Methods for the isolation and purification of ethanol-insoluble,phenolic esters in Mentha arvensis

Previous kinetic, isotopic studies have suggested that ‘insoluble’ phenolic esters may be precursors of lignin. Heretofore, the ‘insoluble’ esters have been detected by the chromatographic examinations of gross hydrolysis products of ethanol-insoluble resides and/or acetone powders. We have developed new methods for the isolation and purification of certain of the ethanol-insoluble, phenolic esters of Mentha arvensis. ‘Insoluble’ conjugates of caffeic, ferulic and p-coumaric acids were purified and were shown to be electro-phoretically and chromatographically homogeneous. These compounds were distinguished on the basis of their anionic mobility at pH 1·9. A second pool of caffeic acid was associated with a high MW fraction. Two acylated anthocyanins containing p-coumaric acid and caffeic acid were also obtained from acetone powders.     

Phenolic Content in Male and Female Carica papaya: A Possible Physiological Marker for Sex Identification of Vegetative Seedlings

The phenolic composition was determined in the leaves, petioles and bark tissues of male and female plants of two papaya cultivars. The same kind of phenolics were isolated from the male and female plants. However, a marked difference was observed between the plant organs of different cultivars. The important free and bound phenolics extracted after acidic and alkaline hydrolysis were caffeic acid, gentisic acid, m-coumaric acid, p-coumaric acid, salicylic acid and quercetin. Four phenolic compounds were not identified. The amounts of free, acid-hydrolysable and alkali-hydrolysable phenolic compounds were considerably higher in male plants.     

Effect and analysis of phenolic compounds during somatic embryogenesis induction in Feijoa sellowiana Berg

Summary. The effect of phenolic compounds on somatic embryogenesis in Feijoa sellowiana was analysed. The results showed that caffeic acid (140–560 μM) significantly increased somatic embryogenesis induction compared with the control. The presence of phloridzin, even at lower concentrations (11.5 μM), or caffeic acid or phloroglucinol at concentrations greater than 140.0 and 197.5 μM, respectively, inhibited somatic embryo development beyond the globular stage. When somatic embryos were transferred to the germination medium, the highest rates of germination (81.9%) were obtained with embryos induced in the presence of phloroglucinol (79.0 μM). At all concentrations tested, somatic embryos induced in medium containing phloroglucinol germinated at higher rates than those induced in the presence of caffeic acid. Histological and ultrastructural studies showed that somatic embryos were formed in close association with phenolic-rich cells which, in more advanced stages of development, formed a zone isolating the embryo from the maternal tissue. A comparative analysis of total phenolic content indicated that phenolics reached a peak by the third week of culture, independently of the medium used. However, after that period, the amount of phenolic compounds was significantly higher in explants cultured in the presence of phloroglucinol than in those cultured in the control or in caffeic acid-containing medium. Attempts to identify the type of phenolic compounds showed that flavan-3-ols and gallic acid derivatives were mainly produced in phloroglucinol-containing medium, whereas flavanones and dihydroflavonols were also present in medium containing caffeic acid. Flavones were the main phenols detected in the control. The ways in which phenolic compounds may affect somatic embryogenesis are discussed. Correspondence: J. M. Canhoto, Departamento de Botanica, Faculdade de Ciências e Tecnologia, Universidade de Coimbra, Cal?ada Martim de Freitas, 3001-455 Coimbra, Portugal.     

Biochemical composition of stigmatic exudate of Eichhornia crassipes (Mart.) solms

The stigmatic exudate of Eichhornia crassipes (Mart.) Solms is moderately soluble in water, but is readily soluble in 80% ethanol. Soluble sugars, fructose, sucrose, total phenol, hydroxy phenol, free amino acids, soluble proteins and free fatty acids were found in the exudate. The exudate is devoid of organic acids and alkaloids, while proteins, soluble sugars, free fatty acids and free amino acids were the principal constituents. The stigma possesses detectable quantities of these compounds and shows a progressive increase in the amount of these substances up to the stage just prior to anthesis. The ultraviolet absorption profile of the stigmatic exudate in ethanol shows characteristic absorption peaks and bathochromatic shifts on addition of NaOH. The peaks probably correspond to those of simple phenolic compounds.     

玉米苗中DIMBOA与几种酚酸类物质抑菌活性比较

本文从室内培养的7日龄玉米幼苗中提取、分离、鉴定了抗性次生化合物丁布(2,4-d ihydroxy-7-m ethoxy-2H-1,4-benzoxazin-3(4H)-one,D IMBOA),并就该物质对玉米纹枯病病原菌立枯丝核菌(Rhizoctonia solani)的活性与三种酚酸类物质(阿魏酸、对羟基肉桂酸和咖啡酸)进行了离体比较研究。结果表明,丁布(D IMBOA),对立枯丝核菌有很强的生物活性,在浓度为50μg/mL时即可抑制立枯丝核菌菌丝的生长,抑制率为18.52%。阿魏酸、对羟基肉桂酸和咖啡酸,这三种酚酸在浓度250μg/mL时对立枯丝核菌菌丝的生长有抑制作用,抑制率分别为26.30%、8.50%和6.30%。不仅如此,丁布与对羟基肉桂酸之间、以及三种酚酸两两组合之间还存在一定的协同作用。在浓度相等的情况下,丁布与对羟基肉桂酸的等量混合液的抑菌率显著高于这两种物质单独存在时的抑菌率之和;同样,对羟基肉桂酸与阿魏酸的等量混合液的抑菌率比单一的对羟基肉桂酸溶液的抑菌率高18.89%,比单一的阿魏酸溶液的抑菌率高13.33%;对羟基肉桂酸与咖啡酸的等量混合液,抑菌率比两者单独试验时分别高9.63%和14.83%;阿魏酸与咖啡酸的混合液,抑菌率比两酸单独试验时分别高11.48%和22.23%。这一结果提示植物体内产生适当比例不同次生化合物的组合对植物抗病性的提高是至关重要的。     

Comparison of the phenolic composition of fruit juices by single step gradient HPLC analysis of multiple components versus multiple chromatographic runs optimised for individual families

After minimal sample preparation, two different HPLC methodologies, one based on a single gradient reversed-phase HPLC step, the other on multiple HPLC runs each optimised for specific components, were used to investigate the composition of flavonoids and phenolic acids in apple and tomato juices. The principal components in apple juice were identified as chlorogenic acid, phloridzin, caffeic acid and p-coumaric acid. Tomato juice was found to contain chlorogenic acid, caffeic acid, p-coumaric acid, naringenin and rutin. The quantitative estimates of the levels of these compounds, obtained with the two HPLC procedures, were very similar, demonstrating that either method can be used to analyse accurately the phenolic components of apple and tomato juices. Chlorogenic acid in tomato juice was the only component not fully resolved in the single run study and the multiple run analysis prior to enzyme treatment. The single run system of analysis is recommended for the initial investigation of plant phenolics and the multiple run approach for analyses where chromatographic resolution requires improvement.     

COMPOUNDS FROM THE STIGMAS OF TEN SPECIES

Extracts of the intact stigmatic surface of 10 species from different families contained little or no free sugar. The principal components of the extracts consisted of phenolic (anthocyanins, flavonoids, cinnamic acids) and lipid compounds. The phenolic compounds occurred as glycosides or esters. Phenolic compounds could inhibit insects and diseases, stimulate or inhibit pollen germination, or provide specificity to the pollen selection-germination process of the stigma by interacting with growth-regulators or related compounds.     

In Vitro Antioxidant Profile of Phenolic Acid Derivatives

Several caffeic acid esters isolated from propolis exhibit interesting antioxidant properties, but their in vivo use is compromised by hydrolysis of the ester bond in the gastrointestinal tract. Therefore, a series of caffeic acid amides were synthesized and their in vitro antioxidant profile was determined. A series of hydroxybenzoic acids, hydroxycinnamic acids, and the synthesized caffeic acid amides were tested for both their 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging and microsomal lipid peroxidation-inhibiting activity. Some of the highly active antioxidants were further tested by means of electron paramagnetic resonance for their hydroxyl radical scavenging activity. Since a promising antioxidant compound should show a lipid peroxidation-inhibiting activity at micromolar level and a low cytotoxicity, the cytotoxicity of the phenolic compounds was also studied. In all the assays used, the caffeic acid anilides and the caffeic acid dopamine amide showed an interesting antioxidant activity.     

In vitro antioxidant profile of phenolic acid derivatives

Several caffeic acid esters isolated from propolis exhibit interesting antioxidant properties, but their in vivo use is compromised by hydrolysis of the ester bond in the gastrointestinal tract. Therefore, a series of caffeic acid amides were synthesized and their in vitro antioxidant profile was determined. A series of hydroxybenzoic acids, hydroxycinnamic acids, and the synthesized caffeic acid amides were tested for both their 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging and microsomal lipid peroxidation-inhibiting activity. Some of the highly active antioxidants were further tested by means of electron paramagnetic resonance for their hydroxyl radical scavenging activity. Since a promising antioxidant compound should show a lipid peroxidation-inhibiting activity at micromolar level and a low cytotoxicity, the cytotoxicity of the phenolic compounds was also studied. In all the assays used, the caffeic acid anilides and the caffeic acid dopamine amide showed an interesting antioxidant activity.     

Interaction of Moisture Stress and Three Phenolic Compounds on Lettuce Seed Germination

No interactions between water stress and three phenolic acids(p-coumaric, caffeic and ferulic acids) on lettuce (Lactucasativa L. var. Grand Rapids) seed germination were found. Probitanalysis indicated that mechanisms of action of water stressand the phenolic inhibitors were similar. The relative effectivenessof the compounds was p-coumaric > ferulic > caffeic. Nointeraction was found between p-coumaric and ferulic acid, whereasantagonism was found between caffeic acid and each of the othertwo phenolic acids. Lactuca sativa L., lettuce, germination, phenolic compounds, moisture stress, allelopathy, seed     

Comparison of the phenolic composition of fruit juices by single step gradient HPLC analysis of multiple components versus multiple chromatographic runs optimised for individual families

After minimal sample preparation, two different HPLC methodologies, one based on a single gradient reversed-phase HPLC step, the other on multiple HPLC runs each optimised for specific components, were used to investigate the composition of flavonoids and phenolic acids in apple and tomato juices. The principal components in apple juice were identified as chlorogenic acid, phloridzin, caffeic acid and p-coumaric acid. Tomato juice was found to contain chlorogenic acid, caffeic acid, p-coumaric acid, naringenin and rutin. The quantitative estimates of the levels of these compounds, obtained with the two HPLC procedures, were very similar, demonstrating that either method can be used to analyse accurately the phenolic components of apple and tomato juices. Chlorogenic acid in tomato juice was the only component not fully resolved in the single run study and the multiple run analysis prior to enzyme treatment. The single run system of analysis is recommended for the initial investigation of plant phenolics and the multiple run approach for analyses where chromatographic resolution requires improvement.     

The phenolic vir gene inducer ferulic acid is O-demethylated by the VirH2 protein of an Agrobacterium tumefaciens Ti plasmid

Some or possibly all Ti plasmids of Agrobacterium tumefaciens encode a bicistronic operon designated virH, which encodes two proteins, VirH1 and VirH2, that resemble a family of cytochrome P450-type monooxygenases. Expression of this operon is induced by a family of phenolic compounds that induce all other operons within the vir regulon. We hypothesized that either or both of these proteins might metabolize some or all of these phenolic compounds. We therefore tested induction of a vir promoter by a variety of phenolic compounds in isogenic strains that express or lack virH1 and virH2. Although some compounds were equally effective inducers regardless of the virH status, other compounds induced vir expression far more effectively in the virH mutant than in the virH-proficient host. For all tested compounds, VirH2 appeared to be solely responsible for this effect. One such compound, ferulic acid, was chosen for biochemical analysis. Ferulic acid was degraded by a VirH-proficient host but not by a VirH mutant. The wild-type strain released large amounts of a more hydrophilic compound into the cell supernatant. This compound was tested by mass spectroscopy, nuclear magnetic resonance and UV spectroscopy and found to consist of caffeic acid. This indicates that wild-type strains convert virtually all added ferulic acid to caffeic acid, and that VirH2 is essential for this O-demethylation reaction. Ferulic acid was far more toxic than caffeic acid to the wild-type strain, although the wild-type strain was more resistant to ferulic acid than was the virH mutant. Caffeic acid was slowly removed from the broth, suggesting further metabolic reactions.     

DNA-breaking versus DNA-protecting activity of four phenolic compounds in vitro

Given the paradoxical effects of phenolics in oxidative stress, we evaluated the relative pro-oxidant and antioxidant properties of four natural phenolic compounds in DNA nicking. The phenolic compounds differed dramatically in their ability to nick purified supercoiled DNA, with the relative DNA nicking activity in the order: 1,2,4-benzenetriol (100% nicking) > gallic acid > caffeic acid > gossypol (20% nicking). Desferrioxamine (0.02 mM) decreased DNA strand breakage by each phenolic, most markedly with gallate (85% protection) and least with caffeic acid (26% protection). Addition of metals accelerated DNA nicking, with copper more effective (～5-fold increase in damage) than iron with all four phenolics. Scavengers revealed the participation of specific oxygen-derived active species in DNA breakage. Hydrogen peroxide participated in all cases (23–90%). Hydroxyl radicals were involved (32–85%), except with 1,2,4-benzenetriol. Superoxide participated (81–86%) with gallic acid and gossypol, but not with caffeic acid or 1,2,4-benzenetriol. With 1,2,4-benzenetriol, scavengers failed to protect significantly except in combination. Thus, in the presence of desferrioxamine, catalase or superoxide dismutase inhibited almost completely. When DNA breakage was induced by Fenton's reagent (ascorbate plus iron) the two catechols (caffeic acid and gossypol) were protective, whereas the two triols (1,2,4-benzenetriol and gallic acid) exacerbated damage.     

Bacteria from wheat and cucurbit plant roots metabolize PAHs and aromatic root exudates: Implications for rhizodegradation

The chemical interaction between plants and bacteria in the root zone can lead to soil decontamination. Bacteria that degrade polycyclic aromatic hydrocarbons (PAHs) have been isolated from the rhizospheres of plant species with varied biological traits; however, it is not known what phytochemicals promote contaminant degradation. One monocot and two dicotyledon plants were grown in PAH-contaminated soil from a manufactured gas plant (MGP) site. A phytotoxicity assay confirmed greater soil decontamination in rhizospheres when compared to bulk soil controls. Bacteria were isolated from plant roots (rhizobacteria) and selected for growth on anthracene and chrysene on PAH-amended plates. Rhizosphere isolates metabolized 3- and 4-ring PAHs and PAH catabolic intermediates in liquid incubations. Aromatic root exudate compounds, namely flavonoids and simple phenols, were also substrates for isolated rhizobacteria. In particular, the phenolic compounds—morin, caffeic acid, and protocatechuic acid—appear to be linked to bacterial degradation of 3- and 4-ring PAHs in the rhizosphere.     

DNA-breaking versus DNA-protecting activity of four phenolic compounds in vitro

Given the paradoxical effects of phenolics in oxidative stress, we evaluated the relative pro-oxidant and antioxidant properties of four natural phenolic compounds in DNA nicking. The phenolic compounds differed dramatically in their ability to nick purified supercoiled DNA, with the relative DNA nicking activity in the order: 1,2,4-benzenetriol (100% nicking) > gallic acid > caffeic acid > gossypol (20% nicking). Desferrioxamine (0.02 mM) decreased DNA strand breakage by each phenolic, most markedly with gallate (85% protection) and least with caffeic acid (26% protection). Addition of metals accelerated DNA nicking, with copper more effective (approximately 5-fold increase in damage) than iron with all four phenolics. Scavengers revealed the participation of specific oxygen-derived active species in DNA breakage. Hydrogen peroxide participated in all cases (23-90%). Hydroxyl radicals were involved (32-85%), except with 1,2,4-benzenetriol. Superoxide participated (81-86%) with gallic acid and gossypol, but not with caffeic acid or 1,2,4-benzenetriol. With 1,2,4-benzenetriol, scavengers failed to protect significantly except in combination. Thus, in the presence of desferrioxamine, catalase or superoxide dismutase inhibited almost completely. When DNA breakage was induced by Fenton's reagent (ascorbate plus iron) the two catechols (caffeic acid and gossypol) were protective, whereas the two triols (1,2,4-benzenetriol and gallic acid) exacerbated damage.     

The role of ion-lipid interactions and lipid packing in transient defects caused by phenolic compounds

The transient disruption of membranes for the passive permeation of ions or small molecules is a complex process relevant to understanding physiological processes and biotechnology applications. Phenolic compounds are widely studied for their antioxidant and antimicrobial properties, and some of these activities are based on the interactions of the phenolic compound with membranes. Ions are ubiquitous in cells and are known to alter the structure of phospholipid bilayers. Yet, ion-lipid interactions are usually ignored when studying the membrane-altering properties of phenolic compounds. This study aims to assess the role of Ca²⁺ ions on the membrane-disrupting activity of two phenolic acids and to highlight the role of local changes in lipid packing in forming transient defects or pores. Results from tethered bilayer lipid membrane electrical impedance spectroscopy experiments showed that Ca²⁺ significantly reduces membrane disruption by caffeic acid methyl ester and caffeic acid. As phenolic acids are known metal chelators, we used UV-vis and fluorescence spectroscopy to exclude the possibility that Ca²⁺ interferes with membrane disruption by binding to the phenolic compound and subsequently preventing membrane binding. Molecular dynamics simulations showed that Ca²⁺ but not caffeic acid methyl ester or caffeic acid increases lipid packing in POPC bilayers. The combined data confirm that Ca²⁺ reduces the membrane-disrupting activity of the phenolic compounds, and that Ca²⁺-induced changes to lipid packing govern this effect. We discuss our data in the context of ion-induced pores and transient defects and how lipid packing affects membrane disruption by small molecules.     

Feruloyl esterases as a tool for the release of phenolic compounds from agro-industrial by-products

Agro-industrial by-products are a potential source of added-value phenolic acids with promising applications in the food and pharmaceutical industries. Here two purified feruloyl esterases from Aspergillus niger, FAEA and FAEB were tested for their ability to release phenolic acids such as caffeic acid, p-coumaric acid and ferulic acid from coffee pulp, apple marc and wheat straw. Their hydrolysis activity was evaluated and compared with their action on maize bran and sugar beet pulp. The specificity of both enzymes against natural and synthetic substrates was evaluated; particular attention was paid to quinic esters and lignin monomers. The efficiency of both enzymes on model substrates was studied. We show the ability of these enzymes to hydrolyze quinic esters and ester linkages between phenolic acids and lignin monomer.     

Variability in the composition of anti-oxidant compounds in Echinacea species by HPLC

A fast and reliable HPLC method for the determination of caffeic acid derivatives (caftaric acid, chlorogenic acid, caffeic acid, cynarin, echinacoside and cichoric acid) in various species of the genus Echinacea has been developed. Extraction of root samples by magnetic stirring with 80% methanol aqueous solution at room temperature allowed the complete recovery of all compounds of interest. Root extracts were analysed on a reversed-phase column with gradient elution and photodiode array detection. Caffeic acid derivatives showed differential qualitative and quantitative distributions in Echinacea species. The total amount of phenolic compounds ranged from 33.95 to 0.32 mg/g. The highest contents of caffeic acid derivatives were found in E. paradoxa var. paradoxa, E. paradoxa var. neglecta and E. purpurea, followed by E. angustifolia var. angustifolia, E. simulata, E. pallida and E. laevigata, whilst E. tennesseensis, E. sanguinea and E. atrorubens had low amounts of phenolic compounds. The radical scavenging activities of methanolic extracts of roots of Echinacea species was evaluated in vitro using the DPPH* radical scavenging method. The EC50 values of the samples ranged from 122 to 1223 microg/mL. The radical scavenging activities of the root extracts were correlated with the content of phenolic compounds, with a correlation coefficient (r2) of 0.923.     

Changes in the composition of phenolic compounds and antioxidant properties of grapevine roots and leaves (Vitis vinifera L.) under continuous of long-term drought stress

Grapevine seedlings Vitis vinifera L. were grown in a greenhouse under optimum conditions (soil moisture ca 70 %) and under drought stress (soil moisture ca 30 %). Drought stress caused reduction in total phenolic compounds in grapevine leaves and roots, where were identified tree phenolic acids: caffeic acid, p-coumaric acid and ferulic acid. All acids found in leaves and roots occurred in the ester-bound form. Only caffeic acid in leaves appeared in the free and ester-bound form. Caffeic acid was present in the highest concentrations. The content of ferulic acid was the lowest in both tissues. The levels of all phenolic acids in leaves and roots decreased significantly under the drought stress. All the extracts from grapevine leaves and roots had antioxidative properties, but the antiradical activity of the extracts obtained from roots subjected to drought stress was lower to the control. The results of the analysis revealed that long-term drought stress caused a decrease in selected elements of secondary metabolism in such a different plant tissues that are the leaves and roots of the grapevine.     

3-Desoxyanthocyanin and other phenolics in the water fernAzolla

The phenolic compounds ofAzolla imbricata andA. japonica have been examined in the present study. Both species were found to contain luteolinidin 5-glucoside and several phenolic compounds, particularly chlorogenic acid, aesculetin, caffeic acid 3,4-diglucoside and 6-(3′-glucosylcaffeoyl)-aesculetin. In addition, glucose esters ofp-coumaric acid and glucose, 1,6-diester of caffeic and chlorogenic acids were present in a small amount. The acid- and alkali-hydrolyzates ofAzolla plants yielded caffeic acid and aesculetin present at the level of about 0.047% and 0.012% in fresh plants, and a large part of the caffeic acid seems to be present as the ester.