Searching for new compounds with anti-inflammatory activity from native plant species belonging to the Oleaceae family with the use of in vitro and in vivo models, together with an indication of the molecular mechanisms determining the observed activity.

Objective and expected results: The aim of the project is to find, among plants from the Oleaceae family, compounds with potential activity promoting inflammation suppression by inhibiting neutrophil infiltration, inhibiting the production of pro-inflammatory cytokines, stimulating phagocytosis of apoptotic neutrophils and inducing the secretion of anti-inflammatory factors by macrophages. The well-documented traditional use of plants from the Oleaceae family in the treatment of inflammatory diseases, combined with their photochemical diversity, allows for a hypothesis that these plants can be a rich source of interesting new compounds with multidirectional anti-inflammatory effects. In order to verify the hypothesis, comprehensive photochemical tests will be carried out, combined with the isolation of biologically active compounds. A thorough characterization of the biological activity of the isolated compounds in vitro and in vivo will allow to determine their anti-inflammatory mechanism at the molecular level. Research method The planned research will concern 32 extracts (water, 60% ethanol, dichloromethane (DCM)) obtained from various parts of plants belonging to three genera of the Oleaceae family: Fraxinus (1 species), Syringa (1 species) and Forsythia (3 species). The work plan was divided into two parts: photochemical studies and biological studies. The main task of the phytochemical part will be to analyze the obtained extracts and fractions using the HPLC-DAD-MS / MS method. The most active extracts will be divided into fractions of different polarity. From the most active extracts / fractions, the compounds responsible for the observed activity will be isolated using the 'bio-guided isolation' method. The study of biological activity will consist of the following steps: - Preliminary evaluation of the anti-inflammatory activity of the extracts will be established based on the examination of the inhibition of oxygen burst, the release of proteases (elastase and MMP-9) and cytokines (IL-8 and ILip) in the model of human neutrophils as well as influence on the production of pro-inflammatory cytokines (IL-6 and TNF-a) and anti-inflammatory IL-10 as well as phagocytosis of apoptotic neutrophils by macrophages obtained from the THP-1 cell line. Assessment of cytotoxicity of extracts on both cell models. - For selected extracts / fractions with outstanding activity, tests will be carried out using a wider range of concentrations. Additionally, the effect on the surface expression of adhesion molecules and apoptosis of neutrophils will be determined. More in-depth studies of the effects on the production of anti-inflammatory factors (TGF-P) as well as efferocytosis and phagocytosis will be carried out on macrophages obtained from the THP-1 cell line. - In order to carry out 'bio-guided isolation', an individual model will be selected for each fraction, in which the strongest or outstanding activity was observed in the previous stages. - The isolated active compounds will be comprehensively tested on other models, which will allow to indicate their selectivity or pleiotropic effect. - The molecular mechanism of action of selected single active compounds will be determined by the impact on the NFKB, JAK / STAT, MAPK / ERK and IP3 pathways and the expression of mRNA encoding previously determined cytokines (RT-PCR, qRT-PCR) - For selected promising compounds (1-3 ) the influence on the activation state of the immune system caused by arterial hypertension in an animal experimental model will be investigated. Effect of the results Plant extracts containing secondary metabolites, which are a group of compounds with a very diverse chemical structure, and therefore generating a large number of pharmacophores with a unique spatial structure, have long been a source of new drugs. In addition, compounds of natural origin, unlike synthetic molecules, due to their biochemical functions in the plant, offer a great chance for potential interaction with proteins and the ability to penetrate intercellularly. Currently, due to the rapid development of phytochemical methods, metabolomics, biological models and rationalization of the selection of plant material, there is a renaissance of interest in a natural source in the search for new drugs. This project is in line with the trend of modern pharmacognosy / pharmacy and will allow the selection of compounds with selective or pleiotropic action that can reduce the symptoms of chronic inflammation.