Introduction

Lichens, a major component of alpine and arctic vegetation, are self-supporting symbiotic associations formed by a fungus with one or several photoautotrophs (algae and/or cyanobacteria), as primary partners, and with other microorganisms as further colonizers. Since the fungal constituent usually dominates the association, lichens traditionally have been considered as a life style of fungi. The lichenous lifestyle is maintained by c. 18,800 known species (Feuerer and Hawksworth 2007). In contrast to many fungi that are embedded within their substrates, most lichens expose their vegetative parts at the substrate surface, enabling the photobiont to harvest energy from solar radiation. The compact light-exposed vegetative bodies of lichens, called thalli, are among the most complex and aesthetically pleasing morphologies evolved by fungi during the past 600 million years (Yuan et al. 2005). Many lichens are highly extremotolerant which allows them to live as pioneers in the alpine zone and other cold environments. Life under these conditions correlates with the production of a variety of compound classes. Several of these are already found in plants or in other fungi, but about 80% reach highest diversity in lichens (Huneck and Yoshimura 1996). The valuation of these metabolites for medicinal purposes is generating an increasing interest as their activities are varied and sometimes significant. Only few lichen compounds are commercially available whereas the thousand known compounds are only partly or not at all investigated for their medicinal potential (Stocker-Wörgötter 2008). We review herein on the biological activities of some lichen compounds and focus on those encountered in lichen species growing in the polar or high mountain areas.

The alpine zone and lichens

The alpine zone is commonly recognised to correspond to the zone above the forest line with the nival zone at the upper limit (at about 2,800 m as in the North French Alps). The altitude may vary according to the latitudinal range and climate type resulting in some varied ecological features. In the Bavarian Alps, it is considered to start around 2,000 m in elevation compared to 4,700–5,000 m of the Andes of Southern Peru or Northern Bolivia (Troll 1973). Differences can also be determined by South or North exposure but such a zone is mainly characterised by an environmental harshness with a cold climate, drastic and rapid changes, lower oxygenated atmosphere and lower soil nutrient content, the mineral support increasing while the organic material is becoming scarce. To some extent, a correlation can be observed of altitudes and latitudes, although light regime varies across latitudes: the vegetation at a 2,000 m altitude for European Alps resembles the tundra vegetation, and that of 3,000 m altitude is similar to the one found in the High Arctic. Similar psychrophilic biological communities are shared by alpine and arctic habitats. There is also some evidence that microbial diversity is high in these habitats (Neufeld and Mohn 2005). The relative amount of microscopic fungi is increasing with altitude (Margesin et al. 2009) and recent analysis carried on subglacial sediment-rich ice from Arctic glaciers showed an unexpected high occurrence and diversity of filamentous Penicillium species (Sonjak et al. 2006). Similar richness in cold habitats has been observed for lichens (Asta 1984; Elvebakk and Bjerke 2006)

In arctic-alpine habitats, lichens are pioneer organisms. Often dominating as communities on rocks and stable soils, they form extensive biosurfaces a striking ability to survive in harsh conditions, in cases for several thousand years (Denton and Karlén 1973). Most lichens are therefore recognized by bare eye from a distance as they form bright colored marks on their substrates. Some lichens grow underneath the rock surface in the weathering rind as cryptoendolithic associations. Therefore, they are also particularly studied as models for life survival strategies in extreme terrestrial environments (Wynn-Williams and Edwards 2000; Edwards et al. 2003; de la Torre et al. 2007). Their resistance to extreme temperatures, desiccation and UV radiation stress is sustained by various biochemical processes and synthesis of protective molecules but above all to their capacity to survive in the stage of anhydrobiosis (Beckett et al. 2008). In lichens, all the control mechanisms respond with high sensitivity to excess light (Heber et al. 2000). In the cold environments, the symbiotic association allows a downshift of the photosynthetic gas exchange rates to optimum around 0°C while each of the photobiont or the mycobiont have a range optimum temperature above 15°C (Friedmann and Sun 2005). Such lichens are still active with some liquid water which can be found within the lichen thallus as combined with polyols, sugars or hydrated calcium oxalates, allowing low but functional respiration to temperature of a −20°C. The cellular and intracellular structures, which integrity are particularly exposed through freeze-thawing cycles, are preserved by polyols, polysaccharides, nucleotides, proteins and membrane lipids (Aubert et al. 2007). Extreme desiccation and irradiation increase the formation of ROS in the fungi and alga but protective mechanisms of the whole organism are highly more effective than those of the isolated partners (Kranner et al. 2005). The metabolic processes sustaining tolerance involve the gluthatione redox system and gluconate 6-P accumulation, which is of importance for reviviscence (Aubert et al. 2007). These mechanisms can also be involved to protect lichens from high UV irradiance (Bartak et al. 2004).

Other protective strategies as physical light scattering with oxalates or UV absorbers accumulated in the external layer such as xanthophylls, carotenoids (Singh et al. 2008) or other pigments (e.g., anthraquinones, melanins, chromones) may be involved to help them to tolerate such deleterious conditions. Some of these compounds such as dibenzofurans (e.g., usnic acid), depsides (e.g., atranorin), depsidones (e.g., lobaric acid) or shikiminate derivatives (e.g., calycin) are very commonly found in lichens (Fig. 1). Most of these secondary metabolites are accumulated as crystals on the external surfaces of hyphae (extrolites) in the upper cortex or in the internal medulla of the thallus. One or two of the cortex “lichen metabolites” are usually accumulated in higher amount. They may represent sometimes half of the 5–20% of the extractable material in the organic solvents.

Fig. 1
figure 1

Phenolic compounds from alpine lichen species with phototoprotective and/or biological activities

Full size image

Colourless substances of internal or basal thallus parts can vary, and chemical races are known for many species. Variation often occurs within substance classes (chemosyndromatic variation, Elix and Stocker-Wörgötter 2008). It has been controversially discussed for long whether the chemical races could represent separate species. Chemical variation is variably patterned in lichens and quantities of compounds can in some crustose species even vary among the areoles of a single thallus. Also, altitudinal gradients of chemical compound composition can sometimes be observed, e.g., in the crustose species Dimelaena oreina, which comprises 7 described chemotypes. Sampling across an altitudinal transect from 700 to 2,700 m revealed four chemotypes at lower elevations, but only two types at higher altitudes (Leuckert et al. 1981). To what extent this correlates with evolutionary patterns is not yet clear. In the search for new compounds such a chemical diversity can be considered as an opportunity. Alpine lichens offer an original material to investigate, particularly with fruticose or foliose species but also with some crustose species, at least when their macroscopic thalli can be scraped from their support. Lichen material for extraction in phytochemical studies needs to be gathered in dozens of grams (or from in vitro culture). Such studies will likely reveal a variety of further and uncommon compounds, but will be limited to species, which are easy to collect and available in large quantities. One example is presented in the following.

Cetraria islandica L. (Ach.): an alpine lichen

Cetraria islandica, commonly known also as “Islandic moss” is probably one of the best known alpine lichens used in folk medicine. The species develops extensive carpets composed of brownish cushions with more or less erect strap-like branches in boreal to subarctic tundra or above the tree-line in the Alps (see http://jlcheype.free.fr/imagesw/Lichens/Cetraria-islandica.htm). It preferentially occurs over acidic soil at wind-swept habitats, where the lichen is exposed to rapid and often drastic climatic fluctuations, ranging from over-hydration to almost complete desiccation that is correlated also with extreme temperature changes. Besides typical lichen polysaccharides such as lichenan and isolichenan, Cetraria islandica contains several secondary metabolites, including the paraconic acids (chiral trisubstituted γ-butyrolactones bearing a carboxylic acid group in the β-position and a lipophilic alkyl chain in the γ-position) protolichesterinic acid (Fig. 2) and roccellaric acid (Horhant et al. 2007) or the depsidone fumarprotocetratric acid (and traces of protoceraric acid), and quite often reddish quinone-like compounds at the senescing bases. There is also a chemical gradient between the growing tips and the senescing basis: the tips contain significantly more paraconic acids, whereas the basis contains higher amounts of fumarprotocetraric acids than detected in the tips, as assessed by TLC (W. Obermayer, unpublished data). In the Kolyma Upland and the Badjal mountains of far Eastern Russia, lichens growing in the highly rigorous climate have changed their metabolism and appearance with regard to European species. For instance, Cetraria islandica var. polaris do not contain lichesterinic acids but is characterized by bright red thallus tips due to an accumulation of polyhydroxynaphtaquinoid pigments like islandoquinone (Stepanenko et al. 2002).

Fig. 2
figure 2

Miscelleanous compounds from alpine lichen species with biological activities

Full size image

Protective compounds as a response to environment

Many lichen symbioses tolerate extreme environmental conditions unfavorable to the survival of the individual partners and higher accumulation of secondary protective metabolites is recognized as a response to environmental stress, such as high sun exposure (Gauslaa and Solhaug 2001). In field experiments, the screening of “visible and UV” radiations on Cetraria islandica thalli during 3 months induced a 40% average decrease in the level of phenolic compounds, the biosynthesis of most of them being particularly influenced by UV-B radiations (Asta et al. 1999). Both the terricolous Flavocetraria nivalis and the crustose Ophioparma ventosa are common light-exposed species in the alpine zone and circumpolar areas. In these species, compound production is influenced by UV and photoactive radiation, particularly affecting production of usnic acid (Bjerke et al. 2002). Comparatively large quantities of some compounds, such as 12.7% of divaricatic acid in the Ophioparma lapponicun dry weight or 10% of gyrophoric acid (Fig. 1) in Umbilicaria rossica collected in mountains or permafrost areas of far East Russia, may indicate that they are stress-related compounds (Stepanenko et al. 2002). Seasonal changes in usnic acid accumulation in Flavocetraria nivalis (up between 4 and 8% of the thallus dry weight), both of Arctic-alpine habitat or Patagonian heathland, show the adaptability of lichens (Bjerke et al. 2005). In alpine zones of Chile or in Antarctica where the ozone layer is depleted, an increase of rhizocarpic acid accumulation was found to correlate with the increasing UV-B radiation levels (Rubio et al. 2002). In many alpine species, this shikimic acid derivative is synthesized as well as other shikimic acid derivatives (e.g., calycin) characterized by a conjugated lactone, suggesting a chemical acclimatization of high levels of irradiance. The presence of the UV-absorbing groups can be frequently observed in many cortical metabolites such as depsidones or usnic acid (Rancan et al. 2002; Fernandez et al. 2006) and the anthraquinonic parietin pigment (Fig. 1) synthesis is also described to be UV-B induced in Xanthoria elegans (Nybakken et al. 2004). In sunlit steppes on southern slopes of the Mongolian mountain taiga, green-algal lichens with either yellow or orange lichen substances (usnic acid, parietin, pulvinic acid derivatives) or melanin in the cortex dominate, probably because they efficiently absorb both ultraviolet radiations and visible light (Hauck et al. 2009). The cyanolichens (photobiont = cyanobateria ± green algae) are more particularly restricted to moist microsites, the foliose Peltigera and Solorina species are frequently encountered while gelatinous lichens (e.g., Leptogium, Collema) are less common. In such lichens, the protective UV compounds are mainly amino acid derivatives, such as the UV-B absorbing mycosporine collemin (Fig. 3) found in Collema cristatum (Torres et al. 2004). All these compounds are considered as UV filter candidates and studied for their photoprotective properties (Hidalgo et al. 2005). Indeed most of them reach the physicochemical criterions as UV filters (Rancan et al. 2002) and can be considered for preventive use against sunburns but also skin cancers, including the highly aggressive melanoma.

Fig. 3
figure 3

Compounds from lichen-derived cultures (photobiont, mycobiont or an associated microorganism)

Full size image

More ubiquitous protective compounds such as carotenoids and xanthophylls (e.g., asthaxanthin) are also commonly encountered in lichen species. The 22 species collected in the Himalayan region and analysed by Czeczuga and colleagues (1996) contained a variety of these compounds. It can also be frequently observed that paraconic acids corresponding to butyrolactones which are substituted by a long carbon chain are commonly described in species found in alpine areas such as Ural Mountains (Rezanka and Guschina 2000, 2001) or in polar areas (Bodo and Molho 1980). Protolichesterinic acid and lichesterinic acids (Fig. 2) are the most common of these aliphatic acids and found abundant in Cetraria islandica. Some of these compounds are also described to form unusual macrolactone glycosides (Rezanka and Guschina 2001) and acetylenic lipids are also encountered in these lichens (Rezanka and Dembitsky 1999). Such compounds may account for a role in membrane integrity and favour liquid water maintain in low temperature conditions. Polyols and polysaccharides which may correspond to up to 57% of the extractable lichen compounds (Baron et al. 1991 cited in Olafsdottir and Ingolfdottir 2001; Podterob 2008) are also likely involved as freeze protectants. Interestingly, it can be noted that lichen derived antifreeze-proteins have also been used as food preservatives (see Oksanen 2006).

Lichen polysaccharides are high molecular weight compounds extractable with polar solvents, and generally with boiling water. Subsequently, the different types of polysaccharides can be separated according to their solubility in cold water (e.g., the soluble isolichenan compared to lichenan) or in alkaline versus acidic solutions, prior to precipitation with alcohols (e.g., methanol). Additionally various membrane filtration devices and gel filtration or ion exchange chromatography systems are used to separate lichen polysaccharides (Smestad Paulsen et al. 2002). Among the dozens of lichens studied for their polysaccharide content, some species are frequently found in cool montane to alpine zones. These are Cladonia species (Iacomini et al. 1985), Cetraria islandica (Ingolfsdottir et al. 1994b), Thamnolia vermicularis (Olafsdottir et al. 1999, 2007), Peltigera species (Fukuoka et al. 1968; Omarsdottir et al. 2006b) and Umbilicaria species (Nishikawa et al. 1969; Carbonero et al. 2006). They produce a variety of polysaccharides, including α- and β-glucans, galactomannanns and some heteroglycans, the β-glucans being often found in high amounts (Olafsdottir and Ingolfdottir 2001). The different structural types of lichen polysaccharides appear to be quite specific at the family level (e.g., pustulan for Umbilicaria species Carbonero et al. 2006 or for a given species such as thamnolan formed by Thamnolia subuliformis, the worm lichen (Olafsdottir et al. 1999).

Bioactivities of some lichen compounds found in alpine or polar regions

Lichen substances exhibit a great diversity of biological effects, including antimicrobial, anti-inflammatory, antioxidant, antiproliferative and cytotoxic activities. Activities described from secondary lichen metabolites encountered in alpine or polar lichen species are given in Tables 1 and 2. Recent reviews have discussed the pharmaceutical potential of lichen substances (Huneck 1999; Muller 2001; Boustie and Grube 2005; Takahashi et al. 2005; Oksanen 2006; Podterob 2008; Muggia et al. 2009). There is a growing interest in the pharmaceutical properties of compounds derived from lichens. However, relatively few lichen substances have been screened in detail for biological activity and therapeutic potential, due principally to difficulties in obtaining them in quantities and purities sufficient for structural elucidation and pharmacological testing. Additionally, precise lichen determination is essential and requires taxonomic expertise. Consequently, very few marketed medicines contain lichen compounds or lichen extracts. One example of commercial success are the riminophenazine antibiotics, exemplified by clofazimine (Lamprene®), which were developed as antimycobacterial drugs (Reddy et al. 1999). Their synthesis followed from identification of the antituberculous activity of derivatives of diploicin, a depsidone produced by the overspread lichen Diploicia (Buellia) canescens (Nolan et al. 1948; Barry and Twomey 1950).

Table 1 Active phenolic compounds from alpine lichen species [values correspond to 50% Inhibitory Concentrations (IC50), 50% Effective Doses (ED50), 50% Lethal Doses (LD50) or Minimal Inhibitory Concentrations (MIC)]
Full size table
Table 2 Active miscellaneous compounds from alpine lichen species (abbreviations as in Table 1)
Full size table

The alpine Cetraria islandica (Icelandic lichen, Lichen islandicus, or Islandic moss) is one of the rare lichen entering the formula of cold remedies [Broncholind® by MCM Klosterfrau (Köln, Germany) Isla-Moos® by Engelhard Arzneimittel (Niederdorfelden, Germany)]. The activity of Cetraria extracts is reported to be linked to the presence of polysaccharides as lichenan which helps to maintain hydration in human respiratory routes lowering the mucosal irritation. A small number of cosmetic, nutraceutical (http://www.iceherbs.is/ind_ensk.html), or pharmaceutical firms (e.g., Ichimaru Pharcos; Gifu, Japan) also manufacture products containing lichen substances or extracts. In any case, it should be taken into account that lichen compounds may have side effects particularly when misused. Allergenic reactions have been described for some (Thune and Solberg 1980; Hausen et al. 1993) as so as severe liver failures following about 500 mg/day ingestion of usnic acid in nutraceuticals marketed as weight-loss aids (Durazo et al. 2004; Neff et al. 2004). Thus, applications derived from activities described for lichen compounds have to be thoroughsly studied, particularly in the antimicrobial, anticancer and anti-inflammatory domains.

Antibiotic and antifungal activities

The antibacterial properties of lichen extracts are known for long (Burkholder et al. 1945) and still assessed (Piovano et al. 2002; Rankovic et al. 2007; Paudel et al. 2008; Schmeda-Hirschmann et al. 2008; Micheletti et al. 2009). Gram-positive Staphyloccocus aureus and Bacillus subtilis were sensitive to methanolic extracts of four different Antarctic lichen species (Paudel et al. 2008). These Antarctic lichens were considered to be an enriched source of effective antibacterial agents. Three new depsidones isolated from a Neuropogon collected on the Antarctic Livingstone Island showed a moderate activity on a Mycobacterium vaccae strain (Ivanova et al. 2002). MICs values obtained from monoaromatic phenols (methyl beta-orsellinate, methyl and ethyl orsellinates) derived from various Icelandic species were found equal or higher than usual preservatives (methyl and propyl-p-hydroxybenzoates, o-cresol) (Ingolfsdottir et al. 1985). Usnic acid is one of the most common substances found in lichens and encountered in one enantiomeric form (+)-usnic acid in Usnea species, (−)-usnic acid in some Cladonia or Alectoria species, both isomers being, however, described to occur in two alpine species Flavocetraria nivalis and Flavocetraria cucculata (cited in Stocker-Wörgötter 2008). This substance and others may interfere with palatability or with the digestive flora of the gut of ruminants in the sub polar regions (Iacomini et al. 1985; Vegar Storeheier et al. 2002). Lichens known for their nutritional value as reindeer lichens (e.g., Alectoria ochroleuca, Cladonia spp., Cetraria spp.) species contain high amounts of usnic acid and, in fact, resistant microorganisms such as newly described Eubacterium rangiferinum (Clostridiales) have recently been isolated from the reindeer rumen (Sundset et al. 2008). Usnic acid has been used as antibiotic (e.g., Binan®, Usno®), and is still available as a topical antiseptic in some products (e.g., Gessato® shaving treatment from Italy, Camillen 60 Fudes spray and nail oil from Germany). It is suggested for application in medical devices, since usnic acid inhibits bacterial biofilm formation on polymer surfaces (Francolini et al. 2004). This compound or derivatives are valuable active compounds against serious pathogens such as vancomycin-resistant enteroccocci, methicillin-resistant Staphylococcus aureus (Elo et al.), mycobacteria (Ingolfsdottir et al. 1998) or Listeria monocytogenes (Tomasi et al. 2006). Some anthraquinone derivatives have also shown weak activity on Bacillus subtilis (Ivanova et al. 2000) but a more specific lichen compound, protolichesterinic acid exhibit a very interesting antibacterial activity against Helicobacter pylori (Ingolfsdottir et al. 1997b). This species is found responsible of ulcers and this antibiosis combined with a gel-forming protective effect of polysaccharides from the Islandic lichen may account for the traditional anti-ulcer use of Cetraria islandica (Ingolfsdottir et al. 1997b) or the experimental gastric-protective effect of an alcoholic extract reported in (Podterob 2008). (+)-Protolichesterinic acid which presents antibiotic activities against various bacteria (Borkowski et al. 1964; Ingolfsdottir et al. 1998; Turk et al. 2003), might also impair quorum sensing systems of bacteria (Riedel et al. 2008, preliminary data). Antiviral activities have been also reported (Pengsuparp et al. 1995; see Table 2).

Antifungal activities have been reported for parietin and other anthraquinone derivatives isolated from the foliaceous-crustose Xanthoria parietina (Manojlovic et al. 1998). More recently, extracts of the Andean lichens Protousnea poeppigii and Usnea rigida where found inactive against several yeast and Aspergillus strains but divaricatinic acid and the new isodivaricatic acid (Fig. 1) isolated from P. poeppigii displayed interesting activities against Microsporum and Trichophyton pathogen dermatophytes (Schmeda-Hirschmann et al. 2008).

Cytotoxic, antimutagenic and antitumor activities for potential anticancer agents

Usnic acid is the most ancient lichen compound also found in alpine species (Cladonia, Flavocetraria) and considered for antitumor activity with ten to hundred micromolar IC50 units on several cancer cell lines (Bazin et al. 2008). Apoptosis induction features have been revealed for usnic acid on L1210 cell lines (Bézivin et al. 2004) through caspase 3 induction (Bazin et al. 2008) but in cellular tests as for in vivo experiments, the solubilisation of this substance appear to be of importance. 2-Hydroxypropyl-β-cyclodextrine was found to be suitable for solubilising usnic acid (Kristmundsdottir et al. 2002) and IC50 obtained on malignant cells are in the same range of the one obtained with a diaminooctane derivative (Bazin et al. 2008). A slight increase of its antitumor effect has been obtained with encapsulating this hydrophobic compound in microspheres (da Silva Santos et al. 2006). However, in vivo assays show (−)-usnic acid to have a weak, if any, antitumoral effect against Lewis Lung carcinoma and P388 leukemia (Kupchan and Kopperman 1975; Takai et al. 1979). As (+)-usnic acid was found active against Epstein-Barr Virus activation, it could therefore constitute a valuable candidate as antitumor promoters (Yamamoto et al. 1995) and the recent activities described in a p53-independent manner on three cancer cell lines led the authors to consider usnic acid as a non-genotoxic anti-cancer agent that makes it a potential candidate for novel cancer therapy (Mayer et al. 2005).

Series of depsides and depsidones isolated from Antarctic lichens were tested along with colchicine in cell cultures of lymphocytes (Correché and Carrasco 2002) or with usnic acid for their cytotoxic and apoptotic effects on hepatocytes (Correché and Carrasco 2002; Correché et al. 2004). Pannarin, 1′-chloropannarin and sphaerophorin were found more cytotoxic than colchicine and on primary cultures of hepatocytes, the depside sphaerophorin has higher cytotoxic effects than usnic acid while the depsidones salazinic acid, stictic acid, and psoromic acid displayed the more significant apoptotic activities. Pannarin and sphaerophorin are also reported to inhibit cell growth and to induce apoptosis in human prostate carcinoma DU-145 (Russo et al. 2006) and human melanoma M14 cells (Russo et al. 2008). Although active at higher doses than the anticancer alkaloid vinblastine, the depsidone lobaric acid was recently found to exhibit a remarkable inhibition of tubulin polymerization (Morita et al. 2009). This effect may be linked to the moderate but constant effect of lobaric acid (IC50 # 12–65 μg ml−1) obtained on fourteen cancer cell lines while normal skin fibroblasts appear to be less sensitive (Ogmundsdottir et al. 1998; Haralsdottir et al. 2004). For some lichen compounds (i.e., gyrophoric, usnic acid) inhibiting HaCat keratinocytes at low concentrations, the antiproliferative activity was considered to be due to a cytostatic effect rather than a cytotoxic effect (Kumar and Muller 1999). Recent antiproliferative assays carried out on MM98 malignant mesothelioma cells, on A431 vulvar carcinoma cells compared to HaCat keratinocytes with (+)-usnic, salazinic, vulpinic, gyrophoric and evernic acids confirm the higher toxicity of usnic acid and afford interesting remarks about the disconnection of cell migration stimulation and mitosis inhibition (Burlando et al. 2009). The combination of usnic acid and gyrophoric acid, both used at subtoxic doses, was therefore stimulating keratinocyte tissue regeneration with possible use in wound healing (Burlando et al. 2009).

The antiproliferative activities of tenuiorin (a tridepside, as gyrophoric acid found in Umbilicaria species) and methyl orsellinate extracted from Peltigera leucophlaebia were tested on human breast (T-47D), pancreatic (PANC-1), and colon (WIDR) cancer cell lines; the former compound caused a weak-to-moderate effect on the pancreatic and colon cells, whereas methyl orsellinate was without effect (Ingolfsdottir and Gudmundsdottir 2002). A moderate cytotoxicity (IC50 # 0–13 μM) on three carcinoma cell lines has been recently described for stereocalpin A, a new cyclic depsipeptide (Fig. 2) isolated from Stereocaulon alpinum that was collected in Antarctica (Seo et al. 2008b).

A butyrolactone, protolichesterinic acid, present in Cetraria islandica was also found to act as antiproliferative against a series of fourteen cancer cell lines (IC50 < 10 μg/ml for most of them; Ogmundsdottir et al. 1998; Haralsdottir et al. 2004). Its activity against Ehrlich solid tumor has been confirmed, while other butyrolactone derivatives e.g., nephrosteranic acid had weak activities (Hirayama et al. 1980).

Lichen glucans do not exhibit antiproliferative activities with impressive IC50 values on cancer cells as exemplified by the galactomannose substituted α (1 → 3), α (1 → 4) glucan isolated from Cladonia furcata (IC50 # 500–800 μg/ml). Apoptosis induction and a telomerase activity diminution demonstrate their potential in anticancer adjuvants (Lin et al. 2003). Indeed, these lichen polysaccharides have been investigated in antitumor assays for 50 years (see Boustie and Lohézic-Le Dévéhat 2008) including lichenan derivatives (Demleitner et al. 1991) and other structural features (Olafsdottir and Ingolfdottir 2001). Although the per os absorption of these high molecular weight compounds is poor, they are recognized to act on the immune system and are generally classified as Biological Response Modifiers (BRMs), more particularly stimulating the innate immunity through macrophage activation. These effects may result in a better defense of the organisms receiving these compounds with application as anti-tumor adjuvants but also promoting infectious resistance, and wound healing. Anti-tumor lichen PS have been characterized in in vivo tests as lichenan inducing highest scores for complete tumor regression when compared to isolichenan or pustulan (Fukuoka et al. 1968; Nishikawa et al. 1969). Thus, the polysaccharide sub-structure has to be considered, the reticuloendothelial system activation being observed with a heteroglycan (Ki-M-7) isolated from Cetraria islandica but not with the lichenan (3:7) (Ingolfsdottir et al. 1994b) and with O-acylated pustulan isolated from Umbilicaria esculenta (Nishikawa and Ohno 1981). In a recent study carried on the methanolic extracts of 8 polar lichens from Antarctica (Choi et al. 2009), the methanol extract of the crustose Caloplaca regalis was found to be the most active on rat peritoneal macrophages. Although the composition of the extract was not indicated, some lichen polysaccharides may have been be present along with the secondary metabolites to account for the tumoricidal activity. The macrophage activation resulting in TNF-α and NO production, was suggested to be mediated via the p38 MAPK and the NF-κB pathway (Choi et al. 2009).

Antiinflammatory activities

Some lichen compounds such as longissimone A have clearly demonstrated anti-inflammatory responses comparable to aspirin (Choudhary et al. 2005). The cyclooxygenase (COX) and lipoxygenase (LOX) pathways resulting in the production of prostaglandins and leukotrienes formation are involved in many inflammatory processes but also in tumor growth and promotion (Ara and Teicher 1996). Most assays have therefore been carried for these key enzymes, mainly with lichen compounds isolated from material collected in cold places as in Iceland. In a COX screening with depsides and depsidones, 4-O-methylcryptochlorophaeic acid (IC50 = 0.34 μM) was found to be the most active depside (Sankawa et al. 1982) and the monoaromatic phenol lichen compound methyl-β-orsellinate was also found to inhibit phospholipase A2 (Kim et al. 1996) and 5-LOX (Ingolfsdottir and Gudmundsdottir 2002). The trimeric depside tenuiorin isolated from Peltigera aphthosa (Ingolfsdottir and Gudmundsdottir 2002), the depsidones baeomycesic acid and lobaric acid, respectively isolated from Thamnolia vermicularis (Ingolfsdottir et al. 1997c) and Stereocaulon alpinum (Bucar et al. 2004) were also found active against 5-LOX. Although a dual inhibition of the inflammatory targets could result in more effective drugs, a selective inhibition of LOX with regard to COX, as for protolichesterinic acid (Ingolfsdottir et al. 1994a), can be of interest as the gastroprotective effect of prostaglandins would be theorically maintained. An alkamide, cis-9-octadecenamide (Fig. 2) isolated from Stereocaulon alpinum, also showed an inhibition of COX from the microsomes of sheep seminal vesicle (Ingolfsdottir et al. 1997a).

The anti-complementary activities of some PS lichens (Olafsdottir and Ingolfdottir 2001; Olafsdottir et al. 2003) may also result in an anti-inflammatory effect and particularly with the beta-glucans upregulating IL-10 secretion by the dendritic cells (Omarsdottir et al. 2006a). Recent in vivo experiments with sub cunaneously injected aqueous extracts of Cetraria islandica suggest the significant reduction of the BSA-induced arthritis in rats is related to the presence of lichenan (Freysdottir et al. 2008).

Antioxidant activities

Due to the harsh conditions in alpine or polar environments with sudden rehydration enhancing the production of reactive oxygen species (ROS), efficient protective mechanisms are expected to be encountered in such exposed lichens. The burst of intracellular ROS is accompanied by a decrease of water soluble low molecular-weight antioxidants such as gluthatione (Weissman et al. 2005b) and adjustment of enzymatic activities (Kranner et al. 2005; Weissman et al. 2005a). Although mainly hydrophobic, the polyphenolic nature of the major secondary metabolites of lichens is expected to afford antioxidant properties. Cuculloquinone, a bisnaphthoquinone of Flavocetraria cucullata was found to inactivate DPPH to a 80% extent while the BHT that was used as a standard antioxidant was twofold less active (Stepanenko et al. 2002). A recent DPPH autoradiography assay with extracts of five lichens collected in Antarctica showed that 33–50% of the compounds were antioxidants with a 17–47 mg/g phenolic content in the lichen extracts (Bhattarai et al. 2008). However, the total phenolic content and antioxidant potency did not always correlate (Odabasoglu et al. 2005). Although reports on crude lichen extracts (e.g., Gulcin et al. 2002; Behera et al. 2003) are quite promising, the antioxidant properties of isolated compounds are not so impressive. For instance, two depsidones were found only slightly more active than the commercial quercetine in a superoxide scavenging assay with IC50 # 600 μM (Lohézic-Le Dévéhat et al. 2007) and none of the orcinol or orsellinate derivatives were as active as the commercial gallic acid to reduce DPPH (Lopes et al. 2008).

Products of axenically cultured mycobionts and photobionts

The main lichen partners can be dissociated to be cultivated separately, allowing a biotechnological approach for the metabolite production (Oksanen 2006). The mycobiont obtained from lichen spores or mechanical dissociation of the thallus has to be cultivated in aposymbiotic conditions, mainly on solid support and the growing rate is quite limited. However, lichen mycobionts synthesize significant quantities and variety of secondary metabolites only under permissive conditions, as has been demonstrated repeatedly in cultivation experiments (e.g., Hamada and Ueno 1987; Leuckert et al. 1990; Yamamoto et al. 1995; Hamada et al. 1996). Alpine lichen mycobionts were for example not growing at temperatures of 20°C and above (Yamamoto et al. 1987). Therefore, cultured mycobionts can produce new, interesting metabolites not encountered in nature, and synthesis of these novel products may be enhanced by varying the type and concentration of nutrients in the growth medium, or by simulating stress situations (ultraviolet irradiation, changes in thermal or osmotic conditions). For instance, different carbon sources in the culture medium can switch secondary metabolite biosynthesis from the polyketide pathway to the fatty acid biosynthetic pathway (Molina et al. 2003). Appropriate conditions are in some cases attributed to elevated osmotic conditions in vitro, since high levels of sugars, e.g., mannitol, often are needed to initiate secondary metabolite production in axenic fungal cultures. Alteration of secondary metabolite production was reported for the mycobiont of Xanthoria elegans grown on different media (Brunauer et al. 2007). Permissive conditions appear to include those mimicking the temporally changing conditions to which lichens are exposed in nature, since constant culture conditions often do not promote production of lichen-specific secondary metabolites (Stocker-Wörgötter 2008). In resynthesis experiments with Cladia retipora and Dactylina arctica, a complete thalli yielded typical lichen substances (Stocker-Wörgotter and Elix 2006), whereas resyntheses of Lecanora rupicola also produced additional substances, not found in the natural lichen, nor in the mycobiont cultures conditioned to produce the natural chemical diversity (Brunauer et al. 2006).

Secondary metabolite production by lichen photobionts warrants further investigations Among the very few such compounds reported are some with moderate cytotoxic activities: the nostoclides (Yang et al. 1993), corresponding to γ-butyrolactones, which are found in a Nostoc sp. (Fig. 3) from the lichen Peltigera canina. Microcystins, hepatotoxic cyclic heptapeptides were produced by a Nostoc sp. isolated from the lichen Pannaria pezizoides (Oksanen et al. 2004), and β-N-methylamino-l-alanine, a neurotoxic amino acid is produced by a Nostoc sp. from a Peltigera sp. (Cox et al. 2005). Further cryptophycins, peptolides with promising antineoplastic activities, are present in Nostoc strain ATCC53789, which was isolated from a lichen collected on Aaron Island in Scotland (Magarvey et al. 2006).

Secondary compounds from microbial associates of lichens

Lichens harbor not just the traditionally recognized cyanobacterial, algal, and fungal symbionts but also diverse lichen-inhabiting (lichenicolous and endophytic) fungi, as well as a plethora of bacteria. The species diversity of these co-inhabiting fungi is well-studied: more than a thousand species parasitic for lichens have been documented (Lawrey and Diederich 2003). They can be producers of interesting metabolites in their own right. For example, ambuic acid and its acetyl derivative isolated from the endolichenic fungus Pestalotiopsis sp. inhabiting Multiclavula lichen species are active against Staphylococcus aureus (Ding et al. 2009). In the same way, the bisnaphtopyrone lichenicolin A isolated from an unidentified lichen-inhabiting fungus (He et al. 2005) was active against Gram-positive bacteria, including strains of methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium. Morever, the new bioactive structures of preussomerins produced by endolichenic fungi are considered as lead compounds to be optimized by chemists (Weerapreeyakul et al. 2007). They were originally detected in the lichenicolous fungus Microsphaeropsis sp. (Seephonkai et al. 2002).

Gonzales et al. (2005) studied the biosynthetic potential of actinomycetes associated with lichens by DNA fingerprinting. High frequencies were obtained for PKS I (62.6%), PKS II (64.7%) and non-ribosomal peptide synthetases (58.5%). Associated bacteria in lichens from arctic habitats apparently had lower biosynthetic potential than those of tropical habitats. So far, species-rich and detailed studies of bacterial secondary metabolite biosyntheses from arctic-alpine species are still pending. Such studies could be highly interesting for pharmaceutical research. For example, a streptomycete isolate from a reindeer lichen (Cladonia uncialis) in British Columbia revealed uncialamycin as a new enediyne (Davies et al. 2005). Enedyines are cytotoxic compounds, and not surprisingly, uncialamycin shows strong antibacterial activity against both Gram-positive and Gram-negative human pathogens, including Burkholderia cepacia (MIC, 0.001 μg/ml) and Staphylococcus aureus (MIC, 0.0064 μg/ml). This strain also produces a series of cladoniamides (Fig. 3) corresponding to new alkaloids related to the indolocarbazole structural pattern (Williams et al. 2008). As staurosporine and rebeccamycin are two indolocarbazole heterosides used as lead compounds for new anticancer treatment, these Streptomyces metabolites are of high value for biogenetics and biotechnological production or for pharmacological tests. In addition, six new aminocoumarins, angucycline and a new butenolide were recently isolated from lichen-derived Streptomyces spp. (Cheenpracha et al. 2010; Motohashi et al. 2010).

The complex interaction between all these organisms remains unclear but we can assume that these compounds could be involved in defence and chemical communication pathways. Yet, associated bacteria might have been of profound evolutionary importance by contributing genetic material to lichen fungi. Schmitt and colleagues showed that lichen fungal genomes contain 6-methylsalicylic acid synthase-like genes which are related to bacterial genes. These genes could be responsible to contribute the basic phenolic units (orsellinic acid, resorcinol) for typical lichen compounds. The authors suggested that these genes could have been acquired by past horizontal transfer from bacterial genomes (Schmitt et al. 2008). Such horizontal transfer might be plausible, given the ubiquity of associated bacterial communities. Diverse and abundant bacteria have also been observed in some alpine and antarctic lichens (Grube et al. 2009; Selbmann et al. 2010).

Conclusions and future prospects

Investigations on the natural product composition of whole lichen thalli or lichen constituents, and their biosynthetic machineries, lag behind those of non-lichenous bacteria and fungi. The range of bioactivities identified to date, however, are quite promising for further research aiming at commercial production and exploitation of pharmaceutically interesting substances. The score of activities (with regard to the number of lichen or lichen-associated compounds tested) is in a good range for natural products (Cragg and Newman 2009). This is sustained by the structural diversity of the metabolites produced as a result of the symbiosis and magnified by the metabolic diversity of the hosted organisms. The extremophilic conditions under which alpine lichens thrive are also enhancing the chances to obtain new bioactive chemotypes. New compounds have therefore been reported recently from lichens collected from psychrophilic habitats. Usimines corresponding to natural new enamino derivatives of usnic acid have been reported from the polar lichen Ramalina terebrata: they exhibit a moderate inhibitory activity against the protein tyrosine phosphatase 1B (Seo et al. 2008a). This enzyme is a major non-transmembrane phosphatase involved in human tissues as a negative regulator of the insulin-stimulated signal transduction pathways and a target for new type 2 antidiabetes treatment. Better activities are obtained with lobaric acid and other new pseudodepsidones isolated from Stereocaulon alpinum collected in the same Antarctic region (Seo et al. 2009).

A deeper investigation of arctic-alpine lichens can still reveal quite interesting discoveries. Recently, an arctic Lecanora (griseofulva) was described which contained (+)-griseofulvin (1) and (+)-dechlorogriseofulvin, as well as traces of alternariol (Elix and Øvstedal 2004). The presence of such antifungal spirobenzofuranones is unique in lichens so far (many lichens, though, are able to produce the griseofulvin precursor norlichenxanthone). These compounds are instead often produced by non-lichenized fungi (such as Penicillium and Alternaria). Interestingly, the recently described Penicillium lanosum, which has also been isolated from Lecanora, and other psychrophilic Penicillia are able to produce griseofulvin (Frisvad et al. 2005). Further studies are required in our opinion to evaluate whether unusual compounds are produced by lichens or their associated fungi. In any case we suppose that mold fungi with interesting secondary compounds can be isolated from lichens living in cold habitats.

For some potent active compounds the limitations for collection can be overcome through chemical synthesis or in vitro production as for hybocarbone, a naphtazarine-dimer derivative with potent activities on cancer cells (IC50 = 1 nM, MCF-7). Hybocarpone (Fig. 3) has been synthetised (Gray and Nicolaou 2004) and also isolated from a mycobiont culture of Lecanora hybocarpa (Ernst-Russel et al. 1999). Culturing approaches could be extended now to other lichen-associated microrganisms that produce compounds of interest (e.g., uncialamycin in Fig. 3). In the case, biotechnological approaches might be facilitated as most of the microorganisms allow high fermentation rates. Recent progress has been achieved for lichen cultivation particularly with synchrotron chambers where the natural environmental conditions can be reproduced and the metabolite production triggered with specific stresses (Stocker-Wörgötter 2008), while biotechnological approaches with lichen fungi are still in its infancy. These require the establishment of appropriate expression systems as previous work has not succeeded in production of lichen compounds so far (Chooi et al. 2008; Gagunashvili et al. 2009). It is anticipated that the genetic engineering and combinatorial biosynthesis approaches currently used to expand the repertoire of polyketides from “conventional” bacteria and fungi will soon become applicable to lichen PKS genes of (cyano-)bacterial and fungal origin. When these technological hurdles are solved we will be able to fully open the lichen’s treasure chest of biosynthetic potential.