1. Introduction
Skin is an essential feature of the human body which provides a shield from external foreign particles [
1]. Skin, on the other hand, is much more susceptible to bacterial infection [
2]. Skin illnesses such as eczema, cellulitis, and antibacterial infections caused by bacteria such as
Streptococci,
Staphylococci,
Escherichia coli, and others have become common in today’s world [
3,
4]. Fungi are another type of invading organism that can cause serious fungal infections such as vaginal and superficial candidiasis, ringworm, and athlete’s foot [
5]. The most common forms of fungal skin infections are caused by fungi that flourish in moist, humid settings.
Candida albicans,
Candida glabrata,
Candida krusei,
Trichophyton rubrum,
Tinea capitis,
Tinea barbae,
Malassezia furfur, and
Cryptococcus neoformans are only a few of the fungi that cause such illnesses [
6,
7]. Topical dosage forms such as creams, gels, sprays, and lotions are widely available and contain synthetic active pharmaceutical ingredients (APIs) such as triazole derivatives (fluconazole, eberconazole, ketoconazole, and luliconazole); in addition, there are oral antifungal medications such as tablets, capsules, and suspensions containing fluconazole, itraconazole, and micafungin [
8]. However, due to the progressive emergence of fungal resistance and its side effects, as well as treatment costs, the use of conventional antibiotics now shows major limitations for antifungal treatment. On the other hand, essential oils, apart from having lower toxicity and better biodegradability, are eco-friendly in nature as compared with conventional antibiotics [
9,
10,
11].
Micro-emulgels, being a combination of microemulsion and gel, are a unique and novel dosage form that can cope with the problems associated with conventional dosage forms and have advantages in gel and microemulsion form. Compared to cream, micro-emulgels have more penetration power in skin. Being a dual mechanism using an emulsion and gel, micro-emulgels (
Figure 1) are regarded as one of the most promising technologies among innovative drug delivery systems and have emerged as a novel approach for topical delivery of drugs [
12]. Additionally, adding gel to an emulsion boosted its stability [
13]. The outstanding solubilizing and skin penetration properties of the microemulsion technology were the deciding factors during its formulation development [
14]. The primary requirement for the formulation of an emulgel is the appropriate selection of the oil phase, emulsifier, and gelling agent Therefore, oils, surfactants, and co-surfactants must be carefully screened and optimized during the preparation of micro-emulgels. The choice of these is made based on the solubility profile of the API. The API penetrates the skin more deeply when there is oil present. A large interfacial area is created by the creation of tiny droplets in the microemulsion, which increases the surface area available for medication absorption [
15].
Candidiasis is the most prevalent fungal disease, including a wide range of infections from superficial to systemic with high mortality rates.
Candida albicans is the most studied species for candidiasis infection and is becoming resistant towards existing antifungal drugs. It is a diploid yeast, which means it has two sets of eight chromosomes. Its genome is around 16 megabytes in size and has a total of 6159 coding genes. It is a frequent cause of invasive fungal infections and is also considered as one of the main agents responsible for opportunistic pathogenic infections [
16,
17]. Citronella and cinnamon oils are essential plant oils obtained from
Cymbopogon winterianus and
Cinnamon cassia, respectively [
18]. Citronella is a monoterpenoid that is the main component of citronella oil and gives it its distinct lemon aroma. It is a monoterpene as well as an aldehyde and acts as an antifungal agent both single as well as with its metabolite. Citronella oil is water-soluble, ethanol-soluble, and glycerol-insoluble. Cinnamon is a common spice that has been used for thousands of years by various cultures all over the world [
19]. It is derived from several components of a tropical evergreen tree of the genus Cinnamomum. Several in vivo and in vitro investigations have revealed that cinnamon essential oils and their primary constituents have significant inhibitory effects against a variety of fungi, including
Coriolus versicolor,
Laetiporus sulphureus,
Eurotium spp.,
Aspergillus spp., and
Penicillium spp. [
20,
21,
22,
23]. Fungicidal and inhibitory efficacy of citronella and cinnamon oils on Candida albicans biofilms has already been reported in the literature [
24,
25]. Recently, it was reported that the antimicrobial and anticancer properties of citronella oil were promisingly improved once it was used as a nanoemulsion form of formulation [
26]. Therefore, the current study proposes to develop and analyze a citronella oil-loaded microemulsion-based gel to enhance its solubility and permeability and for improved antifungal efficacy by using carbopol 940 as the gel foundation.
3. Conclusions
In this study, a total of five formulations of CITRO-microemulgel were prepared by using citronella oil as an active pharmaceutical ingredient with different concentrations of 2, 4, 5, 3, and 1% (w/v), whereas cinnamon oil was used as an antifungal oil and penetration enhancer. Among the five formulations, F3 showed the best organoleptic properties in terms of clarity, color, consistency, and phase separation. F3 showed the most favorable pH (6.5 ± 0.12) and contained the highest percentage of drug content, which was 87.05 ± 0.03%. According to the statistics provided, formulation F3 of CITRO-microemulgel had the highest drug release rate of around 87.05% within 4 h. The model with the highest r2 value was chosen as the best-fit model for the formulation. The Korsmeyer– Peppas model with n=0.82, which is between 0.5–1, had the highest r2 value, indicating that release followed non-Fickian/anomalous diffusion and providing a better dimension for all of the formulations. The optimized CITRO-microemulgel (F3) showed a highest zone of inhibition diameter of 35 mm for Candida albicans, which confirms that this can be a promising agent for eradicating infection.
4. Material and Methods
The citronella oil and cinnamon oil were purchased from Allin exporters (Noida, Uttar Pradesh, India). Tween 80, carbopol 940, and methyl paraben were obtained from CDH Fine Chemicals (New Delhi, India). The PEG-200 was from Seva Fine Chemicals (Ahmedabad, India), while the triethanolamine (TEA) was from Fisher Scientific (Loughborough, United Kingdom). Highly pure deionized water was used for aqueous solution preparation during formulation development analysis. All other organic solvents and chemicals used for formulation development and analysis were of analytical and chromatographic grade.
4.1. Preparation of Citronella Oil-Loaded Microemulsion
By using the phase titration method, the microemulsion of citronella oil was prepared using tween 80 as a surfactant and PEG 200 as a co-surfactant, respectively. The different combinations of surfactant and co-surfactant in different ratios referred to as Smix ratios, oil, and water were used to investigate the phase behavior by using a simple titration method. Oil-to-Smix ratios of 1:1, 1:2, 1:3, 1:4, 4:1, and 5:1 were used to create a total of 6 ternary phase diagrams. To create a pseudo-ternary phase diagram for each Smix ratio, solutions comprising oil and Smix were made with volume ratios ranging from 1:9 to 9:1 and titrated drop by drop with water (kept in continuous stirring) at room temperature until turbidity appeared.
4.2. Preparation of CITRO-Microemulgel
The gelling agents impact the consistency of dosage form, spreading coefficient, viscosity of the formulation, drug release from the formulation, and stability of a system. Generally, gelling agents are used to increase the consistency of any formulation. The gel phase was made by dispersing carbopol 940 (a gelling agent) in water with the use of a mechanical stirrer at 1000 rpm until no lumps remained. The cinnamon oil was added as an antifungal oil and penetration enhancer. After that, drop by drop, TEA was added to adjust the pH to 6–6.5. Finally, using a mechanical stirrer set to 1000 rpm for 15 min, the produced emulsion was disseminated in the gel system in a 1:1 ratio [
27]. The schematic flow chart for preparation of CITRO-microemulgel is presented in
Figure 9.
4.3. Evaluation Parameters
4.3.1. Organoleptic Properties and Physical Appearance
The prepared CITRO-microemulgel was kept for 24 h at different specified temperature conditions, i.e., 8 °C, 25 °C, and 40 °C with 75% relative humidity (RH), and was visually evaluated for assessment of their organoleptic (color and odor) and physical stability parameters (grittiness, clarity, phase separation, and consistency) [
28].
4.3.2. pH
The pH values of 1% aqueous solutions of the prepared gellified emulsion were measured by a digital pH meter. The prepared one gram of CITRO-microemulgel was dissolved in 25 mL of distilled water, and the electrode was immersed in the solution for 30 min until a steady reading was obtained. Each formulation’s pH was measured three times, and the average values were calculated [
29].
4.3.3. Spreadability
The distribution of the CITRO-microemulgel formulation was examined after 48 h of preparation. A petri dish with 1 g of emulgel was placed inside a 1 cm diameter circle pre-marked on a glass plate, followed by a 75 g glass plate. On a glass plate, the weights were then left to rest for 5 min. The spread CITRO-microemulgel resulted in an increase in diameter, which was noticed. It was calculated by the formula [
30]:
where
S = Spreadability
m = Weight of an upper glass plate and weight put on it (g)
l = Diameter of spreading emulgel (cm)
t = Time taken to spread emulgel (min)
4.3.4. Drug Content Determination
One gram of CITRO-microemulgel was dissolved in a suitable solvent and filtered to obtain a clear solution. Its absorbance was determined by using a UV spectrophotometer. A conventional drug plot was made in the same solvent. Concentration and drug content were determined by plotting absorbance values on the same standard plot [
31].
4.4. In Vitro Drug Release
The CITRO-microemulgel in vitro diffusion was studied using a dialysis membrane. The membrane was carefully clamped to one end of the dialysis cell’s hollow glass tube after being soaked in phosphate buffer, pH 7.4 (PB) for 6–8 h (2.3 cm diameter; 4.16 cm area). A beaker containing 250 mL of phosphate buffer was employed as the receptor compartment. The membrane was then evenly coated with 1 g of CITRO-microemulgel. The donor compartment remained in contact with the receptor compartment, and the temperature was fixed at 37.5 °C. Externally driven Teflon-coated magnetic bars agitated the liquids on the receptor side. An amount of 5 mL of solution was pipetted out from the receptor compartment at predefined time intervals and immediately replaced with fresh 5 mL phosphate buffer. The drug concentration in the receptor fluid was measured spectrophotometrically in comparison to an acceptable blank. The experiment was conducted three times [
32].
4.5. Microbial Assay
Trench plates were used as a method. They are a technique for figuring out whether a substance has bacteriostatic or fungistatic properties and are mostly utilized in formulations for semisolids. The previously produced agar-dried plates of Sabouraud were used. Three grams of gellified emulsion were placed in a trench that had been carved in the plate. Freshly made culture loops were smeared across the agar at a straight angle from the ditch to the edge of the plate. Following 18 to 24 h of incubation at 25 °C, the fungal growth was checked, and the percentage of inhibition was estimated as follows [
33].
where
L1 = Total length of the streaked;
L2 = Length of Inhibition.