Study On Quantitative Analysis Of Proanthocyanidins And Application Of Antioxidant Efficacy

As a natural product with great anti-oxidation properties,proanthocyanidins has become a leading star in the fields which of food,medicine and biomedical research with its remarkable free radical scavenging ability,extensive pharmacological activity and excellent safety.At home and abroad,the extraction,measurement and development of proanthocyanidins have been studied fiercely.In the research of proanthocyanidins detection method,due to the relatively complex structure of proanthocyanidins,there are still some shortcomings in the research of quantitative methods,and there is no uniform standard,thus limiting the development and utilization of proanthocyanidins and the standardized management of the market.At the same time,the research on the antioxidant activity of proanthocyanidins also needs more in-depth research and comprehensive investigation.In order to promote the rational and effective development and application of procyanidins,this paper aims to establish a method for rapid quantitative analysis of proanthocyanidins.In order to promote the rational and effective development and application of proanthocyanidins,this paper aims to establish a method for rapid quantitative analysis of proanthocyanidins,and at the same time,it focuses on the confirmation of the anti-oxidative effects of proanthocyanidins in vitro and in vivo.The main work is as follows:1.Establishment of Microwave Rapid Assisted Porter-HPLC Method for Determination of Proanthocyanidins.In the Porter method,n-butanolhydrochloric acid and iron salt were used as the reaction medium.Compared with the original method,we improved the hydrolysis method,and the water bath reflux hydrolysis was replaced by microwave hydrolysis.A method for improving the determination of proanthocyanidin content by high performance liquid chromatography was established.The results show that the best hydrolysis is achieved in the microwave reactor at 640 w power for 75 s.A calibration curve ranged from 10 ?g/mL to 150 ?g/mL was established for proanthocyanidin ion by external standard method,the linearity was 0.9999,and the linearity was good.The limits of detection(LODs)and limits of quantitation(LOQs)were 0.53 g/mL and 1.61 g/mL for proanthocyanidin ion,respectively.At the same time,we have comprehensively examined the precision and recovery rate intra-and interday for the three types of health products such as capsules,tablets and tea.RSD values of intra-and inter-day is between 1.5% and 3.1%,and the recovery rate is between 91.40% and 107.43%.The method is convenient,rapid and efficient for quantitative analysis of various proanthocyanidin health products.2.Determination of antioxidant activity of proanthocyanidins in vitro.The antioxidant properties of proanthocyanidins in vitro were determined by ABTS,DPPH free radical scavenging method and pyrogallol autooxidation method.The time of three methods for free radical scavenging reaction and the effect of sample concentration on free radical scavenging effects were investigated.It was found from the three detection methods that the proanthocyanidins were most effective in the DPPH free radical scavenging assay.In the ABTS free radical scavenging test,the effects of proanthocyanidins and vitamin C on scavenging free radicals are relatively small.In the pyrogallol autooxidation test,the operation stability is inferior to the other two methods due to the difficulty in controlling and grasping the rate of auto-oxidation.In summary,three in vitro antioxidant assays have confirmed the significant antioxidant effects of proanthocyanidins.3.Determination of antioxidant activity of proanthocyanidins in vivo.One of the most representative and stable DNA oxidative damage markers in human body was selected,8-hydroxydeoxyguanosine,and it was used as the detection object.A quantitative analysis method for the determination of 8-hydroxydeoxyguanosine in urine samples by HPLC-MS was established.A quantitative analysis method for the determination of 8-hydroxydeoxyguanosine in urine samples by HPLC-MS was established.The urine sample of the population after taking a procyanidin and vitamin C+E tablets for a certain period of time was collected,and it was processed for batch determination of the content of 8-OHdG.In the experiment,90 people were selected to take health care tablets for the subjects,which were divided into three groups.They were A: control group(no pill group);B: grape seed proanthocyanidin tablets group;C: vitamin C+ E group,urine samples collected in these 3 groups of people after 0,3,and 6 months.By collecting and detecting the content of in the urine of human samples,the final statistical analysis revealed: With the increase of medication time,the content of 8-OHdG in the proanthocyanidin tablets group was significantly different from that in the blank control group and the self-control group(P<0.05),which can be concluded that proanthocyanidins have a antioxidant effect.From the comparison between the medication groups,after treatment for 3 months and 6 months,the content of 8-hydroxydeoxyguanosine in the proanthocyanidin group was lower than that in the vitamin C+E group,and and P<0.05 was significant in each group.That is,The antioxidant effect is superior to the vitamin C+E group.