The Effects of Nature-Inspired Synthesis on Silver Nanoparticle Generation.

A wide range of methods can be used for nature-inspired metallic nanoparticle (NP) synthesis. These syntheses, however, are ongoing in the presence of diverse mixtures of different chemical compounds, and all or only a few of these contribute to resultant particle properties. Herein, the linden (Tilia sp.) inflorescence leachate and pure citric and protocatechuic acids were chosen for Ag-AgCl nanoparticle (NP) synthesis, and the resultant particles were then compared. We focused on the following four issues: (1) preparation of Ag-AgCl NPs using the Tilia sp.-based phytosynthetic protocol, (2) analytical determination of the common phenolic, nonphenolic, and inorganic profiles of three Tilia sp. types from different harvesting locations, (3) preparation of Ag-AgCl NPs using a mixture of citric and protocatechuic acids based on chromatographic evaluation, and (4) comparison of Tilia-based and organic acid-based syntheses. Our research confirms that the Tilia organic and inorganic profiles in biomasses are influenced by the harvesting location, and the three sites influenced both the morphology and final NP size. Our processing method was uniform, and this enabled great Ag-AgCl NP reproducibility for each specific biomass. We were then able to prove that the simplified organic acid-based synthesis produced even smaller NPs than Tilia-based synthesis. These findings provide better understanding of the significant influence on NP final properties resulting from other organic acids contained in the linden.

Analytical methods in strigolactone research.

Strigolactones (SLs) are important plant hormones that are produced via the carotenoid biosynthetic pathway and occur at extremely low concentrations in various plant species. They regulate root development, play important roles in symbioses between higher plants and mycorrhizal fungi, and stimulate germination of plant-parasitic Orobanche and Striga species. Chemical analysis is central to research on the biochemistry of SLs and their roles in developmental biology and plant physiology. Here we summarize key issues relating to the identification and quantification of SLs isolated from plant tissues and exudates. The advantages and drawbacks of different protocols used for strigolactone analysis are discussed, and guidelines for selecting a procedure that will minimize losses during isolation and purification prior to final analysis are proposed. Hyphenated techniques suitable for SL analysis such as GC-MS and LC-MS/MS are also discussed, and newer ambient techniques such as HR-DART-MS and DESI-MS are highlighted as tools with considerable potential in SL research. A key advantage of these methods is that they require only simply sample preparation.

Assessment of Antioxidants in Selected Plant Rootstocks.

The service tree (Sorbus domestica) is a wild fruit tree with immense medicinal and industrial value. This study aimed at determining the four major groups of antioxidants (flavonoids, phenolic acids and aldehydes, catechin and procyanidin) in rootstocks of Crataegus laevigata (genotypes O-LE-14 and O-LE-21), Aronia melanocarpa (genotypes O-LE-14 and O-LE-21), Chaenomeles japonica (genotype O-LE-9) and Cydonia oblonga (BA 29) (genotypes O-LE-14 and O-LE-21). Hyperoside (Quercetin 3-D-galactoside) was the most abundant flavonoid compound, since its average content in the rootstocks of Crataegus laevigata (O-LE-21) was 180.68 ± 0.04 µg·g-1. Dihydrokaempherol was the least frequently found flavonoid compound, with an average concentration of 0.43 ± 0.01 µg·g-1 in all the rootstocks of plants considered in this study. Among the phenolic compounds, the most represented one was protocatechuic acid, with 955.92 ± 10.25 µg·g-1 in the rootstocks of Aronia melanocarpa (O-LE-14). On the other hand, the least represented p-Coumaric acid exhibited the average concentration of 0.34 ± 0.01 µg·g-1 in the plant rootstocks. Epicatechin was the most abundant catechin compound, with a content of 3196.37 ± 50.10 µg·g-1 in the rootstocks of Aronia melanocarpa (O-LE-14). The lowest represented catechin compound was epigallocatechin, with the average concentration of 0.95 ± 0.08 µg·g-1 in the screened plant rootstocks. From the procyanidin compounds, the most abundant one was procyanidin b2 in the rootstocks of Crataegus laevigata (O-LE-14), with a concentration of 5550.40 ± 99.56 µg·g-1. On the contrary, procyanidin a2, with an average concentration of 40.35 ± 1.61 µg·g-1, represented the least frequent procyanidin compound in all the plant rootstocks screened herein.

Lycorine and UV-C stimulate phenolic secondary metabolites production and miRNA expression in Chlamydomonas reinhardtii.

Studying stress pathways on the level of secondary metabolites that are found in very small concentration in the cells is complicated. In the algae, the role of individual metabolites (such as carotenoids, phenolic compounds, organic acids, and vitamins) and miRNAs that participate in plant's defence are very poorly understood during stressful conditions. Therefore, in the present experiment, the model organism Chlamydomonas reinhardtii was exposed to stress conditions (Lyc and UV-C irradiation) to detect these substances, even at very low concentrations. The purpose was to monitored changes at each response level with a future view to identifying their specific roles under different stress factors. In stress-treated cultures, numerous transcriptomic and metabolomic pathways were triggered in C. reinhardtii. Although Lyc significantly decreased the concentration of AA, suggesting that Lyc has a similar function in C. reinhardtii as in plants. The negative effect of UV-C radiation was based on the production of ROS and enhancement of antioxidant responses, resulting in increased levels of polyphenols and simple phenolic compounds. Both treatments did lead to extensive changes in transcript levels and miRNA expression patterns.

Sarcosine is a prostate epigenetic modifier that elicits aberrant methylation patterns through the SAMe-Dnmts axis.

DNA hypermethylation is one of the most common epigenetic modifications in prostate cancer (PCa). Several studies have delineated sarcosine as a PCa oncometabolite that increases the migration of malignant prostate cells while decreasing their doubling time. Here, we show that incubation of prostate cells with sarcosine elicited the upregulation of sarcosine N-demethylation enzymes, sarcosine dehydrogenase and pipecolic acid oxidase. This process was accompanied by a considerable increase in the production of the major methyl-donor S-adenosylmethionine (SAMe), together with an elevation of cellular methylation potential. Global DNA methylation analyses revealed increases in methylated CpG islands in distinct prostate cell lines incubated with sarcosine, but not in cells of nonprostate origin. This phenomenon was further associated with marked upregulation of DNA methyltransferases (Dnmts). Epigenetic changes were recapitulated through blunting of Dnmts using the hypomethylating agent 5-azacytidine, which was able to inhibit sarcosine-induced migration of prostate cells. Moreover, spatial mapping revealed concomitant increases in sarcosine, SAMe and Dnmt1 in histologically confirmed malignant prostate tissue, but not in adjacent or nonmalignant tissue, which is in line with the obtained in vitro data. In summary, we show here for the first time that sarcosine acts as an epigenetic modifier of prostate cells and that this may contribute to its oncometabolic role.

Antioxidant, gene expression and metabolomics fingerprint analysis of Arabidopsis thaliana treated by foliar spraying of ZnSe quantum dots and their growth inhibition of Agrobacterium tumefaciens.

Nanotechnology, new fascinating field of science, is bringing many application's options. However, it is necessary to understand their potential environmental risk and toxicity. Zinc selenide quantum dots (ZnSe QDs) are getting valuable due to wide industrial usage, mainly as cadmium free diodes or stabilizing ligand. Thanks to unique properties, they could also open the possibilities of application in the agriculture. Their effects on living organisms, including plants, are still unknown. Therefore, the attention of this work was given to antioxidant response of Arabidopsis thaliana to 100 and 250 µM ZnSe QDs foliar feeding. ZnSe QDs treatment had no statistically significant differences in morphology but led to increased antioxidant response in the leaves at the level of gene expression and production secondary antioxidant metabolites. Concurrently, analysis of growth properties of Agrobacterium tumefaciens was done. 250 µM ZnSe solution inhibited the Agrobacterium tumefaciens viability by 60%. This is the first mention about effect ZnSe QDs on the plants. Although QDs induced oxidative stress, the apply treatment dose of ZnSe QDs did not have significant toxic effect on the plants and even no morphological changes were observed. However, the same amount of ZnSe QD induced an inhibitory effect on Agrobacterium tumefaciens.

The strong reaction of simple phenolic acids during oxidative stress caused by nickel, cadmium and copper in the microalga Scenedesmus quadricauda.

This work has analysed the influence of CdCl2, NiCl2 and CuCl2 on simple phenolic acids, such as the 3PPP (phenylpyruvic, phenylacetic, and 4-hydroxyphenylpyruvic) and 2DR (3,4-dihydroxyphenylacetic and rosmarinic) phenolic acids for the first time and studied their interactions with antioxidant systems and the glutathione-ascorbate cycle in the freshwater green microalga Scenedesmus quadricauda. The compounds investigated are related to both the catabolic and anabolic pathways of l-phenylalanine and l-tyrosine, the main molecules in the biosynthesis of polyphenols. The concentrations of the simple phenolic acids responded significantly to NiCl2, CdCl2 and CuCl2 at 5, 20 and 40 µM concentrations. The 3PPP phenolic acid concentrations after 24 h were always higher in metal-treated cells than in controls, while the 2DR concentrations were significantly lower in the metal-treated cells than the controls. The GSH/GSSG ratio was lower in all experimental groups treated with the selected metals (especially so at 40 µM). Pearson correlation analysis indicated a strong negative correlation between ascorbate and rosmarinic acid content (-0.670; p < 0.05) in NiCl2-treated samples and reduced glutathione and 3,4-dihydroxyphenylacetic content (-0.700; p < 0.05) in CdCl2-treated samples. The GSSG content in samples exposed to CuCl2 was correlated with the concentrations of all of the investigated phenolic acids (4 negative, 1 positive).

The effects of 5-azacytidine and cadmium on global 5-methylcytosine content and secondary metabolites in the freshwater microalgae Chlamydomonas reinhardtii and Scenedesmus quadricauda.

Epigenetic changes are important mechanisms in the regulation of chromatin structure and gene expression. Cytosine methylation is one of the major epigenetic modifications, mediated by DNA methyltransferases, which transfer methyl groups from S-adenosyl-L-methionine (SAM) to the fifth carbon of cytosine. Various external environmental conditions can change the global hypo/hypermethylation pattern of DNA. These alterations may affect the organism's response to stress conditions. In this study, for the first time, we investigated the effects of 5-azacytidine, a DNA methyltransferase inhibitor, and cadmium, a toxic metal and environmental pollutant, on the growth, biosynthesis of secondary metabolites (phenols, flavonoids, carotenoids), SAM, S-adenosylhomocysteine, 5'-methylthioadenosine and global 5-methylcytosine (5-mC) in the green microalgae Chlamydomonas reinhardtii and Scenedesmus quadricauda. The studied species showed major differences in 5-mC content, secondary metabolite content, and antioxidant activity. Cadmium increased GSH (glutathione) content in C. reinhardtii by 60% whereas 5-azacytidine did not affect GSH. The biosynthesis of GSH in S. quadricauda in response to the stressors was the opposite. Global 5-mC content of C. reinhardtii was 1%-1.5%, and the content in S. quadricauda was 3.5%. Amount of some investigated methionine cycle metabolites (SAM, S-adenosyl homocysteine [SAH], methionine) in S. quadricauda distinctly exceeded C. reinhardtii as well. However, chlorophylls a and b, carotenoids, total phenolic content, total flavonoid content and, antioxidant activity were significantly higher in C. reinhardtii than S. quadricauda. Therefore, in further studies it would be advisable to verify whether methylation of cytosine affects the expression of genes encoding certain secondary metabolites.

Chromatin architecture changes and DNA replication fork collapse are critical features in cryopreserved cells that are differentially controlled by cryoprotectants.

In this work, we shed new light on the highly debated issue of chromatin fragmentation in cryopreserved cells. Moreover, for the first time, we describe replicating cell-specific DNA damage and higher-order chromatin alterations after freezing and thawing. We identified DNA structural changes associated with the freeze-thaw process and correlated them with the viability of frozen and thawed cells. We simultaneously evaluated DNA defects and the higher-order chromatin structure of frozen and thawed cells with and without cryoprotectant treatment. We found that in replicating (S phase) cells, DNA was preferentially damaged by replication fork collapse, potentially leading to DNA double strand breaks (DSBs), which represent an important source of both genome instability and defects in epigenome maintenance. This induction of DNA defects by the freeze-thaw process was not prevented by any cryoprotectant studied. Both in replicating and non-replicating cells, freezing and thawing altered the chromatin structure in a cryoprotectant-dependent manner. Interestingly, cells with condensed chromatin, which was strongly stimulated by dimethyl sulfoxide (DMSO) prior to freezing had the highest rate of survival after thawing. Our results will facilitate the design of compounds and procedures to decrease injury to cryopreserved cells.

Separation, identification and quantification of carotenoids and chlorophylls in dietary supplements containing Chlorella vulgaris and Spirulina platensis using High Performance Thin Layer Chromatography.

In this study, 14 commercial products (dietary supplements) containing alga Chlorella vulgaris and cyanobacteria Spirulina platensis, originated from China and Japan, were analysed. UV-vis spectrophotometric method was applied for rapid determination of chlorophylls, carotenoids and pheophytins; as degradation products of chlorophylls. High Performance Thin-Layer Chromatography (HPTLC) was used for effective separation of these compounds, and also Atomic Absorption Spectrometry for determination of heavy metals as indicator of environmental pollution. Based on the results obtained from UV-vis spectrophotometric determination of photosynthetic pigments (chlorophylls and carotenoids), it was confirmed that Chlorella vulgaris contains more of all these pigments compared to the cyanobacteria Spirulina platensis. The fastest mobility compound identified in Chlorella vulgaris and Spirulina platensis using HPTLC method was ß-carotene. Spectral analysis and standard calibration curve method were used for identification and quantification of separated substances on Thin-Layer Chromatographic plate. Quantification of copper (Cu2+, at 324.7 nm) and zinc (Zn2+, at 213.9nm) was performed using Flame Atomic Absorption Spectrometry with air-acetylene flame atomization. Quantification of cadmium (Cd2+, at 228.8 nm), nickel (Ni2+, at 232.0nm) and lead (Pb2+, at 283.3nm) by Electrothermal Graphite Furnace Atomic Absorption Spectrometry; and quantification of mercury (Hg2+, at 254nm) by Cold Vapour Atomic Absorption Spectrometry.

Anticarcinogenic Effect of Spices Due to Phenolic and Flavonoid Compounds-In Vitro Evaluation on Prostate Cells.

This study shows the effects of spices, and their phenolic and flavonoid compounds, on prostate cell lines (PNT1A, 22RV1 and PC3). The results of an MTT assay on extracts from eight spices revealed the strongest inhibitory effects were from black pepper and caraway seed extracts. The strongest inhibitory effect on prostatic cells was observed after the application of extracts of spices in concentration of 12.5 mg·mL-1. An LC/MS analysis identified that the most abundant phenolic and flavonoid compounds in black pepper are 3,4-dihydroxybenzaldehyde and naringenin chalcone, while the most abundant phenolic and flavonoid compounds in caraway seeds are neochlorogenic acid and apigenin. Using an MTT assay for the phenolic and flavonoid compounds from spices, we identified the IC50 value of ~1 mmol·L-1 PNT1A. The scratch test demonstrated that the most potent inhibitory effect on PNT1A, 22RV1 and PC3 cells is from the naringenin chalcone contained in black pepper. From the spectrum of compounds assessed, the naringenin chalcone contained in black pepper was identified as the most potent inhibitor of the growth of prostate cells.

Development of new efficient method for isolation of phenolics from sea algae prior to their rapid resolution liquid chromatographic-tandem mass spectrometric determination.

The extraction of phenolic compounds from 4 different sea algae samples, three brown algae (Cystoseira abies-marina, C. abies-marina grinded under cryogenic conditions with liquid nitrogen, Undaria pinnatifida and Sargassum muticum) and one red algae (Chondrus crispus) via solid phase extraction using micro-elution solid-phase extraction (µ-SPE) plate method was studied. Prior to µ-SPE, 50mg of algae with 80% methanol mixture was extracted in hyphenated series by various extraction techniques, such as pressurized liquid extraction and Ika Ultra-Turrax® Tube Drive, in combination with ultrasound assisted extraction. The µ-SPE plate technique reduced the time of sample pre-treatment thanks to higher sensitivity and pre-concentration effect. Selected groups of benzoic acid derivatives (p-hydroxybenzoic, protocatechuic, gallic, vanillic, and syringic acids), hydroxybenzaldehydes (4-hydroxybenzaldehyde, and 3,4-dihydroxybenzaldehyde), and cinnamic acid derivatives (p-coumaric, caffeic, ferulic, sinapic, and chlorogenic acids) were determined using rapid resolution liquid chromatography coupled to mass spectrometry detection with negative ion electrospray ionization (RRLC-ESI-MS) using multiple reactions monitoring. LOQs of measured samples varied in the range 0.23-1.68ng/mL and LODs in the range 0.07-0.52ng/mL. The applied method allowed a simultaneous determination of phenolics (i.e. free, esters soluble in methanol, glycosides, and esters insoluble in methanol) in less than 5min (including alkaline or acidic hydrolysis of raw extracts) from sea algae extracts.

Determination of Phytoestrogen Content in Fresh-Cut Legume Forage.

The aim of the study was to determine phytoestrogen content in fresh-cut legume forage. This issue has been much discussed in recent years in connection with the health and safety of feedstuffs and thus livestock health. The experiments were carried out on two experimental plots at Troubsko and Vatín, Czech Republic during June and July in 2015. Samples were collected of the four forage legume species perennial red clover (variety "Amos"), alfalfa (variety "Holyne"), and annuals Persian clover and Alexandrian clover. Forage was sampled twice at regular three to four day intervals leading up to harvest and a third time on the day of harvest. Fresh and wilted material was analyzed using liquid chromatography-mass spectrometry (LC-MS). Higher levels ( p < 0.05) of isoflavones biochanin A (3.697 mg·g (-1) of dry weight) and formononetin (4.315 mg·g (-1) of dry weight) were found in red clover than in other species. The highest isoflavone content was detected in red clover, reaching 1.001% of dry matter ( p < 0.05), representing a risk for occurrence of reproduction problems and inhibited secretion of animal estrogen. The phytoestrogen content was particularly increased in wilted forage. Significant isoflavone reduction was observed over three to four day intervals leading up to harvest.

Two New Faces of Amifostine: Protector from DNA Damage in Normal Cells and Inhibitor of DNA Repair in Cancer Cells.

Amifostine protects normal cells from DNA damage induction by ionizing radiation or chemotherapeutics, whereas cancer cells typically remain uninfluenced. While confirming this phenomenon, we have revealed by comet assay and currently the most sensitive method of DNA double strand break (DSB) quantification (based on γH2AX/53BP1 high-resolution immunofluorescence microscopy) that amifostine treatment supports DSB repair in γ-irradiated normal NHDF fibroblasts but alters it in MCF7 carcinoma cells. These effects follow from the significantly lower activity of alkaline phosphatase measured in MCF7 cells and their supernatants as compared with NHDF fibroblasts. Liquid chromatography-mass spectrometry confirmed that the amifostine conversion to WR-1065 was significantly more intensive in normal NHDF cells than in tumor MCF cells. In conclusion, due to common differences between normal and cancer cells in their abilities to convert amifostine to its active metabolite WR-1065, amifostine may not only protect in multiple ways normal cells from radiation-induced DNA damage but also make cancer cells suffer from DSB repair alteration.

Age affects not only metabolome but also metal toxicity in Scenedesmus quadricauda cultures.

Responses of Scenedesmus quadricauda grown in vitro and differing in age (old culture-13 months, young culture-1 month) to short-term cadmium (Cd) or nickel (Ni) excess (24h) were compared. Higher age of the culture led to lower amount of chlorophylls, ascorbic acid and glutathione but higher signal of ROS. Surprisingly, sucrose was detected using DART-Orbitrap MS in both old and young culture and subsequent quantification confirmed its higher amount (ca. 3-times) in the old culture. Cd affected viability and ROS amount more negatively than Ni that could arise from excessive Cd uptake which was also higher in all treatments than in respective Ni counterparts. Surprisingly, nitric oxide was not extensively different in response to age or metals. Strong induction of phytochelatin 2 is certainly Cd-specific response while Ni also elevated ascorbate content. Krebs cycle acids were more accumulated in the young culture but they were rather elevated in the old culture (citric acid under Ni excess). We conclude that organic solid 'Milieu Bristol' medium we tested is suitable for long-term storage of unicellular green algae (also successfully tested for Coccomyxa sp. and Parachlorella sp.) and the impact of age on metal uptake may be useful for bioremediation purposes.

Drought-Induced Responses of Physiology, Metabolites, and PR Proteins in Triticum aestivum.

The impact of severe drought stress (13% soil moisture) on the physiological responses, metabolic profile, and pathogenesis-related (PR) proteins in wheat above- and below-ground biomass after 20 days of treatment was studied. Drought depleted growth, assimilation pigments, and majority of free amino acids in the shoots (but proline increased considerably, +160%). On the contrary, root growth parameters were elevated, and free amino acids did not decrease, indicating investment of metabolites into the growth of roots under water deficiency. Mineral nutrients were only slightly influenced. Profiling of pathogenesis-related (PR) proteins revealed that chitinases (EC 3.2.1.14) and glucanases (EC 3.2.1.39) were activated in wheat by drought. Individual isoforms and their activity were rather stimulated under drought, especially in shoots. The expression of selected genes is in agreement with enzymatic data and suggests an organ (tissue) specific- and opposing behavior of these two types of defense components in drought-stressed wheat. Metabolic analyses at the level of phenolics showed an increase in the free and bound fraction of phenolic acids almost exclusively in the shoots and flavonoid isoorientin increased considerably: protective action against oxidative stress and dehydration of the leaves seems to be the main reason for this finding. The role of PR proteins and phenolics in drought-stressed tissue is discussed.

Chromium speciation and biochemical changes vary in relation to plant ploidy.

Uptake of trivalent chromium (Cr(III)-chloride), Cr speciation and consequences for the metabolism in chamomile plants with two ploidy levels have been studied. Depletion of fresh biomass, tissue water content and soluble proteins in response to high (120 µM) Cr(III) was ploidy-independent. Cr mainly accumulated in the roots (only negligibly in the shoots) and total root Cr amount was higher in tetraploid ones including the proof with specific fluorescent indicator (naphthalimide-rhodamine) of Cr(III). Quantification of Cr(VI) detected its higher content in tetraploid roots (up to 4.2% from total Cr), indicating partial oxidation of applied Cr(III). Higher H2O2 presence but lower activities of peroxidases were observed in tetraploid roots while nitric oxide, superoxide dismutase and glutathione reductase activities did not differ extensively. Soluble phenols, lignin, non-protein thiols, individual thiols (glutathione and phytochelatin 2) and ascorbic acid responded to high Cr(III) similarly in both cultivars while decrease of minerals was more pronounced in tetraploid ones. It seems that Cr(III)-induced oxidative stress arises from high root Cr uptake and Cr(VI) presence and is related to depletion of thiols. Assay of Krebs cycle acids confirmed rather depletion under 120 µM Cr(III) in both cultivars but increase in citric acid may indicate its involvement in root Cr chelation. Subsequent comparison of Cr(III)-chloride and Cr(III)-nitrate showed similar influence on Cr accumulation and majority of biochemical responses while different impact on phytochelatin 2 amount was the most distinct feature.

Determination of pyruvate and lactate as potential biomarkers of embryo viability in assisted reproduction by capillary electrophoresis with contactless conductivity detection.

Human-assisted reproduction is increasing in importance due to the constantly rising number of couples suffering from infertility issue. A key step in in vitro fertilization is the proper assessment of embryo viability in order to select the embryo with the highest likelihood of resulting in a pregnancy. This study proposes a method based on CE with contactless conductivity detection for the determination of pyruvate and lactate in spent culture media used in human-assisted reproduction. A fused-silica capillary of 64.0 cm total length and 50 µm inner diameter was used. The inner capillary wall was modified by the coating of successive layers of the ionic polymers polybrene and dextran sulfate to reverse EOF. The BGE was composed of 10 mM MES/lithium hydroxide, pH 6.50. The sample was injected by pressure 50 mbar for 18 s, separation voltage was set to -24 kV, and capillary temperature to 15°C. The presented method requires only 2 µL of the culture medium, with LODs for pyruvate and lactate of 0.03 and 0.02 µM, respectively. The results demonstrated the method's suitability for the analysis of spent culture media to support embryo viability assessment by light microscopy, providing information about key metabolites of the energy metabolism of a developing embryo.

Lanthanum rather than cadmium induces oxidative stress and metabolite changes in Hypericum perforatum.

Physiology, oxidative stress and production of metabolites in Hypericum perforatum exposed to moderate Cd and/or La concentration (10 µM) were studied. La evoked increase in reactive oxygen species, malondialdehyde and proline but suppressed growth, tissue water content, glutathione, ascorbic acid and affected mineral nutrient contents more than Cd while the impact of Cd+La was not synergistic. Similar trend was observed at the level of superoxide dismutase gene expression. Shoot Cd amount increased in Cd+La while only root La increased in the same treatment. Extensive quantification of secondary metabolites revealed that La affected phenolic acids more pronouncedly than Cd in shoots and roots. Flavonols were suppressed by La that could contribute to the appearance of oxidative damage. Procyanidins increased in response to La in the shoots but decreased in the roots. Metabolic responses in Cd+La treatment resembled those of La treatment (almost identically in the roots). Phenylalanine ammonia-lyase activity was mainly suppressed by La. The presence of La also depleted amount of hypericin and expression of its putative gene (hyp-1) showed similar trend but accumulation of hyperforin increased under Cd or La excess. Clear differences in the stem and root anatomy in response to Cd or La were also found. Overall, H. perforatum is La-sensitive species and rather Cd ameliorated negative impact of La.

Direct analysis in real time--high resolution mass spectrometry as a valuable tool for the pharmaceutical drug development.

In this study, direct analysis in real time-mass spectrometry (DART-MS) was assessed for the analysis of various pharmaceutical formulations with intention to summarize possible applications for the routine pharmaceutical development. As DART is an ambient ionization technique, it allows direct analysis of pharmaceutical samples in solid or liquid form without complex sample preparation, which is often the most time-consuming part of the analytical method. This makes the technique suitable for many application fields, including pharmaceutical drug development. DART mass spectra of more than twenty selected tablets and other common pharmaceutical formulations, i.e. injection solutions, ointments and suppositories developed in the pharmaceutical industry during several recent years are presented. Moreover, as thin-layer chromatography (TLC) is still very popular for the monitoring of the reactions in the synthetic chemistry, several substances were analyzed directly from the TLC plates to demonstrate the simplicity of the technique. Pure substance solutions were spotted onto a TLC plate and then analyzed with DART without separation. This was the first DART-MS study of pharmaceutical dosage forms using DART-Orbitrap combination. The duration of sample analysis by the DART-MS technique lasted several seconds, allowing enough time to collect sufficient number of data points for compound identification. The experimental setup provided excellent mass accuracy and high resolution of the mass spectra which allowed unambiguous identification of the compounds of interest. Finally, DART mass spectrometry was also used for the monitoring of the selected impurity distribution in the atorvastatin tablets. These measurements demonstrated DART to be robust ionization technique, which provided easy-to-interpret mass spectra for the broad range of compounds. DART has high-throughput potential for various types of pharmaceutical analyses and therefore eliminates the time for sample cleanup and chromatographic separation.