Evaluation of the Biological Activities of Marine Bacteria Collected from Jeju Island, Korea, and Isolation of Active Compounds from their Secondary Metabolites

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  • ABSTRACT

    To explore marine microorganisms with medical potential, we isolated and identified marine bacteria from floats, marine algae, animals, and sponges collected from Jeju Island, Korea. We isolated and identified 21 different strains from the marine samples by 16S rRNA analysis, cultured them in marine broth, and extracted them with ethyl acetate (EtOAc) to collect secondary metabolite fractions. Next, we evaluated their anti-oxidative and anti-inflammatory effects. Among the 21 strains, the secondary metabolite fraction of Bacillus badius had both strong antioxidant and anti-inflammatory activity, and thus was selected for further experiments. An antioxidant compound detected from the secondary metabolite fraction of B. badius was purified by preparative centrifugal partition chromatography (n-hexane:EtOAc:methanol:water, 4:6:4:6, v/v), and identified as diolmycin A2. Additionally, diolmycin A2 strongly inhibited nitric oxide production. Thus, we successfully identified a significant bioactive compound from B. badius among the bacterial strains collected from Jeju Island.


  • KEYWORD

    Marine microorganism , Secondary metabolite , Jeju Island , Bacillus badius , Diolmycin A2 , Biological activities

  • 1. Aruoma OI 1998 Free radicals, oxidative stress, and antioxidants in human health and disease [J Am Oil Chem Soc] Vol.75 P.199-212 google doi
  • 2. Bae MH, Kim HG, Shin YH, Kim BY, Lee SK, Oh KB, Shin JH, Oh DC 2013 Separacenes A-D, novel polyene polyols from the marine actinomycete, Streptomyces sp [Mar Drugs] Vol.11 P.2882-2893 google doi
  • 3. Bringmann G, Lang G, Steffens S, Gunther E, Schaumann K 2003 Evariquinone, isoemericellin, and stromemycin from a sponge derived strain of the fungus Emericella variecolor. [Phytochemistry] Vol.63 P.437-443 google doi
  • 4. Bringmann G, Lang G, Gulder TAM, Tsuruta H, Muhlbacher J, Maksimenka K, Steffens S, Schaumann K, Stohr R, Wiese J, Imhoff JF, Perovic-Ottstadt S, Boreiko O, Muller WEG 2005 The first sorbicillinoid alkaloids, the antileukemic sorbicillactones A and B, from a sponge-derived Penicillium chrysogenum strain [Tetrahedron] Vol.61 P.7252-7265 google doi
  • 5. Collins MD, Jones D 1981 Distribution of isoprenoid quinone structural types in bacteria and their taxonomic implication [Microbiol Rev] Vol.45 P.316-354 google
  • 6. Conner EM, Grisham MB 1996 Inflammation, free radicals and antioxidants [Nutrition] Vol.12 P.274-277 google doi
  • 7. Faulkner DJ 2000 Marine pharmacology [Antonie Van Leeuwenhoek] Vol.77 P.135-145 google doi
  • 8. Hayashi M, Kim YP, Takamatsu S, Enomoto A, Shinose M, Takahashi Y, Tanaka H, Komiyama K, Omura S 1996 Madindoline, a novel inhibitor of IL-6 activity from Streptomyces sp. K93-0711. I. Taxonomy, fermentation, isolation and biological activites [J Antibiot] Vol.49 P.1091-1095 google doi
  • 9. Hirata Y, Uemura D 1986 Halichondrins-antitumor polyether macrolides from a marine sponge [Pure Appl Chem] Vol.58 P.701-710 google
  • 10. Holmstrom C, Kjelleberg S 1999 Marine Pseudoalteromonas species are associated with higher organisms and produce biologically active extracellular agents [FEMS Microbiol Ecol] Vol.30 P.285-293 google doi
  • 11. Kim Sk, Ravichandran YD, Khan SB, Kim YT 2008 Prospective of the Cosmeceuticals Derived from Marine Organisms [Biotechnology and Bioprocess Engineering:BBE] Vol.13 P.511-523 google doi
  • 12. Kochanowska-Karamyan AJ, Hamann MT 2010 Marine indole alkaloids: potential new drug leads for the control of depression and anxiety [Chem Rev] Vol.110 P.4489-4497 google doi
  • 13. Kodali VP, Perali RS, Sen R 2011 Purification and partial elucidation of the structure of an antioxidant carbohydrate biopolymer from the probiotic bacterium Bacillus coagulans RK-02 [J Nat Prod] Vol.74 P.1692-1697 google doi
  • 14. Lee JH, KO JY, Samarakoon K, Oh JY, Heo SJ, Kim CY, Nah JW, Jang MK, Lee JS, Jeon YJ 2013 Preparative isolation of sargachromanol E from Sargassum siliquastrum by centrifugal partition chromatography and its anti-inflammatory activity [Food Chem Toxicol] Vol.62 P.54-60 google doi
  • 15. Lee JH, Ko JY, Oh JY, Kim CY, Lee HJ, Lim J, Jeon YJ 2014 Preparative isolation and purification of phlorotannins from Ecklonia cava using centrifugal partition chromatography by one-step [Food Chem] Vol.158 P.433-437 google doi
  • 16. Liu R, Cui CB, Duan L, Gu QQ, Zhu WM 2005 Potent in vitro anticancer activity of metacycloprodigiosin and undecylprodigiosin from a sponge-derived actinomycete Saccharopolyspora sp. nov [Arch Pharm Res] Vol.28 P.1341-1344 google doi
  • 17. Mearns-Spragg AM, Bregu M, Boyd KG, Burgess JG 1998 Cross-species induction and enhancement of antimicrobial activity produced by epibiotic bacteria from marine algae and invertebrates, after exposure to terrestrial bacteria [Lett Appl Microbiol] Vol.27 P.142-146 google doi
  • 18. Muller HE 1985 Detection of hydrogen peroxide produced by microorganism on ABTS-peroxidase medium [Zentralbl Bakteriol Mikrobiol Hyg A] Vol.259 P.151-154 google
  • 19. Nakae K, Yoshimoto Y, Sawa T, Homma Y, Hamada M, Takeuchi T, Imoto M 2000 Migrastatin, a new inhibitor of tumor cell migration from Streptomyces sp. MK929-43F1. Taxonomy, fermentation, isolation and biological activities [J Antibiot] Vol.53 P.1130-1136 google doi
  • 20. Pimentel-Elardo SM, Kozytska S, Bugni TS, Ireland CM, Moll H, Hentschel U 2010 Anti- parasitic compounds from Streptomyces sp. strains isolated from Mediterranean sponges [Mar Drugs] Vol.8 P.373-380 google doi
  • 21. Sakai K, Nisijima H, Ikenaga Y, Wakayama M, Moriguchi M 2000 Purification and characterization of nitrite-oxidizing enzyme from heterotrophic Bacillus badius 1-73, with special concern to catalase [Biosci Biotechnol Biochem] Vol.64 P.2727-2730 google doi
  • 22. Schmitt S, Tsai P, Bell J, Fromont J, Ilan M, Lindquist N, Perez T, Rodrigo A, Schupp PJ, Vacelet J, Webster N, Hentschel U, Taylor MW 2012 Assessing the complex sponge microbiota: core, variable and species-specific bacterial communities in marine sponges [ISME J] Vol.6 P.564-576 google doi
  • 23. Sohn MJ, Zheng CJ, Kim WG 2008 Macrolactin S, a new antibacterial agent with Fab G-inhibitory activity from Bacillus sp. AT28 [J Antibiot] Vol.61 P.687-691 google doi
  • 24. Soria-Mercado IES, Davo AP, Jensen PR, Fenical W 2005 Antibiotic terpenoid chloro-dihydroquinones from a new marine actinomycete [J Nat Prod] Vol.68 P.904-910 google doi
  • 25. Suarez-Jimenez GM, Burgos-Hernandez A, Ezquerra-Brauer JM 2012 Bioactive peptides and depsipeptides with anticancer potential: sources from marine animals [Mar Drugs] Vol.10 P.963-986 google doi
  • 26. Sunazuka T, Tabata N, Nagamitsu T, Tomoda H, Omura S 1993 Synthesis of diolmycin analogs and their anticoccidial activities [J Antibiot] Vol.47 P.1178-1180 google
  • 27. Tabata N, Sunazuka T, Tomoda, H, Nagamitsu T, Iwai Y, Omura S 1993 Diolmycins, new anticoccidial agents produced by Streptomyces sp, II. Structure elucidation of diolmycins A1, A2, B1 and B2, and synthesis of diolmycin A1 [J Antibiot] Vol.46 P.762-769 google doi
  • 28. Zheng L, Chen H, Han X, Lin W, Yan X 2005 Antimicrobial screening and active compound isolation from marine bacterium NJ6-3-1 associated with the sponge Hymeniacidon perleve [World J Microbiol, Biotechnol] Vol.21 P.201-206 google doi
  • 29. ZoBell CE, Johnson FH 1949 The influence of hydrostatic pressure on the growth and viability of terrestrial and marine bacteria [J Bacteriol] Vol.57 P.179-189 google
  • [Table 1.] Scientific names of the 21 marine bacteria species isolated from marine samples
    Scientific names of the 21 marine bacteria species isolated from marine samples
  • [Fig. 1.] Hydrogen peroxide scavenging (A) anti-inflammatory activities and cytotoxicity (B) of the secondary metabolites fractions from marine bacteria. Hydrogen peroxide effect was measured by the above described protocol. To evaluation of anti-inflammation, Raw 264.7 cells were pretreated with the secondary metabolites fractions for 2 h and then the produced nitric oxide (NO) was measured after incubation with lipopolysaccharide (LPS) for 24 h. Names of all bacteria species were expressed using the number indicated in Table 1. Each value indicates the mean ± standard error from three independent experiments.
    Hydrogen peroxide scavenging (A) anti-inflammatory activities and cytotoxicity (B) of the secondary metabolites fractions from marine bacteria. Hydrogen peroxide effect was measured by the above described protocol. To evaluation of anti-inflammation, Raw 264.7 cells were pretreated with the secondary metabolites fractions for 2 h and then the produced nitric oxide (NO) was measured after incubation with lipopolysaccharide (LPS) for 24 h. Names of all bacteria species were expressed using the number indicated in Table 1. Each value indicates the mean ± standard error from three independent experiments.
  • [Fig. 2.] Anti-inflammatory activities and cytotoxicity of Bacillus badius (075-1) secondary metabolites fraction against lipopolysaccharide (LPS)- induced RAW 264.7 cells. RAW 264.7 cells were pretreated with the secondary metabolites fraction for 2 h, and then incubated with LPS for 24 h. Each value indicates the mean ± standard error (SE) from three independent experiments. Experiments were performed in triplicate and the data are expressed as mean ± SE. *P < 0.1, **P <0.05.
    Anti-inflammatory activities and cytotoxicity of Bacillus badius (075-1) secondary metabolites fraction against lipopolysaccharide (LPS)- induced RAW 264.7 cells. RAW 264.7 cells were pretreated with the secondary metabolites fraction for 2 h, and then incubated with LPS for 24 h. Each value indicates the mean ± standard error (SE) from three independent experiments. Experiments were performed in triplicate and the data are expressed as mean ± SE. *P < 0.1, **P <0.05.
  • [Fig. 3.] ABTS+ on-line HPLC chromatogram of secondary metabolites fraction from Bacillus badius. The mobile phase comprised acetonitrile? distilled water (ACN-DW) in gradient mode as follows: ACN-DW (0-50 min: 20:80→60:40, v/v; 50-60 min: 60:40, v/v; and 60-70 min: 100:0, v/v). The flow rate was 0.2 mL/min, and the UV absorbance was detected at 280 nm. Peak 1 represents compound 1.
    ABTS+ on-line HPLC chromatogram of secondary metabolites fraction from Bacillus badius. The mobile phase comprised acetonitrile? distilled water (ACN-DW) in gradient mode as follows: ACN-DW (0-50 min: 20:80→60:40, v/v; 50-60 min: 60:40, v/v; and 60-70 min: 100:0, v/v). The flow rate was 0.2 mL/min, and the UV absorbance was detected at 280 nm. Peak 1 represents compound 1.
  • [Fig. 4.] Chromatograms of the secondary metabolites fraction. The CPC chromatogram of secondary metabolites fraction (A), HPLC chromatograms of the secondary metabolites fraction (B), and the compound 1 (C) from Bacillus badius. For operation of CPC, stationary phase: upper organic phase, mobile phase: lower aqueous phase, flow rate: 2 mL/min, rotation speed: 1000 rpm, and sample: 500 mg dissolved in 6 mL mixture of lower phase and upper phase (1:1, v/v) of the solvent system. For operation of HPLC, column: SUNFIRE? C18 5μm ODS column (250 × 4.6 mm i.d.; Waters, Milford, MA, USA); mobile phase: acetronitrile (20:80 v/v to 60:40 v/v at 0-50 min, 60:40 v/v to 100:0 v/v at 50-60 min, 100:0 v/v to 100:0 v/v at 60-70 min); flow rate: 1 mL/min, monitored at 280 nm. Peak 1 represents compound 1.
    Chromatograms of the secondary metabolites fraction. The CPC chromatogram of secondary metabolites fraction (A), HPLC chromatograms of the secondary metabolites fraction (B), and the compound 1 (C) from Bacillus badius. For operation of CPC, stationary phase: upper organic phase, mobile phase: lower aqueous phase, flow rate: 2 mL/min, rotation speed: 1000 rpm, and sample: 500 mg dissolved in 6 mL mixture of lower phase and upper phase (1:1, v/v) of the solvent system. For operation of HPLC, column: SUNFIRE? C18 5μm ODS column (250 × 4.6 mm i.d.; Waters, Milford, MA, USA); mobile phase: acetronitrile (20:80 v/v to 60:40 v/v at 0-50 min, 60:40 v/v to 100:0 v/v at 50-60 min, 100:0 v/v to 100:0 v/v at 60-70 min); flow rate: 1 mL/min, monitored at 280 nm. Peak 1 represents compound 1.
  • [Fig. 5.] Mass spectrometry (MS) and MS/MS analysis data (A) and anti-oxidative effect (B) of diolmycin A2 from the secondary metabolite fraction of B. badius. The production of nitric oxide (NO) was assayed in the culture medium of cells stimulated with lipopolysaccharide (LPS, 1 μg/mL) for 24 h in the presence of diolmycin A2. Cytotoxicity was determined using the lactate dehydrogenase (LDH) method. Experiments were performed in triplicate and the data are expressed as mean ± standard errors. *P < 0.1, **P < 0.05, ***P < 0.01.
    Mass spectrometry (MS) and MS/MS analysis data (A) and anti-oxidative effect (B) of diolmycin A2 from the secondary metabolite fraction of B. badius. The production of nitric oxide (NO) was assayed in the culture medium of cells stimulated with lipopolysaccharide (LPS, 1 μg/mL) for 24 h in the presence of diolmycin A2. Cytotoxicity was determined using the lactate dehydrogenase (LDH) method. Experiments were performed in triplicate and the data are expressed as mean ± standard errors. *P < 0.1, **P < 0.05, ***P < 0.01.