ROS-Induced DNA-Damage and Autophagy in Oral Squamous Cell Carcinoma by Usnea barbata Oil Extract-An In Vitro Study.

Oxidative stress is associated with aging, cancers, and numerous metabolic and chronic disorders, and phenolic compounds are well known for their health-promoting role due to their free-radical scavenging activity. These phytochemicals could also exhibit pro-oxidant effects. Due to its bioactive phenolic secondary metabolites, Usnea barbata (L.) Weber ex. F.H. Wigg (U. barbata) displays anticancer and antioxidant activities and has been used as a phytomedicine for thousands of years. The present work aims to analyze the properties of U. barbata extract in canola oil (UBO). The UBO cytotoxicity on oral squamous cell carcinoma (OSCC) CLS-354 cell line and blood cell cultures was explored through complex flow cytometry analyses regarding apoptosis, reactive oxygen species (ROS) levels, the enzymatic activity of caspase 3/7, cell cycle, nuclear shrinkage (NS), autophagy (A), and synthesis of deoxyribonucleic acid (DNA). All these studies were concomitantly performed on canola oil (CNO) to evidence the interaction of lichen metabolites with the constituents of this green solvent used for extraction. The obtained data evidenced that UBO inhibited CLS-354 oral cancer cell proliferation through ROS generation (316.67 × 104), determining higher levels of nuclear shrinkage (40.12%), cell cycle arrest in G0/G1 (92.51%; G0 is the differentiation phase, while during G1 phase occurs preparation for cell division), DNA fragmentation (2.97%), and autophagy (62.98%) than in blood cells. At a substantially higher ROS level in blood cells (5250.00 × 104), the processes that lead to cell death-NS (30.05%), cell cycle arrest in G0/G1 (86.30%), DNA fragmentation (0.72%), and autophagy (39.37%)-are considerably lower than in CLS-354 oral cancer cells. Our work reveals the ROS-mediated anticancer potential of UBO through DNA damage and autophagy. Moreover, the present study suggests that UBO pharmacological potential could result from the synergism between lichen secondary metabolites and canola oil phytoconstituents.

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.

Design, Characterization, and Anticancer and Antimicrobial Activities of Mucoadhesive Oral Patches Loaded with Usnea barbata (L.) F. H. Wigg Ethanol Extract F-UBE-HPMC.

The oral cavity's common pathologies are tooth decay, periodontal disease, and oral cancer; oral squamous cell carcinoma (OSCC) is the most frequent oral malignancy, with a high mortality rate. Our study aims to formulate, develop, characterize, and pharmacologically investigate the oral mucoadhesive patches (F-UBE-HPMC) loaded with Usnea barbata (L.) F.H. Wigg dry ethanol extract (UBE), using HPMC K100 as a film-forming polymer. Each patch contains 312 µg UBE, with a total phenolic content (TPC) of 178.849 µg and 33.924 µg usnic acid. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) were performed for their morphological characterization, followed by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), and thermogravimetric analysis (TGA). Pharmacotechnical evaluation involved the measurement of the specific parameters for mucoadhesive oral patches as follows: weight uniformity, thickness, folding endurance, tensile strength, elongation, moisture content, pH, disintegration time, swelling rate, and ex vivo mucoadhesion time. Thus, each F-UBE-HPMC has 104 ± 4.31 mg, a pH = 7.05 ± 0.04, a disintegration time of 130 ± 4.14 s, a swelling ratio of 272 ± 6.31% after 6 h, and a mucoadhesion time of 102 ± 3.22 min. Then, F-UBE-HPMCs pharmacological effects were investigated using brine shrimp lethality assay (BSL assay) as a cytotoxicity prescreening test, followed by complex flow cytometry analyses on blood cell cultures and oral epithelial squamous cell carcinoma CLS-354 cell line. The results revealed significant anticancer effects by considerably increasing oxidative stress and blocking DNA synthesis in CLS-354 cancer cells. The antimicrobial potential against Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27353, Candida albicans ATCC 10231, and Candida parapsilosis ATCC 22019 was assessed by a Resazurin-based 96-well plate microdilution method. The patches moderately inhibited both bacteria strains growing and displayed a significant antifungal effect, higher on C. albicans than on C. parapsilosis. All these properties lead to considering F-UBE-HPMC suitable for oral disease prevention and therapy.

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.

Evaluation of Usnea barbata (L.) Weber ex F.H. Wigg Extract in Canola Oil Loaded in Bioadhesive Oral Films for Potential Applications in Oral Cavity Infections and Malignancy.

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 Calimani 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 Secondary Metabolites and Antiradical and Antibacterial Activities of Different Extracts of Usnea barbata (L.) Weber ex F.H.Wigg from Calimani 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.

Antioxidant, Cytotoxic, and Rheological Properties of Canola Oil Extract of Usnea barbata (L.) Weber ex F.H. Wigg from Calimani Mountains, Romania.

Usnea genus (Parmeliaceae, lichenized Ascomycetes) is a potent phytomedicine, due to phenolic secondary metabolites, with various pharmacological effects. Therefore, our study aimed to explore the antioxidant, cytotoxic, and rheological properties of Usnea barbata (L.) Weber ex F.H. Wigg (U. barbata) extract in canola oil (UBO) compared to cold-pressed canola seed oil (CNO), as a green solvent used for lichen extraction, which has phytoconstituents. The antiradical activity (AA) of UBO and CNO was investigated using UV-Vis spectrophotometry. Their cytotoxicity was examined in vivo through a brine shrimp lethality (BSL) test after Artemia salina (A. salina) larvae exposure for 6 h to previously emulsified UBO and CNO. The rheological properties of both oil samples (flow behavior, thixotropy, and temperature-dependent viscosity variation) were comparatively analyzed. The obtained results showed that UBO (IC50 = 0.942 ± 0.004 mg/mL) had a higher 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity than CNO (IC50 = 1.361 ± 0.008 mg/mL). Both UBO and CNO emulsions induced different and progressive morphological changes to A. salina larvae, incompatible with their survival; UBO cytotoxicity was higher than that of CNO. Finally, in the temperature range of 32-37 °C, the UBO and CNO viscosity and viscoelastic behavior indicated a clear weakening of the intermolecular bond when temperature increases, leading to a more liquid state, appropriate for possible pharmaceutical formulations. All quantified parameters were highly intercorrelated. Moreover, their significant correlation with trace/heavy minerals and phenolic compounds can be observed. All data obtained also suggest a possible synergism between lichen secondary metabolites, minerals, and canola oil phytoconstituents.

Usnic Acid and Usnea barbata (L.) F.H. Wigg. Dry Extracts Promote Apoptosis and DNA Damage in Human Blood Cells through Enhancing ROS Levels.

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.

In Vitro Anticancer Activity and Oxidative Stress Biomarkers Status Determined by Usnea barbata (L.) F.H. Wigg. Dry Extracts.

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 Calimani 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.

Antioxidant and Cytotoxic Activities of Usnea barbata (L.) F.H. Wigg. Dry Extracts in Different Solvents.

Lichens represent a significant source of antioxidants due to numerous metabolites that can reduce free radicals. Usnea barbata (L.) F.H. Wigg. has been recognized and used since ancient times for its therapeutic effects, some of which are based on its antioxidant properties. The present study aims to analyze the phytochemical profile and to evaluate the antioxidant and cytotoxic potential of this lichen species. Five dry extracts of U. barbata (UBDE) in different solvents (acetone, ethyl acetate, ethanol, methanol, water) were prepared by refluxing at Soxhlet to achieve these proposed objectives and to identify which solvent is the most effective for the extraction. The usnic acid content (UAC) was quantified by ultra-high performance liquid chromatography (UHPLC). The total polyphenols content (TPC) and tannins content (TC) were evaluated by spectrophotometry, and the total polysaccharides (PSC) were extracted by a gravimetric method. The 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) free radical method was used to assess the antioxidant activity (AA) and the Brine Shrimp Lethality (BSL) assay was the biotest for cytotoxic activity evaluation. The ethyl acetate extract had the highest usnic acid content, and acetone extract had the highest content of total polyphenols and tannins. The most significant antioxidant effect was reported to methanol extract, and all the extracts proved high cytotoxicity. The water extract has the lowest cytotoxicity because usnic acid is slightly soluble in this solvent, and it was not found at UHPLC analysis. All extracts recorded a moderate correlation between the content of usnic acid, polyphenols, tannins, and AA; furthermore, it has been observed that the cytotoxicity varies inversely with the antioxidant effect.

Elemental Analysis and In Vitro Evaluation of Antibacterial and Antifungal Activities of Usnea barbata (L.) Weber ex F.H. Wigg from Calimani Mountains, Romania.

This study aims to complete our research on Usnea barbata (L.) Weber ex F.H. Wigg (U. barbata) from the Calimani Mountains, Romania, with an elemental analysis and to explore its antibacterial and antifungal potential. Thus, we analyzed twenty-three metals (Ca, Fe, Mg, Mn, Zn, Al, Ag, Ba, Co, Cr, Cu, Li, Ni, Tl, V, Mo, Pd, Pt, Sb, As, Pb, Cd, and Hg) in dried U. barbata lichen (dUB) by inductively coupled plasma mass spectrometry (ICP-MS). For the second study, we performed dried lichen extraction with five different solvents (ethyl acetate, acetone, ethanol, methanol, and water), obtaining five U. barbata dry extracts (UBDE). Then, using an adapted disc diffusion method (DDM), we examined their antimicrobial activity against seven bacterial species-four Gram-positive (Staphylococcus aureus, Enterococcus casseliflavus, Streptococcus pyogenes, and Streptococcus pneumoniae) and three Gram-negative (Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa)-and two fungi species (Candida albicans and Candida parapsilosis). Usnic acid (UA) was used as a positive control. The ICP-MS data showed a considerable Ca content (979.766 µg/g), followed by, in decreasing order, Mg, Mn, Al, Fe, and Zn. Other elements had low levels: Ba, Cu, Pb, and Cr (3.782-1.002 µg/g); insignificant amounts (<1 µg/g) of Hg and V were also found in dUB. The trace elements Ag, As, Cd, Co, Li, Tl, Mo, Pd, Pt, and Sb were below detection limits (<0.1 µg/g). The DDM results-expressed as the size (mm) of the inhibition zone diameter (IZs)-proved that the water extract did not have any inhibitory activity on any pathogens (IZs = 0 mm). Gram-positive bacteria displayed the most significant susceptibility to all other UBDE, with Enterococcus casseliflavus showing the highest level (IZs = 20-22 mm). The most susceptible Gram-negative bacterium was Pseudomonas aeruginosa (IZs = 16-20 mm); the others were insensitive to all U. barbata dry extracts (IZs = 0 mm). The inhibitory activity of UBDE and UA on Candida albicans was slightly higher than on Candida parapsilosis.

Evaluation of the Cytotoxic Activity of the Usnea barbata (L.) F. H. Wigg Dry Extract.

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.