Molecular actions of flavonoids conjugates

Many papers have suggested the health-beneficial activity of natural dietary polyphenols to prevent chronic diseases and aging processes in humans. It is generally recognized that polyphenols are absorbed from the intestines and metabolized into the phase-II conjugates, i.e., the glucuronides and sulfates. For example, a major dietary flavonoid, quercetin, abundant in onion and buckwheat, is metabolized after oral intake into its conjugates, such as quercetin-3-O-glucuronide and quercetin-3’-O-sulfate, whereas no aglycone was found in the human plasma. Therefore, to understand the mechanisms of the biological activity of quercetin in vivo, we should focus on the molecular actions of these conjugates. In the last decade, we have demonstrated the unique actions of quercetin-3-O-glucuronide at sites of inflammation, including specific accumulation in macrophages and the following deconjugation into active aglycone, catalyzed by the macrophage-derived β-glucuronidase. This review summarizes recent findings regarding the anti-inflammatory mechanisms of quercetin conjugates in macrophages and propose a possible strategy for the effective utilization of natural polyphenols in our daily diet for prevention of age-related chronic diseases

ATP/P2X7R signaling as a target of natural polyphenols

Innate immune cells, such as macrophages, respond to pathogen-associated molecular patterns, such as a lipopolysaccharide (LPS), to secrete various inflammatory mediators. Recent studies have suggested that damage-associated molecular patterns (DAMPs), released extracellularly from damaged or immune cells, also play a role in the activation of inflammatory responses. In this study, to prevent excess inflammation, we focused on DAMPs-mediated signaling that promotes LPS-stimulated inflammatory responses, especially adenosine 5’-triphosphate (ATP)-triggered signaling through the ionotropic purinergic receptor 7 (P2X7R), as a potential new anti-inflammatory target of natural polyphenols. We focused on the phenomenon that ATP accelerates the production of inflammatory mediators, such as nitric oxide, in LPS-stimulated J774.1 mouse macrophages. Using an siRNA-mediated knockdown and specific antagonist, it was found that the ATP-induced enhanced inflammatory responses were mediated through P2X7R. We then screened 42 polyphenols for inhibiting the ATP/P2X7R-induced calcium influx, and found that several polyphenols exhibited significant inhibitory effects. Especially, a flavonoid baicalein significantly inhibited ATP-induced inflammation, including interleukin-1β secretion, through inhibition of the ATP/P2X7R signaling. These findings suggest that ATP/P2X7R signaling plays an important role in excess inflammatory responses and could be a potential anti-inflammatory target of natural polyphenolic compounds

DHA Hydroperoxides as a Potential Inducer of Neuronal Cell Death: a Mitochondrial Dysfunction-Mediated Pathway

During the lipid peroxidation reaction, lipid hydroperoxides are formed as primary products. Several lines of evidence suggest that lipid hydroperoxides can trigger cell death in many cell types, including neurons. In a screening of lipid hydroperoxides which can induce toxicity in neuronal cells, we found docosahexaenoic acid hydroperoxides (DHA-OOH) induced much severe levels of reactive oxygen species generation and cell death in human neuroblastoma SH-SY5Y cells compared to the hydroperoxides of linoleic acid and arachidonic acid. Therefore, we focused on DHA-OOH, and demonstrated that DHA-OOH apparently induced an apoptosis in the neuronal cells through several apoptotic hallmarks including nuclei condensation, DNA fragmentation, poly (ADP-ribose) polymerase cleavage and increased activity of caspase-3. We also found the signaling changes in mitochondria-mediated apoptosis, such as cytochrome c release and increased expression of Bcl-2, as well as a dose-dependent attenuation of mitochondrial membrane potential in the DHA-OOH treated cells. These data indicated DHA hydroperoxide as a potential inducer of apoptosis in human neuroblastoma SH-SY5Y cells, which may be mediated by mitochondria dysfunction pathway

Approach to novel functional foods for stress control : 5. Antioxidant activity profiles of antidepressant herbs and their active components

Oxidative stress is frequently mentioned in relation to the neurodegenerative diseases. This study examined the effect of three herb extracts, Hypericum perforatum, Ginkgo biloba L. and Apocynum venetum L., and their components on lipid hydroperoxide-induced oxidative stress in PC-12cells. Among them, the extract of Apocynum venetum and its components showed the remarkable inhibitory effect, indicating that this herb extract serves as a protective agent against lipid peroxidation-related oxidative stress in CNS. Oxidative stress may be associated with the progress of depression, as this extract has been proposed to be an effective antidepresant herb

Sesamin catechol glucuronides exert anti-inflammatory effects by suppressing IFN-β and iNOS expression through the deconjugation in macrophage-like J774.1 cells

Sesamin, a representative sesame lignan, has health-promoting activities. Sesamin is converted into catechol derivatives and further into their glucuronides or sulfates in vivo, whereas the biological activities of sesamin metabolites remain unclear. We examined the inhibitory effects of sesamin metabolites on the lipopolysaccharide (LPS)-induced NO production in mouse macrophage-like J774.1 cells and found that a mono-catechol derivative SC1, (7α,7'α,8α,8'α)-3,4-dihydroxy-3',4'-methylenedioxy-7,9':7',9-diepoxylignane, has a much higher activity than sesamin and other metabolites. The inhibitory effects of SC1 glucuronides were time-dependently enhanced, associated with the intracellular accumulation of SC1 and the methylated form. SC1 glucuronides and SC1 attenuated the expression of inducible NO synthase (iNOS) and upstream interferon-β (IFN-β) in the LPS-stimulated macrophages. The inhibitory effects of SC1 glucuronides against NO production were canceled by the β-glucuronidase inhibitor and enhanced by the catechol- O-methyltransferase inhibitor. Our results suggest that SC1 glucuronides exert the anti-inflammatory effects by inhibiting the IFN-β/iNOS signaling through macrophage-mediated deconjugation

UVA ユウドウ ヒフ ヒカリロウカ ニオケル シシツ カサンカブツ ノ ヤクワリ ト コウサンカ ブッシツ セッシュ ノ エイキョウ

Role of ultraviolet A（UVA）on oxidative damage has attracted much attention in relation to skin photoaging process. In this study, we investigated effect of cholesterol hydroperoxides（Chol- OOHs）, stable products of reactive oxygen species（ROS）-induced lipid peroxidation, on matrix metalloproteinase（MMP）activation responsible for wrinkle formation in hairless mouse skin and then estimated the inhibition of UVA-induced MMP activation by dietary β-carotene. Hairless mice were exposed to UVA irradiation for 8 weeks. Chol-OOHs content in the skin was found to increase significantly by the exposure of UVA. In addition, the activity of MMP-9 and its protein expression were elevated with wrinkle formation. This activation was also induced by intracutaneous injection of Chol-OOHs. Interestingly, dietary β-carotene（500 mg/kg diet） and α-tocopherol （100 mg/kg diet）suppressed the accumulation of Chol-OOH as well as MMP activation. These results suggest that Chol-OOHs formed by the exposure of UVA to skin contribute to the activation of MMPs resulting in skin photoaging. Dietary antioxidants may prevent skin photoaging through, at least partly, the suppression of MMP activation due to UVA-induced lipid peroxidation

Metal-catalyzed formation of 4-oxo-2-alkenals

Lipid peroxidation products react with cellular molecules, such as DNA bases, to form covalent adducts, which are associated with aging and disease processes. Since lipid peroxidation is a complex process and occurs in multiple stages, there might be yet unknown reaction pathways. Here, we analyzed comprehensively 2’-deoxyguanosine (dG) adducts with oxidized arachidonic acid using liquid chromatography-tandem mass spectrometry and found the formation of 7-(2-oxo-hexyl)-etheno-dG as one of the major unidentified adducts. The formation of this adduct was reproduced in the reaction of dG with 2-octenal and predominantly with 4-oxo-2-octenal (OOE). We also found that other 2-alkenals (with five or more carbons) generate corresponding 4-oxo-2-alkenal-type adducts. Importantly, it was found that transition metals enhanced the oxidation of C4-position of 2-octenal, leading to the formation of OOE-dG adduct. These findings demonstrated a new pathway for the formation of 4-oxo-2-alkenals during lipid peroxidation and might provide a mechanism for metal-catalyzed genotoxicity

Approach to novel functional foods for stress control : 4. Regulation of serotonin transporter by food factors

Serotonin transporters (SERTs) are pre-synaptic proteins specialized for the clearance of serotonin following vesicular release at central nervous system (CNS) and enteric nervous system synapses. SERTs are high affinity targets in vivo for antidepressants such as serotonin selective reuptake inhibitors (SSRIs). These include ‘medical’ psychopharmacological agents such as analgesics and antihistamines, a plant extract called St John’s Wort (Hypericum) . Osteoclasts are the primary cells responsible for bone resorption. They arise by the differentiation of osteoclast precursors of the monocyte/macrophage lineage. The expression of SERTs was increased in RANKL-induced osteoclast-like cells. Using RANKL stimulation of RAW264.7 cells as a model system for osteoclast differentiation, we studied the direct effects of food factor on serotonin uptake. The SSRIs (fluoxetine and fluvoxamine) inhibited markedly (～95%) in serotonin transport in differentiated osteoclast cells. The major components of St. John’s Wort, hyperforin and hypericine were significantly decreased in serotonin transport activity. Thus, a new in vitro model using RANKL-induced osteoclast-like cells may be useful to analyze the regulation of SERT by food factors and SSRIs

Effects of forced swimming stress on rat brain function

Chronic stress has been reported to be an essential factor for depression. In this study, the effect of forced swimming stress on neurotransmitters and cellular signaling pathway contributing to brain functions was investigated using the forced swimming test (FST) in order to understanding of mechanisms to regulate stress signals in brain. Antidepressant drug, imipramine, significantly reduced the immobility time of male rats in the FST by 85% at a dose of 15mg/kg for 2 weeks. This result indicated that the swimming stress caused a depressed state in the rats without administration of imipramine. Swimming stress significantly lowered the serotonergic ratio and also markedly enhanced the phosphorylation of ERK1/2 in the hypothalamus region compared to the rats without FST. These phenomena maybe included in key mechanisms of the development of depression

Antioxidant capacity of albumin-bound quercetin metabolites after onion consumption in humans

Quercetin is a major dietary flavonoid found in onions and other vegetables. It is known that dietary quercetin is metabolized in the intestinal mucosa and the liver and is present as its glucuronide/sulfate conjugates with or without methylation. Although quercetin is known to possess strong antioxidant activity, there are only limited reports on the antioxidant activity of its metabolites. In this study, the antioxidant capacity of quercetin metabolites under physiological conditions was investigated. After consumption of cooked onion, more than 80% of quercetin metabolites were localized in the human plasma fraction containing concentrated serum albumin. Other lipoprotein fractions contained only small amounts of quercetin metabolites. Addition of quercetin 3-O-β-glucuronide to the lipoprotein-eliminated plasma fraction generated antioxidant activity against LDL oxidation in a dose-dependent manner. However, onion consumption failed to enhance the antioxidant activity of the lipoprotein-eliminated plasma fraction against LDL oxidation, probably because the amount of quercetin metabolites bound to albumin was less than the effective level in an ex vivo study. The physiological role of plasma albumin in retaining quercetin metabolites needs to be further clarified