Indeed, Ab induces cell death via p75NTR in various types of cells, including neurons

the active ingredients for bone metabolism have not been identified. To address this question is useful for not only developing the high efficient dietary supplements but also finding novel drug seeds. Here we show that delphinidin is the most potent inhibitor of osteoclastogenesis and will be an effective agent for preventing in vivo bone degradation. Materials and Methods Anthocyanins and Anthocyanidins Bilberon-25, a concentrated extract of bilberry, was purchased from Tokiwa Phytochemical Co., Ltd.. Cassis extract-35, a concentrated extract of blackcurrant, was generously donated by Tama Biochemical Co., Ltd.. Cyanidin chloride, delphinidin chloride and peonidin chloride were from Extrasynthese and Sigma-Aldrich, respectively. Epicatechin was purchased from Kurita Analysis Service Co., Ltd.. Cell Culture RAW264.7 cells, a mouse macrophage cell line, were used as osteoclast precursor cells and maintained in a modified essential medium supplemented with 10% fetal bovine serum at 37uC and 5% CO2. For osteoclast induction, cells were plated in a 96-well plate at a density of 46103 cells/well and stimulated with 100 ng/ml RANKL for 4 days. For the inhibition study, cells were pre-incubated in a-MEM supplemented with vehicle or with various concentrations of anthocyanin-rich extracts and anthocyanidins, 1 h before the addition of RANKL. To confirm multinucleated osteoclast formation, the cultured cells were fixed in 10% formalin for 3 minutes, and then stained with an osteoclast marker enzyme, tartrate-resistant acid phosphatase. Effects of anthocyanins and anthocyanidins on osteoclast formation were evaluated by morphological observations and the intensity of TRAP staining was measured at 520 nm using a spectrophotometer. Osteoblasts were isolated from newborn get GSK-126 calvariae of C57BL/6J mice, as described previously with slight modifications. Briefly, calvariae were minced and sequentially digested with collagenase solution at 37uC. Cells retrieved from the osteogenic cell fractions were separately cultured in a-MEM supplemented with 10% FBS and antibiotics. After 24 h, cells were pooled and grown in multi-well plates in the same medium containing 50 mg/ ml of ascorbic acid, 10 mM dexamethasone and 10 mM b-glycerophosphate with or without anthocyaninrich extracts. After two weeks culture, cells were stained with von Kossa’ s staining to determine the matrix mineralization, as described previously. . Twelve mice were intraperitoneally injected with GST-RANKL twice at interval of 3 days. First injection was performed at 3 days after starting of delphinidin-treatment. For six mice, delphinidin treatment via gavage started 3 days before the first injection of GST-RANKL, and continued for 14 days. Another six mice received the same volume of vehicle, a mixture of dimethyl sulfoxide and water. We further assessed the effect of delphinidin using OVX mice. Eight-week-old female C57BL/6 mice were purchased from Charles River Laboratory Japan. Thirty PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19655565 mice were either sham-operated or OVX. OVX mice were divided into four groups: OVX control, low-dose delphinidin, intermediate-dose delphinidin, and high-dose delphinidin groups. After OVX, delphinidin was administrated orally, as mentioned above, for 28 days. OVX-control mice received the same volume of vehicle, a mixture of DMSO and water. All mice were housed in an animal room with free access to food and water. At the end of treatment, the mice were sacrificed. Femora were removed and fixed with 3.7%