Intestinal anti-inflammatory effects of Phlomis purpurea extract in the TNBS model of rat colitis.

F Algieri1, P Zorrilla1, N Garrido-Mesa1, A Rodriguez-Nogales1, ME Rodriguez-Cabezas1, MP Utrilla1, M Toral1, L Perez2, M Olivares2, MR Gonzalez-Tejero3, M Casares3, J Molero3, A Zarzuelo1, J Galvez1. 1CIBER-EHD University of Granada, Department of Pharmacology, Spain, 2Biosearch Life, Department of Immunology and Animal Sciences, Spain, 3University of Granada, Department of Botany, Spain

Introduction: Phlomis purpurea whole plant is traditionally used in mediterranean traditional medicine as an anti-inflammatory remedy. It contains polyphenols, which display antioxidant properties interesting for the treatment of inflammatory pathologies associated with oxidative stress in humans, including inflammatory bowel disease (IBD). The aim of this study was to evaluate the intestinal anti-inflammatory properties of a hydroalcoholic extract of P. purpurea that contains at least 14 % of polyphenolic compounds, in the trinitrobenzenesulphonic acid (TNBS) model of rat colitis, a well characterized model with some resemblance to human IBD.

Material and Methods: Female Wistar rats (200 ± 10 g) were assigned to five groups (n=8): non-colitic, control colitic (without treatment) and treated colitic groups: two treated with P purpurea extract (10 and 25 mg/kg/day) (provided by Biosearch Life) and the remaining received sulphasalazine (SAZ) (200 mg/kg/day), as a positive control. Treatments started the same day of TNBS colitis induction, and rats were sacrificed one week after. Colonic damage was assessed macroscopically (score 0-10) and biochemically: myeloperoxidase activity (MPO), glutathione content (GSH), as well as IL-1β, IL-17, CINC-1, villin, MUC-2 and TFF-3 expressions by qPCR. Statistical analyses was carried out with Statgraphics 5.0, with statistical significance set at p<0.05 using a one-way analysis of variance (ANOVA) and post hoc least significance tests.

Results: The administration of P. purpurea extract resulted in an intestinal anti-inflammatory effect, at doses of 10 and 20 mg/kg/day, as evidenced macroscopically by a reduction in the extension of damage and in the weight/length ratio (an index of edema tissue) in comparison with control colitic rats (Table 1). Biochemically, it was observed a decrease in colonic MPO activity over 50% in both doses studied, which indicates a lower neutrophil infiltration in the inflamed tissue, and a 55% increment in glutathione content, in the dose of 10 mg/kg, thus improving the altered oxidative status in colitic rats (Table 1). When the expression of the cytokines was evaluated by qPCR, the treatment with 25 mg/kg of P. purpurea extract resulted in decreased colonic expression of the proinflammatory cytokines IL-1β and IL-17, indicating an inhibitory effect on the upregulated immune response that characterizes the colonic inflammatory process. In addition, this dose of the extract reduced the expression of chemokine CINC-1, which may justify the lower neutrophil infiltration that occurs in the colonic specimens obtained from treated colitic rats. In addition, this dose of the extract was able to significantly upregulate the expression of markers of intestine epithelial integrity, like MUC-2, TFF-3 and villin (Table 1). When the dose of 10 mg/kg of the extract was evaluated, it significantly reduced the expression of IL-17, and increased that of the mucin MUC-2.

Table 1. Effects of Phlomis purpurea extract in TBNS rat colitis

Data are expressed as mean ± SEM. *p<0.05 vs. Control group.

Conclusion: The extract of Phlomis purpurea showed intestinal anti-inflammatory activity in the TNBS model of rat colitis, in which the antioxidant properties, the downregulation of the immune response as well as an improvement in the intestine epithelial barrier may contribute to its beneficial effect.