Identification and quantification of caffeic and rosmarinic acid in complex plant extracts by the use of variable-temperature two-dimensional nuclear magnetic resonance spectroscopy

A combination of advanced nuclear magnetic resonance (NMR) methodologies for the analysis of complex phenolic mixtures that occur in natural products is described, with particular emphasis on caffeic acid and its ester derivative, rosmarinic acid. The combination of variable-temperature two-dimensional proton-proton double quantum filter correlation spectroscopy (1H-1H DQF COSY) and proton-carbon heteronuclear multiple quantum coherence (1H-13C HMQC) gradient NMR spectroscopy allows the identification and tentative quantification of caffeic and rosmarinic acids at 243 K in extracts from plants of the Lamiaceae family, without resorting to previous chromatographic separation of the components. The use of proton-carbon heteronuclear multiple bond correlation (1H-13C HMBC) gradient NMR spectroscopy leads to the complete assignment of the correlations of the spins of H2a and H3a with the ester and carboxyl carbons of rosmarinic and caffeic acid, even at room temperature, and confirms the results of the above methodology Quantitative results are in reasonable agreement with reverse phase HPLC measurements.     

Megastigmane and phenolic components from Laurus nobilis L. leaves and their inhibitory effects on nitric oxide production

Laurus nobilis L. leaves are widely used in cooking and in folk medicine. Five new megastigmane glucosides (2-4, 7, and 9) named laurosides A-E and a new phenolic glucoside 12 were isolated from the methanolic extract of L. nobilis L. leaves, along with 10 known components: megastigmane (5), megastigmane glucosides (1, 6, 8, 10, and 11), aromatic compounds (13 and 14), and flavonoids (15 and 16). The structures and relative stereochemistry have been elucidated by one- and two-dimensional nuclear magnetic resonance experiments ((1)H and (13)C NMR, DEPT, correlation spectroscopy, heteronuclear multiple quantum correlation, heteronuclear multiple bond correlation, and nuclear Overhauser enhancement spectroscopy) and by chemical derivatization. The effect of isolated compounds on nitric oxide production in lipopolysaccharide-activated murine macrophages were examined.     

NMR and molecular mechanics study of pyrethrins I and II.

Bioassay-directed fractionation of the organic extract of the Kenyan pyrethrum flowers (Chrysanthemum cinerariaefolium Vissiani) resulted in the isolation of two natural pyrethrin esters, pyrethrin I (PI) and pyrethrin II (PII) as the major constituents. These esters elicited inhibition of the multiple drug resistant (MDR) Mycobacterium tuberculosis. The high-field (1)H and (13)C nuclear magnetic resonance (NMR) chemical shifts of PI and PII were unequivocally assigned using modern two-dimensional (2D) proton-detected heteronuclear multiple-quantum coherence (HMQC) and heteronuclear multiple-bond correlation (HMBC) experiments. The conformations of both esters were deduced from (1)H-(1)H vicinal coupling constants and confirmed by 2D nuclear Overhauser effect spectroscopy (NOESY). Computer molecular modeling (MM) studies revealed that PI and PII molecules adopt a "love-seat" conformation in chloroform (CDCl(3)) solution.     

Isolation, Characterization, and NO Inhibitory Activities of Sesquiterpenes from Blumea balsamifera

Blumea balsamifera belongs to the family Compositae, and its leaves have been used as a flavoring ingredient and a tea. A phytochemical investigation of the aerial parts of B. balsamifera led to the isolation of 10 new (1-10) and 1 known (11) sesquiterpenes. Their structures were elucidated on the basis of extensive one- and two-dimensional nuclear magnetic resonance (heteronuclear multiple-quantum coherence, heteronuclear multiple-bond correlation, (1)H-(1)H correlation spectroscopy, and nuclear Overhauser effect spectrometry) spectroscopic data analyses, and the structure of compound 1 was confirmed by X-ray crystallography. The inhibitory activities on lipopolysaccharide-induced NO production in murine microglial BV-2 cells of these sesquiterpenes were evaluated, and all of the compounds showed inhibitory effects.     

Epicatechin carbonyl-trapping reactions in aqueous maillard systems: Identification and structural elucidation

Recently, our group reported via labeling experiments that epicatechin in Maillard reaction aqueous glucose-glycine model systems formed adduct reaction products with C2, C3, and C4 sugar fragments. In the current study, we investigated the identity of the sugar fragment precursors responsible for adduct generation by directly comparing the liquid chromatography-mass spectrometry properties of these reported epicatechin (EC)-sugar fragments adducts with those generated from reactions consisting of only EC and well-known Maillard-generated glucose fragments (i.e., glyoxal, glycolaldehyde, methylglyoxal, glyceraldehyde, etc.). The structural properties of an EC-methylglyoxal adduct reaction product were also analyzed by NMR. The most likely precursors for the C2, C3, and C4 sugar moiety of the EC-sugar fragment adducts were identified as glyoxal, hydroxyacetone, and erythrose, respectively. 1H NMR analysis of the EC-methylglyoxal indicated that the analyte underwent rapid conformational/constitutional exchange. Using cold temperature (-25 degrees C) two-dimensional NMR analyses (heteronuclear multiple bond coherence, heteronuclear multiple quantum coherence, and 1H-(1)H correlation spectroscopy), the structure of one of the isomers was reported to consist of a covalent linkage between the C1 position of the methylglyoxal and either the C6 or the C8 position of the EC A ring, presumably generated by hydroxyalkylation and aromatic substitution reactions.     

Classification of Italian honeys by 2D HR-NMR

The importance of honey has been recently increased because of its nutrient and therapeutic effects, but the adulteration of honey in terms of botanical origin has increased, too. The floral origin of honeys is usually determined using melisso-palynological analysis and organoleptic characteristics, but the application of these techniques requires some expertise. A number of papers have confirmed the possibility of characterizing honey samples by selected chemical parameters. In this study high-resolution nuclear magnetic resonance (HR-NMR) and multivariate statistical analysis methods were used to identify and classify honeys of five different floral sources. The 71 honey samples (robinia, chestnut, citrus, eucalyptus, polyfloral) were analyzed by HR-NMR using both 1H NMR and heteronuclear multiple bond correlation spectroscopy (HMBC). Spectral data were analyzed by application of unsupervised and supervised pattern recognition and multivariate statistical techniques such as principal component analysis (PCA) and general discriminant analysis (GDA). The use of 1H-(13)C HMBC coupled with appropriate statistical analysis seems to be an efficient technique for the classification of honeys.     

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.     

Phenylphenalenone type compounds from the leaf fibers of abaca (Musa textilis)

A series of phenylphenalenone type compounds, known to play a role as phytoalexins in plants of the Musaceae family, have been identified for the first time in the leaf fibers of abaca (Musa textilis). Among the phenylphenalenone type compounds identified, the structure of a novel compound, (1R)-2,3-dihydro-4,9-dihydroxy-8-methoxy-1-phenylphenalene, has also been described in abaca fibers. Its structure was elucidated by analysis of one- and two-dimensional NMR (correlation spectroscopy, heteronuclear single quantum correlation, and heteronuclear multiple bond correlation) spectroscopic data.     

Analysis of plant complex matrices by use of nuclear magnetic resonance spectroscopy: St. John's wort extract

The efficiency of two-dimensional homonuclear (1)H--(1)H correlated spectroscopy and two-dimensional reverse heteronuclear shift correlation spectroscopy (i.e., heteronuclear multiple quantum correlation) in characterizing and evaluating the relative content of herbal extract constituents is demonstrated. These experiments are able to fully assign the proton and carbon resonances of all three classes of constituents present in dried commercial extract of St. John's wort, that is, flavonols, phloroglucinols, and naphthodianthrones, with particular regard to the very unstable phloroglucinols. In addition, shikimic and chlorogenic acids, sucrose, lipids, polyphenols, and traces of solvents of the extractive process (methanol) were also identified. These experiments can be considered to be a very simple and fast analytical method for determining the quality and stability of the titled commercial extract. They represent a generally applicable technique for a rapid screening and a specific measurement of other commercial phytochemicals or, in selected cases, an alternative to the classical analytical techniques such as high-performance thin-layer chromatography, high-performance liquid chromatography, capillary gas chromatography, and electrophoresis.     

Antifungal activity and biotransformation of diisophorone by Botrytis cinerea

Diisophorone (1) was tested against two strains of the necrotrophic plant pathogen Botrytis cinerea.Fungal sensitivity varied according to the strain. B. cinera 2100 was more sensitive than B. cinereaUCA992: its mycelial growth was significantly inhibited at concentrations of 50 ppm and above. Although diisophorone (1) showed an effective control of B. cinerea, a detoxification mechanism was present. The detoxification of racemic diisophorone (1) by B. cinerea was investigated. Incubation with two strains of B. cinerea gave one and four biotransformation products (2-5), respectively. Their structures were established as the known 8beta-hydroxydiisophorone (2), 6alpha-hydroxydiisophorone (3), 6beta-hydroxydiisophorone (4) and 8beta,14beta-dihydroxydiisophorone (5) on the basis of their spectroscopic data, including two-dimensional NMR analysis [heteronuclear multiple quantum coherence (HMQC), heteronuclear multiple bond correlation (HMBC), and nuclear Overhauser enhancement spectroscopy (NOESY)] and an X-ray crystallographic study.     

Sesamolinol glucoside, disaminyl ether, and other lignans from sesame seeds

The application of a procedure based on XAD-4 adsorption resin permitted the obtainment of an enriched polyphenolic extract from Sesamum indicum seeds. Chemical analysis of the obtained extract led to the identification of 12 lignans. Among them, 2 lignans, (+)-sesamolinol-4'-O-β-D-glucoside and disaminyl ether, are reported for the first time as natural compounds. Their structure has been determined by spectroscopic methods, mainly by the application of one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR) techniques [heteronuclear multiple-quantum coherence (HMQC), heteronuclear multiple-bond correlation (HMBC), and nuclear Overhauser effect spectrometry (NOESY)] and mass spectroscopy. The isolated compounds were evaluated for their antimutagenic activity. Among the tested lignans, the most active lignan was found to be sesamolin, followed by sesamolinol and samin, against H(2)O(2). Additionally, some of the tested lignans showed desmutagenic activity against benzo[a]pyrene (BaP).     

Triterpene saponins from barrel medic (Medicago truncatula) aerial parts

Triterpene saponins from Medicago truncatula aerial parts have been separated and their structures determined by the extensive use of 1D- and 2D-NMR experiments including 1H-1H (DQF-COSY, 1D-TOCSY) and 1H-13C (HSQC, HMBC) spectroscopy along with ESIMS. Fifteen individual compounds were isolated that included seven medicagenic acid and eight zanhic acid glycosides. Additionally, two soyasapogenol B and soyasapogenol E glycosides were identified by MS/MS and TLC. Four medicagenic acid glycosides (5, 11, 12, 14) and eight zanhic acid glycosides (1-4, 6-9) are reported here for the first time. The common feature of M. truncatula aerial part saponins is the (1-->3) linkage between the two glucose units at C-3 of medicagenic and zanhic acids, which is different from that found in alfalfa (Medicago sativa), where this linkage was always (1-->2). This may suggest differences in glucosyltransferases between these two Medicago species.     

Enzymatic dehydrogenative polymerization of urushiols in fresh exudates from the lacquer tree, Rhus vernicifera DC

Fresh exudates from the lacquer tree, Rhus vernicifera DC, were extracted with acetone and the solution was chromatographed to isolate monomer, dimer, trimer, and oligomer fractions of urushiols. Constituents of the monomeric and dimeric fractions were then identified by two-dimensional (2D) 1H-13C heteronuclear multiple quantum coherence (HMQC) and heteronuclear multiple bond coherence (HMBC) NMR spectroscopic techniques. The results showed that the monomeric fraction contained 3-[8'Z,11'E,13'Z-pentadecatrienyl]catechol (1), 3-[8'Z,11'Z,14'-pentadecatrienyl]catechol (2), and 3-pentadecanyl]catechol (3), which was verified by HPLC analysis. The dimeric fraction contained 8'-(3' ',4' '-dihydroxy-5' '-alkenyl)phenyl-3-[9'E,11'E,13'Z-pentadecatrienyl]catechol (4), 14'-(3' ',4' '-dihydroxy-5' '-alkenyl)phenyl-3-[8'Z,10'E,12'E-pentadecatrienyl]catechol (5), 2-hydroxyl-3- or -6-alkenylphenyl ethyl ether (6), 14'-(3' ',4' '-dihydroxy-2' '-alkenyl)phenyl-3-[8'Z,10'E,12'E-pentadeca-trienyl]catechol (7), 15'-(2' '-hydroxy-3' '- or -6' '-alkenyl)phenyloxy-3-[8'Z,11'Z,13'E)-pentadecatrienyl]catechol (8), 14'-(2' ',3' '-dihydroxy-4' '-alkenyl)phenyl-3-[8'Z,10'E,12'E-pentadecantrienyl]catechol (9), 1,1',2,2'-tetrahydroxy-6,6'-dialkenyl-4,3'-biphenyl (10), 1,1',2,2'-tetrahydroxy-6,6'-dialkenyl-4,4'-biphenyl (11), 1,1',2,2'-tetrahydroxy-6,6'-dialkenyl-5,4'-biphenyl (12), and 1,2,1'-trihydroxy-6,6'-dialkenyldibenzofuran (13) as constituents. In addition, dimeric ethers and peroxides, such as compounds 14 and 15, were produced by autoxidation of monomeric urushiols in atmospheric air. The possible reaction mechanisms for the dehydrogenative polymerization of urushiols by Rhus laccase present in the fresh raw exudates under the atmospheric oxygen are discussed on the basis of structures identified. This is of primary importance because the use of the urushi exudates as coating materials does not involve organic solvents and is an environmentally friendly process.     

Triterpenoid saponins from the shells of Argania spinosa seeds

Two new oleanene saponins were isolated from the MeOH extract of the shell of Argania spinosa.They possess protobassic acid and 16alpha-protobassic acid as aglycons. The disaccharide moiety linked to C-3 of the aglycon is made up of two glucose units; the pentasaccharide moiety linked to C-28 is made up of arabinose, xylose, and three rhamnose units. Their structures were elucidated by 1D and 2D NMR experiments including (1)H-(1)H (DQF-COSY, 1D TOCSY, and 2D HOHAHA) and (1)H-(13)C (HSQC and HMBC) spectroscopy along with mass spectrometry.     

Quantitative characterization of a hardwood milled wood lignin by nuclear magnetic resonance spectroscopy

The structure of Eucalyptus grandis milled wood lignin (MWL) was investigated by 2D 1H-13C HSQC, HMQC, and 1H-1H TOCSY correlation NMR techniques and by quantitative 13C NMR as well as by the permanganate oxidation degradation technique. The combination of 2D NMR and quantitative 13C NMR spectroscopy of nonacetylated and acetylated lignin preparations allowed reliable identification and calculation of the amount of different lignin structures. About 85% of side-chain moieties were estimated on the structural level. This information was substantiated by data on the quantity of various functional groups and interunit linkages as a whole. A modified method for calculation of the h:g:s ratio has been suggested and compared with previously suggested approaches. E. grandis MWL has been determined to have an h:g:s ratio of 2:36:62. The amounts of various phenolic/etherified noncondensed/condensed guaiacyl and syringyl moieties were approximately estimated. E. grandis MWL contained approximately 0.60/Ar of beta-O-4 moieties along with small amounts of other structural units such as pino/syringyresinol (0.03/Ar), phenylcoumaran (0.03/Ar), and spirodienone (0.05/Ar). The degree of condensation was estimated at approximately 21%; the main condensed structures are 4-O-5 moieties (approximately 0.09/Ar). The structure of E. grandis MWL was compared with those of other lignin preparations isolated from various hardwoods.     

Acetylation of 5-amino-1H-[1,2,4]triazole revisited

The products of the acetylation reactions of the common herbicide 5-amino-1H-[1,2,4]triazole were investigated using HPLC, GC-MS, 1H NMR, and FTIR spectroscopy. The conventional annular monoacetylation procedures with acetyl chloride are not regioselective and furnish a mixture of isomers. Traditional diacetylation in neat acetic anhydride under reflux produces a mixture of di-, mono-, and triacetylated derivatives. By using equivalent amounts of acetic anhydride in a dimethylformamide solution, a rapid and selective annular monoacetylation of 5-amino-1H-[1,2,4]triazole was achieved. The monoacetylation proceeds via the formation of the intermediate, 1-acetyl-3-amino-1H-[1,2,4]triazole, which had not been observed previously and which undergoes transformation into the known 1-acetyl-5-amino-1H-[1,2,4]triazole. Neat acetic anhydride at room temperature affords the diacetylated derivative, 1-acetyl-3-(acetylamino)-1H-[1,2,4]triazole both from 5-amino-1H-[1,2,4]triazole itself and from either 1-acetyl-5-amino-1H-[1,2,4]triazole or 5-(acetylamino)-1H-[1,2,4]triazole. The atypical product of the second acetylation, 1-acetyl-5-(acetylamino)-1H-[1,2,4]triazole, has been identified. These results may be useful in the development of effective and selective preparative procedures for the acetylation of 5-amino-1H-[1,2,4]triazole.     

Reactivity of anthocyanins and pyranoanthocyanins. Studies on aromatic hydrogen-deuterium exchange reactions in methanol

Reactivity studies involving anthocyanin structures and their equilibrium forms will lead to better understanding of the properties of these antioxidants. Hydrogen-deuterium (H --> D) exchange reactions at various sites of the 3-glucosides of delphinidin (1), petunidin (2), malvidin (3), and the corresponding 3-glucosides of carboxypyranodelphinidin (4), carboxypyranopetunidin (5), carboxypyranomalvidin (6), and the flavonol quercetin 3-O-(6-alpha-rhamnopyranosyl-beta-glucopyranoside)(7) have been examined at room temperature in pure CD 3OD and in CD 3OD acidified with CF 3CO 2D. The H --> D exchange rate constants of H-6 and H-8 of 2 determined from (1)H NMR integration data were found to be independent upon pigment concentration (up to 4 x 10 (-2) M) and trifluoroactic acid concentration (0-15%, v/v), respectively. This suggest that these reactions follow first-order kinetics and unexpectedly to be independent of the acid concentration. H-6 and H-8 of the flavylium cation A-rings of 1- 3, and in the corresponding hydrogens of the hemiketal forms, exchanged with half-lives of approximately 100 h ( 1) and approximately 50 h ( 2 and 3), respectively. The pyranoanthocyanins (4-6) experienced no H --> D exchange for the analogous hydrogens, but H --> D exchange of H-beta (H-4)(t 1/2 approximately 25 h) for these compounds was observed. Only H-8 underwent significant H --> D exchange in 7. It is concluded that a stabilization of the sigma-complexes, assumed to be the intermediates in the reactions, takes place for the common anthocyanins (1-3) contrary to the pyranoanthocyanins (4-6).     

Heteronuclear single-quantum coherence nuclear magnetic resonance (HSQC NMR) characterization of acetylated fir (Abies sachallnensis MAST) wood regenerated from ionic liquid

An ionic liquid, 1-butyl-3-methylimidazolium chloride ([Bmim]Cl), was used to dissolve Japanese fir (Abies sachallnensis MAST) wood. Milled woods prepared by planetary ball-milling for 8 h dissolved completely in [Bmim]Cl at 100 °C in 2 h. The dissolved woods were then subjected to in situ acetylation, and the fully acetylated woods were regenerated from [Bmim]Cl. (1)H-(13)C correlation heteronuclear single-quantum coherence (HSQC) nuclear magnetic resonance (NMR) experiments were successfully conducted with the acetylated woods in dimethyl sulfoxide (DMSO)-d(6). The acetylated lignin and polysaccharide signals dispersed reasonably well on the 2D spectra. Characterization of the NMR signals for the whole cell-wall components, including lignin, cellulose, and hemicelluloses, was achieved by comparison with isolated lignin and commercial cellulose and hemicelluloses (arabinoxylan, galactomannan, and glucomannan). The procedure used here is applicable for the characterization of cell-wall components in various plant biomasses.     

Studies on the constituents of Cyclanthera pedata fruits: isolation and structure elucidation of new flavonoid glycosides and their antioxidant activity.

The isolation of six flavon glycosides (1-6), among them four new natural compounds (1-4), from the CHCl(3)/MeOH extract of the fruits of Cyclanthera pedata is reported. All of the structures were elucidated by spectroscopic methods, including the concerted application of one-dimensional ((1)H, (1)H TOCSY, (13)C, and (13)C DEPT-NMR) and two-dimensional NMR techniques (DQF-COSY, HSQC, and HMBC). For all of the isolated compounds the antioxidant activity was determined by measuring the free radical scavenging activity, using the Trolox equivalent antioxidant capacity (TEAC) method, and the coupled oxidation of beta-carotene and linoleic acid.     

Characterization of oligomeric procyanidins and identification of quercetin glucuronide from lotus ( Nelumbo nucifera Gaertn.) seedpod

Procyanidins are a class of polyphenols in the plant kingdom. Lotus ( Nelumbo nucifera Gaertn.) seedpods, the inedible part of lotus and a byproduct during the production of lotus seeds, were found to be a new source rich in procyanidins. Detailed information about oligomeric procyanidins in lotus seedpods remains unknown. In this study, lotus seedpods were extracted using 60% aqueous methanol and characterized with phloroglucinolysis and liquid chromatography (mass spectrometry with an electrospray ionization source). The results indicate that the oligomeric and polymeric fraction had a mean degree of polymerization of 3.2 and 15.4, respectively, and consisted of (+)-catechin (m/z 289), gallocatechin or epigallocatechin (m/z 305), quercetin glycoside (m/z 463), quercetin glucuronide (m/z 477), procyanidin dimers (m/z 577.1), proanthocyanidin dimer gallate (m/z 593.3), prodelphinidin dimers (m/z 609.1), procyanidin trimers (m/z 865.1), etc. Quercetin glucuronide was further purified using flash chromatography and identified as quercetin-3-O-β-glucuronide by determining its exact mass using ion-trap time-of-flight mass spectrometry and 1H and 13C nuclear magnetic resonance, 1H-detected heteronuclear single-quantum coherence, and 1H-detected heteronuclear multiple-bond correlation analyses.