| Blackberry(Rubus fruticosus L.)belongs to the genus Rubus in the rose family.Since it contains a large amount of natural antioxidant anthocyanins,there is a globally big market for its consumption.However,the anthocyanins in blackberry show intrinsically chemical unstability which is a big limition on its extraction,processing,storage,and transport for its commercialization in industry.The stability of anthocyanins in blackberry is vulnerable to light,high p H and high temperature shown by numerous literatures.Therefore,many strategies were proposed to improve anthocyanins stability,such as chemical modification,microencapsulation and metal complexation.However,the urgent unmet need of maintaining enough stability and antioxidant activity of anthocyanins in industry has not been satisfied yet.Therefore,in order to preserve anthocyanin good activity,we would like to improve the convenience of the industrialization and commercialization of anthocyanins by discovering the new and safe copigments,natural amino acids,to exert efficient copigmentation effect on anthocyanins.The thesis was divided into four parts.Firstly,five different natural amino acids were evaluated their copigmentation effects on in-house blackberry anthocyanins extract.L-proline,as th best one,was further evaluated its effect on improving in vitro antioxidant activity of blackberry anthocyanins extract.Secondly,cyanidin-3-O-glucoside,as one of the main activity components in blackberry anthocyanins,was used as an example to investigate whether L-proline could improve anthocyanins' antioxidant activity and chemical stability as a new copigment.Thirdly,CCl4 induced acute chemical liver injury mouse model was used to evaluate the in vivo copigmentation effect of L-proline on cyanidin-3-O-glucoside.Finally,the molecular mechanism of copigmentation between L-proline and cyanidin-3-O-glucoside was investigated and the hypothesis was proposed to better understand the atomic level interactions.The main results are shown as follows:(1)Amino acids to improve in vitro stability and antioxidant activity of blackberry anthocyanins extractFive different representative amino acids were evaluated whether they could improve blackberry anthocyanins extract stability as new copigments by measuring anthocyanins content change at high temperature 90°C for 6 hours.The outcome showed L-proline performed best.Blackberry anthocyanins extract without L-proline degraded by 52.0% at high temperature 90 °C for 6 hours while blackberry anthocyanins extract with L-proline degraded by only 34.2%.Therefore,in vitro antioxidant activity assay was used to further evaluate the copigmentation effect of L-proline on blackberry anthocyanins extract.Similarly,at high temperature 90°C for 6 hours,blackberry anthocyanins extract with L-proline showed better antioxidant activity than blackberry anthocyanins extract alone in DPPH·,ABTS·+ and Hydroxyl free radicals scavenging experiments.It was improved by 14.7% in DPPH·and improved by 20.3% in ABTS·+ and improved by 27.5% in Hydroxyl free radicals scavenging assay(2)Amino acids to improve in vitro stability and antioxidant activity of cyanidin-3-O-glucosideBesides blackberry anthocyanins extract,cyanidin-3-O-glucoside,as one of the main activity components in blackberry anthocyanins,was used as purer sample to further validate the outcome shown in last section.Similarly,five different representative amino acids were evaluated again whether they could improve cyanidin-3-O-glucoside stability as new copigments by more accurate kinetic study,the half life(t1/2),at high temperature 90°C for 6 hours.Again,L-proline was demonstrated the best one.Then,the capacity of L-proline copigmentation effect on stability and antioxidant activity of cyanidin-3-O-glucoside was further evaluated at room temperature for 20 days by kinetic study and in vitro antioxidant activity assays to further confirm the copigmentation effect of L-proline on cyanidin-3-O-glucoside stability and antioxidant capacity.L-proline could significantly extend the half-life of cyanidin-3-O-glucoside degradation in a dose-dependent way.When the molar ratio between cyanidin-3-O-glucoside and L-proline was 1:100,half-life was extended from 350.1min to 474.8 min at high temperature 90°C and from 23814.4min to 28875.0 min at room temperature.L-Proline could significantly(p<0.05)improve cyanidin-3-O-glucoside in vitro antioxidant activity in a dose-dependent way.Heated for 6 hours at 90°C or at room temperature for 20 days,cyanidin-3-O-glucoside with L-proline showed better antioxidant activity in both DPPH· and ABTS·+ free radicals scavenging assays than cyanidin-3-O-glucoside alone.It was improved by 38.5% in DPPH· free radicals scavenging assay and improved by 26.8% in ABTS·+ free radicals scavenging assay.(3)Efficacy of L-proline cyanidin-3-O-glucoside complex on acute chemical liver injury mouse model induced by CCl4.Animal experiment test was used to evaluate whether L-proline cyanidin-3-O-glucoside complex could alleviate acute chemical liver injury in mice induced by CCl4 by measuring MDA,SOD and GSH-px content change in liver homogenate.The outcome showed that cyanidin-3-O-glucoside copigmented with high concentration of L-proline could improve acute chemical liver injury(p<0.05).(4)Copigment mechanism and interaction pattern between L-proline and cyanidin-3-O-glucosideUV,FT-IR,and in vitro antioxidant activity assays were exploited to investigate interaction mechanism between L-proline and cyanidin-3-O-glucoside while cyclopentanecarboxylic acid was used as negative control to confirm the important role of introgen of L-proline.The results showed that,first of all,the specific UV absorption was lower in cyanidin-3-O-glucoside with additive L-proline than cyanidin-3-O-glucoside alone,which suggested cyanidin-3-O-glucoside participated in the interaction with L-proline.Secondly,IR of absorption of additive L-proline with cyanidin-3-O-glucoside shifted to 1731.06cm-1 which was not existed in the IR of absorption of L-proline or cyanidin-3-O-glucoside alone,but the IR of absorption of additive cyclopentane-carboxylic acid with cyanidin-3-O-glucoside did not show the similar phenomenon.Finally,cyclopentanecarboxylic acid could not improve the antioxidant activity of cyanidin-3-O-glucoside in in intro assay.Based on molecular recognition concept and the outcome shown above,we proposed that the positive charged nitrogen of L-proline could form hydrogen bonds with two oxygens of cyanidin-3-O-glucoside sugar ring to stabilize the specific binding mode to help the carboxylic acid of L-proline form charge-charge interaction with the positive charged oxygen of cyanidin-3-O-glucoside;at the same time,hydrophobic interactions between the carbon atoms of L-proline ring and the aromatic rings of cyanidin-3-O-glucoside could further stabilize the complex.These interactions play important roles in L-proline and cyanidin-3-O-glucoside recognition and copigmentation.Cyclopentanecarboxylic acid also suggested the specific and unique role of the nitrogen of L-proline in the copigmentation effect. |
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