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.

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.

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.

Protective effect of S-allyl-L-cysteine, a garlic compound, on amyloid beta-protein-induced cell death in nerve growth factor-differentiated PC12 cells.

Aged garlic extract (AGE) contains several neuroactive compounds, including S-allyl-L-cysteine (SAC) and allixin. We characterized cell death induced by amyloid beta-protein (Abeta), 4-hydroxynonenal (HNE), tunicamycin, an endoplasmic reticulum (ER) stressor, or trophic factor deprivation, and investigated whether and how SAC could prevent this in nerve growth factor (NGF)-differentiated PC12 cells, a model of neuronal cells. Exposure of the cells to amyloid beta-protein(1-40) (Abeta(1-40)) decreased the extent of [3-(4,5)-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium (MTT) reduction activity and loss of neuronal integrity, but these effects were not prevented by Ac-DEVD-CHO, a caspase-3 inhibitor. Simultaneously applied SAC protected the cells against Abeta-induced cell death in a concentration-dependent manner. It also protected them against tunicamycin-induced neuronal death. In contrast, it afforded no protection against cell death induced by HNE and trophic factor deprivation, which is mediated by a caspase-3-dependent pathway. These results suggest that SAC may selectively protect cell death induced by Abeta and tunicamycin, which may be triggered by ER dysfunction in NGF-differentiated PC12 cells.