Study On Physicochemical Properties And Biological Activity Of Polysaccharides From Green Tea By Different Methods

As a kind of organic macromolecules be rich in organisms, active polysaccharides play the roles of energy supplier and structural matter, or participate various physiological activities. Active polysaccharides are regarded as functional compounds which are considered to be able to regulate the physiological function in human body. Tea polysaccharide conjugates (TPC) is a heteropolysaccharide, in which proteins are bounded with the polysaccharide chain. TPC are considered to have a wide range of biological activities such as hypoglycemic effect, immune regulation, etc. The extraction, purification, physicochemical and functional activity of TPC have been researched extensively in recent years, while their exploitation and application is still limited. To some extent, this might due to their low content in tea and complicated compositions, more over they are difficult to isolate and purify. Lack of reliable quality accordance and functionality evaluation approaches might be another negative factor.In this study, green tea was chosen as the temple because it is most popularly consumed in China. Fresh leaves and its stir-drying products derived from Longjin-43 (a type of tea tree) in May were used as the materials to prepare the TPC. Various approaches were applied to prepare the TPC, including crude tea polysaccharide conjugates extracted with enzymes (CTPC-E), purified CTPC-E (TPC-E), water-extracted tea polysaccharides conjugates (TPC-W), alkali-extracted tea polysaccharides conjugates (TPC-A) and tea polysaccharide conjugates prepared from fresh leaves (TPC-L). The physical and chemical properties of the various TPC were investigated to provide technical norms for the producing and quality controlling of TPC. More over, this study also investigated the application of TPC in impaired glucose tolerance (impaired glucose tolerance, IGT) of type I diabetes and prodromal stage of type II diabetes, and their inhibitory effects on cell multiplication of HepG2 (human liver cancer cell). The current research would afford a theoretical basis for the development and application of TPC products. The main results include following.(1) There are 6 or 7 kinds of neutral monosaccharides contained in the TPC obtained through different approaches, include rhamnose, fucose, arabinose, xylose, mannose, glucose and galactose, respectively. Rhamnose, arabinose and galactose were the three major monosaccharides and they totally made up 73-79%(mole number) of the TPC. The average mole ratio of the three monosaccharide in different TPC was 1:1.4:1.6. A mixed solution containing rhamnose, arabinose and galactose at mole ration of 1:1.4:1.6 should be used as the standard solutions when determine the neutral monosaccharide content in green tea. The acidic monosaccharide contained in green tea TPC is galacturonic acid, which should be used as the standard composition in determination of the acidic monosaccharide content in green tea TPC.(2) According to the results, there were 15 kinds of amino acids existed in CTPC-E and TPC-E contained,16 kinds of amino acids existed in TPC-W and TPC-A cantained, and 15 kinds of amino acids existed in CTPC-L. No free amino acids had been detected in all of the green tea TPC, indicated that the amino acids might existed as protein or peptide state.(3) Zeta potential measurement showed that all the TPC were negatively charged and no apparent isoelectric point presented during a wide range of pH value, although there are bound protein existed in the TPC. UV-Vis scanning spectra showed that the TPC did not have the distinctive ultraviolet absorption of protein or peptide. Furthermore, there were no protein (or peptide) band was observed by SDS-PAGE electrophoresis. The above results suggested that most of the physical or chemical properties of the protein were shielded, such as the surface charge, UV-Vis adsorption, and binding capacities with Coomassie brilliant blue (G-250). It could be considered that the hydrophilic polysaccharide chains of the green tea TPC are the side-chain of the protein or peptide which exposed at the outside of the conjugates. As a result, the physical or chemical properties of the protein or peptide could not be observed.(4) The pigment components existed in the green tea TPC can lead to the UV-Vis light adsorption. Therefore, the light adsorption spectrum during a wavelength of 200-400 nm of the TPC could be applied to evaluate the decoloring degree of the products.(5) Temperature would be the crisis factor that affect the composition and conformation of the green tea TPC. Combination of HPGPC-ELSD and Circular Dichroism (CD) technology could accurately detect the variation in composition and conformation of TPC after thermal experience. This technology could be applied to analysis and control the quality of TPC conjugates which contained covalent bound protein.(6) The environment humidity for TPC storage should below 50 %, because that the TPC obtained by all approaches have a strong hygroscopicity and water binding capacity. Results of zeta-potential measurement showed that all the TPC were negative charged acidic polysaccharides, and high stability could be obtained when they were dissolved in the aqueous phase with pH 5.0～pH 8.0.(7) TPC from the fresh tea leaves could improve the starch sugar tolerance of mice by inhibit the intracorporal degradation (hydrolysis) of starch. TPC-W at a feeding level of 50mg·kg-1·d-1, or TPC-A at a feeding level of 150mg·kg-1·d-1 could delay the blood sugar rises and reduce the disease incidence of NOD mice. Both of TPC-W and TPC-A have a prevention effects on type 1 diabetes, while TPC-W was more effective than TPC-A. Results showed that four of the TPC (TPC-E, TPC-W, TPC-A and TPC-L) displayed a significant inhibit capacity on HepG2 liver cancer cells, and exhibited a dosage effect.