Extraction of Lipophilic and Hydrophilic Antioxidants A modified method was used to separate the lipophilic and hydrophilic extracts of tomato (Toor and Savage 2005; Teow et al. 2007; Kotíková et al. 2011; Riahi and Hdider 2013). In brief, 1 g of freeze-dried sample powder in 20 mL hexane was ultrasonicated by ultrasound assisted extraction (Ultrasonic Sonicator, RF103H, Bandelin Sonorex, Germany) for 20 min, and the mixture was transferred to centrifuge tube and centrifuged at 4000 rpm for 10 min. The supernatant was filtered through Whatman filter paper No. 42. The filtrate was evaporated to dryness at 50oC using a vacuum evaporator. The residue was then redissolved in 5 mL acetone and vortexes to get homogenous samples. The lipophilic
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The residue after hexane extraction was then extracted with 20 mL of 50% (v/v) methanol in water and ultrasonicated for 20 min. The mixture was transferred to a centrifuge tube followed by centrifugation at 4000 rpm for 10 min. The supernatant was filtered once and transferred to another tube prior to the determination of hydrophilic antioxidant activity. The lipophilic and hydrophilic antioxidant activity was measured in triplicates for each extract. Four variables that could potentially affect the extraction efficiency were studied using the same procedure mentioned above. The experiments were carried out at three types of organic solvents for lipophilic fractions (ethyl acetate, hexane, 50% acetone in water) and four types of solvent for hydrophilic fractions (0.1M phosphate buffer pH7.4, acetone: water: acetic acid (70:29.5:0.5), 50% methanol in water, 7% acetic acid in 80% methanol). The amount between solid (sample) and liquid (organic solvents) was carried out at the ratios of 1:10, 1:15, 1:20 and 1:25 g:mL. The ultrasonication times were varied at six extraction time levels (10, 20, 30, 40, 50, and 60 min). …show more content…
This method was carried out with slight modifications (Hossaina et al. 2008; Li et al. 2012). The FRAP method measures the ability of the antioxidants to reduce ferric–tripyridyl-triazine (Fe3+-TPTZ) complex to the blue colored ferrous form which absorbs light at 593 nm. The ferric-TPTZ reagent was prepared by mixing 300 mM acetate buffer, pH 3.6, 10 mM TPTZ in 40 mM HCl and 20 mM FeCl3.6H2O in the ratio of 10:1:1 (v/v/v). The FRAP reagent was freshly prepared before each experiment. Briefly, 60 µL of different concentrations of the reference standard or the sample extractwere mixed with 1000 µL FRAP reagent and incubated at 37oC for the duration of the reaction. The absorbance readings were taken at 593 nm at 30 min. Increasing absorbance of the reaction mixture indicates an increase of reduction capability. Six concentrations of 500, 600, 700, 800, 900 and 1000 µM were used to prepare the standard curve of ascorbic acid. The antioxidant activities of the tomato extracts were expressed as µmol AEAC/100 g DW while the antioxidant activities of six reference standards were expressed as EC1, the concentration of antioxidant that reduced Fe3+-TPTZ equal to 1 mM
The residue after hexane extraction was then extracted with 20 mL of 50% (v/v) methanol in water and ultrasonicated for 20 min. The mixture was transferred to a centrifuge tube followed by centrifugation at 4000 rpm for 10 min. The supernatant was filtered once and transferred to another tube prior to the determination of hydrophilic antioxidant activity. The lipophilic and hydrophilic antioxidant activity was measured in triplicates for each extract. Four variables that could potentially affect the extraction efficiency were studied using the same procedure mentioned above. The experiments were carried out at three types of organic solvents for lipophilic fractions (ethyl acetate, hexane, 50% acetone in water) and four types of solvent for hydrophilic fractions (0.1M phosphate buffer pH7.4, acetone: water: acetic acid (70:29.5:0.5), 50% methanol in water, 7% acetic acid in 80% methanol). The amount between solid (sample) and liquid (organic solvents) was carried out at the ratios of 1:10, 1:15, 1:20 and 1:25 g:mL. The ultrasonication times were varied at six extraction time levels (10, 20, 30, 40, 50, and 60 min). …show more content…
This method was carried out with slight modifications (Hossaina et al. 2008; Li et al. 2012). The FRAP method measures the ability of the antioxidants to reduce ferric–tripyridyl-triazine (Fe3+-TPTZ) complex to the blue colored ferrous form which absorbs light at 593 nm. The ferric-TPTZ reagent was prepared by mixing 300 mM acetate buffer, pH 3.6, 10 mM TPTZ in 40 mM HCl and 20 mM FeCl3.6H2O in the ratio of 10:1:1 (v/v/v). The FRAP reagent was freshly prepared before each experiment. Briefly, 60 µL of different concentrations of the reference standard or the sample extractwere mixed with 1000 µL FRAP reagent and incubated at 37oC for the duration of the reaction. The absorbance readings were taken at 593 nm at 30 min. Increasing absorbance of the reaction mixture indicates an increase of reduction capability. Six concentrations of 500, 600, 700, 800, 900 and 1000 µM were used to prepare the standard curve of ascorbic acid. The antioxidant activities of the tomato extracts were expressed as µmol AEAC/100 g DW while the antioxidant activities of six reference standards were expressed as EC1, the concentration of antioxidant that reduced Fe3+-TPTZ equal to 1 mM