1. Introduction
The fish industry produces large amounts of fish by-products such as skin, heads, frames, fins, viscera, and trimmings. While always being discarded, the fish by-products have a high content of valuable nutritional compounds, including essential fatty acids. Some of these fatty acids, such as long-chain n-3 polyunsaturated fatty acids (PUFAs), are beneficial to human health [
1,
2] and could be a potential source of oil for various industries. Fish oil has been widely reported as a developing supplement to improve the severity of skin disorders such as dermatitis, photoaging, allergy, skin cancer, melanogenesis, and cutaneous wounds [
3]. Omega-3 polyunsaturated fatty acids (PUFAs), docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA) are said to be responsible for the relationship of fish oil with skin protection and homeostasis [
3]. Owing to their nutrients, fish by-products have been used to produce products for topical administration. However, fish oils are usually associated with problems such as being inherently unstable and highly susceptible to oxidation, which imperils the oil quality [
4].
Citrus limonum, often known as lemon oil, is a highly volatile essential oil that can be applied therapeutically due to its anti-oxidative, anti-proliferative, antibacterial, and anti-cancerous merits [
5,
6]. Lemon essential oil’s antioxidant properties benefit human skin as it undergoes environmental and chronological ageing [
7]. Other than that, lemon oil could help to restore the skin’s natural balance and nourish damaged skin while hydrating the skin. However, the physicochemical properties of lemon oil, such as high volatility, poor stability and rapid degradability upon exposure, lamentably, impede its potency [
6]. Thus, to overcome the issue of oil degradation, one of the methods to stabilize the oil is through nanoemulsions formulation. Studies have shown that fish oil characteristics could be improved through this system [
8,
9].
Nanoemulsion (NE) is a colloidal particulate system in the submicron size range that consists of oil, water, and emulsifiers or surfactants. NE particle size can vary substantially across different studies, with particle sizes as small as 5 nm to 200 nm [
10]. These carriers are solid spheres whose surface is amorphous and lipophilic with a negative charge. Their small size results in a greater surface area providing a greater absorption enabling NE to effortlessly find its way into commercial preparations in many industries. Ample studies have been done as NE has numerous advantages over other technologies, such as ease of manufacture, adjustable particle sizes, and excellent kinetic stability [
11]. Since NEs consist of oil, water, and interfacial regions, lipophilic, hydrophilic, and amphiphilic components can be easily integrated into the formulation. Hence, they are suitable to be used in various products. Because of their flexibility, a broad range of delivery formulations with various functional qualities and improved sensorial properties can be developed.
Currently, low and high-energy emulsification methods can be utilized to fabricate NEs. The low-energy method transforms a water-in-oil (W/O) emulsion into an oil-in-water emulsion (O/W NE). By changing the experimental parameters, such as the composition or the temperature, phase inversion can be achieved to produce fine oil droplets at the inversion point where there is very little interfacial tension [
11]. On the other hand, high-energy procedures use specific mechanical tools, such as high-pressure homogenizers, ultrasonicator, or microfluidizers, to break the droplets into extremely small particles. The ultrasonication method is frequently employed because of its appealing features where the cavitation principles are utilized. In this method, when an asymmetric cavity collapses, strong turbulence is produced by ultrasound, which starts the emulsification process by creating droplets in the acoustic field. The bigger droplets are fragmented into smaller ones, which disperse into the continuous phase as a result [
8]. In recent years, research has explored ultrasonication to produce nanoemulsions with various components for various purposes. For example, Nirmal et al. [
12] focused on different essential oils to produce nanoemulsions, while Nirmala et al. [
13] employed ultrasonication to study the anticancer and antibacterial activities of celery oil-based nanoemulsions.
Considering that the selected oils are composed of chemical components that are sensitive to high temperatures, light, and the presence of oxygen; and that nanoemulsion systems have improved the stability and quality of many essential oils, this research aimed to produce oil-in-water nanoemulsion of lemon and fish by-product oils by ultrasonication technique for topical application. The physicochemical properties of the obtained nanoemulsions were characterized, and their antioxidant, anti-inflammatory and antibacterial activity were also assessed. This nanoemulsion produced is suitable as a topical application for skin treatment and cosmetics. It could be used as nanocarriers to improve skin penetration and provide a controlled release of active compounds for cosmetic products. Although it is not reported in this study, this formulation is currently in development for drug delivery for the rapid metabolism of active pharmaceutical ingredients and improve their bioavailability, especially the hydrophobic drugs.
3. Materials and Methods
3.1. Materials
2,2-diphenyl-1-picrylhydrazyl (DPPH) reagent and Folin–Ciocalteu reagent were obtained from Merck, Darmstadt, Germany. Sodium linoleate, soybean lipoxygenase enzyme solution and nordihydroguaiaretic acid (NDGA) for anti-inflammatory studies were purchased from Merck. Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Life Technologies Corporation, Carlsbad, CA, USA. Human skin fibroblast cells (HSF 1184) were obtained from Life Technologies Bio Diagnostic, Malaysia. South African lemon oil was obtained from a local supplier (Wellness Original Ingredient, Malaysia). Catfish by-product oil was prepared in the laboratory as previously described by Djaeni and Listyadevi [
59]. The anionic co-surfactants used in the study are not stated as it is under a patent application. All other solvents and chemicals used were analytical and/or pharmaceutical grade.
3.2. Preparation of Nanoemulsion of Fish by-Product and Lemon Oils (NE-FLO)
The method for ultrasonication is adopted from Espinosa-Andrews and Paez-Hernandez [
60]. A two-step process was used to prepare the oil-in-water nanoemulsion of fish by-products and lemon oils (NE-FLO). Briefly, the continuous phase, known as the aqueous phase, was prepared by adding 10 wt% of Tween 80 and 27.7% cosurfactant to pure distilled water and stirred for 15 min. The oil phase consisted of 2 wt% fish oil and 8% lemon oil. The aqueous and the oil phases were mechanically stirred at 500 rpm until both mixtures were well combined. The oil phase was then added dropwise into the continuous aqueous phase while stirring at the same speed. A T25 digital Ultra-Turrax homogenizer was used to combine the mixture for 3 min at 20,000 rpm to form a coarse oil-in-water emulsion. The coarse emulsion was ultrasonicated at a predetermined amplitude using an ultrasonicator (LABSONIC M, Sartorius, Teltow, Germany) for 42 min to obtain the nano-sized droplet. The ultrasonication time is based on the optimization of this study. The sonication was set at 80% of the maximum output of 100 W.
There is no pulse used; however, to avoid an increase in temperature, the beaker is put inside a water-filled beaker. Thus, even when the samples were sonicated for a long time, there was no increase in temperature detected. Tween 80 was used as a surfactant as it is a hydrophilic non-ionic surfactant that is well-established in the emulsification and dispersion of substances in pharmaceutical and food products. Other than that, Tween 80 was also listed as one of the generally regarded safe agents (GRAS). For the co-surfactant, it was proven that it can form hydrogen bonds with water, lowering the water−oil interfacial tensions and improving the interface’s fluidity. The percentage of the surfactant and co-surfactant used were optimized.
Figure 7 shows the schematic diagram of NE-FLO preparation using the ultrasonication method.
3.3. Measurement of Particle Size, Polydispersity Index (PDI) and Zeta Potential
The determination of particle size, PDI and Zeta potential of NE-FLO were analyzed using a particle size analyzer (Malvern Instruments, Malvern, UK) at 25 °C. Dilution using distilled deionized water at a ratio of 1:9 of all samples was carried out to prevent back-scattering phenomena. The particles and water refractive indices employed were 1.54 and 1.33, respectively. Each measurement was repeated in triplicate.
3.4. Morphological Analysis by Microscopic Techniques
Scanning electron microscopy (Vega3 Tescan, Kohoutovice, Czech Republic) was used to observe the morphology of the NE-FLO following a method by Krithika and Preetha [
31]. NE-FLO samples were mounted on the stabs with adhesive tape, and SEM images were captured at a 20 kV accelerating voltage with a magnification of ×100 k. To examine the NE-FLO images, multiple resolutions ranging from magnification ranging from 20× to approximately 30,000× were used accordingly.
3.5. Stability Assessment
The emulsion stability, or an emulsion’s ability to withstand changes in its properties over time, was studied by storing the NEs at various temperatures. Measurements were carried out at 0, 7, 14, 21, 28, and 90 days of storage. The visual instability (creaming, phase separation, or flocculation) was observed and recorded (if any). The determination of particle size of NE-FLO at day 0 and day 90 were analyzed using a particle size analyzer (Malvern Instruments, Malvern, UK) at 25 °C. The stability was also evaluated by monitoring changes in pH at different intervals of time using a pH meter (Mettler Toledo, Zurich, Switzerland).
3.6. Antioxidant Evaluation
The in vitro antioxidant activity of the NE-FLO was conducted using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. DPPH solution at an amount of 2.5 mL (60 µmol·L−1 in ethanol) was combined with 0.3 mL of the NE-FLO and 0.2 mL of ethanol in a 10 mL test tube with a final volume of 3.0 mL. The absorbance of the solution was measured at 517 nm after allowing the mixture to stand for 30 min at room temperature.
3.7. Antibacterial Evaluation
The disc diffusion test (DDT) and minimum inhibitory concentration (MIC) are the most used tests for antimicrobial properties against Gram-positive and Gram-negative bacteria. In this study, seven strains of Gram-negative bacteria and seven strains of Gram-positive bacteria were used. All the bacterial strains were first precultured by placing a loopful of cells into 10 mL of autoclaved tryptone soy broth (TSB) and incubated at 37 °C for 24 h. Then, the cells were spread evenly on the agar plates using a sterile cotton bud. A sterile filter paper (Whatman No. 1, 6 mm diameter) was immersed in NE-FLO and equilibrated before being placed on the seeded plates before incubation. The plates were then allowed for incubation (24 h, 37 °C). The diameter of the inhibition zones was measured to quantify the antimicrobial activity. The analysis was repeated in triplicate for each of the evaluated bacterial strains. The positive and negative controls used are tetracycline at 5 mg·mL
−1 and dimethyl sulfoxide (DMSO), respectively [
45].
MIC is the minimum concentration of an antibacterial agent that completely inhibits the microorganism’s growth in tubes or microdilution wells detected by the unassisted eye, and it was observed through 96-well agar plates. The antibacterial activity of the NEs was assessed with a broth micro-dilution assay, using a serial dilution in sterile 2 mL 96-well microplates [
61]. Each of the rows was filled with 0.1 mL sterilized tryptone soy broth. Wells 2–11 were topped up with 0.1 mL of NE-FLO and diluted serially to form a sequence of concentrations from 500 mg·mL
−1 to 0.488 mg·mL
−1. The first well served as growth control. Then, 10 µL of the culture medium was placed in each well containing NE-FLO. The microplates were then incubated (37 °C, 24 h). The turbidity assessment was carried out at the time of incubation,
tT = 0, and
t = 24 by reading the optical density at 600 nm (UV–vis spectrophotometer, Thermo fisher, Massachusetts, USA). The analysis was done in triplicate for each assay. Minimum bactericidal concentration (MBC) is the minimum concentration of an antibacterial agent needed to kill and prevent the growth of a bacterium over a fixed and extended time, such as 18 h or 24 h, under a specific set of conditions. A loophole from each well that is equal and higher than MIC is streaked on Mueller–Hinton agar to validate the MIC value. All plates were incubated at 37 °C for 24 h. The minimum bactericidal concentration (MBC) is expressed as the concentration at which no apparent and visible growth of bacteria occurs. The test was performed in triplicates.
3.8. Anti-Inflammatory Evaluation
The activity of lipoxygenase inhibitors was accurately determined by slightly altering the spectrometric approach described by Azhar-Ul-Haq et al. [
62]. In the protocol of the assay, 160 µL of sodium phosphate buffer (100 mM, pH 8.0), 20 µL of soybean lipoxygenase enzyme solution, and 10 µL of the NE-FLO were thoroughly mixed and incubated at 25 °C for 10 min, without shaking. The addition of 10 µL sodium linoleate (0.3 mM) as substrate solution initiated the enzyme reaction, and it was incubated once again in a spectrophotometer at 25 °C for 10 min. Linoleic acid was converted to form (9Z,11E)-(13S)-13-hydroperxyoctadeca-9,11-dienoate (13-HPODE) following the incubation period and measured by spectrophotometer at 234 nm. The initial reaction rate was measured by spectrophotometry, and the inhibitory activity of a sample was measured by the reduction of this initial rate [
63]. The test compounds and the positive control were dissolved in DMSO because this universal aprotic polar solvent can dissolve NE-FLO as a test sample. All the reactions were performed in triplicates, and the absorbance was measured at 234 nm. Nordihydroguaiaretic acid (NDGA) was used as the reference standard. The percentage of inhibition was calculated using Equation (1).
where OD
control is the optical density of control, and OD
sample is the optical density of the sample.
3.9. Cell Cytotoxicity Study
Cell lines used in this study, human skin fibroblast cells (HSF 1184), were cultivated in Dulbecco’s Modified Eagle’s Medium (DMEM), with 1% penicillin with streptomycin and 10% fetal bovine serum. The cells were maintained at 37 °C in a humidified atmosphere of 5% CO
2. The reduction of MTT dye by viable cells to yield purple formazan products was used to assess the NE-FLO cytotoxicity to the cell according to the method by Azrini et al. [
64]. Firstly, fibroblast cells were seeded at a density of 1 × 104 cells using a 96-well plate, followed by incubation in a culture medium until 100% confluence. Next, the varied concentrations of NE-FLO were used to treat the cells for 24 h. Different concentrations of NE-FLO were used to determine the concentration at which the NE-FLO is toxic to the cells. After 24 h, the media was withdrawn and washed with phosphate-buffered saline. The cell was incubated in the dark with 20 µL of 5 mg·mL
−1 MTT solution for four hours at 37 °C. Next, 100 µL of DMSO was added to each well. The absorbance was measured at 570 nm using a Tecan M200 Pro Multiplate reader. The percentage of viable cells was determined with the assumption that the cell viability without treatment was 100%.