The action of dietary phytochemicals quercetin, catechin, resveratrol and naringenin on estrogen-mediated gene expression

Hepatic expression of apolipoprotein (apo) II is in part modulated by estrogen-mediated stabilization of its mRNA. This stabilization is due to the estrogen-regulated mRNA stabilizing factor (E-RmRNASF) expressed in the liver in response to estrogen (Ratnasabapathy, 1995, Cell. Mol. Biol. Res, 41: 583-594). E-RmRNASF protects the RNA from targeted endonucleolytic degradation. The hepatic expression of E-RmRNASF is modulated by certain estrogenic and antiestrogenic nonsteroidal environmental xenobiotics (Ratnasabapathy et al. 1997, Biochem. Pharmacol., 53: 1425-1434). To determine whether dietary phytochemicals purported to prevent hormone-dependent breast and prostate cancers, and atherosclerosis, acted via the estrogen-cell-signaling pathway, roosters were administered increasing doses up to 1 mmole/kg of resveratrol, quercetin, catechin or naringenin parenterally and tested for hepatic expression of E-RmRNASF. Besides estrogen, the expression of E-RmRNASF in the liver was stimulated by resveratrol and catechin, indicating these agents to be estrogenic. A lack of E-RmRNASF expression was seen with the roosters treated with the vehicle, naringenin or quercetin. To determine whether the agents exerted partial agonistic or antagonistic effects, roosters were administered combinations of estrogen and increasing doses of the above phytochemicals. Resveratrol showed agonistic activity at all concentrations (10-1000 micromol/kg) tested. Catechin showed partial agonistic activity, while quercetin and naringenin appeared to be antagonistic.     

Mechanisms of antioxidant activities of quercetin and catechin

Abstract

The seleno-organic compound ebselen mimics the glutathione-dependent, hydroperoxide reducing activity of glutathione peroxidase. The activity of glutathione peroxidase determines the rate of hydroperoxide-induced Ca²⁺ release from mitochondria. Ebselen stimulates Ca²⁺ release from mitochondria, accelerates mitochondrial respiration and uncoupling, and induces mitochondrial swelling, indicating a deterioration of mitochondrial function. These manifestations are abolished by cyclo-sporine A, a potent inhibitor of the mitochondrial permeability transition. However, when ebselen-induced Ca²⁺ cycling is prevented with ruthenium red, an inhibitor of the Ca²⁺ uniporter, or by chelation of extramitochondrial Ca²⁺ by EGTA, no detectable elevation of swelling or uncoupling is observed. The release of Ca²⁺ from mitochondria is delayed in the absence of rotenone, i.e. when pyridine nucleotides are maintained in the reduced state due to succinate-driven reversed electron flow. We suggest that ebselen induces Ca²⁺ release from intact mitochondria via an NAD⁺ hydrolysis-dependent mechanism.     

A theoretical study of the different radical-scavenging activities of catechin, quercetin, and a rationally designed planar catechin

To improve the radical-scavenging activity of catechin, a planar catechin analogue was designed and synthesized by Fukuhara [J. Am. Chem. Soc. 124 (2002) 5952]. Although the planar catechin is less active than quercetin, it is much more active than catechin in its ability to scavenge galvinoxyl radical, suggesting that the rational design was successful. However, an interesting question remains: what is the basis for the enhanced radical-scavenging activity of the planar catechin? By DFT calculations, we determined that the galvinoxyl radical is scavenged through an electron-transfer mechanism rather than a hydrogen-atom-transfer mechanism. Moreover, the antioxidant anion, derived from proton dissociation, plays a key role in the radical-scavenging process. Hence, the different radical-scavenging activities of the three antioxidants may result from the different ionization potentials of their anions.     

Comparison of the bioavailability of quercetin and catechin in rats

Quercetin and catechin are present in noticeable amounts in human diet and these polyphenolic compounds are supposed to exert beneficial effects on human health. However, their metabolic fates in the organism have never been compared. In the present study, rats were fed a 0.25% quercetin or a 0.25% catechin diet. Quercetin and catechin metabolites were analyzed in plasma and liver samples by high-performance liquid chromatography coupled to an ultraviolet or a multielectrode coulometric detection. All plasma metabolites were present as conjugated forms, but catechin metabolites were mainly constituted by glucuronidated derivatives, whereas quercetin metabolites were sulfo- and glucurono-sulfo conjugates. Quercetin was more intensively methylated than catechin in plasma. The plasma quercetin metabolites are well maintained during the postabsorptive period (approximately 50 microM), whereas the concentration of catechin metabolites dropped dramatically between 12- and 24-h after an experimental meal (from 38.0 to 4.5 microM). In the liver, the concentrations of quercetin and catechin derivatives were lower than in plasma, and no accumulation was observed when the rats were adapted for 14 d to the supplemented diets. The hepatic metabolites were intensively methylated (90-95%), but in contrast to plasma, some free aglycones could be detected. Thus, it clearly appears that studies dealing with the biological impact of these polyphenols should take into account the feature of their bioavailability, particularly the fact that their circulating metabolites are conjugated derivatives.     

Comparative reactivity of myeloperoxidase-derived oxidants with mammalian cells

Myeloperoxidase is an important heme enzyme released by activated leukocytes that catalyzes the reaction of hydrogen peroxide with halide and pseudo-halide ions to form various hypohalous acids. Hypohalous acids are chemical oxidants that have potent antibacterial, antiviral, and antifungal properties and, as such, play key roles in the human immune system. However, increasing evidence supports an alternative role for myeloperoxidase-derived oxidants in the development of disease. Excessive production of hypohalous acids, particularly during chronic inflammation, leads to the initiation and accumulation of cellular damage that has been implicated in many human pathologies including atherosclerosis, neurodegenerative disease, lung disease, arthritis, inflammatory cancers, and kidney disease. This has sparked a significant interest in developing a greater understanding of the mechanisms involved in myeloperoxidase-derived oxidant-induced mammalian cell damage. This article reviews recent developments in our understanding of the cellular reactivity of hypochlorous acid, hypobromous acid, and hypothiocyanous acid, the major oxidants produced by myeloperoxidase under physiological conditions.     

Combined resveratrol, quercetin, and catechin treatment reduces breast tumor growth in a nude mouse model

Grape polyphenols can act as antioxidants, antiangiogenics, and selective estrogen receptor (ER) modifiers and are therefore especially relevant for gynecological cancers such as breast cancer. The major polyphenols of red wine (resveratrol, quercetin, and catechin) have been individually shown to have anticancer properties. However, their combinatorial effect on metastatic breast cancers has not been investigated in vivo. We tested the effect of low dietary concentrations of resveratrol, quercetin, and catechin on breast cancer progression in vitro by analyzing cell proliferation and cell cycle progression. The effects of these compounds on fluorescently tagged breast tumor growth in nude mice were assessed using in situ fluorescence image analysis. Individual polyphenols at 0.5 microM neither decreased breast cancer cell proliferation nor affected cell cycle progression in vitro. However, a combination of resveratrol, quercetin, and catechin at 0.5, 5, or 20 microM each significantly reduced cell proliferation and blocked cell cycle progression in vitro. Furthermore, using in situ image analysis, we determined that combined dietary polyphenols at 0.5, 5, or 25 mg/kg reduced primary tumor growth of breast cancer xenografts in a nude mouse model. Peak inhibition was observed at 5 mg/kg. These results indicate that grape polyphenols may inhibit breast cancer progression.     

Peroxidative metabolism of apigenin and naringenin versus luteolin and quercetin: glutathione oxidation and conjugation

GSH was readily depleted by a flavonoid, H(2)O(2), and peroxidase mixture but the products formed were dependent on the redox potential of the flavonoid. Catalytic amounts of apigenin and naringenin but not kaempferol (flavonoids that contain a phenol B ring) when oxidized by H(2)O(2) and peroxidase co-oxidized GSH to GSSG via a thiyl radical which could be trapped by 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) to form a DMPO-glutathionyl radical adduct detected by ESR spectroscopy. On the other hand, quercetin and luteolin (flavonoids that contain a catechol B ring) or kaempferol depleted GSH stoichiometrically without forming a thiyl radical or GSSG. Quercetin, luteolin, and kaempferol formed mono-GSH and bis-GSH conjugates, whereas apigenin and naringenin did not form GSH conjugates. MS/MS electrospray spectroscopy showed that mono-GSH conjugates for quercetin and luteolin had peaks at m/z 608 [M + H](+) and m/z 592 [M + H](+) in the positive-ion mode, respectively. (1)H NMR spectroscopy showed that the GSH was bound to the quercetin A ring. Spectral studies indicated that at a physiological pH the luteolin-SG conjugate was formed from a product with a UV maximum absorbance at 260 nm that was reducible by potassium borohydride. The quercetin-SG conjugate or kaempferol-SG conjugate on the other hand was formed from a product with a UV maximum absorbance at 335 nm that was not reducible by potassium borohydride. These results suggest that GSH was oxidized by apigenin/naringenin phenoxyl radicals, whereas GSH conjugate formation involved the o-quinone metabolite of luteolin or the quinoid (quinone methide) product of quercetin/kaempferol.     

Co-administration of quercetin and catechin in rats alters their absorption but not their metabolism

Quercetin and catechin are among the major flavonoids in plant foods and their intake has been associated to a risk reduction in several degenerative diseases. The aim of the present study was to bring data on the bioavailability of quercetin and catechin when administered simultaneously. The study was performed on rats adapted to diets containing (i) 0.25% quercetin, or (ii) 0.25% catechin, or (iii) 0.25% quercetin+0.25% catechin. Quercetin, catechin and their metabolites were determined in plasma, urine and liver by HPLC with UV or coulometric detection. When quercetin and catechin were fed in association, their respective plasma concentration significantly decreased (-35% and -28% respectively), whereas the urinary and hepatic concentrations were only affected for quercetin (-36%). These data may be explained by a competitive interaction between quercetin and catechin at the digestive level, leading to a reduction of the intestinal absorption of quercetin and a possible delaying of catechin absorption over time. The simultaneous administration of quercetin and catechin had no effect on the formation of their glucurono and sulfo conjugates, indicating the absence of competition between quercetin and catechin for the corresponding conjugative enzymes.     

Neuroprotective properties of the natural phenolic antioxidants curcumin and naringenin but not quercetin and fisetin in a 6-OHDA model of Parkinson's disease

Although the cause of dopaminergic cell death in Parkinson's disease (PD) remains unknown, oxidative stress has been strongly implicated. Because of their ability to combat oxidative stress, diet derived phenolic compounds continue to be considered as potential agents for long-term use in PD. This study was aimed at investigating whether the natural phenolic compounds curcumin, naringenin, quercetin, fisetin can be neuroprotective in the 6-OHDA model of PD. Unilateral infusion of 6-OHDA into the medial forebrain bundle produced a significant loss of tyrosine hydroxylase (TH)-positive cells in the substantia nigra (SN) as well as a decreased of dopamine (DA) content in the striata in the vehicle-treated animals. Rats pretreated with curcumin or naringenin showed a clear protection of the number of TH-positive cells in the SN and DA levels in the striata. However, neither pretreatment with quercetin nor fisetin had any effects on TH-positive cells or DA levels. The ability of curcumin and naringenin to exhibit neuroprotection in the 6-OHDA model of PD may be related to their antioxidant capabilities and their capability to penetrate into the brain.     

Ultrasensitive assay for three polyphenols (catechin,quercetin and resveratrol) and their conjugates in biological fluids utilizing gas chromatography with mass selective detection

The concentrations of three polyphenols ((+)-catechin, quercetin and trans-resveratrol) in blood serum, plasma and urine, as well as whole blood, have been measured after their oral and intragastric administration, respectively, to humans and rats. The method developed for this purpose utilized ethyl acetate extraction of 100 μl samples and their derivatization with bis(trimethylsilyl)trifluoroacetamide (BSTFA) followed by gas-chromatographic analysis on a DB-5 column followed by mass selective detection employing two target ions and one qualifier ion for each compound. Total run time was 17 min with excellent resolution and linearity. The limits of detection (LOD) and quantitation (LOQ) were an order of magnitude less than for any previously published method, being 0.01 μg/l and 0.1 μg/l, respectively, for all compounds. Recovery at 1 μg/l and 10 μg/l was >80% in all instances but one, and was >90% in 50%. Imprecision was acceptable at 0.25 and 1.0 μg/l, concentrations below the LOQ of previous methods. Aglycones released from conjugates after hydrolysis were easily measurable. Optimal conditions for hydrolysis were established. After oral administration of the three polyphenols to humans, their conjugates vastly exceeded the concentrations of the aglycones in both plasma and urine. Concentrations peaked within 0.5–1.0 h in plasma and within 8 h in urine. During the first 24 h, 5.1% of the (+)-catechin and 24.6% of the trans-resveratrol given were recovered in the urine (free plus conjugated). This method can be proposed as the method of choice to assay these polyphenols and their conjugates in biological fluids.     

On the ability of four flavonoids, baicilein, luteolin, naringenin, and quercetin, to suppress the fenton reaction of the iron-ATP complex

Four flavonoids, baicilein, luteolin, naringenin, and quercetin were investigated for their ability to suppress the Fenton reaction characteristic of the iron-ATP complex. Absorption spectroscopy indicates that under the conditions of 18.75% aqueous methanol, 0.0625 mM HEPES pH 7.4 buffer and 1.5:1 quercetin/iron-ATP ratio a mix ligand complex formed. All four flavonoids were found to interfere with the voltammetric catalytic wave associated with the iron-ATP complex in the presence of H₂O₂. Quercetin and luteolin were able to completely suppress the catalytic wave of the iron-ATP/H₂O₂ system when a minimum ratio of 1.5:1 of the flavonoid to iron-ATP was reached. At this ratio, the ability of the studied series of flavonoids to suppress the Fenton reaction characteristic of iron-ATP follows as quercetin luteolin > naringenin baicilein. Both quercetin and luteolin contain catechol on the B ring, which may enhance the iron chelation of these species over baicilein and naringenin. The common structural feature of all of these flavonoids is the 4-keto, 5-hydroxy region, which may also contribute to the chelation of iron.     

Formation of novel bioactive metabolites from the reactions of pro-inflammatory oxidants with polyphenolics

Dietary polyphenolics such as those in soy or red wine can have beneficial effects on the development of chronic human diseases. The mechanisms of action of isoflavones have been diverse and include their roles as weak estrogens, inhibitors of tyrosine kinase-dependent signal transduction processes and as antioxidants. Recent insights into the oxidative stress model of atherosclerosis suggest an interesting synthesis of these concepts. Sites of inflammation are associated with the formation of complex mixtures of reactive oxygen, nitrogen and halogenating species capable of modifying both endogenous biomolecules and polyphenolics. Of particular significance are the halogenation reactions mediated by myeloperoxidase that can modify key amino acids such as arginine and polyphenolics such as genistein. Hypochlorite, the reaction product of myeloperoxidase can halogenate polyphenolics to form stable derivatives with modified biological activity. Thus the in situ metabolism at sites of inflammation is unique and generates novel pharmacophores with potentially distinct modes of action from the parent compounds.     

Comparative QSPR studies with molecular connectivity,molecular negentropy and TAU indices

Molecular thermochemical properties (heats of formation and atomization) of diverse functional acyclic compounds have been correlated with TAU indices and the relations have been compared to those involving molecular negentropy and first order valence molecular connectivity indices to unravel the diagnostic feature of the TAU scheme and to explore the relative suitability of the scheme in describing physicochemical parameters. For both the properties it was found that TAU relations could satisfactorily explain the variances of the thermochemical parameters and the relations were comparable to those involving molecular negentropy and molecular connectivity. Moreover, specific contributions of functionality, branchedness, shape and size factors to the thermochemical properties could be found from the relations involving TAU parameters. Figure Different topochemical (TAU) indices     

Interactions studies of CYP2D6 with quercetin and hyperoside by spectral analysis and molecular dynamics simulations

Some ingredients from herbal medicine can significantly affect the activity of CYP2D6, thus leading to serious interactions between herbs and drugs. Quercetin and hyperoside are active ingredients widely found in vegetables, fruits, and herbal medicines. Quercetin and hyperoside have many biological activities. In this work, the characteristic bindings of CYP2D6 with quercetin/hyperoside are revealed by multi-spectroscopy analysis, molecular docking, and molecular dynamics simulations. The fluorescence of CYP2D6 is statically quenched by quercetin and hyperoside. The binding constant (K_a) values of CYP2D6–quercetin/hyperoside range from 10⁴ L mol⁻¹, which indicates that these two flavonoids bind moderately to CYP2D6. Meanwhile, quercetin has a stronger quenching ability to CYP2D6 than that of hyperoside. The secondary structure of CYP2D6 is obviously changed by binding with quercetin/hyperoside. The docking results reveal that the quercetin/hyperoside enters the active site of CYP2D6 near heme and binds to CYP2D6 by hydrogen bonds and van der Waals forces. The molecular dynamics simulation results indicate that the binding of quercetin/hyperoside can stabilize the two complexes, enhance the flexibility of CYP2D6 backbone atoms, and make a more unfolded and looser structure of CYP2D6.     

Comparative molecular field analysis (CoMFA) for sulfoxidation reactions in Mortierella isabellina ATCC 42613 and Helminthosporium sp. NRRL 4671

Previous models for mechanisms of enzymatic sulfoxidation have been somewhat limited by a lack of knowledge of the essential features of substrate-enzyme versus product-enzyme relationships. Computerized methods for modeling ligand-protein (substrate-enzyme) interactions can overcome some of these limitations. Specifically, CoMFA (comparative molecular field analysis) provided a useful general approach in which to evaluate substrate-enzyme and product-enzyme relationships. The present investigation examined the relationship between substrate and product structure in predicting enantioselective sulfoxidation reactions using CoMFA for two species of microorganisms that have been used as models for mammalian metabolism, Mortierella isabellina and Helminthosporium sp. The overall enantioselectivity observed was based on the composite stereoselectivity of sulfoxide formation, sulfone formation (from the sulfoxide), and sulfoxide reduction back to the achiral substrate (sulfide).     

Revealing chemical reactions of coal pyrolysis with GPU-enabled ReaxFF molecular dynamics and cheminformatics analysis

The complex chemistry of coal pyrolysis is difficult to be captured by experimental techniques or simulated with the quantum mechanics computational methods. The emerging of both the large-scale coal models and the promising capability of reactive molecular dynamics (ReaxFF MD) motivated us to develop a new methodology by combining graphics processing unit (GPU)-enabled high performance computing with cheminformatics analysis in order to explore the coal pyrolysis mechanisms using ReaxFF MD. The methodology is rooted in two new software tools, GMD-Reax, the first GPU-enabled ReaxFF MD codes that make it practical to simulate large-scale models (～10,000 atoms) on desktop workstations, and visualisation and analysis of reactive molecular dynamics (VARMD), the first software dedicated to analysis of detailed chemical reactions from the trajectories of ReaxFF MD simulation. With this methodology, reasonable product profiles and gas generation sequences of pyrolysis for bituminous coal models ranging from ～1000 to ～10,000 atoms (including the system with 28,351 atoms, one of the largest systems used in ReaxFF MD) have been obtained. The complex and detailed chemical reactions directly revealed by VARMD can provide further information on radical behaviours and their connection with pyrolysates. The methodology presented here offers a new and promising approach to systematically understand the complex chemical reactions in thermolysis of very complicated molecular systems.     

Effect of heated naringenin on immunomodulatory properties and cellular antioxidant activity

Naringenin is one of the most popular flavonoids derived from citrus. It has been reported to be an effective anti-inflammatory compound. Citrus fruit may be used raw, cooked, stewed, or boiled. The present study was conducted to investigate the effect of thermal processes on naringenin in its immunomodulatory and cellular antioxidant activities. The effects of flavonoids on B and T cell proliferation were assessed on splenocytes stimulated or not with mitogens. However, their effects on cytotoxic T lymphocyte (CTL) and natural killer (NK) activities were assessed in splenocytes co-incubated with target cells. The amount of nitric oxide production and the lysosomal enzyme activity were evaluated in vitro on mouse peritoneal macrophages. Cellular antioxidant activity in splenocytes and macrophages was determined by measuring the fluorescence of the dichlorofluorescin (DCF). Our findings revealed that naringenin induces B cell proliferation and enhances NK activity. The highest concentration of native naringenin exhibits a significant proliferation of T cells, induces CTL activity, and inhibits cellular oxidation in macrophages. Conversely, it was observed that when heat-processed, naringenin improves the cellular antioxidant activity in splenocytes, increases the cytotoxic activity of NK cells, and suppresses the cytotoxicity of T cells. However, heat treatment maintains the anti-inflammatory potency of naringenin.     

Comparative molecular field analysis and comparative molecular similarity indices analysis of human thymidine kinase 1 substrates

Thymidine kinase 1 (TK1) is a key target for antiviral and anticancer chemotherapy. Three-dimensional quantitative structure-activity relationship (3D-QSAR) using comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) techniques was applied to analyze the phosphorylation capacity of a series of 31 TK1 substrates. The optimal predictive CoMFA model with 26 molecules provided the following values: cross-validated r(2) (q(2))=0.651, non-cross-validated r(2)=0.980, standard error of estimate (s)=0.207, F=129.3. For the optimal CoMSIA model the following values were found: q(2)=0.619, r(2)=0.994, s=0.104, F=372.2. The CoMSIA model includes steric, electrostatic, and hydrogen bond donor fields. The predictive capacity of both models was successfully validated by calculating known phosphorylation rates of five TK1 substrates that were not included in the training set. Contour maps obtained from CoMFA and CoMSIA models correlated with the experimentally developed SAR.     

Synthesis,biological evaluation and SAR analysis of O-alkylated analogs of quercetin for anticancer

O-Alkylated quercetin analogs were synthesized and their anticancer activities were assessed by a high-throughout screening (HTS) method. The structure–activity relationships (SAR) showed that introduction of long alkyl chain such as propyl group at the C-3 OH position or short alkyl chain such as ethyl group at the C-4′ OH position were very important for keeping inhibitory activities against the 16 cancer cell lines. Furthermore, when the two n-butyl groups were introduced into the C-3, C-7 or C-4′, C-7 positions, the anticancer activity was enhanced.     

Studies of inclusion complexes of natural and modified cyclodextrin with (+)catechin by NMR and molecular modeling

The aim of this paper is to describe the inclusion properties and the factors affecting the complexation selectivity and stabilization of catechin (CA) into beta-cyclodextrin (beta-CD) and two of its derivatives, namely Heptakis 2,6-di-O-methyl-beta-cyclodextrin (DM-beta-CD) and 2 hydroxypropyl-beta-cyclodextrin (HP-beta-CD). Analysis of the proton shift change using the continuous variation method confirm the formation of a 1:1 stoichiometric complex for catechin and the different CDs in aqueous medium. The formations constant obtained by diffusion-ordered spectroscopy (DOSY) techniques indicated the following trend upon complex formation: beta-CD>HP-beta-CD>DM-beta-CD. The detailed spatial configuration is proposed based on 2D NMR methods. These results are further interpreted using molecular modeling studies. The latter results are in good agreement with the experimental data. The models confirm that when CA-beta-CD is formed, the catechol moiety in the complex is oriented toward the primary rim; however when CD is derivatized to HP-beta-CD and DM-beta-CD this ring is oriented toward the secondary rim.