Investigation of the mechanism of action of the Propolis-Punica granatum mixture as an additional therapeutic source for rheumatoid arthritis treatment

Rheumatoid arthritis (RA) [1] is a chronic autoimmune disease characterized by the production of diverse inflammatory factors. Since prolonged use of classical anti-inflammatory drugs exert several side effects, the identification of natural products capable of relieving RA symptoms could pave the way for new therapies for this disease. We have therefore devised and assayed a herbal preparation based on a traditional formulation from Campania region that has long been used against local and systemic inflammation, and composed by propolis and pomegranate peel. Firstly, propoli-pomegranate peels mixtures (PB) in different ratios were prepared and tested for their anti-inflammatory ability to reduce IL-6 secretion on human synoviocytes, a cell line suitable to study RA disease. Mixture PB 1:2 (w/w) was identified as the preparation exerting the most effective anti-inflammatory activity (IC50 = 25 μg/mL). In order to identify the compounds responsible for the observed effect, this mixture was subjected to chromatographic separation, collecting eight fractions (PB/1-8), whose anti-inflammatory activity was assayed at 12.5, 25, and 50 μg/mL by ELISA assay. Fraction PB/8, the most active one, was able to induce a significant and dose- dependent inhibition of IL-6 secretion (IC50 = 50 μg/mL). Therefore, a phytochemical characterization of this fraction was performed, leading to the identification of pinobanksin and phenethyl caffeate as the most representative constituents. To investigate the biological activity of PB/8, firstly its effect on the expression of inflammatory proteins was analyzed, observing that synoviocytes incubation with 25 mg/mL PB/8 caused STAT-3 cleavage and a significant over-expression of inducible-COX-2. It is noteworthy that the expression level of the above markers was restored when PB/8 concentration increased to 50 mg/mL. To elucidate the molecular mechanism underlying these effects, a chemical-proteomic approach, Drug Affinity Responsive Target Assay[2], was performed on PB/8 and its major constituents. Obtained results suggested several potential proteins belonging to MAPK signaling as putative targets. Finally, the anti-inflammatory activity of these components was further investigated in 3D-model of synoviocytes cells. References 1. JB Smith, MK Haynes (2002). Ann Intern Med, 136(12), 908-22 2. B Lomenick, G Jung, JA Wohlschlegel and J Huang. Curr Protoc Chem Biol, 3(4), 163–180