Usnea aurantiaco-atra (Jacq) Bory: Metabolites and Biological Activities.

BACKGROUND: Lichens are complex symbiotic associations between a fungus and an alga or cyanobacterium. Due to their great adaptability to the environment, they have managed to colonize many terrestrial habitats, presenting a worldwide distribution from the poles to the tropical regions and from the plains to the highest mountains. In the flora of the Antarctic region, lichens stand out due to their variety and development and are a potential source of new bioactive compounds. METHODS: A phytochemical study of the Antarctic lichen Usnea aurantiaco-atra (Jacq) Bory was conducted with the intention of determining the most important metabolites. In addition, the cytotoxic and antioxidant activities of its extracts were determined. RESULTS: Cytotoxicity studies revealed that the hexane extract contains usnic acid as a majority metabolite, in addition to linoleic acid, ergosterols and terpenes, and demonstrates cytotoxic activity against an A375 melanoma cell line. On the other hand, the presence of total phenols in the extracts did not influence their antioxidant activity. CONCLUSIONS: U. aurantiaco-atra contains mainly usnic acid, although there are terpenes and ergosta compounds that could be responsible for its cytotoxic activity. The presence of phenols did not confer antioxidant properties.

ROS-Induced DNA-Damage and Autophagy in Oral Squamous Cell Carcinoma by Usnea barbata Oil Extract-An In Vitro Study.

Oxidative stress is associated with aging, cancers, and numerous metabolic and chronic disorders, and phenolic compounds are well known for their health-promoting role due to their free-radical scavenging activity. These phytochemicals could also exhibit pro-oxidant effects. Due to its bioactive phenolic secondary metabolites, Usnea barbata (L.) Weber ex. F.H. Wigg (U. barbata) displays anticancer and antioxidant activities and has been used as a phytomedicine for thousands of years. The present work aims to analyze the properties of U. barbata extract in canola oil (UBO). The UBO cytotoxicity on oral squamous cell carcinoma (OSCC) CLS-354 cell line and blood cell cultures was explored through complex flow cytometry analyses regarding apoptosis, reactive oxygen species (ROS) levels, the enzymatic activity of caspase 3/7, cell cycle, nuclear shrinkage (NS), autophagy (A), and synthesis of deoxyribonucleic acid (DNA). All these studies were concomitantly performed on canola oil (CNO) to evidence the interaction of lichen metabolites with the constituents of this green solvent used for extraction. The obtained data evidenced that UBO inhibited CLS-354 oral cancer cell proliferation through ROS generation (316.67 × 104), determining higher levels of nuclear shrinkage (40.12%), cell cycle arrest in G0/G1 (92.51%; G0 is the differentiation phase, while during G1 phase occurs preparation for cell division), DNA fragmentation (2.97%), and autophagy (62.98%) than in blood cells. At a substantially higher ROS level in blood cells (5250.00 × 104), the processes that lead to cell death-NS (30.05%), cell cycle arrest in G0/G1 (86.30%), DNA fragmentation (0.72%), and autophagy (39.37%)-are considerably lower than in CLS-354 oral cancer cells. Our work reveals the ROS-mediated anticancer potential of UBO through DNA damage and autophagy. Moreover, the present study suggests that UBO pharmacological potential could result from the synergism between lichen secondary metabolites and canola oil phytoconstituents.

Anticancer Activity of 2-O-caffeoyl Alphitolic Acid Extracted from the Lichen, Usnea barbata 2017-KL-10.

Colorectal cancer is one of the life-threatening ailments causing high mortality and morbidity worldwide. Despite the innovation in medical genetics, the prognosis for metastatic colorectal cancer in patients remains unsatisfactory. Recently, lichens have attracted the attention of researchers in the search for targets to fight against cancer. Lichens are considered mines of thousands of metabolites. Researchers have reported that lichen-derived metabolites demonstrated biological effects, such as anticancer, antiviral, anti-inflammatory, antibacterial, analgesic, antipyretic, antiproliferative, and cytotoxic, on various cell lines. However, the exploration of the biological activities of lichens' metabolites is limited. Thus, the main objective of our study was to evaluate the anticancer effect of secondary metabolites isolated from lichen (Usnea barbata 2017-KL-10) on the human colorectal cancer cell line HCT116. In this study, 2OCAA exhibited concentration-dependent anticancer activities by suppressing antiapoptotic genes, such as MCL-1, and inducing apoptotic genes, such as BAX, TP53, and CDKN1A(p21). Moreover, 2OCAA inhibited the migration and invasion of colorectal cancer cells in a concentration-dependent manner. Taken together, these data suggest that 2OCAA is a better therapeutic candidate for colorectal cancer.

Evaluation of the Cytotoxic Activity of the Usnea barbata (L.) F. H. Wigg Dry Extract.

The secondary metabolites of lichens have proven to be promising sources of anticancer drugs; one of the most important of these is usnic acid, which is a phenolic compound with dibenzofuran structure that is responsible for the numerous biological actions of lichens of genus Usnea. As a result, in this study, we related to this phenolic secondary metabolite. The aim of the present study is the evaluation of the cytotoxic activity of Usnea barbata (L.) F. H. Wigg dry acetone extract (UBE). In advance, the usnic acid content was determined in various extracts of Usnea barbata (L.) F. H. Wigg: the liquid extracts were found in water, ethanol, acetone, and the dry acetone extract; the highest usnic acid quantity was found in the dry acetone extract. First, the cytotoxic action of UBE was assessed using Brine Shrimp Lethality (BSL) test; a significant lethal effect was obtained after 24 h of treatment at high used concentrations of UBE, and it was quantified by the high mortality rate of the Artemia salina (L.) larvae. Secondly, in vitro cytotoxicity of UBE was evaluated on human tongue squamous cells carcinoma, using CAL 27 (ATCC® CRL-2095™) cell line. The most intense cytotoxic effect of UBE on CAL 27 cells was registered after 24 h; this response is directly proportional with the tested UBE concentrations. The obtained results have been reported regarding usnic acid content of UBE, and the data show that CAL 27 cells death was induced by apoptosis and high oxidative stress.

Trace elements baseline levels in Usnea antarctica from Clearwater Mesa, James Ross Island, Antarctica.

Clearwater Mesa is a pristine area located SE of Croft Bay in James Ross Island, at the northeast of the Antarctic Peninsula. The closest anthropic construction is the summer Mendel station located 25 km to the NE. Clearwater is a remote basalt volcanic mesa, (64° 01' S, 57° 42' W) located at ~ 250 m a.s.l., with around 50 small shallow lakes, a well-developed lichens flora and scarce fauna. The mesa offers a good opportunity to use lichens, like Usnea antarctica, as a biomonitor species. The aim of this study was to determine the baseline levels of trace elements in Usnea antarctica and their relationships with geographical parameters and climatological aspects, to use them as reference levels in futures studies. Usnea antarctica was collected in 23 sites in two transects: perpendicular and parallel to the shore. We found high contents of the elements associated with long-distance transport like Sm, La, Sc, Fe, Co, Hg, and Ca. The elements linked to the marine biogenic cycle as Sr, Rb, Cs, K, Na, Sb, As, Se, and Br were less significant. The distance to the sea was the factor that related the element contents. Hg, Se, As, and Br content in Usnea antarctica increased from the inland towards the sea coast.

New insights into the Usnea cornuta aggregate (Parmeliaceae, lichenized Ascomycota): Molecular analysis reveals high genetic diversity correlated with chemistry.

Biological processes such as hybridization, incomplete lineage sorting and gene flow can obscure the recognition of distinct evolutionary lineages, particularly in groups of organisms that have recently diverged. Therefore, compiling pieces of evidence from diverse data sources is critical to accurately assess species boundaries in such groups. The increasing availability of DNA sequence data allows for a much deeper understanding of diversification and speciation processes and their consequences on biodiversity. In this study, we applied an integrative approach based on DNA sequence, chemical, geographic and morphological data to attempt to define species boundaries in the lichen-forming genus Usnea (Parmeliaceae), particularly the U. cornuta aggregate, a cosmopolitan species group. We provide the first species delimitation for this group in the neotropics based on the multispecies coalescent (MSC) model. Using ITS rDNA and two protein-coding genes, Mcm7 and RPB1, we estimated the species tree under the MSC model in a Bayesian framework using STACEY. Our results indicate that at least nine strongly supported distinct lineages coexist in the U. cornuta aggregate, which are well chemically characterized. Additionally, we found evidence for the polyphyly of three morphospecies, Usnea brasiliensis, U. cornuta and U. dasaea.

Therapeutic effects of beard lichen, Usnea barbata extract against Lactococcus garvieae infection in rainbow trout (Oncorhynchus mykiss).

In this study, therapeutic effects of aqueous methanolic extracts of beard lichen (Usnea barbata) against Lactococcus garvieae (ATCC 43921) in rainbow trout (Onchorhynchus mykiss) were investigated. Six different experimental groups [0 mg/100 µL (Control), 4 mg/100 µL, 8 mg/100 µL, 12 mg/100 µL, 6 mg/100 µL florfenicol (positive control), 6 mg/100 µL erythromycin (positive control)] were set up to determine effects of lichen extract on immune responses and survival rate. In the study, superoxide radical production was increased in fish treated with 12 mg beard lichen extract, florfenicol and erythromycin compared to that of control (P < 0.05). Lysozyme activity was generally decreased (P < 0.05) or no differences were observed in all experimental groups compared to that of control (P > 0.05). Myeloperoxidase was significantly increased in all antibiotic treated groups. No differences were observed in liver histology of experimental groups compared to control. Cytokine gene expressions were elevated in all experimental groups compared to that of control (P < 0.05), except IL-1ß expression at 10th day sampling time. Other immune related genes (IL-8, TGF- ß, IL-12 Beta, TNFα1, IL-10, COX-2, IL-6, TLR5, C3, IGM, MHC-II, iNOS, IgT, IFN1, IFN2, IFN reg) were also elevated in all experimental groups compared to that of control group. The survival rates obtained in 4 mg beard lichen treated group, 8 mg beard lichen treated group and erythromycin treated group were 73.08, 65.38 and 80.77% respectively. Our results suggest that beard lichen methanolic extract could be an effective therapeutic agent to be used against L. garvieae infection in rainbow trout at the dose of 4 mg/17.41 ±â€¯0.3 g body weight/day.

Inhibition of growth of U87MG human glioblastoma cells by Usnea longissima Ach.

Herbal medicines are efficient to reduce side effects in the fight against glioblastoma, which plays a critical role within brain cancer species. The recent studies designated for testing the effects of lichens that have shown numerous anticancer activities on glioblastoma so far. In the present study, different concentrations of water extract obtained from Usnea longissima Ach. were used in order to determine cytotoxic (via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and lactate dehydrogenase tests), antioxidant (via total antioxidant capacity test), pro-oxidant (via total oxidant status test) and genotoxic (via 8-hydroxy-2'-deoxyguanosine test) effects of them on human U87MG-glioblastoma cancer cell lines. Primary mixed glial-neuronal non-cancerous cells from Sprague-Dawley rats were also utilized to measure the effects of treatments on non-cancerous cells. Based on median inhibitory concentration values, the data belonged to non-cancerous cells (2486.71 mg/L) showed distinct towering compared to U87MG (80.93 mg/L) cells. The viability of non-cancerous and U87MG cells exposed to extract is decreased in a dose dependent manner. It was also showed that low concentrations of extract notably increased total antioxidant capacity on non-cancerous cells. In addition, various phenolic compounds in extract were detected through high-performance liquid chromatography. The recent results encourage that extract will be able to have therapeutic potential against glioblastoma.

Usnic Acid Activity on Oxidative and Nitrosative Stress of Azole-Resistant Candida albicans Biofilm.

Several studies report that (+)-usnic acid, a lichen secondary metabolite, inhibits growth of different bacteria and fungi; however, the mechanism of its antimicrobial activity remains unknown. In this study, we explored the ability of usnic acid, obtained from Usnea amblyoclada, as an antibiofilm agent against azole-resistant and azole-sensitive Candida albicans strains by studying the cellular stress and antioxidant response in biofilms. The biofilm inhibitory concentration of usnic acid (4 µg/mL) exhibited a significant biofilm inhibition, 71.08 % for azole-resistant and 87.84 % for azole-sensitive C. albicans strains. Confocal scanning laser microscopy showed that the morphology of mature biofilm was altered (reduced the biomass and thickness) in the presence of usnic acid. The antifungal effect was mediated by an oxidative and nitrosative stress, with a significant accumulation of intracellular and extracellular reactive oxygen species detected by confocal scanning laser microscopy and by nitro blue tetrazolium, respectively. In fact, azole-resistant and azole-sensitive C. albicans biofilms treated at the biofilm inhibitory concentration of usnic acid presented 30-fold and 10-fold increased reactive oxygen species measurements compared to basal levels, respectively, and important nitric oxide generation, showing 25-fold and 60-fold increased reactive nitrogen intermediates levels with respect to the controls, respectively. Nonenzymatic and enzymatic antioxidant defenses were increased in both strains compared to biofilm basal levels as response to the increase of oxidant metabolites. The present study shows for the first time that usnic acid can alter the prooxidant-antioxidant balance, which may be the cause of the irreversible cell damage and lead to cell death. Our results suggest that usnic acid could be an alternative for the treatment of Candida infections, which deserves further investigation.

Secondary Metabolite Profiling of Species of the Genus Usnea by UHPLC-ESI-OT-MS-MS.

Lichens are symbiotic associations of fungi with microalgae and/or cyanobacteria, which are considered among the slowest growing organisms, with strong tolerance to adverse environmental conditions. There are about 400 genera and 1600 species of lichens and those belonging to the Usnea genus comprise about 360 of these species. Usnea lichens have been used since ancient times as dyes, cosmetics, preservatives, deodorants and folk medicines. The phytochemistry of the Usnea genus includes more than 60 compounds which belong to the following classes: depsides, depsidones, depsones, lactones, quinones, phenolics, polysaccharides, fatty acids and dibenzofurans. Due to scarce knowledge of metabolomic profiles of Usnea species (U. barbata, U. antarctica, U. rubicunda and U. subfloridana), a study based on UHPLC-ESI-OT-MS-MS was performed for a comprehensive characterization of their secondary metabolites. From the methanolic extracts of these species a total of 73 metabolites were identified for the first time using this hyphenated technique, including 34 compounds in U. barbata, 21 in U. antarctica, 38 in U. rubicunda and 37 in U. subfloridana. Besides, a total of 13 metabolites were not identified and reported so far, and could be new according to our data analysis. This study showed that this hyphenated technique is rapid, effective and accurate for phytochemical identification of lichen metabolites and the data collected could be useful for chemotaxonomic studies.

The contents and distributions of cadmium, mercury, and lead in Usnea antarctica lichens from Solorina Valley, James Ross Island (Antarctica).

Lichens are efficient and cost-effective biomonitors of the environment. Their geographic distribution together with their slow growth rate enable investigation of the deposition patterns of various elements and substances. In this research, levels of cadmium, lead, and mercury in Usnea antarctica lichens in the area of James Ross Island, Antarctica, were investigated. The lichens were microwave-digested, and the metals were determined by means of atomic absorption spectrometry with graphite furnace and a direct mercury analyzer. Median total contents of Cd, Hg, and Pb were 0.04, 0.47, and 1.6 mg/kg in whole lichens, respectively. The bottom-up distributions of these metals in the fruticose lichen thalli were investigated, and it was revealed that the accumulation patterns for mercury and lead were opposite to that for cadmium. The probable reason for this phenomenon may lie in the inner structure of thalli. The total contents of metals were comparable with those published for other unpolluted areas of maritime Antarctica. However, this finding was not expected for mercury, since the sampling locality was close to an area with some of the highest mercury contents published for Antarctic lichens. In short, lichens proved their usability as biological monitors, even in harsh conditions. However, the findings emphasize the need to take into account the distributions of elements both in the environment and in the lichen itself.

In vivo anti-inflammatory and anti-platelet aggregation activities of longissiminone A, isolated from Usnea longissima.

Secondary metabolite, longissiminone A (1) was isolated from a lichen, Usnea longissima. It was screened for its' in vivo anti-inflammatroy and anti-platelet aggregation activities. Compound 1 showed moderate in vivo anti-inflammatory activity as well as moderately active against the aggregation induced by arachidonic acid at different doses.

Usnic Acid Derivatives with Cytotoxic and Antifungal Activities from the Lichen Usnea longissima.

Eight usnic acid derivatives, that is, usenamines A-F (1-6), usone (7), and isousone (8), together with the known (+)-usnic acid (9), were isolated from the lichen Usnea longissima. Their structures were elucidated using 1D and 2D NMR and MS data, and the absolute configurations of compounds 1 and 2 were defined by single-crystal X-ray diffraction analyses. Compounds 1, 2, and 8 showed inhibitory effects on the growth of human hepatoma HepG2 cells with IC50 values of 6.0-53.3 µM compared with methotrexate as the positive control, which had an IC50 value of 15.8 µM. Furthermore, 1 induced apoptosis of HepG2 cells in a dose-dependent manner at concentrations of 0-15.0 µM. The isolated compounds were also evaluated for their antifungal and antibacterial activities, with 7 and 8 exhibiting weak inhibitory effects on fungal Trichophyton rubrum spp. with an MIC value of 41.0 µM.

Effects of (+)-usnic acid and (+)-usnic acid-liposome on Toxoplasma gondii.

Toxoplasma gondii pathogen is a threat to human health that results in economic burden. Unfortunately, there are very few high-efficiency and low-toxicity drugs for toxoplasmosis in the clinic. (+)-Usnic acid derived from lichen species has been reported to have anti-inflammatory, antibacterial, anti-parasitology, and even anti-cancer activities. Herein, the systematic effect of (+)-usnic acid and (+)-usnic acid-liposome on toxoplasma were studied in vitro and in vivo. The viability of toxoplasma tachyzoite was assayed with trypan blue and Giemsa staining; while the invasive capability of tachyzoite to cardiofibroblasts was detected using Giemsa staining. The survival time of mice and the changes in tachyzoite ultrastructure were studied in vivo. The results showed that (+)-usnic acid inhibited the viability of tachyzoite; pretreatment with (+)-usnic acid significantly decreased the invasion of tachyzoite to cardiofibroblasts in vitro; (+)-usnic acid and (+)-usnic acid-liposome extensively prolonged the survival time of mice about 90.9% and 117%, respectively; and improved the ultrastructural changes of tachyzoite, especially in dense granules, rhoptries, endoplasmic reticulum, mitochondria and other membrane organelles. In summary, these results demonstrate that (+)-usnic acid and (+)-usnic acid-liposome with low toxicity have an inhibitory effect on the viability of toxoplasma tachyzoite, and mainly destructed membrane organelles which are connected with the virulence of toxoplasma. These findings provide the basis for further study and development of usnic acid as a potential agent for treating toxoplasmosis.

Norstictic Acid Inhibits Breast Cancer Cell Proliferation, Migration, Invasion, and In Vivo Invasive Growth Through Targeting C-Met.

Breast cancer is a major health problem affecting the female population worldwide. The triple-negative breast cancers (TNBCs) are characterized by malignant phenotypes, worse patient outcomes, poorest prognosis, and highest mortality rates. The proto-oncogenic receptor tyrosine kinase c-Met is usually dysregulated in TNBCs, contributing to their oncogenesis, tumor progression, and aggressive cellular invasiveness that is strongly linked to tumor metastasis. Therefore, c-Met is proposed as a promising candidate target for the control of TNBCs. Lichens-derived metabolites are characterized by their structural diversity, complexity, and novelty. The chemical space of lichen-derived metabolites has been extensively investigated, albeit their biological space is still not fully explored. The anticancer-guided fractionation of Usnea strigosa (Ach.) lichen extract led to the identification of the depsidone-derived norstictic acid as a novel bioactive hit against breast cancer cell lines. Norstictic acid significantly suppressed the TNBC MDA-MB-231 cell proliferation, migration, and invasion, with minimal toxicity to non-tumorigenic MCF-10A mammary epithelial cells. Molecular modeling, Z'-LYTE biochemical kinase assay and Western blot analysis identified c-Met as a potential macromolecular target. Norstictic acid treatment significantly suppressed MDA-MB-231/GFP tumor growth of a breast cancer xenograft model in athymic nude mice. Lichen-derived natural products are promising resources to discover novel c-Met inhibitors useful to control TNBCs.

[Phenolic constituents from lichen Usnea longissima].

Sixteen compounds were isolated from lichen Usnea longissima using of various chromatographic techniques including silica gel, Sephadex LH-20, ODS, and semi-preparative HPLC. By spectroscopic data analyses, their structures were identified by as useanol(1), lecanorin(2), 3-hydroxy-5-methylphenyl 2-hydroxy-4-methoxy-6-methylbenzoate(3), lecanorin E(4), 3'-methylevernic acid(5), evernic acid(6), barbatinic acid(7), 3,7-dihydroxy-1,9-dimethyldibenzofuran(8), orcinol(9), O-methylorcinol(10), methyl orsellinate(11), methyl everninate(12), 2,5-dimethyl-1,3-benzenediol(13), 2-hydroxy-4-methoxy-3,6-dimethyl benzoic acid(14), ethyl everninate(15), and ethyl 2,4-dihydroxy-6-methylbenzoate(16). Compound 1 was obtained as a natural product for the first time, and 3,4, 8,10,12, and 13 were isolated from Usneaceae family for the first time. Compound 1, 8, and 13 showed significant anti-inflammatory activity against NO production in RAW 267.4 cells with IC50 values of 6.8, 3.9 and 4.8 µmol•L⁻¹, respectively, compared with the positive controls curcumin(IC50 15.3 µmol•L⁻¹) and indomethacin(IC50 42.9 µmol•L⁻¹).

Quercetin sensitizes fluconazole-resistant candida albicans to induce apoptotic cell death by modulating quorum sensing.

Quorum sensing (QS) regulates group behaviors of Candida albicans such as biofilm, hyphal growth, and virulence factors. The sesquiterpene alcohol farnesol, a QS molecule produced by C. albicans, is known to regulate the expression of virulence weapons of this fungus. Fluconazole (FCZ) is a broad-spectrum antifungal drug that is used for the treatment of C. albicans infections. While FCZ can be cytotoxic at high concentrations, our results show that at much lower concentrations, quercetin (QC), a dietary flavonoid isolated from an edible lichen (Usnea longissima), can be implemented as a sensitizing agent for FCZ-resistant C. albicans NBC099, enhancing the efficacy of FCZ. QC enhanced FCZ-mediated cell killing of NBC099 and also induced cell death. These experiments indicated that the combined application of both drugs was FCZ dose dependent rather than QC dose dependent. In addition, we found that QC strongly suppressed the production of virulence weapons-biofilm formation, hyphal development, phospholipase, proteinase, esterase, and hemolytic activity. Treatment with QC also increased FCZ-mediated cell death in NBC099 biofilms. Interestingly, we also found that QC enhances the anticandidal activity of FCZ by inducing apoptotic cell death. We have also established that this sensitization is reliant on the farnesol response generated by QC. Molecular docking studies also support this conclusion and suggest that QC can form hydrogen bonds with Gln969, Thr1105, Ser1108, Arg1109, Asn1110, and Gly1061 in the ATP binding pocket of adenylate cyclase. Thus, this QS-mediated combined sensitizer (QC)-anticandidal agent (FCZ) strategy may be a novel way to enhance the efficacy of FCZ-based therapy of C. albicans infections.

Genotoxic and antigenotoxic potentials of two Usnea species.

For ages, lichens have long been investigated popularly for biological roles, mainly antitumor, antimicrobial and antioxidant activities. Many positive results were obtained in these previous research. Thus, in this study, we aimed to determine whether extracts of Usnea articulata (UAE) and Usnea filipendula (UFE) possessing a protection against aflatoxin B1 (AFB1)-induced genotoxic and oxidative damage. The results of our studies showed that 5 µM concentrations of AFB1 increased the frequencies of sister chromatid exchange (SCE) and the level of malondialdehyde (MDA) and decreased the activities of superoxide dismutase (SOD), glutathione (GSH) and glutathione peroxidase (GPx). However, when 5, 10 and 20 µg/mL concentrations of UAE and UFE was added to AFB1, the frequencies of SCE and MDA level were decreased and SOD, GSH and GPx level were increased. The Ames (Salmonella typhimurium TA1535, TA1537) and WP2 (Escherichia coli) test systems carried out evinced that UAE and UFE possess any mutagenicity, but have antimutagenic effects. Consequently, the results of this experiment have clearly shown that UAE and UFE have strong antioxidative and antigenotoxic effects that are associated with its antioxidant nature. A detailed study can be performed to determine the antioxidant properties of each compound that will extend the use of lichen extracts in food and pharmacy industries.

[Chemical Constituents from Usnea longgisima, a Traditional Mongolian Medicine(II)].

OBJECTIVE: To study the chemical constituents of traditional Mongolian medicine Usnea longissima. METHODS: The compounds were isolated and purified by the methods of solvent extraction and chromatographic technique, and their structures were identified on the basis of the analyses of spectral data. RESULTS: Three compounds were obtained and identified as 4-hydroxy-2-[ (2-hydroxy-4-methoxy-6-methylbenzoyl) oxy]-6-methylbenzoic acid (1), dibutyl phthalate (2) and diisobutyl phthalate (3). CONCLUSION: Compound 1 is a new compound and named as isoevernic acid, compounds 2 and 3 are isolated from Usnea longissima for the first time.

[Simultaneous determination of 3 phenolic acids in Usnea by HPLC-ESI-MS/MS].

A quick HPLC-ESI-MS/MS method was established for simultaneous determination of three chemical compositions in Usnea, including usnic acid, diffractaic acid, and ramalic acid. The separation was performed on a chromatographic column of Agilent ZORBAX SB-C, (4.6 mm x 250 mm, 5 µm), and the mobile phase was methanol (0.05% formic acid)-0.05% formic acid solution (4 mmol ammonium acetate), with an isocratic elution at a flow rate of 0.8 ml · min⁻¹. Multiple reaction monitoring scanning mode (MRM) was performed combined with the ion switching technology in positive and negative ion switching mode to apply for the quantitative determination. The calibration curves for the above three compounds were linear in corresponding injection amount. Their average recoveries were 95.0%-105.1%, with RSDs of 1.1%-5.2%. The method was simple, rapid, accurate with high repeatability, which could provide a reference for overcalling evaluation the quality of Usnea.