Development, optimization and characterization of nanoemulsion loaded with clove oil-naftifine antifungal for the management of tinea

Figures & data

Table 1. Independent variables and their levels, along with dependent variables and their constraints in the Box–Behnken designs of plain and medicated nanoemulsion formulations.

	Levels
	Low	High
Plain SNEDDS
Independent variables
A = Clove oil %	10%	20%
B = Smix ratio (surfactant: co-surfactant) %	40%	60%
C = Water %	20%	50%
Dependent variables	Constrains
Y1 = Droplet size (nm)	Minimize
	Levels
Independent variables	−1	0	1
A = Clove oil %	10%	13.5%	17%
B = Naftifin amount (mg)	10	20	30
C = Smix ratio (surfactant: co-surfactant)	1:1	1:2	1:3
Medicated SNEDDs
Dependent variables	Constrains
Y1 = Droplet size (nm)	Minimize
Y2 Ex vivo % naftifine permeated	Maximize
Y3 = zone of inhibition against   Trichophyton rubrum (mm)	Maximize
Y4 = Interleukin-31 Level (pg/mL)	Minimize

Table 2. Box–Behnken design responses of plain NE formulation.

Run	A	B	C	Response 1
	Clove Oil %	Smix %	Water%	Globule Size (nm)	PDI
1	0.15	50.0	35.0	142	0.23
2	15.0	60.0	24.9	147	0.19
3	14.6	48.4	36.8	138	0.10
4	20.0	50.3	29.6	195	0.33
5	10.0	57.0	32.9	85	0.35
6	20.0	44.3	35.6	185	0.25
7	10.0	47.7	42.2	96	0.11
8	13.7	42.0	44.2	123	0.35
9	20.0	60.0	20.0	175	0.14
10	10.0	57.0	32.9	86	0.29
11	19.2	40.0	40.7	167	0.30
12	15.0	60.0	24.9	146	0.17
13	19.2	40.0	40.7	166	0.11
14	14.6	48.4	36.8	136	0.24
15	15.3	54.9	29.7	153	0.34
16	14.6	48.4	36.8	136	0.16
17	20.0	55.1	24.8	200	0.26
18	10.0	40.0	50.0	145	0.30

Table 3. Box–Behnken design responses of NF-CO-SENDDs.

Run	A	B	C	Y1	Y2	Y3	Y4	PDI
	A:Clove Oil %	B:Naftifine amount (mg)	C:Smix ratio	Globule size (nm)	Ex vivo % naftifine permeated (%)	Zone of inhibition against Trichophyton rubrum (mm)	Interleukin-31 Level (pg/mL)
1	1	−1	−1	164	56	5	120	0.12
2	−1	1	−1	200	34	12	750	0.23
3	−1	−1	−1	119	52	3	190	0.22
4	1	1	−1	295	52	13	340	0.30
5	0	0	0	171	65	16	300	0.15
6	0	0	−1.68	161	69	10	255	0.14
7	−1.68	0	0	128	18	12	590	0.32
8	−1	−1	1	141	44	14	200	0.40
9	1	−1	1	186	52	15	175	0.19
10	0	−1.68	0	146	60	6	100	0.25
11	0	0	0	172	63	16	300	0.36
12	0	1.68	0	310	41	22	680	0.31
13	0	0	1.68	178	59	21	385	0.21
14	1	1	1	289	44	24	385	0.22
15	−1	1	1	190	25	22	800	0.17
16	1.68	0	0	255	35	19	260	0.10
17	0	0	0	173	62	17	300	0.20

Figure 1. (A) Contour plot, (B) 3D surface plot, and (C) overlay plot showing the effects of different independent variables on the globule size of plain nanoemulsion.

Figure 2. Main effect diagram, contour, and 3D response surface plots representing the effect of the studied variables on droplet size (Y1). (A) Main effect diagram of clove oil (%); (B) main effect diagram of naftifine amount (mg); (C) main effect diagram of Smix ratio (%); (D) contour plot showing the effect of clove oil and naftifine on globule size; (E) 3D surface plot representing the effects of the clove oil % and naftifine amount (mg) on droplet size.

Figure 3. Main effect diagram, contour, and 3D response surface plots representing the effect of the studied variables on ex vivo % naftifine permeated (Y2). (A) Main effect diagram of clove oil (%); (B) main effect diagram of naftifine amount (mg); (C) main effect diagram of Smix ratio (%); (D) Contour Plot showing the effect of clove oil and naftifine on ex vivo % naftifine permeated; (E) 3D Surface plot representing the e effects of the clove oil % and naftifine amount (mg) on ex vivo % naftifine permeated.

Figure 4. Main effect diagram, contour, and 3D response surface plots representing the effect of the studied variables on the zone of inhibition against Trichophyton rubrum (Y3). (A) Main effect diagram of clove oil (%); (B) main effect diagram of naftifine amount (mg); (C) main effect diagram of Smix ratio (%); (D) contour plot showing the effect of clove oil and naftifine on zone of inhibition against Trichophyton rubrum; (E) 3D surface plot representing the effects of the clove oil % and naftifine amount (mg) on zone of inhibition against Trichophyton rubrum.

Figure 5. Main effect diagram, contour, and 3D response surface plots representing the effect of the studied variables on IL-31 level (Y4). (A) Main effect diagram of clove oil (%); (B) main effect diagram of naftifine amount (mg); (C) main effect diagram of Smix ratio (%); (D) contour plot showing the effect of clove oil and naftifine on IL-31 levels; (E) 3D surface plot representing the effects of the clove oil % and naftifine amount (mg) on IL-31 levels.

Table 4. Regression analysis results for Y1, Y2, Y3, and Y4 responses.

Dependent variables	R^2	Adjusted R^2	Predicted R^2	p-Value	F-value	Adequate precision
Y1	0.9988	0.9973	0.9913	.0001	184.78	480.27
Y2	0.9953	0.9893	0.9714	.0001	326.04	97.03
Y3	0.9283	0.9118	0.8777	.0001	56.13	74.16
Y4	0.9930	0.9840	0.9460	.0001	110.8	40380.18

Figure 6. Desirability ramp and bar chart for optimization. (A) Desirability ramp shows the levels for independent variables and predicted values for the responses of the optimum formulation. (B) Bar chart shows the desirability values for the combined responses.

Table 5. Actual and experimental values of the optimized nanoemulsion formulation.

Solution	Clove oil %	NF amount (mg)	Smix ratio	Droplet size (nm	Ex vivo % naftifine permeated	Zone of inhibition against Trichophyton rubrum (mm)	Interleukin-31 Level (pg/mL)	desirability
Predicated value	14	12.5	3:1	158.7	62.3	15.1	185	0.80
Experimental value	14	12.5	3:1	161.0	64.0	16.0	180	0.80

Figure 7. Overlay plot for the optimal NF-CO SNEDDS region determination.

Table 6. Composition, actual, and predicted responses of the optimal nanoemulsion formulation.

Factor	Optimal value	Response Variable	Actual value	Predicted value	% Prediction error^a
A:Clove oil %	14	Droplet size (nm)	161	158.7	0.006
B:NF amount (mg)	12.5	Ex vivo % naftifine permeated	64	62.3	0.026
C:Smix ratio	3:1	Zone of inhibition against Trichophyton rubrum (mm)	16	15.1	0.056
		Interleukin-31 Level (pg/mL)	180	185	−0.027

^aCalculated as [Actual − predicted/Actual] × 100.

Table 7. Permeation parameters of optimum NF-CO-SENDDs.

Permeation parameters	Optimized formulation	Commercially available cream
Cumulative amount permeated (μg/cm^2)	3300 ± 423	1633 ± 212
Steady state flux, Jss, (μg/cm^2 min)	9.23 ± 1.33	3.12 ± 0.62
Permeability coefficient, P, (cm/min)	1.30 × 10^−3	0.22 × 10^−3
Diffusion coefficient, D, (cm^2/min)	16.12 × 10^−5	5.87 × 10^−5
Enhancement factor (EF)	2.02

Table 8. Scoring of skin sensitivity test results of optimum NF-CO-SENDDs.

	Itching	Erythema	Papule	Flakiness	Dryness	Average Sensitivity Score
Control	0	1	0	0	0	1/20
Optimized formulation	1	0	0	0	1	2/20
Commercial cream	3	3	2	2	2	12/20

0: no sign of inflammation; 1: mild; 2: moderate; 3: severe.

Table 9. Physical stability parameters of stored and freshly prepared NF-CO-SENDDs.

	Emulsification ability	Precipitation	pH	Globule size (nm)	PDI
Freshly prepared optimized formulation	Simultaneous emulsification	No	6.64 ± 0.21	161 ± 6	0.21
Optimized formulation after stability test	Simultaneous emulsification	No	6.49 ± 0.19	164 ± 4	0.23