Viscosity

The viscosity of the products was determined by using the Brookfield Viscometer (DV-E1 model, Middleboro, MA, USA) at 0.5 rpm and using the no. 64 spindle in the temperature range of 25 ± 1 ◦C. The experiment was carried out in a triplicate manner.

### Spreadability

On the principle of slip–drag, the spreadability of the gel formulations was determined. The required amount of the formulation was placed on the ground slide, and the test material was prepared such that it was sandwiched by another glass slide which comprised

a hook system. A pressure of 1 kg of mass was applied to the slides to remove the air entrapped between the thin films. The excess content of the formulation was swiped off from the edges of the slide, and the top slide was dragged with a force equivalent to 50 g intensity. The time required for the top slide to cover 7.5 cm distance was estimated and concluded from the following formula:

$$\mathbf{S} = \mathbf{M} \times \mathbf{L} / \mathbf{T}$$

where S = Spreadability coefficient, M = weight applied, L = length moved by the glass slides, and T = time taken to separate the glass slides completely from each other [38].

### 4.6.2. Keratin and Genistein Contents

In each formulation, the drug content was estimated by taking 0.1 mL of sample in a 10 mL volumetric flask and making up the volume with double-distilled water to prepare a stock solution. Further dilutions were prepared and analysed spectrophotometrically at λmax 222 nm and 280 nm, respectively. The experiment was performed in a triplicate manner.

### 4.6.3. HPTLC

A previously discussed method, as stated above, with the mobile phase in butanol: acetic acid: water in the ratio of 4:1:1 *v*/*v*/*v* for keratin gel and chloroform: methanol in the ratio of 10:1 *v/v* for genistein gel was followed.

### 4.6.4. Short-Term Stability Study

The formulation was subjected to accelerated conditions of temperature (40 ± 2 ◦C) and humidity (75 ± 5%) for the duration of 90 days. The formulations were packed in aluminium foil and kept inside the PVC bottle. After the completion of the study, the essential physical parameters and drug content were determined at pre-determined intervals of 30 days, 60 days, and, finally, 90 days. The obtained observations/parameters were then compared with the pharmacopoeia guidelines.

### *4.7. Animals*

Male albino rats [(n = 30) (age: 5 to 6 weeks; weight range: 200–300g)] were randomly divided into five groups. There were four hydrogel-treated experimental groups, including (1) feather keratin hydrogel, (2) genistein hydrogel, (3) keratin–genistein hydrogel, and (4) marketed preparation, while the fifth group was a non-treated control group (six rats in each group) after obtaining animal study permission from the institutional animal ethical committee (IAEC) as per the Committee for Control and Supervision of Experiments on Animals (CCSEA) guidelines and histopathological studies with approval numbers 1426/PO/Re/S/11/CPCSEA. The animals were kept in the animal house, provided free access to water, also fed standard food, and maintained under a controlled environment (temperature 22 ± 2 ◦C; humidity 50–60% RH; and 12/12 h light and dark conditions) with proper hygiene. All animals were housed under standard environmental conditions to prevent any significant antibacterial interference with the wound due to external factors [39–41].

### *4.8. Wound Healing Study*

For evaluating the wound-healing properties of the feather keratin, genistein, and keratin–genistein hydrogel, and marketed preparation, a full-thickness excision model was applied for the evaluation. All animals were anaesthetised with an intra-peritoneal ketamine injection (100 mg/mL) and xylazine, ilium-xylazine-20 (20 mg/mL). For anaesthetic administration, 7.5 mL of ketamine and 5 mL of xylazine were diluted with 7.5 mL of double-distilled water. A dosage of 0.2 mL/100 g body weight was used for the induction of complete anaesthesia. The dorsal hair was removed with a hair-removing cream (Anne French, Ahmedabad, Gujarat, India). A partial-thickness skin wound of dimension 1.5 × 1.5 cm was prepared via excision of the dorsal skin of the animal by using surgical

scissors and forceps. Subsequently, the excised wound of the experimental group of rats was covered with the tested hydrogel (applied 1 fingertip unit (FTU)), and the bare wound was kept as a negative control. The probable changes observed in the wound area with time progression were measured by using a method of transparency-based digital imaging. On the 7th and 14th postoperative days, the reconstructed skins of the wounds were excised and fixed in 4% paraformaldehyde for histological observations and collagen deposition determination. At the selected post-wound intervals, photographs were taken. The wound closure was estimated according to the following equation [42,43]:

$$\text{Wound closure } (\%) = [\text{A}\_0 - \text{A}\_t] / \text{A}\_0 \times 100$$

where A<sup>0</sup> is the initial wound area and at is the wound area at the same time interval of "t" days.
