Absorption and Metabolism of Luteolin in Rats and Humans in Relation to in Vitro Anti-inflammatory Effects.

Luteolin is a flavonoid present in plants in the form of aglycone or glucosides. In this study, luteolin glucosides (i.e., luteolin-7- O-ß-d-glucoside, luteolin-7- O-[2-(ß-d-apiosyl)-ß-d-glucoside], and luteolin-7- O-[2-(ß-d-apiosyl)-6-malonyl-ß-d-glucoside]) prepared from green pepper leaves as well as luteolin aglycone were orally administered to rats. Regardless of the administered luteolin form, luteolin glucuronides were mainly detected from plasma and organs. Subsequently, luteolin aglycone, the most absorbed form of luteolin in rats, was orally administered to humans. As a result, luteolin-3'- O-sulfate was mainly identified from plasma, suggesting that not only luteolin form but also animal species affect the absorption and metabolism of luteolin. When LPS-treated RAW264.7 cells were treated with luteolin glucuronides and luteolin sulfate (the characteristic metabolites identified from rats and humans, respectively), the different luteolin conjugates were metabolized in different ways, suggesting that such difference in metabolism results in their difference in anti-inflammatory effects.

Metabolic Fate of Luteolin in Rats: Its Relationship to Anti-inflammatory Effect.

Luteolin is a naturally occurring flavone that reportedly has anti-inflammatory effects. Because most luteolin is conjugated following intestinal absorption, free luteolin is likely present at low levels in the body. Therefore, luteolin metabolites are presumably responsible for luteolin bioactivity. Here we confirmed that luteolin glucuronides, especially luteolin-3'-O-glucuronide, are the major metabolites found in plasma after oral administration of luteolin (aglycone) or luteolin glucoside (luteolin-7-O-glucoside) to rats. Luteolin-4'-O-glucuronide and luteolin-7-O-glucuronide were also detectable together with luteolin-3'-O-glucuronide in the liver, kidney, and small intestine. Next, we prepared these luteolin glucuronides and compared the anti-inflammatory effects of luteolin and luteolin glucuronides on gene expression in lipopolysaccharide-treated RAW264.7 cells. Luteolin glucuronides, especially luteolin-7-O-glucuronide, reduced expression of inflammatory genes in the cells, although their effects were weaker than those of luteolin. These results indicate that the active compound responsible for the anti-inflammatory effect of luteolin in vivo would be luteolin glucuronide and/or residual luteolin.

Quercetin suppresses immune cell accumulation and improves mitochondrial gene expression in adipose tissue of diet-induced obese mice.

SCOPE: To examine the effect of dietary quercetin on the function of epididymal adipose tissue (EAT) in Western diet-induced obese mice. METHODS AND RESULTS: C57BL/6J mice were fed a control diet; a Western diet high in fat, cholesterol, and sucrose; or the same Western diet containing 0.05% quercetin for 18 weeks. Supplementation with quercetin suppressed the increase in the number of macrophages, the decrease in the ratio of CD4(+) to CD8(+) T cells in EAT, and the elevation of plasma leptin and tumor necrosis factor α levels in mice fed the Western diet. Comprehensive gene expression analysis revealed that quercetin suppressed gene expression associated with the accumulation and activation of immune cells, including macrophages and lymphocytes in EAT. It also improved the expression of the oxidative stress-sensitive transcription factor NFκB, NADPH oxidases, and antioxidant enzymes. Quercetin markedly increased gene expression associated with mitochondrial oxidative phosphorylation and mitochondrial DNA content. CONCLUSION: Quercetin most likely universally suppresses the accumulation and activation of immune cells, including antiinflammatory cells, whereas it specifically increased gene expression associated with mitochondrial oxidative phosphorylation. Suppression of oxidative stress and NFκB activity likely contributed to the prevention of the accumulation and activation of immune cells and resulting chronic inflammation.

Isoenzyme-specific up-regulation of glutathione transferase and aldo-keto reductase mRNA expression by dietary quercetin in rat liver.

The impact of quercetin on the mRNA expression of hepatic enzymes involved in drug metabolism was evaluated with a DNA microarray and real-time PCR. Male Sprague-Dawley rats were fed an experimental diet containing either 0, 2.5, 5, 10, or 20 g/kg of quercetin for 15 days. The DNA microarray analysis of the gene expression profile in pooled RNA samples from rats fed diets containing 0, 5, and 20 g/kg of quercetin revealed genes of some isoenzymes of glutathione transferase (Gst) and aldo-keto reductase (Akr) to be activated by this flavonoid. Real-time PCR conducted with RNA samples from individual rats fed varying amounts of quercetin together with the microarray analysis showed that quercetin caused marked dose-dependent increases in the mRNA expression of Gsta3, Gstp1, and Gstt3. Some moderate increases were also noted in the mRNA expression of isoenzymes belonging to the Gstm class. Quercetin also dose-dependently increased the mRNA expression of Akr1b8 and Akr7a3. However, it did not affect the parameters of the other Gst and Akr isoenzymes. It is apparent that quercetin increases the mRNA expression of Gst and Akr involved in drug metabolism in an isoenzyme-specific manner. Inasmuch as Gst and Akr isoenzymes up-regulated in their gene expression are involved in the prevention and attenuation of cancer development, this consequence may account for the chemopreventive propensity of quercetin.

Isolation of an isoflavone-metabolizing, Clostridium-like bacterium, strain TM-40, from human faeces.

Recently, the biological effects of isoflavones have attracted much attention. Intestinal microbiota plays an important role in the metabolism and bioavailability of isoflavones. However, few reports have discussed intestinal bacteria that metabolize daidzein into dihydrodaidzein. In this study, we isolated the dihydrodaidzein-producing intestinal bacterium TM-40 from a healthy boy's faeces. The bacteria from faecal samples were incubated with daidzein. Among all tested bacteria, one strain (strain TM-40) produced dihydrodaidzein both from daidzein and daidzin. However, in our experimental conditions, strain TM-40 did not produce equol from daidzein. The 16S rRNA partial sequence of strain TM-40 (AB249652) exhibited a 93% similarity to that of Coprobacillus catenaformis (AB030218). This strain seems to be a new species.

Antiproliferative effect on human cancer cell lines after treatment with nimbolide extracted from an edible part of the neem tree (Azadirachta indica).

Nimbolide, a triterpenoid extracted from the flowers of the neem tree (Azadirachta indica), was found to have antiproliferative activity against some cancer cell lines. Treatment of cells with 0.5-5.0 microm concentrations of nimbolide resulted in moderate to very strong growth inhibition in U937, HL-60, THP1 and B16 cell lines. Flow cytometric analysis of U937 cells showed that nimbolide treatment (1-2.5 microm) resulted in cell cycle disruption by decreasing the number of cells in G0/G1 phase, with initial increases in S and G2/M phases. Cells exposed to a higher dose of nimbolide for a longer period displayed a severely damaged DNA profile, resulting in a remarkable increase in the number of cells in the sub-G1 fraction, with a reciprocal decrease of cells in all phases. Quantification of the expression of phosphatidylserine in the outer cell membrane showed that doses of nimbolide higher than 0.4 microm exerted remarkable lethality, with over 60% of cells exhibiting apoptotic features after exposure to 1.2 microm nimbolide. The antiproliferative effect of nimbolide and its apoptosis-inducing property raise hope for its use in anticancer therapy by enhancing the effectiveness of cell cycle disruption.

Comparative studies of some phenolic compounds (quercetin, rutin, and ferulic acid) affecting hepatic fatty acid synthesis in mice.

The physiological activities of some phenolic compounds affecting hepatic fatty acid synthesis in mice were compared. Male ICR mice were fed an experimental diet containing 1% quercetin dihydrate, rutin, or ferulic acid or a control diet free of phenolic compounds for 15 days. Quercetin significantly lowered serum cholesterol and phospholipid levels in mice. Also, the serum triacylglycerol level was considerably lower in mice fed the quercetin-containing diet than in those fed a diet free of phenolic compounds, although the difference was not significant. Rutin and ferulic acid did not affect these parameters. Quercetin significantly reduced the activity and mRNA levels of various enzymes involved in hepatic fatty acid synthesis. Rutin reduced a few of the parameters for lipogenesis, but ferulic acid did not affect any of the parameters. It was suggested that a reduction in hepatic lipogenesis is the mechanism underlying the hypolipidemic effect of quercetin.

Inhibition of colon cancer (HT-29) cell proliferation by a triterpenoid isolated from Azadirachta indica is accompanied by cell cycle arrest and up-regulation of p21.

Nimbolide, a natural triterpenoid present in the edible parts of the neem tree ( Azadirachta indica), was found to be growth-inhibitory in human colon carcinoma HT-29 cells. Nimbolide treatment of cells at 2.5 - 10 microM resulted in moderate to very strong growth inhibition. Flow cytometric analysis of HT-29 cells showed that nimbolide treatment (2.5 microM, 12 h) caused a 6.5-fold increase in the number of cells (55.6 %) in the G2/M phase compared with the control cells (8.8 %). At 48 h, the cell population in the G2/M phase decreased to 18 %, while that in the G0/G1 phase increased to 52.3 %. Western blot analysis revealed that nimbolide-mediated G2/M arrest was accompanied by the up-regulation of p21, cyclin D2, Chk2; and down-regulation of cyclin A, cyclin E, Cdk2, Rad17. At G0/G1 cell cycle arrest, modulation in the expression of the cell cycle regulatory molecules was also observed. We found that nimbolide-induced growth inhibition and cell cycle arrest were not associated with cellular differentiation. Quantification of cells with respect to the expression of phosphatidylserine in the outer cell membrane showed an increase in apoptotic cells by about 13 % after 48 h of nimbolide treatment.

Apoptosis, necrosis and cell proliferation-inhibition by cyclosporine A in U937 cells (a human monocytic cell line).

The immunosuppressive drug cyclosporine A (CsA) has been used in both organ transplantation and the treatment of autoimmune disorders. However, the drug causes adverse effects in the kidney, liver and nervous system, characterized by cellular loss in the affected area. Apoptosis has been shown to play a role in CsA-induced cytotoxicity. Because permeabilization of the mitochondrial membrane is a common criterion in most apoptotic settings in vertebrate cells, here we evaluated whether CsA causes loss of mitochondrial function in the pathway leading to cellular cytotoxicity. We found that CsA caused a concentration- and time-dependent loss of cell viability in the U937 cell line. Treatment of cells at a dose of 10 microM CsA resulted in G0/G1 arrest with a concurrent decrease in the number of cells in the S and G2/M phases of the cell cycle. In mechanistic studies related to the loss of viability, treating cells with 10 microM CsA for 24 h resulted in both DNA fragmentation and an increase of annexin-V-positive cells. CsA treatment also increased activity of the cysteine protease caspase-3, decreased the mitochondrial membrane potential and induced the release of cytochrome c into the cytosol. Furthermore, CsA treatment increased the number of cells in the sub-G0/G1 peak, indicative of a reduction in DNA, although this increase was not observed when cells were pre-treated with a broad caspase inhibitor. In the study, we also found that a higher dose of CsA induces LDH release when the cells were incubated for a longer period. Taken together, these data suggest that the mode of cell death induced by CsA is dose- and time-dependent. Short-term incubation with lower doses of CsA arrests cell growth; this arrest overlaps with the occurrence of apoptosis and then with necrosis after longer treatment periods with higher doses of CsA.

Procyanidin trimers to pentamers fractionated from apple inhibit melanogenesis in B16 mouse melanoma cells.

The effects of apple polyphenols on melanogenesis in B16 mouse melanoma cells were investigated. The inhibitory effect of apple polyphenols was stronger than that of arbutin or kojic acid. Three polyphenol fractions (phenolic acid derivatives, procyanidins and other flavonoids) were isolated, and the procyanidins were fractionated according to the degree of polymerization using normal-phase chromatography. The procyanidin trimer-to-pentamer fractions were found to have the most pronounced effect on melanogenesis. Furthermore, each procyanidin fraction inhibited mushroom tyrosinase. No correlation between the degree of procyanidin polymerization and tyrosinase inhibitory activity was observed. Nevertheless, these observations suggest that procyanidins are effective inhibitors of tyrosinase.

Coumarin and flavone derivatives from estragon and thyme as inhibitors of chemical mediator release from RBL-2H3 Cells.

Estragon and thyme extracts showed potent inhibitory activities against chemical mediator release from rat basophilic leukemia RBL-2H3 cells. 7-Methoxycoumarin was isolated from estragon, and 5,4'-dihydroxy-6,7,3'-trimethoxyflavone, 5,4'-dihydroxy-6,7,8,3'-tetramethoxyflavone, 5-hydroxy-6,7,8,3',4'-pentamethoxyflavone and luteolin were isolated from thyme as active components. Structure-activity relationship studies among the active isolates and their related compounds indicated that the oxygen-containing functional group at the 7-position of the coumarin structure was advantageous for the inhibitory activity and that methylation of the hydroxyl group at the 4'-position of the flavone structure was disadvantageous. It was also found that coumarin derivatives inhibited an earlier step than intracellular calcium release and proteinkinase C activation, while flavones inhibited a later step or both earlier and later steps.

Quercetin-4'-glucoside is more potent than quercetin-3-glucoside in protection of rat intestinal mucosa homogenates against iron ion-induced lipid peroxidation.

Quercetin is a typical antioxidative flavonoid found in vegetables, which is more commonly present as its glucosides, quercetin-3-glucoside (Q3G) and quercetin-4'-glucoside (Q4'G). The main aim of this study was to estimate the antioxidant activity of Q3G and Q4'G on iron ion-driven lipid peroxidation of the gastrointestinal mucosa. Q4'G markedly suppressed the lipid peroxidation when rat gastrointestinal mucosa homogenates were incubated with Fe(NO3)3 and ascorbic acid. Its effectiveness was greater as compared to that of Q3G and comparable to that of quercetin aglycone. Furthermore, Q4'G yielded higher amounts of quercetin aglycone than Q3G on incubation with the homogenates. However, Q4'G showed a lower chelating activity in comparison to Q3G. These results indicate that Q4'G, even though it has a low chelating activity, because of its efficient conversion to antioxidative aglycone on exposure to the mucosa, can act as a powerful antioxidant on iron ion driven lipid peroxidation in the intestinal mucosa. Thus, vegetables rich in Q4'G, such as onion, are likely to serve as favorable antioxidant sources for suppressing iron-induced oxidative stress in the intestinal tract.

Induction of apoptosis in cancer cells by Bilberry (Vaccinium myrtillus) and the anthocyanins.

Among ethanol extracts of 10 edible berries, bilberry extract was found to be the most effective at inhibiting the growth of HL60 human leukemia cells and HCT116 human colon carcinoma cells in vitro. Bilberry extract induced apoptotic cell bodies and nucleosomal DNA fragmentation in HL60 cells. The proportion of apoptotic cells induced by bilberry extract in HCT116 was much lower than that in HL60 cells, and DNA fragmentation was not induced in the former. Of the extracts tested, that from bilberry contained the largest amounts of phenolic compounds, including anthocyanins, and showed the greatest 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity. Pure delphinidin and malvidin, like the glycosides isolated from the bilberry extract, induced apoptosis in HL60 cells. These results indicate that the bilberry extract and the anthocyanins, bearing delphinidin or malvidin as the aglycon, inhibit the growth of HL60 cells through the induction of apoptosis. Only pure delphinidin and the glycoside isolated from the bilberry extract, but not malvidin and the glycoside, inhibited the growth of HCT116 cells.

An improved method for measuring vascular permeability in rat and mouse skin.

INTRODUCTION: We have improved a rodent vascular permeability measurement method employing fluorescent dye-labeled bovine serum albumin. METHODS: The incubation duration for direct fluorescent detection of skin injected with an inflammatory agent was decided based on regression curve parameters with the correlation coefficient obtained from the least squares method. RESULTS: A suitable incubation time was determined to be 2-6 h. The recovery of FITC-BSA from the skin sample was very good, and the correlation coefficient of the linear regression curve was .99. The linear relation between the previous dye extraction method using brilliant blue 6B and the new and improved fluorescence method was very high. In mice, histamine-induced serum exudation in the back skin increased from 0.31 to 1.25 microg/site in a dose-dependent manner and reached a plateau at 1.25-2.5 microg/site. The serum exudation caused by histamine increased to 10 microg/site and almost reached a plateau at 10-40 microg/site in rats. The time required for the measurement of fluorescence intensity was very short because a microplate reader was used as the measurement apparatus. CONCLUSION: The improved method is easy to use and sensitive and does not necessitate extraction of dye from the skin.