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Beni-Suef University Journal of Basic and Applied Sciences
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Uncovering and evaluating coconut oil-loaded silica nanoemulsion as anti-viral, bacterial, and fungal: synthesis, fabrication, characterization, and biosafety profiles

Beni-Suef University Journal of Basic and Applied Sciences volume 13, Article number: 56 (2024) Cite this article

Abstract

Background

Coconut oil, a natural component abundant in terpenoids, possesses various physiological functions. The global concern over the spread of viral infections and antimicrobial-resistant bacteria and fungi has highlighted the need for novel treatments. Coconut oil, with its known antimicrobial properties, presents an attractive candidate for combating these pathogens. This study aims to investigate the potential of coconut oil-loaded silica nanoemulsion (ON@SiO2) as a novel therapeutic agent against viral, antimicrobial-resistant bacteria, and fungal pathogens.

Results

The study synthesized coconut oil-loaded silica nanoemulsion (ON@SiO2) using an eco-friendly, cost-effective method with native coconut oil (CO). Characterization confirmed successful synthesis on the nanoscale with good distribution. Three nanoemulsion samples (ON-1@SiO2, ON-2@SiO2, and ON-3@SiO2) were prepared, with average particle sizes of 193 nm, 200 nm, and 325 nm, respectively. Evaluation of cytotoxicity on Vero-E6 cell lines indicated safety of ON-0@SiO2 and ON-3@SiO2, with CC50 values of 97.5 mg/ml and 89.1 mg/ml, respectively. ON-3@SiO2 demonstrated anti-Herpes I and II (HSV1 and HSV2) activity, with IC50 values of 1.9 mg/ml and 2.1 mg/ml, respectively. Additionally, ON-3@SiO2 exhibited promising antibacterial activity against E. coli, P. aeruginosa, S. aureus, and B. subtilis, with MIC values of 25 mg/ml, 12.5 mg/ml, 25 mg/ml, and 3.12 mg/ml, respectively.

Conclusions

ON-3@SiO2 showed potential antifungal activity against C. albicans, a unicellular fungus, with an MIC of 12.5 mg/ml. Overall, ON@SiO2 possesses antiviral, antibacterial, and antifungal properties.

1 Background

Antimicrobial resistance (AMR) is one of the top global public health and development threats. It is estimated that bacterial AMR was directly responsible for 1.27 million global deaths in 2019 and contributed to 4.95 million deaths [1]. Unfortunately, the number of deaths attributable to antimicrobial resistance is expected to exceed those caused by cancer by 2050 [2]. The development of drugs resistant to the microbial effect stimulates the search for new alternatives with unique action modes. Virgin coconut oil is derived from the plant species Cocos nucifera Linn (Arecaceae), primarily found in tropical regions [3]. The oil mainly consists of medium-chain fatty acids, particularly lauric acid, and other fatty acids, including oleic and linoleic acids. Coconut oil has been observed to possess a notable capacity for absorption, exhibiting a propensity for enhancing cellular metabolism, promoting wound moisture, and mitigating inflammatory manifestations [4]. Hydrolyzing coconut oil often forms monoglycerides, diglycerides, and free fatty acids. Coconut oil exhibits potential for wound-healing formulations due to its antibacterial and antifungal properties, mainly attributed to monoglycerides, free fatty acids, lauric acid, and monolaurin [5].

Commercial coconut oil the lack of antimicrobial activity [6] corroborates the idea of catalytic need in the antimicrobial properties of coconut oil and coconut milk-derived products. A nano-sized emulsion can enhance functional compounds, such as bioactive lipids, antioxidants, and antimicrobials [7]. Nanoemulsions (NEs) refer to heterogeneous formulations consisting of two immiscible liquids, commonly oil, and water that are stabilized by surfactants, which are surface-active agents. During the process of emulsification, surfactants play a crucial role in minimizing or preventing the coalescence of oil droplets by adsorbing at the interface between oil and water, hence reducing the surface tension. Surfactants have a role in promoting the separation or disruption of dispersed phases by creating a film at the interface between oil and water, which effectively delays mechanisms that lead to destabilization. The selection of surfactants plays a crucial role in the formulation of nanoemulsions since it determines the desired physiochemical characteristics of the final product. Nanoemulsions (NEs) exhibit droplet sizes within the range of 10 to 1000 nm, rendering them kinetically stable but thermodynamically unstable systems. This instability arises from the larger free energy present within the system in comparison with the individual phases [8]. Consequently, the process of phase separation in homogenized NEs is influenced by the presence of kinetic energy barriers. This phenomenon unfolds gradually over time and gives rise to destabilizing processes such as Ostwald ripening and flocculation. Nevertheless, it is possible for nanoemulsions (NEs) to exhibit extended durability or shelf-life, provided the chosen system components are rationalized according to their physiochemical characteristics. The stability of nanoemulsions (NEs) is a critical feature in medical applications since any changes in their physiochemical properties can significantly affect the pharmacological properties of the formulation. NEs are a burgeoning category of colloidal carriers that possess distinct characteristics enabling their effective and cost-efficient utilization in several fields, such as cosmetics [9], food [10], pharmaceuticals [8, 11], and agriculture [12].

Infectious viral diseases and outbreaks are continuously evolving global threats. Despite successive advances in the medical field, the prevention and treatment of viral outbreaks remain challenging due to the innate characteristics of viral particles, such as the high dynamic rate of genetic mutations [13]. Viruses and microorganisms' ability to resist drugs is a problem in chemotherapy. Viruses have many genetic variations and mechanisms for their survival [14, 15]. New methods for developing antiviral drugs are required due to the emergence of virus resistance. As a result, advances in novel medications and antiviral therapeutic techniques continue. New drug research has many prospects thanks to nanotechnologies. The antiviral activity of nonfunctionalized nanoparticles is a crucial finding [16] of specific interest, mainly because the watched activity probably results from a new mechanism of action. The experimental evidence for no functionalized nanoparticles' antiviral activity [17] and the findings shown in this communication allow for an agent based on the proximity interaction of nanoparticles with the viral envelope and its constituent parts.

HSV1 and HSV2 are common human diseases in the alpha herpes virus family [18]. Every herpes virus has an enveloped virion with an icosahedral nucleocapsid that contains the genome's double-stranded linear DNA. HSV's neurovirulence and latency directly impact humans and the illness's progression. This can lead to severe neurological consequences, such as HSV encephalitis and profound disease. HSV creates a prolonged infection by dodging the host's immunological response. The HSV genome is kept in a suppressed condition in the neural cells throughout latency. The viral genome may then be activated and transmitted to the skin by the neuron's axon, leading to viral replication and the regrowth of herpetic sores. Due to the rise in immunocompromised individuals and sexual activity, HSV incidence and severity have grown over the past few decades. Close intimate touch is when HSV1 and HSV2 transmission happens. Most people get latently infected with HSV1 in infancy or early adolescence and carry it with them for the rest of their lives. HSV2 spreads mainly through sexual contact. Although it aids in infection management, administering antiviral medications after latency development will not result in the virus' eradication. Herpes labialis, herpetic gingivostomatitis, herpes genitalis, keratitis, encephalitis, and newborn herpes infection are all brought on by HSV [19]. After initial infection, the virus creates a lifelong latent infection in the sensory and autonomic brain ganglia. HSV can cause life-threatening conditions in infants and those with weakened immune systems, physical disabilities, social marginalization, and psychological suffering [20]. HSV1 and HSV2 infections have affected, respectively, 3.7 billion and 417 million persons worldwide, according to a 2012 World Health Organization (WHO) study [21].

HSV infections and reactivation are treated with acyclovir (ACV), penciclovir (PCV), and related pro-drugs [22]. The emergence of medication resistance in high-risk populations, including immunocompromised individuals and transplant recipients, motivated researchers to look for new therapeutic agents with a distinct mode of action [19]. Additionally, antimicrobial resistance occurs when fungi and bacteria evolve to resist the medications created to kill them. The overuse of antibiotics, which has given microorganisms the capacity to form biofilms embedded in the extracellular matrix that make it difficult for antibiotics to enter cells, is one of the primary causes of this resistance [23, 24]. The discovery of innovative approaches for treating infections linked to antimicrobial resistance and biofilm formation in afflicted individuals has been prompted by the capacity of some strains of bacteria and fungi to resist the actions of conventional antibiotics and antifungals. Antimicrobial nanoemulsions are detergent, oil, and water combinations that have been emulsified. They exhibit overall antibacterial effectiveness against fungi, enveloped viruses, and bacteria; the size of the droplets ranges from 100 to 800 nm [25].

In the present study, coconut oil nanoemulsion was prepared with three different concentrations using 1.5, 3, and 6 ml of native coconut oil. An attempt has been made to evaluate them as anti-HSV, antibacterial and antifungal.

2 Methods

2.1 Materials

Coconut oil (CO) was obtained from Tropicana oil, Thailand. Tween®60 was purchased from WIN Lab Co. India. Tetraethyl orthosilicate (TEOS) was purchased from ALPHA CHEMIKA, Mumbai, India.

The chosen strains for evaluating the inhibitory activity of ON-0@SiO2 and ON-3@SiO2 include Escherichia coli ATCC 8739 and Pseudomonas aeruginosa ATCC 15442, which are gram-negative (G −) bacteria, Staphylococcus aureus ATCC 25923 and Bacillus subtilis ATCC 6633 which are gram-positive (G +) bacteria. At the same time, Candida albicans ATCC 10231 is a fungus strain. The strains used in the testing were from the import company Sigma-Aldrich's from the American Type Culture Collection.

2.2 Preparation of coconut oil nanoemulsion

The nanoemulsions containing coconut oil (CO) were prepared as follows: The oil phase was formed by adding (1.5, 3, and 6 ml) of CO to 30 ml H2O containing 1.5 ml of Tween® 60 as surfactant. Meanwhile, the aqueous phase was prepared by dissolving 3 ml of TEOS in 30 H2O. The two phases were separately stirred for 15 min using a homogenizer (1500 rpm). After that, the oil phase was added drop by drop to the aqueous phase (TEOS solution) and vigorously stirred using a high-speed homogenizer for 15 min. At the end of stirring time, the color of the whole solution was changed due to the preparation of CO-loaded SiO2 nanoemulsion. The blank nanoemulsion without oil (ON-0@SiO2) was prepared as previously mentioned, except for adding CO. The other samples of SiO2 loaded with three CO concentrations (1.5, 3, and 6 ml) are coded as ON-1@SiO2, ON-2@SiO2, and ON-3@SiO2, respectively. The formed nanoemulsions were kept in the refrigerator for further characterization and biological studies. The nanoemulsion characterization includes particle shape and topographical features.

2.3 Characterization of coconut oil nanoemulsion

2.3.1 Gas chromatography–mass spectrometry analysis (GC–MS) for phytochemical identification of coconut oil

The phytochemical constituents of coconut oil were determined using GC–MS analysis. After its solubilization in chloroform, the sample was injected into the GC apparatus. The investigation carried out at the Central Laboratories Network, Cairo, Egypt, utilized a GC–MS system from Agilent Technologies. The system consisted of a gas chromatograph (model 7890B) and a mass spectrometer detector (model 5977A). A gas chromatography apparatus was furnished with an HP-5MS column measuring 30 m in length, 0.25 mm in film thickness, and 0.25 mm in internal diameter. With a flow rate of 2.0 ml/min and a splitless injection volume of 1 µl, hydrogen gas was employed as the carrier gas. The investigation commenced with an initial temperature of 50 °C, which was maintained for a period of 5 min. Following this, the temperature was progressively raised until it reached 100 °C, after which it remained at this point for an interval of 0 min at a rate of 5 °C/min. In the end, the temperature was elevated to 320 °C and maintained at that level for 20 min, at a rate of 10 °C/ min. The temperatures of the injector and detector were maintained at 250 °C and 320 °C, correspondingly. With a solvent delay of 4 min, electron ionization at an energy level of 70 eV produced mass spectra spanning the range of m/z 25–700. Despite the Quad's recorded temperature of 150 °C, the mass temperature was determined to be 230 °C. By conducting a comparison between the fragmentation pattern of a spectrum and the data contained in the Wiley and NIST Mass Spectral Libraries, many constituents can be identified.

To determine the particle shape of the prepared nanoemulsion samples (ON-1@SiO2, ON-2@SiO2, and ON-3@SiO2), TEM (JEOL, 2100, Japan) was used. Firstly, the nanoemulsion samples were placed on a copper grid coated with a thin carbon film and dried in the air before TEM investigation. Meanwhile, the droplet size determination of the three prepared nanoemulsion samples was evaluated using Zetasizer Nano-ZS, Malvern (UK).

2.4 Antiviral activity

The Vero-E6 cells (105 cells/well) were plated on a 6-well tissue culture plate. Following two washes with 1X fetal bovine serum (FBS) at 90% to 100% confluence (one-day post-seeding), the virus was diluted tenfold in a medium containing 2% FBS (Lonza, USA) and 2% antibiotic–antimycotic combination (Lonza, Walkersville. MD, USA). Four hundred microliters of infection media was combined with 100 ul of each dilution before being used to inoculate a single well of Vero-E6 cells. A one-hour incubation period was performed at 37°C and 5% carbon dioxide. To get rid of any lingering inoculum, the wells were sucked. Afterward, 2 ml of 1:1 2X medium and 2% agarose type 1 overlay was quickly poured on each well (Lonza, Basel, Switzerland). The plates then spent three days in a 37°C, 5% CO2 incubator. The plaques were monitored daily under a microscope as they formed. Plaques were seen, and then 1 ml of 10% formaldehyde was added to each well and left there for 2 h at room temperature (TR) to kill the virus and fix the cells. Finally, the plates were washed with water and dried once the formaldehyde had been removed. After adding 1 ml of the staining solution (1% crystal violet and 20% methanol in distilled water) to each well and letting them sit at room temperature for 5 min, the plaques could be seen [26,27,28]. Then, the number of viral plaques was used to determine the titer of the virus using the following formula:

$${\text{Plaque}}\;{\text{forming}}\;{\text{unit}}\;\left( {{\text{PFU}}} \right){\text{/ml}} = {\text{Virus}}\;{\text{dilution}} \times {\text{number}}\;{\text{of}}\;{\text{plaques}} \times {\text{volume}}\;{\text{of}}\;{\text{inoculum}}\; \times \;{\text{multiplicand}}\;{\text{number}}\;{\text{to}}\;{\text{complete}}\;{\text{the}}\;{\text{inoculum}}\;{\text{volume}}\;{\text{to}}\;{\text{one}}\;{\text{ml}}{.}$$

2.4.1 Cytotoxicity by MTT protocol (CC50)

The specimens were diluted by DMEM (Dulbecco's modified Eagle's medium). The test chemicals were dissolved in 10% DMSO in dd H2O to create stock solutions. The cytotoxic activity of the extracts was evaluated on Vero-E6 cells using a modified version of the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay [29, 30]. Briefly, the cells were plated on 96 plates (100 ul/well at a density of 3*105 cells/ml) and incubated at 37 °C and 5% CO2 for 24 h. After 24 h, cells were treated in triplicate with varying doses of the test substances. After a further 72 h, the supernatant was removed, and the cell monolayers were washed three times with 1X phosphate-buffered saline (PBS) before MTT solution (20 ul of a 5 mg/ml stock solution) was added to each well and incubated at 37°C for 4 h, followed by medium aspiration. In each well, 200 ul of DMSO (0.04 M HCl in 100% isopropanol = 0.073 ml HCl in 50 ml isopropanol) was used to dissolve the formazan crystals. Using a multi-well plate reader, the absorbance of formazan solutions was measured at a max of 540 nm and 620 nm as the reference wavelength. The plot of percent cytotoxicity against sample concentration was used to determine the concentration at which 50% cytotoxicity was observed. “The % of cytotoxicity compared to the untreated cells was determined with the following equation:

$$\% \;{\text{cytotoxicity}} = ({\text{absorbance}}\;{\text{of}}\;{\text{cells}}\;{\text{without}}\;{\text{treatment}} - {\text{absorbance}}\;{\text{of}}\;{\text{cells}}\;{\text{with}}\;{\text{treatment}}) \times 100\;{\text{absorbance}}\;{\text{of}}\;{\text{cells}}\;{\text{without}}\;{\text{treatment}}$$

2.4.2 Screening for antiviral activity by plaque reduction assay

MDCK cells were cultivated for 24 h in a six-well number plate at 37°C. The growth medium was removed from the cell culture plates, and the virus human herpes HSV1 and HSV2 viruses were inoculated (100 µL/well) and incubated at 37°C for one hour. Afterward, the numerous concentrations of ON-0@SiO2, ON-1@SiO2, ON-2@SiO2, and ON-3@SiO2 (100 µl/ well) were injected for one h and incubated at 37°C onto the infected cells. Next, one hour contact time, 1.5 ml of DMEM supplemented with 2% agarose was included in the cell monolayer; plates were left to solidify and incubated at 37°C to prepare the creation of viral plaques (through 3 days). Formalin (10%) was inserted for two hours. Plates were stained with 0.1% crystal violet in distilled water. Control wells were included where the untreated virus was incubated with cells, as studied by [31, 32]. Finally, plaques were enumerated, and the plaque reduction % relative to the control was recorded: \(\text{Inhibition}= {\text{Viral} \text{count}}_{\left(\text{untreated}\right)}- \frac{{\text{Viral} \text{count}}_{\left(\text{treated}\right)}}{{\text{Viral} \text{count}}_{\left(\text{untreated}\right)}}\times 100\).

2.4.3 Inhibitory concentration 50 (IC50) determination

Vero-E6 cells spread equally in a 96-well tissue culture plate (2.4 × 104) and incubated for 24 h in a humidified incubator at 37°C and 5% CO2. At room temperature, the monolayer cells were washed with subject and 1 × PBS for adsorption of a virus. Additionally, the monolayer cells were soaked in DMEM (100 ul), which contained oil-loaded silica nanoemulsion, and incubated for 72 h at 37°C and 5% CO2. Cells were stained with crystal violet (0.1%) for 15 min at RT in distilled water after being fixed in paraformaldehyde (100 ul of 4%) for 20 min. Then, crystal violet dye per well was dissolved by absolute methanol (100 ul) and measured at 570 nm of optical density. The oil-loaded silica nanoemulsion IC50 is a concentration of 50% of virus-induced cytopathic effects reduction relative to virus control [26,27,28].

2.4.4 Mode of action of virus inhibition

The possible mode of action of virus inhibition by the coconut oil-loaded silica nanoemulsion was examined at three potential mechanisms of action: (i) Inhibition of viral replication and budding. (ii) the direct effect of coconut oil-loaded silica nanoemulsion to inactivate the virus viability (virucidal activity) and (iii) The ability of coconut oil-loaded silica nanoemulsion to infected cells-membrane fusion to inhibit the attachment of the virus known as blocking the viral entry (protective activity or viral adsorption). For consistency, each action was repeated three times individually of each other, and the average of the three experiments of each activity was posed. Additionally, the above-mentioned mode of action could account for the recorded antiviral.

2.4.5 Infection Post-infection treatment (viral replication)

The 105 cells/ml vero-E6 cells (6-well plate) were incubated at 37°C for 24 h. The virus was transferred to the cells after being diluted to 10 PFU per well and incubated at 37°C for one hour to ensure complete adsorption. Additionally, wash cells with free medium supplements three times to eliminate unabsorbed viral particles. After one hour of oil-loaded silica nanoemulsion, the cell monolayer was overlaid with DMEM medium supplemented (3 ml) with agarose (2%). The viral plaques were shown after the hardening of plates, which were incubated at 37°C. The cell monolayers were stained with crystal violet dye after being fixed for two hours in 10% formalin. In the negative control well, the virus was incubated with vero-E6 cells without being treated with the oil-loaded silica nanoemulsion; on the other hand, remdesivir was used as positive control. Finally, we recorded the percentage of plaque reduction formation relative to control wells [26,27,28].

2.4.6 Cell pretreatment (protective activity) viral adsorption:

This protocol showed that the virus enters the host cell by preventing attachment to the cell surface. In the 6-well plate, Vero-E6 cells were cultivated at 37°C for 24 h for assay of viral adsorption. The oil-loaded silica nanoemulsion in different concentrations was applied in a medium (200 ul), and then the cell was incubated at 4°C for two hours. The oil-loaded silica nanoemulsion unabsorbed was eliminated by washing three times with a supplemented free medium. The diluted virus was set for one hour with the pretreated cell, adding DMEM increased with agarose (1%). The viral plaques formed after the plates hardened and incubated at 37°C. Fixed, stained, and recorded results, as mentioned above [26,27,28].

2.4.7 Virus pretreatment (virucidal):

In a 6-well plate, the vero-E6 monolayer cells were propagated at 37°C for 24 h for virucidal assy. DMED (200 ul) containing virus was added to nonlethal concentrations of oil-loaded silica nanoemulsion (1:1 V/V). After virus adsorption, incubated for one hour, diluting mixture by free medium each tenfold, whereas viral particles still grow on vero-E6 cells but no excess of oil-loaded silica nanoemulsion was present, then added 100 ul of each dilution to monolayer cells. DMEM was added to the top of the cell monolayer. The viral plaques formed after the plates solidified and incubated at 37 °C. Fixed, stained, and recorded results, as mentioned above [26,27,28].

2.5 Antibacterial and antifungal

2.5.1 Antimicrobial Activity

The antimicrobial activity of ON-0@SiO2 and ON-3@SiO2 against bacteria and fungi of clinical interest was tested. The technique was healthy diffusion using a slightly modified variation of the procedure mentioned by [33, 34]. The Petri dishes used for the test were sterilized and prepared with Sabouraud agar (SDA) medium for the growth of C. albicans and Muller–Hinton agar (MHA) medium for bacterial strain growth. The agar surface was inoculated using a sterile spreader with a microbe suspension adjusted to 0.5 McFarland's standard (equivalent to 108 CFU/ml for bacteria and 106 CFU/ml for C. albicans). A 6 mm-diameter hole was made using a sterile cork borer, and 100 μl of each microbe suspension was introduced to the well using a micropipette. Four concentrations of ON-0@SiO2 and ON-3@SiO2 were investigated, containing 100, 50, 25, and 12.5 mg/ml. The negative control was carried out with saline (0.85%), while the positive control was carried out by tetracycline for bacteria and ketoconazole for fungi. For bacterial strains, the plates were incubated at 37°C for 24 h and at 28°C for 72 h; for fungus, zones of inhibition for all plates were determined.

2.5.2 Determination MIC (minimum inhibitory concentration):

Using a standard broth microdilution method according to [35] in Muller–Hinton broth medium and a slightly altered form of the technique in Sabouraud broth medium for tested microorganisms.

Sterile distilled water was added to the wells, serving as the negative control using the Broth Micro dilution procedure. The same sort of suitable broth medium for bacteria and fungus was added to 100 µl (ul) plates to prepare them. A concentration of 100 to 3.12 mg/ml of the tested chemical was obtained by serial dilution. The control wells were left out, and each well received 20 ul of microbial suspension (0.5 McFarland standards) (control wells contained broth only and distilled water only). The absorbance of the plates was measured before and during incubation at 37°C after 24 h for bacteria and after 48 h at 28°C for fungi using an automated ELISA microplate reader (Sun Rise-TECAN, Inc. ®, USA) set at 600 (nm). The data were plotted versus concentration to assess the absorbencies and identify any changes in growth. Inhibiting bacterial or fungal growth at the lowest measured extract concentration was recorded as the MIC [35].

2.5.3 Determination of minimum bactericidal concentration (MBC) and minimum fungicidal concentration (MFC):

The wells of MICs obtained from the previous experiment were taken to be re-cultured on new media of MHA for bacteria and SDA for fungi. Plates of bacteria and fungi were incubated for appropriate time and temperature, depending on the organism type. The bactericidal endpoint (MBC) is the lowest concentration and eliminates 99.9% of the remaining germs [36]. MFC is the lowest drug concentration, killing ≥ 99.9% of cells [37]. The determination of MBC and MFC may be clinically more significant than the MIC of some antibacterial and antifungal drugs to effectively treat bacterial and fungal infections.

3 Results

3.1 GC–MS for the phytochemical identification of coconut oil

The study of bioactive phyto-compounds in coconut oil involved GC–MS analysis, revealing 17 compounds with diverse chemical structures and potential biological activities (Table 1 and Fig. 1). These compounds include 2(3H)-Furanone, dihydro-5-pentyl-, dodecanoic acid, oleic acid, 9,12-Octadecadienoyl chloride, dodecanoic acid, 3-hydroxy, oxiraneundecanoic acid, 7-Methyl-Z-tetradecen-1-ol acetate, 9-Hexadecenoic acid, 3-(Octanoyloxy)propane-1,2-diyl bis(decanoate), dodecanoic acid, 1-(hydroxymethyl)-1,2-ethanediyl ester, laurin, 1,2,3-propanetriyl ester, cyclobarbital, cyclobarbital, cyclobarbital, dodecyl cis-9,10-epoxyoctadecanoate, and Octadecanal, 2-bromo.

Table 1 The bioactive phytochemical compounds detected in coconut oil using GC–MS analysis
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Fig. 1
figure 1

GC–MS chromatogram of bioactive compounds in the coconut oil

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3.2 Characterization of coconut oil nanoemulsion

Our naked eye first investigated the as-prepared coconut oil nanoemulsion with different concentrations. It was observed that the prepared nanoemulsion samples were formed as a milky color with one phase. The absence of separation between nanoemulsion components confirmed the successful preparation. A high-speed homogenizer can potentially prepare coconut oil nanoemulsion with good distribution and blending of nanoemulsion components (TEOS, coconut oil, and Tween®60). Then, TEM was used to examine the shape of nanoemulsion particles (Fig. 2). Some samples were taken at two different magnifications to investigate the distribution of particles. It can be observed that the nanoemulsion sample (ON-1@SiO2) that was prepared using a low concentration of coconut oil (1.5 ml) has a spherical shape with uniform distribution (Fig. 2a, b) and is free to disperse without co free to distribute without aggregation. Increasing the concentration of coconut oil to 3 ml for the preparation of ON-2@SiO2 (Fig. 2c) leads to the formation of particles with nearly spherical shapes but with less distribution than ON-1@SiO2. However, high-speed homogenizer still has an efficient power to prepare uniformly nanoemulsion with small size. As observed from (Fig. 2a, c), there is no significant change regarding the particle shape or their distributions. On the contrary, increasing the concentration of coconut oil to 6 ml significantly affects the particle shape and distribution of the formed nanoemulsion. It can be observed that ON-3@SiO2 (Fig. 2d) does not have a specific condition as in the other two samples (ON-1@SiO2 and ON-2@SiO2). As seen from the TEM image (Fig. 2d), these particles have formed large aggregations, which can be attributed to using coconut oil with a high concentration (6 ml).

Fig. 2
figure 2

TEM of a, b ON-1@SiO2, c ON-2@SiO2 and d ON-3@SiO2

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Dynamic light scattering was used to calculate the average size (200 nm) of coconut oil nanoemulsions (ON-1@SiO2 and ON-2@SiO2 and ON-3@SiO2), and the data were obtained as shown in Fig. 3a–c. It was observed that ON-1@SiO2 (Fig. 3a) has an average particle size of around 193.1 nm with a high percentage (97.5%), and there are about 2.5%with 4985 nm. The average particle size of the ON-2@SiO2 sample is 200 nm with a high percent (87.1%), and the other peak illustrates another size (5560 nm) with 12.9%. Moving on to the third nanoemulsion sample (ON-3@SiO2), it was found that the volume had increased significantly and recorded a value of 325.7 nm, with a percentage of 84.7%. While it was found that the sample has another size at 4985 nm, but with a small rate (2.5%), from the preceding, it can be said that the obtained results are broadly consistent with the results of TEM.

Fig. 3
figure 3

Particle size analyzer for a ON-1@SiO2, b ON-2@SiO2, and c ON-3@SiO2

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3.3 Cytotoxicity and antiviral of nanoemulsion evaluations

3.3.1 Cytotoxicity

Vero cell monolayers were incubated with various concentrations (0, 20, 50, 80, 150 mg/ml) of coconut oil and (0, 20, 50, 80, and 100 mg/ml) of ON-0@SiO2 and ON-3@SiO2 for multiple periods to rule out the possibility that the cellular toxicity of ON-0@SiO2 and ON-3@SiO2 brought on the reduction in infectivity. Data indicate that three experiments express values, value, mean, and standard deviation. After 48-h incubation, the MTT findings showed that coconut oil, ON-0@SiO2, and ON-3@SiO2 at concentrations up to 0, 20, 50, and 80 mg/ml did not cause cell death.

The study measured the cytotoxic activity of ON-0@SiO2 and ON-3@SiO2 in Vero-E6 cells using an MTT assay. The concentration which exhibited 50% cytotoxic concentration (CC50) was calculated and was found to be equal to 107.6 mg/ml for coconut oil (Fig. 4A), 97.5 mg/ml for ON-0@SiO2 (Fig. 4B), while CC50 was 89.1 mg/ml for ON-3@SiO2 (Fig. 4C).

Fig. 4
figure 4

Cytotoxicity percentage and CC50 of A coconut oil, B ON-0@SiO2, (C) ON-3@SiO2 by MTT assay, used with CC50 of 107.6, 97.5 and 89.1 mg/ml, respectively

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3.4 Antiviral evaluation

In the current study, in searching for new anti-HSV1 and anti-HSV2 agents, the antiviral effects of ON-0@SiO2, ON-1@SiO2, ON-2@SiO2, and ON-3@SiO2 compared with Acyclovir (stander drug) after being measured using a plaque reduction assay. The antiviral effects were 28.3, 60.9, 73.9, and 76% for HSV1 with concentrations of 0, 20, 50, and 80 mg/ml, respectively, compared with Acyclovir was 80.4%. On the other hand, they were 21.1, 68.9, 75.3, and 76.4% for HSV2 with concentrations of 0, 20, 50, and 80 mg/ml, respectively, compared with Acyclovir at 87.9% (Table 2).

Table 2 Plaque reduction assay for coconut oil-loaded silica nanoemulsion against Herpes viruses
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3.5 Mode of action

The possible mode of action for ON-0@SiO2, ON-1@SiO2, ON-2@SiO2, ON-3@SiO2, and Acyclovir may be investigated at three different levels: inhibition of viral replication, virucidal activity, and viral adsorption. Herein, the mode of action for the coconut oil-loaded silica nanoemulsion with different safe concentrations was illustrated for ON-0@SiO2, ON-1@SiO2, ON-2@SiO2, and ON-3@SiO2 (Fig. 5).

Fig. 5
figure 5

Modes of action for the silica nanoemulsion and coconut oil-loaded silica nanoemulsion against HSV1 and HSV2

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Percent inhibition for each mode of action is shown in Fig. 5. Interestingly, the results showed that the best mechanism of action for antiviral activity was the adsorption effect for ON-3@SiO2. ON-3@SiO2 had a combination of viral inhibitory effects on HSV1 and HSV2 at different viral stages. ON-3@SiO2 showed 68 and 63% virucidal results, indicating that it possibly acts directly on the virion, causing inactivation. Additionally, it showed 86 and 90% inhibitory effects during the viral adsorption stage. It indicated 62 and 67% inhibitory effects during the viral replication stage against HSV1 and HSV2, respectively. Furthermore, the ON-2@SiO2 exhibited a virucidal impact with a 66 and 62% viral inhibitory effect. Additionally, it showed 83 and 82% inhibitory effects during the viral adsorption stage. It indicated 50 and 61% inhibitory effects during the viral replication stage against HSV1 and HSV2, respectively. ON-1@SiO2 showed almost 37 and 40% inhibitory effects on virucidal and 49 and 51% virus adsorption. And 21 and 29 virus replications against HSV1 and HSV2, respectively. Conversely, ON-0@SiO2 displayed the virucidal effect with a 26 and 31% viral inhibitory effect, 50 and 48% virus adsorption, and 28 and 20 virus replications against HSV1 and HSV2, respectively. This result confirmed the results of the crystal violet assay, which gave IC50 equal 1.9 and 5.5 mg/ml for ON-3@SiO2, ON-0@SiO2, for HSV1, and 2.1 and 6.2 for HSV2, respectively (Table 3).

Table 3 Antiviral activity of ON-0@SiO2 and ON-3@SiO2 against Herpes viruses
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3.6 Evaluations of antimicrobial activity

Figure 6 clearly shows that the coconut oil showed an effect against both E. coli and B. subtilis at a concentration of 100 mg/ml, but it did not show any effect against P. aeruginosa, S. aureus, and C. albicans. Antimicrobial susceptibility tests were performed against bacterial and fungal strains to examine the antimicrobial activity of the synthesized ON-0@SiO2 and ON-3@SiO2. Firstly, ON-0@SiO2 showed a weak effect against only E. coli, S. aureus, and B. subtilis (Fig. 7). In contrast, ON-3@SiO2 showed the highest antimicrobial activity against all the tested bacteria and fungi with a dose-dependent manner of the following zone sizes: 27, 26, 17, and 0 mm for E. coli; 13, 11, 7, and 1 mm for P. aeruginosa; 12, 10, 1 and 0 mm for S. aureus; 27, 26, 23 and 21 mm for B. subtilis and 13, 10, 7 and 2 mm for C. albicans at concentrations 100, 50, 25 and 12.5 mg/ml, respectively. All strains responded to ON-3@SiO2 in a dose-dependent manner. As the concentration of ON-3@SiO2 was diluted, the size of the inhibition zone decreased significantly with all bacterial and fungal strains. Both E. coli and B. subtilis are more sensitive to ON-3@SiO2 than P. aeruginosa, S. aureus, and C. albicans. Negative control does not show any effect, while positive control showed a higher impact against gram-negative strains than gram-positive ones. The present study evaluated the MIC, MBC, and MFC of ON-3@SiO2 against several microorganisms. Generally, the MIC, MBC, and MFC values for the tested bacteria and fungi were found to be 3.12 to 25 mg/ml. The MIC against E. coli, P. aeruginosa, S. aureus, B. subtilis, and C. albicans were 25, 12.5, 25, 3.12, and 12.5 mg/ml, respect regarding MBC, all bacterial strains showed an MBC value of 25 mg/ml except for B. subtilis. Regarding MBC, all bacterial strains showed an MBC value of 25 mg/ml except for B. subtilis, which had an MBC value of 6.25 mg/ml. Also, C. albicans showed an MFC value of 25 mg/ml. All these findings are summarized in Tables 4 and 5 and Figs. 7, 8, and 9.

Fig. 6
figure 6

Antimicrobial activity of coconut oil against bacterial and fungal strains. PC = positive control; 1 = 100 mg/ml, 2 = 50 mg/ml; 3 = 25 mg/ml; 4 = 12.5 mg/ml

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Fig. 7
figure 7

Antimicrobial activity (zone of inhibition) of ON-0@SiO2, ON-3@SiO2, and positive control (PC) against bacterial and fungal strains

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Table 4 MIC (mg/ml) values of ON-3@SiO2 against different tested microorganisms
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Table 5 MBC and MFC of ON-3@SiO2 (mg/ml) after 24 h
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Fig. 8
figure 8

Antibacterial activity of ON-0@SiO2 and ON-3@SiO2 against both gram-positive and gram-negative strains. 1 = 100 mg/ml of ON-3@SiO2, 2 = 50 mg/ml of ON-3@SiO2; 3 = 25 mg/ml of ON-3@SiO2; 4 = 12.5 mg/ml of ON-3@SiO2

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Fig. 9
figure 9

Antifungal activity of ON-0@SiO2 and ON-3@SiO2 against C. albicans. PC; positive control, NC; negative control; 1 = 100 mg/ml of ON-3@SiO2, 2 = 50 mg/ml of ON-3@SiO2; 3 = 25 mg/ml of ON-3@SiO2; 4 = 12.5 mg/ml of ON-3@SiO2

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4 Discussion

Coconut oil contains several phyto-compounds with antiviral properties, as discovered by GC–MS research. Laurine, also known as monolaurin or 2-capri-1,3-di, is an ester of lauric acid and has been shown to be effective against lipid-enveloped viruses like influenza, HIV, and herpes simplex virus (HSV). It disrupts the lipid bilayer of the viral envelope, preventing viral growth and penetration into host cells [38]. Dodecyl gallate, also known as dodecyl cis-9,10-epoxyoctadecanoate, has shown antiviral efficacy against various viruses, including enteroviruses, hepatitis C virus (HCV), and herpes viruses. Its mechanism is believed to be interfering with glycoproteins used by the virus to bind to cell receptors [39]. Glycerol monolaurate, also known as glycerol monolaurate or monolaurin, has broad-spectrum antiviral action against enveloped viruses like HIV, HSV, and respiratory syncytial virus [40]. Notably, Furfural, also known as 2(3H)-Furanone, has antiviral properties against influenza, adenovirus, and HSV by inhibiting viral protein synthesis and replication.

Decanoic acid (DA) is a saturated medium- fatty acid, also known as capric acid [41]. Its antiviral properties are attributed to its ability to disrupt the lipid bilayer of the viral enveloped, similar to lauric acid and its derivative monolaurin. Decanoic acid has been studied for its potential antiviral effects, particularly against herpes simplex virus (HSV) and other lipid-enveloped viruses [42].

Virus mutations and bacterial antibiotic resistance are serious problems that are common worldwide, so most researchers try to solve this problem by discovering novel drugs [14, 43]. It is worth noting that HSV1 and HSV2 herpes viruses pose a significant risk to public health worldwide and can spread globally and start pandemics [22, 44]. The discovery of novel and potent anti-herpes medicines is urgently needed since the effectiveness of the present antiviral treatments against herpes is compromised by their severe side effects and the rising occurrence of drug-resistant strains during treatment [45]. Using nanoparticles for therapeutics has several benefits over traditional therapies, including potent antiviral efficacy against drug-resistant viruses, cost-effective manufacturing, and compatibility with various coating types [23, 46,47,48].

The MTT findings showed that coconut oil, ON-0@SiO2 and ON-3@SiO2 at concentrations up to 0, 20, 50, and 80 mg/ml did not cause cell death [49], and also our result indicated that according to the results of the cytotoxicity assay, the doses of 80, 50, and 20 mg/ml as different safe concentrations were selected to begin assays of plaque reduction for ON-0@SiO2 and ON-3@SiO2, both Human herpes HSV1 and HSV2 viruses’ propagation, as well as antibacterial and antifungal activities [50].

It is worth noting that HSV1 and HSV2 herpes viruses pose a significant risk to public health worldwide and can spread globally and start pandemics. The discovery of novel and potent anti-herpes medicines is urgently needed since the effectiveness of the present antiviral treatments against herpes is compromised by their severe side effects and the rising occurrence of drug-resistant strains during treatment [51,52,53]. Using nanoparticles for therapeutics has several benefits over traditional therapies, including potent antiviral efficacy against drug-resistant viruses, cost-effective manufacturing, and compatibility with various coating types [23, 46,47,48].

Generally, antiviral compounds inhibit the formation of new viruses by blocking crucial steps of viral replication or directly acting on the virus [54]. Understanding of the antiviral mechanism of action of nanoemulsion is therefore essential for target-oriented and efficient development of new antiviral strategies exploiting nanoparticles. Nevertheless, their antiviral mechanisms are still under investigation and seem to depend on the synthesis of nanoemulsion, with many factors such as size, shape and surface functionalization influencing the antiviral activity. This suggested that the nanoemulsion acted as an inhibitor as it emulated cellular receptors, or virus receptor inducing the virus to bind nanoemulsion. These nanoemulsions have been applied to an HSV inhibition test, but they potentially can be used to hinder other viruses, since functional groups present on the nanoemulsion surface and different basic materials can attract and inhibit different types of viruses [23].

Our results indicated that coconut oil-loaded silica nanoemulsion inhibited herpes viruses; additionally, nanoemulsion may decrease viral replications in host cells by blocking viral attachment on host cells. However, it has been proposed that the antiviral action of nanoemulsion is caused by two different processes that prevent viral replication: 1. nanoemulsion may interact with viral components and double-strand breaks when they traverse cell membranes. 2. Nanoemulsion can attach to sulfur-containing residues on the surface glycoproteins of viruses, preventing viral-host penetration and cell binding and leaving the virus in the extracellular space where it cannot spread [55,56,57,58]. Nano-loaded natural products have been recognized as a significant therapeutic approach within traditional medicine, particularly in addressing the worldwide pandemic and facilitating the recuperation of those afflicted with COVID-19 [59]. Numerous plants synthesize secondary metabolites and several other compounds as a defense mechanism against the intrusion of pathogens, including viruses. NEs have been shown to specifically target certain types of infections, while others effectively deliver active antivirals and vaccinations [60, 61]. According to previous studies [62, 63], it has been found that Leptospermum scoparium and Chamomilla essential oils and nanoemulsion have notable efficacy as antiviral agents specifically targeting the herpes simplex viruses HSV1 and HSV2, respectively.

Our results indicated that the coconut oil-loaded silica nanoemulsion has antiviral properties against HSV1 and 2, which prevent the virus from entering the cell by blocking the cell or virus receptors, so the high results appear in adsorption mode. Viruses are classified as a diminutive obligatory intracellular parasite. Extracellular virions facilitate the dissemination of these entities outside host cells, where they exist in an inert state. The interactions between the pathogen and the host cell mostly involve receptor-ligand interactions [64]. The antiviral properties of several natural items have demonstrated significant potential in inhibiting the entry of pathogenic viruses into cells and interacting with their pathogenicity [62]. Viral features, which are essential for the entrance into host cells or adsorption, might be either concealed or directly impeded to hinder viral infection [65, 66].

Despite the coconut oil having antimicrobial activity against many bacterial and fungal strains [67], it showed a weak effect on the isolates used compared to the positive control. Antimicrobial resistance pathways can be divided into four major groups as shown in Fig. 10. First, reducing medication uptake by gram-negative bacteria's lipopolysaccharide (LPS) layer [45]. Secondly, modifying a drug target, e.g., inhibiting cell wall synthesis, ribosomal mutation, inhibiting metabolic pathways via enzyme mutations, and modifying DNA gyrase or topoisomerase [68]. Thirdly, there are two main ways to inactivate a drug: by actual drug degradation or by adding a chemical group to the drug. Acetyl, phosphoryl, and adenyl groups are the most frequently added [69], or the drug is hydrolyzed by, e.g., β-lactamase [70]. Fourthly, active drug efflux [71]. Gram-negative bacteria use all four main mechanisms [72]. Our data showed that ON-0@SiO2 showed little effect on the tested strains; this may be due to the need for materials with potent activity against pathogenic bacteria in antibacterial treatments or surfaces may be the reason for this. Nude silica nanoparticles showed no detrimental effects on bacteria [73]. In contrast, all bacterial and fungal strains responded to ON-3@SiO2; this may be due to the energy held in the oil-and-detergent emulsion being released and destabilizing the lipid membrane, leading to the antibacterial activity of Nanoemulsions, which function by fusing with lipid bilayers of cell membranes. [74]. The tested bacteria and fungi are responsible for many surface and local infections, which need specific treatment for each condition, and contrary to antibiotics, Nanoemulsions antibacterial activity is nonspecific, allowing for broad-spectrum activity while reducing the development of resistance. Due to these characteristics, Nanoemulsion can treat wounds and clean surfaces [74].

Fig. 10
figure 10

Mechanisms of microorganism's resistance to antibiotics. Gram-negative (G −); gram-positive (G +)

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5 Conclusion

Coconut oil-loaded silica nanoemulsion was prepared using Tween 60 as a surfactant and stabilizing agent. In addition, a high-speed homogenizer was used for dispersion. It was concluded that the nanoemulsions nominated as ON-1@SiO2 and ON-2@SiO2 were obtained with uniform distribution and spherical shape. The size of both nanoemulsions was around 200 nm. Our results showed that ON-3@SiO2 had promised as anti-HSV1 and HSV2 because the selective index is 51.3 and 42.4 and inhibition of them 76.0 and 76.4%. ON-3@SiO2 had a combination of viral inhibitory effects on HSV1 and HSV2 at different viral stages. it showed 86 and 90% inhibitory effects during the viral adsorption stage. On other hand, ON-3@SiO2 showed the highest antimicrobial activity against all the tested bacteria and fungi with a dose-dependent manner of the following zone sizes: 27, 26, 17, and 0 mm for E. coli; 13, 11, 7, and 1 mm for P. aeruginosa; 12, 10, 1 and 0 mm for S. aureus; 27, 26, 23 and 21 mm for B. subtilis and 13, 10, 7 and 2 mm for C. albicans at concentrations 100, 50, 25 and 12.5 mg/ml, respectively. However, the final formulation of coconut oil-loaded silica nanoemulsion exhibited significant activity against HSV1, HSV2, P. aeruginosa, C. albicans, B. subtilis, and E. coli, and it could be new antiviral, antibacterial, and antifungal agents with significant potential usage in both wound treatment and surface decontamination.

Availability of data and materials

Not applicable.

Abbreviations

MTT:

3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide

ACV:

Acyclovir

CLSI:

Clinical and Laboratory Standards Institute

CO:

Coconut oil

ON@SiO2 :

Coconut oil-loaded silica nanoemulsion

CFU:

Colony-forming unit

CC:

Cytotoxicity concentration

DW:

Distilled water

DMEM:

Dulbecco’s modified eagle’s medium

ELISA:

Enzyme-linked immunosorbent assay

FBS:

Fetal bovine serum

GC–MS:

Gas chromatography–mass spectrometry analysis

HCV:

Hepatitis C virus

HSV:

Herpes virus

HIV:

Human immunodeficiency virus

IC:

Inhibitory concentration

LPS:

Lipopolysaccharide

MDCK:

Madin–Darby canine kidney

MBC:

Minimum bactericidal concentration

MFC:

Minimum fungicidal concentration

MIC:

Minimum inhibitory concentration

MHA:

Muller–Hinton agar

MDR:

Multidrug-resistant

NEs:

Nanoemulsions

nm:

Nanometer

NCCLS:

National Committee for Clinical Laboratory Standards

NC:

Negative control

ON:

Oil nanoemulsion

PCV:

Penciclovir

PBS:

Phosphate-buffered saline

PFU:

Plaque forming unit

PC:

Positive control

RT:

Room temperature

SDA:

Sabouraud agar

TEOS:

Tetraethyl orthosilicate

TEM:

Transmission electron microscopy

WHO:

World Health Organization

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Authors and Affiliations

  1. Botany and Microbiology Department, Faculty of Science, Al-Azhar University, Nasr City, 11884, Cairo, Egypt

    Mohamed E. Elnosary, Ahmed R. Sofy, Ahmed A. Hmed & Ehab E. Refaey

  2. Botany and Microbiology Department, Faculty of Science, Al-Azhar University, Assiut, 71524, Egypt

    Hesham A. Aboelmagd

  3. Microbiology Department, National Institute of Oceanography and Fisheries (NIOF), Aswan Research Station, Aswan, Egypt

    Sayeda M. Ali

  4. Pharmaceutical Technology Department, National Research Centre, Dokki, Giza, Egypt

    Mayssa Abdel Hady

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Contributions

M. E. Elnosary; H. A. Aboelmagd; A. R. Sofy; A. A. Hmed, E.E. Refaey; S. M. Ali; and M. Abdel Hady contributed to the study of conception and design. Material preparation, data collection and analysis were performed by M. E. Elnosary; H. A. Aboelmagd; and Mayssa Abdel Hady. The first draft of the manuscript was written by M. E. Elnosary; H. A. Aboelmagd; and Mayssa Abdel Hady and M. E. Elnosary; H. A. Aboelmagd; A. R. Sofy; A. A. Hmed, E. E. Refaey; S. M. Ali; and M. Abdel Hady commented on previous versions of the manuscript. M. E. Elnosary; H. A. Aboelmagd; A. R. Sofy; A. A. Hmed, E. E. Refaey; S. M. Ali; and M. Abdel Hady read and approved the final manuscript.

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Elnosary, M.E., Aboelmagd, H.A., Sofy, A.R. et al. Uncovering and evaluating coconut oil-loaded silica nanoemulsion as anti-viral, bacterial, and fungal: synthesis, fabrication, characterization, and biosafety profiles. Beni-Suef Univ J Basic Appl Sci 13, 56 (2024). https://doi.org/10.1186/s43088-024-00513-w

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