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 ERβ in the phytoestrogen activity of these anthocyanins. Blackcurrant anthocyanin induced ERβ-mediated transcriptional activity, and the IC₅₀ of ERβ was lower than that of ERα, indicating that blackcurrant anthocyanins have a higher binding affinity to ERβ. In silico docking analysis of cyanidin and delphinidin, the core portions of the compound that fits within the ligand-binding pocket of ERβ, showed that similarly to 17β-estradiol, hydrogen bonds formed with the ERβ residues Glu305, Arg346, and His475. No fitting placement of glucoside or rutinoside sugar chains within the ligand-binding pocket of ERβ-estradiol complex was detected. However, as the conformation of helices 3 and 12 in ERβ 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 ERβ docking structures revealed that the selectivity for ERβ is higher than that for ERα, similar to genistein. These results show that blackcurrant anthocyanins exert phytoestrogen activity via ERβ.

Blackcurrants (Ribes nigrum L.) contain high levels of anthocyanin polyphenols, which have beneficial effects on health, owing to their antioxidant and anticarcinogenic properties. Phytoestrogens are plant-derived substances with estrogenic activity, which could have beneficial effects on the skin. Estradiol secretion decreases during menopause, reducing extracellular matrix (ECM) component production by skin fibroblasts. Using a normal human female skin fibroblast cell line (TIG113) and ovariectomized rats, the present study investigated whether an anthocyanin-rich blackcurrant extract (BCE) and four blackcurrant anthocyanins have novel phytoestrogenic activities that could benefit the skin in menopausal women. In TIG113 cells, a microarray and the Ingenuity^® Pathway Analysis showed that 1.0 μg/mL of BCE upregulated the expression of many estrogen signaling-related genes. A quantitative RT-PCR analysis confirmed that BCE (1.0 or 10.0 μg/mL) and four types of anthocyanins (10 μM) altered the mRNA expression of ECM proteins and enzymes involved in ECM turnover. Immunofluorescence staining indicated that the anthocyanins stimulated the expression of ECM proteins, such as collagen (types I and III) and elastin. Dietary administration of 3% BCE to ovariectomized rats for 3 months increased skin levels of collagen, elastin, and hyaluronic acid. This is the first study to show that blackcurrant phytoestrogens have beneficial effects on skin experimental models.

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.

Most models of hereditary hypotrichosis are due to alterations in growth factors and transcription factors, and the examples of causative mutations in hair keratin genes are limited. The Hirosaki hairless rat (HHR) is a mutant strain spontaneously derived from Sprague-Dawley rats (SDRs). In this study, the locus of the responsible gene was examined by linkage analysis and mapped on chromosome 7q36. Because many basic keratin genes are clustered on 7q36, their expression was examined. Reverse transcription-PCR and genomic PCR indicated that the Kb21 (Krt81), -23 (Krt83), and -26 (Krt86) genes encoding basic hair keratins were not expressed and were deleted. Furthermore, 80-kb genomic DNA ranging from exon 9 of Kb25 (Krt85) to exon 9 of Krt2-25 was deleted. The breakpoints of these genes were within a 95-bp portion shared by the two genes, suggesting that deletion due to non-allelic homologous recombination occurred. Proteins identified as Kb21, Kb23, and Krt2-25 in SDR hairs by mass spectrometry were not detected in HHR. Instead, the product of a fusion gene became dominant in HHR. Because fusion occurred between the exons of the two genes with the same sequences, the product was identical to the wild-type Kb25 protein. By using immunohistochemistry, Kb21 was not detected in HHR hair follicles. Kb25 was expressed in the cortex in HHRs, whereas it was in the medulla in SDRs. This study clearly illustrates the importance of hair keratin genes in hair growth.