Study On The Preparation Of TFDG And The Differentiation Effect Of Theaflavins On C2C12 Cells

Theaflavin-3,3’-digallate（TFDG）is a theaflavin in black tea which is condensed by ECG and EGCG under the catalysis of polyphenol oxidase,not only has an important impact on the quality of black tea,It also has a variety of biological activities such as lowering blood fat,lowering blood pressure,and antioxidant,and has broad application prospects in the fields of food,health products and natural medicine.At present,the preparation of total theaflavins on the market mainly uses the catechin in vitro simulated oxidation synthesis process.Its principle is similar to that of black tea processing.The reaction conditions are mild and the reaction substrates are all food-grade,which is effective for the industrial preparation of total theaflavins.There have been many reports on the approach,reaction mechanism and condition screening.However,due to the low content of TFDG in black tea and the similar structure to other theaflavins monomers,it is more difficult to obtain high-purity TFDG directly from black tea or from commercially available total theaflavins,and it cannot meet the purity and requirements of food and pharmaceutical research.Due to the quantity requirement,in view of the current purification technology of the tea group catechin EGCG and ECG monomers is relatively mature,and the cost is relatively controllable,two catechin monomers are directly selected for the enzymatic synthesis of TFDG It is expected to break through the bottleneck of industry preparation in this field.On the other hand,sarcopenia and cachexia caused by aging and chronic diseases inhibit the myogenic differentiation of patients,lose the quality and strength of skeletal muscle,and seriously reduce the quality of life of patients.According to reports,theaflavins can enhance the performance of skeletal muscles by promoting the differentiation of skeletal muscle stem cells,but there is no research to systematically study the activity of theaflavins to promote muscle differentiation.Substances（total content>57%）contribute to the work of screening myogenic differentiation activity.The main results are:1.In vitro simulated oxidation catalysis of EGCG and ECG to synthesize TFDG,the optimal reaction condition is 300 g crown pear homogenate with a molar ratio of 1:1 EGCG（1.55 g）and ECG（1.5 g）at37℃,oxygen 1 L·min^-1,stirring for 100 r·min^-1,and reacting for 40 min.Under this reaction condition,0.61 g of TFDG with a content of 36.34%can be generated with a yield of 21%.2.In this study,HP-20 was selected from 6 common macroporous resins to purify TFDG.The TFDG with 71%content could be prepared after the resin was purified twice and subjected to initial crystallization.3.Interfere with different concentrations of theaflavins to interfere the differentiation of mouse skeletal myoblasts（C2C12 cells）,count the number,length and diameter of differentiated mature myotubes,and detect the m RNA of myotube differentiation markers My OG,My OD and My HC Expression,from the two aspects of cell phenotype and gene expression level to evaluate the strength of theaflavins to promote myoblast differentiation activity.The results show that theaflavins mixture（TFs>57%）can enhance the differentiation ability of skeletal muscle stem cells,significantly improve the cell surface morphology,reduce its adhesion,stiffness and Young’s modulus,etc.,and the optimal concentration is 5μM.