Formation of a seed germination promoter from carbohydrates and amino acids

The ability of plant-derived smoke to act as a germination cue in many species has led to widespread interest in this aspect of seed biology. Recently, 3-methyl-2H-furo[2,3-c]pyran-2-one was identified as the main germination cue from smoke. Here, we report on the formation of this compound from reactions of sugars with amino acids. Heating proteins or amino acids with sugars at 180 degrees C for 30 min produces water soluble extracts that promote germination. High-performance liquid chromatography indicated that the active compound(s) derived from these reactions coeluted with the active fraction from a smoke solution. Gas chromatography-mass spectroscopy showed that the active constituent is identical to the germination cue from plant-derived smoke. The results presented in this paper provide evidence for the formation of the major germination cue found in smoke from ubiquitously occurring organic compounds.     

Vicinal diketone formation in yogurt: (13)C precursors and effect of branched-chain amino acids

Addition of branched-chain amino acids (BCAA) or an inhibitor of the BCAA biochemical pathways during fermentation of milk with a lac(-) mutant of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus strongly influenced the formation of two aroma-impact compounds, 2,3-butanedione and 2,3-pentanedione, as well as their direct precursors 2-acetolactate and 2-acetohydroxybutyrate. This suggests a connection between vicinal diketone formation and BCAA biosynthesis in yogurt bacteria. A recently developed static-and-trapped headspace technique combined with gas chromatography-mass spectrometry demonstrated incorporation of (13)C from [U-(13)C(6)]-D-glucose and [U-(13)C(4)]-L-threonine into both vicinal diketones. For 2,3-butanedione, glucose is the major precursor via pyruvate and activated acetaldehyde. For 2, 3-pentanedione, L-threonine is a precursor via 2-ketobutyrate, but glucose is the major contributor via activated acetaldehyde and, possibly, also via 2-ketobutyrate, which is a degradation product of 3-methylaspartate, an intermediate in glutamate synthesis.     

Improved RP‐HPLC and anion‐exchange chromatography methods for the determination of amino acids and carbohydrates in soil solutions

In spite of their low concentrations in soil solutions, low–molecular weight organic substances (LMWOS) such as amino acids, sugars, and uronic acids play a major role in the cycles of C and N in soil. With respect to their low concentrations and to possible matrix interferences, their analysis in soil leachates is a challenging task. We established two HPLC (high‐performance liquid chromatography) methods for the parallel determination of amino acids and carbohydrates in soil leachates. The pre‐column derivatization of amino acids with an o‐phthaldialdehyde (OPA) mercaptoethanol solution yields quantitation limits between 0.03 and 0.44 µmol L^–1 and S_D values of <8.3% (n = 9). High‐performance anion‐exchange chromatography (HPAEC) on a Dionex CarboPac PA 20 column with a NaOH acetate gradient combined with pulsed amperometric detection (PAD) was used for the determination of carbohydrates. The calibration curves obtained for 11 carbohydrates showed excellent linearity over the concentration range from 0.02 to 50.0 mg L^–1. Recovery studies revealed good results for all analytes (89%–108%). Interferences from Hg(II) salts and chloroform used for stabilization of the leachates did not occur with both chromatographic methods. The optimized method was successfully used for quantitative determinations of amino acids and carbohydrates in soil leachates.     

Thermal generation of 3-amino-4,5-dimethylfuran-2(5H)-one, the postulated precursor of sotolone, from amino acid model systems containing glyoxylic and pyruvic acids

4,5-Dimethyl-3-hydroxy-2(5H)-furanone (sotolone), a naturally occurring flavor impact compound, can be isolated from various sources, especially fenugreek seeds. It can also be thermally produced from intermediates generated from the Maillard reaction such as pyruvic and ketoglutaric acids, glyoxal, and 2,3-butanedione. A naturally occurring precursor of sotolone, 3-amino-4,5-dimethyl-2(5H)-furanone, was thermally generated for the first time from pyruvic acid and glycine or from glyoxylic acid and alanine model systems. Isotope labeling studies have implicated 4,5-dimethylfuran-2,3-dione as an intermediate that can be converted into 3-amino-4,5-dimethyl-2(5H)-furanone through Strecker-like interaction with any amino acid. Furthermore, these studies have also indicated the presence of two pathways for the formation of 4,5-dimethylfuran-2,3-dione, one requiring pyruvic acid and a formaldehyde source and the other requiring glyoxylic acid and acetaldehyde. Self-aldol condensation of pyruvic acid followed by lactonization and further aldol reaction with formaldehyde can generate the same intermediate as the self-aldol addition product of acetaldehyde with glyoxylic acid followed by lactonization. The pyruvic acid pathway was found to be a more efficient route than the glyoxylic acid pathway. Furthermore, the pyruvic acid/glycine model system was able to generate sotolone in the presence of moisture, and in the presence of ammonia, commercial sotolone was converted back into 3-amino-4,5-dimethyl-2(5H)-furanone.     

Supercritical fluid extraction of bioavailable amino acids from soils and their liquid chromatographic determination with fluorometric detection

A new procedure with supercritical CO2 modified with 0.5 mL of water and 0.75 mL of 0.1 M HCl in situ and 0.75 mL of water on-line at 15 MPa and 50 degrees C for 45 min was applied for the extraction of bioavailable amino acids from soil samples. Total extraction time was 60 min, but more favorable conditions are even possible for selected groups of amino acids. All analytes were trapped into 20 mL of methanol with satisfactory recovery (94-104%) and determined using high-performance liquid chromatography with fluorometric detection on a Zorbax Eclipse column (4.6 x 75 mm, 3.5 microm) with Na2HPO4 and acetonitrile/methanol/water as a mobile phase. Linear calibration curves were obtained (r > 0.999 except 0.99823 for Ile) with lower limits of detection (S/N = 3) in the range from 1.54 pg (Gly) to 13.5 pg (Cy2) or from 18.6 fmol (Ser) to 64.8 fmol (Lys). Validation and repeatability data are also given. Comparable results were obtained with a robust, commonly used extraction method (0.5 M ammonium acetate, 60 min in shaker, followed by filtration and lyophilization). Limiting values of artificial release of amino acids were also determined for each soil sample to eliminate any false results to ensure that all extracted amino acids originate from soil solution and exchangeable bound positions of soil samples.     

Isolation and structure of pulcherrimine, a novel bitter-tasting amino acid, from the sea urchin (Hemicentrotus pulcherrimus) ovaries

A novel sulfur-containing amino acid, pulcherrimine, has been isolated as a bitter principle from ovaries of the sea urchin Hemicentrotus pulcherrimus. The structure was elucidated as 4-(2'-carboxy-2'-hydroxy-ethylthio)-2-piperidinecarboxylic acid by spectroscopic and chemical methods. Absolute stereochemistry was determined by NOE experiments and chiral HPLC analysis. Pulcherrimine exhibited bitterness with a threshold value of 0.306 mM.     

Synthesis and herbicidal activities of novel 3-N-substituted amino-6-methyl-4-(3-trifluoromethylphenyl)pyridazine derivatives

4-(3-Trifluoromethylphenyl)pyridazine is a new series of compounds with bleaching and herbicidal activities. Starting from ethyl 2-(3-trifluoromethylphenyl)acetate, an important intermediate 7 was synthesized in five steps with a moderate total yield of 51.5% in a safe and practical way. Twenty-six novel 3-N-substituted amino-6-methyl-4-(3-trifluoromethylphenyl)pyridazine derivatives were synthesized and evaluated through a Spirodela polyrrhiza test and greenhouse test. Some compounds can completely inhibit Chl at 1 microg/mL and exhibit equal or higher herbicidal activities with the commercial bleaching herbicide diflufenican against dicotyledonous plants at a rate of 75 g/ha.     

Gas chromatography-mass spectrometry method for the determination of free amino acids as their dimethyl-tert-butylsilyl (TBDMS) derivatives in animal source food

The suitability of a one-step derivatization procedure using N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide for the simultaneous assay of 22 free amino acids and its application for their analysis in six animal source foods (pork, dry cured ham, chicken stock, fresh cheese, ripened cheese, and dry salted sardine) by GC-MS were studied. All 22 free amino acid derivatives were correctly detected and resolved. Reproducibility (%RSD) of the method was in the range of 1.9-12.2%. Detection and quantitation limits of the analytical procedure ranged from 0.01 to 0.46 mg/100 g dry weight and from 0.02 to 1.55 mg/100 g dry weight, respectively. The calibration curves were linear within the range 0.1-15.0 mg/100 g with correlation coefficient values (R(2)) from 0.9891 to 0.9983. All analyzed food products showed free amino acid contents similar to those found in the scientific literature. The proposed GC-MS method for the determination of free amino acids in animal source food can be used in routine for both analytical and research purposes.     

Inhibitory effect(s) of polymeric black tea polyphenol fractions on the formation of [(3)H]-B(a)P-derived DNA adducts

Five polymeric black tea polyphenol fractions (PBP-1-5) were isolated from a popular brand of black tea. The effect of these PBPs and epigallocatechin gallate (EGCG), a major green tea polyphenol, was studied on the formation of [(3)H]-B(a)P-derived DNA adducts in vitro, employing rat liver microsomes. PBP-1-3 inhibited microsome-catalyzed [(3)H]-B(a)P-derived DNA adduct formation in vitro in a dose-dependent manner. This inhibition was further enhanced on preincubation of microsomes with each of the PBPs. PBP-4 was not effective per se and required preincubation with microsomes to exhibit its inhibitory effect, whereas PBP-5 remained ineffective with or without preincubation with microsomes. Further investigations revealed that the observed decrease in [(3)H]-B(a)P-DNA adduct formation was due to inhibition of isozymes of CYP450s by PBPs. Overall, results suggest that polymeric black tea polyphenol fractions retain one of the chemopreventive effects exhibited by the monomeric green tea polyphenol EGCG in vitro.     

3-Hydroxy-4,5-dimethyl-2(5H)-furanone (Sotolon) causing an off-flavor: elucidation of its formation pathways during storage of citrus soft drinks.

Gas chromatography/olfactometry (GCO) and gas chromatography-mass spectrometry (HRGC-MS) revealed 3-hydroxy-4, 5-dimethyl-2(5H)-furanone (sotolon) to be responsible for the "burnt" and "spicy" off-flavor observed in citrus soft drinks during storage. Among the ingredients of citrus soft drinks, ethanol and ascorbic acid were identified as the essential precursors of sotolon. Two formation pathways were postulated by studies using (2)H (D)- and (13)C-labeled ethanol and ascorbic acid; i.e., sotolon is formed from two molecules of ethanol and carbons 2 and 3 of ascorbic acid (pathway 1), or it is generated from one molecule of ethanol and carbons 3-6 of ascorbic acid (pathway 2).     

Inhibition of lactoperoxidase-catalyzed 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) and tyrosine oxidation by tyrosine-containing random amino acid copolymers

Oxidation of 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) by lactoperoxidase was found to be inhibited by tyrosine-containing random amino acid copolymers but not by tyrosine. Both electrostatic effects and polymer size were found to be important by comparison of negatively and positively charged copolymers of varying lengths, with poly(Glu, Tyr)4:1 ([E 4Y 1] approximately 40) as the strongest competitive inhibitor (EC 50 approximately 20 nM). This polymer did not form dityrosine in the presence of lactoperoxidase (LPO) and peroxide. Furthermore, incubation with tert-butyl hydroperoxide, as opposed to hydrogen peroxide, resulted in a peculiar long lag phase of the reaction between the redox intermediate compound II and [E 4Y 1] approximately 40, indicating a very tight association between enzyme and inhibitor. We propose that interactions between multiple positively charged areas on the surface of LPO and the polymer are required for optimal inhibition.     

Enzymatic synthesis of [3'-O-methyl-(3)H]malvidin-3-glucoside from petunidin-3-glucoside

Malvidin-3-glucoside has been labeled by enzymatic synthesis in a single-step experiment. Catechol-O-methyl transferase catalyzed the B-ring methylation of petunidin-3-glucoside, and S-Adenosyl-L-[methyl-(3)H] methionine was the methyl donor. Solid phase extraction and preparative high-performance liquid chromatography were necessary to separate [3'-O-methyl-(3)H]malvidin-3-glucoside from an isomer and the starting material. The specific activity was 2.2 Ci mmol(-1), and the yield of incorporation was 1.1%. A possible application of the labeled material is the study of anthocyanin reactions in complex mixtures such as red wine where products are difficult to isolate and analyze.     

Structure determination of partially deuterated carotenoids from intrinsically labeled vegetables by HPLC-MS and 1H NMR

The structures of biosynthetic deuterated carotenoids in labeled vegetables were investigated: (all-E)-lutein and (all-E)-beta-carotene from spinach, and (all-E)-beta-carotene and (all-E)-alpha-carotene from carrots. The vegetables were grown hydroponically using a nutrient solution enriched with deuterium oxide (D(2)O) and were extracted using matrix solid-phase dispersion (MSPD). Deuterium enrichment in the carotenoid molecules was determined by liquid chromatography-mass spectrometry (LC-MS). (all-E)-Lutein and (all-E)-beta-carotene in spinach showed partial deuteration from (2)H(1) to (2)H(12), with the abundance maximum at (2)H(5). (all-E)-beta-Carotene and (all-E)-alpha-carotene from carrots showed partial deuteration from (2)H(1) to (2)H(17), with the abundance maximum at (2)H(11). The (1)H NMR spectra of the four deuterated carotenoids showed additional signals for all methyl groups and decreased signal intensity for the olefinic protons and the methylene protons in the ring. These differences are due to isotopic effects and are based on the substitution of protons by deuterium atoms. The deuteration was distributed randomly throughout the carotenoid molecules.     

Isolation, structure determination, synthesis, and sensory activity of N-phenylpropenoyl-L-amino acids from cocoa (Theobroma cacao)

Application of chromatographic separation and taste dilution analyses recently revealed besides procyanidins a series of N-phenylpropenoyl amino acids as the key contributors to the astringent taste of nonfermented cocoa beans as well as roasted cocoa nibs. Because these amides have as yet not been reported as key taste compounds, this paper presents the isolation, structure determination, and sensory activity of these amino acid amides. Besides the previously reported (-)-N-[3',4'-dihydroxy-(E)-cinnamoyl]-3-hydroxy-L-tyrosine (clovamide), (-)-N-[4'-hydroxy-(E)-cinnamoyl]-L-tyrosine (deoxyclovamide), and (-)-N-[3',4'-dihydroxy-(E)-cinnamoyl]-L-tyrosine, seven additional amides, namely, (+)-N-[3',4'-dihydroxy-(E)-cinnamoyl]-L-aspartic acid, (+)-N-[4'-hydroxy-(E)-cinnamoyl]-L-aspartic acid, (-)-N-[3',4'-dihydroxy-(E)-cinnamoyl]-L-glutamic acid, (-)-N-[4'-hydroxy-(E)-cinnamoyl]-L-glutamic acid, (-)-N-[4'-hydroxy-(E)-cinnamoyl]-3-hydroxy-L-tyrosine, (+)-N-[4'-hydroxy-3'-methoxy-(E)-cinnamoyl]-L-aspartic acid, and (+)-N-[(E)-cinnamoyl]-L-aspartic acid, were identified for the first time in cocoa products by means of LC-MS/MS, 1D/2D-NMR, UV-vis, CD spectroscopy, and polarimetry, as well as independent enantiopure synthesis. Using the recently developed half-tongue test, human recognition thresholds for the astringent and mouth-drying oral sensation were determined to be between 26 and 220 micromol/L (water) depending on the amino acid moiety. In addition, exposure to light rapidly converted these [E]-configured N-phenylpropenoyl amino acids into the corresponding [Z]-isomers, thus indicating that analysis of these compounds in food and plant materials needs to be performed very carefully in the absence of light to prevent artifact formation.     

Azaphilones, furanoisophthalides, and amino acids from the extracts of Monascus pilosus-fermented rice (red-mold rice) and their chemopreventive effects

Six azaphilones, monascin (1), ankaflavin (2), rubropunctatin (3), monascorburin (4), rubropunctamine (5), and monascorburamine (6), two furanoisophthalides, xanthomonasin A (7) and xanthomonasin B (8), and two amino acids, (+)-monascumic acid (9) and (-)-monascumic acid (10), isolated from the extracts of Monascus pilosus-fermented rice (red-mold rice) were evaluated for their inhibitory effects on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation in mice, on the induction of Epstein-Barr virus early antigen (EBV-EA) by TPA in Raji cells, and on the activation of (+/-)-(E)-methyl-2[(E)-hydroxy-imino]-5-nitro-6-methoxy-3-hexemide (NOR 1), a nitric oxide (NO) donor. Among the compounds tested, seven compounds (1-6 and 10) on TPA-induced inflammation, and six compounds (1, 3-5, 9, and 10) on EBV-EA activation, exhibited potent inhibitory effects. All of the compounds tested showed moderate inhibitory effects on NOR 1 activation.     

Degradation studies on benzoxazinoids. Soil degradation dynamics of (2R)-2-O-beta-D-glucopyranosyl-4-hydroxy-(2H)- 1,4-benzoxazin-3(4H)-one (DIBOA-Glc) and its degradation products, phytotoxic allelochemicals from Gramineae

Wheat (Triticum aestivum L.) has been found to possess allelopathic potential and studies have been conduced to apply wheat allelopathy for biological weed control. 2,4-Dihydroxy-(2H)-1,4-benzoxazin-3(4H)-one (DIBOA) is a common product found in wheat, corn, and rye exudates and it can be released to the environment by that way. In this report, the stability of DIBOA is studied in two soils from crop lands of wheat cv. Astron and cv. Ritmo. These varieties were selected by their concentrations of DIBOA and 2,4-dihydroxy-7-methoxy-(2H)-1,4-benzoxazin-3(4H)-one (DIMBOA) from aerial parts and by the bioactivities of their aqueous extracts in the growth of wheat coleoptile sections. The degradation rate of DIBOA in these soils was measured in laboratory tests during 90 h by high-pressure liquid chromatography methods. These analyses demonstrate that DIBOA was transformed primarily into 2-benzoxazolinone (BOA). This transformation was similar in both soil types with an average half-life of 43 h. The degradation studies for BOA show its biotransformation to 2-aminophenoxazin-3-one (APO) with a half-life of 2.5 days. Therefore, BOA is an intermediate product in the biotransformation from DIBOA to APO in these wheat crop soils and is consistent with previous findings. APO was not degraded after three months in soil, suggesting that its degradation rate in soil is very slow.