TANNINS FROM RUBUS AND FRAGARIA BERRIES FOR THE CONTROL OF GASTRIC INFLAMMATION: IN VITRO AND IN VIVO STUDIES

Gastritis and ulcers are very common inflammatory-based diseases which can be caused by Helicobacter pylori (H. pylori) infection, chemical factors or immunological disorders[3]. H. pylori is the leading cause of gastritis[1], it colonizes the gastric mucosa of over 80% of human population in developing countries[3] and at least 50% of the world’s human population[36]. Gastric epithelial cells, during H. pylori infection, show increased levels of cytokines/chemokines including IL-1β, IL-6, TNFα and IL-8[66]. Epithelial cells stimulated with TNFα, IL-1β or bacterial infection, release a variety of cytokines (TNFα, IL-1β, IL-8) and increase expression of cyclooxygenase-2 (COX-2) and inducible NO synthase (iNOS). The activation of nuclear factor κB (NF-κB) is deeply involved in the control of transcription of inflammatory mediators in the stomach[95]. Ellagitannins (ET) have shown anti-inflammatory and anti-H.pylori properties; however, their anti-inflammatory activity at gastric level was not previously investigated. Rubus berries (raspberries and blackberries) and Fragaria berries (strawberries) are considered a rich source of phenolic compounds, including anthocyanins, tannins, ET and ellagic acid (EA) derivatives conjugated[308]. The aim of the present research was to evaluate the effects of tannins, in particular ET, from Rubus and Fragaria berries, the most common sources of ET in the European diet, on gastric inflammation. Tannin enriched extracts (TEs) were prepared from Rubus fruticosus L. (blackberry), Rubus idaeus L. (raspberry), Fragaria X ananassa Duch. (strawberry) and Fragaria vesca L. (wild strawberry). The anti-inflammatory activity was tested in vitro on gastric cell line AGS stimulated by TNFα and IL-1β. TEs inhibited TNFα-induced NF-κB driven transcription and reduced NF-kB nuclear translocation. TEs inhibited also IL-8 secretion induced by TNFα and IL-1β at low concentrations (IC50 range: 0.5–8.8 µg/mL). The effect of an in vitro gastric digestion on TEs activity was also evaluated. Sanguiin H-6, lambertianin C, (the major ET present in Rubus berries) and agrimoniin (ET from Fragaria berries), were found to be responsible, at least in part, for the effect of the mixtures. In vivo the protective effect of TEs was evaluated in a rat model of ethanol-induced gastric lesions. Rats were treated orally for ten days with 20 mg/kg/day of TEs, and ethanol was given one hour before the sacrifice. Gastric mucosa was isolated and used for the determination of IL-8 release, NF-κB nuclear translocation, Trolox equivalents, superoxide dismutase and catalase activities. TEs of blackberry and raspberry decreased Ulcer Index by 88% and 75% respectively and protected from the ethanol-induced oxidative stress in rats. CINC-1 (the rat homologue of IL-8) secretion in the gastric mucosa was reduced in the animals receiving blackberry and raspberry TEs. The effect of TEs on CINC-1 was associated to a decrease of NF-κB nuclear translocation in TEs treated animals. The anti-inflammatory activity exerted by TEs occurs at extremely low concentrations, even below the concentration of 1 μg/mL, values that are considered easy to reach by the ingestion of a portion of berries (approximately 5 mg/mL). These results show that tannins interfere with the metabolic cascade deriving from the activation and translocation of NF-κB that in turn activates the transcription of target genes involved in inflammation, including IL-8. ET do not require any absorption to reach the stomach and their concentrations are not reduced by metabolic processes in this district. In this study was demonstrated for the first time that ET from blackberries and raspberries are able to protect the stomach against the gastric lesions caused by ethanol. The outcome of this research suggests the use of ET as integration in dietary regimens designed for treat or prevent inflammatory gastric diseases.