Is also governed by the equilibrium concentrations achieved before their corresponding apparent reductions and rapid degradations [24]. The optimum extraction time for phenolic compounds and flavonoids, perhaps due to similar degrees of polymerization and solubilities of common phenolic flavonoids, required as much timeTable 2. Regression models fitted to the experimental data of response variables.Response TPC (mg GAE/g DW) TFC (mg RE/g DW) DPPH radical scavenging capability ( )Model equationa Y = 15.013364+4.513759X1+0.923033X2+2.182736X323.334297X1223.004297X2222.129297X3222.185563X1X3 Y = 41.384322+18.636959X1+3.438491X2+3.676343X3211.249728X1227.534728X2229.244728X32 Y = 83.864603+10.942177X1+3.247395 X 2+8.151488X3215.882841X1228.517841X22211.097841X32210.931425X1XProbability of lack of fit 0.1597 0.1040 0.1594 0.R0.9937b 0.9877b 0.9822b 0.9729bABTS radical scavenging Y = 90.535606+11.522496X1+3.896676X2+7.59029X3212.494807X1225.484807X2224.584807X3227.517612X2X32 capability ( ) 17.Title Loaded From File 610938X1X326.433863X1Xa X1, EtOH ( ); X2, Time (min); X3, T (uC). Coded values. b P,0.001. doi:10.1371/journal.pone.0068392.tExtraction of Antioxidants from C. cyrtophyllumFigure 2. Response surface plots of TPCs of C. cyrtophyllum leaf extracts as affected by ethanol concentration, temperature, and time in UAE. (A) Ethanol Title Loaded From File concentration and time (temperature 60uC); (B) ethanol concentration and temperature (time 80 min); (C) temperature and time (40 ethanol). doi:10.1371/journal.pone.0068392.gand antioxidant capability, an optimal temperature of 60uC, was used for RSM optimization.Optimization 23148522 by RSMCentral composite rotatable design (CCRD) was used to further optimize the extraction conditions with respect to the concentration of antioxidant compounds in C. cyrtophyllum leaf extracts. An ethanol concentration of 40 (v/v), an extraction time of 80 min, and extraction temperature 18055761 of 60uC were chosen from previous single-factor experiments. The response values of TPC, TFC, DPPH, and ABTS radical-scavenging of extracts obtained undervarious experimental conditions are shown in Table 1. Maximum recovery of TPC (16.260.2 mg GAE/g DW) and TFC (48.161.5 mg RE/g DW) was recorded during Run No. 8, and maximum radical-scavenging capacity of DPPH (85.660.7 ) and ABTS (91.860.5 ) were recorded during Run No. 18. The lowest TPC (6.660.1 mg GAE/g DW), DPPH (54.061.0 ) and ABTS (55.961.8 ) radical-scavenging capacities were observed in Run No. 1. The lowest TFC (12.760.6 mg RE/g DW) was detected at Run No. 10.Table 3. Predicted and experimental values of response variables under optimal conditions.ResponsesOptimum extraction conditions EtOH ( ) Time (min) 85.4 82.9 85.1 81.3 T (6C) 63.3 63.0 63.9 63.Maximum value Experimentala 16.860.2 49.360.4 86.860.3 92.960.5 Predicted 16.7 49.4 86.4 93.TPC(mg GAE/g DW) TFC(mg RE/g DW) DPPH radical scavenging ability ( ) ABTS radical scavenging ability ( )a Responses are the means 6 SD (n = 3). doi:10.1371/journal.pone.0068392.t60.9 67.7 48.8 50.Extraction of Antioxidants from C. cyrtophyllumTable 4. Correlation between response variables under different extraction conditions.rEtOH ( ) TPC TFCaTime (min) DPPH TPC 0.6031 0.8763b 0.b c NST (6C)TFC DPPH TPC 0.8329 0.5413NS 0.NS NS cTFCDPPHTFC DPPH ABTS0.0.7537c 0.c0.2258NS 0.7617 0.c0.9375a 0.5449 0.NS0.8101c 0.3599NS 0.3121NSa P,0.005, bP,0.01, cP,0.05; NS: non-significant; r: correlation coefficient. doi:10.1371/journal.pone.0068392.tFitting the modelMultiple regression analysis.Is also governed by the equilibrium concentrations achieved before their corresponding apparent reductions and rapid degradations [24]. The optimum extraction time for phenolic compounds and flavonoids, perhaps due to similar degrees of polymerization and solubilities of common phenolic flavonoids, required as much timeTable 2. Regression models fitted to the experimental data of response variables.Response TPC (mg GAE/g DW) TFC (mg RE/g DW) DPPH radical scavenging capability ( )Model equationa Y = 15.013364+4.513759X1+0.923033X2+2.182736X323.334297X1223.004297X2222.129297X3222.185563X1X3 Y = 41.384322+18.636959X1+3.438491X2+3.676343X3211.249728X1227.534728X2229.244728X32 Y = 83.864603+10.942177X1+3.247395 X 2+8.151488X3215.882841X1228.517841X22211.097841X32210.931425X1XProbability of lack of fit 0.1597 0.1040 0.1594 0.R0.9937b 0.9877b 0.9822b 0.9729bABTS radical scavenging Y = 90.535606+11.522496X1+3.896676X2+7.59029X3212.494807X1225.484807X2224.584807X3227.517612X2X32 capability ( ) 17.610938X1X326.433863X1Xa X1, EtOH ( ); X2, Time (min); X3, T (uC). Coded values. b P,0.001. doi:10.1371/journal.pone.0068392.tExtraction of Antioxidants from C. cyrtophyllumFigure 2. Response surface plots of TPCs of C. cyrtophyllum leaf extracts as affected by ethanol concentration, temperature, and time in UAE. (A) Ethanol concentration and time (temperature 60uC); (B) ethanol concentration and temperature (time 80 min); (C) temperature and time (40 ethanol). doi:10.1371/journal.pone.0068392.gand antioxidant capability, an optimal temperature of 60uC, was used for RSM optimization.Optimization 23148522 by RSMCentral composite rotatable design (CCRD) was used to further optimize the extraction conditions with respect to the concentration of antioxidant compounds in C. cyrtophyllum leaf extracts. An ethanol concentration of 40 (v/v), an extraction time of 80 min, and extraction temperature 18055761 of 60uC were chosen from previous single-factor experiments. The response values of TPC, TFC, DPPH, and ABTS radical-scavenging of extracts obtained undervarious experimental conditions are shown in Table 1. Maximum recovery of TPC (16.260.2 mg GAE/g DW) and TFC (48.161.5 mg RE/g DW) was recorded during Run No. 8, and maximum radical-scavenging capacity of DPPH (85.660.7 ) and ABTS (91.860.5 ) were recorded during Run No. 18. The lowest TPC (6.660.1 mg GAE/g DW), DPPH (54.061.0 ) and ABTS (55.961.8 ) radical-scavenging capacities were observed in Run No. 1. The lowest TFC (12.760.6 mg RE/g DW) was detected at Run No. 10.Table 3. Predicted and experimental values of response variables under optimal conditions.ResponsesOptimum extraction conditions EtOH ( ) Time (min) 85.4 82.9 85.1 81.3 T (6C) 63.3 63.0 63.9 63.Maximum value Experimentala 16.860.2 49.360.4 86.860.3 92.960.5 Predicted 16.7 49.4 86.4 93.TPC(mg GAE/g DW) TFC(mg RE/g DW) DPPH radical scavenging ability ( ) ABTS radical scavenging ability ( )a Responses are the means 6 SD (n = 3). doi:10.1371/journal.pone.0068392.t60.9 67.7 48.8 50.Extraction of Antioxidants from C. cyrtophyllumTable 4. Correlation between response variables under different extraction conditions.rEtOH ( ) TPC TFCaTime (min) DPPH TPC 0.6031 0.8763b 0.b c NST (6C)TFC DPPH TPC 0.8329 0.5413NS 0.NS NS cTFCDPPHTFC DPPH ABTS0.0.7537c 0.c0.2258NS 0.7617 0.c0.9375a 0.5449 0.NS0.8101c 0.3599NS 0.3121NSa P,0.005, bP,0.01, cP,0.05; NS: non-significant; r: correlation coefficient. doi:10.1371/journal.pone.0068392.tFitting the modelMultiple regression analysis.
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