- Email: [email protected]
Chemical composition of three Parmelia lichens and antioxidant, antimicrobial and cytotoxic activities of some their major metabolites
Phytomedicine 19 (2012) 1166–1172
Contents lists available at SciVerse ScienceDirect
Phytomedicine journal homepage: www.elsevier.de/phymed
Chemical composition of three Parmelia lichens and antioxidant, antimicrobial and cytotoxic activities of some their major metabolites Nedeljko Manojlovic´ a , Branislav Rankovic´ b , Marijana Kosanic´ b,∗ , Perica Vasiljevic´ c , Tatjana Stanojkovic´ d a
Department of Pharmacy, Medical Faculty, University of Kragujevac, 34000 Kragujevac, Serbia Department of Biology, Faculty of Science, University of Kragujevac, 34000 Kragujevac, Serbia c Department of Biology and Ecology, Faculty of Sciences and Mathematics, University of Niˇs, 18000 Niˇs, Serbia d Institute of Oncology and Radiology of Serbia, 11000 Belgrade, Serbia b
a r t i c l e
i n f o
Keywords: Lichens HPLC-UV Chemical composition Biological activities
a b s t r a c t The aim of this study is to investigate chemical composition of acetone extracts of the lichens Parmelia caperata, P. saxatilis and P. sulcata and antioxidant, antimicrobial and anticancer activities of some their major metabolites. The phytochemical analysis of acetone extracts of three Parmelia lichens were determined by HPLC-UV method. The predominant phenolic compounds in these extracts were protocetraric and usnic acids (P. caperata) and depsidone salazinic acid (other two species). Besides these compounds, atranorin and chloroatranorin, were also detected in some of these extracts. Antioxidant activity of their isolated metabolites was evaluated by free radical scavenging, superoxide anion radical scavenging and reducing power. As a result of the study salazinic acid had stronger antioxidant activity than protocetraric acid. The antimicrobial activity was estimated by determination of the minimal inhibitory concentration by the broth microdilution method. Both compounds were highly active with minimum inhibitory concentration values ranging from 0.015 to 1 mg/ml. Anticancer activity was tested against FemX (human melanoma) and LS174 (human colon carcinoma) cell lines using MTT method. Salazinic acid and protocetraric acid were found to be strong anticancer activity toward both cell lines with IC50 values ranging from 35.67 to 60.18 g/ml. The present study shows that tested lichen compounds demonstrated a strong antioxidant, antimicrobial, and anticancer effects. That suggest that these lichens can be used as new sources of the natural antimicrobial agents, antioxidants and anticancer compounds. © 2012 Elsevier GmbH. All rights reserved.
Introduction Lichens are complex symbiotic associations between a fungus (mycobiont) and photobiont which can be either an alga or cyanobacteria (Bates et al. 2011). They are proven as the earliest colonizers of terrestrial habitats on the earth with a worldwide distribution from artic to tropical regions and from the plains to the highest mountains. Their specific, even extreme, conditions existence, slow growth and long life are the reason for producing of numerous protective compounds against different physical and biological influences (Mitrovic´ et al. 2011). Lichens synthesize a variety of secondary metabolites, mostly from fungal metabolism. They are crystals deposited on the surface of hiphes. They are poorly soluble in water and can usually be
∗ Corresponding author. Tel.: +381 34336223; fax: +381 34335040. ´ E-mail address: [email protected] (M. Kosanic). 0944-7113/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.phymed.2012.07.012
isolated from a lichen by organic dilutants (Otzurk et al. 1999). More than one hundred secondary metabolites, mainly monoaromatics, depsides, depsidones, pulvinates, dibenzofurans, anthraquinones and xanthones, characteristic of lichen have been detected and isolated (Molnar and Farkaˇs 2010). Chemicals structures of these classes of compounds are similar and identification is often very difficult.For a long time, some lichen species have been used in traditional medicine in the treatment of numerous infectious diseases (Bown 2001). The use of lichens in medicine is based on the fact that they contain unique and varied biologically active substances. Lichen substances exert a wide variety of biological actions including antibiotic, antimycotic, antiviral, anti-inflammatory, analgesic, antipyretic, antiproliferative and cytotoxic effects (Kosanic´ et al. 2012a; Manojlovic´ et al. 2010). Thus, the aim of the present work was to identify of secondary metabolites of P. caperata, P. saxatilis and P. sulcata by HPLC-UV and to evaluate the antioxidant capacity, antimicrobial and anticancer activities of the acetone extracts from this lichen as well as their major secondary metabolites.
N. Manojlovi´c et al. / Phytomedicine 19 (2012) 1166–1172
Material and methods
to ascorbic acid and usnic acid. The DPPH radical concentration was calculated using the following equation:
Lichen samples DPPH scavenging effect (%) = Lichen samples of P. caperata (L.) Ach. P. sulcata (Taylor) and P. saxatilis (L.) Ach. were collected from Kopaonik, Serbia, in September of 2011. The demonstration samples are preserved in facilities of the Department of Biology and Ecology of Kragujevac, Faculty of Science. Determination of the investigated lichens was accomplished using standard methods. Preparation of the lichen extracts Finely dry ground thalli of the investigated lichens (100 g) were extracted using acetone in a Soxhlet extractor. The extracts were filtered and then concentrated under reduced pressure in a rotary evaporator. The dry extracts were stored at −18 ◦ C until they were used in the tests. The extracts were dissolved in 5% dimethylsulfoxide (DMSO) for the experiments. High performance liquid chromatography (HPLC) analysis Dry lichen extracts were redissolved in 500 l of acetone and analyzed on an 1200 Series HPLC (Agilent Technologies) instrument with C18 column (C18; 25 cm × 4.6 mm, 10 m) and a UV spectrophotometric detector with methanol–water–phosphoric acid (75:25:0.9, v/v/v) solvent. Methanol was of HPLC grade and was purchased from Merck (Darmstadt, Germany). Phosphoric acid was analytical-grade reagent. Deionized water used throughout the experiments was generated by a Milli-Q academic water purification system (Milford, MA, USA). The flow rate was 1.0 ml/min. The sample injection volume was 10 l. The standards used were obtained from the following sources: salazinic acid (SAL, tR = 3.01 ± 0.20 min) was isolated from lichen Lobaria pulmonaria, protocetraric acid (PRO, tR = 3.62 ± 0.20 min) isolated from lichen Toninia candida, usnic acid (USN, tR = 10.53 ± 0.30 min), atranorin (ATR, tR = 11.72 ± 0.10 min) and chloroatranorin (CHL, tR = 13.23 ± 0.20 min) from lichen Evernia prunastri. Isolation of lichen metabolites Usnic acid (190 mg) was isolated from the acetone extract of P. caperata (500 mg) by precipitation in benzene, and in this study it has been used as a standard compound. Protocetraric acid was isolated from the filtrate residue using a silica gel column (0.149–0.074 mm; 100–200 mesh) with methanol–chloroform gradient solvent (10:1 and 5:1). Salazinic acid (300 mg) was isolated from acetone extract of P. saxatilis (500 mg) by precipitation in acetone. All isolated compounds were identified by their melting points and spectroscopic data (Huneck and Yoshimura 1996). Antioxidant activity Scavenging DPPH radicals The free radical scavenging activity of samples was measured by 1,1-diphenyl-2-picryl-hydrazil (DPPH). The method used is similar to the method previously used by some authors (Ibanez et al. 2003; Dorman et al. 2004) but was modified in details. Two milliliters of methanol solution of DPPH radical in the concentration of 0.05 mg/ml and 1 ml of test samples (1000, 500, 250, 125 and 62.5 g/ml) were placed in cuvettes. The mixture was shaken vigorosly and allowed to stand at room temperature for 30 min. Then the absorbance was measured at 517 nm in spectrophotometer (“Jenway”, UK). Free radical scavenging activity was compared
1167
A − A 0 1 A0
× 100
where A0 is the absorbance of the negative control and A1 is the absorbance of reaction mixture or standard. The inhibition concentration at 50% inhibition (IC50 ) was the parameter used to compare the radical scavenging activity. Reducing power The reducing power of samples was determined according to the method of Oyaizu (1986). One milliliter of test samples (1000, 500, 250, 125 and 62.5 g/ml) were mixed with 2.5 ml of phosphate buffer (2.5 ml, 0.2 M, pH 6.6) and potassium ferricyanide (2.5 ml, 1%). The mixtures were incubated at 50 ◦ C for 20 min. Then, trichloroacetic acid (10%, 2.5 ml) was added to the mixture and centrifuged. Finally, the upper layer was mixed with distilled water (2.5 ml) and ferric chloride (0.5 ml; 0.1%). The absorbance of the solution was measured at 700 nm in spectrophotometer (“Jenway”, UK). Higher absorbance of the reaction mixture indicated that the reducing power is increased. Reducing power was compared to ascorbic acid and usnic acid. Superoxide anion radical scavenging activity The superoxide anion radical scavenging activity of samples was detected according to the method of Nishimiki et al. (1972). Briefly, 0.1 ml of test samples (1000, 500, 250, 125 and 62.5 g/ml) was mixed with 1 ml nitroblue tetrazolium (NBT) solution (156 M in 0.1 M phosphate buffer, pH 7.4) and 1 ml nicotinamide adenine dinucleotide (NADH) solution (468 M in 0.1 M phosphate buffer, pH 7.4). The reaction was started by adding 100 L of phenazine methosulfate (PMS) solution (60 M in 0.1 M phosphate buffer, pH 7.4). The mixture was incubated at room temperature for 5 min, and the absorbance was measured at 560 nm in spectrophotometer (“Jenway”, UK) against blank samples. Decreased absorbance indicated increased superoxide anion radical scavenging activity. Superoxide anion scavenging activity was compared to ascorbic acid and usnic acid. The percentage inhibition of superoxide anion generation was calculated using the following formula: Superoxide anion scavenging activity (%) =
A − A 0 1 A0
× 100
where A0 is the absorbance of the negative control and A1 is the absorbance of reaction mixture or standards. The inhibition concentration at 50% inhibition (IC50 ) was the parameter used to compare the radical scavenging activity. Antimicrobial activity Microorganisms and media The following bacteria were used as test organisms in this study: Bacillus mycoides (ATCC 6462), Bacillus subtilis (ATCC 6633), Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922) and Klebsiella pneumoniae (ATCC 13883). All the bacteria used were obtained from the American Type Culture Collection (ATCC). Their identification was confirmed at the Microbiological Laboratory of Kragujevac, University of Kragujevac, Department of Biology. The fungi used as test organisms were: Aspergillus flavus (ATCC 9170), Aspergillus fumigatus (DBFS 310), Candida albicans (ATCC 10231), Penicillium purpurescens (DBFS 418) and Penicillium verrucosum (DBFS 262). They were from the from the American Type Culture Collection (ATCC) and the mycological collection maintained by the Mycological Laboratory within the Department of Biology of Kragujevac University’s Faculty of Science (DBFS). Bacterial cultures were maintained on Müller-Hinton agar substrates (Torlak, Belgrade).
1168
N. Manojlovi´c et al. / Phytomedicine 19 (2012) 1166–1172
Fungal cultures were maintained on potato dextrose (PD) agar and Sabourad dextrose (SD) agar (Torlak, Belgrade). All cultures were stored at 4 ◦ C and subcultured every 15 days. The sensitivity of microorganisms to tested samples was tested by determining the minimal inhibitory concentration (MIC). Bacterial inoculi were obtained from bacterial cultures incubated for 24 h at 37 ◦ C on Müller-Hinton agar substrate and brought up by dilution according to the 0.5 McFarland standard to approximately 108 CFU/ml. Suspensions of fungal spores were prepared from fresh mature (3–7-day old) cultures that grew at 30 ◦ C on a PD agar substrate. Spores were rinsed with sterile distilled water, used to determine turbidity spectrophotometrically at 530 nm, and then further diluted to approximately 106 CFU/ml according to the procedure recommended by NCCLS (1998). Minimal inhibitory concentration (MIC) The minimal inhibitory concentration (MIC) was determined by the by the broth microdilution method with using 96-well micro-titer plates (Sarker et al. 2007). A series of dilutions with concentrations ranging from 4 to 0.00181 mg/ml for test samples was used in the experiment against every microorganism tested. The starting solutions of test samples were obtained by measuring off a certain quantity of extract and dissolving it in DMSO. Two-fold dilutions of test samples were prepared in Müller-Hinton broth for bacterial cultures and SD broth for fungal cultures. The minimal inhibitory concentration was determined with resazurin. Resazurin is an oxidation–reduction indicator used for the evaluation of microbial growth. It is a blue non-fluorescent dye that becomes pink and fluorescent when reduced to resorufin by oxidoreductases within viable cells. The boundary dilution without any changing color of resazurin was defined as the minimal inhibitory concentration (MIC) for the tested microorganism at the given concentration. As a positive control of growth inhibition, streptomycin, ketoconazole and usnic acid were used. A DMSO solution was used as a negative control for the influence of the solvents. All experiments were performed in triplicate. Cytotoxic activity Cell lines The human melanoma FemX and human colon carcinoma LS174 cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA). Both cancer cell lines were maintained in the recommended RPMI-1640 medium supplemented with 10% heat-inactivated (56 ◦ C) fetal bovine serum, l-glutamine (3 mM), streptomycin (100 mg = ml), penicillin (100 IU = ml), and 25 mM HEPES and adjusted to pH 7.2 by bicarbonate solution. Cells were grown in a humidified atmosphere of 95% air and 5% CO2 at 37 ◦ C. Treatment of cell lines Stock solutions (100 mg/ml) of test samples, made in dimethylsulfoxide (DMSO), were dissolved in corresponding medium to the required working concentrations. Neoplastic FemX cells (5000 cells per well) and neoplastic LS174 cells (7000 cells per well) were seeded into 96-well microtiter plates, and 24 h later, after the cell adherence, five different, double diluted, concentrations of investigated compounds, were added to the wells. Final concentrations applied to target cells were 200, 100, 50, 25 and 12.5 g/ml, except to the control wells, where only nutrient medium was added to the cells. Nutrient medium was RPMI 1640 medium, supplemented with l-glutamine (3 mM), streptomycin (100 lg/ml), and penicillin (100 IU/ml), 10% heat inactivated (56 ◦ C) fetal bovine serum (FBS) and 25 mM Hepes, and was adjusted to pH 7.2 by bicarbonate solution. The cultures were incubated for 72 h.
Determination of cell survival (MTT test) The effect of test samples on cancer cell survival was determined by MTT test (microculture tetrazolium test), according to Mosmann (1983) with modification by Ohno and Abe (1991) 72 h upon addition of the compounds, as it was described earlier. Briefly, 20 l of MTT solution (5 mg/ml PBS) were added to each well. Samples were incubated for further 4 h at 37 ◦ C in 5% CO2 and humidified air atmosphere. Then, 100 l of 10% SDS were added to extract the insoluble product formazan, resulting from the conversion of the MTT dye by viable cells. The number of viable cells in each well was proportional to the intensity of the absorbance of light, which was then read in an ELISA plate reader at 570 nm. Absorbance (A) at 570 nm was measured 24 h later. To get cell survival (%), A of a sample with cells grown in the presence of various concentrations of the investigated test samples was divided with control optical density (the A of control cells grown only in nutrient medium), and multiplied by 100. It was implied that A of the blank was always subtracted from A of the corresponding sample with target cells. IC50 concentration was defined as the concentration of an agent inhibiting cell survival by 50%, compared with a vehicle-treated control. As a positive control were used cis-diamminedichloroplatinum (Cis-DDP) and usnic acid. All experiments were done in triplicate. Flow cytometry analysis Cellular DNA content and cell distribution were quantified by flow cytometry using propidium iodide (PI). Cells (3 × 105 cells/well) were seeded in 6-well plates and incubated with or without IC50 concentration of investigated compounds for 24 h. After treatment, the cells were collected by trypsinization, and fixed in ice-cold 70% ethanol at −20 ◦ C overnight. After fixation, the cells were washed in PBS and pellets obtained by centrifugation was treated with RNase (100 lg/ml) at 37 ◦ C temperature for 30 min and then incubated with propidium iodide (PI) (40 lg/ml) for at least 30 min. DNA content and cell cycle distribution were analyzed using a Becton Dickinson FAC-Scan flow cytometer. Flow cytometry analysis was performed using a CellQuestR (Becton Dickinson, San Jose, CA, USA), on a minimum of 10,000 cells per sample (Clothier 1995). Statistical analyses Statistical analyses were performed with the EXCEL and SPSS softwares package. To determine the statistical significance of antioxidant activity, Student’s t-test was used. All values are expressed as mean ± SD of three parallel measurements. Results and discussion Chromatograms for standards and P. caperata, P. sulcata, and P. saxatilis acetone extracts eluted by HPLC are represented in Figs. 1 and 2. As it is evidenced in the chromatograms, there were the presence of depsidones, depsides and dibenzofuran as the most abundant substance classes in the extracts examined. The HPLC chromatogram of the acetone extracts of P. sulcata and P. saxatilis showed that the highest peaks belong to the depsidone salazinic acid (SAL) (tR = 3.01 ± 0.20 min). On the other hand, protocetraric acid (PRO) (tR = 3.62 ± 0.20 min) and usnic acid (USN) (tR = 10.53 ± 0.30 min) were detected as the major secondary metabolites in the acetone extract of P. caperata. Identification of these compounds was achieved by comparison of their tR values with the standard substances previously isolated from lichens. The UV absorbance spectral data (200–400 nm) also corresponded with those of standards and found in Yoshimura et al. (1994). Table 1 shows the retention time of the detected lichen substances and their absorbance maxima (nm). The structures of the detected compounds are shown in Fig. 3. Some of the compounds are identified for the first time in this lichen. After detection of the present
N. Manojlovi´c et al. / Phytomedicine 19 (2012) 1166–1172
1169
Fig. 1. Chromatogram of the standards used for identification of the compounds present in Parmelia species.
compounds in lichens, major lichen metabolites in these species (salazinic acid, protocetraric acid and usnic acid – which was used as a standard compound) were isolated and used for antioxidant, antimicrobial and anticancer investigations. The scavenging of DPPH radicals and superoxide anion radicals by the tested compounds is shown in Table 2. There was a statistically significant difference between samples and control (p < 0.05). Salazinic acid showed stronger DPPH radicals and superoxide anion radicals scavenging activity than protocetraric acid. The IC50 values were 91.57 and 138.23 g/ml for salazinic acid and 119.10 and
177.60 g/ml for protocetraric acid (for DPPH radicals and superoxide anion radicals scavenging activity, respectively). As a shown in Table 3, salazinic acid also demonstrated greater reducing power. Various antioxidant activities were compared to ascorbic acid and usnic acid. The results showed that standards have stronger activity than tested samples. The tested lichen compounds have a strong antioxidant activity against various oxidative systems in vitro. The isolated components belong phenols which indicate an important role of phenol in the antioxidant activity for lichens. In fact, number of previous studies
Fig. 2. HPLC chromatograms acquired at 254 nm of the acetone extracts of P. caperata, P. sulcata and P. saxatilis. Chromatographic peaks identities are reported in Table 1.
1170
N. Manojlovi´c et al. / Phytomedicine 19 (2012) 1166–1172
Fig. 3. Structures of the identified compounds.
found that the lichens where found the higher content of phenols exert stronger antioxidant activity (Behera et al. 2009; Kosanic´ et al. 2012a), which means that phenols are important antioxidants. Antioxidant effect of some other lichen compounds was also studied by other researchers. For example, antioxidant activity was found for lecanoric acid, salazinic acid, stictic acid, usnic acid (Luo et al. 2009; Amo de Paz et al. 2010). The antimicrobial activity of the lichen components against the test microorganisms are shown in Table 4. Salazinic acid and protocetraric acid showed similar antimicrobial activity, but antibacterial activity was stronger than antifungal activity for both
Table 1 Retention time of the examined lichen compounds and their absorbance maxima (nm). Peaks
Compound
Retention time (tR ± SD)a (min)
SAL PRO USN ATR CHL
Salazinic acid Protocetraric acid Usnic acid Atranorin Chloroatranorin
3.01 3.62 10.53 11.72 13.23
± ± ± ± ±
0.20 0.20 0.30 0.10 0.20
Absorbance maxima (nm) UV spectrum 213, 238, 312 212, 240, 320m 234, 282 210, 252, 321m 213, 252, 315m , 350
m, minor absorbance maximum. a Values are the means of three determinations ± SD.
compounds. The MIC for components relative to the tested microorganisms ranged from 0.015 to 1 mg/ml. The antimicrobial activity was compared with the standard antibiotics, streptomycin (for bacteria) and ketoconazole (for fungi) and usnic acid. As shown in Table 2, standards have stronger activity than tested samples. In a negative control, DMSO had no inhibitory effect on the tested organisms. Previously, numerous lichen compounds were screened for antimicrobial activity in search of the new antimicrobial agents (Candan et al. 2006; Kosanic´ and Rankovic´ 2011; Turk et al. 2006). In our experiments, the tested lichen compounds show very strong antimicrobial activity. Antibacterial activity was stronger than antifungal. This observation is in accordance with other studies (Yang
Table 2 DPPH radical scavenging activity and superoxide anion scavenging activity of isolated compounds. Lichen compounds
DPPH radical scavenging IC50 (g/ml)
Superoxide anion scavenging IC50 (g/ml)
Salazinic acid Protocetraric acid Usnic acid Ascorbic acid
91.57 138.23 60.73 6.42
119.10 177.60 97.28 115.61
Ascorbic acid and usnic acid were used as a standards.
N. Manojlovi´c et al. / Phytomedicine 19 (2012) 1166–1172
1171
Table 3 Reducing power of isolated compounds. Lichen compounds
Salazinic acid Protocetraric acid Usnic acid Ascorbic acid
Absorbance (700 nm) 1000 g/ml
500 g/ml
250 g/ml
125 g/ml
62.5 g/ml
0.2722 0.1209 0.6723 2.113
0.1842 0.0903 0.5468 1.654
0.0758 0.0721 0.2692 0.9571
0.0534 0.0502 0.1249 0.4783
0.0321 0.0312 0.0901 0.2472
Ascorbic acid and usnic acid were used as a standards. Table 4 Minimum inhibitory concentration (MIC) of isolated compounds. Lichen compounds
Salazinic acid
Protocetraric acid
Usnic acid
S
K
B. mycoides B. subtilis E. coli K. pneumoniae S. aureus A. flavus A. fumigatus C. albicans P. purpurescens P. verrucosum
0.015 0.0312 1 0.5 0.125 1 1 0.25 1 0.5
0.015 0.015 1 0.5 0.015 1 0.25 0.25 1 0.5
0.0008 0.0008 0.25 0.0625 0.125 0.5 0.25 0.125 0.5 0.5
7.81 7.81 31.25 31.25 15.72 – – – – –
– – – – – 3.9 3.9 1.95 3.9 3.9
Values given as mg/ml for lichen compounds and as g/ml for antibiotics. Values are the mean of three replicate. S, streptomycin; K, ketoconazole and usnic acid were used as a standards.
and Anderson 1999; Kosanic´ et al. 2012b), focused on the antimicrobial activity which have demonstrated that bacteria are more sensitive to the antimicrobial activity than the fungi due to differences in the composition and permeability of the cell wall. The cytotoxic activity of the tested compounds against the tested cell lines is shown in Table 5. The tested samples exhibited high cytotoxic activity against the target cells in vitro. The IC50 value for both compounds relative to the tested cells ranged from 35.67 to 60.18 g/ml. The best cytotoxic activity was exhibited the salazinic acid. As shown in this table, Cis-DDP and usnic acid have slightly better cytotoxic activity than tested samples. The effect of protocetraric acid and salazinic acid on cell cycle progression was investigated in FemX and LS174 cells. Cell cycle was assessed by cytofluorimetric analysis, using propidium iodide to label DNA. Results are given in Table 6. Cells treated for 24 h with IC50 (g/ml) concentrations of both compounds showed a significant increase in the number of cells in sub-G1 phase, and an reduction in the number of cells in S phase. Specially, incubation of the cell line FemX with salazinic acid induced G0-G1 and G2-M cell cycle blockade. In addition, treatment LS174 cells of protocetraric acid and salazinic acid leads to a somewhat more pronounced increase in sub-G1 phase and concomitant decrease in G2/M were observed, supporting a G1 phase arrest. An increase in cells containing sub-G1 amounts of DNA was observed, indicating that the compounds was inducing cell death. Some literature data reported that lichen components are responsible for anticancer activities of lichens (Bucar et al. 2004; Burlando et al. 2009). However, it is difficult to determine the contribution of individual components for the overall anticancer effect.
Table 5 Growth inhibitory effects of isolated compounds on FemX and LS174 cell lines CisDDP and usnic acid were used as a standards. Lichen compounds
Apoptotic cells sub-G1
G1
S
G2/M
FemX Control Salazinic acid Protocetraric acid Usnic acid
0.15 16.23 16.20 10.18
62.65 43.74 43.81 54.38
16.69 13.64 15.30 14.64
16.64 24.93 18.62 20.37
LS174 Control Salazinic acid Protocetraric acid Usnic acid
3.68 20.88 22.90 12.89
51.68 41.37 39.71 52.77
12.61 14.27 16.96 15.06
30.19 15.90 23.92 19.28
Effect of compounds on cell cycle phase distribution. FemX and LS174 cell lines were exposed to compounds (IC50 g/ml) for 24 h and then collected for analysis of cell cycle phase distribution using flow cytometry. Percentage of cells under different stages of cell cycles (sub-G1, G1, S, G2/M) is shown. Usnic acid was used as a standard compound.
Often, the activity of lichens may be the result of synergistic or antagonistic effect of several compounds. In conclusion, it can be stated that tested lichen compounds have a strong antioxidant, antimicrobial and anticancer activity in vitro, which suggest that lichens could be good natural antioxidant, antimicrobial and anticancer agents. Acknowledgement This work was financed in part by the Ministry of Science, Technology, and Development of the Republic of Serbia and was carried out within the framework of projects no. 173032, 175011 and 172015. References
IC50 (g/ml) FemX
Salazinic acid Protocetraric acid Usnic acid Cis-DDP
Table 6 Effect of isolated compounds on cell cycle progression.
39.02 58.68 12.72 0.94
LS174 ± ± ± ±
0.56 2.11 0.35 0.35
35.67 60.18 15.66 2.3
± ± ± ±
1.89 0.59 1.45 0.31
Amo de Paz, G., Raggio, J., Gomez-Serranillos, M.P., Palomino, O.M., Gonzales-Burgos, E., Carretero, M.E., Crespo, A., 2010. HPLC isolation of antioxidant constituents from Xanthoparmelia spp. Journal of Pharmaceutical and Biomedical Analysis 53, 165–171. Bates, S.T., Cropsey, G.W., Caporaso, J.G., Knight, R., Fierer, N., 2011. Bacterial communities associated with the lichen symbiosis. Applied and Environment Microbiology 77, 1309–1314.
1172
N. Manojlovi´c et al. / Phytomedicine 19 (2012) 1166–1172
Behera, B.C., Verma, N., Sonone, A., Makhija, U., 2009. Optimization of culture conditions for lichen Usnea ghattensis G. Awasthi to increase biomass and antioxidant metabolite production. Food Technology and Biotechnology 47, 7–12. Bown, D., 2001. Encyclopedia of Herbs and Their Uses. Dorling Kindersley, London. Bucar, F., Schneider, I., Ogmundsdottir, H., Ingolfsdottir, K., 2004. Antiproliferative lichen compounds with inhibitory activity on 12(S)-HETE production in human platelets. Phytomedicine 11, 602–606. Burlando, B., Ranzato, E., Volante, A., Appendino, G., Pollastro, F., Verotta, L., 2009. Antiproliferative effects on tumour cells and promotion of keratinocyte wound healing by different lichen compounds. Planta Medica 75, 607–613. Candan, M., Yilmaz, M., Tay, T., Kivanc, M., Turk, H., 2006. Antimicrobial activity of extracts of the lichen Xanthoparmelia pokornyi and its gyrophoric and stenosporic acid constituents. Zeitschrift fur Naturforschung C 61, 319–323. Clothier, R.H., 1995. The FRAME cytotoxicity test. Methods in Molecular Biology 43, 109–118. Dorman, H.J., Bachmayer, O., Kosar, M., Hiltunen, R., 2004. Antioxidant properties of aqueous extracts from selected Lamiaceae species grown in Turkey. Journal of Agricultural and Food Chemistry 52, 762–770. Huneck, S., Yoshimura, I., 1996. Identification of Lichen Substances. Springer-Verlag, Heidelberg, Berlin. Ibanez, E., Kubatova, A., Senorans, F.J., Cavero, S., Reglero, G., Hawthorne, S.B., 2003. Subcritical water extraction of antioxidant compounds from rosemary plants. Journal of Agricultural and Food Chemistry 51, 375–382. ´ M., Rankovic, ´ B., Stanojkovic, ´ T., 2012a. Antioxidant, antimicrobial and antiKosanic, cancer activity of 3 Umbilicaria species. Journal of Food Science 77, T20–T25. ´ B., 2011. Antioxidant and antimicrobial properties ´ M., Rankovic, Kosanic, of some lichens and their constituents. Journal of Medicinal Food 14, 1624–1630. ´ M., Rankovic, ´ B., Stanojkovic, ´ T., 2012b. Antioxidant, antimicrobial, and antiKosanic, cancer activities of three Parmelia species. Journal of the Science of Food and Agriculture, http://dx.doi.org/10.1002/jsfa.5559. Luo, H., Yamamoto, Y., Kim, J.A., Jung, J.S., Koh, Y.J., Hur, J.S., 2009. Lecanoric acid, a secondary lichen substance with antioxidant properties from Umbilicaria antarctica in maritime Antarctica (King George Island). Polar Biology 32, 1033–1040.
´ N., Vasiljevic, ´ P., Gritsanapan, W., Supabphol, R., Manojlovic, ´ I., 2010. Manojlovic, Phytochemical and antioxidant studies of Laurera benguelensis growing in Thailand. Biological Research 43, 169–176. ´ T., Stamenkovic, ´ S., Cvetkovic, ´ V., Nikolic, ´ M., Toˇsic, ´ S., Stojiˇcic, ´ D., 2011. Mitrovic, Lichens as source of versatile bioactive compounds. Biologica Nyssana 2, 1–6. Molnar, K., Farkaˇs, E., 2010. Current results on biological activities of lichen secondary metabolites: a review. Zeitschrift fur Naturforschung C 65, 157–173. Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Journal of Immunological Methods 65, 55–63. NCCLS (National Commitee for Clinical Laboratory Standards)., 1998. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Conidiumforming Filamentous Fungi: Proposed Standard M38-P. NCCLS, Wayne, PAUSA. Nishimiki, M., Rao, N.A., Yagi, K., 1972. The occurrence of super-oxide anion in the reaction of reduced phenazine methosulfate and molecular oxygen. Biochemical and Biophysical Research Communications 46, 849–853. Ohno, M., Abe, T., 1991. Rapid colorimetric assay for the quantification of leukemia inhibitory factor (LIF) and interleukin-6 (IL-6). Journal of Immunological Methods 145, 199–203. Otzurk, S., Guvenc, S., Arikan, N., Yylmaz, O., 1999. Effect of usnic acid on mitotic index in root tips of Allium cepa L. Lagascalia 21, 47–52. Oyaizu, M., 1986. Studies on products of browning reaction prepared from glucoseamine. Japanese Journal of Nutrition 44, 307–314. Sarker, S.D., Nahar, L., Kumarasamy, Y., 2007. Microtitre plate-based antibacterial assay incorporating resazurin as an indicator of cell growth, and its application in the in vitro antibacterial screening of phytochemicals. Methods 42, 321–324. Turk, H., Yılmaz, M., Tay, T., Turk, A.O., Kıvanc, M., 2006. Antimicrobial activity of extracts of chemical races of the lichen Pseudevernia furfuracea and their physodic acid, chloroatranorin, atranorin, and olivetoric acid constituents. Zeitschrift fur Naturforschung C 61, 499–507. Yang, Y., Anderson, E.J., 1999. Antimicrobial activity of a porcine myeloperozidase against plant phatgenic bacteria and fungi. Journal of Applied Microbiology 86, 211–220. Yoshimura, I., Kinoshita, Y., Yamamoto, Y., Huneck, S., Yamada, Y., 1994. Analysis of secondary metabolites from lichen by high performance liquid chromatography with a photodiode array detector. Phytochemical Analysis 5, 197–205.
Contents lists available at SciVerse ScienceDirect
Phytomedicine journal homepage: www.elsevier.de/phymed
Chemical composition of three Parmelia lichens and antioxidant, antimicrobial and cytotoxic activities of some their major metabolites Nedeljko Manojlovic´ a , Branislav Rankovic´ b , Marijana Kosanic´ b,∗ , Perica Vasiljevic´ c , Tatjana Stanojkovic´ d a
Department of Pharmacy, Medical Faculty, University of Kragujevac, 34000 Kragujevac, Serbia Department of Biology, Faculty of Science, University of Kragujevac, 34000 Kragujevac, Serbia c Department of Biology and Ecology, Faculty of Sciences and Mathematics, University of Niˇs, 18000 Niˇs, Serbia d Institute of Oncology and Radiology of Serbia, 11000 Belgrade, Serbia b
a r t i c l e
i n f o
Keywords: Lichens HPLC-UV Chemical composition Biological activities
a b s t r a c t The aim of this study is to investigate chemical composition of acetone extracts of the lichens Parmelia caperata, P. saxatilis and P. sulcata and antioxidant, antimicrobial and anticancer activities of some their major metabolites. The phytochemical analysis of acetone extracts of three Parmelia lichens were determined by HPLC-UV method. The predominant phenolic compounds in these extracts were protocetraric and usnic acids (P. caperata) and depsidone salazinic acid (other two species). Besides these compounds, atranorin and chloroatranorin, were also detected in some of these extracts. Antioxidant activity of their isolated metabolites was evaluated by free radical scavenging, superoxide anion radical scavenging and reducing power. As a result of the study salazinic acid had stronger antioxidant activity than protocetraric acid. The antimicrobial activity was estimated by determination of the minimal inhibitory concentration by the broth microdilution method. Both compounds were highly active with minimum inhibitory concentration values ranging from 0.015 to 1 mg/ml. Anticancer activity was tested against FemX (human melanoma) and LS174 (human colon carcinoma) cell lines using MTT method. Salazinic acid and protocetraric acid were found to be strong anticancer activity toward both cell lines with IC50 values ranging from 35.67 to 60.18 g/ml. The present study shows that tested lichen compounds demonstrated a strong antioxidant, antimicrobial, and anticancer effects. That suggest that these lichens can be used as new sources of the natural antimicrobial agents, antioxidants and anticancer compounds. © 2012 Elsevier GmbH. All rights reserved.
Introduction Lichens are complex symbiotic associations between a fungus (mycobiont) and photobiont which can be either an alga or cyanobacteria (Bates et al. 2011). They are proven as the earliest colonizers of terrestrial habitats on the earth with a worldwide distribution from artic to tropical regions and from the plains to the highest mountains. Their specific, even extreme, conditions existence, slow growth and long life are the reason for producing of numerous protective compounds against different physical and biological influences (Mitrovic´ et al. 2011). Lichens synthesize a variety of secondary metabolites, mostly from fungal metabolism. They are crystals deposited on the surface of hiphes. They are poorly soluble in water and can usually be
∗ Corresponding author. Tel.: +381 34336223; fax: +381 34335040. ´ E-mail address: [email protected] (M. Kosanic). 0944-7113/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.phymed.2012.07.012
isolated from a lichen by organic dilutants (Otzurk et al. 1999). More than one hundred secondary metabolites, mainly monoaromatics, depsides, depsidones, pulvinates, dibenzofurans, anthraquinones and xanthones, characteristic of lichen have been detected and isolated (Molnar and Farkaˇs 2010). Chemicals structures of these classes of compounds are similar and identification is often very difficult.For a long time, some lichen species have been used in traditional medicine in the treatment of numerous infectious diseases (Bown 2001). The use of lichens in medicine is based on the fact that they contain unique and varied biologically active substances. Lichen substances exert a wide variety of biological actions including antibiotic, antimycotic, antiviral, anti-inflammatory, analgesic, antipyretic, antiproliferative and cytotoxic effects (Kosanic´ et al. 2012a; Manojlovic´ et al. 2010). Thus, the aim of the present work was to identify of secondary metabolites of P. caperata, P. saxatilis and P. sulcata by HPLC-UV and to evaluate the antioxidant capacity, antimicrobial and anticancer activities of the acetone extracts from this lichen as well as their major secondary metabolites.
N. Manojlovi´c et al. / Phytomedicine 19 (2012) 1166–1172
Material and methods
to ascorbic acid and usnic acid. The DPPH radical concentration was calculated using the following equation:
Lichen samples DPPH scavenging effect (%) = Lichen samples of P. caperata (L.) Ach. P. sulcata (Taylor) and P. saxatilis (L.) Ach. were collected from Kopaonik, Serbia, in September of 2011. The demonstration samples are preserved in facilities of the Department of Biology and Ecology of Kragujevac, Faculty of Science. Determination of the investigated lichens was accomplished using standard methods. Preparation of the lichen extracts Finely dry ground thalli of the investigated lichens (100 g) were extracted using acetone in a Soxhlet extractor. The extracts were filtered and then concentrated under reduced pressure in a rotary evaporator. The dry extracts were stored at −18 ◦ C until they were used in the tests. The extracts were dissolved in 5% dimethylsulfoxide (DMSO) for the experiments. High performance liquid chromatography (HPLC) analysis Dry lichen extracts were redissolved in 500 l of acetone and analyzed on an 1200 Series HPLC (Agilent Technologies) instrument with C18 column (C18; 25 cm × 4.6 mm, 10 m) and a UV spectrophotometric detector with methanol–water–phosphoric acid (75:25:0.9, v/v/v) solvent. Methanol was of HPLC grade and was purchased from Merck (Darmstadt, Germany). Phosphoric acid was analytical-grade reagent. Deionized water used throughout the experiments was generated by a Milli-Q academic water purification system (Milford, MA, USA). The flow rate was 1.0 ml/min. The sample injection volume was 10 l. The standards used were obtained from the following sources: salazinic acid (SAL, tR = 3.01 ± 0.20 min) was isolated from lichen Lobaria pulmonaria, protocetraric acid (PRO, tR = 3.62 ± 0.20 min) isolated from lichen Toninia candida, usnic acid (USN, tR = 10.53 ± 0.30 min), atranorin (ATR, tR = 11.72 ± 0.10 min) and chloroatranorin (CHL, tR = 13.23 ± 0.20 min) from lichen Evernia prunastri. Isolation of lichen metabolites Usnic acid (190 mg) was isolated from the acetone extract of P. caperata (500 mg) by precipitation in benzene, and in this study it has been used as a standard compound. Protocetraric acid was isolated from the filtrate residue using a silica gel column (0.149–0.074 mm; 100–200 mesh) with methanol–chloroform gradient solvent (10:1 and 5:1). Salazinic acid (300 mg) was isolated from acetone extract of P. saxatilis (500 mg) by precipitation in acetone. All isolated compounds were identified by their melting points and spectroscopic data (Huneck and Yoshimura 1996). Antioxidant activity Scavenging DPPH radicals The free radical scavenging activity of samples was measured by 1,1-diphenyl-2-picryl-hydrazil (DPPH). The method used is similar to the method previously used by some authors (Ibanez et al. 2003; Dorman et al. 2004) but was modified in details. Two milliliters of methanol solution of DPPH radical in the concentration of 0.05 mg/ml and 1 ml of test samples (1000, 500, 250, 125 and 62.5 g/ml) were placed in cuvettes. The mixture was shaken vigorosly and allowed to stand at room temperature for 30 min. Then the absorbance was measured at 517 nm in spectrophotometer (“Jenway”, UK). Free radical scavenging activity was compared
1167
A − A 0 1 A0
× 100
where A0 is the absorbance of the negative control and A1 is the absorbance of reaction mixture or standard. The inhibition concentration at 50% inhibition (IC50 ) was the parameter used to compare the radical scavenging activity. Reducing power The reducing power of samples was determined according to the method of Oyaizu (1986). One milliliter of test samples (1000, 500, 250, 125 and 62.5 g/ml) were mixed with 2.5 ml of phosphate buffer (2.5 ml, 0.2 M, pH 6.6) and potassium ferricyanide (2.5 ml, 1%). The mixtures were incubated at 50 ◦ C for 20 min. Then, trichloroacetic acid (10%, 2.5 ml) was added to the mixture and centrifuged. Finally, the upper layer was mixed with distilled water (2.5 ml) and ferric chloride (0.5 ml; 0.1%). The absorbance of the solution was measured at 700 nm in spectrophotometer (“Jenway”, UK). Higher absorbance of the reaction mixture indicated that the reducing power is increased. Reducing power was compared to ascorbic acid and usnic acid. Superoxide anion radical scavenging activity The superoxide anion radical scavenging activity of samples was detected according to the method of Nishimiki et al. (1972). Briefly, 0.1 ml of test samples (1000, 500, 250, 125 and 62.5 g/ml) was mixed with 1 ml nitroblue tetrazolium (NBT) solution (156 M in 0.1 M phosphate buffer, pH 7.4) and 1 ml nicotinamide adenine dinucleotide (NADH) solution (468 M in 0.1 M phosphate buffer, pH 7.4). The reaction was started by adding 100 L of phenazine methosulfate (PMS) solution (60 M in 0.1 M phosphate buffer, pH 7.4). The mixture was incubated at room temperature for 5 min, and the absorbance was measured at 560 nm in spectrophotometer (“Jenway”, UK) against blank samples. Decreased absorbance indicated increased superoxide anion radical scavenging activity. Superoxide anion scavenging activity was compared to ascorbic acid and usnic acid. The percentage inhibition of superoxide anion generation was calculated using the following formula: Superoxide anion scavenging activity (%) =
A − A 0 1 A0
× 100
where A0 is the absorbance of the negative control and A1 is the absorbance of reaction mixture or standards. The inhibition concentration at 50% inhibition (IC50 ) was the parameter used to compare the radical scavenging activity. Antimicrobial activity Microorganisms and media The following bacteria were used as test organisms in this study: Bacillus mycoides (ATCC 6462), Bacillus subtilis (ATCC 6633), Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922) and Klebsiella pneumoniae (ATCC 13883). All the bacteria used were obtained from the American Type Culture Collection (ATCC). Their identification was confirmed at the Microbiological Laboratory of Kragujevac, University of Kragujevac, Department of Biology. The fungi used as test organisms were: Aspergillus flavus (ATCC 9170), Aspergillus fumigatus (DBFS 310), Candida albicans (ATCC 10231), Penicillium purpurescens (DBFS 418) and Penicillium verrucosum (DBFS 262). They were from the from the American Type Culture Collection (ATCC) and the mycological collection maintained by the Mycological Laboratory within the Department of Biology of Kragujevac University’s Faculty of Science (DBFS). Bacterial cultures were maintained on Müller-Hinton agar substrates (Torlak, Belgrade).
1168
N. Manojlovi´c et al. / Phytomedicine 19 (2012) 1166–1172
Fungal cultures were maintained on potato dextrose (PD) agar and Sabourad dextrose (SD) agar (Torlak, Belgrade). All cultures were stored at 4 ◦ C and subcultured every 15 days. The sensitivity of microorganisms to tested samples was tested by determining the minimal inhibitory concentration (MIC). Bacterial inoculi were obtained from bacterial cultures incubated for 24 h at 37 ◦ C on Müller-Hinton agar substrate and brought up by dilution according to the 0.5 McFarland standard to approximately 108 CFU/ml. Suspensions of fungal spores were prepared from fresh mature (3–7-day old) cultures that grew at 30 ◦ C on a PD agar substrate. Spores were rinsed with sterile distilled water, used to determine turbidity spectrophotometrically at 530 nm, and then further diluted to approximately 106 CFU/ml according to the procedure recommended by NCCLS (1998). Minimal inhibitory concentration (MIC) The minimal inhibitory concentration (MIC) was determined by the by the broth microdilution method with using 96-well micro-titer plates (Sarker et al. 2007). A series of dilutions with concentrations ranging from 4 to 0.00181 mg/ml for test samples was used in the experiment against every microorganism tested. The starting solutions of test samples were obtained by measuring off a certain quantity of extract and dissolving it in DMSO. Two-fold dilutions of test samples were prepared in Müller-Hinton broth for bacterial cultures and SD broth for fungal cultures. The minimal inhibitory concentration was determined with resazurin. Resazurin is an oxidation–reduction indicator used for the evaluation of microbial growth. It is a blue non-fluorescent dye that becomes pink and fluorescent when reduced to resorufin by oxidoreductases within viable cells. The boundary dilution without any changing color of resazurin was defined as the minimal inhibitory concentration (MIC) for the tested microorganism at the given concentration. As a positive control of growth inhibition, streptomycin, ketoconazole and usnic acid were used. A DMSO solution was used as a negative control for the influence of the solvents. All experiments were performed in triplicate. Cytotoxic activity Cell lines The human melanoma FemX and human colon carcinoma LS174 cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA). Both cancer cell lines were maintained in the recommended RPMI-1640 medium supplemented with 10% heat-inactivated (56 ◦ C) fetal bovine serum, l-glutamine (3 mM), streptomycin (100 mg = ml), penicillin (100 IU = ml), and 25 mM HEPES and adjusted to pH 7.2 by bicarbonate solution. Cells were grown in a humidified atmosphere of 95% air and 5% CO2 at 37 ◦ C. Treatment of cell lines Stock solutions (100 mg/ml) of test samples, made in dimethylsulfoxide (DMSO), were dissolved in corresponding medium to the required working concentrations. Neoplastic FemX cells (5000 cells per well) and neoplastic LS174 cells (7000 cells per well) were seeded into 96-well microtiter plates, and 24 h later, after the cell adherence, five different, double diluted, concentrations of investigated compounds, were added to the wells. Final concentrations applied to target cells were 200, 100, 50, 25 and 12.5 g/ml, except to the control wells, where only nutrient medium was added to the cells. Nutrient medium was RPMI 1640 medium, supplemented with l-glutamine (3 mM), streptomycin (100 lg/ml), and penicillin (100 IU/ml), 10% heat inactivated (56 ◦ C) fetal bovine serum (FBS) and 25 mM Hepes, and was adjusted to pH 7.2 by bicarbonate solution. The cultures were incubated for 72 h.
Determination of cell survival (MTT test) The effect of test samples on cancer cell survival was determined by MTT test (microculture tetrazolium test), according to Mosmann (1983) with modification by Ohno and Abe (1991) 72 h upon addition of the compounds, as it was described earlier. Briefly, 20 l of MTT solution (5 mg/ml PBS) were added to each well. Samples were incubated for further 4 h at 37 ◦ C in 5% CO2 and humidified air atmosphere. Then, 100 l of 10% SDS were added to extract the insoluble product formazan, resulting from the conversion of the MTT dye by viable cells. The number of viable cells in each well was proportional to the intensity of the absorbance of light, which was then read in an ELISA plate reader at 570 nm. Absorbance (A) at 570 nm was measured 24 h later. To get cell survival (%), A of a sample with cells grown in the presence of various concentrations of the investigated test samples was divided with control optical density (the A of control cells grown only in nutrient medium), and multiplied by 100. It was implied that A of the blank was always subtracted from A of the corresponding sample with target cells. IC50 concentration was defined as the concentration of an agent inhibiting cell survival by 50%, compared with a vehicle-treated control. As a positive control were used cis-diamminedichloroplatinum (Cis-DDP) and usnic acid. All experiments were done in triplicate. Flow cytometry analysis Cellular DNA content and cell distribution were quantified by flow cytometry using propidium iodide (PI). Cells (3 × 105 cells/well) were seeded in 6-well plates and incubated with or without IC50 concentration of investigated compounds for 24 h. After treatment, the cells were collected by trypsinization, and fixed in ice-cold 70% ethanol at −20 ◦ C overnight. After fixation, the cells were washed in PBS and pellets obtained by centrifugation was treated with RNase (100 lg/ml) at 37 ◦ C temperature for 30 min and then incubated with propidium iodide (PI) (40 lg/ml) for at least 30 min. DNA content and cell cycle distribution were analyzed using a Becton Dickinson FAC-Scan flow cytometer. Flow cytometry analysis was performed using a CellQuestR (Becton Dickinson, San Jose, CA, USA), on a minimum of 10,000 cells per sample (Clothier 1995). Statistical analyses Statistical analyses were performed with the EXCEL and SPSS softwares package. To determine the statistical significance of antioxidant activity, Student’s t-test was used. All values are expressed as mean ± SD of three parallel measurements. Results and discussion Chromatograms for standards and P. caperata, P. sulcata, and P. saxatilis acetone extracts eluted by HPLC are represented in Figs. 1 and 2. As it is evidenced in the chromatograms, there were the presence of depsidones, depsides and dibenzofuran as the most abundant substance classes in the extracts examined. The HPLC chromatogram of the acetone extracts of P. sulcata and P. saxatilis showed that the highest peaks belong to the depsidone salazinic acid (SAL) (tR = 3.01 ± 0.20 min). On the other hand, protocetraric acid (PRO) (tR = 3.62 ± 0.20 min) and usnic acid (USN) (tR = 10.53 ± 0.30 min) were detected as the major secondary metabolites in the acetone extract of P. caperata. Identification of these compounds was achieved by comparison of their tR values with the standard substances previously isolated from lichens. The UV absorbance spectral data (200–400 nm) also corresponded with those of standards and found in Yoshimura et al. (1994). Table 1 shows the retention time of the detected lichen substances and their absorbance maxima (nm). The structures of the detected compounds are shown in Fig. 3. Some of the compounds are identified for the first time in this lichen. After detection of the present
N. Manojlovi´c et al. / Phytomedicine 19 (2012) 1166–1172
1169
Fig. 1. Chromatogram of the standards used for identification of the compounds present in Parmelia species.
compounds in lichens, major lichen metabolites in these species (salazinic acid, protocetraric acid and usnic acid – which was used as a standard compound) were isolated and used for antioxidant, antimicrobial and anticancer investigations. The scavenging of DPPH radicals and superoxide anion radicals by the tested compounds is shown in Table 2. There was a statistically significant difference between samples and control (p < 0.05). Salazinic acid showed stronger DPPH radicals and superoxide anion radicals scavenging activity than protocetraric acid. The IC50 values were 91.57 and 138.23 g/ml for salazinic acid and 119.10 and
177.60 g/ml for protocetraric acid (for DPPH radicals and superoxide anion radicals scavenging activity, respectively). As a shown in Table 3, salazinic acid also demonstrated greater reducing power. Various antioxidant activities were compared to ascorbic acid and usnic acid. The results showed that standards have stronger activity than tested samples. The tested lichen compounds have a strong antioxidant activity against various oxidative systems in vitro. The isolated components belong phenols which indicate an important role of phenol in the antioxidant activity for lichens. In fact, number of previous studies
Fig. 2. HPLC chromatograms acquired at 254 nm of the acetone extracts of P. caperata, P. sulcata and P. saxatilis. Chromatographic peaks identities are reported in Table 1.
1170
N. Manojlovi´c et al. / Phytomedicine 19 (2012) 1166–1172
Fig. 3. Structures of the identified compounds.
found that the lichens where found the higher content of phenols exert stronger antioxidant activity (Behera et al. 2009; Kosanic´ et al. 2012a), which means that phenols are important antioxidants. Antioxidant effect of some other lichen compounds was also studied by other researchers. For example, antioxidant activity was found for lecanoric acid, salazinic acid, stictic acid, usnic acid (Luo et al. 2009; Amo de Paz et al. 2010). The antimicrobial activity of the lichen components against the test microorganisms are shown in Table 4. Salazinic acid and protocetraric acid showed similar antimicrobial activity, but antibacterial activity was stronger than antifungal activity for both
Table 1 Retention time of the examined lichen compounds and their absorbance maxima (nm). Peaks
Compound
Retention time (tR ± SD)a (min)
SAL PRO USN ATR CHL
Salazinic acid Protocetraric acid Usnic acid Atranorin Chloroatranorin
3.01 3.62 10.53 11.72 13.23
± ± ± ± ±
0.20 0.20 0.30 0.10 0.20
Absorbance maxima (nm) UV spectrum 213, 238, 312 212, 240, 320m 234, 282 210, 252, 321m 213, 252, 315m , 350
m, minor absorbance maximum. a Values are the means of three determinations ± SD.
compounds. The MIC for components relative to the tested microorganisms ranged from 0.015 to 1 mg/ml. The antimicrobial activity was compared with the standard antibiotics, streptomycin (for bacteria) and ketoconazole (for fungi) and usnic acid. As shown in Table 2, standards have stronger activity than tested samples. In a negative control, DMSO had no inhibitory effect on the tested organisms. Previously, numerous lichen compounds were screened for antimicrobial activity in search of the new antimicrobial agents (Candan et al. 2006; Kosanic´ and Rankovic´ 2011; Turk et al. 2006). In our experiments, the tested lichen compounds show very strong antimicrobial activity. Antibacterial activity was stronger than antifungal. This observation is in accordance with other studies (Yang
Table 2 DPPH radical scavenging activity and superoxide anion scavenging activity of isolated compounds. Lichen compounds
DPPH radical scavenging IC50 (g/ml)
Superoxide anion scavenging IC50 (g/ml)
Salazinic acid Protocetraric acid Usnic acid Ascorbic acid
91.57 138.23 60.73 6.42
119.10 177.60 97.28 115.61
Ascorbic acid and usnic acid were used as a standards.
N. Manojlovi´c et al. / Phytomedicine 19 (2012) 1166–1172
1171
Table 3 Reducing power of isolated compounds. Lichen compounds
Salazinic acid Protocetraric acid Usnic acid Ascorbic acid
Absorbance (700 nm) 1000 g/ml
500 g/ml
250 g/ml
125 g/ml
62.5 g/ml
0.2722 0.1209 0.6723 2.113
0.1842 0.0903 0.5468 1.654
0.0758 0.0721 0.2692 0.9571
0.0534 0.0502 0.1249 0.4783
0.0321 0.0312 0.0901 0.2472
Ascorbic acid and usnic acid were used as a standards. Table 4 Minimum inhibitory concentration (MIC) of isolated compounds. Lichen compounds
Salazinic acid
Protocetraric acid
Usnic acid
S
K
B. mycoides B. subtilis E. coli K. pneumoniae S. aureus A. flavus A. fumigatus C. albicans P. purpurescens P. verrucosum
0.015 0.0312 1 0.5 0.125 1 1 0.25 1 0.5
0.015 0.015 1 0.5 0.015 1 0.25 0.25 1 0.5
0.0008 0.0008 0.25 0.0625 0.125 0.5 0.25 0.125 0.5 0.5
7.81 7.81 31.25 31.25 15.72 – – – – –
– – – – – 3.9 3.9 1.95 3.9 3.9
Values given as mg/ml for lichen compounds and as g/ml for antibiotics. Values are the mean of three replicate. S, streptomycin; K, ketoconazole and usnic acid were used as a standards.
and Anderson 1999; Kosanic´ et al. 2012b), focused on the antimicrobial activity which have demonstrated that bacteria are more sensitive to the antimicrobial activity than the fungi due to differences in the composition and permeability of the cell wall. The cytotoxic activity of the tested compounds against the tested cell lines is shown in Table 5. The tested samples exhibited high cytotoxic activity against the target cells in vitro. The IC50 value for both compounds relative to the tested cells ranged from 35.67 to 60.18 g/ml. The best cytotoxic activity was exhibited the salazinic acid. As shown in this table, Cis-DDP and usnic acid have slightly better cytotoxic activity than tested samples. The effect of protocetraric acid and salazinic acid on cell cycle progression was investigated in FemX and LS174 cells. Cell cycle was assessed by cytofluorimetric analysis, using propidium iodide to label DNA. Results are given in Table 6. Cells treated for 24 h with IC50 (g/ml) concentrations of both compounds showed a significant increase in the number of cells in sub-G1 phase, and an reduction in the number of cells in S phase. Specially, incubation of the cell line FemX with salazinic acid induced G0-G1 and G2-M cell cycle blockade. In addition, treatment LS174 cells of protocetraric acid and salazinic acid leads to a somewhat more pronounced increase in sub-G1 phase and concomitant decrease in G2/M were observed, supporting a G1 phase arrest. An increase in cells containing sub-G1 amounts of DNA was observed, indicating that the compounds was inducing cell death. Some literature data reported that lichen components are responsible for anticancer activities of lichens (Bucar et al. 2004; Burlando et al. 2009). However, it is difficult to determine the contribution of individual components for the overall anticancer effect.
Table 5 Growth inhibitory effects of isolated compounds on FemX and LS174 cell lines CisDDP and usnic acid were used as a standards. Lichen compounds
Apoptotic cells sub-G1
G1
S
G2/M
FemX Control Salazinic acid Protocetraric acid Usnic acid
0.15 16.23 16.20 10.18
62.65 43.74 43.81 54.38
16.69 13.64 15.30 14.64
16.64 24.93 18.62 20.37
LS174 Control Salazinic acid Protocetraric acid Usnic acid
3.68 20.88 22.90 12.89
51.68 41.37 39.71 52.77
12.61 14.27 16.96 15.06
30.19 15.90 23.92 19.28
Effect of compounds on cell cycle phase distribution. FemX and LS174 cell lines were exposed to compounds (IC50 g/ml) for 24 h and then collected for analysis of cell cycle phase distribution using flow cytometry. Percentage of cells under different stages of cell cycles (sub-G1, G1, S, G2/M) is shown. Usnic acid was used as a standard compound.
Often, the activity of lichens may be the result of synergistic or antagonistic effect of several compounds. In conclusion, it can be stated that tested lichen compounds have a strong antioxidant, antimicrobial and anticancer activity in vitro, which suggest that lichens could be good natural antioxidant, antimicrobial and anticancer agents. Acknowledgement This work was financed in part by the Ministry of Science, Technology, and Development of the Republic of Serbia and was carried out within the framework of projects no. 173032, 175011 and 172015. References
IC50 (g/ml) FemX
Salazinic acid Protocetraric acid Usnic acid Cis-DDP
Table 6 Effect of isolated compounds on cell cycle progression.
39.02 58.68 12.72 0.94
LS174 ± ± ± ±
0.56 2.11 0.35 0.35
35.67 60.18 15.66 2.3
± ± ± ±
1.89 0.59 1.45 0.31
Amo de Paz, G., Raggio, J., Gomez-Serranillos, M.P., Palomino, O.M., Gonzales-Burgos, E., Carretero, M.E., Crespo, A., 2010. HPLC isolation of antioxidant constituents from Xanthoparmelia spp. Journal of Pharmaceutical and Biomedical Analysis 53, 165–171. Bates, S.T., Cropsey, G.W., Caporaso, J.G., Knight, R., Fierer, N., 2011. Bacterial communities associated with the lichen symbiosis. Applied and Environment Microbiology 77, 1309–1314.
1172
N. Manojlovi´c et al. / Phytomedicine 19 (2012) 1166–1172
Behera, B.C., Verma, N., Sonone, A., Makhija, U., 2009. Optimization of culture conditions for lichen Usnea ghattensis G. Awasthi to increase biomass and antioxidant metabolite production. Food Technology and Biotechnology 47, 7–12. Bown, D., 2001. Encyclopedia of Herbs and Their Uses. Dorling Kindersley, London. Bucar, F., Schneider, I., Ogmundsdottir, H., Ingolfsdottir, K., 2004. Antiproliferative lichen compounds with inhibitory activity on 12(S)-HETE production in human platelets. Phytomedicine 11, 602–606. Burlando, B., Ranzato, E., Volante, A., Appendino, G., Pollastro, F., Verotta, L., 2009. Antiproliferative effects on tumour cells and promotion of keratinocyte wound healing by different lichen compounds. Planta Medica 75, 607–613. Candan, M., Yilmaz, M., Tay, T., Kivanc, M., Turk, H., 2006. Antimicrobial activity of extracts of the lichen Xanthoparmelia pokornyi and its gyrophoric and stenosporic acid constituents. Zeitschrift fur Naturforschung C 61, 319–323. Clothier, R.H., 1995. The FRAME cytotoxicity test. Methods in Molecular Biology 43, 109–118. Dorman, H.J., Bachmayer, O., Kosar, M., Hiltunen, R., 2004. Antioxidant properties of aqueous extracts from selected Lamiaceae species grown in Turkey. Journal of Agricultural and Food Chemistry 52, 762–770. Huneck, S., Yoshimura, I., 1996. Identification of Lichen Substances. Springer-Verlag, Heidelberg, Berlin. Ibanez, E., Kubatova, A., Senorans, F.J., Cavero, S., Reglero, G., Hawthorne, S.B., 2003. Subcritical water extraction of antioxidant compounds from rosemary plants. Journal of Agricultural and Food Chemistry 51, 375–382. ´ M., Rankovic, ´ B., Stanojkovic, ´ T., 2012a. Antioxidant, antimicrobial and antiKosanic, cancer activity of 3 Umbilicaria species. Journal of Food Science 77, T20–T25. ´ B., 2011. Antioxidant and antimicrobial properties ´ M., Rankovic, Kosanic, of some lichens and their constituents. Journal of Medicinal Food 14, 1624–1630. ´ M., Rankovic, ´ B., Stanojkovic, ´ T., 2012b. Antioxidant, antimicrobial, and antiKosanic, cancer activities of three Parmelia species. Journal of the Science of Food and Agriculture, http://dx.doi.org/10.1002/jsfa.5559. Luo, H., Yamamoto, Y., Kim, J.A., Jung, J.S., Koh, Y.J., Hur, J.S., 2009. Lecanoric acid, a secondary lichen substance with antioxidant properties from Umbilicaria antarctica in maritime Antarctica (King George Island). Polar Biology 32, 1033–1040.
´ N., Vasiljevic, ´ P., Gritsanapan, W., Supabphol, R., Manojlovic, ´ I., 2010. Manojlovic, Phytochemical and antioxidant studies of Laurera benguelensis growing in Thailand. Biological Research 43, 169–176. ´ T., Stamenkovic, ´ S., Cvetkovic, ´ V., Nikolic, ´ M., Toˇsic, ´ S., Stojiˇcic, ´ D., 2011. Mitrovic, Lichens as source of versatile bioactive compounds. Biologica Nyssana 2, 1–6. Molnar, K., Farkaˇs, E., 2010. Current results on biological activities of lichen secondary metabolites: a review. Zeitschrift fur Naturforschung C 65, 157–173. Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Journal of Immunological Methods 65, 55–63. NCCLS (National Commitee for Clinical Laboratory Standards)., 1998. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Conidiumforming Filamentous Fungi: Proposed Standard M38-P. NCCLS, Wayne, PAUSA. Nishimiki, M., Rao, N.A., Yagi, K., 1972. The occurrence of super-oxide anion in the reaction of reduced phenazine methosulfate and molecular oxygen. Biochemical and Biophysical Research Communications 46, 849–853. Ohno, M., Abe, T., 1991. Rapid colorimetric assay for the quantification of leukemia inhibitory factor (LIF) and interleukin-6 (IL-6). Journal of Immunological Methods 145, 199–203. Otzurk, S., Guvenc, S., Arikan, N., Yylmaz, O., 1999. Effect of usnic acid on mitotic index in root tips of Allium cepa L. Lagascalia 21, 47–52. Oyaizu, M., 1986. Studies on products of browning reaction prepared from glucoseamine. Japanese Journal of Nutrition 44, 307–314. Sarker, S.D., Nahar, L., Kumarasamy, Y., 2007. Microtitre plate-based antibacterial assay incorporating resazurin as an indicator of cell growth, and its application in the in vitro antibacterial screening of phytochemicals. Methods 42, 321–324. Turk, H., Yılmaz, M., Tay, T., Turk, A.O., Kıvanc, M., 2006. Antimicrobial activity of extracts of chemical races of the lichen Pseudevernia furfuracea and their physodic acid, chloroatranorin, atranorin, and olivetoric acid constituents. Zeitschrift fur Naturforschung C 61, 499–507. Yang, Y., Anderson, E.J., 1999. Antimicrobial activity of a porcine myeloperozidase against plant phatgenic bacteria and fungi. Journal of Applied Microbiology 86, 211–220. Yoshimura, I., Kinoshita, Y., Yamamoto, Y., Huneck, S., Yamada, Y., 1994. Analysis of secondary metabolites from lichen by high performance liquid chromatography with a photodiode array detector. Phytochemical Analysis 5, 197–205.