Influence of constitutive phenolic compounds on the response of grapevine (Vitis vinifera L.) leaves to infection by Plasmopara viticola

Flavonols and hydroxycinnamic acids are known to contribute to plant resistance against pathogens, but there are few reports on the implication of flavonols in the resistance of grapevine against Plasmopara viticola, and none on the involvement of hydroxycinnamic acids. In order to analyze the effect of flavonols on P. viticola infection, variable amounts of flavonols were induced by different light conditions in otherwise phenologically identical leaves. Differences in content of leaf hydroxycinnamic acids were induced at the same time. A non-invasive monitoring of flavonols and hydroxycinnamic acids was performed with Dualex leaf-clip optical sensors. Whatever the light condition, there were no significant changes in flavonol or in hydroxycinnamic acid contents for control and inoculated leaves during the development of P. viticola until 6 days after inoculation. The violet-blue autofluorescence of stilbenes, the main phytoalexins of grapevine that accumulate in inoculated leaves, was used as an indicator of infection by P. viticola. The implication of leaf constitutive flavonols and hydroxycinnamic acids in the defence of Vitis vinifera against P. viticola could be investigated in vivo thanks to this indicator. The increase in stilbene violet-blue autofluorescence started earlier for leaves with low flavonol content than for leaves with higher content, suggesting that constitutive flavonols are able to slow down the infection by P. viticola. On the contrary, constitutive hydroxycinnamic acids did not seem to play a role in defence against P. viticola. The non-destructive nature of the methods used alleviates the major problem of destructive experiments: the large variability in leaf phenolic contents.     

Antioxidant Properties of the Major Polyphenolic Compounds in Broccoli

We have examined the antioxidant activity of the major phenolic compounds in Broccoli: two flavonol glycosides (quercetin 3-O-sophoroside and kaemp-ferol 3-O-sophoroside) and four hydroxycinnamic acid esters (1,2'-disinapoyl-2-feruloyl gentiobiose, 1-sinapoyl-2-feruloyl gentiobiose, 1,2,2'-trisinapoyl gentiobiose and 1,2-disinapoyl gentiobiose). The Trolox C equivalent antioxidant capacity (TEAC) and inhibition of iron/ascorbate-induced lipid per-oxidation of phosphatidyl choline vesicles were measured. In the aqueous phase TEAC assay, the two flavonol glycosides were less active than their respective aglycones. TEAC values for the hydroxycinnamic acid esters were less than the sum of their constituent hydroxycinnamic acids on a molar basis. Quercetin 3-O-sophoroside was a potent inhibitor of lipid peroxidation, in contrast to kaempferol 3-O-sophoroside. The hydroxycinnamic acid esters were highly effective at preventing lipid damage with the exception of 1,2,2'-trisinapoyl gentiobiose. The six compounds analysed herein demonstrate the antioxidant activity of the major phenolics in broccoli and indicate the effect on antioxidant activity of sugar substitutions in the phenolic B ring.     

Effect of wounding on chalcone synthase and pathogenesis related <Emphasis Type="Italic">PR-10</Emphasis> gene expression and content of phenolic compounds in bilberry leaves

The influence of artificial wounding on biosynthesis of flavonoids and hydroxycinnamic acids was studied in bilberry leaves using two separate wounding experiments. In the first experiment bilberry leaves were wounded by cutting. The expression of the first gene from flavonoid pathway, chalcone synthase (CHS) and a wound induced pathogenesis related PR-10 gene was analysed from samples collected immediately and after 3, 6, 24 h and 4 d from the wounding treatment. In the second experiment annual shoots were removed. Proanthocyanidins, flavonol glycosides and hydroxycinnamic acids were quantified in leaf samples after 0–5 d (experiment 1) and 5 weeks (experiment 2) from the treatment. In the first experiment, no change was observed in the expression of CHS whereas increase in expression of PR-10 gene was detected after 6 h of wounding treatment. In both experiments, the contents of flavonol glycosides and hydroxycinnamic acids were not influenced by the wounding treatment and the contents of proanthocyanidins were decreased.     

Phenolic compounds and the hypersensitivity reaction in nicotiana tabacum infected with tobacco mosaic virus

The hypersensitivity of Nicotiana tabacum cv. Xanthi to tobacco mosaic virus infection leads to the production and accumulation of a great number of phenolics (flavonol glycosides, caffeoylquinic, feruloylquinic and p-coumaroylquinic acids, glucose esters and glucosides of cinnamic and benzoic acids). An increase in temperature inhibits the hypersensitive reaction, resulting in the disappearance of these substances. The differences between the healthy and infected leaves become important when the synthesis of the virus is practically brought to completion and the hypersensitivity taken hold. The phenolic compounds do not appear to be responsible for the necrotic hypersensitivity and their production is one of the secondary effects of the virus infection.     

Phenolic compounds in berries and flowers of a natural hybrid between bilberry and lingonberry (Vaccinium × intermedium Ruthe)

Hybridization between species plays an important role in the evolution of secondary metabolites and in the formation of combinations of existing secondary metabolites in plants. We have investigated the content of phenolic compounds in berries and flowers of Vaccinium × intermedium Ruthe, which is a rare natural hybrid between bilberry (Vaccinium myrtillus L.) and lingonberry (Vaccinium vitis-idaea L.). The berries and flowers of the hybrid showed characteristics inherited from both parent species in the distribution and contents of phenolic compounds. Bilberry is known as one of the richest sources of anthocyanins and to have a profile of 15 major forms combining cyanidin, delphinidin, petunidin, peonidin and malvidin with galactose, glucose and arabinose. Lingonberry contains only cyanidin glycosides. Hybrid berries contained all bilberry anthocyanins with pronounced cyanidin content. With regard to proanthocyanidins and flavonol glycosides, the hybrid inherited diverse profiles combining those of both parental species. The distribution of hydroxycinnamic acids was quite uniform in all studied berries. Of the identified compounds, 30 were detected in lingonberry, 46 in bilberry, 53 in hybrid berries and 38 in hybrid flowers. Hence, compared with the parent species, hybrid berries possess a more diverse profile of phenolic compounds and, therefore, can offer interesting material for breeding purposes.     

On the reported molluscicidal activity from Tephrosia vogelii leaves

The rotenoids deguelin and tephrosin were isolated from leaves of Tephrosia vogelii, together with three flavonol glycosides, rutin, isoquercitrin and quercetin 3-O-arabinoside. Although T. vogelii leaves are reportedly toxic to aquatic snails, deguelin and tephrosin were found to have no significant molluscicidal activity.     

Hairy roots of Hypericum perforatum L.: a promising system for xanthone production

Hypericum perforatum L. is a common perennial plant with a reputed medicinal value. Investigations have been made to develop an efficient protocol for the identification and quantification of secondary metabolites in hairy roots (HR) of Hypericum perforatum L. HR were induced from root segments of in vitro grown seedlings from H. perforatum, after co-cultivation with Agrobacterium rhizogenes A4. Transgenic status of HR was confirmed by PCR analysis using rolB specific primers. HR had an altered phenolic profile with respect to phenolic acids, flavonol glycosides, flavan-3-ols, flavonoid aglycones and xanthones comparing to control roots. Phenolics in control and HR cultures were observed to be qualitatively and quantitatively distinct. Quinic acid was the only detectable phenolic acid in HR. Transgenic roots are capable of producing flavonol glycosides such as quercetin 6-C-glucoside, quercetin 3-O-rutinoside (rutin) and isorhamnetin O-hexoside. The HPLC analysis of flavonoid aglycones in HR resulted in the identification of kaempferol. Transformed roots yielded higher levels of catechin and epicatechin than untransformed roots. Among the twenty-eight detected xanthones, four of them were identified as 1,3,5,6-tetrahydroxyxanthone, 1,3,6,7-tetrahydroxyxanthone, γ-mangostin and garcinone C were de novo synthesized in HR. Altogether, these results indicated that H. perforatum HR represent a promising experimental system for enhanced production of xanthones.     

Structurally different flavonol glycosides and hydroxycinnamic acid derivatives respond differently to moderate UV-B radiation exposure

The aim of this study was to investigate the modifying influence of moderate ultraviolet-B (UV-B) radiation exposure on structurally different flavonol glycosides and hydroxycinnamic acid derivatives during pre-harvest using kale, a leafy Brassica species with a wide spectrum of different non-acylated and acylated flavonol glycosides. Juvenile kale plants were treated with short-term (1 day), moderate UV-B radiation [0.22-0.88 kJ m?2 day?1 biologically effective UV-B (UV-B(BE))]. Twenty compounds were quantified, revealing a structure-specific response of flavonol glycosides and hydroxycinnamic acid derivatives to UV-B radiation. A dose- and structure-dependent response of the investigated phenolic compounds to additional UV-B radiation was found. The investigated quercetin glycosides decreased under UV-B; for kaempferol glycosides, however, the amount of sugar moieties and the flavonol glycoside hydoxycinnamic acid residue influenced the response to UV-B. Monoacylated kaempferol tetraglucosides decreased in the investigated UV-B range, whereas the monoacylated kaempferol diglucosides increased strongly with doses of 0.88 kJ m?2 day?1 UV-B(BE) . The UV-B-induced increase in monoacylated kaempferol triglucosides was dependent on the acylation pattern. Furthermore, the hydroxycinnamic acid glycosides disinapoyl-gentiobiose and sinapoyl-feruloyl-gentiobiose were enhanced in a dose-dependent manner under UV-B. While UV-B radiation treatments often focus on flavonol aglycones or total flavonols, our investigations were extended to structurally different non-acylated and acylated glycosides of quercetin and kaempferol.     

Phenolic compounds of Sophora flavescens Soland. of Russian origin

A qualitative composition and a quantitative content of phenolic compounds of underground and above-ground parts of Sophora flavescens Soland. (the Fabaceae family) growing in Russia (Transbaikalia, Primorsky Krai, Aga Buryat Autonomous District) were studied. Eleven compounds were isolated from the roots and rhizomes: kushenol A, isokurarinone, kuraridine, sophoraflavanone G, kurarinone, isoxanthohumol, umbeliferon, and, for the first time, scopoletin, ferulic, caffeic, and chlorogenic acids. Ten phenolic compounds were identified in the herb of S. flavescens: cynaroside, cosmosiin, caffeic acid, and, for the first time, apigenin, luteolin, quercetin, umbelliferone, rutin, chlorogenic, and neochlorogenic acids. Dominant compounds in the underground part were kurarinone and sophoraflavanone G, and in the above-ground part, cynaroside and rutin. It was shown that the maximum content of flavonoids in the underground part of S. flavescens was accumulated in the epidermal layers of rhizomes. The dynamics of flavonoids accumulation in S. flavescens was studied.     

Diverting phenylpropanoid pathway flux from sinapine to produce industrially useful 4-vinyl derivatives of hydroxycinnamic acids in Brassicaceous oilseeds

Sinapine (sinapoylcholine) is an antinutritive phenolic compound that can account for up to 2% of seed weight in brassicaceous oilseed crops and reduces the suitability of their protein-rich seed meal for use as animal feed. Sinapine biosynthesis draws on hydroxycinnamic acid precursors produced by the phenylpropanoid pathway. The 4-vinyl derivatives of several hydroxycinnamic acids have industrial applications. For example, 4-vinyl phenol (4-hydroxystyrene) is a building block for a range of synthetic polymers applied in resins, inks, elastomers, and coatings. Here we have expressed a modified bacterial phenolic acid decarboxylase (PAD) in developing seed of Camelina sativa to redirect phenylpropanoid pathway flux from sinapine biosynthesis to the production of 4-vinyl phenols. PAD expression led to a ∼95% reduction in sinapine content in seeds of both glasshouse and field grown C. sativa and to an accumulation of 4-vinyl derivatives of hydroxycinnamic acids, primarily as glycosides. The most prevalent aglycone was 4-vinyl phenol, but 4-vinyl guaiacol, 6-hydroxy-4-vinyl guaiacol and 4-vinylsyringol (Canolol) were also detected. The molar quantity of 4-vinyl phenol glycosides was more than twice that of sinapine in wild type seeds. PAD expression was not associated with an adverse effect on seed yield, harvest index, seed morphology, storage oil content or germination in either glasshouse or field experiments. Our data show that expression of PAD in brassicaceous oilseeds can supress sinapine accumulation, diverting phenylpropanoid pathway flux into 4-vinyl phenol derivatives, thereby also providing a non-petrochemical source of this class of industrial chemicals.     

A survey of mangiferin and hydroxycinnamic acid ester accumulation in coffee (Coffea) leaves: biological implications and uses

Background and Aims

The phenolic composition of Coffea leaves has barely been studied, and therefore this study conducts the first detailed survey, focusing on mangiferin and hydroxycinnamic acid esters (HCEs).

Methods

Using HPLC, including a new technique allowing quantification of feruloylquinic acid together with mangiferin, and histochemical methods, mangiferin content and tissue localization were compared in leaves and fruits of C. pseudozanguebariae, C. arabica and C. canephora. The HCE and mangiferin content of leaves was evaluated for 23 species native to Africa or Madagascar. Using various statistical methods, data were assessed in relation to distribution, ecology, phylogeny and use.

Key Results

Seven of the 23 species accumulated mangiferin in their leaves. Mangiferin leaf-accumulating species also contain mangiferin in the fruits, but only in the outer (sporophytic) parts. In both leaves and fruit, mangiferin accumulation decreases with ageing. A relationship between mangiferin accumulation and UV levels is posited, owing to localization with photosynthetic tissues, and systematic distribution in high altitude clades and species with high altitude representatives. Analyses of mangiferin and HCE content showed that there are significant differences between species, and that samples can be grouped into species, with few exceptions. These data also provide independent support for various Coffea lineages, as proposed by molecular phylogenetic analyses. Sampling of the hybrids C. arabica and C. heterocalyx cf. indicates that mangiferin and HCE accumulation may be under independent parental influence.

Conclusions

This survey of the phenolic composition in Coffea leaves shows that mangiferin and HCE accumulation corresponds to lineage recognition and species delimitation, respectively. Knowledge of the spectrum of phenolic accumulation within species and populations could be of considerable significance for adaptation to specific environments. The potential health benefits of coffee-leaf tea, and beverages and masticatory products made from the fleshy parts of Coffea fruits, are supported by our phenolic quantification.Key words: Arabica coffee, C. arabica, C. canephora, chlorogenic acids, Crop Wild Relatives (CWRs), coffee-leaf tea, hybridization, hydroxycinnamic acids, mangiferin, phenolic compounds, phylogeny, robusta coffee     

The polyphenolic content of fruit and vegetables and their antioxidant activities. What does a serving constitute?

Analysis of the major flavone, flavonol, anthocyanidin and hydroxycinnamic acid constituents (and their glycosides) of onion, tomato, egg plant and apple has been undertaken and the antioxidant activities of the phenolic extracts determined. The major phenolic antioxidant components of egg plant are chlorogenic acid in the flesh and a delphinidin conjugate in the skin. In the case of apple, the major phenolic antioxidants detected are chlorogenic acid, procyanidins/catechin compounds, rutin and phloridzin. Quercetin glycosides are well-known to be the major phenolic components of onion. Assessment of the antioxidant activities of a serving of 100 g fresh weight fruit, vegetable and comparison with previously reported findings for 150 ml beverage (500 ml portion in the case of beer), expressed in μmol Trolox equivalents show that the antioxidant activities of 1 glass (150 ml) red wine ≡ 12 glasses white wine ≡ 2 cups of tea ≡ 4 apples ≡ 5 portions of onion ≡ 5.5 portions egg plant ≡ 3.5 glasses of blackcurrant juice ≡ 3.5 (500 ml) glasses of beer ≡ 7 glasses of orange juice ≡ 20 glasses of apple juice (long life).     

Changes in content and composition of phenolic acids during growth of xylem cells of Scots pine

The content and composition of alcohol soluble phenolic acids (PhAs) were studied during cell xylem growth in course of wood annual increment formation in the trunks of Scots pine. Cells of the cambium zone, two stages of expansion growth, and outset of secondary thickening zone (before lignification) within the period of formation of early wood xylem were subsequently isolated from trunk segments of 25-year-old trees with constant anatomical and histochemical control. The amount of free and bound forms of phenolic acids extracted from tissues by 80% ethanol, as well as their ethers and esters, were calculated both per dry weight and per cells. The substantial alteration in content, proportion of fractions and composition of acids has been found between the cambium zone and the outset of secondary thickening of tracheids, and the character of variation depended on the calculation method. The amount of free and bound PhAs and esters and especially ethers calculated per cell had increased at the first stage of extension growth, reduced at the second, and increased in the outset of secondary wall deposition. The pool of bound acids was more than acids by 2–5 times depending on the stage of development of the cells. Sinapic and ferulic acids dominate among free hydroxycinnamic acids. The composition and the content of hydroxycinnamic acids in esters and ethers also depended on the stage of development of the cells. p-Coumaric and sinapic acids were the main aglycons in ethers in the cambium and sinapic and caffeic acids were in the other stages. The esters from cambium included mostly p-coumaric acid and those at other stages of development were sinapic and ferulic acids. The esters included benzoic acid at the first stages of growth. The pool of these esters decreased from the first phase of growth until the outset of cell wall thickening. The level of free benzoic acid increased respectively.     

Phenolic compounds (hydroxycinnamic acids,flavan-3-ols,flavonols) profile in fruit of Italian peach varieties

Interest in phenolic compounds of fruit is growing due to their positive effects on reducing the risk of cardiovascular and carcinogen diseases. The role of the flesh colour, of the tissue (exocarp or mesocarp) and of the cultivar on the content of phenolic compounds (hydroxycinnamic acids, flavan-3-ols, flavonols) was evaluated in peach, Prunus persica (L.) Batsch, with a HPLC-DAD analysis, on 10 varieties deriving from the Italian breeding programmes, never previously characterized by this approach. The flavan-3-ols (catechin, epicatechin and procyanidin B1) were the most abundant class of phenolic compounds in peach cultivars analysed. Among hydroxycinnamic acids, neochlorogenic and chlorogenic acids were also identified and measured in the peach cultivars analysed. Quercetin-3-O-rutinoside was the only flavonol found in the exocarp of all the cultivars evaluated, but in the mesocarp of only some cultivars. Content of phenolic compounds was confirmed to be strongly dependent on the cultivar and, in general, the content was significantly higher in exocarp than in mesocarp. An overall significant difference in phenolic compounds as associated with flesh colour was not observed.     

Distribution of charged flavones and caffeylshikimic acid in Palmae

Identification of the phenolic constituents in flowers of nine palm species has revealed that charged C-glycosylflavones and caffeylshikimic acid are characteristically present. Flavonol glycosides are also common; the 3-glucosides, 3-rutinosides and 3,4′-diglucosides of quercetin and isorhamnetin and the 7-glucoside and 3,7-diglucoside of quercetin are all variously present. Tricin 7-glucoside, luteolin 7-rutinoside and several unchanged C-glycosylflavones were also detected. Male flowers of Phoenix canariensis differ from female flowers in having flavonol glycosides. As expected, in most species studied, flavonoid patterns in the flowers vary considerably from those found in the leaves.     

Identification of Terpene Lactones and Flavonol Glycosides in Preparations Based on Ginkgo Biloba Extract and a New Way of Semi-Quantitative Determination of Flavonol Glycosides by <Superscript>1</Superscript>H NMR Spectroscopy

The composition of terpen lactones and flavonol glycosides of commercial preparation series based on Ginkgo biloba extracts was investigated by ¹H NMR spectroscopy. The content of individual terpen lactones was determined using DMSO-d₆ and acetone-d₆ solvents. The effect of the structure of flavonol glycosides on the signal of the hydroxyl proton at a position 5 of the ring A was examined. A new approach was proposed for semiquantitative determination of the total amount of flavonol glycosides by the integral intensity of this signal, which is a superposition of the singlets in the region of 12.5–12.65 ppm of individual flavonoids in DMSO-d₆. Since the corresponding signals of aglycones (quercetin, kaempferol, isorhamnetin), which are minor components of the Ginkgo biloba extracts, appear separately in a slightly different region (12.45–12.48 ppm), the proposed method can also be used for detecting adulteration of Ginkgo biloba extracts by means of the addition into them of relatively cheap aglycones or rutin as well as for assessment of the content of flavonoids of similar structure in some types of plant raw materials.     

The Antioxidant Activity of the Russian Far East Representatives of the <Emphasis Type="Italic">Spiraea</Emphasis> L. (Rosaceae Juss.) Genus

Biologically active substances and antioxidant activity of extracts from leaves and inflorescences of nine representatives of the genus Spiraea L. growing on the territory of the Far East of Russia were investigated. Widespread species of the genus Spiraea (S. salicifolia, S. media var. media, S. betulifolia and S. ussuriensis subsp. ussuriensis) have the highest levels of biologically active substances. The inflorescences of spiraeas there contain more flavonols (up to 3.9%), oxycinnamic acids (up to 1.2%), catechins (up to 5.7%) and saponins (up to 5.1%) compared to their leaves, and there are more tannins (up to 11.6%) in the leaves. Among the Far Eastern representatives of the genus Spiraea, S. betulifolia and S. beauverdiana (section Calospira), S. humilis and S. salicifolia (section Spiraria), S. pubescens and S. media var. media (section Chamaedryon) are promising antioxidants. Plants of the genus Spiraea probably contain water-soluble antioxidant compounds of phenolic type, because the antioxidant activity of aqueous extracts in the leaves and inflorescences of spiraeas is higher (0.16–2.79 mg/g) than that of water-alcoholic compounds (0.06–2.54 mg/g). The antioxidant activity in the leaves of spiraeas is generally higher than that in the inflorescence. A reliable positive correlation is observed between the antioxidant activity of aqueous extracts from the organs of spiraeas and a content of oxycinnamic acids.     

The effect of nitrate assimilation deficiency on the carbon and nitrogen status of Arabidopsis thaliana plants

Carbon (C) and nitrogen (N) metabolism are integrated processes that modulate many aspects of plant growth, development, and defense. Although plants with deficient N metabolism have been largely used for the elucidation of the complex network that coordinates the C and N status in leaves, studies at the whole-plant level are still lacking. Here, the content of amino acids, organic acids, total soluble sugars, starch, and phenylpropanoids in the leaves, roots, and floral buds of a nitrate reductase (NR) double-deficient mutant of Arabidopsis thaliana (nia1 nia2) were compared to those of wild-type plants. Foliar C and N primary metabolism was affected by NR deficiency, as evidenced by decreased levels of most amino acids and organic acids and total soluble sugars and starch in the nia1 nia2 leaves. However, no difference was detected in the content of the analyzed metabolites in the nia1 nia2 roots and floral buds in comparison to wild type. Similarly, phenylpropanoid metabolism was affected in the nia1 nia2 leaves; however, the high content of flavonol glycosides in the floral buds was not altered in the NR-deficient plants. Altogether, these results suggest that, even under conditions of deficient nitrate assimilation, A. thaliana plants are capable of remobilizing their metabolites from source leaves and maintaining the C–N status in roots and developing flowers.     

Phenolics during early development of <Emphasis Type="Italic">Betula pubescens</Emphasis> seedlings: inhibition of phenylalanine ammonia lyase

We studied phenolic metabolism and plant growth in birch seedlings at the beginning of their development by inhibiting phenylalanine ammonia lyase (PAL), which is the first committed step in phenylpropanoid metabolism. Betula pubescens (Ehrh.) seeds were germinated in inhibitor-free media and the seedlings were transferred to hydroponic culture at the cotyledon stage. They were 6 days old at the start of the experiment, which lasted for 3 weeks. PAL activity was inhibited by three different concentrations of 2-aminoindane-2-phosphonic acid monohydrate (AIP) in the growing media. At the end of 3 weeks, phenolics in all plant parts (roots, stem, cotyledons, first, second and third true leaves) were determined. AIP inhibited strongly the accumulation of phenolic acids, salidroside, rhododendrins, ellagitannins and their precursors, flavan-3-ols, and soluble condensed tannins. The accumulation of lignin and flavonol glycoside derivatives was moderately inhibited. The accumulation of flavonol glycosides, such as quercetin glycosides and kaempferol glycosides, was not generally inhibited, even in leaves that emerged during the experiment, while the accumulation of insoluble condensed tannins was inhibited only slightly and not in all plant parts. This suggests that flavonol glycosides, which may have a UV-B protective role, and insoluble condensed tannins, which may have structural functions, are prioritized in seedling development. Inhibition of PAL with AIP decreased seedling growth and possible reasons for this are discussed. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.