Xperimental data for response variables for optimization of C. cyrtophyllum leaf extracts.RunProcess variables ?real and (coded) valuesResponsesa DPPH radical- scavenging capacity ( ) 54.061.0 72.961.2 61.060.9 1317923 76.461.3 76.560.7 78.760.2 79.960.4 81.760.5 77.260.5 55.961.2 76.660.6 71.260.9 79.560.5 63.161.7 82.860.3 83.861.1 83.561.1 85.660.7 ABTS radical- scavenging capacity ( ) 55.961.8 87.662.7 73.263.0 88.061.5 89.260.7 90.062.0 91.161.3 87.960.5 89.660.7 66.160.6 89.060.2 80.761.1 90.961.6 80.661.3 89.460.8 91.060.7 90.161.0 91.860.X1, EtOH ( )1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17aX2, Time (min)60 (21) 60 (21) 100 (1) 100 (1) 60 (21) 60 (21) 100 (1) 100 (1) 80 (0) 80 (0) 114 (1.68) 46 (21.68) 80 (0) 80 (0) 80 (0) 80 (0) 80 (0) 80 (0)TPC (mg GAE/g X3, T (6C) DW) TFC (mg RE/ 50 (21) 70 (1) 50 (21) 70 (1) 50 (21) 70 11967625 (1) 50 (21) 70 (1) 60 (0) 60 (0) 60 (0) 60 (0) 77 (1.68) 6.660.1 11.060.1 7.860.1 12.260.3 13.660.3 15.060.6 14.860.5 16.260.2 15.960.5 7.360.3 12.760.6 11.260.2 14.660.5 14.360.2 20.360.4 21.860.8 25.260.7 41.560.4 44.060.3 42.560.8 48.161.5 46.160.2 12.760.6 36.260.6 30.060.5 35.060.6 27.860.4 42.860.3 40.760.6 41.460.5 40.860.20 (21) 20 (21) 20 (21) 20 (21) 60 (1) 60 (1) 60 (1) 60 (1) 74 (1.68) 6 (21.68) 40 (0) 40 (0) 40 (0) 40 (0) 40 (0) 40 (0) 40 (0) 40 (0)43 (21.68) 11.060.1 60 (0) 60 (0) 60 (0) 60 (0) 14.860.8 15.360.3 15.060.6 15.060.Responses are the means 6 SD (n = 3). doi:10.1371/journal.pone.0068392.tExtraction of Antioxidants from C. cyrtophyllumFigure 1. Effect of (A) FCCP chemical information ethanol concentration (extraction time 60 min, extraction temperature 606C), (B) extraction time (40 ethanol, extraction temperature 606C), (C) extraction temperature (40 ethanol, extraction time 80 min) on TPC, TFC, DPPH and ABTS radical scavenging capacity from C. cyrtophyllum leaf extracts. doi:10.1371/journal.pone.0068392.RE-640 biological activity gExtraction of Antioxidants from C. cyrtophyllumWe found that TPC, TFC, and antioxidant capacities were not optimized when extracted with pure water or 100 ethanol, a finding that agrees with previous reports which suggest that a binary solvent system was superior to a mono-solvent system when extracting phenolic antioxidants and preserve their relative polarity [16,22]. Similar polarity was detected for the phenolic and flavonoid components of C. cyrtophyllum leaves. Maximum recovery was observed at 60 ethanol. These components are highly soluble in hydroalcoholic solutions, especially when in a glycoside form, which may explain recovery variations. More flavonoids were recovered than phenols. This could be because flavonoids comprise a majority of the total phenols. The remainder of the plant’s metabolic flavonoids are glycosides and derivatives with non-phenolic hydroxyl groups. Antioxidant capacity was sensitive to solvent polarity, and a single ethanol concentration recovered all individual phenolic and antioxidant plant compounds. Extracts obtained at low ethanol concentrations (40 ) had a greater scavenging capacity for both DPPH and ABTS radicals. Antioxidant contents of C. cyrtophyllum are more hydrophilic, so high-ethanol solvents may solubilize more lipophilic compounds. Briefly, a high yield of individual phenolic compounds does not necessarily indicate maximal antioxidant capacity. Phenolic compounds with the best antioxidant capacities were of intermediate polarity and their solubility was sensitive to solvent polarity. Because excessive quantities of solvent can compromise the extraction of phenolic.Xperimental data for response variables for optimization of C. cyrtophyllum leaf extracts.RunProcess variables ?real and (coded) valuesResponsesa DPPH radical- scavenging capacity ( ) 54.061.0 72.961.2 61.060.9 1317923 76.461.3 76.560.7 78.760.2 79.960.4 81.760.5 77.260.5 55.961.2 76.660.6 71.260.9 79.560.5 63.161.7 82.860.3 83.861.1 83.561.1 85.660.7 ABTS radical- scavenging capacity ( ) 55.961.8 87.662.7 73.263.0 88.061.5 89.260.7 90.062.0 91.161.3 87.960.5 89.660.7 66.160.6 89.060.2 80.761.1 90.961.6 80.661.3 89.460.8 91.060.7 90.161.0 91.860.X1, EtOH ( )1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17aX2, Time (min)60 (21) 60 (21) 100 (1) 100 (1) 60 (21) 60 (21) 100 (1) 100 (1) 80 (0) 80 (0) 114 (1.68) 46 (21.68) 80 (0) 80 (0) 80 (0) 80 (0) 80 (0) 80 (0)TPC (mg GAE/g X3, T (6C) DW) TFC (mg RE/ 50 (21) 70 (1) 50 (21) 70 (1) 50 (21) 70 11967625 (1) 50 (21) 70 (1) 60 (0) 60 (0) 60 (0) 60 (0) 77 (1.68) 6.660.1 11.060.1 7.860.1 12.260.3 13.660.3 15.060.6 14.860.5 16.260.2 15.960.5 7.360.3 12.760.6 11.260.2 14.660.5 14.360.2 20.360.4 21.860.8 25.260.7 41.560.4 44.060.3 42.560.8 48.161.5 46.160.2 12.760.6 36.260.6 30.060.5 35.060.6 27.860.4 42.860.3 40.760.6 41.460.5 40.860.20 (21) 20 (21) 20 (21) 20 (21) 60 (1) 60 (1) 60 (1) 60 (1) 74 (1.68) 6 (21.68) 40 (0) 40 (0) 40 (0) 40 (0) 40 (0) 40 (0) 40 (0) 40 (0)43 (21.68) 11.060.1 60 (0) 60 (0) 60 (0) 60 (0) 14.860.8 15.360.3 15.060.6 15.060.Responses are the means 6 SD (n = 3). doi:10.1371/journal.pone.0068392.tExtraction of Antioxidants from C. cyrtophyllumFigure 1. Effect of (A) ethanol concentration (extraction time 60 min, extraction temperature 606C), (B) extraction time (40 ethanol, extraction temperature 606C), (C) extraction temperature (40 ethanol, extraction time 80 min) on TPC, TFC, DPPH and ABTS radical scavenging capacity from C. cyrtophyllum leaf extracts. doi:10.1371/journal.pone.0068392.gExtraction of Antioxidants from C. cyrtophyllumWe found that TPC, TFC, and antioxidant capacities were not optimized when extracted with pure water or 100 ethanol, a finding that agrees with previous reports which suggest that a binary solvent system was superior to a mono-solvent system when extracting phenolic antioxidants and preserve their relative polarity [16,22]. Similar polarity was detected for the phenolic and flavonoid components of C. cyrtophyllum leaves. Maximum recovery was observed at 60 ethanol. These components are highly soluble in hydroalcoholic solutions, especially when in a glycoside form, which may explain recovery variations. More flavonoids were recovered than phenols. This could be because flavonoids comprise a majority of the total phenols. The remainder of the plant’s metabolic flavonoids are glycosides and derivatives with non-phenolic hydroxyl groups. Antioxidant capacity was sensitive to solvent polarity, and a single ethanol concentration recovered all individual phenolic and antioxidant plant compounds. Extracts obtained at low ethanol concentrations (40 ) had a greater scavenging capacity for both DPPH and ABTS radicals. Antioxidant contents of C. cyrtophyllum are more hydrophilic, so high-ethanol solvents may solubilize more lipophilic compounds. Briefly, a high yield of individual phenolic compounds does not necessarily indicate maximal antioxidant capacity. Phenolic compounds with the best antioxidant capacities were of intermediate polarity and their solubility was sensitive to solvent polarity. Because excessive quantities of solvent can compromise the extraction of phenolic.