The Anti-tumor Angiogenesis Effect Of Active Ingredient From Sanguisorba Officinalis L And Mechanism Study

Objective:To screen the anti-tumor angiogenesis active ingredient from sanguisorba officinalis L, investigate the anti-tumor angiogenesis effect of the active ingredient and the mechanism of Ellagic Acid on anti-tumor angiogenesis.Methods:① The anti-tumor angiogenesis active extract screen from sanguisorba officinalis L. The preparation of five kinds of extracts from sanguisorba officinalis L were investigated using HUVEC, MCF-7, Bel-7402 proliferation model by SRB assay. ②Isolation of fraction, screen of the active ingredient and structure identification. The isolation of fraction were studied using HPLC assay; The anti-tumor angiogenesis active ingredient screen was investigated using HUVEC, MCF-7, Bel-7402 proliferation models by SRB assay. Using area normalization method to detect the purity of fraction; The structure of the active fraction was determinated by MS and H-NMR. ③The anti-tumor angiogenesis effect study of active fraction (Ellagic Acid): The effect of Ellagic Acid on proliferation, migration and tube formation of HUVEC were studied using SRB assay, wound healing assays and the tube formation respectively; Mice bearing S180 and H22 cells were randomly grouped into 5 groups: The model group(5%CMC),Cyclophosphamide group(positive control group), Ellagic Acid high-dose group (200 mg/kg), Ellagic Acid middle-dose group (100 mg/kg) and Ellagic Acid low-dose group (50 mg/kg),the drugs were successive administration for 10 d.Then the tumor inhibitory ratios,body weight,the thymus and spleen index were measured respectively. The effect of Ellagic Acid on microvasculardensity was explored using immunohistochemicalassay. ④ The anti-tumor angiogenesis mechanism study of Ellagic Acid:RT-PCR and ELISA assays were used to investigate the effect of ellagic acid on the expression of PDGFB mRNA and the secretion of PDGFB in the supernatant. The expression of PDGFB, STAT3 and p-STAT3 were studied using Western blot analysis. The effect of Ellagic Acid on expression of PDGFB,p-STAT3 and STAT3 in tumor were explored by immunohistochemicalassay.Results: ①DY extract of N-butyl alcohol significantly inhibited the proliferation of HUVEC, MCF-7 and Bel-7402 (P<0.01 or P<0.05). ②22 fraction (1-22#) were isolation from DY extract of N-butyl alcohol and 5# significantly inhibited the proliferation of HUVEC, MCF-7 and Bel-7402 (P<0.01 or P<0.05).5# is Ellagic Acid. ③ Ellagic Acid (20、10、5μg/mL) inhibited the proliferation, migration and tube formation of HUVEC significantly(P<0.01 or P<0.05),which inhibited HUVEC proliferation in a dose-dependent and time-dependent manner and migration and tube formation of HUVEC in a time-dependent manner.④ Ellagic Acid (200、100、50 mg/kg) inhibited the tumor growth of S180 and H22 significantly while have no influence on body weight. The spleen index of high-dose group was higher than the model group (P<0.05) and the micro vascular density of high-dose group and middle-dose group on S180 and H22 were less than model group (P<0.05). ⑤ Ellagic Acid (20、10、5 μg/mL) down-regulated the gene and protein expression of PDGFB (P<0.01 or P<0.05)and inhibited the phosphorylation of STAT3(P<0.01 or P<0.05)in HUVEC while have no influence in total STAT3 protein expression. The expression of PDGFB,p-STAT3 and STAT3 of Ellagic Acid dosage groups were lower compared to the model group in tumor.Conclusion:The anti-tumor angiogenesis active ingredient of sanguisorba officinalis L is Ellagic acid and it has an definite anti-tumor angiogenesis, which may related to the decreased expression of PDGFB which further affect the expression of STAT3 and the phosphorvlation of STAT3.