Study On The Antitumor Active Constituents Of Vernonia Patula Merr.

Vernonia patula Merr.is one of the Vernonia plant with large quantities,widely distributed,common plant.Related pharmacological studies have shown that Vernonia patula have a good antitumor activity.Therefore,In this paper,we make more systematic study for Vernonia patula based on the study of anti-tumor active compounds of Vernonia patula.In this paper,MTT colorimetric method was used to screen the antitumor active sites of Vernonia patula,and then the active parts were separated and purified by using a variety of modern chromatographic isolation techniques to obtain the active compounds with high purity and further study the anti-tumor activity of the monomer compounds.On this basis,it will lay a theoretical foundation for the development and utilization of Vernonia patula in the future through further study of the extraction process and establishment of quality standard method.In this experiment,the anti-tumor active site of Vernonia patula was screened by small experiment.A small amount of Vernonia patula powders was carried out by98%industrial ethanol and the ethanol extraction was extracted with petroleum ether,chloroform,ethyl acetate and n-butanol respectively.The extract of four parts was obtained,after alcohol extraction,the drying Vernonia patula continue to be extracted by water and the aqueous extraction was concentrated to obtain water extraction.MTT assay was used to determine the inhibitory effect of various parts on human hepatocellular carcinoma cells,human gastric cancer cells and human breast cancer cells.The results showed that the ethyl acetate site of Vernonia patula has a strong inhibitory effect on three human tumor cells and is the best site of antitumor activity.In order to obtain the maximum amount of ethyl acetate and be helpful to separate and purify the chemical constituents,using the quality of crude extract as the reference standard,the influence of ethanol concentration,ratio of material to solution,extraction time and extraction times on the quality of crude extract were studied.The orthogonal experimental design of four factors was established to explore the influencing factors and determine the optimal extraction scheme.The results showed that the factors affecting the extraction effect of Vernonia patula were the ratio of solid-liquid ratio>ethanol concentration>extraction times>extraction time,and determined the best extraction scheme is 90%ethanol,solid-liquid ratio is 1:10,extraction time is 30min/min,extraction times is 3 times.In order to study the chemical constituents of the ethyl acetate fraction,the ethyl acetate extract were extracted and purified by normal phase silica gel column chromatography,reversed phase silica gel column chromatography(ODS),macroporous adsorption resin(D101)and gel column chromatography(Sephadex LH-20).The structure of the four high purity monomer compounds were identified by physical and chemical properties and spectroscopic data.Four purity high compound are 2-nonene(I)、8?-(3-chloro-2-hydroxy-2-methylpropanoyloxy)hirsutinolide(II)、linoleic acid(III)、(2S,3S,4R,10E)-2-[(2R)-2-hydroxytetradecanoylamino]-10-octadecane-3,4-triol(IV)respectively.The antitumor activity of the monomeric compounds I、II、III、IV was me asured by MTT assay.The results showed that 8?-(3-chloro-2-hydroxy-2-methylpropanoyloxy)hirsutinolide(II)and(2S,3S,4R,10E)-2-[(2R)-2-hydroxytetrad ecanoylamino]-10-octadecane-1,3,4-Triol(IV)had agood inhibitory activity against three human cancer cells,which was the main component of anti-tumor activity of ethyl acetate.Establishing the quality standard for the Vernonia patula.In the experiment,(2S,3S,4R,10E)-2-[(2R)-2-hydroxytetradecanoylamino]-10-octadecane-1,3,4-Triol(IV)was used as the reference substance,The content of IV in the extract of Vernonia patula was determined by benzoylation-UV detection method.Further analysis was carried out on the determination of precision test,stability test,repeatability test and sample recovery rate.The results show that IV had a good linear relationship in the range of 8-128μg/ml,the regression equation isy(28)12.423x(10)14.486(R~2(28)0.999).The content in the extract of Vernonia patula is 0.086μg/mg.The average recoveries are 97.56%and RSD is 1.02%.