Grape Processing Waste: Effects on Inflammatory Bowel Disease and Colorectal Cancer

ABSTRACT Inflammatory Bowel Diseases (IBD) are inflammatory conditions characterized by bowel disruption and the involvement of the immune system and can be associated with the development of colorectal cancer. Conventional therapeutic approaches are based on administering drugs capable of modulating the inflammatory response; however, many patients are irresponsive to conventional therapies. Grapes are largely used in producing wine, juice, jam, and other products. However, the waste from the production process represents an environmental problem. On the other hand, grape by-products are rich in bioactive compounds such as proanthocyanidins, anthocyanins, phenolic acids, stilbenes, and flavonols. These compounds can help in the prevention or in treatment of several conditions such as IBD and colorectal cancer. Grape by-products can promote remarkable effects in reducing pro-inflammatory, pro-oxidative, and proliferative actions in IBD and CRC both in vivo and in vitro. These effects are related to the improvement of epithelial integrity, reduction of Disease Activity Index, mucosal barrier reinforcement, induction of strong chemopreventive actions, reduction of tumor incidence, and cell proliferation.


Introduction
Inflammatory Bowel Diseases (IBD) are inflammatory conditions mainly represented by Crohn's Disease (CD) and Ulcerative colitis (UC).They can occur in any age and are characterized by bowel disruption and the involvement of immune cells and molecules, including chemokines, cytokines, and proteases.[3][4] The main symptoms are abdominal pain, diarrhea, bleeding, and weight loss, and extraintestinal manifestations may be observed in the skin, eyes, and joints.7] There is a growing incidence of IBD around the world, mainly pronounced in newly industrialized countries.In China, the incidence went from a rare to a common occurrence, accounting for a significant proportion of hospitalizations recently.In the United States, the frequency is 70-150 cases per 100,000 people and is ranked as the 5 th most costly gastrointestinal condition to healthcare systems. [6,8,9]ne major complication for IBD patients is the development of colorectal cancer (CRC).Although this complication occurs only 1%-2% of the patients, it is associated with 15% of IBD-related mortality, and the risk is particularly high in patients with extensive UC.CRC is shown to be the second most common cause of deaths and the third most common type of cancer worldwide.Most cases are observed in Western countries, but the incidence is increasing in several countries.[12][13][14] Besides IBD, other conditions are risk factors for CRC such as genetics environment, food-borne mutagens, smoking, obesity, alcohol abuse, physical inactivity, imbalance in gut microbiome, and poor diet.17][18] The target of IBD is remission, avoiding recurrences. [19,20]Conventional clinical therapeutic approaches are based on administering drugs capable of modulating the inflammatory response such as steroids, aminosalicylates, methotrexate, antibiotic, immunomodulators, and thiopurines.Biologic therapies include monoclonal antibodies anti-Tumor Necrosis Factor and related biosimilar as infliximab and adalimumab.Other drugs like JAK inhibitors, Interleukin-12/ Interleukin-23, and integrin antagonists can be also considered.23][24][25][26] Besides the high costs and the presence of adverse events related to the conventional drugs, some patients are refractory to these medications.For these reasons, various alternative and complementary therapies have been searched such as plant extracts and plant bioactive compounds.[29][30][31][32][33][34] Grape phenolic compounds are among the potential agents for the therapeutic approach for IBD patients since are related to the reduction of inflammatory processes and OS. [35,36]henolic compounds such as anthocyanins, quercetin, resveratrol, catechin, epicatechin, naturally occurring in plant and are associated with odor, color and flavor in fruits and vegetable, can exert antioxidant and anti-inflammatory action, prevent DNA damage, induce DNA repair, slow cancer cell growth, lead to cancer cell death, and boost immune function.For these reasons, a plethora of studies have considered phenolic compounds to treat several diseases including IBD and CRC. [15,37,38]Grape is among the many fruits with this potential.This rich and very fruit possesses several bioactive compounds (proanthocyanidins, anthocyanins, phenolic acids, stilbenes, and flavonols) whose amount could vary considerably in grape pulp, skin and seed.Although the fruit can be eaten in natura, it is also used as raw material to the production of different products, such as juice, wine, and jelly.The grape byproducts such as grape pomace and seed can be used for many purposes in the food industry and for cosmetics. [39]rapes are one of the most common cultivated plants in the world and are distributed over the world due to the low requirements for soil and climatic conditions.The total area under vines reached, in 2019, 7.4 million hectares (Spain, China, France, Italy, and Turkey represent more than 50% of the world's vineyard).[42][43] For these reasons, this study aims to review the potential role of grape by-products on IBD and colorectal cancer.This is the first study, to our knowledge, that considered the grape by-products on the therapeutic approach of intestinal inflammatory conditions.

Focused question
The focal question for this study was: what are the effects of grape and its phenolic compounds on Inflammatory Bowel Disease?

Studies selection
We searched PubMed, Embase, Google Scholar, and Cochrane databases and did not restrict a period for the survey of articles.The search for animals, in vitro and human studies followed Preferred Reporting Items for a Systematic Review and Meta-Analysis (PRISMA) guidelines. [44]Figure 1 shows the selection of the studies.
The keywords and mesh terms for the search were Inflammatory Bowel Disease or Crohn's Disease or Ulcerative Colitis or colitis and grape or phenolic compounds or poplyphenols or flavonoids or anthocyanins or stilbene or resveratrol or anthoxantins or flavans or flavons or flavonols or quercetin or catechin or epicatechin or tannin or phenolic acids.

Eligibility criteria
Eligibility criteria for the literature survey for human studies were based on PICO (Population, Intervention, Comparison, and Outcomes) format.

Study selection
We selected only studies in English to perform this review.Two independent authors (SMB, and LFL) consulted the databases and independently retrieved the studies that were in accordance with the mesh terms.Full-text articles were studied to support decision-making.Disagreements between the two reviewers were evaluated by the other two reviewers (RAG and RD).

Inclusion criteria
We included studies that investigated the effects of grape and its phenolic compounds on Crohn's Disease and Ulcerative Colitis.

Exclusion criteria
Abstracts, clinical guidelines, poster presentations, and articles not in English were excluded from this review.

Investigation of the risk of bias
The risk of biases in the included clinical trials were carefully considered and evaluated according to the selection, detection, and reporting of bias in each trial.Risks of quality and bias in the inclusion of patients, interventions, evaluation of outcomes, missing information and data were also investigated.Quality assessments of bias and quality were performed according to the directives of the Cochrane Handbook for Systematic Reviews of Interventions. [45]The descriptive results of bias identified in animal models followed SYRCLE guidelines. [46]

Results
The flow diagram found in Fig. 1 shows the study selection.61] These studies show several beneficial effects of grape by-products such as reduction of pro-inflammatory markers (TNF-α, IL-1β, IL-6, IFN-γ), reduction of the Disease Activity Index, reduction of pro-oxidative enzymes, increase of antioxidant enzymes (glutathione peroxidase, catalase, and superoxide dismutase) reduction of oxidative stress, improvement of epithelial integrity and mucosal barrier reinforcement (modulation of claudins, OCCL and ZO-1 tight junction genes and proteins, MYO9B and tight junction regulators and MUC2 gene), an increase of mRNA levels of occludin, and claudin-1 of colon tissue; reduction of NLRP3 inflammasome in colon tissue.Rebalance of colitisdamaged gut microbiota (with reduction of Bacteroidetes, Dubosiella, and Veillonella and increase of Firmicutes to Bacteroidetes ratio).Reduction of COX, MMP-2, and MMP-9; suppression of TLR-2 and TLR-4 genes; and inhibition of MAPKs and NF-kB gene expression.

Grape by-products as sources for polyphenolic compounds
Grape (Vitis vinifera L.) is a fruit with a long cultivation history and is among the top five produced fruits around the world.Its annual production reaches approximately 75 million tons, and the bigger producers are Europe (41%), Asia (29%) and the Americas (21%).Approximately 45% of grapes production is used in natura or in fresh derivatives, while 55% is fermented to wine production. [79,80]he immensity of bioactive compounds found in fresh grapes and products derived from them are related to numerous actions that can protect the body against different pathologies.These compounds exhibit activities such as anti-inflammatory and antioxidant.They, therefore, are closely related to the protection against numerous chronic-degenerative diseases such as diabetes, obesity, dyslipidemia, cardiovascular diseases and cancer, which are among the main cause of deaths.The bioactive compounds from grapes are especially polyphenols, consisting principally of proanthocyanidins, anthocyanins, flavonols, phenolic acids, and stilbenes. [39,40,81,82]Figure 2 shows the main polyphenols of grapes.
Vitis vinifera grapes are extremely used in processing wine and juice and are very rich in bioactive components.Grape wine shows a continuous rising worldwide, currently reaching 25 billion liters, and it is almost unbelievable to say that to produce 50 mL of wine, approximately 1,00 kg of grapes are used. [83]With this information in hand, it is not necessary to point that these industrial processes produce a large amount of biodegradable waste that can harm the environment if not treated properly. [84]During the production of wine, a solid waste (grape pomace or grape marc) results from the extraction of the juice, white wine, and red wine.[87] Table 1 shows some bioactive compounds found in grape by-products.
Besides fibers, essential oils, minerals, fats, proteins and carbohydrates, grape seeds contain 5 to 8% of polyphenols.The seed is rich in extractable phenolic compounds such as phenolic acids, flavonoids,

Stalk and leaves p-coumaric acid
Antioxidant, anti-inflammatory, antimicrobial [94,95]   Stalk Caffeic acid Antioxidant, anti-inflammatory, antimicrobial [96-98]   Stalk Ferulic acid Antioxidant, anti-inflammatory, antimicrobial [99]   Peel, stalk and leaves Caftaric acid Antioxidant, anti-inflammatory, antimicrobial [100,101]   Peel and stalk p-coutaric acid Antioxidant, antimicrobial [102,103]   Peel Fertaric acid antimicrobial activity; antioxidant; liver protection [104]   (Continued) proanthocyanidins, resveratrol, and flavonoids, being the class of polyphenols with the highest concentration in grape seeds.The skin is rich in anthocyanins.][113] Figure 3 shows the main chemical compounds of grape pomace and Figure 4 shows the main use of grapes in food industry and the waste resulted from the industrial process.Moreover, it shows the possibility of applications in several food and cosmetic products The evaluation of the concentration of total phenolics in grape seed extracts showed a great variation among the analyzed cultivars (88.11 to 667.8 mg•GAE•g − 1 •dw). [114,115]However, the determination of total phenolic compounds in grape skins showed little variation (up to 12 mg•GAE g − 1) in the different varieties studied. [116]On the other hand, the total phenolic contents in the stems varied from 26.88 to 35.9 mg•GAE•g − 1 among the grape cultivars. [117]rape seeds have in their composition: omega-6 fatty acid, vitamins and phenolics compounds (up to 70% of the total polyphenols).However, the flavonoid contents in grapes are concentrated in the seeds, skin, and stem. [43,118]A study showed that the seeds of white grape varieties had high proportions of gallic acid and protocatechuic acid. [119]onzález-Centeno et al. [120] observed an average content of 71% (dry matter) of insoluble alcoholic residues in the stem of the grapes, however, they did not find differences between the white and red varieties.Regarding the phenolic presence of the grape stem, the following have been described: flavan-3-ols, hydroxycinnamic acids, monomeric and oligomeric flavonols, and stilbenes. [121,122]he composition of grape leaves was described by Doshi et al. [123] and Xia et al. [124] who verified levels of organic acids, phenolic acids, flavonols, tannins, procyanidins, anthocyanins, lipids, enzymes, vitamins, carotenoids, terpenes, and reducing or non-reducing sugars.
Peptic substances are polymers present in big amounts in the cell walls of grape pomace, with a polysaccharide content ranging from 37 to 54% and cellulose ranging from 27 to 37%, in addition to other compounds such as protein, fat, and minerals. [120,125,126]The marc grape is composed of 50% of the grape skins, however the skin seed ratio may vary with growing conditions, grape variety, etc.The antioxidant action of grape skins is obtained by the presence of anthocyanins, hydroxycinnamic acids, catechins, and flavonols, acting to inhibit the oxidation of low-density lipoproteins (LDL). [127]henolic antioxidants extracted from grape pomace represent 10 to 11% of the dry weight.The phenolic composition is variable, with red grapes being rich in proanthocyanidins, while white grapes do not have significant levels of these substances.Studies show that the skins of red grapes have higher levels of fiber and phenolic compounds when compared to the skins of white grapes and that the marc grapes of red grapes have high levels of fiber but tiny proportions of total phenolics when compared to white grapes. [128]roanthocyanidins are flavan-3-ols, a class of compounds synthesized by the association of flavanol monomers.In grape, four main proanthocyanidins were observed: (+)-catechin, (−)-epicatechin, procyanidin B1 and procyanidin B2.Flavan-3-ols are the main flavonoids in grapes and are present in high concentrations in the seeds, but detected in low concentrations in the pulps.Flavonoids may also include anthocyanins, concentrated in purple or red grapes skin. [129,130]egarding the flavonol group, quercetin-3-O-glucuronide is the predominant compound in marc grape.It was also found that malvidin-3-O-glucoside is the anthocyanin found in the highest concentration in the skin and grape pomace. [131]henolic acids and stilbenes are the main compounds in grapes that are not part of the flavonoid class.The phenolic acids present in high concentrations are hydroxybenzoic (4-hydroxybenzoic, gallic, protocatechuic, vanillic and syringic acids) and hydroxycinnamic acids (p-coumaric, caffeic, ferulic, caftaric, p-coutaric, and fertaric acids). [112,132]Resveratrol is the main stilbene of grape skin. [133]ccording to a study by Wongnarat and Srihanam, [134] the total phenolic compound content in grape seeds is 130 times higher than in pulp (mg gallic acid eq (GAE)/g dry weight).The composition of polyphenols depends on maturation, variety, time, place of cultivation of the grapes, genetic potential, etc. [134][135][136] Iuga and Mironeasa [137] found that the marc grape is a source of fiber and antioxidants in baked goods and pasta.The authors also verified that the chemical composition of the bagasse will depend on the grape type/variety.

Inflammatory bowel diseases: general pathological aspects
The incidence for CD is increasing at a bigger rate than other IBD types in Asia, and the peak of occurrence is in the second and fourth decades of life.Due to the gravity of the symptoms, CD is linked to poor quality of life and a high rate of hospitalizations, surgical operations, and morbidity. [5,138]The prevalence of UC is in higher proportions in the USA and Europe and can vary from approximately 500 patients per 100,000 inhabitants. [139,140]espite the crescent increase in the occurrence of IBD worldwide, the pathogenesis is still not fully understood.However, several authors have demonstrated that IBD can result of different interactions involving environment, genetics, immunological imbalance, intestinal dysbiosis, and other factors such as psychological issues.Under homeostasis, the intestinal epithelium is represented by a physical and biochemical barrier between luminal bacteria and mucosal immune cells.Dysregulation of epithelial cell proliferation and cell death lead to exacerbation of intestinal damage.Overactivation of the apoptosis of intestinal epithelial cells is commonly found in both CD and UC, leading to disruptions of the integrity on intestinal barrier, allowing the infiltration of bacteria from the lumen into the bowel wall, triggering an inflammatory process, and the synthesis of pro-inflammatory cytokines. [2,141,142]here are several genetic factors and gene loci involved with the pathogenesis of IBD.[145] The increase in the luminal permeability contributes to the exacerbated immune response.Antigens can activate macrophages, dendritic cells, and nuclear factor kappa B (NFkB) pathways resulting in an overloaded production of pro-inflammatory cytokines.Besides that, disruption in tight junctions, desmosomes, and adherens junctions seem to have a role in IBD pathogenesis. [146,147]urthermore, CD and UC show different cell activation, and a T helper (Th)1 response is related to CD development, and a non-conventional Th2 and Th9 response is linked to the genesis of UC.And the main release cytokines Interleukin-5 (IL-5) and IL-13.Mast cells (also linked to the Th2 arm of immunity) have a pivotal role in the development of UC.150][151][152] Th1 and Th17 are essential for intestine integrity via preventing pathogenic cells invasion and regulating intestinal epithelial cells.Nevertheless, during IBD flare, both Th1 and Th17 change the pattern of intestinal homeostasis to intestinal mucosa destruction.Pathogenic Th17 cells can result in intestinal epithelial cells injury by stimulating IBD susceptibility expression of several genes and specifically killing intestinal epithelial cells.The co-expressing of IFN-γ and IL-17A leads to Th17 cells with pathogenicity.The resulting scenario is the impairment of apoptosis, and recruiting immune cells, leading to adhesion molecules expression.Moreover, Th1 associated with pathogenic Th17 cells can work in cooperation to induce colitis.This relationship is seen in the disease's occurrence and is related to the worsening of mucosal inflammation. [153]162] CD and UC shows some anatomopathological differences.While CD is represented by transmural lesions affecting the entire length of the gastrointestinal tract, UC is characterized by the presence of lesions restricted to the mucosa and affecting only the colon and rectum. [163- 165]Clinically, both share similar manifestations, and the primary complaints are abdominal pain, diarrhea, and hematochezia. [166,167]Figure 5 shows the main pathways involved in the pathogenesis of CD and UC and the consequences for the affected individual.

Colorectal cancer and IBD: general pathological aspects
CRC occurs by virtue of a complex interaction between several conditions such as environment, lifestyle, and genetic factors.However, the molecular pathogenesis seen in IBD-CRC has particularities regarding sporadic CRC. [168]The incidence of IBD-CRC is quite different between the two major types of IBD and my reach 2% to 18% at 10 to 0 years after the onset of UC.The occurrence in CD is debatable, lower if compared to UC, and may reach and estimated risk of 3% at 0 years, 5.5% at about 0 years, and 8.3% at about 0 years in these patients.However, several factors like duration of follow-up, and geographical distribution can influence these estimates.The estimative is that CRC has almost two million new cases every year and is the third most frequent cancer worldwide.More than 860,000 patients die from this type of cancer each year.][171] Figure 4 shows CRC as a consequence of IBD.
The proliferation processes required to the repair of the epithelial layer damage augments the risk of dysplasia.Furthermore, the mucosa-associated bacteria that can produce a higher mass of biofilm and extracellular matrix may be found in IBD patients.74]  Oxidative stress and the inflammatory process are among the main causes of CRC development and the intensity of inflammation.These processes can trigger chromosomal and microsatellite instability resulting in mucosal dysplasia. [175,176]The microenvironment alterations observed due to chronic inflammation are related to increase the risk.Many pro-inflammatory cytokines are related to the pathogenesis of CRC.IL-6 can activate a plethora of cells stimulating pathways that increase the expression and nuclear translocation of STAT3 (due to the activation of JAK), stimulating antiapoptotic genes, such as Bcl-xl, c-myc, Mcl1, surviving and VEGF, resulting in apoptosis-resistance of lamina propria T-cells, contributing to chronic intestinal inflammation and carcinogenesis.Besides that, PI3K mediated activation of FOXO (Forkhead box O3) inhibits gene transcription, whereas PI3K mediated activation of mTOT results in oncogene transcription-mediated oncogenesis.Other effects are also observed for the IL-6 family cytokines, as self-renewal, proliferation, invasion, and angiogenesis. [177]IL-17 contributes to the inflammatory process due to the maintenance of NKT cells that synthesizes IL-17, through the PI3K/AKT/mTOR pathway. [178,179]IL-21 shows a dual role in the inflammatory scenario.Its deficiency is linked to an early-onset IBD in humans followed by problems in B-cell.Increased IL-21 levels have a role in maintaining the tissue-damaging immune responses.Associated with the effects of IL-21, the increased expression of IL-32 by tumor microenvironment reveals that expression augmented the control tumor growth.Modifications of IL-21 and IL-32 and alterations in immune cell density indicate they play an important role in tumor growth and invasion. [180]IL-3 R actions can interfere with the disease risk and increased IL-23 release.Moreover, in colitis murine models, inhibiting IL-23 p19, IL-12/IL-23 p40, or IL-3 R function suppressed gut inflammation.This result was related to the decreased stimulation of IL-23 target cells (innate lymphoid cells type 3, granulocytes, T helper 17 cells, and NKT (natural killer cells)) and imbalanced release of proinflammatory cytokines. [181,182]TNF-α can trigger systemic inflammation and participates of the acute phase reaction in IBD patients.TNF-α regulates the stimulation of MET transcriptional regulator (MACC1) via NF-κB and the c-Jun transcription factor in cancer cells.Furthermore, it can interfere with the expression of transcriptional factors that stimulate epithelial to mesenchymal transitions, contributing to the dissemination of tumor cells. [183,184]nt signaling has a crucial role in regulating and developing carcinogenesis and was most prominently described for CRC.The Wnt can be divided into β-catenin dependent (canonical) and independent (non-canonical) signaling, and aberrant Wnt signaling is observed in several other cancer entities. [185]The Dickkopf (DKK)family constitute a family of inhibitory molecules in this signaling and some evidence suggests that the members of DKK family have effects on the incidence and development of tumors.DKKs genes encodes the glycoprotein as inhibitory marker of the typical Wnt pathway. [169]Some authors have shown that Wnt pathway is activated in the early stages of colitisassociated CRC, and in approximately 50% of IBD associated neoplasia cases.Another relevant discover is that the pathway was also activated in the margins of dysplasia associated with IBD, in a phenomenon named field-cancerization.These results suggest that the evaluation of β-catenin could be used as a marker for colonic cancerization, favoring the early detection of neoplasia in colonic surveillance and prognosis. [168,186]

Phenolic compounds, antioxidant, anti-inflammatory, and anticancer effects
As pointed out before, oxidative and inflammatory processes are related to developing several diseases, such as IBD and CRC.
OS occurs due to an imbalanced status between antioxidation and oxidation in the body and has a strong relationship with degenerative processes and accelerating aging.The results are an increased risk of developing several metabolic diseases. [187,188]In this sense, the consumption of grapes can reduce OS since their biological compounds exhibit antioxidant ability.Grapes show a strong antioxidant activity, mainly by augmenting the activity of the antioxidant enzymes (such as superoxide dismutase, glutathione-reductase, and glutathione peroxidase) and by regulating signaling pathways, such as erythroid-derived 2)-like 2 (Nrf2) and PI3K/Akt pathways.Maurer et al [53] showed that the (Continued) consumption of powder from grape peel could ameliorate 2,4,6-trinitrobenzene sulfonic acid (TNBS)induced oxidative harm in rats due to the restoration of the activity of these antioxidant enzymes and reduce oxidation and NO in the colon.Different grape fractions can exhibit different antioxidant actions according to the content of polyphenols.Some authors investigated the antioxidant activities of peel, pulp, peel and seeds of grape varieties and showed that the seeds possessed the strongest antioxidant capacity, followed by peel and pulp since the majority of polyphenolic compounds were concentrated in the seeds, followed by peel and pulp. [189,190]As pointed before, proanthocyanidins are among the most relevant phenolic compounds contributing to the antioxidant actions of grapes. [39]urthermore, the antioxidants obtained from grapes can avoid lipid peroxidation by scavenging peroxyl radicals.Therefore, resveratrol can be stored in erythrocytes and can work as electron donor and thus, can activate erythrocyte membrane redox system, reducing extracellular oxidants and recycling oxidized ascorbate.193]  Grape also exhibits potent anti-inflammatory actions and can inhibit the release of proinflammatory cytokines and downregulate the related signaling pathways.For instance, a study showed increased TNF-α and IL-1β in TNBS-induced colitis in Wistar rats, and consuming grape peel powder reduced the inflammatory response by downregulating the NFκB pathway. [53]Moreover, the supplementation with 8% grape seed in a dextran-sulfate-sodium-induced inflammation in pig colon led to the inhibition of mitogen-activated protein kinase (MAPK) signaling cascades, downregulated the expression of NFκB gene and reduced the release of TNF-α, TNF-β, IL-6, IL-8 and macrophage inflammatory protein-1α. [50]In summary, the anti-inflammatory actions produced by grape consumption are mainly associated with reduced release of cytokines, such as IL-1β.196] Grapes and their bioactive compounds also show activity against various cancers and the underlying mechanisms; for example, grapes exhibit antiproliferative and cell-cycle-arrest-inducting abilities.A study with Ehrlich ascites carcinoma in mice showed that the treatment with grape seed and grape skin prevented the tumor development by 47% and substantially reduced the tumor volume and weight by respectively 93.9% and 86.3%.These effects were linked to cell proliferation inhibition and cell cycle arrest, and apoptosis stimulation. [197]Moreover, grape seed proanthocyanidin and anthocyanidin could inhibit the proliferation of the cell cycle in the G2/M phase, lead to apoptosis by increasing ROS level, and induce to apoptosis of hepatocellular carcinoma cells, blocking the MAPK/Akt pathways. [198,199]urthermore, grape seed proanthocyanidins could effectively block the migration and incursion of bladder cancer cells by reversing epithelial-mesenchymal transition by suppressing the Transforming Growth Factor (TGF)-β signaling pathway. [200]In general, grape by-products and its bioactive components are promising therapeutic adjuvants to the prevention and treatment of a different types of cancer due to several mechanisms such as suppression of cell proliferation, stimulation of cell cycle arrest, reduction of angiogenesis, induction of apoptosis, inhibition of migration and invasion of cells, and reducing multidrug resistance. [39,201]

Grape by-products in inflammatory bowel diseases and colorectal cancer
Grape is a fruit with an excellent source of bioactive compounds, especially polyphenols enabling it to play enormous possibilities of biological activities.The same can be said about its by-products.The included studies (Tables 2 and 3) show several benefits promoted by grape by-products, such as improvement of inflammatory and oxidant processes, reduction of the Disease Activity Index, improvement of epithelial barrier integrity and mucosal barrier reinforcement, reduction of NLRP3 inflammasome in colon tissue, and rebalance of colitis-damaged gut microbiota.
Besides the studies incorporated in the above-referenced tables, other studies investigated isolated grape compounds on IBD.The use of proanthocyanidins extracted from grape seeds (GSPE) can bring benefits to the treatment of UC, as shown by a study that used GSPE (100, 200, 00 mg/kg/day for seven days) in TNBS-treated rats.The use of GSPE-induced recovery of pathologic changes in the colon significantly decreased the colonic macroscopic and microscopic damage scores and reduced myeloperoxidase activity.There was also a reduction in IL-β in the colon tissues.There was a significant improvement in IL-2 and IL-4 in the colon tissues.The authors concluded that GSPE promoted antiinflammatory effects in the acute phase of induced colitis due to the downregulation of biomarkers related to the intestinal inflammatory response, reducing oxidation damage and inflammatory cell infiltration and promoting tissue repair to recover from the colonic oxidative stress. [207]Wang et al [208] also investigated the effects of proanthocyanidins from grape seeds (GSPE) in an animal model of colitis.In this interesting study, the authors induced recurrent colitis with TNBS on the sixteenth day after the first induction the condition.Animals received GSPE (00 mg/kg/day) for seven days after twice-induced colitis.Sulfasalazine was used as a positive control drug.GSPE improved recovery of pathologic damages in the colon after induction of recurrent colitis since there was reduction in colonic weight/length ratio and micro and macroscopic damage scores.The activity of myeloperoxidase and iNOS with nitric oxide and malondialdehyde levels in both serum and colon tissues were

Colorectal cancer
Water extract of grape pomace (WEGP)

Increasing GSE concentrations
Both treatments ↓SCD1, ratio phospho-Rac1/Cdc42/Rac1/ Cdc42 ratio, Vimentin, Cofilin, and phospho-Paxillin (mainly in Caco2 cells).GSEs blocked the membrane fluidity and cell migration through a mechanism of action related to structural cellular components. [67] Grape pomace and grape seed extracts CCC cells (Caco-2, HT-29) and fibroblasts NA ↓viability and proliferation of Caco-2 (not with HT-29 cells); grape seed purified extracts were more potent and specific on Caco-2 cells (HT-29 cells were more sensitive to purified extracts).Downregulation of Myc gene in HT-29 and upregulation of Ptg2 in Caco-2 cells. [68] (Continued) Grape Seed Extract SW480 and HCT116 cells 0-00 μg/mL Apoptosis induction due to loss of mitochondrial membrane potential, oxidative stress, modulation of pro-and antiapoptotic proteins, and modulation of caspasedependent/independent apoptotic pathways.[77]   Grape Seed Extract from 3 different cultivars: Italia, Palier and red Globe Caco2 and HCT-8 colon cancer cells 25, 50, and 00 μg/mL The 3 extracts induced growth inhibition and apoptosis in Caco2 and HCT-8 cells (in a considerably higher proportion than epigallocatechin, procyanidins and their association).[75]   Grape Seed Extract HT29 cells 25-00 μg/mL Induction of p21 up-regulation (essential for G1 arrest effect).[72]   (Continued) significantly reduced in the GSPE treated group.Moreover, GSPE treatment increased antioxidant enzymes such as glutathione peroxidase and superoxide dismutase.The treatment also protected the damages promoted by recurrent colitis by modulating the inflammatory response, antioxidation damage, and reducing inflammatory cell infiltration.In a similar model, Wang et al [209] evaluated the effects of GSPE in different doses (100, 200, and 00 mg/kg/seven days) and showed a remarkable augment in the SOD and GSH-Px activities in the treated groups.GSPE also significantly decreased the expression amounts for p-IKKα/β, p-IκBα, and TNF-α, and the NF-κB translocation in the colon mucosa.GSPE could modify inflammatory response and could p repair of damaged tissue to ameliorate colonic oxidative stress.Li e t al [210] also investigated the effects of GSPE in TNBS-induced colitis in rats.It showed that the treatment could reduce the expression of pIκBα NF-κB, and IκK in the colon, exerting beneficial effects in IBD due to the inhibition of NF-κB signal transduction pathways.The use of GSPE in C57BL/6 mice (0 mg per kg per day/1 days) reduced the Disease Activity Index scores, oxidative stress, and the amounts of TNF-α and IL-1β.On the other hand, it increases occludin, and claudin-1 mRNA levels of colon tissue; reduces NLRP3 inflammasome mRNA levels of colon tissue.A rebalance of DSS-damaged gut microbiota is also observed due to the reduction of Bacteroidetes, Dubosiella, and Veillonella, and an increment of Firmicutes to Bacteroidetes ratio. [52]ore recently Wang et al [211] showed that the use of GSPE could reduce pathological damage to the colonic mucosa in a dose-dependent manner; reduce serum and colonic tissue levels of IL-1β, IL-6, TNF-α, MDA, NF-κB and NO; and could increase the levels of Superoxide Dismutase, Nrf2 and HO-1 proteins in DSS-induced UC in mice.Grape Seed Extract Caco-2 cell 00 mg/L/2 h a day Inhibition of cell growth; stimulation of alkaline phosphatase activity.The study performed by Kim et al [212] investigated the effects of a polyphenol found in grapes, piceatannol (3,5,3',4'-tetrahydroxy-trans-stilbene).The authors used BALB-c mice-DSS (dextran sulfate sodium)-induced colitis treated with this piceatannol (1, 2.5, 5, or 0 mg/kg) for seven days and found that compared to control, the animals treated with the grape compound could show improvement of clinical signs and body weight.These effects were associated with a profound amelioration of the disruption of the colonic architecture, a reduction in colonic myeloperoxidase activity, and a reduction in the release of pro-inflammatory biomarkers such as nitric oxide and prostaglandin E2.The increases in iNOS and COX-2 were reduced by 85% and 69%, respectively.There was also a significant reduction in the mRNA expression of IL-6, TNF-α, MCP-1 (monocyte chemoattractant protein-1), and KC (keratinocyte chemoattractant).
Martin et al [213] evaluated the actions of resveratrol when used in the colon injury caused by the use of TNBS (trinitrobenzenesulphonic acid) in rats and found the treatment with this phenolic compound (5-0 mg/kg/day) significantly decreased colonic injury, neutrophil infiltration index and the production of cytokines such as IL-1β.Moreover, resveratrol significantly decreases the prostaglandin-2 concentration and stimulates apoptosis.COX-2 expression was also dropped.The authors concluded that this grape compound could reduce the damage of induced colitis, alleviate oxidative events and promote apoptosis.Sánches-Fidalgo et al [214] also investigated resveratrol's effects in a colitis model (DSS-treated mice) that received an enriched diet with resveratrol (0 mg/kg).After 0 days, the animals were exposed to DSS for developing acute colitis that cursed to severe inflammation.The treatment with resveratrol significantly reduced the clinical signs of the disease (diarrhea, body weight loss, and rectal bleeding), ameliorating disease activity indices.Moreover, the animals that received resveratrol survived until the end of the treatment, but the control group presented a 40% of mortality rate.There were significant reductions of IL-1β and TNF-α and an increase of IL-10.The therapy with resveratrol also decreased prostaglandin E synthase-1, COX-2, and inducible iNOS proteins expression (due to the downregulation of the MAPK signaling pathway.Panaro et al [215] evaluated the effects of resveratrol on the inflammatory responses induced by lipopolysaccharide treatment in human intestinal Caco-2 and SW480 cell lines and showed that it can, in a dose-dependent manner, inhibit the expression of iNOS mRNA and the protein expression, leading to reduced production of NO.The treatment also significantly reduced the expression of Toll-like receptor-4.Besides these results, the authors showed that resveratrol curbed the phosphorylation and the degradation of the IκB complex.The reduction of LPS-induced inflammation promoted by resveratrol occurred thanks to inhibiting the activation of NF-κB-dependent mechanisms. Epicatechin extracted from grape seeds can also promote benefits to IBD, as shown by a study that examined the effects of this compound on DSS-induced UC C57BL/6J mice.The authors showed that the treatment could reduce the colon macroscopic damage scores and the Disease Activity Index.Besides that, there was a reduction in body weight loss and significant relief of colon crypt damage and contracture.The proportions of Nitric Oxide, IL-6, TNF-α, malondialdehyde, and Myeloperoxidase were reduced, and the activity of superoxide dismutase, glutathione peroxidase, and catalase showed a substantially increased activity.Inhibition of NF-κB activation was also observed both in vivo and in vitro. [216]rape compounds can be associated with positive effects against CRC.Resveratrol has relevant products, possibly due to its anti-cancer activity resulting from the modulation of several transduction signaling pathways, controlling cell division and growth, and contributing to apoptosis stimulation via CD95, Bax, and Bcl-2. [217]In CRC, it can act in several pathways and molecules such as AMPK, BMP7, COX-2, caspases, NF-κB, NO, P53, P21, SIRT1, ROS, TNFs, Wnt, and pentose phosphate pathway. [218]It can arrest the cell cycle and can attenuate neoplastic transformation.][221] Resveratrol may reduce polyamine biosynthesis which is related to being augmented in cancer cells by inhibiting ornithine decarboxylase.Furthermore, it can hinder CaCO-2 cell line growth and proliferation, down-regulating Cyclin D1/Cdk4 complex.- [222]Ji et al. showed that resveratrol inhibited the proliferation, invasion, and metastasis of HCT116 and LoVo cell lines. [223]IN HCT116 and CaCO2 carcinoma cell lines, resveratrol can also reduce vascular endothelial growth factor (VEGF) and activate the caspase 3 and 8 apoptotic markers promoting apoptotic and anti-angiogenic actions. [224]nthocyanins also have several antitumor effects that are linked to their structures, bioavailability, and how they are metabolized. [15]In CRC, there is an inverse association between high consumption and risk reduction.A multicentric study showed a significant contrary association between anthocyanidins consumption and CRC. [225]The other two studies also have an inverse relationship between anthocyanidins and CRC risk shown. [226,227]A survey with iron nanoparticles of isolated proanthocyanidin produced with grape seed showed cytotoxicity actions against CRC cell lines such as COLO320DM and HT29 cells showing promising anticancer effects. [228]Figure 6 shows the impact of grape by-products in CRC.

Bioavailability of grape by-products
As pointed out above, grape pomace is a waste consisting mainly of stalks, skin residues, broken cells with pulp remains, and seeds.Despite being a residue, it has large amounts of bioactive compounds such as flavonols, anthocyanins, and catechins.However, these bioactive compounds' beneficial actions largely depend on their bioavailability.Bioavailability is related to the gastrointestinal system's absorption and metabolization, tissue metabolism, and cellular distribution.However, it is necessary to improve the bioavailability of grape by-products due to the high amounts of lignified cell walls and tannins, both antinutritional components that can reduce nutrient absorption.Mostly polyphenols are generally found in the form of glycosides, esters, or polymers that are not absorbed in their native form, and for these reasons, they must be enzymatically hydrolyzed before absorption.231][232][233][234] Chedea et al [235] investigated the presence, and the bioavailability of polyphenols from grape pomace in IPEC cells and in vivo in piglet's colon and duodenum fed a diet supplemented with 5% of grape pomace.Their results showed that the polyphenols underwent oxidation in the cellular and extracellular medium, and procyanidin trimer C2 was the compound accumulated in the duodenum.Moreover, there were correlations between in vitro and in vivo analysis concerning the qualitative quantification of polyphenols in the cells and the gut as oxidated products such as quinones.The authors also found unmetabolized procyanidin trimers in the colon and duodenum tissues.The in vivo evaluation showed that the supplemented diet increased the total antioxidant status of piglets and reduced lipid peroxidation in the duodenum and colon.Furthermore, they observed an augmented SOD activity in duodenum and catalase and Glutathione peroxidase activity in the colon.These actions were modulated by the absorption of different polyphenols, mainly catechin and procyanidin trimers on one side and, on the other side, by polyphenols metabolites.
The bioactive components' bioavailability can be increased depending on the food excipient particularly containing phytochemicals that can modulate their absorption and metabolization.As examples, we can cite quercetin which can modify catechin methylation by binding catechol-O-methyltransferase; piperine can improve the absorption of catechin from green tea (epigallocatechin gallate), and incubation of milk with green tea augmented the absorption of catechins from green tea in Caco-2 cell.In this sense, the existence of the other polyphenols can influence the absorption of catechin.Moreover, the differences in the quantitative and qualitative catechins absorption in the duodenum and colon may also be modulated by gut microbiome that can bio-transform unabsorbed procyanidins to smaller phenolic components, which are more bioavailable. [236,237]esides that, the half-life of catechin in plasma is longer after oral administration compared to intravenous administration (possibly because of the slower rate of absorption).Furthermore, epigallocatechin gallate shows higher antioxidant activity than other flavanol compounds because it is available mainly in free form, while other catechins types are associated with glucaldehyde. [238,239]terostilbene is a natural analog of resveratrol that many species, including grapes, can produce.Since resveratrol has relatively poor oral bioavailability and suffers rapid first-pass metabolism, methylated polyphenols such as PTS possess better intestinal absorption and higher hepatic stability.The bioavailability of pterostilbene is increased with a 2-hydroxypropyl-β-cyclodextrin solution. [240,241]he study of Parekh et al [242] incorporated Nasco grape (Vitis vinifera L.) pomace extract into a nanovesicle system named nutriosomes (composed of phospholipids and water-soluble maltodextrin).Nutriosomes are homogeneously dispersed and biocompatible with intestinal epithelial cells (Caco-2); they are rich in antioxidant polyphenols such as catechin, epicatechin, gallic acid, procyanidin B2, and quercetin.These authors used these structures to explore the effects of grape pomace extract in a mouse model of Parkinson's Disease and showed the effectiveness of the nutriosome as a new nanodrug delivery system for in vivo administration.These results show the potential for use in other diseases such as IBD.
The potential biological activities of grape by-products that could be used to treat IBD, mainly due to their antioxidant and anti-inflammatory properties, make them impressive candidates as supplements that could be used for prevention or as adjuvants in the therapeutic approach.

Figure 1 .
Figure 1.Flow diagram showing the study selection (according to Preferred Reporting Items for a Systematic Review and Meta-Analysis (PRISMA) guidelines, Page et al, 2020).

Figure 2 .
Figure 2. Main phenolic compounds found in grape pulp, seeds, and skin.

Figure 3 .
Figure 3.Chemical composition of grape by products.Grape pomace can include skin, leaf, seeds and stalk that can possess many bioactive compounds.

Figure 4 .
Figure 4. Industrial use of grapes result in different by-products rich in several bioactive compounds that can be used in many products.

Figure 5 .
Figure 5. Main pathophysiological aspects of Inflammatory Bowel Diseases.Imbalance of the intestinal barrier, increased entry of microorganisms and abnormal activation of the immune system leading to an intense inflammatory process and increased risk of colorectal cancer.IL: interleukin; TNF-α: Tumor Necrosis Factor-α; TGF-β: Transforming Growth factor-β; Th: T helper cell; TLR: Toll-like receptor.

Figure 6 .
Figure 6.Initiating events in colorectal cancer formation and the effect of polyphenols such as resveratrol on cancer cell apoptosis.

Table 1 .
Main compounds present in grape by-products.

Table 2 .
In vivo biological and pharmacological activities of grape by-products on Inflammatory Bowel Diseases and colorectal cancer.

Table 3 .
In vitro biological and pharmacological activities of grape by-products on Inflammatory Bowel Diseases and colorectal cancer. [61]