**1. Introduction**

Topical fungal infections greatly affect skin health in developing and developed countries. *Candida* species are the most common fungi for superficial skin infections. Fungal infections infect the skin superficially and then enter the deeper layers of the skin by desquamation; hence, agents that can permeate deep into the skin are needed to treat these infections [1]. Topical therapies are the preferred treatment for dermal fungal infections because of their targeted action and reduced side effects [1,2]. Various synthetic drugs such as itraconazole, ketoconazole, and clotrimazole are used as conventional topical drugs for fungal infections. Although they have advantages such as localized effects, increased bioavailability, and good patient compliance, conventional drugs also have disadvantages, such as the potential to trigger allergies and eczema [1]. The repeated use of synthetic drugs also increases toxicity and resistance [3].

**Citation:** Noor, A.; Jamil, S.; Sadeq, T.W.; Mohammed Ameen, M.S.; Kohli, K. Development and Evaluation of Nanoformulations Containing Timur Oil and Rosemary Oil for Treatment of Topical Fungal Infections. *Gels* **2023**, *9*, 516. https:// doi.org/10.3390/gels9070516

Academic Editor: Esmaiel Jabbari

Received: 1 June 2023 Revised: 16 June 2023 Accepted: 20 June 2023 Published: 26 June 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

The utilization of essential oils to combat microbial infections is gaining importance. Timur oil (*Zanthoxylum armatum*), of the family *Rutaceae*, is an aromatic, annual herb, mostly growing in the hot valleys of the subtropical Himalayas, from Indus areas to Bhutan, up to an altitude of 2400 m. It also grows in the Khasi Hills from 700 m to 1000 m. Essential oils and fatty acids are thought to be the main constituents of this medicinal plant. The essential oils obtained from fruits of *Zanthoxylum armatum* have antifungal, antibacterial, and anthelmintic properties. Numerous scientific investigations have shown the antifungal [4] and mosquito-repelling properties [5] of Timur oil. According to Tiwary et al. [6], the main ingredients of the essential oil of *Zanthoxylum armatum* are linalool (57%) and limonene (19.8%). The linalool content in essential oils has been shown to mainly account for their antifungal properties [7]. However, limonene has modest antibacterial activity while also exhibiting effective antimicrobial action [4]. Prakash et al. [8] reported that the essential oil of a different variety of Zanthoxylum possesses a wide range of antifungal activity against molds such as *Aspergillus flavus*, *niger*, *terreus*, *candidus*, *sydowi*, *fumigates*, *Alternaria alternate*, *Cladosporium cladosporioides*, *Curvular ialunata*, *Fusarium nivale*, *Penicillium italicum*, and *Trichoderma virdie*. Zanthoxylum alatum showed a zone of inhibition 18 mm in diameter against *Alternaria alternate* [9].

Rosemary is a member of the mint family [10], and it possesses various antioxidants such as carnosic acid, carnosol, and rosmarinic acid [11,12]. Additionally, it has significant concentrations of flavonoids, phenolic acid, diterpene, and triterpene. Furthermore, studies have revealed that rosemary oil exerts antifungal [10], antibacterial, anti-inflammatory [13] antimicrobial [14], and antileishmanial activities [15]. The protective/beneficial effects of rosemary essential oil on candidiasis have been explored in a few studies [16].

Recent findings have revealed that natural herbal products can be used to treat major health issues such as cancer, microbial infections, and cardiovascular disorders. Therefore, novel drug delivery systems are required to tackle the challenges associated with these natural compounds to increase their effectiveness [17]. Nanoemulsion is a novel approach to producing stable preparations with increased solubility. Producing nanoemulsions is a good technique for developing formulations that contain essential oils as active ingredients for antifungal activity [18]. The spontaneous emulsification method (titration method) for nanoemulsion is very simple. It includes gently swirling while combining oil, water, surfactant, and co-surfactant in an ideal ratio [19]. To determine this ideal ratio, a phase diagram can be constructed using the aqueous titration technique. This method produces particles that range in size from 20 to 200 nm [20,21]. The hydrophilic character of the formulation makes it simple to remove the NEG from the application site when the necessary efficacy has been achieved. The thixotropic NEG facilitates easy spreadability at the target site and prolongs retention at the application site due to its mucoadhesive property [22,23]. In order to increase the thickness, lower the interfacial tension, and improve the stability, a nanoemulsion is added to the hydrogel matrix, such as a carbomer (Carbopol), to create a nanoemulgel, which functions as a drug reservoir [24]. The nanoemulgel increases the perforation of the oil into the skin [25]. When oily particles exit the skin's gel matrix intact and enter its layers, they reach their bodily targets [26].

The study aimed to use a rosemary-mediated nanoemulgel containing Timur oil as the main oil to effectively treat topical fungal infections. In this study, Timur oil is nanoemulsified in Smix and rosemary oil is used as carrier oil on the basis of its miscibility with Timur oil. Considering geographical factors as important parameters for herbal products, the extraction of two varieties of Timur seeds from different geographical sources (India and Nepal) was carried out to determine the percentages of linalool in both varieties by GC analysis. As this combination was new, there has not yet been research on combining these two essential oils for antifungal activity. In this study, Tim–Ros–NEG is the developed rosemary-mediated nanoemulgel containing Timur oil and its topical antifungal activity was evaluated against *C. albicans*, compared with that of a 1% ketoconazole cream (marketed preparation).

### **2. Results and Discussion 2. Results and Discussion**

### *2.1. Extraction of Timur Oil and Characterization 2.1. Extraction of Timur Oil and Characterization*

ketoconazole cream (marketed preparation).

*Gels* **2023**, *9*, x FOR PEER REVIEW 3 of 28

In order to develop a nanoformulation containing Timur oil and rosemary oil, Timur oil was extracted from the fresh seeds of *Zanthoxylum Armatum* (Timur) of Indian and Nepali origin, with a 56% percentage of active constituents (linalool) of Nepali origin; the yield was 1.5 percent/200 g. The Timur oil was characterized by FTIR, HPTLC, and GC. The FTIR functional group was identified, and the results are shown in Figure 1 and Table 1. The quantification of linalool was carried out by GC, and the area for linalool (Standard) and the area for linalool present in the Timur oil were found to be 272,960,345 and 51,550,363, respectively. The retention times for linalool (Standard) and linalool in the extracted Timur oil were observed to be 15.583 and 15.531 (Table 2), respectively. HPTLC analysis showed that the linalool content in the Nepali variety (56%) was higher than that in the Indian variety (36%). Therefore, the variety from Nepal was selected for further studies. In order to study the release of the developed formulation, solubility studies for Timur and rosemary oil were carried out in distilled water, methanol, ethanol, toluene, and phosphate buffer at pH values of 5.5 and 7.4. Figure 2 represents the maximum solubility of rosemary oil in buffer at pH 7.4, while Timur oil was soluble in acetone and buffer at pH 5.5 and 7.4. It was observed that the rosemary oil and Timur oil have significantly (*p* < 0.05) higher solubility in pH 7.4 phosphate buffer compared to the other solvents. In order to develop a nanoformulation containing Timur oil and rosemary oil, Timur oil was extracted from the fresh seeds of *Zanthoxylum Armatum* (Timur) of Indian and Nepali origin, with a 56% percentage of active constituents (linalool) of Nepali origin; the yield was 1.5 percent/ 200 g. The Timur oil was characterized by FTIR, HPTLC, and GC. The FTIR functional group was identified, and the results are shown in Figure 1 and Table 1. The quantification of linalool was carried out by GC, and the area for linalool (Standard) and the area for linalool present in the Timur oil were found to be 272960345 and 51550363, respectively. The retention times for linalool (Standard) and linalool in the extracted Timur oil were observed to be 15.583 and 15.531 (Table 2), respectively. HPTLC analysis showed that the linalool content in the Nepali variety (56%) was higher than that in the Indian variety (36%). Therefore, the variety from Nepal was selected for further studies. In order to study the release of the developed formulation, solubility studies for Timur and rosemary oil were carried out in distilled water, methanol, ethanol, toluene, and phosphate buffer at pH values of 5.5 and 7.4. Figure 2 represents the maximum solubility of rosemary oil in buffer at pH 7.4, while Timur oil was soluble in acetone and buffer at pH 5.5 and 7.4. It was observed that the rosemary oil and Timur oil have significantly (*p* < 0.05) higher solubility in pH 7.4 phosphate buffer compared to the other solvents.

antifungal activity was evaluated against *C. albicans*, compared with that of a 1%

**Figure 1.** FT-IR Spectra of Timur oil and chemical structure of linalool. **Figure 1.** FT-IR Spectra of Timur oil and chemical structure of linalool.



**Table 2.** GC analysis of Timur oil.

**Figure 2.** (**A**) Mean solubility (mg/mL) of rosemary oil at 25 °C. (**B**) Mean solubility (mg/mL) of Timur oil at 25 °C. An asterisk (\*) indicates that the value is significantly different at *p* < 0.05 compared to the other group. **Figure 2.** (**A**) Mean solubility (mg/mL) of rosemary oil at 25 ◦C. (**B**) Mean solubility (mg/mL) of Timur oil at 25 ◦C. An asterisk (\*) indicates that the value is significantly different at *p* < 0.05 compared to the other group.

#### **Table 2.** GC analysis of Timur oil. *2.2. Construction of Pseudoternary Phase Diagram*

**Parameter Linalool Content in Timur Oil (Nepali Variety) Standard (Linalool)** Area 51,550,363 272,960,345 Retention time 15.531 15.583 *2.2. Construction of Pseudoternary Phase Diagram* Timur oil, rosemary oil, Transcutol P (cosurfactant), and Tween 80 (surfactant) were combined at various concentrations to create ternary phase diagrams to identify the best formulation, which resulted in a nanoemulsion with a PDI of 0.3 and a droplet size of less Timur oil, rosemary oil, Transcutol P (cosurfactant), and Tween 80 (surfactant) were combined at various concentrations to create ternary phase diagrams to identify the best formulation, which resulted in a nanoemulsion with a PDI of 0.3 and a droplet size of less than 200 nm (Figure 3). Pseudoternary phase diagrams were constructed by preparing various Smix ratios (1:0, 1:1, 1:2, 2:1, 3:1, and 4:1). First, placebo formulations were prepared using the selected oil/surfactant/co-surfactant in the above-mentioned Smix ratio. After the formation of the placebo, drug loading was performed for the placebo. Timur oil was used as the oil phase, Tween 80 as the surfactant, Transcutol P as the co-surfactant, and distilled water as the aqueous phase during phase diagram formation. The Smix ratio of 4:1 occupied the largest area out of all the ratios. Thus, it was determined that the 4:1 Smix ratio produced the largest possible nanoemulsion area.

than 200 nm (Figure 3). Pseudoternary phase diagrams were constructed by preparing various Smix ratios (1:0, 1:1, 1:2, 2:1, 3:1, and 4:1). First, placebo formulations were prepared using the selected oil/surfactant/co-surfactant in the above-mentioned Smix ratio. After the formation of the placebo, drug loading was performed for the placebo. Timur oil was used as the oil phase, Tween 80 as the surfactant, Transcutol P as the cosurfactant, and distilled water as the aqueous phase during phase diagram formation. The An O/W nanoemulsion was prepared by using the titration method. It is well established that, the higher the quantity of the surfactant, the greater the potential for toxicity toward the skin and irritation. Therefore, an attempt was made to increase the oil concentration and decrease the concentration of the Smix. Therefore, from the titration chart, the suitable oil/Smix ratios were selected, which were 5:5, 6:4, and 7:3. Out of these, the 5:5 oil/Smix ratio was selected.

### Smix ratio of 4:1 occupied the largest area out of all the ratios. Thus, it was determined that the 4:1 Smix ratio produced the largest possible nanoemulsion area. Physicochemical Characterization of Nanoemulsion

the 5:5 oil/Smix ratio was selected.

An O/W nanoemulsion was prepared by using the titration method. It is well established that, the higher the quantity of the surfactant, the greater the potential for toxicity toward the skin and irritation. Therefore, an attempt was made to increase the oil concentration and decrease the concentration of the Smix. Therefore, from the titration chart, the suitable oil/Smix ratios were selected, which were 5:5, 6:4, and 7:3. Out of these, The prepared nanoemulsion was examined for globule size using a Malvern Zetasizer [27]. The droplet size of the developed nanoemulsion (Smix ratio: 5:5) was 93.12 nm. In contrast, the polydispersity index of the same formulation was 0.243, which shows a better distribution of the globules of the dispersed phase into the dispersion system (Figure 4). The ideal range of the PDI lies between 0.1 and 0.5.

**Figure 3.** Pseudoternary phase diagram showing o/w nanoemulsion regions for the Smix ratio (1:0, 1:1, 1:2, 2:1, 3:1, and 4:1). **Figure 3.** Pseudoternary phase diagram showing o/w nanoemulsion regions for the Smix ratio (1:0, 1:1, 1:2, 2:1, 3:1, and 4:1). *Gels* **2023**, *9*, x FOR PEER REVIEW 6 of 28

Physicochemical Characterization of Nanoemulsion

**Figure 4.** (**A**) Droplet size of developed nanoemulsion. (**B**) Bar diagram for droplet size of developed nanoemulsion. (**C**) Zeta potential of developed nanoemulsion. (**D**) Bar diagram for zeta potential of developed nanoemulsion. **Figure 4.** (**A**) Droplet size of developed nanoemulsion. (**B**) Bar diagram for droplet size of developed nanoemulsion. (**C**) Zeta potential of developed nanoemulsion. (**D**) Bar diagram for zeta potential of developed nanoemulsion.

and the results were determined. It was found that, upon increasing the polymer concentration from 0.5 % *w*/*v*, the particle size, as well as the polydispersity index of the respective formulations, decreased but only up to a polymer concentration of 1.8% *w*/*v*, and beyond this concentration, the particle size and PDI further increased (shown in Figure 5). Therefore, it was discovered that a Carbopol-940 concentration of 1.8% *w*/*v* was appropriate for creating an optimal nanoemulgel formulation. It was observed that the nanoemulgel containing 0.5% *w*/*v* polymer concentration significantly (*p* < 0.05) reduced the droplet size compared to the nanoemulgel containing 1%, 1.5%, 1.8% & 2% *w*/*v*

*2.3. Fabrication of Timur Oil Nanoemulgel Formulation*

polymer concentrations.

An appropriate zeta potential ensures that the nanoformulation system is intense and stable [27,28]. Nanoemulsions typically have a zeta potential value between +100 mV and −100 mV. Neutral nanoemulsions correspond to those with zeta potential values between −10 and +10 mV [29]. The developed nanoemulsion formulation showed a zeta potential value of −14.32 mv, indicating good stability (Figure 4).

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

Five nanoemulgel formulations were prepared using the same drug concentration but different concentrations of polymer (Carbopol-940), i.e., 0.5, 1.0, 1.5, 1.8, and 2.0% *w*/*v*, and the results were determined. It was found that, upon increasing the polymer concentration from 0.5% *w*/*v*, the particle size, as well as the polydispersity index of the respective formulations, decreased but only up to a polymer concentration of 1.8% *w*/*v*, and beyond this concentration, the particle size and PDI further increased (shown in Figure 5). Therefore, it was discovered that a Carbopol-940 concentration of 1.8% *w*/*v* was appropriate for creating an optimal nanoemulgel formulation. It was observed that the nanoemulgel containing 0.5% *w*/*v* polymer concentration significantly (*p* < 0.05) reduced the droplet size compared to the nanoemulgel containing 1%, 1.5%, 1.8% & 2% *w*/*v* polymer concentrations. *Gels* **2023**, *9*, x FOR PEER REVIEW 7 of 28

**Figure 5.** Influence of various Carbopol concentrations on droplet size. An asterisk (\*) indicates that the value is significantly different at *p* < 0.05 compared to the other group. **Figure 5.** Influence of various Carbopol concentrations on droplet size. An asterisk (\*) indicates that the value is significantly different at *p* < 0.05 compared to the other group.

2.3.1. Physicochemical characterization of Nanoemulgel 2.3.1. Physicochemical characterization of Nanoemulgel

The various nanoemulgel formulations were prepared, and their mean particle sizes were determined (Table 3 and Figure 6) [17]. The results revealed that the developed formulation (i.e., **FD**) showed a mean droplet size of 139 nm and a lower PDI. The value of the PDI should lie between 0.1 and 0.5. The developed formulation showed a PDI of 0.309, which shows a good size distribution for the droplets. The various nanoemulgel formulations were prepared, and their mean particle sizes were determined (Table 3 and Figure 6) [17]. The results revealed that the developed formulation (i.e., FD) showed a mean droplet size of 139 nm and a lower PDI. The value of the PDI should lie between 0.1 and 0.5. The developed formulation showed a PDI of 0.309, which shows a good size distribution for the droplets.

This technique determines the charge on the globule surface of the dispersed phase. **Table 3.** Different nanoemulgel formulations with different polymer and drug concentrations.


**(%** *w***/***v***) Concentration**

**FA 1 2 0.5** 255 0.590 9 FB 0.5 0.5 1.0 195 0.521 10 FC 1.0 1.0 1.5 166 0.412 11 FD 2.0 1.0 1.8 139 0.309 15 FE 1.0 0.5 2.0 172 0.389 10

**Particle Size (nm)**

**Resultant** 

**(%)**

**Drug Concentration Carbopol 940** 

**Formulation Code**

**Figure 6.** (**A**) Particle size of developed nanoemulgel. (**B**) Bar diagram for particle size of developed nanoemulgel. (**C**) Zeta potential for developed nanoemulgel. (**D**) Bar diagram for zeta potential of developed nanoemulgel. **Figure 6.** (**A**) Particle size of developed nanoemulgel. (**B**) Bar diagram for particle size of developed nanoemulgel. (**C**) Zeta potential for developed nanoemulgel. (**D**) Bar diagram for zeta potential of developed nanoemulgel.

2.3.2. Mechanical Strength of NEG Patient acceptability depends on creating a gel with the necessary physical characteristics, such as excellent pourability, spreadability, and acceptable hardness and viscosity. A texture analyzer was used to investigate the mechanical characteristics of Tim–Ros–NEG. The findings showed that the characteristics varied with the concentration of Carbopol 940 (0.5%, 1%, 1.5%, and 1.8%). With an increase in hydroxypropyl methylcellulose content, gel cohesiveness was observed to rise by Karavana et al. [32] and This technique determines the charge on the globule surface of the dispersed phase. The zeta potential value of a nanoemulgel typically ranges from +100 mV to −100 mV [27,28,30,31]. The zeta potential of the developed nanoemulgel formulation was found to be −21.99 mV, −16.54 mV, and −18.85 mV, respectively. The mean zeta potential of the developed nanoemulgel formulation was observed as −19.12 ± 2.73 mV and we have shown these results in the bar chart (Figure 6D). We have considered a zeta potential value of −21.99 mV of the developed nanoemugel formulation (FD), indicating good formulation stability, as shown in Figure 6C.

the same observation was recorded by Cevher et al. [33] for Carbopol gels. The developed

### nanoemulgel's hardness was found to be 783.01 g force. Firmness or hardness refers to the 2.3.2. Mechanical Strength of NEG

formulation's deformational flexibility under stress, while cohesiveness refers to the number of cross-links between gel molecules and their ability to maintain shape. The higher the value, the better the gel strength [23]. The findings demonstrated Tim–Ros– NEG's resistance to deformation. However, our spreadability results demonstrated that Tim–Ros–NEG is spreadable even though it is more resistant to deformation. Typically, increased hardness results in less spreading. The developed Tim–Ros–NEG was determined to have a consistency of 898.34 g.s. The term "consistency" refers to a product's viscosity and characterizes its texture and hardness. A high consistency score suggests that Tim–Ros–NEG was consistent, according to the investigation. Cohesiveness is just a measure of how effectively a product resists a second deformation in comparison to how well it resists a first deformation. The greatest force represents a sample's stickiness or adhesiveness. According to the findings of our research, Tim–Ros–NEG is more cohesive or sticky than other materials since it demonstrated high cohesiveness (−459.81 g force) and an index of viscosity of 688.84 g/s. Higher Tim–Ros–NEG adhesion translates into a longer skin contact duration, which might result in less frequent nanoemulgel application. Osmałek et al. reported that Opokan® , a commercial product, Patient acceptability depends on creating a gel with the necessary physical characteristics, such as excellent pourability, spreadability, and acceptable hardness and viscosity. A texture analyzer was used to investigate the mechanical characteristics of Tim–Ros–NEG. The findings showed that the characteristics varied with the concentration of Carbopol 940 (0.5%, 1%, 1.5%, and 1.8%). With an increase in hydroxypropyl methylcellulose content, gel cohesiveness was observed to rise by Karavana et al. [32] and the same observation was recorded by Cevher et al. [33] for Carbopol gels. The developed nanoemulgel's hardness was found to be 783.01 g force. Firmness or hardness refers to the formulation's deformational flexibility under stress, while cohesiveness refers to the number of cross-links between gel molecules and their ability to maintain shape. The higher the value, the better the gel strength [23]. The findings demonstrated Tim–Ros–NEG's resistance to deformation. However, our spreadability results demonstrated that Tim–Ros–NEG is spreadable even though it is more resistant to deformation. Typically, increased hardness results in less spreading. The developed Tim–Ros–NEG was determined to have a consistency of 898.34 g.s. The term "consistency" refers to a product's viscosity and characterizes its texture and hardness. A high consistency score suggests that Tim–Ros–NEG was consistent,

according to the investigation. Cohesiveness is just a measure of how effectively a product resists a second deformation in comparison to how well it resists a first deformation. The greatest force represents a sample's stickiness or adhesiveness. According to the findings of our research, Tim–Ros–NEG is more cohesive or sticky than other materials since it demonstrated high cohesiveness (−459.81 g force) and an index of viscosity of 688.84 g/s. Higher Tim–Ros–NEG adhesion translates into a longer skin contact duration, which might result in less frequent nanoemulgel application. Osmałek et al. reported that Opokan®, a commercial product, had the lowest textural characteristics with a hardness of more than twice that of other gels [34]. Mohapatra et al. reported that Black Cohosh loaded ethosomal gel for the treatment of menopause. They have used Carbopol® 971P NF as a gelling agent. They have considered firmness, consistency, cohesiveness, and work of cohesion as evaluation parameters [35]. Hasan et al. developed combined therapy of 5-fluorouracil (5-FU) and cannabidiol (CBD)-loaded nanostructured lipid carrier gel (FU-CBD-NLCs gel) for the effective treatment of non-melanoma skin cancer. In this study, they have reported textural properties of 5-fluorouracil (5-FU) and cannabidiol (CBD)-loaded nanostructured lipid carrier gel (FU-CBD-NLCs gel). They have used Carbopol 934 as a gelling agent. The results of our investigation are in accordance with the published research article [36]. The findings of FD's texture analysis (Table 4 and Figure 7) indicated that the formulation was sufficiently firm and cohesive, showing that it would be user-friendly during application. Both formulations showed thermosensitivity, as indicated by a negative index of viscosity values.

### **Table 4.** Texture analysis of developed formulation.


**Figure 7. Figure 7.** Texture Analysis of developed nanoemulgel showing the resultant force versus time Texture Analysis of developed nanoemulgel showing the resultant force versus time.

### 2.3.3. In Vitro Release Study

The cumulative % release experiments of the Timur oil from developed nanoemulgel formulation were determined using phosphate buffer (with pH 5.5) and compared with that of the prepared nanoemulsion and pure Timur oil. Due to the essential oil's encapsulation in the lipid portion of the nanoemulgel, the essential oil from the developed nanoemulgel was released slowly over time. Within 24 h, the developed nanoemulgel formulation showed a considerable release (70%) while the developed nanoemulsion formulation exhibited an 85% release (Figure 8). A 100% release was seen in the case of pure Timur oil within 4 h [3]. *Gels* **2023**, *9*, x FOR PEER REVIEW 10 of 28 formulation exhibited an 85% release (Figure 8). A 100% release was seen in the case of pure Timur oil within 4 h [3].

**Figure 8.** Comparison of in vitro release profile between pure Timur oil, nanoemulsion, and nanoemulgel. An asterisk (\*) indicates that the value is significantly different at *p* < 0.05 compared to the other group. **Figure 8.** Comparison of in vitro release profile between pure Timur oil, nanoemulsion, and nanoemulgel. An asterisk (\*) indicates that the value is significantly different at *p* < 0.05 compared to the other group.

Any drug's therapeutic effectiveness depends on how readily it is released from its pharmaceutical formulation [37]. The viscosity, surfactant content, polymer, and drug concentration are only a few of the variables that affect how quickly a topical pharmaceutical formulation releases a drug [38]. The development of a viscous formulation due to the synthetic polymer Carbopol 940's large molecular weight delays the release profile of pharmaceuticals from topical preparations like nanoemulgel [39]. Additionally, it has been stated previously that emulgel serves as a reservoir for the drug, releasing it first from the internal phase to the exterior phase before entering the skin [40]. In contrast to nanoemulsion, which requires the oil droplets to pass through a hydrophilic phase transition, nanoemulgel allows the oil droplets to first be released into the gel matrix and then they can pass directly into the skin's subcutaneous layer [41]. Additionally, among all the formulations, the nanoemulgel showed a superior sustained release effect, as shown in Figure 8 [42]*.* Therefore, based on this study, it was concluded that the Timur oil release with the developed formulation having a polymer concentration of 1.8% *w/v* was considerably higher, with a sustained release, compared to the pure Timur oil. Therefore, based on this study, it was concluded that among all the prepared nanoemulgel formulations, (**FD**) was the optimal formulation and can be considered for further in vivo studies. The antimicrobial action is due to the oil components and the drug delivery system, we used. The developed nanoemulgel formulation showed a Any drug's therapeutic effectiveness depends on how readily it is released from its pharmaceutical formulation [37]. The viscosity, surfactant content, polymer, and drug concentration are only a few of the variables that affect how quickly a topical pharmaceutical formulation releases a drug [38]. The development of a viscous formulation due to the synthetic polymer Carbopol 9400 s large molecular weight delays the release profile of pharmaceuticals from topical preparations like nanoemulgel [39]. Additionally, it has been stated previously that emulgel serves as a reservoir for the drug, releasing it first from the internal phase to the exterior phase before entering the skin [40]. In contrast to nanoemulsion, which requires the oil droplets to pass through a hydrophilic phase transition, nanoemulgel allows the oil droplets to first be released into the gel matrix and then they can pass directly into the skin's subcutaneous layer [41]. Additionally, among all the formulations, the nanoemulgel showed a superior sustained release effect, as shown in Figure 8 [42]. Therefore, based on this study, it was concluded that the Timur oil release with the developed formulation having a polymer concentration of 1.8% *w*/*v* was considerably higher, with a sustained release, compared to the pure Timur oil. Therefore, based on this study, it was concluded that among all the prepared nanoemulgel formulations, (FD) was the optimal formulation and can be considered for further in vivo studies. The antimicrobial action is due to the oil components and the drug delivery system, we used. The developed nanoemulgel formulation showed a significantly (*p* < 0.05) higher release of Timur oil compared to the in vitro release of pure Timur oil.

#### Timur oil. 2.3.4. Release Kinetics

2.3.4. Release Kinetics The release data were examined to determine the goodness of fit in various kinetic models in order to establish a suitable Tim–Ros–NEG release pattern. The release data The release data were examined to determine the goodness of fit in various kinetic models in order to establish a suitable Tim–Ros–NEG release pattern. The release data were run through various release kinetic models, including zero-order, first-order, Higuchi matrix, and Korsmeyer–Peppas models, and the best-fitting model was chosen based on the

were run through various release kinetic models, including zero-order, first-order, Higuchi matrix, and Korsmeyer–Peppas models, and the best-fitting model was chosen

nanoemulgel, having an R<sup>2</sup> value of 0.9807 (Figure 9) [43]**.** The value of n was found below

) value. The highest R<sup>2</sup> value was 0.9807, while the

significantly (*p* < 0.05) higher release of Timur oil compared to the in vitro release of pure

regression coefficient (R<sup>2</sup> ) value. The highest R<sup>2</sup> value was 0.9807, while the lowest R<sup>2</sup> value was 0.8879. The Higuchi matrix model was the best-fitting model for the nanoemulgel, having an R<sup>2</sup> value of 0.9807 (Figure 9) [43]. The value of *n* was found below 0.5 (i.e., 0.1561), showing that Timur oil release from the developed nanoemulgel formulation follows Fickian diffusion [44]. *Gels* **2023**, *9*, x FOR PEER REVIEW 11 of 28 0.5 (i.e., 0.1561), showing that Timur oil release from the developed nanoemulgel formulation follows Fickian diffusion [44].

**Figure 9.** Various release kinetic models of the nanoemulgel: (**A**) zero-order kinetics; (**B**) first-order kinetics; (**C**) Higuchi matrix; (**D**) Korsmeyer–Peppas. **Figure 9.** Various release kinetic models of the nanoemulgel: (**A**) zero-order kinetics; (**B**) first-order kinetics; (**C**) Higuchi matrix; (**D**) Korsmeyer–Peppas.

### 2.3.5. Drug Deposition Study 2.3.5. Drug Deposition Study

The skin retention properties of the nanoemulgel were determined using a drug deposition study. The skin retention of the Timur oil from the fourth nanoemulgel formulation (with the formulation code **FD**) was found to be 56.45 µg of the dose applied, whereas pure Timur oil was found to be 34.56 µg of the dose applied on the skin. The gel used for topical application has to have the right formulation properties to make it easy to apply and remain in contact with the skin for a long time. Tim–Ros–NEG at 1.8% had a greater viscosity and remained stuck to the skin for longer compared to Tim–Ros–NEG at 0.5%, 1.0%, and 1.5%. This nanoemulgel showed greater drug localization in the skin. Therefore, the nanoemulgel (**FD**) formulation resulted in more medication being retained in the skin, as demonstrated in Table 5*.* The skin retention properties of the nanoemulgel were determined using a drug deposition study. The skin retention of the Timur oil from the fourth nanoemulgel formulation (with the formulation code FD) was found to be 56.45 µg of the dose applied, whereas pure Timur oil was found to be 34.56 µg of the dose applied on the skin. The gel used for topical application has to have the right formulation properties to make it easy to apply and remain in contact with the skin for a long time. Tim–Ros–NEG at 1.8% had a greater viscosity and remained stuck to the skin for longer compared to Tim–Ros–NEG at 0.5%, 1.0%, and 1.5%. This nanoemulgel showed greater drug localization in the skin. Therefore, the nanoemulgel (FD) formulation resulted in more medication being retained in the skin, as demonstrated in Table 5.

**Table 5.** Drug retention of pure Timur oil and nanoemulgel on rat skin. **Table 5.** Drug retention of pure Timur oil and nanoemulgel on rat skin.


2.3.6. Morphological Studies/Internal Composition Study 2.3.6. Morphological Studies/Internal Composition Study

TEM is the most effective characterization technique that uses high magnification to anticipate morphological characteristics and examine samples' surfaces [31,45,46]. TEM was used to study the morphology of the globules of the developed nanoemulgel. The TEM is the most effective characterization technique that uses high magnification to anticipate morphological characteristics and examine samples' surfaces [31,45,46]. TEM was used to study the morphology of the globules of the developed nanoemulgel. The

globule size was in accordance with the results obtained using the Zetasizer*.* TEM images of the developed formulation (having the formulation code **FD**) indicated spherical globes

globule size was in accordance with the results obtained using the Zetasizer. TEM images of the developed formulation (having the formulation code FD) indicated spherical globes with slight roughness, and no aggregation was observed among the globules (Figure 10). of the developed formulation (having the formulation code FD) indicated spherical globes with slight roughness, and no aggregation was observed among the globules (Figure 10).

TEM is the most effective characterization technique that uses high magnification to

anticipate morphological characteristics and examine samples' surfaces [31,45,46]. TEM was used to study the morphology of the globules of the developed nanoemulgel. The globule size was in accordance with the results obtained using the Zetasizer*.* TEM images

*Gels* **2023**, *9*, x FOR PEER REVIEW 12 of 29

2.3.6. Morphological Studies/Internal Composition Study

**Figure 10.** TEM of developed nanoemulgel (scale bar = 100 nm; 92,000× frame). **Figure 10.** TEM of developed nanoemulgel (scale bar = 100 nm; 92,000× frame).

2.3.7. Confocal Laser Scanning Microscopy

2.3.7. Confocal Laser Scanning Microscopy The CLSM investigation evaluated the depth and intensity of permeation through the skin of the rats treated with a marker-loaded commercial gel, the developed nanoemulsion, and the developed nanoemulgel. The outcomes were contrasted with those for the skin from the group that received the control treatment. The breadth and depth of the carrier system's penetration into the skin were similar to the intensity and depth of the marker's penetration. Figure 11A–D display the photomicrographs of rat skin treated with the control, commercial gel, developed nanoemulsion, and optimal nanoemulgel formulations. Compared to the control group, the treatment group treated The CLSM investigation evaluated the depth and intensity of permeation through the skin of the rats treated with a marker-loaded commercial gel, the developed nanoemulsion, and the developed nanoemulgel. The outcomes were contrasted with those for the skin from the group that received the control treatment. The breadth and depth of the carrier system's penetration into the skin were similar to the intensity and depth of the marker's penetration. Figure 11A–D display the photomicrographs of rat skin treated with the control, commercial gel, developed nanoemulsion, and optimal nanoemulgel formulations. Compared to the control group, the treatment group treated with nanoemulgel-based formulations showed dramatically enhanced depth and intensity of penetration into the skin. This showed that formulations based on developed carriers have more potential for skin penetration.

with nanoemulgel-based formulations showed dramatically enhanced depth and intensity of penetration into the skin. This showed that formulations based on developed car-

riers have more potential for skin penetration.

**Figure 11.** *Cont*.

**Figure 11.** Confocal laser scanning microscopy images of excised rat skin treated with (**A**) plain dye solution, (**B**) dye-loaded marketed gel, (**C**) dye-loaded developed nanoemulsion, and (**D**) dyeloaded developed nanoemulgel formulation. **Figure 11.** Confocal laser scanning microscopy images of excised rat skin treated with (**A**) plain dye solution, (**B**) dye-loaded marketed gel, (**C**) dye-loaded developed nanoemulsion, and (**D**) dye-loaded developed nanoemulgel formulation.

### 2.3.8. Ex Vivo Skin Permeation Study

philic globules also made this possible.

2.3.8. Ex Vivo Skin Permeation Study In order to compare the permeability of the nanoemulgel with that of a pure Timur oil, ex vivo permeation tests were carried out. Franz diffusion cells and rat skin were used in the study. Figure 12 shows the outcomes for the ex vivo permeation of Timur oil from the Tim–Ros–NEG and pure Timur oil. The Tim–Ros permeation from the two formulations is compared, and it is shown that the permeation of Timur oil from the created NEG In order to compare the permeability of the nanoemulgel with that of a pure Timur oil, ex vivo permeation tests were carried out. Franz diffusion cells and rat skin were used in the study. Figure 12 shows the outcomes for the ex vivo permeation of Timur oil from the Tim–Ros–NEG and pure Timur oil. The Tim–Ros permeation from the two formulations is compared, and it is shown that the permeation of Timur oil from the created NEG formulation (2265.01 <sup>±</sup> 0.01 <sup>µ</sup>g/cm<sup>2</sup> ) was considerably greater (*p* < 0.05) than that from the pure Timur oil (12.96 <sup>±</sup> 0.33 <sup>µ</sup>g/cm<sup>2</sup> ) after 24 h (Figure 12). For the Tim–Ros–NEG, the %

formulation (2265.01 ± 0.01 µg/cm2) was considerably greater (*p* < 0.05) than that from the pure Timur oil (12.96 ± 0.33 µg/cm2) after 24 h (Figure 12). For the Tim–Ros–NEG, the %

at 6 h, 57.89% at 12 h, and 79.11% at 24 h, whereas the % DP of the pure Timur oil was 4.08% at 0.5 h, 5.71% at 2 h, 5.89% at 6 h, 12% at 12 h, and 15.03% at 24 h (Figure 12). The nanosized oil globules possessing Timur oil enhance the penetration, and may accelerate drug permeation through the skin's lipophilic layers [47–49]. As the permeation parameters were examined simultaneously, it was discovered that the permeation flux was considerably increased (*p* < 0.05) with Tim–Ros–NEG (484.12 ± 0.065 µg cm−2 h−1) compared to the pure Timur oil (3.21 ± 0.074 µg cm−2 h−1). The nanoemulgel showed a better permeability profile, as the flux was 94.947 µg/cm2/h, whereas the flux of the pure Timur oil was 24.504 µg/cm2/h, much lower than that of the nanoemulgel. The nanoemulgel permeability was four times better than that of the pure Timur oil. When comparing the apparent penetration and flux of the two formulations, it was discovered that there was a four-fold augmentation ratio between the Tim–Ros–NEG and the pure Timur oil. This could be because the skin permeability increased due to Carbopol-940 [50], and the nanometric lipo-

drug permeation for the nanoemulgel was found to be 21.7% at 0.5 h, 26.42% at 2 h, 44.73% at 6 h, 57.89% at 12 h, and 79.11% at 24 h, whereas the % DP of the pure Timur oil was 4.08% at 0.5 h, 5.71% at 2 h, 5.89% at 6 h, 12% at 12 h, and 15.03% at 24 h (Figure 12). The nanosized oil globules possessing Timur oil enhance the penetration, and may accelerate drug permeation through the skin's lipophilic layers [47–49]. As the permeation parameters were examined simultaneously, it was discovered that the permeation flux was considerably increased (*<sup>p</sup>* < 0.05) with Tim–Ros–NEG (484.12 <sup>±</sup> 0.065 <sup>µ</sup>g cm−<sup>2</sup> <sup>h</sup> −1 ) compared to the pure Timur oil (3.21 <sup>±</sup> 0.074 <sup>µ</sup>g cm−<sup>2</sup> <sup>h</sup> −1 ). The nanoemulgel showed a better permeability profile, as the flux was 94.947 µg/cm2/h, whereas the flux of the pure Timur oil was 24.504 µg/cm2/h, much lower than that of the nanoemulgel. The nanoemulgel permeability was four times better than that of the pure Timur oil. When comparing the apparent penetration and flux of the two formulations, it was discovered that there was a four-fold augmentation ratio between the Tim–Ros–NEG and the pure Timur oil. This could be because the skin permeability increased due to Carbopol-940 [50], and the nanometric lipophilic globules also made this possible. NEG, the % drug permeation for the nanoemulgel was found to be 21.7% at 0.5 h, 26.42% at 2 h, 44.73% at 6 h, 57.89% at 12 h, and 79.11% at 24 h, whereas the % DP of the pure Timur oil was 4.08% at 0.5 h, 5.71% at 2 h, 5.89% at 6 h, 12% at 12 h, and 15.03% at 24 h (Figure 12). The nanosized oil globules possessing Timur oil enhance the penetration, and may accelerate drug permeation through the skin's lipophilic layers [47–49]. As the permeation parameters were examined simultaneously, it was discovered that the permeation flux was considerably increased (*p* < 0.05) with Tim–Ros–NEG (484.12 ± 0.065 µg cm−<sup>2</sup> h−<sup>1</sup> ) compared to the pure Timur oil (3.21 ± 0.074 µg cm−<sup>2</sup> h−<sup>1</sup> ). The nanoemulgel showed a better permeability profile, as the flux was 94.947 µg/cm<sup>2</sup> /h, whereas the flux of the pure Timur oil was 24.504 µg/cm<sup>2</sup> /h, much lower than that of the nanoemulgel. The nanoemulgel permeability was four times better than that of the pure Timur oil. When comparing the apparent penetration and flux of the two formulations, it was discovered that there was a four-fold augmentation ratio between the Tim–Ros–NEG and the pure Timur oil. This could be because the skin permeability increased due to Carbopol-940 [50], and the nanometric lipophilic globules also made this possible.

**Figure 11.** Confocal laser scanning microscopy images of excised rat skin treated with (**A**) plain dye solution, (**B**) dye-loaded marketed gel, (**C**) dye-loaded developed nanoemulsion, and (**D**) dye-

In order to compare the permeability of the nanoemulgel with that of a pure Timur oil, ex vivo permeation tests were carried out. Franz diffusion cells and rat skin were used in the study. Figure 12 shows the outcomes for the ex vivo permeation of Timur oil from the Tim–Ros–NEG and pure Timur oil. The Tim–Ros permeation from the two formulations is compared, and it is shown that the permeation of Timur oil from the

) was considerably greater (*p* < 0.05) than

) after 24 h (Figure 12). For the Tim–Ros–

*Gels* **2023**, *9*, x FOR PEER REVIEW 14 of 28

loaded developed nanoemulgel formulation.

created NEG formulation (2265.01 ± 0.01 µg/cm<sup>2</sup>

that from the pure Timur oil (12.96 ± 0.33 µg/cm<sup>2</sup>

2.3.8. Ex Vivo Skin Permeation Study

**Figure 12.** (**A**) Comparative % drug permeation profiles of the drug nanoemulgel and pure Timur oil. (**B**) Cumulative drug permeation profile of the nanoemulgel and pure Timur oil. An asterisk (\*) indicates that the value is significantly different at *p* < 0.05 compared to the other group. **Figure 12.** (**A**) Comparative % drug permeation profiles of the drug nanoemulgel and pure Timur oil. (**B**) Cumulative drug permeation profile of the nanoemulgel and pure Timur oil. An asterisk (\*) indicates that the value is significantly different at *p* < 0.05 compared to the other group.

#### 2.3.9. Skin Irritation Study 2.3.9. Skin Irritation Study

A skin irritancy test was performed to verify the safety of the gel formulation. The formulation exhibited no erythemal or edematous scores even after 72 h. The average skin irritation score was 0 (no erythema in a rat), which is less than 5, demonstrating that the gel does not irritate Wistar rat skin when applied to it (Figure 13) [20]. Table 6 presents the results of this study. According to Draize et al. (33), a primary irritancy index (PII) value of less than two (2) denotes that the applied formulation is not irritating to human skin. As a result, because the PII for optimised nanoemulgel and optimised nanoemulsion was less than 2, they were considered to be non-irritants. Developing an appropriate nanoemulgel formulation depends heavily on the choice of surfactant. To compare ionic and nonionic surfactants, Tween 80, Tween 20, Span 80, polyethylene glycol 400, and PEG A skin irritancy test was performed to verify the safety of the gel formulation. The formulation exhibited no erythemal or edematous scores even after 72 h. The average skin irritation score was 0 (no erythema in a rat), which is less than 5, demonstrating that the gel does not irritate Wistar rat skin when applied to it (Figure 13) [20]. Table 6 presents the results of this study. According to Draize et al. (33), a primary irritancy index (PII) value of less than two (2) denotes that the applied formulation is not irritating to human skin. As a result, because the PII for optimised nanoemulgel and optimised nanoemulsion was less than 2, they were considered to be non-irritants. Developing an appropriate nanoemulgel formulation depends heavily on the choice of surfactant. To compare ionic and nonionic surfactants, Tween 80, Tween 20, Span 80, polyethylene glycol 400, and PEG 200 (a nonionic surfactant) were chosen to develop the Tim–Ros–NEG. These nonionic surfactants generate homogeneous, superior droplets with a low critical micelle concentration, which aids in the quick absorption and release of the nanoemulgel. They also have a minimal risk of causing irritation and show low toxicity compared to other materials [19]. Therefore, it was determined that all the excipients included in the formulation were non-irritating and safe for topical usage.

formulation were non-irritating and safe for topical usage.

**Figure 13.** (**A**) Irritation study on nude rat skin conducted by applying the developed nanoemulsion. (**B**) Irritation study on nude rat skin conducted by applying the developed nanoemulgel. **Figure 13.** (**A**) Irritation study on nude rat skin conducted by applying the developed nanoemulsion. (**B**) Irritation study on nude rat skin conducted by applying the developed nanoemulgel.

200 (a nonionic surfactant) were chosen to develop the Tim–Ros–NEG. These nonionic surfactants generate homogeneous, superior droplets with a low critical micelle concentration, which aids in the quick absorption and release of the nanoemulgel. They also have a minimal risk of causing irritation and show low toxicity compared to other materials [19]. Therefore, it was determined that all the excipients included in the


**Table 6.** Mean erythema scores for various formulations obtained at the end of 24, 48 and 72 h. **Table 6.** Mean erythema scores for various formulations obtained at the end of 24, 48 and 72 h.

### 2.3.10. Antifungal Activity 2.3.10. Antifungal Activity

The results for the in vitro antifungal efficacy of Smix, Timur oil, lavender oil, rosemary oil, ketoconazole (standard), Timur:rosemary (2:1), the developed nanoemulsion, and a 1.8% nanoemulgel obtained using the cylinder plate method are presented in Figures 14 and 15, and in Table 7. The antifungal activity of each sample or formulation was assessed at a concentration of 10 µg/mL (Table 7). It was found that the developed (Tim–Ros–NEG) formulations exhibited promising antifungal activity against *C. albicans*. It was revealed that the Tim–Ros–NEG significantly suppressed the growth of *C. albicans* compared to the other test preparations. The results for the in vitro antifungal efficacy of Smix, Timur oil, lavender oil, rosemary oil, ketoconazole (standard), Timur:rosemary (2:1), the developed nanoemulsion, and a 1.8% nanoemulgel obtained using the cylinder plate method are presented in Figures 14 and 15, and in Table 7. The antifungal activity of each sample or formulation was assessed at a concentration of 10 µg/mL (Table 7). It was found that the developed (Tim–Ros–NEG) formulations exhibited promising antifungal activity against *C. albicans*. It was revealed that the Tim–Ros–NEG significantly suppressed the growth of *C. albicans* compared to the other test preparations.

**Figure 14.** (**A**) Comparison of the ZOIs (mm) of the test samples with the ZOI of ketoconazole (Standard drug). (**B**) Comparison of the ZOI (mm) of the nanoemulgel formulation with that of ketoconazole (Standard drug). An asterisk (\*) indicates that the value is significantly different at *p* < 0.05 compared to the other group. **Figure 14.** (**A**) Comparison of the ZOIs (mm) of the test samples with the ZOI of ketoconazole (Standard drug). (**B**) Comparison of the ZOI (mm) of the nanoemulgel formulation with that of ketoconazole (Standard drug). An asterisk (\*) indicates that the value is significantly different at *p* < 0.05 compared to the other group. **Figure 14.** (**A**) Comparison of the ZOIs (mm) of the test samples with the ZOI of ketoconazole (Standard drug). (**B**) Comparison of the ZOI (mm) of the nanoemulgel formulation with that of ketoconazole (Standard drug). An asterisk (\*) indicates that the value is significantly different at *p* < 0.05 compared to the other group.

**Figure 15.** (**A**) ZOIs (mm) of the developed Smix (**5**), Timur oil (**6**), lavender oil (**7**), and rosemary oil (**8**) against *C*. *albicans.* (**B**) ZOIs (mm) of the placebo nanoemulgel (**5**), Timur (**2**): rosemary (**1**) (**6**), **Figure 15.** (**A**) ZOIs (mm) of the developed Smix (**5**), Timur oil (**6**), lavender oil (**7**), and rosemary oil (**8**) against *C*. *albicans.* (**B**) ZOIs (mm) of the placebo nanoemulgel (**5**), Timur (**2**): rosemary (**1**) (**6**), **Figure 15.** (**A**) ZOIs (mm) of the developed Smix (5), Timur oil (6), lavender oil (7), and rosemary oil (**8**) against *C*. *albicans.* (**B**) ZOIs (mm) of the placebo nanoemulgel (5), Timur (2): rosemary (1) (6), Timur oil (7), and developed nanoemulsion (8). (**C**) ZOIs (mm) of the placebo nanoemulsion (5), ketoconole standard (6), Timur (2): rosemary (1) (7), and 1.8% nanoemulgel (8) against *C. albicans*.


**Table 7.** Assessment of antifungal activity of samples against *C. Albicans*.

<sup>a</sup> Figure 15A; <sup>b</sup> Figure 15B; <sup>c</sup> Figure 15C. An asterisk (\*) indicates that the value is significantly different at *p* < 0.05 compared to the other formulations.

The developed nanoemulsion formulation was selected for the fabrication of the nanoemulgel with different Carbopol Ultrez 21 and Carbopol 940 concentrations (viz., 0.5%, 1%, 1.5%, 1.8%, and 2% gels). The developed Tim–Ros–NEG formulation was notably effective as an antifungal agent.

Timur oil exhibited an inherent antifungal effect reflected by the zone of inhibition for the developed nanoemulsion. Due to the synergistic effects of Timur oil and rosemary oil when delivered together using nanoemulsion technology, which permitted the intensive diffusion of the drug-containing oil globules, Tim–Ros–NEG's action was enhanced. Timur oil, lavender oil, Timur:rosemary (2:1), and the developed nanoemulsion produced results comparable to those for the known antifungal essential oil and the reference drug (ketoconazole). The Tim–Ros nanoemulsion showed statistically significant growth suppression [51]. As a result, it was discovered that the nanoemulsion platform using Timur oil as the oil core had superior efficacy against *C. albicans* species. When both Timur oil and rosemary oil were used in combination, better antifungal activity than ketoconazole against *C. albicans* was observed. Timur oil and rosemary oil were combined in different ratios, and they gave comparable results when compared to the reference drug (ketoconazole). Both Timur oil and rosemary oil have antifungal activity themselves. When we used a 2:1 ratio of Timur:rosemary oil, the oils showed more synergistic activity compared with the other ratios of Timur oil and rosemary oil, as described in Table 7 and Figure 14. It was apparent that Tim–Ros nanoemulsion and Tim–Ros–NEG were active against *C. albicans*, and showed a significantly greater zone of inhibition compared to that resulting from placebo NEG and the marketed formulation (*p* < 0.05).

### 2.3.11. Histopathological Study

Histopathological analysis was carried out to evaluate the toxicity profile of the developed formulation (Tim–Ros–NEG). The animal groups were divided into four groups: normal control, formalin solution treatment (positive control), blank NEG, and Tim–Ros– NEG. The rat skin was observed in positive control, blank NEG, and Tim–Ros–NEG groups and was compared with the control group. A histopathological examination of skin from the rat's dorsal skin was performed to look for any indications of inflammatory reactions. Pathological alterations, including a thicker, deteriorated epidermis (E), intercellular edema, and inflammatory cell infiltrate were visible in the formalin-treated group (Figure 16). The Tim–Ros–NEG-treated groups did not exhibit aberrant alterations in the treated rat

skin tissue compared to controls, other than a slightly thicker epidermis. Overall, the findings showed that the Tim–Ros–NEG was safe for topical administration and within the skin's tolerance limit. No macrophages or lymphocytes were observed on the rat's dorsal skin, indicating that there was no major inflammation in the surrounding tissues (Figure 16). This suggests that Tim–Ros–NEG might be safely administered topically using the formulated nanoemulgel. aberrant alterations in the treated rat skin tissue compared to controls, other than a slightly thicker epidermis. Overall, the findings showed that the Tim–Ros–NEG was safe for topical administration and within the skin's tolerance limit. No macrophages or lymphocytes were observed on the rat's dorsal skin, indicating that there was no major inflammation in the surrounding tissues (Figure 16). This suggests that Tim–Ros–NEG might be safely administered topically using the formulated nanoemulgel.

Timur oil (**7**), and developed nanoemulsion (**8**). (**C**) ZOIs (mm) of the placebo nanoemulsion (**5**), ketoconole standard (**6**), Timur (**2**): rosemary (**1**) (**7**), and 1.8% nanoemulgel (**8**) against *C. albicans*.

Histopathological analysis was carried out to evaluate the toxicity profile of the developed formulation (Tim–Ros–NEG). The animal groups were divided into four groups: normal control, formalin solution treatment (positive control), blank NEG, and Tim–Ros–NEG. The rat skin was observed in positive control, blank NEG, and Tim–Ros– NEG groups and was compared with the control group. A histopathological examination of skin from the rat's dorsal skin was performed to look for any indications of inflammatory reactions. Pathological alterations, including a thicker, deteriorated epidermis (E), intercellular edema, and inflammatory cell infiltrate were visible in the formalin-treated group (Figure 16). The Tim–Ros–NEG-treated groups did not exhibit

*Gels* **2023**, *9*, x FOR PEER REVIEW 18 of 28

2.3.11. Histopathological Study

**Figure 16.** Histological micrographs of hematoxylin-eosin-stained skin section showing the normal epidermis, dermis tissues of (**A**) untreated rat skin, (**B**) formalin-exposed rat skin showed thickened degenerated epidermis (E) and inflammatory cells infiltrate(+). (**C**) Rat skin treated with conventional oil (Rosemary oil). (**D**) Rat skin treated with nanoemulgel enriched with Timur oil (2): Rosemary oil (1). The florescent blue arrow indicates blood vessel. E: Epidermis; D: Dermis; HF: Hair follicle. **Figure 16.** Histological micrographs of hematoxylin-eosin-stained skin section showing the normal epidermis, dermis tissues of (**A**) untreated rat skin, (**B**) formalin-exposed rat skin showed thickened degenerated epidermis E and inflammatory cells infiltrate(+). (**C**) Rat skin treated with conventional oil (Rosemary oil). (**D**) Rat skin treated with nanoemulgel enriched with Timur oil and Rosemary oil. The florescent blue arrow indicates blood vessel. E: Epidermis; D: Dermis; HF: Hair follicle.

### **3. Conclusions**

**3. Conclusions** In the present study, the topical application of a Timur–rosemary oil nanoemulgel (Tim–Ros–NEG) showed antifungal activity against *C. Albicans* count, improving its effect compared to the action of pure Timur oil. Moreover, we observed a 15 ± 2.9 mm ZOI from the Timur oil:rosemary oil (2:1) nanoemulgel in comparison to the pure Timur oil (11 ± 0.7 mm) and standard drug ketoconazole (13 ± 0.8 mm). The Timur oil:rosemary oil (2:1) nanoemulgel exhibited a synergistic effect. We believe that, if we used it in combination with ketoconazole, it would also have a synergistic effect. Therefore, we could reduce the In the present study, the topical application of a Timur–rosemary oil nanoemulgel (Tim–Ros–NEG) showed antifungal activity against *C. Albicans* count, improving its effect compared to the action of pure Timur oil. Moreover, we observed a 15 ± 2.9 mm ZOI from the Timur oil:rosemary oil (2:1) nanoemulgel in comparison to the pure Timur oil (11 ± 0.7 mm) and standard drug ketoconazole (13 ± 0.8 mm). The Timur oil:rosemary oil (2:1) nanoemulgel exhibited a synergistic effect. We believe that, if we used it in combination with ketoconazole, it would also have a synergistic effect. Therefore, we could reduce the therapeutic dose of ketoconazole, thereby reducing the toxicity caused by ketoconazole.

therapeutic dose of ketoconazole, thereby reducing the toxicity caused by ketoconazole. Natural substances such as Timur oil and rosemary oil have been considered to be suitable options for topical and even oral treatments because synthetic drugs can have undesirable effects and some infections caused by *Candida* are resistant to them. It was possible to create a nanoemulgel by adding Carpobol 940, a hydrogel material, at a concentration of 1.8% to a nanoemulsion containing Timur oil, rosemary oil, Tween 80, Natural substances such as Timur oil and rosemary oil have been considered to be suitable options for topical and even oral treatments because synthetic drugs can have undesirable effects and some infections caused by *Candida* are resistant to them. It was possible to create a nanoemulgel by adding Carpobol 940, a hydrogel material, at a concentration of 1.8% to a nanoemulsion containing Timur oil, rosemary oil, Tween 80, and Transcutol P. This method allows for greater penetration through the skin, improving the topical bioavailability and increasing the retention time.

When Timur oil and rosemary oil were added to the mixture, the tested microbial strains were suppressed more effectively due to better penetration of the oil globules from the NEG. The developed formulation was confirmed to be safe when tested on the skin of Wistar rats. The animals treated with Tim–Ros–NEG showed no indications of erythema. Additionally, the histopathological investigation showed no toxicity on the skin of the Wistar rats, indicating that the formulation is safe and effective for topical use. Consequently, the data suggest that the use of Tim–Ros–NEG might be a promising approach for the safe, highly effective, targeted delivery of Timur oil.

### **4. Materials and Methods**

### *4.1. Materials*

Tween 80, Carbopol 940, Carbopol Ultrez 21, acetone, chloroform, propylene glycol, disodium hydrogen phosphate, and Span 80 were obtained from S D Fine Chemicals

Ltd., Mumbai, India. Methanol was purchased from Merck, Mumbai, India. Mueller– Hinton agar (which is produced by Becton, Dickinson, and company, Franklin lakes, NJ, United States) was used in the culture media. Marketed Timur oil was purchased from Moksha lifestyle, New Delhi (India). The rosemary oil and lavender oil were obtained from Pharmacos, Faridabad, Haryana, India. The Indian variety of Timur seeds and Nepali variety of Timur seeds were obtained from Hari Gokul, Khari Baoli, Old Delhi, India, and Khopra, Nepal, respectively. Both Indian and Nepali varieties of Timur seeds, authenticated by Prof. Javed Ahmad, Ex-head Department of Botany, School of Chemical & Life Sciences, Jamia Hamdard, New Delhi, has been deposited in the Herbarium of the herbal garden, Jamia Hamdard, New Delhi.

### *4.2. Extraction of Timur Oil from Timur Seeds*

A total of 200 g of Timur seeds were weighed and crushed using a mortar and pestle. Then, the crushed Timur seeds were transferred to the clean round-bottomed flask of the Clevenger apparatus setup. A hydro-distillation process lasting 6–7 h was conducted. After completion of the distillation process, the Timur oil was collected. The collected Timur oil was dried using sodium sulfate to absorb the excess water. Then, it was filtered and the obtained oil was stored at 2 ◦C to 7 ◦C [52].

### 4.2.1. Identification of Timur Oil

Fourier Transform Infrared (FTIR) Spectroscopy

A Perkin–Elmer 591B spectrophotometer was used to determine the FTIR spectra of the Timur oil and Rosemary oil with KBr pellets or Nujol films, and scanning was performed at wavelengths of 4000 to 400 (cm−<sup>1</sup> ) to obtain the characteristic spectra.
