Comparative mutagenesis of plant flavonoids in microbial systems.

The plant flavonoids quercetin (3,5,7,3',4'-pentahydroxyflavone), morin (3,5,7,2',4'-pentahydroxyflavone), kaempferol (3,5,7,4'-tetrahydroxyflavone), chrysin (5,7-dihydroxyflavone), fisetin (3,7,3',4'-tetrahydroxyflavone), myricetin (3,5,7,3',4',5'-hexahydroxyflavone), myricitrin (myricetin-3-rhamnoside), hesperetin (3',5,7-trihydroxy-4'-methoxyflavanone), quercitrin (quercetin-3-L-rhamnoside), rutin (quercetin-3-rhamnosylglucoside or quercetin-3-rutinoside), and hesperidin (hesperetin-7-rutinoside) have been assayed for mutagenicity in the Salmonella/microsomal activation system. Quercetin, morin, kaempferol, fisetin, myricetin, quercitrin and rutin were mutagenic in the histidine reversion system with the frameshift strain TA98. The flavonols quercetin and myricetin are mutagenic without metabolic activation, although more effective when a rat liver microsomal preparation (S-9) is included; all others require metabolic activation. Flavonoids are common constituents of higher plants, with extensive medical uses. In addition to pure compounds, we have examined crude extracts of tobacco (snuff) and extracts from commonly available nutritional supplements containing rutin. Mutagenic activity can be detected and is correlated with the flavonoid content.     

Flavonols from Pterogyne nitens and their evaluation as myeloperoxidase inhibitors

A myeloperoxidase inhibitory kaempferol derivative, namely pterogynoside (1), was isolated from fruits of Pterogyne nitens, along with six known flavonols, kaempferol, afzelin, kaempferitrin, quercetin, isoquercetrin and rutin. The structures of all compounds were elucidated primarily from 1D and 2D NMR spectroscopic analyses, as well as by high resolution mass spectrometry. All flavonols were screened to identify secondary metabolites as potential myeloperoxidase (MPO) inhibitors, and at concentrations of 0.50–50 nM, quercetin (5), isoquercitrin (6) and rutin (7) exhibited strong inhibitory effects with IC₅₀ values of 1.22 ± 0.01, 3.75 ± 0.02 and 3.60 ± 0.02, respectively. The MPO activity detected for the new derivative 1 was markedly decreased (IC₅₀ 10.3 ± 0.03) when compared with known flavonols 5–7, and interestingly increased when tested against ABTS scavenging activity.     

Flavonoids of Abies amabilis needles

Seventeen flavonol glycosides were identified from needles of Abies amabilis and these were based on 6 aglycone types: syringetin, isorhamnetin, kaempferol, quercetin, laricytrin and myricetin. Glycosides were 3-O-rutinosides, 3-O-glucosides, 3-O-galactosides or 3-O-rhamnosides. Also identified as needle constituents were rhamnosylvitexin and dihydroquercetin.     

Flavonol and drimane-type sesquiterpene glycosides of Warburgia stuhlmannii leaves

An investigation of methanolic extract of Warburgia stuhlmannii leaves has led to the isolation of two new drimane-type sesquiterpene glycosides characterized as mukaadial 6-O-beta-D-glucopyranoside, mukaadial 6-O-alpha-L-rhamnopyranoside together with two other novel flavonol glycosides identified as 3',5'-O-dimethylmyricetin 3-O-beta-D-2",3"-diacetylglucopyranoside and 3'-O-methylquercetin 3-O-beta-D-2",3",4"-triacetylglucopyranoside. The known compounds; mukaadial, deacetylugandensolide, quercetin, kaempferol, kaempferol 3-O-alpha-L-rhamnopyranoside, quercetin 3-O-beta-D-glucopyranoside, kaempferol 7-O-beta-D-glucopyranoside, myricetin 3-O-alpha-L-rhamnopyranoside, quercetin 3-O-alpha-L-rhamnopyranoside, quercetin 3-O-sophoroside and isorhamnetin 3-O-beta-D-glucopyranoside were also isolated from the same extract.     

Flavonoids of Leptarrhena pyrolifolia

The flavonoids of Leptarrhena pyrolifolia comprise (+)-dihydromyricetin and mono-, di-and triglycosides of kaempferol, quercetin, isorhamnetin and myricetin. This is the first report of a dihydroflavonol in the Saxifragaceae.     

Terpenoids and flavonoids of Bridelia ferruginea

The terpenoid and flavonoid constituents of the hitherto unexamined medicinal plant Bridelia ferruginea are reported. Quercetin, quercetin 3-glucoside, rutin, myricetin 3-glucoside and myricetin 3-rhamnoside were identified.     

Flavonol glycosides of Limnanthes douglasii

Eighteen flavonol glycosides were isolated from petal and leaf-stem of Limnanthes douglasii. There were six aglycones: kaempferol, quercetin, isorhamnetin, myriectin, syringetin and a new flavonol, myricetin 3′-methyl ether. Each occurred as the 3-rutinoside, 3-rhamnosylrutinoside and 3-rutinoside-7-glucoside.     

Effect of Ginkgo biloba extract on rat hepatic microsomal CYP1A activity: role of ginkgolides, bilobalide, and flavonols

The present study investigated the in vitro effect of Ginkgo biloba extracts and some of the individual constituents (ginkgolides, bilobalide, and flavonols such as kaempferol, quercetin, isorhamnetin, and their glycosides) on CYP1A-mediated 7-ethoxyresorufin O-dealkylation in hepatic microsomes isolated from rats induced with beta-naphthoflavone. G. biloba extract competitively inhibited CYP1A activity, with an apparent Ki value of 1.6 +/- 0.4 microg/mL (mean +/- SE). At the concentrations present in the G. biloba extracts, ginkgolides A, B, C, and J and bilobalide did not affect CYP1A activity, whereas kaempferol (IC50 = 0.006 +/- 0.001 microg/mL, mean +/- SE), isorhamnetin (0.007 +/- 0.001 microg/mL), and quercetin (0.050 +/- 0.003 microg/mL) decreased this activity. The monoglycosides (1 and 10 microg/mL) and diglycosides (10 microg/mL) of kaempferol and quercetin but not those of isorhamnetin also inhibited CYP1A activity. The order of inhibitory potency was kaempferol approximately equal to isorhamnetin > quercetin, and for each of these flavonols the order of potency was aglycone > monoglycoside > diglycoside. In summary, G. biloba extract competitively inhibited rat hepatic microsomal CYP1A activity, but the effect was not due to ginkgolides A, B, C, or J, bilobalide, kaempferol, quercetin, isorhamnetin, or the respective flavonol monoglycosides or diglycosides.     

Flavonoids from the genus Taxus

From the needles of Taxus baccata the following flavonoids were isolated: 3-O-rutinosides quercetin, myricetin and kaempferol, 7-O-glucosides kaempferol and quercetin, kaempferol, quercetin, myricetin. The composition of flavonols and biflavones in some of the species of the genus Taxus, namely T. celebica, T. cuspidata, T. media and cultivar varieties T. baccata 'Aurea', T. baccata 'Aurea decora', T. baccata 'Elegantissima', T. baccata 'Fastigiata', T. baccata 'Pyramidalis', T. media 'Hatfieldii' were compared by HPLC separation.     

Scavenger effect of flavonols on HOCl-induced luminol chemiluminescence.

Hypochlorous acid (HOCl), the main product of the myeloperoxidase system, is a strong oxidant and a potent chlorinating agent, which can damage host tissues. In the present work, the scavenger effect of three aglycone flavonols (myricetin, quercetin and kaempferol) and of the natural glycoside flavonol, rutin, was studied towards HOCl using luminol-dependent chemiluminescence (CL). At 1 micro mol/L fi nal concentration, rutin was the most powerful scavenger of HOCl with an inhibitory luminol oxidation of 91.4% +/- 3.2%. Quercetin, kaempferol and myricetin inhibited the luminol-dependent CL at the same concentration only by 75.9% +/- 3.4%, 57.7% +/- 5.3% and 43.3% +/- 3.5%, respectively. With increasing concentration of these flavonols, a dose-dependent inhibition of luminol CL was observed. In order to prove to what extent flavonols scavenge HOCl, their concentrations that gave 50% inhibition of luminescence (IC50) were compared to IC50 values of the sulphur-containing compounds N-acetyl cysteine (NAC) and taurine. The scavenging activities of compounds tested decrease in the order: rutin > NAC > quercetin > kaempferol > taurine. The present study revealed that rutin was the most effective scavenger agent.     

Identification of the potential active components of Abelmoschus manihot in rat blood and kidney tissue by microdialysis combined with ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry

In this paper, microdialysis combining with ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) was applied to simultaneously identify components in blood and kidney dialysis after oral administration of Abelmoschus manihot extract. Microdialysis probe was implanted in the jugular vein and the kidney medulla, respectively; microdialysis samples were collected continuously, transferred to microtubes and analyzed by UPLC-Q-TOF/MS. The components in microdialysis samples were separated by an UPLC HSS T3 column and eluted with acetonitrile and water (containing 0.1% formic acid) at a flow rate of 0.4 mL/min. The results showed that unbound constituents in blood circulation of the rat include hyperoside, isoquercitrin, quercetin monoglucuronide, quercetin-3'-O-glucoside, quercetin, myricetin, and hibifolin while unbound constituents in kidney are hyperoside, isoquercitrin, quercetin monoglucuronide, which might be the potential active components in vivo. The developed method was simple and reliable, and could be adopted to rapidly screen and identify potential active components contributing to pharmacological effects of TCM and to better clarify its action mechanism.     

Chemosensory behaviour and ciliary cyclic GMP-dependent protein kinase in Tetrahymena thermophila

The role of protein kinase, in particular cyclic GMP-dependent protein kinase (PKG), in the control of chemotaxis was studied in Tetrahymena thermophila using the membrane-permeable cGMP analogue 8-bromo-cGMP and the NO-generator sodium nitroprusside (SNP) that stimulates cGMP production by activating guanylate cyclase. Stimulation of chemoattraction was observed in the presence of 8-bromo-cGMP and nitroprusside when used in 10–100 μM concentrations in vivo. In vitro stimulation of ciliary membrane PKG activity was observed when using similar concentrations of cGMP or 8-bromo-cGMP to those in the in vivo experiments. In contrast, the protein kinase flavonol inhibitors quercitin and kaempherol block chemoattraction and reduce ciliary membrane PGK activity in vitro. For the inhibition of PKG, the IC-50 s for quercitin and kaempherol are 22 and 19 μM, respectively. The results suggest a modulating function of PKG on adaptory processes in cilia-mediated chemotaxis.

The ciliary membrane-associated PKG was partially characterized. Without added external protein kinase substrate in vitro, an endogenous ciliary membrane kinase activity showed phosphorylation of 55 and 97 kDa Triton-X-100 soluble proteins when analyzed by SDS-PAGE under reducing conditions and with ³²P-γ-ATP as phosphorylation donor. Phosphoamino acid analysis of PKG-phosphorylated proteins showed ³²P-phosphate labeling of serine and threonine residues. Ciliary membrane-associated PKG was further purified by carboxy-methyl-sephadex-column chromatography. The membrane enzyme was Mg²⁺⁺-dependent and had a pH optimum at 6.4. The carboxy-methyl-sephadex-eluted PKG was analyzed by electrophoresis on sodium dodecyl sulphate polyacrylamide gels showing a molecular weight of 70–75 kDa.     

In vitro degradation of the flavonol quercetin and of quercetin glycosides in the porcine hindgut

Abstract

The present study investigated the microbial degradation of the plant flavonol quercetin and its naturally occurring glycosides isoquercitrin and rutin in the porcine hindgut. The experiments were carried out with the semicontinuous colon-simulation technique. The fluid and particle phase of pig hindgut contents from freshly slaughtered animals were used for the in vitro incubations. Following a five-day equilibration period, quercetin, isoquercitrin or rutin were administered to fermentation vessels and their turnover rate was determined. None of the flavonols affected parameters of microbial fermentation like pH, redox potential or VFA production. The turnover rate for isoquercitrin was seven times higher than the turnover for the fermentation fluid. The turnover rates for quercetin and rutin were four and twofold higher than fluid turnover, respectively. After administration of isoquercitrin or rutin, their aglycone quercetin was detected as an intermediary metabolite. Under sterile conditions using autoclaved incubation fluids and hindgut contents, turnover rates for quercetin and rutin were still higher than the fluid turnover in the fermentation vessels. This indicates a certain chemical instability of the flavonols and/or adsorption to ingesta particles. Thus, flavonols are subjected to microbial metabolism in the porcine hindgut. The glycosidic structure strongly influences the rate of metabolism.     

Flavonoids and other phenolics of Cichorium and related members of the Lactuceae (Compositae)

Extraction of the leaves of chicory, Cichoriumintybus , revealed the presence for the first time of the 3-glucuronides of kaempferol, quercetin and isorhamnetin, the 3-glucoside of kaempferol and the 7-glucuronide of luteolin. The same compounds appear to occur throughout the genus. An aglycone survey of the leaves of 240 other members of the Lactuceae failed to reveal any other source of isorhamnetin, although quercetin and kaempferol were found as occasional constituents in 12 other genera. In Lactuca , a survey of 12 species showed eight with quercetin present. In general, though, flavonols are more uncommon than flavones and thus have more potential as taxonomic markers within the tribe. The coumarins cichoriin and aesculin and the caffeoyl-tartaric acid ester, chicoric acid, were also found to be present widely in the tribe.     

Flavonoids from Zea mays pollen

The flavonol glycosides of quercetin, isorhamnetin and kaempferol were isolated from Zea mays pollen. The most prominent flavonols were diglycosides of quercetin and isorhamnetin. Flavonol 3-O-glucosides of quercetin, isorhamnetin and kaempferol, and triglucosides of quercetin and isorhamnetin, were minor components. The flavonoid pattern of maize pollen is characterized by the accumulation of quercetin and isorhamnetin diglycosides and by the absence of flavones, which are common in other maize tissues.     

Flavonoid ingredients of Ginkgo biloba leaf extract regulate lipid metabolism through Sp1-mediated carnitine palmitoyltranferase 1A up-regulation

Background

Lipid accumulation is the primary evidence of non-alcoholic fatty liver disease (NAFLD). Ginkgo biloba extract (GBE) and its flavonoid ingredients (quercetin, kaempferol, and isorhamnetin) could lessen the lipid accumulation associated with up-regulation of the rate-limiting enzyme, carnitine palmitoyltransferase 1A (CPT1A), in the β-oxidation of long-chain fatty acids. In this study, we investigated the mechanisms by which GBE and its flavonoids induced expression of CPT1A.

Results

CPT1A inhibition with RNAi resulted in triglyceride accumulation in HepG2 cells. Through deletion and mutation analysis of CPT1A’s promoter combined with electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) experiments, the CPT1A promoter region (−50 to −5 nt) was determined to contain two putative Sp1 binding sites, namely Sp1a and Sp1b, which might act as the GBE regulation response DNA element. Sp1 might be induced to transfer from cytoplasma to nucleus to bind the promoter region of −50 to −5 nt by GBE. The regulatory effects of GBE on CPT1A were also verified on the flavonoid ingredients quercetin, kaempferol, and isorhamnetin.

Conclusion

Sp1 was crucial in regulating CPT1A expression with GBE and its flavonoid ingredients, and the −50 to −5 nt region of CPT1A promoter played important roles in Sp1 binding.

Electronic supplementary material

The online version of this article (doi:10.1186/s12929-014-0087-x) contains supplementary material, which is available to authorized users.     

In vitro degradation of the flavonol quercetin and of quercetin glycosides in the porcine hindgut

The present study investigated the microbial degradation of the plant flavonol quercetin and its naturally occurring glycosides isoquercitrin and rutin in the porcine hindgut. The experiments were carried out with the semicontinuous colon-simulation technique. The fluid and particle phase of pig hindgut contents from freshly slaughtered animals were used for the in vitro incubations. Following a five-day equilibration period, quercetin, isoquercitrin or rutin were administered to fermentation vessels and their turnover rate was determined. None of the flavonols affected parameters of microbial fermentation like pH, redox potential or VFA production. The turnover rate for isoquercitrin was seven times higher than the turnover for the fermentation fluid. The turnover rates for quercetin and rutin were four and twofold higher than fluid turnover, respectively. After administration of isoquercitrin or rutin, their aglycone quercetin was detected as an intermediary metabolite. Under sterile conditions using autoclaved incubation fluids and hindgut contents, turnover rates for quercetin and rutin were still higher than the fluid turnover in the fermentation vessels. This indicates a certain chemical instability of the flavonols and/or adsorption to ingesta particles. Thus, flavonols are subjected to microbial metabolism in the porcine hindgut. The glycosidic structure strongly influences the rate of metabolism.     

Effects of flavonoid on calcium content in femoral tissue culture and parathyroid hormone-stimulated osteoclastogenesis in bone marrow culture in vitro

The effect of various flavonoids, which are present in food and plants, on bone calcium content and osteoclastogenesis were investigated to compare action of flavonoid on bone formation and bone resorption in vitro. Rat femoral-diaphyseal (cortical bone) and -metaphyseal (trabecular bone) tissues were cultured for 48 h in Dulbecco’s modified Eagle’s medium (high glucose) supplemented with antibiotics and bovine serum albumin. Amoung quercetin, myricetin, kaempferol, isorhamnetin, curcumin, hesperidin, or astaxanthin in the range of 10⁻⁷–10⁻⁵ M, culture with quercetin (10⁻⁶ or 10⁻⁵ M) caused a significant increase in diaphyseal calcium content. Such an effect was not seen in other compounds. Mouse bone marrow cells were cultured for 7 days in the presence of parathyroid hormone (PTH; 10⁻⁷ M), a bone-resorbing factor, in vitro. Culture with PTH caused a significant increase in osteoclast-like cell formation. This increase was significantly inhibited in the presence of quercetin, myricetin, kaempferol, isorhamnetin, or curcumin in the range of 10⁻⁸–10⁻⁶ M. Such an effect was not seen in the case of hesperidin or astaxanthin. In addition, culture with PTH (10⁻⁷ M) caused a significant decrease in diaphyseal calcium content. This decrease was completely prevented in the presence of quercetin, myricetin, kaempferal, or isorhamnetin of 10⁻⁶ M. This study demonstrates that various flavonoids have a potent inhibitory effect on osteoclastogenesis and bone resorption rather than bone formation in vitro. Among various flavonoids, quercetin had a stimulatory effect on bone formation and an inhibitory effect on bone resorption in vitro.     

Comparison of chemical composition of Hypericum perforatum and H. maculatum in Estonia

Of numerous species belonging to the medicinally important genus Hypericum, only H. perforatum L. and H. maculatum Crantz grow widely in Estonia. A comparative biochemical systematics study of hypericins, hyperforins and other phenolics within Hypericum spp. growing in Estonia was performed. For comprehensive metabolomic investigation, 42 samples of H. perforatum and 16 samples of aerial parts of H. maculatum were collected in two consecutive years from various locations; methanolic extracts were prepared from airdried leaves and flowers. The concentrations of a quinic acid derivative, caffeic acid glucoside, vanillic acid glucoside, neochlorogenic acid, chlorogenic acid, catechin, epicatechin, myricetin glucoside, hyperoside, isoquercitrin, rutin, quercetin pentoside, quercitrin, kaempferol glucoside, kaempferol rutinoside, quercetin, hyperforin, adhyperforin, protopseudohypericin, pseudohypericin, and hypericin were determined by LC-DAD-MS/MS. All the aforementioned compounds were detected in both species, although some at very different levels – H. maculatum contained rutin and hyperforins only in trace amounts and overall tended to contain more phenolic compounds. The level of total hypericins was the same for both species. These results constitute a further contribution to the systematic knowledge about the Hypericum spp. Results of principal component analysis (PCA) demonstrated distinct between-years and between-species diversity in the chemical composition of the plants studied. Between-years diversity in Hypericum spp. has not been addressed before.