Study On The Anti-pancreatic Cancer Active Quassinoids From Brucea Javanica And Their Structure-Activity Relationship

Our research is based on the project of National Natural Fund of China,the study of the structure-activity relationship and mechanism of the antipancreatic cancer effect of the quassinoids from Brucea javanica.We mainly studied the quassinoids in Brucea javanica.Firstly,quassinoids were isolated and purified from Fructus Brucea,then their structures were identified.The inhibitory activity of qassinoids on pancreatic cell lines was detected by MTT assays,and the structure-activity relationship of quassinoids against pancreatic cancer was analyzed.Then flow cytometry was used to detect the effect of quassinoids on apoptosis of pancreatic cell line and immunofluorescence was used to detect the expression of p38 protein in pancreatic cancer cells.The paper is divided into three chapters.The contents and main results of each chapter are summarized as follows:Chapter 1.Literature ResearchThe chapter is divided into three sections.In the first section the chemical structure type of quassinoids in Simarubaceae was reviewed and the structure characteristics of quassinoids in Brucea javanica was classified based on the specificity of A-ring in structures.In the second section the characteristics of13 C-NMR spectrum data of A-ring were summarized based on the structure characteristics of quassinoids in Brucea javanica,which provides a convenient reference for component identification.In the last section anticancer activities of quassinoids in Brucea javanica and its structure-activity relationship were summarized.Chapter 2.isolation and identification of the anti-pancreatic cancer active components of quassinoids in Brucea javanica.Extraction fraction of ethyl acetate from ethanol extract of Brucea javanica was separated and purified by column chromatography.Fifteen quassinoids were isolated from Brucea javanica and were identified as bruceine B,bruceine A,bruceantarin,yadanziolide B,bruceantin,bruceantinol,bruceine J,brusatol,yadanziolide S,bruceine E,bruceine D,yadanzioside K,yadanziolide A,yadanziolide C,and lbruceoside A.Chapter 3.Anti-pancreatic cancer activity evaluation and structure-activity relationship of quassinoids Brucea javanica.Section 1.Cytotoxicity of quassinoids in Brucea javanica on pancreatic cancer cellsFour kinds of human pancreatic cancer cell lines(MIA-PACA-2,ASPC-1,PANC-1,SW1990)were evaluated by MTT assays.The IC50 values of quassinoids on different pancreatic cancer cells were caculated.The results showed that the trend of proliferation inhibition on the different pancreatic cancer cells were basically similar.MIA-PACA-2 cells were more sensitive to quassinoids than the other three cell lines with IC50 values rang from 0.07 ?M to 4.780 ?M.Brusatol,bruceine B,bruceine A,bruceantinol and bruceantin showed significant inhibitory effects on cell proliferation,while yadanziolide S,bruceoside A and bruceine J show little poroliferation inhibition on pancreatic cancer cells(IC50>50?M).Based on the analysis of proliferation inhibitory activity and structure characteristics,the general characteristics of the anti-pancreatic cancer activities of quassinoids are summarized as follows:an ?.?-unsaturated ketene structure in A-ring is essential;substitution site of carbonyl group in A-ring has effects on the activities of quassinoids,for example,quassinoids which have a carbonyl in C-2 position show higher anti-pancreatic cancer activity than in other positions;hydroxyl in C-6 position of C-ring makes quassinoids more active;substituent groups in C-21 position may have influence on activities;the oxygen bridge structure in C-ring in necessary;ester substituents in C-15 position of D-ring enhance the anti-pancreatic cancer activity of quassinoids,meanwhile the length of carbon chain may not be too long or too large.Section 2.Evaluation of apoptotic effect of quassinoids in Brucea javanica on pancreatic cancer cellsTwelve active quassinoids were tested based on MTT assay's.Flow cytometry was used to detect the effects of these components on apoptosis of MIA-PACA-2 cells.It was found that quassinoids could induce apoptosis of MIA-PACA-2 cells in a dose-dependent manner.The degree of apoptotic induction of these compounds was similar to that of in vitro activity,suggesting that quassinoids might inhibit the growth of pancreatic cancer cells by inducing apoptosis.Compounds brusatol,bruceantin,Bruceine B,bruceantinol,bruceine A,bruceine D and yadanziolide B showed the most significant apoptotic induction effect,while bruceine E show little apoptotic induction effect.The common characteristics of quassinoids inducing apoptosis of pancreatic cancer cells are summarized as follows:an ?.?-unsaturated ketene structure in A-ring is essential;the carbonyl group in A-ring can enhance the apoptosis induced by quassinoids;hydroxyl in C-6 position of C-ring makes quassinoids more active in induction of cell apopsis;the lipophilicity of substituent groups in C-21 position may enhance apoptotic induction;the ester substituents in C-15 position of D-ring do contribution to the induction apopsis of quasionids,and high steric hindrance of substituents can decrease the activity of quassinoids;the oxidation of C-15 side chain can reduce the apoptotic induction of quassinoids.Section 3,Evaluation of phosphorylation of p38 protein activated by quassinoids in Brucea javanicaWestern blot was used to detect the expression of phosphorylated p38 protein induced by quassinoids in MIA-PACA-2 cells.The results showed that brusatol,bruceine B,bruceantinol,bruceine D,and bruceantin can significantly active the phosphorylation p38 protein in pancreatic cancer cellsl,especially brusatol,suggesting that these compounds may induce apoptosis of pancreatic cancer cells by activating the phosphorylation of p38 protein.However some quassinoids such as bruceine A induce cell apopsis through other pathway.The common characteristics of quassinoids stimulating phosphorylation of p38 protein in pancreatic cancer cells are summarized as follows:an a,?-unsaturated ketene structure in A-ring is essential;the lipophilicity of substituent groups in C-21 position may enhance apoptotic induction;ester substituents and unsaturated double bond in C-15 position may enhance the activity of quassinoids;2-5 carbons in aliphatic chain of C-15 position is appropriate and steric hindrance will decrease the activity of quassinoids.