Inhibition of iron-stress reactions in sunflower by bicarbonate

In pot culture experiments using a calcareous soil the growth rate of sunflowers was depressed by latent iron deficiency. Iron-stress reactions, reflected by enhanced uptake rate of Fe-59 after short-term supply of ⁵⁹FeEDDHA were observed under these conditions. These reactions, however, were delayed and much less distinct than those observed under latent iron deficiency in water culture experiments. Addition of MgCO₃ to this soil increased the iron deficiency of the plants and caused chlorosis, but prevented these iron-stress reactions. In contrast to the soil experiments, distinct iron-stress reactions could be observed with latent iron-deficiency in sand culture experiments in which varied amounts of inorganic Fe-III were supplied. These reactions were only observed, however, in absence of bicarbonate. Even 4 meq bicarbonate severely inhibited these iron-stress reactions which were almost totally inhibited at 10 meq bicarbonate. Bicarbonate depressed both short-term uptake of Fe-59 as well as total iron content of the leaves. There was no evidence for an additional “inactivation” of iron within the leaves due to bicarbonate treatment. Bicarbonate also strongly depressed the manganese content of the young leaves. The results demonstrate that the regulatory mechanism of so-called “iron-efficient” plant species like sunflower under iron-stress - increase in H⁺ efflux and reducing capacity of the roots and thus enhanced uptake of iron (and manganese) - is severely inhibited or even blocked by high bicarbonate concentrations. “Lime chlorosis”, caused by high bicarbonate concentrations on calcareous substrates is therefore also widespread in so-called “iron-efficient” species.     

Enzymatic synthesis of vanillin.

Due to increasing interest in natural vanillin, two enzymatic routes for the synthesis of vanillin were developed. The flavoprotein vanillyl alcohol oxidase (VAO) acts on a wide range of phenolic compounds and converts both creosol and vanillylamine to vanillin with high yield. The VAO-mediated conversion of creosol proceeds via a two-step process in which the initially formed vanillyl alcohol is further oxidized to vanillin. Catalysis is limited by the formation of an abortive complex between enzyme-bound flavin and creosol. Moreover, in the second step of the process, the conversion of vanillyl alcohol is inhibited by the competitive binding of creosol. The VAO-catalyzed conversion of vanillylamine proceeds efficiently at alkaline pH values. Vanillylamine is initially converted to a vanillylimine intermediate product, which is hydrolyzed nonenzymatically to vanillin. This route to vanillin has biotechnological potential as the widely available principle of red pepper, capsaicin, can be hydrolyzed enzymatically to vanillylamine.     

Structure-function analysis of the vanillin molecule and its antifungal properties

The aim of the present study was to evaluate which structural elements of the vanillin molecule are responsible for its observed antifungal activity. MICs of vanillin, its six direct structural analogues, and several other related compounds were determined in yeast extract peptone dextrose broth against a total of 18 different food spoilage molds and yeasts. Using total mean MICs after 4 days of incubation at 25 degrees C, the antifungal activity order was 3-anisaldehyde (1.97 mM) > benzaldehyde (3.30 mM) > vanillin (5.71 mM) > anisole (6.59 mM) > 4-hydroxybenzaldehyde (9.09 mM) > phenol (10.59 mM) > guaiacol (11.66 mM). No correlation was observed between the relative antifungal activity of the test compounds and log P(o/w). Furthermore, phenol (10.6 mM) was found to exhibit a greater activity than cyclohexanol (25.3 mM), whereas cyclohexanecarboxaldehyde (2.13 mM) was more active than benzaldehyde (3.30 mM). Finally, the antifungal order of isomers of hydroxybenzaldehyde and anisaldehyde was found to be 2- > 3- > 4- and 3- > 2- > 4-, respectively. In conclusion, the aldehyde moeity of vanillin plays a key role in its antifungal activity, but side-group position on the benzene ring also influences this activity. Understanding how the structure of natural compounds relates to their antimicrobial function is fundamentally important and may help facilitate their application as novel food preservatives.     

Phenolic antioxidants and the protection of low density lipoprotein from peroxynitrite-mediated oxidations at physiologic CO2

Dietary phenolic antioxidants have been shown to prevent LDL modifications mediated by several physiologic oxidants including peroxynitrite. However, more recent data demonstrated that CO(2) affected the fate of peroxynitrite in biological fluids and significantly reduced peroxynitrite scavenging by polyphenols, raising doubts concerning their antioxidant activity. We found that the oxidation of LDL lipids mediated by peroxynitrite decreased in the presence of bicarbonate, while Trp oxidation and 3-nitroTyr formation increased, suggesting a redirection of peroxynitrite reactivity toward the protein moiety. We therefore evaluated the protective activity of some phenolic antioxidants (quercetin, oleuropein, resveratrol, (+)-catechin, (-)-epicatechin, tyrosol, alpha- and gamma-tocopherol, ascorbate) on peroxynitrite-mediated oxidation of LDL aromatic residues. Some of these phenols protected LDL Trp from oxidation better than ascorbate or alpha-tocopherol, although protection at 100 microM did not exceed 30-40%. However, the same phenolic antioxidants were more active in inhibiting 3-nitroTyr formation and those with a catechin structure provided significant protection (IC(50%) 40-50 microM). Red wine, a polyphenol-rich beverage, showed a protective effect comparable to that of the most active phenolic antioxidants. Direct EPR studies showed that bicarbonate significantly increased the peroxynitrite-dependent formation of O-semiquinone radicals in red wine, supporting the hypothesis that polyphenols are efficient scavengers of radicals formed by peroxynitrite/CO(2). Ascorbate was a poor inhibitor of peroxynitrite/CO(2)-induced LDL tyrosine nitration, but the simultaneous addition to the most active polyphenols halved their IC(50%). In conclusion, although cooperation with other antioxidants can further decrease the IC(50%) of polyphenolics, as demonstrated for ascorbate, their antioxidant activity appears to occur at concentrations at least 1 order of magnitude higher than their bioavailability.     

Inhibition of herbicide photodegradation by plant products

Pesticide reactivity toward light is rarely considered at the leaf surface after crop treatment; regardless, these degradation reactions directly impact the pesticide effectiveness. The use of sunscreen adjuvants to overcome photodegradation has presented some limitations so far. Raw hydroalcoholic plant extracts have been recently proposed to be used as photoprotecting adjuvants; on a model system they significantly decreased the photodegradation of pesticide. Here it is demonstrated that their use makes possible a dose reduction. Sulcotrione, a selective herbicide for use in maize, was tested in a growth chamber equipped with simulated solar light against a typical weed in maize. Sprayed weeds were monitored by biometrical and physiological parameters. Sulcotrione minimum dose required for a good herbicidal efficacy (ED(50), corresponding to 50% of chlorophyll content decay) was estimated to be 55 g ha(-1). In the presence of grape extract added in a 3-fold excess compared to the herbicide, the ED(50) decreased to 34 g ha(-1). The use of grape extract allows extension of sulcotrione herbicidal activity and reduction of the dose by 35% in controlled conditions. This is a promising result for the effective dose field adjustment.     

Inhibition of acetylcholinesterase activity by bicyclic monoterpenoids

Inhibition of acetylcholinesterase (AChE) activity by 17 kinds of bicyclic monoterpenoids was investigated. Bicyclic monoterpenoids are contained in many kinds of essential oils. Inhibition of AChE was measured according to the colorimetric method. 3.1.1 and 4.1.0 bicyclic hydrocarbons with allylic methyl group showed strong inhibition. (+)- and (-)-alpha-pinene and (+)-3-carene were potent inhibitors of AChE. 3.1.1 and 2.2.1 bicyclic alcohols and ketones showed weak inhibition. 3.1.1 and 4.1.0 bicyclic hydrocarbons with allylic methyl group were found to be uncompetitive inhibitors.     

Inhibition of soil urease by organophosphorus insecticides

The effect of three organophosphorus insecticides on soil urease was examined. Inhibition of urea hydrolysis, some 60 days after application of 1000 parts/10⁶ of insecticide to a sandy clay loam, approached 40% (accothion) and exceeded 50% in the case of malathion and thimet. Similar inhibitory effects were recorded using a silt loam soil with which 200 parts/10⁶ application also produced inhibition ranging from 14% (accothion) to 23% (thimet) after 10 days. With lower concentrations of insecticide (50 parts/10⁶) inhibition, though again significant, was of a more ephemeral nature.All three insecticides, at a concentration of 1000 parts/10⁺⁶, prevented almost any hydrolysis of urea by jack bean urease. Ureolytic microorganisms, isolated from the soils under investigation, were inhibited by the organophosphates to a greater or lesser extent but the development of tolerance was common.It is suggested that the application of insecticides to control soil-borne insect pests may be a factor in determining the efficiency of urea fertilizer mineralization.     

Inhibition of malate dehydrogenase by humic acids

Malate dehydrogenase (from pig heart mitochondria) is markedly inhibited by a number of humic acids extracted from different types of soil or prepared synthetically. The inhibitors were found to be competitive for both substrates of the forward reaction, whereas inhibition is of the mixed type with NADH in the reverse reaction. In both directions, inhibition is strictly dependent on the number of phenolic hydroxyl groups on the humic acids. The affinity of the inhibitors for the enzyme is increased in such a way that it is readily reversible in the forward reaction but becomes more irreversible in the opposite direction.Studies on complex formation between humic acids and NAD⁺ led to the conclusion that different effects on both reactions are due to the formation of such a complex. Enzyme activity could be recovered in the presence of calcium ions. This is assumed to be due to changes in the electron structure of the humic acid particles.     

Authentication of the origin of vanillin using quantitative natural abundance 13C NMR

The use of 13C isotopic distribution as an efficient means to determine the origin of vanillin has been substantiated. Using quantitative 13C NMR, the 13C/12C ratios at all eight carbon positions can be exploited. On a set of 21 samples of vanillin from five different origins, complete discrimination can be achieved. It is shown that, for many purposes, a rapid analysis in which only five sites are used is sufficient. However, improved discrimination using all eight sites is preferable to differentiate between different methods of production from natural ferulic acid or between natural and lignin-derived vanillin on the basis of the 13C/12C ratios characteristic of different origins. The C1 and C8 positions are demonstrated to be the most significant sites for discrimination using principle component analysis. However, aromatic carbon positions make an essential contribution, notably in differentiating between natural and lignin-derived vanillin.     

Kinetics of interaction of vanillin with amino acids and peptides in model systems

Model systems were used to study the reaction kinetics of vanillin and pentalysine, lysine, glutathione, cysteine, aspartame, or phenylalanine (molar ratio 1:1) in phosphate buffer. The buffer pH was adjusted to the pK(a)(2) of the available alpha-amino group of each amino acid or peptide. Reductions of vanillin followed first-order kinetics at 55, 65, and 75 degrees C in the presence of each of the amino acids or peptides used. The reaction rates were accelerated as the temperature increased. The rate constants were highest for pentalysine followed by lysine, phenylalanine, glutathione/cysteine, and aspartame. The reduction of phenylalanine followed first-order kinetics, whereas the formation of its reaction product followed zero-order kinetics. The activation energy (E(a)) for the reaction ranged from 5.6 to 14.5 kcal/mol.     

Inhibition of nitrification in soil by gaseous hydrocarbons

Summary Recent work has shown that gaseous hydrocarbons such as methane, ethane, and ethylene are competitive inhibitors of the monooxygenase enzyme responsible for oxidation of ammonia by chemoautotrophic nitrifying microorganisms such as Nitrosomonas europaea. Because methane, ethane, and ethylene are produced by microbial activity in soil, we studied the possibility that they may inhibit oxidation of ammonia by the nitrifying soil microorganisms. We found that all three of these gaseous hydrocarbons inhibited nitrification in soil and that their ability to inhibit nitrification decreased in the order: ethylene > ethane > methane. Ethylene was much more effective than ethane or methane for inhibiting nitrification of ammonium in soil, but it was much less effective than acetylene, and it seems unlikely that the amounts of ethylene produced in soils will be sufficient to cause significant inhibition of nitrification by soil microorganisms.     

Inhibition of trypsin by condensed tannins and wine

Phenolic compounds are abundant vegetable secondary metabolites in the human diet. The ability of procyanidin oligomers and wine polyphenols to inhibit trypsin activity was studied using a versatile and reliable in vitro method. The hydrolysis of the chromogenic substrate N-benzoyl-d,l-arginine-p-nitroanilide (BApNA) by trypsin was followed by spectrophotometry in the presence and absence of condensed tannins and wine. A clear relationship between the degree of polymerization of procyanidins and enzymatic inhibition was observed. Trypsin activity inhibition was also detected in several types of wine. In general, the inhibition increased with the concentration of phenolic compounds in wines. These results may be relevant when considering these compounds as antinutritional factors, thereby contributing to a reduced absorption of nutrients.     

Inhibition of low-density lipoprotein oxidation by carnosine histidine

Carnosine is a beta-alanylhistidine dipeptide found in skeletal muscle and nervous tissue that has been reported to possess antioxidant activity. Carnosine is a potential dietary antioxidant because it is absorbed into plasma intact. This research investigated the ability of carnosine to inhibit the oxidation of low-density lipoprotein (LDL) in comparison to its constituent amino acid, histidine. Carnosine (3 microM) inhibited Cu2+-promoted LDL (20 of protein/mL) oxidation at carnosine/copper ratios as low as 1:1, as determined by loss of tryptophan fluorescence and formation of conjugated dienes. Carnosine (6 microM) lost its ability to inhibit conjugated diene formation and tryptophan oxidation after 2 and 4 h of incubation, respectively, of LDL with 3 microM Cu2+. Compared to controls, histidine (3 microM) inhibited tryptophan oxidation and conjugated diene formation 36 and 58%, respectively, compared to 21 and 0% for carnosine (3 microM) after 3 h of oxidation. Histidine was more effective at inhibiting copper-promoted formation of carbonyls on bovine serum albumin than carnosine, but carnosine was more effective at inhibiting copper-induced ascorbic acid oxidation than histidine. Neither carnosine nor histidine was a strong inhibitor of 2,2'-azobis(2-amidinopropane) dihydrochloride-promoted oxidation of LDL, indicating that their main antioxidant mechanism is through copper chelation.     

Inhibition of polyphenol oxidases activity by various dipeptides

In an effort to develop natural and nontoxic inhibitors on the activity of mushroom polyphenol oxidase (PPO) the effect of various glycyl-dipeptides (GlyAsp, GlyGly, GlyHis, GlyLeu, GlyLys, GlyPhe, GlyPro, GlyTyr) was investigated. The inhibition study with dihydroxyphenylalanine (DOPA) as substrate is based on separation of the enzymatic reaction components by reversed phase HPLC and the UV detection of the dopachrome formed. The results have evidenced that several of tested dipeptides inhibited PPO activity in the range of 20-40% while GlyPro and GlyLeu had no effect. The study has also permitted the characterization of the following kinetic pattern: a linear-mixed-type mechanism for GlyAsp, GlyGly, GlyLys, and GlyPhe and a hyperbolic-mixed-type for GlyTyr. It was not possible to identify the inhibition mechanism for GlyHis, although it affects PPO activity. In addition the effects of GlyAsp, GlyLys and GlyHis were evaluated for lessening the browning of fresh Golden Delicious apple and Irish White Skinned potato. The effectiveness of such inhibitors was determined by the difference between the colors observed in the dipeptide-treated sample and the controls using the color space CIE-Lab system. The % browning inhibition on potato (20-50%) was greater than of apple (20-30%) by the all tested dipeptides. Only GlyLys presented the significant value of 50%.     

Enzymatic extraction and transformation of glucovanillin to vanillin from vanilla green pods.

Glucovanillin was extracted from green pods and simultaneously transformed to vanillin by a combination of enzyme activities involving cell wall degradation and glucovanillin hydrolysis. The reaction is best carried out with 47.5% v/v aqueous ethanol solution during 8 h at 70 degrees C, in a two-step enzymatic reaction using Viscozyme followed by Celluclast, two commercial enzymatic products containing mainly pectinase and cellulase activities, respectively. The extractive reaction proceeded with high efficiency with an amount of extracted vanillin 3.13 times higher than the one obtained with the Soxhlet method. The classical curing/extraction process results in 1.1-1.8 g of vanillin/100 g of dry pods. It is concluded that the enzymatic reaction may substitute the microbial process involved in tissue fermentation previous to vanillin extraction with the simultaneous hydrolysis of glucovanillin.     

Soil invertebrates and the degradation of vanillin,cinnamic acid,and lignins

A range of 8–25% of fed cinnamic 2-[¹⁴C] acid and 9–14% of injected vanillin 5-[¹⁴C] were oxidised to ¹⁴CO₂ at 15 C over 7 and 6 days respectively in an isopod, Oiuscus asellus; a millipede, Pseudopolydesmus serratus; a slug, Deroceras reticulatum; a snail, Oxychilus draparnaldi; and an earthworm, Eisenia foetida. Approximately 2–10% of nonmetabolised and 13–48%, of metabolised vanillin were present in the animal tissues after 6 days. Correspondingly. 1–4% and 22–66% of these materials were found in egesta.A millipede (Oxidus gracilis), O. asellus. D. reticuluttum. O. draparnaldi, and E. foetida were found unable to degrade ring [¹⁴C]-, methoxy [¹⁴C]- and side chain [¹⁴C]-lignin to ¹⁴CO₂ over 10 days, thus providing very strong evidence that these invertebrates are incapable of degrading liginin.     

Authentication of natural vanilla flavorings: isotopic characterization using degradation of vanillin into guaiacol

The isotopic investigation of vanillin has been extended to the new sources of natural precursors of vanillin recently introduced with a view of obtaining natural vanillin by biotechnological processes. To check the consistency of the isotopic composition of vanillin with that of the corresponding aromatic fragment, a selective degradation reaction into guaiacol was carried out. The reaction was shown to proceed without significant isotopic fractionation at the sites of interest, and an optimized procedure was defined from the results of an experimental design involving the quantity of reagent and the temperature and duration of the experiment. Guaiacol, which can be easily obtained in a reasonable time, is an interesting isotopic probe for carbon- and oxygen-isotope ratio mass spectrometry (IRMS) determinations. It provides (13)C information specific to the aromatic fragment and, combined with delta(13)C values measured on vanillin itself, it improves the authentication potential of carbon-IRMS. Thus, the natural status of ferulic acid may be characterized by significant (13)C depletion at the formyl site. Similarly, the oxygen-18 content of guaiacol is a better authentication tool than delta(18)O of vanillin because it does not suffer the drawback of being altered by chemical exchange of the sp(2) oxygen atom with water in industrial or laboratory procedures. Although collaborative studies are still necessary to improve the interlaboratory reproducibility of the delta(18)O parameters, consistent results can be obtained in an intralaboratory context. It is shown in particular that chemical oxidation of ferulic acid is characterized by a relative enrichment of the aromatic moiety of vanillin.     

Molecularly imprinted photonic hydrogels as colorimetric sensors for rapid and label-free detection of vanillin

A novel colorimetric sensor for the rapid and label-free detection of vanillin, based on the combination of photonic crystal and molecular imprinting technique, was developed. The sensing platform of molecularly imprinted photonic hydrogel (MIPH) was prepared by a noncovalent and self-assembly approach using vanillin as a template molecule. Morphology characterization by scanning electron microscope (SEM) showed that the MIPH possessed a highly ordered three-dimensional (3D) macroporous structure with nanocavities. The vanillin recognition events of the created nonocavities could be directly transferred into readable optical signals through a change in Bragg diffraction of the ordered macropores array of MIPH. The Bragg diffraction peak shifted from 451 to 486 nm when the concentration of the vanillin was increased from 10?12 to 10?3 mol L?1 within 60 s, whereas there were no obvious peak shifts for methyl and ethyl vanillin, indicating that the MIPH had high selectivity and rapid response for vanillin. The adsorption results showed that the hierarchical porous structure and homogeneous layers were formed in the MIPH with higher adsorption capacity. The application of such a label-free sensor with high selectivity, high sensitivity, high stability, and easy operation might offer a potential method for rapid real-time detection of trace vanillin.     

Inhibition of nitrification in soils by volatile sulfur compounds

Carbon disulfide, dimethyl disulfide, methyl mercaptan, dimethyl sulfide, and hydrogen sulfide retard nitrification of ammonium in soils incubated in closed systems. The inhibitory effects of these volatile sulfur compounds on nitrification decrease in the order listed. Hydrogen sulfide is a relatively weak inhibitor of nitrification, but carbon disulfide is considerably more effective than patented nitrification inhibitors (N-Serve. AM, and ST) for inhibition of nitrification in closed systems.It is concluded from the work reported that the inhibitory effects of methionine, cystine, cysteine, and other nonvolatile organic sulfur compounds on nitrification in soils may be at least partly due to decomposition of these compounds by soil microorganisms with formation of volatile sulfur compounds that retard nitrification.