Analysis,Enrichment And Bioactivity Of Nucleoside Components From The Primary Cultivars Edible-medicinal Fungi

Nucleoside components are the basic of cells to maintain life activities,widely existing in animals,plants,fungi and other organisms.Nucleoside components have multiple biological activities such as antivirus,anti-tumor,bacteriostasis,and have been widely used in the field of medicine.Edible-medicinal fungi are an important source of natural small molecule active compounds.Some studies have been carried out on the unique nucleoside components found in edible-medicinal fungi,but there are few systematic studies of nucleoside components in different edible-medicinal fungi.In order to clarify the information of the nucleoside components in the main cultivated edible-medicinal fungi in China,this study used high performance liquid chromatography(HPLC)to detect the nucleoside components in the edible-medicinal fungi,combined with its species classification and fingerprint technique.The characteristics were systematically analyzed and compared,and the process of obtaining the macroporous resin to separate and enrich the nucleoside components was optimized.Based on the preparation of the nucleoside components enriched products of different edible-medicinal fungi,the anti-tumor cell proliferation activity was evaluated.The research of this thesis mainly includes the following three aspects:(1)Analysis of nucleoside components of common cultivated edible-medicinal fungi in China25 common edible-medicinal fungi of 11families and 19 genera in the market were collected,and the contents of 13 nucleoside components were determined by HPLC and analyzed by fingerprint technique.The results showed that the three nucleosides,adenosine,uridine and guanosine,were the most common and highest-weighted components in edible-medicinal fungi.Even so,there are significant differences in the content of nucleoside components between different types of edible-medicinal fungi.There are 11 edible fungi with a similarity of more than 80%,including Lentinus edodes,Pleurotus citrinopileatus,Agaricus blazei Murr.,Pleurotus eryngii,Hypsizygus marmoreus,white Hypsizygus marmoreus,Flammulina velutipes and Panellus serotius,have high similarity and content of nucleoside components in the range of 4.31mg/g-12.84mg/g.The contents of Cordyceps militaris,Grifola frondose,Morchella esculenta and Pholiota nameko are 12.97mg/g,11.66mg/g,9.55mg/g and 8.90mg/g respectively.The content of nucleosides in Ganoderma,Tremella,and Auricularia is generally low,ranging from 0.17mg/g to 3.17mg/g,and there are certain differences in the types of nucleosides in edible fungi of the genus Tremella and Auricularia.The similarity between Agrocybe aegerita and most of the edible-medicinal fungi is low.Compared with the eluting profile of HPLC,it is found that the nucleoside composition of Agrocybe aegerita is better.The edible-medicinal fungi with nucleoside content above1mg/g were selected for principal component analysis,and the comprehensive evaluation score of each variety was calculated by the evaluation function F=0.549F₁+0.276F₂+0.175F₃.Among the 19 edible-medicinal fungi,L.edodes had the highest comprehensive score of 5.4017,and the comprehensive scores of Pleurotus citrinopileatus and Grifola frondosa were more than 5,indicating that the comprehensive quality of the nucleoside components was relatively high.However,the edible-medicinal fungi of Ganoderma have low comprehensive scores,with the content ranging from 0.17mg/g to 3.17mg/g,indicating that the comprehensive quality of the nucleoside components of Ganoderma is low.(2)Separation and enrichment of nucleoside components in edible-medicinal fungiL.edodes was selected as the material,and the extraction conditions of nucleoside components were optimized.The results showed that the concentration of nucleosides by ultrasonic extraction for 2 hours was 4 times that of boiling water extraction,and the concentration of cytidine,guanosine,uridine and adenosine were higher.The NKA-?macroporous resin exhibited the excellent characteristics for enrichment of nucleoside components,and its adsorption rate and desorption rate were 86.29%and 80.86%respectively.The extracted solution was adsorbed for 2h under the condition of 20:100(g:mL)ratio of resin addition to volume of extracted solution,and then desorbed with30%ethanol for 0.5h.For twice to obtain the enriched fraction with yield of 8.39%and the total concentration of known nucleosides of 21.16%.Then 14 kinds of edible-medicinal fungi with yield of 4.96%-10.70%were prepared,and the total known content of nucleoside components was 7.72%?25.95%.The unknown characteristic peaks in the enriched product were calibrated,and a total of 10 unknown characteristic peaks(Peak1-10)were obtained by screening.(3)Study on tumor growth inhibition test of enriched nucleoside components from edible-medicinal fungiThe tumor growth inhibition test of the enriched nucleoside products of different edible-medicinal fungi was evaluated.The results showed that the IC₅₀ values of the enriched products of Flammulina velutipes,white Hypsizygus marmoreus,Lentinus edodes,Agaricus blazei Murr.,Ganoderma leucocontextum,Cordyceps militaris,Coprinus comatus,Grifola frondosa,Pleurotus citrinopileatus,Pholiota nameko and Morchella esculenta were 54.807?g/mL,71.796?g/mL,83.060?g/mL,106.639?g/mL,141.781?g/mL,202.474?g/mL,240.651?g/mL,390.358?g/mL,462.025?g/mL,587.900?g/mL,821.688?g/mL,respectively.The enriched nucleoside products of Panellus serotius,Pleurotus eryngii and Agrocybe aegerita had a weak inhibition ability on K562 cells.A comprehensive comparison of the total content of nucleoside components and fingerprint characteristics of the enriched product reveals that its tumor growth inhibition activity is related to the content and type of nucleoside components.Adenosine,uridine and guanosine,as the main nucleoside components in edible and medicinal fungi,had no significant effect on the tumor growth inhibition activity.