Dietary exposure biomarkers and metabolic profiling technology to support a standardised validation methodology for functional foods

Herbs have been used as the basis of medicines for centuries. Although there is often only anecdotal evidence linking specific phytochemicals to health benefits there is currently a great deal of interest in the development of foods containing plant 'bioactive' metabolites (functional foods). To improve the possibility of validating health claims associated with any novel food requires a standardised methodology to be place to assess compositional qualities of any food raw materials and to confirm consumption, compound bioavailability and to assess any possibility of differential metabolism in food intervention trials. Salvia sclareoides brot. is a wild species native to the Iberic Peninsula and is currently, being investigated for use in functional foods by ERVITAL, a company specialising in the commercialisation of medicinal plants/herbs. Previous research efforts have studied its composition, targeting compounds such as rosmarinic acid that is likely to provide potential health benefits; however, its metabolome is completely unexplored. Metabolomics methodologies were applied initially to investigate metabolome diversity and stability of of different organs of Salvia sclareoides brot in ERVITAL field trial in Portugal. Initial experiments indicated the metabolomes of harvested rosette leaves and most parts of flowering shoots to be very similar. The field location was also found to have little impact on the metabolome. Metabolome analysis of plant material collected from the field in two different years indicated considerable variability, potentially associated with changeable weather conditions. The impact of environmental stress on the Salvia sclareoides metabolome was proposed to be studied under greenhouse conditions in Aberystwyth using seeds harvested by ERVITAL. The seeds exhibited poor germination rates and so seedlings were vegetatively propagated and allowed to flower and set seed. Due to the low number of replicates more seed was requested and set to grow. The new growing seedlings exhibited substantial morphological differences to the wild transplanted material in Portugal and further examination revealed them to actually be a different species of Salvia (Salvia verbenaca) also growing at the same site. These presented a significantly better germination rate and allowed for the study to continue. Commonly encountered abiotic stresses, such as temperature and drought had no impact on the levels of rosmarinic acid, whilst global metabolome analysis highlighted significant changes. Functional enrichment modelling conducted using the close relative Sesame indicum KEGG ID highlighted 18 potential pathways that were altered with the hot and drought treatments. It was possible to observe that sugar concentration in the drought treatments remained similar to the control while regularly water plants in hot treatment depleted its starch reserves. A food intervention study was designed to evaluate methodology that might be used to validate any health claims associated with the consumption of sage products, using commercially available 'sage' teas approved for consumption in the UK. 3-Methylrosmarinate was found to be a key urinary biomarker for consumption of "sage" related products. Urine samples from a large fruit and vegetable intervention study (FAVRIT) was used to help develop a semi-quantitative method for non-targeted metabolite profiling by LC-MS in order to screen human populations for major metabotype clusters. The method utilized natural chemical structural analogues as internal standards and applied a normalization step in the pre-processing of the urine samples to allow alignment of data sets. It is concluded that metabolite fingerprinting using LC-MS has value for optimising growth conditions to achieve consistent levels of targeted 'bioactive' secondary metabolites from wild plant species. In the case of 'sage' species 3-methylrosmarinic acid can be used to monitor compliance in food intervention trials aimed at validating health claims. LC-MS methodology has been developed that should allow rapid screening of individuals in clinical trials for evidence of any differential metabolism of 'bioactive' that could impact on bioavailability.