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研究生:
江偉萍
研究生(外文):
Wei-Ping Chiang
論文名稱:
中草藥-水錦花萃取物顯示抗發炎、抗氧化和抗動脈粥樣硬化的活性
論文名稱(外文):
A Chinese herbal medicine, Hibiscus syriacus extracts, exhibits anti-oxidative, anti-inflammatory and anti-atherosclerotic activities
指導教授:
鄭志鴻
指導教授(外文):
TZU-HURNG,CHENG
學位類別:
碩士
校院名稱:
中國醫藥大學
系所名稱:
生物科技學系碩士班
學門:
生命科學學門
學類:
生物科技學類
論文種類:
學術論文
論文出版年:
2013
畢業學年度:
101
語文別:
中文
論文頁數:
47
中文關鍵詞:
水錦花
、
抗氧化
、
動脈粥狀硬化
外文關鍵詞:
Hibiscus syriacus
、
anti-oxidative
、
atherosclerotic
相關次數:
被引用:0
點閱:159
評分:
下載:0
書目收藏:0
背景:水錦花(Hibiscus syriacus Linn),分佈在亞洲東、南部,在中國傳統的中草藥中普遍用作解熱、驅蟲以及抗真菌劑。過去的研究顯示,從溶於氯仿的水錦花根皮萃取物中分離出syriacusin A-C,具有抑制脂質過氧化作用活性。在水錦花的根皮也分離出一種furanosesquiterpenoid的衍生物Hydroxyhibiscone A,和hibiscone D一起可以經由抑制人類嗜中性粒細胞彈性蛋白酶的活性和MMP-1和MMP-2的表現,防止因光害而形成的皮膚皺紋。此外,水錦花根皮的丙酮萃取物經由活化p53和細胞凋亡誘導因子誘導細胞凋亡,對人類肺癌細胞不管在體外或體內都發揮抑制增生的作用。近來,在水錦花的花瓣色素提取物也發現具有清除自由基的活性。水錦花的親代及其突變型(Ggoma)根皮的甲醇萃取物中含有抗菌物質nonanoic acid,具有治療腳癬的功效。然而,除了在植物的不同部位分離出多元化的生物活性,不同的萃取過程也可能產生多種活性化合物。在本研究中,我們的目標是進一步分離水錦花的生物活性成分,特別是從莖使用不同極性溶劑進行萃取,調查在多種細胞類型的抗氧化,抗發炎和抗動脈粥樣硬化的影響,並闡明其相關機制。
方法與結果:將水錦花的莖切塊用乙醇(Ethanol)加熱萃取,經過減壓濃縮得到第一個粗萃物(HS-EtOH),再分別用不同極性的溶劑(已烷(Hexane)、乙酸乙脂(Ethyl acetate)、正丁醇(BuOH)和水)分離得到四個萃取物。將extract 3透過離子交換樹脂純化,再以HPLC分析經過NMR鑑定出β-sitosterol、2-Linoleodistearin、Linoleic acid、Syriacusin B、Tricetin 3’,5’-dimethyl ester等化合物。對初萃物和純化物的生物活性,在培養臍靜脈內皮細胞和血管平滑肌細胞進行了抗氧化、抗發炎和抗動脈粥樣硬化篩選。
由於動脈粥狀硬化是一種慢性發炎疾病,我們進一步研究其在內皮細胞抗動脈粥狀硬化的影響。我們已經證實,在內皮細胞中LPS會增加蛋白酶活化受體-2 (PAR-2)的基因表現,活化ERK路徑和之後的單核球趨化蛋白(MCP-1)表現。在這裡,我們證明以LPS(0.5μg/ml)預處理內皮細胞再用胰蛋白酶(5μg/ml)刺激,2-Linoleodistearin以不同濃度處理(1、3、5、7μg/ml)呈現劑量依賴性地抑制ERK的磷酸化和MCP-1蛋白表現。接下來,我們以MTT和wound healing實驗測試粗萃物和純化物抑制以第二型血管收縮素(Ang II;100 nM)所誘導的血管平滑肌細胞增生和爬行的能力。粗萃物(不同濃度10, 30, 50, 70, 100μg/ml)和純化物2-Linoleodistearin(不同濃度0.5, 1, 2, 3μg/ml)會抑制血管平滑肌細胞的增生和爬行,可用於動脈重塑和機械性損傷引起的再狹窄。雖然HS-EA在內皮細胞以過氧化氫處理後利用流式細胞儀進行分析,顯示其清除自由基能力微弱,2-Linoleodistearin對細胞內的活性氧分子(ROS)沒有抑制作用,表明其抑制LPS在內皮細胞活化的ERK途徑和Ang II所誘導的血管平滑肌細胞增生和爬行作用不是經由抗氧化活性所調控,在未來需要進一步調查的相關機制。
結論: 水錦花莖的萃取物HS-EA和純化物2-Linoleodistearin表現出對抗發炎、抗動脈粥狀硬化和抑制增生有很強效的作用。這些發現提供水錦花萃取物治療動脈粥狀硬化和冠狀動脈疾病術後再狹窄的可能性。
Background: Hibiscus syriacus Linn (H.syriacus L), widely distributed in eastern and southern Asia is an ornamental and medicinal plant that has been popularly used as an antipyretic, anthelmintic, and antifungal agent in the Chinese traditional herbal medicine. Previous studies revealed that three naphthalene compounds, syriacusin A-C were isolated from the CHCl3-soluble extract of the root bark of H. syriacus, exerting the inhibitory effect on lipid peroxidation. Furthermore, hydroxyhibiscone A, a new furanosesquiterpenoid, together with hibiscone D, was isolated from the root bark of H. syriacus could protect against skin wrinkle formation by photodamage via suppression of activity of human neutrophil elastase (HNE) and the expression of MMP-1 and MMP-2. The acetone extract of the root bark of H. syriacus exerts antiproliferative effects on human lung cancer cells in vitro and in vivo via induction of apoptosis by activating p53 and apoptosis-induced factor. Recently, the pigment extract from H. syriacus L petals has free radical scavenging activities. The methanol extracts of the root bark of both H. syriacus parent type (malvaceae) and its mutant type (Ggoma) contain antifungal substance, nonanoic acid for treatment of athlete’s foot. However, in addition to the diversity of bioactive constituents isolated from different parts of a medicinal plant, different extraction processes may also produce a variety of active compounds with different concentrations. In this study, we aim to further isolate the bioactive components of H. syriacus L, especially from the stem bark using various solvents with different polarity, investigate their potential anti-oxidative, anti-inflammatory and anti-atherosclerotic effects on a variety of cell types, and elucidate their underlying mechanisms.
Method: The stem of H. syriacus was extracted with ethanol, and then followed by consecutive partitioning with n-hexane (HS-Hex), ethyl acetate (HS-EA), n-butanol (HS-BuOH), and H2O (HS- H2O), respectively. The HS-EA extract was purified by Diaion HP20 resin and silica gel column and then analyzed by HPLC. We further identified pure compounds from HS-EA extract using mass spectrometry combined with NMR spectral analysis. Bioactivities of crude extracts and pure constituents were screened for anti-oxidation, anti-inflammation and anti-atherosclerosis in cultured human umbilical vascular endothelial cells (HUVECs) and vascular smooth muscle cells (VSMCs).
Result: As atherosclerosis is a chronic inflammatory disease of the vascular wall, we further investigate its anti-atherosclerotic role in vascular endothelial cells. We have previously demonstrated that LPS aggravates vascular inflammation via induction of PAR2 (protease activated receptor 2) gene expression, activation of ERK pathway, and subsequent expression of pro-inflammatory chemokine, MCP-1 in vascular endothelial cells. Here, we showed that treatment with 2-linoleodistearin (at various concentrations of 1, 3, 5, and 7μg/ml) dose-dependently attenuates ERK phosphorylation and MCP-1 protein level in LPS (0.5μg/ml)-pretreated HUVECs in response to trypsin (5μg/ml) stimulation.
Next, we examined the potential inhibitory effects of various extracts and pure compounds on both VSMCs’ proliferation and migration stimulated with angiotensin II (100 nM) using MTT and wound healing assays respectively. The crude extracts (at various concentrations of 10, 30, 50, 70, and 100μg/ml) and pure compound, 2-linoleodistearin (at various dosages of 0.5, 1, 2, and 3μg/ml) suppressed VSMCs’ proliferation and migration in a concentration manner implicating their usage on arterial remodeling and mechanical injury-induced restenosis.
Although HS-EA extract revealed weak free radical scavenging ability assessed with flow cytometry in HUVECs exposed to H2O2 treatment, 2-linoleodistearin had non-inhibitory effect on the intracellular ROS levels suggesting its inhibition of LPS-stimulated activation of ERK pathway in HUVECs and suppression of Ang II-induced proliferation and migration of VSMCs were not mediated by the anti-oxidative activity. Additional investigation for the underlying mechanisms is required in the future.
Conclusion: The HS-EA extract of the stem of H. syriacus and the pure compound, 2-linoleodistearin isolated from the extract exhibited potent ability of anti-inflammation, anti-atherosclerosis as well as anti-proliferation. These findings may provide therapeutic potential of extracts of H. syriacus for inflammatory bowal disease, atherosclerosis and balloon-injured restenosis of coronary artery disease.
中文摘要 I
Abstract IV
縮寫表 VII
致謝 VIII
第一章、前言 1
第一節、研究背景 1
一、水錦花 (Hibiscus syriacus Linn) 簡介 1
二、水錦花在近代醫學上的研究 2
三、動脈粥狀硬化 (Atherosclerosis) 5
四、血管收縮素系統: 7
五、脂多糖 (Lipopolysaccharides, LPS) 與動脈粥狀硬化: 8
第二節 研究目的 9
第二章 材料與方法 10
第一節 水錦花的粗萃取 10
第二節 水錦花的生物活性純化物(pure compound)的分離 10
第三節 研究方法 15
一、細胞培養 15
二、四唑鹽比色法(MTT assay) 17
三、傷口癒合(scratch wound healing) 19
四、流式細胞儀 (Flow cytometry) 20
五、西方點墨法(Western blotting) 20
第四節 統計方法 23
第三章 研究結果 24
第一節 水錦花萃取物對內皮細胞的抗氧化能力 24
第二節 水錦花萃取物對平滑肌細胞的細胞毒性 24
第三節 水錦花萃取物的抗動脈粥樣硬化能力 25
第四章 結論 27
第五章 討論 30
第六章 參考文獻 33
第七章 圖表 35
圖一、水錦花粗萃流程圖 35
圖二、不同濃度的extract 2抑制ROS的產生 36
圖三、不同濃度的extract 3抑制ROS的產生 37
圖四、不同濃度的extract 5抑制ROS的產生 38
圖五、不同濃度的extract 2、3抑制ROS產生的量化圖 39
圖六、不同compound抑制ROS的產生 40
圖七、水錦花萃取物對平滑肌細胞的毒性作用 41
圖八、水錦花萃取物抑制平滑肌細胞爬行的能力 43
圖九、水錦花萃取物抑制平滑肌細胞爬行的能力 45
圖十、水錦花萃取物抑制平滑肌細胞增生的能力 46
圖十一、水錦花萃取物抑制內皮細胞ERK和MCP-1的蛋白表現 47
Cheng, Y.L., Lee, S.C., Harn, H.J., Huang, H.C., and Chang, W.L. (2008). The extract of Hibiscus syriacus inducing apoptosis by activating p53 and AIF in human lung cancer cells. The American journal of Chinese medicine 36, 171-184.
Dzau, V.J., Braun-Dullaeus, R.C., and Sedding, D.G. (2002). Vascular proliferation and atherosclerosis: new perspectives and therapeutic strategies. Nature medicine 8, 1249-1256.
Inagami, T., and Eguchi, S. (2000). Angiotensin II-mediated vascular smooth muscle cell growth signaling. Brazilian journal of medical and biological research = Revista brasileira de pesquisas medicas e biologicas / Sociedade Brasileira de Biofisica [et al] 33, 619-624.
Jang, Y.W., Jung, J.Y., Lee, I.K., Kang, S.Y., and Yun, B.S. (2012). Nonanoic Acid, an Antifungal Compound from Hibiscus syriacus Ggoma. Mycobiology 40, 145-146.
Mingjiang, G. (2012). Free radical scavenging activities of pigment extract from Hibiscus syriacus L. petals in vitro. African Journal of Biotechnology 11, 429-435.
Ross, R. (1999). Atherosclerosis--an inflammatory disease. The New England journal of medicine 340, 115-126.
Ryoo, I.J., Yun, B.S., Lee, I.K., Kim, Y.H., Lee, I.S., Ahn, J.S., Bae, K., and Yoo, I.D. (2010). Hydoroxyhibiscone A, a novel human neutrophil elastase inhibitor from Hibiscus syriacus. Journal of microbiology and biotechnology 20, 1189-1191.
Yoo, I.D., Yun, B.S., Lee, I.K., Ryoo, I.J., Choung, D.H., and Han, K.H. (1998). Three naphthalenes from root bark of Hibiscus syriacus. Phytochemistry 47, 799-802.
Yun, B.S., Ryoo, I.J., Lee, I.K., Park, K.H., Choung, D.H., Han, K.H., and Yoo, I.D. (1999). Two bioactive pentacyclic triterpene esters from the root bark of Hibiscus syriacus. Journal of natural products 62, 764-766.
Carey, R. M., et al. (2000). "Role of the angiotensin type 2 receptor in the regulation of
blood pressure and renal function." Hypertension 35(1 Pt 2): 155-163.
Ross, R. (1999). "Atherosclerosis--an inflammatory disease." N Engl J Med 340(2): 115-126.
Mattila, K. J., et al. (1998). "Role of infection as a risk factor for atherosclerosis, myocardial infarction, and stroke." Clin Infect Dis 26(3): 719-734.
Brunet, P. and Y. Berland (2000). "Water quality and complications of haemodialysis." Nephrol Dial Transplant 15(5): 578-580.
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