2.3.4. Antitryptic Activity (ATA)

This method is also known to have anti-arthritic activity in vitro. The analysis was carried out using the method described by Oyedapo and Femurewa [16], with minor modifications as described by Manolov et al. [15].

Trypsin, 2 mL 0.06 mg/mL, 1 mL Tris-HCl buffer (20 mM, pH 7.4), and 1 mL test sample/standard of various concentrations (20–1000 μg/mL) were added to the reaction mixture. For 5 min, the mixture was incubated at 37 ◦C. Following that, 1 mL of human albumin (4% *v*/*v*) was added. The mixture was then incubated for an additional 20 min. To stop the reaction, 2 mL of 70% perchloric acid was added to the mixture. The cloudy suspension was cooled and centrifuged for 20 min at 5000 rpm. A spectrophotometer (Camspec M508, Leeds, UK) was used to measure the absorbance of the supernatant at 280 nm in comparison to the control solution. The control solution consisted of various concentrations of sample/standard in methanol. As a baseline, ibuprofen was used. The analysis was carried out three times. By comparing the samples to a blank sample, the percentage of antitryptic activity (ATA) was determined. The blank sample was prepared in the same way as the test sample, with the exception that perchloric acid was added before the albumin.

$$\%ATA = \left[\frac{A\_{blank} - (A\_{TS} - A\_{CS})}{A\_{blank}}\right] \ast 100\tag{2}$$

where *Ablank* is the absorbance of the blank sample, *ACS* is the absorbance of the control solution (test sample in different concentrations), and *ATS* is the absorbance of the test samples. The mean IC50 values were estimated by means of interpolating the graphical dependence of ATA on concentration.
