天然物抗過敏活性篩選與土半夏化學成份及其生物活性研究

Anti-allergic Screening of Natural Products & Chemical and Biological Investigation on Typhonium blumei and T. roxburghii

柯麥可(Michal Kořinek)

指導教授 ： 張芳榮

共同指導教授 ： 陳炳宏 ； 吳永昌(Yang-Chang Wu)

高雄醫學大學/藥學院/天然藥物研究所博士班/博士(2017年)

摘要

過敏是一種嚴重影響生活品質的免疫疾病。近年來越來越複雜的生活環境導致患有過敏疾病的人越來越多，因此尋找更有效治療過敏的藥物變成當務之急。我們利用活性導引分離法篩選一些有潛力的抗過敏植物，並且分離出這些植物有抗過敏潛力的萃取物或分子。在這些植物中又發現到一些植物中的抗過敏成分。
土半夏Typhonium blumei Nicolson & Sivadasan (Araceae) 是一種台灣民間用來治療癌症和發炎的民俗用藥,但目前為止對於土半夏的成分分析和藥理研究還相當稀少。首先我們透過 RBL-2H3細胞的脫顆粒實驗發現低極性的土半夏和金慈菇 (T. roxburghii，台灣南部常用來當土半夏的替代品)萃取物都有抗過敏的效果。進一步我們發現土半夏的低極性萃取物會經由 PI3K/PLCγ2抑制細胞鈣離子內流,但不會影響FcεRI的表現、IL-4 和 MCP-1 細胞激素的 mRNA表現以及MAPK的蛋白表現。我們利用1H NMR 和 GC-MS分析發現此兩種萃取物中都富含palmitic、linoleic 和 α-linolenic acids， 之前研究就發現土半夏和金慈菇中富含這些脂肪酸成分,我們研究證實民間利用金慈菇來當作土半夏的替代品是有根據的。
脂肪酸對於人體脂質恆定和發炎作用是很重要的營養成分。我們從土半夏的地下部分離出四個成分12R-hydroxyoctadec-9Z,13E-dienoic acid methyl ester (2)、10R-hydroxyoctadec-8E,12Z-dienoic acid methyl ester (3)、9R-hydroxy-10E-octadecenoic acid methyl ester (4) 和12R*-hydroxy-10E-octadecenoic acid methyl ester (5)。我們利用這四個成分和一系列其它飽和、不飽和及oxygenated脂肪酸一起來評估其細胞毒、抗發炎和抗過敏活性。研究結果顯示，游離不飽和脂肪酸的抗炎活性不受脂肪酸長鏈羥基影響，但脂肪酸長鏈羥基對於抗過敏活性卻很重要。我們進一步利用ChemGPS-NP來研究脂肪酸結構和抗過敏活性的關聯，結果顯示這些具有抗過敏活性的脂肪酸和目前臨床使用的抗過敏藥物結構關聯性很低。土半夏這個台灣具有歷史的民俗用藥其生物活性可能和其脂肪酸和脂肪酸代謝物含量有關。

關鍵字

土半夏 ； 羥基脂肪酸 ； 抗過敏 ； 抗發炎 ； 天然物化學導航系統

並列摘要

Allergy is an immune disorder causing a serious burden on the quality of life. Increasing prevalence of allergic diseases with an inadequate variety of treatment drives forward search for new alternative drugs. We conducted screening for new potent anti-allergic plants, further evaluating subfractions and/or pure compounds of active extracts, with the aim to understand the chemical and molecular basis of the observed activity. Several plant materials were found to possess promising anti-allergic properties.
Typhonium blumei Nicolson & Sivadasan (Araceae) is used as anti-cancer and anti-inflammatory folk medicine in Taiwan. Phytochemical and biological studies on T. blumei are scanty. We compared biological activity and composition of T. blumei, the originally used herb, with T. roxburghii, its common substitution in southern Taiwan. The nonpolar fractions of both species showed potent inhibitory activity in A23187 induced degranulation assay in RBL-2H3 cells. They also inhibited superoxide production and elastase release in human neutrophils. T. blumei nonpolar fractions inhibited antigen-induced β-hexosaminidase and histamine release. The nonpolar fractions of T. blumei significantly inhibited calcium influx upon activation with either A23187 or an antigen. T. blumei fractions did not affect FcεRI expression, mRNA level of IL-4 and MCP-1 cytokine production or MAPK proteins expression, but did suppress the calcium signaling pathway via PI3K/PLCγ2. 1H NMR and gas chromatography-mass spectrometry (GC-MS) analyses revealed that the active fractions were rich in fatty acids with palmitic, linoleic and α-linolenic acids identified as the major fatty acids in both plants. Thus, the obtained results support the traditional use of T. blumei in the treatment of inflammatory diseases as well as its substitution with T. roxburghii.
Fatty acids, abundant in nature, are regarded as important bioactive compounds and powerful nutrients playing an important role in lipid homeostasis and inflammation. Phytochemical study on T. blumei rhizomes, yielded four oxygenated fatty acids, 12R-hydroxyoctadec-9Z,13E-dienoic acid methyl ester (2), 10R-hydroxyoctadec-8E,12Z-dienoic acid methyl ester (3), 9R-hydroxy-10E-octadecenoic acid methyl ester (4), and 12R*-hydroxy-10E-octadecenoic acid methyl ester (5). Isolated fatty acids together with a series of saturated, unsaturated and oxygenated fatty acids were evaluated for their cytotoxic, anti-inflammatory and anti-allergic activities in vitro. Unsaturated (including docosahexaenoic and eicosapentaenoic acids) as well as hydroxylated unsaturated fatty acids exerted strong anti-inflammatory activity in superoxide anion generation (IC50 2.14 – 3.73 μM) and elastase release (IC50 1.26 – 4.57 μM) assays. On the other hand, in the anti-allergic assays, the unsaturated fatty acids were inactive, while hydroxylated fatty acids showed promising inhibitory activity in A23187- and antigen-induced degranulation assays (e.g. 9S-hydroxy-10E,12Z-octadecadienoic acid, IC50 92.4 and 49.7 μM, respectively). According to our results, the presence of a hydroxy group in the long chain did not influence the potent anti-inflammatory activity of free unsaturated acids. Nevertheless, hydroxylation of fatty acids (or their methyl esters) seems to be a key factor for the anti-allergic activity. Moreover, ChemGPS-NP was explored to predict the structure-activity relationship of fatty acids. The anti-allergic fatty acids formed different cluster distant from clinically used drugs. Thus, the bioactivity of T. blumei, which is historically utilized in folk medicine, might be related to the content of fatty acids and their metabolites.

並列關鍵字

Typhonium blumei ； hydroxy fatty acids ； anti-allergic ； anti-inflammatory ； ChemGPS-NP

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