U 2000 uv vis spectrophotometer

Diluted extracts of H. foliosum (Hf. E, Hf. DM, and Hf. W) (300 µL) in different concentrations were mixed with 3 mL of reagent solution containing 0.6 M sulfuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate. After 90 min of incubation at 95 °C, the absorbance was measured at 695 nm with a Hitachi U-2000 UV–Vis spectrophotometer (Tokyo, Japan). Ascorbic acid was used as a standard, and the results were expressed as the inhibitory concentration (IC₅₀ value).

Urinary creatinine was used to normalize the measured levels of the four biomarkers, and the creatinine concentration was measured as the creatinine-picrate complex with a U-2000UV/VIS spectrophotometer (Hitachi, Tokyo, Japan) at a wavelength of 520 nm. The urinary levels of each analyte were expressed as μg/g creatinine.

The influence of ACP treatment on the sulfhydryl (SH) content has been detected as mentioned by Nikoo, Benjakul [23 (link)] with slight modifications. One mL trypsin solution (1 mg/mL) was dissolved in 20 mM Tris-HCl buffer (pH 8.0), then 8 mL of dissociating buffer (5.2 g Tris-HCl, 3.45 g glycine, 240 g urea, 0.6 g EDTA in 500 mL distilled water, pH 8.0) and 0.5 mL of DTNB (4 mg/mL of 5, 5-dithiobis-(2-nitrobenzoic acid)) were added. The mixture was incubated in a dark place at room temperature for 10 min. After incubation, the absorbance was read at 412 nm using a Hitachi U-2000 UV-Vis spectrophotometer (Tokyo, Japan). An 8 mL mixture of the dissociating buffer and 0.5 mL DTNB without trypsin solution was considered as a control and handled in the same pattern. Calculations for SH concentration in the samples involved a molar absorbance coefficient of 13,600 (mol/L)⁻¹ cm⁻¹, and results were expressed as nmol total SH/mg of protein.

The antioxidant properties of red and white Portuguese AoBTHPs were evaluated using three different spectrophotometric methods: DPPH method, FRAP assay, and TAC using the phosphomolybdenum method.
All values were determined in 3 sets of experiments and evaluated in triplicate using a Hitachi U-2000 UV-Vis spectrophotometer (Tokyo, Japan). The results are presented as mean ± SD.

Specified tissues (100 mg) were sampled and ground separately in liquid nitrogen for the enzyme extraction. The in vitro determination of acid invertase activities of specific tissues were performed, as described previously [76 (link)]. Preparations (100 μL) were mixed with 100 μL sucrose (100 mM) up to 300 μL and incubated for 1 h at 37 °C. The reaction was terminated by 30 μL sodium phosphate (1 M, pH 7.5), and incubated for 5 min at 95 °C. The assay was conducted in quadruplicate, one of which was neutralized and boiled immediately after the addition of sucrose. Sample activity was subtracted from the activity of the others as background absorption. Liberated glucose was measured in a coupled enzymatic-optical assay. In this assay, 100 μL of the reaction, 20 μL 30 mM ATP, 20 μL 30 mM NADP, and 2 μL suspension (340 U/mL HK, 170 U/mL G6P-DH, Roche) mixed up to 1 mL with buffer (40 mM triethanolamine, 8 mM MgSO₄, pH 7.5) and incubated for 5 min at room temperature. Synthesis of NADPH was detected by U-2000 UV/VIS spectrophotometer (Hitachi, Japan) at 340 nm, and finally, the released glucose was calculated while using the Lambert-Beer law. Invertase activity was defined as expression in nkat/g fresh weight (1 nkat=1 nmole Glcose liberated/second).

The TBARS (thiobarbituric acid-reactive substances) assay was performed as described by Steels et al. [27 (link)] with modifications. Next, 10% (w/v) trichloroacetic acid (final concentration) was added to the crude cell extracts, which were then centrifuged for 20 min at 10,000× g. The supernatant was mixed with 0.1 mL EDTA (0.1 M) and 0.6 mL 1% (w/v) thiobarbituric acid in 0.05 M NaOH. Blank samples contained lysis buffer instead of supernatant. The reaction mixture was incubated in a water bath (90 °C) for 15 min and, after rapid cooling, the absorption was measured at 532 nm using the Hitachi UV–VIS (U-2000) spectrophotometer. The results are expressed as nmoles of malondialdehyde (MDA) equivalents/mg protein and then assigned as the mean ± SD of the ratio between the treated cells and control cells (untreated) from three independent measurements (CV% = 1.1, n = 5). If the TBARS concentration is lower in oxidant-treated cells supplemented with phenolic compounds than that in treated cells without added phenolic compounds, the phenolic compound has acted as an antioxidant.

Total protein content (TPC) in the crude cell extract was determined by the Bradford protein assay [24 (link)] and the absorbance of CBBG was measured at 595 nm using the Hitachi UV–VIS (U-2000) spectrophotometer (Tokyo, Japan). BSA was used as a standard in a concentration range of 0.07–1.4 mg/mL of protein. According to the standard calibration curve, the value of TPC for each crude cell extract was presented as mg/mL. Data are presented as mean (±SD) of three independent measurements (CV% = 1.3, n = 5).