Study On Extraction,Purification,Characterization And Bioactivity Of Grifola Frondosa Polysaccharide

Grifola frondose is widely planted all over the world.It is not only a kind of delicious,crisp and nutritious traditional food loved by the majority of consumers,but also has a long history of medicine.As an important nutritional component of Grifola frondosa,polysaccharide has been widely concerned by researchers because of a series of biological activities,such as antioxidant,anti-inflammatory,anti-virus,anti radiation,liver protection,immune regulation,hypoglycemic,blood pressure lowering,blood lipid lowering and anti-tumor biological activities.This thesis aims to establish a simple and efficient extraction method of polysaccharide from Grifola frondosa,and to study the antioxidant and antitumor activities and mechanism of Grifola frondosa polysaccharide and its purified components,so as to provide a theoretical basis for better utilization of Grifola frondosa resources and research and development of Grifola frondosa polysaccharide related healthy food.The main results of this thesis are as follows:1.Vc-H₂O₂ assisted extraction method was established to extract polysaccharide from Grifola frondosa,and the processing parameters were optimized through single factor and response surface experiments.The final optimum extraction conditions are as follows:liquid material ratio 34 m L/g,Vc/H₂O₂ concentration 18.40 mmol/L,Vc/H₂O₂ action temperature50℃.Under these conditions,the extraction rate of Grifola frondosa polysaccharide（CGFP）was 14.80%,which was higher than the traditional hot water extraction method（8.91%）.GFP,obtained from CGFP after protein removal,was separated and purified by DEAE-52 ion exchange chromatography column,followed by dextran Sephadex G-100 chromatography,providing three polysaccharide components,GFP0,GFP1 and GFP3,with a yield of 11.64%,33.81%and 4.31%respectively（Calculated with GFP as starting material）.2.The total sugar contents of GFP,GFP0,GFP1 and GFP3 were determined to be 72.67%,59.38%,78.56%and 62.42%respectively.There were small amount of reducing sugar,sulfate group and protein in the four polysaccharides.With the exception of GFP0 fraction,the other three polysaccharides also contain a small amount of uronic acid.The results of monosaccharide composition assay showed that GFP,GFP0,GFP1 and GFP3 were mainly composed of glucose,galactose,mannose and fucose.FTIR and ¹H NMR spectra assays indicated that the four polysaccharides containedαandβglycosidic bond.Congo red experiment showed that there were three-dimensional helical conformations in GFP,GFP0,GFP1 and GFP3.3.The chemical antioxidant capacity of GFP,GFP0,GFP1 and GFP3 was evaluated by measuring their free radical scavenging capacity and reducing power.The results indicated the IC₅₀ values of GFP,GFP0,GFP1 and GFP3 against DPPH radical scavenging were 0.252,1.809,0.682 and 0.117 mg/m L respectively;The IC₅₀ values of hydroxyl radical clearance were 1.652,15.779,2.684 and 0.918 mg/m L respectively;The IC₅₀ values of ABTS radical scavenging rate were 0.739,7.689,3.035 and 0.736 mg/m L respectively;at a concentration of 3 mg/m L,the iron reducing power of GFP and GFP3 was stronger than that of GFP0 and GFP1.4.In the macrophage RAW 264.7 oxidative stress model induced by H₂O₂,GFP,GFP0,GFP1 and GFP3 showed significant protective activity against oxidative stress.At a concentration of 100μg/m L,compared with the model group,the levels of ROS in GFP,GFP0,GFP1 and GFP3 groups decreased by 63.11%,73.98%,103.11%and 72.11%respectively;compared with the model group,the activity of SOD was increased from 6.52U/mg prot to 9.84,8.46,10.60 and 11.22 U/mg prot,respectively;compared with the model group,CAT enzyme activity increased from 1.51 U/mg prot to 2.84,2.46,2.60 and 2.80 U/mg prot;compared with the model group,the content of MDA decreased from 2.14 nmol/mg prot to 0.88,1.00,0.78and 0.75 nmol/mg prot.At a concentration of 100μg/m L,it was found that the expression levels of SOD1,CAT,HO-1 and Nrf-2 m RNA in GFP,GFP1 and GFP3 treatment groups were significantly increased,while such effect of GFP0 treatment group was not obvious.5.The antitumor activity of GFP1 was investigated using MTT assay.It was found that GFP1 significantly inhibited the proliferation of lung cancer cell H1975 in a concentration-dependent manner.Scratch test,migration test and invasion test showed that GFP1 could inhibit the migration of H1975 cells.Hoechst 33258 staining showed that H1975 cells treated with GFP1 showed typical morphological characteristics of apoptosis;flow cytometry analysis further confirmed that GFP1 could induce the apoptosis of H1975 cell.GFP1 treatment was also able to increase the expression level of ROS,decrease the mitochondrial membrane potential and increase the activity of Caspase 3 in H1975 cells.6.The effect of GFP1 treatment on the m RNA expression of multiple genes in H1975 cells was investigated.The results showed that GFP1 could significantly down regulate the m RNA expression levels of PI3K,ERK2,ERK1,FAK and RAF1,suggesting that the inhibition of Ras/Raf/MEK/ERK signal pathway and PI3K/Akt/m TOR signal pathway may be the important antitumor mechanisms of GFP1.