Usnic Research Articles

Usnic acid as potential inhibitors of BCL2 and P13K protein through network pharmacology-based analysis, molecular docking and molecular dynamic simulation

Usnic acid (UA) lately piqued the interest of researchers for its extraordinary biological characteristics, including anticancer activity. Here, the mechanism was clarified through network pharmacology,molecular docking and molecular dynamic simulation. Sixteen proteins were selected through network pharmacology study as they are probable to interact with UA. Out of these proteins, 13 were filtered from PPI network analysis based on their significance of interactions (p < 0.05). KEGG pathway analysis has also aided us in determining the three most significant protein targets for UA, which are BCL2, PI3KCA and PI3KCG. Therefore molecular docking and molecular dynamic (MD) simulations throughout 100 ns were performed for usnic acid onto the three proteins mentioned. However, UA's docking score in all proteins is lower than their co-crystalised ligand, especially for BCL2 (−36.5158 kcal/mol) and PI3KCA (−44.5995 kcal/mol) proteins. The only exception is PI3KCG which has comparable results with the co-crystallised ligand with (−41.9351 kcal/mol). Furthermore, MD simulation has also revealed that usnic acid does not stay fit in the protein throughout the simulation trajectory for PI3KCA protein evident from RMSF and RMSD plots. Nevertheless, it still poses good ability in inhibiting BCL2 and PI3KCG protein in MD simulation. In the end, usnic acid has exhibited good potential in the inhibition of PI3KCG proteins, rather than the other proteins mentioned. Thus further study on structural modification of usnic acid could enhance the ability of usnic acid in the inhibition of PI3KCG as anti-colorectal and anti-small cell lung cancer drug candidate.Communicated by Ramaswamy H. Sarma

Safety in Rats of a Novel Nasal Spray Formulation for the Prevention of Airborne Viral Infections.

Hexedra+® is a nasal spray containing hydroxypropyl methylcellulose, beta-cyclodextrin, and usnic acid. It has been developed with the aim of reducing the risk of transmission of airborne viral infections, with particular reference to influenza and COVID-19. As part of the preclinical development of the product, we carried out a study on thirty male Wistar rats divided into three study groups and treated with Hexedra+, an alternative formulation containing a double concentration of usnic acid (0.015% instead of 0.0075%) or saline solution. Products were administered at the dose of 30 μL into each nostril, three times a day for seven consecutive days by means of a micropipette. By the end of the treatment period, no significant changes were observed in body weight. Histological examination of nasal mucosa and soft organs did not show any significant difference in the three study groups. Serum transaminase level remained in the normal limit in all the animals treated. The serum level of usnic acid was measured in order to assess the absorption of the molecule through the nasal mucosa. By the end of the study period, the usnic acid serum level was negligible in all the animals treated. In conclusion, the safety profile of Hexedra+ appears favorable in the animal model studied.

Identification of Pyrazole Derivatives of Usnic Acid as Novel Inhibitor of SARS-CoV-2 Main Protease Through Virtual Screening Approaches.

The infection produced by the SARS-CoV-2 virus remains a significant health crisis worldwide. The lack of specific medications for COVID-19 necessitates a concerted effort to find the much-desired therapies for this condition. The main protease (Mpro) of SARS-CoV-2 is a promising target, vital for virus replication and transcription. In this study, fifty pyrazole derivatives were tested for their pharmacokinetics and drugability, resulting in eight hit compounds. Subsequent molecular docking simulations on SARS-CoV-2 main protease afforded two lead compounds with strong affinity at the active site. Additionally, the molecular dynamics (MD) simulations of lead compounds (17 and 39), along with binding free energy calculations, were accomplished to validate the stability of the docked complexes and the binding poses achieved in docking experiments. Based on these findings, compound 17 and 39, with their favorable projected pharmacokinetics and pharmacological characteristics, are the proposed potential antiviral candidates which require further investigation to be used as anti-SARS-CoV-2 medication.

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 β- 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 β- 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.

Inhibition of Autophagy Increases Cell Death in HeLa Cells through Usnic Acid Isolated from Lichens.

The Western Ghats, India, is a hotspot for lichen diversity. However, the pharmacological importance of lichen-associated metabolites remains untapped. This study aimed to evaluate the cytotoxic potential of lichens of this region. For this, sixteen macrolichens were collected and identified from two locations in the Western Ghats. The acetone extract of Usnea cornuta (UC2A) showed significant cytotoxicity towards multiple human cancer cell lines. Interestingly, co-treatment with chloroquine (CQ), an autophagy inhibitor, increased the cytotoxic potential of the UC2A extract. A gas chromatography mass spectrometry (GCMS) study revealed usnic acid (UA), atraric acid and barbatic acid as the dominant cytotoxic compounds in the UC2A extract. Further, UA was purified and identified from the UC2A extract and evaluated for cytotoxicity in HeLa cells. The monodansyl cadaverine and mitotracker red double staining revealed the autophagy-inducing activities of UA, and the inhibition of autophagy was confirmed via CQ treatment. Autophagy inhibition increased the cytotoxicity of UA by 12-16% in a concentration-dependent manner. It also increased lipid peroxidation, ROS levels and mitochondrial depolarization and decreased glutathione availability. A decrease in zeta potential and a 40% increase in caspase 3/7 activity were also noted after CQ treatment of UA-treated cells. Thus, cytotoxicity of UA can be increased by inhibiting autophagy.

Influence of Tyrosyl-DNA Phosphodiesterase 1 Inhibitor on the Proapoptotic and Genotoxic Effects of Anticancer Agent Topotecan

To date, various strategies have been proposed to increase the efficiency of cancer therapy. It is known that the action of DNA repair system can determine the resistance of cancer cells to DNA-damaging chemotherapy and radiotherapy, and one of these ways to increase therapeutic efficiency is the search for inhibitors of enzymes of the DNA repair system. Inhibition of the DNA repair enzyme tyrosyl-DNA phosphodiesterase1 (Tdp1) leads to an increase in the effectiveness of the topoisomerase 1 (Top1) inhibitor, the anticancer drug topotecan. Covalent complexes Top1-DNA, which are normally short-lived and are not a threat to the cell, are stabilized under the influence of topotecan and lead to cell death. Tdp1 eliminates such stabilized complexes and thus weaken the effect of topotecan therapy. We have previously shown that the use of the usnic acid hydrazonothiazole derivative OL9-119 in combination with topotecan increased the antitumor and antimetastatic efficacy of the latter in a mouse model of Lewis lung carcinoma. In this work, it was shown that the combined use of topotecan and Tdp1 inhibitor, the hydrazonothiazole derivative of usnic acid OL9-119, leads to an increase in the DNA-damaging effect of topotecan which is used in the clinic for the treatment of cancer. The study of the proapoptotic effect of the compound OL9-119 showed that the compound itself does not induce apoptosis, but increases the proapoptotic effect of topotecan. The results of the study could be used to improve the effectiveness of anticancer therapy and/or to reduce the therapeutic dose of topotecan and, therefore, the severity of side effects.

Usnic Acid Isolated from Lichen Usnea sp. from Ciwidey, West Java

Indonesia is a tropical country that has a variety of plants. Plants are one potential source of medicine. One of them is Lichen Usnea sp., which is used by the community to treat several diseases, so further research is needed to study the chemical composition contained and other potentials of this plant. Research on the chemical constituent of Lichen Usnea sp. consists of various stages: sample preparation, maceration using methanol, fractionation by Vacum Liquid Chromatography (VLC) and radial chromatography guided by Thin Layer Chromatography (TLC) eluting with n-hexane, chloroform and ethyl acetate obtaining an isolated compound. The isolated compound was analyzed by various spectroscopic data (UV-Vis, FT-IR, 1H-NMR and 13C-NMR). Based on the spectra, the isolated compound was usnic acid.Keywords: Usnic acid; Lichen; Usnea sp.ABSTRAKIsolat Asam Usnat dari Lichen Usnea sp. Asal Ciwidey, Jawa BaratIndonesia adalah negara tropis dengan banyaknya spesies tumbuhan. Tumbuhan adalah salah satu sumber bahan baku yang potensial untuk obat–obatan. Salah satunya Lichen Usnea sp. yang digunakan masyarakat dalam pengobatan beberapa penyakit, sehingga diperlukan penelitian lebih lanjut untuk mengetahui senyawa kimia yang terkandung dan potensi lainnya dari tanaman ini. Penelitian kandungan kimia dari Lichen Usnea sp. asal Ciwidey, Jawa Barat, terdiri dari berbagai tahapan, yaitu, persiapan sampel, maserasi dengan pelarut metanol, fraksinasi dan pemurnian dengan metode Kromatografi Cair Vakum (KCV) dan kromatografi radial yang dipandu dengan Kromatografi Lapis Tipis (KLT) dengan eluen n-heksana, kloroform dan etil asetat dan menghasilkan satu senyawa. Senyawa yang diperoleh tersebut dianalisis dengan berbagai data spektroskopi (UV, IR, 1H-NMR, dan 13C-NMR). Hasil karakterisasi menunjukkan bahwa senyawa tersebut adalah asam usnat.Kata kunci: Asam usnat; Lichen; Usnea sp.

Divergent reactivity of usnic acid and evaluation of its derivatives for antiproliferative activity against cancer cells

Natural products continue to be an inspiration for new drugs to treat debilitating diseases such as cancer. Usnic acid is a secondary metabolite isolated predominately from lichen species and has been shown to exhibit antiproliferative properties, however its application is limited by poor drug-like properties and low specificity. We report our work on investigating the reactivity of usnic acid for incorporating heterocyclic rings and the divergent reactivity that can be obtained by simply altering the reaction solvent and temperature. The synthesised derivatives were then tested against HeLa cancer cells for their antiproliferative properties. A number of promising compounds were obtained including 4, 5 and 9 that showed an IC50 of 878, 311 and 116 nM, respectively, against HeLa cancer cells after 48 h of treatment.

Biochemical and Neurobehavioral Changes in the Rat Model of Parkinson�s Disease with Suprathreshold Exercise and Usnic Acid

Finding a solution for Parkinson's disease has been a challenge to researchers despite advances in medicinal research. None of the options available so far has been able to reverse dopaminergic damage or increase endogenous dopamine production from existing dopaminergic cells in Parkinson's disease. Research in Parkinson�s disease has not so far resulted in finding better solutions for reversing dopaminergic damage or increasing dopamine production. Above-the-threshold (suprathreshold) exercises producing improvement have been documented with supporting clinical and imaging findings but without any biochemical evidence. Above-the-threshold (suprathreshold) exercises producing improvement in Parkinson's disease have been documented with supporting clinical and imaging findings. To the best of our knowledge, the biochemical basis of this improvement has not been studied in detail. Recently, biomolecules like usnic acid have been shown to improve dopamine levels through protective and regenerative effects. This study aimed at understanding the biochemical basis of the improvement following suprathreshold exercises and usnic acid. The objectives were to assess whether the suprathreshold exercise produces improvement in PD rat models and, if so, to understand the biochemical basis of improvement and compare the mechanism with that of usnic acid.The effect of suprathreshold exercise and usnic acid on Parkinson�s disease Hence, this study was conceived to understand whether the exercise produces results due to a similar mechanism that had never been reported before. Parkinson's disease was compared with rotenone alone, rotenone and high-fat diet models, and control. Induced with rotenone and a high-fat diet, the effect of suprathreshold exercise was compared to that of usnic acid, the rotenone alone model, and control. Locomotor behaviour was assessed with an actophotometer and rotarod. Western blotting assessed the blood biochemical parameters, antioxidant and dopamine levels in addition to a-synuclein, tyrosine hydroxylase, Nurr1, and HSP70 levels. Usnic acid decreased oxidative stress markers like MDA, peroxynitrite and nitric oxide, indicating lesser oxidative stress, increased dopamine and serotonin levels and decreased a-synuclein expression. But the suprathreshold exercise did not significantly affect oxidative stress markers nor increased dopamine levels. However, a-synuclein expression was decreased. Compared with usnic acid, lower Nurr1 levels in the suprathreshold group demonstrate lesser protective and regenerative effects on dopaminergic neurons. HSP70 levels indicate that improvement in suprathreshold exercise resulted from preventing misfolding of a-synuclein or through a noncanonical interaction, and comparable locomotor behaviour was observed in both groups. Biomolecules like brain-derived neurotropic factors produced by suprathreshold exercises acting centrally and in muscles might have also contributed to improvement in this group. When usnic acid increased dopamine levels in the brain through protective effects on dopaminergic neurons, antioxidant effects, and reduced expression of a-synuclein, suprathreshold exercise did not increase dopamine, decreased a-synuclein and had less antioxidant effects. This study biochemically proves that suprathreshold exercise alleviates Parkinson�s disease symptoms by preventing a-synuclein misfolding and not by mechanisms other than increasing dopamine, neuroprotection or regeneration of dopaminergic neurons, which require further studies in humans.

THE ROLE OF PLANT EXTRACTS IN THE REPAIR OF Rattus Norvegicus MANDIBULAR ALVEOLAR BONE IN A PERIODONTITIS MODEL

The evaluated herbs plants (α-Mangostin, usnic acid, Piper betle L, and Daemonorops draco) have potency in the alveolar bone repair of mandibular on the periodontitis modeling. Its herbs were evaluated with the flavonoid and phenolic, and ionic values identified by a multi-tester meter. Mol-inspiration software was used to calculate the miLogP value of -mangostin and usnic acid. The evaluation of alveolar bone repair is based on H and E staining profiles of osteoblasts and osteoclasts. The α-Mangostin and Piper beetle L had higher total flavonoid and phenolic concentrations than usnic acid and Daemonorops draco. Based on the miLogP value, usnic acid has better absorption than α-mangostin. The ionic amounts of dissolved Oxygen, conductivity, and total dissolved solids have correlated with osteoblast and osteoclast expression in the bone repair process. The herbs extract can increase the stimulation to produce osteoclast and stability in 7 and 14 days. The ionic values, flavonoids, and phenolic totals of herbs affect the mandibular alveolar bone repair, which is marked to increase the new bone formation (osteoblast) and bone resorption (osteoclast).

Antioxidant Activity of Usnic Acid Compound from Methanol Extract of Lichen Usnea sp.

Lichen Usnea sp. is one of the sources of natural bioactive compounds which are currently being developed as medicinal ingredients. The purpose of this study was the isolation and identification of secondary metabolites from methanol extract, toxicity test and antioxidant activity of Usnea sp. Lichen was isolated by maceration using methanol solvent, then separated by liquid-liquid partition and separation using vacuum chromatography. Based on the results of the study, NMR-1D spectral data and FTIR spectrum is the presence of functional groups showed the presence of F15 compound is usnic acid consisting of 18 carbons with 3 carbons from the C=O carbonyl group. The results of the toxicity test showed that all of them were active against Artemia salina L. shrimp larvae with LC50 values of 0.820 µg/mL (Usnea sp.), 1.030 µg/mL (n-hexane), 1.056 µg/mL (ethyl acetate), and 1.236 µg/mL (methanol extract). The results of the antioxidant activity test showed that the inhibitory activity of usnic acid isolate was very active with an IC50 value of 11.696 µg/mL. Meanwhile, methanol extracts and ethyl acetate showed antioxidant activity with IC50 values of 18.098 and 26.917 µg/mL, respectively.

Virtual screening, identification and in vitro validation of small molecule GDP-mannose dehydrogenase inhibitors.

Upon undergoing mucoid conversion within the lungs of cystic fibrosis patients, the pathogenic bacterium Pseudomonas aeruginosa synthesises copious quantities of the virulence factor and exopolysaccharide alginate. The enzyme guanosine diphosphate mannose dehydrogenase (GMD) catalyses the rate-limiting step and irreversible formation of the alginate sugar nucleotide building block, guanosine diphosphate mannuronic acid. Since there is no corresponding enzyme in humans, strategies that could prevent its mechanism of action could open a pathway for new and selective inhibitors to disrupt bacterial alginate production. Using virtual screening, a library of 1447 compounds within the Known Drug Space parameters were evaluated against the GMD active site using the Glide, FRED and GOLD algorithms. Compound hit evaluation with recombinant GMD refined the panel of 40 potential hits to 6 compounds which reduced NADH production in a time-dependent manner; of which, an usnic acid derivative demonstrated inhibition six-fold stronger than a previously established sugar nucleotide inhibitor, with an IC50 value of 17 μM. Further analysis by covalent docking and mass spectrometry confirm a single site of GMD alkylation.

Analyzing the Impact of Ramalina digitellata, R. fastigiata, R. fraxinea, and R. polymorpha's Usnic Acid Concentration on Antioxidant, DNA-Protective, Antimicrobial, and Cytotoxic Properties.

The present study is focused on the antimicrobial, antioxidant, cytotoxic, and DNA protective effects of methanol extract obtained from R. digitellata, R. fastigiata, R. fraxinea, and R. polymorpha species that are distributed in Turkey. The highest total phenol content was determined in R. digitellata (144.6 mgGAE/gextract ), and the highest total amount of flavonoids was found in R. fastigiata (20.40 mgGAE/gextract ). The content of usnic acid was determined by High-Performance Liquid Chromatography (HPLC) and the highest amount was found in R. digitellata. DPPH (1,1-diphenyl-2-picrylhydrazyl) and ABTS [2,2'-azinobis(3-ethylbenzathiazoline-6-sulfonic acid)] radical scavenging methods were used for antioxidant activity. R. fraxinea showed the highest DPPH⋅ and ABTS+ ⋅ scavenging activity. In addition, the DNA protective effect was investigated using pBR322 plasmid DNA, and; all studied species were found to have DNA protective effects. The antibacterial activity was investigated using the disc diffusion method, and the R. digitellata methanol extract showed the best results with a 12.35 mm zone on Proteus mirabilis. On the human lung cancer (A549) and breast cancer (MDA-MB-231) cell lines, cytotoxic activity was assessed using an MTT assay. All lichen extracts were found to have a significant cytotoxic effect on both cancer cell lines at 1000 μg/mL concentration. These results suggest that Ramalina species may be potential candidates for developing new phytopharmaceuticals and functional components.

Pharmacophore-based Molecular Docking of Usnic Acid Derivatives to Discover Anti-viral drugs Against Influenza A Virus

Pharmacophore-based Molecular Docking of Usnic Acid Derivatives to Discover Anti-viral drugs Against Influenza A Virus

Usnic acid based thiazole-hydrazones as multi-targeting inhibitors of a wide spectrum of SARS-CoV-2 viruses

We have identified new usnic acid derivatives that are active against a wide range of strains of the SARS-CoV-2 virus and have multi-targeting effects.

PCL/Collagen/UA Composite Biomedical Dressing with Ordered Microfiberous Structure Fabricated by a 3D Near-Field Electrospinning Process.

In this work, a functionalized polycaprolactone (PCL) composite fiber combining calf-type I collagen (CO) and natural drug usnic acid (UA) was prepared, in which UA was used as an antibacterial agent. Through 3D near-field electrospinning, the mixed solution was prepared into PCL/CO/UA composite fibers (PCUCF), which has a well-defined perfect arrangement structure. The influence of electrospinning process parameters on fiber diameter was investigated, the optimal electrospinning parameters were determined, and the electric field simulation was conducted to verify the optimal parameters. The addition of 20% collagen made the composite fiber have good hydrophilicity and water absorption property. In the presence of PCUCF, 1% UA content significantly inhibited the growth rate of Gram-positive and negative bacteria in the plate culture. The AC-PCUCF (after crosslinking PCUCF) prepared by crosslinking collagen with genipin showed stronger mechanical properties, water absorption property, thermal stability, and drug release performance. Cell proliferation experiments showed that PCUCF and AC-PCUCF had no cytotoxicity and could promote cell proliferation and adhesion. The results show that PCL/CO/UA composite fiber has potential application prospects in biomedical dressing.

-Based Virtual Screening Towards The Discovery of Usnic Acid Derivatives as Novel mTOR Inhibitor to Treat Breast Cancer

In this study, mTOR is chosen as the main target for breast cancer treatment. While the existing drugs still pose severe side effects, research on finding new anti-cancer drug should be done continuously. Usnic acid (UA) has been studied for its wide range of biological properties and potential in pharmaceutical research. A structure-based virtual screening approach is applied since it could reduce production time, cost and environmental issues. This study comprises molecular docking simulation, ADMET filtration and drug-likeness prediction. 340 UA derivatives were retrieved from literature and used to build an in-house database. The resulting compounds from docking were then filtered using ADMET prediction which comprises human intestine absorption, aqueous solubility, plasma protein binding, cytochrome P450 2D6 (CYP2D6) and hepatotoxicity parameters to identify the most potent UA derivatives with favourable physicochemical characteristics. After all, the hit compound, 118, was further stimulated in order to forecast its drug-like features. The chalcone-based scaffold of 118 resembled the reported breast cancer compound’s chemical structure strengthening the results obtained from this study. Thus, it is concluded that the structure-based virtual screening was an efficient and effective approach in the discovery of UA derivative, 118, as a potential novel mTOR inhibitor to treat breast cancer.

Rhizoplaca ouimetensis sp. nov. (Lecanoraceae) from Ontario, the first sorediate species in the genus

Rhizoplaca ouimetensis is described new to science, growing on outcrops of diabase sills in the Lake Superior region of Ontario, Canada. It is the first known sorediate species of the genus, and a phylogenetic reconstruction based on the ITS and mtSSU markers place it in the R. chrysoleuca group. Morphologically, however, it resembles sorediate, yellow-green species of Lecanora with usnic acid, e.g., L. handelii and L. soralifera, but differs from those in forming larger, often pulvinate or minutely peltate areoles with a well-developed upper cortex and a medulla densely filled with calcium oxalate crystals.

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.

Uncovering the Anticancer Potential of Lichen Secondary Metabolites

Lichens produce a plethora of primary and secondary metabolites. Secondary metabolites have several biological functions that can be used for human health. Recent studies have described their antioxidant, anti-inflammatory, antimycotic, and antibiotic/antiviral activities. However, attention has mainly been focused on their antiproliferative, cytotoxic, and anticancer effects. Because there are many publications describing the molecular mechanisms leading to the anticancer effects of lichen secondary metabolites, the aim of this review is to summarize results from current research with the main emphasis on atranorin, usnic and gyrophoric acid.