Study On Active Metabolites Of Symbiotic Streptomyces Tanashiensis BYF-112 From Odontotermes Formosanus

Insect symbiotic microbes are a kind of typical microorganisms that have co-evolved with insects for a long time.In recent years,numerous studies have shown that insect symbiotic microorganisms are an important source of novel compounds of new antibacterial,herbicidal,and anticancer active precursors.In this paper,the symbiotic actinomyce BYF-112 from Odontotermes formosanus was used as the research object,and the morphological observation and molecular biology methods were used to identify it.The quality of crude extract and nuclear magnetic resonance(NMR)of five different cultures were preliminarily analyzed to determine the most suitable cultural medium.A total of 18 compounds were isolated and purified by thin layer chromatography,silica gel column chromatography,sephadex LH-20 gel column chromatography and recrystallization.The structures of 12 compounds were identified by 1D-NMR(1H-NMR、13C-NMR、DEPT135),2D-NMR(1H-1H COSY、HMQC、HMBC)high-resolution electrospray mass spectrometry(HR-ESI-MS)and related databases search as well as literature comparison,of which 4 were new alkaloid metabolites;The cytotoxic,antibacterial and herbicidal activities of some compounds were further studied.The results are summarized as follows:The symbiotic actinomycete BYF-112 of O.formosanus was identified by morphological observation and molecular biology methods.The 16S rDNA sequence result showed that the similarity between BYF-112 and S.tanashiensis was 99.9%.Combining morphological observation,the strain was initially identified as S.tanashiensis.The quality and NMR spectrum of five different cultures of S.tanashiensis crude was analysised.After seven days of fermentation(200 ml),the heaviest quality of crude culture was that of Gauze’s No.1(0.045 g),and the lightest was that of ISP1(0.002 g).The thin-layer chromatography and nuclear magnetic hydrogen spectrum results of the five crude extracts showed that the secondary metabolites of Gauze’s No.1 medium were the most abundant.Therefore,Gauze’s No.1 was recognized the most suitable medium for fermentation of S.tanashiensis BYF-112.A total of 18 compounds were isolated from S.tanashiensis BYF-112,and 12 compounds(1-12)were identified,Including 4 new alkaloid compounds vegfrecine A(1),vegfrecine B(2),exfoliazone A(3),venezueline H(7)and 8 known compounds texazone(4),viridobruunine A(5),exfoliazone(6),N-(2-Hydroxy-5-methylphenyl)acetamide(8),3-acetylamino-4-hydroxybenzaldehyde(9),3-acetamido-4-hydroxybenzoic acid(10),viridomvcin A(11),X-14881E(12).The viridobrunnine(5),exfoliazone(6),viridomvcin A(11)and X-14881E(12)showed significant cytotoxicity against human malignant melanoma cell line(A375),with IC50 values of 7.32,5.85,5.46 and 8.20 μM,respectively,which were comparable to those of referenced adriamycin,with IC50 value of 1.01 μM.The viridobrunnine(5),exfoliazone(6),viridomvcin A(11)and X-1488 1E(12)showed moderate cytotoxicity against human ovarian cancer cell lines(SKOV-3),with IC50 values of 6.09,8.76,11.09 and 19.28 μM,respectively,which were comparable to those of referenced adriamycin,with IC50 value of 0.72 μM.The viridobrunnine(5),exfoliazone(6),viridomvcin A(11)and X-14881E(12)showed weaker cytotoxicity against human ovarian cancer cell lines(SKOV-3),with IC50 values of 4.61,7.58,13.57,11.36 μM,respectively,which were comparable to those of referenced adriamycin,with IC50 value of 0.48 μM.These results suggest that viridobrunnine(5)and exfoliazone(6)viridomvcinA(11)and X-14881E(12)have potential to be used as anti-cancer lead compounds.The antibacterial activity showed that compound 11 had moderate inhibitory activity against growth of Mlicrococcus tetragenus,with the diameter of the inhibition zone of 13.3 mm at the concentration of 30 μg/filter paper.The phytotoxic activity results showed that compounds 11 and 6 had moderate activity against the growth of Echinochloa crusgalli at the concentration of 100 μg/mL,with the inhibition rate of 55.3%and 41.7%,respectively.The above studies indicated that the symbiotic actinomycete of O.formosanus can produce new secondary metabolites with wide biological activity,which was valueable for further research.