title: In vitro anti-angiogenesis effects and active constituents of the saponin fraction from Gleditsia sinensis.
abstract: BACKGROUND
  The anomalous fruits of Gleditsia sinensis Lam. (Leguminosae), a crude drug in China, have long been used in traditional Chinese medicine for the treatment of various diseases. The saponin fraction isolated from the fruits (SFGS) is considered as the active component for the antitumor activity of this crude drug.
  
  
  OBJECTIVE
  The present study was performed to investigate the anti-angiogenesis activities and active constituents of SFGS.
  
  
  METHODS
  Human umbilical vein endothelial cells (HUVECs) were treated with SFGS in the presence or absence of basic fibroblast growth factor (bFGF) in vitro. The proliferation, migration, and tube formation were studied by MTT, Transwell, and 2D Matrigel assays, respectively. The cell cycle and apoptosis were analyzed by flow cytometry. Enzyme-linked immunosorbent assay for protein expression of vascular endothelial growth factor (VEGF) and western blot analysis for caspase-3, caspase-8, and caspase-9 as well as Fas were performed. In addition, the effects of 13 saponin compounds isolated from SFGS on the tube formation of HUVECs were screened, and the structure-activity relationships were discussed.
  
  
  RESULTS
  SFGS, at concentrations (1, 3, and 10 µg/mL) without significant cytotoxicity on endothelial cells, significantly inhibited the proliferation, migration, and tube formation of HUVECs induced by bFGF (10 ng/mL). It moderately arrested the cell cycle to G1 phase but greatly induced cell apoptosis and increased the expressions of caspases-3, caspase-8, and Fas but not caspase-9 in HUVECs. Moreover, SFGS did not affect the bFGF-induced autosecretion of VEGF from endothelial cells. Among the 13 saponin compounds tested, gleditsiosides B, I, J, O, and Q showed inhibition of the tube formation at a concentration of 3 µM, and only gleditsioside B exerted significant inhibition at 1 µM.
  
  
  CONCLUSIONS
  SFGS is substantially able to prevent angiogenesis by interfering with multiple steps. The findings provide a new explanation for the antitumor effects of G sinensis fruits. Gleditsiosides B, I, J, O, and Q are probably the main active constituents of SFGS.
authors.affiliation: China Pharmaceutical University, Nanjing, China.
authors.forename: Dan
authors.initials: D
authors.lastname: Lu
authors.affiliation: China Pharmaceutical University, Nanjing, China.
authors.forename: YuFeng
authors.initials: Y
authors.lastname: Xia
authors.affiliation: China Pharmaceutical University, Nanjing, China.
authors.forename: Bei
authors.initials: B
authors.lastname: Tong
authors.affiliation: China Pharmaceutical University, Nanjing, China.
authors.forename: ChaoFeng
authors.initials: C
authors.lastname: Zhang
authors.affiliation: China Pharmaceutical University, Nanjing, China.
authors.forename: Rong
authors.initials: R
authors.lastname: Pan
authors.affiliation: China Pharmaceutical University, Nanjing, China.
authors.forename: Huan
authors.initials: H
authors.lastname: Xu
authors.affiliation: China Pharmaceutical University, Nanjing, China.
authors.forename: Xue
authors.initials: X
authors.lastname: Yang
authors.affiliation: China Pharmaceutical University, Nanjing, China yuedaicpu@hotmail.com.
authors.forename: Yue
authors.initials: Y
authors.lastname: Dai
mesh_terms: D020533:Angiogenesis Inhibitors / Q000008:administration & dosage / Q000302:isolation & purification / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D002453:Cell Cycle / Q000187:drug effects; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism; D005434:Flow Cytometry; D005638:Fruit; D031501:Gleditsia / Q000737:chemistry*; D061307:Human Umbilical Vein Endothelial Cells / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D012503:Saponins / Q000008:administration & dosage / Q000302:isolation & purification / Q000494:pharmacology*
chemical_list: D020533:Angiogenesis Inhibitors; D012503:Saponins; D016222:Fibroblast Growth Factor 2