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

Highlights

• First report of the antimicrobial activity of micromeric acid (1) against MRSA.

• Positive correlations of isolated compounds to the bioactivity.

• Putative identifications of additional compounds using bioinformatics.

• Advantage of using both bioassay-guided fractionation and bioinformatics.

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.

Introduction

The use of natural products, including medicinal plants, remains widely popular today with approximately 80 % of the world’s population relying on herbal products and related supplements as part of their health care regimen [1]. Rosmarinus officinalis L., or rosemary, synonymously known as Salvia rosmarinus Spenn., is an aromatic shrubby herb that is a member of Lamiaceae, native to the Mediterranean region [2]. R. officinalis extract has been reported to demonstrate antimicrobial activity against the bacterial pathogen methicillin-resistant Staphylococcus aureus (MRSA), although the chemical compounds responsible for that activity were not identified [3]. According to the 2019 CDC report, MRSA is one of the most common antibiotic-resistant bacterial pathogens and is estimated to cause more than 323,000 cases and 10,600 deaths annually in the United States alone [4].

In this study, our goal was to use two different approaches, bioassay-guided fractionation and bioinformatics, to evaluate the antimicrobial profile of R. officinalis against MRSA USA300 LAC strain AH1263 [5], with the long-term objective being the utilization of this combination approach to efficiently improve the antimicrobial screening processes in natural products.

Bioassay or bioactivity-guided fractionation has been the gold standard in natural products, in which the extracts are subjected to chemical fractionations and bioassays to simplify the complexity and pinpoint certain compounds with significant biological effects [6]. It was reported in 2012 that bioassay-guided fraction was used in more than 1500 published articles in ISI Web of Science, with hundreds more citations applying various forms of the same method [6]. While the bioassay-guided fractionation method can be employed to effectively isolate the most abundant antimicrobials, it also comes with several limitations in that the botanical materials contain thousands of individual compounds and the methodology tends to overlook some of the bioactive constituents that are present in low amounts [7].

Here we sought to demonstrate the use of bioinformatics approaches to correlate the chemical constituents with the bioactivity of R. officinalis and identify the additional antimicrobials that are not identified through bioassay-guided fractionation. Both orthogonal partial least square-discriminant analysis (OPLS-DA) and selectivity ratio (SR) analysis were utilized toward this goal. OPLS-DA is an extension of partial least square-discriminant analysis (PLS-DA) and utilizes orthogonal signal correction filter to remove variabilities that are not appropriate to the dataset, which in turn allows the analysis to be targeted and robust against noise [7]. SR plots compare the correlation and covariance to the residual variance and provide a numerical scale to differentiate between constituents that correlate with biological activity and those that do not [8].