Antimicrobial and Antioxidant Potentials of Non-cytotoxic Extracts of Corticolous Lichens Sampled in Armenia

doi:10.21203/rs.3.rs-541914/v1

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Antimicrobial and Antioxidant Potentials of Non-cytotoxic Extracts of Corticolous Lichens Sampled in Armenia

https://doi.org/10.21203/rs.3.rs-541914/v1

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published 29 Jul, 2021

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Due to wide range of secondary metabolites, lichens were used from antiquity as sources of colorants, perfumes and medicaments. This research focuses on exploring the antioxidant, antimicrobial and cytotoxic activities of methanol, ethanol, acetone extracts and aqueous infusions of corticolous lichens sampled from Armenia.

Methanol, ethanol and acetone extracts from all tested lichens were active against Gram-positive bacterial strains. The most effective solvent to retrieve antimicrobial compounds was methanol. Aqueous infusions of tested lichens didn’t show any significant antibacterial and antifungal activity. The highest antimicrobial activity was observed for methanol extract of Ramalina sinensis. The minimum inhibitory concentration of methanol extract of Ramalina sinensis were 0.9 to 1.8 mg mL^-1. Pseudevernia furfuracea demonstrated antifungal activity (Ø 12 mm). Methanol extract of Parmelia sulcata demonstrated largest DPPH radical scavenging activity (71%). The cytotoxicity was measured on human HeLa (cervical carcinoma) cell lines using microculture tetrazolium test assay. The IC₅₀ values estimated for methanol extracts of Peltigera praetextata, Evernia prunastri, R. sinensis and R. farinacea species in HeLa cell line were within 1.8-2.8 mg mL^-1 and considered as non-cytotoxic. Obtained results suggest that studied lichens can be prospective in biotechnologies as alternative sources of antimicrobial and antioxidant substances.

Health Economics & Outcomes Research

Lichens

Crude extracts

Antimicrobial activity

Antioxidant activity

Cytotoxic activity

About 20000 species of lichens growing on wide variety of substrates like rocks, walls, exposed soil surfaces and as epiphytes on the bark of trees and leaves have been recorded worldwide [1]. They are well adapted to survive in various geographical zones, from sea level to high elevations and from equator to polar regions. They are composite organisms consisting fungi as mycobiont and photosynthetic green algae and/or cyanobacteria as photobiont. Recently it was shown that besides cyanobacteria other bacteria also exist in lichen thalli and take part in mutualistic relationship [2-4]. Due to this multiparty mutualism they adapted to survive even in extreme environments characterized by high or low temperatures, periodic desiccation, high levels of UV radiation and salinity. To withstand extreme conditions, lichens synthesized metabolites (e.g., UV screens, cryoprotectants, osmolytes) which are valuable sources to develop new biotechnologies [5]. Ability to produce wide range of unique chemical compounds approves usage of lichens from ancient times as sources of colorants, cosmetics and remedies [5-7]. For example, Parmelia sulcata have been used to treat diseases of respiratory system, while Xanthoria parietina and Letharia vulpina were used against to cure jaundice and gastrointestinal disorders, respectively [8,9].

To date more than 800 secondary metabolites are identified for lichens. The continuing trends in compounds isolated from lichens approved their importance as a source of new natural products [6, 7, 10]. Long time lichens were out of attention by pharmaceutical industry reasons of which were their slow-growing nature and difficulties to cultivate in laboratory conditions [7, 11]. For the same reason it is difficult to obtain pure lichen metabolites in needful quantity for checking out their biological activities [6, 7, 12].

Many species of lichens in form of infusions, tinctures and different extracts have been historically used in folk medicine of many countries [9]. During the last decades, pharmaceutical potential (i.e. antifungal, antibacterial, antiviral, antitumor, cytotoxic, analgesic, antipyretic properties) of lichens sampled from different regions of the glob has been investigated [6, 13-15]. Despite its small territory Armenia is a crossroad for variety of rare lichens’ species [16,17], biodiversity and biotechnological potential of which, still remains unexplored.

The purpose of this study was to determine antioxidant, antimicrobial and cytotoxic activities of different extracts of eight corticolous lichens distributed on the territory of Armenia.

Collection and identification of lichens

Corticolous lichens samples of Ramalina sinensis Jatta, R. farinacea (L.) Ach., Flavoparmelia caperata (L.) Hale, Evernia prunastri (L.) Ach., Punctelia subrudecta (Nyl.) Krog, Pseudevernia furfuracea (L.) Zopf, Peltigera praetextata (Sommerf.) Zopf, Parmelia sulcata Taylor were collected from the Dilijan National Park (40°39′23″N, 45°01′17″E) and “Zikatar” Environmental Center (41°07′19.02″N, 44°55′32.52″E), which are located in the Tavush province, Armenia. The area mostly covered by temperate deciduous forests predominating by Oriental Beech (Fagus orientalis), Hornbeam (Carpinus spp.) and Oak (Quercus spp.).

Species identification was performed by standard methods and according to the common identification guides and keys [18]; [19]; [20]. Voucher specimens of all lichens were deposited in the publicly available Herbarium of Yerevan State University (YSU, Yerevan, Armenia) where serial numbers of five lichens were given; R. sinensis Jatta (ERHM 11071), R. farinacea (L.) Ach. (ERHM 11072), E. prunastri (L.) Ach. (ERHM 11073), P. furfuracea (L.) Zopf (ERHM 11074), P. sulcata Taylor (ERHM 11070).

Preparation of lichen extracts

Lichen thalli (10 g) were grinded using automatic grinder, until obtaining powder-like state. Grinded thalli were drenched with methanol, ethanol and acetone separately at 10:1 solvent-to-sample ratio (v/w). The mixtures with methanol, ethanol and acetone were left for extraction for 24 hours on magnetic stirrer, centrifuged (15 min, 12000 rpm) and then concentrated under reduced pressure in a rotary evaporator (BOV-50 V vacuum drying oven, Biobase Meihua Trading, China) at 37 °C temperature to dry. Extraction process was repeated three times to retrieve active compounds as much as possible. To obtain aqueous infusions the grinded lichens were dissolved in distilled water, then left for extraction for 24 hours on magnetic stirrer under heat conditions not exceeding the boiling point. Aqueous infusions filtered through 0.22 μm sterile filter (Millipore). Dried extracts was weighted and stored at -18 °C until they were used in the tests. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich) was used to prepare stock solutions. The extracts were diluted by sterile water up to 5% DMSO for the experiments.

Antimicrobial activity

The microbial strains used in this study were following: Gram-positive bacteria Bacillus subtilis WT-A1, Staphylococcus aureus MDC 5233, Gram-negative bacteria Escherichia coli VKPM-M17, Pseudomonas aeruginosa GRP3 and Salmonella typhimurium MDC 1754 and a yeast Candida albicans WT-174. Microbial strains were from microbial culture collection maintained by the Department of Biochemistry, Microbiology and Biotechnology, YSU.

Agar disc diffusion method was used to evaluate antimicrobial activity of lichens. The bacterial strains were inoculated in Müller-Hinton broth (MHB) and incubated overnight. Bacteria were subcultured in MHB liquid medium at 37 °C to OD600 = 0.2. Then 100 μL of inoculum was spread on a Petri dish with Müller-Hinton agar (MHA). Yeasts were inoculated in Sabourad dextrose (SD) broth and incubated overnight. After incubation 100 μL of culture was spread on SD agar. Sterilized Whatman filter paper discs (5 mm diameter) were infiltrated by 5 μL (500 μg mL⁻¹) of extracts and placed on MHA or SD agar plates containing appropriate microbial strain. Diameter of inhibition zones (IZ) formed around discs after incubation at 37 °C for 24 h was measured. The experiments were conducted at least thrice and the average of three measurements was accepted as an index of antibacterial activity. As positive controls gentamicin (10 μg mL⁻¹) (for bacteria) and nystatin 20 μg mL⁻¹ (for yeast) were used; while 5 % DMSO was used as a negative control.

Broth microdilution method was applied to determine minimum inhibitory concentration (MIC) [21]. A series of dilutions ranging from 0.9 to 7.5 mg mL^-1 for extracts were used in the experiment. The highest dilution of samples without visible growth after 24 h incubation at 37 °C was considered as MIC. To check sterility of crude extracts all its dilutions were cultured in agar media. The minimum bactericidal/fungicidal concentration (MBC/MFC) values was determined by sub-culturing samples from the tubes with concentrations above the MIC on new plates with MHA and SD agar for bacteria and yeast, respectively. The values obtained were the average data of experiments performed at least three time.

Antioxidant activity

Free radical scavenging method based on 1,1-diphenyl-2-picryl-hydrazil (DPPH) was used to measure antioxidant activity of lichen extracts [22]. The reactive solution contained 1mg mL^-1 ethanol extract, to which was added 0.1 mM DPPH. The absorbance was measured spectrophotometerically (λ 517 nm). As positive control ascorbic acid was used. In negative control, the extract was replaced by ethanol. The following equation was used to evaluate radical scavenging activity (RSA): RSA (%) = [(A₀ − A₁)/A₀] × 100, where A₀- absorbance of the negative control, A₁ - absorbance of reaction mixture or standard [23].

Determination of total phenolic compounds (TPC)

TPC was measured by the Folin-Ciocalteu method [24]. 1 mL of methanol extracts of 1 mg mL^-1 aliquots were mixed with 5 mL of Folin-Ciocalteu reagent (diluted 1:10) and 15 mL of 20 % (w/v) sodium carbonate solution. The mixture was incubated at room temperature in the dark for 1 hour and absorbance was measured at 765 nm. TPC was calculated as gallic acid equivalents (GAE) per 100 g of lichen thalli (mg GAE/100 g).

Determination of total flavonoid content (TFC)

TFC was determined by the Dowd method [24]. The mixture containing 2 mL of methanol extracts (1 mg mL^-1) and 2 mL of methanol solution of aluminum trichloride (2 %, v/w) was incubated at room temperature for 30 min. The absorbance was measured at 420 nm. Negative control, without extract was used as a blank. TFC was determined as microgram of catechin equivalent by using an equation that was obtained from standard catechin graph. The result was expressed as mg of catechin equivalents per 100 g of lichen thalli (mg CE/100 g).

Cytotoxic activity

The human HeLa (cervical carcinoma cell line) cells from the European Collection of Authenticated Cell Cultures, UK were used in experiments. The cancer cell lines were routinely maintained and cultivated in Dulbecco's Modified Eagle's medium (Sigma Aldrich, Germany) supplemented with 10 % fetal bovine serum (HyClone, UK), 2 mM L-glutamine (Sigma Aldrich, Germany), 100 IU mL^-1 penicillin (Sigma Aldrich, Germany) and 100 μg mL^-1 streptomycin (Sigma Aldrich, Germany). Cells were incubated in humidified atmosphere containing of 95 % air and 5 % CO₂ at 37 °C.

Microculture tetrazolium test (MTT) assay was used to determine the effect of extracts on cancer cell survival [25]. The cells were seeded at the density of 0.1x10⁶ cell/ mL^-1 into 96-well plates (Greiner, Germany). After incubation for 24 h, different concentration of extracts obtained by diluting the stock solution (1:10, 1:50, 1:100, 1:200) were added to the cell cultures. The stock solution concentrations were for R. sinensis 177 mg mL^-1, for R. farinacea 391 mg mL^-1, for E. prunastri 600 mg mL^-1, and for P. praetextata 328 mg mL^-1. The cells treated with DMSO (Sigma Aldrich, Germany) were used as vehicle control. After further incubation for 48 h, the MTT dye (Sigma Aldrich, Germany) was added to each well (500 μg mL^-1 final concentration) and incubated for 4 hours at 37 °C. Then the supernatant was removed and 100 μL of DMSO was added. Enzyme-linked immunosorbent assay (ELISA) plate reader (Human Reader HS, Germany) was used to measure the absorbance (λ 570 nm). Cell viability was expressed as a percentage of the negative control (cell cultures with no treatment). To reveal the cytotoxicity of the extracts doses inducing 50 % inhibition of cell viability (IC₅₀ value) were determined.

Data processing

GraphPad Prism 5.01 (GraphPad Software, USA) was used to perform data analysis. All experiments were conducted in triplicates. Values were expressed as means ±standard error (SE). Data were analyzed by repeated measures ANOVA. Dunn’s post-hoc test was used to determine differences between groups. p < 0.05 values were considered as the statistically significant.

Antibacterial activity

Results of antimicrobial activity of alcoholic extracts and aqueous infusions of tested lichens against tested bacteria and yeast are summarized in the Table 1. Diameters of IZ around used paper disks were measured to evaluate antimicrobial activity qualitatively. Aqueous infusions of all tested lichens did not demonstrate any significant antibacterial and anticandidal activity. Methanol, ethanol and acetone extracts from all tested lichens were demonstrated antibacterial activity against Gram-positive bacterial strains i.e. B. subtilis and S. aureus, while they were not able to inhibit the growth of tested Gram-negative bacteria.

The most antibacterial activity was observed for methanol extracts, which even at low concentrations were able to inhibit all tested Gram-positive bacteria. The methanol and ethanol extracts of R. sinensis showed the largest IZs (25 mm and 23 mm, respectively) against the B. subtilis, while largest IZs (19 mm and 16 mm, respectively) of the same extracts against S. aureus was observed in case of F. caperata. The same pattern is observed in case of acetone extracts of the same lichens, where largest IZs against to B. subtilis and S. aureus were 21 mm and 14 mm, respectively.

Although all alcoholic extracts of the lichen P. fufuracea manifested lowest antibacterial activity, it was only lichen, methanol extract of which, demonstrated antifungal activity against C. albicans (Ø, 12 mm).

Since the maximum antimicrobial activity by agar disc diffusion tests was found in methanol extracts, antimicrobial activities subsequently were quantitatively evaluated by MICs and MBCs/MFCs values only for methanol extracts. MICs values were determined for each bacterium. Methanol extract from R. sinensis was shown relatively high antibacterial effect (MIC 0.9 mg mL^-1) at the concentrations used. MIC for B. subtilis varied from 0.9 to 7.5 mg mL^-1, while MIC for S. aureus ranged from 0.9 to more than 7.5 mg mL^-1. MBC were determined for B. subtilis (in case of R. sinensis it was 0.9 mg mL^-1) and S. aureus (ranged from 1.8 to > 7.5 μg mL⁻¹). MFC value observed only for methanol extract of P. fufuracea was > 7.5 μg mL⁻¹.

Gentamycin and nystatin were used as standard antibiotics to compare antimicrobial activities obtained for bacteria and yeast, respectively. The results confirmed that antimicrobial activities were several time higher in case of standard antibiotics. DMSO didn’t show inhibitory effect on the tested organisms.

Antioxidant activity

The scavenging activity of DPPH radicals of lichen extracts is shown in Figure 1. Methanol extracts showed a good radical scavenging activity. The highest activity showed methanol extract of P. sulcata with 71 % activity, which was only slightly lower compared with ascorbic acid (96 ). Methanol extract of P. preatextata was also demonstrated promising scavenging activity (44 %). The methanol extract of other tested lichens showed slightly weaker DPPH radical scavenging activities (< 30 %). Relatively higher scavenging activity was observed also of acetone extracts of lichens P. praetextata and R. sinensis. Ethanol and aqueous extracts were not shown significant scavenging activity of DPPH radicals. Surprisingly, aqueous extracts of P. praetextata, R. sinensis and R. farinacea have shown more antiradical activity then ethanol extract of the same lichens. These results were somewhat unexpected, since usually the ethanol extracts exhibiting higher radical scavenging abilities.

TPC and TFC of tested methanol extracts are shown in Table 2. Highest phenolic compounds was identified in extract of P. sulcata 3811 mg GAE per 100 g lichen dry weight, while extract of P. subrudecta showed the lowest content (608 mg GAE per 100 g lichen dry weight). Relatively high phenolic compounds was determined also for E. prunastri (3585 mg GAE/100 g).

TFC for methanol extracts of P. sulcata, F. caperata and R. sinensis were 700, 567 and 523 mg CE/100 g lichen dry weight, respectively. The methanol extract of P. sulcata showed highest TFC among all lichen extracts, the lowest content of flavonoids was observed for P. subrudecta extracts (222 mg CE/100 g lichen dry weight).

Cytotoxic activity

The statistically significant and dose-dependent decrease in cell viability was shown for methanol extracts of all tested lichens started from lower tested concentrations (Fig. 2). The IC₅₀ values of R. sinensis and R. farinacea extracts were almost similar 1.8±0.1 mg mL^-1 and 1.75±0.4 mg mL^-1, respectively. However, at higher tested concentrations, the cytotoxic profile of mentioned extracts was different, since 10% of viability was observed at the concentration of 18 mg mL^-1 for R. sinensis and 40 mg mL^-1 for R. farinacea extracts. The similar cytotoxic activity was shown for P. praetextata and E. prunastri extracts at all tested concentrations and the IC₅₀values were 2.8±0.3 mg mL^-1and 2.4±0.2 mg mL^-1, respectively.

Armenia is a South-Caucasian landlocked mountainous country with climate contradictions. Divers of bio-geographical and climatic conditions, well-defined vertical zonation, as well as active tectonic processes in Armenia have contributed to the formation of ecosystems with rich biodiversity and endemic species. In this context Armenia is a crossroad also for variety of rare and still unexplored lichens’ species [17].

Continuous and uncontrolled use of synthetic medicaments often causes numerous side effects. This evidence forces scientists to look for new preparations of natural origin. Lichens as alternative sources can be used to search of new bioactive substances [6, 7]. Since biotechnological potential of lichens distributed on the territory of Armenia still remains unexplored, we aimed to study of bioactivity of the aqueous and different alcoholic extracts of the corticolous lichens sampled from Armenia. In the present study antimicrobial, antioxidant and cytotoxic activities of crude methanol, ethanol and acetone extracts and aqueous infusions from R. sinensis, R. farinacea, F. caperata, E. prunastri, P. subrudecta, P. furfuracea, P. praetextata and P. sulcata were examined.

In our experiments, aqueous infusions of all tested lichens lack of antibacterial and antifungal activities, which coincides with literature data [26]. Weak activity of aqueous infusions is probably result of insolubility or poor solubility of antimicrobial metabolites in water [27]. Despite this generally accepted opinion, some researchers have also shown antimicrobial activity of some aqueous extracts of lichens. Thus, [28] have shown that aqueous extracts of R. farinacea and some species belonging to the genera Anaptychia, Peltigera, Umbilicaria, Xanthoria and Xanthoparmelia exhibited potent inhibition toward E. coli, B. subtilis and S. aureus. Recently it was shown aqueous extracts from Ecuadorian lichens Usnea sp. possessed antibacterial activity against B. subtilis [29]

In contrast to aqueous extracts, alcoholic extracts of lichens in our experiments demonstrated relatively high antibacterial activity. The quality of the antibacterial effect depended on the species of lichen. Within tested lichen extracts only R. sinensis has demonstrated significant bactericidal activity against B. subtilis. Probably R. sinensis possessed activity against endospores as well. The methanol and ethanol extracts of R. sinensis was shown the maximum antibacterial activity. The methanol extract of F. caperata was active against S. aureus.

Only static activity was observed against other tested microbes. MBC/MFC almost in all cases were higher than respective MIC values. According to the results obtained Gram-positive bacteria were more sensitive against the crude extracts of tested lichens. Such selective inhibition by extracts can be explained by composition and structural peculiarities of bacterial cell walls. Gram-positive bacterial cell walls endowed with higher permeability than Gram-negative bacterial ones [26].

Antimicrobial features of different extracts of lichens’ species belonging to genus Ramalena were investigated by other researchers too. Thus, ethanol extract of R. farinacea sampled from New Zealand showed inhibitory effect toward bacilli and some Gram-negative bacteria [30]. Both tested Gram-positive and Gram-negative bacteria were sensitive against ethanol extract of R. farinacea sampled from Turkey [28]. Lichen species sampled from Antarctic also demonstrated high antibacterial properties against to S. aureus and B. subtilis [31]. Mitrovic et al. [32] also reported about strong inhibitory effect of methanol extracts of P. sulcata, F. caperata and E. prunastri against mainly Gram-positive bacteria. In general results obtained in this study are in agreement with literature data and confirmed high antibacterial activity of tested lichens.

Among tested lichen demonstrating the antifungal activity was only P. fufuracea, methanol extract of which, has inhibited C. albicans (Ø, 12 mm). There are some reports revealing high resistance of fungi against antimicrobial agents of lichen origin. Presumably it depends on specific composition and permeability of its cell wall [26].

In contrast to our results it was shown that aqueous and different polar and nonpolar extracts of many lichens also demonstrated antifungal activity. For instance, Karabuluti and Ozturk [33] reported that some extracts of E. prunastri, P. sulcata and P. furfuracea demonstrated significant antifungal activity against species of genera Aspergillus, Botrytis, Fusarium, Macrophomina, Penicillium and Rhizoctonia. Using disc diffusion method Turkan et al. [34] was shown anticandidal activity of acetone and chloroform extracts of P. furfuracea. Similar investigation carried out using nonpolar fractions of P. furfuracea exhibited significant antifungal activity especially against [35].

It was shown earlier lichens’ thalli comprise numerous secondary metabolites with antibacterial and antifungal activity [6, 7, 9, 13, 15]. The type of extracting solvent is also has a decisive significance. Considering this we aimed also evaluate solvents efficiency to extract bioactive compounds from lichens’ thalli. As mentioned above methanol extracts demonstrated the highest antimicrobial activities. Second strongest antimicrobial activities were recorded in case of ethanol extracts, followed by acetone. Thus, we assumed that in our investigation methanol was efficient solvent to extract phenolic and/or other compounds with antimicrobial activity.

The tested lichen methanol extracts also expose relatively strong antioxidant activities against DPPH radical in vivo. The strong antioxidant activity is probably connected with the substances extracted by methanol [36]. It is distinctive that water extracts of P. praetextata, R. sinensis, R. farinacea and E. prunastri also derive relatively strong antioxidant activity. Moreover, the antioxidant activity of aqueous infusions of P. praetextata, R. sinensis and R. farinacea exceeds the activity of ethanol extracts of the same species. The scavenging activity is possible associated with secondary metabolites which are unique for that species and type of solvent. In this case, as it was mentioned the most efficient solvent was methanol.

Kumar et al. [37] reported existence of correlation between some secondary metabolites (mainly phenols) in lichen thalli and its antioxidant properties. Correlation between phenolic and flavonoid compounds of the tested extracts and free radical scavenging activity were shown in our study too. The tested methanol extracts of P. sulcata exhibited the highest radical scavenging activity with the greatest amount of phenolic and flavonoid contents. However, recently some deviations from this pattern have been also shown [38]. This evidence allowed to assume that antioxidant activity can be conditioned other, non-phenol components. Gulcin et al. [39] were shown strong antioxidant activity of aqueous extracts of Cetraria islandica. Stanly et al. [40], studding some Malaysian lichens have found contradiction between antioxidant activity and total phenol content. In contrast to this, methanol extracts of the lichen species P. sulcata, F. caperata,E. prunastri, Hypogymnia physodes and Cladonia foliacea collected from southeast of Serbia demonstrated high antioxidant activities [32]. In our studies, we also clearly showed that not only alcoholic solvents (which usually extracts phenolic compounds), but aqueous extracts also demonstrated antioxidant activity. Moreover, methanol extract of P. preatextata demonstrated relatively high DPPH radical radical scavenging activity (44 %), but TPC and TFC were low. Despite of high flavonoid and phenolic content the radical scavenging activity of methanol extract of E. prunastri was very low (Table 2. Fig. 1).

There are other reports regarding to antioxidant effect of lichens. Thus, Rankovic et al. [8] were shown free radical scavenging activity (94.7% inhibition) for acetone extract of Lecanora atra. In this study, the highest activity was observed for methanol extract of P. sulcata with 71% activity. For comparison, it should be noted that methanol extract of another representative of the genus Parmelia (P. saxatilis) had free radical-scavenging activity with 55.3% inhibition [41]. To our knowledge it is first report about high antioxidant activity observed for methanol extracts of P. sulcata.

It was established that some lichen secondary metabolites (usnic acid, lecanoric acid, lobaric acid, evernic acid, vulpinic acid and so on) have cytotoxic properties [12]. Cytocidal effect of mentioned metabolites displays by cell cycle arrest, apoptosis, necrosis, and inhibition of angiogenesis [42].

Earlier the cytotoxicity of E. prunastri extracts was analyzed in different cell lines [43]. The weak cytotoxic effect (IC₅₀=120.89 μg mL^-1) was shown for acetone extract of E. prunastri in FemX and LS 174 cell lines lines [43]. Non-cytotoxic properties of E. prunastri methanol extract was revealed on colon cancer adenocarcinoma cell line HCT-116 (IC₅₀=295.64 μg mL^-1) [32]. A crude extract of Xanthoria parietina significantly inhibited growth of Murine myeloma P3X63-Ag8.653 cells [44]. Only few publications are available demonstrating anticancer activity of lichen extracts. Ari et al. [45] were reported significant anticancer effect (IC₅₀ values 16.5 μg mL^-1) for methanol extract of P. sulcata against Human Breast cancer cell lines MDAMB-231. Viable cell number of Human colon cancer cell (HT-29) line was decreased after treatment them by acetone and methanol extracts of Lethariella zahlbruckneri [46].

In present study, the IC₅₀ values of methanol extracts of studied lichens were in the range of 1.8-2.8 mg mL^-1. According to the American National Cancer Institute, a crude extract is considered as active for an IC₅₀< 30 μg mL^-1 in the preliminary assay [47]. Following this 1criterion, the methanol extracts of studied lichen species (P. praetextata, E. prunastri, R. sinensis, R. farinacea) cannot be considered as cytotoxic. Since compounds possessing potential antimicrobial and antioxidant activities may not be useful in pharmacological preparations if they possess cytotoxic effect, the non-cytotoxic profile of extracts studied in our work proves their safety and extracts can be recommended for further studies. Results obtained are stated strong antioxidant, antimicrobial activity and non-cytotoxic profile of tested lichen extracts. Lichens stand as organisms with high biotechnological potential, which was proven before by various authors, but was reported for the first time for lichens distributed on the territory of Armenia.

Funding/Acknowledgements

The work was supported by the Science Committee of RA, in the frames of the research project № 20AA-1F018 and was partially supported by grants from the Norwegian Cooperation Program in Higher Education with Eurasia (CPEA LT 2016/10095, CPEA-LT-2017/10061) to collect samples and obtain reagents for analysis.

Authors' contributions

RS and AG carried out sampling and identification of lichens, RS prepared of extracts, carried out the antimicrobial and antioxidant activities, generated figures, tables. GT carried out cytotoxic activities and performed data analysis. HP designed and supervised the study, performed all data analysis, coordinated and drafted the manuscript. AT provided critical comments and edited the manuscript. All authors read and approved the final manuscript.

Consent for publication

Not applicable.

Conflicts of interest/Competing interests

The authors declare that they have no conflicts of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Availability of data and material

Lichens are available in the Herbarium of Yerevan State University (Yerevan, Armenia). Materials and data of this study are available upon request.

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Table 1. Disc diffusion inhibition zones (IZ) minimum inhibitory concentrations (MIC) and minimum bactericidal/fungicidal concentrations (MBC/MFC) of tested lichen crude extracts.

Lichens species

Crude extracts^a

Test microbes, IZ (Ø (mm)), MIC (mg mL⁻¹) and MBC/MFC (mg mL⁻¹)

B. subtilis

S. aureus

E. coli

P. aeruginosa

S. typhymurium

C. albicans

MIC

MBC

MIC

MBC

MIC

MBC

MIC

MBC

MIC

MBC

MIC

MFC

R. sinensis

25±0.5

0,9

15±0.5

1,8

1.5±0.3

>7.5

2.0±0.3

>7.5

1.5±0.3

>7.5

23±0.5

12±0.5

1.0±0.3

1.2±0.3

21±0.5

13±0.5

1.0±0.3

R. farinacea

23±0.5

1,8

15±0.5

3,75

1.2±0.3

>7.5

1.5±0.3

>7.5

1.0±0.3

>7.5

21±0.5

11±0.5

1.0±0.3

1.2±0.3

19±0.5

10±0.5

1.0±0.3

E. prunastri

19±0.5

3,75

17±0.5

3,75

1.3±0.3

>7.5

15±0.5

14±0.5

1.1±0.3

10±0.5

9±0.5

1.0±0.3

F. caperata

17±0.5

7,5

>7,5

19±0.5

3,75

1.0±0.3

1.8±0.3

>7.5

15±0.5

16±0.5

1.2±0.3

12±0.5

14±0.5

1.0±0.3

P. subrudecta

15±0.5

7,5

>7,5

10±0.5

>7,5

1.0±0.3

>7.5

1.6±0.3

>7.5

10±0.5

8±0.5

1.3±0.3

1.2±0.3

9±0.5

7±0.5

1.5±0.3

1.3±0.3

P. furfuracea

15±0.5

7,5

>7,5

8±0.5

>7.5

1.5±0.3

>7.5

1.2±0.3

>7.5

12±0.5

3.75

>7.5

7±0.5

6±0.5

1.0±0.3

1.2±0.3

9±0.5

3±0.5

1.2±0.3

1.0±0.3

P. sulcata

13±0.5

7,5

>7,5

9±0.5

7,5

>7,5

1.4±0.3

>7.5

1.2±0.3

>7.5

9±0.5

8±0.5

1.0±0.3

8±0.5

5±0.5

1.0±0.3

P. praetextata

13±0.5

7,5

>7,5

10±0.5

7,5

>7,5

1.2±0.3

>7.5

9±0.5

1.0±0.3

9±0.5

7±0.5

1.0±0.3

PC^b

32±0.5

0.25

0.5

28±0.5

0.3

0.5

20±0.5

0.5

1.5

25±0.5

0.3

0.5

23±0.5

2.5

20±0.5

1.75

^aExtracts: M-Methanol, E-Ethanol, A-Acetone, W-Water,

^bPC: positive control, for IZ: gentamicin (10 μg mL⁻¹) for bacteria, nystatin 20 μg mL⁻¹ for yeasts; for MIC and MBC/MFC: gentamicin (range 2–0.003 μg mL⁻¹) for bacteria, nystatin (range 4–0.125 μg mL⁻¹) for yeasts.

nd - not determined

Table 2. TPC and TFC of methanol extracts of studied lichens.

Lichen species	TFC (mg CE/ 100g dw*)	TPC (mg GAE/ 100g dw*)
P. sulcata	700 ± 7.1	3811 ± 71.25
E. prunastri	373 ± 4.2	3585 ± 69.30
P. preatextata	310 ± 3.7	1648 ± 72.3
F. caperata	567 ± 6.3	1522 ± 67.2
R. farinacea	295 ± 2.1	1128 ± 70
R. sinensis	523 ± 5.1	786.5 ± 56
P. subrudecta	222 ± 2.3	608 ± 42.1

*dw-dry weight.

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Antimicrobial and Antioxidant Potentials of Non-cytotoxic Extracts of Corticolous Lichens Sampled in Armenia

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