**5. In vitro, in vivo and ex vivo studies on the benefits of PACs for intestinal dysfunction**

During the last decade, the beneficial properties of PACs for intestinal function have been reported in several studies performed with cell-culture models and experimental animals (**Tables 1** and **2**). This experimental data indicate that PACs contribute to maintaining the intestinal barrier and improving mucosal inflammation induced by environmental insults. However, there are few studies on the effect of PACs on human intestinal health, although epidemiological studies connect PAC-rich food consumption with a lower risk of colorectal cancer [88].

**275**

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

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

**Table 1.** *Interaction*

 *of PACs with intestinal permeability*

 *markers in cell culture and animal models [10, 76, 82–87].*


**1.***InteractionofPACswithintestinalpermeabilitymarkers in cellcultureandanimalmodels[10,76,82–87].*

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

limited and is highly dependent on DP, and that the permeation of larger oligomers (DP > 5) and polymers is negligible. No PAC transporter has been identified in the enterocyte membrane in the small intestine. Thus, dimers to tetramers are passively transported across the intestinal epithelium essentially by paracellular diffusion. Although transcellular passive diffusion is not likely to occur due to the hydrophilic nature of PACs conferred by the multiple hydroxyl groups, uptake might be possi-

In humans, a study assessed the contribution of the ingested cocoa flavan-3-ols and procyanidins to the systemic pool, and found that the plasma (�)- epicatechin came from the orally administered cocoa (�)- epicatechin and not from their oligomers or polymers [67]. This is in agreement with the evidence obtained with rats that suggests that PACs from different sources do not depolymerize to mono-

mers after ingestion [68, 69]. Stalmach et al. [56] conducted a study with ileostomized patients who were administered green tea, and found 70% of the ingested flavan-3-ol in the ileal fluid after 24 h. Altogether, these findings suggest that substantial amounts of ingested flavan-3-ol monomers and PACs remain unabsorbed in the small intestine and reach the colon. There, they are efficiently transformed by the colonic microbiota into low molecular weight phenolic com-

In vitro fermentation of purified procyanidin dimers with human fecal

bly to the inhibition of digesting enzymes or to the antibacterial properties

**5. In vitro, in vivo and ex vivo studies on the benefits of PACs**

effect of PACs on human intestinal health, although epidemiological studies connect PAC-rich food consumption with a lower risk of colorectal

During the last decade, the beneficial properties of PACs for intestinal function

have been reported in several studies performed with cell-culture models and experimental animals (**Tables 1** and **2**). This experimental data indicate that PACs contribute to maintaining the intestinal barrier and improving mucosal inflammation induced by environmental insults. However, there are few studies on the

ized cross-over study in healthy humans found that a great portion of the ingested (�)- epicatechin and procyanidin B1 was metabolized by the colonic microbiota to produce phenyl-γ-valerolactones as the major microbial metabolites [60]. In this study, microbial degradation of larger procyanidins was substantially lower, possi-

exhibited by these compounds. Other human studies analyzing the bioavailability of flavan-3-ols, reported high levels of phenyl-γ-valerolactones in the circulation and urinary excretion after ingestion of a red grape pomace drink [71] and apple juice [72]. In the colonocytes and hepatocytes, these microbial products undergo further metabolism by phase II enzymes to produce conjugated derivatives. Margalef et al. [73] analyzed the tissue distribution of metabolites derived from a grape-seed proanthocyanidin extract (GSPE) 2 h after ingestion by rats. These authors detected a few microbial metabolites (methyl conjugated phenols) at low concentrations in the colon tissue, while most phase II metabolites (glucuronidated and methylglucuronidated forms) were found in the kidneys and liver. In humans, the major contributors to the excretion of phenyl-γ-valerolactones after ingestion of a red grape pomace drink, are sulfated and glucuronidated conjugates of 5-(3<sup>0</sup>

,4<sup>0</sup>



,4<sup>0</sup> -

ble by endocytic mechanisms [62].

*Weight Management*

pounds that can be absorbed by colonocytes [57].

microbiota has shown to produce mainly 2-(3<sup>0</sup>

dihydroxyphenyl)-γ-valerolactone [71].

**for intestinal dysfunction**

cancer [88].

**274**

5-(3<sup>0</sup> ,4<sup>0</sup>


**Table 2.**

**277**

*Interaction*

 *of PACs with permeability*

 *and* 

*inflammatory*

 *markers in animal models of intestinal dysfunction*

 *[8, 13–15, 88–94].*

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

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


**Table 2.**

*Interaction of PACs with permeability and inflammatory markers in animal models of intestinal dysfunction [8, 13–15, 88–94].*

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

**276**

*Weight Management*

In vitro models of inflammation have been fundamental in the comprehension of cellular mechanisms driving physiological effects of bioactive molecules. Studies on intestinal dysfunction have employed human colon carcinoma cell lines, being Caco-2 the most well-established and widely used model of the human intestine barrier ([89] and **Table 1**). Mucus producer [79], macrophages [90], and B cell lines [91] have been employed in co-culture systems to explore the interaction between cell populations. Although there is a strong trend in the industry toward replacing animal experiments with human cell-culture based models [92, 93], there are no in vitro models of the human intestine that replicate the complex interplay between cell types and the regulation of the barrier function by the mucosal innate and adaptive immunity. Therefore, most physiologically relevant data on intestinal dysfunction comes from the animal model. Most in vivo studies testing the effect of PAC supplementation on intestinal health have been performed in diet-induced obesity models and chemical-induced colitis models. The first resemble intestinal alterations seen in humans with metabolic syndrome [43]. The latter closely mimic histopathological features of human colitis and are frequently used to study the pathophysiology of IBD and the effectiveness of novel therapeutic drugs [94]. Notably, PAC-rich grape-seed extracts (GSPE) are among the most studied botanical extracts, mainly by in vivo approaches in rodents (**Table 2**).

changes did not reduce TEER loss. Altogether, these results suggest that the ability of PACs to strengthen the intestinal barrier integrity depends on the degree of

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

The cafeteria (CAF) diet is a self-selected high-saturated fat/high-refined sugar diet that stimulates hyperphagia and a rapid weight gain in experimental animals [97, 98]. In this feeding regime, highly palatable and energy dense foods commercially available, such as muffins, biscuits, bacon, sausages, and sugared milk, are offered *ad libitum* [15, 99]. A long-term CAF diet (62% carbohydrate (mostly sugar), 23% lipid, and 13% protein) has negative effects on intestinal function in rodents, increasing intestinal permeability, and inducing mucosal inflammation [43]. We have described the beneficial effects of administering GSPE against the intestinal dysfunction induced by a long-term CAF diet (17–18 weeks) in Wistar rats [8, 14, 15]. The composition of the GSPE used in these studies has been analyzed in detail [100]. Both nutritional (5–50 mg kg<sup>1</sup> [14]) and pharmacological (100–500 mg kg<sup>1</sup> [8, 15]) doses of GSPE administered orally as a preventive [8] or counteractive treatment [14, 15], tended to reduce intestinal inflammatory markers such as TNF-α release or myeloperoxidase (MPO) activity (an indicator of neutrophil infiltration in tissues). The reduction of plasma ovalbumin (OVA), an in vivo marker of intestinal permeability, was supported by (1) the increase in TEER in small and large intestine segments. This parameter is determined *ex vivo* by UChbased protocols [8, 15]; and (2) by the upregulation of TJ proteins such as ZO-1 [14] and claudin-1 [8, 15]. Notably, the protective effect of GSPE in the intestinal barrier function was linked to the amelioration of metabolic endotoxemia (reduction of plasma LPS) and systemic inflammation (reduction of plasma TNF-α) in obese rats [15, 101]. Other authors have also reported the upregulation of ZO-1 and claudin-1 TJ proteins in high-fat fed rats supplemented with other PAC-rich extracts [81].

**5.2 In vivo studies of diet-induced intestinal permeability**

**5.3 In vivo studies of chemical-induced intestinal dysfunction**

comparable to those of sulfasalazine (200 mg kg<sup>1</sup> d<sup>1</sup>

Chemical agents administered orally to induce colitis in rodents include trinitrobenzene sulfonic acid (TNBS) and DSS. These agents erode the colonic mucosal lining and produce the loss of the intestinal barrier function and colonic inflammation. In these models, the severity of outcomes depends on the dose of the chemical agent and the frequency of administration. Li et al. [102] found that intragastric administration of GSPE in rats at pharmacological doses (100–

attenuated macro- and microscopic tissue damage scores in the colon. This protective effect was accompanied by a reduction in oxidative stress (malondialdehyde; MDA), inflammation (IL-1β), and neutrophil infiltration (MPO activity) in colonic tissues. Remarkably, the beneficial effects of low to high doses of GSPE were

κB. Subsequent studies carried out by these authors with the same model, confirmed the role of the GSPE down-regulating NF-κB response [83, 84]. A preventive effect of procyanidin B2 was also evidenced in a mouse model of DSS-induced

metalloproteinase-9 (MMP-9), a marker of macrophage infiltration. In addition, inhibition of the NF-κB signaling and of NLRP3 inflammasome activation was observed, with a concomitant reduction in the gene expression of pro-inflammatory cytokines. Overall, the benefits of procyanidin B2 administration, especially at the

colitis [85]. Administration of procyanidin B2 (10–40 mg kg<sup>1</sup> d<sup>1</sup>

severity of tissue damage in the colon and reduced the levels of matrix

) prior to TNBS-induced recurrent colitis, reduced weight loss, and

), a potent inhibitor of NF-

) attenuated the

polymerization (DP).

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

400 mg kg<sup>1</sup> d<sup>1</sup>

**279**

#### **5.1 In vitro studies of barrier integrity**

The data available on the interaction between PACs and permeability and inflammation markers in cell models of intestinal dysfunction are summarized in **Table 1**. Caco-2-based models have shown to be responsive to pro-inflammatory stimulation, producing a wide range of inflammatory mediators and increasing the paracellular permeability. Pro-inflammatory agents such as LPS, phorbol 12 myristate 13-acetate (PMA), and cytokines (TNF-α and IL-1β) have been used in multiple studies testing the effect of PAC molecules and PAC-rich botanical extracts on Caco-2 cells [10, 74, 77, 78]. Stimulated-Caco-2 cell monolayers incubated with PACs generally show a reduction in gene expression and secretion of TNFα, IL-6, and IL-8 [10, 74, 75, 77], which is often linked to the downregulation of NF-κB signaling at different levels [10, 76, 77]. An increased expression of antioxidant enzymes, such as glutathione peroxidase (GPx), superoxidase dismutase (SOD), and hemeoxygenase 1 (HO-1), has also been reported [10].

When permeable support systems such as transwell or Ussing chamber (UCh) are used, alterations in barrier integrity and paracellular permeability of epithelial cell monolayers are evaluated by transepithelial electrical resistance (TEER), an electrophysiological parameter that measures ion conductance across the monolayer, and by the transepithelial transport of molecular markers such as Lucifer yellow (LY) and fluorescently labeled dextrans (FD) [95, 96]. Some in vitro studies have associated PACs with increased TEER and decreased transport of permeability markers in the context of barrier dysfunction [77, 78, 80]. The expression levels of TJ proteins (claudins, occludins, and ZOs) often correlate, but not always [79], with intestinal permeability and are also considered markers of epithelial integrity. Bitzer et al. [78] found that the dextran sodium sulfate (DSS)-induced loss of barrier function in Caco-2 cells was significantly inhibited by polymeric PACs of cocoa but not by oligomers. Moreover, a higher barrier-protective activity was determined in PACs with DP ≥ 7, which were able to reduce the detrimental effect of DSS in a dose-dependent fashion [78]. Effectiveness of procyanidin B2 ameliorating dextran sodium sulfate (DSS)-induced permeability alterations was examined using a Caco-2/HT29-MTX co-culture model [79]. Although procyanidin B2-incubated cells showed increased levels of the TJ proteins claudin-7, occludin, and ZO-1, these

changes did not reduce TEER loss. Altogether, these results suggest that the ability of PACs to strengthen the intestinal barrier integrity depends on the degree of polymerization (DP).
