Characterisation of anthocyanidin reductase from Shuchazao green tea

Abstract

Flavan-3-ols, which account for approximately 700-800 g kg(-1) of tea polyphenols, exert many health-promoting effects. Anthocyanidin reductase (ANR) is an important enzyme involved in the biosynthesis of flavan-3-ols in the tea plant. The purpose of this study was to establish a suitable method for the determination of ANR activity. Thin layer chromatography (TLC), high-performance liquid chromatography (HPLC) and mass spectrometry (MS) analyses showed that cyanidin and delphinidin were converted into epicatechin and epigallocatechin respectively via ANR by using reduced nicotinamide adenine dinucleotide phosphate (NADPH) as a coenzyme in the tea plant. In order to measure ANR activity via NADPH concentration changes at 340 nm, several interference factors were studied. The interferences from the high background absorbance of substrate and coenzyme and the oxidation reaction of substrate and product were excluded by devising control experiments, decreasing substrate and coenzyme concentrations or adding antioxidants. The optimal pH and concentrations of substrate and NADPH were chosen such that the ANR assays were carried out at 45 °C for 25 min in a total volume of 1.5 mL of reaction mixture containing 0.1 mol L(-1) phosphate buffer (pH 6.5), 0.0667 mmol L(-1) cyanidin, 1 mmol L(-1) NADPH, 0.53 mmol L(-1) ascorbic acid and 150 µg total protein. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analysis showed that the trends in ANR gene expression corresponded with the enzyme activity in leaves at different development stages. The proposed method is simple, rapid, sensitive and suitable for the determination of ANR activity in the tea plant.