Capturing the antimicrobial profile of Rosmarinus officinalis against methicillin-resistant Staphylococcus aureus (MRSA) with bioassay-guided fractionation and bioinformatics

Manead Khin^a,b, Sonja L. Knowles^a, William J. Crandall^a,c, Derick D. Jones Jr.^a, Nicholas H. Oberlies^a, Nadja B. Cech^a, Joëlle Houriet^a

Department of Chemistry and Biochemistry, University of North Carolina at Greensboro, Greensboro, NC, 27402, United States.

Abstract

Natural products have been a primary source of medicines throughout the history of human existence. It is estimated that close to 70 % of small molecule pharmaceuticals on the market are derived from natural products. With increasing antibiotic resistance, natural products remain an important source for the discovery of novel antimicrobial compounds. The plant rosemary (Rosmarinus officinalis), has been widely and commonly used as a food preservative due to its antimicrobial potential. To evaluate the antimicrobial profile of this plant, we used bioassay-guided fractionation and bioinformatics approaches. Through bioassay-guided fractionation, we tested in vitro activities of a R. officinalis extract and fractions thereof, as well as pure compounds micromeric acid (1), oleanolic acid (2), and ursolic acid (3) against methicillin-resistant Staphylococcus aureus (MRSA). Compounds 1 and 3 showed complete inhibition of MRSA (with MIC values of 32 μg/mL and 8 μg/mL, respectively) while compound 2 displayed only partial inhibition (MIC > 64 μg/mL). In addition, we utilized orthogonal partial least square-discriminant analysis (OPLS-DA) and selectivity ratio (SR) analysis to correlate the isolated compounds 1-3 with the observed antimicrobial activity, as well as to identify antimicrobials present in trace quantities. For mass spectrometry (MS) data collected in the negative ionization mode, compound 1 was the most positively correlated with activity, while for MS data collected in the positive ion mode, compounds 2-3 had the highest positive correlation. Using the bioinformatics approaches, we highlighted additional antimicrobials associated with the antimicrobial activity of R. officinalis, including genkwanin (4), rosmadial (5a) and/or 16-hydroxyrosmadial (5b), rosmanol (6), and hesperetin (7). Compounds 1-3 resulting from the bioassay-guided fractionation were identified by MS-MS fragmentation patterns and 1H NMR spectra. Among the compounds highlighted by the biochemical analysis, compound 6 was identified by comparison with its commercial standard by employed ultra-performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS), while 4, 5a-b and 7 were putatively identified based on MS data and in comparison with the literature. This is the first reported antimicrobial activity of micromeric acid (1) against MRSA.

Keywords: Rosmarinus officinalis, Rosemary, Bioassay-guided fractionation, Bioinformatics, Biochemometrics, MRSA, Natural products.