Versamax plate reader

The scavenging activity of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals was measured as previously described [13 (link)]. Briefly, each sample (250 µL) of EtSCT was mixed with 500 µL of 0.2 mM DPPH (Sigma-Aldrich Co.) in 95% ethanol solution or 100 µL of 95% ethanol solution, then incubated for 30 min at room temperature. The absorbance of the reaction mixture was subsequently measured at 517 nm using a Versa-max plate reader (Molecular Devices). The DPPH radical scavenging activity of the EtSCT was expressed as the percent decrease in absorbance relative to the control.
The scavenging activity of nitric oxide (NO) was measured as previously described [14 (link)]. Briefly, each sample of EtSCT (500 µL) was mixed with 500 µL of 10 mM sodium nitroprusside (Sigma-Aldrich Co.), then incubated at 25℃ for 150 min. This mixture was subsequently added to 500 µL of 1% sulfanilamide solution and 500 µL of 0.1% N-(1-naphthyl) ethylenediamine dihydrochloride solution and incubated at room temperature for 10 min. Next, the absorbance of the reaction mixture was measured at 546 nm using a Versamax plate reader (Molecular Devices). The NO scavenging activity of the EtSCT was expressed as the percentage absorbance relative to a control treated with dimethyl sulfoxide (DMSO, 3047-4460, DAEJUNG Chemicals & Metals Co., LTD, Siheung, Korea).

To measure the total phenolic contents, we used the Folin-Ciocalteu method with slight modification¹⁷ . Briefly, 1 ml of EtSCT solution was mixed with 5 ml of Folin-Ciocalteu reagent (Sigma-Aldrich Corporation, St. Louis, MO, USA) and then incubated at room temperature for 5 min. The mixture was subsequently added to 15 ml of 20% Na₂CO₃ and vortexed for 30 sec, after which the absorbance was repeatedly measured at 765 nm using a VersaMax plate reader (Molecular Devices, Sunnyvale, CA, USA). A standard calibration curve was made using different concentrations of gallic acid (Sigma-Aldrich Corporation), and the concentration of total phenolic contents in EtSCT was presented as mg gallic acid equivalent of extract.
Flavonoid contents were measured as previously described¹⁸ . Briefly, several different concentrations of EtSCT (200 µl) were mixed with 60 µl of 5% NaNO₂ (Sigma-Aldrich Corporation) and 60 µl of 10% AlCl₃ (Sigma-Aldrich Corporation). Following incubation at 25°C for 5 min, the mixture was added to 400 µl of 1 M NaOH, and the absorbance was repeatedly measured at 510 nm using a VersaMax plate reader (Molecular Devices). A standard calibration curve was then made using different concentrations of catechin (Sigma-Aldrich Corporation). The final concentration of flavonoid contents in EtSCT was presented as mg catechin equivalent of extract.

Total phenolics were measured by the Folin-Ciocalteu method, with slight modification [17 ]. Briefly, 1 mL of EEtRLP solution was mixed with 5mL of Folin-Ciocalteu reagent (Sigma-Aldrich Co., St. Louis, MO, USA) and then incubated at room temperature for 5 min. The mixture was subsequently added to 15 mL of 20% Na₂CO₃ and vortexed for 30 sec, after which the absorbance was repeatedly measured at 765 nm using a Versa-max plate reader (Molecular Devices, Sunnyvale, CA, USA). A standard calibration curve was made using different concentrations of gallic acid (Sigma-Aldrich Co.), and the concentration of total phenolic contents in EEtRLP was presented as mg gallic acid equivalent of extract.
The total flavonoid contents were measured as previously described [18 ]. Briefly, 200 µL of several different concentrations of EEtRLP were mixed with 60 µL of 5% NaNO₂ (Sigma-Aldrich Co.) and 60 µL of 10% AlCl₃ (Sigma-Aldrich Co.). Following incubation at 25℃ for 5 min, the mixture was added to 400 µL of 1M NaOH and the absorbance was repeatedly measured at 510 nm using a Versa-max plate reader (Molecular Devices). A standard calibration curve was then made using different concentrations of catechin (Sigma-Aldrich Co.). The concentration of flavonoid contents in EEtRLP was presented as mg catechin equivalent of extract.