Bryophytes, a source of inspiration for active ingredients discovery

Bryophytes are the second largest group of terrestrial plants with more than 20 000 species divided into Marchantiophyta (liverworts), Bryophyta (mosses), and Anthocerotophyta (hornworts). Bryophytes are small plants with simple body structures; however, they present a high degree of biochemical complexity. They produce rare and diverse specialized metabolites reported with various biological activities. Their arsenal of bioactive natural products highlights them as a rich source of natural products with commercial potential.

Therefore, this Ph.D. study investigated the biological activities of bryophytes extracts as a novel source of natural ingredients for cosmetics or pharmaceutical applications. The main objective was the in vitro screening of their inhibitory activities towards skin aging-related enzymes (collagenase and elastase) and pigmentation-related enzymes (tyrosinase) as well as antioxidant and antimicrobial activities. Also, the extracts were screened for their antiinflammatory properties by the inhibition of induced nitric oxide production in RAW 264.7 murine macrophage cells. The dereplication of extracts and natural products included a combination of analytical tools such as ultra-high performance liquid chromatography mass spectrometry (UHPLC-MS), tandem mass spectrometry (MS/MS), nuclear magnetic resonance (NMR), and database searching.

The first chapter provides a theoretical background of the subjects addressed in this thesis. The chapter presents bryophytes as a source of bioactive natural products, which is further discussed in the review paper. Besides, the chapter introduces an overview of the biological target enzymes (tyrosinase, collagenase and elastase) and activities (antioxidant, antimicrobial and anti-inflammatory) investigated throughout this thesis. Finally, the chapter covers key techniques applied for the investigation of natural products and bioactive compounds.

The second chapter includes two subchapters: 2.1 explores the anti-inflammatory activity of a variety of bryophytes species and 2.2 combines the initial screening of bryophytes extracts for in vitro collagenase and elastase inhibitory activities. Among the analyzed extracts, the mosses Dicranum majus and Thuidium delicatulum exhibited anti-inflammatory activity on lipopolysaccharide (LPS) stimulated RAW264.7 cell lines. Active extracts from the moss Polytrichum formosum and the liverwort Bazzania trilobata showed inhibitory activity towards collagenase and were subjected to a second level of screening and chemical investigation of bioactive constituents as presented in the following chapters.

In the third chapter, the extracts from the moss P. formosum were analyzed in vitro for their inhibitory properties on collagenase and tyrosinase activity. A specific ligand-protein approach, Target Binding®, was used to retrieve candidate molecules for both enzymatic inhibitory activities. The candidate compounds ohioensin A, ohioensin C, communin B, and a new compound named nor-ohioensin D were isolated from P. formosum and tested towards the target enzymes. Ohioensin A showed anti-collagenase and anti-tyrosinase activities. The new compound nor-ohioensin D also showed collagenase inhibitory potential.

In the fourth chapter, the extracts of the liverwort B. trilobata were investigated for in vitro biological activities of cosmetic interest. The results showed that the extracts of this liverwort exhibited anti-collagenase and anti-tyrosinase activity. Moreover, the extracts showed antioxidant and antimicrobial properties. Lignans, coumarins and bis-bibenzyls were the major classes of phenolic constituents tentatively identified by mass spectrometry. In addition, drimenyl caffeate was isolated for the first time in B. trilobata and its structure was confirmed by Nuclear Magnetic Resonance (NMR) spectroscopy. The chapter includes section 4.2 with additional investigation of bioactive compounds of B. trilobata extracts.

The final part of this thesis presents a general discussion and conclusions of the significant findings of this work.