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Antibacterial and antifungal compounds from Kigelia pinnata.

A biologically monitored fractionation of the methanolic extracts of the root and fruits of Kigelia pinnata D.C. led to the isolation of the naphthoquinones kigelinone (1), isopinnatal (2), dehydro-alpha-lapachone (3), and lapachol (4) and the phenylpropanoids p-coumaric acid (5) and ferulic acid (6) as the compounds responsible for the observed antibacterial and antifungal activity of the root and kigelinone (1) and caffeic acid (7) from the fruits of this plant.

Anthraquinones↗

15-LOX-1: a novel molecular target of nonsteroidal anti-inflammatory drug-induced apoptosis in colorectal cancer cells.

BACKGROUND: Nonsteroidal anti-inflammatory drugs (NSAIDs) appear to act via induction of apoptosis-programmed cell death-as potential colorectal cancer chemopreventive agents. NSAIDs can alter the production of different metabolites of polyunsaturated fatty acids (linoleic and arachidonic acids) through effects on lipoxygenases (LOXs) and cyclooxygenases. 15-LOX-1 is the main enzyme for metabolizing colonic linoleic acid to 13-S-hydroxyoctadecadienoic acid (13-S-HODE), which induces apoptosis. In human colorectal cancers, the expression of this enzyme is reduced. NSAIDs can increase 15-LOX enzymatic activity in normal leukocytes, but their effects on 15-LOX in neoplastic cells have been unknown. We tested the hypothesis that NSAIDs induce apoptosis in colorectal cancer cells by increasing the protein expression and enzymatic activity of 15-LOX-1. METHODS: We assessed 15-LOX-1 protein expression and enzymatic activity, 13-S-HODE levels, and 15-LOX-1 inhibition in association with cellular growth inhibition and apoptosis induced by NSAIDs (primarily sulindac and NS-398) in two colorectal cancer cell lines (RKO and HT-29). All P values are two-sided. RESULTS: Sulindac and NS-398 progressively increased 15-LOX-1 protein expression in RKO cells (at 24, 48, and 72 hours) in association with subsequent growth inhibition and apoptosis. Increased 13-S-HODE levels and the formation of 15-hydroxyeicosatetraenoic acid on incubation of the cells with the substrate arachidonic acid confirmed the enzymatic activity of 15-LOX-1. Inhibition of 15-LOX-1 in RKO cells by treatment with caffeic acid blocked NS-398-induced 13-S-HODE production, cellular growth inhibition, and apoptosis (P =. 007, P<.0001, and P<.0001, respectively); growth inhibition and apoptosis were restored by adding exogenous 13-S-HODE (P<.0001 for each) but not its parent compound, linoleic acid (P = 1.0 for each). Similar results occurred with other NSAIDs and in HT-29 cells. CONCLUSIONS: These data identify 15-LOX-1 as a novel molecular target of NSAIDs for inducing apoptosis in colorectal carcinogenesis.

Anti-Inflammatory Agents, Non-Steroidal↗

Influence of cooking process on phenolic marker compounds of vegetables.

Phenolic compounds are secondary plant metabolites which have long been associated with flavor and color characteristics of fruits and vegetables. These phenolic compounds attract great interest due to their postulated health protecting properties. However, adequate intakes and absorption rate of phenolic compounds are necessary for these beneficial effects. Until now, little is known about alterations of phenolic compounds content by the cooking process. In the present study, the influence of different volumes of cooking water on the amount of selected phenolic marker compounds resting in the vegetables was assessed. In zucchini, rutin was quantified as a marker for flavonoid glycosides. Chlorogenic acid, representative of phenolic acids was analyzed in carrots. In beans, rutin and quercitrin, both belonging to flavonoid glycosides, were investigated. In potatoes, chlorogenic and caffeic acid were determined. The cooking of zucchini, beans and carrots with smaller amounts of water resulted in significant higher content of phenolic phytochemicals in the vegetables compared to cooking with larger water volumes. For potatoes, which showed great variations in content of phenolic acids after cooking, no significant differences in phenolic acids was observed. It can be concluded from these observations, that real intakes of phenolic compounds from cooked vegetables are lower and that the amounts consumed are therefore overestimated.

Caffeic Acids↗

Phenolic acids inhibit chloroplast mutagenesis in Euglena gracilis.

The mutagenicity (bleaching activity) of ofloxacin (43 microM) and acridine orange (AO) (13.5 microM) in Euglena gracilis is inhibited by plant phenolics. Caffeic acid (CA), p-coumaric acid (PCA), ferulic acid (FA) and gentisic acid (GA) (25, 50, 100 and 250 microM) exhibited a significant concentration-dependent inhibitory effect against ofloxacin-induced mutagenicity, which was very effectively eliminated by the highest concentration of all four of those phenolic acids. The mutagenicity of AO was also significantly reduced in the presence of CA, PCA and FA (25, 50, 100 and 250 microM). However, GA exhibited no significant activity, even at the concentration of 250 microM. Based on the UV spectrophotometric measurements, we suggest that the antimutagenic effect of CA, PCA, FA and GA resulted from the scavenging of reactive oxygen species (ROS) produced by ofloxacin. On the other hand, the reduction of AO-induced mutagenicity correlates with the binding capabilities of CA, PCA and FA, with the exception of GA.

Acridine Orange↗

In vitro and in vivo evaluation of caffeic and ferulic acids as topical photoprotective agents.

Topically-applied antioxidant drugs represent a successful strategy for protecting the skin against UV-mediated oxidative damage. However, they can afford to the skin a satisfactory photoprotection only if able to permeate through the stratum corneum and thus to reach deeper cutaneous layers. Caffeic and ferulic acids, dissolved in saturated aqueous solutions at pH 3 or 7.2, have been tested for their capability to permeate through excised human skin mounted in Franz cells. At both pH values, ferulic and, at a lower degree, caffeic acids appeared able to permeate through the stratum corneum. The known higher lipophilicity of ferulic acid may explain the fact that it permeates through the stratum corneum better than caffeic acid. However, vehicle pH values proved to have no influence on biophenol skin permeation profile; this observed lack of pH effect may reflect the drug higher concentration attainable in saturated solutions at high pH. On the basis of the findings obtained in these in vitro experiments, we designed the schedule of a series of in vivo experiments, carried out to evaluate the ability of caffeic and ferulic acids to reduce, in healthy human volunteers, UVB-induced skin erythema, monitored by means of reflectance spectrophotometry. Caffeic and ferulic acids, dissolved in saturated aqueous solution pH 7.2, proved to afford a significant protection to the skin against UVB-induced erythema. To conclude, we have confirmed, by means of in vitro and in vivo experiments, that caffeic and ferulic acids may be successfully employed as topical protective agents against UV radiation-induced skin damage; however their skin absorption is not influenced by the pH of the formulation.

Administration, Topical↗

Purification and characterization of a novel esterase induced by growth of Aspergillus niger on sugar-beet pulp.

An inducible esterase has been isolated from a liquid culture of Aspergillus niger grown on sugar-beet pulp. The enzyme was active on methyl esters of cinnamic acids, caffeic > p-coumaric > ferulic, and is therefore termed a cinnamoyl esterase. The enzyme was not active on methyl sinapinate, a good substrate for ferulic acid esterase III, which was purified previously from A. niger [Faulds and Williamson (1994) Microbiology 140, 779-787]. With methyl caffeate as substrate the enzyme had temperature and pH optima of 50 degrees C and 6.0 respectively, and a specific activity of 96.9 units per mg of protein. The purified protein (native molecular mass 145 000 Da) gave a single heavily stained band on SDS/PAGE, suggesting the protein was a dimer, and seemed to be heavily glycosylated. Isoelectric focusing gave a single band corresponding to a pl of 4.80. The pure enzyme was free of other carbohydrase activities. The activity of the pure enzyme was inhibited by more than 99% after treatment with the serine-specific protease inhibitor aminoethylbenzenesulphonylfluoride (1 mM) for 12 h. The enzyme was capable of releasing ferulic acid from sugar beet pulp.

Ammonium Sulfate↗

Isolation and characterization of Streptomyces sp. NL15-2K capable of degrading lignin-related aromatic compounds.

Strain NL15-2K was isolated from soil by screening for bacteria capable of catabolizing lignin-related aromatic acids. This isolate was identified as a Streptomyces sp. on the basis of morphology and an analysis of its 16S rRNA gene sequence. NL15-2K utilized caffeic acid, coniferyl alcohol, ferulic acid, isovanillic acid, protocatechuic acid, vanillic acid, vanillin, and veratric acid as sole carbon sources.

Benzaldehydes↗

Synthesis of selenium-containing polyphenolic acid esters and evaluation of their effects on antioxidation and 5-lipoxygenase inhibition.

Six novel selenium-containing polyphenolic acid esters were synthesized and evaluated as antioxidants and 5-lipoxygenase inhibitors. Synthesis of the title compounds involved the Mitsunobu reaction of polyphenolic acids with 2-phenylselenoethanol. Compounds and were found to be very effective antioxidants and 5-lipoxygenase inhibitors with activity comparable to or better than caffeic acid (3,4-dihydroxycinnamic acid) phenethyl ester (CAPE).

Antioxidants↗

Modified 2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (abts) method to measure antioxidant capacity of Selected small fruits and comparison to ferric reducing antioxidant power (FRAP) and 2,2'-diphenyl-1-picrylhydrazyl (DPPH) methods.

The measurement of antioxidant capacity in fruits differs from that of other biological samples due to their low pH and very low lipophilic antioxidant capacity. In this report, we present a modified 2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) method for fruits and compare its performance with the other commonly used antioxidant methods of 2,2'-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP). The antioxidant capacity and reaction kinetics of four phenolic compounds, two antioxidant standards, and five fruits were also investigated. The modified ABTS method prepared at a pH of 4.5 with sodium acetate buffer is highly stable and easily applied to fruit samples as compared to the standard (pH 7.4) version. The measured antioxidant capacity of samples varied with the assay method used, pH, and time of reaction. Traditional antioxidant standards (trolox, ascorbic acid) displayed stable, simple reaction kinetics, which allowed end point analysis with all of assays. Of the phenolic compounds examined, chlorogenic and caffeic acids exhibited the most complex reaction kinetics and reaction rates that precluded end point analysis while gallic acid and quercetin reached stable end points. All fruit extracts exhibited complex and varied kinetics and required long reaction times to approach an end point. Because the antioxidant capacity of fruit extracts is a function of the array of individual antioxidants present, accurate comparisons among fruit samples require that reaction times be standardized and of sufficient length to reach steady state conditions and that more than one assay be used to describe the total antioxidant activity of fruit samples.

Antioxidants↗

LC/MS/MS characterization of phenolic constituents in dried plums.

Dried plums are known as a healthy food in the West and used as medicine in India. They have been characterized by high concentrations of phenolic compounds, which are believed to play a crucial role in protection against various age-related diseases. Liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) with four different conditions was used to analyze the phytochemicals in commercial dried plums. The major components were neochlorogenic acid and cryptochlorogenic acid. Forty minor components were characterized by their MS/MS spectra and LC retention time. Six of them are novel ester isomers formed by two caffeic acids and one quinic acid. The diagnostic fragmentation patterns of different phenolics are presented on the basis of electrospray ionization (ESI) MS/MS data of components in dried plums and fourteen authentic standards.

Caffeic Acids↗

Molecular cloning, characterization, and expression of a redox-responsive cutinase from Monilinia fructicola (Wint.) Honey.

cDNA clones encoding a cutinase expressed in cutin-induced cultures of the plant pathogen Monilinia fructicola were isolated using a protein-based strategy. The largest cDNA (Mfcut1) was found to contain an open reading frame of 603 bp that predicted a 20.2-kDa protein of 201 amino acids with a 20-amino-acid secretory signal peptide and a pI of 8.4. The predicted protein contained cutinase/lipase consensus sequences with active site serines and potential protein kinase phosphorylation sites. Comparison of the deduced amino sequence from Mfcut1 with other fungal cutinase sequences revealed new features, which include conserved cysteines, C-terminal aromatic residues, and a novel histidine substitution in the D-H active site motif. The presence in the growth medium of antioxidants, such as caffeic acid, suppressed mRNA accumulation and enzyme activity of a cutinase from M. fructicola. MFCUT1 was expressed at high levels as a His-tagged fusion protein in Pichia pastoris and purified to apparent homogeneity in a single step by Ni(2+)-nitrilotriacetic acid affinity chromatography. Analysis of variant MFCUT1 mutants in which the novel serine and histidine residues were replaced by site-directed mutagenesis indicated that these residues had an important effect on enzyme activity.

Amino Acid Sequence↗

Biochemical studies on cloned Bacillus sp. BP-7 phenolic acid decarboxylase PadA.

Sequence analysis of a Bacillus sp. BP-7 recombinant clone coding for a previously described carboxylesterase revealed the presence of an additional ORF with homology to bacterial hydroxycinnamic acid decarboxylases. Analysis of the amino acid sequence of the encoded enzyme revealed the presence of a single, highly conserved domain of 161 amino acids, with a predicted molecular mass of 19,143 Da and a pI of 5.5. Crude cell extracts from the recombinant clone displayed activity on ferulic, p-coumaric and caffeic acids, with no need for added cofactors. The cloned enzyme, named PadA, displayed maximum activity at 40 degrees C and pH 5.5, being stable over a broad range of pH and up to 45 degrees C. HPLC analysis of the products of catalysis revealed the conversion of phenolic acids to their aromatic 4-vinyl derivatives, with no accumulation of other by-products. PadA was found as a homodimer in the parental Bacillus sp. BP-7 strain and its expression was induced by both hydroxycinnamic acids and their corresponding derivative products. The results obtained suggest that the enzyme could be involved in a stress response for conversion of toxic hydroxycinnamic acids released after plant cell wall degradation.

Bacillus↗

Cinnamic acid derivatives as matrices for ultraviolet laser desorption mass spectrometry of proteins.

The paper reports the discovery of three new matrices for the matrix-assisted laser desorption of proteins. These new matrices (sinapinic, ferulic and caffeic acids) are cinnamic acid derivatives that have several practical advantages over the nicotinic acid matrices previously used. These materials form much less intense photochemically generated adduct peaks in the protein quasimolecular ion signal and the adduct peaks that are present are easier to resolve. These matrices produce intense protonated-molecule ions from all of the proteins (over 50) so far examined. These new matrices are also very stable in a vacuum, allowing for their convenient use in very high vacuum applications (e.g., Fourier transform ion cyclotron resonance mass spectrometry).

Animals↗

[Occurrence of chlorogenic acids in potatoes].

Ten varieties of potato cultivated under the same conditions were analyzed for the occurrence of chlorogenic acids by high performance liquid chromatography. 3-O-caffeoylquinic acid (n-chlorogenic acid) is the predominant compound with 22-71 ppm, (on average of 41 ppm) depending on variety. 4-O-caffeoylquinic acid (phi 11 ppm), 5-O-caffeoylquinic acid (phi 7 ppm), 3,4-dicaffeoylquine acid (phi 3 ppm), and at times 3,5-dicaffeoylquinic acid (in traces) were also demonstrated. Caffeic acid compounds are not distributed uniformly in the potato tuber. They occur in strongly decreasing concentration from the outer sections to the inner ones. About 50% of these compounds are located in the potato peel and the adjoining tissue.

Chemical Phenomena↗

Phenolic compounds protect HepG2 cells from oxidative damage: relevance of glutathione levels.

In the present work, the potential hepatoprotective effects of five phenolic compounds against oxidative damages induced by tert-butyl hydroperoxide (t-BHP) were evaluated in HepG2 cells in order to relate in vitro antioxidant activity with cytoprotective effects. t-BHP induced considerable cell damage in HepG2 cells as shown by significant LDH leakage, increased lipid peroxidation, DNA damage as well as decreased levels of reduced glutathione (GSH). All tested phenolic compounds significantly decreased cell death induced by t-BHP (when in co-incubation). If the effects of quercetin are given the reference value 1, the compounds rank in the following order according to inhibition of cell death: luteolin (4.0) > quercetin (1.0) > rosmarinic acid (0.34) > luteolin-7-glucoside (0.30) > caffeic acid (0.21). The results underscore the importance of the compound's lipophilicity in addition to its antioxidant potential for its biological activity. All tested phenolic compounds were found to significantly decrease lipid peroxidation and prevent GSH depletion induced by t-BHP, but only luteolin and quercetin significantly decreased DNA damage. Therefore, the lipophilicity of the natural antioxidants tested appeared to be of even greater importance for DNA protection than for cell survival. The protective potential against cell death was probably achieved mainly by preventing intracellular GSH depletion. The phenolic compounds studied here showed protective potential against oxidative damage induced in HepG2 cells. This could be beneficial against liver diseases where it is known that oxidative stress plays a crucial role.

Cell Line, Tumor↗

Identification of six phenylpropanoids from garlic skin as major antioxidants.

The extract of garlic skins (peels) showed strong antioxidant activity, and some responsible constituents were isolated and identified. Garlic (Allium sativum L.) has been used as an herbal medicine, but there is no report on the health benefits of the skin or peel. In this study, the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity of garlic skin extract was evaluated. Using chromatographic techniques, the active constituents were isolated and subsequently identified. Analyses by high-performance liquid chromatography coupled with a photodiode array detector (HPLC-PDA) suggested that these compounds were phenylpropanoids, which had a characteristic absorbance at 300-320 nm. Liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance analyses allowed the chemical structures of the isolated constituents to be postulated. The proposed compounds were subsequently synthesized and compared with the constituents in the extract using HPLC-PDA and LC-MS. N-trans-Coumaroyloctopamine, N-trans-feruloyloctopamine, guaiacylglycerol-beta-ferulic acid ether, and guaiacylglycerol-beta-caffeic acid ether were identified as were trans-coumaric acid and trans-ferulic acid. Also, the antioxidant activities of these compounds were determined.

Antioxidants↗

Retrobiosynthetic NMR studies with 13C-labeled glucose. Formation of gallic acid in plants and fungi.

The biosynthesis of gallic acid was studied in cultures of the fungus Phycomyces blakesleeanus and in leaves of the tree Rhus typhina. Fungal cultures were grown with [1-13C]glucose or with a mixture of unlabeled glucose and [U-13C6]glucose. Young leaves of R. typhina were kept in an incubation chamber and were supplied with a solution containing a mixture of unlabeled glucose and [U-13C6]glucose via the leaf stem. Isotope distributions in isolated gallic acid and aromatic amino acids were analyzed by one-dimensional 1H and 13C NMR spectroscopy. A quantitative analysis of the complex isotopomer composition of metabolites was obtained by deconvolution of the 13C13C coupling multiplets using numerical simulation methods. This approach required the accurate analysis of heavy isotope chemical shift effects in a variety of different isotopomers and the analysis of long range 13C13C coupling constants. The resulting isotopomer patterns were interpreted using a retrobiosynthetic approach based on a comparison between the isotopomer patterns of gallic acid and tyrosine. The data show that both in the fungus and in the plant all carbon atoms of gallic acid are biosynthetically equivalent to carbon atoms of shikimate. Notably, the carboxylic group of gallic acid is derived from the carboxylic group of an early intermediate of the shikimate pathway and not from the side chain of phenylalanine or tyrosine. It follows that the committed precursor of gallic acid is an intermediate of the shikimate pathway prior to prephenate or arogenate, most probably 5-dehydroshikimate. A formation of gallic acid via phenylalanine, the lignin precursor, caffeic acid, or 3,4, 5-trihydroxycinnamic acid can be ruled out as major pathways in the fungus and in young leaves of R. typhina. The incorporation of uniformly 13C-labeled glucose followed by quantitative NMR analysis of isotopomer patterns is suggested as a general method for biosynthetic studies. As shown by the plant experiment, this approach is also applicable to systems with low incorporation rates.

Carbon Isotopes↗

A Na(+)-dependent mechanism is involved in mucosal uptake of cinnamic acid across the jejunal brush border in rats.

Phenolic acids are present in all plant-derived foods and in most diets. Indirect evidence indicates substantial absorption of phenolic monomers from the gastrointestinal tract. However, the mechanisms involved in the absorptive process are unknown. The present study investigates mucosal uptake of radioactively labeled cinnamic acid as a model substance for monomeric cinnamic acid derivatives (e.g., cinnamic, ferulic or caffeic acid) in the rat jejunum using an in vitro mucosal uptake technique. The results indicate the existence of a Na(+)-dependent saturable transport mechanism for uptake of cinnamic acid across the jejunal brush border membrane. The observed Na+ dependence of jejunal cinnamate uptake seems not to be related to the activity of the Na+,H+ exchanger. Lowering the pH of the incubation medium resulted in a pronounced increase in mucosal cinnamate uptake that can be only partially explained by an increase in nonionic diffusion of cinnamic acid. Furthermore, jejunal uptake of cinnamate seems to be influenced by intracellular HCO3- and/or pH, since the addition of methazolamide to a HCO3(-)- and CO2-free incubation medium significantly inhibited mucosal cinnamate uptake, whereas methazolamide was without an effect in the presence of HCO3- and CO2 in the incubation medium. Unlabeled cinnamic and ferulic acid as well as short-chain fatty acids (acetic, propionic and butyric acid) significantly inhibited Na(+)-dependent uptake of radioactivity labeled cinnamic acid.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗