Isolation And Fine Structure Of Acidic Polysaccharide From Green Tea

Tea is the shoot of tea plants, belonging to Camellia, Theaceae. As the hometown of tea, China is one of largest tea producing countries. Tea was initially considered to be as a medicinal tea, and later to bas as drink. Studies show that tea contains a variety of active chemical ingredients, including polysaccharides, proteins, amino acids, polyphenols, alkaloids, etc. Green tea is rich in Wuyuan in Jiangxi province. However, lots of crude tea was wasted during the manufacture process of tea. We need to utilize the crude tea more effectively. Polysaccahride is considered to be one of main bioactive components in the tea. Study on tea polysaccharide is an important aspect for the research on tea. This paper intends to analyze phycico-chemical properties and fine structural characteristics of polysaccharide from Wuyuan rough old tea. The polysaccharide was extracted from the tea by boiling water, and further purified. The main conclusions are as follows:1. The crude polysaccharide was prepared by water extraction and ethanol precipitation from Wuyuan rough old tea. After the sample powder was soaked in 80% ethanol and dried in the open air, the crude tea was extracted with water at 96 oC for 4 h as material-water ratio of 1:10 g/m L. The extraction procedure was repeated twice. The combined extract was concentrated, and subsequently precipitated with ethanol at its final concentration of 80%. The yield of obtained tea polysaccharide was 5.6% under these conditions. Sugar content of TPSR was measured by phenol-sulfuric acid method, the corrected factor was 2.87. Phenol-sulfuric acid method was verified to be accuracy and reliability to determine the suger content of tea polysaccharide, by the tests of stability, precision, reproducibility and recoveries. Therefore, a simple and accurate method for sugar content determination of tea polysaccharide was established.2. The polysaccharide was purified by Sevag method, and physic-chemical properties of the purified polysaccharide were determined. Its purity and molecular weight(Mw) were determined by high performance gel permeation chromatography(HPGPC). The content of neutral polysaccharide is 55.1%, the content of protein is 1.8%. It was mainly composed of rhamnose, ribose, arabinose, mannose, glucose, galactose in the molar ratio of 1.26:3.18:4.08:1.00:1.52:3.92, by GC method. The mainly uronic acid is galacturonic acid with the content of 33.5% from HPAEC results. 13 kinds of amino acids were detected, and alanine was found to be the most. It was confirmed that TPS was an acidic glycoprotein by the UV and IR spectra. Pyranose ring could be found in the polysaccharide.3. Primary structure of TPS was determined. Methylation analysis results showed that TPS was mainly composed of T-linked Araf(2.8%), T-linked Ribf(2.1%), T-linked Glcp(3.3%), T-linked Galp(5.1%), 1, 3-linked Araf(4.9%), 1,2,4-linked Rhap(3.7%), 1,4,6-linked Glcp(3.1%), 1,4-linked GalpA(33.5%), 1,6- linked Glcp(9.6%) and 1,3,4-linked GalpA(32.2%), ect. Gal was mainly existed as T-linked Galp, 1,4-linked GalpA, 1,3,4-linked GalpA. It was concluded that Gal was probably linked by 1,4-linkage, and its O-3 was substituted by branched chain.TPS was hydrolyzed by 0.05 M TFA to get the fraction of TPS-H, which was inside of dialysis bag. TPS-H was mainly composed of galactose(37.4%), arabinose(16.2%) and glucose(15.9%). The results of HPGPC showed that the component of TPS-H was homogeneous, and its molecular weightwas about 101726. The methylation results of TPS-H showed that T-linked Araf content was decreased, comparing with that of TPS. It indicated the T-linked Araf residues in the end chain were hydrolyzed. The content of 1,3,4-linked GalpA was also decreased, from 32.2% to 16.4%. The high levels of 1,3,4-linked Galp and 1,4-linked GalpA residues indicated that the main residues in the chain were �' 4)-GalpA-(1 �' with substitution at O-3. The main terminal residues were T-linked Glcp(10.5%) and T-linked Galp(9.8%). NMR analysis showed galactose mainly was existed as β-configuration, and arabinose mainly was existed as α-configuration. The majority residues of TPS were β-1,4-linked GalpA、β-1,3,4-linked Galp、α-T-linked Glcp and β-T-linked Galp.