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

ABSTRACT: 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.

SUBMITTER: Popovici V

PROVIDER: S-EPMC8388874 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:This study aims to complete our research on Usnea barbata (L.) Weber ex F.H. Wigg (U. barbata) from the Călimani 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.

Project description: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.

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

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