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研究生:林宗賢
研究生(外文):LIN, TSUNG-HSIEN
論文名稱:馬鞭草化學成分及藥理活性之研究
論文名稱(外文):Study on Chemical Constituents and Pharmacological Activities of Verbena officinalis.
指導教授:張溫良林漢欽林漢欽引用關係
指導教授(外文):CHANG, WEN-LIANGLIN, HANG-CHING
口試委員:張溫良林漢欽嚴逸釗張自忠石麗仙
口試委員(外文):CHANG, WEN-LIANGLIN, HANG-CHINGYEH, I-ChuanChang, Tsu-ChungSHI,LI-SHIAN
口試日期:2018-05-18
學位類別:碩士
校院名稱:國防醫學院
系所名稱:藥學研究所
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:2018
畢業學年度:106
語文別:中文
論文頁數:122
中文關鍵詞:馬鞭草香菸煙霧萃取人類黏附型肺上皮細胞株
外文關鍵詞:Verbena officinalis.Cigarette crude extractA549 cells
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根據台灣國家衛生福利部 2013 年統計結果顯示,惡性腫瘤佔台灣總死因 29.0%,其惡性腫瘤中,又以氣管、支氣管及肺部癌症為首,其吸菸者罹 患肺癌的機率遠高出不吸菸者 20 倍。 根據研究顯示香菸燃燒後進入人體會產生氧化壓力(oxidative stress)。其 氧化壓力會造成 Glutathione (GSH)抑制,GSH 是保護細胞和線粒體免受氧 化損傷之抗氧化劑,並且氧化壓力會增 NO 產生,使線粒體超極化 (mitochondria hyperpolarization) 進一步導致發炎反應的產生以及細胞自噬 能力的降低,除此之外氧化壓力還會產生超氧化物造成後天免疫系統抗體 IgG 異常修飾與 DNA、內源性蛋白的再修飾導致細胞突變產生癌症。
馬鞭草為傳統中藥,具有活血散瘀,解毒,利水消腫。用於經閉痛經,瘧疾,喉痹,水腫,熱淋。近年研究指出馬鞭草萃取物具有抗發炎及氧化 壓力之作用。 在初步試驗中發現經由香菸粗萃物(Cigarette crude extract; CSE)誘導人 類肺上皮細胞(A549),在低濃度會促進細胞增生,在高濃度會導致細胞毒殺。
而經馬鞭草水粗萃物藥物治療後,發現其具有抑制低濃度促進細胞增生或 高濃度導致細胞毒殺之活性。 因此,將馬鞭草以水萃取並依極性大小作區分,分別為水層、20%酒精 層、40%酒精層以及 95%酒精層。將各分層進行進一步藥理活性試驗,結果顯示其藥理活性較佳的為 40% E-VEOF,並且根據高效液相層析儀分析後,發現具抗氧化活性之主成分 verbaascoside 位於 40% E-VEOF,因此,以 40% E-VEOF 進一步進行純化分離,將 40%酒精層利用逆向管柱分離純化得到 9 種化合物,分別為 verbenalin (1)、hastatoside (2)、gelsemiol (3) 、 scutellarein-7-glucuronide (4)、 R/S- campneoside Ⅱ (5) (6)、verbascoside (7) Verbenalinoside (8)與 isoverbascoside (9)。其中 Verbenalinoside (8)為新化合物。
將上述所得化合物分別以5% CSE進行誘導A549細胞增生試驗與70% CSE 進行誘導 A549 細胞毒殺試驗,得知其化合物在抑制 A549 受 5% CSE 誘導增生之活性效劑量皆約在 0.5~25 μM,其中化合物 VO-K02、03、04 以 及 07 在濃度 0.25 μM 亦具有抑制活性,並且發現所有化合物高濃度抑制效 果相較低濃度差,而在抑制 A549 受 70% CSE 誘導毒殺之活性試驗中,化 合物有效劑量皆約在 1~50 μM 除 VO-K04 不具顯著藥理活性。其中化合物 VO-K08 以及 09 在濃度 0.25 μM 亦具有抑制活性。
由以上藥理活性測試結果顯示,其馬鞭草粗萃取及部分化合物對細胞 受 CSE 誘導增生具有良好抑制效果,而在 CSE 細胞毒殺試驗上,馬鞭草萃 取之化合物亦具有抑制毒殺之活性。


According to statistics from the National Health and Welfare Ministry of Taiwan in 2013, malignant tumors accounted for 29.0% of the total cause of death in Taiwan. Among the malignant tumors, cancers of the trachea, bronchus, and lungs are the leading causes. The chance of lung cancer among smokers is 20 times more than that of non-smokers.
According to research, oxidative stress (ROS) occurs when cigarettes enter the body after burning. Oxidative stress can cause Glutathione (GSH) inhibition. GSH is an antioxidant that protects cells and mitochondria from oxidative damage, and oxidative stress increases NO production, making mitochondrial hyperpolarization further contributes to the production of inflammatory responses and cells. In addition to the decrease in autophagy, in addition to oxidative stress, superoxide is also produced, which results in an abnormal modification of the immune system, antibody IgG, and the re-modification of DNA and endogenous proteins, leading to cell mutations that cause cancer.
Herba verbenae, the dried aerial parts of Verbena officinalis L. (Verbenaceae), is a traditional Chinese medicine used as a hemostatic, antimalarial, and diuretic agents. In recent research, various biological activities have been reported on the extracts of V. officinalis including ant-inflammatory and antibacterial activities
In preliminary test, the water extracts of Herba Verbenae exhibited a significantly protective activity in the high concentration of cigarette crude extracts (CSE) inducer, which induced cell damage and decreased cell proliferation, in the low dose of CSE inducer, which induced cell proliferation, in lung cancer cell line (A549 cell). It is worthy to further develop as lung protector from this herb.
The water extracts of the dried aerial parts of V. officinalis was passed through an Diaion HP-20 column eluted with EtOH/H2O system to give H2O layer, 20% EtOH layer, 40% EtOH layer and 95% EtOH layer, respectively. According to the analysis by CSE pharmacological activies and high performance liquid chromatography, it was found that the best pharmacological activies in the four layers is 40% alcohol layer (figures 5-6, 5-7) and analysis the ingredient verbascoside with antioxidant activity was located in the 40% alcohol layer, so the inverse was first used with 40% alcohol layer.
In the experiment, 9 compounds were separated and purified by reversed-phase column chromatography from the 40% alcohol layer, which was verbenalin (1), hastatoside (2), gelsemiol (3), scutellarein-7-glucuronide (4), R/S-campneoside II (5) (6), verbascoside (7), Verbenalinoside (8) and isoverbascoside (9). Among them, Verbenalinoside (8) is a new compound.
Those compounds were tested by A549 cell proliferation induced by 5% CSE and cell toxicity induced by 70% CSE. The results show that the effective dose of those compound in inhibiting A549 by 5% CSE-induced proliferation was about 0.5~25 μM. Among them, the compounds VO-K02, 03, 04 and 07 also have inhibitory activity at a concentration of 0.25 μM, and in inhibiting the activity of A549 by 70% CSE-induced toxicity, The effective dose of those compound is about 1~50 μM excepting VO-K04 which has no significant pharmacological activity. Among them, the compounds VO-K08 and 09 also have an inhibitory activity at a concentration of 0.25 μM.
From the pharmacological activity test, those compounds from Verbena has an inhibitory effect on cells induced by CSE at low concentration. In the cytotoxicity test, the compounds isolated from Verbena also has a protective activity on cell toxicity induced by 70% CSE.
第一章 緒論
第一節 研究背景
第二節 研究目的
第二章 馬鞭草植物介紹
第一節 植物與藥材概述
第二節 化學成分回顧
第三節 藥理活性回顧
第三章 馬鞭草化學成分之抽取與分離
第一節 抽取分層方法
第二節 純化分離方法
第四章 化合物之結構鑑定
第一節 Verbenalin (1)之結構鑑定
第二節 Hastatoside (2)之結構鑑定
第三節 Gelsemiol (3)之結構鑑定
第四節 Scutellarin-7-glucuronide (4)之結構鑑定
第五節 R/S-Campneoside Ⅱ (5) (6)之結構鑑定
第六節 Verbascoside (7)之結構鑑定
第七節 Verbenalinoside (8)之結構鑑定
第八節 Isoverbascoside (9)之結構鑑定
第五章 藥理作用之結果與討論
第一節 馬鞭草水萃物對細胞存活率之試驗
第二節 香菸粗萃取物對 A549 細胞之細胞毒殺試驗
第三節 馬鞭草粗萃物對 5%香菸粗萃物誘導細胞增生活性試驗
第四節 馬鞭草粗萃物對 70%香菸粗萃物誘導細胞毒殺活性試驗
第五節 馬鞭草分層粗萃物對 A549 細胞存活試驗
第六節 馬鞭草分層粗萃物對 5%香菸粗萃物誘導細胞增生活性試驗
第七節 馬鞭草分層粗萃物對 70%香菸粗萃物誘導細胞毒殺活性試驗
第八節 馬鞭草純化物對 A549 細胞存活試驗
第九節 馬鞭草純化物對 5%香菸粗萃物誘導細胞增生活性試驗
第十節 馬鞭草純化物對 70%香菸粗萃物誘導細胞毒殺活性試驗
第六章 實驗方法
第一節 材料與儀器
第二節 抽取與萃取
第三節 分離與純化
第四節 細胞實驗方法
第五節 各化合物之物理及光譜數據
第七章 結論
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