Phytochemical studies and evaluation of anti-inflammatory and antibacterial properties of extracts prepared from the pharmacopoeial plant substance of linden blossom (Tiliae flos).

Linden flower (Tiliae flos) is a popular herbal remedy used in the form of infusions to soothe and heal symptoms of the common cold, such as sore throats, coughs and high temperature. Tiliae flos has its monograph in European Pharmacopoeia 8.0, which defines this plant substance as whole, dried flowers obtained from broad-leaved lime (Tilia platyphyllos L.), small-leaved lime (Tilia cordata Mill.) and Dutch (Tilia x vulgaris Hayne) or being a mixture of the above-mentioned three species. Although the linden flower is considered a well-known plant material, the available literature still lacks research on the chemical composition and biological activity of extracts prepared from flowers obtained from pharmacopoeial linden species. The main goal of the presented project is a comprehensive phytochemical analysis of extracts prepared from linden flowers collected from pharmacopoeial linden species and the assessment of their anti-inflammatory activity using a model of human neutrophils. During the project, comparisons of the composition of individual extracts will be carried out, taking into account the chemometric analysis of the obtained results and a comparison of the biological activity of the tested material obtained from three pharmacopoeial linden species (T. platyphyllos, T. cordata, T. x vulgaris) and two species that are not the source of the pharmacopoeial plant substance - Tilia americana L. and Tilia tomentosa Moench. The planned research will be carried out with the use of linden flower samples collected from the natural state in spring / summer 2014, 2015 and 2016. The research will focus on samples obtained from three pharmacopoeial species of this plant (T. platyphyllos, T. cordata and T. x vulgaris) and two non-pharmacopoeial species (T. tomentosa and T. americana) listed as the most common contaminants for this drug substance. Commercial linden blossom samples will also be purchased and analyzed. Phytochemical analysis will be based on modern analytical techniques such as UHPLC-DAD-MS / MS and HPTLC. Both methods will be optimized in the course of the conducted research. Infusions prepared from selected five species of linden will be characterized in terms of their components based on the obtained data. All extracts, with particular emphasis on extracts intended for biological research, will be standardized using the validated UHPLC method and simpler chemical methods to quantify individual compounds and groups of compounds present in them. The isolation part of phytochemical studies will concern flowers obtained from T. cordata.By using chromatographic techniques such as column chromatography, flash chromatography and preparative HPLC, the main components present in the extract prepared from T. cordata flowers will be isolated. Purified compounds will be identified using spectroscopic and spectrometric methods such as 1D and 2D NMR, UV-Vis or HR-MS. The results of phytochemical tests will be subjected to chemometric analysis using statistical methods such as principal component analysis (PCA) or Czekanowski's diagrams method (HCA). This will allow for the identification of differences in the chemical composition between the infusions obtained from the flowers of five selected linden species and to determine whether the observed differences are statistically significant. During biological tests, the anti-inflammatory potential of the prepared extracts will be assessed. The effect of extracts on the pro-inflammatory functions of human neutrophils, such as the release of reactive oxygen species, proteolytic enzymes (elastases, metalloproteinases) will be determined using spectrophotometric methods used in the project lead's laboratory. The effect on the production of cytokines such as interleukin-8 and 1β will also be tested using ready-made ELISA tests. The research will also determine the antibacterial properties of the analyzed extracts. Using strains of bacteria from the streptococcal group, the minimum concentrations that inhibit the growth of bacteria and the minimum bactericidal concentrations will be determined. The proposed project is focused on basic research on the chemical composition of the pharmacopoeial herbal substance - Tiliae flos. The obtained results will contribute to a significant expansion of the current knowledge about the chemical composition of this drug, and will indicate whether there are significant differences in the chemical composition of flowers from five selected species of this plant. The planned research will indicate which standardization method should be used for the linden flower, which will have a direct impact on increasing the safety and effectiveness of using infusions from this plant substance. Biological tests will show whether linden flower infusions have the ability to regulate the inflammatory response of human neutrophils and the ability to inhibit the growth of bacteria from the streptococcal group. The results of the conducted research may contribute to the confirmation of the legitimacy of using linden flower infusions as a mild anti-inflammatory and antibacterial agent.