The combined use of constrained total-line-shape 1H NMR and LC–MS/MS for quantitative analysis of bioactive components in yellow onion

Onions (Allium cepa L.) are an important source of bioactive compounds including flavonoids, fructo-oligosaccharides (FOS), thiosulfinates and other sulfur compounds, and many of these compounds have potential beneficial properties for human health. Onion metabolites have traditionally been analyzed separately using high-performance liquid chromatography (HPLC) and mass spectrometry (MS). However, it has recently been found that nuclear magnetic resonance (NMR) spectroscopy represents a powerful tool for the simultaneous analysis of a large number of compounds from plants. Although the NMR quantification method provides excellent quantification of compounds with separated spectral lines, the method often produces overlapping signals, and an improved quantification method is needed. Here, we used a constrained total-line-shape (CTLS) approach to quantify overlapping metabolites from onion proton nuclear magnetic resonance (¹H NMR) spectra. This method permitted the identification and quantification of amino acids, organic acids, and sugars; however, aromatic compounds excluding aromatic amino acids were difficult to analyze by NMR spectroscopy because of the small signal intensities produced by flavonols and their sugar derivatives. Metabolites present at low concentrations were successfully analyzed using the developed mass spectrometry method operating in the positive mode. The ¹H NMR method developed here is an efficient technique for use in simultaneously analyzing compounds in a food matrix.     

NMR-metabolic methodology in the study of GM foods

The 1H-NMR methodology used in the study of genetically modified (GM) foods is discussed. Transgenic lettuce (Lactuca sativa cv "Luxor") over-expressing the ArabidopsisKNAT1 gene is presented as a case study. Twenty-two water-soluble metabolites (amino acids, organic acids, sugars) present in leaves of conventional and GM lettuce were monitored by NMR and quantified at two developmental stages. The NMR spectra did not reveal any difference in metabolite composition between the GM lettuce and the wild type counterpart. Statistical analyses of metabolite variables highlighted metabolism variation as a function of leaf development as well as the transgene. A main effect of the transgene was in altering sugar metabolism.     

铝电解车间粉尘有机提取物及其组分遗传毒性的研究

采用化学分离与体外微核试验，姐妹染色单体交换试验的方法，研究了铝电解车间粉尘有机提取物及其５个组分的遗传毒性。结果表明粉尘有机提取的及其有机酸，多环芳烃，极性化合物可使微核率和ＳＣＥ率显著增高，并有一定的剂量－反应关系。     

Studies on synthesis and pharmacological activities of 3,6-disubstituted-1,2,4-triazolo[3,4-b]-1,3,4-thiadiazoles and their dihydro analogues

Several 3,6-disubstituted-1,2,4-triazolo[3,4-b]-1,3,4-thiadiazole and their dihydro analogues were synthesized from hetero aromatic acids and hetero aromatic aldehydes, respectively, by microwave-assisted dry media and conventional methods. Elemental analysis, IR, (1)H NMR, (13)C NMR and mass spectral data elucidated the structures of all newly synthesized compounds. Synthesized compounds are studied for their antibacterial, antifungal, anti-inflammatory and analgesic activities. Some of the tested compounds showed significant pharmacological activities.     

Metabolic profiling of organic acids in honey by stable isotope labeling assisted liquid chromatography-mass spectrometry

Organic acids in honey have great nutritive and medicinal value such as antimicrobial and antioxidant activities. In this study, profiling of organic acids in honey is carried out by sequential liquid-liquid extraction assisted isotope labeling-liquid chromatography-mass spectrometry. A total of 493 potential organic acids are detected in honey, and 165 of those are confirmed by comparing with authentic standards and interpretation of obtained MSⁿ spectra. Findings from this study further expand our understanding about the number of organic acids in honey, increasing from 120 (previously reported) to 258. Moreover, we find that the composition of organic acids detected in rape, acacia and Chinese astragalus honey is quite similar; but their contents show certain variations. This study provides basic knowledge about the number and nature of organic acids in honey and could be stepping stone to explore the link between organic acids and nutritional and medicinal characteristics of honey.     

Synthesis and antimicrobial activities of some new triazolothiadiazoles bearing 4-methylthiobenzyl moiety

A series of substituted triazolothiadiazoles have been synthesized by condensing 4-amino-3-[4-methylthiobenzyl]-4H-1,2,4-triazole-5-thiol (5) with substituted aryl furoic acids/aromatic acids in the presence of POCl3. The triazole (5) was obtained by the fusion of 4-methylthiophenyl acetic acid with thiocarbohydrazide. The structures of newly synthesized compounds are characterized by elemental analysis, IR, 1H NMR and mass spectroscopic studies and were screened for their antimicrobial activities. The preliminary results revealed that some of the compounds exhibited promising antimicrobial activities.     

Heterocyclic system containing bridgehead nitrogen atom: synthesis and pharmacological activities of some substituted 1,2,4-triazolo[3,4-b]-1,3,4-thiadiazoles

Several 3,6-disubstituted-1,2,4-triazolo[3,4-b]-1,3,4-thiadizoles were prepared by the condensation of 4-amino-3-aryl/aralkyl substituted-5-mercapto-1,2,4-triazoles 3(a-c) with various substituted aromatic/hetero aromatic acids through a single step reaction. Elemental analysis, IR, 1H NMR and mass spectral data confirmed the structure of the newly synthesized compounds. Synthesized triazolo thiadiazoles investigated for their antibacterial, antifungal, anti-inflammatory and analgesic activities. Some of the tested compounds showed moderate antimicrobial activity against various tested bacterial and fungal strains. None of the synthesized compounds have significant anti-inflammatory and analgesic activities.     

Solid state C CP-MAS NMR and FT-IR spectroscopic analysis of cuticular fractions of berries and suberized membranes of potato

Cuticular fractions of the important northern berries sea buckthorn (Hippophaë rhamnoides) and black currant (Ribes nigrum), and the suberized membrane of red peel potato (Solanum tuberosum var. Asterix), were investigated for the first time with solid state spectroscopic methods. In addition, the ester-bonded aliphatic monomers were analyzed as TMS derivatives by GC-MS after depolymerization with NaOMe catalyzed methanolysis. The fractions inspected by solid state ¹³C CP-MAS NMR and FT-IR spectroscopy included isolated cuticular and suberin-enriched membranes, i.e. raw cutin and suberin, the non-depolymerizable fractions and further cellulase- and pectinase-resistant fractions. Spectroscopic results supported the findings of the monomeric composition of ester-bound cutin, confirmed the presence of polysaccharides and an aromatic, possibly lignin-like, fraction and also suggested the presence of a non-degradable cutan-type polymer in the berry cuticular fraction. Black currant peel cutin and receptacle cutin were found to differ from each other in composition, notably in terms of the abundance of monomeric, aromatic and polysaccharide fractions. Some effect of latitudinal growth on the composition of cuticular fractions was observed for black currants grown in northern and southern Finland. The spectra of potato suberin were similar to the spectra of black currant receptacles, but the berry had even higher proportions of aromatic components.     

Synthesis and anticonvulsant activity of some potential thiazolidinonyl 2-oxo/thiobarbituric acids

A series of 5-[(N-substituted benzylidenylimino)amino]-2-oxo/thiobarbituric acids (3a-3h) have been synthesized by the condensation of 5-hydrazino-2-oxo/thiobarbituric acids (2a-2b) with various aromatic aldehydes. Cycloaddition of thioglycolic acid to 3a-3h, yielded 5-[(2'-substituted phenyl-4'-oxothiazolidin-3'-yl)amino]-2-oxo/thiobarbituric acids (4a-4h). All these compounds were screened, in vivo, for their anticonvulsant activity and acute toxicity studies. Compounds 4f and 4g were found to be most potent compounds of this series and were compared with the reference drugs, phenytoin sodium, lamotrigine and sodium valproate. The structures of these compounds have been established by IR, 1H NMR and mass spectroscopic data.     

A sulfur amino acid-free meal increases plasma lipids in humans

The content of sulfur amino acid (SAA) in a meal affects postprandial plasma cysteine concentrations and the redox potential of cysteine/cystine. Because such changes can affect enzyme, transporter, and receptor activities, meal content of SAA could have unrecognized effects on metabolism during the postprandial period. This pilot study used proton NMR ((1)H-NMR) spectroscopy of human plasma to test the hypothesis that dietary SAA content changes macronutrient metabolism. Healthy participants (18-36 y, 5 males and 3 females) were equilibrated for 3 d to adequate SAA, fed chemically defined meals without SAA for 5 d (depletion), and then fed isoenergetic, isonitrogenous meals containing 56 mg·kg(-1)·d(-1) SAA for 4.5 d (repletion). On the first and last day of consuming the chemically defined meals, a morning meal containing 60% of the daily food intake was given and plasma samples were collected over an 8-h postprandial time course for characterization of metabolic changes by (1)H-NMR spectroscopy. SAA-free food increased peak intensity in the plasma (1)H-NMR spectra in the postprandial period. Orthogonal signal correction/partial least squares-discriminant analysis showed changes in signals associated with lipids, some amino acids, and lactate, with notable increases in plasma lipid signals (TG, unsaturated lipid, cholesterol). Conventional lipid analyses confirmed higher plasma TG and showed an increase in plasma concentration of the lipoprotein lipase inhibitor, apoC-III. The results show that plasma (1)H-NMR spectra can provide useful macronutrient profiling following a meal challenge protocol and that a single meal with imbalanced SAA content alters postprandial lipid metabolism.     

The influence of thermal and chemical processes on the content and structure of humic acids--the basic ingredient of peat preparations

Water extracts with supplement NaOH, Na2CO3 and NaCl obtained in different temperatures from peat of low type were examined. These extracts are the basic ingredient of peat preparation applicable in cosmetics and therapeutics (pastes, gels, emulsions, oinments, tonics, creams. The continuous absorption spectra of humid compounds in the UV-VIS and LR range in the extracts were developed. In the differential 4th degree absorption spectra in the range UV-VIS the typical wavelengths were compared with separated absorption peaks of examined and standard humic acids. The quantitative proportions among acids in these extracts were determined. Also absorptions bands corresponding to characteristic functional groups and chemical bonds were determined. On the basis of the analysis of UV-VIS and IR absorption spectra the influence of temperature and non organic compounds supplementation on the structure (the bonds types, functional groups, molecular helicity inversion) and the content of humic compounds in water extracts was evaluated.     

Noncovalent interactions between aromatic compounds and dissolved humic acid examined by nuclear magnetic resonance spectroscopy

We examined the molecular-level interactions of aromatic compounds with a humic acid that was extensively characterized with one- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy. Interactions of target compounds were evaluated by monitoring their NMR relaxation properties. Solid-state NMR revealed that the humic sample contains paraffinic carbon (31%), substituted aliphatic carbon (30%), and aromatic carbon (28%). The liquid-state experiments further identified amino acids, a range of carbohydrates, methoxylated aromatics (likely derived from lignin), and a series of aliphatic chains. The 13C spin-lattice relaxation time (T1) of site-specifically labeled naphthalene, 1-naphthol, and quinoline was found to decrease with increasing additions of humic acid. The 1H T1 values also were measured and demonstrated that the association with the humic acid was not specific because all the protons acquired the T1 value of the humic acid at the same rate. The lack of a chemical shift change and an increase in signal line broadening indicates that the interaction between these compounds and humic acid is noncovalent. These interactions were detected at low humic concentrations (5 mg C/L) and suggest that low concentrations of humic material, which are prevalent in both aquatic and terrestrial systems, will significantly affect the fate and transport of contaminants in the environment.     

Changes in the amino acid composition of Bogue (Boops boops) fish during storage at different temperatures by 1H-NMR spectroscopy

Nuclear magnetic resonance spectroscopy was employed to obtain information about the changes occurring in Bogue (Boops boops) fish during storage. For this purpose, (1)H-NMR spectra were recorded at 600 MHz on trichloroacetic acid extracts of fish flesh stored over a 15 days period both at 4 °C and on ice. Such spectra allowed the identification and quantification of amino acids, together with the main organic acids and alcohols. The concentration of acidic and basic free amino acids was generally found to increase and decrease during storage, respectively. These concentration changes were slow during the first days, as a consequence of protein autolysis, and at higher rates afterward, resulting from microbial development. Two of the amino acids that showed the greatest concentration change were alanine and glycine, known to have a key role in determining the individual taste of different fish species. The concentration of serine decreased during storage, as highlighted in the literature for frozen fish samples. Differences in the amino acids concentration trends were found to be related to the different storage temperatures from day 4 onwards.     

含癌细胞的血清吸收光谱研究

目的探讨癌细胞对血清光谱的影响。方法采用拟合的方法对加入癌细胞的血清吸收光谱进行分析研究。结果血清吸收光谱的峰强与所加癌细胞的数量呈非线性关系,癌细胞的加入对酪氨酸和色氨酸的影响有相同的趋势,但对色氨酸的含量和吸光度的影响更为显著,而且癌细胞的加入没有破坏其双键结构。结论利用光谱技术研究癌细胞的生长分裂过程有其重要意义;把吸收光谱与荧光光谱结合起来,对癌症的早期诊断也有一定的参考价值。     

Mechanisms regulating bioavailability of phenanthrene sorbed on a peat soil-origin humic substance

The organic matter-mineral complex plays an important role in regulating the fate of hydrophobic organic compounds (HOCs) in the environment. In the present study, the authors investigated the microbial bioavailability of phenanthrene (PHE) sorbed on the original and demineralized humic acids (HAs) and humin (HM) that were sequentially extracted from a peat soil. Demineralization treatment dramatically decreased the 720-h mineralized percentage of HM-sorbed PHE from 42.5 ± 2.6% to 3.4 ± 1.3%, whereas the influence of this treatment on the biodegradability of HA-associated PHE was much lower. Degradation kinetics of HA- and HM-sorbed PHE showed that its initial degradation rate was negatively correlated with the aromatic carbon content of humic substances (p<0.05). This was attributed to the strong interactions between PHE and the aromatic components of humic substances, which hampered its release and subsequent biodegradation. The 720-h mineralized percentage of PHE was inversely correlated with the estimated thickness of the organic matter layer at the surfaces of HAs and HMs. Therefore, in a relatively long term, diffusion of PHE within the organic matter layer could be an important factor that may limit the bioavailability of PHE to bacteria. Results of the present study highlight the molecular-scaled mechanisms governing bioavailability of PHE sorbed on humic substances.     

家庭厨房油烟冷凝物化学成分及对果蝇经口染毒遗传毒性的研究

采用气相色谱－质谱仪（ＧＣ／ＭＳ）对家庭厨房吸油烟机油杯中冷凝物的总化学成分进行了分析,发现样品中有７４种化合物,其中包括烃类、醇酚类、醛酮类,酯类、羧酸类、杂环稠环类、甾醇类等,其中有些化合物具有致癌作用。同时还用该样品对果蝇经口染毒的细胞遗传毒性进行了研究,果蝇伴性隐性致死试验（ＳＬＲＬ）的实验结果表明：厨房油烟冷凝物具有较强的致突变作用。其１１０、３２０、９６０ｍｇ／Ｌ三个浓度组的总突变率分别为０．１７３２％、０．４３０６％、０．１７０７％,实验中第Ⅱ窝的３２０ｍｇ／Ｌ浓度组与第Ⅲ窝１１０ｍｇ／Ｌ浓度组的致突变率分别达到０．５３０％和０．５４０％（Ｐ＜０．００１）。呈现有较高的隐性致死作用。此外,本次实验还表明油烟冷凝物对果蝇的生殖系统有一定的作用（Ｐ＜０．０５）,其中３２０ｍｇ／Ｌ组有非常显著的差异（Ｐ＜０．０１）。三个浓度组第Ⅰ窝的不育率分别为２．５６４％、２．０５６％和２．８４５％,与对照组相比统计学上有显著性差异（Ｐ＜０．０５）。     

Evaluation of sample preparation methods for nuclear magnetic resonance metabolic profiling studies with Eisenia fetida

The earthworm Eisenia fetida is frequently used in ecotoxicological studies; however, it has not yet been investigated using proton nuclear magnetic resonance ((1)H NMR) metabolic profiling methods. The present study investigates the impact of depuration time, sample homogenization, and different extraction solvents on the quality and reproducibility of the (1)H NMR spectra of E. fetida with the goal of determining whether this species is suitable for future metabonomic studies. A depuration time of 96 h, followed by intact lyophilization before homogenization and extraction into a deuterium oxide (D(2)O)-based phosphate buffer, was found to produce extracts with excellent (1)H NMR reproducibility. The D(2)O buffer extracted the largest quantity of the widest variety of earthworm metabolites, which is consistent with the results from other studies using different earthworm species. Nuclear magnetic resonance assignments of the major metabolites in the D(2)O-based buffer also were performed and found to be similar to those for other earthworm species, such as Eisenia veneta, but also to have characteristic attributes in E. fetida. The major metabolites identified include amino acids (alanine, arginine, glutamic acid, glutamine, glycine, leucine, lysine, phenylalanine, serine, tyrosine, and valine), two sugars (glucose and maltose), the sugar alcohol mannitol, and the polyalcohol inositol. Two other earthworm species (Lumbricus rubellus and Lumbricus terrestris) also were examined using protocols developed for E. fetida, and of the three species, the (1)H NMR spectra of E. fetida had the least variation, indicating this species is well-suited for future metabolomic-based ecotoxicity studies.     

Antioxidative properties of water extracts obtained from herbs of the species Lamiaceae

Essential oils and extracts of aromatic herbs obtained by organic solvents have been extensively studied for their antioxidant activity in lipid substrates. Very little is known about the possible presence of antioxidants in polar extracts from herbs used in preparation of infusions and decoctions. In this work water extracts of six different herbs of the Lamiaceae family (dittany, lemon balm, mint, sage, sideritis and sweet marjoram) were prepared. The extracts were examined for their effect against lipid oxidation in comparison to a tea water extract. Sweet marjoram, sage and dittany extracts were found to have a remarkable capacity in retarding lipid oxidation. Examination by thin-layer chromatography of the freeze-dried extracts, before and after hydrolysis, showed that the extracts were rich in bound forms of phenolic compounds such as hydroxycinnamic acids and flavonoids. Rosmarinic and caffeic acids were detected in all extracts with the exception of those from mint and sideritis. These results indicate that certain plants used for the preparation of infusions could be further studied like tea as sources of antioxidants.     

Antioxidative properties of water extracts obtained from herbs of the species Lamiaceae

Essential oils and extracts of aromatic herbs obtained by organic solvents have been extensively studied for their antioxidant activity in lipid substrates. Very little is known about the possible presence of antioxidants in polar extracts from herbs used in preparation of infusions and decoctions. In this work water extracts of six different herbs of the Lamiaceae family (dittany, lemon balm, mint, sage, sideritis and sweet marjoram) were prepared. The extracts were examined for their effect against lipid oxidation in comparison to a tea water extract. Sweet marjoram, sage and dittany extracts were found to have a remarkable capacity in retarding lipid oxidation. Examination by thin-layer chromatography of the freeze-dried extracts, before and after hydrolysis, showed that the extracts were rich in bound forms of phenolic compounds such as hydroxycinnamic acids and flavonoids. Rosmarinic and caffeic acids were detected in all extracts with the exception of those from mint and sideritis. These results indicate that certain plants used for the preparation of infusions could be further studied like tea as sources of antioxidants.     

新装修居室内空气中有机污染物的定性分析

目的建立可以同时测定空气中多种有机污染物的吸附-热解吸-气相色谱-质谱方法,分析新装修居室内空气中有机污染物的成分.方法以Tenax TA为吸附剂,利用吸附-热解吸-气相色谱-质谱方法对新近装修的2个典型居室内空气进行定性分析.结果新装修居室内空气中共检测出乙酸、乙二醇、甲苯等挥发性有机物和苊、蒽、十八烷等半挥发性有机物60多种,包括芳香烃、多环芳烃、脂肪烃、含氧烃等,以苯系物和脂肪烃为主.结论该方法可用于定性分析室内空气中的有机污染物.