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A microwave-assisted extraction technique was developed to optimize the extraction of phenolic compounds from grape seeds. The microwave power (300-150W) and time of extraction (20-200s) were varied during the optimization process. The polyphenol content of the resulting extracts were measured as mg of tannic acid equivalent per gram of crude extract (mg TAE/g of crude extract), using a Folin-Ciocalteau reagent. In general, neither the time nor the power had a significant effect on the overall % yield (average of 13.5%) and on the polyphenol content (392 mg TAE/g of crude extract) of the extracts. However, when the solvent polarity was changed by the addition of 10% water, the yield increased to 15.2% and the polyphenol content increased to 429 mg TAE/g of crude extract.

A Microwave-Assisted Process (MAP) solvent extraction procedure was used in conjunction with GC/MS analysis to investigate the chemical composition of dried silkworm, dried earthworm, silkworm droppings, and cicada nymph skin. Selected insect samples were also analyzed by pyrolysis-GC-MS. Silkworm and earthworm generated fatty acids, sterols and phenol derivatives. Cicada nymph skin was analyzed by pyrolysis/GC/MS and generated mainly heterocyclic compounds. The data indicated that insects can retain environmental contaminants such as 2,6-bis(1,1-dimethylethyl)-4-methyl phenol (BHT) and as such could be used as biological indicators. In addition, some of the therapeutic characteristics associated with insects could be attributed to the chemicals ingested from plant sources.

The influence of the reaction phase on the mechanism of formation of Maillard products was studied by comparison of (13)C-label incorporation patterns of the common products formed in model systems consisting of labeled glycine and D-glucoses subjected to both pyrolysis and heating in aqueous solutions. Pyrolysis experiments were performed at 250 degrees C for 20 s, and aqueous model systems were heated in sealed vials for 3 h at 120 degrees C followed by GC/MS analysis. Label incorporation patterns of the following compounds were analyzed: cyclotene, furanmethanol, acetylpyrrole, 5-methyl-pyrrole, trimethylpyrazine, acetic acid, 3-hydroxy-2-butanone, 2,3-butanedione, and 2-methyl-4, 5-dihydro-3(2H)-furanone. Although pyrolysis reaction produced higher number of products, however, the major pathways of formation of variety of important Maillard products followed the same mechanism under both pyrolytic and aqueous systems. Furthermore, contrary to literature speculations, 2-methyl-4, 5-dihydro-3(2H)-furanone was shown to be formed by ring contraction of 2,3-dihydro-3,5-dihydroxy-6-methyl-4(H)-pyran-4-one, through benzilic acid rearrangement, followed by decarboxylation.

Electrospray ionization mass spectrometry (ESI-MS) was used to study the glycation of lysozyme by D-glucose (LZM-G) and by D-fructose (LZM-F) under dry heating conditions in the presence and in the absence of oxygen. ESI-MS proved to be a precise method for monitoring protein glycation with respect to following the extent of glycation and changes in the glycoconjugate profile with time. The ESI-MS spectrum of glycated LZM revealed a heterogeneous distribution of glycoforms of LZM at different reaction stages. D-Glucose showed a higher level of reactivity with the amino groups of LZM than D-fructose, both in the presence and in the absence of oxygen. The presence of oxygen in the reaction system induced oxidative side reactions, which competed with and slowed the initial rate of formation of Amadori or Heyns products. The more reactive glycoxidation products formed during the initial stages of incubation in the presence of oxygen accelerated the rate of glycation during the later stages of incubation and increased the involvement of arginine residues of LZM in the glycation reaction. The interaction between the initial glycoxidation product(s) of the reducing sugars and intact lysozyme during the later stages of incubation was observed by the appearance of a different cluster of glycoconjugates in the mass spectrum during the latter stages of incubation. The molecular weight differences between the molecular ions of the new cluster of glycoconjugates are consistent with the formation of D-glucosone from the autoxidation of D-glucose or from the oxidative cleavage of the glucose-lysozyme imine adduct in the lysozyme-glucose system. The effect of oxygen-induced glycoxidation on the glycation reaction was also more pronounced in the LZM-G system compared with that in the LZM-F system.

Maillard model systems consisting of labeled D-[(13)C]glucoses and L-[(13)C]alanines have been utilized to identify the origin of carbon atoms in glycolaldehyde, pyruvaldehyde, 1-hydroxy-2-propanone (acetol), 2,3-butanedione, 3-hydroxy-2-butanone, 2,3-pentanedione, and compounds containing C(5) and C(6) intact glucose carbon chains. The origin of carbon atoms in glycolaldehyde and pyruvaldehyde was inferred from the analysis of label incorporation pattern of methyl and dimethylpyrazines. The origin of carbon atoms in the remaining compounds was determined by direct analysis. The data indicated that glycolaldehyde incorporated intact C5-C6 and C1-C2 carbon chains of glucose. Acetol and pyruvaldehyde incorporated intact C1-C2-C3 and C4-C5-C6 carbon chains of glucose. On the other hand, 2, 3-butanedione and 3-hydroxy-2-butanone incorporated intact C3-C4-C5-C6 carbon chain of glucose. In addition, analysis of compounds containing intact glucose C(5) carbon chains have indicated that glucose in the presence of L-alanine can lose either C-1 atom to produce a pentitol moiety responsible for the formation of furanmethanol or it can lose the C-6 atom to produce a pentose moiety responsible for the formation of furfural. Plausible mechanisms, consistent with the observed label incorporation, were proposed for the formation of sugar degradation products.

Analysis of the pyrolysis products of [1-(13)C], [2-(13)C], and [3-(13)C]-labeled L-serines has indicated the presence of three initial degradation pathways. Decarboxylation followed by deamination produces aminoethanol and acetaldehyde, respectively; a retro-aldol pathway generates formaldehyde and glycine. Dehydration of L-serine can lead to the formation of pyruvic acid, which eventually can be converted into the amino acid alanine. Formation of alanine and glycine was confirmed due to the detection of 2, 5-diketo-3,6-dimethylpiperazine and cycloglycylalanine. Most of the advanced decomposition products of L-serine can be rationalized on the basis of these initial degradation products. Label incorporation studies have elucidated the origin of carbonyl precursors of methyl- and 2,3-dimethylpyrazines formed in the thermal decomposition mixture of L-serine. Three mechanistic pathways were identified for the formation of carbonyl precursors of methyl- and 2, 3-dimethylpyrazines. The major pathway (70%) for the formation of the precursor of methylpyrazine involved aldol addition of formaldehyde to glycolaldehyde to form glyceraldehyde. On the other hand, the major pathway (60%) for the formation of the precursor of 2,3-dimethylpyrazine involved an aldol condensation of acetaldehyde with glycolaldehyde to form 2,3-butanedione.

A colorimetric method was developed for the determination of total azadirachtin-related limonoids (AZRL) in neem seed kernel extracts. The method employed acidified vanillin solution in methanol for the colorization of the standard azadirachtin or neem seed kernel extracts in dichloromethane. Through the investigation of various factors influencing the sensitivity of detection, such as the concentration of vanillin, acid, and the time required for the formation of color, optimum conditions were selected to perform the assay. Under the optimum conditions, a good linearity was found between the absorbance at 577 nm and the concentration of standard azadirachtin solution in the range of 0.01-0.10 mg/mL. In addition, different extraction procedures were evaluated using the vanillin assay. The HPLC analysis of the extracts indicated that if the extractions were performed in methanol followed by partitioning in dichloromethane, approximately 50% of the value determined by the vanillin assay represents azadirachtin content.

Glycation of bovine serum albumin by D-glucose and D-fructose under dry-heating conditions was studied. The reactivities of D-glucose and D-fructose, with respect to their ability to utilize primary amino groups of proteins, to cross-link proteins, to develop Maillard fluorescence, and to reduce protein solubility in the presence and absence of air (molecular oxygen) were investigated. D-Glucose showed a higher initial rate of utilization of primary amino groups than D-fructose, both in the presence and in the absence of oxygen. Subsequent reactions of the Amadori and Heyns rearrangement products, cross-linking, development of Maillard fluorescence, oxidation, and fragmentation, indicated that the alpha-hydroxy carbonyl group of Amadori products is more reactive than the aldehydo group of Heyns products. D-Fructose showed a greater sensitivity than D-glucose toward the presence of oxygen at the initial stages of the Maillard reaction. The presence or absence of oxygen in the glycation mixture did not seem to have an influence on the nature of products generated in the glycation mixtures during the advanced stages of the Maillard reaction.

Model studies using independently labeled D-[(13)C]glucoses and L-[(13)C]alanines have indicated that 2,3-butanedione is formed by a single pathway involving only glucose carbon atoms, whereas 2, 3-pentanedione is formed by two pathways, one involving glucose carbon atoms (10%) and the other (90%) through the participation of C2'-C3' atoms of L-alanine and a C(3) carbon unit from D-glucose. Analysis of label incorporation into selected mass spectral fragments of 2,3-pentanedione have indicated that the C(3) carbon unit originates either from C1-C2-C3 or from C4-C5-C6 fragments of D-glucose. In addition, model studies with pyruvaldehyde and glyceraldehyde have implicated these intermediates as plausible C(3) glucose carbon units capable of producing 2,3-pentanedione upon reaction with L-alanine. The labeling studies have also confirmed a previously identified chemical transformation of alpha-keto aldehydes affected by the amino acid that leads to the addition of the C-2 atom of the amino acid to the aldehydic carbon atom of alpha-keto aldehydes.

Infrared absorption bands characteristic of the aldehydo, keto, and enediol forms of 1-hydroxy-2-propanone (acetol) were identified and used to study the effect of solvent on the absorption frequencies and the effect of temperature and acid/base catalysis on the enolization reactions. The data indicated that, in addition to water, acids and bases can catalyze the enolization of 1-hydroxy-2-propanone and that the temperature inversely effects the rate of enolization under basic conditions. However, under acidic conditions, increasing the temperature favors the enolization process. In addition, the reaction of 1-hydroxy-2-propanone with a primary and a secondary amine was also monitored by Fourier transform infrared spectroscopy. The data indicated that at room temperature the rate of amine reaction was faster than the rate of its catalysis of enolization; however, below room temperature, the rate of base-catalyzed enolization became comparable with the rate of carbonyl-amine reaction forming both Heyns and Amadori adducts.

Model studies with pyrolysis/GC/MS using labeled [13C] glucoses with labeled [15N/13C]glycines and proline have indicated that the Maillard model systems consisting of glucose and glycine or proline generate similar C2, C3, C4 fragments such as acetic acid, and pyruvaldehyde. Furthermore, the labeling studies have enabled the identification of the origin of these reactive intermediates and their stable end-products such as N-acetylpyrrolidine, 1-(1'-pyrrolidinyl)-2-propanone amd 1-(1'-pyrrolidinyl)-2-butanone in proline model system and pyrazines and pyrazinones in glycine. In glycine model system, pyruvaldehyde and 2,3-butandione were found to be formed either from the degradation of the carbohydrate moiety (90 and 35%, respectively) or by an aldol-type interaction of glycine with alpha-ketoaldehydes. The same intermediates in proline system are formed exclusively from the carbohydrate degradation pathway.

The chemistry of the key intermediate in the Maillard reaction, the Amadori rearrangements product, is reviewed covering the areas of synthesis, chromatographic analyses, chemical and spectroscopic methods of characterization, reactions, and kinetics. Synthetic strategies involving free and protected sugars are described in detail with specific synthetic procedures. GC- and HPLC-based separations of Amadori products are discussed in relation to the type of columns employed and methods of detection. Applications of infrared (IR) and nuclear magnetic resonance (NMR) spectroscopy for structural elucidation of Amadori products are also reviewed. In addition, mass spectrometry of free, protected, and protein-bound Amadori products under different ionization conditions are presented. The mechanism of acid/base catalyzed thermal degradation reactions of Amadori compounds, as well as their kinetics of formation, are critically evaluated.