Study On Two Actinobacteria Lived In Special Environment And Their Active Secondary Metabolites Based On OSMAC Strategy

Actinomycetes metabolites are the important sources of active lead compounds because of their rich variety and structural diversity.In the process of long-term adaptation to the special environment,actinomycetes could evolve unique metabolic pathway and metabolize more active substances.Up to now,about 70%of the antibiotics have been derived from actinomycetes.For all this,only a small part of the genes controlling microbial metabolism have been expressed,most of them are silent.Studies have found that OSMAC strategy can be used as one of the effective methods to solve this problem.In this study,actinomycetes isolated from Hainan Island were cultured under different conditions to activate their potential to metabolize more active secondary products.In the meantime,antibacterial,antioxidant and cytotoxic activities of the metabolites were assayed.17 fungus and 47 actinomycetes were isolated from 8 samples:Termite mud,three kinds of Lichens,Dendrobium nobile,Cattleya hybrida,Sansevieria trifasciata var.laurentii and Synsepalum dulcificum,which were from Hainan Island.Through ¹H NMR,TLC,HPLC and bacteriostatic analysis,J-1 and J-5were selected as the dominant strains and identified as Streptomyces by 16S r DNA sequencing.From the culture of the dominant strains and purification by silica gel and Sephadex LH-20 dextran gel,10compounds were obtained.Among them,three compounds were characterized as Actinomycin D（L-1）,5-hydroxy-3,4,7-triphenyl-2,6-benzofurandione（L-2）,kojic acid（L-3）by NMR and ESI-MS.J-1 was selected as the target strain.Based on OSMAC strategy,we experienced co-culture,ratio screening of solid-liquid mixed medium and culture medium screening.The mixed medium containing 20g rice and 100 m L Gaucher No.1 medium was selected for mass fermentation by means of yield,TLC,HPLC and bacteriostatic analysis.Then,21 compounds were obtained,and 15 of them were characterized,which were Actinomycin D（L-1）,Actinomycin V（L-4）,8-O-Methylsolaniol（L-5）,8-O-methyljavanicin（L-6）,8-O-methylbostrycoidin（L-7）,Epicatechin（L-8）,N-（tert-Butoxycarbonyl）-L-prolinol（L-9）,N-[（Phenylmethoxy）carbonyl]-D-valine（L-10）,Uracil（L-11）,Kaempferol（L-12）,Boc-phenylalaninol（L-13）,citridone B/B’（L-14）,Adenosine（L-15）,4-hydroxyphenylacetic acid（L-16）and N-methylhydroxylamine（L-17）,respectively.Among them,the yield of L-1 was increased by 30 times,which indicated that the use of solid-liquid mixed medium could increase the yield and abundance of metabolites of J-1.All compounds were assayed for their antibacterial,antioxidant and cytotoxic activities,and the results showed as follows:1.SA and E.coli were used as test bacteria to investigate the antibacterial activities:L-1 and L-4 were highly sensitive to SA and E.coli at low concentration of 6.25μM,and their inhibition was about 20 times that of chloramphenicol.L-1 inhibited E.coli better than SA,with MIC values of 0.005μM and 0.625μM,respectively.The inhibitory effect of L-4 on SA was better than that of E.coli with MIC values of 0.0012μM and 0.0195μM,respectively.L-2 was moderately sensitive to SA and E.coli at 2 m M with MIC value of 500μM.The inhibitory effect of L-12 on E.coli was better than that of SA with MIC values of 68.125μM and 272.5μM,respectively.2.The total antioxidant capacity of the compounds was evaluated by the production of Prussian blue:When the concentration was 0～4 m M of L-1,L-3,L-8,L-12 and 0～2 m M of L-4,the antioxidant capacity was positively correlated with the concentration,and the compounds could neutralize excessive free radicals while playing antibacterial effects.However,the antioxidant capacity of L-4 tended to be stable when the concentration was greater than 2 m M,due to the poor solubility of L-4 in water.3.HL-7702,HCT-116,He La,4T1 and MCF-7 were used as test cells to investigate the cytotoxicity of the compounds:The IC₅₀ values of L-1 and L-4 were247.16±27.47μM and 287.45±34.11μM for HL-7702 cells,3.22±0.15μM and 2.88±0.21μM for HCT-116 cells,respectively.It was superior to cisplatin(IC₅₀ value 11.07±1.23μM),which indicated that the toxicity of L-4 to normal liver cells was less than L-1,and the inhibition of L-4 to colon cancer cells was greater than L-1.The inhibitory effect of L-1 and L-4 on MCF-7 cells was positively correlated with concentration.When the concentration of L-1 and L-4 was 5～160μM,the inhibitory effect on 4T1 cells increased with the increase of the concentration,but when the concentration was 320～1000μM,the inhibitory effect on 4T1 cells decreased.Compound L-8 can promote the proliferation of HL-7702.