Advanced analysis of polyphenols and phytosterols from grapes and grape byproducts
thesisposted on 07.02.2017, 22:55 authored by Hashim, Siti Nur Nazathul Shima
In the food and nutraceutical industries, increasing emphasis is being placed on value-added products. This high worldwide demand for health-beneficial natural compounds can be met by intensifying the focus on using phytochemicals for enriching food. Epidemiological studies have shown that phytochemicals, including polyphenols and phytosterols, derived from vegetables and fruits are substantial sources of bioactive compounds that are beneficial to human health. The polyphenols phytoalexin (E)-resveratrol has attracted considerable interest because of its remarkable pharmacological activity in the prevention of various human illnesses such as cancer and cardiovascular disease. Several studies have shown that phytosterols such as stigmasterol are useful in the prevention of some cancers, including ovarian, prostate, breast and colon cancers, in addition to serving as a precursor in the manufacture of synthetic progesterone. However, the determination of polyphenols and phytosterols from plants has proven to be an extremely challenging analytical task, hindered by the complexity of the numerous chemicals in natural extracts that necessitates tedious separation methodologies. In this thesis, a new method for the separation and enrichment of health-beneficial polyphenols and phytosterols using green chemical techniques adaptable to analysis and isolation is described. This method utilises new molecularly imprinted polymers (MIPs) in a solid-phase extraction (SPE) format, known as MISPE, for the selective extraction of valuable (E)-resveratrol and stigmasterol from grape pressings, which are an abundant waste product in the wine industry currently used for tartrate and alcohol extraction or compost. MIPs are synthetic materials designed to have selectivity for specific molecular targets and are synthesized from self-assembled pre-polymerization complexes of functional monomers around a molecular template by either thermal- or photo-polymerisation, followed by a post-synthetic template removal. The resulting polymer possesses recognition sites complementary to the size, shape or/and functionality of the chemical or biological template (imprint) molecule. A selectivity evaluation of the recognition sites in MIPs is normally performed in batch-binding and dynamic binding experiments using the templates and template analogues with a non imprinted polymer as control, on the basis of the quantitative information derived from liquid chromatography supernatants or eluates. This evaluation process is frequently limited by the requirement for sufficiently large quantities of the template analogues, which must be chemically synthesised if they are not commercially available. MIPs have many advantages including predetermined selectivity, reusability, low cost of preparation, and stability in highly acidic and basic conditions. The MIPs for the selective capture of (E) resveratrol was synthesised using the non-covalent imprinting technologies and the MIP for stigmasterol proved to be most effective prepared using covalent imprinting. For both systems, comprehensive dynamic binding evaluations in aqueous media were performed using non-target imprinted polymers (NIPs). The optimised (E) resveratrol-templated MIP in the SPE format enabled the capture of the target compound with 99% recoveries and high enrichment factor (IF) from red wine (IF = 32), fresh grape pressing residue extract (IF = 43), aged grape pressing residue extract (IF = 18), and extracts of grape skins (IF = 72) and grape seeds (IF = 26). The stigmasterol-templated MIP achieved 96% recovery and 12-fold enrichment of the target compound from grape seed extract. Finally a new method to enrich the health-beneficial resveratrol, procyanidin and epicatechin as well as stigmasterol and campesteryl glycoside from one single grape pressing feedstock with two MIPs and green chemistry methods was elaborated. Furthermore, a systematic separation methodology for the identification and quantification of polyphenols (twenty compounds) and phytosterols (eight compounds) was developed using reversed-phase high performance liquid chromatography (RP-HPLC) in combination with electrospray ionisation ion trap tandem mass spectrometry (ESI-MS/MS) using environmentally benign mobile phases. This rapid and high-resolution method can be used for binding site characterisation, selectivity optimisation and monitoring of the performance of MIPs intended for solid-phase extraction of bioactive molecules and nutraceuticals from diverse complex mixtures. These investigations demonstrated the application of MISPE for the selective enrichment of health-beneficial compounds from wines and grape byproducts, providing a new, efficient, cost-effective and sustainable approach for utilisation of natural resources.