Studies On The Secondary Metabolites And Their Functions Of Cockroach Endosymbiont Aspergillus Sp.

Insect symbiotic microbe is a major microbe who live together with other organisms. As the increasing number of researches on insect symbiotic microbe, the secondary metabolites of symbiotic microbe active ingredients have been isolated, purified and identified, which has become an important source and pathway of new compounds. Due to the long-term coevolutionary with the host, they have a special metabolic pathway. There for the symbiotic microbes are likely to produce some secondary metabolites with special structure and significant physiological activity. Previous studies confirmed that the secondary metabolites can produce secondary metabolites with a novel skeleton, and they have many physiological functions, such as antibacterial, anti-malaria, anti-tumor, immunity, weeding and so on.In previous work, we had completed the symbiotic bacteria separation and purification work of the five kind of insects as cockroaches, termites, butterflies, bees, crickets. There are 42 strains, including 20 strains of fungi, 13 strains of actinomycetes and 9 strains of bacteria. Through the in vitro activity test, two strains with stronger activity were screened out. On this basis, we share from which a cockroach sample symbiotic fungus FL-F5(Aspergillus, Aspergillus sp.) as the research object, its fermentation broth secondary metabolites extraction, separation, purification and structural identification, and the function and biological activity of the isolated compounds were studied. The main research contents are as follows:1. Comprehensive use of various modern chromatographic separation to extract, separate and purify the secondary metabolites from fermentation broth, then we get nine monomer compounds, identify five compounds' structure. Among them, compound 1 was new skeleton compound Aspergirin A(1), compounds 2-5 were known compounds: E-4-hydroxycinnamic acid methyl ester(2),6,8-dihydroxy-3,4,5-trimethylisocoumarin(3),E-4-hydroxycinnamic acid(4),tricin(5).2. By fluorescence spectroscopy on the interaction of these four compounds(2-5) with various metal ions were studied and found that compound 3 and 5 have a good recognition effect with Pb2+ ion. By the Job's plot analysis and Benesi-Hildebrand method, we found these two compouds and Pb2+ formation of 1:1 complex, the binding constants were 1.62×104 and 1.84×104.3. The inhibitory effect of four compounds on 5-lipoxygenase(5-LOX) activity was investigated, the results showed that compound 2, 3 and 4 without enzyme inhibitory effected to 5-LOX, compound 5 had the weak inhibitory effect to 5-LOX, and IC50 value was 2.05 mmol/L. Then the inhibitory effect of four compounds we studied on the activity of tyrosinase. The results showed that compound 2 had no inhibition, compounds 3, 4 and 5 had inhibition effect, with IC50 value 0.94 mmol/L, 0.41 mmol/L and 0.66 mmol/L respectively. The inhibitory effect of these three compounds on tyrosinase was equivalent to the positive control Vc, which IC50 was 0.31 mmol/L, Kinetic experiments showed that the inhibition of tyrosinase by compound 3 was reversible inhibition, and the type of inhibition was competitive inhibition.4. The four compounds isolated from the fermentation broth were tested the cytotoxic activity by using the MTT method with six kinds of tumor cells, such as human hepatocellular carcinoma cell line HepG-2, human breast cancer Hela, K562 cells, MCF-7 cells, SMMC-7721 cells and MDA-MB-231 cells. The results showed that compound 2 exhibited moderate inhibition agains Hela, with IC50 values of 52.59 ?mol/L, compound 3 exhibited weak inhibition agains SMMC-7721, with IC50 values of 154.36 ?mol/L, compound 4 exhibited weak inhibition agains HepG-2, with IC50 values of 121.6 ?mol/L, compound 5 exhibited moderate inhibition agains Hela and SMMC-7721, with IC50 values of 13.19 ?mol/L and 46.17 ?mol/L.