Isolasi, karakterisasi dan optimasi media produksi senyawa aktif kapang endofit untuk menghambat proliferasi kanker payudara MCF-7 secara in vitro

Abstract

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 about 22 fold compared to un-optimized media.

Kapang endofit adalah kapang yang dalam periode waktu tertentu dalam siklus hidupnya mengkolonisasi jaringan hidup tanaman inangnya tanpa menimbulkan gejala apapun. Kapang endofit dari tanaman obat adalah salah satu kelompok kapang endofit yang mempunyai prospek bagus. Telah banyak dilaporkan bahwa metabolit sekunder yang dihasilkan oleh kapang endofit mempunyai struktur dan khasiat yang menyerupai metabolit sekunder tanaman inangnya. Dalam penelitian ini telah dilakukan rekayasa proses produksi senyawa aktif kapang endofit dari tanaman obat yang diambil dari Lombok Timur propinsi Nusa Tenggara Barat. Hasil isolasi menggunakan metode sterilisasi permukaan dan media Corn meal malt extract agar menghasilkan 75 isolat kapang endofit yang terdiri dari 57 isolat dari sampel daun dan 18 isolat dari sampel batang yang berasal dari 34 jenis tanaman obat. Hasil isolasi menunjukkan tingkat kolonisasi kapang pada daun mencapai 70% dari total sampel, sedangkan tingkat kolonisasi pada batang hanya 32% dari total sampel. Dari 75 isolat kapang yang berhasil diisolasi telah dilakukan penapisan sitotoksisitas terhadap 36 kapang hasil isolasi terhadap Artemia salina dengan menggunakan metode Brine shrimp lethality test. Penapisan pertama dengan konsentrasi ekstrak 1000 mg/L mendapatkan 9 ekstrak aktif dari 108 ekstrak yang diuji. Penapisan lanjutan terhadap 9 ekstrak dengan menghitung LC50 menunjukkan bahwa ekstrak etil asetat dari isolat ENLT 74.3d.2 adalah ekstrak yang menunjukkan aktivitas tertinggi dibandingkan ekstrak yang lain dengan LC50 sebesar 30,21 mg/L. Kapang ENLT 74.3d.2 diisolasi dari tanaman Cibotium barometz dan secara morfologi sesuai dengan deskripsi fungi Curvularia lunata. Isolated aktif disimpan dalam koleksi kultur dan diberi nomer BioMCC FE-00283.