The Studies Of High Polysaccharide Agaricus Blazei Murrill And Grifola Frondosa Varieties And The Biological Activities Of Their Polysaccharides

This research collected 26 Agaricus blazei Murill varieties and 29 Grifola frondosa varieties,screened all the varieties to select high polysaccharide content varieties,and studies the biological activities of the polysaccharides,the main results are as follows:1.Using RAPD,SRAP,ISSR molecular markers and ITS-PCR,the polymorphism analysis was done to 26 Agaricus blazei Murill varieties(A01～A29). Based on the results of four kinds of molecular markers,using UPGMA for the clustering analysis,Agaricus blazei Murill varieties were temporarily clustered into 4 sorts.The difference coefficient among A20,A21 and A22 was 0;The difference coefficient among A15,A16 and A17 was 0;The difference coefficient between A08 and A10 was 0.These mushroom varieties could be the same variety with different names.Using RAPD,SRAP and ISSR molecular markers,we clustered 29 Grifola frondosa varieties(G01～G29)were clustered into 8 sorts.The difference coefficient between G15 and G16 was 0;The difference coefficient between G07 and G10 was 0; The difference coefficient among G14,G18,G21,G26 and G27 was 0.These mushroom varieties could be the same variety with different names.2.Using the orthogonal test,we carried out the research on Agaricus blazei Murill and Grifola frondosa polysaccharide extraction by the methods including hot water method,ultrasonic and hot water method,microwave and hot water method. The suitable conditions for hot water extraction of Agaricus blazei Murill were pH 7.5, the ratio between material and water 1:20,extraction temperature 100℃,extraction time 4h,ethyl alcohol concentration 100%.The suitable conditions for ultrasonic and hot water extraction method were ultrasonic power 600W,4min for ultrasonic,2h for extraction,which improved 13.06%of extraction rate of Agaricus blazei Murill polysaccharide,while the polysaccharide content was decreased 9.87%.The suitable conditions for microwave and hot water extraction method were medium power,4min for microwave,2h for water extraction,which improved 16.10%of Agaricus blazei Murill polysaccharide extraction,while little influence on polysaccharide content.The suitable conditions for hot water extraction of Grifola frondosa were pH 7.5, the ratio between material and water 1:20,extraction temperature 100℃,extraction time 4h,ethyl alcohol concentration 95%.The suitable conditions for ultrasonic and hot water extraction method were ultrasonic power 400W,6min for ultrasonic,2h for extraction,which improved 13.56%of extraction rate of Grifola frondosa polysaccharide,while the polysaccharide content was decreased 14.89%.The suitable conditions for microwave and hot water extraction method were high power,4min for microwave,2h for water extraction,which improved 29.52%of Grifola frondosa polysaccharide extraction,while little influence on polysaccharide content.3.The polysaccharides from 4 Agaricus blazei Murill varieties and 8 Grifola frondosa varieties were extracted and analyzed.We selected high polysaccharide Agaricus blazei Murill "AB01",whose polysaccharide content in submerged fermentation liquid was 2.05mg/ml,1.61 times to the average value(1.27mg/ml),3.36 times to the lowest polysaccharide content variety "AB03"(0.61mg/ml).The output of this variety's submerged fermentation was high,the intracellular polysaccharide content was 12.04%,and the extracellular polysaccharide content was 1.54 mg/ml.We selected high polysaccharide Grifola frondosa "GF06",whose polysaccharide content in submerged fermentation liquid was 1.65mg/ml,2.79 times to the average value(0.59mg/ml),7.50 times to the lowest polysaccharide content variety "GF03"(0.22mg/ml).This variety's intracellular polysaccharide content was 7.22%and the extracellular polysaccharide content was 0.98 mg/ml.4.Through setting up mouse S₁₈₀tumor-bearing models,we carded out the research on the anti-tumor functions of the polysaccharide(AB01-P)from fermentation liquid of "AB01","AB03-P" from fermentation liquid of "AB03", "AB01-P-1" from intracellular polysaccharide,"AB01-P-2" from extracellular polysaccharide,and polysaccharide "GF06-P" from the fermentation liquid of "GF06", "GF06-F-1" from intracellular polysaccharide,"GF06-P-2" from etracellular polysaccharide and compound polysaccharide "AG-P" containing "AB01" and "GF06".In the group of Agaricus blazei Murill:AB01-P has obvious function on the suppression of the S180 tumor's activity,relating with the dosage present.High dosage group's tumor suppression rate was 45.36%,which was higher than AB03-P's highest rate 25.14%,AB01-P-1's highest rate 36.61%,AB01-P-2's highest rate 30.05%.In the group of Grifola frondosa,GF06-P had obvious function on the suppression of the S₁₈₀tumor's activity,relating with the dosage present.High dosage group's tumor suppression rate was 42.08%,which was higher than GF03-P's highest rate 26.78%,GF06-P-1's highest rate 38.25%,GF01-P-2's highest rate 39.89%. Compound polysaccharide(AG-P)'s tumor suppression was related with low and medium dosage,high dosage didn't bring high tumor suppression rate comparing to medium dosage,medium dosage group's tumor suppression rate was 49.18%.The results indicated that compound polysaccharide had a little higher tumor suppression rate comparing to single polysaccharide.Comparing with the control group,all of Agaricus blazei Murill,Grifola frondosa and compound polysaccharide could obviously or extremely obviously enhanced the S₁₈₀tumor-bearing mouse's thymus index and the spleen index.5.We used Annexin the V/PI double dyeing flow cell sorter to study the influence on the early death of human usteosarcoma cell line(MG-63)by AB01-P, GF06-P and AG-P.The results indicated that AB01-P induced MG-63 cell death, relating to the dosage present.The group of 2.0mg/ml had the death rate 30.98%. GF06-P had no influence on MG-63 cell death.AG-P induced some MG-63 cell death, and the rate was 16.35%,not relating to the dosage present.6.Using the RT-PCR technology,we studied the influence on granulocyte-macrophage colony-stimulating factor(GM-CSF)gene expression by "AB01-P" and "GF06"."AB01-P" induced granulocyte mononuclear cell to express GM-CSF,the most suitable concentration was 2.0μg/ml,and thus confirmed its molecular mechanism of promotion of granulocyte-macrophage cells and regulation of animals' immune system.GF06-P could not obviously induce the mononuclear cell GM-CSF gene expression.7.We carried out the fruit fly survival experiments to test the days of half death, average lifetime and the longest average lifetime,and also SOD value,MDA total content and total anti-oxidation value in fruit fly homogenate.We studied the anti-senile function and its mechanism in fruit fly by AB01-P,AB03-P,AB01-P-1, AB01-P-1 and GF06-P,GF03-P,GF06-P-1,GF06-P-2 and compound polysaccharide AG-P.AB01-P was the best polysaccharide elongating the fruit fly's average lifetime, elongating male fruit fly's lifetime 17.32%,and female fruit fly's lifetime 11.40%;the elongation of half death time were 10.81%(male)and 10.21%(female);The elongation of fruit fly's the longest average lifetime were 15.91%(male)and 13.11% (female).GF06-P obviously elongated the average lifetime of female fruit fly,which was 22.00%,and 17.67%for male.The elongation of half death time were 13.31% (male)and 19.71%(female);The elongation of fruit fly's the longest average lifetime were 12.74%(male)and 28.32%(female).Compound polysaccharide AG-P elongated the lifetime 23.78%for male and 13.06%for female;AG-P also obviously elongated the longest average lifetime 20.77%fro male and 19.01%for female.AB01-P extremely obviously enhanced the male fruit fly's total anti-oxidation ability 28.89%(p＜0.01),while no influence on female fruit fly.AB01-P-2 extremely obviously enhanced SOD value 42.77%(p＜0.01)to male fruit fly,while no influence on female fruit fly;it also extremely obviously enhanced total anti-oxidation 68.89% (male)and 40.54%(female)(p＜0.01).GF06-P obviously enhanced male fruit fly's total anti-oxidation 17.78%(p＜0.05);it extremely obviously enhanced the female fruit fly's total anti-oxidation 48.64%(p＜0.01).GF06-P-1 obviously decreased MDA content in female fruit fly and extremely obviously decreased MDA content in male fruit fly.GF06-P-1 obviously enhanced female fruit fly's total anti-oxidation 18.92% (p＜0.05).GF06-P-2 obviously enhance male fruit fly's SOD value 12.26%,and 38.41%for female fruit fly(p＜0.01).Compound polysaccharide AG-P obviously decreased female fruit fly's MDA value,while no obvious influence on male fruit fly.