Isolation and Purification of Anthocyanins from Blood Oranges by Column Chromatography

In this study, isolation and purification of anthocyanins from blood oranges by column chromatography were investigated, and then the anthocyanins of blood orange were identified. The behaviors of static adsorption and desorption, dynamic adsorption and desorption of 12 kinds of resins were compared. The results indicated that NKA-9 macroporous resin was optimum for isolation of blood orange anthocyanins, and the optimal elution reagent was 50% ethanol with citric acid （pH 2.5）. Toyopearl TSK HW-40S column was employed to separate and purify the anthocyanin extracts from blood orange. The best separation of Toyopearl TSK HW-40S column was obtained using a mobile phase of 35% methanol with 2% formic acid at a flow-rate of 0.6 mL min-~. Three kinds of anthocyanins were purified from blood orange. Then, the anthocyanins of blood orange were identified by HPLC-ESUMS analysis. The results showed that cyanidin-3-glucoside （35.2%） and cyaniding-3-（6＂-malonyl） glucoside （42.9%） were the major anthocyanins of blood orange. Furthermore, cyanidin-3-（3＂-malonyl） glucoside, cyanidin 3-（6＂-dioxalyl） glucoside and cyanidin-3-glucoside adduct：4-vinylcatechol were identified in blood orange. The combination of NKA-9 macroporous resin and Toyopearl TSK HW-40S column chromatography for isolation and purification of blood orange anthocyanins was an effective method, and HPLC-ESI/MS analysis was a convenient, rapid and effective method for identification of anthocyanins from blood orange.

Thermal Degradation Kinetics of Three Kinds of Representative Anthocyanins Obtained from Blood Orange

Cyanidin-3-glucoside, cyanidin-3-（6′′-malonyl）-glucoside, and cyanidin-3-glucoside-derived pyranoanthocyanins which were three major anthocyanins of blood orange, were obtained using a Toyopearl TSK HW-40S column chromatography. Then, thermal degradation kinetics of the three major anthocyanins was studied at selected temperatures （70, 80, and 90°C）. Degradation parameters such as k and t1/2 values were determined. The activation energy （Ea） values for cyanidin- 3-glucoside, cyanidin-3-（6′′-malonyl）-glucoside and cyanidin-3-glucoside-derived pyranoanthocyanin were 75.4, 79.5, and 81.7 kJ mol-1, respectively. Ea values suggested that cyanidin-3-glucoside-derived pyranoanthocyanin had the highest stability, followed by cyanidin-3-（6′′-malonyl）-glucoside and cyanidin-3-glucoside. However, t1/2 values indicated cyanidin- 3-glucoside-derived pyranoanthocyanin degraded faster than cyanidin-3-（6′′-malonyl）-glucoside and cyanidin-3-glucoside at selected temperature.

Thermal Degradation Kinetics of Anthocyanins and Visual Color of Blood Orange Juice

Thermal degradation kinetics of anthocyanins and visual color （Hunter α value） of blood orange juice were studied at selected temperatures （70-90℃）. Results indicated that both the thermal degradation of anthocyanin and visual color all followed first-order reaction kinetics, and they could be expressed by Arrhenius equation. The activation energy values for the anthocyanins degradation and visual color degradation were 55.81 and 47.51 kJ tool-1, respectively. The linear relationship between visual color and anthocyanin content was obtained. Furthermore, during thermal processing of blood orange juice, the formulas about the linear relationships showed no significant difference at selected temperatures. So, the relationships between visual color and anthocyanins content during thermal processing at selected temperatures could be described by the same equation： α＊/αo＇=0.559（C/Co）＋0.43. It might be inferred that visual color measured instantaneously by tristimulus colorimeters for on-line quality control, could be used to predict the anthocyanins degradation during thermal processing of blood orange juice.

Changes in phenolic content,composition,and antioxidant activity of blood oranges during cold and on-tree storage

Citrus fruits are rich in phenolic compounds that possess several health benefits.However,few studies have focused on the changes in phenolic compounds in citrus fruits during postharvest storage.This study dynamically monitored the phenolic content,components and antioxidant activity of‘Tarocco’blood oranges during a period of 12-week cold storage and on-tree storage,respectively.We investigated the alteration mechanism of phenolic compounds in blood oranges by evaluating phenylpropanoid pathway-related enzyme activities and gene expression.Results showed that flavanones were the main phenolic compounds in blood oranges.Both storage methods mainly stimulated the accumulation of phenolic acids to improve total phenolic content,which reached the maximum at week 12.Nonetheless,blood oranges had a higher phenolic content and antioxidant activity under on-tree storage than cold storage.Furthermore,the enzyme activities and gene expression of the phenylpropanoid pathway demonstrated that the accumulation of phenolics in blood oranges during storage was highly related to the activation of the phenylpropanoid pathway.These results demonstrate that on-tree storage is a potential approach for extending the supply period of blood orange from the perspective of phenolic compounds.