Are metabolites of flavonoids responsible for the health benefits of fruit and vegetable consumption?

Finding strategies to maintain population health during ageing represents a major challenge. Flavonoids are plant compounds found in fruits, vegetables, berries, wine, chocolate, tea, etc., and scientific reports reveal associations between intake and increased likelihood of surviving to old age in optimal health. In addition, the news, internet and food and health magazines contain countless reports describing the health effects of flavonoid-rich foods. However, flavonoid-rich foods do not have official health claims associated with them and the government does not endorse their increased intake for providing population health benefit. Why is this the case, when flavonoid-rich foods may be able to help delay the onset of age-related diseases and disorders? Oddly enough it's because we actually know very little about how flavonoids work, despite their extensive study. In fact, we are just now forming a basic understanding of their metabolism (i.e., how the body breaks them down and processes them), and the impact this has on their activity. Unfortunately, presently we don't even have the basic tools to study the products of flavonoid metabolism (i.e., commercially available metabolites) and therefore, are unable to establish the extent to which metabolism impacts their biological activity. In the present study, we hope to remedy this by producing flavonoid metabolites using organic synthesis techniques. These metabolites can then be used as a tool for investigating the physiological abundance of flavonoid metabolites, their correlation with dietary consumption (i.e., markers of intake), the impact of metabolism on their activity and their molecular mechanisms of action.

Considering recent reports proposing the importance of flavonoid metabolites (Rodriguez-Mateos et al., 2014), this project is particularly timely, and our group is well-situated to lead this research, as we are one of a few groups globally who are exploring the activity of flavonoid metabolites. After identifying a 40-fold higher abundance of flavonoid metabolites relative to their unmetabolised counterparts (Czank et al., 2012; De Ferrars et al., 2013 & 2014), we have recently been investigating the activity of a select number of these metabolites in vascular and inflammatory cell culture models (di Gesso et al., 2015; Amin et al., 2015). We have observed often greater and differential bioactivity relative to their unmetabolised forms found in food. It's clear that the key to unlocking the functional activity of flavonoids lies with their metabolites. However, flavonoids are metabolised to an extensive array of metabolites, and studying each individually would be a mammoth undertaking; particularly as over 4000 unique flavonoid structures have been identified and one individual flavonoid could be metabolised to form greater than 20-30 distinct metabolic by-products. A technique called structure activity relationship (SAR) analysis, which is commonly used for drug discovery can speed this process along. SAR establishes the data necessary to predict the effect of metabolism on bioactivity without having to test all the metabolites individually. This will provide valuable information required to drive future clinical validation of flavonoids, which is paramount to establishing how these compounds contribute to health.

In this project we will: develop flavonoid metabolites using synthetic chemistry and identify metabolite activity (Objective 1, synthesis and activity screening); further synthetically modify the metabolites to establish the effect of altering their structure on their function (Objective 2, SAR analysis); establish the effects of metabolism on biological mechanism of action by identifying the impact of the active compounds (identified in Objective 1 & 2) on biological pathways associated with inflammation and vascular function.

In our recent vascular and anti-inflammatory bioactivity screen of flavonoids and metabolites (in HUVEC, SMC and THP-1 cells), many common flavonoid metabolites were shown to be bioactive, whereas in most cases, the flavonoids themselves were not. In the present study, we aim to use organic synthesis (both solid and solution phase) to generate common previously identified metabolites (conjugates of hydroxybenzoic, vanillic and ferulic acid) and synthetically modified conjugates (amide and ester) to establish structure activity relationships (SAR), and mechanisms of action.

We will utilise techniques common to drug development, such as ester and amide addition and the incorporation of peptidic and carbohydrate motifs which are likely to result in improved cellular uptake, kinetics and activity. We will establish the SAR of the target conjugates utilising IC50 and a previously validated vascular (in endothelial cells) and anti-inflammatory (in monocytes) activity screen. This synthetic modification and SAR model is required to provide the information needed to predict the effect of metabolism on flavonoid bioactivity, without having to test all known metabolites individually (of which there are over 150 reported to date), which would not be a virtuous use of resources. The target conjugates will be subjected to further mechanistic bioactivity analysis, utilising previously validated in-house methodologies, including cell based analysis of toxicity, vascular and inflammatory pathways. Outputs include VCAM-1, IL-6, HO-1, TNF-a, IL-1B, Nrf2, Akt/PI3K activity, chemokine regulation, p38/TACE activity and functional assays in monocytes and vascular endothelial cells. These cellular targets were predicted from previous screening analysis as the most suitable pathways of focus (BB/I006028/1).

The present project cuts across the Food and Health priorities of the BBSRC and demonstrates that our understanding of the nutritive value of flavonoid- and phenolic acid-rich foods (including such agricultural commodities as berries and cereal crops) has the potential to drive the discovery of alternative preventative strategies and even provide therapeutic leads for drug discovery. This combination of nutrition-focused research with lead therapeutic discovery is a unique focus in health research, addressing the 'bioscience for health' strategic priority of the BBSRC. This proposal therefore holds strong government, industry and public value as it aims to identify and validate the activity of a family of food components which are potentially beneficial to health.

Public. This project will establish mechanistic targets required to inform the design of clinical interventions directed at addressing dietary change in the UK for health benefit. Our assessment of biological impact will focus on markers of vascular health, including vascular reactivity and inflammatory status. These markers are particularly relevant as cardiovascular disease is the leading cause of death in the UK. Therefore, this project could have impact on the health of the populace, potentially reducing future co-morbidity and health care burden, particularly if the outputs lead to the development of diets or products with enhanced nutritive value.

Government. Our assessment of biological impact will focus on markers and pathways associated with vascular health, including vascular function and inflammatory status. These processes are particularly relevant as cardiovascular disease is the leading cause of death in the UK. In addition, the European Food Standards Agency (EFSA) and Department of Health (DOH) requires robust documentation on bioavailability, backed by strong clinical substantiation of bioactivity in order to establish health claims for foods. However, the identification of suitable biomarkers of dietary intake has been recognized as a significant gap in recent nutrition and health research. We aim to provide this foundation of evidence for flavonoid-rich foods, by providing evidence of the potential health impact of flavonoid consumption, leading to the refinement of existing knowledge regarding bioavailability and bioactivity. The findings will therefore be beneficial to government agencies for developing more informed policies regarding dietary guidelines for the improvement of public health.

Industry. This proposal encompasses research aimed at identifying and validating a family of food components which hold strong industrial and agricultural relevance. The research will be of particular benefit to producers of fruits, vegetables and processed foods rich in flavonoids, and could provide a unique opportunity to improve marketing strategies. The outputs of this project may ultimately enhance the competitiveness of UK food industry, potentially leading to increased sales as a result of marketing the health effects of flavonoid-rich foods. In addition, this project will explore how dietary flavonoids exert their health effects through understanding the impact of metabolism on mechanisms of action. The proposed mechanistic endpoints involve activity within the Nrf2 and NF-kB pathways and PI3K and p38 MAP kinase pathways, which play key roles in oxidative stress and inflammation and are important targets for nutraceutical, functional-food and pharmaceutical industries. Alternatively, the structure activity relationship (SAR) analysis could identify novel therapeutic compounds and lead to the development of preventative or therapeutic products.