TTPC from leaf extracts was measured according to the FolinCiocalteu (FC) procedure [17] as described with some modifications. The FC phenol reagent was prepared according to King’s method [18]. Thus, 10 g K162 biological activity sodium tungstate and 2.5 g sodium molybdate were gently dissolved in 70 mL deionized water, 5 ml 85 phosphoric acid, and 10 mL concentrated hydrochloric acid were subsequently added and allowed to reflux for 10 hr. Then, 1.5 g lithium sulfate and 6 mL hydrogen peroxide were added and refluxed for another 15 min until the color changed to a glassyExtraction of Antioxidants from C. cyrtophyllumRSM experimental designA five-level, three-variable central composite rotatable design [21] was developed to determine the best combinations of extraction conditions for TPC from C. cyrtophyllum leaves. Three 223488-57-1 independent variables selected were ethanol concentration, extraction temperature, and extraction time. From single-factor experiments, 11967625 the range for each independent variable was preliminarily determined and later used in subsequent experiments to test additional independent variables. Eight factorial points were used, six axial points (two axial points on the axis of each design variable at a distance of 1.68 from the design center), and four center points leading to 18 experimental runs. The actual and coded levels of the independent variables used in the experimental design are shown in Table 1. Yield values of TPC, TFC, DPPH and ABTS radical-scavenging capabilities of C. cyrtophyllum leaf extract were evaluated with multiple linear regression to fit the following empiric second-order polynomial model.k X j 1 k X jStatistical analysisResults were expressed as mean 6 standard deviation of replicate solvent extractions and triplicate of assays and analyzed by Statistical Analysis System (SAS, version 9.1). Data were analyzed by ANOVA (p,0.05). Optimal extraction conditions were estimated through three-dimensional response surface 18055761 analyses of the three independent variables and each response variable.Results and Discussion Single-factor experimentsFirst, we investigated whether ethanol concentration, extraction temperature, and time could be optimized for phenolic and flavonoid antioxidant extraction using single-factor experiments to determine appropriate experimental ranges for subsequent analyses.Effects of ethanol concentrationY b0 z bj Xj z bjj Xj2 z XX bij Xi Xjivjwhere Y represents the response function; b0 is an intercept andbi, bii, andbij are the regression coefficients of the linear, quadratic, and interactive terms, respectively; accordingly Xi, Xi2, and XiXj represent the coded independent variables, respectively; k is the number of variables.Ethanol concentrations of 0 , 20 , 40 , 60 , 80 and 100 have been used extraction. Fig. 1A depicts of phenolic yield extracted (TPC and TFC) and the antioxidant capacities of those agents (against ABTS and DPPH radicals), and shows that both were greatly influenced by ethanol concentration. TPC and TFC recovery was parabolic with a maximum value at 60 ethanol. This was followed by a considerable decline with greater concentrations of ethanol. ABTS radical-scavenging capacities initially increased and peaked at 40 ethanol, then decreased considerably. DPPH radical-scavenging capacity declined with further increases in ethanol after the maximum values, 40 and 80 ethanol, respectively, were reached.Table 1. Five-level, three-independent variable central composite rotatable design and e.TTPC from leaf extracts was measured according to the FolinCiocalteu (FC) procedure [17] as described with some modifications. The FC phenol reagent was prepared according to King’s method [18]. Thus, 10 g sodium tungstate and 2.5 g sodium molybdate were gently dissolved in 70 mL deionized water, 5 ml 85 phosphoric acid, and 10 mL concentrated hydrochloric acid were subsequently added and allowed to reflux for 10 hr. Then, 1.5 g lithium sulfate and 6 mL hydrogen peroxide were added and refluxed for another 15 min until the color changed to a glassyExtraction of Antioxidants from C. cyrtophyllumRSM experimental designA five-level, three-variable central composite rotatable design [21] was developed to determine the best combinations of extraction conditions for TPC from C. cyrtophyllum leaves. Three independent variables selected were ethanol concentration, extraction temperature, and extraction time. From single-factor experiments, 11967625 the range for each independent variable was preliminarily determined and later used in subsequent experiments to test additional independent variables. Eight factorial points were used, six axial points (two axial points on the axis of each design variable at a distance of 1.68 from the design center), and four center points leading to 18 experimental runs. The actual and coded levels of the independent variables used in the experimental design are shown in Table 1. Yield values of TPC, TFC, DPPH and ABTS radical-scavenging capabilities of C. cyrtophyllum leaf extract were evaluated with multiple linear regression to fit the following empiric second-order polynomial model.k X j 1 k X jStatistical analysisResults were expressed as mean 6 standard deviation of replicate solvent extractions and triplicate of assays and analyzed by Statistical Analysis System (SAS, version 9.1). Data were analyzed by ANOVA (p,0.05). Optimal extraction conditions were estimated through three-dimensional response surface 18055761 analyses of the three independent variables and each response variable.Results and Discussion Single-factor experimentsFirst, we investigated whether ethanol concentration, extraction temperature, and time could be optimized for phenolic and flavonoid antioxidant extraction using single-factor experiments to determine appropriate experimental ranges for subsequent analyses.Effects of ethanol concentrationY b0 z bj Xj z bjj Xj2 z XX bij Xi Xjivjwhere Y represents the response function; b0 is an intercept andbi, bii, andbij are the regression coefficients of the linear, quadratic, and interactive terms, respectively; accordingly Xi, Xi2, and XiXj represent the coded independent variables, respectively; k is the number of variables.Ethanol concentrations of 0 , 20 , 40 , 60 , 80 and 100 have been used extraction. Fig. 1A depicts of phenolic yield extracted (TPC and TFC) and the antioxidant capacities of those agents (against ABTS and DPPH radicals), and shows that both were greatly influenced by ethanol concentration. TPC and TFC recovery was parabolic with a maximum value at 60 ethanol. This was followed by a considerable decline with greater concentrations of ethanol. ABTS radical-scavenging capacities initially increased and peaked at 40 ethanol, then decreased considerably. DPPH radical-scavenging capacity declined with further increases in ethanol after the maximum values, 40 and 80 ethanol, respectively, were reached.Table 1. Five-level, three-independent variable central composite rotatable design and e.