Sodium carbonate

Analytical grade acetone, acetic acid, anhydrous sodium acetate, ethanol, hexane, hydrochloric acid, potassium chloride, sodium carbonate, and sulfuric acid were obtained from Chem Supply (Port Adelaide, South Australia, Australia). High pressure liquid chromatography (HPLC) grade methanol, acetonitrile, formic acid, and ammonium acetate as well as analytical standards (Folin–Ciocalteu reagent, vanillin, gallic acid, cyanidin-3-O-glucoside, (+)-catechin, coumestrol, formononetin, genistein, and naringenin) were purchased from Sigma Aldrich (Australia).

Total phenolic content (TPC) of ethanol, hexane, methanol and water extracted herb samples were spectrophotometrically analyzed according to Folin–Ciocalteu colorimetric technique [34 ]. Samples were diluted (10–1000 times) with Mili-Q water and 25 µL from each dilution added to the 96-well polystyrene plate (Sarstedt, Nümbrecht, Germany). Several concentrations of gallic acid (3,4,5-Trihydroxybenzoic acid ≥98%, Sigma-Aldrich, St. Louis, MO, USA), 0–100 mg/L, were prepared to construct the standard calibration curve and 25 µL of gallic acid was loaded into the plate. All wells were loaded with 125 µL of Folin-Ciocalteu’s phenol reagent (Sigma-Aldrich, St. Louis, MO, USA), followed by the addition of 125 µL Sodium carbonate (Chem-Supply, Gillman, Australia). The 96-well plate placed in a microplate-reader (Tecan, Grödig, Austria) and shaken for 15 s and the absorbance reading measured at 750 nm after 15 min of incubation in the dark. Calculated results expressed as milligram of gallic acid equivalents per gram of sample dry weight (GAE/g DW).

All reagents used were analytical grade or higher purity. Hydrochloric acid and sodium carbonate was purchased from Chem-Supply. All other reagents including the HPLC grade methanol, were purchased from Sigma-Aldrich (Australia). Some reagents used in the cytotoxicity analysis which included the CellTiter 96^® AQueous Assay (composed of solutions of tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)−5-(3-carboxymethoxyphenyl)−2-(4-sulfophenyl)−2H-tetrazolium, inner salt; MTS(a)] and an electron coupling reagent (phenazine methosulfate; PMS), commonly known as MTS reagent, and Fetal bovine serum (FBS) were obtained from Promega (United States of America) and Cytiva (United States of America), respectively. The Dulbecco’s Modified Eagle’s Medium—high glucose (DMEM), Dulbecco’s Phosphate Buffered Saline (DPBS) solution were kept in the dark at 4 °C, while the other reagents used in the bioassays were frozen until required for use. All dilutions and assay preparations used Milli-Q water.