Studies On Isolation And Metabolism In Vivo And In Vitro Of Alantolactone And Isoalantolactone From Inula Helenium

Elecampane (Inula helenium)'s alias is'Qi mu xiang'in Chinese, which is perennial herbs. The roots of Inula helenium have been traditionally used as an expectorant, antitussive, diaphoresis and bactericidal agent [1]. Recently, it has been reported that the sesquiterpene lactones of it have antiproliferative activity and anti-inflammatory effect. In 1999 and 2002, Charles [2] and Konishi [3] et al. pointed out that theα-methylene-λ-lactone moiety of these sequiterpenes may contribute to the antiproliferative activity and anti-inflammatory effect, which led to quite extensive studies on the Inula helenium.It has been demonstrated that the major active components in I. helenium are alantolactone (AL) and isoalantolactone (IAL) [4], so we study the isolation and metabolism in vivo and in vitro on these two compounds. Meanwhile, isolation, purification and structure identification were also carried out on the metabolites. On this basis, the activity of the AL, IAL and the metabolites on human tumor cell should be screened. This work will organically combine the traditional studies of the medicinal natural product chemistry with the studies of pharmacology. The research provided a significant exploration for pharmacokinetics and therapeutic basis of TCM. In addition, the research of metabolic chemistry profited for discovery of new active constituents and synthesis of new pro-drugs.Part one Study on chemical consituents of Inula helenium (Preparation of AL and IAL reference substance)Object: To isolate and purify the two sesquiterpenes (AL and IAL) from the aerial part of the Inula helenium and screen the activity of them on human tumor cells.Methods: Aerial parts of Inula helenium was chipped and extracted with 95 % EtOH. The ethanolic extract was combined; the solvent was evaporated at reduced pressure. The residue was suspended in water and partitioned with petroleum ether, dichloromethane, and ethyl acetate in turn. The petroleum ether part was subjected to silica gel column chromatography to get compounds in pure form. The spectroscopic methods were used for structural identification.Results: After investigation on extraction of the aerial parts of Inula helenium, 2 compounds were isolated from the petroleum ether part. These compounds were characterized on the basis of spectral analysis as alantolactone (1) and isoalantolactone (2). The purity of alantolactone and isoalantolactone was higher than 98% by normalization method of HPLC. The results of primary bioactive screening on tumor cells showed that compound 2 displayed the strong activity in the test.Conclusions: Alantolactone and isoalantolactone with the similar polarity are difficult in separating, but they will be separated well by AgNO3 silica gel column chromatography. Compound 2 showed strong activity in the test of primary bioactive screening on tumor cells.Part two Metabolism of alantolactone (AL) and isoalantolactone (IAL) in vivo and in vitroObject: To study the biotransformation of alantolactone and isoalantolactone by anaerobic culture in vitro and in vivo for preparing metabolites, identifying their metabolites in rat and checking up their activity on human humor cell.Methods: 1. Culture solution of intestinal bacteria were produced from rat feces, and then incubated with AL and IAL by anaerobic culture to prepare metabolites of AL and IAL. Metabolites of AL and IAL were purified by TLC and silica gel column chromatography and identified by comparison of their ESI-MS, 1D NMR and 2D NMR with those of standard and prepared compounds. 2. After taking orally AL and IAL, blood, urine, feces, contents of stomach and contents of intestine were pretreated and analyzed by HPLC-PDA. The peaks of AL, IAL and its metabolites were validated by comparing their retention times and the maximum wavelengths of UV spectra.Results: Two metabolites of AL and one of IAL were isolated and identified, respectively. Three metabolites were named as D1: 13,13'- Dithiobis (11,13-dihydroalantolactone), d1: 13,13'-Dithiobis (11,13-dihydro- iso-alantolactone), d2: 13,13'-Thiobis (11,13-dihydroalantolactone), respectively. Moreover, it showed that AL and IAL were difficult to be metabolized in stomach and liver from the results in vivo. Except that d2 displayed weak inhabiting activity on human humor cell, the others results of screening did not show obviously inhabiting activity.Conclusions: Metabolites of AL and IAL prepared in vitro were coincident to those in biological samples. So we can prepare metabolites by anaerobic culture with intestinal bacteria. The metabolites of AL and IAL show less or no bioactivity on the inhibition of tumor cell proliferation.