1. Flavonols
Flavonols are the most widespread form of flavonoids in plant foods, especially in onions (Allium cepa), strawberries (Fragaria spp.), spinaches (Spinacia oleracea), blueberries (Vaccinium sect. Cyanococcus), cauliflowers and broccoli (Brassica oleracea). The best-known compounds in this sub-family are quercetin and kaempferol.
In vitro, quercetin has shown anticancer, apoptosis-inducing
[1] and antioxidant activities
[2]. Similarly, kaempferol has been associated with antioxidant, anti-inflammatory, anticancer and antimicrobial effects
[3]. In vivo, several clinical trials have been conducted, finding general associations between flavonols intake and a reduction in cardiovascular risk factors
[4][5]. Among the biochemical properties of flavonols, a marked antibacterial activity has been reported
[6], and they have also recently been proposed as adjuvants to antiviral drugs, acting against SARS-CoV-2
[7] and other viral pathogens in general
[8].
Their structure is characterized by a 3-hydroxyflavone backbone, with the phenolic hydroxyl moieties being responsible for the diversity of this class of polyphenols. They are mostly found in glycosylated forms in foods, predominantly complexed with glucose or rhamnose, and the type of glycosylation affects their metabolism as follows: the higher the number of saccharides in the complex, the slower the metabolism of the compound (especially for the trisaccharide forms)
[9]. Usually, the glycosylated forms are found as O-glycosides, with substitution occurring at many sites of the flavonol backbone. Besides the aglycones quercetin and kaempferol, myricetin is also a widespread flavonol, together with the methylated glycosidic derivative isorhamnetin. When conjugated with saccharides, quercetin and kaempferol glycosides possess hundreds of different glycosidic combinations, dramatically expanding the diversity of flavonols that can be found in food. Notably, rutin is one of the main sources of flavonols in food
[10] and constitutes a complex glycoconjugate form of quercetin.
The breakdown of flavonols begins in the oral cavity, with the saliva and oral microbiota initiating the conversion of the flavonol glycosides to their aglycone form
[11], a process that proceeds in the upper digestive tract. Here, glycosidic forms are poorly absorbed and, in the large intestine, they undergo most of the metabolism through α-rhamnosidase and other glycosidase activities provided by the gut microbiota. In particular, exploiting fluorescence-based single-cell activity measurement coupled with fluorescent activated cell sorting after anaerobic incubation of healthy human fecal microbiota with rutin, Riva ang colleagues
[12] detected an enrichment in the bacterial families
Lachnospiraceae (specifically, the genera
Lachnoclostridium and
Eisenbergiella),
Enterobacteriaceae (Escherichia),
Tannarellaceae (
Parabacteroides) and
Erysipelotricaceae (
Erysipelatoclostridium), suggesting that they carry the metabolic capacity to degrade rutin. Other species such as
Lactobacillus acidophilus,
Lactobacillus plantarum and
Bifidobacterium dentium were reported to have a high rutin-deglycosylation capacity in in vitro fermentation studies, releasing the sugar moiety and the aglycone quercetin
[13][14]. Flavonol aglycones can be partially absorbed by epithelial cells in the upper and lower intestine and undergo phase II metabolism with the production of O-glucuronide and O-sulfate conjugates that can be found in plasma and urine
[15]. However, bioavailability studies recorded only a small amount of the flavonol structure in plasma, mainly because the highest fraction of the aglycone is not absorbed through the epithelium but rather undergoes metabolic processing by the gut microbiota. Multiple components of the intestinal microbial ecosystem, such as the species
Bacteroides fragilis,
Clostridium perfringens,
Eubacterium ramulus,
Lactobacillus spp.,
Bifidobacterium spp. and
Bacteroides spp., showed in mouse models the potential to degrade aglycones, releasing ring-fission phenolic acid end-products, including 3(3,4-dihydroxyphenyl) acetic acid, 3,3-hydroxyphenylpropionic acid, 3,4-dihydroxybenzoic acid and 4-hydroxybenzoic acid
[16], some of which are known to exert free-radical scavenging and antioxidant activity
[17].
2. Flavanones
Flavanones, generally represented by hesperetin and naringenin, are widely distributed in the plant families
Compositae (as lettuce, chicory, artichoke),
Leguminosae (such as chickpeas, peanuts, lentils, peas, beans) and
Rutaceae (such as cedar, tangerine and lemon, belonging to the
Citrus spp. fruits, which are the main source of flavanone glycosides, mainly hesperidin)
[18]. Flavanones possess antioxidant and anti-inflammatory activities
[19] and constitute important scaffolds for the development of anti-inflammatory and anticancer therapeutic agents
[20]. Several clinical trials have tested the in vivo effects of flavanone intake, mainly focusing on their ability to exert protective effects on the cardiac function, but providing contradictory results
[21], or on their ability to improve endothelial function
[22]. Concerning epithelial barrier function,
Citrus extract has been shown to enhance β-carotene uptake in intestinal Caco-2 cells by inducing paracellular permeability and directly interacting with the membrane
[23], thus suggesting that the pleiotropic mechanism of action of flavanones could begin directly in the intestinal lumen.
Flavanones generally show antibacterial potential
[24], with those extracted from
Calcedaria thyrsiflora (a succulent plant commonly called “kalanchoe”) exhibiting inhibitory activity against methicillin-resistant
Staphylococcus aureus (MRSA)
[25]. They also possess antiviral activities and have recently been tested to possibly aid in anti-COVID-19 therapy
[26].
This polyphenol subfamily exists in both glycosidic and aglyconic forms. Hesperetin and naringenin are the best-known aglycone forms, whilst the glycoside counterparts hesperidin and naringin are bound to the disaccharide rutinose and rhamnose-β1,2-glucose, respectively. The deglycosylation process—starting from saliva and continuing in the upper digestive tract—is likely analogous to that of other flavonoids and generally shared among all the glycosylated biomolecules ingested. Once released from the glycoside moiety, the aglycone naringenin has been tested for fermentation by the gut microbiota in rats
[27] and its metabolism yielded a high number of metabolites, mainly phenolic acids,
p-hydroxyphenylacetic acid and 3-(
p-hydroxyphenyl) propionic acid. It has recently been confirmed that also the flavanones hesperidin and eriocitrin (a glycosylated flavanone extracted from lemons, e.g.,
Citrus limon) are first deglycosylated in the upper digestive tract, then metabolized in the lower intestine with a complex series of metabolic transformations and interconversions, involving methylation, followed by glucuronic acid and/or sulfonate conjugation
[28]. Phase II flavanone metabolites naringenin 7-O-glucuronide, hesperetin 3′-O-glucuronide and hesperetin 7-O-glucuronide, together with the microbiota-derived flavanone metabolites hippuric acid and 3-(4-hydroxyphenyl) propionic acid showed anti-inflammatory and antioxidant activities in vitro at physiologically tangible concentration
[29]. Several bacterial species have been associated with the core metabolic steps required to yield flavanone metabolic end-products, such as O-deglycosylation (among which
Parabacteroides distasonis,
Bifidobacterium adolescentis,
Bifidobacterium bifidum,
L. plantarum,
Lactobacillus buchneri), C-deglycosylation (
Eubacterium cellulosolvens), O-demethylation (
Eubacterium limosum and
Blautia sp. MRG-PMF1), dihydroxylation or general C-ring cleavage (
Clostridium butyricum,
E. ramulus,
Flavonifractor plautii)
[30].
3. Isoflavones
Isoflavones are generally found in soybeans (
Glycine max, the richest source of these biomolecules), chickpeas (
Cicer arietinum), pistachios (
Pistacia vera), peanuts (
Arachis hypogaea) and other nuts and legumes. Isoflavones are often found as glycosides, most commonly daidzin, genistin and glycitin, with daidzein, genistein and glycitein being the corresponding aglycones. As a common feature of polyphenols, isoflavones exhibited in vitro antioxidant, antimicrobial, anti-inflammatory and anticancer properties as well
[31]. It is well known that isoflavones act as phytoestrogens by binding to estrogen receptors in mammals
[32], and this has raised several concerns given the potential endocrine-disrupting effect. However, a recent critical review
[33], which considered 417 reports on soybean-derived products and isoflavone phytoestrogen effects on humans, concluded that they do not act as endocrine disruptors, but rather are beneficial for their antioxidant and anti-inflammatory effects. Furthermore, the coupled administration of isoflavones and probiotics has recently shown long-term efficacy in counteracting the loss of bone mineral density in a double-blind, placebo-controlled trial in postmenopausal osteopenic women
[34].
Regarding the antibacterial activity, isoflavones have shown inhibitory effects against biofilms of
Escherichia coli and
Listeria monocytogenes [35], as well as against MRSA
[36]. Moreover, ingestion of isoflavones has been shown to potentially reduce viral infection and mortality of porcine reproductive and respiratory syndrome in pigs, with evident outcomes relevant to the food supply chain
[37].
In vivo, isoflavones are mainly ingested as glycosides, so a high fraction of their content reaches the large intestine, where they are metabolized by the gut microbiota. Conversely, aglycone isoflavones are absorbed by small intestinal endothelial cells and metabolized through phase II reactions, yielding glucuronide and sulfate metabolites, as demonstrated by in vitro experiments
[38]. These conjugates can be excreted in the bile, re-enter the intestine and, here, be converted back into aglycone forms by enzymes of microbial commensals, such as β-glucuronidases. The resulting compounds, mainly dihydrodaidzein and dihydrogenistein, can again be further metabolized by the colonic microbiota. It appears that the configuration of the intestinal microbial community plays a major role in determining the end-product that will be generated, alternatively equol or O-desmethylangolensin (O-DMA)
[39], with
Clostridium spp. And
E. ramulus being among the main bacteria involved in such decision-making balance
[40]. It should be noted that the equol-producer phenotype is less prevalent in the population than the O-DMA-producer one and, given that equol shows better binding properties to α and β estrogen receptors than O-DMA and unprocessed isoflavones, this might result in different physiological effects and health benefits
[41]. For what concerns the effects of equol and O-DMA on the human body, the former showed potential anti-atherogenic effects, possibly preventing stroke
[42], while for the latter, only a few studies have evaluated disease risk factors in relation to being an O-DMA producer, but apparently the O-DMA producer phenotype might be associated with obesity in adults
[43]. This duality kindles the interest in deepening our knowledge of the relationship between the gut microbiota and isoflavones and polyphenols in general—given their widespread presence in our daily dietary foods.
4. Flavones
Flavones are a subfamily of flavonoids commonly reported in parsley (
Petroselinum crispum) and celery (
Apium graveolens), but also present in grains including maize (
Zea mays), wheat (
Triticum spp.), rye (
Secale cereale), barley (
Hordeum vulgare), oats (
Avena sativa), sorghum (
Sorghum spp.) and millet (
Pennisetum glaucum). The vast majority of flavones reported come from cereal grains and exist as various O- or C-glycosides of the aglycones apigenin and luteolin, forming a huge variety of structural variants. In fact, the different glycosylations can generate several glycosidic forms depending on the following: (i) the number of saccharide units; (ii) the type of glycosyl moiety; (iii) the position in which the saccharides bind to the flavone backbone. For example, the saccharides bound to position 7 of the A-ring of apigenin can generate apigetrin (1 unit of glucose), apiin (a disaccharide of furanose and glucose) or rhoifolin (a disaccharide of rhamnose and glucose). Most of the in vitro studies have used aglycones or unprocessed glycosides, yielding controversial results, probably due to the lack of a specific metabolism to make the flavone configuration actually functional. Conversely, results with synthetic derivates of flavones showed consistent and strong effects, mainly associated with antioxidant activity and inhibition of the lipoxygenase enzyme
[44]. Anticancer activity often associated with several flavonoids has also been reported for apigenin and luteolin, inducing apoptosis in tumor cells, but the limited in vivo evidence requires further investigation
[45][46]. Finally, flavones have generally been associated with antiviral, antibacterial and antifungal effects
[8].
As mentioned for the other flavonoid subfamilies, ingested flavones are mainly glycosides and, in such form, they reach the intestine, with partial degradation in the oral cavity and in the upper digestive tract. The aglycone form can be absorbed and found in plasma together with glucuronide and sulfate forms
[47]. An interesting effect is reported for baicalin, a glucuronide form of baicalein—a flavone mainly derived from the roots of
Scutellaria baicalensis—that shows enhanced absorptive and bioactive potential after undergoing glucuronidation
[48]. Both the flavone forms that reach the intestine directly and those that return to the intestine after hepatic metabolism can be further metabolized by the gut microbiota. In particular, flavone O-deglycosylation appears to be widely distributed among gut microbes, in genera such as
Eubacterium [49],
Flavonifractor and
Clostridium [50]. The released aglycones are mostly further fermented by colonic bacteria with C-ring cleavage, releasing 3-(3,4-dihydroxyphenyl) propionic acid, 3-(4-hydroxyphenyl) propionic acid, 3-(3-hydroxyphenyl) propionic acid and 4-hydroxycinnamic acid. Such phenolic acids are absorbed and circulated in the bloodstream until their excretion in the urine
[51]. In particular, for 3-(4-hydroxyphenyl) propionic acid, Xie et al.
[52] reported associations with a reduction in total cholesterol in healthy adults.
On the other hand, it must be said that the introduction of phenolic molecules through the diet is likely to exert an impact on the gut microbiota itself. Tangeretin and nobiletin are other flavones isolated from the tangerine peel (
Citrus reticulata), which are an example of polymethoxyflavones, a subclass of flavones bearing two or more methoxy groups on the basic benzopyrone. Such biomolecules have been shown to positively alter the composition of the mouse gut microbiota after ingestion, with an increase in the relative abundances of the genera
Lactobacillus and
Bifidobacterium [53]. These taxa are in turn involved in polymethoxyflavone metabolism, thus paving the way for studies focused on the bidirectional relationship between polyphenols and gut microbes.
5. Flavan-3-ols
Flavan-3-ols is a collective term for several types of so-called catechins, which are mainly found in apples (
Malus spp.), hops (
Humulus lupulus), tea and black tea (
Camellia sinensis), beer, wine, fruit juices and cranberries (
Vaccinium spp.).
In vitro, flavan-3-ols have been shown to inhibit tumor angiogenesis and TNF-α-related inflammation
[54] and have recently been tested in association with procyanidin B2—an anthocyanin flavonoid—in breast and prostate cancer cell lines, finding out that the combined treatment efficiently enhanced a sensitization mechanism that could be exploited in novel clinical trials
[55]. Sensitization of cancer cells often requires a combination of therapies with multiple drugs and aims to achieve a synergistic induction of cell death in cancer cells. When tumor cells are resistant to therapy, the combination of drugs can enhance the antitumoral activity by modulating one or more mechanisms of resistance. Among the potential chemosensitizers in use, 60% of them are of natural origin
[56][57]. Further evidence of the cancer-protective effects of flavan-3-ols has been reported in a meta-analysis on various cancers, including rectal, oropharyngeal, laryngeal, breast and stomach cancers
[58]. Flavan-3-ols have also shown antioxidant properties, mainly when extracted from natural food matrices
[59]. In case-control studies, the intake of foods rich in flavan-3-ols—mainly tea extract and green tea infusions—was associated with a lower risk of type-2 diabetes
[60] and cardiovascular diseases
[61]. These results, far from being exhaustive, should encourage further research on flavan-3-ols and polyphenols in general as promising molecules.
Flavan-3-ols and, in particular, foods rich in such biomolecules, have also been shown to be effective in counteracting viral infections
[62]. Catechins and derivates exhibited antibacterial properties as well, on both Gram-positive and Gram-negative bacteria. The synergistic effects of antibiotic treatments have also been reported in clinical trials
[63], paving the way for future studies to overcome antibiotic resistance, by reducing antibiotic dosage while maintaining and possibly increasing therapeutic efficacy (with alleviation of the economic burden on healthcare systems).
The flavan-3-ol structure accounts for the ease of free radical scavenging activity, primarily due to the high reactivity of hydroxyl substituents in the flavan-3-ol backbone. The best-known flavan-3-ol compounds are catechin, epicatechin, gallocatechin and epigallocatechin, as well as their galloylated forms. In addition to the possible substituent, the catechin’s potential structural diversity relies on two chirality centers. The epicatechin scaffold carries both chirality centers pointing in the same direction (
cis), whereas that of catechin shows opposite directions (
trans). Galloylation—together with the multiple hydroxyl groups—results in the formation of a hydration shell that determines reduced bioavailability and absorption in the upper digestive tract, thus making flavan-3-ols reliant on gut microbial metabolism—attributed to several
Lactobacillus spp.
[64]—to break down the ester bond and release the catechin flavonic backbone and gallic acid, which is further metabolized into pyrogallol
[65]. On the other hand, the subsequent microbial metabolism of catechin generates phenyl-ɣ-valerolactones—exclusive intermediates of flavan-3-ol degradation—that are further metabolized into hydroxyphenylpropionic acid and hydroxybenzoic acid
[65], whose features—better outlined in the phenolic acid section below—include antioxidant, anti-inflammatory, antiviral, antimicrobial and anticancer properties
[66]. According to recent in vitro fermentation experiments, flavan-3-ols promote the growth of
Bacteroides,
Faecalibacterium,
Parabacteroides and
Bifidobacterium, thus suggesting that these genera might be directly involved in the intestinal metabolism of such biomolecules
[67].
Phenolic acid metabolites such as vanillic acid, homovanillic acid, hippuric acid and
p-coumaric acid have also been related to flavan-3-ol metabolism mediated by the gut microbiota
[68] and are found methylated, glucuronated, sulfated or nucleated in the bloodstream. These end-products have been reported to possess an antiadhesive effect in
E. coli urinary infections in a T24 bladder epithelial cell assay
[69]. Such results further confirm the reported efficacy of cranberry juice—particularly rich in such biomolecules—in reducing the recurrence of urinary tract infections in women
[70].
6. Anthocyanins
Anthocyanins are natural plant pigments that can be easily found in our daily diet as they are responsible for most of the red, blue or purple color of the fruit. Examples include berries, apples, pears, red-skinned grapes and vegetables, such as radishes (
Raphanus sativus), purple tomatoes (
Lycopersicon esculentum ‘
Indigo Rose’) and red cabbage (
Brassica oleracea var. capitata f. rubra). Comparable to other classes of flavonoids, anthocyanins—together with their aglycone counterparts called anthocyanidins—have shown antioxidant properties in vitro, with scavenging effects on free radicals
[71]. A recent study reported an effective reduction of platelet aggregation induced by arachidonic acid in coincubation with anthocyanins
[72], thus supporting the possible supplementation of the latter as an adjunct in the prevention of thrombosis.
In vivo, many anthocyanin compounds have shown neuroprotective and anti-inflammatory effects, comparable to those of acetylsalicylic acid
[73][74]. According to several epidemiological and human intervention studies, anthocyanin administration could be a reducing factor for the risk of cardiovascular diseases, due to their anti-thrombotic effects
[75][76][77][78]. In addition, anthocyanins can inhibit the replication of viruses such as
Herpes simplex, human parainfluenza viruses, respiratory syncytial virus, human immunodeficiency virus (HIV), rotaviruses and adenoviruses
[79].
Anthocyanins are the glycosylated forms of anthocyanidins, carrying one or several saccharides bound to their scaffold structure. Procyanidins can occur in monomeric as well as in polymeric forms, the latter being responsible for the red, purple and blue colors found in fruits and vegetables
[80]. Their backbone structure is the flavylium cation and, depending on the number and position of hydroxyl and methylated groups, various anthocyanidins have been described. The hydration layer constituted by the hydroxyl groups and the polymeric and oligomeric glycone forms contributes to the low bioavailability of anthocyanins in the upper digestive tract. It is therefore very likely that a large numbers of these compounds enter the colon unmodified, where they are processed by the resident microbiota
[81]. In fact, only about 1–2% of ingested anthocyanins retain their original structure in the plasma
[82]. In the intestine, bacterial β-glucosidases and other enzymes involved in the ring-opening lead to a series of degradation products such as phloroglucinol, vanillic acid and protocatechuic acids
[81] known for their antioxidant, antimicrobial and anti-inflammatory properties
[83]. Particular emphasis should be given to phloroglucinol, which, in addition to antioxidant and anti-inflammatory properties, was shown to have a relaxing effect on gut smooth muscle during placebo-controlled human trials, with positive effects on patients suffering from irritable bowel syndrome with diarrhea
[84] and potential application during esophagogastroduodenoscopy
[85]. Other identified microbial end-products include gallic, syringic and
p-coumaric acids, which have been associated with health-promoting properties
[86]; to date, the main bacteria presumably involved have been identified by in vitro microbial cultivations and include
Lactobacillus spp. (i.e.,
L. plantarum,
Lactobacillus casei and
L. acidophilus) and
Bifidobacterium spp. (i.e.,
B. adolescentis,
Bifidobacterium infantis and
B. bifidum)
[87], most likely because these genera typically possess β-glucosidases and ring-fission catabolic activities
[88].