【作者】 赵永明；

【导师】 史清文； 李作平；

【作者基本信息】 河北医科大学 ， 药物化学， 2007， 硕士

【摘要】 土木香(Inula helenium)为菊科(Compositae)旋覆花属植物,多年生草本,根供药用。该属植物约有100种,为多年生稀一或二年生草本。分布于欧洲、非洲及亚洲,以地中海地区为中心。我国有20余种和多种变种[1]。该属有多种植物常供药用。土木香在古方中常用于祛痰、镇咳和抗菌。近年来国内外对土木香化学成分及药理活性研究发现土木香内酯类成分有抗肿瘤细胞增殖作用和抗结核分支杆菌等作用。1999年,Charles等人研究了Inula helenium中土木香内酯类化合物对结核分枝杆菌的抑制作用,提出除α-亚甲基-γ-内酯是抑菌活性的必需功能基外,具有亲脂作用的不饱和双键和环氧基团可增加倍半萜内酯类化合物的体外抑菌活性[2]。2002年,Konishi等人从土木香根的甲醇提取物中分离得到了七个倍半萜,并研究了这些化合物对人胃癌细胞(MK-1)人子宫癌细胞(HeLa)以及小鼠黑素瘤细胞B16F10增殖的抑制作用。通过对比各化合物结构与抗肿瘤细胞增殖作用之间的关系,推测α-亚甲基-γ-内酯部分是该类倍半萜抗肿瘤细胞增殖的功能基[3]。2006年,Chen等人从土木香中首次分离得到Isocostunolide,并发现该化合物对人黑素瘤细胞(A2058),人肝细胞瘤(HT-29),人结肠腺癌细胞(HepG2)三种肿瘤细胞有明显的细胞毒性。并进一步阐明Isocostunolide对A2058的作用机制是通过线粒体依赖性途径诱导癌细胞凋亡[4]。由于土木香内酯类化合物具有多方面的药理活性,近年来有关土木香的研究越来越多,寻找新的化学成分,在此基础上进一步探讨同类化合物的构效关系并以此为指导对其活性成分进行深入研究,具有重要意义。为土木香的进一步开发利用和扩大药用资源提供化学成分方面的科学依据,我们对土木香根的化学成分进行了研究。在分离纯化并确定化合物结构的基础上,对化合物的活性进行筛选,希望发现新的抗菌和抗肿瘤化合物。目的:本课题以土木香(Inula helenium)干燥根为研究对象,利用各种分离手段对其化学成分进行全面系统研究。采用硅胶柱色谱, TLC, pre-TLC, pre-HPLC, Sephadex LH-20等方法对土木香的化学成分进行系统分离,寻找新的化学成分。并运用UV、IR、1H-NMR、13C-NMR、HMQC、HMBC、MS、NOESY等现代方法对所得单体化合物进行结构鉴定,以期发现新化合物及具有生物活性的单体化合物。方法:干燥的土木香(Inula helenium)根3.0 Kg适当粉碎后,用99%的乙醇回流提取三次,每次两小时。过滤,将所得滤液合并后减压浓缩至膏状,得到土木香提取物0.79 Kg。将土木香提取物浸膏悬浮在饱和盐水中,用石油醚、二氯甲烷、乙酸乙酯依次萃取,各部分萃取液分别减压浓缩至膏状。各部位萃取物分别经薄层色谱检识选择适宜的洗脱剂经硅胶柱色谱分离,梯度洗脱,接收到多份洗脱液,各份洗脱液回收溶剂后更换不同溶剂以便固体析出,析出固体后的母液及未析出固体的洗脱液经薄层色谱检识后,进一步用硅胶色谱、制备薄层色谱、制备高效液相色谱和葡聚糖凝胶(Sephadex LH-20)反复分离纯化得到多个单体化合物。分离得到的单体化合物用薄层色谱检识、高效液相检查纯度,再用UV、IR、1H-NMR、13C-NMR、1H-1HCOSY、HMQC、HMBC、MS、NOESY等方法对所得单体化合物进行检测,根据测定的波谱数据分析确定化合物的平面和立体结构。薄层检识中用薄层色谱硅胶为GF254,粘合剂为0.5%的羧甲基纤维素钠。显色剂为10%的硫酸-乙醇溶液。制备型高效液相所用流动相为甲醇-水(45:55),流速为3ml/min.结果:通过对土木香(Inula helenium)的化学成分进行了系统提取分离共得到19个化合物,并对其进行了结构鉴定。确定了12个化合物的化学结构(时间所限其他化合物的结构待鉴定),其中八个土木香内酯类化合物,一个生物碱类化合物,一个咖啡酸酸酐,一个三萜苷和一个甾醇类成分。确定了结构的12个化合物按分离得到的先后顺序排列分别为:isoalantolactone (1), 11,13-dihydroisoalantolactone (2), alantolactone (3),β-stiosterol (4), 3-hydroxy-11,13-dihydro- isoalantolactone (6), 1-hydroxy-11,13-dihydroisoalantolactone (7), 2-acetoxylmethyl-6-(7’-phenyl-2’-indoline)-indoline(8), 3-hydroxy-11,13-dihydroalantolactone (10), macrophylli- lactone E (11), 2-hydroxy-11,13-dihydroisoalantolactone (12) urs-12-en-18α-H-3-O-β-D- glucopyranoside (13), caffeic acid anhydride (14).结论:本文对土木香(Inula helenium)的化学成分进行了系统研究,对其成分的提取溶剂及提取分离方法适当,采用了硅胶柱色谱、TLC、pre-TLC、pre-HPLC、Sephadex LH-20等多种分离纯化手段,共分离得到了19个化合物,运用UV、IR、1H-NMR、13C-NMR、HMQC、HMBC、MS确定了12个化合物的结构(其余的正在鉴定中)。其中,1-hydroxy-11,13-dihydroisoalantolactone (7), 2-acetoxyl- methyl-6-(7’-phenyl-2’-indoline)-indoline (8),和3-hydroxy- 11,13-dihydroalantolactone (10)三个化合物是新化合物。urs-12-en-18α-H-3-O-β-Dglucopyranoside (13)和caffeic acid anhydride (14)是首次从旋覆花属植物中分离得到。更多还原

【Abstract】 Inula helenium (Compositae) is a perennial herb. There exist about 100 species of the genus Inula, which widely distributed in Asia, Europe, predominantly in Mediterranean region, of them, more than 20 species being distributed in China[1]. Many of these have long been used in Chinese folk medicine. The roots of Inula helenium have been traditionally used as an expectorant, antitussive, diaphoresis and bactericidal agent. Recently, the investigations of its chemical constituents and biological activities have shown that the sesquiterpene lactones of it have antiproliferative activity and anti-inflammatory effect.In 1999, Charles et al. isolated several eudesmanolides from Inula helenium, which exhibited significant activity against mycobacterium tuberculosis, and suggested the presence of a second alkylating site, such asα,β-unsaturated carbonyl group or an epoxide function together with a moderate to high lipophilicity, seems to enhance the in vitro antimycobacterial activity of sesquiterpene lactones [1]. In 2002, Knishi et al. isolated seven sesquiterpenes from the MeOH extract of the roots of Inula helenium and studied antiproliferative activities of the isolates against MK-1, HeLa and B16F10 cells. Comparing the structures of these compounds and their antiproliferative activities, they infer that theα-methylene-λ-lactone moiety of these sequiterpenes contributed to the antiproliferative activity [2]. In 2006, Chen et al. isolated isocostunolide from the roots of Inula helenium for the first time, and reported isocostunolide could effectively induce cytotoxicity in three cancer cell lines: Human melanoma cells(A2058), human hepatoma cells(HepG2), human colon adenocarcinoma cells (HT-29). Furthermore, they found this compound induces apoptosis through a mitochondria dependent pathway in A2058 cells [3].Recently, much attention has been paid to Inula helenium due to its diverse biological activities. In order to exploid new bioactive sesquiterpene lactones, we initiated an investigation on the chemical components of the roots of Inula helenium.Object: To study the components in the roots of Inula helenium and search for new bioactive sesquiterpene lactones. Using Silica gel column chromatography to fractionate the crude extracts. Using preparative TLC, reversed phase preparative HPLC, Sephadex LH-20 to separate and purify integrates and to get compounds in pure form. Using spectroscopic methods including UV, IR, 1H-NMR, 13C-NMR, HMQC, HMBC, MS, NOESY to establish the structures of the isolated pure compounds.Methods: Air-dried roots of Inula helenium (3.0Kg), purchased in An-Guo herbal material market, Hebei Province, China, was chipped and refluxed in 99% Ethanol three times. The ethanolic extract was decanted and combined; the solvent was evaporated at reduced pressure. The residue, after removing solvement, was suspended in brine and partitioned with petroleum ether, dichloromethane, ethyl acetate in turn. Flash silica gel column chromatography was used to preliminary fraction, each fraction was monitored with TLC, developed TLC were detected by UV light at 254nm and followed by color reaction with spraying 10% H2SO4-ethanol reagent. Similar fractions were pooled and subjected to silica gel column chromatography, preparative TLC, reversed phase preparative HPLC, Sephadex LH-20 to separate and purify integrates and to get compounds in pure form. The purity was checked up with TLC under different developing system and analytic RP-HPLC. The spectroscopic methods including various of 1D and 2D NMR methods were used for structural identification.Result: After investigation on extraction of the roots of Inula helenium, 19 compounds were isolated from the roots of Inula helenium. These compounds were characterized on the basis of spectral analysis and the structures of 12 compounds were estabilished, including 8 eudesmanolides, an alkaloid, triterpenic glycoside, a caffeic acid anhydride and a sterol. They were isoalantolactone (1), 11,13-dihydroisoalantolactone (2), alantolactone (3), β-stiosterol (4), 3-hydroxy-11,13 -dihydroisoalantolactone (6), 1-hydroxy-11,13-dihydroiso -alantolactone (7), 2-acetoxylmethyl-6-(7’-phenyl-2’-indoline)- indoline (8), 3-hydroxy-11,13-dihydroalantolactone (10), macrophyllilactone E (11), 2-hydroxy-11,13-dihydroiso alantolactone (12), urs-12-en-18α-H-3-O-β-D-glucopyranoside (13), caffeic acid anhydride (14).Conclusion: The result of our experiment indicated that the solvent and methods of extraction used in this experiment are practicable. Column chromatography, preparative TLC and preparative HPLC were employed to isolated and purify the components in the roots of Inula helenium in our experiment, and variety of spectroscopic methods were used to elucidated the structures of the compounds. 1-hydroxy-11, 13-dihydroisoalantolactone (7), 2-acetoxylmethyl-6-(7’-phenyl- 2’-indoline)-indoline (8), and 3-hydroxy-11,13-dihydro- alantolactone (10) were new compounds. Urs-12- en-18α-H-3-O-β-D-glucopyranoside (13), Caffeic acid anhydride (14) was reported for the first time from the genus of Inula.更多还原