Studies On The Methods Of Extraction And Determination Of Two Alantolactions In Inula Helenium L.

Inula helenium L. medicine material is the dry root of Inula helenium L. which belongs to the compositae.Pharmacological studies have shown that Inula helenium L. can inhibit tumor cells, and it also has anthelmintic, antibacterial,antiinflammatory, hypoglycemic, analgesic and other effects. The major bio-active components of Inula helenium L. are volatile oils, and there are also alkaloids, sterols and amino acids in it.In this study, two compounds were isolated from Inula helenium L. By using MS, NMR spectroscopy and other identification means, their chemical structure were indentified as isoalantolactone and alantolactone.The extraction methods of isoalantolactone and alantolactone were studied in this paper, and matrix solid phase dispersion(MSPD) was firstly used to extract the two constituents in Inula helenium L. By comparing with the tradional methods of reflux, Soxhlet, ultrasonic and microwave extraction, it was found that MSPD had the highest extraction rate with advantages of short time extracting and reagents saving.The determination method of isoalantolactone and alantolactone was also established in this experiment by using HPLC with UV detector. The contents of these two alantolactones in Inula helenium L. were determined.1 Extraction, separation and identification of chemical constituents from Inula helenium L.1.1 ExtractionDried roots of Inula helenium L.(5.0 kilograms) were crushed and extracted by heat reflux in 95% ethanol for three times. The volume of the reagent used each time were 40L,30L,30L, and the extraction time were 60min,45min,30min, respectively. The combined extracting solution, after the sovent was evaporated.Then hot water was added to the extract, after suspension, it was extracted with petroleum ether for three times. After recovering petroleum ether,180g extracts was obtained.1.2 Seperation and identificationThe extracts was subjected to silica gel column repeatedly with the mobile phase of petroleum ether and ethyl acetate in the ratio of 12 to 1. All the components were collected and the solvent was removed. Then recrystallization in anhydrous ethanol was done to obtain two compounds. By using MS, NMR spectroscopy and their physical and chemical properties, compoundⅠand compoundⅡwere indentified as isoalantolactone and alantolactone after comparison with the literature.2 The research of extraction methods of isoalantolactone and alantolactone2.1 MSPDThe contents of these two alantolactones were determined by HPLC, the results of which were used as indicators to optimize the extract conditions in the MSPD method. After studying the various factors, such as dispersant, eluting agent, the material ratio, eluent, purification agent and eluting solvent dosage, the best MSPD conditions were found as follow.Diatomite as dispersant, ethyl acetate as eluting agent, neutral alumina as cleaning agent, the dosage of eluting agent 12mL and water as cosolvent.2.2 MAEVarious factors in the microwave-assisted extraction method were studied to find the best extraction conditions as follow.Hexane-ethanol (1:6,V:V) as extraction solvent,80℃as extraction temperature, 3min as extraction time and 1:50 as the liquid ratio.2.3 Traditional extraction methodsThe extraction solvent and times were studied in the method of reflux extraction. As a result, ethanol was used as extraction solvent to extract three times in reflux. The extraction solvent and time were studied in the method of Soxhlet extraction. The results indicated that petroleum ether was used as extraction solvent to extract 4.5h in Soxhlet. The ultrasonic extraction was performed strictly as what was described in Chinese Pharmacopoeia 2010.2.4 The extractions were performed in various metods with the best conditions as periviously selected, and it was found that the MSPD method had the highest extraction rate.3 Contents determination of two alantolactones in the Inula helenium L. by MSPD-HPLC3.1 Chromatographic conditionsHPLC was performed on ZORBAX SB C18 coloum (250mm×4.6mm,5μm), using a mixture of acetonitrile and water as the mobile phase in the ratio of 53 to 47. The flow rate was 1.0 mL-min-11 at 25℃, and the wavelength for determination was 194 nm.3.2 The linear rangeThe linearity performed well in the range of 0.2-20μg, the regression equations for compound I and compound II are as follow.Y=71912X+878.96,R=0.9995(isoalantolactone)Y=79204X+406.53,R=0.9998(alantolactone)3.3 Precision testSolution of isoalantolactone and alantolactone was injected into HPLC for six times in one day, the RSD of peak area for both compounds were 0.22% and 0.21% respectively;The same solution was injected into HPLC three times a day for three days, the RSD of peak area for both compounds were 0.50% and 0.29% respectively;,3.4 RepeatabilitySix copies of the same compound were injected into HPLC, the RSD of peak area for isoalantolactone and alantolactone were 0.30% and 0.15% respectively.3.5 Stability testThe same solution of sample was injected into HPLC once every 2h within 12h, the RSD of peak area for isoalantolactone and alantolactone were 0.32% and 0.31%.3.6 RecovriesThe average recovery for isoalantolactone was 95.4% and the RSD was 1.26%; the average recovery for alantolactone was 99.9% and the RSD was 0.82%.3.7 Determination of the samples of Inula helenium L.from different places By using MSPD extraction and HPLC determination, the results showed that the contents of isoalantolactone and alantolactone in the samples of Inula helenium L. from different places are as follow:1.19% and 1.12% (from Beijing),1.70% and 1.44% (from Anguo of Hebei province),1.28% and 1.29% (from Changchun of Jilin province),1.50% and 1.70%(from Bozhou of Anhui province),respectively. It can be inferred that the contents of the two alantolactones in Inula helenium L.are both more than 1%.In this paper, MSPD method of isoalantolactone and alantolactone from Inula helenium L. were furtherly studied. The quantification determination method of two alantolactones was established by using HPLC. As a new extraction method, it was firstly used to determinate the active principles from Inula helenium L., which performed fast and accurately. The results of this research provided a scientific basis for the improvement of the quality standards for Inula helenium L. In addition, the results can also provide a favorable basis for MSPD method to be used in the fast extraction of low polarity active ingredients from Chinese herbal medicines and their preparations.