| The anthocyanins from red cabbage can be used as a good natural pigment with high water solublilty,health benefits and easy to obtain.The structure analysis,separation and properties studies of red cabbage anthocyanins is importance for its production and application.In this study,the method of high performance liquid chromatography(HPLC)analysis of anthocyanins from red cabbage was established.The adsorption process and modeling for anthocyanin purification from the extrusion of red cabbage was studied.Further more,the stability of anthocyanins in milk were explored and the antioxidation properties were evluated.In the optimization of HPLC analysis of red cabbage anthocyanins,the methanol/water were used to instead of acetonitrile/water system in the traditional analysis.The mobile phase gradient,column temperature,flow rate and injection volume were optimized.The optimal separation conditions were obtained as follows:Ultimate LP-C18 column(4.6 × 250 mm,5 ?m);Phase A:5%formic acid aqueous solution,B:methanol;The elution linear grdient:0-5 min 20-30%B,12 min 33%B,18 min 40%B,25 min 60%B,30 min(stop).The flow rate,injection volume,detection wavelength,column temperature and balance time were 1.0 mL/min,10?L,517 nm,45 ? and 5 min,respectively.The structures of 14 anthocyanins were confirmed by HPLC-ESI-MS/MS,which based on separation conditions of HPLC.All the main structure of red cabbage anthocyanins is cyanidin for red cabbage in this study.Among 14 anthocyanins,there are 19%,48%,and 33%of non-,mono-and di-acylated anthocyanins,respectively.The results showed that the method established in this study can be used for qualitative and quantitative analysis of anthocyanins in red cabbage.The process and models of adsorption process of anthocyanins from red cabbage by using macroporous resin were studied.The adsorption and desorption characteristics of six macroporous resins(AB-8,LX-60,X-5,LX-68,XDA-8 and NKA)were investigated.The X-5 was tested as the best adsorbent for adsorption and desorption.The conditions of elution were optimized,and the results showed that 80%ethanol in pH 4 was the best eluent.The dynamic adsorption conditions of red cabbage anthocyanins using X-5 macroporous resin were studied.The results were obtained as follows:volume and flow rate were 6 BV,1.5 mL/min,respectively,and the resin has high utilization rate;elute volume and flow rate were 4 BV and 1.5 mL/min,respectively,and anthocyanins were eluted completely.The purity of red cabbage anthocyanins powder was found to increase from 0.9%to 20%after purifying by X-5 resin.The static adsorption process of the X-5 macroporous resin was fitted for kinetics and isotherms and thermodynamics models.The R2 of pseudo-second-order model was 0.9968 which showed its good performance.The results showed that pseudo-second-order kinetic model calculated value(Qe.c,24.82 mg/g)was in good agreement with the experimental value(Qe,23.56 mg/g).The adsorption isotherms data fitted the Langmuir model well at low temperature(25 ?,R2=0.9943),and the results showed that the isotherm is favorable when 1/KL between 0 and 1.The thermodynamic parameters of ?H°=-33.96 kJ/mol(|?H°|<43 kJ/mol?G°<0)and ?S°=119.73 J mol-1 K-1 were obtained,which showed that the resin adsorption was the spontaneous,exothermic and physical adsorption process.The method and models provide kinetic and thermodynamic data for the industrial application of purification of red cabbage anthocyanins.The thermal stability of red cabbage anthocyanins in pure milk and aqueous was studied under conditions of 70-90 ?,and the kinetic models were established.Degradation of red cabbage anthocyanins in milk and water were fitted to the first order kinetics model.The degradation rate of red cabbage anthocyanins in pure milk was faster than in aqueous under the same temperatures.The activation energy Ea of red cabbage anthocyanins in water and milk were 28.493 and 64.706 kJ/mol at 70-90 ?,respectively,indicating that red cabbage anthocyanins in milk was more sensitive to temperature.The thermostability of anthocyanins from red cabbage in casein,sodium and calcium solutions in 90 ? was also investigated.The results showed that the degradation of red cabbage anthocyanins was accelerated in casein solution.The results of HPLC analysis showed that monoacylated anthocyanins were more stable than non-and di-acylated in the milk and water system.Finally,the antioxidant activity of red cabbage anthocyanins was investigated by using the DPPH,FRAP and protection of cellular damage methods.The DPPH and FRAP methods showed that the antioxidant capacity of red cabbage anthocyanins increased with the concentration in a certain concentration range.The antioxidant capacity of purified anthocyanins was higher than the unpurified anthocyanins.In the oxidative stress test of HepG2 cells,the protective effect of purified anthocyanins at 1 mg/mL was the best and the cell viability was 82.33%.Intracellular ROS level was reduced significantly by purified red cabbage anthocyanins,and fluorescence intensity was 104.45%,which indicated that purified anthocyanins have strong antioxidant activity. |
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