Otitis media is caused by bacterial infections entering the middle ear via the Eustachian tube. It can arise from a perforation in the eardrum and can be caused by allergies, respiratory infections, or anatomical issues (Schilderet al., 2016; Vanneste & Page, 2019). It can be caused by bacteria, viruses, or co-infections, with Streptococcus pneumoniae, Moraxella catarrhalis, and Haemophilus influenza (Silva & Sillankorva, 2019; Mittalet al., 2018).

Marjoram oil, an essential oil, obtained by extraction from Origana majorana plant is used in aromatherapy, cosmetics, skin care, and treating sore muscles and joints. It has therapeutic properties like antibacterial, antimicrobial, antiviral, anticancer, and anti-inflammatory (Dhiman & Bhasin, 2022; Thanhet al., 2019). Marjoram oil, a popular antimicrobial agent, has the greatest potential for industrial applications and widely extracted worldwide. It contains mixture of many phytochemicals with high percentage of terpinene-4-ol (29.6%) and sabinene hydrate (3%), and thymol (17.47%) and carvacrol (50%). These phytochemicals are responsible for antimicrobial and antifungal effects (Vera, 1999; Kamariet al., 2023; Raina & Negi, 2012). Chemical structure of marjoram oil components shown in Figure 1.

Microemulsions serve as carriers that enhance permeation and penetration through the skin by effectively overcoming the barrier of the stratum corneum. Their unique characteristics enable them to increase the liposolubility of hydrophilic drugs by incorporating a less polar chain into their more polar side, improving the bioavailability of challenging molecules. Similarly, for lipophilic substances, a less polar side can be combined with a more polar chain to enhance their effectiveness (Soutoet al., 2022). These properties of microemulsions are significant in the development of antibacterial microemulsions to penetrate the drug inside bacteria for the effectiveness.

In the view of antimicrobial activity, present work focused to develop microemulsion of marjoram oil in the line of treatment of Otitis media.

Origanum majorana leaves were purchased from a local nursery in Malegaon. Tween 80 (Loba Chemie), propylene glycol (Loba Chemie), and ethanol (Pure Chem) were purchased from chemical suppliers. The materials used in the study were of pharmaceutical and analytical grade.

Plant material of Origanum majorana L. was collected in Malegaon from June to December 2023 and authenticated with voucher specimen number VSPOM-1 by the Botanical Survey of India, Western Circle, in Pune. The leaves were allowed to dry in the shade. The dried, finely ground sweet marjoram sample was a greyish-brown fine powder with a characteristic essence. This powder was used for the extractions. Dried Origanum majorana leaves (100 g) were placed in a 2 L flask containing 1 L of demineralized water. The Clevenger extractor was set up, and the leaves were extracted for 6 hr at 70% heat on a heating mantle. Marjoram oil was obtained using the hydro-distillation method (Ferhatet al., 2006; Dorsafet al., 2010).

The obtained marjoram oil was evaluated for various physicochemical properties, including odour, appearance, solubility, pH, boiling point, viscosity (Cps), and density (g/mL). Odour of marjoram oil was determined by nasal perception. Appearance of oil was visually inspected for colour. Solubility of oil was observed in ethanol. pH was determined using previously calibrated digital pH meter. Refractive index was determined using refractometer. Boiling point was determined using capillary method. Viscosity of oil was determined using Brookfield viscometer (DV II+ Pro) using spindle no 3, rpm 100 and temperature 25°C.

Marjoram oil and the formulation F7 mixture were analyzed by FTIR spectroscopy (JASCO 4600). The samples were screened between 650-4000 cm^-1. Liquid samples were directly analyzed for the FTIR spectrum using an ATR attachment.

Marjoram oil was emulsified with a combination of co-surfactant and surfactant (1:1) i.e. S_mix and water. Oil and S_mix were mixed by vigorous shaking to obtain oil phase. Water was added to the oil phase in small increments and continuously stirred by a magnetic stirrer at room temperature.

For the phase diagram, nine formulations (1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1) of oil and S_mix in various proportions were mixed, as shown in Tables 1 and 2. Tween 80 and propylene glycol in a 1:1 ratio were used to produce S_mix. Under agitation at sixty rpm, a small amount of water was added to the oil and S_mix combination in 0.5% w/w increments, allowing the mixture to equilibrate. The water volume was recorded at the point of the clear-to-turbid change in the microemulsion. The mass percentages of oil, water, and S_mix were recorded at the endpoint since the total mass sum was 100% w/w. To obtain appropriate components and their concentration ranges for the microemulsion, a pseudo-ternary phase diagram was constructed. The microemulsion region was identified and formulations were optimized. The shaded area indicated the transparent or clear microemulsion region, while the remaining area showed the turbid microemulsion. The formulations were observed for one week period for clarity of microemulsions (Kumaret al., 2016; Lavanyaet al., 2016).

Physical Appearance, viscosity and density of stable microemulsion was determined as per reported methods. Physical appearance was observed for clarity of prepared microemulsion (Yadavet al., 2018). Viscosity of the prepared microemulsion was determined using Brookfield viscometer (DV II pro) at 100 rpm, 25°C and Spindle no 3. Density of the microemulsions were determined using density bottles (Badwi et al., 2009).

Formulation F7 was tested for particle size (globule), polydispersibility index and zeta potential by dynamic light scatting detection using Malvern instrument (Zeta sizer). 1 mL of F7 sample was added into 100 mL volumetric flask and diluted with purified water up to the mark. The sample was analysed for particle size (globule), Polydispersibility Index (PDI) and zeta potential (Asmawatiaet al., 2014).

Antimicrobial activity for Moraxella catarrhalis, Streptococcus pneumonia and Haemophiles influenza was performed by using agar disc method. Chloramphenicol ear drop was used as a control sample.

Agar plates were inoculated with a standardized inoculum of the test microorganism. Formulation F7 was applied to filter paper discs and placed on the agar surface at concentrations of 50, 100 and 200 μg/mL. Control sample of concentration 100 mg/mL was applied. The appropriate settings were used to incubate the Petri plates. An antimicrobial drug that diffuses into the agar normally inhibits the test microorganism’s germination and development, and the diameters of the inhibitory growth zones were calculated. The Petri dishes were examined using a Motic 2.0 microscope (Prabuseenivasanet al., 2006; Borseet al., 2020).

Physicochemical characterization of marjoram oil was performed for various tests and are shown in Table 3. The obtained marjoram oil was found aromatic, pale yellow liquid, soluble in ethanol, pH (6.30-6.95), boiling point (164-172°C), density (0.889±0.012 g/mL), viscosity (166-200 cps) and refractive index at 20°C (1.472±0.054).

The ternary phase diagram was obtained from developed formulations F1 to F9 shown in Figure 2. When the oil and S_mix mixture was titrated against water resulted clear and transparent emulsion after stirring. The oil, S_mix (1:1), and water percentages were marked as point in the phase diagram. S_mix composed of 1:1 proportion of Tween 80 and Propylene glycol. Total 9 formulations of different proportions of oil, S_mix and water was developed. The dark region in the phase diagram represents microemulsion zone. It wss observed that formulation F7, and F8 were stable and were not shown separation of oil and water layers. Formulation F8 was stable but become slightly turbid upon storage after 7 days. The formulation F1, F2, F3, F4, F5, F6 and F9 were not stable and showed phase separation after 1 hour storage.

It was observed that S_mix (1:1) at 19.28% concentration was produced stable emulsification of marjoram oil (Formulation F7). It was also observed that at lower S_mix concentration, marjoram oil of higher concentration was produced clear microemulsion in water. Formulation F7 ingredient mixture and marjoram oil sample were analysed by JASCO FTIR-4600 for functional group analysis. The chemical compatibility between marjoram oil and S_mix was studied. FTIR spectrum shown in Figure 3 revealed characteristic peaks associated with the functional groups present in this essential oil at 3482.81, 2960.2, 2925.48, 1733.69, 1445.39, 1369.21 and 1278.57 cm^-1.

Formulation F7 was found stable microemulsion and was further evaluated for physical appearance, viscosity, density, droplet size, polydispersity index and Zeta potential. Prepared microemulsions were found clear and pleasant odor. Formulation F7 was evaluated for viscosity which was found low viscous in the range 102.5 to 124.0 cps at 100 rpm using spindle no 3. Microemulsions were shown density 1.11 to 1.095 g/mL.

The Malvern particle size analyser (Zetasizer Ver. 8.02) was used to measure the mean droplet size, polydispersity index, and zeta potential. The globule size of formulation F7 was found 370.3 nm confirmed the formation of microemulsion which ranges between 100-400 nm. Polydispersibility Index (PDI) is a measure of the heterogeneity of the sample based on size. A polydispersibility index of 0.480 indicated moderate particle size distribution in the microemulsion. A zeta potential of -25.7 mV indicated a strong negative charge at surface of globules in a dispersion. This suggested good stability of micro emulsion and stability against coalescence. Particle size distribution and zeta potential represented graphically in Figure 4.

Figure 1:
Structure of marjoram oil components.

Figure 2:
Ternary phase diagram of marjoram oil microemulsion (The dark area represents the microemulsions existence field where stable, clear and transparent formulations are produced).

Figure 3:
FTIR marjoram oil and Micro emulsion F1.

Figure 4:
Particle size distribution and Zeta Potential of formulation F7.

Figure 5:
Zone of inhibition for formulation F7 against A. Moraxella catarrhalis B. Streptococcus pneumonia and C. Haemophiles influenza.

It was observed that as the concentration of Marjoram oil was increased in the disc method, zone of inhibition was found increased as compared to chloramphenicol control sample. The results of zone of inhibition are shown in Figure 5 and Table 4. Microemulsion formulation (F7) was used at 50 μg/mL, 100 μg/ mL, 200 μg/mL concentration for the respective sample code 1 mL-M Oil, 2 mL-M oil and 3 mL-M Oil. The control sample chloramphenicol ear drop used at 100 μg/mL concentration for antibacterial test. Microemulsion F7 was shown 17 to 29 mm, 09 to 20 mm and 20 to 27 mm concentration dependent zone of inhibition for Moraxella catarrhalis, Streptococcus pneumonia and Haemophiles influenza bacterial species respectively. The zone of inhibition was found increased with increase in concentration of microemulsion against all three bacterial species. The chloramphenicol ear drop (control sample) was shown lower zone of inhibition against Moraxella catarrhalis and Streptococcus pneumonia and better zone of inhibition against Haemophiles influenza.

The most of the volatile oils are transparent or pale yellow due to presence of linalool (Sadgroveet al., 2022). Volatile oils generally showed less density compared to water. Volatile oils are immiscible in water but soluble in organic solvents (Dhifiet al., 2016). Naturally, skin pH values lies between 4.0 to 7.0 and neutral pH value of marjoram oil suggested the no irritation of skin surface (Lamberset al., 2006). The higher boiling point of marjoram oil suggested long lasting aroma, extended release from formulations and more heat stability. The higher boiling point of marjoram oil is due to presence of 4-terpeneol (Warsitoet al., 2017). Low viscosity of oil are easily pourable liquids. Generally, the refractive index value of volatile oils falls between 1.45 to 1.55 (Siejaket al., 2021). The results of physicochemical studies confirmed the purity of marjoram oil and its properties.

Formulation F7 ingredient mixture and marjoram oil sample were analysed by JASCO FTIR -4600 for functional group analysis. The chemical compatibility between marjoram oil and S_mix was studied. FTIR spectrum shown in Figure 3 revealed characteristic peaks associated with the functional groups present in this essential oil at 3482.81, 2960.2, 2925.48, 1733.69, 1445.39, 1369.21 and 1278.57 cm^-1. Peaks around 2900-3000 cm^-1 indicated the stretching vibrations of C-H bonds in aliphatic hydrocarbons, which commonly found in essential oils (Agatonovic-Kustrinet al., 2020). A peak around 1700-1750 cm^-1 indicated the presence of carbonyl groups, which could be from ketones, aldehydes, or esters present in the oil. A broad peak in the region of 3200-3600 cm^-1 indicated the presence of hydroxyl groups typically found in alcohols and phenols. Peaks in the range of 1500-1600 cm^-1 and 800-900 cm^-1 might represent vibrations associated with aromatic ring structures present in the oil (Nandiyantoet al., 2019). All the peaks of marjoram oil were observed in microemulsion F7 with small shift in frequency indicated compatibility between oil and the excipients used.

The marjoram oil was emulsified with the Tween 80 surfactant and propylene glycol co-surfactant. The emulsification was produced due to solubilization of oil in the water at critical micelle concentration of surfactant and co-surfactant effect (Fernández-Peñaet al., 2019). Non-ionic surfactant, Tween 80, stabilizes the emulsions based on dynamic interfacial theory which produces greater resistance to phase disruption and coalescence of oil droplets. Stearic repulsion slows down the droplet flocculation (Roldan-Cruzet al., 2016).

The prepared microemulsion of marjoram oil F7, was shown the globule size 370.3 nm. Generally, an emulsion with average droplet size ≥6000 nm is classified as having poor stability (Adejokun & Dodou, 2020). Formulation F7 produced Zeta potential of -25.7 mV. The zeta potential of the emulsion system is an important parameter for particle – particle aggregation, and stability. The ideal zeta potential between ±10 to 30 mV is considered as stable formulation. The negative potential prevents coalescence of droplets (Kotakadiet al., 2013). The negative charge of microemulsion could be due to presence of anionic groups of fatty acids, and glycols present in oil, surfactant and co-surfactant (Shinde UA et al., 2018). Negatively charged carriers are considered more effective than positive charged for drug accumulation in skin and diffusion (Gilletet al., 2011).

It is investigated that marjoram oil shows antimicrobial activity against S. aureus, S. epidermitis, E. Coli, P. aeruginosa, H. influenza, S. pneumonia due to presence of alcoholic and phenolic monoterpinoids (Karen et al., 2023; Leigh-de et al., 2021; Inouyeet al., 2001). Hydrophobic essential oils components are present in the microemulsion which partition with the lipids present in the cell membrane of bacteria and mitochondria. The penetrated components disturb the bacterial cell structures and lead to death (Deviet al., 2010; Malik & Upadhyay, 2023). The results of antimicrobial activity on marjoram oil microemulsion confirms the effectiveness against Otitis media causing microorganisms.

The developed microemulsion formulation of marjoram oil was shown better antimicrobial effect against otitis media causing micro-organisms than the chloramphenicol control sample (marketed ear drop). This research study reveals that marjoram oil used in microemulsion system showed antibacterial effect and have a potential to use as a drug delivery system. In future, in vivo preclinical and clinical test should be conducted to evaluate toxicity and clinical effectiveness against Otitis media infection.