*4.1. Material*

Keratin was successfully extracted from chicken feathers based on an existing method. Genistein was purchased from a local shop, namely, Green Heaven Lab Ltd., Nagpur, Maharashtra, India. Carbopol 934 was procured from Apex Drug House Ltd., Mumbai, India. LOBA Chemie Ltd., Mumbai, India, supplied analytical-grade methyl paraben and propyl paraben. Cremophor RH-40 was obtained from PIOMA Chemicals, Mumbai, India. The chemicals that were used in the study were of analytical grade and obtained in the highest pure form. Double-distilled water (Borosil, Mumbai, Maharashtra, India) was utilised for the experiment. The standard keratin was obtained from Sigma-Aldrich, St. Louis, MO, USA, with CAS No. 69430-36-0.

### *4.2. Extraction of Keratin from Chicken Feathers*

Chicken feathers of a sufficient quantity were obtained from a nearby poultry farm and soaked in ether for a period of 24 h. The wet feathers were exposed to sunlight until they were completely dried. The dried feathers were further cut uniformly into small pieces and blended. The blended feathers were sealed carefully in a sealed plastic bag. In a 2 L conical flask, 0.5 M of sodium sulphide solution was prepared, and the weighed amounts of 50 g (Shimadzu BL-220H, Kyoto, Japan) of blended chicken feathers were added. The

solution was stirred by a mechanical stirrer (Biotechniques BIPO5B, India) for the duration of 6 hrs at a temperature of 30 ◦C and the target pH range of 10–12. Furthermore, the solution was filtered off to acquire the supernatant liquid. The supernatant liquid was taken into a beaker and stirred. In a conical flask, ammonium sulphate solution (0.7 g/mL) was prepared in double-distilled water with continuous stirring until all particles were dissolved [32,33]. The solution was subsequently filtered, and the obtained filtrate was added drop wise to the previous breaker containing the feather filtrate solution in a ratio of 1:1 with fast stirring. The supernatant precipitates were collected separately. The collected solid particles were added to ethanol (100 mL), and a pH of 12 was maintained with a NaOH solution (0.1 N) with continuous stirring. Additionally, the solution was placed overnight on a mechanical shaker. Finally, the solution was filtered to procure the solid particles and the supernatant liquid. The solid particles were dissolved in 0.5 N NaOH solution. Ultimately, the liquid was collected, and the solid materials were discarded. The liquid sample was then dried, and the keratin crystals were collected [22].

### *4.3. Characterisation of Extracted Keratin*

### 4.3.1. Protein Content

The protein content of the keratin sample was estimated via the Kjeldahl digestion flask method, as reported in the standard literature [34]. Protein content was determined by multiplying nitrogen contents by conventional factor 6.25, as the average nitrogen (N) content of most of the proteins was found to be about 16%. The % total nitrogen content in the sample was determined from the following formula [32]:

% Total Nitrogen = (Blank titration−Sample titration) × 1.4 (constant factor for Nitrogen) × 2 × Exact normality of 0.1N NaOH solution

### 4.3.2. Amino Acid Analysis

The amino acids present in the feather keratin were estimated quantitatively by using the post-column derivatisation method with Ninhydrin applications followed by an amino acid analyser. By using 6 N HCl, the keratin samples (n = 4) were hydrolysed in a glass tube. The hydrolysed amino acids were then characterised via reverse-phase HPLC (RP-HPLC) [35].

### 4.3.3. Scanning Electron Microscopy (SEM)

The morphology of the extracted keratin sample was characterised by electron microscopy (Jeol JSM-6360A, Japan). The dried sample was sprinkled lightly over the aluminium stub (3–5 nm; 75 s; 40 W) connected with double adhesive tape. The sample underwent a gold coating to enhance the electrical conductance. The stub containing the gold-coated sample was scanned randomly, and at 10 kV acceleration voltage, the photomicrographs were recorded [32].

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

The Fourier-transform infrared (FT-IR) spectroscopic (Shimadzu IR-Affinity-1, Kyoto, Japan) investigation of keratin, genistein, Carbopol 934, and a physical mixture in the range of 4000 cm−<sup>1</sup> to 400 cm−<sup>1</sup> was studied to determine the inherent stability and compatibility of the formulation through various chemical groups. Any such changes perceived through the spectra are a clear reflection of some possible interactions between the drug and the polymer [36].

4.3.5. High-Performance Thin Layer Chromatography (HPTLC)

### (a) For Keratin

High-performance thin-layer chromatography (HPTLC) (Camag, North Carolina, USA) was performed for the keratin samples on 10 cm × 10 cm F<sup>254</sup> HPTLC Si-60, diol, CN, NH2, and RP-18 plates. The sample and standard were spotted as bands of 5 mm width by

using the automatic applicator Desaga AS 30 (Heidelberg, Germany). The samples were applied in the triplicate manner in the form of a band of 6 mm in the concentration of 10 µL. The distance between the tracks was kept to 10 mm at an application rate of 110 nL/s for the application of the bands. The plates were developed in a linear ascending mode at 85 mm in a chromatographic horizontal Teflon DS chamber (Chromdes, Lublin, Poland) that was previously saturated with some vapours of the mobile phase of butanol: acetic acid: water in the ratio of 4:1:1 *v*/*v*/*v*. The plates were developed after drying in the warm air streams and observed at 254 nm and 366 nm under a UV lamp. Densitometric scanning was performed by using the TLC scanner-III in the pre-absorbance/reflectance mode [37].

### (b) For Genistein

In the present study, 10 cm × 10 cm F<sup>254</sup> HPTLC Si 60, diol, and CN, NH2, and RP-18 plates were used for thin-layer chromatography (TLC). A Desaga AS 30 automatic applicator (Heidelberg, Germany) was used to identify standards and samples within 5 mm bands. In a chromatographic horizontal Teflon DS chamber (Chromdes, Lublin, Poland), which had been earlier saturated with mobile phase vapours, the plates were developed to an 85 mm thickness. The plates were examined at 254 nm and 366 nm under a UV light after being dried in a stream of hot air. On pre-coated TLC Plates (10 cm × 10 cm) and by using a concentration of 10 µL, the samples were used in triplicates in the shape of a band with a width of 6 mm via a Linomat-V sample applicator. A 110 nL/s rate of application was used to apply bands while maintaining a 10 mm space among the tracks. In a glass chamber with twin troughs that had been saturated with mobile phase, linear ascending progression was performed. By utilising the TLC scanner III in the pre-absorbance/reflectance mode, densitometric scanning was carried out. HPTLC-plates were used to hold the commercial and enzymatic genistein hydrolysates. Chloroform: methanol (10:1 *v*/*v*) was used for the development as long as the solvent front travelled to the highest point of the plate [33,37].
