Nowadays, numerous biomedical studies performed on natural compounds and plant extracts aim to obtain highly selective pharmacological activities without unwanted toxic effects. In the big world of medicinal plants, Usnea barbata (L) F.H. Wigg (U. barbata) and usnic acid (UA) are well-known for their therapeutical properties. One of the most studied properties is their cytotoxicity on various tumor cells. This work aims to evaluate their cytotoxic potential on normal blood cells. Three dry U. barbata extracts in various solvents: ethyl acetate (UBEA), acetone (UBA), and ethanol (UBE) were prepared. From UBEA we isolated usnic acid with high purity by semipreparative chromatography. Then, UA, UBA, and UBE dissolved in 1% dimethyl sulfoxide (DMSO) and diluted in four concentrations were tested for their toxicity on human blood cells. The blood samples were collected from a healthy non-smoker donor; the obtained blood cell cultures were treated with the tested samples. After 24 h, the cytotoxic effect was analyzed through the mechanisms that can cause cell death: early and late apoptosis, caspase 3/7 activity, nuclear apoptosis, autophagy, reactive oxygen species (ROS) level and DNA damage. Generally, the cytotoxic effect was directly proportional to the increase of concentrations, usnic acid inducing the most significant response. At high concentrations, usnic acid and U. barbata extracts induced apoptosis and DNA damage in human blood cells, increasing ROS levels. Our study reveals the importance of prior natural products toxicity evaluation on normal cells to anticipate their limits and benefits as potential anticancer drugs.

Lichens represent an important resource for common traditional medicines due to their numerous metabolites that can exert diverse pharmacological activities including anticancer effects. To find new anticancer compounds with fewer side effects and low tumor resistance, a bioprospective study of Usnea barbata (L.) F.H. Wigg. (U. barbata), a lichen from the Călimani Mountains (Suceava county, Romania) was performed. The aim of this research was to investigate the anticancer potential, morphologic changes, wound healing property, clonogenesis, and oxidative stress biomarker status of four extracts of U. barbata in different solvents (methanol, ethanol, acetone, and ethyl acetate), and also of usnic acid (UA) as a positive control on the CAL-27 (ATCC® CRL-2095™) oral squamous carcinoma (OSCC) cell line and V79 (ATCC® CCL-93™) lung fibroblasts as normal cells. Using the MTT assay and according to IC50 values, it was found that the most potent anticancer property was displayed by acetone and ethyl acetate extracts. All U. barbata extracts determined morphological modifications (losing adhesion capacity, membrane shrinkage, formation of abnormal cellular wrinkles, and vacuolization) with higher intensity in tumor cells than in normal ones. The most intense anti-migration effect was established in the acetone extract treatment. The clonogenic assay showed that some U. barbata extracts decreased the ability of cancer cells to form colonies compared to untreated cells, suggesting a potential anti-tumorigenic property of the tested extracts. Therefore, all the U. barbata extracts manifest anticancer activity of different intensity, based, at least partially, on an imbalance in antioxidant defense mechanisms, causing oxidative stress.

Usnea lichens are known for their beneficial pharmacological effects with potential applications in oral medicine. This study aims to investigate the extract of Usnea barbata (L.) Weber ex F.H. Wigg from the Călimani Mountains in canola oil as an oral pharmaceutical formulation. In the present work, bioadhesive oral films (F-UBO) with U. barbata extract in canola oil (UBO) were formulated, characterized, and evaluated, evidencing their pharmacological potential. The UBO-loaded films were analyzed using standard methods regarding physicochemical and pharmacotechnical characteristics to verify their suitability for topical administration on the oral mucosa. F-UBO suitability confirmation allowed for the investigation of antimicrobial and anticancer potential. The antimicrobial properties against Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27353, Candida albicans ATCC 10231, and Candida parapsilosis ATCC 22019 were evaluated by a resazurin-based 96-well plate microdilution method. The brine shrimp lethality assay (BSL assay) was the animal model cytotoxicity prescreen, followed by flow cytometry analyses on normal blood cells and oral epithelial squamous cell carcinoma CLS-354 cell line, determining cellular apoptosis, caspase-3/7 activity, nuclear condensation and lysosomal activity, oxidative stress, cell cycle, and cell proliferation. The results indicate that a UBO-loaded bioadhesive film’s weight is 63 ± 1.79 mg. It contains 315 µg UBO, has a pH = 6.97 ± 0.01, a disintegration time of 124 ± 3.67 s, and a bioadhesion time of 86 ± 4.12 min, being suitable for topical administration on the oral mucosa. F-UBO showed moderate dose-dependent inhibitory effects on the growth of both bacterial and fungal strains. Moreover, in CLS-354 tumor cells, F-UBO increased oxidative stress, diminished DNA synthesis, and induced cell cycle arrest in G0/G1. All these properties led to considering UBO-loaded bioadhesive oral films as a suitable phytotherapeutic formulation with potential application in oral infections and neoplasia.

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.

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.