**6. Biochemical and molecular mechanisms underlying the barrierprotective and anti-inflammatory properties of PAC in the intestine**

PACs were often considered to be nutritionally undesirable due to their ability to form complexes with macronutrients and reduce the activity of virtually any enzyme implicated in digestion [114, 115]. Nevertheless, based on the anticancerous, anti-mutagenic, and anti-microbial activities these phytochemicals elicited in laboratory experiments, a role in the modulation of the metabolism and immune system was suggested [115]. The ability of PACs to form cross-links with biomolecules can be attributed to the hydroxyl groups and aromatic rings in their structure that can establish hydrogen bonds and hydrophobic interactions [116]. PACs have a significant affinity for proline-rich proteins and peptides [117]. In general, binding to proteins seems to increase with the DP as larger PAC molecules have more potential binding sites for the associations with proline residues [117]. The interaction results in effects determined by the biological function of the target protein. Thus, PACs not only alter enzymatic activity, but they may also prevent ligand-receptor interactions and the binding of transcription factors to their specific sites in DNA. In addition, some PAC molecules can be adsorbed non-specifically onto biomembrane surfaces [118], affecting their physical characteristics, such as fluidity and density, and potentially altering membrane-dependent processes,

including protein receptor activity [119]. Altogether, these effects lead ultimately to the alteration of cell signaling pathways and the modulation of gene expression.

experimental results are not sufficiently consistent [138]. Interestingly, the existence of a colitogenic microbiota was demonstrated in T-bet/ RAG2/ deficient mice whose spontaneous ulcerative colitis was horizontally transmissible to wild-type individuals when co-housed [139]. Although mechanisms by which dysbiosis trigger intestinal dysfunction are not fully understood, it is known that they involve the loss of immune tolerance due to local immune homeostasis disrup-

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

Several authors have suggested that both dietary PACs, which are the substrates of intestinal bacteria, and the metabolites produced during PACs degradation in the colon may modulate and induce oscillations in the composition of the microbiota populations by means of prebiotic and antimicrobial effects against gut pathogenic microorganisms [141–144]. Dietary PACs, specifically longer polymers, reach the distal intestine nearly intact, where they become fermentable substrates for the commensal microbiota [145]. PACs have been associated with prebiotic properties, boosting the composition of several kinds of probiotics such as *Bifidobacterium* spp., *Lactobacillus* spp. [146] and the stimulator of mucus production *Akkermansia muciniphila* [147, 148]. Nevertheless, current evidence is somewhat controversial as effects described in different in vivo studies mainly performed with rodents, do not always agree. This suggests that interactions between PACs and microbiota depend largely on the botanical source, the types of molecules present in the extracts tested

A recent study by Casanova-Marti et al. [150] found that oral administration of GSPE in Wistar rats for 8 days resulted in profound changes in the cecal microbiota composition, reducing diversity indices and the ratio of *Firmicutes* to *Bacteroidetes*.

supplemented with a PAC-rich extract of the *Pyracantha fortuneana* fruit, although in this study an increase in microbiota diversity was also reported [81]. GSPE supplementation in IL-10 deficient mice resulted in an increased abundance of *Bacteroides* and *Lactobacilli* [86]. Xing et al. [148] reported that the administration of procyanidin B2 in rabbits feeding a high-fat-cholesterol diet, promoted an increase in the relative abundance of *Akkermansia*. These authors proposed that the reduction of metabolic endotoxemia found in animals treated with procyanidin B2 was attributed to the ability of *Akkermansia* to retain the thickness of the intestinal mucus layer, thus reducing intestinal permeability and the leakage of LPS into the

Cueva et al. [146] found that in vitro fermentation of grape-seed monomers and PACs in human feces resulted in a reduced abundance of *Clostridium histolyticum*. Inhibition of the growth of some infectious microorganisms, such as the mentioned *C. histolyticum* in the intestine and *Helicobacter pylori* in the stomach [152], may be related to the anti-adherence activity that PACs have demonstrated in in vitro studies [153], as adherence to the epithelium is a prerequisite for colonization and

Finally, phenolic acids and phenyl-γ-valerolactones resulting from the colonic

The health-promoting properties of PACs in the intestine are attributed not only to the antioxidant activity inherent to phenolic compounds, but also to the capacity

fermentation of PACs also exhibit a significant bioactivity in cell models and experimental animals [154]. They therefore may partially account for the beneficial anti-inflammatory effects reported in intestinal and peripheral tissues in vivo. Further research is needed to clarify the importance of these microbial products in

health-promoting properties associated with the intake of PACs.

Similar results were found in diet-induced obese Sprague Dawley rats

infection of the intestinal gastrointestinal mucosa.

**8. Conclusions and future perspectives**

tion and continuous abnormal activation of TLRs [140].

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

and the animal model [149].

circulation [151].

**283**

#### **6.1 Modulation of TJ integrity**

The precise mechanisms underlying the improvement in intestine paracellular permeability due to PACs in inflammation are not yet completely elucidated; however, it is known that they lead ultimately to the upregulation (e.g., ZO-1 and claudin-1 [8, 13]) or downregulation (e.g., claudin-2 [86]) of TJ protein expression. Loss of TJ integrity in the pro-inflammatory state is mediated by the NF-κB signaling pathway and by the activation of protein kinases MAPKs, PI3Ks, AMPK, and MLCK [120]. MLCK is particularly crucial in actomyosin-based cytoskeletal functions and multiple studies highlight its important role in intestinal TJ remodeling [121, 122]. PACs reduce the production of pro-inflammatory mediators (e.g., TNFα) and reactive oxygen species (i.e., iNOS activity) associated with enhancing intestinal permeability by antagonizing the NF-κB signaling pathway. In addition, PACs are potent inhibitors of kinases including MLCK [120, 123]. Contreras et al. [124] also suggested that there is an upstream novel mechanism associated with flavan-3-ols that leads to the prevention of TNF-α-induced intestinal permeability. In this study, TNF-α-stimulated Caco-2 monolayers incubated with () epicatechin showed a reduction of NOX activity, an enzyme that also facilitates activation of TNF-α signaling. This effect was directly associated with the inhibition of ERK1/2 MAPK activity of IκB phosphorylation and of MLCK activation.

#### **6.2 Interaction with bacterial endotoxins**

Delehanty et al. [125] demonstrated that naturally occurring A- and B-type cranberry PACs were able to bind the lipid A moiety of LPS, exhibiting an affinity similar to that of polymyxin B, a potent LPS-binding molecule. In this study, PACs efficiently blocked endocytosis of bacterial LPS in a dose-dependent manner in HEK 293 (human embryonic kidney cells) that expressed receptors TLR4/MD-2 and CD14, thus preventing the induction of the NF-κB signaling pathway without any interaction with cellular components. However, other authors reported that PACs isolated from cocoa beans did not abrogate the binding of LPS to TLR4 in cultivated human dendritic cells [126]. PAC-LPS binding has been linked to the reduction of the post prandial increase in blood LPS associated with the ingestion of a high-fat meal in obese subjects ingesting an oral dose of GSPE [112].

#### **7. PACs modulation of intestinal microbiota**

Diet plays an important role in the composition of intestine microbiota, promoting or inhibiting growth of microorganisms [127]. Alterations in the composition and metabolism of the intestinal microbiota (dysbiosis) have also been associated with the consumption of high-saturated fat diets in rodents and humans [128, 129]. In fact, metagenomic analysis of the intestinal microbiome in Western populations has shown a reduction not only of microbial diversity, but also of functional potential [130]. Dysbiosis is linked to obesity-associated intestinal inflammation, although the "egg or hen" question related to the cause-effect relationship is not answered yet [131]. High-fat intake in rodents often decreases overall diversity of microbiota and the abundance of Bacteroidetes, and increases the relative abundance of Firmicutes [132, 133]. Several human studies have described similar associations [134, 135], but the importance of the ratio Firmicutes to Bacteroidetes remains controversial [136, 137], and some authors state that the

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

experimental results are not sufficiently consistent [138]. Interestingly, the existence of a colitogenic microbiota was demonstrated in T-bet/ RAG2/ deficient mice whose spontaneous ulcerative colitis was horizontally transmissible to wild-type individuals when co-housed [139]. Although mechanisms by which dysbiosis trigger intestinal dysfunction are not fully understood, it is known that they involve the loss of immune tolerance due to local immune homeostasis disruption and continuous abnormal activation of TLRs [140].

Several authors have suggested that both dietary PACs, which are the substrates of intestinal bacteria, and the metabolites produced during PACs degradation in the colon may modulate and induce oscillations in the composition of the microbiota populations by means of prebiotic and antimicrobial effects against gut pathogenic microorganisms [141–144]. Dietary PACs, specifically longer polymers, reach the distal intestine nearly intact, where they become fermentable substrates for the commensal microbiota [145]. PACs have been associated with prebiotic properties, boosting the composition of several kinds of probiotics such as *Bifidobacterium* spp., *Lactobacillus* spp. [146] and the stimulator of mucus production *Akkermansia muciniphila* [147, 148]. Nevertheless, current evidence is somewhat controversial as effects described in different in vivo studies mainly performed with rodents, do not always agree. This suggests that interactions between PACs and microbiota depend largely on the botanical source, the types of molecules present in the extracts tested and the animal model [149].

A recent study by Casanova-Marti et al. [150] found that oral administration of GSPE in Wistar rats for 8 days resulted in profound changes in the cecal microbiota composition, reducing diversity indices and the ratio of *Firmicutes* to *Bacteroidetes*. Similar results were found in diet-induced obese Sprague Dawley rats supplemented with a PAC-rich extract of the *Pyracantha fortuneana* fruit, although in this study an increase in microbiota diversity was also reported [81]. GSPE supplementation in IL-10 deficient mice resulted in an increased abundance of *Bacteroides* and *Lactobacilli* [86]. Xing et al. [148] reported that the administration of procyanidin B2 in rabbits feeding a high-fat-cholesterol diet, promoted an increase in the relative abundance of *Akkermansia*. These authors proposed that the reduction of metabolic endotoxemia found in animals treated with procyanidin B2 was attributed to the ability of *Akkermansia* to retain the thickness of the intestinal mucus layer, thus reducing intestinal permeability and the leakage of LPS into the circulation [151].

Cueva et al. [146] found that in vitro fermentation of grape-seed monomers and PACs in human feces resulted in a reduced abundance of *Clostridium histolyticum*. Inhibition of the growth of some infectious microorganisms, such as the mentioned *C. histolyticum* in the intestine and *Helicobacter pylori* in the stomach [152], may be related to the anti-adherence activity that PACs have demonstrated in in vitro studies [153], as adherence to the epithelium is a prerequisite for colonization and infection of the intestinal gastrointestinal mucosa.

Finally, phenolic acids and phenyl-γ-valerolactones resulting from the colonic fermentation of PACs also exhibit a significant bioactivity in cell models and experimental animals [154]. They therefore may partially account for the beneficial anti-inflammatory effects reported in intestinal and peripheral tissues in vivo. Further research is needed to clarify the importance of these microbial products in health-promoting properties associated with the intake of PACs.

#### **8. Conclusions and future perspectives**

The health-promoting properties of PACs in the intestine are attributed not only to the antioxidant activity inherent to phenolic compounds, but also to the capacity

including protein receptor activity [119]. Altogether, these effects lead ultimately to the alteration of cell signaling pathways and the modulation of gene expression.

The precise mechanisms underlying the improvement in intestine paracellular permeability due to PACs in inflammation are not yet completely elucidated; however, it is known that they lead ultimately to the upregulation (e.g., ZO-1 and claudin-1 [8, 13]) or downregulation (e.g., claudin-2 [86]) of TJ protein expression. Loss of TJ integrity in the pro-inflammatory state is mediated by the NF-κB signaling pathway and by the activation of protein kinases MAPKs, PI3Ks, AMPK, and MLCK [120]. MLCK is particularly crucial in actomyosin-based cytoskeletal functions and multiple studies highlight its important role in intestinal TJ remodeling [121, 122]. PACs reduce the production of pro-inflammatory mediators (e.g., TNFα) and reactive oxygen species (i.e., iNOS activity) associated with enhancing intestinal permeability by antagonizing the NF-κB signaling pathway. In addition, PACs are potent inhibitors of kinases including MLCK [120, 123]. Contreras et al. [124] also suggested that there is an upstream novel mechanism associated with flavan-3-ols that leads to the prevention of TNF-α-induced intestinal permeability.

In this study, TNF-α-stimulated Caco-2 monolayers incubated with () epicatechin showed a reduction of NOX activity, an enzyme that also facilitates activation of TNF-α signaling. This effect was directly associated with the inhibition

of ERK1/2 MAPK activity of IκB phosphorylation and of MLCK activation.

a high-fat meal in obese subjects ingesting an oral dose of GSPE [112].

**7. PACs modulation of intestinal microbiota**

**282**

Delehanty et al. [125] demonstrated that naturally occurring A- and B-type cranberry PACs were able to bind the lipid A moiety of LPS, exhibiting an affinity similar to that of polymyxin B, a potent LPS-binding molecule. In this study, PACs efficiently blocked endocytosis of bacterial LPS in a dose-dependent manner in HEK 293 (human embryonic kidney cells) that expressed receptors TLR4/MD-2 and CD14, thus preventing the induction of the NF-κB signaling pathway without any interaction with cellular components. However, other authors reported that PACs isolated from cocoa beans did not abrogate the binding of LPS to TLR4 in cultivated human dendritic cells [126]. PAC-LPS binding has been linked to the reduction of the post prandial increase in blood LPS associated with the ingestion of

Diet plays an important role in the composition of intestine microbiota, promoting or inhibiting growth of microorganisms [127]. Alterations in the composition and metabolism of the intestinal microbiota (dysbiosis) have also been

associated with the consumption of high-saturated fat diets in rodents and humans [128, 129]. In fact, metagenomic analysis of the intestinal microbiome in Western populations has shown a reduction not only of microbial diversity, but also of functional potential [130]. Dysbiosis is linked to obesity-associated intestinal inflammation, although the "egg or hen" question related to the cause-effect relationship is not answered yet [131]. High-fat intake in rodents often decreases overall diversity of microbiota and the abundance of Bacteroidetes, and increases the relative abundance of Firmicutes [132, 133]. Several human studies have described similar associations [134, 135], but the importance of the ratio Firmicutes to Bacteroidetes remains controversial [136, 137], and some authors state that the

**6.2 Interaction with bacterial endotoxins**

**6.1 Modulation of TJ integrity**

*Weight Management*

of these phytochemicals to interact with multiple biomolecules, including proteins, biomembrane lipids, and endotoxins. Bioactivity of PACs is highly structuredependent and enriched botanical extracts composed by a large variety of molecular structures exert a wide range of unrelated physiological effects. In this way, PACrich extracts can modulate kinase activity, several signal transduction pathways implicated in the inflammatory response and the remodeling of TJs. Flavan-3-ol monomers and short PAC oligomers are absorbed by enterocytes and immune cells and exert a direct action on kinases and transcription factors. Bioactivity of larger oligomers and polymeric PACs do not require direct intestinal absorption and are able to bind protein receptors on the enterocyte and immune cell surfaces as well as luminal bacterial endotoxins, thus inhibiting pro-inflammatory signaling and improving barrier integrity. Due to the negligible absorption of large PAC molecules in the small intestine, phenyl-γ-valerolactones and phenolic acids produced by the microbiota metabolism in the colon are thought to play an important role in these health-promoting effects, and thus need to be further researched.

The barrier-protective properties of PACs are emerging as a potential adjunctive support to current therapies for managing obesity related intestinal dysfunction and IBD. However, there have been no large, well-designed clinical trials establishing the efficacy of these phytochemicals in chronic conditions. At preclinical stages, the use of animal models is the predominant approach for testing novel therapies for intestinal dysfunction, although several strategies for replacing animal experiments have been proposed. As there are still no studies on the impact of PACs on human intestinal health, ex vivo models of the human intestine could be a more physiologically reliable alternative to human cell lines and an alternative to animal experimentation in preclinical development.
