*4.6. Preparation of the Nanoemulsion*

Using Timur–rosemary oil as the oil phase and distilled water as the aqueous phase, the nanoemulsion was formulated. Under continuous stirring at high speed using a vortex mixer at room temperature, rosemary oil was dissolved into the oil phase at room temperature. After the drug had been fully dissolved in the oil phase, Smix (Tween 80 and Transcutol P) was introduced. Under continuous stirring, the water was added drop by drop into the oil phase. The mixture was then subjected to gentle agitation using the vortex for 3 min. The ideal formulation was selected based on the polydispersity index (PDI) and droplet size. Before evaluating each formulation's droplet size, polydispersity index (PDI), and physical characteristics, each formulation was self-emulsified in distilled water with gentle agitation [55,56].

### Physicochemical Characterization of Nanoemulsion

The developed formulations were characterized for their physicochemical parameters, droplet sizes, and PDIs. Equipment from Malvern Instruments was utilized to determine the particle size and PDI. The Timur–rosemary oil was prepared as a nanoemulsion using a titration process [57].

### *4.7. Fabrication of Timur Oil Nanoemulgel Formulation*

The best formulation was chosen to be the nanoemulsion with the greatest concentration of Timur and rosemary oil, the smallest particle size, and the lowest PDI. The developed nanoemulsion formulation was selected for the fabrication in the nanoemulgel with different Carbopol Ultrez 21 and Carbopol 940 concentrations (viz., 0.5%, 1%, 1.5%, 1.8%, and 2% gels). First, Carbopol hydrogels were prepared using Carbopol 940 and Carbopol Ultrez 21 as thickening agents by dispersing Carbopol in purified water and left-over night for swelling, then the pH of the hydrogel was neutralized using 0.05% of triethanolamine (TEA), and DMDM was added as a preservative. Then, the hydrogel matrix was mixed with the developed nanoemulsion (5 mL) with 1% rosemary oil and 2% Timur oil at 100 rpm until the nanoemulgel was formed. In order to achieve uniformity, each formulation was thoroughly blended [58].

### 4.7.1. Physical Characterization of Timur Oil Nanoemulgel Formulation

We visually assessed a wide range of physical characteristics, including consistency, spreadability, homogeneity, phase separation, and visual appearance, when developing the nanoemulgel. Using a pH meter, the pH values were determined (Mettler Toledo Inc., Columbus, OH, USA).

### Zeta Potential

Equipment from Malvern Instruments was used to determine the zeta potential to predict the dispersion stability and surface charge of the particles. The zeta potentials were measured in triplicate [59–63].

### Transmission Electron Microscopy (TEM)

Transmission electron microscopy (TEM) is a high-resolution technology that can be used to investigate specimens at the nanoscale. The developed formulation was studied for its surface morphology with the help of TEM, which was operated at 20–120 kV (Thermo Scientific Talos L120C G2 (S)TEM Microscope, Waltham, MA, USA) and carried out at Jamia Hamdard, New Delhi, India, enabling point-to-point resolution. A droplet of the sample was applied to a 300 mesh grid of copper-coated carbon that had been adequately saturated with water (1:1000), dyed with phosphotungstic acid (2% *w*/*v*), and allowed to air dry for about one minute before evaluation [54].

### Mechanical Properties of NEG

To determine the gel's mechanical attributes, including its hardness, cohesiveness, adhesiveness, gumminess, chewiness, and elasticity, a texture study was conducted. The texture analysis was performed using a texture analyzer (TA-XT Plus, Stable Microsystems, Godalming, UK). Using a probe that dips into the formulation at a certain speed and with a predetermined amount of force, data were collected and analyzed. A beaker free of air bubbles was filled with 100 g of the gel. A disc of Perspex (diameter 40 mm) was attached to the handle, and speeds of 1.0 mm/s before the test, 2.0 mm/s during the test, and 10.0 mm/s after the test were set. A 25 mm diameter cylinder probe was used for the penetration test, with a 5 mm penetration depth and a test speed of 2 mm/s in compression mode. Data computation was performed in Texture Exponent Software. All the tests were performed at room temperature using a triple (mean ± SD) set [62].

### 4.7.2. In Vitro Release Study

The dialysis bag (Sigma, St. Louis, MO, USA) was kept in running water for 3–4 h for the removal of glycerine and then treated with a 0.3% *w*/*v* sodium sulfide solution in water at 80 ◦C for 1 min for the removal of sulfur compounds. It was then washed with hot water at 60 ◦C for 2 min. The procured dialysis bag was acidified with 0.2% *v*/*v* H2SO<sup>4</sup> in distilled water. It was then rinsed with hot water to remove the acid and stored in the dissolution medium in the refrigerator to keep the pores open [63]. The essential oil release from antifungal nanoemulgel was determined in phosphate buffer at pH 5.5 using the dialysis bag method [54,64].

A freshly prepared formulation (0.5 gm of gel) was put in the dialysis bag (MWCO 12KD, Sigma), pre-soaked in double-distilled water for 12 h before use, and sealed. The dialysis bags were put in a beaker containing 100 mL of phosphate buffer at pH 5.5, which acted as a receptor compartment; it was maintained at 37 ◦C and stirred at a speed of 150 rpm. At an appropriate time interval (0.5, 1, 2, 4, 6, up to 24 h), a 3 mL aliquot of the dissolution medium was withdrawn, and the same volume of fresh dissolution medium was then added. A good sink condition was maintained throughout the test. The amount of the drug was evaluated using a UV spectrophotometer at 272 nm for the developed formulation [63].

%Cumulative drug release = concentration(µg/mL) × volume of dissolution media(mL) × Dilution Factor × 100/Initial amount of drug.

### Release Kinetics

The data from the drug release studies were fitted with different kinetic models, such as the zero-order model, first-order model, Higuchi matrix model, and Korsmeyer–Peppas model. The correlation coefficient (R<sup>2</sup> ) for each model was calculated using the following formula. The model resulting in the R<sup>2</sup> closest to 1 was chosen as the best fit for drug release [65].

Zero order model Q = kt First order model log Q = kt/2.303 Higuchi model Q = k√ t Korsmeyer-Peppas model = kt<sup>n</sup>

### 4.7.3. Antifungal Activity of Formulations

The antifungal activity of the rosemary oil, Timur oil, lavender oil (standard), ketoconazole (standard), developed nanoemulsion, and developed nanoemulgel with regard to *C. albicans* was determined following the method demonstrated by Kadimi et al. [66]. A McFarland standard was used for preparing the suspension of the organism. The organism was first grown on sabouraud dextrose agar medium (pH 6.2). The medium was prepared and sterilized in an autoclave for 20 min at 121 ◦C. A sterile borer was used to create wells that were 6 mm in diameter. After 24 h of incubation, the inoculum was firmly swept over the agar plate using a sterile cotton swab to make uniform culture lawns. Quantities of 0.1 gm of different gels (0.5, 1%, 1.5%, 1.8%, and 2%) with different concentrations of Timur oil and rosemary oil were poured into wells with the help of a sterile spatula and incubated for 48 h, and these plates were assessed for clear zones around the wells. The zones of inhibition were assessed by measuring the diameters [67].

### Calculation

Based on the ZOIs of the control and test samples, the % inhibition was calculated using the formula below:

$$\% \text{ inhibition} = \text{AIC} - \text{AIT}/\text{AIC} \times 100$$

where AIC is the area of inhibition for the control and AIT is the area of inhibition for the extracts.

### 4.7.4. Ex Vivo Permeation Study

Male Wistar rats (weighing between 150 and 200 g) were acquired from Institutional Animal Ethical Committee constituted by Jamia Hamdard, under protocol number 1575, which was utilized to prepare the skin for permeation testing. The rats were euthanized by CO<sup>2</sup> inhalation. The skin was removed from the area of the dorsal. With the use of "veet cream", the hairs were plucked. Using isopropyl alcohol and a cotton swab, the fat was eliminated [68]. The skin was then cleaned with distilled water and kept at −21 ◦C for scientific investigation. The skin penetration study was conducted on the dorsal skin of a rat used as the model skin for the Franz diffusion cell. The sample skin was positioned between the donor compartment and the receptor compartment, with the stratum corneum towards the donor compartment. The sample formulation served as the donor medium, while phosphate-buffered saline served as the receptor (pH 5.5). After 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, and 24 h, the receptor medium (1 mL) was removed and replaced with a new medium. After the dilutions, a 0.45 µm membrane filter was used to filter the solutions, and a UV spectrophotometer was used to measure the quantity of drug in the receptor medium [69].

### Data Analysis

After the study, the flux was plotted against time on a graph. The quantity of the drug that permeated was determined using the formula:

Amount of drug permeated (µg/cm<sup>2</sup> ) = concentration (µg/mL) × volume of dissolution media/Area (cm<sup>2</sup> )

The volume of dissolution media was equal to 35 mL, and the area of the diffusion cell was equal to 1.966 cm<sup>2</sup> .

The flux was equal to the slope of the steady-state portion of the plot between the amount of drug permeated per cm<sup>2</sup> vs. time in µg/cm2/h.

The permeability coefficient (Kp) was equal to flux/initial drug concentration (C0).

### 4.7.5. Drug Deposition Study

The quantity of drug held in the skin sample used in permeation testing was used to measure the capacity of the globules to help keep the drug inside the skin layers. The skin placed on the diffusion cell was taken off once the permeation experiment was finished. In order to remove any adherent product, the skin was cleansed with cotton soaked in ordinary saline solution and wiped with tissue paper. In order to extract the medication, the skin sample was then homogenized with 10 mL of phosphate buffer at pH 5.5. The resulting homogenate solution was filtered through a membrane filter (0.45 µm), and its drug content was measured using a UV spectrophotometer at 272 nm. All the readings were collected three times for accuracy [70].

### 4.7.6. Confocal Laser Scanning Microscopy (CLSM)

For confocal microscopy, gels were applied to the dorsal skin of the rats. Formulations containing the vesicles loaded with the fluorescent dye rhodamine were prepared by adding the dye (as a marker) to the nanoemulgel. Confocal pictures were acquired as XY-planes to show the fluorescence distribution (parallel to the plane of the skin surface). The brightest fluorescence with a morphology of stratum corneum surface resembles the skin surface in the imaging plane. The process of creating an XZ-section involves drawing a horizontal line across the area of interest in the Z = 0 µm XY-plane. This line is then "optically sliced" across the digital image data of the subsequent XY sections, yielding (XA-planar) optical cross-sections. All the photos were produced using an identical optical aperture, lens, and scan speed, and were averages of the triplet scans. The CLSM was used to examine the extent and mechanism of the rhodamine-loaded nanoemulgel's skin permeability [71,72].

### 4.7.7. Skin Irritation Studies

This study was carried out on healthy Wistar rats weighing 150–200 gm. By performing a Draize patch test, the irritability potential of the antifungal nanoemulgel was compared with that of the commercialized antifungal cream [73–75]. The protocol (1575) was approved by the Institutional Animal Ethical Committee constituted by Jamia Hamdard for such a purpose. Throughout the whole experimental process, animals were cared for and handled in accordance with CPCSEA criteria. Experimental Wistar rats were used to assess the irritability of the developed nanoemulgel (Tim–Ros–NEG) [76]. With care taken not to harm the skin's top layer, the hair from the dorsal side of the acclimatized animals was removed 24 h before the experiment. The animals were subsequently split into five groups (*n* = 3), with the first group (Group I; the control group) receiving no treatment (no drug treatment) and Group II received 0.8% (*v*/*v*) aqueous formalin solution as a standard irritant. Group III, Group IV, and Group V received the developed nanoemulgel (1% rosemary oil + 2% Timur oil), developed nanoemulsion (1% rosemary oil + 2% Timur oil), and marketed antifungal preparation, respectively. In the drug-free treatment group, an equal amount of blank NEG was applied to the cleansed skin across a 1 cm<sup>2</sup> area instead of the formulations. At 24, 48 and 72 h, the skin of the test animals was examined for any dermal responses, namely erythema or edema [73].

### 4.7.8. Histopathology of Skin

Histopathology studies were performed for the evaluation of the rat skin for any damage resulting from the application of the various drugs on the skin surface. The animals were sacrificed after one week, and the skin was removed from the interscapular region using scissors and forceps. Samples were prepared and sectioned by utilizing a

microtome. Furthermore, hematoxylin and eosin dye were used to stain the sectioned samples. The specimens were then examined using a high-power microscope to assess their integrity and perform stratum corneum and epidermal examinations. The results were compared with those of the rats in the control group [77].

### 4.7.9. Statistical Assessment

The data were acquired in triplicate for each of the established experiments, and the values were presented as mean ± S.D. Statistical significance was considered when the *p*-value was less than 0.05.

**Author Contributions:** Conceptualization, A.N. and K.K.; methodology, A.N.; validation, K.K. and M.S.M.A.; formal analysis, S.J. and T.W.S.; investigation, A.N.; resources, K.K.; writing—original draft preparation, A.N.; writing—review and editing, S.J.; supervision, K.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** This study was conducted after approval from CAHF, Jamia Hamdard, under protocol number 1575.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** The authors are thankful to Jamia Millia Islamia, New Delhi, India, for providing the FTIR facilities. The authors are also thankful to AIIMS, New Delhi, India, for providing the zeta potential and Zetasizer facilities. We thank Jamia Hamdard, New Delhi, for providing the TEM facilities.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**


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