Institute of Food Research · Fl id fl id ihf d d di l ik tli f Flavonoid intervention studies...
Transcript of Institute of Food Research · Fl id fl id ihf d d di l ik tli f Flavonoid intervention studies...
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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 [email protected]
3rd Serbian Conference on Dietary Supplements, 25-27 November 2011, Belgrade
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N&NUH
Norwich Research Park
Institute of Food Research N&NUH
UEAJohn Innes
CentreInstitute of
GenomeAnalysisInstitute of
Food ResearchAnalys sCentre
BUPA PBL
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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
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PART 1
Flavonoids in plants, foods and dietsFlavonoids in plants, foods and diets
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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
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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
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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
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SupplementsSupplements
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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
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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/)
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PART 2
Health benefits of flavonoid consumptionHealth benefits of flavonoid consumption
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Evidence from:
Epidemiologicalstudies
Interventionstudiesstudies studies
OHO
OH
O
OOH
OH
HO
Flavonoids andother phenols
Flavonoid / phenolic-rich foods beveragesother phenols rich foods, beverages
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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
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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)
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‘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
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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%
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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...
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The EFSA health claimevaluation processevaluation process
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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)
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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
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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
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Overall, the evidence from clinical trials is strongest for flavanols (cocoa, tea)
and isoflavones…
i.e. for foods / compounds that have been most studied!
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PART 3
Flavonoid absorption and metabolismFlavonoid absorption and metabolism
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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
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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
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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
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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)
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PART 4
How should we establish evidence for the mechanisms underlying health benefit?
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The classic response to epidemiological findings…
So dietary quercetinprotects against CVD…
How does it do that?
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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.
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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
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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
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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.
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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.
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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.
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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.
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Mechanisms can be demonstrated in vivo
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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)
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Using transcriptomics to identify mechanisms
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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)
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• 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
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Effects of apple procyanidins on migration activity
Vehicle control Procyanidin (dp 3.9)
4 h
24 h
48 h48 h
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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.
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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…)
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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
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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
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Thank youf tt tifor your attention