Separation And Purification,Antioxidant Activitives Of Tea Polyphenols And Tea Polysaccharides From Se-enriched Tea

This paper used old leaves of ziyang se-enriched tea as raw materials.Tea polyphenols and tea polysaccharides were obtained by contiuous extraction with ultrasonic assisted enzyme extraction method.What’s more,tea polyphenols and tea polysaccharides were purified.We studied the antioxidant activity and antimicrobial activity of tea polyphenols.The other task was that studying the antioxidant activity and preliminary structural analysis of tea polysaccharides.1.In the extraction experiment,considering solid-liquid ratio,ultrasonic power,the content of pectase,extraction temperature as the single factor.The process parameters of extraction were optimized by orthogonal experime.Best of continuous extraction conditions: solid-liquid ratio was 1:35 g/mL,ultrasonic power was 250 W,the content of pectase was 0.4%,extraction temperature was 50 ℃.The results showed that the extraction rate of tea polyphenols was 30.37%,the content was 35.69%;the extraction rate of tea polysaccharide was 20.12%,the content was 27.67%.2.Purification of tea polyphenols and tea polysaccharides.(1)The preliminary purification of tea polyphenols were obtained by ultrafiltration,HPD-600 macroporous adsorption resin and polyamide chromatography column.Separation and preparation of EGCG,ECG,EGC and GCG from raw power of catechins were investigated by HPLC.Preparative chromatography conditions : column(10 mm×250 mm,C18,5.0 μm);Mobile phase A: methanol(0-5 min,15%-20%;10-20 min,20%;10-15 min,20%-25%;15-25 min,25%;25-35 min,25%-35%;35-45 min,35%;45-50 min,35%-50%;50-55 min,50%-80%;55-60 min,80%-15%);Mobile phase B: ultra pure water;detective wave-length(278 nm);Injection volume 3.00 g.The collected fractions were concentrated and then re crystallized.The powders of EGCG,ECG,EGC and GCG were obtained by freeze drying.The purity of each substance were 91.28%,91.56%,90.67%,93.41%.(2)Higher purity tea polysaccharides were obtained by successive purification using ultrafiltration,macroporous resin AB-8,polyamide column chromatography,cellulose DEAE-52(fractionation precipitation)and dialysis.What’s more,the effects of DEAE-52 cellulose column elution and fractionation precipitation on the purification of tea polysaccharides were compared.The results showed that tea polysaccharides were composed of LPS-1,LPS-2,LPS-3 and LPS-4 after DEAE-52 cellulose column;tea polysaccharides were composed of LPS-60,LPS-70,LPS-80 and LPS-90 after fractionation precipitation.The contents of these 8 kinds of polysaccharides were 88.63%,86.24%,83.45%,80.89%,70.42%,85.86%,81.47%,79.52%.The polysaccharide fraction LPS-1 was selected for subsequent experiments.3.The structural characterization of tea polysaccharide fraction was evaluated through UV,FT-IR,CE,SEM,AFM,MS in this paper.The result showed that LPS-1 has a significant absorption peak in the wavelength of 200 nm and 260-280 nm,which indicates that it was a kind of glycoprotein.Characterization of LPS-1 by FT-IR analysis showed the typical absorption of polysaccharide.LPS-1 was a homogeneous heteropolysaccharied incluing rhamnose,mannose,glcose and galactose acid.Molar ratio: Rha: Man: Glc: Gal = 0.3: 2.5: 0.9: 1.6.Scanning electron microscopy showed that LPS-1 was columnar,smooth surface.Atomic force microscopy scans showed that LPS-1 molecules were arranged in acompact,accurmulation of circle structure.The characteristic absorption peak of polysaccharide was revealed by the mass spectra of methylated fragments.4.To study the antioxidant activity of crude tea polyphenols,catechins,crude tea polysaccharide and LPS-1 scavenging DPPH free radicals,superoxide anion radicals and hydroxyl radicals in vitro.The result showed that the DPPH radical,the superoxide anion free radical and hydroxyl free radical could be eliminated by Crude tea polyphenols and caffeine.In a certain range,there was a certain effect relationship between the concentration and the scavenging ability.The scavenging activity of crude polyphenols was stronger than that of VC.The scavenging ability of catechu monomer was 1.22-1.45 times stronger than VC.(scavenging ability)EGCG > GCG > ECG > EGC.Well,the antioxidant experiments showed that the DPPH radical,the superoxide anion free radical and hydroxyl free radical could be eliminated by crude tea polysaccharide and LPS-1.In a certain range,there was a certain effect relationship between the concentration and the scavenging ability.LPS-1 had better antioxidant activity than crude tea polysaccharide.Crude tea polyphenols had a certain inhibitory effect on Staphylococcus aureus and Escherichia coli.The inhibitory effect on Staphlococcus aureus was better than that of Escherichia coli.