Penelusuran Senyawa Antikanker Ekstrak Etil Asetat Kultur Fungi Endofit Kode BS1 dari Batang Sirih Merah (Piper crocatum Ruiz. & Pav.) melalui Modulasi Siklus Sel T47D

Willy Tirza Eden, Dr. Puji Astuti, M.Sc., Apt

2014 | Tesis | S2 Ilmu Farmasi

Kanker payudara menduduki peringkat pertama kasus kanker pada wanita di seluruh dunia. Fungi endofit merupakan sumber metabolit antikanker alternatif yang berasal dari tanaman. Ekstrak etil asetat fungi endofit kode BS1 dari batang sirih merah (Piper crocatum Ruiz. & Pav.) terbukti mempunyai aktivitas sitotoksik terhadap sel T47D in vitro. Penelitian ini bertujuan untuk menelusuri senyawa aktif antikanker yang selektif pada ekstrak etil asetat tersebut melalui modulasi siklus sel pada sel T47D. Identifikasi morfologi fungi endofit kode BS1 dilakukan melalui pengamatan mikroskopis. Produksi metabolit sekunder dilakukan melalui fermentasi menggunakan medium PDB selama 14 hari dan medium diekstraksi dengan etil asetat. Metode pemisahan dan pemurnian menggunakan kromatografi lapis tipis (KLT), KLT preparatif dan kromatografi cair. Isolat aktif antikanker diidentifikasi dengan spektrofotometer IR, LC-MS, 1H-NMR, 13C-NMR, dan DEPT. Uji sitotosik terhadap sel T47D dan sel Vero dilakukan dengan metode MTT, dimana IC50 dan Selectivity Index (SI) digunakan sebagai parameter efektivitas antikanker. Distribusi siklus sel ditentukan dengan metode flow cytometry dan dianalisis dengan program Modfit LT 3.0. Berdasarkan analisa mikrokopik, fungi endofit kode BS1 merupakan Aspergillus sp. Spektra IR, LC-MS, 1H-NMR, 13C-NMR, dan DEPT menunjukkan isolat 4 yang didapat dari penelitian adalah N-(3’-chloro-5’-oxobutyl)-1-methyl-5- phenyl-1H-pyrrole-3-carboxamide. Isolat 4 mempunyai aktivitas sitotoksik terhadap sel T47D dan Vero dengan IC50 berturut-turut 9,2 dan 109 μg/mL, dimana mempunyai nilai Selectivity Index 11,8. Isolat 4 pada konsentrasi 0,4 μg/mL menghambat siklus sel T47D pada fase S. Penelusuran jalur biosintesis dan mekanisme molekuler cell cycle arrest isolat 4 masih perlu diteliti lebih lanjut.

Breast cancer is one of the most commonly diagnosed cancers and the leading causes of cancer death in women worldwide. Endophytic fungi become anticancer metabolite sources which alternatively in plants. The ethyl acetate extract from BS1 code of endophytic fungi from red sirih stems (Piper crocatum Ruiz. & Pav.) have cytotoxic activity to T47D cells in vitro. This research objectives to discover active constituent of anticancer which selective in the ethyl acetate extract through cell cycle modulation of T47D cell. Identification of BS1 code endophytic fungi morphology was determine by using microscope. Secondary metabolite production was carried out through fermentation using PDB as medium during two weeks and the medium was extracted with ethyl acetate. Separate and purify methode was done using TLC, TLC preparative, and liquid chromatography. Active isolat was characterized by IR, LC-MS, 1H-NMR, 13C-NMR, and DEPT. In cytotoxic assay, T47D and Vero cells were cultured in the precense of pure isolate and was evaluated by MTT assay, whereas IC50 and Selectivity Index were used as the parameters to evaluate effectivity of anticancer. Cell cycle distribution was determined by flow cytometry and then analized by using ModFit LT 3.0 program. Based on microscopic analysis, BS1 code of endophytic fungi is Aspergillus sp. The infrared, mass spectra, 1H-NMR, 13C-NMR, and DEPT signals confirm that isolate 4 is N-(3’-chloro-5’-oxobutyl)-1-methyl-5-phenyl-1H-pyrrole- 3-carboxamide. Isolate 4 have dose-dependent cytotoxic activity to T47D and Vero cells with their IC50 values of 9,2 and 109 μg/mL, respectively and Selectivity Index is 11,8. Isolate 4 at concentration of 0,4 μg/mL inhibit T47D cell cycle in S-phase. Discovering biosynthetic pathways and molecular mechanisms of cell cycle arrest in isolate 4 still needs to be further investigated.

Kata Kunci : fungi endofit, Aspergillus sp., sitotoksisitas, sel T47D, siklus sel