Non-photochemical quenching may contribute to the dominance of the pale mat-forming lichen Cladonia stellaris over the sympatric melanic Cetraria islandica.

The mat-forming fruticose lichens Cladonia stellaris and Cetraria islandica frequently co-occur on soils in sun-exposed boreal, subarctic, and alpine ecosystems. While the dominant reindeer lichen Cladonia lacks a cortex but produces the light-reflecting pale pigment usnic acid on its surface, the common but patchier Cetraria has a firm cortex sealed by the light-absorbing pigment melanin. By measuring reflectance spectra, high-light tolerance, photosynthetic responses, and chlorophyll fluorescence in sympatric populations of these lichens differing in fungal pigments, we aimed to study how they cope with high light while hydrated. Specimens of the two species tolerated high light equally well but with different protective mechanisms. The mycobiont of the melanic species efficiently absorbed excess light, consistent with a lower need for its photobiont to protect itself by non-photochemical quenching (NPQ). By contrast, usnic acid screened light at 450-700 nm by reflectance and absorbed shorter wavelengths. The ecorticate usnic species with less efficient fungal light screening exhibited a consistently lower light compensation point and higher CO2 uptake rates than the melanic lichen. In both species, steady state NPQ rapidly increased at increasing light with no signs of light saturation. To compensate for less internal shading causing light fluctuations with a larger amplitude, the usnic lichen photobiont adjusted to changing light by faster induction and faster relaxation of NPQ rapidly transforming excess excitation energy to less damaging heat. The high and flexible NPQ tracking fluctuations in solar radiation probably contributes to the strong dominance of the usnic mat-forming Cladonia in open lichen-dominated heaths.

Usnic acid attenuates 7,12-dimethylbenz[a] anthracene (DMBA) induced oral carcinogenesis through inhibiting oxidative stress, inflammation, and cell proliferation in male golden Syrian hamster model.

In this study, we investigated the chemopreventive efficacy of usnic acid (UA), an effective secondary metabolite component of lichens, against 7,12-dimethylbenz[a]anthracene (DMBA)-induced oral squamous cell carcinoma (OSCC) in the hamster model. Initially, the buccal pouch carcinogenesis was induced by administering 0.5% DMBA to the HBP (hamster buccal pouch) region about three times a week until the 10th week. Then, UA was orally treated with different concentrations (25, 50, 100 mg/kg b.wt) on alternative days of DMBA exposure, and the experimental process ended in the 16th week. After animal experimentation, we observed 100% tumor incidence with well-differentiated OSCC, dysplasia, and hyperplasia lesions in the DMBA-induced HBP region. Furthermore, the UA treatment of DMBA-induced hamster effectively inhibited tumor growth. In addition, UA upregulated antioxidant levels, interfered with the elevated lipid peroxidation by-product of thiobarbituric acid reactive substances, and changed the activities of the liver detoxification enzyme (Phase I and II) in DMBA-induced hamsters. Furthermore, immunohistochemical staining of inflammatory markers (iNOS and COX-2) and proliferative cell markers (cyclin-D1 and PCNA) were upregulated in the buccal pouch part of hamster animals induced with DMBA. Notably, the oral administration of UA significantly suppressed these markers during DMBA-induced hamsters. Collectively, our findings revealed that UA exhibits antioxidant, anti-inflammatory, antitumor, and apoptosis-inducing characteristics, demonstrating UA's protective properties against DMBA-induced HBP carcinogenesis.

Oxidative DNA damage contributes to usnic acid-induced toxicity in human induced pluripotent stem cell-derived hepatocytes.

Dietary supplements containing usnic acid have been increasingly marketed for weight loss over the past decades, even though incidences of severe hepatotoxicity and acute liver failure due to their overuse have been reported. To date, the toxic mechanism of usnic acid-induced liver injury at the molecular level still remains to be fully elucidated. Here, we conducted a transcriptomic study on usnic acid using a novel in vitro hepatotoxicity model employing human induced pluripotent stem cell (iPSC)-derived hepatocytes. Treatment with 20 µM usnic acid for 24 h caused 4272 differentially expressed genes (DEGs) in the cells. Ingenuity Pathway Analysis (IPA) based on the DEGs and gene set enrichment analysis (GSEA) using the whole transcriptome expression data concordantly revealed several signaling pathways and biological processes that, when taken together, suggest that usnic acid caused oxidative stress and DNA damage in the cells, which further led to cell cycle arrest and eventually resulted in cell death through apoptosis. These transcriptomic findings were subsequently corroborated by a variety of cellular assays, including reactive oxygen species (ROS) generation and glutathione (GSH) depletion, DNA damage (pH2AX detection and 8-hydroxy-2'-deoxyguanosine [8-OH-dg] assay), cell cycle analysis, and caspase 3/7 activity. Collectively, the results of the current study accord with previous in vivo and in vitro findings, provide further evidence that oxidative stress-caused DNA damage contributes to usnic acid-induced hepatotoxicity, shed new light on molecular mechanisms of usnic acid-induced hepatotoxicity, and demonstrate the usefulness of iPSC-derived hepatocytes as an in vitro model for hepatotoxicity testing and prediction.

Enhancement of the Antitumor and Antimetastatic Effect of Topotecan and Normalization of Blood Counts in Mice with Lewis Carcinoma by Tdp1 Inhibitors-New Usnic Acid Derivatives.

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is an important DNA repair enzyme and one of the causes of tumor resistance to topoisomerase 1 inhibitors such as topotecan. Inhibitors of this Tdp1 in combination with topotecan may improve the effectiveness of therapy. In this work, we synthesized usnic acid derivatives, which are hybrids of its known derivatives: tumor sensitizers to topotecan. New compounds inhibit Tdp1 in the micromolar and submicromolar concentration range; some of them enhance the effect of topotecan on the metabolic activity of cells of various lines according to the MTT test. One of the new compounds (compound 7) not only sensitizes Krebs-2 and Lewis carcinomas of mice to the action of topotecan, but also normalizes the state of the peripheral blood of mice, which is disturbed in the presence of a tumor. Thus, the synthesized substances may be the prototype of a new class of additional therapy for cancer.

The pyrazole derivative of usnic acid inhibits the proliferation of pancreatic cancer cells in vitro and in vivo.

BACKGROUND: Pancreatic cancer is one of the leading causes of cancer death in Western societies. Its late diagnosis and resistance to chemotherapies result in a high mortality rate; thus, the development of more effective therapies for the treatment of pancreatic cancer is strongly warranted. Usnic acid (UA) is a secondary metabolite of lichens that shows modest antiproliferative activity toward cancer cells. Recently, we reported the synthesis of a UA pyrazole derivative, named 5, which was more active than the parent compound toward cervical cancer cells. Here, its anticancer potential has been evaluated in detail in other cancer cells, particularly pancreatic cancer cells. METHODS: The impact of UA and derivative 5 on cell viability, morphology, cell cycle, and death was assessed using the MTT test, electron microscopy, flow cytometry, and immunoblotting, respectively. The calcium ions level was detected fluorometrically. In vivo, the anticancer activity of 5 was evaluated in a murine xenograft model. RESULTS: Derivative 5 inhibited the viability of different cancer cells. Noncancerous cells were less sensitive. It induced the release of calcium ions from the endoplasmic reticulum (ER) and ER stress, which was manifested by cell vacuolization. It was accompanied by G0/G1 cell cycle arrest and cell death of pancreatic cancer cells. When applied to nude mice with xenografted pancreatic cancer cells, 5 inhibited tumor growth, with no signs of kidney or liver toxicity. CONCLUSIONS: UA derivative 5 is superior to UA inhibiting the growth and proliferation of pancreatic cancer cells. ER stress exaggeration is a mechanism underlying the activity of derivative 5.

Aryl-n-hexanamide linked enaminones of usnic acid as promising antimicrobial agents.

Lichen secondary metabolites are well explored medicinal agents with diverse pharmacological properties. One of the important antibiotic lichen secondary metabolites is usnic acid. Its diverse medicinal profiles prompted us to explore it as a potential antitubercular molecule. Towards this direction, continuing our efforts on the discovery and development of new analogs with potent antitubercular properties we designed, synthesized, and evaluated a set of 37 usnic acid enaminone-coupled aryl-n-hexanamides (3-39). The study yielded a 3,4-dimethoxyphenyl compound (13, 5.3 µM) as the most active anti-TB molecule. The docking studies were performed on 7 different enzymes to better understand the binding modes, where it was observed that compound 13 bound strongly with glucose dehydrogenase (Gscore: - 9.03). Further antibacterial investigations revealed compound 2 with potent inhibition on Salmonella typhi and Bacillus subtilis (MIC 3 µM) and MIC values of 7 and 14 µM on Streptococcus mutans and Escherichia coli respectively. Compound 19 (3-F-5-CF3-phenyl) displayed encouraging antibacterial profiles against E. coli, S. typhi and S. mutans with MIC values of 10 µM respectively. Interestingly, compound 20 (2,6-difluorophenyl) also displayed good antibacterial activity against E. coli with an MIC value of 6 µM. These encouraging pharmacological results will help for better designing and developing usnic acid-based semi-synthetic derivatives as potential antimicrobial agents. A set of 37 new usnic acid enaminone-coupled aryl-n-hexanamides were synthesized and evaluated as potential antimicrobial agents. Compound 13 was identified as the most active antitubercular molecule. 13 was further docked against 7 different enzymes of tuberculosis. The molecule displayed maximum binding energy with the enzyme Glucose dehydrogenase (Gscore: - 9.03), indicating that these hexanamides possibly act by inhibiting the glucose metabolic pathway of the bacterium. Surprisingly, the intermediate hexanoic acid 2 was identified as potent antibacterial agent, acting on both gram-positive and gram-negative bacterial strains (3-14 µM). The active compounds may be subjected to structural iterations to develop further leads.

Electrospun Nanofibrous Mesh Based on PVA, Chitosan, and Usnic Acid for Applications in Wound Healing.

Injuries and diseases of the skin require accurate treatment using nontoxic and noninvasive biomaterials, which aim to mimic the natural structures of the body. There is a strong need to develop biodevices capable of accommodating nutrients and bioactive molecules and generating the process of vascularization. Electrospinning is a robust technique, as it can form fibrous structures for tissue engineering and wound dressings. The best way of forming such meshes for wound healing is to choose two polymers that complement each other regarding their properties. On the one hand, PVA is a water-soluble synthetic polymer widely used for the preparation of hydrogels in the field of biomedicine owing to its biocompatibility, water solubility, nontoxicity, and considerable mechanical properties. PVA is easy to subject to electrospinning and can offer strong mechanical stability of the mesh, but it is necessary to improve its biological properties. On the other hand, CS has good biological properties, including biodegradability, nontoxicity, biocompatibility, and antimicrobial properties. Still, it is harder to electrospin and does not possess as good mechanical properties as PVA. As these structures also allow the incorporation of bioactive agents due to their high surface-area-to-volume ratio, the interesting point was to incorporate usnic acid into the structure as it is a natural and suitable alternative agent for burn wounds treatment which avoids an improper or overuse of antibiotics and other invasive biomolecules. Thus, we report the fabrication of an electrospun nanofibrous mesh based on PVA, chitosan, and usnic acid with applications in wound healing. The obtained nanofibers mesh was physicochemically characterized by Fourier transform infrared spectroscopy (FT-IR) and scanning electron microscopy (SEM). In vitro biological assays were performed to evaluate the antimicrobial properties of the samples using the MIC (minimum inhibitory concentration) assay and evaluating the influence of fabricated meshes on the Staphylococcus aureus biofilm development, as well as their biocompatibility (demonstrated by fluorescence microscopy results, an XTT assay, and a glutathione (GSH) assay).

Effects of Usnic Acid to Prevent Infections by Creating a Protective Barrier in an In Vitro Study.

Nasal sprays are medical devices useful for preventing infection and the subsequent spread of airborne pathogens. The effectiveness of these devices depends on the activity of chosen compounds which can create a physical barrier against viral uptake as well as incorporate different substances with antiviral activity. Among antiviral compounds, UA, a dibenzofuran derived from lichens, has the mechanical ability to modify its structure by creating a branch capable of forming a protective barrier. The mechanical ability of UA to protect cells from virus infection was investigated by analyzing the branching capacity of UA, and then the protection mechanism in an in vitro model was also studied. As expected, UA at 37 °C was able to create a barrier confirming its ramification property. At the same time, UA was able to block the infection of Vero E6 and HNEpC cells by interfering with a biological interaction between cells and viruses as revealed also by the UA quantification. Therefore, UA can block virus activity through a mechanical barrier effect without altering the physiological nasal homeostasis. The findings of this research could be of great relevance in view of the growing alarm regarding the spread of airborne viral diseases.

Synthesis, Biological and In Silico Studies of Griseofulvin and Usnic Acid Sulfonamide Derivatives as Fungal, Bacterial and Human Carbonic Anhydrase Inhibitors.

Carbonic anhydrases (CAs, EC 4.2.1.1) catalyze the essential reaction of CO2 hydration in all living organisms, being actively involved in the regulation of a plethora of patho-/physiological conditions. A series of griseofulvin and usnic acid sulfonamides were synthesized and tested as possible CA inhibitors. Since ß- and γ- classes are expressed in microorganisms in addition to the α- class, showing substantial structural differences to the human isoforms they are also interesting as new antiinfective targets with a different mechanism of action for fighting the emerging problem of extensive drug resistance afflicting most countries worldwide. Griseofulvin and usnic acid sulfonamides were synthesized using methods of organic chemistry. Their inhibitory activity, assessed against the cytosolic human isoforms hCA I and hCA II, the transmembrane hCA IX as well as ß- and γ-CAs from different bacterial and fungal strains, was evaluated by a stopped-flow CO2 hydrase assay. Several of the investigated derivatives showed interesting inhibition activity towards the cytosolic associate isoforms hCA I and hCA II, as well as the three γ-CAs and Malassezia globosa (MgCA) enzyme. Six compounds (1b-1d, 1h, 1i and 1j) were more potent than AAZ against hCA I while five (1d, 1h, 1i, 1j and 4a) showed better activity than AAZ against the hCA II isoform. Moreover, all compounds appeared to be very potent against MgCA with a Ki lower than that of the reference drug. Furthermore, computational procedures were used to investigate the binding mode of this class of compounds within the active site of human CAs.

Usnic Acid-Loaded Magnetite Nanoparticles-A Comparative Study between Synthesis Methods.

Since cancer is a continuously increasing concern for the general population, more efficient treatment alternatives ought to be developed. In this regard, a promising direction is represented by the use of magnetite nanoparticles (MNPs) to act both as a nanocarrier for the targeted release of antitumoral drugs and as hyperthermia agents. Thus, the present study focused on improving the control upon the outcome properties of MNPs by using two synthesis methods, namely the co-precipitation and microwave-assisted hydrothermal method, for the incorporation of usnic acid (UA), a natural lichen-derived metabolite with proven anticancer activity. The obtained UA-loaded MNPs were thoroughly characterized regarding their morpho-structural and physicochemical properties through X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FT-IR), dynamic light scattering (DLS) and zeta potential, scanning electron microscopy (SEM), and vibrating sample magnetometry (VSM). Results demonstrated the formation of magnetite as the unique mineralogical phase through both types of synthesis, with increased uniformity regarding the drug loading efficiency, size, stability, and magnetic properties obtained through the microwave-assisted hydrothermal method. Furthermore, the cytotoxicity of the nanostructures against the HEK 293T cell line was investigated through the XTT assay, which further proved their potential for anticancer treatment applications.

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.

Unravelling Novel Phytochemicals and Anticholinesterase Activity in Irish Cladonia portentosa.

Acetylcholinesterase inhibitors remain the mainstay of symptomatic treatment for Alzheimer's disease. The natural world is rich in acetylcholinesterase inhibitory molecules, and research efforts to identify novel leads is ongoing. Cladonia portentosa, commonly known as reindeer lichen, is an abundant lichen species found in Irish Boglands. The methanol extract of Irish C. portentosa was identified as an acetylcholinesterase inhibitory lead using qualitative TLC-bioautography in a screening program. To identify the active components, the extract was deconvoluted using a successive extraction process with hexane, ethyl acetate and methanol to isolate the active fraction. The hexane extract demonstrated the highest inhibitory activity and was selected for further phytochemical investigations. Olivetolic acid, 4-O-methylolivetolcarboxylic acid, perlatolic acid and usnic acid were isolated and characterized using ESI-MS and two-dimensional NMR techniques. LC-MS analysis also determined the presence of the additional usnic acid derivatives, placodiolic and pseudoplacodiolic acids. Assays of the isolated components confirmed that the observed anticholinesterase activity of C. portentosa can be attributed to usnic acid (25% inhibition at 125 µM) and perlatolic acid (20% inhibition at 250 µM), which were both reported inhibitors. This is the first report of isolation of olivetolic and 4-O-methylolivetolcarboxylic acids and the identification of placodiolic and pseudoplacodiolic acids from C. portentosa.

Antimelanoma Potential of Cladonia mitis Acetone Extracts - Comparative in Vitro Studies in Relation to Usnic Acid Content.

In this study, the cytotoxic activity of acetone extracts of Cladonia mitis was assessed with respect to the content of usnic acid, a secondary metabolite commonly present in this species. Following quantitative HPLC analysis of the extracts, usnic acid was isolated by preparative chromatography. The study of cytotoxic activity was performed using the MTT test on three melanoma cell lines - HTB140, A375 and WM793. The selectivity of action was also assessed by comparing the effect towards normal human keratinocytes HaCaT. The results showed a dose-dependent cytotoxic activity of the extracts tested and usnic acid itself, but no relationship was found between the content of usnic acid and the activity of the extracts. Furthermore, the extracts showed varied, but rather low anti-tyrosinase activity. Other in vitro and in vivo studies are necessary to demonstrate that C. mitis extracts may be useful in the adjuvant external treatment of skin melanoma.

Anti-Inflammatory Potential of Compounds Isolated from Tunisian Lichens Species.

The lichen's special symbiotic structure enables it to produce bioactive substances. They have historically been recognized for their aesthetic and medicinal benefits. Furthermore, in recent years, they have performed in various fields, including perfumery, dyeing, and pharmacology due to their rich secondary metabolites. From our study, four compounds were isolated from organic extracts of Parmotrema hypoleucinum, Roccella phycopsis, and Xanthoria parietina and identified by spectroscopic investigation as atranorin, (+)-iso-usnic acid, methyl orsellinate, and parietin, respectively. The anti-inflammatory effects of lichens extracts, and pure compounds were evaluated on RAW 264.7 macrophages cells at different concentrations. At 25 µg/mL all treated samples did not show any effect on cell viability. Atranorin and (+)-iso-usnic acid showed an inhibitory effect on nitric oxide (NO) levels in lipopolysaccharide (LPS)-stimulated macrophages. Nitric oxide (NO) production was measured using Griess reagent, atranorin and (+)-iso-usnic acid showed a high anti-inflammatory potential (75.99 % and 57.27 % at 25 µg/mL). On the other hand, methyl orsellinate and the organic extracts of three lichens showed good anti-inflammatory activity ranging from 29.16 % at 25 µg/mL to 86.91 % at 100 µg/mL.

The Isoxazole Derivative of Usnic Acid Induces an ER Stress Response in Breast Cancer Cells That Leads to Paraptosis-like Cell Death.

Derivatives of usnic acid (UA), a secondary metabolite from lichens, were synthesized to improve its anticancer activity and selectivity. Recently we reported the synthesis and activity of an UA isoxazole derivative, named 2b, against cancer cells of different origins. Herein, the molecular mechanisms underlying its activity and efficacy in vivo were tested. The viability of breast cancer or normal cells has been tested using an MTT assay. Cell and organelle morphology was analyzed using light, electron and fluorescence microscopy. Gene expression was evaluated by RNAseq and protein levels were evaluated by Western blotting. In vivo anticancer activity was evaluated in a mice xenograft model. We found that 2b induced massive vacuolization which originated from the endoplasmic reticulum (ER). ER stress markers were upregulated both at the mRNA and protein levels. ER stress was caused by the release of Ca2+ ions from the ER by IP3R channels which was mediated, at least partly, by phospholipase C (PLC)-synthetized 1,4,5-inositol triphosphate (IP3). ER stress led to cell death with features of apoptosis and paraptosis. When applied to nude mice with xenografted breast cancer cells, 2b stopped tumour growth. In mice treated with 2b, vacuolization was observed in tumour cells, but not in other organs. This study shows that the antiproliferative activity of 2b relates to the induction of ER stress in cancer, not in healthy, cells and it leads to breast cancer cell death in vitro and in vivo.

Polyglycerol Adipate-Grafted Polycaprolactone Nanoparticles as Carriers for the Antimicrobial Compound Usnic Acid.

Nanoparticle (NP) drug delivery systems are known to potentially enhance the efficacy of therapeutic agents. As for antimicrobial drugs, therapeutic solutions against drug-resistant microbes are urgently needed due to the worldwide antimicrobial resistance issue. Usnic acid is a widely investigated antimicrobial agent suffering from poor water solubility. In this study, polymer nanoparticles based on polyglycerol adipate (PGA) grafted with polycaprolactone (PCL) were developed as carriers for usnic acid. We demonstrated the potential of the developed systems in ensuring prolonged bactericidal activity against a model bacterial species, Staphylococcus epidermidis. The macromolecular architecture changes produced by PCL grafted from PGA significantly influenced the drug release profile and mechanism. Specifically, by varying the length of PCL arms linked to the PGA backbone, it was possible to tune the drug release from a burst anomalous drug release (high PCL chain length) to a slow diffusion-controlled release (low PCL chain length). The developed nanosystems showed a prolonged antimicrobial activity (up to at least 7 days) which could be used in preventing/treating infections occurring at different body sites, including medical device-related infection and mucosal/skin surface, where Gram-positive bacteria are commonly involved.

Usnic Acid-Mediated Exchange of Protons for Divalent Metal Cations across Lipid Membranes: Relevance to Mitochondrial Uncoupling.

Usnic acid (UA), a unique lichen metabolite, is a protonophoric uncoupler of oxidative phosphorylation, widely known as a weight-loss dietary supplement. In contrast to conventional proton-shuttling mitochondrial uncouplers, UA was found to carry protons across lipid membranes via the induction of an electrogenic proton exchange for calcium or magnesium cations. Here, we evaluated the ability of various divalent metal cations to stimulate a proton transport through both planar and vesicular bilayer lipid membranes by measuring the transmembrane electrical current and fluorescence-detected pH gradient dissipation in pyranine-loaded liposomes, respectively. Thus, we obtained the following selectivity series of calcium, magnesium, zinc, manganese and copper cations: Zn2+ > Mn2+ > Mg2+ > Ca2+ >> Cu2+. Remarkably, Cu2+ appeared to suppress the UA-mediated proton transport in both lipid membrane systems. The data on the divalent metal cation/proton exchange were supported by circular dichroism spectroscopy of UA in the presence of the corresponding cations.

Advances in Research on Bioactivity, Toxicity, Metabolism, and Pharmacokinetics of Usnic Acid In Vitro and In Vivo.

Lichens are among the most widely distributed plants on earth and have the longest growth cycle. Usnic acid is an abundant characteristic secondary metabolite of lichens and the earliest lichen compound used commercially. It has diverse pharmacological activities, such as anti-inflammatory, antibacterial, antiviral, anticancer, antioxidant, and photoprotective effects, and promotes wound healing. It is widely used in dietary supplements, daily chemical products (fodder, dyes, food, perfumery, and cosmetics), and medicine. However, some studies have found that usnic acid can cause allergic dermatitis and drug-induced liver injury. In this paper, the bioactivity, toxicity, in vivo and in vitro metabolism, and pharmacokinetics of usnic acid were summarized. The aims were to develop and utilize usnic acid and provide reference for its future research.

Inhibition of penicillin-binding protein 2a (PBP2a) in methicillin resistant Staphylococcus aureus (MRSA) by combination of oxacillin and a bioactive compound from Ramalinaroesleri.

Lichens are known to be useful and important in ethanopharmacology since ages and still possess substantial interest in alternative medical practices around the world. The intent of this investigation was to evaluate and to understand the antibacterial potential of usnic acid which was isolated from Himalyan fruticose lichen Ramalina roesleri. Usnic acid is predicted for its pharmaceutical properties through in -silico studies. Binding efficiency of usnic acid with Penicillin binding protein-PBP2a, a protein which is responsible for conferring resistance in Staphylococcus aureus was accessed using in-silico interaction assays comparing with oxacillin and ceftaroline. Further, the validation of in-silico modelling was checked by determining the antibacterial potential of usnic acid against methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates. In total, 28 clinical isolates collected from hospitals/medical students were included in the study and the anti-Staphylococcal activity was determined using agar plate dilution method followed by time-kill kinetics and synergistic studies. The scanning electron microscopic (SEM) pictures were obtained to show the cell wall disruption of MRSA by usnic acid. Docking results clearly indicated the enhanced binding potential of usnic acid (Glide XP G Score: 10.968; Glide energy -64.869) with PBP2a which is better than the energy range of reference compound, oxacillin (Glide XP G Score: 6.596; Glide energy -53.285) and roughly comparable to the co-crystallized ligand ceftaroline (Glide XP G Score: 12.20; Glide energy -70.322). Cefteroline is known to be more active against MRSA compared to oxacillin. The minimum inhibitory concentrations (MICs) of usnic acid against the clinical isolates of MRSA and reference strain (NCTC-6571) were in the range of 32-128 µg/ml. The high affinity of usnic acid to bind with PBP2a which is demonstrated via in-silico studies is further confirmed by the impressive inhibitory activity of usnic acid on MRSA clinical isolates.

(+)-Usnic acid modulates the Nrf2-ARE pathway in FaDu hypopharyngeal carcinoma cells.

Naturally occurring phytochemicals of different origin and structure, arctigenin, bergenin, usnic acid and xanthohumol, were shown to affect Nrf2 pathway in the context of various diseases, but their effect on this pathway in cancer cells was not extensively investigated. This study aimed to evaluate the effect of these compounds on Nrf2 expression and activation in hypopharyngeal FaDu squamous cell carcinoma cells. FaDu cells were treated with 2 or 10 µM arctigenin, bergenin, (+)-usnic acid or xanthohumol for 24 h. While arctigenin, bergenin, and xanthohumol did not affect either Nrf2 expression or activation, (+)-usnic acid treatment increased its transcript level and increased the nuclear/cytosol Nrf2 protein ratio-the measure of Nrf2 pathway activation. Consequently, (+)-usnic acid enhanced the transcription and translation of Nrf2 target genes: NQO1, SOD, and to a lesser extent, GSTP. The treatment of FaDu cells with (+)-usnic acid decreased both GSK-3ß transcript and protein level, indicating its possible involvement in Nrf2 activation. All the tested compounds decreased Bax mRNA but did not change the level of Bax protein. (+)-Usnic acid tended to increase the percentage of early apoptotic cells and LC3 protein, autophagy marker. Significant induction of p53 also was observed after treatment with (+)-usnic acid. In summary, the results of this study indicate that low concentrations of (+)-usnic acid activate Nrf2 transcription factor, most probably as a result of ROS accumulation, but do not lead to FaDu hypopharyngeal carcinoma cells death.