| Organic selenium shows a higher bioavailability and threshold for toxicity compared to inorganic selenium, so organic selenium research has become a central issue. Selenium polysaccharide as a kind of biological macromolecule of organic selenium, its pharmacological activity is generally higher than that of polysaccharide or selenium. But the selenium content in natural polysaccharides cannot meet the requirements of people. Wen-Codonopsis pilosula polysaccharide(W-CPP) is one of mainly active substances of Codonopsis pilosula Nannf.var.modesta (Nannf.) L.T.Shen (Wendang), and possess antitumor activity.The specific research contents as follows:1、Preparation and charcteristic of W-sCPP1 and study on its anti-tumor activity(1) The four Wen-Codonopsis pilosula polysaccharide fractions (W-CPP1, W-CPP2, W-CPP3 and W-CPP4) were collected by our lab, the inhibition rate of above four polysaccharide on A549, BGC-823 and HeLa cell was 47.7%,38%,31.7%; 23.0%,26.9%,5.01%; 20.5%,23%, 20.9%; 21.8%,20%,17.2% in the concentration of 400μg/mL, respectively. The result shown that W-CPP1 posses best antitumor activity, so choose W-CPP1 for further research.(2) HNO3-Na2SeO3 method was performed for the selenylation modification of W-CPP1 (W-sCPPl). orthogonal experimental design combine with single-factor experiments,5h of reaction time,60℃ of reaction temperature, and 2:2 ratio of polysaccharide to Na2SeO3, the amount of BaCl2 is O.lg, concentration of HNO3 is 1.0% were chosen as the optimal selenylation modification conditions. The selenium content of W-sCPP1 and its inhibition rate on A549 cells exhibited moderate correlation(r=0.67).2、Preparation and charcteristic of selenized Wen Codonopsis pilosula pectin polysaccharide (W-CPP1b) and study on its anti-tumor activity(1) Considering W-CPP1 is a complex polysaccharides, W-CPP1 was further purified by DEAE-cellulose filtration medium. And obtain three fractions (W-CPPla, W-CPPlb, W-CPP1c), the grow inhibition rate of A549, BGC-823 and HeLa cell was 43.2%,20.96%,23.09%; 55.8%, 48.84%,45.13%; 26.87%,11.97%,15.23% in the concentration of 400μg/mL, respectively.The result shown that W-CPP1b posses best antitumor activity.(2) A HNO3-Na2SeO3 method was performed for the selenylation modification of W-CPPlb (W-sCPPlb). The result of orthogonal experimental design shown optimal selenylation modification conditions of W-CPPlb like that of W-CPP1, the validation experiments completed under the optimal conditions gave the mean selenium content and yield of W-sCPPlb were 0.48mg/g and 86%, respectively. Selenylation modification can significantly increase the antitumor activity of W-CPPlb in vitro.(3) The FT-IR of W-sCPPlb indicated that the form of W-CPPlb attaching Se are mainly as ; The result of thermogravimetric analysis (TG) shown that thermal stability of W-CPPlb has already change; High-performance gel permeation chromatography indicated that W-sCPPlb was a homogeneous polysaccharide and W-sCPPlb were a random string; Uronic acid content of W-sCPPlb is 78%. Based on the structure of W-CPPlb, we speculated the structure of W-sCPP1b as follows:3、Studying on antitumor mechanism of W-sCPPlb and W-CPPlbA549 cell was employed for in vitro studies on changes of morphology with Wright’s dye, cell migration with wound healing method, cycle distribution and induction of apoptosis with Annexin V-FITC/PI apoptosis detection kit, and expression of apoptosis-associated proteins with Western blot after treatment of W-sCPPlb and W-CPPlb. The results demonstrated that the mechanism of W-sCPPlb and W-CPPlb induced apoptosis involves a caspase-mediated pathway. And the antitumor activity of W-sCPP1b stronger than that of W-CPP1b. |
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