Abstract
Plant oils have been applied for numerous purposes. Developing the composition of oils through nanotechnology has become a requirement, whether from a medical or industrial point of view. In this study, nanoemulsions (NEs) of olive and peanut oils were evaluated. GC-MS was used to determine the saturated and unsaturated fatty acids contents in both oils. Based on the area %, cis-8,11,14-eicosatrienoic acid (54.0%), myristic acid (30.7%), and arachidonic acid (23.1%) were the greatest constituents in peanut oil, while arachidonic acid (23.2%), cis-11,14,17-eicosatrienoic acid (22.7%), and cis-11-eicosenoic acid (11.4%) were the greatest constituents in olive oil. TEM examination indicated that the diameter of peanut oil NEs (14.6 nm) was less than that of olive oil NEs (24.5 nm). Olive oil and its NEs exhibited more antioxidant activity than peanut oil and its NEs had IC50 values of 158.6, 102.5, 435.1, and 291.5 µg/mL, respectively. Negligible hemolysis was observed using olive oil, unlike peanut oil, while hemolysis based olive oil NEs was increased compared with hemolysis based peanut oil NEs, particularly at high concentrations of 600 to 1000 µg/mL. Molecular docking investigation offered the structure–activity correlation and binding modes of cis-8,11,14-eicosatrienoic acid with Salmonella typhi (5ZXM) enzymes.
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Nanoemulsions of Some Edible Oils and their Antimicrobial, Antioxidant, and Anti-hemolytic Activities
Aisha M. H. Al-Rajhi,a and Tarek M. Abdel Ghany b,*
Plant oils have been applied for numerous purposes. Developing the composition of oils through nanotechnology has become a requirement, whether from a medical or industrial point of view. In this study, nanoemulsions (NEs) of olive and peanut oils were evaluated. GC-MS was used to determine the saturated and unsaturated fatty acids contents in both oils. Based on the area %, cis-8,11,14-eicosatrienoic acid (54.0%), myristic acid (30.7%), and arachidonic acid (23.1%) were the greatest constituents in peanut oil, while arachidonic acid (23.2%), cis-11,14,17-eicosatrienoic acid (22.7%), and cis-11-eicosenoic acid (11.4%) were the greatest constituents in olive oil. TEM examination indicated that the diameter of peanut oil NEs (14.6 nm) was less than that of olive oil NEs (24.5 nm). Olive oil and its NEs exhibited more antioxidant activity than peanut oil and its NEs had IC50 values of 158.6, 102.5, 435.1, and 291.5 µg/mL, respectively. Negligible hemolysis was observed using olive oil, unlike peanut oil, while hemolysis based olive oil NEs was increased compared with hemolysis based peanut oil NEs, particularly at high concentrations of 600 to 1000 µg/mL. Molecular docking investigation offered the structure–activity correlation and binding modes of cis-8,11,14-eicosatrienoic acid with Salmonella typhi (5ZXM) enzymes.
DOI: 10.15376/biores.18.1.1465-1481
Keywords: Nanoemulsions; Olive; Peanut; Oils; Antimicrobial; Antioxidant; Hemolysis
Contact information: a: Department of Biology, College of Science, Princess Nourah bint Abdulrahman University P.O. Box 84428, Riyadh 11671, Saudi Arabia; b: Botany and Microbiology Department, Faculty of Science, Al-Azhar University, Nasr City, Cairo 11725, Egypt;
* Corresponding author: tabdelghany.201@azhar.edu.eg; amoalrajhi@pnu.edu.sa
INTRODUCTION
The development of efficient natural additives that may be used in place of synthetic alternatives has received much attention because there is growing interest from consumers as well as food and pharmaceutical industrial companies in the exploration of extremely safe natural ingredients (Al-Rajhi et al. 2022a). Although essential oils (EOs) have many applications in the fields of food, medicine, and cosmetics, there is still a problem of instability that prevents these uses. Scientists have been made aware of employing nanotechnology to solve these difficulties as a result of the growth of nanoscience (Abdelghany et al. 2018; Ganash et al. 2018; Al-Rajhi et al. 2022b; Yahya et al. 2022). Not all oils were covered in the publications that discussed the evolution of emulsions into nano-emulsions (NEs). The characteristics of NEs, which range in size from 20 to 100 nm, include long-term physical stability, high bioavailability, a high surface-to-volume ratio, and simple digestion. Due to this, two EOs – peanut and olive oils – were used to prepare NEs for the current investigation. NEs have attracted a growing interest in numerous fields, including materials sciences, chemistry, and in medical and pharmaceutical sciences (Atanase 2022).
Tropical and sub-tropical nations are well suited for the production of peanuts (Arachis hypogaea L.) (Bhatti et al. 2020). India is the world’s largest important yielder of peanuts, followed by China, West Africa, and the USA.
According to Rodrigues et al. (2011), peanut oil is used in the production of margarines, surfactants, medicines, and cosmetics. From peanut oil’s biological activities, including its ability to repress the bacteria growth such as Staphylococcus aureus, Listeria ivanovii, L. innocua, Bacillus cereus, Enterococcus hirae, and Pseudomonas aeruginosa (Sebei et al. 2013), antioxidant potential resulting from its great content of total phenolic compounds (Matthäus and Özcan 2015; Zio et al. 2021), and anti-aging due to γ-tocopherol, vitamin E, α-tocopherol and phytosterols (Matthäus et al. 2015).
According to Lin et al. (2017), olive oil has appealing biological properties that include improving ROS removal, lowering the risk of cardiovascular disease, and improving memory and cognitive performance in the aged. Olive oil’s antibacterial potential against different species including B. cereus, B. subtilis, E. coli, and S. aureus was recently reported by Wang et al. (2021). Olive oil had the highest concentrations of squalene and β-sitosterol, which were major contributors to its antibacterial effects (Wang et al. 2021).
For confirmation of the efficacy of the any NEs preparation, its activities were compared with bulk oil. Moghimi et al. (2016) demonstrated that the NEs of sage (Salvia officinalis) reflected higher antibacterial activity than bulk oil. Another study was performed on the antioxidant and antibacterial activity of olive oil NEs, indicating stronger activity than bulk oil (Lu et al. 2020). Also, the influence of a number NEs of oils on the microbial contamination of muscle foods was reviewed recently by Aziz et al. (2022). It was shown that olive oil NEs reduced the proliferation of mesophilic and psychrophilic bacteria, besides lactic acid bacteria under storage conditions. At the same time, no changes were observed on muscle foods as a result of NEs treatment. Nano-emulsions (NEs) of olive oils planned for the intravenous drug delivery was established (Karami et al. 2019) and NEs displayed low hemolysis (4.6%); therefore, it was considered safe for intravenous administration. Recently, a successful trial for oral S. aureus, S. epidermidis, Chromobacterium voilaceum treatment was documented using oils NEs (Ullah et al. 2022). Also, NEs were prepared and experimented against phytopathogenic bacteria by means of in vitro and in vivo investigations. Abdelrasoul et al. (2018) converted monoterpenes to NEs to enhance their antibacterial potential for suppression of Pectobacterium carotovorum and Ralstonia solanacearum. Compared with the bulk peanut oil, the proliferation of A549 lung cancer cells was more influenced by its NEs (Parastoo et al. 2021). In olive oil, there are only a few kinds of fatty acids, but the quantities of each strongly effect the nutritive and characteristics of the oil such as oil stability (Ghanbari et al. 2012). Also, polyunsaturated fatty acids have been found to play an essential role in the rancidification of numerous oils. Olive oil nutritional value, as well as its health utilities are attributed to the presence of a great quantity of oleic acid as a monounsaturated fatty acid and valuable minor components (Al-Bachir and Sahloul 2017). From the earlier literature, peanut oil commonly consists of triglycerides, including eight fatty acids. Oleic acid and linoleic represent nearly 80% of these fatty acids. Moreover, the content of phospholipids in the crude peanut oil can represent from 0.6 to 2%, depending on the peanuts maturity (Akhtar et al. 2014; Yang et al. 2022).
Based on the development of nanotechnology and vital role of EOs, the current investigation was designed to formulate NEs from two edible oils, namely olive and peanut oils, with determination of its antioxidant, antimicrobial, and hemolytic potentialities.
EXPERIMENTAL
Materials
All chemicals were analytical grade and purchased from Sigma-Aldrich (St. Louis, MO, USA), except microbial growth media and oils (Al- Gomhuria Company, Cairo, Egypt) and dimethyl sulfoxide (DMSO) (Riedel-de-haen, Seelze, Germany).
Fatty Acid Analysis of Olive and Peanut Oils via GC-MS
The extraction solvent was composed of chloroform and methanol (2/1, v/v). One mL of oil was extracted by 20 mL of the solvent, followed by the addition of salt solution to separate the lower and higher phases. The lower phase was separated with a separation funnel, followed by concentration of the fatty acids. Fatty acid methylation was performed by adding H2SO4-methanol 2% (v/v) to the vial containing fatty acid extract. The vial was heated at 80 °C under slow shaking. Then, 0.25 mL of 1 M NaOH was added to neutralize the solution under slow shaking. The sample was subjected to fatty acid analysis (Liu et al. 2018).
Nano-emulsion Preparation and Visualization by Transmission Electron Microscopy (TEM)
Polysorbate 80 (Tween 80) surfactant was dispersed into a homogeneous suspension at 2% v/v in distilled water to prepare 100 mL. Olive or peanut oil (1:100) was added slowly to the suspension with constant stirring for 10 min. The EMs were prepared according to the method described previously (Salvia-Trujillo et al. 2013; Moradi and Barati 2019), with some modifications. The prepared EMs were sonicated by probe ultrasonic homogenizer (Silent Crusher M, Heidolph, Germany) for 20 min to obtain translucent NEs. Phosphotungstic acid was mixed with one drop of oil emulsion, which was then fixed on a copper grid. The shape and size of the prepared NEs of the olive and peanut oils in the dispersion NEs system (via Brownian diffusion) were examined via transmission electron microscopy (JEOL JEM-1200, Tokyo, Japan) at 200 kV with a tungsten source.
Microbial Inactivation Assay of Bulk Oils and its NEs
The well diffusion method was used to evaluate the antimicrobial activity of olive, peanut oils, and other specimens. Agar plates were inoculated with the test bacteria (Salmonella typhi, Bacillus cereus, Escherichia coli, and Staphylococcus aureus) and fungi (Candida albicans and Aspergillus flavus). Agar plugs were removed via sterilized cork borer (6 mm), and 100 µL of the tested compounds were added into the well. Under appropriate temperature (37 °C for bacteria and 30 °C for fungi), the agar plates were incubated for 24 h and 3 days for bacteria and fungi, respectively. The visualized clear zone diameters around each well were measured. The activity of tested compounds was compared with a positive control represented with standard antibiotic (Gentamycin) and antifungal (Fluconazole). Another control (as negative control) was represented by DMSO without oils. The experiments were repeated three times (Al-Rajhi et al. 2022c).
DPPH Scavenging for Determine the Antioxidant Activity
The reaction mixture composed of 2 mL of 1,1-diphenyl-2-picryl hydrazyl (DPPH) dissolved in DMSO with separate doses of tested compound ranged from 3.9 up to 1,000 μg/mL. Using a vortex, the tubes containing the reaction mixture were shaken vigorously, then kept for 24 h without light. Then, the absorbance (517 nm) of the reaction mixture was measured by spectrophotometer (UV-VIS Milton Roy). DPPH solution free from the tested compound was applied as a control, while DMSO was utilized as a blank. A comparison with a standard antioxidant was conducted, and thus, ascorbic acid was utilized with the same procedures required to determine the antioxidant of tested compound. The inhibition of 50% (IC50 value) (by undetermined quantity of the tested compound) of the DPPH free radical was determined through log dose inhibition curve (Qanash et al. 2022). The DPPH scavenging activity (%) was estimated via calculation of the following expressions
(1)
where Ac is the absorbance of the control and At is the absorbance of the tested compound.
Assay of Hemolytic Activity of Bulk Oils and its NEs
There were a few modifications of the protocol of Bulmus et al. (2003) to determine the hemolytic activity of olive and peanut bulk oils and its NEs. Briefly, in a sterile tube, a blood sample (five mL) was taken from healthy humans (non-suffering from any disease) and then centrifuged at 2,500 rpm for 10 min in order to separate the plasma from cells. Then, the cells were collected and washed three times using 150 mM of NaCl, followed by centrifugation to remove NaCl and collect the cells. Cells suspension was prepared as 2% in phosphate buffer saline (PBS) with pH 7.4. Different doses of tested samples were added to the cells suspension then completed by the PBS to 1 mL, followed by incubation at 37 °C for 1 h in water bath. Finally, at the same prior condition, the cells suspension was centrifuged, and the absorbance of the collected supernatant was recorded at 541 nm. The positive control and blank sample were represented by deionized water and PBS, respectively. The hemolysis % was estimated via the next expression:
(2)
Experimental: Molecular Docking
In the current research, molecular docking was carried out to explore the probable molecular mechanisms underlying the antibacterial activity of cis-8,11,14-eicosatrienoic acid. The molecular interactions were studied for binding affinities of selected compounds of oil with bacterial receptors that play key roles in cell growth and DNA duplication. The crystal structures of the proteins identified for the Salmonella typhi (5ZXM) were supplied from the bank of protein data (http://www.rcsb.org/pdb, accessed on 20 June 2021). The protein’s surrounding water molecules were eliminated, and hydrogen atoms were then added. The MMFF94x force field was used to assign the parameters and charges. Cis-8,11,14-eicosatrienoic acid (the main detected fatty acid in peanut oil) was docked in the active site using the DOCK module of MOE after alpha-site spheres were created using the site finder module of MOE. The London dG scoring function, placement: triangle matcher, retain: 10, and refinement: forcefield were used to determine the dock scoring in the MOE programme. RMSD values, binding energies and binding modes with the selected residues were considered to identify the leading conformations of the docked ligands (Gurung et al. 2021; Qanash et al. 2022).
Statistical Analysis
All experiments were achieved in triplicate, and the findings are described as the mean to calculate standard deviation of the obtained results.
RESULTS AND DISCUSSION
Fatty Acids Analysis of Oils
GC-MS analysis of the olive and peanut oils indicated its richness in fatty acids (Tables 1 and 2). GC-MS examination of peanut oil reflected the occurrence of 16 and 19 saturated and unsaturated fatty acids, respectively (Table 1).
Table 1. Detected Peanut Oil Fatty Acids by GC/MS
Cis-8,11,14-eicosatrienoic acid, myristic acid, and arachidonic acid represented 54.0%, 30.7%, and 23.1%, respectively. Other important fatty acids included lauric acid, erucic acid, and caprylic acid. Lauric acid and caprylic acid have antibacterial and antiviral activity (Fischer et al. 2012; Matsue et al. 2019). They have low inhibitory potential toward commensal lactic acid bacteria, while high inhibitory potential toward Clostridium and Bacteroides (Matsue et al. 2019). The mechanism of antimicrobial activity was previously reported (Bergsson et al. 1998): the cell wall or membrane of bacterial pathogens are disrupted by these acids. Seven saturated and 10 unsaturated fatty acids were recognized in olive oil (Table 2). Most of the unsaturated fatty acids were detected with high area%, such as arachidonic acid (23.2 %), cis-11,14,17-eicosatrienoic acid (22.7 %), and cis-11-eicosenoic acid (11.4 %). All fatty acids detected in olive oil were detected also in peanut oil. Kanlayavattanakul and Lourith (2011) reported that the natural sources containing linoleic acid exhibited anti-inflammatory activity and were used in skin ulcer treatment. According to Kapseu (2009), the stability of peanut oil is due to the composition of fatty acids including more than 47% of monounsaturated fatty acids.
Table 2. Detected Olive Oil Fatty Acids by GC/MS
TEM Characterization of the Prepared NEs
Despite the many applications of the essential oils, there are some problems that impede the performance of these applications, such as limited water solubility of oils and excessive sensitivity of oils to storage or industrial conditions associated to oxygen, heat, and light. Conversion of oils to NEs may represent a great solution to overcome these problems. The diameter of the NEs was 14.6 nm for peanut and 24.5 nm for olive (Fig. 1). The NEs of olive oils that were created in another study (Karami et al. 2019) had a spherical shape and diameter of 40 nm. The size of NEs may be influenced by quantities and type of the applied surfactants (Campolo et al. 2020).
Fig. 1. TEM of NEs of olive and peanut oils. Magnification, 10000 X; Scale bar, 100 nm
Biological Activity
The tested microorganisms varied in their susceptibility towards the same oil and its NEs (Table 3 and Fig. 2). There was a slight change in the inhibition zone of B. cereus, and A. flavus treated with peanut oil compared with its NEs. The sensitivity of other tested organisms particularly S. typhi was higher (inhibition zone was 20.17 mm) when treated with NEs than when treated with peanut bulk oil (Inhibition zone was 13.17 mm). These outcomes are similar to those of Moghimi et al. (2016), who reported that bulk oil of sage (Salvia officinalis) had less bacteriostatic potential than NEs using E. coli as a test bacterium.
The conversion of olive oil to NEs enhanced its antimicrobial activity against all tested organisms; the inhibition zones of B. cereus, S. aureus, E.coli, S. typhi, C. albicans, and A. flavus were 25.8, 20.2, 20.3, 19.3, 20.8, and 18.2 mm using NEs compared to bulk oil with 21.3, 13.2, 13.3, 14.8, 16.8, and 15.5 mm, respectively (Table 3). In previous reports, NEs of some oils were tested against food-borne and human pathogens compared to bulk oils. For example, NEs of sage oil showed better bactericidal potential against Shigella dysentery, Salmonella typhi, and Escherichia coli (Moghimi et al. 2016), with MIC being four-times higher for the bulk oil than for the NEs.
While many other studies demonstrated that the antibacterial activity of EOs was enhanced when it was converted into NEs (Salvia-Trujillo et al. 2015; Zahi et al. 2015; Maté et al. 2016; Lu et al. 2018), in some reports there was no alteration in the antimicrobial activity when the essential oils were converted into NEs (Chang et al. 2012; Xue et al. 2015). The mechanism of the bactericidal potential may be due to the electrostatic interaction among positively charged NEs and the negatively charge of the walls (Majeed et al. 2016).
Table 3. Antimicrobial Activity of Peanut Oil, Olive Oil, and NEs
The antioxidant activity of olive and peanut oils and their NEs are shown in Table 4. The increased concentration of olive and peanut and their NEs was accompanied by increased antioxidant activity. The achieved results indicated that NEs of the two oils, olive and peanut, exhibited more antioxidant activity with IC50 102.5 µg/mL and 291.5 µg/mL than bulk oils with IC50 158.6 µg/mL and 435.08 µg/mL, respectively. Olive oil and its NEs reflected the highest antioxidant activity compared to the antioxidant activity to oil and its NEs.
According to previous literature, the richen lipids with unsaturated fatty acids exhibited good antioxidant activity compared with the less saturated fatty acids containing lipids (Banskota et al. 2019). These results were unlike those in the literature, probably due to the presence of certain fatty acids.
Fig. 2. Antimicrobial activity of olive oil, peanut oil and its NEs. Negative control (1), positive control (2), Oil (3), and NEs of oil (4). Positive control represented with standard antibiotic (Gentamycin) and antifungal (Fluconazole). The negative control was represented by DMSO without oils.
Table 4. Antioxidant Activity of Olive Oil, Peanut Oil, and NEs
A gradual rise in hemolysis% was recorded using olive oil at all used concentrations up to 1000 µg/mL (Table 5). Olive oil caused 2.7% hemolysis, while its NEs caused 19.8% hemolysis and NEs of peanut oil caused less 9.5% hemolysis than peanut oil (26.8% hemolysis) at 1000 µg/mL. Up to 200 µg/mL of the two oils and its NEs, the hemolysis % did not exceed 2%, so the inhibition of RBC lysis was near 98% with using NEs of olive oil (0.2 % hemolysis) followed by olive oil (0.9 % hemolysis), followed by NEs peanut oil (1.1 % hemolysis), and followed by NEs peanut oil (2.0% hemolysis). Some fatty acids of the oils prevent the membrane damage of RBC resulting from oxidative stress through scavenging of the generated hydrogen peroxide and peroxide radicals. The developed NEs of olive oils by Karami et al. (2019) exhibited only 4.6% of hemolysis with safe application when used for intravenous delivery.
Table 5. Hemolytic Activity of Olive Oil, Peanut Oil, and NEs