2026-11-092026-11-09https://scholars.csmu.edu.tw/handle/123456789/38482Polyphenols are ubiquitous in fruits, vegetables, and herbs, especially in Morus alba L. leaves. Our laboratory has isolation several compounds in Morus alba L. leaves extracts(MLE) (supplementary Fig. 1). We determined the antioxidative effects of MLE on the oxidative modification of LDL induced by copper (supplementary Fig. 2). We also demonstrated that MLE inhibit proliferation and migration of VSMC (supplementary Fig. 3~5). The aim of this study is to establish the hypolipidemia activity of polyphenol isolated from Mulberry leaves (MLPE). First year, we will determine the effect on MLPE scavenged the DPPH radical. Then, we precede a qualitative analysis to determine MLPE contains the component using HPLC experiment. We then determine the copper-induced oxidative modification of human LDL. Oxidation of LDL is measured the formation thiobarbituric acid reacting substances (TBARS), apoB fragmentation, elctrophoretic mobility. More importantly, we will use high cholesterol diet induced animal model for NZW rabbit to determine the anti-atherosclerosis effects of MLPE. Then we will investigate to test whether the MLPE can inhibit foam cell formation and by what mechanism. Second year, we will demonstrate whether MLPE can inhibit smooth muscle cell migration by wound healing assay, boyden chamber and MMP zymogrphagy assay or suppress serum-induced smooth muscle cell proliferation by cell number counting and flow cytometric analysis. Also we will take several studies in how MLPE inhibit on VSMC by immunoblotting and immunoprecipitation. These results will help us to understand that whether MLPE has cytostatic functions in the vessel wall and whether can protect against vascular diseases through inhibition of cell signaling events associated with growth factor-induced smooth muscle cell migration and proliferation. Third year, we will ask whether MLPE can protect against the oxLDL-induced dysfunction in endothelial cells. To test whether MLPE inhibit the reactive oxygen species (ROS) generation, intracellular calcium accumulation, and the subsequent mitochondrial membrane potential collapse, cytochrome c release and caspase-3 activation induced by oxLDL. We will also analysis what mechanism of MLPE protect endothelial cells. However, our results will show that the effects polyphenols in mulberry leaves prevent atherosclerosis in hypercholesterolemic rabbits, foam cell formation, VSMC proliferation and migration, and protection of endothelial cell dysfunction. Also, we hope these observations will be useful for clinical implications in the prevention of atherosclerotic vascular disease.桑葉（Morus alba L. leaves）來自於一種天然植物桑科落葉喬木，具有豐富的 多酚成分，本實驗室已分離各桑葉成分（附件一）及抗LDL 氧化（附件二），及 抑制VSMC 增生及位移（附件三~五），因為眾多的多酚及酚酸，可能具有抑制心 血管病變的功能，因此本研究探討（1）桑葉及多酚之萃取物（MLE, MLPE）降 血脂及抑制低密度脂蛋白氧化及心血管病變等作用；進一步探討（2）抑制氧化型 低密度脂蛋白（ox-LDL）所誘導之泡沫細胞的形成；及（3）探討抑制血管平滑 肌細胞（VSMC）之增生與遷移作用；（4）是否具有保護血管內皮細胞，避免血 管內皮細胞死亡之作用。 第一年之計劃擬探討MLE 與MLPE 抑制血脂肪及對抗LDL 之氧化作用，分別 在cell free system 與animal model 當中以thiobarbituric acid reactive substances （TBARS），electrophoretic mobility（EM）及apoB 斷裂試驗評估LDL 被氧化的 情形；並以紐西蘭白兔的動物模式，誘導產生動脈粥狀硬化病變來評估MLE 之降 血脂及降低血管病變的作用；由於ox-LDL 誘導巨噬細胞形成泡沫細胞，在早期 動脈粥狀硬化發展過程當中，扮演一個相當重要的指標，我們將進一步誘導 J774A.1 巨噬細胞形成泡沫細胞來評估MLE 與MLPE 之降血脂作用之機制，同 時，我們將以免疫染色觀察MLE 對兔子血管內皮細胞傷害之抑製作用。而第二年 的工作，我們擬探討MLE 與MLPE 抑制血管平滑肌細胞之不正常增生與遷移現 象，利用誘導A7r5 細胞株產生增生與遷移，再利用細胞計數、流式細胞儀、傷口 癒合、boyden chamber 分析MLE 與MLPE 之抑製作用，並搭配免疫沉澱、西方點 墨法、訊息傳遞抑制劑等，分析抑制增生及位移其中相關的機轉，並利用動物試 驗之檢體配合免疫染色（anti-PCNA, anti-α actin,）觀察VSMC 之不正常增生與遷 移現象，驗證我們在細胞實驗得到的結果；第三年的工作，我們擬探討MLE 與 MLPE 對於血管內皮細胞之保護作用，觀察MLE 與MLPE 是否具有抵抗HUVEC 細胞死亡，降低自由基、減少黏著蛋白及維持其正常功能之效用，並利用免疫沉 澱、西方點墨法分析抑制內皮細胞傷害相關的分子機轉，並利用動物試驗之檢體 配合免疫染色（anti-ICAM, anti-VCAM）觀察內皮細胞損傷之情形，驗證我們在 細胞實驗所得到的結果。 從以上的結果，我們預期MLE 與MLPE 具有（1）降血脂、抗LDL 氧化作用； （2）抑制ox-LDL-induced atherosclerosis 是經由抑制泡沫細胞之生成；（3）抑制 VSMC 細胞增生是經由MLE 與MLPE 是造成cell cycle arrest 達到抑制細胞生長， 並有效地調控VSMC 之遷移作用；（4）MLE 與MLPE 具有抗氧化，降低自由基 之生成，保護血管內皮細胞之能力，以上的試驗也經由動物實驗之組織染色證明。 因此，從以上的研究，可說明MLE 與MLPE 能夠預防心血管疾病之作用，具有 發展為一種優良的保健食品之潛力。桑葉多酚萃取物降血脂作用抑制血管病變抑制泡沫細胞生成細胞遷移抑制增生抑制內皮細胞傷害Mulberry leavespolyphenolatherosclerosissmooth muscle cellfoam cell formationendotheial cell dysfunctionProject