Judul: Isolation, characterization and medium optimization for production of active compound produce by endophytic fungi inhibiting breast cancer cell MCF-7 proliferation
Abstrak: An endophytic fungus is the fungus that lives in close association with living plant tissues. Medicinal endophytic fungi are one group of prospecting endophytic fungi. These fungi reported producing secondary metabolite which has structure and activity similar with the host plant. In this study; isolation, characterization and medium optimization for the production of active compound produced by endophytic fungi from medicinal plant originated from East Lombok, West Nusa Tenggara has been conducted. Seventy five endophytic fungi have been isolated from 34 medicinal plants that consist of 57 isolate from leave sample and 18 isolate from stem. The result showed that colonization endopyhitic fungi in leave (70%) was higher compared to stem (32%) from total samples. Thirty six isolates from total 75 isolates were screened for citotoxicity against Artemia salina using brine shrimp lethality test method. First screening using 1000 mg/L concentration of crude extract showed that 9 extracts were active out of 108 extracts assayed. Further screening for the 9 active extracts using LC50 showed that ethyl acetate extract from fungus ENLT 74.3d.2 was the most active compared to the others with LC50 of 30,21 mg/L. Fungus ENLT 74.3d.2 was isolated from Cibotium barometz and morphological descript as Curvularia lunata. This active fungus was deposited and labelled as BioMCC FE-00283. Active compound purification of endophytic fungi C. lunata BioMCC FE-00283 was conducted by bioassay guided. Preliminary bioassay of the extract and fractions was conducted by Brine Shrimp Lethality Test, while citotoxicity of the pure active compound was conducted by MTT assay against human carcinoma breast MCF-7 cell. First purification of active compound was done by coloumn cromatography using silica gel 60 as stationary phase and eluted by step gradient method using hexane, ethyl acetate, and methanol subsequently. Further purification was done by HPLC using C18 column and eluted by gradient system of acetonitril water from 15% up to 100% for 25 minutes. Bioassay showed that ethyl acetate fraction was the most active (LC50 = 4.69 mg/L), while the pure compound from HPLC on concentration 5 mg/L could inhibit 28 % proliferation of MCF-7. Elucidation of formula and structure using spectroscopy IR, LC-MS, 1H NMR, 13C NMR and MS-MS resulted that active compound of endophytic fungi C. lunata BioMCC FE-00283 is 8-hydroxy 9,12-octadecadienoic acid (8-HODE) which molecule formula is C18H32O3. Glucose, monosodium glutamate and corn oil were media components which showed significant effect toward 8-HODE production by C. lunata BioMCC FE-00283. Composition optimization of these three medium components was performed by RSM and full factorial CCD was used design the experiment. Maximum 8-HODE production predicted by the quadratic model was 11.78 mg/L; which was verified experimentally to be 10.78 ± 0.872 mg/L and had rendement (YP/S) 0.001. This maximum 8-HODE production reached in media composition that consist of 5.87 g/L glucose, 11.05 g/L monosodium glutamate and 1.00 ml/L corn oil. Eventhough experimentally verification showed lower result compared to model prediction, however it was higher compare to 8-HODE production using basal media which only reach concentration 0.491 ± 0.072 mg/L and rendemen 4.879 x 10-5. This verification showed that optimization increased 8- HODE production
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