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Neuroprotective activity and cytotoxic potential of two Parmeliaceae lichens: identification of active compounds.

ABSTRACT

Background: Lichens are symbiotic organisms capable of producing unique secondary metabolites, whose pharmacological activities are attracting much interest.

Purpose: The present study aimed to investigate the in vitro neuroprotective effects and anticancer potential of methanol extracts of two Parmeliaceae lichens: Cetraria islandica and Vulpicida canadensis. The chemical composition of the two lichens was also determined.

Methods: Neuroprotective activity was studied with respect to the antioxidant properties of the extracts: radical scavenging tests (ORAC and DPPH assays) were performed and oxidative stress markers (intracellular ROS production, caspase-3 activity, MDA and glutathione levels) were assessed in a hydrogen peroxide-induced oxidative stress model in astrocytes. Cytotoxic activity was tested against human HepG2 (hepatocellular carcinoma) and MCF-7 (breast adenocarcinoma) cell lines.

Results: Cell viability studies identified a single concentration for each extract that was subsequently used to measure oxidative stress markers. Lichen extracts were able to reverse the oxidative damage caused by hydrogen peroxide, thus promoting astrocyte survival. Both lichen extracts also had anticancer activity in the cell lines, with [IC.sub.50] values of 19.51-181.05 [micro]g/ml. The extracts had a high total phenolic content, and the main constituents identified by HPLC were fumarprotocetraric acid in Cetraria islandica, and usnic, pinastric and vulpinic acids in Vulpicida canadensis. The biological activities of the lichen extracts can be attributed to these secondary metabolites.

Conclusion: The lichen species studied are promising sources of natural compounds with neuroprotective activity and cytotoxic potential, and warrant further research.

Keywords:

Lichens

Parmeliaceae

Neuroprotective activity

Cytotoxic potential

Phytochemical analysis

HPLC

Introduction

Lichens are symbiotic organisms consisting of at least one fungus and one photosynthetic partner (alga or cyanobacterium). This association is highly successful since lichens inhabit most ecosystems. The pharmacological interest in lichens lies in their capacity to produce an array of unique secondary metabolites, mostly derived from fungal metabolism, with biological activity that can be exploited for human use (Huneck and Yoshimura, 1996). Most lichen substances are phenolic compounds, dibenzofurans, depsides, depsidones, lactones and pulvinic acid derivatives. They are mainly synthesized by polymalonate, shikimic acid and mevalonic acid pathways (Boustie and Grube, 2005). Parmeliaceae (Ascomycota, Lecanorales) is one of the best studied and widespread families among the lichenised fungi, comprising more than 2500 species grouped in 85 genera. Markedly increasing numbers of new species in this family have been identified in the last decade due to the availability of molecular data (Crespo et al., 2010).

Oxidative stress (OS), defined as a redox imbalance between reactive oxygen species (ROS) and the antioxidant defence system, results in the damage of cellular contents, including proteins, lipids and DNA. It plays a crucial role in neuronal damage, and is responsible for the development of several neurodegenerative diseases (Halliwell, 2001; Sayre et al., 2008). The antioxidant potential of nervous system cells can be exploited as a therapeutic tool for delaying and preventing neurodegeneration. Several intracellular mechanisms help counteract OS; for instance, antioxidant compounds that upregulate the Nrf2-ARE pathway promote the induction of cytoprotective genes, such as detoxifying antioxidant phase-II enzymes (Zhang et al., 2013). Various natural products have demonstrated neuroprotective activities in in vitro and in vivo models of neuronal cell death and neurodegeneration. These involve antioxidant mechanisms, such as those considered her, and others (Kelsey et al., 2010).

Our knowledge of the pharmacological properties of lichens is still poor compared with that of other natural products. In previous investigations, some lichen species have demonstrated antioxidant properties arising from their phenolic content. For example, antioxidant activities of several depsides and depsidones isolated from various lichen species (de Paz et al., 2010; Manojlovic et al., 2012) and in vitro properties of some crude lichen extracts (Kosanic et al., 2013; Stojanovic et al., 2010) have been described. Nevertheless, there are few studies of intracellular ROS modulation by lichen extracts and metabolites (Paudel et al., 2011), and none has focused on their role as protective agents in nervous system-like cells under OS conditions.

In view of this, our research attempts to identify and isolate lichen metabolites with potential antioxidant activities and the capacity to protect against OS in models of nervous system-like cell lines. The present work focuses for the first time on the possible neuroprotective and anticancer properties of the methanol extracts from two Parmeliaceae lichens from the cetraroid clade: Cetraria islandica and Vulpicida canadensis. We also identify the main constituents of the extracts.

Material and methods

Reagents

Dulbecco's modified Eagle's medium (DMEM), RPMI 1640 medium, foetal bovine serum (FBS), PBS and gentamicin were purchased from Cibco (Invitrogen, Paisley, UK). Dimethyl sulphoxide (DMSO) and high-performance liquid chromatography (HPLC)-grade methanol were supplied by Panreac (Barcelona, Spain). Hydrogen peroxide solution (30% w/w), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT), 1,1-diphenyl-2-picrylhydrazyl (DPPH), 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (TROLOX), 2,7-dichloro-dihydrofluorescein diacetate (DCFH-DA), caspase-3 substrate (AC-DEVD-AMC), 2,2-azobis(2m-ethylpropionamidine) dihydrochloride (AAPH) and all other reagents were obtained from Sigma-Aldrich (St Louis, MO, USA).

The astrocyte cell line U373 MG was provided by the University of Alcala (Madrid, Spain), CAI Medicina Biologia, Unidad de Cultivos. Cancer cell lines (MCF-7 and HepG2) were obtained from the NCI-Frederick Cancer DCTD Tumor/Cell line Repository (Frederick National Laboratory for Cancer Research, National Cancer Institute).

Lichen samples

The specimens of lichen species studied were collected and authenticated by a taxonomist and then deposited in the Herbarium of the Faculty of Pharmacy, University Complutense of Madrid, with the following identifying data:

--Cetraria islandica (L. Ach.), Slovakia, Presovsky Kraj, prov. Poprad, Popradske pleso, August 2006 (MAF-LICH 17201).

--Vulpicida canadensis (Rasanen)J.-E. Mattson & M.J. Lai., USA, Oregon, Jefferson County, Cold Spring Campground, May 2010 (MAF-LICH 4263).

Preparation of lichen extracts

Around 30 mg of dry lichen thallus (accurately weighted) were extracted in 2 ml of methanol for 1 h and subsequently filtered (de Paz et al., 2010). Methanol was then evaporated at room temperature. Dry extracts were finally weighted and the yield of the extraction was calculated for each lichen specimen.

Determination of total phenolic compounds

The total amounts of soluble phenolic compounds in the lichen extracts were determined by the Folin-Ciocalteu method (Saura-Calixto et al., 2007) using gallic acid (GA) as a standard. Briefly, 0.5 ml of the lichen extract (1 mg/ml) was mixed in a volumetric flask with 0.5 ml of Folin-Ciocalteu reagent, and then 10 ml of [Na.sub.2]C[O.sub.3] solution (75 g/1) and 14 ml of distilled water were added. The mixture was shaken thoroughly, then incubated in darkness for 1 h). Finally, its absorbance was measured at 760 nm in a spectrophotometer (Uvikon 930, Kontron Instruments, Bardsey, UK).

Phytochemical analysis

Dry lichen extracts were dissolved in methanol to give a concentration of 250 [micro]g/ml and then subjected to HPLC, performed with an Agilent 1260 instrument (Agilent Technologies, CA, USA) with a reversed-phase Mediterranea Sea18 column (150 mm x 4.6 mm, 3 [micro]m particle size; Teknokroma, Barcelona, Spain). The mobile phase consisted of 1% orthophosphoric acid in milli-Q water (A)/methanol (B), and elution was performed by a gradient method (de Paz et al., 2010). A 20 [micro]l volume of sample was injected, and a flow rate of 0.6 ml/min and a temperature of 40[degrees] were established. Analyses were monitored by a photodiode array detector (190-800 nm) throughout the entire run. The main peaks were scanned in the UV spectrum between 190 and 400 nm. The standards used were obtained by isolating fumarprotocetraric acid from Bryoria sp., protocetraric acid from Flavoparmelia caperata (L.) Hale, vulpinic acid from Letharia vulpina (L.) Hue. and pinastric acid from Cetraria pinastri (Scop.) S. Gray. Usnic acid was purchased from Sigma Aldrich (MO, USA).

Evaluation of neuroprotective activities

Radical scavenging activities

--Oxygen radical antioxidant capacity (ORAC) assay: this was carried out as previously described (Davalos et al., 2004). Dilutions of samples and Trolox (reference antioxidant, water-soluble vitamin E analogue) were incubated in opaque 96-well plates for 10 min at 37[degrees]C with fluorescein. After this period, AAPH was added to the mixture. Fluorescence was read every 56 s for 98 min using a FLUOstar Optima fluorimeter (BMC Labtech, Ortenberg, Germany) ([[lambda].sub.exc] 485 nm and [[lambda].sub.em] 520 nm). The area under the curve (AUC) was calculated for each sample and compared with that of Trolox. ORAC values are expressed as [micro]mol Trolox equivalent (TE)/mg sample.

--DPPH assay: this was conducted following the previously described DPPH method (Amarowicz et al., 2004) with slight modifications. In brief, different concentrations of the extracts were placed in a 96-well plate and a DPPH solution (50 [micro]M) was added to make up a volume of 225 [micro]l/well. The resulting solutions were incubated in darkness for 30 min and their absorbances read at 517 nm in a FLUOstar Optima apparatus.

Astrocyte culture and treatments

Astrocytes from the U373-MG cell line (human astrocytoma) were maintained in DMEM supplemented with 10% FBS and 0.5% gentamicin in a humidified atmosphere with 5% C[O.sub.2] at 37[degrees]C. [H.sub.2][O.sub.2] was used as an OS inductor. Cells were treated with lichen extracts at different concentrations for 24 h. Extracts and [H.sub.2][O.sub.2] were dissolved in PBS for corresponding dilutions.

Morphological study

Cellular morphology changes after treatments were examined by phase-contrast microscopy (Nikon TMS). Photographs were taken using a Motic Moticam 2500 camera.

Assessment of cell viability

MTT assay. Mitochondrial integrity and activity, and cell viability indicators, were determined by the MTT assay (Mossman, 1983), with minor variations. Cells were plated at a density of 5x [10.sup.4] cells/well in 96-well plates overnight and then treated with different concentrations of lichen extracts (ranging from 0.5 to 250 [micro]g/ml) for 24 h. Triton X-100 5% was used as the negative control. Finally, 2 mg/ml MTT was added and plates were incubated for 1 h at 37[degrees]C. After removal of the medium, DMSO was added to dissolve the dark-blue formazan crystals. Absorbance was then measured at 550 nm using a Digiscan 340 microplate reader (ASYS Hitech GmbH, Eugendorf, Austria).

LDH assay. The lactate dehydrogenase (LDH) release assay was carried out to measure the loss of cell membrane integrity, following the method of Lopez et al. (2003). Cells were seeded in 24-well plates ([10.sup.5] cells/well) and treated with four concentrations of the extracts for 24 h. The supernatant was then removed and stored, and sodium phosphate buffer 0.1 mM (pH 7.4) with 0.5% Triton X-100 was added to produce cellular lysis and to release the remaining LDH. The activity of the enzyme released from intracellular to extracellular medium after treatments was measured spectrophotometrically at 340 nm every 1 min for 10 min using a FLUOstar Optima instrument. Measurements are expressed as a percentage of total enzyme activity.

Protection against [H.sub.2][O.sub.2]-induced toxicity

To assess the possible neuroprotective effect against OS, cells were exposed to 1 mM [H.sub.2][O.sub.2] for 30 min after treatment with the extracts (Gonzllez-Burgos et al., 2012), and the MTT assay was conducted as described (Section MTT assay). In the LDH assay, cells were exposed to 1 mM [H.sub.2][O.sub.2] in 1% DMEM for 150 min (model with significant cell necrosis).

Intracellular ROS production assay

ROS production was evaluated by the DCFH-DA method (Lebel et al., 1992), with some modifications, as defined by Gonzalez-Burgos et al. (2012).

Determination of caspase-3 activity

Inhibition of [H.sub.2][O.sub.2]-mediated apoptosis by lichen extracts was investigated through the titration of caspase-3 enzyme activity and measurement of the cleavage of a fluorogenic substrate (Garcimartin et al., 2014).

Glutathione levels

Levels of GSH and GSSG were determined by the method of Hissin and Hilf (1976), which is described in full in Gonzalez-Burgos et al. (2013). The GSH/GSSG ratio was obtained from the amounts (nmol/mg) of the GSH and GSSG proteins.

Measurement of lipid peroxidation

Malondialdehyde (MDA) levels were analysed by HPLC (Grotto et al., 2007). An Eclipse plus C18 column (150 mm x 4.6 mm, 5 [micro]m particle size; Agilent Technologies) was used. The UV detector wavelength was set at 268 nm, and the elution was performed isocratically at 40[degrees]C for 10 min with a mixture of Milli-Q. water and methanol (50/50) at a flow rate of 0.5 ml/min. The MDA content of samples was calculated using an MDA standard curve, and is expressed as nmol MDA/mg protein.

Cytotoxic activities

Cell culture

The cytotoxic potential was evaluated in two human cancer cell lines: the hepatocellular carcinoma cell line HepG2, and the breast adenocarcinoma cell line MCF-7. Cultures were maintained in RPMI 1640 medium, supplemented with 10% FBS and 0.5% gentamicin in a humidified atmosphere with 5% C[O.sub.2] at 37[degrees]C.

Assessment of cell viability

Cells were treated with nine concentrations of lichen extracts for 24 h, and their abilities to reduce cell survival were determined by the MTT assay (Section MTT assay).

Statistical analysis

Results are expressed as means and standard deviations (SDs) of at least three independent experiments. Statistical differences between groups were determined by one-way ANOVA followed by Tukey's test for multiple comparisons, using Statgraphics Centurion XVI software. Values of p < 0.05 were considered statistically significant.

Results and discussion

Lichen extraction and phenolic content

Final yields (% w/w) of the methanol extractions of lichen specimens and their content in polyphenols, expressed as [micro]g GA/mg dry extract, are summarised in Table 1. The total concentration of phenolic compounds in the extracts was calculated as [micro]g GA equivalent/mg dry extract, using a standard graph for GA. The highest content of phenolic compounds was found in Ci; however, Vc yielded the greatest dry weight, indicating the presence of other non-phenolic substances in the extract.

Radical scavenging activities

We observed that the chemoluminescence induced by the peroxyl radical generation, initiated by AAPH in the ORAC assay, decreased following addition of lichen extracts; ORAC values were 0.77 [micro]mol TE/mg sample for Vc and 3.06 [micro] mol TE/mg sample for Ci, indicating the lichens' different capacities for scavenging peroxyl radicals. A different pattern of DPPH free radical scavenging activity was seen, whereby Vc had the highest antiradical activity with the lowest [IC.sub.50] (Table 1). The distinct behaviours of the extracts in these assays may be explained by the fundamentally different nature of the methods used. The ORAC and DPPH assays are respectively based on hydrogen atom transfer (HAT) and electron transfer (ET) (Apak et al., 2007). The lichen extracts may therefore mediate their radical scavenging activities through different mechanisms.

Phytochemical analysis

Using an HPLC-UV method we determined the main lichen secondary metabolites in the extracts of the species under study. Very good resolution of chromatographic peaks and baseline separation of all compounds were obtained after optimising the separation on the reversed-phase for 65 min. Comparing the retention times ([t.sub.R]) and UV spectra (190-400 nm) of the main peaks with those of reference substances previously isolated from lichens confirmed that the methanol extract of Cetraria islandica contains the depsidone fumarprotocetraric acid ([t.sub.R] = 26.397 min) as its main constituent (> 90% of total integrated area); it also contains traces of the related depsidone protocetraric acid ([t.sub.R] = 21.769 min). On the other hand, two pulvinic acid derivatives, vulpinic acid ([t.sub.R] = 30.336 min) and pinastric acid ([t.sub.R] = 32.350 min), and dibenzofuran usnic acid ([t.sub.R] = 39.463 min) were the predominant metabolites in the Vulpicida canadensis methanol extract. Usnic acid presented the highest peak (45.56% of the total area), followed by pinastric acid (30.54%) and vulpinic acid (18.41%). Chromatograms of both methanol extracts, under the analytical conditions described in Section Phytochemical analysis, are presented in Fig. 1A (Ci) and B (Vc). Table 2 shows the UV absorbance spectrum data obtained from each compound in the lichen extracts. The UV absorbance spectrum data correspond to those of the standards and those presented in Yoshimura et al. (1994).

The phytochemical analysis of our lichen specimens is consistent with the findings of other studies dealing with the issue, such as the recent work conducted by Igoli et al. (2014), which found both fumarprotocetraric and protocetraric acid to be present in Cetraria islandica. Similarly, the secondary chemistry described for Vulpicida candensis is confirmed by the phytochemical study of our specimens (Mattson and Lai, 1993).

It is now possible to correlate the concentration-dependent antioxidant activity, demonstrated for the extracts in the chemical assays (Section Radical scavenging activities) with their components. Results obtained for Ci and Vc are in agreement with those obtained by other researchers, since both fumarprotocetraric and usnic acid are known to display moderate to strong antioxidant activity in radical scavenging tests in vitro (Behera et al., 2012; Lohezic-Le Devehat et al., 2007).

Assessments of cell viability, protection against [H.sub.2][O.sub2]-induced toxicity and morphological studies

Nine concentrations of each extract, ranging from 0.5 to 250 [micro]g/ml, were tested to determine the effects of single extracts in the MTT assay. Results obtained for the effect of Ci and Vc on U373MG cell viability are shown in Fig. 2A and B respectively, and expressed as the percentage of cell viability, taking the optical density of untreated control cells to be 100%.

Significant loss of cell viability was observed for Ci at 25 [micro]g/ml and higher concentrations, while for Vc only the highest concentration (250 [micro]g/ml) reduced astrocyte viability. At this point, five concentrations for each extract were chosen to assess their capacity to protect against OS and the cellular toxicity of [H.sub.2][O.sub.2]. [H.sub.2][O.sub.2] decreased cell viability to approximately 60% of control, but Ci at 10-25 [micro]g/ml and Vc at 25 [micro]g/ml significantly reversed that effect and enhanced astrocyte viability (Fig. 2C and D). The concentration offering the greatest protection against [H.sub.2][O.sub.2] was then chosen for each extract (10 [micro]g/ml for Ci and 25 [micro]g/ml for Vc) and assayed in subsequent experiments. The morphology was also studied at these concentrations. Cells treated only with [H.sub.2][O.sub.2] lost their normal morphology, becoming brighter (less viable and attached to culture dish) and more rounded. Pretreatment with both extracts partially prevented these deleterious effects (see pictures in Fig. 2E).

The LDH release assay was used to evaluate the integrity of the cell membrane as another parameter reflecting cell viability. The results complement those of the MTT assay and are expressed as LDH released after treatments (taking total intracellular LDH to be 100%). Control cells released 15% of total intracellular LDH (basal conditions), and cells treated with [H.sub.2][O.sub.2] alone exhibited a greater release, of up to 40% of total LDH. This elevation was partially attenuated at certain extract concentrations.

Results of the LDH release assay confirmed the range of concentrations over which Ci extract affects cell viability by itself (Fig. 3A), and that over which it protects against [H.sub.2][O.sub.2] damage (Fig. 3C). A different effect at several concentrations of Vc was found when comparing their effects on LDH release and MTT reduction; although Vc at 50-100 [micro]g/ml did not affect cell viability in the MTT assay, it provoked significant LDH release (Fig. 3B). Similarly, concentrations between 5 and 10 [micro]g/ml diminished [H.sub.2][O.sub.2]-induced LDH release (Fig. 3D) even though they did not protect against the [H.sub.2][O.sub.2]-induced decrease in MTT reduction. The different activities in the two experiments reflect the different natures of the methods used, whereby the LDH assay assesses cell membrane integrity while the MTT test evaluates mitochondrial reductase functionality.

Intracellular ROS production assay

The effect of exogenous [H.sub.2][O.sub.2] on intracellular ROS level was assessed by measuring 2,7-dichlorofluorescein fluorescence. Fig. 4A and B show that U373-MG cells exposed to [H.sub.2][O.sub.2] presented intracellular ROS levels that markedly increased to approximately 20% in comparison with control cells (100% ROS generation), from the beginning to the end of the experiment. These results confirm that [H.sub.2][O.sub.2], under established experimental conditions, induces OS. Moreover, none of the lichen extracts caused intracellular ROS production when compared with control cells, indicating that methanol extracts per se neither induced ROS generation nor reacted with components of the culture medium favouring ROS formation (Halliwell, 2003). However, pretreatments with Ci and Vc significantly inhibited [H.sub.2][O.sub.2]-induced intracellular ROS generation. These findings may explain the protective role of the two lichen extracts through the reduction of OS.

Determination of caspase-3 activity

Caspase-3 is a key enzymatic mediator in external and internal apoptosis pathways (Marks et al., 1998). Direct suppression of active caspase-3 contributes to the cellular protection against OS (Ozben, 2007). Once it was known that [H.sub.2][O.sub.2] could induce cellular death through necrosis, the possibility that it could also promote cell death via apoptosis was examined. We evaluated the effects of lichen extracts on caspase-3 activity by fluorimetry. As shown in Table 3, exposure of U373-MG cells to [H.sub.2][O.sub.2] produced a remarkable increase of over 300% in caspase-3 activity relative to control cells. However, pre-treatment with 0.1 mM Trolox (the reference antioxidant) was able to significantly revert this elevation, but not to basal levels. When treated with Ci and Vc, cells did not show a significant decrease in caspase-3 activity compared with those exposed to [H.sub.2][O.sub.2] alone (although the mean caspase activity was slightly lower). Consequently, it cannot be concluded that the protective effects of lichen extracts are partially mediated by the inhibition of apoptosis.

Glutathione levels

Antioxidant glutathione (GSH) and its oxidised disulphide form (GSSG), which may be considered were measured by fluorimetry as other important OS markers. GSH was mostly found in control cells, at a concentration 11 times higher than GSSG; however, when threatened by [H.sub.2][O.sub.2]-induced OS, U373MG cells presented a markedly reduced antioxidant capacity at higher concentrations of GSSG, and the GSH/GSSG ratio was reduced to half the initial value. On the other hand, pretreatments with both Ci and Vc before [H.sub.2][O.sub.2] exposure increased the GSH/GSSG ratio, indicating that extracts can enhance the antioxidant response in these cells, thereby partially restoring normal GSH and GSSG levels. Antioxidant effects displayed by both Ci and Vc turned out to be statistically significant (see Table 3).

The ameliorative effect of Vc in the glutathione system of U373 cells might have been expected, given the presence of usnic acid in the extract; this dibenzofuran is known to have beneficial effects on antioxidant enzymes and to increase the content of GSH in a model of indomethacin-induced decline of antioxidant capacity in gastric tissue of rats (Odabasoglu et al., 2006).

Lipid peroxidation

Lipid peroxidation is a key mechanism of cellular damage caused by ROS, and malondialdehyde (MDA) is one of the best known secondary products of lipid peroxidation, being widely used as an indicator of cell injury. To quantify it, we determined MDA levels by HPLC in the different groups of cells. As illustrated in Table 3, the MDA concentration was significantly higher in [H.sub.2][O.sub.2]-treated U373MG cells than in control cells (5.60 versus 2.01 nmol/mg protein, respectively). Pretreatments with lichen extracts significantly inhibited the [H.sub.2][O.sub.2]induced increase of lipid peroxidation in these cells. Ci was the most active extract and reduced the lipid peroxidation almost to the basal levels found in control cells.

Cytotoxic activities

To consider their anticancer potential, particularly given the published reports of such activity in lichen components (Bucar et al., 2004; Burlando et al., 2009), we evaluated the cytotoxic effects of Cetraria islandica and Vulpicida canadensis on two human cancer cell lines (HepG2 and MCF-7). A wide range of concentrations was tested and, as reflected in Fig. 5, MCF-7 cells appeared to be more sensitive to both lichen extracts than those of the HepG2 cell line. Ci had [IC.sub.50] values of 181.05 and 19.51 [micro]g/ml, and Vc had values of 58.02 and 148.42 [micro]g/ml, respectively for the HepG2 and MCF-7 cell lines.

In fact, Ci affected the viability of HepG2 cells at concentrations of 50 [micro]g/ml and above (Fig. 5A) and of MCF-7 cells at concentrations of 10 [micro]g /ml or more (Fig. 5C). Since the latter concentration (10 [micro]g /ml) did not affect cell viability in astrocytes and had shown promising results in previous experiments assessing OS markers, it is interesting to note its cytotoxic potential against human breast adenocarcinoma cells. This effect may be due to the presence in the extract of fumarprotocetraric acid, a compound that exerts antiproliferative activity against other human cancer cell lines via induction of apoptosis (Kosanic et al., 2014).

Regarding Vc, an acetone extract of the lichen has recently been shown to effectively reduce the viability of Raji cells in a trypan blue exclusion assay (Srestha et al., 2015). In our study of its methanol extract, it reduced the cell viability of both cell lines at lower concentrations than those needed for Ci, implying that it has stronger cytotoxic activity (see Fig. 5B and D). Similarly, results obtained from Vc at a concentration of 25 [micro]g/ml are remarkable, since it had previously demonstrated antioxidant and protective effects in astrocytes; it has antiproliferative potential in both types of cancer cell lines, especially against the hepatocarcinoma cell model. The antiproliferative capacity of Vc may be related to its usnic acid content. This compound has already been identified as being of interest in cytotoxicity studies, since both enantiomers of usnic acid inhibited the growth of the cancer cell lines T-47D and Capan-2 via inhibition of DNA synthesis and mitochondrial dysfunction (Einarsdottir et al., 2010).

Conclusions

For the first time, the neuroprotective activities of methanol extracts of Cetraria islandica and Vulpicida canadensis have been investigated, with respect to their antioxidant actions, in a model of OS in nervous system-like cells (astrocyte model); such a model was chosen due to the increasingly acknowledged importance of glial cells in physiological and pathological diseases (Colangelo et al., 2014).

Both extracts demonstrated interesting activities in two in vitro radical scavenging assays (ORAC and DPPH), suggesting this to be a possible mechanism accounting for their antioxidant capacity. We then assessed the antioxidant potential at the intracellular level and its involvement in neuroprotection.

Cell viability assays enabled us to determine optimal concentrations for each extract (10 [micro]g/ml Ci and 25 [micro]g/ml Vc), which were selected on the basis of their cytoprotective actions against [H.sub.2][O.sub.2], and then tested in the aforementioned OS marker experiments. In general, our results indicate that these Parmeliaceae lichens can partially reverse the [H.sub.2][O.sub.2]-induced deleterious effects on redox status in astrocytes; in fact, they were able to reduce intracellular ROS formation, attenuate changes in the glutathione system and lower lipid peroxidation. However, it seems that they could not significantly protect cells from OS-induced apoptosis. Furthermore, we have demonstrated a promising cytotoxic potential for Ci and Vc towards human hepatocarcinoma and breast adenocarcinoma cell lines whose mechanisms of action deserve further investigation.

With respect to their phytochemistry, the chromatograms confirmed that depsidones and dibenzofurans were the most abundant classes of substance in the extracts. These molecules include phenolic groups, indicating an important role for phenolic compounds in the antioxidant activity of lichens, as noted in another study (Kosanic et al., 2013). All biological activities of Cetraria islandica demonstrated in this work can be attributed to the fumarprotocetraric acid, the predominant metabolite in the extract (more than 90%). However, in Vulpicida canadensis, three components are present in relatively high proportions; although usnic acid is the most abundant, it is difficult to determine the contribution of the individual components to the overall bioactivity.

In conclusion, the lichen species tested have promising neuroprotective properties, based on their antioxidative effects, and interesting cytotoxic activities against cancer cells. Considered as a whole, our results suggest that lichens could be a good source of natural antioxidant, neuroprotective and anticancer agents. They merit further investigation, including an exhaustive study of the biological activities of the compounds isolated here.

Conflict of interest

The authors declare that they have no conflicts of interest.

ARTICLE INFO

Article history:

Received 6 February 2015

Revised 10 June 2015

Accepted 12 June 2015

Abbreviations: Ci, methanol extract of Cetraria islandico: Vc, methanol extract of Vulpicida canadensis; OS, oxidative stress; ROS, reactive oxygen species; HPLC, high-performance liquid chromatography; Nrf2-ARE, nuclear erythroid factory-antioxidant response element; DMEM, Dulbecco's modified Eagle's medium; FBS, foetal bovine serum; PBS, phosphate saline buffer; DMSO, dimethyl sulphoxide; MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide; DPPH, 1,1-diphenyl-2-picrylhydrazyl; DCFH-DA, 2,7-dichloro-dihydrofluorescein diacetate; AAPH, 2,2'-azobis(2-methylpropionamidine) dihydrochloride; GA, gallic acid; ORAC, oxygen radical antioxidant capacity; AUC, area under the curve; TE, Trolox equivalent; LDH, lactate dehydrogenase; GSH, reduced glutathione; GSSG, oxidized glutathione; MDA, malondialdehyde; SD, standard deviation; tR, retention time.

Acknowledgments

This work was supported by project CGL2013-42498-P from the Spanish Ministry of Economy awarded to Prof. Dr. Ana Crespo, and a doctoral grant from the Spanish Ministry of Education, Culture and Sports (FPU), awarded to Carlos Fernandez-Moriano (No. FPU12/03824).

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Carlos Fernandez-Moriano (a), Pradeep Kumar Divakar (b), Ana Crespo (b), M. Pilar Gomez-Serranillos (a,) *

(a) Department of Pharmacology, Faculty of Pharmacy, University Complutense of Madrid, Plaza Ramon y Cajal s/n, 28040 Madrid, Spain

(b) Department of Plant Biology II, Faculty of Pharmacy, University Complutense of Madrid, Plaza Ramon y Cajal s/n, 28040 Madrid, Spain

* Corresponding author: Tel.: +34 91 394 1767; fax: +34 91 394 2276.

E-mail address: pserra@ucm.es, pserra@farm.ucm.es (M.P. Gomez-Serranillos).

http://dx.doi.org/10.1016/j.phymed.2015.06.005

Table 1
Results obtained for both extracts in the methanol extraction,
phenolic content determination and free radical scavenging
assays (ORAC and DPPH).

                                            Phenolic content
Lichen specie          Yield (% w/w)        ([micro]g GA/mg)

Cetraria islandica     3.44 [+ or -] 0.19   57.34 [+ or -] 3.30
Vulpicida Canadensis   7.83 [+ or -] 0.43   34.93 [+ or -] 1.40

                       ORAC value            DPPH [IC.sub.50]
Lichen specie          (TE/mg dry extract)   ([micro]g/ml)

Cetraria islandica     3.06 [+ or -] 0.31    1183.55
Vulpicida Canadensis   0.77 [+ or -] 0.08      99.83

Table 2
Retention times (mean [+ or -] SD) of the examined lichen
compounds and their UV-absorbance spectral data.

                               Retention            Absorbance maxima
Peak  Compound                 time (min)           (nm) UV spectrum

PRO   Protocetraric acid       21.93 [+ or -] 0.14  212, 244, 318
FUM   Fumarprotocetraric acid  26.73 [+ or -] 0.35  212, 240, 318
VUL   Vulpinic acid            30.34 [+ or -] 0.01  202, 234, 276, 354
PIN   Pinastric acid           32.35 [+ or -] 0.01  202, 246, 394
USN   Usnic acid               39.04 [+ or -] 037   204, 232, 282

Table 3
Effects of Ci and Vc pretreatments on OS markers (concentrations: 10
[micro]g/ml for Ci, 25 [micro]g/ml for Vc and for Trolox 0.1 mM; 24
h). Cells were treated with lichen extracts and [H.sub.2][O.sub.2]
(1 mM). Means [+ or -] SD, (#) p < 0.05 Vs [H.sub.2][O.sub.2].

Cell treatment     Caspase-3 activity          Ratio GSH/GSSG
                   (% activity)

Control cells      100                             11.67 [+ or -] 1.30
[H.sub.2]              302.25 [+ or -] 30.1         5.85 [+ or -] 0.68
  [O.sub.2] 1 mM
Ci [+ or -]            267.43 [+ or -] 36.35    7.93 (#) [+ or -] 0.65
  [H.sub.2]
  [O.sub.2] 1 mM
Vc + [H.sub.2]         272.65 [+ or -] 31.29   10.11 (#) [+ or -] 1.86
  [O.sub.2] 1 mM
Trolox [+ or -]    209.25 (#) [+ or -] 29.93   10.03 (#) [+ or -] 0.74
  [H.sub.2]
  [O.sub.2] 1 mM

Cell treatment     MDA levels
                   (nmoles/mg protein)

Control cells          2.01 [+ or -] 0.47
[H.sub.2]              5.60 [+ or -] 031
  [O.sub.2] 1 mM
Ci [+ or -]        2.25 (#) [+ or -] 0.22
  [H.sub.2]
  [O.sub.2] 1 mM
Vc + [H.sub.2]     4.15 (#) [+ or -] 0.32
  [O.sub.2] 1 mM
Trolox [+ or -]        5.19 [+ or -] 0.69
  [H.sub.2]
  [O.sub.2] 1 mM
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Author:Fernandez-Moriano, Carlos; Divakar, Pradeep Kumar; Crespo, Ana; Gomez-Serranillos, M. Pilar
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Date:Aug 15, 2015
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