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33646054301
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2. Osteoblast differentiation was induced by changing the medium containing 50 μg/ml ascorbic acid and cells were treated with vehicle or compounds to be tested. After 3 days, medium was changed with fresh medium containing each compound and maintained for further 4 days.
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33646026748
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2O 50:20:30, 2 ml/min, rt: 14.30 and 13.04 min, respectively) to yield compounds 2 (15 mg) and 3 (4 mg). Fr. 5 was applied on Sephadex LH-20 (25-100 μm, Pharmacia) using MeOH to yield compound 6 (4 mg). The purity of each compound was higher than 95.0%, respectively, as measured by HPLC.
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33646034091
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note
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For the assessment of ALP activity, cells were rinsed with phosphate-buffered saline and lysed in 0.01% sodium dodecyl sulfate in PBS followed by sonication. After clarification by centrifugation, cell lysates were assayed for ALP activity using the Alkaline Phosphate Assay Kit (Youngdong Pharmaceutical Co., Korea). Each value was normalized with the protein content of cell lysate, measured using bicinchoninic acid with bovine serum albumin as a standard. The evaluation of statistical significance was determined by the one-way ANOVA with a value of p < 0.05 or less considered to be statistically significant.
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29
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33646020433
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note
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Biflavonoids to be tested were dissolved in dimethylsulfoxide (DMSO). Our preliminary study showed that DMSO at a final concentration of 0.1% in media did not affect the cell viability or differentiation. Osteoblast differentiation was induced by changing the medium containing 50 μg/ml ascorbic acid and cells were then treated with vehicle or compounds to be tested. After 3 days, medium was changed with fresh medium containing each compound and maintained for further 4 days.
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30
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33646035266
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2 for 2 h in the dark.
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31
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33646068802
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Sowa H., Kaji H., Yamaguchi T., Sugimoto T., and Kazuo C. J. Biol. Chem. 39 (2002) 36204
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Sowa, H.1
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Kazuo, C.5
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32
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33646027646
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note
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To evaluate the effects of biflavonoids on collagen synthesis, osteoblasts were treated with vehicle or biflavonoids to be tested in the presence of ascorbic acid and grown for 2 weeks by changing the medium containing compounds every 3 days.
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33
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33646049755
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Cultured osteoblasts were washed with PBS, followed by fixation with Bouin's fluid for 1 h. After fixation, the fixation fluid was removed and the culture dishes were washed by immersion in running tap water for 15 min. The culture dishes were air-dried and stained by Sirius red dye reagent for 1 h under mild shaking on a shaker. Thereafter, the solution was removed and the cultures were washed with 0.01 N HCl to remove nonbound dye. The stained material was dissolved in 0.1 N NaOH and absorbance was measured at 550 nm against 0.1 N NaOH as a blank.
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35
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33646022293
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note
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To evaluate the effects of biflavonoids on calcium deposition, osteoblasts were treated with vehicle or biflavonoids to be tested in the presence of 50 μg/ml ascorbic acid and 10 mM β-glycerophosphate, and grown for 2 weeks by changing the medium containing compounds every 3 days.
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36
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33646037395
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note
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For Alizarin red staining, the cells were rinsed with PBS and fixed with ice-cold 70% ethanol for 1 h. The ethanol was removed and rinsed with deionized water. The cells were then stained with 40 mM Alizarin red S in deionize d water (adjusted to pH 4.2) for 10 min at room temperature. The Alizarin red S solution was removed and rinsed with deionized water and PBS. The stained material was extracted in DMSO and the absorbance was measured at 562 nm.
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