The major aim of this study was to assess the hypothesis that plant extracts rich in polyphenols are able to inhibit the inflammatory process in the duodenum of pigs. For this purpose, we used GSGME, a by-product of wine/grape juice processing, as a plant extract rich in flavonoids. The most abundant polyphenols in grape seeds (GS) are gallic acid, catechin, epigallocatechin-3-gallate, epigallocatechin, epicatechin-3-gallate, epicatechin, and proanthocyanidins . Grape marc (GM) contains less procyanidins (with the exception of the procyanidin dimer B1) but contains significant amounts of anthocyanins, such as cyanidine 3-glucoside, malividin 3-glucoside, cyaniding and peonidin, which are absent in GS . For technical reasons, we were not able to characterise the polyphenol spectrum in the product used. According to the supplier, the total polyphenol content of the GSGME product used was about 8.5%. The high content of polyphenols in this product was confirmed by HPTLC-determination of malvidin 3-glucoside, the quantitative most important pigment in red grape, whose mean concentration was 681 μg/g.
In accordance with the hypothesis underlying this study, we observed that feeding the polyphenol-rich GSGME reduces transactivation of NF-κB in the duodenal mucosa and in turn lowers transcript levels of various NF-κB target genes involved in the inflammatory process. These findings implicate that GSGME exerts anti-inflammatory effects in the duodenum of pigs. Although the study does not directly provide evidence for this, it is likely that the anti-inflammatory effect of the plant extract might have been induced by its high content of polyphenols. Thus, this study suggests that feeding of plants rich in polyphenols such as GSGME could provide a useful dietary strategy to suppress the inflammation process in the small intestine frequently occurring in pigs, particularly after weaning. The finding that polyphenols are able to suppress the inflammation process in duodenum of pigs agrees with several in vitro-studies using intestinal epithelial cells and in vivo-studies which were mainly performed in rodent models of acute or chronic colitis . While most of these studies have been performed with green tea polyphenols, there are also some studies which showed inhibitory effects on the inflammation process of anthocyanins from grapes or GS in either intestinal epithelial cells [23, 27] or intestine of rats with ulcerative colitis . Based on these findings, supplementation of polyphenols has been even proposed as a complementary medicinal approach for treatment of inflammatory bowel disease . Notably, in the present study, we did not use pigs with an experimentally induced intestinal inflammation due to ethical reasons. Based on the findings in rodent models of acute or chronic intestinal inflammation, we assume that GSGME might exert anti-inflammatory effects also in pigs with an acute intestinal inflammation process.
There are several studies showing that polyphenols have the potential to activate Nrf2 and in turn to enhance the expression of several antioxidative and cytoprotective genes in the small intestine [30, 31]. Surprisingly, in the present study administration of the polyphenol rich GSGME did not increase but even reduced transactivation of Nrf2 and gene expression of several Nrf2 target genes in duodenum. Based on the fact that Nrf2 is activated by either ROS or by pro-inflammatory cytokines [32, 33], we assume that the inhibition of Nrf2 signalling in the small intestine of pigs administered GSGME was simply due to its strong anti-oxidative and anti-inflammatory properties which might have suppressed the local production of ROS and pro-inflammatory cytokines in the surrounding of intestinal cells.
Several studies have shown that diverse polyphenols, particularly those present in green tea, are able to inhibit NF-κB and to activate Nrf2 in the liver [10, 34]. Furthermore, studies exist dealing with the effects of polyphenols from GS or GM on the antioxidant system in the liver, mostly in rats or rabbits [35–37]. However, to our knowledge, less information is available about effects of polyphenols from GS or GM on the signalling pathways in the liver. In order to find out whether feeding GSGME could also have protective effects on the liver, we determined relative mRNA abundances of NF-κB and Nrf2 target genes in the liver of the pigs. However, no alterations of mRNA abundances of these genes were observed, indicating that feeding GSGME had no effect on NF-κB and Nrf2 signalling in the liver. A low intestinal bioavailability might be one possible reason for the lack of effect in this respect. Although there is only limited information available in the literature regarding the bioavailability of polyphenolic compounds, it has been suggested that the bioavailability of polyphenols might be in the range between 10 and 50%, depending on their chemical structure, the dose applied, the form of application and the species studied . As the absorption of polymeric proanthocyanidins is negligible [39, 40], the bioavailability of total polyphenols of GSGME might be comparably low.
Polyphenols have a great antioxidative potential. Therefore, it has been suggested that feeding diets rich in polyphenols could improve the antioxidative status of plasma and tissues and increase tocopherol concentrations due to a vitamin E-sparing effect . However, the published literature regarding the effects of dietary polyphenols on tocopherol concentrations and the antioxidative status in plasma and tissues is inconclusive. While some studies in rats reported an increase of plasma and tissue tocopherol concentrations by feeding various types of flavonoids [42–44], other studies observed no effects [45–47]. In pigs, a tocopherol-sparing effect of quercetin has been observed under condition of a low dietary vitamin E concentration . In contrast, in pigs fed diets with nutritionally adequate vitamin E concentrations, no increases of plasma and tissue tocopherol concentrations by supplementing dietary flavonoids were observed [49, 50]. The study of Wiegand et al.  moreover shows that dietary flavonoids do not alter the expression of hepatic genes involved in transfer of tocopherols into plasma lipoproteins, decomposition and excretion into the bile, meaning that metabolism of vitamin E remained unchanged. The present study confirms that dietary polyphenols from GS and GM do not improve the vitamin E status, and do not improve the antioxidant status of pigs with an adequate supply of dietary vitamin E (of around 100 mg α-tocopherol acetate/kg diet).
In this study, we found that administration of GSGME improves the gain:feed ratio in growing pigs suggesting that either digestibility of nutrients from the diets or intermediary utilisation of nutrients was improved. To our knowledge, there are no other comparable studies in pigs available in the literature dealing with the effects of moderate amounts of dietary grape by-products on the growth of pigs. There are, however, a few studies in broiler chicks showing beneficial effects of polyphenol-rich grape products on digestibility of nutrients and feed efficiency. In the study of Viveros et al. , feeding polyphenol-rich grape pomace extract (60 g/kg diet) improved the gain:feed ratio in broilers at 21 d of age. In that study, also an increase of the villus height:crypt depth ratio at the jejunum and a shift in the ileal bacterial populations (increase in beneficial bacteria such as Enterococcus, decrease in potential pathogens such as Clostridium) was observed in broilers fed the grape pomace extract. In the study of Brenes et al. , feeding diets containing 0.6 to 3.6 g GS extract did not improve the gain:feed ratio but improved ileal protein digestibility in 21 d-old broilers. In the study of Wang et al. , administration of a GS proanthocanidin extract lowered mortality and increased growth in broilers infected with E. tenella. Weaning in pigs is associated with a strong decrease in the villus height:crypt depth ratio in the small intestine which in turn leads to a reduced digestive capacity . Interestingly, the present study is in agreement with the broiler study of Viveros et al.  in observing an increased villus height:crypt depth ratio in the small intestine by feeding a polyphenol rich GS by-product. It is assumed that an increased villus height leads to an improvement of digestive and absorptive function of the intestine as a result of increased absorptive surface, expression of brush border enzymes and nutrient transport systems [54, 55]. Thus, although we did not determine the digestibility of nutrients in this study, it is possible that the increase in the gain:feed ratio of pigs administered GSGME was due to an improvement of the digestibility of nutrients. Based on the observations in broilers , the effects on the microflora could contribute to the beneficial effects of GSGME.