Isolation of antioxidant constituents from Combretum apiculatum subsp. apiculatum

Species of the family Combretaceae are used extensively in traditional medicine against inflammation and infections, and although antibacterial activity has been reported in non-polar extracts, further rationale for the widespread use of the Combretaceae is expected to exist. Methanol extracts of leaves of ten different Combretum species were evaluated for antioxidant activity by spraying TLC chromatograms of each leaf extract with 2,2-diphenyl-1-picrylhydrazyl (DPPH). Compounds with antioxidant activity were detected by bleaching of the purple DPPH colour. Leaf extracts of Combretum apiculatum subsp. apiculatum had the most antioxidant compounds. This species was consequently selected for phytochemical investigation. A DPPH assay-directed fractionation of the leaf extracts of C. apiculatum led to the isolation of four antioxidant compounds from the ethyl acetate and butanol soluble fractions. The structures of the compounds were determined by spectroscopic analyses (¹H-NMR, ¹³C-NMR and MS) and identified as: cardamonin (1), pinocembrin (2), quercetrin (3) and kaempferol (4). In a quantitative antioxidant assay, the more polar fractions (ethyl acetate and butanol) obtained by solvent–solvent fractionation had the highest antioxidant activity among the solvent fractions obtained from C. apiculatum, with EC₅₀ values of 3.91 ± 0.02 and 2.44 ± 0.02 μg/ml respectively. Of the four isolated compounds, quercetrin (4) and kaempferol (3) had the strongest antioxidant activity, with EC₅₀ values of 11.81 ± 85 and 47.36 ± 0.03 μM respectively. Cardamonin (1) and pinocembrin (2) did not demonstrate strong activity. L-ascorbic acid was used as standard antioxidant agent (EC₅₀ = 13.37 ± 0.20 μM or 2.35 μg/ml). The cytotoxicity of cardamonin and pinocembrin was evaluated on Vero kidney cells using the MTT (3-(4,5-dimethylthiazol)-2,5-diphenyl tetrazolium bromide) assay with berberine as positive control. At concentrations higher than 50 μg/ml of cardamonin or pinocembrin, the cells were not viable. Cardamonin was more toxic (LC₅₀ = 1.97 μg/ml) than pinocembrin (LC₅₀ = 29.47 μg/ml) and even the positive control, berberine (LC₅₀ = 12.35 μg/ml).     

Two acylated flavanone glycosides from Nierembergia hippomanica

Two new acylated flavanone glycosides have been isolated from Nierembergia hippomanica and identified by spectral data as pinocembrin 7-O-β-(3?-O-acetyl)neohesperidoside and pinocembrin 7-O-β-(6″-O-acetyl)neohesperidoside.     

Improved pinocembrin production in <Emphasis Type="Italic">Escherichia coli</Emphasis> by engineering fatty acid synthesis

The development of efficient microbial processes for pinocembrin production has attracted considerable attention. However, pinocembrin biosynthetic efficiency is greatly limited by the low availability of the malonyl-CoA cofactor in Escherichia coli. Fatty acid biosynthesis is the only metabolic process in E. coli that consumes malonyl-CoA; therefore, we overexpressed the fatty acid biosynthetic pathway enzymes β-ketoacyl-ACP synthase III (FabH) and β-ketoacyl-ACP synthase II (FabF) alone and in combination, and investigated the effect on malonyl-CoA. Interestingly, overexpressing FabH, FabF or both enzymes in E. coli BL21 (DE3) decreased fatty acid synthesis and increased cellular malonyl-CoA levels 1.4-, 1.6-, and 1.2-fold, respectively. Furthermore, pinocembrin production was increased 10.6-, 31.8-, and 5.87-fold in recombinant strains overexpressing FabH, FabF and both enzymes, respectively. Overexpression of FabF, therefore, triggered the highest pinocembrin production and malonyl-CoA levels. The addition of cerulenin further increased pinocembrin production in the FabF-overexpressing strain, from 25.8 to 29.9 mg/L. These results demonstrated that overexpressing fatty acid synthases can increase malonyl-CoA availability and improve pinocembrin production in a recombinant E. coli host. This strategy may hold promise for the production of other important natural products in which cellular malonyl-CoA is rate limiting.     

Kaempferol,a new nutrition-derived pan-inhibitor of human histone deacetylases

Kaempferol is a natural polyphenol belonging to the group of flavonoids. Different biological functions like inhibition of oxidative stress in plants or animal cells and apoptosis induction have been directly associated with kaempferol. The underlying mechanisms are only partially understood. Here we report for the first time that kaempferol has a distinct epigenetic activity by inhibition of histone deacetylases (HDACs). In silico docking analysis revealed that it fits into the binding pocket of HDAC2, 4, 7 or 8 and thereby binds to the zinc ion of the catalytic center. Further in vitro profiling of all conserved human HDACs of class I, II and IV showed that kaempferol inhibited all tested HDACs. In clinical oncology, HDAC inhibitors are currently under investigation as new anticancer compounds. Therefore, we studied the effect of kaempferol on human-derived hepatoma cell lines HepG2 and Hep3B as well as on HCT-116 colon cancer cells and found that it induces hyperacetylation of histone complex H3. Furthermore, kaempferol mediated a prominent reduction of cell viability and proliferation rate. Interestingly, toxicity assays revealed signs of relevant cellular toxicity in primary human hepatocytes only starting at 50 μM as well as in an in vivo chicken embryotoxicity assay at 200 μM.In conclusion, the identification of a novel broad inhibitory capacity of the natural compound kaempferol for human-derived HDAC enzymes opens up the perspective for clinical application in both tumor prevention and therapy. Moreover, kaempferol may serve as a novel lead structure for chemical optimization of pharmacokinetics, pharmacology or inhibitory activities.     

(—)-Rubranine from Aniba rosaeodora

Aniba rosaeodora wood was shown to contain (—)-rubranine. An optically inactive form of this citrylidene-chalcone was previously considered to be an artifact of extraction. The constitution of rubranine was confirmed by pyrolysis and syntheses, involving previously reported pyridine-catalysed condensation of pinocembrin and citral, whereby novel intermediates were prepared, and the acid-catalysed condensation of pinocembrin with geraniol followed by oxidation.     

In vivo evolution of metabolic pathways by homeologous recombination in mitotic cells

We describe a rapid and highly efficient method for the assembly, recombination, targeted chromosomal integration and regulatable expression of mosaic metabolic pathways by homeologous recombination in DNA repair deficient yeast cells. We have assembled and recombined 23 kb pathways containing all the genes encoding enzymes for the production of flavonoids, a group of plant secondary metabolites of nutritional and agricultural value. The mosaic genes of the pathways resulted from pair-wise recombination of two nonidentical (homeologous) wild-type genes. The recombination events occurred simultaneously in the cell. Correctly assembled mosaic gene clusters could only be observed in DNA repair deficient strains. Thus, libraries of intragenic mosaic pathways were generated. Randomly isolated clones were screened for their ability to produce flavonoids such as kaempferol, phloretin and galangin. Thus, the functionality of the recombinant pathways was proven. Additionally, significant higher concentrations of metabolites such as naringenin, pinocembrin and dihydrokaempferol were detected. Further analysis also revealed the production of different aromatic compounds such as styrene, hydroxystyrene, phloretic acid and other molecules. We show that the in vivo homeologous recombination strategy can generates libraries of intragenic mosaic pathways producing a high diversity of phenylpropanoid compounds.     

Foliar Essential Oil Glands of Eucalyptus Subgenus Eucalyptus (Myrtaceae) Are a Rich Source of Flavonoids and Related Non-Volatile Constituents

The sub-dermal secretory cavities (glands) embedded within the leaves of Eucalyptus (Myrtaceae) were once thought to be the exclusive repositories of monoterpene and sesquiterpene oils. Recent research has debunked this theory and shown that abundant non-volatile compounds also occur within foliar glands. In particular, glands of four species in subgenus Eucalyptus contain the biologically active flavanone pinocembrin. Pinocembrin shows great promise as a pharmaceutical and is predominantly plant-sourced, so Eucalyptus could be a potential commercial source of such compounds. To explore this we quantified and assessed the purity of pinocembrin in glands of 11 species of E. subg. Eucalyptus using Electro-Spray Ionisation Liquid Chromatography Mass Spectrometry of acetonitrile extracts and Gas Chromatography Mass Spectrometry analyses of hexane extracts of isolated glands which were free from other leaf tissues. Our results showed that the glands of subgenus Eucalyptus contain numerous flavanones that are structurally related to pinocembrin and often present in much greater abundance. The maximum concentration of pinocembrin was 2 mg g^-1 dry leaf found in E. stellulata, whereas that of dimethylpinocembrin (5,7-dimethoxyflavanone) was 10 mg g^-1 in E. oreades and that of pinostrobin (5-hydroxy-7-methoxyflavanone) was 12 mg g^-1 in E. nitida. We also found that the flavanones are exclusively located within the foliar glands rather than distributed throughout leaf tissues. The flavanones differ from the non-methylated pinocembrin in the degree and positions of methylation. This finding is particularly important given the attractiveness of methylated flavonoids as pharmaceuticals and therapeutics. Another important finding was that glands of some members of the subgenus also contain flavanone O-glucosides and flavanone-β-triketone conjugates. In addition, glands contain free β-triketones, β-triketone heterodimers and chromone C-glucosides. Therefore, the foliar glands of this taxonomically distinct group of plants are a rich source of a range of flavonoids and other biologically active compounds with great commercial potential.     

Flavonoidmuster als systematisches Merkmal in der Gattung Populus

Buds of almost every species of popular excrete lipophilic “coating” which contains many flavonoid and related derivatives. The compounds found in this “bud coat” of eighty taxa of Populus have been examined and nearly 30 compounds have been identified and their distribution related to the taxonomy of the genus. In sections Aigeiros and Tacamahaca and hybrids of these, chrysin, galangin and pinocembrin form the major components which, in most cases, are accompanied by lesser amounts of tectochrysin, izalpinin, pinostrobin, galangin-3-methylether and pinobanksin-3-acetate. Variations are shown by presence of apigenin and its monomethyl ethers, kaempferol methyl ethers, rhamnetin, quercetin-3,7- and 3,3′-dimethylether, the unusual flavanone “P4” and two chalcones. Myricetin, genkwanin and kaempferol-7.4′-dimethylether are each found only in one species. In the sections Leuce and Leucoides, the major compounds are cinnamic acid derivatives. Flavonoid patterns, as analysed on polyamide TLC, allow species of the sections Aigeiros and Tacamahaca and some hybrids to be characterized, provided several individuals are examined, It is not possible by this method to differentiate clones within a species.     

Flavonoid glycosides from fiveCyathea species

The leaves of 5 fern species of the genusCyathea, i.e.C. fauriei, C. mertensiana, C. leichhardtiana, C. podophylla andC. hancockii, have been chemically analysed. The former 3 species have kaempferol 3-sophoroside (sophoraflavonoloside) and kaempferol 7-rhamnoglucoside as glycosidic components, and the latter 2 species contain kaempferol 3-galactoside (trifolin) and kaempferol 3-rhamnoglucoside (nicotiflorin). In addition, vitexin, orientin, kaempferol 3-glucoside (astragalin), kaempferol 3-rhamnoside (afzelin) and kaempferol 7-arabinoside are detected as common constituents in all the 5 species analysed.     

4‴-O-acetylsarotanoside,a novel flavanone glycoside from Nierembergia hippomanica

A new flavanone glycoside was isolated from the aerial parts of Nierembergia hippomanica and identified as the 4?-acetate of pinocembrin 7-neohesperidoside.     

Effect of kaempferol on IgE-mediated anaphylaxis in C57BL/6 mice and LAD2 cells

BackgroundImmunoglobulin E (IgE)-mediated mast cell (MC) activation is crucial in multiple allergic diseases. Parkinson disease protein 7 (DJ-1) and Lyn kinase were reported as the receptor-proximal events in IgE receptor (FcεRI) signals in human MC. Kaempferol, a natural flavonol mainly derived from the rhizome of traditional Chinese herb Kaempferia galanga L. (Zingiberaceae), has been known to inhibit allergic reactions, but it was limited to the receptor-distal signals on rat basophilic leukemia cells. A thorough investigation of the inhibitory effects of kaempferol on human MC has not been done.PurposeTo investigate the inhibitory effects of kaempferol on IgE-mediated anaphylaxis in vivo and in human MCs, as well as the mechanism underlying its effects, especially the receptor-proximal signals.MethodsIgE-mediated passive cutaneous anaphylaxis and systemic anaphylaxis model were applied to elucidate the antiallergic activity of kaempferol in vivo. The degranulation assay, calcium imaging, the release of cytokines and chemokines on the laboratory of allergic disease 2 (LAD2) cells were used to evaluate the antiallergic effect of kaempferol in vitro. Western blot analysis was performed to investigate the DJ-1/Lyn signaling pathway and downstream molecules. Kinase activity assay, immunofluorescence, and molecular docking were conducted to confirm the influence of kaempferol on DJ-1/Lyn molecules.ResultsKaempferol dose-dependently attenuated ovalbumin/IgE-induced mice paw swelling, primary MC activation from paw skin, as well as rehabilitated the hypothermia, and reduced the serum concentrations of histamine, tumor necrosis factor-alpha, interleukin-8, and monocyte chemo-attractant protein-1. Additionally, kaempferol suppressed IgE-mediated LAD2 cell degranulation and calcium fluctuation. Remarkably, kaempferol was found to bind with DJ-1 protein, and initially prevented DJ-1 from translocating to the plasma membrane, thereby inhibited full activation of Lyn, and eventually restrained those receptor-distal signaling molecules, involved Syk, Btk, PLCγ, IP3R, PKC, MAPKs, Akt and NF-κB.ConclusionKaempferol could be used as a DJ-1 modulator for preventing MC-mediated allergic disorders through attenuating Lyn activation.     

Species-specific kaempferol derivatives in ferns of the appalachian Asplenium complex

A series of kaempferol derivatives have been identified in fronds of three parental species of the Appalachian Asplenium complex. Asplenium platyneuron is characterised by the presence of the 7-glucoside of kaempferol 3,4′-dimethyl ether and also contains kaempferol 3,7-diglucoside, free and with an aliphatic acyl attachment. By contrast, A. rhizophyllum contains a remarkable caffeoyl complex of kaempferol glycosides, which appears to be chromatographically homogenous. However, on deacylation, the complex yields caffeic acid and the 7-glucoside, 3,7-diglucoside, 3-sophoroside-7-glucoside and 7,4′-diglucoside of kaempferol. Asplenium montanum, in addition to having previously characterised glycosylxanthones, has two further kaempferol derivatives. It has been confirmed that these various species specific flavonoids are inherited in an additive fashion in three interspecific hybrids.     

The synthesis of afzelin,paeonoside and kaempferol 3-O-β-rutinoside

The structure of afzelin (kaempferol 3-O-α-l-rhamnopyranoside) and paeonoside (kaempferol 3,7-bis-O-β-d-glucopyranoside) has been confirmed by total synthesis. Synthetic kaempferol 3-O-β-rutinoside had a mp of 190–192° suggesting that those natural kaempferol 3-O-rhamnoglucosides which melt in the same range are also 3-O-β-rutinosides.     

An acylated flavonoid from Nierembergia hippomanica

A new acylflavonoid has been isolated from Nierembergia hippomanica and identified by chemical and spectral data as pinocembrin 7-O-β-(2?-O-acetyl)neohesperidoside.     

Flavonol glycosides from Asplenium bulbiferum

From aerial parts of the fern Asplenium bulbiferum, besides kaempferol 3,7-diglucoside and kaempferol 3-O-rhamnoside- 7-O-glucoside, the new glycoside kaempferol 3-O-β-glucoside-7-O-β-galactoside has been characterized.     

Chemotaxonomic value of flavonoids in Chromolaena congesta (Asteraceae)

The phytochemical investigation on the aerial parts of Chromolaena congesta led to the isolation of nine flavonoids, known in the literature as genkwanin (1) kumatakenin (2) acacetin (3), kaempferol 3-methyl ether (4), apigenin (5), apigenin 5,7-dimethyl ether (6), apigenin 5-methyl ether (7), luteolin (8) and kaempferol (9). The chemical structures were established on the basis of spectral evidence. All the compounds were isolated from this species for the first time. The results from the present study provide further information about the flavonoids as taxonomic marker of the genus Chromolaena, and the chemotaxonomic significance of these compounds were also summarized.     

Flavonoids from the leaf resin of Adenostoma sparsifolium

Four flavonoids were identified from the external leaf extract of Adenostoma sparsifolium: the two new flavones 3, 7-dihydroxy-5, 6-dimethoxyflavone and 3, 5, 7-trihydroxy-8-methoxyflavone and the known compounds galangin and pinocembrin.     

Preparation of Water and Ethanolic Extracts of Propolis and Evaluation of the Preparations

Propolis was extracted using water and various concentrations of ethanol as solvents. The extracts were investigated by measurement of absorption spectrum with a UV spectrophotometer, reversed phase-high pressure thin-layer chromatography and reversed phase-HPLC. Maximum absorption of all extracts was 290 nm, resembling flavonoid compounds, and the 80% ethanolic extract showed highest absorption at 290 nm. The most isosakuranetin, quercetin, and kaempferol were extracted from mixtures of propolis and 60% ethanol, while 70% ethanol extracted the most pinocembrin and sakuranetin, but 80% ethanol extracted more kaempferide, acacetin, and isorhamnetin from propolis. The 60 to 80% ethanolic extracts of propolis strongly inhibited microbial growth and 70 and 80% ethanolic extracts had the greatest antioxidant activity and 80% ethanolic extract strongly inhibited hyaluronidase activity.     

Further studies of flavonols of Clibadium (compositae)

The flavonoids of an additional eight species of Clibadium have been determined. The compounds are derivatives of kaempferol, quercetin and quercetagetin. O-Methylated quercetagetin derivatives were found in several taxa with the possibility that 6-methoxykaempferol may also exist in one collection. Kaempferol and quercetin exist as 3-O-glucosides, galactosides, rhamnosides, rutinosides and diglucosides although not all glycosides occur in each taxon. Quercetagetin derivatives occur as 7-O-glucosides. Observations on these newly investigated species confirm previous work in the genus that three types of flavonoid profiles exist: (1) kaempferol and quercetin 3-glycosides; (2) kaempferol and quercetin 3-glycosides plus quercetagetin 7-glucoside; and (3) kaempferol and quercetin 3-glycosides plus quercetagetin 7-glucoside and O-methylated derivatives of quercetagetin.     

Kaempferol and its glycosides from Equisetum silvaticum L. from the Khanty-Mansi autonomous area

Three flavonoids were isolated from the aerial part of the wood horsetail (Equisetum silvaticum L.); two of them were found for the first time. The compounds were identified as kaempferol, kaempferol 3-O-β-D-galactopyranosyl-7-O-α-L-rhamnopyranoside and kaempferol 3-O-rutinosyl-7-O-L-rhamnopyranoside on the basis of the chemical transformations and IR, UV, ¹H-NMR and mass spectra.