2.5. Hyaluronidase Inhibition

The enzymatic assay for the inhibition of hyaluronidase was conducted as described by Algul et al. [28], with some modifications. The plant extracts were diluted to DMSO to a final concentration of 0.30 mg/mL. The inhibition activity was calculated inversely proportional of the production of N-acetyl-D-glucosamine. Values of 100 μL of acetate buffer (0.1 M NaCl, pH 3.5), 150 μL of tested sample and 50 μL of hyaluronidase solution 1% w/v (dissolved in acetate buffer) were added in eppendorfs. Afterwards, 100 μL of BSA solution 0.2% w/v (dissolved in ddH₂O) was added in each eppendorf and incubated for 20 min at 37 °C. Then, 50 μL of hyaluronic acid solution 0.5% w/v (dissolved in ddH₂O) was added and incubated for 60 min at 37 °C; 45 μL from each eppendorf was transferred into new eppendorfs containing 10 μL of sodium tetraborate solution 0.8 M (dissolved in ddH₂O), and heated for 3 min at 100 °C and cooled down on ice. In each tube 300 μL of dimethylaminobenzaldehyde (DMAB) solution was added (10% w/v dissolved in 10 N HCl and then dissolved 10 times in acetic acid glacial) and incubated for 20 min at 37 °C. Finally, 200 μL from the last tube was transferred into a 96-well microplate and measured at 586 nm in the Infinite 200 PRO series plate reader (Tecan GmbH, Crailsheim, Germany). Tannic acid was used as positive control and DMSO instead of sample for negative control. Experiments were performed in triplicate.

The activity was calculated depending on the change in absorption using the following equation: Activity (%) = [A − (B − C)]/A ∗ 100,where A is the absorbance of the negative control, B is the absorbance of the tested sample and C is the absorbance of zero value.