Lichens in Pharmacological Action: What Happened in the Last Decade?

Lichens are a unique group of organisms, which can produce compounds that are named secondary metabolites and rarely or are not produced in other organisms. Lichens possess pharmacological actions related to their secondary metabolites. Our knowledge of lichens and their pharmacological actions rapidly increases as new technologies and devices, which facilitate the investigation of the chemical profile and biological activities of lichens, are introduced and become more readily available. In addition, new methods and perspectives, as well as suggestions for pharmacological mechanisms, accumulate daily. Furthermore, lichen substances stand as a relatively untapped source of natural products. Accordingly, researchers investigate the pharmacological actions of lichen-derived material more frequently than it was in the past. This review focused on the pharmacological activities of lichens published in the last 11 years (2012-2022). Literature data obtained from WebOfScience and PubMed databases using related search keywords revealed that anti-genotoxicity, anti-cancer, and anti-microbial activity studies have constantly been conducted. More recently, immunomodulatory and inflammation-related studies took to the stage. Enzyme inhibition actions were popular as well. Our selection was based on the novelty and mechanistic insight that papers presented.


Introduction
Lichens are typically described as symbiotic organizations composed of a fungus and a photosynthetic symbiont, generally a green alga or cyanobacteria, or both.However, recent discoveries of microorganisms (bacteria or fungi) constantly present in or over lichen thalli led scientists to coin the more accurate term "miniature ecosystem." In this "ecosystem, " chemical interactions occur between any partners, enriching the pool of lichen-derived substances.This richness eased the way of lichens into ethno botan ical/ ethno pharm acolo gical use throughout history. 1 Lichen secondary metabolites are mainly depsides and depsidones, but also monocyclic aromatic compounds, dibenzofurans, depsones, amino-acid derivatives, aliphatic acids, macrocyclic lactones, sugar alcohols, quinones, xanthones, chromones carotenoids, and terpenoids were discovered in lichens. 2 Pharmacological actions of lichens are unambiguously related to their secondary metabolite content.The quantity and quality of lichen substances present in any thalli were not easy to determine until recently.The advancements in analytical techniques and the rapid spread of modern analytical devices stimulated phytochemical analysis of lichens all over the world, increasing the number of purified compounds and bioactivity tests related to these compounds.In addition, the accumulated chemical data regarding lichen substances became readily available to anyone who asks for it.Considered together, these alterations in status led to folds of increase in lichen substances' bioactivity-related papers.more recent papers.A summary of pharmacological actions of lichens is presented in Table 1.Following is the account of our selection of papers in a pharmacological activity-oriented layout.

Pharmacological Activities of Lichens
Anti-Mutagenic and Antioxidant Activity A methanolic Cladonia foliacea extract was evaluated for antigenotoxic and antioxidant activities in various test systems.Investigators reported that the extract was not mutagenic and had antigenotoxic effect as well as a poor antioxidant capacity. 3 Total extracts from Letharia vulpina and Vulpicida pinastri were evaluated for antigenotoxic and antioxidant capacity in human lymphocytes treated with aflatoxin B1.Authors reported that lichen extracts decreased malondialdehyde levels and micronuclei frequencies and increased glutathione peroxidase, glutathione, and superoxide dismutase levels. 4thanolic extracts of Peltigera horizontalis and Peltigera praetextata were assessed for mutagenic, anti-mutagenic, and antioxidant activity.The results indicated that lichen extracts had no mutagenic activity.On the contrary, they had anti-mutagenic and antioxidant activity in all tested doses. 5traria aculeata, Cetrelia olivetorum, and Cladonia chlorophaea extracts were shown to have significant anti-mutagenic effects in N-met hyl-N ′-nit ro-N-nitro sogua nidin e, aflatoxin B1, sodium azide, and acridin induced mutagenesis tests.The extracts also improved superoxide dismutase and glutathione peroxidase activities and decreased malondialdehyde and glutathione amounts. 6ing single-cell gel electrophoresis, researchers assessed antigenotoxic effects of lobaric, diffractaic, vulpinic, and lecanoric acids, on human lymphocytes in vitro.Authors reported that all lichen acids mediated a considerable decrease in total DNA damage at all tested concentrations, and vulpinic acid was best among them. 7tioxidant and cytotoxic potential of Pseudevernia furfuracea-derived olivetoric and physodic acids were examined on cultured human amnion fibroblasts.Researchers revealed that these metabolites were not genotoxic, while they exhibited strong antioxidant potential at low doses. 8searchers found that usnic acid (UA) had no mutagenic effects on human lymphocytes, and low concentrations (1 and 5 µg/mL) of UA increased total antioxidant capacity in cultured human blood cells but had no effect on total oxidative status. 9timicrobial Activity In an interesting study, Lauinger et al 10 chemically targeted liver-stage parasites of Plasmodium berghei, the malaria parasite, with (+)-usnic, vulpic, evernic, and psoromic acids (PA).They found that UA exhibited the highest liver stage activity and stage specificity (liver stage IC50: 2.3 μM, blood stage IC50: 47.3 μM).The authors reported that other 3 compounds inhibited at least 1 enzyme (PfFabZ, PfFabG, PfFabI) from the plasmodial fatty acid biosynthesis (FAS-II) route, a possible target route.In addition, the compounds were also assessed against whole cells and FabI homologs of Staphylococcus aureus, Mycobacterium tuberculosis, and Escherichia coli.According to in silico studies, compounds acted indirectly by attaching to allosteric sites on the protein surface of the FAS-II enzymes.
Three UA derivatives and their metal salts were synthesized and tested against 10 pathogenic microorganisms.In addition, antimutagenic activity of these complexes was evaluated.Researchers reported that UA derivatives had potent antimutagenic effects and metal complexes had potent antimicrobial effects. 11erapeutic action of UA against coccidiosis was tested in broilers and was compared to that of toltrazuril.Authors reported that 100 mg/kg dose of UA exhibited anticoccidial activity compared to toltrazuril. 12 an extensive study, Shrestha et al 13 investigated antibiotic effects of 34 North American lichen extracts against gram (+) S. aureus, Pseudomonas aeruginosa, and methicillin-resistant S. aureus and gram (−) E. coli.Acetone extracts of Vulpicida canadensis, Letharia vulpina, and L. columbiana demonstrated antibacterial activity against E. coli.Except for Lobaria pulmonaria, all lichen extracts exhibited inhibitory effects against gram (+) bacteria with a range 3.9-500 µg/mL of MIC values.The effects of Rhizoplaca marginalis and Usnea hirta against methicillin-resistant S. aureus (7.8 µg/mL MIC) are noteworthy.
Basile et al 14 investigated acetone extract of Xanthoria parietina' s antifungal, antibacterial, and antiproliferative activities and its major secondary metabolite, parietin.They found that both exhibited antibacterial potential against 9 pathogenic bacteria including standard ATCC (American Type Culture Collection) and multidrug-resistant strains.Parietin performed better in antifungal test than acetone extract.The acetone extract also inhibited proliferation of human breast cancer cells and triggered apoptosis.This inhibition came with modification of production of cell cycle adjusting genes p16, p27, cyclin A, and cyclin D1.The extract triggered apoptosis through activation of external and internal cell death pathways, modifying various molecular processes.
Protocetraric acid isolated from Usnea albopunctata was found to have a supreme antimicrobial effect against a range of bacteria and fungi.It produced inhibition zones comparable to ciprofloxacin in disc diffusion tests and MIC (Minimum Inhibitory Concentration) values were better than those of ciprofloxacin for some of the tested bacteria.Especially, 0.5 µg/ mL MIC value for Salmonella typhi is impressive.In case of fungi, protocetraric acid performed comparably to amphotericin B in disc diffusion test, but MIC values were much higher than those of amphotericin B, except for Trichophyton rubrum, where protocetraric acid had a MIC value of 1 µg/mL and amphotericin B had a MIC value of 4 µg/mL. 15thak et al 16 tested acetone extract of Usnea orientalis against 6 dermatophyte species and found that the extract produced MIC values between 9 and 1040 µg/mL.Authors revealed the presence of usnic and salazinic acids in the extract.
According to a recent study, 17 Usnea steineri acetone extract displayed strong antibacterial effect (<10 µg/mL) against resistant Staphylococcus epidermidis and Enterococcus faecalis strains.Authors isolated (+) UA, which displayed potent activity against S. aureus and S. haemolyticus (MIC: 12.5 µg/mL) and particularly S. epidermidis (MIC: 3.12 µg/mL).Another interesting finding was the absence of synergistic antimicrobial effects in combinations of (+)-UA with penicillin and tetracycline.
The authors studied 27 secondary lichen metabolites isolated from 28 lichen species for their ability to inhibit RecA, an essential protein for genetic recombination, from E. coli.They found that 9 compounds exhibited over 80% inhibition of ATP hydrolytic activity of RecA protein.Among those 9 (IC50 values ranging from 14.2 to 42.6 µM) compounds, authors put forward epiphorellic acid, isolated from Cornicularia epiphorella, as a propitious candidate for the development of more potent RecA inhibitors. 18ragoz et al 19 tested extract fractions of Bryoria capillaris against numerous human and plant

Atraric acid Monocyclic Aromatic
Himantormia lugubri pathogens.They found that fractions rich in barbatolic and alectorialic acids were effective antimicrobial agents against tested bacteria in liquid but not in solid media.
In a recent study 20  Researchers investigated the effect of Cladonia uncialis acetone extract (CUE) and UA enantiomers on 10 skin infection-causing clinical microbial strains.At the same concentrations, (-)-UA alone was less active than CUE containing (-)-UA and squamatic acid.Cladonia uncialis acetone extract exhibited an activity that was similar to that of (+)-UA observed for S. epidermidis and E. faecium but did not display any activity against fungal strains.In addition, CUE showed low cytotoxicity against HaCaT (immortal human keratinocyte line) keratinocytes, compared to UA enantiomers, which is essential for its therapeutic use. 22e extracts of 12 lichen species were tested for their antibacterial potential by broth microdilution assay against 5 bacterial strains.None of the extracts were effective against gram (−) E. coli.On the other hand, Cladonia borealis, Cladina confusa, Stereocaulom ramulosum, and Canoparmelia cryptochlorophaea had 7.8 µg/mL MIC values against clinical isolates of S. aureus (resistant to clindamycin, erythromycin, and penicillin G) and E. faecium (resistant to vancomycin). 23

Antiviral Activity
Lai et al 24 explored activity of sekikaic acid (SA) isolated from Ramalina farinacea on the respiratory syncytial virus.They found that sekikaic acid strongly (IC50: 5.69 µg/mL) inhibited a recombinant rg respiratory syncytial virus strainand respiratory syncytial virus A2 strain (IC50: 7.73 µg/mL).In addition, they investigated viability of HEp2 and Vero cells treated with sekikaic acid.The time of addition assay uncovered a clear interference of sekikaic acid with viral replication at a viral post-entry step.Sekikaic acid was more effective than the control ribavirin (over 1.3-fold), at 4 hours post-infection addition.
Researchers concluded that the inhibitory action of PA against HSV-1 DNA polymerase is involved in its inhibitory action on HSV-1.

Enzyme Inhibition
Cheng et al 26 reported that anziaic acid isolated from Hypotrachyna sp. was able to inhibit bacterial topoisomerase I.In in vitro assays, anziaic acid exhibited antibacterial activity against Bacillus subtilis and E. coli.The authors also found anziaic acid to function as an inhibitor of human topoisomerase II but had little effect on human topoisomerase I.
Luo et al 27 isolated biruloquinone from Cladonia macilenta and it was shown to inhibit eel acetylcholinesterase with an IC50 value of 83.1 µM.According to the authors, biruloquinone was a mixed-II inhibitor on acetylcholinesterase and it improved the viability of the H 2 O 2 -and β-amyloid-injured PC12 cells at 1-25 µg/mL.
Bessadottir et al 28 isolated (+)-protolichesterinic acid((+)-PA), a known anti-proliferative agent, from Cetraria islandica and tested whether the anti-proliferative activity of (+)-PA is associated with effects on fatty acid synthase (FASN), human epidermal growth factor receptor 2 (HER2), and major signaling pathways in SK-BR-3 and T-47D breast cancer cell lines.They revealed that treatment with (+)-PA increased FASN in SK-BR-3 cells, implying a compensatory response to the inhibition of this enzyme.Human epidermal growth factor receptor 2 expression was decreased suggesting secondary downregulation.ERK1/2 and AKT signaling pathways were inhibited, probably due to reduced levels of HER2.
Authors investigated inhibition effect of 5 lichen acids (diffractaic, evernic, lobaric, lecanoric, and vulpinic acid) on mitochondrial hioredoxin reductase purified from rat lung in vitro and found that lichen acids' (particularly lecanoric and vulpinic acid) IC50 values were significantly lower than IC50 values of cisplatin and doxorubicin. 31kmak and Gulcin 32 reported that UA had significant antioxidant activity in various tests and was a good inhibitor of eel acetylcholinesterase and butyrylcholinesterase.
Researchers found that UA was able to inhibit purified human paraoxonase, glutathione reductase, and glutathione S-transferase enzymes in a favorable fashion.The authors concluded that UA could be useful in drug development studies. 33searchers investigated the mechanisms of cytotoxicity of parietin, atranorin, UA, and gyrophoric acid on A2780 and HT-29 cancer cell lines.According to the results, UA and atranorin were able to initiate an extensive loss in the mitochondrial membrane potential, along with caspase-3 activation (only in HT-29 cells) and phosphatidylserine externalization in both tested cell lines.The authors concluded that UA and atranorin are activators of programmed cell death in A2780 and HT-29, possibly via the mitochondrial pathway. 38canicin isolated from Psoroma dimorphum and PA isolated from Rhizoplaca melanophthalma were investigated for antigrowth and pro-apoptotic activity in androgen-sensitive (LNCaP) and androgen-insensitive (DU-145) human prostate cancer cells.Both compounds showed a doseresponse relationship in the range of 6.25-50 µM concentrations in DU-145 and LNCaP cells, activating an apoptotic process. 39 et al 40 42 Antitumor potential of olivetoric, physodic, and PAs was investigated on human glioblastoma multiforme (U87MG-GBM) cell lines and primary rat cerebral cortex (PRCC) cells.Investigators found that olivetoric acid was a promising agent with high toxicity against cancer cells and low toxicity against healthy cells.They also reported that all metabolites presented favorable antioxidant profiles (i.e., higher total antioxidant capacity in healthy cells and higher total oxidative status in cancer cells). 43isdelli et al 44 investigated the influence of PA on anti-proliferative activity of doxorubicin in HeLa, SH-SY5Y, and K562 cells and found that PA synergically increased the in vitro activity of doxorubicin against HeLa cancer cell line but not against SH-SY5Y and K562 cells.Protolichesterinic acid-promoted increase in cytotoxicity in HeLa cells was caused by a proapoptotic effect and was associated with caspase-3, 8, and 9 activation.
Three doses (50, 100, and 200 mg/kg body weight) of diffractaic acid isolated from Usnea longissima were found to have anti-tumoral activity against Ehrlich ascites carcinoma cells inoculated to mice.The best activity was observed with 200 mg/kg dose.Diffractaic acid did not have significant effect on hematological parameters. 45 another study, the methanol extract from Cladonia pocillum was able to inhibit cell proliferation while inducing programmed cell death in MCF-7 human breast cancer cells.The extract was found to prevent the oxidative damage to some extent.Also, the methanol extract of C. pocillum had antimicrobial activity, although not more than that of the chloroform extract. 46searchers investigated the cytotoxicity of atranorin, gyrophoric acid, and physodic acid on A375 melanoma cancer cells.The depsidone physodic acid showed a dose-response relationship in the range of 6.25-50 μM concentrations in A375 cells, activating an apoptotic process that probably involves the reduction of Hsp70 expression. 47searchers evaluated the anticancer capacities of ramalin, a secondary metabolite from the Antarctic lichen Ramalina terebrata, in the human colorectal cancer cell line HCT116.Ramalin significantly suppressed proliferation and induced apoptosis in a dose-dependent manner.In addition, it inhibited the migration and invasion of colorectal cancer cells.Cellular data revealed that, at both transcription and translation level, ramalin caused a gradual increase in the expression of tumor protein p53 and its downstream gene cyclin-dependent kinase inhibitor 1A, while decreasing the expression of cyclin-dependent kinase 1 and cyclin B1 and induced cell cycle arrest in the gap 2/mitosis phase. 48hyl acetate extract of Usnea longissima prevented gastric and esophageal carcinogenesis induced in rats with N-met hyl-N '-nit ro-N-nitro sogua nidin e (MNNG).The extract was not cytotoxic up to 2000 mg/kg dose.Its prominent anticarcinogenic activity at 50 and 100 mg/kg doses suggested that it was selective to the cancer tissue. 49 recent studies, researchers found lichen substances vulpinic 50 and diffractaic 51 acids to be promising inhibitors of thioredoxin reductase 1, an overexpressed enzyme in breast cancer cells.These substances also exerted potent antiproliferative and antimigration effects on MCF-7 and MDA-MB-453 cell lines.
Diffractaic, lobaric, and (+)-UAs were tested for proliferation change, oxidative status, and DNA damage potentials against human glioblastoma multiforme (U87MGGBM) and PRCC cells.Authors found that lobaric acid highly inhibited the proliferation of PRCC and U87MG cells.
Diffractaic and (+)-UAs exerted cytotoxic activity on both cells.The latter 2 was speculated to have minimal damage to healthy cells while reducing the number of cancerous cells. 52searchers isolated tsavoenones A, B, and C from Parmotrema tsavoense.They evaluated tsavoenones A for its cytotoxicity against K562 and HepG2 tumor cell lines using doxorubicin as a positive control.The results showed that tsavoenones A exerts a moderate cytotoxicity against human myelogenous leukemia K562 cell line with an IC50 value of 66 ± 1.7 µg/mL. 53vestigators showed that an unidentified polysaccharide extracted from Umbilicaria esculenta was able to inhibit the growth of A875 and A375 melanoma cells, without obvious toxicity to normal vascular endothelial cells.The generation of ROS in A875 cells was significantly elevated after 24 hours treatment with the polysaccharide.In addition, the expression of caspase-3 and -9 also increased as compared to the controlled group, which resulted in the apoptosis of A875 melanoma cells. 54ddy et al 55  Researchers assessed the antihyperglycemic activity as well as the antihyperlipidemic capacity of Evernia prunastri in normal and streptozotocin-induced diabetic rats.The results revealed that both single and repeated oral doses of E. prunastri (60 mg/kg) significantly reduced blood glucose, triglycerides, and very-low-density lipoprotein levels in diabetic rats.Furthermore, repeated oral administration of E. prunastri for 7 days ameliorated the liver function by increasing its glycogen content and improving its histological architecture in treated diabetic rats.E. prunastri extract showed antioxidant activity and proved richness in phenolic acids and flavonoids. 72

Hepatoprotective Activity
Researchers investigated the possible effects of UA on liver metabolism in livers of male Wistar rats and found that UA stimulated oxygen consumption at low concentrations, diminished the cellular ATP levels, increased the cytosolic but diminished the mitochondrial NADH/NAD + ratio, strongly inhibited gluconeogenesis, stimulated glycolysis, fructolysis, glycogenolysis, and ammonia genesis, and inhibited ureogenesis. 73 the other hand, hepatoprotective effect of diffractaic acid isolated from Usnea longissima was investigated against hepatic damage induced by carbon tetrachloride, and according to the authors, 50 mg/kg daily dose of diffractaic acid could be considered to have hepatoprotective effect by ameliorating the studied biochemical parameters and tissue histological structures. 74 another study, 75 researchers investigated hepatoprotective effect of hydroalcoholic Cladonia rangiferina extract in ethanol-induced liver damage in rats.The extract showed a significant restoration of altered biochemical parameters toward normal in both in vitro and in vivo conditions.It restored alkaline phosphatase (ALP), alanine transaminase (ALT), aspartate aminotransferase (AST), total protein content, γ-glutamyl transferase, glutathione S-transferase, glutathione, malondialdehyde, and glutathione reductase levels almost back to normal at 50 and 100 mg/kg bw dose.Authors concluded that C. rangiferina could be a good treatment for alcohol liver disease.
Kim et al 76 investigated the hepatoprotective properties of ramalin (RM) isolated from Ramalina terebrata, against hepatic fibrosis in vitro and in vivo.Ramalin suppressed hepatic stellate cell (HSC) activation in vitro without any significant signs of adverse effects on the cells tested, and the accumulation of extracellular matrix (ECM) was dramatically reduced in the liver tissue.Authors concluded that RM could attenuate hepatic fibrosis progression in dimethyl nitrosamine (DMN)-induced rats, potentially via the Nrf2/ARE pathway.

Immunomodulatory and Inflammation-Related Activities
The researchers examined in vitro ability of stereocalpin A isolated from Ramalina terebrata to suppress the tumor necrosis factor-alpha (TNFα)-induced expression of adhesion molecules in vascular smooth muscle cells (VSMCs).Two hours of stereocalpin A treatment at nontoxic concentrations (0.1-10 μg/mL) inhibited cell adhesion and expression of adhesion molecules.Stereocalpin A reduced TNF-α-induced production of intracellular reactive oxygen species and phosphorylation of signal and response pathways.Stereocalpin A inhibited TNF-αinduced activation and translocation of nuclear factors.Authors speculated that stereocalpin A has the potential to exert a protective effect by modulating inflammation within the atherosclerotic lesion. 77ur polysaccharide fractions were isolated from Umbilicaria esculenta.They were mainly composed of glucose, galactose, and mannose with different molar ratio.In the in vitro immunomodulatory assay, all the polysaccharide fractions (20-500 μg/mL) were able to increase the NO production and phagocytic activity of RAW 264.7 cells in a dose-dependent manner. 78Four polysaccharide fractions were isolated from Umbilicaria esculenta.They were made up from different molar ratios of glucose, galactose, and mannose.Around 20-500 μg/mL doses of all 4 polysaccharides increased phagocytic activity and NO production in RAW 264.7 cells in a dose-dependent manner. 78searchers isolated lobastin from Stereocaulon alpinum and examined its effect on the expression of vascular cell adhesion molecules (VCAM-1) induced by TNF-α in MOVAS-1 (the cultured mouse vascular smooth muscle cells).Lobastin was able to inhibit VCAM-TNF-α-induced expression of VCAM-1.Lobastin also inhibited TNF-α-stimulated production of intracellular ROS, abolished TNF-α-induced phosphorylation of p38 and ERK 1/2, but not JNK, and inhibited TNF-α-promoted NF-κB activation.In addition, lobastin suppressed TNF-α-induced IκB kinase activation, subsequent degradation of IκBα and nuclear translocation of p65 NF-κB.Authors interpreted that lobastin downregulated the TNF-α-mediated induction of VCAM-1 in vascular smooth muscle cells by inhibiting intracellular ROS generation of p38, ERK ½, and NF-κB signaling pathways and intracellular ROS generation.They concluded that lobastin could be an important regulator of inflammation in the atherosclerotic lesion and be a novel therapeutic drug for the treatment of atherosclerosis treatment. 79 et al 80 isolated 16 metabolites from Usnea longissima and tested some of them for NO production inhibition in RAW 267.4 cells.Compounds useanol, 3,7-d ihydr oxy-1 ,9-di methy ldibe nzofu ran, and 2,5-d imeth yl-1, 3-ben zened iol showed significant anti-inflammatory activity against NO production with IC50 values of 6.8, 3.9, and 4.8 μmol/L, respectively, compared with the positive controls curcumin (IC50 15.3 μmol/L) and indomethacin (IC50 42.9 μmol/L).
In a recent study, 81 the inhibitory activity of ethyl acetate extract of Bryoria sp. on the proliferation of CD8(+) T cells and the mixed lymphocytes reaction was evaluated in vitro.Authors suggested that Bryoria sp.extract inhibited mixed lymphocytes reaction via the suppression of IL-2Rα expression in CD8(+) T cells, and the extract had the potential to be developed as an anti-immunosuppression agent for organ transplants.
Researchers synthesized 16 new (+)-UA-based triazole hybrids and evaluated their anti-inflammatory potential against the cytokine proteins TNF-α and IL-1β on human U937 cells.Intermediates 2a, 2b, 3a, and 3b and triazoles 4f, 4g, 4h, 5f, 5g, and 5h exhibited promising anti-inflammatory activity against the TNF-α with IC50 values between 1.40 and 5.70 μM, while IL-1β inhibitions were insignificant.Prednisolone' s IC50 value was 0.52 µM.Authors suggested that compounds 5f and 5h were possible candidates for novel anti-inflammatory drug development. 82 an extensive work, acetone extract of Everniastrum vexans was investigated for antimigratory activity against human lung cancer cell A549.The extract induced a potent inhibitory activity.Atranorin was determined as the active compound and further analyzed in various tests.Authors reported that atranorin significantly inhibited tumorigenesis in vitro and in vivo, and atranorin may inhibit lung cancer cell motility and tumorigenesis by affecting AP-1, Wnt, and STAT signaling and suppressing RhoGTPase activity. 83e anti-inflammatory activities of lobaric acid and pseudodepsidones isolated from Stereocaulon paschale were investigated.Lobaric acid was found to inhibit the NF-κB activation and the secretion of pro-inflammatory cytokines (IL-1β and TNF-α) in lipopolysaccharide (LPS)stimulated macrophages.Inhibition and docking simulation experiments provided evidence that lobaric acid binds to PPAR-γ between helix H3 and the beta sheet, similarly to partial PPAR-γ agonists.Authors concluded that lobaric acid reduced the expression of pro-inflammatory cytokines by blocking the NF-κB pathway via the activation of PPAR-γ.Activities of pseudodepsidones were not as strong as lobaric acid. 84searchers used Maillard reaction-based fluorescence assay to evaluate the in vitro inhibitory effects of secondary lichen metabolites on the formation of pentosidine-like advanced glycation end products.Of the tested 37 natural and 5 synthetically modified compounds, 18 exhibited IC50 values in the range of 50-700 µM.Best inhibitions were demonstrated by variolaric, pannaric, and leprapinic acids (IC50 values 50, 60, and 80 µM, respectively). 85ngissiminone A was isolated from Usnea longissima and screened for its in vivo anti-inflammatory and anti-platelet aggregation activities.It showed moderate in vivo anti-inflammatory activity in paw edema compared to aspirin as well as moderately active against the aggregation induced by arachidonic acid at different doses. 86uthors isolated longissiminone A from Usnea longissima and evaluated its in vivo anti-platelet aggregation and anti-inflammatroy potential.It exhibited moderate in vivo anti-inflammatory effect in paw edema compared to aspirin and was moderately active against the aggregation induced by arachidonic acid at different doses. 86 a recent study, 87 the effect of lobaric acid isolated from Stereocaulon alpinum and its mechanism on LPS-induced inflammatory responses in macrophages were investigated.Researchers revealed that lobaric acid decreased nitric oxide production and the expression of cyclooxygenase-2 and prostaglandin E2 in LPS-stimulated macrophages.Significant reduction in the production of TNF-α and interleukin (IL)-6 was also recorded, and the underlying mechanism was suggested to inhibit the activation of mitogenactivated protein kinases (MAPKs) and nuclear factor-kappa B (NF-κB).Additionally, lobaric acid inhibited NLRP3 inflammasome activation in LPS/ATP-stimulated cells and extended the production of IL-1β and IL-18, as well as caspase1 maturation.Authors' interpretation was that LA could inhibit inflammation by downregulating NF-κB/MAPK pathways and NLRP3 inflammasome activation in activated macrophages.
Researchers tested the antiarthritic property of methanolic extract of Parmotrema tinctorum in Freund' s complete adjuvant (CFA)-induced arthritic rat model.They investigated effects of the extracts on paw edema, arthritic score, radiological analyzes, blood parameters, and tissue enzyme and marker analyses.Constant administration of extract diminished the complications associated with arthritis by inhibiting the edema formation and arthritic score considerably.The altered biochemical parameters were restored with an enhancement in free radical scavenging ability after treatment.The authors reported that levels of especially TNF-α, C-reactive protein, and rheumatoid factor (RF) along with ALP have reverted to near normal. 88searchers isolated a polysaccharide, CSL-0.1, from Usnea longissima and evaluated its effects on intestinal immunity and antioxidant activity.According to the results, CSL-0.1 dose dependently increased the spleen and thymus indices and presented immunomodulation on reversing the Th1/Th2-related cytokine imbalance in cyclophosphamide (CP)-induced immunosuppressed mice.CSL-0.1 also enhanced the levels of secretory immunoglobulin A in CP-injected mice.Additionally, the antioxidant levels in the liver and intestine of the mice were increased from 20% to 50% after intragastric injection by CSL-0.1. 89m et al 90 91 Researchers isolated stereocalpin A, stereocalpin B, and an unnamed dibenzofuran (CAS registry number 674786-23-3) from Ramalina terebrata and investigated their biological activities.They found that all isolated substances exhibited moderate antimicrobial activities against E. coli, with the IC50 values ranging from 18 to 30 µg/mL.Stereocalpin A exhibited cell growth inhibition against HCT116 cell lines, with the IC50 value of 20 ± 1.20 µM.Both stereocalpins also showed potent anti-inflammatory activities against LPS-induced RAW 264.7 macrophages with the IC50 values ranging from 5 to 7 µM. 92uroprotective Activity Researchers reported the neurotrophic activity of atranorin (ATR), perlatolic acid (PER), PHY, and (+)-UA in a Neuro2A neurite outgrowthbased preliminary screening.No cytotoxicity was observed except for UA.Perlatolic acid also exerted acetylcholinesterase inhibition and strong proneurogenic activity.Atranorin, PER, and PHY modulated the gene expression of brain-derived neurotrophic factor and nerve growth factor.Perlatolic acid also showed increased protein levels of acetyl H3 and H4 in Neuro2A cells.These lichen substances showed neuroactive properties in vitro (Neuro2A cells) and ex vivo (primary neural stem or progenitor cells), indicating their potential to treat central nervous system disorders. 93ieteiglesias et al 94  Cazarin et al 95 investigated UA enantiomers regarding cognitive-enhancing and anti-neuroinflammatory effects.The interaction of UA and acetylcholinesterase (AChE) was assessed by molecular docking and its inhibitory capability on AChE was assessed in vitro.In vivo effects of UA enantiomers were investigated in mice intra cereb roven tricu larly (i.c.v.) exposed to Aβ1−42 peptide (400 pmol/mice).Additionally, UA antioxidant capacity and neuroinflammatory biomarkers were measured in the cortex and hippocampus of mice.The results revealed that UA enantiomers evoked complex-receptor interaction with AChE-like galantamine in silico.Usnic acid improved the learning and memory of the animals and in parallel decreased the myeloperoxidase activity and the lipid hydroperoxides (LOOH) in the cortex and hippocampus and reduced the IL-1β levels in the hippocampus.
Studzińska-Sroka et al 96 used A-172, T98G, and U-138 MG glioblastoma cell lines to evaluate Hypogymnia physodes (HP) acetone extract and its major component PHY in terms of anticancer and neuroprotective activity.Hypogymnia physodes and PHY were able to strongly inhibit cell proliferation and hyaluronidase activity of glioblastoma.Hypogymnia physodes reduced tyrosinase and COX-2 activity.However, the acetylcholinesterase and butyrylcholinesterase inhibitions exerted by HP and PHY were moderate.Researchers demonstrated that PHY crossed the blood-brain barrier.They concluded that PHY and Hypoymnia physodes should be considered as promising agents with anticancer, chemopreventive, and neuroprotective activities, especially concerning the central nervous system diseases.

Lipid Metabolism
Zhu et al 97 targeted to uncover the impacts of water and ethyl alcohol extracts of Usnea diffracta on the adjustment of lipid metabolism, investigate the possible mechanism, and compare the effects with those of simvastatin.Rats were fed a high-fat diet for 45 days to induce hyperlipidemia.In serum, the extracts significantly reduced levels of total cholesterol, triglyceride (TG), and low-density lipoprotein cholesterol (LDL-C) and significantly increased the contents of high-density lipoprotein cholesterol.They decelerated weight gain and reduced TG and LDL-C content in liver.Serum levels of ALT and AST were reduced by the extracts.Total bile acid content in serum and liver was also reduced.In addition, researchers performed histopathological examinations and protein expression studies in liver tissues.They found that extracts could relatively improve cell degeneration and significantly reduce protein expressions of sterol regulatory element-binding proteins-1c and liver X receptor α (LXR-α).Furthermore, aqueous extract could substantially increase hepatic lipase activity and promote apoprotein A5 (ApoA5) protein expression.The authors concluded that ethanol extract exhibited higher activity and needed further studies, while aqueous extract could regulate lipid metabolism through increased ApoA5 expression via inhibition of the LXR-α signal pathway.

Bone Metabolism
Kim et al 98 studied whether UA affected receptor activator of NF-κB ligand (RANKL)mediated osteoclastogenesis and found that it significantly inhibited RANKL-mediated osteoclast formation and function by reducing the transcriptional and translational expression of nuclear factor of activated T cells, cytoplasmic 1 (NFATc1), a master regulator of osteoclastogenesis.In addition, it prevented lipop olysa cchar ides-induc ed bone erosion in mice.

Conclusions
In this review, experimental papers from the last 11 years about pharmaceutical activities of lichens were covered.It is evident that early papers from this period focused on antioxidant and anti-genotoxic activities of lichens.The focus shifted to anti-cancer activities and later enzyme inhibitory activities received remarkable attention.
There are topics that deserve much more attention and nano technologic use of lichens is absolutely one of them.The reader is referred to Hamida et al 99 for a comprehensive review of the subject.Although the present review contains much data about anti-cancer activities of lichens, it is not possible to cover this subject along with other activities.Solarova et al 100

Table 1 .
Summary of Pharmacological Activities of Lichen-Derived Extracts and Compounds

Table 1 .
Summary of Pharmacological Activities of Lichen-Derived Extracts and Compounds (Continued)
An interesting result from this study was the decrease in body mass index from 31 to 30 in the third month and retained the same at the sixth month in BAFS group, while it decreased from 30 to 29 in third month in the placebo group and raised back to 30 in sixth month.
71Researchers investigated apoptotic effects and proliferative properties of extracts of Lobaria pulmonaria, Evernia divaricata, Cladonia fimbriata, anticancer potential of a tropical lichen Dirinaria consimilis (DCME).In vitro antioxidant studies showed promising ROS and reactive nitrogen species scavenging potential of DCME.The in vitro antiproliferative study revealed that DCME was cytotoxic toward human breast cancer cell line MCF-7 (IC50: 98.58 ± 6.82 μg/mL) and In a randomized clinical trial, Kershengolts et al71investigated the effects of a Cladonia sp.-based biologically active food supplement (BAFS) on a population of 150 type 2 diabetes 101ently reviewed this subject.Finally, ethno botan ical/ ethno pharm acolo gical aspect of lichens was not covered in this review, but very recently Adenubi et al101published an excellent review on ethnobotanical uses of lichens.Author Contributions: Concept -Y.K.; Design -Y.K.; Supervision -Y.K.; Data Collection and/or Processing -Y.K., B.K.; Analysis and/or Interpretation -Y.K., B.B.; Literature Review -Y.K., B.K.; Writing Manuscript -Y.K., B.K.; Critical Review -Y.K., B.K. Declaration of Interests: The authors have no conflicts of interest to declare Funding: The authors declared that this study has received no financial support Peer-review: Externally peer-reviewed.