Study On Separation And Anticancer Activity Of Compounds From Tagetes Erecta L. Residue

Marigold,one of the most well known species of Tagetes L.,had been applied to extract lutein.In this study,marigold residue was chosen as object and studied from the following four aspects used by an offline two-dimensional preparative high performance liquid chromatography(HPLC)method guided by a real time cell analysis system: selecting the extraction procedure,establishing various modes of two-dimensional liquid chromatography(2D-LC)methods,separating compounds and pharmacodynamics study of anticancer activity.The study aimed to establish a separation method for active compounds guided by bioassay and provide worthy information for drug development.By studying the factors that affected the extraction of marigold flower residue,such as time,number,temperature and liquid ratio of extraction,the extraction process was optimized to get the best conditions.The best extraction process of marigold residue was extracted by ethanol,1: 5 solid-liquid ratio and hot reflux in 60 min with third at 60?.The ethanol extract of marigold residue was separated by first dimensional HPLC.The first dimensional separation was carried out by normal phase and reversed phase HPLC and the best preparation method was determined ultimately.By the normal phase preparative chromatography,9 fractions were collected.Samples were initially divided,which improved the efficiency of the preparation for the subsequent two-dimensional separation.Three anticancer active fractions were chosen for second dimension by preliminary screening.The system of 2D-HPLC was established using Innoval silica column and YMC Diol column.The results showed that the method possessed the properities of good selectivity and retention for flavonoids from marigold extracts.In second dimension,8 compoounds were obtained and their purities were above 96%.The structures of these compouds were identified by NMR data.The resuls indicated that they were mainly belonged to flavonoids and organic acids.Moreover,6-hydroxykaempferol was firstly found in Tagetes L..The result of preliminary screening of compounds showed Compounds 2 and 3 showed significant anticancer activity against A549 and Hep-G2 cells.Compounds 4 and 5 were effective against A549 cells,but had no obvious effect on Hep-G2 cells.The safety evaluation and anticancer activity ofcompound 5(quercetagetin)with the highest productionand inhibition on A549 cells were further investigated in vivo.The results suggested that the mice were living in good conditions without toxitoxic reaction and death after quercetagetin adiministration at dose in the range of 0.63 g/kg and 10.0 g/kg.The anicancer activity of quercetagetin were evaluated using Lewis lung tumor and H22 liver tumor mice models.Quercetagetin treatment effectively inhibited the lung and liver tumor,improved the quality of life and prolonged lifetime.In conclusion,this bioassay-guided offline two-dimensional preparative HPLC method established in this study played a vital role on getting active compounds from marigold quickly and effectively.It also provided a new train of thought for separation of active compounds from crude drugs.