Preparation And Antioxidant Activity Of Anthocyanin From Kunlun Coreopsis Tinctoria Nutt

Anthocyanin is an important water-soluble pigment,because of its natural,non-poisonous,non-side effect.It has a great diversity of bio-activities,and become a hot topic already.Coreopsis tinctoria Nutt?C.tinctoria?has been a tea in usual because of its particular growing environment and the multiple protections of stomach,spleen,skin,blood and so on.Nowadays,the researches about C.tinctoria are concentrated on its total flavonoids,but the studies of C.tinctoria anthocyanin and protection pathways are limited.The best situation of preparation and purification will be detected,and exploration of antioxidant and the protection against H₂O₂-induced will be researched in this paper.It will provide the experimental and theoretical basis,which it can be produced in industry and used in antioxidants.The best preparation technology of C.tinctoria anthocyanin by ultrasonic assisted with enzymatic method were studied,the hydrolysis temperature,enzyme activity,time of ultrasonic and ethanol content were chosen as single factors.The response surface experiments were used to optimize the extraction conditions.With the help of AB-8 macroporous resin,the crude products were purified by static and dynamic methods,the best purified condition was obtained by single factors and the color value were investigated,the effects of methods were compared.To ensure the activities of C.tinctoria anthocyanin,the stability was measured through three levels,physical factors,chemical factors and metal ions.Finally,the antioxidants of C.tinctoria anthocyanin from three levels were demonstrated.On Chemical level,the DPPH,·O₂^-and·OH^-can be cleared by C.tinctoria anthocyanin through radical scavenging experiments.On cellular level,the cell morphological observation,MTT,LDH,DCFH-DA and Hoechst 33258 assays were used to indicate the influence of C.tinctoria anthocyanin on INS-1 cells.On oxidation mechanism,the RT-PCR and western blot experiments were selected to prove the protection mechanism of anthocyanin.Experiments results manifested that ultrasonic assisted with enzymatic method is a proper method to prepare C.tinctoria anthocyanin,the optimum extracting process:hydrolysis temperature 55?,enzyme activity 105 U,ultrasonic 20 min and ethanol content 60%.Under this circumstance,the best extraction ratio is?1.234±0.023?mg/g,purification through AB-8 macroporous resin,the best adsorption conditions of static purification:adsorption time 5 h,macroporous resin content 8 g,the crude products concentration 8 mg/mL and pH 3.The best desorption conditions:eluent50%ethanol,desorption time 3 h.The best adsorption conditions of dynamic purification:adsorption velocity 2 mL/min,the crude products concentration 8mg/mL,pH 2 and beam filtered 4 times;the best desorption conditions:desorption velocity 2mL/min,eluent 50%ethanol.The dynamic purification was better than static purification,the crude products were purified 10 times.The C.tinctoria anthocyanin storage need avoid the appearance of high temperature,oxidants and Ca²⁺.In addition,C.tinctoria anthocyanin has a strong antioxidant activity,it can savage the DPPH,·OH^-and·O₂^-ions.In INS-1 cells,it can protected INS-1 cells against H₂O₂-induced damage,several assays indicated that the quantities of adherence cells,survival,LDH,ROS,etc better than the H₂O₂ group under C.tinctoria anthocyanin protection.The protect mechanism of C.tinctoria anthocyanin is though Nrf2pathway,and enhance the expression of antioxidant enzymes,so that oxidant injury is decreased.This study demonstrated that C.tinctoria anthocyanin has a strong antioxidant function,it's an evidence for C.tinctoria anthocyanin used in antioxidant medicine.