Studies On The Separation And Purification Of Secondary Metabolites And The Activity Of Secondary Metabolites In Sanghuangporous Baumii And Spectrum-Activity Relationship

Sanghuangporous spp.is a traditional Chinese medicine.It is recorded in Compendium of Materia Medica written by Li Shizhen in the Ming Dynasty that"The Sanghuangporous spp.is also known as the parasitic fungus on the mulberry,the mulberry fungus and the fungus on the mulberry.Cool the blood to stop bleeding,activate the blood and disperse the knot;Treating epistx,blood in urine,hemorrhoid,leakage,throat lump,and lump accumulation.Its activity was studied by scholars at home and abroad for a long time.Its main efficacy is anti-tumor,anticancer,liver protection,blood glucose lowering,diabetes treatment,anti-oxidation,anti-inflammatory and antibacterial.Recently,it has been widely recognized as a good anticancer drug due to its excellent anticancer activity.There have also been reports on active substances.According to the reports,the active ingredients in Sanghuangporous spp.mainly include polysaccharides,sterols,triterpenes,flavonoids,pyranones and polyphenols.Although a large number of compounds have been reported,most of them are determined by liquid-quality ratio.There is no specific means of separation and purification,and many compounds have not been able to obtain a single pure product.In this paper,in order to explore the main active components of Sanghuangporous spp.and obtain the related single pure products through compound separation and purification,as well as the correlation between fingerprint and activity,the following three aspects of experiments were designed,in order to explain the activity of Sanghuangporous spp.and its corresponding active substances,and to establish the spectral efficiency relationship of Sanghuangporous spp..1.Composition and activity comparison of secondary metabolites in mycelia and fruiting bodies of Sanghuangporous spp.Sanghuangporous spp.mycelium and fruiting body in petroleum ether,chloroform,ethyl acetate,n-butanol extraction,four by four different extraction phase,four different extraction phase for the liquid phase analysis,compares the difference between their retention time and peak area,to determine the mulberry huang mycelium and fruit body of secondary metabolites on the composition and content of the specific difference;Activity mainly from two aspects,anti-oxidation and anti-tumor are analyzed:clear in antioxidant capacity are compared in DPPH·free radicals and ABTS⁺free radical ability difference,first of all,from the result of the removal ability of DPPH·free radicals,mycelium and fruit body were ethyl acetate and n-butanol phase had good antioxidant activity,in addition,the mycelium of four phase extraction to remove DPPH·free radical ability is higher than that of fruiting body four extraction phase.Secondly,from the results of ABTS⁺free radical scavenging ability,mycelium and fruiting bodies also had better antioxidant activity in the ethyl acetate phase and n-butanol phase.In addition,the DPPH·free radical scavenging ability of the four extraction phases of mycelium was higher than that of the frustum except the petroleum ether phase.The antioxidant capacity of two kinds of free radical scavenging experiments showed the difference of antioxidant capacity between different extractive phases of mycelium and fruiting body.In terms of tumor observation,differences were compared in their ability to inhibit Hep G2 and MCF-7cells.From the perspective of the ability of mycelium and fruiting body to inhibit the growth of the two tumor cells,the four extraction phases of mycelium and fruiting body had certain inhibitory effect and dose effect on the growth of the two tumor cells in the higher concentration group.Contrary to the antioxidant capacity,the inhibition rate of each extractive phase of the fruiting body on Hep G2 proliferation was higher than that of the mycelium.In terms of inhibiting Hep G2 cell growth,both mycelium and frustum had the highest inhibition rate of proliferation of chloroform phase,followed by ethyl acetate phase.In terms of inhibiting the growth of MCF-7 cells,the proliferation inhibition rate of chloroform in each extraction phase of the fruiting body was still the highest,while that of petroleum ether in each extraction phase of the mycelium was the highest.2.Isolation,purification and structural identification of secondary metabolites in Sanghuangporous spp.The separation and purification of secondary metabolites of Sanghuangporous spp.can be divided into two parts according to different raw materials,respectively using mycelium and fruiting body as raw materials.First of all,the mycelium as the raw material for the separation and purification of the material is extracted through the first chapter of the ethyl acetate phase.In order to screen the suitable solvent system for the separation of high-speed countercurrent chromatography,60 different solvent systems were analyzed,and the solvent system suitable for the ethyl acetate phase samples was finally determined as hexane:ethyl acetate:methanol:Water,and their ratio is 2:5:2:4,1:3:1:2,2:4:12:8,1:5:2:7,4:10:4:9,8:12:7:11,8:27:10:16.The value of K is suitable for the separation of high-speed counter-current chromatography.Through the experimental results of high-speed counter-current chromatography,the final ratio is 4:12:5:8,and the separation effect is the best.The components of 71%target peak purity 1 and 64%target peak purity 2were separated and enriched by high speed counter-current chromatography.The two components are then purified by preparation of a liquid phase,and the resulting pure product is analyzed by liquid phase and nuclear magnetic resonance analysis,The second is the separation and purification by using the fruiting body as raw material.Fruiting body by 80%ethanol extraction enrichment,dry after 24 hours,the samples obtained large diameter silica gel column separation,silica gel column separation shall be carried out in accordance with the test tube branch collection,at the same time each tubes and TLC thin layer chromatographic analysis,according to the results of the analysis of the merger,the merger of the component concentration and dry,the samples obtained small diameter of silica gel column separation,repeat the above operation,incorporating tube sample concentration after drying for the preparation of liquid phase separation,preparation of liquid phase process is optimized,and then a large number of preparation,according to the peak time for collecting samples and eventually won the six components.After liquid phase analysis,the purity of two components is between 70%and 80%.To further purification,the two components through TLC separation,preparation of TLC first screening the suitable expansion agent,and then for the preparation of collection,the TLC plate of silica gel blow down after using the elution agents,and then put the elution of the sample solution for enrichment,after repeated three times,the components through the liquid phase analysis,determine their concentration was 85%,and nuclear magnetic analysis,3.Study on the relationship between spectral effects and antitumor and antioxidation of Sanghuangporous spp.The HPLC analysis was carried out on 32 Sanghuangporous spp.fruiting bodies as raw materials,and the chromatogram was processed by the traditional Chinese medicine fingerprint analysis software to establish the HPLC fingerprint of Sanghuangporous spp..As in chapter 1,anti-oxidation and anti-tumor experiments were carried out on 32 samples,namely,the ability to eliminate DPPH·free radicals and ABTS⁺free radicals,and the ability to inhibit Hep G2 cells and MCF-7 cells,respectively.The peak area in fingerprint was taken as independent variable and the IC₅₀ value of antioxidant capacity and anti-tumor capacity as dependent variable for regression analysis,and the regression equation of their correlation was determined.In order to verify the accuracy of the models,with seven other mulberry yellow samples HPLC analysis,at the same time,anti-oxidation and anti-tumor activity experiment was carried out to the seven samples of peak area into the prediction of the regression equation obtained active value and the actual experimental comparative analysis of the proceeds of the activity,in order to compare with the actual and estimated values,introduced the concept of similarity,the calculation is drawn and the actual and estimated values of the XY scatterplot and Y=X in a straight line,Q=1-(SS_res)/(SS_tot)=1-（∑_i（y_i-f_i）²）/（∑_i（y_i-（?））²）,the calculated value is more close to 1 shows that the higher the similarity.