Uv vis 1800

A dependent dilution series of albumin standard (2.125–200 mg/dL) was prepared. This was used for the generation of albumin standard curve [13 ]. Albumin standard of 2.0 mL was acidified with 50 μL of trichloroacetic acid followed by 100 μL of SSA at 25 ± 2°C. Proteins were allowed to precipitate for three minutes, and the absorbance was measured at 600 nm against reagent blank in a double beam spectrophotometer (SHIMADZU 1800 UV-VIS, Japan) using manual procedures. Standard curves were generated for 3%, 6%, and 25% SSA assays by plotting absorbance verses concentration of albumin standards.

The in vitro release profile of each formulation was estimated using two different dissolution media. A phosphate buffered saline of pH 7.4 with 20% of MeOH and 2.5% of Tween 80 was used as medium 1 (M1), and water was used as medium 2 (M2). An amount of 1 mL of each formulation or 1.0 mg of the free curcuminoid was placed in 80 mL of the respective medium maintained at 37 ± 0.5 °C and 150 rpm constant agitation in a Labnet 211 DS shaking incubator. At specific time intervals, a volume of 4 mL of each solution was withdrawn, and this medium volume was not replaced. The collected solutions were centrifuged at 6000 rpm for 10 min using a Thermo Scientific Sorvall ST 16R centrifuge at a controlled temperature of 37 °C. The concentrations of CUR, DMC, and BDM were determined using a double beam spectrophotometer Shimadzu 1800 UV-Vis at a wavelength of 420 nm. The dissolution profiles of pure CUR, DMC, and DMC were evaluated in the two media for comparison with curcuminoid release from the developed nanoparticles. The sampling was performed in triplicate.

For the effect of initial concentrations of AgNO₃, four different concentrations of AgNO₃ were chosen (0.005, 0.050, 0.010, and 0.100 M). A mixture of 0.005 M AgNO₃ (15 mL) and E. spiralis extract (1.5 g: 150 mL) was added in an Erlenmeyer flask and stirred at 400 rpm using a magnetic stirrer at ~52°C. During the Ag-NPs synthesis process, 10 mL of E. spiralis/Ag-NO₃ solution was taken using a pipette after 15, 30, 60, 90, 120, 180, 240, 360, 480, and 600 min. All of the solutions were then put in a vial sample and kept at 4°C for further characterization studies. The aluminum foil was used to cover the flask used during the Ag-NPs biosynthesis process because of the light-sensitivity of Ag-NPs. The reduction of Ag⁺ ions to Ag⁰ was then analyzed using an UV-visible spectrophotometer (UV-vis 1800, Shimadzu). The color changes of the solution, after the formation of Ag-NPs, were also observed. The same procedure was then repeated for the initial AgNO₃ concentration at 0.050, 0.010, and 0.100 M.

Fibers were immersed in Nisin Z at an initial concentration of 3 × MIC (pH 6) and left under slow stirring (120 rpm) for 72 h at RT. This concentration was selected to guarantee a proper functionalization of the fibers with the peptide, reaching values of at least 2 × MIC, and was determined based on data collected in previous studies [2 (link),34 ]. In addition, in these works it was also seen that 2 × MIC or MIC concentrations did not reach similar results to those obtained with the free peptide. Thus, in order to approximate that data, 3 × MIC was required. The pH of the Nisin Z solution (pH 6) was not adjusted so that the peptide maintained its positively charged nature and electrostatic interactions with the polymers would be facilitated [18 (link)]. Aliquots of the solution were collected, and their absorbance was measured using an UV–visible spectrometer (UV–VIS 1800, Shimadzu, Switzerland), every 24 h, at 205 nm as stated in the literature [35 (link),36 (link)]. This wavelength was also confirmed as the one in which nisin Z presents the maximum absorbance value by scans ranging from 200 to 800 nm. The percentage of Nisin Z incorporated within the fibers was determined by measuring the difference of absorbance at times 0 and 72 h. After this period, the fibers were washed in dH₂O for 5 min, dried and stored for future testing (Table 1).

Total flavonoid content (TFC) was determined by colorimetric method using aluminum chloride (AlCl₃) as reported by Chandra et al.^{16 (link)} with minor modifications. 25 mg of ethanolic extracts of O. stamineus leaf or stem was dissolved in 25 mL ethanol. 1 mL of the solution was mixed with 0.5 mL of 10% AlCl₃ and incubated at room temperature for 74 min. The absorbance was measured at 410 nm with a Uv–Vis spectrophotometer (Shimadzu Uv–Vis 1800, Japan). Quercetin standard was measured ranging from 5 to 20 µg/mL as the standard for total flavonoid concentration. TFC was calculated as μg/g quercetin equivalent (QE) of dried extract. All measurements were carried out in triplicates.