The genus Usnea: a potent phytomedicine with multifarious ethnobotany, phytochemistry and pharmacology

Prateeksha† ^a, B. S. Paliya†^a, R. Bajpai^b, V. Jadaun^a, J. Kumar^a, S. Kumar^a, D. K. Upreti^b, B. R. Singh‡ ^c, S. Nayaka^b, Y. Joshi^d and Brahma N. Singh*^a
aPharmacognosy & Ethnopharmacology Division, CSIR-National Botanical Research Institute, Lucknow – 226001, U.P., India. E-mail: bn.singh@nbri.res.in
^bLichenology Laboratory, Plant Biodiversity and Conservation Biology Division, CSIR-National Botanical Research Institute, Lucknow – 226001, U.P., India
^cCentre of Excellence in Materials Science (Nanomaterials), Z. H. College of Engineering & Technology, Aligarh Muslim University, Aligarh-202002, India
^dDepartment of Botany, S. S. J. Campus Almora-263601, Uttarakhand, India

First published on 28th January 2016

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1. Introduction

Lichens are an obligate mutualism between a fungus (mycobiont) and one or more photosynthetic organisms, an alga or cyanobacterium (photobiont).^1,2 Typically the fungal partner delivers most of the composite organism’s structure and mass, hence trading physical protection for carbohydrates manufactured by the photosynthetic partner. These organisms represent a unique division in the plant kingdom and are the most successful symbiotic organisms in nature, dominating 8% or more of the earth’s terrestrial area.^2–4

Lichens have been shown to produce a number of primary and secondary metabolites that may protect them against physical stresses or biological attack.^5,6 Some lichen species and their metabolites have been used for medicinal and industrial purposes.^7–10 Among the medicinal lichens, the genus Usnea Adans. (Parmeliaceae; lichenized Ascomycetes) is edible and is utilized in the preparation of traditional foods and medicines in both Eastern and Western countries.^11,12 This genus is regarded as one of the taxonomically most difficult genera of macro-lichens.¹³ Most of the species are globally distributed with more than 350 species and highly variable in morphology. Many species are also very variable in chemistry, and may include several chemotypes.¹⁴ In India, 57 species of Usnea are known which grow luxuriantly in higher regions of Western Ghats and Himalaya.^15,16 Many species are also very variable in chemistry, and may include several chemotypes. The species of Usnea are known to be used in traditional medicines, in dyeing and in spices in various parts of the country.^15,17 The first recorded use of the Usnea species in traditional Chinese medicine dates to 101 B.C., when it was used as an antimicrobial agent under the Chinese name of Song Lo. Song Lo tea or its decoction has also been recorded for internal and external detoxification of the liver, treatment of malaria, wounds, snake bite, and cough.¹⁸ In Unani literature, the medicinal uses of Usnea species are mentioned as astringent, antidote, analgesic, cardiotonic, resolvent and stomachic.^17,19–21

Along with its emerging position in the herbal market, primary as well as secondary metabolites (extrolites), the chemical constituents of Usnea species have been broadly investigated. Usnic acid, protocetraric acid, barbatic acid, norstictic acid, salazinic acid, and stictic acid were characterized as the main bioactive chemical constituents in Indian species.^17,22,23 All the major secondary metabolites in Usnea belong to aromatic products formed from β-orcinol units, while ceparatic acid and protolichesterinic acid belong to higher aliphatic acids. Most of these metabolites are unique to Usnea lichens, being of great significance for systematics and phylogeny, and are employed at different taxonomic levels from species and subspecific to generic and higher ranks.^24,25 Furthermore, these bioactive metabolites play important ecological roles in nature such as UV protection and defense against predators and pathogenic microorganisms.^1,26

Significant research has been done on Usnea and its metabolites which confirm various biological activities including anti-microbial, anti-oxidant, anti-tumor, anti-viral, anti-inflammatory, cardiovascular protective, and hepatoprotective properties.^{17,20,22,27–33} These are closely correlated with the ethno-medicinal uses. Recent pharmacological studies have revealed significant anti-cancer, anti-genotoxic, anti-proliferative, and anti-neoplastic activities and these potentials have further put Usnea under the spotlight.^29,34–36 The aim of this review is to summarize the recent advances in phytochemistry and pharmacology of the genus Usnea. Phylogenetic and toxicological aspects are also given in brief.

2. Botanical characterization and distribution

The genus Usnea is highly diverse, with more than 350 estimated species, distributed in polar, temperate and tropical regions. This genus is characterized by fruticose habit and especially by the presence of a cartilaginous central axis. Dillenius (1742) first proposed the name Usnea in Historia muscorum.³⁷ The genus was placed in family Usneaceae until studies on apothecial ontogeny and ascus apical structures proved that Usnea belongs to the family Parmeliaceae.³⁸ One can easily recognize Usnea in fields by its shrubby to pendent greenish yellow thallus, radial symmetry and presence of central cartilaginous axis.¹⁵ There are three forms of fruticose thallus in Usnea, erect/bushy when the thallus is small (U. orientalis), a long pendulous thallus hanging from tree branches (U. angulate and U. longissima) or a sub-pendent thallus of intermediate length (U. aciculifera and U. rubicunda). Dominant branching patterns including dichotomous, sub-dichotomous and sympodial are observed. The basic characteristics used for identification of Usnea up to species level include morphological features like the habit of the thallus, branching pattern, pigmentation of basal part, presence or absence of sorelia together with its morphology, isidia, pseudocyphellae, papillae, tubercles, fibrils, faveolae and shape of branches; anatomical features like the ratio of thickness of the cortex (C), medulla (M) and central axis (A), the compactness of fungal hyphae in medulla and the presence or absence of specific secondary metabolites. A combination of morphological, anatomical and chemical characters can be used to delimit species.

Usneasensu lato comprises of an assemblage of approximate 350 species worldwide.¹⁵ The species belonging to the genus Usnea contain usnic acid, a bioactive compound, which imparts a yellow colour to the thalli. Using modern concepts, the taxonomist divided Usnea into three genera, i.e., genus Dolichousnea, genus Eumitria and genus Usnea sensu stricto. The genus Dolichousnea is characterized by an annular pseudocyphellae between the segments, solid central axis and positive iodine reaction of central axis. The genus Eumitria is characterized by a fistulose central axis whereas genus Usnea sensu stricto is characterized by the absence of annular pseudocyphellae and a solid, I – central axis.

Usnea is a cosmopolitan genus occurring on all continents. Species diversity, however, is low in arid and arctic areas and is highest in humid regions of temperate latitudes. U. aciculifera is found in Eastern Asia and U. angulate is distributed in Australia, America and West Africa. U. baileyi is found in pantropical countries in world. U. compressa is widely distributed in India and Nepal. U. fragilis is known in the South-East Asian region. U. himalayana is from the Himalayas, Western Ghats and Africa. U. indica is endemic to North-West Himalayas and found in Uttarakhand in India. U. ghattensis is endemic to Western Ghats. U. luridorufa is found in North and South Asia. U. nepalensis is found in Himalayas and Western Ghats in India. Several species including U. orientalis, U. pangiana, U. sinensis, and U. perplexans are widely distributed in North-East Asia. U. pseudosinensis and U. robusta are restricted only to the Himalayas. U. subflorida is distributed in East Africa and North Asia, while U. subfloridana is found in Europe and North East Asia. U. undulata is found in South and East Africa.^39–41

U. longissima is distributed throughout the Northern temperate zones, such as the sub-arctic and the coastal rainforests of Europe, Asia and North America.^41,42 In India, the species is distributed in North-Eastern Himalayan regions between 1500–4000 m altitudes in moist old mixed forests of Quercus and Pinus. Seven chemosyndromes of U. longissima are reported from India which include barbatic acid, squamatic acid, diffractaic acid, evernic acid, fumaroprotocetraric acid and usnic acid strains. U. longissima is characterized by fruticose, a pendulous thallus of 60 cm long or more, of pale yellow-greyish green to light brown, a 0.5–1.0 mm diameter main branch, a 2–5 cm long, dense perpendicular usually decorticated or pulverulent to powdery lateral branches, sorediate or isidiate, with a colorless central lattice. Apothecia range from being rare to up to 5 mm in diameter, with ciliate margin. The species has more than seven chemotypes containing barbatic, squamatic, diffractiatic, evernic, fumarprotocetraric, and usnic acids.⁴¹

3. Phylogeny and classification

The phylogeny and classification of Usnea have been a matter of debate, given the lack of phenotypic characters to describe phylogenetic clades and the low degree of resolution of phylogenetic trees.³⁷ Motyka (1936–38) proposed a classification of Usnea, in which all fruticose lichens with an inner, cartilaginous tissue are included. He identified six subgenera: Euusnea, Protousnea, Lethariella, Chlorea, Neuropogon, and Eumitria.⁴³ Later Protousnea and Lethariella (including Chlorea) were elevated to generic rank by Krog (1976).⁴⁴ The position of Neuropogon as a subgenus to Usnea was accepted by several authors.⁴³ Krog (1982) suggested a classification of genera (usneoid e.g. Neuropogon, Protousnea, Evernia, Letharia, Lethariella).⁴⁵ In this hypothesis Neuropogon and Usnea are sister groups. Protousnea and Evernia form together the sister to the clade, comprising of Neuropogon and Usnea. Finally, Letharia and Lethariella form the sister group to the other usneoid genera. But gradually many diverse classifications have been proposed due to a lack of phenotypic characters.

A study based on the ITS sequence data supported the subgenera Eumitria and Usnea, and revealed a new subgenus, Dolichousnea.⁴⁶ The authors also concluded that Usnea contains at least three taxa at subgeneric level, Usnea, Eumitria, and Dolichousnea. Neuropogon was not included in this study and the position remained unclear. Molecular phylogenies based on the ITS-LSU nrDNA and part of the β-tubulin region have been used to examine the position of Neuropogon in Usnea s. lat.⁴³ Bayesian inference and maximum parsimony strongly supported the monophyly of Neuropogon. Subgenus Usnea and Neuropogon form a strongly supported group with subg. Eumitria and subg. Dolichousnea is a consecutive monophyletic sister group. The following generic classification was proposed: Usnea (subgenus Usnea only), Neuropogon, Eumitria, and Dolichousnea. Dolichousnea is elevated to generic rank. The following new combinations are made: Dolichousnea Articus, D. longissima (Ach.) Articus, D. trichodeoides (Vain.) Articus, D. diffracta (Vain.) Articus, and Eumitria pectinata (Taylor) Articus.⁴³ Recently, the phylogenetic relationships of 52 Usnea species from across the genus, based on ITS rDNA, nuLSU, and two protein-coding genes RPB1 and MCM7 have been investigated. The phylogeny based on the concatenated dataset revealed that the genus Usnea is subdivided into four highly-supported clades, corresponding to the traditionally circumscribed subgenera Eumitria, Dolichousnea, Neuropogon and Usnea.⁴⁷ However, characteristics that have been used to describe these clades are often homoplasious within the phylogeny and their parallel evolution is suggested. The study has suggested that combinations of phenotypic characters are suitable discriminators for delimitating species, but are inadequate to describe generic subdivisions.

4. Traditional uses and ethnopharmacology

The species of lichen genus Usnea is used for the treatment of various diseases such as diarrhea, ulcer, urinary infection, tuberculosis, pneumonia, stomachache, anti-fungal, human pathogens, and cattle fungal diseases.^48–51 Some other uses of the species are for strengthening, hair growth, sterility cure, flavoring agent, pulmonary disease, antiseptic, anti-tuberculosis and anti-viral diseases are summarized in Table 1.^52–60 The Usnea species are the most common source of antibiotic and antifungal lichen acids, particularly usnic acid. The species have widespread potential for medicinal applications. Usnea is used for weight loss, pain relief, fever control, and wound healing; and to make phlegm easier to cough up.^61,62 It was recorded that Usnea had been used directly on the skin for sore mouth and throat.

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U. longissima grows commonly on bark, mostly on the twigs of trees, bushes and over soil and rock in temperate and alpine regions of India, it is known locally as “Syara” by the Bhotia and Garhwalis of remote areas of Uttarkashi district of Uttarakhand, India and is used for making pillows. The Baiga tribes of Madhya Pradesh, India used the species along with other ingredients for treating bone fractures.^62,63 Likewise the ancient Greeks used lichen as medicines. Hippocrates recommended a lichen, perhaps U. barbata, for uterine complaints. The Chinese used U. longissima as an expectorant and as a powder application to heal external ulcers in the name “Sun-Lo”.¹ It is also a major ingredient of Chinese medicine.⁵⁷ In China, this species of Usnea is also called as “Lao-tzu’s” beard, “Pine gauze” and used for stopping sweating dizziness cold, pain and phlegm. U. longissima is still utilizing today as a tincture to treat tuberculosis lymphadenitis. In the Bolivian Andes, U. longissima is commercially sold as a folk medicine for cough and hoarseness.^7,12 The Nitinaht Indians of Vancouver also used the species for wound dressing in Turkey.⁶³ U. longissima is used in the treatment of gastric ulcers by the Anatolians as a folk medicine. This species is also used as to strain impurities from hot pitch before the pitch was used as medicine. In Unani medicine, it has been described to stimulate menstruation or induce abortion, taken orally and inserted into the vagina.⁶³ However, it was used for treating cancer, tuberculosis, and ulcers in Turkey.⁶⁴ It has also been used as a decongestant and for the local treatment of ulcers and tuberculosis by Chinese people.⁵⁷

U. barbata is used to treat mammary infections in cattle. The udder is washed several times with a decoction of lichen and used for indigestion in humans, where the tincture or decoction taken orally several times daily.⁶⁵ In the Philippines, it has been used for wounds, chopped, and mixed with coconut oil, spread over the wound and for abdominal pain where the decoction is used as a drink.⁶⁶ However, in Europe it was used for internal and external bleeding, whooping cough, jaundice, and growing hair.⁶⁷ Spanish people used it as drying agent and an antiseptic for cracks and irritations of the feet.⁶⁸ In China U. aciculifera was used to treat bladder infection, painful urination, urinary retention, swelling, and edema in the heart and kidneys.⁵⁷ In Tanzania, U. articulate was used for the treatment of stomachache.⁶⁹ A handful of U. articulate and U. gigas are chewed fresh and the bitter juice swallowed, relieving pain after a time. In China, U. ceratina was used for coughs, inflamed lungs, pulmonary tuberculosis, hepatitis, heat related headaches, infection due to injury, inflamed lymph channels, mastitis, and snakebites.⁵⁷

In traditional Argentinian medicine, teas of U. densirostra and U. durietzii were used externally as astringents, antiseptics, and anti-inflammatory agents.^70,71 In China, U. diffracta has been applied to treat a range of problems such as cough, tuberculosis of neck or lungs, headache, dizziness, sweating, dim vision, swelling, pus oozing from breasts or sores, burns and scalds, snakebite, traumatic injuries, bone fracture, bleeding from external injuries, vomiting blood, blood in feces, bleeding from uterus, menstrual disorders, vaginal discharge, swelling of female genitalia, urinary tract afflictions, and ascarid or schistosoma parasitic infections.^72,73 The same species of Usnea were used to cure fevers of the lungs, liver, and heart and fever caused by poisoning in Tibet⁷⁴ while in Korea, the species was used to induce menstruation (Pusan) and treat tuberculosis of the neck.⁷⁵

The traditional Chinese herbal medicine, U. florida has been used for aching in sinews and bones, stopping bleeding or infection from external injuries, skin diseases, painful urination, coughs, tuberculosis of lungs or neck, heart palpitations, and edema.⁷⁶ The decoction of U. florida was also used for colds and coughs in Europe,⁷⁷ while in Chile, its infusion is used for diarrhea.⁴⁹ U. himalayana is burned as a “lichen cigarette” in Japan.⁷⁸ U. hirta has been used by European people to heal wounds and to prevent hair loss.⁷⁷ U. laevis has been widely used to treat different kind of microbial infections including dermatosis, fungal infections, tuberculosis, and pneumonia.⁵⁰ In Canada, most of the Usnea species were used for wound dressing, but U. longissima is preferred by wrapping around the wound.⁷⁹ It was recorded that U. pectinata had been used in China for stopping bleeding from external injuries, relieving pain, bloody feces, and swelling.⁷⁶

U. plicata used as an astringent for internal and external use,⁸⁰ also for whooping cough,⁸¹ jaundice, strengthening stomach and abdominal cavity, and restraining abortion in Europe.⁸² As the traditional Indian herbal medicine, U. sikkimensis has been used for treating lung troubles, hemorrhages and asthma.⁸³ It has also been used to bandage surface wounds, skin eruptions, and boils.⁸⁴ The concoction of U. strigosa was taken orally for the treatment of headaches.⁵⁸ Moreover, in Ireland U. subfloridana was used to treat sore eyes. In China, it was used to treat painful and reddened eyes, bleeding from external injuries, and swelling.⁷⁶ The traditional Chinese herbal medicine, U. trichodeoides has been used to treat coughs, pulmonary tuberculosis, headaches, blurred vision, inflamed cornea, swellings, sores, and pus discharge, bleeding from external injuries, bloody feces, uterine bleeding, menstrual disorders, and vaginal discharge.

5. Phytochemistry

Recent investigations have revealed that lichens are slow-growing organisms that produce a wide array of secondary metabolites with different pharmacological activities.¹ Lichen secondary metabolites are mostly synthesized from the fungal metabolism. These extrolites are usually deposited as crystals on the surface of cortical and medullary hyphal cell walls, which poorly dilute in water and can usually be isolated from lichen by organic diluents.⁸⁵ The chemistry of Usnea is cynosure for all applied field researchers because of its wide range of medicinally important primary and secondary metabolites, only known in lichens, with significant variety in biological and biomedical properties. Until now, more than 60 compounds have been identified from Usnea species which belong to various classes such as depsidones, depsides, depsones, lactones, quinines, polyphenolics, polysaccharides, fatty acids, and dibenzofurans. Fig. 1–7 show the chemical structure of active compounds collected in Table 2.

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5.1. Primary metabolites

To date, few primary metabolites of Usnea have been analyzed, however these metabolites have valuable standing compared to other classes of plants due to its medicinal properties which are described below.

5.2. Secondary metabolites

Lichens had to evolve diverse biosynthetic pathways to produce such complex arrays of extrolites. The polyketide biosynthetic pathway appears to be responsible for most of the classes of lichen compounds, whereas pulvinic acids are shikimate derivatives, and the Abundance of di- and triterpenoids found in lichens are formed via the mevalonate pathway. There are large numbers of studies reporting the isolation and characterization of individual components of lichen extracts.⁹¹ Lichens are a rich source of unique secondary metabolites which are synthesized by the acetyl-polymalonate pathway (APP), shikimic acid pathway (SAP), and mevalonic acid pathway (MAP). All pathways are initiated by a central precursor molecule, acetyl-co A which is the main product of glucose catabolism but the most important pathway for lichen is APP which exerts unique metabolites i.e. depsides, depsidones etc. MAP and SAP derive more commonly occurring metabolites such as terpenes, terpenoids, steroids and pulvinic acid derivatives (Fig. 8).⁹²

5.3. Miscellaneous

Two new phenolic compounds, longissiminone A (50) and longissiminone B (51) have been extracted from U. longissima.⁹⁹ A new O-deoxyglycoside of dimeric tetrahydroxanthane and hirtusneanoside (52) identified in U. hirta¹⁰³ and anthraquinone and longissimausnone (53) have been isolated from U. longissima.¹⁰⁴ Yellow pigments such as eumitrins A1, A2, and B were also isolated from U. bayleyi.¹⁰⁵ A new flavanoid glycoside, apigenin 7-O′-D-glucuronide (54) and primary phenolic compounds such as atranol (55) and orcinol (56) have been characterized in the extract of U. longissima,¹⁷ which plays a key role in the synthesis of depsides and depsidones. Recently, our group has isolated and identified an anti-candidal flavonoid, quercetin (57) from U. longissima.²⁸ Two alcoholic compounds, arabitol (58) and octanol (59) were also isolated from U. longissima.¹⁷ Two new phenylalanine diketopiperazines have also been found, ambewelamide A (60) and B (61) from the chloroform extract of Usnea species.¹⁰⁶

6. Pharmacological properties

Usnea species have been used as anti-microbial agents in different regions of the world and a number of formulations were developed as modern pharmaceuticals just prior to the advent of the penicillin antibiotics. Numerous investigations on the pharmacological properties of the Usnea species have enlightened their efficacious remedy for various illnesses. Tables 3 and 4, respectively, report the pharmacological activities of extracts and bioactive constituents obtained from different species of Usnea.^107–110

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6.1. Antimicrobial activity

6.2. Antioxidant

The polyphenolic nature of the major secondary metabolites of the Usnea species is expected to afford antioxidant activity and a range of in vitro investigations have already been carried out on this issue with promising results. In general, antioxidant activity has been mainly evaluated based on some chemical in vitro assays, such as free radical quenching activity, reducing power and lipid peroxidation inhibition. Among organic solvent, ethanol and methanol have been used as the most efficient and suitable solvents for the extraction of metabolites with antioxidant properties from Usnea. Usnic and psoromic acids were extracted from the submerged cultivation of U. complanata. Different organic solvents including ethanol, methanol, ethyl acetate, and acetone were used for the preparation of extracts to determine their antioxidant activity. Except for the methanolic extract, other extracts exhibited anti-oxidative action in terms of free radical scavenging activity (FRSA), nitric oxide radical scavenging activity, and anti-lipid peroxidation potential with IC₅₀ values ranging from 22.86 to 25.0, 141.3 to 149.1, and 125 to 157.9 μg mL⁻¹, respectively. Isolated bioactive compound usnic acid showed FRSA with IC₅₀ values ranging from 0.174 to 0.271 mg mL⁻¹.²⁰ Antioxidant and hepato-protective activities of a cultured lichen U. ghattensis have also been observed.¹²¹ The obtained results revealed that at 20 μg mL⁻¹ concentration the methanolic extract exhibits 67% inhibition of lipid peroxidation and 86% trolox equivalent antioxidant capacity. At the same concentration, it also showed superior superoxide (O₂˙⁻), 1,1-diphenyl-2-picrylhydrazyl, nitric oxide, and hydroxyl (˙OH) free radical scavenging activities of 89%, 89.6%, 94.8%, and 89.6%, respectively, compared to the synthetic antioxidants, butylated hydroxytoluene, butylated hydroxyanisol, and quercetin. O₂˙⁻ scavenging activity and inhibition of lipid peroxidation potential of U. longissima was reported. The results were presented in terms of IC₅₀ for O₂˙⁻ (0.45 mg mL⁻¹) and lipid peroxidation (1.57 mg mL⁻¹).¹²²

Usnic acid was isolated from the acetone extract of U. barbata and its in vitro antioxidant potential was examined. The IC₅₀ values for the O₂⁻˙ scavenging and reducing power were 102.65 and 130.73 μg mL⁻¹, respectively.¹⁹ The methanol extract of U. ghattensis has shown good antioxidant potential by inhibiting lipid peroxidation and scavenging free radicals with pretty similar values to those of reference antioxidant compounds.¹²³ Moreover, U. longissima methanol extract was found to increase the level of antioxidant enzymes and inhibit lipid peroxidation^124,125 and its water extract was able to revert the effects of indomethacin in vivo through activation of SOD and GST activities and a decrease of CAT activity.⁵³ A depside, diffractaic acid was characterized as a major metabolite of U. longissima and also showed in vivo antioxidant properties.¹²⁶ Cakir and colleagues demonstrated that the methanol extracts of U. articulata and U. lipendula showed a protective role against AFB1 in human lymphocytes by enhancing SOD and GPx enzymatic activity and by decreasing lipid peroxidation.⁶⁴ Polysaccharides of U. longissima (PUS) scavenge the superoxide anion free radical (O₂⁻˙) and hydroxyl free radical (˙OH) with considerable IC₅₀ values of 0.45 mg mL⁻¹ and 1.57 mg mL⁻¹, respectively.¹²¹ Authors concluded that PUS weakly inhibits the lipid peroxidation of the hepatocyte homogenate of mice. Various extracts of U. complanata showed DPPH free radical quenching properties (IC₅₀: 22.86–25 mg mL⁻¹), nitric oxide radical scavenging activity (72.52–149.1 mg mL⁻¹) and lipid peroxidation inhibition (74.58–157.9 mg mL⁻¹). Usnic and psoromic acids were identified as the active substances of the cultured symbiont. Usnic acid demonstrated better radical quenching potential while psoromic acid presented higher lipid peroxidation inhibition.²⁰

6.3. Anti-cancer

Several crude extracts and isolated compounds from Usnea lichens have been screened against different cancer cell lines showing promising anti-cancer and cytotoxic activities.^127,128 The anti-proliferative effect of U. filipendula Stirt. on different human cancer cell lines, including lung cancer (A549 and PC3), liver cancer (Hep3B), and rat glioma (C6) was investigated. In a dose-dependent manner (1.56–100 μg mL⁻¹), the methanolic extract was observed to induce apoptotic cell death with a significant increase in genetic damage in the test cell lines.¹²⁹ The (+)-usnic acid and diffractaic acid isolated from the lichens U. subcavata and Usnea species were evaluated against melanoma UACC-62 and B16-F10 cells. The data from rgw sulforhodamine B assay revealed significant cytotoxic activity of diffractaic acid and usnic acid towards UACC-62 cells with IC₅₀ values of 24.7 and 36.6 μg mL⁻¹, respectively. Moreover, IC₅₀ values of diffractaic acid and usnic acid against B16-F10 cells were 24.0 and 25.4 μg mL⁻¹, respectively.¹³⁰ The bioactive metabolites in the acetone extract of U. barbata were investigated for their anticancer activity against FemX (human melanoma) and LS174 (human colon carcinoma) cell lines using the microculture tetrazolium test. Usnic acid was found to be potentially active against human melanoma FemX cells and human colon carcinoma S174 cells with IC₅₀ values of 12.72 and 15.66 μg mL⁻¹, respectively.¹⁹ U. longissima thallus strongly suppressed Epstein Barr Virus (EBV)-induced tumor promotion. Usnic acid, barbatic acid, 4-O-dimethyl-barbatic acid, diffractaic acid and evernic acid were responsible for this activity. Of these, usnic acid displayed the highest inhibitory activity (IC₅₀ 1.0 mM).¹³¹

Two new heptaketides, including corynesporol (1) and 1-hydroxydehydroherbarin (2) along with herbarin (3) were isolated from an endolichenic fungal strain, Corynespora species BA-10763, associated with U. cavernosa. Aerial oxidation of corynesporol (1) yielded herbarin (3). The structures of 1–3 were elucidated from their spectroscopic data. Acetylation of 1 produced the naphthalene derivative 4, whereas acetylation of 3 yielded the corresponding naphthoquinone 6 and dehydroherbarin (5). All compounds were evaluated for their cytotoxicity and observed inhibitory effect on the migration of human metastatic breast cancer MDA-MB-231 and prostate cancer PC-3 cell lines.¹³² Diffractaic acid, a novel proapoptotic agent extracted from U. longissima and determined its in vivo anticancer activity. The orally and locally administered diffractaic acid showed the induction of apoptosis in tissues of titanium-implanted rabbits by activating initiator caspases (Cas-2, -8 and -9) and executioner caspase (Cas-3). It also showed strong effect on myeloperoxidase and inducible nitric oxide synthase activities, providing an alleviating effect.¹³³ The in vitro cytotoxicity assay of two new derivatives of phenylalanine diketopiperazine, ambewelamide A and B was examined against murine leukemia P388 cells. Only ambewelamde A exhibited significant cytotoxicity with IC₅₀ value 8.6 ng mL⁻¹.¹⁰⁶

Usnic acid obtained from U. barbata was examined for its anti-proliferative activity. L-Usnic acid caused moderate inhibition of murine P388 leukemia cells and also exhibited cytotoxic potential against cultured mouse leukemia Ll210 cells. It was inferred that the p-tri-ketone moiety was essential for optimum activity.¹³⁴ On the other hand, D-usnic acid (50 μg mL⁻¹) was found to reduce the cell counts of leukemic K-562 cells and endometrial carcinoma HEC-50 cells.^135,136 Different extracts of U. fasciata containing usnic acid and isolichenin showed moderate anti-cancer activity against sarcoma 180 and Ehrlich tumor cells. However, high anti-tumoral activity, near 90% inhibition, was observed with the fraction containing raffinose.⁸⁷ Recently, Zuo and colleagues have elucidated the molecular mechanism through which usnic acid mediates anti-cancer activity. Usnic acid selectively killed the human breast cancer MCF-7 cells by inducing the generation of reactive oxygen species (ROS), which triggered the activation of c-Jun-N-terminal kinase (JNK), loss of mitochondrial membrane potential (MMP), release of cytochrome-c, and activation of the caspase-cascade.¹³⁷ Eventually, usnic acid was found to inhibit tumor growth in MCF-7 tumor-bearing mice without inducing significant toxicity. The authors suggested that usnic acid stimulated apoptosis through an ROS-dependent mitochondrial pathway in MCF-7 cells. Eumitrin A1, isolated from U. blepharea was evaluated for its cytotoxic activity against Murine Leukemia P388 cells. According to the observed IC₅₀ value (4.5 μg mL⁻¹), it is reported as a very active toxic compound for cancer cell lines.¹³⁸

6.4. Anti-inflammatory

Usnic acid extracted from U. barbata using supercritical fluid method has shown anti-inflammatory properties by inhibiting ultraviolet-B induced prostaglandin E2 synthesis and cyclooxygenase-2 (COX-2) expression in HaCaT keratinocytes. Moreover, a crude extract also inhibited prostaglandin E2 production at a half-maximal concentration of 60 μg mL⁻¹ which contains 2.4 μg mL⁻¹ of usnic acid. However, the extract did not affect the UVB-induced upregulation of COX-2, suggesting an effect on enzymatic activity rather than on protein expression.¹³⁹ Choudhary et al. succeeded in isolating new compounds, including longissiminone A, longissimone B and glutinol, from U. longissima and evaluated them for their anti-inflammatory activity. Longissimone A showed potential anti-inflammatory activity in comparison to standard drugs with IC₅₀ 165.74 μg mL⁻¹.⁹⁹ Usnic acid has been demonstrated to be a potent anti-inflammatory agent.¹⁴⁰ Lichen metabolites such as atranorin, diffractaic, and protolichesterinic acids were found to attenuate LTB₄ biosynthesis in polymorphonuclear leukocytes, due to specific enzyme interaction rather than nonspecific redox mechanism.¹⁴¹ The phenolic compound longissimone A, isolated from U. longissima displayed anti-inflammatory responses comparable to standard aspirin in a cell-based contemporary assay.⁹⁹

6.5. Genotoxic, anti-genotoxic and anti-mutagenic

The genotoxic and anti-genotoxic potentials of two lichen methanolic extracts, U. articulata (UAE) and U. filipendula (UFE) against aflatoxin B1 (AFB1)-induced genotoxic and oxidative damage were studied. It was observed that the methanolic extracts of UAE and UFE decrease the frequencies of sister chromatid exchange and malondialdehyde level and increase the level of antioxidant enzymes such as superoxide dismutase, glutathione, and glutathione peroxidase in a concentration-dependent manner (5 to 20 μg mL⁻¹).³⁴ A concentration-dependent anti-mutagenic potential of usnic acid ligands (L1, L2 and L3) and their complexes were examined for the first time against known mutagens, NaN3, 9-AA and MNNG in S. typhimurium TA1535, TA1537, and E. coli WP2uvrA, respectively. The results were evaluated using the standard plate incorporation method. The results showed that the ligands and their coordination compounds exhibited various anti-mutagenic effects ranging from 25.2–82.5%.¹¹² Usnic acid, a major bioactive compound of the Usnea genus was evaluated for genotoxicity and MMS-induced genotoxicity in a conc. dependent manner in in vivo (Swiss mice) and in vitro systems (V79 cells). It was demonstrated that usnic acid exhibits a protective effect against MMS-induced genotoxicity by reducing the frequencies of micronuclei and DNA damage.¹⁴²

6.6. Anti-platelet and anti-thrombotic

The antiplatelet and antithrombotic properties of a methanolic extract of U. longissima were determined. The test was performed on platelet aggregation in vitro and on pulmonary thrombosis in vivo.¹⁴³ A concentration dependent inhibitory effect was seen on ADP-induced platelet aggregation, with an IC₅₀ value of 3.6 mg mL⁻¹. For the in vivo studies, a thrombotic model was used in which mice were injected intravenously with a mixture of collagen and epinephrine. The oral administration of the extract prior to the injection produced a significant inhibition of thrombotic death or paralysis at 100–200 mg kg⁻¹ body weight. The results revealed that the antithrombotic activity of U. longissima extract might be due to antiplatelet activity rather than anti-coagulant activity.¹⁴³

6.7. Others

Many other biological activities for the Usnea species have also been reported. Diffractaic and usnic acids of U. diffracta were identified as analgesic and antipyretic components in mice.¹⁰⁹ Halici et al. confirmed the potential gastroprotective effect of the aqueous extract of U. longissima against indomethacin-induced gastric lesions in rats through an antioxidant mechanism.¹⁴⁴ The acetic acid-induced writhing and tail-pressure methods were performed to examine the effects. Both compounds showed an analgesic effect for acetic acid-induced writhing and tail-pressure methods in mice. Diffractaic acid showed significant effects at 500 mg kg⁻¹ and 1 g kg⁻¹, while usnic acid was found to be effective at 100 mg kg⁻¹.¹⁴⁴ One year later, the same research group isolated the diffractaic and the usnic acids from an organic extract of U. longissima as promising anti-ulcerogenic agents.^145,146 This effect was investigated using indomethacin-induced ulcer models in rats by comparing the negative (treated only with indomethacin) and positive (ranitidine) control groups. The extract showed significant anti-ulcerogenic activity compared to the negative control groups in a dose-dependent manner. The highest activity (79.8%) was observed with 100 mg kg⁻¹ body. It was associated with the inhibition of oxidative damage and neutrophil infiltration.

The methanolic extract of U. longissima was also applied to determine in vitro melanogenesis inhibitory effects.¹⁴⁷ The extract was found to reduce melanin formation in human melanoma cells in concentration-dependent manner. Inhibition of melanin content by 51.1% and 34.9% were recorded at 0.01% and 0.1% solutions of the extract, respectively. The obtained results were compared to ascorbic acid. It has also determined that the extract affected the activity of tyrosinase via inhibition of glycosylation process.¹⁴⁷

7. Toxicity

Apart from the analysis of phyto-constituents as a traditional medicine, researchers have also carried out toxic studies on Usnea species. Until now, usnic acid, a major constituent of the genus Usnea was reported only for severe hepatotoxicity and allergic cross reaction, but because it is poorly and slowly absorbed when in either a tea or alcoholic solution, there is little cause for concern. The LD₅₀ is 25 mg kg⁻¹ in mice. It is best not to swallow the alcoholic tincture without diluting it, as it can be irritating. Large quantities of a strong tea of some lichens could cause gastro-intestinal upset, because of the irritating nature of the lichen compounds. Recently diffractic acid, isolated from U. longissima was investigated for carbon tetrachloride-induced hepatic damage in vivo and all biochemical and histopathologically assays were performed. Diffractic acid was found to be hepato-protective agent at low dose 50 mg kg⁻¹ daily but at high dose (100 and 200 mg kg⁻¹) it showed hepatotoxicity.¹⁴⁸ Dobrescu and colleagues studied about the acute toxicity of U. barbata and U. hirta. The hydro-alcoholic extracts of U. barbata and U. hirta exhibited toxicity with LD₅₀ values of 22.53 g kg⁻¹ and 21.02 g kg⁻¹, respectively after intraperitoneal administration in rats. LD₅₀ values of 7.43 g kg⁻¹ for U. barbata and 4.52 g kg⁻¹ for U. hirta were recorded after intravenous administration.¹⁴⁹ Recently, in Spring 2003, several reports appeared indicating that ingestion of usnic acid, as the suspect compound in LipoKinetix (a product of Syntrax Innovations, USA), caused liver failure that was complicated by cerebral edema in one individual who took the product, and liver damage in six other cases. The duration of ingestion for this adverse effect is just a few weeks. The first FDA warning appeared in 2001 and was updated in spring of 2002,¹⁵⁰ and formally reported in Annals of Internal Medicine at the same time.¹⁵¹ There may have been other cases of liver damage from this same product,¹⁵² based on retrospective studies.

Chemical constituents of Usnea species exhibit acute toxicity against larvae of the polyphagous insect herbivore Spodoptera littoralis revealed the LD₅₀ at 8.6 μM for (−)-usnic acid, 90.8 μM for (+)-usnic acid and 111.0 μM for vulpinic acid.¹⁵³ Pramyothin et al. demonstrates that usnic acid showed no serum transminase activity when inducing swell in liver mitochondria and endoplasmic reticulum at a dose level of 50 or 200 mg kg⁻¹ intraperitoneally for 5 days. Meanwhile a dose level of 1 mM usnic acid in rat primary hepatocytes triggered the release of hepatic transaminases, decreased the content of reduced glutathione, and caused a loss of cell membrane integrity.¹⁵⁴ It was observed that the administration of 5 μM usnic acid for 16 h in mouse primary hepatocytes exhibited 98% necrosis rather than apoptosis by generating oxidative stress and acting directly on the uncoupling of oxidative phosphorylation of the electron transport chain in mitochondria. Usnic acid administration in sheep triggered serum creatine kinase, aspartate aminotransferase, and lactate dehydrogenase activities. It was also estimated that 485 and 647 mg kg⁻¹ d⁻¹ median toxic doses (ED₅₀) in domestic sheep.¹⁵⁵ Sheu and colleagues studied allergic content dermatitis by applying lichen acid mixture and usnic acid to four patients and observed that all patients showed positive results for patch test.¹⁵⁶

8. Conclusions and future prospects

Within the fungus kingdom, lichens produce a wide array of both primary (intracellular) and secondary (extracellular) compounds with different biological properties. However, the knowledge of the biological potential of many lichens and their metabolites is very narrow compared to other fungi. Moreover, this knowledge is very recent and limited investigations have been conducted for a deeper understanding of the mechanism and cellular sites of action of lichen substances responsible for the different pharmacological properties described so far. Thus, the aim of this review is to provide up-to-date information about traditional uses, phylogeny, phytochemistry, pharmacology, and toxicology of the most numerous and widespread genus of lichens, Usnea which comprises of approximately 350 species based on scientific literatures.

Usnea is a lichen; a combination of an algae and a fungus growing together. Usnea species are endemic to many parts of Asia, Africa, Europe, and America and are widely used in traditional medicine for various applications. It is used to treat stomachache, bronchitis, sterility, pneumonia, pulmonary diseases, strep throat, colds, flues, urinary tract, kidney, and bladder infections. Usnea is also beneficial for women with yeast infections, trichonomosas, bacterial vaginosis, and chlamydia. It could be useful for people with chronic fatigue, HIV, herpes, and other chronic conditions related to depressed immune systems. The phytochemical results have indicated a significantly diversity of structural types of chemical constituents. Pharmacological studies indicated that Usnea lichens and their bioactive constituents possess various biological properties, especially in the areas of anti-microbial, anti-cancer, anti-proliferative, anti-oxidant, anti-inflammatory, anti-ulcer, hepatoprotective, and anti-genotoxicity etc. To a certain extent, pharmacological results have shown that (a) traditional uses for the treatment of flues, gastroenteritis and bacterial/fungal infections, and strep throat were related to antimicrobial activities; (b) use for wounds, ulcers, and fevers were associated with anti-inflammatory activity; (c) the anti-cancer activity was due to the regulation of molecular targets including caspases; (d) the anti-oxidant, anti-ulcer, and anti-genotoxic properties have been investigated by in vitro and in vivo experiments. Regarding the constituents contributing to therapeutic values, the findings indicated that depsides, depsidones, and benzofurans are key phytochemicals for the treatment of microbial infections, oxidative stress, cancer, ulcer, and inflammation. It is imperative to discuss the stereo-chemistry and structure–activity relationships of depsides, depsidones, and benzofurans for evaluating medicinal properties of Usnea species. However, relating mode of action with chemical structure is difficult since, up to date, the number of investigated chemical constituents for each of these substance classes and pharmacological activities is not very large and the techniques applied and parameters estimated are very variable. As an example, for assessing cytotoxic activity, most of the current research evaluated the capacity of crude extracts and active compounds to inhibit cancer cell proliferation, without focusing on the mechanism of action. For anti-microbial activity, some researchers have focused on Gram positive bacteria, others on Gram negative bacteria, on fungi, on mycobacteria and some other on mixture of them. For estimating antioxidant activity, some researchers have examined the capacity of certain active compounds to scavenge free radicals, others assess their anti-lipidperoxidative property and others determine the level of endogenous antioxidants including CAT, SOD, and GST. Moreover, existing information on the relationships between chemical structure and pharmacological mechanism of action is very limited within natural products, taking as an example the plant kingdom, which is much more known than fungus.

It is noteworthy that current studies on the chemical constituents and pharmacological mechanisms of Usnea species lack depth and more investigations on phytochemistry and the mechanisms of the main active ingredients in demonstrating certain biological activities should be encouraged to fully understand the compounds responsible for the pharmacological effects and the mechanisms of action. The great progress on the phytochemistry and pharmacology of the genus Usnea that has been made confirm its traditional uses. However, there is a pressing need to investigate more conclusive molecular and clinical studies on the safety, efficacy, and toxicity of extracts as well as pure phytochemicals to gain a better understanding of this genus. Furthermore, a significant proportion of the collected pharmacological research has been performed on lichen extracts with promising results, being of interest for the determination of active principles.

Several hurdles were initially faced in the in vitro culture of lichens in order to obtain substantial quantities of lichen substances for various applications. However, advanced techniques such as mycobionts under adjusted culture conditions and heterologous expression of polyketide synthase gene in filamentous fungi, yeasts, and bacteria have recently contributed to great progress in lichen research. These could contribute to future pharmaceutical applications of selected substances of Usnea species, obtained in suitable amounts.

Acknowledgements

This work was financially supported by Twelfth Five-year Plan program (BSC-0106) sponsored by the Council of Scientific and Industrial Research (CSIR) and research grant (GAP 3304) received from the Department of Science and Technology (DST), New Delhi, India. Authors are also grateful to the Director CSIR-National Botanical Research Institute, Lucknow, India for his support and encouragement.

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Footnotes

† These authors contributed equally.

‡ Present address: TERI-Deakin Nano Biotechnology Centre, The Energy and Resources Institute (TERI), Darbari Seth Block, IHC complex, Lodhi Road, New Delhi – 110003, India.

This journal is © The Royal Society of Chemistry 2016