Study On Isolation, Purification, Structure And Structure-activity Relationship Of Tea Polysaccharides

On the basis of systematic research of isolation, purification, physiochemical properties and structure of tea polysaccharides, a comparison of hypoglycemic activity and radical-scavenging activity was made between these fractionated tea polysaccharides, meanwhile, chemical modifications were also made on tea polysaccharides, and structure-activity relationship were studied between these polysaccharides with different structure, which was academically and practicably important for opening out relationship between structure and bioactivity of tea polysaccharide, making full use of the crude, outdated tea resource, promoting health of human being. The main contents and conclusions are listed as follows:1. By using deionized water, oxalate ammonium solution, hydroxide sodium solution to extract tea polysaccharides gradually, comparing their monosaccharide components and hypoglycemic activities, it was found that the TPS extracted by oxalate ammonium solution had the highest content of uronic acids;the TPS extracted by deionized water had the best hypoglycemic activity with the lowest yield. By using the mixed cellulase solution to extract the tea residues left by deionized water, the extraction rate of TPS compared with tea leaves is 2.7 times more than that of the TPS extracted by deionized water. The hypoglycemic activity didn't differ greatly between the TPS extracted by water and by enzyme solution.2. The application of ion exchange resin in TPS isolation was studied. Firstly, it was found that weak-base anion exchange resin D315 selected from seven ion exchange resins and adsorbent resins had the best decolorizing effect of TPS, meanwhile it can also remove the crude protein very well. Secondly, the processing technology about TPS isolation by D315 was optimized. NTPS with low content of Gal A and ATPS with high content of Gal A were obtained respectively by D315 isolation. It was suggested by monosaccharide composition analysis that NTPS was mainly composed of neutral sugar, namely galactose, ATPS was mainly composed of rhamnose , arabinose, galactose and galcuronic acid. DEAE-52 isolation showed that neutral polysaccharides were main fractions of NTPS and that acidic polysaccharides were main fractions of ATPS.3. NTPS and ATPS were further isolated into four fractionates, namely NTPS1, ATPS2,ATPS3 and ATPS4.Through the analysis of HPGPC, Sephadex G-150 and sepharose electrophoresis, it was concluded that NTPS 1 ,ATPS2 and ATPS4 were all the homogenouspolysaccharides, and ATPS3 was composed of two polysaccharides. By monosaccharide composition analysis, NTPS1 had no GalA, which was mainly composed of galactose. ATPS2,ATPS3 and ATPS4 all had Rha, Ara, Gal, GalA. It was indicated by FTIR that NTPS was neutral polysaccharides which was mainly composed of pyranose and had P epimer, ATPS2, ATPS3 and ATPS4 were all acidic polysaccharides which contained GalA, pyranose ,furanose and had a epimer. By HPGPC, Mp of NTPS 1 was around 21247;Mp of ATPS2 was 4430;Mp of ATPS3 was 13387 and 7011;Mp of ATPS4 was 19562.4.By hyperiodic acid oxidation-Smith degradation analysis, ID NMR and 2DNMR analysis, primary structures of NTPS K ATPS2 and ATPS4 were presumed. NTPS1 was a p-1, 4 linked galactan;ATPS2 and ATPS4 were all pectin-like polysaccharides. The backbone of NTPS 1 was ->4)- p-D-Galp -(1-+4)- p-D-Galp-(l->. The backbone of ATPS2 proved to consist of a-l,4-D-galactopyranosylunan blocks interconnected by 1,2-linked rhamnose residues involved in the linear sugar chain. The backbone of ATPS2 could be expressed by [—-4)-a-D-GalpA- (1—4) -a-D-GalpA- (1 —]?- [—4)- a-D-GalpA-(l-2)- a-L-Rhap-(l —]m, There were sidechains at the C4 of Rha in ATPS2 which composed of furanose a-L-Ara and pyranose P-D-Gal. GalpA of ATPS2 formed into methyl ester, and there was a relatively high content of acetyl group in ATPS2. The backbone of mainchain of ATPS4 was composed of repeat unit —>2)-a-L-Rhap-(l—>4)- a-D-GalpA-(l—?. The methyl esterificated degree of GalpA in ATPS4 was lower than that in ATPS2. The content of acetyl group in ATPS4 was more than that in ATPS2.What's more, the substitution position of acetyl group for ATPS4 was more complex than that for ATPS2.5. Through AFM observations of NTPS1,ATPS2 and ATPS4, it was found that NTPS1 aggregated into well-proportioned, little round particles, with diameter about 10-40 run and height about 1-6 nm. ATPS2, ATPS4 aggregated into bad-proportioned, little round particles, and average height of ATPS2, ATPS4 was respectively about 1-4 nm, 5-15 nm, average diameter of ATPS2, ATPS4 was respectively about 15-40 nm, 10-80 nm. NTPS1. ATPS2 and ATPS4 all have blame-like protuberance.6. Sulfonic esterificated tea polysaccharides were synthesized by pyridine-sulfonic acid method. The characteristic absorbance of the sulfonic esters was determined by FTIR.ATPS formed into metal-ATPS complex with Fe ,Ca .Chelating ways and chelating capacity of TPS with different metal were variable. Through AFM observations of ultra-micro structure of TPS before and after chemical modification, it was found that both thickness of polysaccharide chain and protuberance of polysaccharide surface were all different before and after chemical modification. It was suggested that after sulfonic esterification and metal chelation, advanced structure of TPS changed. After hydroxyl group of TPS being substituted by sulfonic group, conformation of saccharide ring could distort or transform, and repulsion between sulfonic groups resulted in the conformation transiting from random coil to stretchedstate and rigid state. After TPS being chelated with metal ions, entangled molecular chains of TPS increased. Thus ATPS-Ca was formed into tightly-arranged "egg box" structure;ATPS-Fe aggregated into approximately fastigium-like "pagoda" shape and its local flame-like protuberance changed into "rod"-like structure. Exothermic peaks and endothermic peaks of TPS determined by DSC after esterification and metal chelation also changed greatly.7. It was suggested by animal experiment that both NTPS and ATPS had remarkable hypoglycemic activity. After sulfonic esterification, hypoglycemic activity of NTPS improved greatly, but that of ATPS changed no markedly. After chelation of ATPS with Ca2+ and Fe3+, hypoglycemic activity of ATPS-Ca reduced, but that of ATPS-Fe could maintain for relatively long time, which was probably related with the active sites such as non-branched smooth region which composed of 1,4-linked a-D-GalpA being occupied and the spacial structure being altered to facilitate to interact polysaccharides acceptor after chelation. NTPS1, ATPS2, ATPS3 and ATPS4 all had hypoglycemic activity, but acidic heteropolysaccharide ATPS2, ATPS3 and ATPS4 had better hypoglycemic activity than NTPS 1. Among the three, ATPS2 had the best hypoglycemic activity. It was possibly related with the structural arrangement which contained a smooth region of GalA ,a rhamnose /galacuronic acid center branched with neutral sugar sidechains and the acetyl group.8. Radical-scavenging activity of TPS was studied by using chemical luminescence system to produce radicals. Results showed that radical-scavenging activity of ATPS was higher than that of NTPS, which was suggested that radical-scavenging activity of TPS might be related with its uronic acids content. After sulfonic esterification, radical-scavenging activity of NTPS improved greatly, but that of ATPS reduced. It was showed that radical-scavenging activity of polysaccharide was probably related with negatively-charged functional groups linked with polysaccharide, but it seemed that there was not great relationship between the activity and kinds of those negatively-charged groups. After ATPS being chelated with Fe3\ Ca2+, radical-scavenging activity of ATPS-Ca reduced much less than that of ATPS , but the activity of ATPS-Fe was close to that of ATPS. It was probably related with whether the active sites being occupied and the metal ions themselves being reacted with radicals or not after chelation. Among NTPS1, ATPS2, ATPS3 and ATPS4, acidic heteropolysaccharide ATPS2, ATPS3 and ATPS4 had better radical-scavenging activity than neutral heteropolysaccharide NTPS1, and ATPS2 had the best radical-scavenging activity among these four TPS. It was indicative that relatively high GalA content was probably the main reason of strong radical-scavenging activity.