The marine fungi investigated in this study were predominantly isolated from red seaweed (marine alga). Three strains were isolated from Kappaphycus alvarezii (Doty) Doty ex P.C. Silva (synonym: Kappaphycus cottonii (Weber-van Bosse) Doty ex P.C. Silva), two strains from Eucheuma edule (Kützing) Weber-van Bosse and one strain from Gracilaria sp. Surface sterilization of the algae followed by cultivation using agar media containing antibiotics was the best way to isolate pure strains of fungi.

All marine fungi were cultivated using different culture media to induce sporulation. Finally, some fungi producing spores could be identified by their morphological characteristics. Thus, three isolates were identified as Aspergillus sp. (KT13), Lasiodiplodia theobromae (Pat.) Griffon et Maubl. (KT26) and Epicoccum nigrum Link (KT28). Two algicolous isolates were identified as Xylaria psidii J.D. Rogers & Hemmes (KT30) and Coniothyrium sp. (KT33). Six strains could not be identified, five of which did not form spores under any of the applied culture conditions.

As it is difficult to obtain sexually reproducing forms, molecular biology-based methods such as sequencing rDNA can be a relevant strategy [6]. The rapid taxonomic assessment of fungal strains might be useful for drug discovery studies based on such organisms because it could reduce the risk of repetitive isolation of known substances. In the future this task will require more attention.

Salinity is one of the environmental factors that affect fungal growth as well as production of secondary metabolites. In an attempt to select the suitable culture conditions for the production of bioactive compounds, the culture medium salinity was varied using marine salt. Results from these tests were taken into account in further cultivation of each strain.

From fungus KT29 one compound (1) was isolated and investigated further to determine its chemical structure. It was obtained as brown crystals by evaporation of methanol solution. The molecular formula of compound 1 was determined to be C₁₂H₁₀O₄ by HR-FTICR-MS with the molecular ion peaks at m/z 241.04721 ([M + Na]⁺) in positive-ion mode and 217.05042 ([M − H]⁻) in negative-ion mode.

In order to elucidate the compound’s structure, NMR measurements were carried out in CD₃OD. Interpretation of ¹H and ¹³C-NMR spectra confirmed the presence of 12 carbon atoms and 10 proton signals, including methyl and hydroxyl groups. The presence of hydroxyl groups was confirmed by the IR spectrum showing an absorption band at 3439 cm⁻¹. The IR (KBr) spectrum also exhibits the presence of a carbonyl group (1623 cm⁻¹), aromatic system (1584 cm⁻¹) and olefinic bond (1384 cm⁻¹). The complete structural elucidation of 1 as well as the unambiguous assignment of all ¹H and ¹³C NMR signals was based on 2D NMR experiments (HMBC, HSQC and ROESY). In particular, the attachment of the OH group at C-1 and not at C-2 is based on the HMBC correlation of H-3 with the carbonyl signal at 176.9 ppm (C-11) as well as on the high-field shift of C-9 (117.8 ppm) due to two OR substituents in ortho position. The ¹H and ¹³C-NMR data of compound 1 are presented in Table 7.

According to the spectroscopic data, compound 1 was determined as 2-carboxy-8-methoxynaphthalene-1-ol (Figure 1). To date, this compound was only known as an intermediate in the synthesis of a naphthalene carboxylic acid (C₂₀H₁₈O₈) using naphthalenediol (C₁₀H₈O₂) as starting material [23,24]. Thus, the discovery of compound 1 as a natural product is new.

In agar diffusion assays, compound 1 showed no antimicrobial activity against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli at 100 μg/disc and Candida maltosa at 200 μg/disc. Compound 1 was also tested in vitro against the human bladder carcinoma cell line 5637 with etoposide as the positive control. The results showed that the compound possesses a negligible cytotoxic activity with an IC₅₀ value of 0.34 mM, compared to etoposide with an IC₅₀ value of 0.6 μM. Against Cladosporium cucumerinum, the compound was not active at a concentration of 200 μg/spot. In contrast to our results, many other naphthalene derivatives, mainly naphthoquinones, possess strong antibacterial, antiviral and further activities [25]. This discrepancy might be due to the following reasons: Compound 1 does not possess a chinoid structure (as do many of the active naphthalene derivatives), and it can be easily metabolized by esterification or etherification of the hydroxyl group at C-1 and the carboxyl group at C-2. The carboxyl group itself renders the compound quite polar and thereby massively reduces transmembrane transport, resulting in a low (cellular) availability.

In addition to the described new substance another isolated compound displayed stronger activity. Unfortunately due to a very low yield the structural elucidation of this compound has not been completed.

NMR spectra were recorded on a Varian VNMRS at 600 MHz for ¹H and at 150 MHz for ¹³C. The samples were dissolved in CD₃OD. Chemical shifts were referenced to internal TMS (δ = 0 ppm, ¹H) and CD₃OD (δ = 49.0 ppm, ¹³C), respectively. ESI-MS and HR-ESI-MS were performed using Bruker Apex III 70 e FT-ICR-MS; spectra were expressed in m/z. Analytical TLC was performed on Merck precoated silica gel 60 F₂₅₄. Spots were visualized by UV light (λ 254 and 366 nm) and spraying with anisaldehyde/sulfuric acid reagent (1% in a solution of 10 mL of AcOH in 10 mL of a 15% methanolic H₂SO₄) followed by heating. Column chromatography was carried out using Sephadex LH-20 (Amersham Biosciences) using methanol as mobile phase. Fractions were collected and processed responding to their chemical composition. IR Spectra were measured on a Perkin Elmer System 2000 FT-IR; wave numbers were expressed in cm⁻¹.

Fungal host samples were collected from Indonesian marine and coastal areas, located in East and West Java, and North Jakarta in April-May 2007 and from South Sulawesi in May 2008. The samples included driftwoods, mollusc shells, sand foam and algae. Eleven fungal strains were isolated consisting of six algicolous fungi (voucher No. KT28, KT29, KT30, KT31, KT32, KT33), two lignicolous fungi (voucher No. KT03 and KT26), two isolates from sandy habitats (voucher No. KT13 and KT19) and one strain from mollusc shell (voucher No. 15). The isolates are deposited in the Institute of Pharmacy, Greifswald University, Germany. Five isolates were morphologically identified as Aspergillus sp. (KT13), Lasiodiplodia theobromae (Pat.) Griffon et Maubl. (KT26), Epicoccum nigrum Link (KT28), Xylaria psidii J.D. Rogers & Hemmes (KT30), Coniothyrium sp. (KT33). Six other strains have not been identified until now.

Cultivation was carried out in liquid shake cultures in 500 mL Erlenmeyer flasks containing 200 mL of Hagem medium (0.5 g of ammonium succinate; 0.5 g of KH₂PO₄; 0.5 g of MgSO₄.7H₂O; 0.5 mL of FeCl₃ (1%); 5 g of glucose; 5 g of malt extract; 1000 mL of aqua destillata; pH 7.5). The sterilized media were inoculated with a homogenized pre-culture of fungal strain and incubated on a rotary shaker (120 rpm) for 14–21 days at room temperature.

Finally, mycelia and culture broth were separated, and the filtered culture broth was extracted with EtOAc (3 × 500 mL). The resulting EtOAc broth extracts were dried in a vacuum evaporator and the residue was extracted with ethanol. The mycelia were lyophilized and subsequently extracted with DCM, MeOH and water using a Soxhlet apparatus. After drying (Na₂SO₄), the organic layers were concentrated using a vacuum evaporator to afford the respective crude extracts.

All fungal extracts were screened for their biological activities. However, only the ethyl acetate extracts of the fungal culture broth exhibited considerable antibacterial and cytotoxic activities. Therefore, the present study was focused on EtOAc broth extracts.

The ethyl acetate extract (525 mg) of KT29 was fractionated on Sephadex LH-20 and using 100% MeOH. Based on TLC analysis, subfractions were combined into 10 fractions. On TLC, fraction F5 showed a major band with a blue fluorescence both in short and long wavelength UV detection. Subsequently, F5 was refractionated on Sephadex LH-20 using methanol as mobile phase to afford compound 1 (13.9 mg; R_f = 0.45 on Si60 F₂₅₄ plate (Merck) using toluene/ethyl formate/formic acid, 10:5:3, v/v). The purity of the compound was confirmed by RP-HPLC (YMC ODS-A 120, 5 μm) with a linear gradient of 2–100% aqueous ACN (0.1% TFA) for 35 min; the retention time was 13.833 min.

Agar diffusion assays to determine antibacterial activity were performed according to the Kirby-Bauer method as described by Boyle et al. [26]. Ampicillin, gentamicin and oxytetracycline were used as positive controls for antibacterial tests and nystatin and benomyl for antifungal tests. Gram-positive bacteria Bacillus subtilis ATCC 6051 and Staphylococcus aureus ATCC 6538 as well as Gram-negative bacteria Escherichia coli ATCC 11229, Pseudomonas aeruginosa ATCC 22853, Vibrio anguillarum DSMZ 11323, Aeromonas salmonicida ATCC 51413 and Yersinia ruckeri ATCC 29493 were used as test organisms. Candida maltosa SBUG 700 and Cladosporium cucumerinum were used as test organisms in antifungal assays.

The activity against the phytopathogenic fungus C. cucumerinum was carried out by the method of Gottstein et al., a semiquantitative test that allows a relative estimation of the activity of the compounds with similar diffusion characteristics [27]. Briefly, the crude extracts were loaded on TLC plates (glass plate, 20 cm × 20 cm, silica gel 60 HF₂₅₄, thickness 0.5 mm) using a microliter syringe. Loaded samples give 10 mm diameter (area of 78.5 mm²). The dried plates were sprayed with 10 mL spore suspension of C. cucumerinum (spore density ca. 2.5 × 10⁶ spores/mL) and dried at room temperature for about 15 min. The dried plates were then placed in a TLC chamber containing moist filter paper and the fungus was cultivated in an incubator at 25 °C for two days. Fungicidal activity was measured as inhibition area (mm²). A larger area correlates with higher activity and mobility of fungicidal constituents.

Cytotoxic activity of the crude extracts was determined by Neutral Red Uptake Assay (NRU-Test) according to Bohrenfreund and Puerner [28] using the cultivated human bladder carcinoma cell line 5637 (ATCC HTB-9).

For assays, 5637 cells were seeded at 2.5 × 10³ cells/mL in 96-well plates (TPP; Trasadingen, CH) and left undisturbed overnight. After washing, fresh medium containing test substances or controls (etoposide, vehicle) was added. Extracts and controls were diluted in assay medium using a stock solution (20 mg/mL in vehicle). Final vehicle concentration did not exceed 0.05%. Cells were allowed to grow for 72 h at 37 °C. Finally, plates were washed and incubated with RPMI 1640 containing 3.3 μg/mL neutral red for 3 h. After removing the supernatant and extensive washing, cells were lysed in acidic ethanol and OD at 540 nm was measured. Cell viability was calculated as percentage of vehicle control after background reduction. All experiments were carried out twice with six replicates for each concentration tested. Where applicable, IC₅₀ values were calculated by linear regression using MS Excel.