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The ability to functionally express proteins in hosts is a precondition to an advanced understanding of the biosynthetic pathways that are responsible for producing life's complex molecules. The study of secondary metabolites in lichen‐forming fungi has long been hampered by slow growth. This study, reports on heterologous expression trials of four polyketide synthase (PKS) genes from C. uncialis in Aspergillus oryzae NSAR1. Isolation of mRNA and RT‐PCR demonstrated that A. oryzae can transcribe all lichen genes and remove introns to produce translationally‐coherent mRNA. Transformation of A. oryzae with a codon‐optimized PKS did not result in metabolite production, nor did co‐expression of a number of accessory genes restore function to any lichen PKS. Genes encoding an orsellinic acid synthase (OAS) from Fusarium sp. and a 6‐methylsalicylic acid synthase (6MSAS) from Penicillum sp. were transformed into A. oryzae. Readily detectable amounts of de novo orsellinic acid and 6‐methylsalicylic acid biosynthesis were observed in A. oryzae when transformed with these non‐lichen PKS genes. However, transformation with functionally homologous PKS genes from C. uncialis produced no detectable product. This work demonstrates that lichen PKS genes are correctly transcribed by A. oryzae but that polyketide biosynthesis failed for a reason that is presently unknown but may be attributable to a fault of translation [ABSTRACT FROM AUTHOR]

• Usnic acid is a lichenic secondary metabolite useful for the treatment of burn wounds. • The poor solubility limits the use of usnic acid in health care products. • Usnic acid was formulated in adhesive polymeric films to improve wounds treatment. • Film F2 is the most effective against usnic acid sensitive bacteria. • Film F2 is safe towards human keratinocytes and fibroblasts. Usnic acid (UA) is a lichenic secondary metabolite useful for the treatment of burn wounds thanks to its antimicrobial activity, particularly toward strains responsible for their infections. However, the poor solubility is the main factor limiting the activity and thus its use in health care products. Adhesive polymeric films were designed to improve UA use by enhancing its bioavailability in the wounded tissues. Three different NaCMC hydrogel films, NaCMC 2% alone (F1), mixed to PVP K90 0.1% (F2) or to Carbopol 971 P 0.1% (F3), were prepared by casting method. Ex vivo experiments performed on pig skin samples showed their suitable adhesion capacity. in vitro release test, performed using the extraction cell, showed that film F2 provides the highest UA concentrations. Differential scanning calorimetry and X-ray analyses performed on the three films highlighted that UA is present in a more soluble form in F2. The in vitro antibacterial activity studies demonstrated that F2 is the most effective film against UA sensitive bacteria S. Epidermidis , E. Faecalis , B. Cereus and S. Pyogenes. In vitro cytotoxicity assays on human keratinocytes and fibroblasts showed that cells viability is not compromised. [ABSTRACT FROM AUTHOR]

Abstract Thymol and usnic acid as the important secondary metabolites of respectively Artemisia haussknechtii and Protoparmeliopsis muralis were used for reduction and stabilizing of AgNO 3 and CuSO 4 in metal nanoparticles (MNPs) biosynthesis process. Antibacterial effects of prepared Ag-thymol (ATNPs), Ag-usnic acid (AUNPs), Cu-thymol (CTNPs), and Cu-usnic acid (CUNPs) on multi drug resistant (MDR) bacteria including methicillin-resistant Staphylococcus aureus (MRSA) (gram positive), Acinetobacter baumannii (A52), and Klebsiella pneumonia (K38) (gram negative) were compared with thymol, usnic acid, AgNO 3 , CuSO 4 , and tetracycline. Results of this study showed higher antibacterial activities of usnic acid, CUNPs, and CTNPs with MIC/MBC values (20, 40, and 40 μg/mL, respectively) than ATNPs and AUNPs against MRSA bacteria. Leakage of macromolecules involving nucleic acids and proteins from bacteria under stress of MNPs, thymol, and usnic acid proved significant antibacterial activities of usnic acid, and Cu NPs. In addition, SEM images illustrated different patterns of aggregation in biofilms resulted from interactions of these antibacterial agents with bacterial macromolecules. Totally, this investigation illustrated new green method of Ag and Cu NPs biosynthesis with suitable antibacterial properties. [ABSTRACT FROM AUTHOR]

Abstract Usnic acid (UA), an old antibiotic and one of the first described mitochondrial uncouplers, has demonstrated many beneficial activities, such as antimicrobial, antiviral, antitumour and anti-inflammatory properties. Here, we performed a thorough investigation of effects of usnic acid and its analogues on artificial planar bilayer lipid membrane (BLM), rat liver mitochondria and bacteria. Surprisingly enough, all of the three hydroxyl groups of UA appeared to be involved in its proton-shuttling activity on BLM. We ascribed this fact to an ability of UA to form complexes with calcium ions, aiding it in cycling protons across the membrane. Actually, the addition of calcium ions markedly stimulated the UA-induced electrical current across BLM. By using the calcium ionophore A23187, we proved the involvement of calcium ions in the UA uncoupling action on isolated rat liver mitochondria. The calcium-chelating property of UA was demonstrated here by the method of extracting metal ions into a hydrophobic phase. Modification of any of the hydroxyl groups in UA dramatically reduced not only the UA-induced current across BLM and the UA-mediated calcium extraction, but also the uncoupling activity of UA in mitochondria and the inhibiting effect of UA on the growth of Bacillus subtilis. The ability of UA to cause dissipation of membrane potential in isolated liver mitochondria and bacterial cells was shown here for the first time. In view of the data obtained, the protonophoric activity of UA is considered to make a significant contribution to its antibacterial action. Graphical abstract Unlabelled Image Highlights • Proton conductance of planar BLM induced by usnic acid is stimulated by calcium ions. • The charged membrane-permeant proton-shuttling form of usnic acid is an anionic dimer. • All the 3 OH groups of UA are important for its protonophoric and uncoupling action. • Modification of any of OH groups of usnic acid suppresses its antibacterial activity. • Protonophoric activity of UA is apparently essential for its antibacterial action. [ABSTRACT FROM AUTHOR]

Leishmaniasis is a neglected disease caused by a trypanosomatid protozoan of the genus Leishmania. Most drugs used to treat leishmaniasis are highly toxic, and the emergence of drug‐resistant strains has been observed. Therefore, new therapeutic targets against leishmaniasis are required. Several isoprenoid compounds, including dolichols or ubiquinones, have been shown to be important for cell viability and proliferation in various trypanosomatid species. Here, we detected the biosynthesis of tocopherol in Leishmania (L.) amazonensis promastigotes in vitro through metabolic labelling with [1‐(n)‐3H]‐phytol. Subsequently, we confirmed the presence of vitamin E in the parasite by gas chromatography–mass spectrometry. Treatment with usnic acid or nitisinone, inhibitors of precursors of vitamin E synthesis, inhibited growth of the parasite in a concentration‐dependent manner. This study provides the first evidence of tocopherol biosynthesis in a trypanosomatid and suggests that inhibitors of the enzyme 4‐hydroxyphenylpyruvate dioxygenase may be suitable for use as antileishmanial compounds. Database: The amino acid sequence of a conserved hypothetical protein [Leishmania mexicana MHOM/GT/2001/U1103] has been deposited in GenBank (CBZ28005.1) Isoprenoid compounds have been shown to be important for cell viability and proliferation in various trypanosomatids. We detected tocopherol biosynthesis in Leishmania (L.) amazonensis and report that the tocopherol may play an important antioxidant role. Inhibitors of tocopherol biosynthesis inhibited growth of the parasite. We supply evidence of tocopherol biosynthesis in trypanosomatids and propose a new drug target based on the results. [ABSTRACT FROM AUTHOR]

This review is focused on the comparison of the biological and pharmacological activities of usnic acid enantiomers. Most of the available data refer to (+)-usnic acid, while the left-handed isomer has been less often significantly studied. Special attention was paid to the experiments comparing both (+)- and (−)-usnic acid at the same time, the results of which indicated interesting differences, however no tendency as to which enantiomer was more potent could be observed. Nevertheless, more studies, especially on (−)-usnic acid, are needed to give a final explanation for the similarities and differences between both usnic acid enantiomers. These should be especially directed to steric structure–activity relationship of the enantiomers, tested under the same experimental conditions, which may help to explain the possible mechanisms of their actions. [ABSTRACT FROM AUTHOR]

Highlights • The genotoxic effect of usnic acid was only observed 1 h after oral administration. • (+)- and (−)-usnic acid doses of 100 and 50 mg/kg resulted in DNA damage in the liver and kidney cells. • The genotoxic effect of usnic acid is associated with oxidative stress. • There were no significant differences in the effects of (+)- and (−)-enantiomers. Abstract Usnic acid, which is the most widespread and well-studied secondary lichen compound, has antibacterial and cytotoxic effects. Usnic acid is present in lichens as the (+)- and (-)-enantiomers, which have different biological activities. We used a DNA-comet assay to determine the genotoxic effect of (+)- and (−)-usnic acid in the liver and kidney cells of mice. The genotoxic effect of usnic acid was only observed 1 h after oral administration. Usnic acid doses of 100 and 50 mg/kg resulted in DNA damage in the liver and kidney cells. The genotoxic effect of usnic acid is associated with oxidative stress in cells. There were no significant differences in the effects of (+)- and (−)-enantiomers. [ABSTRACT FROM AUTHOR]

Graphical abstract Abstract The intention of present research work is to formulate usnic acid (UA) loaded heparin modified gellan gum (HAG) nanoparticles (NPs). HAG copolymer based conjugation was synthesized and characterized by 1H NMR and FT-IR spectroscopy. Plain and UA loaded HAG NPs were prepared via nanoprecipitation technique. NPs were typified and further characterized for particle size, polydispersity index, entrapment efficiency, zeta potential, atomic force microscopy, differential scanning calorimetry, X-ray diffraction analysis, and in-vitro release. In-vitro tube formation assay, tumorsphere assay, autophagy assay, DNA cleavage assay, internalization by confocal and FACS based internalization analysis, caspase assay and cell cycle assay were performed for biological activity. Obtained experimental results explored that HAG NPs displayed a sustained release of UA (95.67% in 48 h) compared to gellan gum NPs (96.12% in 8 h). In cytotoxicity studies, UA loaded HAG NPs exhibited an enormous cytotoxic potential against A549 cancer cells. In the in vivo bio-distribution study, using albino rat model the free UA concentration was found 7.09 ± 0.9%, 2.7 ± 1.5%, 7.5 ± 2.1, 9.2 ± 2%, and 6.25 ± 1.3% post two hours of intravenous administration, however, in the case of UA loaded HAG NPs the obtained level was 4.1 ± 1.10, 7.7 ± 1.30%, 2.21 ± 0.29%, 1.85 ± 0.25%, 2.2 ± 0.78%, 2.9 ± 1.21% respectively, in heart, lung, liver, spleen, intestine and kidney. The overall anticancer study and result of internalization deciphered the higher anticancer potential of UA loaded HAG NPs. [ABSTRACT FROM AUTHOR]

Usnic acid (UA) is a multifunctional bioactive lichen secondary metabolite with potential anti-cancer properties. Although the promising therapeutic effects of UA have been investigated in different cancer cell lines, the mechanism driving UA-induced cell death has yet to be elucidated. As the type of cell death (apoptosis or autophagy) induced by UA may vary depending on the cancer cell type, we first studied the cytotoxic effects of UA in HEPG2 (HBV(−)) and SNU-449(HBV(+)) hepatocellular carcinoma (HCC) cell lines. HCC cell viability was considerably reduced in a dose-dependent manner at 12, 24, and 48 h after treatment with UA (p < 0.05). However, SNU-449 cells were more sensitive to UA than HEPG2 cells. UA also induced apoptotic cell death in HCC cells with cell cycle arrest at G0/G1 and G2/M phase depending on the genetic profile of each cell type. On the other hand, we observed acidic vesicular organelles in HCC cells after 36 h of UA treatment. Taken together, these findings suggest that UA stimulates apoptosis and autophagy in HEPG2 and SNU-449 cells without damaging normal control cells. Thus, UA might be a potential therapeutic compound for HCC treatment. However, there is a need for further studies investigating the death-promoting or preventing roles for autophagy and the molecular signaling mechanisms induced by UA treatment. [ABSTRACT FROM AUTHOR]

Abstract The druggability of the tyrosyl-DNA phosphodiesterase 1 (Tdp1) enzyme was investigated in conjunction with topoisomerase 1 inhibition. A novel class of thiazole, aminothiazole and hydrazonothiazole usnic acid derivatives was synthesized and evaluated as Tdp1 inhibitors and their ability to sensitize tumors to topotecan, a topoisomerase inhibitor in clinical use. Of all the compounds tested, four hydrazinothiazole derivatives, 20c, 20d, 20h and 20i , inhibited the enzyme in the nanomolar range. The activity of the compounds was verified by affinity experiments as well as supported by molecular modelling. The most effective Tdp1 inhibitor, 20d , was ton-toxic and increased the effect of topotecan both in vitro and in vivo in the Lewis lung carcinoma model. Furthermore, 20d showed significant increase in the antitumor and antimetastatic effect of topotecan in mice. The results presented here justify compound 20d to be considered as a drug lead for antitumor therapy. Graphical abstract Image 1 Highlights • Hydrazinothiazole derivatives of usnic acid are excellent inhibitors of Tdp1. • The most effective Tdp1 inhibitor showed significant increase in the antitumor activity of topotecan in mice. • The most effective Tdp1 inhibitor showed significant increase in the antimetastatic effect of topotecan in mice. • The inhibitor proved to have no own acute toxicity at the concentration used. [ABSTRACT FROM AUTHOR]

Highlights • We investigated the effects of UA on hypertrophic scarring in a rabbit ear model. • UA reduced the scar elevation index and ameliorated collagen accumulation. • UA inhibited HUVEC migration and tube formation and hypertrophic scar angiogenesis. • UA inhibited proliferation of both HUVECs and hypertrophic scar fibroblasts. • UA inhibits hypertrophic scar formation via suppression of scar angiogenesis. Abstract Hypertrophic scarring is a common condition in the Chinese population; however, there are currently no satisfactory drugs to treat the disorder. Previous studies showed that angiogenesis plays an important role in the early phase of hypertrophic scarring and inhibition of angiogenesis has been reported as an effective strategy for anti-hypertrophic scar therapy. A recent study showed that usnic acid (UA), an active compound found mainly in lichens, inhibited tumor angiogenesis both in vivo and in vitro. To investigate the therapeutic effects of UA on hypertrophic scarring and to explore the possible mechanism involved, a rabbit ear hypertrophic scar model was established. Scars were treated once a week for four weeks with UA, DMSO or triamcinolone acetonide acetate. Histological evaluation of hematoxylin and eosin staining indicated that UA significantly inhibited hypertrophic scar formation, with obvious reductions in scar height and coloration. The scar elevation index (SEI) was also evidently reduced. Masson's trichrome staining showed that UA significantly ameliorated accumulation of collagen tissue. Immunohistochemical analysis of CD31 expression showed that UA significantly inhibited scar angiogenesis. In vitro, UA inhibited endothelial cell migration and tube formation as well as the proliferation of both human umbilical vein endothelial cells and scar fibroblast cells. These results provide the first evidence of the therapeutic effectiveness of UA in hypertrophic scar formation in an animal model via a mechanism that involves suppression of scar angiogenesis. [ABSTRACT FROM AUTHOR]

During the years, many usnic acid (UA) conjugates have been synthesized to obtain potent endowed with biological properties. Since (S)-UA is less abundant in nature than (R)-enantiomer, it is difficult to source, thus precluding a deeper investigation. Among the lichens producing UA, Cladonia foliacea is a valuable (S)-UA source. In the present work, we report on a rapid HPLC-UV/PAD-CD protocol suitable for the analysis and the identification of the main secondary metabolites present in C. foliacea extract. Best results were achieved using XBridge Phenyl column and acetonitrile and water, which were both added with formic acid as mobile phase in gradient elution. By combining analytical, spectroscopical, and chiroptical analysis, the most abundant analyte was unambiguously identified as (S)-UA. Accordingly, a versatile microwave-assisted extractive (MAE) protocol, assisted by a design of experiment (DoE), to quantitatively recover (S)-UA was set up. The best result in terms of UA extraction yield was obtained using ethanol and heating at 80 °C under microwave irradiation for 5 min. Starting from 100 g of dried C. foliacea, 420 mg of (S)-UA were achieved. Thus, our extraction method resulted in a suitable protocol to produce (S)-UA from C. foliacea for biological and pharmaceutical investigation or commercial purposes.

• Peziculone, a usnic acid derivative, has strong antimicrobial activity against several Gram-positive bacterial pathogens, including methicillin-resistant Staphylococcus aureus. • Clustered regularly interspaced short palindromic repeats interference sequencing analysis together with membrane fluorescent staining was used to demonstrate that peziculone destabilizes the cell wall of Gram-positive bacteria. • Peziculone treatment impairs cell surface appendages and biofilm formation by S. aureus. Usnic acid, a representative dibenzofuran metabolite, is known to have antimicrobial properties. However, despite considerable interest as an antimicrobial agent, the mechanism by which usnic acid and its derivatives exert their action is not fully characterized. This article describes the synthesis of peziculone, a 5:1 equilibrium mixture of two inseparable usnic acid derivatives: peziculone A and peziculone B. The antibacterial activity of peziculone against several Gram-positive bacterial pathogens was found to be significantly better compared with usnic acid. Clustered regularly interspaced short palindromic repeats interference sequencing analysis and membrane fluorescent staining were used to demonstrate that peziculone destabilizes the cell walls of Gram-positive bacteria. Additionally, peziculone 2.5 and 3.5 µg/mL impaired cell surface appendages and biofilm formation by Staphylococcus aureus. Taken together, these data demonstrate that peziculone, a derivative compound of usnic acid, has significant antimicrobial activity against Gram-positive bacteria by targeting the cell walls; this provides a platform for development of novel antibacterial drugs. [ABSTRACT FROM AUTHOR]

Hyaluronic acid, curcumin, and usnic acid are separately utilized as effective biological agents in medicine, and materials based on its blend are considered to have wider therapeutic effects than individual ones. In this study, for the first time, native hyaluronic acid-based fibers containing curcumin and usnic acid with an average diameter of 298 nm were successfully prepared by the electrospinning technique and characterized. Additionally, unstable and hydrophobic curcumin and usnic acid were loaded into the hydrophilic hyaluronic acid matrix without utilizing the activating (catalyzing) agents, resulting in the formation of an electrospinnable solution. Only the binary mixture deionized water—dimethyl sulfoxide (50:50)—was used as solvent. The presence of small amounts of dimethyl sulfoxide in the fibrous materials was expected to provide the materials with local anesthetic and antiseptic activity. The effect of electric voltage on the electrospinning process, diameter, and morphology of hyaluronic acid/curcumin/usnic acid fibers was investigated in detail. The impact of curcumin and usnic acid on the stability of fiber formation was observed. The investigation of fibers based on pure hyaluronic acid without additional polymers and with active pharmaceutical ingredients will lay the groundwork for the development of highly effective wound dressings and new drug delivery scaffolds.

The increasing global emergence of multidrug resistant (MDR) pathogens is categorized as one of the most important health problems. Therefore, the discovery of novel antimicrobials is of the utmost importance. Lichens provide a rich source of natural products including unique polyketides and polyphenols. Many of them display pharmaceutical benefits. The aim of this study was directed towards the characterization of sunflower oil extracts from the fruticose lichen, Usnea barbata. The concentration of the major polyketide, usnic acid, was 1.6 mg/mL extract as determined by NMR analysis of the crude mixture corresponding to 80 mg per g of the dried lichen. The total phenolics and flavonoids were determined by photometric assays as 4.4 mg/mL (gallic acid equivalent) and 0.27 mg/mL (rutin equivalent) corresponding to 220 mg/g and 13.7 mg/g lichen, respectively. Gram-positive (e.g., Enterococcus faecalis) and Gram-negative bacteria, as well as clinical isolates of infected chickens were sensitive against these extracts as determined by agar diffusion tests. Most of these activities increased in the presence of zinc salts. The data suggest the potential usage of U. barbata extracts as natural additives and mild antibiotics in animal husbandry, especially against enterococcosis in poultry.

The present study aimed to prepare usnic acid (UA)-loaded chitosan (CS) nanoparticles (UA-CS NPs) and evaluate its antibacterial activity against biofilm-forming pathogenic bacteria. UA-CS NPs were prepared through simple ionic gelification of UA with CS, and further characterized using Fourier transform infrared spectroscopy, X-ray diffraction, and field-emission transmission electron microscopy. The UA-CS NPs presented a loading capacity (LC) of 5.2%, encapsulation efficiency (EE) of 24%, and a spherical shape and rough surface. The maximum release of UA was higher in pH 1.2 buffer solution as compared to that in pH 6.8 and 7.4 buffer solution. The average size and zeta potential of the UA-CS NPs was 311.5 ± 49.9 nm in diameter and +27.3 ± 0.8 mV, respectively. The newly prepared UA-CS NPs exhibited antibacterial activity against persister cells obtained from the stationary phase in batch culture, mature biofilms, and antibiotic-induced gram-positive and gram-negative pathogenic bacteria. Exposure of sub-inhibitory concentrations of UA-CS NPs to the bacterial cells resulted in a change in morphology. The present study suggests an alternative method for the application of UA into nanoparticles. Furthermore, the anti-persister activity of UA-CS NPs may be another possible strategy for the treatment of infections caused by biofilm-forming pathogenic bacteria.

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.

Usnic acid is one of the most investigated lichen secondary metabolites, with several proven biological properties with potential medical relevance. However, its unfavorable physico-chemical properties, as well as observed hepatotoxicity, have discouraged wide-range utilization of usnic acid as a promising therapeutic agent. In accordance with the growing research interest in the development of nanotechnology, especially in the arena of preparations based on natural sources of medicinal compounds, usnic acid incorporated into nano- and microsized colloidal carriers has been a subject of a large number of publications. Therefore, this review discusses the overall results of the studies dealing with usnic acid encapsulated into lipid-based, polymeric and nonorganic micro- and/or nanocarriers, as potential drug delivery systems for this natural compound, in an attempt to introduce its usage as a potential antitumor, antimicrobial, wound-healing, antioxidative and anti-inflammatory drug.

ABSTRACT The treatment of infections caused by resistant microorganisms is limited, and vancomycin (VAN) treatment failures for methicillin-resistant Staphylococcus aureus (MRSA) bacteremia are not uncommon, even when MRSA clinical isolates are susceptible to VAN. Thus, this study proposed the association of VAN with usnic acid and β-lapachone encapsulated into liposomes as a novel therapeutic option for infections caused by MRSA. Liposomes containing β-lap (β-lap-lipo) or usnic acid (UA-lipo) were prepared by the thin lipid film hydration method followed by sonication. Antimicrobial activity against MRSA clinical isolates was investigated by the microdilution method according to the Clinical and Laboratory Standards Institute (CLSI). The interaction studies were carried out using the checkerboard method and epsilometer test (Etest). The interaction between VAN and β-lap or β-lap-lipo was synergistic (FICI = 0.453 and FICI = 0.358, respectively). An additive interaction between VAN and UA (FICI = 0.515) was found. UA-lipo resulted in synergism with VAN (FICI = 0.276). The Etest reproduced the results obtained by the checkerboard method for approximately 82% of the analysis. Thus, the present study demonstrated that VAN in combination with UA-lipo, β-lap or β-lap-lipo synergistically enhanced antibacterial activity against MRSA.

Lichens are a form of symbiont between a fungus and an alga or cyanobacterium, which contains a wide variety of organic compounds with certain secondary metabolite classes typical of these organisms. The Ramalina genus has approximately 246 species distributed around the World, of which in this review approximately 118 species with published chemical or biological activity studies of extracts or isolated compounds were cited. From the 153 mentioned compounds, only 27 passed were tested for biological activity, being usnic acid the most studied compound and the one showing the best results in almost all in vitro tests performed, although other compounds also presented excellent results as antimicrobial, antitumor and anti-inflammatory agents, among others. Extracts of several species also presented significant results in performed biological tests, demonstrating the potential that these organisms have, in particular, the gender Ramalina, to produce bioactive molecules that can be used as a model for the production of pharmaceuticals.

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., (© 2023. BioMed Central Ltd., part of Springer Nature.)

In a conventional Mannich reaction using piperidine, hydroxypiperidines, morpholine, and N‐methylpiperazine with usnic acid, a deacetylation was observed resulting in a substitution at C‐2, a loss of an acetyl group, and a Mannich base with a stabilized enol. The enol has a hydrogen bond to the nitrogen of the secondary amine. The structure was investigated by nuclear magnetic resonance and deuterium isotope effects on 13C chemical shifts as well as with density functional theory calculations to study the changed hydrogen bond pattern. It was found that the hydrogen bond involving the OH‐9 group in chloroform forms a strong hydrogen bond than in usnic acid itself and that this hydrogen bond becomes even stronger in the more polar solvent, dimethylsulfoxide. Tautomerism was observed in the Mannich base as demonstrated by deuterium isotope effects on chemical shifts. The position of the tautomeric equilibrium depends on the solvent, and the position of the equilibrium governs the strength of the OH‐9...O═C hydrogen bond. A deacetylation reaction is occurring in a traditional Mannich reaction. The product shows both intramolecular hydrogen bonding and tautomerism as demonstrated by deuterium isotope effects on 13C chemical shifts. Other hydrogen bonds are modified. [ABSTRACT FROM AUTHOR]

Abstract Usnic acid is a lichen compound which is extensively studied due to its cytotoxic, antiproliferative, antimicrobial, antiviral, antiprotozoal, and anti-inflammatory activities. Despite a broad spectrum of biological properties, usnic acid is a hepatotoxic agent, thus its potential use as a drug is limited. Certain hepatotoxic drugs may act by generating reactive metabolites that damage the liver. The aim of the study was to predict the biotransformation of usnic acid enantiomers to reactive products using a trapping assay with glutathione in human, rat, and mice liver microsomes. Our results indicate that each enantiomer forms two reactive metabolites; in turn, these metabolites form adducts with glutathione, which may partially explain the toxicity of usnic acid. In silico analysis indicated structural alerts for the generation of reactive metabolites in usnic acid formula. This study proposes a novel mode of the hepatic toxicity of usnic acid enantiomers; it also provides some useful suggestions for designing safer usnic acid derivatives. Highlights • Biotransformation of usnic acid leads to the formation of its reactive metabolites, detectable by trapping assay. • There are differences between usnic acid enantiomers and between microsomal models in the formation of its reactive products. • Reactive metabolites formation may partially explain usnic acid hepatotoxicity. [ABSTRACT FROM AUTHOR]

Efforts by lichenologists to characterize lichen polyketide synthases (PKS) through heterologous expression experiments have so far proved unfruitful. A determination of systematic causes of failure is therefore required. Three hypotheses involving the ketosynthase (KS) domain of lichen polyketide synthases (PKS) from Cladonia uncialis are tested: (1) Horizontal versus vertical gene transfer; (2) Typical versus atypical active site residues; (3) Typical versus atypical tertiary protein structure and active site architecture. Phylogenetics, amino acid sequence alignment, and protein modelling indicate that C. uncialis PKS evolved through vertical transfer from Ascomycota fungi, possess Cys-His-His catalytic triads typical of KS from most organisms, and possess protein and catalytic site architecture identical to well-characterized KS from non-lichen organisms. Though the reason for lack of functional activity in heterologous hosts remains unknown, complications involving the KS are ruled out as a likely explanation. Heterologous translation of lichen PKS (or parts thereof) have not been reported. We demonstrate heterologous translation of two lichen KS domains in E. coli . [ABSTRACT FROM AUTHOR]

Purpose: Some phytochemicals have shown the potential of being radiomodifiers, especially phenolic compounds, such as lichenic secondary metabolites. To evaluate the phytochemical usnic acid as a radiomodifier, embryonic cells of molluscs have been used due to their ease of collection, high sensitivity to physical and chemical agents, well-known embryology and low cost for analysis. Materials and methods: This study aimed to assess the radiosensitizing action of usnic acid on Biomphalaria glabrata embryos. Samples were irradiated with 4 Gy of gamma rays from a 60Co source (dose rate 2.906 Gy/h). An acute toxicity test was performed using B. glabrata embryos in the blastula stage, in order to determine the toxicity of usnic acid and to establish the lethal Concentration for 50% (LC50). Subsequently, the radiomodifing capacity of usnic acid was estimated using assays with B. glabrata embryos. Results: Irradiation increased the number of non-viable embryos compared to unirradiated controls. Additionally, it was observed that embryos exposed to a non-toxic concentration of usnic acid (0.6 µg/mL) before irradiation showed a further enhancement in non-viable embryos when compared with exposure to ionizing radiation alone. Conclusion: The results presented here indicate that usnic acid makes cells more sensitive to the damaging effects of radiation. [ABSTRACT FROM AUTHOR]

The improved bactericidal activity of new composites for wound dressing prototypes represents an important strategy for development of more efficient devices that make use of synergistic interaction between components. The doping level of polyaniline represents a critical parameter for its corresponding biologic activity. In this work, it is explored the doping effect of usnic acid on undoped polyaniline, that introduces important advantages namely, improved bactericidal activity of polyaniline and the anti-biofilm properties of lichen derivative. The deposition of the resulting material on polyurethane foam potentializes its applicability as wound dressing, characterizing a new platform for application against Escherichia coli and Staphylococcus aureus . [ABSTRACT FROM AUTHOR]

Usnic acid is a dibenzofuran derivate produced by some lichens species primarily Usnea sp., that has several pharmacological activities. Based on Biopharmaceutical Classification System (BCS), usnic acid belongs to class II that has high permeability but low solubility. Therefore, the purpose of this research was to increase the solubility of usnic acid by preparing solid dispersions (SD) using hydrophilic polymer HPMC 2910. Usnic acid and HPMC at ratio 1:1 (w/w) were prepared for solid dispersion using spray drying and freeze drying techniques. Physical mixture at the same ratio was prepared as comparison. Characterization for intact materials, physical mixture and solid dispersions were done using powder X-ray diffraction (PXRD), differential scanning calorimetry (DSC), scanning electron microscopy (SEM), fourier transform infra-red (FT IR) spectroscopy, and solubility test. The result of PXRD showed a decrease of peak intensity for solid dispersions. Thermal properties of solid dispersions showed lower melting point compared to usnic acid and physical mixture. The morphology of solid dispersion depicted different shape compared to intact materials and physical mixture. The infra-red spectrum showed the slightly shift at wave numbers of functional groups but there was no new functional groups formed. The solubility of solid dispersions significantly increased, which the solubility test result were 55±0.023, 227±0.034, 567± 0.020, and 932±0.053 (µg/ml) for intact usnic acid, physical mixture, SD by spray drying and SD by freeze drying, respectively. In conclusion, solid dispersions of usnic acid - HPMC 2910 were able to modify physicochemical properties and increased the solubility. [ABSTRACT FROM AUTHOR]

Objective: To prepare a new nanosystem of usnic acid (UA) with higher solid content and higher bioavailability. Methods: Usnic acid nanocrystal suspensions were prepared by the wet milling method, and then the particle size distributions and zeta potential were determined with the Nano ZS90 laser diffraction particle size analyzer. The particles morphology of UA-NCS were observed by scanning electron microscopy method. In addition, solubility and dissolution of UA-NCS in water and phosphate buffer solution were determined in vitro, analyzed by the HPLC method, and then the cellular uptake and pharmacokinetic were carried out on the Caco-2 cells and rats, analyzed by the UPLC-MS/MS method. Results: Particle size distributions and zeta potential of the UA nanocrystal suspension were 268.7 ± 4.0nm and -23.1 ± 0.7mV, respectively. About the dissolution rate of UA, nanosuspension were significantly faster and higher than common suspension in water and phosphate buffer. And in cellular uptake experiments, the ratio of the maximum amount of drug in unit protein of UA nanosuspension to common suspension was 2.8 times. In rats, oral absorption of nanocrystal UA were superior to the ordinary groups, with the 348% of the maximum concentration and 181% of the AUC after the same dosage administration. Conclusion: The wet-milling technique was suitable for the preparation of UA nanocrystal suspension, and a new nanosystem of UA with higher solid content and higher bioavailability was achieved. [ABSTRACT FROM AUTHOR]