Wound Healing Activity of Opuntia ficus-indica Fixed Oil Formulated in a Self-Nanoemulsifying Formulation

Figures & data

Table 1 The Composition of OFI-SNEDDSs Prepared Based on D-Optimal Mixture Design and Their Measured Globule Size

Run #	Mixture Components Percentages*			Globule Size (nm)
	OFI; X1	Tween 20; X2	PEG 200; X3
1	40	40	20	168
2	30	50	20	176
3	26.67	36.67	36.66	152
4	23.33	43.33	33.34	130
5	30	40	30	162
6	20	50	30	155
7	40	40	20	171
8	10	50	40	50
9	30	50	20	188
10	40	30	30	245
11	26.67	46.67	26.66	154
12	30	40	30	165
13	30	30	40	228
14	40	35	25	218
15	40	30	30	248
16	30	40	30	177
17	30	40	30	180
18	20	40	40	109
19	10	50	40	53
20	30	30	40	223

Note: *The percentages of each run are summed up to 100.

Table 2 Model Fit Statistics of OFI-SNEDDSs Globule Size

Model	Model P-value	Lack of Fit P-value	SD	R^2	Adjusted R^2	Predicted R^2	PRESS
Linear	< 0.0001	< 0.0001	26.41	0.7840	0.7586	0.6740	17,892.90
Quadratic	0.0026	0.1207	8.20	0.9829	0.9767	0.9693	1686.15
Special cubic	0.4717	0.0983	8.33	0.9836	0.9760	0.9542	1859.62

Abbreviations: R², multiple correlation coefficient; PRESS, predicted residual error sum of squares.

Figure 1 Diagnostic plots for globule size of OFI-SNEDDSs (A) Box-Cox for power transforms (B) externally studentized residuals vs predicted values (C) externally studentized residuals vs run number and (D) predicted vs actual globule sizes.

Figure 2 2D contour plot (A) and 3D response surface plot (B) for the influence of mixture components on the globule size of OFI-SNEDDSs.

Table 3 Skin Permeation Parameters of OFI-Oil and OFI-SNEDDS Through Rat Abdominal Skin

Parameter	Unit	OFI -OIL	OFI-SNEDDS
Steady-state flux	ng/cm^2·h	0.17±1.32	0.22±2.12*
Permeability coefficient	cm/h	0.00069±0.001	0.00089±0.003
Diffusion coefficient (D)	cm^2/h	0.00056±0.002	0.0011±0.001*

Note: *Significant variance (P-value <0.05).

Figure 3 Percentage of cumulative OFI permeation from OFI-SNEDDS gel and from OFI–OIL gel through abdominal rat skin.

Figure 4 (A) Photographs showing wound closure in rats treated with topical application of OFI-SNEDDS on day 7 and day 14. (B) Effects of different treatments on percent wound closure. Data are presented as Mean ± SD (n = 6). Statistical analysis was performed by one-way ANOVA followed by Tukey’s test. *Significant difference from Untreated Control group at p < 0.05. ^#Significant difference from vehicle-treated group at p < 0.05. ^†Significant difference from OFI group at p < 0.05. ^‡Significant difference from OFI-SNEDDS group at p < 0.05.

Figure 5 Histopathology effect of OFI and OFI-SNEDDS on wound healing of rats (X200). Panels A & B represent skin sections from wound areas collected at day 7 and stained with H&E and MT, respectively. Panels C & D represent skin sections from wound areas collected at day 14 and stained with H&E and MT, respectively. Arrows indicate healthy skin (black arrows), collagen deposition (red arrows), inflammatory infiltration (blue arrows), Congestion (yellow arrows).

Table 4 Histological Features of Wound Healing in Animals Treated Topically with OFI-SNEDDS on Day 7

	RE	GT	CD	IC	Inflammation Phase	Proliferation Phase	Remodeling Phase
Untreated Control	–	+	+	++	++	++	–
Vehicle-treated	–	++	+	++	++	++	–
OFI	+	+++	+	±	+	+++	++
OFI-SNEDDS	+	+	++	–	+	++	+++
Positive Control	+	++	++	±	++	+++	++

Notes: (–) Absence of the histological feature, (+) Low-degree, (++) Intermediate-degree, (+++) High-degree, (±) Absence or Low-degree feature.

Abbreviations: RE, Re-epithelization; GT, Granulation tissue; CD, Collagen deposition; IC, Inflammatory cell infiltration.

Table 5 Effect of OFI-SNEDDS on Oxidative Stress in Wounded Skin of Rats

	MDA (nmol/mg Protein)	GSH (nmol/mg Protein)	SOD (Unit/mg Protein)	GPx (nmol/mg Protein)
Untreated Control	7.60 ± 0.88	1.55 ± 0.18	6.43 ± 0.31	2.93 ± 0.47
Vehicle-treated Control	7.20± 0.82	1.70 ± 0.19	6.46 ± 0.57	3.31 ± 0.47
OFI	5.30 *^# ± 0.62	2.10 * ± 0.26	7.80 *^# ± 0.81	3.79 * ± 0.39
OFI-SNEDDS	4.31 *^# ± 0.42	2.60 *^#† ± 0.23	8.73 *^# ± 0.88	4.06 *^# ± 0.31
Positive Control	5.11 *^# ± 0.57	2.30 *^# ± 0.30	7.79 *^# ± 0.88	3.90 *^# ± 0.46

Notes: Data are presented as Mean ± SD (n = 6). Statistical analysis was performed by one-way ANOVA followed by Tukey’s test. *Significant difference from Untreated Control group at p < 0.05. ^#Significant difference from vehicle-treated control at p < 0.05. ^†Significant difference from OFI group at p < 0.05.

Figure 6 Effect of OFI-SNEDDS on inflammation in wounded skin of rats. (A) represents IL-6 content and (B) represents TNF-α content. Data are presented as Mean ± SD (n = 6). Statistical analysis was performed by one-way ANOVA followed by Tukey’s test. *Significant difference from Untreated Control group at p < 0.05. ^#Significant difference from vehicle-treated group at p < 0.05. ^†Significant difference from OFI group at p < 0.05.

Figure 7 Effect of OFI-SNEDDS on collagen formation in wounded skin of rats. (A) represents hydroxyproline content and (B) represents Col 1 relative mRNA expression. Data are presented as Mean ± SD (n = 6). Statistical analysis was performed by one-way ANOVA followed by Tukey’s test. *Significant difference from Untreated Control group at p < 0.05. ^#Significant difference from vehicle-treated group at p < 0.05. ^†Significant difference from OFI group at p < 0.05. ^‡Significant difference from OFI-SNEDDS group at p < 0.05.

Figure 8 Effect of OFI-SNEDDS on expression of transforming growth factor-beta (TGF-β) by immunohistochemical staining. (A) Untreated Control; (B) Vehicle-treated Control; (C) OFI; (D) OFI-SNEDDS; (E) Positive Control. Negative photographs are put as inserts in their respective ones with strong positive reaction (C, D and E). (F) Quantitative image analysis for TGF-β immuno-histochemical staining, expressed as optical density (OD). Data are presented as Mean ± SD (n = 6). Statistical analysis was performed by one-way ANOVA followed by Tukey’s test. *Significant difference from Untreated Control group at p < 0.05. ^#Significant difference from vehicle-treated group at p < 0.05. ^†Significant difference from OFI group at p < 0.05.

Figure 9 Effect of OFI-SNEDDS on expression of vascular endothelial growth factor (VEGF) by immunohistochemical staining. (A) Untreated Control; (B) Vehicle-treated Control; (C) OFI; (D) OFI-SNEDDS; (E) Positive Control. Negative photographs are put as inserts in their respective ones with strong positive reaction (C, D and E). (F) Quantitative image analysis for TGF-β immuno-histochemical staining, expressed as optical density (OD). Data are presented as Mean ± SD (n = 6). Statistical analysis performed by one-way ANOVA followed by Tukey’s test. *Significant difference from Untreated Control group at p < 0.05. ^#Significant difference from vehicle-treated group at p < 0.05. ^†Significant difference from OFI group at p < 0.05.

Figure 10 Total ion chromatogram of GC/MS analysis of hexane fraction of OFI seeds.