Determination of total phenolic content, total flavonoid content, and antioxidant activity of plant extract fractions

Total phenolic content (TPC) of eight plant extract fractions was measured using the Folin–Ciocalteu test as previously described²⁵. Briefly, 0.1 mL of each fraction (re-solubilized as 1 mg/mL in 50% methanol) was mixed with 0.75 mL of the Folin–Ciocalteu reagent (diluted tenfold with deionized water before use) and incubated for 5 min at 22 °C. Next, 0.75 mL of 7.5% (w/v) sodium carbonate was added and the mixture was incubated for an additional 60 min at 22 °C. The absorbance at 725 nm was then measured using a LAMBDA XLS spectrophotometer (PerkinElmer). A calibration curve was made using 0–0.6 mg/mL gallic acid in 50% methanol; TPC was calculated as gallic acid equivalents in milligrams per gram of Z. bungeanum fractionated extracts.

Total flavonoid content (TFC) was determined using the aluminum chloride method as described previously²⁶. Quercetin standards (0–25 µg/mL) or test samples were prepared in methanol. Standard or sample dilution (1 mL) was mixed with 1 mL 2% AlCl₃, agitated for 30 s, and left to stand for 15 min at 22 °C. The absorbance of the mixtures at 430 nm was then measured, and TFC was calculated as quercetin equivalents in milligrams per gram of extract.

Total antioxidant activity was determined using the ABTS assay as described previously²⁷ and in Supplementary materials. This assay monitors the antioxidant-dependent reduction of ABTS·+ radical cation as a change in absorbance at 734 nm. Z. bungeanum fractions were prepared at 0.1–1 mg/mL concentrations in 50% ethanol; 0.9 mL of ABTS·+ working solution was added to 0.1 mL of sample and incubated for 6 min at 22 °C. Absorbance was then measured using Trolox as a standard. Experiments were performed in triplicate. The total antioxidant activity of each sample was expressed as percent inhibition (PI) as defined in Eq. (¹):

where A734_ABTS·+ is the initial absorbance of diluted ABTS·+ at 734 nm and A734_sample is the absorbance of the sample. The potency of each sample was expressed as the ability to scavenge 50% of ABTS radicals (IC₅₀, in mg/mL).