DISTRIBUTION AND ACCUMULATED cytotoxic compounds AGAINST BREAST CANCER CELLS IN Brucea T47D javanica (L.) MERR
ABSTRACT: Brucea javanica (L.) Merr (Buah Makasar) has been traditionally used by Indonesian people for various diseases treatments including cancer. Isolation of bioactive compounds by bioassay guided isolation method and accumulation of its bioactive compounds in organs Brucea javanica (L.) Merr have not been reported. Therefore the objective of this study were : 1 ) to identify the structure of toxic compound of breast cancer cells (T47D cell) and the index selectivity against breast cancer cells (MCF7 cell) and its effect on normal cells (Vero cell), 2) reviewing the distribution and accumulation of the most toxic compounds against breast cancer cells (T47D cell) in generative and vegetative organs of Brucea javanica (L.) Merr. The materials used roots, stems, leaves and fruits of Brucea javanica (L.) Merr taken from Betung Karihun National Park, West Kalimantan. Breast cancer cell used was T47D cell line. Samples were socletated using chloroform and methanol and boiled using water. Extracts were tested using cytotoxicity test performed by MTT assay. Furthermore, the most potent extract with the lowest IC50 was fractionated by Vaccum Liquid Chromatographi (VLC) method followed by Thin Layer Chromatographi (TLC). The profile of similar fractions were combined. The cytotoxicity of the combined fractions were tested by the same method as previous method, the most toxic fraction was then performed to Preparative-TLC (PTLC). Isolates were tested for MTT assay test to obtain the most toxic isolates. The most toxic isolate was then run in the PTLC for the second times and again tested for the MTT assay. The most toxic isolated of the second P-TLC were determined using a spectrum of spectroscopic UV, FT-IR, LC-ESI-ToF-MS, FT-NMR (1H-NMR and 13C-NMR ). Doxorubicine was used as a reference drug. The index selectivity was determined on breast cancer cells (T47D cell and MCF7 cell) and normal cells (Vero cell) The results of the cytotoxicity test of fifteen extracts showed that chloroform extracts of ripe fruit showed the highest cytotoxic activity : IC50 96.85±0.24 μg/mL. Fractionation resulted in three combined fractions, and the hexan and chloroform combined fraction showed the highest cytotoxic activity (IC50 63.32±2.35 μg/mL). PTLC most potent fraction produced three isolates, and middle isolates showed the highest cytotoxic activity (IC50 47.04±0.69 μg/mL). PTLC most toxic isolates resulted 2 isolates, and isolates upper produced the most potent cytotoxic activity (IC50 25.82±0.23 μg/mL). Selectivity index (SI) of upper isolates on breast cancer cells (T47D cell) (11.3) was greater than SI the breast cancer cells (MCF7 cell) (2,58). Based on spectral data of LC-ESI-ToF-MS, the compound had the molecular formula C14H26O2 (m/z 226.355). Molecular structure elucidation by UV spectra, FT-IR, LC-ESI-ToF-MS, FT-NMR (1HNMR and 13C-NMR) showed that the compound was tetradecane-4-enoic acid. The identification of the compound distribution in various plant organs showed xxi that tetradecane-4-enoic acid accumulated in the leaves, juvenile fruits and mature fruits with relative concentration (RC) 6.68%; 4.63% and 7.67%. In fruit endocarp (RC 9,56%) and embryo (RC 29,58%) were the place for accumulation. Based on the results, we can infer that the tetradecane-4-enoic acid was potential to be developed as anticancer agent. Keywords: Brucea javanica, T47D breast cancer, cytotoxicity, accumulation, tetradecane-4-enoic acid
