2.3. Characterisation of the interaction between α-La and CA or EGCG
2.3.1. Determination of total phenolic content
The total phenolic contents of α-La–CA or EGCG covalent complex and controls of α-La, CA and EGCG were determined according to the Folin–Ciocalteu method with CA or EGCG as a standard. Briefly, 0.5 ml of the appropriately diluted sample was mixed with 2.5 ml of freshly prepared Folin–Ciocalteu reagent (10 N) at room temperature. After 5 min, 2 ml of 7.5% (w/v) sodium carbonate solution were added, the mixture was vortexed and stood for 2 h at room temperature in the dark. The absorbance was measured with a UV-1800 UV–vis spectrophotometer (Shimadzu, Japan) at 760 nm. Measurements were carried out in triplicate and data were expressed as means ± SEM (standard error of mean) (He et al., 2011).