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

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– 400 mg kg<sup>1</sup> d<sup>1</sup> ) prior to TNBS-induced recurrent colitis, reduced weight loss, and 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 comparable to those of sulfasalazine (200 mg kg<sup>1</sup> d<sup>1</sup> ), a potent inhibitor of NFκB. 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 colitis [85]. Administration of procyanidin B2 (10–40 mg kg<sup>1</sup> d<sup>1</sup> ) attenuated the severity of tissue damage in the colon and reduced the levels of matrix 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

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 botan-

ical extracts, mainly by in vivo approaches in rodents (**Table 2**).

and hemeoxygenase 1 (HO-1), has also been reported [10].

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),

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

**5.1 In vitro studies of barrier integrity**

*Weight Management*

**278**

highest dosage (40mg kg<sup>1</sup> ), were comparable to those of mesalazine (200 mg kg<sup>1</sup> ), a COX inhibitor. The authors suggest that these effects were largely due to the reduction in activated macrophages infiltrating colonic tissues, probably driven by ROS clearance.

A feature of ex vivo models is that screening of drug effects does not compromise

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

Translation of doses of PAC-rich extracts used in rodent models of intestinal dysfunction to human equivalent doses (HED) indicates that pharmacological doses (up to 5 g d<sup>1</sup> for a 60 kg person) could be required to achieve beneficial effects in clinical trials [14, 15]. Thus, the first uncertainty involved in assessing the use of

proanthocyanidin-rich extracts have been subjected to toxicological tests in rats to determine their safety for use in functional foods [109–111]. In these studies, the median lethal dose (LD50) was found to be greater than 5000 mg kg<sup>1</sup> bw (HED of ≈50 g) when administered once by oral gavage, and 1400–2000 mg kg<sup>1</sup> d<sup>1</sup> (HED

for systemic toxicity in sub-chronic administration. A recent study evaluated the

adults for a 4-week period and found a good tolerability without adverse effects on

To date, there are few clinical studies that evaluate the influence of PACs on intestinal inflammatory conditions. A clinical study revealed that the postprandial increase of plasma LPS associated with the intake of a high-fat meal was significantly reduced in obese subjects who consumed 1 g of GSPE [112]. As translocation of LPS to the circulation is considered an indicator of intestinal permeability and a critical factor in the appearance of systemic low-grade inflammation in patients with metabolic syndrome [113], reduction of postprandial endotoxemia could be particularly interesting from a therapeutic perspective. Large double-blind clinical studies need to be conducted to provide more information on PAC clinical efficacy in intestinal dysfunction so that these phytochemicals can be used therapeutically to

**6. Biochemical and molecular mechanisms underlying the barrier-**

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,

**protective and anti-inflammatory properties of PAC in the intestine**

PACs were often considered to be nutritionally undesirable due to their ability to

) was found to be the no-observed-adverse-effect level (NOAEL)

) in a small number of healthy

the patients by exposing them to unknown outcomes.

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

safety and tolerability of GSPE intake (up to 2.5 g d<sup>1</sup>

improve intestinal health in obese and IBD individuals.

hematological or biochemical parameters [47].

PACs as therapy agents in humans, is safety. Grape seed and skin

**5.6 Clinical trials**

of ≈14–20 g d<sup>1</sup>

**281**
