*4.4. Characterisation of Genistein*

A pre-formulation study of the drug moiety was performed to determine the authenticity of the drug and to estimate its associative properties that may influence formulation development. As per the methods that were comprehensively described in the previous section, the λmax of the compound through UV-Vis spectroscopy, FT-IR, and HPTLC were performed.

### *4.5. Formulation Development (Gels)*

### 4.5.1. Optimisation of Gel Base

A lack of adequate activity of antioxidants, tissue injury, or healing of wounds may both be impaired. In-process quality control (IPQC) is a vital phase in the formulation of keratin protein wound-healing gel. In order to ensure that the final goods remain uniform from run to run, efficient over a period of time, and risk-free to be utilised, certain particular tests are carried out at different stages of the process of production. The first step that is performed is to verify the unprocessed products to determine if they agree with the standards that have already been established. As a result, the pH, viscosity, and amount of drug of the test formulation were examined. Certain distinct quality control criteria, such as texture profile, must be properly taken into account in the production of a gel (a semisolid formulation), with the aim of increasing stability, elegance, and subsequent approval from customers. The 1% gel formulation was prepared by taking 1 g of Carbopol 934, which was further added to 100 mL of distilled water and hydrolysed for duration of 24 h.

### 4.5.2. Formulation Development of 2% Keratin Gel

In total, 1 g of keratin was taken (powder form obtained through freeze-drying technique) and triturated with 3 mL of Cremophor RH-40 (as a solubiliser) in a mortar and pestle. Furthermore, 50 g of hydrated Carbopol 934 was added to the above content. The preservatives (propyl paraben and methyl paraben) were added to the required amount. The pH of the formulation was maintained by adding Triethanolamine to form the gel product (Table 7).

**Table 7.** Composition of keratin gel, genistein gel, and keratin–genistein gel (50 g quantity).


### 4.5.3. Formulation Development of 1% Genistein Gel

In total, 50 g of hydrated Carbopol 934 was taken, and 0.5 g of genistein was added. The content was mixed vigorously with a stirrer, and in required quantities, the preservatives (propyl paraben and methyl paraben) were added. The pH of the product was maintained by using triethanolamine (Table 7).

### 4.5.4. Formulation Development of Keratin–Genistein Combination Gel

In total, 0.5 g genistein was dissolved in 50 mL of hydrated Carbopol 934 (Sample 1). A total of 1 g keratin crystal was mixed with 3 mL of the solubiliser Cremophor RH-40 in a mortar and pestle (Sample 2). Sample 2 was added to sample 1, and preservatives (propyl paraben and methyl paraben) were added. The pH of the gel formulation was maintained by using Triethanolamine (Table 7).

### *4.6. Characterisation of the Gel Formulation*

### 4.6.1. Physical Evaluation

### Gel Strength

The gel strength was tested by the gel strength apparatus in accordance with the method. The 100 mL measuring cylinder was filled with the gel formulations, and then the gel was loaded with a 50 g piston. The gel strength was determined from the time (in seconds) required to move the piston 5 cm down through the gel. In this case, it took more than 5 min to drop the apparatus into the gel strength, and it was described by the minimum weights that pushed the apparatus 5 cm down through the gel.

### pH

The pH of the gel formulations was determined by utilising the digital type of pH meter (Global-pH-DPH-507, Delhi, India) in the calibrated range of 4 and 7. In total, 1 g of the gel was placed into double-distilled water, and both the reference electrode and the glass electrode were dipped completely into the formulations to achieve the pH of the products. The experiment was executed in a triplicate form.
