Dossier: Polyphenols: diversity and bioavailability

Absorption and metabolism of polyphenols in the gut and impacton health

Augustin Scalbert *, Christine Morand, Claudine Manach, Christian Rémésy

Laboratoire des maladies métaboliques et micronutriments, INRA, 63122 Saint-Genes-Champanelle, France

Received 12 May 2002; accepted 14 May 2002

Abstract

Polyphenols are the most abundant antioxidants in the human diet. They show a considerable structural diversity, which largely influencestheir bioavailability. Phenolic acids like caffeic acid are easily absorbed through the gut barrier, whereas large molecular weight polyphenolssuch as proanthocyanidins are very poorly absorbed. Once absorbed, polyphenols are conjugated to glucuronide, sulphate and methyl groupsin the gut mucosa and inner tissues. Non-conjugated polyphenols are virtually absent in plasma. Such reactions facilitate their excretion andlimit their potential toxicity. The polyphenols reaching the colon are extensively metabolised by the microflora into a wide array of lowmolecular weight phenolic acids. The biological properties of both conjugated derivatives and microbial metabolites have rarely beenexamined. Their study will be essential to better assess the health effects of dietary polyphenols. Alternatively, some health effects ofpolyphenols may not require their absorption through the gut barrier. Their role as iron chelators in the gut lumen is briefly discussed.© 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved.

Keywords: Polyphenols; Flavonoids; Bioavailability; Metabolism

1. Introduction

Polyphenols form a very complex group of moleculespresent in most plants. Several thousand molecules havebeen identified in various plant species, where they haveseveral important functions: they inhibit the development ofpathogens and decay microorganisms, and they provideprotection against UV radiation and oxidative stress. Tan-nins deter herbivores from feeding on astringent fruits.Phenolic compounds such as salicylic acid also serve assignalling molecules, and lignins provide mechanical sup-port [50,56].

Polyphenols are regular constituents of human food. Thedifferent classes of polyphenols and some of the mostcommon compounds are shown inTable 1. The averageconsumption of polyphenols with the diet is 1 g/d[39,57].The main sources are fruits, beverages such as tea, coffee,

wine and fruit juices, chocolate and, to a lesser extent,vegetables, cereals and legume seeds. Particular types ofpolyphenols may be present in a large number of foods:tannins give astringency or bitterness to different fruits,wine, cider and tea, and anthocyanins give colour to redfruits such as strawberry, blackcurrant and grape. On theother hand, others such as isoflavone phytoestrogens in soyaare restricted to a given food source.

Today, dietary polyphenols receive considerable interestfor their presumed role in the prevention of various dege-nerative diseases such as cancers and cardiovascular dis-eases. This presumed role is based on numerous animalstudies and some clinical and epidemiological studies.These views are strengthened by the identification ofpossible mechanisms of action. These mechanisms may begeneric or specific to a particular phenolic compound:

• All polyphenols are reducing agents. As such, they mayscavenge free radicals, participate in the regenerationof other antioxidants such as vitamin E and protect cellconstituents against oxidative damage. Their chemical

* Corresponding author.E-mail address: scalbert@clermont.inra.fr (A. Scalbert).

Biomed Pharmacother 56 (2002) 276–282

© 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved.PII: S 0 7 5 3 - 3 3 2 2 ( 0 2 ) 0 0 2 0 5 - 6

structures influence their redox potential, and differentin vitro tests have been developed to compare theirantioxidant capacity. Polyphenols with two vicinalhydroxyl groups on an aromatic residue are better freeradical scavengers than polyphenols with a singlehydroxyl group per aromatic residue [36,53]. However,the magnitude of these differences is much lower thanthe differences in gut absorption. Therefore, it isessential to evaluate the bioavailability of the differentpolyphenols to explain their respective health effects.

• Some polyphenols show specific effects. Isoflavonephytoestrogens, associated with a lower risk ofhormone-dependent diseases [1], have well-documented pseudoestrogenic properties [40]. Biologi-cal activities are often measured on cultured cells orisolated tissues using polyphenol compounds in theirform present in food. However, polyphenols are exten-sively metabolised both in tissues and by the colonicmicroflora (Fig. 1). It is thus necessary to identify theirmetabolites and test their own biological properties.

We present here some data recently obtained on thebioavailability of polyphenols. They are discussed in viewof their biological effects and site of action.

2. Absorption of polyphenols through the gut barrier

Indirect evidence of their absorption through the gutbarrier is the increase of the antioxidant capacity of theplasma following the consumption of polyphenol-richfoods. This has been observed for a wide array of foodstuffsrich in polyphenols [26,46,60,65,68,69]. Recovery in urineafter ingestion of given amounts of a particular polyphenolallows the comparison of the bioavailability of the differentmolecules present in diets (Fig. 2). The few studies inhumans show that the quantities of polyphenols found intactin urine vary from one phenolic compound to another [57].Among flavonoids, recovery is particularly low for querce-

tin and rutin, a glycoside of quercetin (0.3–1.4%), butreaches higher values (3–27%) for catechins in green tea,isoflavones in soya, flavanones in citrus fruits or anthocya-nidins in red wine. The highest recovery values wereobserved for caffeic acid (27%) [35] and the lowest for teatheaflavins (0.0006%) [48].

This low recovery in urine for some polyphenols is notexplained by chemical degradation in the gut. Severalphenolic compounds like caffeic acid and quercetin glyco-sides were shown to be stable in gastric or intestinal juices[27,30,49]. The stability of proanthocyanidins was followedin man by regularly sampling the gastric juice with a gastricprobe after ingestion of proanthocyanidin-rich chocolate.They were not degraded in the acidic conditions of thestomach [54]. Another explanation could be that thepolyphenols not recovered in urine were excreted in the bile[18]. The extent of biliary excretion of polyphenols has sofar not been assessed in man.

Table 1Classification of dietary polyphenols according to their chemical structures

Class Examples

FlavonoidsFlavonols Quercetin, kaempferol, myricetinFlavones Luteolin, apigeninIsoflavones Daidzein, genisteinFlavanones Hesperetin, naringeninFlavanols

Catechins (monomers) Catechin, epicatechin, gallocatechinProanthocyanidins (polymers) Procyanidins, prodelphinidins

Anthocyanins Cyanidin, delphinidin

Phenolic acidsCinnamic acids Caffeic acid, ferulic acid, chlorogenic acidBenzoic acids Gallic acidEllagitannins Casuarictin, sanguin H6

.

Fig. 1. Routes for dietary polyphenols and their metabolites in humans.

A. Scalbert et al. / Biomed Pharmacother 56 (2002) 276–282 277

Polyphenol structure has a major impact on intestinalabsorption. The most widely discussed structural parametersare molecular weight, glycosylation and esterification. Thehigh molecular weight of tea theaflavins (M = 568) shouldexplain their low recovery in urine. Another important classof high molecular weight polyphenols is that of proantho-cyanidins (syn. condensed tannins). Proanthocyanidins areflavonoid polymers with varying degrees of polymerisationand molecular weights of 578 or above. It is clear today thatthey are virtually not absorbed in the gut [17,20,33].

Most flavonoids, with the exception of catechins andproanthocyanidins, are glycosylated in food, and this gly-cosylation influences absorption through the gut barrier. Theabsorption of quercetin glycosides has been measured inileostomised volunteers. The absorption of the quercetinglucosides contained in onions was higher (52%) than thatof quercetin aglycone (24%) [30]. The 3-O-�-glucoside ofquercetin is also better absorbed than quercetin, as shown bythe threefold higher plasma concentration 4 h after theiradministration in the meal of rats [47]. In contrast, rham-nosides of quercetin were poorly absorbed in the sameconditions. The gut absorption of these rhamnosides likelyrequires deglycosylation by the colonic microflora, as sug-gested by their delayed absorption as compared to theglycosides [31,45]. The facilitated absorption of quercetinglucosides might be ascribed to their hydrolysis by thelactase phlorizin hydrolase or the cytosolic �-glycosidase inthe enterocyte [58]. However, the facilitation of absorptionby glycosylation observed for quercetin was not observedfor other flavonoids such as naringenin [25] and phlorizin[13].

Intestinal absorption is also influenced by esterificationwith gallic acid for catechins and with quinic acid for caffeicacid. The recovery of galloylated catechins in human urineafter black tea consumption was about 10-fold lower thanthat of non-galloylated catechins [67]. Similar results were

obtained in rats given decaffeinated green tea [12]. Caffeicacid is also much better absorbed than chlorogenic acid, itsester with quinic acid, common in many fruits and veg-etables and particularly abundant in coffee. The intestinalabsorption reached 95% and only 33% for caffeic acid andchlorogenic acid, respectively, in ileostomised human sub-jects, and the recovery of intact chlorogenic acid in urine didnot exceed 3% of the dose ingested [49]. The amounts ofcaffeic acid and its methylated metabolite, ferulic acid,recovered in urine after administration of chlorogenic acidby gastric intubation were 100-fold lower than those ob-served after administration of caffeic acid [3].

Most dietary polyphenols are quickly eliminated in bothurine and bile after ingestion. In man, a post-prandial peakis observed 1–2 h after ingestion of various flavonols andflavanols but is longer for isoflavones and other polyphenolsonly absorbed after partial degradation by the colon micro-flora [58]. With regard to rutin, the maximum concentrationof quercetin in the plasma is reached 9 h after ingestion[32]. For most flavonoids absorbed in the small intestine,the plasma concentration then rapidly decreases (elimina-tion half-life period of 1–2 h). The maintenance of a highconcentration in plasma thus requires a repeated ingestionof the polyphenols over time, as has been observed withvolunteers consuming tea every 2 h [66].

3. Polyphenol metabolism

Polyphenols are extensively metabolised either in tissues,once they are absorbed through the gut barrier, or, for thenon-absorbed fraction and the fraction re-excreted in thebile, by the colonic microflora. All polyphenols are conju-gated to form O-glucuronides, sulphate esters and O-methylether. This conjugation first occurs in the gut barrier, as hasbeen shown in experiments where quercetin was perfused inthe gut of living rats [14]. Quercetin glucuronides wereformed in the gut mucosa and secreted back either to the gutlumen or to the serosal side. In the rat, the highest level ofglucuronyl transferase activity was observed in the intestine[52]. These conjugates then reach the liver, where they arefurther metabolised. For example, catechin is extensivelymethylated in the liver. When it was perfused in the gut ofrats, only half of the catechin circulating in the mesentericplasma before reaching the liver was O-methylated, whereas99% of the catechin excreted in the bile was O-methylated[19].

Virtually all circulating polyphenols are glucuronidatedand/or sulphated, and no free aglycones are found in plasma[6,44], except for particular flavonoids such as phloretin,which was present in both conjugated (90%) and non-conjugated (10%) forms in rat plasma [13]. Free flavonoidaglycones were also detected in studies in which pharma-

Fig. 2. Recovery of various dietary polyphenols in urines after dietaryintake in humans. From Refs. [10,24,29,32,33,41,48,49].

278 A. Scalbert et al. / Biomed Pharmacother 56 (2002) 276–282

cological doses were administered, indicating a possiblesaturation of the conjugation pathways [15]. The formationof anionic derivatives by conjugation with glucuronides andsulphate groups facilitates their urinary and biliary excretionand explains their rapid elimination. Although the conjuga-tion of polyphenols has been recognised for many years,most of the biological studies have only been carried withpolyphenol aglycones, and very little is known on thebiological properties of conjugated derivatives due to thelack of commercial standards. Sulphate esters and glucu-ronides were shown to retain part of their antioxidantproperties and still delay the in vitro oxidation of lowdensity lipoproteins [44]. However, two other studiesshowed that glucuronidation of flavonoids reduces theirbiological potency. Daidzein and genistein glucuronideshad, respectively, a 10- and 40-fold lower affinity forestrogenic receptors as compared to their aglycones, but stillshowed weakly estrogenic activity at physiological concen-trations [70]. An epicatechin glucuronide differed fromepicatechin and 3’ -O-methylepicatechin as it failed to pro-tect primary cultures of neurones and fibroblasts fromdamage induced by hydrogen peroxide [63]. More suchstudies are needed to properly evaluate the in vivo proper-ties of polyphenol conjugates.

The extent of polyphenol methylation should also affectthe biological properties of polyphenols. Most dietarypolyphenols have catechol groups in their structures, whichcan be oxidised in vivo and form toxic quinones. Similarquinones derived from drugs or endogenous estrogens andcatecholamines are known to be toxic for cells through theelicitation of redox-cycling and the formation of superoxideradical or through the reaction with nucleophilic constitu-ents of the cell [4,5,11]. A variable proportion of thecatechol group of polyphenols such as catechin [21,52],quercetin [47] or caffeic acid [3,8] are O-methylated in vivo.This reaction was proposed to explain the absence ofcarcinogenicity of quercetin in vivo, despite its well-established in vitro mutagenicity [72].

Polyphenol conjugates could also exert biological activi-ties after deconjugation at the cellular level. Phenolicestrogen conjugates were deglucuronidated and demethy-lated in the presence of lysosomes isolated from hamsterkidney and liver [71]. Similarly, estrone sulphates werehydrolysed by mammary cancer cell lines [51]. They alsoshowed estrogenic potency with a 10-fold lower activitythan that of the non-conjugated estrone, which depended ontheir deconjugation. Similarly, a glucuronide of a flavone(luteolin) was shown to be deconjugated in the plasma ofrats following induction of inflammation with lipopolysac-charide [61]. The absence of any effects of epicatechinglucuronide on neurones and fibroblasts, as compared toepicatechin aglycone [63], raises doubts on the capacity of

tissues to deconjugate polyphenols in non-inflammatoryconditions.

Polyphenols, once they reach the colon, are extensivelymetabolised by the microflora. The flavonoid glycosidessuch as rutin not absorbed in the upper part of the intestinaltract can be hydrolysed and the aglycone absorbed [7]. Theflavonoid glucuronides excreted in the bile can also behydrolysed by the microflora and the resulting aglyconereabsorbed, thus entering into an enterohepatic cycle [2].Aglycones are also further metabolised to a wide array oflow molecular weight aromatic acids, well absorbed throughthe colonic barrier [59]. These aromatic acids are phenylva-leric, phenylpropionic, phenylacetic and benzoic acids. It isrealised today that their yields can be high. The cumulativeurinary excretion of phenylvalerolactones, the main metabo-lites of catechins, was higher than that of their precursorsafter green tea consumption by volunteers [42]. The variousaromatic acid metabolites were estimated in the 24-h urineof rats fed a diet supplemented with either catechin or winepolyphenols (Gonthier et al., in preparation). The highestyields were observed with the wine extract, rich in poorlyabsorbed proanthocyanidins, which reach the colon, wherethey are metabolised into phenolic acids [16]. Therefore, thelowest is the absorption of polyphenols in the small intes-tine; the highest should be the amount of substrates reachingthe colon and the tissue exposure to their microbial metabo-lites.

4. Polyphenol bioavailability and health effects

Most studies on the biological properties of polyphenolshave been carried out on flavonoids in their native form.They were shown to interact with receptors, to inhibitenzymes and to induce various responses in cultured cells.However, many of their biological effects observed inanimal experiments or clinical studies may equally beexplained by their microbial metabolites. The biologicalproperties of the polyphenol microbial metabolites haverarely been explored. Because of their phenolic nature, theyshould contribute to the protection against oxidative stress.Other reported activities include the inhibition of plateletaggregation: the 3,4-dihydroxyphenylacetic and4-hydroxyphenylacetic acid metabolites were more activethan their precursors rutin or quercetin [37]. Equol, ametabolite of daidzein isoflavone, showed a higher affinityfor estrogen receptors than daidzein itself [62]. The activi-ties of the various microbial metabolites should be exploredin more detail. Just as short chain fatty acids may explainpart of the health benefits of fibre intake, they could beresponsible for some of the health effects of polyphenols,particularly of those poorly absorbed through the smallintestine.

A. Scalbert et al. / Biomed Pharmacother 56 (2002) 276–282 279

Alternatively, some health effects of polyphenols maynot require their absorption through the gut barrier. Thehighest local concentration of polyphenols is found in thegut lumen [55]. They may have a direct impact on the gutmucosa and protect it against oxidative stress or the actionof carcinogens. The poorly absorbed wine and tea polyphe-nols given orally to rats were shown to limit DNA oxidativedamage in caecal mucosal cells [28,43] and reduced thenumber of tumours in rats treated with azoxymethane [9].

Polyphenols also interact with nutrients in the gut lumen.They form stable complexes with non-heme dietary iron andlimit its absorption in the gut [34]. It is advisable forpopulation groups most susceptible to developing irondeficiency (infants, children and pregnant women) to avoidan excessive intake of polyphenol-rich beverages such astea or coffee or to avoid their consumption together with themeals, the source of iron. On the other hand, the consump-tion of polyphenol-rich beverages or supplements has beensuggested as a strategy for reducing iron absorption inpatients with iron overload disorders and may providehealth benefits for persons with high body iron stores. Ahigh level of serum ferritin, a marker of iron status, has beenassociated with a lower risk of myocardial infarction in menand in women over 55 years old [38,64]. The consumptionof black tea was shown to reverse endothelial dysfunction inpatients with coronary artery disease, a mechanism that mayreduce the risk of cardiovascular diseases [23]. Such aneffect could be explained by a reduction of iron bioavail-ability, as similar effects on endothelial dysfunction wereobserved by arterial infusion of deferoxamine, an ironchelator that decreased the level of serum iron [22].

5. Conclusions

Much progress has been recently made in the field ofpolyphenol bioavailability. It is now essential that allbiologists integrate this knowledge in the conception oftheir experiments and in the interpretation of the results.Numerous experiments have been carried out on the effectson cultured cells derived from inner tissues with dietarypolyphenols such as proanthocyanidins, which are notabsorbed through the gut barrier. The value of such datamust seriously be questioned. Great attention should also bepaid to the dose and mode of administration of polyphenolswhen interpreting the results of animal experiments.

Not all polyphenols are equal. It is still difficult today todetermine which particular polyphenols are the most pro-tective against the different degenerative diseases such ascancers or cardiovascular diseases. If some generic proper-ties of polyphenols are responsible for these effects, theywill be largely influenced by the highly variable bioavail-ability and, more particularly, by their absorption in the gut.

Their metabolism will also alter their specific properties andthe biological responses at the cellular levels. Much re-search effort is still needed to evaluate the biological effectsof the conjugated derivatives and microbial metabolites ofpolyphenols. This will allow the determination of the activefraction of phenolic compounds among all those circulatingin the organism, and to determine their best dietary precur-sors and dietary sources. This knowledge will contribute tothe development of diets with optimal health benefits.

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