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

SANKET  KUMAR

The genus Usnea Adans. (Parmeliaceae; lichenized Ascomycetes) is a typical group of mostly pale grayish-green fruticoselichens that grow as leafless mini-shrubs. More than 360 species of Usnea are known in the world. Usnea has long been thought to have treat various illnesses in addition to its historical use as dyes, cosmetics, preservatives, and deodorants, particularly in eastern countries such as China, Japan, Taiwan, India and Europe. The current review focuses on the traditional uses and phytochemistry aspects of different Usnea species, and discusses the pharmacological findings and toxicology of their extracts and isolated compounds. The available compilation of data will provide a new base for future perspectives and highlight the need for further studies of this potent herbal source to harvest more beneficial therapeutic drugs. Nineteen species of the genus Usnea are found to be important folk medicines all over the world. It is evident from the comparative analysis of the searched literature that the genus Usnea has been used for various purposes for centuries and its long and traditional medicinal history was well documented in the past. As per ancient records and recent scientific literature, the species of genus Usnea have been used as promising traditional medicines, exerting an array of therapeutic properties to relieve sore throats, bronchitis, cold, flu, infection, and indigestion. Phytochemical analysis confirms the general presence of a wide range of metabolites, polysaccharides, fatty acids, phenolic acids, flavonoids, terpenes, sterols, depsides, depsidones, and benzofurans. As specific constituents, usnic acid, polyphenols, and depsides have been considered as main efficacy component for antibacterial and antifungal activities. In addition, pharmacological analysis also revealed that other pure compounds and crude extracts of Usnea species prove to be significant anti-cancer, anti-proliferative, anti-oxidant, anti-viral, anti-inflammatory, anti-ulcer, hepatoprotective, and anti-genotoxic agents. However, there is a need for more precise investigations to examine the clinical value of both isolated pure compounds and crude extracts and to elucidate their mechanisms of action. Apart from clinical validation and elucidation of their mechanism of action, biosafety studies of the compounds are also important to legitimately use the potential bioactive compounds for the further development of future lead drugs.

RSC Advances REVIEW Cite this: RSC Adv., 2016, 6, 21672 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. Joshid and Brahma N. Singh*a The genus Usnea Adans. (Parmeliaceae; lichenized Ascomycetes) is a typical group of mostly pale grayishgreen fruticoselichens that grow as leaﬂess mini-shrubs. More than 360 species of Usnea are known in the world. Usnea has long been thought to have treat various illnesses in addition to its historical use as dyes, cosmetics, preservatives, and deodorants, particularly in eastern countries such as China, Japan, Taiwan, India and Europe. The current review focuses on the traditional uses and phytochemistry aspects of diﬀerent Usnea species, and discusses the pharmacological ﬁndings and toxicology of their extracts and isolated compounds. The available compilation of data will provide a new base for future perspectives and highlight the need for further studies of this potent herbal source to harvest more beneﬁcial therapeutic drugs. Nineteen species of the genus Usnea are found to be important folk medicines all over the world. It is evident from the comparative analysis of the searched literature that the genus Usnea has been used for various purposes for centuries and its long and traditional medicinal history was well documented in the past. As per ancient records and recent scientiﬁc literature, the species of genus Usnea have been used as promising traditional medicines, exerting an array of therapeutic properties to relieve sore throats, bronchitis, cold, ﬂu, infection, and indigestion. Phytochemical analysis conﬁrms the general presence of a wide range of metabolites, polysaccharides, fatty acids, phenolic acids, ﬂavonoids, terpenes, sterols, depsides, depsidones, and benzofurans. As speciﬁc constituents, usnic acid, polyphenols, and depsides have been considered as main eﬃcacy component for antibacterial and antifungal activities. In addition, pharmacological analysis also revealed that other pure compounds and Received 16th November 2015 Accepted 21st January 2016 crude extracts of Usnea species prove to be signiﬁcant anti-cancer, anti-proliferative, anti-oxidant, antiviral, anti-inﬂammatory, anti-ulcer, hepatoprotective, and anti-genotoxic agents. However, there is a need for more precise investigations to examine the clinical value of both isolated pure compounds and crude extracts and to elucidate their mechanisms of action. Apart from clinical validation and DOI: 10.1039/c5ra24205c elucidation of their mechanism of action, biosafety studies of the compounds are also important to www.rsc.org/advances legitimately use the potential bioactive compounds for the further development of future lead drugs. 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 Pharmacognosy & Ethnopharmacology Division, CSIR-National Botanical Research Institute, Lucknow – 226001, U.P., India. E-mail: bn.singh@nbri.res.in a Lichenology Laboratory, Plant Biodiversity and Conservation Biology Division, CSIRNational Botanical Research Institute, Lucknow – 226001, U.P., India b Centre of Excellence in Materials Science (Nanomaterials), Z. H. College of Engineering & Technology, Aligarh Muslim University, Aligarh-202002, India c Department of Botany, S. S. J. Campus Almora-263601, Uttarakhand, India d † 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. 21672 | RSC Adv., 2016, 6, 21672–21696 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 macrolichens.13 Most of the species are globally distributed with more This journal is © The Royal Society of Chemistry 2016 Review 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 rst 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 detoxication 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 b-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 signicance for systematics and phylogeny, and are employed at diﬀerent taxonomic levels from species and subspecic 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 Signicant research has been done on Usnea and its metabolites which conrm various biological activities including anti-microbial, anti-oxidant, anti-tumor, anti-viral, anti-inammatory, cardiovascular protective, and hepatoprotective properties.17,20,22,27–33 These are closely correlated with the ethno-medicinal uses. Recent pharmacological studies have revealed signicant anti-cancer, anti-genotoxic, antiproliferative, 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) rst 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 This journal is © The Royal Society of Chemistry 2016 RSC Advances recognize Usnea in elds 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 identication 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, brils, 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 specic 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 stulose 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. suborida is distributed in East Africa and North Asia, while U. suboridana 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 RSC Adv., 2016, 6, 21672–21696 | 21673 RSC Advances 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 classication 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 classication of Usnea, in which all fruticose lichens with an inner, cartilaginous tissue are included. He identied 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 classication 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 classications 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 b-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 classication 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 oen homoplasious within the phylogeny and their parallel 21674 | RSC Adv., 2016, 6, 21672–21696 Review 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, avoring agent, pulmonary disease, antiseptic, antituberculosis 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. 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 This journal is © The Royal Society of Chemistry 2016 Review Table 1 RSC Advances Traditional uses of Usnea spp. Species/folk name Country Uses References Usnea spp. Dill. ex Adans. ushna Unani medicine of India 157 U. aciculifera Vain. China U. articulata (L.) Hoﬀm. hewas Tanzania U. atlantica Vain. barbas U. baileyi (Stirt.) Zahlbr. Canary Islands India U. barbata (L.) Weber ex F.H. Wigg. USA Used for heart troubles, for reducing inammation, for promoting digestion and improving appetite, as an antidote, as an astringent, and as an analgesic. It also helps wounds heal and lactation in women if applied as a paste on breast Used for bladder infection, painful urination, urinary retention, swelling, and edema in heart and kidneys Used to treat stomachache. A handful of hewasis chewed fresh and the juice swallowed, it is bitter but relieves the pain Used as a disinfectant Mixed with other aromatic herbs, such as Valeriana jatamansi for favoring and curing tobacco Used to treat fungal infections of the mouth, stomach, intestines, anus, vagina, nose, ear, skin as well as “systematic fungal infection” Applied to treat mammary infections in cattle, the udder is washed several times with decoction of lichen. Also used for indigestion in humans Endangered medicinal lichen banned from raw export Used for wounds, chopped and mixed with coconut oil, spread over wound. Also utilized for abdominal pain, it used as drink decoction Used for colds and strengthening aer connement Used to treat insomnia, nausea, and the uterus, also used for internal and external bleeding, whooping cough, jaundice, and growing hair Utilized as drying agent and antiseptic for cracks and irritations of the feet Liquid made from it is given to women to cure sterility Unspecied medicine Adopted to treat coughs, inamed lungs, pulmonary tuberculosis, hepatitis, and headache due to heat, infection due to injury, inamed lymph channels, mastitis, and snakebites Tea applied externally as astringent, antiseptic, and anti-inammatory South Africa Nepal Philippines tagahumok puti West Malaysia Europe memby rakúı́ja Spain Brazil U. campestris R. Sant barba de piedra U. ceratina Ach. Argentina China U. densirostra Taylor, U. durietzii Mot. yerba de la piedra; barba de piedra U. diﬀracta Vain. lao-jun-xu, Lao Tzu’s beard, pine gauze, or female gauze Argentina China This journal is © The Royal Society of Chemistry 2016 Utilized to cure 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, 76 158 159 160 11 65 161 162 163 82 164 165 166 100 167 100 RSC Adv., 2016, 6, 21672–21696 | 21675 RSC Advances Table 1 Review (Contd. ) Species/folk name Country song-nag Korea gser.skud Tibet U.durietzii Mot. [syn. Neuropogon durietzii] Usnea lipendula Stirt. [syn. Usnea dasypoga] U. orida (L.) F. H. Wigg. Argentina Russia China South central Chile Europe U. himalayana C. Bab. nayonayo saruogase U. hirta (L.) F. H. Wigg. Chile Japan Europe U. laevis (Eschw.) Nyl. barba de piedra or tusinya USA U. longissima Ach. India China sun-lo Mongolia Madhya Pradesh, India Turkey Indo-Tibetan Himalayas urmil Canada U. nidica Taylor uru nū Rarotongan 21676 | RSC Adv., 2016, 6, 21672–21696 Uses References vomiting, blood in feces, bleeding from uterus, menstrual disorders, vaginal discharge, swelling of female genitalia, urinary tract aﬄictions, parasitic infections, when it used as a drink decoction; or apply decoction or powdered lichen to aﬀected area Used to induce menstruation and treated tuberculosis of the neck Cured fevers of the lungs, liver, and channels and fever caused by poisoning Same as Argentine use of U. densirostra Powdered form used to treat wounds and some infections 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. Drink decoction; or apply decoction or powdered lichen to aﬀected area Infusion taken for management of diarrhea Decoction used for colds and coughs Infusion used for diarrhea Burned as a “lichen cigarette” Used for heal wounds and to prevent hair loss Utilized to treat dermatosis, fungal infections, tuberculosis, and pneumonia Used as a simple drug to stimulate menstruation or induce abortion, taken orally and inserted into the vagina Used in Chinese medicine especially as an expectorant and in the treatment of ulcers, stop sweating, dizziness cold, pain, phlegm, and stop swelling in female genitalia. Also applied as a decongestant for treatment of ulcers and tuberculosis Used medicinally Used to treat bone fractures, along with other ingredients Applied for treating cancer, tuberculosis, and ulcers Used to heal bone fractures. Washed, air-dried, soaked overnight in salted water, and placed over aﬀected part Used to strain impurities out of hot pitch when making medicine, and for other unspecied medicines 11 74 168 48 57 and 169 49 51 49 3 51 170 171 57,99 and 102 7 63 52 and 53 172 173 174 This journal is © The Royal Society of Chemistry 2016 Review Table 1 RSC Advances (Contd. ) Species/folk name Country U. pectinata Taylor China U. plicata (L.) Weber Libya scı́ba Europe U. strigosa (Ach.) Eaton Papua New Guinea U. sikkimensis Biswas sp. nov. darimataghosa India U. strigosa (Ach.) Eaton oleazu Kimi U. suboridana Stirt. Ireland China U.subsordida Stirt. ayurvedic medicine U. trichodeoides Vain. India China Africa, Mt. Kilmanjaro 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 aer a time. In China, U. ceratina was used for coughs, inamed lungs, pulmonary tuberculosis, hepatitis, heat related headaches, infection due to injury, inamed 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-inammatory agents.70,71 In China, U. diﬀracta has been applied to treat a range of problems such as cough, This journal is © The Royal Society of Chemistry 2016 Uses References Thallus chewed and applied to cuts (to stop bleeding) and stings Used for stopping bleeding from external injuries, relieving pain, bloody feces, and swelling Used as an ingredient in medicinal decoction called scıba An astringent for internal and external use for whooping cough, jaundice, strengthening stomach and abdominal cavity, and restraining abortion Concoction taken orally for headaches Used for lung troubles, hemorrhages, and asthma; powdered and used to strengthen hair. Also used to bandage surface wounds, skin eruptions, and boils, when it inserted into nostril to stop nose bleeds; put in shoes to prevent or treat blisters Concoction taken orally for curing headaches Applied for treating sore eyes, mixed with tobacco and butter, boiled, cooled, and applied as lotion to eyes Used for painful and reddened eyes, bleeding from external injuries, and swelling Same as ayurvedic use of U. baileyi Used for coughs, pulmonary tuberculosis, headaches, blurred vision, inamed cornea, swellings, sores, uterine bleeding, menstrual disorders, and vaginal discharge Used as an ingredient in herbal tea given by African guide to relieve altitude sickness 100 54 and 55 56 58 83 and 84 58 59 57 160 57 60 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. orida 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. orida was also used for colds RSC Adv., 2016, 6, 21672–21696 | 21677 RSC Advances 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. suboridana 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, inamed 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 slowgrowing 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 eld researchers because of its wide range of medicinally important primary and secondary metabolites, only known in lichens, with signicant variety in biological and biomedical properties. Until now, more than 60 compounds have been identied 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. 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. 5.1.1. Polysaccharides. Polysaccharides, present in the thallus of Usnea are categorized into glucan type [b-(1/3)(1/ 4)], lichenan homoglucan with b-(1/3)(1/4) linkage and 21678 | RSC Adv., 2016, 6, 21672–21696 Review pustulan [b-(1/6)]. Shahiba and colleagues described that U. barbata, U. longissima, and U. bayleyi contain lichenan (1) homoglucan with b-(1/3) and (1/4) linkages,86 while U. fasciata produces isoliichenan (2).87 Sumanarathna worked on the extraction and isolation of polysaccharides from Usnea species and identied the polymers of glucose, galactose and mannose having various linkages such as glucans [(1,3)-b-glucopyranosyl/ (1,3)-b-glucopyranosyl] and galactomananns [(O-2)-a-D-galactopyranosyl (O-4)-a-D-galactopyranosyl (16)-a-Dmanopyranosyl].188 5.1.2. Fatty acids. A new fatty acid, methyl 3,4-dicarboxy-3hydroxy-19-oxoeicosanoate (3) has been isolated from U. meridensis.88 Additionally, a few more fatty acids isolated from Usnea species are bourgeanic acid (4) from U. esperatiana,21 and U. orida,89 caperatic acid (5) from U. lapponica, U. nipparensis, U. orientalis, and U. orida and murolic acid complex from U. hirta.90 Recently, isomuronic acid (6), murotic acid, lichesterinic acid (7), neuropogolic acid (8), protolichesteric acid (9) and 18Rhydroxydihydroalloprotolichensterinic acid have been isolated and identied from U. longissima.7 so the complete fatty acid proling of Usnea species is not yet available. 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 5.2.1. Depsides. Depsides, the major components of Usnea are polyphenolic compounds with 2 or more aromatic cyclic rings joined by ester linkage. Aciculiferin A, atranorin (10), baeomycesic acid (11), barbatic acid (12), diﬀractaic acid (13), squamatic acid (14), evernic acid (15), 4-O-demethylbarbatic (16), methyl beta-orsellinate (17), ethyl orsellinate (18), thamnolic acid (19), barbatolic acid (20), barbatinic acid, ethyl hematommate (21), methyl hematommate (22), alectorialic acid (23), 7-hydroxy-5-methoxy-6-methylphthalide (24), methyl-2,4dihydroxy-3,6-dimethylbenzoate (25), and methyl beta orcinol carboxylate (26) have been found in various species of Usnea, presented in Table 2.93–97 5.2.2. Depsidones. The depsidones are not only composed of two or more aromatic cyclic rings but also bonded by ether linkage. To date, various species of Usnea were explored for the This journal is © The Royal Society of Chemistry 2016 Review Fig. 1 Chemical structures of compounds 1–9 from Usnea spp. Fig. 2 Chemical structures of compounds 10–19 from Usnea spp. This journal is © The Royal Society of Chemistry 2016 RSC Advances RSC Adv., 2016, 6, 21672–21696 | 21679 RSC Advances Fig. 3 Review Chemical structures of compounds 20–29 from Usnea spp. identication of depsidone compounds and observed to synthesize galbinic acid (27), hypoconstictic acid (28), menegazziaic acid (29), norstictic acid (30), constictic acid (31), virensic acid, salazinic acid (32), lobaric acid (33), protocetraric acid (34), psoromic acid (35), and 2-O-methylhypostictic acid (36), stictic acid (37), fumaprotocetraric acid (38), and cryptostitic acid (39) (Table 2).52,93,98 5.2.3. Terpenes and terpenoids. Two terpenes, (glutinol (40) and beta-amyrin (41)) and three terpenoids, (friedelin (42), oleanolic acid (43), and zeorin (44)) were extracted and characterized from U. longissima.99,100 5.2.4. Sterols. Laxinamujila and colleagues extracted two sterols, 5,8-epidioxy-5alpha, 8alpha-ergosta-6,22E-dien-3beta-ol (45) and ergosterol (46) from U. longissima.17 Moreover, betasitosterol (47) has also been isolated from the same lichen, the most common phytosterol.101 5.2.5. Benzofurans. A most signicant benzofuran, usnic acid (48) is found in all species of Usnea. Some new benzofurans such as ethyl 2-(3,3-bis(7-acetyl-4,6-dihydroxy-3,5-dimethylbenzofuran-2-yl)acryloyl),102 ethyl 4-(7-acetyl-4,6-dihydroxy-3,5-dimethyl2-oxo-2,3-dihydrobenzofuran-3-yl)-4-(7-acetyl-4,6-dihydroxy-3,5dimethylbenzofuran-2-yl)-3 oxo-butanoate (49), (Z)-2-acetyl-5,5bis(7-acetyl-4,6-dihydroxy-3,5-dimeth-ylbenzofuran-2-yl)-4-hydroxypenta-2,4-dienal, (7-acetyl-C(7-acetyl-2,3-dihydro-4,6-dihydro-3,5dimethyl-2-oxo)-3-benzofuranyl)-4,6-dihydroxy-3,5-dimethyl-b-oxoethyl ester, (4aR,9bS)-2,6-diactyl-3,4a,7,9-tetrahydroxy-8,9b-dimethyl-1-oxo-1,4,4a,9b-tetrahydrodibenzo[b,d] furan diethanone, 21680 | RSC Adv., 2016, 6, 21672–21696 longiusnine,17 and 2-benzofuranbutanoic acid102 have recently been isolated from U. longissima. 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) identied 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 avanoid glycoside, apigenin 7-O0 -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 identied an anti-candidal avonoid, 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 This journal is © The Royal Society of Chemistry 2016 Review RSC Advances Fig. 4 Chemical structures of compounds 30–38 from Usnea spp. 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 6.1. Antimicrobial activity 6.1.1. Anti-fungal. Our group has isolated a dietary avonoid quercetin (QC) from U. longissima, which sensitizes uconazole (FCZ)-resistant C. albicans to induce FCZ-mediated cell death by modulating the quorum sensing (QS) system. QC (200 mg mL1) inhibited the secretion of C. albicans virulence factors, namely biolm formation, hyphal development, phospholipase, proteinase, esterase, and hemolysin. It has also demonstrated that the sensitizing eﬀect of QC was associated with the production of farnesol, a QS molecule that acts as a regulator of virulence factors of C. albicans.28 Protocetaric acid (PA) was characterized from ethyl acetate extract of U. albopunctata using spectroscopic methods. PA was found to be a broad spectrum antimicrobial agent against medically important human pathogenic microbes. At 1 mg mL1 of concentration, ethyl acetate extract showed signicant antifungal activity against Trichophyton rubrum, compared to reference antifungal agents such as PA and amphotericin B.22 The results suggested that U. This journal is © The Royal Society of Chemistry 2016 albopunctata may contain also other antifungal compounds which show synergistic action. Two new metabolites, depside and isodivaricatic acid and three known metabolites, 5-propylresorcinol, divaricatinic acid and usnic acid were isolated from U. orida. These metabolites displayed antimicrobial activity against human pathogenic fungi Microsporum gypseum, Trichophyton mentagrophytes, and T. rubrum. Among them, isodivaricatic and divaricatinic acids exhibited antifungal eﬀect towards M. gypseum, T. mentagrophytes, and T. rubrum with minimum inhibitory concentration (MIC) values of 50, 50, and 100 mg mL1, respectively. However, isodivaricatic acid was found to be eﬀective against Leishmania amazonensis, L. brasiliensis, and L. infantumpromastigotes by inducing 100% lysis at 100 mg mL1.111 6.1.2. Antibacterial. The novel multifunctional hydroxyphenylimino ligands such as L1, L2, and L3 were synthesized through the condensation of 2-aminophenol, 3-aminophenol, and 4-aminophenol with usnic acid, respectively.112 The synthesized ligands and their complexes, Cu(II), Co(II), Ni(II) and Mn(II) were characterized using FT-IR, UV-Vis, (1)H-NMR, (13)CNMR, 1D- and 2D NMR (DEPT, COSY, HMQC and HMBC), LCMS, and TGA. The ligands and their complexes were tested against ten important pathogenic microorganisms, such as Enterobacter aerogenes, Brevibacillus brevis, Micrococcus luteus, RSC Adv., 2016, 6, 21672–21696 | 21681 RSC Advances Fig. 5 Review Chemical structures of compounds 39–48 from Usnea spp. Escherichia coli, Bacillus megaterium, Pseudomonas aeruginosa, E. cloacae, Streptococcus aureus, C. albicans, and Saccharomyces cerevisiae. The metal complexes of the ligands were found to be more eﬀective against all of the microorganisms tested, exhibiting 11–32 mm inhibition zones around the ligands. On the other hand, a broad spectrum antimicrobial activity was observed for the Mn(II) and Cu(II) complexes of the hydroxyphenylimino ligand (L3) with usnic acid.112 Extracts of U. ghattensis were prepared using diﬀerent organic solvents and their antibacterial activity was determined using a disc diﬀusion assay. The ethanolic extract was most eﬀective against B. cereus, P. aeruginosa, S. aureus, and Streptococcus faecalis with MIC values of 3.125, 200, 6.25, and 25 mg mL1, respectively. Acetone and methanolic extracts presented almost similar eﬀect against S. aureus.113 L-()-Usnic acid was isolated from U. suboridana and showed promising antibacterial against methicillin-resistant S. aureus (MRSA). The MIC of 1 L-()-usnic acid against MRSA was recorded by 50 mg mL . Similarly, a combined eﬀect of L-()-usnic acid and 7.5% sodium chloride resulted in a reduced number of viable cells within 24 h compared to the control.114 Furthermore, an in vivo study showed that L-()-usnic acid signicantly (p < 0.001) reduced the microbial load of rat spleen in a dose-dependent manner (1 to 5 mg kg1). 21682 | RSC Adv., 2016, 6, 21672–21696 The antibacterial activity of U. steineri was evaluated against Mycobacterium tuberculosis, M. kansasii, and M. avium. The (+)-usnic acid rich acetone extract displayed promising MIC values of 32 mg mL1 for M. tuberculosis and 62 mg mL1 for both M. kansasii, and M. avium.21,115 Acetone and methanol extracts of U. lapponica were screened against four pathogenic bacteria, namely S. aureus, E. coli, P. aeruginosa, and MRSA. The extracts inhibited growth of all tested bacteria except E. coli. Usnic acid was identied as the major active antimicrobial compound in the extracts. The acetone extract was found to be particularly active against MRSA and P. aeruginosa with a MIC value of 15.6 mg mL1.116 A new formulation of U. barbata extract was developed using alkyl polyglucoside surfactants as a vehicle to examine the antimicrobial potential for skin infections. This formulation has implausible potential against Gram positive bacteria.117 A study was performed to assess the in vitro eﬀect of usnic acid isolated from U. dasypoga against clinical isolates and standard Helicobacter pylori strains. The dual susceptibility rate to usnic acid and clarithromycin was detected as very high (97.3%). Usnic acid had a strong and dose-dependent activity against H. pylori strains. The synergism between usnic acid and clarithromycin was also observed and it may be eﬀective in the treatment of H. pylori infection.118 Ethanolic and methanolic This journal is © The Royal Society of Chemistry 2016 Review Fig. 6 RSC Advances Chemical structures of compounds 49–59 from Usnea spp. extracts of Usnea species showed a zone of inhibition against some pathogenic bacterial strains, S. aureus, P. aeruginosa, Klebsiella pneumoniae, Salmonella typhiand, and E. coli.112,119 6.1.3. Anti-viral. The acetone extract of U. complanta exhibited signicant antiviral activity against herpes simplex viruses (HSV) at a concentration non-toxic to the Vero cell line using cytopathic eﬀect inhibition and virus yield reduction assays. The recorded IC50 value was 100 mg mL1.120 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 antioxidative 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 Fig. 7 Chemical structures of compounds 59–61 from Usnea spp. This journal is © The Royal Society of Chemistry 2016 RSC Adv., 2016, 6, 21672–21696 | 21683 RSC Advances Table 2 Review Bioactive constituents of Usnea spp. S. No. Chemical constituent Source References (A) Polysachharides 1 Lichenan 2 Isolichenin U. barbata, U. longissima, U. bayleyi U. fasciata 86 87 U. longissima U. longissima U. longissima U. longissima U. longissima U. esperatiana, U. orida U. lapponica, U. angulata, U. nipparensis, U. orientalis, U. orida, U. sinensis U. meridensis 175 175 102 149 149 4 and 89 89 (B) Fatty acids 3 18R-Hydroxydihydroallopr-otolichensterinic acid 4 Murotic acid 5 Iso-muronic acid 6 Lichesterinic acid 7 Neuropogolic acid 8 Bourgeanic acid 9 Caperatic acid 10 Methyl 3,4-dicarboxy-3-hydroxy-19-oxoeicosanoate (C) 11 12 13 14 15 Depsides Aciculiferin A Atranorin Baeomycesic acid 4-O-Demethylbarbatic acid Barbatic acid 16 Diﬀractaic acid 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 3b-Hydroxy-glutin-5-ene 7-Hydroxy-5-methoxy-6-methylphthalide Alectorialic acid Methyl hematommate Ethyl hematommate Ethyl orsellinate Evernic acid Barbatinic acid Barbatolic acid Methyl orsellinate Methyl b-orsellinate Methyl-2,4-dihydroxy-3,6-dimethylbenzoate Thamnolic acid Squamatic acid 4-O-Demethylbarbatic Methyl b-orcinol carboxylate Decarboxy stenosporic acid (D) Depsidones 34 2-O-Methylhypostictic acid 35 Menegazziaic acid 36 Norstictic acid 37 Constictic acid 38 Protolichesterinic acid 39 Protocetraric acid 40 Psoromic acid 41 Hypocon stictic acid 42 Lobaric acid 43 Salazinic acid 21684 | RSC Adv., 2016, 6, 21672–21696 U. aciculifera U. aciculifera, U. articulate U. pacicana U. longissima U. diplotypes, U. fulvoreaquens, U. lapponica, U. pacicana, U. substerilis, U. wasmuthii, U. pangiana, U. dendritica, U. fragilis, U. norketti, U. nilgirica, U. certaina U. longissima, U. baileyi, U. aciculifera, U. certaina, U. fulvoreagens, U. diﬀracta U. longissima U. aciculifera U. dendritica, U. orida, U. suborida U. aciculifera U. longissima U. longissima U. madeirensis, U. longissima U. longissima, U. aciculifera U. barbata U. longissima, U. undulate, U. aciculifera U. aciculifera, U. undulate U. longissima U. suboridana, U. hirta, U. orida U. pacicana, U. suboridana, U. fragilescens, U. orida, U. longissima U. dendritica, U. longissima U. articulate U. diﬀracta U. undulate U. undulate, U. aciculifera U. baileyi, U. hakonensis, U. undulata, U. cornuta, U. ammea, U. frgilescens, U. fulvoreagens, U. hirta, U. wirthi, U. aciculifera, U. angulate, U. vulneraria, U. suboridana U. aciculifera U. albopunctata U. albopunctata, U. articulta, U. glabrata, U. madeirensis, U. rmula, U. dasaea, U. maculate, U. trichodeoides U. complanata, U. bornmuelleri, U. dasaea, U. inermis, U. pseudosinensis, U. suboridana U. undulate U. orida, U. barbata U. rubrotincta, U. baileyi, U. trichodeoides, U. pangiana, U. longissima, U. complanata, U. compressa, U. corallina, U. dendritica, U. dasaea, U. himalayana, U. luridorufa, U. norketti, U. orientalis, U. pangiana, U. perplexans, U. picta, U. rigidula, U. robusta, U. sordida, U. rubicunda, U. splendens, U. suboridana, U. trichodeoides, U. undulate 88 95 95 and 96 20 109 22 22,97 and 109 104 95 22 95 104 104 97 95 and 104 87 95 and 104 95 and 176 104 22 and 87 22 36 93 15 97 and 98 97 95 and 97 36 22 and 87 87 22,87 and 177 97 51 87 and 97 This journal is © The Royal Society of Chemistry 2016 Review Table 2 RSC Advances (Contd. ) S. No. Chemical constituent Source References 97 22,87 and 176 46 Fumarprotocetraric acid U. undulate U. aciculifera, U. cornuta, U. ammea,U. frgilescens, U. fulvoreagens, U. bismolliuscula, U. complanata, U. dasaea, U. eumitrioides, U. scheri, U. himalayana, U. himantodes, U. indica, U. lucea, U. luridorufa, U. picta, U. pectinata, U. nipparensis, U. pseudojaponica, U. rigidula, U. rubicunda, U. spinosula, U. stigmatoides, U. stigmata U. articulate, U. glabrata 22 (E) Terpenoids and triterpenes 47 b-Amyrin 48 Zeorin 49 Oleanolic acid 50 Friedelin 51 Glutinol U. longissima U. longissima U. longissima U. longissima U. longissima 104 104 104 104 99 U. longissima 102 U. longissima 175 U. longissima 175 U. longissima 175 U. longissima 175 44 Galbinic acid 45 Static acid (F) Benzofurans 52 Ethyl 2-(3,3-bis(7-acetyl-4,6-dihydroxy-3,5dimethylbenzofuran-2-yl)acryloyl) 53 7-Acetyl-C((7-acetyl-2,3-dihydro-4,6-dihydro-3,5-dimethyl-2oxo)-3-benzofuranyl)-4,6-dihydroxy-3,5-dimethyl-B-oxo-, ethyl ester 54 Ethyl 4-(7-acetyl-4,6-dihydroxy-3,5-dimethyl-2-oxo-2,3dihydrobenzofuran-3-yl)-4-(7-acetyl-4,6-dihydroxy-3,5dimethylbenzofuran-2-yl)-3-oxobutanoate 55 (4aR,9bS)-2,6-Diactyl-3,4a,7,9-tetrahydroxy-8,9b-dimethyl-1oxo-1,4,4a,9b-tetrahydrodibenzo[b,d] furan diethanone 56 (Z)-2-Acetyl-5,5-bis(7-acetyl-4,6-dihydroxy-3,5-dimethylbenzofuran-2-yl)-4-hydroxypenta-2,4-dienal 57 3,6-Diacetyl-2,7,9-trihydroxy-8,9b-dimethyl-1[9bH]dibenzofuranone (longiusnine) 58 Usnic acid 59 2-Benzofuranbutanoic acid 102 and 104 U. orida, U. barbata, U. longissima, U. rigida, U. hirta, U. suborida, 97 and 109 U. undulate U. longissima 7 (G) 60 61 62 U. longissima U. longissima U. longissima 104 175 175 U. longissima U. aciculifera U. longissima U. longissima U. hirta U. longissima U. longissima U. baileyi U. longissima 99 178 28 104 103 175 175 36 175 Sterols b-Sitosterol Ergosterol 5,8-Epidioxy-5alpha,8alpha-ergosta-6,22E-dien-3beta-ol (H) Others 63 Longissiminone A & B 64 Atranol 65 Quercetin 66 Longissimausnone 67 Hirtusneanoside 68 Orcinol 69 Apigenin 7-O0 -D-glucuronide 70 Eumitrin B, eumitrin A2, eumitrin A1 71 Arabitol U. longissima 25.0, 141.3 to 149.1, and 125 to 157.9 mg mL1, respectively. Isolated bioactive compound usnic acid showed FRSA with IC50 values ranging from 0.174 to 0.271 mg mL1.20 Antioxidant and hepato-protective activities of a cultured lichen U. ghattensis have also been observed.121 The obtained results revealed that at 20 mg mL1 concentration the methanolic extract exhibits 67% inhibition of lipid peroxidation and 86% trolox equivalent antioxidant capacity. At the same concentration, it also showed This journal is © The Royal Society of Chemistry 2016 superior superoxide (O2c), 1,1-diphenyl-2-picrylhydrazyl, nitric oxide, and hydroxyl (cOH) 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. O2c scavenging activity and inhibition of lipid peroxidation potential of U. longissima was reported. The results were presented in terms of IC50 for O2c (0.45 mg mL1) and lipid peroxidation (1.57 mg mL1).122 RSC Adv., 2016, 6, 21672–21696 | 21685 RSC Advances Fig. 8 Secondary metabolites of Usnea spp. synthesized by acetylpolymalonate pathway, shikimic acid pathway and mevalonic acid pathway. Usnic acid was isolated from the acetone extract of U. barbata and its in vitro antioxidant potential was examined. The IC50 values for the O2c scavenging and reducing power were 102.65 and 130.73 mg mL1, 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 peroxidation124,125 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 (O2c) and hydroxyl free radical (cOH) with considerable IC50 values of 0.45 mg mL1 and 1.57 mg mL1, 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 mL1), nitric oxide radical scavenging activity (72.52–149.1 mg mL1) and lipid peroxidation inhibition (74.58–157.9 mg mL1). Usnic and psoromic acids were identied 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 21686 | RSC Adv., 2016, 6, 21672–21696 Review The anti-proliferative eﬀect of U. lipendula 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 mg mL1), the methanolic extract was observed to induce apoptotic cell death with a signicant 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 signicant cytotoxic activity of diﬀractaic acid and usnic acid towards UACC-62 cells with IC50 values of 24.7 and 36.6 mg mL1, respectively. Moreover, IC50 values of diﬀractaic acid and usnic acid against B16-F10 cells were 24.0 and 25.4 mg mL1, 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 mg mL1, 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 1hydroxydehydroherbarin (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 signicant cytotoxicity with IC50 value 8.6 ng mL1.106 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 mg mL1) was found to reduce the cell counts of leukemic K-562 cells and endometrial carcinoma HEC-50 cells.135,136 Diﬀerent extracts of This journal is © The Royal Society of Chemistry 2016 Review Table 3 RSC Advances Pharmacological properties of extracts obtained from Usnea spp. S. No. Extract/compound (a) In vitro studies 1 Methanol extract Source Bioactivity U. lipendula Antipopulation Methanol, acetone extracts 3 Acetone extract 4 Acetone extract 5 Methanol extract 6 Acetone, methanol extracts 7 Acetone extract 8 Polysaccharide U. longissima Anti-lipid peroxidation 9 Methanol extract U. artarctica Anti-oxidant, Human lymphocytes anti-genotoxic U. barbata AntiHaCaT keratinocytes inammatory Antioxidant In vitro system FemX (human melanoma) and LS174 (human colon carcinoma) Reduces cell viability U. complanta Anti-viral Herpes simplex viruses Exhibits cytopathic eﬀect (HSV) U. longissima Melanogenesis Human melanoma cells Inhibits tyrosinase glycosylation inhibition Kills bacteria U. lapponica Anti-bacterial S. aureus, E. coli, P. aureginosa and Methicillin resistant S. aureus U. barbata AntiMycobacterium Inhibits growth of pathogenic bacteria mycobacterial tuberculosis, M. kansasii and fungi and M. avium In vitro system 12 Acetone, methanol, Antimicrobial aqueous extracts Antimicrobial 13 Supercritical CO2extract 14 Ethanol, methanol U. ghattensis Antioxidant In vitro system U. fasciata Cytotoxic 15 Diethyl ether, acetone, methanol, aqueous extract 16 Methanol extract U. lipendula Anti-oxidant, anti-genotoxic 17 Methanol, aqueous U. longissima Antioxidant extracts 18 Acetone, methanol, U. ghattensis Antibacterial ethanol extracts Sarcoma 180 and Ehrlich tumor cells Malassezia furfur, S. aureus In vitro system Human lymphocytes In vitro system Dose References 1.56–100 mg mL1 129 IC50: 1 mg 179 mL1 18 IC50: 102.65 and 130.73 mg mL1 IC50: 100 120 mg mL1 0.1% 147 MIC: 15.6 116 mg mL1 18 MIC: 32 mg mL1 and 62 mg mL1 0.45–1.57 121 Scavenges free oxygen radicals and hydroxyl radical oxygen and reduces DNA mg mL1 damage 34 Inhibits lipid peroxidation and enhances 5–20 mg antioxidant enzyme activities mL1 Inhibits prostaglandin E2 synthesis and 60 mg 139 cyclooxygenase-2 (COX-2) expression mL1 Scavenges free radicals 0.0008 to 18 0.5 mg mL1 Inhibits the growth of bacteria and fungi IC50: 0.1 60 mg mL1 Inhibits the growth of bacteria and yeasts — 180 with dermatological relevance Inhibits lipid peroxidation 20 mg 123 mL1 Decreases cell viability — 87 Inhibits lipid peroxidation and enhances 5–20 mg antioxidant enzyme activities mL1 Scavenges free radicals — Inhibits the growth of bacteria Antibacterial Human pathogenic bacteria Inhibits the growth of bacteria Inhibits lipid peroxidation and scavenges 0.2 mg free radicals mL1 Scavenges superoxide radicals 2–20 mg mL1 Quenches diﬀerent types of free radicals 20 mg mL1 — Antioxidant In vitro system Antioxidant In vitro system 22 Methanol extract Antioxidant In vitro system U. longissima This journal is © The Royal Society of Chemistry 2016 Human blood cells 34 124 113 MIC: 3– 200 mg mL1 MIC: 5–10 181 mg mL1 Human pathogenic bacteria 19 Acetone, dimethyl sulphoxide, methanol, light petroleum extracts 20 Diﬀerent extracts of cultured mycobiont 21 Methanol extract 23 Methanol extract Mode of action Human lung cancer Induces apoptosis like cell death (A549, PC3), liver cancer (Hep3B) and rat glioma (C6) cells In vitro system Scavenges free radicals 2 10 Supercritical CO2extract 11 Acetone extract U. artarctica, Antioxidant U. auranticoatra U. barbata Anti-cancer, anti-oxidant Target/system 123 182 183 125 RSC Adv., 2016, 6, 21672–21696 | 21687 RSC Advances Table 3 Review (Contd. ) S. No. Extract/compound Source 24 Acetone extract Antigenotoxic, antioxidant U. rubicunda Antitumor In vitro system Bioactivity Cytotoxic Target/system Mode of action Dose References — 131 Cancer cell lines Inhibits lipid peroxidation and induces antioxidant enzyme levels Inhibits tumor promoter-induced Epstein–Barr virus activation Reduces cell viability IC50: 20 mg 184 mL1 25 n-Hexane, diethyl ether, methanol extracts 26 Methanol extract U. siamensis Antifungal C. albicans Inhibits the growth of fungus — 185 (b) In vivo studies 1 Aqueous extract U. longissima Anti-ulcer Rats — 100 mg kg1 100–200 mg kg1 52 5–20 mg mL1 33 20 mg mL1 122 2 Methanol extract 3 Methanol extract 4 Methanol extract Mice U. longissima Anti-platelet, antithrombotic U. articulate, Anti-oxidative, Rats U. lipendula anti-genotoxic U. ghattensis Hepatoprotective Rats 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 anticancer 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-Nterminal 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 signicant 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 mg mL1), it is reported as a very active toxic compound for cancer cell lines.138 6.4. Anti-inammatory Usnic acid extracted from U. barbata using supercritical uid method has shown anti-inammatory 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 mg mL1 which contains 2.4 mg mL1 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 21688 | RSC Adv., 2016, 6, 21672–21696 Anti-platelet activity Increases the activities of superoxide dismutase, glutathione and glutathione peroxidase and decreases malondialdehyde formation Inhibits lipid peroxidation and induces antioxidant enzymes 143 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 antiinammatory activity. Longissimone A showed potential antiinammatory activity in comparison to standard drugs with IC50 165.74 mg mL1.99 Usnic acid has been demonstrated to be a potent anti-inammatory agent.140 Lichen metabolites such as atranorin, diﬀractaic, and protolichesterinic acids were found to attenuate LTB4 biosynthesis in polymorphonuclear leukocytes, due to specic enzyme interaction rather than nonspecic redox mechanism.141 The phenolic compound longissimone A, isolated from U. longissima displayed antiinammatory 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. lipendula (UFE) against aatoxin 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 concentrationdependent manner (5 to 20 mg mL1).34 A concentrationdependent anti-mutagenic potential of usnic acid ligands (L1, L2 and L3) and their complexes were examined for the rst 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 This journal is © The Royal Society of Chemistry 2016 Review RSC Advances Table 4 Pharmacological properties of chemical constituents isolated from Usnea spp. S. No. Extract/compound (a) In vitro studies 1 Quercetin 2 Isodivaricatic acid 3 Usnic acid 4 Usnic and diﬀractaic acids Source Bioactivity Anti-fungal U. longissima Target/system Candida albicans Mode of action Suppressor of biolm formation and hyphal formation U. orida Anti-fungal Microsporum gypseum, Inhibits growth of Trichophyton mentagrophytes and human pathogenic T. rubrum, C. albicans, C. tropicalis, fungal pathogens Saccharomyces cerevisiae, Aspergillus niger, A. avus and A. fumigates Anti-bacterial E. aerogenes, B. brevis, M. luteus, E. Increases the synthesis U. longissima coli, B. megaterium, P. aeruginosa, of some novel multifunctional E. cloacae, S. aureus, C. albicans hydroxyphenylimino and S. cerevisiae ligands (L1, L2 and L3) U. Anti-proliferative UACC-62 and B16-F10 melanoma Increases genetic subcavata cells damage in the cell lines Dose References 0.2–1.0 mg mL1 28 50–100 mg mL1 111 0.25–2 mg mL1 112 IC50: 24.7– 130 36.6 mg mL1 (UACC-62) and 25.4 mg mL1 (B16F10) 5 Usnic, psoromic acids U. Anti-oxidant In vitro system Scavenges free radicals IC50: 22.86 to 19 25.0 mg mL1 complanata Inhibits the migration 5.0 mM 132 6 Heptaketides, U. Anti-cancer Human metastatic breast and corynesporol, 1cavernosa prostate cancer cell lines including of cancer cells hydroxydehydroherbarin MDA-MB-231 and PC-3M MDAMB-231 and PC-3M 7 Heptaketides, Anti-cancer Human metastatic breast and U. 132 Inhibits the migration 5.0 mM corynesporol, 1cavernosa prostate cancer cell lines including of cancer cells hydroxydehydroherbarin MDA-MB-231 and PC-3M MDAMB-231 and PC-3M 8 L-Usnic, D-usnic acids U. barbata Anti-proliferative Leukemic cells (K-562) and Reduces cell viability 50 mg mL1 135,136 endometrial carcinoma cells (HEC-50) 9 Usnic acid U. Anti-helicobacter Helicobacter pylori MIC: 0.128–2 118 dasypoga pylori mg mL1 Anti-mutagenic S. typhimurium TA1535, TA1537 Prevents mutation 20–100 mg 112 10 Usnic acid U. longissima and E. coli WP2uvrA per plate Cytotoxic P388 cells Inhibits cell viability 4.5 mg mL1 138 11 Eumitrin A1 U. blepharea 12 Ambewelamide A, B Usnea sp. Cytotoxic Cancer cell lines Reduces cell viability — 106 >1 mM 131 U. Antitumor Tissue culture Inhibits tumor 13 Barbatic acid, 4-Opromoter-induced demethylbarbatic acid, longissima Epstein–Barr virus diﬀractaic acid activation Cytotoxic UACC-62 and B16-F10 melanoma Decreases cell viability 24.7 to 36.6 130 U. 14 Diﬀractaic, usnic, subcavata cells and 3T3 normal cells mg mL1 norstictic, psoromic acids Antimycobacterial Mycobacterium tuberculosis Inhibits the growth of 15.5–125 mg 186 U. 15 Diﬀractaic, norstictic, bacterium mL1 subcavata usnic, hypostictic, protocetraric acids 187 Scavenges free radicals — U. Antioxidant, DPPH radical system Gram16 Diﬀractaic, norstictic, and inhibits the growth longissima antimicrobial positive and Gram-negative usnic, hypostictic, of pathogenic microbes bacteria and fungi protocetraric acids Antibacterial pro- Bacillus licheniformis Inhibits the growth of 0.005–0.01% 187 17 Usnic acid U. ghattensis apoptotic bacteria >1 mM 131 19 Evernic acid U. Antitumor Tissue culture Inhibits tumor longissima promoter-induced Epstein–Barr virus activation 20 Galbinic acid U. undulata Antimicrobial B. cereus, B. subtilis, S. epidermidis Shows inhibitory eﬀect 31–62.5 mg 176 mL1 This journal is © The Royal Society of Chemistry 2016 RSC Adv., 2016, 6, 21672–21696 | 21689 RSC Advances Review Table 4 (Contd. ) S. No. Extract/compound 21 Gautinol 22 Hirtusneanoside 23 20 -O-Methyl hypostictic acid 24 Psoromic acid 25 Norstictic acid 26 Usnic acid 27 Methyl b-orsellinate (b) In vivo studies 1 Diﬀractaic acid 2 Usnic acid 3 Ambewelamide A 4 Diﬀractaic acid 5 Diﬀractaic, usnic acids Source Bioactivity U. Antilongissima inammatory, cytotoxic U. hirta Antibacterial U. undulata Antimicrobial U. Antioxidant, camplanata cardiovascular protective U. undulata Antimicrobial AntiU. longissima inammatory U. undulata Antibacterial U. Pro-apoptotic longissima Usnea species Usnea species U. longissima U. diﬀracta Target/system Mode of action Dose Spectroscopic model system Reduces cell viability 200 mg mL1 99 Gram-positive bacteriaS Shows growth — inhibitory activity B. cereus, B. subtilis, S. epidermidis Shows inhibitory eﬀect 31–62.5 mg mL1 In vitro system Scavenges free radicals 0.174–0.271 and inhibits lipid mg mL1 peroxidation B. cereus, B. subtilis, S. epidermidis Shows inhibitory eﬀect 31–62.5 mg mL1 IC50: 12.8 mM Decreases the TNFLPS-stimulated RAW264.7 alpha level macrophages B. cereus, B. subtilis, S. epidermidis Shows inhibitory eﬀect 31–62.5 mg mL1 Rabbits Anti-genotoxicity Mice Cytotoxicity Swiss mice and V79 cells Hepatoprotective Mice Analgesic, antipyretic 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 mL1. 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 signicant inhibition of thrombotic death or paralysis at 100–200 mg kg1 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 21690 | RSC Adv., 2016, 6, 21672–21696 Induces levels of antioxidant enzymes — Mice 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 Activates the expressions of initiator caspases (Cas-2, -8 and -9) and executioner caspase (Cas-3) Modulation of enzyme activity (ALT and AST) Reduces cell growth 30 mg kg1 References 103 176 20 97 140 97 133 142 60–120 mg mL1 8.6 ng mL1 106 50 mg kg1 148 500–1 g kg1 109 6.7. Others Many other biological activities for the Usnea species have also been reported. Diﬀractaic and usnic acids of U. diﬀracta were identied as analgesic and antipyretic components in mice.109 Halici et al. conrmed the potential gastroprotective eﬀect of the aqueous extract of U. longissima against indomethacininduced 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 signicant eﬀects at 500 mg kg1 and 1 g kg1, while usnic acid was found to be eﬀective at 100 mg kg1.144 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 signicant antiulcerogenic activity compared to the negative control groups in a dose-dependent manner. The highest activity (79.8%) was observed with 100 mg kg1 body. It was associated with the inhibition of oxidative damage and neutrophil inltration. This journal is © The Royal Society of Chemistry 2016 Review 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 kg1 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 kg1 daily but at high dose (100 and 200 mg kg1) 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 kg1 and 21.02 g kg1, respectively aer intraperitoneal administration in rats. LD50 values of 7.43 g kg1 for U. barbata and 4.52 g kg1 for U. hirta were recorded aer 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 rst 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 mM for ()-usnic acid, 90.8 mM for (+)-usnic acid and 111.0 mM 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 kg1 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 mM usnic acid for 16 h This journal is © The Royal Society of Chemistry 2016 RSC Advances 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 kg1 d1 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 scientic 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, ues, urinary tract, kidney, and bladder infections. Usnea is also benecial 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 signicantly 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, antiproliferative, anti-oxidant, anti-inammatory, anti-ulcer, hepatoprotective, and anti-genotoxicity etc. To a certain extent, pharmacological results have shown that (a) traditional uses for the treatment of ues, gastroenteritis and bacterial/fungal infections, and strep throat were related to antimicrobial activities; (b) use for wounds, ulcers, and fevers were associated with anti-inammatory 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 ndings indicated that depsides, depsidones, and benzofurans are key phytochemicals for the treatment of microbial infections, oxidative stress, cancer, ulcer, and inammation. It RSC Adv., 2016, 6, 21672–21696 | 21691 RSC Advances 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 antilipidperoxidative 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 conrm 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 signicant 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 lamentous 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 nancially supported by Twelh Five-year Plan program (BSC-0106) sponsored by the Council of Scientic 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- 21692 | RSC Adv., 2016, 6, 21672–21696 Review National Botanical Research Institute, Lucknow, India for his support and encouragement. 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