Antioxidant assay kit

The total antioxidant capacity (TAOxC,) was measured in lysed HepG2 cells using an Antioxidant Assay kit (CS0790, Sigma-Aldrich, South Africa) according to the manufacturer’s instructions. The kit is based on the ability of antioxidants present in the sample to inhibit the oxidation of 2,2′-azino-bis-3-ethylbenzothiazoline (ABTS) to ABTS+ by ferryl myoglobin. HepG2 cells were seeded at a density of 1×10⁶ cells per flask and treated with 20 mM APAP and various concentrations (62.5–250 μg/mL) of the PPRFs for 24 h. After treatment, the cell pellets were homogenized on ice in 1 mL of cold assay buffer before being centrifuged at 12,000 g for 15 min, at 4 °C. The supernatant of the lysed cells was used to measure TAOxC. Absorbance in the well was measured after 5 min at 405 nm. The scavenging activity was expressed as Trolox equivalents (TEAC) from the Trolox calibration curve.

TAC was assessed by a method adapted from Rice-Evans and Miller [33 (link)] which is based on the antioxidant-induced inhibition of the absorbance of the radical cation of 2,2′-azino-bis (3-ethylbenzothiazoline 6-sulphonate) (ABTS). The ABTS radical cation is formed by the interaction of 150 μM ABTS with the ferrylmyoglobin radical species, generated by the activation of 2.5 μM metmyoglobin with 75 μM H₂O₂. Antioxidant agents suppress the formation of the ABTS radical cation. The assay was performed with the use of the Antioxidant Assay Kit (Sigma-Aldrich, USA). Briefly, ABST, myoglobin, and 10 μL of serum were mixed, and the reaction was initiated by the addition of H₂O₂. Following the incubation for 5 min at 21°C, the absorbance of the product was read at 735 nm and compared to a calibration curve prepared using Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), a water soluble analogue of vitamin E commonly used in antioxidant assays as a standard antioxidant [31 (link)]) (0.0–2.0 mM; r² = 0.98) and given as mM Trolox equivalents. Three technical replicates were performed for each serum sample.

Whole blood was challenged without or with bacteria (1 × 10⁶ CFU/mL of blood) for 3, 20 and 60 minutes. LPS (1 μg/mL) was used as a positive control. Samples were centrifuged for 15 minutes at 1000 g, room temperature, to obtain plasma. Supernatants were partitioned and immediately frozen and stored at −80°C until required for analysis. Samples were analysed for total antioxidant capacity using an antioxidant assay kit according to the manufacturer's instructions (Sigma Aldrich). Briefly, metmyoglobin and H₂O₂ form a ferryl myoglobin radical that oxidizes ABTS (2,2′‐azino‐bis(3‐ethylbenzthiazoline‐6‐sulfonic acid)) to a green radical cation. Any antioxidants present in the sample will suppress the formation of the green radical in a concentration‐dependent manner. Absorbance was read at 450 nm using an ELISA plate reader.

TAC was measured with a spectrophotometer in 96-well plates,
using the Antioxidant Assay Kit (Sigma) as per the manufacturer's
instructions. After 5 min incubation, reaction was stopped
and the endpoint absorbance at 405 nm was determined using
a microplate reader (SpectraMax 340PC, 384 — Molecular Devices). All samples
were performed in duplicate. A reference curve based on the soluble
antioxidant Trolox was used, and TAC was expressed in Trolox concentration
(mM).

Measurement of malondialdehyde (MDA) was made using the thiobarbituric acid method (Ohkawa et al. 1979 (link)). The total antioxidant capacity measured using a commercial Antioxidant Assay Kit (Antioxidant Assay Kit, Catalog Number CS0790, Sigma-Aldrich, Inc.). Liver tissue samples were homogenized on cold ice and buffered and then centrifuged at 12,000g for 15 min at 4 °C. The supernatant was removed for study and placed on plates. The principle of the antioxidant assay relies on the formation of a ferryl myoglobin radical from metmyoglobin and hydrogen peroxide. A soluble chromogen, green in color, can be determined spectrophotometrically at 405 nm (Huang et al. 2005 (link)). The blood samples were centrifuged at 5000g at 4 °C and the supernatants were removed. Serum Alanine aminotransferase (ALT), Aspartate Aminotransferase (AST), Alkaline phosphatase (ALP), Gamma-glutamyl transpeptidase (GGT), and total bilirubin and direct bilirubin levels were determined.

Fasting blood glucose level was determined on days 1, 7, 14 and 21 using OneTouch electronic glucometer (OneTouch). On day 21 after overnight fasting, the blood was collected after decapitation of rats under mild ether anesthesia. The serum was separated (centrifugation at 1500 ×g for 5 min) and used to determine the concentration of total cholesterol, triglycerides, and HDL-cholesterol using enzymatic colorimetric method (kits, Human Co., Germany). The serum level of LDL-cholesterol was calculated [15 (link)]. To investigate the effect of MTPE on liver and kidney function, the level of creatinine was determined with kits (ELITech Diagnostics, France) as well as the activities of aspartate transaminase (AST) and alanine aminotransferase (ALT) using enzymatic methods (kits, TECO diagnostic, USA). The in vivo antioxidant property of the MTPE was also carried out through the determination of the lipid peroxidation (MDA); reduced glutathione (GSH); and the total antioxidant status (TAOS) (Antioxidant Assay Kit, Sigma Co. (Catalog number CS0790).