Chapter 3

Lichen Secondary Metabolites as Potential

Antibiotic Agents

Marijana Kosanić and Branislav Ranković

Abstract It is well known that pathogenic microbes pose serious threats to human
health and are increasing in prevalence in institutional health-care settings due to
the growing resistance that infectious agents have developed against antibiotics.
Therefore, new alternatives for combating the spread of infection through
antibiotic-resistant microbes are necessary for keeping pace with the evolution of
“super” pathogens. Natural products are proposed as a therapeutic alternative to
conventional antimicrobial treatment. Among them, lichen-derived product and
their antibiotic properties are of special interest to scientists as up to 50 % of all
lichens have been reported to possess antibiotic activities. A great number of
reports concerning the antimicrobial screening of lichens have appeared in the
literature. According to published data, the lichens and their secondary metabolites
exhibited the activity against a great number of microorganisms. Therefore, the
present study represents lichens as very interesting source of bioactive compounds
which provide unlimited opportunities for new antimicrobial agents.

3.1 Needing for New Antibiotics from Nature

Before the introduction of antibiotics in the 1940s, infections were rare, but rapidly
increased in frequency as the use of antibiotics increased. In fact, most antibiotics
that were first used in the 1940s and 1950s are no longer used clinically because
nowadays the resistance of infectious beings to these antibiotics is very common.
Over time they have been developing new antibiotics, and with the introduction of
each, new drug-resistant bacteria appeared rapidly. The process of resistance is
augmented by short generation times of bacteria enabling rapid mutation and
selection of resistant strains and a horizontal transfer of resistance genes. Bacterial
pathogens resistant to more than one, or even most clinically used antibiotics, have
become common. Today, we moved the mode of use and prescription of antibiotics

M. Kosanić (*) • B. Ranković

Department of Biology, Faculty of Science, University of Kragujevac, 34000 Kragujevac,
Serbia
e-mail: marijanakosanic@yahoo.com

© Springer International Publishing Switzerland 2015 81

B. Ranković (ed.), Lichen Secondary Metabolites,
DOI 10.1007/978-3-319-13374-4_3
82 M. Kosanić and B. Ranković

in order to try to slow the relentless pace of bacterial evolution but not yet found a
solution to this problem. Microbiologists continue to study how bacteria evolve so
that we can predict how they will respond to medical treatment and so we can better
manage the evolution of infectious diseases.
Bacteria are able to resist the effects of antimicrobials through preventing
intracellular access, immediately removing antimicrobial substances through efflux
pumps, modifying the antimicrobial agent through enzymatic breakdown, or modi-
fying the antimicrobial targets within the bacterial cell to render the substance
ineffective. Successful development of resistance often results from a combination
of two or more of these strategies (Sheldon 2005).
The first antibiotic resistance mechanism described was that of penicillinase. Its
presence and activity were first reported by Abraham and Chain in 1940 shortly
after its discovery (Abraham and Chain 1940).
Antimicrobial resistance traits are genetically coded and can either be intrinsic
or acquired. Intrinsic resistance is due to innately coded genes which create natural
“insensitivity” to a particular antibiotic. Innate resistance is normally expressed by
virtually all strains of that particular bacterial species. Acquired resistance is gained
by previously susceptible bacteria either through mutation or horizontally obtained
from other bacteria possessing such resistance via transformation, transduction, or
conjugation. Acquired resistance is limited to subpopulations of a particular bacte-
rial species and may result from selective pressure exerted by antibiotic usage.
The drug resistance of human and animal pathogens is one of the best
documented in biological evolution and a serious problem in both developed and
developing countries. The consumption of more than one ton daily antibiotics in
some European countries has resulted in resistance to bacterial populations, thus
causing a serious public health problem. In view of this scenario, the search for new
antimicrobial substances from natural sources, including lichens, has gained impor-
tance in pharmaceutical companies.
Since lichens produce a variety of substances with antimicrobial properties, it is
expected that screening programs discover candidate compounds for the develop-
ment of new antibiotics. However, scientific research to determine the therapeutic
potential of lichens is limited, and there is a lack of scientific studies that confirm
the possible experimental antibiotic properties of a large number of lichens. It is
expected that compounds that reach targets different from those used by known
antibiotics may be active against resistant pathogens.

3.2 Antimicrobial Activity and Probable Mechanisms

of Action of Lichens

Antimicrobial activity of lichens is understood as their ability to eliminate micro-

organisms or to inhibit their growth.
3 Lichen Secondary Metabolites as Potential Antibiotic Agents 83

The antimicrobial properties of lichen extracts and their secondary metabolites

are known for long (Burkholder and Evans 1945; Stoll et al. 1950; Vartia 1973;
Piovano et al. 2002; Ranković et al. 2007a; Paudel et al. 2008; Schmeda-
Hirschmann et al. 2008; Micheletti et al. 2009) and still assess the mechanisms of
their effects.
The probable mechanisms of antimicrobial action of lichens are:
Inhibition of cell wall synthesis The peptidoglycan layer is important for cell wall
structural integrity, being the outermost and primary component of the wall.
Inhibitors of cell wall synthesis act by inhibiting the synthesis of the peptidoglycan
layer of bacterial cell walls and thus come to degradation of the cell wall.
Inhibition of protein synthesis (translation) Protein synthesis inhibitors act at
the ribosome inhibiting the synthesis of proteins of the pathogen to occur, mis-
reading the sequence of amino acids, and thus inhibit the functioning of the patho-
genic cells.
Alteration of cell membranes Injury bacterial plasma membranes lead to cell
death through leakage of the cell contents and associated disruption of the cross-
membrane potential (which essentially are ion concentration gradients).
Inhibition of nucleic acid synthesis Nucleic acid inhibitors act by inhibiting the
production of nucleic acids (DNA and RNA).
Antimetabolite activity Antimetabolites prevent a cell from carrying out a meta-
bolic reaction. Antimetabolites function by competitive inhibition of enzymes and
by erroneous incorporation into nucleic acids. In both cases, the cells become
unable to normally function.
The secondary metabolites of the lichen are active substances against pathogenic
microorganisms. Most known lichen substances with antimicrobial activity are
usnic acid, phenolic compounds, triterpenes, steroids, anthraquinones, depsides,
depsidones, and dapsones, and most of them are known mechanisms of their
antibiotic action.
Usnic acid Usnic acid, a compound produced by various lichen species, has been
demonstrated previously to inhibit growth of different bacteria and fungi; however,
the mechanism of its antimicrobial activity has not been fully explained.
The mechanisms of the antibiotic activity of usnic acid against Gram-positive
bacteria were attributed to its protonophoric properties as an uncoupler of oxidative
phosphorylation (Abo-Khatwa et al. 1996). This effect was remarkably more
pronounced than that produced by dinitrophenol, a well-known uncoupler agent
that increases the permeability of mitochondria to protons, reducing the electro-
chemical potential and thus inhibiting adenosine triphosphate synthesis. Two
research groups found that usnic acid played an active role in the transport of
protons through the membranes of isolated mouse liver mitochondria, uncoupling
the electron flow through the respiratory electron transport chains from the gener-
ation of an acidic gradient and thereby inhibiting the synthesis of adenosine
84 M. Kosanić and B. Ranković

triphosphate (Abo-Khatwa et al. 1996; Bouaid and Vicente 1998). The ability of
usnic acid to shuttle protons through the membranes was confirmed by studies with
artificial phospholipid membranes (Bačkor et al. 1998). A model for the function of
usnic acid in the control of the intracellular pH of lichens was recently proposed
(Hauck and Jurgens 2008) that involves two hypotheses: (1) at the optimum pH near
the pKa1 value of usnic acid (4, 4), buffering is assumed to be compensated by the
usnic acid-mediated proton transport into the cell and (2) at low pH (<3.5), the
equilibrium between usnic acid and usneate shifts toward the usnic acid, and
protons are increasingly shuttled into the cells, considering that usnic acid dissoci-
ates to usneate at cytosolic pH 7.4. This implies that more protonated molecules
would be able to cross the membrane and release protons into the cell. As a result,
the intracellular pH would decrease and lead to the death of the cells (Hauck and
Jurgens 2008).
In addition, Macia˛g-Dorszyńska et al. (2014) assume that inhibition of RNA
synthesis may be a general mechanism of antibacterial action of usnic acid, with
additional direct mechanisms, such as impairment of DNA replication in B. subtilis
and S. aureus.
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 and
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 enterococci,
methicillin-resistant Staphylococcus aureus (Elo et al. 2007), mycobacteria
(Ingolfsdottir et al. 1998), or Listeria monocytogenes (Tomasi et al. 2006).
Phenols Most lichen substances with antibiotic activity are phenolic metabolites.
Phenols are one of the largest classes of secondary biomolecules, which are
characterized by the presence of aromatic rings with hydroxyl group bonded
directly to an aromatic hydrocarbon group. Although they are firstly identified in
plants (Cowan 1999), their presence was also observed in lichens (Odabasoglu
et al. 2004; Kekuda et al. 2011; Ranković et al. 2010, 2014; Mitrović et al. 2011;
Kosanić and Ranković 2011; Manojlović et al. 2012). In recent years, there was a
causal relationship between the total contents of these compounds with biological
activities recorded in a large number of lichens, which include anti-inflammatory,
antiallergic, anticancer, antihypertensive, antirheumatic, and antibacterial activity.
Antimicrobial properties of phenolics are explained by the presence of phenol
hydroxyl groups, the number of which is in correlation with their toxicity toward
microorganisms (Cowan 1999). The possible mechanisms of their action include
inhibition of extracellular microbial enzymes, deprivation of the substrates required
for microbial growth, or direct action on microbial metabolism through inhibition
of oxidative phosphorylation, by sulfhydryl groups and some nonspecific inter-
actions (Cowan 1999).
3 Lichen Secondary Metabolites as Potential Antibiotic Agents 85

A correlation between phenolic constituents and antimicrobial activity has been

established. Gulluce et al. (2006) found that the content of total phenolic of the
extracts of Parmelia saxatilis, Platismatia glauca, Ramalina pollinaria, Ramalina
polymorpha, and Umbilicaria nylanderiana was strongly related with their anti-
microbial activity. A positive correlation was seen between the phenolic content
and antibiotic activity of Lasallia pustulata, Parmelia sulcata, Umbilicaria crustulosa,
and Umbilicaria cylindrica (Ranković et al. 2007a). Ranković et al. (2007b) found a
high correlation between antimicrobial efficacy of macrolichens Cladonia furcata,
Parmelia caperata, Parmelia pertusa, Hypogymnia physodes, and Umbilicaria
polyphylla and their phenolic content. Many other studies also have shown a direct
correlation between the phenolic content and the antimicrobial activity (Vartia 1973;
Ingolfsdottir et al. 1998; Ranković et al. 2009, 2011).
Terpenes In recent years, more data indicate the presence of terpenes in numerous
representatives of lichens (Culberson 1970; Rundel 1978; Abdullah et al. 2007).
One of the many functions of these compounds is their antimicrobial activity, but
the mechanism of action of terpenes on microorganisms is not fully understood
(Cowan 1999). According to their lipophilic nature, it is assumed to act by
disrupting membrane functions of microbial cells (Cowan 1999), and some authors
believe that they may cause nonspecific cell membrane permeability increase for
the antibiotic molecule (Byron et al. 2003).
Steroids These compounds are highly often present in lichens. Steroids have been
reported to have antibacterial properties; the correlation between membrane lipids
and sensitivity for steroidal compound indicates the mechanism in which steroids
specifically associate with the membrane lipid and exert its action by causing
leakages from liposomes (Epand et al. 2007; Mohammed 2013).
Depsides, tridepsides, and tetradepsides consist of two, three, and four
hydroxybenzoic acid residues linked by ester groups. These are the most numerous
classes of secondary metabolites in lichens. More than one hundred lichen com-
pounds are depsidones, which have an additional ether bond between aromatic
rings. Depsidones in lichen are believed to arise by oxidative cyclization of
depsides. It has been found that depsidone and depside compounds such as
atranorin, divaricatic acid, lecanoric acid, evernic acid, salazinic acid, physodic
acid, and stictic acid possess important antimicrobial activity (Manojlović
et al. 2012; Kosanić et al. 2013, 2014a; Ranković et al. 2014).
Anthraquinones and xanthones are also important constituents of many
lichens. Anthraquinones such as parietin, parietinic acid, emodin, fallacinol, and
fallacinal were shown to have a high antimicrobial effect (Manojlović et al. 2002).
Manojlovic et al. (2010) found that the antimicrobial activity of lichen Laurera
benguelensis is mainly related to the presence of lichexanthone.
No clear mechanisms have been identified that specifically indicate how
depsides, tridepsides, tetradepsides, depsidones, anthraquinones, and xanthones
target microbial invasion.
86 M. Kosanić and B. Ranković

3.3 Methods to Detect Antimicrobial Activity of Lichens

Methods to detect antimicrobial activity are in vitro procedures used to detect

antimicrobial resistance in individual microbial isolates.
For lichens, antimicrobial activity can be examined both for their extracts and
for their secondary metabolites.
Extraction is a process of separation of active compounds from plant material
using different solvents. Extract can be prepared using various methods, such as
sonification, heating under reflux, Soxhlet extraction, maceration, and others.
Different solvent systems are available to extract the bioactive compound from
natural products. The solvent systems used in extraction are selected on the basis of
their capacity to dissolve the maximum amount of desired active constituents and
the minimum amount of undesired constituents. The extraction of hydrophilic
compounds uses polar solvents such as methanol, ethanol, or ethyl acetate. For
extraction of more lipophilic compounds, dichloromethane and a mixture of
dichloromethane/methanol are used (Sasidharan et al. 2011). Due to the fact that
extracts usually occur as a combination of various types of bioactive compounds or
phytochemicals with different polarities, their separation to obtain pure compounds
using different separation techniques such as TLC, column chromatography, flash
chromatography, Sephadex chromatography, and HPLC is still required. The pure
compounds are then used for the determination of structure and antimicrobial
activity.
Currently, several methods have been applied to measure the in vitro antimicro-
bial activity of lichens, such as disk diffusion, agar dilution, and broth microdilution
(Fig. 3.1).
The disk diffusion method allows for the simultaneous testing of a large number
of antimicrobials in a relatively easy and flexible manner. In this method, the
bacterial inoculum is adjusted to certain concentration, inoculated onto the entire
surface of a Mueller-Hinton agar (MHA) plate with a sterile cotton-tipped swab to
form an even lawn. The paper disks impregnated with diluted antibiotic solution
were placed on the surface of each MHA plate using a sterile pair of forceps. Then
the plates were incubated aerobically and the diameter of zone inhibition was
measured by a ruler or caliper. Based on the diameter of the inhibition zone, the
results are then assigned to three categories, susceptible, intermediate, or resistant.
The bigger the diameter of the inhibition zone, the more susceptible is the micro-
organism to the antimicrobial.
Agar dilution is a quantitative susceptibility testing method because MIC values
can be obtained using the method. In this method, twofold serial dilutions of an
antibiotic made in MHA medium and then bacterial suspensions were inoculated on
the MHA using a Cathra replicator with 1 mm pins. It has been studied extensively
as a method for the bacteria growing aerobically. The advantages of agar dilution
include the ability to simultaneously test the susceptibility of a number of bacteria
in one plate and the ability to test susceptibility of fastidious organisms since the
agar with supplements is able to adequately support the bacterial growth. However,
3 Lichen Secondary Metabolites as Potential Antibiotic Agents 87

Fig. 3.1 Disk diffusion (a), agar dilution (b), and broth microdilution (c) methods to measure the
in vitro antimicrobial activity of test samples

agar dilution is not commonly used in most microbiology laboratories because it is

time-consuming and labor-intensive.
Broth microdilution is another quantitative reference method routinely used in
clinical laboratories. In this method, susceptibility panel in 96-well microtiter plates
was containing various concentrations of antimicrobial agents. Then, standardized
numbers of bacteria were inoculated into the wells of 96-well microtiter and
incubated overnight at 35
C. This method is often used to obtain minimal inhi-
bitory concentration (MIC) values. The MIC value was observed as the lowest
concentration where no viability was observed in the wells of 96-microwell plates
after incubation. It is a widely utilized method, allowing for the simultaneous
testing of multiple antimicrobials with ease particularly when commercially pre-
pared microtiter trays are used. Compared with agar-based method, broth
microdilution can decrease much labor and time. However, limitations of the
method primarily are associated with the lack of or poor growth of many anaerobic
microorganisms. Testing some fastidious anaerobes gives inconsistent and
88 M. Kosanić and B. Ranković

unreliable results because of poor growth of strains due to excessive exposure to

oxygen during the setup procedure (CLSI 2009).
Among the three methods, disk diffusion seems to be the most popular method
used to examine the antimicrobial activity of natural antimicrobials including
lichens. Although the method is relatively inexpensive and easy to perform, there
are several disadvantages. Since disk diffusion measures the inhibition zone size
which is then converted to categories of susceptible/intermediate/resistant, this
method is unable to obtain MIC values (Dickert et al. 1981). Also, it has been
reported (Klancnik et al. 2010) that this method is not always reliable for deter-
mining the antimicrobial activity of natural antimicrobials, i.e., lichen extract,
because the polarity of the natural compounds can affect the diffusion of com-
pounds onto the culture medium. Compounds with less polarity diffused slower
than more polar ones (Moreno et al. 2006). Due to these concerns, disk diffusion
may not be a suitable one to determine the antimicrobial activity of natural
compounds. Besides, similar to other agar-based methods, disk diffusion is labor-
intensive and time-consuming (Klancnik et al. 2010).
In contrast, agar dilution and broth microdilution methods are able to overcome
some of the limitations of the disk diffusion method. Not only are they more
convenient for routine antimicrobial susceptibility testing of bacteria in clinical
laboratories, they are capable of drawing quantitative conclusions by determining
the MIC values for antimicrobials, as opposed to qualitative data generated by the
disk diffusion method (Kim and Kim 2007).

3.4 Lichen Extracts as Potential Antibacterial

and Antifungal Agents

The screening of lichen extracts has been of great interest to scientists for the
discovery of new compound effective in the treatment of microbial infection. There
are various reports on the antimicrobial activity of crude lichen extracts.
The first study on the antibiotic properties of lichens was carried out by
Burkholder et al. (1944). He tested 42 lichens for antibiotic property and 27 were
reported to inhibit growth of bacteria. A number of lichen extracts were screened
for antibacterial activity in the 1950s, and in many cases activity was confirmed
against mycobacteria and Gram-positive organisms (Stoll et al. 1950). A review of
the work performed during this period is presented in Vartia (1973). More recent
reports include a study describing the antimicrobial screening of lichen extracts and
subsequent isolation of compounds with a broad spectrum of activity against
filamentous fungi, yeast, as well as Gram-positive and Gram-negative bacteria.
Extracts of Andean lichens Protousnea poeppigii and Usnea florida demon-
strated antimicrobial activity against the pathogenic fungi Microsporum gypseum,
Trichophyton mentagrophytes, and T. rubrum. Gram-positive Staphylococcus
aureus and Bacillus subtilis were sensitive to methanolic extracts of four different
3 Lichen Secondary Metabolites as Potential Antibiotic Agents 89

Antarctic lichen species (Paudel et al. 2008). Ranković et al. (2007a, b) tested
aqueous, acetone, and methanol extracts of Cladonia furcata, Parmelia caperata,
Parmelia pertusa, Hypogymnia physodes, Umbilicaria polyphylla, Lasallia
pustulata, Parmelia sulcata, Umbilicaria crustulosa, and Umbilicaria cylindrica
from Serbia on six species of bacteria and ten species of fungi. The strongest
activity was observed with methanol extracts of Parmelia pertusa and Parmelia
sulcata, and the weakest activity was manifested by Parmelia caperata and
Umbilicaria cylindrica. Aqueous extracts of all tested lichen species were inactive.
Bacillus mycoides was the most sensitive bacterial species tested, whereas Candida
albicans was the most sensitive fungal species examined. Other studies monitored
Ramalina farinacea and 69 species of lichens from New Zealand and showed their
inhibitory effect against a lot of bacteria such as Bacillus, Pseudomonas, E. coli,
Streptococcus, Staphylococcus, Enterococcus, and Mycobacterium (Esimone and
Adikwn 1999; Perry et al. 1999). Behera et al. (2005) reported that acetone,
methanol, and light petroleum extracts of lichen Usnea ghattensis were effective
against Bacillus licheniformis, B. megaterium, B. subtilis, and S. aureus. Also,
Karagoz et al. (2009) evaluated aqueous and ethanol extracts of 11 different species
from Turkey and determined potent antibacterial activity of aqueous extract of
Peltigera polydactyla and ethanol extract of Ramalina farinacea. Recently,
Mitrović et al. (2011) studied antibacterial and antifungal activity of methanol
extracts of five lichen species (Parmelia sulcata, Flavoparmelia caperata, Evernia
prunastri, Hypogymnia physodes, and Cladonia foliacea). The analysis of their
antibacterial potential was performed on 15 strains of bacteria and revealed the
strongest inhibitory effect, especially on Gram-positive bacteria, of Hypogymnia
physodes and Cladonia foliacea. In the case of fungi, Evernia prunastri exerted the
best effect on yeasts, while Hypogymnia physodes were better on filamentous fungi.
Similarly, acetone extracts of the lichens Cladonia furcata, Lecanora atra, and
Lecanora muralis were studied for their antimicrobial potential (Ranković
et al. 2011). The antimicrobial activity was estimated by determination of the
minimal inhibitory concentration by the broth microdilution method against six
species of bacteria and ten species of fungi. The extract of Cladonia furcata was the
most active antimicrobial agent with minimum inhibitory concentration values
ranging from 0.78 to 25 mg/ml, while the lowest activity was shown by Lecanora
muralis. In similar research, antifungal activity of hexane, ethyl acetate, and
methanol extracts of Parmelia reticulata was evaluated against soilborne patho-
genic fungi, namely, Sclerotium rolfsii, Rhizoctonia solani, R. bataticola, Fusarium
udum, Pythium aphanidermatum, and P. debaryanum by Goel et al. (2011). Maxi-
mum antifungal activity was exhibited by hexane and ethyl acetate extracts against
most of the test pathogens.
Acetone, diethyl ether, and ethanol extracts of the lichen Cetraria aculeata for
their antimicrobial activity have been evaluated. The extracts were found active
against Escherichia coli, Staphylococcus aureus, Aeromonas hydrophila, Proteus
vulgaris, Streptococcus faecalis, Bacillus cereus, Bacillus subtilis, Pseudomonas
aeruginosa, and Listeria monocytogenes. However, no antimicrobial activity
against the fungi was detected (Türk et al. 2003). The lichen extract almost
90 M. Kosanić and B. Ranković

increased by twofold in the presence of the stock solution of the colloidal silver
concentrate. The ointment containing the extract of lichen Ramalina farinacea
exhibited antimicrobial activities against Escherichia coli, Salmonella typhi, Asper-
gillus niger, and Candida albicans (Ofokansi and Esimone 2005).
The aqueous and ethanol extracts prepared from some lichens species were
evaluated for antibacterial activity against six standard strains (Escherichia coli,
Pseudomonas aeruginosa, Bacillus subtilis, Klebsiella pneumoniae, Staphylo-
coccus aureus, and Staphylococcus epidermidis) and (Aeromonas) that were iso-
lated from different lakes. The aqueous and ethanol extracts showed a variable
range of antibacterial activity to both standard strains and environmental strains.
Similarly the aqueous extract of Peltigera polydactyla and the ethanol extract of the
Ramalina farinacea exhibited potent antibacterial activities (Karagoz et al. 2009).
The antimicrobial activity of the acetone, diethyl ether, and ethanol extracts of
the lichen Cetraria aculeata tested against different pathogenic bacteria and fungi
showed only with bacteria but not with fungi. In a related study, Roccella
belangeriana were extracted from different solvents like acetone, methanol, diethyl
ether, ethanol, ethyl acetate, petroleum ether, chloroform, and aqueous extracts and
tested against 12 bacterial strains. A maximum antibacterial activity was observed
from chloroform extracts against Enterococci sp., and minimum activity was
observed from ethyl acetate extract against Klebsiella pneumoniae, Enterococci
sp., Salmonella sp., and Shewanella sp. (Karthikaidevi et al. 2009). In addition,
antibacterial and antifungal activity of the acetone, methanol, and aqueous extracts
of the lichens Lecanora frustulosa and Parmeliopsis hyperopta have been screened
in vitro against the Bacillus mycoides, Bacillus subtilis, Staphylococcus aureus,
Enterobacter cloacae, Escherichia coli, Klebsiella pneumoniae, Aspergillus flavus,
Aspergillus fumigatus, Botrytis cinerea, Candida albicans, Fusarium oxysporum,
Mucor mucedo, Paecilomyces variotii, Penicillium purpurascens, Penicillium
verrucosum, and Trichoderma harzianum. Tested lichen species also showed strong
activity against both bacteria and fungi (Kosanić et al. 2010). This would perhaps
indicate that the lichens would be used in the treatment of various diseases.
According to Schmeda-Hirschmann et al. (2008), dichloromethane and metha-
nol extracts of Protousnea poeppigii had strong antifungal effects against the fungal
pathogens Microsporum gypseum, Trichophyton mentagrophytes, and T. rubrum.
The extracts were also active against the yeasts Candida albicans, C. tropicalis, and
Saccharomyces cerevisiae and the filamentous fungi Aspergillus niger, A. flavus,
and A. fumigatus but with much higher strength. In the same assay, extracts of
Usnea florida also showed strong antifungal properties. Methanol extracts of five
lichens from Antarctica (Caloplaca regalis, Caloplaca sp., Lecanora sp., Ramalina
terebrata, Stereocaulon alpinum) exhibited target-specific antibacterial activity,
especially strong against Gram-positive bacteria, compared to previously described
lichens (Paudel et al. 2008). Whiton and Lawrey (1982) reported that ascospore
germination of Sordaria fimicola was significantly inhibited by evernic and
vulpinic acids. Aqueous, ethanol, and ethyl acetate extracts of Alectoria sarmentosa
and Cladonia rangiferina were found to have moderate antifungal action against
different species of fungi, including human pathogens (Ranković and Mišić 2007),
3 Lichen Secondary Metabolites as Potential Antibiotic Agents 91

ethanol extracts showing the highest activity. Halama and Van Haluwin (2004)
reported that acetone extracts of Evernia prunastri and Hypogymnia physodes
showed a strong inhibitory effect on the growth of some plant pathogenic fungi,
i.e., Phytophthora infestans, Pythium ultimum, and Ustilago maydis.
Antimicrobial features of acetone, methanol, and aqueous extracts of lichens of
Cladonia furcata, Parmelia caperata, Parmelia pertusa, Hypogymnia physodes,
and Umbilicaria polyphylla were investigated by Ranković et al. (2009) by two
different methods at the same time. Testing of antimicrobial activities of extracts
from five species of lichens was performed by disk diffusion test in relation to
Gram-positive and Gram-negative bacteria and fungal organisms and through
determination of minimal inhibitory concentration (MIC) by broth tube dilution
method. They found that acetone and methanol extracts of all investigated lichens
in different concentrations manifested selective antibacterial and antifungal activ-
ity. That activity was more evident in relation to Gram-positive than Gram-negative
bacteria and fungal organisms. Acetone and methanol extracts of lichens Parmelia
pertusa, Hypogymnia physodes, and Umbilicaria polyphylla inhibited the growth of
all tested microorganisms, most of all of lichens Cladonia furcata and Parmelia
caperata. Although the methanol extracts were generally the most active against
the test organisms, the lowest MIC value was measured for acetone extract of
species Cladonia furcata 0.39 mg/ml in relation to bacterium Bacillus subtilis.
Aqueous extracts of investigated lichens were inactive against all tested organisms.
Santiago et al. (2010) examined antibacterial activity of 63 lichens collected
from different sites in Luzon Island, Philippines. Lichen crude extracts were then
tested against Gram-positive bacteria (Bacillus subtilis and Staphylococcus aureus)
and Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa) using
the paper disk diffusion assay. Their results showed that all 45 tested extracts
inhibited at least one of the test bacteria. However, only 38 extracts were found
to be very active (>19 mm zone of inhibition) against Gram-positive bacteria.
Similarly, Santos et al. (1964) observed that 30 of the 38 lichen extracts tested
inhibited at least one of the 12 test microorganisms, particularly the Gram-positive
test bacteria. Other studies also confirmed several lichens to be active against
Gram-positive microorganisms. For example, Saenz et al. (2006) found that four
species of lichens, namely, Ramalina canariensis, R. subfarinacea, Cladonia firma,
and Lecanora muralis, were most active against Gram-positive bacteria.
In vitro antifungal activity of acetone, methanol, and chloroform extracts of
Parmotrema tinctorum was investigated against ten plant pathogenic fungi, viz.,
Aspergillus niger, A. flavus, A. fumigatus, Alternaria alternata, Fusarium
oxysporum, F. solani, F. roseum, Ustilago sp., Albugo candida, and Penicillium
citrinum, with reference to commercially available synthetic antifungal drug keto-
conazole (positive control) using disk diffusion assay (Tiwari et al. 2011). Methanol
extract was most effective against all investigated fungi followed by acetone and
chloroform extract. Principal component analysis (PCA) concluded that though
ketoconazole was effective against five of the investigated fungi, the extracts of
Parmotrema tinctorum were more effective against the rest of the five broad-
spectrum plant pathogenic fungi (A. fumigatus, F. solani, F. roseum, P. citrinum,
and Ustilago spp.).
92 M. Kosanić and B. Ranković

In a study described by Karthikaidevi et al. (2011), antimicrobial activity of the

mangrove lichen Roccella belangeriana collected from the Gulf of Mannar Bio-
sphere Reserve area was tested. The lichen was extracted in different solvents,
acetone, methanol, diethyl ether, ethanol, ethyl acetate, petroleum ether, chloro-
form, and water and tested against 14 bacterial strains and trifungal strains by well
diffusion assay. Regarding antibacterial activity, the maximum zone of inhibition
was recorded in methanol extracts against Vibrio cholerae, and the minimum zone
of inhibition was in ethyl acetate extract against Klebsiella pneumoniae, Entero-
cocci sp., Salmonella sp., and Shewanella sp. Regarding the antifungal activity, the
maximum zone of inhibition was recorded against Aspergillus niger, and the
minimum was noted against Rhizopus sp.
Five common lichens (Cladonia sp., Everniastrum sp., Parmelia sp.,
Stereocaulon sp., and Usnea sp.) of Darjeeling hills were extracted from different
solvents like ethanol, methanol, petroleum ether, chloroform, and aqueous extracts
and tested against four Gram-positive and four Gram-negative bacterial strains.
Ethanol extracts exerted stronger inhibitory action followed by methanol extracts.
Aqueous extracts manifested less activity to the tested microorganisms (Sharma
et al. 2012).
Srivastava et al. (2013) investigated antimicrobial activity of the acetone, metha-
nol, and ethanol extracts of some common lichen species such as Usnea longissima,
Everniastrum cirrhatum, Peltigera polydactylon, and Sulcaria sulcata which were
screened in vitro against six clinically important pathogenic bacteria, Staphylo-
coccus aureus, Streptococcus faecalis, Bacillus cereus, Pseudomonas aeruginosa,
Salmonella typhimurium, and Escherichia coli by the Kirby-Bauer technique of
disk diffusion method. Minimum inhibitory concentration was taken out by broth
microdilution method according to the NCCLS guidelines. It was found that
acetone, methanol, and ethanol extracts of the investigated lichens showed rela-
tively strong antimicrobial activity against all the Gram-positive bacteria and two
Gram-negative bacteria. The lowest MIC value was observed to be as low as
6.25 μg/ml against B. cereus and U. longissima.
Antibiotic properties of acetone and methanol extracts from 34 North American
lichens were screened against four pathogenic bacteria by Shrestha et al. (2014).
The microwell dilution method was used to determine the minimum inhibitory
concentration. Most of the lichen extracts demonstrated inhibitory effects against
Staphylococcus aureus, Pseudomonas aeruginosa, and methicillin-resistant
S. aureus with MIC values ranging from 3.9 to 500 μg/ml. In addition, extracts
from three species, Letharia columbiana, Letharia vulpina, and Vulpicida
canadensis (MIC ¼ 125–500 μg/ml), were also effective against Escherichia coli.
Generally, acetone extractions were found to be more effective than methanol
extractions.
In the study described by Vivek et al. (2014), antibacterial potential of three
Parmotrema species, viz., P. tinctorum, P. grayanum, and P. praesorediosum from
India, was tested against three Gram-positive and five Gram-negative bacteria by
agar well diffusion assay. The lichen extracts showed dose-dependent antibacterial
activity. Overall, the lichen extracts were more inhibitory to Gram-positive bacteria
than Gram-negative bacteria. P. grayanum displayed high inhibitory activity
3 Lichen Secondary Metabolites as Potential Antibiotic Agents 93

against test bacteria. Balaji and Hariharan (2007) reported marked antimicrobial
efficacy of dichloromethane extract of P. praesorediosum. Kumar et al. (2010)
showed the antibacterial activity of methanol extract of P. pseudotinctorum. Sinha
and Biswas (2011) reported the antibacterial efficacy of solvent extracts of
P. reticulatum from Sikkim, India. Verma et al. (2011) found antibacterial efficacy
of solvent extracts of P. nilgherrensis and P. sanctiangelii collected from Karna-
taka, India. Chauhan and Abraham (2013) showed the inhibitory effect of methanol
extract of Parmotrema sp. against clinical isolates of bacteria. In addition, Javeria
et al. (2013) showed the inhibitory efficacy of solvent extracts of P. nilgherrense
against drug-resistant bacteria.
In the study described by Ranković et al. (2012), acetone lichen extracts
obtained from Usnea barbata showed a moderate antibacterial and antifungal
activity. It inhibited the microorganisms tested at concentrations from 0.125 to
12.5 mg/ml. The acetone extract from T. candida inhibited all the tested micro-
organisms, but at higher concentrations. In related research, Evernia prunastri and
Pseudoevernia furfuraceae lichens were screened for their antimicrobial effects by
Kosanić et al. (2013) who found varying antimicrobial success in the inhibition of
Gram-positive and Gram-negative bacteria and fungi, and Pseudoevernia
furfuraceae was found to be the most effective.
Kosanić et al. (2014a, b) extracted with acetone the three Cladonia species
(C. furcata, C. rangiferina, and C. pyxidata) in order to investigate their antimicrobial
effect. As test organisms in this study, Bacillus mycoides, B. subtilis, Staphylococcus
aureus, Escherichia coli, Klebsiella pneumonia, Aspergillus flavus, A. fumigatus,
Candida albicans, Penicillium purpurascens, and P. verrucosum were used. They
obtained results showing that extracts from C. furcata and C. rangiferina showed
similar antibacterial and antifungal activity. They inhibited the microorganisms
tested at concentrations from 0.78 to 25 mg/ml, while extracts from C. pyxidata
inhibited all the tested microorganisms, but at higher concentrations.
Lecanora muralis, Parmelia saxatilis, Parmeliopsis ambigua, Umbilicaria
crustulosa, and Umbilicaria polyphylla were tested for their antibacterial and
antifungal activity (Kosanić et al. 2014a, b). The antimicrobial activity was esti-
mated by determination of the minimal inhibitory concentration by the broth
microdilution method against six species of bacteria and ten species of fungi, and
it has been found that of the lichens tested, Umbilicaria polyphylla had the largest
antimicrobial activity with minimum inhibitory concentration values ranging from
0.78 to 1.56 mg/ml.

3.5 Studies on Antimicrobial Activities of Lichen

Secondary Metabolites

Such as the abovementioned, there are many studies on the antimicrobial activity of
crude lichen extracts. However, studies on antimicrobial activity of lichen com-
pounds are scarce and scattered. Lichens have been found to contain a variety of
secondary lichen substances with strong antimicrobial activity (Table 3.1).
94 M. Kosanić and B. Ranković

Table 3.1 List of lichen secondary metabolites used to evaluate antimicrobial activity
Lichen compounds References
Lecanoric acid Gomes et al. (2003), Ranković and Mišić (2008), Honda et al. (2010)
Atranorin Kumar and Müller (1999), Yilmaz et al. (2004), Turk et al. (2006),
Ranković et al. (2008, 2014), Kosanić et al. (2014a, b)
Zeorin Kosanić et al. (2010)
Gyrophoric acid Candan et al. (2006), Ranković et al. (2008)
Stenosporic acid Candan et al. (2006)
Protocetraric acid Tay et al. (2004), Ranković and Mišić (2008), Manojlović et al. (2012)
Fumarprotocetraric Yilmaz et al. (2004), Ranković and Mišić (2008), Kosanić et al. (2014a,
acid b)
Stictic acid Ranković and Mišić (2008)
Salazinic acid Candan et al. (2007), Manojlović et al. (2012)
Usnic acid Lauterwein et al. (1995), Perry et al. (1999), Yilmaz et al. (2003),
Ivanova et al. (2004), Tay et al. (2004), Ranković et al. (2008, 2012,
2014), Schmeda-Hirschmann et al. (2008), Ranković and Mišić (2009),
Paudel et al. (2010), Ramos and Silva (2010)
Vulpinic acid Whiton and Lawrey (1982), Lawrey (1986), Lauterwein et al. (1995)
Evernic acid Whiton and Lawrey (1982), Lawrey (1986), Halama and Van Haluwin
(2004), Kosanić et al. (2013)
Lobaric acid Ingolfsdottir et al. (1998), Piovano et al. (2002), Sundset et al. (2008)
Physodic acid Turk et al. (2006), Ranković et al. (2008, 2014), Kosanić et al. (2013)
Protolichesterinic Ingolfsdottir et al. (1998), Türk et al. (2003)
acid
Norstictic acid Tay et al. (2004), Honda et al. (2010), Ranković et al. (2014)
Ramalin Paudel et al. (2008, 2010)
Barbatic acid Martins et al. (2010)
Divaricatic acid Piovano et al. (2002), Kosanić et al. (2010)
Diffractaic acids Piovano et al. (2002), Honda et al. (2010)
Umbilicaric acid Buçukoglu et al. (2013)
Homosekikaic acid Sisodia et al. (2013)
Sekikaic acid Sisodia et al. (2013)
Parietin Manojlović et al. (2002, 2005)
Parietinic acid Manojlović et al. (2002)
Emodin Manojlović et al. (2002)
Fallacinal Manojlović et al. (2002)
Fallacinol Manojlović et al. (2002)
Isodivaricatic acid Schmeda-Hirschmann et al. (2008)
Divaricatinic acid Schmeda-Hirschmann et al. (2008)
Hirtusneanoside Renzaka and Sigler (2007)
Neuropogonines A, Ivanova et al. (2002)
B, and C
Hypostictic acid Honda et al. (2010)
Norstictic acid Honda et al. (2010)
Secalonic acid Honda et al. (2010)
Psoromic acid Tasdemir and Franzblau (2007)
Vulpic acid Tasdemir and Franzblau (2007)
Usimines A, B, and C Paudel et al. (2010)
3 Lichen Secondary Metabolites as Potential Antibiotic Agents 95

Literature sources mentioning data for numerous lichen substances with examined
antimicrobial activity are mentioned below.
Atranorin (from Physcia aipolia), fumarprotocetraric acid (from Cladonia
furcata), gyrophoric acid (from Umbilicaria polyphylla), lecanoric acid (from
Ochrolechia androgyna), physodic acid (from Hypogymnia physodes), proto-
cetraric acid (from Flavoparmelia caperata), stictic acid (from Parmelia
conspersa), and usnic acid (from Flavoparmelia caperata) showed relatively strong
antimicrobial effects against six bacteria and ten fungi, among which were human,
animal, and plant pathogens, mycotoxin producers, and food-spoilage organisms
(Ranković and Mišić 2008; Ranković et al. 2008). Usnic acid was found to be the
strongest antimicrobial agent (comparable to streptomycin) and physodic and
stictic acids the weakest.
The antifungal activity of ten depsidones and five depsides was evaluated, as
well as the antibacterial of these compounds and three additional depsides and one
diarylether; all of them were isolated from lichens growing in Chile (Piovano
et al. 2002). Obtained results showed, in general, negative activity against yeast
and filamentous fungi at concentrations of 250 mg/ml. Nevertheless, divaricatic and
diffractaic acids, and to a lesser degree lobaric acid, presented a moderate but
significant activity against Microsporum gypseum, Trichophyton mentagrophytes,
T. rubrum, and Epidermophyton floccosum, all of them being dermatophyte fungi
which cause skin infections. Regarding antibacterial activity, results indicated that
against Gram-negative bacteria, the 19 compounds are inactive. In contrast against
Gram-positive bacteria, a marked action can be observed for seven compounds.
Anthraquinones (parietin, parietinic acid, emodin, fallacinal, and fallacinol)
from Caloplaca schaereri were tested for antimicrobial activity using Bacillus
subtilis, Staphylococcus aureus, Escherichia coli, Pseudomonas fluorescens, Can-
dida albicans, Trichoderma harzianum, Aspergillus niger, and Penicillium
verrucosum (Manojlović et al. 2002). All the anthraquinones tested showed potent
antibacterial activity against B. subtilis, S. aureus, and P. fluorescens (MIC 20–
320 μg/ml), but only parietinic acid showed any activity against E. coli (MIC
160 μg/ml). Their effects are generally most potent on B. subtilis and P. fluorescens.
Fallacinol was most potent against S. aureus. Fallacinol was the most active
(potent) of the isolated compounds against all the fungi tested but was particularly
active against T. harzianum, A. niger, and P. verrucosum (MIC 10–40 μg/ml).
Potent antifungal effects on the fungi tested also showed parietinic acid (MIC 20–
80 μg/ml), while parietin had MIC values of 80, 40, and 20 μg/ml for C. albicans,
P. verrucosum, A. niger, and T. harzianum, respectively. Emodin showed MIC
values of 20–40 μg/ml for A. niger, T. harzianum, and P. verrucosum but was much
less effective against C. albicans (MIC 80 μg/ml).
According to Schmeda-Hirschmann et al. (2008), isodivaricatic acid,
divaricatinic acid, and usnic acid, the main lichen metabolites in Protousnea
poeppigii, displayed antifungal action against Microsporum gypseum,
Trichophyton mentagrophytes, and T. rubrum, usnic acid being less active.
Divaricatic acid and zeorin constituents of Lecanora frustulosa and Parmeliopsis
hyperopta have been screened in vitro against the Bacillus mycoides, Bacillus
96 M. Kosanić and B. Ranković

subtilis, Staphylococcus aureus, Enterobacter cloacae, Escherichia coli, Klebsiella

pneumoniae, Aspergillus flavus, Aspergillus fumigatus, Botrytis cinerea, Candida
albicans, Fusarium oxysporum, Mucor mucedo, Paecilomyces variotii, Penicillium
purpurascens, Penicillium verrucosum, and Trichoderma harzianum. Divaricatic
acid and zeorin showed strong activity against both bacteria and fungi (Kosanić
et al. 2010). Bacillus subtilis, B. cereus, Staphylococcus aureus, Streptococcus
faecalis, Proteus vulgaris, Listeria monocytogenes, Aeromonas hydrophila, Can-
dida albicans, and Candida glabrata growth were inhibited by usnic acid,
atranorin, and fumarprotocetraric acid constituents of Cladonia foliacea (Yilmaz
et al. 2004). Similarly, anti-Gram-positive activities have been reported for evernic
acid, vulpinic acid, and hirtusneanoside (Renzaka and Sigler 2007).
Antimicrobial activity of salazinic acid isolated from Parmelia sulcata has been
screened against 28 foodborne bacteria and fungi, and it has been found that these
compounds showed activity against Pseudomonas aeruginosa and Salmonella
typhimurium as well (Candan et al. 2007). Antimicrobial activity of fumarproto-
cetraric acid, lecanoric acid, protocetraric acid, and stictic acid isolated from the
lichen of Cladonia furcata, Ochrolechia androgyna, Parmelia caperata, and
Parmelia conspersa was studied in relation to Bacillus mycoides, Bacillus subtilis,
Staphylococcus aureus, Enterobacter cloacae, Escherichia coli, Klebsiella
pneumoniae, Aspergillus flavus, Aspergillus fumigatus, Botrytis cinerea, Candida
albicans, Fusarium oxysporum, Mucor mucedo, Paecilomyces variotii, Penicillium
purpurascens, Penicillium verrucosum, and Trichoderma harzianum (Ranković
and Mišić 2008). The antimicrobial activity was estimated by determining the
minimal inhibited concentration by the broth tube dilution method. The researched
lichen components inhibited the growth of all the tested microorganisms. The
lowest MCI value (0.031 mg/ml) was measured for the fumarprotocetraric acid
related to the Klebsiella pneumoniae species. The weakest antimicrobial activity
was found in stictic acid, which inhibited most of the tested microorganisms at
significantly higher concentrations.
Whiton and Lawrey (1982) reported that ascospore germination of Sordaria
fimicola was significantly inhibited by evernic and vulpinic acids. Usnic acid,
evernic acid, and vulpinic acid inhibited the growth of the Gram-positive bacteria
Staphylococcus aureus, Bacillus subtilis, and Bacillus megaterium, but had no
effect on the Gram-negative bacteria Escherichia coli or Pseudomonas aeruginosa
(Lawrey 1986). Three new depsidones (neuropogonines A, B, and C) isolated from
a Neuropogon showed a moderate activity on a Mycobacterium vaccae strain
(Ivanova et al. 2002). Two years later, the same authors from this lichen isolated
usnic acid and further investigated for antimicrobial activity against pathogenic
Gram-positive and Gram-negative bacteria as Streptococcus pyogenes, Staphylo-
coccus epidermidis, Bacillus cereus, Listeria monocytogenes, Yersinia
enterocolitica, Klebsiella pneumoniae, and Salmonella typhimurium (Ivanova
et al. 2004).
Manojlovic et al. (2005) reported antifungal activity of the anthraquinone
parietin isolated from Caloplaca cerina. A potent fungitoxic compound, lecanoric
acid, was isolated from Parmotrema tinctorum lichen and tested against the fungus
3 Lichen Secondary Metabolites as Potential Antibiotic Agents 97

Cladosporium sphaerospermum (Gomes et al. 2002). In addition, MIC values

obtained from monoaromatic phenols (methyl beta-orsellinate and methyl and
ethyl orsellinates) derived from various Icelandic lichen species were found equal
or higher than usual preservatives (methyl- and propyl-p-hydroxybenzoates, o-
cresol) (Ingolfsd
ottir et al. 1985).
The antimicrobial activity of gyrophoric acid and stenosporic acid constituents
of the Xanthoparmelia pokornyi lichen has been screened against some foodborne
bacteria and fungi. Both acids showed antimicrobial activity against Aeromonas
hydrophila, Bacillus cereus, Bacillus subtilis, Listeria monocytogenes, Proteus
vulgaris, Staphylococcus aureus, Streptococcus faecalis, Yersinia enterocolitica,
Candida albicans, and Candida glabrata while were inactive against the tested
filamentous fungi (Candan et al. 2006).
Tasdemir and Franzblau (2007) investigated four commercially available lichen
metabolites, (+)-usnic acid, evernic acid, psoromic acid, and vulpic acid, for their
in vitro antitubercular effects. H37Rv strain of Mycobacterium tuberculosis and the
well-established microplate alamar blue assay were used for the determination of
the MIC values. (+)-Usnic acid proved to be the most potent antituberculotic agent
(MIC ¼ 5.2 μg/ml), followed by psoromic acid (MIC ¼ 44 μg/ml) and vulpic acid
(MIC ¼ 140 μg/ml). Evernic acid was found to be inactive at highest concentrations
tested (MIC > 200 μg/ml). Honda et al. (2010) described the extraction and identifi-
cation of several classes of phenolic compounds from the lichens Parmotrema
dilatatum, Parmotrema tinctorum, Pseudoparmelia sphaerospora, and Usnea
subcavata and determined their antitubercular activity. The depsides (atranorin,
diffractaic, and lecanoric acids), depsidones (protocetraric, salazinic, hypostictic,
and norstictic acids), xanthones (lichexanthone and secalonic acid), and usnic acid,
as well as seven orsellinic acid esters, five salazinic acid 80 ,90 -O-alkyl derivatives,
and four lichexanthone derivatives, were evaluated for their activity against Myco-
bacterium tuberculosis. Diffractaic acid was the most active compound (MIC value
15.6 mg/ml, 41.6 mM), followed by norstictic acid (MIC value 62.5 mg/ml,
168 mM) and usnic acid (MIC value 62.5 mg/ml, 182 mM). Hypostictic acid
(MIC value 94.0 mg/ml, 251 mM) and protocetraric acid (MIC value 125 mg/ml,
334 mM) showed moderate inhibitory activity. The other compounds showed lower
inhibitory activity on the growth of M. tuberculosis, varying from MIC values of
250–1,370 mM.
Cladonia verticillaris lichen lectin was evaluated by Ramos et al. (2014) for its
antimicrobial potential, and it showed activity against Gram-positive (Bacillus
subtilis, Staphylococcus aureus, and Enterococcus faecalis) and Gram-negative
(Escherichia coli and Klebsiella pneumoniae) assayed strains, with greater inhi-
bitory effect on the growth of E. coli (MIC of 7.18 μg/ml). The lowest minimum
bactericidal concentration (MBC, 57.4 μg/ml) was detected against E. faecalis. The
antifungal assay performed with Trichophyton mentagrophytes, Microsporum
gypseum, Trichophyton rubrum, Trichosporon cutaneum, and Trichosporon asahii.
Cladonia verticillaris lichen lectin was the most active against T. rubrum with an
inhibition percentage of 35 % compared to negative control.
98 M. Kosanić and B. Ranković

A total of five compounds, usnic acid, usimine A, usimine B, usimine C, and

ramalin, were isolated by bioactivity-guided fractionation of the methanol extract
of Ramalina terebrata (Paudel et al. 2010). The qualitative antibacterial activities
of the isolated compounds were determined by the disk diffusion method, while the
minimum inhibitory concentration (MIC) determination assay gave the quantitative
strength of the test samples. All the test samples showed antibacterial activity
against Bacillus subtilis, and usnic acid showed antibacterial activity against
Staphylococcus aureus. The MIC values of the isolated compounds against
B. subtilis were in the range of 1–26 μg/ml.
Manojlović et al. (2012) investigated antibacterial and antifungal activities for
protocetraric acid from Parmelia caperata lichen and depsidone salazinic acid from
Parmelia saxatilis species. Antioxidant activities of these isolated metabolites were
evaluated against Bacillus mycoides, Bacillus subtilis, Staphylococcus aureus,
Escherichia coli, Klebsiella pneumoniae, Aspergillus flavus, Aspergillus fumigatus,
Candida albicans, Penicillium purpurascens, and Penicillium verrucosum. As a
result of the study, salazinic acid and protocetraric acid showed similar antimicro-
bial activity, but antibacterial activity was stronger than antifungal activity for both
components.
In the study described by Ranković et al. (2012), lichen compounds norstictic
acid isolated from Toninia candida and usnic acid from Usnea barbata demon-
strated very strong antimicrobial activity. The MIC for different components
relative to the tested microorganisms ranged from 0.0008 to 1 mg/ml. The strongest
antimicrobial activity was found in usnic acid, which in extremely low amounts
inhibited all species of bacteria and fungi. Similarly, 2 years later, antimicrobial
activities of major lichen metabolites in Hypogymnia physodes lichen (physodic
acids, atranorin, and usnic acid) were studied by Ranković et al. (2014). They found
that usnic acid and physodic acid showed very strong and similar antimicrobial
activity, followed by atranorin. Antibacterial activity was stronger than antifungal
activity for all compounds.
Evernic acid and physodic acid from Evernia prunastri and Pseudoevernia
furfuraceae lichens were screened for their antimicrobial effects by the broth
microdilution method (Kosanić et al. 2013), and physodic acid was found to be
the most effective. One year later, in a related experiment, Kosanić et al. (2014a, b)
investigated antimicrobial activity for atranorin and fumarprotocetraric acid iso-
lated from Cladonia lichen. Antimicrobial activity was studied in relation to five
species of bacteria and five species of fungi. The isolated lichen components
demonstrated very strong antimicrobial activity. The MIC for different components
relative to the tested microorganisms ranged from 0.015 to 1 mg/ml. The strongest
antimicrobial activity was found in fumarprotocetraric acid, which in extremely
low amounts inhibited all the species of bacteria and fungi.
The results obtained in studies related to the antimicrobial activity of lichens
indicate differences in activity between extracts depending on the species of lichen
and as a function of the type of extracting solvent. These results are in agreement
with the suggestion of Oloke and Kolawole (1998) that bioactive components of
any medical plant have different solubility in different extracting solvents.
3 Lichen Secondary Metabolites as Potential Antibiotic Agents 99

Numerous researchers found lower antimicrobial activity of aqueous extracts in

comparison with acetone, methanol, or other organic solvent extracts (Land and
Lundstrom 1998; Madamombe and Afolajan 2003; Ranković et al. 2008; Kosanić
et al. 2010). The reason for the weak activity of aqueous extracts is that active
substances present in the thalli of lichens are insoluble or poorly soluble in water
(Kinoshita et al. 1994).
In general, in studies regarding antimicrobial activity of lichens, fungi and
Gram-negative bacteria were more resistant than Gram-positive ones. The differ-
ence of sensitivity between Gram-positive and Gram-negative bacteria and fungi
can be ascribed to morphological differences between these microorganisms, above
all to differences in permeability of the cell wall (Nostro et al. 2000). The cell walls
of Gram-positive bacteria are made of peptidoglycans and teichoic acids, while
those of Gram-negative bacteria are made of peptidoglycans, lipopolysaccharides,
and lipoproteins (Heijenoort 2001; Ranković et al. 2008). The cell walls of fungi are
poorly permeable and consist of polysaccharides such as hitchin and glucan (Farkaš
2003).
Since microorganisms have developed resistance to many antibiotics, pharma-
cologists need to pursue new sources for antimicrobial agents. All these results
suggest that lichens and their metabolites yield significant new bioactive substances
for the treatment of various diseases caused by microorganisms. New compounds
are to be described from poorly studied lichens, and even in species that are
considered chemically well known, new chemical strains are still detected (e.g.,
Stocker-Worgotter et al. 2004). Many lichens are known which contain structurally
unknown lichen products. Here we opened the entrance door of a vast and inter-
esting field of research.

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