Uv vis spectrophotometer

Absorption and fluorescence spectra were recorded in quartz glass cuvettes with either 10 mm or 2 mm optical path length (Hellma Analytics) on a Jasco V‐650 UV‐vis spectrophotometer and a Hitachi F‐4500 fluorometer. Absorption spectra were measured at an optical density (OD) close to 1. OD values for fluorescence spectra measurements were set to 0.1 to 0.15. Data for ON1 was obtained on a Tecan Infinite M200 Pro plate reader.
Uncaging quantum yields were determined using our recently published fulgide actinometry setup controlled by our in‐house programmed software PHITS (Photoswitch Irradiation Test Suite) written with LabVIEW.^[43] A concentrated indolylfulgide photoswitch solution in toluene served as a reference for the chemical actinometry. Its absorption spectrum was tracked (Ocean Optics DH‐mini light source, Ocean Optics USB4000 or Thorlabs CCS200/M detector) while converting the fulgide from its closed form to the Z‐form through irradiation with a 530 nm LED (M530 L3, Thorlabs). The caged oligonucleotide was then irradiated in the same setup with known photon flux. Last, the photolysis rate was determined by integration of the RP‐HPLC signals.

Optimization of HIP complex was conducted on the basis of complexation efficiency. Thus, insulin concentration in supernatant was analyzed spectrophotometrically at λmax 270 nm (UV-vis spectrophotometer, Hitachi) as a reflection of unbound insulin concentration and used for the estimation of optimum insulin/SDS molar ratio by using the following equation: $$\mathrm{Percentage} \mathrm{Complexation} \mathrm{Efficiency}=\frac{I_T-I_F}{I_T}\times 100$$
where $I_T$ is the amount of insulin initially added and $I_F$ is the amount of insulin present in the supernatant.

Total flavonoid content of the C. inophyllum extract was assessed by the aluminum chloride colorimetric assay as described previously [20 (link)]. 0.2 ml of either C. inophyllum extract or standard was mixed with 4.8 ml of ultrapure water in a test tube. 0.3 ml of 5% NaNO₂ was topped up and mixed well by using a vortex mixer. After that, the mixture was left at room temperature for 5 min prior to the addition of 0.3 ml of 10% AlCl₃ 6H₂O as well as 2 ml of 1 M NaOH solution into each tube. The ultrapure water was dispensed to reach the final volume of 10 ml. The optical density values at 415 nm were recorded using a UV-VIS spectrophotometer (Hitachi, Japan). Quercetin was used as a standard. The total flavonoid content was expressed as mg of quercetin equivalents per gram of dry extract (mg QE/g DW) using the calibration curve of quercetin.

The acetylcholinesterase (AChE) activity was examined via the approach, with slight modifications, described by Ellman et al. [23 (link)]. Briefly, the plasma, RBC, or brain tissue homogenate was mixed with a reaction mixture in phosphate buffer (0.1 M, pH 7.4) containing 5,5-dithiobis-2 nitrobenzoate and acetylthiocholine iodide. The total mixture was then left at 25 °C for 30 min. The degradation of acetylthiocholine iodide was read at 412 nm using a UV–Vis spectrophotometer (Hitachi, Tokyo, Japan). The results were calculated and presented as U/mL for plasma sample, U/g hemoglobin for RBC sample, and U/g protein for brain tissue homogenates following the company protocol.

The determination of the tyrosinase enzyme inhibitory activity refers to [13 (link)] with modifications. The assay consists of the tyrosinase enzyme (EC 1.14.18.1; lyophilized powder; ≥1000 unit/mg solid, Sigma-Aldrich), L-DOPA as a substrate, and kojic acid as the positive controls. First, a solution of kojic acid was prepared in a phosphate buffer (pH 6.5) from a concentration of 7.8, 15.63, 31.25, 62.5, and 125 ppm. Then, 1000 ppm of PSC and HC stock solutions were separately diluted in a phosphate buffer (pH 6.5) to obtain a solution with concentrations of 100, 150, 200, 250, 300, and 350 ppm. As much as 350 μL of each sample was taken and added to 300 μL of tyrosinase solution. The plates were incubated at room temperature for 5 min and 550 μL of 2 mM L-DOPA substrate was added; they were then reincubated for 30 min at room temperature. The absorbance was measured at λ = 492 nm using a UV-VIS spectrophotometer (Hitachi, Tokyo, Japan).

Using the cell-free lysates, cytosolic LDH (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) enzymatic activity was assayed by following the oxidation of NADH at 340 nm as a function of time as described previously (Place and Powers, 1984 (link)). Briefly, the LDH activity was assayed in 1 ml [sodium phosphate buffer (0.1 M, pH 7.5), 1 mM sodium pyruvate (Sigma-Aldrich, P2256) and 0.17 mM NADH (SRL, 77268)]. Then the commencement of the reaction was followed by the addition of 150 μg of cell lysate. The depletion of NADH was recorded for 5 min at 340 nm using a UV–Vis Spectrophotometer (Hitachi). The absorption coefficient used for NADH was 6220 M⁻¹cm⁻¹. The reaction rate without the addition of cell lysate was subtracted for the calculation of the specific activity in each set.

Five milliliters of blood was drawn from each patient arm veins with a catheter on the seventh morning of hospitalization within 7–8 a.m. in a stable state. The heparin anticoagulant tube was used to collect 4–5 ml peripheral blood during fasting. The blood sample was centrifuged at 1500 rpm for 10 min. The upper plasma was collected and placed in four centrifuge tubes, and placed in −20°C refrigerator. Malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-PX) and T-AOC kits were purchased from Jiancheng Co. Ltd. (Nanjing, China). Plasma levels of MDA, SOD, GSH-PX and T-AOC were measured using corresponding kits. MDA, SOD, GSH-PX and T-AOC was measured spectrophotometrically on a UV-Vis spectrophotometer (Hitachi, Japan) at 535, 560, 340 and 520 nm according to manufacturer’s instructions, respectively.