Effect of Flavanol-Rich Cacao Extract on the Profile of Mood State in Healthy Middle-Aged Japanese Women: A Randomized, Double-Blind, Placebo-Controlled Pilot Study.

To investigate the effects of flavanol-rich cacao extract on healthy middle-aged women's fatigue and mood conditions, we conducted a randomized, double-blind, placebo-controlled study in women aged 40-60 years who had reported fatigue and had shown high levels of a serum oxidative stress marker. We randomized the participants (n = 60) into equal groups receiving either a beverage containing cacao flavanols (240 mg/200 mL/day) or a placebo for 8 weeks. Before and after the 8-week treatment, we determined the participants' Chalder fatigue scale (CFS) scores, various mood states, autonomic nervous system (ANS) activity levels, and their ANS balance. The results demonstrated that among the mood states, the indicators of negative mood (e.g., depression, fatigue, and anger) and the total mood disturbance score were significantly lower in the cacao group compared to the placebo group after the treatment (p < 0.05). The change in the index of positive mood (i.e., vigor) from baseline to 8 weeks was significantly higher in the cacao group versus the placebo group (p < 0.05). There were no significant between-group differences in the changes in the CFS score or ANS activity level. The consumption of flavanol-rich cacao extract both suppressed negative moods and promoted positive moods in healthy middle-aged women. These results suggest that cacao flavanols may be a useful food material that can improve variable mood conditions in middle-aged women and support their active lives.

Cacao liquor procyanidins prevent postprandial hyperglycaemia by increasing glucagon-like peptide-1 activity and AMP-activated protein kinase in mice.

Procyanidins have been reported to possess potential for the prevention of hyperglycaemia. However, there are very few data for procyanidins about the difference the degree of polymerisation (DP) has on anti-hyperglycaemic effects. Moreover, the underlying molecular mechanisms by which procyanidins suppress hyperglycaemia are not yet fully understood. In the present study, we prepared procyanidin fractions with different DP, namely low-DP (DP≤3) and high-DP (DP≥4) fractions, from a cacao liquor procyanidin-rich extract (CLPr). These fractions were administered orally to Institute of Cancer Research (ICR) mice and their anti-hyperglycaemic effects were examined. We found that CLPr and its fractions prevent postprandial hyperglycaemia accompanied by an increase in the plasma glucagon-like peptide-1 (GLP-1) level with or without glucose load. In the absence of glucose load, both fractions increased the plasma insulin level and activated its downstream signalling pathway in skeletal muscle, resulting in promotion of the translocation of GLUT4. Phosphorylation of AMP-activated protein kinase (AMPK) was also involved in the promotion of GLUT4 translocation. High- and low-DP fractions showed a similar activation of insulin and AMPK pathways. In conclusion, cacao liquor procyanidins prevent hyperglycaemia by promoting GLUT4 translocation in skeletal muscle, and both the GLP-1-activated insulin pathway and the AMPK pathway are involved in the underlying molecular mechanism.

Fructooligosaccharides suppress high-fat diet-induced fat accumulation in C57BL/6J mice.

Two experiments were performed to examine the effects of fructooligosaccharides (FOS) on the development of obesity. In the first experiment, Wistar rats were orally administered a 2.5 g/kg body weight lipid emulsion containing FOS, and the subsequent elevation of plasma triglycerides was significantly suppressed compared with that in rats receiving lipid emulsion alone. In the second experiment, C57BL/6J male mice were fed a high-fat "western" diet with or without 2.5% FOS supplementation (n = 10/group) ad libitum for 12 weeks. Body weight and percent body fat were lower in mice fed FOS than in controls. Furthermore, the weight of the visceral adipose tissue, and the weight and triglyceride content of the liver were significantly lower in the high-fat + FOS group. Fecal excretion of lipids was markedly enhanced by FOS consumption. These results indicate that dietary FOS suppress high-fat diet-induced body fat accumulation, and inhibit intestinal absorption of dietary fat.

Cacao polyphenol extract suppresses transformation of an aryl hydrocarbon receptor in C57BL/6 mice.

Dioxins enter the body through the diet and cause various toxicological effects through transformation of an aryl hydrocarbon receptor (AhR). Plant extracts and phytochemicals including flavonoids are reported to suppress this transformation. This paper investigates the suppression by a cacao polyphenol extract (CPE) of AhR transformation in vivo. The CPE was administered orally to C57BL/6 mice at 100 mg/kg of body weight, followed 1 h later by 3-methylcholanthrene (MC), an AhR agonist, injected intraperitoneally at 10 mg/kg of body weight. CPE suppressed the MC-induced transformation to the control level by inhibiting the formation of a heterodimer between AhR and an aryl hydrocarbon receptor nuclear translocator in the liver at 3 h postadministration. It also suppressed MC-induced cytochrome P4501A1 expression and NAD(P)H:quinone-oxidoreductase activity, whereas it increased glutathione S-transferase activity at 25 h. CPE constituents and their metabolites might contribute, at least in part, to the suppression of AhR transformation. The results indicate that the intake of CPE suppressed the toxicological effects of dioxins in the body.

Plasma LDL and HDL cholesterol and oxidized LDL concentrations are altered in normo- and hypercholesterolemic humans after intake of different levels of cocoa powder.

Cocoa powder is rich in polyphenols, such as catechins and procyanidins, and has been shown in a variety of subject models to inhibit oxidized LDL and atherogenesis. Our study evaluated plasma LDL cholesterol and oxidized LDL concentrations following the intake of different levels of cocoa powder (13, 19.5, and 26 g/d) in normocholesterolemic and mildly hypercholesterolemic humans. In this comparative, double-blind study, we examined 160 subjects who ingested either cocoa powder containing low-polyphenolic compounds (placebo-cocoa group) or 3 levels of cocoa powder containing high-polyphenolic compounds (13, 19.5, and 26 g/d for low-, middle-, and high-cocoa groups, respectively) for 4 wk. The test powders were consumed as a beverage after the addition of hot water, twice each day. Blood samples were collected at baseline and 4 wk after intake of the test beverages for the measurement of plasma lipids. Plasma oxidized LDL concentrations decreased in the low-, middle-, and high-cocoa groups compared with baseline. A stratified analysis was performed on 131 subjects who had a LDL cholesterol concentrations of > or =3.23 mmol/L at baseline. In these subjects, plasma LDL cholesterol, oxidized LDL, and apo B concentrations decreased, and the plasma HDL cholesterol concentration increased, relative to baseline in the low-, middle-, and high-cocoa groups. The results suggest that polyphenolic substances derived from cocoa powder may contribute to a reduction in LDL cholesterol, an elevation in HDL cholesterol, and the suppression of oxidized LDL.

Continuous intake of polyphenolic compounds containing cocoa powder reduces LDL oxidative susceptibility and has beneficial effects on plasma HDL-cholesterol concentrations in humans.

BACKGROUND: Cocoa powder is rich in polyphenols such as catechins and procyanidins and has been shown in various models to inhibit LDL oxidation and atherogenesis. OBJECTIVE: We examined whether long-term intake of cocoa powder alters plasma lipid profiles in normocholesterolemic and mildly hypercholesterolemic human subjects. DESIGN: Twenty-five subjects were randomly assigned to ingest either 12 g sugar/d (control group) or 26 g cocoa powder and 12 g sugar/d (cocoa group) for 12 wk. Blood samples were collected before the study and 12 wk after intake of the test drinks. Plasma lipids, LDL oxidative susceptibility, and urinary oxidative stress markers were measured. RESULTS: At 12 wk, we measured a 9% prolongation from baseline levels in the lag time of LDL oxidation in the cocoa group. This prolongation in the cocoa group was significantly greater than the reduction measured in the control group (-13%). A significantly greater increase in plasma HDL cholesterol (24%) was observed in the cocoa group than in the control group (5%). A negative correlation was observed between plasma concentrations of HDL cholesterol and oxidized LDL. At 12 wk, there was a 24% reduction in dityrosine from baseline concentrations in the cocoa group. This reduction in the cocoa group was significantly greater than the reduction in the control group (-1%). CONCLUSION: It is possible that increases in HDL-cholesterol concentrations may contribute to the suppression of LDL oxidation and that polyphenolic substances derived from cocoa powder may contribute to an elevation in HDL cholesterol.

Absorption, metabolism, degradation and urinary excretion of rosmarinic acid after intake of Perilla frutescens extract in humans.

BACKGROUND: Rosmarinic acid (RA) is a natural polyphenolic substance contained in many Lamiaceae herbs such as Perilla frutescens. Previous studies have shown RA has antioxidative and anti-inflammatory activity. However, little is known on the absorption, metabolism, degradation and excretion of RA. AIM OF THE STUDY: The aim of this study in healthy humans was to determine the absorption, metabolism, and urinary excretion of RA after a single intake of perilla extract (PE). METHOD: Six healthy men (mean age 37.2 +/- 6.2 y and mean body mass index 22.0 +/- 1.9 kg/m(2)) were enrolled in the study that was a crossover design involving single intakes of PE containing 200 mg RA and placebo with a 10 day interval between treatments. Blood samples were collected before intake and at designated time intervals, while urine samples were collected over the periods 0-6 h, 6-24 h and 24-48 h after intake. RA and its related metabolites in plasma and urine were measured by LC-MS. RESULTS: RA, methylated RA (methyl-RA), caffeic acid (CAA), ferulic acid (FA) and a trace of m-coumaric acid (COA) were detected in the urine after intake of PE. In plasma, RA, methyl-RA and FA were detected, with maximum levels obtained 0.5, 2 and 0.5 h after intake of PE, respectively. The majority of these components in both plasma and urine were present as conjugated forms (glucuronide and/or sulfated). The proportion of RA and its related metabolites excreted in the urine was 6.3 +/- 2.2% of the total dose, with approximately 75% of these components being excreted within 6 h after intake of PE. CONCLUSIONS: RA contained in PE was absorbed, conjugated and methylated following intake, with a small proportion of RA being degraded into various components, such as conjugated forms of CAA, FA and COA. These metabolites were then rapidly excreted in the urine.

Extract of Perilla frutescens enriched for rosmarinic acid, a polyphenolic phytochemical, inhibits seasonal allergic rhinoconjunctivitis in humans.

Extract of Perilla frutescens enriched for rosmarinic acid, a polyphenolic phytochemical, suppresses allergic immunoglobulin responses and inflammation caused by polymorphonuclear leukocytes (PMNL) in mice. However, few placebo-controlled clinical trials have examined the efficacy and safety of polyphenolic phytochemicals for treatment of allergic inflammatory diseases in humans. The present study determined whether oral supplementation with rosmarinic acid is an effective intervention for patients with seasonal allergic rhinoconjunctivitis (SAR). In this 21-day, randomized, double-blind, age-matched, placebo-controlled parallel group study, patients with mild SAR were treated daily with extract of Perilla frutescens enriched for rosmarinic acid (200 mg [n=10] or 50 mg [n=9]) or placebo (n=10). Patients recorded symptoms daily in a diary. Profiles of infiltrating cells and concentrations of eotaxin, IL-1beta, IL-8, and histamine were measured in nasal lavage fluid. Serum IgE concentrations and routine blood tests were also examined. As compared with placebo supplementation, supplementation with extract of Perilla frutescens enriched for rosmarinic acid resulted in a significant increase in responder rates for itchy nose, watery eyes, itchy eyes, and total symptoms (P<0.05). Active treatment significantly decreased the numbers of neutrophils and eosinophils in nasal lavage fluid (P<0.05 vs. placebo). Patients reported no adverse events, and no significant abnormalities were detected in routine blood tests. In conclusion, extract of Perilla frutescens enriched for rosmarinic acid can be an effective intervention for mild SAR at least partly through inhibition of PMNL infiltration into the nostrils. Use of this alternative treatment for SAR might reduce treatment costs for allergic diseases.

Rosmarinic acid inhibits epidermal inflammatory responses: anticarcinogenic effect of Perilla frutescens extract in the murine two-stage skin model.

Perilla frutescens extract showed marked reduction on tumorigenesis in a murine, two-stage skin carcinogenesis model. In this model, cancer is initiated by application of 7,12-dimethylbenz[a]anthracene (DMBA) and promoted by application of 12-tetradecanoylphorbol 13-acetate (TPA). Following tumor initiation with DMBA, topical application of a perilla-derived fraction (PF) at doses of 2 mg/mouse/application resulted in significant inhibition of tumorigenesis. The efficacy of each fraction was correlated with rosmarinic acid (RA) and luteolin concentration. Topical application of perilla extract (PE) that contained 68% RA or an equivalent amount of commercially available RA showed nearly identical antiinflammatory activity 5 h after TPA treatment. Application of luteolin had less anti-inflammatory activity. Marked neutrophil infiltration was observed in TPA-challenged skin by histological examination using hematoxylin-eosin. This change was greatly reduced by pre-treatment with PE or RA. Myeloperoxidase activity, a marker of neutrophil recruitment, was also increased in TPA-challenged skin and was significantly decreased in the PE and RA treated groups. Intercellular adhesion molecule 1 and vascular cell adhesion molecule-1 mRNA expression levels were reduced by pre-treatment with PE or RA. TPA-induced increases in synthesis of the chemokines KC and macrophage inflammatory protein-2 were significantly decreased by pre-treatment with PE or RA. Prostaglandin E2 and leukotriene B4 levels were slightly increased 5 h after TPA treatment. These levels were only numerically decreased in the PE and RA treated groups. However, induction of cyclooxygenase-2 mRNA expression was obviously reduced by pre-treatment with PE or RA. Reactive oxygen radical production, detected as thiobarbituric acid reactive substance and lipid peroxide, by double treatment of TPA was reduced by pre-treatment with PE or RA. Production of 8-hydroxy-2'deoxyguanosine, which was detected immunohistochemically, was also induced by double treatment with TPA. This adduct was barely visible in PE or RA treated mice. Thus, we conclude that part of the anticarcinogenic effects of P.frutescens extract is due to RA via two independent mechanisms: inhibition of the inflammatory response and scavenging of reactive oxygen radicals.

Rosmarinic acid inhibits lung injury induced by diesel exhaust particles.

Epidemiological and experimental studies have suggested that diesel exhaust particles (DEP) may be involved in recent increases in lung diseases. DEP has been shown to generate reactive oxygen species. Intratracheal instillation of DEP induces lung inflammation and edema in mice. Rosmarinic acid is a naturally occurring polyphenol with antioxidative and anti-inflammatory activities. We investigated the effects of rosmarinic acid on lung injury induced by intratracheal administration of DEP (500 microg/body) in mice. Oral supplementation with administration of rosmarinic acid (2 mg/body for 3 d) inhibited DEP-induced lung injury, which was characterized by neutrophil sequestration and interstitial edema. DEP enhanced the lung expression of keratinocyte chemoattractant (KC), interleukin-1beta, monocyte chemoattractant protein-1, and macrophage inflammatory protein-1alpha, which was inhibited by treatment with rosmarinic acid. DEP enhanced expression of iNOS mRNA and formation of nitrotyrosine and 8-OHdG in the lung, which was also inhibited by rosmarinic acid. These results suggest that rosmarinic acid inhibits DEP-induced lung injury by the reduction of proinflammatory molecule expression. Antioxidative activities of rosmarinic acid may also contribute to its protective effects.

Chemoprevention of lung carcinogenesis by cacao liquor proanthocyanidins in a male rat multi-organ carcinogenesis model.

The effects of cacao liquor proanthocyanidins (CLPr) on tumorigenesis were investigated using a multi-organ carcinogenesis model in male F344 rats receiving combined treatment with a single i.p. injection of diethylnitrosamine (100 mg/kg body wt), four i.p. injections of N-methylnitrosourea (20 mg/kg body wt), four s.c. injections of dimethylhydrazine (40 mg/kg body wt), along with 0.05% N-butyl-N-(4-hydroxybutyl)nitrosamine and then 0.1% 2,2'-dihydroxy-di-n-propylnitrosamine, both in the drinking water, for 2 weeks each, during the initial 4-week period (DMBDD treatment). Starting 1 week thereafter, rats were administered CLPr at a dose of 0.025% or 0.25% and the experiment was terminated at week 36. The final survival rate for the DMBDD+0.25% CLPr group was significantly greater than for the DMBDD alone group. In the lung, significant reduction in the incidence and multiplicity of carcinomas was also observed, and in the thyroid, quantitative values for adenomas also tended to decrease in a CLPr dose-dependent manner. No significant modification in the small intestine, colon or kidney was evident. These results indicate that CLPr exerts chemopreventive effects in the lung without any promoting influence in other major organs.

Effects of cacao liquor proanthocyanidins on PhIP-induced mutagenesis in vitro, and in vivo mammary and pancreatic tumorigenesis in female Sprague-Dawley rats.

The effects of cacao liquor proanthocyanidins (CLPr) on 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced mutagenesis in vitro and on in vivo carcinogenesis in female Sprague-Dawley (SD) rats were investigated. In the Ames assay using Salmonella typhimurium TA98, CLPr showed strong antimutagenic effects against PhIP when assayed in the presence of S-9 mixture. For determination of the influence on initiation and subsequent development of lesions, CLPr (0.025% or 0.25%) were fed during the period of PhIP application (100 mg/kg given to rats via gastric tubes eight times over 4 weeks), or thereafter until the termination at 48 weeks. CLPr treatments did not affect body or organ weights. The incidences, multiplicities and volumes of mammary tumors in the 0.25% CLPr (post-initiation) group showed a tendency to decrease as compared to PhIP alone group values, although without statistical significance. The incidences of preneoplastic eosinophilic foci in the exocrine pancreas were significantly (P<0.05) decreased in a dose-dependent manner when CLPr were given during the initiation period. These results indicate that CLPr inhibit in vitro mutagenicity of PhIP, as well as rat pancreatic carcinogenesis in the initiation stage, but not mammary carcinogenesis induced by PhIP.

Proanthocyanidin glycosides and related polyphenols from cacao liquor and their antioxidant effects.

Purification of polar fractions from cacao liquor extracts gave 17 phenolics including four new compounds. The new compounds were characterized as a C-glycosidic flavan, an O-glycoside of a dimeric and two O-glycosides of trimeric A-linked proanthocyanidins, on the basis of spectroscopic data. Isolated polyphenols showed inhibitory effects on nicotinamide adenine dinucleotide phosphate-dependent lipid peroxidation in microsomes and on the autoxidation of linoleic acid. These effects were attributed to the radical-scavenging activity in the peroxidation chain reactions, based on the findings that the cacao polyphenols effectively scavenged the 1,1-diphenyl-2-picrylhydrazyl radical.

Catechins and their oligomers linked by C4 --> C8 bonds are major cacao polyphenols and protect low-density lipoprotein from oxidation in vitro.

In vitro effects of catechins and their oligomers linked by C4 --> C8 bonds are major antioxidative components of chocolate and cocoa. Their effects on the susceptibility of human low-density lipoprotein (LDL) to oxidation were evaluated. The strength of the antioxidative activity was measured using copper ions as the radical generator as compared by weight varied in the following order: (+)-catechin > procyanidin B2 > or = (-)-epicatechin > or = procyanidin C1 > cinnamtannin A2. Using 2,2'-azobis (4-methoxy-2,4-dimethylvaleronitrile) (MeO-AMVN) as the radical generator, the order was (-)-epicatechin > or = procyanidin B2 > or = procyanidin C1 > (+)-catechin > or = cinnamtannin A2. It is suggested that these compounds contribute to the activity of cacao products to protect LDL from oxidation.