Studies On Bioactive Components In The Secondary Metabolites Of Streptomyces Qinlingensis SP. Nov

Screening bio-active compounds from microbial metabolites is an important way of pesticide research and development. The total number of known microbial metabolites was more than fifty thousands, in which at least 22,000 compounds exhibited biological activities, and the bioactive compounds were mainly isolated from fungi, bacteria and actinomycetes. Since streptomycin was isolated from the broth of Streptomyces griseus by Wakesman in 1945, actinomycetes has became the primary source of antibiotics, the total number of known bioactive compounds metabolized from this group is over 10000, 47% of all microbial metabolites.An actinomycetes strain isolated from a soil sample collected in Qinling mountain area, was identified by traditional sorting process and molecular biology technique as a new Streptomyces, named as Streptomyces qinlingensis sp. nov. The fermentation product of Streptomyces qinlingensis exhibited extraordinary inhibition activity against many species of fungi and bacteria, and moderate insecticidal activity against Plutella xylostella. As a potential candidate, the studies on the pesticidal components in the fermentation broth of this microorganism have been carryed out from 2003 to 2007. The main results are as follows:1. Purified by macroporous adsorption resin, silica gel column chromatograph and pre-HPLC orderly, three insecticidal compounds, 2-1, 2-2 and 2-3, were isolated from the fermentation products. The LC50 value of 2-1, 2-2 and 2-3 against 3rd Plutella xylostella by means of leaves immersion was 3.72, 0.40 and 3.60μg/mL respectively. Investigated by HR-ESI -MS and NMR techniques, the active compounds were identified as avermectin B2a,B1a and A1a. The results showed that Streptomyces qinlingensis was a new producer of insecticidal antibiotic avermectins.2. Purified by macroporous adsorption resin, silica gel column chromatograph and pre-HPLC orderly, four cyclic dipeptides compounds, 3-1, 3-2, 3-3 and 3-4, were isolated from the fermentation products finally. Investigated by HR-ESI-MS and NMR techniques, these compounds were identified as cyclo-(Gly-Pro), cyclo-(Ala -Pro), cyclo-(Val-Pro) and cyclo-(Gly-Leu), but these compounds had no insecticidal or fungicidal activity. A novel macrocycle-lactam compound was designed and synthesized, which showed the potential of becoming a novel fungicidal lead compound.3. In the process of isolating fungicidal components by silica gel column chromatograph, compound 4-1, a colorless crystal was obtained, which identified as diacetonamine by HRMS and NMR techniques. Diacetonamine exhibited inhibitory activities against many species of crop pathogenic fungus, such as Fusarium oxysporium, Fusarium. Azysporum and Alternaria Solani, it could also inhibite some Gram-positive bacteria, such as Bacillus cereus and Bacillus subtills. This was the first report of fungicidal activity of diacetonamine. A novel diacetonamine-Cu complex was synthesized, and its fungicidal activities were also investigated primarily, the complex had the potential to be used as an agro-fungicide.4. Purified by anion-exchange resin and ion-pair reversed-phase HPLC, ten fungicidal compounds, 5-1~5-10, were isolated from the metabolites of Streptomyces qinlingensis. Compound 5-1, 5-3 and 5-9 were identified as streptothricin D,F and N-Acetyl-streptothricin D respectively, others were novel compounds isolated from nature products.5. The anti-bacteria activity of compounds 5-1~5-10 against Bacillus subtilis, Staphyloco- ccus aureus, Escherichis coli,Bacillus cereus and Pseudomonas aeruginosa were tested by micro-dilution, the sensitivity was tested by paper disc diffusion. The results showed that Bacillus subtilis, Staphylococcus aureus and Escherichis coli were more sensitive to tested compounds, compound 5-1 and 5-3 were the primary fungicidal ingredients. The,minimum inhibitory concentration (MIC) of 5-1 against three species sensitive bacteria was 3.1,6.3 and 3.1μg·mL-1, MIC of 5-3 were 6.3,12.5 and 3.1μg·mL-1. The results of inhibiting spore germination against Curvulavia lunata, Botrytis cinerea, Fusarium coeruleum and Glomerella cingulata showed that 5-1 and 5-3 were more active than other compounds, the IC50 value of 5-1 were 11.8,0.6,0.7,0.9 and 0.9μg·mL-1,the IC50 value of 5-3 were 12.6,0.8,0.6,3.4 and 0.7μg·mL-1.6. The pathway of fragment forming and cleavage principle of compound 5-1~5-10 were investigated by ESI MS/MS, a LC/MS method used to identify streptothricins in the process of screening novel antibiotics was developed. Compared with the characteristic peak in ms spectrum of three pairs of isomer, 5-1 and 5-2,5-3 and 5-4,5-6 and 5-7, a simple and reliable method of identifying streptothricin isomers by ESI MS/MS was developed. The fragment pathway of streptothricin was summarized as three characters and they were validated by parent ion-product ion scan. In the process of analyzing the fermentation product of Streptomyces qinlingensis by LC/MS, a novel compound 7-1 was identified as N-methyl-streptothricin D-aicd by the characteristics mentioned above, the possible structure of another compound 7-2 , N-acetyl-streptothricin C-acid, was also presumed in the same way. Both of them were novel compounds.7. A silica gel Thin-Layer-Chromatograph method, which used for distinguishing streptothricin quickly, was developed, in which the optimal solvent system was methanol mixed with ammonia at the ratio of two to one, and the sample was checked by ninhydrin reaction. A direct ion-pair reversed-phase HPLC method for quantitative analysis of streptothricin in fermentation broth was studied. Chromatographic seperation on a ODS2 column was achieved using a mobile phase contained acetonitrile, water, 0.03mol·L-1 1-Heptanesulfonic acid sodium and 0.1mol·L-1 disodium hydrogen phosphate, the pH was adjusted by phosphoric acid to 3.5. The developed method was validated in terms of linearity, accuracy and precision. The linear regression data showed a good linear relationship for 5-1 and 5-3 over a concentration range of 15～500μg·mL-1, the recovery and RSD for 5-1 were 98.1～100.8% and 0.85%, for 5-3 were 98.9～99.6%and 0.77%.