Cent. Eur. J. Biol.• 5(5) • 2010 • 649-655 DOI: 10.2478/s11535-010-0043-z

Central European Journal of Biology

Antioxidant and antimicrobial properties of the Anaptychya ciliaris, parile, Ochrolechia tartarea and Parmelia centrifuga

Research Article Branislav Ranković1,*, Darko Ranković2, Marijana Kosanić1, Danijela Marić1

1Department of Biology, Faculty of Science, University of Kragujevac, 34000 Kragujevac, Serbia 2Faculty of Medicine, University of Kragujevac, 34000 Kragujevac, Serbia Received 05 December 2009; Accepted 01 April 2010

Abstract: The aim of this study was to characterize the antioxidant and antimicrobial activity of the methanol extracts of: Anaptychya ciliaris, , Ochrolechia tartarea and Parmelia centrifuga. The methanol extract of the P. centrifuga showed a strong antioxidant activity, in comparison to the extracts from A. ciliaris, O. tartarea and N. parile which were relatively weaker. Furthermore, the methanol extract of the P. centrifuga was shown to contain the highest total phenol content (54.19 mg/g of the dry extract). Interestingly, a statistically significant positive relationship between the antioxidant activity and the total phenol content was observed. The minimum inhibitory concentration (MIC) against six bacterial and eleven fungi was established for the methanol extracts from each of species of lichens. The methanol extracts of the lichens P. centrifuga and O. tartarea showed the strongest both antibacterial and antifungal activity. Taken together, the results from this study suggest that the lichens P. centrifuga and O. tartarea may be used as a natural source of antioxidants in addition to providing certain antimicrobial features. Keywords: Lichens • Antioxidant activity • Antimicrobial activity • Methanol extract © Versita Sp. z o.o.

1. Introduction due to the growing resistance of microorganisms to existing antibiotics, the antimicrobial activity of lichens In living organisms, free radicals that can cause tissue has been intensively explored [11-13]. damage, are continuously produced and may eventually Therefore, the aim of this study is to undertake lead to various diseases [1-2]. While antioxidants can an in vitro assessment of the antioxidant and the interact with free radicals to prevent any associated antimicrobial activity of the methanol extract derived damage occurring, many synthetic antioxidant from four previously uncharacterized species of drugs have deleterious side effects. Consequently, lichens notably Anaptychia ciliaris, Nephroma parile, an alternative solution is to use naturally occurring Ochrolechia tartarea and Parmelia centrifuga. antioxidants as additives in food as well as traditional medicines [3-5]. In accordance with identifying a suitable natural antioxidant, the antioxidant activity of 2. Experimental Procedures many organisms has been demonstrated which includes some species of lichens [6-9]. Lichens Anaptichya ciliaris (L.) Körber, Nephroma Lichens are symbiotic organisms consisting of a parile (Ach.) Ach., Ochrolechia tartarea (L.) Massal. and partner and a photosynthetic organism (either Parmelia centrifuga (L.) Ach. were collected on Mount an alga or ). The secondary metabolites Kopaonik during the summer of 2008. Determination from several lichens have been shown to have antiviral, of the investigated lichen was accomplished using antibacterial, anti-tumor and anti-allergic potential as standard methods [14-17] and lichen samples are well as inhibitory activity against the plant growth, and stored in the Department of Biology, Faculty of Science are often used in alternative medicines [10]. Interestingly, at Kragujevac University.

* E-mail: [email protected] 649 B. Ranković et al.

2.1 Preparation of the methanol extract 2.4 Reducing power Finely pulverized dried lichen thalli (100 g) were The total reducing power of lichen extract was extracted with 1 L of methanol using a Soxhlet extractor determined according to the method of Oyiazu [20]. for 72 h at a temperature not exceeding the boiling point Briefly, 1 ml of of the metanolic lichen extracts were of the solvent. The extracts were filtered using Whatman mixed with 2.5 ml of phosphate buffer (0.2 M, pH 6.6) filter paper (No.1) and then concentrated under reduce and 2.5 ml of potassium fericyanide [K3Fe(CN)6] (1%). pressure in a rotary evaporator. The dry extracts were The mixture was then incubated at 50˚C in a water bath stored at the temperature of -18ºC until required for for 20 min before being cooled rapidly, spiked with 2.5 ml further experiments. of 10% trichloroacetic acid and centrifuged (3000 rpm for 10 min). The supernatant (5 ml) was then mixed with 2.2 Antioxidant activity assay an equal amount of destilled water and 1 ml of 0.1%

Antioxidant activities of lyophilized lichen extracts ferric chloride (FeCl3). After 10 min, the absorbance of were determined using the thiocyanate method [18]. 700 nm was then measured. The higher the absorbance Briefly, 1 mg of dry extracts were resuspended in represents the stronger reducing power. The assay were 1 ml distiled water and mixed with 5 ml linoleic acid carried out in triplicate and the results were expressed emulsion (0.02 M, pH 7.0) and 5 ml phosphate buffer as mean values ± standard deviations. (0.2 M, pH 7.0). Linoleic acid emulsion was prepared by mixing 0.5608 g of linoleic acid with 0.5608 g of 2.5 Antimicrobial activity Tween 20 as emulsifer, and 100 ml phosphate buffer, 2.5.1 Bacterial and fungal strains and the mixture was then homogenized. The reaction Methanol extracts were individually tested against mixture was then incubated at 37°C and 0.1 ml aliquots 6 bacteria: Enterococcus faecalis (ATCC 29212), were taken at different intervals during incubation. Escherichia coli (ATCC 25922) Klebsiella pneumoniae The degree of oxidation was measured according to (ATCC 700603), Micrococcus lysodeikticus (ATCC Mitsuda et al. [18], by sequentially adding 4.7 ml ethanol 15800), Pseudomonas aeruginosa (ATCC 27853), (75%), 0.1 ml ammonium thiocyanate (30%), 0.1 ml Staphylococcus aureus (ATCC 25923) and 11 fungi: sample solution, and 0.1 ml ferrous chloride (0.02 M, Alternaria alternata (ATCC 13963), Aspergillus flavus in 3.5% HCl), incubating for 3 min and determining the (ATCC 9170), Aspergillus niger (ATCC 6275), Candida peroxide value by reading the absorbance at 500 nm albicans (ATCC 10259), Cladosporium cladosporioides (using a UV-visible spectrophotometer, Jenway G105, (ATCC 11278), Mucor mucedo (ATCC 48559), U.K). A control was performed with linoleic acid but Paecilomyces variotii (ATCC 22319), Acremonium without the extract. Trolox and ascorbic acid solutions, chrysogenum (DBFS 401), Fusarium oxysporum were used as positive control. Inhibition % was (DBFS 292), Penicillium verrucosum (DBFS 418) and calculated using the following equation: Trichoderma harzianum (DBFS 379). The fungi were I% = (1-absorbance of sample at 500 nm/absorbance obtained from the mycological collection maintained of control at 500 nm) x 100. by the mycological laboratory within the Department of All experiments were carried out in triplicate. Biology of were University’s Faculty of Science (DBFS). Bacterial cultures were maintained on Müller-Hinton 2.3 Detrmination of Total Phenolic agar substrates (Torlak, Belgrade). All cultures were Total phenolic content in the extracts was determined stored at 4°C and subcultured every 15 days. with Folin-Ciocalteu reagent according to the method The antimicrobial activity of the methanol soluble of Slinkard and Sigleton [19], using gallic acid as a extracts was evaluated by measuring the zone of standard. A 1 ml extract solution containing 5 mg of inhibition against the test microorganism using the disc dried methanol soluble extract was mixed with 1 ml diffusion method. In addition, the minimal inhibitory of Folin-Ciocalteu reagent and shaken vigorously. concentrations (MICs) were also studied for the After incubation at room temperature for 5 min, microorganisms that were determined as sensitive in

2 ml of 2% Na2CO3 was added and allowed to stand the disc diffusion method. for 2 h with intermittent shaking. Absorbance was Inoculums were obtained from bacterial cultures measured at 760 nm. The concentration of total incubated for 24 h at 37°C and diluted according to the phenolic compounds in the extract was determined 0.5 McFarland standard to approximately 1x108 colony- as milligrams of gallic acid equivalent per gram of forming units (CFU)/mL. The bacterial suspension the dry extract. All experiments were carried out in is compared to the 0.5 McFarland standard. The triplicate. turbidity standard should be agitated on a vortex mixer

650 Antioxidant and antimicrobial properties of the lichens Anaptychya ciliaris, Nephroma parile, Ochrolechia tartarea and Parmelia centrifuga

immediately prior to use. If the bacterial suspension dilutions ranging from 30 to 0.12 mg/ml was used does not appear to the same density as the McFarland for each extract against all microorganism tested. 0.5, the turbidity can be reduced by adding sterile broth The initial concentration of 30 mg/ml was obtained or increased by adding more bacterial suspension. by measuring a quantity of extract and dissolved it in Suspensions of fungal spores were prepared from DMSO. Two-fold dilutions of extracts were prepared in fresh mature (three to seven days old) cultures grown Müller-Hinton for bacterial cultures and in SD broth for at 30°C on a Potato dextrose agar (PDA) substrate. fungal cultures. Spores were rinsed with sterile distilled water and culture The MIC was determined by establishing visible media turbidity was evaluated spectrophotometrically at growth of the microorganisms. The last dilution to 530 nm, and then diluted to approximately 106 CFU/mL show no visible growth was defined as the MIC for according to the procedure recommended by the National the tested microorganism at the given lichen extract Committee for Clinical Laboratory Standards [21]. concentration. Streptomycin and ketoconazole were used as positive controls. All experiments were 2.6 Disc diffusion assay performed in triplicate. The dried lichen extracts were dissolved in methanol to a final concentration of 30 mg/mL and filter sterilized (0.45 μm Millipore filters). Antimicrobial tests were 3. Results then carried out by the disc diffusion method [22], using 100 μL of suspension containing 1x108 CFU/mL 3.1 Antioxidant activity of bacteria, 1x106 CFU/mL of fungi spread on Müller- The antioxidant activity of the methanol extracts of A. Hinton agar (for bacteria) or Sabouraud Dextrose ciliaris, N. parile, O. tartarea and P. centrifuga on the agar (for fungi). The discs (6 mm in diameter) were peroxidation of the linoleic acid are shown in Table 1 impregnated with 10 μL of the methanol solution of the as a percent (%) of the inhibition. Interestingly, the dried lichen extract (300 μg /disk) and placed onto the oxidation of the linolenic acid was inhibited by all lichen corresponding media. Streptomycin (for bacteria) and extracts. The methanol extract of P. centrifuga showed ketoconazole (for fungi) were used as controls. Negative a relatively strong antioxidant activity with 54.19% of the controls were prepared with the same solvents used to inhibition of the linolenic acid, whereas a comparatively dissolve the lichen extract one strain/isolate in each weaker activity was shown with O. tartarea (26.61%), bacterial species tested. The inoculated plates were A. ciliaris (22.60) and with N. parile (3.63%). The total incubated at 37ºC for 24 h for the bacterial strains, and content of the phenol was also determined and given 72 h at 27ºC for the fungi isolates. Antimicrobial activity as the equivalent of the gallic acid (Table 1). The extract was evaluated by measuring the zone of inhibition of P. centrifuga had the highest total phenol content, against the test organisms. Each assay was repeated followed by the O. tartarea and A. ciliaris with similar three times. content, whereas N. parile had an extremely low total content of the phenol. The highest reducing power was 2.7 Minimal Inhibitory Concentration (MIC) demonstrated in the methanol extract of P. centrifuga. The MIC was determined by the broth microdilution Interestingly, significant correlation between the anti- method using 96-well micro-titer plates [23]. Serial oxidative activity and the total phenol content, r=0.990

Antioxidant activitya Total phenolic contenta Reducing power a

Lichen Mean % Inhibition (mg Gallic acid/g liophylisate) (A700 nm) A500 nm (48 h) Anaptychia ciliaris 22.60 1.014 ± 0.047 27.6 ± 1.012 0.195 ± 0.014 Nephroma parile 3. 63b 1.195 ± 0.052 6.2 ± 1.202 0.098 ± 0.012 Ochrolechia tartarea 26.61 0.910 ± 0.030 29.4 ± 1.605 0.202 ± 0.003 Parmelia centrifuga 54.19 0.568 ± 0.058 49.8 ± 1.296 0.375 ± 0.015 Trolox 99.60 0.005 ± 0.001 - - Ascorbic acid 35.01 0.805 ± 0.058 - - Control - 1.240 ± 0.018 - -

Table 1. Antioxidant activity, total phenolic content and reducing power of the lichens Anaptychia ciliaris, Nephroma parile, Ochrolechia tartarea and Parmelia centrifuga. a The values are presented as mean ± SD (n = 3)

651 B. Ranković et al.

60 60 50 50 40 40 r=0,990 r=0,978 30 30

20 20

Gallic acid/g liophylisate) acid/g Gallic 10 10 Gallic acid/g liophylisate) acid/g Gallic Total phenolics conent (mg conent phenolics Total Total phenolics conent (mg conent phenolics Total 0 0 0 20 40 60 0 0.1 0.2 0.3 0.4 Antioxidant activity (%) Reducing power (mean Abs. 700 nm)

Figure 1. Correlation between antioxidant activity and total phenolic Figure 2. Correlation between reducing power and total phenolic conent of lichens Anaptychia ciliaris, Nephroma parile, conent of lichens Anaptychia ciliaris, Nephroma parile, Ochrolechia tartarea and Parmelia centrifuga. Ochrolechia tartarea and Parmelia centrifuga.

Anaptychia Nephroma Ochrolechia Parmelia Antibiotic (S)c ciliaris parile tartarea centrifuga Tested organisms ZIa MICb ZI MIC ZI MIC ZI MIC ZI MIC

Enterococcus faecalis 27 1.87 - - 25 1.87 14 7.5 18 7.5 Escherichia coli - - - - 8 7.5 11 7.5 14 15 Klebsiella pneumoniae 17 3.75 13 7.5 21 1.87 26 1.87 28 1.87 Micrococcus lysodeikticus 25 1.87 11 7.5 35 0.94 28 1.87 22 7.5 Pseudomonas aeruginosa 30 0.94 15 3.75 35 0.23 36 0.23 31 1.87 Staphylococcus aureus 10 15.00 - - 10 7.5 19 3.75 10 30.00

Table 2. Antibacterial activities of methanolic extracts of Anaptychia ciliaris, Nephroma parile, Ochrolechia tartarea and Parmelia centrifuga against the organisms tested based on disk-diffusion and Broth microdilution assay.

a ZI = Inhibition zones of grown in diameter (mm) around the discs (6 mm) impregnated with 300 μg/disc of metanol extract and 30 μg/ml for antibiotics. Values are the mean of three replication. b Minimal inhibitory concentracio (MIC); values given ag mg/ml for lichen extract and as μg/ml for antibiotics. c Antibiotics (S)= Streptomycin μg/ml.

Anaptychia Nephroma Ochrolechia Parmelia Antibiotic (K)c ciliaris parile tartarea centrifuga Tested organisms ZIa MICb ZI MIC ZI MIC ZI MIC ZI MIC

Acremonium chrysogenum 7 15 - - - - 7 30 18 7.5 Alternaria alternata 8 15 - - 14 7.5 28 1.73 32 1.87 Aspergillus flavus ------19 7.5 Aspergillus niger - - - 7 30 6 15 21 7.5 Candida albicans 10 15 8 30 12 7.5 19 3.75 25 3.75 Cladosporium cladosporioides 15 7.5 10 15 22 3.75 28 1.53 29 3.75 Fusarium oxysporum - - - - 7 30 8 30 30 3.75 Mucor mucedo ------12 30 Paecilomyces variotii 12 7.5 9 7.5 18 3.75 17 3.75 34 1.87 Penicillium verrucosum ------24 7.5 Trichoderma harzianum ------7 30 18 7.5

Table 3. Antifungal activities of methanolic extracts of Anaptychia ciliaris, Nephroma parile, Ochrolechia tartarea and Parmelia centrifuga against the organisms tested based on disk-diffusion and Broth microdilution assay.

a ZI = Inhibition zone of growth in diameter (mm) around the discs (6 mm) impregnated with 300 μg/disc of metanol extract and 30 μg/ml for antibiotics. Values are the mean of three replication. b Minimal inhibitory concentracio (MIC); values given ag mg/ml for lichen extract and as μg/ml for antibiotics. c Antibiotics (K)= Ketoconazole μg/ml.

652 Antioxidant and antimicrobial properties of the lichens Anaptychya ciliaris, Nephroma parile, Ochrolechia tartarea and Parmelia centrifuga

(Figure 1), as well as between the reducing power The extracts showed different levels of the and the total phenol content, r=978 (Figure 2) were antioxidant activity. P. centrifuga and O. tartarea showed observed. a high level of the activity towards the inhibition of the peroxide of the linoleic acid. The antioxidant activity of 3.2 Antimicrobial activity the methanol extract of P. centrifuga was higher than The antimicrobial activity of the methanol extracts the one of the ascorbic acid (positive control). A strong of the lichens A. ciliaris, N. parile, O. tartarea and antioxidant activity of different lichens was also recorded P. centrifuga was analyzed against seventeen by other researchers [24-26]. microorganisms using the disc diffusion method and by In most lichens, phenols including depsides, measuring the MIC values (Table 2,3). The methanol depsidones and dibenzofurans are, important extract of P. centrifuga had the strongest antimicrobial components with antioxidant activity [7,9,27]. In activity, inhibiting the growth of all the bacteria tested this study, we found that the content of phenol in the (diameters of growth inhibition zones ranged from 11 to methanol extract of different species is very different. 36 mm and MIC values were 0.23 to 7.5 mg/ml). The The highest value was seen in the methanol extract of P. extract of this lichen had a strong antifungal activity, centrifuga. It was established that there was a high level too, inhibiting growth in eight out of the eleven fungal of correlation between the content of phenol and the total species. antioxidant activity, as well as between the total phenols The methanol extract of O. tartarea also inhibited and the reducing power. Overall, in lichen extracts with the growth of all the tested bacteria with the zones of a higher content of the phenols (P. centrifuga), a greater inhibition from 28 mm for Pseudomonas aeruginosa to antioxidant activity and reducing power was observed. 8 mm for Escherichia coli and relatively low. MIC values These results support findings from Behera et al. [28] from 0.23 to 7.5 mg/ml of the extract for the same which demonstrated a significant correlation between microorganisms. Related to the fungi, this extract had the content of the total phenols and the antioxidant a selective activity, inhibiting the growth in 6 out of 11 activity of the methanol extract of the lichen Usnea tested species with the diameter of the inhibited zones ghattensis. Moreover, similar results have also been ranging from 7 to 18 mm. The MIC values were from shown with other lichen and plant species [8,29-31]. 3.75 to 30 mg/ml of the extract related to the sensitive Interestingly however, Odabasoglu et al. [8] reported microorganisms. that in the methanol extract of some lichens (Lobaria The methanol extract of A. ciliaris had a moderate pulmonaria and Usnea longissima) no correlation was antimicrobial activity, inhibited the growth of five out observed between the total phenol and the antioxidant of six tested species of bacteria and five out of eleven activity, suggesting that the phenols from different tested species of fungi. lichens can have different antioxidant activity that may The methanol extract of N. parile showed the weakest depend on other, non-phenol components. antibacterial and antifungal activity, inhibiting growth of Lichens are unique organisms which synthesize only half the bacteria species tested and three out of numerous secondary metabolites that have high 11 fungal species, with small values of the diameter of biological activity, and are used in pharmaceutical the inhibition of growth of the sensitive microorganisms preparations [32]. The antimicrobial activity of the (8-15 mm) and the high MIC values up to 30 mg/ml of methanol extracts of the examined lichens showed a the extract. range of activity, depending on the species of lichen, In a negative control, DMSO in applied concentration concentration of the extract and the tested organism. The of lichen extract solvent had no inhibitory effect on the extract of P. centrifuga had the strongest antimicrobial tested organisms. Streptomycin, used as a positive activity among the tested species in this study, inhibiting control, inhibited growth of all the tested bacteria, and most of the bacteria and fungi tested. It is possible that ketoconazole of fungi. the observed differences in the antimicrobial activities may reflect the presence of different components with the antimicrobial activity in the extracts of different 4. Discussion lichen species [14]. The extracts of all the examined lichens in the same In the present study, in vitro antioxidant, antibacterial concentrations showed a stronger antibacterial than and antifungal activities of methanol extracts from antifungal activity. Extracts of P. centrifuga and O. tartarea the lichens Anaptychia ciliaris, Nephroma parile, had inhibitory activity against all the bacteria tested, while Ochrolechia tartarea and Parmelia centrifuga were A. ciliaris acted on five andN. parile on three out of the six examined for the first time. species tested. Overall, antifungal activity of extracts was

653 B. Ranković et al.

weaker: P. centrifuga acted on eight, O. tartarea on six, In addition, the lichen extracts showed a selective A. ciliaris on five, andN. parile on three out of the eleven and moderate inhibitory effect on the growth of fungi, tested species. The stronger antibacterial than antifungal whose cell wall is made of polisaccharides such as activity of extracts of different lichen species was reported chitin and glucane [37]. All extracts tested, also in by numerous researchers [11,12,33], while some other higher concentrations, inhibited a smaller number observed strong antibacterial activity of some lichens of fungi than bacteria [11,12,33]. Higher resistance and no inhibtory effects against filamentous fungi. The of fungi to the antimicrobial agents contained in the extracts used in this study, had a stronger antibacterial lichen extracts was observed by many researchers and activity on Gram-positive bacteria (Micrococcus is explained by the poor permeability of their cell wall lysodeikticus, Enterococcus fecalis) compared to [35,37]. Gram-negative bacteria (Escherichia coli, Klebsiella In summary, our data suggest that the extracts pneumoniae). This observation is in accordance with of the tested lichens P. centrifuga, O. tartarea and A. other studies [33,34], focused on the antibacterial activity ciliaris could be used as potential natural sources of which have demonstrated that Gram-positive bacteria are antioxidants. more sensitive to the antibacterial activity than the Gram- negative bacteria due to differences in the composition and permeability of the cell wall [35]. The cell wall of Acknowledgements Gram-positive bacteria is made of peptidoglucanes and teichoic acids, while the cell wall of Gram-negative This work was financed in part by the Ministry of Science, bacteria is made of peptidoglucanes, lipopolysacharides Technology and Development of the Republic of Serbia and lipoproteins [36,37]. and is the result of studies in Project 143041.

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