Study On The Isolation,Purification And Biological Activity Of Secondary Metabolites From Cladonia Amaurocraea (Fl(?)rke) Schaer

Lichen is a symbiotic complex formed by symbiotic bacteria and symbiotic algae in the long-term growth process,and is considered to be the most perfect model of mutualistic symbiosis.Cladonia amaurocraea（Fl（?）rke）Schaer belongs to the genus Cladonia of Cladoniaceae Cladonia of the order Lecanorales.It has the functions of improving eyesight,calming the nerves,clearing heat and moistening dryness,and hemostasis in traditional medicine.A large number of studies have shown that there are many biologically active substances in lichens,which show great application potential in modern medical fields such as antioxidant,antibacterial,antitumor and antiviral.In order to further develop the potential value of Cladonia amaurocraea（Fl（?）rke）Schaer in the field of modern medicine,this thesis systematically studies the biological activity and chemical composition of the metabolites and secondary metabolites of Cladonia amaurocraea（Fl（?）rke）Schaer.（1）Taking Cladonia amaurocraea（Fl（?）rke）Schaer as the research object,using 4different solvents（cold water,hot water,absolute ethanol and diethyl ether）as extractants,the antioxidant activity（the ability of ABTS free radical scavenging ability,the ability to DPPH）was improved.The biological activities of the obtained four types of extracts were evaluated in terms of free radical scavenging ability and total reducing ability)and antibacterial activity.The results showed that the antioxidant activity of the hot/cold water extract was better than that of the ethanol/ether extract;while the antibacterial activity showed the opposite result,compared with the hot/cold water extract,the ethanol/ether extract better antibacterial activity.Further through the chemical composition system pre-test experiment,the chemical composition of the four types of extracts was preliminarily analyzed.Different solvent extracts contain different chemical compositions:the cold water extract mainly contains amino acid polypeptides;the hot water extract mainly contains amino acids and polypeptides.Contains tannin components;ethanol extracts mainly contain phenolic components;ether extracts mainly contain volatile oils.（2）Using the experimental strategy similar to the above,taking the fermentation broth of Cladonia amaurocraea（Fl（?）rke）Schaer as the research object,dichloromethane,ethyl acetate and n-butanol were used to extract the active components in the fermentation broth of Cladonia amaurocraea（Fl（?）rke）Schaer.The chemical composition of the fermentation broth of Cladonia amaurocraea（Fl（?）rke）Schaer symbiotica was studied by chemical system pre-test method.Dichloromethane,ethyl acetate and n-butanol were used to extract the active components from the fermentation broth of Cladonia amaurocraea（Fl（?）rke）Schaer,and the biological activities of the three extracts and fermentation broth were evaluated,mainly including antioxidant activity and antibacterial activity.active.The results showed that the fermentation broth of Cladonia amaurocraea（Fl（?）rke）Schaer contains four active components:tannin,flavonoids,phenolic components and volatile oils.Compared with other solvent extracts,ethyl acetate extract showed better effect in both antioxidant activity and antibacterial activity,and more importantly,its scavenging ability and total reducing ability of DPPH radicals were better than positive control group.A comprehensive analysis of the biological activities of Cladonia amaurocraea（Fl（?）rke）Schaer and secondary metabolites of symbiotic bacteria fermentation broth,in the tested extract,the ethyl acetate extract of secondary metabolites of symbiotic bacteria fermentation broth obtained with ethyl acetate as a solvent has The best life activity,the next step will take this as the research object,and the structure identification of the effective chemical components will be carried out.（3）Taking the ethyl acetate extract of the fermentation broth of Cladonia amaurocraea（Fl（?）rke）Schaer as the research object,the preparation conditions of the fermentation broth were optimized.Under the best conditions,the obtained ethyl acetate extract was further processed by silica gel column chromatography The biological activity of the obtained 6 components was evaluated.The two components with the best biological activity were selected from them,and after preparative liquid phase separation,the composition of the compounds was analyzed and identified by combining mass spectrometry and nuclear magnetic resonance spectroscopy.This chapter takes the ethyl acetate extract as the research object,uses silica gel column chromatography for preliminary separation,and studies the antioxidant activity of the obtained groups,mainly including the research of ABTS free radical scavenging ability,DPPH free radical scavenging ability and total reducing ability.Guided by activity,the two groups with the best antioxidant activity were separated and purified by preparative liquid phase,and the obtained compounds were analyzed and identified by mass spectrometry and nuclear magnetic resonance spectroscopy.The results showed that 6groups of F₁,F₂,F₃,F₄,F₅and F₆were obtained by preliminary silica gel column chromatography.Through the evaluation of three antioxidant indexes,it showed that the antioxidant activity of F₂and F₆groups was the best.Through preparative liquid phase separation and purification,5 compounds were obtained,and the structure of compound1 was analyzed and identified.The molecular formula of compound 1 was C₂₁H₂₀O₉,the relative molecular mass:416.1,and it was determined to be daidzein,an isoflavone component.