Design expert software v 8

The experiment was planned using Box-Behnken design (BBD) in Design Expert software v. 8.0.6 (Stat-Ease, Minneapolis, MN, USA). The ranges of design parameters (independent variables) were: glycerol concentration (X₁, 10–90%, w/w), temperature (X₂, 20–70°C), and drug weight (X₃, 0.6–1g) used for the extraction. TP content, TP/X₃ ratio (TPy), as well as the Gla and Iso content of the extracts were dependent variables. Response-surface methodology was used to find the relationship between dependent and independent variables. Experimental data was analyzed by multiple regression analysis and fitted to the appropriate polynomial models. The validity of the model was confirmed by the analysis of variance (ANOVA). p values < 0.1 were considered statistically significant.

Design-Expert software v. 8.0.6 (Stat-Ease, Minneapolis, MN, USA) was used for the experimental design preparation (Box–Behnken) and validation (ANOVA) of Box–Behnken results. For evaluation of antioxidant and enzyme inhibiting activity, the results were presented as the mean ± standard deviation of three measurements. IC₅₀ values were calculated using regression analysis. For wound healing assay, two independent experiments were performed, using three wells for each treatment. Statistical comparisons were made between the extracts using Students’ t-test (GraphPad Prism) and Dunnett’s post hoc test was used for comparison with the control. p-values < 0.05 were considered statistically significant.