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.     

Sensory-guided decomposition of roasted cocoa nibs (Theobroma cacao) and structure determination of taste-active polyphenols

Sequential application of solvent extraction, gel permeation chromatography, and RP-HPLC in combination with taste dilution analyses, followed by LC-MS and 1D/2D-NMR experiments and thiolytic degradation, revealed that, besides theobromine and caffeine, the flavan-3-ols epicatechin, catechin, procyanidin B-2, procyanidin B-5, procyanidin C-1, [epicatechin-(4beta-->8)](3)-epicatechin, and [epicatechin-(4beta-->8)](4)-epicatechin were among the key compounds contributing to the bitter taste as well as the astringent mouthfeel imparted upon consumption of roasted cocoa. In addition, a series of quercetin, naringenin, luteolin, and apigenin glycopyranosides as well as a family of not previously identified amino acid amides, namely, (+)-N-[4'-hydroxy-(E)-cinnamoyl]-L-aspartic acid, (+)-N-[3',4'-dihydroxy-(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, have been identified as key astringent compounds of roasted cocoa. Furthermore, (-)-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, reported previously as antioxidants, have been found as contributors of cocoa's astringent taste. By means of the half-tongue test, the taste thresholds of flavan-3-ols and glycosides have been determined.     

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.     

Effects of commercial processing on levels of antioxidants in oats (Avena sativa L.)

The effects of various commercial hydrothermal processes (steaming, autoclaving, and drum drying) on levels of selected oat antioxidants were investigated. Steaming and flaking of dehulled oat groats resulted in moderate losses of tocotrienols, caffeic acid, and the avenanthramide Bp (N-(4'-hydroxy)-(E)-cinnamoyl-5-hydroxy-anthranilic acid), while ferulic acid and vanillin increased. The tocopherols and the avenanthramides Bc (N-(3',4'-dihydroxy-(E)-cinnamoyl-5-hydroxy-anthranilic acid) and Bf (N-(4'-hydroxy-3'-methoxy)-(E)-cinnamoyl-5-hydroxy-anthranilic acid) were not affected by steaming. Autoclaving of grains (including the hulls) caused increased levels of all tocopherols and tocotrienols analyzed except beta-tocotrienol, which was not affected. Vanillin and ferulic and p-coumaric acids also increased, whereas the avenanthramides decreased, and caffeic acid was almost completely eliminated. Drum drying of steamed rolled oats resulted in an almost complete loss of tocopherols and tocotrienols, as well as a large decrease in total cinnamic acids and avenanthramides. The same process applied to wholemeal made from groats from autoclaved grains resulted in less pronounced losses, especially for the avenanthramides which were not significantly affected.     

Isolation and characterization of structurally novel antimutagenic flavonoids from spinach (Spinacia oleracea).

Thirteen compounds, isolated from spinach (Spinacia oleracea), acted as antimutagens against the dietary carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline in Salmonella typhimurium TA 98. The antimutagens were purified by preparative and micropreparative HPLC from a methanol/water (70:30, v/v) extract of dry spinach (commercial product) after removal of lipophilic compounds such as chlorophylls and carotenoids by solid-phase extraction (SPE). Pure active compounds were identified by instrumental analysis including FT-IR, (1)H and (13)C NMR, UV-vis spectroscopy, and mass spectrometry. All of these compounds were flavonoids and related compounds that could be attributed to five groups: (A, methylenedioxyflavonol glucuronides) 5,3'-dihydroxy-4'-methoxy-6,7-methylenedioxyflavonol 3-O-beta-glucuronide (compound 1), 5,2',3'-trihydroxy-4'-methoxy-6,7-methylenedioxyflavonol 3-O-beta-glucuronide (compound 2), 5-hydroxy-3',4'-dimethoxy-6,7-methylenedioxyflavonol 3-O-beta-glucuronide (compound 3); (B, flavonol glucuronides) 5,6,3'-trihydroxy-7,4'-dimethoxyflavonol 3-O-beta-glucuronide (compound 4), 5,6-dihydroxy-7,3',4'-trimethoxyflavonol 3-O-beta-glucuronide (compound 5); (C, flavonol disaccharides) 5,6,4'-trihydroxy-7,3'-dimethoxyflavonol 3-O-disaccharide (compound 6), 5,6,3',4'-tetrahydroxy-7-methoxyflavonol 3-O-disaccharide (compounds 7 and 8); (D, flavanones) 5,8,4'-trihydroxyflavanone (compound 9), 7,8,4'-trihydroxyflavanone (compound 10); (E, flavonoid-related compounds) compounds 11, 12, and 13 with incompletely elucidated structures. The yield of compound 1 was 0.3%, related to dry weight, whereas the yields of compounds 2-13 ranged between 0.017 and 0.069%. IC(50) values (antimutagenic potencies) of the flavonol glucuronides ranged between 24.2 and 58.2 microM, whereas the flavonol disaccharides (compounds 7 and 8), the flavanones (compounds 9 and 10), and the flavonoid-related glycosidic compounds 11-13 were only weakly active. The aglycons of compounds 7 and 8, however, were potent antimutagens (IC(50) = 10.4 and 13.0 microM, respectively).     

Protective effects of 2',4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone to PC12 cells against cytotoxicity induced by hydrogen peroxide

Oxidative stress has been considered as a major cause of cellular injuries in various clinical abnormalities. One of the possible ways to prevent reactive oxygen species (ROS)-mediated cellular injury is dietary or pharmaceutical therapies to augment the endogenous antioxidant defense capacity. The present study found that 2',4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone (DMC), a chalcone isolated from the buds of Cleistocalyx operculatus, possessed cytoprotective activity in PC12 cells treated with H(2)O(2). The results showed that DMC could effectively increase cell viability [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) reduction], decrease the cell apoptotic percentage [annexin V/propidium iodide (AV/PI) assay], prevent the membrane from damage [lactate dehydrogenase (LDH) release], scavenge ROS formation, reduce caspase-3 activity, and attenuate the decrease of mitochondrial membrane potential (MMP) in PC12 cells treated with H(2)O(2). Meanwhile, DMC increased the catalytic activity of superoxide dismutase (SOD) and the cellular amount of glutathione (GSH), decreased the cellular amount of malondialdehyde (MDA), and decreased the production of lipid peroxidation in PC12 cells treated with H(2)O(2).     

Hepatoprotective effects of 2',4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone on CCl4-induced acute liver injury in mice

In this paper, the hepatoprotective effects of 2',4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone (DMC) on CCl(4)-induced acute liver injury in Kunming mice were investigated. DMC was administered intraperitoneally (ip) (5, 10, or 20 mg/kg of body weight) for 7 days prior to the administration of CCl(4) (0.1%, ip). Pretreatment with DMC significantly decreased activities of serum hepatic enzymes, namely alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, alkaline phosphatase, γ-glutamyl transferase, and total bilirubin, and decreased the elevation of lipid peroxidation, malondialdehyde, reactive oxygen species, and protein carbonyl content. Pretreatment with DMC markedly increased activities of enzymatic antioxidants such as superoxide dismutase, catalase, glucose-6-phosphate dehydrogenase, glutathione peroxidase, glutathione S-transferase, and glutathione reductase and increased levels of nonenzymatic antioxidant markers such as reduced glutathione, total sulfhydryl groups, vitamin C, and vitamin E in liver. These results combined with liver histopathology demonstrate that DMC has potential hepatoprotective effects, which may be related to the attenuation of oxidative stress, accelerating the antioxidant cascade and inhibition of lipid peroxidation.     

Structural analysis of a novel antimutagenic compound, 4-Hydroxypanduratin A, and the antimutagenic activity of flavonoids in a Thai spice, fingerroot (Boesenbergia pandurata Schult.) against mutagenic heterocyclic amines.

Six compounds were isolated from fresh rhizomes of fingerroot (Boesenbergia pandurata Schult.) as strong antimutagens toward 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) in Salmonella typhimurium TA98. These compounds were 2',4',6'-trihydroxychalcone (pinocembrin chalcone; 1), 2',4'-dihydroxy-6'-methoxychalcone (cardamonin; 2), 5,7-dihydroxyflavanone (pinocembrin; 3), 5-hydroxy-7-methoxyflavanone (pinostrobin; 4), (2,4,6-trihydroxyphenyl)-[3'-methyl-2'-(3' '-methylbut-2' '-enyl)-6'-phenylcyclohex-3'-enyl]methanone (5), and (2,6-dihydroxy-4-methoxyphenyl)-[3'-methyl-2'-(3' '-methylbut-2' '-enyl)-6'-phenylcyclohex-3'-enyl]methanone (panduratin A; 6). Compound 5 was a novel compound (tentatively termed 4-hydroxypanduratin A), and 1 was not previously reported in this plant, whereas 2-4 and 6 were known compounds. The antimutagenic IC(50) values of compounds 1-6 were 5.2 +/- 0.4, 5.9 +/- 0.7, 6.9 +/- 0.8, 5.3 +/- 1.0, 12.7 +/- 0.7, and 12.1 +/- 0.8 microM in the preincubation mixture, respectively. They also similarly inhibited the mutagenicity of 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). All of them strongly inhibited the N-hydroxylation of Trp-P-2. Thus, the antimutagenic effect of compounds 1-6 was mainly due to the inhibition of the first step of enzymatic activation of heterocyclic amines.     

Identification of 5,7,3',4'-tetramethoxyflavone metabolites in rat urine by the isotope-labeling method and ultrahigh-performance liquid chromatography-electrospray ionization-mass spectrometry

5,7,3',4'-Tetramethoxyflavone (TMF), one of the major polymethoxyflavones (PMFs) isolated from Kaempferia parviflor , has been reported possessing various bioactivities, including antifungal, antimalarial, antimycobacterial, and anti-inflammatory activities. Although several studies on the TMF have been reported, the information about the metabolism of TMF and the structures of TMF metabolites is still not yet clear. In this study, an isotope-labeling method was developed for the identification of TMF metabolites. Three isotope-labeled TMFs (5,7,3',4'-tetramethoxy[3'-D(3)]flavone, 5,7,3',4'-tetramethoxy[4'-D(3)]flavone, and 5,7,3',4'-tetramethoxy[5,4'-D(6)]flavone) were synthesized and administered to rats. The urine samples were collected, and the main metabolites were monitored by ultrahigh-performance liquid chromatography-electrospray ionization-mass spectrometry. Five TMF metabolites were unambiguously identified as 3'-hydroxy-5,7,4'-trimethoxyflavone, 7-hydroxy-5,3',4'-trimethoxyflavone sulfate, 7-hydroxy-5,3',4'-trimethoxyflavone, 4'-hydroxy-5,7,3'-trimethoxyflavone, and 5-hydroxy-7,3',4'-trimethoxyflavone.     

Inhibitory activity against tobacco mosaic virus (TMV) replication of pinoresinol and syringaresinol lignans and their glycosides from the root of Rhus javanica var. roxburghiana

Four new diepoxylignan glycosides, pinoresinol-4'-O-[6' '-O-(E)-feruloyl]-beta-D-glucopyranoside (1), pinoresinol-4'-O-[4' ',6' '-O-(E)-diferuloyl]-beta-D-glucopyranoside (2), pinoresinol-4'-O-[3' ',6' '-O-(E)-diferuloyl]-beta-D-glucopyranoside (3), and syringaresinol- 4'-O-[4' ',6' '-O-(E)-diferuloyl]-beta-D-glucopyranoside (4), together with three known compounds, pinoresinol (5), syringaresinol (6), and pinoresinol-4'-O-beta-D-glucopyranoside (7), were isolated from the n-butanol extract of Rhus javanica var. roxburghiana, and their structures were established using various spectroscopic techniques. Three glycosides (2-4) of the lignans showed moderate inhibition of multiplication of the tobacco mosaic virus.     

N-acetylglutamate and N-acetylaspartate in soybeans (Glycine max L.), maize (Zea mays L.), [corrected] and other foodstuffs

N-Acetylglutamate (NAG) and N-acetylaspartate (NAA) are amino acid derivatives with reported activities in a number of biological processes. However, there is no published information on the presence of either substance in foodstuffs. We developed a method for extracting and quantifying NAG and NAA from soybean seeds and maize grain using ultra performance liquid chromatography-electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS). The lower limit of quantification for both NAG and NAA was 1 ng/mL. The method was then utilized to quantify NAG and NAA in other foodstuffs (fruits, vegetables, meats, grains, milk, coffee, tea, cocoa, and others). Both NAG and NAA were present in all of the materials analyzed. The highest concentration of NAG was found in cocoa powder. The highest concentration of NAA was found in roasted coffee beans. Both NAG and NAA were found at quantifiable concentrations in all foods tested indicating that these two acetylated amino acids are common components of the human diet.     

Bioactive lignans from a cultivar of Helianthus annuus

A bioassay-guided fractionation of water extracts from Helianthus annuus cv. SH-222 was carried out. Ten lignans and a phenylpropanoid were isolated from the polar bioactive fractions of H. annuus. This study is the first to report lignans as constituents of sunflower and is the first time that tanegool has been isolated as a natural aglycone. Additionally, we report biological activities of the isolated compounds. The general bioactivity has been evaluated using the wheat coleoptiles bioassay. The phytotoxic activities of compounds pinoresinol, lariciresinol, dihydro-dehydrodiconiferilic alcohol, and l-(4'-hydroxy-3'-methoxyphenyl)-2-[4' '-(3hydroxypropyl)-2' '-methoxyphenoxy]propane-l,3-diol were also evaluated in a bioassay on the standard target species. The structure-activity relationships are discussed.     

Phytotoxic activity of bibenzyl derivatives from the orchid Epidendrum rigidum

A whole plant chloroform-methanol extract of the orchid Epidendrum rigidum inhibited radicle growth of Amaranthus hypochondriacus seedlings (IC50 = 300 microg/mL). Bioassay-guided fractionation furnished four phytotoxins, namely, gigantol (1), batatasin III (2), 2,3-dimethoxy-9,10-dihydrophenathrene-4,7-diol (9), and 3,4,9-trimethoxyphenanthrene-2,5-diol (11), along with the known flavonoids apigenin, vitexin, and isovetin and the triterterpenoids 24,24-dimethyl-9,19-cyclolanostane-25-en-3beta-ol (14) and 24-methyl-9,19-cyclolanostane-25-en-3beta-ol (15). Stilbenoids 1, 2, 9, and 11 inhibited radicle growth of A. hypochondriacus with IC50 values of 0.65, 0.1, 0.12, and 5.9 microM, respectively. Foliar application of gigantol (1) at 1 microM to 4 week old seedlings of A. hypochondriacus reduced shoot elongation by 69% and fresh weight accumulation by 54%. Bibenzyls 1 and 2, as well as synthetic analogues 4'-hydroxy-3,3',5-trimethoxybibenzyl (3), 3,3',4',5-tetramethoxybibenzyl (4), 3,4'-dihydroxy-5-methoxybibenzyl (5), 3'-O-methylbatatasin III (6), 3,3',5-trihydroxybibenzyl (7), and 3,4',5-trihydroxybibenzyl (8), were tested for phytotoxicity in axenic cultures of the small aquatic plant Lemna pausicostata. All bibenzyls derivatives except 7 and 8 inhibited growth and increased cellular leakage with IC50 values of 89.9-180 and 89.9-166 microM, respectively. The natural and synthetic bibenzyls showed marginal cytotoxicity on animal cells. The results suggest that orchid bibenzyls may be good lead compounds for the development of novel herbicidal agents.     

Prenylated flavanones isolated from flowers of Azadirachta indica (the neem tree) as antimutagenic constituents against heterocyclic amines

Four prenylated flavanones were isolated from the methanol extract of the flowers of Azadirachta indica (the neem tree) as potent antimutagens against Trp-P-1 (3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole) in the Salmonella typhimurium TA98 assay by activity-guided fractionation. Spectroscopic properties revealed that those compounds were 5,7,4'-trihydroxy-8-prenylflavanone (1), 5,4'-dihydroxy-7-methoxy-8-prenylflavanone (2), 5,7,4'-trihydroxy-3',8-diprenylflavanone (3), and 5,7,4'-trihydroxy-3',5'-diprenylflavanone (4). All isolated compounds were found for the first time in this plant. The antimutagenic IC(50) values of compounds 1-4 were 2.7 +/- 0.1, 3.7 +/- 0.1, 11.1 +/- 0.1, and 18.6 +/- 0.1 microM in the preincubation mixture, respectively. These compounds also similarly inhibited the mutagenicity of Trp-P-2 (3-amino-1-methyl-5H-pyrido[4,3-b]indole) and PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine). All of the compounds 1-4 strongly inhibited ethoxyresorufin O-dealkylation activity of cytochrome P450 1A isoforms, which catalyze N-hydroxylation of heterocyclic amines. However, compounds 1-4 did not show significant inhibition against the direct-acting mutagen NaN(3). Thus, the antimutagenic effect of compounds 1-4 would be mainly based on the inhibition of the enzymatic activation of heterocyclic amines.     

Formation of aflatoxin derivatives on thin layer chromatographic plates.

Rapid confirmation of the presence of aflatoxins B-1 and G-1 in foods is provided by reaction with trifluoroacetic acid at the origin of a thin layer chromatographic plate. The procedure has been used successfully with various nuts, grains, coffee and cocoa beans, and other foods.     

Flavone C-glycoside,phenolic acid,and nitrogen contents in leaves of barley subject to organic fertilization treatments

From the leaves of barley, Hordeum vulgare, one new flavone C-glucoside and three known flavone glucosides were isolated and characterized by (1)H and (13)C NMR and MALDI-TOF-MS. The novel flavone C-glucoside was isovitexin 7-O-beta-[6' "-O-(E)-p-coumaroyl]glucoside (6' "-coumaroylsaponarin), and the known compounds were isovitexin 7-O-beta-[6' "-O-(E)-feruloyl]glucoside, isoorientin 7-O-beta-[6' "-O-(E)-feruloyl]glucoside, and tricin 7-O-beta-glucoside. The sum of all the flavone glycosides and soluble phenolic acids in the leaves decreased with increased rate of plant nutrients given in animal manure and with increased crop yield. All of the major phenylpropanoids showed the same general response to nutrient level. The concentration of nitrogen in the leaves was not directly related to nutrient application or to contents of phenylpropanoids.     

Further investigation into maple syrup yields 3 new lignans, a new phenylpropanoid, and 26 other phytochemicals

Maple syrup is made by boiling the sap collected from certain maple ( Acer ) species. During this process, phytochemicals naturally present in tree sap are concentrated in maple syrup. Twenty-three phytochemicals from a butanol extract of Canadian maple syrup (MS-BuOH) had previously been reported; this paper reports the isolation and identification of 30 additional compounds (1-30) from its ethyl acetate extract (MS-EtOAc) not previously reported from MS-BuOH. Of these, 4 compounds are new (1-3, 18) and 20 compounds (4-7, 10-12, 14-17, 19, 20, 22-24, 26, and 28-30) are being reported from maple syrup for the first time. The new compounds include 3 lignans and 1 phenylpropanoid: 5-(3″,4″-dimethoxyphenyl)-3-hydroxy-3-(4'-hydroxy-3'-methoxybenzyl)-4-(hydroxymethyl)dihydrofuran-2-one (1), (erythro,erythro)-1-[4-[2-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-1-(hydroxymethyl)ethoxy]-3,5-dimethoxyphenyl]-1,2,3-propanetriol (2), (erythro,threo)-1-[4-[2-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-1-(hydroxymethyl)ethoxy]-3,5-dimethoxyphenyl]-1,2,3-propanetriol (3), and 2,3-dihydroxy-1-(3,4- dihydroxyphenyl)-1-propanone (18), respectively. In addition, 25 other phenolic compounds were isolated including (threo,erythro)-1-[4-[(2-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-1-(hydroxymethyl)ethoxy]-3-methoxyphenyl]-1,2,3-propanetriol (4), (threo,threo)-1-[4-[(2-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-1-(hydroxymethyl)ethoxy]-3-methoxyphenyl]-1,2,3-propanetriol (5), threo-guaiacylglycerol-β-O-4'-dihydroconiferyl alcohol (6), erythro-1-(4-hydroxy-3-methoxyphenyl)-2-[4-(3-hydroxypropyl)-2,6-dimethoxyphenoxy]-1,3-propanediol (7), 2-[4-[2,3-dihydro-3-(hydroxymethyl)-5-(3-hydroxypropyl)-7-methoxy-2-benzofuranyl]-2,6-dimethoxyphenoxy]-1-(4-hydroxy-3-methoxyphenyl)-1,3-propanediol (8), acernikol (9), leptolepisol D (10), buddlenol E (11), (1S,2R)-2-[2,6-dimethoxy-4-[(1S,3aR,4S,6aR)-tetrahydro-4-(4-hydroxy-3,5-dimethoxyphenyl)-1H,3H-furo[3,4-c]furan-1-yl]phenoxy]-1-(4-hydroxy-3-methoxyphenyl)-1,3-propanediol (12), syringaresinol (13), isolariciresinol (14), icariside E4 (15), sakuraresinol (16), 1,2-diguaiacyl-1,3-propanediol (17), 2,3-dihydroxy-1-(4-hydroxy-3,5-dimethoxyphenyl)-1-propanone (19), 3-hydroxy-1-(4-hydroxy-3,5-dimethoxyphenyl)propan-1-one (20), dihydroconiferyl alcohol (21), 4-acetylcatechol (22), 3',4',5'-trihydroxyacetophenone (23), 3,4-dihydroxy-2-methylbenzaldehyde (24), protocatechuic acid (25), 4-(dimethoxymethyl)pyrocatechol (26), tyrosol (27), isofraxidin (28), and 4-hydroxycatechol (29). One sesquiterpene, phaseic acid (30), which is a known metabolite of the phytohormone abscisic acid, was also isolated from MS-EtOAc. The antioxidant activities of MS-EtOAc (IC(50) = 75.5 μg/mL) and the pure isolates (IC(50) ca. 68-3000 μM) were comparable to that of vitamin C (IC(50) = 40 μM) and the synthetic commercial antioxidant butylated hydroxytoluene (IC(50) = 3000 μM), in the diphenylpicrylhydrazyl radical scavenging assay. The current study advances scientific knowledge of maple syrup constituents and suggests that these diverse phytochemicals may impart potential health benefits to this natural sweetener.     

Regioselective dimerization of ferulic acid in a micellar solution.

Dehydrodimers of hydroxycinnamates play an important role in the cross-linking of plant cell walls. An aqueous solution of quaternary ammonium salts with a long aliphatic chain is known to spontaneously organize itself into micelles with the ionic part at the outer sphere. It is shown that regioisomeric ferulic acid dehydrodimers can be obtained in one step from trans-ferulic acid after attachment to these micelles and using the biomimetic peroxidase-H2O2 system. The surfactant hexadecyltrimethylammonium hydroxide yielded trans-4-(4-hydroxy-3-methoxybenzylidene)-2-(4-hydroxy-3-methoxyphenyl)-5-oxotetrahydrofuran-3-carboxylic acid (25%), (E,E)-4,4'-dihydroxy-5,5'-dimethoxy-3,3'-bicinnamic acid (21%), and trans-5-[(E)-2-carboxyvinyl]-2-(4-hydroxy-3-methoxyphenyl)-7-methoxy-2,3-dihydrobenzofuran-3-carboxylic acid (14%), whereas the surfactant tetradecyltrimethylammonium bromide gave 4-cis, 8-cis-bis(4-hydroxy-3-methoxyphenyl)-3,7-dioxabicyclo[3.3.0]octane-2,6-dione (18%) as the main product. The use of micelles appears to be not only a new way to synthesize regioisomeric ferulic acid dehydrodimers but may also help to understand the regiospecificity of dimeric hydroxycinnamate formation in vivo.     

Anti-inflammatory properties of clovamide and Theobroma cacao phenolic extracts in human monocytes: evaluation of respiratory burst, cytokine release, NF-κB activation, and PPARγ modulation

There is a great interest in the potential health benefits of biologically active phenolic compounds in cocoa (Theobroma cacao) and dark chocolate. We investigated the anti-inflammatory potential of clovamide (a N-phenylpropenoyl-L-amino acid amide present in cocoa beans) and two phenolic extracts from unroasted and roasted cocoa beans, by evaluating superoxide anion (O(2)(-)) production, cytokine release, and NF-κB activation in human monocytes stimulated by phorbol 12-myristate 13-acetate (PMA). The effects of rosmarinic acid are shown for comparison. Clovamide and rosmarinic acid inhibited PMA-induced O(2)(-) production and cytokine release (with a bell-shaped curve and maximal inhibition at 10-100 nM), as well as PMA-induced NF-κB activation; the two cocoa extracts were less effective. In all tests, clovamide was the most potent compound and also enhanced peroxisome proliferator-activated receptor-γ (PPARγ) activity, which may exert anti-inflammatory effects. These findings indicate clovamide as a possible bioactive compound with anti-inflammatory activity in human cells.     

Preparation and promotion of fruit growth in kiwifruit of fluorinated N-phenyl-N'-1,2,3-thiadiazol-5-yl ureas

Seventeen phenyl-fluorinated analogues of thidiazuron [N-phenyl-N'-(1,2,3-thiadiazol-5-yl)urea, TDZ] have been prepared and characterized. The effects of each fluorinated urea on growth and quality of kiwifruits (Actinidia deliciosa) were evaluated by comparison with untreated (control) and TDZ-treated fruits. The results obtained showed a clear dependence of the growth-promoting activity of these fluorinated ureas on the pattern and degree of fluorine substitution in the phenyl ring. The most effective for promoting fruit growth was N-(2,3,5,6-tetrafluorophenyl)-N'-(1',2',3'-thiadiazol-5'-yl)urea at 25 ppm (at harvest, treated fruits were 58% heavier than untreated ones) followed by N-(3,5-difluorophenyl)-N'-(1',2',3'-thiadiazol-5'-yl)urea at 10 ppm (50%). Comparatively, TDZ-treated fruits were 31% (10 ppm) and 38% (25 ppm) heavier than untreated ones. The results also indicate that the effects of the more active phenyl-fluorinated ureas on some standard quality parameters of fruits, for example, percent of fruit dry matter content, soluble solids contents, total titratable acids, shape, and internal structure, are similar to those of TDZ. Quantitative structure-activity relationships have been derived for the fruit growth promoting activity of the phenyl-fluorinated analogues of TDZ.