Phytochemical, In Vitro, In Vivo, and In Silico Research on the Extract of Ajuga chamaepitys (L.) Schreb.

The present study focuses on the chemical characterization of a dry extract obtained from the species Ajuga chamaepitys (L.) Schreb, evaluating its antioxidant properties, toxicity, and in silico profile. Quantitative analysis of the dry extract revealed a notable amount of phytochemical compounds: 59.932 ± 21.167 mg rutin equivalents (mg REs)/g dry weight, 45.864 ± 4.434 mg chlorogenic acid equivalents (mg ChAEs)/g dry weight and, respectively, 83.307 ± 3.989 mg tannic acid equivalents (TAEs)/g dry weight. By UHPLC-HRMS/MS, the following were quantified as major compounds: caffeic acid (3253.8 µg/g extract) and kaempherol (3041.5 µg/g extract); more than 11 types of polyphenolic compounds were quantified (genistin 730.2 µg/g extract, naringenin 395 µg/g extract, apigenin 325.7 µg/g extract, galangin 283.3 µg/g extract, ferulic acid 254.3 µg/g extract, p-coumaric acid 198.2 µg/g extract, rutin 110.6 µg/g extract, chrysin 90.22 µg/g extract, syringic acid 84.2 µg/g extract, pinocembrin 32.7 µg/g extract, ellagic acid 18.2 µg/g extract). The antioxidant activity was in accordance with the amount of phytochemical compounds: IC50DPPH = 483.6 ± 41.4 µg/mL, IC50ABTS•+ = 127.4 ± 20.2 µg/mL, and EC50FRAP = 491.6 ± 2 µg/mL. On the larvae of Artemia sp., it was found that the extract has a low cytotoxic action. In silico studies have highlighted the possibility of inhibiting the activity of protein kinases CDK5 and GSK-3b for apigenin, galangin, and kaempferol, with possible utility for treating neurodegenerative pathologies and neuropathic pain. Further studies are warranted to confirm the predicted molecular mechanisms of action and to further investigate the therapeutic potential in animal models of neurological disorders.

Bioactive Potential of Carrot-Based Products Enriched with Lactobacillus plantarum.

The primary goal of this study was to generate different kinds of functional products based on carrots that were supplemented with lactic acid bacteria. The fact that carrots (Daucus carota sp.) rank among the most popular vegetables in our country led to the convergence of the research aim. Their abundance of bioactive compounds, primarily polyphenols, flavonoids, and carotenoids, offers numerous health benefits. Among the obtained products, the freeze-dried carrot powder (FDCP) variation presented the highest concentrations of total carotenoids (TCs) and ß-carotene (BC) of 26.977 ± 0.13 mg/g DW and 22.075 ± 0.14 mg/g DW, respectively. The amount of total carotenoids and ß-carotene significantly increased with the addition of the selected lactic acid bacteria (LAB) for most of the samples. In addition, a slight increase in the antioxidant activity compared with the control sample for the FDCP variant, with the highest value of 91.74%, was observed in these functional food products. The content of polyphenolic compounds varied from 0.044 to 0.091 mg/g DW, while the content of total flavonoids varied from 0.03 to 0.66 mg/g DW. The processing method had an impact on the population of L. plantarum that survived, as indicated by the viability of bacterial cells in all the analyzed products. The chromatographic analysis through UHPLC-MS/MS further confirmed the abundance of the bioactive compounds and their corresponding derivatives by revealing 19 different compounds. The digestibility study indicated that carotenoid compounds from carrots followed a rather controlled release. The carrot-based products enriched with Lactobacillus plantarum can be considered newly functional developed products based on their high content of biologically active compounds with beneficial effects upon the human body. Furthermore, these types of products could represent innovative products for every related industry such as the food, pharmaceutical, and cosmeceutical industries, thus converging a new strategy to improve the health of consumers or patients.

Antioxidant and Wound Healing Bioactive Potential of Extracts Obtained from Bark and Needles of Softwood Species.

Interest in the extraction of phytochemical bioactive compounds, especially polyphenols from biomass, has recently increased due to their valuable biological potential as natural sources of antioxidants, which could be used in a wide range of applications, from foods and pharmaceuticals to green polymers and bio-based materials. The present research study aimed to provide a comprehensive chemical characterization of the phytochemical composition of forest biomass (bark and needles) of softwood species (Picea abies L., H. Karst., and Abies alba Mill.) and to investigate their in vitro antioxidant and antimicrobial activities to assess their potential in treating and healing infected chronic wounds. The DPPH radical-scavenging method and P-LD were used for a mechanistic explanation of the biomolecular effects of the investigated bioactive compounds. (+)-Catechin, epicatechin, rutin, myricetin, 4 hydroxybenzoic and p-cumaric acids, kaempherol, and apigenin were the main quantified polyphenols in coniferous biomass (in quantities around 100 µg/g). Also, numerous phenolic acids, flavonoids, stilbenes, terpenes, lignans, secoiridoids, and indanes with antioxidant, antimicrobial, anti-inflammatory, antihemolytic, and anti-carcinogenic potential were identified. The Abies alba needle extract was more toxic to microbial strains than the eukaryotic cells that provide its active wound healing principles. In this context, developing industrial upscaling strategies is imperative for the long-term success of biorefineries and incorporating them as part of a circular bio-economy.

Experimentally Induced Burns in Rats Treated with Innovative Polymeric Films Type Therapies.

Considering that microbial resistance to antibiotics is becoming an increasingly widespread problem, burn management, which usually includes the use of topical antimicrobial dressings, is still facing difficulties regarding their efficiency to ensure rapid healing. In this context, the main objective of this research is to include new oxytetracycline derivatives in polymeric-film-type dressings for the treatment of wounds caused by experimentally induced burns in rats. The structural and physico-chemical properties of synthesized oxytetracycline derivatives and the corresponding membranes were analyzed by FT-IR and MS spectroscopy, swelling ability and biodegradation capacity. In vitro antimicrobial activity using Gram-positive and Gram-negative bacterial strains and pathogenic yeasts, along with an in vivo study of a burn wound model induced in Wistar rats, was also analyzed. The newly obtained polymeric films, namely chitosan-oxytetracycline derivative membranes, showed good antimicrobial activity noticed in the tested strains, a membrane swelling ratio (MSR) of up to 1578% in acidic conditions and a biodegradation rate of up to 15.7% on day 7 of testing, which are important required characteristics for the tissue regeneration process, after the production of a burn. The in vivo study proved that chitosan-derived oxytetracycline membranes showed also improved healing effects which contributes to supporting the idea of using them for the treatment of wounds caused by burns.

Application of Ionic Liquids as Mobile Phase Additives for Simultaneous Analysis of Nicotine and Its Metabolite Cotinine in Human Plasma by HPLC-DAD.

Nicotine and cotinine are very polar basic molecules, which makes it difficult to analyze them by reversed-phase liquid chromatography (RPLC), especially in biological samples. Additives with an ionic character have been traditionally used in RPLC as silanol suppressors. The aim of our study was to investigate the potential of selected ionic liquids in improving chromatographic performance in comparison with common additives. The experimental design was conducted using the following ionic liquids as the mobile phase modifiers: 1-butyl-3-methylimidazolium tetrafluoroborate, BMIM[BF4] and 1-butyl-3-methylimidazolium hexafluorophosphate BMIM[PF6], with a C18 chromatographic column. The separation of these alkaloids on silica-based RPLC stationary phases was successfully conducted by the addition of BMIM[BF4] in an acetonitrile:phosphate-buffer-based mobile phase in a pH range of 2.3-5.2. The presented chromatographic method can be used as alternative for monitoring studies or pharmacokinetic application necessary for the evaluation of tobacco smoke exposure.

Lethal toxicity induced by combined ingestion of dietary acetic acid and carbamazepine.

Acetic acid is an organic acid that can be used in the food industry, which normally has an insignificant rate of adverse reactions when used rationally. However, irrational use can cause serious toxic effects and even death. In this context, the case of a death of a 52-year-old woman, involving the suspected voluntary use of food acetic acid, is presented, while toxicological and histopathological aspects were addressed for death mechanism elucidation. In this case, the pH value of 6.75 in blood, has shown severe metabolic acidosis after the ingestion of the large quantity of dietary acetic acid - about a liter. Also, the victim suffers from mental illness, carbamazepine being one of the treatment drugs. Liver damage, demonstrated by histopathological examination may be a consequence of both massive accumulation of carbamazepine in the liver and toxicity of food acetic acid. In conclusion, the hepatotoxicity induced by high level of carbamazepine was suspected of increasing the risk of multiple organ failure, in the context of acetic acid acute toxicity, highlighting the particularities of the case.

Distribution, sources and ecological risk assessment of polycyclic aromatic hydrocarbons in waters and sediments from Olt River dam reservoirs in Romania.

This study provides important data on the distribution, sources and ecological risks of polycyclic aromatic hydrocarbons (PAHs), in surface waters and sediments collected from dam reservoirs on middle and lower course of the Olt River, the main tributary of the Danube, until the discharge into the Black Sea. A wide variation range of total PAHs concentrations in water (from 1.3 to 46.2 ng/L) and sediment (from 1.78 to 614.04 µg/kg) samples was emphasized by the results. The highest average PAHs concentration in water was recorded in the cold season and the lowest in the summer. In sediments, no differences were observed depending on the sampling period. Spatial distribution of PAHs in waters and sediments was correlated with the main anthropogenic activities along the river course. Regardless of the method used to attribute PAH sources (diagnostic ratios of specific PAHs, principal component analysis and hierarchical cluster analysis), it was confirmed that the potential anthropogenic sources of PAHs were both pyrogenic (incomplete combustion of biomass and coal) and pyrolytic (incomplete combustion of liquid fossil fuels and vehicle exhaust emissions), with a dominant pyrolytic input. Ecological risk assessment based on environmental quality standards, mean effect range-median quotient (m-ERM-Q), toxic equivalency factors (TEFs) and risk quotient (RQ) methods indicated potentially low ecological risks from PAHs. The ecological status of the Olt river waters poses no potential risk, and pollution of surface sediments can be classified as low polluted, except for two sites near industrial activities classified as moderately polluted. Therefore, a regular monitoring of PAHs concentration in the waters and sediments should be performed to prevent further contamination of PAHs in the studied area, especially in densely populated industrial areas.

Chitosan Microparticles Loaded with New Non-Cytotoxic Isoniazid Derivatives for the Treatment of Tuberculosis: In Vitro and In Vivo Studies.

Lately, in the world of medicine, the use of polymers for the development of innovative therapies seems to be a major concern among researchers. In our case, as a continuation of the research that has been developed so far regarding obtaining new isoniazid (INH) derivatives for tuberculosis treatment, this work aimed to test the ability of the encapsulation method to reduce the toxicity of the drug, isoniazid and its new derivatives. To achieve this goal, the following methods were applied: a structural confirmation of isoniazid derivatives using LC-HRMS/MS; the obtaining of microparticles based on polymeric support; the determination of their loading and biodegradation capacities; in vitro biocompatibility using MTT cell viability assays; and, last but not least, in vivo toxicological screening for the determination of chronic toxicity in laboratory mice, including the performance of a histopathological study and testing for liver enzymes. The results showed a significant reduction in tissue alterations, the disappearance of cell necrosis and microvesicular steatosis areas and lower values of the liver enzymes TGO, TGP and alkaline phosphatase when using encapsulated forms of drugs. In conclusion, the encapsulation of INH and INH derivatives with chitosan had beneficial effects, suggesting a reduction in hepatotoxicity and, therefore, the achievement of the aim of this paper.

Comparison of the Polyphenolic Profile of Medicago sativa L. and Trifolium pratense L. Sprouts in Different Germination Stages Using the UHPLC-Q Exactive Hybrid Quadrupole Orbitrap High-Resolution Mass Spectrometry.

Identification and quantification of polyphenols in plant material are of great interest since they make a significant contribution to its total bioactivity. In the present study, an UPLC-Orbitrap-MS/MS approach using the variable data acquisition mode (vDIA) was developed and applied for rapid separation, identification, and quantification of the main polyphenolic compounds in Medicago sativa L. and Trifolium pratense L. sprouts in different germination stages. Based on accurate MS data and fragment ions identification strategy, a total of 29 compounds were identified by comparing their accurate masses, fragment ions, retention times, and literatures. Additionally, a number of 30 compounds were quantified by comparing to the reference standards. Data were statistically analysed. For both plant species, the sprouts of the third germination day are valuable sources of bioactive compounds and could be used in phytotherapy and nutrition. Although Trifolium pratense L. (Red Clover) is considered to be a reference for natural remedies in relieving menopause disorders, alfalfa also showed a high level of biological active compounds with estrogenic activity.

High sensitive multiresidue analysis of pharmaceuticals and antifungals in surface water using U-HPLC-Q-Exactive Orbitrap HRMS. Application to the Danube river basin on the Romanian territory.

The occurrence of 67 pharmaceutical and antifungal residues in the Danube river on the Romanian territory was studied by using solid-phase extraction (SPE) and LC-Q Exactive Orbitrap high resolution MS in both full scan (FS) MS and targeted MS/MS modes. A single-laboratory validation procedure was carried out for the determination of 67 compounds in FSMS mode evaluating selectivity, sensitivity, linearity, precision and accuracy. The method showed satisfactory analytical performance. The evaluation of the recovery concluded that 75% of the compounds show recoveries between 85 and 115% and 10% of the compounds show recoveries between 85% and 65%. The level of detection was lower than 5 ng l(-1) for 66% of the compounds, between 5 and 10 ng l(-1) for 22% and between 10 and 25 ng l(-1) for 14% of the compounds. The coefficients of determination R(2) were higher than 0.99 for 79% of the compounds, over a linearity range of 2.5-50 ng l(-1). Targeted MS/MS analysis, performed in addition to the full scan acquisition was used for confirmatory purpose. Twenty samples of Danube water and three of the main tributaries were collected in May, July, August and October 2014. Analysis of the selected water samples revealed the occurrence of 23 compounds such as diclofenac, carbamazepine, sulfamethoxazole, tylosin, indomethacin, ketoprofen, piroxicam, together with antifungals like thiabendazole, and carbendazim. Carbamazepine was detected in 17 samples, the maximum concentration being 40 ng l(-1). The highest concentration reached was 166 ng l(-1) for diclofenac.

Quantitative modelling to estimate the transfer of pharmaceuticals through the food production system.

Use of pharmaceuticals in animal production may cause an indirect route of contamination of food products of animal origin. This study aimed to assess, through mathematical modelling, the transfer of pharmaceuticals from contaminated soil, through plant uptake, into the dairy food production chain. The scenarios, model parameters, and values refer to contaminants in emission slurry production, storage time, immission into soil, plant uptake, bioaccumulation in the animal's body, and transfer to meat and milk. Modelling results confirm the possibility of contamination of dairy cow's meat and milk due the ingestion of contaminated feed by the cattle. The estimated concentration of pharmaceutical residues obtained for meat ranged from 0 to 6 ng kg(-1) for oxytetracycline, from 0.011 to 0.181 µg kg(-1) for sulfamethoxazole, and from 4.70 to 11.86 µg kg(-1) for ketoconazole. The estimated concentrations for milk were: zero for oxytetracycline, lower than 40 ng L(-1) for sulfamethoxazole, and from 0.98 to 2.48 µg L(-1) for ketoconazole. Results obtained for the three selected pharmaceuticals indicate a minor risk for human health. This study showed that supply chain modelling could be an effective tool in assessing the indirect contamination of feedstuff and animal products by residues of pharmaceuticals. The model can easily be adjusted to other contaminants and supply chain and, in this way, present a valuable tool to underpin decision making.

Uptake of oxytetracycline, sulfamethoxazole and ketoconazole from fertilised soils by plants.

This study was performed to investigate the potential for a set of two antibiotics and one antifungal compound to be taken up from the soil by plants. Plants are used for animal or human consumption, and so the measured concentrations in the plant material will be used to model potential human exposure to these compounds. The uptake by two types of plants (grass and watercress) from two types of soil was studied. The compounds used for these experiments were sulfamethoxazole, oxytetracycline and ketoconazole at concentrations of 5 and 10 mg kg(-1) in the soil. The compounds of interest were extracted out of the plant matrix by applying accelerated solvent extraction. Analyses were carried out by a LC-MS/MS. From the results, it was concluded that the plant materials used for this study were able to take up sulfamethoxazole and ketoconazole when the soil was contaminated with these compounds at a concentration ranging from 5 to 10 mg kg(-1). Sulfamethoxazole was detected in all samples, at levels ranging from 7 to 21 µ kg(-1) for grass and 4 to 7.5 µ kg(-1) for watercress. For ketoconazole, the results showed low absorption. Oxytetracycline was not detected in any sample. A partition-limited model approach was applied for the comparison of experimental and estimated data, and the relationship between physicochemical properties of the compounds and plant uptake was highlighted.

Accurate mass screening of pharmaceuticals and fungicides in water by U-HPLC-Exactive Orbitrap MS.

The use of pharmaceuticals in livestock production is a potential source of surface water, groundwater and soil contamination. Possible impacts of antibiotics on the environment include toxicity and the emergence of antibiotic resistance. Monitoring programs are required to record the presence of these substances in the environment. A rapid, versatile and selective multi-method was developed and validated for screening 43 pharmaceutical and fungicides compounds, in surface and groundwater, in one single full-scan MS method, using benchtop U-HPLC-Exactive Orbitrap MS at 50,000 (FWHM) resolution. Detection was based on calculated exact masses and on retention time. Sample volume, pH conditions and solid-phase extraction (SPE) sample clean-up conditions were optimized. In the final method, 74 % of the compounds had recoveries higher than 80 %, 15 % of the compounds had recoveries between 60 % and 80 %, and 7 % of the compounds had recoveries between 40 % and 50 %. One of the compounds (itraconazole) had a recovery lower than 10 % and nystatin was not detected. The level of detection was 10 ng L(-1) for 61 % of the compounds, 50 ng L(-1) for 32 % and 100 ng L(-1) for 5%. In-house validation, based on EU guidelines, proves that the detection capability CCß is lower than 10 ng L(-1) (for ß error 5 %) for 37 % of the compounds, lower than 50 ng L(-1) for 35 % of the compounds and lower than 100 ng L(-1) for 14 % of compounds. This study demonstrates that the ultra-high resolution and reliable mass accuracy of Exactive Orbitrap MS permits the detection of pharmaceutical residues in a concentration range of 10-100 ng L(-1), applying a post target screening approach, in the multi-method conditions.

Ultrasonic or accelerated solvent extraction followed by U-HPLC-high mass accuracy MS for screening of pharmaceuticals and fungicides in soil and plant samples.

Different veterinary pharmaceuticals are used in agricultural livestock becoming a source of environment contamination. Furthermore, no regulation exists for the concentration limits of pharmaceuticals in soil or water. Monitoring programs for environment contamination with pharmaceuticals are needed, requiring new sensitive and selective screening methods. The present study focuses on developing a method for the simultaneous scanning of forty-two compounds (pharmaceuticals, azole biocides and fungicides) in soil and plant material samples. For extraction purposes the use of ultrasonic assisted and accelerated solvent extraction (ASE) were compared. The extract was purified and concentrated by applying a solid phase extraction step followed by ultra-high-performance-chromatographic separation and accurate-mass spectrometric detection using Exacte Orbitrap technology (FWHM 50,000). The effects of the different extraction solvents and conditions on the extraction efficiency were tested. Although both extraction approaches are applicable the optimal extraction efficiency was obtained by applying accelerated solvent extraction using solvent mixtures containing acetone for soil and methanol for plant samples. An ASE process has been validated for the determination of selected pharmaceuticals and fungicides in soil and in plant material. The recoveries from soil samples were >70% for more than 68% of the compounds. The levels of detection were ≤10 µg kg(-1) for 93% of the compounds tested. The recoveries from plant material were >70% for 64% of the compounds tested. The levels of detection were ≤10 µg kg(-1) for 66% of the compounds. The developed method was used to screen soil and plant material collected throughout the Netherlands and oxytetracycline residues were detected.