Antimicrobial efficacies of Brassica Napus L. essential oils/ nanoparticles composites

INTRODUCTION
Organic substances such as essential oils (EOs) are categorized as GRAS by the U.S. Food and Drug Administration as well as the European Legislation for materials intended to be in contact with food. Thyme, cinnamon, clove, basil, oregano, garlic, and basil oils have been intensively explored for the development of food packaging films due to their excellent antibacterial properties against foodborne pathogens [1-3]. A range of both biodegradable and non-biodegradable polymer films, such as chitosan, fish skin, whey protein isolate, low-density polyethylene, ethylene vinyl alcohol, poly(ethylene terephthalate), and polypropylene films, have either coated or incorporated with EO and evaluated for their antibacterial effectiveness [4]. Surprisingly, some studies are available in the literature on the use of PLA-based films as carriers of EOs for antibacterial packaging applications. In addition, there is lack of information with regards to the application of PLA-based EO films, especially on food systems such as cheese and meats. Recently, antibacterial activity of PLA-based films incorporated with cinnamaldehyde against gram-negative E. coli and gram-positive S. aureus was revealed [5]. Tests with these developed films were also found to be effective in reducing the mesophilic and psychrophilic bacterial counts of button mushrooms and extend its postharvest life [6]. In another similar recent study, PLA films containing an extract of Allium spp. EO were shown to have inhibitory effect against several molds, yeast, and pathogenic bacteria and those films were also observed to be effective for inhibiting the growth of aerobic, enterobacteriaceae, yeast, and mold on ready-to-eat salads for up to 7 days under refrigerated storage conditions [7-8]. Rapeseed, an annual plant belonging to the Cruciferous family (Brassicaceae), is one of the cultivated medicinal plants in Central Asia, North Africa and Western Europe [9,10]. Rapeseed oil helps lower blood cholesterol and strengthens blood vessels, preventing blood clots. Monounsaturated and polyunsaturated fatty acids, vitamins E, A, PP, B1, B2 and phytosterols were found in the chemical composition of rapeseed oil [11]. That is why it is considered as healthy, edible oil: the ratio of linoleic to linolenic acid amounts 2 and is higher balanced than in soybean oil [12]. This 7% of saturated fatty acids from canola oil is about half the level present in corn oil, olive oil or cottonseed oil. The most important in nature is the monounsaturated fatty acid (MUFA), an oleic acid. Tocopherols content in canola oil ranges from 0.5 to 0.9% [13]. Some researches show that Brassica extracts have a whitening effect and a skin-beautifying effect based on a melanin production inhibitory action and an anti-inflammatory action are specially mentioned. Like all oils with a high content of oleic acid and tocopherols, it has an accelerating healing and tissue regeneration effect, is suitable for general improvement of dry skin condition, relieves inflammation and irritation, restores elasticity, promotes better nutrition and moisturizing of the skin.

MATERIALS AND METHODS
Plant material was collected at the Kazakh Research Institute of Agriculture and Crop Production in Almaty during the ripening of seeds. Rapeseed extract was obtained by subcritical CO2 extraction.

Study of chemical compounds
The study of the chemical composition of rapeseed extract was carried out by gas chromatography with mass spectrometric detection equipped with an Agilent 7890B / 5977A, WAXetr column (30 m × 0.25 mm, thickness 0.25 mm). Data processing included determining retention times, peak areas, and processing of spectral information obtained using a mass spectrometric detector. 

Antimicrobial Study 
The analysis of antimicrobial activity was carried out by the method of two serial dilutions in a liquid nutrient medium [14,15]. A 108-well plate was used to determine antimicrobial activity. In all wells from 1 to 8, the nutrient broth MBH (for testing bacteria) or Saburo broth (for testing fungi) was poured in an amount of 0.5 ml. The working solution (in this case, the initial extract) was made in pure form (in a volume of 0.5 ml) in the 1st test tube. Next, serial dilutions were made, which were carried out by sampling the mixture (MBB (0.1 ml) + test drug (0.5 ml)) from a 2nd test tube in an amount of 0.5 ml into a third test tube already containing 0, 5 ml of broth. Thoroughly mixed and transferred 0.5 ml of the test sample in the broth from the 3rd tube to the 4th, also containing initially 0.5 ml of broth. This procedure was repeated until the required number of dilutions was achieved. 0.5 ml of the mixture is removed from the last tube. Thus, the following dilutions were obtained: 1: 1; 1: 2; 1: 4; 1: 8; 1:16; 1:32; 1:64; 1: 128; which corresponds to wells from the 1st to 8th test tube control culture. After a series of dilutions, 0.05 ml of test strains of microorganisms at a concentration of 1.5 × 106 CFU / ml were added to all tubes. The procedure was repeated for all test samples. All samples were incubated for 18-24 hours at 37 ± 1 ° C. After the incubation time, seeding on Petri dishes was performed to determine living cells. After seeding, the plates were placed in a thermostat for 18-24 hours at 37 ± 1 ° C. The results were taken into account by the presence of visible growth of microorganisms on the surface of a dense nutrient medium. The minimum bactericidal concentration (MBC) was considered the lowest concentration in the test tube, which inhibited the growth of microorganisms. Used reagents, solutions and culture media: Muller-Hintton Agar (MHA); Mueller-Hinton Broth (ICB); Saburo Bouillon (Sab); 0.9% sodium chloride solution (saline).
Equipment used: Densitometer DEN-1 (Latvia), Comfort thermal shaker (Germany), analytical balance LB 210-A (Russia), pH meter PB11 (Germany), vertical autoclave SystecV-120 (Germany), thermostat BD-115 (Germany), BioIIA / G laminar box (Spain), IKAMS3 Digital shaker (Germany), Eppendorf dispenser (1-10 ml, 100-1000 μl, 20-200 μl, 0.5-10 μl) (Germany), HaakeP14 thermal bath (Germany), Arium611 VF water treatment system (Germany).

RESULTS AND DISCUSSIONS
Obtaining and chemical composition of CO2-extract of seed Brassica napus 
The CO2-extract from rapeseed was developed and obtained at ZhanaPharm LLP and has valuable pharmacological properties. At present, the base of ZhanaPharm LLP is a unique production in the Republic of Kazakhstan, which receive CO2 extracts in pre-critical conditions from plant materials. 
The extraction of rapeseed is carried out with the following parameters: Extraction mass is 3 kg; Working pressure is 45-51 atm; The temperature of extraction is 18-21 ˚C; The extraction time is 11 h; The extract amount is 29.74 g.
Fig. 1 presents the encapsulation of the extracted essential oil into nanoparticles. Fig. 2 presents the SEM image of the as-prepared essential oil – nanoparticle hybrid sphere. The image reveals the well-formed sphere shape of the prepared nanocomposite.

GC-FID Analysis of the extract
Analysis was performed on rapeseed extract obtained by subcritical CO2 extraction. Chromatographic analysis conditions: sample volume 1.0 μl, sample inlet temperature 240 °C, flow division 1:10. Separation was carried out using a WAXetr chromatographic capillary column with a length of 30 m, an inner diameter of 0.25 mm and a film thickness of 0.25 μm at a constant carrier gas (helium) speed of 1 ml / min. The chromatographic temperature is programmed from 40 °C (exposure 0 min) to 260 °C with a heating rate of 10 °C/min (exposure 20 min). Detection is carried out in the SCAN m/z 34-850 mode. To control the gas chromatography system, register and process the obtained results and data, Agilent MSD ChemStation software (version 1701EA) was used. Data processing included determination of retention times, peak areas, and processing of spectral information obtained using a mass spectrometric detector (Fig. 3). For decoding the obtained mass spectra, the Wiley 7th edition and NIST’02 libraries were used (the total number of spectra in the libraries was more than 550 thousand) (Table 1).
According to the results of the study, in the composition of the rapeseed extract in large quantities, chemical compounds were found: 1,3,6,10-Cyclotetradecatetraene, 3,7,11-trimethyl-14- (1-methylethyl) - 30,07%, Cyclohexanone, 5 -methyl-2- (1-methylethylidene) - 12.91%, 3,4-Methylenedioxypropiophenone - 9,67%, Hexadecanoic acid, ethyl ester - 8.28%, Octacosanol - 5,50%, 11,15-Tetramethylhexadeca-1,3,6,10,14-pentaene - 4,55% and 1,6,10,14-Hexadecatetraen-3-ol, 3,7,11,15-tetramethyl – 3,14 % (Table 2). 

Antimicrobial Properties
The results of a study of the antibacterial and fungicidal activity of rapeseed extract against four strains of pathogenic microorganisms S. aureus ATCC 6538-P, E. coli ATCC 8739, P. aeruginosa ATCC 9027 and C. albicans ATCC 10231 are presented in Table 3.
From the data presented in table 2 it is seen that the rapeseed extract exhibits the expected biological activity against strains of C. albicans ATCC 10231 at a dilution of 1:32 and E. coli ATCC 8739 at a dilution of 1: 8, respectively. In relation to P. aeruginosa ATCC 9027 and S. aureus ATCC 6538-P, no antivicity was detected.
Fig. 5 presents the general antimicrobial activity test of the as-prepared nanomaterials based essential oil extract. 

CONCLUSIONS
We conducted a study of the chemical composition of CO2 of the rape seed extract (Brassica napus) by gas chromatography with mass spectrometric detection. The analysis was carried out using a WAXetr capillary column, in the detection mode SCAN m / z 34-850. The retention time and peak areas were determined using a detector. As a result, 39 types of chemical compounds were revealed in the composition of rape extract, in which terpenoids, diterpenes, sesquiterpenes, and other organic substances predominate. The tested Rapeseed extract exhibits the expected biological activity against yeast fungi of the genus Candida. The obtained results of the antimicrobial activity of the tested samples indicate the prospect of their further study for use as anti-infective (anti-inflammatory) drugs in medicine. The study indicated that nanomaterial-based EOs were effective against tested microorganisms. The PLA-based films formulated with the oil extract showed excellent antibacterial efficacy against both gram-positive and gram-negative pathogens.

CONFLICT OF INTEREST
The authors declare that there are no conflicts of interest regarding the publication of this manuscript.