Antioxidant capacity changes and phenolic profile of Echinacea purpurea, nettle (Urtica dioica L.), and dandelion (Taraxacum officinale) after application of polyamine and phenolic biosynthesis regulators

The changes of the antioxidant (AOA) and antiradical activities (ARA) and the total contents of phenolics, anthocyanins, flavonols, and hydroxybenzoic acid in roots and different aerial sections of Echinacea purpurea, nettle, and dandelion, after treatment with ornithine decarboxylase inhibitor, a polyamine inhibitor (O-phosphoethanolamine, KF), and a phenol biosynthesis stimulator (carboxymethyl chitin glucan, CCHG) were analyzed spectrophotometrically; hydroxycinnamic acids content was analyzed by RP-HPLC with UV detection. Both regulators increased the AOA measured as inhibition of peroxidation (IP) in all herb sections, with the exception of Echinacea stems after treatment with KF. In root tissues IP was dramatically elevated mainly after CCHG application: 8.5-fold in Echinacea, 4.14-fold in nettle, and 2.08-fold in dandelion. ARA decrease of Echinacea leaves treated with regulators was in direct relation only with cichoric acid and caftaric acid contents. Both regulators uphold the formation of cinnamic acid conjugates, the most expressive being that of cichoric acid after treatment with CCHG in Echinacea roots from 2.71 to 20.92 mg g(-1). There was a strong relationship between increase of the total phenolics in all sections of Echinacea, as well as in the studied sections of dandelion, and the anthocyanin content.     

Calculation of the total nitrate uptake of lettuce (Lactuca sativa L.) by use of a mathematical model to simulate nitrate inflow

Lettuce was grown in nutrient solution in a climate chamber under varied levels of temperature and radiation and in the greenhouse under fluctuating climatic conditions. A model to calculate maximum nitrate inflow (Imax) in relation to relative growth rate (RGR) and root:shoot ratio (RSR) was used for computing total-N uptake. For doing this, developing of submodels to calculate RGR and RSR was necessary. The good agreement between calculated N uptake and measured total-N taken up by the plants supports the assumption that Imax reflects the inflow necessary to meet the demand of plants.     

Carbon costs of nitrate reduction in broad bean (Vicia faba L.) and pea (Pisum sativum L.) plants

The present study aimed at the assessment of carbon (C) costs for nitrate reduction by measuring the additional CO₂ amounts released from roots of nitrate‐fed plants in comparison with urea‐fed plants. Only roots were suitable for these determinations, because nitrate reduction in illuminated shoots is fed nearly exclusively by reducing equivalents coming directly from photosynthetic processes. Therefore, in a first experiment, the sites of nitrate reduction were determined in nodule‐free broad bean (Vicia faba L.) and pea (Pisum sativum L.) plants grown in pots filled with quartz sand and supplied with KNO₃. The extent of nitrate reduction in the various plant organs was determined by measuring in vitro nitrate reductase activity and in situ ¹⁵NO reduction. Only between 9% and 16% of nitrate were reduced in roots of German pea cultivars, whilst 52% to 65% were reduced in broad bean roots. Therefore, C costs of the process could be determined only in broad bean, using an additional pot experiment. The C costs amounted to about 4.76 mol C (mol N)^–1 or 4 mg C (mg N)^–1, similar to those measured earlier for N₂ fixation. The high proportion of nitrate reduction in shoots of pea plants implies that only very little C is required for this nitrate fraction. This can explain the better root growth of nitrate‐nourished pea plants in comparison with N₂‐fixing organisms, which need C compounds for N₂ reduction in roots. Moreover, a different availability of photosynthates in roots of plant genotypes could explain physiologically the occurrence of “shoot and root reducers” in nature.     

Kinetics of 15N-labelled nitrate uptake by maize (Zea mays L.) root segments

Abstract

Isotherms and kinetic constants of nitrate uptake by excised root segments from the apical root zone of 6-d-old maize seedlings pretreated with nitrate were investigated using ¹⁵N-labelled nitrate. The isotherms were resolved into two systems namely a multiphasic saturable system at substrate concentrations lower than 2 mol m^?-3 and a linear system at higher concentrations. The detailed analysis of the multiphasic saturable system suggested the existence of at least three phases, which followed the Michaelis-Menten kinetics. The I _max and K _m of each phase increase from the lower phase to the upper phase. The distance from the root tip and the presence of stele affected considerably the linear system but only slightly the saturable system.     

High-performance liquid chromatography separation and purification of cacao (Theobroma cacao L.) procyanidins according to degree of polymerization using a diol stationary phase

A new chromatographic approach for separating cacao procyanidins according to their degree of polymerization has been developed. It utilizes diol stationary phase columns operating in normal phase mode with a binary gradient of acidified acetonitrile and methanol-water. Performance of the diol stationary phase was evaluated on an analytical scale utilizing classical chromatographic conditions for the normal phase separation of procyanidins according to their degree of polymerization. The new separation approach was developed on an analytical scale but further extended to the preparative scale. These newly developed analytical and preparative high-performance liquid chromatography procedures were successfully applied to the separation, as well as isolation, of cacao procyanidins from unfermented cacao seeds. The degree of polymerization associated with each molecular weight fraction was determined by mass spectrometry.     

Effect of genotype and growing season on nitrate accumulation and expression patterns of nitrate transporter genes in potato (Solanum tuberosum L.)

Nitrate (NO₃^?) is the most important source of mineral nitrogen (N) affecting the vegetative growth, the tuber yield and the quality of potato (Solanum tuberosum L.). The screening of 16 potato cultivars for low nitrate content was reported and the effects of genotype and experimental factors on nitrate accumulation were studied. Both genotype and growing season had a highly significant effect on dry weight, number of tubers/plant, yield and nitrate accumulation in potato which varied significantly from one genotype to another. Furthermore, nitrate accumulation varies with the season, being higher in the first season (autumn) than in the second season (spring), for all the genotypes. The cultivars Safrane and Universa followed by Actrice and Alaska produced the highest tuber yield/plant during the first and second season, respectively, accumulating higher quantities of nitrate compared to other cultivars. Ponomera cultivar was of interest from a breeding perspective due to both combining best tuber yield/plant and low nitrate accumulation for both growing season. Expression pattern of nitrate transporter genes (StNRT1.2, StNRT1.5 and StNRT2.1) has been reported. Thus, these genes were differentially expressed which could be explained in part by the difference in nitrate accumulation rate between the studied cultivars. The results showed that high accumulator genotype got significant higher expression level of StNRT1.2, StNRT1.5 or StNRT2.1 which lead to the higher nitrate uptake rate and consequently higher nitrate accumulation than low accumulator genotype.     

Analysis and stability of sucralose in a milk-based confection by a simple planar chromatographic method

Sucralose used as high potency sweetener in foods was determined in burfi, a milk-based confection produced in-house. Therefore planar chromatography was employed as a preferred method because of a reagent-free derivatization step. Sucralose was determined on HPTLC amino plates whose amino groups reacted with sucralose to fluorescent zones by just heating the plate after chromatography. Thus derivatization was simultaneously performed for 22 separations per plate, and with ease, over 300 runs can be performed within a day of labor. The within-run precision (%RSD) of sucralose determination in milk-based confection was 4.2% (n = 5), and the mean recovery 88% +/- 4.7% (n = 6). LOD via fluorescence measurement was 6 ng/band for standard solutions and 1 mg/kg for the milk-based matrix. According to European legislation, the limits for sucralose addition ranged between 10 and 3000 mg/kg for various foods and thus were fully met with this method. The fluorescence measurement at 366/>400 nm turned out to be slightly more robust and intense than the absorbance measurement at UV 254 nm. The stability of sucralose in milk-based confection was proved under the usual storage conditions at 5, 30, and 45 degrees C for up to 28 days. Potential hydrolysis products of sucralose caused by various modes of storing the confection were not observed up to 28 days.     

氨基酸部分替代硝态氮对小白菜生长、硝酸盐累积及大量元素含量的影响

A hydroponic experiment was carried out to determine the influence of replacing 20% of nitrate-N in nutrient solutions with 20 individual amino acids on growth, nitrate accumulation, and concentrations of nitrogen （N）, phosphorus （P）, and potassium （K） in pak-choi （Brassica chinensis L.） shoots. When 20% of nitrate-N was replaced with arginine （Arg） compared to the full nitrate treatment, pak-choi shoot fresh and dry weights increased significantly （P ≤ 0.05）, but when 20% of nitrate-N was replaced with alanine （Ala）, valine （Val）, leucine （Leu）, isoleucine （Ile）, proline （Pro）, phenylalanine （Phe）, methionine （Met）, aspartic acid （Asp）, glutamic acid （Glu）, lysine （Lys）, glycine （Gly）, serine （Ser）, threonine （Thr）, cysteine （Cys）, and tyrosine （Tyr）, shoot fresh and dry weights decreased significantly （P ≤ 0.05）. After replacing 20% of nitrate-N with asparagine （Asn） and glutamine （Gin）, shoot fresh and dry weights were unaffected. Compared to the full nitrate treatment, amino acid replacement treatments, except for Cys, Gly, histidine （His）, and Arg, significantly reduced （P ≤0.05） nitrate concentrations in plant shoots. Except for Cys, Leu, Pro, and Met, total N concentrations in plant tissues of the other amino acid treatments significantly increased （P ≤ 0.05）. Amino acids also affected total P and K concentrations, but the effects differed depending on individual amino acids. To improve pak-choi shoot quality, Gln and Asn, due to their insignificant effects on pak-choi growth, their significant reduction in nitrate concentrations, and their increase in macroelement content in plants, may be used to partially replace nitrate-N.     

Estimation of nitrate retention in a Ferralsol by a transient-flow method

Anion retention is important in highly weathered soils that contain large amounts of iron and aluminium oxides with surfaces of variable charge. Sorption mechanisms retard anionic solute transfer through these soils. We determined the retardation factor for nitrate in highly weathered Ferralsols from New Caledonia from dynamic experiments using a transient‐flow method, and we evaluated the effect of soil solution concentration and organic matter content. A simple method with sectionable tubes was used to determine the nitrate isotherm during non‐steady‐state water flow under unsaturated conditions. The topsoil retarded the movement of nitrate, and the sorption followed a linear isotherm. In subsoils, retardation factors were larger and increased from 1.15 to 2.05 at soil pH as the NO₃^–‐N concentration of the input solution decreased from 71.43 to 0.35 mm , indicative of a non‐linear isotherm. Positive surface charge sites were considered to be of two types: one with strong affinity for nitrate at small concentrations and one with weak affinity for adsorption of nitrate at larger concentrations. This type of isotherm with high‐ and low‐energy sites is similar to those found for oxyanions and heavy metals. The related anion exchange capacity was larger than that usually observed in soils of variable charge. Not all exchange sites were detected with our method, and some sites were obviously not available for nitrate retention.     

The effect of chloride and sulphate salts on the nitrate content in lettuce plants (Lactuca sativa L.)

The effect of chloride and sulphate on the nitrate accumulated in lettuce plants (Lactuca sativa L., cv. deci minor) under low light conditions was examined. The plants were grown on a complete nutrient solution for circa 45 days and then chloride was substituted for the nitrate in the nutrient solution. In another experiment, sulphate was substituted for the nitrate in the nutrient solution. Approximately 5 days later a reduction in growth occurred. The nitrate in the plants was not completely exhausted and was very slowly available for reduction both in the leaf blades and midribs. It was immaterial whether sulphate or chloride was substituted for nitrate in the nutrient solution. Chloride was taken up from the solution but sulphate was not. In plants in both treatments, sugars accumulated concomitantly with the decrease in nitrate content.

The actual nitrate reductase activity in the leaf blades decreased several days after the chloride and sulphate treatments began and was negligible on day 12. Nevertheless, the potential nitrate reductase activity of the treated plants was still 50% of the control.

It is suggested that the availability of nitrate from the storage pool is the limiting process for nitrate reductase activity, and is independent of the metabolic demand. For commercial growers it may be of advantage to use hydroponics in which the nitrate can be removed easily several days before harvest, to reduce high nitrate contents in vegetables, particularly under low light conditions.     

Rapid and reversible nitrate inhibition of nodule growth and N2 fixation activity in soybean (Glycine max (L.) Merr.)

Nodulated soybean (Glycine max. (L) Merr. cv. Williams) plants were hydroponically cultured, and various combinations of 1-week culture with 5 or 0 mm nitrate were applied using 13-d-old soybean seedlings during three successive weeks. The treatments were designated as 0-0-0, 5-5-5, 5-5-0, 5-0-0, 5-0-5, 0-5-5, and 0-0-5, where the three sequential numbers denote the nitrate concentration (mm) applied in the first-second-third weeks. The size of the individual nodule was measured periodically using a slide caliper. All the plants were harvested after measurement of the acetylene reduction activity (ARA) at the end of the treatments. In the 0-0-0 treatment, the nodules grew continuously during the treatment period. Individual nodule growth was immediately suppressed after 5 mm nitrate supply. However, the nodule growth rapidly recovered by changing the 5 mm nitrate solution to a 0 mm nitrate solution in the 5-0-0 and 5-5-0 treatments. In the 5-0-5 treatment, nodule growth was completely inhibited in the first and the third weeks with 5 mm nitrate, but the nodule growth was enhanced in the second week with 0 mm nitrate. The nodule growth response to 5 mm nitrate was similar between small and large size nodules. After the 5-5-5, 5-0-5, 0-0-5, and 0-5-5 treatments, where the plants were cultured with 5 mm nitrate in the last third week, the ARA per plant was significantly lower compared with the 0-0-0 treatment. On the other hand, the ARA after the 5-0-0 and 5-5-0 treatments was relatively higher than that after the 0-0-0 treatment, possibly due to the higher photosynthate supply associated with the vigorous vegetative growth of the plants supplemented with nitrate nitrogen. It is concluded that both soybean nodule growth and N₂ fixation activity sensitively responded to the external nitrate level, and that these parameters were reversibly regulated by the current status of nitrate in the culture solution, possibly through sensing of the nitrate concentration in roots and / or nodules.     

增施CO2降低小白菜硝酸盐积累的机理研究

以低硝酸盐积累基因型（东妃）和高硝酸盐积累基因型（高雄甜脆）两种小白菜为材料,采用溶液培养法研究了增施CO2降低蔬菜硝酸盐积累的生理机制。结果表明,CO2浓度升高能显著提高2种基因型小白菜的生物量和硝酸还原酶活性,并降低根、茎叶各部位的硝酸盐含量。CO2浓度升高不仅促进了植株对硝态氮的吸收,而且植株吸收硝酸盐的累积量增幅均高于鲜重的增幅。由此可见,除了鲜重增加的稀释作用,处理后生理机制的变化也可能是CO2浓度升高引起硝酸盐含量降低的重要原因。研究还表明,增施CO2后“东妃”的硝酸盐含量降低百分率与硝酸还原酶活性的增加百分率呈极显著相关,而“高雄甜脆”的硝酸盐含量降低百分率则与鲜重的增加百分率的相关性达极显著水平。说明增施CO2后植株各部位硝酸还原酶活性提高及鲜重的增加均为引起硝酸盐含量降低的重要原因,但贡献率具有明显的基因型差异。     

Priming effects of 15N-labelled ammonium nitrate on uptake of soil N by wheat (Triticum aestivum L.) under field conditions

Summary The uptake of labelled and unlabelled N by wheat was measured in a field experiment using ¹⁵N-labelled ammonium nitrate fertilizer. The dry matter yield and N yields were significantly increased with fertilizer N application compared to those from unfertilized soil. The uptake of applied N by wheat ranged between 25 and 34%. Fertilizer N application increased the uptake of unlabelled soil N which was attributed to a positive priming effect or added N interaction. The added N interaction observed by applying 20, 60, and 120 kg fertilizer N was 11.4, 19.1, and 27.9 kg, corresponding to 26, 44 and 64%, respectively of the N taken up from unfertilized soil. The A values did not alter with the increase in fertilizer N application. The observed added N interaction may have been the result of pool substitution whereby added labelled fertilizer N stood proxy for unlabelled soil N. A significant correlation coefficient (r=0.996^**) between the uptake of soil N and the dry matter yield showed that soil N was more important than fertilizer N in wheat production.     

Xanthophylls in commercial egg yolks: quantification and identification by HPLC and LC-(APCI)MS using a C30 phase

The xanthophylls lutein and zeaxanthin have attracted a lot of interest since it was presumed that an increased nutritional uptake may prevent adult macula degeneration (AMD). Although egg yolks serve as an important dietary source of lutein and zeaxanthin, data on xanthophyll concentrations in commercial egg yolks are not available. Thus, an high-performance liquid chromatography-diode array detector (HPLC-DAD) method was developed allowing for simultaneous separation of eight xanthophylls used to fortify poultry feed. Peak identification was carried out by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry [LC-(APCI)MS]. Egg yolks of four types of husbandry (seven batches each) were examined. Lutein and zeaxanthin were the predominant xanthophylls in egg yolks produced in accordance with ecological husbandry (class 0) because the concentrations of these xanthophylls ranged from 1274 to 2478 microg/100 g and from 775 to 1288 microg/100 g, respectively. Analysis of variance (ANOVA) proved that both mean lutein and mean zeaxanthin concentrations of eggs from class 0 were statistically discriminable from mean lutein and zeaxanthin concentrations from eggs of all other classes (P < 0.01). Mean concentrations of synthetic xanthophylls in eggs of classes 1 (free range), 2 (barn), and 3 (cage) were as follows: canthaxanthin, 707 +/- 284 microg/100 g; beta-apo-8'-carotenoic acid ethyl ester, 639 +/- 391 microg/100 g; and citranaxanthin, 560 +/- 231 microg/100 g. Experiments with boiled eggs proved that beta-apo-8'-carotenoic acid ethyl ester was the xanthophyll with the highest stability, whereas lutein was degraded to the largest extent (loss of 19%). Detailed knowledge about the xanthophyll amounts in eggs is indispensable to calculate the human uptake.     

Antioxidant activity of chemical components from sage (Salvia officinalis L.) and thyme (Thymus vulgaris L.) measured by the oil stability index method

A new abietane diterpenoid, 12-O-methyl carnosol (2), was isolated from the leaves of sage (Salvia officinalis L.), together with 11 abietane diterpenoids, 3 apianane terpenoids, 1 anthraquinone, and 8 flavonoids. Antioxidant activity of these compounds along with 4 flavonoids isolated from thyme (Thymus vulgaris L.) was evaluated by the oil stability index method using a model substrate oil including methyl linoleate in silicone oil at 90 degrees C. Carnosol, rosmanol, epirosmanol, isorosmanol, galdosol, and carnosic acid exhibited remarkably strong activity, which was comparable to that of alpha-tocopherol. The activity of miltirone, atuntzensin A, luteolin, 7-O-methyl luteolin, and eupafolin was comparable to that of butylated hydroxytoluene. The activity of these compounds was mainly due to the presence of ortho-dihydroxy groups. The 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity of these compounds showed the similar result.     

Effect of drying method on the volatiles in bay leaf (Laurus nobilis L.)

The effect of different drying treatments on the volatiles in bay leaf (Laurus nobilis L.) was studied. Simultaneous distillation extraction (SDE) and solid-phase microextraction (SPME) were compared by gas chromatography-mass spectrometry (GC-MS) of the volatile components in bay leaves. SDE yielded better quantitative analysis results. Four drying treatments were employed: air-drying at ambient temperature, oven-drying at 45 degrees C, freezing, and freeze-drying. Oven drying at 45 degrees C and air-drying at ambient temperature produced quite similar results and caused hardly any loss in volatiles as compared to the fresh herb, whereas freezing and freeze-drying brought about substantial losses in bay leaf aroma and led to increases in the concentration levels of certain components, e.g., eugenol, elemicin, spathulenol, and beta-eudesmol.     

Residual nitrogen effects on a succeeding oat (Avena sativa L.) Crop of clover species and ryegrass (Lolium perenne L.) undersown in winter wheat (Triticum aestivum L.)

The aim of the present investigation was to study the effect of white clover (var. Milka and Donna), red clover (var. Fanny) and ryegrass (var. Tove) undersown in winter wheat on a succeeding oat crop. Under the climatic conditions prevailing in Sweden, growing a catch crop after winter cereals is of particular interest because the latter are usually followed by a spring sown crop, leaving the ground bare during autumn and winter. Field trials were carried out during three growing seasons in an integrated farming system and for one year in an organic farming system. Competition from the dense wheat crop in the integrated farming system had a negative effect on the undersown species, and at harvest of the wheat they showed quite poor growth in all three years, with nitrogen contents under 5 kg ha^?1. No significant yield increase was measured without added nitrogen when the averages for all three years were calculated. On average, the grain yields were improved by 750 kg ha^?1 (14% moisture content) for the treatments with undersown clover for all three years when 90 kg N ha^?1 were added. The experiment within the organic system showed a different pattern with a better development of the undersown clover species, with nitrogen contents approximately 25 kg ha^?1 and an improvement in oat grain yield, from around 2?000 kg ha^?1 for the control to almost 3?500 kg ha^?1 with clover undersown the previous year.     

Evaluation of denitrification losses by the acetylene inhibition technique in a permanent ryegrass field (Lolium perenne L.) fertilized with animal slurry or ammonium nitrate

In a field experiment, the effect of animal slurry, (with and without the nitrification inhibitor dicyandiamide on total denitrification losses estimated by the C₂H₂ inhibition technique was measured over 2 years (1989–1990). During this period, four different plots (each with four replicates) were fertilized six times with 150 kg N ha^-1 in the form of cattle-pig slurry or NH₄NO₃. Soil samples (0–20 cm) were analysed at regular intervals for NH _inf4 ^sup+ and NO _inf3 ^sup– concentrations. The soil water content was determined gravimetrically. During the first year (1989) total denitrification losses from unfertilized, mineral-fertilized, and animal slurry-amended plots (with or without dicyandiamide) were estimated as 0.2, 3.1, 0.7, and 0.6 kg N ha^-1, respectively. During the second year (1990) the denitrification losses were 0.4, 1.3, 0.7, and 0.7 kg N ha^-1, respectively. There was a clear relationship between the NO _inf3 ^sup– concentration or soil water content and the denitrification rate. The results are siteund experiment-specific and cannot be generalized so far.     

Influence of the earthworm Aporrectodea trapezoides (Lumbricidae) on the colonization of alfalfa (Medicago sativa L.) roots by Rhizobium meliloti L5-30R and the survival of R. meliloti L5-30R in soil

A greenhouse study was performed to examine the ability of the earthworms Microscolex dubius and Aporrectodea trapezoides to transport Rhizobium meliloti L5-30R through soil. When R. meliloti L5-30R was inoculated into either ezi-mulch (a pelleted formulation of cereal-pea straw), oat hay, pea hay, or sheep dung and placed on the soil surface together with either A. trapezoides or M. dubius, >10⁴ colony-forming units (CFU) of R. meliloti L5-30R g^-1 soil were detected at 90 mm soil depth after 18 days. In the absence of earthworms, R. meliloti L5-30R was not detected at 90 mm soil depth after this time. In a second experiment using ezi-mulch as the inoculant material and in the presence of A. trapezoides (in a number equivalent to 471 or 785 m^-2), ca. 10³ CFU of R. meliloti L5-30R per 10 mm of alfalfa root were detected at 0–30, 30–60, and 60–90 mm soil depth after 18 days, while <3 CFU were detected per 10 mm of root in the absence of A. trapezoides. In a third experiment in which R. meliloti L5-30R was distributed evenly through soil at the start of the experiment, A. trapezoides (in a number equivalent to 157, 471, or 785 m^-2) significantly decreased the survival of L5-30R in soil after 40 days of incubation at 15°C, but not after 20 days. The decrease in survival of R. meliloti L5-30R was correlated with the density of A. trapezoides. These results demonstrate that A. trapezoides can increase root colonization of alfalfa by R. meliloti L5-30R, but may also reduce the ability of R. meliloti L5-30R to survive in soil.