Study Of The Immunomodulatory And Anti-tumor Effects Of Forsythia Suspensa Extracts

Objectives:1.To study the anti-tumor effect of Forsythia suspensa extracts in vitro and to preliminarily screen for active fraction of Forsythia suspensa extracts with anti-tumor function.2.To investigate the inhibitory effect of Forsythia suspensa extracts on the proliferation of mouse splenocytes in vitro and to preliminarily screen for active fraction of Forsythia suspensa extracts with immunomodulatory function.3.To explore the immunomodulatory function of active fraction of Forsythia suspense extracts.Methods:1.The models of cell proliferation of mouse S-180 tumor cell line and splenocytes were used to preliminarily screen the hot water extract of Forsythia suspensa for the anti-tumor and immunomodulatory active fractions.(1) Crude Forsythia suspense was extracted for three times by 12-fold distilled water and the three portions of hot water extracted solution were combined and concentrated by vacuum evaporation to obtain the condensed hot water extract of Forsythia suspense(FSE).(2)The condensed hot water extract of Forsythia suspense was then further extracted with petroleum ether,aether and ethyl acetate one by one.The residue was precipitated with 80%alcohol.Obtained by this way were petroleum ether fraction (FSⅠ),aether fraction(FSⅡ),ethyl acetate fraction(FSⅢ),alcohol-soluble fraction (FSⅣa) and alcohol-insoluble fraction(FSⅣb).(3) MTT assay was used to study the effect of the hot water extract of Forsythia suspensa and its chemical fractions mentioned above on the proliferation of mouse S-180 tumor cells and splenocytes in vitro so as to find out the active fractions.2.Several in vitro and in vivo experimental models were used to study the immunomodulatory effects of active fractions of the hot water extract of Forsythia suspense,including mouse splenocyte proliferation induced by Con A which was used to test the function of T lymphocytes,mouse splenocyte proliferation induced by LPS to test the function of B lymphocytes,MTT assay to measure the metabolic capability of mouse peritoneal exudate cells,ELISA assay to evaluate TNF-αlevel produced by mouse peritoneal macrophages,ELISA assay to assess the levels of INF-γand IL-2 secreted by mouse splenocytes,phagocytosis assay using chicken red blood cells to estimate the phagocytic activity of mouse peritoneal macrophage(MΦ) in vivo,limited dilution combined with micro-hemolysis to determine the level of hemolysin in mouse serum and mouse ear swelling to estimate the extent of delayed type hypersensitivity. Results:1.The hot water extract of Forsythia suspensa and its alcohol-soluble fraction (FSIVa) had marked inhibitory effect on the proliferation of mouse S-180 tumor cells in vitro and the difference was significant(P＜0.05) compared with experimental control group.And all the other chemical fractions had little inhibitory effect on the proliferation of mouse S-180 tumor cells.2.The hot water extract of Forsythia suspensa and its alcohol-soluble fraction (FSⅣa) had strong inhibitory effect on the proliferation of splenocytes in vitro and the difference was significant(P＜0.05) compared with experimental control group. And its alcohol-insoluble fraction(FSⅣb) in high concentration(8mg·mL^-1) also had marked inhibitory effect on the splenocyte proliferation in mice in vitro and its difference had significance(P=0.008) compared with the experimental control group. And all the other chemical fractions had little inhibitory effect on the proliferation of splenocytes.3.The experimental results showed that the alcohol-soluble fraction(FSⅣa) of the hot water extract of Forsythia suspensa had various immunological inhibitory effects on mice such as inhibitory effect on splenocyte proliferation induced by Con A or LPS,on MTT metabolic capability of peritoneal exudate ceils in mice,on TNF-αsecretion by peritoneal macrophages in mice,on secretion of INF-γand IL-2 by splenocytes in mice,on the phagocytosis of peritoneal macrophage(MΦ) in mice in vivo,on the level of hemolysin in mouse serum and on delayed type hypersensitivity in mice. Conclusion:1.The hot water extract of Forsythia suspensa has strong inhibitory effect on the proliferation of mouse S-180 tumor cells in vitro.Its main active fraction is its alcohol-soluble fraction(FSⅣa).2.The hot water extract of Forsythia suspensa also has strong inhibitory effect on the proliferation of mouse splenocytes in vitro.Its main active fraction is its alcohol-soluble fraction(FSⅣa) as well.3.The hot water extract of Forsythia suspense(FSⅣa) has some inhibitory effect on the specific immunity and the nonspecific immunity in mice.