Determination Of Four Flavanoids In Scutellariae Barbatae By HPLC With The Effects On U937 Cells Proliteration And Apoptosis

BackgroundThere had always been the saying of genuine regional drug in the science of Chinese materia medical since ancient times,which shows that the origin of medicinal herbs,the environment and other objective factors have an impact on the quality of Chinese herbal medicine and the content of effective components are different.With the constant appearance of analysis instrument and the deepening of analysis techniques,such as High Performance Liquid Chromatography(HPLC)which has the features of high efficiency,fast speed,high sensitivity and automatic operation,the kinds of chemical components contained in herbal medicines and the content of testing components can be reflected so that the medicine quality can be controlled overall.Herba Scutellariae Barbatae is the whole dry herbs of Labiatae plant,Scutellaria barbatae D.Don.In the clinical practice of Chinese Medicine,it's mainly used together with other medicines which clear away heat and toxic materials in the treatment of liver cancer,lungs cancer and other diseases.Flavonoids are active substances which contain the largest and most important Herba Scutellariae Barbatae.They can inhibit the proliferation of tumor cells,influence the medicine's metabolizing enzyme activity and induce cell apoptosis.This paper chooses four main Scutellariae Barbatae flavonoids(SBF)in Herba Scutellariae Barbatae,respectively cutellarin,baicalein,luteolin and Apigenin.The HPLC method which test the contents of the four components at the same time is established.And,the contents of the four Scutellariae Barbatae flavonoids in 12 batches of Herba Scutellariae Barbatae from different origins were examined to evaluate the medicine quality of Herba Scutellariae Barbatae.Leukemia is one of the most common hematological diseases.The main treatment methods are chemotherapy,bone marrow or stem cell transplantation.But,the side effects are high and the recurrence rate is high.Besides,it is difficult to transplant,and the cost is high.Herba Scutellariae Barbatae is often used in Traditional Chinese Medicine to treat leukemia.The study shows that Herba Scutellariae Barbatae extract can inhibit the proliferation and of K562 cells and induce their apoptosis and reduce the multidrug resistance of tumor cells.Multiple medicine's flavonoids components can inhibit the proliferation and apoptosis of leukemia cells.Generally,there are fewer reports on the treatment effect of Herba Scutellariae Barbatae on leukemia.This study first explored the influence of Herba Scutellariae Barbatae flavonoids on leukemia U937 cells' proliferation and apoptosis and discussed the possible function mechanism to provide theoretical basis for clinical treatment.ObjectiveThis study established the HPLC method which test the SBF content at the same time,tested the contents of four SBF in 12 batches of Herba Scutellariae Barbatae from different origins,studied the influence of SBF on the proliferation,apoptosis and cycle of leukemia U937 cells,tested the expression of SALL4 genes and downstream target genes c-myc and CCND1 in the signaling pathway of Wnt/?-Catenin using RT-PCR and explored the possible function mechanismMethodUse HPLC method to test the contents of four main SBFs and perform methodological investigation.Perform in-vitro culture of leukemia cells U937 and observe the influence of SBF on the form of U937 cells with inverted phase contrast microscope.Adopt CCK-8 to test the influence of SBF on the survival rate of U937.Use flow cytometry to test the influence of SBF on cell cycle and apoptosis;use RT-PCR to test the influence of SBF on the apoptosis-related gene expression in U937Results1.The testing method of HPLC is established to test the contents of the four flavonoids in Herba Scutellariae Barbatae.The methodological investigation is great.2.The observation result of the inverted phase contrast microscope shows that with the increase of time and concentration,cell numbers is decreased.The volume shrinkage becomes smaller and the number of fragments increases.3.Based on time and dose,SBF can inhibit the U937 cells proliferation significantly.4.After SBF solution acts on U937 cells fro 24h,it can block U937 cells in the G0/G1 stage.With the increase of concentration,the effect of its blocking effect is more obvious(P<0.01).Compared with the control group,SBF solution can induce U937 cells'apoptosis(P<0.01).5.Compared with the control group,SBF can down regulate the gene expression of SALL4,C-myc and CCND1.The differences have statistic significance(P<0.01).Conclusions1.The content testing method of four flavonoids components of Scutellariae Barbatae flavonoids is established to provide a means of quality control for it.2.SBF can inhibit the U937 cells proliferation significantly based on the concentration and time,down regulate the gene expression levels of SALL4,c-myc and CCND1 in U937 cells,induce cell apoptosis and block cells in the G0/G1 stage.