Antioxidant Activity and Mechanisms of Action of Natural Compounds Isolated from Lichens: A Systematic Review

Chronic diseases such as cancer, diabetes, neurodegenerative and cardiovascular diseases are characterized by an enhanced state of oxidative stress, which may result from the overproduction of reactive species and/or a decrease in antioxidant defenses. The search for new chemical entities with antioxidant profile is still thus an emerging field on ongoing interest. Due to the lack of reviews concerning the antioxidant activity of lichen-derived natural compounds, we performed a review of the antioxidant potential and mechanisms of action of natural compounds isolated from lichens. The search terms “lichens”, “antioxidants” and “antioxidant response elements” were used to retrieve articles in LILACS, PubMed and Web of Science published until February 2014. From a total of 319 articles surveyed, 32 met the established inclusion and exclusion criteria. It was observed that the most common isolated compound studied was usnic acid, cited in 14 out of the 32 articles. The most often described antioxidant assays for the study of in vitro antioxidant activity were mainly DPPH, LPO and SOD. The most suggested mechanisms of action were scavenging of reactive species, enzymatic activation and inhibition of iNOS. Thus, compounds isolated from lichens are possible candidates for the management of oxidative stress, and may be useful in the treatment of chronic diseases.


Introduction
Oxidative stress is characterized as an imbalance between the production of reactive species and antioxidant defense activity, and its enhanced state has been associated with many of the chronic diseases such as cancer, diabetes, neurodegenerative and cardiovascular diseases [1].Based on that, many research groups have driven efforts to assess the antioxidant properties of natural products.These properties have been investigated through either chemical (in vitro) or biological (in vivo) methods, or both [2].The results of these researches have led some to suggest that the long-term consumption of food rich in antioxidants can retard or avoid the ocurrence of such diseases [3,4].
According to Brewer [5], the effectiveness of a large number of antioxidant agents is generally proportional to the number of hydroxyl (OH) groups present in their aromatic ring(s).Based on that, the natural compounds would seem to have better antioxidant activity than the currently used synthetic antioxidants, making them a particularly attractive ingredient for commercial foods [5].
Despite the large number of natural products that are currently consumed as antioxidant agents, the search for new chemical entities with antioxidant activity still remains a burgeoning field.In this context, the lichens have played an important role as a source for new antioxidant agents.
Lichens are symbiotic organisms consisting of a fungus and one or more photosynthetic partners, the latter usually being either a green alga or a cyanobacterium [6,7].They are found in a wide variety of natural habitats or in places with low temperatures, prolonged darkness, drought and continuous light [8].Lichens produce characteristic and unique secondary metabolites, and most of them occur exclusively in these symbiotic organisms [9].The most common lichen compounds are aromatic polyketides, particularly depsides, depsidones, depsones, dibenzofurans, and chromones [10].
Lichens have been used in the folk medicine for numerous purposes, among them as astringents, laxatives, anticonvulsives, antiemetics, antiasthmatics, anti-inflammatories, antibiotics, and also for the treatment of cardiovascular, respiratory, and gastric disorders [11].Furthermore, pharmacological and biotechnological studies have been carried out in order to test and to develop biomaterials containing lichen-isolated natural compounds for humans use [12,13].
Therefore, based on that, and also due to the lack of reviews concerning the antioxidant activity of lichen-isolated natural compounds, we have performed, for the first time, a systematic review of the literature that provides an overview of the antioxidant properties and mechanisms of action of natural compounds isolated from lichens.

Results and Discussion
A total of 319 abstracts/citations was identified for preliminary review from electronic and manual searches.The primary search identified 319 articles, with 214 from PubMed, 24 from LILACS, 71 from Web of Science and 10 from manual selection.After the removal of duplicates and screening for relevant titles and abstracts, a total of 89 articles was submitted for a full-text review.Thirty two articles met the inclusion and exclusion criteria established.A flow chart illustrating the progress of study selection and number of articles at each stage were performed as described in Barreto et al. [14] (Figure 1).The characteristics of included studies were summarized in the Table 1.The 32 studies were developed between 2000 and 2014, in 15 different countries, being eight from Asia, seventeen from Europe and six from South America.Among them, Serbia was the country with the largest number of studies, represented by six studies, followed by Brazil with five.Among these articles, it was observed that the most common isolated compound studied was usnic acid, which was cited in 14 of the total 32 articles.Besides usnic acid, another nine compounds were frequently referred to: atranorin, diffractaic acid, lecanoric acid, lobaric acid, stictic acid, salazinic acid, fumarprotocetraric acid, physodic acid and the orsellinates.
On the other hand, protective effects were found by De Paz et al. [21] against hydrogen peroxide-induced damage in U373 MG cells (human glioblastoma astrocytoma).UA showed a strong antioxidant capacity in the oxygen radical absorbance capacity (ORAC) assay, indicating significantly reduced radical oxygen species (ROS) production.These data indicate that UA (5-50 µg/mL) could act as an antioxidant agent against neurodegenerative disorders associated with oxidative damage, such as Alzheimer's and Parkinson's disease.In a study by Santos et al. [18], usnic acid induced the greatest release of NO in peritoneal macrophages, promoting a immunostimulatory effect.
Polat et al. [26], assessing the genotoxic and antioxidant effects of UA in human blood cells, observed that UA did not induce mutagenic effects on human lymphocytes, and increased total antioxidant capacity (TAC) at low doses (1 and 5 µg/mL) and in total oxidative status (TOS), at a high dose (200 µg/mL).However, at this high dose, UA significantly decreased TAC levels.
Conversely, no antioxidant action of UA was observed by Dévéhat et al. [16] and Thadhani et al. [7] on the DPPH assay.The radical-scavenging effect of antioxidants on DPPH is a simple and reliable method to quantify the hydrogen donating potency of chemicals.Since no activity of UA was observed in the DPPH, it does not seem to have labile hydrogen atoms.As for the contradictory data in the LPO assay, the different concentrations utilized could have influenced the test results.
Furthermore, in a study conducted by Rabelo et al. [27], who tested the UA redox properties against different reactive species (RS) generated in vitro, and evaluated its action on SH-SY5Y neuronal-like cells upon hydrogen peroxide (H2O2) exposure, it was observed that UA could display significant antioxidant properties in the TRAP/TAR and OH radical scavenging activity tests.It also induced cell detachment and loss of viability of SH-SY5Y cells at higher concentrations (20 µg/mL) alone or in the presence of H2O2 or 1% of FBS, related to the increase of intracellular ROS, inducing an oxidative stress scenario, potentiated in the presence of H2O2.The pro-oxidant properties in biological systems might be responsible for the potential neurotoxicological effects of UA.
The heterocyclic structure composed by conjugated dienes and polar OH groups of UA suggests that this molecule is able to act as a redox-active agent, thus interacting with different RS as observed in some in vitro assays by the works described above.Nonetheless, the results observed in different biological assays indicate that UA may exert either pro-oxidant or antioxidant effects in different cell types and tissues, thus other mechanisms such as modulation of antioxidant enzymes and cell detoxification systems must be further investigated to address the mechanism of its redox actions.Also, UA may influence the polarity of the inner mitochondrial membrane [20], which may be reflected in changes in basal RS production to varying degrees in different cell types.As the profile of mitochondria expression and activity varies according the cell type, the effect of UA on mitochondrial integrity and activity should be further investigated in the different cell models studied.
Stronger cytotoxic activity was verified by atranorin, in the MTT assay, in several human cancer cell lines such as U937, HL-60 [24], FemX, LS174 [28], A2780, MCF-7, SK-BR-3, HT-29, HCT-116 p53 −/− , HCT-116 p53 +/+ and Jurkat [25].In the study conducted by Kosanić et al. [28], the antiproliferative activity was accompanied by a stronger increase in the percentage of the sub-G1 population and concomitant decrease in G2/M, leading to a G0/G1 cell cycle block and inducing apoptosis in a cell cycle-dependent manner.Bačrová et al. [25] also identified higher pro-apoptotic activity (except on the A2780 cell line), supported by the inhibition of clonogenic ability and cell proliferation.On the other hand, Melo et al. [8] demonstrated no cytotoxic effect on the SH-SY5Y cells, bestowing atranorin with the capacity to induce cytoprotection in the presence of toxic concentrations of H2O2.
A peroxyl radical scavenging effect of atranorin (0.1 to 100 µg/mL) in TRAP/TAR assays was also observed by Melo et al. [8].It also presented a significant superoxide dismutase-like activity, evidencing an antioxidant potential against superoxide radicals.Conversely, it presented a pro-oxidant capacity in a lipid-rich system, enhancing TBARS formation induced by thermolysis of 2,20-azo-bis-(2-amidinopropane) dihydrochloride (AAPH) incubation.In assays where the antioxidant potential against NO and H2O2 was evaluated, atranorin was also shown to enhance the production of such species, acting as a pro-oxidant molecule, but only at higher concentrations.
Hydrogen peroxide is known to induce cell death by oxidative stress-dependent necrosis and apoptosis, which results from severe oxidative damage to DNA, lipids and proteins.It is very likely that the pro-oxidative observed by Melo et al. [8] is related to the concentration range tested, since in the study of Marante et al. [24] lower doses (100-250 µM) acted reducing H2O2/FeCl2 and inhibiting LPO, protecting from oxidative damage by the inhibition of both ROS and free radicals [24].These last findings were corroborated by Papadopoulou et al. [29] and Kosanić et al. [28], whom also observed a noteworthy antioxidant activity of atranorin (0.012-0.017 mg/mL) on the Co(II)/EDTA-induced luminol plateau chemiluminescence assay and very strong antioxidant activity in the DPPH, SAS and reducing power assays, respectively.These antioxidant properties could be contributing to its pharmacological effects, such as to reduce the damage effects on skin and to modulate the healing process of wounds [30].
Although antioxidant effects were described by Melo et al. [8], Marante et al. [24], Kosanić et al. [28] and Papadopoulou et al. [29], neither Dévéhat et al. [16] nor Thadhani et al. [7] found any antioxidant activity of atranorin on SOR and SAS, respectively.The presence of at least one free OH group, attached to either ring A or B, is necessary for the SOR activity; atranorin has a deactivating aldehyde group at C-3 of ring A [7].The quantity of free OH might also be involved in the superoxide scavenging-effect [5].Other similarly substituted compounds should be tested to determine whether these substituents are associated with the activity.

Lecanoric Acid
Lecanoric acid (Figure 4) belongs to the depsidone family and can be isolated from several lichens, including Usnea subvacata Motyka, Parmotrema stuppuem, Parmotrema tinctorum and Parmotrema grayana [18,28,31,32].Research conducted by Thadhani et al. [7], demonstrated a high SOR activity of lecanoric acid, comparable to that of the standards propyl gallate (PG) and butylated hydroxyanisole (BHA).They also verified a moderate activity in NOR and DPPH assays, corroborating the report of Lopes et al. [32].Jayaprakasha and Rao [31] also observed moderate antioxidant potential in the β-carotene-linoleate model system.The moderate antioxidant activity of lecanoric acid could be justified by its electron-attracting properties due to the two hydrogen bonds between the 2'-OH and 1'-COOCH3/COOH groups and the 2-OH and 1-COO-groups and also due to the presence of the COO-group, conjugated with an aromatic ring, which is also electron-attracting [31].Anyhow, these studies suggest that lecanoric acid is potentially able to interact with superoxide radicals.
Research conducted by Bayir et al. [33] verified that different doses of diffractaic acid (25, 50, 100, and 200 mg/kg) decreased MPx and iNOS and increased cNOS activities, suggesting that it could play an inhibitory role in neutrophil infiltration into gastric mucosal tissues, resulting in a gastroprotective effect on indomethacin-induced gastric lesions in rats.It also increased levels of SOD, GPx, GSH and decreased the effect on LPO, indicating an enhancing effect on the antioxidant defense system against oxidative tissue damage.It is not known, however, whether these effects resulted from transcriptional or post-translational effects on these enzymes.Molecules presenting polyphenolic structures may influence the oxidation state of intracellular thiol groups, thus affecting the activation of redox-sensitive transcription factors such as Nrf2, which modulates the transcription of genes involved in the antioxidant response [34,36].
In the study of Odabasoglu et al. [36], it was demonstrated that oral treatment of rabbits with diffractaic acid (30 mg/kg), in olive oil, can exert pro-apoptotic effects in tissue surrounding titanium implants, through the activation of initiator caspases (caspases 2, 8, and 9), executioner caspase (caspase 3), SOD activity and GSH levels, and the inhibition of the enzymatic activities of iNOS and MPx.These data indicate that this could be a possible mechanism involved in the protection towards cancer development in several tissues, by means of natural chemically induced apoptosis.The modifications on the levels of endogenous antioxidants and enzymes involved in the antioxidant response along with the activation of initiator caspases also suggest that the effect of diffractaic acid on mitochondrial homeostasis should be investigated in detail.

Anticarcinogenic
Santos et al. [18], on the other way, observed a high activity of diffractaic acid in the release of NO in mice macrophage cells, which could contribute to the production and extracellular release of ROS, bringing forth immunostimulatory effects.
Brisdelli et al. [23] demonstrated that diffractaic acid (2.5-100 µM) presents good antiproliferative activity against HCT-116 cells and reduction of viability in MCF-7 and HeLa cells, through the MTT assay.However, no free radical scavenging activity was observed, and the lichen metabolites did not significantly increase the intracellular ROS level and did not prevent oxidative injury induced by t-butyl hydroperoxide in HeLa cells.These findings suggest that the cytotoxic effect was not triggered by reactive oxygen overproduction, but could be related to diffractaic acid ability to induce programmed cell death through a caspase-dependent pathway.

Lobaric Acid
Lobaric acid (Figure 6) is a member of the depsidone family and can be isolated from several Antarctic lichens, including Sterocaulon alpinum e Cladonia sp.[7,37].In the study of Bhattarai et al. [37], this compound (0-4000 µM) was shown to have antibacterial activity against Gram-positive bacteria Staphylococcus aureus and Bacillus subtilis, through a MIC assay.Nevertheess, moderate antioxidant activity by reducing DPPH free radicals in a dose-dependent manner without any toxic effects was also observed [37].
Brisdelli et al. [23] identified lobaric acid effects on cytotoxic activity against HeLa and HCT cells lines only at higher concentrations (100 µM) on an MTT assay.However, no free radical scavenging activity was observed in the DPPH assay and no increase in cellular ROS, suggesting that cytotoxic action was not triggered by ROS formation.These results are in accordance with previous research conducted by Thadhani et al. [7], in which no activity on DPPH and NOR was observed.
Although no dose or concentration was specified by Thadhani et al. [7], the contradictory responses of lobaric acid on the DPPH assay between Bhattarai et al. [37] and Brisdelli et al. [22] could be related to the concentration range, since Bhattarai et al. [37] used much higher doses than Brisdelli et al. [22] and the DPPH activity was found to be dose-dependent.

Antioxidant Neuroprotective
In the study of De Paz et al. [21], stictic acid showed protective effects against U373MG cell line (5 and 10 µg/mL) by decreasing ROS production induced by hydrogen peroxide in an ORAC assay, inducing neuroprotection through their antioxidant activity.It also presented a noteworthy antioxidant activity (concentration range 0.012-0.017mg/mL) according to the radical scavenging Co(II)/EDTA-induced luminol plateau chemiluminescence assay [29].However, no antioxidant activity was demonstrated by Dévéhat et al. [26] (at 187.5-3000 µM) through the DPPH and SAS assays.A possible explanation for this is the molecular conformation, which could be affecting their antiradical activity, intrinsically related to DPPH.

Anticarcinogenic Immunoestimulatory
Research about its properties revealed a stronger antioxidant (DPPH, SAS and reducing power assays) [16,28], antimicrobial (MIC assay) and anticarcinogenic (MTT assay) activity against the FemX and LS174 cell lines, consistent with the induction of apoptosis in a cell cycle-dependent manner [28].In another study conducted by Santos et al. [18], furmaprotocetraric acid stimulated an increase of NO release in macrophage cells.Macrophages are known to play an important role in host defense mechanisms.In the immune system, reactive oxygen intermediates (ROI) often function together with nitric oxide (NO), for example in macrophage killing of bacteria and tumors cells, therefore inducing immunostimulatory effects [18].

Salazinic Acid
From the depsidone class, salazinic acid (Figure 9) can be isolated from Xanthoparmelia camtschadalis, Rimelia cetrata and Parmelia caperata [18,21,38].This compound can be used as an antioxidant agent in Alzheimer's disease for its benefits in decreasing ROS production in U373MG cells by hydrogen peroxide in the ORAC assay, inducing neuroprotection through their antioxidant ability in astrocytes and protecting against oxidative stress [21].Santos et al. [18] also demonstrated that salazinic acid was able to activate the release of H2O2 and NO in a culture of mice peritoneal macrophages.These releases could involve the so-called oxidative burst (O2 − ), a sequence of biochemical reactions that ends with the production and extracellular release of ROS, playing an important role in macrophage killing of bacteria and tumors, and inducing immunostimulatory effects [18].Furthermore, salazinic acid, in a study conducted by Manojlović et al. [38], demonstrated stronger antioxidant properties due to high activity on scavenging DPPH radicals, superoxide anion radical scavenging and reducing power, besides high antibacterial properties against B. mycoides, B. subtilis, E. coli, K. pneumonia, S. aureus, A. flavus, A. fumigatus, C. albicans, P. purpurescens and P. verrucosum (MIC assay).Cytotoxic activity was also verified against the Femx and LS174 cell lines in the MTT assay.The authors suggest that these activities could be due to its higher phenol content.
Kosanić et al. [40] demonstrated high antioxidant activity in the reducing power and SAS assays, correlated with a high content of total phenolics of the acetone extracts of the species from which physodic acid was isolated.Likewise, very strong antimicrobial (MIC) assay activity against B. mycoides, B. subtilis, E. coli, K. pneumoniae, S. aureus, A. flavus, A. fumigatus, C. albicans, P. purpurescens and P. verrucosum was observed along with cytotoxic activities against human melanoma FemX and human colon carcinoma LS 174 cell lines (MTT assay and flow cytometry).However, no mechanism of action was clarified in this study.Stojanović et al. [41] also observed a certain anticarcinogenic activity through a significant decrease in the viability and proliferation of HeLa cells, which could be explained by the substitution of positions 1 and 6 in these compounds with long nonpolar substituents.Moreover, Pavlović et al. [39] verified that physodic acid decreased rat thymocytes proliferation, mediated by increased cytotoxicity that could be due to the increase of ROS levels and decrease of mithochondrial membrane potential (MMP), therefore, inducing immunoprotective effects.

Orcinol, Orsellinic Acid and other Orsellinates
Orsellinic acid and methyl orsellinate (Figure 11), were isolated from Parmotrea stuppeum by Jayaprakasha and Rao [31] and from Heterodermia obscurata by Thadhani et al. [7], together with methyl-β-orcinolcarboxylate and orcinol (benzoic acid derivatives and a benzenoid, respectively).Both studies observed antioxidant activity of the compounds in the β-carotene-linoleate model system [31] and in the NOR assay [7].Orsellinate methyl-β-orsellinate or methyl-β-orcinolcarboxylate demonstrated antibacterial activity against Staphilococcus aureus in the MIC assay [24] and high activity in the NOR assay [7].However, lower activity was demonstrated on the SOR assay and low or no effect was verified in the DPPH assay [27].

Antioxidant Antibacterial
The relative inactivity in the SOR assay can be rationalized by observing the structures of the standards propyl gallate (PG) and butylated hydroxyanisole (BHA) which themselves possess only one aromatic ring.The latter two compounds, although mononuclear aromatic ones, have ortho and para OH/OR groups as compared to the meta relationship between OH/OR in the orsellinates, leading to higher SOR activity.On the other hand, mononuclear aromatic compounds were more active in the NOR assay suggesting that the redox potentials (LUMO) of simple aromatics are more compatible with the NO radical HOMO [7].

Other Compounds
Besides these major compounds, other less cited substances were also isolated from lichens and tested for antioxidant activity in the articles reviewed herein.We can classify all these compounds according to their antioxidant, antimicrobial and anticarcinogenic properties.

Antioxidant
The depside evernic acid, isolated in research conducted by Kosanić et al. [40], demonstrated high antioxidant activity in reducing power and SAS assays, correlated with a high content of total phenolics of the acetone extracts of the species from which it was isolated.Other two depsides, sekikiac acid and erythrin, were isolated by Thadhani et al. [7], demonstrating good activity in SOR assay and NOR assays respectively.
Among the depsidones, norstistic acid, 8'-methylmenegazziaic, psoromic and protocetraric acid were the most active.Norstistic acid, isolated in the study of Dévéhat et al. [16] showed better SAS activity than quercetin.These results are in accordance with Ranković et al. [19] which also observed activity in the SAS and in the DPPH and reducing power assays.8'-methylmenegazziaic exhibited higher antioxidant activity in the Co(II)/EDTA-induced luminol plateau chemiluminescence assay [29].Meanwhile, psoromic acid showed antioxidant activity in the FRSA, NOR and LPO assays, and cardiovascular-protective effects in a concentration-dependent manner, in HMGR and ACE inhibition assays [17].Protocetraric acid demonstrated higher activity in increasing NO release in macrophage cells, playing an important role in host defense mechanisms [18].
Kinoshita et al. [42] isolated four quinone derivatives from the lichens Lethariella sernanderi, L. cashmeriana and L. sinensis.Their antioxidant activity was assessed using a commercial assay kit based on Cu(II) reducing activity.Among them, 7-chlororubrocashmeriquinone showed the strongest potential, although canarione and 7-chlorocanarione also demonstrated high antioxidant activities, which suggested that the 1,2-quinone and 7-Cl moieties were important for antioxidant activity.Ramalin (γ-glutamyl-N'-(2-hydroxyphenyl)hydrazide), a nitrogen compound, was isolated from the Antarctic lichen Ramalina terebrata [39].According to Paudel et al. [43], ramalin presented scavenging activity against DPPH, 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid free radicals (ABTS•+), and superoxide anion radicals, and Fe 3+ to Fe 2+ ion reducing capacity.Furthermore, ramalin was able to inhibit the tyrosinase enzyme activity and showed no or very little cytotoxicity in human keratinocyte and fibroblast cells at its antioxidant concentration.These data suggest that ramalin had strong hydrogen-and electron-donating capacity, which may be the source of its very strong non-toxic antioxidant potential [43].TRamalin could therefore be viewed as a potential product for future cosmetic and therapeutic applications.
Marante et al. [24] used a phytotoxicity-based extraction and fractionation to separate allelochemicals contained in an extract of Lethariella canariensis.Among benzoic acid and its derivatives isolated, atranol, chloroatranol, methyl hematommate and ethyl hematommate exhibited a dose-dependent antioxidant activity in a LPO assay, protecting tissue against oxidative stress.Methyl haematommate also demonstrated NOR activity, along with another benzoic acid derivative, montagnetal, in the study of Thadhani et al. [7].Given the large amounts of p-substituted polyphenolic compounds found in lichens and their antioxidant activity, it is conceivable that they contribute to the antioxidant defense mechanisms of these organisms [7].

Anticarcinogenic
From the depside class, Kosanić et al. [40] described evernic acid's activity against FemX and LS174.Bačrová et al. [20] verified that gyrophoric acid was highly effective, cytotoxic and slightly pro-apoptotic against the HL-60, A2780 and Jurkat cell lines, which correlated with a cell cycle variation, represented by the accumation of cells in S-phase at the expense of the G1/G0-phase.Another depside, sphaerophorin, was able to trigger apoptotic death in melanoma cancer cells.In fact, a high DNA fragmentation (Comet and TUNEL Assays), reinforced by a significant increase in the caspase-3 enzyme activity, and not correlated to lactic dehydrogenase release, a marker of membrane breakdown, occurred in melanoma cells treated with these natural compound.Also, increased ROS formation was observed in a concentration-dependent manner, which could amplify the apoptosis cascades, besides a dose-dependent superoxide scavenging effect [45].
Also from the research of Russo et al. [45], the depsidone pannarin was isolated.Its activity was the same as that observed for sphaerophorin.These findings provide evidence that pannarin and sphaerophorin prevent UV light and nitric oxide-mediated plasmid DNA damage, and attenuate the growth of melanoma cells, at least in part, by triggering an apoptotic process.Antiproliferative activity correlated with an increase in the number of cells in the sub-G1 phase whiled the percentage of cells in the S-phase and G2/M phase remained unchanged compared to the controls.Interestingly, LS174 cells treated with the tested samples showed a significant increase of the sub-G1 phase and concomitant decrease in G2/M was observed, supporting a G1 phase arrest.These results suggested that the compound have a prominent ability to induce apoptosis in FemX and LS174 cells.Other compounds from the depsidone family were also mentioned.Protocetraric acid demonstrated cytotoxicity against the FemX and LS174 cell lines [38].Norstictic acid also presented antiproliferative activity against FemX and LS174 which may occur through an apoptosis pathway [18].Physodalic acid significantly decreased rat thymocyte proliferation, mediated by increased cytotoxicity that could be due to the increase of ROS levels and decrease of mithochondrial membrane potential (MMP) [39].3-Hydroxyphysodic acid also decreased rat thymocyte proliferation and HeLa cell lines, however, it is speculated that its application to cell culture may not induce ROS generation, crucial for cell death, but at certain concentrations may lead to low intracellular energy levels with resulting cytotoxicity [39,41].Vicanicin induced a significant loss of viability in a dose-dependent manner in HeLa and HCT-116 cells and protolichesterinic acid induced apoptosis in HeLa cells by activating an extrinsic caspases-8/-3-mediated as well as intrinsic caspases-9/-3-mediated pathway [23].
Concerning the quinones, in the study of Bačrová et al. [25], parietin demonstrated significant cytotoxicity in the A2780, Jurkat and HT-29 cell lines.Although it did affect cell proliferation, the impact on cell viability and percentage of floating cells was insignificant.Therefore, the action of parietin may best be regarded as cytostatic.Biruloquinone, an ortho-phenanthraquinone compound was isolated by Luo et al. [46].The AChE inhibitory assay classified this compound as a mixed-II inhibitor.An MTT assay verified that biruloquinone improved the viability of the H2O2 and β-amyloid injured PC12 cells.Due to its potent antioxidant activity, the authors suggested that biruloquinone may be used in the treatment of Alzheimer's disease patients for enhancing their cognition and slowing the symptoms by protecting the injured neurons.

Final Considerations
Within the 32 studies here reviewed, eight classes of secondary metabolites were described: dibenzofurans, represented by usnic acid; depsides, represented by 13 different compounds; depsidones, by 28 compounds; benzoic acid derivatives, by 20 compounds; xanthones, by seven compounds; quinones, by six compounds; terpenoids, by two compounds; and nitrogen compound, represented by ramalin.The most cited substance was the usnic acid, mentioned in 14 studies, followed by atranorin, described in eight.
The pharmacological and other biological activities of lichen substances in this review could be divided into the following categories: antimicrobial activity, anticarcinogenic and antioxidant, which includes all the enzyme inhibitory activities involved in cytoprotective, cardioprotective, gastroprotective, and immunostimulatory properties.The wide variety of biological activities of lichens is generally correlated to their special ecological circumstances.Geographic, altitudinal or microhabitat conditions and even the presence or absence of light can cause differentiations in lichen physiology and metabolism.The phenolic content and distribution is also modified according to direct UV irradiation [47,48].Consequently, the response of each isolated compound in the enormous variety of specific tests can differ among the different lichen species or related groups.
For the antioxidant assays it was observed that the DPPH, SAS, SOR and NOR tests were the most common ones used in the articles discussed here.However, other major assays were also used such as the Co(II)/EDTA-induced luminol plateau chemiluminescence assay, ORAC assay, TRAP/TAR, OHRS, TBARS, DFCH-DA, TAC/TOS, β-carotene/linoleate model, HMGR and AChE inhibition, reducing power, total phenolic content, ROS determination, H2O2 and NO measurements, CCK-8, ABTS •+ and the determination of some enzymes as SOD, CAT, GR, GPx, MPx, NOS, cNOS, iNOS, LPO and GSH.Most antibacterial activity was assessed by the MIC assay, apart from the paper disk infusion assay described in the study of Bhattarai et al. [37].As for the anticarcinogenic tests, MTT was the most described cytotoxicity assay, used in almost all the studies discussed herein, except for those of Odabasoglu et al. [36] and Russo et al. [46] in which TUNEL and Comet assays were utilized instead.However, other tests were assessed in a way to describe the compound's mechanism of action, such as HTCA, cell viability, cell proliferation, cell cycle transition, flow cytometry and caspases activities.
Although several studies utilized the same assays for the same compounds, there were discrepancies among the results observed, more specifically for the antioxidant assays.One possible explanation would concern the IC50 values.The several studies presented different standard or control compounds and also divergent IC50 values considered statistically significant.Thus, contrasting responses should be expected.For the reason, the activity of a compound must always be assessed with different tests, in order to identify different mechanisms and to establish in which degree a given compound interacts with the different reactive species.
Concerning the mechanisms of action by which these lichen compounds exert such activities, most of them are still not clear.However, it seems important that the higher phenol content in these compounds could be responsible for its antioxidant properties.Also, the number and position of OH groups in the aromatic ring(s) of the compounds is a common feature that may also account for some of their activities.Most of the lichenic compounds currently studied present a polyphenolic structure combined with acidic groups capable of exchanging protons with the environment.

Methods
This systematic review was conducted in accordance with the guidelines of Transparent Reporting of Systematic Reviews and Meta-Analyses (PRISMA statement) [49].

Search Strategy
Three internet sources were used to search for appropriate papers that met the study purpose.These included the National Library of Medicine, Washington, D.C. (MEDLINE-PubMed), Web of Science and LILACS (Latin American and Caribbean Health Sciences), using different combinations of the following keywords in medical subjects heading (MeSH) included: "Lichens", "Antioxidant Response Elements", and "Antioxidants".The databases were searched for studies conducted in the period up to and including February, 2014.The structured search strategy was designed to include any published paper that evaluated the use of natural compounds obtained from lichens in antioxidant activity to identify those that show therapeutic potential.We did not contact investigators and did not attempt to identify unpublished data.

Study Selection
All electronic search titles, selected abstracts and full-text articles were independently reviewed by a minimum of two reviewers (J.S.S.Q. and M.R.V.S.).Disagreements on study inclusion/exclusion were resolved with a consensus reaching.The following inclusion criteria were applied: antioxidant activity studies and the use of natural compounds obtained from Lichens.Studies were excluded according to the following exclusion criteria: studies in humans, studies using extracts, mixtures of lichen compounds, synthetic or derived compounds, review articles, meta-analyses, abstracts, conference proceedings, editorials/letters and case reports (Figure 1).

Data Extraction
Data were extracted by one reviewer using standardized forms and were checked for completeness and accuracy by a second reviewer.Extracted information included data regarding the substance, source (lichen species), chemical classes of secondary metabolites, assay type, pharmacological activity studied, and effect and/or mechanism of action suggested.

Conclusions
Lichens are clearly an interesting and rich source of compounds; their properties clearly indicate their potential for pharmaceutical purposes, although some activities of lichens still need consideration.Further studies are still needed to clarify the molecular processes and signaling pathways involved in their activity.Thus, new compounds will certainly be described from poorly studied lichens, and even in species that are commonly used and chemically well known, new chemical components are still being detected.

Figure 1 .
Figure 1.Flowchart of included studies.Studies were excluded according to the following exclusion criteria: studies in humans, studies of mixtures of substances or extracts from lichens, review articles, meta-analyses, abstracts, conference proceedings, editorials/letters, case reports.

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, 2008, China Usnea longissma In vitro: MTT, TNF-α and NO Anti-inflammatory Dose-dependent inhibitory effect on LPS-induced TNF-α and NO production in macrophages RAW 264.7, associated with decreased synthesis of TNF-α mRNA and iNOS protein De Paz et al. species (ROS) production on hydrogen peroxide-induced damage in U373 MG cells Bačkorová et al. [25], 2011, Slovakia Purchased from Sigma Chemical In vitro: MTT, HTCA, viability, cell proliferation and detachment, cell cycle transition and apoptotic nuclear morphology Anticarcinogenic Anti-proliferative action on A2780, MCF-7, SK-BR-3, HT-29, HCT-116 p53 −/− , HL-60 and Jurkat.Higher pro-apoptotic activity, supported by the suppression of viability and cell proliferation, correlated more strongly with an increased number of floating cells Thadhani et al. antioxidant activity, concentration-dependent manner, on the FRSA, NOR) and in LPO.Poor fobrinolytic potencial HL-60, A2780, MCF-7, SK-BR-3, HT-29, HCT-116 p53 −/− , HCT-116 p53 +/+ and Jurkat, triggered by higher pro-apoptotic activity (except on A2780 cell line), and supported by the inhibition of clonogenic ability and cell proliferation Thadhani et al.Superoxide dismutase-like and scavenging activity of peroxyl radicals.Induce cytoprotection in the presence of toxic concentrations of H 2 O 2 on the SH-SY5Y cells.Conversely, it presented a pro-oxidant capacity in a lipid-rich system, enhancing TBARS and also enhanced production of NO and H 2 O 2 in higher concentrations Barreto et al. [30], 2013, Brazil Cadina Kalbi In vivo: Myofibroblast field and macroscopic and histological analyses Wound healing Topical application of atranorin reduced wound areas, induced earlier granulation tissue formation, increased cell proliferation, improved collagenization and modulated the myofi broblasts differentiation when compared to control animals.
antimicrobial activities.Antiproliferative activity accompanied by a stronger increase in the percentage of the sub-G1 population and concomitant decrease in G2/M of FemX and LS174 cell lines, inducing apoptosis in a cell cycle

[ 19 ]
, 2012, Serbia Toninia candida In vitro:DPPH, SAS, reducing power, MIC, MTT and flow cytometry Antioxidant, antimicrobial and anticarcinogenic Stronger antioxidant and antimicrobial activities.Antiproliferative activity correlated with an increase in the number of cells in the sub-G1 phase whiled the percentage of cells in the S-phase and G2/M phase remained unchanged compared to the controls.Interestingly, LS174 cells treated with the tested samples showed a significant increase of the sub-G1 phase and concomitant decrease in G2/M was observed, supporting a G1 phase arrest.These results suggested that the compound have a prominent ability to induce apoptosis in FemX and

Table 1 .
Characteristics of Included Studies.