Marine-derived fungi have been shown in recent years to produce a plethora of new bioactive secondary metabolites, some of them featuring new carbon frameworks hitherto unprecedented in nature. These compounds are of interest as new lead structures for medicine as well as for plant protection. The aim of this protocol is to give a detailed description of methods useful for the isolation and cultivation of fungi associated with various marine organisms (sponges, algae and mangrove plants) for the extraction, characterization and structure elucidation of biologically active secondary metabolites produced by these marine-derived endophytic fungi, and for the preliminary evaluation of their pharmacological properties based on rapid 'in house' screening systems. Some results exemplifying the positive outcomes of the protocol are given at the end. From sampling in marine environment to completion of the structure elucidation and bioactivity screening, a period of at least 3 months has to be scheduled.

Bioactive natural products from endophytic fungi, isolated from higher plants, are attracting considerable attention from natural product chemists and biologists alike as indicated by the steady increase of publications devoted to this topic during recent years (113 research articles on secondary metabolites from endophytic fungi in the period of

Two new 10-oxo-10H-phenaleno[1,2,3-de]chromene-2-carboxylic acids, xanalteric acids I (1) and II (2), and 11 known secondary metabolites were obtained from extracts of the endophytic fungus Alternaria sp., isolated from the mangrove plant Sonneratia alba collected in China. The metabolites were confirmed to be of fungal origin, and the structures of the new natural products were unambiguously elucidated on the basis of extensive one- and two-dimensional NMR spectroscopic studies and mass spectrometric analysis. The two new compounds 1 and 2 exhibited weak antibiotic activity against multidrug-resistant Staphylococcus aureus. Altenusin (3) displayed broad antimicrobial activity against several additional multidrug-resistant bacterial and fungal strains.

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Six new chromones, named pestalotiopsones A-F (1-6), and the known derivative 7-hydroxy-2-(2-hydroxypropyl)-5-methylchromone (7) were obtained from the mycelia and culture filtrate of the mangrove endophytic fungus Pestalotiopsis sp., which was isolated from leaves of the Chinese Mangrove plant Rhizophora mucronata. Their structures were elucidated on the basis of spectroscopic data. Pestalotiopsones A-F are chromones having both an alkyl side chain substituted at C-2 and a free or substituted carboxyl group at C-5. Compound 6 exhibited moderate cytotoxicity against the murine cancer cell line L5178Y, whereas the other investigated compounds proved to be inactive.

This review highlights recent findings of our group on bioactive marine natural products isolated from marine sponges and marine derived fungi. The activated chemical defence of the Mediterranean sponge Aplysina aerophoba is introduced as an example of a dynamic response of marine sponges to wounding. Following tissue disrupture preformed brominated isoxazoline alkaloids are enzymatically cleaved and thereby give rise to aeroplysinin-1 which is believed to protect sponges from invasion of pathogenic bacteria. A preliminary characterization of the membrane bound enzyme(s) involved in the cleavage reaction is presented. Bromotyrosine derived, oxime group bearing peptides, the so called bastadins, obtained from the sponge Ianthella basta and some of their synthetic derivatives were shown to exhibit pronounced antifouling activity against larvae of the barnacle Balanus improvisus. The antifouling activity could be traced to the oxime group as an important pharmacophore that was also found to be present in other sponge derived natural products exhibiting antifouling activity. Marine derived fungi that reside within invertebrates such as sponges or inside Mangrove plants are emerging as a new source of bioactive metabolites as demonstrated for Aspergillus ustus and Alternaria sp. that were isolated from the sponge Suberites domuncula or from the Mangrove plant Sonneratia alba, respectively. The former fungus yielded new moderately cytotoxic sesquiterpenoids of the drimane type whereas the latter was found to produce polyketides such as alternariol that exhibited strong and selective inhibitory activity against several protein kinases, for instance Aurora A and B which are targets for anticancer chemotherapy.

Chemical investigation of the EtOAc extract of the endophytic fungus Bionectria ochroleuca, isolated from the inner leaf tissues of the plant Sonneratia caseolaris (Sonneratiaceae) from Hainan island (China), yielded two new peptides, pullularins E and F (1 and 2) together with three known compounds (3–5). The structures of the new compounds were unambiguously determined on the basis of one- and two-dimensional NMR spectroscopy as well as by high-resolution mass spectrometry. The absolute configurations of amino acids were determined by HPLC analysis of acid hydrolysates using Marfey’s method. The isolated compounds exhibited pronounced to moderate cytotoxic activity against the mouse lymphoma cells (L5178Y) with EC50 values ranging between 0.1 and 6.7 µg/mL.

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