Molecular characterization around a glassy transition of starch using (1)H cross-relaxation nuclear magnetic resonance

The aim of this work was to characterize the glassy-rubbery transition in starch gels using molecular (NMR) techniques. Proton cross-relaxation ((1)H CR) NMR spectra of gelatinized starch ( approximately 50% mc) were obtained by cooling stepwise from 20 to -30 degrees C. A significant line broadening was observed in the CR spectra between 0 and -10 degrees C. Deconvolution of the spectra into its component curves (broad and narrow) yielded a peak amplitude, width at half-height, and peak area for each curve. Between 0 and -10 degrees C (temperatures around T(g)), a significant line width change in the broad component (rigid solid) was apparent. These observed qualitative changes may be evidence of a glassy-rubbery transition at a molecular (short-range) level which are strengthened by a similar transition temperature range found previously with (13)C CP-MAS and DMA tan delta(T) measurements. However, the increase in the relative quantity of rigid protons observed by (1)H CR NMR spectra could also be attributed to ice. The (1)H CR NMR method showed its potential application for probing solid components in gels using a simple and economical NMR spectrometer, without the need for a solid-state instrument.     

Two-dimensional 1H-13C nuclear magnetic resonance (NMR)-based comprehensive analysis of roasted coffee bean extract

Coffee was characterized by proton and carbon nuclear magnetic resonance (NMR) spectroscopy. To identify the coffee components, a detailed and approximately 90% signal assignment was carried out using various two-dimensional NMR spectra and a spiking method, in which authentic compounds were added to the roasted coffee bean extract (RCBE) sample. A total of 24 coffee components, including 5 polysaccharide units, 3 stereoisomers of chlorogenic acids, and 2 stereoisomers of quinic acids, were identified with the NMR spectra of RCBE. On the basis of the signal assignment, state analyses were further launched for the metal ion-citrate complexes and caffeine-chlorogenate complexes. On the basis of the signal integration, the coffee components were successfully quantified. This NMR methodology yielded detailed information on RCBE using only a single observation and provides a systemic approach for the analysis of other complex mixtures.     

Nondestructive observation of bovine milk by NMR spectroscopy: analysis of existing States of compounds and detection of new compounds

In this study were successfully observed the one- (1H, 13C) and two-dimensional (1H-13C, 1H-15N, 1H-31P) NMR spectra of milk directly without any pretreatment. The signals of each NMR spectrum were assigned, and their existing states were also analyzed. Lactose existed in a free state in milk. The signals due to the butyric acid chain can be assigned among the other fatty acid chains. Monounsaturated fatty acid (oleic acid chains) and polyunsaturated fatty acid chains (linoleic and linolenic acid) were assigned by their characteristic signals. The signals from citrate, N-acetylcarbohydrates, and lecithin could be observed directly in the 1H-13C HSQC NMR spectra; the assignment of their signals was made through the 1H-13C, 1H-15N, and 1H-31P HMBC spectra of extracted milk. Signals from creatine and N-acetylcarbohydrates were detected for the first time.     

1H NMR studies on Italian balsamic and traditional balsamic vinegars

In the present work Principal Component Analysis applied to (1)H NMR spectra of balsamic and traditional balsamic vinegars is used to establish a simple and rapid aging determination protocol. Chemical composition of vinegar is dominated by carbohydrates even though several small components can be clearly observed in the proton NMR spectrum. Quantitative determination of some selected metabolites such as ethanol, acetic acid, malic acid, glucose, and HMF, considered as potential aging indicators, has been performed. (1)H NMR spectroscopy provides noninvasive characterization of such compounds, and our data demonstrate the validity of this approach, giving very promising results for assessing the quality of the final product.     

Nondestructive quantification of organic compounds in whole milk without pretreatment by two-dimensional NMR spectroscopy

In this study, various organic compounds in commercial whole milk were quantified simultaneously by 1H 1D and 1H - 13C HSQC 2D NMR spectra without any pretreatment. 2D NMR spectroscopy was applied to quantification of milk compounds for the first time. Milk fat content was easily determined to be 3.6 +/- 0.1%, and the lactose content was 47.8 +/- 1.0 mg/mL by 1H NMR spectra. From 1H-13C HSQC spectra, the concentrations of citrate, N-acetylcarbohydrates, and trimethylamine were determined to be 3.2 +/- 0.2, 2.9 +/- 0.1, and 4.0 +/- 0.6 mM, respectively. The latter two compounds were quantified in milk for the first time. Butyric acid, total monounsaturated fatty acids, and total polyunsaturated fatty acids of triacylglycerols were 6.2 +/- 0.5, 9.1 +/- 0.9, and 2.9 +/- 0.3 mM, respectively. The fatty acid compositions (mol %) of triacylglycerols were then calculated and were observed to be in good agreement with reference values. The results indicated that 1H 1D and 1H-13C HSQC 2D NMR spectroscopy is useful for the rapid and nondestructive determination of various compounds in milk.     

Quantitative (13)C NMR analysis of lignins with internal standards

Novel protocols for acquiring quantitative (13)C NMR spectra of lignins have been developed using the internal reference compounds 1,3,5-trioxane and pentafluorobenzene. Trioxane offers a convenient internal standard for collecting inverse gated proton decoupled (13)C NMR spectra for lignins, whereas pentafluorobenzene can be used to provide information on the amount of methine carbon using the DEPT experiment. In each case, the internal reference compounds provide single, un-overlapped sharp signals in the middle of the spectral region, permitting facile integration. These integrals could be used to determine the amounts of different structural features of lignins, expressed in absolute units of millimoles per gram. The optimum parameters for these experiments were validated for a variety of spectrometer platforms, and standard errors were determined for different spectral areas using lignin model compounds and "standard" lignins. In addition, the data derived for the International Round Robin "standard" lignins showed good agreement with the data from quantitative (31)P NMR spectroscopy and published data, obtained by independent laboratories using independent methods of analysis.     

Isolation and identification of molecular species of phosphatidylcholine and lysophosphatidylcholine from jojoba seed meal (Simmondsia chinensis)

A mixture of lysophosphatidylcholine (LPC) and phosphatidylcholine (PC) has been isolated by column chromatography from a jojoba meal (Simmondsia chinensis) extract. The molecular species of both classes could be separated and isolated by C18 reversed phase HPLC. The two major compounds were identified by 1D and 2D (1)H and (13)C NMR, by MS, and by GC-MS as 1-oleoyl-3-lysophosphatidylcholine and 1,2-dioleoyl-3-phosphatidylcholine. Eight other molecular species of LPC and four other molecular species of PC could be assigned by comparison of the mass spectra of the isolated compounds with the spectra of the two major compounds. Complete characterization of the individual molecular species was achieved by GC and GC-MS analysis of the fatty acyl composition from the isolated compounds. The PC/LPC proportion in the phospholipid mixture from three different samples is 1.6 +/- 0.1. LPC is considered to be an important bioactive compound; the results of this study suggest further research for the evaluation of potential health benefits of jojoba meal phospholipids.     

Soil organic matter transformations induced by Hieracium pilosella L. in tussock grassland of New Zealand

 To study the effect of Hieracium pilosella L. invasion on the transformations of soil organic matter of New Zealand tussock grassland soils (Ustochrepts), plant material and soils underneath Hieracium, the surrounding halo, and the adjacent herbfield (depleted tussock grassland) were examined for their chemical composition. An attempt was made to reveal possible changes in chemical composition of the soil organic matter induced by H. pilosella invasion. Small differences were detected by solid-state ¹³C nuclear magnetic resonance (NMR) spectroscopy in the composition of the plant and soil materials from these zones. Most of the differences in soil organic matter occurred due to differences in the amount and quality of plant-residue inputs. Comparable amounts of phenolic C were detected in the solid-state ¹³C NMR spectra of H. pilosella and herbfield vegetation, while alkaline CuO oxidation yielded considerable lower lignin oxidation products for H. pilosella. A slightly higher proportion of these compounds in H. pilosella soil revealed an accumulation and a low degradation rate of lignin compounds under H. pilosella. The HCl hydrolysis and solid-state ¹⁵N NMR spectroscopy showed similar chemical compositions of the N fractions of the three different soils. The absence of ¹⁵N NMR signal intensity assignable to aniline derivatives or aromatic heterocyclic N indicates that the condensation of phenolic compounds with N groups plays a minor role in N sequestration in these soils. Received: 6 September 1999     

Free radical scavenging active components from Cedrus deodara

An activity-directed fractionation and purification process was used to identify the antioxidant components of Cedrus deodara. Dried heartwood powder of C. deodara was first defatted with petroleum ether and then extracted with chloroform. The chloroform extract showed strong antioxidant activity on 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical. This fraction was then subjected to separation and purification using silica gel column chromatography. Three compounds with potent antioxidant activity were isolated in significant yields and identified by spectroscopic methods ((1)H NMR, (13)C NMR, IR, and MS). They were identified as (-)-matairesinol, (-)-nortrachelogenin, and a dibenzylbutyrolactollignan (4,4',9-trihydroxy-3,3'-dimethoxy-9,9'-epoxylignan). This is the first report of the occurrence of these compounds in C. deodara.     

Two new Monascus metabolites with strong blue fluorescence isolated from red yeast rice

Red yeast rice obtained as cultures of Monascus AS3.4444 on rice was extracted and analyzed by high-performance liquid chromatography (HPLC). Two new Monascus metabolites with similar fluorescence spectra (lambda ex = 396 nm, lambda em = 460 nm) and UV absorption spectra (lambda max = 386 nm) were detected. They were isolated by rechromatography on a silica gel column and semipreparative HPLC, and two strong blue fluorescent compounds were obtained. Their structures were elucidated by electrospray ionization mass spectrometry (ESI-MS), electrospray ionization tandem mass spectrometry (ESI-MS/MS), intensive ESI-MS, and nuclear magnetic resonance spectroscopy ( (1)H NMR, (13)C NMR, COSY, and HMBC) studies. High-resolution mass spectrometry indicated the molecular formulas C 21H 24O 5 and C 23H 28O 5. The two new compounds, named monasfluore A and monasfluore B, respectively, contain a alkyl side chain, gamma-lactone, and propenyl group, whereas the more lipophilic compound, monasfluore B, is a higher homologue of monasfluore A, with the more lipophilic octanoyl instead of the hexanoyl side chain.     

Identification of amino acids in wines by one- and two-dimensional nuclear magnetic resonance spectroscopy

Amino acids are minor compounds in wines, but they have a profound influence on wine quality, and amino acids composition can be used to differentiate wines according to the vine variety, geographical origin, and year of production. The NMR signals of amino acids in NMR spectra are overlapped by the signals of other compounds present and especially by the signals of dominant compounds such as water, ethanol, and glycerol. In this work we used 1D (1)H and (13)C, 2D homonuclear COSY, TOCSY, and 2D heteronuclear HSQC and HMQC pulse sequences, also with an incorporated WET pulse sequence element that allows the simultaneous suppression of several frequencies. Complete (1)H and (13)C NMR assignments for 17 amino acids commonly present in wine and of gamma-aminobutyric acid at pH 3 have been achieved in wine sample of Sauvignon from the Coastal wine-growing region of Slovenia, vintage 1994.     

Mosquitocidal, nematicidal, and antifungal compounds from Apium graveolens L. seeds

The methanolic extract of Apium graveolens seeds was investigated for bioactive compounds and resulted in the isolation and characterization of mosquitocidal, nematicidal, and antifungal compounds sedanolide (1), senkyunolide-N (2), and senkyunolide-J (3). Their structures were determined by 1H and 13C NMR spectral methods. Compounds 1-3 gave 100% mortality at 25, 100, and 100 microg mL(-1), respectively, on the nematode, Panagrellus redivivus. Compound 1 showed 100% mortality at 50 microg mL(-1) on nematode, Caenorhabditis elegans, and fourth-instar mosquito larvae, Aedes aegyptii. Also, it inhibited the growth of Candida albicans and Candida parapsilasis at 100 microg mL(-1). Compounds 2 and 3 were isolated for the first time from A. graveolens. This is the first report of the mosquitocidal, nematicidal, and antifungal activities of compounds 1-3.     

Biflavonoids isolated from Selaginella lepidophylla inhibit photosynthesis in spinach chloroplasts

Through bioactivity-guided chemical analysis of Selaginella lepidophylla, biflavonoids robustaflavone (1), 2,3-dihydrorobustaflavone (2), and 2,3-dihydrorobustaflavone-5-methyl ether (3) were isolated and their structures confirmed by spectroscopic and spectrometric analyses. Their NMR resonances were unambiguously assigned from HMBC, NOESY, and NOESY-1D experiments, and absolute configurations of 2 and 3 were established. Compound 3 has not been reported, and although structure of 2 was described before, the (13)C NMR assignment does not correlate with the structure reported. Therefore, this is the first report of 2. All compounds inhibited ATP production. Compounds 1 and 2 behaved as Hill reaction inhibitors. 1 interacted with photosystem II, transforming the reaction centers to silent centers at 300 and 600 microM. The interaction and inhibition target of 2 was located on Cyt b6f to PC. The three compounds also behaved as energy transfer inhibitors, 3 being the most active.     

New bioactive orange pigments with yellow fluorescence from Monascus-fermented dioscorea

Red mold dioscorea (RMD) is a fermented product of Monascus purpureus NTU 568 using dioscorea as culture substrate. To investigate the bioactive components of RMD, six orange pigments including four new azaphilones with yellow fluorescence, monapilol A-D (1-4), and known monascorubrin (5) and rubropunctatin (6) were isolated and characterized. Structural elucidation of new isolates was based on nuclear magnetic resonance ((1)H NMR, (13)C NMR, COSY, HMQC, and HMBC) and other spectroscopic analyses. The structures of monapilols (1-4) were similar to those of monascorubrin (5) and rubropunctatin (6); however, the hydroxyl group (8-OH) in compounds 1-4 substituted for the C-8 carbonyl in compounds 5 and 6. Biological evaluation indicated that compounds 1-4 inhibited nitric oxide (NO) production on lipopolysaccharide-stimulated RAW 264.7 cells. Compounds 1-4 also exhibited antiproliferative activities against human laryngeal carcinoma (HEp-2) and human colon adenocarinoma (WiDr).     

Rice Quality by Spectroscopic Analysis: Precision of Three Spectral Regions

Three types of spectroscopy were used to examine rice quality: near infrared (NIR), Raman, and proton nuclear magnetic resonance (¹H NMR). Samples from 96 rice cultivars were tested. Protein, amylose, transparency, alkali spreading values, whiteness, and degree of milling were measured by standard techniques and the values were regressed against NIR and Raman spectra data. The NMR spectra were used for a qualitative or semiquantitative assessment of the amylose/amylopectin ratio by determining the 1–4 to 1–6 ratio for glucans. Protein can be measured by almost any instrument in any configuration because of the strong relationship between the spectral response and the precision of the reference method. Amylose has an equally strong relationship to the vibrational spectra, but its determination by any reference method is far less precise, resulting in a 10× increase in the standard error of cross‐validation (SECv) or standard error of performance (SEP) with R ² values equal to that of the protein measurement.     

Investigation of different apple cultivars by high resolution magic angle spinning NMR. A feasibility study

(1)H HR-MAS NMR spectroscopy was applied to apple tissue samples deriving from 3 different cultivars. The NMR data were statistically evaluated by analysis of variance (ANOVA), principal component analysis (PCA), and partial least-squares-discriminant analysis (PLS-DA). The intra-apple variability of the compounds was found to be significantly lower than the inter-apple variability within one cultivar. A clear separation of the three different apple cultivars could be obtained by multivariate analysis. Direct comparison of the NMR spectra obtained from apple tissue (with HR-MAS) and juice (with liquid-state HR NMR) showed distinct differences in some metabolites, which are probably due to changes induced by juice preparation. This preliminary study demonstrates the feasibility of (1)H HR-MAS NMR in combination with multivariate analysis as a tool for future chemometric studies applied to intact fruit tissues, e.g. for investigating compositional changes due to physiological disorders, specific growth or storage conditions.     

Novel fatty acid esters of p-coumaryl alcohol in epicuticular wax of apple fruit

Hexane extracts of epicuticular wax from cv. Gala apples were noted to have an unusual, broad absorbance maximum at approximately 258 nm, which led us to isolate and identify the primary UV-absorbing compounds. Column and thin-layer chromatography yielded a fraction that gave a series of paired, 260-nm-absorbing peaks on C(18) HPLC. These were shown to be a family of phenolic fatty acid esters, for which retention times increased with increasing fatty acid chain length, and paired peaks were esters of two related phenolics with the same fatty acid moiety. Alkaline hydrolysis of the esters released two water-soluble phenolics separable by C(18) HPLC. Electrospray ionization mass spectrometry gave a molecular mass of 150 for both, and (1)H NMR plus UV absorbance spectra identified them as E and Z isomers of p-coumaryl alcohol. Alkaline cleavage of the fatty acid esters in the presence of methanol or ethanol resulted in partial derivatization of E-p-coumaryl alcohol to the corresponding gamma-O-methyl or O-ethyl ether. Gradient HMQC NMR of the HPLC-purified stearate ester of E-p-coumaryl alcohol indicated that fatty acid esterification occurs at the gamma-OH rather than at the 4-OH on the phenyl ring. This is the first report of fatty acid esters of monolignols as a natural plant product.     

Insect pest management agents: hormonogen esters (juvenogens)

The chemical part of this investigation focused on designing structures and synthesizing a series of six new esters (juvenogens), derived from biologically active insect juvenile hormone bioanalogues (juvenoids, JHAs) and unsaturated short-chain linear and branched fatty acids for possible application as biochemically targeted insect hormonogen agents. The structures of the new compounds were assigned on the basis of a detailed NMR analysis of their (1)H and (13)C NMR spectra. The biological part of this investigation focused on introductory biological screening tests with these compounds against the red firebug (Pyrrhocoris apterus), termites (Reticulitermes santonensis and Prorhinotermes simplex), and the blowfly (Neobellieria bullata). The biological activity of the juvenogens was studied in relation to the fatty acid functionality in the structures. Notable biological activity in topical tests and medium activity in peroral tests was found for the juvenogens 3 and 7 with P. apterus. The compounds 6 and 8 showed the lowest activity in both topical and oral assays with P. apterus. Considerable effect of all tested juvenogens was observed in P. simplex; however, the juvenogens 5 and 6 (derivatives of the only branched short-chain fatty acid) showed no activity against R. santonensis. The effect of the compounds 3-8 on larval hatching of N. bullata was only moderate (larval hatching 80-90%); however, the proliferation effect caused by 5, 6, and 8 is more pronounced than the effect caused by 3, 4, and 7.     

NMR experiments of oleuropein biomimetic hydrolysis.

The oleuropein hydrolytic conversion, under biomimetic conditions, induced by the endogenous enzymatic system of the olive fruit, has been monitored by the intermediate derivative formation through (1)H and (13)C NMR spectra; the assigned molecular structures reveal the identity of new bioactive biophenolic metabolites, the hemiacetal aglycon, and the two epimeric dialdehydes, which influence the pathogen antagonism of olive fruits and the hedonistic-sensorial characteristics of olive oil.