2.1.2. Amino Acid Profiling

As per the literature, total amino acid contents must exceed 70% of the keratin obtained from the chicken feathers. The keratin sample displayed the presence of aspartic acid, alanine, glutamic acid, serine, threonine, and tyrosine in the range of 0.36 to 12.1 g/100 g. These values corresponded with the reference range of standard keratin (Table 1). Serine, glutamic acid, and aspartic acid were found in higher concentrations than tyrosine, alanine, and threonine [16].


### 2.1.3. Fourier-Transform Infrared (FT-IR) Spectroscopy

The absorption bands that were observed in the keratin sample are mainly because of the amide bonds (-CONH-). Among nine amide bands, five were prominently observed, viz., amide-I, II, III, A and B. The signal at approximately 3300 cm−<sup>1</sup> corresponds to amide A. The Amide B band has been reported and appeared within 3056 to 3075 cm−<sup>1</sup> . The strong peaks at 1654 cm−<sup>1</sup> were assigned to amide-I, which is closely related to the carbonyl groups. Amide–II was observed within 1480–1580 cm−<sup>1</sup> , which is related to symmetric bending vibrations of –NH and –CN. The bands of approximately 1230–1240 cm−<sup>1</sup> were observed and associated with β-sheet of amide-III. All the characteristic bands that were observed in the FT-IR spectrum of extracted keratin were indicative of its similar secondary structure as that of the standard [23].

The FT-IR spectrum of pure genistein shows a broad band at 3464.15 cm−<sup>1</sup> , corresponding to the phenolic–OH group, and carbonyl stretching vibrations were observed at 1625.99 cm−<sup>1</sup> . The C-H aromatic band was observed at 3041.74 cm−<sup>1</sup> , whereas the band of carbonyl functionality C=O was observed at 1159.22, and that of the C-O stretching band was seen at 1022.27 cm−<sup>1</sup> . Aromatic C=C stretching bands were observed in the range of 1600–1400 cm−<sup>1</sup> . All the characteristic bands were observed in the FT-IR spectrum of pure genistein, confirming its identity and purity [24,25]

The FT-IR spectrum of the physical mixture of keratin and genistein with Carbopol 940 and other excipients was found to retain all major peaks shown in pure keratin and genistein. It revealed no interaction of the drug component with the excipients, and thus, the formulation is considered stable and compatible, as shown in Figure 1. *Gels* **2023**, *9*, x FOR PEER REVIEW 4 of 19

**Figure 1.** FT-IR spectra of (**A**) reference keratin; (**B**) extracted keratin; (**C**) genistein; (**D**) Carbopol 934; and (**E**) physical mixture. **Figure 1.** FT-IR spectra of (**A**) reference keratin; (**B**) extracted keratin; (**C**) genistein; (**D**) Carbopol 934; and (**E**) physical mixture.

value of 0.63 for the extracted keratin, which was also found in the keratin gel with an alike Rf value (Table 2). The chromatogram of the keratin gel produced the related characteristic peaks with a% corresponding area of 0.57 and 0.95, respectively (Figure 2). The most abundant constituents of keratin were found in the final formulation, which repre-

**(Rf)** 

**End Height** 

**(AU) Area (AU) Area %** 

**(AU) Max % End Position** 

sents the intactness of the keratin in the gel.

**Max Position (Rf)** 

**Peak Start Position (Rf)** 

**Start Height (AU)** 

**Table 2.** HPTLC interpretation of various gel formulations.

**Keratin extract and keratin gel formulation**  3 0.62 6.5 0.63 12.8 2.52 0.64 0.4 90.3 0.57 7 0.62 7.7 0.63 18.9 3.54 0.65 6.4 159.5 0.95 **Genistein extract and genistein gel formulation**  6 0.56 42.9 0.56 49.2 8.81 0.60 38.7 802.0 11.03 5 0.48 26.8 0.50 41.4 13.37 0.51 25.6 448.2 13.34 **Keratin, genistein extract and keratin, genistein combination gel (Mobile Phase: butanol: acetic acid: water)**  10 0.56 3.5 0.58 14.3 2.33 0.60 9.8 175.0 1.46 9 0.49 5.9 0.51 13.0 2.35 0.55 1.2 135.9 1.78 **Keratin, genistein extract and keratin, genistein combination gel (Mobile Phase: chloroform: methanol)** 4 0.73 21.3 0.74 27.7 6.09 0.75 19.5 279.2 3.68 8 0.68 15.2 0.70 25.4 7.02 0.72 14.2 271.8 4.87

**Max Height** 

2.1.4. High-Performance Thin Layer Chromatography (HPTLC)

### 2.1.4. High-Performance Thin Layer Chromatography (HPTLC)

The solvent system butanol: acetic acid: water in the ratio of 6.5:3.5:1 presented Rf value of 0.63 for the extracted keratin, which was also found in the keratin gel with an alike Rf value (Table 2). The chromatogram of the keratin gel produced the related characteristic peaks with a% corresponding area of 0.57 and 0.95, respectively (Figure 2). The most abundant constituents of keratin were found in the final formulation, which represents the intactness of the keratin in the gel.


**Figure 2.** HPTLC of (**A**) Comparative chromatograms of extracted keratin and keratin gel; (**B**) extracted keratin; and (**C**) keratin gel. **Figure 2.** HPTLC of (**A**) Comparative chromatograms of extracted keratin and keratin gel; (**B**) extracted keratin; and (**C**) keratin gel.

2.1.5. Scanning Electron Microscopy (SEM) 2.1.5. Scanning Electron Microscopy (SEM)

keratin at 1000×.

*2.2. Characterisation of Genistein* 

The morphology of the keratin crystals at both 5000× (Figure 3B) and 1000× (Figure 3C) was investigated via SEM analysis. The microphotographs presented a region of dense fibrous keratin with an internal porous network (Figure 3A). The morphology of the keratin crystals at both 5000× (Figure 3B) and 1000× (Figure 3C) was investigated via SEM analysis. The microphotographs presented a region of dense fibrous keratin with an internal porous network (Figure 3A).

**Figure 3.** Microphotograph of (**A**) reference keratin; (**B**) extracted keratin at 5000×; and (**C**) extracted

The solvent system chloroform: methanol in the ratio of 10:1 presented an Rf value of 0.49, which was also found in genistein gel (Rf = 0.50) (Table 2). The chromatogram of the genistein gel produced the related characteristic peaks with a % corresponding area of 11.03 and 13.34, respectively (Figure 4). The most abundant constituents of genistein were found in the final formulation, which represents the intactness of the genistein in the gel.

2.2.1. High-Performance Thin Layer Chromatography (HPTLC)

**Gel Type** 

Keratin– Genistein gel <sup>4</sup>

**Gel Strength (Sec)** 

**Drug Content Mean ± SD** 

93.21 ± 0.764 (Keratin) 92.79 ± 0.7543 (Genistein)

Keratin gel 4 96.07 ± 0.7214 7.6 ± 0.15 6.8 ± 0.15 470,913 ±

same (3.68 and 4.87) (Figure 5).

**Figure 3.** Microphotograph of (**A**) reference keratin; (**B**) extracted keratin at 5000×; and (**C**) extracted keratin at 1000×. **Figure 3.** Microphotograph of (**A**) reference keratin; (**B**) extracted keratin at 5000×; and (**C**) extracted keratin at 1000×.

**Figure 2.** HPTLC of (**A**) Comparative chromatograms of extracted keratin and keratin gel; (**B**) ex-

The morphology of the keratin crystals at both 5000× (Figure 3B) and 1000× (Figure 3C) was investigated via SEM analysis. The microphotographs presented a region of dense fibrous keratin with

#### *2.2. Characterisation of Genistein 2.2. Characterisation of Genistein*

tracted keratin; and (**C**) keratin gel.

2.1.5. Scanning Electron Microscopy (SEM)

an internal porous network (Figure 3A).

2.2.1. High-Performance Thin Layer Chromatography (HPTLC) High-Performance Thin Layer Chromatography (HPTLC)

The solvent system chloroform: methanol in the ratio of 10:1 presented an Rf value of 0.49, which was also found in genistein gel (Rf = 0.50) (Table 2). The chromatogram of the genistein gel produced the related characteristic peaks with a % corresponding area of 11.03 and 13.34, respectively (Figure 4). The most abundant constituents of genistein were found in the final formulation, which represents the intactness of the genistein in the gel. The solvent system chloroform: methanol in the ratio of 10:1 presented an Rf value of 0.49, which was also found in genistein gel (Rf = 0.50) (Table 2). The chromatogram of the genistein gel produced the related characteristic peaks with a % corresponding area of 11.03 and 13.34, respectively (Figure 4). The most abundant constituents of genistein were found in the final formulation, which represents the intactness of the genistein in the gel. *Gels* **2023**, *9*, x FOR PEER REVIEW 6 of 19

**Figure 4.** HPTLC of (**A**) comparative chromatograms of extracted genistein and genistein gel; (**B**) extracted genistein; and (**C**) genistein gel. **Figure 4.** HPTLC of (**A**) comparative chromatograms of extracted genistein and genistein gel; (**B**) extracted genistein; and (**C**) genistein gel.

### *2.3. Characterisation of Gel Formulations 2.3. Characterisation of Gel Formulations*

### 2.3.1. General Characterisation 2.3.1. General Characterisation

The initial batches of the gel base were characterised for their consistency, pH, viscosity, and spreadability. Based on the study base formula, F1 was found to be difficult and not acceptable. The viscosity was also found to be quite high. In contrast, the F2 showed a slightly acidic nature and sticky content. The formulation F3 represented a nonsticky consistency, which favoured the ease of its application to the skin surface, and the pH was found to be neutral. Furthermore, the drug content study was performed to determine the content of the drug that was present in the optimised formulation. An optimised gel formulation was found to be a satisfactory drug content that ranges from 91 to 100% of keratin and genistein extract (Table 3). The initial batches of the gel base were characterised for their consistency, pH, viscosity, and spreadability. Based on the study base formula, F1 was found to be difficult and not acceptable. The viscosity was also found to be quite high. In contrast, the F2 showed a slightly acidic nature and sticky content. The formulation F3 represented a non-sticky consistency, which favoured the ease of its application to the skin surface, and the pH was found to be neutral. Furthermore, the drug content study was performed to determine the content of the drug that was present in the optimised formulation. An optimised gel formulation was found to be a satisfactory drug content that ranges from 91 to 100% of keratin and genistein extract (Table 3).

1978.7

2411.4

3040.3

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

**Viscosity (Cps) Mean ± SD** 

**Pre-Stability Post-Stability Pre-Stability Post-Stability Pre-Stability Post-Stability** 

433,342 ±

706,920 ±

700,446 ±

**Spreadability (gm.cm/s) Mean ± SD** 

9495.1 9.78 ± 1.0 8.4 ± 0.85

1126.8 14 ± 1.4 13.65 ± 0.63

6777.86 12.56 ± 0.92 10.83 ± 1.2

7.2 ± 0.2 6.5 ± 0.26 714,547.53 ±

2.3.2. High-Performance Thin Layer Chromatography (HPTLC)

**Table 3.** Post-stability characterisation of gel formulations.

**pH Mean ± SD** 


**Table 3.** Post-stability characterisation of gel formulations.
