In recent years, nanotechnology has been used in various fields of science because of its great potential [1, 2]. Nanomaterials have become very widespread due to their small size and large surface area [3, 4]. Today, the widespread application of plant science in nanotechnology has increased [5, 6]. In recent years, the application of nanoemulsion has attracted much interest in various areas such as cosmetics, pharmaceutical compounds, food and agriculture industry [7, 8]. Nanoemulsions are classified between classical emulsions and microemulsions with a size range of 20 to 500 nm and generally include water-in-oil and oil-in-water [9, 10]. The O/W nanoemulsions can contain a lipophilic bioactive ingredient as an oil phase and an aqueous medium [11, 12]. Nanoemulsion can be prepared by homogenization method as the high energy technique in the presence of emulsifier . Recently, the herbal nanoemulsions have been developed for medical applications [14, 15].
Newly, the use of saffron has expanded despite its high price due to the importance of using natural products and medicinal food with therapeutic properties and anti-bacteria activity . Also, sesame oil is used as an herbal medicine in the tropics [17, 18]. The antimicrobial activity of sesame has become an interesting and expanding application . The high prevalence of bacterial infections and the increasing resistance of bacteria expand the need for new antibiotics . Nanotechnology is increasingly using nanomaterials and nanostructures for antibacterial applications and is considered as an approach to eliminate or reduce the activity of microorganisms [21, 22]. Medicinal plants can be used as natural antibacterial agents to increase efficacy and inhibit bacterial growth [23, 24]. Essential oil as one of the compounds derived from the plant can have antimicrobial activity. The antimicrobial activity of this natural substance has been greatly increased, mainly in response to consumer concerns about the safety of synthetic oral additives . Recently, the use of essential oils in nanoemulsions has had a special place [26-28]. These volatile substances can cause therapeutic and pharmacological properties [29, 30]. In the present study, nanoemulsion was prepared containing sesame oil, saffron aqueous solution and PEG and evaluated antibacterial activity against E. coli Gram-negative bacterial and cytotoxicity on HEK239 human cell line.
MATERIALS AND METHODS
All chemicals used had analytical grade. Ultra-pure water was used to prepare all reactant solutions. Span 80 and PEG 1000 were purchased from Merck Millipore (Darmstadt, Germany). Sesame essential oil and saffron bought from Adonis Gol Daro (Iran).
The size and size distribution of nanoparticles were evaluated by dynamic light scattering (ZEN314, England). The morphology and size were investigated by atomic force microscopy (Nanosurf, Switzerland). The viscosity was studied by rheological properties based on spindle velocity for nanoemulsions using Reologica InstrumentsAB (Lund, Sweden). Disc diffusion method was used to evaluate the antibacterial activities of samples against Escherichia coli bacteria, ATCC 1399, that procured from Islamic Azad University. MTT assay was applied to investigate the cytotoxicity of nanoemulsions for HEK239 human cell line at three different concentrations for 1 and 5 days.
The optimal nanoemulsion was prepared from a mixture of 4 mL sesame oil, 0.25 g saffron in aqueous solution and 2 mL span 80 surfactants by homogenizing at 15000 rpm for 15 min in presence or the absence of 2 g PEG (Table. 1).
RESULTS AND DISCUSSION
Dynamic light scattering was used to determine the mean particle diameter and the particle size distribution of the nanoemulsions. Based on the results, a sharp peak with a narrow distribution was observed.
Based on these results, the size was observed for the nanoemulsions including 62 nm (I), 71 nm (II), 75 nm (III) and 134 nm (IV) and the presence of saffron and PEG was caused to the increase of the size. The DLS results are based to previous report .
Fig. 2 was shown the AFM images for the nanoemulsions with spherical morphology. The AFM result approved a narrow distribution of the DLS results. AFM results are according to previous research .
The viscosity was investigated for nanoemulsions (Table. 2). In the same spindle speed, the viscosity of nanoemulsions was as follows:
nanoemulsions (I) < nanoemulsions (II) = nanoemulsions (III) < nanoemulsions (IV).
The presence of saffron and PEG increased the viscosity of nanoemulsions. According to previous studies, the presence of the polymer increases the rheological properties [7, 32]. This work is the first report on the increase of rheological properties by saffron.
The antibacterial activity was investigated by determination of inhibition zone diameter of samples against a Gram-negative bacterial. The Escherichia coli cells were cultivated on Mueller-Hinton Agar and incubated at 37 ºC for 1 day. The inhibition zone diameter of nanoemulsion was 2 mm (I), 5 mm (II), 8 mm (III) and 9 mm (IV). According to the result, saffron and PEG increased the antibacterial activity of nanoemulsion. The antibacterial activity is reported in the presence of nanoemulsion , PEG  and saffron .
MTT assay was used to investigate the cytotoxicity of nanoemulsion for HEK239 human cell line at 0.25, 0.5, and 1% (v/v) concentrations for 1 and 5 days. Based on the result, the two effective factors in the cytotoxicity of nanoemulsions were concentration and time. Increasing concentration and time increased cytotoxicity due to decreased cell viability. Based on the analysis of the results, on the fifth day, the rate of cell death increased in samples 3, and this increase in death is somewhat significant. The advantages of the nanoemulsion include acceptable toxicity over a longer period of time compared to a previous report by researchers .
The present study showed that the nanoemulsion with sesame oil and saffron aqueous solution in the presence of PEG could be a good candidate for antibacterial activity against Escherichia coli with low cytotoxicity for HEK239 human cell. The presence of saffron and PEG leads to increased size, viscosity and antibacterial activity. Nanoemulsion can be a cost-effective method for future use in food and pharmaceutical applications.
The author declares there are no conflicts of notice in preparing the manuscript.