Ethnobiology, Phytochemistry and Pharmacology of Usnea Longissima: A Review

Lichenized fungi or lichens are believed to be one of the most inconspicuous living forms on the earth. The traditional knowledge of numerous benefits which lichens possess has been transcended over the centuries within and among the humans. All the different growth forms of lichens have been used by man in one way or the other. These find their use as source of different medicines, food, fodder, dye and as ingredients in perfumery. They are pioneers in ecological succession and also serve as indicators of pollution. Usnea longissima is a fruticose form of lichen that has been used as a traditional medicine for centuries. Scientific studies have proved that the pharmacological properties of the species are due to presence of several important secondary metabolites. These secondary metabolites possess antioxidant, anticancer, antimicrobial activity and thus can be used for the control and treatment of several diseases. The present communication gives a review of the multifarious benefits associated with Usnea longissima along with the emphasis on its pharmacological properties.

Pradesh uses the lichen along with some other ingredients for treatment of bone fractures [8,25]. In Uttaranchal, Bhotia and Grahwal tribes use Usnea longissima as a spice, for stuffing nets and even as special ingredient of poultice for bone setting [26]. It is one of the important dietary components of the Musk deer found in Garhwal Himalayas region in India [27].

Pharmacological properties
Usnea longissima is considered to be a potent lichen with multifarious phytochemical and pharmacological properties [35].
Antimicrobial and Antifungal activity: The ethanolic extracts of Usnea longissima exhibited antibacterial activity against gram +ve bacteria namely Staphylococcus aureus and several gram -ve bacteria such as Pseudomonas aeruginosa, Klebsiella pneumonia, Shigella dysenteriae, Salmonella typhii and Escherichia coli [36]. Likewise, the methanol extract of Usnea longissima showed antibacterial activity against Salmonella typhii, Staphylococcus aureus and Escherichia coli [37]. The methanolic extract possessed antifungal properties against Trichoderma viridae and Candida albicans [2]. The secondary metabolites usone and isousone exhibited inhibitory effect on the fungus Trichophyton rubrum [38]. Another study conducted by Oran and his coworkers revealed that Escherichia coli isolates were resistant to methanolic extracts of Usnea longissima and other two species as well [34]. Usnic acid can effectively inhibit the formation of bacterial biofilm on the surfaces of polymer [39]. Thus, usnic acid proves to be more effective against penicillin. The usnic acid extracted from Usnea longissima inhibited the growth of  some bacterial strains such as Pneumococcus, Streptococcus and Staphylococcus in dilutions of 1:20,000. Moreover, at dilutions of 1:200,000 -1:2000,000 the growth of some strains of human TB was weakened and at 1:20,000 -1:50,000 the growth of human TB was inhibited [36]. HPLC analysis of the usnic acid isolated from Usnea longissima collected from Anatolia and Turkey was performed. The acetone extract of usnic acid exhibited significant antimicrobial activity against Escherichia coli (ATCC 35218), Enterococcus faecalis (RSKK 508), Proteus mirabilis (Pasteur Ens. 235), Staphylococcus aureus, Bacillus subtilis and Bacillus megaterium [40]. The activity of novel multifunctional hydroxyphenylimino ligands (namely L1, L2 and L3) synthesized from usnic acid as well as their complexes (namely Cu(II), Co(II), Ni(II) and Mn(II) salts) was tested against some pathogen microbes by disc diffusion method. The ligand complexes of the metals showed significant zone of inhibition i.e. 11 -32 mm against ten pathogenic microorganisms. The Mn(II) and Cu(II) complexes of the hydroxyl phenyl imino ligand along with the usnic acid showed the maximum antimicrobial activity [41].
In vitro microbial synthesis of bioactive nanoparticles from Usnea longissima exhibited potent activity against several humanoid pathogenic fungi. These nanoparticles effectively controlled the fungal infections [42].

Insecticidal effects:
The secondary metabolites of the lichen when tested against Sitophilus granarius (Coleoptera: Curculionidae) showed insecticidal effects on them causing a high mortality rate [43].

Antiplatelet and Antithrombotic activities:
The methanol extracts of Usnea longissima were investigated in vitro on platelet aggregation and in vivo on pulmonary thrombosis for determining antiplatelet and antithrombotic activities. The study showed concentration dependent inhibitory effects with an IC50 value of 3.6 mg /m of the extracts on the ADP-induced platelet aggregations shown in Figure 2 [56]. Longissiminone A isolated from U. longissima exhibited anti-inflammatory activity in a cell-based contemporary assay [31].

Antiulcerogenic effect:
The antiulcerogenic activity of water extract of Usnea longissima was determined by using Indomethacin -induced ulcer models in six rats. The negative and positive controls were treated with indomethican and rantidine respectively and the body weight doses administered were 50, 100 and 200 mg/kg. Although all the doses exhibited antiulcerogenic activity yet the highest activity was observed in case of 100 mg/kg body weight doses (i.e.79.8%). In the stomach tissues of rats the activities of antioxidant enzymes such as superoxide dismutase, catalase and glutathione S -transferase were also determined. This study was done in order to compare the effects of antioxidant enzymes on antiulcerogenic activity. The study also shows that the Indomethacin reduced the Superoxide dismutase and glutathione S -transferase enzymes activities. The activities of these enzymes were activated by the water extracts of Usnea longissima. However in case of CAT activity an opposite trend was observed which was increased by indomethacin and decreased by all doses of Usnea longissima and ranitidine [44].

Growth Inhibitory affect:
The growth inhibitory-activity of some compounds isolated from Usnea longissima on lettuce seedling was observed. Depsides and orcinol derivatives which exhibited growth-inhibitory activity against lettuce seedlings were isolated. 4-O-demethylbarbatic acid exhibited the highest activity among the eight depsides compounds. However, β-Orcinol type depsides showed higher activity than orcinol type depsides. The nature of the hydroxy or methoxy substituents at the 2-and 4-positions affected the solubility in acetone and their biological activity. 3-α-Hydroxydiffractaic acid exhibited 90% radicle length and 60% hypocotyl length inhibitory activity to control at 4.0 x 10 -4 M [45].

Melanogenesis Inhibitory Effect:
The total phenolic compounds of methanolic extract of Usnea longissima were 1.46% and the electron donating abilities as well as the lipid peroxidation rates were also determined. The EDA values measured by the reduction of 1.1'-diphenyl-2-picrylhydrazyl (DPPH) was 80.7% for the extracts, with median scavenging concentration (SC50) of 1.03±0.06 mg/ml. Measurements of EDA using DPPH were conducted via the direct estimation method for antioxidation activity. Extracts of Usnea esculenta and Usnea longissima evidenced 72.8% and 80.7% electron-donation effects, respectively, at a concentration of 6 mg/ml (Figure 3). The rates of inhibition of lipid peroxidation using linoleic acid was 97.3%, with IC 50 (median inhibitory concentration) value of 0.53 ± 0.06 mg/ml, as shown in (Figure 4). The methanolic extracts of Usnea longissima showed direct tyrosinase inhibition via inhibition of tyrosinase glycosylation thus exhibiting prominent melanogenesis inhibitory effects occurring through multiple routes. The inhibitory rate of extracts against tyrosinase was observed to be 84.8%. The extracts reduced melanin formation in human melanoma cells. Melanin contents in the samples treated with 0.01% and 0.1% Usnea longissima were 51.1% and 34.9%, respectively, whereas a value of 54.0% was observed when ascorbic acid was used as a positive control [46]. Activity against oxidative damage: Usnea longissima exhibited potent antioxidant activity [47]. The methanolic extracts of Usnea longissima suppressed the mutagenic effects of aflatoxin B 1 [48]. For evaluating antioxidative effect of methanolic extracts of Usnea longissima species, the activities of superoxide dismutase, glutathione peroxidase and malondialdehyde level were measured. This was followed by aflatoxin B 1 treatment which showed an increase of malondialdehyde level with a decrease of superoxide dismutase and glutathione peroxidase activities. The methanolic extract of lichen eliminated the genotoxicity and lipid peroxidation of aflatoxin B 1 by increasing the level of antioxidant enzymes activities. The adverse effects of aflatoxin B 1 in human blood cells is modified by methanol extract of lichen and shows strong antioxidative and antigenotoxic effects. This study reveals the antioxidant and free radical activity of ethanolic extract of Usnea longissima [49]. Polysaccharides of Usnea longissima were evaluated for their scavenging action against superoxide anion free radical (O 2-) using xanthine-xanthine oxidase system. Fenton reaction was used to study the Scavenging action on hydroxyl free radical (OH) and anti-lipid peroxidation effects. The scavenging action was characterized by the scavenging content of 50% for all the free radical (IC50). The IC50 for O 2and OH were 0.45 mg/ml and 1.57 mg/ml respectively [50].

Gastro-protective Effect:
A study conducted by Bayir showed that the gastro-protective effect of diffractaic acid could be attributed to its enhancing effects on antioxidant defense systems and reducing effects of neutrophil infiltration [51]. The antiulcerogenic effect of diffractaic acid isolated from Usnea longissima on indomethacin induced gastric lesions was investigated in rats. Doses of 25, 50, 100 and 200 mg/kg of diffractaic acid and 50 mg/kg of ranitidine when administered reduced the gastric lesions in rats by 43.5%, 52.9%, 91.4%, 96.7% and 72.7% respectively. In all the treated groups of rats, in vivo activities of the antioxidant enzymes namely superoxide dismutase, catalase, glutathione peroxidase were evaluated. Also the levels of reduced glutathione and lipid peroxidation were evaluated. Indomethican caused oxidative stress in rats by decreasing the levels of glutathione peroxidase, Superoxide dismutase and glutathione as compared to healthy rats. Indomethican induced the gastric mucosal damage throughout the development in rats which also led to changes in activities of gastric mucosal nitric oxide synthases. However the gastric damage tissues induced by indomethican resulted in an increase in inducible nitric oxide synthases activity and decrease in constitutive nitric oxide synthases activity. The administered dose of 100 mg/kg of diffractaic acid and rantidine reversed the activities of inducible nitric oxide synthases and constitutive nitric oxide synthases. Usnic acid isolated from Usnea longissima showed gastro-protective and antioxidant effects on indomethicaninduced gastric ulcer in rats [16].
Hepatoprotective activity: The methanolic extracts of Usnea longissima showed powerful hepato-protective and antioxidant activity against several experimental animals [52]. Hepatotoxicity study was carried out, in which ethanolic extract of U. longissima at doses 200 and 400 mg/kg body weight) were compared with Silymarin (25 mg/kg body weight). Hepatotoxicity was induced by CCl 4 (1ml/kg) and the study showed that the liver weights were significantly decreased. However ULE showed a dose dependent protection in liver weight. The result of the high dose (400 mg/kg) was compared with standard drug Silymarin (25 mg/kg) ( Figure 5). The hematological and antioxidant activity of chemical constituents isolated from Usnea longissima against CCl 4 iatrogenic acute liver damage in rats was investigated. This was disclosed by changes in level of LPO and GSH concentration in liver, additionally to the elevation of SOD, CAT and GPx activity. Usnea longissima extract ameliorates acute liver damage by the improvement in histopathological changes. The extract of Usnea longissima either causes stabilization of cellular membrane or showed anti-peroxidase activity. The study revealed that the extract of Usnea longissima exhibits powerful anti-oxidant and hepato-protective activity. Diffractic acid isolated from Usnea longissima at 3 doses 50, 100 and 200 mg/kg against CCl 4 induced hepatic fibrosis in Wistar rats whereas the daily dose of 50 mg/kg produced hepato-protective effect on Wistar albino rats and rest of the doses exhibited hepatotoxic effects. Diffractic acid is therefore potentially hepatotoxic at a low dose of 50mg/kg against acute liver toxicity induced by CCl 4 [53].
Anti-cancerous properties: Usnic acid shows antiproliferative activity against breast cancer cell lines namely the wild-type p53 (MCF7) and the nonfunctional p53 (MDA-MB-231) as well as against lung cancer cell line H1299. This non-genotoxic anti-cancer activity of usnic acid in a p53independent manner needs to be further investigated. Thus, usnic acid has the potential for the treatment of tumors either as a systemic therapy or as a topical agent [54]. Cancer chemoprevention assay were designed using usnic acid to detect potential inhibitors of tumor promotion. It was shown that usnic acid unveiled strong inhibitory effects (ED 50 1.0µg/ml) against Epstein -Barr virus activation induced by teleocidin B -4 potent tumor promoter [55].
Antimutagenic activity: Condensation of 2aminophenol, 3-aminophenol and 4-aminophenol was done with usnic acid isolated from Usnea longissima to synthesize novel multifunctional hydroxyphenylimino ligands namely L1, L2 and L3. FT-IR, UV-Vis, (1)H-NMR, (13)C-NMR, 1D-and 2D NMR (, LC-MS and TGA,FT-MIR/FAR, UV-Vis, elemental analysis, ICP-OES and TG/DTA techniques were used to characterize the synthesized ligands and their Cu(II), Co(II), Ni(II) and Mn(II) complexes. The antimutagenic activities of all the ligands as well as their metal complexes were detected against Ames-Salmonella and E. coli WP2 microbial assay systems. The study revealed that the ligand complexes of Co and Mn possess potent antimutagenic activity [41].

II. CONCLUSIONS
The biochemical and physiological activities associated with the chemical entities present in Usnea longissima reveal their tremendous potential benefits which are yet to be explored fully. With the aid of modern equipments and latest biotechnological interventions, isolation and characterization of active compounds as well as elucidation of mechanism of their action has also become easier. Therefore, the secondary metabolites in lichens which are of natural origin can be isolated and utilized for the treatment of several diseases and ailments. Medicinal properties associated with Usnea longissima make it a very promising entity for drug development and in turn providing a great boon to the pharmaceutical industry