Microreactor synthesis of labelled polyphenols : a route to antibacterial modes of action in important hospital pathogens

Betts, Jonathan, Ph.D.

Chemistry
October 2011

Thesis or dissertation


Rights
© 2011 PhD Jonathan Betts. All rights reserved. No part of this publication may be reproduced without the written permission of the copyright holder.
Abstract

The aims of the PhD were to synthesize deuterium labelled epicatechin in a microreactor, discover if theaflavin and epicatechin possess antibacterial action against important hospital pathogens and to determine if synergy exists between polyphenols and between polyphenol and current antibiotics. Other aims were to determine the minimum inhibitory concentrations of theaflavin and a theaflavin:epicatechin combination against the bacteria Stenotrophomonas maltophilia, to synthesize theaflavin and deuterium labelled theaflavin in a microreactor and to link the continuous flow process to antimicrobial testing for future mode of action analysis.

A 2-step microreactor synthesis using in a T-shaped microreactor successfully produced deuterium labelled epicatechin in position 8. Mass spectrometry and H-NMR confirmed deuterium labelled epicatechin had been produced. The 2- step synthesis produced a yield in excess of 90%. A 4-step micro reactor synthesis of deuterium labelled epicatechin, was found to be unsuccessful after step 2 of the synthesis. A 1-step method for the microreactor production of planar epicatechin was also shown to be unsuccessful in a microreactor.

Antimicrobial testing of theaflavin, epicatechin and a 2:1 combination of theaflavin and epicatechin was performed against 4 clinical isolates of MRSA, 6 clinical isolates of Acinetobacter baumannii and 6 clinical isolates of Stenotrophomonas maltophilia. Results from the disc diffusion assay confirmed that epicatechin produced no antibacterial action and theaflavin produced strong antibacterial action. The combination of theaflavin and epicatechin produced higher antibacterial activity than theaflavin alone indicating synergy between the two polyphenols. Minimum inhibitory concentrations (MICs) of theaflavin and the theaflavin:epicatechin combination (2:1) were determined against 9 clinical isolates and one control isolate of Stenotrophomonas maltophilia using a microtiter assay. Results from the microtiter assay used indicated that the MIC for theaflavin was between 400 and 800 μg/mL. The MIC for the theaflavin:epicatechin combination (2:1) was between 200 and 400 μg/mL.

A 1-step microreactor synthesis of theaflavin from epicatechin and epigallocatechin using extracted polyphenol oxidase was shown to be successful, producing high yields. Using the same methodology, the synthesis of deuterium labelled theaflavin was undertaken using epigallocatechin and the deuterium labelled epicatechin. However, this reaction was shown to be unsuccessful. The antibacterial action of microreactor synthesized theaflavin against Acinetobacter baumannii in a continuous flow process was investigated. Bacterial viability was tested using the resazurin indicator method. No viable cells were observed from bacterial samples exposed to greater than or equal to 4 hours of the continuous flow of theaflavin products. This indicated that the theaflavin produced antibacterial action after this time of exposure. At exposure times less than 4 hours, viable cells were detected.

Publisher
Department of Chemistry, The University of Hull
Supervisor
Haswell, S. J. (Stephen John), 1954-; Kelly, S. M. (Stephen Malcolm); Kitney, Stuart Paul; Murphy, Christine
Qualification level
Doctoral
Qualification name
PhD
Language
English
Extent
Filesize: 4 MB
Identifier
hull:5612
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