1800 uv visible spectrophotometer

Ferric reducing antioxidant power (FRAP) was determined following the protocol described by Radenkovs et al. [55 (link)]. The FRAP reagent was prepared daily from a 100 mL acetate buffer (0.3 mol L⁻¹; pH 3.6), a 2,4,6-tripyridyl-s-trizine (TPTZ) solution in 10 mmol L⁻¹ of HCl, and FeCl₃ (20 mmol L⁻¹). The three solutions were mixed together at a ratio of 10:1:1 (v/v/v), respectively, and then heated at 37 °C. Then, a 0.15 mL aliquot of each sample or standard was mixed with the 2.85 mL FRAP reagent and vortex mixed for 1 min. The resulting absorbance was measured at a wavelength of 593 nm after 10 min against a blank sample (UPW), which was used as the reference. The assay was performed using a UV-1800–Visible Spectrophotometer (Shimadzu Corp., Kyoto, Japan) The results were calculated using a calibration curve of the standard and expressed as mg CGA mL⁻¹.

The total phenolics content (TPC) was determined using the colorimetric Folin–Ciocalteu method [53 ]. The stock solutions of extracts and AgNPs were prepared from 0.2 mL aliquot of each extract and AgNPs by mixing with 1.8 mL with ultrapure water (UPW) and subsequent vortex mixing and ultrasonication for 1 and 5 min at room (22 ± 1 °C) temperature, respectively. Since the dissolution of AgNPs can vary depending on the AgNPs’ surface charge and capping agent used, resulting in the underestimation of antiradical activity, under a separate trial, both the prepared extracts and AgNPs were dissolved in acidified 80% MeOH (MeOH:H₂O:hydrochloric acid ratio 89:19:0.1 v/v/v) for comparative purposes. Afterward, a 0.5 mL aliquot of each sample (from stock) or standard or blank (UPW) was mixed with 2.5 mL of a 10-fold diluted Folin–Ciocalteu reagent and 2.0 mL of 7.5% Na₂CO₃ with subsequent vortex mixing for 1 min. The prepared solutions were incubated for 30 min at room temperature. Finally, the absorbance was measured at 760 nm using a UV-1800–Visible Spectrophotometer (Shimadzu Corp., Kyoto, Japan). The results were expressed as chlorogenic acid (CGA) equivalents per g of raw material (mg CGA g⁻¹).

The scavenging activity of ABTS^•+ was determined according to the method of Du et al. [56 (link)] with some modifications. The synthetic radical ABTS^•+ was prepared by mixing 0.15 mL of the 7.4 mM ABTS^•+ solution with 0.15 mL of 2.6 mM potassium persulfate (K₂S₂O₈). The prepared solution was allowed to react for 16 h at room temperature in the dark. The ABTS^•+ solution was then diluted with phosphate-buffered saline (PBS) with an adjusted pH of 7.4 to an absorbance of 0.70 ± 0.02 at 734 nm. An aliquot of 0.15 mL of each sample, standard, or blank (PBS) was added to 2.85 mL of diluted ABTS^•+ and left to react for 10 min at 22 ± 1 °C in the dark, and absorbance at 734 nm was measured using a UV-1800–Visible Spectrophotometer (Shimadzu Corp., Kyoto, Japan). The results were calculated using a calibration curve of the standard expressed as mg CGA g⁻¹.