Beneficial Effects of Proanthocyanidins on Intestinal Permeability and Its Relationship with Inflammation

*Carlos González-Quilen, Esther Rodríguez-Gallego, Raúl Beltrán-Debón, Montserrat Pinent, Anna Ardévol, Maria Teresa Blay and Ximena Terra*

## **Abstract**

The intestinal barrier is constantly exposed to potentially harmful environmental factors including food components and bacterial endotoxins. When the intestinal barrier function and immune homeostasis are compromised, inflammatory conditions may be developed and impact overall health. Evidence from experimental animal and cell-culture studies suggests that exposure of intestinal mucosa to proanthocyanidin-rich plant products may contribute to maintain the barrier function and to ameliorate the inflammation present in prevalent pathologies such as diet-induced obesity and inflammatory bowel disease. In this review, we aim to update the current knowledge on the bioactivity of PACs in experimental models of altered intestinal permeability and in humans, emphasizing the beneficial effects of grape-seed proanthocyanidin extracts in intestinal health and giving insights into the subjacent biochemical and molecular mechanism.

**Keywords:** gut, permeability, inflammation, metabolic endotoxemia, obesity, IBD, flavonoid, flavan-3-ol, condensed tannin, procyanidin

#### **1. Introduction**

The primary function of the intestinal tract is to digest food components and absorb nutrients and water from the lumen to the systemic circulation. The intestine is also a physical barrier that is in contact with the environment. As a result, the intestinal epithelium is constantly exposed to potentially pathogenic microorganisms, toxins, and harmful components of the diet. When there are disturbances in the barrier function and mucosal immune homeostasis, the influx of intestine luminal content triggers barrier dysfunction and an exaggerated mucosal immune response [1]. Ultimately, chronic exposition to these detrimental environmental stimuli may lead to the development of local and systemic inflammatory conditions [2, 3] that contribute to barrier dysfunction.

Natural products have been recognized as a source of therapeutic agents for many years [4]. Some plant-derived phenolic compounds show promising

anti-inflammatory effects and have been associated with the prevention of certain chronic diseases [5]. Proanthocyanidins (PACs), also known as condensed tannins, are oligo- and polymeric end products of the flavonoid biosynthesis pathway in plants [6]. There has been extensive laboratory research into the effects of both pure PAC molecules and PAC-rich extracts on overall health. These phytochemicals show a wide range of physiological activities [7], including anti-inflammatory and barrier-protective effects in the intestine [8–10], which may be interesting in the context of diet-induced obesity and inflammatory bowel disease (IBD).

various inflammatory conditions of the gut [24]. Previous studies [25–28] have shown that myosin light chain kinase (MLCK) plays a central role in the regulation of intestinal TJ permeability. The activation of MLCK catalyzes the phosphorylation of myosin light chain (MLC), inducing contraction of the peri-junctional actinmyosin filaments and the opening of the TJ barrier. In contrast, inhibition of MLCK

*Beneficial Effects of Proanthocyanidins on Intestinal Permeability and Its Relationship…*

*DOI: http://dx.doi.org/10.5772/intechopen.91212*

activation prevents this effect [27]. It has been suggested that the cytokinemediated barrier dysfunction could be mediated by an increase in Nuclear

translation and transcription of inflammatory mediators [30].

(**Figure 1**).

**Figure 1.**

**271**

Factor (NF)-kB, which, in turn, activates MLCK gene and protein expression [29]

Once intestinal bacteria and endotoxins enter the portal vein and/or lymphatic system, they can reach other tissues and organs, leading to a cascade response modulated by inflammatory mediators. This situation can induce a systemic inflammatory response, which further damages the function of the intestinal barrier [30]. The endotoxin-signaling pathway includes the binding of LPS to LPS-binding protein (LBP) and its subsequent transfer to the CD14 receptor. LBP-bound LPS initiates inflammation via TLRs associated with membrane-anchored CD14 [31]. TLRs are a family of pattern-recognition receptors that play a key role in the innate immune system. Among all, the TLR4 is expressed at high levels in the intestinal tract, and given that LPS is its specific ligand, TLR4 could be considered the first barrier for recognition of bacterial presence in the gastrointestinal tract. NF-kB is the final effector transcription factor of the TLR4 signaling pathway. It promotes the development of many intestinal diseases and also plays a pivotal role in the

In mammals, the NF-kB family comprises five proteins, including p65 (RelA), RelB, c-Rel, p105/p50 (NF-kB1), and p100/p52 (NF-kB2), which associate with each other to form transcriptionally distinct homo- and heterodimeric complexes; the p65:p50 heterodimer is the most abundant and the most relevant for inflammation [32]. In resting cells, the p65:p50 NF-kB heterodimer is sequestered in the cytoplasm by binding to its inhibitory protein, IkappaB (IkB). In response to an inflammatory stimulus, such as LPS, the classical NF-kB activation pathway leads to the activation of the IkB kinase (IkkB), a member of the IKK complex, triggering IkB-a phosphorylation (pIkB-a). Then, pIkB-a is recognized by the ubiquitin ligase

machinery, resulting in its polyubiquitination and subsequent proteasomal

*Protective properties of PACs in the intestinal barrier function. (A) Chronic exposition to detrimental environmental stimuli may lead to dysbiosis, breakdown of the intestinal barrier, influx of bacterial endotoxins and mucosal inflammation. (B) PACs ameliorate loss of barrier function blocking the activation of MLCK*

*mediated by NF-κB and MAPK signaling. See text for details.*

We have reported previously that grape-seed PACs and other flavonoids have beneficial effects on inflammation [11–13] and protect the intestine against alterations associated with diet-induced obesity in rats [8, 9, 14, 15]. In addition, research conducted during the last decade with cell culture and animal models has made significant progress in determining the underlying mechanism of the healthpromoting properties of PACs in the gastrointestinal tract and peripheral tissues.

### **2. Altered networks in intestinal dysfunction: barrier integrity and inflammatory response**

The intestinal epithelium is a single cell-layer responsible for separating underlying mucosal tissues from the environment and is the largest exposed surface area in the body [16]. As there is a prolific commensal microbial community in the intestinal lumen (intestinal microbiota), epithelial integrity plays a pivotal role in maintaining overall health [16, 17]. The intestinal epithelium is integrated by several cell types with specialized functions. The enterocytes are responsible for the absorptive function and constitute the most abundant epithelial cell lineage. The goblet cells are implicated in the synthesis of secretory mucin glycoproteins that form the mucus layer [18]. Other cellular types integrating the epithelium, microfold (M) [19], Paneth and enteroendocrine cells are specialized in antigen sampling and presentation to dendritic cells, synthesis of antimicrobial peptides, and secretion of hormones, respectively.

The first strategy the host tissue has to maintain its homeostatic relationship with the intestinal microbiota is to minimize the physical interaction with microorganisms, thus limiting microbial translocation and physiological inflammation [20, 21]. The thick mucus layer secreted by goblet cells represents a primary defense line against environmental insults [18]. In addition, the enterocytes are joined together forming an intricately and well-regulated barrier sustained by intercellular junctions linked to the cell cytoskeleton, such as tight junctions (TJs), desmosomes, and adherent junctions. TJs partially seal the paracellular space and prevent passive transport of large molecules, including microbial components and other potentially harmful agents [1, 22].

The paracellular and transcellular pathways are the two major pathways mediating transmembrane transfer of intestinal bacterial substances. Both mechanisms may be involved in intestinal mucosal barrier damage and bacterial translocation. The paracellular pathway is integrated by tight junctions (TJs), consisting of zonulin/zonula occludens (ZO)-1, occludin, claudins, junction adhesion molecules (JAMs), and actin-myosin cytoskeletal proteins. Previous studies have shown that inflammatory cytokines and bacterial antigens can affect the expression level and assembly of these elements, thereby exerting an influence on TJ functions [23]. Immune cells, including neutrophils, dendritic cells, and monocytes, have also been directly implicated in inducing disturbances in TJ barrier function. It has been postulated that pro-inflammatory cytokine-induced opening of the intestinal TJ barrier is an important mechanism contributing to the TJ barrier defects present in

#### *Beneficial Effects of Proanthocyanidins on Intestinal Permeability and Its Relationship… DOI: http://dx.doi.org/10.5772/intechopen.91212*

various inflammatory conditions of the gut [24]. Previous studies [25–28] have shown that myosin light chain kinase (MLCK) plays a central role in the regulation of intestinal TJ permeability. The activation of MLCK catalyzes the phosphorylation of myosin light chain (MLC), inducing contraction of the peri-junctional actinmyosin filaments and the opening of the TJ barrier. In contrast, inhibition of MLCK activation prevents this effect [27]. It has been suggested that the cytokinemediated barrier dysfunction could be mediated by an increase in Nuclear Factor (NF)-kB, which, in turn, activates MLCK gene and protein expression [29] (**Figure 1**).

Once intestinal bacteria and endotoxins enter the portal vein and/or lymphatic system, they can reach other tissues and organs, leading to a cascade response modulated by inflammatory mediators. This situation can induce a systemic inflammatory response, which further damages the function of the intestinal barrier [30]. The endotoxin-signaling pathway includes the binding of LPS to LPS-binding protein (LBP) and its subsequent transfer to the CD14 receptor. LBP-bound LPS initiates inflammation via TLRs associated with membrane-anchored CD14 [31]. TLRs are a family of pattern-recognition receptors that play a key role in the innate immune system. Among all, the TLR4 is expressed at high levels in the intestinal tract, and given that LPS is its specific ligand, TLR4 could be considered the first barrier for recognition of bacterial presence in the gastrointestinal tract. NF-kB is the final effector transcription factor of the TLR4 signaling pathway. It promotes the development of many intestinal diseases and also plays a pivotal role in the translation and transcription of inflammatory mediators [30].

In mammals, the NF-kB family comprises five proteins, including p65 (RelA), RelB, c-Rel, p105/p50 (NF-kB1), and p100/p52 (NF-kB2), which associate with each other to form transcriptionally distinct homo- and heterodimeric complexes; the p65:p50 heterodimer is the most abundant and the most relevant for inflammation [32]. In resting cells, the p65:p50 NF-kB heterodimer is sequestered in the cytoplasm by binding to its inhibitory protein, IkappaB (IkB). In response to an inflammatory stimulus, such as LPS, the classical NF-kB activation pathway leads to the activation of the IkB kinase (IkkB), a member of the IKK complex, triggering IkB-a phosphorylation (pIkB-a). Then, pIkB-a is recognized by the ubiquitin ligase machinery, resulting in its polyubiquitination and subsequent proteasomal

#### **Figure 1.**

anti-inflammatory effects and have been associated with the prevention of certain chronic diseases [5]. Proanthocyanidins (PACs), also known as condensed tannins, are oligo- and polymeric end products of the flavonoid biosynthesis pathway in plants [6]. There has been extensive laboratory research into the effects of both pure PAC molecules and PAC-rich extracts on overall health. These phytochemicals show a wide range of physiological activities [7], including anti-inflammatory and barrier-protective effects in the intestine [8–10], which may be interesting in the

We have reported previously that grape-seed PACs and other flavonoids have beneficial effects on inflammation [11–13] and protect the intestine against alterations associated with diet-induced obesity in rats [8, 9, 14, 15]. In addition, research conducted during the last decade with cell culture and animal models has made significant progress in determining the underlying mechanism of the healthpromoting properties of PACs in the gastrointestinal tract and peripheral tissues.

The intestinal epithelium is a single cell-layer responsible for separating underlying mucosal tissues from the environment and is the largest exposed surface area in the body [16]. As there is a prolific commensal microbial community in the intestinal lumen (intestinal microbiota), epithelial integrity plays a pivotal role in maintaining overall health [16, 17]. The intestinal epithelium is integrated by several cell types with specialized functions. The enterocytes are responsible for the absorptive function and constitute the most abundant epithelial cell lineage. The goblet cells are implicated in the synthesis of secretory mucin glycoproteins that form the mucus layer [18]. Other cellular types integrating the epithelium, microfold (M) [19], Paneth and enteroendocrine cells are specialized in antigen sampling and presentation to dendritic cells, synthesis of antimicrobial peptides,

The first strategy the host tissue has to maintain its homeostatic relationship with the intestinal microbiota is to minimize the physical interaction with microorganisms, thus limiting microbial translocation and physiological inflammation [20, 21]. The thick mucus layer secreted by goblet cells represents a primary defense line against environmental insults [18]. In addition, the enterocytes are joined together forming an intricately and well-regulated barrier sustained by intercellular junctions linked to the cell cytoskeleton, such as tight junctions (TJs), desmosomes, and adherent junctions. TJs partially seal the paracellular space and prevent passive transport of large molecules, including microbial components and other potentially

The paracellular and transcellular pathways are the two major pathways mediating transmembrane transfer of intestinal bacterial substances. Both mechanisms may be involved in intestinal mucosal barrier damage and bacterial translocation. The paracellular pathway is integrated by tight junctions (TJs), consisting of zonulin/zonula occludens (ZO)-1, occludin, claudins, junction adhesion molecules (JAMs), and actin-myosin cytoskeletal proteins. Previous studies have shown that inflammatory cytokines and bacterial antigens can affect the expression level and assembly of these elements, thereby exerting an influence on TJ functions [23]. Immune cells, including neutrophils, dendritic cells, and monocytes, have also been directly implicated in inducing disturbances in TJ barrier function. It has been postulated that pro-inflammatory cytokine-induced opening of the intestinal TJ barrier is an important mechanism contributing to the TJ barrier defects present in

context of diet-induced obesity and inflammatory bowel disease (IBD).

**2. Altered networks in intestinal dysfunction: barrier integrity**

**and inflammatory response**

*Weight Management*

and secretion of hormones, respectively.

harmful agents [1, 22].

**270**

*Protective properties of PACs in the intestinal barrier function. (A) Chronic exposition to detrimental environmental stimuli may lead to dysbiosis, breakdown of the intestinal barrier, influx of bacterial endotoxins and mucosal inflammation. (B) PACs ameliorate loss of barrier function blocking the activation of MLCK mediated by NF-κB and MAPK signaling. See text for details.*

degradation. After pIkB-a degradation, the p65:p50 heterodimers are able to translocate to the nucleus, where they bind to the kB motif found in the promoter or enhancer regions of numerous pro-inflammatory genes to induce their expression [33].

NF-kB target genes include cytokines (e.g., tumor necrosis factor (TNF)-α and interleukins), adhesion molecules, acute phase proteins, and inducible enzymes (inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX2)), among others [11]. All of these genes contain verified NF-kB binding sites in their sequences, providing strong experimental evidence for their direct control by NF-kB [34]. Among all of these genes, the expression of iNOS and COX2 has been widely studied in relation to intestinal inflammation. In this regard, sustained high nitric oxide (NO) production by iNOS plays a role in the pathology of chronic inflammatory bowel disease [35, 36]. During the last decade, it has become increasingly clear that NO overproduction by iNOS is deleterious to intestinal function [37], thus contributing significantly to gastrointestinal immunopathology. Cyclooxygenases are enzymes that are responsible for the metabolism of arachidonic acid, converting it into prostaglandins. These products influence a wide variety of biological processes, ranging from homeostasis to inflammation [38]. There are two cyclooxygenase isoforms: the constitutive COX1 isoform and the inducible COX2 isoform [38, 39]. As a result of COX2 induction, prostaglandin E2 levels increase at the site of inflammation and can also be detected systemically.

Multiple environmental factors have been identified as potential triggers of intestinal inflammatory conditions, including Western dietary habits [40]. It has been described that saturated fats play a direct role in inflammatory signaling. Saturated fatty acids (SFA) such as lauric (C12:0) and palmitic (C16:0) directly induce NF-κB activation, acting as non-microbial TLR2 and TLR4 agonists in macrophages [41]. Data suggest that activation of TLRs by SFA is mediated by TLR dimerization and recruitment into lipid rafts [42]. We have reported mild intestinal inflammation and increased permeability in rats feeding on a cafeteria diet consisting of high-saturated fat/high-refined sugar food products [43]. This enhanced permeability has been shown to favor bacterial LPS and other potentially pro-inflammatory molecules entering the systemic circulation, which is known as metabolic endotoxemia [15].

Dietary assessment studies have shown that PACs, especially procyanidins are among the most abundant polyphenols in the human diet [6], as they are present in a variety of botanical sources and plant food products such as tea, fruits, nuts, cacao products, legumes, and cereal grains [1, 2]. However, PAC intake varies widely between geographical regions and cultures and is greatly dependent on eating habits, lifestyle behaviors, and socioeconomic status [48]. The daily PAC (dimers to polymers) intakes in adult populations from Korea, the U.S., Mexico, and EU were estimated as 71 [49], 73 [48], 103 [50], and 123–180 mg [51, 52], respectively, but intakes up to 230 mg d<sup>1</sup> have been reported in some regions of Spain and

*Chemical structures of PACs. Flavan-3-ol monomers differ based on the hydroxylation pattern and their cis- or trans-configuration. Dimers A1/A2 and B1/B2 are shown as example of A- and B-type PACs, respectively.*

*Beneficial Effects of Proanthocyanidins on Intestinal Permeability and Its Relationship…*

*DOI: http://dx.doi.org/10.5772/intechopen.91212*

Flavan-3-ols are remarkably stable during gastric transit in humans [54]. Monomers such as (+)- catechin and ()- epicatechin are readily absorbed in the upper sections of the small intestine [55, 56], recognized as xenobiotics and then subjected to an extensive phase II metabolism that generates glucuronidated, sulfated, and methylated conjugates [57]. Flavan-3-ol monomers and their conjugated metabolites reach peak plasma concentration 1–4 h after flavan-3-ol-rich food consumption

[58–60]. Studies conducted in cultivated epithelial monolayers [61–63], rats [64, 65], and humans [60, 66] indicate that PAC absorption is conversely more

Norway [53].

**273**

**Figure 2.**

**4. The fate of PACs after ingestion**

Taken together, these data suggest that HF diet-induced changes in the intestinal microbiota could be responsible for metabolic endotoxemia and for the onset of the corresponding diseases. The causative link between changes in intestinal bacteria populations, endotoxemia, and metabolic disease needs further assessment [44], but the mechanisms likely include altered epithelial permeability, translocation of bacterial products, and upregulation of pro-inflammatory cytokines and hormones produced by gut endocrine cells, mechanisms which might be modulated by PACs.
