A rationale for the shift in colour towards blue in transgenic carnation flowers expressing the flavonoid 3',5'-hydroxylase gene

Recently marketed genetically modified violet carnations cv. Moondust and Moonshadow (Dianthus caryophyllus) produce a delphinidin type anthocyanin that native carnations cannot produce and this was achieved by heterologous flavonoid 3',5'-hydroxylase gene expression. Since wild type carnations lack a flavonoid 3',5'-hydroxylase gene, they cannot produce delphinidin, and instead accumulate pelargonidin or cyanidin type anthocyanins, such as pelargonidin or cyanidin 3,5-diglucoside-6"-O-4, 6"'-O-1-cyclic-malyl diester. On the other hand, the anthocyanins in the transgenic flowers were revealed to be delphinidin 3,5-diglucoside-6"-O-4, 6"'-O-1-cyclic-malyl diester (main pigment), delphinidin 3,5-diglucoside-6"-malyl ester, and delphinidin 3,5-diglucoside-6",6"'- dimalyl ester. These are delphinidin derivatives analogous to the natural carnation anthocyanins. This observation indicates that carnation anthocyanin biosynthetic enzymes are versatile enough to modify delphinidin. Additionally, the petals contained flavonol and flavone glycosides. Three of them were identified by spectroscopic methods to be kaempferol 3-(6"'-rhamnosyl-2"'-glucosyl-glucoside), kaempferol 3-(6"'-rhamnosyl-2"'-(6-malyl-glucosyl)-glucoside), and apigenin 6-C-glucosyl-7-O-glucoside-6"'-malyl ester. Among these flavonoids, the apigenin derivative exhibited the strongest co-pigment effect. When two equivalents of the apigenin derivative were added to 1 mM of the main pigment (delphinidin 3,5-diglucoside-6"-O-4,6"'-O-1-cyclic-malyl diester) dissolved in pH 5.0 buffer solution, the lambda(max) shifted to a wavelength 28 nm longer. The vacuolar pH of the Moonshadow flower was estimated to be around 5.5 by measuring the pH of petal. We conclude that the following reasons account for the bluish hue of the transgenic carnation flowers: (1). accumulation of the delphinidin type anthocyanins as a result of flavonoid 3',5'-hydroxylase gene expression, (2). the presence of the flavone derivative strong co-pigment, and (3). an estimated relatively high vacuolar pH of 5.5.     

Reversal of P-glycoprotein-mediated MDR by 5,7,3',4',5'-pentamethoxyflavone and SAR

During screening for the flavonoid chemosensitizers, it was found that 5,7,3',4',5'-pentamethoxyflavone (PMF) was equipotent to verapamil in vitro with respect to the chemosensitizing effect. PMF appears to have a chemosensitizing effect not only by increasing the intracellular accumulation of the drugs without competition in a binding site of azidopine but also by interfering with the substrate-stimulated ATPase activity. Structure-activity relationship suggests that methoxylated substitution and its numbers or sites of the rings are more important than its hydroxylated counterparts in chemosensitization. Overall, PMF is anticipated to be a novel and highly potent second-generation flavonoid chemosensitizer because PMF has significant advantages of having a high therapeutic index, of being a non-transportable inhibitor, and of having a low possibility of drug interactions at the azidopine-binding site of Pgp.     

Flavonoids from shoots and roots of Trifolium repens (white clover) grown in presence or absence of the arbuscular mycorrhizal fungus Glomus intraradices

White clover (Trifolium repens) plants were grown in the presence or absence of the arbuscular mycorrhizal fungus Glomus intraradices. Flavones, 4',5,6,7,8-pentahydroxy-3-methoxyflavone and 5,6,7,8-tetrahydroxy-3-methoxyflavone, as well as two flavones 3,7-dihydroxy-4'-methoxyflavone and 5,6,7,8-tetrahydroxy-4'-methoxyflavone never previously reported in plants, were isolated. The known 3,5,6,7,8-pentahydroxy-4'-methoxyflavone, 2',3',4',5',6'-pentahydroxy-chalcone, 6-hydroxykaempferol, 4',5,6,7,8-pentahydroxyflavone and 3,4'-dimethoxykaempferol were also obtained. Analysis of extracts obtained from roots and shoots revealed that the compositions of the flavonoid mixtures varied with growing conditions. Quercetin, acacetin and rhamnetin accumulated in roots of inoculated plants, whereas they were not detected in non-inoculated plants.     

Acylated flavone glycosides from Veronica

A survey of the flavonoid glycosides of selected taxa in the genus Veronica yielded two new acylated 5,6,7,3',4'-pentahydroxyflavone (6-hydroxyluteolin) glycosides and two unusual allose-containing acylated 5,7,8,4'-tetrahydroxyflavone (isoscutellarein) glycosides. The new compounds were isolated from V. liwanensis and V. longifolia and identified using NMR spectroscopy as 6-hydroxyluteolin 4'-methyl ether 7-O-alpha-rhamnopyranosyl(1"'-->2")[6"-O-acetyl-beta-glucopyranoside] and 6-hydroxyluteolin 7-O-(6"-O-(E)-caffeoyl)-beta-glucopyranoside, respectively. Isoscutellarein 7-O-(6"'-O-acetyl)-beta-allopyranosyl(1"'-->2")-beta-glucopyranoside was obtained from both V. intercedens and V. orientalis and its 4'-methyl ether from V. orientalis only. Complete 1H and 13C NMR spectral assignments are presented for both isoscutellarein glycosides. Two iridoid glucosides new to the genus Veronica (melittoside and globularifolin) were also isolated from V. intercedens.     

Triterpene saponins and flavonoids in the seeds of Trifolium species

Seeds of 57 species of the genus Trifolium have been studied for the occurrence and concentration of soyasapogenol B glycosides and flavonoids. It was shown that all tested species contained soyasaponin I and in some species astragaloside VIII and/or 22-O-glucoside and 22-O-diglucoside of soyasaponin I were also present. Total concentration of saponins ranged from trace amounts up to 10 mg/g(DM). It was suggested that soyasapogenol B glycosides could be recognized as chemotaxonomic character of Fabaceae family. All but three tested species contained flavonoids. The majority of species contained quercetin as a sole flavonoid or in the mixture with a number of unidentified flavonoid components. Concentration of quercetine in some species was at the level of about 3 mg/g(DM). This high quercetin concentration and soyasaponin occurrence makes the seeds of some Trifolium species a potential source of health beneficial phytochemicals, to be used in human nutrition.     

Taxonomic significance of flavonoid variation in temperate species of Nothofagus

Forty-two flavonoids and a stilbene (pinosylvin) were identified in the leaf exudates of 11 temperate species of Nothofagus from South America, Australia and New Zealand. The flavonoid profiles demonstrate significant taxonomic value at the subgeneric level. Most species of subgenus Fuscospora are characterized by the presence of pinosylvin, galangin and galangin methyl ethers. Kaempferol-type flavonols are abundant in subgenus Lophozonia while these flavonols are largely absent from species of subgenus Fuscospora. The flavonoid patterns are largely in agreement with a recent subgeneric classification of Nothofagus.     

Variations in lipophilic and vacuolar flavonoids among European Pulicaria species

Four European Pulicaria species, P. odora, P. paludosa, P. sicula and P. vulgare, were analysed for their surface and vacuolar constituents for comparison with previous data obtained for P. dysenterica. Each species had a distinct flavonoid pattern with notable differences between leaf and inflorescence. 6-Hydroxyflavonols were the major lipophilic components in all of the species and tissues except in the leaves of P. paludosa and P. vulgare, where scutellarein 6-methyl ether was the main constituent. In the leaves of P. sicula a more unusual flavone, 6-hydroxyluteolin 5,6,7,3',4'-pentamethyl ether, was a major component. Pulicaria odora was distinguished by the presence of a series of methylated 6-hydroxykaempferol derivatives including a 3,5,6,7,4'-pentamethyl ether. Quercetagetin hexamethyl ether occurred in both tissues of P. sicula together with the 3,7,3,4'-tetra methyl ether and other quercetagetin derivatives, which were 5-methylated. Quercetagetin 3,7,3'-methyl ether was present in all species except P. odora. Flavonol glucuronides were characteristic vacuolar constituents of all the taxa studied. Two rare glycosides, patuletin and 6-hydroxykaempferol 6-methyl ether 7-glucuronides were identified in the inflorescence of P. odora. Pulicaria vulgaris, a rare plant of southern England, had the vacuolar flavonoid profile most similar to the other more abundant British plant, P. dysenterica.     

The antileishmanial activity assessment of unusual flavonoids from Kalanchoe pinnata

The importance of flavonoids for the antileishmanial activity of Kalanchoe pinnata was previously demonstrated by the isolation of quercitrin, a potent antileishmanial flavonoid. In the present study, the aqueous leaf extract from the medicinal plant K. pinnata (Crassulaceae) afforded a kaempferol di-glycoside, named kapinnatoside, identified as kaempferol 3-O-alpha-L-arabinopyranosyl (1-->2) alpha-L-rhamnopyranoside (1). In addition, two unusual flavonol and flavone glycosides already reported, quercetin 3-O-alpha-L-arabinopyranosyl (1-->2) alpha-L-rhamnopyranoside (2) and 4',5-dihydroxy-3',8-dimethoxyflavone 7-O-beta-D-glucopyranoside (3), have been isolated. Their structures were determined via analyses of mono and bi-dimensional (1)H and (13)C NMR spectroscopic experiments and HR-MALDI mass spectra. Because of its restricted occurrence and its abundance in K. pinnata, flavonoid (2) may be a chemical marker for this plant species of high therapeutic potential. The three flavonoids were tested separately against Leishmania amazonenis amastigotes in comparison with quercitrin, quercetin and afzelin. The quercetin aglycone - type structure, as well as a rhamnosyl unit linked at C-3, seem to be important for antileishmanial activity.     

Intra- and interspecific variations in vacuolar flavonoids among Ficus species from the Budongo Forest,Uganda

Leaves of 14 species of Ficus growing in the Budongo Forest, Uganda, were analysed for vacuolar flavonoids. Three to six accessions were studied for each species to see whether there was intraspecific chemical variation. Thirty-nine phenolic compounds were identified or characterised, including 14 flavonol O-glycosides, six flavone O-glycosides and 15 flavone C-glycosides. In some species the flavonoid glycosides were acylated. Ficus thonningii contained in addition four stilbenes including glycosides. Most of the species could be distinguished from each other on the basis of their flavonoid profiles, apart from Ficus sansibarica and Ficus saussureana, which showed a very strong intraspecific variation. However, on the whole flavonoid profiles were sufficiently distinct to help in future identifications.     

Scutellarein 4'-methyl ether glycosides as taxonomic markers in Teucridium and Tripora (Lamiaceae,Ajugoideae)

The flavonoid profiles of two monotypic genera, Teucridium and Tripora, have been studied by analytical methods. These genera were formerly placed in the Verbenaceae, but are now classified in the Lamiaceae, subfamily Ajugoideae. The major flavonoids of both genera were identified as glycosides of scutellarein 4'-methyl ether (5,6,7-trihydroxy-4'methoxyflavone) and acacetin (5,7-dihydroxy-4'-methoxyflavone). The new flavone glycoside, scutellarein 4'-methyl ether 7-O-rutinoside, was isolated from Teucridium parvifolium and the rare scutellarein 4'-methyl ether 7-O-glucuronide from Tripora divaricata. The latter compound has only been reported previously in the related genus Clerodendron. The potential of these flavonoids as taxonomic markers for the tribe Ajugoideae is discussed.     

Two aurone glycosides from heartwood of Pterocarpus santalinus

Two new aurone glycosides, 6 hydroxy 5 methyl 3',4',5' trimethoxy aurone 4-O-alpha-L-rhamnopyranoside and 6,4' dihydroxy aurone 4-O-rutinoside have been isolated from the ethanolic extract of the wood of Pterocarpus santalinus. Their structures were determined on the basis of chemical and spectroscopic analysis (UV, IR, EIMS, (1)H and (13)C NMR).     

The application of multiplex fluorimetric sensor for the analysis of flavonoids content in the medicinal herbs family Asteraceae,Lamiaceae, Rosaceae

Background

The aim of our research work was to quantify total flavonoid contents in the leaves of 13 plant species family Asteraceae, 8 representatives of family Lamiaceae and 9 plant species belonging to family Rosaceae, using the multiplex fluorimetric sensor. Fluorescence was measured using optical fluorescence apparatus Multiplex(R) 3 (Force-A, France) for non-destructive flavonoids estimation. The content of total flavonoids was estimated by FLAV index (expressed in relative units), that is deduced from flavonoids UV absorbing properties.

Results

Among observed plant species, the highest amount of total flavonoids has been found in leaves of Helianthus multiflorus (1.65 RU) and Echinops ritro (1.27 RU), Rudbeckia fulgida (1.13 RU) belonging to the family Asteraceae. Lowest flavonoid content has been observed in the leaves of marigold (Calendula officinalis) (0.14 RU) also belonging to family Asteraceae. The highest content of flavonoids among experimental plants of family Rosaceae has been estimated in the leaves of Rosa canina (1.18 RU) and among plant species of family Lamiaceae in the leaves of Coleus blumei (0.90 RU).

Conclusions

This research work was done as pre-screening of flavonoids content in the leaves of plant species belonging to family Asteraceae, Lamiaceae and Rosaceae. Results indicated that statistically significant differences (P > 0.05) in flavonoids content were observed not only between families, but also among individual plant species within one family.     

Flavonoid glycosides from Centaurea pseudoscabiosa subsp. pseudoscabiosa from Turkey

Three flavonoid glycosides were isolated and characterized, together with a further 13 substances belonging to various classes of compounds, in particular two phenolic acids, a coumarin, a sugar and nine flavonoids from the flowered aerial parts of Centaurea pseudoscabiosa subsp. pseudoscabiosa Boiss. et Buhse (Asteraceae). Some considerations about their evolutionary meaning are provided.     

Leaf flavonoid glycosides as chemosystematic characters in Ocimum

Thirty-one accessions of nine species belonging to three subgenera of Ocimum (basil, family Lamiaceae) were surveyed for flavonoid glycosides. Substantial infraspecific differences in flavonoid profiles of the leaves were found only in O. americanum, where var. pilosum accumulated the flavone C-glycoside, vicenin-2, which only occurred in trace amounts in var. americanum and was not detected in cv. Sacred. The major flavonoids in var. americanum and cv. Sacred, and also in all other species investigated for subgenus Ocimum, were flavonol 3-O-glucosides and 3-O-rutinosides. Many species in subgenus Ocimum also produced the more unusual compound, quercetin 3-O-(6″-O-malonyl)glucoside, and small amounts of flavone O-glycosides. The level of flavonol glycosides produced was reduced significantly in glasshouse-grown plants, but levels of flavone glycosides were unaffected. A single species investigated from subgenus Nautochilus, O. lamiifolium, had a different flavonoid glycoside profile, although the major compound was also a flavonol O-glycoside. This was identified as quercetin 3-O-xylosyl(1‴→2″)galactoside, using NMR spectroscopy. The species investigated from subgenus Gymnocimum, O. tenuiflorum (=O. sanctum), was characterised by the accumulation of flavone O-glycosides. These were isolated, and identified as the 7-O-glucuronides of luteolin and apigenin. Luteolin 5-O-glucoside was found in all nine species of Ocimum studied, and is considered to be a key character for the genus.     

Comparative phytochemical analysis of four Mexican Nymphaea species

Four Mexican Nymphaea species, N. ampla, N. pulchella, N. gracilis and N. elegans belonging to subgenera Brachyceras were analyzed. In this work two 5-glycosyl isoflavones, 7,3',4'-trihydroxy-5-O-beta-D-(2'-acetyl)-xylopyranosylisoflavone (1) and 7,3',4'-trihydroxy-5-O-alpha-L-rhamnopyranosylisoflavone (2), were isolated from N. ampla and N. pulchella, respectively, together with other known 3-glycosyl flavones and triterpene saponins from the same four species. The structures were elucidated by 1D and 2D NMR, FABMS, and other spectroscopic analyses. These results confirmed that the four species were different from each other and established that N. pulchella represents a different taxa than N. ampla. In addition, the 5-glycosyl isoflavones could be considered as a taxonomic character of this group of plants.     

Systematic relationships among Lutzomyia sand flies (Diptera: Psychodidae) of Peru and Colombia based on the analysis of 12S and 28S ribosomal DNA sequences

Lutzomyia spp. are New World phlebotomine sand flies, many of which are involved in the transmission of human diseases, such as leishmaniases and bartonellosis. The systematic classification of the approximately 400 species in the genus has been based on morphological characters, but the relationships within the genus are still very much in question. We have inferred phylogenies of 32 species of phlebotomine sand flies belonging to seven sub-genera and two species groups, by using fragments of the mitochondrial small subunit (12SrRNA) and of the nuclear large subunit (28SrRNA) ribosomal gene sequences. The subgenus Helcocyrtomyia and the Verrucarum species group, prominent representatives of the Peruvian sand fly fauna, were represented by 11 and 7 species, respectively. Although based on a limited number of taxa, the resulting phylogenies, based on 837 characters, provide an initial phylogenetic backbone for the progressive reconstruction of infrageneric relationships within Lutzomyia.     

Veronica: Acylated flavone glycosides as chemosystematic markers

HPLC/DAD and LC–MS of an extract of Veronica spicata (subgenus Pseudolysimachium, Plantaginaceae) revealed the presence of six 6-hydroxyluteolin glycosides acylated with phenolic acids, three of which are new compounds and which we called spicosides. A flavonoid survey of seven more species belonging to V. subgenus Pseudolysimachium and eight species of V. subgenus Pentasepalae showed that all the Pseudolysimachium species and four of the Pentasepalae species produced 6-hydroxyflavone glycosides, whereas the remaining four Pentasepalae species contained acetylated 8-hydroxyflavone glycosides instead. Spicosides appeared to be common in subgenus Pseudolysimachium (detected in five out of eight species), but we did not find them in subgenus Pentasepalae. Previously, acetylated 8-hydroxyflavone glycosides have been isolated from or detected in eight species of V. subgenus Pentasepalae (in 13 species altogether including the new results) and seven species of V. subgenus Pocilla. However, 6-hydroxyflavone glycosides occur in some other species of these subgenera. The results are discussed in an evolutionary context.     

Flavonoid profiles in the Heliohebe group of New Zealand Veronica (Plantaginaceae)

The Heliohebe group of Veronica (sect. Hebe) consists of five species occurring in the South Island of New Zealand. These species and a hybrid were analysed for their flavonoids. Five flavone glycosides were isolated and identified by NMR spectroscopy and three additional glycosides were detected by LC–UV–MS. Luteolin 7-O-, 3′-O- and 4′-O-glucosides and apigenin 7-O-glucoside were present in all six taxa investigated, 6-hydroxyluteolin glycosides were found in five and a luteolin caffeoylglycoside in four taxa, while a hypolaetin 7-O-glycoside was detected only in Veronica pentasepala. The 3′-O- and 4′-O-glucosides of luteolin are also common in other species of Veronica sect. Hebe (restricted to the Southern Hemisphere), but are rare in Northern Hemisphere species of Veronica and thus act as good chemotaxonomic markers for the section. The relatively simple flavonoid profiles found in the Heliohebe group are plesiomorphic and consistent with the group's status as sister to the Hebe clade. Based on the detected flavonoids, two groups could be distinguished within the Heliohebe clade: (1) Veronica hulkeana, Veronica lavaudiana and Veronica raoulii, characterised by luteolin caffeoylglycoside, and (2) V. pentasepala and Veronica scrupea, where this compound is replaced by a 6-hydroxyluteolin dihexoside.     

Abnormal growth of polyamine-deficient Escherichia coli mutant is partially caused by oxidative stress-induced damage

Polyamines participate in numerous cellular processes and are required for normal cell growth in Escherichia coli. In this study, we constructed a new polyamine-deficient E. coli mutant and investigated the physiological function of polyamines during normal aerobic growth conditions. We showed that the requirement for sulfur-containing, branched chain, and aromatic amino acids, which was exhibited in the sodA sodB double mutant faced with severe oxidative stress, was also true of the polyamine-deficient mutant during normal aerobic cell growth. Sorbitol, sucrose, mannose, 1,2-dihydroxybenzene-3,5-disulfonic acid (Tiron), an antioxidant that functions as an oxygen radical scavenger including z.rad;O(2)(-), and thiamine partially relieved the cell growth defect caused by polyamine depletion in a dose-dependent manner. As was the case for the cells treated with paraquat, the mutant had an elongated shape compared with the polyamine-proficient wild type. Decreased aeration also relieved the cell growth defect of the polyamine-deficient mutant. Finally, we confirmed that chloromethyl-2('),7(')-dichlorofluorescin diacetate (DCFH-DA), which is oxidized in a fluorescent product in the presence of various oxidants, also fluoresce in the polyamine-deficient cells. These results showed that abnormal growth of the polyamine-deficient E. coli mutant results partially from oxidative stress-induced damage and the mutant thus exhibits the requirement for antioxidant or specific nutritional amino acid during normal aerobic growth.