*4.2. Experimental Methods*

### 4.2.1. Solubility of Babchi Oil

The solubility of Babchi oil in surfactants (rhamnolipid) and co-surfactants (propylene glycol) was observed by dissolving the oil within these in excessive amounts. To achieve equilibrium, the sample was constantly swirled for ten minutes in a vortex mixer and preserved at room temperature in an isothermal shaker for seventy-two hours. The samples that were equilibrated were then centrifuged at 3000 rpm for fifteen minutes. The supernatant acquired underwent filtration from a 0.45 µm filter membrane and was diluted using the mobile phase. The content of the drug was determined by using a UV-VIS spectrophotometer of Shimadzu-1700, Japan set at 260 nm.

### 4.2.2. Screening of Nanoemulsion Components

Based on solubility experiments, Babchi oil has the best solubilization ability. Surfactant and co-surfactant screening were performed using the percentage of transmittance. The capacity of surfactants (rhamnolipid) to emulsify was tested by the addition of 300 mg into the Babchi oil. To accomplish homogeneity, the sample was carefully heated at forty to forty-five degrees Celsius for thirty seconds. In order to formulate a fine emulsion, a measurement of 50 mg of the sample was taken and then blended with 50 mL of double distilled water. The overall turbidity of the final sample was assessed visually. After two hours, transmittance tests through the UV-VIS spectrophotometer of Shimadzu-1700, Japan were performed on the emulsions at 700 nm and a blank of water with double distillation as a narrow spectrum focused more on particles with shorter wavelengths. Out of a variety of co-surfactants that were screened, propylene glycol was used for the nanoemulsion formulation as it is considered safe. Propylene glycol (mg), rhamnolipid (mg), and Babchi oil (mg) formulations were created and analyzed in a similar method as indicated in the screening technique of surfactants.

### 4.2.3. Construction of Phase Diagrams

Titration methods were used to construct diagrams for pseudoternary phases. To create the surfactant, co-surfactant, and oil phase we used rhamnolipid, propylene glycol, and Babchi oil. Ratios of surfactant and co-surfactant weights that were used in the study were 2:1, 1:1, and 1:0 and underwent optimization to determine the exact ratio at which maximum area for nanoemulsion existence could be achieved.

These formulations underwent titration in presence of water through micro syringing drops until the separation of phase or turbidity began. Within the other batch, similar ratios of surfactant, co-surfactant, and water were made and parameters were visualized. The formulations were briskly agitated for long enough periods for homogenization to occur in both situations. The endpoints of the formulations were monitored visually through a darkened screen by lighting the formulations using white light. To ensure repeatability, the tests were carried out thrice. The percent of mass components of Babchi oil and the combination of surfactant and co-surfactant along with the water was determined and represented on coordinates in the triangular form to build the diagrams for the pseudoternary phase from the endpoint.

### 4.2.4. Nanoemulsion Formulation

Babchi oil was added to the samples of surfactant and co-surfactant with different ratios that were observed from diagrams of the pseudoternary phase. Drop by drop, an adequate quantity of water was poured into the sample. The Babchi oil nanoemulsion was thus created by constantly stirring the formulation at the temperature of the room. The nanoemulsions that were prepared were kept at room temperature for future research.

### 4.2.5. Nanoemulsion Optimization

Optimization of nanoemulsion was performed through 3 levels and 3-factor software of Design expert, namely Box–Behnken version 12 design of Stat-Ease, USA. The impacts of several processing factors such as concentration, sonication, zeta potential, size of particles, and efficiency of entrapment along with the in vitro release of nanoemulgel were comprehensively examined. The different levels used were observed with axial-α, low, medium, high, axial + α of oil and Smix with dependent variables to be particle size, zeta potential (ZP), and entrapment efficiency. Table 3 depicts the variables used during the CCRD to optimize the formulation. The design included a formulation run with several combinations at three points of center to evaluate the impact of the variables. Several models are provided by polynomial equations along with response plots for the surface to assess the influence of different variables and factors such as quadratic and linear factors. Of all the models, both individual and combined influence on dependent variables was best demonstrated by the quadratic model.

**Table 3.** Variables used in CCRD to optimize the formulation.


### Nanoemulsion Structure and Morphology

TEM (Transmission electron microscope) of Hitachi H7500, Japan was used to evaluate the shape and small structures of loaded nanoemulsion drugs. Water was used to dissolve the nanoemulsion compositions in a 1:10 ratio. Thereafter, a dissolved nanoemulsion droplet was implemented on the holey grid of film, dyed, and dried with a 1% aqueous solution of phosphotungstic acid.

### Nanoemulsion Micromeritics

Dynamic light dispersion with zeta-sizer HSA 3000 was used to analyze the size of globules of the nanoemulsion and index of polydispersity (PDI) of Malvern Instruments Ltd., UK. Before determining the PDI and size of globules, all the formulations were sonicated.

### Nanoemulsion Conductivity and Viscosity

At twenty-five degrees, the electrical conductance of the nanoemulsion was measured through an EC Testr 11+, USA conductance meter. This test was carried out thrice for reproducibility.

The nanoemulsion prepared was observed for viscosity using the viscometer of Brookfield DV-II+ Pro without dilution. The formulation was placed within the beaker for five minutes before being measured at 0.5, 1, 2.5, and 5 rpm with a spindle. The accompanying dial was read on and recorded from the viscometer.

### Nanoemulsion-Based Hydrogel (Nanoemulgel) Formulation

Nanoemulgel was formed when each of the formulations was observed to be nanosized and so integrated inside the matrix of gel. The matrix of the gel base was chosen to

be carbomer 940. The phase for oil was created by combining Babchi oil, rhamnolipid, and propylene glycol. Swelling of carbomer 940 was performed in a small amount of water for twenty-four hours to generate solutions of high viscosity. During this duration, the oily phase was progressively added into the viscous carbomer 940 formulations during the magnetic stir. After adjusting the pH from 6 to 9 using triethanolamine, nanoemulgel was produced.

### 4.2.6. Nanoemulsion Characterization

Particle Size and Polydispersity Index (PDI)

Rhamnolipid as surfactant and propylene glycol as cosurfactant was mixed with distilled water to produce an O/W type of nanoemulsion with Babchi oil. The nanoemulsion was prepared and the combination was sonicated for twenty minutes in an ice bath with the usage of a sonicator of 20 kHz with a peak power of 750 Watts. To detect the diameter of the mean droplet, the Litesizer-500 particle size analyzer was used with PDI.

### Drug Content Determination

By dissolving 100 mg of the created nanoemulgel in 10 mL of distilled water, the quantity of medication included in the nanoemulgel was evaluated. This combination was tested using a UV spectrophotometer at 260 nm against distilled water as a blank control. It was then quantified by spreadability.

The drug content was determined by the following formula:

Concentration = Absorbance/(E1 cm1%) × Dilution factor × 10.

### pH Determination

As the formulation would be administered to the skin, pH monitoring was required to guarantee that it was non-irritating. A digital pH meter was used to analyze the formulation pH and was measured at room temperature.

### Spreadability

for consistency.

Log analysis on both sides is expressed as follows:

consistency, and *n* is the index of flow.

4.2.7. Ex Vivo Drug Studies on Permeation

and the dermal side facing the receiver segment.

4.2.8. Determination of Nanoemulgel Drug Content

before being inspected for integrity and then utilized.

4.2.9. DPPH Scavenging Activity of Nanoemulgel

for consistency.

The potential spreading capacity of the nanoemulgels was measured 48 h after preparation. The spreadability was measured by evaluating the nanoemulgel diameter after spreading it across two glass plates for one minute. The mass of nanoemulgel was positioned upon the glass slide with one a pre-marked circle diameter of one centimeter, and a second glass slide had been placed. The diameter was observed to be grown as a result of the masses introduced, causing the gels to spread. The formulation may be used to calculate spreadability: *Gels* **2022**, *8*, x FOR PEER REVIEW 18 of 21

S = (m · l)/t. *Gels* **2022**, *8*, x FOR PEER REVIEW 18 of 21

> The initial S is the ability to spread, m is the higher slide weight, l is the upper slide length, and t is the required time. The initial S is the ability to spread, m is the higher slide weight, l is the upper slide length, and t is the required time. The initial S is the ability to spread, m is the higher slide weight, l is the upper slide

#### Measurements of Viscosity and Rheological Management Measurements of Viscosity and Rheological Management length, and t is the required time.

The produced preparations were evaluated for viscosity through spindle number 4 of Brookfield DV-II+ Pro viscometer at various angular speeds at 31.0 ± 0.1 ◦C. The rheological behavior of the formulation of nanoemulgel was assessed using plate and cone configurations of a 40 mm cone with a 2.5-degree cone angle. Rheology experiments were carried out at 25 ◦C with shear rates ranging from 53.21–496.5 s−<sup>1</sup> . The produced preparations were evaluated for viscosity through spindle number 4 of Brookfield DV-II+ Pro viscometer at various angular speeds at 31.0 ± 0.1 °C. The rheological behavior of the formulation of nanoemulgel was assessed using plate and cone configurations of a 40 mm cone with a 2.5-degree cone angle. Rheology experiments were carried out at 25 °C with shear rates ranging from 53.21–496.5 s−1 . Measurements of Viscosity and Rheological Management The produced preparations were evaluated for viscosity through spindle number 4 of Brookfield DV-II+ Pro viscometer at various angular speeds at 31.0 ± 0.1 °C. The rheological behavior of the formulation of nanoemulgel was assessed using plate and cone

The power law equation was used to determine the consistency and flow indexes: The power law equation was used to determine the consistency and flow indexes: configurations of a 40 mm cone with a 2.5-degree cone angle. Rheology experiments were

Ʈ = . = *Krn*. carried out at 25 °C with shear rates ranging from 53.21–496.5 s−1 . The power law equation was used to determine the consistency and flow indexes:

The symbol Ʈ refers to shear stress, *r* refers to shear rate, *K* refers to the index of consistency, and *n* is the index of flow. Log analysis on both sides is expressed as follows: The symbol Ʈ = . The symbol Ʈ refers to shear stress, *r* refers to shear rate, *K* refers to the index of the control. At 517 nm, the formulations were examined in a spectrophotometer. refers to shear stress, *r* refers to shear rate, *K* refers to the index of consistency, and *n* is the index of flow.

Log Ʈ= log K+ n log r.

Therefore, the slope of the graph of logs of shear stress versus logs of shear rate was

Ex vivo studies for permeation were conducted using the Franz cell for diffusion, which is a proven approach for predicting the delivery of drugs through the skin. The skin of Wistar rats was removed for this research. The sacrificed rats' hair from their dorsal part was detached using a surgical blade of 24 numbering in the region from tail to head. The shaved skin was split, and unwanted fat along with the connective tissues was excluded with a scalpel. The skin was further removed and cleaned with saline and inspected for its integrity to be used. The skin of the rats was placed on an assembly cell with a 10 cm<sup>2</sup> high area for diffusion, with the stratum corneum facing the donor segment

Through the lower back direction using 24 surgical blades, the animal was sacrificed through the dorsal side. A knife was used to remove superfluous fat and connective tissue from the shaved area of the animal skin. The removed skin was cleaned with normal saline

The DPPH (2, 2 diphenyl- 1- picryl hydrazyl) technique developed by Williams et al. was used to assess the overall radical scavenging activity of Babchi oil emulsions before and following encapsulation [31]. The donating capacity of electrons of the anti-oxidants causes the violet hue of the solution to become colorless at normal temperature. Upon dissolution of the sample of 0.5 within methanol of 3 mL, the resultant solution was processed using the methanolic solution of 0.3 mL for DPPH. During the progression of the reaction, this formulation was held inside a darkened room for an hour. The changes in the color indicated the properties of antioxidants in the formulation due to the donating capacity of hydrogen. The sample of 0.3 mL and 3.3 mL of methanol was included within the blank while 0.3 mL of DPPH reagent along with 3.5 mL of methanol was included in

the control. At 517 nm, the formulations were examined in a spectrophotometer.

4.2.7. Ex Vivo Drug Studies on Permeation

selected as the flow index, while the inverse function of the Y-intercept provided the index

Log Ʈ= log K+ n log r.

and the dermal side facing the receiver segment.

Ex vivo studies for permeation were conducted using the Franz cell for diffusion, which is a proven approach for predicting the delivery of drugs through the skin. The skin of Wistar rats was removed for this research. The sacrificed rats' hair from their dorsal part was detached using a surgical blade of 24 numbering in the region from tail to head. The shaved skin was split, and unwanted fat along with the connective tissues was excluded with a scalpel. The skin was further removed and cleaned with saline and inspected for its integrity to be used. The skin of the rats was placed on an assembly cell with a 10 cm<sup>2</sup> high area for diffusion, with the stratum corneum facing the donor segment

4.2.8. Determination of Nanoemulgel Drug Content

before being inspected for integrity and then utilized.

4.2.9. DPPH Scavenging Activity of Nanoemulgel

The DPPH (2, 2 diphenyl- 1- picryl hydrazyl) technique developed by Williams et al. was used to assess the overall radical scavenging activity of Babchi oil emulsions before and following encapsulation [31]. The donating capacity of electrons of the anti-oxidants causes the violet hue of the solution to become colorless at normal temperature. Upon dissolution of the sample of 0.5 within methanol of 3 mL, the resultant solution was processed using the methanolic solution of 0.3 mL for DPPH. During the progression of the reaction, this formulation was held inside a darkened room for an hour. The changes in the color indicated the properties of antioxidants in the formulation due to the donating capacity of hydrogen. The sample of 0.3 mL and 3.3 mL of methanol was included within the blank while 0.3 mL of DPPH reagent along with 3.5 mL of methanol was included in

Through the lower back direction using 24 surgical blades, the animal was sacrificed through the dorsal side. A knife was used to remove superfluous fat and connective tissue from the shaved area of the animal skin. The removed skin was cleaned with normal saline

Log analysis on both sides is expressed as follows: carried out at 25 °C with shear rates ranging from 53.21–496.5 s−1 The power law equation was used to determine the consistency and flow indexes:

*Gels* **2022**, *8*, x FOR PEER REVIEW 18 of 21

Measurements of Viscosity and Rheological Management

length, and t is the required time.

$$\text{Log } \mathsf{T} = \log \mathsf{K} + \mathsf{n} \, \log \mathsf{r}.$$

.

The initial S is the ability to spread, m is the higher slide weight, l is the upper slide

The produced preparations were evaluated for viscosity through spindle number 4 of Brookfield DV-II+ Pro viscometer at various angular speeds at 31.0 ± 0.1 °C. The rheological behavior of the formulation of nanoemulgel was assessed using plate and cone configurations of a 40 mm cone with a 2.5-degree cone angle. Rheology experiments were

The symbol Ʈ refers to shear stress, *r* refers to shear rate, *K* refers to the index of consistency, and *n* is the index of flow. Log analysis on both sides is expressed as follows: Therefore, the slope of the graph of logs of shear stress versus logs of shear rate was selected as the flow index, while the inverse function of the Y-intercept provided the index for consistency.

### Log Ʈ= log K+ n log r. 4.2.7. Ex Vivo Drug Studies on Permeation

Therefore, the slope of the graph of logs of shear stress versus logs of shear rate was selected as the flow index, while the inverse function of the Y-intercept provided the index for consistency. 4.2.7. Ex Vivo Drug Studies on Permeation Ex vivo studies for permeation were conducted using the Franz cell for diffusion, which is a proven approach for predicting the delivery of drugs through the skin. The skin of Wistar rats was removed for this research. The sacrificed rats' hair from their dorsal part was detached using a surgical blade of 24 numbering in the region from tail to head. Ex vivo studies for permeation were conducted using the Franz cell for diffusion, which is a proven approach for predicting the delivery of drugs through the skin. The skin of Wistar rats was removed for this research. The sacrificed rats' hair from their dorsal part was detached using a surgical blade of 24 numbering in the region from tail to head. The shaved skin was split, and unwanted fat along with the connective tissues was excluded with a scalpel. The skin was further removed and cleaned with saline and inspected for its integrity to be used. The skin of the rats was placed on an assembly cell with a 10 cm<sup>2</sup> high area for diffusion, with the stratum corneum facing the donor segment and the dermal side facing the receiver segment.

### The shaved skin was split, and unwanted fat along with the connective tissues was excluded with a scalpel. The skin was further removed and cleaned with saline and in-4.2.8. Determination of Nanoemulgel Drug Content

spected for its integrity to be used. The skin of the rats was placed on an assembly cell with a 10 cm<sup>2</sup> high area for diffusion, with the stratum corneum facing the donor segment and the dermal side facing the receiver segment. Through the lower back direction using 24 surgical blades, the animal was sacrificed through the dorsal side. A knife was used to remove superfluous fat and connective tissue from the shaved area of the animal skin. The removed skin was cleaned with normal saline before being inspected for integrity and then utilized.

### 4.2.8. Determination of Nanoemulgel Drug Content 4.2.9. DPPH Scavenging Activity of Nanoemulgel

Through the lower back direction using 24 surgical blades, the animal was sacrificed through the dorsal side. A knife was used to remove superfluous fat and connective tissue from the shaved area of the animal skin. The removed skin was cleaned with normal saline before being inspected for integrity and then utilized. 4.2.9. DPPH Scavenging Activity of Nanoemulgel The DPPH (2, 2 diphenyl- 1- picryl hydrazyl) technique developed by Williams et al. was used to assess the overall radical scavenging activity of Babchi oil emulsions before and following encapsulation [31]. The donating capacity of electrons of the anti-oxidants causes the violet hue of the solution to become colorless at normal temperature. Upon dissolution of the sample of 0.5 within methanol of 3 mL, the resultant solution was pro-The DPPH (2,2 diphenyl-1-picryl hydrazyl) technique developed by Williams et al. was used to assess the overall radical scavenging activity of Babchi oil emulsions before and following encapsulation [31]. The donating capacity of electrons of the anti-oxidants causes the violet hue of the solution to become colorless at normal temperature. Upon dissolution of the sample of 0.5 within methanol of 3 mL, the resultant solution was processed using the methanolic solution of 0.3 mL for DPPH. During the progression of the reaction, this formulation was held inside a darkened room for an hour. The changes in the color indicated the properties of antioxidants in the formulation due to the donating capacity of hydrogen. The sample of 0.3 mL and 3.3 mL of methanol was included within the blank while 0.3 mL of DPPH reagent along with 3.5 mL of methanol was included in the control. At 517 nm, the formulations were examined in a spectrophotometer.

cessed using the methanolic solution of 0.3 mL for DPPH. During the progression of the

### reaction, this formulation was held inside a darkened room for an hour. The changes in 4.2.10. In Vitro Release and Permeation Studies

the color indicated the properties of antioxidants in the formulation due to the donating capacity of hydrogen. The sample of 0.3 mL and 3.3 mL of methanol was included within the blank while 0.3 mL of DPPH reagent along with 3.5 mL of methanol was included in the control. At 517 nm, the formulations were examined in a spectrophotometer. The process of dialysis bag was used to examine the in vitro drug release from conventional and Babchi oil nanoemulgels. The dialysis bags that were activated beforehand were carefully secured after adding 1 mL of the conventional and similarly 1 mL Babchi oil into the gel. The bag of dialysis was then immersed in a 200 mL medium of phosphate buffer along with methanol for dissolving at pH 6.8. This was maintained at room temperature and stirred at 400 rpm. At preset intervals, HPLC (High-Performance Liquid Chromatography) was used to evaluate all the formulations. Different models such as the zero-order release model, first-order release model, Higuchi model, and Korsmeyer Peppas model were used for analysis. The total quantities of Babchi oil released from the membrane were plotted as diffusion areas per time.

### 4.2.11. Release of Babchi Oil Nanoemulgel

The process involved entailed replacing a dialysis bag with the rat skin section. Any extra hairs were removed from the rat skin and were washed with a solution of Tyrode. These skin segments were immersed in 1 mL of Babchi oil nanoemulgel and conventional formulation. The study was conducted with the placement of applied rat skin in 100 mL of

solution of Tyrode at room temperature with constant agitation on Hanson Research SR8 plus of California, USA at the speed of 100 rpm. This was observed at various time points, and a sample of 2 mL was removed and a quantity similar to the solution of Tyrode was included as a preservative. The Babchi oil concentration was observed at 218 nm through a spectrophotometer and the procedures were repeated thrice for reproducibility.

### 4.2.12. Dermatokinetic Studies

Application of Babchi oil nanoemulgel on rat skin was observed with Franz Diffusion Cell (FDC) according to the literature search on in vitro skin permeation studies. Using the tool, we analyzed the content and concentration of Babchi oil and its formulated mixture at different periods of 0, 0.5, 1, 1.5, 2, 3, 4, 5, 6, 7, and 8 hours with the entire skin obtained from FDC. Excess nanoformulation was removed from the skin and rinsed using saline of pH 7.4. It was then dipped in the mildly warm water of temperature 60 ◦C for three minutes. We then separated the dermis and epidermis layers of skin using forceps. These layers were cut into small parts and kept in methanol (5 mL) for a day to obtain the Babchi oil content. The solution of methanol left after removing the layers underwent membrane filtration and the Babchi oil content was measured through HPLC. Separate concentrations per cm<sup>2</sup> of Babchi oil from dermis and epidermis layers were observed with time and parameters of T skin max, C skin max, AUC 0–8 h, and Ke were analyzed.

### 4.2.13. Stability Studies

Assessment of stability of the created formulation was performed by keeping it for three months at different temperatures of 30 ± 2 ◦C and 40 ± 2 ◦C with a humidity level of 60 ± 5%. According to the Iqubal et al. method, the samples were observed at different intervals of 0, 1, 2, and within 3 months to observe their appearance, separation of phases, size of globules, EE and PDI. These measurements were done thrice to determine the repeatability [32].

### 4.2.14. Statistical Analysis

The measurements obtained for the experiment were in triplicate values and were presented in values of mean ± SD (standard deviation). The analysis of statistical difference was performed on the flux at the stable stage and the permeation of ex vivo on intervals that were pre-determined from the formulations. Unpaired *t*-test was utilized with a *p*-value < 0.05 as the significance level.

**Author Contributions:** Conceptualization, A.A. and M.R.; methodology, M.R., M.A.S. and A.A.; software, M.R.; validation, M.H.A., A.I.F. and M.R.; formal analysis, A.A. and M.A.S.; investigation, A.A. and M.R.; resources, M.H.A., A.I.F. and A.A.; data curation, M.R. and M.A.S.; writing—original draft preparation, A.A.; writing—review and editing, M.H.A., A.I.F. and M.R.; visualization, M.H.A. and A.I.F.; supervision, A.A. and M.A.S.; project administration, A.A. and M.R.; funding acquisition, A.A. and A.I.F. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Deputyship for Research and Innovation, Ministry of Education in Saudi Arabia through the project number-IF-PSAU-2022/03/22592 and the APC was funded by IF-PSAU.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** There is no conflict of interest.

### **References**


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