Study On The Chemical Constituents And Pharmacological Activities Of Brucea Javanica

Brucea javanica is dried ripe fruits of Brucea javanica（L.）Merr.,which is mainly used for treatment of various cancers.At present,Brucea javanica oil emulsion has been well developed and applied,and Brucea javanica oil emulsion or soft capsule has been marketed as the first-line clinical anticancer drug in China.Although Brucea javanica has a clear pharmacological effect,but the substance basis of its efficacy is not clear;There is a lot of residue left in the production of Brucea javanica oil emulsion,so it can not give fully medicinal value of Brucea javanica.Therefore,based on the present research situation of Brucea javanica,the Brucea javanica oil was removed from the fruit of Brucea javanica,and we study the chemical constituents and biological activities of the remaining portions of the aqueous extract and the alcohol extract.In this study,the traditional extraction and separation methods were combined with modern separation techniques,such as preparative liquid chromatography,to separate the chemical constituents from the 20%ethanol elution part of HP20 macroporous resin.Eleven compounds were separated by macroporous resin HP20,MCI column chromatography,Sephadex LH-20 gel column chromatography and semi-preparative HPLC.The structures of 9 compounds were determined by ESI-MS,¹H-NMR,¹³C-NMR,etc.including yadanzigan（1）,bruceine E（2）,20-hydroxy-yadanzigan（3）,bruceine F（4）,bruceine H（5）,bruceine D（6）,isovanillic acid（7）,p-Hydroxybenzoic acid（8）,pyrocatechol（9）.Among them,20-hydroxy-yadanzigan was a new compound and isovanillic acid was reported from the Brucea javanica for the first time.In this study,the water extract from Brucea javanica was treated by modern separation and purification methods.Crude polysaccharide of Brucea javanica was dialyzed（MW:3000）to get out small molecule impurity,and according to the difference of surface charge and molecular size of polysaccharide,The crude polysaccharides were separated and purified by DEAE-52 and Sephadex G-200.Two homogeneous polysaccharides,BPS1 and BPS2,were obtained.The single peaks of BPS1 and BPS2by High performance gel permeation chromatography indicate that they were homogeneous polysaccharides.By Monosaccharide Composition Analysis,Methylation Analysis and other chemical methods,combined with GC-MS,Atomic Force Microscope（AFM）and Nuclear Magnetic Analysis（NMR）,the structure of two homogenous polysaccharides of Brucea javanica were characterized,including their monosaccharide compositions and sugar residue link type.The monosaccharide composition of BPS1:Xylose and Glucose,with molar ratio 5:7,the average molecular weight of it（MV）is 13.4 kDa;the monosaccharide composition of BPS2 is xylose and glucose.It molar ratio is 3:4 and the average molecular weight（MV）is 18.6 kDa.Three residues of 1,4-Xylp→,→1,3-Glup→and→1,4,6-Glup→were detected in BPS1;Three residues of T-Xylp→,→1,4-Glup→and→1,4,6-Glup→were detected in BPS2.In addition,based on the chemical research,the anti-complement and anti-inflammatory activities of some compounds were studied in vitro according to the literature and existing experimental conditions.In order to evaluate the anticomplement activity of compounds of Brucea javanica（homopolysaccharide and monomers）,the hemolytic activity of some compounds was determined in the classical pathway of complement.Quassinoids compounds had strong anti-complement activity(CH₅₀=0.027⁰.058 mg/mL),which was slightly stronger than that of the positive control heparin sodium(CH₅₀=0.066±0.003 mg/mL).In this experiment,the anti-complement active components of Brucea javanica were preliminarily screened,which laid a foundation for the study of the mechanism of the antipyretic and detoxifying activities of the Quassinoids compounds in vivo.We determined the anti-inflammatory activity of the isolated Quassinoids compounds.The results showed that all of the six compounds had anti-inflammatory effects on LPS induced inflammatory model in RAW264.7 cells.Compound 1（yadanzigan）has significantly effect(IC₅₀=29.69μM)and is almost non-toxic to RAW264.7 cells.Therefore,we selected compound 1 for further experimental study,using ELISA kit to detect the release of related cytokines and western blotting to detect the expression of various proteins related to inflammatory pathways.Result indicated that yadanzigan inhibited the expression of iNOS,COX-2,TNF-αand IL-6 in LPS induced RAW264.7cells in a concentration dependent manner.yadanzigan inhibited inflammation by inhibiting the activation of NF-κB pathways.In this study,the anti-inflammatory components of Brucea javanica were initially screened,which laid a foundation for the study of the mechanism of the anti-inflammatory activity of the Quassinoids compounds in vivo.