possessed a strong cytotoxic activity on both MCF-7 and MDA-MB-231 cell lines, and those IC 50 values were 28.67 µg/mL and 29.23 µg/mL, respectively [12]. The authors stated that the oil could not only responsible for chemoprevention but also for chemotherapeutic to endocrine insensitive breast tumors. Therefore, EO of Mongolian Scotch pine needle may seem to contain some compounds or fraction with a high potential to be developed as candidates for prevention or therapeutic adjuvants to breast cancer.

CONCLUSIONS
For the first time, the present work provides the research results on the chemical compositions and some biological activities of the essential oil of Pinus sylvestris. var mongolica. As compared with the previous studies [26,29,37], the large differences in antimicrobial and antioxidant activities seem not related to the major chemical components in the essential oil, rather with observed possibility of the synergistic effects of the minor components to the major components. Besides, our results showed the essential oil could be an easily accessible source of the potential candidate for developing novel therapeutic anticancer agents. Given our findings that this essential oil has such profound activity against MCF-7 cancer cells, a further investigation concerning the full extent of the anticancer activities of this essential oil seems warranted. Furthermore, given the promising antimicrobial effects of this essential oil against various bacterial species, an investigation concerning its effect against drugresistant bacteria would be of immense interest. A detailed study on its anticancer and antibiotic activity against drug resistant bacteria is required based on the results in this work.