2.3.2. High-Performance Thin Layer Chromatography (HPTLC)

The most abundant constituents of keratin and genistein were found in the final formulation. The mobile phase system comprised butanol: acetic acid: water (6.5:3.5:1 *v*/*v*/*v*); the keratin extract showed an Rf value of 0.58, which was also found in the keratin–genistein gel (Rf value of 0.51) (Table 2). The most abundant constituents of keratin and genistein were found in the final formulation. The mobile phase system comprised chloroform: methanol (10:1 *v*/*v*); the genistein extract demonstrated an Rf value of 0.74, which was equivalent to the keratin–genistein gel (Rf value of 0.70) (Table 2). However, only genistein runs in this solvent system. The chromatogram of keratin, genistein gel revealed similar characteristic peaks to that of genistein, which represented the intactness of the genistein in the gel formulation. The corresponding % area was found to be about the same (3.68 and 4.87) (Figure 5). *Gels* **2023**, *9*, x FOR PEER REVIEW 7 of 19

**Figure 5.** HPTLC of (**A**) comparative chromatograms of extracted keratin, genistein, and keratin– genistein combination gel; (**B**) extracted keratin, genistein; and (**C**) keratin–genistein gel under mobile phase system chloroform: methanol (10:1 v/v). **Figure 5.** HPTLC of (**A**) comparative chromatograms of extracted keratin, genistein, and keratin– genistein combination gel; (**B**) extracted keratin, genistein; and (**C**) keratin–genistein gel under mobile phase system chloroform: methanol (10:1 *v*/*v*).

### *2.4. Stability Studies 2.4. Stability Studies*

*2.5. Wound-Healing Activity* 

[27].

The stability studies were carried out for the optimised formulation at 40 ± 2 °C temperature and 65 ± 5% RH for a period of 90 days [26]. The formulation presented good stability over the time regimen with no remarkable change in the pH, viscosity, and spreadability test profile (Table 4). The stability studies were carried out for the optimised formulation at 40 ± 2 ◦C temperature and 65 ± 5% RH for a period of 90 days [26]. The formulation presented good stability over the time regimen with no remarkable change in the pH, viscosity, and spreadability test profile (Table 4).

genistein (87.40%) gel formulation (Table 5). Furthermore, the keratin–genistein-combination gel formulation presented a comparable activity to that of the marketed hydrogel product (96.66%), as well as the control group (85.98%) (Figure 6). The stages of healing by the keratin–genistein-combination gel formulation in the experimental animal model are described in Figure 7. The present observations and potential roles of genistein in promoting wound healing were found to be comparable with the results stated in previous reports on early-stage cutaneous wound healing, refractory wound healing in type-1 diabetes, skin wound repair in an incision model, and wound healing in ovariectomised mice

**Mean ± SD** 

Keratin gel 54.29 ± 4.170 87.57 ± 5.00 Genistein gel 48.51 ± 7.88 87.40 ± 3.57

Standard gel 54.99 ± 13.63 96.66 ± 5.77 Control 48.15 ± 9.32 85.98 ± 3.53

**Day 14 (% Wound Healing) Mean ± SD** 

**Groups Day 7 (% Wound Healing)** 

Keratin and genistein combination gel 51.18 ± 2.043 94.65 ± 4.64


**Table 4.** Short-term (90 days) stability studies of gel formulations. **Table 4.** Short-term (90 days) stability studies of gel formulations.

**Table 5.** Wound healing after 7 days and 14 days.

### *2.5. Wound-Healing Activity*

Keratin–genistein gel formulation showed higher degrees of wound healing (94.65%) for the duration of 14 days as compared to the keratin gel formulation (87.57%) and genistein (87.40%) gel formulation (Table 5). Furthermore, the keratin–genistein-combination gel formulation presented a comparable activity to that of the marketed hydrogel product (96.66%), as well as the control group (85.98%) (Figure 6). The stages of healing by the keratin–genistein-combination gel formulation in the experimental animal model are described in Figure 7. The present observations and potential roles of genistein in promoting wound healing were found to be comparable with the results stated in previous reports on early-stage cutaneous wound healing, refractory wound healing in type-1 diabetes, skin wound repair in an incision model, and wound healing in ovariectomised mice [27]. *Gels* **2023**, *9*, x FOR PEER REVIEW 8 of 19

**Figure 6.** Percentage of wound closure after treatment with various gel formulations. **Figure 6.** Percentage of wound closure after treatment with various gel formulations.


### *2.6. Histopathological Investigations*

(**A**) 7 days; (**B**) 14 days; and (**C**) 21 days.

After 14 days, the wound-healing activity produces a normal architecture of the skin. As compared to the genistein gel and keratin gel, the keratin–genistein combination gel produced proper development of the epidermis, along with the proliferation of fibrous connective tissue and the growth of hair follicles. After 14 days, the normal functioning of the kidney and liver was studied, where wound healing does not produce any toxic effect. Genistein purposely solved the development of epidermal growth on the skin. The control sample of the skin showed failing to develop an epidermis and no proliferation of fibrous connective tissue. The keratin sample presented the development of epidermis and the proliferation of fibrous connective tissue, indicating wound healing. The genistein gel sample revealed the development of epidermis and excessive proliferation of fibrous connective tissue, demonstrating wound healing [23,28].

**Figure 7.** This figure indicates morphological studies of wound-healing activity of formulation after

**Figure 6.** Percentage of wound closure after treatment with various gel formulations.

**Figure 7.** This figure indicates morphological studies of wound-healing activity of formulation after (**A**) 7 days; (**B**) 14 days; and (**C**) 21 days. **Figure 7.** This figure indicates morphological studies of wound-healing activity of formulation after (**A**) 7 days; (**B**) 14 days; and (**C**) 21 days.

The combination of keratin and genistein showed proper development of epidermis, along with the proliferation of fibrous connective tissue and growth of hair follicles. The standard sample displayed the best wound healing, with development of epidermis and proliferation of fibroblastic cells and hair follicles. The control sample of the liver revealed vacuolar degenerative changes in hepatocytes around the central vein. The keratin, the genistein, the standard, and the combination sample showed the normal architecture of hepatocytes around the central vein. The control sample of the kidney revealed the inflamed architecture of glomeruli and tubules. In contrast, keratin, genistein, standard, and the combination sample showed the normal architecture of glomeruli and tubules (Figure 8).

In the case of vital organ toxicity studies, the histological section of liver shows normal architecture of hepatocytes around the central vein as compared to vacuolar degenerative changes in positive control group. Kidney histology shows the normal architecture of glomeruli and tubules. This result proved the safety of keratin and genistein for medicinal use.

8).

**Figure 8.** This figure shows that histopathological studies of skin, liver, and kidney. **Figure 8.** This figure shows that histopathological studies of skin, liver, and kidney. *2.7. Biochemical Investigations*  The biochemical investigations through ELISA and qRT-PCR studies strongly advo-

#### In the case of vital organ toxicity studies, the histological section of liver shows nor-*2.7. Biochemical Investigations* cated that the keratin–genistein combination gel-formulation-treated group produced a

*2.6. Histopathological Investigations* 

nective tissue, demonstrating wound healing [23,28].

After 14 days, the wound-healing activity produces a normal architecture of the skin. As compared to the genistein gel and keratin gel, the keratin–genistein combination gel produced proper development of the epidermis, along with the proliferation of fibrous connective tissue and the growth of hair follicles. After 14 days, the normal functioning of the kidney and liver was studied, where wound healing does not produce any toxic effect. Genistein purposely solved the development of epidermal growth on the skin. The control sample of the skin showed failing to develop an epidermis and no proliferation of fibrous connective tissue. The keratin sample presented the development of epidermis and the proliferation of fibrous connective tissue, indicating wound healing. The genistein gel sample revealed the development of epidermis and excessive proliferation of fibrous con-

The combination of keratin and genistein showed proper development of epidermis, along with the proliferation of fibrous connective tissue and growth of hair follicles. The standard sample displayed the best wound healing, with development of epidermis and proliferation of fibroblastic cells and hair follicles. The control sample of the liver revealed vacuolar degenerative changes in hepatocytes around the central vein. The keratin, the genistein, the standard, and the combination sample showed the normal architecture of hepatocytes around the central vein. The control sample of the kidney revealed the inflamed architecture of glomeruli and tubules. In contrast, keratin, genistein, standard, and the combination sample showed the normal architecture of glomeruli and tubules (Figure

mal architecture of hepatocytes around the central vein as compared to vacuolar degenerative changes in positive control group. Kidney histology shows the normal architecture of glomeruli and tubules. This result proved the safety of keratin and genistein for medicinal use. The biochemical investigations through ELISA and qRT-PCR studies strongly advocated that the keratin–genistein combination gel-formulation-treated group produced a comparatively high impact over the IL-6 expression via the downregulation of the overexpressed IL-6 gene in contrast to the working control group (Figure 9). In comparison with other pro-inflammatory factors such as COX-2, IL-2, IL-10, and IL-1β expressed in the liver tissues, genistein and keratin, both in combination, have the capacity to regulate the higher expression of mRNA of IL-6 in inflamed liver tissues and, therefore, play an imperative role in the suppression of inflammation [29,30]. The exploration of the underlying mechanism(s) supported our hypothesis that the combination markedly normalised (*p* < 0.001) the upregulation of the IL-6 gene and exerted an anti-inflammatory effect by modulating the molecular targets (Table 6). The efficacy of the gel combination was observed to be quite comparable with the positive control (marketed standard formulation) at an analogous dose. comparatively high impact over the IL-6 expression via the downregulation of the overexpressed IL-6 gene in contrast to the working control group (Figure 9). In comparison with other pro-inflammatory factors such as COX-2, IL-2, IL-10, and IL-1β expressed in the liver tissues, genistein and keratin, both in combination, have the capacity to regulate the higher expression of mRNA of IL-6 in inflamed liver tissues and, therefore, play an imperative role in the suppression of inflammation [29,30]. The exploration of the underlying mechanism(s) supported our hypothesis that the combination markedly normalised (*p* < 0.001) the upregulation of the IL-6 gene and exerted an anti-inflammatory effect by modulating the molecular targets (Table 6). The efficacy of the gel combination was observed to be quite comparable with the positive control (marketed standard formulation) at an analogous dose.

**Figure 9.** This figure shows mRNA expression levels of COX-2, IL-2, IL-6, IL-10, and IL1-β in the liver through qRT-PCR. [WC: Working Control; PC: Positive Control; \*\*\* represents the level of significance in comparision with working control]. **Figure 9.** This figure shows mRNA expression levels of COX-2, IL-2, IL-6, IL-10, and IL1-β in the liver through qRT-PCR. [WC: Working Control; PC: Positive Control; \*\*\* represents the level of significance in comparision with working control].

**Table 6.** ELISA measurement of interleukins (IL-2, IL-6, IL-1β, and IL-10) and COX-2 levels in liver

Legends: NC: Normal Control; WC: Working Control; PC: Positive Control. \*\*\* Represents the level

The inflammation-reducing potential of genistein by reducing the levels of cytokines (IL-1β, IL-2, IL-6, and IL-10) was found to have complied with the previous reports on the protective actions of this natural product in gut inflammation, inflammation that is related to colitis, TNF-α-induced endothelial inflammation, and vascular inflammation [23,31].

A safe and effective keratin, genistein wound-healing combined gel formulation was successfully developed and comprehensively characterised through sophisticated

**Genistein** 

of significance in comparision with normal control/working control.

**3. Conclusions** 

COX-2 (pg/mL) 188.78 ± 8.09 449.24 ± 12.95 210.84 ± 10.51 \*\*\* 240.97 ± 8.46 \*\*\* 223.79 ± 11.49 \*\*\* 196.25 ± 9.61 \*\*\* IL-2 (pg/mL) 492.27 ± 12.03 1103.32 ± 19.37 594.38 ± 12.45 \*\*\* 620.31 ± 16.85 \*\*\* 601.76 ± 18.17 \*\*\* 519.37 ± 11.51 \*\*\* IL-6 (pg/mL) 148.91 ± 4.61 327.94 ± 8.16 179.24 ± 5.34 \*\*\* 204.37 ± 9.17 \*\*\* 191.38 ± 7.42 \*\*\* 163.84 ± 7.19 \*\*\* IL-10 (pg/mL) 630.94 ± 20.37 1294.01 ± 25.79 764.85 ± 23.14 \*\*\* 804.59 ± 22.67 \*\*\* 789.41 ± 11.64 \*\*\* 681.93 ± 9.43 \*\*\* IL1-β (pg/mL) 552.37 ± 18.19 1109.86 ± 24.17 624.87 ± 12.37 \*\*\* 655.49 ± 11.27 \*\*\* 634.59 ± 9.73 \*\*\* 611.48 ± 10.70 \*\*\*

after the application of keratin, genistein, and keratin–genistein gel.


**Table 6.** ELISA measurement of interleukins (IL-2, IL-6, IL-1β, and IL-10) and COX-2 levels in liver after the application of keratin, genistein, and keratin–genistein gel.

Legends: NC: Normal Control; WC: Working Control; PC: Positive Control. \*\*\* Represents the level of significance in comparision with normal control/working control.

The inflammation-reducing potential of genistein by reducing the levels of cytokines (IL-1β, IL-2, IL-6, and IL-10) was found to have complied with the previous reports on the protective actions of this natural product in gut inflammation, inflammation that is related to colitis, TNF-α-induced endothelial inflammation, and vascular inflammation [23,31].

### **3. Conclusions**

A safe and effective keratin, genistein wound-healing combined gel formulation was successfully developed and comprehensively characterised through sophisticated analytical techniques. The major active components that were needed for the woundhealing activity were identified, along with other components, by using TLC, FTIR, and amino acid profiling techniques. The present investigation revealed that the fabrication of keratin gel showed excellent wound-healing properties, and with the combination of the inhibitor of EGF- induced proliferation, genistein showed noteworthy wound-closure activity. The fabricated formulations demonstrated good spreadability with a pH suitable for human skin application. The formulations also retained a good stability condition over a period of 90 days. The ability of the natural-component-containing gel product as an effective wound healer was confirmed via histopathological studies. From all the biochemical studies, it may be concluded that the novel keratin, genistein wound-healing gel effectually reduced the pro-inflammatory factors (IL-2, IL-6, IL-1β, IL-10, and COX-2), which led to acute lessening of inflammation. Comparing the gel to other commercially available formulations, its desirable formulation texture characteristics, in addition to its viscosity, pH, spreadability, and wound-healing function, contributed to the quality and stability attributes of the gel.

### **4. Material and Methods**
