The collective bioactivity of dietary flavonoids: importance of specific structural characteristics for cardiovascular benefits
Lead Research Organisation:
University of East Anglia
Department Name: Norwich Medical School
Abstract
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.
Technical Summary
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.
Planned Impact
What is the benefit of this research? 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 findings of this project will aid in the design of future feeding interventions directed at addressing dietary change in the UK for cardiovascular health benefit. Who will benefit from this research? This proposal holds strong government and public relevance as it encompasses research aimed at identifying and validating a family of food components which are potentially beneficial to cardiovascular health. In addition, this work promotes a nutritional strategy to improve cardiovascular health using a 'natural' dietary approach. How will they benefit from this research? The proposed research will potentially help to establish multiple food sources which may reduce CVD risk. This research strategy could reinforce the beneficial health effects of the 5 a day regime, and push future governmental policy, education and marketing in this area; which is important given the current low intake of fruits and vegetables in the UK diet. As the current under consumption of fruits and vegetables is most likely a major contributor to the burden of diet-related ill health, the evidence provided could directly benefit the health and welfare of the population, by 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. Therefore, the findings of this project could be beneficial to the Food Standards Agency and other EU government agencies for developing more informed policies regarding dietary guidelines for the prevention of CVD and improvement of public health. This holds direct government and public relevance as CVD is a condition directly attributed to poor diet and represents the largest cost to health care in the UK. What will be done to ensure they benefit? Dissemination to public & health authorities will advance the overall state of knowledge regarding the findings of this project and the importance of flavonoid-rich foods for health and well being. The knowledge gained through this project will be communicated through annual reports to the BBSRC, ILSI symposiums, nutrition society meetings, media briefings, public presentations, and national and international conferences. Who else will benefit from this research? 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 flavonoid-rich processed foods and could provide a unique opportunity to improve marketing strategies. The results of this study are relevant to agricultural industries as levels and types of flavonoids in food crops can be easily modified 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 of a number of flavonoids will also aid in establishing functional health claims for many products already high in flavonoids. Dissemination to industry and government agencies is planned through presentations to the DRINC partners, Biotech, Life-science, food and agricultural industries (including ILSI Europe), planned workshops (i.e., BBSRC/DRINC consortium, BNF, FSA) and industry led seminars (i.e, Leatherhead Foods Int. training conferences and supporting partner workshops, including GSK and Unilever).
Publications
Warner EF
(2016)
Common Phenolic Metabolites of Flavonoids, but Not Their Unmetabolized Precursors, Reduce the Secretion of Vascular Cellular Adhesion Molecules by Human Endothelial Cells.
in The Journal of nutrition
Warner EF
(2017)
Signatures of anthocyanin metabolites identified in humans inhibit biomarkers of vascular inflammation in human endothelial cells.
in Molecular nutrition & food research
Skinner M
(2013)
Bioactives in Fruit - Health Benefits and Functional Foods
Schär MY
(2015)
Orange juice-derived flavanone and phenolic metabolites do not acutely affect cardiovascular risk biomarkers: a randomized, placebo-controlled, crossover trial in men at moderate risk of cardiovascular disease.
in The American journal of clinical nutrition
Kay CD
(2017)
Anthocyanins and Flavanones Are More Bioavailable than Previously Perceived: A Review of Recent Evidence.
in Annual review of food science and technology
Kay CD
(2015)
Rethinking paradigms for studying mechanisms of action of plant bioactives.
in Nutrition bulletin
Jessica Di Gesso (Author)
(2013)
Metabolism of dietary flavnoids alters their effect on tumor necrosis factor-alpha
Description | Parent/unmetabolised flavonoids at physiologically relevant concentrations were less active across the screening model than their respective metabolites, namely phenolic acid conjugates (methyl, glucuronide, sulfate) of protocatechuic acid, vanillic acid and benzoic acid. Specifically, conjugates of protocatechuic acid, vanillic acid and benzoic acid reduced LPS induced TNF-? protein secretion in monocytes, while in vascular endothelial cells, they inhibited TNF-? induced VCAM and increased basal eNOS. Interestingly, in LPS stimulated monocytes, when these metabolites were treated as mixtures of compounds, they were often as active, and in some cases more active than when used in isolation. Metabolites were also active on IL-1ß production in monocytes and HO-1 production in vascular endothelial cells, however, only 4-hydroxybenzoic acid actively induced transcription factor Nrf2 at dietary achievable concentration and no compounds were active on NF-?B pathway inhibition. |
Exploitation Route | The compounds investigated are found in abundance in commonly consumed fruits and vegetables in the UK. As these compounds are found in such a diverse array of fruits and vegetables, beverages and other processed foods, the outputs from this project have widespread implications at both at the industrial and agricultural level. Evidence gathered from this study will help us to identify which natural products found within the U.K. diet have specific beneficial effects on human health (e.g. anti-inflammatory, lowing CVD risk, and the development of a healthy ageing phenotype). This will allow the development and informed marketing of products with known quantities of these bioactive components as 'functional-foods' for health and wellbeing. In addition, ascertaining the combined bioactivity of flavonoids will offer commercial benefit by establishing their functional significance, thus providing a foundation of evidence for future health claims for many products. By assessing the effects of combinations of these compounds and their metabolites we can also help inform future clinical intervention studies by having already established the safety and bioactivity in cell/animal models. These data could help lead to the development of large-scale, long-term dietary interventions to determine the exact of effects these natural compounds have on chronic disease outcomes. Furthermore, findings from this study will provide evidence on the benefits to public health of consuming particular flavonoid rich items (e.g. berries, tea, apples) already prevalent in the U.K. diet; which is important given the current low intake of fresh fruits and vegetables in the UK. Ultimately, the evidence provided from this research could be used to establish optimal dietary levels, safety, efficacy and marketing strategies, and help regulatory agencies develop more specific policies regarding dietary guidelines for CVD prevention. The project is currently providing valuable information regarding the bioactivity of important, dietary available, flavonoids and their metabolites as potential cardioprotective agents. This will ultimately provide valuable evidence for academics, supporting the importance of studying the bioactivity of dietary flavonoids and metabolites both individually and collectively (i.e., in combination). This project is of particular importance to phytochemcial researchers as the study design outlines an experimental approach for defining the active compounds responsible for the health benefits of certain foods; an approach which may be applied to future studies on other food-based bioactives. Establishing levels and efficacy of bioactive flavonoids and metabolites will also help determine optimal dietary levels, and clinical endpoints for use in future interventions aimed at establishing safety and bioactivity in cell/animal models. These findings will ultimately help the design of large-scale, long-term dietary interventions to determine effects on chronic disease outcomes. |
Sectors | Healthcare Pharmaceuticals and Medical Biotechnology |
Description | BBSRC DRINC Travel Fund Award |
Amount | £5,000 (GBP) |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 03/2013 |
End | 06/2013 |
Description | BBSRC DRINC round-3 award |
Amount | £357,230 (GBP) |
Funding ID | BB/I006028/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 01/2010 |
End | 10/2013 |
Description | BBSRC Doctoral Training ICASE Partnership - Norwich Research Park - Partner Unilever |
Amount | £115,000 (GBP) |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 09/2016 |
End | 09/2021 |
Description | BBSRC Response Mode |
Amount | £497,277 (GBP) |
Funding ID | BB/N008448/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 03/2016 |
End | 03/2019 |
Description | Norwich Rsearch Park Enterprise and Engagement award, 2014 |
Amount | £3,000 (GBP) |
Organisation | Norwich Research Park |
Sector | Private |
Country | United Kingdom |
Start | 09/2014 |
End | 10/2015 |
Description | Medicinal Chemistry Collaboration UEA |
Organisation | University of East Anglia |
Department | School of Chemistry |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | outputs and publications derived from a previously funded BBSRC DRINC proposal exploring the activity of flavonoid metabolites led to contact and collaboration in the development of a BBSRC full response mode application |
Collaborator Contribution | collaboration in the development of a successful BBSRC full response mode application |
Impact | successful BBSRC full response mode application |
Start Year | 2014 |
Description | PepsiCo Collaboarative Meeting at Cranfield University, Oct 2014 |
Organisation | PepsiCo |
Country | United States |
Sector | Private |
PI Contribution | Proposal outlined to members of PepsiCo research and nutrition divisions exploring development of processing technology |
Collaborator Contribution | Met for a 4 hour meeting to explore future engagement and TSB application |
Impact | None as of yet |
Start Year | 2014 |
Description | School of Biology University of East Anglia, |
Organisation | University of East Anglia |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Collaboration formed during the course of this project exploring transcription factor activation; ultimately improving analysis efficiency, and increasing potential outputs. |
Start Year | 2012 |
Description | Unilever collaboration for ICASE studentship |
Organisation | Unilever |
Country | United Kingdom |
Sector | Private |
PI Contribution | Data and publications following BBSRC Project exploring the activity of flavonoid metabolites led to both parties approaching one another to develop a collaboration which initiated with the application of a BBSRC ICASE studentship |
Collaborator Contribution | intellectual and contribution to part-fund the studentship and consumables. Student interviews still to commence |
Impact | successful studentship application to the UEA NRP DTP ICASE awards; Unilever visit to UEA with presentation of seminar to the Department of Nutrition |
Start Year | 2015 |
Description | University of Dundee, RW |
Organisation | University of Dundee |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Collaboration formed during the course of this project exploring the effects of flavonoids upon transcription factor activation using a high throughput microplate system; ultimately improving analysis efficiency, and increasing potential outputs and therefore dissemination capacity. |
Collaborator Contribution | Method optimisation |
Impact | Helped establish optimised high-throughput in-house method |
Start Year | 2012 |
Description | Internal Postgraduate Research Day Poster presentation to the University of East Anglia, Norwich |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Postgraduate students |
Results and Impact | 50 PhD students attended an Internal UEA Postgraduate Research Day where we a Poster 'Targeting the underlying mechanisms of flavonoids cardiovascular bioactivity' was presented by PhD student Emily Warner no actual impacts realised to date |
Year(s) Of Engagement Activity | 2013 |
Description | Invited Keynote presentation, Wild Blueberry Association (WBANA) 16th Annual Summit, Bar Harbor, Maine, 2014 |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Other academic audiences (collaborators, peers etc.) |
Results and Impact | 40 academics, marketing, industrial research and distribution employees attended the three day workshop to promote future research, marketing and development of the wild blueberry industry. contributed to future marketing strategies of the wild blueberry association and developed new international collaborations and research funding opportunities |
Year(s) Of Engagement Activity | 2014 |
Description | Invited Seminar to MARS research and the department of Nutrition, University of California, 2014 |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Other academic audiences (collaborators, peers etc.) |
Results and Impact | 35 Academics, PhD students and Research associates attended the seminar 'Establishing the effectiveness of dietary flavonoids in improving health, through understanding the structural activity relationships of their metabolites' Presented to the Department of Nutrition & Mars Research Division Sept 25, 2014, UC Davis, CA. no notable impacts |
Year(s) Of Engagement Activity | 2014 |
Description | Invited Speaker and Session Chair : Experimental Biology, March 28-April 1, Beltsville, MD. - The bioactivity of flavonoids is likely the result of cumulative low exposure to a variety of structurally similar phenolic metabolites. |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Health professionals |
Results and Impact | talk sparked many questions from policy makers |
Year(s) Of Engagement Activity | 2015 |
Description | Invited Speaker: Food Matters Live, November 19, 2015, London UK. Naturally functional |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Public/other audiences |
Results and Impact | talk sparked questions for discussion with industry and KTN attendees unknown |
Year(s) Of Engagement Activity | 2015 |
Description | Invited Speaker: National Berry Health Benefits Symposium, October 13-15, Madison, Wisconsin. Newly discovered bioactive metabolites of berry anthocyanins: potential health effects and implications for future research |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Other academic audiences (collaborators, peers etc.) |
Results and Impact | sparked questions relating to the future of phytochemical research and health claims for foods rich in flavonoids Unknown |
Year(s) Of Engagement Activity | 2015 |
Description | Invited Speaker: Nutraformulate, March 17-18, Birmingham, UK. Exploring the impact of commercial and biological processing on the functionality of flavonoids: a summary of the findings and implications of recent BBSRC DRINC-funded research |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Public/other audiences |
Results and Impact | sparked talks with industry representatives unknown |
Year(s) Of Engagement Activity | 2015 |
Description | Invited Speaker: Wild Blueberry Association of North America (WBANA) 18th Annual Summit, Aug 5-7, Bar Harbor, Maine. Unravelling Bioavailability and Human Health: |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Other academic audiences (collaborators, peers etc.) |
Results and Impact | talk changing way industry markets product unknown |
Year(s) Of Engagement Activity | 2015 |
Description | Oral Presentation BBSRC DRINC 11th Dissemination Event |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | Yes |
Geographic Reach | Regional |
Primary Audience | Other academic audiences (collaborators, peers etc.) |
Results and Impact | 45 academics and members of the food and drink industry attended a two day workshop Future collaboration planned with PepsiCo |
Year(s) Of Engagement Activity | 2014 |
Description | Plenary Lecture - International Workshop on Anthocyanins (IWA), 8th International Workshop on Anthocyanins, September 16-18, 2015, Montpellier France. Newly Discovered Metabolism & Health Effects of Anthocyanins: implications for future research and healt |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Other academic audiences (collaborators, peers etc.) |
Results and Impact | talk sparked questions and discussion regarding future of phytochemical research and development of health claims Unknown |
Year(s) Of Engagement Activity | 2015 |
Description | UEA Medical School Postgraduate Research Day. Norwich, 6 March 2014 |
Form Of Engagement Activity | Participation in an open day or visit at my research institution |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Postgraduate students |
Results and Impact | 40 PhD students and academics attended a poster presentation - UEA Medical School Postgraduate Research Day. Norwich, 6 March 2014 "Exploring the cytoprotective effects of flavonoids and their metabolites in human endothelial cells" PhD student poster presentation by EW NA |
Year(s) Of Engagement Activity | 2014 |
Description | UEA School of Pharmacy Postgraduate Research Day. Norwich, 19 June 2014 |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Postgraduate students |
Results and Impact | 40 PhD students and academics attended an internal research workshop -UEA School of Pharmacy Postgraduate Research Day. Norwich, 19 June 2014 'How does the consumption of dietary flavonoids modulate inflammatory diseases?' PhD student oral presentation by JdG NA |
Year(s) Of Engagement Activity | 2014 |
Description | University of East Anglia internal conference Norwich UK. |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Postgraduate students |
Results and Impact | 60-100 PhD students and academics attended an internal conference at UEA - poster presented on project findings no actual impacts realised to date |
Year(s) Of Engagement Activity | 2012 |