Ab102530

In order to evaluate the effect of cinnamon samples on the activity of HepG-2 and BJ-1, cells were seeded in RPMI-1640 medium containing 25 mg/mL of each cinnamon sample, with one 106 cell per flask, and incubated for 48 h at 37 °C under a humidified atmosphere of 5% CO₂. After this, the cell medium was converted to serum-free medium (SFM) comprising 10 µL/mL of yogurt extract. Then, trypsin 0.05%/0.53 mM EDTA solutions were used to trypsinize the cell cultures after incubation. After washing in PBS, the cells were centrifuged at 2000 rpm for 5 min at 4 °C and resuspended in 1 mL PBS containing 0.1% Triton X-100. Using a 1.5 mL micro centrifuge tube, cells were sonicated twice for 10 s at 100 Hz (Vibra-cell, Sonics & Material) and centrifuged for 30 min at 4 °C at 14,000 rpm. The cells were sonicated for 2 min at 100 Hz in a 1.5 mL micro centrifuge tube placed on ice, then centrifuged at 14,000 rpm for 30 min at 4 °C after the sonication (Vibra-cell, Sonics & Material). Enzyme activity was tested in the supernatant of the tube. We measured the enzyme activity of SOD, CAT, GSH, and GPx in cell culture according to the manufacturer’s instructions for colorimetric kits ab65354, ab83464, ab142044, and ab102530 Abcam.

The activity of GPx was measured with a commercially available kit(ab102530, Abcam, Cambridge, United Kingdom) [40 (link)–44 (link)]. In the assay, GSSG produced after GPx oxidizes GSH during H₂O₂ reduction. GSSG is reduced back to GSH by GR with the aid of nicotinamide adenine dinucleotide phosphate (NADPH). The reduction of NADPH is proportional to GPx activity and can be measured colorimetrically at 340 nm. The kit reagents were dissolved as described in the manual. A standard curve was prepared as described in the kit [45 (link)]. Measurements were performed according to a previous study [46 (link)]. The activity of GPx is expressed as mU/ml.

Malondialdehyde (MDA; ab238537), glutathione peroxidase (GPx; ab102530), and glutathione content (GSH; ab239727) ELISA kits were procured from Abcam Inc. (Cambridge, UK). Superoxide dismutase (SOD; MBS036924) and catalase (CAT; MBS726781) ELISA kits were acquired from My BioSource (San Diego, CA, USA) and performed in agreement with the manufacturer’s directions.

A thiobarbituric acid reactive substances (TBARS) assay kit was used to measure lipid peroxidation in the form of malondialdehyde (MDA) using tissue homogenate, according to the method of [43 ]. The resultant TBARS absorbance was calibrated at 532 nm using a spectrophotometer, and the outcomes were presented as nmol MDA/mg protein. Assays for antioxidant enzymes such as glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase (CAT) were performed on the tissue homogenate via a catalase activity assay kit (ab83464, Abcam, UK), a SOD assay kit (706002, Cayman chemical, Ann Arbor, MI, USA), and a glutathione peroxidase assay kit (ab102530, Abcam, UK), respectively.

GPx activity was assessed using a colorimetric assay kit (ab102530, Abcam, Cambridge, MA, USA) following the manufacturer’s instructions. In this assay, cells were washed with cold phosphate-buffered saline and resuspended in 200 μL of cold Assay Buffer. The samples were homogenized quickly by pipetting up and down a few times on ice. Cells were centrifuged at 4 °C at 10,000× g for 15 min using a cold microcentrifuge to remove any insoluble material. The supernatant was collected, transferred to a clean tube, and stored on ice before starting the assay. Total protein concentration in the samples was measured using the Pierce™ BCA protein analysis kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. The GPx activities were normalized by the protein concentrations.