Chemical Study On The Components Of The Inula Helenium

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.