Secondary Metabolites From A Soft Coral And A Gorgonian-Derived Fungus And Their Zebrafish Toxicity

Corals and their symbiotic microbes are an important source of marine natural products. Secondary metabolites with various structures have been found from corals including terpenoids, steroids, prostanoids, and alkaloids, and a large number of these compounds showed significant biological activities, with a broad medicinal development prospects. Marine microorganisms have been a hot spot for development of marine natural products and shown to produce prolific novel bioactive compounds. In particular, symbiotic microbes probably take part in the biosynthesis of some bioactive marine secondary metabolites isolated from the host organisms and have aroused extensive concern. It is noteworthy that large number of symbiotic microbes also exist in corals but rare studies have been focused on these microbes. In this paper, a soft coral and a gorgonian-derived fungus have been selected for chemical investigation. Compounds isolated from corals and their symbiotic fungi were evaluated on zebrafish (Danio rerio) toxicity microplate model, and based on the data from the assay, their structure-activity relationships were analyzed.Secondary metabolites of soft coral Sarcophyton sp.(XW-4) were isolated by repeated column chromatography on silica gel, Sephadex LH-20and semi-preparative HPLC, and identified by modern spectroscopy methods of NMR, MS, UV and IR. Seven cembranoids were identified as compound1,(-)-marasol (2),(+)-11,12-epoxy-11,12-dihydrocembrene-C (3),(+)-11,12-epoxysarcophytol A (4), Sarcophytol-B (5),(1Z,3E,7E,11Z)-19-hydroxy-cembra-1,3,7,11-tetraen-10,20-lactone (6),(2E,7E)-4,11-dihydroxy-1,12-oxidocembra-2,7-dien (7). Compound1was a new cembrane diterpenoid. Compounds2,4-7have been mainly obtained from the soft corals of the family Alcyoniidae, while compounds2and6have only been recorded before from the genus Sarcophyton. These cembranoids should be characteristic compounds to the chemotaxonomy for the family Alcyoniidae. Terpenoids have been considered to be the largest components of the genus Sarcophyton with the most abundant type of structures, most of them showed strong biological activity.From the gorgonian-derived fungus Peniophora sp.(ZJ-XS-2009001),12compounds including11steroids and1diphenyl ether were identified as4-hydroxy-17R-methylincisterol (8), ergosterol (9), episterol (10), ergosta-7,22-diene-3β-ol (11),(3β,5a,6β,22E)-6-methoxyergosta-7,22-diene-3,5-diol (12), cerevisterol (13),(3β,5a,6a,7a,22E)-5,6-epoxyergosta-8,22-dien-3,7-diol (14),(3β,5a,6a,7a,22E)-5,6-epoxyergosta-8(14),22-dien-3,7-diol (15), ergosta-4,6,8,22-tetraen-3-one (16), ergosterol peroxide (17) and9,11-dehydroergosterol peroxide (18), and Penicillide (19). Compounds8,12-19were the first report in the genus Peniophora, and among them8,12,16were isolated from coral-derived fungus for the first time. It was found that the main compositions of gorgonian genus Echinogorgia were steroids which showed a variety of biological activities such as antitumor, antibacterial, antioxidant activities. This study suggested that symbiotic fungi probably take part in the biosynthesis of steroids in the host gorgomian.Zebrafish (D. rerio) toxicity microplate model combined with other screening models, including lethal activity toward brine shrimp Artemia salina, antifouling against barnacle Balanus amphitrite and antibacterial(Bacillus subtilis, Bacillus cereus, Staphylococcus aureus, Micrococcus tetragenus, etc), were used to evaluate bioactivities. The results show that compounds12,13and19exhibited potent fish toxicity toward zebrafish embryo. Compound12showed zebrafish embryo lethal toxicity with the EC50value of16.34μg·mL-1(24h), compound13exhibited potent fish toxicity causing embryo notochord malformation of zebrafish D. rerio with a EC50value of7.83μg·mL-1(72h), and showed zebrafish embryo lethal toxicity with the EC50value of10.22μg·mL-1(24h). Compound3exhibited modest antibacterial activity against B. cereus, compounds4and6showed weak antibacterial activity towards B. subtilis, S. aureus and M. tetragenus. Compound6showed potent antifouling activity against the larval settlement of the barnacle B. amphitrite. Compound8exhibited significant lethal activity toward brine shrimp A. salina.Zebrafish toxicity microplate model was used to evaluate the activity and toxicity of115secondary metabolites isolated from corals and coral-derived fungus. The analysis of structure-activity relationships indicated that biphenylene oxides and isocoumarins exhibited obvious teratogenic and lethal effects on zebrafish embryo, with EC50values of3-15μg·mL-1. The existence of hydroxyl group has an important contribution to activity. Part of anthraquinones also showed strong fish toxicity causing embryo notochord malformation and eggs condensation of zebrafish D. rerio. The presence of C-2hydrogenated pyran ring enhance the activity of these compounds.In conclusion, the isolated secondary metabolites of soft coral Sarcophyton sp. and gorgonian-derived fungus have provided compounds foundation for chemotaxonomic studies of marine species, and offered reference for exploring the relationship between the hosts and their symbiotic microbes. Studies on zebrafish toxicity and structure-activity relationship of indole alkaloids, biphenylene oxides, anthraquinones, and isocoumarins have provided basic information for the discussion of structures and functions of marine chemical defense compounds.