Methyl thiazolyl tetrazolium (mtt)

The MTT (3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide) (Nacalai Tesque) assay was performed to evaluate cell viability following viral infection according to methods previously described [46 (link)]. Camostat, E-64d, nafamostat (FUJIFILM Wako Pure chemical) and remdesivir (MedChemExpress) were serially diluted 2-fold increments in duplicates and plated on 96-well microplates in MEM containing 2% FBS. Vero-TMPRSS2 were infected with either WT or S mutants of SARS-CoV-2 at 4–10 TCID₅₀ and added to the plates. Plates were incubated at for 3 days, and CPE was determined for calculation of 50% endpoints using MTT assay. The concentration achieving 50% inhibition of cell viability (effective concentration; EC₅₀) was calculated.

Gallic acid of 97% purity, iron oxide coated with polyethylene glycol (PEG) nanocarrier (Fe₃O₄-PEG) and gallic acid-iron oxide coated with PEG nanocomposite (Fe₃O₄-PEG-GA) were provided by the Material Synthesis and Characterization Laboratory, Institute of Advance Technology University Putra Malaysia (Serdang Selangor Malaysia). All three drugs were used for the preliminary screening of the effectiveness between gallic acid nanocomposite and pure gallic acid against normal cell and three different types of cancer cell lines. To prepare the stock solution, 5 mg of each drug was initially dissolved in 200 µL of dimethyl sulfoxide (DMSO) before the mixture was vortexed and sonicated for at least 30 min to ensure that the drug was completely dissolved. Upon sonication, 800 µL of RPMI 1640 (Nacalai Tesque, Kyoto, Japan) was added and then vortexed for 2 min to make the total volume of 1 mL. The drug sub stock was further diluted to a series of concentrations (0.47–200 µg/mL) and was used on the same day it was prepared. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and Trypsin-EDTA (Ethylene diamine tetraacetate) (0.25%) were purchased from Nacalai Tesque (Kyoto, Japan).

Methanol, n-hexane, n-butanol, ethyl acetate, chloroform, Triton X-100 and Tween 20 were purchased from Fisher Scientific (Loughborough, Leicestershire, UK). Linoleic acid, gallic acid, quercetin, β-carotene (Type I synthetic, 95%), bovine serum albumin (BSA), ABTS, potassium persulfate, ferrous sulfate, hydrogen peroxide, AAPH, tetramethylchroman-2-carboxylic acid (trolox), ethylenediamine tetra acetic acid (EDTA), DPPH, dichloro-dihydro-fluorescein diacetate (DCFH-DA), Folin–Ciocalteu’s phenol reagent and ferrozine were supplied by Sigma-Aldrich Co. (St. Louis, MO, USA). Dulbecco’s Modified Eagle’s Medium (DMEM), gentamicin and fetal bovine serum (FBS) were purchased from GIBCO (Grand Island, NY, USA). The 8-iso prostaglandin F2α ELISA kit was bought from Cusabio (Wuhan, China). MTT and neutral red were obtained from Nacalai Tesque (Kyoto, Japan). All of the other chemicals were purchased from Sigma-Aldrich Co.

The lung adenocarcinoma cell line (A549) was obtained from the Japanese Collection of Research Bioresources (JCRB) Cell Bank, National Institute of Biomedical Innovation. Institute of Tropical Medicine, Nagasaki University supplied us with L. major, which was used as endorsed by the ethical board of the Institute of Tropical Medicine, Nagasaki University, Japan. M199 and Dulbecco’s Modified Eagle’s Media, kanamycin, dimethyl sulfoxide (DMSO), etoposide, miltefosine, fetal bovine serum, and MTT were obtained from Nacalai Tesque in Kyoto, Japan. The 96-well plates were purchased from Becton Dickinson (Franklin Lakes, NJ, USA). Solvents for the extraction, fractionation, and mass analysis were obtained from Sigma-Aldrich Inc., St. Louis, MO, USA.