Fitoterapia 82 (2011) 53–66

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Fitoterapia

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Review

The intestinal microbiome: A separate organ inside the body with themetabolic potential to influence the bioactivity of botanicals

Sam Possemiers a,⁎, Selin Bolca a,b, Willy Verstraete a, Arne Heyerick b

a Laboratory of Microbial Ecology and Technology (LabMET), Ghent University, Coupure Links 653, B-9000 Gent, Belgiumb Laboratory of Pharmacognosy and Phytochemistry, Faculty of Pharmaceutical Sciences, Ghent University, Harelbekestraat 72 B-9000, Gent, Belgium

a r t i c l e i n f o

Abbreviations: ADME, absorption–distribution–me8-prenylnaringenin; SECO, secoisolariciresinol; SHIME⁎ Corresponding author. Tel.: +32 9 264 59 76; fax

E-mail address: sam.possemiers@ugent.be (S. Poss

0367-326X/$ – see front matter © 2010 Elsevier B.V.doi:10.1016/j.fitote.2010.07.012

a b s t r a c t

Article history:Received 26 May 2010Accepted in revised form 13 July 2010Available online 23 July 2010

For many years, it was believed that the main function of the large intestine was the resorptionof water and salt and the facilitated disposal of waste materials. However, this task definitionwas far from complete, as it did not consider the activity of the microbial content of the largeintestine. Nowadays it is clear that the complexmicrobial ecosystem in our intestines should beconsidered as a separate organ within the body, with a metabolic capacity which exceeds theliver with a factor 100. The intestinal microbiome is therefore closely involved in the first-passmetabolism of dietary compounds. This is especially true for botanical supplements, which arenow marketed for various health applications. Being of natural origin, their structural buildingblocks, such as polyphenols, are often highly recognized by the human and especially theintestinal microbial metabolism machinery. Intensive metabolism results in often lowcirculating levels of the original products, with the consequence that final health effects ofbotanicals are often related to specific active metabolites which are produced in the bodyrather than being related to the product's original composition. Understanding how suchmetabolic processes contribute to the in situ exposure is therefore crucial for the properinterpretation of biological responses. A multidisciplinary approach, characterizing the foodand phytochemical intake as well as the metabolic potency of the gut microbiota, whilemeasuring biomarkers of both exposure and response in target tissues, is therefore of criticalimportance. With polyphenol metabolism as example, this review describes how theincorporation of microbial metabolism as an important variable in the evaluation of the finalbioactivity of botanicals strongly increases the relevance and predictive value of the outcome.Moreover, knowledge about intestinal processes may offer innovative strategies for targetedproduct development.

© 2010 Elsevier B.V. All rights reserved.

Keywords:PolyphenolsGut bacteriaPhytoestrogensBioavailabilityDegradationNutraceuticals

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 542. The gut as a metabolic organ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54

2.1. Composition of the intestinal microbiome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 542.2. Metabolic potential of the intestinal microbiota . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 552.3. Intestinal microbiota and polyphenol metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

tabolism–excretion; E2, 17β-estradiol; ER, estrogen receptor; GF, germ-free; IX, isoxanthohumol; 8-PN,, Simulator of the Human Intestinal Microbial Ecosystem.: +32 9 264 62 48.emiers).

All rights reserved.

54 S. Possemiers et al. / Fitoterapia 82 (2011) 53–66

3. Bioavailability of polyphenols: role of degradation reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 574. Bioavailability of polyphenols: role of intestinal activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

4.1. Phytoestrogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 584.2. Intestinal activation of phytoestrogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 594.3. Making the relation between bacterial metabolism and health effects: the need for well-designed multidisciplinary studies . 614.4. Strategies to increase bioavailability of active compound: the concept of functional probiotics . . . . . . . . . . . . . 62

5. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

1. Introduction

With a rapidly increasing consumer's understanding on thelink between health and lifestyle in general, and diet inparticular, it is not surprising that the global market ofnutraceuticals, estimated at 120 billion dollars in 2007, isexpected to keep on growing at a compound annual growthrate of 7.4% [1]. The fashionable term ‘nutraceutical’ is a blendoftwowords, nutrition and pharmaceutical and is used to refer toany substance that is a food or a part of a food and providesmedical or health benefits, including disease prevention andtreatment. Typically, these products include functional foods,administered in the form of fresh or processed foods, anddietary supplements, which are provided in oral single dosageforms such as pills, capsules, powders or liquids and typicallycontain vitamins, minerals, amino acids, essential fatty acids,botanicals, or mixes thereof. Furthermore, nutraceutical ingre-dients, and especially botanicals, also find application inpharmaceutical products such as herbal medicines (phytother-apeutics). It is often difficult to make a clear-cut distinctionbetween a botanical dietary supplement and its correspondingherbal medicinal product (if there is any difference at all),particularly because of the many different legislations acrossthe globe. Most commonly, the herbal medicine provides astronger claim based on the evidence provided in theregistration dossier. Still, many botanical dietary supplementscarry health-related claims based on more limited amount ofevidence.

In view of the importance to provide the consumers withaccurate information on the health benefits of nutraceuticalsin general and botanical dietary supplements in particular, itis highly desirable to generate conclusive results using thegold standard approach of the randomized clinical trial.However, it should be taken into account that the conven-tional pharmaceutical approach is not most suitable fornutraceutical studies. Conventional pharmaceutical clinicalstudies have a much higher possible success rate because ofhigher specific dosages, disease-specific clinical endpoints,and a specific patient population. As botanical dietarysupplements are not intended to treat a disease but ratherto modify an imbalance within the normal physiologicalboundaries, it can be expected that randomized clinical trialswill be harder to organize. The ‘patient’ population is less welldefined (all are healthy individuals), the dosages are typicallylow and interference with components from the diet may behighly significant, and markers of bioactivity may not yet bestrictly related to a specific disease-risk reduction effect.Combining these factors results in the requirement for largepopulations to be studied that will be too cost-intensive for

any company pursuing products based on conclusive clinicalevidence.

Furthermore, natural products such as botanicals aretypically much more susceptible to interindividual variationin ADME (absorption–distribution–metabolism–excretion)-characteristics. Their common natural origin and rather limitedstructural building blocks, such as polyphenols, are highlyrecognized by the human and especially the intestinalmicrobial metabolismmachinery. Intensive host and especiallymicrobial metabolism results in often low circulating levels ofthe originally administered products, with the importantconsequence that final health effects of botanicals are oftenrelated to specific activemetaboliteswhich are produced in thebody rather than being related to the product's originalcomposition. This further complicates the execution of clinicaltrials with botanical dietary supplements.

In this review, we will therefore focus on the intestinalmicrobiome as an important metabolic site for botanicalproducts. We will show that variable intestinal metabolism ofsuch products results in variable exposure to active substancespresent in the botanical or to specific active metabolites. Assuch interindividual variation in exposure to active ingredientscould have a profound impact on the final clinical outcomeupon intake of the product under investigation, understandingof microbial metabolic processes in the intestine is a crucialaspect of clinical research towards botanical dietary supple-ments. Moreover, such in-depth knowledge may offer innova-tive strategies for new product developments.

2. The gut as a metabolic organ

2.1. Composition of the intestinal microbiome

The composition of the microbial community in the gut isgoverned by age, diet, environment and phylogeny (i.e. co-evolution of the gut microbes with their host) [2–4] and theecosystem contains all three domains of life: bacteria, archaeaand eukarya (fungi, yeasts and protozoa), with the largestcommunity residing in the colon. Indeed, the human colonharbors a highly complex microbial ecosystem of about 200 gliving cells, at concentrations of 1012 microorganisms pergram gut content, the highest recorded for any microbialhabitat [5]. Together, all 6.5 billion humans on earth repre-sent a gut reservoir of 1023–1024 microbial cells, which is justfive orders of magnitude less than the world's oceans (1029

cells) [6]. Therefore, the human gut constitutes a substantialhabitat in our biosphere. Despite such high numbers, themicrobial diversity is however relatively limited. Although 55and 13 divisions of respectively bacteria and archaea have

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been described, only 8 bacterial divisions have been identifiedso far in the gastrointestinal tract and the gut microbiome isdominated by only two bacterial divisions, Firmicutes andBacteroidetes, that make up over 90% of the intestinalmicrobiota. The remainder consists of Actinobacteria [7] and,to a lesser extent, Proteobacteria, Verrucomicrobia, andCyanobacteria [6,8,9]. Further, only two archaeal specieshave been described with Methanobrevibacter smithii beingmore predominant than Methanosphaera stadtmanae [8]. Atthis level, the intestinal communities of all humans thereforeappear quite similar. However, within these divisions, alimited number of lineages terminate in broad, shallowradiations comprising hundreds of species and thousands ofstrains, making the microbiota of an individual as personal-ized as a fingerprint [6,9].

The unique shape of this microbial ecosystem is related toa specific functional organization. The structure and compo-sition of this ecosystem reflects a natural selection at bothmicrobial and host levels, which promotes a mutual cooper-ation in the search of a functional stability [10]. It has beenproposed that such equilibrium requires a series of evolved,nested equilibria to achieve the overall homeostasis, inanalogy with the ‘Evolutionary Stable Strategy’ theory. Inother words, microbial success in a host requires the ability togrow and overcome the host's defenses, yet cannot affect thefitness of the host, as this would ultimately reduce thenumber of hosts and thereby lead to loss of the bacteria's ownhabitat [10]. The diversity found in the gastrointestinal tract,namely a few divisions represented by very tight clusters ofrelated bacteria, may therefore reflect strong host selectionfor specific bacteria whose emergent collective behavior isbeneficial for the host [9].

Due to themultitude of direct and indirect interactionswithits host, the intestinal microbiome is closely involved in thehost's health, for instance by the extraction of energy fromotherwise indigestible compounds [11], the stimulation of thegut immune system [12], the regulation of cell proliferation[13], the synthesis of essential vitamins K and B [14] and byenhancingpathogen resistance [15]. On the other hand, specificcommunity assemblagesmay also be considered as a risk factorcontributing to a disease state. This is shown by recent reportslinking intestinal bacteria with diseases ranging from allergies[16] to bowel inflammation [17] and obesity [18].

2.2. Metabolic potential of the intestinal microbiota

In addition to the above-mentioned health effects, theintestinalmicrobiotamay also play an important role in humanhealth by means of its metabolic potential. Indeed, it isestimated that the collection of all microbial genomes in thegut comprises between 2 million and 4 million genes, which is70–140 times more than that of their host [19]. This ‘micro-biome’ encompasses all genes that are responsible for numer-ous processes such as substrate breakdown, protein synthesis,biomass production, production of signaling molecules, anti-microbial compounds and it encodes biochemical pathwaysthat humans have not evolved [20]. The intestinal microbiotacan thereforebe regarded asa separate organwithin thehumanhost, that is capable of even more conversions than the humanliver, and we can view ourselves as a composite of human cellsand bacteria and our genetic landscape as a ‘metagenome,’ an

amalgam of genes embedded in our genome and in thegenomes of all our microbial partners [21].

As the colonic microbial community receives a largediversity of non- or partly digested food components and hostexcretion products as nutrient and energy source, the set ofbacterial genes that code for metabolic enzymes is highlydiverse and redundant to ensure survival under fluctuatingnutritional conditions. Carbohydrates, resistant to digestion,drive colonic fermentation and the resulting end products(short-chain fatty acids) are considered beneficial for the host.In contrast, when proteins are fermented, the end productsinclude toxic compounds, such as amines and phenols [22].Besides carbohydrates and proteins, many other components,such as phytochemicals, food or environmental contaminantsare also exposed to the gut microbiota. It has been shown thatthegutmicrobial communityhas theability tometabolize theseso-called xenobiotics far more extensively than any other partof the body (reviewed by [23]). This microbial factor in themetabolism of ingested chemicals has often been overlooked,yet the last decade of scientific research has brought newinsights and surprising findings. Ilett demonstrated that gutbacterial metabolism has a strong potential for both bioactiva-tion and detoxification of xenobiotics [24]. Table 1 presents thedifferent types of microbial enzymes in the gut. In contrast tothe oxidative and conjugative nature of liver metabolism,which generates hydrophilic high molecular weight biotrans-formation products, the metabolic nature of the gut microbialcommunity in an anaerobic environment is mainly reductiveand hydrolytic, generating non-polar low molecular weightbyproducts [23].

Additionally, the intestinal microbiota also interfere withthe human biotransformation process through the enterohe-patic circulation of xenobiotic compounds. Compounds thathave been absorbed in the intestine and subsequentlydetoxified are usually conjugated with polar groups (glucuro-nic acid, glycine, sulfate, glutathion and taurine) in theepithelium or liver. Such metabolites may enter the bloodstream prior to excretion in the urine, but depending on thecompound a considerable fractionmay also enter again into theintestine via secretion with the bile [24]. Once released in theintestinal lumen, these conjugates may be hydrolyzed again bybacterial enzymes such as β-glucuronidases, sulfatases andglucosidases. McBain and MacFarlane [25] estimated that1010–1012 bacteria/mL intestinal content produce β-glucosi-dase and 107–1011 produce β-glucuronidase, showing theimportance of intestinal bacteria in this deconjugation process.This would negate the detoxification cycle and delay theexcretion of many exogenous compounds since the originalcompounds or phase I metabolites are more prone to intestinalabsorption than their phase II conjugates.

2.3. Intestinal microbiota and polyphenol metabolism

Health effects from plant-derived products are oftenattributed to their polyphenol content. Indeed, a largenumber of mechanistic in vitro and animal studies withpolyphenols in pure form or occurring in natural extractshave indentified among others oxidative stress, inflammationand endothelial function as important targets where poly-phenols may exert their beneficial effects. However, theemergence of more and more studies on the bioavailability of

Table 1Metabolic potency of human gastrointestinal microbiota.After: Ilett et al. [24].

Reactions Enzyme Bacterial species/origin of sample

HydrolysisGlucuronides β-glucuronidase E. coliGlycosides β-glucosidase Enterococcus faecalis, Eubacterium rectale, Clostridium sphenoidesAmides Amide hydrolase E. coli, B. subtilisEsters Deacetylase Enterococcus faecalisSulfamates Arylsulfotransferase Clostridia, enterobacteria, enterococci

ReductionAzo-compounds Azoreductase Clostridia, lactobacilliUnsaturated lacton Unsat. glycoside hydrogenase Eubacterium lentumAliphatic double bounds Unsat. fatty acid hydrogenase Enterococcus faecalisNitro-compounds Nitroreductase E. coli, bacteroidesN-oxides N-oxide reductase Human colonS-oxides Sulfoxide reductase E. coliKetones Hydrogenase Rat caecumHydroxylamines Nitroreductase Rat GIT

DehydroxylationDemethylaton Demethylase Enterococci, lactobacilli, clostridiaDeamination Deaminase Bacteroides, clostridiaDecarboxylation Decarboxylase Enterococcus faecalisDehydrogenation Dehydrogenase Clostridium welchiiDehalogenation Dehalogenase E. coli, Aerobacter aerogenes

Synthetic reactionsEsterification Acetyltransferase E. coliN-nitrosation Enterococcus faecalis, E. coli

Other reactionsOxidation Oxidase E. coli, Enterococcus faecalisIsomerization Isomerase Eubacterium rectale, clostridium sphenoidesFission aliphatic Tryptophase E. coli, Bacillus alveiFission ring C-S lyase Pig GIT, Eubacerium aerofaciens

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polyphenols has created increasing doubt about the physio-logical relevance of such findings. Indeed, it has become clearthat the bioavailability of polyphenols, as they occur in ourdiet, is highly variable between individuals and generally fartoo low to explain direct antioxidant effects in vivo. Otherspecific mechanisms of action have been proposed, yet alsohere the concern has been raised that the bioavailability ofthe polyphenols in the forms as they occur in dietary formatsis probably very low. It has been pointed out that biocon-verted forms of polyphenols, conjugated forms of intactpolyphenols resulting from phases I and II metabolism, mayprobably have more physiological importance than the nativefree form in which they were present in the diet, yet also herelevels of circulating species tend to be low. A far more likelyreason for the low bioavailabilty of many of the polyphenolaglycones and conjugates is therefore related to theirmetabolic fate in the intestine.

A general scheme of the different aspects which areinvolved in the bioavailability of polyphenols is given inFig. 1. This shows that in order to reach the target tissues, thenative polyphenols have to pass a multitude of barriers and areexposed to numerousmetabolic processes. The result of all thisis that the target tissues are unlikely exposed to only theingested polyphenol, if exposed at all, but rather to a complexmixture of metabolites from various origin. It is thereforeessential to understand the nature and extent of the different

metabolic processes in order to predict which compoundsmayreach their molecular targets in the body and therefore whichhealth effects may occur.

While many recent studies focus on the absorption ofpolyphenols from the small intestine and how phase I and IImetabolism may affect their final biological activity, it is oftenforgotten that a major fraction of ingested polyphenols in factreaches the colon directly or through enterohepatic circulation.Whereas some polyphenols occur in food as free aglycones,many are present as glycosides, bound to sugar moieties, or aspolymers with varying chain length [26]. For instance,naringenin is present in citrus fruits bound to glucose orrutinose molecules, but in tomatoes it is present as freenaringenin [27].Moreover, different structuresmaybegroupedtogether in so-calledmacromolecules, which is for instance thecase for lignans in flax [28]. In general, aglycones and a fewglycosides can readily be absorbed in the small intestine anddepending on the sugarmoiety and position on the polyphenol,deglycosilation of polyphenol glycosides can occur by mam-malian β-glucosidases in the small intestine, followed byabsorption of the aglycon [29–31]. This is indicated by a rapidpeak in plasma concentration of the compounds. Othershowever, such as narirutin (naringenin-O-rhamnoglucoside),the predominant naringenin glycoside in oranges, reach thecolon intact where deglycosilation occurs by bacterial enzymes[32–34]. Due to extensive phases I and II metabolism during

Fig. 1. General scheme of different factors influencing the bioavailability of polyphenols. A part of the ingested fraction may be absorbed from the small intestine andmay be conjugated with glucuronic acid or sulfate during absorption and in the liver. It can further be metabolized, circulate in the bloodstream or enter intoenterohepatic circulation. The fraction of the ingested polyphenols which reaches the colon directly or indirectly can be exposed to the intestinal microbiota and itsextensive metabolic pathway. Despite the high initial structural variations, overlapping pathways result in the production of a relatively small number of metabolites.

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and after absorption, a major fraction of the absorbedpolyphenols is excreted back into the intestine through thebile as glucuronides and/or sulfates and reaches the colonwhere bacterialβ-glucuronidases and sulfatases can release thepolyphenol aglycon [35–37]. Absorption of the releasedaglycone leads to enterohepatic circulation, indicated by asecond plasma peak (7–10 h post ingestion) [38].

The fraction of the polyphenols that reaches the colondirectly or indirectly can subsequently be absorbed from thecolon or act as substrate for the indigenous bacterial commu-nity with their extensive metabolic potential. In many casesbacterial metabolism of polyphenols leads to a decrease in

biological activity. However, in some cases specific bacterialtransformations give products with increased biological prop-erties compared to the parent compounds. Therefore, bacterialdegradation and specific transformation will be discussedseparately.

3. Bioavailability of polyphenols: role of degradationreactions

Colonic metabolism of dietary polyphenols has beenextensively studied and associationsbetweenurinary excretionof simple phenolic structures, such as hippuric acid derivatives,

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and bacterial polyphenol degradation was shown in manystudies [39–42]. It is clear that the metabolites are formed bymicrobial action, as lower recovery was found in human andanimal studies where antibiotics were dosed prior to flavonoiduptake [43,44] and as phenolic metabolites were not detectedin germ-free rats [45].

Withflavonoids as example, bacterialflavonoid degradationfollows a very general pattern in which many non-specificmetabolites are formed, such as hydroxylated phenylpropionicacids. Already in 1956 it was shown that flavonoid degradationinvolves ringfission of theheterocyclic C ring [46].More studieshave elucidated the complete degradation pathway [32,47–49].With naringenin as example, the degradation starts withisomerization of the C ring at the hetero atom into thecorresponding chalcone phloretin (Fig. 2). After reductioninto a dihydrochalcone, further splicing takes place at thecarbonyl moiety, yielding phloroglucinol and 3-(4-hydroxy-phenyl)propionic acid. The latter may be dehydroxylated into3-phenylpropionic acid and the mixture can be absorbed fromthe colon. Bothcomponents areoften recovered inurineas suchor conjugated [50,51], butmay also be subjected to β-oxidationand glycination in the liver, yielding hippuric acid and4-hydroxyhippuric acid [52]. In contrast, phloroglucinol hardlyever is recovered as final metabolite, as it can be degraded intoacetate, butyrate and CO2 [53,54]. Although bacteria can gainsomeenergy fromthedegradation, themain rationalewouldbedetoxification, as many flavonoids exert antibacterial activity[55,56].

As consequence of the general polyphenol degradationpatterns, only a relatively small number of phenolic degradationproducts are formed in the colon from the extremely diversegroup of natural flavonoids. This is illustrated in Fig. 1. Despitethe initially high degree of structural difference in polyphenolstructures, overlapping metabolic pathways result in theformation of a limited number of intermediate products. Theseintermediates subsequently enter a general degradation path-way, inwhich the typical end products, such as phenylpropionicacid, phenylacetic acid and bezoic acid derivatives, are formed.These compounds may be recovered in the blood or urine assuch, or may further be metabolized in the liver, yielding forinstance the corresponding aroylglycines, upon glycination.

Fig. 2. Degradation of naringenin by intestinal bacteria. Naringenin degradation yformer may further be transformed to 3-phenylpropionic acid, the latter is comple

In theory, one could therefore conclude that understandingthe bioavailability and bioactivity of polyphenols is relativelystraightforward, as overlappingmetabolic pathways result in thesystemic circulation of a limited number of metabolites.However, one crucial additional aspect of bacterial metabolismof polyphenols should be taken into account, i.e. the hugeinterindividual variation in the rate and extent of intestinalmicrobial metabolism. The consequence is therefore that thefinal circulating metabolite patterns highly vary in bothconcentration and composition, resulting in highly varyingbioactivity in different individuals.

The importance of this variation can be illustrated with 2examples from recent work. For instance, the quercetindegradation pathway by the gut microbiota is quite wellunderstood, and results in the formation of a number of typicalendmetabolites [52], such as shown in Fig. 3. In an in vitro studyinwhich the interindividual variation in polyphenolmetabolismwas investigated, black tea extract was incubated with fecalmicrobiota from 10 different individuals and differences in themetabolic patterns of specific polyphenols were calculated [57].Fig. 3 illustrates that both the extent and rate by which theintestinal microbiota produce the different metabolites stronglyvaries among individuals. With a similar aim, Van Velzen et al.performed a placebo-controlled human intervention studywith20 healthy volunteers in which urinary profiles of polyphenolmetabolism were recorded over a period of 48 h uponconsumption of polyphenol-rich black tea [58]. Fig. 4 showsthe excretion profile of a typical metabolite, 1,3-dihydroxyphe-nyl-2-O-sulfate, upon placebo or tea intervention. This furtherillustrates the importance of interindividual variation in meta-bolic transformations, resulting in very individual exposureprofiles to active metabolites.

4. Bioavailability of polyphenols: role of intestinalactivation

4.1. Phytoestrogens

Phytoestrogens are hormone-like compounds found in awide range of plants, which have a unique diphenolicstructure [59]. Due to their structural similarity to the female

ields 3-(4-hydroxyphenyl)propionic acid and phloroglucinol. Whereas thetely degraded by intestinal bacteria.

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hormone E2, phytoestrogens have the ability to bind toestrogen receptors (ERs) [60]. The presence of these hor-mone-like compounds in plants was first realized over60 years ago, when fertility problems were observed insheep that had been grazing pastures containing red clover[61,62]. Later it became clear that molecules with estrogenicactivity are present in a broad range of plants. In the eightiesthe list already contained over 300 species [63] and thenumber keeps increasing [64,65]. However, the principalphytoestrogens in human diet are restricted to a limitednumber of molecules.

The isoflavones daidzein and genistein, mainly present inhuman diet in various soy products, are by far themost studiedphytoestrogens [66–68]. Next to this, lignans are ubiquitous inhumandiet [69].Whereas the highest concentrations are foundin flaxseed [70], the most important dietary sources of lignansare whole-grain cereals, legumes and various vegetables andfruits [59,71]. Most common lignans found in our diet aresecoisolariciresinol (SECO), matairesinol, lariciresinol andpinoresinol [72]. Other groups of phytoestrogens include thecoumestans (soy and clover) [73] and stilbenes (e.g. resveratrolin red wine and peanuts) [74].

Recently, a new important group of phytoestrogens wasadded to this list. Hops (Humulus lupulus L.) are known sinceancient times for its medicinal properties and nowadaysmainly used in the beer industry. These applications arerelated to the large amounts of favorable secondary metabo-lites which are produced in so-called lupulin glands. Althoughseveral hundreds of metabolites are being produced in theseunique glands, the most important compounds for thebrewing industry are the bitter acids, which give beer itstypical bitter flavor and ensure foam and bacterial stability,and the hop essential oils, which give beer its typical “hoppy”aroma. Yet, since 1953 hops have been studied as a possiblesource of estrogenically active compounds [75]. According tothese authors, hops contain “the equivalent of 20–300 μgestradiol/g.” In those days, hops were picked by hand and theneed for massed labor at harvest time led to the migration ofmany families from the entire region surrounding the hopregion to the hop fields. According to folk legends, womenbegan to menstruate 2 days after starting picking hopsmanually, indicating the presence of an estrogenic substance.However, the final identification of the active phytoestrogensin hops was only established when Miligan et al. [76]identified 8-prenylnaringenin (8-PN) as one of the mostpotent phytoestrogens identified so far.

8-PN belongs to the group of prenylated flavonoids [77],which are accumulated in high amounts in the female hopcone. Xanthohumol is the principal flavonoid, present at highconcentrations in the lupulin glands (0.1–1% of cone dryweight) with large differences depending on the variety andflowering stage [78]. This prenylated chalcone is accompa-nied by at least 13 related chalcones and a number ofprenylated flavanones, from which isoxanthohumol (IX),8-PN and 6-prenylnaringenin are the most important [79,80].

In vitro and in vivo studies showed that 8-PN was the firstERα selective phytoestrogen with only 25-fold lower affinityfor the latter receptor than 17β-estradiol (E2), the endoge-nous female reproductive hormone [81]. Its in vitro biologicalactivity on the ERα receptor would be in the range of 5–600times weaker than E2 [76,82], making it far more active

compared to the other known phytoestrogens. Hop-contain-ing dietary supplements are now marketed to reducemenopausal complaints [83,84] and for breast enhancement[85].

4.2. Intestinal activation of phytoestrogens

Despite their different structure and origin, the 3predominant classes of phytoestrogens in our diet share aunique feature. Indeed, isoflavones, lignans and prenylflavo-noids have the common characteristic that an importantfraction of the orally ingested amount reaches the colon,where they are exposed to the metabolic activity of the gutmicrobiota. And instead of being degraded into smallphenolics, their structure is only slightly modified bymicrobial enzymes, resulting in metabolites with increasedbiological activity, which can subsequently be absorbed andmay exert systemic biological effects. Phytoestrogens there-fore act as the perfect example to illustrate that bacterialmetabolism of plant compounds not necessarily decreasestheir activity, but may in contrast also increase theirbioactivity profile and related health effects.

The isoflavones daidzein and genistein are presentpredominantly as glucosides in most soy products. Glucosidesoriginating from food which escaped deconjugation in thesmall intestine, as well as phase II glucuronides and sulfatesexcreted in the gut through enterohepatic circulation, can bedeconjugated [86]. Further microbial metabolism may lead toextensive degradation and therefore low bioavailability, as isnoted for genistein. Many reports have shown however that,in contrast to genistein, daidzein not only can partially bedegraded into O-demethylangolensin, but may also betransformed into equol. The production of equol, namedafter equus (horse) as it was first found in horse urine [87],was shown to be performed exclusively by bacteria asantibiotic treatment decreased equol production [88] and asequol was not produced in germ-free rats [89]. As many invitro [68,90,91] and in vivo studies (reviewed in Atkinson etal. [92]) indicate that equol would exert increased healthbeneficial effects, the intestinal bacteria have a crucial role inthe activation of soy phytoestrogens.

A similar story is true for lignans. These plant compoundsare present in foods as inactive glycoside precursors [93] butcan be activated inside the human intestine into the activeenterolignans enterodiol and enterolactone [94,95]. As thistransformation againwas shown to be performed uniquely bybacteria [89] and as the enterolignans would have increasedbiological activity [96–98], it can be concluded that intestinalbacteria also determine health effects related to lignan intake.

For hop prenylflavonoids, a similar phenomenon wasrecently observed. IX is the prevailing prenylflavonoid inbeers and hop extracts, being 10 to 30 times more abundantcompared to 8-PN [99]. After administering IX to 2 men,Schaefer et al. [100] recovered small amounts of 8-PN inurine, indicating the activation of IX into the phytoestrogen8-PN. The authors associated this process with liver metab-olism, as Nikolic et al. [101] recovered some 8-PN afterincubating human liver microsomes with IX. However, recentfindings [102] showed that not only the liver but also theintestinal community may be responsible for the productionof 8-PN after IX consumption. Incubation of fecal cultures

Fig. 3.Metabolic degradation pathway of quercetin. Kinetic curves of quercetin metabolites are displayed for 10 individuals from the incubation with black tea polyphenols. The color coding of the kinetic curves refers to theresults from the 10 different individuals.Redrafted based on the work of Gross et al. [57].

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with IX led to efficient production of 8-PN through microbialO-demethylation of IX. As 8-PN was the major microbialtransformation product, whereas only minor amounts of8-PN were recovered after liver metabolization of IX, it couldbe concluded that the gut may be the principal site ofactivation of IX.

Using a dynamic in vitro model of the intestinal tract,designated the Simulator of the Human Intestinal MicrobialEcosystem (SHIME), we further unravelled this process andshowed that activation of IXmainly occurs in the distal part ofthe colon, leading up to 80% 8-PN production [103].Moreover, the definitive role of bacterial enzymes in thisprocess was recently shown by the fact that no 8-PNproduction was noted in germ-free rats [104] and that recentuse of antibiotics was negatively correlated with theintestinal IX-bioactivation capacity in a human interventionstudy [105]. Therefore, we were the first to show thatintestinal processes not only determine the activity ofisoflavones and lignans, but that this is also valid for thisnew group of phytoestrogens, the prenylflavonoids.

But we discovered also another interesting parallel withthe other groups of phytoestrogens. For isoflavones andlignans it is well known that the extent of bacterialmetabolism is highly variable between individuals. It hasbeen observed that only about 30–50% of humans have theintestinal metabolic potential to produce equol [106,107]. Aswide ranges in the production of the mammalian lignans alsohave been observed [107–109], interindividual differences inthe intestinal bacterial community result in interindividualdifferences in the exposure to certain phytoestrogenmetabolites.

Very similar observations were made for hop phytoestro-gens. In a study with 100 fecal samples, only about 35% of invitro tested intestinal microbial communities producedmedium or high amounts of 8-PN from IX [102,103],separating individuals in high, moderate and low 8-PNproducers. Furthermore, a recent dietary intervention trialwith fifty healthy postmenopausal Caucasian women, con-firmed the important role of the intestinal microbiota in theexposure to 8-PN after uptake of IX-containing hop extracts[105]. The subjects could be classified into poor (60%),moderate (25%) and strong (15%) 8-PN producers based oneither urinary excretion or microbial bioactivation capacityand a significant correlation could be found between theintestinal IX-activation capacity and the urinary 8-PNexcretion.

4.3. Making the relation between bacterial metabolism and healtheffects: the need for well-designed multidisciplinary studies

The role of the gut bacteria in the intestinal activation ofphytoestrogens is summarized in Fig. 5. Specific modifica-tions of the original polyphenol structure by bacterialenzymes lead to the production of metabolites with increasedbioactivity. However, the extent of intestinal metabolismstrongly varies among individuals, as illustrated by thevariable bioactivation in an in vitro study with 100 fecalsamples incubated with either daidzein, SECO or IX (Fig. 5)[110]. This shows that, when given an identical dose ofprecursor, some persons are exposed to higher, while othersto lower levels of estrogenically active compounds than

expected based on their intake. Moreover, the story maybecome even more complex when considering that humannutrition and an increasing number of food supplementscontain mixtures of various phytoestrogens instead ofisolated molecules. A unique human intervention trial, inwhich the circulating levels of the active metabolites of eitherseparately or simultaneously dosed isoflavones, lignans, andprenylflavonoids were evaluated, revealed how the microbialpotential to activate various phytoestrogens within anindividual increases, adds an extra dimension of variabilityand complexity [111]. Co-administration of different pre-cursors was shown to influence the intestinal activation ofeach of the precursors, thereby also impacting the exposureto phytoestrogen-derived E2 equivalents.

The example of intestinal phytoestrogen activation furthersuggests that incorporation of microbial metabolism as animportant variable in the evaluation of the final bioavailabil-ity and bioactivity of botanical extracts would stronglyincrease the relevance and predictive value of the outcome.A multidisciplinary approach, controlling and characterizingthe food and phytochemical intake as well as the metabolicpotency of the gut microbiota, while measuring biomarkers ofboth exposure and response in target tissues, is thereforerecommended to evaluate these endpoints. For instance,given the complexity of the estrogen-like activities of dietaryphytoestrogens, such an approach, combining knowledgefrom both in vivo and in vitro studies, is mandatory toproperly evaluate their potential impact on breast carcino-genesis. Responding to the safety concerns on phytoestrogen-induced breast cell proliferation and the subsequent need toidentify subpopulations which may be at risk, Bolca et al.[112,113] dosed, prior to an esthetic breast reduction, well-characterized nutritional doses of soy- and hop-derivedphytoestrogens to healthy women with a controlled dietarybackground, known hormonal profile and phenotyped mi-crobial bioactivation potential, and measured the levels ofisoflavones and prenylflavonoids that actually reach thebreast tissue in a bioactive form. As phases I and II reactionsalter the pharmacological profiles of phytoestrogens [114]and as cell-type specific responses to estrogen exposure havebeen reported [115], not only the concentration, but also thenature of the metabolites and the biodistribution wereassessed and compared to the endogenous estrogen expo-sure. Taking into account the relative estrogenic potenciestowards ERα and ERβ compared to E2 [76,116,117], attenu-ation due to phases I and II metabolism[114,118,119], anddose-addition [120], isoflavones were found to reach expo-sure levels at which ERβ agonistic, putative protective effectsmay occur, whereas estrogenic responses through the ERα-agonism of 8-PN, which was only detected in breast tissue ofmoderate and strong 8-PN producers at low pmol/g-concen-trations, were estimated to be unlikely. Nevertheless, thesevalues are derived from theoretical concepts based on invitro-data, and, therefore, the clinical implications of thesefindings require further investigation. Therefore, the nextstep is to characterize the differential activation of estrogen-responsive genes between dietary phytoestrogens and che-mopreventive therapeutics such as the SERMs tamoxifen andraloxifene. Integration of human in situ-exposure data(phytoestrogens, selective estrogen receptor modulators,endogenous estrogens, and metabolites in breast tissue, as

Fig. 4. The cumulative urinary excretion of 1,3-dihydroxyphenyl-2-O-sulfateduring 48 h after black tea intervention and placebo intervention. A capsulecontaining either 2500 mg of dried black tea extract powder (black lines) or aplacebo (sucrose, red lines) was administered to 20 healthy individuals (s1to s20) in a cross-over design with a 10d wash-out period. Excretion of1,3-dihydroxyphenyl-2-O-sulfate was analyzed using NMR profiling.Figure used with permission from Van Velzen et al. [58].

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well as their overall estrogenicity towards ERα and ERβ),transcriptomic profiles (estrogen and phytoestrogen targetgene transcription in mammary gland epithelial cells), and

Fig. 5. Variable metabolic activation of phytoestrogens by intestinal bacteria.prenylflavonoid isoxanthohumol (B) and the lignans secolariciresinol andmatairesinwith either daidzein, isoxanthohumol or secolariciresinol, a high interindividual varseparating individuals into low, moderate and efficient converters.Redrafted after Possemiers et al. [110].

epigenetic patterns provides a rigorous assessment of themechanisms of action of these exogenous estrogens inhealthy breast tissue.

The above example of a multidisciplinary approach withincorporation of the microbial aspect is especially true for thespecific case of phytoestrogens, but given the extensivemetabolic potential of the gut microbiota, may very well beexpanded to various other food products and supplements,such as botanicals.

4.4. Strategies to increase bioavailability of active compound:the concept of functional probiotics

The previous chapters showed that microbial metabolismin the intestine strongly increases the complexity of predict-ing the bioavailability and health outcome of dietary inter-ventions with for instance botanical supplements. The highvariability in intestinal degradation or activation reactionssupport the development of personalized treatment strate-gies based on pre-screening of individual metabolic patterns.However, in-depth knowledge on such microbial metabolicpatterns may also offer unique opportunities to developstrategies to overcome such inherent variability and to createnew functional products with high, standardized biologicalactivity.

Again, phytoestrogens from hops can be used to supportthis hypothesis. Indeed, if we accept that an individual'sphytoestrogen exposure depends on variable microbialactivation of IX into 8-PN, the next question would logicallybe: how can one change the phytoestrogenmetabolism statusin individuals, making all individuals exposed to similaramounts of the active metabolites? The answer to thisquestion could be simple: by introducing bacteria which are

The specific microbial modifications of the isoflavone daidzein (A), theol are indicated in color. In a study in which 100 fecal samples were incubatediability in the efficiency of the metabolic conversions was noted (bar charts)

,

Table 2Comparison of ion intensities of IX-glucuronides and 8-PN-glucuronides in liver, kidney and uterus of rats dosed with IX in the absence (Expt. 1) or presence (Expt.2) of E. limosum. 1

After: Possemiers et al. [104].

Expt. 1 (IX) Expt. 2 (IX+E. limosum)

IX-glucuronide 8-PN-glucuronide IX-glucuronide 8-PN-glucuronide

Male Female Male Female Male Female Male Female

Liver Hop+ ++ ++ ++ ++ ++ ++ ++ ++Hop− ++ ++ − − ++ ++ +++ +++GF +++ +++ − − +++ ++ ++ ++

Kidney Hop+ + + − + ++ ++ − ++Hop− + + − − + ++ + ++GF + + − − + − + −

Uterus Hop+ − + − +Hop− − − − −GF ++ − + +

1 Ion intensities are scored as follows: high, +++; medium, ++; low, +; not detected, −.

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capable of metabolizing the precursor (such as IX) into theintestine of individuals who lack the appropriate intestinalmicrobiota to activate the precursor. This inoculation wouldlead to efficient production of the active metabolite. Wesuccessfully showed the efficiency of this strategy, which werefer to as the use of ‘functional probiotics,’ i.e. the introduc-tion of specific bacteria in the intestine with the aim toimprove host health, by the supplementation of a specificadditional metabolic function into the microbial ecosystem.

A first attempt to apply this strategy to phytoestrogenmetabolism has been made by Decroos et al. who isolated aconsortium capable of equol production [121,122]. Thesepositive results encouraged us to apply the same strategy forhop phytoestrogens. After screening a number of candidatedemethylating bacterial strains (as the activation of IX into8-PN is a demethylation of a methoxy-group on the A-ring),only one bacterium, Eubacterium limosum, was found whichcould efficiently convert IX into 8-PN after a selectionprocedure. Administration of the strain to fecal samplesincreased the 8-prenylnaringenin production in these sam-ples [102]. In recent years, this bacterium has gainedincreasing attention because of its beneficial effects on thecolonic environment in inflammatory bowel disease, possiblyattributed to its butyrate-producing capacity [123,124].Therefore, the bacterium would be a suitable candidateprobiotic to increase and standardize intestinal 8-prenylnar-ingenin production. Administration of the bacterium to theSHIME and rats confirmed this potential application [104].

To create a rat model for high- and low-8-PN producingindividuals, germ-free (GF) rats were colonized with fecalmicrobiota from subjects with either a high (Hop+) or a low(Hop-) 8-PN production status. GF rats acted as negativecontrol. Two separate experiments were designed. Thepurpose of Expt. 1 was to assess the formation of 8-PN fromIX in GF, Hop− and Hop+ rats. Therefore, all rats weregavaged every morning for 4 days with IX (2 mg/kg body wt).Expt. 2was designed to assess the formationof 8-PN from IX inGF, Hop− and Hop+ rats supplemented with E. limosum.Therefore, all rats were gavaged everyday for 4 days with theIX solution and for 6 days (starting 2 days before the IXadministration) with E. limosum (109 CFU/rat). As expected,

after administration of only IX (Expt. 1), highest 8-PN titerswere detected in the intestine, urine and plasma of the ratscolonizedwithHop+microbiota and no 8-PNwas detected inany of the samples of the GF control rats. However, combinedadministration of IX and E. limosum triggered 8-PNproductionin the GF rats and increased 8-PN production in the ratscolonizedwithHop−microbiota and 8-PNexcretionwas nowsimilar in all rats. Further confirmation was obtained uponanalysis of organ samples of the different rats in Expt. 1 andExpt. 2 (Table 2).Whereas the glucuronides of 8-PN could onlybe detected in the organs of the rats colonized with Hop+microbiota when only IX was dosed, 8-PN-glucuronides weredetected in the organs of all rats upon combined administra-tion of IX and E. limosum. Therefore, the possible use of E.limosum to balance the exposure and possible health effects of8-PN in all individuals was definitively shown.

In addition to the probiotic strategy, other applications of E.limosum are currently being investigated to standardize 8-PNexposure, including theprefermentationof IX in thehopproductitself. Application of this alternative strategy removes beforeingestion the source of variability, i.e. variable activation of IX inthe intestine, by converting IX into 8-PN in the original product.

5. Concluding remarks

With polyphenols as examples, the aim of this review wasto show that research towards gut microbial metabolism ofnutraceuticals, such as botanical extracts, is of criticalimportance when trying to characterize health effects ofsuch products, yet that it adds another important degree ofcomplexity to the story. Indeed, it is a particularly challengingtask to study polyphenol–microbiota interactions and theirrelevance to human health, considering the high diversity ofpolyphenols and their variable bioavailability and activities,the large interindividual variability in both composition andactivity of gut microbiota, and the numerous molecularmechanisms by which they may trigger health responses.Moreover, there is a non-exhaustive list of interfering factors,which can impact the final outcome at each stage of theabsorption, metabolism and health response process.

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Understanding how these different factors interact andcontribute to the in situ exposure is crucial for the properinterpretation of biological responses, or the lack thereof, inobservational and intervention studies. Furthermore, knowledgeof the critical parameters involved will help to identifysubpopulations thatmay benefit or be at risk and to substantiatehealth claims. As shown with the example of phytoestrogens, itmay even offer innovative opportunities for new productdevelopment. New strategies which target specific metabolicprocesses in the gut can influence the bioavailability of activecompounds ormetabolites into a desired direction and thereforehave a profound effect on the final health response towardspecific products.

However the high degree of complexity implies thatcontrolling and evaluating all, often interrelated, variableswithin one single experimental setting will not be feasible.Consequently, to get the whole picture, a multidisciplinaryapproach, combining both in vitro- and in vivo studies, is the keyto success. The more recent studies applying overall morecomprehensive and less biased measurement technologies incombination with novel pattern recognition techniques to allsystems involved, i.e. diet, microbiota, and host, clearly demon-strated potential of such multidisciplinary approaches todescribe the complexity of polyphenol–microbiota interactionsand provide more comprehensive insights into their physiolog-ical relevance. New hypotheses on the molecular and cellularmechanisms of action have already been proposed and there isno doubt that many other will rapidly emerge from theincreasing use of nutrigenomic approaches in the near future[125].

Acknowledgement

Sam Possemiers benefits from a post-doctoral grant fromthe Research Foundation — Flanders (FWO-Vlaanderen).

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