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Bryophytes: Active Ingredients Discovery - Ph.D. Thesis

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Bryophytes, a source of inspiration for active ingredients discovery
Volpatto Marques, Raíssa
Publication date:
2021
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Citation (APA):
Volpatto Marques, R. (2021). Bryophytes, a source of inspiration for active ingredients discovery. DTU
Bioengineering.
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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. The
applied docking in this study is the flexible-rigid one (flexible ligand and rigid receptor) in
which all rotatable bonds of ligands are subjected to free rotation [167]. Pymol software was
used for the visualization of the interactions between ligands and proteins (Schrodinger. The
PyMOL Molecular Graphics System, Version 1.8 (2015)).
36
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46
1.9 Natural products from bryophytes: from basic biology to biotechnological
applications
Introduction to the chapter
This section includes a review on the ecological distribution, chemical diversity, novel genetic
tools and biotechnological applications of bryophytes.
My contribution to this manuscript was the collection of literature and the writing of Chapter
5 and table 5. 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. All authors reviewed and added
corrections to the manuscript. Y.L. contributed the graphical visualization and N.C. provided
the photos for Figure 1.
Acknowledgements
This work was supported by Marie- Skłodowska -Curie Actions Innovative Training Networks
under the Horizon 2020 programme under grant agreement n° 765115 – MossTech. We thank
to Gróa Valgerður Ingimundardóttir for giving us permission to use her photos in Figure 1.
78
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Chapter 2
Screening of bryophytes extracts for
biological activities of pharmaceutical
and cosmetic interest
100
Introduction to the chapter
This chapter is devoted to the screening of bryophytes extracts for biological activities of
pharmaceutical and cosmetic interest. 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.
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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).
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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. The NMR Center • DTU and the Villum
Foundation are acknowledged for access to the 800 MHz spectrometers.
Conflicts of Interest: The authors declare no conflict of interest.
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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. Moreover, in vitro cultivation
systems enable a sustainable supply of active ingredients and the conservation of endangered
species [54].
198
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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.
Thereby, the work presented in this thesis contributes to the knowledge of the biological
activities of bryophytes, opening up new possibilities for their applications in the cosmetics
and pharmaceutical sectors.
204
References
[1]
Frahm JP. Recent developments of commercial products from bryophytes. Bryologist 2004; 107:
277–283. doi:10.1639/0007-2745(2004)107[0277:rdocpf]2.0.co;2
[2]
Lebermoosextrakt, Pflanzenstärkungsmittel. . Im Internet: https://www.jean-puetzprodukte.de/lebermoosextrakt-pflanzenstaerkungsmittel-100-ml-p-103.html; Stand: 03.09.2021
[3]
Hanano Akinori, Akira H, Seki Taizo, et al. Cell activator, collagen production promoter,
melanin production inhibitor, hyaluronic acid production promoter and skin care preparation. JP
Pat 2003321376A, 2003
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