The enzyme-oriented regulation of theaflavin-3, 3′-digallate synthesis and the accurate determination of its yield

Theaflavin-3,3 '-digallate (TFDG) is the main product formed by oxidative condensation between (-)-Epigallocatechin-3-gallate (EGCG) and (-)-Epicatechin-3-gallate (ECG). Some sub-products, such as homo- and hetero-oligomers of EGCG and ECG, are also produced simultaneously, which significantly decreases the yield of TFDG. In general, the optimum temperature for TFDG synthesis is 20 degrees C below which can reduce its generation resulting in the coexistence of a large amount of unreacted substrates (ECG and EGCG). Therefore, a higher possibility of increasing the formation of TFDG could be achieved if a suitable synthetic method can be developed at a low temperature. Our results showed that simultaneous dropwise addition of both EGCG and ECG could markedly enhance the content of TFDG at ice bath. Using such technique, the production of TFDG increased by 47.60% as compared to the group synthesised under the optimum temperature. The absorbance of TFDG at 278 nm is 2.36 times higher than that of ECG with equimolar concentration. Therefore, a rapid, simple and accurate calculation method of TFDG yield was developed via the spectral correlation. Based on the counting method, the yield of TFDG was 30.58% by using the novel synthetic method of TFDG.