Effects of an environmental endocrine disruptor, para-nonylphenol on the cell growth of Euglena gracilis: association with the cellular oxidative stress.

Effects of an environmental endocrine disruptor, para-nonylphenol (NP) on the cell growth of a photosynthetic eukaryotic microorganism, Euglena gracilis were analysed under different cell culture conditions. Although NP did not show significant inhibitory effects on the cell growth of E. gracilis (Z and SM strains) under light culture condition, NP exhibited significant suppressive effects under dark culture condition. Exogenous supplementation with lipophilic antioxidants (α-tocopherol, ß-carotene or 6-O-palmitoyl-ascorbic acid) to E. gracilis caused strong preventive effects against NP-induced cell growth inhibition under dark culture condition, but hydrophilic antioxidants [ascorbic acid, glutathione and epigallocatechin gallate (EGCG)] did not show significant preventive effects. NP caused significant generation of reactive oxygen species (ROS) in E. gracilis under dark culture condition, but E. gracilis under light culture condition did not show significant increase in ROS generation. Supplementation with lipophilic antioxidants to E. gracilis caused significant suppressive effects against NP-induced cellular ROS generation under dark culture condition, but hydrophilic antioxidants did not show significant suppressive effects. Furthermore, the productivities of typical cellular antioxidants (α-tocopherol, ß-carotene and ascorbic acid) in E. gracilis under light culture conditions were much higher than those under dark culture conditions.

Identification and characterization of a dipeptidyl peptidase IV inhibitor from aronia juice.

Aronia berries have many potential effects on health, including an antioxidant effect, effect for antimutagenesis, hepatoprotection and cardioprotection, an antidiabetic effect and inhibition of cancer cell proliferation. Previous human studies have shown that aronia juice may be useful for treatment of obesity disorders. In this study, we found that aronia juice has an inhibitory effect against dipeptidyl peptidase IV (DPP IV) (EC 3.4.14.5). DPP IV is a peptidase that cleaves the N-terminal region of incretins such as glucagon-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1). Inactivation of incretins by DPP IV induces reduction of insulin secretion. Furthermore, we identified that cyanidin 3, 5-diglucoside as the DPP IV inhibitor in aronia juice. DPP IV was inhibited more strongly by cyanidin 3, 5-diglucoside than by cyanidin and cyanidin 3-glucoside. The results suggest that DPP IV is inhibited by cyanidin 3, 5-diglucoside present in aronia juice. The antidiabetic effect of aronia juice may be mediated through DPP IV inhibition by cyanidin 3, 5-diglucoside.

Resveratrol inhibits hypoxia-inducible factor-1α-mediated androgen receptor signaling and represses tumor progression in castration-resistant prostate cancer.

Androgen-dependent prostate cancer inevitably progresses to incurable castration-resistant prostate cancer (CRPC) after androgen deprivation therapy. Because castration-induced hypoxia-inducible factor (HIF)-1α enhances the transcriptional activity of androgen receptor (AR) at low androgen levels mimicking the castration-resistant stage, HIF-1α is expected to be a promising target for suppression of growth of CRPC. We investigated the effect of resveratrol (3,4',5-trihydroxy-trans-stilbene) on the growth of human prostate cancer LNCaP xenografts in castrated male BALB/cSlc-nu/nu mice (5 wk old). The mice were administered a control diet or a resveratrol diet (4 g/kg diet) for 40 d. The resveratrol diet significantly suppressed tumor growth compared to the control diet. In LNCaP xenografts, dietary resveratrol decreased the protein level of HIF-1α, but not the AR coactivator ß-catenin, and reduced the mRNA levels of androgen-responsive genes. In the control group, ß-catenin was predominantly localized in the nucleus with HIF-1α in LNCaP xenografts, whereas dietary resveratrol inhibited the nuclear accumulation of ß-catenin. In hypoxic LNCaP cells at a low androgen level mimicking the castration-resistant stage, hypoxia-induced nuclear accumulation of ß-catenin was inhibited by resveratrol. Furthermore, resveratrol repressed the expression level of HIF-1α even in the presence of a proteasome inhibitor and suppressed hypoxia-enhanced AR transactivation. These results indicate that dietary resveratrol represses nuclear localization of ß-catenin by decreasing the HIF-1α expression, perhaps in a proteasome-independent manner, and inhibits ß-catenin-mediated AR signaling; this contributes to suppression of tumor growth of CRPC.

S-equol enantioselectively activates cAMP-protein kinase A signaling and reduces alloxan-induced cell death in INS-1 pancreatic ß-cells.

S-Equol is enantioselectively produced from the isoflavone daidzein by gut microflora and is absorbed by the body. An increase of pancreatic ß-cell death is directly associated with defects in insulin secretion and an increased risk of type 2 diabetes mellitus. In the present study, we demonstrate that only the S-enantiomer has suppressive effects against alloxan-induced oxidative stress in INS-1 pancreatic ß-cells. S-Equol reduced alloxan-induced cell death in a dose-dependent manner, whereas R-equol had no effects. In contrast, no significant differences were observed between the enantiomers in estrogenic activity. The cytoprotective effects of S-equol were stronger than those of its precursor daidzein and were blocked by the protein synthesis inhibitor cycloheximide. The cytoprotection was diminished when cells were incubated with a protein kinase A (PKA) inhibitor (H89), but not an estrogen receptor inhibitor. S-Equol increased intracellular cAMP levels in an enantioselective manner. S-Equol, but not R-equol, induced phosphorylation of cAMP-response element-binding protein at Ser 133, and induced cAMP-response element-mediated transcription, both of which were diminished in the presence of H89. Taken together, these results show that S-equol enantioselectively increases the survival of INS-1 cells presumably through activating PKA signaling. Thus, S-equol might have applications as an anti-type 2 diabetic agent.

Resveratrol reduces the hypoxia-induced resistance to doxorubicin in breast cancer cells.

Resveratrol (3,4',5-trihydroxy-trans-stilbene) is known to enhance the cytotoxicity of the anticancer drug doxorubicin. On the other hand, breast cancer MCF-7 cells acquire resistance to doxorubicin under hypoxic conditions. In this study, we investigated the effect of resveratrol on hypoxia-induced resistance to doxorubicin in MCF-7 cells. Resveratrol and its derivative 3,5-dihydroxy-4'-methoxy-trans-stilbene, but not 3,5-dimethoxy-4'-hydroxy-trans-stilbene, cancelled hypoxia-induced resistance to doxorubicin at a concentration of 10 µM. Carbonyl reductase 1 (CBR1) catalyzes the conversion of doxorubicin to its metabolite doxorubicinol, which is much less effective than doxorubicin. Hypoxia increased the expression of CBR1 at both mRNA and protein levels, and knockdown of CBR1 inhibited hypoxia-induced resistance to doxorubicin in MCF-7 cells. Knockdown of hypoxia-inducible factor (HIF)-1α repressed the hypoxia-induced expression of CBR1. Resveratrol repressed the expression of HIF-1α protein, but not HIF-1α mRNA, and decreased hypoxia-activated HIF-1 activity. Resveratrol repressed the hypoxia-induced expression of CBR1 at both mRNA and protein levels. Likewise, 3,5-dihydroxy-4'-methoxy-trans-stilbene decreased the hypoxia-induced expression of CBR1 protein, but not 3,5-dimethoxy-4'-hydroxy-trans-stilbene. Furthermore, resveratrol decreased the expression of HIF-1α protein even in the presence of the proteasome inhibitor MG132 in hypoxia. Theses results indicate that in MCF-7 cells, HIF-1α-increased CBR1 expression plays an important role in hypoxia-induced resistance to doxorubicin and that resveratrol and 3,5-dihydroxy-4'-methoxy-trans-stilbene decrease CBR1 expression by decreasing HIF-1α protein expression, perhaps through a proteasome-independent pathway, and consequently repress hypoxia-induced resistance to doxorubicin.

Identification of carbonyl reductase 1 as a resveratrol-binding protein by affinity chromatography using 4'-amino-3,5-dihydroxy-trans-stilbene.

The mechanisms by which resveratrol (3,4',5-trihydroxy-trans-stilbene) elicits diverse health benefits remain unclear because the intracellular target molecules of resveratrol are poorly defined. We screened resveratrol-binding proteins from lysates of MCF-7 breast cancer cells using resveratrol-affinity resin, which was constructed by immobilizing 4'-amino-3,5-dihydroxy-trans-stilbene on activated CH-Sepharose. On SDS-PAGE, two bands were detected as proteins that specifically bound to the resveratrol-affinity resin. One of these, a 30-kDa protein, was identified as human carbonyl reductase 1 (CBR1) by hybrid linear ion trap/time-of-flight mass spectrometry. Similarly, recombinant CBR1 bound to the resveratrol-affinity resin in the absence of resveratrol, but not in the presence of resveratrol. Among its activities, CBR1 catalyzes a NADPH-dependent reduction of the anticancer drug doxorubicin to the cardiotoxin doxorubicinol. The effects of doxorubicin on viability of MCF-7 cells were enhanced by resveratrol, 3,5-dihydroxy-4'-methoxy-trans-stilbene, 3,4'-dihydroxy-5-methoxy-trans-stilbene, and 4'-amino-3,5-dihydroxy-trans-stilbene at concentrations of 1 and 10 µM. Resveratrol and these derivatives inhibited CBR1 activities to a similar degree at concentrations of 100 and 200 µM. However, 3,5-dimethoxy-4'-hydroxy-trans-stilbene and m-hydroquinone had no influence on doxorubicin cytotoxicity or CBR1 activity. Resveratrol inhibited CBR1 activity through an apparent mix of competitive (Ki=55.8 µM) and noncompetitive (αKi=164 µM; α=2.98) inhibition kinetics. These results indicate that (i) resveratrol enhances the cytotoxic effects of doxorubicin on MCF-7 cells; (ii) the moiety that contains the 3,5-dihydroxyl groups of resveratrol, but not the m-hydroquinone structure alone, is required to bind CBR1; and (iii) resveratrol acts as a mixed-type inhibitor of CBR1 activity on doxorubicin.

A yeast bioassay for androgenic and anti-androgenic compounds based on the NH2- and COOH-terminal interaction of androgen receptor.

Androgenic compounds induce an interaction between the NH(2)- and COOH-terminal regions (N-C interaction) of androgen receptor (AR). We describe a rapid yeast bioassay for androgenic and anti-androgenic compounds based on androgen-dependent ß-catenin-enhanced N-C interaction. The bioassay was also effective at detecting compounds that inhibit the N-C interaction in ways that do not involve binding to the ligand-binding domain.

Characterization of methylmalonyl-CoA mutase involved in the propionate photoassimilation of Euglena gracilis Z.

Significant accumulation of the methylmalonyl-CoA mutase apoenzyme was observed in the photosynthetic flagellate Euglena gracilis Z at the end of the logarithmic growth phase. The apoenzyme was converted to a holoenzyme by incubation for 4 h at 4 degrees C with 10 microM 5'-deoxyadenosylcobalamin, and then, the holoenzyme was purified to homogeneity and characterized. The apparent molecular mass of the enzyme was calculated to be 149.0 kDa +/- 5.0 kDa using Superdex 200 gel filtration. SDS-polyacrylamide gel electrophoresis of the purified enzyme yielded a single protein band with an apparent molecular mass of 75.0 kDa +/- 3.0 kDa, indicating that the Euglena enzyme is composed of two identical subunits. The purified enzyme contained one mole of prosthetic 5'-deoxyadenosylcobalamin per mole of the enzyme subunit. Moreover, we cloned the full-length cDNA of the Euglena enzyme. The cDNA clone contained an open reading frame encoding a protein of 717 amino acids with a calculated molecular mass of 78.3 kDa, preceded by a putative mitochondrial targeting signal consisting of nine amino acid residues. Furthermore, we studied some properties and physiological function of the Euglena enzyme.

Cobalamin deficiency results in an abnormal increase in L-methylmalonyl-co-enzyme-A mutase expression in rat liver and COS-7 cells.

The aim of the present study was to examine the effects of cobalamin (Cbl) on the activity and expression of L-methylmalonyl-CoA mutase (MCM) in rat liver and cultured COS-7 cells. The MCM holoenzyme activity was less than 5% of the total (holoenzyme+apoenzyme) activity in the liver although rats were fed a diet containing sufficient Cbl. When weanling rats were maintained on a Cbl-deficient diet, the holo-MCM activity became almost undetectable at the age of 10 weeks. In contrast, a marked increase in the total-MCM activity occurred under the Cbl-deficient conditions, and at the age of 20 weeks it was about 3-fold higher in the deficient rats than in the controls (108 (SD 14.5) v. 35 (SD 8.5) nmol/mg protein per min (n 5); P<0.05). Western blot analysis confirmed that the MCM protein level increased significantly in the Cbl-deficient rats. However, the MCM mRNA level, determined by real-time PCR, was rather decreased. When COS-7 cells were cultured in a medium in which 10% fetal bovine serum was the sole source of Cbl, holo-MCM activity was barely detected. The supplementation of Cbl resulted in a large increase in the holo-MCM activity in the cells, but the activity did not exceed 30% of the total-MCM activity even in the presence of Cbl at 10 micromol/l. In contrast, the total-MCM activity was significantly decreased by the Cbl supplementation, indicating that Cbl deficiency results in an increase in the MCM protein level in COS-7 cells as well as in rat liver.

Effects of cacao liquor polyphenols on cardiovascular and autonomic nervous functions in hypercholesterolaemic rabbits.

Many epidemiological studies have shown that polyphenols can reduce the risk of mortality from cardiovascular diseases. This study tested the hypothesis that cacao liquor polyphenols have the properties to restore the cardiovascular and autonomic nervous function in an animal model of familial hypercholesterolaemia. Male Kurosawa and Kusanagi-hypercholesterolaemic rabbits were housed in individual cages in a room where a 12-hr light:dark cycle (lights-on at 8:00 and lights-off at 20:00) was maintained. At 3 months of age, they were divided into two groups (standard diet and cacao liquor polyphenol) and the animals received 100 g of the respective diets per day and were provided with tap water ad libitum. Heart rate and blood pressure were measured by a telemetry system. To clarify the autonomic nervous function, power spectral analysis of heart rate variability, baroreflex sensitivity and autonomic nervous tone were measured. After 6 months of dietary administration of cacao liquor polyphenols, heart rate and blood pressure were lowered but plasma lipid concentrations were unchanged. The area of atherosclerotic lesions in the aorta in the cacao liquor polyphenol group was significantly smaller than that in the standard diet group. The high-frequency power of heart rate variability in the rabbits in the standard diet group was significantly decreased with ageing, but that in the cacao liquor polyphenol group was not different between short-term and long-term treatment. Moreover, cacao liquor polyphenols preserved parasympathetic nervous tone, although that in the standard diet group was significantly decreased with ageing. We conclude that cacao liquor polyphenols may play an important role to protect cardiovascular and autonomic nervous functions.

Relationship between the usage of carbapenem antibiotics and the incidence of imipenem-resistant Pseudomonas aeruginosa.

Metallo-beta-lactamase (MBL)-producing Pseudomonas aeruginosa isolates are resistant to almost all broad-spectrum beta-lactams and carbapenems. We investigated 389 P. aeruginosa isolates, collected from 29 hospitals in the Tohoku area of Japan, to determine their susceptibilities to ten antimicrobial drugs, and the rates of MBL-producing P. aeruginosa among them. Two hundred and one P. aeruginosa strains were isolated from small (group S)hospitals that had adopted imipenem as a carbapenem antibiotic, and 188 were isolated from general (group G) hospitals, which employed three or four carbapenems. MBL genes were analyzed by polymerase chain reaction (PCR) in all isolates for which the sodium mercaptoacetic acid (SMA) disk method gave positive results. The antimicrobial agents tested were imipenem, meropenem, biapenem, panipenem, piperacillin, ceftazidime, sulbactam/cefoperazone, amikacin, arbekacin, and ciprofloxacin. Sixteen (8.0%) of the 201 isolates from group S hospitals and three (1.6%) of the 188 isolates from group G hospitals were MBL-producing P. aeruginosa. In this study, the proportion of MBL-producing P. aeruginosa in group S was significantly higher than that found in group G (P < 0.01). The use of only one agent as a carbapenem antibiotic may have been one of the factors contributing to the high detection rate of MBL-producing P. aeruginosa observed in group S hospitals.

Antidiabetic effect of long-term supplementation with Siraitia grosvenori on the spontaneously diabetic Goto-Kakizaki rat.

Siraitia grosvenori Swingle (SG) is a traditional Chinese fruit used as a folk medicine. Its extract (SG-ex) contains potent sweet elements with a sweetness several hundred times higher than table sugar. We investigated the antidiabetic effect of SG-ex in the type 2 diabetic Goto-Kakizaki (GK) rat. Diabetic 7-week-old GK rats were fed a diet supplemented with 0.4 % of the SG-ex for 13 weeks, and its antidiabetic effects were evaluated. SG-ex had no effect on food intake or body weight. In oral glucose tolerance tests (OGTT), SG-ex supplementation improved the insulin response at 15 min (control, 63 (sem 6) pm; SG-ex, 107 (sem 20) pm; P < 0.05) and reduced the plasma glucose level at 120 min after the glucose administration (control, 18.5 (sem 0.8) mm; SG-ex, 14.8 (sem 0.7) mm; P < 0.05). The total amount of insulin in whole pancreas taken from fasting rats was higher in the SG-ex-supplemented group, which may explain the greater capacity to secrete insulin during the OGTT. Thiobarbituric acid-reactive substances in both the liver and the plasma were lower in the SG-ex-supplemented group, suggesting that an absorbable component in SG-ex has an antioxidative effect on lipid peroxidation, thereby counteracting the oxidative stress caused by a diabetic state. Excreted urine volume and urinary albumin level for 24 h were both reduced in the SG-ex-supplemented group, suggesting the attenuation of kidney damage that is caused by diabetes. These data indicate that SG-ex supplementation may prevent complications and attenuate pathological conditions for type 2 diabetes, along with its sweet characteristics.

Maternal vitamin B12 deficiency affects spermatogenesis at the embryonic and immature stages in rats.

To evaluate the role of cobalamin (Cbl) on spermatogenesis, the effect of dietary vitamin B(12) deficiency on early spermatogenesis was histologically investigated in male fetuses and newborns in the first filial generation (F(1) males) of rats. There was no difference in the number of gonocytes and supporting cells of Sertoli in the gonad in male fetuses on day 16 of gestation and in the testes in F(1) males at 0 days of age between vitamin B(12)-deficient (VB12-D) and vitamin B(12)-supplemented (VB12-S) groups. However, at 21 days of age, a decreased number of spermatogonia and no spermatocytes were observed in the VB12-D group. Numerous TUNEL positive cells were located among spermatocytes of the spermatogenic epithelium. The ultrastructural features examined using transmission electron microscopy were considered to be indicative of apoptosis. The incidence of seminiferous tubules having apoptotic cells was 51.5% in the VB12-D group. At 60 days of age, aplasia of the spermatids and spermatozoa was detected in the VB12-D group. In the connective tissue between the seminiferous tubules, many interstitial Leydig cells and blood vessels were observed in the VB12-D group, as compared with the VB12-S group. These changes produced by vitamin B(12) deficiency can be reversed by providing a VB12-S diet after weaning at 21 days of age. From these findings, such a vitamin B(12) deficiency during gestation and lactation could affect the germ cells and especially damage spermatocytes in F(1) male rats, which indicates that Cbl may be an essential constituent in the meiosis of spermatogenesis.

Purification and characterization of a corrinoid-compound in an edible cyanobacterium Aphanizomenon flos-aquae as a nutritional supplementary food.

The vitamin B12 concentration of the dried cells of Aphanizomenon flos-aquae was determined by both microbiological method with Lactobacillus delbrueckeii ATCC7830 and chemiluminescence method with intrinsic factor. The Aphanizomenon cells contained 616.3 +/- 30.3 micro g (n = 4) of vitamin B12 per 100 g of the dried cells by the microbiological method. The values determined with the chemiluminescence method, however, were only about 5.3% of the values determined by the microbiological method. A corrinoid-compound was purified from the dried cells and characterized. The purified corrinoid-compound was identified as pseudovitamin B12 (an inactive corrinoid-compound for humans) by silica gel 60 TLC, C18 reversed-phase HPLC, ultraviolet-visible spectroscopy, and 1H NMR spectroscopy. The results suggest that the Aphanizomenon cells are not suitable for use as a vitamin B12 source, especially in vegans.

Abnormal increase in the expression level of proliferating cell nuclear antigen (PCNA) in the liver and hepatic injury in rats with dietary cobalamin deficiency.

Dietary cobalamin (Cbl; vitamin B12) deficiency resulted in severe growth retardation in rats, and body weight in the Cbl-deficient rats at 20 wk of age was significantly lower compared with the age-matched Cbl-sufficient control rats. In contrast, liver weight, when normalized to body weight, was greater in the Cbl-deficient rats than in the controls (p<0.05). The expression level of proliferating cell nuclear antigen (PCNA), a marker for cell proliferation, in the liver was significantly enhanced in the deficient rats, suggesting that cell proliferation is abnormally activated in the liver under Cbl-deficient conditions. In addition, plasma alanine aminotransferase (ALT) activity, a marker for hepatic injury, was also significantly elevated in the deficient rats. When L-carnitine, which is used clinically for the treatment of Cbl-deficient patients with methylmalonic aciduria, was administered to the Cbl-deficient rats by intraperitoneal injection twice per day for 2 wk (each 0.5 mmol), the amount of methylmalonic acid excreted into the urine was significantly reduced, and the plasma ALT activity was lowered to a normal level. However, the PCNA expression in the liver was barely influenced by the treatment with carnitine. In contrast, when the deficient rats were fed an L-methionine-supplemented diet (4 g of L-methionine per kg of the diet) for 2 wk, the increased expression of PCNA was normalized.

Eucalyptus leaf extract inhibits intestinal fructose absorption, and suppresses adiposity due to dietary sucrose in rats.

Sucrose is more lipogenic than starch, and the extreme ingestion of sucrose induces adiposity and obesity. The aim of this study was to examine the effect of the eucalyptus (Eucalyptus globulus) leaf extract (ELE) on adiposity due to dietary sucrose in rats. In addition, in this study, the effect of ELE on intestinal fructose absorption was also examined. Rats were fed a high-sucrose diet (75 % in calorie base) with or without ELE (10 g/kg diet) for 5 weeks. Body weight was lower in the rats receiving ELE than in the controls (342 (sd 37.9) v. 392 (sd 26.0) g (n 7); P<0.05). Furthermore, ELE resulted in decreases in the triacylglycerol concentrations in the plasma (1.44 (sd 0.448) v. 2.79 (sd 0.677) mmol/l (n 7); P<0.05) and liver (19.1 (sd 5.07) v. 44.1 (sd 16.28) micromol/g (n 7); P<0.05). In contrast, ELE did not show any significant effects in the rats fed a starch diet. When rats were orally given ELE 10 min before fructose administration, the intestinal fructose absorption, which was examined by measuring the elevated concentration of fructose in the portal vein at 30 min after the fructose administration, was significantly inhibited in a dose-dependent manner. Furthermore, in rats fed a high-fructose diet, the plasma and hepatic triacylglycerol concentrations were significantly decreased by ELE. These results indicate that ELE, which inhibits the intestinal fructose absorption, can suppress adiposity in rats that ingest large amounts of sucrose or fructose.

Triterpene glycosides of Siraitia grosvenori inhibit rat intestinal maltase and suppress the rise in blood glucose level after a single oral administration of maltose in rats.

The effect of the crude extract from Siraitia grosvenori Swingle (SG-ex) on the postprandial rise in blood glucose level was investigated. The increase in plasma glucose level in response to the oral administration of maltose was significantly suppressed in rats when SG-ex was given orally 3 min before the maltose administration. There was, however, no effect when glucose was administered instead, suggesting that the antihyperglycemic effect of SG-ex is elicited by inhibition of maltase in the small intestinal epithelium. In vitro, SG-ex inhibited rat small intestinal maltase. Similar effects were also observed both in vivo and in vitro when the concentrate of the sweet elements (triterpene glycosides) prepared from SG-ex was used. Furthermore, the main sweet element of SG-ex, mogroside V, and some minor elements such as mogroside IV, siamenoside I, and mogroside III also exhibited maltase inhibitory effect with IC50 values of 14, 12, 10, and 1.6 mM, respectively. These results suggest that SG-ex exerts anti-hyperglycemic effects in rats by inhibiting maltase activity and that these effects are at least partially exerted by its sweet elements, triterpene glycosides.

Suppressive effects of cacao liquor polyphenols (CLP) on LDL oxidation and the development of atherosclerosis in Kurosawa and Kusanagi-hypercholesterolemic rabbits.

We investigated the properties of cacao liquor polyphenols (CLP), which have an antioxidative effect on low-density lipoprotein (LDL) and an anti-atherosclerotic effect in the spontaneous familial hypercholesterolemic model, the Kurosawa and Kusanagi-hypercholesterolemic (KHC) rabbit. After 6 months of dietary administration of CLP at 1% (w/w) to the KHC rabbits, a higher total cholesterol concentration was observed in the treatment group compared to the control group. However, no other effects were noted in lipid profiles in plasma or lipoproteins. The plasma concentration of thiobarbituric acid reactive substances (TBARS), which is a lipid-peroxidation index, was significantly decreased 1 month after the start of CLP administration compared to that of the control group. The antioxidative effect of CLP on LDL was observed from 2 to 4 months of administration. The area of atherosclerotic lesions in the aorta in the CLP group (32.01+/-1.58%) was significantly smaller than that in the control group (47.05+/-3.29%), and the tissue cholesterol and TBARS concentrations were lower in the CLP group than in the control group. The anti-atherosclerotic effect of CLP was confirmed both rheologically and histopathologically. An in vitro study using KHC rabbit-derived LDL revealed that CLP significantly prolonged the lag time of LDL oxidation that was induced by a lipophilic azo-radical initiator, 2,2'-azobis(4-methoxy)-2,4-dimethylvaleronitrile (V-70), or Cu(2+) from a low concentration of 0.1 microg/mL. The antioxidative effect of CLP was superior to those of the well-known antioxidative substances, vitamin C, vitamin E and probucol. Therefore, CLP suppressed the generation of atherosclerosis, and its antioxidative effect appeared to have an important role in its anti-atherosclerotic activity.

Characterization of corrinoid compounds from a Japanese black tea (Batabata-cha) fermented by bacteria.

A Japanese fermented black tea (Batabata-cha) contained a considerable amount of vitamin B(12) (456 +/- 39 ng per 100 g dry tea leaves and 2.0 +/- 0.3 ng per 100 mL of tea drink). A corrinoid compound was partially purified and characterized from the tea leaves. The patterns of the purified compound by the silica gel 60 thin-layer chromatography and C18 reversed phased high-performance liquid chromatography were identical to those of authentic vitamin B(12). When 20 week old vitamin B(12) deficient rats, which excreted substantial amounts (about 250 mg/day) of methylmalonic acid in urine as an index of vitamin B(12) deficiency, were fed the tea drink (50 mL/day, 1 ng of vitamin B(12)) for 6 weeks, urinary methylmalonic acid excretion (169 +/- 29 mg/day) of the tea drink-supplemented 26 week old rats decreased significantly relative to that (250 +/- 32 mg/day) of the deficient rats. The results indicate that the vitamin B(12) found in the fermented black tea is bioavailable in mammals.

Effect of carotenoids and ascorbic acid of Japanese persimmons on cellular lipid peroxidation in HepG2 cells.

In this experiment, we examine the functional property of carotenoids; beta-cryptoxanthin (Cry), zeaxanthin (Zea), beta-carotene (Car)) and ascorbic acid (AsA). The accumulation amounts of Cry, Zea and Car in HepG2 cells cultured in the high concentration medium were larger than that in a low concentration. Further those accumulation amounts in long incubation time within 24 hours were greater than that in a shorter time. When the added carotenoid concentration, with or without hydrogen peroxide, increased from 0 to 5 microM in the culture medium, the thiobarbituric acid reaction substance (TBARS) values in the HepG2 cells decreased significantly (p < 0.05). The decrease of TBARS values shows the antioxidative property of the carotenoids. When AsA and Tocopherol(Toc) were added to the medium from 0 to 20 microM, the TBARS values, with or without hydrogen peroxide, decreased significantly with increasing concentrations of AsA and Toc respectively (p < 0.05). The decreased amount of TBARS in 5 microM Cry compared with control(0 microM) was the largest among 6 antioxidants (Cry, Car, Zea, Retinol(Ret), AsA, Toc) used in this experiment.