Commercially Available Viola odorata Oil, Chemical Variability and Antimicrobial Activity

Viola odorata L. oil is frequently recommended in the aromatherapeutic literature for treating respiratory, urinary, and skin infections; however, antimicrobial evidence is lacking. In addition, in aromatherapy, combinations of essential oils are predominantly utilized with the goal of achieving therapeutic synergy, yet no studies investigating the interaction of essential oil combinations with V. odorata oil exists. This study thus aimed to address these gaps by investigating the antimicrobial activity of three Viola odorata oil samples, sourced from different suppliers, independently and in combination with 20 different commercial essential oils, against micro-organisms involved in respiratory, skin, and urinary tract infections associated with global resistance trends. These pathogens include several of the ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp.) The chemical profile of the oils was determined using gas chromatography coupled with mass spectrometry. The minimum inhibitory concentrations (MIC) were determined using the broth micro-dilution method. The interactive profiles for the combinations were assessed by calculating the fractional inhibitory concentration index (ΣFIC). The main compounds varied across the three samples, and included phenethyl alcohol, isopropyl myristate, 2-nonynoic acid, methyl ester, α-terpineol, α-cetone, and benzyl acetate. The V. odorata oil samples displayed overall poor antimicrobial activity when tested alone; however, the antimicrobial activity of the combinations resulted in 55 synergistic interactions where the combination with Santalum austrocaledonicum resulted in the lowest MIC values as low as 0.13 mg/mL. The frequency of the synergistic interactions predominantly occurred against Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, and Enterococcus faecium with noteworthy MIC values ranging from 0.25–1.00 mg/mL. This study also reports on the variability of V. odorata oils sold commercially. While this warrants caution, the antimicrobial benefit in combination provides an impetus for further studies to investigate the therapeutic potential.


Introduction
The use of natural products is of particular interest to "green consumers" who have a preference for eco-friendly products which are naturally derived [1]. Essential oils have gained popularity over the years, originating from the practice of aromatherapy, which is an alternative medical therapy incorporating the use of volatile/plant essential oils for disease management [2][3][4][5]. Essential oils have further been shown to demonstrate antimicrobial activity, including against antimicrobial-resistant strains [6][7][8], and they have been proven to display activity in clinical studies [9,10].
Viola odorata L., commonly known as English or sweet violet, is a member of the Violaceae family and is indigenous to Europe, North Africa, and western Asia [11]. The therapeutic potential of the plant has gained attention in recent years [12]. Viola odorata essential oil is recommended for the treatment of various ailments and is also frequently recommended in combination with other commercial essential oils (Table 1). Combining essential oils is a common practice as it is believed that the different properties contributed by each essential oil may achieve a synergistic therapeutic benefit [13]. Despite the use of this oil in aromatherapy, scientific evidence validating the antimicrobial activity alone and in combination against the pathogens involved in the recommended infectious diseases to be treated by this oil (Table 1) is deficient, which is surprising if one considers how often it is cited and recommended for use in combination [12,[14][15][16][17]. Added to that, V. odorata is also often available and used as an absolute oil which is a highly aromatic liquid extracted via solvent extraction as opposed to distillation with the perceived benefit of a stronger scent. This may result in chemical variability between the absolute and essential oil [18]. Table 1. Use of V. odorata oil in the aromatherapeutic literature and recommended combinations [12,[14][15][16][17].

System Indicated Use Recommended Combinations
Respiratory Bronchitis, chesty and painful coughs, sore throat and throat infections, inflammation of throat and pleurisy, irritating coughs, whooping cough.
While there have been some studies on the antimicrobial activity and variation in the chemistry of V. odorata [19,20], the majority of the studies were acutely focused on simple analysis (disk diffusion assays) [19,21]. Further studies that depicted antimicrobial activity were limited to other Viola spp., such as V. calcarata and V. dubyana [22] or the extracts (non-volatiles) of the V. odorata plant [23][24][25][26][27][28].
In terms of chemistry, the variability of major compounds of V. odorata oil appears to vary across previous studies. Pentane 2,3,4-trimethyl, N-hexadecanoic acid, 10-undecyn-1-l and pentadecanoic acid is reported as the main compounds in the oil by one study [20], and butyl-2-ethylhexylphtalate and 5,6,7,7a-tetrahydro-4,4,7a-trimethyl-2(4H)-benzofuranone was reported in another [19]. This indicates that investigations of this natural product may be beneficial by including multiple collections to identify how variable the chemistry is, and its effects on the antimicrobial activity. Based on the variation of previously reported oil samples, emphasis is placed on the importance of analytical (GC-MS) data to accompany the bio-activity results. Thus, this study aimed to investigate the chemical composition and the antimicrobial profile of three different commercially available Viola odorata oil samples, independently and in combination with a range of commercial essential oils.

Chemical Analysis
The chemical analysis of three V. odorata oil samples (designated 1-3) is shown in Table 2. The chemical analysis of the commercials oils that were combined with the V. odorata oil samples has been previously reported [6,29]. While all the V. odorata oil samples were commercially available, a clear quantitative and qualitative variation exists across all three oils. The V. odorata 1 and V. odorata 2, procured from The United States, comprises of a high percentage of the compound phenethyl alcohol, which is a known essential oil compound with a distinct floral note, found in various essential oils such as Rosa damascena Mill. (rose otto) [30,31]. Oil sample 1 also had 2-Nonynoic acid, methyl ester (also known as methyl 2-nonynoate [32]) as a main compound, which is responsible for the floral scent of V. odorata [33]. Oil sample 2 had isopropyl myristate as a main compound, which suggests possible adulteration. Isopropyl myristate is a fatty acid derivative composed of isopropyl alcohol and myristic acid (fatty acid), which is a common additive in cosmetics [34]. This is not the first study to identify variations in the chemistry in Viola spp., as a study on the chemical composition of V. calcarata and V. dubyana identified fatty acids in the oil samples which had been distilled by the authors of that study [22]. The main compounds of our oil sample 3 were α-terpineol, a known essential oil compound, benzyl acetate which is a natural product found in Vitis rotundifolia Michx. and Tanacetum parthenium L., and lastly α-cetone [35]. Other studies have reported dominant compounds such as 1-hexadecene, 1-octadecene, 1-ecosene and hexade-canoic acid in V. odorata from Toulouse, France [36], butyl-2-ethylhexylphtalate, 5,6,7,7 αtetrahydro-4,4,7 α-trimethyl-2(4 H)-benzofuranone from Iran [19], pentane 2,3,4-trimethyl, N-hexadecanoic acid, 10-undecyn-1-l, pentadecanoic acid from Kermanshah [20,37], 1phenyl butanone, linalool, benzyl alcohol, α-cadinol, globulol, viridiflorol from Tunisia [38], and terpineol, benzyl acetate, methyl salicylate, eugenol, pentadecanoic acid ethyl ester, pentaoxahexadecan-1-ol, tetraoxahexadecan-1-ol, octadecadienal, octadecatrienoic acid ethyl ester, pentaoxanonadecan-1-ol and hexadecanoic acid, all from Egypt [39]. These previous studies have all identified different major compounds across the V. odorata oil samples, and this trend continues with the findings reported in this study. However, several minor compounds appear to be present here or across some of the reported studies such as α-terpineol, methyl benzoate, geraniol, terpinen-4-ol, α-pinene, β-ionone, α-hexyl cinnamaldehyde [19,38,40]. Unlike most commercial essential oils, this variation across samples makes it difficult to define what can be expected from the chemistry of V. odorata. It is known that the chemical composition may vary according to harvest and geographical location of the essential oils; however, this rampant variation across V. odorata oil would be in the percentage of compounds composition, as opposed to variations in types of compounds present.
What was also not common for these tested V. odorata oil samples is the presence of a synthetic compound such as Lilial present in two of the samples that are sold as pure essential oils. This would indicate adulteration of the types of products available to consumers where synthetic compounds are added to the oils to enhance certain properties.

Antimicrobial Activity
The minimum inhibitory concentration (MIC) activity of the three V. odorata oil samples and the combination with commercial essential oils is given in Table 3. Noteworthy antimicrobial activity was observed for V. odorata 1 and V. odorata 2 (USA samples) against Cutibacterium acnes ATCC 6919 and Candida albicans ATCC 10231 (MIC values range 0.50-0.67 mg/mL). Viola odorata 3 (derived from Turkey) displayed noteworthy antimicrobial activity against Staphylococcus epidermidis ATCC 14990. The overall antimicrobial activity, however, was moderate to poor, which is surprising considering the popularity of this oil (Table 1). What is interesting to observe, however, is the correlation between the geographical location (1 and 2) and consistent antimicrobial activity pattern.
Several of the main compounds across the chemotypes, such as phenethyl alcohol, α-terpineol and terpinen-4-ol have been reported to display bactericidal activity against micro-organisms such as Staphylococcus aureus, Enterococcus faecium, Escherichia coli and Pseudomonas aeruginosa [41][42][43]. Previous studies investigating the Viola spp. essential oils were limited to investigating the antimicrobial activity (minimum inhibitory concentrations) against Klebsiella pneumoniae (0.13 mg/mL), Staphylococcus epidermidis (0.50 mg/mL) and Bacillus subtilis (0.50-31.00 mg/mL) [19,20]. Against K. pneumoniae and S. epidermidis, V. odorata was previously reported to display stronger antimicrobial activity than that reported in this study. The difference in antimicrobial activity of V. odorata in the current study and that reported in the literature is not surprising, considering the difference in chemistry between this study and those previously reported.
For the other commercial oils, Cutibacterium acnes ATCC 6919 was the most susceptible micro-organism, with 16 essential oils inhibiting the reference strain at noteworthy concentrations (0.09-1.00 mg/mL). Klebsiella pneumoniae ATCC 13883 was clearly the most resilient of the strains tested. The Santalum spp. and Vetiveria zizanioides are highlighted as essential oils with predominantly noteworthy antimicrobial activity across the selected micro-organisms. This is congruent with previous findings [6]. Table 3. Antimicrobial activity (mg/mL) of essential oils against the selected pathogen reference strains (n = 3). While V. odorata mostly displayed poor antimicrobial activity when tested independently, in combination (the preferred application of essential oil use in aromatherapy), there were several synergistic interactions where the antimicrobial activity was enhanced (Table 4). A summary of the synergy is provided in Figure 1, where V. odorata samples 1 and 2, which both have similar chemical profiles, have 14 synergistic interactions, while the third sample having a different chemical profile, has almost double the synergistic interactions and least number of antagonistic interactions. Figure 1 provides a summary of the combinations with each of the V. odorata samples analysed independently and then grouped to include all three V. odorata samples.

Essential Oil Selection
Three commercially available V. odorata oil samples (V. odorata 1, V. odorata 2 and V. odorata 3) samples were investigated. The selection of the 20 commercial essential oils investigated in combination with the V. odorata samples was based on the aroma-therapeutic literature available to the layman [14][15][16][17], as well as commercially popular essential oils. A selection of commercial oils based on a range of antimicrobial activity, representative of noteworthy, moderate, and poor antimicrobial activity, was selected from our previous studies [6,29]. The essential oils were obtained from Scatters Oils (Johannesburg, South Africa) and Prana Monde (Midrand, South Africa).

Chemical Analysis
Gas chromatography coupled with mass spectrometry (GC-MS) was used to analyse the chemistry of the three V. odorata oil samples. The GC system (Agilent 6890 N GC, Santa Clara, CA, USA) was coupled directly to a 5973 MS equipped with an HP-Innowax polyethylene glycol column (60 m × 250 μm i.d. × 0.25 μm film thickness). A volume of 1 μL was injected (using a split ratio of 200:1 in hexane) with an auto-sampler at 24.79 psi and an inlet temperature of 250 °C. The GC oven temperature was maintained at a temperature of 60 °C for 10 min, then 220 °C at a rate of 4 °C/min for 10 min, followed by a temperature of 240 °C at a rate of 1 °C/min. Helium, the carrier gas, was at a constant flow of 1.2 mL/min. Spectra were obtained on electron impact at 70 eV, scanning from m/z 35 to 550. The percentage composition of the individual components was then quantified by integration measurements using flame ionization detection (FID, 250 °C), and n-alkanes were used as reference points in the calculation of relative retention indices (RRI). Component The frequency of the synergistic interactions is mostly against the reference organisms K. pneumoniae, P. aeruginosa, A. baumannii, and E. faecium. Each of these form part of the resistant ESKAPE pathogens [44] and thus hold the potential to combat antimicrobial resistance. The former three are also Gram-negative micro-organisms, which have been reported to generally be less susceptible to the inhibition of essential oils [6].
The essential oils L. cubeba, L. scoparium, S. album, and S. austrocaledonicum were each involved in at least five synergistic combinations with the V. odorata samples. A previous study, albeit different essential oils, has also noted the frequency of S. austrocaledonicum in synergistic interactions [45].

Essential Oil Selection
Three commercially available V. odorata oil samples (V. odorata 1, V. odorata 2 and V. odorata 3) samples were investigated. The selection of the 20 commercial essential oils investigated in combination with the V. odorata samples was based on the aroma-therapeutic literature available to the layman [14][15][16][17], as well as commercially popular essential oils. A selection of commercial oils based on a range of antimicrobial activity, representative of noteworthy, moderate, and poor antimicrobial activity, was selected from our previous studies [6,29]. The essential oils were obtained from Scatters Oils (Johannesburg, South Africa) and Prana Monde (Midrand, South Africa).

Chemical Analysis
Gas chromatography coupled with mass spectrometry (GC-MS) was used to analyse the chemistry of the three V. odorata oil samples. The GC system (Agilent 6890 N GC, Santa Clara, CA, USA) was coupled directly to a 5973 MS equipped with an HP-Innowax polyethylene glycol column (60 m × 250 µm i.d. × 0.25 µm film thickness). A volume of 1 µL was injected (using a split ratio of 200:1 in hexane) with an auto-sampler at 24.79 psi and an inlet temperature of 250 • C. The GC oven temperature was maintained at a temperature of 60 • C for 10 min, then 220 • C at a rate of 4 • C/min for 10 min, followed by a temperature of 240 • C at a rate of 1 • C/min. Helium, the carrier gas, was at a constant flow of 1.2 mL/min. Spectra were obtained on electron impact at 70 eV, scanning from m/z 35 to 550. The percentage composition of the individual components was then quantified by integration measurements using flame ionization detection (FID, 250 • C), and n-alkanes were used as reference points in the calculation of relative retention indices (RRI). Component identifications were made by comparing mass spectra from the total ion chromatogram and retention indices using NIST ® Version 2.2 and Mass Finder ® Version 4 libraries. The chemical analysis of the 20 commercials oils that were combined with the V. odorata samples was previously reported [6,29].

Preparation of Cultures
The ten micro-organisms tested were from the American Type Culture Collection (ATCC) available within the Department of Pharmacy and Pharmacology of the University of the Witwatersrand (Johannesburg, South Africa). These included reference strain pathogens related to the infections for which V. odorata oil is most commonly recommended, such as respiratory infections (Klebsiella pneumoniae ATCC 13883, Streptococcus pyogenes ATCC 12344), skin infections (Pseudomonas aeruginosa ATCC 27858, Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25924, Staphylococcus epidermidis ATCC 14990 and Cutibacterium acnes ATCC 6919), opportunistic nosocomial infections (Acinetobacter baumannii ATCC 17606 and Enterococcus faecium ATCC 8739) and a fungal pathogen reference strain (Candida albicans ATCC 10231). The aerobic bacteria and C. albicans were cultured in Tryptone Soya broth (TSB) (Oxoid, Basingstoke, UK) at 37 • C for 24 h and 48 h, respectively. Cutibacterium acnes was inoculated into Thioglycolate broth (TGB) (Oxoid) under anaerobic conditions using a CO 2 incubator (8.4% CO2) for seven days at 37 • C, and S. pyogenes was inoculated into a Haemophilus broth (HB) (Oxoid), supplemented with nicotinamide adenine dinucleotide (NAD) (Oxoid), at 37 • C for 24 h. A waiver for the use of these micro-organisms was granted by The University of the Witwatersrand Human Research Ethics Committee (Reference W-CJ-131026-3).

The Minimum Inhibitory Concentration (MIC)
The broth microdilution method was used to determine the MIC of the essential oils alone and in combination [29]. First, 100 µL of appropriate media was aseptically added into all 96 wells of a micro-titre plate, followed by 100 µL of the sample (or 50 µL V. odorata samples with 50 µL of a commercial essential oil for the combinations), diluted to a starting concentration of 32.00 mg/mL in acetone, and added into the first well. This was serially diluted descending down the micro-titre plate. Antimicrobial susceptibility was confirmed using 0.01 mg/mL ciprofloxacin (Sigma Aldrich ® , St. Louis, MO, USA) (for bacteria) or 0.10 mg/mL amphotericin B (Sigma Aldrich ® ) (for C. albicans). A negative control of 32.00 mg/mL water in acetone was included to ensure the antimicrobial activity was not due to the solvent. The respective growth media with the reference strain was also included to ensure microbial viability. After the preparation of an approximate inoculum concentration of 1 × 10 6 colony forming units per ml (CFU/mL) for each micro-organism, 100 µL was added to each well. The sterile adhesive sealing film was used to seal each micro-titre plate, and the plates were incubated accordingly. After incubation, 40 µL of 0.04% (w/v) p-iodonitrotetrazolium violet solution (INT) (Sigma Aldrich ® ) was added to each well. Microbial growth was indicated by a colour change of the indicator to pink or purple, and the lowest concentration displaying no growth was taken as the MIC. Each sample was tested in triplicate, and the average was taken as the MIC value. An MIC value less than or equal to 1.00 mg/mL is considered noteworthy [13]. The fractional inhibitory concentration index (ΣFIC) was calculated for the combinations according to Equation (1) * where (a) is the MIC value of the V. odorata oil sample in the combination and (b) is the MIC value of the commercial essential oil. An ΣFIC of ≤0.5 was interpreted as synergy, an ΣFIC of between >0.5 and ≤1.0 was indicative of an additive interaction, >1.0-≤4.0 indicated indifference and >4.0 indicated antagonism [46].

Conclusions
While V. odorata oil is often cited in therapeutic aromatherapy to be used in combination, this study (to the best of our knowledge) is the first to explore the antimicrobial effects in combination. Santalum austrocaledonicum combined with the V. odorata oil samples could be identified as the combinations that resulted in predominantly noteworthy antimicrobial activity, having the highest frequency of synergistic interactions. The frequency of synergy with low MIC values highlights the potential of these oils and their combinations for further research against antimicrobial-resistant strains.
This study is also the first to highlight variations between the V. odorata oil samples sold commercially and emphasizes the importance of including the GC-MS data within aromatic product studies. While some compounds identified appear to be of unnatural origin, it is important to note that these are sold and used for aromatherapy, and whether they are true essential oils or not, the therapeutic value is of importance if used for medical practises. Future studies investigating the comparative chemical profiles of V. odorata oils across multiple sources are recommended to allow for a better understanding of what is considered acceptable and non-toxic. Institutional Review Board Statement: Ethical review and approval were waived for this study due to no human or animal tissue being used (W-CJ-131026-3) from the University of the Witwatersrand human research ethics committee (medical).

Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.