Study On The Extraction And Identification Of Polyphenols From Elaeagnus Pungens Thunb.and Their Antioxidant Activity

Clinical studies have shown that oxidative stress is one of the inducements of many chronic diseases.Polyphenols can effectively prevent many chronic diseases caused by oxidative stress.Fruits are one of the important sources of human ingestion of polyphenols.Elaeagnus pungens Thunb.which is a wild drupe plant belongs to the Elaeagnus genus of the Elaeagneaceae family.Its rhizomes,leaves and fruits can be used as medicine,and it has the functions of anti-oxidation,treatment of asthma,anti-inflammatory and anti-cancer.The fruit is rich in a variety of biologically active substances,such as:polyphenols,flavonoids,terpenes,alkaloids,etc.It is widely distributed in our country,such as Yunnan,Hubei,Sichuan and so on,but its utilization and development is low,and it is often used as a fresh fruit.In this paper,Elaeagnus pungens Thunb.was picked from three different areas,extracted with different methods.The extract was purified with macroporous resin,and explored its antioxidant capacity,components and content.And content testing.Study on the protective effect of Elaeagnus pungens Thunb.extract on HUVEC cells from oxidative stress,hoping to provide reference for the research and development of Elaeagnus asiatica.The main results are as follows:The effects of ultrasonic-assisted extraction and solvent extraction on the extraction rate of fruit polyphenols and flavonoids were examined,and the results showed that the ultrasonic-assisted extraction method is better,the total content of polyphenols(TPC)from fruit of Lincang,Baoshan,and Dehong was 1.01±0.01,1.17±0.03,and 1.33±0.02 mg gallic acid(GAE)/g fresh weight(FW),which were significantly higher than the polyphenols obtained by solvent extraction method,and the same trend was discovered in flavonoids.The contention are 80.26±1.47、85.74±0.23、138.39±2.24 μg catechin(CE)/g FW.The adsorption and desorption capacity of different types of macroporous resins on the extract was determined,and LS-46D was selected to purify the extract.After purification,TPC of the fruit was 0.70±0.02,0.81±0.03,0.83±0.03 mg GAE/g FW.Measuring the antioxidant activity with DPPH,ABTS,FRAP,ORAC,the overall performance is:Dehong(2.66±0.04～11.40±1.16 μmol TE/g FW)>Baoshan(2.81 ±0.06)～10.05±0.75 μmol TE/g FW)>Lincang(2.08±0.01～7.10±0.71 μmol TE/g FW),consistent with the trend of TPC,the antioxidant activity of Dehong Elaeagnus with the highest polyphenol content.After purification,the measured results of DPPH,ABTS,FRAP and ORAC are 1.73±0.08,2.14±0.10,1.66±0.06,5.48±0.45 μmol TE/g FW,respectively.UPLC-ESI-MS/MS technology was used to identifiy 15 phenolic compounds from the extract,including 7 hydroxylated benzoic acids:gallic acid,ellagic acid,p-hydroxybenzoic acid,vanillin,quinic acid,shikimic acid,methyl gallate;1 hydroxycinnamic acid:ferulic acid;7 flavonoids:dihydromyricetin,quercetin,myricetin,polydatin,isoquercitrin,rutin,epi gallic catechins.These substances can be detected in the fruit from three areas but the contention is different.The gallic acid in Lincang Elaeagnus was 1434.9052±59.8886 ng/g FW,Baoshan,Baoshan,Dehong Elaeagnus contains the most dihydromyricetin,8274.39±266.33 and 31202.27±406.39 ng/g FW respectively;the highest TPC is Dehong and the lowest TPC is Lincang.Cellular anti-oxidation was used to evaluate the antioxidant effect of the extract.The oxidative stress damage model of HUVEC caused by H2O2 was used to explore whether the extract can play an anti-oxidant effect.The study of the cytotoxic effect of the extract on HUVEC shows that samples are non-toxic within a certain concentration range and has a protective effect on cell oxidative stress damage.Compared with the model group,the cell survival rate is higher with the addition of extract.Some intracellular indicators for measuring oxidative stress damage also have a trend of change.Compared with the model group,the expression of ROS and MDA in the cells of the extract group decreased,LDH release also weakened,and SOD showed an upward trend.With the increase of the concentration,the changes of these indicators became more significant.Dehong showed the best protective effect,followed by Baoshan,and Lincang Elaeagnus last.