Modification of MTT colorimetric bacterial assays and investigation of antibacterial compounds from selected Myrtaceae plants : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University, Manawatū, New Zealand
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2024-11-21
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Massey University
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Abstract
This thesis presented a pivotal component of a comprehensive bioactivity screening project conducted by our research group, focusing on the improvement and validation of established antibacterial techniques. This investigation aimed to enhance various 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)-based bacterial assays, resulting in the attainment of several noteworthy accomplishments, which include: (1) the development of an equation that establishes a correlation between colony-forming units (CFU) and the quantity of formazan converted by bacteria, enabling accurate calculation of viable bacteria number and precise measurement of the colorimetric microdilution assay; (2) a modification to the existing bacterial MTT assay protocol; (3) an enhanced direct TLC-bioautography protocol tailored for osmotically vulnerable bacteria; and (4) a suggested method for determining the minimum effective dose (MED) using direct TLC-bioautography.
The efficacy of these improved protocols was confirmed through the utilization of feijoa (Feijoa sellowiana) fruit peel and steam-distilled mānuka (Leptospermum scoparium) leaves and branches, which are two locally available waste materials from the Myrtaceae family. The antibacterial components, flavone and grandiflorone, were targeted using direct TLC-bioautography, and subsequently identified using UHPLC-HR-MS in conjunction with Compound Discoverer. These compounds were then purified for NMR verification. To assess their antibacterial properties, the direct TLC-bioautography-based MED determination method, as proposed in this study, was employed, and accompanied by conventional antibacterial assay for comparison. The results have shown that the MED of flavone was 4.2-5.2 μg/cm² against the S. aureus and 5.2-6.1 μg/cm² against the E. coli. Similarly, the MED of grandiflorone was observed to be 0.29 - 0.59 μg/cm² against S. aureus and 2.34 - 4.68 μg/cm² against E. coli.
Untreated New Zealand and Chinese mānuka samples were also studied for comparison to the steam-distilled samples. A total of 22 compounds were identified through these samples, including five β-triketones, thirteen flavonoids, three phloroglucinol derivatives, and one exotic plant growth retardant only presented in Chinese mānuka. In addition to three volatile β-triketones, there is little difference in the main chemical composition between steam-distilled and untreated New Zealand mānuka. However, most of the compounds identified from New Zealand mānuka, especially β-triketones, were not present in Chinese mānuka.
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MTT assay; Formazan; HPLC; LC-MS; Viable count; Staphylococcus aureus; Escherichia coli; TLC-bioautography; Compound Discoverer; NMR; antibacterial activity; Feijoa sellowiana; Mānuka; Leptospermum scoparium; Flavone; Grandiflorone, Bioactive compounds, Analysis, Myrtaceae, Microbiology, Antibiotics, Colorimetric analysis