Abstract: To overcome the difficulty of isolating theasinensin A (TSA) from tea, polyphenol oxidase (PPO) extracted from Fengshui pears was employed to catalyze the low-temperature oxidative conversion of epigallocatechin gallate (EGCG) into TSA. High-purity TSA was subsequently obtained by dynamic axial compression chromatography. Its inhibitory effects on enzymes involved in carbohydrate and lipid metabolism, as well as its behavior during in vitro simulated digestion, were systematically evaluated. The results showed that the purity of the prepared TSA was as high as 98.92%. TSA had a molecular mass of 914 Da and a molecular formula of C44H34O22. One-dimensional proton nuclear magnetic resonance spectroscopy (1H NMR) and carbon-13 nuclear magnetic resonance spectroscopy ( 13C NMR), together with two-dimensional NMR analysis, confirmed its symmetric dimeric structure. The half-maximal inhibitory concentration (IC50) of TSA against α-amylase was (0.34 ± 0.01) mg/mL, and its maximum inhibitory rate reached (86.45 ± 0.78)%, which was significantly higher than those of EGCG and acarbose (P < 0.05). The inhibitory rate of TSA against α-glucosidase remained below 50% throughout the tested concentration range, with an IC50 value greater than 100 μg/mL. The IC50 values of TSA against lipase and cholesterol esterase were (29.96 ± 2.28) and (32.77 ± 2.54) mg/mL, with maximum inhibitory rates of (73.79 ± 1.74)% and (81.00 ± 2.17)%, respectively. These inhibitory effects were significantly stronger than those of EGCG and orlistat (P < 0.05). In vitro simulated digestion showed that the inhibitory activities of TSA against α-amylase and lipase varied with digestion stages, remaining relatively high during the oral and gastric phases but decreasing significantly during the intestinal phase. This pattern may be attributed to the relatively high digestive stability conferred by the dimeric structure of TSA. These findings provide a theoretical basis for the green and efficient preparation of TSA and its potential application in the development of functional food products.

Key words: theasinensin A; low-temperature enzymatic synthesis; dynamic axial compression chromatography; glucose and lipid metabolic enzymes; inhibitory effect; in vitro simulated digestion