Bioassay-guided In vitro Study of the Antimicrobial and Cytotoxic Properties of the Leaves from Excoecaria Lucida Sw

Abstract

Background:

Excoecaria lucida Sw. ( Euphorbiaceae) is a plant conventionally used throughout the Caribbean in the treatment of infectious diseases.

Objective:

To evaluate, using bioassay-guided fractionation, the in vitro cytotoxicity and antimicrobial activity of E. lucida leaves.

Materials and Methods:

A 95% ethanol crude extract was dried and fractionated by solid-liquid separation in four phases (hexane, dichloromethane, ethyl acetate, and butanol). Antimicrobial activity (3 bacteria, 6 yeasts, and 2 fungi) was evaluated by the dilution method with resazurin (2048, 512, 128, 32, and 8 μg/mL). The cytotoxicity assays were evaluated in two cell lines: MRC-5 and RAW 264.7; calculating the selectivity index. Assays were performed for the total extract, the isolated compound with the highest yield, and the ethyl acetate and butanol phases. Isolated compounds were characterized by nuclear magnetic resonance and mass spectrometry techniques.

Results:

Fractionation process led to the isolation of ellagic acid (784.29 mg), 3,3',4'-tri-O-methyl ellagic 4-O-β-D-glucopyranoside acid (6.1 mg), and corilagin (6.91 mg). The most active were ethyl acetate phase and ellagic acid with IC ₅₀= 128 μg/mL against seven and five different species of microorganisms, respectively. The total extract (IC ₅₀=512 μg/mL) and the ethyl acetate phase (IC ₅₀=128 μg/mL) were cytotoxic in both cell lines, while butanol phase and ellagic acid both with IC ₅₀>2048 μg/mL seemed to be safer.

Conclusions:

The results obtained indicate that the Excoecaria leaves can be conventionally used as antimicrobial, but it should be present that some cytotoxicity could appear. In addition, the three identified compounds were reported for the first time in the species.

SUMMARY

Excoecaria lucida leaves (Euphorbiaceae) are used by the Cuban population due to their antimicrobial activity. This ethnopharmacological knowledge is confirmed by the integrated antibacterial and antifungal in vitro screening developed, using the bioassay-guided fractionation method.

Abbreviations Used: MRC-5-SV2: Diploid human lung fibroblasts cells, RAW 264.7: Murine macrophages cells, IC50: Inhibitory Concentration 50%, ATCC: American Type Culture Collection, CCEBI: Culture Collection of Industrial Biotechnology Center, CECT: Spanish Culture Collection Type, CFU: Colony forming units, CC50: 50% cytotoxic concentration, CO2: Carbon dioxide, SI: Selectivity index, IR: Infrared spectroscopy, ¹H NMR: Nuclear Magnetic Resonance of hydrogen, ¹³C NMR: Nuclear Magnetic Resonance of carbon, HMQC: Heteronuclear Multiple-Quantum Correlation, HMBC: Heteronuclear Multiple Bond Correlation, COSY: Correlation Spectroscopy, NOESY: Nuclear Overhauser Effect Spectroscopy, KBr: Potassium bromide, DMSO-D ₆: Deuterated dimethyl sulfoxide, LC.MS: Liquid Chromatography-Mass Spectrometry, [α] _D: Optical rotation, EL1: ellagic acid, EL2: 3,3’,4’-tri-O-methyl ellagic 4-O-β-D-glucopyranoside acid, EL3: corilagin, Active (+), inactive (-).