Puerarin Palmitate Purification Protocol

The obtained product, puerarin palmitate, was purified by liquid–liquid extraction. The immobilized enzyme in the reaction mixture that reacted under optimal conditions was isolated through filtration, and the reaction solution was dried using a vacuum desiccator. The powder obtained by drying was dissolved in 35 mL of an acetonitrile/n-heptane (2:5, v/v) solution at 60 °C. The unreacted acyl donor in the n-heptane phase was then removed by separating the acetonitrile phase. The separated acetonitrile phase was then dried in a vacuum desiccator. To remove unreacted puerarin, the obtained powder was dissolved in 40 mL of an ethyl acetate/water (3:5, v/v) solution at 60 °C. Puerarin palmitate was finally obtained by separating and drying the ethyl acetate phase in which the puerarin ester had been dissolved. All the processes were repeated three times to improve the purity of the product. Afterwards, it was purified using thin-layer chromatography (TLC) and column chromatography on silica to obtain a higher-purity product. A mixture of ethyl acetate:chloroform:methanol (5:4:1, v/v/v) was used as the eluent.