Abstract
Objective: This study was focused on isolation of brazilein from the dried heartwood of Secang (Caesalpinia sappan L.) followed by its characterization using Infrared (IR) spectroscopy, liquid chromatography-mass spectrometry (LC-MS) with electrospray ionization (ESI), proton (1H), carbon-13 (13C) nuclear magnetic resonance (NMR) and two dimensional (2D)-NMR, evaluation the cytotoxic activity of brazilein in MCF-7 resistant doxorubicin (MCF-7/DOX) cells and evaluate the interaction between brazilein and ATP with P-glycoprotein (Pgp) in silico using molecular docking.Methods: Brazilein was isolated and purified from ethyl acetate fraction by flash silica gel column chromatography, eluting with chloroform, ethyl acetate and methanol in gradient concentration. In the cytotoxicity assay, MCF-7/DOX cells were cultured in the presence of brazilein for 24 hour (h) and cell viability was evaluated by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Interactions between brazilein and the target proteins were evaluated and calculated in silico by molecular docking using PLANTS.Results: The infrared, mass spectra with a molecular weight of 284 and NMR signal confirmed that was brazilein. MTT assay showed a dose-dependent inhibition of MCF-7/DOX cell proliferation with brazilein IC50 value of 43 µM. The docking score of brazilein was-71,45 kcal/mol and ATP value-96,23 kcal/mol.Conclusion: Brazilein has a potent cytotoxic value on MCF-7/DOX and high affinity in Pgp protein target. Brazilein can be developed as anticancer especially in cancer resistance incidence. Further study must be established to evaluate the molecular mechanism of brazilein inhibit MCF-7/DOX cell proliferation in vitro.
Highlights
Brazilein has been already isolated and purified from methanolic extract heartwood of secang by preparative HPLC using ODS-3 column and acetonitrile: isopropanol (65:35 v/v) as mobile phase [1]
The methanolic extract of kayu secang was obtained 10.24% b/b and this extract was applied to fractionation using liquid-liquid extraction (LLE) with n-hexan and ethyl acetat
Isolation and purification of brazilein in kayu secang by silica gel column chromatography Vacuum column chromatography by using silica gel and chloroformethyl acetate-methanol as a mobile phase was performed to separate the components in the ethyl acetate fraction
Summary
Brazilein has been already isolated and purified from methanolic extract heartwood of secang by preparative HPLC using ODS-3 column and acetonitrile: isopropanol (65:35 v/v) as mobile phase [1]. Flash column chromatography has several advantages than preparative HPLC such as less expensive, this method do not require the help of expensive machinery, low maintenance, cost effective and available in many laboratories. MCF-7/DOX cells were cells model of breast cancer cell resistance to chemotherapeutic agents [10]. In MCF-7 breast cancer cell cases, overexpression of Pgp could be reduced the chemotherapy agent concentration intracellular that influence decreasing its cytotoxicity activity through efflux mechanism [14]. To our knowledge and literature survey, there is no discussion and evaluation about the cytotoxic activity of brazilein on MCF-7/DOX cells through Pgp inhibition. This study was conducted to isolate brazilein from the dried heartwood of secang and to evaluate its cytotoxic effect on resistant breast cancer cells using MCF-7/DOX cells by inhibition of pgp
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