A coumarin with an unusual structure from Cuphea ignea, its cytotoxicity and antioxidant activities.

Phenolic metabolite profiling using two dimensional paper chromatographic analysis (2 DPC) was used for assaying the complex mixture of phenolics of an aqueous ethanol aerial part extract of Cuphea ignea (Lytheraceae). A coumarin with a rare structure, namely, 7-hydroxy 3-methoxy coumarin 5-O-ß-glucopyranoside was isolated from the investigated extract. The structure was elucidated by conventional methods and spectral analysis, including one and two dimensional NMR (1D and 2D NMR), as well as by interpretation of the spectra obtained by high resolution electrospray ionization mass technique (HRESIMS). The rare coumarin significantly inhibited reactive oxygen species production with an ED50 value of 6.31±1.64 µg/ml and 5.78±0.66 µg/ml as determined by the the free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) and the oxygen radical absorption capacity (ORAC) assay respectively. The isolated coumarin presented a cytotoxic activity assessed by using the neutral red assay (NRU) against lung cancer cell line (H23) with IC50 of 40.38±2.75 µg/ml.

A cytotoxic flavonol glycoside from Melaleuca leucadendra leaves extract with immunostimulant activity.

Leaves of Melaleuca leucadendra contain the novel flavonol glycoside, myricetin 3-O-ß-4C1-galactopyranuronoid. In addition, known fifteen phenolics were identified. All isolates are characterized for the first time from this plant. Structures were established by conventional methods and confirmed by spectral methods of analysis, including one and two-dimensional nuclear magnetic resonance spectroscopy (1D and 2D-NMR) and high resolution electro-spray ionization mass spectrometry (HRESIMS), as well. Assessment of some immunological and biological efficacy, of the extract in combination with a parallel cytotoxicity evaluation, using the method of cellular reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) technique was carried out. Besides, evaluation of the antioxidant effectiveness, using the free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) and the oxygen radical absorption capacity (ORAC) methods was performed. In addition, the cytotoxicity against liver (Huh-7), breast (MCF-7) and prostate (PC-3) cancers using the neutral red assay (NRU) technique for the extract and the new flavonol glycoside also, was assessed.

Phenolic profiling of an extract from Eugenia jambos L. (Alston)--the structure of three flavonoid glycosides--antioxidant and cytotoxic activities.

Phenolic metabolite profiling and identification using high performance liquid chromatography (HPLC) coupled to high resolution accurate mass spectrometry (HR-ESI-MS) with detection of negative ions was used for assaying the complex mixture of phenolics of an aqueous ethanol leaf extract of Eugeniajambos L. (Myrtaceae). Eight known polyphenolics were tentatively identified, and, in addition, three hitherto unknown flavonol-O-glycosides were detected in the extract. These unknowns were taken as the targets and isolated by means of consecutive polyamide S6, MCI gel and repeated Sephadex LH-20 column fractionation. The isolation and purification were monitored by HPLC/ESI-MS. The isolates were subsequently identified as quercetin 3-O-xylosyl-(1"' --> 2")-O-xyloside, myricetin 7-methylether 3-O-xylosyl-(1"' --> 2")-rhamnoside and myricetin 3',5'-dimethyl ether 3-O-xylosyl-(1"'-->* 2")-O-rhamnoside. All known metabolites were also separated by applying the same chromatographic techniques. ESI-MS, ¹H and ¹³C NMR spectra were then recorded, completely interpreted and confirmed by HR-ESI-MS and 2D NMR spectroscopy. In order to get information about biological activities of E. jambos the extract was tested for radical scavenging activity by DPPH and ORAC assay. In addition, its cytotoxicity was assessed by the neutral red assay against non-tumorigenic HaCaT keratinocytes and the human bladder carcinoma cell line 5637.

Polyphenols in Ammania auriculata: structures, antioxidative activity and cytotoxicity.

Chemical and biological investigations of the extract of Ammania auriculata (Lytheraceae) resulted in the identification of eight polyphenols (1 - 8) for the first time from this plant, including the gallotannin, 2,3,6-tri-O-galloyl-(α,ß)-4C1-glucopyranose (8), for which 1D and 2D-NMR spectra were recorded and assigned for the first time. The structures of all isolates (1 - 8) were elucidated by conventional methods, spectroscopic analysis, including 1D and 2D NMR, and by HR-ESIMS as well. All of the isolated compounds were evaluated for their antioxidant activities, determined by the DPPH and ORAC methods and for their cytotoxicity against the keratinocyte cell line HaCaT using the neutral red assay (NRU) and cell cycle analysis. Compounds 1, 3, 4, 5, and 6 significantly inhibited reactive oxygen species production with ED50 values between 3.22 and 9.79 µg/ml. Compounds 1, 3, 4, and 5 showed cytotoxic activity against HaCaT cells with IC50 values between 30.7 and 84.1 µg/ml. The new galloyl glucose (8) was found not cytotoxic. Ellagitannins, 2,3-hexahydroxy-((α/ß)-glucopyranose (1) and 1 -0-galloyl 2,3-hexahydroxy-(α)-glucopyranose (5) possess remarkable antioxidative and comparably weak cytotoxic activity.

Application of metal-coded affinity tags (MeCAT): absolute protein quantification with top-down and bottom-up workflows by metal-coded tagging.

As the quantification of peptides and proteins extends from comparative analyses to the determination of actual amounts, methodologies for absolute protein quantification are desirable. Metal-coded affinity tags (MeCAT) are chemical labels for peptides and proteins with a lanthanide-bearing chelator as a core. This modification of analytes with non-naturally occurring heteroelements adds the analytical possibilities of inductively coupled plasma mass spectrometry (ICPMS) to quantitative proteomics. We here present the absolute quantification of recombinantly expressed aprotinin out of its host cell protein background using two independent MeCAT methodologies. A bottom-up strategy employs labeling of primary amino groups on peptide level. Synthetic peptides with a MeCAT label which are externally quantified by flow injection analysis (FIA)-ICPMS serve as internal standard in nanoHPLC-ESI-MS/MS. In the top-down approach, protein is labeled on cysteine residues and separated by two-dimensional gel electrophoresis. Flow injection analysis of dissolved gel spots by ICPMS yields the individual protein amount via its lanthanide label content. The enzymatic determination of the fusion protein via its ß-galactosidase activity found 8.3 and 9.8 ng/µg (nanogram fusion protein per microgram sample) for batches 1 and 2, respectively. Using MeCAT values of 4.0 and 5.4 ng/µg are obtained for top-down analysis, while 14.5 and 15.9 ng/µg were found in the bottom-up analysis.

Distribution profiles of nitroxide spin probes in human skin--a combined study using spatially resolved electron spin resonance spectroscopy and mass spectrometry.

Electron spin resonance spectroscopy and mass spectrometry are two analytical methods that are very rarely used in combination. In this paper, we will show that the methods complement one another in the example of the distribution of stable nitroxide radicals in human skin, including the spatial resolution of these distribution processes. There are many ESR investigations dealing with this subject, but unfortunately, they are all limited to the detection of paramagnetic species. The combination with MS allows the successful examination of the distribution profile of the main biotransformation product of the nitroxide radicals, the respective "ESR-silent" hydroxylamines. In order to maintain the biological state of the sample material as far as possible, atmospheric pressure matrix-assisted laser desorption/ionization with ion trap detection has been used for the mass spectrometric investigations. The results validate the former findings of the strong reduction of stable free radicals by biological material; moreover, the diamagnetic species formed during these processes have been identified.

Development of an in vitro modified skin absorption test for the investigation of the follicular penetration pathway of caffeine.

The Organization for Economic Cooperation and Development (OECD) recommends caffeine as a reference substance for in vitro skin absorption tests using Franz diffusion cells (FDC). However, it has not been possible to investigate the follicular penetration pathway using this method until now. The aim of this study was to develop a technique to allow the examination of the follicular penetration pathway of a substance penetrating into the skin. The OECD standard method was therefore combined with the follicle closing technique (FCT), an established in vivo method. By using test skin of varying follicular densities, different penetration values were obtained for the test substance caffeine. The follicular penetration rate was determined by an indirect calculation after modifying the in vivo FCT for use in the in vitro FDC. This method is the first to allow the differentiation of penetration pathways by combining the OECD standard method (using the FDC) and the FCT. Caffeine showed a surprisingly high rate of penetration through the follicular shunts in vitro.

Bone mineralization enhancing activity of a methoxyellagic acid glucoside from a Feijoa sellowiana leaf extract.

The capability of an aqueous methanol extract obtained from the leaves of Feijoa sellowiana Berg. on possible prevention and treatment of osteoporosis has been examined by evaluating its stimulating effect on the two human osteoblastic cell lines HOS58 and SaOS-2. The extract was found to increase significantly the mineralization of cultivated human bone cell, whereby a clear increment (15.3 +/- 2.7%) in von Kossa positive area was determined when administering 25 microg/ml leaf extract. A phytochemical investigation of the extract has demonstrated the high phenolic content and led to the isolation and identification of twenty three of them, among which the new 3-methoxyellagic acid 4-O-beta-glucopyranoside was fully identified. All structures were elucidated on the basis of conventional analytical methods and confirmed by FTMS, 1D- and 2D-NMR data. The new compound was found to cause a significant increase of mineralized area at 20 microg/mL, while at lower concentrations the effect was not significant. However, an increase of the number of mineralized spots (nodules) at all tested concentrations of the compound was observed.

Aphyllin, the first isoferulic acid glycoside and other phenolics from Tamarix aphylla flowers.

The first glycosylated isoferulic acid, isoferulic acid 3-O-beta-glucopyranoside, together with the new phenolics, tamarixetin 3,3'-di-sodium sulphate and dehydrodigallic acid dimetyl ester have been characterized from a flower extract of Tamarix aphylla. The structures were established on the basis of spectral data. The extract exhibited a distinct radical scavenging effect and to improve the viability of human keratinocytes (HaCaT cells). Also, the known isoferulic acid and ferulic acid which have been determined to be the major components of the investigated extract by HPLC/ESI mass spectrometric screening have been separated, characterized and evaluated as active antioxidants and as cell activity stimulating agents as well.

Destabilizing the interplay between miR-1275 and IGF2BPs by Tamarix articulata and quercetin in hepatocellular carcinoma.

Insulin-like growth factor-2 binding proteins (IGF2BPs) are oncogenic RNA-binding proteins, highly up-regulated in HCC, and were recently validated as direct targets of the tumour suppressor miR-1275. It is worth noting that around 47% of FDA approved anticancer drugs are derived from plants. Modulation by miRNAs and their cellular signalling cascades could constitute new pathways by which these phytochemicals exert their effects. This study aimed to investigate the potential use of Tamarix articulata, quercetin and epigallocatechin gallate (EGCG) in HCC and how these phytochemicals could epigenetically modulate the IGF axis using their impact on miR-1275. T. articulata ethyl acetate fraction significantly reduced the viability of Huh-7 cells compared to control cells. Treatment with T. articulata ethyl acetate fraction, quercetin and EGCG significantly enhanced miR-1275, while suppressed IGF2BP1 and IGF2BP3 mRNA expression levels. In summary, T. articulata, quercetin and EGCG have important implications for HCC molecular-targeted therapy through destabilizing the interplay between miR-1275 and the IGF axis.

Ericifolin: an eugenol 5-O-galloylglucoside and other phenolics from Melaleuca ericifolia.

Ericifolin, an eugenol 5-O-beta-(6'-O-galloylglucopyranoside) possessing the naturally unknown phenolic moiety, 5-hydroxyeugenol, together with the two new phenolics, 2-O-p-hydroxybenzoyl-6-O-galloyl-(alpha/beta)-4C1-glucopyranose and 3-methoxyellagic acid 4-O-rhamnopyranoside have been isolated from the antibacterial leaves extract of Melaleuca ericifolia. In addition, 19 known phenolics were also separated and characterized. All structures were elucidated on the basis of analysis of 1H, 13C NMR, HMQC, HMBC and FTMS spectral data.

Fragmentation behavior of DOTA complexes under different activation conditions.

We have investigated the fragmentation behavior of a number of DOTA-metal complexes under collision-induced dissociation, infrared-multiphoton dissociation and higher-energy collisional dissociation activation conditions. Both, positive and negative ion mode electrospray ionization was applied. The results show that characteristic fragmentations were obtained for the metal-complexes under the investigated conditions. All elemental compositions of fragment ions have been unambiguously identified by high resolution-accurate mass measurements. Certain trends, for instance eliminations of carbon dioxide, alkyl and amine residues, were observed that coincide with the size of the metal and its location within the periodic table. Additionally, lanthanide, aluminium and indium species with even oxidation state or containing a radical have been detected. To further investigate the observed water capture during activation, deuterium labeling experiments have also been carried out. Copyright © 2017 John Wiley & Sons, Ltd.

Application of higher energy collisional dissociation (HCD) to the fragmentation of new DOTA-based labels and N-termini DOTA-labeled peptides.

1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) derivatives are applied in quantitative proteomics owing to their ability to react with different functional groups, to harbor lanthanoides and hence their compatibility with molecular and elemental mass spectrometry. The new DOTA derivatives, namely Ln-MeCAT-Click and Ln-DOTA-Dimedone, allow efficient thiol labeling and targeting sulfenation as an important post-translational modification, respectively. Quantitative applications require the investigation of fragmentation behavior of these reagents. Therefore, the fragmentation behavior of Ln-MeCAT-Click and Ln-DOTA-Dimedone was studied using collision-induced dissociation (CID), infrared multiphoton dissociation (IRMPD) and higher-energy collision dissociation (HCD) using different energy levels, and the efficiency of reporter ion production was estimated. The efficiency of characteristic fragment formation was in the order IRMPD > HCD (normal energy level) > CID. On the other hand, the application of HCD at high energy levels (HCD@HE; NCE > 250%) resulted in a significant increase in reporter ion production (33-54%). This new strategy was successfully applied to generate label-specific reporter ions for DOTA amino labeling at the N-termini and in a quantitative fashion for the estimation of amino:thiol ratio in peptides. Copyright © 2017 John Wiley & Sons, Ltd.

Membrane lipids of Rhodopseudomonas viridis.

In search of the precyanobacterial origin of the typical thylakoid lipids found in cyanobacteria and chloroplasts, we analyzed the polar lipids of the anaerobic phototrophic bacterium Rhodopseudomonas viridis. Glycolipids (monogalactosyl-, digalactosyl- and glucuronosyl diacylglycerol), phospholipids (phosphatidyl choline, -ethanolamine, -glycerol and cardiolipin) and an ornithine lipid were isolated and identified by NMR (1H, 13C, 31P) and mass spectrometry. Positional distribution and pairing of fatty acids in molecular species show small, but significant differences between glyco- and phospholipids. In this context, a new enzymatic method is described for assigning the enantiomeric structure of the diacylglycerol moiety in glyco- and phospholipids. 14C-Labelling studies suggest that monogalactosyl diacylglycerol is formed by galactosylation of diacylglycerol as in chloroplasts and not by glucosylation followed by epimerization as in cyanobacteria. The two 1,6-linked galactopyranose residues of digalactosyl diacylglycerol are both in beta-linkage and thus differ from the corresponding chloroplast lipid with its alpha-beta-sequence. R. viridis does not contain the sulfolipid, and even phosphate starvation does not induce the synthesis of this most characteristic thylakoid lipid, which on the other hand is present in other anaerobic phototrophic bacteria.

Quantitative determination of DNA adducts using liquid chromatography/electrospray ionization mass spectrometry and liquid chromatography/high-resolution inductively coupled plasma mass spectrometry.

The quantitative determination of nucleotides from DNA modified by styrene oxide is described using a combination of inductively coupled plasma high-resolution mass spectrometry (ICP-HRMS) and electrospray ionization mass spectrometry (ESI-MS), both interfaced to reversed-phase high-performance liquid chromatography (HPLC). LC/ICP-MS (resolution > 1500 to discriminate against 15N16O+ and 14N16OH+) was employed to determine quantitatively the content of modified nucleotides in standard solutions based on the signal of phosphorus; phosphoric acid served as an internal standard. By means of the standard addition technique the sensitivity of the LC/ESI-MS approach was subsequently determined. Since a comparison of UV, ICP and ESI-MS data suggested that in ESI-MS the ionization efficiency of the adducts is identical within the error limits, quantitative determination of all adducts is possible. For LC/ESI-MS with single ion monitoring, the detection limit for styrene oxide adducts of nucleotides was determined to be 20 pg absolute or 14 modified in 10(8) unmodified nucleotides in a 5 micrograms DNA sample, which comes close to the best methods available for the detection of chemical modifications in DNA.

Determination of styrene oxide adducts in DNA and DNA components.

The reaction of styrene oxide with DNA components was studied using separation by capillary zone electrophoresis (CZE) and detection by negative-ion electrospray mass spectrometry (MS). The CZE-MS interface was built for a sector field mass spectrometer. The reaction of styrene oxide with mononucleotides (dGMP, dAMP) was used to optimize the relevant separation parameters and to gather the first information about the behaviour of the possible products. With these mixtures, sample stacking procedures were developed and the scope of collision-induced dissociations were studied. From the fragments recorded, information about the reaction sites in the nucleotides was obtained. Further, the reaction with intact calf thymus DNA was investigated. The DNA was digested into oligonucleotides using the previously described approach with Benzonase, an unspecific nuclease, and alkaline phosphatase. Styrene oxide mono-adducts in dinucleotides, trinucleotides and tetranucleotides were detected, whereas pentanucleotides exhibit mono- and discernible amounts of di-adducts. The hexanucleotides were generally modified twice. The alkylated species moved faster than the unmodified oligomers.

Polyphenolic constituents of Callistemon lanceolatus leaves.

Two new flavonol glycosides, kaempferol 3-O-beta-D-galacturonopyranoside and quercetin 3-O-(2"-O-galloyl)-beta-D-glucoronopyranoside, were isolated, from leaves of Callistemon lanceolatus DC, as well as eighteen known polyphenols (phenolic acids, flavonoids and three tannins). All structures were established mainly on the basis of chemical and spectroscopic analysis (UV, 1D NMR and negative ESI-MS) and finally confirmed by 2D NMR experiments (HMQC and HMBC), in the case of flavonoid glycosides and tannins.

Styrene oxide DNA adducts: in vitro reaction and sensitive detection of modified oligonucleotides using capillary zone electrophoresis interfaced to electrospray mass spectrometry.

Styrene is one of the most important synthetic chemicals in the world and is subject to investigations concerning carcinogenicity and mutagenicity due to the active metabolite, styrene-7,8-oxide. This epoxide shows a tendency to react, among others, with DNA and DNA constituents. The in vitro reaction of styrene oxide with DNA was investigated by cleaving incubated calf thymus DNA with two different enzymes, namely Benzonase and alkaline phosphatase, to obtain oligonucleotides of the type n-nucleotide-(n-1)-phosphate with chain length from 2 to 8 bases. Alkylated and nonalkylated nucleotides were separated in groups according to their chain length using capillary zone electrophoresis and were detected with electrospray mass spectrometry. This improvement in sensitivity made it possible to obtain new information about the reaction of styrene oxide with DNA, especially to detect unknown reaction products. The results indicate that primarily purine bases were alkylated by styrene oxide before pyrimidine bases, which react with higher concentrations of styrene oxide. This means that in addition to the already reported adducts in DNA at the N-7-, O6- and N2-position of guanine also adducts at the nucleophilic sites of adenine can be found using mass spectrometry. We anticipate for the future this procedure will allow us to investigate base sequence specific reactions as well as interactions from xenobiotics and cytostatic drugs, since reaction products would directly be detectable.

A new mass spectrometric approach to detect modifications in DNA.

A new approach is described for the enzymatic digestion of DNA yielding oligonucleotides ranging from dinucleoside monophosphates to octanucleoside heptaphosphates. DNA was digested by means of the benzon nuclease, an unspecific nuclease, and alkaline phosphatase to remove the terminal phosphate. The mixture of oligonucleotides was separated using capillary-zone electrophoresis with a buffer system, yielding a rather strong electro-osmotic flow. The oligomers are separated into groups with nucleotides of the same chain length. The separation capillary was used as the innermost capillary of an electrospray spraying system. Negative molecular ions of the nucleotides were recorded using a home-built interface and ion source for a sector-field mass spectrometer. This approach allows the facile detection of DNA modifications since they lead not only to differences in mass, but also can possess altered electrophoretic mobility. For modifying reactions which exhibit sequence specificity, the information is retained in the oligomers. Thus, reactions of DNA with electrophiles can be evaluated at different levels, since in longer chains, even complex sequence specificity may be apparent. Results from calf thymus DNA digests and preliminary experiments with DNA adducts with styrene oxide are discussed.

Characterization of a capillary zone electrophoresis/electrospray-mass spectrometry interface.

A dependable and stable CZE/ESI-MS interface has been constructed. To avoid instabilities in both, the capillary electrophoretic separation and the electrospray, the second of the three concentric capillaries in the three-layered sprayer has been replaced by an aluminum-coated fused-silica capillary with an inner diameter only slightly greater than the outer diameter of the separation capillary. By this means, the otherwise often observed destruction of the separation capillary ("electrodrilling") can be avoided completely due to the suppression of electrochemical processes leading to gas bubble formation at the tip of the sprayer. With some examples taken from different biochemical areas and by separation of natural compounds, the capability and the reliability of the modified sprayer as the central part of the interface are demonstrated.