3.6. In Vitro Evaluation of CoNiPr (x ≤ 0.10) NSFs)

Colorectal carcinoma HCT-116 and embryonic kidney cells HEK-293 were purchased from American Type Culture Collection (ATCC), Manassas, VA, USA. The cells were considered for anticancer activity after treatment with CoNiPr (x ≤ 0.10) NSFs) to examine cell viability. The cells (20,000/well) were first grown in a 96-well culture plate in the DMEM media comprised of fetal bovine serum + L-glutamine + penicillin + streptomycin + selenium in a CO₂ incubator at 37 °C. Once the cells achieved 70–80% confluence, they were treated with CoNiPr (x ≤ 0.10) NSFs) with dosages (2.0 µg/mL to 500 µg/mL) for 48 h and were taken for the MTT assay [41]. The control group was not treated with CoNiPr (x ≤ 0.10) NSFs). Thereafter, cells were immersed in the MTT solution (5.0 mg/mL) and were subsequently treated with DMSO (1%). The optical density (OD) was read with an absorbance value of 570 nm on the microplate reader (Biotek Instruments) and % cell viability was calculated using Equation (2) [42]. The inhibitory concentration (IC50) was calculated using GraphPad Prism 8. The unit for the inhibitory concentration (IC₅₀), expressed in µg/mL+, is the Standard Deviation. The statistical data presented were considered from triplicates, and the data were analyzed by using GraphPad Prism 10 Software, San Diego, CA, USA.