3. Electronic Theses and Dissertations (ETDs) - All submissions
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Item The antimicrobial and toxicity properties of essential oil compounds combined with carrier oils(2024) Moola, SalehahEssential 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.Item The combination of medicinal dyes with conventional antimicrobials: potential for synergy in topical skin infections(2024) Ramfol, RheaMedicinal dyes have been used for centuries to treat ailments, however, research into their use as an antimicrobial has been lacking in recent years and most clinical work predates to 1945 and has now been overshadowed by antibiotics. Many antibiotics have become ineffective in treating the simplest of infections thereby sparking a new interest in combination therapy. This study aimed to explore potential synergistic effects of medicinal dyes and antimicrobials against pathogens (reference, resistant and clinical strains) responsible for skin infections. To test the activity of the medicinal dyes and commercial antimicrobials, minimum inhibitory concentrations (MIC) and minimum bactericidal concentration (MBC) assays were conducted against Gram-positive and Gram-negative bacteria, and yeasts. Combination studies were conducted in equal ratios (1:1) and the fractional inhibitory index (ΣFIC) was calculated. Varied ratio combinations were conducted on selected combinations and represented visually as isobolograms. Gentian violet demonstrated the strongest antimicrobial activity against all pathogens with a notably strong MBC value of 0.000031 µg/ml against Staphylococcus epidermidis (Clinical BA16), 0.01µg/ml against Pseudomonas aeruginosa (ATCC 27858 and DSM 46316) and Escherichia coli (ATCC 8379). It also demonstrated the strongest activity against Candida albicans with an MIC value of 0.00025 µg/ml. Among the antibiotics, Mupirocin and Fusidic acid noted the strongest antimicrobial activity with an MIC/MBC of 0.04 µg/ml against S. epidermidis (Clinical BA16 and ATCC 51625), followed by Betadine® with an MIC/MBC of 1.56 µg/ml against E. coli ATCC 8379 and DSM 22314) and an MIC/MBC of 1.30 µg/ml against C. albicans (ATCC 10231). Individually, medicinal dyes noted stronger antimicrobial activity compared to conventional antimicrobials. Combination studies (1:1 ratio) noted 26% of dye-antibiotic combinations were synergistic against the Grampositive strains, 15% against the Gram-negative strains and 14% against the yeasts. In the varied ratio studies, the Mercurochrome: Betadine® combination noted synergy among all the Staphylococcus aureus strains with the 1:1 ΣFIC values ranging from 0.05 to 0.48. Fuchsine with Gentamycin noted the most synergistic ΣFIC value of 0.001 against P. aeruginosa (DSM 46316) strain. Methylene blue with Ketoconazole demonstrated the best synergistic ΣFIC value of 0.01 among the yeasts. The brine shrimp lethality assay was then undertaken to determine the toxicity of the medicinal dyes, commercial antimicrobials, and selected combinations (used in varied ratio studies). Four dyes (Iodine tincture, Gentian violet, Malachite green and Mercurochrome) demonstrated high toxicity at 24 and 48 hrs. All four dyes demonstrated a percentage mortality of 100% at 48 hrs. The majority of the commercial antibiotics were non-toxic at both 24 and 48 hrs. Combination studies (1:1) on selected (50%) synergistic combinations observed a decrease in toxicity. The combination of Gentian violet with Gentamycin noted a fifteen-fold decrease in toxicity – the most synergistic combination. Malachite green in combination with Neomycin noted the second highest decrease in toxicity. The selectivity index (SI) of the medicinal dyes, commercial antimicrobials and selected combinations were calculated based on the brine shrimp lethality assay together with the MIC. A low SI (<10.00) was observed amongst the majority of the dyes tested. The lowest SI was demonstrated by Iodine tincture with a SI value of 0.0001 against all S. aureus strains, Gramnegative strains, and yeasts. The commercial antimicrobials noted higher SI values in comparison with the medicinal dyes. The highest SI value observed was 12500.00 against S. epidermidis (ATCC 12228) when calculated against Fusidic acid. Gentamycin demonstrated high SI values among majority of the Gram-negative strains. All three antifungals demonstrated high SI ratios among C. albicans (ATCC 10231 and A100). For the combinations tested, Gentian violet with Gentamycin demonstrated the highest SI of 977.50 against the E. coli (DSM 22314) strain. Time-kill studies were conducted on the combinations with the highest safe SI value and lowest safe SI value: Gentian violet with Gentamycin and Malachite green with Neomycin respectively. Both medicinal dyes demonstrated bacteriostatic activity over a period of 24 hrs. When combined, Malachite green with Neomycin demonstrated bactericidal activity. This study has demonstrated that selected dye: antimicrobial combinations (20.46%) potentially act synergistically, however, when the antimicrobial activity and toxicity of these combinations have been investigated, two combinations demonstrate more promise in comparison to the others: Gentian violet with Gentamycin and Malachite green with Neomycin. These combinations have the potential for further clinical studies as a future treatment for skin infections. This study also highlights the importance of further evaluation of the antimicrobial properties of medicinal dyes in combination with commercial antimicrobials in order to determine viable options to treat skin infections.Item An antimicrobial and phytochemical validation of southern African plants used as soap substitutes and formulation of herbal soap(2024) Mzimba, Nyiko FortunateA major proportion of the population in southern Africa relies on medicinal plants commonly known as soapy plants for bathing and washing. There is limited scientific research that assess the effectiveness of southern African soap plants. Therefore, this study investigated the phytochemistry, antimicrobial activity, and toxicity of plants used in southern Africa as soap substitutes. Thereafter, an effective antimicrobial herbal soap was formulated and assessed for its antimicrobial efficacy. A comprehensive literature review was conducted to gather information on plants used as soap substitutes in southern Africa. A total of 59 plant species were identified to be used for bathing and washing. A total of 26 plant species were collected based on availability at Walter Sisulu Botanical Garden, Random Harvest Indigenous Nursery and University of Johannesburg herbarium and University of the Witwatersrand storage. The organic and aqueous extracts were prepared and screened for the presence of alkaloids, terpenoids, and saponins. Methanol and acetone were the optimal solvents to extract alkaloids from 62.07% of plant extracts. Terpenoids were best extracted with ethanol (75.86% of plant extracts), followed by methanol (68.97% of plant extracts). Saponins were highly detectable using water (93.10% of plant extracts) and ethanol (82.76% of extracts). The qualitative evaluation of saponins using thin layer chromatography displayed a variety of saponins, including steroidal saponins that had Rf-values comparable to diosgenin (a steroidal aglycone used as a standard). Sideroxylon inerme subsp. inerme (16.3%) had a high percentage yield of saponins. Hermannia cuneifolia displayed the highest saponin content (262.41 ± 1.90 mg/g), followed by Sideroxylon inerme subsp. inerme (71.34 ± 1.01 mg/ml), Acalypha glabrata (70.48 ± 2.05 mg/g), and Noltea africana (68.53 ± 2.43 mg/g). The organic and aqueous extracts of each of the selected soap plants were tested for their antimicrobial activity against skin-relevant pathogens. Pelargonium peltatum demonstrated the best antimicrobial activity against Brevibacterium linens and Cutibacterium acnes with an MIC value of 0.06 mg/ml. Calodendrum capense (leaves), Noltea africana (leaves), Olea europaea (leaves), Pelargonium peltatum (leaves), Plectranthus ciliatus (leaves), Ptaeroxylon obliquum (bark), and S. inerme subsp. inerme (leaves) organic extracts displayed noteworthy antimicrobial activity against the pathogen C. acnes with an MIC value of 0.06 mg/ml. The plants that demonstrated notable broad-spectrum activity against most of the tested pathogens were Calodendrum capense (leaves), Pelargonium peltatum, Plectranthus ciliatus, and Ptaeroxylon obliquum (bark). The toxic profiles of the organic and aqueous extracts were evaluated to assess the safety of the plant species using brine-shrimp lethality assay (BSLA). Aqueous plant extracts were more toxic (65.52%) compared to organic plant extracts (62.07%). Acalypha glabrata (leaves), Aloe maculata (leaves), Bauhinia bowkeri (leaves), Deinbollia oblongifolia (leaves), Ledebouria luteola (bulb), Pouzolzia mixta (leaves), and Sideroxylon inerme subsp. inerme (leaves) organic and aqueous extracts demonstrated the lowest toxic effects at 24 and 48 h. Aristaloe aristata (leaves), Calodendrum capense (leaves) and P. obliquum (bark) organic extracts were nontoxic, and Hermannia cuneifolia (leaves), Plectranthus ciliatus (leaves), and Ptaeroxylon obliquum (leaves) aqueous extracts were non-toxic. Crinum bulbispermum (bulb), Haemanthus albiflos (bulb) and Ilex mitis (leaves) were highly toxic, with LC50 values > 250 µg/ml after 48 h. Pelargonium peltatum displayed low toxicity at a concentration of 125 µg/ml. The extracts of Calodendrum capense, Pelargonium peltatum, and Ptaeroxylon obliquum were then used for soap-making by the basic saponification reaction, and the physicochemical parameters and antimicrobial activity of the soaps were evaluated. The Calodendrum capense herbal soap had the lowest pH (10.79), moisture content (28%), and free caustic alkali (0.03%). Pelargonium peltatum and Calodendrum capense herbal soaps were categorized as first-grade soaps (84 and 80%, respectively). The antimicrobial efficacy of the soaps was determined by inoculating selected skin micro-organisms on agar containing soap formulations using the multipoint inoculator. Gram-positive micro-organisms were inhibited (MIC values of ≤ 1.57 mg/ml). All the tested micro-organisms except for Enterobacter cloacae were inhibited at a concentration of 12.5 mg/ml, which is comparable to the control, Protex® commercial soap. The findings herein of the antimicrobial properties, phytochemistry, and toxicity contribute to the knowledge gaps that exist in the ethnobotanical literature of some southern African soap plants and provide evidence for their incorporation into soap formulation.Item An optimal polyherbal formulation with antimicrobial and anti-oxidant properties(2024) Mapeka, Tsholofelo MavlineIntroduction: Since ancient times, spices and herbs have been traded, valued, and used for the enhancement of taste, flavour, and colour of food and beverages. Their role in preservation has been investigated in several in vitro and in vivo studies. Many herbs and spices, other than their culinary uses, have health-promoting properties and have been used by different cultures and in various traditions to prevent and manage illnesses and diseases. Such health benefits among others include antibacterial, antifungal, antiviral, anti-oxidant, anti-inflammatory, antidiabetic, and anticancer activities. A number of studies have demonstrated synergism as an effective strategy to enhance the bioactivity of plant extracts and essential oils. Thus, using this framework, this study was conducted to effectively determine the ideal combination of crude extracts or neat essential oils of some common culinary herbs and spices that would exhibit the most favourable anti-oxidant and antimicrobial activities, and incorporate these into an optimal polyherbal formulation. Materials and methods: A total of 51 crude extracts (methanol, dichloromethane, water) and 15 neat essential oils were chemically profiled using ultra-performance liquid chromatography coupled with mass spectrometry (UPLC-MS) and gas chromatography coupled with mass spectrometry (GC-MS), respectively. The major constituents in the extracts and essential oils were identified based on the available data from the literature. The anti-oxidant activities of the extracts and essential oils were evaluated independently, and in combination (1:1) using 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2-azinobis(3- ethylbenzothiazoline-6-sulphonic acid) (ABTS), and ferric reducing anti-oxidant power (FRAP) assays. Whereas the minimum inhibitory concentration (MIC) assay was used to determine the antibacterial activities of the individual extracts and essential oils and their combinations (1:1) against three Gram-positive and three Gram-negative bacteria. The antimicrobial and anti-oxidant interactions were determined by calculating the sum of fractional inhibitory concentration index (ΣFICI). The Design of Experiments (DOE) (MODDE 9.1®) software analysis was used to optimize synergy potential of the herbal extracts by generating a statistically significant model that predicted the most effective ratio required for the polyherbal combination, as validated experimentally. A polyherbal capsule was formulated from blending various spice extracts based on DOE model predictions. The formulation was evaluated for toxicity using Brine shrimp lethality assay (BSLA), and the antioxidant and antibacterial activity of the formulation was also determined. Results: The major compounds that were identified in the analysed essential oils were limonene, linalool, 1,8-cineole, β-caryophyllene, α-pinene, β-pinene, eugenol, estragole, cinnamaldehyde, thymol, citral, terpinene-4-ol, trans-sabinene hydrate, γ- terpinene, thujone (α, β), menthone and menthol. A total of 45 bioactive compounds , mainly phenolic acids and flavonoids were identified in the analysed crude extracts by comparing mass spectra and retention time (Rt) with those of commercially available reference standards. Where standards were not available, the compounds were tentatively identified by comparing mass spectra in terms of m/z fragments with data reported in the literature. Compounds such as apigenin, caffeic acid, proanthocyanidins, catechin, and rosmarinic acid were common in numerous analysed extracts. Each extract and essential oil displayed variable anti-oxidant activity. A total of 27 combinations were assessed for anti-oxidant interactions using the DPPH, ABTS, and FRAP assays. From these combinations, 3.8% was synergistic in the DPPH assay, 7.7% in the FRAP and ABTS assays, whilst 50.0% of the combinations were additive using the DPPH and FRAP assays, and 19.2% with the ABTS assay. Antagonism was shown in 11.1% with the DPPH assay, and 3.7% with ABTS and FRAP assays. Many of these combinations had an indifferent effect (37.0% with DPPH and FRAP assays, and 66.7% with ABTS assay). Out of the 14 combinations of the extracts assessed for antibacterial interactions, 16.7% were synergistic, additive, and antagonistic, whilst 50.0% of the combinations had an indifferent effect. A total of 21 essential oil combinations were evaluated for antibacterial interactions. of these combinations, 66.7% were antagonistic, whereas 33.3% were indifferent. None of the essential oils combinations showed additive or synergy interactions. The optimum combination for anti-oxidant properties was predicted by DOE to be the mixture containing crude methanol extracts of Mentha piperita L. (55.0%), Thymus vulgaris L.(44.0%), and Zingiber officinale Roscoe (1.0%). The optimal antimicrobial mixture contained crude methanol extracts of Rosmarinus officinalis L.(59.5%), Salvia officinalis L.(40.0%), and 1.8 (0.5%). A polyherbal capsule containing a mixture of R. officinalis, S. officinalis, and S. aromaticum methanol extracts was formulated successfully. The formulation demonstrated a dosedependent mortality to the brine shrimps. The formulation exhibited less than 50% mortality at concentrations below 500 µg/mL. The formulation demonstrated notable anti-oxidant and antibacterial activity Conclusion: An optimal polyherbal capsule containing R. officinalis (59.5%), S. officinalis (40.0%), and S. aromaticum (0.5%) methanol extracts, with antimicrobial and anti-oxidant properties was formulated successfully by calculating ΣFICI and using Design of Experiment (DOE).