The Secondary Metabolites And Antifouling Activity Of Three Gorgonian-derived Fungi From The South China Sea

The competitions between marine organisms which live in coral reef ecosystemsare very common and cruel. Many invertebrates, such as gorgonians and soft corals,are lack of effective means of physical capability against their potential predators, andrely on their chemical defensive system to survive in the fierce environments. Theecology studies found that the microorganisms isolated from the marine invertebratesmay be involved in the biosynthesis of chemical defensive compounds in the host,such as sea anemone toxins and conotoxins. The special marine environment withhigh-salt, high-pressure, low-temperature, oligotrophic and low light conditions madea unique metabolic mechanism for the epiphytic microbial beings able to producemany structurally unique and biologically active metabolites. Many of which aresecondary metabolites with chemical ecology effects, including anti-predators,antifouling, ichthyootoxin, and also with pharmacologically activities, such asanti-bacterial, anti-tumor, and anti-viral activities. Therefore, the studies on secondarymetabolites from the epiphytic microorganisms derived from marine invertebrates areimportant for the screening and discovery for chemical defensive compounds and thelead compounds for marine drugs.We isolated the epiphytic fungi from corals and other invertebrates. Three fungaltrains of Aspergillus sp. XS-2009-0B15, Aspergillus sp. XS-2009-0066, and Eurotiumsp. XS-2009-00E6were chosen for chemical investigation under the guidance ofchemical defensive effects, especially antifouling activity against the larval settlementof barnacle Balanus amphitrite. The secondary metabolites were isolated from theactive fungal fermentation by column chromatography on si-gel, Sephadex LH-20,and semi-HPLC, and identified by spectroscopy methods of NMR, MS, UV, CD, IR,and X-ray, combined with a variety of chemical methods. From these fungi,83 compounds were obtained, including31new compounds. More than10structuraltypes were found, including6peptides,4quinolinone alkaloids,2butyrolactones,14benzaldehyde derivatives,24indole alkaloids,4nucleoside derivatives,16anthraquinone derivatives,2cytochalasins,5steriods, and6diphenyl ethers.Compounds44and47were chemically modified by acylation, alkylation, andbromination reactions to give8diphenyl ether derivatives (44a–41g,47a).Precursors feeding experiments with amino acids were carried out to the strain ofAspergillus sp. XS-20090B15. The results showed that the fungus could produce anew lumazine peptide1with the precursor L-methionine, and the yield of the mainmetabolite5was also increased, suggestting that the L-methionine was involved in thebiosynthesis of this type of lumazine peptides. The absolute configurations of theamino acid residues in the peptides were determined by Marfey’s method and otherchemical methods. The sequence of the amino acids of the cyclic peptide6wascomfirmed by tandem mass spectrometry. The absolute configurations of22and32were confirmed by single-crystal X-ray diffraction analysis using Cu Kα radiation forthe first time. The absolute configurations of compounds23and24were revised as3Sby the CD spectra combined with the X-ray structure of22. Benzaldehyde derivativeswith the δ-lactone ring moiety were isolated for the first time and were proved to beracemates, which were separated by chiral-HPLC.With an emphasis on antifouling activity against the larval settlement of barnacleB. amphitrite, the antifouling activity of the compounds were screened resulting infinding of several bioactive compounds. For example, quinolinone alkaloid10hasantifouling activity against the larvae settlement of barnacle with an EC50value of lessthan0.02μg/mL, but low toxic to the barnacle larvae (LC50=6.25μg/mL);butyrolactone12is also a natural product with high efficiency (EC50=2.10μg/mL)and low toxicity (LC50=50μg/mL) to the barnacle larvae. Other types of compoundssuch as biphenyl ethers (42–47), cytochalasins (13,14), and xanthone derivative (56)also showed potent antifouling activity. In addition, other chemical ecology effects,including toxicity against zebrafish, growth-inhibition of microalgae, and lethalitytoward brine shrimp were also evaluated for these active compounds. It was found that the antifouling active compounds generally displayed some other chemicalecology activities. For example, diphenyl ether47showed zebrafish Danio rerio eggcondensation at the concentrate of4.4μg/mL (48h), zebrafish lethal at3.0μg/mL (72h), and aslo showed potent growth-inhibition activity towards Karenia mikimotoi andHeterosigma akashiwo with EC50of1.16and4.36μg/mL, respectively. These activecompounds have a comprehensive chemical ecology effect.In summary, in the present study, the secondary metabolites from thegorgonian-derived fungi have been isolated under the guidance of chemical defensiveeffects. Antifouling active compounds found by our research provided bioresourceand compounds source for the research and development of efficient, non-toxic, andnatural marine antifouling agents.