Silver Nanoparticles Synthesized from Abies alba and Pinus sylvestris Bark Extracts: Characterization, Antioxidant, Cytotoxic, and Antibacterial Effects.

In recent years, phytofunctionalized AgNPs have attracted great interest due to their remarkable biological activities. In the present study, AgNPs were synthesized using Abies alba and Pinus sylvestris bark extracts. The chemical profile of these bark extracts was analyzed by LC-HRMS/MS. As a first step, the synthesis parameters (pH, AgNO3 concentration, ratio of bark extract and AgNO3, temperature, and reaction time) were optimized. The synthesized AgNPs were characterized by ATR-FTIR spectroscopy, DLS, SEM, EDX, and TEM. Their antioxidant, cytotoxic, and antibacterial properties were evaluated by the DPPH, ABTS, MTT, and broth microdilution assays, respectively. Abies alba and Pinus sylvestris bark extract-derived AgNPs were well-dispersed, spherical, small (average particle size of 9.92 and 24.49 nm, respectively), stable (zeta potential values of -10.9 and -10.8 mV, respectively), and cytotoxic to A-375 human malignant melanoma cells (IC50 = 2.40 ± 0.21 and 6.02 ± 0.61 µg/mL, respectively). The phytosynthesized AgNPs also showed antioxidant and antibacterial effects.

Cytogenetic and Biochemical Responses of Wheat Seeds to Proton Irradiation at the Bragg Peak.

The present study aimed to evaluate the morphological, cytogenetic and biochemical changes in wheat seedlings as affected by seed exposure to a proton beam at the Bragg peak. The average energy of the proton beam was of 171 MeV at the entrance into the irradiator room while at the point of sample irradiation the beam energy was of 150 MeV, with the average value of the Linear Energy Transfer of 0.539 keV/µm and the dose rate of 0.55 Gy/min, the radiation doses being of the order of tens of Gy. Cytogenetic investigation has revealed the remarkable diminution of the mitotic index as linear dose-response curve as well as the spectacular linear increase of the aberration index. Analyzing some biometric parameters, it was found that neither dry matter nor water content of wheat seedlings was influenced by proton beam exposure. Studying the biochemical parameters related to the antioxidant defense system, we found that the irradiation caused the slight increasing tendency of peroxidase activity as well as the decreasing trend in the activity of superoxidedismutase in the seedlings grown from the irradiated seeds. The level of malonedialdehyde (MDA) and total polyphenols showed an increasing tendency in all seedling variants corresponding to irradiated seeds, compared to the control. We conclude that the irradiation clearly induced dose-response curves at the level of cytogenetic parameters together with relatively slight variation tendency of some biochemical parameters related to the antioxidant defense system while imperceptible changes could be noticed in the biometric parameters.

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.

Impact of Proton Beam Irradiation on the Growth and Biochemical Indexes of Barley (Hordeum vulgare L.) Seedlings Grown under Salt Stress.

The present paper examines the effects of salt stress on the growth, pigments, lipid peroxidation and antioxidant ability of barley (Hordeum vulgare L.) seedlings raised from proton beam irradiated caryopses. In order to assess the effects of radiation on the early stages of plant growth and analyze its possible influence on the alleviation of salinity, 3 and 5 Gy doses were used on dried barley seeds and germination occurred in the presence/absence of NaCl (100 mM and 200 mM). After treatment, photosynthetic pigments increased in the 5 Gy variant, which registered a higher value than the control. Among the antioxidant enzymes studied (SOD, CAT, and POD) only CAT activity increased in proton beam irradiated seeds germinated under salinity conditions, which indicates the activation of antioxidant defense. The malondialdehyde (MDA) content declined with the increase of irradiation doses on seeds germinated at 200 mM NaCl. On the other hand, the concentration of 200 mM NaCl applied alone or combined with radiation revealed an increase in soluble protein content. The growth rate suggests that 3 Gy proton beam irradiation of barley seeds can alleviate the harmful effects of 100 mM NaCl salinity, given that seedlings' growth rate increased by 1.95% compared to the control.

Antitumoral Drug: Loaded Hybrid Nanocapsules Based on Chitosan with Potential Effects in Breast Cancer Therapy.

Cancer remains one of the world's most devastating diseases and is responsible for more than 20% of all deaths. It is defined as uncontrolled proliferation of cells and spreads rapidly to healthy tissue. Controlled drug delivery systems offers great opportunities for the development of new non-invasive strategies for the treatment of cancers. The main advantage of these systems is their capacity to accumulate in tumors via enhanced permeability and retention effects. In the present study, an innovative hybrid drug delivery system based on nanocapsules obtained from the interfacial condensation between chitosan and poly(N-vinyl pyrrolidone-alt-itaconic anhydride) and containing both magnetic nanoparticles and an antitumoral drug was developed in order to improve the efficiency of the antitumoral treatment. Using dynamic light scattering, it was observed that the mean diameter of these hybrid nanocapsules was in the range of 43 to 142 nm. SEM confirmed their nanometric size and their well-defined spherical shape. These nanocapsules allowed the encapsulation of an increased amount of 5-fluorouracil and provided controlled drug release. In vitro studies have revealed that these drug-loaded hybrid nanocapsules were able to induce a cytostatic effect on breast carcinoma MCF-7 cell lines (Human Caucasian breast adenocarcinoma - HTB-22) comparable to that of the free drug.

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.

Iron phthalocyanine-sensitized magnetic catalysts for BPA photodegradation.

The catalytic behavior of iron phthalocyanine (FePc)-sensitized magnetic nanocatalysts was evaluated for their application in the oxidative treatment of Bisphenol A (BPA) under mild environmental conditions. Two types of FePc (Fe(II)Pc and Fe(III)Pc), which are highly photosensitive compounds, were immobilized on the surface of functionalized magnetite. The nanomaterials were characterized by high resolution transmission electron microscopy (HR-TEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and thermogravimetric analyses (TGA). The generation of singlet oxygen by nanomaterials was also investigated. In the presence of UVA light exposure (365 nm) and 15 mM H2O2, the M@Fe(III)Pc photocatalyst gave the best results; for a catalyst concentration of 2.0 g L - 1, around 60% BPA was removed after 120 min of reaction. These experimental conditions were further tested under natural solar light exposure, for which also M@Fe(III)Pc exhibited enhanced oxidative catalytic activity, being able to remove 83% of BPA in solution. The water samples were less cytotoxic after treatment, this being confirmed by the MCF-7 cell viability assay.

Aptamer-Functionalized Liposomes as a Potential Treatment for Basal Cell Carcinoma.

More than one out of every three new cancers is a skin cancer, and the large majority are basal cell carcinomas (BCC). Targeted therapy targets the cancer's specific genes, proteins, or tissue environment that contributes to cancer growth and survival and blocks the growth as well as the spread of cancer cells while limiting damage to healthy cells. Therefore, in the present study AS1411 aptamer-functionalized liposomes for the treatment of BCC were obtained and characterized. Aptamer conjugation increased liposome size, suggesting that the presence of an additional hydrophilic molecule on the liposomal surface increased the hydrodynamic diameter. As expected, the negatively charged DNA aptamer reduced the surface potential of the liposomes. Vertical Franz diffusion cells with artificial membranes were used to evaluate the in vitro release of 5-fluorouracil (5-FU). The aptamer moieties increase the stability of the liposomes and act as a supplementary steric barrier leading to a lower cumulative amount of the released 5-FU. The in vitro cell viability, targeting capability and apoptotic effects of liposomes on the human dermal fibroblasts and on the basal cell carcinoma TE 354.T cell lines were also evaluated. The results indicate that the functionalized liposomes are more efficient as nanocarriers than the non-functionalized ones.

"In vitro" behaviour of aptamer-functionalized polymeric nanocapsules loaded with 5-fluorouracil for targeted therapy.

New type of nanocapsules based on carboxymethyl chitosan functionalized with AS1411 aptamer and poly(N-vinylpyrrolidone-alt-itaconic anhydride) loaded with 5-Fluorouracil (5-FU) were developed, with the potential to improve the treatment of cancer. Functionalization of nanocapsules with AS1411 aptamer will enhance their recognition by tumor cells, due to the interaction with nucleolin, and subsequent endocytosis. Nanocapsules were prepared by interfacial condensation method in the absence of any toxic crosslinking agents. The condensation reaction took place at the interface between the organic and aqueous phases by opening the anhydride cycles from the copolymer, under the action of the NH2 groups from mixture of chitosan/aptamer-functionalized carboxymethyl chitosan. The nanocapsules diameter varied between 100 and 267 nm as a function of the molar ratio of the polymers. SEM images have revealed that nanocapsules were spherical and presented relatively low dimensional polydispersity. Nanocapsules swelling degree was found between 1000 and 1680% in PBS solution (pH = 7.4) and they allowed the encapsulation of an important amount of 5-Fluorouracil (5-FU). The release efficiency of 5-FU was studied, the processes being controlled by the drug diffusion through the polymeric membrane, as confirmed by the theoretical analysis of the drug release. The cytotoxicity and haemolysis tests performed on the nanocapsules proved their lack of toxicity and their excellent hemocompatibility. The obtained results were encouraging, showing that these original 5-FU-loaded nanocapsules were able to induce a more pronounced cytotoxic effect on neoplastic MCF-7 cells, the occurrence of dead cells being more rapidly than in the case of free 5-FU.

Graphene oxide effects in early ontogenetic stages of Triticum aestivum L. seedlings.

Nanomaterials are being used increasingly in various areas such as electronic devices manufacture, medicine, mechanical devices production, and even food industry. Therefore, the evaluation of their toxicity is mandatory. Graphene oxide (GO) has been shown to have both positive as well as negative impact on different crop plants, depending on species, dose, and duration of exposure. The current study evaluated the impact of GO sheets at different concentrations (500, 1000 and 2000 mg/L) on physiological, biochemical and genetic levels to determine the possible toxic action. Wheat caryopses were treated with GO for 48 h and 7 days. The germination rate and roots elongation decreased in a dose-response manner, except the sample treated with GO at a concentration of 1000 mg/L. Mitotic index has ascendant trend; its increase may be due to the accumulation of prophases GO induced significant accumulation of the cells with aberrations, their presence suggests a clastogenic/aneugenic effect of these carbon nanomaterials. Regarding enzymatic and non-enzymatic antioxidant system defence, the activity varied depending on the dose of GO. Thus, chlorophyll a pigments content decreased significantly at high dose (2000 mg/L), while the carotenoid pigments had lower content at 500 mg/L of GO, and no statistical difference encountered in case of chlorophyll b amount. The antioxidant enzyme activity (CAT, POD, and SOD) was higher at low dose of GO, indicating the presence of oxidative stress generated as a response to the GO treatment. Also, the free radical scavenging activity of the polyphenolic compounds was enhanced upon GO exposure. The GO accumulation has been identified by transmission electron microscopy only at plumules level, near the intercellular space.

HPLC-DAD-ESI-Q-TOF-MS/MS profiling of Verbascum ovalifolium Donn ex Sims and evaluation of its antioxidant and cytogenotoxic activities.

INTRODUCTION: Plant species of Verbascum genus have been intensively investigated in the last decades but most studies focused on evaluation of their biological activities; there are only few studies dealing with their chemical characterisation. OBJECTIVE: Detailed investigation of the qualitative and quantitative chemical composition, antioxidant and cytogenotoxic activities of a previously non-studied Verbascum species (V. ovalifolium Donn ex Sims). METHODS: Qualitative analysis of secondary metabolites was performed by HPLC-DAD-ESI-Q-TOF-MS/MS, whereas quantitative data were obtained through HPLC-DAD. Antioxidant activity was evaluated using in vitro assays; cytotoxic and genotoxic effects were assessed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium (MTT) and comet assays, respectively. RESULTS: More than 50 secondary bioactive metabolites belonging to various classes (iridoids, phenylethanoids, flavonoids, phenolic acids) were detected in the methanolic extract of V. ovalifolium and its fractions. The fragmentation pathways of acylated catalpol-type iridoid diglycosides are thoughtfully described herein. The extracts showed good free radical scavenging and ferric ion reducing properties correlated with phenolic, flavonoid, chlorogenic acid and verbascoside contents. Moreover, 24 h treatment of SK-MEL-2 cells with V. ovalifolium extracts produced significant changes in terms of tumour cell viability. The crude extract and the ethyl acetate fraction showed no important signs of cytogenotoxicity in non-tumour cells. CONCLUSION: The performed phytochemical and biological analyses contribute to the preclinical knowledge about V. ovalifolium and they could help exploiting it in novel herbal medicinal products.

Performances of Pichia kudriavzevii in decolorization, biodegradation, and detoxification of C.I. Basic Blue 41 under optimized cultural conditions.

The aim of this study was to evaluate the performances of Pichia kudriavzevii CR-Y103 yeast strain for the decolorization, biodegradation, and detoxification of cationic dye C.I. Basic Blue 41, a toxic compound to aquatic life with long-lasting effects. Under optimized cultural conditions (10.0-g L-1 glucose, 0.2-g L-1 yeast extract, and 1.0-g L-1 (NH4)2SO4), the yeast strain was able to decolorize 97.86% of BB41 (50 mg L-1) at pH 6 within 4 h of incubation at 30 °C under shaken conditions (12,238.00-µg h-1 average decolorization rate) and 100% within 12 h. The UV-Vis spectral analysis, high-performance liquid chromatography (HPLC), and Fourier-transform infrared spectroscopy (FTIR) analysis confirmed the complete decolorization and degradation of the BB41 dye by P. kudriavzevii CR-Y103. Also, other seven yeast strains, isolated from soil, as P. kudriavzevii (CR-Y108, CR-Y119, and CR-Y112), Candida tropicalis CR-Y128, Cyberlindnera saturnus CR-Y125, and Candida solani CR-Y124 have shown a promising decolorizing potential of azo-dye BB41 (99.89-76.09% decolorization). Phytotoxicity, cytotoxicity, and genotoxicity assays on Trifolium pratense and Triticum aestivum seedlings confirmed the high toxicity of BB41 dye (500 ppm), with inhibition on germination rate (%), root and shoot elongation, decreasing of mitoxic index value (with 34.03% in T. pratense and 40.25% in T. aestivum), and increasing the frequency of chromosomal aberrations (6.87 times in T. pratense and 6.25 times in T. aestivum), compared to control. The same biomarkers indicated the nontoxic nature of the BB41 degraded metabolite (500 ppm) obtained after P. kudriavzevii CR-Y103 treatment. Moreover, the healthy monkey kidney cells (Vero cells) had a low sensitivity to BB41 biodegraded products (250 µg mL-1) (MTT cell viability assay) and revealed minor DNA damage (comet assay) compared to BB41 dye treatment. These findings show that P. kudriavzevii could be used in eco-friendly bioremediation technologies, applicable for reducing the toxicity of basic azo-dyes containing wastewaters.

Antioxidant and antigenotoxic potential of Ramaria largentii Marr & D. E. Stuntz, a wild edible mushroom collected from Northeast Romania.

Ramaria largentii Marr & D. E. Stuntz (orange coral mushroom) is a wild edible mushroom whose chemical composition and bioactivity have not been investigated. Herein, we present a study on the phenolic constituents, antioxidant and antigenotoxic effects of a hydromethanolic extract of the fruiting bodies. Total phenolic content, estimated by Folin-Ciocalteu assay, was found to be 42.33 ± 0.18 mg GAE/g. Protocatechuic and vanillic acids were detected by HPLC-DAD-ESI-MS. The extract showed good free radical scavenging and reducing capacities (EC50 = 64.3 ± 0.2 and 61.54 ± 0.46 µg/mL, respectively). In normal Vero cells, the extract (100, 200 and 300 µg/mL) showed no genotoxic potential and moreover, almost completely protected DNA against H2O2-induced damage (2.09-7.91% tail DNA) (24 and 48 h pre-treatment). Taken together, the results of our study show that Ramaria largentii extract is devoid of genotoxicity and has a remarkable DNA protective activity against H2O2-induced damage.