Synthesis and fungicidal activity of novel 4,4'-bis(2' '-aryl-5' '-methyl/unsubstituted-4' '-oxo-thiazolidin-3' '-yl) bibenzyl

Reduction followed by nitration of benzil I yielded 4,4'-dinitrobibenzyl (III) which by reduction furnished quantitatively and analytically pure 4,4'-diaminobibenzyl (IV) which on condensation with different carbonyl compounds gave 4,4'-bis (benzylideneamino) bibenzyls (Va-f). Compounds (Va-f) on cycloaddition with mercaptoacetic acid/2-mercaptopropionic acid yielded the corresponding 4-oxothiazolidin-3-yl bibenzyls (VIa-l). The compounds VIg-l have two chiral centers in each thiazolidinone moiety so two diastereomers are possible, but on crystallization and repeated chromatography, one diastereomer was obtained. The absolute configuration of the diastereomer was tentatively assigned on the basis of (1)H NMR spectra. (1)H NMR spectra of the product showed a distinct doublet at delta 1.22 for C(5)-CH(3) of thiazolidinone ring (22, 23) and a distinct quartet at delta 4.20 for the C(5)-H proton. Similarly, the C(2) proton showed an independent singlet at delta 5.95, so the diastereomers obtained were assigned trans configuration. Compounds Va-f and VIa-l were evaluated in vitro for their fungitoxicities against Fusarium oxysporium and Penicillium citrinum. All the compounds were found to be antifungal active. Some of the compounds displayed activities comparable with that of the commercial fungicide Dithane M-45. Structure-activity relationships for the screened compounds are discussed.     

Liquid chromatography/mass spectrometry and liquid chromatography/nuclear magnetic resonance as complementary analytical techniques for unambiguous identification of polymethoxylated flavones in residues from molecular distillation of orange peel oils (Citrus sinensis)

Liquid chromatography/mass spectrometry and liquid chromatography/nuclear magnetic resonance techniques with ultraviolet/diode array detection were used as complementary analytical tools for the reliable identification of polymethoxylated flavones in residues from molecular distillation of cold-pressed peel oils of Citrus sinensis. After development of a liquid chromatographic separation procedure, the presence of several polymethoxy flavones such as sinensetin, nobiletin, tangeretin, quercetogetin, heptamethoxyflavone, and other derivatives was unambiguously confirmed. In addition, proceranone, an acetylated tetranortriterpenoid with limonoid structure, was identified for the first time in citrus.     

Urinary excretion of 5-(3',4'-dihydroxyphenyl)-gamma-valerolactone, a ring-fission metabolite of (-)-epicatechin, in rats and its in vitro antioxidant activity

There is great interest in the nutritional potential of (-)-epicatechin, a common polyphenolic constituent of many foods and beverages, because of its potent antioxidant capacity. To better evaluate the biological role of (-)-epicatechin, we studied the urinary excretion of 5-(3',4'-dihydroxyphenyl)-gamma-valerolactone, a ring-fission metabolite of (-)-epicatechin by intestinal microflora, in rats as well as its antioxidant activity in vitro. The method for measuring the urinary levels of (-)-epicatechin and 5-(3',4'-dihydroxyphenyl)-gamma-valerolactone was based on the enzymatic hydrolysis of beta-glucuronidase and sulfatase, and was subsequently determined by HPLC coupled to an electrochemical detector. Following administration of (-)-epicatechin at doses of 0, 20, 40, and 80 mumol per rat, (-)-epicatechin and 5-(3',4'-dihydroxyphenyl)-gamma-valerolactone were excreted into the urine within 24 h in a dose-dependent manner. Urinary 5-(3',4'-dihydroxyphenyl)-gamma-valerolactone was mostly in the conjugated form, with a higher ratio of conjugation than (-)-epicatechin. We assessed the relative antioxidant potentials for scavenging radicals in the aqueous phase as expressed in the Trolox equivalent antioxidant capacity (TEAC). The results demonstrated that the degradation of (-)-epicatechin into 5-(3',4'-dihydroxyphenyl)-gamma-valerolactone attenuated the antioxidant ability of the former. However, 5-(3',4'-dihydroxyphenyl)-gamma-valerolactone showed stronger antioxidant activity than l-ascorbic acid. These results led us to suppose that 5-(3',4'-dihydroxyphenyl)-gamma-valerolactone, a microbial metabolite of (-)-epicatechin, circulating in the body may also at least be biologically active in terms of contributing to its combined antioxidant effect.     

Immunoadjuvant activity, toxicity assays, and determination by UPLC/Q-TOF-MS of triterpenic saponins from Chenopodium quinoa seeds

The adjuvant activity of Chenopodium quinoa (quinoa) saponins on the humoral and cellular immune responses of mice subcutaneously immunized with ovalbumin (OVA) was evaluated. Two quinoa saponin fractions were obtained, FQ70 and FQ90, and 10 saponins were determined by UPLC/Q-TOF-MS. Mice were immunized subcutaneously with OVA alone or adjuvanted with Quil A (adjuvant control), FQ70, or FQ90. FQ70 and FQ90 significantly enhanced the amount of anti-OVA-specific antibodies in serum (IgG, IgG1, and IgG2b) in immunized mice. The adjuvant effect of FQ70 was significantly greater than that of FQ90. However, delayed type hypersensitivity responses were higher in mice immunized with OVA adjuvanted with FQ90 than mice treated with FQ70. Concanavalin A (Con A)-, lipopolysaccharide-, and OVA-stimulated splenocyte proliferation were measured, and FQ90 significantly enhanced the Con A-induced splenocyte proliferation. The results suggested that the two quinoa saponin fractions enhanced significantly the production of humoral and cellular immune responses to OVA in mice.     

Stability of ferric complexes with 3-hydroxyflavone (flavonol), 5,7-dihydroxyflavone (chrysin), and 3',4'-dihydroxyflavone

The acid dissociation and ferric stability constants for complexation by the flavonoids 3-hydroxyflavone (flavonol), 5,7-dihydroxyflavone (chrysin), and 3',4'-dihydroxyflavone in 50:50 (v/v) ethanol/water are determined by pH potentiometric and spectrophotometric titrations and the linear least-squares curve-fitting program Hyperquad. Over the entire range of pH and reagent concentrations spanning the titration experiments, the stoichiometry for iron-flavonoid complex formation was 1:1 for all three flavonoids examined. The three flavonoids were chosen for their hydroxy substitution pattern, with each possessing one of the three most commonly suggested sites for metal binding by the flavonoids. On the basis of the calculated stability constants, the intraflavonoid-binding site competition is illustrated as a function of pH via speciation curves. The curves indicate that the binding site comprised of the 3',4'-hydroxy substitutions, the catecholic site, is most influential for ferric complexation at the physiological pH of 7.4. The possibility for antioxidant activity by flavonoid chelation of ferric iron in the presence of other competitive physiological complexing agents is demonstrated through additional speciation calculations.     

Determination of vanillin in orange, grapefruit, tangerine, lemon, and lime juices using GC-olfactometry and GC-MS/MS

The presence of vanillin in orange, tangerine, lemon, lime, and grapefruit juices has been identified and confirmed using high-resolution GC retention index values, mass spectra, and aroma quality. The impact of vanillin on the flavor score for grapefruit juice is discussed and reported to be minimal. Vanillin concentrations are determined to be in the low parts-per-million range for the various citrus juices. The calculated concentrations in the orange, tangerine, lemon, lime, and grapefruit juices are 0.20, 0.35, 0.41, 0.35, and 0.60 ppm, respectively. Pasteurization produced an average 15% increase in the concentration of vanillin in grapefruit juices. Vanillin did not correlate well with the overall flavor score despite a rather intense signal using OSME gas chromatography-olfactometry software.     

Simultaneous measurement of S, macronutrients, and heavy metals in the soil microbial biomass with CHCl3 fumigation and NH4NO3 extraction

The study was carried out to investigate whether 1 M NH₄NO₃ extraction is a useful alternative to 10 mM CaCl₂ extraction for estimating soil microbial biomass S and whether the data of CHCl₃-labile NH₄NO₃-extractable macronutrients and heavy metals are useful and in agreement with the available data on element concentrations in soil microorganisms. Microbial biomass C was followed by microbial biomass S after CaCl₂ extraction with an average C/S ratio of 82, and by microbial biomass S after NH₄NO₃ extraction with an average C/S ratio of 57. The mean contribution of CHCl₃-labile metals in relation to the NH₄NO₃-extractable fraction from non-fumigated soils ranged from 0.1 to 112% in the order potassium < magnesium < cadmium < sodium < zinc + nickel < manganese < copper. The mean contribution of CHCl₃-labile metals in relation to the microbial biomass C ranged from 0.03 to 22‰ in the order cadmium < nickel < zinc < manganese < magnesium < copper < sodium < potassium. These relative contributions varied within the different metals from a 4-fold (Na⁺) to a more than 200-fold range (Cu²⁺). Significant positive correlations with microbial biomass C were observed for CHCl₃-labile zinc, sodium and especially potassium. The concentration of all elements except copper in relation to microbial biomass C were in the range known from the limited literature on fungi grown on heavy metal contaminated soils.     

Studies on the antioxidant activity of pomegranate (Punica granatum) peel and seed extracts using in vitro models.

Antioxidant-rich fractions were extracted from pomegranate (Punica granatum) peels and seeds using ethyl acetate, methanol, and water. The extracts were screened for their potential as antioxidants using various in vitro models, such as beta-carotene-linoleate and 1,1-diphenyl-2-picryl hydrazyl (DPPH) model systems. The methanol extract of peels showed 83 and 81% antioxidant activity at 50 ppm using the beta-carotene-linoleate and DPPH model systems, respectively. Similarly, the methanol extract of seeds showed 22.6 and 23.2% antioxidant activity at 100 ppm using the beta-carotene-linoleate and DPPH model systems, respectively. As the methanol extract of pomegranate peel showed the highest antioxidant activity among all of the extracts, it was selected for testing of its effect on lipid peroxidation, hydroxyl radical scavenging activity, and human low-density lipoprotein (LDL) oxidation. The methanol extract showed 56, 58, and 93.7% inhibition using the thiobarbituric acid method, hydroxyl radical scavenging activity, and LDL oxidation, respectively, at 100 ppm. This is the first report on the antioxidant properties of the extracts from pomegranate peel and seeds. Owing to this property, the studies can be further extended to exploit them for their possible application for the preservation of food products as well as their use as health supplements and neutraceuticals.     

Comparative studies on the interaction of genistein, 8-chlorogenistein, and 3',8-dichlorogenistein with bovine serum albumin

Chlorination can significantly enhance the antioxidant and antitumor activity of genistein. In this paper, genistein, 8-chlorogenistein, and 3',8-dichlorogenistein were selected to investigate the binding to bovine serum albumin (BSA) using fluorescence spectroscopy and circular dichroism (CD). The results showed that chlorination, especially at position 3', had significant effects on the binding constant value of chlorinated genistein derivatives to BSA; however, the binding site and the binding number were slightly affected. The thermodynamic parameters indicated that hydrophobic and electrostatic forces played important roles in the binding process and the enhanced binding affinity mainly associated with the increase of the hydrophobicity caused by the chlorine atom substitution. Furthermore, the CD data demonstrated that the conformation of BSA was slightly altered in the presence of genistein, 8-chlorogenistein, and 3',8-dichlorogenistein, with different reduced α-helix contents. The results obtained herein will be of biological significance in toxicology investigation and genistein derivative drug design.     

Design, synthesis, structure, and acaricidal/insecticidal activity of novel spirocyclic tetronic acid derivatives containing an oxalyl moiety

A series of novel spirocyclic tetronic acid derivatives containing an oxalyl moiety was designed and synthesized via the key intermediate 3-(2,4,6-trimethyl)-2-oxo-1-oxaspiro[4.4]-decyl-3-en-4-ol. The target compounds were identified by (1)H NMR and elemental analysis or high-resolution mass spectrum (HRMS). The results of bioassays indicated that most of the target compounds possessed excellent acaricidal activities against carmine spider mite larvae and eggs. Especially, diisopropylamino oxalyl compound 7g and piperidine oxalyl compound 7h were 1.4- and 2.3-fold as high as the activities of commercial Spiromesifen, respectively, against spider mite eggs. Moreover, most of the target compounds exhibited insecticidal activities against Lepidoptera pest. Interestingly, compounds containing alkylamino-substituted oxalyl moiety showed obvious selectivity between spider mite larvae and eggs because the activities against spider mite eggs of 7g and 7h were 25-fold those against spider mite larvae, whereas Spiromesifen had no significant differences in these activities. This meant that the introduction of an oxalyl moiety to spirocyclic tetronic acid might lead to novel biological activity characteristics.     

Antioxidant activity of 3-dehydroshikimic acid in liposomes,emulsions, and bulk oil

The antioxidant activity of 3-dehydroshikimic acid (DHS), an intermediate in the biosynthesis of aromatic amino acids, was evaluated in three assay systems: bulk oil (lard), liposomes, and a 10% corn oil-in-water emulsion. Upon initiation of peroxidation in the liposome or emulsion systems, DHS exhibited weak antioxidant activity. In contrast, DHS displayed strong antioxidant activity in lard, suppressing peroxidation with activity comparable to that of tert-butylhydroquinone, propyl gallate, and gallic acid and superior to that of alpha-tocopherol. Two major DHS oxidation products, gallic acid and protocatechuic acid, were identified by gas chromatography/mass spectral analysis of lard extracts; both compounds are effective antioxidants in the bulk oil system. In the liposome system, DHS remained intact throughout the assay period. A small amount of gallic acid was observed in extracts of the emulsion; however, protocatechuic acid was not detected. A mechanism to explain the different activities of DHS in the three lipid systems is proposed.     

Fluxes of CO2, CH4, and N2O in an alpine meadow affected by yak excreta on the Qinghai-Tibetan plateau during summer grazing periods

To assess the impacts of yak excreta patches on greenhouse gas (GHG) fluxes in the alpine meadow of the Qinghai-Tibetan plateau, methane (CH₄), carbon dioxide (CO₂), and nitrous oxide (N₂O) fluxes were measured for the first time from experimental excreta patches placed on the meadow during the summer grazing seasons in 2005 and 2006. Dung patches were CH₄ sources (average 586 μg m⁻² h⁻¹ in 2005 and 199 μg m⁻² h⁻¹ in 2006) during the investigation period of two years, while urine patches (average −31 μg m⁻² h⁻¹ in 2005 and −33 μg m⁻² h⁻¹ in 2006) and control plots (average −28 μg m⁻² h⁻¹ in 2005 and −30 μg m⁻² h⁻¹ in 2006) consumed CH₄. The cumulative CO₂ emission for dung patches was about 36-50% higher than control plots during the experimental period in 2005 and 2006. The cumulative N₂O emissions for both urine and dung patches were 2.1-3.7 and 1.8-3.5 times greater than control plots in 2005 and 2006, respectively. Soil water-filled pore space (WFPS) explained 35% and 36% of CH₄ flux variation for urine patches and control plots, respectively. Soil temperature explained 40-75% of temporal variation of CO₂ emissions for all treatments. Temporal N₂O flux variation in urine patches (34%), dung patches (48%), and control (56%) plots was mainly driven by the simultaneous effect of soil temperature and WFPS. Although yak excreta patches significantly affected GHG fluxes, their contributions to the whole grazing alpine meadow in terms of CO₂ equivalents are limited under the moderate grazing intensity (1.45 yak ha⁻¹). However, the contributions of excreta patches to N₂O emissions are not negligible when estimating N₂O emissions in the grazing meadow. In this study, the N₂O emission factor of yak excreta patches varied with year (about 0.9-1.0%, and 0.1-0.2% in 2005 and 2006, respectively), which was lower than IPCC default value of 2%.     

Overwintering management on upland pasture causes shifts in an abundance of denitrifying microbial communities, their activity and N2O-reducing ability

Pasture soils used for cattle overwintering may represent significant sources of N₂O emissions from soils. Therefore, the long-term effect of cattle overwintering on the abundance and activity of a denitrifying community was explored. The study was performed at a cattle overwintering area in South Bohemia (Czech Republic), where three sites differing in the degree of animal impact were selected: severely impacted (SI) and moderately impacted (MI), as well as a control site with no impact (NI). N₂O flux measurement and soil sampling were performed in spring and fall of 2005. The activity was measured in terms of potential denitrification activity. Bacterial nirK, nirS and nosZ genes were used as functional markers of the denitrifying communities; abundance was analyzed using a real-time PCR assay. Surprisingly, in situ N₂O emissions were the highest in spring at MI and significantly differed from those at SI and NI, while in autumn, rates of emissions generally decreased. In contrast potential denitrification rates were highest at SI, followed by MI, and the lowest at NI. An overall significant shift in N₂O/N₂ molar ratio was shown in cattle impacted sites. The highest abundance of all genes measured at both sampling times was found at site SI, whereas at site MI increased numbers were observed only in spring. Our results indicate a strong influence of cattle on the abundance as well as the activity of microbes involved in denitrification.     

Quantification of soy isoflavones, genistein and daidzein, and conjugates in rat blood using LC/ES-MS.

Genistein is the principal soy isoflavone to which the putative beneficial effects of soy consumption have been attributed; however, the possibility of adverse biological effects (e.g., estrogenic, antithyroid) has also been raised. This paper describes development and validation of a simple and sensitive analytical method for the determination of genistein in the blood of rats receiving dietary genistein (<0.5-1250 microg of genistein aglycone/g of chow). The method uses serum/plasma deproteination, liquid-liquid extraction, deuterated genistein and daidzein internal standards, isocratic LC separation, and electrospray mass spectrometric quantification using selected ion monitoring. Extraction efficiency is approximately 85%, the detection limits for genistein and daidzein from 50 microL of rat blood are approximately 5 nM, and the limit of quantification is approximately 15 nM. Interassay precision (relative standard deviation 4.5-4.6%) and intraassay precision (3.3-6.7%) were determined from replicate analysis of a spiked control and an incurred serum sample. The distribution of conjugated and unconjugated forms of genistein in the blood of rats was determined using selective enzyme hydrolysis. The glucuronide was the predominant metabolite (>90%), and only small amounts of the sulfate conjugate and the aglycone were observed at all dose levels. No evidence for additional metabolites was obtained. The 7- and 4'-glucuronide conjugates of genistein were identified using electrospray mass spectrometry and (1)H NMR. Total blood genistein ranged from <15 nM in animals fed soy-free control diet to as high as 8.9 microM in male rats fed 1250 microg of genistein/g of chow and encompasses blood isoflavone levels observed in humans consuming a typical Asian diet and nutritional supplements (0.1-1 microM) and infants consuming soy formulas (2-7 microM).     

Effect of grazing intensity on protozoan community,microbial biomass,and enzyme activity in an alpine meadow on the Tibetan Plateau

Purpose

The alpine meadow has received mounting attention due to its degradation resulting from overgrazing on the Tibetan Plateau. However, belowground biotic characteristics under varied grazing stresses in this ecosystem are poorly understood.

Materials and methods

Here, the responses of soil protozoan abundance, community composition, microbial biomass, and enzyme activity to five grazing patterns including (1) artificial grassland without grazing (AG), (2) winter grazing (WG), (3) grazing for 7 months within a fence (GF), (4) continuous grazing for a whole year (CG), and (5) natural heavy grazing (HG) were investigated for two continuous years. Soil protozoan community composition was investigated using the most possible number (MPN) method, and soil microbial biomass and enzyme activity were analyzed using chloroform fumigation extraction and substrate utilization methods, respectively. Multivariate statistical analysis, the analysis of variance (ANOVA), multiple comparisons, and correlation analysis were together performed.

Results and discussion

The WG treatment had the highest abundance of total protozoa (2342–2524 cell g^?1). Compared with AG treatment, HG treatment significantly reduced the abundance of soil total, flagellate and ciliate protozoa, and protease activities in 2012 and 2013. Significantly, lower soil microbial biomass nitrogen (MBN) was also observed in the HG (6.60 and 14.6 mg N kg^?1) than those in other four treatments (22.3–82.9 mg N kg^?1) both in 2012 and 2013, whereas significantly higher microbial biomass carbon (MBC) was observed in HG than that in AG treatment in 2012. Moreover, significantly positive correlations were detected between the abundance of soil protozoa and soil moisture, pH, organic C, total N, and MBN. Our results indicated that soil protozoa showed a negative response to increasing grazing intensities and therefore, suggesting that aboveground grazing practices also exerted strong impact on belowground protozoa, not only on soil microbial characteristics.

Conclusions

Soil protozoan community composition was apparently different between the HG treatment and other four grazing patterns and was potentially impacted by altered soil properties and MBC and/or MBN. Our results suggested that moderate grazing may sustain better belowground biotic diversity and ecosystem functioning in this alpine meadow on the Tibetan Plateau.

     

Cloning and characterization of an 11S legumin, Car i 4, a major allergen in pecan

Among tree nut allergens, pecan allergens remain to be identified and characterized. The objective was to demonstrate the IgE-binding ability of pecan 11S legumin and characterize its sequential IgE-binding epitopes. The 11S legumin gene was amplified from a pecan cDNA library and expressed as a fusion protein in Escherichia coli. The native 11S legumin in pecan extract was identified by mass spectrometry/mass spectrometry (MS/MS). Sequential epitopes were determined by probing the overlapping peptides with three serum pools prepared from different patients' sera. A three-dimensional model was generated using almond legumin as a template and compared with known sequential epitopes on other allergenic tree nut homologues. Of 28 patients tested by dot blot, 16 (57%) bound to 11S legumin, designated Car i 4. MS/MS sequencing of native 11S legumin identified 33 kDa acidic and 20-22 kDa basic subunits. Both pecan and walnut seed protein extracts inhibited IgE binding to recombinant Car i 4, suggesting cross-reactivity with Jug r 4. Sequential epitope mapping results of Car i 4 revealed weak, moderate, and strong reactivity of serum pools against 10, 5, and 4 peptides, respectively. Seven peptides were recognized by all three serum pools, of which two were strongly reactive. The strongly reactive peptides were located in three discrete regions of the Car i 4 acidic subunit sequence (residues 118-132, 208-219, and 238-249). Homology modeling of Car i 4 revealed significant overlapping regions shared in common with other tree nut legumins.     

Development of stable isotope dilution assays for the simultaneous quantitation of biogenic amines and polyamines in foods by LC-MS/MS

Microbial amino acid metabolism may lead to substantial amounts of biogenic amines in either spontaneously fermented or spoiled foods. For products manufactured with starter cultures, it has been suggested that certain strains may produce higher amounts of such amines than others; however, to support efforts of food manufacturers in mitigating amine formation, reliable methods for amine quantitation are needed. Using 10 isotopically labeled biogenic amines as the internal standards, stable isotope dilution assays were developed for the quantitation of 12 biogenic amines and of the 2 polyamines, spermine and spermidine, in one LC-MS/MS run. Application of the method to several foods revealed high concentrations of, for example, tyramine and putrescine in salami and fermented cabbage, whereas histamine was highest in Parmesan cheese and fermented cabbage. On the other hand, ethanolamine was highest in red wine and Parmesan cheese. The results suggest that different amino acid decarboxylases are active in the respective foods depending on the microorganisms present. The polyamine spermine was highest in salami and tuna.     

Trophic structure of a macroarthropod litter food web in managed coniferous forest stands: a stable isotope analysis with δN and δC

We studied the composition of a litter detrital community in a temperate coniferous forest using stable isotopes of nitrogen and carbon. Samples of mineral soil, bulk litter material, macroarthropods and understory plants were collected from ten experimental forest stands. Half of the stands were previously thinned 17–42 years ago, the other half served as controls. Values of δ¹⁵N and δ¹³C were based on the analysis of almost 500 individuals of at least 22 species in 11 arthropod families. The isotopic analysis showed a significant increase in δ¹⁵N and δ¹³C values with soil depth. Isotopic signatures of macroarthropods ranged from −26.51‰ to −20.52‰ for δ¹³C and −2.85‰ to 5.10‰ for δ¹⁵N. All consumers showed levels of ¹³C enrichment substantially higher than those of primary producers and litter. Predators were generally significantly more ¹⁵N enriched than detritivores and herbivores, but their δ¹³C levels were similar to those of primary consumers. Our data indicate that this community consists of at least 2–3 trophic levels with a considerable amount of variation in the ¹⁵N enrichment among detritivores and predators. We suggest that the spread of δ¹⁵N values of predators likely reflects the diversity of potential prey among detritivores and a varying degree of intraguild predation among different species. Our findings generally agree closely with the results of similar studies from other forest litter communities. Thinning did not appear to influence the overall isotopic composition of the detrital food web. Extensive omnivory and intraguild predation among litter consumers may buffer long-term effects of thinning on the trophic structure of these species-rich communities.