Anthocyanin-rich blackcurrant extract improves long-term memory impairment and emotional abnormality in senescence-accelerated mice.

Alzheimer's disease (AD) is associated with a progressive worsening in cognitive function, which is often accompanied by emotional disturbance. Recent studies revealed that anthocyanin-rich blackcurrant extract (BCE) can impart health benefits, but it is not known whether BCE is useful in the prevention and/or treatment of AD. Here, we examined the effects of BCE using a senescence-accelerated mouse prone 8 (SAMP8) model. Dietary BCE supplementation for 9 weeks was found to both improve the diminished long-term recognition memory and normalize the anxiety levels of SAMP8 mice. RNA sequencing demonstrated that dietary supplementation with anthocyanin-rich BCE significantly altered the gene expression profile in the hippocampus. According to enrichment analysis, genes regulated by BCE were related to cellular component terms such as "smooth endoplasmic reticulum," "axon," and "glutamatergic synapse." Real-time PCR verified alterations in the expression of AD-related genes. These findings indicate that anthocyanin-rich BCE may be valuable for the prevention and/or treatment of AD. PRACTICAL APPLICATIONS: Blackcurrant contains an abundance of polyphenols, especially anthocyanins. This study demonstrated that anthocyanin-rich BCE improves the long-term recognition memory impairment and emotional abnormality of SAMP8 mice, a mouse model characterized by several pathological features of AD. These findings indicate that anthocyanin-rich BCE may be a useful food supplement or ingredient for the prevention of AD.

Potential Vasculoprotective Effects of Blackcurrant (Ribes nigrum) Extract in Diabetic KK-Ay Mice.

Polyphenols are bioactive compounds found naturally in fruits and vegetables; they are widely used in disease prevention and health maintenance. Polyphenol-rich blackcurrant extract (BCE) exerts beneficial effects on vascular health in menopausal model animals. However, the vasculoprotective effects in diabetes mellitus (DM) and atherosclerotic vascular disease secondary to DM are unknown. Therefore, we investigated whether BCE is effective in preventing atherosclerosis using KK-Ay mice as a diabetes model. The mice were divided into three groups and fed a high-fat diet supplemented with 1% BCE (BCE1), 3% BCE (BCE2), or Control for 9 weeks. The mice in the BCE2 group showed a considerable reduction in the disturbance of elastic lamina, foam cell formation, and vascular remodeling compared to those in the BCE1 and Control groups. Immunohistochemical staining indicated that the score of endothelial nitric oxide synthase staining intensity was significantly higher in both BCE2 (2.9) and BCE1 (1.9) compared to that in the Control (1.1). Furthermore, the score for the percentage of alpha-smooth muscle actin was significantly lower in the BCE2 (2.9%) than in the Control (2.1%). Our results suggest that the intake of anthocyanin-rich BCE could have beneficial effects on the blood vessels of diabetic patients.

Psychological and Antibacterial Effects of Footbath Using the Lindera umbellata Essential Oil.

Lindera umbellata (Lu) essential oil primarily contains linalool and has relaxation properties. We investigated the psychological and antibacterial effects of footbath with Lu essential oil. The participants included 20 women without medical history and received two intervention plans: footbath without any essential oil and footbath using Lu essential oil. Next, questionnaires regarding impressions and mood states were provided for them to answer. In addition, their autonomic nervous system activity was measured, and the aerobic viable of count on the feet was determined. The high-frequency value reflecting the parasympathetic nervous system activity significantly increased after footbath using Lu essential oil. In the questionnaire about the mood states, the subscale scores of tension-anxiety, depression, fatigue, and confusion after intervention were lower than those before intervention regardless of the use of the essential oil. Conversely, the anger-hostility score decreased only in the group using Lu essential oil. Furthermore, the decrease in aerobic viable count after intervention was not significantly different between the two groups. Footbath using Lu essential oil increased the parasympathetic nervous system activity and relieved anger. Taken together, we suggest that footbath using Lu essential oil has a relaxation effect.

Hypocholesterolemic Effect of Blackcurrant (Ribes nigrum) Extract in Healthy Female Subjects: A Pilot Study.

Blackcurrant extract (BCE) ameliorates dyslipidemia in menopausal model animals and in elderly women at a risk of dyslipidemia. However, it is unknown whether the daily intake of BCE can prevent lipid abnormalities in healthy individuals. Lipids are essential for the body, but they also cause arteriosclerosis. In this noncomparative pilot study, we examined the effects of BCE administered for 29 days on serum lipids in young healthy women. Blood samples were collected before and on days 4 and 29 after BCE intake, and 20 lipoprotein fractions in the serum were separated using a gel-permeation high-performance liquid chromatography method to measure the triacylglycerol and cholesterol levels in lipoproteins. There were no effects on lipids on day 4 of BCE intake, but the total cholesterol level decreased on day 29. Furthermore, the levels of total very-low-density lipoprotein (VLDL) cholesterol, small VLDL cholesterol, and large low-density lipoprotein cholesterol were significantly decreased. These results suggest that the daily intake of BCE has a hypocholesterolemic effect in healthy women, and that it is effective in preventing atherosclerosis.

Blackcurrant (Ribes nigrum L.) Extract Exerts Potential Vasculoprotective Effects in Ovariectomized Rats, Including Prevention of Elastin Degradation and Pathological Vascular Remodeling.

Estrogen exerts cardioprotective effects in menopausal women. Phytoestrogens are plant-derived substances exhibiting estrogenic activity that could beneficially affect vascular health. We previously demonstrated that blackcurrant (Ribes nigrum L.) extract (BCE) treatment exerted beneficial effects on vascular health via phytoestrogenic activity in ovariectomized (OVX) rats, which are widely used as menopausal animal models. Here, we examined whether BCE treatment reduced elastin degradation and prevented pathological vascular remodeling in OVX rats fed a regular diet (OVX Control) or a 3% BCE-supplemented diet (OVX BCE), compared with sham surgery rats fed a regular diet (Sham) for 3 months. The results indicated a lower staining intensity of elastic fibers, greater elastin fragmentation, and higher α-smooth muscle actin protein expression in OVX Control rats than in OVX BCE and Sham rats. Pathological vascular remodeling was only observed in OVX Control rats. Additionally, we investigated matrix metalloproteinase (MMP)-12 mRNA expression levels to elucidate the mechanism underlying elastin degradation, revealing significantly upregulated MMP-12 mRNA expression in OVX Control rats compared with that in Sham and OVX BCE rats. Together, we identify BCE as exerting a vascular protective effect through reduced MMP-12 expression and vascular smooth muscle cell proliferation. To our knowledge, this is the first report indicating that BCE might protect against elastin degradation and pathological vascular remodeling during menopause.

Comparison of Chemical Composition between Kuromoji (Lindera umbellata) Essential Oil and Hydrosol and Determination of the Deodorizing Effect.

Kuromoji (Lindera umbellata) is a tree that grows throughout Japan. The components of kuromoji essential oil have antitumor and aromatherapy effects. However, the composition of the hydrosol, obtained as a by-product of the essential oil process, is unknown. Furthermore, it is unknown whether kuromoji essential oil has a deodorizing effect. Therefore, the purpose of the current study was to compare the chemical composition of kuromoji essential oil and hydrosol, as well as evaluate the deodorizing effect of the former. The chemical composition of samples was evaluated using gas chromatography-mass spectrometry (GC-MS). Additionally, the deodorizing effect of Kuromoji essential oil was investigated with the detector tube method using ammonia, hydrogen sulfide, methyl mercaptan, and isovaleric acid. Linalool was the most abundant component in both the essential oil and hydrosol; however, its proportion was higher in the hydrosol (57.5%) than in the essential oil (42.8%). The hydrosol contained fewer chemical components, but higher proportions of trans-geraniol and ethanol. Moreover, the essential oil eliminated 50% of ammonia and 97.6% or more of isovaleric acid. Interestingly, linalool was soluble in the hydrosol and did not irritate the skin. This suggests that the hydrosol may be an effective foot care product.

Blackcurrant (Ribes nigrum) Extract Prevents Dyslipidemia and Hepatic Steatosis in Ovariectomized Rats.

Estrogen is involved in lipid metabolism. Menopausal women with low estrogen secretion usually gain weight and develop steatosis associated with abnormal lipid metabolism. A previous study showed that blackcurrant (Ribes nigrum L.) extract (BCE) had phytoestrogen activity. In this study, we examined whether BCE improved lipid metabolism abnormalities and reduced liver steatosis in ovariectomized rats, as a menopausal animal model. Twelve-week-old ovariectomized (OVX) rats were fed a regular diet (Ctrl) or a 3% BCE supplemented diet while sham rats were fed a regular diet for three months. Body weight, visceral fat weight, levels of serum triglycerides, total cholesterol, and LDL cholesterol decreased in the BCE-treated OVX and sham rats, but not in OVX Ctrl rats. The results of hematoxylin and eosin staining revealed that BCE decreased the diameters of adipocytes and the nonalcoholic fatty liver disease activity score. Furthermore, quantitative RTPCR indicated a decreased expression of hepatitis-related genes, such as tumor necrosis factor-α, IL-6, and IL-1ß in OVX rats after BCE treatment. This is the first study that reported improvement of lipid metabolism abnormalities in OVX rats by BCE administration. These results suggest that the intake of BCE alleviated dyslipidemia and prevented nonalcoholic steatohepatitis during menopause in this animal model.

Effects of Blackcurrant Anthocyanin on Endothelial Function and Peripheral Temperature in Young Smokers.

BACKGROUND: Blackcurrant anthocyanin (BCA) is expected to repair endothelial dysfunction, but it remains unclear whether beneficial effects are present in young healthy persons. This study examines whether supplements containing blackcurrant anthocyanin improve endothelial function and peripheral temperature in young smokers. METHODS: Young, healthy male nonsmokers (N group: n = 11; mean age 22 ± 2 years) and smokers (S group: n = 13; mean age 21 ± 1 years) were enrolled. A randomized and double-blind trial was designed to compare the effects of no supplement, a supplement containing 50 mg of blackcurrant anthocyanin (supplement A), and a supplement containing 50 mg of blackcurrant anthocyanin plus vitamin E (supplement B) on flow-mediated dilatation (FMD) and skin temperature. RESULTS: Under no supplement, FMD was unchanged during the 2 h period after smoking in the N group, whereas it was decreased during the 2 h period after smoking in the S group. Under the A supplement, FMD was decreased 1 h after smoking and returned to the baseline level 2 h after smoking in the S group. The skin temperature in the area of the foot dorsum was decreased in the S group after smoking compared with that in the N group, who did not smoke, whereas under A and B supplements, it was higher in the S group compared with that in the N group. CONCLUSIONS: BCA could attenuate the smoking-induced acute endothelial dysfunction and improve peripheral temperature in young smokers.

Phytoestrogenic Effects of Blackcurrant Anthocyanins Increased Endothelial Nitric Oxide Synthase (eNOS) Expression in Human Endothelial Cells and Ovariectomized Rats.

Phytoestrogens are plant-derived chemicals that are found in many foods and have estrogenic activity. We previously showed that blackcurrant extract (BCE) and anthocyanins have phytoestrogenic activity mediated via estrogen receptors (ERs), and anthocyanins may improve vascular function. BCE contains high levels of anthocyanins, but their health-promoting effects are unclear. This study examined the effects of BCE on the regulation of endothelial nitric oxide synthase (eNOS) expression and nitric oxide (NO) synthesis in human endothelial cells as key regulators in cardiovascular disease. The results showed that eNOS mRNA levels were significantly upregulated in BCE- or anthocyanin-treated human vascular endothelial cells but decreased in cells treated with fulvestrant, an ER antagonist. These results corresponded with NO levels, suggesting that BCE and anthocyanin may regulate NO synthesis via eNOS expression. Thus, the phytoestrogenic effects exerted by BCE via ERs influenced eNOS mRNA expression and NO synthesis. In vivo, we investigated whether anthocyanin-rich BCE upregulated eNOS protein expression in ovariectomized (OVX) rats, a widely used animal model of menopause. Our results showed that anthocyanin-rich BCE significantly upregulated eNOS mRNA levels and NO synthesis through phytoestrogenic activity and therefore promoted blood vessel health in OVX rats as a postmenopausal model.

Blackcurrant Extract with Phytoestrogen Activity Alleviates Hair Loss in Ovariectomized Rats.

Ancocyanin-rich blackcurrant extract (BCE) has phytoestrogen activity; however, its effect on hair follicles is unknown. Additionally, hair loss is known to occur during menopause in women owing to decreased estrogen secretion. This study examined whether BCE alleviated female pattern hair loss using a rat model. RNA was extracted and analyzed using a microarray and ingenuity pathway analysis. A quantitative polymerase chain reaction revealed that 1 µg/mL BCE altered many genes downstream of beta-estradiol in human hair dermal papilla cells. Additionally, the expression of the hair follicle stem cell marker keratin 19 was greatly enhanced. In a menopause model, ovariectomized rats were fed a diet containing 3% BCE for three months. An analysis of the number of hair shafts revealed that BCE increased the number of hairs by 0.5 hairs/follicular unit. Moreover, immunostaining revealed that the expression of Ki67 also increased by 19%. Furthermore, fluorescent immunostaining showed that the expression of other stem cell markers, including keratin 15, CD34, and keratin 19, was induced in rat hair follicular cells. In conclusion, these findings suggest that BCE has phytoestrogen activity in hair follicles and contributes to the alleviation of hair loss in a menopausal model in rats.

Phytoestrogenic Activity of Blackcurrant Anthocyanins Is Partially Mediated through Estrogen Receptor Beta.

Phytoestrogens are plant compounds with estrogenic effects found in many foods. We have previously reported phytoestrogen activity of blackcurrant anthocyanins (cyanidin-3-glucoside, cyanidin-3-rutinoside, delphinidin-3-glucoside, and delphinidin-3-rutinoside) via the estrogen receptor (ER)α. In this study, we investigated the participation of ERß in the phytoestrogen activity of these anthocyanins. Blackcurrant anthocyanin induced ERß-mediated transcriptional activity, and the IC50 of ERß was lower than that of ERα, indicating that blackcurrant anthocyanins have a higher binding affinity to ERß. In silico docking analysis of cyanidin and delphinidin, the core portions of the compound that fits within the ligand-binding pocket of ERß, showed that similarly to 17ß-estradiol, hydrogen bonds formed with the ERß residues Glu305, Arg346, and His475. No fitting placement of glucoside or rutinoside sugar chains within the ligand-binding pocket of ERß-estradiol complex was detected. However, as the conformation of helices 3 and 12 in ERß varies depending on the ligand, we suggest that the surrounding structure, including these helices, adopts a conformation capable of accommodating glucoside or rutinoside. Comparison of ERα and ERß docking structures revealed that the selectivity for ERß is higher than that for ERα, similar to genistein. These results show that blackcurrant anthocyanins exert phytoestrogen activity via ERß.

Anthocyanin­rich blackcurrant extract inhibits proliferation of the MCF10A healthy human breast epithelial cell line through induction of G0/G1 arrest and apoptosis.

Blackcurrants (Ribes nigrum L., Grossulariaceae) possess a high content of anthocyanin polyphenols, which have been demonstrated to exhibit beneficial effects on health due to their antioxidant and anticarcinogenic prope-rties. The present study investigated novel functions of anthocyanin­rich blackcurrant extracts (BCEs) in a healthy mammary epithelial cell line, MCF10A. The percentages of viable cells were 85, 75, 53 and 31% following exposure to 50, 100, 200 and 400 µg/ml BCE, respectively. The half­maximal response concentration of BCE was 237.7 µg/ml. Microarray and Ingenuity® Pathway Analysis demonstrated that BCE downregulated cell cycle signaling, including upstream genes with mitotic roles such as polo­like kinase signaling. BCE increased the number of cells in the G0/G1 phase and decreased the number of cells in the S and G2/M phases. Alkaline comet assays demonstrated that 50 and 100 µg/ml BCE induced DNA damage in a dose­dependent manner. Cultures treated with 0, 50, and 100 µg/ml BCE contained 4.6, 13.4 and 16.0% apoptotic cells, respectively. As compared with the untreated cultures (1.9%), the number of necrotic cells increased in the 100 µg/ml BCE­treated cultures (from 1.9 to 4.3%) but not in the 50 µg/ml BCE­treated cultures. Reverse transcription­quantitative polymerase chain reaction analysis demonstrated that BCE reduced mRNA expression of the genomic caretaker lysine­specific demethylas  5B (KDM5B). The results suggested that blackcurrant anthocyanins may act as cell arrest and death inducers via KDM5B downregulation in healthy breast cells.

Phytoestrogenic activity of blackcurrant (Ribes nigrum) anthocyanins is mediated through estrogen receptor alpha.

SCOPE: Blackcurrants (Ribes nigrum L., Grossulariaceae) contain high amounts of anthocyanin polyphenols, which have antioxidant and anti-carcinogenic health benefits. This study analyzed the potential phytoestrogenic effects of blackcurrant extract (BCE) in breast cancer (MCF-7) and human endometrial cancer (Ishikawa) cell lines that over-express estrogen receptor alpha (ERα), as well as in immature female rats. METHODS AND RESULTS: Microarray analysis and Ingenuity® Pathway Analysis showed that BCE activated the ERα pathway, whereas quantitative-PCR confirmed that BCE and four types of anthocyanins up-regulated genes downstream of ERα. BCE (0.1-1.0 µg/mL) and anthocyanins (0.1-10 µM) induced MCF-7 cell proliferation; however, this effect was blocked by ER antagonist fulvestrant. Flow cytometry showed that anthocyanins reduced and increased the number of MCF-7 cells in the G0/G1 and G2/M phases, respectively. Anthocyanins stimulated ERα transcriptional activity in human ERα reporter assays and induced alkaline phosphatase activity in Ishikawa cells. Competition assays and in silico analysis indicated that anthocyanins bind to ERα. Finally, BCE focally induced stratification of columnar epithelial cells in the rat uterus and increased cytoplasmic mucin levels in these cells. CONCLUSION: These results suggest that blackcurrant anthocyanins act as phytoestrogens in vitro and in vivo.

Protein adsorption of ultrafine metal oxide and its influence on cytotoxicity toward cultured cells.

Many investigations about the cellular response by metal oxide nanoparticles in vitro have been reported. However, the influence of the adsorption ability of metal oxide nanoparticles toward cells is unknown. The aim of this study is to understand the influence of adsorption by metal oxide nanoparticles on the cell viability in vitro. The adsorption abilities of six kinds of metal oxide nanoparticles, namely, NiO, ZnO, TiO2, CeO2, SiO2, and Fe2O3, to Dulbecco's modified Eagle's medium supplemented with a 10% fetal bovine serum (DMEM-FBS) component such as serum proteins and Ca2) were estimated. All of the metal oxide nanoparticles adsorbed proteins and Ca2+ in the DMEM-FBS; in particular, TiO2, CeO2, and ZnO showed strong adsorption abilities. Furthermore, the influence of the depletion of medium components by adsorption to metal oxide nanoparticles on cell viability and proliferation was examined. The particles were removed from the dispersion by centrifugation, and the supernatant was applied to the cells. Both the cell viability and the proliferation of human keratinocyte HaCaT cells and human lung carcinoma A549 cells were affected by the supernatant. In particular, cell proliferation was strongly inhibited by the supernatant of TiO2 and CeO2 dispersions. The supernatant showed depletion of serum proteins and Ca2+ by adsorption to metal oxide nanoparticles. When the adsorption effect was blocked by the pretreatment of particles with FBS, the inhibitory effect was lost. However, in NiO and ZnO, which showed ion release, a decrease of inhibitory effect by pretreatment was not shown. Furthermore, the association of the primary particle size and adsorption ability was examined in TiO2. The adsorption ability of TiO2 depended on the primary particle size. The TiO2 nanoparticles were size dependently absorbed with proteins and Ca2+, thereby inducing cytotoxicity. In conclusion, the adsorption ability of metal oxide nanoparticles is an important factor for the estimation of cytotoxicity in vitro for low-toxicity materials.

Nuclear localization of STAT5A modified with O-linked N-acetylglucosamine and early involution in the mammary gland of Hirosaki hairless rat.

Hirosaki hairless rat (HHR) is a mutant strain spontaneously derived from Sprague-Dawley rats (SDR), and its inheritance is autosomal recessive. In addition to hair loss, female HHRs show involution of the mammary gland at an early stage of lactation. In the present study we investigated the mammary gland development in HHR. Morphological examinations revealed that HHR mammary glands are underdeveloped in virgins and exhibit distended alveoli on day 1 of lactation (L1), followed by involution. Milk secretion was observed on L1 in HHR. Whey acidic protein and other proteins were increased in milk of HHR and heterozygous rats on SDS-polyacrylamide gel electrophoresis. Terminal deoxynucleotide transferase-mediated dUTP nick-end labeling assay revealed apoptosis induction in HHRs at an early stage of lactation. By Western blotting, signal transducer and activator of transcription (STAT) 5A levels in cytoplasmic and nuclear fractions of the mammary glands were not different between HHR and SDR on L1 and L7. Nuclear localization of STAT5A in HHR and SDR was confirmed by immunohistochemistry. Tyr-phosphorylated STAT5A was not detected in HHR but was detected in SDR nuclear fractions. Several proteins modified with O-linked N-acetylglucosamine (O-GlcNAc) were detected in HHR nuclear extract on L1, although not in SDR or heterozygous rats by Western blotting. When HHR nuclear extract was applied to wheat germ agglutinin-agarose, a part of STAT5A was recovered in bound fractions. STAT5A of SDR or heterozygous rat nuclei were not bound to the lectin. Electrophoretic mobility shift assay revealed that STAT5A modified with O-GlcNAc is bound to the STAT5-responsive element. These results indicate that the mammary glands of HHR showed terminal differentiation for a short period, followed immediately by involution. In HHR, STAT5A is modified with O-GlcNAc but is not Tyr-phosphorylated. This type of glycosylation is suggested to be involved in the transient activation of STAT5A in HHR.