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標題: | 第一部分:穗花香苦草、短柄香苦草及香苦草種子化學成分之研究 第二部分:Aporphine及phenanthrene衍生物之製備暨其抗乙醯膽鹼酶與黃嘌呤氧化酶之探討 Part I Chemical investigation of Hyptis spicigera, H. brevipes, and the seed of H. suaveolens Part II Preparation of aporphine and phenanthrene derivatives as acetylcholinesterase inhibitors and xanthine oxidase inhibitors |
作者: | Fu-Chun Hsu 許富鈞 |
指導教授: | 李水盛(Shoei-Sheng Lee) |
關鍵字: | 穗花香苦草,短柄香苦草,香苦草,山粉圓,黃嘌呤氧化酶,阿朴芬類生物鹼,litebamine型生物鹼,phenanthrene型衍生物,乙醯膽鹼酶, Hyptis spicigera,H. brevipes,H. suaveolens seed,xanthine oxidase,aporphine alkaloids,litebamine alkaloids,phenanthrene derivatives,acetylcholinesterase, |
出版年 : | 2020 |
學位: | 博士 |
摘要: | 本論文包含兩個部分: 第一部分:穗花香苦草、短柄香苦草及香苦草種子化學成分之研究 臨床上常使用黃嘌呤氧化酶抑制劑於預防痛風復發,但目前常用的抑制劑-allopurinol-有引發嚴重過敏反應之可能,因此仍須開發具低副作用的抑制劑。本實驗室過去自頭花香苦草 (Hyptis rhomboides) 地上部及香苦草 (H. suaveolens) 莖部發現具有抑制黃嘌呤氧化酶的成分。因此本研究旨在運用高效液相層析-固相萃取-細管轉移-核磁共振儀串聯技術 (HPLC-SPE-TT-NMR),分析同為香苦草屬的穗花香苦草 (H. spicigera)、短柄香苦草 (H. brevipes) 及香苦草種子 (山粉圓) 是否含有抑制黃嘌呤氧化酶的成分,並期望賦予山粉圓更高的應用價值。 穗花香苦草葉部、短柄香苦草地上部及香苦草種子之乙醇萃取物,分別經液相-液相分配進行極性分割後,取乙酸乙酯與正丁醇可溶部分,經Sephadex LH-20 column分群,再運用HPLC-SPE-TT-NMR及HPLC-MS進行分離與分析,自穗花香苦草葉部鑑定出2個酚類成分 (1及2)、4個黃酮類成分 (4、5、17及18) 與12個苯丙烷類成分 (3及6-16),其中,sagecoumarin methyl ester (13) 為一新化合物;自短柄香苦草地上部鑑定出1個酚類成分 (1)、3個黃酮類成分 (4、24及25) 與10個苯丙烷類成分 (3、6、7、10、12及19-23),其中,shimobashiric acid C (19)、lithospermic acid B (20) 及其甲酯化衍生物 (21及22) 為四聚體苯丙烷類,此為首次自香苦草屬植物中發現;最後,自香苦草種子鑑定出6個酚類成分 (3、26-29及38) 與9個苯丙烷類成分 (3及30-37),與過去自其莖部發現之成分差異極大,且化合物30-37為dicaffeoylquinic acid類成分。透過定量實驗揭示香苦草種子富含dicaffeoylquinic acid類成分,且於黃嘌呤氧化酶抑制試驗呈現中等抑制效果 (30、31、35及37),可見其具有保健食品之應用價值。 第二部分:Aporphine及phenanthrene衍生物之製備暨其抗乙醯膽鹼酶與黃嘌呤氧化酶之探討 乙醯膽鹼酶抑制劑常用於改善阿茲海默症的認知及行為功能異常之症狀。本實驗室過去發現litebamine (40) 具有抑制乙醯膽鹼酶 (AChE) 之效果,其結構上N原子與4號位置之O原子間之距離與乙醯膽鹼接近,使得4-OMe被認為是重要的藥效基團。然而,進一步移除aporphine衍生物之2-OH,維持1-OH或-OMe;或移除litebamine之3-OH,維持4-OH或-OMe (43-47) 卻導致抑制活性下降。因此本研究旨在製備去除1-OMe且保留2-OH或-OMe之aporphine衍生物 (50-52), 及保留3-OH且去除4-OMe之litebamine衍生物 (54及56),繼續探討其活性。其中,以化合物56具有最佳的抗AChE活性 (IC50 6.3 μM c.f. galantamine 1.1 μM)。本研究結果釐清此類結構之結構與活性關係,以aporphine衍生物2號及litebamine衍生物3號位置的氧原子對於AChE抑制活性較為重要;此外,litebamine衍生物之6-OMe亦為重要基團,此結果將有助於開發衍自此類生物鹼之AChE抑制劑。 另一方面,本實驗室之阿朴芬類生物鹼於電腦分子模擬中與黃嘌呤氧化酶的結合力佳,但與實際酵素測試結果並不相符,推測可能是模擬時胺基自動質子化,而與酵素產生離子鍵,但實測時 (pH 7.5) 質子化卻未發生有關。本研究為探討此效應,以台灣雅楠富含之生物鹼laurolitsine為起始物,製備含四級銨之phenanthrene衍生物74;此外,亦同時製備含羥基 (68、80及96) 及羧基 (76及93) 之衍生物,並進行黃嘌呤氧化酶抑制實驗以評估其抑制效果。然而,具四級銨之目標產物74於100 μg/mL之濃度下僅達18%抑制率,無法有效提供離子鍵作用力。因此,此類阿朴芬生物鹼於電腦分子模擬與實際酵素實驗結果不符之原因尚需進一步研究釐清。 Part 1. Chemical investigation of Hyptis spicigera, H. brevipes, and the seed of H. suaveolens Xanthine oxidase inhibitors are used in clinic to prevent recurrent gout. As some severe side effects of allopurinol, a common xanthine oxidase inhibitor, were reported, the discovery of safer xanthine oxidase inhibitors is still in medical need. Several unusual phenylpropanoids had been isolated from the aerial part of H. rhomboides and the stem of H. suaveolens from our lab. They displayed good inhibitory activity against xanthine oxidase. Whether Hyptis spicigera, H. brevipes, and the seed of H. suaveolens (san fen yuan) from the same genus contain similar chemical constituents is the aim of current study. Our efforts led to the identification of 18 compounds (1-18) from the ethanol extract of H. spicigera leaves, 14 (1, 3, 4, 6, 7, 10, 12, and 19-25) from H. brevipes aerial parts, and 15 (3, and 26-38) from H. suaveolens seeds by separation and analysis through liquid-liquid partitioning, Sephadex LH-20 chromatography, and HPLC-SPE-TT-NMR and HPLC-MS analyses. Among these isolates, sagecoumarin methyl ester (13) is new and four known, i.e. shimobashiric acid C (19), lithospermic acid B (20), lithospermic acid B 9''-methyl ester (21), and lithospermic acid B 9'''-methyl ester (22), were firstly reported tetrameric phenylpropanoids from Hyptis plants. Besides, H. suaveolens seed is abundant in dicaffeoylquinic acids (30, 31, 35, and 37) which displayed moderate inhibitory activity against xanthine oxidase, determined by quantitative reverse-phase HPLC analysis. Thus, this discovery demonstrated the potential of H. suaveolens seed as nutraceutical. Part 2. Preparation of aporphine and phenanthrene derivatives as acetylcholinesterase inhibitors and xanthine oxidase inhibitors Acetylcholinesterase inhibitors are able to improve the cognitive and behavioral abnormalities of Alzheimer’s disease. Litebamine (40) was demonstrated to possess good inhibitory activity against acetylcholinesterase (AChE) previously. The distance between N and O4 atoms of litebamine was close to that of the corresponding ones in acetylcholine. Thus, this structure moiety was considered to be the pharmacophore. However, aporphines with 1-OH or -OMe but without 2-OH, and litebamines with 4-OH or -OMe but without 3-OH (43-47) were demonstrated poor AChE inhibitory activity by a lab colleague previously. In this study, aporphine derivatives without 1-OMe but with 2-OH or -OMe (50-52), and litetamine derivatives without 4-OMe but with 3-OH (54 and 56) were prepared to verify their anti-AChE activity furtherly. Among these derivatives, 56 displayed the best anti-AChE activity (IC50 6.3 μM c.f. galantamine 1.1 μM). This study discloses partial structure–activity relationship of aporphine and litebamine derivatives. The O2 of aporphine and O3 of litebamine skeletons are essential to possess AChE inhibitory activity, in contrast to that suggested before, i.e. O4. Besides, the 6-OMe substitution of litebamine derivatives was found essential as well. The current result provides useful information for the exploration of AChE inhibitors derived from these alkaloids. Several aporphine alkaloids have been demonstrated good docking scores by molecular docking, which, however, did not correlate well with their inhibitory activity against xanthine oxidase. The nitrogen atom of these aporphines was automatically protonated during docking study, providing additional ionic interaction to the binding. This phenomenon, however, might not occur under the bioassay condition (pH 7.5). To clarify whether such effect is the main cause of inconsistency between bioassay and molecular docking, this study is aimed to prepare phenanthrene derivatives from laurolitsine with either inherent ammonium (74), or hydroxy (68, 80, and 96), or carboxylic (76 and 93) group in the side chain. Unfortunately, the target compound 74 possessing the inherent quaternary ammonium group didn’t possess good inhibitory activity against xanthine oxidase (18% inhibition at 100 μg/mL). In conclusion, the current study could not verify the initial assumption and such issue remains to be clarified. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/78324 |
DOI: | 10.6342/NTU202002369 |
全文授權: | 有償授權 |
電子全文公開日期: | 2023-08-04 |
顯示於系所單位: | 藥學系 |
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