A grape (Vitis vinifera L.) pomace water extract modulates inflammatory and immune response in SW‐480 cells and isolated mouse colon

Grape (Vitis vinifera L.) pomace is a residue derived from the winemaking process, which contains bioactive compounds displaying noteworthy health‐promoting properties. The aim of the present study was to investigate the phenolic composition and protective effects of a water extract of grape pomace (WEGP) in colorectal cancer cell line SW480 and in isolated mouse colon exposed to Escherichia coli lipopolysaccharide (LPS). The extract decreased SW‐480 cell viability, as well as vascular endothelial factor A (VEGFA), hypoxia‐induced factor 1α (HIF1α), and transient receptor potential M8 (TRPM8) LPS‐induced gene expression. Moreover, the extract inhibited mRNA levels of nuclear factor kB (NFkB), cyclooxygenase (COX)‐2, tumor necrosis factor (TNF)α, interleukin (IL)‐6, IL‐1β, IL‐10, inducible nitric oxide synthase (iNOS), and interferon (IFN)γ, in isolated colon. Conversely, WEGP increased the gene expression of antioxidant catalase (CAT) and superoxide dismutase (SOD), in the same model. The modulatory effects exerted by WEGP could be related, at least in part, to the phenolic composition, with particular regards to the catechin level. Docking calculations also predicted the interactions of catechin toward TRPM8 receptor, deeply involved in colon cancer; thus further suggesting the grape pomace as a valuable source of bioactive extracts and phytochemicals with protective effects in the colon.

In this context, a wide body of evidence hinted some biological activities exerted by grape polyphenols, including antioxidant, cardioprotective, anticancer, antiinflammatory, antiaging, and antimicrobial properties (Peixoto et al., 2018). In addition, the health-promoting effects induced by the phenolic compounds of red wine are well known (Boussetta, Vorobiev, Le, Cordin-Falcimaigne, & Lanoisellé, 2012;Renaud & De Lorgeril, 1992). Grape seed extract and its constituents were found to exert protective effects on TNBSS-or DSS-induced ulcerative colitis in rodents (Cheah et al., 2013;Li, Cai, Qin, & Wu, 2008). Moreover, dietary grape seed extract supplementation has been previously considered to ameliorate inflammatory bowel disease symptoms in interleukin (IL)-10 knockout mice (Wang et al., 2013), as well as to exert preventive effects against colorectal carcinogenesis (Tian et al., 2020). Considering these findings, in the present study, we aimed to evaluate the effects of a water extract of grape pomace (GPWE) in colorectal cancer cell line SW480 and in an ex vivo experimental model of colon inflammation constituted by isolated mouse colon challenged with Escherichia coli lipopolysaccharide (LPS) (Menghini et al., 2016). The grape pomace was the by-product of the vintage (year 2020) of a DOC red wine, the Montepulciano d'Abruzzo variety (Villamagna doc).

| Plant material and extraction
Pomace was obtained from the grapes kindly provided by a local farm  . Specifically, The optimal conditions to obtain the higher yield of the most abundant metabolites include high solvent: plant ratio (12 v/w), corresponding to 12 ml/g.
Detailed protocols were reported as supplementary material.
Absorbance of each well was quantified at 540 and 690 nm, using a Isolated colon specimens were maintained in a humidified incubator with 5% CO 2 at 37 C for 4 h (incubation period), in RPMI buffer with added bacterial LPS (10 μg/ml), as previously described Recinella et al., 2021). During the incubation period, the tissues were challenged with scalar concentrations of GPWE (1-100 μg/ml).

| Statistical analysis
Statistical analyses were performed using GraphPad Prism version 5.01 software (San Diego, CA). Means ± SEM were determined for each experimental group and analyzed by one-way analysis of variance (ANOVA), followed by Newman-Keuls comparison multiple test.
Statistical significance was set at p < .05. The number of animals randomized for each experimental group was calculated on the basis of the "resource equation" N = (E + T)/T (10 ≤ E ≤ 20) (Charan & Kantharia, 2013).

| Characterization of the extract
In the present study, a water extract from grape pomace was examined to determine whether it could have beneficial effects on colon cancer and inflammation. This extract from plant material collected in the vintage of 2020 has been investigated for the content in phenolic compounds, with total phenols, total flavonoids, flavonols, and antocyanins assayed via colorimetric assays (Supplementary material: Table S1). Whereas 19 phytochemicals were identified through HPLC-DAD-MS, in comparison with pure standards (Table 1). The ultrasound-assisted extraction conditions in water solvent were selected according to a recent study of ours , and catechin was found to be the prominent flavonoid (Figures 1 and   2). This is also consistent with the phytochemical profile of the water extract from the grape pomace collected during the 2019 vintage. The extract was also found effective in exerting scavenging/reducing and enzyme inhibition effects (Supplementary material: Table S2), which are consistent, albeit partially, with its content in phenolic compounds (Chatatikun et al., 2020;Meserole, 2002); thus supporting further pharmacological investigations for exploring health-promoting effects, as described below.

| Effects of the extract on colon cancer (SW480) cells
The extract was tested on colon cancer (SW480) cells for determining the effects on cell viability, in both basal and LPS-induced inflammatory condition. In parallel, the extract was also tested in human fibroblast The dietary supplementation with grape pomace also demonstrated preventive effects against the onset of colon cancer in mice exposed to azoxymethane and dextran sulfate sodium (Tian et al., 2020). In the present study, the grape pomace extract (0.1-1,-000 μg/ml) was able to induce a significant reduction of SW-480 cell viability in both basal and LPS-induced inflammatory conditions (Table 2); and the effects were relevant starting from the concentrations of 100 μg/ml, at which the cell viability was under the limits of biocompatibility (70% viability compared with the control group; p < .0001). By contrast, the HFF-1 cell line viability was not modified by the extract (Table 2) The gene expression of endothelia factor A (VEGFA) and the hypoxia-induced factor 1α (HIF1α) was assessed as well, in SW-480 F I G U R E 1 Chromatographic analysis of the water extract from the Vitis vinifera pomace. The chromatographic analysis confirmed the presence of 19 phytochemicals. The prominent compound was catechin (peak #4). The other main phytochemicals were gallic acid (peak #1), caftaric acid (peak #3), and epicatechin (peak #11)  (Panyathep, Punturee, & Chewonarin, 2021). Together with the HIF1α, VEGFA is a well-known angiogenesis-stimulating factor, and is considered a key mediator of the so-called inflammatory to cancer transition (Chen et al., 2019). The inhibition of VEGFA and HIF1α gene expression (p < .0001) was also related to the cytotoxic effects induced by thalidomide in SW480 cells (Zhang & Luo, 2018). In the present study, the gene expression of both factors was downregulated by the extract, and the effect was more relevant when the cells were challenged with LPS (Table 3).
In colon cancer, there is increasing evidence of the involvement of transient receptor potential (TRP) M8 (TRPM8), whereas its block has been related, albeit partially, to the inhibition of carcinogenesis, in mice (Borrelli et al., 2014;Liu, Li, & Xu, 2022). Additionally, in melanoma cells, the activation of TRPM8 was related to the inhibition of VEGFA-induced transactivation of TRV1 (Walcher et al., 2018). In
Overall, the present findings agree with the present and recent findings of intrinsic scavenging/reducing and anti-inflammatory effects by grape pomace extract . The pattern of phenolic composition, with particular regards to the content of catechin, could explain, albeit partially the modulatory effects on the abovementioned biomarkers of inflammation and oxidative stress (Fan, Sang, & Jiang, 2017).

| CONCLUSIONS
The results of the study indicated the efficacy of the water extract from grape pomace in reverting the burden of inflammation and oxidative stress occurring in isolated colon specimens exposed to LPS.
Whereas, the reduction of human colon cancer SW-480 cell viability, and the modulation of pattern of gene expression of proteins involved in carcinogenesis, further support protective effects in the colon. The mechanism underlying these effects could involve more than one phytochemical. However, in SW480 cells the prominent phenolic compound, namely catechin, could be the main responsible of the inhibitory effects on VEGFA, HIF1α, and TRPM8 gene expression.
Docking calculations also predicted the interactions of catechin towards TRPM8 receptor, deeply involved in colon cancer; therefore further suggesting the potential of grape pomace as a valuable source of bioactive extracts and phytochemicals with protective effects in the colon.

ACKNOWLEDGEMENTS
The present study is also part of the third mission activities of the botanical garden "Giardino dei Semplici" of "G. d'Annunzio" University. The authors would like to express their gratitude to Prof.