Design expert software version 8

The ketalization reaction of the glycerol using AAC-CC was optimized using face centred composite design (FCCD) of RSM, Design-Expert software version 8.0 (STAT-EASE Inc., Minneapolis, USA) in terms of process parameters, the molar ratio (X1); time (X2); temperature (X3); catalyst amount (X4) to provide us with the maximum glycerol conversion (RSY) as a response (Y)^{50 (link)}. Software provided 30 no. of reactions consisting of 24 non-central and 6 central axial points (α = 1). Central (axial) points were defined for the authenticity of the model via pure error variance. After completion of each reaction, the samples were collected in microcentrifuge tubes and then centrifuged using a 5430 R centrifuge to remove the traces of catalyst present in the sample. The centrifuged samples were then diluted 100 times and were analyzed using high-performance liquid chromatography (HPLC—Agilent Technologies model 1260 Infinity and glycerol conversion was calculated using equation (Eq. 1), from the graph obtained from HPLC. $$\mathrm{Glycerol}\mathrm{Conversion}\left(\%\right)=\left[\frac{\mathrm{Peak}\mathrm{of}\mathrm{product}}{\mathrm{Peak}\mathrm{of}\mathrm{reactant}+\mathrm{Peak}\mathrm{of}\mathrm{product}}\right]\times 100$$

As an effective statistical method, RSM is usually used to generate the optimal experimental conditions, e.g., an optimization extraction process. According to the results of single-factor experiments, the liquid-solid ratio (A), ethanol concentration (B), extraction time (C) and ultrasound extraction temperature (D) were selected as independent variables for RSM, while the concentration of steroidal saponins was used as the response (dependent) variable (Y). The optimization was carried out through a three-level, four-factor Box–Behnken design (BBD) project consisting of 29 experimental runs including five replicates at the central point. The three different levels were set as −1 (low), 0 (medium) and +1 (high). The coded and actual values of the experimental factors for the BBD are shown in Table 7. The extraction procedure and determination methods were the same as those described above. Analysis of variance (ANOVA) was carried out to determine individual linear, quadratic and interaction regression coefficients using Design-Expert software version 8.0.6 (Stat-Ease, Inc.). The coefficient of determination (R²) was used to assess the fitness of the quadratic polynomial equation to the experimental responses, and the significance of the model and independent variables was evaluated by computing the F value at a p value < 0.05.

The Design-Expert software version 8.0.6 (Stat-Ease, Inc., Minneapolis, MN, USA) was used for the experimental design and data analysis of RSM. The results of the antioxidant assay were reported as mean ± SD of three replicates. Data were statistically analyzed by One-Way analysis of variance (ANOVA) procedure with SPSS software version 19.0 (IBM SPSS, Inc., Chicago, IL, USA), followed by the Duncan test. P-value of less than 0.05 was regarded as significance.

Selection of the extraction solvent was performed according to the IV-optimal mixture design, using Design-Expert software version 8.0.6 (Stat-Ease, Minneapolis, MN, USA). The independent variables were as follows (the content is expressed as the weight ratio, while the values in the brackets represent the minimum and the maximum values, respectively): PPG (0.100–0.890 g), water (0.100–0.890 g), and LA (0.010–0.100 g). The dependent variables were the phenolic compounds (oleuropein and total phenols). For the preparation of the extracts according to the optimal mixture design, 0.2 g of powdered olive leaf was added to 10 g of the solvent. The mixtures were stirred at 25 °C for 3 h at 300 rpm using a magnetic stirrer (2mag MIX 15 eco multi-position, Munich, Germany). After extraction, the mixture was filtered using folded filter papers (Filtrak, 80 g/cm³, grade 6). The remaining solutions were immediately used for the determination of phytochemical composition. Based on the maximum content of the selected dependent variables (TP and oleuropein content) in the prepared extracts, two different ternary solvent mixtures, solvent A (10% PPG, 89% water, 1% LA) and solvent B (28.6% PPG, 63.6% water, 7.8% LA), were chosen for further optimization of the extraction procedure, including extraction duration, technique, and optimal HM/SR.

The preparation of the two BBDs, regression analysis and optimization of the results was performed using Design Expert software version 8.0.6 (Stat-Ease, Minneapolis, MN, USA). The range of values for the three independent variables, as well as the corresponding codes, were chosen as presented in Table 1. TP was selected as the response (Table 2). Experimental data were fitted to a quadratic polynomial model as described in: ${\mathrm{Y}=\mathrm{A}}_0+{{\displaystyle \sum }}_{\mathrm{i}=1}^{\mathrm{k}}{\mathrm{A}}_{\mathrm{i}}{\mathrm{X}}_{\mathrm{i}}+{{\displaystyle \sum }}_{\mathrm{i}=1}^{\mathrm{k}}{\mathrm{A}}_{\mathrm{ii}}{\mathrm{X}}_{\mathrm{i}}^2+{{\displaystyle \sum }}_{\mathrm{i}=1}^{\mathrm{k}-1}\times {{\displaystyle \sum }}_{\mathrm{j}=1+1}^{\mathrm{k}}{\mathrm{A}}_{\mathrm{ij}}{\mathrm{X}}_{\mathrm{i}}{\mathrm{X}}_{\mathrm{j}},$
where Y is the dependent variable; A₀, A_i, A_ii, and A_ij are the regression coefficients for intercept, linearity, square and interaction, respectively; Xi and Xj are the independent variables.