The Anti-tumor Metastasis Effect Of Ethyl Acetate Fraction Of Scirpus Yagara

Scirpus yagara belongs to the family of Cyperaceae,its tubers have been used in traditional Chinese medicine for the treatment of blood stasis and abdominal mass lump.Modern pharmacological studies showed that S.yagara has many biological activities,including anti-viral,anti-tumor,hepatoprotective and anti-inflammatory.In recent years,studies have shown that inflammatory microenvironment is the seventh factor in the occurrence and development of cancer.Chronic uncontrolled inflammation is closely related to the occurrence and progress of a variety of malignant tumors.TLRs is a biological pattern recognition receptor,whose active component released by tumor cells is the dominant factor in the persistent inflammatory reaction in tumor microenvironment,which promoted tumor occurrence and metastasis.In this study,the anti-tumor metastasis effect of S.yagara was evaluated.The efficacy is a hot and difficult point in the research of traditional Chinese medicine(TCM),which is the key to reveal the material basis of TCM.Xiao-he Xiao firstly put forward"target component knockout/research plan in medicinal material",and the key components of TCM were locked quickly and accurately.In this research,components of anti-cancer metastasis in alcohol extract of S.yagara based on the component knockout mode were studied.Alcohol extract knockouted petroleum ether,ethyl acetate and n-butanol and water fractions,respectively.MTT assay was performed to measured B16 cell viability of target components and target components of negative samples.And cell migration and invasion were tested by wound-healing and transwell invasion assay,respectively.The results showed that B16 cell viability had no significant effect when the concentration of ethanol extracts less than 25 ?g/mL.The contribution rate of petroleum ether,ethyl acetate,n-butanol and water fraction in inhibition LPS-induced B16 cells migration,was 10.78%,42.87%,12.76%and 4.6%respectively.In addition,the contribution rate of petroleum ether,ethyl acetate,n-butanol and water fraction in inhibition LPS-activated B16 cells invasion,was about-44.82%?45.38%?-60.11%?-55.61%,respectively.In addition,the result indicated that ethyl acetate fraction has a better inhibitory activity on LPS-induced cell migration and invasion than that of other fraction.There is obvious antagonism between petroleum ether,ethyl acetate,n-butanol and water fraction on inhibition LPS-induced B16 cell migration and invasion of alcohol extract of S.yagara.Further,we studied the anti-tumor metastasis effect of ethyl acetate fraction of B16 cells LPS-induced in vivo and in vitro.MTT assay was performed to measured B16 cell viability of ethyl acetate.Then,cells migration and invasion were detected by wound-healing and transwell assay,respectively.In vivo experiment,the mouse lung metastasis model and the liver metastasis model were established by injecting the tumor cells through the caudal vein and the spleen.The results showed that B16 cell viability had no significant effect when the concentration of ethyl acetate fraction less than 50 ?g/mL.Ethyl acetate fraction dose-dependently reduced LPS-induced B16 cells migration and invasion.In the lung metastasis model,50 ?g/mL ethyl acetate fraction significantly reduced the number of metastatic nodules on the surface of lungs,lung weight,and expression of inflammatory factor TNF-and IL-6 in the serums and lung tissues.HE experiments further revealed 50?g/mL ethyl acetate fraction could reduce lung metastasis.In the experiment of liver metastasis,50 ?g/mL ethyl acetate fraction can significantly inhibited the number of metastatic nodules on the surface of livers,liver weight,and expression of inflammatory factor TNF-and IL-6 in the liver tissues.To further exploration the material basis of ethyl acetate fraction in anti-tumor metastasis activities,we determined the main compositions of ethyl acetate by HPLC.According to the structure type,selecting representative components to study B16 cells LPS-or Pam3csk4-induced migration and invasion by wound-healing and transwell assay.The results showed that ethyl acetate fraction contained a variety of components,including p-hydroxycinnamic acid,ferulic acid,2-hydroxyl xanthone and other ingredients.According to the structure type,selecteing 8 representative components,respectively,including p-hydroxycinnamic acid(p-HA),ferulic acid(FA),dehydrophyllodulcin(DPL),2-hydroxy xanthone(2-HX),diphenyl acetylene(JCB8),Sciryagarol I(J1),Sparstolonin B(SsnB)and SanLeng diphenyllactone(SA2).Further,we studied their anti-cancer metastasis and their synergistic effect.The result showed isocoumarins(DPL,SsnB and SA2),xanthones(2-HX)and stilbenes(J1)significantly inhibited LPS-and Pam3csk4-induced B16 cells migration and invasion,and synergism of different type compounds had a better inhibitory effect than single drug.Phenolic acids(p-HA and FA)had no significant inhibitory effects on LPS-or Pam3csk4-stimulated B16 cells migration and invasion.