Optimization Of Extraction Process, Separation And Purification Of Polysaccharides From Tartary Buckwheat And Its Protection Against Damage Of HUVECs Cells

Buckwheat (Fagopyrum tataricum (L.) Gaertn) belongs to polygonaceae Fagopyrum dicotyledonous plants. The seeds of buckwheat could be processed into natural plant drinks through screening, baking and other machining processes. Present studies have found that buckwheat tea has good biological health-care effects, such as increasing immunity, lowering blood sugar and blood fat, anti-oxidation, and anti-cancer. There are already a large number of researches on flavonoids and proteins of buckwheat tea, but studies on polysaccharide of buckwheat tea are rare indeed. In order to elucidate the mechanism of multiple health-care effects of buckwheat tea more clearly, we studied tartary buckwheat tea polysaccharide (TBTP) in this article. Firstly, ultrasonic was applied to optimize the extraction process of TBTP; Secondly, cellulose column and dextran gel column were used to purify the TBTP, and monosaccharide composition of those polysaccharide were analysised by HPLC; Finally, we studied the protective effects of TBTP on HUVECs (human umbilical vein endothelial cell) which were damaged by HG/HF (high sugar/high fat,25 mM DMEM+ 500 ?M palmitate).The main data and results obtained in this experiment are showed as following:(1) In the single factor experiments, we can see that every single factor is corelative to the extraction rate of TBTP. The extraction rate of TBTP is significantly changed with the change of a single factor parameter values which including extraction times, ultrasonic power, solid-liquid ratio and extraction temperature. We applied the BBD to optimize the extraction process, and the results showed that when the ultrasonic power is 300 W, extracted times is three, liquid to solid ratio is 24.7:1, extracted temperature is 59.2 ?, and extracted time is 74.3 minutes, the extraction rate of TBTP reaches the maximum, which is 8.26%. Then, DEAE-52 cellulose column and Sephadex G-100 were used to isolated and purified TBTP. During this process, we got three kinds of homogeneous polysaccharide, which were named TBTP-1, TBTP-2 and TBTP-3. Finally, we used HPLC to analysis the monosaccharide composition of those polysaccharide, and the results showed that:The composition of TBTP are D-Man, D-Rib, L-Pha, D-Glcua, D-Glaua, D-Gla, D-Xyl, D-Gal, L-Ara and the relative molar percentage of these monosaccharide are 4.47%,3.09%,8.55%,2.52%,6.28%,22.08%, 12.49%,26.88%,13.64%, respectively; The composition of TBTP-1 are D-Man, L-Rha, D-Glaua, D-Gla, D-Xyl, D-Gal, L-Ara and the relative molar percentage of these monosaccharide are 1.66%,2.31%,0.60%,75.43%,1.96%,6.08%,11.97%, respectively; The composition of TBTP-2 are D-Man, L-Rha, D-Glcua, D-Glaua, D-Gla, D-Xyl, D-Gal, L-Ara and the relative molar percentage of these monosaccharide are 4.66%,4.39%,2.33%,9.51%,1.61%,25.86%,35.97%,15.67%, respectively; The composition of TBTP-3 are D-Man, L-Rha, D-Glcua, D-Glaua, D-Gla, D-Xyl, D-Gal, L-Ara and the relative molar percentage of these monosaccharide are 7.86%,8.35%, 4.41%,3.97%,3.74%,20.07%,46.53%,5.07%, respectively.(2) The cell experiments results showed that:after TBTP treatment, the cell survival rates of HG/HF+TBTP group was significantly higher (P?0.01) than HG/HF group, and the single treatment of TBTP group had no obvious difference with the control group; The apoptosis induced by HG/HF was blunted by TBTP, during this process, the expression of Bax was inhibited while Bcl-2 was increased, and the ratio of Bcl-2/Bax was lower than the control group, but was significantly higher compared to the HG/HF group (P?0.01); The expression of Cleaved Caspase-3 was also significantly inhibited; Moreover, TBTP can significantly reduce the abnormally elevated of ROS levels in HUVECs cells which were induced by HG/HF.The results indicate that the ultrasonic assisted extraction can significantly increase the extraction rate of TBTP. There are remarkable differences among TBTP, TBTP-1,TBTP-2, and TBTP-3, which lies not only in the monosaccharide composition, but also in the composition ratio of each monosaccharide. TBTP reduces oxidative stress and apoptosis induced by HG/HF. These findings provide the theoretical basis for further exploitation and application of TBTP.