Imark 168 1130

The free radical scavenging assay was determined as described by Adedapo and Jimoh [25 ]. FRAP reagent in 25 mL of acetate buffer (300 mM, pH 3.6), 2.5 mL of tripyridyltriazine (10 mM), and 2.5 mL of FeCl₃·6H₂O (20 mM) was warmed and kept in a water bath at 37 °C. Then, 30 μL volume of 1 mg/mL sample was mixed with 900 μL FRAP solution, and then, the mixtures were allowed to stand in the dark for 30 min. Using a microplate absorbance reader (iMark 168–1130, Bio-Rad laboratories, Hercules, CA, USA), the absorbance of the colored result, a ferrous tripyridyltriazine complex, was measured at 595 nm and was expressed as mg gallic acid equivalent/g.

Both normal colon (FHC; ATCC^® CRL-1831™) and colon cancer (HCT-116; ATCC^® CCL-247™)) cells were procured from the American Type Culture Collection (ATTC) and maintained in Dulbecco’s Modified Eagle Medium/F-12 (DMEM⁄F12; cat no. D0547.DMEM⁄F12, Sigma-Aldrich, Saint Louis, MO, USA). Both cell types were provided with 2 mM L-glutamine (Catalog #: BE17-605E Lonza, Bornem, Belgium) and 10% fetal bovine serum (FBS) purchased from (CAT.NO.12103C Sigma-Aldrich, St. Louis, MO, USA), 1% penicillin/streptomycin (Lonza, Belgium). Cell incubation was done at 37 °C in a 5% carbon dioxide atmosphere (NuAire). Cell plating at a density equal to 5000 cells was undertaken in triplicate in a plate of 96 wells [46 ]. On the second day, cells were treated with 5-FU or nitazoxanide at the (0.01, 0.1, 1, 10, and 100 µM) concentrations. Cell viability was assessed after 48 h using MTT solution (Promega, Madison, WI, USA) [47 (link)]. 20 μL of MTT dye were transferred to wells and then incubation of the plate was allowed for a period of three hours. Colour intensity was subsequently read at 570 nm employing a BIO-Rad enzyme-linked immunosorbent assay (ELISA) microplate reader (iMark™ #1681130, BIO-Rad, CA, USA). The viability was calculated relative to a control and the IC50 values were determined using the GraphPad prism 7 as previously reported [48 (link),49 (link)].

Blood and salivary glucose levels were determined using the glucose oxidase peroxidase (GOD-POD) method. Blood and saliva samples were separated into three test tubes and labeled “Blank”, “Standard”, and “Test”. The following steps were implemented: first, add 50 μL of different concentrations of standards to the “Standard; add 40 μL of sample diluent to the wells of the samples to be tested, and then add 10 μL of the samples to be tested; add 100 μL of enzyme-labeled reagents to each well, except for the “Blank”; seal the plate with a sealing film and incubate at 37 °C for 60 min; dilute the 20-fold concentrated washing solution with 20-fold distilled water for later use; remove the sealing membrane, discard the liquid, spin dry, fill each well with washing solution, let set aside for 30 s and discard, repeat 5 times, and pat dry; add 50 μL of color developer A to each well, then add 50 μL of color developer B, gently shake and mix, and develop color at 37 °C for 15 min in the dark; stop adding to each well; terminate the reaction was by adding 50 μL of solution; finally, set the “Blank” to zero, and measure the absorbance (OD value) of each well in sequence at a wavelength of 450 nm with a microplate reader (Bio-Rad iMark, 168-1130, Tokyo, Japan).