Oral supplementation with collagen hydrolysate (CH) has been shown to improve the condition of the skin in humans and experimental animals. Several hydroxyproline-containing oligo-peptides were previously detected in human peripheral blood after the ingestion of CH, and the two dipeptides, prolyl-hydroxyproline (PO) and hydroxyprolyl-glycine (OG), have been proposed to have beneficial effects on human health. When HR-1 hairless mice were fed a HR-AD diet, which lacked magnesium and zinc, transepidermal water loss (TEWL) increased and water content of stratum corneum decreased. In the present study, we investigated the effects of dietary PO and OG on skin barrier dysfunction in HR-1 hairless mice. Mice were fed a HR-AD diet with or without PO (0.15%) and OG (0.15%) for 35 consecutive days. The administration of PO and OG significantly decreased TEWL, and significantly increased water content of stratum corneum. A DNA microarray analysis of the dorsal skin revealed differences in gene expression between the group administered PO and OG and the control group. We also identified muscle-related Gene Ontology as a result of analyzing the up-regulated genes. These results suggested that the administration of PO and OG improved skin barrier dysfunction and altered muscle-related gene expression.

Prolyl-hydroxyproline (Pro-Hyp) is one of the major constituents of collagen-derived dipeptides. The objective of this study was to investigate the effects of Pro-Hyp on the proliferation and differentiation of MC3T3-E1 osteoblastic cells. Addition of Pro-Hyp did not affect MC3T3-E1 cell proliferation and matrix mineralization but alkaline phosphatase activity was significantly increased. Furthermore, cells treated with Pro-Hyp significantly upregulated gene expression of Runx2, Osterix, and Col1α1. These results indicate that Pro-Hyp promotes osteoblast differentiation. This study demonstrates for the first time that Pro-Hyp has a positive effect on osteoblast differentiation with upregulation of Runx2, Osterix, and Collα1 gene expression.

Prolyl-hydroxyproline (Pro-Hyp) is one of the major constituents of collagen-derived dipeptides. We previously reported that Pro-Hyp promotes the differentiation of osteoblasts by increasing Runx2, osterix and Col1α1 mRNA expression levels. Here, to elucidate the mechanism of Pro-Hyp promotion of osteoblast differentiation, we focus on the involvement of Foxo1 in osteoblast differentiation via Runx2 regulation and the role of Foxg1 in Foxo1 regulation. The addition of Pro-Hyp had no effect on MC3T3-E1 cell proliferation in Foxo1- or Foxg1-knockdown cells. In Foxo1-knockdown cells, the addition of Pro-Hyp increased ALP activity, but in Foxg1-knockdown cells, it had no effect on ALP activity. An enhancing effect of Pro-Hyp on the Runx2 and osterix expression levels was observed in Foxo1-knockdown cells. However, no enhancing effect of Pro-Hyp on osteoblastic gene expression was observed when Foxg1 was knocked down. These results demonstrate that Pro-Hyp promotes osteoblastic MC3T3-E1 cell differentiation and upregulation of osteogenic genes via Foxg1 expression.

Collagen-derived dipeptide prolyl hydroxyproline directly binds to Foxg1 to change its conformation and inhibit the interaction with Runx2 Collagen-derived dipeptide prolyl hydroxyproline (Pro-Hyp) is involved in the proliferation and differentiation of various types of cultured cells. To elucidate the mechanism underlying Pro-Hyp actions during osteoblast differentiation, we hypothesized that proteins binding to Pro-Hyp serve to mediate cellular signaling, affecting Runx2 expression. Recently, we performed the characterization of Foxg1, that it enhances Runx2 expression in the presence of Pro-Hyp. Our findings indicate that Pro-Hyp directly binds to the Foxg1 recombinant protein, which leads to the structural alteration of the Foxg1 protein. In addition, Foxg1 appears to interact with Runx2 in the absence of Pro-Hyp, with Pro-Hyp disrupting the interaction between Foxg1 and Runx2. Collectively, our results indicate that the Pro-Hyp bound Foxg1 alters the structured conformation of Foxg1, resulting in conformational changes that lead to dissociation from Runx2. These novel findings suggest that during osteoblast differentiation, Pro-Hyp mediates Runx2 activity though directly binding to Foxg1 and increases Runx2 expression.

Mangiferin is a polyphenolic compound present in Salacia reticulata. It has been reported to reduce bone destruction and inhibit osteoclastic differentiation. This study aimed to determine whether mangiferin directly affects osteoblast and osteoclast proliferation and differentiation, and gene expression in MC3T3-E1 osteoblastic cells and osteoclast-like cells derived from primary mouse bone marrow macrophage cells. Mangiferin induced significantly greater WST-1 activity, indicating increased cell proliferation. Mangiferin induced significantly increased alkaline phosphatase staining, indicating greater cell differentiation. Reverse transcription-polymerase chain reaction (RT-PCR) demonstrated that mangiferin significantly increased the mRNA level of runt-related transcription factor 2 (RunX2), but did not affect RunX1 mRNA expression. Mangiferin significantly reduced the formation of tartrate-resistant acid phosphatase-positive multinuclear cells. RT-PCR demonstrated that mangiferin significantly increased the mRNA level of estrogen receptor β (ERβ), but did not affect the expression of other osteoclast-associated genes. Mangiferin may inhibit osteoclastic bone resorption by suppressing differentiation of osteoclasts and promoting expression of ERβ mRNA in mouse bone marrow macrophage cells. It also has potential to promote osteoblastic bone formation by promoting cell proliferation and inducing cell differentiation in preosteoblast MC3T3-E1 cells via RunX2. Mangiferin may therefore be useful in improving bone disease outcomes.

BackgroundRecent studies suggest that some of the clinical effectiveness of soy or daidzein, which is a type of isoflavone, may be attributed to a person’s ability to produce equol from daidzein. Equol, which is a metabolite of one of the major soybean isoflavones called daidzein, is produced in the gastrointestinal tract by certain intestinal microbiota where present. Habitual dietary patterns may alter the intestinal bacterial profile, and influence the metabolism of isoflavones and the production of equol. Fructooligosaccharides (FOS) have a prebiotic activity as well as being a dietary fibre. The purpose of the present study was to determine whether FOS supplementation increases equol production in equol producers and stimulates equol production in equol non-producers in Japanese postmenopausal women.MethodsA soy challenge was used to assess equol-producer status prior to the start of the study in healthy postmenopausal Japanese women. The study involved 4 separate groups in randomised crossover design. First, subjects were classified as equol producers (n = 25) or non-producers (n = 18), and then they were randomly assigned to the FOS or control group. All subjects received a daily dose of 37 mg isoflavone conjugates in the capsule (21 mg aglycone form) and either FOS (5g/day) or sucrose as control, in a randomised crossover study design. Equol -production was assessed by testing the serum and urine before and after the 2-week supplementation period.ResultsThe analyses were conducted on 34 subjects completed the study, 21 (61.8%) were classified as equol producers, and 13 (38.2%) as non-producers. Significant differences were observed in the interaction effect of time × equol state after 1 week of intervention (p = 0.006). However there were no effects after 2 weeks of intervention (p = 0.516). Finally, in both equol producers and non-producers, FOS supplementation did not affect the serum equol concentration or the urinary equol to daidzein concentration ratios.ConclusionsWe have reported that FOS intervention (5 g/day for 2 weeks) does not significantly modulate the capacity of intestinal microbiota to produce equol in postmenopausal Japanese women, in either equol producers or non-producers in this pilot study. Further larger investigations that explore the roles of specific intestinal microbiota in equol production will enable the establishment of dietary conditions that are required to enhance equol production.

This study demonstrates clearly that nomilin has inhibitory effects on osteoclastic differentiation in vitro. These findings indicate that nomilin-containing herbal preparations have potential utility for the prevention of bone metabolic diseases.

We compared the effects of the S-enantiomer and racemic forms of equol on bone using ovariectomized (OVX) mice. Femoral bone mineral density and bone strength decreased in the OVX mice, but not in OVX mice administered 0.5 mg/d S-equol. This, however, did not hold for racemic equol. Serum and urine S-equol concentrations were higher in the mice administered S-equol than in those administered racemic equol. These results suggest that the inhibitory effects of S-equol on bone fragility in OVX mice are greater than those of racemic equol.