Mechanisms of gut bacterial metabolism of dietary polyphenols into bioactive compounds

ABSTRACT

The fruits and vegetables we consume as part of our diet are rich in bioactive metabolites that can prevent and ameliorate cardiometabolic diseases, cancers, and neurological conditions. Polyphenols are a major metabolite family that has been intensively investigated in this context. However, for these compounds to exert their optimal bioactivity, they rely on the enzymatic capacity of an individual’s gut microbiota. Indeed, for most polyphenols, the human host is restricted to more basic metabolism such as deglycosylation and hepatic conjugation. In this review, we discuss the mechanisms by which gut bacteria metabolize the core scaffold of polyphenol substrates, and how their conversion into bioactive small molecules impacts host health.

1. Introduction

Polyphenols are an abundant family of secondary metabolites produced by plants, and they are mainly studied for their various health benefits¹. The polyphenol family of compounds is very large and structurally diverse and includes flavonoids, tannins and lignans, stilbenes, and phenolic acids. Among these different polyphenol classes, flavonoids have received most of the attention to date. Due to the presence of aromatic chromophores in their structure, flavonoids are the pigments that are responsible for the distinctive colors of many fruits, vegetables, and flowers. For plants, they play important roles in pollination and reproduction, as well as in protection from pathogens and UV radiation.^2,3 The basic structure of flavonoids consists of two aromatic rings, designated A and B, which are connected by a central heterocyclic pyrene C-ring.⁴ Depending on the degree of oxidation and saturation of the C-ring, as well as the position and number of hydroxyl and other functional groups on the A- and B-rings, flavonoids are further classified into different subgroups, including flavones, flavanols, flavanones, and isoflavones (Figure 1).¹

Figure 1.

Flavonoids are classified according to the oxidation state of their central C-ring into six major classes: flavones, flavonols, flavanones, isoflavones, anthocyanidins, and chalcones.

Flavonoids are widely consumed in the human diet through colorful fruits and vegetables, red wine, chocolate, and tea, accounting for an average daily intake of 200–600 mg in adults. It is estimated that there are over 4,000 dietary flavonoids, of which several dozen are investigated for their potential positive effects in preventing and managing various diseases, including cardiovascular and metabolic diseases,^5–7 cancer,^8–10 and Alzheimer’s disease.^11,12 Flavonoids act in a variety of ways, including serving as antioxidant,^13,14 anti-inflammatory,^15,16 antimutagenic,^8,17–19 antiatherogenic,^20,21 antidiabetic,^22–24 and antithrombotic compounds.^25–27 It is worth noting that most of these epidemiological studies involving a dietary intervention are not designed to specifically test the health benefits of individual compounds and might be confounded by additional variables such as a higher dietary fiber content. Therefore, additional controlled intervention studies will be required help elucidate the health benefits of individual dietary compounds, and the potential role of the gut microbiota in their bioactivity.

Despite their various attributed health benefits, the bioavailability of polyphenols in humans is low, as the majority of ingested polyphenols are not absorbed in the small intestine.²⁸ Host metabolism of flavonoids and other polyphenols is limited to select deglycosylations, as well as hepatic phase I conjugation with glucuronic acid, sulfate, or glutathione.^29,30 These conjugations render the metabolites more hydrophilic and facilitate their urinary excretion from the body or their enterohepatic circulation to the intestine, where they can be further metabolized by the gut bacteria. To date, no mammalian enzymes have been identified that can break down the core flavonoid three-ring structure.

The bulk of ingested polyphenols reaches the colon relatively unmodified, where they present as a substrate for catabolism by certain gut microbiota.³¹ The metabolism of polyphenols by the gut microbiota involves a series of enzymatic reactions, including hydrolysis, reduction, and dihydroxylation,^32–35 which result in the formation of various metabolites such as monophenolic acids, dihydroflavonols, and urolithins.^36–38 These metabolites can be absorbed by the colonic epithelium and reach the systemic circulation, where they can exert their biological activities. Our group recently demonstrated that bacterial flavonoid catabolite 4-hydroxyphenylacetic acid was sufficient to revert diet-induced hepatic steatosis in mice.⁷

The metabolism of polyphenols by the gut microbiota is a complex process that is influenced by various factors, including the structure and oxidation state of the molecule itself, as well as the composition of the gut microbiota, and their enzymatic repertoire.³⁹ Gut bacterial enzymes are essential for the metabolism and processing of a wide range of dietary components that are not digestible by human enzymes.⁴⁰ In this review, we focus on the different gut microbiota and their enzymatic activities involved in polyphenol catabolism characterized to date (Table 1). Whereas several microbes and enzymes have been identified, we’ve likely only scratched the surface in this field, leaving many pathways to be discovered.

Table 1.

Gut microbial enzymes involved in polyphenol metabolism.

Substrate	Enzyme	Products	Corresponding gut bacteria
Deglycosylation
O-linked flavonoid glycosides	Glycosidase	Flavonoid aglycone	Bifidobacteriaceae, Lactobacillaceae, and Lachnospiraceae41–47
C-linked flavonoid glycosides	Glycosidase	Flavonoid aglycone	Lachnospiraceae, Enterococcaceae,Streptococcaceae.44,45,48,49
Flavonoid glycosides	β-glucosidase	Flavonoid aglycone	Lactobacillus crispatus, Companilactobacillus paralimentarius, Lacticaseibacillus paracasei, Lactiplantibacillus plantarum Levilactobacillus hammesii Furfurilactobacillus milii50
Flavones
Flavone	Flavone reductases	Dihydroflavone	F. plautii ATCC 49,53135 Clostridium difficile DSM 630 and Clostridium perfringens ATCC 13,12435
Dihydroflavone	Chalcone isomerase	Chalcone	E. ramulus DSM 16,296, F. plautii51
Chalcone	Enoate reductase	Dihydrochalcone	E. ramulus32, F. plautii ATCC 49,53135
Dihydrochalcone	Phloretin hydrolase	Phloroglucinol +3-(4-hydroxyphenyl)propionic acid	E. ramulus,32F. plautii ATCC 49,53135
Flavonol
Flavonol	Flavone reductases	Dihydroflavonol	E. ramulus,32F. plautii ATCC 49,53135
Dihydroflavonol	Chalcone isomerase	Hydroxyaurone	E. ramulus,32F. plautii ATCC 49,53135
Hydroxyaurone		Phloroglucinol +4-Hydroxyphenylacetic acid	E. ramulus,32F. plautii ATCC 49,53135
Quercetin
Quercetin	Quercetinases	Protocatechuic acid + 2,4,6-Trihydroxybenzoic acid	Bacillus subtilis52–54
Isoflavones
Daidzin	Daidzein reductase	(R)-Dihydrodaidzein + (S)- Dihydrodaidzein	Eggerthella sp. strain YY7918 (99% identity), and Slackia (only 43% identity).55,56 Lactococcus 20-92 Lactobacillus sp. Niu-O16, Eggerthella sp. Julong 732, Lactococcus garvieae 20–92, Slackia isoflavoniconvertens, and Adlercreutzia equolifaciens57–60
(R)-Dihydrodaidzein	Dihydrodaidzein racemase	(S)- Dihydrodaidzein	Lactobacillus sp. Niu-O16, Eggerthella sp. Julong 732, Lactococcus garvieae 20–92, Slackia isoflavoniconvertens, and Adlercreutzia equolifaciens57–60
(S)- Dihydrodaidzein	Dihydrodaidzein reductase	(3S, 4 R)-tetrahydrodaidzein	Lactobacillus sp. Niu-O16, Eggerthella sp. Julong 732, Lactococcus garvieae 20–92, Slackia isoflavoniconvertens, and Adlercreutzia equolifaciens57–60
(3S, 4 R)-tetrahydrodaidzein	Tetrahydrodiadzein reductase	(S)-equol	Slackia isoflavoniconvertens and Slackia sp. strain NATTS Lactococcus sp.Eggerthella sp. strain YY791856 Lactobacillus sp. Niu-O16, Eggerthella sp. Julong 732, Lactococcus garvieae 20–92, Slackia isoflavoniconvertens, and Adlercreutzia equolifaciens57–60
Hydroxycinnamic acids
Chlorogenic acids	Hydroxycinnamic acid esterase	hydroxycinnamic acid + Quinic acid	L. plantarum,61L.johnsonii,62Lactobacillus acidophilus63, B. longum64 and B. animalis .65
Hydroxycinnamic acid	Hydroxycinnamate reductase	Phenylpropionic acid	L. plantarum .66 L. fermentum L. plantarum enzyme .67 L. rossiae L. plantarum reductase ,67 L. kunkeei ,68 and Weissella cibaria .67
Hydroxycinnamic acids	Phenolic acid decarboxylase	vinyl derivatives	Lactobacillus spp69
Vinyl derivatives	vinyl phenol reductase	Ethyl derivatives	Lactobacillus plantarum WCFS170
Urolithins
Urolithin M5	10-dehydroxylase	Urolithin D	Entrocloster spp71
Urolithin M5	9-dehydroxylase	Urolithin E	Entrocloster spp71
Urolithin M5	4-dehydroylase	Urolithin 6	Ellagibacter spp38
Urolithin M6	10-dehydroxylase	Urolithin C	Gordonibacter urolithinfaciens (DSM 27213T)36
Urolithin M6	8-dehydroxylation	Isourolithin A	Ellagibacter38
Urolithin D	4-dehydroylase	Urolithin C	Gordonibacter urolithinfaciens (DSM 27213T)36
Urolithin D	4-dehydroylase	Urolithin G	Entrocloster spp71
Urolithin C	8-dehydroxylation	Isourolithin A	Ellagibacter38
Urolithin C	4-dehydroylase	Urolithin A	E. bolteae CEBAS S4A9 and DSM 15670T, E. asparagiformis DSM 15981T, and E. citroniae DSM 19261T
Urolithin M7	10-dehydroylase	urolithin A	E. clostridioformis DSM 933T
Isourolithin A	9-dehydroxylation	Urolithin B	Ellagibacter38,72

2. Deglycosylation: a key first step in flavonoid metabolism

Flavonoids occur predominantly in plants as either O- or C-glycosides as this enhances their solubility. O-glycosylated flavonoids contain mono- or disaccharides attached to the hydroxyl groups on the backbone A or C rings, while C-glycosides involve the attachment of a sugar molecule to the carbon atom of the A-ring.⁷³ Deglycosylation of flavonoids by the host or intestinal microbiota is critical for their absorption and further phase I hepatic metabolism. On the host side, the O-deglycosylation process can be facilitated by enzymes such as lactase-phlorizin hydrolase and β-glucosidase expressed in the intestinal epithelium,^74,75 whereas flavonoid C-glycosides are refractory to host enzymes (Figure 2). Hydrolysis of flavonoid O-glycosides by the gut microbiota is well documented across several genera of gut microbes, including Bifidobacteriaceae, Lactobacillaceae, and Lachnospiraceae.^41–47 Cleavage of C-glycosides is far less common and relatively understudied but has been described for specific species of Lachnospiraceae, Enterococcaceae, and Streptococcaceae.^44,45,48,49 Food-fermenting lactobacilli encode a wealth of enzymes specifically capable of phytochemical β-glucoside hydrolysis, including phospho-β-glucosidases (for example encoded by Lactobacillus crispatus, Companilactobacillus paralimentarius, and Lactiplantibacillus plantarum), and 3β-glucosidases (for example from Levilactobacillus hammesii, and Furfurilactobacillus milii).⁵⁰

Figure 2.

Deglycosylation of C-linked flavonoid glycosides is exclusively catalyzed by the small intestinal microbiota, whereas cleavage of O-linked sugars can also be performed in the stomach and small intestine by host enzymes such as lactase-phlorizin hydrolase and β-glucosidase.

Since both the host and the gut microbiota are capable of deglycosylation, there is some controversy regarding the relative role of microbial enzymes and the need for deglycosylation to ensure flavonoid absorption. Some studies report direct uptake of glycosylated flavonoids into the bloodstream, facilitated by glucose transporters in the intestinal lumen. For example, quercetin glucosides were shown to be transported through sodium-glucose co-transporters (SGLTs) and GLUT2 within 30 minutes after oral ingestion, and the amount of absorbed quercetin was enhanced when conjugated to glucose.^76–79 In a separate study, intestinal transport of anthocyanidins like delphinidin 3-rutinoside, cyanidin 3-rutinoside, and cyanidin 3-glucoside, also resulted in increased plasma concentrations a few hours after ingestion.⁸⁰ Interestingly, this systemic uptake does not seem to work analogous for glycosylated isoflavones, which rely on the gut microbiome for metabolism.^81,82

Overall, the deglycosylation of flavonoids in the gut involves both human and bacterial enzymes, and it represents an important first step for their further metabolism, as well as has implications for their bioavailability and associated health effects.

3. Enzymatic conversion of the flavonoid core

Gut microbial catabolism of the core structure of flavanols, flavanonols, flavones, and flavanones is catalyzed by similar enzymatic processes, yet can yield different products depending on the bacteria involved and their enzymatic repertoire. An additional source of catabolite variation is caused by differences in B-ring decorations of the parent flavonoid. Flavones, such as apigenin and luteolin, are converted to 3-(3-hydroxyphenyl)propionic acid and 3,4-dihydroxyphenylacetic acid by different gut bacteria, including Eubacterium ramulus, Blautia glucerasea, and Eggerthella lenta.^83,84 Flavanones, such as hesperetin and naringenin, are converted to 3-(3-hydroxyphenyl)propionic acid and phloroglucinol by strains of Bacteroides thetaiotaomicron and Flavonifractor plautii (previously classified as Clostridium orbiscindens).^85,86 The catabolism of flavones into monophenolic acids involves a four-step metabolic pathway, initiated by C-ring reduction and subsequent cleavage, followed by further reduction of the resulting α,β-unsaturated ketone moiety, and finally, a hydrolysis reaction that cleaves the molecule.

The enzymes involved in these reactions and their corresponding genes have been identified in studies with E. ramulus and F. plautii^35,47 and are discussed in more detail below (Table 2).

Table 2.

Uniprot identifiers for functionally characterized polyphenol metabolizing enzymes.

Enzyme	Uniprot ID
Lactase/phlorizin hydrolase	P09848
Chalcone isomerase	V9P0A9
Enoate reductase	V9P074
Phloretin hydrolase	Q715L4
Quercetin 2,3-dioxygenase	P42106
Daidzein reductase	E1CIA4
Dihydrodaizein racemase	I7H868
Tetrahydrodaidzein reductase	E7FL40
Phenolic acid decarboxylase	Q9KHI7

3.1. Reduction of the central C-ring by flavone reductase

Flavone reductases catalyze the first step in the catabolic pathway of flavone and flavonol aglycones, using a flavin mononucleotide (FMN) cofactor molecule. While catalyzing the first step in the pathway, this novel class of ene-reductases enzymes was only recently functionally characterized in F. plautii ATCC 49,531.³⁵ The activity of the F. plautii flavone reductase (FLR) was demonstrated in an in vitro assay using apigenin as a substrate and involves the transfer of a hydride ion from the FMNH₂ cofactor to the C2=C3 double bond in the flavone’s C-ring. This reduction converts apigenin to its dihydroflavone form, naringenin (Figure 3).

Figure 3.

The catabolism of flavones into monophenolic acids involves a four-step metabolic pathway. Flavone reductase (FLR) initiates by reducing the C-ring, followed by chalcone isomerase (chi)-mediated cleavage, and subsequent enoate reductase (EnoR)-mediated further reduction of the resulting α,β-unsaturated ketone moiety. Finally, phloretin hydrolase (PHY) catalyzes a second hydrolysis reaction that cleaves the molecule.

The crystal structure of the FLR holoenzyme in complex with apigenin, a flavone substrate, revealed the location of the active site and the residues involved in catalysis.³⁵ A reaction mechanism was proposed and supported by circular dichroism spectrum analysis. FLR consists of a compact N-terminal compact α-β-α domain and an extended C-terminal domain. FLR forms a homodimer and binds the FMN cofactor at the dimer interface, which is formed involving parts of both the N-terminal and C-terminal domains and burying about a third of the enzyme’s surface area. While functionally similar to Old-Yellow-Enzymes (OYEs), FLR differs in two notable ways.^32,87 Firstly, FLR lacks a suitable residue that forms direct hydrogen bonds to the C4-carbonyl group of apigenin for substrate activation. Instead, a hydrogen-bond relay is formed through the hydroxyl of Thr279 via an H₂O molecule, and the hydride from FMNH- is transferred to apigenin’s β-carbon. Secondly, FLR does not contain a direct proton donor to reduce the α-carbon of apigenin, which contrasts with OYEs that utilize a tyrosine residue as a general acid. Since the crystal structure revealed that the apigenin α-carbon is exposed to the bulk solvent, the required proton for the formation of the naringenin product likely comes from a water molecule.³⁵

Interestingly, this reduction catalyzed by FLR is reversible, yet the reduction of flavone/flavonol, is favored over the oxidation of dihydroflavones. The substrate promiscuity of FLR was demonstrated in the reduction of other flavone and flavonol substrates, including quercetin, kaempferol, myricetin, chrysin, luteolin, and diosmetin. Interestingly, the F. plautii FLR is unable to reduce flavones that contain methoxy groups, such as tangeretin and nobiletin. FLR-like genes were identified in members of the human gut microbiome, as well as in pathogenic gut bacteria such as Clostridium difficile DSM 630 and Clostridium perfringens ATCC 13,124.³⁵ Because of their unique fold and function, flavone reductases were proposed to form a novel enzyme class separate from previously identified reductases or flavone synthases.

3.2. Chalcone isomerase opens the central C-ring

The conversion of dihydroflavone to chalcone is the second step in the flavonone catabolic pathway, and the chalcone isomerase (CHI) catalyzing this reaction has been extensively studied in E. ramulus DSM 16,296 (exemplified by the conversion of naringenin to naringenin chalcone in Figure 3).⁸⁸ Purified CHI was next demonstrated to participate in the catabolism of various flavonols and flavanonols, including taxifolin and dihydrokaempferol. The E. ramulus CHI crystal structure has a hexameric organization, consisting of three dimer subunits.³² Enzymatic conversion efficiencies for various flavanone substrates indicated that a B-ring containing a para hydroxyl group is crucial for CHI activity. Molecular docking of naringenin to the active site confirmed that this hydroxyl forms hydrogen bonds with Asp79 and Gln101, ensuring proper substrate orientation. The substrate specificity of CHI is relatively relaxed, allowing flavanones with B-rings that contain hydroxyl or methoxy groups at the meta position. His33 is a critical amino acid located in the active site of CHI, which is postulated to play a crucial role in the acid-base catalysis-mediated opening of the heterocyclic C-ring. The proton transfer from the C-3 carbon of the flavonoid intermediate to His33 generates a negative charge, which facilitates ring opening. The key catalytic role of His33 was confirmed by site-specific mutations, which resulted in loss of CHI activity.^51,89 It is worth noting that both the A- and B-rings are vital for the formation and stability of the CHI enzyme-substrate complex. CHI homologs have since been identified across various other genera of flavonoid-catabolizing bacteria, including F. plautii.⁵¹

3.3. Reduction of the chalcone double bond by enoate reductase

The third step in the flavone/flavonol catabolic pathway is the reduction of the C=C bond, which was introduced as a result of the CHI reaction. This reduction converts the chalcones to their respective dihydrochalcones, and is catalyzed by the enzyme enoate reductase^32,89,90(Figure 3). Similar to FLR -which initiates the flavone catabolic pathway-, enoate reductase also belongs to the enzyme class of ene-reductases. An important distinction between the two types of enzymes is that enoate reductase uses NADH as a reducing agent, whereas FLR is incapable of using NAD(P)H.⁹⁰ The E. ramulus enoate reductase crystal structure revealed that it has a multidomain structure, consisting of a barrel domain and an oxygen-sensitive domain with an iron-sulfur cluster.³² The activity of this enzyme was demonstrated by converting naringenin chalcone to phloretin. To date, E. ramulus and F. plautii are the only documented gut bacteria that can produce the dihydrochalcone phloretin.

3.4. Final cleavage of the dihydrochalcone by phloretin hydrolase

The final step in flavone/flavanone catabolism is catalyzed by phloretin hydrolase (PHY). Anecdotally, the E. ramulus phy gene was the first gene to be discovered in the bacterial flavonoid degradation pathway. Phy is transcribed from an 822 base-pair open reading frame and forms a homodimer in solution, consisting of two 30 kDa subunits.³² PHY hydrolyzes the C-C bond at the A-ring of phloretin, yielding phloroglucinol and the monophenolic acid 3-(4-hydroxyphenyl)propionic acid (Figure 3).⁹¹ This C-C bond cleavage occurs through a reverse Claisen mechanism and is facilitated by a 6-membered transition state.⁹² Tryptophan, which is part of the substrate binding site, plays a vital role in catalysis.⁹¹ The phloretin hydrolase enzyme in F. plautii ATCC 49,531 shares 46.92% amino acid sequence similarity with the PHY enzyme in E. ramulus DSM 16,296.³⁵

4. An alternative pathway for flavonoid catabolism using bacterial dioxygenases

An alternative, oxygen-dependent pathway for microbial flavonoid catabolism was discovered in a series of in vitro degradation studies of the flavonol quercetin by the Bacillus subtilis enzyme YxaG. Gut microbial catabolism of the quercetin typically yields protocatechuic acid (3,4-dihydroxybenzoic acid) and 3,4-dihydroxyphenylacetic acid as primary metabolites. The enzymatic release of protocatechuic acid from quercetin can be facilitated by YxaG-like bacterial quercetin dioxygenases, also known as quercetinases.^52–54 Quercetinase uses molecular oxygen to catalyze the simultaneous cleavage of two carbon-carbon bonds in the heterocyclic C-ring, resulting in the formation of an ester that links the two aromatic A- and B-rings the release of carbon monoxide. The enzymatic mechanism requires a divalent cation to chelate the 4-keto/2,3-enol moiety of quercetin, thereby promoting the 3,4-diketo tautomer. First, the hydrogen atom at position 2 is removed, and the resulting carbon 2-centered radical next reacts with superoxide to form a five-membered endo-peroxide. The endo-peroxide is finally rearranged through the conversion of the 3-keto into an acylium ion, leading to the formation of a 2-protocatechuoyl-phloroglucinol carboxylic acid ester, and the release of carbon monoxide. The ester can subsequently be hydrolyzed into protocatechuic acid and 2,4,6-trihydroxybenzoic acid, while the latter can be further decarboxylated to form the gut microbial metabolite phloroglucinol^52,92,93 (Figure 4). Further investigations into gut microbial dioxygenase-dependent metabolism of quercetin and other flavonols will elucidate if these oxygen-dependent reactions are relevant in the anaerobic intestinal environment and whether they are perhaps localized to the small intestine or close to the mucosal surface, as well as identify potential enzymes involved in the latter hydrolysis and decarboxylation steps of this pathway.

Figure 4.

An alternative pathway for opening the flavonoid C-ring involves the action of bacterial dioxygenases, like quercetinase, which require molecular oxygen for their activity.

5. Enzymatic conversion of isoflavones

Isoflavones represent a remarkable structural and functional subclass of polyphenols because several have been characterized as phytoestrogens because they are chemically and structurally similar to 17β-estradiol.^94–96 Due to this similarity, isoflavones are investigated for their modulatory effect in certain hormone-dependent diseases, such as breast cancer, colon cancer, and cardiometabolic diseases.^97–99

Daidzein is one of the most intensively investigated isoflavones and is present primarily in soybeans. It is primarily found in its glycosylated form, daidzin.^100–102 Like other flavonoids, isoflavones are poorly absorbed by the small intestine due to their bulky structure.^103–105 This provides ample substrate availability for the gut microbiota, which have an important role in their metabolism and bioactivity. Different types of gut microbial products of daidzein include (S)-equol and O-desmethylangolensin (O-DMA).^57,104–106 The human host does not produce enzymes capable of metabolizing daidzein, and depending on the gut microbial enzymatic repertoire, individuals are either (S)-equol producers or non-producers.¹⁰⁷ An estimated 30–50% of the total human population harbors (S-)-equol producing bacteria in their gut microbiome, while approximately 40–50% of people’s microbiota are estimated to be capable of O-DMA production.^97,108

Only a few bacterial strains have been characterized to metabolize daidzein to (S)-equol, including Lactobacillus sp. Niu-O16, Eggerthella sp. Julong 732, Lactococcus garvieae 20–92, Slackia isoflavoniconvertens, and Adlercreutzia equolifaciens .^57–60 In addition, certain bacterial strains are incapable of fully converting daidzein to (S)-equol, likely due to the absence of one or two enzymes in the pathway. It is of note that these partial converters may work together with different strains in the gut microbiota that complement their capabilities and can still result in an (S)-equol producer individual.^59,60

A characterized pathway for the metabolism of daidzein to (S)-equol involves four subsequent enzymatic steps.^48,104,109 Daidzein is made available to (S)-equol producing microbes after deglycosylation of daidzin, via β-glucosidase enzymes that could be produced by either host intestinal epithelial cells, or the gut microbiota.^110,111 A portion of this aglycone can get absorbed by the basolateral membrane of the intestine, and become conjugated in the liver.¹¹² Some conjugated daidzein can, in turn, reenter the blood circulation, while a large proportion is re-introduced in the small intestine via enterohepatic circulation, and can be further metabolized by the gut microbiota.¹¹³

5.1. Daidzein reductase initiates the conversion of non-glycosylated isoflavones

The first step in the gut bacterial daidzein conversion pathway is catalyzed by the oxidoreductase called daidzein reductase (DZR) and yields dihydrodaidzein (Figure 5).³⁴ This enzyme was initially identified in Lactococcus 20–92, and the dzr gene that encodes it has been shown to be upregulated by the presence of daidzein.¹¹⁴ Functional homologs were later identified in (S)-equol producers Eggerthella sp. strain YY7918 (99% identity), and Slackia (only 43% identity).^55,56

Figure 5.

Gut microbial conversion of isoflavonoids to phytoestrogens (S)-equol and O-desmethylangolensin (O-DMA) involves a series of reduction steps and is more efficient if a racemase is present in the microbiota. DZR = daidzein reductase; DDRC = dihydrodaidzein racemase; DDR = dihydrodaidzein reductase; TDR = tetrahydrodaidzein reductase.

Daidzein reductase has a cysteine-rich [4Fe-4S] cluster domain, which assists in the reduction of the C2-C3 double bond to yield mainly the ®-enantiomer of dihydrodaidzein.^33,115 Three cofactor binding motifs for NAD(P)H, FAD, and FMN were identified, yet daidzein reductase mainly uses NADPH as a cofactor resulting in its classification as a NADP(H)-dependent daidzein reductase.³⁴

5.2. Dihydrodaidzein racemase provides an (S)-enantiomer supply for the subsequent step

Due to an asymmetric carbon atom at the C-3 position, dihydrodaidzein has two enantiomers, (R)-dihydrodaidzein and (S)-dihydrodaidzein.¹¹⁶ The next step in the conversion pathway involves dihydrodaidzein racemase (DDRC), which is capable of dihydrodaidzein conversation into its mirror-image isomer, thereby producing a racemic mixture.¹⁰⁹ This reaction catalyzed by dihydrodaidzein racemase involves an enolization and epimerization (Figure 5).¹¹⁵ The gene ddrc encoding for this enzyme is found in all of the characterized bacterial strains capable of the conversion of daidzein to (S)-equol, except Slackia sp. NATTS .¹¹⁷ Dihydrodaidzein racemase was the last enzyme to be functionally characterized, thereby completing the mapping of enzymes onto the conversion pathway of daidzein to (S)-equol.¹¹⁵ Since the production of (S)-equol is a stereospecific process, dihydrodaidzein racemase, in essence, ensures the supply of (S)-enantiomer substrate from the total dihydrodaidzein pool.

5.3. Dihydrodaidzein reductase is key for stereospecific product formation

Dihydrodaidzein reductase (DDR) catalyzes the third step in the daidzein conversion pathway, the reduction of dihydrodaidzein to tetrahydrodaidzein (Figure 5). Dihydrodaidzein reductase operates primarily by involving the transfer of electrons from a cofactor molecule, such as NADH or NADPH, to its (S)-dihydrodaidzein substrate, reducing the ketone group on position C-4 to an alcohol.¹¹⁴ This reduction results in the stereospecific production of (3S,4 R)- tetrahydrodiadzein.¹¹⁷ Dihydrodaidzein reductase belongs to the short-chain dehydrogenase/reductase (SDR) superfamily, which is characterized by a conserved NAD(P)H-binding domain involved in electron transfer during oxidation-reduction reactions.^{55,56,114,118}

A subset of individuals harbor gut microbes capable of performing the first enzymatic reactions in the pathway (DZR and DDRC), but lack DDR and hence cannot produce (S)-equol. For these people, the intermediate metabolite (S)-dihydrodaidzein can be transformed into O-desmethylangolensin (O-DMA) via cleavage of the C-ring. Individuals with an (S)-equol-producing microbiota could, in addition, produce O-DMA, but analysis of urinary samples suggests that the conversion pathway to (S)-equol is predominant.^119,120

5.4. Tetrahydrodaidzein reductase produces (S)-equol

The final step in the daidzein conversion pathway is the reduction of tetrahydrodaidzein to (S)-equol, catalyzed by the glycyl radical enzyme tetrahydrodaidzein reductase (TDR, Figure 5).¹¹⁴ TDR from Slackia isoflavoniconvertens and Slackia sp. strain NATTS contains a characteristic RVXG glycyl radical motif, which is not completely conserved in the TDR enzymes of (S)-equol producers Lactococcus sp. strain 20–92 and Eggerthella sp. strain YY7918.⁵⁶ Glycyl radical enzymes only function in the absence of oxygen and catalyze the abstraction of substrate hydrogen atoms in a mechanism involving a cation radical intermediate.^33,121 First, TDR is proposed to remove a hydrogen atom from tetrahydrodaidzein’s C3 position, forming a stable benzylic radical. Subsequently, the benzylic radical mediates the loss of the hydroxyl group located at the C4 position, leading to the formation of a delocalized radical cation. This radical cation is next reduced by the addition of a hydride donor at the C4 position. Finally, a hydrogen atom is added back to the C3 position, producing (S)-equol.^{33,56,122,123}

A study of the TDR from Eggerthella sp. strain YY7918 indicated that the enzyme might not function as a reductase, but rather as a novel type of dismutase. The enzyme is proposed to function in the absence of cofactors such as NAD(P)H to transform tetrahydrodaidzein into (S)-equol and produce dihydrodaidzein as a by-product via a disproportionation reaction.⁵⁵ Further investigations will shed more light on the different proposed TDR mechanisms in Slackia and Eggerthella.

While the key enzymes in the bacterial pathway for conversion of daidzein to (S)-equol have been identified, several questions remain unresolved. In particular, the role of dihydrodaidzein racemase (DDRC) has received little attention thus far.⁵⁵ Previous studies have demonstrated that the presence of the ddrc gene in combination with the three reductase genes (dzr, ddr, and tdr) results in higher (S)-equol production levels compared to when the ddrc racemase gene is absent.^115,124 However, since lower levels of (S)-equol production are still observed with only the three reductases present,³⁴ this suggests that the racemase is not essential yet makes the production process more efficient. It is tempting to speculate that this increase in efficiency is due to the increased supply of (S)-dihydrodaidzein for further stereospecific reduction by DDR, thereby depleting the total pool of (RS)-dihydrodaidzein. In the absence of the racemase, both dihydrodaidzein enantiomers are formed and only the (S)-dihydrodaidzein half of the pool can be metabolized further, resulting in lower yields.

6. Enzymatic conversion of hydroxycinnamic acids

Hydroxycinnamic acids are a widely distributed family of nonflavonoid phenolic acids which are characterized by their double bond-containing carboxylic acid side chain linked to the benzene ring (Figure 6). Hydroxycinnamic acids have a variety of beneficial effects on host health, attributed to their antioxidant, antimicrobial, and anti-inflammatory functions and their plasma concentrations are correlated with a reduced risk of cardiovascular diseases, diabetes, hypertension, and colorectal cancer.^125–127

Figure 6.

Hydroxycinnamic acids occur as simple free fatty acids or in an esterified form. Examples of simple hydroxycinnamic acids include caffeic acid, ferulic acid, sinapic acid, and coumaric acid. The family of hydroxycinnamic acids that are esterified to quinic acid is named chlorogenic acids.

Hydroxycinnamic acids are most abundantly present in berries, potatoes, coffee, tea, and wine¹²⁸ and they occur in free acid or conjugated forms. Examples of non-conjugated hydroxycinnamic acids include caffeic acid (3,4-dihydroxycinnamic acid), ferulic acid (4-hydroxy-3-methoxycinnamic acid), sinapic acid (4-hydroxy-3,5-dimethoxycinnamic acid), and coumaric acid (4-hydroxycinnamic acid). Hydroxycinnamic acids can also occur in an esterified form, bound to sugars like arabinose or galactose, or in case of the chlorogenic acids bound to quinic acid, yielding 5-O-caffeoylquinic acid, 5-O-feruloylquinic acid, and 5-O-coumaroylquinic acid (Figure 6).¹²⁹

Hydroxycinnamic acids can be absorbed through passive diffusion and the monocarboxylic acid transporter system, or it can be absorbed post-deconjugation. After absorption, hydroxycinnamic acids can be sulfated, methylated, or glucuronidated by phase II enzymes in the liver or small intestine. Alternately, the fraction of non-absorbed hydroxycinnamic acids can be metabolized by the gut microbiome.^130,131

6.1. Hydrolysis of hydroxycinnamic acid esters by feruloyl esterase (FAE)

Several lactic acid bacteria, including members of the lactobacilli and bifidobacteria, produce hydroxycinnamic esterases, also known as feruloyl esterases (FAEs; Figure 7). The hydrolysis reaction catalyzed by these esterases cleaves chlorogenic acids, yielding quinic acid and hydroxycinnamic acid products, or sugar residues and hydroxycinnamic acid. Hydroxycinnamic esterases have been characterized in L. plantarum ,⁶¹ L. johnsonii ,⁶² Lactobacillus acidophilus ⁶³, B. longum ,⁶⁴ and B. animalis .⁶⁵

Figure 7.

Gut microbial conversion of different chlorogenic acids to corresponding hydroxycinnamic acid and quinic acid. FAE = feruloyl esterase.

6.2. Reduction of hydroxycinnamic acids to phenylpropionic acid by hydroxycinnamate reductase

Hydroxycinnamate reductase (HcrB, also known as phenolic acid reductase), is a heterodimeric NADH-dependent reductase that reduces the double bonds in hydroxycinnamic acids to form phenylpropionic acids while re-oxidizing the reduced NADH co-factor to NAD⁺ .¹³² Hence, caffeic acid is reduced to dihydrocaffeic acid, coumaric acid to phloretic acid, and ferulic acids to dihydroferulic acids.⁶⁷

Similar to FAE enzymes, HcrB is also present in lactic acid bacteria, and has been characterized in L. plantarum .⁶⁶ The L. fermentum hydroxycinnamic acid reductase is encoded by the hcrF gene and shares 63% amino acid homology with HcrB from L. plantarum.⁶⁷ The L. rossiae hydroxycinnamate reductase, Par1, shares an even lower sequence identity of ~ 25% compared to the functional homologues found in L. plantarum reductase ,⁶⁷ L. kunkeei ,⁶⁸ and Weissella cibaria .⁶⁷

6.3. Decarboxylation of hydroxycinnamic acids to vinyl phenol derivatives via phenolic acid decarboxylase (PAD)

Decarboxylation of hydroxycinnamic acids is an important mechanism to alleviate their potential antimicrobial effects. This reaction is catalyzed by enzymes of the phenolic acid decarboxylase (PAD) family, members of which are well-characterized and upregulated in the presence of caffeic acid. Decarboxylation results in the formation of vinyl derivatives that exert less cytotoxic stress on the lactobacillus cell wall. The decarboxylation of caffeic acid yields vinyl catechol, while the decarboxylation of ferulic acid and coumaric acid result in vinyl guaiacol and vinyl phenol respectively⁶⁹ (Figure 8). Several Lactobacillus species are capable of hydroxycinnamic acid decarboxylation, yet select strains are devoid of a pad homolog, including Lactobacillus delbrueckii, and Lacticaseibacillus casei BL23.¹³²

Figure 8.

Further metabolism of hydroxycinnamic acids to their corresponding phenolic metabolites by the gut bacteria. PAD = phenolic acid decarboxylase; VprA = vinyl phenol reductase; HcrB = hydroxycinnamate reductase.

6.4. Further reduction of vinyl phenols to their ethyl derivatives by vinyl phenol reductase

The L. plantarum WCFS1 vinyl phenol reductase, VprA, is capable of reducing vinyl catechol to 4-ethyl catechol, vinyl guaiacol to 4-ethyl guaiacol, and vinyl phenol to 4-ethyl phenol.⁷⁰ Interestingly, the phylogenetic distribution of the vprA gene appears to be limited to lactobacilli, indicating that this last conversion step might be more specialized.^70,132 The vinyl phenol reductase reaction has a prime use in the food industry, where it is used to modulate the release of volatile molecules, thereby improving the aroma and sensory characteristics of fruit and vegetable fermentations.¹³³ Ethyl phenols produced by Lactobacillus spp. in the human gut microbiome have demonstrated beneficial effects on brain activity, anxiety, and colon cancer.^134,135

7. Enzymatic conversion of ellagitannins and ellagic acid

In the past decade, urolithins have gained significant attention due to their profound anti-obesity and antitumor properties. It is of note that urolithin A has been approved by the Food and Drug Administration as a Generally Recognized as Safe oral supplement, and clinical trials have been performed to study its potential benefits on muscle strength in middle-aged adults.^39,136–141 Urolithins are a family of dibenzopyran-6-one metabolites that are formed from the microbial metabolism of the non-flavonoid phenolics ellagitannin and ellagic acid, which are present in pomegranates, raspberries, strawberries, and nuts. The microbial involvement in urolithin metabolism was demonstrated using germ-free rats, which lack gut microbiota. These animals did not produce urolithin metabolites after being fed an ellagitannin and ellagic acid-rich diet, highlighting the essential role of gut microbial metabolism.^{36,136,138,142–144}

Ellagitannins, a class of polyphenols, structurally consist of one or more hexahydroxydiphenic acid esters bound to a single glucose. However, some ellagitannins appear to as a monomer in structure, whereas others appear more dimeric. Due to these variabilities within their structure, ellagitannins cannot be absorbed in the small intestine without first undergoing extensive metabolism to smaller compounds, which have improved bioavailability.¹⁴⁵

Host enzymes in the jejunum hydrolyze the glucose moiety, after which hexahydroxydiphenic acid spontaneously lactonizes to form ellagic acid (Figure 9). Ellagic acid itself can also occur in the diet since it is present in berries and nuts. A small fraction of ellagic acid is absorbed in the upper small intestine, which explains the presence of dimethyl ellagic acid-glucuronide in bile, portal plasma, and urine.¹³⁶ The majority of the intestinal ellagic acid pool can provide a substrate for further metabolism by the microbiota. Metabolism of ellagic acid starts in the small intestine, where a series of transformations is mediated by gut microbial catechol dehydroxylases. The initial dehydroxylation in the catabolic pathway is the result of a decarboxylation reaction. One of the lactone moieties undergoes hydrolysis, leading to the formation of a free carboxylic acid and keto-enol tautomerization of the p-hydroxy group. The resulting carboxylic acid is then decarboxylated and subsequently, the p-hydroxy group leaves as water.

Figure 9.

Ellagitannins (such as pedunculagin) are hydrolyzed by host enzymes in the jejunum. The resulting hexahydroxydiphenic acid products undergo spontaneous lactonization to ellagic acid, which is the main substrate for urolithin metabolism by the gut microbiota.

Further enzymatic removal of hydroxyl groups results in the production of a variety of urolithin intermediates, such as urolithin D and urolithin C.¹³⁶ These intermediates are further metabolized to generate urolithin A, isourolithin A, and urolithin B, which are detected in high concentrations in plasma, urine, and other tissues.^41,146,147 Urolithin A is the most potent of these metabolites, and the only one detectable in feces due to its rapid absorption and subsequent glucuronidation or sulfation in the liver. To date, a total of 25 urolithins have been identified.³⁶

The metabolism of ellagic acid into urolithins is dependent upon specific gut bacteria, whose presence drastically varies among individuals (Figure 10).¹⁴⁸ This variability in gut microbial enzymatic capacity gives rise to differences in both the types and amounts of urolithins. A study of urinary urolithin excretion revealed that, after ingestion of walnut and pomegranate extracts, urolithin A is predominantly present and in its glucuronidated form.¹³⁸ A subset of participants failed to produce urolithin A and instead produced isourolithin A and urolithin B. A noticeable fraction of the study did not produce any detectable urolithins, most likely indicating the absence of an appropriate urolithin-metabolizing microbiome.¹³⁹ These corroborating observations led to the classification of healthy individuals into three phenotypes, or “metabotypes” based on their ability to produce and excrete different urolithin variants. Individuals of phenotype A produce only urolithin A, whereas phenotype B produce urolithin A, isourolithin A, and urolithin B. In individuals with “phenotype 0”, no production of bioactive urolithin is detected due to the lack of any gut microbiota to fully metabolize ellagic acid.^136,140 Interestingly, individuals with chronic diseases characterized by dysbiosis, such as obesity, diabetes, and colon cancer, were mostly phenotype B. A similar trend was observed in the elderly population.¹³⁸

Figure 10.

Ellagic acid catabolism capacity depends on an individual’s microbiota enzymatic repertoire, and can be classified into three main ‘phenotypes’. Individuals with phenotype 0 do not produce bioactive urolithin metabolites (shaded red). The majority of the population has phenotype a and can produce urolithin A, as well as various intermediates (yellow). The main metabolic pathway in phenotype B individuals culminates in production of urolithin B due to the presence of Ellagibacter spp. with 8-dehydroxylase capability (blue). Individuals belonging to phenotype a or B that harbor bacteria with 3-dehydroxylases can produce R-urolithins in small quantities (green).

Further in-depth investigations revealed the metabolic pathways involved in ellagic acid breakdown for the different phenotypes.¹⁴⁰ Phenotype A, which represents the majority of the population at around 70%, is characterized by gut microbiota that can readily catabolize ellagic acid by hydrolyzing a lactone ring and catalyzing three consecutive dehydroxylation reactions to yield urolithin A as the ultimate product.¹³⁶ On the other hand, phenotype B, which accounts for approximately 20% of the population, utilizes a similar pathway as phenotype A but with an additional dihydroxylation step leading to the production of urolithin B. In contrast, phenotype 0, which is relatively rare in Chinese and Spanish populations at 10% but more prevalent at 60% in the United States, is characterized by a gut microbiota that can only initiate the metabolic pathway of ellagic acid by opening one of the lactone rings to produce the urolithin M5 precursor.¹⁴⁹

Several bacterial species have now been isolated and characterized from humans, which contribute to the ellagic acid catabolic pathway of ellagic acid in humans. Notable converters Gordonibacter urolithinfaciens DSM 27213T and Gordonibacter pamelaeae DSM 19378T belong to the Eggerthellaceae family and are capable of producing intermediate urolithin metabolites. Gordonibacter urolithinfaciens (DSM 27213T) can dehydroxylate urolithin D and urolithin M6, yielding urolithin C as the final metabolite through its 4-dehydroylase and 10-dehydroxylase activities, respectively.³⁶ However, Gordonibacter lacks the ability to produce urolithin A from urolithin C, or urolithin B from urolithin A, or any other urolithin conversion involving dehydroxylation at the 9-position.^36,136 Despite this observation, a positive correlation was identified between the presence of urolithin A in feces and the presence of Gordonibacter urolithinfaciens, as well as a negative correlation between this bacterium and isourolithin A and urolithin B in feces.^140,150

The closely related species Ellagibacter ³⁸ is capable of producing isourolithin from ellagic acid by performing a distinct a 8-dehydroxylation of urolithins M6 and urolithin C (Figure 10). While this species is a key member of phenotype B gut microbiota, it is incapable of performing this dehydroxylation to produce urolithin B. Additionally, Ellagibacter can be present in phenotype A, indicating the requirement for another bacterial species to convert isourolithin A into urolithin B.^38,72 Several new urolithin metabolites, including urolithin M6R, urolithin M7R, urolithin CR, and urolithin AR, have been detected in small quantities in the feces of individuals that harbor bacteria with a 3-dehydroxylase. Since previously identified urolithins all maintained their 3-hydroxyl group these R-urolithins represent a new arm of the ellagic acid metabolic pathway and their bioactive role remains to be determined.¹⁴³

A recent study identified complementary bacterial species with 9-dehydroxylase and 10-dehydroxylase activity, which are required for the final steps of converting urolithin C into urolithin A and isourolithin A into urolithin B. Four different Enterocloster strains (E. bolteae CEBAS S4A9 and DSM 15670T, E. asparagiformis DSM 15981T, and E. citroniae DSM 19261T) were identified that can convert urolithin C to urolithin A. In addition, E. clostridioformis DSM 933T was able to yield urolithin A via converting urolithin M6 to urolithin M7. However, none of these species are capable of metabolizing ellagic acid, for which they depend on a different member of the microbiota such as Gordonibacter. These Enterocloster species can also produce the novel metabolite, 3,4,8-trihydroxy-urolithin, also known as urolithin G.⁷¹

Co-culturing experiments investigated the discrepancies in urolithin metabolism between those with phenotype A versus B. Co-culturing G. urolithinfaciens DSM 27213T with E. bolteae CEBAS S4A9 strains resembled the urolithin profile to that of individuals with phenotype A, whereas co-culturing E. isourolithinifaciens with E. bolteae CEBAS S4A9 strains resembled the urolithin metabolic profile of individuals with the phenotype B.⁷¹ Other Enterocloster species which were tested exhibited similar results, except for Enterocloster citroniae DSM 19261T.

8. Health benefits of gut bacterial polyphenol-derived metabolites

Gut microbial bioconversion of dietary polyphenols into bioactive metabolites might well account for a significant share of the beneficial health effects attributed to their parent compounds and dietary sources. There is a growing interest in characterizing the responsible enzymes from the microbiota to better understand their role in disease progression and leverage their potential therapeutic properties.

Gut microbial flavonoid degradation products have received a large part of the attention and in vitro as well as select in vivo studies have highlighted health benefits for several of these metabolites. The common flavonoid metabolite phloroglucinol has been investigated as a medication for the reduction of digestive, biliary, and urinary spasms.¹⁵¹ In this capacity, phloroglucinol is promising for relieving the symptoms of irritable bowel syndrome,^152,153 and protecting the gastric mucosa from ethanol-induced injury.¹⁵⁴ Phloroglucinol might also be promising for treatment for nonalcoholic fatty liver disease by lowering inflammation, fat accumulation, and apoptosis in liver cells.¹⁵⁵

Several monophenolic acid products of gut microbial flavonoid catabolism have beneficial effects on host health, either localized to the intestine or impacting distal targets. Phloretic acid (3-(4-hydroxyphenyl)propionic acid) reduced inflammation in Dextran Sulfate Sodium (DSS)-induced colitis in mice, and decreased the development of atherosclerosis by preventing plasma lipid accumulation and inhibiting foam cell formation.^156,157 Protocatechuic acid (PCA) has a protective effect on distal organs attributed to its antioxidant activity, its ability to scavenge free radicals and reactive oxygen species,¹⁵⁸ chelate metals,¹⁵⁹ and induce the enzymatic activity of glutathione and superoxide dismutase.^5,160 PCA has pro-apoptotic activity in breast, cervix, liver, prostate, bone, stomach and lung cancer,^161–164 as well as prevents accumulation of mutations by blocking carcinogens from binding to DNA.^165,166 In addition, PCA has antidiabetic effects, stimulating the insulin signaling pathway in human adipocytes, which leads to a reduction in plasma glucose levels.^22,167 PCA also reduces diabetes-associated complications, including dyslipidemia, fatty liver disease, and retinopathy.^5,168–170 PCA has an anti-inflammatory effect,^164,171,172 and can act as a neuroprotective agent in animal models of neurodegenerative diseases.^173–175 Additional research will be required to uncover the molecular mechanisms and pathways impacted by PCA across this plethora of conditions. Nonetheless, the versatile beneficial effects of PCA make it a good therapeutic option for diseases that are characterized by oxidative stress, including metabolic diseases. 3,4-Dihydroxyphenylacetic acid, 3-hydroxyphenylacetic acid, and 4-hydroxyphenylpropionic acid exhibit antiproliferative activity for prostate and colon cancer,^176,177 in addition to their antioxidant activity.^177,178 Furthermore, 3-hydroxyphenylacetic acid reduces alcohol-induced liver damage,¹⁷⁹ acts as a vasodilator to decrease blood pressure,¹⁸⁰ and reduces dysregulated spermatogenesis of old mice.¹⁸¹ 4-Hydroxyphenylacetic acid can reverse obesity-induced hepatic steatosis, and this effect is associated with the activation of AMP-activated protein kinase α (AMPKα).¹⁰ 4-Hydroxyphenylacetic acid also improves inflammation and lipid accumulation in alcohol-induced liver disease,¹⁸² protects the liver against acetaminophen-induced liver injury,¹⁸³ and attenuates sepsis-induced acute kidney injury.¹⁸⁴

Among the various polyphenolic gut microbial metabolites, the bioactivity of the isoflavonoid-derived metabolite (S)-equol and the urolithin compound family have been most intensely investigated. (S)-equol helps improve postmenopausal symptoms, such as hot flashes, osteoporosis.^185–190 It can also decrease the risk of hormone-associated cancers, including breast cancer^191,192 and prostate cancer.^193–195 (S)-equol can also help in the amelioration of cardiometabolic diseases, by reducing liver steatosis,¹⁹⁶ improving arterial stiffness and blood pressure,^197,198 and increasing lipid metabolism in an obese population.¹⁹⁹ Alleviation of several aging-associated conditions has been attributed to (S)-equol, including prevention of cognitive impairment in dementia and Alzheimer patients,^200–202 as well as reducing inflammation and bone erosion-induced rheumatoid arthritis in mice.²⁰³ Urolithins are investigated for their capability to reduce inflammation in the small intestine and colon^204–206 as well as their antioxidant and anticancer properties.¹⁴⁵ Urolithin B is mainly of interest for its anti-cancer properties^206–212 acting on cell cycle arrest, aromatase inhibition, and promotion of autophagy as well as senescence.²¹³ In addition to potential anti-cancer activity, urolithin B can inhibit cardiomyocyte apoptosis in ischemic heart disease,²¹⁴ and reduce inflammation and oxidative stress in neurodegenerative diseases.^147,215 The closely related compound urolithin A has a variety of complementary bioactivities, including triggering neurogenesis resulting in improved cognition in mice²¹⁶ and protection against ischemic neuronal injury.^217–219 Urolithin A also ameliorates cardiometabolic disease¹⁴⁵ and induces adipocyte browning, improving lipid metabolism.²⁰⁴ Finally, urolithin D has potential protective effects in cancer, as it inhibits ephrin type-A receptor 2 (EPHA2) phosphorylation in a cell-based assay.²²⁰

9. Conclusion and outlook

The discovery of polyphenol-metabolizing bacteria and their diverse metabolic pathways has shed light on the health-promoting effects of different classes of polyphenols and their bioactive metabolites, emphasizing the crucial role of gut microbiota in the transformation of a healthy diet to more bioavailable, and easily absorbed compounds in the small intestine. In this review, we described each enzymatic step in the metabolic pathway of different gut microbiota members that are necessary for the breakdown of flavonoids, ellagic acid, and ellagitannins to monophenolic acids, phloroglucinol, dihydroflavonols, and urolithins. Further investigations are needed to fully understand the mechanisms behind this process, but overall, the study of polyphenols metabolism and the identification of gut bacteria involved in that mechanism is a promising avenue for the development of new nutraceuticals and therapies for various diseases, as well as a way the development of probiotic interventions.

Funding Statement

This study was supported by a Research Grant from the Prevent Cancer Foundation [PCF2019-JC], an American Cancer Society Institutional Research Grant [IRG-16-186-21] and a Jump Start Award [CA043703] from the Case Comprehensive Cancer Center, and seed funding from the Cleveland Clinic Foundation (JC). JC is additionally supported by a National Institutes of Health grant [R01 AI153173], and a research grant from the Harold and Leila Mathers Foundation [HLMCJC1022].

Disclosure statement

No potential conflict of interest was reported by the author(s).

Author contributions

Writing of the original draft: SA. Figures: SA, JC. Writing, review and interpretation: SA, JC.