吳茱萸活性成分之研究

Studies on the biologically active components of Wu-Chu-Yu

柯漢傑(Han-Chieh Ko)

指導教授 ： 陳繼明 ； 李慶國 ； 林淑娟

臺北醫學大學/藥學院/藥學研究所/博士(2007年)

https://doi.org/10.6831/TMU.2007.00017

摘要

吳茱萸為芸香科 (Rutaceae) 植物吳茱萸Evodia rutaecarpa ( Juss.) Benth.[E.R.] 之未成熟乾燥果實，為我國傳統常用藥物之一。神農本草經將吳茱萸列為草部中品，可治胃痛、頭痛、腹痛、痢疾、產後出血及停經等疾病，現代藥理學研究發現其藥效成分dehydroevodiamine (DeHE)、evodiamine (Evo)、rutaecarpine (Rut)有降血壓、抗心律不整、刺激內皮細胞及抑制巨噬細胞釋放一氧化氮之作用，而synephrine (Syn) 具有升血壓之作用。本研究利用高效液相層析法(HPLC)，包括HP-1100高效液相層析儀系統， C18 (Reversed-phase C18 column ) 分離管柱，選擇acetonitrile，water，sodium dodecyl sulfate，orthophosphoric acid solution的混合緩衝液為移動相，以線性梯度程式沖提，270 nm波長來分析不同地區之吳茱萸及相關品種，在不同採收時間，不同炮製，及含吳茱萸之不同方劑中DeHE、Evo、Rut及Syn之含量，並以大白鼠主動脈血管及人體嗜中性白血球，分別評估不同吳茱萸標本之舒張血管及抗發炎作用。本實驗所用HPLC方法可在60分鐘內測定DeHE、Evo、Rut及Syn，並發現 (a) 吳茱萸植物中DeHE、Evo和Rut之含量，隨著花果成長時間延長而增加，但是達到果實成熟時期其含量有下降的趨勢；而Syn 只在中國大陸產之吳茱萸出現；(b) 吳茱萸生藥材及不同炮製品比較時，四種活性成分含量檢測顯示炮製品之DeHE、Syn (親水性成份)下降，Evo、Rut (疏水性成份)上升；(c) 實驗室自行配方之吳茱萸湯(吳茱萸28%、人參17%、大棗22%和生薑33%)單煎或合煎，其 DeHE、Evo、Rut及Syn含量，以合煎者較高；(d) 另外蒐集八家GMP 廠產品，發現廠與廠間的品質有所差異；(e) 大陸吳茱萸及在台灣所採集的吳茱萸和賊仔樹樣品，其甲醇萃取液對phenylephrine引起的大白鼠主動脈血管收縮有劑量相關性的抑制作用，台灣吳茱萸雖然其生物鹼成分Evo、DeHE和Rut含量較低，但對老鼠主動脈血管舒張作用而言，作用反而較強；(f) 在抑制體外人類嗜中性白血球及微膠細胞
之自由基之活化等抗發炎實驗中，發現吳茱萸萃取物及其活性成份對活細胞的抑制作用，強於對酵素(NADPH oxidase)的直接抑制作用，很可能是藥物作用於蛋白質激酶C (PKC) 或其下游之訊息傳導層次。
本研究顯示，中藥或中藥製劑之品質變數很多，而且已知主要藥效成分外，其他成分也可能與已知之藥效成分有相互作用，甚至在不同生物活性檢測上呈現不同結果，所以測定植物藥之藥效成分或指標成分，仍不足作為品質管制的依據，必須補以多樣生物活性的實驗。

關鍵字

吳茱萸, 賊仔樹, 採收, 炮製, 舒張作用, 抗發炎作用, 高效液相層析.

並列摘要

Wu-Chu-Yu, the unriped fruit of Evodia rutaecarpa (Juss.) Benth (E.R.)., is a commonly used Chinese herbal drug for the treatment of gastrointestinal disorders, headache, abdominal pain, dysentery, postpartum hemorrhage and amenorrhea and has been classified as “middle class” in Shen-Nong-Ben-Cao-Jing. Previously, it was found that three alkaloids, dehydroevodiamine (DeHE), evodiamine (Evo), and rutaecarpine (Rut), obtained from Wu-Chu-Yu, possess hypotensive and antiarrhythmic effects, stimulate endothelial cells and inhibite macrophages nitric oxide release, and another bioactive compound, synephrine (Syn) possess hypertensive effect.
In this study, the high performance liquid chromatographic (HPLC), by using HP-1100 HPLC, a C18 column (Reversed-phase C18 column ), a buffer solution composed of acetonitrile, water, sodium dodecyl sulfonate (SDS), and orthophosphoric acid as mobile phase, utilizing linear gradient program and UV (270 nm), under 1.0 ml/min of flow rate, was used to simultaneous detect the contents of DeHE, Evo, Rut, and Syn in Evodia and related species, collected at different locations, harvest time, processing, and in the various prescriptions which composed of E.R. within 60 minutes. The vasorelaxant and anti-inflammatory effects of these preparations were also evaluated in isolated rat aorta and human neutrophil.
The results demonstrated that (a) DeHE, Evo and Rut were contained in E.R. obtained from mainland China, Taiwan, and Tetradium glabrifolium (T.G.) obtained from Taiwan. While, Syn was only detected in E.R. obtained from mainland China. The contents of DeHE, Evo, Rut were the highest in the unripe fruit and comparably lower in ripe fruit；(b) DeHE and Syn (hydrophilic) were decreased and Evo and Rut (hydrophobic) were increased after various processing；(c) in the Wu-Chu-Yu Soup, ( composed by E.R., 28% ; Panax ginseng, 17%; Zizyphus jujube, 22% ; Zingiber officinale, 33% ), the contents of DeHE, Evo, Rut and Syn were higher when these four

herbal drugs cooking together as compared with E.R. which was cooked alone；(d) there were significant variations on the contents of DeHE, Evo, Rut and Syn among the E.R. contained preparations obtained from eight GMP herbal drug companies；(e) vasorelaxant effect of methanol extract of E.R. obtained from mainland China, Taiwan and T.G. in phenylephrine induced constriction of isolated Sprague-Dawley aortas, all were dose dependently, and was greater in sample obtained from Taiwan than that from mainland China in spite of lower levels of the relaxing alkaloids DeHE, Evo and Rut; (f) phorbol-12-myristate-13-acetate (PMA) and N-formyl-methionyl-leucyl-phenylalanine (fMLP) induced reactive oxygen species production, firm adhesion and transmigration of neutrophil (PMN) was more nonspecific inhibited by E.R. crude extract than DeHE, Evo, Rut and Syn in the tested cells than in the NADPH oxidase. Thus, protein kinase C or its downstream signaling pathway was involved in this anti-inflammatory effect.
Our results demonstrated that the chemical compositions of E.R. and related species were affected by different variables, the lack of correction between chemical composition and relaxing or anti-inflammatory activities of crude extract of different samples of E.R. showed the significant contribution of drug interaction and/or the minor components. Thus, besides chemical standardization, multiple biological functions evaluation is the more adequate way to ensure the quality control of botanical products.

並列關鍵字

Evodia rutaecarpa, Tetradium glabrifolium, Dehydroevodiamine,

參考文獻

54. Chen, G. L.; Yuan, Y. C.; Li, Y. F.; Lee, A. R.; Chen, C. F. A stability indicating method for dehydroevodiamine chloride by high-performance liquid chromatography/diode array detector, J Chin Pharm, 1991, 43, 75-80.

60. Chen, C. F.; Chiou, W. F.; Chou, C. J.; Liao, J. F.; Lin, L. C.; Wang, G. J.; Ueng, Y. F. Pharmacological effects of Evodia rutaecarpa and its bioactive components, Chin Pharm J, 2002, 54, 419-435.

3. Chen, C. F.; Chen, S. M.; Lin, M. T. and Chou, S. Y. In vitro and in vivo studies on the mechanism of cardiovascular effects of Wu-Chu-Yu (Evodiae Fructus), Am J Med Chin 1981, 9, 39-47.

4. Chiou, W. F.; Chou, C. J.; Liao, J. F.; Shum A. Y. C.; Chen, C. F. The mechanism of the vasodilatory effect of rutaecarpine, an alkaloid isolated from Evodia rutaecarpa, Eur J Pharmacol, 1994, 257, 59-66.

5. Chiou, W. F.; Chou, C. J.; Shum A. Y. C.; Chen, C. F. The vasorelaxant effect of evodiamine in rat isolated mesenteric arteries mode of action, Eur J Pharmacol, 1992, 215, 277-283.