3. Electronic Theses and Dissertations (ETDs) - All submissions

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Subject: search.filters.subject.Essential oils

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    The antimicrobial and toxicity properties of essential oil compounds combined with carrier oils
    (2024) Moola, Salehah
    Essential oils contain a number of biologically active compounds that have been identified as alternative antimicrobials, however, their use is often limited due to their toxic nature. Carrier oils can reduce the toxicity of essential oils, which raises the question as to whether such activity would extend to the essential oil compounds if used in combination. Thus, this study aimed to investigate the toxicity, and the antimicrobial activity of 21 essential oil compounds in combination with six carrier oils against the ESKAPE pathogen group. The antimicrobial properties of the essential oil compounds, alone and in combination with carrier oils, were determined using the broth microdilution assay to determine the minimum inhibitory concentration (MIC) against Enterococcus faecium (ATCC 27270), Staphylococcus aureus (ATCC 25923), Klebsiella pneumoniae (ATCC 13883), Acinetobacter baumannii (ATCC 17606), Pseudomonas aeruginosa (ATCC 27853) and Escherichia coli (ATCC 8739) reference strains. A yeast reference strain, Candida albicans (ATCC 10231), was also included. The toxicity was determined using the brine shrimp lethality assay. The interactive profiles of the combinations of the compounds and carrier oils was determined by calculating the fractional inhibitory concentration index (ΣFIC) (MIC studies) and the fractional percentage mortality index (ΣFPM) (toxicity studies). The selectivity index (SI) of the combinations showing synergy in the broth microdilution assay was investigated. The time-kill effects of the essential oil compound: carrier oil combination that was synergistic in the broth microdilution assay and that demonstrated reduced toxicity, was further evaluated. Of the combinations tested in the broth microdilution assay, 3% resulted in synergy (ΣFIC ≤ 0.50), with the compound thymoquinone and the carrier oil Prunus armeniaca demonstrating broad-spectrum synergistic activity. The carrier oils reduced the toxicity of the compounds, where at 24 and 48 hrs, the combinations showed 8% and 6% synergy, respectively. Calendula officinalis and P. armeniaca carrier oils were responsible for most of the reduced toxicity observed. The compound thymoquinone was present most often in combinations which showed SI values > 4. The combination of Aloe vera with α-terpinene demonstrated synergy in the broth microdilution assay (ƩFIC value of 0.41), as well as reduced toxicity (ƩFIC value of 0.49) and was thus evaluated in the time-kill assay. The combination provided bacteriostatic activity over 6 hrs. This study provides evidence of the essential oil compound: carrier oil interactions where favourable several combinations such as A. vera with α-terpinene, P. armeniaca with thymoquinone, P. americana with thymoquinone and H. perforatum with p-cymene, could be identified as ideal candidates for further research into developing novel combination therapy against the ESKAPE pathogens. Furthermore, the interactions demonstrate the added value of carrier oils in combination with several essential oil compounds.
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    The prediction of synergy in essential oil combinations used in the treatment of respiratory infections through experimental design and chemometric modeling
    (2024) De Rapper, Stephanie Leigh
    The therapeutic use of essential oils, or aromatherapy, is a popular practice of complementary and alternate medicine for the treatment and management of respiratory infections. Essential oils are most often applied topically via massage or via inhalation. Fifty-five essential oils and 369 combinations were identified for the management of respiratory infections. As scientific evidence supporting these indications was lacking, this study intended to explore this avenue of research. The minimum inhibitory concentration (MIC) was recorded for each identified essential oil against nine test organisms (Staphylococcus aureus ATCC 25924, Streptococcus agalactiae ATCC 55618, Streptococcus pneumoniae ATCC 49619, Streptococcus pyogenes ATCC 12344, Haemophilus influenzae ATCC 19418, Klebsiella pneumoniae ATCC 13883, Moraxella catarrhalis ATCC 23246, Mycobacterium smegmatis ATCC 19420 and Cryptococcus neoformans ATCC 14116). Gas chromatography coupled to mass spectrometry (GC-MS) was utilised to confirm purity of the essential oils and to identify the chemical profiles. The data from the GC-MS and antimicrobial studies was then analysed using chemometrics, a multivariate tool used to determine structure-activity relationships. Orthogonal projections to latent structure models were created and the compounds responsible for antimicrobial activity identified. The antimicrobial and toxic effects of the essential oils were then studied in 1:1 combinations using the MIC and brine shrimp lethality assay (BSLA), respectively. The interactive profiles of the combinations (antimicrobial and toxicity) were determined by calculating the fractional inhibitory concentration (ΣFIC) where either synergy, additivity, indifference or antagonism could be identified. Forty-nine essential oils and twentyfour essential oil combinations (1:1) were studied further for anti-inflammatory effects due to the favorable results of these oils in the MIC assay and BSLA. The anti-inflammatory activity was assessed by measuring the inhibition of nitric oxide (NO), an inflammatory mediator, production in LPS-activated RAW 264.7 macrophages. These oils were further investigated for potentially toxic effects using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) against human lung cell lines (A549). Based on the results of the antiinflammatory and cell viability assessment against A549 cell lines, five essential oil combinations were found to be promising in equal combinations. These five combinations were then studied further by means of variable ratios using MIC analysis against the nine micro- xii organisms associated with the respiratory tract. The variable ratios were plotted on isobolograms and then expanded where the MIC results were assessed using the synergy prediction software, SynergyFinder. The results of the isobolograms were compared to the SynergyFinder plots and an optimal blend of essential oils determined. The optimum blend of essential oils was then formulated into a nanoemulsion using the self-emulsification technique. The antimicrobial potential of the nanoemulsed essential oil formulation was validated by means of MIC analysis against the same nine micro-organisms and the results of the formulation compared to those of the pure essential oils in optimal ratios. This study confirmed the antimicrobial potential of essential oils, with noteworthy inhibition observed for 31.9% of essential oils against all the pathogens tested. Amyris balsamifera L. (amyris), Coriandrum sativum L. (coriander) and Cinnamomum zeylanicum Blume (ceylon cinnamon) showed the broadest spectrum of activity. Computational software was applied to define the chemistry responsible for individual antimicrobial effect. Eugenol was identified as the most frequently reoccurring biomarker that contributed to broad-spectrum noteworthy antimicrobial activity of essential oils; while linalyl acetate, α-pinene and β-pinene were frequently responsible for poor antimicrobial activity. The chemometric tool was assessed for prediction accuracy by means of antimicrobial validation. The prediction accuracy relating to the chemometric outcomes of this study was 92.3% for active biomarker predictions and 61.9% for inactive biomarker predictions. Following the analysis of individual essential oils, combinations were assessed for antimicrobial potential in 1:1 and varying ratios. The prediction tool, SynergyFinder was implemented and compared to isobolograms generated for essential oil blends. Prediction accuracy for synergy by SynergyFinder was on average 34% across all nine ratios studied. This low outcome is primarily due to differences in mathematical theory applied to the definition of synergy between the isobologram and SynergyFinder. An optimal blend of essential oil was identified. This blend included the Hyssopus officinalis var. angustifolius (M.Bieb.) Benth. (hyssop) in combination with Rosmarinus officinalis var. angustifolius (Mill.) DC. (rosemary) essential oil in blends of 49.57% of H. officinalis to 50.43% of R. officinalis. This blend of essential oil was developed into a pharmaceutical nanoemulsion formulation and assessed against the same panel of pathogens. The formulation provided a six-fold enhanced antimicrobial effect as compared to the neat essential oil blend. Essential oils, as investigated here, display antimicrobial potential alone and in combination against pathogens of the respiratory tract. Chemometric analysis was proven to be an effective xiii tool for identifying active compounds that could potentially become novel lead compound for new antimicrobials. The blended essential oils further demonstrated potential for holistic treatment effects, proving anti-inflammatory and non-toxic activity when equally blended. It was further determined that the SynergyFinder prediction software, critical in preclinical formulation design, provides valuable supporting tools to isobolograms when optimizing essential oil combinations for antimicrobial effect. This study has also gone on to show the opportunity to combine essential oils into novel pharmaceutical formulations for enhanced antimicrobial effects. Therefore, this study has been majorly successful in identifying unique resources for antimicrobial optimization of essential oils and creates an exciting starting point for future research in this field.
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