The Nutraceutical Properties of Sumac (Rhus coriaria L.) against Gastritis: Antibacterial and Anti-Inflammatory Activities in Gastric Epithelial Cells Infected with H. pylori

Sumac (Rhus coriaria L.) is a spice and medicinal herb traditionally used in the Mediterranean region and the Middle East. Since we previously demonstrated Sumac biological activity in a model of tumor necrosis factor alpha (TNF-α)-induced skin inflammation, the present work is aimed at further demonstrating a potential role in inflammatory disorders, focusing on gastritis. For this purpose, different polar extracts (water-W, ethanol-water-EW, ethanol-E, ethanol macerated-Em, acetone-Ac, ethylacetate-EtA) were investigated in gastric epithelial cells (GES-1) challenged by TNF-α or H. pylori infection. The ethanolic extracts (E, EW, Em) showed the major phenolic contents, correlating with lower half maximal inhibitory concentrations (IC50s) on the release of interleukin-8 (IL-8, <15 μg/mL) and interleukin-6 (IL-6, <20 μg/mL) induced by TNF-α. Similarly, they inhibited IL-8 release (IC50s < 70 μg/mL) during Helicobacter pylori (H. pylori) infection and exhibited a direct antibacterial activity at comparable concentrations (minimum inhibitory concentration (MIC) = 100 μg/mL). The phenolic content and the bioactivity of EW were maintained after simulated gastric digestion and were associated with nuclear factor kappa B (NF-κB) impairment, considered the main putative anti-inflammatory mechanism. On the contrary, an anti-urease activity was excluded. To the best of our knowledge, this is the first demonstration of the potential role of Sumac as a nutraceutical useful in H. pylori-related gastritis.


Introduction
Rhus coriaria L. (Sumac) belongs to the Anacardiaceae family, widely grown throughout the Mediterranean region. Leaves and fruits have a remarkable medicinal value in Middle Eastern herbal medicine [1]. The brown/red fruits of Rhus coriaria are used as a very popular spice in food production for their sour lemony taste. Phytochemical characterization of berries showed the occurrence of different antioxidants belonging to several classes of polyphenols, among which flavonoids and gallotannins are the most abundant [2].
Red fruits are traditionally used in Persian medicine to treat diarrhea, hemorrhoids, gout, and decrease cholesterol, uric acid, and blood sugar levels, and for a variety of other biological activities recently revised by Elagbar et al. [1]. Much evidence supports the pharmacological and nutraceutical properties of Sumac in a variety of diseases [3][4][5][6][7][8]. Moreover, additional studies demonstrated interesting effects in inflammatory conditions, including the reduction of pro-inflammatory mediators and the inhibition of the nuclear factor kappa B (NF-κB) activation in human keratinocytes challenged with tumor necrosis factor alpha (TNF-α) [5].
The total phenol content, measured as gallic acid equivalents/g of extract, showed a significant amount of polyphenols (above 200 mg/g) in E, EW, Em, and Ac, whereas the amount for W and EtA was found significantly lower (under 100 mg/g). In general, the presence of ethanol as a solvent increased the total phenol content, which was maximum following maceration (Table 1). The preliminary characterization through high-performance liquid chromatography ultraviolet/with diode-array detection (HPLC-UV/DAD) analysis, previously published by our group, showed the presence of a significant amount of flavonoids, tannins, and anthocyanins in the EW and Em extracts. Both extracts showed similar amounts of flavonoids (0.23%, flavonoids expressed as quercetin-3-O-glucoside) and tannins (4.54% and 4.33%, tannins expressed as gallic acid, for EW and Em, respectively), while EW showed a significantly higher anthocyanin content (0.207%, anthocyanins expressed as cyanidin-3-O-glucoside) than the Em extract (0.031%, anthocyanins expressed as cyanidin-3-O-glucoside) [5].

Total Phenol Content Assay
Total polyphenol content was measured by Folin-Ciocâlteu's method. Briefly, the extracts were dissolved in water (5 mg/mL), then 20 µL were diluted to a final volume of 800 µL, corresponding to 100 µg of extracts weight. Then, 50 µL of 2 N Folin-Ciocâlteu reagent (Merck Life Science, Milan, Italy) and 150 µL of 20% (w/v) sodium carbonate (Na 2 CO 3 ) were added. After 30 min of incubation at 37 • C, the absorbance of the samples was measured with a Jasco V630 Spectrophotometer (JASCO International Co. Ltd., Tokyo, Japan) at 765 nm. The total phenol content was measured using a calibration curve of gallic acid. Results were expressed as mg of gallic acid equivalents per g of extract.

Cell Treatment
To measure the release of the pro-inflammatory cytokines and the activation of NF-κB, cells were seeded in 24-or 6-well-plates (Falcon ® , Corning Life Science, Amsterdam, The Netherlands) at a density of 3 × 10 5 cells/well and 10 5 cells/well, respectively. After 72 h, GES-1 were treated with the pro-inflammatory stimulus TNF-α (10 ng/mL) or the bacterium H. pylori (bacterium:cell ratio of 50:1) along with the extracts at different concentrations. In the co-treatment with the bacterium, serum starvation was performed using 0.5% serum medium, added with 1% L-glutamine and 1% penicillin/streptomycin, 24 h before. Regarding H. pylori infection, all the treatments were conducted with serum and antibiotic-free medium in the co-culture with, while TNF-α treatments were conducted with serum-free medium. During the treatment, cells were maintained in incubator at 37 • C and 5% CO 2 . After 6 h for the release of the pro-inflammatory cytokines or 1 h for NF-κB activity, culture media or cell lysates were collected for biological assessments.

Cytotoxicity Assay
The correct cell morphology was verified by light microscope inspection before and after treatment. Cell viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2-5diphenyltetrazolium bromide (MTT) method (Merck Life Science, Milan, Italy) at the end of the treatments (6 h) [18]. This method is an undirect index of viability, since it evaluates the activity of a mitochondrial enzyme, the succinate dehydrogenase. Briefly, the medium was discarded, then 200 µL of MTT solution (0.1 mg/mL, phosphate buffered saline (PBS) 1X) were added to each well (45 min, 37 • C) and kept in darkness. Then, MTT solution was discarded and the purple salt included into the cells was dissolved by isopropanol:dimethyl sulfoxide (DMSO) (90:10 v/v), and the absorbance was read at 595 nm (Victor TM X3, Perkin Elmer, Walthman, MA, USA).

Measurement of IL-8 and IL-6 Release
The pro-inflammatory mediators IL-8 and IL-6 were quantified in cell media after 6-h treatments with TNF-α or H. pylori as stimuli and the extracts, by an enzyme-linked immunosorbent assay (ELISA), using two sandwich ELISA kits: Human Interleukin-8 ELISA Development Kit and Human Interleukin-6 ELISA Development Kit (Peprotech, London, UK), according to Nwakiban et al. [19] and manufacturer instructions. Briefly, clear plates (Corning enzyme immunoassay/radioimmunoassay (EIA/RIA) plates, 96-well, Merck Life Science, Milan, Italy) were coated with the capture antibody from the ELISA kit (overnight, room temperature (r.t.)). The non-specific binding sites were blocked with albumin 1% for 1 h and then a total of 100 µL of samples in duplicate were transferred into wells at room temperature for 2 h. The pg/mL of IL-8 and IL-6 was detected through the colorimetric reaction due to horseradish peroxidase (HRP)-conjugated biotinylated antibody and 3,3 ,5,5 -tetramethylbenzidine (TMB) substrate (Merck Life Science, Milan, Italy). The absorbance was obtained at 450 nm 0.1 s by multiplate reader (Victor TM X3, PerkinElmer, Waltham, MA, USA). Data were expressed as percentage relative to stimulated control, which was arbitrarily assigned the value of 100%.

NF-κB Activation
The activation of NF-κB was measured by western blot and immunofluorescence techniques. Western blot was employed to measure the activation of phospho-p65 in the GES-1 cells, while immunofluorescence was employed to reveal the translocation of p65 in the nucleus of GES-1 cells.

Immunofluorescence
Immunofluorescence technique was used to assess the translocation of NF-κB from cytoplasm to nucleus of GES-1 cells, challenged with H. pylori and treated with extracts. Cells were seeded on coverslips placed in 24-well plates at the density of 30,000 cells/well. Before treatment H. pylori was stained with carboxyfluorescein succinimidyl ester (CFSE) 5 mM (CellTrace™, Cell Proliferation kits; Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) diluted 1:500 v/v and incubated for 20 min at 37 • C. Subsequently, fetal bovine serum (FBS) was added to the bacterial suspension to bind the excess of CFSE, followed by three washes with PBS and centrifugation at 6000× g for 5 min to remove the excess of CFSE not bound to the bacterium. After 1 h treatment, co-cultures were washed (PBS 1X) and fixed with 4% formaldehyde solution for 15 min at r.t. A 5% BSA blocking solution was added to the well and incubated at room temperature for 1 h. Cells were incubated with the primary antibody (NF-κB p65 (D14E12) XP ® Rabbit mAb #8242, Cell Signaling Technology, Danvers, MA, USA) diluted 1:400 v/v overnight at 4 • C and then with the secondary antibody (Alexa Fluor 647 conjugated with anti-rabbit immunoglobulin G (IgG) (heavy + light (H + L)), F(ab')2 Fragment #4414, Cell Signaling Technology, Danvers, MA, USA) diluted 1:1000 v/v. After 2 h, coverslips were washed with PBS and mounted on slides with a drop of ProLong Gold Antifade Reagent with 4 ,6-diamidino-2-phenylindole (DAPI) (#8961, Cell Signaling Technology, Danvers, MA, USA), and then were imaged with a confocal laser scanning microscope (LSM 900, Zeiss, Oberkochen, Germany).

Minimum Inhibitory Concentration (MIC)
The microbroth dilution method was performed as per the recommendations of Clinical and Laboratory Standards Institute (CLSI) [20] and was used to determine MIC. Extracts at different concentration and positive control (tetracycline 0.125 µg/mL) were prepared in Brucella broth (BBL™, BD, Franklin Lakes, NJ, USA) supplemented with 5% FBS, and 100 µL of each sample were placed in a 96-well U-bottom plate (Greiner Bio-One™, Rome, Italy). Then, 100 µL of H. pylori suspension prepared in the same medium in a dilution adjusted to O.D. value = 0.1 were added to each well. After well-mixing, the 96-well plate was incubated at 37 • C in a 5% CO 2 incubator under microaerophilic condition. After 72 h, the assay plate was read visually for growth inhibition at 600 nm 0.1 s, using a multi-detection microplate reader (Victor™ X3, PerkinElmer, Waltham, MA, USA).

Urease Activity
The urease activity of H. pylori was measured using a phenol red solution (9.1 g/L potassium dihydrogen phosphate (KH 2 PO 4 ), 9.5 g/L disodium hydrogen phosphate (Na 2 HPO 4 ), 0.01 g/L phenol red) and the substrate urea (2 g/L), according to the protocol used by Svane et al. and Korona-Glowniak et al. [21,22]. In the presence of the bacterium, urea was converted to ammonia (NH 3 ), modifying the pH and changing the color of the solution. In a 96-well plate 100 µL of extracts and positive controls were prepared, then H. pylori suspension (final concentration O.D. value = 0.1) and 100 µL of phenol red/urea solution were added. After 1 h of incubation at 37 • C, the absorbance was measured with a spectrophotometer at 570 nm 0.1 s (EnVision Plate reader, Perkin Elmer, Waltham, MA, USA).

In Vitro Simulated Gastric Digestion
The gastric digestion was mimicked by an in vitro simulation, as previously described [23]. In brief, EW extract (100 mg) was incubated for 5 min at 37 • C with simulated saliva (6 mL), then gastric juice (12 mL) was added, and the sample was incubated for 2 h at 37 • C. Then, the suspension was centrifuged (5 min, 3000 g) and the supernatant was freeze-dried.

Statistical Analysis
All data were expressed as mean ± standard deviation (SD) of at least three independent experiments; the interval of confidence related to the half maximal inhibitory concentrations (IC 50 s) calculation (% confidence interval (C.I.)) was reported in the Results session.Data were elaborated by unpaired ANOVA test and Bonferroni post-hoc analysis. Statistical measures were conducted by GraphPad Prism 8.0 software (GraphPad Software Inc., San Diego, CA, USA). Values of p < 0.05 were considered statistically significant. The IC 50 s were calculated by GraphPad Prism 8.0 software.

Cytotoxicity of the Extracts in Human Gastric GES-1 Cells
All the extracts were tested for cytotoxicity (MTT assay) in GES-1 cells, incubating increasing concentrations of extracts in presence of TNF-α (Supplementary Materials Figure  S1A) or H. pylori (Supplementary Materials Figure S1B) for six hours. In both cases, all the extracts did not show any cytotoxicity in the range 10-200 µg/mL. Concentrations used for the biological assays were chosen accordingly. The MTT assay was also performed on the EW extract subjected to in vitro simulated gastric digestion (EWd), at concentrations ranging between 10 and 200 µg/mL. No cytotoxicity was detected in cells treated with each concentration tested in the presence of TNF-α or H. pylori as pro-inflammatory stimuli (Supplementary Materials Figure S2A,B).

Effect of the Extracts on the TNF-α-Induced IL-8 and IL-6 Release in GES-1 Cells
Several cytokines are involved in gastric inflammation including IL-8, IL-6, and TNF-α. IL-8 and IL-6 are produced by gastric epithelial cells following inflammation [10]. The release of TNF-α by gastric epithelial cells and macrophages feeds the inflammatory process in the gastric mucosa, activating the cytokine cascade. To investigate the activity of Rhus coriaria in modulating the inflammatory process, GES-1 cells were treated for six hours with the extracts and TNF-α, then IL-8 or IL-6 release was measured through ELISA assay.
All the extracts showed a concentration-dependent inhibition of the TNF-α-induced IL-8 release; the aqueous (W), ethanolic (E), or hydroethanolic (EW) extracts showed the highest inhibitory activity (IC 50 ranging from 12.1 to 14.7 µg/mL, Table 2) whereas the activity of the acetonic (Ac) and ethylacetate (EtA) ones was lower (IC 50~3 0 µg/mL, Table 2). For all the extracts, the first concentration which showed statistically significant inhibition of IL-8 release was 10 µg/mL (Figure 1). All the extracts showed a concentration-dependent inhibition of the TNF-α-induced IL-8 release; the aqueous (W), ethanolic (E), or hydroethanolic (EW) extracts showed the highest inhibitory activity (IC50 ranging from 12.1 to 14.7 μg/mL, Table 2) whereas the activity of the acetonic (Ac) and ethylacetate (EtA) ones was lower (IC50 ~ 30 μg/mL, Table  2). For all the extracts, the first concentration which showed statistically significant inhibition of IL-8 release was 10 μg/mL (Figure 1).   IL-6 is known to require NF-κB activation during H. pylori-related gastritis. Thus, the potential ability of the extracts to control gastric inflammation acting on IL-6 release was further verified. Likewise, the results on the IL-8 release, all the extracts inhibited the TNF-α-induced IL-6 release in a concentration-dependent fashion (Figure 2). IL-6 is known to require NF-κB activation during H. pylori-related gastritis. Thus, the potential ability of the extracts to control gastric inflammation acting on IL-6 release was further verified. Likewise, the results on the IL-8 release, all the extracts inhibited the TNFα-induced IL-6 release in a concentration-dependent fashion (Figure 2).
The IC50 values paralleled those obtained on the IL-8 release, with the aqueous, ethanolic, or hydroethanolic extracts showing the most potent effect (IC50s ranging from 10.3 to 19.3 μg/mL, Table 2) compared to the acetonic and ethylacetate ones (IC50: 69.9 and 85.4 μg/mL, respectively; Table 2). Similar effects were obtained by the aqueous and ethanolic extracts in inhibiting IL-8 and IL-6.   Table 2) compared to the acetonic and ethylacetate ones (IC 50 : 69.9 and 85.4 µg/mL, respectively; Table 2). Similar effects were obtained by the aqueous and ethanolic extracts in inhibiting IL-8 and IL-6.

Effect of the Extracts on the H. pylori-Induced IL-8 and IL-6 Release in GES-1 Cells
According to the literature, expression of both the NF-κB dependent IL-8 and IL-6 genes occurs during H. pylori-induced gastric inflammation [12][13][14][15][16]. Thus, cells were treated with H. pylori and the extracts (50-200 µg/mL), as referred to by the Materials and Methods section, and release of IL-8 or IL-6 was assessed through an ELISA assay. All the extracts were able to affect IL-8 release to a different extent (Figure 3), with IC 50 s ranging between 62.0 and 183.1 µg/mL (Table 3).

Effect of the Extracts on the H. pylori Growth
The exploration of novel compounds with antibacterial activity and a favorable safety profile, based on long-standing consumption as a food, is an attractive therapeutic approach to prevent or ameliorate several gastric inflammatory conditions. Thus, the effect of the extracts (25-400 μg/mL) on the H. pylori growth after 72 h of incubation was investigated. All the extracts, except for the aqueous one, showed a concentration-dependent inhibition of the bacterial growth (Figure 4), with a MIC value of 100 μg/mL for all the active extracts; however, EW and Em showed to better impair bacterial growth since it was completely abolished at 100 μg/mL.  The highest activity was found for the ethanolic or hydroethanolic extracts compared to the water extract. On the opposite, all the extracts showed the same negligible activity against IL-6 release, with IC 50 s above 200 µg/mL (Table 3).

Effect of the Extracts on the H. pylori Growth
The exploration of novel compounds with antibacterial activity and a favorable safety profile, based on long-standing consumption as a food, is an attractive therapeutic approach to prevent or ameliorate several gastric inflammatory conditions.
Thus, the effect of the extracts (25-400 µg/mL) on the H. pylori growth after 72 h of incubation was investigated. All the extracts, except for the aqueous one, showed a concentration-dependent inhibition of the bacterial growth (Figure 4), with a MIC value of 100 µg/mL for all the active extracts; however, EW and Em showed to better impair bacterial growth since it was completely abolished at 100 µg/mL.

Effect of the In Vitro Digested Hydroethanolic Extract (EWd) on the TNF-α-or H. pyloriinduced IL-8 Release in GES-1 Cells
In the next steps, we focused on the EW extract, the choice of which was based on the following experimental indications: the extract showed a high amount of phenols and was among the most active extracts able to inhibit IL-8 release, induced by both H. pylori or TNF-α; it was also active in inhibiting H. pylori growth and the release of IL-6; it is reason-  : 72 h), which was arbitrarily assigned value of 100%. The reference antibiotic (black color) used was tetracycline 0.125 µg/mL (100% inhibition). * p < 0.05; ** p < 0.01; *** p < 0.001 versus H. pylori.

Effect of the In Vitro Digested Hydroethanolic Extract (EWd) on the TNF-α-or H. pylori-induced IL-8 Release in GES-1 Cells
In the next steps, we focused on the EW extract, the choice of which was based on the following experimental indications: the extract showed a high amount of phenols and was among the most active extracts able to inhibit IL-8 release, induced by both H. pylori or TNFα; it was also active in inhibiting H. pylori growth and the release of IL-6; it is reasonable to consider the combination of water and ethanol as a safer and cheaper extraction solvent than ethanol alone.
Thus, the extract was firstly subjected to in vitro simulated gastric digestion with the purpose of assessing its stability in the gastric environment, then total phenol content and IL-8 release induced by H. pylori or TNF-α were assessed.
The EW extract subjected to digestion (EWd) showed no decrease in the phenol content compared to the undigested extract (251.8 vs. 258.5 µg/mL, respectively); moreover, once again the extract inhibited both the TNF-α-and H. pylori-induced IL-8 release ( Figure 5), with low IC 50 s (7.73 and 43.92 µg/mL, respectively).  EGCG 20 μM (−74%) was used as reference compound (black color). Data are reported as percentage in comparison to the stimulated control, which was arbitrarily assigned to 100% value. ** p < 0.01, *** p < 0.001 versus TNF-α or H. pylori. EWd, EW extract subjected to in vitro simulated gastric digestion.

EW and EWd Extracts Impair the NF-κB Pathway in GES-1 Cells
It has been extensively reported in the literature that the activation of the NF-κB pathway is able to promote the release of IL-8 and IL-6, which in turn lead to the amplification of the gastric inflammatory process [10,11]. Since the EW extract showed interesting effects as an inhibitor of the release of these cytokines we decided to investigate if the NF-κB pathway could be affected by extracts as well, through western blot and immunofluorescence assays. EW and EWd extracts (200 μg/mL) impaired the NF-κB pathway, as evident from the reduction of p65 nuclear accumulation ( Figure 6) and phosphorylation at the upstream level (Figure 7). Accordingly, EW extract also reduced the TNF-α-induced NF-κB driven transcription in GES-1 cells at lower concentrations (IC50 50.16 μg/mL, data not shown).

EW and EWd Extracts Impair the NF-κB Pathway in GES-1 Cells
It has been extensively reported in the literature that the activation of the NF-κB pathway is able to promote the release of IL-8 and IL-6, which in turn lead to the amplification of the gastric inflammatory process [10,11]. Since the EW extract showed interesting effects as an inhibitor of the release of these cytokines we decided to investigate if the NF-κB pathway could be affected by extracts as well, through western blot and immunofluorescence assays. EW and EWd extracts (200 µg/mL) impaired the NF-κB pathway, as evident from the reduction of p65 nuclear accumulation ( Figure 6) and phosphorylation at the upstream level ( Figure 7). Accordingly, EW extract also reduced the TNF-α-induced NF-κB driven transcription in GES-1 cells at lower concentrations (IC 50 50.16 µg/mL, data not shown).

Effect of EWd on H. pylori Growth and Urease Activity
To investigate if the gastric digestion could influence the EW extract activity, the bacterium was incubated for 72 h with increasing concentrations of the EWd extract (25-400 µg/mL), and the growth of H. pylori was assessed. The extract elicited a concentration-dependent inhibition of the bacterial growth ( Figure 8A) with a MIC of 100 µg/mL, the same of the non-digested extract, suggesting that the simulated gastric digestion did not affect the activity; however, both the extracts did not affect the urease activity at 400 µg/mL ( Figure 8B). as an inhibitor of the release of these cytokines we decided to investigate if the NF-κB pathway could be affected by extracts as well, through western blot and immunofluorescence assays. EW and EWd extracts (200 μg/mL) impaired the NF-κB pathway, as evident from the reduction of p65 nuclear accumulation ( Figure 6) and phosphorylation at the upstream level (Figure 7). Accordingly, EW extract also reduced the TNF-α-induced NF-κB driven transcription in GES-1 cells at lower concentrations (IC50 50.16 μg/mL, data not shown).

Effect of EWd on H. pylori Growth and Urease Activity
To investigate if the gastric digestion could influence the EW extract activity, the bacterium was incubated for 72 h with increasing concentrations of the EWd extract (25-400 μg/mL), and the growth of H. pylori was assessed. The extract elicited a concentrationdependent inhibition of the bacterial growth ( Figure 8A) with a MIC of 100 μg/mL, the same of the non-digested extract, suggesting that the simulated gastric digestion did not affect the activity; however, both the extracts did not affect the urease activity at 400 μg/mL ( Figure 8B).  terium was incubated for 72 h with increasing concentrations of the EWd extract (25-400 μg/mL), and the growth of H. pylori was assessed. The extract elicited a concentrationdependent inhibition of the bacterial growth ( Figure 8A) with a MIC of 100 μg/mL, the same of the non-digested extract, suggesting that the simulated gastric digestion did not affect the activity; however, both the extracts did not affect the urease activity at 400 μg/mL ( Figure 8B).

Discussion
Rhus coriaria L. (Sumac) fruits are widely used as a spice; previous phytochemical characterizations showed the presence of several antioxidants including flavonoids and gallotannins [5].
A few papers have reported anti-inflammatory activities of Sumac in a variety of diseases. Ahmad et al. reported the anti-ulcer effect of a hydroalcoholic extract (145 and 248 mg/kg) from Sumac in rodent models of stress, ethanol, and indomethacin-induced gastritis [24][25][26]; however, the anti-inflammatory activities in H. pylori-related gastritis, as well as the direct effect on H. pylori growth, deserved further investigation. In the present study, we aimed at assessing which different Rhus coriaria L. fruit extracts, obtained with various solvents, could be the best candidate to be used as an ingredient of food supplements intended as adjuvants against gastric inflammatory conditions. All the extracts, except for the aqueous one, showed anti-H. pylori activity with similar MIC, regardless of the solvent used for extraction. Since the MIC values correlated with the phenolic content (lower in aqueous and ethylacetate extract), we suppose that the efficiency of polyphenol extraction, more than the presence of specific polyphenols, may account for the antibacterial activity. For this reason, the attribution of the antibacterial effect to specific compounds or their combination and the related mechanism of action may deserve dedicated studies. Our results are in line with another work present in the literature, in which the direct anti-H. pylori effect of ethanol extract from Rhus coriaria showed comparable MIC value (214.28 µg/mL) [27]. Of note, our experiments were conducted using a well-characterized bacterial strain (ATCC 26695), while the mentioned work assessed the antibacterial activity on clinical strains, thus limiting the comparability of results. Another Rhus species (Rhus typhina L.), widely used in China to treat gastrointestinal disorders, has been previously reported to impair H. pylori growth in vitro [28], thus suggesting that antibacterial compounds could be common into the Rhus genus. Hydroalcoholic extracts of Rhus coriaria L. showed antibacterial activity against a variety of Gram-positive and Gram-negative food-borne bacteria, but the anti-H. pylori effect was not investigated [29]. This is the first study assessing the anti-H. pylori potential of different Sumac extracts made up with different solvents with a broad range of polarity; the study shows that the hydroethanolic extract maintains a similar MIC value after in vitro simulated gastric digestion.
Although the EW extract impaired H. pylori growth, both EW extracts, non-digested and subjected to simulated gastric digestion, did not influence in vitro urease activity in our experimental conditions. These results seem to be in contrast with the study by Mahernia et al. [30] in which a hydroalcoholic extract from Rhus coriaria L. fruits showed an anti-urease activity, with an IC 50 value of 80.29 µg/mL; however, the different extract used in the study (methanol:water 80:20 v/v) as well as the different H. pylori strain may account for this discrepancy.
Rhus coriaria extracts show a strong anti-inflammatory activity, inhibiting IL-8 release induced by H. pylori or TNF-α. The effect on IL-8 release was more pronounced when cells were challenged with TNF-α (range: 14.1-32.5 µg/mL); a similar trend was recorded for IL-6 release. This may be due to the complexity of pathways modulated by H. pylori, with respect to TNF-α alone, activating a variety of factors including NF-κB and activator protein 1 (AP-1).
Since during H. pylori-induced gastric inflammation there is a massive release of TNF-α from infiltrated immune cells, which activates and prolongs the inflammatory cascade, it is likely that these effects may add up in vivo. NF-κB plays a key role in the release of several cytokines including IL-8 and IL-6 which, in turn, lead to the amplification of the gastric inflammatory processes; thus, we selected NF-κB signalling as a relevant target to assess the anti-inflammatory properties of EW and the matched digested extracts. EW extract was able to impair the NF-κB pathway during TNF-α induction. Accordingly, immunofluorescence showed the inhibition of the H. pylori-induced NF-κB (p65) translocation from the cytoplasm into the nucleus for EW and EWd extracts, which was in line with the inhibition of p65 phosphorylation at the upstream level observed in western blots. Consequently, our data suggest that polyphenols from Rhus coriaria L. may exert anti-inflammatory effect by interfering with NF-κB at gastric level.

Conclusions
In conclusion, the antibacterial and anti-inflammatory effects of the hydroalcoholic extract, which is maintained also when subjected to in vitro simulated gastric digestion, suggest the beneficial properties of Sumac towards inflammation of the gastric epithelium and its possible use as ingredient of food supplements able to prevent inflammatory-based gastric diseases induced by H. pylori; the extract may be also able to limit bacterial growth by acting directly against the bacterium. In addition, taking into consideration the plausible dietary intake of grams of fruits and the extraction yields, the bioactivity was observed at concentrations (101-102 µg/mL) easily achievable following oral consumption of the fruits or the corresponding enriched polar extracts.
Future studies will be devoted to better clarify the role of specific classes of polyphenols present in Rhus coriaria L. fruits to recommend standardized products with potential antigastritis effect in vivo.