Extraction And Isolation Of Alkaloids From Metaplexis Japonica Makino And Its Effects On Human Liver Cancer SMMC-7721 Cells

ObjectiveThe purpose of this experiment was to optimize the extraction process of alkaloids from the seeds of Metaplexis japonica,to isolate and purify alkaloids,and study its effect on human liver cancer SMMC-7721 cells.It provided an experimental basis for the research and development of alkaloids in seeds.MethodsThe alkaloids of Metaplexis japonica seeds were extracted by acid water extraction method,and the alkaloid extraction rate was used as a standard.Through single-factor experiments,selected the appropriate range of four factors(p H of aqueous hydrochloric acid solution,material-liquid ratio,extraction temperature,and extraction time)that had a significant impact on the total alkaloid extraction rate.Designed and optimized the extraction process of alkaloids from Metaplexis japonica seeds to determine the optimal extraction conditions.The silica gel column separation method was used to separate and purify the extracted alkaloids.The molecular weight and structure of the separated and purified alkaloids were identified by mass spectrometer and nuclear magnetic resonance spectroscopy(NMR,Nuclear Magnetic Resonance).The effects of isolated N-Nornuciferine on the viability,apoptosis,and inhibition of migration of human liver cancer SMMC-7721 cells were studied by MTT method,Flow cytometry,Western blot and Scratch test.ResultsOptimization of the extraction process by single factor experiments and orthogonal experiments determined the optimal extraction conditions for thealkaloids of Metaplexis japonica seeds: p H of aqueous hydrochloric acid solution 2,material-liquid ratio 1:20(g/m L),extraction time 2.0 h,extraction under the conditions of 70 ℃,the extraction rate of alkaloids from Metaplexis japonica seeds was 2.91%.A monomeric alkaloid was separated and purified through a silica gel column.The alkaloid was identified as N-Nornuciferine with a molecular weight of 281.3 by mass spectrometry and nuclear magnetic resonance.MTT experiments showed that N-Nornuciferine can inhibit the proliferation of SMMC-7721 hepatoma cells;The flow cytometry was performed using the AV-PI double staining method.The results showed that compared with the control group,N-Nornuciferine can induce obvious apoptosis of SMMC-7721 cells,and it showed a certain concentration dependence;Using JC-1 kit combined with flow cytometry to test,the results confirmed that N-Nornuciferine can reduce mitochondrial membrane potential in SMMC-7721 cells significantly.Western Blot results showed that Bcl-2 protein expression decreased,BAX and Cleaved PARP protein expression increased,indicating that N-Nornuciferine induced SMMC-7721 cell apoptosis.Scratch experiments and Western Blot confirmed that N-Nornuciferine can reduce SMMC-7721 cell migration.ConclusionsThe extraction process optimized by single factor experiment and orthogonal experiment improved the extraction rate of alkaloids from Metaplexis japonica seeds.A silica gel column was used to separate and purify the crude alkaloids to obtain a monomeric alkaloid N-Nornuciferine with a molecular weight of 281.3.Cell experiments showed that N-Nornuciferine can inhibit the proliferation of SMMC-7721 cells,induce the apoptosis of SMMC-7721 cells,and reduce the migration of SMMC-7721 cells.