Why do we expect flavonoids to function as antioxidants in vivo?

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1 Why do we expect flavonoids to function as antioxidants in vivo? Catherine Rice-Evans PhD, DSc, FRCPath Antioxidant Research Group Wolfson Centre for Age-Related Diseases Guy s, King s & St. Thomas s School of Biomedical Sciences King s College, University of London

2 FLAVNIDS: FCUS F MUC CURRENT NUTRITINAL AND TERAPEUTIC INTEREST CARDIPRTECTIN Role for flavonoid-rich dietary components in reduction in risk of cardiovascular disease NEURPRTECTIN Anthocyanin-rich fruit associated with protection against age-related decline in cognitive function CEMPREVENTIN

3 Flavonoids: naturally occurring low molecular wt phenols consisting of 2 benzene rings linked via a heterocyclic pyrone or pyran ring -> patterns and substitutions comprising the sub-classes: Anthocyanin - berries Flavanone - citrus Flavanol - red wine teas chocolate fruit Flavonol - fruit vegetables ydroxycinnamates - most fruit & some vegetables

4 Flavonol e.g. quercetin onion, cranberry, red apple many fruit and vegetables Flavanol e.g. epicatechin red wine, green tea, as procyanidins in apple, chocolate Flavanone e.g. hesperetin Citrus fruit, orange Anthocyanidin C 3 ydroxycinnamate e.g. cyanidin major constituents of dark red fruit berries e.g. raspberries e.g. caffeic acid most fruit especially tomato, apple some vegetables e.g. egg plant grains C +

5 SMALL DIFFERENCES IN STRUCTURE LARGE CANGES IN BILGICAL ACTIVITIES Number and specific positions of groups / nature of substitutions determine whether flavonoids function as: antioxidant, anti-inflammatory, inflammatory, cytotoxic or antimutagenic agents in vitro or in vivo. Antioxidant/pro-oxidant activities Enzyme induction / inhibition Cell proliferation / growth inhibition Lipophilicity / polarity - cellular access

6 PRTECTIVE PRPERTIES F FLAVNIDS AGAINST XIDATIVE STRESS ARE STRUCTURE-DEPENDENT Scavengers of reactive oxygen species- -donating abilities Transition metal chelators catechol requirement? Scavengers of reactive nitrogen species nitric oxide, peroxynitrite etc nitration or oxidation? Non-antioxidant mechanisms - modulation of signaling pathways, gene expression

7 STRUCTURAL REQUIREMENTS FR -DNATING ANTIXIDANT ACTIVITY: ortho-dihydroxy substitution in B-ring 2,3-unsaturation in C-ring 4-carbonyl group Bors et al. 1990; Rice-Evans et al. 1996; A C QUERCETIN B

8 SCREENING FLAVNIDS FR ANTIXIDANT ACTIVITY: INFLUENCE F B-RING STRUCTURE Reduction potentials E 7 Antioxidant activity TEAC CATECLS quercetin epicatechin MNYDRXY B-RING kaempferol hesperetin ALKYLPERXYL RADICAL 1.06 VITAMIN C 0.25 Jovanovic et al. 1998; Rice-Evans et al. 1996

9 STRUCTURAL DETERMINANTS F CYTTXICITY Ease of oxidation catechol vs monophenolic lipophilicity

10 XIDATIN F QUERCETIN Quercetin Damage through adduct formation with proteins, GS, RNA and DNA Quercetin-7-quinone methide o-quinone Quercetin-5-quinone methide

11 STRUCTURAL DEPENDENCE F PERXIDATIVE METABLISM F FLAVNIDS monophenolic B-ring Flav + ferryl radical Flav Phenoxyl radical Flav + GS GS Thiyl radical GS + 2 Reactive oxygen species GSSG Galati et al. 2002

12 WAT S APPENING IN VIV? STRUCTURAL CANGES N ABSRPTIN Influence of conjugation and metabolism on structural parameters governing biological properties

13 MAJR METABLIZING ENZYMES: small intestine / liver / colon Glucosidases ydrolases UDP-glucuronosyl transferases Catechol--methyl transferases Sulfotransferases Esterases Cytochrome P450s TERS: Glutathione-S transferases Quinone reductases

14 Absorption and Biotransformation of Dietary Flavonoids In Vivo SKIN AND BRAIN Stomach ligomers cleaved cells ligomeric Flavonoids Monomeric units jejunum Small Intestine ileum Phase I and II metabolism Portal vein Further metabolism -methylated glucuronides Sulphates Liver Colon glucuronides Kidney Flavonoid Gut microflora Phenolic acids Urine Renal excretion of glucuronides

15 PTENTIAL MLECULAR SITES F METABLIC MDIFICATIN glucuronidation sulphation methylation oxidation cleavage

16 EFFECTS F METABLISM N FLAVNID STRUCTURES IMPLICATINS FR BILGICAL PRPERTIES C methyl-epicatechin C methyl-epicatechin epicatechin-7-b-d-glucuronide C epicatechin-7-b-d-glucuronide C epicatechin-7-b-d-glucuronide C 4 --methyl-epicatechin-7-b-d-glucuronide C 3 C 4 --methyl-epicatechin-7-b-d-glucuronide C 3 C 4 --methyl-epicatechin-7-b-d-glucuronide C 3 C epicatechin A C B ' 2' 3' 4' 5' 6' epicatechin A C B ' 2' 3' 4' 5' 6'

17 STRUCTURAL FACTRS INFLUENCING INTRACELLULAR ANTIXIDANT PRPERTIES Reduction potentials of resulting conjugates Cellular access and partition coefficients Intracellular/extracellular metabolism and structural modifications

18 FLAVNIDS CAN BE EXTENSIVELY METABLISED BY cytp450s -> metabolites with modified biological activities human liver microsomes II Kaempferol 3'-hydroxylation Quercetin Tamarixetin C 3 Quercetin 4 -demethylation Isorhamnetin C 3 Isorhamnetin C 3 No demethylation Breinholt et al. 2002

19 STRUCTURAL CNSEQUENCES F INTRACELLULAR METABLISM quercetin Q 4 --methyl quercetin 3MeQ 3 --methyl quercetin Me 3MeQ Me?? Q demethylation glucuronide/ glucoside C 3 C 3 GS, cys, protein thiol GS, cys, protein thiol?? S Glutathione Glucose

20 nm ? Quercetin 55.4 Querc 56.8? MeQ Relative Abundance RT: [M + + ] + : AU (320 nm) Quercetin 3 --Me-quercetin RT (min) Relative Abundance m/z RT: C 3 [M + + ] + : Spencer et al m/z

21 CLNIC BITRANSFRMATIN WAT S APPENING IN TE CLN? Majority of ingested flavonoids undergo colonic metabolism

22 G. Gibson, University of Reading UK

23 Pathway of the colonic degradation of rutin - implications for properties of in vivo metabolites -Rutinose Deglycosylation Quercetin Further degradation C Rutin Dehydoxylation C 3,4-dihydroxyphenylacetic acid Ring fission, water elimination, dehydroxylation Protocatechuic acid C 3-hydroxyphenylacetic acid Absorption from the colon C 3-(3-hydroxyphenyl)- propionic acid ß-xidation N C C + glycination 3-hydroxyhippuric acid

24 MAJR CLNIC METABLITES 3,4-dihydroxyphenyl acetic acid 3-(3-hydroxyphenyl)propionic acid 3-(4-hydroxyphenyl)propionic acid ydroxybenzoates

25 S D WE EXPECT FLAVNIDS T BE ANTIXIDANTS IN VIV? IT DEPENDS: on what we mean by antioxidation on the extent and structural consequences of conjugation and metabolism

26 BIAVAILABILITY AND METABLISM F FLAVNIDS Less bioavailable than ascorbate and tocopherols MDIFIED by metabolism on absorption Less extensively absorbed and circulating levels in vivo much lower

27 PLASMA LEVELS F FLAVNID CNJUGATES Flavan-3-ol: Wine catechins Procyanidin: Chocolate/cocoa Flavanone grapefruit/orange Anthocyanin berry juices 100 nm 4 um; 0.26 um; 0.7 um < 4 um 100 nm; 147 nm METYL + SULPATE +GLUCURNIDE EPICATECIN SULPATE + GLUCURNIDE NARINGENIN /ESPERETIN GLUCURNIDE ANTCYANIN GLYCSIDES Donovan et al., Keen et al., Baba et al., Ameer et al, Miyazawa et al.

28 IN VIV METABLITE FRMS VERSUS CELLULAR XIDATIVE STRESS Apoptotic Stimulus Caspase-3 acetyl-asp-glu-val-asp p-nitroanilide Pro-caspase-3 p-nitroaniline 0.25 Increase in Absorbance (405 nm) over control cells Control 2 2 (50 µm) All 30µM *** *** EC MeEC EC Gluc 2 2 (50 µm) 405 nm METYLATED METABLITE Lower -donating potential modified catechol group Similar protective effects against oxidative stressinduced cell death GLUCURNIDE METABLITE Marginally lower -donating potential No protective effects against oxidative stress-induced cell death- inaccessibility or substituted A-ring? SPENCER et al. 2001

29 PRTECTIN F NEURNS FRM XIDATIVE STRESS- INDUCED CELL DEAT BY EPICATECIN I II III IV I Control neurons II Neurons exposed to oxidative stress III Control neurons treated with epicatechin IV Neurons pretreated with epicatechin prior to oxidative stress Schroeter et al. 2000

30 CNCLUSINS: BIACTIVITY F FLAVNIDS in vivo MAY NT DEPEND N TEIR ACTIVITIES AS DIRECT SCAVENGERS F REACTIVE XYGEN R NITRGEN SPECIES PER SE BUT RATER N TE INFLUENCE F TEIR IN VIV FRMS N TE MDULATIN F ENZYME / PRTEIN FUNCTINS, INTRACELLULAR CELL SIGNALLING AND RECEPTR ACTIVITIES