The collective bioactivity of dietary flavonoids: importance of specific structural characteristics for cardiovascular benefits

A high intake of fruits and vegetables reduces heart disease risk, but the components of the fruits and vegetables that provide these protective effects remains unclear. Growing evidence supports a role of fruit and vegetable components called flavonoids and research shows that people who consume high intakes of flavonoid-rich foods have improved cardiovascular health. In particular, research shows that some flavonoids and flavonoid-rich foods exert direct protective effects on blood vessel walls, and since cardiovascular diseases (CVD) and specifically stiffness of the arteries results from accumulated damage to blood vessel walls, these data suggest that flavonoids can help restore and optimise vessel function; therefore reducing CVD risk. When researchers measure the amount of any one type of flavonoid in the blood after we eat flavonoid-rich foods, their levels are too low to explain the observed cardiovascular benefit. Therefore, it is likely that the health benefits of flavonoid consumption results from the collective effects of consuming multiple types of flavonoids at the same time. We therefore believe that the health benefits resulting from flavonoid consumption must be the result of many different types of flavonoids working together, as happens when we eat them in our normal diet. Complex combinations of flavonoids are found in our blood and vessels as a result of the consumption of a diversity of flavonoid-rich foods, such as fruits, vegetables, chocolate, juice, wine, and tea. However, although we know that flavonoids are consumed as mixtures of compounds, to date, researchers have studied their impacts in isolation and using levels of flavonoids that are well above that which could be obtained through eating a normal diet. These existing findings are therefore likely to be inaccurate, making it difficult to use these findings to provide dietary advice for the health benefits of flavonoid consumption. We plan to study the cardiovascular benefits of consuming multiple flavonoids as would be consumed in the normal UK diet to provide more accurate data which could be used in the future to provide advice on the true health benefits of flavonoid consumption. Aims and objectives: We aim to use an advanced cell culture model involving both vascular and immune cells to establish if the beneficial effects of flavonoids on cardiovascular health are the result of many types of flavonoid working together. We will also apply flavonoids and their metabolites in both isolation and in combination to provide a more biologically realistic model. These studies will explore the activities of the flavonoids found in the highest abundance in the present UK diet as established using survey data from the National Diet and Nutrition Survey. As this survey established the current UK consumption of fruits and vegetables to be around 3 portions per day on average (and 2 portions below recommended intake levels), we will test the effects of consuming doses relative to 3, 5 and 7 servings of fruits and vegetables per day. Implications: This proposal promotes a nutritional strategy to improve cardiovascular health using a 'natural' dietary approach, involving flavonoids from multiple food sources which hold strong industrial, agricultural and public relevance. This project will provide evidence of the health impact of our current dietary consumption of flavonoid-rich foods, in relation to reaching optimal levels of 5-a-day and above; and will aid in the design of future feeding interventions directed at addressing dietary change in the UK for health benefit. This project will also provide 'proof of concept' for the design of future flavonoid intervention trials by establishing optimal cardiovascular endpoints, flavonoids and doses. Lastly, establishing a shared and cumulative bioactivity of a number of flavonoids will aid in establishing future functional health claims for many products high in flavonoids.

We hypothesise that flavonoids share common biological activities, which are based on structural similarities between species and their reported 'health effects' are the result of multiple compounds working collectively. The bioactivity-directed screening model we have developed will use complimentary cell systems to investigate the structure-activity-relationships of flavonoids (objective 1), flavonoid metabolites (objective 2) and combinations of flavonoids and metabolites (objective 3), focusing on their effects on vascular and inflammatory markers (primary endpoints) and oxidative stress and anti-oxidant gene expression (secondary endpoints) in HUVEC, SMC and THP-1 cells. This model will test the flavonoids catechin, cyanidin, quercetin, naringenin, peonidin and hesperetin, and their common metabolic by-products (13 metabolites) at levels between 0.1 and 100uM. Our treatment matrix consists of 48 treatments across 5 concentrations and 3 replicates, for a combined 720 initial bioactivity screens. The model contains 5 levels of bioactivity: global cellular bioactivity utilising the WST-1 and H2DCFDA assays (Model Level 1); cardiovascular bioactivity relative to vascular, anti-inflammatory and anti-oxidant activity, utilising NADPH oxidase and eNOS bioactivity assays and the LPS induced pro-inflammatory cytokine assay (Model Level 2); biomarker gene (Model Level 3) and protein (Model Level 4) expression for eNOS, NADPH-oxidase, haem oxygenase-1, NQO1, glutathione regulating enzymes TNF-alpha, IL-1beta, tissue factor and VCAM-1; and DNA binding activity of the redox-sensitive transcription factors NF-kappaB or Nrf2 (Model Level 5). At each level of the model, as the more targeted assays become increasingly more time and resource 'expensive', the initial treatment numbers will be refined as the treatments displaying neutral or negative responses will not be taken to the next level of the model.

This project will provide evidence on the cardiovascular benefits of flavonoids as found in a diversity of fruits, vegetables and flavonoid-rich processed foods. The proposed experimental model will utilise the most common flavonoids found in the UK diet and test them at physiologically appropriate concentrations which reflect doses achievable at the current consumption level of the UK public (approximately 3/day), the projected optimal level (5 a day), and above (as recommended by other European and North American countries). Uniquely, we will also examine the biological activity of the main metabolites, which will be synthesised at St Andrews. The results will aid in the design of future feeding interventions directed at establishing common biological activities of flavonoids in fruits, vegetables and flavonoid-rich processed foods to address dietary change in the UK for cardiovascular health benefit. This proposal holds strong government and public relevance encompassing research aimed at identifying and validating a family of food components potentially beneficial to cardiovascular health and promoting a nutritional strategy to improve cardiovascular health using a 'natural' dietary approach. The proposed research will potentially help to establish multiple food sources which may reduce cardiovascular disease (CVD) risk. This strategy could reinforce the beneficial health effects of the 5 a day regime, and guide future governmental policy, education and marketing; which is important given the current low intake of fruits and vegetables in the UK. Current under consumption of fruits and vegetables is most likely a major contributor to the burden of diet-related ill health, so evidence provided by this project could directly benefit the health and welfare of the population, providing valuable data for the design of future large-scale dietary interventions aimed at addressing the issues of fruit and vegetable consumption in relation to the cardiovascular health burden. The findings of this project could be beneficial to the Food Standards Agency and other EU agencies for developing more informed policies regarding dietary guidelines for CVD prevention and improvement of public health. This holds direct government and public relevance as CVD is directly attributed to poor diet and represents the largest cost to UK health care. Dissemination to Public & health authorities will advance the overall state of knowledge regarding the findings of this project, the health effects of dietary flavonoids and importance of flavonoid-rich foods for health and well being. The knowledge gained through this project will therefore be communicated through various channels, including annual reports to the BBSRC, international life Sciences Institute (ILSI) symposiums, nutrition society meetings, media briefings, public presentations and workshops, University websites, local presentations and lectures, and national and international conferences. This proposal aims to identify and validate a family of food components with strong industrial and agricultural relevance. The results are relevant to agricultural industries as levels of flavonoids in food crops can be easily increased using breeding strategies and pre and post harvest manipulation. This project is particularly relevant to the processed food and beverage industry, as many processed foods are extremely rich sources of flavonoids (chocolate, juice, tea, wine) and could easily be marketed as vehicles for the delivery of beneficial mixtures of cardioprotective flavonoids for health. Establishing a shared and cumulative bioactivity for a number of flavonoids will aid in establishing functional health claims for many products already high in flavonoids. Dissemination to industry and government agencies is planned through presentations to DRINC partners, ILSI Europe Annual Symposium, workshops and industry led seminars (i.e, Leatherhead Foods Int. training conferences).