耐辐射球菌清除活性氧自由基及对DNA的保护作用

用化学发光法分析了耐辐射球菌 (D .radiodurans)的两个菌株 (R1、KD830 1 )和大肠杆菌对超氧阴离子自由基、羟基自由基和过氧化氢的清除能力。通过羟基自由基诱导的DNA氧化损伤化学发光模型考察了耐辐射球菌细胞提取物对自由基氧化导致DNA氧化损伤的保护作用。结果表明 ,耐辐射球菌提取物能显著清除O-2·、H2 O2 和·OH ,清除能力明显高于大肠杆菌 ,并能有效地防止自由基所致DNA氧化损伤 ,表现出超强的抗氧化和耐辐射能力 ,这归因于耐辐射球菌中具有较高的抗氧化酶活性     

Isolation and identification of mosquito bite deterrent terpenoids from leaves of American (Callicarpa americana) and Japanese (Callicarpa japonica) beautyberry

Essential oil extracts from Callicarpa americana and Callicarpa japonica were investigated. Bioassay-guided fractionation of C. americana extracts using the yellow fever mosquito, Aedes aegypti, led to the isolation of alpha-humulene, humulene epoxide II, and intermedeol and a newly isolated terpenoid (callicarpenal). Similar work involving C. japonica resulted in the isolation of an additional compound, spathulenol, as well as the four compounds isolated from C. americana. Structure elucidation was performed on all isolated compounds using a combination of gas chromatography-mass spectrometry-electron ionization, high-resolution liquid chromatography-MS-electrospray ionization, and one- and two-dimensional NMR experiments. Heretofore, 13,14,15,16-tetranorclerodane, callicarpenal, has never been identified from natural sources. Complete (1)H and (13)C NMR assignment data are provided for this compound. In bite deterrent studies, spathulenol, intermedeol, and callicarpenal showed significant repellent activity against A. aegypti and Anopheles stephensi.     

H_2S对低温胁迫下甜樱桃柱头和子房AsA-GSH循环的响应

为了探究H_2S对甜樱桃花器官低温伤害的缓解机理,以甜樱桃品种早大果为试材,分析了不同浓度H_2S供体NaHS对低温胁迫下甜樱桃柱头和子房AsA-GSH循环系统的影响。结果表明,喷施0.02、0.05、0.1 mmol·L~(-1)的NaHS均可降低低温胁迫下甜樱桃柱头和子房超氧阴离子(O_2~-)、过氧化氢(H_2O_2)、丙二醛(MDA)含量,显著提高还原型抗坏血酸(AsA)和还原型谷胱甘肽(GSH)含量(P0.05),降低氧化型抗坏血酸(DHA)和氧化型谷胱甘肽(GSSG)含量,使AsA/DHA和GSH/GSSG比值显著升高(P0.05),以0.05 mmol·L~(-1)NaHS效果最显著;0.02、0.05、0.1 mmol·L~(-1)的NaHS均显著提高低温胁迫下柱头和子房AsA-GSH循环中抗坏血酸过氧化物酶(APX)、脱氢抗坏血酸还原酶(DHAR)、单脱氢抗坏血酸还原酶(MDAR)、谷胱甘肽还原酶(GR)、谷胱甘肽过氧化物酶(GPX)和谷胱甘肽-S-转移酶(GST)的活性(P0.05),以0.05 mmol·L~(-1)NaHS提高上述酶活性的幅度最大。NaHS的浓度大于0.2 mmol·L~(-1)不再提高低温胁迫下AsA-GSH循环效率;低温胁迫下添加NaHS的同时添加H_2S清除剂HT可解除H_2S的效果。综上所述,喷施适量外源H_2S可有效降低低温胁迫下O_2~-、H_2O,和MDA积累,提高AsA-GSH循环效率,减轻低温对甜樱桃柱头和子房的氧化伤害。本研究结果为明确H_2S缓解甜樱桃花器官低温伤害作用机制以及生产上花期低温伤害预防提供了理论依据。     

Lactoperoxidase-induced protein oxidation in milk

The reaction between lactoperoxidase (LPO) and H(2)O(2) in the presence of bovine serum albumin (BSA), beta-lactoglobulin, or casein was investigated for the formation of protein radicals by freeze-quench electron spin resonance (ESR) and by the formation of the protein oxidation product, dityrosine. The presence of BSA resulted in a dramatic change after 1 min of reaction in the obtained ESR spectrum compared with the spectrum obtained for LPO and H(2)O(2) alone. Furthermore, experiments employing BSA or beta-lactoglobulin resulted in the formation of long-lived protein radicals detectable 10 min after initiation of the reaction. The presence of casein resulted in a minor change in the fine structure of the ESR spectrum after 1 min of reaction compared with LPO and H(2)O(2) alone, but no difference between the two reaction mixtures could be observed after 10 min of reaction. The formation of dityrosine could be detected in reaction mixtures containing LPO and H(2)O(2) after 1 and 10 min of incubation at 25 degrees C both in the absence and in the presence of BSA, beta-lactoglobulin, or casein. The presence of casein resulted in an increased dityrosine concentration compared with the reaction with LPO and H(2)O(2) alone. Endogenous LPO in unpasteurized milk was activated at 25 degrees C by adding 1 mM H(2)O(2). Radical species could be detected directly in the milk by freeze-quench ESR during the initial phase of the reaction, and dityrosine could be measured after 4 h of incubation. The role of LPO activity in the formation of ESR detectable radical species and dityrosine in milk was further verified in ultrahigh temperature (UHT) milk with no endogenous enzyme activity, as the formation of ESR detectable radical species and dityrosine took place in UHT milk only upon the addition of both H(2)O(2) and exogenous LPO.     

Identification and quantification of major steviol glycosides in Stevia rebaudiana purified extracts by 1H NMR spectroscopy

The use of (1)H NMR spectroscopy for the characterization of Stevia rebaudiana extracts is presented. The developed method allows qualitative and quantitative determination of the major steviol glycosides in purified extracts and fractions obtained from various stages of the purification process. Moreover, it proved to be a powerful tool to differentiate between glycosides which are naturally occurring in the stevia plant and artifacts formed in the course of the manufacturing process. Identification of steviol glycosides was achieved by the use of 2D NMR techniques, whereas quantification is based on qHNMR using anthracene as internal standard. The solvent mixture pyridine-d(5)-DMSO-d(6) (6:1) enabled satisfactory separation of the signals to be integrated. Validation of the method was performed in terms of specificity, precision, accuracy, linearity, robustness, and stability. Quantitative results were compared to those obtained with the JECFA HPLC-UV method and were found to be in reasonable agreement. NMR analysis does not rely on the use of reference compounds and enables significantly faster analysis compared to HPLC-UV. Thus, NMR represents a feasible alternative to HPLC-based methods for the quality control of Stevia rebaudiana extracts.     

Identification and quantification of caffeic and rosmarinic acid in complex plant extracts by the use of variable-temperature two-dimensional nuclear magnetic resonance spectroscopy

A combination of advanced nuclear magnetic resonance (NMR) methodologies for the analysis of complex phenolic mixtures that occur in natural products is described, with particular emphasis on caffeic acid and its ester derivative, rosmarinic acid. The combination of variable-temperature two-dimensional proton-proton double quantum filter correlation spectroscopy (1H-1H DQF COSY) and proton-carbon heteronuclear multiple quantum coherence (1H-13C HMQC) gradient NMR spectroscopy allows the identification and tentative quantification of caffeic and rosmarinic acids at 243 K in extracts from plants of the Lamiaceae family, without resorting to previous chromatographic separation of the components. The use of proton-carbon heteronuclear multiple bond correlation (1H-13C HMBC) gradient NMR spectroscopy leads to the complete assignment of the correlations of the spins of H2a and H3a with the ester and carboxyl carbons of rosmarinic and caffeic acid, even at room temperature, and confirms the results of the above methodology Quantitative results are in reasonable agreement with reverse phase HPLC measurements.     

Optimization of sample ph and temperature for phosphorus‐31 nuclear magnetic resonance spectroscopy of poultry manure extracts

Abstract

Organic phosphorus (P) compounds can be characterized using nuclear magnetic resonance (NMR) spectroscopy provided conditions are suitable for detecting the NMR signal. The objective of the research was to optimize pH and temperature conditions for turkey manure extracts prior to analysis of organic P compounds using NMR. Samples of turkey manure were extracted with 0.25 MNaOH + 0.05 MEDTA. The extracts were lyophilized and resolubilized in distilled H₂O before analysis on a General Electric GN500 NMR spectrometer. Initial ³¹P NMR experiments were run to determine the optimal instrumental parameters for ³¹P studies. Samples were titrated to seventeen pH values ranging from 4.0 to 13.2. Samples adjusted to pH 10.0 had the greatest spectral resolution. A seven‐by‐three factorial experiment was used to investigate the effect of seven temperatures (5,10,20,30,40,50, and 60°C) on three separate samples at pH 6.5,9.0, or 10.0. Spectra resolution was greatest at pH 10.0 and 20°C.     

Identification and synthesis of a novel selenium-sulfur amino acid found in selenized yeast: Rapid indirect detection NMR methods for characterizing low-level organoselenium compounds in complex matrices

After proteolytic digestion, aqueous extraction, and derivatization with diethyl pyrocarbonate or ethyl chloroformate, HPLC-inductively coupled plasma (ICP)-MS, GC-atomic emission detection (AED), and GC-MS analysis of high-selenium yeast stored at room temperature for more than 10 years showed selenomethionine as the major Se product along with substantial amounts of selenomethionine selenoxide hydrate and the previously unreported selenoamino acid having a Se-S bond, S-(methylseleno)cysteine. The identity of the latter compound was confirmed by synthesis. The natural product was shown to be different from a synthetic sample of the isomeric compound Se-(methylthio)selenocysteine. Selenium-specific NMR spectroscopic methods were developed to directly analyze the aqueous extracts of the hydrolyzed selenized yeast without derivatization or separation. Selenomethionine and S-(methylseleno)cysteine were identified by 77Se-1H HMQC-TOCSY experiments.     

Analysis of acid-soluble glycogen in pork extracts of two PRKAG3 genotypes by 1H liquid-state NMR spectroscopy and biochemical methods

Meat extracts with acid-soluble glycogen (macroglycogen) from M. longissmus dorsi of carriers and noncarriers of the PRKAG3 mutation (RN(-) and rn(+) genotype) were analyzed by both (1)H liquid-state NMR spectroscopy and a biochemical method. The (1)H NMR analysis revealed that shorter polymers (dimers, trimers, etc.) of α-1,4-linked glucose were generated 24-48 h post-mortem. This is not possible to elucidate with the biochemical method, by which only the total amount of hydrolyzed glucose residues is determined. The shorter polymers were primarily formed in carriers of the PRKAG3 mutation, suggesting different post-mortem glycogen degradation mechanisms in the two genotypes.     

Chemical characterization of Cuban propolis by HPLC-PDA, HPLC-MS, and NMR: the brown, red, and yellow Cuban varieties of propolis

Sixty-five samples of propolis were collected from eleven regions of Cuba; methanolic extracts of propolis were prepared from all samples, and a classification method was developed using a combination of NMR, HPLC-PDA, and HPLC-ESI/MS techniques. The analysis of (1)H and (13)C NMR spectra and chromatographic profiles of all propolis extracts allowed the definition of three main types of Cuban propolis directly related to their secondary metabolite classes: brown Cuban propolis (BCP), rich in polyisoprenylated benzophenones, red Cuban propolis (RCP), containing isoflavonoids as the main constituents, and yellow Cuban propolis (YCP), probably with aliphatic compounds. Subsequently, the principal compounds of the brown and red types were characterized by HPLC-ESI/MS analysis. Instrumental techniques used are complementary; NMR was shown to be a quick and informative tool for the rapid analysis of crude propolis polar extracts and allowed the identification of the main class of secondary metabolites, while LC-PDA and LC-MS techniques were useful tools for qualitative and quantitative analysis of marker compounds of Cuban propolis.     

Purification of fusaproliferin from cultures of Fusarium subglutinans by preparative high-performance liquid chromatography

Thirty-six Fusarium strains were grown on cracked yellow corn and evaluated for optimum fusaproliferin production, with Fusarium subglutinans E-1583 producing the highest levels (1600 microg/g). Three solvent systems were tested for extracting fusaproliferin from the cultures of F. subglutinans E-1583. Methanol gave the highest fusaproliferin recovery, followed by methanol/1% aqueous NaCl (55:45, v/v) and acetonitrile/methanol/H(2)O (16:3:1, v/v/v). Hexane partitioning was effective in removing many impurities from the crude fusaproliferin extracts prior to the liquid chromatography step. Fusaproliferin samples were further purified by high-performance liquid chromatography (HPLC) with a C18 preparatory column using a mobile phase of acetonitrile/H(2)O (80:20, v/v). The purity of the fusaproliferin was verified by analytical HPLC, GC/MS, (1)H NMR spectroscopy, and electrospray ionization (ESI) MS. The isolated fusaproliferin was shown to be free of impurities and can be used as a standard for routine analysis. Fusaproliferin was shown to be temperature-sensitive when samples were stored at room temperature (20-24 degrees C) for more than several days. After 30 days at 4 degrees C, approximately 8% of the fusaproliferin had been transformed to deacetyl-fusaproliferin; however, samples stored at -20 degrees C for 1 year contained only trace amounts of the deacetylated form.     

1H NMR, a rapid method to monitor organic acids during cupuassu (Theobroma grandiflorum Spreng) processing

The development of an analytical method using 1H nuclear magnetic resonance (1H NMR) spectrometry to monitor cupuassu (Theobroma grandiflorum Spreng) bean fermentation, drying, and roasting processes is reported. The analysis of organic acids and alcohols of crude water extracts of cupuassu ground kernels were monitored by HPLC and 1H NMR spectroscopy. The residual protein signals caused deleterious effects on acid and alcohol quantifications. Therefore, the analytical procedures were optimized by sample cleanup and water suppression pulse sequences in order to obtain compatible data using HPLC and 1H NMR. The quantification of lactic acid, acetic acid, and 2,3-butanediol by NMR is 5- to 10-fold faster than by HPLC, with the advantage of providing the identification of several chemical species in a single experiment. Application of these analytical conditions to some cupuassu samples revealed that this methodology can be applied to the quality profiles of fermentation and roasting processes.     

Inhibition of reactive nitrogen species effects in vitro and in vivo by isoflavones and soy-based food extracts

Recent studies have shown that soy isoflavone inhibits inducible nitric oxide (NO) synthase activities and is reported to have peroxynitrite scavenging ability. Consequently, we investigated whether isoflavones (daidzein and genistein) and extracts from soy-based products (miso, soymilk, tofu, soy sprout, black soybean, soybean, and yuba) would inhibit the reactive nitrogen species (RNS) effect in vitro and in vivo. In the in vitro experiments [including the protection of cellular DNA from peroxynitrite or sodium nitroprusside damage, an inhibitory effect on nitric oxide production from lipopolysaccharide (LPS)-induced RAW 264.7 cells, and nitric oxide scavenging ability], extracts from soy-based foods showed a potent antioxidant activity and an inhibiting effect on RNS activity. These effects were correlated with total isoflavone content. In the in vivo experiments, rats were given isoflavones (4.0 mg/kg bw) or soy-based product extracts (1.0 g/kg bw) orally for 1 week and were injected with vehicle H(2)O (1 mL/kg bw) or LPS (10 mg/kg bw) on the day 7. Twelve hours after treatment, the rats were killed, and blood serum was collected for analysis. The intraperitoneal administration of LPS resulted in an increase in serum nitrite, nitrate, and nitrotyrosine concentrations. These are stable metabolite end products of nitric oxide, to 4-, 16-, and 5-fold levels, (4, 10 microM and 58 +/- 14 pmol/mL), of the placebo control, respectively. Results showed that oral administration of isoflavones and extracts from soy-based products significantly decreased serum nitrite, nitrate, and nitrotyrosine levels in LPS-induced rats. This study demonstrates that soy isoflavone supplementation may inhibit RNS-induced oxidation both in vitro and in vivo.     

Molecular water motions of skim milk powder solutions during acidification studied by 17O and 1H nuclear magnetic resonance and rheology

The molecular motion of water was studied in glucono-δ-lactone-acidified skim milk powder (SMP) solutions with various pH values and dry matter contents. NMR relaxometry measurements revealed that lowering the pH in SMP solutions affected 17O and 1H T2 relaxation rates almost identically. Consequently, the present study indicates that the proteins present in the samples do not affect the 1H relaxation behavior markedly, even at relatively high SMP concentrations (15-25%). Comparison of rheological measurements and NMR measurements suggested that the collapse of κ-casein during acidification could contribute to the initial decrease in 17O and 1H relaxation rate in the pH range between 6.6 and 5.5 for 15% SMP and in the pH range between 6.6 and 5.9 for 25% SMP. However, below pH 5.5 the viscosity and 17O and 1H NMR relaxation rates did not correlate, revealing that the aggregation of casein micelles, which increases viscosity below pH 5.5, does not involve major repartitioning of water.     

High-field nuclear magnetic resonance (NMR) study of truffles (Tuber aestivum vittadini)

A high-field NMR technique was used to analyze aqueous and organic extracts of truffles (Tuber aestivum vittadini) to characterize their chemical composition. Water-soluble metabolites belonging to different classes such as sugars, polyols, amino acids, and organic acids were almost completely assigned by means of one- and two-dimensional experiments (1H-1H COSY, TOCSY, 1H-13C HSQC, 1H-13C HMBC, and 1H-31P HMBC). The 1H spectral assignment of the cell membrane components such as lipids, sterols, and fatty acids extracted in organic solvents was also performed.     

Stimulation of acidic reduction of nitrite to nitric oxide by soybean phenolics: possible relevance to gastrointestinal host defense

This study aimed to evaluate the potential of soybean-promoted acidic nitrite reduction and to correlate this activity with the content of phenolics and with the bactericidal activity against Escherichia coli O157:H7. Extracts of embrionary axes and cotyledons enriched in phenolics increased ?NO formation at acidic pH at values that were 7.1 and 4.5 times higher, respectively, when compared to the reduction of the nonenriched extracts. Among the various phenolics accumulated in the soybean extracts, five stimulated nitrite reduction in the following decreasing order of potency: epicatechin gallate, chlorogenic acid, caffeic acid, galic acid and p-coumaric acid. Extracts of embrionary axes presented higher contents of epicatechin gallate and caffeic acid, compared to that of cotyledons, indicating a positive correlation between activity of the extracts and content of phenolics with regard to nitrite reducing activity. Soybean extracts enriched in phenolics interacted synergistically with acidified nitrite to prevent E. coli O157:H7 growth. The results suggest that soybean phenolics may interfere with the metabolism of ?NO in an acidic environment by accelerating the reduction of nitrite, with a potential antimicrobial effect in the stomach.     

Chinese cabbage extracts and sulforaphane can protect H2O2-induced inhibition of gap junctional intercellular communication through the inactivation of ERK1/2 and p38 MAP kinases

The cruciferous vegetables such as Chinese cabbages and broccoli are known to have anticancer phytochemicals, and the consumption of cruciferous vegetables has been proposed to protect against various cancers. The anticarcinogenic properties of some Chinese cabbage extracts and sulforaphane glucosinolate (SFN) were assessed by examining their ability to prevent the inhibition of gap junctional intercellular communication (GJIC) induced by hydrogen peroxide (H2O2) in WB-F344 normal rat liver epithelial cells. The cells were preincubated with Chinese cabbage extracts and SFN for 24 h followed by cotreatment with cells and H2O2 (750 microM) for 1 h. Chinese cabbage extracts and SFN prevented the inhibition of GJIC and phosphorylation of gap junction protein connexin43 (Cx43) by H2O2 treatment. Chinese cabbage extracts and SFN were able to prevent the inhibition of GJIC through the blocking of Cx43 phosphorylaton and inactivation of ERK 1/2 and p38 MAP kinase. The results suggest that cruciferous vegetables and their components, SFN, may exert the anticancer effect by targeting the GJIC as a functional dietary chemopreventive agent.     

Characterization and optimization of the preparation procedure for solution P-31 NMR analysis of organic phosphorus in sediments

Purpose

The single-step sodium hydroxide?Cethylenediaminetetraacetic acid (NaOH?CEDTA) extraction is currently the most common preparation technique for the measurement of organic phosphorus (P) composition in sediments using solution ³¹P nuclear magnetic resonance (NMR). In this study, detailed investigations were conducted to evaluate the performance of this technique, with an objective of finding an optimal procedure for the measurement of sediment organic P.

Materials and methods

A single-step extraction with NaOH?CEDTA was investigated on two types of sediment, i.e., Fe/Al-rich sediment and calcareous sediment. The influence of different sediment preparation methods, NaOH?CEDTA compositions, solid:solution ratios, extraction time, pre-concentration techniques, and NMR sample frozen storage time on P extraction and solution ³¹P NMR analysis were investigated.

Results and discussion

Both air- and freeze-drying increased organic P extraction for the calcareous sediment. An extraction time of 16?h was sufficient for quantitative recovery of extracted organic P for both Fe/Al-rich and calcareous sediments. The use of a 1:8 solid:solution ratio achieved stronger NMR signals and greater P diversity than the use of a 1:20 ratio. Extraction of the two sediments with 0.25 NaOH?C50?mM EDTA favored ³¹P NMR detection by reducing the relaxation times required and the risk of organic P degradation compared to the use of stronger NaOH?CEDTA solutions. Rotary evaporation was a better technique for pre-concentration of the NaOH?CEDTA extracts than freeze drying. The concentrated extracts could be preserved by freezing (?20?°C) for at least 2?months.

Conclusions

An optimized procedure was developed based on these investigations, including freeze-drying of fresh sediments, extraction with 0.25?M NaOH?C50?mM EDTA for 16?h using a solid:solution ratio of 1:8, pre-concentration of the extract to the level of ??10 times of its original concentration using rotary evaporation, and storage of the NMR sample at ?20?°C until ³¹P NMR analysis.     

IR-, 1H-NMR- und 13C-NMR-spektroskopische Untersuchungen dioxanlöslicher Substanzen in Coniferenstreu

IR, ¹H and ¹³C NMR spectroscopic studies of dioxane soluble substances in coniferous litter Litter of mixed spruce and larch stands was extracted with dioxane. The extracts were studied by IR, ¹H NMR and ¹³C NMR spectroscopy. The NMR spectra revealed the dominance of aliphatic hydrocarbons. NMR peaks attributed to aromatic structures intensified after pressure extraction of the litter with dioxane. There is no conformity of the spectra of our dioxane soluble substances with spectra of lignin. Results of FTIR spectroscopic studies are in agreement with the NMR results.     

Spectroscopic (IR,NMR) characterization of water-soluble organic substances extracted from straw,straw incubated with Pleurotus ostreatus,and straw compost

Water extracts from fresh wheat and barley straw, straw incubated with Pleurotus ostreatus, and straw compost were studied by IR, ¹H NMR, and ¹³C NMR spectroscopy. During incubation lignin was degraded, water extractability increased, and water extracts were rich in polysaccharides. After composting solubility decreased and the water extracts were rich in aromatic, methoxyl, and carboxyl C, but poor in O-alkyl C indicating that during composting mainly polysaccharides had been mineralized. GPC revealed that water extracts from straw compost contained polysaccharides, peptides, C- and O-substituted aromatics, and alkyl compounds.