title: [Establishment and application of co-transfection screening method for phytoestrogen active constituents].
abstract: OBJECTIVE
  To establish a highly sensitive screening method for phytoestrogen active constituents and to primarily screen the phytoestrogenic active constituents from the chickpea extractions by the method.
  
  
  METHODS
  Human ERalpha cDNA was cloned using MCF-7 total RNA as the template by RT-PCR and then was constructed into a pcDNA3 and named as pERalpha. The cell line MCF-7 was co-transfected with pERalpha and the reporter plasmid pERE-Luc which carrying the estrogen response element (ERE) plus the luciferase reporter gene. The luciferase activity was then assayed. The model was optimized by changing the ratio of two plasmids. The feasibility of the optimized model was further proved by the several known phytoestrogen compounds including fermononetin, biochanin A and genistein, et al. As an application of the model, the phytoestrogen activity of the extracts of the chickpea was assayed.
  
  
  RESULTS
  The recombinant plasmid (pERalpha) can enhance luciferase activities of pERE-Luc transfected MCF-7 cells. The highest transfection efficiency and luciferase activity were found at the ratio of 10:1 (pERE-Luc: pERalpha), the luciferase activity was improved five times as high as the unique pERE-Luc transfection. The co-transfection screening model also indicated that fermononetin, biochanin A and genistein could induce ERE-driven luciferase activity and ICI 182,780 suppressed the induced transcription. As the application of the model, the results showed that the ethanol (70%) total extraction, the ethyl acetate extraction and the ligarine extraction of the chickpea can induce ERE-driven luciferase activity. Concurrent treatment with ICI 182,780 abolished the induced luciferase activity.
  
  
  CONCLUSIONS
  A phytoestrogen active constituent screening mode have been established based on co-transfection method. It is sensitive to assay the phytoestrogen active constituents and can be applied to screen the active component of phytoestrogens.
authors.affiliation: Key Laboratory of Plant Resources and Chemistry of Arid Zone, Xinjiang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Urumqi 830011, China. weihuabojack@sina.com
authors.forename: Huabo
authors.initials: H
authors.lastname: Wei
authors.forename: Abulimiti
authors.initials: A
authors.lastname: Yili
authors.forename: Qingling
authors.initials: Q
authors.lastname: Ma
authors.forename: Dina
authors.initials: D
authors.lastname: Mai
authors.forename: Zhenhua
authors.initials: Z
authors.lastname: Wang
authors.forename: Hairong
authors.initials: H
authors.lastname: Ma
mesh_terms: D045744:Cell Line, Tumor; D029863:Cicer / Q000737:chemistry* / Q000378:metabolism; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D047628:Estrogen Receptor alpha / Q000235:genetics / Q000378:metabolism; D017930:Genes, Reporter / Q000187:drug effects; D005822:Genetic Vectors / Q000378:metabolism; D019833:Genistein / Q000737:chemistry / Q000494:pharmacology; D006801:Humans; D008156:Luciferases / Q000187:drug effects* / Q000378:metabolism; D048789:Phytoestrogens / Q000032:analysis* / Q000494:pharmacology*; D010936:Plant Extracts / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D010957:Plasmids / Q000187:drug effects / Q000378:metabolism; D014162:Transfection / Q000379:methods
chemical_list: C506487:ESR1 protein, human; D047628:Estrogen Receptor alpha; D048789:Phytoestrogens; D010936:Plant Extracts; D019833:Genistein; D008156:Luciferases; C004541:biochanin A