Antimicrobial Screening and HPLC-DAD-MS Characterization of the Flavonoid–Rich Fractions of the Methanol Leaf-Extract of Lawsonia inermis Linn

Abstract

Flavonoids are known for their wide biological activities, including antimicrobial activity. This study was aimed at investigating the antimicrobial potential of the flavonoid-rich fractions of Lawsonia inermis leaf extract. The ethylacetate fraction from the methanol extract of L. inermis leaves was subjected to Vacuum Liquid Chromatography (VLC) using binary combinations of Hexane: Ethylacetate and Dichloromethane:Methanol to obtain fractions F1-F14. Fractions F11 and F12 which contain flavonoids were further subjected to High Performance Liquid Chromatography–Diode Array Detector (HPLC-DAD) and Liquid Chromatography-Mass Spectrometry (LC-MS) analysis, and subsequently subjected to in vitro test for antimicrobial activity against laboratory strains of Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Aspergillus niger, Candida albicans using Agar well diffusion method at concentrations of 0.0625-1 mg/mL. Erythromycin (50 μg/mL) and Miconazole (50μg/mL) were used as positive controls for bacteria and fungi, respectively. HPLC-DAD and LC-MS analysis of the flavonoid-rich fraction (F12) led to the detection and identification of four flavone pigments namely; vitexin, luteolin 4-glucoside, apigenin monoglycoside and isoorientin (luteolin 6C-β-glucoside). Fraction F11 (constituents not identified) and fraction F12 (the flavone- rich fraction) showed moderate antibacterial activity, with E. coli being the most susceptible bacteria with Minimum Inhibition Concentrations (MIC) of 62.5 μg/mL for F11 and 500 μg/mL for F12. Very mild anti-fungal activity was only observed against C. albicans but not against A. niger. The unidentified polar compounds (the major components of F11) and luteolin 6C-β-glucoside (the major flavones detected in F12) may contribute to the observed antimicrobial activity of L. inermis leaf extract.