Thesis

Isolation, identification and pharmacological evaluation of the uterine actives of ficus exasperata vahl (moraceae) and the development and application of metabolomic techniques in drug discovery

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Awarding institution
  • University of Strathclyde
Date of award
  • 2014
Thesis identifier
  • T13845
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Abstract
  • In the search for new, safe and efficacious uterine active agents, the plant Ficus exasperata was subjected to phytochemical screening and pharmacological analysis. Ethyl acetate and methanolic leaf extracts of F. exasperata were fractionated and purified by a series of chromatographic techniques. The isolation process was guided by in vitro functional uterine assays involving the use of C57Bl/6 female mice. Identification of the active chemical constituents was performed by several spectroscopic techniques which included 1D and 2D nuclear magnetic resonance (NMR) and high resolution mass spectrometry (HRMS). The uterine effects of these compounds were investigated on lidocaine-induced, spontaneous, oxytocin-induced and high KCl-induced contractions using isolated uterine segments of non-pregnant female mice. The activity of different compounds on the amplitude (maximum tension above basal force) and frequency of uterine contractions were simultaneously measured and then statistically analysed. The structure-activity relationships were also examined where possible. These studies led to the identification of some new phytochemical derivatives. Pharmacological assays revealed the presence of both uterine stimulatory and inhibitory constituents. The new pheophytin/pheophorbide derivatives, flavonoids, fatty acids and glycerol derivatives significantly reduced the frequency and amplitude of uterine contraction, while KCl salt, pyrimidine and pheophorbide-b derivatives significantly augmented both spontaneous and agonist-induced contractions. This study has demonstrated that F. exasperata generates secondary metabolites which have proven effective in the significant inhibition of uterine contractions and thus a potential source of new tocolytic agents.;Additionally, uterine stimulatory constituents were also generated some of which may be potential drugs for contraception and/or labour facilitation. Lead compounds generated from this study are the pheophytin/pheophorbide derivatives, pyrimidine derivatives and flavonoid derivatives. The rather low yield of compounds frequently experienced in the course of bioassay-guided phytochemical screening has made it almost impossible to investigate the possible mechanism(s) of action of active fractions and/or compounds. This necessitated study into the development of high throughput analytical tools combining metabolomics and pharmacology in the investigation of the function of drugs. This study involved the application of liquid chromatography coupled to high resolution Fourier transform mass spectrometry (LC-HRFTMS) and proton NMR (1H-NMR) as analytical platforms in the determination of myometrial function. An initial study was performed to determine the success of the method and this was achieved by assessment of mouse myometrial metabolites altered in response to oxytocin and ritodrine. The myometrial tissues and bath fluids were extracted at the peak of activity and subjected to LC-HRFTMS analysis. The use of the bath fluids in this study was an innovative approach in sampling. The resulting data were preprocessed and analyzed via a pair-wise chemometric comparison model. Pathway analyses following metabolite identification confirmed previously known mechanism(s) thus validating the method while revealing new insights and creating knowledge-driven hypotheses for future research. This study therefore enabled the development of a technique which combines metabolomics with in vitro pharmacology for the rapid detection of compelling myometrial metabolites in drug function and was successfully applied in the determination of possible mechanism(s) of the active constituents of F. exasperata.
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DOI
Date Created
  • 2014
Former identifier
  • 1039467

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