臺灣本土植物於人類成骨細胞之活性影響

Effects of Taiwanese native plants in human osteoblast cells

毛怡文(Yi-Wen Mao)

指導教授 ： 李美賢

臺北醫學大學/藥學院/藥學系(碩博士班)/博士(2015年)

摘要

在成骨作用 (osteogenesis) 的過程中，成骨細胞的生長、分化以及礦化扮演著很重要的關鍵。本論文乃利用類人類成骨細胞株 (MG-63細胞株) 建立體外活性篩選平臺，針對成骨作用過程中早期及晚期的生化指標進行臺灣本土植物之活性篩選試驗。我們藉由成骨作用相關活性試驗篩選了135種臺灣本土植物萃取物，其中包含了細胞存活率試驗 (cell viability) 、鹼性磷酸酶活性檢測 (ALP activity assay) 及骨質礦化染色分析 (alizan-red S staining)。在結果中發現有9種臺灣本土植物可顯著提高MG-63細胞株早期分化之ALP活性，以及晚期之骨礦化活性作用。其中所篩選出的兔尾草 (Uraria crinita (L.) Desv. ex DC.) 根部為傳統藥用植物，在臺灣常用作治療小兒發育不良等症狀，是良好的食補養身藥材，基於其未來的臨床運用價值，我們選用人類初代成骨細胞 (HOb cells)，針對成骨作用過程中相關之生化指標及mRNA的表現作用，以探討兔尾草95%乙醇萃取物及其成分對於HOb細胞的成骨作用活性影響。在結果中可見，兔尾草95%乙醇萃取物及其成分dalbergioidin (1) 、apigenin 6-C-β-D-apiofuranosyl(1→2)-α-D -xylopyranoside (3) 、adenosine (7) 及byzantionoside B (9)皆可透過BMP-2/Runx2 訊息傳遞路徑，進而提升HOb細胞ALP的活性、刺激OPN mRNA以及骨基質的礦化表現。進一步地，我們探討兔尾草在使用不同萃取方式下對於HOb細胞的成骨作用活性的影響，由結果中發現兔尾草之50%乙醇萃取物相較於95%乙醇萃取物更能提高HOb細胞的骨基質礦化活性表現。50%乙醇萃取之兔尾草所分離出來的六個具有成骨作用之活性成分salicylic acid (2)、apigenin 6-C-β-D-apiofuranosyl(1→2)-α-D-xylopyranoside (3)、vitexin (4)、2′-hydroxygenistein (12)、genistein (13) 及5,7-dihydroxy-3',5'-dihydroxyisoflavone (14)，顯示其可調控HOb細胞分化及礦化的活性表現。我們運用液相層析串聯質譜儀 (LC-MS/MS)的分析技術，進行指紋圖譜及定量分析實驗。亦證實了此六個活性成分在50%乙醇萃取兔尾草之活性EtOAc層之存在。希望以上之成骨活性作用及品管相關分析的研究結果，對於兔尾草在日後於刺激骨骼生成及再生的研究領域上有良好的發展。

關鍵字

成骨作用 ； 臺灣本土植物 ； MG-63細胞株 ； 兔尾草 ； 人類成骨細胞

並列摘要

Osteoblast proliferation, differentiation, and mineralization play critical roles in the process of osteogenesis. We have established an in vitro screening platform for Taiwanese native plants screening by determining the early and late biomarkers of osteogenesis in human osteoblast-like cell line (MG-63). The extracts from 135 species of Taiwanese native plants were evaluated by various osteogenesis bioassays, including cell viability assay, alkaline phosphatase (ALP) activity assay, and alizan-red S staining. We found that 9 species of Taiwanese native plants significantly increased ALP activity and calcium contents of the mineralized matrix. Among them, Uraria crinita (L.) Desv. ex DC. is an edible herb used as a natural food in childhood skeletal dysplasia. The 95% ethanolic extracts of U. crinita and its isolated compounds were evaluated for their effects on osteogenesis-related biomarkers and mRNA expression in primary human osteoblast cells (HOb). Results showed that the 95% ethanol extract of U. crinita and its bioactive compounds, including dalbergioidin (1), apigenin 6-C-β-D-apiofuranosyl(1→2)-α-D-xylopyranoside (3), adenosine (7), and byzantionoside B (9), significantly increased ALP activity, calcium contents of the mineralized matrix, as well as osteopontin, bone morphogenetic protein-2 (BMP-2), and runt-related transcription factor 2 (Runx2) mRNA expression through activation of the BMP-2/Runx2 pathway. We further investigated the influence of different extraction methods on the osteogenic activity of U. crinita. We found that 50% ethanolic extracts of U. crinita were more effective than 95% ethanolic extracts in matrix mineralization-inducing activity in HOb cells. Six compounds, salicylic acid (2), apigenin 6-C-β-D-apiofuranosyl(1→2)-α-D -xylopyranoside (3), vitexin (4), 2ʹ-hydroxygenistein (12), genistein (13), and 5,7-dihydroxy-3′,5′-dihydroxyisoflavone (14), were isolated from 50% ethanolic extracts and showed osteogenesis-inducing activity by regulating differentiation and mineralization in HOb cells. Data of quantitative and chemical fingerprint analysis obtained after liquid chromatography tandem mass spectrometry (LC-MS/MS) suggested the presence of six osteogenesis-inducing active compounds in an ethyl acetate (EtOAc) fraction from the active extracts of U. crinita. These osteogenesis-inducing activities and quality control results could enhance the efficacy of the U. crinita for stimulating bone formation and regeneration.

並列關鍵字

Osteogenesis ； Taiwanese native plants ； MG-63 cell ； Uraria crinita ； human osteoblast cells

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