Study On The Extraction Of Total Flavonoids From Vernonia Amygdalina Del.and Its Antitumor Activity In Vitro

Vernonia amygdalina Del.(Bitter Leaf),which is native to tropical Africa,belongs to the branch of Asteraceae(or Compositae)of Vernonia.It is used as herbal medicine and traditional vegetable for local people.The active components of Bitter Leaf include saponins,alkaloids,terpenoids,steroid,coumarin,flavonoids,phenolic acids,lignans,xanthene,anthraquinones and sesquterpenoids.It has been found that Bitter Leaf has various effects such as cancer prevention,anti-cancer,antibacterial and anti-mutation effects.Its main components are hot topics of pharmacologists in recent years.Among them,flavonoids show outstanding efficacy and it has made certain achievements for the treatment of liver cancer,breast cancer,cervical cancer,leukemia,et al.through variety of targets and diversified channels.To continue to expand anticancer spectrum plays an important role in promoting the development and utilization of its medicinal value.Esophageal cancer is a common digestive tract tumor.About 300 thousand people died from it every year in the world,and China is also one of the high incidence areas.Although there are many studies on Bitter Leaf,there have been no studies on its effects for esophageal cancer.In this study,the total flavonoids from Bitter Leaf planted in Fujian Agriculture and Forestry University were extracted,and their antibacterial activities were studied.In addition,its anti-tumor activity and the mechanism were investigated by study its effects on ECA109 cells.The main investigations and experimental results in this paper were listed as follows:1.The ultrasonic extraction method was used to extract total flavonoids in Bitter Leaf.The optimized reaction conditions were 50 ℃,51 mins,75%ethanol,ratio of liquid to material 40:1 mL/g.The extract was concentrated and then extracted by petroleum ether.After 24h,water phase was processed by HPD-722 macroporous adsorption column to remove sugar and impurities.Subsequently,70%ethanol elution,concentration and freeze drying were performed to obtain 15.33 g flavonoids.The content of total flavonoids was 192 mg/g.2.Under the optimum conditions,the total flavonoids were obtained by extraction and separation from Bitter Leaf.The results of bacteriostasis experiment showed it has a certain inhibitory effect on three kinds of bacteria including staphylococcus aureus,Escherichia coli and bacillus subtilis.In particular,the inhibited effect for the Escherichia coli is especially obvious.3.The results of CCK8 method for screening of resistant tumor cells showed that the total flavonoids of Bitter Leaf have strong inhibitory activity on four kinds of tumor cells,such as human hepatocellular carcinoma cell line(HepG-2),human esophageal cancer cell(ECA109),human gastric carcinoma cell line(BGC-823)and human breast cancer cells(MDA-MB-231),of which for ECA109 is strongest.Moreover,the inhibition effect has a relationship with quantity and aging.After 72 hours,the maximum inhibition rate reached 86.9%.4.Further study was made to investigate its anti-tumor mechanism in vitro with ECA109 cells.The cell morphology was observed by inverted microscope,found that the number of cells decreased,cells showed cytoplasmic retraction,strong refraction and narrow cell.The apoptosis of ECA109 cells was detected by the method of PI single dye flow cytometry.The results revealed that the DNA content of G0/G1 phase decreased,while increased significantly in S phase and G2/M phase,and the cell cycle blocked in S and G2/M phase.It can be seen that total flavonoids can inhibit the proliferation of ECA109 cells and induce apoptosis,which can effectively control the development of tumor.The results indicated that the inhibitory effect of total flavonoids extracted from Bitter Leaf on Escherichia coli was the best.In vitro,it has strong inhibitory effect on ECA109 cells of esophageal cancer and can induce apoptosis.This study laid the foundation for the research and development of total flavonoids of Bitter Leaf for the treatment of esophageal cancer and new anticancer drugs,and the subsequent studies in vitro and toxicology.All above properties made it possible to be developed into a new anticancer drug.