Molecular Aspects of Medicine

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1 Molecular Aspects of Medicine 31 (2010) Contents lists available at ScienceDirect Molecular Aspects of Medicine journal homepage: Review Bioavailability of dietary flavonoids and phenolic compounds Alan Crozier a,, Daniele Del Rio b, Michael N. Clifford c a Plant Products and Human Nutrition Group, Graham Kerr Building, School of Medicine, University of Glasgow, Glasgow G12 8QQ, UK b Human Nutrition Unit, Department of Public Health, University of Parma, Via Volturno 39, Parma, Italy c Food Safety Research Group, Centre for Nutrition and Food Safety, Faculty of Health and Medical Sciences, University of Surrey, Guildford, Surrey GU2 7XH, UK article info abstract Keywords: Flavonoids Chlorogenic acids Ellagitannins Bioavailability Metabolism Small intestine absorption Colonic degradation This paper reviews recent human studies on the bioavailability of dietary flavonoids and related compounds, including chlorogenic acids and ellagitannins, in which the identification of metabolites, catabolites and parent compounds in plasma, urine and ileal fluid was based on mass spectrometric methodology. Compounds absorbed in the small intestine appear in the circulatory system predominantly as glucuronide, sulfate and methylated metabolites which seemingly are treated by the body as xenobiotics as they are rapidly removed from the bloodstream. As a consequence, while analysis of plasma provides valuable information on the identity and pharmacokinetic profiles of circulating metabolites after acute supplementation, it does not provide accurate quantitative assessments of uptake from the gastrointestinal tract. Urinary excretion, of which there are great variations with different classes of flavonoids, provides a more realistic figure but, as this does not include the possibility of metabolites being sequestered in body tissues, this too is an under estimate of absorption, but to what degree remains to be determined. Even when absorption occurs in the small intestine, feeding studies with ileostomists reveal that substantial amounts of the parent compounds and some of their metabolites appear in ileal fluid indicating that in volunteers with a functioning colon these compounds will pass to the large intestine where they are subjected to the action of the colonic microflora. A diversity of colonic-derived catabolites is absorbed into the bloodstream and passes through the body prior to excretion in urine. There is growing evidence that these compounds, which were little investigated until recently, are produced in quantity in the colon and form a key part of the bioavailability equation of dietary flavonoids and related phenolic compounds. Ó 2010 Elsevier Ltd. All rights reserved. Contents 1. Introduction Flavan-3-ols Green tea flavan-3-ol monomers Proanthocyanidins Flavonols nion quercetin--glucosides Tomato juice quercetin-3--rutinoside range juice flavanones Isoflavones Corresponding author. address: Alan.Crozier@glasgow.ac.uk (A. Crozier) /$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi: /j.mam

2 A. Crozier et al. / Molecular Aspects of Medicine 31 (2010) Anthocyanins Strawberries and blackberries Blueberries and blackcurrants Accumulation of anthocyanins in body tissues Stability of anthocyanins Ellagitannins Chlorogenic acids Conclusions References Introduction The protective effects of diets rich in fruits, vegetables and derived beverages are due not only to fiber, vitamins and minerals, but also to a diversity of plant secondary metabolites, in particular phenolic compounds and flavonoids. The bioavailability of these compounds after dietary intake has been a topic of increasing research in recent years. Following the ingestion of dietary flavonoids which, with the notable exception of flavan-3-ols and proanthocyanidins, exist in planta predominantly as glycoside conjugates, absorption of some but not all components into the circulatory system occurs in the small intestine (Donovan et al., 2006b). Typically, the absorption at this site is associated with hydrolysis, with release of the aglycone as a result of the action of lactase phloridzin hydrolase (LPH) in the brush-border of the small intestine epithelial cells. LPH exhibits broad substrate specificity for flavonoid--b-d-glucosides and the released aglycone may then enter the epithelial cells by passive diffusion as a result of its increased lipophilicity and its proximity to the cellular membrane (Day et al., 2000). An alternative hydrolytic step is mediated by a cytosolic b-glucosidase (CBG) within the epithelial cells. In order for CBG-catalysed hydrolysis to occur, the polar glucosides must be transported into the epithelial cells, possibly with the involvement of the active sodium-dependent glucose transporter SGLT1 (Gee et al., 2000). Thus, it has been accepted that there are two possible routes by which the glucoside conjugates are hydrolysed and the resultant aglycones appear in the epithelial cells, namely LPH/diffusion and transport/cbg. However, an investigation, in which SGLT1 was expressed in Xenopus laevis oocytes, has indicated that SLGT1 does not transport flavonoids and that glycosylated flavonoids, and some aglycones, have the capability to inhibit the glucose transporter (Kottra and Daniel, 2007). Using Caco-2 cells Johnson et al. (2005) found that glucose uptake into cells under sodium-dependent conditions was inhibited by flavonoid glycosides and non-glycosylated polyphenols whereas aglycones and phenolic acids were without effect. Prior to passage into the blood stream the aglycones undergo metabolism forming sulfate, glucuronide and/or methylated metabolites through the respective action of sulfotransferases (SULT), uridine-5 0 -diphosphate glucuronosyltransferases (UGTs) and catechol--methyltransferases (CMT). There is also efflux of at least some of the metabolites back into the lumen of the small intestine and this is thought to involve members of the adenosine triphosphate (ATP)-binding cassette (ABC) family of transporters, including multidrug resistance protein (MRP) and P-glycoprotein (P-gp). nce in the portal bloodstream, metabolites rapidly reach the liver, where they can be subjected to phase II metabolism with further conversions and enterohepatic recirculation may result in some recycling back to the small intestine through bile excretion (Donovan et al., 2006b). Analysis of ileal fluid collected from ileostomists after the ingestion of various foodstuffs indicate that even when dietary flavonoids are absorbed in the small intestine, substantial quantities none-the-less pass from the small to the large intestine (Kahle et. al., 2005, 2006; Jaganath et al., 2006; Marks et al., 2009) where the colonic microbiota will cleave conjugating moieties and the resultant aglycones will undergo ring fission leading to the production of smaller molecules, including phenolic acids and hydroxycinnamates. These can be absorbed and may be subjected to phase II metabolism in the liver before being excreted in urine in substantial quantities that, in most instances, are well in excess of the flavonoid metabolites that enter the circulatory system via the small intestine (Jaganath et al., 2006; Roowi et al., 2009, 2010; Stalmach et al., 2010a,b). Manach et al. (2005) published a detailed and subsequently much quoted review on the bioavailability of polyphenols in humans. Much of the research covered involved feeding volunteers a single supplement and monitoring the levels of flavonoids in plasma and urine over a 24 h period. As flavonoid metabolites were and, indeed, still are rarely available, analysis almost invariably involved treatment of samples with mollusc glucuronidase/sulfatase preparations and the subsequent quantification of the released aglycones by HPLC using either absorbance, fluorescence or electrochemical detection. Some more recent bioavailability studies have analysed samples directly by HPLC with tandem mass spectrometric (MS) detection without recourse to enzyme hydrolysis. The availability of reference compounds enables specific metabolites to be identified by HPLC MS 2 and MS 3 (Mullen et al., 2006). In the absence of standards it is not possible to distinguish between isomers and ascertain the position of conjugating groups on the flavonoid skeleton. None-the-less, a metabolite that in reality is pelargonidin-3--glucuronide can be partially identified as a pelargonidin--glucuronide on the basis of its MS fragmentation pattern (Mullen et al., 2008b). The use of MS in this way represents a powerful HPLC detection system as, with low ng quantities of sample, it provides structural information on analytes of interest that is not obtained with other detectors. Quantification of partially identified metabolites by MS using consecutive reaction monitoring (CRM) or selected ion monitoring (SIM) is, of necessity, based on a calibration curve of a related compound, which in the instance cited above could

3 448 A. Crozier et al. / Molecular Aspects of Medicine 31 (2010) Fig. 1. Concentrations of (epi)gallocatechin--glucuronide (EGC-GlcUA), methyl-(epi)gallocatechin--glucuronide (4 0 -Me-EGC-GlcUA), methyl- (epi)gallocatechin--sulfates (4 0 -Me-EGC-S), ( )-epicatechin glucuronide (EC-3 0 -GlcUA), and methyl-(epi)catechin--sulfates (Me-EC-S), ( )-epigallocatechin-3--gallate (EGCg) and ( )-epicatechin-3--gallate (ECg) in the plasma of human subjects 0 8 h after the ingestion of 500 ml of green tea. Data expressed as mean values with their standard errors (n = 10) depicted by vertical bars. Note that no flavan-3-ols or their metabolites were detected in plasma collected 24 h after ingestion of the green tea. be pelargonidin-3--glucoside as it can be purchased from commercial sources. In such circumstances, as the slopes of the glucoside and glucuronide SIM dose response curves will not necessarily be identical there is a potential source of error in the quantitative estimates and there is a view that quantitative estimates based on enzyme hydrolysis are, therefore, much more accurate. We do not share this opinion. The glucuronidase/sulfatase preparations are characterised by various enzyme activities and there can be substantial batch-to-batch variation in their specificity (Donovan et al., 2006b). There are no reports of flavonoid bioavailability studies using glucuronidase/sulfatase preparations where information on the identity, number and quantity of the individual sulfate and glucuronide conjugates in the samples of interest has been obtained. As a consequence, there are no direct data on the efficiency with which the enzymes hydrolyse the individual metabolites and release the aglycone. This introduces a varying, unmeasured error factor. The accuracy of quantitative estimates based on the use of glucuronidase/sulfatase preparations are, therefore, probably no better than those based on HPLC CRM/SIM. The fact that enzyme hydrolysis results in very reproducible data does not reflect the validity of the method as it is a measure of precision not accuracy (Reeve and Crozier, 1980). These shortcomings of analyses based on enzyme hydrolysis apply to bioavailability studies with all dietary flavonoids and it is interesting to note that the one publication on the subject to date reports that the use of enzyme hydrolysis results in an underestimation of isoflavone metabolites (Gu et al., 2005). Even if MS is used only to locate and characterise the conjugate and quantification is performed using the UV or visible absorbance an error can still occur when an aglycone is used for calibration because the relevant conjugate is not available. This review concentrates principally on post-2005 human bioavailability studies where metabolites and related compounds were identified by mass spectrometric-based methods without recourse to the use of enzyme hydrolysis prior to analysis. 2. Flavan-3-ols 2.1. Green tea flavan-3-ol monomers Green tea, produced by aqueous infusion of young leaves of Camellia sinensis, is a rich source of several flavan-3-ol monomers, typically, with ( )-epigallocatechin-3--gallate, ( )-epigallocatechin and ( )-epicatechin predominating (Del Rio

4 A. Crozier et al. / Molecular Aspects of Medicine 31 (2010) Table 1 Pharmacokinetic analysis of flavan-3-ols and their metabolites detected in plasma of healthy volunteers following the ingestion of 500 ml of green tea. * Flavan-3-ols (number of isomers) C max (nmol/l) T max (h) (Epi)gallocatechin--glucuronide (1) 126 ± ± Methyl-(epi)gallocatechin--glucuronide (1) 46 ± ± Methyl-(epi)gallocatechin--sulfates (2) 79 ± ± 0.2 (Epi)catechin--glucuronide (1) 29 ± ± 0.2 (Epi)catechin--sulfates (2) 89 ± ± 0.2 -Methyl-(epi)catechin--sulfates (5) 90 ± ± 0.2 ( )-Epigallocatechin-3--gallate (1) 55 ± ± 0.1 ( )-Epicatechin-3--gallate (1) 25 ± ± 0.2 * Data expressed as mean values ± SE (n = 10). Table 2 Quantification of the major groups of flavan-3-ol metabolites excreted in urine 0 24 h after the ingestion of 500 ml of green tea by ten human volunteers. * Flavan-3-ol metabolites (number of isomers) 0 24 h excretion (lmol) (Epi)gallocatechin--glucuronide (1) Methyl-(epi)gallocatechin--glucuronide (1) Methyl-(epi)gallocatechin--sulfates (2) 19.8 (Epi)gallocatechin--sulfates (3) 2.6 Total (epi)gallocatechin metabolites 33.3 (11.4%) (Epi)catechin--glucuronide (1) 1.5 ± 0.3 (Epi)catechin--sulfates (2) 6.7 ± 0.7 -Methyl-(epi)catechin--sulfates (5) 10.9 ± 1.2 Total (epi)catechin metabolites 19.1 (28.5%) Total flavan-3-ol metabolites 52.4 (8.1%) * Data expressed as mean values in lmol ± standard error (n = 10). Italicised figures in parentheses indicate amount excreted as a percentage of intake. et al., 2004). In a recent study, ten healthy human subjects, who had been on a low flavonoid diet for two days, consumed 500 ml of green tea containing 648 lmol of flavan-3-ols after which plasma and urine were collected over a 24 h period and analysed by HPLC MS 2 (Stalmach et al., 2009b). Plasma contained a total of twelve metabolites, in the form of -methylated, sulfated and glucuronide conjugates of (epi)catechin and (epi)gallocatechin along with the native green tea flavan-3-ols ( )- epigallocatechin-3--gallate and ( )-epicatechin-3--gallate. 1 The concentrations of these compounds in plasma after green tea intake are presented in Fig. 1 and a pharmacokinetic analysis of the profiles is presented in Table 1. None of the flavan-3-ols were present in the circulatory system before tea intake, but they were present in detectable quantities 30 min after. The main component to accumulate was an (epi)gallocatechin--glucuronide with C max of 126 nmol/l and a T max of 2.2 h while an (epi)catechin--glucuronide, probably the conjugate, attained a C max of 29 nmol/l with a 1.7 h T max. The unmetabolised flavan-3-ols, ( )-epigallocatechin-3--gallate and ( )-epicatechin-3--gallate, have C max values of 55 and 25 nmol/l after 1.6 and 2.3 h, respectively. The T max values ranged from 1.6 to 2.3 h (Table 1) and all the flavan-3-ols and their metabolites were present only in trace quantities after 8 h and were not detected in the 24 h plasma. This indicates a small intestine absorption, a fact confirmed when a similar flavan-3-ol metabolite plasma profile was obtained after the ingestion of green tea by humans subjects with an ileostomy, having had their colon removed surgically (Stalmach et al., 2010a). Urine collected 0 24 h after green tea consumption by healthy subjects with a functioning colon contained a similar spectrum metabolites of (epi)catechin and (epi)gallocatechin to plasma except for the appearance of two (epi)catechin--sulfates and an absence of unmetabolised flavan-3-ols (Table 2). The overall metabolite excretion was equivalent to 8.1% of the 648 lmol flavan-3-ol intake. However, there was notable distinction between the excretion of (epi)catechin and (epi)gallocatechin metabolites. The recovery of (epi)gallocatechin metabolites was 11.4% while that of (epi)catechin metabolites was 28.5% of the ( )-epicatechin and (+)-catechin intake (Table 2). These high levels of excretion are also in keeping with recoveries obtained in other studies with green tea, cocoa and related products (Baba et al., 2000; Auger et al., 2008; Mullen et al., 2009) confirming that ( )-epicatechin and (+)-catechin, in particular, are highly bioavailable being absorbed and excreted to 1 Analysis of flavan-3-ols and their metabolites is somewhat more subtle than is generally appreciated. For instance, without reference compounds which can be separated by reversed phase HPLC, MS is unable to distinguish between epicatechin and catechin metabolites and also epigallocatechin and gallocatechin metabolites. We, therefore refer to metabolites as (epi)catechins or (epi)gallocatechins. To complicate matters further, although chiral chromatography, using a mobile phase that is not compatible with on-line MS, can separate (+) and ( ) flavan-3-ol enantiomers, they co-chromatograph when analysed by reversed phase HPLC (Donovan et al. 2006a). Although, some degree of interconversion may occur between optical isomers with, for instance ( )- epicatechin forming (+)-epicatechin (Gotti et al. 2006), for simplicity we have assumed that unmetabolised green tea flavon-3-ols detected in teas, plasma and ileal fluid have not undergone such a change.

5 450 A. Crozier et al. / Molecular Aspects of Medicine 31 (2010) H ( )-epigallocatechin gallate (IF) pyrogallol (F,U) H ( )-epigallocatechin (IF) H 1-(3',4',5')-trihydroxyphenyl)- 3-(2'',4'',6''-trihydroxy)propan-2-ol* H H ( )-epicatechin (IF) 1-(3',4')-dihydroxyphenyl)- 3-(2'',4'',6''-trihydroxy)propan-2-ol* pyrocatechol (F,U) ( )-5-(3',4',5')-trihydroxyphenyl - -valerolactone (F,U) ( )-5-(3',4')-dihydroxyphenyl - valerolactone (F) HC 5-(3,4,5)-trihydroxyphenyl- -valeric acid* HC 5-(3,4)-dihydroxyphenyl- -valeric acid (F) HC 4-hydroxyphenylacetic acid (F,U) HC 3-(3-hydroxyphenyl)propionic acid (F,U) HC CH 3 3-methoxy-4-hydroxyphenylacetic acid (U) HC 4-hydroxybenzoic acid (U) HC 3-hydroxyphenylhydracrylic acid (U) HC H N hippuric acid (U) Fig. 2. Proposed pathways involved in the colonic catabolism and urinary excretion of green tea flavan-3-ols. Following consumption of green tea more than 50% of the ingested ( )-epicatechin, ( )-epigallocatechin and ( )-epigallocatechin-3--gallate (blue structures) pass into the large intestine. When incubated with fecal slurries these compounds are catabolised by the colonic microflora probably via the pathways illustrated with red structures. Analysis of urine after green tea consumption indicates that some of the colonic catabolites enter the circulatory and undergo further metabolism before being excreted in urine. Green structures indicate catabolites detected in urine but not produced by fecal fermentation of ( )-epicatechin, ( )-epigallocatechin or ( )-epigallocatechin-3--gallate. The dotted arrow between pyrogallol and pyrocatechol indicate this is a minor conversion. Double arrows indicate conversions where the intermediate(s) did not accumulate and are unknown, although metabolism of 4-hydroxyphenylacetic acid to 3-methoxy-4- hydroxyphenylacetic acid probably proceeds via 3,4-dihydroxyphenylacetic acid. Compounds detected in ileal fluid after green tea consumption (IF); catabolites detected in fecal slurries (F) and in urine (U); potential intermediates that did not accumulate in detectable quantities in fecal slurries( * ).

6 A. Crozier et al. / Molecular Aspects of Medicine 31 (2010) a much greater extent than other flavonoids with the possible exception of isoflavones (Donovan et al., 2006b; Crozier et al., 2009; Manach et al., 2005). Despite the relatively high absorption of flavan-3-ols in the small intestine, Stalmach et al. (2010a) report that after ileostomists drank green tea containing 634 lmol of flavan-3-ols, 69% of the intake was recovered in 0 24 h ileal fluid as a mixture of native compounds and metabolites. Thus, in volunteers with a functioning colon most of the ingested flavan-3-ols will pass from the small to the large intestine where their fate is a key part of the overall bioavailability equation. To mimic these events two sets of experiments were carried out (Roowi et al., 2010). Firstly, 50 lmol of ( )-epicatechin, ( )-epigallocatechin and ( )-epigallocatechin-3--gallate were incubated under anaerobic conditions in vitro with fecal slurries and their degradation to phenolic acid by the microbiota monitored. A limitation of in vitro fermentation models is that it may not fully depict the in vivo conditions. The use of fecal material may alter the bacterial composition and, thus, may not fully represent the microbiota present in the colonic lumen and mucosa, where catabolism occurs in vivo. bviously, the accumulation and retention of the degradation products in the fermentation vessel makes collection, identification and quantification of the metabolites easier but is not necessarily representative of the events that occur in vivo where the actual concentration of a metabolite at any time interval is dependent on the combined rates of catabolism and absorption. However, the use of an in vitro model provides information on the types of breakdown products formed, helps elucidate the pathways involved, and the rate of catabolism can be determined. To complement the in vitro incubations, phenolic acids excreted in urine 0 24 h after (i) the ingestion of green tea and water by healthy subjects in a cross-over study, and (ii) the consumption of green tea by ileostomists, was also investigated (Roowi et al., 2010). The data obtained in these studies provided the basis for the operation of the catabolic pathways that are illustrated in Fig. 2. Some of these catabolites, such as 4-hydroxyphenylacetic acid and hippuric acid, were detected in urine from subjects with an ileostomy indicating that they are produced in the body by additional routes unrelated to colonic degradation of flavan-3-ols. It is, for instance, well known that there are pathways to hippuric acid from compounds such as benzoic acid, quinic acid (Clifford et al., 2000), tryptophan, tyrosine and phenylalanine (Self et al., 1960; Grumer, 1961; Bridges et al., 1970). None-the-less, the elevated urinary excretion of hippuric acid and 4-hydroxyphenylacetic acid, occurring after green tea consumption, is likely to be partially derived from flavan-3-ol degradation. Earlier research showing statistically significant increases in urinary excretion of hippuric acid after consumption of both green and black tea by human subjects (Clifford et al., 2000; Mulder et al., 2005) supports this hypothesis. Quantitative estimates of the extent of ring fission of the flavan-3-ol skeleton are difficult to assess because, as discussed above, the production of some of the urinary phenolic acids was not exclusive to colonic degradation of flavan-3-ols. If these compounds, along with pyrogallol and pyrocatechol which are derived from cleavage of the gallate moiety from ( )-epigallocatechin-3--gallate rather than ring fission, are excluded, the excretion of the remaining urinary phenolic acids, namely 4-hydroxybenzoic acid, 3-methoxy-4-hydroxyphenylacetic acid, 3-hydroxyphenylhydracrylic acid and 5-(3 0,4 0,5 0 -trihydroxyphenyl)-c-valerolactone, after ingestion of green tea was 210 lmol compared to 38 lmol after drinking water. The 172 lmol difference between these figures corresponds to a 39% of the 439 lmol of flavan-3-ols entering the colon after consumption of green tea. Added to this is the ca. 8% excretion of glucuronide, sulfate and methylated flavan-3-ols originating from absorption in the small intestine. This estimate of a 47% recovery is nonetheless a minimum value because with the analytical methodology used some urinary catabolites will have escaped detection (Roowi et al., 2010). This will include glucuronide and sulfate metabolites of ( )-5-(3 0,4 0,5 0 -trihydroxyphenyl)-c-valerolactone, ( )-5-(3 0,4 0 -dihydroxyphenyl)-c-valerolactone and ( )-5-(3 0,5 0 -dihydroxyphenyl)-c-valerolactone which were detected after green tea consumption with a cumulative 0 24 h excretion corresponding to 16% of flavan-3-ol intake (Li et al., 2000; Meng et al., 2002; Sang et al., 2008). More recently, in a similar study in which urine was collected for 24 h after green tea intake, valerolactone metabolites were excreted in quantities equivalent to 36% of intake (Del Rio et al., 2010). When added to the 47% recovery of Roowi et al. (2010) this gives a total of 83% of intake. While this figure is obviously an approximation because of factors such as different volunteers, flavan-3-ol intakes and analytical methodology, it does demonstrate that, despite substantial modification as they pass through the body, there is a very high urinary recovery of flavan-3-ols, principally in the form of colon- derived catabolites Proanthocyanidins Proanthocyanidins (condensed tannins), the oligomeric forms of flavan-3-ols, are among the most widespread polyphenols in plants (Ferreira and Slade, 2002) and also in the human diet (Gu et al., 2004) occurring at significant levels in foods including cocoa, grapes, apples, strawberries, and red wine. The average dietary intake of proanthocyanidins in the United States has been estimated at 58 mg/day (Gu et al., 2004), but there is good evidence that this might be an underestimate because of problems associated with extraction from the food matrix prior to quantification (Tarascou et al., 2010). Procyanidins are the commonest type of proanthocyanidin with (+)-catechin and ( )-epicatechin their main constituent units (Ferreira and Slade, 2002; Gu et al., 2004), but (epi)gallocatchin and (epi)afzelchin units are also found. There are numerous feeding studies with animals and humans indicating that the oligomeric and polymeric flavan-3-ols, the proanthocyanidins, are not absorbed. Most pass unaltered to the large intestine where they are catabolised by the colonic microflora yielding a diversity of phenolic acids (Selma et al., 2009) including 3-hydroxyphenylhydracrylic acid and 4-methyl-gallic acid (Déprez et al., 2000; Gonthier et al., 2003; Ward et al., 2004) which are absorbed into the circulatory system and excreted in urine. There is one report based on data obtained in an in vitro model of gastrointestinal conditions, that procyanidins degrade yielding more readily absorbable flavan-3-ol monomers (Spencer et al., 2000) Subsequent studies have

7 452 A. Crozier et al. / Molecular Aspects of Medicine 31 (2010) not supported this conclusion (Donovan et al., 2002; Rios et al., 2002; Tsang et al., 2005). There are two reports of minor quantities of procyanidin dimers B1 and B2 being detected in human plasma after the respective consumption of a grape seed extract (Sano et al., 2003) and a flavan-3-ol-rich cocoa (Holt et al., 2002). In the latter study, the levels of the B2 dimer in plasma were ca. 100-fold lower than those of flavan-3-ol monomers. There is also evidence after oral dosing of dimethylated procyanidin dimers in the plasma of volunteers (Duweler and Rohdewald, 2000). Recent studies using pure synthetic procyanidin B2 and [ 14 C]procyanidin-B2, the commonest dimer consisting of two ( )-epicatechin units with a 4 8 linkage, have helped clarify their in vitro gut flora catabolism (Appeldoom et al., 2009; Stoupi et al., 2010a,b) and rodent pharmacokinetics (Stoupi et al., 2010c). After oral dosing approximately 60% of the radioactivity was excreted in the urine after 96 h. Comparison of the total clearance and volume of distribution following oral and i.v. doses has established in rodents that the vast majority of the radioactivity absorbed after oral dosing is in a form(s) very different from the intact procyanidin dosed. This observation is consistent with the in vitro studies that show extensive catabolism by the gut microflora. The scission of the interflavan bond represents a minor route (Appeldoom et al., 2009; Stoupi et al., 2010a), and the dominant products are a series of phenolic acids having one or two phenolic hydroxyls and between one and five aliphatic carbons in the side chain. There are, in addition, some C 6 C 5 catabolites with a side chain hydroxyl, and associated lactones, and several diaryl-propan-2-ols, most of which are also produced from the flavan-3-ol monomers. In vitro, procyanidin B2 also yields 24 dimeric catabolites, i.e. having a mass greater than the constituent monomer ( )-epicatechin (290 a.m.u), which early in the incubation collectively accounted for some 20% of the substrate. Clearly, these catabolites retain the interflavan bond. ne was identified tentatively as either 6- or 8-hydroxy-procyanidin B2. Thirteen were characterised as having been microbially reduced in at least one of the epicatechin units. Five contained an apparently unmodified epicatechin unit but in at least one case this was shown to consist of the B-ring of the upper epi- Fig. 3. Concentration of (A) quercetin sulfate, quercetin-3--glucuronide (B) a quercetin--glucuronide--sulfate, isorhamnetin-3--glucuronide and a quercetin--diglucuronide in plasma from six human volunteers collected 0 6 h after the ingestion of onions containing 275 lmol of flavonol glucosides. Data expressed as mean values in nmol/l ± standard error (n = 6). Note that no quercetin metabolites were present in detectable amounts in plasma collected 24 h after supplementation.

8 A. Crozier et al. / Molecular Aspects of Medicine 31 (2010) catechin unit and the A-ring of the lower. It is not known whether these unique catabolites are produced in vivo, and if so, whether they are absorbed (Stoupi et al., 2010c). The biological effects of procyanidins are generally attributed to their more readily absorbed colonic breakdown products, the phenolic acids, although there is a lack of detailed study in this area. There is, however, a dissenting view as trace levels of procyanidins, in contrast to ( )-catechin and (+)-epicatechin, inhibit platelet aggregation in vitro and suppress the synthesis of the vasoconstriction peptide, endothelin-1 by cultured endothelial cells (Corder, 2008). Supporting this view is a study in which individual procyanidins were fed to rats after which dimers through to pentamers were detected in plasma which was extracted with 8 mol/l urea, rather than the more traditional methanol/acetonitrile, which it was proposed prevented the irreversible binding of procyanidins to plasma proteins (Shoji et al., 2006). The procyanidins were, however, administered by gavage at an extremely high dose, 1 g/kg body weight and it remains to be determined if procyanidins can be similarly detected in urea-extracted plasma after the ingestion of more nutritionally relevant quantities by humans. 3. Flavonols 3.1. nion quercetin--glucosides nions are a rich source of quercetin glucoside and quercetin-3,4 0 --diglucoside and Mullen et al. (2006) reported on an acute human feeding study with 270 g of lightly fried onions containing a total of 275 lmol of flavonol glucosides with the main constituents being 143 lmol of the glucoside and 107 lmol of the 3,4 0 --diglucoside. Plasma and urine collected over 24 h post-supplementation were analysed by HPLC MS 2. Five principal quercetin metabolites, quercetin sulfate, quercetin-3--glucuronide, isorhamnetin-3--glucuronide, a quercetin--glucuronide--sulfate, and a quercetin--diglucuronide were detected in plasma and their 0 6 h concentration profiles are illustrated in Fig. 3. No quercetin metabolites were detected in plasma collected either prior to consumption or 24 h after supplementation. A pharmacokinetic analysis of the five plasma metabolites is summarised in Table 3 with information on maximum post-ingestion plasma C max, T max and the elimination half-life (T 1/2 ). The two major metabolites, quercetin sulfate (C max 665 nmol/l) and quercetin-3--glucuronide (C max 351 nmol/l) appeared in plasma within 30 min of the ingestion of onions, both had T max values lower than 1 h and T 1/2 values of 1.71 and 2.33 h, respectively (Fig. 3, Table 3) (Mullen et al., 2006). The quercetin--diglucuronide had a lower C max and similar T max and T 1/2 values. The pharmacokinetic profiles of isorhamnetin-3--glucuronide and the quercetin--glucuronide--sulfate were somewhat different in that both had a much longer T 1/2 and the glucuronide sulfate also had a much delayed T max (Table 3). However, the total contribution of these two compounds to the overall absorption profile was minimal, having no effect on the T max and only extending the T 1/2 to 2.61 h. This T 1/2 is much shorter than values obtained in earlier flavonol absorption studies (Hollman et al., 1996), almost certainly a consequence of the enhanced accuracy of analytical data available with improved chromatographic resolution and the application of HPLC MS 2. In keeping with the rapid T max and short plasma T 1/2 values, most urinary excretion of quercetin metabolites occurred within the first 8 h after ingestion of the onions and over the 0 24 h collection period a total of 12.9 lmol of metabolites were excreted which corresponds to 4.7% of intake (Mullen et al., 2006). The urinary metabolite profile was very different from that of the plasma as shown in Table 4. The main plasma metabolite, quercetin sulfate was excreted only in trace quantities while isorhanmetin-3--glucuronide and quercetin--diglucuronide that were minor components in plasma were major urinary metabolites. Several other metabolites, including quercetin glucuronide and isorhamnetin-4 0 -glucuronide, which were present in trace quantities or absent from plasma were excreted in urine in substantial amounts (Table 4). The data obtained by Mullen et al. (2006) indicate absorption in the proximal part of the small intestine but provide no information on the mechanisms involved or the efficiency with which quercetin glucoside and quercetin-3,4 0 --diglucoside are hydrolysed and the aglycone enters the enterocyte. n the basis of the plasma metabolite profile, it is evident that, following the release of the aglycone, quercetin is subjected to sulfation, glucuronidation and methylation. Arguably, the short T max times of quercetin sulfate, quercetin-3--glucuronide and the quercetin--diglucuronide may be indicative of sulfation and glucuronidation occurring in the enterocyte prior to passage of the metabolites into the circulatory system. The longer plasma T 1/2 value of isorhamnetin-3--glucuronide could reflect post-absorption methylation of Table 3 Pharmacokinetic analysis of quercetin metabolites in the plasma of volunteers after the consumption of 270 g of fried onions containing 275 lmol of flavonol glucosides. * Metabolites C max (nmol/l) T max (h) T 1/2 (h) Quercetin sulfate 665 ± ± Quercetin-3--glucuronide 351 ± ± Isorhamnetin-3--glucuronide 112 ± ± Quercetin--diglucuronide 62 ± ± Quercetin--glucuronide--sulfate 123 ± ± * Data presented as mean values ± standard error (n = 6).

9 454 A. Crozier et al. / Molecular Aspects of Medicine 31 (2010) Table 4 Quercetin metabolites detected in plasma and urine after the consumption of 270 g of fried onions containing 275 lmol of flavonol glucosides by six human volunteers. * Metabolites Plasma C max (nmol/l) 0 24 h Urinary excretion (nmol) Quercetin-3--glucuronide 351 ± ± 149 Quercetin sulfate 665 ± 82 Trace Isorhamnetin-3--glucuronide 112 ± ± 27 Quercetin--glucuronide--sulfate 123 ± ± 190 Quercetin--diglucuronide 51 ± ± 417 Quercetin glucuronide Trace 1845 ± 193 Isorhamnetin glucuronide Trace 700 ± 11 Quercetin--glucuronide--sulfate n.d ± 163 Quercetin--glucoside--sulfates n.d ± 156 Quercetin--glucoside--glucuronide n.d. 163 ± 23 Methylquercetin--diglucuronides n.d ± 156 * Data presented as mean values ± standard error (n = 6) n.d. not detected. Trace compound detected but not in sufficient amounts for routine quantification. Fig. 4. Concentration of quercetin-3--glucuronide and isorhamnetin-3--glucuronide in the plasma of six healthy human subjects 0 8 h after the consumption of tomato juice containing 176 lmol of quercetin-3--rutinoside. Data expressed as mean values in nmol/l ± standard error (n = 6). Note neither metabolite was present in detectable amounts in plasma collected 24 h after supplementation. Table 5 Pharmacokinetic analysis of quercetin metabolites in the plasma of six human volunteers after the consumption of 250 ml of tomato juice containing 176 lmol of quercetin-3--rutinoside. * Metabolites C max (nmol/l) T max (h) Quercetin-3--glucuronide 12 ± ± 0.3 Isorhamnetin-3--glucuronide 4.3 ± ± 0.2 * Data presented as mean values ± standard error (n = 6). quercetin-3--glucuronide in the liver. Likewise, the delayed T 1/2 of the quercetin--glucuronide--sulfate could be a consequence of post-absorption sulfation of quercetin-3--glucuronide and/or glucuronidation of quercetin sulfate. The unusually marked differences in the plasma and urinary metabolite profiles are suggestive of extensive phase II metabolism and rapid turnover and removal from the circulatory system via the kidneys, but where in the body these conversions occur remains to be determined Tomato juice quercetin-3--rutinoside In a human study parallel to that carried out with quercetin glucosides in onions, the bioavailability of quercetin-3-rutinoside was investigated by feeding 250 ml of tomato juice containing 176 lmol of the quercetin rhamnose glucose disaccharide (Jaganath et al., 2006). In this instance only two metabolites were detected in plasma, quercetin-3--glucuronide and isorhamnetin-3--glucuronide (Fig. 4). They were present in ca. 25-fold lower quantities than in the onion study with respective C max values of 12 and 4.3 nmol/l. The T max times were extended to ca. 5 h (Table 5) indicating absorption in

10 A. Crozier et al. / Molecular Aspects of Medicine 31 (2010) Fig. 5. Proposed pathway for colon bacteria-mediated catabolism of quercetin-3--rutinoside in the large intestine resulting in the production of 3,4- dihydroxyphenylacetic acid and smaller quantities of 3-hydroxphenylacetic acid with the subsequent hepatic conversion of 3,4-dihydroxyphenylacetic acid to 3-methoxy-4-hydroxyphenylacetic acid prior to urinary excretion. Dotted arrow indicates a minor pathway. the large rather than the small intestine. A total of nine methylated and glucuronidated quercetin metabolites were detected in urine but some volunteers excreted only 3 4 metabolites. The overall level of metabolite excretion ranged from 0.02% to 2.8% of quercetin-3--rutinoside intake, which is probably a reflection of variations in the colonic microflora of the individual volunteers. Confirmation of large intestine absorption was obtained in a separate feeding study using subjects with an ileostomy. Unlike the subjects with a functioning colon, neither plasma nor urinary metabolites were detected and ileal fluid collected post-tomato juice consumption contained 86% of the ingested quercetin-3--rutinoside. The urine collected from the ileostomy volunteers as well as not containing quercetin metabolites, also lacked the phenolic acid catabolites 3,4-dihydroxyphenylacetic acid, 3-hydroxyphenylacetic acid, and 3-methoxy-4-hydroxyphenylacetic acid. These catabolites were present in the urine of the volunteers with a functioning colon in quantities corresponding to 22% of quercetin-3--rutinoside intake (Jaganath et al., 2006). It was proposed that the rutinose sugar moiety of quercetin-3--rutinoside is not cleaved by the action of either LPH or CBG during passage through the small intestine and that, as a consequence, following the release of quercetin through the action of colonic bacterial enzymes, low level production and absorption of methylated and glucuronidated quercetin metabolites takes place in the large intestine. However, most of the quercetin undergoes ring fission, releasing substantial quantities of 3,4-dihydroxyphenylacetic acid and smaller quantities of 3-hydroxphenylacetic acid. Subsequent methylation of 3,4-dihydroxyphenylacetic acid yields 3-methoxy-4-hydroxyphenylacetic acid (Fig. 5), with all three catabolites being excreted in urine. Most of these steps probably occur in the large intestine mediated by local colonic microbes. Enterobacter species are among the colonic bacteria that are able to hydrolyse a rhamnosyl moiety by releasing a- and b-rhamnosidases to cleave the sugar moiety (Braune et al., 2005). However, some post-absorption metabolism may also occur as a result of the involvement of hepatic enzymes, such as -methyltransferases. It is of interest to note that after feeding tomato juice containing quercetin-3--rutinoside, where absorption is restricted to the large intestine, no quercetin sulfates were detected either in plasma or urine. In marked contrast, after feeding onions containing quercetin--glucosides that are transformed in the small intestine, quercetin sulfate was the major plasma metabolite and other sulfated metabolites accumulated in urine, as described in Section 3.1. This indicates that sulfation of quercetin occurs exclusively in the wall of the small intestine and that data obtained in ex vivo studies in which quercetin- 3--glucuronide was converted to quercetin-3--sulfate by liver cell-free preparations ( Leary et al., 2003) may not accurately reflect in vivo sulfation. It also suggests that formation of mixed conjugates such as quercetin--glucuronide--sulfate might occur following glucuronide excretion in bile and re-absorption in the large intestine, and this would be consistent with T max values of <1 h for quercetin-3--glucuronide compared with ca. 3 h for the mixed conjugate (Mullen et al., 2006).

11 456 A. Crozier et al. / Molecular Aspects of Medicine 31 (2010) Table 6 Quantities of hesperetin and naringenin metabolites excreted in the urine of eight human volunteers 0 24 h after the consumption of 250 ml of orange juice containing 168 lmol of hesperetin-7--rutinoside and 12 lmol of naringenin-7--rutinoside. * Metabolites nmol Hesperetin-7--glucuronide 1373 ± 471 Hesperetin--glucuronide 3662 ± 1483 Hesperetin--glucuronide 2319 ± 420 Hesperetin--diglucuronide 767 ± 361 Hesperetin--glucuronide--sulfates 2841 ± 699 Total hesperetin metabolites 10,962 (6.5%) Naringenin-7--glucuronide 1001 ± 344 Naringenin glucuronide 976 ± 389 Naringenin--diglucuronide 98 ± 46 Total naringenin metabolites 2075 (17.3%) * Data expressed as mean values ± standard error (n = 8). Italicised figures in parentheses indicate excretion of hesperetin and naringenin metabolites as a percentage on intake. 4. range juice flavanones Several early studies, where analyses involved the use of enzyme hydrolysis, have shown that orange juice flavanone rutinosides are absorbed in the large intestine (Erlund et al., 2001; Manach et al., 2003). In a more recent study metabolites were analysed by HPLC MS 2 after volunteers consumed 250 ml of orange juice containing 168 lmol of hesperetin-7--rutinoside and 12 lmol of naringenin-7--rutinoside (Mullen et al., 2008). The hesperetin-7--rutinoside dose was therefore very similar to that of quercetin-3--rutinoside in the study outlined in Section 3. Plasma contained hesperetin-7--glucuronide and a second unassigned hesperetin--glucuronide and the combined C max for both metabolites was 922 nmol/l at a T max of 4.4 h. The two hesperetin metabolites were also excreted in urine along with a third hesperetin--glucuronide, two hesperetin-glucuronide--sulfates and a hesperetin--diglucuronide. These marked differences in the plasma and urinary hesperetin metabolite profiles demonstrate that substantial post-absorption phase II metabolism is occurring. The quantities of these metabolites excreted 0 24 h after ingestion corresponded to 6.5% of hesperetin-7--rutinoside intake. Although no naringenin metabolites were detected in plasma, urine contained naringenin-7--glucuronide, narigenin glucuronide and a naringenin--diglucuronide in amounts equivalent to 17.3% of the ingested naringenin-7--rutinoside (Table 6) (Mullen et al., 2008a). The differing levels of excretion of hesperetin and naringenin metabolites, relative to the amounts ingested, is a trend that has been observed in some but not all flavanone feeding studies (Manach et al., 2005). While it could be a dose effect reflecting the higher intake of the hesperetin conjugate, it is more likely to be due to naringenin-7--rutinoside being more bioavailable than hesperetin-7--rutinoside indicating that the 3 0 and 4 0 substituents impact on absorption. Although both are absorbed in the large intestine, the 922 nmol/l C max of the hesperetin--glucuronides is more than 50-fold higher than that of the quercetin-3--rutinoside metabolites (Table 5), and the amount fed were extremely similar. This, coupled with the higher level of excretion of the orange juice metabolites, indicates that hesperetin-7--rutinoside is absorbed from the large intestine much more effectively than quercetin-3--rutinoside. This may be a consequence of the hesperetin-7--rutinoside being converted to glucuronides in the large intestine more efficiently than quercetin-3--rutinoside, perhaps because it is less prone to degradation by colonic bacteria. Among the flavanone metabolites excreted in quantity were two hesperetin--glucuronide--sulfates (Table 6). This contrasts with the absence of sulfated naringenin metabolites and with metabolites derived from large intestine absorption of quercetin-3--rutinoside in the tomato juice feed (Section 3.2). Thus, there appears to be clear differences in the substrate specificity of flavonoid SLTs in the large intestine and/or the liver. Analysis of phenolic acids excreted in urine after the ingestion of orange juice indicates that the hesperetin, released through colonic bacteria-mediated deglycosylation, as well as being glucuronidated, undergoes ring fission and is catabo- Table 7 Quantities of key phenolic acids excreted in human urine 0 24 h after drinking 250 ml of water and 250 ml of orange juice, containing 168 lmol hesperetin-7- -rutinoside and 12 lmol naringenin 7--rutinoside, with and without yoghurt. * 0 2 h 2 5 h 5 10 h h Total (0 24 h) Water 1.8 ± ± ± 0.2 a 2.7 ± 0.5 a 6.7 ± 1.8 a range juice 0.5 ± ± ± 2 b 34 ± 12 b 62 ± 18 b range juice with yoghurt 0.5 ± ± ± 2 b 34 ± 12 b 62 ± 18 b * Data were expressed in lmol as mean values ± standard error (n = 5). Quantifications based on the combined levels of 3-hydroxyphenylacetic acid, 3-hydroxyphenylhydracrylic acid, dihydroferulic acid and 3-methoxy-4-hydroxyphenylhydracrylic acid and 3-hydroxyhippuric acid presented in Table 2. Means in columns followed by different superscript letters are significantly different at p < 0.05.

12 A. Crozier et al. / Molecular Aspects of Medicine 31 (2010) H H H H H hesperetin-7--rutinoside CH 3 CH 3 H -demethylation hesperetin H 2 -demethylation -methylation H H 2 -demethylation CH 3 H2 H H 2 HC 3-methoxy-4-hydroxyphenyl hydracrylic acid -demethylation HC 3-hydroxyphenyl hydracrylic acid conjugation C 3-hydroxyphenyl acetic acid H 2 CH 3 conjugation H 2 HC HC NH 4-hydroxy-3-methoxyphenylpropionic acid (dihydroferulic acid) 3-hydroxyhippuric acid Fig. 6. Proposed catabolism of hesperetin-7--rutinoside in humans. lised producing 3-hydroxyphenylhydracrylic acid of undetermined chirality, 3-hydroxyphenylacetic acid, 3-methoxy-4- hydroxyphenylhydracrylic acid, dihydroferulic acid and 3-hydroxyhippuric acid. (Roowi et al., 2009). The overall level of the five phenolic acids excreted 0 24 h after drinking water was 6.7 lmol, and this rose to 62 lmol, equivalent to 37% of the ingested flavanones, following orange juice consumption (Table 7). When the 250 ml of orange juice was ingested with 150 ml of full-fat natural yoghurt, phenolic acid excretion fell back markedly to 9.3 lmol. This did not appear to be due to a

13 458 A. Crozier et al. / Molecular Aspects of Medicine 31 (2010) difference in mouth to caecum transit time of the head of the meal, as measured with breath hydrogen production. Arguably, there may have been a slowing of the bulk of the meal reaching the large intestine, which may then have altered the catabolism of the flavanones to phenolic acids by the colonic microbiota. Exactly how this is brought about is a topic that requires further investigation, especially as it is an event that is not exclusive to flavanones and yoghurt, as Urpi-Sarda et al. (2010) have reported when cocoa was consumed with milk rather than water there was reduced excretion of nine out of fifteen phenolic acids derived from colonic degradation of flavan-3-ols. The phenolic acids that accumulated in urine after orange juice consumption may have originated from the breakdown of hesperetin-3--rutinoside via the pathways illustrated in Fig. 6 (Roowi et al., 2009). In this scheme, degradation of hesperetin-7--rutinoside starts with deglycosylation to form hesperetin (Schoefer et al., 2003). The C-ring is then opened by cleavage of the ether--linkage followed by dehydrogenation resulting in the formation of 3-methoxy-4-hydroxyphenylhydracrylic acid. This C C cleavage probably occurs between the ether--linkage and the A-ring and between C4 and the A-ring as illustrated in Fig. 6. As no phloroglucinol (1,3,5-trihydroxybenzene) was detected in urine, cleavage of the ether--linkage and C2, and of C4 and the A-ring, are unlikely. 3-Hydroxyphenylhydracrylic acid may be produced from the same C C cleavage of the hesperetin C-ring followed by -demethylation. Alternatively, it could also arise from - demethylation of 3-methoxy-4-hydroxyphenylhydracrylic acid. These two compounds may then link to dihydroferulic acid, 3-hydroxyhippuric acid and 3-hydroxyphenylacetic acid via the routes indicated in Fig. 6. Most of these steps probably occur in the large intestine mediated by the colonic microflora where Enterobacteriaceae, along with a number of other human intestinal bacteria, including Eubacterium limosum and Escherichia coli, possess -demethylase activity (Grbic-Galic, 1986; DeWeerd et al., 1988; Hur and Rafii, 2000). However, some post-absorption metabolism may also occur as a result of the involvement of hepatic enzymes, such as -methyltransferases. 5. Isoflavones Isoflavones, though not a major component of the European diet, have always been considered as the better absorbed dietary flavonoids, with urinary excretion of metabolites typically being 20 50% of intake (Manach et al., 2005; Donovan et al., 2006b). A study by Rüfer et al. (2008) in which seven male volunteers were given either pure daidzein or pure daidzein-7--glucoside, both at a dose of 3.9 lmol/kg body weight, has demonstrated that the plasma C max, at ca. 8 9 h, was three to six times greater after consumption of the glucoside, which is dominant in soya compared with the aglycone, the main component in fermented soya products. The metabolites, quantified after deconjugation, included dihydrodaidzein, -desmethylangolensin, 6-hydroxy-daidzein, 8-hydroxy-daidzein and 3 0 -hydroxy-daidzein. ne of the seven volunteers also produced equol. The bioavailability reported in this study contrasts markedly with the results obtained when tablets containing a crude preparation of soya saponins and either daidzein and genistein aglycone or their mixed glycosides was given to eight volunteers at doses of 0.11 and 1.7 mmol (Izumi et al., 2000). At the higher dose, the isoflavone aglycone mixture produced plasma C max concentrations up to five times higher than the preparation containing the daidzein and genistein glycosides. The T max in this study was ca. 4 h which is much earlier than the 8 9 h reported by Rüfer et al. (2008). These differences are not easily explained but a possible role for the saponins is suggested. A study in which two volunteers consumed 50 g of kinako (baked soya bean powder) containing 66 lmol of daidzein, 106 lmol of genistein, 120 lmol of diadzein-7-glucoside and 205 lmol of genistein-7--glucoside suspended in 300 ml of cow s milk, used HPLC MS to establish the presence of daidzein, genistein, daidzein glucuronide, genistein glucuronide, daidzein-7--glucuronide, genistein-7--glucuronide, daidzein sulfate, genistein sulfate, daidzein-7-sulfate and genistein-7--sulfate in plasma in the 1 7 h period post-consumption (Hosoda et al., 2008). Traces of the glucosides of genistein and daidzein were also detected in plasma 1 h post-consumption. The short duration of the study prevented determination of T max, C max and T 1/2. The aglycone concentration never exceeded ca. 200 nmol/l with genistein exceeding daidzein for one volunteer but the reverse for the other. Within the period studied the isoflavone metabolites never exceeded ca. 3 lmol/l in total, and no single metabolite exceeded 0.8 lmol/l. Conjugation for both isoflavones occurred preferentially at the C7 position, but the ratio of glucuronides to sulfates varied with time. Although, as mentioned above, traces of the glucosides have been detected in plasma most absorption occurs after deconjugation. The first phase of absorption, up to one hour, is impaired in lactose malabsorbers, suggesting a role for LPH, but overall this is compensated by microbial hydrolysis, and total absorption was not significantly affected by lactose malabsorption (Tamura et al., 2008). The ability of ileostomists to absorb isoflavone glycosides, not significantly different from volunteers with an intact colon, confirms that absorption occurs in the upper gastrointestinal tract. However, urinary excretion of the microbial metabolites equol, dihydrodaidzein and dihydrogenistein by ileostomists was lower than that of healthy subjects, and the ileostomy group contained fewer equol-producers. Equol was characterised as the S-enantiomer (Wang et al., 2005; Walsh et al., 2007). 6. Anthocyanins Anthocyanins, for people who eat berries and drink red wine on a routine basis, are major dietary components. Although there are exceptions, unlike other flavonoids that are absorbed and excreted, most anthocyanins do not appear to undergo extensive metabolism of the parent glycosides to glucurono, sulfo or methyl derivatives (McGhie et al., 2003; Miyazawa