*2.4. Cytotoxicity Assay*

Cell morphology was checked by light microscope inspection. Cell viability was measured, after 6 h co-treatment with the stimulus (TNF α, 10 ng/mL) and the extracts, by the 3,4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT) method (Sigma-Aldrich, St. Louis, MA, USA) [28]. This method evaluates the succinate dehydrogenase activity, which is an index of cell viability. Six hours later, the medium was removed from each well and 0.1 mg/mL of MTT solution (200 μL) was added for 45 min at 37 ◦C, in dark conditions. Violet formazan salt was extracted from the cells with 200 μL of a mixture isopropanol:DMSO (90:10), and the absorbance was read at 570 nm (Envision, PerkinElmer, Waltham, MA, USA). The extracts were assessed in the range 0.1–20 μg/mL.

## *2.5. Cell Treatment*

To investigate the release and gene expression of the pro-inflammatory mediators, the NF-κB driven transcription, and IL-8 promoter activity, cells were seeded in 24-well plates (DB FalconTM) at the density of 30,000 cells/well. After 72 h, cells were co-treated with the pro-inflammatory stimulus (TNF α, 10 ng/mL) and the extracts for 6 h, using serum-free medium: DMEM/F12 or RPMI 1640 medium (Gibco, Thermo Fisher Scientific, Waltham, USA), supplemented with L-glutamine 2 mM (Gibco, Thermo Fisher Scientific, Waltham, USA), penicillin 100 units/mL (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), and streptomycin 100 mg/mL (Gibco, Thermo Fisher Scientific, Waltham, USA). Then, the medium or the cell lysate was collected for the biological assays. To assess the e ffect of the extracts on ROS generation, AGS and GES-1 cells were seeded in black 96-well plates (PerkinElmer, USA) at the density of 10,000 cells/well; after 72 h, cells were pre-treated with the extracts for 24 h in serum-free medium, and subsequently challenged with H2O2 for 2 h.

## *2.6. ROS Production*

The intracellular ROS level was measured, in GES-1 and AGS cells, with the oxidant-sensitive fluorescence probe CM-H2DCFDA (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). After the treatment, cells were incubated for 30 min with the fluorescent probe (10 μM), washed with PBS and stimulated with H2O2 for 2 h. The fluorescence intensity was read at an excitation wavelength of 485 nm and an emission wavelength of 535 nm using a plate reader (Envision, Perkin Elmer, Waltham, MA, USA). Trolox (500 μM) was used as reference compound. Data were expressed as mean ± SD of at least three experiments.

#### *2.7. Transient Transfection Assays*

GES-1 and AGS cells were transiently transfected in 24-well plates with two di fferent reporter plasmids, the NF-κB-Luc (50 ng/well) or the IL-8-Luc (100 ng/well) [29]. The NF-κB-Luc contains the luciferase gene under control of the E-selectin promoter characterized by three κB responsive elements, while IL-8-Luc contains the luciferase gene under control of a fragment of the native promoter of the human IL-8. The plasmid NF-κB-Luc was a kind gift of Dr. N. Marx (Department of Internal Medicine-Cardiology, University of Ulm, Ulm, Germany)m whereas the native IL-8-Luc promoter was kindly provided by Dr. T. Shimohata (Department of Preventive Environment and Nutrition, University of Tokushima Graduate School, Tokushima, Japan). GES-1 cells were transfected using Lipofectamine ® (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), whereas AGS cells by the calcium phosphate method. Sixteen hours later, the cells were treated for 6 h with extracts in the presence of the pro-inflammatory mediators (TNF α 10 ng/mL). Six hours later, cells were harvested and the luciferase assay was carried out using the BriteliteTM Plus reagen<sup>t</sup> (PerkinElmer Inc., Waltham, MA, USA), according to the manufacturer's instructions. The results (mean ± SD of at least three experiments) were expressed as percentage, relative to stimulated control, which was arbitrarily assigned the value 100%.

#### *2.8. IL-8 and IL-6 Release*

IL-8 and IL-6 were quantified in cell media after the treatment with TNF α and plant extracts, using two di fferent ELISA kits, a Human Interleukin-8 ELISA Development Kit and a Human Interleukin-6 ELISA Development Kit (Peprotech Inc., London, UK). Briefly, Corning 96-well EIA/RIA plates (Sigma-Aldrich, St. Louis, USA) were coated with the corresponding antibody provided by the ELISA Kit (Peprotech Inc., London, UK) and incubated overnight at room temperature. The non-specific binding sites were blocked with albumin 1%. A total of 200 μL of samples was transferred in duplicate into wells at room temperature for 2 h. The amount of IL-8 and IL-6 in the samples was detected at 450 nm by measuring the colorimetric reaction between horseradish peroxidase enzyme and 3,3,5,5-tetramethylbenzidine substrate (Sigma-Aldrich, St. Louis, MO, USA). The absorbance

was read at 450 nm using a spectrophotometer (Victor X3, PerkinElmer, USA). IL-8 and IL-6 were quantified by optimization of the standard curve provided with the ELISA kit (8.0–1000 pg/mL for IL-8 and 32–2000 pg/mL for IL-6). Epigallocatechin-3-O-gallate (EGCG, 20 μM) was used as a reference molecule able to inhibit TNFα-induced IL-8 and IL-6 secretion. The results (mean ± SD of at least three experiments) were expressed as percentage, relative to stimulated control, which was arbitrarily assigned the value 100% (values around 1000 and 1500 pg/mL, respectively for IL-8 and IL-6 under stimulated conditions).
