Study On Isolation, Purification And Anticoagulant Activity Of Tea Polysaccharides

Tea polysaccharides were found to be mostly glycoconjugates in which a protein carries on or more carbohydrate chains covalently attached to a polypeptide backbone, usually via N-or O-linkages. It has been reported that they have showed various bioactivities, such as antioxidant, hypoglycemic and immunological activities, and so on. In this study, we attempted to investigate the extraction, deproteination, purification, characterization of structure, anticoagulant activities and to find a potential pharmaceutical value of the natural anticoagulant polysaccharides. The main results were as follows:1. Extraction and optimization of the tea polysaccharidesThe crude polysaccharides were extracted from the leaves of Camellia sinensis using distilled water. Respond surface methodology (RSM), based on Box-Behnken design was employed to obtain the best possible combination of extraction time, water to the raw material ratio and extraction temperature for maximum polysaccharide extraction. The experimental data were fitted to a second-order polynomial equation using multiple regression analysis and also analyzed by appropriate statistical method (ANOVA). The optimum extraction conditions were as follows:extraction time of106min, water to the raw material ratio of30and extraction temperature of64℃. Under these conditions, the experimental yield was13.26%, which is well close agreement with the value predicted by the model13.14%.2. Deproteination of the tea polysaccharidesThe deproteination of TPS was compared by the method of Sevag, trichloroacetic acid and polamide. The method of polyamide was not only for the best deproteination but also minial polysaccharide loss. Through the efficiency of deproteination and decolorization were similar, the polysaccharide loss of the dynamic deproteination was larger than static. In contrast, the method of static polyamide was better than dynamic state. The efficiency of deproteination and polysaccharides holding were88.8%and90.3%, respectively.3. Isolation and purification of the tea polysaccharidesThe TPS were further separated and purified by anion exchange chromatograph on a DEAE sepharose CL-6B. Four major fractions eluted with0,0.1,0.2,0.5mol/L sodium chloride were collected, respectively, and then concentrated, dialyzed and lyophilized to obtain TPS-1, TPS-2, TPS-3and TPS-4. The yield were13.4%、27.4%、15.9%and17.1%, respectively. TPS-1was known as neutral polysaccharides, and TPS-2, TPS-3and TPS-4were known as acidic polysaccharides.4. Preliminary structural characterization of the TPS fractionsTPS-4had a positive respond to absorption at280and260nm in the UV-visible spectrum, indicating presence of protein and nucleic acid. However, no absorption at280and260nm were observed for TPS-1, TPS-2and TPS-3. Characterization of TPS fractions by FT-IR analysis showed the typical absorption of polysaccharides. TPS-1, TPS-2and TPS-3were shown as single symmetrical peak in their respective HPGPC chromatograms, indicating that they were homogeneous. The average molecular weight of TPS-1, TPS-2and TPS-3were20760,24230and250643Da, respectively. However, the average molecular weights of TPS-4were689113and4150Da, suggesting it was heterogeneous. The monosaccharide compositions were analyzed by HPAEC-PAD after the hydrolysis of TFA. The main constitutes of TPS-1was arabinose, galactose, glucose and mannose. The results implied the dominance of rhamnose, arabinose, galactose, galacturonic acid and glucuronic acid in TPS-2. The monosaccharide compositions of the TPS-3and TPS-4fractions were similar. Both TPS-3and TPS-4were rich in rhamnose, arabinose, galactose, glucose, galacturonic acid and glucuronic acid. 5. Anticoagulant activity of TPS fractions in vitroThe anticoagulant activities of the polysaccharides from the leaves of Camellia sinensis were investigated by the classical coagulation assays APTT, PT and TT, and meanwhile the saline and heparin were used as negative and positive control, respectively. All the polysaccharide fractions prolonged APTT compared to saline control a dose-dependent manner. Although activity less than heparin, TPS-4exhibited a little stronger anticoagulant activity than TPS, TPS-1, TPS-2and TPS-3with the raise of concentration in APTT experiment and showed a significant different (P<0.05) from negative control. Meanwhile TPS-1and TPS-4showed increasing clotting time in the TT assays. But, according to the PT assay the polysaccharide fractions were no significant differences in modulating pro thrombin time. The results implied that TPS-4inhibited both the intrinsic and thrombin activity of controlling the fibrinogen to fibrin conversion.