Studies On Optimized Extracting Technology Of Crude Polysaccharides From Oviductus Ranae And Its Pharmacological Activities

In this paper, Oviductus Ranae from the Changbai Mountains was used to study extracting technology, antineoplastic activity and fatigue resistence under laboratory conditions.1. Comparative test of enzymes:to hydrolyze the dry Oviductus Ranae, three proteases including pepsin, neutral protease and trypsinase were used to compare the extracting output of crude polysaccharides. Water fraction was set as 1:100, corresponding zymohydrolysis pH was adjusted, trypsinase concentration was 5% and zymohydrolysis time was 2h. After alcohol deposition, extracting output of crude polysaccharides was:0.19%,2.16% and 2.3%. The result showed that trypsinase was a ideal enzyme for crude polysaccharides extrcting from Oviductus Ranae.2. Optimizing test of extracting technology:first, water fraction, trypsinase concentration, zymohydrolysis time and zymohydrolysis pH were taken as factors to evaluate the best intervals by single-factor examination. Then, further L9(34) orthogonal design trials were carried out. The extracting output and purity of crude polysaccharides were examined. Results showed that extracting condition had significant (P<0.05) effect on crude polysaccharides output. The most effective condition was water fraction, trypsinase concentration took the second place, the third was zymohydrolysis pH, and the last was zymohydrolysis time, or A>B>D>C. Optimum conditions for the highest output, which was 5.36% and purity was17.40%, was A2B2C3D2, which reffered that the ratio of water to sample was 1:150, trypsinase concentration was 7%; zymohydrolysis time was 8h, and zymohydrolysis pH was 8. Each condition of purity was insignificant (P>0.05), The most important factor was water fraction, zymohydrolysis time came the second, the third was zymohydrolysis pH, and the last was trypsinase concentration, or A>C>D>B. Optimum conditions for the highest purity, which was 24.19% and output was 4.17%, was A3,B3,C3,D3, which reffered that the ratio of water to sample was 1:200, trypsinase concentration was 9%; zymohydrolysis time was 8h, and zymohydrolysis pH was 8.5. 3. Test of antineoplastic activity:to investigate the effect of crude polysaccharides from Oviductus Ranae on SKOV-3 Cells of ovarian cancer, the density of HEC-1B cells with logarithmic phase were regulated to 5×106/ml,we took the blank for the negative control, and crude polysaccharides for test group.and its density were set as 0.08,0.4,2,10,50,100ug/ml, using 96 hole board, and six holes for each density. After 24h and 48h of culturing, SKOV-3cell viability was detected by MTT method to record OD value,and verified by SRB. Useed the different methord to measuring the effect of crude polysaccharides from Oviductus Ranae on SKOV-3 Cells of ovarian cancer,and results showed that each dose of crude polysaccharides from Oviductus Ranae had little or no effect on its cell livability,and had no significant antineoplastic activity.4. Fatigue resistence test:activity of Oviductus Ranae and its crude polysaccharides were determined by mouse swimming performance test. Experimental animals were randomly divided into 7 groups:negative control group was given physiological saline, positive control group was Oviductus Ranae, and 5 experimental groups were given different dosages of crude polysaccharides (800,400,200,100 and 50mg/kg). Room temperature was at 25±1℃. Each mouse was intragastrically administrated 0.5ml everyday, for 10 days. Swimming performance test was carried out one hour later from the last administration. Iron wire of 5% of its body weight was tied on the mouse - tail. The swimming time was recorded when the mouse could not stick its head out of water for 10 seconds. Results showed that Polysaccharides from Oviductus Ranae can greatly increased the fatigue esistence of mouse, and the effect was related with dosages of crude polysaccharides.the 100mg/kg dose was the best, extremely significant difference (P<0.01), swimming time extension was the highest, and it was 71.9%. the effect was insignificant above 200mg/kg dose, and the higher doses were lower than negative control.