8-Prenylnaringenin tissue distribution and pharmacokinetics in mice and its binding to human serum albumin and cellular uptake in human embryonic kidney cells.

8-Prenylnaringenin (8-PN), a hop flavonoid, is a promising food substance with health benefits. Compared with nonprenylated naringenin, 8-PN exhibits stronger estrogenic activity and prevents muscle atrophy. Moreover, 8-PN prevents hot flushes and bone loss. Considering that prenylation reportedly improves the bioavailability of flavonoids, we compared the parameters related to the bioavailability [pharmacokinetics and tissue distribution in C57/BL6 mice, binding affinity to human serum albumin (HSA), and cellular uptake in HEK293 cells] of 8-PN and its mother (non-prenylated) compound naringenin. C57/BL6 mice were fed an 8-PN or naringenin mixed diet for 22 days. The amount of 8-PN (nmol/g tissue) in the kidneys (16.8 ± 9.20), liver (14.8 ± 2.58), muscles (3.33 ± 0.60), lungs (2.07 ± 0.68), pancreas (1.80 ± 0.38), heart (1.71 ± 0.27), spleen (1.36 ± 0.29), and brain (0.31 ± 0.09) was higher than that of naringenin. A pharmacokinetic study in mice demonstrated that the of 8-PN (50 mg/kg body weight) was lower than that of naringenin; however, the plasma concentration of 8-PN 8 h after ingestion was higher than that of naringenin. The binding affinity of 8-PN to HSA and cellular uptake in HEK293 cells were higher than those of naringenin. 8-PN bioavailability features assessed in mouse or human model experiments were obviously different from those of naringenin.


Suppressive effects of quercetin on hydrogen peroxide-induced caveolin-1 phosphorylation in endothelial cells

Caveolin-1 is a major protein of the caveolae structure in vascular endothelial cell membrane. Phosphorylation of caveolin-1 is one of the initial events leading to exacerbation of vascular permeability caused by oxidative stress. Although quercetin is known to be an anti-atherosclerosis factor that acts as a dietary antioxidant, little is known about its role in the regulation of caveolin-1 phosphorylation. In this study, we investigated the inhibitory effect of quercetin on hydrogen peroxide-induced caveolin-1 phosphorylation in human umbilical vein endothelial cells. Quercetin inhibited caveolin-1 phosphorylation in cells pretreated with quercetin for 24 h and then exposed to hydrogen peroxide. However, quercetin 3--β-glucuronide, a conjugated metabolite of quercetin, did not exert this inhibitory effect. Exposure to hydrogen peroxide increased vascular permeability and reduced mRNA expression of the intercellular adhesion protein, vascular endothelial cadherin (VE-cadherin). By contrast, pretreatment with quercetin suppressed the increase in vascular permeability and decreased VE-cadherin expression. These results indicate that deconjugated quercetin can play a role in the prevention of altered vascular permeability under oxidative stress by suppressing caveolin-1 phosphorylation. Thus, dietary quercetin may be beneficial for the maintenance of endothelial cell function.

Journal of Clinical Biochemistry and Nutrition


Chocolate as a food matrix reduces the bioavailability of galloylated catechins from green tea in healthy women.

In this study, we evaluated the food matrix effects of chocolate on the absorption of green tea catechins (GTCs), (-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECg), and (-)-epigallocatechin gallate (EGCg), in five healthy 22-year-old women. In the single-intake experiment, the plasma concentrations of ECg (P < 0.05, at 1.5 h) and EGCg (P < 0.05, at 6 h) but not those of EC and EGC were reduced by the chocolate matrix. Regardless of the chocolate matrix, ECg and EGCg were mainly present as their aglycones in the plasma, whereas EGC and EC were found mostly as conjugated metabolites. After daily intake of GTCs mixed with chocolate for 14 days followed by overnight fasting, ECg but not EGCg was detected in the plasma. To compare the plasma profiles of ECg and EGCg, a mixture containing approximately equal amounts of ECg and EGCg was administered to nine rats for 14 days. Following treatment and overnight food deprivation, the plasma content of ECg was higher than that of EGCg. After a single injection of the same mixture in seven rats, ECg levels were higher than those of EGCg, and a greater amount of conjugated metabolites of ECg than those of EGCg was detected in the plasma 10 h after administration. In conclusion, the chocolate matrix affects the plasma profiles of GTCs, particularly ECg. ECg appears to persist in the plasma for a longer period, regardless of the chocolate matrix.


ATP/P2X7 receptor signaling as a potential anti-inflammatory 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.


Inhibitory effect of catecholic colonic metabolites of rutin on fatty acid hydroperoxide and hemoglobin dependent lipid peroxidation in Caco-2 cells.

To determine the preventive effect of dietary rutin on oxidative damages occurring in the digestive tract, 13-hydroperoxyoctadecadienoic acid and hemoglobin were exposed to Caco-2 intestinal cells after the pretreatment with colonic rutin metabolites. Among four catechol-type metabolites, quercetin and 3,4-dihydroxytoluene exerted significant protection on 13-hydroperoxyoctadecadienoic and hemoglobin-dependent lipid peroxidation of this epithelial cell. Compared with quercetin, a much lower concentration allowed 3,4-dihydroxytoluene to maximize the protective effect, though it needed a longer pre-incubation period. Neither quercetin nor 3,4-dihydroxytoluene affected the expression of peroxiredoxin-6 protein, which comprises the cellular antioxidant defense system. It is concluded that 3,4-dihydroxytoluene is a plausible rutin colonic metabolite that can suppress oxidative damages of intestinal epithelial cells by directly inhibiting lipid peroxidation. This result may illuminate the preventive role of dietary rutin against colorectal cancer incidence in relation to the consumption of red and processed meat.


8-Prenylnaringenin promotes recovery from immobilization-induced disuse muscle atrophy through activation of the Akt phosphorylation pathway in mice.

8-Prenylnaringenin (8-PN) is a prenylflavonoid that originates from hop extracts and is thought to help prevent disuse muscle atrophy. We hypothesized that 8-PN affects muscle plasticity by promoting muscle recovery under disuse muscle atrophy. To test the promoting effect of 8-PN on muscle recovery, we administered an 8-PN mixed diet to mice that had been immobilized with a cast to one leg for 14 days. Intake of the 8-PN mixed diet accelerated recovery from muscle atrophy, and prevented reductions in Akt phosphorylation. Studies on cell cultures of mouse myotubes in vitro demonstrated that 8-PN activated the PI3K/Akt/P70S6K1 pathway at physiological concentrations. A cell-culture study using an inhibitor of estrogen receptors and an in vivo experiment with ovariectomized mice suggested that the estrogenic activity of 8-PN contributed to recovery from disuse muscle atrophy through activation of an Akt phosphorylation pathway. These data strongly suggest that 8-PN is a naturally occurring compound that could be used as a nutritional supplement to aid recovery from disuse muscle atrophy.

American Journal of Physiology. Regulatory, Integrative and Comparative Physiology


Effect of quercetin and its metabolite on caveolin-1 expression induced by oxidized LDL and lysophosphatidylcholine in endothelial cells.

Oxidized low-density lipoprotein contributes to atherosclerotic plaque formation, and quercetin is expected to exert anti-atherosclerotic effects. We previously reported accumulation of conjugated quercetin metabolites in the aorta of rabbits fed high-cholesterol diets with quercetin glucosides, resulting in attenuation of lipid peroxidation and inhibition of lipid accumulation. Caveolin-1, a major structural protein of caveolae in vascular endothelial cells, plays a role in atherosclerosis development. Here we investigated effects of oxidized low-density lipoprotein, quercetin and its metabolite, quercetin 3-O--glucuronide, on caveolin-1 expression. Oxidized low-density lipoprotein significantly upregulated caveolin-1 mRNA expression. An oxidized low-density lipoprotein component, lysophosphatidylcholine, also induced expression of both caveolin-1 mRNA and protein. However, lysophosphatidylcholine did not affect the location of caveolin-1 proteins within caveolae structures. Co-treatment with quercetin or quercetin 3-O--glucuronide inhibited lysophosphatidylcholine-induced caveolin-1 expression. Quercetin and quercetin 3-O--glucuronide also suppressed expression of adhesion molecules induced by oxidized low-density lipoprotein and lysophosphatidylcholine. These results strongly suggest lysophosphatidylcholine derived from oxidized low-density lipoprotein contributes to atherosclerotic events by upregulating caveolin-1 expression, resulting in induction of adhesion molecules. Quercetin metabolites are likely to exert an anti-atherosclerotic effect by attenuating caveolin-1 expression in endothelial cells.


Anti-inflammatory effects and molecular mechanisms of 8-prenyl quercetin.

PQ as a potential inhibitor revealed anti-inflammatory effect in both cell and animal models at least by targeting SEK1-JNK1/2 and MEK1-ERK1/2.


Preventive effect of dietary quercetin on disuse muscle atrophy by targeting mitochondria in denervated mice.

Quercetin is a major dietary flavonoid in fruits and vegetables. We aimed to clarify the preventive effect of dietary quercetin on disuse muscle atrophy and the underlying mechanisms. We established a mouse denervation model by cutting the sciatic nerve in the right leg (SNX surgery) to lack of mobilization in hind-limb. Preintake of a quercetin-mixed diet for 14days before SNX surgery prevented loss of muscle mass and atrophy of muscle fibers in the gastrocnemius muscle (GM). Phosphorylation of Akt, a key phosphorylation pathway of suppression of protein degradation, was activated in the quercetin-mixed diet group with and without SNX surgery. Intake of a quercetin-mixed diet suppressed the generation of hydrogen peroxide originating from mitochondria and elevated mitochondrial peroxisome proliferator-activated receptor- coactivator 1 mRNA expression as well as NADH dehydrogenase 4 expression in the GM with SNX surgery. Quercetin and its conjugated metabolites reduced hydrogen peroxide production in the mitochondrial fraction obtained from atrophied muscle. In C2C12 myotubes, quercetin reached the mitochondrial fraction. These findings suggest that dietary quercetin can prevent disuse muscle atrophy by targeting mitochondria in skeletal muscle tissue through protecting mitochondria from decreased biogenesis and reducing mitochondrial hydrogen peroxide release, which can be related to decreased hydrogen peroxide production and/or improvements on antioxidant capacity of mitochondria.


Dietary Supplementation with Isoflavones Prevents Muscle Wasting in Tumor-Bearing Mice.

Proinflammatory cytokines contribute to the progression of muscle wasting caused by ubiquitin-proteasome-dependent proteolysis. We have previously demonstrated that isoflavones, such as genistein and daidzein, prevent TNF--induced muscle atrophy in C2C12 myotubes. In this study, we examined the effect of dietary flavonoids on the wasting of muscle. Mice were divided into the following four groups: vehicle-injected (control) mice fed the normal diet (CN); tumor-bearing mice fed the normal diet (TN); control mice fed the isoflavone diet (CI); and tumor-bearing mice fed the isoflavone diet (TI). There were no significant differences in the intake of food or body weight gain among these four groups. The wet weight and myofiber size of gastrocnemius muscle in TN significantly decreased, compared with those in CN. Interestingly, the wet weight and myofiber size of gastrocnemius muscle in TI were nearly the same as those in CN and CI, although isoflavone supplementation did not affect the increased tumor mass or concentrations of proinflammatory cytokines, such as TNF- and IL-6, in the blood. Moreover, increased expression of muscle-specific ubiquitin ligase genes encoding MAFbx/Atrogin-1 and MuRF1 in the skeletal muscle of TN was significantly inhibited by the supplementation of isoflavones. In parallel with the expression of muscle-specific ubiquitin ligases, dietary isoflavones significantly suppressed phosphorylation of ERK in tumor-bearing mice. These results suggest that dietary isoflavones improve muscle wasting in tumor-bearing mice via the ERK signaling pathway mediated-suppression of ubiquitin ligases in muscle cells.

Journal of Nutritional Science and Vitaminology


Capric Acid Up-Regulates UCP3 Expression without PDK4 Induction in Mouse C2C12 Myotubes

Uncoupling protein 3 (UCP3) and pyruvate dehydrogenase kinase 4 (PDK4) in skeletal muscle are key regulators of the glucose and lipid metabolic processes that are involved in insulin resistance. Medium-chain fatty acids (MCFAs) have anti-obesogenic effects in rodents and humans, while long-chain fatty acids (LCFAs) cause increases in body weight and insulin resistance. To clarify the beneficial effects of MCFAs, we examined UCP3 and PDK4 expression in skeletal muscles of mice fed a MCFA- or LCFA-enriched high-fat diet (HFD). Five-week feeding of the LCFA-enriched HFD caused high body weight gain and induced glucose intolerance in mice, compared with those in mice fed the MCFA-enriched HFD. However, the amounts of UCP3 and PDK4 transcripts in the skeletal muscle of mice fed the MCFA- or LCFA-enriched HFD were similar. To further elucidate the specific effects of MCFAs, such as capric acid (C10:0), on lipid metabolism in skeletal muscles, we examined the effects of various FAs on expression of UCP3 and PDK4, in mouse C2C12 myocytes. Although palmitic acid (C16:0) and lauric acid (C12:0) significantly induced expression of both UCP3 and PDK4, capric acid (C10:0) upregulated only UCP3 expression via activation of peroxisome proliferator-activated receptor-δ. Furthermore, palmitic acid (C16:0) disturbed the insulin-induced phosphorylation of Akt, while MCFAs, including lauric (C12:0), capric (C10:0), and caprylic acid (C12:0), did not. These results suggest that capric acid (C10:0) increases the capacity for fatty acid oxidation without inhibiting glycolysis in skeletal muscle.


Effect of Processed Onions on the Plasma Concentration of Quercetin in Rats and Humans.

Onion is a popular source of antioxidative flavonoid quercetin and its vascular function attracts considerable attention in relation to anti-atherosclerotic effect. The present study estimated the effect of food processing on the bioavailability of onion quercetin aglycone and its glucosides provided through the consumption of onion products. The intake of a peel powder-containing meal showed a significantly higher bioavailability than the peel extract, bulb powder, bulb extract, and bulb sauté containing meals. Hence, food processing of onion peel may enhance the health impact of onion quercetin by elevating its bioavailability.


Flavones Inhibit LPS-Induced Atrogin-1/MAFbx Expression in Mouse C2C12 Skeletal Myotubes.

Muscle atrophy is a complex process that occurs as a consequence of various stress events. Muscle atrophy-associated genes (atrogenes) such as atrogin-1/MAFbx and MuRF-1 are induced early in the atrophy process, and the increase in their expression precedes the loss of muscle weight. Although antioxidative nutrients suppress atrogene expression in skeletal muscle cells, the inhibitory effects of flavonoids on inflammation-induced atrogin-1/MAFbx expression have not been clarified. Here, we investigated the inhibitory effects of flavonoids on lipopolysaccharide (LPS)-induced atrogin-1/MAFbx expression. We examined whether nine flavonoids belonging to six flavonoid categories inhibited atrogin-1/MAFbx expression in mouse C2C12 myotubes. Two major flavones, apigenin and luteolin, displayed potent inhibitory effects on atrogin-1/MAFbx expression. The pretreatment with apigenin and luteolin significantly prevented the decrease in C2C12 myotube diameter caused by LPS stimulation. Importantly, the pretreatment of LPS-stimulated myoblasts with these flavones significantly inhibited LPS-induced JNK phosphorylation in C2C12 myotubes, resulting in the significant suppression of atrogin-1/MAFbx promoter activity. These results suggest that apigenin and luteolin, prevent LPS-mediated atrogin-1/MAFbx expression through the inhibition of the JNK signaling pathway in C2C12 myotubes. Thus, these flavones, apigenin and luteolin, may be promising agents to prevent LPS-induced muscle atrophy.


Biological impacts of resveratrol, quercetin, and N-acetylcysteine on oxidative stress in human gingival fibroblasts.

In periodontitis, production of reactive oxygen species (ROS) by neutrophils induces oxidative stress and deteriorates surrounding tissues. Antioxidants reduce damage caused by ROS and are used to treat diseases involving oxidative stress. This study summarizes the different effects of resveratrol, quercetin, and N-acetylcysteine (NAC) on human gingival fibroblasts (HGFs) under oxidative stress induced by hydrogen peroxide. Real-time cytotoxicity analyses reveals that resveratrol and quercetin enhanced cell proliferation even under oxidative stress. Of the antioxidants tested, resveratrol is the most effective at inhibiting ROS production. HGFs incubated with resveratrol and quercetin up-regulate the transcription of type I collagen gene after 3 h, but only resveratrol sustained this up-regulation for 24 h. A measurement of the oxygen consumption rate (OCR, mitochondrial respiration) shows that resveratrol generates the highest maximal respiratory capacity, followed by quercetin and NAC. Simultaneous measurement of OCR and the extracellular acidification rate (non-mitochondrial respiration) reveals that resveratrol and quercetin induce an increase in mitochondrial respiration when compared with untreated cells. NAC treatment consumes less oxygen and enhances more non-mitochondrial respiration. In conclusion, resveratrol is the most effective antioxidant in terms of real-time cytotoxicity analysis, reduction of ROS production, and enhancement of type I collagen synthesis and mitochondrial respiration in HGFs.

Journal of Clinical Biochemistry and Nutrition


N-myristoylated ubiquitin ligase Cbl-b inhibitor prevents on glucocorticoid-induced atrophy in mouse skeletal muscle.

A DGpYMP peptide mimetic of tyrosine(608)-phosphorylated insulin receptor substrate-1 (IRS-1), named Cblin, was previously shown to significantly inhibit Cbl-b-mediated IRS-1 ubiquitination. In the present study, we developed N-myristoylated Cblin and investigated whether it was effective in preventing glucocorticoid-induced muscle atrophy. Using HEK293 cells overexpressing Cbl-b, IRS-1 and ubiquitin, we showed that the 50% inhibitory concentrations of Cbl-b-mediated IRS-1 ubiquitination by N-myristoylated Cblin and Cblin were 30 and 120M, respectively. Regarding the DEX-induced atrophy of C2C12 myotubes, N-myristoylated Cblin was more effective than Cblin for inhibiting the DEX-induced decreases in C2C12 myotube diameter and IRS-1 degradation. The inhibitory efficacy of N-myristoylated Cblin on IRS-1 ubiquitination in C2C12 myotubes was approximately fourfold larger than that of Cblin. Furthermore, N-myristoylation increased the incorporation of Cblin into HEK293 cells approximately 10-folds. Finally, we demonstrated that N-myristoylated Cblin prevented the wet weight loss, IRS-1 degradation, and MAFbx/atrogin-1 and MuRF-1 expression in gastrocnemius muscle of DEX-treated mice approximately fourfold more effectively than Cblin. Taken together, these results suggest that N-myristoylated Cblin prevents DEX-induced skeletal muscle atrophy in vitro and in vivo, and that N-myristoylated Cblin more effectively prevents muscle atrophy than unmodified Cblin.


Effects of dietary soy protein on skeletal muscle volume and strength in humans with various physical activities

Background: In recent years, the number of bedridden people is rapidly increasing due to aging or lack of exercise in Japan. This problem is becoming more serious, since there is no countermeasure against it. In the present study, we designed to investigate whether dietary proteins, especially soy, had beneficial effects on skeletal muscle in 59 volunteers with various physical activities. Methods: We subjected 59 volunteers with various physical activities to meal intervention examination. Persons with low and high physical activities were divided into two dietary groups, the casein diet group and the soy diet group. They ate daily meals supplemented with 7.8 g of powdered casein or soy protein isolate every day for 30 days. Bedridden patients in hospitals were further divided into three dietary groups: the no supplementation diet group, the casein diet group and the soy diet group. They were also subjected to a blood test, a urinalysis, magnetic resonance imaging analysis and muscle strength test of the knee before and after the meal intervention study. Results: Thirty-day soy protein supplementation significantly increased skeletal muscle volume in participants with low physical activity, compared with 30-day casein protein supplementation. Both casein and soy protein supplementation increased the volume of quadriceps femoris muscle in bedridden patients. Consistently, soy protein significantly increased their extension power of the knee, compared with casein protein. Although casein protein increased skeletal muscle volume more than soy protein in bedridden patients, their muscle strength changes by soy protein supplementation were bigger than those by casein protein supplementation. Conclusions: The supplementation of soy protein would be one of the effective foods which prevent the skeletal muscle atrophy caused by immobilization or unloading. J. Med. Invest. 62: 177-183, August, 2015

The Journal of Medical Investigation : JMI


Comparison of Isoflavone Absorption by Soybean Products in Humans

大豆の加工形態によるイソフラボン生体吸収量の比較

We investigated the bioavailability of soy isoflavones from processed soybean products. Five healthy male volunteers ingested soy milk, soy curd (tofu) , boiled soybeans (nimame) , or fermented soybeans (natto) in a single dose, and blood was collected 90 min later for measurement of the plasma isoflavone concentration. Each subject ingested test meals containing an isoflavone equivalent of 50 mg aglycone. The total amount of each isoflavone was determined by HPLC analysis after deconjugation of conjugated metabolites using sulfatase H-1. The concentration of glycitein was below the detection limit before and after intake of the soybean products. The concentrations of daidzein and genistein were elevated in all subjects after ingestion of all the test meals, and the concentration of genistein was higher than that of daidzein except after ingestion of fermented soybeans. Ingestion of boiled soybeans appeared to result in a higher concentration of total isoflavones (daizein and genistein) than ingestion of soy curd. These results suggest that the bioavailability of isoflavones from soybeans varies according to the type of processing.

本試験は, 大豆イソフラボンの吸収性を大豆の加工形態の違いから比較した．5名の健常人男性が豆乳, 豆腐, 大豆煮豆, 納豆をそれぞれ単回摂取し, 摂取90分後の血中イソフラボン濃度の上昇値を吸収量とした．試験食中に含まれるダイゼイン, ゲニステイン, グリシテインとこれらの配糖体であるダイジン, ゲニスチン, グリシチンの合計をアグリコン換算で50 mg含むように摂取量を調整した．摂取前後の血中イソフラボンはアグリコンとして定量した．ダイゼイン, ゲニステインは試験食摂取後すべての被験者で上昇し, 摂取量に対する上昇値は納豆を除いてゲニステインがダイゼインより高い値であった．アグリコン換算した血漿中の総イソフラボン濃度の上昇は煮豆が豆腐に比べて大きい傾向を示した．以上の結果は大豆の加工形態がイソフラボンの生体吸収性に影響することを示唆するものである．

Journal of Japanese Society of Nutrition and Food Science


Location of a-tocopherol and a-tocotrienol to heterogeneous cell membranes and inhibition of production of peroxidizied cholesterol in mouse fibroblasts.

α-Tocopherol (α-T) and α-tocotrienol (α-T3) are well recognized as lipophilic antioxidants. Nevertheless, there is limited knowledge on their location in heterogeneous cell membranes. We first investigated the distribution of α-T and α-T3 to the cholesterol-rich microdomains (lipid rafts and caveolae) of heterogeneous cell membranes by incubating these antioxidants with cultured mouse fibroblasts. Levels of cellular uptake for α-T and α-T3 were adjusted to the same order, as that of the latter was much more efficient than that of the former in the cultured cells. After ultracentrifugation, α-T and α-T3 were partitioned to the microdomain fractions. When the distribution of α-T and α-T3 was further confirmed by using methyl-β-cyclodextrin (which removes cholesterol from membranes), α-T was suggested to be distributed to the microdomains (approx. 9% of the total uptake). The same treatment did not affect α-T3 content in the microdomain fractions, indicating that α-T3 is not located in these cholesterol-rich domains. However, α-T and α-T3 significantly inhibited the production of peroxidized cholesterol when cells were exposed to ultraviolet-A light. These results suggest that α-T and α-T3 can act as membranous antioxidants against photo-irradiated cholesterol peroxidation irrespective of their distribution to cholesterol-rich microdomains.


Cellular uptake of quercetin and luteolin and their effects on monoamine oxidase-A in human neuroblastoma SH-SY5Y cells


Specific localization of quercetin-3-O-glucuronide in human brain.

In recent years, many papers have suggested that dietary flavonoids may exert beneficial effects in the brain tissue for the protection of neurons against oxidative stress and inflammation. However, the bioavailability of flavonoids across the blood-brain barrier and the localization in the brain remain controversial. Thus, we examined the localization of quercetin-3-O-glucuronide (Q3GA), a major phase-II metabolite of quercetin, in the human brain tissues with or without cerebral infarction by immunohistochemical staining using anti-Q3GA antibody. A significant immunoreactivity was observed in the epithelial cells of the choroid plexus, which constitute the structural basis of the blood-cerebrospinal fluid (CSF) barrier, and in the foamy macrophages of recent infarcts. The cellular accumulation of Q3GA was also reproduced in vitro in macrophage-like RAW264, microglial MG6, and brain capillary endothelial RBEC1. It is of interest that a common feature of these cell lines is the deconjugation of Q3GA, resulting in the cellular accumulation of non-conjugated quercetin and the methylated forms. We then examined the anti-inflammatory activity of Q3GA and the deconjugated forms in the lipopolysaccharide-stimulated macrophage cells and revealed that the deconjugated forms (quercetin and a methylated form isorhamnetin), but not Q3GA itself, exhibited inhibitory effects on the inflammatory responses through attenuation of the c-Jun N-terminal kinase pathway. These results suggested that a quercetin glucuronide can pass through the blood-brain barrier, perhaps the CSF barrier, accumulate in specific types of cells, such as macrophages, and act as anti-inflammatory agents in the brain through deconjugation into the bioactive non-conjugated forms.


The First Synthesis of Uralenol, 5'-Prenylated Quercetin via Palladium-catalyzed O-dimethylallylation Reaction with Concurrent Acetyl Migration


Cholesterol hydroperoxides and their degradation mechanism.

Cholesterol is one of the oxidizable lipids constituting biomembranes and plasma lipoproteins. Cholesterol hydroperoxides (Chol-OOH) are the primary products if cholesterol is subjected to attack by reactive oxygen species. In particular, singlet molecular oxygen reacts with cholesterol to yield cholesterol 5α-hydroperoxide as the major hydroperoxide species. Chol-OOH may accumulate in biological systems because of its resistance to glutathione-dependent enzymatic detoxification reactions. Their degradation products (including hydroxycholesterol and 7-ketocholesterol) participate in the pathophysiological functions of oxysterols. Highly reactive cholesterol 5,6-secosterol present in atherosclerotic lesions can be derived from the degradation of cholesterol 5α-hydroperoxide. Chol-OOH themselves may affect the lipid rafts of biomembranes, thereby leading to the modification of signal transduction pathways.


Mitochondrial dysfunction leads to deconjugation of quercetin glucuronides in inflammatory macrophages.

Dietary flavonoids, such as quercetin, have long been recognized to protect blood vessels from atherogenic inflammation by yet unknown mechanisms. We have previously discovered the specific localization of quercetin-3-O-glucuronide (Q3GA), a phase II metabolite of quercetin, in macrophage cells in the human atherosclerotic lesions, but the biological significance is poorly understood. We have now demonstrated the molecular basis of the interaction between quercetin glucuronides and macrophages, leading to deconjugation of the glucuronides into the active aglycone. In vitro experiments showed that Q3GA was bound to the cell surface proteins of macrophages through anion binding and was readily deconjugated into the aglycone. It is of interest that the macrophage-mediated deconjugation of Q3GA was significantly enhanced upon inflammatory activation by lipopolysaccharide (LPS). Zymography and immunoblotting analysis revealed that -glucuronidase is the major enzyme responsible for the deglucuronidation, whereas the secretion rate was not affected after LPS treatment. We found that extracellular acidification, which is required for the activity of -glucuronidase, was significantly induced upon LPS treatment and was due to the increased lactate secretion associated with mitochondrial dysfunction. In addition, the -glucuronidase secretion, which is triggered by intracellular calcium ions, was also induced by mitochondria dysfunction characterized using antimycin-A (a mitochondrial inhibitor) and siRNA-knockdown of Atg7 (an essential gene for autophagy). The deconjugated aglycone, quercetin, acts as an anti-inflammatory agent in the stimulated macrophages by inhibiting the c-Jun N-terminal kinase activation, whereas Q3GA acts only in the presence of extracellular -glucuronidase activity. Finally, we demonstrated the deconjugation of quercetin glucuronides including the sulfoglucuronides in vivo in the spleen of mice challenged with LPS. These results showed that mitochondrial dysfunction plays a crucial role in the deconjugation of quercetin glucuronides in macrophages. Collectively, this study contributes to clarifying the mechanism responsible for the anti-inflammatory activity of dietary flavonoids within the inflammation sites.


Prenylation enhances quercetin uptake and reduces efflux in Caco-2 cells and enhances tissue accumulation in mice fed long-term.

Prenyl flavonoids are widely distributed in plant foods and have attracted appreciable attention in relation to their potential benefits for human health. Prenylation may enhance the biological functions of flavonoids by introducing hydrophobic properties in their basic structures. Previously, we found that 8-prenyl naringenin exerted a greater preventive effect on muscle atrophy than nonprenylated naringenin in a mouse model. Here, we aimed to estimate the effect of prenylation on the bioavailability of dietary quercetin (Q). The cellular uptake of 8-prenyl quercetin (PQ) and Q in Caco-2 cells and C2C12 myotube cells was examined. Prenylation significantly enhanced the cellular uptake by increasing the lipophilicity in both cell types. In Caco-2 cells, efflux of PQ to the basolateral side was <15% of that of Q, suggesting that prenylation attenuates transport from the intestine to the circulation. After intragastric administration of PQ or Q to mice or rats, the area under the concentration-time curve for PQ in plasma and lymph was 52.5% and 37.5% lower than that of Q, respectively. PQ and its O-methylated form (MePQ) accumulated at much higher amounts than Q and O-methylated Q in the liver (Q: 3400%; MePQ: 7570%) and kidney (Q: 385%; MePQ: 736%) of mice after 18 d of feeding. These data suggest that prenylation enhances the accumulation of Q in tissues during long-term feeding, even though prenylation per se lowers its intestinal absorption from the diet.


Influence of fatty acid patterns on the intestinal absorption pathway of quercetin in thoracic lymph duct-cannulated rats.

Since it is known that dietary fats improve the bioavailability of the flavonol quercetin, we purposed to investigate whether this effect is due to increased lymphatic transport of quercetin. In rats with implanted catheters in the thoracic lymph duct, we administered quercetin into the duodenum with TAG emulsions containing either long-chain fatty acids (LCT) or medium-chain fatty acids (MCT). Controls received quercetin together with a glucose solution. LCT administration increased the lymphatic output of quercetin (19.1 (SEM 1.2) nmol/8 h) as well as the lymph-independent bioavailability of the flavonol, determined as area under the plasma concentration curve (1091 (SEM 142) microM x min). Compared with glucose administration, MCT neither increased the lymphatic output (12.3 (SEM 1.5) nmol/8 h) nor the bioavailability of quercetin (772 (SEM 99) microM x min) significantly (glucose group: 9.8 (SEM 1.5) nmol/8 h and 513 (SEM 55) microM x min, respectively). Because LCT are released within chylomicrons into the intestinal lymph while MCT are mainly released into the portal blood, we conclude from the present results that dietary fats that are mainly composed of LCT improve quercetin bioavailability by increasing its transport via the lymph, thereby circumventing hepatic first-pass metabolism of the flavonol. In addition, LCT could enhance quercetin absorption by improving its solubility in the intestinal tract.


Non-selective distribution of isomeric cholesterol hydroperoxides to microdomains in cell membranes and activation of matrix metalloproteinase activity in a model of dermal cells.

Cholesterol hydroperoxides (ChOOHs) are included as lipid peroxidation products in the skin exposed to ultraviolet (UV) light irradiation. They may exert physicochemical actions affecting biomembrane rigidity because cholesterol is one of the major components of cell membranes. We investigated the distribution of isomeric ChOOHs in heterogeneous cell membranes with different lipid profiles using mouse fibroblast NIH-3T3 cells as a model of the dermis. Before and after UVA irradiation in the presence of hematoporphyrin, cell membranes were partitioned to microdomains (lipid rafts and caveolae) containing a higher amount of cholesterol and non-microdomains (containing a lower amount of cholesterol) by ultracentrifugation. By a combination of diphenylpyrenylphosphine-thin-layer chromatography blotting analyses and gas chromatography-electron ionization-mass spectrometry/selected ion monitoring analyses, ChOOH isomers were determined as their trimethylsilyloxyl derivatives. Cholesterol 5α-, 7α- and 7β-hydroperoxide were found as isomeric ChOOHs before irradiation. The amounts of the three ChOOH isomers increased significantly after photoirradiation for 2h. No difference was observed between microdomains and non-microdomains with regard to the ratio of the amounts of isomeric ChOOHs to that of cholesterol, suggesting that these ChOOH isomers were distributed equally in both parts depending on cholesterol content. When cells were treated with a purified mixture of ChOOH isomers, cell membranes incorporated ChOOHs into microdomains as well as non-microdomains evenly. Cellular matrix metalloproteinase-9 (MMP-9) activity was elevated by treatment with the purified mixture of ChOOH isomers. These results strongly suggest that ChOOHs accumulate in cell membranes irrespective of the heterogeneous microstructure and promote MMP activity if dermal cells are exposed to photodynamic actions.


Soy Glycinin Contains a Functional Inhibitory Sequence against Muscle-Atrophy-Associated Ubiquitin Ligase Cbl-b.

Background. Unloading stress induces skeletal muscle atrophy. We have reported that Cbl-b ubiquitin ligase is a master regulator of unloading-associated muscle atrophy. The present study was designed to elucidate whether dietary soy glycinin protein prevents denervation-mediated muscle atrophy, based on the presence of inhibitory peptides against Cbl-b ubiquitin ligase in soy glycinin protein. Methods. Mice were fed either 20% casein diet, 20% soy protein isolate diet, 10% glycinin diet containing 10% casein, or 20% glycinin diet. One week later, the right sciatic nerve was cut. The wet weight, cross sectional area (CSA), IGF-1 signaling, and atrogene expression in hindlimb muscles were examined at 1, 3, 3.5, or 4 days after denervation. Results. 20% soy glycinin diet significantly prevented denervation-induced decreases in muscle wet weight and myofiber CSA. Furthermore, dietary soy protein inhibited denervation-induced ubiquitination and degradation of IRS-1 in tibialis anterior muscle. Dietary soy glycinin partially suppressed the denervation-mediated expression of atrogenes, such as MAFbx/atrogin-1 and MuRF-1, through the protection of IGF-1 signaling estimated by phosphorylation of Akt-1. Conclusions. Soy glycinin contains a functional inhibitory sequence against muscle-atrophy-associated ubiquitin ligase Cbl-b. Dietary soy glycinin protein significantly prevented muscle atrophy after denervation in mice.


Plasma HDL reduces nonesterified fatty acid hydroperoxides originating from oxidized LDL.

The antioxidant property of plasma high-density lipoprotein (HDL) is thought to be involved in potential anti-atherogenic effects but the exact mechanism is not known. We aimed to reveal the contribution of HDL on the elimination of lipid hydroperoxides (LOOH) derived from oxidized low-density lipoprotein (LDL). Oxidized LDL prepared by copper ion-induced oxidation contained nonesterified fatty acid hydroperoxides (FFA-OOH) and lysophosphatidylcholine (lysoPtdCho), in addition to cholesteryl ester hydroperoxides (CE-OOH) and phosphatidylcholine hydroperoxides (PtdCho-OOH). A platelet-activating factor-acetylhydrolase (PAF-AH) inhibitor suppressed formation of FFA-OOH and lysoPtdCho in oxidized LDL. Among LOOH species, FFA-OOH was preferentially reduced by incubating oxidized LDL with HDL. HDL exhibited selective FFA-OOH reducing ability if it was mixed with a liposomal solution containing FFA-OOH, CE-OOH and PtdCho-OOH. Two-electron reduction of the hydroperoxy group to the hydroxy group was confirmed by the formation of 13-hydroxyoctadecadienoic acid from 13-hydroperoxyoctadecadienoic acid in HPLC analyses. This reducing effect was also found in apolipoprotein A-1 (apoA-1). FFA-OOH released from PtdCho-OOH due to PAF-AH activity in oxidized LDL undergo two-electron reduction by the reducing ability of apoA1 in HDL. This preferential reduction of FFA-OOH may participate in the mechanism of the antioxidant property of HDL.


Non-specific protein modification by a phytochemical induce heat shock response for self-defense.

Accumulated evidence shows that some phytochemicals provide beneficial effects for human health. Recently, a number of mechanistic studies have revealed that direct interactions between phytochemicals and functional proteins play significant roles in exhibiting their bioactivities. However, their binding selectivities to biological molecules are considered to be lower due to their small and simple structures. In this study, we found that zerumbone, a bioactive sesquiterpene, binds to numerous proteins with little selectivity. Similar to heat-denatured proteins, zerumbone-modified proteins were recognized by heat shock protein 90, a constitutive molecular chaperone, leading to heat shock factor 1-dependent heat shock protein induction in hepa1c1c7 mouse hepatoma cells. Furthermore, oral administration of this phytochemical up-regulated heat shock protein expressions in the livers of Sprague-Dawley rats. Interestingly, pretreatment with zerumbone conferred a thermoresistant phenotype to hepa1c1c7 cells as well as to the nematode Caenorhabditis elegans. It is also important to note that several phytochemicals with higher hydrophobicity or electrophilicity, including phenethyl isothiocyanate and curcumin, markedly induced heat shock proteins, whereas most of the tested nutrients did not. These results suggest that non-specific protein modifications by xenobiotic phytochemicals cause mild proteostress, thereby inducing heat shock response and leading to potentiation of protein quality control systems. We considered these bioactivities to be xenohormesis, an adaptation mechanism against xenobiotic chemical stresses. Heat shock response by phytochemicals may be a fundamental mechanism underlying their various bioactivities.


Cbl-b is a critical regulator of macrophage activation associated with obesity-induced insulin resistance in mice.

We previously reported the potential involvement of casitas B-cell lymphoma-b (Cbl-b) in aging-related murine insulin resistance. Because obesity also induces macrophage recruitment into adipose tissue, we elucidated here the role of Cbl-b in obesity-related insulin resistance. Cbl-b(+/+) and Cbl-b(-/-) mice were fed a high-fat diet (HFD) and then examined for obesity-related changes in insulin signaling. The HFD caused recruitment of macrophages into adipose tissue and increased inflammatory reaction in Cbl-b(-/-) compared with Cbl-b(+/+) mice. Peritoneal macrophages from Cbl-b(-/-) mice and Cbl-b-overexpressing RAW264.7 macrophages were used to examine the direct effect of saturated fatty acids (FAs) on macrophage activation. In macrophages, Cbl-b suppressed saturated FA-induced Toll-like receptor 4 (TLR4) signaling by ubiquitination and degradation of TLR4. The physiological role of Cbl-b in vivo was also examined by bone marrow transplantation and Eritoran, a TLR4 antagonist. Hematopoietic cell-specific depletion of the Cbl-b gene induced disturbed responses on insulin and glucose tolerance tests. Blockade of TLR4 signaling by Eritoran reduced fasting blood glucose and serum interleukin-6 levels in obese Cbl-b(-/-) mice. These results suggest that Cbl-b deficiency could exaggerate HFD-induced insulin resistance through saturated FA-mediated macrophage activation. Therefore, inhibition of TLR4 signaling is an attractive therapeutic strategy for treatment of obesity-related insulin resistance.


Isoflavones derived from soy beans prevent MuRF1-mediated muscle atrophy in C2C12 myotubes through SIRT1 activation.

Proinflammatory cytokines are factors that induce ubiquitin-proteasome-dependent proteolysis in skeletal muscle, causing muscle atrophy. Although isoflavones, as potent antioxidative nutrients, have been known to reduce muscle damage during the catabolic state, the non-antioxidant effects of isoflavones against muscle atrophy are not well known. Here we report on the inhibitory effects of isoflavones such as genistein and daidzein on muscle atrophy caused by tumor necrosis factor (TNF)- treatment. In C2C12 myotubes, TNF- treatment markedly elevated the expression of the muscle-specific ubiquitin ligase MuRF1, but not of atrogin-1, leading to myotube atrophy. We found that MuRF1 promoter activity was mediated by acetylation of p65, a subunit of NFB, a downstream target of the TNF- signaling pathway; increased MuRF1 promoter activity was abolished by SIRT1, which is associated with deacetylation of p65. Of interest, isoflavones induced expression of SIRT1 mRNA and phosphorylation of AMP kinase, which is well known to stimulate SIRT1 expression, although there was no direct effect on SIRT1 activation. Moreover, isoflavones significantly suppressed MuRF1 promoter activity and myotube atrophy induced by TNF- in C2C12 myotubes. These results suggest that isoflavones suppress myotube atrophy in skeletal muscle cells through activation of SIRT1 signaling. Thus, the efficacy of isoflavones could provide a novel therapeutic approach against inflammation-related muscle atrophy.

Journal of Nutritional Science and Vitaminology


Blueberry intervention improves vascular reactivity and lowers blood pressure in high fat, high cholesterol fed rats

Growing evidence suggests that intake of flavonoid-containing foods may exert cardiovascular benefits in human subjects. We have investigated the effects of a 10-week blueberry (BB) supplementation on blood pressure (BP) and vascular reactivity in rats fed a high-fat/high-cholesterol diet, known to induce endothelial dysfunction. Rats were randomly assigned to follow a control chow diet, a chow diet supplemented with 2 % (w/w) BB, a high-fat diet (10 % lard; 0·5 % cholesterol) or the high fat plus BB for 10 weeks. Rats supplemented with BB showed significant reductions in systolic BP (SBP) of 11 and 14 %, at weeks 8 and 10, respectively, relative to rats fed the control chow diet (week 8 SBP: 107·5 (SEM 4·7) v. 122·2 (SEM 2·1) mmHg, P= 0·018; week 10 SBP: 115·0 (SEM 3·1) v. 132·7 (SEM 1·5) mmHg, P< 0·0001). Furthermore, SBP was reduced by 14 % in rats fed with the high fat plus 2 % BB diet at week 10, compared to those on the high-fat diet only (SBP: 118·2 (SEM 3·6) v. 139·5 (SEM 4·5) mmHg, P< 0·0001). Aortas harvested from BB-fed animals exhibited significantly reduced contractile responses (to L-phenylephrine) compared to those fed the control chow or high-fat diets. Furthermore, in rats fed with high fat supplemented with BB, aorta relaxation was significantly greater in response to acetylcholine compared to animals fed with the fat diet. These data suggest that BB consumption can lower BP and improve endothelial dysfunction induced by a high fat, high cholesterol containing diet.


Evaluation of the inhibitory effects of quercetin-related flavonoids and tea catechins on the monoamine oxidase-A reaction in mouse brain mitochondria.

Quercetin, a typical dietary flavonoid, is thought to exert antidepressant effects by inhibiting the monoamine oxidase-A (MAO-A) reaction, which is responsible for regulation of the metabolism of the neurotransmitter 5-hydroxytryptamine (5-HT) in the brain. This study compared the MAO-A inhibitory activity of quercetin with those of O-methylated quercetin (isorhamnetin, tamarixetin), luteolin, and green tea catechins ((-)-epicatechin, (-)-epicatechin gallate, (-)-epigallocatechin, and (-)-epigallocatechin gallate) by measuring the formation of the oxidative deamination product of 5-HT, 5-hydroxyindole aldehyde (5-HIAL), in mouse brain mitochondria. Quercetin was inferior to luteolin in the inhibition of MAO-A activity, whereas isorhamnetin, tamarixetin, and tea catechins scarcely exerted inhibitory activity. Quercetin did not affect MAO-A activity in mouse intestinal mitochondria, indicating that it does not evoke side effects on the metabolism of dietary monoamines in the gut. These data suggest that quercetin is a weak (but safe) MAO-A inhibitor in the modulation of 5-HT levels in the brain.

Journal of Agricultural and Food Chemistry


Prevention of disuse muscle atrophy by dietary ingestion of 8-prenylnaringenin in denervated mice

Flavonoids have attracted considerable attention in relation to their effects upon health. 8-Prenylnaringenin (8-PN) is found in the common hop (Humulus lupulus) and assumed to be responsible for the health impact of beer consumption. We wanted to clarify the effects of prenylation on the physiological functions of dietary flavonoids by comparing the effects of 8-PN with that of intact naringenin in the prevention of disuse muscle atrophy using a model of denervation in mice. Consumption of 8-PN (but not naringenin) prevented loss of weight in the gastrocnemius muscle further supported by the lack of induction of the protein content of a key ubiquitin ligase involved in muscle atrophy, atrogin-1, and by the activation of Akt phosphorylation. 8-PN content in the gastrocnemius muscle was tenfold higher than that of naringenin. These results suggested that, compared with naringenin, 8-PN was effectively concentrated into skeletal muscle to exert its preventive effects upon disuse muscle atrophy. It is likely that prenylation generates novel functions for 8-PN by enhancing its accumulation into muscle tissue through dietary intake.


An Efficient Method for C8-Prenylation of Flavonols and Flavanones


Effect of quercetin and its glucuronide metabolite upon 6-hydorxydopamine-induced oxidative damage in Neuro-2a cells.

Quercetin is ubiquitously distributed in plant foods. This antioxidative polyphenol is mostly converted to conjugated metabolites in the body. Parkinson disease (PD) has been suggested to be related to oxidative stress derived from abnormal dopaminergic activity. We evaluated if dietary quercetin contributes to the antioxidant network in the central nervous system from the viewpoint of PD prevention. A neurotoxin, 6-hydroxydopamine (6-OHDA), was used as a model of PD. 6-OHDA-induced H(2)O(2) production and cell death in mouse neuroblastoma, Neuro-2a. Quercetin aglycone suppressed 6-OHDA-induced H(2)O(2) production and cell death, although aglycone itself reduced cell viability at higher concentration. Quercetin 3-O-β-d-glucuronide (Q3GA), which is an antioxidative metabolite of dietary quercetin, was little incorporated into the cell resulting in neither suppression of 6-OHDA-induced cell death nor reduction of cell viability. Q3GA was found to be deconjugated to quercetin by microglial MG-6 cells. These results indicate that quercetin metabolites should be converted to their aglycone to exert preventive effect on damage to neuronal cells.


Self-reported seafood intake and atopy in Japanese school-aged children.

The effects of fish consumption and n-3 poly-unsaturated fatty acid (PUFA) levels on atopic disorders are inconsistent in previous reports, but few studies have investigated the effects of both fish and n-3 PUFA. The aim of the present study was to investigate whether erythrocyte fatty acids and the consumption of fish are associated with atopic diseases in pre- and early adolescents. A total of 135 students with eczema, 136 students with asthma, and 137 healthy control students were selected from fifth and eighth grades in Shunan, Japan. Atopic disorders and dietary intake were evaluated with questionnaires, and total serum IgE was measured using an enzyme-linked immunosorbent assay. In addition, erythrocyte membrane levels of PUFA were assessed via gas chromatography. Total IgE was significantly elevated in the atopic subjects (P < 0.001). The intake of fatty and dried fish or seafood was significantly associated with eczema (odds ratios of the highest quartiles: 0.46, 95% confidence interval (95%CI): 0.22-0.94; 0.34, 95%CI: 0.16-0.71, respectively). Additionally, only erythrocyte eicosapentaenoic acid (EPA) level had a negative association with eczema (P= 0.048). For asthma, the effect of fish consumption was not significant. Fish consumption was related to a low prevalence of eczema, but not asthma in Japanese pre- and early adolescents. EPA may be involved in this mechanism.


Unloading stress disturbs muscle regeneration through perturbed recruitment and function of macrophages.

Skeletal muscle is one of the most sensitive tissues to mechanical loading, and unloading inhibits the regeneration potential of skeletal muscle after injury. This study was designed to elucidate the specific effects of unloading stress on the function of immunocytes during muscle regeneration after injury. We examined immunocyte infiltration and muscle regeneration in cardiotoxin (CTX)-injected soleus muscles of tail-suspended (TS) mice. In CTX-injected TS mice, the cross-sectional area of regenerating myofibers was smaller than that of weight-bearing (WB) mice, indicating that unloading delays muscle regeneration following CTX-induced skeletal muscle damage. Delayed infiltration of macrophages into the injured skeletal muscle was observed in CTX-injected TS mice. Neutrophils and macrophages in CTX-injected TS muscle were presented over a longer period at the injury sites compared with those in CTX-injected WB muscle. Disturbance of activation and differentiation of satellite cells was also observed in CTX-injected TS mice. Further analysis showed that the macrophages in soleus muscles were mainly Ly-6C-positive proinflammatory macrophages, with high expression of tumor necrosis factor- and interleukin-1, indicating that unloading causes preferential accumulation and persistence of proinflammatory macrophages in the injured muscle. The phagocytic and myotube formation properties of macrophages from CTX-injected TS skeletal muscle were suppressed compared with those from CTX-injected WB skeletal muscle. We concluded that the disturbed muscle regeneration under unloading is due to impaired macrophage function, inhibition of satellite cell activation, and their cooperation.


Bioavailability of orally administered water-dispersible hesperetin and its effect on peripheral vasodilatation in human subjects: implication of endothelial functions of plasma conjugated metabolites.

Hesperetin is an aglycone of citrus flavonoids and is expected to exert a vasodilatation effect in vivo. We developed water-dispersible hesperetin by the process of micronization to enhance the bioavailability of hesperetin. This study aimed to assess the effect of this process on the bioavailability of hesperetin and to estimate its efficiency on vasodilatation-related functions using endothelial cells in vitro and a human volunteer study at a single dose in vivo. We found that water-dispersible hesperetin was absorbed rapidly, with its maximum plasma concentration (C(max)) being 10.2 ± 1.2 μM, and that the time to reach C(max), which is within 1 h if 150 mg of this preparation was orally administered in humans. LC-MS analyses of the plasma at C(max) demonstrated that hesperetin accumulated in the plasma as hesperetin 7-O-β-D-glucuronide (Hp7GA), hesperetin 3'-O-β-D-glucuronide (Hp3'GA) and hesperetin sulfate exclusively. Similar to hesperetin, Hp7GA enhanced nitric oxide (NO) release by inhibiting nicotinamide adenine dinucleotide phosphate-oxidase (NADPH oxidase) activity in a human umbilical vein endothelial cell culture system, indicating that plasma hesperetin metabolites can improve vasodilatation in the vascular system. A volunteer study using women with cold sensitivity showed that a single dose of water-dispersible hesperetin was effective on peripheral vasodilatation.These results strongly suggest that rapid accumulation with higher plasma concentration enables hesperetin to exert a potential vasodilatation effect by the endothelial action of its plasma metabolites. Water-dispersible hesperetin may be useful to improve the health effect of dietary hesperetin.


Tissue distribution of hesperetin in rats after a dietary intake.

Hesperetin, the aglycone of hesperidin present in citrus fruits, possesses various biological activities. We assessed the tissue distribution of hesperetin in rats fed with a 0.2% hesperetin diet for 4 weeks. Its highest concentration was found in the liver, and the second highest was in the aorta. The aorta is assumed to be one of the main target tissues of hesperetin for exerting its functions.

Bioscience, Biotechnology, and Biochemistry


Pharmacology in health food: Metabolism of quercetin in vivo and its protective effect against arteriosclerosis

Quercetin, a member of the bioflavonoids family, has been proposed to have anti-atherogenic, anti-inflammatory, and anti-hypertensive properties leading to the beneficial effects against cardiovascular diseases. It was recently demonstrated that quercetin 3-O-β-D-glucuronide (Q3GA) is one of the major quercetin conjugates in human plasma, in which the aglycone could not be detected. Although most of the in vitro pharmacological studies have been carried out using only the quercetin aglycone form, experiments using Q3GA would be important to discover the preventive mechanisms of cardiovascular diseases by quercetin in vivo. Therefore we examined the effects of the chemically synthesized Q3GA, as an in vivo form, on vascular smooth muscle cell (VSMC) disorders related to the progression of arteriosclerosis. Platelet-derived growth factor-induced cell migration and proliferation were inhibited by Q3GA in VSMCs. Q3GA attenuated angiotensin II-induced VSMC hypertrophy via its inhibitory effect on JNK and the AP-1 signaling pathway. Q3GA scavenged 1,1-diphenyl-2-picrylhydrazyl radical measured by the electron paramagnetic resonance method. In addition, immunohistochemical studies with monoclonal antibody 14A2 targeting the Q3GA demonstrated that the positive staining specifically accumulates in human atherosclerotic lesions, but not in the normal aorta. These findings suggest Q3GA would be an active metabolite of quercetin in plasma and may have preventative effects on arteriosclerosis relevant to VSMC disorders.


Effect of quercetin and glucuronide metabolites on the monoamine oxidase-A reaction in mouse brain mitochondria.

OBJECTIVE: Quercetin is a flavonoid found in plant foods and herbal medicines. It possesses antidepressant-like effects in forced swimming test-loaded rodents. We wanted to clarify the mechanism of action of dietary quercetin for exerting antidepressant-like effects. The effect of quercetin and its antioxidative metabolite quercetin 3-glucuronide (Q3GA) on the activity of mouse brain mitochondrial monoamine oxidase-A (MAO-A) was evaluated by measuring the deamination product of serotonin, 5-hydroxyindole acetaldehyde (5-HIAL). METHODS: An ultraviolet high-performance liquid chromatographic analysis was applied to measure the 5-HIAL generated by the reaction of MAO-A with serotonin. The inhibitory effect of quercetin and Q3GA on mitochondrial MAO-A activity was estimated by the content of 5-HIAL and hydrogen peroxide accompanied by the MAO-A reaction. RESULTS: Quercetin (but not Q3GA) decreased the production of 5-HIAL by MAO-A activity. Q3GA inhibited the generation of hydrogen peroxide from the MAO-A reaction with serotonin. A periodic forced swimming test in mice increased brain mitochondrial MAO-A activity. Brain mitochondrial MAO-A activity was decreased in mice administered quercetin for 7 d, but its effect was much weaker than that of the selective MAO-A inhibitor clorgyline. CONCLUSION: Quercetin is effective in the modulation of serotonergic activity by attenuating mitochondrial MAO-A activity in the brain. Its antioxidative metabolite Q3GA attenuates oxidative stress by interrupting the generation of hydrogen peroxide accompanying the MAO-A reaction.


Rantes secreted from macrophages disturbs skeletal muscle regeneration after cardiotoxin injection in Cbl-b-deficient mice

Deficiency of the Cbl-b ubiquitin ligase gene activates macrophages in mice. This study aimed to elucidate the pathophysiological roles of macrophages in muscle degeneration/regeneration in Cbl-b-deficient mice. We examined immune cell infiltration and cytokine expression in cardiotoxin-injected tibialis anterior muscle of Cbl-b-deficient mice. Ablation of the Cbl-b gene expression delayed regeneration of cardiotoxin-induced skeletal muscle damage compared with wild-type mice. CD8-positive T cells were still present in the damaged muscle on day 14 after cardiotoxin injection in Cbl-b-deficient mice, but there was dispersal of the same cells over that time-frame in wild-type mice. Infiltrating macrophages in Cbl-b-deficient mice showed strong expression of RANTES (regulated-on-activation, normal T cell expressed and secreted), a chemokine for CD8-positive T cells. In turn, a neutralizing antibody against RANTES significantly suppressed the infiltration of CD8-positive T cells into the muscle, resulting in restoration of the disturbed muscle regeneration. Cbl-b is an important regulatory factor for cytotoxic T-cell infiltration via RANTES production in macrophages.


Conjugated quercetin glucuronides as bioactive metabolites and precursors of aglycone in vivo

Quercetin is a typical anti-oxidative flavonoid ubiquitously distributed in vegetables. It is likely to act as a bioactive compound by exerting reactive oxygen species (ROS)-scavenging activity and/or binding to specific proteins such as oxidative enzymes and transcriptional factors in signal transduction pathways. Its absorption and metabolism (as well as its molecular targets) have been extensively explored from the viewpoint of its potential for disease prevention. It is known that glucuronide and/or sulfate conjugates with or without O-methylation exclusively circulate in the human bloodstream after intake of a quercetin-containing diet. We propose that glucuronide conjugates of quercetin function not only as detoxified metabolites but hydrophilic bioactive agents to various ROS-generating systems and precursors of hydrophobic aglycone. Quercetin aglycone is assumed to emerge in the target site by the action of β-glucuronidase activity under oxidative stress such as inflammation. The cardiovascular system and central nervous system seem to be the major targets of conjugated quercetin glucuronides circulating in the human bloodstream.


Singlet molecular oxygen-quenching activity of carotenoids: relevance to protection of the skin from photoaging.

Carotenoids are known to be potent quenchers of singlet molecular oxygen [O(2) ((1)Δ(g))]. Solar light-induced photooxidative stress causes skin photoaging by accelerating the generation of reactive oxygen species via photodynamic actions in which O(2) ((1)Δ(g)) can be generated by energy transfer from excited sensitizers. Thus, dietary carotenoids seem to participate in the prevention of photooxidative stress by accumulating as antioxidants in the skin. An in vivo study using hairless mice clarified that a O(2) ((1)Δ(g)) oxygenation-specific peroxidation product of cholesterol, cholesterol 5α-hydroperoxide, accumulates in skin lipids due to ultraviolet-A exposure. Matrix metalloproteinase-9, a metalloproteinase family enzyme responsible for the formation of wrinkles and sagging, was enhanced in the skin of ultraviolet-A -irradiated hairless mice. The activation of metalloproteinase-9 and the accumulation of 5α-hydroperoxide, as well as formation of wrinkles and sagging, were lowered in mice fed a β-carotene diet. These results strongly suggest that dietary β-carotene prevents the expression of metalloproteinase-9 (at least in part), by inhibiting the photodynamic action involving the formation of 5α-hydroperoxide in the skin. Intake of β-Carotene therefore appears to be helpful in slowing down ultraviolet-A -induced photoaging in human skin by acting as a O(2) ((1)Δ(g)) quencher.

Journal of Clinical Biochemistry and Nutrition


Kinetic study of the quenching reaction of singlet oxygen by carotenoids and food extracts in solution. Development of a singlet oxygen absorption capacity (SOAC) assay method.

A kinetic study of the quenching reaction of singlet oxygen (1O2) with eight kinds of carotenoids and α-tocopherol was performed in ethanol/chloroform/D2O (50:50:1, v/v/v) solution at 35 °C. The overall rate constants, kQ (=kq+kr, physical quenching+chemical reaction), for the reaction of carotenoids with 1O2 were measured, using the competition reaction method, where endoperoxide was used as a singlet oxygen generator, 2,5-diphenyl-3,4-benzofuran (DPBF) as an UV-vis absorption prove, and α-tocopherol as a standard compound. The rate constants, kQ (S) and kQ (t1/2), were determined by analyzing the first-order rate constant (S) and the half-life (t1/2) of the decay curve of DPBF with carotenoids, respectively, showing good accordance with each other. Similar measurements were performed for tomato and carrot extracts. From the results, a new assay method that can quantify the singlet oxygen absorption capacity (SOAC) of antioxidants, including carotenoids, α-tocopherol, and vegetable extracts, has been proposed.

Journal of Agricultural and Food Chemistry


Quercetin Prevents Unloading-Derived Disused Muscle Atrophy by Attenuating the Induction of Ubiquitin Ligases in Tail-Suspension Mice.

The effects of quercetin (1) were investigated on disused muscle atrophy using mice that underwent tail suspension. Periodic injection of 1 into the gastrocnemius muscle suppressed muscle weight loss and ubiquitin ligase expression. Compound 1 reduced the enhancement of lipid peroxidation in the muscle. Injection of N-acetyl-l-cysteine, but not flavone (2), also prevented muscle weight loss and enhancement of lipid peroxidation. These findings demonstrate that 1 can prevent disused muscle atrophy by attenuating the expression of ubiquitin ligases and that such prevention originates from its antioxidant activity.


alpha-Oligoglucosylation of a sugar moiety enhances the bioavailability of quercetin glucosides in humans.

Dietary intake of quercetin is suggested to be potentially beneficial for the prevention of various diseases. We examined the effect of alpha-oligoglucosylation of the sugar moiety of quercetin monoglucoside on its bioavailability in humans. Enzymatically modified isoquercitrin (EMIQ) was prepared by enzymatic deglycosylation and the subsequent of alpha-oligoglucosylation of quercetin 3-O-beta-rutinode (rutin). The plasma level of quercetin metabolites was instantly increased by oral intake of EMIQ and its absorption efficiency was significantly higher than that of isoquercitrin (quercetin 3-O-beta-glucoside; Q3G), and rutin. The profile of plasma quercetin metabolites after EMIQ consumption did not differ from that after Q3G consumption. The apparent log P of EMIQ indicated that EMIQ is more hydrophilic than Q3G but less than quercetin 3,4'-O-beta-diglucoside. These data indicated that enzymatic alpha-oligoglucosylation to the sugar moiety is effective for enhancing the bioavailability of quercetin glucosides in humans.


Ingested quercetin but not rutin increases accumulation of hepatic beta-carotene in BALB/c mice.

Beta-carotene is a carotenoid with a range of reported health benefits besides vitamin A activity. If the enzymatic conversion of beta-carotene to retinal is suppressed in the digestive tract, residual beta-carotene that reaches the tissues increases. We evaluated the function of quercetin and rutin (quercetin-3-rutinoside) to increase the accumulation of beta-carotene in vitro and in vivo in BALB/c mice. When the conversion of beta-carotene by a preparation of the murine small intestine was measured in vitro, the addition of quercetin or rutin considerably inhibited the conversion. When the levels of hepatic beta-carotene and retinoids were measured among three groups of mice fed a diet supplemented with beta-carotene plus quercetin or rutin or beta-carotene alone (four to six mice per group), quercetin increased the level of beta-carotene and decreased the level of retinol, whereas rutin did not. These results demonstrate that quercetin can suppress the conversion of beta-carotene which develops in the cytosol of small intestinal epithelial cells, and that rutin whose rutinose-moiety prevents being absorbed in the small intestine cannot suppress the conversion in vivo. This study offers a novel insight into the interaction between flavonoids and carotenoids with respect to the health benefits from the latter.


Consultation clinics for complementary and alternative medicine at Japanese university hospitals: An analysis at Tokushima University Hospital

Here, we report on a Consultation Clinic for Complementary and Alternative Medicine (CAM) which we established at Tokushima University Hospital in July of 2007 with the aim of providing person-to-person information on CAM, though not CAM therapy itself. In December of 2008, we received 55 applications for consultation, 37% concerning health foods, 37% Japanese herbal medicine (Kampo), and 26% various other topics. The consultants (nutritionists and pharmacists) communicated individually with 38 applicants; malignancies (26%) and cardiovascular disease (24%) were the main underlying concerns. To promote the quality of consultation, data was collected by means of focus group interviews concerning the perspective of the consultants. Safe and effective use of CAM requires a network of communication linking individuals, consultation teams, physicians, primary care institutions and university hospitals. To advance this goal, we plan to broaden the efforts described herein. Our findings indicate that the specific role of the consultation clinic in promoting the scientific use of CAM merits further study.


Association of serum carotenoids and tocopherols with atopic diseases in Japanese children and adolescents.

The present study assessed whether serum carotenoids and tocopherols are associated with atopic diseases (eczema and asthma) in 10- and 13-yr-olds in a Japanese community. Of 2796 students attending schools in Shunan, Japan, in 2006, 396 students were randomly selected for this study using nested case-control design. Atopic diseases and dietary food intake were assessed using self-administered questionnaires, and serum antioxidants were analyzed using high-performance liquid chromatography. We found no associations between serum carotenoids and atopic diseases. However, odds ratios (OR)s for the third and fourth quartiles of serum alpha-tocopherol with atopic eczema were 0.33 (95% confidence interval: 0.15-0.73) and 0.36 (0.14-0.89), respectively, and the trend was negatively significant (P(trend) = 0.048). We did not find a significant association for asthma. In conclusion, serum alpha-tocopherol was negatively associated with the prevalence of eczema. Serum carotenoids did not show definitive protective effects in Japanese youth.


Evaluation of tolerable levels of dietary quercetin for exerting its antioxidative effect in high cholesterol-fed rats.

The tolerable level of dietary quercetin for exerting its antioxidative effect was evaluated in high cholesterol-fed rats, using quercetin-containing diets (31-1260 mg quercetin/kg body weight/day) and onion diets (19-94 mg quercetin aglycone equivalent/kg body weight/day), from the viewpoint of a safety assessment. After feeding for 4 weeks, the urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) levels of the quercetin-containing diet groups fed more than 157 mg quercetin/kg body weight/day were higher than the group fed a quercetin-free diet, although the plasma quercetin metabolite levels and plasma antioxidative activity were elevated depending on the amounts of quercetin or onion diet intake. No significant effect on body weight gain by quercetin-containing diets or onion diets was observed. However, ratios of the liver and kidney weights to the body weight were significantly increased in the quercetin-containing diet groups fed more than 314 mg and 157 mg quercetin/kg body weight/day, respectively, and in the onion diet groups fed more than 47 mg quercetin aglycone equivalent/kg body weight/day. These results indicated that the tolerable level for dietary quercetin for exerting its antioxidative effect was between 126 and 157 mg/kg/day for the quercetin diet and between 19 and 34 mg/kg/day for the onion diet.


Quercetin metabolites and protection against peroxynitrite-induced oxidative hepatic injury in rats.

Quercetin has strong antioxidant potency. Quercetin-3'-O-sulphate (Q3'S) and quercetin-3-O-glucuronide (Q3GA) are the main circulating metabolites after consumption of quercetin-O-glucoside-rich diets by humans. However, information about how these quercetin metabolites function in vivo is limited. Hence, this study evaluated the efficacy of Q3'S and Q3GA for the protection of oxidative injury using in vitro and in vivo experiments. Peroxynitrite-mediated hepatic injury in rats was induced by administration of galactosamine/lipopolysaccharide (GalN/LPS). Twenty-four hours after GalN/LPS treatment, plasma ALT and AST levels delta increased significantly. However, pretreatment with 4(G)-alpha-D-glucopyranosyl rutin, a quercetin glycoside (30 mg/kg body weight), prevented these increases and reduced nitrotyrosine formation, indicating that consumption of quercetin glycosides prevent oxidative hepatotoxicity. Moreover, physiological levels of Q3'S and Q3GA (1 microM) effectively prevented peroxynitrite-induced nitrotyrosine formation in human serum albumin in in vitro experiments. These findings indicate peroxynitrite-induced oxidative hepatotoxicity is protected by the in vivo metabolites of quercetin, Q3'S and Q3GA.


Carotenoid, tocopherol, and fatty acid biomarkers and dietary intake estimated by using a brief self-administered diet history questionnaire for older Japanese children and adolescents.

We investigated the association between nutrient biomarkers and dietary intake estimated using a brief self-administered dietary history questionnaire (BDHQ) for Japanese children and adolescents. Blood samples were collected from 398 subjects (5th graders of elementary school aged 10-11 y, and 2nd graders of secondary schools aged 13-14 y) randomly selected from among students in Shunan City, Japan, who were then required to answer two questionnaires. Spearman correlations were calculated between dietary intake and the corresponding biomarkers (serum carotenoids, tocopherols, and erythrocyte fatty acids). Correlations with beta-carotene and beta-cryptoxanthin were significant in the 13- and 14-y age group (r=0.220-0.333, p<0.030) and the 10- and 11-y age subgroup who answered the questionnaire with assistance (r=0.295-0.299, respectively, p=0.006). Consumption of green-yellow vegetables and fruits was significantly correlated with beta-carotene and beta-cryptoxanthin levels (r=0.205-0.341, p<0.047). In the 13- and 14-y age group, correlations with eicosapentaenoic and docosahexaenoic acids were between 0.215 and 0.473 (p<0.040). Total seafood intake was significantly correlated with marine n-3 polyunsaturated fatty acids (PUFAs; r=0.239-0.420, p<0.023). In the 10- and 11-y age subgroup who completed the questionnaire with assistance, seafood intake was significantly correlated with marine n-3 PUFAs (r=0.239-0.243, p<0.032). In conclusion, dietary intake assessed using the BDHQ reflects the corresponding biomarkers for 13- and 14-y-olds; however, when used for elementary school children, caution is necessary in interpreting the results.

Journal of Nutritional Science and Vitaminology


Suppressive effect of quercetin on acute stress-induced hypothalamic-pituitary-adrenal axis response in Wistar rats.

The flavonoid quercetin is considered to have beneficial effects on human health. We recently have shown that quercetin-enriched foods reduced the duration of immobility time in a rat forced swimming test, indicating that dietary quercetin is promising as an antidepressant-like factor, whereas its mechanism of action is poorly understood. The aim of this study is to investigate the effects of quercetin on water immersion-restraint (WIR), stress-induced hypothalamic-pituitary-adrenal (HPA) axis activation, which is a major component of stress response and plays an important role in the pathology of depression. Quercetin administration to rats significantly suppressed WIR stress-induced increase of plasma corticosterone and adrenocorticotropic hormone levels as well as the mRNA expression of corticotropin-releasing factor (CRF) in the hypothalamic region. In addition, quercetin modulated the DNA binding activities of glucocorticoid receptor and phosphorylated cyclic adenosine 3',5'-monophosphate (cAMP) response element binding protein as well as the phosphorylation of extracellular signal-regulated kinase 1/2 in the hypothalamic region, all of which are known to regulate the expression of CRF mRNA. Taken together, these results suggest that dietary quercetin attenuates the HPA axis activation by the suppression of the CRF mRNA expression.


Different profiles of quercetin metabolites in rat plasma: comparison of two administration methods.

The bioavailability of polyphenols in human and rodents has been discussed regarding their biological activity. We found different metabolite profiles of quercetin in rat plasma between two administration procedures. A single intragastric administration (50 mg/kg) resulted in the appearance of a variety of metabolites in the plasma, whereas only a major fraction was detected by free access (1% quercetin). The methylated/non-methylated metabolites ratio was much higher in the free access group. Mass spectrometric analyses showed that the fraction from free access contained highly conjugated quercetin metabolites such as sulfo-glucuronides of quercetin and methylquercetin. The metabolite profile of human plasma after an intake of onion was similar to that with intragastric administration in rats. In vitro oxidation of human low-density lipoprotein showed that methylation of the catechol moiety of quercetin significantly attenuated the antioxidative activity. These results might provide information about the bioavailability of quercetin when conducting animal experiments.

Bioscience, Biotechnology, and Biochemistry


Polyphenols prevent clinorotation-induced expression of atrogenes in mouse C2C12 skeletal myotubes.

Oxidative stress is a key factor in stimulating the expression of atrogenes, which are muscle atrophy-related ubiquitin ligases, in skeletal muscle, and it induces muscle atrophy during unloading. However, the effects of antioxidative nutrients on atrogene expression have not been demonstrated. We report on the inhibitory effects of polyphenols, such as epicatechin (EC), epicatechin gallate (ECg) and epigallocatechin gallate (EGCg) and quercetin, on atrogene expression up-regulated by three dimensional (3D)-clinorotation or glucocorticoid. These treatments markedly elevated the expression of atrogenes, including atrogin-1 and MuRF-1, in mouse C2C12 myoblasts and myotubes. Interestingly, EC, ECg, EGCg and quercetin significantly decreased the expression of atrogin-1 and MuRF-1 up-regulated by 3D-clinorotation, whereas they hardly affected atrogene expression induced by dexamethasone. ERK signaling is a well known MAPK pathway to mediate oxidative stress. Therefore, we also investigated the effect of these polyphenols on phosphorylation of ERK in C2C12 myotubes. As expected, EC, ECg, EGCg, and quercetin significantly suppressed phosphorylation of ERK, corresponding to the up-regulation of atrogenes induced by 3D-clinorotation. These results suggest that antioxidative nutrients, such as catechins and quercetin, suppress atrogene expression in skeletal muscle cells, possibly through the inhibition of ERK signaling. Thus, catechins and quercetin may prevent unloading-mediated muscle atrophy.

The Journal of Medical Investigation : JMI


(-)-Epicatechin gallate accumulates in foamy macrophages in human atherosclerotic aorta: implication in the anti-atherosclerotic actions of tea catechins.

The localization and target sites of tea catechins underlying their biological activity including anti-atherosclerotic activity have not yet been fully understood. To identify the target sites of catechins in vivo, we have developed a novel monoclonal antibody (mAb5A3) specific for (-)-epicatechin-3-gallate (ECg), one of the major tea catechins. The immunoreactive materials with mAb5A3 were detected in the human atherosclerotic lesions but not in the normal aorta, and were specifically localized in the macrophage-derived foam cells. In vitro experiments using macrophage-like cell lines also showed the significant accumulation of ECg in the cells. We also demonstrated that ECg could suppress the gene expression of a scavenger receptor CD36, a key molecule for foam cell formation, in macrophage cells. These results, for the first time, showed the target site of a tea component ECg in the aorta and might provide a mechanism for the anti-atherosclerotic actions of the catechins.

Biochemical and Biophysical Research Communications


Peroxidized cholesterol-induced matrix metalloproteinase-9 activation and its suppression by dietary beta-carotene in photoaging of hairless mouse skin.

The activation of matrix metalloproteinase (MMP)-9 leading to the formation of wrinkle and sagging of skin is an essential step in the skin photoaging on exposure to ultraviolet A (UVA). This study attempted to elucidate the role of peroxidized cholesterol including cholesterol hydroperoxides (Chol-OOHs), primary products of lipid peroxidation in biomembranes, in MMP-9 activation and the effect of dietary beta-carotene in MMP-9 activation. Hairless mice were subjected to periodic UVA irradiation for 8 weeks. The amount of peroxidized cholesterol detected as total hydroxycholesterol in the skin was increased significantly by the exposure. The activity and protein level of MMP-9 were elevated with wrinkling and sagging formation. MMP-9 activity was also enhanced by the intracutaneous injection of Chol-OOHs into the mouse skin. Adding beta-carotene to the diet of the mice during the period of irradiation suppressed the activity and expression of MMP-9 as well as the wrinkling and sagging formation. The amount of cholesterol 5alpha-hydroperoxide, a singlet molecular oxygen oxygenation-specific peroxidized cholesterol, was significantly lowered by the addition of beta-carotene to the diet. These results strongly suggest that Chol-OOHs formed on exposure to UVA contribute to the expression of MMP-9, resulting in photoaging. Dietary beta-carotene prevents the expression of MMP-9, at least partly, by inhibiting photodynamic action involved in the formation of Chol-OOHs.


Flavonoids as Substrates and Inhibitors of Myeloperoxidase : Molecular Actions of Aglycone and Metabolites

Myeloperoxidase (MPO), secreted by activated neutrophils and macrophages at the site of inflammation, may be implicated in the oxidation of protein/lipoprotein during the development of cardiovascular diseases. Flavonoids have been suggested to act as antioxidative and anti-inflammatory agents in vivo; however, their molecular actions have not yet been fully understood. In this study, we examined the molecular basis of the inhibitory effects of dietary flavonoids, such as quercetin, and their metabolites on the catalytic reaction of MPO using a combination of biological assays and theoretical calculation studies. Immunohistochemical staining showed that a quercetin metabolite was colocalized with macrophages, MPO, and dityrosine, an MPO-derived oxidation product of tyrosine, in human atherosclerotic aorta. Quercetin and the plasma metabolites inhibited the formation of dityrosine catalyzed by the MPO enzyme and HL-60 cells in a dose-dependent manner. Spectrometric analysis indicated that quercetin might act as a cosubstrate of MPO resulting in the formation of the oxidized quercetin. Quantitative structure-activity relationship studies showed that the inhibitory actions of flavonoids strongly depended not only on radical scavenging activity but also on hydrophobicity (log P). The requirement of a set of hydroxyl groups at the 3, 5, and 4'-positions and C2-C3 double bond was suggested for the inhibitory effect. The binding of quercetin and the metabolites to a hydrophobic region at the entrance to the distal heme pocket of MPO was also proposed by a computer docking simulation. The current study provides the structure-activity relationships for flavonoids as the anti-inflammatory dietary constituents targeting the MPO-derived oxidative reactions in vivo.


Antidepressant-like Effect of Ginseng (Panax ginseng C.A. Meyer) in Behavioral Models

Journal of Clinical Biochemistry and Nutrition


Multitargeted cancer prevention by quercetin.

Quercetin is an anti-oxidative flavonoid widely distributed in the plant kingdom. Phenolic hydroxyl groups at the B-ring and the 3-position are responsible for its free radical-scavenging activity. Quercetin is commonly present as a glycoside and is converted to glucuronide/sulfate conjugates during intestinal absorption and only conjugated metabolites are therefore found in circulating blood. Although metabolic conversion attenuates its biological effects, active aglycone may be generated from the glucuronide conjugates by enhanced beta-glucuronidase activity during inflammation. With respect to its relationship with molecular targets relevant to cancer prevention, quercetin aglycone has been shown to interact with some receptors, particularly an aryl hydrocarbon receptor, which is involved in the development of cancers induced by certain chemicals. Quercetin aglycone has also been shown to modulate several signal transduction pathways involving MEK/ERK and Nrf2/keap1, which are associated with the processes of inflammation and carcinogenesis. Rodent studies have demonstrated that dietary administration of this flavonol prevents chemically induced carcinogenesis, especially in the colon, whilst epidemiological studies have indicated that an intake of quercetin may be associated with the prevention of lung cancer. Dietary quercetin is, therefore, a promising agent for cancer prevention and further research is warranted.


Chiisanoside is not absorbed but inhibits oil absorption in the small intestine of rodents.

Chiisanoside is the main component of Acanthopanax sessiliflorus leaves. Simultaneous administration of chiisanoside resulted in a decrease in the plasma TG level and increase of undigested TG in the intestinal lumen after oil gavage to mice. This suggests that chiisanoside has the potential to prevent obesity as a lipase inhibitor which suppresses fat absorption in vivo.

Bioscience, Biotechnology, and Biochemistry


Occurrence of singlet oxygen oxygenation of oleic acid and linoleic acid in the skin of live mice.

To assess the contribution of singlet molecular oxygen [O(2) ((1)Delta(g))] to lipid peroxidation in vivo, this study combined gas chromatography-mass spectrometry with thin layer chromatography to analyse peroxidized lipids in the skin of hairless mice. Hydroxyoctadecenoate isomers and unconjugated hydroxyoctadecadienoate isomers derived from peroxidized oleic acid and linoleic acid, respectively, which are specific to O(2) ((1)Delta(g))-dependent oxygenation, were detected in the skin of live mice under ordinary feeding conditions. Short-term ultraviolet A (UVA)-irradiation of the skin in vivo elevated levels of the unconjugated hydroxyoctadecadienoate isomers significantly, whereas the irradiation of skin homogenate in vitro increased levels of all isomers derived from both O(2) ((1)Delta(g)) and free radical-dependent oxygenation to a much greater extent. This is the first report to demonstrate the occurrence of O(2) ((1)Delta(g))-specific oxygenation of unsaturated fatty acids in living animals.


Immunochemical detection of flavonoid glycosides: development, specificity, and application of novel monoclonal antibodies.

Flavonoid-rich diets are expected to decrease the risk of cardiovascular diseases. The localization and target sites of flavonoids underlying the protective mechanism in vivo have not been fully investigated because the methods for detection of flavonoids have been limited to chemical analysis such as high-performance liquid chromatography. To further understand the actions of flavonoids in vivo, we developed a novel methodology that immunochemically evaluates flavonoids using specific antibodies. Quercetin-3-glucuronide (Q3GA), a major metabolite in human plasma, was coupled with keyhole limpet hemocyanin. Alternatively, the sugar moiety of quercetin-3-glucoside (Q3G) was succinylated and then coupled with a carrier protein. Using these two immunogens, we finally obtained two monoclonal antibodies, mAb14A2 and mAb11G6, from the immunogen using Q3GA and Q3G, respectively. Competitive enzyme-linked immunosorbent assay showed the unique difference in the specificity between the two similar antibodies: mAb14A2 recognized several quercetin-3-glycosides including Q3G and rutin but mAb11G6 was highly specific to the Q3G structure. The macrophage-derived foam cells in human atherosclerotic lesions were significantly stained with mAb14A2 but scarcely with mAb11G6. These results showed that the anti-flavonoid glycoside antibodies are useful tools for evaluating their localization in tissues and that the specificities strongly depend on the immunogen design for synthesizing the hapten-protein conjugates.


Macrophage as a target of quercetin glucuronides in human atherosclerotic arteries: implication in the anti-atherosclerotic mechanism of dietary flavonoids.

Epidemiological studies suggest that the consumption of flavonoid-rich diets decreases the risk of cardiovascular diseases. However, the target sites of flavonoids underlying the protective mechanism in vivo are not known. Quercetin represents antioxidative/anti-inflammatory flavonoids widely distributed in the human diet. In this study, we raised a novel monoclonal antibody 14A2 targeting the quercetin-3-glucuronide (Q3GA), a major antioxidative quercetin metabolite in human plasma, and found that the activated macrophage might be a potential target of dietary flavonoids in the aorta. Immunohistochemical studies with monoclonal antibody 14A2 demonstrated that the positive staining specifically accumulates in human atherosclerotic lesions, but not in the normal aorta, and that the intense staining was primarily associated with the macrophage-derived foam cells. In vitro experiments with murine macrophage cell lines showed that the Q3GA was significantly taken up and deconjugated into the much more active aglycone, a part of which was further converted to the methylated form, in the activated macrophages. In addition, the mRNA expression of the class A scavenger receptor and CD36, which play an important role for the formation of foam cells, was suppressed by the treatment of Q3GA. These results suggest that injured/inflamed arteries with activated macrophages are the potential targets of the metabolites of dietary quercetin. Our data provide a new insight into the bioavailability of dietary flavonoids and the mechanism for the prevention of cardiovascular diseases.


Antidepressant-like effect of onion (Allium cepa L.) powder in a rat behavioral model of depression.

The present study evaluated the antidepressant-like effect of the quercetin-rich vegetable, onion, by using the rat behavioral model of depression, the forced swimming test (FST). Daily administration of onion powder at a dosage of 50 mg/kg of body weight/day for 14 days significantly reduced the immobility time in FST without changing the motor dysfunction, indicating that the daily consumption of onion exerted antidepressant-like activity. The plasma corticosterone level was elevated after an FST trial, and pretreatment with onion powder did not modulate this elevation. Although the FST trial tended to increase the dopaminergic activity in the rat hypothalamus, the administration of onion powder (50 mg/kg) suppressed the increase in the turnover of this neurotransmitter. However, the same prevention was also observed with a higher dosage of onion, in which no significant antidepressant effect was apparent. The results of the present study suggest that onion exerted antidepressant-like activity in a behavioral model that acted independently of the hypothalamic-pituitary-adrenal axis.


Vegetable flavonoids and cardiovascular disease.

Studies have suggested that dietary flavonoids are helpful in the prevention of atherosclerosis and cardiovascular disease. Antioxidant activity should be noted as underlying mechanism of their health impact in the vascular system, as atherosclerosis is closely related to oxidative events such as oxidized LDL accumulation in the macrophages. Vegetables contain a variety of flavonoids, such as flavonols, flavones and anthocyanidins. We focused on quercetin (3,3',4',5,7- pentahydroxyflavone), a major flavonoid in onion, and its anti-atherosclerotic effect was examined from the aspect of the bioavailability and translocation to the target site. Although quercetin exists as its glucoside form in onion, it is metabolized into several glucuronides and/or sulfate conjugates with or without methylation during its intestinal absorption. We found that these metabolites circulating in the human blood stream were mostly localized in plasma albumin fraction, but not LDL fraction. Onion consumption failed to enhance the antioxidant activity of plasma fraction against LDL oxidation, indicating that the level of quercetin metabolites bound to albumin is insufficient to exert the antioxidative effect in vivo. In contrast, we discovered that quercetin metabolites accumulate in the aorta tissue and exerted their antioxidant activity, when rabbits were fed with quercetin glucoside and high cholesterol diet. Furthermore, quercetin metabolites were detected in human atherosclerotic aorta exclusively. These imply that quercetin metabolites are incorporated into the atherosclerotic region and act as complementary antioxidants, when oxidative stress is loaded in the vascular system. It is likely that plasma albumin is a carrier for translocation of quercetin metabolites to vascular target.

Asia Pacific Journal of Clinical Nutrition


Effect of excessive intake of quercetin on the vitamin E level and antioxidative enzyme activities of mouse liver under paraquat-induced oxidative stress

The liver alpha-tocopherol level of the paraquat fed mice group was lower than that of the control diet-fed group. An excessive intake of quercetin lowered the liver alpha-tocopherol level of the control diet-fed mice group, but did not affect it in the paraquat-fed mice group. The same quercetin intake significantly increased the superoxide dismutase and glutathione peroxidase activities in the liver of both groups, indicating that excessive quercetin intake can either promote or attenuate oxidative stress in the liver.


Combination of TLC blotting and gas chromatography-mass spectrometry for analysis of peroxidized cholesterol

We have established a sensitive and convenient method for analysis of cholesterol hydroperoxides (Chol-OOHs) as trimethylsilyloxyl derivatives using diphenylpyrenylphosphine (DPPP)-thin-layer chromatography (TLC) blotting and gas chromatography-electron ionization-mass spectrometry/selected-ion monitoring (GC-EI-MS/SIM). Chol-OOH standards were prepared by photosensitized oxidation and azo radical-induced peroxidation of cholesterol. Trimethylsilyloxyl derivatives of cholesterol 5alpha-hydroperoxide (Chol 5alpha-OOH), cholesterol 7alpha-hydroperoxide (Chol 7alpha-OOH), and cholesterol 7beta-hydroperoxide (Chol 7beta-OOH) could be separated from one another in the SIM chromatogram using a fragment ion with elimination of trimethylsilanol from the molecular ion. This method was used to characterize peroxidized cholesterol from azo radical-exposed human low-density lipoprotein and UVA-irradiated human keratinocytes in the presence of hematoporphyrin. Finally, we succeeded in the quantification of each Chol-OOH isomer present in hairless mouse skin with and without UVA irradiation by use of beta-sitosterol hydroperoxide as internal standard. The accumulation of Chol 5alpha-OOH with Chol 7alpha/betaOOH in the skin indicates that singlet molecular oxygen ((1)O(2)) participated in the peroxidation of skin cholesterol, because Chol 5alpha-OOH is known to be a (1)O(2) specific cholesterol peroxidation product. We concluded that the combination of DPPP-TLC blotting and GC-EI-MS/SIM is useful for quantifying peroxidized cholesterol in biological samples and confirming the participation of (1)O(2) in oxidative stress.


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-beta-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.

The Journal of Medical Investigation : JMI