Downloaded from orbit.dtu.dk on: Jun 15, 2024 Bryophytes, a source of inspiration for active ingredients discovery Volpatto Marques, Raíssa Publication date: 2021 Document Version Publisher's PDF, also known as Version of record Link back to DTU Orbit Citation (APA): Volpatto Marques, R. (2021). Bryophytes, a source of inspiration for active ingredients discovery. DTU Bioengineering. General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. Users may download and print one copy of any publication from the public portal for the purpose of private study or research. You may not further distribute the material or use it for any profit-making activity or commercial gain You may freely distribute the URL identifying the publication in the public portal If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Bryophytes, a source of inspiration for active ingredients discovery Ph.D. Thesis Raíssa Volpatto Marques Academic Supervisor: Associate Professor Henrik Toft Simonsen Industrial Supervisor: Frédéric Bourgaud Submitted: September 2021 Preface The present industrial Ph.D. thesis describes the research carried out in the Photosynthetic Cell Factories group at the Department of Biotechnology and Biomedicine, Technical University of Denmark (DTU) and with the industrial partner Plant Advanced Technologies (Vandoeuvrelès-Nancy, France) in the period of April 2018 – April 2021. The work was supervised by the academic supervisor Associate Professor Henrik Toft Simonsen and by the industrial supervisor Frédéric Bourgaud. The project was funded by Marie Sklodowska Actions Innovative Training Networks under the Horizon 2020 program under grant agreement n° 765115– MossTech. During this project, I had the opportunity to learn and be involved in many aspects of the basic and applied research within the bioactive natural products discovery. This was a challenging but worthwhile journey for my professional and personal development. I had a chance to develop a fascinating research topic within the unexplored world of bryophytes. These tiny plants showed me that we have to be persistent, patient, and adapt to the environment around us. I am very grateful to be able to contribute a little more to the scientific research on the biological properties of mosses and liverworts. I would like to thank my supervisor Henrik Toft Simonsen for giving me the opportunity to work in his research group. I appreciate his scientific advice and support throughout my project. I would like to thank my industrial supervisor Frédéric Bourgaud for the opportunity to develop my research in the company Plant Advanced Technologies (PAT). The almost two years of work at PAT was a great chance to experience the research and development sector of a biotech company. I appreciate his support and scientific orientation during this project. Thank you Sissi Miguel for the guidance and great scientific discussions. I would like to thank Professor Nils Cronberg from the Department of Biology, Faculty of Sciences, Lund University (Lund, Sweden) for the identification and collection of bryophytes. I am also grateful for all the collaborations within this project. I would like to thank the NMR Center at DTU and Associate Professor Charlotte Held Gotfredsen and Kasper EnemarkRasmussen for the NMR structural elucidation. I would like to thank the ABC PlatformRaphaël E. Duval, Nicolas Hocquigny, and Arnaud Risler at the University of Lorraine 2 (Vandœuvre-lès-Nancy, France) for the antimicrobial screening. Also, the Molecular Engineering and Articular Physiopathology (IMoPA) research unit of the University of Lorraine and the French National Center for Scientific Research (CNRS) (Vandœuvre-lèsNancy, France) for the anti-inflammatory screenings, in special David Moulin, Stefania Sestito, and the entire group. Also, I would like to extend my gratitude to all my colleagues, friends and professors from DTU, PAT, and Mosstech. Thank you all for the great scientific discussions, field trips, and nice work environment. Thank you to the most important person of my life, my mother Irene Volpatto Marques. Thank you for encouraging me to follow my dreams. Thank you my brother Marcelo Volpatto Marques for all the discussions, for inspiring me, and to my father Carlos Alberto Silva Marques for being present in my memories and my journey. Thank you for all the great time, help, understanding and support during this period. Thank you my boyfriend Charles Gosserez for always being there and my new French family – Anne, Patrick, Louise, Guillaume and Gero. Finally, I am very glad for having nice friends that were very important during this period. Thank you Renata Galetti and Fabiano Contesini for the nice moments in Denmark and for being so helpful during my Ph.D. journey. Thank you Jessica Vidor, Thais Lopes, Julio Garighan and MossTech friends Isidora Lončarević and Yi Lu for the support throughout my studies. Raíssa Volpatto Marques September 2021 Summary 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. 4 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. Dansk Resumé Bryofytter er den næststørste gruppe af landbaserede planter med mere end 20 000 arter opdelt i Marchantiophyta (levermosser), Bryophyta (mosser) og Anthocerotophyta (hornmosser). Bryofytter er små planter med enkle strukturer, som dog har en høj grad af biokemisk kompleksitet. De producerer sjældne og forskellige naturstoffer, der har mange forskellige biologiske aktiviteter. Deres arsenal af bioaktive naturstoffer fremhæver dem som en rig kilde til naturstoffer med kommercielt potentiale. I dette Ph.d. har jeg undersøgt de biologiske aktiviteter af bryofytekstrakter som en ny kilde til naturlige ingredienser til kosmetik eller farmaceutiske anvendelser. Hovedformålet har været in-vitro screening af ekstrakternes hæmmende aktiviteter over for hudaldringsrelaterede enzymer (collagenase og elastase) og pigmenteringsrelaterede enzymer (tyrosinase) samt antioxidant- og antimikrobielle aktiviteter. Ekstrakterne blev også screenet for deres antiinflammatoriske egenskaber ved hæmning af induceret nitrogenoxidproduktion i RAW 264.7 murine makrofagceller. Dereplikering af ekstrakter og naturstoffer omfattede en kombination af analyseværktøjer såsom ultrahøj ydeevne væskekromatografi- massespektrometri (UHPLC-MS), tandemmassespektrometri (MS/MS), nuklear magnetisk resonans (NMR) og databasesøgning. Det første kapitel giver en teoretisk baggrund for de emner, der behandles i dette studie. Kapitlet beskriver bryofytter, som en kilde til bioaktive naturstoffer, hvilket diskuteres yderligere i review artiklen. Desuden introducerer kapitlet en oversigt over de biologiske enzymer (tyrosinase, collagenase og elastase) samt aktiviteter (antioxidant, antimikrobielle og antiinflammatoriske) der er undersøgt i denne afhandling. Endelig dækker kapitlet centrale teknikker, der anvendes til undersøgelse af naturstoffer og bioaktive forbindelser. Det andet kapitel indeholder to underkapitler: 2.1 Undersøger den antiinflammatoriske aktivitet af en række bryofytter og 2.2 kombinerer den indledende screening af bryofytekstrakter for invitro kollagenase- og elastasehæmmende aktiviteter. Blandt de analyserede ekstrakter udviste moserne Dicranum majus og Thuidium delicatulum antiinflammatorisk aktivitet på lipopolysaccharid (LPS) stimulerede RAW264.7 cellelinjer. Aktive ekstrakter fra mosen Polytrichum formosum og levermosen Bazzania trilobata viste hæmmende aktivitet mod collagenase og blev udsat for et andet niveau af screening og kemisk undersøgelse af bioaktive bestanddele som præsenteret i de følgende kapitler. 6 I det tredje kapitel blev ekstrakterne fra mosen P. formosum analyseret in-vitro for deres hæmmende egenskaber på collagenase- og tyrosinaseaktivitet. En specifik ligand-protein binding, Target Binding®, blev brugt til at opdage kandidatmolekyler til begge enzym hæmmende aktiviteter. Stofferne ohioensin A, ohioensin C, communin B og en ny forbindelse kaldet nor-ohioensin D blev isoleret fra P. formosum og testet på enzymerne. Ohioensin A udviste anti-collagenase og anti-tyrosinase aktivitet. Den nye forbindelse nor-ohioensin D udviste også et anti-collagenase-hæmmende potentiale. I det fjerde kapitel blev ekstrakterne af levermosen B. trilobata undersøgt for biologiske aktiviteter af kosmetisk interesse. Resultaterne viste, at ekstrakterne af denne levermos udviste anti-collagenase og anti-tyrosinase aktivitet. Desuden udviste ekstrakterne antioxidant og antimikrobielle egenskaber. Lignaner, kumariner og bis-bibenzyler er hovedklasserne af de phenoliske naturstoffer, der foreløbigt blev identificeret ved hjælp af massespektrometri. Desuden blev drimenylcaffeat isoleret for første gang fra B. trilobata, og dets struktur blev bekræftet af Nuclear Magnetic Resonance (NMR) spektroskopi. Kapitlet indeholder afsnit 4.2 med yderligere undersøgelse af bioaktive forbindelser af B. trilobata ekstrakter. Den sidste del af dette speciale præsenterer en generel diskussion og konklusioner af de væsentlige resultater af dette arbejde. List of publications 1. Armin Horn, Arnaud Pascal, Isidora Lončarević, Raíssa Volpatto Marques, Yi Lu, Sissi Miguel, Frédéric Bourgaud, Margrét Thorsteinsdóttir, Nils Cronberg, Jörg D. Becker, Ralf Reski, Henrik T. Simonsen. “Natural Products from Bryophytes: From Basic Biology to Biotechnological Applications.” Critical Reviews in Plant Sciences 2021; 40:3, 191-217. 2. Raíssa Volpatto Marques, Stefania E. Sestito, Frédéric Bourgaud, Sissi Miguel, Sophie Rahuel-Clermont, Sandrine Boschi-Muller, Henrik Toft Simonsen, David Moulin. “Anti‑inflammatory activity of bryophytes extracts in LPS-stimulated RAW264.7 murine macrophages”. Manuscript in preparation for the journal Frontiers in Bioscience-Landmark. Special issue: Bioactive Phytochemicals and Botanicals in Health and Disease. 3. Raíssa Volpatto Marques, Agnès Guillaumin, Ahmed B. Abdelwahab, Aleksander Salwinski, Charlotte H. Gotfredsen, Frédéric Bourgaud, Kasper Enemark-Rasmussen, Sissi Miguel, Henrik Toft Simonsen, 2021. “Collagenase and tyrosinase inhibitory effect of isolated constituents from the moss Polytrichum formosum”. Plants 2021; 10(7):1271. This article belongs to the Special Issue: Advances in Research with Bryophytes. 4. Raíssa Volpatto Marques, Aleksander Salwinski, Kasper Enemark-Rasmussen, Charlotte H. Gotfredsen, Yi Lu, Nicolas Hocquigny, Arnaud Risler, Raphaël E. Duval, Sissi Miguel, Frédéric Bourgaud, Henrik Toft Simonsen. “Extracts from the liverwort Bazzania trilobata with potential dermo-cosmetic properties”. Manuscript in preparation for Phytochemistry Letters 8 Abbreviations ANOVA Analysis of variance AP-1 Activator protein-1 CID Colission-induced dissociation COSY Homonuclear correlation spectroscopy DAD Diode array detector DAMPs Damage-associated molecular patterns DPPH 1,1- Diphenyl-2-Picryl- Hydrazyl radical DQF-COSY Double-quantum filtered correlation spectroscopy ECM Extracellular matrix EIC Extracted ion chromatogram ESI Electrospray ionization FID Free induction decay HMBC Heteronuclear multiple bond correlation HPLC High resolution mass spectrometry HSQC Heteronuclear single quantum correlation hPrx Human peroxiredoxin IC50 Half maximal inhibitory concentration J Coupling constant LC Liquid chromatography LC-MS Liquid chromatography coupled with mass spectrometry L-DOPA L-3,4-dihydroxyphenylalanine LPS Lipopolysaccharides MMPs Matrix metalloproteinases MS Mass spectrometry MS/MS Tandem mass spectrometry NF-κB Nuclear factor-kappa B NMR Nuclear Magnetic Resonance NO Nitric oxide PAMPs Pathogen-associated molecular patterns QTOF Quadrupole time-of-flight ROS Reactive oxygen species RT Retention time TB® Target Binding® TOF Time of flight UHPLC Ultra-high-performance liquid chromatography UHPLC-HRMS Ultra-high performance liquid chromatography-high resolution mass spectrometry UHPLC-MS Ultra-high performance liquid chromatography- mass spectrometry UV-VIS 10 Ultraviolet-Visible Table of Contents Preface ......................................................................................................................................... 2 Summary...................................................................................................................................... 4 Dansk Resumé ............................................................................................................................. 6 List of publications ...................................................................................................................... 8 Abbreviations............................................................................................................................... 9 Chapter 1 .................................................................................................................................. 13 Introduction ............................................................................................................................... 13 1.0 General Introduction .......................................................................................................... 14 1.1 1.1.1 1.2 1.2.1 Bryophytes as a source of bioactive natural products ...................................................... 14 Potential use of bryophytes in cosmetics .............................................................. 18 Target enzymes of pharmaceutical and cosmetic importance .......................................... 19 Tyrosinase- the main enzyme in skin pigmentation .............................................. 20 1.2.2 Collagenase and elastase- proteinases responsible for the regulation of dermal matrix proteins ........................................................................................................................... 21 1.2.3 An overview of target inhibitors ........................................................................... 22 1.3 Antioxidants .................................................................................................................... 24 1.4 Natural antimicrobials in cosmetics ................................................................................. 25 1.5 Overview of skin inflammation and inhibitory drug targets ............................................ 26 1.6 Tools for the investigation of natural products ................................................................ 28 1.6.1 Liquid chromatography ........................................................................................ 28 1.6.2 Mass spectrometry................................................................................................ 30 1.6.3 Nuclear magnetic resonance (NMR) spectroscopy ............................................... 32 1.7 Affinity-based screening approach for the identification of bioactive compounds .......... 34 1.8 Molecular docking for the investigation of bioactive compounds.................................... 35 1.9 Natural products from bryophytes: from basic biology to biotechnological applications 47 Chapter 2 ................................................................................................................................ 100 Screening of bryophytes extracts for biological activities of pharmaceutical and cosmetic interest ........................................................................................................................... 100 Chapter 3 ................................................................................................................................ 134 Collagenase and tyrosinase inhibitory effect of isolated constituents from the moss Polytrichum formosum ....................................................................................................................... 134 Chapter 4 ................................................................................................................................ 158 Extracts from the liverwort Bazzania trilobata with potential dermo-cosmetic properties ...... 158 General Discussion ................................................................................................................. 190 Conclusion .............................................................................................................................. 204 12 Chapter 1 Introduction 1.0 General Introduction Plants are a valuable source of a range of specialized metabolites that have significant medicinal value for drug development and cosmetics applications. One important group of plants are the bryophytes, which comprise mosses, liverworts, and hornworts. Although the phytochemistry of bryophytes is less explored than higher plants, they have proved to be an essential source of biologically active substances with original chemical structures. It highlights the economic importance of bryophytes in pharmaceutical and cosmetic applications. The investigation of new active ingredients such as plant extracts or individual molecules with significant roles in preventing skin aging, pigmentation, and inflammation is fundamental for the pharmaceutical and cosmetic industry. Therefore, this thesis aimed to explore a collection of bryophytes, including mosses and liverworts, as novel sources of bioactive extracts and natural products for cosmetics or pharmaceutical applications. The objectives of this study include the evaluation of bryophytes extracts with skin anti-aging, anti-pigmentation, and antiinflammatory activities. Antioxidant and antimicrobial activities were also investigated in selected active extracts. In order to investigate the bioactive compounds towards the target enzymes, the Target Binding® approach was applied for the most promising extracts. The active extracts were subjected to dereplication studies and isolation of candidate molecules for biological evaluation and structural elucidation. In this thesis, this first chapter provides a theoretical background of the subjects addressed in this Ph.D. study. The results are discussed in three different chapters. The second chapter introduces the screening of bryophytes extracts for biological activities of cosmetic and pharmaceutical interest. The third and fourth chapters focus on investigating the active plants selected from the previous biological screenings. The final part presents the overall discussion and conclusions of this thesis. 1.1 Bryophytes as a source of bioactive natural products Bryophytes are the second largest group of terrestrial plants and among the earliest plants to colonize land [1]. They are divided into Marchantiophyta (liverworts), Bryophyta (mosses), and Anthocerotophyta (hornworts) which comprise over 20 000 species [2]. Mosses and liverworts are the most predominant groups with around 13 000 and 7486 species described, respectively [3,4]. They grow in different ecological niches such as rocks, tree trunks, soil and are present on all continents. Bryophytes lack true vascular tissues in their leaves and do not 14 have roots, which characterize them with small and simple body structures [5]. However, bryophytes have developed biochemical adaptation strategies, producing specialized metabolites to survive in diverse ecosystems [6]. Despite their small size, which makes them difficult to collect and identify, bryophytes draw the attention of researchers due to their rare and new chemical constituents [7]. Natural products isolated from bryophytes, mainly terpenoids, flavonoids, (bis)bibenzyls, and lipids, have shown important biological activities (e.g. antimicrobial, antiviral, anti-inflammatory, and anticancer) and are reported in many scientific papers [7,8]. Therefore, chapter 1.9 (Paper 1) of this thesis summarizes the recently discovered compounds and novel biological activities reported from bryophytes in the last 10 years. In the third chapter of this thesis (Paper 3), the moss Polytrichum formosum has shown potential in vitro inhibitory activities on collagenase and tyrosinase, two cosmetic target enzymes associated respectively with skin aging and hyperpigmentation conditions (see chapter 1.2). The bioactive compounds of P. formosum were identified as ohioensins from the family of benzonaphthoxanthenones, only isolated from moss species. Ohioensin A is an aromatic compound isolated for the first time from the ethanol extract of Polytrichum ohioense (Polytrichaceae) that exhibited cytotoxicity against murine leukemia (PS) and breast (MCF-7) tumor cells lines at ED50 (effective dose) of 1.0 and 9.0 µg/mL, respectively [9]. Ohioensins have been isolated from Polytrichum species and show a wide range of biological activities (Table 1 and Figure 1). The biogenesis of ohioensins was suggested to include the condensation of o-hydroxycinnamate with hydroxylated phenanthrenes or 9,10-dihydrophenanthrenenes originated via acetate-malonate and shikimic acid pathways (Figure 2) [10]. Table 1. Overview of bioactive ohioensins. Origin Compound Bioactivity Ref. Polytrichum alpinum ohioensin A, ohioensin C, ohioensin F and ohioensin G Anti-tyrosine phosphatase 1B (PTP1B) [11] P. alpinum ohioensin F Anti-inflammatory [12] P. alpinum ohioensin F and ohioensin G Antioxidant [13] Polytrichum formosum ohioensin A Anti-collagenase and antityrosinase This study Origin Compound Bioactivity Ref. P. formosum nor-ohioensin D Anti-collagenase This study Polytrichum ohioense ohioensin A, ohioensin B, ohioensin C, ohioensin D and ohioensin E Cytotoxicity against human tumor cell lines [10] Polytrichum pallidisetum 1-O-methylohioensin B; 1-O-methyldihydroohioensin B and 1,14-di-Omethyldihydroohioensin B Cytotoxicity against human tumor cell lines [14] Figure 1. Chemical structures of bioactive ohioensins. 16 Figure 2. Suggested biogenesis of ohioensins, adapted from [10,15]. In the fourth chapter of this thesis (Paper 4), the liverwort Bazzania trilobata showed in vitro antioxidant, antimicrobial, collagenase, and tyrosinase inhibitory activities. The bioactive B. trilobata extracts showed that major constituents were phenolics (lignans, coumarins, and bisbibenzyls classes) and sesquiterpenoids (sesquiterpene caffeate). The most characteristic group of specialized metabolites from B. trilobata are the rare chlorinated bis-bibenzyls. Bazzanins are chlorinated macrocyclic bis-bibenzyls of the isoplagiochin C/D type isolated from the liverworts B. trilobata and Lepidozia incurvata [16–18]. An exception is bazzanin K that possesses a bibenzyl and a phenanthrene moiety [19]. Bazzanins A-J have been isolated from B. trilobata [16] along with bazzanin S [19]. Other chlorinated bis-bibenzyls derivatives from isoplagiochin were isolated from the species of liverworts Plagiochila sp., Herbertus sakuraii, and Mastigophora diclados [6,18]. Bazzanin B, bazzanin S, and the non-chlorinated bis- bibenzyls, isoplagiochin D, were reported with antifungal activity against phytopathogenic fungi (Figure 3) [20]. Figure 3. Antifungal isoplagiochin D and bazzanins from B. trilobata. Isoplagiochin biosynthesis was proposed to occur by the dimerization of monomeric bibenzyls precursors such as lunularin, which is found in most liverworts, through the phenylpropanoid pathway [21,22]. Moreover, to explore the biogenesis of chlorinated bis-bibenzyls, the chlorination of isoplagiochin C has been obtained in vitro with a chloroperoxidase enzyme from Caldariomyces fumago [23]. The chloroperoxidase type enzyme was later detected for the first time in bryophytes, namely in the liverwort B. trilobata [24]. Sesquiterpenoids are another important group of bioactive compounds widely found in Bazzania species. In particular, sesquiterpene caffeates are rare compounds only reported from the Bazzania genus [6]. These compounds have been reported with cytotoxic [25], superoxide anion release [26], and nitric oxide production [27] inhibitory activities. In brief, the biosynthetic pathways of specialized metabolites in bryophytes remain poorly understood. Both precursors of phenylpropanoid and terpenoid biosynthetic pathways are conserved over land plants [22,28,29]. The phenylpropanoid pathway gives rise to many bioactive compounds found in bryophytes, such as bibenzyls, bis-bibenzyls, and flavonoids. Furthermore, the terpenoid pathway generates the largest group of specialized metabolites from bryophytes. 1.1.1 Potential use of bryophytes in cosmetics Bioactive extracts from bryophytes have provided a new source of rare active ingredients for commercial applications. In 2018, the first moss-based cosmetic ingredient, MossCellTec No.1, launched by Mibelle AG Biochemistry won the gold innovation award for the best 18 ingredient at in-cosmetics® global based on a novel anti-aging target and a new ingredient source [30]. The active extract ingredient of Physcomitrella patens has been shown to improve nucleus health markers in aged skin cells (keratinocytes) and to enhance the resilience of the skin against environmental changes [31]. In 2021, the same company developed the MossCellTec™ Aloe ingredient with the active extract of the moss Aloina aloides with the promise to harmonize the skin’s moisture flow and reduce the volume and depth of wrinkles [32]. Moreover, extracts of the moss Sphagnum magellanicum have also applications in skincare products [33]. In addition, extracts from plants of the family Polytrichaceae were patented with skin anti-aging activities by promoting collagen and hyaluronic acid production and melanin inhibition [34]. 1.2 Target enzymes of pharmaceutical and cosmetic importance Skin is the largest organ of the human body consisting of three primary layers: the epidermis, the dermis, and the hypodermis (Figure 4) [35]. The skin has a vital role in maintaining homeostasis and protecting the body as a barrier against external factors. Intrinsic and extrinsic factors can affect the skin structure and physiology, accelerating the skin aging process [36,37]. Intrinsic aging is the natural progression of cell maturation. In contrast, extrinsic aging is associated with external elements such as the exposition to air pollution and sun radiation, smoking, dietary habits, and many others. The effect of ultraviolet (UV) radiation on the skin is referred to as photo-aging. It is the major stimulator associated with the overproduction of reactive oxygen species (ROS) and oxidative stress [38,39]. This leads to increased activation of extracellular matrix (ECM)-degrading proteases that contribute to the appearance of premature skin wrinkles [40]. Moreover, sun exposure and ROS increase the activation of the main enzyme of the melanin pathway leading to skin hyperpigmentation disorders [41,42]. Figure 4. Simplified schema of the human skin structure. Illustration adapted from the reference [43]. 1.2.1 Tyrosinase- the main enzyme in skin pigmentation The production of melanin pigments occurs in melanosomes, the organelles present in the melanocytes, situated on the basal layer of the epidermis (Figure 4) [44]. Melanosomes are transported to keratinocytes resulting in the pigmentation of the skin [45]. Tyrosinase is the rate-limiting enzyme in melanogenesis or melanin biosynthesis. It is a type 3 copper-containing glycoprotein enzyme with two copper ions into the active site and located in the membrane of the melanosome [46]. Tyrosinase has monophenolase and diphenolase activities. The first activity catalyzes the hydroxylation of monophenols (L-tyrosine) into o‐diphenols (L-DOPA), while the second oxidizes o-diphenols into o-quinone (DOPAquinones) derivatives, which are precursors of melanin pigments. The catalytic properties of tyrosinase are linked to the oxidation states of the active site that can be present in deoxy-, oxy-, met- and deact-tyrosinase alternative forms [47]. Pheomelanin and eumelanin are types of melanin found in human skin; however, eumelanin (brown eumelanin and black eumelanin) is the most common. These pigments are essential for human skin protection against the harmful effects of UV radiation [41]. However, the overproduction and accumulation of melanin are associated with various hyperpigmentation disorders, e.g., melasma, ephelides (freckles), solar lentigines (age spots) [48]. Therefore, tyrosinase became the most explored target for the development of skinwhitening agents. 20 1.2.2 Collagenase and elastase- proteinases responsible for the regulation of dermal matrix proteins Fibroblasts are the cells present in the dermal connective tissue and are responsible for the synthesis of components of the extracellular matrix such as collagen and elastin fibers (Figure 4) [49]. Collagen and elastin are important structural proteins that provide firmness, tensile strength, flexibility, and elasticity of the skin. Under normal conditions, these proteins are synthesized and degraded to keep the homeostasis of the cell and the remodelling process of the ECM [50]. However, excessive and uncontrolled protein breakdowns are related to diseases and the emergence of premature aging. Collagen, type I, is the main structural protein of the skin, and its depletion is one of the leading causes of wrinkle formation [51]. Collagenases are proteinases responsible for cleaving collagen. They belong to the group of matrix metalloproteinases (MMPs), a family of zincdependent endopeptidases, which comprises MMP-1 (collagenase-1), MMP-8 (collagenase-2), and MMP-13 (collagenase-3) [52]. The dysregulation of collagenases is involved in a wide range of pathological conditions such as inflammation, rheumatoid arthritis, cardiovascular diseases, and cancer [53,54]. MMPs are secreted from keratinocytes, fibroblasts, and cells of the immune system [55,56]. In the skin, these cells release mainly MMP-1 that breaks down mostly fibrillar collagen type I and III [40,55,57]. The production of MMPs is increased by ROS via the mitogen-activated protein kinase (MAP-kinase) pathway inducing a signaling cascade and activation of main transcription factors (e.g. AP-1 and NF-κB) that regulate the MMPs gene expression [40]. Elastin, another important protein found within the ECM, promotes elasticity and resilience to the skin; however, its active degradation can contribute to skin aging and diseases [58]. Elastases degrade elastin fibers and are classified in many families such as serine, MMPs, or cysteine proteases. Serine proteases such as the human leukocyte elastase (HLE), also called neutrophil elastase, are a potent protease widely involved in the degradation of elastin and other matrix extracellular components (e.g. decorin, collagen) [59,60]. Moreover, neutrophil elastase is secreted by neutrophils in the acute inflammation phase after UV irradiation [58,60]. Thereby, one of the key targets in the cosmetic industry is the discovery of natural inhibitors of aging-related enzymes. 1.2.3 An overview of target inhibitors In general, tyrosinase inhibitors are characterized by their similar structure to that of the natural substrates of tyrosinase (L-tyrosine, monophenol and L-DOPA, diphenol) [61]. Hydroquinone, the most popular skin-whitening agent, was described to act as both as tyrosinase inhibitor and substrate [62]. However, there are safety controversies over its use [63]. In Europe and many other countries, hydroquinone has been banned due to reported side effects [64]. Thus, an alternative source of tyrosinase inhibitors from natural sources such as bacteria, plants, and fungi has been explored [61]. Recently, phytochemicals ingredients have gained greater attention in the cosmetic market. The use of naturally derived ingredients has become more popular and fashionable among consumers. Consequently, there is a growing demand for natural ingredients, especially in the European cosmetic market [65]. Some of the most common tyrosinase inhibitors are shown in Table 2. Kojic acid is a lightening skincare ingredient in cosmetic products and treats skin hyperpigmentation disorders [66]. However, kojic acid has been reported with poor efficacy, low stability, and toxicity [66–68]. Arbutin is a glycosylated hydroquinone produced by many plants with tyrosinase inhibitory activity [69]. It is considered a safer alternative compared to hydroquinone [70]. Polyphenolic compounds have been reported as a large class of tyrosinase inhibitors [61,71] (e.g. apigenin, EGCG, glabridin, quercetin; Table 2). These compounds also play an important role as inhibitors of collagenase and elastase activity [72]. For instance, flavonoids are effective metal chelating agents that can chelate the zinc ion in the active site of MMPs, inhibiting their activity [73]. Several active constituent-rich plant extracts have skin lightening and anti-aging potential. For example, the extracts from licorice roots have skin-lightening activity in which glabridin was reported as its main active compound (Table 2) [74]. Green tea extract contains active polyphenolic compounds, including the major component epigallocatechin gallate (EGCG, Table 2) that is widely applied to anti-aging cosmetic products [75]. Hence, the constituents from the active plant extracts can enhance the desired biological activity by additive and synergetic effects or as individual active components. In the literature, few studies report the inhibitory effects of bryophytes extracts or individual metabolites on these target enzymes [76]. 22 Table 2. Natural source of tyrosinase, collagenase and elastase inhibitors. Name Origin Target Chemical structure Ref. Apigenin Produced in many Tyrosinase, [61,77– vegetables (parsley, collagenase 79] Tyrosinase [61,80,81] Green tea (Camellia Collagenase, [75,82,83] sinensis L.) elastase, celery, onions), fruits (oranges), herbs (chamomile, thyme, oregano, basil), tea Arbutin Arctostaphylos uvaursi (L.) Spreng. (Ericaceae); Bergenia crassifolia (L.) Fritsch Epigallocatechin gallate (EGCG) tyrosinase Glabridin Licorice Tyrosinase [61,74] Tyrosinase [66,84,85] (Glycyrrhiza glabla L.) Kojic acid Fungal metabolite produced by many species of Aspergillus and Penicillium Name Origin Target Quercetin Produced in many Tyrosinase, fruits, vegetables, collagenase Chemical structure Ref. [78,79,86] tea, black tea, onions, apples 1.3 Antioxidants Reactive oxygen species (ROS) include free radicals (atoms or molecules with one or more unpaired electrons in their atomic orbital) such as hydroxyl radical (OH·) and superoxide (O2·-) as well as non-radical species such as hydrogen peroxide (H2O2). ROS are byproducts of oxygen metabolism and are important for normal cellular function. However, increased levels of ROS can cause cell dysregulation and damage to lipids, proteins, and nucleic acids [87]. Free radicals are highly reactive and unstable substances that tend to capture electrons from other molecules leading to a chain reaction of oxidative propagation [87]. Oxidative stress is the result of the imbalance between excess ROS production and the impaired cell antioxidant defense. ROS may be generated by endogenous sources (from biochemical reactions) and exogenous stimulated sources (agents including environmental pollutants, sun radiation, smoke, diet, etc.) [88]. Thus, oxidative stress lead to cell and tissue damage and is related to many diseases such as neurodegenerative diseases [89], cancer [90], inflammation [91], and many others. Besides that, oxidative stress plays a major role in the skin aging process. Oxidative stress affects collagen synthesis in human dermal fibroblasts [92]. Moreover, ROS activate skin ECM-degrading proteases and pigmentation-related enzymes as described in chapter 1.2. Cells contain several defense mechanisms known as antioxidants that neutralize free radicals and prevent from its harmful effects. Key components from the antioxidant defense are enzymes (superoxide dismutase, catalase, and glutathione peroxidase) and non-enzymatic molecules (e.g., lipoic acid, uric acid, glutathione) [93]. Antioxidant compounds react with free radicals through different chemical mechanisms like donating electrons (single electron transfer mechanisms (SET)) or hydrogen (atom transfer mechanisms (HAT)) forming stable compounds and consequently interrupting the oxidative 24 chain reaction [94]. Antioxidants also can chelate metal ions, such as iron and copper, preventing the production of free radicals in the cells. Antioxidant molecules are usually obtained by diet or nutritional supplementation. Plants comprise a great diversity of bioactive compounds acting as antioxidants agents that include, for example, vitamins (A, C, E), carotenoids, and phenolic compounds. Phenolic compounds are the major antioxidants from plants; in general, their antioxidant activity is directly related to their chemical structures such as by number and position of hydroxyl groups in their aromatic rings as well as double bond conjugations and resonance effects [95–97]. Plant-derived antioxidants have been applied with distinct biological and therapeutic activities. Plant-derived extracts from green tea, grapes, berries, and various other plants along with pure natural antioxidants (e.g., quercetin, resveratrol, curcumin, etc.) are popularly applied as active ingredients in cosmetics to improve skin appearance [98]. 1.4 Natural antimicrobials in cosmetics In the cosmetic industry, preservatives are added to cosmetic formulations to prevent the growth of microorganisms, keeping away from product spoilage, then increasing their shelf life and ensuring their safety to consumers. The safety of cosmetic products is evaluated through the international cosmetic challenge test, also known as the preservative efficacy test regulated by ISO (International Organization for Standardization) standards 11930:2019 [99]. Briefly, the growth of microorganisms is monitored at predefined time intervals for 28 days by inoculating the cosmetic product with selected strains of pathogenic microorganisms. The strains include Staphylococcus aureus (gram-positive bacteria), Pseudomonas aeruginosa (gram-negative bacteria), Escherichia coli (gram-negative bacteria), Candida albicans (fungus), and Aspergillus brasiliensis (fungus) [100]. Common cosmetic preservatives are antibacterial and antifungal agents including formaldehyde releasers (e.g. imidazolidinyl urea, diazolidinyl urea), halogenated preservatives (e.g. chloroacetamide), organic acids (e.g. benzoic acid, sorbic acid), quaternary ammonium salts (e.g. benzalkonium chloride), isothiazolinones (e.g. methylisothiazolinone), and alcohols and their derivatives (e.g. phenoxyethanol, methylparaben, ethylparaben) [101]. Conventional preservatives are associated with undesirable side effects and risks to human health, such as allergy and irritations, and might even exhibit toxicity, increasing consumer concern [101]. There are alternatives to replace preservatives in cosmetics that depend on specific characteristics of product formulation. These may include using dry formulas, highly acidic or alkaline pH, oil mixtures, alcohol content, enzymes (e.g. glucose oxidase and lactoperoxidase), and proper product packing [101]. Moreover, the antimicrobial potential of natural bioactive substances such as essential oils, natural extracts, and derived active constituents is being explored to minimize synthetic preservatives in cosmetic products. These substances are known as preservative boosters or self-preserving ingredients and are usually multifunctional agents presenting bioactive properties (e.g. antioxidant, anti-inflammatory, anti-aging) [102]. Several studies have reported the efficacy of plant-derived metabolites as antimicrobials. Some examples of ingredients usually included in cosmetic products are essential oils such as thyme oil (active phenolic components) [103], tea tree oil (terpene-rich) [104,105], and rosemary oil (terpene-rich) [106,107] as well as plant extracts such as grapefruit seed extracts (mix of polyphenolics, tocopherols, citric acid, etc.) [108]. The mechanisms of action of antimicrobial plant-derived ingredients are many. They can include disruption and depolarization of cell membrane, inhibition of cell wall, protein and nucleic acid synthesis, and inhibition of metabolic pathways. [109–111]. 1.5 Overview of skin inflammation and inhibitory drug targets Skin inflammation can occur due to an immune response triggered by different factors such as infections (bacterial, fungal, and viral), allergic reactions (pollen, medications, food), physical injuries (UV radiation), chemical compounds (ROS), and disorders of the immune system. Inflammation may be acute or chronic based on the duration and the response of the body to the damage. Skin acute inflammation is a short-term response induced by specific stimuli such as UV radiation, microbial infection, and allergens [112]. Whereas chronic conditions are longterm responses as revealed by many common skin diseases such as psoriasis (red and itchy skin patches), atopic dermatitis (eczema; red and itchy skin), and seborrheic dermatitis (skin flakesdandruff) [113]. Inflammation is a complex process involving different cells (e.g. mast cells, phagocytes, and granulocytes) and many signalling molecules (e.g. cytokines, chemokines, and growth factors) [114]. The acute inflammatory response starts after specific stimuli mediated by immune cells, cytokines, and other molecules. This activates important signals promoting the migration of cells from innate immunity to the area of inflammation. In chronic skin inflammation, primary immune cells are involved in the increased expression and secretion of pro-inflammatory cytokines (e.g. interferons and interleukins) as well as other molecules, which cause significant tissue destruction and damage to the skin [115]. 26 The most common drugs used in the treatment of skin inflammation diseases are topical or oral corticosteroids, however, they can cause adverse side effects on the skin (e.g. atrophy, striae, rosacea-like dermatitis, perioral dermatitis) [116]. Another critical main adverse effect of corticosteroids is the depletion of the immune system that can lead to an increased risk of infections [117,118]. Therefore, researchers are focusing on new sources of compounds that have potent anti-inflammatory properties. Some examples of plant-derived anti-inflammatory compounds are shown in Table 3. Compounds that can block critical inflammatory mediators are essential in drug development for the treatment of inflammatory diseases [56,119]. Table 3. Plants with anti-inflammatory properties. Sources Constituents/classes Targets Reference Curcuma longa L. Curcumin Cytokines (TNF-α); [120] (Zingiberaceae) (polyphenol) interleukines (IL-1, IL-6); enzymes (Rhizomes) (MMP-1 and MMP-3; iNOS), etc. Many sources like Resveratrol Nuclear factor kappa mulberries, peanuts, (polyphenol) B (NF-kB), MAPK, grapes [121] iNOS, etc. Vegetables and edible Quercetin and Pro-inflammatory fruits like apples, nuts, derivatives cytokines in atopic herbs, onions, (polyphenol) dermatitis EGCG NF-kB, neutrophil [122] grapevines Green tea (polyphenol) [123] elastase, proinflammatory cytokines, etc. In chapter 2 (Paper 2), several extracts of bryophytes were screened for anti-inflammatory activities assessed by their ability to inhibit induced-nitric oxide (NO) production in macrophage cells. NO, a small gaseous messenger, is an important pro-inflammatory mediator in the Toll-like receptor (TLR) signaling pathway [119]. TLRs are transmembrane proteins that play a key role in both innate and adaptive immune responses [119,124]. They recognize pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs) inducing the production of inflammatory mediators [119]. NO synthases (neuronal (nNOS), endothelial (eNOS), and inducible (iNOS)) synthesize NO from L-arginine and oxygen by an oxidoreductase reaction that gives L-citrulline and NO [125]. NO plays an essential function in various physiological processes, however, increased levels are involved in inflammatory disorders such as skin diseases (e.g. psoriasis, atopic dermatitis, allergic dermatitis) [126]. The inflammatory process can also induce and contribute to skin aging [79]. Additionally, inflammatory events are associated with the activation of extracellular matrix (ECM)degrading proteases (see chapter 1.2.2) [56]. Previous studies have shown that MMP-1 upregulation is induced by several inflammatory cytokines such as TNF-α (tumor necrosis factoralpha), interleukins, and many others mediators [127]. Moreover, neutrophil elastases are released from neutrophils during inflammation [58,60]. 1.6 Tools for the investigation of natural products 1.6.1 Liquid chromatography Liquid chromatography (LC) is a technique commonly used to separate the constituents of a mixture for either identification, quantification, or purification. It is a technique with a wide range of applications, such as for the analysis and quality control of food, pharmaceuticals, cosmetics, environmental samples, and many others, including studies of metabolites from plants, microorganisms, animals, and humans. In general, the separation of constituents occurs based on the interactions of the sample with two phases, one stationary and the other mobile [128]. Liquid chromatography is classified into liquid-liquid chromatography (LLC) and liquid-solid chromatography (LSC) [129]. In LSC, the mobile phase is a liquid and the stationary phase is typically a solid held in place in a column or a plane (such as a plate of glass, plastic, or a sheet of paper). In liquid-solid chromatography, there are two types of separation systems available: normal and reverse-phase. In normal-phase chromatography, the sample constituents are carried by the mobile phase through the column and are retained on the polar stationary phase by adsorption (such as silica gel or polar-bonded silica gel) and nonpolar mobile phases are employed [129]. The polar compounds with more affinity to the stationary phase are eluted slowly and non-polar compounds are eluted first. Opposite, the reverse phase chromatography includes the use of hydrophobic stationary phases (such as 28 octadecylsilyl groups (ODS groups or C18 groups), phenyl) and polar mobile phases in which the most polar compounds are eluted first [129]. Reverse-phase chromatography has become more used than the normal phase because it has a wider coverage range for separating most organic compounds [130]. High-performance liquid chromatography (HPLC) and ultra-high-performance liquid chromatography (UHPLC) are advanced types of LC that can significantly improve the speed, resolution, and sensitivity of separation. In HPLC and UHPLC, high pressure is applied from a pump to push solvents through the column generating a specific flow rate of the mobile phase, which reduces the time for the separation of individual components. UHPLC operates at higher pressure (15 000-22 000 psi) and consists of columns filled with lower particles size (≤ 2 µm) allowing more efficient and faster separation than in HPLC systems [131]. HPLC combined with large columns and high flow rates are applied for the purification of target compounds. It can be classed in semi-preparative (< 0.5 g), preparative (> 0.5 g) and industrial (g to kg) liquid chromatography depending on the amount of compound needed [132,133]. The basic LC system consists of the following instrumentation: the mobile phase reservoir, the pump, sample injector, column (packed with stationary phase), detector, computer data collection station, and waste as illustrated in Figure 5 [134]. Figure 5. Basic high-performance liquid chromatography scheme system. Adapted from the reference [134]. The eluted samples can be observed through various detectors, which are selected based on the nature of the constituents of the sample. The most applied detectors are the ultraviolet or visible light (UV-vis) detectors with fixed or variable wavelengths such as the diode array detectors (DAD, PDA: Photodiode Array Detector) that enable the detection of various wavelengths of light simultaneously or mass spectrometry (see chapter 1.6.2) [131]. For that, the components should be capable of absorbing light in the UV-vis region (from 190-600 nm); a characteristic of certain molecules or parts of molecules that contains chromophores [135]. UV-vis light that hits the chromophore can thus be absorbed through electron transition by exciting an electron from its ground state into an excited state. The absorption peaks are characteristic of compounds with conjugated double bonds (conjugated systems) and associated heteroatoms present; aromatic compounds mostly absorb strongly around 260 nm [135]. UV-vis spectroscopy is a non-destructive method, which allows after its detection the collection or discard of the eluate. 1.6.2 Mass spectrometry Mass spectrometry (MS) is an analytical tool also usually coupled to separation techniques such as LC for measuring the mass-to-charge ratio (m/z) of a compound or its fragments. Mass spectrometry instrumentation usually consists of four principal components including a sample inlet, an ionization source, a mass analyzer, and an ion detection system as illustrated in Figure 6 [136]. The sample is introduced into the ion source through the sample inlet and converted to ions and separated according to their m/z by subjecting them to electrostatic fields in the mass analyzer. The result is a mass spectrum, which is a plot of relative abundance against the mass-to-charge ratio, used to determine the molecular weight or structural information of a compound. Figure 6. Basic components of a mass spectrometer instrument. Adapted from the reference [136]. 30 Electrospray ionization (ESI) is a common soft ionization technique (little fragmentation) suitable for high to medium hydrophilic compounds and large biomolecules. It produces ions by applying a high voltage, which can be either negative or positive, to a liquid sample to create a fine spray of charged droplets [137]. The mass analyzer is responsible for the separation of the charged fragments based on their m/z. Many mass analyzers are currently available that can differentiate in accuracy, resolution, and sensitivity [138]. Quadrupole mass analyzer consists of four parallel cylindrical metal rods where a radio frequency voltage is applied between one pair of opposing rods. The ions passing through the analyzer are separated based on their stable flight trajectories in an oscillating electrical field. Only ions of a certain mass-to-charge ratio reach the detector and the other ions will collide with the rods [138]. Quadrupoles can be placed in tandem, known as MS/MS or MS2, commonly set up as a triple quadrupole; the first (Q1) and third (Q3) quadrupoles are mass analyzers (mass filters) and the middle one acts as a collision cell (q2). In the collision cell, the selected parent ion(s) from Q1 is fragmented in the presence of neutral gas (Ar, He, or N2) by collision-induced dissociation (CID), and then the produced ions travel to Q3 for m/z selection [138,139]. This process enables the identification of ion fragmentations applied for the structural elucidation of a compound. The quadrupole mass analyzer can be combined with time of flight (TOF) technology known as quadrupole time of flight (Q-TOF) resulting in highresolution mass spectrometers [138]. The Q1 selects specific ions and Q2 can act as in either collision cell or without further ion fragmentations. In TOF, a controlled electric field is applied to accelerate ions with the same kinetic energy through a flight tube for mass separation. The time of the ion to reach the detector is related to its mass; the ion mass-to-charge ratio is determined through a time of flight measurement [140]. Mass spectrometry coupled to chromatography techniques represents an important tool for natural products investigation. It is applied for the dereplication, i.e. identification of known compounds and the discovery of novel compounds. The chemical formula of a compound can be determined based on accurate mass and isotope pattern distribution along with tandem MS (MS/MS) fragmentation for structural elucidation [141,142]. Natural products databases are critical for the dereplication of natural products and numerous are available including Reaxys, Scifinder, Dictionary of Natural Products (DNP), Massbank, MS-DIAL, and Global Natural Product Social Molecular Networking (GNPS) to name a few [143,144]. There are available many commercial (from MS vendors machine) and open-source software such as MZmine, MS-DIAL, GNPS, and many others for mass spectrometry data processing, interpretation, and compounds annotation [145–147]. Computational MS approaches are also applied for tentative annotation of compounds that is crucial when no reference mass spectra match with an experimental derived spectrum. Many available computational approaches combine different algorithms and rules for fragment prediction and comparison against experimental mass spectra data. Computational approaches include combinatorial fragmentation (MetFrag and MAGMA), molecular spectral fingerprinting (ChemDistiller and CSI:FingerID), spectra similarity (CFM-ID) and hydrogen rearrangement rules (MS-FINDER) [142,148,149]. In this Ph.D. study (Paper 4), the in silico fragmentation tool MS-FINDER was applied for the dereplication of plant extracts. MS-Finder is a free software available for the identification of compounds and structure elucidation that comprises comparison and ranking of computergenerated MS/MS fragmentation spectra to experimental MS/MS data of a target compound [150]. The molecular formulas of precursor ions are determined from accurate mass, isotope ratio, and product ion formation that are retrieved from metabolome databases. The structure is ranked by specific rules including bond dissociation energies, mass accuracies, fragment linkages, and nine hydrogen rearrangement rules [150,151]. 1.6.3 Nuclear magnetic resonance (NMR) spectroscopy Nuclear Magnetic resonance (NMR) spectroscopy is a preeminent technique applied for the structure determination of organic compounds and is routinely employed in dereplication strategies [152]. It is based on the absorption of electromagnetic radiation in the radiofrequency region by the nuclei of the atoms to promote transitions between nuclear energy levels. The theory behind NMR is that many nuclei have spin (nuclei that contain an odd number of protons and/or neutrons such as 1H and 13C show the magnetic properties required for NMR) and since the nuclei are electrically charged, they generate a magnetic field. The spins are randomly oriented in the absence of an external magnetic field. Upon exposure to a magnetic field, the nuclei align themselves either with or against the applied field. Nuclei subjected to a radiofrequency pulse will absorb energy (excited state) and when the spin returns to its base level (relaxation), energy is emitted as an observed free induction decay (FID) [152]. The FIDs are deconvoluted by Fourier transformation to generate the NMR spectra, which is displayed as a plot of the intensity of NMR signals versus the frequency (chemical shifts). The NMR basic instrumentation comprises a superconducting magnet, which produces a homogeneous magnetic field where samples are exposed to radio waves, a spectrometer to transmits and receives the radio-frequency waves, and a computer for instrumental control and data processing (Figure 7) [152,153]. 32 Figure 7. Simplified scheme of an NMR spectrometer. Adapted from the reference [153]. The structure of a compound can be assigned by the interpretation of the NMR spectra by the analysis of chemical shift, integration, spin multiplicity, and coupling constants parameters [152]. Briefly, chemical shift is the difference between the resonant frequency of the observed nucleus relative to an internal reference compound (e.g., tetramethylsilane (TMS) and deuterochloroform) in a magnetic field. Integration corresponds to the relative number of hydrogens at a given shift and multiplicity shows the number of neighbouring hydrogens to the hydrogen peak. The coupling constant (J) is a measure of the distance between the peaks in a multiplet, which provides important information on the connectivity of chemical bonds [152]. Structural elucidation of complex molecules and new compounds can be determined using twodimensional (2D) NMR spectroscopy, in which signals are represented as a function of two frequency axes rather than one. 2D NMR spectroscopy has the advantage of distinguishing signals that are superimposed in 1D NMR spectroscopy. In general, 2D NMR can be divided into two types, homonuclear and heteronuclear [152,154]. Homonuclear through-bond correlation methods include correlation spectroscopy (COSY) that provides information about homonuclear correlations between 1H atoms separated by three bonds and associated carbons in the ¹H NMR spectra. There are many variants of COSY such as the improved DoubleQuantum Filtered COSY (DQF-COSY) with a higher resolution spectrum. Heteronuclear through-bond correlation methods provide signals based on the correlation of two different nuclei being usually proton and carbon (1H-13C). Common heteronuclear experiments include heteronuclear single quantum correlation (HSQC) and heteronuclear multiple bond correlation (HMBC) that provides correlation of nuclei separated by two or more chemical bonds. In addition, there are methods based on space correlation where it reveals the nuclei close to each other in space, separated by less than 5Å. These methods include nuclear Overhauser effect spectroscopy (NOESY) and Rotating-frame nuclear Overhauser effect spectroscopy (ROESY) that are useful for determining the stereochemistry of a molecule [154]. 1.7 Affinity-based screening approach for the identification of bioactive compounds The classical bioassay-guided fractionation approach is usually used to identify bioactive compounds in complex plant extracts. This procedure relies on successive rounds of extract fractionation and biological activity screening whereby only active fractions are selected in each round until the isolation of active molecules [155]. This technique is labor-intense, timeconsuming, and has a lower detection to low concentrated compounds. Therefore, other methods have been developed to identify bioactive natural products from complex extracts. The affinity-based approach is an efficient way to identify possible active constituents (ligands) to macromolecular targets based on their ligand-target affinity [156]. In chapters 3 and 4, the affinity-based approach was applied to determine which constituents from the active plant extracts were involved with the observed in vitro biological activities. The Target Binding® is a patented affinity-based method that enables the pre-screening of candidate inhibitors of enzymes of interest [157,158] (Figure 8). It consists of a binding step where the desired target is incubated with a complex sample allowing the formation of targetligand complexes. Subsequently, several washing steps eliminate the compounds that were not bound. The release of the ligands from the target is achieved by enzyme denaturation. The original extract and the sample containing the specific target’s ligands are analyzed by liquid chromatography coupled with mass spectrometry (LC-MS). The relative affinities between ligands and target are calculated based on the quantity associated with each ligand peak (area) in the chromatograms. The advantage of this technique is that the further isolation and biological screening analysis are limited to the compounds that show specific affinity to the target. 34 Figure 8. Target binding® workflow. Illustration by Aleksander Salwinski. 1.8 Molecular docking for the investigation of bioactive compounds Molecular docking is a method applied for the prediction of the interactions between a ligand and a biological target (e.g. protein) obtained by computational methods. Docking is an important tool frequently used in computer-aided drug design (CADD) approaches to assist in the discovery, design, and analysis of drugs and bioactive molecules [159]. The method includes the prediction of the conformation and orientation of a ligand relative to the targetbinding site, described as binding modes (poses), and the docking score with the estimated binding free energy and binding affinity of the ligand-protein interactions. Binding energy estimation between a ligand and its target is influenced by intermolecular interactions (e.g. hydrogen bonding, Van der Waals forces, electrostatic and hydrophobic interactions), desolvation, and entropic effects [159]. Various docking software are currently available applying different search algorithms and scoring functions for molecular docking studies (e.g. AutoDock, AutoDock Vina, DOCK, GOLD, FlexX, GLIDE, RDOCK) [160]. In Chapter 3 (paper 3), the mode of action of the bioactive compounds (inhibitors) against the 3D structures of the target enzymes collagenase from Clostridium hytolyticum and tyrosinase from Agaricus bisporus was investigated by docking studies. Clostridial collagenase and mushroom tyrosinase are routinely used for in vitro and in silico studies of potential inhibitors of these target enzymes [161–163]. Therefore, the crystal structures of collagenase (PDB ID 2Y6I) and tyrosinase (PDB ID 2Y9W) were obtained from RCSB protein data bank (http://www.rcsb.org). The structure of collagenase (Collagenase G) was obtained cocrystallized with isoamylphosphonyl-Gly-Pro-Ala. The structure of tyrosinase was found in the protein database as apoenzyme. Therefore, the known inhibitor EGCG was selected as a docking reference to generate the parameters that would be applied in the molecular docking trials. Clostridial collagenases are classified into class I (ColG) and class II (ColH) [164]. They are multi-domain enzymes composed of an N-terminal domain with the catalytic zinc, polycystic kidney disease (PKD)-like domains, collagen-binding domains (CBD) and pre-domain containing the export signal [165]. In this study, chain A containing the catalytic Zn2+ within the peptidase domain was used for the molecular docking. Crystallographic studies of mushroom tyrosinase revealed that the enzyme exhibits four protein chains (A, B, C and D) that corresponds to the H2L2 tetramer [166]. The H subunit was considered to be responsible for catalytic activity of enzyme that contains a binuclear copperbinding site each coordinate by three histidine residues while the function of the L subunit is yet unknown. In this study, chain A from the H-subunit was selected for molecular docking. The molecular modeling procedures combine the selection of 3D structure of target proteins, preparation of protein and ligand, characterization of the binding site, automated docking of the ligand into the binding site, and the evaluation of the strength of ligand-target interaction. The most plausible binding mode of the bioactive compounds could be revealed through the analysis of binding energy values (lowest binding energy), the number of formed hydrogen bonds between the ligands and target enzymes, and the reproducibility of the binding mode. In the current study, the software Autodock vina [167] was employed for the prediction of the interactions of the bioactive compounds within the binding site of the target enzymes. Autodock vina is a popular docking tool of flexible-rigid and flexible-flexible docking. 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I further contributed to the outline and design of the manuscript. This is an ‘Original Manuscript’ of an article published by Taylor & Francis Group in Critical Reviews in Plant Sciences on 07 Jun 2021, available online: https://doi.org/10.1080/07352689.2021.1911034 Natural Products from Bryophytes: From Basic Biology to Biotechnological Applications Armin Horn, 3Arnaud Pascal*, 4,5Isidora Lončarević*, 6Raíssa Volpatto Marques*, 5,6Yi Lu*, 7Sissi Miguel, 7Frederic Bourgaud, 5,8Margrét Thorsteinsdóttir, 4Nils Cronberg, 1,2Jörg D. Becker, 3,9Ralf Reski, 6Henrik T. Simonsen 1,2 *these Authors contributed equally Affiliation: 1Instituto Gulbenkian de Ciência, R. Q.ta Grande 6, 2780-156 Oeiras, Portugal; [email protected]; [email protected] 2ITQB NOVA - Instituto de Tecnologia Química e Biológica António Xavier, Av. da República, 2780-157 Oeiras, Portugal 3Plant Biotechnology, Faculty of Biology, University of Freiburg, Schaenzlestr. 1, 79104 Freiburg, Germany; [email protected]; [email protected] 4Biodiversity, Department of Biology, Lund University, Ecology Building, Sölvegatan 37, 22362, Lund, Sweden; [email protected]; [email protected] 5ArcticMass, Sturlugata 8, IS-101, Reykjavik, Iceland 6 Department of Biotechnology and Biomedicine, Technical University of Denmark, Søltofts Plads 223, 2800 Kongens Lyngby, Denmark; [email protected]; [email protected]; [email protected] 7Plant Advanced Technologies, 19 Avenue de la Forêt de Haye, 54500 Vandoeuvre, France; [email protected]; [email protected] 8 Faculty of Pharmaceutical Sciences, University of Iceland, Hagi, Hofsvallagata 53, 107 Reykjavik, Iceland; [email protected] 9Signalling Research Centres BIOSS and CIBSS, University of Freiburg, Schaenzlestr. 18, 79104 Freiburg, Germany Abstract Natural products from plants have served mankind in a wide range of applications, such as medicines, perfumes, or flavoring agents. For this reason, synthesis, regulation and function of plant-derived chemicals as well as the evolution of metabolic diversity has attracted researchers all around the world. In particular, vascular plants have been subject to such analyses due to prevalent characteristics such as appearance, fragrance, and ecological settings. In contrast, bryophytes, constituting the second largest group of plants in terms of species number, have been mostly overlooked in this regard, potentially due to their seemingly tiny, simple and obscure nature. However, the identification of highly interesting chemicals from bryophytes with potential for biotechnological exploitation is changing this perception. Bryophytes offer a high degree of biochemical complexity, as a consequence of their ecological and genetic diversification, which enable them to prosper in various, often very harsh habitats. The number of bioactive compounds isolated from bryophytes is growing rapidly. 48 The rapidly increasing wealth of bryophyte genetics opens doors to functional and comparative genomics approaches, including disentangling of the biosynthesis of potentially interesting chemicals, mining for novel gene families and tracing the evolutionary history of metabolic pathways. Throughout the last decades, the moss Physcomitrella (Physcomitrium patens) has moved from being a model plant together with Marchantia polymorpha in fundamental biology into an attractive host for the production of biotechnologically relevant compounds such as biopharmaceuticals. In the future, bryophytes like the moss P. patens might also be attractive candidates for the production of novel bryophyte-derived chemicals of commercial interest. This review provides a comprehensive overview of natural product research in bryophytes from different perspectives together with biotechnological advances throughout the last decade. Keywords: Bryophytes, Physcomitrella patens, Natural Products, Plant Biotechnology, Industrial Biotechnology 1. Introduction Bryophytes are the closest modern relatives to the ancestors of the first plants that succeeded to adapt to life on land approximately 470 to 515 million years ago (Morris et al., 2018). They have diversified early into three distinct extant phyla: Marchantiophyta (liverworts), Bryophyta (mosses) and Anthocerotophyta (hornworts). The most recent phylogenomic analyses provides evidence for monophyly of bryophytes, (Harris et al., 2020), with mosses and liverworts as sister groups (setaphyte hypothesis), separate from hornworts (which lack seta) (Renzagllia et al., 2018). Like all land plants (embryophytes), bryophytes have a life cycle with alternating generations. In contrast to other embryophytes, whose diploid sporophyte generation is dominant, in bryophytes, the haploid gametophyte generation is the dominant and persevering stage, whereas the unbranched sporophyte generation is diploid and short-lived (Horst and Reski, 2016). Bryophytes can be found in almost all climatic regions on all continents, where they are important components of many terrestrial ecosystems. At regional level, bryophytes are often most species-rich in cool and humid habitats (Ignatov, 2004; Prendergast et al., 1993). This is probably a consequence of the poikilohydric nature of bryophytes, meaning that they have a poor capacity to regulate internal water content and thus are passively dependent on ambient water availability. They also need water for reproduction, because water enables the motile sperm to swim to the egg cell. Bryophytes are small-sized and morphologically simple but chemically complex (Asakawa et al., 2013). They are rarely consumed by animals (Gerson, 1982), which is likely due to specific chemical constituents that exhibit protective effects. Ricciocarpin, a sesquiternenoid isolated from the liverwort Ricciocarpos natans has molluscicidal activity against the freshwater snail Biomphalaria glabrata (Asakawa and Ludwiczuk, 2018). Acetylenic oxylipins extracted from the moss Dicranum scoparium showed antifeeding activity again herbivorous slugs (Rempt and Pohnert, 2010). Crude extracts of some bryophytes have already been utilized by ancient tribes as medicine due to their beneficial chemical profile (Flowers, 1957; Sabovljević et al., 2016). Several bryophyte species have been used in Chinese traditional medicine, Marchantia polymorpha (DiFuPing) is used for external ailments such as burns and cuts. Sphagnum teres is used for eye diseases and skin irritation. Rhodobryum giganteum (HuiXinCao) is used for minor heart problems (Harris, 2008). Different biologically active chemicals have been described with antimicrobial (Neomarchantins A and B, and Marchantin C), antifungal (Plagiochin E, Viridiflorol), anticancer (Marchantin A, Porellacetals A-D) , antibacterial (mastigophorene C , herbertene-1,2-diol and Sacculatal), and/or antiviral (Marchantin A, B and E) properties (Asakawa et al., 2013; Beike et al., 2010; Commisso et al., 2021; Klavina et al., 2015; Ludwiczuk and Asakawa, 2019; Vollár et al., 2018) In parallel, an increasing availability of genomic resources has paved the way for gene mining approaches to identify genes involved in specialized metabolism that are absent in seed plants. For example, microbial terpene synthase-like (MTPLs), a novel group of metabolic genes exclusive to non-seed plants, has been identified (Jia et al., 2016). All these traits make bryophytes a fascinating group of plants to study, with a high potential for the discovery of desirable natural products amenable by biotechnological tools. The moss P. patens has already been shown to have a high biotechnological potential as an alternative green cell factory. Foremost, for the production of valuable proteins such as biopharmaceuticals (Campos et al., 2020; Decker and Reski, 2020; Reski et al., 2018, 2015). Recently, the first moss-made drug candidate (moss-aGal) successfully passed stage 1 clinical trials (“First moss-made drug,” 2015; Hennermann et al., 2019). During recent years, metabolic engineering has successfully evolved from synthesis of biopharmaceuticals to heterologous production of natural compounds such as commercially relevant terpenoids, e.g. artemisinin, patchoulol and santalene (Khairul Ikram et al., 2017; Zhan et al., 2014). Some of the key 50 features of this sustainable cell factory platform are relatively fast axenic growth in simple mineral media, an established photobioreactor system, progressively up-scaled, currently to 500L, and a well-established procedure for cryopreservation in cell culture banks (Schulte and Reski, 2004). In particular the haploid condition of the gametophyte phase and the relative ease of transformation via homologous recombination with yeast-like efficiency have made this system attractive for transgenic approaches (Hohe et al., 2004; Schween et al., 2005). In this review we show how the ecological and genetic diversity of bryophytes is reflected by chemical diversity. We summarize how this knowledge can lead to the discovery of novel bioactive products of commercial interest and how past decades of research focusing on the moss P. patens have qualified it not just as a model system in evolutionary developmental and cell biology (Rensing et al., 2020), but also as a prime cell factory for heterologous production of valuable natural products. 2. Ecological diversity of bryophytes Bryophytes qualify as the most diverse group of plants after angiosperms with regards to their numbers of species, geographical distribution, and habitat diversification (Tuba et al., 2010). They include around 20 000 species (Shaw et al., 2011), thereof mosses around 13 000 (Magill, 2014), liverworts 6000 and hornworts 200 (Söderström et al., 2016), whereas angiosperms encompass ca. 295 000 species (Christenhusz and Byng, 2016). Even though bryophytes possess lower species diversity and less complex morphology than the more recently diverged angiosperms, they exhibit as much genomic diversity as tracheophytes (including angiosperms), expressed in a broad assemblage of physiological and biochemical adaptations, which are still poorly explored (Glime, 2013). Some of the biochemical adaptations in bryophytes appear to have evolved as a consequence of their often slow growth and small size, protecting them from herbivory (Glime, 2013) and modulating interactions with microbiota and other plants. Chemical interaction, for example by allelopathic substances, may be especially important during early successional stages of bryophyte development. Bryohytes can cope with environments across all climatic regions on the planet, where water is present, from the Antarctic and Arctic permafrost areas to the warm and humid tropical forests, including regions and substrates which are uninhabitable for vascular plants (Tuba et al., 2010). Thus, the apparent simplicity of bryophyte vegetative bodies (gametophytes) contrast to their complex genomic architecture. This is exemplified in a recent study of vegetative (gametophytic) transcriptomes from two morphologically similar species (P. patens and Funaria hygrometrica) (Rahmatpour et al., 2021). These closely related species display quite high genomic divergence, with most innovations being in metabolic genes of F. hygrometrica (encoding for copper chaperone, copper ion binding, universal stress protein, heat stress transcription factor, riboflavin biosynthesis protein, sulfur compound metabolic process, inorganic cofactors, some defensive mechanisms, etc.), supporting the hypothesis that moss evolution is driven by metabolic and physiological adaptations to different environments. 2.1. Ecological roles All ecosystems on earth, except marine and permanently frozen ecosystems (Vanderpoorten and Goffinet, 2009) are occupied by bryophytes. Their ecosystem functions include primary production, nutrient cycling (including mycorrhizal relationships and nitrogen fixation as hosts for cyanobacteria), water retention, primary and secondary colonization and animal interactions (Tuba et al., 2010). By hosting nitrogen fixing cyanobacteria feather mosses Pleurozium schreberi and Hylocomium splendens are an important source of nitrogen input to natural boreal forests and this association could be an asset in forest management (Stuiver et al., 2015). Bryophytes have an essential role in global biogeochemical cycles, by sequestrating substantial quantities of carbon as peat, notably in wetlands and mires dominated by peat mosses, Sphagnum spp. (Figure 1), thus influencing the global climate. In tropical forests, especially montane cloud rain forests, epiphytic bryophytes have a major role in controlling water and nutrient flow, having an overall water holding capacity equivalent to as much as a 20 mm precipitation event (Ah-Peng et al., 2017). Among natural environments, they have the largest standing biomass and productivity in peatlands, fens, bogs, Arctic and Antarctic tundra, alpine ecosystems, especially above tree line and moist forests (Tuba et al., 2010; Vanderpoorten and Goffinet, 2009). Despite occupying only 3% of the global land area, peatlands contain about 25% (600 GtC) of the global soil C stock, which is equivalent to twice the amount in the world’s forests (Loisel et al., 2021; Yu et al., 2010). Bryophytes are also able to inhabit cities, where some species may serve as indirect or even direct (in situ) bioindicators of air pollution, because of their ability to adsorb and accumulate high concentrations of heavy metals (Rühling et al., 1970; Stanković et al., 2018). 52 Figure 1. Some of the more extreme bryophyte habitats: a) – Moss cover on thatched roof. Reconstructed bronze age building, Tanums Hede, Sweden 2007.; b) - Lava field with Racomitrium lanuginosum as dominant component in the vegetation, Ölfus, Iceland, 2018; c) and d) – Mosses on the volcanic rocks and palagonite tuff, as part of pioneering vegetation, Surtsey volcanic island, UNESCO world heritage site, Iceland, 2018; e) - Peat mosses, Sphagnum warnstorfii and S. teres, rich fen, Björnekullakärret, Sweden, 2005; f) - Polar desert with the barren vegetation almost completely composed by bryophytes, Ellef Ringnes Island, Canadian Arctic Archipelago, 1999. (a, b, e, f – photos by Nils Cronberg; c, d – photos by Gróa Valgerður Ingimundardóttir). 2.2. Habitat diversity Bryophytes can grow on a wide range of natural substrates (soil, rock, bark, tree trunks, rotting wood, dung, animal cadavers or leaf cuticles) forming diverse microhabitats, and many bryophytes are actually reliable indicators for specific sets of substratum-related conditions (Townsend, 1964). They can also colonize somewhat more specialized substrates, such as ashes after forest fire, lava and tephra after volcano eruptions, some saline environments (but few are true halophytes) or heavy-metal rich soils (metallophytes) (Ingimundardóttir et al., 2014; Townsend, 1964). They interact with other plants and can promote soil formation and development. Some species even occur on bare volcanic soils and rocks (Figure 1), thus generating environments habitable for vascular plants and facilitating their development (Ingimundardóttir et al., 2014). Bryophytes have been classified according to life strategy (During, 1979), ranging from short-lived fugitives and shuttle species to long-lived perennial stayers. Many species are ecological pioneers or fugitives appearing on substrates with little competition on roofs, soils, rocks and trees (Figure 1). The fugitive life strategy occurs in spatially highly unpredictable environments that exists for short time, where species have fast life span, frequent sexual reproduction and long-lived small spores, such as in F. hygrometrica, Shuttle species occur in habitats with regular disturbance regimes, selecting for fast reproduction and large dispersal agents. Perennial stayers are competitive together with vascular plants in habitats such as forest floor, wetlands and various types of heathland, including arctic tundra (Figure 1). Such species (e.g., Sphagnum spp. and Hylocomium splendens) live in persistent, late successional environments and have long life span, low level of sexual reproduction and dominant vegetative proliferation. Like tracheophytes, bryophytes possess endophytic fungi (K. H. Chen et al., 2018; Nelson and Shaw, 2019; Nelson et al., 2018; Yu et al., 2014), but their functional role in bryophyte ecology is yet to be investigated (Davey and Currah, 2006). Fungal endophytes may provide bryophyte hosts with greater tolerance to extreme pH or promote vegetative growth or adaptation to the extreme environment, as it is found in Antarctic bryophytes (Pressel et al., 2014). 2.3. Biogeographic distribution Despite the considerable differences in ecophysiology, distribution patterns and dispersal between bryophytes and vascular plants, biogeographic distributions of bryophytes are largely consistent with those reported in other taxonomic groups (Patiño and Vanderpoorten, 2018). Bryophytes are present in all five major phytobiogeographic regions of the world. 2.3.1. Endemism Spatial analyses of genetic structure in bryophytes suggest higher long-distance dispersal capacity than for angiosperms due to smaller diaspores (wind-dispersed spores), resulting in lower speciation and endemism (Shaw et al., 2015). From a biogeographic point of view, bryophytes are characterized by low rates of endemism, with clearly different regional endemism patterns compared to angiosperms. Several temperate areas, including Patagonia, the Pacific Northwest American region, and Tasmania exhibit high levels of bryophyte endemism, differing from the most important hotspots for angiosperms located in tropic or subtropic climates such as the Mediterranean and Central American regions (Patiño and Vanderpoorten, 2018). 54 2.3.2. Ecotypes/cryptic species It has sometimes been advocated that bryophyte species, in contrast to the majority of seed plants, do not tend to develop ecotypes, geographic populations genotypically adapted to specific environmental conditions. They rather display an intrinsic broad ability to cope with environmental variation (Patiño and Vanderpoorten, 2018), i.e. individuals display wide physiological and morphological plasticity (Reynolds and McLetchie, 2011) which would then overrule any tendency for local adaptation. Furthermore, many species display low genetic differentiation across large distribution areas, suggesting efficient gene flow through winddispersed spores (summarized in Patiño and Vanderpoorten 2018), which may counteract local differentiation. However, a high gene flow does not necessarily prevent local adaptation as a response to strong selection pressures and few studies have really tested presence of adaptive local differentiation in a rigorous way. Several genomic studies (Myszczyński et al., 2017; Shaw, 2001; Yousefi et al., 2017) have shown that broadly defined morphological species can be separated into “cryptic species”, lineages with distinctly differentiated genomes but obscure or overlapping morphological differentiation. Although these cryptic lineages show geographical or ecological separation at varying degree, most of the genomic differentiation appear to be manifested at the biochemical level and these lineages therefore passed un-noticed in earlier taxonomic revisions based on morphology. However, new discriminating morphological characters are often revealed that enable separation of such cryptic species (Shaw 2001). 2.3.3. Diversity gradient World tropical regions were for a long time considered poorer in bryophyte species compared to temperate areas, suggesting unclear relationship between latitude and diversity in bryophytes (Vanderpoorten and Goffinet, 2009). There was even some evidence for inverse latitudinal diversity gradient at narrower spatial scales, e.g. in Europe (Mateo et al., 2016). However, recent analyses of the distribution of liverworts and hornworts (Söderström et al., 2016), showed that global species richness of tropical areas is markedly higher than that of the extratropical ones, indicating a positive latitudinal diversity gradient (LDG) in hornworts and liverworts (J. Wang et al., 2017). Equally diverse temperate and tropical regions are sometimes reported for mosses (Geffert et al., 2013), which seems to be in conflict with the paradigm of low moss diversity in the tropics and the presence of inverse LDG in moss species. In general, tropical regions are less investigated than temperate regions and taxonomic revisions of many tropical taxa are missing, so the estimation of the species richness and distribution pattern in mosses needs to be re-investigated, requiring a new critical world checklist of mosses (Geffert et al., 2013; Patiño and Vanderpoorten, 2018). An interesting feature unique for mosses among bryophytes, is that they possess high levels of endopolyploid nuclei, which occur in specialized tissues, suggesting an increase in gene copy number and ability to produce an assortment of cell sizes, which in turn could affect other morphological and physiological factors influencing ecology and distribution of mosses (Bainard et al., 2020). The worldwide diversification of bryophytes is paralleled by a huge chemical diversity of specialized metabolites (Asakawa et al., 2013), which provide protection from abiotic and biotic stresses (Xie and Lou, 2009), shaped during their long evolutionary history. 3. Chemical diversity of bryophytes More than 2200 chemical constituents have been described from bryophytes and the number is growing rapidly. The natural products isolated from bryophytes are mainly terpenoids (including mono-, sesqui- and diterpenoids), flavonoids, (bis)bibenzyls (exclusively produced by liverworts), and lipids (Sabovljević et al., 2016). Selected natural product structures from different chemical groups isolated from bryophytes are shown in Figure 2. Several hundreds of these isolated compounds exhibit antimicrobial, antifungal, anticancer, antibacterial and/or antiviral bioactivity. The majority of these compounds have been extensively described before (Asakawa et al., 2013; Jia et al., 2018; Ludwiczuk and Asakawa, 2019). Thus, only recently discovered compounds or novel bioactivities from already known compounds reported in the last ten years are summarized in this chapter (Table 1). 56 Figure 2. Selected chemical structures of natural products found in bryophytes. 3.1. Terpenoids Terpenoids, the largest group of natural products, present in all living species, mediate diverse biochemical and ecological processes in bryophytes Chen et al., 2018). Like in other plants, they contribute to the physiological regulation, as shown for the diterpenoids ent-kaurene and derivatives in P. patens (Hayashi et al., 2010). These are involved in protonemal differentiation and spore development Chen et al., 2018; Hayashi et al., 2010; Vesty et al., 2016). Some terpenoids also act as UV-B absorbers and enhance desiccation tolerance by modulating cytoplasmic osmotic potential Chen et al., 2018). Liverworts produce a larger variety of terpenoids than mosses and hornworts. Over the past 40 years, more than 1600 terpenoids have been isolated and identified from liverworts (including lipophilic mono-, sesqui- and diterpenoids), while only around 100 sesquiterpenoids, and a few mono- and diterpenoids, have been identified in mosses (Ludwiczuk and Asakawa, 2019). This may be because of the presence of the oil bodies exclusively in liverworts where terpenoids are stored. Terpenoids from bryophytes have versatile bioactivities such as anti-bacterial, antiinflammatory, antifungal, phytotoxicity, and insect antifeedant activities (Asakawa et al., 2013; F. Chen et al., 2018). Asakawa et al. (2013) demonstrated that terpenoids and other aromatic compounds are responsible for the antibiotic and antifungal properties in liverworts, although Chen et al. (2018) expressed some uncertainty about to what degree these compounds really repress infestation. Tosun et al. (2015) tested the essential oils of three moss species, Pseudoscleropodium purum, Eurhynchium striatum, and Eurhynchium angustirete for antimicrobial activity. Their minimum inhibitory concentrations (MIC) ranged from 278.2 to 2225 µg/mL, with α-pinene (16.1%), 3-octanone (48.1%), and eicosane (28.6%) as main components, respectively. Bacterial and fungal infections do occur in mosses, whereas this is very rare in liverworts due to the contributions of their large pool of anti-bacterial and antifungal terpenoids Chen et al., 2018). There is evidence of allelopathic effects of terpenoids extracted from liverworts and mosses (reviewed by Whitehead et al., 2018). Momilactones B is a diterpenoid phytoalexin first isolated from the moss Hypnum plumaeforme (Figure 2) (Nozaki et al., 2007), which showed allelopathic activity against angiosperms, mosses, and liverworts. Interestingly, momilactones have only been found in rice before and have shown cytotoxic and antitumor activity against human colon cancer cells (Kim et al., 2007). Repellent odor and bitter taste of bryophyte terpenoids, and sometimes cytotoxicity may serve an anti-herbivore function as well, which may explain why relatively few animals feed on bryophytes, especially liverworts (Asakawa et al., 2013). Compared with mosses and liverworts, hornworts are chemically scarcely studied. Previous studies concluded that the chemical constituents of hornworts are very distinct from liverworts and mosses (Asakawa et al., 2013). Several terpenes have been characterized in hornworts (Xiong et al., 2018), however, bioactive chemicals unique to hornworts have not been reported. 3.2. Phenylpropanoids 3.2.1. Flavonoids The flavonoid pathway (starting from the larger phenylpropanoid pathway) is one of the best characterized among plants, with significant biological and ecological functions (Davies et al., 2020). Flavonoids are widely distributed in mosses, liverworts, and vascular plants (YonekuraSakakibara et al., 2019) and flavonoid biosynthetic ability was also reported in divergent evolutionary lineages of microalgae and bacteria (Goiris et al., 2014; Jiao et al., 2020), 58 suggesting that the ability for flavonoid production originated earlier during evolution than previously thought (Yonekura-Sakakibara et al., 2019). To our knowledge, no flavonoids have been reported from hornworts. Either because the divergence of hornworts occurred before flavonoid pathway evolved, or the hornwort ancestor acquired mutations that caused loss of the flavonoid biosynthetic ability and subsequently caused flavonoid loss in this lineage (Davies et al., 2020). The derivatives of the cinnamic acid, which is the central intermediate in the biosynthesis of flavonoids and other phenylpropanoids, are reported in the hornwort Anthoceros agrestis (Soriano et al., 2018; Wohl and Petersen, 2020). Major classes of flavonoids in bryophytes are flavones, flavonols, isoflavonoids, aurones, 3deoxyanthocyanins, anthocyanins, and recently discovered auronidins exclusive for liverworts (Berland et al., 2019). Flavonoids play diverse roles in bryophyte lifecycle, such as UV-B radiation protection (Li et al., 2019; Waterman et al., 2017), protection against desiccation and extreme temperature fluctuations (mostly due to anthocyanins), and defense against pathogens (sesquiterpenoids have the same function) (Peters et al., 2019). Flavonoids also support the growth of hydroids and leptoids of mosses (which have similar functions as tracheids and sieve cells in vascular plants) by the activity of a few methoxyphenols and cinnamic acids as part of proto-lignin constituents (Peters et al., 2019; Townsend, 1964). Common flavonoids in liverworts and mosses are luteolin and apigenin and their derivatives (Asakawa et al., 2013), these flavonoids and derivatives are present in vascular plants as well. Bi- and tri-flavonoids are more common in Bryophyta, and bioflavonoids are thought to be chemotaxonomic marker of mosses. Pigments such as cell-wall bound red flavonoids riccionidin (an auronidin) (Berland et al., 2019) and sphagnorubin (Vowinkel, 1975), have been reported from liverworts and peat mosses (Figure 1-e), respectively. Auronidins constitute an unreported flavonoid class thus far (Berland et al., 2019) and are unrelated to anthocyanins, which are the main red pigments present in angiosperms. Carella et al. (2019) reported that M. polymorpha accumulated red pigmented Riccionidin A into the thallus cell walls during biotic stress (upon oomycete pathogen infection), mediated by R2R3-MYB transcription factor, which led to largely increased liverwort resistance. R2R3-MYB activation of flavonoid production in the same species during abiotic stress has also been delineated (Albert et al., 2018). Some species of other thalloid liverworts roll their thalli over the dorsal surface when dried out, so that dark pigmented ventral side is left exposed (Davies et al., 2020; Reeb et al., 2018), whereas some desiccation-tolerant leafy liverworts also tend to be dark pigmented (Vitt et al., 2014). The adaptive value of these pigmentations of assumed auronidin type is still subject for debate, light screening, ROS scavenging, strengthening of the cell wall and biotic stress defense are mentioned as possible functions (Davies et al., 2020). Like terpenoids, allelopathic activity has been reported for flavonoid compounds in bryophytes (Whitehead et al., 2018). For example, 3-hydroxy-ß-ionone isolated from the moss Rhynchostegium pallidifolium demonstrated allelopathic activity by inhibiting the growth of several vascular plants (Kato-Noguchi et al., 2010). 3.2.2. Bibenzyls/bisbibenzyls Liverworts (Marchantiophyta) are copious producers of bibenzyls and bisbibenzyls with 103 characterized compounds so far (Yoshida et al., 2016). Their physiological and ecological roles are not fully understood. Marchantin A is one of the well-studied bisbibenzyls, isolated from Marchantia species, whose antibacterial and antifungal activities have been confirmed (Niu et al., 2006). Subsequently, it was reported that Marchantin A inhibited proliferation of protozoan species such as Plasmodium falciparum NF54 with IC50 = 3.41 uM and K1 with IC50 = 2.02 uM; and showed cytotoxic activity against Trypanosoma brucei rhodesiense, T. cruzi and Leishmania donovani with IC50 values 2.09, 14.90 and 1.59 uM, respectively (Jensen et al., 2012). Marchantin A also showed malaria prophylactic potential with moderate inhibitory activity against enzymes of P. falciparum (Jensen et al., 2012). Marchantin A, as well as marchantin B and E, and other marchantin-related phytochemicals from liverworts, inhibit influenza PA endonuclease activity in vitro and exert anti-influenza activity in culture cells (Iwai et al., 2011). Radula is another interesting liverwort genus because it produces not only bibenzyls and bis-bibenzyls but also bibenzyl cannabinoids cis-perrottetinene (cis-PET) (Asakawa et al., 2013), which structurally resembles (−)-△9-trans-tetrahydrocannabinol (△9-trans-THC) from Cannabis sativa (Toyota et al., 2002). The precursor of THC in C. sativa is olivetolic acid, whereas stilbene acid is the precursor of PET in R. marginata (Hussain et al., 2019, 2018). This natural product cis-PET was proven to be psychoactive by mimicking the action of the endocannabinoid 2-arachidonoyl glycerol and provoked a significant decrease of brain prostaglandin levels in a CB1 receptor– dependent manner in mice (Chicca et al., 2018). So far, R. chinensis, R. campanigera, R. laxiramea, R. marginata, R. perrottetii and a Peruvian unidentified species have been proven to contain cis-PET (Asakawa et al., 2020). 60 3.2.3. Lipids and lipid-derivatives High amounts of arachidonic acid (AA, C20:4) and eicosapentaenoic acid (EPA, C20:5) were detected in bryophytes (Beike et al., 2014). These very long chain unsaturated fatty acids are uncommon in higher plants but abundant in bryophytes because of the presence of Δ6desaturase, Δ5-desaturase (first identified by Girke et al., 1998) and Δ6-elongase. AA is synthesized from linoleic acid (C18:2) via ω -6 pathway and EPA from α-linolenic acid (C18:3) via ω-3 pathway (Kaewsuwan et al., 2006). The presence of AA and EPA appear to be an ancestral chemical trait that links bryophytes to charophyte algae, since very long chain unsaturated fatty acids are rarely found in tracheophytes but are commonly produced in algae (Resemann et al., 2019). Lu et al. (2019) summarized the fatty acid compositions and contents in several moss and liverwort species from previous studies. Recently, it was reported that the lipidome of P. patens protonema comprising 733 molecular species derived from glycerolipids, sterol lipids and sphingolipids, whereas Arabidopsis plants harbour only about 54% of this diversity (Resemann et al., 2021). Moreover, a sphingolipid-modifying enzyme was identified that contributes to pathogen defence and cold tolerance, but has no homolog in seed plants (Resemann et al., 2021). Large amounts of polyunsaturated C20 fatty acids in bryophytes imply that they can produce a broad range of oxylipins (Scholz et al., 2012). P. patens has an enzyme lipoxygenase with fatty acid chain-cleaving lyase activity, which uses C18-fatty acids and C20fatty acids as substrates for producing more oxylipins than angiosperms (de León et al., 2015; Senger et al., 2005) whose pathway is activated during bacterial infection (Alvarez et al., 2016). High amounts of long unsaturated fatty acids and existence of oxylipins represent a metabolic difference between mosses and angiosperms, that might have been advantageous for mosses in terms of tolerance to abiotic stress (Mikami and Hartmann, 2004) and protection from pathogens (Ponce de León and Montesano, 2017). Oxylipins are also involved in plant signaling. Common oxylipins, such as phytohormone jasmonic acid (JA), have been found in all vascular plants but not in bryophytes, in which the JA biosynthesis pathway stops at its precursor 12-oxophytodienoic acid (OPDA) (Stumpe et al., 2010; Wasternack and Feussner, 2018). Acetylenic fatty acid derived oxylipin may serve as a putative precursor of volatile oxylipin and can be triggered by mechanical wounding (Abay et al., 2015). Dicranin, an acetylenic fatty acid, which is found almost exclusively in the Dicranaceae family, has slug anti-feeding activity (Rempt and Pohnert, 2010). In addition, acetylenic acids sometimes appear as part of triacylglycerol to maximize energy conservation when growth space is limited (Dembitsky, 1993). Tocopherol plays an important role as antioxidants for long chain unsaturated fatty acids and terpenoids. 266 out of 726 (36.3%) liverwort species were shown to accumulate α-tocopherol (Asakawa et al., 2013). In Porella and Pellia this percentage is even higher (64% and 60%, respectively) (Asakawa et al., 2020). Table 1. Some selected chemical compounds characterized and isolated from bryophytes with different bioactivities and their actions. Compounds Type Species Bioactivities Actions β-phellandrene, β-caryophyllene Terpenoid Porella cordaeana Antimicrobial MIC 0.5-2 mg/mL for yeast, (Bukvicki et b-bazzanene Sesquiterpene Scapania nemorea Antimicrobial isobazzanene aromadendrene References 1-3 mg/mL for bacteria al., 2012) MIC 0.5-3 mg/mL for (Bukvicki et bacteria al., 2014) 0.2-1 mg/mL for yeasts Antioxidative Main conponents: α-pinene (16.1%), Terpenoid Pseudoscleropodium 3-octanone (48.1%), and eicosane purum, Eurhynchium (28.6%) striatum and antimicrobial MIC ranging from 278.2 to (Tosun et 2225 µg/mL al., 2015) Eurhynchium angustirete Porellacetals A-D Pinguisane Porella cordaeana Anti-cancer (Tan et al., (Terpenoid) Jamesoniellides Q−S (1S,3E,7E,11S,12S)-12-hydroxy- Diterpenoid Terpenoid 2017) Jamesoniella Anti- 50–80% maximum (Y. Li et al., autumnalis inflammatory inhibition rate 2018) Lepidozia reptans Anti- Suppress nitric oxide (NO) (S. Li et al., inflammatory production 2018) dolabella-3,7-dien-6-one, (1S,3E,7Z,11S,12S)-12-hydroxydolabella-3,7- dien-6-one, (3S,5S,8R,10R,13S,16R)-3,13dihydroxy-ent-kauran-15-one, (4R,5R,7S)-7-hydroxy-7-isopropyl1,4-dimethylspiro [4.4] non-1-ene-2carbaldehyde, (6R,7S,10R)-6,7dihydroxy-3-oxo-eudesma-4E-ene Scapanacins A–D Terpenoid Scapania carinthiaca Antihypertensive (Qiao et al., and antitumor 2018) Antiproliferative (−)-cis-perrottetinene (cis-PET) Bibenzyl Radula Structurally resembles (−)- (Chicca et △9-trans- al., 2018) tetrahydrocannabinol (△9trans-THC) from Cannabis sativa Marchantin A Bisbibenzyl Marchantia Anti-plasmodial NF54 (IC50 = 3.41 uM) and (Jensen et polymorpha K1 (IC50 = 2.02 uM) against Plasmodium falciparum 62 al., 2012) Marchantin A Bisbibenzyl M. emarginata Anti-cancer subsp.tosana Antioxidant IC50 of 4.0 ug/mL on human ((Huang et MCF-7 breast cancer cells al., 2010) free radical-scavenging (Huang et activity (EC50 =20 ug/mL) al., 2010) M. polymorpha and M. Anti-influenza Inhibition of PA (Iwai et al., paleacea var. diptera endonuclease activity, 2011) Radula kojana inhibitory properties towards Bisbibenzyl Plagiochila sciophila the growth of influenza A Bisbibenzyl Lunularia cruciata Marchantin A, B and E Bisbibenzyl Plagiochin A Bisbibenzyl Perrottetin F Phenanthrene compound and B Anti-cancer Lunularia cruciata Cytotoxic activity against (Novakovic A549 lung cancer cell line et al., 2019) with IC50 values of 5.0 and 5.0 μM (±)-Rasumatranin A−D, M and N Bibenzyl-based Radula Cytotoxicity Against human cancer cells meroterpenoid sumatrana Dicranenone Acetylenic Dicranum scoparium (X. Wang et al., 2017) Antifeeding (Rempt and Oxylipin Pohnert, 2010) Hexane extract Polytrichastrum formosum Anti-insect 70.33% against Sitophilus (Abay et al., granarius 2013) 4. Genodiversity of bryophytes Whereas the metabolic diversity of bryophytes is increasingly recognized, the genetics underlying this chemical diversity largely remain to be described. To date, a small number of (draft) genomes (including P. patens, Ceratodon purpureus, Fontinalis antipyretica, Pleurozium schreberi, M. polymorpha, Marchantia inflexa, Sphagnum fallax, Sphagnum magellanicum, A. agrestis, Anthoceros punctatus, Anthocerus angustus, H. plumaeforme) are available (http://phytozome.jgi.doe.gov/) ((Bowman et al., 2017; Lang et al., 2018; Li et al., 2020; Mao et al., 2020; Marks et al., 2019; Pederson et al., 2019; Rensing et al., 2008; Weston et al., 2018; Zhang et al., 2020). However, within the next 5 years this number is expected to increase significantly. The OneKP database encompasses transcriptome resources of 74 species (7 hornworts, 41 mosses, 26 liverworts) (https://sites.google.com/a/ualberta.ca/onekp/), which are mostly obtained from gametophores (2019). The NCBI database currently lists a total of 443 transcriptome datasets, some of which also capture the P. patens transcriptome under abiotic stress (https://www.ncbi.nlm.nih.gov, Beike et al., 2015; Richardt et al., 2010). For P. patens, a comprehensive gene atlas encompassing all developmental stages and the impact of some abiotic stresses, is available (Ortiz-Ramírez et al., 2016; Perroud et al., 2018). Generally, the advancement of next generation sequencing techniques, especially the long-read sequencing technology, has resulted in an accelerating accumulation of genomic data for bryophytes the last few years, thus paving the road for functional approaches and comparative genomics. 4.1. Genome sizes and transcriptome complexities of bryophytes The genome size in bryophytes ranges between 122 - 20,006 Mbp, which coincidences with the lower spectrum of Angiosperms (Michael, 2014). Despite their small stature, some bryophytes like P. patens surpass angiosperms such as the model plant A. thaliana in genome size. Liverworts exhibit a higher degree of genome size diversity compared to mosses and hornworts (see Figure 3a). Compared to other plant lineages, bryophytes carry a remarkably high number of protein-encoding genes relative to their size. This transcriptome complexity can be utilized as a valuable source for mining of novel genes. For example, the moss P. patens contains more genes than the flowering plant Catharanthus roseus, which is known for its specialized metabolite characteristics, at comparable genome size (see table 2). Figure 3. Genome size of different classes of bryophytes (Bainard et al., 2020). 64 Table 2: Genome size in comparison to protein-encoding genes in selected plants. Organism Genome Protein-encoding Genes Size Gene Density Reference (per Mbp) P. patens 500 35000 70 (Lang et al., 2018) H. plumaeforme 434 32195 74,18 (Mao et al., 2020) M. polymorpha 220 19138 87 (Bowman et al., 2017) A. punctatus 132,8 25800 194,3 (Li et al., 2020) A. agrestis 122,9 24700 201 (Li et al., 2020) A. angustus 119 14629 122,9 (Zhang et al., 2020) Selaginella moellendorfi 100 27793 277,9 (Banks et al., 2011) A. thaliana 135 27655 204,9 (Zimmer et al., 2013) Nicotiana benthamiana 3136 50516 14,4 (Schiavinato et al., 2019) Picea abies 19600 30000 1,5 (Nystedt et al., 2013) Catharanthus roseus 500 33258 66,5 (She et al., 2019) Liverworts generally have 8 to 9 chromosomes with little variation, thus genome duplications are unlikely unless very ancient. A small number of (allo)polyploids are known to occur in some genera (e.g., Porella baueri) (Boisselier-Dubayle et al., 1998). Despite the sister relationships between liverworts and mosses, it seems that the larger genomic size range has evolved independently in liverworts and is not a trait shared with mosses (Bainard et al., 2020). Genome duplications have not been found in hornworts yet (Li et al., 2020). By contrast, the moss P. patens underwent two whole genome duplication events about 40-48 million years ago and 27-35 million years ago (Lang et al., 2018). An excess of duplicate metabolic genes have been retained after these events, which may explain abundance of such genes in the P. patens genome (Lang et al., 2005; Rensing et al., 2007). Interestingly, an abundancy of some gene families encoding specialized metabolites could also be observed in the genome of M. polymorpha and A. angustus (Bowman et al., 2017; Zhang et al., 2020). 4.2. Conservation of precursor routes of specialized metabolism In general, the biosynthesis of specialized metabolites is scarcely studied in bryophytes. Throughout the last years, initial insights into the terpenoid and phenylpropanoid pathway have been obtained. It has been suggested that both the terpenoid precursor pathways, mevalonate (MVA) and methylerythritol 4-phosphate pathway (MEP), are conserved throughout land plants based on similar copy number of pathway genes in M. polymorpha, P. patens, A. thaliana and Oryza sativa Chen et al., 2018). This pattern can also be found in the recently annotated genomes from the Anthoceros genus (Li et al., 2020; Zhang et al., 2020). However, neither genes associated with major bottlenecks in the pathways, 3-hydroxy-3-methylglutarylcoenzyme A reductase (HMGR) and 1-deoxy-D-xylulose-5-phosphate synthase (DXS) nor isopentenyl diphosphate isomerase (IDI), that predominantly controls the DMAPP:IPP flux, which plays a significant role in the synthesis of different isoprenoid classes, have been functionally studied yet. The few described terpenoid synthases (see Table 3) indicate a partial terpenoid pathway conservation across land plants but also the absence of downstream enzymes catalyzing the synthesis of eminently important products for tracheophytes (e.g. gibberellic acid; see Table 3). Most recently, this perception has been challenged via a comparative computational approach, that revealed the presence of gibberellin biosynthesis and inactivation genes in bryophytes (Cannell et al., 2020). Table 3. Catalytic functions of TPSs from bryophytes that have been characterized. *The Monoterpene content varies amongst species. *CcSS was identified from a European species, CcBPPS from a North American species. Species Enzyme Substrate Products Reference M. polymorpha MpDTPS1 GGPP ent-Atisanerol (Kumar et al., 2016) MpDTPS3 GGPP ent-copalyl diphosphate MpDTPS4 Ent-copalyl diphosphate ent-Kaurene CcSS GPP Sabiniene C. conicum* CcBPPS (Adam and Croteau, 1998) Bornyl Acetate H. plumaeforme HpDTC1 GGPP syn-Pimara-7,15-diene (Okada et al., 2016) P. patens PpCPS/KS GGPP ent-Beyerene (Hayashi et al., 2006; Zhan et al., 2015) ent-Sandaracopimaradiene ent-Kaur-16-ene 16-hydroxy-ent-kaurene R. natans GPP 4S-(-)-Limonene (Adam et al., 1996) Such as the terpenoid precursor pathway, a conservation of the early phenylpropanoid pathway genes (PAL, C4H, 4CL, CHS, CHIL) is indicated across all land plants (Davies et al., 2020; Tohge et al., 2013). Downstream enzymes in the flavonoid pathway occur in accordance with the respective flavonoid profile in mosses and liverworts, but have not been detected in hornworts yet. Nevertheless, 21 copies of flavonoid 3’ monooxygenase genes and 11 of flavonoid,3’,5’, hydroxylases have been identified in A. angustus most recently (Zhang et al., 2020). To date, their functional roles have not been investigated. In contrast to seed plants and liverworts, mosses produce flavonoids, although a chalcone isomerase (CHI) gene is missing (Cheng et al., 2018; Clayton et al., 2018). The exact metabolic pathway remains unknown so far. 66 Chalcone synthase (CHS), belonging to the class III Polyketide synthase (PKS) superfamily, is considered as metabolic gatekeeper of flavonoid biosynthesis in plants. Functional characterization of a CHS from Plagiochasma appendiculatum confirmed for the first time this key role in the flavonoid biosynthesis of thalloid liverworts (Yu et al., 2015). Eight CHS gene classes have been reported from the moss P. patens, but they remain to be functionally characterized (Koduri et al., 2010). 4.3. Partial conservation of regulatory mechanisms targeting specialized metabolism Studies of metabolic regulation mostly target developmental processes or transcriptomic dynamics under various stressors. Comparative expression profiling have revealed evolutionary conservation of transcriptional regulation under stress conditions in bryophytes (Beike et al., 2015a; Richardt et al., 2010). Genomic data suggests that members of the Anthoceros genus and M. polymorpha have a small transcription factor repertoire (Bowman et al., 2017; Li et al., 2020). Considering the high genome size range in liverworts, this repertoire might fluctuate significantly and might be linked to specialized metabolism. For example, the liverwort Radula marginata carries a significantly larger number of transcription factors compared to M. polymorpha and to some mosses (Hussain et al., 2018). Comparative studies revealed six transcription factor (TF) families unique to this organism; possibly related to its cannabinoid metabolism (Hussain et al., 2018; Hussain and Kayser, 2019). In the moss P. patens, this transcription factor repertoire is even larger due to ancient genome duplications, although the TF response under salt stress appeared rather limited compared to A. thaliana (Rensing et al., 2007; Richardt et al., 2010). It was hypothesized that one of the reasons may be the partial absence of biosynthetic routes, e.g. parts of the jasmonic acid signaling pathway. Predominant co- and post-transcriptional regulation, which has been suggested on the basis of distinct 5’UTR-intron characteristics, is also a possible explanation (Richardt et al., 2010; Zimmer et al., 2013). Interestingly, the first genes to be transcribed upon cold stress in P. patens are predominantly moss- or even species-specific and of yet unknown function (Beike et al., 2015a). Conservation of the terpenoid precursor pathway has been anticipated in bryophytes, but functional studies targeting its regulation are lacking. Phytochrome interacting factors (PIFs) have been reported as regulators in the MEP pathway by regulating genes encoding the key limiting enzymes DXS and 1-deoxy-D-xylulose 5phosphate reductoisomerase (DXR) as well as phytoene synthase (PSY), the gatekeeper of carotenoid biosynthesis (Chenge-Espinosa et al., 2018). Functional conservation of PIFs across seed plants, mosses and liverworts has been reported (Lee and Choi, 2017; Possart et al., 2017). Table 4. Transcription factors that have been linked to pathway regulation of aromatic chemicals TF Pathway Species Reference MpMYB02 Anthocyanidins, Bisbenzyls M. polymorpha (Kubo et al., 2018) MpMYB14 Anthocyanidins, Phenylpropanoids M. polymorpha (Albert et al., 2018) MpMYB14 Phenylpropanoids M. polymorpha (Kubo et al., 2018) MpHY5 Phenylpropanoids M. polymorpha (Clayton et al., 2018) MpRUP1 Phenylpropanoids M. polymorpha (Clayton et al., 2018) PaBHLH Bisbibenzyls, Phenylpropanoids P. appendiculatum (Wu et al., 2018) PaBHLH1 Phenylpropanoids P. appendiculatum (Y. Zhao et al., 2019) MpBHLH12 Phenylpropanoids M. polymorpha (Arai et al., 2019) Initial insights into the regulation of chemicals derived from the phenylpropanoid pathway in liverworts reveal similarities to seed plants (Table 4). R2R3-MYB transcription factors have a key role in the stress-related regulation of flavonoid biosynthesis. Recently, two R2R3-MYB analogs (MpMYB02, MpMYB14) in M. polymorpha have been characterized, indicating a conservation of this regulatory feature across land plants (Albert et al., 2018; Kubo et al., 2018). On the other hand, central parts of the UV-mediated response in flavonoid biosynthesis like the central activator ELONGATED HYPOCOTYL5 (HY5), and negative feedback regulation by REPRESSOR OF UV‐B PHOTOMORPHOGENESIS1 (RUP1) are conserved between A. thaliana and M. polymorpha (Clayton et al., 2018). In addition, three BHLH transcription factors from P. appendiculatum (PaBHLH, PaBHLH1) and M. polymorpha (MpBHLH12) with regulatory roles in bisbibenzyl and phenylpropanoid biosynthesis have been identified (Arai et al., 2019; Wu et al., 2018; Y. Zhao et al., 2019). Most interestingly, the overexpression of PaBHLH and PaBHLH as well as MpMYB02 and MpMYB14 lead to the accumulation of significantly higher levels of flavonoids, bisbibenzyls and anthocyanidins, respectively (Kubo et al., 2018; Wu et al., 2018; Y. Zhao et al., 2019). All in all, the small repertoire of transcriptional factors makes bryophytes prime candidates to study the basic mechanisms of pathway regulation as well as pathway evolution in comparative genomic approaches. The identification and functional elucidation of the regulatory steps of specialized metabolites will not only provide insights into the evolutionary machinery, but also reveal key knowledge for a successful biotechnological exploitation. 68 4.4. Gene duplication events cause expansion of gene families encoding specialized metabolites The increasing availability of genomic resources allows new insights into the genomic complexity of bryophytes, including expansion of different gene families in the major bryophyte lineages (Linde et al., 2017). Comparative approaches suggest that genes encoding specialized metabolites are particularly abundant in liverworts (Davies et al., 2020), possibly reflecting a more pronounced chemical diversity compared to mosses and hornworts. Besides, some gene families encoding specialized metabolites are more expanded in bryophytes than in other land plants. A good example is the polyphenol oxidase (PPO) gene family, which occurs in low copy number in seed plants, and is even absent in some species such as A. thaliana. PPOs cause a typical browning reaction in damaged tissues, and there is evidence supporting its role in plant defense (Constabel and Barbehenn, 2008), but in general, the physiological role of PPOs is not well studied. They occur in high copy numbers in mosses and in even higher numbers in liverworts (Davies et al., 2020; Tran et al., 2012). Interestingly, the recent genome assembly of A. angustus revealed a high number of protein-encoding PPOs in hornworts as well (Zhang et al., 2020), indicating that all bryophyte phyla have expanded the PPO genes (see Figure 4a). The presence of PPO genes in M. polymorpha is linked to tandem repeats and gene clusters (TAGs) and notably, 66% of the PPO genes were associated with TAGs, in contrast to 5.9% TAG presence throughout the rest of the Marchantia genome. Functional studies are lacking, but it has been speculated that expansion of PPOs is related to ecological diversification and specialized metabolism (Davies et al., 2020). Figure 4. a) Number of transcribed polyphenol oxidases per genome *number of gene copies on genome (Bowman et al., 2017; Zhang et al., 2020). b) Average of expressed MTPSL genes per genome on basis of transcriptomic analysis across plant groups known to express MTPSLs (adopted from Jia et al. 2016). Beside TAGs, a role of gene clustering in specialized metabolism has been described most recently in momilactone biosynthesis of the moss H. plumaeforme. This is the first evidence of gene clustering of biosynthetic pathway genes of specialized metabolites in bryophytes, and emphasizes the significance of the genomic architecture in the synthesis of specialized metabolites not only in vascular plants but also in bryophytes (Mao et al., 2020). Other examples of large gene families involved in the synthesis of specialized metabolites are Dirigent proteins or PKS. Particularly type III PKS may be a significant driver of metabolic diversity in plants (Yonekura-Sakakibara et al., 2019). 24 PKS-like genes have been found in M. polymorpha, which is a substantially higher number compared to seed plants like A. thaliana (4), Malus domestica (10), Vitis vinifera (13) and Populus tremula (14) (Su et al., 2017). Most of the copies seem to be a result of an ancient CHS/PAL gene pair duplication. Besides CHS, the functional role of most PKS remains unknown (Bowman et al., 2017; Davies et al., 2020; Fischer et al., 1995). In R. marginata, stilbene synthase, which evolved from a CHS gene (Tropf et al., 1994), has been recently identified as one of the precursors involved in the biosynthesis of the psychoactive cannabinoid (-)-cis- perrottetinene (cis-PET)(Hussain et al., 2018). 70 4.5. Mining of novel gene families The progressive availability of genomic resources allows for mining of genes that are absent in seed plants, which is particularly interesting with regard to the synthesis of specialized metabolites. For example, transcriptome-mining in M. polymorpha revealed a novel group of mono- and sesquiterpene-like synthases, most of which resemble microbial terpene synthases, motivating the name microbial terpene synthase-like genes (MTPSLs) (Kumar et al., 2016). MTPSLs contain a single α-domain, in contrast to a typical plant TPS which is comprised of either two (αβ-type) or three structural domains (αβγ-type) (Jia et al., 2018). Comparative transcriptome-mining across the plant kingdom have confirmed the exclusive occurrence of this novel class in non-seed plants (see Figure 4b). Liverworts showed by far the highest MTPSL-richness (Jia et al., 2016), although MTPSLs are wide-spread amongst all groups of bryophytes; more than two-thirds of transcriptomes include MTPSLs and members of all four MTPSL clades occurs (Jia et al., 2016). Some of the MTPSL products are identical or similar to terpenoids previously shown to be products of classical TPS and assumed to take part in the protection against abiotic and biotic stresses (Jia et al., 2018, 2016; Kumar et al., 2016; Xiong et al., 2018). At present, a small number of MTPSLs have been functionally characterized and there is a high potential for future discovery (Jia et al., 2016). 5. Biotechnology In order to utilize bryophytes for the commercial production of natural compounds, sophisticated ways are needed to cultivate and generate large amounts of biomass rapidly. Another crucial factor concerns development of tools that allow fast and reliable metabolic engineering. Therefore, this chapter summarizes different approaches that have emerged in the last decade to support the transformation of bryophytes and foremost the moss P. patens, into an alternative production platform for natural compounds. 5.1. Cultivation and scale-up production A range of species have already been established as axenic cultures, generally by being grown photoautotrophically in simple low-cost inorganic media without the supplementation of microelements, vitamins and phytohormones (Beike et al., 2010; Hohe and Reski, 2005). As part of the Mosstech.eu project around 50 species have been brought into axenic culture, which shows that many bryophytes can be cultivated in cell cultures (www.mosstech.eu). Out of these, 15 species are at present deposited at the International Moss Stock Center (IMSC, https://www.moss-stock-center.org/) with the following accession numbers: 40096, 40097, 40098, 40099, 40100, 41101, 41102, 41103, 41104, 41248, 41254, 41255, 41212, 41249, 41246. In vitro cultures of bryophytes can be initiated from surface-sterilized spores, gemmae or vegetative fragments (Beike et al., 2010). Compared to seed plants, bryophytes possess simple body plans and unique regeneration capacity from fragments and even from single cells. Byophytes can be axenically cultured on solid agar-based media or in agitated flask liquid cultures from a few milliliters up to several hundreds of liters (Figure 5a/b) (Decker et al., 2014). Efficient protocols for protoplast cultures and growth of whole plants have been established for several bryophytes (Bach et al., 2014; Hohe and Reski, 2002; Li et al., 2005). Growth characteristics and conditions of cultivation for some bryophyte species are presented in Table 5. Among the different developmental stages, the suspension-cultured protonemal tissue is the most suitable for biotechnological approaches because of its genetic stability, reduced somaclonal variation and high homologous recombination rate during genetic transformation (see Figure 5a/b) (Decker et al., 2014; Decker and Reski, 2012, 2008; Reski, 1998). For high-throughput production of moss biopharmaceuticals, disposable 100 L and 500 L wave-bag bioreactors are applied (Niederkrüger et al., 2019) (https://www.elevabiologics.com). A protocol for cryopreservation of bryophytes (more than 140.000 specimens) was developed by Schulte and Reski (2004) and subsequently used to establish the International Moss Stock Center (IMSC https://www.moss-stock-center.org/), which ensures longevity and stability of a bryophyte collection (Rowntree et al., 2011). 72 Figure 5. Cultivation of bryophytes in photobioreactors. a) Photobioreactor for small-scale production of P. patens. b) Photobioreactor for small-scale production of peat moss (Sphagnum palustre). a+b) source: ReskiLab, University of Freiburg, http://www.plant-biotech.net, CC BY-SA 3.0 c) Photobioreactor100L wave-bag photobioreactor for industrial applications (source: eleva GmbH). Table 5. Established cultivation systems for different bryophyte species. Bryophytes Cultivation System Relevant features References Protonema-derived Growth rate in a mixotrophic condition was (μmax 0.27 (Ruiz-Molina suspension cultures at d−1) three times greater than in autotrophic and et al., 2016) shake-flask scale heterotrophic conditions. Mosses Polytrichum juniperinum Protonema cultures tolerated a wide initial medium pH range (4.5–8). Sphagnum palustre Sphagnum squarrosum Gametophores Sucrose and ammonium nitrate, added in the media, (Beike et al., cultivation in flasks and were able to increase the biomass by around 10- to 30- 2015b) photobioreactor fold within 4 weeks. Protonema-proliferation Detailed time schedule of the thallose protonema (W. Zhao et al., liquid culture regeneration and subsequent developmental processes 2019) was stablished. Peat mosses (Sphagnum L.) Solid and suspension Establishment of axenic in-vitro cultures of 19 (Heck et al., cultures Sphagnum species. 2020) Marchantia linearis Lehm. and Temporarily immersed Time Lindenb. cultures using Rita® development was reduced substantially using liquid Murugan, bioreactor culture media in RITA®. 2014) All the three species grew well in half strength Knop’s (Awasthi et al., macronutrients + Nitsch’s trace elements with 10 ppm 2012) Liverworts Conocephalum conicum (L.) Lindenb., Reboulia hemispherica (L.) Raddi and paleacea Bertol. Marchantia Solid cultivation agar-based required for thallus regeneration, and freshly prepared ferric citrate under the continuous illumination of 4,500–5,000 lux at 20 ± 2°C temperature (Krishnan and 5.2. Elicitation of production of natural products in bryophytes Enhancing the yield of compounds is a major step for large-scale production, which is either done through abiotic and biotic elicitation or through genetic manipulation (see chapter 5.3). Elicitation is mainly performed by abiotic (light, temperature, salt, etc.) and biotic (bacteria, fungus, proteins, etc.) stimuli that induce biosynthesis of specialized metabolites (Thakur et al., 2019). In the moss P. patens, genes encoding enzymes involved in important defense pathways such as phenylpropanoids, were induced by infection with Pectobacterium carotovorum bacteria (Alvarez et al., 2016). The chemical response to the induction was not analyzed. In P. patens ultraviolet (UV)-B irradiation induced genes that encode for enzymes for flavonoid biosynthesis (Wolf et al., 2010), and in the liverwort M. polymorpha, UV-C induced the synthesis of the bisbibenzyls isoriccardin C, marchantin C, and riccardin F, through the abscisic acid (ABA) signaling pathway (Kageyama et al., 2015). Production of phytoalexins momilactone A and B were also induced by UV and, jasmonic acid- and cantharidin-treatments in the moss H. plumaeforme (Kato-Noguchi, 2009). Moreover, it was shown that intracellular flavonoid level in M. linearis were induced by the application of methyl jasmonate, 2-(2-fluoro6-nitrobenzylsulfa-nyl) pyridine-4-carbothioamide and 2,4-Dichlorophenoxyacetic acid (Krishnan et al., 2014). Wounding stress induces the production of the compounds luteolin, apigenin and isoriccardin C in M. polymorpha, biosynthesized through the phenylpropanoid pathway (Yoshikawa et al., 2018), which is interesting since blending is often applied during cultivation. 5.3. Metabolic engineering in bryophytes Bryophytes display many features that make them attractive biotechnological platforms for the production of specialized compounds, lipids and recombinant biopharmaceutical proteins. These advantages include standardized cultivation methods under sterile conditions in bioreactors (see chapter above) and efficient transformation methods for genetic engineering. The methods for genetic engineering have been developed since 1991 when the first method was published for P. patens (Schaefer et al., 1991). It has been followed with methods for engineering of Ceratodon purpureus (Thümmler et al., 1992) and Marchantia polymorpha (Nasu et al., 1997). The methods for engineering are mainly polyethylene glycol-mediated or involve the use of Agrobacterium tumefaciens (see Table 6). The methods employ all the modern transformation technologies, in-vivo DNA assembly (King et al., 2016), CRISPR 74 (Collonnier et al., 2017), which now also include multiplexing (Mallett et al., 2019), and TALEN (Kopischke et al., 2017). It has also been shown that using contemporary synthetic biological parts is also possible in bryophytes (Peramuna et al., 2018). The use of mosses and liverworts has recently been reviewed in several papers (Decker and Reski, 2020; Ishizaki et al., 2016; Patron, 2020; Yongabi Anchang and Simonsen, 2019). Table 6. Overview of bryophytes transformation achievements Marker gene Description apt adeninephosphoribosyl transferase P. patens nptII, aphIV Neomycin phosphotransferase, Aminoglycoside Otransferase P. patens GFP GFP expression Green fluorescence P. patens nptII Neomycin phosphotransferase G418 resistance P. patens human β(1,4)galactosyltransferase human β(1,4)galactosyltransferase Humanised Nglycosilation pattern Adenine Phosphoribosyl transferase; Hygromycin-B-4Okinase Loci: Pp3c8_18830V3.1, Pp3c18_4770V3, Pp3c22_15110V3, Pp3c4_16430V3, Pp3c8_18850V3, Pp3c23_15670V3 2fluoroadenine resistance; Hygromycin resistance P. patens P. patens PpAPT; HPH Effect Kanamycin, Hygromycin, G418 resistance G418 resistance; hygromycin resistance Goal Transfection Reference random integration PEG (Schaefer et al., 1991) targeted integration into locii 108, 420, 213 PEG (Schaefer and Zrÿd, 1997) M.I. (Brücker et al., 2000) Biolistic (Šmídková et al., 2010) PEG (Huether et al., 2005) PEG (Collonnier et al., 2017) expression plasmid, stability unknown random integration; integration targeted into lea2 In vivo assembly knock in, in α(1,3)fucosyltransferase and β(1,2)xylosyltransferase locii CRISPR-Cas9 mediated PpATP knock-out and knock-in (HPH introduction) Cas9 mediated mutation, causing detectable locus size variation CRISPR-Cas9 mediated multiplex targeted mutation of 6 locii PEG (Mallett et al., 2019) P. patens 6 genomic sites M. polymorpha nptII Neomycin phosphotransferase G418 resistance random integration A.T. (Nasu et al., 1997) M. polymorpha hpt, GUS Hygromycin phosphotransferase; βglucuronidase Hygromycin resistance; XGluc marker random integration A.T. (Ishizaki et al., 2008) M. polymorpha hpt; aadA Hygromycin phosphotransferase; aminoglycoside-3″adenyltransferase Hygromycin resistance; Spectinomycin resistance random integration Biolistic (Chiyoda et al., 2008) M. polymorpha hpt; NOP1 Hygromycin phosphotransferase; not mentioned targeted integration (NOP1 knockout) PEG (Ishizaki et al., 2013) M. polymorpha NOP1; not mentioned Talen mediated mutation of NOP1 A.T. (Kopischke et al., 2017) Hygromycin resistance; impaired air chamber formation Impaired air chamber formation C. purpureus nptII Neomycin phosphotransferase Kanamycin resistance C. purpureus GFP GFP expression Green fluorescence C. purpureus Heme oxygenase Heme oxygenase expression C. purpureus APT adeninephosphoribosyl transferase restores phototropic response Kanamycin, Hygromycin, G418 expression of oat PhyA expression plasmid, stability unknown expression plasmid, stability unknown Gene targeting PEG (Thümmler et al., 1992) M.I. (Brücker et al., 2000) M.I. (Brücker et al., 2000) PEG (Trouiller et al., 2007) The moss P. patens has a potential to be a production host for commercially valuable metabolites and proteins. The precursors for the anticancer diterpene, paclitaxel (Taxol TM) were obtained by expressing taxadiene synthase gene from Taxus brevifolia in P. patens. Taxa4(5),11(12)-diene could be produced at a yield of up to 0.05% of the plant fresh weight (Anterola et al., 2009). The anti-malarial drug artemisinin (a sesquiterpene lactone), was obtained with a yield of 0.21 mg/g dry weight after only 3 days of cultivation by engineering of five artemisinin biosynthetic pathway genes into P. patens, which is equivalent to the levels in the original plant A. annua (Khairul Ikram et al., 2017). P. patens is also used for the production of valuable ingredients for the perfume industry, such as the sesquiterpenoids patchoulol and β-santalene. The yield of patchoulol achieved was 1.34 mg/g dry weight (Zhan et al., 2014). The diterpenoid sclareol, another valuable component in fragrances, was obtained in P. patens at the yield of 2.84 mg/g dry weight (2.28 mg/l culture) in liquid media after 18 days of cultivation (Pan et al., 2015). The heterologous production was enhanced using traditional terpenoid metabolic engineering steps like heterologous expression of HMGR using the truncated version of yeast HMGR and enhancing the storage compartments (Zhan et al., 2014). A modular approach for the production of a range of diterpenes was reported in P. patens. Three class II and two class I diterpene synthases (diTPS) enzymes were combined to generate industrially important diterpenes (Banerjee et al., 2019). P. patens has shown promising results as cell factory for the production of terpenoids. As a result, a fragrant moss-based product was developed by Mosspiration Biotech (see Figure 6) (https://www.mossebelle.com). Bryophytes contain high amounts of polyunsaturated fatty acids (see chapter 3.2.3), where most of them are very long-chain polyunsaturated fatty acids (LC-PUFAs). LC-PUFAs are important components of human diet and are mainly obtained from fish and algal oils of limited availability, which stresses the need of a sustainable source of these compounds for human 76 utilization (Lu et al., 2019). Metabolically engineered P. patens producing important very longchain polyunsaturated fatty acids were obtained by encoding 5-elongase from the marine algae Pavlova sp associated with vegetable oil supplementation to enhance biomass and LCPUFAs production (Chodok et al., 2012). The biosynthesis of docosatetraenoic acid or adrenic acid (ADA) and n-3 docosapentaenoic acid (DPA) was obtained from arachidonic acid (ARA) and EPA, produced by P. patens. The transgenic moss produces DPA that is a new source of docosahexaenoic acid (DHA) precursors for human diet (Chodok et al., 2012). Likewise, the liverwort M. polymorpha accumulates ARA, from which prostaglandin F2a, prostaglandin E2 and prostaglandin D2 were generated through heterologous expression of a cyclooxygenase gene from the red alga Gracilaria vermiculophylla (Takemura et al., 2013). In addition, the bioproduction of prostaglandins was increased using an in vitro reaction system and transgenic M. polymorpha offers the first bioproduction of PGs in plant species (Takemura et al., 2013). Figure 6. Air freshener moss-based product. (https://www.mosspirationbiotech.com). Perspectives In the past three decades, biotechnologies around bryophytes have gone from being simple and research laboratory scale only to become an industrial used technology. To our knowledge, only few companies currently use bryophytes in biotech production, but several other bryophyte-based products are marketed based on harvest from wild populations, which show that chemical wealth of bryophytes is slowly but surely being exploited. In the future, it is expected that many new products will arise from the ongoing bryophyte research including the use of contemporary synthetic biology technologies. From ongoing research, it can be expected that products within cosmetics, herbal remedies, perfumes and pharmaceutics will come to market within the next ten years, all based on bryophytes. The establishment of certified goodmanufacturing-practice for production in bioreactors (up to 500L) ensure that lucrative products can also be made within the lucrative market of pharmaceutics. Thus, it is foreseen that use of bryophytes as new production platforms for plant-derived and environmentally friendly products will increase in the next decade and allow for development of novel technologies that can also be applied to vascular plants. Author Contributions A.H. provided the conceptualization and outline under the supervision of H.T.S. A.H., A.P., I. L., Y.L., R.V.M and H.T.S. designed and wrote the manuscript. 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The chapter includes two subchapters: 2.1 explores the anti-inflammatory activity of thirty-two species of bryophytes, presented as paper 2, and 2.2 combines the initial screening of extracts for in vitro collagenase and elastase inhibitory activities. The screenings were carried out on crude extracts of bryophytes collected in different geographic locations with some species belonging to the same family or genus. The criteria for plant collection were mainly based on the availability of plant biomass in the field, respecting its regulatory aspects and the international agreement of access to genetic resources and benefit-sharing from the Nagoya Protocol (https://www.cbd.int/abs/). Furthermore, the bryophytes selected for the screenings were not well investigated chemically or biologically, in particular for the biological activities addressed in this thesis. 2.1 Anti-inflammatory activity of bryophytes extracts The anti-inflammatory activity of bryophytes extracts was assessed by their ability to inhibit induced nitric oxide production in macrophage cells as shown in paper 2. The mosses Dicranum majus (Figure 1A) and Thuidium delicatulum (Figure 1B) exhibited significant antiinflammatory properties. The investigation of nitric oxide inhibitory properties was realized in collaboration with the Molecular Engineering and Articular Physiopathology (IMoPA) research unit of the University of Lorraine located in Vandœuvre-lès-Nancy, France. I contributed to this study by collecting the plants, preparing the extractions/fractions, assisting in data analysis, and finally writing the manuscript. Figure 1. (A) Dicranum majus Turner and (B) Thuidium delicatulum (Hedw.) Schimp. Anti‑inflammatory activity of bryophytes extracts in LPS-stimulated RAW264.7 murine macrophages § Raíssa Volpatto Marques1, §Stefania E. Sestito2, Frédéric Bourgaud3,4, Sissi Miguel4, Sophie RahuelClermont2, Sandrine Boschi-Muller2, Henrik Toft Simonsen1*, David Moulin2 * § these authors contributed equally. * HTS and DM are co-corresponding authors 1 Department of Biotechnology and Biomedicine, Technical University of Denmark, Søltoft Plads 223, 2800 Kongens Lyngby, Denmark. 2 UMR 7365 CNRS-University of Lorraine Ingénierie Moléculaire et Physiopathologie Articulaire IMoPA, Biopole of the University of Lorraine, Avenue de la Forêt de Haye, 54500 Vandœuvre-lèsNancy, France. 3 Plant Advanced Technologies, 19 Avenue de la Forêt de Haye, 54500 Vandœuvre-lès-Nancy, France. 4 Cellengo, 19 Avenue de la Forêt de Haye, 54500 Vandœuvre-lès-Nancy, France *Corresponding authors, email: [email protected]; [email protected] 1. Abstract Bryophytes produce rare and bioactive compounds with a broad range of therapeutic potential, and many species are reported in ethnomedicinal uses. However, there are not that many studies on their potential as natural anti-inflammatory medicine. The present study investigates the anti‑inflammatory effects of thirty-two species of bryophytes, including mosses and liverworts, on Raw 264.7 murine macrophages stimulated with lipopolysaccharide (LPS) or human peroxiredoxin (hPrx1). The 70% ethanol extracts of bryophytes were screened for their potential to reduce the production of nitric oxide (NO), an important pro-inflammatory mediator. Among the analyzed extracts, two moss species significantly inhibited LPS‑induced NO without cytotoxic effects. The bioactive extracts of Dicranum majus and Thuidium delicatulum inhibited NO production in a concentration-dependent manner with IC50 values of 1.04 and 1.54 µg/mL, respectively. The crude 70% ethanol and ethyl acetate extracts were then partitioned with different solvents in increasing order of polarity (n-hexane, diethyl ether, chloroform, ethyl acetate, and n-butanol). The fractions were screened for their inhibitory effects on NO production stimulated with LPS at 1 ng/mL or 10 ng/mL. The NO production levels were significantly affected by the fractions of decreasing polarity such as n-hexane and diethyl ether ones. Therefore, the potential of these extracts to inhibit the LPS-induced NO pathway suggests their effective properties in attenuating inflammation and could represent a perspective for the development of innovative therapeutic agents. Keywords: bryophytes, mosses, Dicranum majus, Thuidium delicatulum, anti-inflammatory activity, nitric oxide. 102 2. Introduction The medicinal use of many species of bryophytes, including mosses and liverworts, has been reported in traditional Chinese medicine (TCM), Indian, Native American and also in traditional European use (1). Bryophytes have shown many ethnomedicinal applications such as for the treatment of skin diseases, inflammation, microbial infections, and many others (1, 2). Bryophytes produce important specialized metabolites, particularly terpenoids (mono-, sesqui- and diterpenoids) and aromatic compounds (mainly flavonoids, (bis)bibenzyls) as well as lipids, which have shown important biological activities (3). Although bryophytes are a valuable source of bioactive molecules, their biological properties and chemical constituents remain relatively unexplored. Inflammation is the physiological response of the body to overcome and contain infections (microbial) and injuries (physical, chemical, etc.) (4). Inflammatory reactions are generally acute but can become chronic leading to many diseases (5, 6). Pathogen-associated (PAMPs) and damage-associated (DAMPs) molecular pattern molecules are derived from microorganisms or released from damaged cells which are then recognized by pattern recognition receptor (PRR)-bearing cells activating the inflammatory response (7, 8). Lipopolysaccharides (LPSs) are examples of PAMPs found in the outer membrane of Gramnegative bacteria (7). In addition, peroxiredoxins (Prxs) act as alarmins and have been reported to play important roles in innate immunity by activating macrophages and promoting DAMPsassociated inflammatory diseases (9–12). Activated macrophage cells release a wide range of inflammatory mediators including nitric oxide (NO) and pro-inflammatory cytokines such as tumor necrosis factor-α (TNF‐α), interleukin (IL)-6, IL‐1β, and IL‐12 that are important signaling molecules in the inflammatory reaction (13). NO is an indicator of the inflammatory response and is synthesized by three types of NO synthases (NOS) (endothelial, neuronal, and inducible NOS (iNOS)) (14). During inflammation, increased levels of NO produced by iNOS have an important pathological role in many inflammatory diseases (15). Therefore, the NO inflammatory mediator is an important target for the development of drugs for anti-inflammatory therapy. In this study, the 70% ethanol extracts of thirty-two species of bryophytes including mosses and liverworts were evaluated for attenuating the NO production induced by LPS and hPrx1 molecules. The bioactive extracts were further partitioned into nonpolar to polar fractions and tested for their nitric oxide inhibitory activity. We demonstrated that the extracts and fractions of the mosses Dicranum majus and Thuidium delicatulum exhibited significant inhibitory effects on NO production in LPS-induced RAW 264.7 cells. 3. Material and methods 3.1 Plant material Thirty specimens of bryophytes were collected from different locations, including Germany, Denmark, Sweden, and Iceland. Three other species of mosses were purchased from a moss provider (Bryoflor, Paris, France) (http://www.bryoflor.com/). The list of species is found in the Supplementary Materials, Table 1. The specimens were identified by Professor Dr. Nils Cronberg (Department of Biology, Faculty of Sciences, Lund University, Lund, Sweden). Voucher specimens of Dicranum majus (ID no MTRaMa30) and Thuidium delicatulum (ID no MTRaMa34) were sent for deposition at the Lund University Botanical Museum (LD). The whole plants were dried at room temperature (samples from Germany) or in an oven at 40 oC and ground to a fine powder using a bead mill. 3.2 Recombinant hPrx1 production Recombinant wild-type hPrx1 was produced in Escherichia coli as an N-terminal fusion with a His-tag. hPrx1 was produced and purified as described by Kriznik et al. (16). 3.3 Extraction of small molecules for screening activities The powdered plants were homogenized in 70% ethanol (v/v) in water (1:10 g/mL of dry weight to solvent ratio) for the extraction of small molecules. The extractions were performed in an ultrasound bath (Blacksonic 275H) at 40 °C for 30 min followed by 24 h in an agitation mixer, adapted from previously reported protocol (17). Polytrichum formosum and Bazzania trilobata were extracted by maceration for 30 min in a rotating mixer at room temperature. After centrifugation, the supernatant was collected and used for analysis. The 70% ethanolic extracts were diluted to the concentrations as indicated in each experiment. 3.4 Extraction and fractionation of bioactive bryophytes According to the results of the screenings, the bioactive plant extracts were selected for further analysis. A second round of extractions was performed in which the powdered plants were homogenized in 70% ethanol (v/v) in water or ethyl acetate (1:10 g/mL of dry weight to solvent ratio) for the extraction of small molecules as previously described. After extractions, the solvents were evaporated under vacuum conditions. The remaining dry crude extracts of 70% ethanol and ethyl acetate extracts were suspended in water and successively partitioned with n-hexane, diethyl ether, chloroform, ethyl acetate, and n-butanol (Table 1). The crude 70% 104 ethanol and ethyl acetate extracts and their derivative fractions were evaporated and suspended in ethanol absolute for analysis. Table 1. Fractions yield (%) of bioactive crude extracts. Fractions yield (%) D. majus D. majus T. delicatulum T. delicatulum (70% ethanol (Ethyl acetate (70% ethanol (Ethyl acetate crude extract) crude extract) crude extract) crude extract) n-Hexane 7 13 5 8 Diethyl Ether 16 30 56 29 Chloroform 15 16 10 18 Ethyl Acetate 7 9 8 19 n-Butanol 12 10 10 23 3.5 Cell culture Murine macrophages Raw 264.7 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco Fisher Scientific) high glucose supplemented with 10% fetal bovine serum, Penstrep 1X, glutamine (2 mM), HEPES 20 mM pH 7.3, at 37 °C, 5% CO2, 95% humidity. Cells were washed in warm PBS, detached using a cell scraper and the cell concentration was estimated by using Trypan Blue (Sigma-Aldrich). 3.6 MTT Cell viability assay Raw 264.7 cells were seeded in a 96-well plate in 100 μL of DMEM without Phenol Red at a density of 1x104 cells/well and incubated overnight at 37 °C, 5% CO2, 95% humidity. Then, the medium was removed and the cells were treated with 100 µg/mL of the 70% ethanol extracts diluted in complete DMEM (90 µL/well, final solvent concentration =0.1%). DMSO 10% and PBS 10% were included as negative and positive controls, respectively. After overnight incubation, 10 μL of MTT solution (5 mg/mL in PBS) were added to each well. After 3 h incubation (37 °C, 5% CO2, 95% humidity), formazan crystals were dissolved with 100 µL/well of HCl 0.1 N in 2-propanol. Formazan concentration was determined by measuring the absorbance at 570 nm (Varioskan, Thermofisher). The results were normalized on untreated control (PBS) and expressed as the mean of percentage ± standard deviation of two independent experiments (n= 3-6). 3.7 Measurement of Nitric Oxide Raw 264.7 cells (5-7×105 cells/well) were seeded in 96-well plates in 150 μL of complete DMEM and incubated for 24 h. Then, the medium was removed and the cells were treated with 100 µg/mL of crude extracts or 10 µg/mL of extracts/fractions (190 µL/well), in triplicate. One hour later, the cells were stimulated with 1 ng/mL or 10 ng/mL LPS from Salmonella typhimurium or 300 nM of hPrx1 (10 µL/well) for 18h. The supernatants were collected and the Griess reaction was performed for nitrite quantification. The plate reading was assessed by using a spectrophotometer at 540 nm. Nitrite quantification was estimated by interpolating the standard curve and then normalized on untreated and stimulated cells. The bioactive extracts were tested in dose-response by applying the same test. 3.8 Statistical analysis Statistical analyses were performed using Prism Software (GraphPad Software, Inc., La Jolla, CA, USA). Differences between the mean values were assessed by one-way analysis of variance (ANOVA) followed by Bonferroni multiple comparison test. P<0.05 was considered statistically significant. The IC50 values were determined by non-linear regression analysis (GraphPad Prism software). 4. Results and Discussion 4.1 Effect of extracts on the viability of RAW 264.7 murine macrophage cells Before performing the activity screening, metabolic effects and, consequently, cytotoxicity of 70% ethanol extracts from bryophytes were evaluated at 100 µg/mL on RAW 264.7 macrophage cells by MTT assay, setting the threshold of cell viability at 70% (Figure 1). The extracts did not show cytotoxicity at the indicated concentration. 106 Figure 1. Effect of 70% ethanol extracts on the viability of RAW 264.7 cells determined by MTT assay. Cells were treated with 100 µg/mL of extracts for 24 h. Data represent mean ± standard deviation (n= 3-6) and values are normalized on control (PBS). The tested extracts revealed no significant difference with P-values > 0.05, calculated with ANOVA. The dashed line represents 70% of cell viability. 4.2 Anti‑inflammatory effects of extracts in hPrx1 or LPS stimulated RAW264.7 murine macrophage cells Nitric Oxide (NO) is a pro-inflammatory mediator and typical marker of inflammation produced in response to a pathogen as well as DAMP, such as hPrx1 (12). Intracellular hPrx1 is a peroxidase involved in the redox signaling in physiological conditions, but it was proposed to function as DAMP by activating Toll-like receptor (TLR) 4. Diverse stress conditions, including cerebral ischemia (12, 18, 19), induce the release of hPrx1 in the extracellular environment with increased expression of TLR4, nuclear translocation of nuclear factor κB (NF-κB) p65 and production of pro-inflammatory mediators (NO, TNF-α and IL-6). The anti-inflammatory activity of 70% ethanol extracts of a range of bryophytes was determined by assessing their potential to inhibit the production of NO induced by hPrx1 at 300 nM in RAW cells using Griess reagent (Figure 2). However, none of the tested whole extracts was considered bioactive in these conditions. Figure 2. A) NO-induced hPrx1. Cells were stimulated with hPrx1 300 nM and the amount of induced NO was quantified by Griess reaction. After 18 h incubation, an amount of 10 µM of NO was measured in the supernatants. The graph represents mean ± standard deviation; ANOVA analysis and Tukey’s test were used for the analysis (****P < 0.0001). B) Effect of 70% ethanol extracts on hPrx1-induced NO in RAW 264.7 cells. Cells pre-treated with 100 µg/mL of extracts for 1 h were stimulated with hPrx1 (300 nM) for 18 h. The NO content of the culture medium was analyzed by the Griess reagent method. Data represent means ± standard deviation (n= 3) and values are normalized on hPrx1. The dashed line represents the level of NO in cells stimulated by hPrx1 alone. Sterile inflammation is not the unique responsible for NO production in cells, but also bacterial endotoxins can induce strong and wide immune responses. Thus, the anti-inflammatory effect of 70% ethanol extracts in reducing NO level was evaluated by performing Griess reaction on supernatants of lipopolysaccharides (LPS)-stimulated RAW cells (Figure 3). Cells were treated 108 with the extracts at 100 µg/mL and then stimulated for 18h with LPS from Salmonella typhimurium at 1 ng/mL. After incubation, nitrite quantification was used as an indicator to estimate NO level in the medium. Among the tested samples, the extracts of the mosses Dicranum majus and Thuidium delicatulum significantly inhibited the production of NO in LPS-stimulated cells, at 68% and 41%, respectively (Figure 3). Figure 3. A) LPS- NO induction in murine macrophages. The amount of NO in cells treated with LPS 1 ng/mL for 18 h is significant different (****P < 0.0001) compared to untreated cells. The graph represents the mean ± standard deviation of three independent experiments. ANOVA and Tukey’s test were used for statistical analysis. B) Effect of 70% ethanol extracts on LPS-induced NO in RAW 264.7 cells. Cells pre-treated with 100 µg/mL of extracts for 1 h were stimulated with LPS (1 ng/mL) for 18h. The NO content of the culture medium was analyzed by the Griess reagent method. Data represent mean ± standard deviation (n= 7-11) and values are normalized on LPS. The tested extracts revealed a significant difference with P-values < 0.05, calculated with ANOVA. **P < 0.01, ****P < 0.0001 indicate significant differences compared to the control. The dashed line represents the level of NO in cells stimulated by LPS alone. The bioactive extracts were tested again at increasing concentrations finding that they decrease the NO level in a concentration-dependent manner with IC50 values of 1.04 µg/mL and 1.54 µg/mL for D. majus and T. delicatulum extracts, respectively (Figure 4). Figure 4. Dose-response effect and IC50 values of 70% ethanol extract from (A) D. majus and (B) T. delicatulum, on LPS-induced NO in RAW264.7 cells. Data represent mean ± standard deviation (n=23 of cells treated with extracts) and values are normalized on LPS (1 ng/mL). 4.3 Anti‑inflammatory effects of fractionated extracts in LPS-stimulated RAW264.7 murine macrophage cells The dried 70% ethanol extracts of D. majus and T. delicatulum were further partitioned into gradient of non-polar to polar fractions through a series of organic solvents (n-hexane, diethyl ether, chloroform, ethyl acetate, and n-butanol). The fractions at 10 µg/mL were screened for their inhibitory effects on NO production in RAW 264.7 cells stimulated with LPS at 1 ng/mL 110 or 10 ng/mL for 18 h. The fractions obtained for solvent of low polarity, such as n-hexane and diethyl ether, showed the maximum effect of NO reduction induced with 1 ng/mL and 10 ng/mL doses (Figure 5A and 5B). For both mosses, the inhibitory activity of the fractions on LPS (10 ng/mL)-induced NO was found to be more significant as compared to the 70% ethanol crude extracts. The inhibition of n-hexane fractions on LPS (10 ng/mL)-induced NO was observed to be 78% and 66% for D. majus and T. delicatulum, respectively (Figure 5A and 5B). In addition, the diethyl ether fractions of D. majus and T. delicatulum showed 53% and 52% of NO inhibition, respectively. Figure 5. Effect of the fractions of the 70% ethanol crude extracts from (A) D. majus and (B) T. delicatulum on LPS-induced NO in RAW264.7 cells. Cells pre-treated with 10 µg/mL of extracts for 1 h were stimulated with LPS at 1 ng/mL or 10 ng/mL for 18 h. The NO content was analyzed by the Griess reagent method. Data represent mean ± standard deviation (n= 2-3). Values are normalized on LPS. The tested extracts revealed a significant difference with P-values < 0.05, calculated with ANOVA. *P<0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 indicate significant differences compared to the control. The bioactive plants were subsequently extracted with ethyl acetate and both crude extracts and derived fractions (n-hexane, diethyl ether, chloroform, ethyl acetate, and n-butanol) were screened for NO inhibition stimulated with LPS at 1 ng/mL or 10 ng/mL for 18h. As previously, the results indicated that the inhibitory effect of D. majus was prevalent in the fractions prepared with less polar solvents (Figure 6A). The inhibition of n-hexane and diethyl ether fractions of D. majus on LPS (10 ng/mL)-induced NO was observed to be 65% (Figure 6A). In addition, the ethyl acetate crude extract of D. majus exhibited the most potent LPS (10 ng/mL)-induced NO inhibition (60%) as compared to 70% ethanol crude extract (20%). In general, T. delicatulum ethyl acetate crude extracts and obtained fractions exhibited lower potential to decrease NO production in both induced LPS 1 and 10 ng/mL doses. Among the tested fractions, the NO was decreased below 36% in LPS (10 ng/mL)-stimulated cells (Figure 6B). There was no significant difference in the NO inhibition potential among the fractions derived of ethyl acetate crude extract on LPS (10 ng/mL)-induced cells; as indicated by the percent inhibition of ethyl acetate crude extract (29%), n-hexane (33%) and diethyl ether (26%) fractions. 112 Figure 6. Effect of the fractions of the ethyl acetate crude extracts from (A) D. majus and (B) T. delicatulum on LPS-induced NO in RAW264.7 cells. Cells pre-treated with 10 µg/mL of extracts for 1 h were stimulated with LPS at 1 ng/mL or 10 ng/mL for 18 h. The NO content of the culture medium was analyzed by the Griess reagent method. Data represent mean ± standard deviation (n= 2-3). Values are normalized on LPS. The tested extracts revealed a significant difference with P-values < 0.05, calculated with ANOVA. *P<0.05, ***P < 0.001, ****P < 0.0001 indicate significant differences compared to the control. To our knowledge, this study is the first to report the potential anti-inflammatory activities of the mosses D. majus and T. delicatulum. The results indicate the presence of bioactive compounds in both 70% ethanol and ethyl acetate extracts and fractions possessing significant anti-inflammatory activity. It was observed that the non-polar fractions had a higher antiinflammatory activity. Those different levels of activity may be related to the concentration of bioactive compounds as well as their polarity. Among the explored biological properties of these plants, D. majus has been reported with antibacterial activity (20). In previous studies, the dichloromethane extract from Dicranum scoparium, which belongs to the same family of D. majus, was reported with anti-inflammatory activity by inhibiting 90% of 15-lipoxygenase (15-LOX) at 100 µg/mL, which has an important role in inflammatory diseases (21, 22). The bioactive compound dicranin was then isolated from D. scoparium and exhibited potent inhibition of 15-LOX (21). However, the extract from D. scoparium was not bioactive in this study. Thuidium spp. has been reported as an antibacterial and anti-inflammatory agent in China (23). Moreover, a terpenoid-rich fraction of the methanol crude extract of Thuidium tamariscellum has exhibited anti-inflammatory activities by inhibiting the activity of enzymes involved in inflammatory pathways such as cyclooxygenase, LOX, and myeloperoxidase and also by decreasing the levels of LPS-induced NO (24). Investigations of the biological properties of the ethanol extract and fractions (acetone, chloroform, and water) from T. delicatulum have been reported with antibacterial and antifungal activities (25). Other studies report the anti-inflammatory activity from extracts and isolated constituents of bryophytes on NO production inhibition. Hence, in previous investigations, the treatment with 50 μg/mL of peat moss (Sphagnum sp.) aqueous extract inhibited the production of NO in LPSstimulated (500 ng/mL, 24 h) RAW 264.7 cells (26). Nevertheless, the extracts of Sphagnum teres and Sphagnum fimbriatum analyzed in the present study showed no anti-inflammatory activities. The methanol extract of Polytrichum commune (Polytrichaceae) was reported to inhibit the NO production induced by the treatment of LPS (1 µg/mL, for 24 h) with an IC50 of 65.15 µg/mL (27). Although, the extract from Polytrichum formosum (Polytrichaceae) showed no anti-inflammatory activities in our screening. Differences in anti-inflammatory properties between plant species may be due to their different chemical constituents, which may also vary depending on the species’ geographical origin and exposure to various environmental factors (e.g., season, soil, climate, etc.) (28, 29). Among the natural products investigated from bryophytes, many bis-bibenzyls isolated from liverworts, including riccardins, marchantins, perrottetins and marchantins derivatives, have been shown to inhibit LPS-induced NO production with IC50 values ranging from 1.44 to 62.16 µM (30). Sesquiterpenoids such as cuparenes from Bazzania decrescens and herbertane from Mastigophora diclados showed inhibition of LPS-induced NO production with IC50 values in the range of 4.1 - 76 µM (30). Myltaylane-type sesquiterpenoid from Bazzania nitida also has shown potent inhibition of NO production with IC50 of 6.3 µM (31). 114 5. Conclusion The present study investigated the anti‑inflammatory effects of thirty-two species of bryophytes on human peroxiredoxin (hPrx1) or lipopolysaccharide (LPS) stimulated RAW264.7 murine macrophage cells. The 70% ethanol extracts were screened for their potential to reduce the production of nitric oxide (NO). Although the extracts showed no inhibition of NO stimulated by hPrx1, two species of mosses significantly inhibited LPS‑induced NO. The bioactive extracts of D. majus and T. delicatulum inhibited NO in a concentration-dependent manner with IC50 values of 1.04 and 1.54 µg/mL, respectively. Among the tested fractions of the crude extracts, the n-hexane and diethyl ether fractions reduced NO production more efficiently. The potential of the extracts to inhibit LPS-induced NO pathway indicates their effective properties in attenuating the inflammatory response. The inhibitory properties of these extracts may present new sources of natural ingredients for antiinflammatory drug discovery. Further studies on their efficacy activities, mode of actions and identification of bioactive compounds should be performed. 6. Author contributions Conceptualization and supervision of the project– D. M., S.R.C, S.B.M, S.M., H.T.S and F.B.; Collection of plant material and extractions/fractionations – R.V.M.; Cell viability and nitric oxide measurements-S.E.S.; Analysis and data interpretation– S.E.S, D.M., R.V.M.; writing— original draft preparation– R.V.M.; writing—review and editing– S.E.S.; D.M., S.R.C, S.B.M, S.M. and H.T.S. 7. Acknowledgement The research leading to these results has received funding from the Marie Curie Actions of the European Union's Horizon 2020 under grant agreement n° 765115 – MossTech. The authors thank Professor Nils Cronberg, Lund University, Sweden, for support in plant identification. The authors acknowledge financial support from the "Impact Biomolecules" project of the "Lorraine Université d'Excellence" (Investissements d’avenir – ANR). 8. Conflict of interest The authors declare no conflict of interest. References 1. MS Sabovljević; AD Sabovljević; N kusaira K Ikram; A Peramuna; H Bae; HT Simonsen. Bryophytes-an emerging source for herbal remedies and chemical production. Plant Genet Resour Characterisation Util 14, 314–327 (2016) 2. S Chandra; D Chandra; A Barh; Pankaj; RK Pandey; IP Sharma. Bryophytes: Hoard of remedies, an ethno-medicinal review. J Tradit Complement Med 7, 94–98 (2017) 3. 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Prog Chem Org Nat Prod 95, 1–796 (2013) 31. L Harinantenaina; Y Asakawa. Chemical constituents of Malagasy liverworts. 6. A myltaylane caffeate with nitric oxide inhibitory activity from Bazzania nitida. J Nat Prod 70, 856–858 (2007) 118 Supplementary Materials Table 1. List of species screened for anti-inflammatory activity and information on collection area. Group of bryophytes Family Species Countrycollection place GPS coordinates Latitude Date of collection Longitude Mosses Amblystegiaceae Warnstorfia fluitans Iceland 64.14197491 -20.23437432 12 June 2019 64.14389823 -20.22702004 12 June 2019 47.917959 8.075349 28 April 2018 Denmark, Rude 55.84145 Skov 12.47838 24-25 2018 (Hedw.) Loeske Bartramiaceae Philonotis fontana Iceland (Hedw.) Brid. Brachytheciaceae Brachythecium rutabulum (Hedw.) Germany, Black Forest Schimp. Cirriphyllum crassinervium October (Taylor) Loeske & M. Fleisch. Homalothecium Sweden 55.7168043 13.7214639 10 2019 Germany, Black Forest 47.917733 8.075181 28 April 2018 Moss provider - - Purchased on lutescens (Hedw.) H. September Rob. Homalothecium sericeum (Hedw.) Schimp. Kindbergia praelonga (Hedw.) Ochyra Bryaceae Bryum pseudotriquetrum 120 (Bryoflor, Paris, France) Germany, Black Forest 20 February 2019 47.912221 8.081158 28 April 2018 (Hedw.) P. Gaertn., B. Mey. & Scherb. Dicranaceae Dicranum majus Sweden 56.02397 13.13074 27 October 2018 Dicranum scoparium Denmark, Rude 55.84072 Skov Hedw. 12.47253 24-25 2018 Racomitrium Turner Grimmiaceae aciculare (Hedw.) October Germany, Black Forest 47.910660 8.093621 28 April 2018 Sweden 55.7264294 13.70606602 10 2019 Moss provider - Brid. Racomitrium elongatum Ehrh. ex September Frisvoll Hedwigiaceae Hedwigia ciliata (Hedw.) P. Beauv. Purchased on 20 February 2019 (Bryoflor, Paris, France) Hylocomiaceae Hylocomium splendens Sweden 55.7223757 13.7066895 10 2019 Germany, Black Forest 47.911177 8.092482 28 April 2018 Denmark, Rude 55.84071 Skov 12.47371 24-25 2018 Iceland -20.28594921 13 June 2019 (Hedw.) September Schimp. Rhytidiadelphus loreus (Hedw.) Warnst. Rhytidiadelphus squarrosus (Hedw.) October Warnst. Rhytidiadelphus triquetrus Warnst. 122 (Hedw.) 64.32743787 Rhytidiadelphus triquetrus Sweden 55.721833 13.7021155 10 2019 September Denmark, Rude 55.84201 Skov 12.4748 24-25 2018 October Moss provider - Purchased on (Hedw.) Warnst. Hypnaceae Hypnum cupressiforme Hedw. Leucobryaceae Campylopus introflexus (Hedw.) Brid. Mniaceae Mnium Plagiomnium T.J. Kop. (Bryoflor, Paris, France) 20 February 2019 hornum Denmark, Rude 55.84161 Skov Hedw. undulatum - (Hedw.) Sweden 55.72548924 12.47275 24-25 2018 October 13.7051406 10 2019 September Neckeraceae Thamnobryum alopecurum (Hedw.) Germany, Black Forest 47.917927 8.074989 28 April 2018 Denmark, Rude 55.84915 Skov 12.4582 24-25 2018 Germany, Black Forest 47.911223 8.092431 28 April 2018 Denmark, Rude 55.84113 Skov 12.48188 24-25 2018 Germany, Black Forest 8.089162 28 April 2018 Nieuwl. ex Gangulee Plagiotheciaceae Plagiothecium undulatum (Hedw.) October Schimp. Polytrichaceae Polytrichum formosum Hedw. Pylaisiaceae Calliergonella cuspidata (Hedw.) October Loeske Ptilium crista-castrensis (Hedw.) De Not. 124 47.911766 Sphagnaceae Sphagnum 12.47348 24-25 2018 64.32743787 -20.28594921 13 June 2019 Sweden 56.02422 13.12932 27 October 2018 Germany, Black Forest 47.917927 8.074989 28 April 2018 trilobata Germany, Black Forest 47.911223 8.092431 28 April 2018 fimbriatum Wilson Sphagnum Denmark, Rude 55.84098 Skov teres Iceland October (Schimp.) Ångström Thuidiaceae Thuidium delicatulum (Hedw.) Schimp. Liverworts Plagiochilaceae Plagiochila asplenioides (L.) Dumort. Lepidoziaceae Bazzania (L.) Gray Metzgeriaceae Metzgeria (L.) Corda 126 furcata Denmark, Rude 55.84182 Skov 12.47596 24-25 2018 October 2.2 Preliminary screening of collagenase and elastase inhibitory activities of bryophytes extracts Plant extracts or derived molecules find important applications as anti-aging ingredients in skincare formulations. Therefore, in the search for active extracts of cosmetic interest, bryophytes were tested for their inhibitory effects on collagenase and elastase activities, two important enzymes involved in skin aging. The 70% ethanol extracts were tested for in vitro inhibitory activities on collagenase from Clostridium histolyticum and elastase from porcine pancreas, which have extensive use in biological screening studies [1–3]. 2.2.1 Overview of experimental work 2.2.1.1 Plant material and preparation of the extracts The plant material used in this work was sampled in the field or acquired commercially. The plants were identified by Professor Dr. Nils Cronberg (Department of Biology, Faculty of Sciences, Lund University, Lund, Sweden). Information about collection sites is found in the supplementary material of chapter 2.1 (paper 2). Briefly, the plants were first cleaned to eliminate soil or insects and then dried (room temperature or 40 oC) for the analysis. The dried plants were ground into a fine powder using a laboratory mill for further extraction of small molecules. The solvent selected for the extraction procedure was 70% ethanol (v/v) in water. The detailed extraction preparation is described in chapter 2.1 (paper 2). 2.2.1.2 In vitro collagenase and elastase inhibitory activities The anti-collagenase activity of crude extracts at a final concentration of 8.33 mg/mL was determined as the procedure described in papers 3 and 4. The anti-elastase activity of Polytrichum formosum and Bazzania trilobata extracts at a final concentration of 2.66 mg/mL was determined according to the method in paper 4. The remaining extracts were screened for elastase inhibitory activity at a final concentration of 8.33 mg/mL as the following protocol: Elastase inhibitory activity was determined based on the detection of enzymatic-driven conversion of N-succinyl-Ala-Ala-Ala-p-nitroanilide (SAAApNA; Sigma-Aldrich, ref. S4760) to p-nitroanilide (pNA). The SAAApNA conversion rate into pNA is proportional to the activity of the enzyme and inversely proportional to the inhibitory properties of the test sample. Briefly, the reaction mixture contained 100 μL of elastase synthetic substrate: SAAApNA (1.5 mM in 50 mM Tris buffer containing, 10 mM CaCl2 and 400 mM NaCl, pH 7,5) and 10 μL of plant extract (test sample) or pure solvent of the sample (blank, control). Enzymatic conversion was initiated by addition of 10 μL of 0.05 mg/mL of porcine pancreatic elastase (SigmaAldrich, ref. E7885-5MG) in the same buffer as its substrate SAAApNA. To determine the rate of enzymatic conversion, the content of pNA was quantified at the beginning of the reaction and 30 min after its initialization by stopping the enzymatic conversion of SAAApNA by H2O, pH 1. To stop the enzymatic conversion, a volume of 50 µL of the test sample was mixed with a volume of 50 µL of H2O, pH 1. The content of pNA in the samples was then quantified by the UHPLC method optimized to separate SAAApNA and pNA. Briefly, the analysis was conducted using Kinetex Biphenyl reverse phase column (150 mm × 2.1 mm, 2.6 µm, Phenomenex, Torrance, CA, USA), maintained at 40 oC. The mobile phase was composed of water containing 0.1% vol. of formic acid (A) and pure acetonitrile (B), delivered at 0.5 mL/min with the gradient of B phase as follows: 5–41% (0–9 min); 41– 90% (9–9.05 min); hold at 90% (9.05–11.50 min); 90–5% (11.50–11.55 min), hold at 5% (11.55–14.50 min). Sample injection volume was 5 µL and SAAApNA/pNA detection at 400 nm. Residual activity (ActR%) and the inhibition degree (Inh%) of elastase in the presence of test sample X were then calculated using the equations: pNA, 400nm ActR%EchX = pNA, 400nm AUC EchX,T=30 −AUC EchX,T=0 pNA, 400nm AUC BL,T=30 pNA, 400nm x 100% − AUC BL,T=0 Inh%EchX = 100% − ActR %EchX where: ActR%—residual activity; Inh%—inhibition degree; AUC—peak area of pNA; EchX—tested sample; BL—blank. 2.2.2 Results In this study, the first screening of collagenase and elastase inhibitory activities was carried out on the hydroalcoholic plant extracts. The screening, reported in Table 1, allowed the selection of two active extracts that showed inhibitory activity only towards collagenase with the percentage of inhibition equal to or higher than 40%. The two extracts from the moss Polytrichum formosum and the liverwort Bazzania trilobata were subjected to a second level 128 of screening and chemical investigation of bioactive constituents as presented in two papers in chapter 3 and 4. Table 1. Species screened for inhibitory effects on collagenase and elastase activities. The whole plant was used for the study. The table indicates the percentage of enzyme inhibitory activity tested with bryophyte extracts at a final concentration of 8.33 mg/mL. ND: Not detected. *tested at a final concentration of 2.66 mg/mL. Group of bryophytes Family Species Amblystegiaceae Warnstorfia fluitans (Hedw.) Loeske Bartramiaceae Philonotis fontana (Hedw.) Brid. Brachytheciaceae Brachythecium rutabulum (Hedw.) Schimp. Collagenase Inhibitory activity Elastase Inhibitory activity ND ND ND ND ND ND ND ND ND ND ND ND ND ND Mosses Cirriphyllum crassinervium (Taylor) Loeske & M.Fleisch. Homalothecium lutescens (Hedw.) H. Rob. Homalothecium sericeum (Hedw.) Schimp. Kindbergia praelonga (Hedw.) Ochyra Bryaceae Bryum pseudotriquetrum (Hedw.) P. Gaertn., B. Mey. & Scherb. Dicranaceae Dichodontium palustre (Dicks.) M. Stech ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND (Collected: Iceland; GPS 64.37287118/ 20.13756961; Voucher ID no MTRaMa55) Dicranum majus Turner Dicranum scoparium Hedw. Grimmiaceae Racomitrium aciculare (Hedw.) Brid. Racomitrium elongatum Ehrh. ex Frisvoll Hedwigiaceae Hylocomiaceae Hedwigia ciliata (Hedw.) P. Beauv. Hylocomium splendens (Hedw.) Schimp. Rhytidiadelphus loreus (Hedw.) Warnst. 130 Rhytidiadelphus squarrosus (Hedw.) Warnst. Rhytidiadelphus triquetrus (Hedw.) Warnst. (from Iceland) Rhytidiadelphus triquetrus (Hedw.) Warnst. (from Sweden) Hypnaceae Hypnum cupressiforme Hedw. Leucobryaceae Campylopus introflexus (Hedw.) Brid. Mniaceae Mnium hornum Hedw. Plagiomnium undulatum (Hedw.) T.J. Kop. Neckeraceae Thamnobryum alopecurum (Hedw.) Nieuwl. ex Gangulee Plagiotheciaceae Plagiothecium undulatum (Hedw.) Schimp. Polytrichaceae Polytrichum formosum Hedw. ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 71% *ND Pylaisiaceae Calliergonella cuspidata (Hedw.) Loeske Ptilium cristacastrensis (Hedw.) De Not. Sphagnaceae Sphagnum fimbriatum Wilson Sphagnum teres (Schimp.) Ångström Thuidiaceae Thuidium delicatulum (Hedw.) Schimp. Plagiochilaceae Plagiochila asplenioides (L.) Dumort. Lepidoziaceae Bazzania trilobata (L.) Gray Metzgeriaceae Metzgeria furcata (L.) Corda ND ND ND ND ND ND ND ND ND ND ND ND 40% *ND ND ND Liverworts 132 References [1] Chiocchio I, Mandrone M, Sanna C, et al. Screening of a hundred plant extracts as tyrosinase and elastase inhibitors, two enzymatic targets of cosmetic interest. Ind Crops Prod 2018; 122: 498–505. doi:10.1016/J.INDCROP.2018.06.029 [2] Lee KE, Bharadwaj S, Yadava U, et al. Evaluation of caffeine as inhibitor against collagenase, elastase and tyrosinase using in silico and in vitro approach. J Enzyme Inhib Med Chem 2019; 34: 927–936. doi:10.1080/14756366.2019.1596904 [3] Mungmai L, Preedalikit W, Pintha K, et al. Collagenase and Melanogenesis Inhibitory Effects of Perilla Frutescens Pomace Extract and Its Efficacy in Topical Cosmetic Formulations. Cosmetics 2020; 7: 69. doi:10.3390/COSMETICS7030069 Chapter 3 Collagenase and tyrosinase inhibitory effect of isolated constituents from the moss Polytrichum formosum 134 Introduction to the chapter The research presented in this chapter was performed throughout the Ph.D. thesis. This chapter describes the in vitro inhibitory properties of the extracts and isolated metabolites from the moss Polytrichum formosum Hedw. (Figure 1) on collagenase and tyrosinase activities. The results of this study show that P. formosum offer a source of new chemical structures with novel biological activities for biotechnological applications. I contributed to this study by collecting P. formosum, performing the extraction of metabolites from P. formosum and tested them for in vitro inhibitory enzymatic activities. I also have investigated the bioactive compounds by the Target Binding® technology, performed their purification, and tested their bioactivities. Finally, I have written the manuscript. Figure 1. Polytrichum formosum Hedw. 136 138 140 142 144 146 148 150 152 154 156 Chapter 4 Extracts from the liverwort Bazzania trilobata with potential dermocosmetic properties 158 Introduction to the chapter The research presented in this chapter was performed throughout the Ph.D. thesis. This chapter describes the in vitro biological properties of the extracts from the liverwort Bazzania trilobata (L.) Gray (Figure 1) on key biological activities of cosmetic interest. The phytochemical constituents of the bioactive extracts were also explored. The chapter includes two sections, section 4.1 with paper 4 and section 4.2 with the additional investigation of bioactive compounds of B. trilobata extracts. I contributed to this study by collecting B. trilobata, performing the extractions, analyzing the total phenolic content, antioxidant and inhibitory enzymatic activities. I also performed the investigation of chemical constituents by UHPLC-MS, Target binding®, and purification of compounds. Finally, I have written the manuscript. Figure 1. Bazzania trilobata (L.) Gray. 4.1 Extracts from the liverwort Bazzania trilobata with potential dermocosmetic properties Raíssa Volpatto Marques1, Aleksander Salwinski2, Kasper Enemark-Rasmussen3, Charlotte H. Gotfredsen3, Yi Lu1, Nicolas Hocquigny4,5, Arnaud Risler4, Raphaël E. Duval4,5, Sissi Miguel6, Frédéric Bourgaud2,6, Henrik Toft Si-monsen1* 1 Department of Biotechnology and Biomedicine, Technical University of Denmark, Søltoft Plads 223, 2800 Kongens Lyngby, Denmark; [email protected]; [email protected]; [email protected] 2 Plant Advanced Technologies, 19 Avenue de la Forêt de Haye, 54500 Vandœuvre-lès-Nancy, France; [email protected]; [email protected] 3 Department of Chemistry, Technical University of Denmark, Lyngby, Denmark; [email protected]; [email protected] 4 Université de Lorraine, CNRS, L2CM, F-54000 Nancy, France; [email protected]; [email protected] 5 ABC Platform®, Faculté de Pharmacie, F-54505 Vandœuvre-lès-Nancy, France; 6 Cellengo, 19 Avenue de la Forêt de Haye, 54500 Vandœuvre-lès-Nancy, France; [email protected] *Correspondence: [email protected] Abstract Bazzania trilobata (L.) Gray is a leafy liverwort from the family of Lepidoziaceae, well known for their antifungal properties. In this study, the 70% ethanol and methanol extracts of B. trilobata were investigated for new in vitro biological activities of cosmetic interest. The results showed that the total phenol content, the DPPH (1,1- Diphenyl-2-Picryl- Hydrazyl) free radical scavenging activity and the anti-collagenase activity of the 70% ethanol extract were higher than for methanol. The methanol extract showed mild tyrosinase inhibitory activity and antimicrobial properties towards the Gram-positive bacteria Enterococcus faecalis. Lignans, coumarins, and bis-bibenzyls were the major classes of phenolic constituents tentatively identified in both extracts. In addition, a known drimenyl caffeate was identified for the first time in B. trilobata and its structure was confirmed by NMR spectroscopy. These results suggest that extracts from B. trilobata could be exploited as an interesting new source of natural active ingredients for cosmetic applications. Keywords: Bazzania trilobata, Lepidoziaceae, liverwort, antioxidant, antimicrobial, collagenase inhibitory activity, tyrosinase inhibitory activity, chemical constituents, drimenyl caffeate 160 1. Introduction Bazzania trilobata (L.) Gray (Lepidoziaceae) is a leafy liverwort with a circumboreal distribution, including western Europe, eastern and western USA, and Japan, which grows in extensive gametophyte mats [1]. B. trilobata has been described for its anti-tumor [2] and antifungal properties [3,4]. There is already a commercial antifungal and antibacterial product in Germany based on an ethanol extract of B. trilobata [5,6]. Sesquiterpenes and bis-bibenzyls have been reported as antifungal constituents from B. trilobata [4]. Extracts and isolated compounds from other species of Bazzania have shown therapeutic potential with antitumor [7,8], antimicrobial [9,10] and inhibitory effects on nitric oxide production [11,12]. Thus, Bazzania spp. are a source of valuable bioactive compounds. However, the knowledge of biological activities available from Bazzania spp. and other bryophytes is little compared to that of higher plants [13–15]. Therefore, this study provides additional knowledge on the new potential biological properties of extracts from B. trilobata. Bioactive plant extracts have found valuable applications, especially within cosmetics and herbal remedies. Plant extracts rich in polyphenols are an important source of natural antioxidant ingredients for the protection of the skin against free radicals [16]. Plant metabolites are also applied as anti-wrinkle and skin lightening agents. One of the key targets in the cosmetic industry is the discovery of inhibitors of aging-related enzymes such as collagenase and elastase. These enzymes, when overexpressed, can lead to accelerated proteolytic degradation of collagen and elastin fibers in the extracellular matrix that impact the integrity and elasticity of the skin [17]. Another important target is tyrosinase, the main enzyme in melanin synthetic pathway. Inhibition of its activity is one of the ways of preventing skin hyperpigmentation disorders [18]. Furthermore, it is an advantage to obtain extracts of cosmetic interest with additional antimicrobial activity. These ingredients are called preservative boosters and can contribute to lowering the concentration of the synthetic preservatives in final cosmetic formulations [19]. In this study, the inhibitory effects of 70% ethanol and methanol extracts from B. trilobata towards skin aging and pigmentation-related enzymes, as well as their antioxidant and antimicrobial properties were investigated. The phytochemical constituents of both extracts were tentatively identified. 2. Results and Discussion 2.1. Determination of Phenolic Content of B. trilobata Extracts Plants extracts containing polyphenols have shown significant redox properties with antioxidants and health benefits for humans [20]. Polyphenolic extracts have found valuable applications as active ingredients in cosmetic formulations due to their range of properties such as antioxidants, antimicrobial, anti-inflammatory and anti-aging activities [21]. Thus, the total phenolic content (TPC) of the 70% ethanol and methanol extracts of B. trilobata was determined based on the colorimetric Folin-Ciocalteu method. The TPC was expressed as gallic acid equivalents (Table 1). The TPC of 70% ethanol-based was shown to be higher by 38% than the methanol-based equivalent, which is possible due to the difference in the solvent polarity that provides a better phenol extraction efficiency (Table 1). Polyphenolic compounds have shown to be abundantly present in liverworts [22]. B. trilobata was described as a source of rare cyclic bis-bibenzyls and chlorinated bis-bibenzyls, e.g. isoplagiochin C and bazzanins (Table 2) [4,23,24]. Other polyphenolic constituents such as lignans are also highly present in B. trilobata, e.g. trilobatins (Table 2) [25,26]. Moreover, coumarins were already reported from B. trilobata extracts, e.g. 7,8-dihydroxy-7-O-β-D-glucuronide (Table 2) [4,27]. Table 1. Total phenolic content (TPC) and DPPH free radical-scavenging activity. Extracts 70% Ethanol Methanol Ascorbic Acid TPC (mg GAE/100 mg)1 1.30 0.95 - DPPH radical scavenging activity, IC50 (µg/mL) 82 122 2 1 TPC values were expressed as gallic acid equivalents (GAE) in mg per 100 mg of dry plant material. 2.2. Antioxidant Activity of B. trilobata extracts Bryophytes have developed efficient antioxidant machinery to overcome biotic and abiotic stresses, this leads to a promising alternative source of antioxidants compounds [28]. Antioxidants are molecules that neutralize free radicals, which play an important role in the prevention of various diseases and skin aging [29]. In the antioxidant screening, 70% ethanol and methanol extracts of B. trilobata were investigated by the DPPH (1-diphenyl-2picrylhydrazyl) scavenging assay. Both extracts revealed a reducing power, however, the 162 DPPH radical scavenging ability of the 70% ethanol (IC 50 82 µg/mL) and methanol (IC50 122 µg/mL) extracts were lower than ascorbic acid used as a positive control [30] (Table 1). These results agree with the TPC of the extracts indicating that the 70% ethanol extract richer in phenols have stronger antioxidant properties. Phenolic compounds have key role as antioxidants and their activity is mainly related to the number and arrangement of hydroxyl groups in their molecular structure [16]. 2.3. Collagenase, Elastase and Tyrosinase inhibitory Activity To expand the knowledge of the biological activities of the extracts from B. trilobata, we attempted to investigate their potential as skin anti-aging and anti-pigmentation ingredients in cosmetic formulations. Therefore, the ability to inhibit the activity of three target enzymes of cosmetic interest was investigated. The results showed that the 70% ethanol extract inhibited 40% of collagenase activity at the final concentration of 8.33 mg/mL whereas the methanol extract inhibited 20% at final concentration of 6.66 mg/mL. The extracts exhibited limited anticollagenase activity compared to that of the positive control EDTA (94% at 1.49 mg/mL) (Figure 1A). Both extracts were tested for tyrosinase activity inhibition at the final concentration of 5.33 mg/mL together with the positive control kojic acid at 0.04 mg/mL. Only the methanol extract showed moderate tyrosinase inhibition of 43%, however, lower than kojic acid that showed 99% (Figure 1B). Furthermore, both extracts showed no elastase inhibitory activity at the final concentration of 2.66 mg/mL. This is the first report of the effects of B. trilobata extracts on collagenase and tyrosinase activities. Indeed, few studies have reported the activity of extracts or isolated metabolites from bryophytes on these target enzymes. Recently, the n-hexane and chloroform extracts at 2 mg/mL of the in vitro culture of the liverwort Marchantia polymorpha L. were reported to inhibit tyrosinase activity (69.54% and 69.10%, respectively) [31]. Figure 1. Inhibitory effect of the 70% ethanol and methanol extracts on (A) collagenase activity and (B) tyrosinase activity. For collagenase activity the 70% ethanol extract was tested at the final concentration of 8.33 mg/mL and the methanol extract at final concentration of 6.66 mg/mL. For tyrosinase activity both extracts were tested at final concentration of 5.33 mg/mL. The positive control experiments were conducted using 1.49 mg/mL for EDTA (collagenase) and 0.04 mg/mL for kojic acid (tyrosinase). The results are expressed as the mean ± standard deviation (n=2-3). 2.4. Antimicrobial Activity Within the cosmetic market, there is a growing demand for skincare products containing natural antimicrobial ingredients as an alternative source to the standard synthetic preservatives. In the literature, antimicrobial activity has been reported in various species of bryophytes, particularly, in liverworts [32–36]. Bryophytes are reported not to be infected by microorganisms due to their ability to produce specialized protective molecules [5,37]. Thus, the antibacterial activity of the methanol extract was evaluated against Gram-positive (Staphylococcus aureus, Enterococcus faecalis, Staphylococcus epidermidis) and Gramnegative (Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Enterobacter cloacae) bacteria of pathogenic interest. Antifungal activity of B. trilobata is well established towards phytopathogenic fungi [4]. Then, the antifungal potential against Candida albicans and Aspergillus brasiliensis was also tested. In this work, the 164 antimicrobial activity of the methanol extract was only detected towards E. faecalis that completely inhibited the bacterial growth at 512 µg/mL. In other investigated species of Bazzania, the ethanol extract of Bazzania tridens evaluated with a different method showed intermediate (125 to 500 µg/mL) minimum inhibitory concentration (MIC) values towards S. aureus, P. aeruginosa and E. coli [9]. Moreover, the sesquiterpenoid, chiloscyphenol A, isolated from the Chinese Bazzania albifolia showed antifungal activity against Candida species with MIC values of 8–32 μg/mL [10]. 2.5. Chemical Constituents of B. trilobata extracts The composition of specialized metabolites of the 70% ethanol and methanol extracts were analyzed by UHPLC-HRMS. The annotation of ten known compounds, based on mass spectrometry and spectral data are shown in Table 2. The comparison of experimental MS/MS and in silico spectra were analyzed using the fragmentation tool MS-FINDER [38] (Figure S1). Figure 2 shows the extracted ion chromatogram of compounds 1-10. Phenolic compounds were the major constituents identified in the extracts; the main classes include lignans (1,2,4 and 5), coumarins (3) and bis-bibenzyls (6-9). Table 2. Predicted compounds from Bazzania trilobata extracts. Peak RT (min) Molecular Formula Experimental (m/z) [M-H]- Theoretical (m/z) [M-H]- Error (ppm) 1 2 2.09 2.43 C18H12O10 C26H26O14 387.0348 561.1252 387.0358 561.1250 -2.58 0.36 3 2.67 C15H14O10 353.0515 353.0514 0.28 4 2.73 C35H28O17 719.1249 719.1254 -0.70 Trilobatin K [26] 5 3.24 C27H20O12 535.0870 535.0882 -2.24 Trilobatin C [25] 6 6.99 C28H22O4 421.1437 421.1445 -1.90 Isoplagiochin C [24] 7 8 9 10 8.01 8.55 8.57 8.89 C28H20Cl2O4 C28H19Cl3O4 C29H20Cl2O4 C24H32O4 489.0658 523.0269 501.0650 383.2231 489.0666 523.0276 501.0666 383.2228 -1.64 -1.34 -3.19 0.78 Bazzanin B [23] Bazzanin C or D [23] Bazzanin K [23] Drimenyl caffeate* [39] * Chemical structure confirmed by NMR. Tentative identification Jamesopyrone [26] Trilobatin A [25] 7,8-dihydroxy-7-O-β-Dglucuronide [4] Figure 2. Extracted ion chromatogram of compounds 1-10 from B. trilobata extracts. Jamesopyrone (1) and the trilobatins A (2), C (5) and K (4) have previously been isolated from B. trilobata [25,26]. Lignans have been associated with a broad range of biological properties including antioxidant, antimicrobial, antiviral, antitumor, anti-inflammatory, and antineurodegenerative activities [40]. The coumarin 7,8-dihydroxy-7-O-β-D-glucuronide (3) has been identified in B. trilobata, although coumarins are less common in liverworts [4]. Coumarin and its derivatives are well-known to have important biological activities [41]. The macrocyclic bis-bibenzyl isoplagiochin C (6) and isoplagiochin D are known constituents from bryophytes proposed as parent compounds of chlorinated bis-bibenzyls of the bazzanin type [24]. Bazzanin B (7) and bazzanin S are bioactive chlorinated cyclic bis-bibenzyls from B. trilobata along with the bis-bibenzyl isoplagiochin D, and they have shown antifungal activities towards phytopathogenic fungi [4]. Several other chlorinated bis-bibenzyls such as bazzanins C/D (8), K (9) are biosynthesized in B. trilobata [23,24], and bibenzyls and bis-bibenzyls from liverworts exhibit a variety of therapeutic properties like anti-cancer, antioxidant, antimicrobial, and nitric oxide inhibitory activities [42]. We also identified for the first time in B. trilobata a sesquiterpene caffeate, drimenyl caffeate (10), which was first isolated from the liverwort Bazzania fauriana [39]. Compound 10 was isolated by preparative liquid chromatography and its structure was determined by 1D and 2D NMR spectra (Figure 3 and Table S1). 166 Figure 3. HMBC (arrows) and COSY (bold bonds) correlations of drimenyl caffeate (10). The trans-caffeate part of compound 10 was evident from the large 3JHH coupling (roughly 16.5 Hz) between H2’ and H3’, the characteristic meta-coupling pattern for H5’and H9’, and ortho-coupling between H8’ and H9’, and observed 1H-13C HMBC correlation peaks between H2’-C1’, H8’-C6’ and H9’-C7’. The HMBC correlation peak between H11 and C1’ and the H11-H9 COSY correlation peak then established the other side of the ester bridge. HMBC correlation peaks between H11-C8 and H11-C10 confirmed positioning of C8 and C10, while HMBC correlation peaks between H13-C10 and H12-C8 identified the position of these two methyl groups. The double-ring sys-tem was then further assigned using COSY correlation peaks to identify the segments C5-C6-C7 and C3-C2-C1. Lastly, shared HMBC correlation peaks to C4 for H3, and the methyl groups H14 and H15 together with an observed correlation peak between the methyl H13 and C10 completed the structure assignment. The observed 1 H and 13C chemical shifts are in good agreement with previously published data [39]. Sesquiterpene caffeates in liverworts have only been identified in Bazzania spp. [27]. The sesquiterpenoid cyclomyltaylyl-3-caffeate isolated from Bazzania japonica showed superoxide anion release inhibitory activity [43] and the myltaylane caffeate from Bazzania nitida showed potent inhibition of nitric oxide production [11]. Also, naviculyl caffeate was reported as a cytotoxic sesquiterpenoid isolated from the liverwort Bazzania novae-zelandiae [7]. 3. Materials and Methods 3.1. Plant Material Bazzania trilobata (L.) Gray was collected in the Black Forest, Germany (Lat. 47.911223/ Long. 8.092431) in April 2018 and identified by the Professor Dr. Nils Cronberg (Department of Biology, Lund University, Lund, Sweden). The specimen is identical to the voucher specimen with ID no MTRaMa13 sent for deposition at the Lund University Botanical Museum (LD). In this study, the whole plant was used for analysis. 3.2. Extraction Preparation B. trilobata was dried at room temperature and ground to a fine powder using a bead mill. The dried powder was homogenized in 70% ethanol (v/v) in water and methanol for the extraction of small molecules. The solution (1:10 g/mL of dry weight to solvent ratio) was macerated for 30 min by rotating mixer at room temperature. After centrifugation, the supernatant was collected and used for analysis. The 70% ethanol and methanol extracts were tested at the final concentrations as indicated in each experiment. For the antimicrobial analysis, the methanol extract was evaporated and the dry extract was dissolved in dimethyl sulfoxide (DMSO; Carlo Erba) at a concentration of 20.5 mg/mL. 3.3. Determination of Total Phenolic Content The total phenolic content (TPC) was determined by the Folin–Ciocalteu’s method [44]. Briefly, 20 μL of plant extracts (1/4 diluted), water (blank) and diluted gallic acid standard solutions (Sigma-Aldrich, ref. G7384; 0.4, 0.2, 0.1, 0.05, 0.025, 0.0125, 0.00625, 0.003125 mg/mL) were added to a microplate. Next, 100 μL of 10% Folin–Ciocalteu (Sigma-Aldrich; ref. F9252) and 80 μL of 7.5% sodium carbonate (Merck; ref. 1.06392.0500) were added to the samples, and the absorption was measured by spectrophotometer (Synergy HT, BioTek®) at 760 nm for 30 minutes at 25 °C. The TPC was estimated from a standard curve of gallic acid. The results were expressed in terms of milligrams of gallic acid equivalent per 100 mg of dry plant material [44]. The assay was conducted in triplicate. 3.4. DPPH Free Radical Scavenging Assay The DPPH (1,1- Diphenyl-2- Picryl-Hydrazyl; Sigma-Aldrich) free radical scavenging activity of the extracts was determined based on the methods previously described [45]. The samples were prepared at eight different concentrations, then, 70 µL of each dilution was mixed to 140 µL of methanolic DPPH solution (0.6 x10-4 M). The same procedure was realized for the 168 positive (ascorbic acid, Sigma-Aldrich; ref. A7506-100G) and negative (methanol) controls. In separated wells, the extracts and ascorbic acid dilutions were also mixed to 140 µL of 100% methanol for the sample's absorbance corrections. The samples were incubated for 30 min at 25 °C and the absorbance was measure at 517 nm. The IC50 values were estimated by the linear regression method. − 𝐷𝑂 (𝑏𝑙𝑎𝑛𝑘) DPPH radical scavenging activity (%) = 1 − 𝐷𝑂𝐷𝑂 𝑥 100 (100%) Where, DO: extraction solution + DPPH solution; DO (blank): extraction solution + methanol and DO (100%): methanol + DPHH solution. 3.5. In vitro Collagenase Inhibition Assay Collagenase inhibition activity was measured by following the enzymatic conversion of the synthetic substrate FALGPA (N-[3-(2-Furyl)acryloyl]-Leu-Gly-Pro-Ala) (Bachem; ref. 4006713.0025) to FAL (N- (3[2-Furyl]acryloyl)-Leu) + Gly-Pro-Ala (GPA). The collagenase activity from Clostridium histolyticum (type IA, Sigma-Aldrich, ref. C9891, specific activity ≥ 125 CDU/mg solid) was determined by the procedure previously described by Chajra et al. [46]. Ethylenediaminetetraacetic acid (EDTA) disodium salt dihydrate (purity ≥ 99%, Alfa Aesar; ref. A15161) was used as the control. 3.6. In vitro Elastase Inhibition Assay The elastase inhibitory activity was determined by a spectrophotometric method using a microplate reader, Synergy HT (Biotek). The assay is based on the detection of enzymaticdriven conversion of N-succinyl-Ala-Ala-Ala-p-nitroanilide (SAAApNA; Sigma-Aldrich, ref. S4760) to p-nitroanilide (pNA) that strongly absorbs at 420 nm. The reaction mixture contained 170 μL of elastase synthetic substrate: SAAApNA (1.5 mM in 50 mM Tris buffer containing, 10 mM CaCl2 and 400 mM NaCl, pH 7,5) and 20 μL of plant extract (test sample) or pure solvent of the sample (blank, control). Enzymatic conversion was initiated by addition of 10 μL of 0.05 mg/mL of porcine pancreatic elastase (Sigma-Aldrich, ref. E7885-5MG) in the same buffer as its substrate SAAApNA. Elastase-driven conversion of SAAApNA to pNA was followed for 25 minutes at 25 °C by measuring an increase of the sample’s absorption at 420 nm, proportional to pNA concentration. 3,4-dichloroisocoumarin (3,4-DCIC) (Sigma Aldrich, D7910, purity ≥ 98% ) was used as a positive control. The points in the linear range of the absorbance versus time plots were applied to calculate the slopes, directly proportional to elastase activity. Then, the values of elastase inhibition, expressed as the percent of the activity of the test samples versus the control experiment (pure solvent) were calculated for all samples according to the following equation: 𝑆𝑙𝑜𝑝𝑒 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 Elastase activity (EA%) = 𝑆𝑙𝑜𝑝𝑒 𝑜𝑓 𝑏𝑙𝑎𝑛𝑘 𝑥 100 Elastase inhibition activity (%) = 100%- EA% 3.7. In vitro Tyrosinase Inhibition Assay The mushroom tyrosinase inhibitory activity was determined by a spectrophotometric method using a microplate reader, Synergy HT (Biotek), based on Kamkaen et al. [47] with modifications. Tyrosinase-driven conversion of L-Tyr to dopachrome was followed for 25 minutes at 25 °C by measuring an increase of the sample’s absorption at 475 nm, proportional to dopachrome concentration. Kojic acid (purity 99%, Alfa Aesar) was used as a positive control. The tyrosinase activity was determined by the procedure described by Marques et al. [48]. 3.8. Antimicrobial Assay Antimicrobial activities were screened with the concentration of the methanol extract at 512 μg/mL. 3.8.1. Antibacterial Assay The antibacterial activity was determined by broth microdilution method based on ISO 207761:2006 standard [49], in accordance with CLSI [50] and EUCAST [51] guidelines. The method was previously described [52]. The following bacteria have been used in this work: Escherichia coli ABC5 (ATCC 25922), Staphylococcus aureus ABC1 (ATCC 29213), Pseudomonas aeruginosa ABC4 (ATCC 27853), Klebsiella pneumoniae ABC12 (ATCC 700603), Staphylococcus epidermidis ABC91 (clinical origin), Enterococcus faecalis ABC 3 (ATCC 29212), Acinetobacteur baumannii ABC 14 (ATCC 19606), Enterobacter cloacae ABC 45 (clinical origin). Briefly, the screening test conditions were performed as below: Growth control: 75 μL MHB-CA (Mueller-Hinton Broth, Cations- Adjusted) with bacteria, + 25 μL H2O. 5 [2-8].105 CFU/mL per well. 8 replicates/bacteria/microplate. Negative control: 75 μL MHB-CA (without bacteria) + 25 μL H2O. 8 replicates/microplate. Sample control: 75μL MHB-CA (without bacteria) + 25μL of sample. 1 control/test. 170 Test: 75μL MHB-CA (Mueller-Hinton Broth, Cations- Adjusted) with bacteria, + 25 μL of sample. 5 [2-8].105 CFU/mL per well. 1 test / bacteria. Incubation 24h 35 °C. 3.8.2. Antifungal Assay Candida albicans ABC F1 (clinical origin) and Aspergillus brasiliensis ABC F16 (ATCC 16404) were used in this study. To investigate the antifungal activities of the extract, the antifungal activity was determined by broth microdilution method according to EUCAST guidelines [53]. Briefly, the screening test conditions were performed as below: Growth control: 100 μL 2X RPMI 1640 + 50 μL fungi suspension + 50 μL H2O. Candida: [1-5].105 UFC/mL per well; Aspergillus: [0.5-2.5].105 spores/mL per well. 8 replicates/fungi/microplate. Negative control: 100 μL 2X RPMI 1640 + 100 μL H2O. 8 replicates/microplate. Sample control: 100 μL 2X RPMI 1640 + 50 μL of sample + 50 μL H2O. 1 control/test. Test: 100 μL 2X RPMI 1640 + 50 μL fungi suspension + 50 μL of sample. Candida: [1-5].105 UFC/mL per well; Aspergillus: [0.5-2.5].105 spores/mL per well. 1 test / fungi. Incubation 24h 35 °C for Candida, 48h 35 °C for Aspergillus. 3.9. UHPLC-HRMS Analysis The extracts were diluted at 10 mg/mL in ethanol absolute and a volume of 1 μl of samples were injected for analysis. Ultra-high Performance Liquid Chromatography-High Resolution Mass Spectrometry (UHPLC-HRMS) was realized on Agilent 1290 Infinity II UHPLC (Agilent Technologies) with diode array detector (DAD) coupled to an Agilent 6545 QTOF with Agilent Dual Jet Stream electrospray ion source with a drying gas temperature of 325 oC, a gas flow of 8 L/min, and a sheath gas temperature of 300 oC and flow of 12 L/min. Capillary voltage was set to 4000 V and a nozzle voltage to 500 V. Analyses were performed in negative ion mode. Mass spectra were recorded at centroid mode for m/z 100–1700 in MS mode and m/z 30–1700 in MS/MS mode, with an acquisition rate of 10 spectra/s using fixed collision energies of 10, 20, and 40 eV and maximum three selected precursor ions per cycle. The separation was performed on a reversed-phase column Agilent Poroshell 120 Phenyl Hexyl column (150 x 2.1 mm, 1.9 µm), using water/acetonitrile mobile phase, both containing 20 mM formic acid (phase A/B respectively). Phase B increase from 10% to 100% in 10 min, then held at 100% B for 2 min, returned to 10% in 0.1 min and equilibrated for 2 min at a flow rate of 350 µL/min, and column temperature of 40 °C. The LC-MS/MS raw data was processed by the open-source software MS-DIAL (version 4.60), enabling ion chromatograms extraction and peak deconvolution [54]. The processed data (mass spectrometry and spectral data) were used to tentatively identify by matching the mass spectral data of the compounds 1-9 against the records of the MS-FINDER databases (Version 3.50) [38] (http://prime.psc.riken.jp/). 3.10. Purification by Preparative Liquid Chromatography Compound 10 was purified from the commercial Lebermooser extract (Niem-Handel, Gernsheim, Germany). The dry crude extract (3 g) was partitioned in distilled water and ethyl acetate. The ethyl acetate phase was evaporated and 226 mg of dry extract was dissolved in 4 mL of ethanol absolute and 1 mL distillated water. The resulting solution was used to separate the compound 10 by preparative liquid chromatography (LC) Armen Spot Prep II (Armen) with a C18 column (250 mm × 50 mm, 10 µm, Vydac Denali; Grace). The fractions were purified using water containing 0.1% vol. of formic acid (A) and pure ACN (B) with the gradient mobile phase of B of 70% (0–20 min), 80–100% (20–21 min), 100% (21 min-25 min) at a flow rate of 120 mL/min and an UV detection at 238 and 324 nm (Figure S2). The fractions containing the purified compound were combined, evaporated under vacuum and 6.28 mg (purity (average UV-vis between 210-600 nm) >95%) of compound was obtained. The isolate was analyzed using the HPLC Agilent 1200 system (Agilent) with an Agilent 1260 Infinity Diode array Detector (applied range: 210-600 nm) coupled to a mass spectrometer Agilent 6120 Quadrupole LC/MS (electrospray ionization and atmospheric pressure chemical ionization in negative or positive ion mode, m/z 100-1000), using a Vydac Denali C18 reversephase column (250 mm × 4,6 mm, 10 µm; Grace) maintained at 25 °C during all analyses. The mobile phase was composed of water containing 0.1% vol. of formic acid (A) and pure ACN (B), delivered at 1.5 mL/min with the gradient of B phase as follows: 70% (0–20 min), 80– 100% (20–21 min), 100% (21 min-25 min). 3.11. NMR measurement The presented NMR spectra were recorded on an 800 MHz Avance III HD spec-trometer equipped with a 5 mm TCI CryoProbe (Bruker Biospin). 1H and 13C chemical shifts are reported relative to TMS ( (1H) = 0.0 ppm, (13C) = 0.0 ppm) using the solvent signals as 172 secondary reference (MeOD: (1H) = 3.31 ppm and (13C) = 49.0 ppm). The HSQC spectra were acquired using a data matrix of 4096 x 1024 complex points with acquisition times of 200 and 15 ms in F2 and F1, respectively. Adiabatic bilevel 1H decoupling was employed during acquisition. The HMBC spectra were acquired using a data matrix of 4096 x 512 complex points with acquisition times of 220 and 6 ms in F2 and F1, respectively. The DQF-COSY spectra were acquired using a data matrix of 4096 x 1024 complex points with acquisition times of 220 and 53 ms in F2 and F1, respectively. 4. Conclusions This study shows that the extracts from the liverwort B. trilobata have antioxidant, antimicrobial, collagenase and tyrosinase inhibitory activities. In addition, a sesquiterpene caffeate was identified for the first time in B. trilobata. The extracts are rich in phenolic constituents and contain a sesquiterpenoid, which possibly explains most of the biological activities. We demonstrate that B. trilobata has, besides its already known antifungal activities, the potential for new biotechnological applications. These results contribute to the knowledge of medicinal properties from liverworts and in special to the inhibitory effect on aging-related enzymes. Supplementary Materials: Figure S1. Tentatively compounds identification by comparing MS/MS spectrums of experimental spectrums and in silico spectrums from MS-FINDER. Figure S2. Preparative LC chromatogram with purified fraction corresponding to drimenyl caffeate. Table S1. NMR spectroscopic data (800 MHz in MeOD-d4) for drimenyl caffeate (10). Author Contributions: Conceptualization and supervision of the project, F.B., S.M., H.T.S; data acquisition, R.V.M., C.H.G., K.E-R., N.H.; analysis and data interpretation, R.V.M., A.S., Y.L., C.H.G., K.E-R., N.H.; writing—original draft preparation, R.V.M; writing—review and editing, H.T.S., F.B., A.S., A.R., R.D., Y.L., C.H.G., K.E-R.; All authors have read and agreed to the published version of the manuscript. Funding: This research was supported by Marie Sklodowska –Curie Actions Innovative Training Networks under the Horizon 2020 program under grant agreement n° 765115 – MossTech. Acknowledgments: The authors thank Professor Nils Cronberg, Lund University, Sweden, for support in plant identification and collection. 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Methods 2015, 12, 523–526, doi:10.1038/nmeth.3393. Supplementary Materials Figure S1. Tentatively compounds identification by comparing MS/MS spectrums of experimental spectrums and in silico spectrums from MS-FINDER. 178 180 Figure S2. Preparative LC chromatogram with purified fraction corresponding to drimenyl caffeate. Table S1. NMR spectroscopic data (800 MHz in MeOD-d4) for drimenyl caffeate (10) Annotation 13 1 40.5 2 19.5 3 42.9 4 5 6 33.6 50.9 24.4 7 8 9 10 11 124.5 133.2 54.6 36.8 63.8 12 13 14 15 1’ 2’ 3’ 4’ 5’ 6’ 7’ 8’ 9’ 22.1 15.0 33.7 22.4 169.1 115.1 146.3 127.4 114.9 146.4 149.1 116.2 122.7 C (ppm) 1 1 H-1H COSY (key) 1H-13C HMBC 1.99 1.14 1.58 1.47 1.43 1.20 Multiplicity, J-couplings (Hz) m m m m m m 2 10 2 4 1.24 2.01 1.90 5.53 m m m s, br 6 5, 7 4, 10 2.1 s, br 4.36 4.19 1.68 0.87 0.87 0.91 dd, 11.8 ; 3.1 dd, 11.8 ; 5.8 s s s s 9 6.19 7.49 d, 16.5 d, 16.5 3’ 2’ 1’ 1’ 7.01 d, 1.6 9’ 7’, 9’ 6.77 6.91 d, 8.1 dd, 8.1 ; 1.6 9’ 5’, 8’ 4’, 6’ 5’, 7’ H (ppm) 1, 3 8 7, 8, 9 9, 10 4, 15 4, 14 HMBC (arrows) and COSY (bold bonds) correlations of drimenyl caffeate. 182 1’, 8, 9, 10 4.2 Investigation of bioactive compounds of B. trilobata extracts The bioactive extracts of B. trilobata exhibited collagenase and tyrosinase inhibitory activities as previously showed in paper 4. Therefore, the pre-screen test Target binding® was applied to tentatively identify candidate inhibitors of target enzymes. The phytochemical constituents of 70% ethanol and methanol active extracts were explored for their interaction and affinity for the target’s collagenase and tyrosinase. The comparison of the chromatograms of the raw extracts and after the Target binding experiment allows the identification of constituents retained by collagenase or tyrosinase, as shown in Figure 1. The relative affinity (RA) of individual compounds for collagenase is shown in Table 1. The RA for the reference compound (compound 1) is equal to one. Compounds 1 and 2 show an affinity to collagenase with RA values very close although compound 2 shows a slightly higher affinity. For tyrosinase, only compound 1 was retained by the enzyme. The results suggest compounds 1 and 2 as inhibitors candidates of the target enzymes; however, the inhibitory properties of these compounds need confirmation by classic enzymatic assays. Table 1. Relative Affinities of the components of 70% ethanol extract to collagenase. Relative affinity for collagenase Compound 1 Compound 2 1.0 1.42 Figure 1. UHPLC chromatograms of the crude extracts of B. trilobata and the Target binding® samples. A) UHPLC chromatograms of the 70% ethanol crude extract (top) and the Target binding® sample with compounds binding to collagenase from Clostridium hystolytichum (bottom). B) UHPLC chromatograms of the methanol crude extract (top) and Target binding® sample with compounds binding to mushroom tyrosinase (bottom). The compounds 1 and 2 were then purified by preparative liquid chromatography (Figure 2) and investigated by UHPLC-HRMS as the procedure in paper 4. 184 Figure 2. Preparative LC chromatogram with purified fractions of Fr. 1 (unknown compound) and Fr.2 (drimenyl caffeate). The comparison of experimental MS/MS (Figure 3) and in silico spectra of compound 1 was analyzed with MS-FINDER for tentative compound identification. The molecular formula for compound 1 was predicted to be C29H40O8 based on pseudomolecular ion, [M + H] + of m/z 517.2795 with an accuracy of -0.19 ppm (HRMS) and [M - H] - of m/z 515.2653 with an accuracy of 0.58 ppm (HRMS) and the ultraviolet (UV) spectrum displayed UV λmax 286 and 324 nm, however, its annotation was not accomplished. Figure 3. Negative and positive ion mode ESI-MS/MS spectra of compound 1 (unknown). Compound 2 was identified as drimenyl caffeate determined by NMR studies as previously described in paper 4. The in vitro collagenase inhibitory activity of the unknown compound (1) and drimenyl caffeate (2) was determined at concentrations ranging from 41.66 to 166.66 μM (Figure 4). The unknown compound 1 exhibited no significant inhibition and drimenyl caffeate exhibited 12% of collagenase inhibition at the final tested concentration of 166.66 μM. The compounds were considered not active or with very low inhibition at the tested concentrations. The unknown compound 1 was also tested for tyrosinase inhibitory activity at a final concentration of 200 μM, however, it was not active. 186 Figure 4. Collagenase inhibitory activity of compounds 1 and 2. The results are expressed as the mean ± standard deviation. These results can indicate a non-specific bound of compound 1 from the extracts during the steps of TB® (washing steps) leading to false-positive detection of inhibitor candidates. Then, after carrying out TB® with the individual compounds (mix of compounds 1 and 2 at final concentration of 100 µM), it was confirmed that compound 1 does not bind to both enzymes (Figure 5). Figure 5. UHPLC chromatograms of the test mixture: mixture of compounds 1 and 2 (reference compounds; top) and Target binding® samples. A) UHPLC chromatograms of the reference (top) and the compounds with affinity to collagenase (bottom). B) UHPLC chromatograms of the reference (top) and the compounds with affinity to tyrosinase (bottom). The numbers correspond to the unknown compound (1) and drimenyl caffeate (2). 4.2.1 Overview experimental work 4.2.1.1 Evaluation of collagenase and tyrosinase affinity by Target Binding® technology The affinity of the constituents from the extracts of B. trilobata to collagenase and tyrosinase was investigated by the Target Binding® technology, which is described in detail in paper 3 with few modifications. The ligands and raw extracts were analyzed by UHPLC (Shimadzu Nexera X2, Shimadzu) with a photodiode array detector coupled to the LCMS2020 mass spectrometer (electrospray ionization in negative and positive ion mode). Target Binding® for collagenase and tyrosinase was performed using the analytical method containing water and 0.1% vol. of formic acid (A) 188 and pure acetonitrile (B) in 0.5 mL/min with the gradient mobile phase of B phase as follows: 5–25% (0–6 min); 25–90% (6–15.45 min); 90–95% (15.45–15.50 min) hold at 95% (15.50– 18.90 min); 95-5% (18.90-19 min); hold at 5% (19–21.50 min) in the Kinetex Biphenyl reverse-phase column (150 mm × 2.1 mm, 2.6 µm; Phenomenex), maintained at 40 oC. The affinity of the individual compounds isolated from B. trilobata to collagenase and tyrosinase was also investigated by the Target Binding® technology. The enzymatic solutions of collagenase and tyrosinase was added to the mixture containing compounds 1 and 2 (100 µM final concentration) and incubated with the enzymatic solution following the same procedure as previously described. The ligands and the mixture of compounds were analyzed by UHPLC. Target Binding® for both enzymes was performed using the analytical method containing water and 0.1% vol. of formic acid (A) and pure acetonitrile (B) in 0.5 mL/min with the gradient mobile phase of B phase as follows: 5–45% (0–12.50 min); 45–95% (12.50–17.50 min); hold at 95% (17.50–20.49 min); 95–5% (20.49–20.50); hold at 5% (20.50–22.50 min) in the Kinetex EVO C18 reverse-phase column (150 mm × 2.1 mm, 2.6 µM; Phenomenex), maintained at 40 ◦C. 4.2.1.2 Purification by Preparative Liquid Chromatography Compound 1 (fraction 1) and 2 (fraction 2) were purified from the commercial Lebermooser extract (Niem-Handel, Gernsheim, Germany) by preparative liquid chromatography (LC) Armen Spot Prep II (Armen) with a C18 column (250 mm × 50 mm, 10 µm, Vydac Denali; Grace). The fractions containing the purified compound were combined and evaporated under a vacuum. Fraction 1 (Fr.1, Figure 2) resulted of 3.23 mg (purity, (UV at 286) 62%) and Fraction 2 (Fr.2, Figure 2) resulted of 6.28 mg (purity, (UV) >95%). The method used for the purification is described in paper 4. 4.2.1.3 Collagenase and tyrosinase inhibitory activities The inhibitory activity of isolated compounds on collagenase and tyrosinase activities was determined as the procedure described in papers 3 and 4. General Discussion Bryophytes produce numerous biologically active compounds with a fascinating chemical complexity that increases their commercial interest. Moreover, they are attractive green cell factories for producing specialized metabolites, lipids, and recombinant biopharmaceutical proteins. Moss-produced drugs, moss-based air fresheners, and moss-derived cosmetic ingredients are recent examples of products being developed from bryophytes [1,2]. Therefore, the new source of bryophytes metabolites with potential applications as cosmetics or pharmaceuticals was the crucial motivation of this study. In this thesis, the extracts of bryophytes were primarily screened for anti-inflammatory and inhibitory activities against skin aging-related enzymes. The inhibitory effects towards skin pigmentation-related enzymes as well as antioxidant and antimicrobial properties were also investigated. The active extracts were selected for further screening and identification of active constituents towards the target enzymes using a protein-ligand affinity approach (Target Binding®) and docking studies. 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. In Chapter 2 (paper 2), 70% ethanol extracts of thirty-two species of bryophytes were screened for their anti-inflammatory activities. Importantly, the extracts did not show any cytotoxicity towards RAW264.7 murine macrophages cells. This Ph.D. study is the first to report the anti-inflammatory potential of the mosses Dicranum majus and Thuidium delicatulum. It was observed that the extracts inhibited (lipopolysaccharide) LPS (1 ng/mL)stimulated nitric oxide (NO) production, a marker of inflammation, in a concentrationdependent manner with half-maximal inhibitory concentrations (IC50) of 1.04 and 1.54 µg/mL, respectively. Not only the crude extracts but also their derived fractions exhibited antiinflammatory activity. Non-polar fractions at 10 µg/mL derived from 70% ethanol and ethyl crude extracts exhibited the highest potential to inhibit NO production. Fractions of n-hexane showed significantly higher inhibition on LPS (10 ng/mL)-induced NO compared to 70% ethanol crude extracts at 10 µg/mL. In a previous report, the treatment with 50 μg/mL of peat moss (Sphagnum sp.) aqueous extract inhibited the production of NO in LPS-stimulated (500 ng/mL) RAW 264.7 cells [3]. In our screenings, the mosses from the Sphagnum genus showed no anti-inflammatory activities. Moreover, the methanol extract of Polytrichum commune was reported to inhibit the NO production induced by the treatment of LPS (1 µg/mL) with an IC 50 190 of 65.15 µg/mL [4]. The moss from the same genus Polytrichum formosum also showed no anti-inflammatory activity in our screenings. The anti-inflammatory activity detected in the screenings is consistent with previous studies related to plants extracts and their effects on the inhibition of pro-inflammatory mediators, including NO. The mosses extracts show a significant potential of NO inhibition compared to reported studies from plants of interest in the pharmaceutical or cosmetic industry. However, the variation in experimental procedures seen in many studies, such as the species-specific LPS and LPS induced doses, can influence the levels of activation of inflammatory pathways and should be considered when comparing extracts' anti-inflammatory potency [5]. Similar studies with plants of medicinal and cosmetic interest have been performed, as seen in this small table below: Table 1. Plant extracts with inhibitory effects on LPS-induced NO production Plants Inhibition of LPS-induced NO production by Ref. extracts/fractions Artemisia annua L. 72.39% and 71% at 6.25 μg/mL for ethanol extract [6] and its hexane/ethyl acetate fraction Artemisia lavandulaefolia IC50 of 1.64 µg/mL for ethanol extract fraction [7] DC. Curcuma zedoaria Roscoe IC50 of 23.44 μg/mL for methanol extract [8] rhizomes Pomegranate flower IC50 of 31.80 μg/mL for ethanol extract [9] Punica granatum L. Pueraria lobata (Willd.) IC50 of 31.80 μg/mL for methanol extract and its [10] Ohwi roots fractions: (n-hexane) 5.88 µg/mL; (dichloromethane) 29.98 μg/mL and (ethyl acetate) 0.13 μg/mL Rosemary IC50 of 2.75 μg/mL for the methanol extract and Rosmarinus officinalis L. 2.83 μg/mL for its hexane fraction Viola yedoensis 90% at 150 μg/mL for the 70% ethanol extract [11] [12] Based on the table, the extracts of D. majus (IC50 of 1.04 µg/mL) and T. delicatulum (IC50 of 1.54 µg/mL) exhibit significant inhibitory activity with low IC 50 values for LPS-induced NO production. Due to their effective anti-inflammatory activity, these plants suggest an innovative source of active ingredients for the development of cosmetics and drug discovery. The evaluation of the anti-inflammatory effect of the extracts on other important pro-inflammatory mediators produced in response to LPS, such as the cytokines interleukins (IL)-1β, IL-6, and tumor necrosis factor (TNF-α), is yet to be determined [13,14]. Moreover, the investigation of the extracts in different signaling pathways will be important to understand the molecular mechanisms involved in the anti-inflammatory response. From a cosmetic application perspective, the extracts should be tested on skin cells or in 2D/3D inflammatory skin models to evaluate their protective effects against acute inflammation induced by specific triggers including bacterial LPS, ultraviolet radiation, etc. [15]. In pharmaceutical applications such as for targeting chronic inflammatory diseases, the tests on experimental models should be carried out with candidate molecules derived from the extracts, which still need to be investigated [16]. The extracts of bryophytes were screened for inhibitory activities against skin aging-related enzymes such as collagenase and elastase, which are also involved in a wide range of pathological conditions (e.g. inflammation, cardiovascular diseases, cancer, etc.) [17,18]. The collagenase inhibitory activity was only detected for the 70% ethanol extracts at a final concentration of 8.33 mg/mL from the moss Polytrichum formosum (71%) and the liverwort Bazzania trilobata (40%). The two active extracts were selected for further studies. Even though the extracts of D. majus and T. delicatulum showed anti-inflammatory effects on LPSinduced NO, they did not show collagenase or elastase inhibitory activities. P. formosum and B. trilobata did not show potential anti-inflammatory activities either. It indicates that the bryophytes tested have particular biological properties, suggesting different application perspectives in the pharmaceutical or cosmetic industry. In paper 3, the extracts from the moss P. formosum were investigated as a new source of collagenase inhibitors. Methanol and ethyl acetate extracts of P. formosum were also selected for the investigations; however, they did not show enzymatic inhibition. The active extract revealed collagenase inhibitory activity by 71% at 8.33 mg/mL and IC50 of 4.65 mg/mL. In paper 4, the previously screened liverwort B. trilobata, which presented 40% of collagenase inhibition at 8.33 mg/mL, also showed 20% when tested with the methanol extract at a final concentration of 6.66 mg/mL. In terms of potency, the moss extract showed higher inhibitory properties than the liverwort extracts. The crude bryophyte extracts exhibit important anticollagenase activity and can be exploited as cosmetic ingredients with skin anti-aging properties. A recent study by our industrial partner (Plant Advanced Technologies) reported 192 anti-collagenase activity by 91% for the root extract of Morus alba L. cultivated with nitrogen deprivation (extract enriched in prenylated molecules) at 4.16 mg/mL [19], tested by the same experimental conditions than in this study. Like that, various other higher plants have been reported with collagenase inhibitory properties, however, it is important to highlight that the bioactivity reported for plant extracts may be biased by the assay conditions and therefore it is quite difficult to compare the potency of the extracts between different studies. In papers 3 and 4, the effects of P. formosum and B. trilobata extracts on mushroom tyrosinase activity, a critical target for developing skin-lightening agents [20], were also investigated. The methanol extracts of P. formosum and B. trilobata at the final concentration of 5.33 mg/mL exhibited a mild tyrosinase inhibition of 44% and 43%, respectively. In a previous study, nhexane and chloroform extracts at 2 mg/mL of the liverwort Marchantia polymorpha L. were reported with tyrosinase inhibitory properties higher than in our studies (69.54% and 69.10%, respectively) [21], but still show that there is a potential in bryophytes. Antioxidants have an important role in the prevention of skin aging by neutralizing free radicals and then oxidative damage [22]. Investigations of several species of mosses and liverworts revealed compounds with antioxidant properties such as phenolics and terpenoids [23,24]. Therefore, the antioxidant activity of B. trilobata was investigated (paper 4) by the DPPH (1diphenyl-2-picrylhydrazyl) scavenging assay. The DPPH radical scavenging activity of the 70% ethanol extract (IC50 of 82 µg/mL) was higher than methanol extract (IC50 of 122 µg/mL). In addition, the total phenolic content of the 70% ethanol extract showed to be higher by 38% than the methanol extract. The antioxidant activity of B. trilobata is in accordance with studies of plants as antioxidants. For instance, similar findings from extracts of plants with medicinal and cosmetic properties are shown in the table below: Table 2. Plant extracts with antioxidant activity. Plants DPPH assay - Ref. IC50 value (µg/mL) of extracts Curcuma aromatica rhizome 102.4 [25] (ethanol extract) Curcuma longa rhizome 134.9 (ethanol extract) [25] Curcuma comosa rhizome 137.7 [25] (ethanol extract) Curcuma aeruginosa rhizome 187.4 [25] (ethanol extract) Centella asiatica L. 320 [26] (ethanol extract) Hibiscus sabdariffa L. 350 [26] (ethanol extract) Nelumbo nucifera Gaertn 340 [26] (ethanol extract) Pueraria lobata (Willd.) Ohwi roots 83.30 [10] (methanol extract) Bioactive extracts with additional antimicrobial properties are a growing demand within the cosmetic market as an alternative to limit the amount of synthetic preservatives in formulations [27]. Therefore, B. trilobata was investigated for antibacterial activity against eight different pathogenic strains and antifungal activity against two pathogenic strains. The methanol extract of B. trilobata only showed to inhibit the bacterial growth of Enterococcus faecalis at 512 µg/mL. Therefore, to investigate the active compounds from P. formosum and B. trilobata an affinitybased approach was applied. The inhibitory potential of the phytochemical constituents from the 70% ethanol and methanol extracts towards the targets collagenase and tyrosinase were investigated by the Target Binding® approach (TB®) [19]. TB® was applied to pre-screen inhibitors candidates in the active plant extracts based on their affinity to the target enzymes. The TB® was the approach used rather than the classical bioassay-guided fractionation to enable faster identification of hit compounds avoiding the successive rounds of extract fractionation and biological screenings of the classical system. Moreover, bioassay-guided fractionation has lower detection to low concentrated compounds and hardly detect synergistic effects of natural substances [28]. After TB® investigations, the candidate molecules were isolated by preparative liquid chromatography and identified by UHPLC-MS and NMR. In paper 3, four hits (ohioensin A, nor-ohioensin D, ohioensin C, and communin B) and two hits (ohioensin A and communin B) from the active extracts of P. formosum revealed relative 194 affinity to collagenase and tyrosinase, respectively. The compounds were identified as belonging to the rare benzonaphthoxanthenones (ohioensins) and flavonoids, previously reported from the genus Polytrichum but not for P. formosum [29]. We also identified a new ohioensin named nor-ohioensin D. To confirm if the hits pre-screened by TB® have a biological effect, the purified compounds were tested for enzymatic inhibitory activities. Ohioensin A and nor-ohioensin D were confirmed as the bioactive molecules from P. formosum by inhibiting collagenase activity by 62% and 53% at 166.66 µM and with IC 50 of 71.99 and 167.33 µM, respectively. To our knowledge, this is the first report on the anti-collagenase activities of ohioensins. Ohioensin A had a higher yield of isolation of 4.59 mg (purity >95%) than norohioensin D (1.91 mg; purity of 82%), indicating ohioensin A to be the major collagenase inhibitor of the crude 70% ethanol extract. These results are in agreement with studies reporting phenylpropanoids belonging to different groups of compounds such as stilbenes, flavonoids, coumarins and derivatives as collagenase inhibitors with a wide range of IC 50 values, as shown in the table below: Table 3. Compounds with anti-collagenase activity. Compounds Collagenase inhibition Ref. IC50 values (µM) Stilbenes Flavonoids Coumarins E-astringin 124.9 [30] E-piceid 258.7 [30] Taxifolin 193.3 [30] Taxifolin 3′-O-glucoside 141.4 [30] Isoorientin 84 [31] Vicenin-2 185 [31] Quercetin 286 [32] Scopoletin 1.8 [31] Esculetin 12 [31] Ohioensin A also exhibited week tyrosinase inhibition by 30% at 200 µM compared to the standard tyrosinase inhibitor kojic acid (99% at 300 µM). Molecular docking study was performed to explore the possible mode of action of the active compounds with the target enzymes. Ohioensin A showed moderate affinity toward collagenase equal to –7.6 Kcal/mol and formed 3 hydrogen bonds with Gly493 and Gly494. Nor-ohioensin D showed an affinity equal to –7.4 Kcal/mol and 3 hydrogen bonds with Gly493, Gly494, and Glu555. The compounds are predicted to occupy a similar region within the active center of the enzyme indicating that a competitive binding mode and enzymatic inhibition would be expected. Also, ohioensin A formed three hydrogen bonds with Ala323, His244, and His85 and showed a moderate affinity of –7.1 Kcal/mol toward the tyrosinase-binding site. In previous studies, ohioensin A was found to exhibit cytotoxicity against murine P388 leukemia (9PS) at a median effective dose (ED50) of 1.0 µg/mL and human breast adenocarcinoma cell lines at ED50 of 9.0 µg/mL [33,34]. Ohioensin A also showed potent inhibitory activity against therapeutically targeted protein tyrosine phosphatase 1B (PTP1B) with IC50 of 4.3 µM [35] which generated also a patent [36]. Therefore, ohioensin A exhibits biological activities on more than one target and with higher potency than for collagenase or tyrosinase targets. This indicates that ohioensin A may be less interesting from a pharmaceutical point of view because it exhibits low specificity and activity on the investigated targets. However, ohioensin A can still be explored as a leading compound in drug discovery, whose optimization can increase its activity and specificity, reducing possible off-target effects. Moreover, the evaluation of toxicity of the compounds is necessary before any further studies considering pharmaceutical or cosmetics applications and their regularity requirements for safety assessment. In cosmetic applications, preliminary toxicological investigations should be carried out in skin cells (e.g. keratinocytes and fibroblasts) or skin models [37–39]. Therefore, safety testing must include the evaluation of genotoxicity (DNA damage), skin and eye irritation, skin sensitization, dermal absorption, among others [40]. In chapter 4.2, the TB® for B. trilobata extracts revealed two hits (one unknown compound and drimenyl caffeate) for collagenase and one hit (unknown compound) for tyrosinase. In this study, the known drimenyl caffeate, a sesquiterpene caffeate, was identified for the first time in the species B. trilobata, however, the second compound is unknown. The unknown compound exhibited no inhibition and drimenyl caffeate 12% of collagenase inhibition at the final concentration of 166.66 μM. The compounds were considered not active or with very low inhibition at the tested concentrations. Usually, extracts of plants are reported with more effective biological activities than the individual compounds due to the synergistic or additive combined effects between components [41]. The unknown compound was also not active for tyrosinase inhibition. False positives hits from TB® indicate nonspecific binding of the tested unknown compound. In the TB® approach, the incubation step of the method allows the formation of target-ligand interactions that are deposited on the surface of a cut-off centrifugal 196 filter followed by centrifugation and washing steps to remove non-specific bound compounds. However, it can occur that if not well eliminated, the unbound compounds can be retained nonspecifically on the filter’s membrane leading to false-positive detection such as for the unknown compound. Further investigations by the TB ® method require improved chromatographic separation and detection systems for the improved screening of B. trilobata active compounds. We then attempted to investigate the composition of specialized metabolites from the extracts of B. trilobata by UHPLC-HRMS and in silico MS/MS dereplication. Phenolic compounds were the major constituents tentatively identified in the extracts. The compounds were previously identified from B. trilobata and include lignans, coumarins, and bis-bibenzyls. Bisbibenzyls and chlorinated derivatives are a very characteristic group of compounds produced by B. trilobata and some of them were described with antifungal activity towards phytopathogenic fungi [42]. In general, the classes of specialized metabolites tentatively identified are associated with a broad range of biological activities (e.g. antioxidant, antimicrobial, anti-inflammatory, etc.)[43–45] and possibly explain most of the biological activities found in this work. Moreover, the drimenyl caffeate identified in B. trilobata belongs to the sesquiterpene caffeates, which among liverworts have only been identified in Bazzania spp. The sesquiterpene caffeates identified in some Bazzania spp. have been reported with superoxide anion release inhibitory activity [46], nitric oxide production inhibition [47] and cytotoxic [48] activities. In the current cosmetic sector, natural ingredients are a growing trend, so there is an important demand for novel plant sources and derived phytochemicals [49,50]. Natural ingredients are specially used in skincare cosmetics or cosmeceutical preparations, a term used by the cosmetic industry to refer to cosmetic products with biologically active ingredients exhibiting medicinal or drug-like benefits for skin health [51]. In a recent review of Ferreira et al., [50], the authors indicate increased use of plants in cosmetics with proven anti-aging properties being DNAprotecting action, enzyme-inhibiting, and anti-inflammation activities the most used categories of ingredients. In perspective, mosses and liverworts studied in this thesis can offer innovative botanical sources of rare natural products to develop dermocosmetics ingredients with antiinflammation, skin anti-aging, and anti-pigmentation properties. It is important to mention that the activities were observed in in vitro assays, which allowed the first indications of the activities reported; however, additional studies with skin models and in vivo are needed to prove their safety and efficacy. In addition, a combination of different procedures can be employed to improve the activity of the extracts by increasing the yield of individual active compounds, making them more commercially feasible. Some of these procedures can include the use of different extraction techniques and solvents or elicitation (i.e., compounds that stimulate plant defense inducing the production of specialized metabolites) allied or not to in vitro plant cultivation systems (such as bioreactors for biomass propagation) [52,53]. P. formosum was already established in axenic cultures in our lab and in future work, the yield of ohioensins could be tentatively enhanced by these procedures. 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Mol 2020, Vol 25, Page 2006 2020; 25: 2006. doi:10.3390/MOLECULES25092006 Conclusion In this thesis, three species of mosses (Dicranum majus, Thuidium delicatulum, and Polytrichum formosum) and one liverwort (Bazzania trilobata) are suggested as attractive botanical sources for pharmaceutical or cosmetics applications. D. majus and T. delicatulum exhibited significant inhibitory effects on NO production in LPS-induced RAW 264.7 cells, which suggest their effective properties in attenuating inflammation. P. formosum and B. trilobata showed inhibitory activities towards skin aging-related enzymes (collagenase) and pigmentation-related enzymes (tyrosinase). Moreover, B. trilobata extracts exhibited antioxidant and antimicrobial properties. Therefore, their active extracts might present effective properties for developing cosmetic ingredients. The bryophytes studied in this work, especially D. majus and T. delicatulum, do not have significant scientific literature related to their biological activities or phytochemical constituents. Except for the liverwort B. trilobata whose antifungal properties and derived commercial products are known [1,2]. Extracts of P. formosum and others species of Polytrichaceae have been patented with valuable properties for skin aging prevention [3], however, their active constituents were not described. In this study, a new group of rare compounds from mosses, the ohioensins, are reported with anti-collagenase and anti-tyrosinase activities. In addition, ohioensins and communins B were identified in the species of P. formosum along with a new ohioensin named nor-ohioensin D. In B. trilobata, a sesquiterpene caffeate was for the first time identified in this species. 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