Mediterranean plant Helichrysum italicum, better known as Everlasting plant or Immortal, is a rich source of bioactive compounds synthesized by the plant, which enable it to thrive in very arid environment. Despite numerous studies, very little information on the activity of whole water extracts is available and therefore several applications in traditional use remain to be scientifically validated. The aim of the doctoral thesis was to chemically characterize the bioactive compounds from Helichrysum italicum extracts, to support their use in traditional medicine by laboratory experiments, and to investigate the suitability of using H. italicum as a potential herbal medicine or as a dietary supplement.
Extracts were prepared from two H. italicum subspecies: H. italicum ssp. italicum (HII) in H. italicum ssp. tyrrhenicum (HIT), using the maceration and infusion method. The chemical composition of the prepared extracts was determined using a high-performance liquid chromatography (HPLC) coupled with tandem mass spectrometry (MS/MS). The antioxidant activity of the extracts, and their mutagenic and antimutagenic activity in Salmonella typhimurium and Escherichia coli strains, were tested. Intestinal cancerous cell line Caco-2 and primary colon fibroblasts CCD112CoN were used for in vitro tests. The cytotoxicity of the H. italicum water extracts, the effect on cell proliferation and the potential protective action upon oxidative stress induction were determined. Transcriptome analysis of the CCD112CoN cells was performed aiming to investigate the mode of action of the infusion at the gene level. The role of the most differentially expressed genes (DEGs) was confirmed in functional assays.
The ethanol-water extracts (EVIs) and the methanol-water extracts (MVIs) of the same subspecies were the most similar in chemical composition, while the cold-water extracts (HVIs) stood out the most. HIT HVI had the highest content of total phenolic compounds (TPC) and HII hot-water extract (VVI) the lowest. VVIs (infusions) proved to be comparable with alcohol-water ones in terms of both their antioxidant and antimutagenic activity, although they contained less TPC. None of the extracts showed mutagenic properties for either strain. In the cytotoxicity assay, HII infusion was less toxic for CCD112CoN cells than the HIT infusion, whereas for Caco-2 cells the opposite was true. HII showed a protective effect against oxidative stress for both cell lines, however, it inhibited ROS generation more effectively in the case of the Caco-2 cell line. A total of 217 DEGs were detected by transcriptome analysis after treatment with HII infusion. The role of the individual genes that were most frequently represented in the enriched pathways was examined in detail and linked to the formerly known effects of H. italicum extracts. On the basis of these results, we hypothesized that the main mode of action of the HII infusion is the modulation of the wound healing process, characterized by hemostasis, cytoskeleton reorganization, cell growth and inflammation, which was further confirmed by functional assays. The cell migration assay showed that treatment of Caco-2 cells with HII infusion increased cell migration potential, and conditioned media of CCD112CoN cells exposed to HII infusion for 24 h significantly reduced blood clotting time and prothrombin time. Serum levels of zonulin and the pro-inflammatory cytokines IL-6, IL-1β and MCP-1 were decreased and levels of the anti-inflammatory cytokine IL-33 were increased after 4 weeks of HII infusion consumption. By maintaining the integrity of the intestinal wall, H. italicum extracts could hypothetically prevent or alleviate pathological conditions such as inflammatory bowel disease and metabolic disorders. Furthermore, H. italicum extracts may also affect inflammation, lipid and glucose metabolism directly, by altering the expression of relevant anti-inflammatory and metabolic genes.
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