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.     

Flavonoids of Astilbe and Rodgersia compared to Aruncus

The flavonoid profiles of Astilbe (four taxa studied) and Rodgersia (two taxa studied) are based on simple flavonol glycosides. Astilbe has 3-O-mono-, 3-O-di-, and 3-O-triglycosides of kaempferol, quercetin, and myricetin, while Rodgersia has only mono- and diglycosides of kaempferol and quercetin. Astilbe×arendsii was also shown to accumulate dihydrochalcone glycosides. The flavonoid profile of Rodgersia is the simplest recorded so far in the herbaceous Saxifragaceae. The flavonoids of two species of Aruncus were shown to be based upon kaempferol and quercetin 3-O-mono- and 3-O-diglycosides. One of the species also exhibited an eriodictyol glycoside. The triglycoside differences were not considered important, but the differences in myricetin occurrences were taken as evidence against derivation of Saxifragaceae from an Aruncus-like ancestor. Should such an event be proposed, however, serious consideration would have to be given to the current pattern of myricetin occurrence in the two families.     

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.     

Anthochlors and other flavonoids as honey guides in the compositae

Anthochlor pigments have been recorded for the first time in three species of Helianthus and in Viguiera laciniata. Yellow flavonols based on quercetagetin or gossypetin have been found in yellow flowered species of Eriophyllum and Geraea. In Eriophyllum, all seven species studied contain quercetagetin and/or patuletin 7-glucosides. In those species containing UV honey guides, either yellow flavonoid or colourless quercetin glycosides can be responsible for producing strongly UV absorbent zones on the flowers. Thus in Helianthus, honey guides in some species result from UV absorbance by the chalcone coreopsin and the aurone sulphurein, whilst in H. annuus they result from absorbance by quercetin 3- and 7-glucosides. Furthermore, the occurrence of yellow flavonoids in flowers is not directly correlated with the presence of UV nectar guides. A detailed study of the distribution of chalcone in Coreopsis bigelovii flowers revealed that the anthochlor was present in epidermal cells on both upper and lower surfaces.     

Metabolic profiling of flavonoids in Lotus japonicus using liquid chromatography Fourier transform ion cyclotron resonance mass spectrometry

Flavonoids detected from a model legume plant, Lotus japonicus accessions Miyakojima MG-20 and Gifu B-129, were profiled using liquid chromatography Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR/MS). Five flavonols and two anthocyanidins were detected as aglycones. LC-FTICR/MS facilitated simultaneous detection of 61 flavonoids including compounds that have not been reported previously. Chemical information of the peaks such as retention time, lambdamax, m/z value of the quasi-molecular ion, m/z value of MS/MS fragment ions, and relative intensity of MS/MS fragments was obtained, along with the molecular formulas and conjugate structures. Fourteen were completely identified by comparison with authentic compounds. The high accuracy of m/z values, being 0.081 ppm between observed and theoretical values, allowed prediction of molecular formulas of unknown compounds with the help of isotope peak information for determination of chemical composition. Based on a predicted elemental composition, the presence of a novel nitrogen-containing flavonoid was proposed. A comparison of flavonoid profiles in flowers, stems, and leaves demonstrated that the flowers yielded the most complex profile, containing 30 flower-specific flavonoids including gossypetin glycosides and isorhamnetin glycosides. A comparison of flavonoid profiles between MG-20 and B-129 grown under the same conditions revealed that the accumulation of anthocyanins was higher in B-129 than MG-20, particularly in the stem. Developmental changes in the flavonoid profiles demonstrated that kaempferol glycosides increased promptly after germination. In contrast, quercetin glycosides, predominant flavonoids in the seeds, were not detectable in growing leaves.     

A CHEMOTAXONOMIC STUDY OF LEMNACEAE

One hundred eighty-six clones of Lemnaceae, representing world-wide collections of 22 taxa, were grown in axenic cultures reproduced by clonal subcultures. Following morphological examinations under a range of culture conditions, a “key” clone was selected to represent each taxon. These key clones were completely representative of the qualitative flavonoid chemistry of the taxa as determined from a number of clones. With the exceptions of Spirodela polyrhiza and S. biperforala which produce identical flavonoids, the flavonoid associations of each species of Lemnaceae were unique in the family. Through paper chromatographic comparisons of purified compounds, plus visible and ultraviolet absorption spectroscopy of isolated individual flavonoids, a total of 47 different compounds was described. These substances included: glycosides of anthocyanidins (cyanidin and petunidin); glycosides of flavonols (kaempferol and quercetin); glycosides of flavones (apigenin and luteolin); and several glycoflavones (C-glycosyl-flavones). When grown under equivalent controlled conditions of culture, infraspecific variation in the qualitative production of these 47 flavonoids was detected in only one flavone glycoside of the species Lemna perpusilla. The reliability of the flavonoid patterns under various conditions of culture was investigated. Under 62 different regimes S. oligorhiza and S. polyrhiza showed only minor variations in their flavonoid glycosides. Studies of other taxa supported this generalization. Under controlled conditions, morphological intergradation obscured identification of many collections. Each could be conclusively identified by its flavonoid chemistry.     

Pathway engineering for healthy phytochemicals leading to the production of novel flavonoids in tomato fruit

Flavonoids are a large family of plant polyphenolic secondary metabolites. Although they are widespread throughout the plant kingdom, some flavonoid classes are specific for only a few plant species. Due to their presumed health benefits there is growing interest in the development of food crops with tailor-made levels and composition of flavonoids, designed to exert an optimal biological effect. In order to explore the possibilities of flavonoid engineering in tomato fruits, we have targeted this pathway towards classes of potentially healthy flavonoids which are novel for tomato. Using structural flavonoid genes (encoding stilbene synthase, chalcone synthase, chalcone reductase, chalcone isomerase and flavone synthase) from different plant sources, we were able to produce transgenic tomatoes accumulating new phytochemicals. Biochemical analysis showed that the fruit peel contained high levels of stilbenes (resveratrol and piceid), deoxychalcones (butein and isoliquiritigenin), flavones (luteolin-7-glucoside and luteolin aglycon) and flavonols (quercetin glycosides and kaempferol glycosides). Using an online high-performance liquid chromatography (HPLC) antioxidant detection system, we demonstrated that, due to the presence of the novel flavonoids, the transgenic tomato fruits displayed altered antioxidant profiles. In addition, total antioxidant capacity of tomato fruit peel with high levels of flavones and flavonols increased more than threefold. These results on genetic engineering of flavonoids in tomato fruit demonstrate the possibilities to change the levels and composition of health-related polyphenols in a crop plant and provide more insight in the genetic and biochemical regulation of the flavonoid pathway within this worldwide important vegetable.     

Anti-inflammatory effect of soyasaponins through suppressing nitric oxide production in LPS-stimulated RAW 264.7 cells by attenuation of NF-κB-mediated nitric oxide synthase expression

The anti-inflammatory properties of soyasaponins (especially soyasaponins with different chemical structures) have scarcely been investigated. We investigated the inhibitory effects of five structural types of soyasaponins (soyasaponin A¹, A², I and soyasapogenol A, B) on the induction of nitric oxide (NO) and inducible NO synthase (iNOS) in murine RAW 264.7 cells activated with lipopolysaccharide (LPS). Soyasaponin A¹, A² and I (25-200 μg/mL) dose-dependently inhibited the production of NO and tumor necrosis factor α (TNF-α) in LPS-activated macrophages, whereas soyasapogenol A and B did not. Furthermore, soyasaponin A¹, A² and I suppressed the iNOS enzyme activity and down-regulated the iNOS mRNA expression both in a dose-dependent manner. The reporter gene assay revealed that soyasaponin A¹, A² and I decreased LPS-induced nuclear factor kappa B (NF-κB) activity. Soyasaponin A¹, A² and I exhibit anti-inflammatory properties by suppressing NO production in LPS-stimulated RAW 264.7 cells through attenuation of NF-κB-mediated iNOS expression. It is proposed that the sugar chains present in the structures of soyasaponins are important for their anti-inflammatory activities. These results have important implication for using selected soyasaponins towards the development of effective chemopreventive and anti-inflammatory agents.     

A novel saponin hydrolase from Neocosmospora vasinfecta var. vasinfecta

We isolated a soybean saponin hydrolase from Neocosmospora vasinfecta var. vasinfecta PF1225, a filamentous fungus that can degrade soybean saponin and generate soyasapogenol B. This enzyme was found to be a monomer with a molecular mass of about 77 kDa and a glycoprotein. Nucleotide sequence analysis of the corresponding gene (sdn1) indicated that this enzyme consisted of 612 amino acids and had a molecular mass of 65,724 Da, in close agreement with that of the apoenzyme after the removal of carbohydrates. The sdn1 gene was successfully expressed in Trichoderma viride under the control of the cellobiohydrolase I gene promoter. The molecular mass of the recombinant enzyme, about 69 kDa, was smaller than that of the native enzyme due to fewer carbohydrate modifications. Examination of the degradation products obtained by treatment of soyasaponin I with the recombinant enzyme showed that the enzyme hydrolyzed soyasaponin I to soyasapogenol B and triose [alpha-L-rhamnopyranosyl (1-->2)-beta-D-galactopyranosyl (1-->2)-D-glucuronopyranoside]. Also, when soyasaponin II and soyasaponin V, which are different from soyasaponin I only in constituent saccharides, were treated with the enzyme, the ratio of the reaction velocities for soyasaponin I, soyasaponin II, and soyasaponin V was 2,680:886:1. These results indicate that this enzyme recognizes the fine structure of the carbohydrate moiety of soyasaponin in its catalytic reaction. The amino acid sequence of this enzyme predicted from the DNA sequence shows no clear homology with those of any of the enzymes involved in the hydrolysis of carbohydrates.     

Identification and characterization of glycosyltransferases involved in the biosynthesis of soyasaponin I in Glycine max

Triterpene saponins are a diverse group of compounds with a structure consisting of a triterpene aglycone and sugars. Identification of the sugar-transferase involved in triterpene saponin biosynthesis is difficult due to the structural complexity of triterpene saponin. Two glycosyltransferases from Glycine max, designated as GmSGT2 and GmSGT3, were identified and characterized. In vitro analysis revealed that GmSGT2 transfers a galactosyl group from UDP-galactose to soyasapogenol B monoglucuronide, and that GmSGT3 transfers a rhamnosyl group from UDP-rhamnose to soyasaponin III. These results suggest that soyasaponin I is biosynthesized from soyasapogenol B by successive sugar transfer reactions.     

Betacyanins and phenolic compounds from Amaranthus spinosus L. and Boerhavia erecta L

Stem bark extracts of Boerhavia erecta L. (erect spiderling) and Amaranthus spinosus L. (spiny amaranth), two wild growing weed plants used in traditional African medicine, were characterized with respect to their phenolic profile including the betalains. While the main betalains in A. spinosus were identified as amaranthine and isoamaranthine, the major betacyanins in B. erecta were betanin, isobetanin together with neobetanin. The latter showed higher betalain concentrations amounting to 186 mg/100 g, while the former contained 24 mg betacyanins in 100 g of the ground plant material. Extracts of A. spinosus were found to contain hydroxycinnamates, quercetin and kaempferol glycosides, whereas catechins, procyanidins and quercetin, kaempferol and isorhamnetin glycosides were detected in B. erecta. The amounts of these compounds ranged from 305 mg/100 g for A. spinosus to 329 mg/100 g for B. erecta.     

The leaf flavonoids of the Zingiberales

A survey of flavonoids in the leaves of 81 species of the Zingiberales showed that, while most of the major classes of flavonoid are represented in the order, only two families, the Zingiberaceae and Marantaceae are rich in these constituents. In the Musaceae (in 9 species), Strelitziaceae (in 8 species) and Cannaceae (1 of 2 species) flavonol glycosides were detected in small amount and in the Lowiaceae no flavonoids were fully identified. In the Zingiberaceae kaempferol (in 22%), quercetin (72%) and proanthocyanidins (71%) are distributed throughout the family. The two subfamilies of the Zingiberaceae may be distinguished by the presence of myricetin (in 26%), isorhamnetin (10%) and syringetin (3%) in the Zingiberoideae and of flavone $\text{C-}\text{glycosides}$ (in 86% of taxa) in the Costoideae. A number of genera have distinctive flavonol profiles: e.g. Hedychium species have myricetin and quercetin. Roscoea species isorhamnetin and quercetin and Alpinia species kaempferol and quercetin glycosides. A new glycoside, syringetin 3-rhamnoside was identified in Hedychium stenopetalum. In the Zingiberoideae flavonols were found in glycosidic combination with glucuronic acid, rhamnose and glucose but glucuronides were not detected in the Costoideae or elsewhere in the Zingiberales. The Marantaceae is chemically the most diverse group and may be distinguished from other members of the Zingiberales by the occurrence of both flavone $\text{O-}$ and $\text{C-}\text{glycosides}$ and the absence of kaempferol and isorhamnetin glycosides. The distribution of flavonoid constituents within the Marantaceae does not closely follow the existing tribai or generic limits. Flavonols (in 50% of species). flavones (20%) and flavone $\text{C-}\text{glycosides}$ (40%) are found with similar frequency in the two tribes and in the genera Calathea and Maranta both flavone and flavonol glycosides occur. Apigenin- and luteolin-7-sulphates and luteolin-7,3′-disulphate were identified in Maranta bicolor and M. leuconeura var. kerchoveana and several flavone $\text{C-}\text{glycosides}$ sulphates in Stromanthe sanguinea. Anthocyanins were identified in those species with pigmented leaves or stems and a common pattern based on cyanidin-and delphinidin-3-rutinosides was observed throughout the group. Finally the possible relationship of the Zingiberales to the Commelinales, Liliales, Bromeliales and Fluviales is discussed.     

Enhancement of pheochromocytoma nerve cell growth by consecutive fractionization of <Emphasis Type="Italic">Angelica gigas</Emphasis> Nakai extracts

This work was to investigate the effect of flavonoids from Angelica gigas Nakai on the proliferation and differentiation of PC12 cells. Several solvents including hexane, chloroform, ethyl acetate, butanol and water consecutively partitioned. We determined the ethanol crude extract of Angelica gigas Nakai. The hexane fraction was shown to contain the highest number of flavonoids as follows; 21.48 mg/g and the composition of the flavonoids was as follows: 12.24 mg/g of quercetin, 4.39 mg/g of myricetin and 2.58 mg/g of catechin. In addition, this hexane fraction greatly increased both cell growth and outgrowth of the neurite, and whose effects were three times higher than those of the other fractions. The length of the neurites was measured as ca. 110 μm in adding 50 μg/mL of the hexane fraction, which was about the same as the case of adding 50 ng/mL of NGF as a positive control. This result indicates that the differentiation of PC12 cells by the addition of the hexane fraction was comparable to the case of adding NGF. The hexane fraction was also determined to prevent apoptosis of PC12 cells by suppressing DNA fragmentation. It is interesting that the mixture of three major flavonoids, quercetin, myricetin and catechin showed stronger activity on, both PC12 cell growth and neuritis outgrowth, than when adding each flavonoid alone. We believe this was due to the synergistic effects of the three flavonoids. The activities of these flavonoids from Angelica gigas Nakai are reported for the first time in this study.     

The phylogenetic significance of flavonoids in Crambe L. (Cruciferae)

A total of 21 flavonoid compounds has been detected in 14 species of Crambe. In general, both the sections of the genus and the species can be distinguished easily by their flavonoid patterns. The members of Crambe sections Crambe and Dendrocrambe seem to be the most primitive with their perennial or rhizocarpous habit. They show a diversity of flavonol glycosides, derived from either kaempferol or quercetin. The presumed presence of proanthocyanidins confers an additional primitive character to Crambe section Dendrocrambe. In contrast, members of Crambe section Leptocrambe show a relatively poor pattern where the quercetin glycosides have disappeared. In the case of C. hispanica and C. abyssinica flavonols are completely absent while two flavones, luteolin and apigenin appear in glycosidic form; these are probably the most evolved Crambe species and exhibit an annual habit. A dendrogram exclusively based on data of the presence or absence of flavonoids has been constructed. It is similar to the one that could have been expected from use of morphological data alone, but it does provide some hints on the possible phylogenetic relationships between the species. Flavonoid evidence also supports the hypothesis on an east-west Mediterranean disjunction within the genus.     

Neolignan and flavonoid glycosides in Juniperus communis var. depressa

Two neolignan glycosides (junipercomnosides A and B) were isolated from aerial parts of Juniperus communis var. depressa along with two known neolignan glycosides and seven flavonoid glycosides. The structures of the isolated compounds were determined by spectral analysis, in particular by 2D-NMR analysis. The significance of distribution of flavonoids in the chemotaxonomy of genus Juniperus was also discussed.     

The reaction of flavonoid metabolites with peroxynitrite

There is much interest in the bioactivity of in vivo flavonoid metabolites. We report for the first time the hierarchy of reactivity of flavonoid metabolites with peroxynitrite and characterise novel reaction products. O-Methylation of the B-ring catechol containing flavonoids epicatechin and quercetin, and O-glucuronidation of all flavonoids reduced their reactivity with peroxynitrite. The reaction of the flavanones hesperetin and naringenin and their glucuronides resulted in the formation of multiple mono-nitrated and nitrosated products. In contrast, the catechol-containing flavonoids epicatechin and quercetin yielded oxidation products which when trapped with glutathione led to the production of glutathionyl-conjugates. However, the O-methylated metabolites of epicatechin yielded both mono- and di-nitrated products and nitrosated metabolites. The 3'-O-methyl metabolite of quercetin also yielded a nitrosated species, although its counterpart 4'-O-methyl quercetin yielded only oxidation products. Such products may represent novel metabolic products in vivo and may also express cellular activity.     

The role of lipophilic and polar flavonoids in the classification of temperate members of the Anthemideae

Lipophilic and vacuolar flavonoids were separately identified in representative temperate species of the genera Anthemis, Chrysanthemum, Cotula, Ismelia, Leucanthemum and Tripleurospermum. The four Anthemis species investigated variously produced four main surface constituents, in leaf and flower: santin, quercetagetin 3,6,3′-trimethyl ether, scutellarein 6,4′-dimethyl ether and 6-hydroxyluteolin 6,3′-dimethyl ether. By contrast, surface extracts of disc and ray florets of the species of Chrysanthemum, Cotula, Ismelia, Leucanthemum and Tripleurospermum surveyed yielded five common flavones in the free state: apigenin, luteolin, acacetin, apigenin 7-methyl ether and chrysoeriol. Polar flavonoids were isolated and identified in leaf, ray floret and disc floret of all the above plants. Anthemis species were distinctive in having flavonol glycosides in the leaves, whereas the leaf flavonoids of the other taxa were generally flavone O-glycosides. The 3-glucoside and 3-rutinoside of patuletin were characterised for the first time from Anthemis tinctoria ssp. subtinctoria. Two new flavonol glycosides, the 5-glucuronides of quercetin and kaempferol, were obtained from the leaf of Leucanthemum vulgare, where they co-occur with the related 5-glucosides and with several flavone glycosides. The ray florets of these Anthemideae generally contain apigenin and/or luteolin 7-glucoside and 7-glucuronide, whereas disc florets have additional flavonol glycosides, notably the 7-glucosides of quercetin and patuletin and the 7-glucuronide of quercetin. A comparison of the flavonoid pattern encountered here with those previously recorded for Tanacetum indicate some chemical affinity between Anthemis and Tanacetum. Flavonoid patterns of the other five genera are more distinct from those of Tanacetum and suggest that those genera form a related group. All 14 species surveyed for their flavonoid profiles have distinctive constituents and the chemical data are in harmony with modern taxonomic treatments of the “Chrysanthemum complex” as a series of separate genera.     

Fragmentation pathways of acylated flavonoid diglucuronides from leaves of Medicago truncatula

Introduction – Flavonoids are important plant compounds occurring in tissues mostly in the form of glycoconjugates. Most frequently the sugar moiety is comprised of mono‐ or oligosaccharides consisting of common sugars like glucose, rhamnose or galactose. In some plant species the glycosidic moiety contains glucuronic acid and may be acylated by phenylpropenoic acids. Methodology – Flavonoid glyconjugates were extracted from leaves of Medicago truncatula ecotype R108 and submitted to analysis using high‐performance liquid chromatography combined with high‐resolution tandem (quadrupole‐time of flight, QToF) mass spectrometry. Results – The studied leaf extracts contained 26 different flavonoid glycosides among which 22 compounds were flavone (apigenin, luteolin, chrysoeriol and tricin) glucuronides and 13 were acylated with aromatic acids (p‐coumaric, ferulic or sinapic). The fragmentation pathways observed in positive and negative ion mass spectra differed substantially between each other and from these of flavonoid glycosides which did not contain acidic sugars. The application of high‐resolution MS techniques allowed unequivocal differentiation between ions with the same nominal m/z values containing different substituents (e.g. ferulic acid or glucuronic acid). Eleven of the identified flavonoids have not been reported previously in this species. Perspectives – The presented unique fragmentation pathways of flavonoid glucuronates enable detection of these compounds in tissue extracts from different plant species. Copyright © 2009 John Wiley & Sons, Ltd.     

Flavonoid variation in Lasthenia burkei

The flavonoid chemistry of Lasthenia burkei has been re-examined. The flavonoid profile of the species is based upon quercetin, patuletin and an unidentified aglycone. Glucosides, glucuronides, xylosides, diglucosides and glucosylxylosides were identified. Small amounts of a patuletin triglycoside were also detected. One hundred and eight individual plants, representing five populations, three in Lake County and two in Sonoma County (California), were compared chromatographically. Two flavonoid races were observed based upon occurrence of xylosyl-based glycosides, quercetin and patuletin diglycosides and distribution of quercetin and patuletin glucuronides. Distinctions between the races was not absolute in all cases but the differences in frequencies of occurrence of the various types of compounds is marked.