Preliminary Study On Chemical Constituents, Antitumor Activity And Exometabolism Of Intestinal Bacteria In Croton Branches And Leaves

Croton tiglium L.belongs to the genus Croton of Euphorbiaceae family,Theseeds of C.tiglium L.are well known as ‘Badou' in China,whose roots and leaves arealso used medicinally.C.tiglium L.has been mainly used for the treatment ofgastrointestinal disorders and intestinal inflammation in traditional Chinese medicine.C.tiglium L.mainly contained diterpene and alkaloids,and toxalbumin,which haseffect on lapactic,antitumor,antimicrobial.The previous research in our lab foundthat diterpenoids extracted and isolated from Croton leaves showed potentcytotoxicity against the K562,A549,DU145,H1975,MCF-7,U937,SGC-7901,HL60,Hela,and MOLT-4 cell lines while few of the diterpenoids exhibited cytotoxiceffects on normal human cell lines 293 T and LX-2,respectively.On this basis,furtherseparation of compounds in the ethyl acetate fraction of Croton leaves,and study onmetabolism of Croton leaf extract by Human intestinal bacteria in vitro.Specifically,the main contents of this dissertation are as follows:Part?:Research on the chemical constituents of ethyl acetate parts of crotonleaves.In this paper,HP-20 column chromatography,MCI column chromatography,column chromatography,vacuum column chromatography,column chromatography,gel column chromatography,RP-18 reversed phase column chromatography,thinlayer chromatography and preparative high performance liquid chromatography wereselected to isolate the compounds of ethyl acetate fraction of Croton leaves.Five newtigliane diterpene esters(1-5),highly oxygenated tetracyclic diterpenoid carbonskeleton,together with three known analogues(6-13)were isolated from the leaves ofCroton tiglium which are 4-deoxy-20-oxophorbol-12-tiglyl-13-acetate(1),7-oxo-5-ene-phorbol-13-(2-methylbutyrate)(2),7-hydroxyl-phorbol-5-ene-13-(2-methyl)butyrate(3),7-hydroxyl-phorbol-5-ene-13-isobutyrate(4),13-O-(2-metyl)Butyryl-phorbol(5)12-O-tiglyl-4-deoxy-4a-phorbol-13-acetate(6),Crotignoid E(7),phorbol(8),20-deoxy-20-oxophorbol 12-tiglate 13-(2-methyl)butyrate(9),12-O-acetyl-phorbol-13-isobutyrate(10),12-O-benzoylphorbol-13-(2-methyl)butyrate(11),12-O-tiglyl-7-oxo-5-ene-phorbol-13-O-2-methylbutyrate(12),13-O-(2-metyl)butyryl-4-deoxy-4a-phorbol(13),respectively.Most of the isolated compounds were tested for their cytotoxic and antitubercular activities.Among them,the cytotoxic activities of these tigliane-type diterpenes strongly depend on the species of the tumor cells.Especially,these bioactivity compounds showed strong cytotoxic activities against the K562 cell line,and their inhibition effects were close to the positive control Taxol.Furthermore,compounds 1,2,6,and 7 showed potent antitubercular activities with MIC values of 19.5,20.9,20.5,and 13.4 ?M,respectively.Part ? : Metabolism of Croton leaf extract by Human intestinal bacteria in vitro.UPLC-MS/MS was used to analysis and identification of metabolites of Croton leaves extract ingredients,in order to speculate that the metabolic changes in normal human Croton leaves in intestinal flora.Through qualitative data spectrum analysis,3-Deoxo-3-hydroxyphorbol-12,13,20-triacetate,Crotignoid K,12-O-Tiglylphorbol-13-isobutyrate,Crotignoids J and other phorbol esters compounds of Croton extract in twigs and leaves had been metabolized by human intestinal flora.In conclusion,based on the further separation of compounds in the branches and leaves of Croton,and in vitro metabolism of preliminary research,the result has laid a solid foundation for the development of Croton antitumor drugs.