Cacao liquor proanthocyanidins inhibit lung injury induced by diesel exhaust particles.

Epidemiological and experimental studies have suggested that diesel exhaust particles (DEPs), which generate reactive oxygen species, may be involved in the recent increase in the prevalence of lung diseases. Cacao liquor proanthocyanidins (CPs) are naturally occurring polyphenols with antioxidative activities. We carried out a study in mice to investigate the effects of dietary supplementation of CPs on lung injury induced by intratracheal administration of DEPs (500 microg/body). Dietary supplementation with 1.0 percent CPs inhibited DEP-induced lung injury, characterized by neutrophil sequestration and edema. Immunohistochemical analyses showed that CPs prevented enhanced expression of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 caused by DEPs in the lung injury. Numerous adducts of nitrotyrosine, N-(hexanonyl) lysine, 4-hydroxy-2-nonenal, and 8-OHdG were also observed immunohistochemically in the lungs of mice treated with DEPs. However, these indicators of oxidative stress were barely visible in mice pretreated with CP supplementation. In addition, the level of thiobarbituric acid reactive substances in the lung was decreased by CP supplementation in the presence of DEPs. These results suggest that CPs inhibit DEP-induced lung injury by reducing oxidative stress, in association with a reduction in the expression of adhesion molecules.

Rosmarinic acid in perilla extract inhibits allergic inflammation induced by mite allergen, in a mouse model.

BACKGROUND: Perilla and its constituent rosmarinic acid have been suggested to have anti-allergic activity. However, few studies have examined the effects on allergic asthma. OBJECTIVE: The purpose of this study was to evaluate the effect of oral administration of perilla leaf extract, which contains high amount of rosmarinic acid, on a murine model of allergic asthma induced by house dust mite allergen. METHODS: C3H/He mice were sensitized by intratracheal administration of Dermatophagoides farinae (Der f). Mice were orally treated with rosmarinic acid in perilla extract (PE) (1.5 mg/mouse/day). RESULTS: Der f challenge of sensitized mice elicited pulmonary eosinophilic inflammation, accompanied by an increase in lung expression of IL-4 and IL-5, and eotaxin. Daily treatment with rosmarinic acid in PE significantly prevented the increases in the numbers of eosinophils in bronchoalveolar lavage fluids and also in those around murine airways. Rosmarinic acid in PE treatment also inhibited the enhanced protein expression of IL-4 and IL-5, and eotaxin in the lungs of sensitized mice. Der f challenge also enhanced allergen-specific IgG1, which were also inhibited by rosmarinic acid in PE. CONCLUSION: These results suggest that oral administration of perilla-derived rosmarinic acid is an effective intervention for allergic asthma, possibly through the amelioration of increases in cytokines, chemokines, and allergen-specific antibody.

Absorption and urinary excretion of (-)-epicatechin after administration of different levels of cocoa powder or (-)-epicatechin in rats.

(-)-Epicatechin is a major polyphenol component of cocoa powder. The absorption and urinary excretion of (-)-epicatechin following administration of different levels of either cocoa powder (150, 750, and 1500 mg/kg) or (-)-epicatechin (1, 5, and 10 mg/kg) were evaluated in rats. Both the sum of plasma (-)-epicatechin metabolites at 1 h postadministration and peak plasma concentrations increased in a dose-dependent fashion. The sum of (-)-epicatechin metabolites in urine, excreted within 18 h postadministration, also increased with dose. Moreover, the sum of (-)-epicatechin metabolites excreted in urine reached the same level in both (-)-epicatechin and cocoa powder administration groups for equivalent amounts of (-)-epicatechin. These results suggest that, in the dose range examined in this study, bioavailability of (-)-epicatechin following administration of either (-)-epicatechin or cocoa powder shows dose dependence and that the various compounds present in cocoa powder have little effect on the bioavailability of (-)-epicatechin in cocoa powder.

Anticlastogenic activity of cacao: inhibitory effect of cacao liquor polyphenols against mitomycin C-induced DNA damage.

Oxidative DNA damage has been implicated as a factor playing a role in mutagenesis and carcinogenesis. We investigated the anticlastogenic activity of cacao: the inhibitory effect of cacao liquor polyphenols on DNA strand cleavage induced by mitomycin C (MMC) in vitro and the anticlastogenic effect of cacao liquor extract against formation of micronuclei induced by MMC in bone marrow cells and peripheral blood cells of mice. In the DNA strand cleavage test, cacao liquor polyphenols inhibited cleavage of RFI DNA. In the micronuclei test, the frequency of occurrence of micronucleated cells among bone marrow cells and peripheral blood cells were reduced significantly when cacao liquor extract was administered orally to mice 6 h before intraperitoneal injection of MMC. These findings suggest that cacao liquor polyphenols are effective in preventing DNA damage, and one of the mechanisms of action might involve scavenging of active oxygen radicals generated in reactions initiated by MMC.

In vivo comparison of the bioavailability of (+)-catechin, (-)-epicatechin and their mixture in orally administered rats.

We compared levels of (+)-catechin, (-)-epicatechin, and their metabolites in rat plasma and urine after oral administration. Rats were divided into four groups and given (+)-catechin (CA group), (-)-epicatechin (EC group), a mixture of the two (MIX group) or deionized water. Blood samples were collected before administration and at designated time intervals thereafter. Urine samples were collected 0-24 h postadministration. (+)-Catechin, (-)-epicatechin and their metabolites in plasma and urine were analyzed by HPLC-mass spectrometry after treatment with beta-glucuronidase and/or sulfatase. After administration, absorbed (+)-catechin and (-)-epicatechin were mainly present in plasma as metabolites, such as nonmethylated or 3'-O-methylated conjugates. In the CA and MIX groups, the primary metabolite of (+)-catechin in plasma was glucuronide in the nonmethylated form. In the EC and MIX groups, in contrast, the primary metabolites of (-)-epicatechin in plasma were glucuronide and sulfoglucuronide in nonmethylated forms, and sulfate in the 3'-O-methylated forms. Urinary excretion of the total amount of (-)-epicatechin metabolites in the EC group was significantly higher than the amount of (+)-catechin metabolites in the CA group. The sum of (+)-catechin metabolites in the urine was significantly lower in the MIX group than in the CA group, and the sum of (-)-epicatechin metabolites in the MIX group was also significantly lower than in the EC group. These results suggest that the bioavailability of (-)-epicatechin is higher than that of (+)-catechin in rats, and that, in combination, (+)-catechin and (-)-epicatechin might be absorbed competitively in the gastrointestinal tract of rats.

[Teaching employees].

I explained stress education for employees around the following four points based on Guidelines for Mental Health Promotion at the Workplace. That is, understanding to employee's stress, the necessity for self-care, awareness of their stress condition and how to cope with a stress condition. Finally, the content (Introduction of relaxation, stress-check by using questionnaires, case-studies and Q and A) of the stress education which I was doing was presented.

Preparation of benzene, furan, and thiophene analogs of duocarmycin SA employing a newly-devised phenol-forming reaction.

Five A-ring analogs of duocarmycin SA 9a-e were synthesized in racemic form modifying our second synthetic route toward duocarmycin SA. The problem encountered at the crucial phenol forming step to secure 17a, b from 16a, b under the conventionally used Kuwajima conditions was overcome by devising a more convenient method: simple heating of 16a-c in benzene in the presence of bis(triphenylphosphine)palladium(II) chloride (10 mol%), cesium carbonate (3 eq), and triphenylphosphine (0.3 eq) gave 17a-c in high yields of 86-91%. The intermediates 17a-e were readily led to the A-ring analogs (+/-)-9a-e almost according to the reported route.

Antimutagenic activity of cacao: inhibitory effect of cacao liquor polyphenols on the mutagenic action of heterocyclic amines.

We investigated the effect of polyphenols derived from cacao liquor on the mutagenic action of heterocyclic amines (HCAs) in vitro and ex vivo. In the Ames test, the cacao liquor polyphenols showed antimutagenic effects in bacteria treated with HCA in the presence of an S-9 mixture; however, they showed less efficacy than quercetin. On the other hand, the cacao liquor polyphenols showed potent antimutagenic activity in bacteria treated with activated forms of HCA, compared with quercetin. We also evaluated the effect of these compounds on enzymatic activation of HCA. They weakly suppressed the production of activated HCA. In the host-mediated assay in mice, a method used to estimate the potential carcinogenicity of chemicals ex vivo, oral administration of the cacao liquor polyphenols, reduced the number of colonies of revertant bacteria recovered from the liver. These data suggest that the cacao liquor polyphenols have an antimutagenic effect not only in vitro, but also ex vivo.

[Stress assessment method for workers (Part 2)--The diagnostic analyses and judgements of stressful conditions of stress-dock examinees on the basis of the total score of numerical-valued stressors they have suffered for the past 1 year].

In order to evaluate the grade of reaction to stressors, especially those in the occupational life of workers, the following stress survey was conducted. This survey consisted of 65 stress questionnaires based on the social readjustment rating scale prepared by Holmes and Rahe, including 18 new questionnaires on the occupational environment. The method is as follows. That is, marriage is given a score of 50 in reference-standard for stress strength and these 65 items for 1,630 workers were evaluated by self-rating method ranging in score from 0 to 100. As for each item, we found the average value for the total sample and we called it the stress score. The subject group (1,426 employees who were examined for Stress-Dock) judged their experience during the past 1 year by their stress scores. The stress scores for the stressors experienced in a year were summed (total experienced stress score). We examined whether the total experienced stress score would be an index of the degree of stress, etc. We then analyzed the relationship between the total experienced stress score and stress conditions (i.e. "severe (hereafter, the assumed severe group)", "borderline (borderline group)" and "not severe (not severe group)"). In addition, the relationship between the total experienced stress score and mental disorders (stress related diseases were the majority) diagnosed by ICD-10 was examined. Our findings and conclusions are as follows: 1. We presented two typical cases. Next, the distribution of the total experienced stress score in all subjects was shown. 2. The average total experienced stress score increased from 135 to 219 points and 312 points in the order, not severe group, borderline group and severe group. It was paid noticeable that the severe group had scores 2.3 high times higher the not severe group. 3. The mental disorders group diagnosed with ICD-10 as mental disorders was large including 888 people, and the average total score for experienced stress score was a high 312 points. This was, as many as 94 points higher than in the normal group, and a significant difference was admitted by both. The difference of 1.1 point was admitted in the LCU numbers. Moreover, in statistical analysis, the score method showed a high significant difference according to F41 and F43 compared with the number of LCU items. We thought that the difference of the score method was more comprehensible than that of the number of LCU items as a result. 4. We took the possible total experienced stress score as 100 points, and examined the score for patients with mental disorders. It was 78.8% in the group with a high 400 points or more, but percentage for patients with mental disorders was only 39.3% in less than 100 points. The greater the point increases the higher the frequency of the appearance of disease. 5. We thought that we were able to measure the degree of worker's stress to obtain the total experienced stress score from life event in the above-mentioned way.

Teasterone-3-O-beta-D-glucopyranoside, a new conjugated brassinosteroid metabolite from lily cell suspension cultures and its identification in lily anthers.

The new brassinosteroid conjugate, teasterone-3-O-betaD-glucopyranoside, was found as a metabolite of teasterone in lily cell suspension cultures. Its structure was determined by means of FAB-MS and 1H-NMR upon comparison with the authentic compound. Furthermore, its presence in lily anthers was confirmed by FAB-MS and LC-APCI-SIM data. This is the first natural brassinosteroid conjugate glucosylated at a hydroxyl group in ring A.

Cocoa powder enhances the level of antioxidative activity in rat plasma.

The aims of the present study were to determine the level of (-)-epicatechin (EC) and its metabolites in rat plasma after oral administration of cocoa powder and to evaluate the protective effect of cocoa powder in terms of suppressing the oxidation of plasma components. Rats were orally administered 1 g cocoa powder/kg body weight, containing 7.80 mg EC, and their blood was collected before administration and at designated time intervals thereafter. The EC and its metabolites in plasma were treated with beta-glucuronidase and/or sulfatase, then analysed by HPLC and by liquid chromatography-MS. Several EC-related compounds were detected in plasma such as free EC, and glucuronide, sulfate, and glucuronide-sulfate conjugates of non-methylated or methylated EC. All EC metabolites showed a maximum concentration in plasma at 30-60 min post-administration. Glucuronide conjugates of both non-methylated and methylated EC were found in high concentration in plasma. Moreover, administration of cocoa powder significantly reduced the accumulation of lipid peroxides in plasma and significantly reduced the consumption of alpha-tocopherol in plasma oxidized by treatment with 2,2'-azobis-(2-amidinopropane) dihydrochloride (AAPH (25 mmol/l)) or CuSO(4) (100 micromol/l) compared with that in the case of plasma obtained before administration. The total EC concentration in plasma was negatively correlated with the level of accumulation of lipid peroxides in plasma oxidized by treatment with AAPH (25 mmol/l) and was positively correlated with the level of residual alpha-tocopherol in plasma oxidized by treatment with CuSO(4) (100 micromol/l). These results indicate that EC in cocoa powder was absorbed from the digestive tract, that various conjugated forms of EC were generated in the digestive tract and distributed to the plasma, and that these enhanced the antioxidative activity of plasma.

Antioxidant effects of polyphenols in chocolate on low-density lipoprotein both in vitro and ex vivo.

Cacao is rich in polyphenols such as (-)-epicatechin, and a colored component of cacao (cacao-red) is polyphenol, which is an antioxidant. These properties stimulated an investigation of the effects of cacao liquor polyphenols (CLP) on low-density lipoprotein (LDL) oxidation. The 2.2 '-azobis(4-methoxy-2,4-dimethylvaleronitrile) (AMVN-CH2O)-induced oxidizability of LDL was assessed by monitoring the absorbance at 234 nm. In vitro. 0.1-0.5 mg/dL CLP prolonged the oxidation lag time of LDL in a dose-dependent manner. Compared with the controls, it was prolonged 1.7-fold in the presence of 0.1 mg/dL CLP, 2.9-fold at 0.2 mg/dL, 3.8-fold at 0.3 mg/dL, 5.4-fold at 0.4 mg/dL, and 6.4-fold at 0.5 mg/dL. Furthermore, we enlisted 13 male volunteers to consume 35 g delipidated cocoa. Venous blood samples were taken before and at 2 h and 4 h after consuming the cocoa. The oxidation lag time of LDL before cocoa ingestion was 59.0 +/- 6.3 min, but it was prolonged at 2 h after cocoa (68.3 +/- 6.0 min); before returning to the initial lag time (61.7 +/- 5.7 min) before consumption. Thus we have shown that cocoa inhibited LDL oxidation both in vitro and ex vivo.

Analyses of polyphenols in cacao liquor, cocoa, and chocolate by normal-phase and reversed-phase HPLC.

The antioxidant polyphenols in cacao liquor, a major ingredient of chocolate and cocoa, have been characterized as flavan-3-ols and proanthocyanidin oligomers. In this study, various cacao products were analyzed by normal-phase HPLC, and the profiles and quantities of the polyphenols present, grouped by molecular size (monomers to approximately oligomers), were compared. Individual cacao polyphenols, flavan-3-ols (catechin and epicatechin), and dimeric (procyanidin B2), trimeric (procyanidin C1), and tetrameric (cinnamtannin A2) proanthocyanidins, and galactopyranosyl-ent-(-)-epicatechin (2alpha-->7, 4alpha-->8)-(-)-epicatechin (Gal-EC-EC), were analyzed by reversed-phase HPLC and/or HPLC/MS. The profile of monomers (catechins) and proanthocyanidin in dark chocolate was similar to that of cacao liquor, while the ratio of flavan-3-ols to the total amount of monomeric and oligomeric polyphenols in the case of pure cocoa powder was higher than that in the case of cacao liquor or chocolate.

Bioavailability of (-)-epicatechin upon intake of chocolate and cocoa in human volunteers.

We evaluated the levels of (-)-epicatechin (EC) and its metabolites in plasma and urine after intake of chocolate or cocoa by male volunteers. EC metabolites were analyzed by HPLC and LC/MS after glucuronidase and/or sulfatase treatment. The maximum levels of total EC metabolites in plasma were reached 2 hours after either chocolate or cocoa intake. Sulfate, glucuronide, and sulfoglucuronide (mixture of sulfate and glucuronide) conjugates of nonmethylated EC were the main metabolites present in plasma rather than methylated forms. Urinary excretion of total EC metabolites within 24 hours after chocolate or cocoa intake was 29.8+/-5.3%'; and 25.3+/-8.1% of total EC intake. EC in chocolate and cocoa was partly absorbed and was found to be present as a component of various conjugates in plasma, and these were rapidly excreted in urine.

Effects of cacao liquor polyphenols on the susceptibility of low-density lipoprotein to oxidation in hypercholesterolemic rabbits.

The effects of cacao liquor polyphenols (CLP) on the susceptibility of low-density lipoprotein (LDL) to oxidation in hypercholesterolemic rabbits were examined. Six Japanese white rabbits which had been fed a high cholesterol diet (HCD) for 3 weeks were fed HCD containing 1% CLP for the following 10 days. The susceptibility of LDL to oxidation induced by 2-2'-azobis(4-methoxy-2, 4-dimethylvaleronitrile) (V-70) was evaluated by measuring the production of conjugated dienes and thiobarbituric acid reactive substances (TBARS). The lag time was significantly prolonged from 37.7 min before intake of CLP to 42.9, 44.2 and 45.8 min after 4, 7 and 10 days of CLP intake. TBARS production after intake of CLP was also markedly reduced compared with the level before intake. There was no difference in plasma lipid concentrations comparing the levels before and after CLP intake. In conclusion, in hypercholesterolemic rabbits, orally administered CLP was absorbed and distributed to the blood, and the resistance of LDL to oxidation was thereby increased.

Detection of antifungal activity in Anemarrhena asphodeloides by sensitive BCT method and isolation of its active compound.

Antifungal activity was detected from Anemarrhena asphodeloides by the Bio-Cell Tracer (BCT) method. An active fraction was separated by silica gel column chromatography and reverse-phase HPLC. The molecular weight was determined by GC-MS, and the molecular structure was analyzed by IR, (1)H NMR, and (13)C NMR. The isolated compound was found to be identical to nyasol, (Z)-1, 3-bis(4-hydroxyphenyl)-1,4-pentadiene, which formerly appeared in the literature without any remark on the antifungal activity. This compound showed antimicrobial activity against 38 strains of fungi and five strains of bacteria. The minimum inhibitory concentration (MIC) ranged from 12.5 to 200 microg mL(-)(1), except for two strains based on the broth dilution method.

Attenuated liver fibrosis and depressed serum albumin levels in carbon tetrachloride-treated IL-6-deficient mice.

Chronic intermittent injection of carbon tetrachloride (CCl4) for more than 10 weeks induced liver fibrosis in mice, as evidenced by positive Azan staining and increased intrahepatic collagen content. Preceding the onset of liver fibrosis, interleukin-6 (IL-6) gene expression was enhanced in liver and immunoreactive IL-6 was detected in infiltrating inflammatory cells. To delineate the role of IL-6 in this process, we treated IL-6-deficient mice with CCl4 in a similar manner for 12 weeks, after which fibrotic changes were less evident and serum albumin levels were lower in IL-6-deficient than wild-type mice. Moreover, CCl4-induced expression of transforming growth factor beta1 and hepatocyte growth factor genes in liver was significantly reduced in IL-6-deficient mice. Thus, IL-6 may be vitally involved in fibrotic changes and maintenance of serum albumin levels, partly by modulating intrahepatic expression of these cytokines.

The antioxidative substances in cacao liquor.

The antioxidative substances contained in cacao liquor, which is one of the major ingredients of chocolate, were separated by column chromatography and high-performance liquid chromatography. Three major compounds were purified and two of them were identified by 1H, 13C NMR and mass spectra as (-)-epicatechin (EC) and (+)-catechin (CA). Their antioxidative activity was measured by monitoring the peroxide value of linoleic acid and the thiobarbituric acid-reactive substance values of erythrocyte ghost membranes and microsomes. EC and CA had strong antioxidative effects in all three methods, but one unidentified peak was found to be less effective. Additionally, we analyzed the polyphenol concentration of cacao liquor extractions produced in several countries. The total polyphenol concentration was 7.0 to 13.0%, catechin concentration was 0.31 to 0.49%, and epicatechin concentration was 0.35 to 1.68% in the extractions. It is believed that chocolate is stable against oxidative deterioration on account of the presence of these polyphenolic compounds, and it is also expected to have a protective role against lipid peroxidation in living systems.

Alleviation of monocrotaline-induced pulmonary hypertension by antibodies to monocyte chemotactic and activating factor/monocyte chemoattractant protein-1.

Administration of monocrotaline (MCT) causes pulmonary vascular lesions consisting of monocyte/macrophage infiltration in the early phase and medial thickening in pulmonary arteries and arterioles associated with pulmonary hypertension (PH) in the later phase. However, the molecular mechanism of monocyte/macrophage infiltration and its roles remain elusive. Herein, we have evaluated the role of a potent monocyte chemotactic and activating chemokine/monocyte chemoattractant protein-1 (MCAF/MCP-1) in MCT-induced PH in rats. A single injection of MCT induced PH at Day 21, as evidenced by increases in the ratio of right ventricular to left ventricular and septum weights (RV/LV+S) and right ventricular systolic pressure (RVSP). A significant increase in macrophage number in lungs started at Day 14, reaching a maximum at Day 21. MCAF/MCP-1 levels in bronchoalveolar lavage fluids were elevated significantly at Day 14 and remained high until Day 28, whereas plasma MCAF/MCP-1 levels increased at Day 7, returning to normal levels by Day 21. Immunoreactive MCAF/MCP-1 proteins were mainly detected in macrophages in alveoli and in perivascular regions of pulmonary arterioles and venules. Intravenous administration of anti-MCAF/MCP-1 antibodies with MCT significantly decreased macrophage infiltration and eventually reduced the increases in RV/LV+S and RVSP, as well as medial thickening of pulmonary arterioles. Thus, MCAF/MCP-1 is essentially involved in MCT-induced PH by recruiting and activating macrophages.