Isolation And Purification Of Theaflavins And Their Antioxidative And α-glucosidase Inhibition Activities

Theaflavins（TFs）are a class of natural active substances in black tea.There are more than 20 kinds of theaflavins,including theaflavin,theaflavin-3-gallate,theaflavin-3’-gallate and theaflavin-3,3’-digallate were the most abundant.Theaflavins have antioxidant,hypoglycemic,antibacterial and anti-inflammatory effects.In this paper,we extracted and purified theaflavin from black tea powder to study its antioxidant and hypoglycemic activities.The antioxidant activity was investigated by adding theaflavin into beverages,and theaflavin microcapsules were prepared for gastrointestinal fluid simulation tests and stability tests.The main results of research are as follows:（1）The theaflavins extraction technology were studied based on single-factor and response surface analysis（RSA）.The optimal extraction process was as follows:the extraction time was 75 min,the extraction temperature was 80℃ and the pH was 4.The extraction rate reached 30.57%under these conditions.Extraction solution of theaflavins was graded by polyamide resin.The optimal results showed that the eluent was ethanol:acetone（50:1）,the elution flow rate was 1 mL/min,and the elution volume was 125 mL,while the theaflavins enriched with polyamide resin increased from 15.21%to62.48%.Therefore,the lyophilized sample was separated by high-speed countercurrent chromatography（HSCCC）in order to get its each component.Theaflavin,theaflavin-3-gallate,theaflavin-3’-gallate and theaflavin-3,3’-digallate were obtained,while their purity was 94.09%,93.62%,81.95%and93.84%by the peak-area normalization method of HPLC,respectively.（2）The four monomers of theaflavins were found to have strong in vitro antioxidant activity by DPPH·,ABTS⁺,PTIO·methods.The inhibitory effect of the four monomers of theaflavins onα-glucosidase was examined,among which theaflavin-3,3’-digallate showed the strongest inhibitory effect with an IC₅₀ value of 0.05 mg/mL.The interaction mechanism between theaflavins andα-glucosidase was verified by molecular docking technique,and it was found that theaflavins could bind to the active site ofα-glucosidase and occupy the groove of protein molecules,thus reducing the catalytic efficiency of the enzyme.（3）Comprehensive application of theaflavin was performed.Theaflavin was added to the apple rose vinegar compound beverage.Under the accelerated test conditions of light and dark,the theaflavin-added compound beverage had better clearance rates of ABTS⁺,DPPH·,PTIO·under the dark condition than that without the addition of theaflavin.The average improvement of the 0-20day clearance rate was 28.56%,24.36%,and41.48%,respectively.Experiments such as the simulated digestion of microcapsules in vitro show that theaflavin microcapsules have good temperature stability,acid stability and stability under the oxidant H₂O₂.Microcapsules can slowly release theaflavins in simulated gastric and intestinal fluids.