Standardization of a method for identification of elecampane sesquiterpene lactones

Abstract

Introduction A wide use of elecampane, Inula helenium L. (Asteraceae), in both, folk and formal medicine, is explained by variety of its chemical composition. Rhizomes and roots of elecampane contain an essential oil, the content of which reaches 4%; the composition of the oil includes a mixture of sesquiterpene lactones. The main components of this mixture are alantolactone (0.5-2.0%), isoalantolactone (1.0-2.7%), and their hydrogenated derivatives: dihydroalantolactone, dihydroisoalantolactone, tetrahydroalantolactone, etc. These compounds seem to be responsible for pharmacological action of elecampane-based preparations. Herbal drug (HD) from elecampane is rhizomes and roots. There is no monograph on this type of HD in the State Pharmacopoeia of Ukraine (SPhU), therefore research related to its development is relevant. The mandatory identification test for HDs by the thin-layer chromatography (TLC) method in accordance with the requirements of SPhU is absent in the article GF XI "Rhizomes and roots of elecampane", therefore it is necessary to develop a procedure for identification of the main biological active substances (BAS) of elecampane by TLC-method. Materials and Methods. The rhizomes and roots of Inula helenium L. (Asteraceae) were obtained from various pharmaceutical enterprises of Ukraine during 2016-2018. The following equipment was used: Silica gel 60 F₂₅₄ TLC plates (20x20 cm², 0.25 mm), Merck (Germany); Ultrasonic bath SUPER RK100H «Bandelin», (Germany); Glass vertical chamber; Automatic spraying device ChromaJet DS20. As standard substances have been used alantolactone, isoalantolactone and dihydroisoalantolactone (<95%) - pharmacopoeial reference standards of the State Pharmacopoeia of Ukraine. Results  and discussion . A procedure for identification of elecampane by TLC method for the national monograph of the SPhU "Elecampane roots and rhizomes" has been developed.. The identification method is based on the ability of silver ions to react with unsaturated C-C bonds in the molecules of the isomers at the position of double bonds, which are alantolactone and isoalantolactone. The development of a procedure for identification was carried out in conjunction with its validation in accordance with the requirements of SphU according to the following scheme: 1) the choice of stationary phase (examination of plates with a thin layer of silica gel treated with 1, 3, 5, 7% (m/v) solutions of silver nitrate); 2) the choice of mobile phase (review of unified phases for chromatography of terpenoids); 3) the choice of concentration for markers-substances (study of 0.05, 0.1, 0.2% methanol solutions of alantolactone, iso-alantolactone, dihydroiso-alantolactone and SQTL mixture for preparation of the reference solution); 4) the choice of the method for test solution preparation (study of extraction of herbal material with methanol, followed by concentration of the extract to the ratio of HD-test solution 1: 2, 1: 5, 1: 8); the choice of application volume of the test solution ; 5) the choice of the detection method (review of unified reagents and/or solutions for derivatization of chromatograms). Following conditions for identification have been chosen: the test solutions of HD (1: 5 in methanol), standard solution CRS SPhU alantholactone and isoalantholactone (0.1% solutions in methanol), TLC plates with a thin layer of silica gel treated with 5% silver nitrate solution, a solvent system toluene-ethyl acetate (9: 1), detection is carried out after treatment the plate with anise aldehyde solution and followed by heating. Conclusion. A procedure for identification of elecampane by for the national monograph of the SPhU "Elecampane roots and rhizomes" has been developed. It allows to identify such biologically active substances of the elecampane, as sesquiterpene lactones, which are markers of this species The developed chromatographic conditions allow to reliably chromatographically identify HD of elecampane in the presence on chromatograms of 3 zones of lactones – alantolactone, isoalantolactone and dihydroisoalantolactone.