Highlights • the Biological Activity of Selected Essential Oil Components
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*Highlights (for review) Highlights x The biological activity of selected essential oil components against strains of E. coli and S. aureus are presented. x Structure-activity relationships are derived from minimum inhibitory concentration data and propose important molecular descriptors in essential oil biological activity. x Molecular topography of the components, electrostatic nature and ligand efficiency of the components are highlighted as principal descriptors *Manuscript Click here to view linked References 1 Title: Structure-Activity Modelling of Essential Oils, their Components, and Key Molecular 1 2 2 Parameters and Descriptors. 3 4 5 3 6 7 8 4 Lucy Owena, Katie Lairda and Philippe B. Wilsonb. 9 10 11 5 12 13 a 14 6 Infectious Disease Research Group, The School of Pharmacy, De Montfort University, 15 16 7 Leicester, UK. 17 18 19 8 bThe School of Pharmacy, De Montfort University, Leicester, UK. 20 21 22 9 23 24 25 10 Corresponding Author: Philippe B. Wilson, The School of Pharmacy, De Montfort University, 26 27 11 Leicester, UK, [email protected]. 28 29 30 12 31 32 33 13 34 35 36 14 37 38 39 15 40 41 42 16 43 44 45 17 46 47 48 18 49 50 51 19 52 53 54 20 55 56 21 57 58 59 22 60 61 62 63 64 65 23 Abstract 1 2 3 24 Many essential oil components are known to possess broad spectrum antimicrobial activity, including 4 5 25 against antibiotic resistant bacteria. These compounds may be a useful source of new and novel 6 7 26 antimicrobials. However, there is limited research on the structure-activity relationship (SAR) of 8 9 27 essential oil compounds, which is important for target identification and lead optimization. This study 10 11 12 28 aimed to elucidate SARs of essential oil components from experimental and literature sources. 13 14 29 Minimum Inhibitory Concentrations (MICs) of essential oil components were determined against 15 16 30 Escherichia coli and Staphylococcus aureus using a microdilution method and then compared to 17 18 31 those in published in literature. Of 12 essential oil components tested, carvacrol and cuminaldehyde 19 20 21 32 were most potent with MICs of 1.98 and 2.10 mM, respectively. The activity of 21 compounds 22 23 33 obtained from the literature, MICs ranged from 0.004 mM for limonene to 36.18 mM for α-terpineol. 24 25 34 A 3D qualitative SAR model was generated from MICs using FORGE software by consideration of 26 27 35 electrostatic and steric parameters. An r2 value of 0.807 for training and cross-validation sets was 28 29 30 36 achieved with the model developed. Ligand efficiency was found to correlate well to the observed 31 2 32 37 activity (r =0.792), while strongly negative electrostatic regions were present in potent molecules. 33 34 38 These descriptors may be useful for target identification ofessential oils or their major components in 35 36 39 antimicrobial/drug development . 37 38 39 40 Keywords: Essential oil, antimicrobial, structure-activity relationship, Escherichia coli, 40 41 41 Staphylococcus aureus. 42 43 44 42 45 46 47 43 1. Introduction 48 49 50 44 Antimicrobial resistance has greatly increased in recent years and is now considered a global public 51 52 45 health threat [1]. Novel antimicrobials are needed to continue treating antibiotic resistant infections, 53 54 55 46 yet the production of new antibiotics has stalled [2]. Natural products are a reservoir of structurally 56 57 47 diverse compounds, so may be a source for the development of novel antimicrobial agents. Essential 58 59 48 Oils (EOs) have been the subject of scientific interest over recent decades, with extensive screening 60 61 62 63 64 65 49 indicating that many of these plant extracts and their isolated components possess antimicrobial 1 2 50 activity [3]. 3 4 5 51 EOs are aromatic, oily plant extracts derived by steam distillation. They are complex mixture of 6 7 52 volatile, low molecular weight organic compounds [4]. Terpenes and their oxygenated derivatives, 8 9 53 terpenoids, are the most common EO compounds [5], while phenylpropanoids and benzenoid 10 11 12 54 compounds are less abundant [6]. Numerous EO components inhibit an array of clinically relevant 13 14 55 pathogenic bacteria, including antibiotic resistant isolates, suggesting they may be candidates for the 15 16 56 development of new antimicrobials. For example, carvacrol, thymol and menthol inhibited 11 17 18 57 important foodborne pathogens at MICs ranging 0.02-4.0 μg/mL [7]. The EO compounds 1,8-cineole, 19 20 21 58 carvacrol, terpinen-4-ol, eugenol and cinnamaldehyde inhibited Staphylococcus aureus and 22 23 59 Methicillin Resistant S. aureus (MRSA) at Minimum Inhibitory Concentrations (MICs) ranging 0.006 24 25 60 to 1.6% and Enterococcus faecalis at MICs ranging 0.012 to >3.2% [8]. Wang et al. [9] reported that 26 27 61 hinokitiol inhibited MRSA and Escherichia coli at 60 and 40 μg/ml, respectively. Orhan et al. [10] 28 29 30 62 showed that thirty-five EO components inhibited 11 isolates of Klebsiella pneumoniae, including 31 32 63 extended-spectrum beta-lactamase producing strains, at MICs ranging 8-64 μg/mL. 33 34 35 64 The mechanism by which EO components exert their antimicrobial effect is incompletely understood. 36 37 65 Much of the published research has concluded that EO components change the structure and function 38 39 66 of bacterial cell membranes; it has been proposed that the hydrophobic nature of these compounds 40 41 67 allows them to partition in the membrane [11]. This non-specific mechanism of action is assumed to 42 43 44 68 bypass many antibiotic resistance mechanisms and inhibit antibiotic resistant isolates. Moreover, it 45 46 69 has been hypothesised that the risk of antibiotic resistance developing is lower than other antibiotics 47 48 70 [12], as per other membrane-targeting antimicrobials such as cationic antimicrobial peptides [13]. EO 49 50 71 components could be an attractive class of compounds for the development of new antimicrobial 51 52 53 72 therapies. 54 55 56 73 Understanding the Structure-Activity Relationships (SAR) of antimicrobial agents is important in 57 58 74 antimicrobial development to identify the most potent compounds and allow optimization of lead 59 60 75 compounds, however there are limited published studies on the SAR of EO components as 61 62 63 64 65 76 antimicrobial agents. The hydrophobicity of EO components, measured by octanal/water partitioning 1 2 77 coefficient (log P) has been correlated with their antimicrobial activity [14,15]. For example, Ben 3 4 78 Arfa et al. [15] reported that carvacrol (log P = 3.52) was more antimicrobial than eugenol (logP = 5 6 79 2.73). The positive correlation of hydrophobicity and antimicrobial activity relates to a greater affinity 7 8 9 80 for partitioning in the bacterial cell membrane, this correlation does not hold true at logP greater than 10 11 81 4, indicating that there are other important structural characteristics [15,16] 12 13 14 82 Several published papers have emphasised the role of a phenolic group in activity, for example, 15 16 83 Andrade-Ochoa et al. [14] reported that thymol, which possesses a phenolic group had a significantly 17 18 84 lower MIC than menthol, which has the equivalent structure but is alicyclic. The importance of a 19 20 21 85 hydroxyl moiety alongside a phenolic group was demonstrated by comparing with aromatic 22 23 86 compounds with alkyl substituents [14,16,17]. It is hypothesised that the delocalised electron system 24 25 87 of the phenolic moiety facilitates proton exchange through the hydroxyl group, which dissipates 26 27 88 proton motive force [16]. 28 29 30 89 This study aims to construct a 3D QSAR model of EO component activity from experimental and 31 32 90 literature sources based on electrostatic and steric descriptors to inform future antimicrobial drug 33 34 35 91 target identification. 36 37 38 92 39 40 41 93 42 43 44 94 2. Materials and Methods 45 46 95 47 48 49 96 2.1. EO Components 50 51 52 97 Authentic standards of (-)-β-pinene (99%), carvacrol (98%), cuminaldehyde (98%), linalool (97%), p- 53 54 55 98 cymene (99%), thymol (≥98.5%), β-caryophyllene (≥80%) and γ-terpinene (97%) were obtained from 56 57 99 Sigma Aldrich (Gillingham, UK). 58 59 60 100 2.2. Microorganisms 61 62 63 64 65 101 Escherichia coli NCTC 8003 and Staphylococcus aureus NCTC 12981 were cultured using nutrient 1 2 102 broth and agar (Oxoid, Basingstoke, UK) and grown aerobically at 37˚C for 24 hours. 3 4 5 103 2.3. Minimum Inhibitory Concentrations 6 7 8 104 The MIC of EO components were determined using a broth microdilution method adapted from the 9 10 105 International Organization for Standardisation (ISO) 20776-1 antibiotic susceptibility test [18]. Serial 11 12 106 two-fold dilutions of EO components to yield final concentrations ranging 8-0.01% (v/v) in nutrient 13 14 15 107 broth supplemented with 10% dimethyl sulfoxide (Fisher Scientific, Loughborough, UK) were 16 17 108 prepared in polystyrene 96-well plates (Scientific Laboratory Supplies, Wilford, UK). An equal 18 19 109 volume (75 μl) of bacterial suspension was added to each well to yield a final well concentration of 20 21 110 5x105 colony forming units (CFU)/ml. Controls were antimicrobial free and inoculum free wells. 22 23 24 111 Bacterial growth was determined by measuring optical density (595 nm) of samples using a 25 26 112 Spectramax Plus 384 microplate reader and Softmax Pro version 6.4 software (Molecular Devices, 27 28 113 Sunnyvale, USA) immediately after inoculation and after 24 hr.