Manabu Sami

Dose-dependent hypocholesterolemic actions of dietary apple polyphenol in rats fed cholesterol.

The dose-dependent hypocholesterolemic and antiatherogenic effects of dietary apple polyphenol (AP) from unripe apple, which contains approximately 85% catechin oligomers (procyanidins), were examined in male Sprague-Dawley rats (4 wk of age) given a purified diet containing 0.5% cholesterol. Dietary AP at 0.5 and 1.0% levels significantly decreased the liver cholesterol level compared with that in the control (AP-free dietfed) group. Dietary AP also significantly lowered the serum cholesterol level compared with that in the control group. However, the HDL cholesterol level was significantly higher in the 1.0% AP fed group than in the control group. Accordingly, the ratio of HDL-cholesterol/total cholesterol was significantly higher in the 0.5% AP-fed group and 1.0% AP-fed group than in the control group. Moreover, the atherogenic indices in the 0.5 and 1.0% AP-fed groups were significantly lower than those in the control group. The activity of hepatic cholesterol 7α-hydroxylase tended to be increased by dietary AP in a dose-dependent manner. In accord with this observation, dietary AP increased the excretion of acidic steroids in feces. Dietary AP also significantly promoted the fecal excretion of neutral steroids in a dose-dependent manner. These observations suggest that dietary AP at 0.5 or 1.0% level exerts hypocholesterolemic and antiatherogenic effects through the promotion of cholesterol catabolism and inhibition of intestinal absorption of cholesterol.

Hop-Resistant Lactobacillus brevis Contains a Novel Plasmid Harboring a Multidrug Resistance-Like Gene

The bitter-tasting compounds derived from the flowers of the hop plant (Humulus lupulus L.) protect beer from bacterial spoilage. However, a few lactic acid bacteria, especially lactobacilli, are resistant to these compounds and sometimes cause serious spoilage in the beer industry. It is important to elucidate the mechanisms of hop-resistance in lactic acid bacteria. We selected mutants of Lactobacillus brevis resistant to high concentrations of the hop compounds. The parental strain, L. brevis ABBC45, carries several plasmids. The copy number of one plasmid, termed pRH45, was remarkably increased in one of the hop-resistant mutants compared with that in the wild-type strain. pRH45 (15.0 kb) contains an open reading frame of 1749 b, termed horA, the deduced protein of which includes six putative transmembrane domains and an ATP-binding domain. The amino acid sequence of this putative protein is significantly homologous to half molecules of a mammalian multidrug resistance gene product, P-glycoprotein, and to several bacterial ABC transporters. Furthermore, the hop-resistant mutant was found to be weakly resistant to novobiocin and ethidium bromide, which are structurally and functionally unrelated to the hop compounds. A possible role of the potential drug efflux pump gene in the hop-resistance of L. brevis is discussed.

Evaluation of the alkaline methylene blue staining method for yeast activity determination

Abstract To ensure reliable manufacture of high quality beer, the brewing yeast must be maintained in a highly active state. The conventional methylene blue staining method, which is very simple but not highly sensitive, has been improved to allow sensitive measurement of yeast activity. Two kinds of yeasts with different activity levels were stained with methylene blue solution with pH levels between 4 and 12. The results showed that highly sensitive measurement of yeast activity was possible when the staining solution was alkaline. When cells were stained with methylene blue solution with a pH of 10.6 at 25°C for 15 min (the alkaline methylene blue staining method), the percentage of unstained cells was consistent with viability measurements made using the slide culture method. The alkaline methylene blue staining method makes possible sensitive, quick, and simple measurement of yeast activity, which is essential to the management of fermentation process.

Procyanidins from Apples (Malus pumila Mill.) Extend the Lifespan of Caenorhabditis elegans

Apple polyphenols (AP) mainly consist of procyanidins (PC), which are composed of (-)-epicatechins and (+)-catechins. In order to investigate the antiageing effects of PC, we measured the lifespan of CAENORHABDITIS ELEGANS worms treated with PC. Treatment with 65 µg/mL PC extended the mean lifespan of wild-type N2 and FEM-1 worms by 12.1 % and 8.4 %, respectively, i.e., to a similar extent as resveratrol. In addition, treatment with 100 µg/mL AP also significantly prolonged the mean lifespan of the same worms by 12.0 % and 5.3 %, respectively, i.e., to a similar extent as PC. In contrast, treatment with (-)-epicatechin did not extend the lifespan of the worms. PC did not modify the growth, food intake, or fecundity of C. elegans. Treatment with PC did not extend the lifespan of MEV-1 worms, which show excessive oxidative stress, indicating that PC had no antioxidant ability in the MEV-1 mutant. Moreover, treatment with PC had no effect on the longevity of SIR-2.1 worms, which lack the activity of SIR-2, a member of the sirtuin family of NAD (+)-dependent protein deacetylases. These results indicated that PC has SIR-2.1-dependent antiageing effects on C. elegans.

Apple Polyphenols Regulate Mitochondrial Superoxide Generation and Extend Survival in a Mouse Model of Dilated Cardiomyopathy

Apple polyphenols (AP), which contain procyanidins as major components, have been reported to display potent antioxidant activity and several beneficial health effects. To investigate the protective effect of AP intake against murine cardiomyopathy caused by endogenous oxidative stress, we orally administered AP to heart/muscle-specific manganese- superoxide dismutase (Mn-SOD)-deficient (H/M-Sod2 -/- ) mice (Nojiri et al., 2006). Dietary AP significantly increased the survival of the mutant mice, extending their mean lifespan by 29%. Dietary AP also suppressed the progression of cardiac dilatation and fibrosis in the H/M-Sod2 -/- mice. In vitro experiments revealed that AP treatment strongly suppressed the production of hydrogen peroxide induced reactive oxygen species (ROS) in C2C12 myoblast cells and endogenous superoxide production in Mn-SOD-deficient cells. Furthermore, dietary AP suppressed ROS production in Mn- SOD-deficient cardiomyocytes and oxidative DNA damage in vivo. These results indicate that dietary AP improved the survival and pathology of short-lived mice with cardiomyopathy by suppressing mitochondrial superoxide production.

Ninety-day dietary toxicity study of apple polyphenol extracts in Crl: CD (SD) rats

To examine the safety of Dietary Applephenon® (AP) in feed, Crl: CD (SD) rats of each sex were divided into four groups and given diets containing AP at 0%, 1.25%, 2.5%, or 5.0% for 90 days. All rats survived and toxic changes were not observed throughout the study. Body weight and food efficiency in the 5.0% AP group of both sexes were significantly decreased compared with that in controls. These changes were considered to be caused by the physiological effects of AP (including the inhibitory effects on pancreatic lipase activity). Slight hypertrophy in acinar cells in the parotid and submandibular glands appeared in the 2.5% and 5.0% groups. These were suggested not to be toxicological but physiologic adaptive responses to oral stimuli by the lower pH of AP-containing diets. In conclusion, dietary AP in feed, up to a maximum level of 5.0% for 90 days, given to rats did not induce toxicological effects.

Hypocholesterolemic Effect of Dietary Apple Polyphenol Is Associated with Alterations in Hepatic Gene Expression Related to Cholesterol Metabolism in Rats

Apple polyphenol (AP) mainly consists of procyanidins and has been reported to improve blood cholesterol levels and promote excretion of cholesterol in rats fed high-cholesterol diets. To understand the molecular mechanisms underlying the effects of AP, we investigated whether dietary AP changed the hepatic expression of genes related to cholesterol metabolism and steroid transport. Four-week-old male Sprague-Dawley rats were pair-fed with diets containing 0.5% cholesterol together with 0% (control), 0.2%, and 0.5% AP, respectively, for 30 days. Administration of 0.5% AP was found to improve serum total cholesterol levels (0.69fold vs. control, p < 0.05), and increase hepatic LDL receptor (LDLR) mRNA (1.59-fold vs. control, p < 0.0001). There was a negative correlation between serum non-HDL cholesterol and LDLR mRNA (p < 0.001). Administration of 0.5% AP increased excretion of primary bile acids (2.96-fold vs. control, p < 0.0001) and up-regulated the expression of steroid catabolism genes such as sterol 12αhydroxylase (CYP8B1). However, improvements in cholesterol levels were not associated with the hepatic expression of cholesterol 7α-hydroxylase (CYP7A1), the rate-limiting enzyme in bile acid biosynthesis. Expression of farnesoid X receptor (FXR), which is involved in the regulation of bile acid biosynthesis, was up-regulated by 0.5% AP (1.56-fold vs. control, p < 0.01), and FXR mRNA levels correlated positively with bile acid excretion (p < 0.01). These results show that dietary AP improved blood cholesterol levels with adequate intake of LDL from blood by increasing hepatic LDLR expression and promoting production of bile acid with an upregulation of genes related to steroid catabolism. KeywordsApple Polyphenol; Procyanidin; Cholesterol Metabolism; LDL Receptor