Phytochemicals: Design, Synthesis, Biological Evaluation and Novel Characterisation Methods.

Abstract

The collective theme of this thesis is: Phytochemicals: Design, Synthesis, Biological Evaluation and Novel Characterisation Methods. Contained within this general theme, the research described is divided into two research areas:

1. Development of new analytical methods for biopolymers (soluble fiber) characterisation.

2. Studies on the structure–activity relationship between selected food ingredients (and their semisynthetic analogues) and specific cell lines in order to understand the variables that enhance their bioavailability and biological activity against these targets.

In the context of the application of a new analytical methods, a new approach to bio-polymers (soluble fibre) determination was developed by means of High Performance Thin Layer Chromatography (HPTLC). Quality control is an important aspect in the food industry; however, the lack of rapid and efficient analytical methods frequently hinders adequate and expedient characterisation of foodstuffs. Furthermore, the presence of high levels of soluble fibres is often used to claim prebiotic activity for the food product. However, in the food industry it is common practice to provide technical data sheets wherein information on soluble fibres are given as a difference from other components (fat, protein, etc).

Given that the term “soluble fibre” is specific to certain structure classes e.g. inulins, fructo-oligosaccharides, it is obvious that the characterisation by mass difference is not sufficient to support such claims and a more precise analytical method should be employed. Thus a novel method based on High Performance Thin Layer Chromatography (HPTLC) was developed where samples of Adansonia digitata fruit pulp were used to demonstrate the application of this method to accurately evaluate its soluble fibre content and use this information to gain further insight into its reported prebiotic activity. High Performance Liquid Chromatography (HPLC) was used to validate the results. Samples of Adansonia digitata were also submitted to different enzymatic treatments in order to gain a deeper understanding of the cell wall composition. The polymers released after enzymatic breakdown were hydrolysed to their monomeric components and the resulting sugar mixture analysed through the newly-developed HPTLC method. It was shown that samples of Adansonia digitata dried fruit pulp contained only sucrose, glucose and fructose,

contrary to previous reports where soluble fibre content were assessed by mass difference.

This emphasised the need for more precise methods of composition assessment of fibrerich

foodstuffs and demonstrated our HPTLC method as a valid tool for soluble fibre

determination.

The second part of this thesis focuses on structure–activity relationship between food

ingredients and their semisynthetic analogues on selected targets. Within that research,

new polyphenol analogues were designed, synthesised and evaluated on different targets in

order to determine their potential application as multi-target compounds.

Polyphenols have attracted a strong interest in the field of medicinal chemistry due to their

reported anti-cancer, anti-inflammatory, anti-oxidant, anti-artherosclerotic properties, as

well as neuro- and cardio-protective activities. They have been used in prebiotics,

pharmaceuticals, cosmetics, nutraceuticals and can be extracted from renewable sources

such as tea leafs, grapes, cocoa seeds and vegetables. Unfortunately, due to their inherently

poor solubility profile in aqueous media and rapid in vivo metabolic breakdown, they have

not yet been extensively exploited to their full potential. The aim of this research, was to

find new carriers for those naturally occurring compounds that enhance their

bioavailability, increase their half-life in the blood and facilitate their transport across the

cell membrane which should consequently increase of their biological activity. In the past,

a number of new carriers have been evaluated to address enhance some of these properties

including pegylation, sugar conjugation or inclusion in liposomes. It was therefore decided

to expand the objectives of the project beyond these preliminary studies and thus new

linkers were chosen that were not only passive connection units, but could also serve as

active pharmacophores, thereby expanding the activity profile of the synthesised molecules

and enabling molecular diversity. This was realised by utilising triazole-containing linkers

as the key structural feature of the new analogues

The synthetic design of the new family of compounds was assisted by molecular modelling

and the newly prepared molecules were tested for their activity on eight strains of

dermatophytes and on 5-lipoxygenase (5-LO) – both the isolated enzyme and on cell based assays.

An overall summary of the outcomes and the proposed future works for the projects are presented before the final section which contains the experimental procedures and characterisation data for the new compounds synthesised through these investigations.