Polyphenols, cardiovascular disease and health claims Paul A Kroon

Polyphenols, cardiovascular disease and health claims Paul A Kroon Institute of Food Research, Norwich Research Park, Norwich, UK

Overview Regulation on nutrition and health claims in Europe - Purpose, types of claims Evidence of cardiometabolic health benefits - Polyphenols, bioactive peptides - What health claims were accepted? The FP7-BACCHUS project - Addressing the challenges - Developing tools, resources and know how A few final thoughts elusive mechanisms Conference announcement

Something we all had to wake up to

Regulation on nutrition and health claims Before the EC Regulation on nutrition and health claims came into force in 2007: Some voluntary guidelines No legal power to stop misleading claims Different approaches across EU. Key objectives of the Regulation are to: Protect consumers from misleading claims Encourage innovation in the food industry Harmonise rules on claims in the EU allowing free trade.

European Health claims Divided into categories Article 13.1 are those based on generally accepted scientific evidence (e.g. calcium contributes to normal muscle function) Article 13.5 are those based on newer evidence (e.g. cocoa flavanols help maintain endothelium-dependent vasodilation which contributes to healthy blood flow) and / or for which protection of proprietary data is requested Article 14 are those relating to either: Reduction in disease risk (14a) (e.g. plant sterols have been shown to lower/reduce blood cholesterol. High cholesterol is a risk factor in the development of coronary heart disease) Children's health and development (14b) (e.g. iodine contributes to the normal growth of children).

What evidence is there that polyphenols can beneficially affect cardiovascular health?

Polyphenols - Epidemiological evidence Meta-analysis:- Huxley & Neil (2003) EurJ ClinNutr57, 904-8 7 prospective cohort studies included (1993-2001; 107,000 subjects) 2087 fatal CHD events Combined RR for highest vs lowest = 0.80 (95% CI = 0.69-0.93) Flavonoid intake and CVD mortality in post-menopausal women. Mink, Scrafford et al. (2007) Am J ClinNutr85, 895-909. 34,489 women from Iowa Women s Health Study Flavones Flavanones Anthocyanins Flavonoid sub-classes & hypertension in adults A Cassidy et al. (2011) AJCN93, 338-347. Anthocyanidins Proanthocyanidins Flavan-3-ols Flavanones Flavones 10-13% High vs low Increasing anthocyanin consumption Decreasing risk

FLAVONOIDs - intervention studies FMD sbp, dbp LDLchol LDLchol Am J Clin Nutr 88 (2008), 38-50 sbp LDLchol

Significant reductions in: Insulin resistance (HOMA-IR); insulin Mean arterial pressure FMD NO-met EC-met Grassi et al. (2009) J Hypertens 27, 774-781. Schroeter et al. (2006) PNAS 103, 1024-9. Cognitive function Diabetes NAFLD Metabolic syndrome HDL functionality Reverse cholesterol transport

Conclusion: There is good evidence of health benefits associated with consumption of certain polyphenols But: Lack of studies for numerous polyphenols Need more properly controlled studies Placebos often differed more than flavonoid Active metabolites usually not known Little supporting mechanistic information

What evidence is there that bioactive peptides can beneficially affect CVD risk?

Bioactive peptides: Specific protein fragments that have a positive impact on body functions and conditions and may ultimately influence health Kitts DD and Weiler KA (2003). Bioactive proteins and peptides from food sources applications of bioprocesses used in isolation and recovery. Current Pharmaceutical Design 9, 1309-1323. Can be: Naturally present - e.g. in milk, cheese, fermented meat Produced by controlled proteolysis

Substantial reductions in blood pressure reported in animals 4.6-34 mm Hg But most IC 50 values too high (Eight < 10 µm)

Mixed outcomes in humans - Hernandez Ledesma report: 19 studies with +ve effects on blood pressure Max reduction was 14.1 mm Hg 6 studies with no significant effects on blood pressure 1 study reporting significant reductions in total and LDL-cholesterol Hernandez-Ledesma et al. (2011) Adv Colloid Interface Sci 165, 23-35.

What health claims have been approved?

European Health Claims

EFSA received a lot of dossiers! Many were for bioactive peptides or polyphenols

Bioactive peptides Reports of anti-hypertensive effects in animal models: >50 Reports claiming anti-hypertensive effects in human RCTs: >20 Number of successful health claims: ZERO

Olive oil polyphenols and protection of LDL particles from oxidative damage First accepted health claim for polyphenols! Olive oil standardised to 5mg oleuropein and its derivatives provided by moderate amounts of olive oil, can be easily consumed in the context of a balanced diet Article 13.1 In weighing the evidence, the Panel took into account that a well conducted and powered study, and two smaller-scale studies, showed a dose-dependent and significant effect of olive oil polyphenol consumption (for three weeks) on appropriate markers of LDL peroxidation (oxldl), that these results were supported by one short-term and one acute study, and by supportive markers of LDL peroxidation (conjugated dienes, ex vivo resistance of LDL to oxidation) going in the same direction, and that evidence for a biologically plausible mechanism by which olive oil polyphenols could exert the claimed effect has been provided. EFSA Journal 2011;9(4):2033 [25 pp.]. doi:10.2903/j.efsa.2011.2033. Available online: www.efsa.europa.eu/efsajournal

Cocoa flavanols and maintenance of normal endothelium-dependent vasodilation The Panel concludes that a cause and effect relationship has been established between the consumption of cocoa flavanols and maintenance of normal endothelium-dependent vasodilation. The following wording reflects the scientific evidence: Cocoa flavanols help maintain endothelium-dependent vasodilation, which contributes to normal blood flow. In order to obtain the claimed effect, 200 mg of cocoa flavanols should be consumed daily. This amount could be provided by 2.5 g of high-flavanol cocoa powder or 10 g of high-flavanol dark chocolate, both of which can be consumed in the context of a balanced diet. The target population is the general population. Article 13.5

All the other claims for polyphenols were not given favourable opinions! e.g. General antioxidant claims

The problem is clear. Despite literally tens of thousands of scientific reports containing scientific data from studies concerned with the health benefits of polyphenols and peptides, it seems we still don t have the right evidence to support a health claim.

Beneficial effects of dietary bioactive peptides and polyphenols on cardiovascular health in humans Coordinating organisation: Institute of Food Research, Norwich, UK Coordinator: Paul Kroon Co-coordinator: Paul Finglas Project Manager: Dawn Wright / Rachel Berry / Robert Tickner Programme FP7 Cooperation Work Programme: Food, Agriculture and Fisheries, and Biotechnologies Call identifier: FP7-KBBE-2012-6 Proposal No. 312090 BACCHUS

Overall aim of BACCHUS To develop tools and resources that will facilitate the generation of robust and exploitable scientific evidence that can be used to support claims of a cause and effect relationship between consumption of bioactive peptides and polyphenolsand beneficial effects related to cardiovascular health in humans

Key concepts Focus on dietary bioactives with real potential health benefits but difficulties in properly realising potential Target health / disease outcomes that are tractable i.e. established human biomarkers Pay close attention to EU and EFSA - changing regulatory environment - cause and effect principle - learn from NDA panel responses Involve innovative food SMEs directly - Gain from their experiences - Source of valuable materials and technologies - But ensure benefits reach outside the consortium

Key concepts Focus on dietary bioactives with real potential health benefits but difficulties in properly realising potential Target health / disease outcomes that are tractable i.e. established human biomarkers Pay close attention to EU and EFSA - changing regulatory environment - cause and effect principle - learn from NDA panel responses Bioactive peptides and polyphenols - Both have great potential, but poor progress to obtain health claims - For both, the strongest evidence is for effects on vascular health / CVD risk - Bioavailability, placebos = key issues Cardiovascular disease - PASSCLAIM - EFSA guidance on beneficial physiological effects and on acceptable markers / methods Integral Health Claims Advisory Board - Expertise from EFSA / national panels - Expertise from SMEs submitting dossiers - Best practice guidance in/outside project Involve innovative food SMEs directly - Gain from their experiences - Source of valuable materials and technologies - But ensure benefits reach outside the consortium Direct involvement of innovative SMEs - Developing products used as case studies - Support development of placebos - Real potential to submit dossiers - Adds credibility to industry dissemination

Why cardiovascular health? Look at the size of the problem in the EU! Cause of death: 42% 52% Cost of CVD: 196 billion / year Of the total cost: 54% healthcare 24% productivity 22% informal care

What can we do? Major modifiable risk factors for CVD: High blood pressure (hypertension) High blood cholesterol (and other blood lipids) Type 2 diabetes Obesity Physical inactivity Smoking Can be affected by diet

Reducing risk of CVD with diet 1 Established Replacing saturated fat with unsaturated fat Reducing sodium intake Plant stanols/sterols Fish oils Beta glucan

Reducing risk of CVD with diet 2 Emerging Wholegrains Nuts Polyphenols Bioactive peptides Bacchus is investigating the potential for bioactive peptides and polyphenolsto reduce the risk of CVD

Is the characterisation of the food or food component sufficient? The substance f or which the claim is made must be the same as that for which the evidence is provided Control authorities must be able to determine that the substance f or which the claim is made is the same as that for which the claim is authorised The EFSA investigation (Article 13.5 & 14 applications) EFSA addresses the following: The conditions of use of the claim The proposed wording of the claim In case of a request f or the protection of proprietary data, if the claim could have been approved without these data Is the claimed effect beneficial to health? In terms of the validity of the end-point In terms of the size of the effect In terms of benefit for specific EU population groups For reduction of disease risk claims, the effect must relate to a risk factor Is there a cause-effect relationship between consumption of the food or food component and the claimed effect? With particular f ocus on whether the evidence has been obtained in the same population group the claim is intended f or With particular f ocus on the intake required to obtain the claimed effect ONLY IF A CAUSE AND EFFECT RELATIONSHIP IS ESTABLISHED

Research and Technological Objectives: 1. Best practice guidelines for health claim dossiers 2. Development and characterisation of foods and placebos 3. Extension and exploitation of the ebasis bioactives database and exposure tool and its use in research of bioactive consumption and in populations 4. Consequences of human digestion and metabolism on bioactive peptides and polyphenols 5. Establish plausible mechanisms of action 6. Gold standard trials to test the potential beneficial effects of consuming these products on human health

Beneficiaries: 13 Research and Technological Centres: 1. Institute of Food Research (IFR) 2. University of Leeds (UL) 3. The Scientific and Technological Research Council of Turkey (TUBITAK) 4. The Spanish National Research Council (CSIC) 5. NOFIMA A.S. (NOFIMA) 6. University College Cork (UCC) 7. Institute of Medical Research (IMR) 8. University of Bologna (UNIBO) 9. Ghent University (UGENT) 10.VITO N.V. (VITO) 11.Laboratory of the Government Chemist (LGC) 12.University of Helsinki (UHEL) 13.Catholic University San Antonio (UCAM)

Beneficiaries: 15 SMEs: 14.British Nutrition Foundation (BNF) 15.European Food Information Resource (EuroFIR) 16.PAMIDA International Ltd (PAMIDA) 17.Crème Ltd (CRÈME) 18.VALDYCOMER S.A. (VALDY) 19.Industrias Carnicas Vaquero S.A. (VAQUERO) 20.WagrAlim (WagrAlim) 21.Critical Processes Ltd (CPL) 22.Coressence Ltd (CORRES) 23.Laboratorios Admira S.L. (ADMIRA) 24.BIOAESIS s.r.l. (BIOAESIS) 25.KAMUT Europe Enterprises (KEE) 26.BioActor (BioActor) 27.ProDigest (ProDigest) 28.Nutrika (Nutrika)

Work-package 1: Best practice Guide and Health Claims Advisory Board Provide an introduction to the Nutrition and Health Claims Regulation Review relevant guidance documents Review health claims opinions to identify key issues Identify criteria for successful health claims dossiers. Additional resources: Dossier template Human studies design - guidance

Work-package 2 Bioactive-rich foods and placebos 1. Development and characterisation of foods, beverages, extracts and placebos Suitable bioactive content and composition Placebos 2. New methods of analysis Ellagitannins Apple flavanols including procyanidin oligomers 3. Preparation and characterisation of foods for the RCTs

Preparation and validation of a placebo beverage for Aronia juice Stage 1: Stage 2: Stage 3: Stage 4: Characterisation of Aronia juice nutrients (sugars, organic acids, vitamins & minerals) and non-nutrients (total phenolics and anthocyanins) Development of 3 different placebo formulations (same nutrient composition, differences in artificial colours and flavours) Instrumental measurements and sensory testing of formulations selection of final formulation Testing for placebo safety & inertness on platelet function

Effects of placebo on platelets in vivo Baseline versus 4 weeks daily consumption Platelet activation markers Platelet-monocyte and plateletneutrophil aggregates Result: No significant changes in expression of any platelet activation markers (P-selectin, GPIIbIIIa, platelet-monocyte / neutrophil aggregates)

WP3 New tools for exposure analysis ebasis: Bioactive Composition of Foods National Consumption Data New composition and bio-effects data: Fruits Meats Wheat Models: Intakes in population Food Safety Health Claims

WP4 Understanding absorption and metabolism - To develop a BIOAVAILABILITY PLATFORM: Polyphenols: sweet orange skins (Hesperion TM ) Egg bioactive peptides (Tensiocontrol TM ) To understandmetabolism and absorption of: Bioactive meat peptides (CSIC-IATA) Polyphenols from pomegranate(csic-cebas) Polyphenols from Aronia (Uni Helsinki)

IN VITRO Test platform POLYPHENOLS and BIOACTIVE PEPTIDES orange and egg MICROBIAL CONVERSION: SHIME ANALYSIS IN VIVO HUMAN STUDY POLYPHENOLS and BIOACTIVE PEPTIDES Intestinal matrix INTESTINAL TRANSPORT: Caco-2 Orange flavanones Cell medium Extracts Egg peptides BIOACTIVITY: Organ baths

WP5 Mechanisms of Action Investigate mechanisms that can be linked to the observed/expected effects of the bioactives in humans Use known human metabolites or those identified in WP4 Test concentrations that are physiological Use omics approaches to identify non-predicted mechanisms and pleiotropic effects Develop improved / better characterised models

Focus on mechanisms related to effects in humans

WP6 Partners WP6 Objectives To provide direct evidence from 7 high quality RCTs in human volunteers of the effects of specific bioactive peptides & polyphenol-enriched foods on established biomarkers of cardiovascular risk -Proof of efficacy IRE: UCC - RTD NL: BioActor- SME To establish dose responses and determine doses that are UK: IFR- RTD required in CORRESENCE SME CPL order -SME to cause beneficial physiological effects in humans - Effective doses. To develop a toolbox for GCP for use in food-based intervention SERBIA: IMR- RTD Nutrika -SME TUR: TUBITAK - RTD studies investigating cardio-metabolic health SPAIN: CSIC- RTD UCAM- RTD ADMIRA - SME VALDY - SME VAQUERO SME ITALY: UNIBO - RTD KEE SME BIOAESIS -SME To support training in the BACCHUS Project Integrated: 7 RTD centres 9 SME This research is funded by the European Commission (Grant Agreement 312090)

Food Bioactives Product and SME Pomegranate Apples Aronia Sweet orange Polyphenols e.g. ellagintannins, ellagic acid Polyphenols e.g. (epi)catechins, procyanidins(oligomers), Polyphenols e.g. anthocyanins, phenolic acids Polyphenols e.g. flavone glycosides Ellagitannin-rich extracts (ADMIRA) Evesse TM apple granules Evesse TM EPC Evesse TM -OPC (CORESSENCE) Polyphenol-rich chokeberry juice (Aronia Anti-Oxi ) (NUTRIKA) Cordiart TM sweet orange extract (BIOACTOR) Cured pork Eggs, egg white Bioactive peptides (formed during curing process) Bioactive peptides (generated by controlled proteolysis) Cured hamsand loins, fermented sausages, pork-containing pastes (VAQUERO, VALDYCOMER) Tensiocontrol (BIOACTOR)

Product & intended claim Initial evidence Proof of concept studies Doseresponse studies Proof of efficacy trial(s) Cholesterol reduction BP reduction BP reduction

Contact us: Email: paul.kroon@ifr.ac.uk paul.finglas@ifr.ac.uk Bacchus@ifr.ac.uk Website: http://www.bacchus-fp7.eu/

Some final thoughts. 1. Don t stop once you demonstrate an effect Seek evidence to demonstrate cause and effect Better to have detailed knowledge of limited foods / polyphenols than superficial knowledge of many 2. Use non-targeted approaches to guide you to the underlying mechanisms Microarrays RCT and mir-33a Microarrays VEGF as a new molecular target 3. Health claims are the ultimate demonstration of the impact of our research go for it!

Some final thoughts. 2. An approach to explore mechanisms Randomised controlled trials - Dietary intervention studies in humans or animal models - (Placebo) controlled - Effects on established risk factors / biomarkers e.g. cholesterol, BP, etc Non-targeted approaches - Transcript profiling - Endogenous metabolite profiling - Targeted analyses for predicted metabolites - Use of labelled compounds Obtain high quality evidence of the beneficial effects of food polyphenols on cardiometabolic health Identify underlying mechanisms Determine the nature and concentrations of polyphenol metabolites Consistent with demonstrating cause and effect Feed metabolite precursors directly to demonstrate efficacy Test ability of metabolites to alter molecular markers or cellular functions identified in vivo

2 nd Scientific Workshop COST Action POSITIVe FA1403 September 15-16, 2016, Norwich, United Kingdom Inter-individual variation in response to consumption of plant food bioactives and determinants involved

Acknowledgements All the partners and their representatives in the BACCHUS consortium The European Commission for funding BACCHUS (grant number: 312090) The Biotechnology and Biological Sciences Research Council (UK) for additional funding

The Temple of BACCHUS! Larger than the Parthenon! Thank you Location: Baalbek in Lebanon BACCHUS