A review on the effects of flavan-3-ols, their metabolites, and their dietary sources on gut barrier integrity

Abstract

Impairment of gut barrier integrity is associated with the pathogenesis of gastrointestinal diseases, including inflammatory bowel disease, colorectal cancer, and coeliac disease. While many aspects of diet have been linked to improved barrier function, (poly)phenols, a broad group of bioactive phytochemicals, are of potential interest. The (poly)phenolic sub-class, flavan-3-ols, have been investigated in some detail owing to their abundance in commonly consumed foods, including grapes, tea, apples, cocoa, berries, and nuts. This review summarises studies on the effects of flavan-3-ols, their microbiome-mediated metabolites, and food sources of these compounds, on gut barrier structure. Extensive evidence demonstrates that flavan-3-ol rich foods, individual flavan-3-ols (e.g., (epi)catechin, epi(gallo)catechin-3-O-gallate, and pro(antho)cyanidins), and their related microbiota-mediated metabolites, could be effective in protecting and restoring the integrity of the gut barrier. In this context, various endpoints are assessed, including transepithelial electrical resistance of the epithelial layer and expression of tight junction proteins and mucins, in ex vivo, in vitro, and animal models. The differences in bioactivity reported for barrier integrity are structure–function dependent, related to the (poly)phenolic source or the tested compound, as well as their dose, exposure time, and presence or absence of a stressor in the experimental system. Overall, these results suggest that flavan-3-ols and related compounds could help to maintain, protect, and restore gut barrier integrity, indicating that they might contribute to the beneficial properties associated with the intake of their dietary sources. However, rigorous and robustly designed human intervention studies are needed to confirm these experimental observations.

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1. Introduction

Preservation of a functional gut barrier is fundamental to the effective absorption of nutrients as well as protection against antigens and pathogenic microorganisms present in the intestinal lumen.^1–8 This barrier comprises a single layer of homeostatically renewed epithelial cells,⁴ joined by specific proteins, including tight junction proteins (TJs), adherens and gap junctions, and desmosomes.^1,2,5 It is separated from the gut lumen by a mucus layer formed from mucins, hydrated high molecular weight glycoproteins, creating a physical-immunological protection for the host, as well as an environmental niche and a source of nutrients for a sub-group of gut microorganisms.^6,7 The gut barrier structure is subject to deleterious factors, both endogenous and exogenous, such as intake of diets low in fibres and high in fat, consumption of advanced glycation end products (AGEs), inflammatory responses, obesity and, psychological stress, that could lead to unregulated destruction of TJs and excessive thinning of mucin coverage.⁸ While beneficial factors include components of fruits and vegetables, such as (poly)phenols, fibres, minerals, and vitamins.^8,9

(Poly)phenols are of particular interest in this respect: these are dietary phytochemicals which possess a structure with one or more hydroxylated aromatic rings and sub-categorised based upon number of rings, their linkages, and chemical group moieties.^10,11 Flavonoids and non-flavonoids are the two main families of (poly)phenols.^10,11 The former are abundant in nature with flavan-3-ols representing the main dietary forms consumed.^11,12 Following intake, flavan-3-ols, which include (–)-epicatechin (1), (–)-epigallocatechin-3-O-gallate (2, ECGC), and pro(antho)cyanidins, among others, are extensively metabolised during their transit through the gastrointestinal tract. This review summarises the current evidence on the bioactive potential of flavan-3-ols and of their microbially derived metabolites with respect to gut barrier integrity in the presence or absence of stressors analysed in ex vivo, in vitro and animal models.

2. Flavan-3-ols and their metabolites

Despite the currently available information on the role of nutrition and (poly)phenols in reducing the occurrence and severity of gut barrier impairments,^9,13 a more in-depth analysis of the potential involvement of flavan-3-ols is warranted, especially when considering their dietary abundance. Tea, cocoa-derived products, wine, and pome fruits such as apples or pears, are key contributors to flavan-3-ol intake.^12,14,15 The National Diet and Nutrition Survey rolling programme (2008–2014) analysed the dietary intake of the UK population, noting consumption of flavan-3-ols gradually increases up to 483 ± 293 mg day⁻¹ from childhood to adulthood, with the type of dietary sources differing in contribution between age groups.¹²

From a gastrointestinal perspective, following intake, flavan-3-ols remain relatively unaffected by the action of saliva enzymes^16–19 and limited modifications occur during the gastric phase, as indicated by studies where stomach metabolism was simulated in vitro, or naso-gastric sampling was performed.^20–23 Depending on the degree of polymerisation, and conjugation, up to ∼90% of pro(antho)cyanidins transit through the small intestine without being metabolized or absorbed, while monomers are predominantly absorbed in the small intestine, with a portion being effluxed back into the intestinal lumen as phase II metabolites, a portion possibly through enterohepatic recirculation.^16,23–33 Phase II sulphate, methoxy, and/or glucuronide metabolites occur in the enterocytes and, after transport through the portal vein, in the liver.³⁴ The mechanism of absorption-conjugation-excretion in the small intestine was confirmed by the pivotal study of Actis-Goretta et al.,³⁵ who analysed intestinal perfusate collected following the intra-intestinal administration of (−)-epicatechin (1). Analysis of the (poly)phenolic content in ileal fluids following intake of flavan-3-ol sources by subjects with a ileostomy confirmed their relative stability with total intra-luminal presence of up to 2468 μmol of total flavan-3-ols over 24 h post-ingestion.^{23,25,28–31,33} The (poly)phenolic profile of blood and urine samples collected from subjects with a full gastrointestinal tract after intake of flavan-3-ol-rich sources were in line with these observations with an average bioavailability estimated to be ∼31%.³⁶ The ca. 70% of flavan-3-ol monomers that reach the colon, as either parent compounds or their metabolites, is then subjected to microbiota-mediated catabolism resulting in the production of phenyl-γ-valerolactones and phenylvaleric acids, unique products of flavan-3-ol monomers and pro(antho)cyanidins.^37,38 Phenolic acids including phenylpropanoic, phenylacetic, and benzoic acids can also be produced. All these catabolites, thus, have the potential to exert bioactivity within the colonic lumen as well as at a systemic level. It must be considered that bioavailability of flavan-3-ol compounds has been widely studies in rat and mice models. However, there are significant differences in their metabolism in comparison to humans, e.g., absence of production of sulfated metabolites in rats,³⁹ which could represent a limit in translating findings in animal models to potential similar effects in humans.

3. Assessment of gut barrier integrity

Several direct and indirect methods are available to analyse the status of intestinal barrier integrity via in vitro and ex vivo models. Transepithelial electrical resistance (TEER) is a reference technique to quantitatively evaluate the permeability and integrity of the gut barrier via its electrical resistance,^40,41 thereby indirectly monitoring TJ functionality.⁴¹ This technique can be applied both in vitro and ex vivo. Most commonly, TJ gene and protein expression and cellular localisation are measured, including zonula occludens (ZO), claudins, occludins, and junctional adhesion molecules (JAM). Permeability can be analysed in vitro through paracellular transport of fluorochrome markers, such as fluorescein isothiocyanate–dextran (FITC-Dx),^42,43 and, in animals, by monitoring circulating ovalbumin (OVA), glucagon-like peptide (GLP)-2, lipopolysaccharides (LPS) and LPS-binding protein (LBP) levels,^42,43 and by mannitol^42,44 and its ratio with lactulose.⁴⁵ In particular, these two monosaccharides cross the small intestinal barrier through trans- and para-cellular passage, therefore, their increased urinary excretion represents a marker of impaired permeability of this gastrointestinal section.^42,44 Faecal markers, including myeloperoxidase (MPO), which is specific for the neutrophil activity,⁴⁶ and calprotectin⁴² are also used, while the presence of a healthy mucus layer can be assessed via analysis of mucin status and abundance of goblet cells. The interest in goblet cell density is mainly due to their ability to produce gel-forming mucins, which form the core structure of the mucus layer.⁶ The production of mucus differs between cell lines typically used for in vitro gut barrier studies and influences the type of cell line selected for use, based on the purpose of the study. For example, IPEC-J2 is a porcine intestinal cell line that does not produce mucus, which is a characteristic common to the Caco-2 human epithelial cell line cultured in normal conditions, whereas Caco-2 co-cultured with HT-29-MTX cells are characterised by mucus expression.⁴⁷ As revised by Martel and co-authors,⁸ there are several factors that can trigger gut barrier integrity through several biological mechanisms. Examples are reported in Table 1.

Table 1 Examples of gut barrier stressors and models to simulate them in animals and cell models

Stressor	Model
Stressor tested on a cell model (c), stressor tested on an animal model (a), colorectal cancer (CRC), cyclophosphamide (CTX), dextran sulphate sodium (DSS), interleukin (IL), lipopolysaccharides (LPS), tumour necrosis factor (TNF).
Diabetes	High-fat diet + streptozotocin administration (a)
Inflammation	DSS administration (a), LPS, IL-1β, IL-6, and-or TNF-α exposure (c)
Coeliac disease	Transgenic DQ8 mice (a), gliadin exposure (c)
Obesity	Diet induced obesity (a)
CRC	Cancer treatment CTX (a)
Stress	Heat exposure, or water avoidance stress (a)
Age	Old mice (a)
Diet	High-fat and/or high-sugars diet (a)
Pathogens	L. monocytogenes exposure (c)

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4. Effects of flavan-3-ols, their metabolites, and their dietary sources

The studies covered in this review have been organised by type of (poly)phenolic source tested (i.e., specific food source or individual compounds) with the main outputs summarised in Table 2 with more comprehensive experimental details provided in ESI Table 1.†

Table 2 Overview of studies reporting the effects of products containing flavan-3-ols and derivatives compounds, as well as these individual compounds, on selected markers of the gut barrier integrity, in presence (Y) or absence (N) of gut barrier stressors

PP source/type	Stress	Main significant outcomes	Ref.
BA, benzoic acid; (–)-ECG, (–)-epicatechin-3-O-gallate; EGCG, (–)-epigallocatechin-3-O-gallate; PP, (poly)phenols; TEER, transepithelial electrical resistance; ↑, significant increase; ↓, significant decrease. Gene/protein expression of zonula occludens (ZO), occludin (OCC), claudin(s) (CL), and mucin (Muc) are reported. Results for paracellular or transcellular permeability were reported. For more experimental details see ESI Table S1.†
Cocoa	N	TEER ↑	51
	Y	Permeability ↓, ZO-1 ↑, OCC ↓ or ↑, CL ↑ or ↑, Muc2 ↑	50–54, 156 and 157
Pome fruits	N	TEER ↑, ZO-1 ↑, OCC ↑, CL ↑	57 and 58
	Y	Permeability ↓, ZO-1 ↑, OCC ↑, CL ↑	56, 57, 60 and 158
Berries	N	ZO-1 ↑	159
	Y	Permeability ↓, TEER ↑, ZO-1 ↑, JAM-1 ↑, OCC ↓ or ↑, CL ↑ or ↓, Muc2 ↑, Muc ↑	61–67, 69–73 and 159
Tea	N	Permeability ↓, TEER ↑, ZO-1 ↑ or ↓, OCC ↑ or ↓, CL ↑, Muc2 ↑	77, 81, 83, 93 and 123
	Y	Permeability ↓, TEER ↑, ZO-1 ↓ or ↑, OCC ↓ or ↑, CL ↑, Muc2 ↑	77, 81–83, 85–92 and 160
Grape	N	Permeability ↓ or ↑, TEER ↑, ZO-1 ↑, OCC ↑ or ↓, CL ↓ or ↑, Muc1 ↑, Muc2 ↑	98, 99, 101–106, 123, 161 and 162
	Y	Permeability ↓, TEER ↑, ZO-1 ↑, OCC ↑, CL ↑, E-cad ↑, Muc2 ↑	98, 99, 101, 102, 105–121 and 163
(–)-Epicatechin	N	Permeability ↑, TEER ↑, ZO-1 ↑.	125
	Y	Permeability ↓, TEER ↑, ZO-1 ↑, OCC ↑, CL ↑	1, 128, 133 and 134
(–)-ECG	N	Muc17 ↑	129
(–)-EGCG	Y	Permeability ↓, TEER ↑, ZO-1 ↑, OCC ↑, CL ↑, TJ ↑, Muc2 ↑	130, 132, 135, 160, 164 and 165
(+)-Catechin	N	TEER ↑, ZO-1 ↑ or ↓, OCC ↑, CL ↑	41 and 125
	Y	TEER ↑, ZO-1 ↑, OCC ↑, CL ↑	1, 41, 125, 160 and 166
Procyanidin B2	N	ZO-1 ↓, OCC ↑, CL ↑	41
	Y	ZO-1 ↑	41
Theaflavin-3′-O-gallate	N	Permeability ↓, TEER ↑, ZO-1 ↑, OCC ↑, CL ↑	127
Theaflavin, theaflavin-3,3′-O-digallate, theaflavin-3-O-gallate	N	Permeability ↓	127
Theasinesins A, theasinesins B	N	Permeability ↓	138
Theabrownin	N	ZO-1 ↑, CL-1 ↑, Muc2 ↑	167
Procyanidin A1	Y	Permeability ↓, TEER ↑, ZO-1 ↑, OCC ↑, CL ↑	168
3,4-Dihydroxy-BA	N	TEER ↑	169
2,4,6-Trihydroxy-BA	N	TEER ↓	169
3,4-Dihydroxy-BA	Y	ZO-1 ↑, Muc2 ↑	149
4-Hydroxy-3-methoxy-BA	Y	OCC ↑	170
Hippuric acid	Y	ZO-1 ↑	149
3′,4′-Dihydroxyphenylacetic acid	N	Permeability ↓, TEER ↑	101 and 125
	Y	TEER ↑	125
BA	N	ZO-1 ↑, OCC ↑	143
4-Hydroxy-BA	Y	OCC ↓, E-cad ↓	142
3-(3',4'-Dihydroxyphenyl)propanoic acid	N	TEER ↑, ZO-1 ↑, OCC ↑, CL ↑	125
	Y	TEER ↑	125
1,3,5-Trihydroxybenzene	N	TEER ↑, ZO-1 ↑, OCC ↑, CL ↑	125
	Y	TEER ↑	125
3,4,5-Trihydroxybenzoic acid	N	TEER ↓, CL ↓	144

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4.1. Cocoa (Theobroma cacao L.)

The (poly)phenolic profile of cocoa is characterised by a range of flavan-3-ols, from the unconjugated monomers to procyanidins with a degree of polymerisation >10 units,⁴⁸ and this composition is susceptible to effects of different processing methods.⁴⁹ Bitzer and co-workers⁵⁰ demonstrated in vitro that different fractions of cocoa extracts, containing mainly monomers, procyanidin oligomers (mainly dimers–hexamers), and polymers (mainly heptamers–decamers), exerted significant protection against a dextran sulphate sodium (DSS)-induced increase in permeability in Caco-2 cells. Moreover, the extracts containing mainly high molecular weight procyanidins showed significantly greater protective effects in comparison to the other fractions at the maximal concentration tested (100 μg mL⁻¹) and a protective effect was also observed at lower concentrations (10 and 25 μg mL⁻¹). Similarly, a cocoa extract containing mainly hexamers (10 μM), tested in the same cell model, protected against a decrease in ZO-1 protein expression and its cytoplasmic translocation induced by exposure to a stressor, the secondary bile acid deoxycholic acid (DCA, 0.2 mM).⁵¹ While, in the absence of stress, the extract induced a significant time – (30–120 min) and concentration – (10–20 μM) dependent increase in TEER.⁵¹ The latter effect was proposed to be the result of the adsorption of cocoa procyanidins to the cell membrane, that could lead to prolonged beneficial effects in the gut lumen,⁵¹ with the procyanidins putatively also acting as a physical protective layer between the gut barrier and the intestinal lumen.

Similar positive protective results were reported in mice following prolonged (i.e., 8- or 18-weeks) daily intake of different formulations of cocoa powder (80 mg per g diet per day) with a high-fat diet, assessed via analysis of plasma markers of gut barrier integrity (i.e., GLP-2, LPS, LBP, or FITC-Dx).^52,53 In rats, chocolate (50 mg day⁻¹) alone did not affect barrier functions, but, in combination with probiotics, which prove to have beneficial effects, inhibited the increase in ileum permeability caused by loperamide-induced constipation, a condition linked to possible impairments on the gut barrier structure, indicating an effect of the probiotics rather than the cocoa-derived compounds.⁵⁴

4.2. Pome fruits

Apple consumption contributes to the intake of both fibres and flavan-3-ols, mainly proanthocyanidins.⁵⁵ Like in an earlier study by Bitzer et al.⁵⁰ with cocoa extracts, Wu and co-workers⁵⁶ analysed (poly)phenolic fractions extracted from Granny Smith apples in Caco-2 cells. The extract which contained (–)-epicatechin (1), (+)-catechin (3), 5-O-caffeoylquinic acid (4), and procyanidins B2 (5) and C1(6), attenuated decreases in ZO-1 and occludin protein expression in a concentration-dependent manner (12.5–150 μg L⁻¹) in response to a LPS challenge. Similarly, an increase in TJs (i.e., ZO-1, occludin, and claudin-1) expression was reported using a IPEC-J2 small intestinal cell model incubated with apple (poly)phenols for 24 h.⁵⁷ Another apple (poly)phenolic extract exerted time (0–48 h)- and concentration (0.01–1%)-dependent increases in TEER of the Caco-2 cell monolayer.⁵⁸ While supplementation of pigs (49-day, three times per day) with similar products (400 and 800 mg kg⁻¹) induced an increase in the expression of the same TJ proteins in the small intestine and promoted villi tightness at the ileum and jejunum level.⁵⁷ Furthermore, Swiss mice fed an extract of the Pyracantha fortuneana (Maxim.) fruit rich in flavan-3-ols and other (poly)phenols,⁵⁹ for 8 weeks (0.4–1% dietary intake), led to significant dose-dependent attenuation in high-fat diet-induced decrease in colonic TJ expression, increased urinary lactulose/mannitol excretion, and widening of intestinal villi caused by the challenge diet.⁶⁰

4.3. Berries

Berries contribute to the intake of flavan-3-ols but it should be noted that the levels vary, and that berries are also the main dietary source of other (poly)phenols, most notably anthocyanins and ellagitannins.^10,11 Respectively, açai and aronia berries protected against reductions in TEER induced by LPS-⁶¹ and a pro-inflammatory cytokines mix,⁶² with a general consistent increase in TJ proteins observed in both studies.^61,62 An 8 weeks diet high in sugars and fats fed to obese mice increased serum LPS, an effect that was mitigated by supplementation of three (i.e., cloudberry, alpine bearberry, and lingonberry) of five types of Arctic berries.⁶³ In addition, supplementation of a high fat diet with blueberry powder (10% w/w diet) for 8 weeks decreased circulating LBP, while promoting Muc2 expression at colonic level in a rodent model.⁶⁴ This agrees with protective effects exerted by wild blueberry (poly)phenolic fractions (17–53 mg (poly)phenols per day) against diet-induced thinning of the colonic mucus in mice, and albeit with inconsistent increases in goblet cells density.⁶⁵ Maqui berry-derived products (50–200 mg kg⁻¹ day⁻¹) enhanced barrier integrity, occludin expression, mucin content, and goblet cell number in mice models of colitis (i.e., DSS or TNBS induced), when supplemented intra-gastrically,⁶⁶ or orally.⁶⁷

Ulcerative colitis and Crohn's disease can contribute to gut barrier disruption⁸ and, although enteral nutrition represents one of the possible treatments for these conditions, it has been linked with side effects.^68,69 A significant dose-dependent (8–200 mg per kg BW per day) attenuation in the decrease of intestinal Muc2 expression and goblet cell number was observed following the addition of cranberry proanthocyanidins to enteral nutrition administered to mice (5 days).⁶⁹ Oral feeding of a cranberry extract (200 mg kg⁻¹),⁷⁰ or a freeze-dried cranberries (20% w/w diet)⁷¹ improved barrier function in mice either challenged with a high fat or genetically-predisposed to colon-rectal cancer. Cranberry products not only increased Muc2 expression and content,^69–71 but promoted increases in ZO-1 and colon claudin-3 gene expression,⁷¹ and attenuated the stress-induced increases in plasma levels of LPS.⁷⁰ Similar effects were observed by Heyman-Lindén et al.⁷² after freeze-dried lingonberry supplementation to mice for 11 weeks decreased serum LBP and modulated occludin gene expression, albeit with some batch product variation being evident. Finally, an infusion of Chinese blackberry (Rubus sauvissimus S.) leaves mitigated against LPS-induced intestinal permeability in a mouse model while increasing expression of ZO-1 and JAM-1.⁷³

4.4. Tea (Camellia sinensis L.)

Green and black teas, major sources of flavan-3-ols and their derived products,^74,75 are consumed widely by the adult population of many countries.⁷⁶ Green tea has been reported to alleviate the detrimental effects of coeliac disease on the gut barrier.^77,78 Avoiding gluten intake is the only current treatment against this disease,^77,79,80 but complete adherence to this restricted diet is often not fulfilled.^79,80 In an in vitro Caco-2 model, a decaffeinated green tea extract (1 mg mL⁻¹) improved gut permeability and prevented a gliadin-induced decrease in TEER up to 24 h post-incubation.⁷⁷ Dias et al.⁷⁸ showed that attenuation of gliadin-induced morphological changes in colonic crypts, and villi of gluten-sensitive DQ8 transgenic mice, were reduced by green tea extract consumption (50 mg kg⁻¹ day⁻¹) over a 45-day period. In the absence of stressors, a 12-weeks oral supplementation of a green tea extract (2% w/w diet) did not significantly change colonic TJ expression (i.e., ZO-1, occludin, claudin-1), nor FITC-Dx permeability in mice, although serum and portal vein endotoxemia were decreased significantly.⁸¹ Furthermore, daily supplementation of animals with green tea-derived products significantly prevented or ameliorated impairments of the gut barrier caused by a high-fat diet, in terms of permeability to endotoxin and TJ expression.^81–84 Broadly in line with these results are the outputs reported for fu brick tea,⁸⁵ pu-erh tea⁸⁶ and its ripened version,^87,88 raw bowl tea,⁸⁹ and other tea-derived products.⁸⁶ As with lingonberries,⁷² batch effects were evident in the efficacy of ripened pu-erh tea (year of production 2006 vs. 2010) with respect to attenuation of DSS-induced ZO-1 protein expression decrease, but not in the modulation of MPO activity.⁹⁰

Alcohol is also a known cause of gut barrier impairment,⁸ and an aqueous extract of fu brick tea (400 mg per kg BW per day) was assessed for efficacy in mice supplemented daily for 12-weeks with alcohol (40%).⁹¹ The treatment improved the alcohol-impaired gut barrier function, up-regulating epithelial TJ expression and reducing circulating LPS concentrations.⁹¹ In addition, intra-gastric gavage with a tea flower extract (200 mg per kg BW per day) significantly mitigated against changes in markers of gut barrier disruption caused by intra-peritoneal supplementation of the cancer treatment cyclophosphamide (CTX) in mice.⁹² Furthermore, daily consumption for 22 days of a green tea catechin extract (0.1–0.5% w/v in drinking water) by Wistar rats significantly increased ileal mucins and decreased the sialomucins/sulfomucins ratio, although it did not affect the content of mucins in either the jejunum or colon.⁹³ Similarly, 3,4,5-trihydroxybenzoic acid (7, aka gallic acid), present in tea both as the free acid and conjugated to monomeric and polymeric flavan-3-ols, inhibited the increase in colonic sialic acid containing mucins as a consequence of sub-cutaneous exposure to 1,2-dimethylhydrazine (DMH).⁹⁴ In contrast to (–)-epicatechin (1), EGCG (2), one of the main monomeric flavan-3-ols in green tea, can cross-link with gastric (MUC5Ac) and duodenal (Muc2) porcine mucins in vitro.⁹⁵ Conjugated flavan-3-ols can also form multilayer-EGCG structures via interactions with other similar molecules already adsorbed to the mucus.⁹⁶

4.5. Grape (Vitis vinifera L.)

Cultivated grapes are a dietary source rich in proanthocyanidins,¹¹ which occur predominantly in skin and seeds.⁹⁷ Grape seed (12.5 μg L⁻¹)⁹⁸ and red wine (60 μg mL⁻¹)⁹⁹ extracts decrease the permeability of fluorescent markers and increase TEER in vitro, improving the barrier function of the intestinal cell layer. This permeability decrease was not always associated with increased expression of TJ proteins.^99–101 Similarly, in a porcine model, grape proanthocyanidins fed (100 and 250 mg kg⁻¹) for up to 28 days promoted a decrease in intestinal permeability in weaned piglets,^102,103 while a combined oral and intra-gastric supplementation of grape seed extract decreased faecal calprotectin, but not blood endotoxemia in rats.¹⁰⁴ Despite differences in (poly)phenolic composition, in the concentrations tested of grape-derived products, and in the duration of interventions, there seemed to be a general trend to protect against stress-induced increase in gut barrier permeability^{99,102,105–114} or decreased TJ expression^{99,102,105–119} caused by inflammatory^98,99,113 and oxidative challenges,¹⁰⁵ the microbial pathogen L. monocytogenes,¹¹⁹ ulcerative colitis,^{106,108,116–118,120} antibiotic treatment,¹⁰² weaning,¹⁰⁵ or westernised dietary patterns characterised by a high content of sugars and fats.^{106,107,109–112,114,115} Studies report significant protection mediated by grape-derived products on some of the monitored markers of gut barrier disruption.^{99,109,110,114,116,121} Absence of beneficial protection was observed for a grape skin powder¹¹⁶ and a table grape extract rich in (poly)phenols.¹⁰⁹ However, their effects improved when fractions obtained from the same original products, but differing in flavan-3-ol profiles, were tested.^109,116 Moreover, a decrease in claudin-1 caused by a cafeteria diet was prevented by intermittent supplementation of a grape seed procyanidin extract, but not when the intake of the extract occurred prior to stress exposure,¹¹¹ highlighting the potential importance of frequency of (poly)phenol intakes in the framework of stress exposure.

4.6. Other sources of flavan-3-ols or their metabolites

Studies on the effects of other dietary sources of flavan-3-ols on gut barrier function include peanut skin and other nut or derived products. Procyanidins from peanut skin significantly mitigated gut barrier impairments in mice affected by type 2 diabetes caused by a high-fat diet and streptozotocin.¹²² However, downregulation of claudin-1 caused by stressors of the gut barrier was not prevented by supplementation with peanut skin procyanidins.¹²² In contrast, a chestnut extract exhibited a dose-dependent decrease in TEER, which was accompanied by a significant decrease in claudin-2 expression.¹²³ However, it did not impact other monitored TJs, namely ZO-1, occludin, claudin-3 and -15, JAM-1.¹²³

4.7. Individual compounds and metabolites

When models were analysed in absence of stressor-related perturbations, a general absence of significant changes in markers of gut barrier integrity, TJs or mucin expression has been reported following intestinal cell line incubations, animal supplementation, and ex vivo exposure to (–)-epicatechin (1),^124–128 (–)-epicatechin-3-O-gallate (8),^127,129 and EGCG (2).^{127,130–132} Protective effects of (–)-epicatechin (1–20 μM) occur in vitro independent of the tested stress-source,^{1,128,133,134} while its effect against dysbiosis in the gut barrier of mice caused by high-fat diet was concentration-dependent upon oral supplementation (2–20 mg per kg BW).¹²⁸ Inhibition of stress-induced decrease in TEER and ZO-1 protein expression in mono-cell lines was caused by (+)-catechin (9) (10–50 μM) incubation.^1,125,135 In contrast, these effects were not observed in a more complex Caco-2/HT-29 MTX co-culture model when (+)-catechin was tested at a 50 μM concentration.⁴¹ Moreover, the protective activities exerted by EGCG (2) were dependent on the concentration,^78,132 stressor type,¹³⁰ and route of administration.¹³⁵ Regarding the latter aspect, a study by Wu and co-workers¹³⁵ highlighted that, in mice, the metabolism of EGCG (50 mg per kg BW per day) may represent a critical step for observing significant effects against DSS-induced changes in MPO plasma activity, disruption of TJs, and mucosal damages, as these properties were absent following rectal administration of the gallated flavan-3-ol.

An increase in TEER was reported upon incubation of Caco-2 cells with theaflavin-3′-O-gallate (10) (10–50 μM)¹²⁷ or a mixture of procyanidins with different degrees of polymerisation,¹³⁶ suggesting their possible effects as gut barrier strengthening factors. Indeed, all the studies testing bioactivity of pro(antho)cyanidins on cell models of gut barrier applied these compounds in their free form, and not bound to proteins which can modulate and mediate their bioactive potential.¹³⁷ Moreover, a decrease in the permeability of Caco-2 monolayers has been reported followed incubation with theaflavin-3′-O-gallate, theaflavin (11), theaflavin-3-O-digallate (12), theaflavin-3,3′-O-digallate (13),¹²⁷ theasinesin A (14), and theasinesin B (15).¹³⁸ Similar effects were observed for compounds with lower molecular weight namely 1,3,5-trihydroxybenzene (16, aka phloroglucinol)¹²⁵ and 3′,4′-dihydroxyphenylacetic acid (17).¹ With a combined in vitro and in silico model involving the use of a porcine gastric mucin type III, Brandão and co-workers¹³⁹ observed that a grape seed fraction, containing mainly procyanidin B4 and a tetramer, could interact with the mucins, through hydrogen bonds and hydrophobic interactions, similarly to that reported for EGCG.^95,96

As reviewed by other authors,^37,140,141 flavan-3-ols undergo extensive metabolism during their transit through the gastrointestinal tract and, especially in the colon where they are catabolised to simple phenolic catabolites. Significant dose-dependent mitigation against stress-induced impairment of gut barrier integrity has been reported for, 4-hydroxybenzoic acid (18), (10–40 mg kg⁻¹ day⁻¹),¹⁴² benzoic acid (19) (2–5 g kg⁻¹ diet),¹⁴³ and 3,4,5-trihydroxybenzoic acid (7) (5–50 μM).¹⁴⁴ Moreover, time-dependent protection exerted by phenolic compounds against stressors of this key intestinal structure,^125,145,146 as well as the absence or partial protective effects, have been described.^{1,62,86,142,147–150} For example, none of the simple phenolic compounds individually tested in vitro by Valdez and collaborators⁶² effectively protected Caco-2 cells against an inflammatory insult, suggesting that the (poly)phenolic fraction of aronia berry powder, having a mixture of these compounds, might not be the unique factor responsible for the beneficial effects attributed to the whole food extract. These results confirm the importance of elucidating the contribution of individual compounds to the overall effects observed in complex matrixes as food products and their extracts, as noted by Bianchi et al.⁴¹

5. Conclusions

The studies included in this review were mainly conducted employing cell models, with the colorectal adenocarcinoma Caco-2 cell line being the most frequently utilised, and in animals, typically mice and rats. Modulation or strengthening of the gut barrier was assessed by testing the flavan-3-ols, their metabolites, or their dietary sources in the absence of stress, or prior to a stress challenge. Their capacity to reduce the severity of damage and dysfunction, or to facilitate the re-establishment of gut barrier integrity following stress-induced impairment, were analysed by tests in mixed temporal models, or after challenge exposure. However, it is important that human interventions are carried out with healthy and patient groups to confirm these protective effects. Several human studies are ongoing,⁹ and a recent investigation by Del Bo’ et al.¹⁵¹ analysed the impact of a diet rich in (poly)phenols on the gut barrier integrity of older subjects via monitoring their serum zonulin levels and reported potential promising findings.

Greater elucidation of the potential benefits derived from flavan-3-ol phase II metabolites and their characteristic phenyl-γ-valerolactones (20) and phenylvaleric acid (21) colonic catabolites³⁷ will be essential in the context of person-to-person variations in the fate of flavan-3-ols within the gastrointestinal tract. In vitro and in silico analyses of the mechanisms and stability of the interactions between these compounds and the epithelial and mucus layers are needed to define their putative mechanism(s) of action. While analysis of the stability of tested compounds in the media used for cell models has been identified as a critical step in the assessment of their bioactivity,¹⁵² this has been investigated with a only a limited number of (poly)phenolic sources with the majority of incubations only up to 24 hours duration. Moreover, artifacts due to the addition of these (poly)phenolic compound to the cell models should be also monitored as they can mediate oxidative stress response via, e.g., hydrogen peroxide generation,¹⁵³ as recently reported by Mahmutović et al.¹⁵⁴ Therefore, it might be advisable that future research implements experimental protocols to consider these factors. For this purpose, the analysis of the concentration of metabolites present in the culture media during incubations requires investigation.^41,152 Furthermore, a more complete analysis of the intra-luminal availability of flavan-3-ols and their metabolites, both in terms of chemical structure and available concentrations, could better support future design of studies aiming at investigate the bioactivity of these compounds on the gut barrier. While a harmonised way to report the compounds name and tested quantity would benefit comparison between studies. Finally, the potential synergies of (poly)phenols with the resident microbiota in maintaining gut barrier functions also deserves attention.^65,155 In this regard, designing and testing functional foods or supplements to improve and maintain a healthy epithelial and mucus layer, including probiotics together with flavan-3-ols, represents a field of importance for future research. There are considerable differences in the metabolism of flavan-3-ols between animals and humans.³⁹ Therefore, clinical human studies are needed to further clarify the potential impact of these (poly)phenols on human gut health. The results reported in this review, in terms of possible promising compounds and concentrations to be tested, could represent a starting point for their design.

Author contributions

SD, LKP, NGT, DDR, and CIRG were involved in the generation of the topic. SD was involved in data review and writing of the review article. LKP, NGT, GJMD, GPC, LB, PM, TMA, AC, KMT, DDR, and CIRG critically revised and edited the manuscript. All the authors have read and approved the final manuscript.

Data availability

Refer to literature references for data availability of each study included in the current work. No new data was generated.

Conflicts of interest

The authors declare on conflicts of interest.

Acknowledgements

This work was undertaken as part of a PhD scholarship funded by the Department for the Economy (Northern Ireland). C. I. R. G., A. C., G. P.-C. and T. M. A. were funded by the Distinguished Scientist Fellowship Program (DSFP) of King Saud University, Riyadh, Saudi Arabia. GJMcD is grateful for support from the Rural & Environment Science & Analytical Services Division of the Scottish Government. The funders played no role in the design, analysis or writing of this article.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4fo04721d

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