| Blackberries have been recently reported as a good source of anthocyanins, which were shown to have potential health benefits, including inhibition of qualitative lipid peroxidation and free radical scavenging activity, lower serum cholesterol and neutral fat, have anti-mutation and anti-tumor effects and improve liver function and vision. Blackberry anthocyanins exhibit broad application prospects in the food, pharmaceutical, cosmetics and other fields.The enzymatic hydrolysis technology of blackberry juice was investigated using the mixture of pectinase and multiplexed pectinase in this paper. Blackberry anthocyanins were separated and purified using macroporous resin and gel column chromatography and their structures were analyzed. Thermal stability of blackberry anthocyanins was investigated to explore their degradation mechanism. Copigmentation effects on the blackberry anthocyanins were compared by organic acids including the stability and antioxidant ability. Copigmentation effects of adding different organic acids on the blackberry anthocyanins in the water system were compared. The changes of blackberry anthocyanins before and after co-pigmentation were analyzed. The protective effects of blackberry anthocyanins on human endothelial cell damage induced by H2O2were investigated to illustrate their antioxidant mechanism. Results were shown below:1. To prepare clarified blackberry juice, blackberries were hydrolyzed by the mixture of pectinase and multiplexed pectinase. The clarification effects of the two enzymes was studied. Taking the juice yield as indexes, the conditions of enzymatic reaction were determined and optimized using response surface methodology(RSM). The results showed that the optimum conditions were:the ratio of two enzyme’s activity was1:1, the enzyme additive amount was0.13%, the temperature was43℃and the time was2.5h. Under these conditions, the blackberry juice yield was79.5%. Compared with no enzymatic processing, the blackberry juice yield was increased75.8%and its quality and stability was improved remarkably.2. To obtain highly purified anthocyanins from blackberry, the adsorption and desorption characteristics of6kinds of macroporous resins were carefully compared. Furthermore, the characteristics of static adsorption dynamics, dynamic adsorption and elution dynamics were investigated. The results showed that AB-8macroporous resins exhibited the best performing resin to obtain highly concentrated extracts. The time of absorption and desorption equilibrium was4h and3h at25℃, respectively.75%ethanol solution is the optimum solution for desorption of blackberry anthocyanins. The optimal purification conditions were as following:2BV/h,75%ethanol of5BV as elution phase, and the elution rate1BV/h. Under these conditions, sample yield was1.22%, anthocyanins recovery yield was75.45%and its purity was86.50%. The purification conditions of Sephadex LH-20were as following:the sample flow rate0.6M1/mim,40%,60%and80%ethanol containing0.01%hydrochloric acid as elution liquid. Then the sample was analyzed by HPLC-ESI/MS and the results showed that five anthocyanins were identified, including cyanidin(Cy)-3-O-glucoside, Cy-3-O-arabinoside, Cy-3-O-malonyl-glucoside, Cy-3-O-dioxalyl-glucoside and Cy derivative.3. The effects of pH and temperature on the stability and antioxidant activity of anthocyanins in blackberry juice were studied at selected temperatures (70℃,80℃and90℃) at pH2.0, pH3.0, pH4.0, respectively. Degradation parameters such as half-lives (T1/2), degradation constant (k) and activity energy (Ea)values were determined. Analysis of kinetic data indicated that the thermal degradation of anthocyanins followed first-order reaction kinetics. The values of k, T1/2and Ea decreased obviously with pH and temperature increasing. The Ea of blackberry anthocyanins at pH2.0,3.0and4.0was40.09%28.80and28.71kJ mol-1.and T1/2of blackberry anthocyanins was6.19h,4.28h and2.97h, which indicated their are more stable that lower temperature and pH.Four individual anthocyanin degradation followed first-order reaction kinetics. Their k, T1/2and Ea values were significantly differed from one another (P<0.05). Maximum of the T1/2values were observed with Cy-3-O-dioxalyl-glucoside, which T1/2values was9.48,6.83and3.54h at70,80and90℃. Meanwhile, the T1/2values of Cy-3-O-glucoside was9.04,6.62and3.09h. However, T1/2values of Cy-3-O-arabinoside was similar with Cy-3-O-malonyl-glucoside. Under the same conditions, the amount of the Cy derivative was observed to slightly increase with temperature increased, which showed that anthocyanins derivative formed.The antioxidant capacity of blackberry juice, measured by DPPH assays, was decreased after thermal treatment, indicating that antioxidant activities in the samples was highly correlated with anthocyanins degradation.4. Copigmentation effects of adding different organic acids on thermal stability of blackberry anthocyanins in water system were investigated, as well as their color and antioxidant activity using the purified fraction2of blackberry anthocyanins.The results showed that:malonic acid, malic acid and oxalic acid enhanced the absorbance values obviously (P<0.05), which showed an increased tendency with organic acids concentration. Among the three organic acids, oxalic acid showed the best copigmentation effects, followed by malic acid and malonic acid. However, ferulic acid, caffeic acid and tannins acid of blackberry anthocyanin copigmentation effect were not significant. Organic acids significantly impacted hromatic aberration of blackberry anthocyanins solution, of which L*decreased with the increase of organic acid concentration, while a*, b*and h increased with the increase of organic acid concentration. The best effect was observed with0.09mol/L contration.The activation energy (Ea) of lackberry anthocyanins were improved by malic acid, malonic acid and oxalic acid (P＜0.05). Oxalic could increase the Ea by86%and significantly improve thermal stability of blackberry anthocyanins. From the point of half-life, whether add organic acid or not, thermal stability blackberry anthocyanins reduced with increasing temperature. Under the conditions of70℃, T1/2values of blackberry anthocyanins after adding malic acid, malonic acid and oxalic acid, were22.07,21.46and34.15h, higher than that of the control group, which was19.04h. No anthocyanins derivatives were formed during the copigmentation. It was deduced that non-covalent copigmentation happened between oxalic acid and blackberry anthocyanins.In three antioxidant assay systems, the total antioxidant capacity of blackberry anthocyanins firstly decreased and then increased with co-pigmentation time extended. However, the hydroxyl radicals scavenging capacity and superoxide anion scavenging capacity of those were increased with time extended. After copigmented with0.04mol/L oxalic acid for2h, the total antioxidant capacity of blackberry anthocyanins reached the maximal value68.72U/ml. However, the hydroxyl radicals scavenging capacity and superoxide anion scavenging capacity reached maximum values of22.37U/ml and446.13U/L after copigmented with0.08mol/L oxalic acid for3h, respectively, which were higher than that before copimentation which were15.32U/ml and350U/L, respectively. 5. The radical scavenging capacity of blackberry anthocyanins was investigated in vitro and their protective effects on human endothelial cell damage induced by H2O2were researched illustrate their antioxidant mechanism. The results showed that:DPPH radical scavenging capacity of blackberry anthocyanins were closely related to the concentration from20to100μg/ml, which were less than that of ascorbic acid. However, the hydroxyl radicals scavenging capacity of blackberry anthocyanins were higher than that of ascorbic acid at the same concentrationCell morphology results showed that human endothelial cell treated with H2O2became shrinked and rounded, and their size became smaller and cell gap widened. The majority of cells disrupted shedded. However, pre-treatment with different doses of blackberry anthocyanins have a different degree of protection on cell morphology than the injury group and cell morphology improved compared with damaged group.MTT method was used to evaluate effects of blackberry anthocyanins on the cell viability. The results showed that cell viability was increased from106%to157%after cell pretreated with blackberry anthocyanins at the concentration ranged from50to200ng/ml for12h and then with1.0mmol/L H2O2for anthother12h. However, ascorbic acid did not show protective effects on uman endothelial cell damage induced by H2O2.Cell malondialdehyde (MDA) levels significantly decreased with blackberry anthocyanins concentration increased. But MDA levels did not decrease after ascorbic acid treated under the same oxidative conditions induced by H2O2. Meanwhile, the peroxidase (POD), superoxide dismutase (SOD) and glutathione S-transferase (GSH-ST) activities of endothelial cells pretreated with blackberry anthocyanins were also largely promoted, of which POD was enhanced80%by200μg/ml treatment. Effects of blackberry anthocyanins on cell apoptosis were also studied. It was showed that cell early apoptosis was reduced from70.58%to11.25%and late apoptosis was from7.53%to2.01%with blackberry anthocyanins treatment of the concenration from50to200μg/ml. These results indicated that blackberry anthocyanins could protect endothelial cell damaged from H2O2through decreasing MDA, improving antioxidant enzymes to remain cell system balance and inhibiting cell apoptosis. |
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