Uv vis spectrophotometer

Glucose-regulating enzyme activities were examined in 10% homogenates of liver tissue (0.3 g) in 0.1 M triethanolamine, 0.2 M EDTA and 2 mM dithiothreitol as described previously [46 (link)]. The homogenates were centrifuged twice at 1000× g and at 10,000× g for 15 min at 4 °C, and the supernatant was collected. For GK, the supernatant was incubated with the reaction buffer containing 50 mM Hepes-NaGT (pH 7.4), 100 mM KCl, 7.5 mM MgCl₂, 2.5 mM dithioerythritol, 10 mg/mL albumin, 10 mM glucose, and 4 units glucose-6-phosphate dehydrogenase for 10 min at 37 °C. The activity was measured at 340 nm after incubating with 5 mM ATP at 37 °C for 10 min. For G6pase, the supernatant was measured at 340 nm after incubating with the buffer containing 131.58 mM Hepes-NaGT (pH 6.5), 18 mM EDTA (pH 6.5), 265 mM glucose-6-phosphate, 0.2 M NADP⁺, 0.6 IU/mL mutarotase, and 0.6 IU/mL glucose dehydrogenase for 4 min at 37 °C. For PEPCK, the supernatant was mixed with the buffer containing 72.92 mM sodium Hepes (pH 7.0), 10 mM dithiothreitol, 500 mM NaHCO₃, 10 mM MnCl₂, 25 mM NADH, 100 mM inositol-1,4-diphosphate, 200 mM phosphoenolpyruvate, and 7.2 units malic dehydrogenase. The activity was measured at 340 nm as the reduced absorbance using a UV/Vis spectrophotometer (OPTIZEN POP, Mecasys, Daejeon, Korea). All reagents were purchased from Sigma-Aldrich (St. Louise, MO, USA).

Lipid oxidation was determined by the 2-thiobarbituric acid (TBARS) method described by Kim et al [12 ]. First, 2 g of sample was homogenized at 12,000 rpm for 1 min in 10 mL of 10% trichloroacetic acid and 10 mL of distilled water. After homogenization, the solution was filtered through filter paper (Whatman No. 1, Whatman, Piscataway, NJ, USA). Subsequently, 5 mL of filtered solution was mixed with 5 mL of 2.88 g/L TBARS, and the mixed solution was placed in a 90°C water bath for 10 min. After 30 min of cooling, the absorbance of the solution at 532 nm was determined using a spectrophotometer (UV/Vis Spectrophotometer, Mecasys, Daejeon, Korea). Data are shown as mg of malondialdehyde (MDA) meat/kg. Standard MDA curves determined from 1,1,3,3-tetraethoxypropane were used for calculations.

The KO’s free radical scavenging ability was assessed through the DPPH radical scavenging assay, following the method outlined by Blois [35 (link)]. A 0.2 mM solution of 2,2-diphenyl-1-picryhydrazyl (DPPH; Sigma-Aldrich, St. Louis, MO, USA) in methanol was promptly prepared. Samples were diluted using distilled water to reach final KO concentrations of 0.25, 0.5, 1, 1.5, 2, 4, and 8 mg/mL, or a final L-AA concentration of 1 mg/mL. The DPPH radical scavenging activity was measured at 517 nm with a UV/Vis spectrophotometer (Optizen Pop, Mecasys, Daejeon, Republic of Korea) after a 10-min incubation. The free radical scavenging activity was calculated using the formula: DPPH radical scavenging activity (%) = 100 − [(OD_s/OD_c) × 100], where OD_s represents the sample’s absorbance at 517 nm and ODc is the absorbance of the control treated with the vehicle at 517 nm. The results were expressed as IC₅₀ values, indicating the concentration needed to decrease DPPH by 50%. A positive control of L-AA at 1 mg/mL was employed.

The TBARS assay determines secondary lipid oxidation. Thiobarbituric acid (TBA) reacts with the lipid oxidation product malondialdehyde (MDA) and forms a red chromophore that is measured spectrophotometrically. The UV/Vis spectrophotometer (Mecasys, Daejeon, Korea) was calibrated with a 1,1,3,3-tetraethoxypropane standard curve as described by Buege and Aust [31 ]. A 5-g meat sample was homogenized with 15 mL distilled water and 100 μL BHT at 13,000 rpm for 1 min. Then, 2 mL of the homogenate was reacted with 4 mL trichloroacetic acid/2-thiobarbituric acid reagent in an 80 °C water bath for 15 min. The samples were then cooled in cold water, centrifuged at 2000× g and 25 °C for 10 min, and filtered through Whatman No. 4 filter paper. Optical densities were measured at 531 nm in the aforementioned spectrophotometer.

The metmyoglobin content was determined by the method of Warriss [34 (link)]. Each 4-g sample was homogenized with 20 mL of cooled 0.04 M phosphate buffer (pH 6.8) at 13,000 rpm for 10 s. The homogenate was left to react at 4 °C for 1 h, centrifuged at 5000 g and 5 °C for 30 min, and passed through Whatman No. 1 filter paper. Absorbance was measured in a spectrophotometer (UV/Vis Spectrophotometer, Mecasys, Daejeon, Korea) at 525 nm, 572 nm, and 730 nm. Metmyoglobin content was obtained as follows: