Isovitexin

Mung bean seed coat was obtained from Kittitat Co., Ltd. Dulbecco’s modified Eagle’s medium (DMEM) and Roswell Park Memorial Institute Medium 1640 (RPMI) were obtained from Hyclone (Logan, UT, USA). Fetal bovine serum (FBS), penicillin–streptomycin, nonessential amino acids, and insulin human recombinant zinc were obtained from Gibco (Paisley, UK), whereas 2-deoxy-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-D-glucose (2-NBDG) was obtained from Invitrogen (Waltham, CA, USA) and 2′,7′-dichlorofluorescin diacetate (DCFH-DA), vitexin (VIT) and isovitexin were obtained from Sigma-Aldrich (St. Louis, MO, USA). Lipopolysaccharides (LPS; Escherichia coli O111:B4) were obtained from Sigma-Aldrich (St. Louis, MO, USA). All other chemical reagents used were of analytical grade. Deionized water was used for preparing all solutions. All chemicals used in the study of SCFA and gut microbiota were of analytical or chromatographical grade, from Merck (Darmstadt, Germany) and Sigma.

Folin-Ciocalteau’s phenol reagent (FCR), 2,2′-diphenyl-1-picrylhydrazyl (DPPH) radicals, gallic acid, 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 6-hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxylic acid (Trolox), 2,4,6-tri(2-pyridyl)-s-triazine (TPTZ), β-carotene, linoleic acid, Tween 40, HPLC standards, including caffeic acid, p-coumaric acid, orientin (luteolin 8-C-β-d-glucoside), isoorientin (luteolin 6-C-β-d-glucoside), vitexin (apigenin 8-C-glucoside), isovitexin (apigenin 6-C-glucoside), vicenin-2 (apigenin 6,8-di-C-β-d-glucoside), and schaftoside (apigenin 8-C-α-l-arabinoside 6-C-β-d-glucoside) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Sodium persulfate, ferrous chloride, and the solvents were provided by Avantor Performance Materials (Gliwice, Poland).

Since the previous study [18 (link)] established that the extracts at 100 μg/mL did not induce significant cytotoxicity on human embryonic kidney cells and macrophage, this indicated that the extracts may not have any cytotoxic effect to non-cancerous cells. Hence, concentrations used were up to 100 μg/mL. Cells were plated in 384-well plates for 24 h followed by LP, LN, SP and SN treatment at 1-100 μg/mL for 72 h incubation as per established in previous studies [11 (link)]. As a comparison, we also tested vitexin and isovitexin (Sigma Aldrich, USA), the pure compounds that were commonly isolated Clinacanthus nutans extracts [13 (link)]. CellTitre-Glo® Luminescent Cell Viability Assay kit (Promega, USA) was used to quantify the cell viability based on the luminescence readings recorded using SpectraMax M3 Multi-Mode Microplate Reader (Radnor, USA). IC₅₀ values were calculated based on the dose-response curve generated. The experiments were validated using with 100 μg/mL gemcitabine and 5-fluorouracil, the first line clinically used anti-cancer agents.