[보고서]길경과 갈근을 이용한 골 형성 촉진  및 골다공증 예방 기능성 식품 소재 개발

정혜광

보고서 정보

주관연구기관

충남대학교 산학협력단

연구책임자

정혜광

참여연구자

한은희 , 박진희 , 정형민 , 임지혜 , 도민트롱 , 딜락

보고서유형

최종보고서

발행국가

대한민국

언어

한국어

발행년월

2011-04

과제시작연도

2010

주관부처

보건복지부

사업 관리 기관

한국보건산업진흥원
Korea Health Industry Development Institute

등록번호

TRKO201400002296

과제고유번호

1355061384

사업명

보건의료기술연구개발

DB 구축일자

2014-04-19

키워드

골형성.길경.갈근.기능성 식품 소재.ossification.Platycodon grandiflorum.Pueraria Radix.functional food material.

표 Effect of puerarin, CKS and xanthohumol on alkaline phosphatase in C2C12 cells. Cells were incubated until sub-confluent and then cultured in the presence of puerarin (A; 5, 25, 100 μg/ml), CKS (B; 1 and 2 μg/ml), and xanthohumol (C; XN; 0.01, 0.1, 1 μg/ml). On day 4, the cells were subjected to ALP staining. This figure is representative of three independent experiments.
표 Effect of CKS and xanthohumol on von Korsa staining in C2C12 cells. Cells were incubated until sub-confluent and then cultured in the presence of CKS (A; 2 μg/ml) or xanthohumol (B; XN; 0.1 and 1 μg/ml) and/or BMP2 (5 ng/ml). On day 4, the cells were subjected to von Korsa staining. This figure is representative of three independent experiments.
표 Effect of CKS and xanthohumol on Alizarin red S staining in C2C12 cells. Cells were treated with 1, 10, or 100 ng/mL or 1 μg/mL of xanthohumol and /or BMP2 (5 ng/ml). On day 14, the cells were subjected to Alizarin red S staining. Each figure is representative of three independent experiments.
표 Effect of CKS on gene expression. C2C12 cells were treated with CKS (1, 2, and 4 μ g/ml) for 48 h. The cells were lysed and total RNA was prepared for analysis of ALP, RANK, RANKL and OPG gene expression using RT–PCR. The mRNA expression in treated cells was compared to the expression in untreated cells at each time point. Each bar shows three independent experiments.
표 Xanthohumol increases the expression of osteogenic differentiation factors and RUNX2. (A) C2C12 cells were treated with xanthohumol (100 ng/mL or 1 μg/mL) and/or BMP2 (5 ng/ml). Total RNA was extracted from the cell lysates, and cDNA was prepared from 1 mg of the total RNA. RT-PCR was performed using the cDNA and primers for ALP, collagen type 1a, BSP, OC, and GAPDH (internal control). (B) C2C12 cells were treated with xanthohumol (100 ng/mL) and/or BMP2 (5 ng/ml) in the absence or presence of Runx2. Total RNA was extracted from the cell lysates, and cDNA was prepared from 1 mg of the total RNA. RT-PCR was performed using the cDNA and primers RUNX2, Dlx5, and GAPDH (internal control).
표 Effect of puerarin, CKS, and xanthohumol on OC luciferase activity in C2C12 cells. (A-C) Cells were transfected with the OC-Luc plasmid and treated with puerarin (A; 5, 25, 100 g/ml), CKS (B; 1, 2, and 4 g/ml), xanthohumol μ μ (C; XN, 0.01, 0.1 and 1 μg/mL). Next, luciferase reporter assay was performed 48 h later. (D) Xanthohumol stimulates OC promoter-driven transcription in a time-dependent manner. Each bar represents three independent experiments. *p < 0.05 versus control.
표 Effect of puerarin, CKS, and xanthohumol on ALP luciferase activity in C2C12 cells. Cells were transfected with the ALP-Luc plasmid and treated with puerarin (A; 5, 25, 100 μ g/ml), CKS (B; 1, 2, and 4 g/ml), xanthohumol (C; XN, 0.01, μ 0.1 and 1 μg/mL). Next, luciferase reporter assay was performed 48 h later. Each bar represents three independent experiments. *p < 0.05 versus control.
표 Effect of xanthohumol on BSP luciferase activity in C2C12 cells. Cells were transfected with the BSP-Luc plasmid and treated with xanthohumol (C; XN, 0.01, 0.1 and 1 μg/mL). Next, luciferase reporter assay was performed 48 h later. Each bar represents three independent experiments. *p < 0.05 versus control.
표 Effect of puerarin on RUNX2 protein expression in C2C12 cells. C2C12 cells were transfected with an expression plasmid for HA-tagged RUNX2 and then treated with puerarin at various concentrations. The expression level of RUNX2 (upper panel) was determined by Western blot analysis using anti-HA antibody. Tubulin (lower panel) was used as a loading control. Each blot shown is representative of three independent experiments giving similar results.
표 Effect of CKS on RUNX2 protein expression in C2C12 cells. C2C12 cells were transfected with an expression plasmid for HA-tagged RUNX2 and then treated with CKS at various concentrations. The expression level of RUNX2 (upper panel) was determined by Western blot analysis using anti-HA antibody. Tubulin (lower panel) was used as a loading control. Each blot shown is representative of three independent experiments giving similar results.
표 Effect of xanthohumol (XN) on RUNX2 protein expression in C2C12 cells. C2C12 cells were transfected with an expression plasmid for HA-tagged RUNX2 and then treated with XN at various concentrations. The expression level of RUNX2 (upper panel) was determined by Western blot analysis using anti-HA antibody. Tubulin (lower panel) was used as a loading control. Each blot shown is representative of three independent experiments giving similar results.
표 Effect of puerarin, CKS and xanthohumol (XN) on transcriptional activity of RUNX2 in C2C12 cells. C2C12 cells were transfected with an expression plasmid for HA-tagged RUNX2 and with OC-Luc plasmid, and a luciferase reporter assay was performed 48 h later. Puerarin (A), CKS (B) and xanthohumol (XN; C) stimulated the transcriptional activity of RUNX2 at the OC promoters. Each bar shows three independent experiments. *p < 0.05 versus RUNX2.
표 Effect of puerarin, CKS and xanthohumol (XN) on transcriptional activity of RUNX2 in C2C12 cells. C2C12 cells were transfected with an expression plasmid for HA-tagged RUNX2 and with BSP-Luc plasmid, and a luciferase reporter assay was performed 48 h later. Puerarin (A), CKS (B) and xanthohumol (XN; C) stimulated the transcriptional activity of RUNX2 at the BSP promoters. Each bar shows three independent experiments. *p < 0.05 versus RUNX2.
표 Effect of puerarin, CKS and xanthohumol (XN) on transcriptional activity of RUNX2 in C2C12 cells. C2C12 cells were transfected with an expression plasmid for HA-tagged RUNX2 and with OSE-Luc plasmid, and a luciferase reporter assay was performed 48 h later. Puerarin (A), CKS (B) and xanthohumol (XN; C) stimulated the transcriptional activity of RUNX2 at the OSE promoters. Each bar shows three independent experiments. *p < 0.05 versus RUNX2.
표 Effect of RANKL on osteoclast related factor gene expression. C2C12 cells were treated with RANKL for 1, 2, 3, 4, and 5 days. The cells were lysed and total RNA was prepared for analysis of TRAP, NFATc1, c-Fos, Cathepsin K, OSCAR, and HPRT gene expression using RT–PCR. The mRNA expression in treated cells was compared to the expression in untreated cells at each time point.
표 Effect of CKS and puerarin on osteoclast related factor gene expression. C2C12 cells were pretreated with CKS (2 ug/ml) and puerarin (100 uM) and treated with RANKL for 1, 2, 3, 4, and 5 days. The cells were lysed and total RNA was prepared for analysis of TRAP, NFATc1, and HPRT gene expression using RT PCR. – The mRNA expression in treated cells was compared to the expression in untreated cells at each time point.
표 Effect of CKS and puerarin on osteoclast related factor gene expression. C2C12 cells were pretreated with CKS (2 ug/ml) and puerarin (100 uM) and treated with RANKL for 1, 2, 3, 4, and 5 days. The cells were lysed and total RNA was prepared for analysis of c-Fos, Cathepsin K, and HPRT gene expression using RT–PCR. The mRNA expression in treated cells was compared to the expression in untreated cells at each time point.
표 Effect of CKS and puerarin on MMP-9 secretion in RAW 264.7 cells. Conditioned media was collected and analyzed for the production of MMP-9. Zymography assays were performed, and the gelatinolytic activity of active MMP-9 (92 kDa) was visualized as clear bands against a blue background of stained gelatin.
표 Effect of CKS and puerarin on osteoclast related factor gene expression. C2C12 cells were pretreated with CKS (2 ug/ml) and puerarin (100 uM) and treated with RANKL for 1, 2, 3, 4, and 5 days. The cells were lysed and total RNA was prepared for analysis of OSCAR and HPRT gene expression using RT PCR. The mRNA – expression in treated cells was compared to the expression in untreated cells at each time point.
표 The CKS-enhanced transcriptional activity of RUNX2 was decreased by p38 MAPK and ERK signaling inhibition. (A) C2C12 cells were transfected with an expression plasmid for HA-tagged RUNX2 and then treated with CKS in the absence or presence of kinase inhibitors (SB203580, U0126, and H89). RT-PCR was performed using primers for OC, ALP, ColIa, and BSP with the cDNA produced from 1 μg of total RNA. GAPDH was used as a control.
표 Effect of upstream signal proteins activation on xanthohumol (XN)-enhanced transcriptional activity of RUNX2. C2C12 cells were transfected with an expression plasmid for HA-tagged RUNX2 and then treated with xanthohumol in the absence or presence of kinase inhibitors (SB203580, U0126, H89, AKT inhibitor). RT-PCR was performed using primers for Runx2, Dlx5, ALP, ColI α1, and BSP with the cDNA produced from 1 μg of total RNA. GAPDH was used as a control.
표 Effect of upstream signal proteins activation on puerarin-enhanced transcriptional activity of RUNX2. C2C12 cells were transfected with an expression plasmid for HA-tagged RUNX2 and with the OC-Luc plasmid and treated with puerarin in the absence or presence of kinase inhibitors (SB203580, U0126 and H89). Each bar represents three independent experiments. *, p < 0.05, versus RUNX2. **,p < 0.05, versus Runx2 + puerarin.
표 Effect of upstream signal proteins activation on CKS-enhanced transcriptional activity of RUNX2. C2C12 cells were transfected with an expression plasmid for HA-tagged RUNX2 and with the OC-Luc plasmid and treated with CKS in the absence or presence of kinase inhibitors (SB203580, U0126 and H89). Each bar represents three independent experiments. *, p < 0.05, versus RUNX2. **, p < 0.05, versus Runx2 + CKS.
표 Effect of upstream signal proteins activation on xanthohumol (XN)-enhanced transcriptional activity of RUNX2. C2C12 cells were transfected with an expression plasmid for HA-tagged RUNX2 and with the OC-Luc plasmid and treated with XN in the absence or presence of kinase inhibitors (SB203580, U0126, H89, and AKT inhibitor). Each bar represents three independent experiments. *, p < 0.05, versus RUNX2.**,p < 0.05, versus Runx2 + xanthohumol (XN).
표 Effect of upstream signal proteins activation on CKS-enhanced transcriptional activity of RUNX2. C2C12 cells were transfected with an expression plasmid for HA-tagged RUNX2 and with the BSP-Luc plasmid and treated with CKS in the absence or presence of kinase inhibitors (SB203580, U0126, and H89). Each bar represents three independent experiments. *, p < 0.05, versus RUNX2.**,p < 0.05, versus Runx2 + CKS.
표 Effect of upstream signal proteins activation on CKS-enhanced transcriptional activity of RUNX2. C2C12 cells were transfected with an expression plasmid for HA-tagged RUNX2 and with the OSE-Luc plasmid and treated with CKS in the absence or presence of kinase inhibitors (SB203580, U0126, and H89). Each bar represents three independent experiments. *, p < 0.05, versus RUNX2.**,p < 0.05, versus Runx2 + CKS.
표 Effect of upstream signal proteins activation on xanthohumol (XN)-enhanced transcriptional activity of RUNX2. C2C12 cells were transfected with an expression plasmid for HA-tagged RUNX2 and with the OSE-Luc plasmid and treated with XN in the absence or presence of kinase inhibitors (SB203580, U0126, H89, and AKT inhibitor). Each bar represents three independent experiments. *, p < 0.05, versus RUNX2.**,p < 0.05, versus Runx2 + xanthohumol (XN).
표 Effect of upstream signal protein PKA activation on puerarin-enhanced transcriptional activity of RUNX2. C2C12 cells were transfected with an expression vector for HA-tagged RUNX2, pretreated with PKA inhibitor (H89), and then treated with puerarin. The expression level of RUNX2 (upper panel) was determined by Western blot analysis using anti-HA antibody. Tubulin (lower panel) was used as a loading control. Each blot shown is representative of three independent experiments with similar results.
표 Effect of upstream signal proteins activation on CKS-enhanced transcriptional activity of RUNX2. C2C12 cells were transfected with an expression vector for HA-tagged RUNX2, pretreated with inhibitors (SB 203580, U0126, and H89), and then treated with CKS. The expression level of RUNX2 (upper panel) was determined by Western blot analysis using anti-HA antibody. Tubulin (lower panel) was used as a loading control. Each blot shown is representative of three independent experiments with similar results.
표 Effect of upstream signal proteins activation on xanthohumol (XN)-enhanced transcriptional activity of RUNX2. C2C12 cells were transfected with an expression vector for HA-tagged RUNX2, pretreated with inhibitors (SB 203580, U0126, H89, and Akt inhibitor), and then treated with XN. The expression level of RUNX2 (upper panel) was determined by Western blot analysis using anti-HA antibody. Tubulin (lower panel) was used as a loading control. Each blot shown is representative of three independent experiments with similar results.
표 Effect of upstream signal proteins activation on CKS-enhanced transcriptional activity of RUNX2. Non-transfected or RUNX2-transfected C2C12 cells were incubated until sub-confluent and then cultured with 2 μg/ml of CKS and BMP2 (5 ng) in the absence or presence of inhibitors (SB, U0126, and H89), followed by ALP staining on day 4. This figure is representative of three independent experiments.
표 Effect of upstream signal proteins activation on xanthohumol (XN)-enhanced transcriptional activity of RUNX2. Non-transfected or RUNX2-transfected C2C12 cells were incubated until sub-confluent and then cultured with XN and BMP2 (5 ng) in the absence or presence of inhibitors (SB, U0126, H89, and Akt inhibitor), followed by ALP staining on day 4. This figure is representative of three independent experiments.
표 Xanthohumol stimulates osteogenic differentiation via the activation of RUNX2 by the p38 MAPK and ERK signaling pathway. (A) Xanthohumol increases the expression of ERK and p38 of the phosphorylation form. C2C12 cells were treated with xanthohumol at various concentrations. The expression level of the phospho-form or total protein on ERK or p38 was determined by Western blot analysis using each antibody. Tubulin was used as a loading control.
표 Experimental Protocol. Female ICR mice with 7 weeks of age were purchased from the Samtako and acclimated to conditions for 1 week before the experiment. The mice were either sham or OVX operated at the age of 8 weeks. After recovering for 3 weeks, the mice were randomized into the following four groups: (1) sham-operated mice fed on saline or 0.5% CMC contained 1% EtOH; (2) ovariectomized mice fed on saline or 0.5% CMC contained 1% EtOH; (3) ovariectomized mice fed on CKS 0.5 mg/kg or Puerarin 50 mg/kg; and (4) ovariectomized mice fed on CKS 2 mg/kg or Puerarin 200 mg/kg. The mice were sacrificed 5 weeks after the operation.
표 Effect of CKS or Puerarin on serum calcium levels were assessed by calcium kit. Three weeks after surgery, ovariectomized mice fed on CKS or puerarin group was orally administered at a dose of 0.5 and 2 mg/kg or 50 and 200 mg/kg and control group was orally administered saline. The serum Ca level was used as markers of bone formation. Each value represents the mean ± SD of five mice. #P<0.05, significantly different from the sham group, *P<0.05, significantly different from the OVX group.
표 Effect of CKS or Puerarin on serum PI levels were assessed by PI kit. Three weeks after surgery, ovariectomized mice fed on CKS or Puerarin group was orally administered at a dose of 0.5 and 2 mg/kg or 50 and 200 mg/kg and control group was orally administered saline. The serum PI level was used as markers of bone formation. Each value represents the mean ± SD of five mice. #P<0.05, significantly different from the sham group, *P<0.05, significantly different from the OVX group.
표 Effect of CKS or Puerarin on serum ALP levels were assessed by ALP kit. Three weeks after surgery, ovariectomized mice fed on CKS or Puerarin group was orally administered at a dose of 0.5 and 2 mg/kg or 50 and 200 mg/kg and control group was orally administered saline. The serum ALP level was used as markers of bone formation. Each value represents the mean ± SD of five mice. #P<0.05, significantly different from the sham group, *P<0.05, significantly different from the OVX group.
표 Effect of CKS or Puerarin on serum osteocalcin levels were assessed by osteocalcin ELISA. Three weeks after surgery, ovariectomized mice fed on CKS or Puerarin group was orally administered at a dose of 0.5 and 2 mg/kg or 50 and 200 mg/kg and control group was orally administered saline. The serum osteocalcin level was used as markers of bone formation. Each value represents the mean ± SD of five mice. #P<0.05, significantly different from the sham group, *P<0.05, significantly different from the OVX group.
표 Effect of CKS or Puerarin on serum BMP-2 levels were assessed by BMP-2 ELISA. Three weeks after surgery, ovariectomized mice fed on CKS or Puerarin group was orally administered at a dose of 0.5 and 2 mg/kg or 50 and 200 mg/kg and control group was orally administered saline. The serum BMP-2 level was used as markers of bone formation. Each value represents the mean ± SD of five mice. #P<0.05, significantly different from the sham group, *P<0.05, significantly different from the OVX group.
표 Effect of CKS or Puerarin on body weights were assessed by weighing machine. Three weeks after surgery, ovariectomized mice fed on CKS or Puerarin group was orally administered at a dose of 0.5 and 2 mg/kg or 50 and 200 mg/kg and control group was orally administered saline. Each value represents the mean ± SD of five mice. #P<0.05, significantly different from the sham group, *P<0.05, significantly different from the OVX group.
표 Effect of CKS or Puerarin on heart weights were assessed by weighing machine. Three weeks after surgery, ovariectomized mice fed on CKS or Puerarin group was orally administered at a dose of 0.5 and 2 mg/kg or 50 and 200 mg/kg and control group was orally administered saline. Each value represents the mean ± SD of five mice. #P<0.05, significantly different from the sham group, *P<0.05, significantly different from the OVX group.
표 Effect of CKS or Puerarin on lung weights were assessed by weighing machine. Three weeks after surgery, ovariectomized mice fed on CKS or Puerarin group was orally administered at a dose of 0.5 and 2 mg/kg or 50 and 200 mg/kg and control group was orally administered saline. Each value represents the mean ± SD of five mice. #P<0.05, significantly different from the sham group, *P<0.05, significantly different from the OVX group.
표 Effect of CKS or Puerarin on liver weights were assessed by weighing machine. Three weeks after surgery, ovariectomized mice fed on CKS or Puerarin group was orally administered at a dose of 0.5 and 2 mg/kg or 50 and 200 mg/kg and control group was orally administered saline. Each value represents the mean ± SD of five mice. #P<0.05, significantly different from the sham group, *P<0.05, significantly different from the OVX group.
표 Effect of CKS or Puerarin on spleen weights were assessed by weighing machine. Three weeks after surgery, ovariectomized mice fed on CKS or Puerarin group was orally administered at a dose of 0.5 and 2 mg/kg or 50 and 200 mg/kg and control group was orally administered saline. Each value represents the mean ± SD of five mice. #P<0.05, significantly different from the sham group, *P<0.05, significantly different from the OVX group.
표 Effect of CKS or Puerarin on kidney weights were assessed by by weighing machine. Three weeks after surgery, ovariectomized mice fed on CKS or Puerarin group was orally administered at a dose of 0.5 and 2 mg/kg or 50 and 200 mg/kg and control group was orally administered saline. Each value represents the mean ± SD of five mice. #P<0.05, significantly different from the sham group, *P<0.05, significantly different from the OVX group.
표 Effect of CKS or Puerarin on three-dimensional images were assessed by microcomputerized tomography ( CT). Three weeks after surgery, μ ovariectomized mice fed on CKS or Puerarin group was orally administered at a dose of 0.5 and 2 mg/kg or 50 and 200 mg/kg and control group was orally administered saline for 2 weeks.
표 Effect of CKS or Puerarin on three-dimensional images were assessed by microcomputerized tomography (μCT). Three weeks after surgery, ovariectomized mice fed on CKS or Puerarin group was orally administered at a dose of 0.5 and 2 mg/kg or 50 and 200 mg/kg and control group was orally administered saline for 2 weeks.
표 Effect of CKS or Puerarin on bone mineral density (BMD) was assessed by microcomputerized tomography ( CT). Three weeks after surgery, μ ovariectomized mice fed on CKS or Puerarin group was orally administered at a dose of 0.5 and 2 mg/kg or 50 and 200 mg/kg and control group was orally administered saline. Each value represents the mean ± SD of five mice. #P<0.05, significantly different from the sham group, *P<0.05, significantly different from the OVX group.
표 Effect of CKS or Puerarin on bone volume/total volume (BV/TV) was assessed by microcomputerized tomography (μCT). Three weeks after surgery, ovariectomized mice fed on CKS or Puerarin group was orally administered at a dose of 0.5 and 2 mg/kg or 50 and 200 mg/kg and control group was orally administered saline. Each value represents the mean ± SD of five mice. #P<0.05, significantly different from the sham group, *P<0.05, significantly different from the OVX group.
표 Effect of CKS or Puerarin on Structure model index (SMI) was assessed by microcomputerized tomography (μCT). Three weeks after surgery, ovariectomized mice fed on CKS or Puerarin group was orally administered at a dose of 0.5 and 2 mg/kg or 50 and 200 mg/kg and control group was orally administered saline. Each value represents the mean ± SD of five mice. #P<0.05, significantly different from the sham group, *P<0.05, significantly different from the OVX group.
표 Effect of CKS or Puerarin on Trabecular number (Tb.N) was assessed by microcomputerized tomography (μCT). Three weeks after surgery, ovariectomized mice fed on CKS or Puerarin group was orally administered at a dose of 0.5 and 2 mg/kg or 50 and 200 mg/kg and control group was orally administered saline. Each value represents the mean ± SD of five mice. #P<0.05, significantly different from the sham group, *P<0.05, significantly different from the OVX group.
표 Effect of CKS or Puerarin on Trabecular thickness (Tb.Th) was assessed by microcomputerized tomography (μCT). Three weeks after surgery, ovariectomized mice fed on CKS or Puerarin group was orally administered at a dose of 0.5 and 2 mg/kg or 50 and 200 mg/kg and control group was orally administered saline. Each value represents the mean ± SD of five mice. #P<0.05, significantly different from the sham group, *P<0.05, significantly different from the OVX group.

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길경과 갈근을 이용한 골 형성 촉진 및 골다공증 예방 기능성 식품 소재 개발
Development off functional food material containing ossification stimulation effects by Platycodon Grandiflorum and Pueraria Radix 원문보기

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길경과 갈근을 이용한 골 형성 촉진 및 골다공증 예방 기능성 식품 소재 개발 Development off functional food material containing ossification stimulation effects by Platycodon Grandiflorum and Pueraria Radix 원문보기

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