Synergistic-protective Effect Of Green Tea Polysaccharides And Tea Polyphenols On Pancreatic Cells

During the last century, rapid social development and changes in human lifestyle have resulted in a dramatic increase in the prevalence of diabetes mellitus over the world. Though oral hypoglycemic agents and insulin is the mainstay of treatment of diabetes, it has prominent side effects and fails to significantly alter the course of diabetic complications. So, development and utilization of anti-diabetic plants are becoming more and more necessary for its less toxicity and no-drug-resistance.Crude green tea has been already confirmed to be prophylactic and therapeutic against diabetes and its complications, which is intensively associated with main components: tea polysaccharides and tea polyphenols. In view of insufficient exploration of crude green tea, strengthen the advanced utilization of crude tea resources is the inexorable trend of tea industry development. Nowadays, tea polyphenols have been widely applied in many industries like medicament, foods and daily chemicals, while relative researches on tea polysaccharides still has a long way to go. It has always been a burning problem to explore an effective preparation and purification methods of tea polysaccharides. Moreover, as for the anti-diabetic effect of green tea, previous researches are limited to hypoglycemic effect of tea polysaccharides or tea polyphenols respectively using animal experiments, while rare reports on either cellular level or their interaction on diabetes.Therefore, this research is designed to firstly study the comprehensive extraction and purification of tea polyphenols and tea polysaccharides from crude green tea, then make structure characterization of green tea polysaccharides with relative high purity (TPS) and its fraction components TPS1, TPS2 prepared from saled crude tea polysaccharides sample with serials of safe and effective purification methods, and lastly investigate whether there is a synergistic-protective effect of TPS and EGCG on STZ-induced MIN6 cells damage and preliminarily explore the possible mechanism via the techniques of radioimmunoassay, DCFH-DA and JC-1 as the fluorescent probes, and the single cell gel electrophoresis (SCGE). The main results are as follows:Firstly, the water extraction technique of tea polyphenols and tea polysaccharides from crude Laoshan Green tea is investigated via both single-factor and orthogonal experiments. The optimum extraction parameters are listed as tea-water ratio 1: 20, extraction temperature 80℃, the extraction time 50 min and extracting twice. Then the crude tea polyphenols and polysaccharides were obtained through organic solvent comprehensive extraction, with the yield of 9.75% and5.83%, respectively.Secondly, the separation and purification of tea polysaccharides is investigated using saled crude tea polysaccharides as material. The sample is firstly pre-extracted in hot water and subsequently submitted to ethanol precipitation. The optimum precipitation condition is that ethanol concentration 80%, precipitation time 3h, centrifugal time 10min and treating twic. Then PVPP was applied to adsorb polyphenols and proteins, resulting in the obvious removal rate of 80.8% and 82.7%, respectively, under the optimum conditions: PVPP content 8.0 g/L, temperature 30℃, treating time 30 min and two times. The product is further decolorized via resin adsorption to give TPS, of which two components TPS1 and TPS2 are obtained through Q-Sepharose FF Chromatography.Thirdly, HPGLC, GC, automatic acid analyzer, visible absorption spectra, UV and IR spectra are used to characterize TPS, TPS1 and TPS2. It was concluded that: (1) TPS, TPS1 and TPS2 are all kinds of acid glycoconjugates containing a small amount of protein. Their polysaccharide part is mainly composed of galactose, glucose and arabinose. Protein part is composed of 15~16 normal amino acids, among which Asp, Glu, Leu are the major proportion. Peptide chain contains Ser, Thr and Tyr, with no hydroxyproline or hydroxylysine. (2) Their main difference lies in two aspects. Both TPS1 and TPS2 are relative homogeneous fraction with molecular weight of 12KDa and 10KDa, respectively, while TPS has a relative broad molecular weight range; Additionally, they have different basical chemical composition. The polysaccharides content of TPS, TPS1 and TPS2 is 43.25%,82.48% and 74.57%, respectively, while uronic acid content of 23.36%, 13.01% and 45.75%, respectively, and protein content of 3.30%, 59%, and 5.05%, respectively. No polyphenols is detected. Lastly, synergistic anti-diabetes effect of TPS and EGCG in vivo is investigated in this paper. Results show that: (1) TPS-500μg/mL, EGCG-10μg/mL, TPS-50+EGCG-1.0μg/mL all significantly stimulate insulin releasing from damaged MIN6 cell induced by STZ (P<0.05), among which TPS-50+EGCG-1.0μg/mL reaches the remarkable level(P<0.01); (2) The better protective effect of TPS-50+EGCG-1.0μg/mL on glucose-induced insulin secretion of MIN6 cells, compared with Green tea water extract, reveals that there is practical significance to study the purification of TPS and synergistic anti-diabetic effect of TPS and EGCG. (3) In many aspects of glucose-induced insulin secretion, mitochondria membrane potential (△Ψm), ATP content, Caspase-3 activity and DNA damage, etc., TPS-50+EGCG-1.0μg/mL all shows significant protective effect on MIN6 cells damaged by STZ (P<0.05), consistently superior to that of TPS or EGCG alone, which fully reflects the synergistic-protective function of TPS and EGCG. (4) The possible mechanism of anti-diabetic ability of TPS+EGCG maybe its protection against mitochondrial function. Its mechanism maybe correlated with avoiding high oxidative stress by scavenging ROS, protecting mitochondria and DNA, as well as inhibiting the activity of Caspase-3 to decrease cell apoptosis. (5) It is TPS2 with high uronic acid content that proves the main functional anti-diabetic component in TPS, not TPS1.This research aims at preparing tea polysaccharides with high purity and bioactivity through serials of mild purification process, and further investigating the synergistic effect of tea polysaccharides and tea polyphenols (eg., EGCG) on diabetes in vivo and relative mechanisms, to provide a theoretical basis for comprehensive utilization of crude tea resource, as well as the exploitation of new kinds of anti-diabetic drugs or healthy food.