Usnic Acid Content Research Articles

Controlling Oral Polymicrobial Biofilm Using Usnic Acid on the Surface of Titanium in the Artificial Saliva Media

Background/Objectives: Titanium dental implants, while highly successful, face challenges due to polymicrobial infections leading to peri-implantitis and implant failure. Biofilm formation on implant surfaces is the primary cause of these infections, with factors such as matrix production and cross-kingdom interactions contributing to the microbial accumulation of bacterial and fungal pathogens species. To combat this issue, naturally derived molecules have been reported to overcome the hurdle of antimicrobial resistance against the application of conventional antibiotics and antifungals. Methods: The present study aimed to employ the lichen-derived molecules, usnic acid (UA), to retard the development of biofilms of bacterial and fungal pathogens on the surface of titanium kept in the human artificial saliva (HAS) working as a growth-supporting, host-mimicking media. Results: The minimum inhibitory concentration of UA in HAS towards Candida albicans was >512 µg/mL, whereas against Staphylococcus aureus and Streptococcus mutans, it was determined to be 512 µg/mL. Whereas, in the standard growth media, the MIC value of UA towards S. mutans and S. aureus were 8 and 16 µg/mL; however, against C. albicans, it was 512 µg/mL. UA synergistically enhanced the efficacy of the antibiotics toward bacterial pathogens and the efficacy of antifungals against C. albicans. The antibiofilm results depict the fact that in the HAS, UA significantly reduced both mono-species of S. mutans, S. aureus, and C. albicans and mixed-species biofilm of C. albicans with S. mutans and S. aureus on the surface of the titanium. Conclusions: The present study showed that UA is a promising natural drug that can control oral polymicrobial disease as a result of the application of dental implants.

Electrospun nanofibers for localized drug release of a neuroprotective natural extract of Usnea ghattensis

This research is based on the incorporation of the methanolic extract of the Usnea ghattensis into poly (caprolactone) (PCL) nanofibers (NFs) to investigate the capacity in reducing reactive oxygen species (ROS). PCL-NFs were fabricated by the electrospinning technique and are investigated as potential dressing material focused on the release of usnic acid (PCL-USNIC NFs), and its encapsulation efficiency and kinetic release were analyzed by high performance liquid chromatography (HPLC). This investigation was performed by analyzing the usnic acid concentration as a function of the distance from the mat center point. The kinetic release analysis is also developed with the usnea ghattensis extract (PCL-USNEA NFs), performing a metabolomic analysis of the released molecules as a function of time by nuclear magnetic resonance (NMR). Usnic acid was revealed as the most relevant compound together with other molecules, such as sucrose, mannitol, arabitol or glycerol that generate a positive matrix effect on the release of usnic acid. Finally, we analize the cytotoxicity and the neuroprotective effect of PCL-USNEA and PCL-USNIC NFs using a human neuroblastoma cell line model. Negligible toxicity was appreciated for both polymeric systems, showing high protective effects in presence of highly oxidative environment (e.g. in presence of H2O2).

Identification and determination of usnic acid and fatty acid from various lichen species in arequipa, Peru, as well as antibacterial and antioxidant capacity

Seven species of lichens such us Umbilicaria aff. calvescens, Hypotrachyna enderythraea, Punctelia graminicola, Cladonia chlorophaea, Xanthoparmelia farinose, Psiloparmelia distincta (rock and tree substrates; rs and ts), and Usnea durietzii were collected from the province of Arequipa - Peru. Their usnic acid (UA) and fatty acid (FA) amounts in methanol-acetone were determined by High-performance liquid chromatography with diode-array detection (HPLC-DAD) and Gas chromatography-flame ionization detector (GC-FID). The antimicrobial activities of these extracts were evaluated against Staphylococcus aureus ATCC strains (43300, 29213, 25923 and 700699), Escherichia coli strains (O157:H7 and ATCC 10536), Salmonella enterica sv typhimurium ATCC 14028, Candida albicans ATCC 90028 and Candida. tropicalis ATCC 750T. In addition, antioxidant capacity was also studied by Total phenolic content (TPC), 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical, 2,2-Azino-bis-3-ethylbenzothiazoline-6-sulfonic Acid (ABTS) radical cation and Ferric Reducing Antioxidant Power (FRAP) assays. The UA contents in all the studied lichen species varied between 0.017 and 0.304 %. Among all the tested extracts, Usnea durietzii had the highest total FA concentration (5.81 mg/g). P. distincta (rs) was active against S. aureus strains (MIC = 0.02–0.2 mg/mL) as well as C. albicans (MIC = 4 mg/mL) and C. tropicalis (MIC = 3 mg/mL). P. distincta (ts) displayed moderate total phenol content (TPC = 29.1 ± 1.6 mg GAE/g) and antioxidant capacity evidenced by scavenging DPPH (IC50 = 1.45 ± 0.03 mg/mL) and ABTS (18.2 ± 0.2 mg TE/g) radicals, but it showed high values of FRAP (1953 ± 87 μM Fe2+/g). Our findings indicate that P. distincta (rs) could be considered as a potential antimicrobial metabolites source whereas P. distincta (ts) and Puntelia graminicole for polyphenols with antioxidant compound.

The algal partnership is associated with quantitative variation of lichen specific metabolites in Cladonia foliacea from Central and Southern Europe

AbstractLichen specific metabolites (LSMs) have interesting biological activities and quantitative variations may be present intraspecifically. For example, variations in medullary fumarprotocetraric acid (FA) and cortical usnic acid (UA) were observed in the lichen Cladonia foliacea, but the mechanism of variation is not well understood. The current study aimed to characterise the quantitative variation of FA and UA and to investigate the association between lichen metabolite content and ecological / biological variables. Fungal and algal trees were constructed using fungal (nrITS, RPB2) and algal (nrITS) loci, respectively. Using a chiral chromatographic method, the contents of (-)-UA were determined in 29 C. foliacea specimens and range from 6.88 to 34.27 mg/g dry wt. The FA contents were lower and varied from 1.44 to 9.87 mg/g dry wt. Although the fungal tree showed two well resolved clades, no significant differences of UA or FA contents were found between the two fungal clades. However, a significantly higher UA/FA ratio as well as a unique habitat were found to be associated with specimens which contained the alga Asterochloris lobophora than those specimens associated other Asterochloris algae. Taking all predictive variables into account (i.e. substrate type, elevation, collection season, photobiont identity), the multivariate data analysis indicated that photobiont identity explained most of the variance of LSM contents in C. foliacea. Thus future LSM biosynthetic studies should take the photobiont into consideration when dealing with intraspecific quantitative variation.

Methodological Aspects of Green Extraction of Usnic Acid Using Natural Deep Eutectic Solvents.

Usnic acid (UA) is a compound with multiple biological activities that make it useful in various industries, e.g., pharmaceutical, cosmetic, dentistry, and agricultural sectors. Lichens are the primary source of UA, which is primarily extracted using acetone. This study aimed to investigate the solubility of UA in numerous natural deep eutectic solvents (NADESs) and use a mixture of thymol and camphor as a NADES in the optimization of the UA extraction process with the design of experiments method. For numerical optimization, the following parameters were employed in the experiment to confirm the model: a camphor-to-thymol ratio of 0.3, a liquid-to-solid ratio of 60, and a time of 30 min. The obtained experimental results aligned well with the predicted values, with the mean experimental value falling within the confidence interval, exhibiting deviations between 11.93 and 14.96. By employing this model, we were able to optimize the extraction procedure, facilitating the isolation of approximately 91% of the total UA content through a single extraction, whereas a single acetone extraction yielded only 78.4% of UA.

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.

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.

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.

Revision of Dimelaena Norman (Caliciaceae, Ascomycota) species containing usnic acid reveals a new species and a new combination from China

Dimelaena tibetica is described as a species new to science, characterized by a grayish yellow surface, usually with a covering of white pruina, a crustose thallus, areolate at center, radiate-plicate at margin, adnate apothecia and lacking gyrophoric acid. It differs from other usnic acid-containing Dimelaena species by its plane marginal lobes and adnate apothecia. Based on the study of fresh specimens from the environment of the type locality, Dimelaena altissima is proposed as a new combination. A fresh specimen of Dimelaena oreina (type species of the genus Dimelaena) was collected in the Swiss Alps, in the vicinity of the area where the type material of the species presumably was collected, and a sequence was generated to confirm the phylogenetic position of this genus. All three species share common characters: usnic acid content and a yellowish green upper surface. Descriptions of these species were based on a combination of morphological and phylogenetic analyses. A key and phylogram are provided for these three Dimelaena species which produce usnic acid.

In Vitro Anticancer Activity of Mucoadhesive Oral Films Loaded with Usnea barbata (L.) F. H. Wigg Dry Acetone Extract, with Potential Applications in Oral Squamous Cell Carcinoma Complementary Therapy.

Oral squamous cell carcinoma (OSCC) is the most frequent oral malignancy, with a high death rate and an inadequate response to conventional chemotherapeutic drugs. Medical research explores plant extracts’ properties to obtain potential nanomaterial-based anticancer drugs. The present study aims to formulate, develop, and characterize mucoadhesive oral films loaded with Usnea barbata (L.) dry acetone extract (F-UBA) and to investigate their anticancer potential for possible use in oral cancer therapy. U. barbata dry acetone extract (UBA) was solubilized in ethanol: isopropanol mixture and loaded in a formulation containing hydroxypropyl methylcellulose (HPMC) K100 and polyethylene glycol 400 (PEG 400). The UBA influence on the F-UBA pharmaceutical characteristics was evidenced compared with the references, i.e., mucoadhesive oral films containing suitable excipients but no active ingredient loaded. Both films were subjected to a complex analysis using standard methods to evaluate their suitability for topical administration on the oral mucosa. Physico-chemical and structural characterization was achieved by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), thermogravimetric analysis (TGA), scanning electron microscopy (SEM), and atomic force microscopy (AFM). Pharmacotechnical evaluation (consisting of the measurement of specific parameters: weight uniformity, thickness, folding endurance, tensile strength, elongation, moisture content, pH, disintegration time, swelling rate, and ex vivo mucoadhesion time) proved that F-UBAs are suitable for oral mucosal administration. The brine shrimp lethality (BSL) assay was the F-UBA cytotoxicity prescreen. Cellular oxidative stress, caspase 3/7 activity, nuclear condensation, lysosomal activity, and DNA synthesis induced by F-UBA in blood cell cultures and oral epithelial squamous cell carcinoma (CLS-354) cell line were investigated through complex flow cytometry analyses. Moreover, F-UBA influence on both cell type division and proliferation was determined. Finally, using the resazurin-based 96-well plate microdilution method, the F-UBA antimicrobial potential was explored against Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27353, Candida albicans ATCC 10231, and Candida parapsilosis ATCC 22019. The results revealed that each UBA-loaded film contains 175 µg dry extract with a usnic acid (UA) content of 42.32 µg. F-UBAs are very thin (0.060 ± 0.002 mm), report a neutral pH (7.01 ± 0.01), a disintegration time of 146 ± 5.09 s, and an ex vivo mucoadhesion time of 85 ± 2.33 min, and they show a swelling ratio after 6 h of 211 ± 4.31%. They are suitable for topical administration on the oral mucosa. Like UA, they act on CLS-354 tumor cells, considerably increasing cellular oxidative stress, nuclear condensation, and autophagy and inducing cell cycle arrest in G0/G1. The F-UBAs inhibited the bacterial and fungal strains in a dose-dependent manner; they showed similar effects on both Candida sp. and higher inhibitory activity against P. aeruginosa than S. aureus. All these properties lead to considering the UBA-loaded mucoadhesive oral films suitable for potential application as a complementary therapy in OSCC.

Formulation and Development of Bioadhesive Oral Films Containing Usnea barbata (L.) F.H.Wigg Dry Ethanol Extract (F-UBE-HPC) with Antimicrobial and Anticancer Properties for Potential Use in Oral Cancer Complementary Therapy.

Medical research explores plant extracts’ properties to obtain potential anticancer drugs. The present study aims to formulate, develop, and characterize the bioadhesive oral films containing Usnea barbata (L.) dry ethanol extract (F-UBE-HPC) and to investigate their anticancer potential for possible use in oral cancer therapy. The physicochemical and morphological properties of the bioadhesive oral films were analyzed through Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), Atomic Force Microscopy (AFM), thermogravimetric analysis (TG), and X-ray diffraction techniques. Pharmacotechnical evaluation (consisting of the measurement of the specific parameters: weight uniformity, thickness, folding endurance, tensile strength, elongation, moisture content, pH, disintegration time, swelling rate, and ex vivo mucoadhesion time) completed the bioadhesive films’ analysis. Next, oxidative stress, caspase 3/7 activity, nuclear condensation, lysosomal activity, and DNA synthesis induced by F-UBE-HPC in normal blood cell cultures and oral epithelial squamous cell carcinoma (CLS-354) cell line and its influence on both cell types’ division and proliferation was evaluated. The results reveal that each F-UBE-HPC contains 0.330 mg dry extract with a usnic acid (UA) content of 0.036 mg. The bioadhesive oral films are thin (0.093 ± 0.002 mm), reveal a neutral pH (7.10 ± 0.02), a disintegration time of 118 ± 3.16 s, an ex vivo bioadhesion time of 98 ± 3.58 min, and show a swelling ratio after 6 h of 289 ± 5.82%, being suitable for application on the oral mucosa. They displayed in vitro anticancer activity on CLS-354 tumor cells. By considerably increasing cellular oxidative stress and caspase 3/7 activity, they triggered apoptotic processes in oral cancer cells, inducing high levels of nuclear condensation and lysosomal activity, cell cycle arrest in G0/G1, and blocking DNA synthesis. All these properties lead to considering the UBE-loaded bioadhesive oral films suitable for potential application as a complementary therapy in oral cancer.

Содержание фенольных соединений в лишайниках в зонах тектонических разломов

The paper studies the influence of geoecological factors on the content of phenolic compounds (PCs) in lichens of the genus Cladonia in the Velsko-Ustyansky and Kholmogorsky tectonic knots (TKs). Lichens were sampled from the test plots (TPs) laid out in the center of the knots and outside the knots (control). The geochemical situation, soil-climatic and environmental conditions in the zone of TKs influence the lichens chemical composition. It is found that the ash content of lichens growing in the center of the TK is 1.8–2.3 times higher than in the control, which indicates a significant accumulation of lithogenic elements in the lichen thallus in the tectonic fault zone. Lichens are exposed to multifactorial adverse effects (increased radiation background and heat flux, electromagnetic radiation, electric and magnetic fields, etc.) in the territory of TKs. This provokes excessive generation of active oxygen radicals and contributes to the rearrangement of lichen metabolism, changing the dynamics of accumulation of PCs and their biosynthesis. The content of the water-insoluble fraction of PCs in lichens was found to increase from the control point to the center of the TK, while the relative share of the water-soluble fraction of PCs in the total composition decreases. This is probably due to an increase in oxidative processes and a decrease in reductive processes in lichens under adverse conditions. The paper shows that the biosynthesis of PCs in lichens is most active in the spring-summer (April and July) vegetation period. The impact of geoecological factors enhances the biosynthesis of secondary metabolites of phenolic nature in lichens in the conditions of the TKs: usnic acid (UA) and atranorin. The content of UA in the composition of the water-insoluble fraction of PCs in lichens growing in the center of the TK is 1.5–1.7 times higher during the spring-summer vegetation period than in lichens growing outside the TK. The quantitative distribution of the water-insoluble fraction of PCs depends on the lichen thallus part and has a gradient character. Their content in the upper growing parts of lichens growing in the center of the TK and in the control is 2 and 1.7 times higher than in the old zones, respectively. Thus, PCs in lichens are active metabolites and their content in thallus can serve as a biomarker of the state of the environment.

Phenolic Secondary Metabolites and Antiradical and Antibacterial Activities of Different Extracts of Usnea barbata (L.) Weber ex F.H.Wigg from Călimani Mountains, Romania.

Phenolic compounds represent an essential bioactive metabolites group with numerous pharmaceutical applications. Our study aims to identify and quantify phenolic constituents of various liquid and dry extracts of Usnea barbata (L.) Weber ex F.H. Wigg (U. barbata) from Calimani Mountains, Romania, and investigate their bioactivities. The extracts in acetone, 96% ethanol, and water with the same dried lichen/solvent ratio (w/v) were obtained through two conventional techniques: maceration (mUBA, mUBE, and mUBW) and Soxhlet extraction (dUBA, dUBE, and dUBW). High-performance liquid chromatography with diode-array detection (HPLC-DAD) was performed for usnic acid (UA) and different polyphenols quantification. Then, the total phenolic content (TPC) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) free-radical scavenging activity (AA) were determined through spectrophotometric methods. Using the disc diffusion method (DDM), the antibacterial activity was evaluated against Gram-positive and Gram-negative bacteria known for their pathogenicity: Staphylococcus aureus (ATCC 25923), Streptococcus pneumoniae (ATCC 49619), Pseudomonas aeruginosa (ATCC 27853), and Klebsiella pneumoniae (ATCC 13883). All extracts contain phenolic compounds expressed as TPC values. Five lichen extracts display various UA contents; this significant metabolite was not detected in dUBW. Six polyphenols from the standards mixture were quantified only in ethanol and water extracts; mUBE has all individual polyphenols, while dUBE shows only two. Three polyphenols were detected in mUBW, but none was found in dUBW. All U. barbata extracts had antiradical activity; however, only ethanol and acetone extracts proved inhibitory activity against P. aeruginosa, S. pneumoniae, and S. aureus. In contrast, K. pneumoniae was strongly resistant (IZD = 0). Data analysis evidenced a high positive correlation between the phenolic constituents and bioactivities of each U. barbata extract. Associating these extracts’ properties with both conventional techniques used for their preparation revealed the extraction conditions’ significant influence on lichen extracts metabolites profiling, with a powerful impact on their pharmacological potential.

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.

Fungal metabolite isolated from Mycosphaerella nawae AM20 and its protective role in cerebral ischemia

Usnic acid is one of the important metabolites showed cardiovascular protective properties. The main aim of the study was to analyze antioxidant and cerebral ischemia preventive role in experimental Albino rats. The amount of usnic acid in the Mycosphaerella nawae AM20 culture extract was determined by High Performance Liquid Chromatography. Antioxidant property was evaluated by various methods and usnic acid has potential Fe2+ chelating activity. The influence of usnic acid on special learning and memory was studied in vivo using Albino rats. Morris water maze (MWM) analysis revealed that the experimental ischemic rats treated with usnic acid reached the platform at minimum time than ischemic rats without usnic acid treatment. At 0.1 mL usnic acid concentration, hydroxy-3-methyl-glutaryl-CoA reductase (HMGR) inhibition activity was 18.4 ± 1.1% and it increased as 80.3 ± 2.4% at 0.5 mL concentration in the reaction mixture. Likewise, acetylcholinesterase (ACE) inhibitor activity was observed and was dose dependent. At 0.2 mL concentration, 21.8 ± 1.7% enzyme activities inhibited and it increased as 48.3 ± 1.8% at 0.5 mL sample concentration. Fibrinolytic analysis revealed 13 ± 0 mm zone of clearance at 0.5 mL sample concentration and the standard plasmin showed 17 ± 2 mm zone of clearance. Biochemical analysis revealed that the amount of malondialdehyde (MDA) was higher in the hippocampal region of ischemia group animal (59.4 ± 3.5 μM). Moreover, usnic acid treated ischemia group showed reduced MDA content (29.8 ± 3.5 μM). Superoxide dismutase (SOD) activity was reduced in ischemic albino rat and improved SOD activity was observed in the treated animal.

Usnic acid-induced programmed cell death in ovarian cancer cells

Usnic acid (UA) is a secondary metabolite obtained by lichen species and exerts a significant anti-proliferative effect on different cancer cells. The current study aimed to determine the efficiency of UA on ovarian cancer and reveal the underlying molecular mechanisms of UA mediated anti-cancer activity. In this study, the cytotoxic effect of UA was evaluated by xCELLigence real-time online cell analysis in OVCAR-3, A2780 ovarian cancer cells and L929 non-cancerous cell compared with Carboplatin. After treatment with IC50 concentration of UA, apoptosis, cell cycle analysis, mRNA expression levels, invasion, wound healing and metastasis assays were performed to evaluate of the molecular mechanism of the apoptosis pathway. UA showed more anti-proliferative effect than carboplatin on OVCAR-3 which is the most aggressive cell among ovarian cancer cells. UA significantly decreased the cell proliferation of OVCAR-3 cells through apoptosis and G0/G1 arrest. Additionally, UA inhibited the invasion and particularly the migration of OVCAR-3 cells compared with control. Moreover, the expression level of 63 target genes from 87 apoptosis associated genes in the primary panel was determined after IC50 concentration of UA in OVCAR-3 cells. UA caused apoptotic cell death through the extrinsic pathway and over-expression of p53. Our findings demonstrated that UA-induced early and late apoptosis through G0/G1 arrest and extrinsic apoptosis signaling pathways and inhibited the migration and invasion of OVCAR-3 cells. Therefore, UA as a novel candidate molecule may be used for the treatment of ovarian cancer.Graphical abstractUsnic acid (UA) exerts anti-cancer effects and induces apoptosis through the extrinsic pathway and over-expression of p53. Additionally, UA potentially inhibits the migration ability of OVCAR-3 ovarian cancer cells. (Created with BioRender.com)

Antibacterial Activity and Stability of Methanol Extract from Thamnolia subuliformis in Vitro

The objective of the study was preliminarily explore the antibacterial activity and stability of Thamnolia subuliformis in vitro, the plate drilling method was used to determine the antibacterial activity of methanol extract of Thamnolia subuliformis against common pathogenic bacteria in vitro. The indicator bacteria of Staphylococcus aureus was used to determine the effects of factors such as different temperature, pH and UV irradiation time on antibacterial stability. Petroleum ether, ethyl acetate and n-butanol were used to extract the methanol extract, determine the polarity of its antibacterial active substances, and determine the content of usnic acid. The results showed that the methanol extract of Thamnolia subuliformis had good antibacterial effect on Gram-positive bacteria such as Staphylococcus aureus, Bacillus subtilis, Listeria stephensi, Staphylococcus surface, Listeria evansi, paratyphoid A, paratyphoid B of Gram-negative bacteria and Candida albicans. The minimum inhibitory concentration and minimum bactericidal concentration against Staphylococcus aureus were 0.625 and 10 mg/mL respectively; The results of antibacterial stability test showed that temperature had no significant effect on the antibacterial activity of methanol extract of Thamnolia subuliformis（P>0.05）, and it still had high antibacterial activity after treatment at 100 ℃ for 30 min; The antibacterial activity of UV irradiation for 40 min and 50 min decreased significantly (P 0.05）, showing high antibacterial activity. When the pH was high (8.0, 10.0) or low (2.0, 4.0), the antibacterial activity of the treatment group decreased significantly (P<0.05), and decreased with the increase or decrease of pH, The content of usnic acid in methanol extract and ethyl acetate extract of Thamnolia subuliformis was 0.8613% and 0.9379%, respectively. The results show that the methanol extract of Thamnolia subuliformis has a certain broad-spectrum antibacterial effect, low concentration and good stability. It can be used for the development and utilization of natural food preservatives and plant-derived antibacterial drugs.

Quantitative variations of usnic acid and selected elements in terricolous lichen Cladonia mitis Sandst., with respect to different environmental factors – A chemometric approach

In the present study, quantitative analysis of usnic acid and the selected elements (Ag, Ca, Cd, Cr, Cu, Mg, Mn, Ni, Pb, Zn) in samples of Cladonia mitis thalli collected along a North-Central Europe transect, was performed. Additionally, we analysed geographical and environmental factors that could potentially influence the levels of the analytes. With the constructed partial least square (PLS) chemometric model we aimed to establish the relationships between the analysed factors. Our study is the first to provide comparative quantitative data on usnic acid in C. mitis. We proved this lichen species is a rich source of usnic acid (4.52 ± 0.54–21.58 ± 2.23 mg/g dry wt). Significant differences in usnic acid content between samples collected in the open and forest areas were noted, with the predominance of the former (mean 17.4 ± 2.8 and 8.8 ± 2.3 mg/g dry wt, respectively). Our results supported the hypothesis on the relationship between usnic acid and selected heavy metals content in lichens, as the positive correlation of the compound was noted with Pb. Our PLS model indicated also a positive correlation between usnic acid and Cr content, which was reported for the first time.

Supercritical carbon dioxide extraction of Usnea longissima (L.) Ach.: Optimization by Box-Behnken design (BBD).

Usnic acid (UA) was extracted from Usnea longissima (L.) Ach. in supercritical carbon dioxide (SC-CO2) medium. The selected process parameters were extraction temperature (35–45 °C), amount of co-solvent (0%–5%) and extraction time (5–9 h). These parameters were applied to Box-Behnken design (BBD) belonging to response surface methodology (RSM) to determine optimum process parameters for the highest amount of UA in the extract. g UA/100g lichen, extraction yield % and UA content values were calculated in the range of 0.045–0.317, 2.77–5.4 and 71%–82% in different experimental conditions, respectively. The optimum conditions were predicted as 42 °C, 4.3% (ethanol) and 7.48 h. It was determined that the predicted and experimental values of g UA/100g lichen were compatible, and the suggested model was valid.

Effect of environmental factors on polyketide synthase gene expression, usnic acid biosynthesis, and ground cover of a lichen-fungus, Cladonia uncialis

Lichens are composed of symbiotic slow-growing organisms and are often exposed to extreme microenvironmental conditions, resulting in the production of unique secondary metabolites. One of the most commonly produced secondary metabolites is usnic acid, which is thought to be produced by two genes. The objectives of the present study were to compare polyketide synthase (PKS) gene expression and usnic acid concentration in Cladonia uncialis (L.) Weber ex F.H. Wigg., with two environmental factors. Seventy-five lichen samples were collected from three locations in Newfoundland, Canada, using a strip transect method (×5 transects, ×5 quadrats). Usnic acid concentration was measured using the liquid chromatography tandem mass-spectrometric method and gene expression of two PKS genes (methylphloracetophenone oxidase (MPAO) and methylphloracetophenone synthase (MPAS)) was examined using quantitative real-time PCR. The results showed that percent ground cover of C. uncialis was affected by soil pH level but not soil moisture, and usnic acid concentration was not affected by either soil pH or soil moisture. MPAO gene expression level was significantly affected by soil pH level but not soil moisture, and MPAS gene expression level was not affected by either soil pH level or soil moisture. There was no significant relationship between MPAS and MPAO gene expression levels and usnic acid concentration. These findings suggest that soil pH may be important for the production of usnic acid by C. uncialis but the genes involved require further study.