Evolution 300 uv vis

Seminal plasma was obtained by centrifuging 500 μL of fresh semen at 5°C for 10 minutes at 660 g. The supernatant was recovered and stored at −20°C for further analysis. Measurements were performed based on the rate of substrate consumption in reactions catalyzed by each antioxidant enzyme in a given time interval using a spectrophotometer (Evolution 300 UV-Vis, Thermo Scientific, Waltham, MA, USA). Activities of superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione reductase (GRD) were determined as described previously [24 (link)]. In addition, catalase activity was determined by evaluating the consumption of hydrogen peroxide for 3 minutes at 242 nm, and the 18.6 × 10³ cm⁻¹M⁻¹ molar extinction coefficient was used.

ACaT were prepared through the self-assembly of THZ1, NaALN and Ca²⁺. Firstly, 0.66 g of NaALN·3H₂O and 0.2 g of CaCl₂·2H₂O were dissolved in 75 mL of water, respectively, and mixed them up. Then, 50 mg of THZ1 (4 mg/mL) was added to the above solution. The pH of this mixture was adjusted to 7.0 by NaOH and stirred for 30 min at 4 °C. ACaT nanomedicine was obtained by washing and centrifugation. The morphologies of obtained formulations were observed by TEM and SEM. Particle size and zeta potential were measured by Zetasizer Nano ZS90 (Malvern, Britain). Fourier transform infrared (FTIR) spectra were measured using Thermo Scientific Nicolet iS5 FTIR spectrometer. Chemical state and composition of ACaT was investigated using X-ray photoelectron spectrometer (Thermo Scientific, K-Alpha, UK). To assess the stability of ACaT in PBS, PBS containing 5% and 10% FBS, the particle sizes were measured by DLS persistently at different time. X-ray powder diffraction (XRD) patterns were recorded using an X-ray diffractometer (Rigaku D/max 2550 V, Japan). UV–vis and fluorescence spectra were recorded by Evolution 300 UV–vis (Thermo Scientific, USA) and LS55 luminescence spectrometer (Perkin-Elmer, USA).

The total content of polyphenolic compounds in the fresh and thermally treated turmeric rhizome was determined using a Thermo Fisher Scientific Evolution 300 UV-Vis spectrophotometer (Thermo, Waltham, MA, USA) using the Folin–Ciocalteu reagent method as previously described [40 (link)].
Initially, 0.5 mL of the studied extract was transferred into a 50 mL volumetric flask. Then, 30 mL of purified water and 2.5 mL of the Folin–Ciocalteu reagent were added. The solution was mixed and allowed to stand for 1 min. Then, in no more than 8 min, 7.5 mL of a 20% Na₂CO₃ solution was added and the whole was made up to 50 mL with distilled water. The obtained mixture was set aside for 2 h in a place with limited access to light. The mixture was stirred again and used for a spectrophotometric determination in 1 cm cuvettes, which were placed in an apparatus. The absorbance was measured at 760 nm.
The calibration curve equation was calculated based on the measurement of gallic acid absorbance. The standard solutions were prepared in a similar manner to the studied samples. The concentrations of the standard solutions were 50, 100, 200, 300 and 500 mg/L. Using the prepared calibration curve, the total content of polyphenolic compounds in the studied samples was calculated in gallic acid equivalents.

Proline content was measured according to the previously described protocol [32 (link)]. For this, 0.5 g of the frozen plant materials was homogenized in 10 mL of 3% sulphosalicylic acid, followed by filtration. Two mL of the extract was added with 2 mL acid ninhydrin and 2 mL glacial acetic acid in a test tube and incubated for 1 h at 100 °C, followed by a halt in the ice bath. The chromophore was separated by adding 4 mL of toluene with aggressive mixing for 15–20 s and was kept until the two phases separated. The toluene-containing chromophore was then transferred to another test tube, and the absorbance was measured at 520 nm using a spectrophotometer (Evolution 300 UV-VIS, Thermoscientific, England, UK).

The content of pigments (chlorophylls, carotenoids) was assayed in dry biomass (50 mg) after extraction using 1 mL of 80% acetone (v/v). The samples were then mixed (30 min), sonicated in an ultrasonic bath (5 min), incubated at 40 °C for 30 min with shaking (400 rpm) and centrifuged (3000 rpm, 5 min). The obtained supernatant was dissolved using 80% acetone (v/v) to give an absorbance ≤ 1. The measurements were performed using quartz cuvettes in a spectrophotometer Evolution™ 300 UV-Vis (Thermo Fisher Scientific, USA) at λ = 646.8 nm, 663.2 nm and 470 nm against 80% acetone as a blank. The samples were protected from light during the analysis. The following formula was used to calculate the content of chlorophyll a, chlorophyll b and carotenoids (µg/mL) using 80% acetone (v/v) as a solvent [47 ]: ${\mathrm{C}}_{\mathrm{a}}=12.25{\mathrm{A}}_{663.2} - 2.79{\mathrm{A}}_{646.8}$
${\mathrm{C}}_{\mathrm{b}} =21.5{\mathrm{A}}_{646.8} - 5.1{\mathrm{A}}_{663.2}$
$\mathrm{Cx}+\mathrm{c} = \left(1000{\mathrm{A}}_{470} - 1.82{\mathrm{C}}_{\mathrm{a}} -85.02{\mathrm{C}}_{\mathrm{b}}\right)/198$
where A is an absorbance, C_a is a concentration of chlorophyll a, C_b is a concentration of chlorophyll b and Cx + c is a concentration of total carotenoids.

The absorbance spectrum of the SPION solution was measured using Thermo Scientific Evolution 300 UV-VIS (Waltham, MA, USA). The hydrodynamic diameter and zeta potential of SPIONs and Tx@ReoPro were measured using Malvern NanoZS [3 (link)].