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研究生:阮怡禎
研究生(外文):Yi-Chen Juan
論文名稱:錫蘭七指蕨萃取之Stilbenoid類衍生物UgonstilbeneA於兔子血小板之作用機轉探討
論文名稱(外文):The action mechanisms of stilbenoid derivative ugonstilbene A extracted from Helminthostachys zeylanica (L) Hook in rabbit platelets.
指導教授:楊行義楊行義引用關係蔡維人蔡維人引用關係
指導教授(外文):Hsing-Yi YangWei-Jern Tsai
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:藥理學研究所
學門:醫藥衛生學門
學類:藥學學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:120
中文關鍵詞:錫蘭七指蕨血小板花生四烯酸血小板活化因子血栓素A2血小板細胞膜流動性
外文關鍵詞:Helminthostachys zeylanicaplateletarachidonic acid(AA)platelet-activating factor(PAF)thromboxane A2(TXA2)platelet membrane fluidity
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錫蘭七指蕨(Helminthostachys zeylanica(L))為蕨綱瓶爾小草科七指蕨屬之植物,俗名有倒地蜈蚣,馬來語則稱之為Tukod-langit。其乾燥根莖屬民間的珍貴藥材,在傳統醫學上使用於行血祛瘀,而東南亞一帶則常用於治療咳嗽、氣喘或用作毒蛇咬傷的急救藥。stilbenoid可廣泛自各種植物中抽提出來,已知具有抑制脂質過氧化、抑制cyclooxygenase活性、鈣離子拮抗劑或自由基捕捉的能力,因而可能具有抗血小板、抗發炎、抗氧化等的活性,而自錫蘭七指蕨乾燥根莖中萃取出的ugonstilbene A為trans-stilbene類化合物,亦屬polyphenolic phytoestrogens的一種,目前尚未有此化合物生物活性的相關報導。為了深入了解其作用機轉,利用兔子血小板進行一連串的實驗。本研究中發現,ugonstilbene A可有效抑制platelet-activating factor (PAF)、U46619 (thromboxane A2的結構類似物) 及arachidonic acid (AA)所媒介的血小板凝集,其IC50值分別為11.73 ± 6.26, 12.9 ± 4.7, 6.24 ± 0.64 □M。利用LDH活性分析,發現其不會造成血小板中LDH的滲漏,排除了以上的抑制現象是來自藥物細胞毒性。此外,預處理ugonstilbene A可降低三種刺激劑所造成的細胞內自由鈣離子濃度的上昇,而以PAF作為刺激劑,在不影響cAMP與cGMP的情況下,藥物亦可直接抑制細胞中DTS內鈣離子的釋放與細胞外鈣離子的內流。另一方面,ugonstilbene A可顯著的降低血小板細胞中PAF刺激所造成的IP3的總量與PKC translocation,由此抑制的現象,推論了藥物是藉由影響PLC的活性,進而影響了下游的活性表現。進一步以免疫沉澱的方式,看到了G protein與PLC之間的耦合現象亦受到ugonstilbene A的影響。而利用將ugonstilbene A移除後,觀察血小板的凝集反應,發現原本的抑制現象消失了,因而推論ugonstilbene A的作用點於細胞膜外側,其對血小板的活性屬可逆性的抑制,此外更發現藥物會影響細胞膜之流動性。另一方面,由藥物處理下MDA的測定發現,ugonstilbene A似乎影響了TXA2的生成或脂質的過氧化,經由PGE2與TXB2的產率分析發現兩者皆受到抑制,判斷藥物抑制了COX的活性,而利用ABTS與DPPH的方式證實了藥物具有抗氧化能力。本研究不但佐證ugonstilbene A可能具有上述trans-stilbenoid的活性,更可提供此植物在傳統療法上一個現代醫學之依據。
Helminthostachys zeylanica, a terrestrial fern of the Ophioglossaceae family, also known as Daodi-Ugonn in Taiwan and Tukod-langit in Malay language. The dry rhizomes were traditionally used to remove blood stasis and promote blood circulation. The folkloric therapies in Southeastern Asia also use them to treat cough in tuberculosis, asthma and poisonous snakebites patients. Stilbenoid can be extracted form various plants and previous studies have revealed its bioactivities in lipid peroxidation inhibition, cyclooxygenase inhibition, calcium channel blocking and radical-scavenging. Ugonstilbene A, a new trans- stilbene compound belong to polyphenolic phytoestrogens, was isolated from the rhizomes of Helminthostachys zeylanica (L.) Hook. To understand the mechanisms of ugonstilbene A, that designed a series of experiments using rabbit platelets. The results showed the compound dose-dependently inhibited washed rabbit platelets aggregation induced by platelet-activating factor (PAF), U46619 (analog of thromboxane A2) or arachidonic acid (AA) with IC50 values of 11.73 ± 6.26, 12.9 ± 4.7, 6.24 ± 0.64 □M, respectively. LDH assay excluded the cytotoxicity of this compound. Pretreatment with ugonstilbene A could reduce PAF- U46619- AA-mediated intracellular calcium elevation. Moreover, calcium flux was directly reduced by this compound including intracellular store release and extracellular influx, but not through inducing cAMP and cGMP levels of rabbit platelets. On the other hand, ugonstilbene A significantly decreased PAF-induced IP3 level and PKC translocation, implicating that this compound might inhibit PLC activity. Immunoprecipitation revealed the coupling between G protein and PLC was modulated by this compound. Platelet activity could reversed by removing ugonstilbene A, indicated its action sites were on the membrane. Furthermore,. the membrane fluidity was enhanced by this compound. The inhibition of MDA formation revealed its bioactivity of lipid peroxidation in AA pathway. Both the inhibitions of TXA2 and PGE2 indicated its COX inhibitory role. ABTS and DPPH assay confirmed the antioxidant effects of ugonstilbene A. This study not only confirmed that this stilbenoid compound has the above mentioned abilities but also provided medical evidences of traditional medicine.
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