Extraction, Characterization and Biological Activities of Selected Lichens Growing in Serbia

Abstract

This study presents a comparative analysis of secondary metabolites and antioxidant, antimicrobial, and anticancer activities of acetone extracts obtained from the lichens Lepraria incana and Pertusaria amara. HPLC-UV analysis identified divaric acid, divaricatinic acid, norstictic acid, divaricatic acid and usnic acid in L. incana, and conprotocetraric acid, protocetraric acid, picrolichenic acid and atranorin in P. amara. Free radical scavenging capacity and reducing power assays were employed to assess the antioxidant activity of the extracts. The IC₅₀ values in the free radical scavenging assay were 664.23 μg/mL for L. incana and 750.50 μg/mL for P. amara, while reducing power absorbances varied between 0.0875–0.2562 and 0.0336–0.2011, respectively. Total phenolic contents in L. incana and P. amara extracts were 40.81 and 33.67 μg PE/mg of extract, while total flavonoid contents were 24.74 and 23.61 μg RE/mg of extract, respectively. Antimicrobial activity, determined by the microdilution method, ranged from 156 to 20 × 10³ μg/mL for L. incana and from 312 to 20 × 10³ μg/mL for P. amara. Cytotoxicity was tested using the MTT method. Among the tested samples, the L. incana extract showed the strongest cytotoxic activity toward A549 cells, with an IC₅₀ value of 47.53 μg/mL. Based on the results, the lichens examined demonstrate promise for future studies and potential development in biopharmaceutical applications.

1. Introduction

Lichens are complex organisms consisting of a fungus (mycobiont) and an alga or cyanobacterium (photobiont) that form a symbiotic association [1]. As some of the first organisms to colonize terrestrial habitats, lichens are found worldwide, from Arctic tundra to tropical forests and from plains to mountain peaks. Their remarkable tolerance to extreme conditions, together with slow growth and long lifespans, allows them to synthesize numerous secondary metabolites that protect against both physical and biological challenges [2].

Lichen secondary metabolites, called lichen substances, have high therapeutic values. Numerous studies have demonstrated that lichens and their metabolites have multiple health benefits, ranging from anticancer, immunomodulatory, and anti-inflammatory activities to antidiabetic, antiviral, antioxidant, and antimicrobial effects, along with potential protection against neurodegenerative diseases and cardiovascular risks [3,4,5,6,7].

Given the demand for natural bioactive products that are effective yet safe, lichens represent an important focus of research due to their potential as sources of bioactive compounds with a wide range of biotechnological applications. Although Serbia is a region with a high diversity of lichens, the bioactive properties of many species remain insufficiently studied. For this reason, our research focused on the lichens Lepraria incana and Pertusaria amara growing in Serbia.

Lepraria incana, a dust lichen belonging to the family Stereocaulaceae, has a thallus that ranges in color from green to greyish-green and appears powdery, composed of tiny granules known as soredia. This species typically grows on the bark at the base of trees in moist, partially shaded environments, but it can also colonize dead wood, silica-rich rocks, or soil. It is not highly selective regarding bark type and has been observed on a wide variety of both deciduous and coniferous trees. L. incana demonstrates relative tolerance to air pollution, and several studies have explored its potential use as a biomonitor. P. amara is a common epiphytic lichen from the Pertusariaceae family. It can mostly be found on mesic bark, only rarely on the ground and rocks, marked by white punctiform soralia on a grey thallus. It is common on a wide range of broad-leaved trees, rarely conifers, and occasionally on the ground and low vegetation as well as on sheltered, humid, siliceous rock. Given the widespread occurrence of these two lichen species, understanding their bioactive properties is of considerable importance. Due to the limited and scarce information on their biological activities, this study focused on the chemical characterization of acetone extracts from L. incana and P. amara, as well as their antimicrobial, antioxidant, and anticancer potentials.

2. Materials and Methods

2.1. Sampling of Lichens and Preparation of Acetone Extracts

Samples of Lepraria incana (L.) Ach. and Pertusaria amara (Ach.) Nyl. were collected in Serbia. Species identification was carried out following standard monographs [8,9], and voucher specimens (Voucher Nos. 54 and 55) were deposited at the herbarium of the Department of Biology and Ecology, Faculty of Science, University of Kragujevac.

The collected lichens were air-dried at room temperature for two weeks and then ground into a fine powder. Approximately 100 g of dried thalli were extracted using acetone in a Soxhlet apparatus. The resulting extracts were filtered and concentrated under reduced pressure using a rotary evaporator. Dried extracts of L. incana and P. amara were obtained in amounts of 2.12 g (2.12% yield) and 2.97 g (2.97% yield), respectively.

Extracts were stored at −18 °C until further analysis. For the experimental assays, the extracts were dissolved in 5% dimethyl sulfoxide (DMSO).

2.2. HPLC Analysis

High-performance liquid chromatography (HPLC) with UV detection was used to analyze the secondary metabolites of the acetone extracts. This analysis was conducted using an Agilent 1200 Series (high-performance liquid chromatography system from Agilent Technologies, Santa Clara, CA, USA) equipped with an HPLC column (Zorbax Eclipse, XDB-C18; 25 cm × 4.6 mm; 5 μm). The detection of secondary metabolites was carried out using a diode array detector (DAD) at 280, 330, and 350 nm for compound analysis. UV spectra of compounds were recorded in the range of 200–400 nm. The samples, once dissolved, were filtered through a 0.45 μm pore size filter. The mobile phase consisted of an acetonitrile–water–phosphoric acid solvent system (82:18:0.9, v/v/v) with a flow rate of 1 mL/min, and 10 μL of each sample was injected into the system. The column was kept at a temperature of 30 °C. This procedure was carried out as previously described [10]. Chromatograms and UV spectral data were collected at a wavelength of 254 nm. The correct identification of the secondary metabolites in the acetone extracts of L. incana and P. amara was made by comparing the retention times (t_R) and UV spectra (λ = 200–400 nm) of the compounds with standards previously isolated in our laboratory whose structures had been confirmed by ¹H NMR, ¹³C NMR, and mass spectrometry and compared with those reported in the literature [11,12,13].

2.3. Antioxidative Activity

2.3.1. Free Radical Scavenging Assay

The free radical scavenging activity of the extracts was assessed using 1,1-diphenyl-2-picrylhydrazyl (DPPH) following the method of Dorman et al. [14], with slight modifications. For the assay, 2 mL of a methanolic DPPH solution (0.05 mg/mL) was mixed with 1 mL of the lichen extract at varying concentrations (1000, 500, 250, 125, and 62.5 μg/mL) in cuvettes. The mixtures were vigorously shaken and incubated at room temperature for 30 min. Absorbance was then measured at 517 nm using a spectrophotometer (Bibby Scientific Limited, Stone, UK). Ascorbic acid served as the positive control. The DPPH radical scavenging activity was calculated using the following equation:

$$\mathrm{D}\mathrm{P}\mathrm{P}\mathrm{H} \mathrm{s}\mathrm{c}\mathrm{a}\mathrm{v}\mathrm{e}\mathrm{n}\mathrm{g}\mathrm{i}\mathrm{n}\mathrm{g} \mathrm{a}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}\left(\%\right)={\displaystyle \frac{\left[{\mathrm{A}}_0-{\mathrm{A}}_1\right]}{{\mathrm{A}}_0}}\times 100$$

A₀ is the absorbance of the negative control and A1 represents the absorbance of the mixture or standard. The experiment was repeated three times, and the results are presented as the mean ± standard deviation. The half maximal inhibitory concentration (IC₅₀) was the parameter used to measure the radical scavenging activity. Lower values of IC₅₀ indicate better radical scavenging activity.

2.3.2. Reducing Power Assay

The reducing power of the extracts was determined following the method of Oyaizu [15]. Lichen extracts (1 mL) at concentrations of 1000, 500, and 250 μg/mL were combined with phosphate buffer (2.5 mL; pH 6.6; 0.2 M) and potassium ferricyanide (2.5 mL; 1%). The mixtures were then incubated in a water bath at 50 °C for 20 min. Following incubation, trichloroacetic acid (2.5 mL; 10%) was added, and the samples were centrifuged at 3000 rpm for 10 min (HermLe Labortechnik, Wehingen, Germany). The upper layer (2.5 mL) was collected and mixed with distilled water (2.5 mL) and iron(III) chloride (2.5 mL; 0.1%). Absorbance was measured at 700 nm using a spectrophotometer (Bibby Scientific Limited, Stone, UK). Ascorbic acid served as the positive control, while the negative control contained all reagents except the extract. All experiments were conducted in triplicate, and results are reported as mean ± standard deviation.

2.3.3. Total Phenolic Compounds

Total soluble phenolic compounds in the extracts were quantified using the Folin–Ciocalteu reagent according to the method of Slinkard and Singleton [16], with pyrocatechol employed as the standard. In brief, 1 mL of each extract (1 mg/mL) was placed in a volumetric flask and diluted with 46 mL of distilled water. One milliliter of Folin–Ciocalteu reagent was added, and the mixture was thoroughly mixed. After 3 min, 3 mL of 20% sodium carbonate was introduced, and the solution was allowed to stand for 2 h with intermittent shaking. Absorbance was measured at 760 nm using a spectrophotometer (Bibby Scientific Limited, Stone, UK). The total phenolic content was expressed as micrograms of pyrocatechol equivalent per milligram of dry extract (µg PE/mg), calculated from a standard pyrocatechol calibration curve using the following equation:

$$\begin{gathered} \mathrm{A}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}=0.0057\times \mathrm{t}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l} \mathrm{p}\mathrm{h}\mathrm{e}\mathrm{n}\mathrm{o}\mathrm{l}\mathrm{s}\left[\mathsf{\mu }\mathrm{g} {\displaystyle \frac{\mathrm{P}\mathrm{E}}{\mathrm{m}\mathrm{g}}} \mathrm{e}\mathrm{x}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{s}\right]-0.01646 \\ {(\mathrm{R}}^2=0.9203) \end{gathered}$$

2.3.4. Total Flavonoid Content

The total flavonoid content of the extracts was measured according to the method of Meda et al. [17]. In this assay, 2 mL of 2% aluminum trichloride in methanol was combined with an equal volume of the extract solution (1 mg/mL). The mixture was incubated at room temperature for 10 min, and absorbance was recorded at 415 nm using a spectrophotometer (Bibby Scientific Limited, Stone, UK) against blank samples. The total flavonoid content was expressed as micrograms of rutin equivalent per milligram of dry extract (µg RE/mg), calculated from a standard rutin calibration curve using the following equation:

$$\begin{gathered} \mathrm{A}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}=0.0296\times \mathrm{t}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l} \mathrm{f}\mathrm{l}\mathrm{a}\mathrm{v}\mathrm{o}\mathrm{n}\mathrm{o}\mathrm{i}\mathrm{d}\mathrm{s}\left[\mathsf{\mu }\mathrm{g} {\displaystyle \frac{\mathrm{R}\mathrm{E}}{\mathrm{m}\mathrm{g}}} \mathrm{e}\mathrm{x}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{s}\right]+0.0204 \\ {(\mathrm{R}}^2=0.9992) \end{gathered}$$

2.4. Antimicrobial Activity

Antimicrobial potential of the studied lichens was tested against several species of bacteria and fungi, belonging to the American Type Culture Collection (ATCC). The antimicrobial activity of the extracts was tested on five species of bacteria: Bacillus cereus (ATCC 11778), B. subtilis (ATCC 6633), Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922), Proteus mirabilis (ATCC 12453) and 10 species of fungi: Aspergillus flavus (ATCC 9170), A. niger (ATCC 16888), Mucor mucedo (ATCC 20094), Candida albicans (ATCC 10231), Trichoderma viride (ATCC 13233), Cladosporium cladosporioides (ATCC 11275), Alternaria alternata (ATCC 11680), Fusarium oxysporum (ATCC 62506), Penicillium expansum (ATCC 20466), and P. chrysogenum (ATCC 10106). Bacterial suspensions were made from bacterial cultures incubated at 37 °C for 24 h on Müller–Hinton agar medium. Their density was adjusted to match the 0.5 McFarland’s standard (bio-Mérieux, Marcy Marcy-l’Etoile, France), which is approximately 10⁸ CFU/mL. Fungal inoculi were obtained from fungal cultures (3- to 7-day-old) growing at 27 °C on potato dextrose agar medium. Spores were washed with sterile distilled water and then adjusted to contain approximately 10⁶ CFU/mL.

The microdilution method using 96-well micro-titer plates was used to determine the minimal inhibitory concentration (MIC) and, consequently, the antimicrobial activity of the studied lichens [18]. Initial stock solutions of the lichen extracts were prepared in 5% DMSO. Serial twofold dilutions were then performed to obtain concentrations ranging from 40 to 0.001 mg/mL in sterile microtiter plates containing Müller–Hinton broth for bacterial assays and Sabouraud dextrose broth for fungal assays. The diluted bacterial and fungal suspensions were subsequently added to the corresponding wells, along with resazurin as an indicator of bacterial growth. Plates were incubated at 37 °C for 24 h for bacteria and at 28 °C for 72 h for fungi. The MIC for bacteria was determined visually as the lowest extract concentration that prevented the resazurin color change from blue to pink, whereas for fungi, it was defined as the lowest concentration that visibly inhibited mycelial growth.

Streptomycin and ketoconazole were used as positive controls for bacteria and fungi, respectively, while 5% DMSO served as the negative control.

2.5. Cytotoxic Activity

The cytotoxic effects of selected lichen extracts were evaluated on human epithelial carcinoma (HeLa) cells, human lung carcinoma (A549) cells, human colon carcinoma (LS174) cells, and human fetal lung fibroblasts (MRC-5), all obtained from the American Type Culture Collection (Manassas, VA, USA). Cancer cell lines were maintained as monolayer cultures in Roswell Park Memorial Institute 1640 (RPMI 1640) medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 3 mM L-glutamine, and antibiotics, at 37 °C in humidified air containing 5% CO₂.

The MTT assay (microculture tetrazolium test) was performed according to Mosmann [19] to assess cytotoxic activity. Cell viability was evaluated 72 h after treatment with the extracts. Briefly, 20 μL of MTT solution (5 mg/mL in PBS) was added to each well and incubated for 4 h at 37 °C in humidified air with 5% CO₂. Following incubation, 100 μL of 10% SDS was added to dissolve the formazan crystals. Absorbance, proportional to the number of viable cells, was measured at 570 nm using a microplate reader (Multiskan EX, Thermo Scientific, Vantaa, Finland). Experiments were conducted in triplicate and repeated independently four times.

Cytotoxic selectivity of the tested extracts was determined using a selectivity index (SI) calculated according to the equation:

$$\mathrm{S}\mathrm{I}={\displaystyle \frac{{\mathrm{I}\mathrm{C}}_{50} (\mathrm{h}\mathrm{e}\mathrm{a}\mathrm{l}\mathrm{t}\mathrm{h}\mathrm{y} \mathrm{c}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{s})}{{\mathrm{I}\mathrm{C}}_{50} (\mathrm{c}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{r} \mathrm{c}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{s})}}$$

where SI values higher than 3 were considered to be highly selective [20].

2.6. Statistical Analysis

The results were presented as means ± standard deviations (mean ± SD) of three measurements. Data was analyzed using Microsoft Excel and SPSS software packages (version 21).

3. Results and Discussion

3.1. Chemical Composition of the Lichens Lepraria incana and Pertusaria amara

A review of the existing literature showed that the chemical composition and biological activity of acetone extracts of the lichens Lepraria incana and Pertusaria amara have been very poorly studied [21,22,23]. Figure 1 shows the chromatograms of the acetone extracts of these lichens obtained by HPLC-DAD analysis. Figure 2 shows the structures of identified compounds in lichens L. incana and P. amara, while

Table 1. Retention time (min) and peak area (%) of the identified secondary metabolites from the lichens Lepraria incana and Pertusaria amara.

Compounds	Class of Compound	Retention Times * tR (min)	Absorbance Maxima (nm)	Peak Area (%)
Lepraria incana
Divaric acid	Monocyclic aromatic	2.84 ± 0.02	212, 264, 300	1.51
Divaricatinic acid	Monocyclic aromatic	3.38 ± 0.01	212, 263, 300	32.31
Norstictic acid	Depsidone	4.35 ± 0.01	212, 239, 310	24.23
Divaricatic acid	Depside	17.03 ± 0.02	210, 269, 320	33.47
Usnic acid	Dibenzofurane	19.87 ± 0.01	234, 282	6.57
Pertusaria amara
Conprotocetraric acid	Depsidone	1.81 ± 0.02	213, 241, 320	9.23
Protocetraric acid	Depsidone	2.03 ± 0.02	212, 242, 320	39.89
Picrolichenic acid	Depsone	3.99 ± 0.01	220, 242, 280	38.81
Atranorin	Depside	11.21 ± 0.02	210, 252, 262, 320	10.48

* values are means of three determinations ± CD.

shows the retention times (min) and peak areas (%) of the identified compounds.

From the chromatograms shown, it can be clearly seen that the acetone extracts of lichens L. incana and P. amara contain various secondary metabolites. HPLC-DAD analysis of the acetone extract of the lichen L. incana confirmed the presence of the following metabolites: divaric acid (DVR), divaricatinic acid (DVC), norstictic acid (NOR), divaricatic acid (DIV), and usnic acid (USN). Divaric acid and divaricatinic acid are monohydroxy benzoic acids and norstictic acid is a depsidone. Divaricatic acid belongs to the chemical class of depsides and consists of two units, divaricatinic acid and divaric acid connected by a depside (ester) bond. This secondary metabolite is produced by lichens and is not found in higher plants. Divaricatic acid is commonly found in lichens of the genus Anzia, Haematomma, Evernia, Lecidea, Parmelia and Ramalina. Usnic acid is a well-known lichen metabolite with a dibenzofurane structure, especially abundant in genera like Usnea, Cladonia and Ramalina [24].

The chemical composition of the lichen P. amara is poorly studied. In the literature, it is most often reported that this lichen contains picrolichenic acid and protocetraric acid, which have been identified by thin-layer chromatography [25]. From our second chromatogram shown, it can be seen that the acetone extract of the lichen P. amara from Serbia, in addition to the two compounds mentioned above, also contains conprotocetraric acid and atranorin. Protocetraric acid and conprotocetraric acid belong to the depsidone class and atranorin is a depside, commonly found in many lichen families, namely Cladoniaceae, Parmeliaceae, Lecanoraceae, Streocaulaceae, and others. Picrolichenic acid is a depsone that has an intensely bitter taste, which is where the bitter taste of lichens comes from. Some of the rare lichen species that biosynthesize picrolichenic acid are from the genera Pertusaria and Ochrolechia [24,26,27]. Conprotocetraric acid is a compound that rarely occurs in lichens and is most often a companion to protocetraric acid, as in the case of our studied lichen. This compound has been previously identified in the lichen Usnea trichodeoides, along with protocetraric acid, usnic acid, and virensic acid [28].

3.2. Antioxidative Activity

The antioxidant activities of the tested extracts are summarized in

Table 2. Free radical scavenging activity, reducing power and total phenolic and flavonoid content of the studied lichens.

		Lepraria incana	Pertusaria amara	Ascorbic Acid
DPPH radical scavenging IC50 (µg/mL)		664.23 ± 1.86	750.50 ± 1.98	9.42 ± 0.07
Reducing power Absorbance (700 nm)	1000 µg/mL	0.2562 ± 0.04	0.2011 ± 0.03	2.1158 ± 0.03
	500 µg/mL	0.1781 ± 0.02	0.1101 ± 0.02	1.6509 ± 0.02
	250 µg/mL	0.0904 ± 0.02	0.0552 ± 0.03	0.9609 ± 0.01
	125 µg/mL	0.0875 ± 0.03	0.0336 ± 0.02	0.4155 ± 0.01
Phenolic content (μg PE/mg of extract)		40.81 ± 0.98	33.67 ± 0.86
Flavonoid content (μg RE/mg of extract)		24.74 ± 0.89	23.61 ± 0.93

Values represent the means of three determinations ± SD.

. The DPPH radical scavenging assay indicated that both lichens exhibited moderate activity, with IC₅₀ values of 664.23 µg/mL for L. incana and 750.50 µg/mL for P. amara. As shown in Table 2, the reducing power of the extracts increased in a concentration-dependent manner, with absorbance values ranging from 0.0336 to 0.2562. Among the species tested, L. incana demonstrated the highest reducing power. Additionally, the total phenolic content of the acetone extracts was 40.81 µg PE/mg for L. incana and 33.67 µg PE/mg for P. amara, while the total flavonoid content was 24.74 µg RE/mg and 23.61 µg RE/mg, respectively.

Data concerning the antioxidant capacity of the acetone extracts of L. incana and P. amara are very scarce. Prior to this research, the antioxidant effects of the methanol extract of L. incana were studied by Ahmed et al. [29], who found that this extract exhibited antioxidant activity, showing relatively strong free radical scavenging (DPPH, ABTS) and ferric reducing (FRAP) capacities. Similarly, methanol extracts of Pertusaria sp. and P. leucosora were evaluated by DPPH free radical scavenging capacity [30]. These studies reported that these extracts exhibited strong antioxidant activity with IC₅₀ values of 22.4 and 49.3 µg/mL.

The studied lichens contain divaricatic acid, norstictic acid, usnic acid, protocetraric acid and atranorin, which are known to display potent antioxidant activity [31,32,33,34]. Nevertheless, in the present study, the extracts showed moderate antioxidant activity. This discrepancy can be explained by differences in the concentration of these active constituents in the crude extract, in addition to the effects of other components present in the extract itself. The concentration of phenols was relatively moderate in both extracts, so this could have been one of the limiting factors. Moreover, L. incana contained higher levels of phenols and flavonoids compared with P. amara, suggesting that the antioxidant activity of the extracts may be correlated with their polyphenolic content, consistent with findings reported in other studies [35,36]. In most lichens, phenols, including depsidones, depsides, and dibenzofurans, are important antioxidants because of their ability to scavenge free radicals such as singlet oxygen, superoxide, and hydroxyl radicals [37]. Furthermore, in our experiment, the identified components belong to phenols, indicating an important role of phenol in the antioxidant activity.

3.3. Antimicrobial Activity

In our study, the lichen extracts demonstrated moderate antimicrobial activity (

Table 3. Minimum inhibitory concentration (MIC) of acetone extracts of the studied lichens.

Lichen Species	Lepraria incana	Pertusaria amara	Streptomycin	Ketoconazole
Test Microbes	MIC (µg/mL)
Staphylococcus aureus	156	312	31	/
Bacillus subtilis	625	5 × 103	16	/
Bacillus cereus	312	5 × 103	16	/
Escherichia coli	156	10 × 103	62	/
Proteus mirabilis	625	10 × 103	62	/
Mucor mucedo	10 × 103	10 × 103	/	156
Trichoderma viride	10 × 103	10 × 103	/	78
Cladosporium cladosporioides	20 × 103	10 × 103	/	39
Fusarium oxysporum	10 × 103	20 × 103	/	78
Alternaria alternata	10 × 103	20 × 103	/	78
Aspergillus flavus	10 × 103	20 × 103	/	312
Aspergillus niger	20 × 103	20 × 103	/	78
Candida albicans	156	625	/	39
Penicillium expansum	2.5 × 103	2.5 × 103	/	156
Penicillium chrysogenum	10 × 103	10 × 103	/	78

). Both extracts were effective against all tested bacterial and fungal strains. MIC values ranged from 156 to 20 × 10³ µg/mL for L. incana and from 312 to 20 × 10³ µg/mL for P. amara. The lowest MIC (156 µg/mL), indicating the strongest antimicrobial effect, was observed for L. incana against S. aureus, E. coli, and C. albicans. The antimicrobial activity of the extracts was compared with standard antibiotics, streptomycin for bacteria and ketoconazole for fungi, which showed a higher activity than the lichen extracts. The negative control, 5% DMSO, showed no effect on microbial growth.

Prior to our research, the antimicrobial activity of L. incana and P. amara had been explored by Taylor et al. [38], who found that acetone extracts of these lichens showed antimicrobial activities against Gram-positive and Gram-negative bacteria, as well as dermatophyte fungi. Some researchers found significant antimicrobial effects for certain species of lichen. For example, Gajendra et al. [39], who studied 34 lichen species, reported that most of the tested lichen extracts demonstrated inhibitory effects against the tested microorganisms with MIC values ranging from 3.9 to 500 µg/mL. Compared to their results, our lichen extracts demonstrated moderate antimicrobial activity. However, several previous studies have also produced results on the moderate antimicrobial activity of lichen extracts, similar to ours [2,40]. This variation in the results among different studies may be due to a combination of factors, including the extraction of different lichen species, the solvent used for extraction, and the specific microorganisms. Additional research is required to determine the specific factors influencing the antimicrobial properties of lichen extracts. Lichen components were also found to exhibit considerable antimicrobial effects against various bacteria and fungi (including divaricatic acid, norstictic acid, usnic acid, protocetraric acid and atranorin) that were present in the tested L. incana and P. amara species [32,34,41,42], suggesting that these constituents may be responsible for the observed antimicrobial activity in L. incana and P. amara acetone extracts. It is important to recognize that the extracts are complex mixtures of natural compounds, and their antimicrobial activity may not solely reflect the individual effects of each component, but also the interactions between them, which can influence the overall activity of the extracts.

Generally, in this study, our extracts demonstrated stronger effects on bacteria than fungi. Fungi were assumed to be more resistant to the tested extracts than bacteria due to the more complex structure of the cell wall. This observation aligns with numerous studies on antimicrobial activity, which have shown that differences in cell wall structure and permeability largely account for the varying sensitivities of bacteria and fungi. Gram-positive bacterial cell walls are composed of peptidoglycans and teichoic acids, whereas Gram-negative bacterial walls contain peptidoglycans, lipopolysaccharides, and lipoproteins. Fungal cell walls, in contrast, are relatively impermeable and consist of polysaccharides such as chitin and glucan [43,44]. However, L. incana showed an MIC value of 156 µg/mL against C. albicans, which is more potent than its effect on some bacteria. This can be linked to ergosterol, which is the main sterol component of the yeast cell membrane. Ergosterol plays a key role in maintaining membrane integrity and fluidity and is a key target for many antifungal agents. Numerous natural products have been shown to interact with ergosterol or inhibit its biosynthesis, leading to increased membrane permeability, leakage of intracellular contents, and ultimately cell death [45,46].

Previous research on lichens suggests that their antimicrobial effects may involve multiple targets within microorganisms, disrupting normal cellular processes. Potential mechanisms for the activity of the tested lichen species include the inhibition of cell wall synthesis, interference with protein synthesis, alteration of cell membrane integrity, and disruption of nucleic acid synthesis [47]. These effects ultimately compromise the ability of cells to function properly. To clarify these mechanisms in detail, further studies are necessary to elucidate the precise modes of action of the examined lichens toward microorganisms, especially for L. incana toward C. albicans, which can cause opportunistic infections in humans.

3.4. Cytotoxic Activity

The cytotoxic effects of the lichen extracts on the tested cell lines are summarized in

Table 4. Cytotoxic effects of tested lichens on the Hela, A549 and LS174 cell lines. The values in parentheses indicate the selective index (SI).

Cell Lines	Hela	A549	LS174	MRC-5
Lichen Species	IC50 (μg/mL)
Lepraria incana	62.15 ± 0.73 (3.9)	47.53 ± 0.86 (5.1)	80.57 ± 0.28 (<3)	>200
Pertusaria amara	125.29 ± 1.56 (<3)	151.91 ± 1.79 (<3)	174.69 ± 2.04 (<3)	>200
cis-DDP	0.83 ± 0.19	3.56 ± 0.23	2.58 ± 0.16	2.58 ± 0.16

IC₅₀ values are expressed as the mean ± SD derived from three separate MTT assay experiments.

. Both extracts demonstrated notable cytotoxic activity in vitro. IC₅₀ values for L. incana ranged from 47.53 to 80.57 μg/mL, while those for P. amara ranged from 125.29 to 174.69 μg/mL. The strongest cytotoxic effect was observed for L. incana against A549 cells, with an IC₅₀ of 47.53 μg/mL. Additionally, L. incana exhibited relatively low IC₅₀ values against HeLa and LS174 cells (62.15 and 80.57 μg/mL, respectively). Importantly, the extracts of both L. incana and P. amara showed selective activity, having minimal effects on normal MRC-5 cells. Therefore, both examined species could be proposed as potential anticancer agents, especially the L. incana acetone extract that showed a good cell-selective effect (SI = 5.1) against the A549 line. In contrast, the positive control, cis-diamminedichloroplatinum (II) (cis-DDP), affected both cancerous and normal cells.

In this study, the cytotoxic activity of L. incana and P. amara was investigated for the first time. Previous research has examined the cytotoxic effects of other lichen species. For instance, Kočović et al. [48] reported that C. lepidophora exhibited anticancer activity, showing cytotoxic effects on HeLa and HCT-116 cells after 72 h, with IC₅₀ values of 21.17 and 21.48 μg/mL, respectively. Similarly, Kosanić et al. [49] demonstrated significant growth inhibition by Cladonia fimbriata, C. furcata, C. subulata, C. foliacea, and C. rangiferina against the HeLa, A549, and LS174 cell lines, with IC₅₀ values ranging from 11.69 to 140.13 μg/mL. In the available literature data [13,32,33,50], it was confirmed that the compounds contained in L. incana and P. amara can also exhibit cytotoxic activity against some cancer cells including A549 human lung cancer cells, human melanoma Fem-x, human colon carcinoma LS174 cell line, melanoma cells, etc.

In recent years, lichens have gained attention as potential anticancer agents, indicating their possible use as natural therapeutics in cancer treatment. The specific mechanisms underlying the cytotoxic effects of the tested extracts remain to be elucidated. Further studies are required to fractionate the extracts in order to identify the compounds responsible for the observed antitumor activity and explore strategies for enhancing their efficacy and selectivity.

4. Conclusions

As can be noted, limited information is available on the biological activities of L. incana and P. amara, whereas numerous studies have reported the antimicrobial, antioxidant, and cytotoxic properties of other lichen species. In comparison with these studies, our findings indicate that the tested extracts exhibited moderate antioxidant and antimicrobial activities, alongside pronounced cytotoxic effects. These results highlight the potential medical, pharmaceutical, and chemotaxonomic significance of these lichens. Future research should focus on the isolation, characterization, and evaluation of individual bioactive compounds with enhanced biological activity, particularly focusing on cytotoxic activity, as our results demonstrated significant in vitro cytotoxic effects.