臺灣產植物成分及其對骨質新生活性研究 (1)

The constituents from Taiwanese plant and their osteogenic activity (1)

彭詩雯(Shih-Wen Peng)

指導教授 ： 李美賢

臺北醫學大學/藥學院/生藥學研究所/碩士(2015年)

摘要

本研究乃採集海州骨碎補 (Davallia mariesii Moore ex Bak) 的根莖，以50%乙醇或二次水加熱迴流萃取，得到其乾燥萃取物，利用人類造骨細胞 (human osteoblasts) 進行活性評估：於MTT細胞存活率實驗中，發現二次水萃取物不具細胞毒性；在促進鹼性磷酸酶活性試驗和礦化染色試驗中，相較於控制組 (100%)，發現二次水萃取物對鹼性磷酸酶活性及礦化基質中鈣離子含量具有較好的促進效果 (分別為111.68±4.55%及130.23±4.29%)。因此將具活性之二次水萃取物進一步用多種管柱層析方式進行成分分離，得到三個化合物。經由理化及光譜相關數據分析以及文獻比對後，確認此三個化合物為一個amino acid及兩個glycoside。骨生成相關標誌物部分，過去研究發現，腦源性神經滋養因子 (BDNF) 基因上的phosSNP rs6265可調控造骨細胞的分化，並對骨密度產生影響。本研究發現隨著人類造骨細胞的分化與礦化，BDNF mRNA表現量逐漸增加。進一步利用人類造骨細胞進行此三個化合物之活性及其機轉探討：於MTT細胞存活率實驗中，發現均無細胞毒性；在促進骨生成活性與其機轉實驗中，發現三個化合物均對BDNF mRNA表現無顯著之促進效果，compound 1 可顯著促進骨骼成形蛋白-2 (BMP-2) 和骨唾液酸蛋白 (BSP) mRNA表現，而compound 2 具顯著促進礦化基質中鈣離子含量的能力，compound 3 則對BMP-2、BSP、第一型膠原蛋白 (Col-1) 和骨橋蛋白 (OPN) mRNA表現及礦化基質中鈣離子含量皆有顯著之促進效果。綜合上述結果，海州骨碎補具發展為骨質疏鬆藥物之潛力，而BDNF可能為具促進骨質新生活性的新標誌物。期望本論文之內容對未來骨質疏鬆的研究及其藥物的設計產生貢獻。

關鍵字

海州骨碎補 ； 人類造骨細胞 ； 腦源性神經滋養因子 ； 骨質新生

並列摘要

In this study, we extracted the rhizoma of Davallia mariesii Moore ex Bak with ddH2O or 50% ethanol. Using cell viability assay, alkaline phosphatase (ALP) activity assay and mineralization staining assay, we evaluated the activities of the extracts in human osteoblasts (HOb). Our data showed that ddH2O extract of D. mariesii has better osteogenic activity than 50% ethanol extract. Hence, the ddH2O extract was further isolated and purified with a variety of columns. After bio-guided column chromatography, we obtain three known compounds. These compounds were identified as one amino acid and two glycosides. Brain-derived neurotrophic factor (BDNF) is an neurotrophin which can influence osteoblast differentiation and bone mass. We found that BDNF mRNA expression was upregulated gradually during differentiation and mineralization process in HOb. However, all of the three compounds didn’t regulate BDNF mRNA expression in HOb. Further, we investigate the effects of these compounds on cell viability, matrix mineralization and osteogenesis-related mRNA expression in HOb. The MTT assay showed that all of these compounds are nontoxic to HOb. Compound 1 could upregulate bone morphogenetic protein-2 (BMP-2) and bone sialoprotein (BSP) mRNA expression. Compound 2 and compound 3 significantly increased the calcium contents of the mineralized matrix in HOb, while compound 3 could also upregulate BMP-2, BSP, type I collagen (Col-1) and osteopontin (OPN) mRNA. The present results imply that Davallia mariesii has potential to be developed into osteoporosis drugs and BDNF is a new biomarker for bone research. Hoping that our study contributes to osteoporosis research and drug design in the future.

並列關鍵字

Davallia mariesii Moore ex Bak ； human osteoblasts ； osteogenic activity ； brain-derived neurotrophic factor

參考文獻

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