5.3.3. Homogeneity

Visual inspection was used to verify the homogeneity of the formed gel formulation. They were examined for the existence of aggregates and the appearance of aggregates [23].

### 5.3.4. Spreadability

The therapeutic effectiveness of the formulations was also determined by its spreadability. It was expressed in terms of the time it takes two slides to slip off the gel that is put between the slides, under the direction of a certain load. It was calculated using the following formula: S = M\*L/t, where M denotes the weight attached to the upper slide, L denotes the length of the glass slide, and T denotes the time required to separate the slides [24,25].

### 5.3.5. Drug Content

A specified amount of formulated gel was dissolved in 100 mL of pH 6.8 phosphate buffer. This solution was filtered and spectrophotometrically estimated at 287.0 nm with phosphate buffer (pH 6.8) as a blank.

### 5.3.6. Stability Study

As per the I.C.H. guidelines, the shelf-life of the formulation was determined and any incompatibility within the formulation was identified as changes in appearance and drug content of the stored mucoadhesive gel, which was observed after 1, 2, 3, 4, 5 and 6 months [26].

### 5.3.7. Drug Release Study by Open-Ended Cylinder

A glass cylinder measuring 10 cm in height, 3.7 cm in diameter, and 3.1 cm in diameter with both ends open were used. A glycerol-soaked cellophane membrane was attached to one end of the cylinder. It kept 5 g of the gel under investigation. At the receptor compartment, a beaker containing 100 mL of 6.8 pH buffer solution was used. The sample was submerged to a depth sufficient to keep it below the medium's surface in the receptor compartment. While at 37 ◦C, the medium in the compartment was agitated with a magnetic stirrer. Every 10 min, 5 mL of the sample was removed and measured at 287 nm for 3 h. Every time, the volume extracted was replaced by an equivalent amount of medium [27]. The investigations were carried out in triplicate, and the standard deviation (SD) was determined.

### 5.3.8. Drug Release Study by Diffusion Cell Apparatus

Phosphate buffer was used as the dissolution medium (pH 6.8). The membrane (DURAPORE HVLP 45 µm) was positioned over the receptor compartment. The receptor compartment was packed with 12 mL of dissolution medium and held at 37 ◦C. The dissolution medium was stirred with a magnetic stirrer at 50 rpm. At various intervals, 2 mL aliquots were removed and replaced with an equal volume of receptor medium [28]. The Electrolab diffusion cell apparatus was used for this study. The trials were carried out in triplicate, and the standard deviation (SD) was estimated.

### 5.3.9. Release Kinetics

Dissolution and release of drugs are important phenomena for solid dosage forms such as tablets, capsules, and semisolid dosage forms such as creams, ointments, and implants, which deliver the drugs over the intended period ranging from hours to weeks and years. It is also applicable to the design and optimization of all kinds of modified release dosage forms such as sustained, delayed, controlled release dosage forms and novel drug delivery systems. For the evaluation of *in vitro* release kinetics to investigate the mechanism of release, the data were analyzed with the following mathematical models [29].

To investigate the mechanism of release, the data were analyzed with the following mathematical models: zero-order kinetic (Equation (1)), first-order kinetic (Equation (2)) and Higuchi kinetic (Equation (3)) Korsmeyer-Peppas model (Equation (4)), and Hixson-Crowell cube root law (Equation (5)) [30,31].

$$\mathbf{Q}\_{\mathbf{t}} = \mathbf{K}\_{\mathbf{0}} \mathbf{t} \tag{1}$$

$$
\ln \mathbf{Q}\_t = \ln \mathbf{Q}\_0 - \mathbf{K}\_1 \mathbf{t} \tag{2}
$$

$$\mathbf{Q}\_{\mathbf{l}} = \mathbf{K}\_{\mathbf{h}} \mathbf{t}^{1/2} \tag{3}$$

$$\mathbf{M}\mathbf{t}/\mathbf{M}\boldsymbol{\alpha} = \mathbf{K}\_{\mathbf{P}}\mathbf{t}^{\mathbf{n}} \tag{4}$$

$$\mathbf{Q\_0}^{1/3} - \mathbf{Q\_t}^{1/3} = \mathbf{K\_{HC}} \mathbf{t} \tag{5}$$

### *5.4. In Vitro Antioxidant Studies for Prepared Gel*

### 5.4.1. 2,2-Diphenylpicrylhydrazyl (DPPH) Free Radical Scavenging Test

Various concentrations of standard ascorbic acid and samples, namely 100, 200, 400, 800 and 1000 µg/mL, were prepared in distilled water. An equal volume of different concentrations of standards and DPPH were mixed separately in a clean and labelled tube and the tubes were incubated at room temperature in the dark for 30 min. The absorbance was measured at 517 nm using a UV-VIS spectrophotometer [32]. The percentage inhibition was calculated using the formula:

$$\text{Percentage inhibition} = \text{(Abs control -- Abs sample)} / \text{Abs control} \times 100$$

### 5.4.2. Nitric Oxide Scavenging Assay

A 3 mL reaction mixture containing sodium nitroprusside (10 mM in phosphatebuffered saline) and various concentrations of standard ascorbic acid and samples, namely 100, 200, 400, 800 and 1000 µg/mL, was prepared in distilled water and kept in incubated at 37 ◦C for 4 h. To the incubation solution, 0.5 mL of Griess reagent was added and the absorbance was read at 546 nm [33]. The percentage inhibition was calculated using the formula:

Percentage inhibition = (Abs control − Abs sample)/Abs control × 100
