| Screening of lead compounds from natural products has been an important way of developing new drugs. The compounds in fungi are important resources of clinical small molecule drugs. Many influential drugs, such as penicillin, cephalosporin antibiotics and statins, are developed from fungal metabolites. In recent years, in order to further obtain the diversity of microbial drugs to find metabolites with novel structures and strong activity, scientists try to expand resources of drug-induced fungi, especially those from the extreme environment.Purpose1. To survey of the fungal species and obtain the active strains from the Arctic Pole（here we call it polar fungi）, 2. To study the secondary metabolites of the active strains, focusing on finding novel structures with strong biological activities.To reach the purpose, we need to do the scale fermentation after clearing the active polar fungal strains, to accumulate the quantum of metabolites for the further system isolation. After getting the purified single compounds, we will carry out bioactive assay of these compounds on malignant tumor and anti-fungi, and try to find the target proteins the compounds work on, to clear the main bioactive constituents in the fungal strains. This work is important for systematically elucidate the bioactive constituents and chemical diversity of the Arctic fungi and found the novel chemical structures with antifungal and antitumor activities.MethodsThe detail of this research includes three steps. First, we did a small amount of fermentation of polar fungi to obtain crude extracts; second, we screened biological activity of the extracts to obtain the active strains. The screening methods included antibacterial activity experiment by paper disk diffusion method, anti-fungal activity experiment using broth micro-dilution method, and MTT cytotoxicity assay in vitro. After finding the active strain, we fermented the strain in large scale, isolated the fermentation metabolites, and determined the structures of purified compounds through analysis of NMR spectra, mass spectra and literature comparison. Last, we found the target protein of the compounds in vivo by biological molecular interaction analyzer, and designed some assay experiment by the target protein properties, such as immunosuppressive activity test to prove the compounds function.Results:Firstly, 15 among 78 fungal strains（accounting for 19.23%） showed antibacterial activity after bioassay in vitro. Among them, D-1, Z1-1, W-1-18, S-1-10, S-1-16, S-1-19 and S-1-17-Z1 are better, which can inhibit tested bacteria grown significantly. 40 among 78 fungal strains（accounted for 51.28%） can inhibited the growth of Pyricularia grisea, among them, 12 strains（accounted for 15.38%） with the maximum valid dilution in 128 times. One strains, named B-13, showed positive result in anti-human pathogenic fungi. 24 strains（accounted for 30.77 %） exhibited cytotoxic activity. Among them, S-3-88 and S-3-62 have the best activity. At 50 μg/m L concentration, the extract of S-3-88 strain can inhibit the growth of MCF-7, He La, H446, A549, SW1990 and SGC7901 with the rates at 78.79 %, 76.20 %, 50.38 %, 45.90 %, 53.18 % and 74.66 % respectively. At the same concentration, the extract of S-3-62 strain have the inhibit rates at 70.90 %, 69.8 %, 43.58 %, 40.64 %, 46.30 %, and 35.96 %, respectively to the same 6 kinds of tumor cell lines. 4 strains have two or more than two kinds of biological activity. They are D-1, B-13, S-1-10 and S-1-16.Secondly, 12 single purified compounds were obtained from the pole strain Nectria sp. B-13. They are ilicicolin C（compound 1）, LL-Z 1272ε（compound 2）, deacetylchloronectrin（compound 3）, dimethoxyilicicolin C（compound 4）, ilicicolin D（compound 5）, ilicicolin F（compound 6）, ilicicolin E（compound 7）, collectochlorin B（compound 8）, ilicicolin A（compound 9）, ilicicolin H（compound 10）, 3,5-dihydroxy toluene（compound 11） and 3,5-dihydroxy benzoic alcohol（compound 12）. Among them, compound 4 is a new compound.Thirdly, 7 single purified compounds were obtained from the pole strain Eutypella sp. D-1. They are ergosterol（compound 13）, α- linolenic acid（compound 14）, dehydrosylvic acid（compound 15）, Libertellenone B（compound 16）, eutypenoid A（compound 17）, eutypenoid D（compound 18） and eutypenoid E（compound 19）. Among them, compounds 18 and 19 are new compounds, belonged to pimarane diterpenes. Eutypenoid E has a special structure with a carbonyl substituted in C-11 position which formed an isopimarane diterpene.Fourthly, cytotoxic activity assay showed that compounds 6, 7 and 9 can inhibit lymphocyte proliferation of Jurkat leukemia cells. The inhibition ratio were 59.27%, 73.97%and 71.43% respectively at 10 μM concentration.Last, the interaction of the molecular biological analyzer results showed that compounds 3, 7 and 8 can combine with FKBP12 protein（FK506 binding protein 12）, the binding constants Ka are 7.85, 220 and 7.47 × 104 respectively（Ka of positive control is 8.03 × 104）. Compound 8 have the similar Ka value with positive control rapamycin. Dissociation constants Kd are 1.01 × 10-5,0.026 and 4.17 × 10-6 respectively(Kd of positive control is 1.4×10-3). The average dissociation constant KD are 1.28×10-6,1.18×10-4 and 5.58×10-11 respectively(KD of positive control is 1.75×10-8). Compound 8 has higher protein affinity with FKBP12 protein than that of the positive control rapamycin according to the KD value and indicates immunosuppression activity of compound 8 as same as rapamycin. However, the inhibition immunological activity study showed that the immunosuppressive effect of all tested compounds are faint. The results suggest that the combination of compounds with FKBP12 protein does not affect B and T cell proliferation. These combination may be related to the physiological function of FKBP12 besides immunosuppressive effect. Compound 7 inhibited the immune activity slightly, which may be related to cell toxicity.ConclusionsA number of active strains were obtained by screening of the polar fungal strains. Though the isolation and identification of two active strains metabolites, 19 natural compounds were obtained from the polar fungi. Among them, three were new compounds. The biological active assay results showed that compound 8 had a higher binding affinity with FKBP12（FK506 binding protein 12） protein than that of the positive control. The above research provides a group of natural products with novel structure and biological activity, and also found compound 8 binding protein in vivo. This thesis provide an experimental basis for the further development of drugs from polar fungi. |
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