Antimicrobial evaluation of a methanolic extract of Alnus glutinosa bark and purification of its active compounds by bioactivity-guided fractionation

Resistance to antibiotics by numerous pathogens has become a real public health problem [1]. The antimicrobial compounds isolated from plants can inhibit bacterial growth by mechanisms that are different from those of antibiotics in current use [2]. In this study, the antimicrobial activity of Alnus glutinosa bark collected in the French region Champagne-Ardenne was evaluated against Staphylococcus aureus CIP 53.154 by bioautography. Among three extracts tested (heptane, methanol and methanol/water), the methanolic extract exhibited the most significant antibacterial activity and was therefore selected for further study to isolate and identify its active compounds.

The methanolic extract of Alnus glutinosa bark was further tested against a group of pathogenic micro-organisms including 8 Gram-positive, 9 Gram-negative bacteria and 5 yeasts by using a multiple inoculator (Steers) [3]. The Minimum Inhibition Concentration values were ≤0.3 mg/ml against 16 microbial strains.

Fractionation of the methanolic extract was performed by Centrifugal Partition Chromatography (CPC) using a three-phase solvent system composed of n-heptane, methyl-ter-butyl ether, acetonitrile and water. The parallel evaluation of the obtained fractions by bioautography against Staphylococcus aureus revealed four major antibacterial metabolites including oregonin, hirsutanonol, betulinic acid and alusenone 1a. The MIC determination of the major and most active compound, oregonin, was carried out with the 96-well microtiter plate technique against 22 micro-organisms. The results confirmed a strong inhibitory activity of oregonin.

Fig. 1: The CPC purification of oregonin was optimized, resulting in the production of 890 mg oregonin pure at 94% in a single run of 140 minutes and after injection of 3.75 g of crude extract.

Acknowledgements: C. Grimplet is acknowledged for technical assistance in microbial tests.

Keywords: Alnus glutinosa, Betulaceae, oregonin, Centrifugal Partition Chromatography, bioautography, antimicrobial.

References:

[1] World Health Organisation, Traditional Medicine Strategy, Hong Kong SAR, China, 2013; 2014 – 2023: 23 – 28

[2] Eloff JN. Which extractant should be used for the screening and isolation of antimicrobial components from plants? J Ethnopharmacol 1998; 60: 1 – 8

[3] Abedini A, Roumy, V, Mahieux, S, Gohari A, Farimani MM, Rivière C, Samaillie J, Sahpaz S, Bailleul F, Neut C, Hennebelle T. Antimicrobial activity of selected Iranian medicinal plants against a broad spectrum of pathogenic and drug multiresistant micro-organisms. Lett Appl Microbiol 2014; 59: 412 – 421