Content and Distribution of Usnic Acid Enantiomers in Three Icelandic Lichen Taxa

M.Sc. Thesis in Pharmacy

Content and distribution of usnic acid enantiomers in three Icelandic lichen taxa

Aron Elvar Gylfason

April 2019

Content and distribution of usnic acid enantiomers in three Icelandic lichen taxa

Aron Elvar Gylfason

M.Sc. Thesis in Pharmacy Administrative Supervisor: Bergþóra Sigríður Snorradóttir

Faculty of Pharmaceutical Sciences School of Health Sciences June 2019

This thesis is for a M.Sc. degree in Pharmacy and may not be reproduced in any form without the written permission of the author.

Printing: Svansprent ehf.

Reykjavík, Iceland 2019

Written by Aron Elvar Gylfason

Administrative Bergþóra Sigríður Snorradóttir, Ph.D. Supervisor Assistant Professor Faculty of Pharmaceutical Sciences, University of Iceland

Supervisor Maonian Xu, Ph.D. Post-doc fellow Faculty of Pharmaceutical Sciences, University of Iceland

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ABSTRACT

Content and distribution of usnic acid enantiomers in three Icelandic lichen taxa

Drug discovery has changed dramatically over the past years with the advent of new tools in chemical analytics and molecular biology. Successful discovery of new antibiotics, however, is rare. In addition to this, the overuse of antibiotics threatens their effectiveness due to increased resistance. Lichen species were used as traditional medicine prior to the discovery of the penicillin antibiotics and increased multidrug resistance has generated renewed interest in lichens.

Usnic acid is one of the most common secondary lichen metabolites and is by far the most extensively studied. The (+)-enantiomer of usnic acid has more potent antimicrobial activity (especially against Gram-positive bacteria) whereas the (-)-enantiomer shows mild antifungal activity and strong phytotoxicity. In contrast to the vast number of papers describing the bioactivity of usnic acid, the characterization of usnic acid enantiomers is lagging behind. The aim of this project was to determine the content of total usnic acid and distribution of usnic acid enantiomers in three commonly occurring lichens in Iceland, Flavocetraria nivalis, Cladonia arbuscula and Alectoria ochroleuca.

A UPLC-PDA-MS method was developed and validated for determination of total usnic acid content in lichens. The content of usnic acid ranged from 1.77% to 4.50%. The usnic acid peaks were fractionated using semi-preparative high- performance liquid chromatography (HPLC) and the chiral separation was performed using HPLC-UV. The ratio of (+)- and (-)-usnic acid varied substantially; Flavocetraria nivalis contained an average ratio of 1.76%:98.24%, Cladonia arbuscula contained an average ratio of 96.77%:3.23% and Alectoria ochroleuca contained only (-)-usnic acid. The complexity of usnic acid production has been revealed in our study of three commonly occurring Icelandic taxa.

In light of pronounced antibiotic activities and intrinsic high contents of usnic acid, this work will have biological and ecological importance for future studies focusing on usnic acid-containing lichens.

ÁGRIP

Innihald og dreifing úsnínsýruhandhverfa í þremur íslenskum fléttutegundum

Með tilkomu nýrra verkfæra í efnafræðilegum greiningum og sameindalíffræði hefur uppgötvun lyfja breyst verulega á undanförnum árum. Árangursrík uppgötvun nýrra sýklalyfja er hins vegar sjaldgæf. Þar að auki er ljóst að ofnotkun sýklalyfja ógnar virkni þeirra vegna vaxandi ónæmi. Fléttutegundir voru notaðar sem hefðbundin lyf fyrir uppgötvun pensillínsýklalyfja og aukið fjöllyfja ónæmi hefur endurnýjað áhugann á fléttum.

Úsnínsýra er eitt algengasta og um leið mest rannsakaða innihaldsefni fléttutegunda. (+)-Handhverfan af úsnínsýru hefur öflugri örverudrepandi virkni (sérstaklega gegn Gram-jákvæðum bakteríum) á meðan (-)-handhverfan sýnir væga sveppaeyðandi virkni og mikil plöntueituráhrif. Fjöldi birtra rannsókna sem lýsa eiginleikum úsnínsýruhandhverfa hefur ekki haldið í við fjölda greina sem lýsa lífvirkni úsnínsýru. Markmið verkefnisins var að ákvarða heildarinnihald úsnínsýru og dreifingu úsnínsýruhandhverfa í þremur algengum fléttum á Íslandi, maríugrösum (Flavocetraria nivalis), hreindýramosa (Cladonia arbuscula) og skollakræðu (Alectoria ochroleuca).

UPLC-PDA-MS-aðferð var þróuð og gilduð fyrir ákvörðun á heildarinnihaldi úsnínsýru í fléttum. Innihald úsnínsýru var á bilinu 1,77% til 4,50%. Úsnínsýrutopparnir voru sundurgreindir með undirbúinni hágæðavökvaskiljun (HPLC) og hendniaðskilnaðurinn var framkvæmdur með HPLC-UV. Hlutfall (+)- og (-)-úsnínsýru var verulega breytilegt milli tegunda. Flavocetraria nivalis innihélt meðalhlutfallið 1,76%:98,24%, Cladonia arbuscula innihélt meðalhlutfallið 96,77%:3,23% og Alectoria ochroleuca innihélt einungis (-)-úsnínsýru. Þessi rannsókn sýndi fram á breytileika í magni úsnínsýru innan þessara þriggja algengu íslensku fléttutegunda.

Í ljósi áberandi örverudrepandi virkni og hás innihalds úsnínsýru er framlag þessa verks bæði líffræðilegt og vistfræðilegt. Verkið skiptir jafnframt máli í samhengi framtíðarrannsókna með áherslu á fléttur sem innihalda úsnínsýru.

LIST OF ABBREVIATIONS

AA Acetic acid ACN Acetonitrile ESI Electrospray ionization FA Formic acid FDA Food and Drug Administration HPLC High-performance liquid chromatography MeOH Methanol MS Mass spectroscopy NMR Nuclear magnetic resonance UPLC Ultra-performance liquid chromatography UV Ultraviolet

TABLE OF CONTENTS

1. INTRODUCTION ...... 1

1.1 Lichens ...... 2

1.2 Usnic acid ...... 4 1.2.1 Chemistry and production of usnic acid ...... 4

1.3 Analysis ...... 7 1.3.1 Analysis of usnic acid enantiomers ...... 7 1.3.2 Mass spectrometry (MS) and ultraviolet (UV) analysis of usnic acid...... 7

1.4 Ecological role of usnic acid ...... 8 1.4.1 Traditional use ...... 10

1.5 Biological activity and hepatotoxicity ...... 10 1.5.1 Antimicrobial activities ...... 10 1.5.2 Antitumor activity ...... 12 1.5.3 Anti-inflammatory, analgesic and antipyretic activity ...... 12 1.5.4 Antiprotozoal activity ...... 12 1.5.5 Hepatotoxicity ...... 13

2. AIM ...... 15

3. MATERIALS AND METHODS ...... 16

3.1 Materials...... 16

3.2 Equipment ...... 17

3.3 Methods ...... 18 3.3.1 Sample preparation ...... 18 3.3.2 Usnic acid quantitation with UPLC-PDA-MS ...... 18 3.3.3 Usnic acid fractionation with semi-preparative HPLC ...... 19 3.3.4 Enantiomeric ratio of usnic acid using chiral HPLC ...... 20 3.3.5 Standard operating procedure ...... 20

4. RESULTS ...... 22

4.1 Validation of the UPLC method for usnic acid quantitation...... 22

4.2 Determination of total usnic acid contents using UPLC-PDA-MS ...... 23

4.3 Usnic acid fractionation and enantiomeric ratio determination ...... 26

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5. DISCUSSION ...... 30

5.1 Usnic acid total content ...... 30

5.2 Usnic acid enantiomeric ratio ...... 31

5.3 Chiral separation ...... 31

6. CONCLUSIONS ...... 33

7. ACKNOWLEDGEMENTS ...... 34

REFERENCES ...... 35

APPENDIX A ...... A

APPENDIX B ...... D

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LIST OF TABLES

Table 1. Distribution of usnic acid enantiomers in lichens ...... 6 Table 2. Ratio of usnic acid enantiomers in lichens ...... 7 Table 3. Examples of different bioactivities between usnic acid enantiomers ...... 9 Table 4. List of materials used in this project ...... 16 Table 5. List of equipment used in this project ...... 17 Table 6. Different solvent combinations for the optimization of chiral separation ...... 20 Table 7. Intra-day coefficient of variation values ...... 22 Table 8. Inter-day coefficient of variation values ...... 22 Table 9. Standard curves of three-day analysis ...... 22 Table 10. The dry weight of usnic acid (mg/g) and % of content ± SD in all three species ...... 26 Table 11. Ratio of usnic acid enantiomers ...... 29

LIST OF FIGURES

Figure 1. Development and discovery of antibiotics since 1930 ...... 1 Figure 2. β-lactamase enzymes identified since 1970, showing the increase in resistance ...... 2 Figure 3. Area photographs of some usnic acid-containing lichens in Iceland...... 3 Figure 4. Structures of (+)- and (-)-usnic acid ...... 5

Figure 5. UV spectra (200-400 nm) of (+)-usnic acid (tR = 16.2 min) and (-)-usnic acid (tR = 20.0 min) showing the two maxima at ca. 232 and 282 nm ...... 8 Figure 6. The elution gradient used in the analysis of usnic acid with UPLC-PDA-MS ...... 19 Figure 7. UPLC-PDA chromatograms for (A) Flavocetraria nivalis extract (B) Cladonia arbuscula extract (C) Alectoria ochroleuca extract ...... 23 Figure 8. UV-VIS spectrum (210-500 nm) (A) and MS spectrum (B) of usnic acid ... 24 Figure 9. MS spectra showing base peaks of the two minor compounds in the lichen Alectoria ochroleuca (A) Diffractaic acid (B) 4-O-demethyldiffractaic acid ...... 25 Figure 10. UV-VIS spectra (200-500 nm) of the two minor compounds in the lichen Alectoria ochroleuca (A) Diffractaic acid (B) 4-O-demethyldiffractaic acid ...... 25 Figure 11. Semi-preparative HPLC-UV chromatogram for Flavocetraria nivalis extract ...... 26 Figure 12. Chiral HPLC-UV chromatogram of the usnic acid enatiomer standards under optimized elution conditions (MeOH + 0.1% FA:ACN (83:17 v/v)). (+)-Usnic acid tR = 10.095; (-)-usnic acid tR = 11.740 ...... 27 Figure 13. Usnic acid enatiomer composition in all three species. (A) Flavocetraria nivalis (B) Cladonia arbuscula (C) Alectoria ochroleuca...... 28

Declaration of Contribution The project was carried out at the research facilities of the Faculty of Pharmaceutical Sciences, University of Iceland. The lichen specimens were collected by Starri Heiðmarsson from the Icelandic Institute of Natural History, Akureyri Division. Maonian Xu conceived and designed the project along with Aron Elvar Gylfason. Aron Elvar Gylfason performed the analysis and collected the data after training by the supervisors, with the help of Maonian Xu. Aron Elvar Gylfason wrote the thesis and Bergþóra Sigríður Snorradóttir and Maonian Xu reviewed it and provided guidance and suggestions for its improvement. Aron Elvar Gylfason made the standard operating procedure and Árni Þorgrímur Kristjánsson helped by testing it and providing suggestions for its improvement.

1. INTRODUCTION

Drug discovery has taken dramatic changes over the past years with new technology and modern molecular biology methods (Eder & Herrling, 2016). However, recent development in discovery of new antibiotics is a rare event (Figure 1). Antibiotics are an important drug class, they have revolutionized medicine and made advances in recent years possible.

Figure 1. Development and discovery of antibiotics since 1930. Figure acquired from Antibiotic Research UK (UK, 2019).

However, overuse of these wonder drugs threatens their effectiveness and the ability to treat patients due to the rise and spread of antibiotic resistant pathogens (Figure 2). Therefore, the presently limited stock of drugs to combat these resistant bacteria, some of which are multidrug-resistant, is an object to overcome (Arias & Murray, 2015; Frieri, Kumar, & Boutin, 2017; Richardson, 2017; Zaman et al., 2017). Lichen species, specifically Usnea species, had been used as traditional medicine prior to the discovery and arrival of the penicillin antibiotics, this sparked renewed interest in them in the 1980s because of increased multidrug resistance (Cocchietto, Skert, Nimis, & Sava, 2002).

Figure 2. β-lactamase enzymes identified since 1970, showing the increase in resistance. Figure acquired from Davies & Davies (Davies & Davies, 2010).

1.1 Lichens Lichens are microbial communities consisting of a broad spectrum of microorganisms, such as fungi, bacteria, microalgae and/or cyanobacteria (Grube, Cardinale, Vieira Castro, Müller, & Berg, 2009; Spribille et al., 2016). Together they form a conspicuous vegetative structure called thallus (pl. thalli). Lichens are found all over the world and cover approximately 8% of the earth’s surface (Larson, 1987). Their slow metabolism, production of biologically active metabolites and ability to stay in a metabolic resting state for long periods of time are what makes them capable of surviving and adapting to extreme environmental conditions (Huneck & Yoshimura, 1996; Leavitt et al., 2015; Romeike, Friedl, Helms, & Ott, 2002). The structure and shape of the lichens can differ distinctly, from fruticose lichens with small leafless branches (Figure 3A, 3B) to foliose lichens with flat leaf-like structures (Figure 3C) and other growth forms (Galloway, 1999).

Figure 3. Area photographs of some usnic acid-containing lichens in Iceland. (A) Flavocetraria nivalis; (B) Cladonia arbuscula; (C) Alectoria ochroleuca. – (With permission from Hörður Kristinsson.)

There are at least 19,387 accepted species of lichen-forming fungi from Ascomycota and Basidiomycota (Lücking, Hodkinson, & Leavitt, 2016). With the use of different chromatographic and spectroscopic methods, such as high-performance liquid chromatography (HPLC), ultraviolet spectrophotometer (UV) and nuclear magnetic resonance (NMR), there are now more than 1000 isolated lichen substances (Huneck, 1999; Stocker-Wörgötter, 2008). The hypothesized ecological roles of lichen substances include: protection against bacterial, viral and protozoan parasites, against insects and other animal predators; protection against environmental elements such as UV rays and extreme dryness and regulation of physiological pathways (Cocchietto et al., 2002). Humans have been using lichens and their active compounds for centuries as sources of dye, as food in periods of famine (Cocchietto et al., 2002), floral decorations, perfumery and for their therapeutic properties in medicine (Cocchietto et al., 2002; Ingolfsdottir, 2002). However, testing their ecological roles as well as sustainable production is still very challenging owing to the difficulty of culturing lichens in controlled conditions for a proper amount of time.

1.2 Usnic acid

1.2.1 Chemistry and production of usnic acid Usnic acid [2,6-diactyl-7,9-dihydroxy-8,9b-dimethyl-1,3(2H,9bH)-dibenzo-furandione;

C18H16O7] (Figure 4) was first isolated during the developmental years of organic- and phytochemistry by German scientist W. Knop in 1844, who also described the basic physical and chemical characteristics (Ingolfsdottir, 2002). The molecule has three hydroxyl groups and the enolic 3-OH is the most acidic character (pKa 4.4) because of an inductive effect of the keto group, followed by 9-OH (pKa 8.8) and 6-OH (pKa 10.7) (Galasso, 2010; Ingolfsdottir, 2002). Along with (+)- and (-)-usnic acids, two other isomers also appear in lichens, (+)- and (-)-isousnic acids. They are structural isomers of the two enantiomers but vary in substitution in ring A (Ingolfsdottir, 2002). Usnic acid can be synthesized from methylphloroacetophenone by oxidative coupling (Hawranik, Anderson, Simmonds, & Sorensen, 2009). It has also been confirmed that methylphloroacetophenone is an intermediate in usnic acid biosynthesis in lichens (Taguchi, Sankawa, & Shibata, 1969), and specific biosynthetic genes have been proposed (Abdel-Hameed, Bertrand, Piercey-Normore, & Sorensen, 2016).

Figure 4. Structures of (+)- and (-)-usnic acid.

Usnic acid is one of the most common secondary lichen metabolites and is by far the most extensively studied (Cocchietto et al., 2002; Ingolfsdottir, 2002). It is particularly abundant in genera such as Alectoria, Cladonia, Usnea, Lecanora, Ramalina and Evernia. The content of usnic acid in lichen thalli can reach up to 4-8% (Bjerke, Elvebakk, Domínguez, & Dahlback, 2005). Usnic acid has recently been isolated from bacteria associated with the marine lichen Lichina confinis (Parrot et al., 2016). Closely related dibenzofuran compounds have also been found in plants such as Sorbus aucuparia (Hüttner, Beuerle, Scharnhop, Ernst, & Beerhues, 2010), fungi such as Mycosphaerella nawae (Sassa & Igarashi, 1990) and other ascomycetes (Millot, Dieu, & Tomasi, 2016). As a dibenzofuran it is among the most unique lichen substances along with other chemical classes such as depsides and depsidones (Ingolfsdottir, 2002). The distribution of usnic acid enantiomers has been observed in a few studies (Table 1). All Usnea species, which have been used in traditional herbal medicine, seem to only produce (+)-usnic acid. The selectivity of these lichens has implications on the pharmaceutical potential of (+)-usnic acid.

Table 1. Distribution of usnic acid enantiomers in lichens. Lichenized fungal Lichen species References family (+)-Usnic acid

Cladoniaceae Cladonia arbuscula (Einarsdottir et al., 2010) Cladonia mitis (Kinoshita, Yamamoto, Yoshimura, Kurowaka, & Huneck, 1997) Nephromataceae Nephroma arcticum (Kinoshita et al., 1997) Ophioparmaceae Ophioparma ventosa (Kinoshita et al., 1997) Parmeliaceae Evernia esorediosa (Kinoshita et al., 1997) Flavopermelia caperata (Kinoshita et al., 1997) Hypogymnia hypotrypella (Kinoshita et al., 1997) Parmelia incurva, P. separate (Kinoshita et al., 1997) Usnea diffracta, U. longissimi, U. hirta, (Kinoshita et al., 1997) U. bismolluscula, U. rubuscens, U. rubicunda Usnea steineri (Lucarini et al., 2012) Xanthoparmelia conspersa (Kinoshita et al., 1997) Ramalinaceae Ramalina boninesis, R. pacifica, R. roesleri, (Kinoshita et al., 1997) R. yasudae

(-)-Usnic acid

Alectoriaceae Alectoria lata (Kinoshita et al., 1997) Alectoria ochroleuca (Melgarejo, Sterner, Vila Castro, & Mollinedo, 2008) Cladoniaceae Cladonia uncialis (Studzinska-Sroka et al., 2015) Cladonia pleurota (Kinoshita et al., 1997) Cladonia foliacea (Koparal, 2015) Parmeliaceae Cetraria oakesiana (Kinoshita et al., 1997) Nephromopsis rugosa (Kinoshita et al., 1997) Parmeliopsis ambigua (Kinoshita et al., 1997) Vulpicida juniperinus (Kinoshita et al., 1997)

(+)- and (-)-usnic acid

Cladoniaceae Cladonia stellaris (Smeds & Kytöviita, 2010) Parmeliaceae Flavocetraria cucculata, F. nivalis (Kinoshita et al., 1997) Vulpicida pinastri (Legouin et al., 2017)

Only four lichen taxa from the genera Cladonia, Flavocetraria and Vulpicida have been shown to produce both enantiomers (Table 1), and interestingly, two (i.e. Flavocetraria and Vulpicida) of them belong to the cetrarioid group of the family Parmeliaceae (Nelsen et al., 2011).

1.3 Analysis 1.3.1 Analysis of usnic acid enantiomers In contrast to the vast number of papers describing the bioactivity of usnic acid, the characterization of usnic acid enantiomers is rather lagging behind. This may affect the interpretations of bioactivity screening results – which enantiomer or mixture of enantiomers results in observed activities. Until now only three papers have studied the ratio of these enantiomers in lichens, as shown in Table 2. Both Bjerke et al. (2005) and Smeds & Kytöviita (2010) used HPLC-UV methods but only the latter used a chiral column with a rather long retention time (ca. 1 hour). Legouin et al. (2017) estimated the ratio of enantiomers using electronic circular dichroism. Therefore, there is a huge need to develop a good chiral chromatography method to characterize the enantiomeric ratio in usnic acid-containing lichens.

Table 2. Ratio of usnic acid enantiomers in lichens. Taxon Ratio (+):(-) Analytical method Reference Flavocetraria nivalis 100:1 HPLC-UV with a (Bjerke et al., 2005) C18 column Cladonia stellaris 0.4-10%:99.6-90% HPLC-UV with a (Smeds & Kytöviita, 2010) chiral column Vulpicida pinastri 35%:65% Electronic circular (Legouin et al., 2017) dichroism

1.3.2 Mass spectrometry (MS) and ultraviolet (UV) analysis of usnic acid Although usnic acid is one of the most studied lichen substances, its negative ion mode mass spectrometry pathway has rarely been reported (Musharraf, Kanwal, Thadhani, & Choudhary, 2015) and the fragmentation in negative ion mode appears to be relative to the MS interface (Xu et al., 2017). The electrospray ionization (ESI)-MS spectrum of usnic acid generally displays an adduct ion of m/z = 709 which corresponds to [2M – 2H + Na+]- (Xu et al., 2017). Ionization in either laser desorption ionization (Le Pogam et al., 2015) or fast atom bombardment interface (Holzmann & Leuckert, 1990) generate the major product ion at m/z = 329 [M-CH3]. This is found to be the only product ion in negative ion mode (Huneck & Yoshimura, 1996). Some studies using

ESI however have found the major product ion at m/z = 328 [M-CH4]- instead (Musharraf et al., 2015; Xu et al., 2017).

The UV spectrum of usnic acid has two maxima, at ca. 232 and 282 (Figure 5), varying according to the solvent in use. Liquid chromatography with UV detection at 282 nm reliably quantifies usnic acid in lichen extracts whereas the 232 nm maximum is solvent-dependant and not as selective as 282 nm (Roach, Musser, Morehouse, & Woo, 2006).

Figure 5. UV spectra (200-400 nm) of (+)-usnic acid (tR = 16.2 min) and (-)-usnic acid (tR = 20.0 min) showing the two maxima at ca. 232 and 282 nm. Figure acquired from Kinoshita et al. (1997).

1.4 Ecological role of usnic acid The main role of usnic acid in lichens is speculated to be the protection of the algae/cyanobacteria from radiation (Smeds & Kytöviita, 2010). However, it may also act as an antimicrobial agent against bacteria (Lauterwein, Oethinger, Belsner, Peters, & Marre, 1995) and fungal pathogens (Cardarelli et al., 1997) and as an antifeedant against insects (Pöykkö, Hyvärinen, & Bačkor, 2005) and snails (Gauslaa, 2005). The two enantiomers have somewhat different biological effects. The (+)-enantiomer has more potent antimicrobial activity whereas the (-)-enantiomer shows antifungal activity as well as strong phytotoxicity (Table 3) (Cocchietto et al., 2002; Smeds & Kytöviita, 2010).

Table 3. Examples of different bioactivities between usnic acid enantiomers. Activity (+)-Usnic acid (-)-Usnic acid Reference

Antivirus (Influenza) ED50 = 51.7 µM ED50 = 14.5 µM (Sokolov et al., 2012)

Insecticidial (S.littoralis) LD50 = 90.8 LD50 = 8.6 µmol/g (Emmerich, Giez, Lange, µmol/g & Proksch, 1993)

Antibacterial (S.mutans) MIC50 = 15 µg/ml MIC50 = 85 µg/ml (Ghione, Parrello, & Grasso, 1988)

Cytotoxic (human K562) IC50 = 52.8 µM IC50 = 21.8 µM (Bazin et al., 2008)

Contact dermatitis (skin) Positive Negative (Mitchell, 1966)

Phytotoxic (PPOX enzyme) Not active IC50 = 3 µM (Romagni, Meazza, Nanayakkara, & Dayan, 2000)

Phytotoxic (HPPD enzyme) Not active IC50 = 50 nM (Romagni et al., 2000)

Abbreviations PPOX: protophorphyrinogen HPPD: 4-hydroxyphenylpyruvate dioxygenase

Compared to the research efforts in pharmaceutical properties of usnic acid, its ecological roles have yet to be fully tested. The main reason for this is the difficulty of keeping the lichen thalli in controlled conditions long enough for experimentation (Cocchietto et al., 2002). The majority of papers seems to be focused on the (+)-enantiomer, which could partly be explained by the fact that the (-)-enantiomer occurs less often in lichen species, and (+)-usnic acid is much more commercially available (Galanty, Paśko, & Podolak, 2018). The papers discussing the ecological roles tend to focus on monitoring of the secondary metabolite concentrations and for usnic acid, its concentration has certainly been shown to be strongly influenced by several ecological factors. For example, light, temperature and humidity all play a part in the concentration of lichen acids as well as seasonal changes (Bjerke et al., 2005). The concentration of usnic acid is higher in late spring and early summer compared to autumn and winter, most likely because of light intensity (Bjerke et al., 2005). Younger thalli also showed a significantly higher concentration of usnic acid than older thalli, probably due to higher metabolic activity (Cocchietto et al., 2002; Quilhot, 1991).

1.4.1 Traditional use Hippocrates supposedly used Usnea barbata as a remedy against urinary complaints (Ingolfsdottir, 2002) and the Chinese used Usnea longissimi as an expectorant and for the treatment of wounds under the name “Sun-Lo” (Shibata, Ukita, & Tamura, 1948). Alectoria species and Usnea species have been used a lot in Finland for skin eruptions or for athlete’s foot, where the lichen is placed on an infected or fresh wound. It has also been used in oral doses for sore throat or toothache. Cladonia species has been used in the treatment of pulmonary tuberculosis and cough, generally in the form of so-called lichen milk (Vartia, 1973).

Superstition and archaic medical skill based on nature describes the popular medicinal uses of lichens very well. In the use of lichens for cough or more specifically for pulmonary tuberculosis however, old folklore and traditions exist all over the globe and modern science has also found moderately strong antibiotics against tuberculosis in some of the same species to some extent. Although the concentration of the active substances is low, the effect is increased since the substances mainly exist in outer surface of the thallus in the form of crystals or powder and can therefore diffuse into their environment (Vartia, 1973). Usnea species, or purified usnic acid, are currently components in an assortment of products worldwide. Purified usnic acid and also just the lichen extract has been used in perfumery, sunscreens, cosmetics and a variety of personal hygiene products such as shampoo, deodorant, toothpaste and mouthwash (Guo et al., 2008).

1.5 Biological activity and hepatotoxicity 1.5.1 Antimicrobial activities Both enantiomers have shown activity against mycobacteria and Gram-positive bacteria (Ingolfsdottir, 2002), and numerous clinical trials and research studies have confirmed the antimicrobial properties of usnic acid.

Zn-usnic acid intravaginal formulation for use in adjuvant therapy against genital human papillomavirus was assessed in a clinical study with 100 female patients (aged 18-46). With regards to recurrence of infection and reepithelization of lesions, the outcome showed significantly positive results for patients receiving the Zn-usnic acid formulation compared to a control group receiving no adjuvant therapy. This difference remained significant even over a 6-month period (Scirpa et al., 1999). The

antiviral activity of usnic acid isomers can also be associated with repression of RNA transcription (Campanella, Delfini, Ercole, Iacoangeli, & Risuleo, 2002). The antiviral activity of both usnic acid enantiomers was compared in a study by Sokolov et al. (2012). The in vitro activity of (-)-usnic acid was much stronger against influenza

A(H1N1)pdm09 virus when compared to (+)-usnic acid, with ED50 values 14.5 µM and 51.7 µM respectively (Sokolov et al., 2012).

Additionally, (+)-usnic acid applied on filter paper discs has been shown to inhibit cytopathogenic effects of polio type 1 and herpes simplex type 1 viruses in BS- C-1 cells (Perry et al., 1999). (+)-Usnic acid isolated from U. longissima has been shown to be effective against tumour-promoter-induced Epstein-Barr virus, whereas commercially obtained (-)-usnic acid showed less activity (Yamamoto et al., 1995).

Usnic acid seems to be a more potent antimicrobial agent than other lichen metabolites. According to the available literature, usnic acid, along with orcinol-type depsidones and depsides and vulpinic acid derivatives, is the most active antibiotic lichen substance (Lawrey, 1986). Usnic acid has been shown to be effective against enterococci, staphylococci, anaerobic Gram-negative bacteria, anaerobic Gram-positive bacteria and against a sizable number of isolates regardless of their resistant phenotype. Both enantiomers of usnic acid showed considerable antimicrobial activity against Staphylococcus aureus, Enterococcus faecium and others, but the (+)-enantiomer was more active against Enterococcus faecalis and some Bacteroides species. The activity was similar in comparison with reference antimicrobial agents but in the case of resistant strains the activity was higher (Cocchietto et al., 2002; Ingolfsdottir, 2002; Lauterwein et al., 1995). In a recent study, both enantiomers of usnic acid were effective inhibitors of one reference strain and six isolates of Helicobacter pylori. The (+)-enantiomer was at least twice as active when compared to (-)-usnic acid, apart from the reference stain and one clinical isolate, where the MIC values were the same (Lage et al., 2018).

Chemical modifications that change the hydrogen-bonding properties can have a big impact on the antimicrobial activity of usnic acid. Even just minor structural changes can render the derivatives, usnic acid acetate and dihydrousnic acid, less effective against Staphylococcus aureus (Correche et al., 1998). The antimicrobial activity of (+)-usnic acid is mainly caused by inhibition of DNA and RNA synthesis. The effect is most potent against Gram-positive bacteria but also takes place in

Gram-negative bacteria (Maciąg-Dorszyńska, Węgrzyn, & Guzow-Krzemińska, 2014; Xu et al., 2016).

During a short-term treatment, the effectiveness of an antibiotic-antimycotic combination against Tinea pedis was studied. In total 65 patients were subjected to a treatment with usnic acid salt and undecylenic acid and they exhibited a significant improvement in their conditions (Cocchietto et al., 2002).

1.5.2 Antitumor activity Both enantiomers of usnic acid have similar cytotoxic activity in many cell lines, such as L1210, DU145, T-47D and Capan-2. However, (-)-usnic acid is more effective in certain cell models like human lymphocytes for the induction of cell apoptosis. The application of (+)- and (-)-usnic acids have also shown inhibition of DNA synthesis in human cell lines (T-47D and Capan-2). This effect appeared with the loss of mitochondrial membrane potential in cells (Einarsdottir et al., 2010). Usnic acid seems to have anti-proliferative activity against certain breast cancer cell lines like the wild-type p53 (MCF7) and the non-functional p53 (MDA-MB-231) as well as the lung cancer cell line H1299, which is null for p53. Usnic acid is therefore a potential candidate as a topical agent or for systemic therapy in the treatment of tumors (Mayer et al., 2005).

1.5.3 Anti-inflammatory, analgesic and antipyretic activity (+)-Usnic acid has shown significant activity compared to ibuprofen. Both acute and chronic effects were tested using the rat paw oedema assay and the cotton pellet assay respectively. With an oral dose of 100 mg/kg in rats it showed similar effectiveness as ibuprofen (Vijayakumar et al., 2000). Usnic acid (and diffractaic acid) was also evaluated with regards to analgesic and antipyretic effects in mice. The acetic acid-induced writhing and tail pressure methods were used and resulted in a considerable analgesic effect. Usnic acid also showed significant antipyretic activity against lipopolysaccharide induced hyperthermia (Okuyama, Umeyama, Yamazaki, Kinoshita, & Yamamoto, 1995).

1.5.4 Antiprotozoal activity (-)-Usnic acid showed a substantial inhibitory effect against the pathogenic protozoa Trichomonas vaginalis at a relatively lower concentration than metronidazole (Wu, 12

Zhang, Ding, Tan, & Yan, 1995). (+)-Usnic acid has also exhibited leishmanicidal activity both in vitro and in vivo. Intralesional treatment showed a decrease in lesion weight and parasite load (Fournet et al., 1997).

1.5.5 Hepatotoxicity Usnic acid-induced toxicity occurs more often in the liver than any other organ. Almost all absorbed substances that are ingested will first enter the liver, and the liver is also responsible for the metabolism and elimination of ingested substances (Araujo et al., 2015). A major problem with usnic acid in pharmaceutical products is the potential risk of severe liver damage (hepatotoxicity). Usnic acid has been associated with hepatotoxicity when taken in overdose amounts. The recommended dosage of LipoKinetix®, i.e. 100 mg of usnic acid in each capsule, was 1-2 capsules three times per day, which is three to six times higher than the dosages used in traditional Chinese medicine (Guo et al., 2008). The U.S. FDA has received around 21 reports of hepatotoxicity in people who ingested dietary supplements that contain usnic acid or sodium usnate for the sake of weight loss, thus raising safety concerns. Consumption of those dietary supplements has resulted in one death, seven consumers with liver failure, one liver transplant, ten cases of hepatitis and four cases of mild hepatic toxicity (Favreau, Ryu, Braunstein, & et al., 2002; Neff et al., 2004). Many other cases show similar results of consumers taking dietary supplements containing usnic acid and experiencing hepatotoxicity (Durazo et al., 2004; Sanchez, Maple, Burgart, & Kamath, 2006; Yellapu, Mittal, Grewal, Fiel, & Schiano, 2011).

The primary targets of usnic acid-induced hepatotoxicity seems to be the endoplasmic reticulum, lysosomes and mitochondria, leading to mitochondrial impairment, energy reduction and stress (Bessadottir et al., 2012; Liu, Zhao, Lu, Fan, & Wang, 2012). These products are marketed as dietary supplements and are therefore not regulated as drugs. It is up to the manufacturers and distributers to oversee and regulate the safety of the products. LipoKinetix® is a multi-component dietary supplement containing caffeine, norephedrine hydrochloride and yohimbine hydrochloride (Guo et al., 2008). The interactions of those components are untested and potential drug interactions could increase the hepatotoxicity. Sadly, these products are still available on the market, and the risk of their consumption is still a problem, even though the U.S. FDA has warned consumers to immediately stop the use of

LipoKinetix® and recommended the withdrawal of the products (CFSAN, 2001; Xu et al., 2016).

Hepatotoxicity of usnic acid could be substantially reduced using nanoencapsulation with enhanced antitumor activity (da Silva Santos et al., 2006; Ribeiro-Costa et al., 2004). This provides an interesting approach to reduce usnic acid hepatotoxicity using proper pharmaceutical formulations.

Overall, usnic acid isomers show very interesting bioactivity such as antimicrobial, antiviral, antiproliferative, antiprotozoal, anti-inflammatory and analgesic activity (Ingolfsdottir, 2002). Previous studies have also demonstrated the differences between usnic acid enantiomers (Table 3). To further clarify the activity of usnic acid enantiomers, a good analytical method is needed. Although hepatotoxicity is a major problem, pharmaceutical drug formulations have shown promise in that field (da Silva Santos et al., 2006; Ribeiro-Costa et al., 2004).

2. AIMS

Usnic acid is widely distributed among lichen genera and well recognized for its potent antibacterial, anti-inflammatory, antiviral and anti-proliferative activity. It has two enantiomers and only (+)-usnic acid has been extensively studied and screened for bioactivity, leaving (-)-usnic acid rather neglected. This is mainly due to poor separation and characterization of usnic acid enantiomers in lichens.

The current study aims to determine the content and distribution of usnic acid enantiomers in commonly occurring lichens in Iceland. Specific objectives are:

I. To develop an ultra-high performance liquid chromatography coupled to photo diode array detector and mass spectrometer (UPLC-PDA-MS) method for total usnic acid determination.

II. To determine the ratio of usnic acid enantiomers using a HPLC-UV method with a chiral column.

III. To create a standard operating procedure for the Waters Acquity UPLC-PDA- MS system

3. MATERIALS AND METHODS 3.1 Materials

Table 4. List of materials used in this project. Material Manufacturer Lot number Acetonitrile Riedel-de-Haen H348F Methanol Riedel-de-Haen H272B Formic acid Riedel-de-Haen I240DIL Acetic acid Sigma-Aldrich 83310 (+)-Usnic acid standarda - - (-)-Usnic acid standardb - - a: Isolated in pure form from Cladonia arbuscula (Einarsdottir et al., 2010). b: Isolated in pure form from Alectoria ochroleuca (Einarsdottir et al., 2010).

3.2 Equipment

Table 5. List of equipment used in this project. Equipment Type Manufacturer HPLC system “STEINI” Ultimate 3000 Dionex Solvent Rack SR-3000 - Pump LPG-3400 - Well Plate Sampler - - Column Compartment - - UV-VIS Detector VWD-3400 - UPLC system “Waters” Waters Acquity Quaternary Solvent - - Manager Sample Manager – FTN - - Column Manager - - PDA Detector - - QDa Detector - - HPLC system “THORUNN” Ultimate 3000 Dionex Solvent Rack & Degasser SRD-3400 - Pump HPG-3400A - Well Plate Sampler WPS-3000TSL - Column Compartment TCC-3100 - Photodiode Array PDA-3000 - Detector Column Luna 5u Phenyl-Hexyl, Phenomenex 250x10.00mm 5 micron Column Lux® 3µm Amylose-1, Phenomenex 150x4.6mm Column Luna® Omega, Phenomenex 100x2.1mm, 1.6µm Vials HPLC – 2ml Waters Caps MSQ™ Caps with Supelco septa, 9mm PTFE Septa Teflon® Lined Silicone Supelco Septa Vials Clear Vial – 7ml Supelco Caps Solid cap with PTFE Supelco Liner, for 7ml Vial Pipette 1 100-1,000µl Thermo Scientific Pipette 2 10-100µl Thermo Labsystems Scale Analyze weight Mettler Toledo Sonic bath 5800 Branson MilliQ water Millipore Merck Syringe 1ml Injekt®-F Luer Solo B. Braun Filters Phenex RC 0.45 µm Phenomenex Chromatography data Chromeleon 7 Thermo Fisher Chromatography data Empower 3 Waters Vortex Vortex-Genie 2 Scientific Industries Centrifuge Pico 17 Thermo

3.3 Methods 3.3.1 Sample preparation Fresh lichen samples were picked up in north and west Iceland on the 4th of November. The samples were sorted into species, ground in liquid nitrogen and then freeze dried overnight. The lyophilized lichen material was accurately weighed (20 mg) and macerated with acetonitrile (800 µl). The sample was vortexed and then centrifuged (8,000 rpm, 6 min) and the clear supernatant was collected. The maceration was repeated four more times and the supernatants were combined. Extracts were filtered (RC, 0.45 µm, Phenomenex, USA) and stored at 6°C before chromatographic analysis. Each lichen specimen was prepared in triplicate.

3.3.2 Usnic acid quantitation with UPLC-PDA-MS Usnic acid enantiomeric standards were isolated from a previous study (Einarsdottir et al., 2010). For quantitation of total usnic acid, (+)-usnic acid was used as a standard since usnic acid enantiomers do not differ in UV absorbance (Kinoshita et al., 1997). (+)-Usnic acid standard was weighted and a stock solution (233.00 µg/ml) was prepared in acetonitrile (ACN). The stock solution was diluted to make a series of standard solutions (14.56 µg/ml, 7.28 µg/ml, 3.64 µg/ml, 1.82 µg/ml and 0.91 µg/ml).

Chromatographic separation was performed on a Luna Omega C18 column (100 mm × 2.1 mm, 1.6 µm; Phenomenex, USA). The temperature of the column oven and autosampler was set at 25°C. Solvent A was water + 0.1% formic acid (FA) and solvent B was ACN + 0.1% FA. The elution conditions were as follows: isocratic elution 50% A / 50% B, 0-1 min; linear gradient from 50% A / 50% B to 100% B, 1-3.5 min; isocratic elution 100% B, 3.5-5.5 min; linear gradient to 50% A / 50% B, 5.5-6 min; isocratic elution 50% A / 50% B, 6-8 min; flow rate 0.4 ml/min (Figure 6). Quantitation of total usnic acid was carried out using a photo diode array detector. Detection wavelength was 280 and spectra were monitored from 210 to 500 nm.

MS detection was used to validate peak identities and check peak homogeneity. MS scan was performed on negative ion mode from 100 to 500 Da. Selected ion recording was set as 343.09 Da, which corresponds to the deprotonated molecular ion of usnic acid. Cone voltage was 15 V and capillary voltage 0.8 kV. MS data was not used for the purpose of quantitation.

Figure 6. The elution gradient used in the analysis of usnic acid with UPLC-PDA-MS.

The UPLC method was validated by assessing precision, limit of quantitation, limit of detection, linear range and linearity of standard curves. The precision of the method was evaluated by intra-day and inter-day variations. The intra-day and inter- day variations were performed by analyzing peak areas of standard dilutions in five replicates and variations were reported as coefficient of variation.

The working linear range was determined by analysis of the linear correlation coefficient of the regression curves using standard dilutions. The limit of quantification and the limit of detection were defined by a signal to noise ratio of 9:1 and 3:1 respectively.

3.3.3 Usnic acid fractionation with semi-preparative HPLC Prior to enantiomeric ratio determination, usnic acid peaks were fractionated using semi-preparative HPLC. Semi-preparative separation was carried out on a Luna Phenyl-Hexyl column (250 mm × 10 mm, 5 µm; Phenomenex, USA). Solvent A was water + 0.1% FA and solvent B was ACN + 0.1% FA. The elution conditions were as follows: isocratic elution 50% A / 50% B, 0-1 min; linear gradient from 50% A / 50% B to 100% B, 1-4 min; isocratic elution 100% B; 4-15 min; linear gradient to 50% A / 50% B, 15-16 min; isocratic elution 50% A / 50% B, 16-20 min; flow rate 1.6 ml/min. Detection wavelength was 280 nm (the elution gradient can be seen in Figure 1A in Appendix A).

The effluent corresponding to usnic acid was collected from around 14.5 to 15.5 min. Fractionation was carried out in three replicates for each specimen.

3.3.4 Enantiomeric ratio of usnic acid using chiral HPLC The chiral separation was carried out on a Lux Amylose-1 column (250 mm × 4,6 mm, 3 µm; Phenomenex, USA). Various solvent combinations were tested to optimize the separation as shown in Table 6. The effects of column oven temperature on separation was also assessed by adjusting temperatures to 25°C, 30°C, 35°C and 40°C.

Table 6. Different solvent combinations for the optimization of chiral separation. Methanol (MeOH) Acetonitrile (ACN) Solvent combination 1 100% + 0.1% FA - Solvent combination 2 80% + 0.1% acetic acid (AA) 20% Solvent combination 3 85% + 0.1% AA 15% Solvent combination 4 83% + 0.1% AA 17% Solvent combination 5 83% + 0.5% AA 17% Solvent combination 6 83% + 0.1% FA 17%

The final elution conditions were as follows: isocratic elution MeOH + 0,1% FA:ACN (83:17 v/v), 20 min; flow rate 0.5 ml/min. Detection wavelength was 280 nm.

The usnic acid effluent collected from the semi-preparative HPLC from all three species were injected (20 µl) to the HPLC system with a chiral column to determine the enantiomeric ratio of usnic acid.

3.3.5 Standard operating procedure After using the Waters Acquity UPLC system and learning the different settings, screenshots and instructions were made for each chapter. The chapters included in the SOP are:

1. Overview: Describing the various applications and windows, detailing what settings can be adjusted in each window. 2. Start-up: How to start the instrument. 3. Instrument method: How to make an instrument method and what the various settings do. 4. Sample sequence: How to make a sample sequence and what the different settings do. 5. Processing method: How to make a processing method and how to apply the processing method to results. 6. MS for verification: How to use the MS for verification of compounds.

The Waters Acquity UPLC system software (Empower) was quite different from the software used to run the Dionex HPLC systems (Chromeleon). The software, Empower 3, rewards knowledge of each component and allows for more manual settings whereas Chromeleon is easier for beginners with all the guides and wizards (the SOP can be seen in Appendix B).

4. RESULTS 4.1 Validation of the UPLC method for usnic acid quantitation The UPLC-PDA-MS method was validated for the quantitation of total usnic acid. The variation was small for both intra-day (Table 7) and inter-day (Table 8) variations with average values of 1.67 for the intra-day tests and 1.8 for the inter-day tests. A higher variation was found in low concentrations.

Table 7. Intra-day coefficient of variation values. All area values for intra-day and inter-day variation can be seen in Table 1A in Appendix A. Standard concentration (µg/ml) CV (%) Day 1 Day 2 Day 3 0.91 2.88 1.59 2.87 1.82 3.88 1.26 1.78 3.64 2.25 1.36 0.96 7.28 1.34 0.34 0.32 14.56 2.10 1.50 0.67

Table 8. Inter-day coefficient of variation values. All area values for intra-day and inter-day variation can be seen in Table 1A in Appendix A. Standard concentration (µg/ml) CV (%) 0.91 2.42 1.82 2.50 3.64 1.50 7.28 0.96 14.56 1.63

The working linear range was tested and confirmed from 0.91 µg/ml to 14.56 µg/ml. Standard curves were estimated daily using five standard concentrations (five replicates for each concentration), which displayed good linearity over the three-day analysis (Table 9). The limit of quantitation and limit of detection were determined as 46.88 ng/ml and 23.46 ng/ml, respectively.

Table 9. Standard curves of three-day analysis. Graphs are shown in Figure 2A in Appendix A. Day Standard curves (n=5) 1 y = 59798x – 1192.6 R2 = 0.9995 2 y = 60633x – 533.29 R2 = 0.9999 3 y = 62649x – 5823.7 R2 = 0.9999

4.2 Determination of total usnic acid contents using UPLC-PDA-MS

Usnic acid (tR= 4.35 min) is the major metabolite in the three lichen taxa, as shown in Figure 7. Retention times of usnic acids show good repeatability eluting around 4.349 minutes in all three species.

Figure 7. UPLC-PDA chromatograms for (A) Flavocetraria nivalis extract; (B) Cladonia arbuscula extract; (C) Alectoria ochroleuca extract.

Figure 8. UV-VIS spectrum (210-500 nm) (A) and MS spectrum (B) of usnic acid.

Figure 8A shows the UV-VIS spectrum of usnic acid with two absorbance maxima, at ca. 232 nm and 282 nm. Figure 8B shows the MS spectrum of usnic acid and the base peak at 343 m/z, which corresponds to the deprotonated molecular ion of usnic acid.

As seen in Figure 7C there seems to be at least two other compounds (tR = 3.713 and 4.018) in Alectoria ochroleuca. They were annotated using MS and PDA data. Mass spectra of the two minor peaks (Figure 9) confirm the homogeneity of peaks, representing two individual compounds. MS base peaks in mass spectra show the mass to charge ratios (m/z) of the ionized compounds at 373 and 359. They correspond to the deprotonated molecular ions of diffractaic acid and 4-O-demethyldiffractaic acid (Huneck & Yoshimura, 1996). The latter could also be deduced by the deletion of one methyl group from diffractaic acid. UV-VIS spectra (200-500 nm) shown in Figure 10 also support our annotation that the compounds are highly conjugated. Both spectra show end-absorbance around 220 nm, and a second

maximum absorbance at 308 nm (diffractaic acid) and 274 nm (4-O-demethlydiffractaic acid).

Figure 9. MS spectra showing base peaks of the two minor compounds in the lichen Alectoria ochroleuca. (A) Diffractaic acid; (B) 4-O-demethyldiffractaic acid.

Figure 10. UV-VIS spectra (200-500 nm) of the two minor compounds in the lichen Alectoria ochroleuca. (A) Diffractaic acid; (B) 4-O-demethyldiffractaic acid.

Considerable variation was observed between different species, especially Alectoria ochroleuca (45.05 mg/g) in which the concentration of usnic acid is two times more than in the other two species as seen in Table 10. Usnic acid accounts to 1.77-4.50% of the whole lichen thalli.

Table 10. The dry weight of usnic acid (mg/g) and % of content ± SD in all three species. Exact numerical values can be seen in Table 3A in Appendix A. Taxon Usnic acid content (mg/g) dry weighta Usnic acid content (%)a Flavocetraria nivalis 17.66 ± 0.18 1.77 ± 0.02 Cladonia arbuscula 18.70 ± 0.38 1.87 ± 0.04 Alectoria ochroleuca 45.05 ± 1.02 4.50 ± 0.10 a: Usnic acid content is measured in three replicates for each taxon.

4.3 Usnic acid fractionation and enantiomeric ratio determination

The effluent was collected from around 14.5 to 15.5 minutes (Figure 11) in three replicates for each species, which corresponds to the elution time of the usnic acid peak.

Figure 11. Semi-preparative HPLC-UV chromatogram for Flavocetraria nivalis extract. Fractionation of the usnic acid peak started from 14.5 to 15.5 min. Detection wavelength was set to 280 nm.

Chiral separation of (+)- and (-)-usnic acid was achieved as shown in Figure 12. The two enantiomers where almost baseline-separated within 13 minutes. The solvent

pair MeOH + 0.1% FA:ACN (83:17 v/v) provided the best separation out of all the combinations. Column oven temperature had negligible influence on chiral separation.

Figure 12. Chiral HPLC-UV chromatogram of the usnic acid enatiomer standards under optimized elution conditions (MeOH + 0.1% FA:ACN (83:17 v/v)). (+)-Usnic acid tR = 10.095; (-)-usnic acid tR = 11.740.

Figure 13 shows the chromatograms of usnic acid fractions from the three lichen species. The usnic acid in Flavocetraria nivalis (Figure 13A) contains both (+)- and (-)-usnic acids with (-)-usnic acid being the predominant one. The enantiomers of Cladonia arbuscula (Figure 13B) did not satisfactorily baseline-separate. However, the chromatogram does confirm the presence of (-)-usnic acid in the lichen species. It is apparent that (+)-usnic acid is the predominant enantiomer in Cladonia arbuscula. The Chromatogram for Alectoria ochroleuca (Figure 13C) shows that there is no trace of (+)-usnic acid in the specimen and (-)-usnic acid is the only enantiomer present. The percentages of usnic acid enantiomers in sample lichen taxa are shown in Table 11.

Figure 13. Usnic acid enatiomer composition in all three species. (A) Flavocetraria nivalis; (B) Cladonia arbuscula; (C) Alectoria ochroleuca.

Table 11. Ratio of usnic acid enantiomers. Exact numerical values can be seen in Table 3A in Appendix A. Taxon Ratio (+):(-)b Flavocetraria nivalis 1.76%:98.24% ± 0.98 Cladonia arbusculaa 96.77%:3.23% ± 0.30 Alectoria ochroleuca 0%:100% a: Peaks are not baseline separated and the value is estimated. b: Enantiomeric ratio is measured in three replicates for each taxon.

5. DISCUSSION 5.1 Usnic acid total content It is known that usnic acid content may vary substantially at both intra- and inter- specific levels, ranging from 0.48% to 8% (Bjerke et al., 2005; Smeds & Kytöviita, 2010). The literature has shown that for Usnea, Cladonia, Ramalina, Lecanora, Parmelia and Evernia, the usnic acid content could be as high as 6.49% (Cansaran- Duman, Aras, & Atakol, 2008). Our results on usnic acid contents from three Icelandic lichen taxa fall well within the range, with the highest content found in the lichen Alectoria ochroleuca (4.50%). This is slightly less than the results of Proksa et al. who obtained a concentration of 6.1% in the same lichen species in Slovakia (Proksa, Sturdikova, Prónayová, & Liptaj, 1996). This could be explained by different environmental factors between Iceland and Slovakia. Proksa et al. (1996) do not mention the time of year that the samples were collected which makes it harder to compare the results.

Bjerke et al. (2005) studied the concentration of usnic acid in Flavocetraria nivalis in four populations, three from sub-arctic habitats in the Northern Hemisphere and one from Patagonian heathland in the Southern Hemisphere (South Chile). They found that usnic acid was produced in large quantities, from 4% to 8% of thallus dry weight with a mean concentration of 5.25%. Our results on Icelandic Flavocetraria nivalis (1.77%) was much lower. This is most likely because Bjerke et al. (2005) only analyzed the thallus apices, which are the upper-most 0.5 cm of the tips. These are the parts of the thallus that contain the highest concentration of usnic acid, whereas in our study, the whole thallus was analyzed. It may also be due to natural intraspecific variation of the lichen in different environmental conditions. Our lichen samples were collected in November, and the content is expected to be higher if collected during summer.

Until now, usnic acid content in Cladonia arbuscula has not been determined. The content in its congener, Cladonia stellaris has been reported to be from 0.48% to 3.08% with an average concentration of 1.53%. However, this species has a different predominant enantiomer, i.e. (-)-usnic acid. The lichen samples were also collected without detailing the sampling procedure and without regard to sun exposure (Smeds & Kytöviita, 2010).

5.2 Usnic acid enantiomeric ratio As mentioned in the introduction, only three papers have studied the ratio of these enantiomers in lichens. Bjerke et al. (2005) reported that both (+)- and (-)-usnic acid were present in all Flavocetraria nivalis specimens, with (+)-usnic acid being the major compound. Only very small peaks of (-)-usnic acid that partly overlapped with (+)-usnic acid were detected. The ratio of the enantiomers was generally less than 1:100. This is not supported by our results where (-)-usnic acid was the major compound with an average ratio of 98.24%:1.76% for (-)- and (+)-usnic acid. Our results, however, are fully supported by another study (Kinoshita et al., 1997), which clearly stated the predominance of (-)-usnic acid in Flavocetraria nivalis. Kinoshita et al. (1997) have provided chiral chromatograms for optimized separation of usnic acid enantiomers, but the results from Bjerke et al. (2005) is questionable. The latter did not use a chiral column and no chromatograms were provided to prove the separation of isomers.

Smeds and Kytöviita (2010) reported that both enantiomers of usnic acid were present in all analyzed extracts with the average proportion of (+)-usnic acid of the total amount of usnic acid being 2.0%. The range was from 0.4-10% and the variation was small (CV = 2.3%). They reported for the first time that Cladonia stellaris may contain both enantiomers of usnic acid and that it contrasted with the results of Kinoshita et al. (1997), who reported only the (-)-enantiomer in Cladonia stellaris. Similar to the findings of Smeds and Kytöviita (2010), we also report for the first time that Cladonia arbuscula may contain both enantiomers of usnic acid. Kinoshita et al. (1997) previously reported only the (-)-enantiomer in Cladonia arbuscula. However, our findings showed the presence of both (-)- and (+)-enantiomers with a ratio of 3.23%:96.77%. The primary reason for difference in results might be due to the concentration of injected usnic acid sample solution. If it is too low, it is easy to miss the small peak. This raises a future working hypothesis that the production of major usnic acid enantiomers may differ in Cladonia lichens. Kinoshita et al. (1997) reported that only the (-)-enantiomer of usnic acid was present in Alectoria ochroleuca. This is fully supported by our results, where Alectoria ochroleuca contained 100% (-)-usnic acid.

5.3 Chiral separation As seen in Figure 13B the chiral separation allows for improvement. The major problem was the tailing of the (+)-usnic acid peak in the Cladonia arbuscula sample. The column

is designed for normal phase and the manufacturer recommends using an isocratic elution of Hexane:Isopropanol (90:10 v/v) and the result is expected to improve.

It is important to emphasize that many lichens were believed to be enantiomerically pure, but many previous studies underestimated the complexity of these organisms.

6. CONCLUSIONS A UPLC-PDA-MS method has been developed and validated for the determination of total usnic acid content in lichens. The content of usnic acid ranged from 1.77-4.50%. The ratio of (+)- and (-)-usnic acid enantiomers has been assessed in three lichens commonly occurring in Iceland and the ratio varied substantially; Flavocetraria nivalis contained an average ratio of 1.76%:98.24%, Cladonia arbuscula contained an average ratio of 96.77%:3.23% and Alectoria ochroleuca contained only (-)-usnic acid. In light of the known different biological activities of usnic acid enantiomers, this research will have biological and ecological importance for future studies investigating the role of usnic acid-containing lichens.

7. ACKNOWLEDGEMENTS I would like to offer my thanks to my supervisor Bergþóra Sigríður Snorradóttir for her support, guidance and suggestions during the development of this thesis. I would especially like to thank my instructor Maonian Xu for the tremendous amount of help, support and great advice in the making of the thesis. I would like to thank Árni Þorgrímur Kristjánsson for his help in the making of the standard operating procedure.

I would also like to thank my fellow students, Jóhann Sigurðsson and Guðjón Trausti Skúlason, for the wonderful times we had together during our time in Hagi working on our projects.

Lastly, I would like to thank my family, especially Haukur Dór Bragason and Iris Edda Nowenstein, for their support, encouragement, advice and help in the making of this thesis.

REFERENCES Abdel-Hameed, M., Bertrand, R. L., Piercey-Normore, M. D., & Sorensen, J. L. (2016). Putative identification of the usnic acid biosynthetic gene cluster by de novo whole-genome sequencing of a lichen-forming fungus. Fungal Biology, 120(3), 306-316. doi:https://doi.org/10.1016/j.funbio.2015.10.009 Araujo, A. A., de Melo, M. G., Rabelo, T. K., Nunes, P. S., Santos, S. L., Serafini, M. R., . . . Gelain, D. P. (2015). Review of the biological properties and toxicity of usnic acid. Nat Prod Res, 29(23), 2167-2180. doi:10.1080/14786419.2015.1007455 Arias, C. A., & Murray, B. E. (2015). A New Antibiotic and the Evolution of Resistance. New England Journal of Medicine, 372(12), 1168-1170. doi:10.1056/NEJMcibr1500292 Bazin, M.-A., Lamer, A.-C. L., Delcros, J.-G., Rouaud, I., Uriac, P., Boustie, J., . . . Tomasi, S. (2008). Synthesis and cytotoxic activities of usnic acid derivatives. Bioorganic & Medicinal Chemistry, 16(14), 6860-6866. doi:https://doi.org/10.1016/j.bmc.2008.05.069 Bessadottir, M., Egilsson, M., Einarsdottir, E., Magnusdottir, I. H., Ogmundsdottir, M. H., Omarsdottir, S., & Ogmundsdottir, H. M. (2012). Proton-Shuttling Lichen Compound Usnic Acid Affects Mitochondrial and Lysosomal Function in Cancer Cells. PLOS ONE, 7(12), e51296. doi:10.1371/journal.pone.0051296 Bjerke, J. W., Elvebakk, A., Domínguez, E., & Dahlback, A. (2005). Seasonal trends in usnic acid concentrations of Arctic, alpine and Patagonian populations of the lichen Flavocetraria nivalis. Phytochemistry, 66(3), 337-344. doi:https://doi.org/10.1016/j.phytochem.2004.12.007 Campanella, L., Delfini, M., Ercole, P., Iacoangeli, A., & Risuleo, G. (2002). Molecular characterization and action of usnic acid: a drug that inhibits proliferation of mouse polyomavirus in vitro and whose main target is RNA transcription. Biochimie, 84(4), 329-334. doi:https://doi.org/10.1016/S0300-9084(02)01386- X Cansaran-Duman, D., Aras, S., & Atakol, O. (2008). Determination of Usnic Acid Content in Some Lichen Species Found in Anatolia. Cardarelli, M., Serino, G., Campanella, L., Ercole, P., De Cicco Nardone, F., Alesiani, O., & Rossiello, F. (1997). Antimitotic effects of usnic acid on different biological systems. Cellular and Molecular Life Sciences CMLS, 53(8), 667- 672. doi:10.1007/s000180050086 CFSAN. (2001). Lipokinetix Dear Healthcare Professional Letter. Retrieved from http://wayback.archive- it.org/7993/20170113165217/http://www.fda.gov/Safety/MedWatch/SafetyInfor mation/SafetyAlertsforHumanMedicalProducts/ucm174455.htm Cocchietto, M., Skert, N., Nimis, P., & Sava, G. (2002). A review on usnic acid, an interesting natural compound. Naturwissenschaften, 89(4), 137-146. doi:10.1007/s00114-002-0305-3 Correche, E. R., Carrasco, M., Escudero, M., Velazquez, L., De Guzman, A. M. S., Giannini, F., . . . Giordano, O. S. (1998). Study of the cytotoxic and antimicrobial activities of usnic acid and derivatives. 69, 493-501. da Silva Santos, N. P., Nascimento, S. C., Wanderley, M. S. O., Pontes-Filho, N. T., da Silva, J. F., de Castro, C. M. M. B., . . . Santos-Magalhães, N. S. (2006). Nanoencapsulation of usnic acid: An attempt to improve antitumour activity and reduce hepatotoxicity. European journal of pharmaceutics and biopharmaceutics : official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e.V, 64(2), 154-160. doi:10.1016/j.ejpb.2006.05.018 35

Davies, J., & Davies, D. (2010). Origins and Evolution of Antibiotic Resistance. Microbiology and Molecular Biology Reviews, 74(3), 417. doi:10.1128/MMBR.00016-10 Durazo, F. A., Lassman, C., Han, S. H. B., Saab, S., Lee, N. P., Kawano, M., . . . Busuttil, R. W. (2004). Fulminant Liver Failure Due to Usnic Acid for Weight Loss. American Journal Of Gastroenterology, 99, 950. doi:10.1111/j.1572- 0241.2004.04165.x Eder, J., & Herrling, P. L. (2016). Trends in Modern Drug Discovery. In U. Nielsch, U. Fuhrmann, & S. Jaroch (Eds.), New Approaches to Drug Discovery (pp. 3-22). Cham: Springer International Publishing. Einarsdottir, E., Groeneweg, J., Bjornsdottir, G. G., Harethardottir, G., Omarsdottir, S., Ingolfsdottir, K., & Ogmundsdottir, H. M. (2010). Cellular mechanisms of the anticancer effects of the lichen compound usnic acid. Planta Med, 76(10), 969-974. doi:10.1055/s-0029-1240851 Emmerich, R., Giez, I., Lange, O. L., & Proksch, P. (1993). Toxicity and antifeedant activity of lichen compounds against the polyphagous herbivorous insect Spodoptera littoralis. Phytochemistry, 33(6), 1389-1394. doi:https://doi.org/10.1016/0031-9422(93)85097-B Favreau, J. T., Ryu, M. L., Braunstein, G., & et al. (2002). Severe hepatotoxicity associated with the dietary supplement lipokinetix. Annals of Internal Medicine, 136(8), 590-595. doi:10.7326/0003-4819-136-8-200204160-00008 Fournet, A., Ferreira, M.-E., de Arias, A. R., de Ortiz, S. T., Inchausti, A., Yalaff, G., . . . Hidalgo, M. E. (1997). Activity of Compounds Isolated From Chilean Lichens Against Experimental Cutaneous Leishmaniasis. Comparative Biochemistry and Physiology Part C: Pharmacology, Toxicology and Endocrinology, 116(1), 51-54. doi:https://doi.org/10.1016/S0742- 8413(96)00127-2 Frieri, M., Kumar, K., & Boutin, A. (2017). Antibiotic resistance. Journal of Infection and Public Health, 10(4), 369-378. doi:https://doi.org/10.1016/j.jiph.2016.08.007 Galanty, A., Paśko, P., & Podolak, I. (2018). Enantioselective activity of usnic acid – a comprehensive review and future perspectives. (PHYT-D-18-00200). Phytochemistry Reviews. Galasso, V. (2010). Probing the molecular and electronic structure of the lichen metabolite usnic acid: A DFT study. Chemical Physics, 374(1), 138-145. doi:https://doi.org/10.1016/j.chemphys.2010.07.017 Galloway, D. J. (1999). Lichen Glossary. Retrieved from https://web.archive.org/web/20141206070827/http://www.anbg.gov.au/glossar y/webpubl/lichglos.htm Gauslaa, Y. (2005). Lichen Palatability Depends on Investments in Herbivore Defence. Oecologia, 143(1), 94-105. Ghione, M., Parrello, D., & Grasso, L. (1988). Usnic acid revisited; its activity on oral flora. 7, 302-305. Grube, M., Cardinale, M., Vieira Castro, J., Müller, H., & Berg, G. (2009). Species- specific structural and functional diversity of bacterial communities in lichen symbioses. 3, 1105-1115. Guo, L. E. I., Shi, Q., Fang, J.-L., Mei, N. A. N., Ali, A. A., Lewis, S. M., . . . Frankos, V. H. (2008). Review of Usnic Acid and Usnea Barbata Toxicity. Journal of Environmental Science and Health, Part C, 26(4), 317-338. doi:10.1080/10590500802533392

Hawranik, D. J., Anderson, K. S., Simmonds, R., & Sorensen, J. L. (2009). The chemoenzymatic synthesis of usnic acid. Bioorganic & Medicinal Chemistry Letters, 19(9), 2383-2385. doi:https://doi.org/10.1016/j.bmcl.2009.03.087 Holzmann, G., & Leuckert, C. (1990). Applications of negative fast atom bombardment and MS/MS to screening of lichen compounds. Phytochemistry, 29(7), 2277-2283. doi:https://doi.org/10.1016/0031-9422(90)83052-3 Huneck, S. (1999). The Significance of Lichens and Their Metabolites. Naturwissenschaften, 86(12), 559-570. doi:10.1007/s001140050676 Huneck, S., & Yoshimura, I. (1996). Identification of Lichen Substances (1 ed.): Springer-Verlag Berlin Heidelberg. Hüttner, C., Beuerle, T., Scharnhop, H., Ernst, L., & Beerhues, L. (2010). Differential Effect of Elicitors on Biphenyl and Dibenzofuran Formation in Sorbus aucuparia Cell Cultures. Journal of Agricultural and Food Chemistry, 58(22), 11977-11984. doi:10.1021/jf1026857 Ingolfsdottir, K. (2002). Usnic acid. Phytochemistry, 61(7), 729-736. doi:https://doi.org/10.1016/S0031-9422(02)00383-7 Kinoshita, Y., Yamamoto, Y., Yoshimura, I., Kurowaka, T., & Huneck, S. (1997). Distribution of optical isomers of usnic and isousnic acids analyzed by high performance liquid chromatography. Journal- Hattori Botanical Laboratory(83), 173-178. Koparal, A. T. (2015). Anti-angiogenic and antiproliferative properties of the lichen substances (-)-usnic acid and vulpinic acid. Z Naturforsch C, 70(5-6), 159-164. doi:10.1515/znc-2014-4178 Lage, T. C. A., Maciel, T. M. S., Mota, Y. C. C., Sisto, F., Sabino, J. R., Santos, J. C. C., . . . Modolo, L. V. (2018). In vitro inhibition of Helicobacter pylori and interaction studies of lichen natural products with jack bean urease. New Journal of Chemistry, 42(7), 5356-5366. doi:10.1039/C8NJ00072G Larson, D. W. (1987). The absorption and release of water by lichens. In P. E (Ed.), Progress and Problems in Lichenology in the Eighties (pp. 351-360). Berlin- Stuttgart: J Cramer: Bibliotheca Lichenologia. Lauterwein, M., Oethinger, M., Belsner, K., Peters, T., & Marre, R. (1995). In vitro activities of the lichen secondary metabolites vulpinic acid, (+)-usnic acid, and (-)-usnic acid against aerobic and anaerobic microorganisms. Antimicrobial agents and chemotherapy, 39(11), 2541-2543. Lawrey, J. D. (1986). Biological Role of Lichen Substances. The Bryologist, 89(2), 111-122. doi:10.2307/3242751 Le Pogam, P., Schinkovitz, A., Legouin, B., Le Lamer, A.-C., Boustie, J., & Richomme, P. (2015). Matrix-Free UV-Laser Desorption Ionization Mass Spectrometry as a Versatile Approach for Accelerating Dereplication Studies on Lichens. Analytical Chemistry, 87(20), 10421-10428. doi:10.1021/acs.analchem.5b02531 Leavitt, S. D., Kraichak, E., Nelsen, M. P., Altermann, S., Divakar, P. K., Alors, D., . . . Lumbsch, T. (2015). Fungal specificity and selectivity for algae play a major role in determining lichen partnerships across diverse ecogeographic regions in the lichen-forming family Parmeliaceae (Ascomycota). Molecular Ecology, 24(14), 3779-3797. doi:doi:10.1111/mec.13271 Legouin, B., Lohézic-Le Dévéhat, F., Ferron, S., Rouaud, I., Le Pogam, P., Cornevin, L., . . . Boustie, J. (2017). Specialized Metabolites of the Lichen Vulpicida pinastri Act as Photoprotective Agents. Molecules, 22(7). doi:10.3390/molecules22071162

Liu, Q., Zhao, X., Lu, X., Fan, X., & Wang, Y. (2012). Proteomic Study on Usnic-Acid- Induced Hepatotoxicity in Rats. Journal of Agricultural and Food Chemistry, 60(29), 7312-7317. doi:10.1021/jf2046834 Lucarini, R., Tozatti, M. G., Salloum, A. I. d. O., Crotti, A. E. M., Silva, M. L. A., Gimenez, V. M. M., . . . Cunha, W. R. (2012). Antimycobacterial activity of Usnea steineri and its major constituent (+)-usnic acid. African Journal of Biotechnology, 11(20), 4636-4639. Lücking, R., Hodkinson, B. P., & Leavitt, S. D. (2016). The 2016 classification of lichenized fungi in the Ascomycota and Basidiomycota – Approaching one thousand genera. The Bryologist, 119(4), 361-416. doi:10.1639/0007-2745- 119.4.361 Maciąg-Dorszyńska, M., Węgrzyn, G., & Guzow-Krzemińska, B. (2014). Antibacterial activity of lichen secondary metabolite usnic acid is primarily caused by inhibition of RNA and DNA synthesis. FEMS Microbiology Letters, 353(1), 57- 62. doi:10.1111/1574-6968.12409 Mayer, M., O'Neill, M. A., Murray, K. E., Santos-Magalhaes, N. S., Carneiro-Leao, A. M., Thompson, A. M., & Appleyard, V. C. (2005). Usnic acid: a non-genotoxic compound with anti-cancer properties. Anticancer Drugs, 16(8), 805-809. Melgarejo, M., Sterner, O., Vila Castro, J., & Mollinedo, P. (2008). More investigations in potent activity and relationship structure of the lichen antibiotic (+)-usnic acid and its derivative dibenzoyl usnic acid. Revista Boliviana de Química, 25, 24-29. Millot, M., Dieu, A., & Tomasi, S. (2016). Dibenzofurans and derivatives from lichens and ascomycetes. Natural Product Reports, 33(6), 801-811. doi:10.1039/C5NP00134J Mitchell, J. C. (1966). Stereoisomeric Specificity of Usnic Acid in Delayed Hypersensitivity*. Journal of Investigative Dermatology, 47(2), 167-168. doi:https://doi.org/10.1038/jid.1966.123 Musharraf, S. G., Kanwal, N., Thadhani, V. M., & Choudhary, M. I. (2015). Rapid identification of lichen compounds based on the structure–fragmentation relationship using ESI-MS/MS analysis. Analytical Methods, 7(15), 6066-6076. doi:10.1039/C5AY01091H Neff, G. W., Rajender Reddy, K., Durazo, F. A., Meyer, D., Marrero, R., & Kaplowitz, N. (2004). Severe hepatotoxicity associated with the use of weight loss diet supplements containing ma huang or usnic acid. Journal of Hepatology, 41(6), 1062-1064. doi:https://doi.org/10.1016/j.jhep.2004.06.028 Nelsen, M. P., Chavez, N., Sackett-Hermann, E., Thell, A., Randlane, T., Divakar, P. K., . . . Lumbsch, H. T. (2011). The cetrarioid core group revisited (Lecanorales: Parmeliaceae). The Lichenologist, 43(6), 537-551. doi:10.1017/S0024282911000508 Okuyama, E., Umeyama, K., Yamazaki, M., Kinoshita, Y., & Yamamoto, Y. (1995). Usnic Acid and Diffractaic Acid as Analgesic and Antipyretic Components of Usnea diffracta. 61, 113-115. doi:10.1055/s-2006-958027 Parrot, D., Legrave, N., Intertaglia, L., Rouaud, I., Legembre, P., Grube, M., . . . Tomasi, S. (2016). Cyaneodimycin, a Bioactive Compound Isolated from the Culture of Streptomyces cyaneofuscatus Associated with Lichina confinis. European Journal of Organic Chemistry, 2016(23), 3977-3982. doi:10.1002/ejoc.201600252 Perry, N., H. Benn, M., Downes, N., Burgess, E., Ellis, G., Galloway, D., . . . Tangney, R. (1999). Antimicrobial, Antiviral and Cytotoxic Activity of New Zealand Lichens. 31, 627-636. doi:10.1017/S002428299900081X

Pöykkö, H., Hyvärinen, M., & Bačkor, M. (2005). Removal of Lichen Secondary Metabolites Affects Food Choice and Survival of Lichenivorous Moth Larvae. Ecology, 86(10), 2623-2632. Proksa, B., Sturdikova, M., Prónayová, N., & Liptaj, T. (1996). (-)-Usnic acid and its derivatives. Their inhibition of fungal growth and enzyme activity (Vol. 51). Quilhot, W. (1991). Quantitative variations of phenolic compounds related to thallus age in Umbilicaria antarctica. Santiago de Chile. Instituto Antártico Chileno. Serie científica, 91-97. Ribeiro-Costa, R., Alves, A., P Santos, N., C Nascimento, S., C P Gonçalves, E., H Silva, N., . . . Santos-Magalhaes, N. (2004). In vitro and in vivo properties of usnic acid encapsulated into PLGA-microspheres. 21, 371-384. doi:10.1080/02652040410001673919 Richardson, L. A. (2017). Understanding and overcoming antibiotic resistance. PLOS Biology, 15(8), e2003775. doi:10.1371/journal.pbio.2003775 Roach, J. A. G., Musser, S. M., Morehouse, K., & Woo, J. Y. J. (2006). Determination of Usnic Acid in Lichen Toxic to Elk by Liquid Chromatography with Ultraviolet and Tandem Mass Spectrometry Detection. Journal of Agricultural and Food Chemistry, 54(7), 2484-2490. doi:10.1021/jf052767m Romagni, J. G., Meazza, G., Nanayakkara, N. P. D., & Dayan, F. E. (2000). The phytotoxic lichen metabolite, usnic acid, is a potent inhibitor of plant p- hydroxyphenylpyruvate dioxygenase. FEBS Letters, 480(2), 301-305. doi:https://doi.org/10.1016/S0014-5793(00)01907-4 Romeike, J., Friedl, T., Helms, G., & Ott, S. (2002). Genetic Diversity of Algal and Fungal Partners in Four Species of Umbilicaria (Lichenized Ascomycetes) Along a Transect of the Antarctic Peninsula. Molecular Biology and Evolution, 19(8), 1209-1217. doi:10.1093/oxfordjournals.molbev.a004181 Sanchez, W., Maple, J. T., Burgart, L. J., & Kamath, P. S. (2006). Severe Hepatotoxicity Associated With Use of a Dietary Supplement Containing Usnic Acid. Mayo Clinic Proceedings, 81(4), 541-544. doi:https://doi.org/10.4065/81.4.541 Sassa, T., & Igarashi, M. (1990). Structures of (-)-Mycousnine, (+)-Isomycousnine and (+)-Oxymycousnine, New Usnic Acid Derivatives from Phytopathogenic Mycosphaerella nawae. Agricultural and Biological Chemistry, 54(9), 2231-2237. doi:10.1271/bbb1961.54.2231 Scirpa, P., Scambia, G., Masciullo, V., Battaglia, F., Foti, E., Lopez, R., . . . Mancuso, S. (1999). [A zinc sulfate and usnic acid preparation used as post-surgical adjuvant therapy in genital lesions by Human Papillomavirus]. Minerva Ginecol, 51(6), 255-260. Shibata, S., Ukita, T., & Tamura, T. (1948). Relation between chemical constitutions and antibacterial effects of usnic acid and its derivatives. Japanese Journal of Medicine, 1(2), 152-155. Smeds, A., & Kytöviita, M.-M. (2010). Determination of usnic and perlatolic acids and identification of olivetoric acids in Northern reindeer lichen ( Cladonia stellaris) extracts. 42, 739-749. doi:10.1017/S002428291000037X Sokolov, D. N., Zarubaev, V. V., Shtro, A. A., Polovinka, M. P., Luzina, O. A., Komarova, N. I., . . . Kiselev, O. I. (2012). Anti-viral activity of (−)- and (+)- usnic acids and their derivatives against influenza virus A(H1N1)2009. Bioorganic & Medicinal Chemistry Letters, 22(23), 7060-7064. doi:https://doi.org/10.1016/j.bmcl.2012.09.084 Spribille, T., Tuovinen, V., Resl, P., Vanderpool, D., Wolinski, H., Aime, M. C., . . . McCutcheon, J. P. (2016). Basidiomycete yeasts in the cortex of ascomycete macrolichens. Science, aaf8287. doi:10.1126/science.aaf8287 39

Stocker-Wörgötter, E. (2008). Metabolic diversity of lichen-forming ascomycetous fungi: culturing, polyketide and shikimatemetabolite production, and PKS genes. Natural Product Reports, 25(1), 188-200. doi:10.1039/B606983P Studzinska-Sroka, E., Holderna-Kedzia, E., Galanty, A., Bylka, W., Kacprzak, K., & Cwiklinska, K. (2015). In vitro antimicrobial activity of extracts and compounds isolated from Cladonia uncialis. Nat Prod Res, 29(24), 2302-2307. doi:10.1080/14786419.2015.1005616 Taguchi, H., Sankawa, U., & Shibata, S. (1969). Biosynthesis of Natural Products. VI. Biosynthesis of Usnic Acid in Lichens. (1). A General Scheme of Biosynthesis of Usnic Acid. CHEMICAL & PHARMACEUTICAL BULLETIN, 17(10), 2054- 2060. doi:10.1248/cpb.17.2054 UK, A. R. (2019). About antibiotic resistance. Retrieved from https://www.antibioticresearch.org.uk/about-antibiotic-resistance/ Vartia, K. O. (1973). Chapter 17 - ANTIBIOTICS IN LICHENS. In V. Ahmadjian & M. E. Hale (Eds.), The Lichens (pp. 547-561): Academic Press. Vijayakumar, C. S., Viswanathan, S., Kannappa Reddy, M., Parvathavarthini, S., Kundu, A. B., & Sukumar, E. (2000). Anti-inflammatory activity of (+)-usnic acid. Fitoterapia, 71(5), 564-566. doi:https://doi.org/10.1016/S0367- 326X(00)00209-4 Wu, J., Zhang, M., Ding, D., Tan, T., & Yan, B. (1995). [Effect of Cladonia alpestris on Trichomonas vaginalis in vitro]. Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi, 13(2), 126-129. Xu, M., Heidmarsson, S., Olafsdottir, E. S., Buonfiglio, R., Kogej, T., & Omarsdottir, S. (2016). Secondary metabolites from cetrarioid lichens: Chemotaxonomy, biological activities and pharmaceutical potential. Phytomedicine, 23(5), 441- 459. doi:https://doi.org/10.1016/j.phymed.2016.02.012 Xu, M., Heidmarsson, S., Thorsteinsdottir, M., Eiriksson, F. F., Omarsdottir, S., & Olafsdottir, E. S. (2017). DNA barcoding and LC-MS metabolite profiling of the lichen-forming genus Melanelia: Specimen identification and discrimination focusing on Icelandic taxa. PLOS ONE, 12(5), e0178012. doi:10.1371/journal.pone.0178012 Yamamoto, Y., Miura, Y., Kinoshita, Y., Higuchi, M., Yamada, Y., Murakami, A., . . . Koshimizu, K. (1995). Screening of Tissue Cultures and Thalli of Lichens and Some of Their Active Constituents for Inhibition of Tumor Promoter-Induced Epstein-Barr Virus Activation. CHEMICAL & PHARMACEUTICAL BULLETIN, 43(8), 1388-1390. doi:10.1248/cpb.43.1388 Yellapu, R. K., Mittal, V., Grewal, P., Fiel, M., & Schiano, T. (2011). Acute liver failure caused by 'fat burners' and dietary supplements: a case report and literature review. Canadian journal of gastroenterology = Journal canadien de gastroenterologie, 25(3), 157-160. Zaman, S. B., Hussain, M. A., Nye, R., Mehta, V., Mamun, K. T., & Hossain, N. (2017). A Review on Antibiotic Resistance: Alarm Bells are Ringing. Cureus, 9(6), e1403-e1403. doi:10.7759/cureus.1403

Appendix A

Figure 1A. Elution gradient for the semi-preparative HPLC.

Table 1A. All area values for intra-day and inter-day variation calculations. Days Concentration (µg/ml) Area under curve (µV × sec)

Rep 1 Rep 2 Rep 3 Rep 4 Rep 5

0.91 44,188 47,482 44,542 44,810 45,256

1.82 84,532 89,820 92,034 92,539 93,076

Day 1 3.64 194,664 186,032 189,200 190,130 183,493

7.28 375,059 372,931 377,291 384,563 383,265

14.56 743,823 751,504 756,061 780,033 776,864

Rep 1 Rep 2 Rep 3 Rep 4 Rep 5

0.91 43,939 44,705 45,049 45,823 45,395

1.82 92,789 90,819 90,453 92,951 92,312

Day 2 3.64 191,046 189,224 190,450 188,560 184,506

7.28 383,181 381,464 383,278 384,929 384,258

14.56 754,831 777,670 766,008 780,384 782,085

Rep 1 Rep 2 Rep 3 Rep 4 Rep 5

0.91 44,159 44,930 45,286 46,369 47,519

1.82 93,977 90,190 90,472 92,595 92,897

Day 3 3.64 189,412 188,355 191,197 186,866 186,959

7.28 384,304 381,973 382,603 381,640 384,110

14.56 769,176 771,507 778,613 777,712 781,603

800000

700000 y = 59798x - 1192.6 R² = 0.9995 600000

500000

400000

300000

200000

raudrcre(V×sec) × curve (µV Area under 100000

0 0 2 4 6 8 10 12 14 Concentration (µg/ml)

800000

700000 y = 60633x - 533.29

sec) R² = 0.9999 × 600000

500000

400000

300000

200000

100000 Area underc curve (µV (µV curve underc Area 0 0 2 4 6 8 10 12 14 Concentration (µg/ml)

900000 y = 62649x - 5823.7 800000 R² = 0.9999 sec) 700000 × 600000 500000 400000 300000 200000

Area under curve (µV (µV curve underArea 100000 0 0 2 4 6 8 10 12 14 Concentration (µg/ml)

Figure 2A. Top figure: Regression curve of the standard samples on day 1. Middle figure: Regression curve of the standard samples on day 2. Bottom figure: Regression curve of the standard samples on day 3.

Table 2A. All values for usnic acid content calculations. Lichen Weight (mg) Area under curve (µV × sec)

Flavocetraria nivalis 1 20.42 668,031

Flavocetraria nivalis 2 20.34 678,427

Flavocetraria nivalis 3 20.41 680,915

Cladonia arbuscula 1 20.36 712,609

Cladonia arbuscula 2 20.30 724,733

Cladonia arbuscula 3 20.30 695,960

Alectoria ochroleuca 1 20.30 1,749,082

Alectoria ochroleuca 2 20.08 1,691,977

Alectotia ochroleuca 3 20.23 1,675,260

Table 3A. All values for usnic acid enantiomeric ratio determination. Lichen (+)-Usnic acid (%) (-)-Usnic acid (%)

Flavocetraria nivalis 1 2.89 97.11

Flavocetraria nivalis 2 1.21 98.79

Flavocetraria nivalis 3 1.17 98.83

Cladonia arbuscula 1 96.6 3.40

Cladonia arbuscula 2 96.59 3.41

Cladonia arbuscula 3 97.11 2.89

Alectoria ochroleuca 1 0 100

Alectoria ochroleuca 2 0 100

Alectoria ochroleuca 3 0 100

Appendix B

STANDARD OPERATING PROCEDURE

Title: How to use the Waters Acquity UPLC instrument. Author: Aron Elvar Gylfason Approved by: Árni Þorgrímur Kristjánsson Date of approval: 15.04.19

1. Overview

There are two different pieces of software that you use when operating the Waters Acquity UPLC.

Waters Acquity Local Console Controller and Empower 3 Chromatography Data Software.

A) The Waters Acquity console is used in the start-up of the instrument. There you can find the controls for all the different parts of the instrument.

System overview

This is the system’s tab of the Waters Acquity console.

It gives you an overview of all the different parts of the instrument and their status.

Click on any of the components to switch to their individual overview screen.

QDa Detector

This is the control tab for

the QDa Detector.

The QDa Detector is the 1st thing we turn on when we are going to use this

instrument and it is very

important that you do

nothing else until it is ready. It is also the last thing we turn off when we are

finished.

Sample Manager FTN This is the control tab for the sample manager.

Here you can set the temperature control for the samples and monitor the current temperature.

Remember to also include the temperature control in your instrument method or else it will turn off when you start your sample run.

PDA Detector

This is the control tab for the PDA Detector.

Here you can see how

many lamp hours remain on the PDA lamp. Here you turn on the lamp before running your

samples.

Quaternary Solvent Manager This is the control tab for the QSM (pump).

Here you can monitor system pressure;

change the flow rate; and change the composition

of the solvents.

Column Manager

This is the control tab for the column manager.

Here you can choose which column to use, column 1 (upper), or column 2 (lower).

Here you can also set temperature control on the column.

Just remember to also

select the temperature

control on your instrument method.

B) The Empower 3 Chromatography Data Software is used to make sample sequences, to run your samples, to browse your data and to create your processing methods.

Empower 3

This is the Empower 3 startup page.

Select “Run Samples” to create a sample sequence and run your samples. Choose in which project folder to save your data and which chromatographic systems to use.

Select “Browse Projects” to view your acquired data but also to create your processing method. F

2. Start-up

A) Turn on the QDa Detector. This is the first thing we turn on before we start and the last thing we turn off when we are finished.

A.1) Use the Waters Acquity console to see when the QDa Detector is ready. This takes around 10 minutes. The QDa Detector must be ready before you continue! If the QDa tab does not appear, close the Waters Acquity console and wait for 1 minute and open it again.

B.1) Start priming solvents. Quaternary Solvent Manager  Control  Prime solvents.

You can either prime by solvent line or by composition. We recommend you prime all lines even though you won’t be using them all. Set the timer to 2-4 minutes for each line (we recommend you use 4 minutes but you can use 2 minutes if you used the instrument in the last couple of days).

B) Prime seal wash. Quaternary Solvent Manager  Control  Prime seal wash.

C.1) Prime seal wash for around 2-3 minutes. You need to manually turn it off afterwards. Simply go to Quaternary Solvent Manager  Control  Prime seal wash and press it again to turn it off.

C) Turn on the PDA lamp. PDA Detector  Control  Set lamp  Yes.

If you are going to use temperature control I would suggest turning it on at this point so that it has time to adjust. See the sample manager and column manager windows in the overview on how to do that (To turn the temperature control off after use, simply write “off” in the field instead of a number.)

The instrument is now ready for use.

3. Instrument method

A) Open Empower 3 and press “Run samples”. Choose in which project to acquire your data and which chromatographic systems to use (PDA, QDa or both).

B) After clicking “OK” on the “Run samples” screen you will be met with the “sample sequence” screen.

You will learn more about this screen in the

“Sample sequence”

section of the SOP. You can select an existing instrument method here;

or press the edit button to create a new instrument method.

If you selected an existing instrument method the edit button will allow you to adjust the settings of your method.

Here you can also see how much of the I solvents your sample run will use.

C) This is the instrument method screen. Here at the top you can select the different components of the instrument and change their settings.

C.1) On the first page you can adjust the settings for the Quaternary Solvent Manager. Fill in which solvents you will be using on each line. If your solvents are not on the list, use this tool to add them.

C.2) After selecting which solvents to use you should insert the conditions of your sample run. Insert the appropriate time, flow rate and percentage values of your solvents. Curve can be set from 1-12, with 6 being linear.

You can click on this icon to see the graph of your conditions. If the graph is not correct, save the method and close the window. Open it again and it should be correct.

C.3) This is the page for the settings of the Sample Manager – FTN. Here you will fill in what solvents you will be using for washing and purging.

If you wish to use temperature control on your samples, you can insert the temperature here and select an alarm band if you so choose (if you select 5.0°C the alarm will go off if the temperature goes 2.5°C over or under the selected value).

C.3.1) If you are used to the HPLC instruments in Hagi, there is an advanced setting that is worth knowing about. Click on “Advanced”.

The default needle placement (from bottom) of the Waters Acquity instrument is 4.0 mm. This is different from the Dionex HPLC instruments where the default setting is 2.0 mm. If you have around 500 µl or less of sample in your HPLC vials you might need to change this setting to 2.0 mm.

C.4) This is the page for the settings of the Column Manager.

Here you can select if you wish to use temperature on the column and if you want to use the alarm band (like with the temperature control of the sample manager if you select 5.0°C the alarm will go off if the temperature goes 2.5°C over or under the selected value).

C.5) This is the page for the setting of the PDA Detector.

These are the default settings of the PDA Detector. To select specific wavelengths, click on “2D Channels”. You can then insert the preferred channels by clicking here and adding them.

When finished, it might look something like this:

C.6) This is the page for the settings of the QDa Detector. Here you can select if you wish to use positive or negative scan. To select which ions to record, click on “SIR”.

Insert the name, mass, polarity and cone voltage of the selected ions.

When finished, it might look something like this:

C.7) The last step is to save your instrument method. Click “File  Save with method set” and insert a name for your method and any comments you wish to attach to the method. When finished click “Save”.

This concludes the instrument method section.

4. Sample sequence

A) Open Empower 3 and press “Run samples”. Choose in which project to acquire your data and which chromatographic systems to use (PDA, QDa or both).

B) After clicking “OK” on the “Run samples” screen you will be met with the “sample sequence” screen.

To add samples, click on the “Plates” icon.

C) This is the “Plates” screen. Select which plate you wish to use and then select the sample positions you want to use either by clicking on them or dragging over them. You can change the sequence mode by selecting the different modes here at the top.

C.2) When you have selected the sample positions you want to use, click on “Insert” to add them to the sequence and then press “OK”.

D) The sample sequence should now look something like this.

Here you can change all the different settings if you want. The injection volume; the number of injections; run time; when the instrument starts collecting data; if you want to delay the next injection; and so on.

D.1) If you change the topmost value and then press “Ctrl+D” it will change all the values in the same row.

D.2) Add names to your samples and then select an instrument method. The instrument method here must match the instrument method you selected down below.

Here you can see how long your sample sequence will take.

E) If you wish to save your sample sequence to be able to use it again, click on the “Save” icon to save it.

F) Before you can start your sample run you need to raise the flow to match the flow at the start of instrument method. Click here to open the “Set flow” window.

G) This is the set flow window. Raise the flow rate slowly so that it matches the starting flow rate in your instrument method. This way the flow doesn’t suddenly raise quickly when you start the sample run, which can be bad for the column. Also adjust the solvent percentages to match the beginning of your instrument method.

H) When the pressure is stable you can start your sample run. Click on the “Run” icon.

H.1) When you click on “Run” this window will pop up. Insert a name for your sample set and press “Run”.

Rules: Always wash after use.

Never run overnight, always turn off manually after running.

This concludes the sample sequence section.

5. Processing method

A) Open Empower 3 and press “Browse projects”. Select the project folder you wish to browse.

B) Choose a sample set you wish to use to make the processing method and double-click it. You can apply the method later to other sample sets after creating it.

Highlight the samples, right-click, and press “Review” to open the Review Main Window.

C) This is the Review Main Window. On the left side you can open individual injections to see the results.

Click here to open the “Processing Method Wizard” and to create a processing method.

C.1) This window will open. Choose to “Create a New Processing Method”.

D) Choose which processing type to use and which integration algorithm to use. Waters recommend ApexTrack since it is easier and better than traditional. Press “OK”.

E) Zoom in on the area you wish to integrate, which is only one peak in this case. Press “Next”.

E.1) Use default settings here and press “Next”.

F) Here you can reject peaks based on either area or height if you wish. Zoom in and select the smallest peak of interest and click on either the Minimum Area or Minimum Height box.

F.1) It is recommended to lower the value a bit to not miss out on any peaks. Here you could for example set the Minimum Height to around 20,000. Press “Next”.

G) Use default settings. Press “Next”.

H) Press “Yes” and fill in the names of your compounds. Press “Next”.

I) Enter the amount and the units of your sample. Press “Next”.

J) Use “External Standard Calibration”. Press “Next”.

K) Choose a name for your processing method and add any comments. Press “Finish”.

L) To apply your processing method to your sample run, choose your run, right- click and select “Review”.

L.1) When you have opened the selected sample run, go to “File  Open  Processing Method” and select your processing method.

L.2) To apply the method, press the Integrate icon and then the Calibrate icon.

The processing method should now be applied.

This concludes the processing method section.

6. MS for verification

You can use the MS for verification of compounds. Follow the steps in the instrument method section on how to adjust and select the appropriate settings for the QDa.

When you have finished your sample run and have gotten your results, follow these steps.

A) Select your sample set in the “Browse Projects” window.

B) Select a sample of the compound you wish to verify and open up the channels window by double-clicking the selected sample.

C) Open the QDa full scan.

D) This is an example of a full scan.

E) Zoom in and right-click to extract the spectrum at the selected position.

F) It will give you a plot with the mass of the compound that you can use for verification.

This concludes the MS for verification section.