Discovering anticancer compound of ethyl acetate extract from RL1 code endophytic fungi culture derived by Phyllanthus niruri Linn leaves through cell cycle modulation in T47d cells

Abstract

Breast cancer is one of the most commonly diagnosed cancers and the leading causes of cancer death in women worldwide. Endophytic fungi becomes anticancer metabolite sources which alternatively in plants. The ethyl acetate extract from RL1 code of endophytic fungi from Phyllanthus niruri Linn has cytotoxic activity to T47D cells in vitro. This research objective is to discover active constituent of anticancer which selective in the ethyl acetate extract through cell cycle modulation of T47D cell. Identification of RL1 code endophytic fungi morphology was determined 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 method was done using TLC, TLC preparative, and liquid chromatography. Active isolate was characterized by IR, LC-MS, 1H NMR, 13C-NMR, and DEPT. In cytotoxic assay, T47D and Vero cells were cultured in the presence 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 analysed by using ModFit LT 3.0 program. Based on microscopic analysis, RL1 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-pyrrole3-carboxamide. Isolate 4 have dose-dependent cytotoxic activity to T47D and Vero cells with their IC50 values of 8,3 and 124 μg/mL, respectively and Selectivity Index is 12,2. Isolate 4 at concentration of 0,9 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.