Study On Effect And Mechanism Of Alkaloid Constituents Of Strychnos Nux-Vomica L. On Human Colon Cancer HT-29 Cells

Objective Systematic extracted and separated Strychnos nux-vomica L.,the characteristic fingerprint of total alkaloids of Strychnos nux-vomica L.was establish by High Performance Liquid Chromatography(HPLC).Alkaloids from strychnos nux-vomica L.was observed the effect on the proliferation of human colon cancer HT-29 cell line to search for the main active constituent and further study its mechanism.Methods The alkaloid was obtained with reflux extraction and separated and purified by silica gel column chromatography,thin layer preparation and Pre-HPLC from Strychnos nux-vomica L..The total alkaloids of 10 batches Strychnos nux-vomica L.were obtained by reflux extraction and the characteristic fingerprint of the total alkaloids of Strychnos nux-vomica L.was established by HPLC.MTT was used to screen the alkaloid monomers which had significant inhibitory effect on human colon cancer HT-29 cells,and then obtained the IC50 value.HT-29 cells were treated with main bioactivity of different concentrations and than cultured 24 h,48 h,and 72 h to compare inhibitory effect by MTT Assay.The effects of HT-29 cell apoptosis,cell cycle and mitochondrial transmembrane potential with different experimental groups were detected by flow cytometry.Western Blot assay was used to detect the expression of P53?caspase3?parp?caspase9?bcl-2 protein in HT-29 cells treated with different experimental groups.Results1.Nine alkaloids,including brucine,pseudostrychnine,pseudobrucine,isostrychnine,isobrucine,nvoacine,icajine,and 16-methoxyl-protostrychnine were isolated from the processed seeds of Strychnos nux-vomica L.and determined by extensive analysis of ESI-MS,1H-NMR and 13C-NMR spectra.2.The 10 batches of the total alkaloids of Strychnos nux-vomica L.were compared for the similarity by using Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System(2004A).The HPLC characteristic fingerprint was established,the similarity analysis 10 batches of samples was 0.90 and ten common peaks were identified.The relative retention times were 0.81(No.1,isostrychnine),0.90(No.2,strychnine),0.96(No.3,brucine),1.00(No.4,16-methoxyl-protostrychnine),1.21(No.6,nvoacine),1.32(No.8,icajine).3.Compared with the positive control group,the inhibitory effects of brucine,pseudostrychine,pseudobrucineon and isobrucine were found by MTT assay,the difference wasn't statistically significant.The IC50 values of the four alkaloids were 0.192±0.18,0.207±0.13,0.265±0.43,0.228±0.22(?mol·mL-1),respectively.4.Brucine and pseudostrychnine has inhibitory effect on human colon cancer HT-29 cells,and has dose-dependent and time-dependent the effect.Compared with the blank group,the early apoptosis rate,late apoptosis rate and total apoptosis rate of HT-29 cells treated with different concentrations of brucine and pseudostrychnine for 48h were significantly different(**P<0.01),the changes of mitochondrial transmembrane potential in each group had a significantly decrease(**P<0.01).compared with the blank group,the number of HT-29 cells in G1 phase was significantly increased after treatment with brucine(250 ?mol·L-1),and the number of cells in S phase and G2 phase was significantly increased.After treatment with pseudostrychnine(250?mol·L-1),the number of HT-29 cells in Gi phase increased,the number of S phase cells remained unchanged,and the number of cells in G2 phase decreased.The difference was statistically significant(*P<0.05).With 125 ?mol·L-1 brucine treated,the expression of p53,parp protein was increased.The expression of p53,caspase3,parp,caspase9 was significantly increased with 250?mol·L-1,500?mol·L-1 brucine and pseudostrychnine treated.The expression of bcl-2 protein was significantly decreased in each group.The difference was statistically significant comparing with the blank group(*P<0.05,**P<0.01).Conclusion Ten alkaloids were isolated from Strychnos nux-vomica L.and nine alkaloids were identified by spectral analysis,the HPLC characteristic fingerprint of the total alkaloids of Strychnos nux-vomica L.was simple,quick and reproducible with high precision,which can lay a foundation for further study on the alkaloids constituents of Strychnos nux-vomica L.and quality control.The alkaloids of Strychnos nux-vomica L.has a certain inhibitory effect on human colon cancer HT-29 cell,brucine was the strongest,followed by pseudostrychnine.Brucine and Pseudostrychnine has significant inhibitory effect on human colon cancer HT-29 cells and has dose-dependent and time-dependent the effect.The mitochondrial transmembrane potential of HT-29 cells was decreased.HT-29 cells was arrested at the Gi phase after brucine and pseudostrychnine intervention and apoptosis by increasing the expression of p53,caspase3,parp,caspase 9 and decreasing the expression of bcl-2 protein.