SECONDARY METABOLITES FROM ENDOPHYTIC FUNGI ASPERGILLUS NIGER ASSOCIATED WITH BIEMNA SP. SPONGE ORIGINATED FROM SERIBU ARCHIPELAGO AND THEIR ACTIVITY AGAINST MURINE LEUKEMIA

• Main Author: Hadijah Sabarwati, Sitti
• Format: Dissertations
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/61033
• Institution: Institut Teknologi Bandung

The high biodiversity of sea, including its plants, animals and microorganisms, is a more potential source of secondary metabolites compared to terrestrial biodiversity. The discovery of the first drugs from the sea, namely cytarabine (Cytosar-U®), an anticancer drug), and vidarabin (Vira-A®, an antiviral drug) produced by sponges, cephalosporins (antibiotic drugs) yielded from by marine fungi (microbes) encourage researchers to conduct chemical studies of marine organisms, one of which is marine endophytic fungi. Marine and terrestrial endophytic fungi are fungi that live in the internal tissues of plants or animals (host) without causing disease to the host. Endophytic fungi that are present in the internal tissue of sponges have a mutualism symbiotic relationship with its host, and it can produce and manipulating various secondary metabolism that help sponges to defend against predators and marine environmental. Aspergillus is one of the genus of endophytic fungi in sponges, which produces phenolic chemical compounds including alkaloids, quinones, dimeric naphtho-?-pyrone derivatives, and peptides along with non-phenolic compounds, i.e. terpenes, steroids, and fatty acids. Those compounds showed important and various bioactivities, such as cytotoxic, antibacterial, antifungal, antimalarial, antioxidant and enzymes inhibitor. Indonesia, an archipelagic country with the highest marine biodiversity and microorganisms in the world, has the opportunity to the research chemists to study the chemical compounds of its marine endophytic fungi. Besides that, the research on marine endophytic fungi currently are still limited. Based on those the backgrounds, the objectives of this research were to isolate and characterize the secondary metabolites from endophytic fungi associated with Biemna sp. sponge originated from Seribu Archipelago and evaluated the cytotoxicity of isolated compounds against murine leukemia P-388 cells that have never been previously reported. In this research, endophytic fungi from Biemna sp. sponge were inoculated on PDA (Potato Dextrose Agar) media to yield single isolate fungi, and it was followed by characterization of the fungi based on its sequence of molecular rDNA ITS (Internal Transcribed Spacer). Then, the single endophytic fungi was cultivated on rice solid media and incubated for 21 days (3 weeks) at 28 °C. Next, the fungi grown on rice medium was extracted with ethyl acetate (EtOAc) to yield 11 g of dried EtOAc extract. The EtOAc extract were then fractionated and purified by using various chromatographic techniques, including vacuum liquid chromatography, gravitational column chromatography and radial chromatography to give pure compounds. The structures of the isolated compounds were determined based on spectroscopic data, which included 1D-NMR (1H and 13C), 2D-NMR (HSQC and HMBC), and high resolution mass spectrometry. Meanwhile, the cytotoxic assay of the isolated compounds was examined against murine leukemia cell P-388 using MTT [3-(4,5-dimethylthiazo-2-yl)-2,5- diphenyltetrazolium bromide] method. In this research, a single isolate of endophytic fungi was identified based on ITS rDNA sequence data and reported as Aspergillus niger with 100% homology. Seven pure known compounds were successfully isolated from A. niger endophytic fungi, including five phenolic compounds, namely methyl 2,5-dihydroxyphenyl acetate (1), carbonarone A (2), aurasperone C (3), aurasperone F (4), and dianhydro- aurasperone C (5), as well as two non-phenolic compounds, namely 2,3-dihydroxypropyl acetate (6) and ?-sitosterol (7). One compound, 2,3-dihydroxypropyl acetate (6), was recently reported in this genus. However, six other compounds are common compounds in Aspergillus genus and three compounds, namely aurasperone C (3), aurasperone F (4), and dianhydro- aurasperone (5) are the major compounds in the genus. The cytotoxic activity of isolated compounds (1?7) were examined against P-388 cell lines. Two compounds, 2,5-dihydroxyphenyl acetate (1) and carbonarone A (2), showed very active cytotoxicity with IC50 value of 0.2 and 2.0 µg/mL, respectively. Aurasperone C (3), aurasperone F (4), and dianhydro-aurasperone (5) exhibited moderate cytotoxicity with IC50 values of 6.5, 6.4, 7.8 µg/ml, respectively, whereas 2,3-dihydroxypropyl acetate (6) and ?-sitosterol (7) displayed weak cytotoxicity with IC50 value 54.3 and 16.5 µg/ml, respectively. Based on the results of this research, it can be showed that five of seven isolated compounds (1?5) have cytotoxic activities, but only compound 1 and 2 that have potential as lead compounds for cancer drug candidates. The results of this research also confirm that the endophytic fungus of Aspergillus niger is one of the fungi associated with the sponge Biemna sp. The finding of various compounds in this research can also add information related to the diversity of structures and skeletons of the Aspergillus niger endophytic fungus associated with the Biemna sp. sponge originated from the Seribu Archipelago in Indonesia.