Study On Tumor Metastasis Prevention Capacity And Mechanism Of Flavonoid-like Extractive From Murraya Paniculata (L.) Jack

Purpose:The fetal threat of tumor metastasis to patients after primary tumor treatment was a critical economic,social,scientific and technological problem,which is of urgent need to be solved in the worldwide.The metastasis is regarded to origin from circulating tumor cells(CTCs)presenting in the ’sub-healthy’ cancer survivals.’Anticancer drug’ is invalid to CTCs.In recent years,a large number of studies have showed that flavonoids have good anti-tumor activity in vivo and in vitro.Based on the low toxicity of flavonoids,and the effect of anti-contraception and implantation,anti-platelet activation and vasodilation of Murraya paniculata(L.)Jack,an original plants of traditional Chinese medicine(TCM)Jiulixiang,this thesis studied the anti-tumor metastasis function and mechanism of flavonoids isolated from TCM Jiulixiang(FMs)in vivo and in vitro,and provided a theoretical and experimental basis to further researches on the intervention of M.paniculata extractive for the metastasis cascade.Methods:TCM Jiulixiang(M.paniculata)were coarsely powdered and extracted following silica gel column chromatography.The extractive was divided to eight parts.From which,an active ingredient FMs was screend out basing on pharmacodynamics and HPLC analyses,and subjected chemical identification,DPPH radical scavenging activity test and spectroscopic characterization,such as fluorenscence.The MTT assay and cell morphology observation were carried out to estimate the toxicity of FMs.The adhesion assay,wound healing assay and transwell assay were carried out with HT-29 cells in vitro to estimatethe cell functions relative the initial progresses of metastasis cascade,such as hetero-adhesion,migration and invasion capacity.The melanoma B16-F10 tumor metastasis model was used to estimate the metastasis prevention capacity of FMs in vivo.Analytical flow cytometry was used to survey the effect of FMs on the surface adhesion factors expression of HT-29 cells and endothelial cells(HUVECs),the cells cycle and apoptosis of HT-29,and the uptake of FMs by HT-29 cells.One of the main composition of FMs was isolated by semi-preparative HPLC,with its structure deduced by NMR and MS.Results:A flavonoid ingredient(FMs)which had special fluorescence characteristics and significant antioxidant capacity was screened out from the extractives of M.paniculata.FMs was found to inhibit cell adhesion to HUVECs,migration and invasion capacity of HT-29,without significant impact on cycle distribution,apoptosis and morphology changes,and cell viability of HT-29 cells up to 30 μg/mL.When exposed to 30 μg/mL of FMs,the relative adhesion rate of HT-29 to matrix composition Fn and HUVECs decreased to 43%and 50%,respectively.The inhibition of heter-adhesion was associated with the down-regulation of β1-，a6-integrins and CD44 expression on HT-29 cells and E-selectin expression on HUVECs on dose-dependent manner.In vitro,FMs inhibited the lung metastasis of B16-F10 melanoma cancer in C57BL/6 mice,with statistically significant(p<0.01).In additon,a single compound,hemamethoxy flavanone-o-[rhamnopyranosyl-(l-T4)-rhamnopyranoside](HMFRR)was isolated from M.paniculata for the first time.Conclusion:The Flavonoids in Murrayapaniculata(L.)Jack(FMs)has low toxicity characteristics and can inhibit tumor metastasis in vivo and in vitro.It has potential application in prevention of tumor metastasis.