Institute of Food Research

Health benefits of dietary polyphenols – current evidence for plausible mechanismsevidence for plausible mechanisms

Paul KroonPaul KroonPolyphenols & Health Group

Pl t N t l P d t & H lth PPlant Natural Products & Health Programmepaul.kroon@ifr.ac.uk

3rd Serbian Conference on Dietary Supplements, 25-27 November 2011, Belgrade

N&NUH

Norwich Research Park

Institute of Food Research N&NUH

UEAJohn Innes

CentreInstitute of

GenomeAnalysisInstitute of

Food ResearchAnalys sCentre

BUPA PBL

Presentation overview

Flavonoids in plants, foods & diets

Health benefits of flavonoid consumption:- Evidence from epidemiological & intervention studies

Flavonoid absorption and metabolism• Pharmacokinetics• Phase 2 metabolism• Phase-2 metabolism

How should we establish evidence for the mechanisms underlying health benefit?underlying health benefit?

Mechanisms – an example using transcriptomics

Conclusions, future priorities

PART 1

Flavonoids in plants, foods and dietsFlavonoids in plants, foods and diets

Classification of flavonoids

OHO

OH

BOHO

OH

OHO

OH

O

OOH

HOA C

O

OOH

OH

HO O

OOH

HO

OOH OOH OOH

Flavones Flavonols Flavanones

OHO

OH

OHO

OH OHO

OH

OH

OH

OH OOH OH

Catechins Anthocyanidins IsoflavonesCatechins(flavan-3-ols)

Anthocyanidins(anthocyanins)

Isoflavones

FLAVONOLS (Quercetin, kaempferol)

FLAVONES(Apigenin, luteolin, tangeretin)

FLAVANONES(Naringenin, hesperetin)

ANTHOCYANINS - Cyanidin(pelargonidin, delphinidin)

ISOFLAVONES –D id i i t i l it i

FLAVAN-3-OLS (CATECHINS) –(-)-Epicatechin, (+)-catechin, Daidzein, genistein, glycitein( ) Epicatechin, ( ) catechin,Gallocatechins, galloyl-catechinspro(antho)cyanidins

SIMPLE PHENOLICSHYDROXYBENZOIC ACIDS

HYDROXYCINNAMATESe.g. Ferulic, p-coumaric, sinapic acids

HYDROXYBENZOIC ACIDSe.g. Gallic acid

Esterified soluble formse.g. Caffeic acid-quinic acid esters

Esterified insoluble forms (bound)e g Ferulic acid esterified to cereal fiber

OTHERSe.g. Hydroxytyrosol

e.g. Ferulic acid esterified to cereal fiber

SupplementsSupplements

Flavonoid contents / concentrations in foods

2-3 mg per cup

QUERCETIN

~90 g / kg in cocoa beans5 g / kg in dark chocolate

2.5-20 mg per 100g FW(dessert low, cider high)

~ 5 g / kg in dark chocolate30-50 mg per 100 g FW(yellow and red)

>500 mg / kg FWf id l

57 mg per 100 gin fresh fruitHollands et al (2008) of cider apples

Estimating flavonoid intakes

The future…. online databases

PhenolExplorer• Developed in Augustin Scalbert’s group(INRA, Clermont-Ferand, France)(INRA, Clermont Ferand, France)

• Comprehensive composition data for polyphenols in foods• Data from peer-reviewed publications• Fully searchable on-line database( h l l )(www.phenol-explorer.eu)

EuroFIR BASIS• Developed within the EuroFIR NoEDeveloped within the EuroFIR NoE(http://www.eurofir.net/index/)

• Composition and biological activity data for plants / plant foods• Critically evaluated published data

C f ith E FIR lit t d d (+ L L l t li t)• Conforms with EuroFIR quality standards (+ LanguaL, plant list)• Fully searchable on-line database(http://www.polytec.dk/ebasis/)

PART 2

Health benefits of flavonoid consumptionHealth benefits of flavonoid consumption

Evidence from:

Epidemiologicalstudies

Interventionstudiesstudies studies

OHO

OH

O

OOH

OH

HO

Flavonoids andother phenols

Flavonoid / phenolic-rich foods beveragesother phenols rich foods, beverages

EpidemiologyEvidence of health benefit

Dietary FLAVONOLS and cardiovascular disease:

Meta analysis: Huxley & Neil (2003) Eur J Clin Nutr 57 904 8Meta-analysis:- Huxley & Neil (2003) Eur J Clin Nutr 57, 904-8

• 7 prospective cohort studies included (1993-2001; 107,000 subjects)

• 2087 fatal CHD events2087 fatal CHD events

• Most flavonols from tea, onions and apples

Adj t d Ri k R ti 0 47 0 89 (6) d 1 6 (1)• Adjusted Risk Ratios 0.47-0.89 (6) and 1.6 (1)

• Combined RR for highest tertile vs lowest = 0.80 (95% CI = 0.69-0.93)

Conclusion:

High flavonol intakes may be associated with ↓ risk from CHD mortalityHigh flavonol intakes may be associated with ↓ risk from CHD mortality

Epidemiology - flavanol intake and CVD

Prospective studies on CATECHIN intake and risk of CVDs:

-------------------------------------------------------------------------------------------------------------------------------High vs Outcome # cases Adjusted RR P for

Country low intake (high vs low) trend--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------US1 74.8 vs 3.6 CAD 767 0.85 (0.67, 1.07) -Netherlands2 124.0 vs 25.3 CAD 90 0.49 (0.27, 0.88) 0.02Netherlands 124.0 vs 25.3 Incident stroke 88 0.92 (0.51, 1.68) 0.75--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------1 Arts et al. (2001) Epidemiology 12, 668-675.2 Arts et al. (2001) Am J Clin Nutr 24, 227-232.

Li it d d t il bl• Limited data available• The Zutphen Elderly Study showed an inverse relationship betweencatechin intake and CAD but not stroke

• A 7 5mg ↑ intake of catechins not from tea associated with 20% reduction• A 7.5mg ↑ intake of catechins not from tea associated with 20% reductionin CAD mortality (p = 0.114)

‘Fl id i t k d CVD t lit ti t d i t l ’

Epidemiologic studies‘Flavonoid intake and CVD mortality: a prospective study in post-menopausal women’Mink, Scrafford et al. (2007) Am J Clin Nutr 85, 895-909.

• 34,489 women from Iowa Women’s Health Study

• Used USDA databases → All flavonoid classes included

• Quintiles of flavonoid intake versus CVD, CHD, stroke, total mortality

Food sources grouped into frequency categories• Food sources grouped into frequency categories

AnthocyaninsRel. Risk (95% CI)

------------------------------------------CHD: 0 88 (0 78 0 99)CHD: 0.88 (0.78-0.99)CVD: 0.91 (0.83-0.99)All mortality: 0.90 (0.86-0.95)

Rel. Risk (95% CI)--------------------------------------

Flavanones

Apples/pears CHD --------------------------------------CHD: 0.78 (0.65-0.94)

Flavones

Apples/pears CHD,Red wine CVD

Grapefruit → CHD

→

Rel. Risk (95% CI)--------------------------------------CHD: 0.78 (0.65-0.94)

Strawberries → CHDChocolate → CHD

EpidemiologyFLAVONOID SUBCLASSES and incident hypertension

‘Habitual intake of flavonoid sub-classes and incident hypertension in adults’Cassidy, O’Reilly, Rimm et al. (2011) Am J Clin Nutr 93, 338-347.

• 87,242 women from Nurses Health Study II, 46,672 women from the Nurses87,242 women from Nurses Health Study II, 46,672 women from the NursesHealth Study I, and 23,043 men from the Health Professionals Follow-Up Study

• Updated USDA databases → All flavonoid classes included

• Quintiles of flavonoid intake versus CVD, CHD, stroke, total mortality

• 29,018 and 5629 cases of hypertension in women and men, respectively (14 y)

8%[0.92 (0.86-0.98)] Pooled analyses – individual compounds

Apigenin Catechin

[ ( )]-12% in ≤ 60y-4% in ≥ 60 y

- 5% - 6%

Epidemiology

SummarySummary

Data from numerous epidemiological studies support thenotion that increased consumption of dietary polyphenolsprotects against various chronic diseases

but these studies cannot prove cause and effect...but these studies cannot prove cause and effect...

The EFSA health claimevaluation processevaluation process

Fl id fl id i h f d d di l i k t l i f

Flavonoid intervention studies

Flavonoids, flavonoid-rich foods and cardiovascular risk: a meta-analysis ofrandomized controlled trials. Lee Hooper, Paul Kroon, Eric Rimm, Jeffrey Cohn,Ian Harvey, Kathryn Le Cornu, Jonathan Ryder, Wendy Hall, Aedín Cassidy. Am JClin Nutr 88, 38-50.,

• Structured search strategy (MEDLINE, EMBASE and Cochrane databases)• formal inclusion/exclusion, data extraction, validity assessment; and meta-analysis• 133 trials included (RCTs) CocoanOXTM

↑ FMD after acute intake (+3.99%, 95% CI 2.86 to 5.12, 6 studies)chronic intake (+1.45%, 0.62 to 2.28, 2 studies)

↓ Systolic bp (-5.88mmHg, -9.55 to -2.21, 4 studies)

CocoanOX

↓ y p ( g, , )↓ Diastolic bp (-3.30mmHg, -5.77 to -0.83, 4 studies)

↓ Diastolic bp (-1.99mmHg, -2.86 to -1.12, 9 studies)↓ p ( g, , )↓ LDL cholesterol (-0.19mmol/L, -0.24 to -0.14, 39 studies)ISP

↓ LDL (-0.23mmol/L,↑ Systolic bp (+5.69mmHg; 4 studies)↑ Diastolic bp (+2.56mmHg; 4 studies)

↓ LDL ( 0.23mmol/L,-0.34 to -0.12, 4 studies)

Green & black tea, FMD and blood pressureIntervention studies

Dose Acute FMD-----------------------------------------------

0 mg 7.8%100 mg 9.0% (p=0.0113)100 mg 9.0% (p 0.0113)200 mg 9.1%400 mg 9.6%800 mg 10.3%Overall p

Intervention studiesOrange flavanones and cardiovascular health

Design:• Randomised, double-blind placebo-controlled crossover trial• 24 healthy overweight men (age 50 65 y)• 24 healthy, overweight men (age 50-65 y) • Four weeks of daily intervention with:

- 500 mL orange juice (OJ)- 500 mL control drink plus hesperidin (CDH)500 mL control drink plus hesperidin (CDH)- 500 mL control drink plus placebo (CDP).

Outcomes:• Fasted: OJ and CDH reduced dBP compared to CDP (p

Overall, the evidence from clinical trials is strongest for flavanols (cocoa, tea)

and isoflavones…

i.e. for foods / compounds that have been most studied!

PART 3

Flavonoid absorption and metabolismFlavonoid absorption and metabolism

Small intestine epithelial cells

Smallintestine

lumen

Bloodsupply

PP-sugarPP-sugar

CBGActive transport

Sugar

SGLT-1CBG

g+

PPLPH

PPMetabolising

PP-metabolites(glucuronides

+sugar

PP

Metabolisingenzymes

Passive(glucuronides,sulphates)

PP-sugarT l d i bi l

diffusion

To colon and microbialhydrolysis/metabolism

Flavonol glycosides are not present in plasma after oral dosing

IS370

nmba

nce

at

ve a

bsor

b

Q3Glc

Rel

ativ

QQ4’Glc

10 12 14 16 18 20 22Retention time (min)10 12 14 16 18 20 22Retention time (min)Volunteers consumed 200 g onions, blood collected after 1.5 hDay et al. Free Rad Res 35 (2001) 941-952

Dietary quercetin is present in human plasma as sulfate and glucuronide conjugates of quercetin and methylquercetincg j g q y q

• Efficient extraction methods• HPLC with diode array and LC-MS/MS

Q3’SO4-IS

• Authentic standards (synthesised in IFR)• Sensitivity to enzyme hydrolysis

3’MeQ3GlcA

Q3Gl AQ4’GlcAce

No free quercetinor glucosides

QGlcASO4-

Q3GlcAQ3’GlcA

abso

rban

c

QdiGlcA 3’MeQ4’GlcAUV

a

Time

Bioavailability - Polyphenol structure is vital

GenisteinGenistein (9)(9)

Single dose of 50 mg aglycone equivalentSingle dose of 50 mg aglycone equivalentGallic acid Gallic acid CC 4 5 M4 5 M

Manach et al. (2005) Am J Clin Nutr 81, 230-242.

2.002.00

2.252.25

GenisteinGenistein (9)(9)DaidzeinDaidzein (9)(9)Querc Gluc.Querc Gluc. (7)(7)RutinRutin (6)(6)

Cmax:Cmax: 4.5µM4.5µMTmax:Tmax: 90 min.90 min.

1.251.25

1.501.50

1.751.75

ol /

L )

ol /

L )

EGCgEGCg (11)(11)

HesperidinHesperidin (5)(5)NaringinNaringin (6)(6)

CatechinsCatechins (12)(12)

0.750.75

1.001.00

( µm

o( µ

mo

AnthocyaninsAnthocyanins (6)(6)gg ( )( )

0.000.00

0.250.25

0.500.50

Literature survey, Literature survey, (x)(x) =Number of studies=Number of studies

0.000.00

00 22 44 66 88 1010 1212 1414 1616 1818Time (h)Time (h)

PART 4

How should we establish evidence for the mechanisms underlying health benefit?

The classic response to epidemiological findings…

So dietary quercetinprotects against CVD…

How does it do that?

Paul A Kroon et al. (2004) How should we assess the effects of exposure todietary polyphenols in vitro? Am J Clin Nutr 80 15 21dietary polyphenols in vitro? Am J Clin Nutr 80, 15-21.

To investigate the possible effects of diet-derivedflavonoids on the vascular system we need to know...y

The nature of the flavonoids or

metabolites in bloodTheir

concentrations,etabo tes b ood ,over time

Th i bilit tTheir ability toinfluence cell

function

Impact on cell function - mechanisms

Endothelium:• Lines inner side of blood vessels• Large secretory tissue (720g in human)

P d i di t (NO ET 1

Human Umbilical Vein Endothelial Cells

• Produces various mediators (NO, ET-1,prostacyclin, prostaglandin) that areimportant for haemostasis & fibrinolysis,and regulation of vascular tone.g

• Expresses adhesion molecules involvedin recruitment and binding of monocytes

• Endothelial dysfunction reflects animbalance in the production of mediatorsimbalance in the production of mediators

We have investigated:Adhesion molecules (ICAM 1 VCAM 1 E selectin)• Adhesion molecules (ICAM-1, VCAM-1, E-selectin)

• Inflammatory cytokines (IL6, MCP1)→ inflammatory status

• Vasomodulators (ET-1, cGMP, iNOS, eNOS)→ vasomodulator balance

The three major metabolites inhibited VCAM surfaceexpression in HUVECs at physiological concentrations

150

expression in HUVECs at physiological concentrations

100MFI

)

50CA

M-1

(%M

* * * *

0

50

VC

****

0C I Q Q3'S Q3GlcA IR3GlcA Qmet

2 µmol/L 10 µmol/L

Tribolo et al. (2008), Atherosclerosis 197, 50-56.

None of the physiological metabolites of quercetinretained the ability to inhibit prostaglandin E2 productiony p g p

(stimulated peripheral monocytes)

Loki et al. (2008), J Agric Food Chem 56, 3609-3615Loki et al. (2008), Biochem Pharmacol 75, 1045-1053.

Quercetin-3’-sulfate, but not other quercetin metabolites,retained the ability to inhibit leukotriene B4 synthesis

Effect of Quercetin and its Metabolites

y 4 y

Qon LTB4 production in peripheral neutrophils

90100110

Q

5060708090 Q3'Me

Q3'SQ3GluQ3'Me3Gluhi

bitio

n

01020304050 Q3 Me3Glu

% In

h

0 1 2 3 4 5 6 7 8 9 10-10

0

[Polyphenols] (M)

Loki et al. (2008), J Agric Food Chem 56, 3609-3615Loki et al. (2008), Biochem Pharmacol 75, 1045-1053.

All of the metabolites efficiently inhibited F2-isoprostaneproduction by PMS-activated neutrophilsproduction by PMS-activated neutrophils

MeQ

3’MeQMeQ

Q-3’-S

Q 3-GlcA

Q-3-GlcA

Loki et al. (2008), J Agric Food Chem 56, 3609-3615Loki et al. (2008), Biochem Pharmacol 75, 1045-1053.

Mechanisms can be demonstrated in vivo

Cocoa flavan-3-ols and cardiovascular health

Effects of cocoa beverage on FMD (A), nitric oxide (B), and plasma flavanols (C)

Schroeter et al. (2006) PNAS 103, 1024-9.

• FMD and NO effects appeared to be transient• And were correlated with plasma peak [total epicatechin] and especially[epicatechin and its glucuronide]P re epicatechin elicited similar responses (n 3)• Pure epicatechin elicited similar responses (n=3)

• Vasodilation effects of metabolites confirmed with rabbit aortic rings• Role of NO confirmed using i.v. infusion of L-NMMA (NOS inhibitor)

Using transcriptomics to identify mechanisms

Effects of dp3.9 on HUVECs gene expression profiles

dp3.9 vs DMSO dp3.9 (45’) + TNFα (6h) TNFα vs DMSOvs control

----------------------------------------------------------------------------------------------------------Selected probes 1318 (2.4%) 1036 (1.9%) 2220 (4.0%)

Downregulated 628 572 509(0.09-0.7, 356) (0.1-0.7, 352) (0.09-0.7, 393)(0.09 0.7, 356) (0.1 0.7, 352) (0.09 0.7, 393)

Upregulated 690 464 1711(1.5-13.0, 258) (1.5-11.2, 221) (1.5-95, 1167)

----------------------------------------------------------------------------------------------------------

• No substantial and significant effects of (-)-epicatechin or procyanidin B2

M d 3 9 i d d h d / TNF• Many dp3.9-induced changes occurred + / - TNFα

• 8/8 gene changes confirmed by qRT-PCR• Significant changes in genes and pathways concerned with angiogenesis, cellproliferation, apoptosis, cell growth, and maintenance of vascular tone

Garcia-Conesa et al. (2009), Mol Nutr Food Res 53

Effects of apple procyanidins on migration activity

Vehicle control Procyanidin (dp 3.9)

4 h

24 h

48 h48 h

Vascular Endothelial Growth Factor

• Is the most potent regulator of i iangiogenesis

• Is a protein that stimulates vascular endothelial cell growth, survival and g ,proliferation

• It binds to specific VEGF receptors on the endothelial cell surfacecell surface.

Conclusions

Flavonoids and other polyphenols have good potential to be effectivefunctional food ingredients and nutraceuticalsfunctional food ingredients and nutraceuticals

- multiple activities- huge range of structures and sources

There are also problems / challenges- poor bioavailability, efficient metabolism- organoleptic propertiesg p p p

Current evidence of health benefit is encouraging- >200 well-designed RCTs

In vitro mechanistic research- must consider metabolism

bl t t i t h l i- amenable to post-genomic technologies(transcriptomics, proteomics, metabolomics, etc…)

Future priorities

Focus on establishing ‘cause and effect’

Randomised double-blind placebo-controlled trials- Very well characterised foods- Work with food technologists to develop placebos for foods- Work with food technologists to develop placebos for foods - Pure compounds versus foods- Sufficiently long studies- Longitudinal measurements for more power and informationg p- Established risk factors and markers- Bioavailability- Mechanism in vivo (if possible)- [Eventually, hard end-point studies]

Complementary in vitro mechanistic studies

Use of omics for new biomarkers, new mechanisms

AcknowledgementsIFR:

David Hughes Sandra TriboloFederica Lodi

University of East Anglia:Aedin CassidyLee Hooper

Federica LodiShikha SahaWendy HollandsGary BrettP l N d

University of NottinghamVince WilsonSunita SuriM i T lPaul Needs

Christina Moyle

University of W Australia (Perth)

Moira Taylor

CSIC, Murcia, SpainMaria Teresa Garcia ConesaUniversity of W Australia (Perth)

Kevin CroftJonathan HodgsonWai Mun Loki

Maria Teresa Garcia ConesaPaco Tomas Barberan

Thank youf tt tifor your attention