The estrogenic activity of isoflavones extracted from chickpea Cicer arietinum L sprouts in vitro.

Isoflavones have drawn attention due to their potential therapeutic use. Isoflavones are the important chemical components of the seeds and sprouts of chickpea and higher isoflavones in sprouts than in seeds. However, there have been no previous reports of the estrogenic activity of isoflavones extracted from chickpea Cicer arietinum L sprouts (ICS) in vitro. In this study, which incorporated several in vitro bioassays methods, we systematically evaluated the estrogenic properties of ICS. MTT assay showed that ICS at the low concentration ranges (10(-3)-1 mg/L) promoted MCF-7 cell growth, while at high concentrations, (>1 mg/L) inhibited cell proliferation, indicating ICS worked at a diphasic mechanism. Flow cytometric analysis further calculated the proliferation rate of ICS at low concentration (1 mg/L). ERα/Luc trans-activation assay and then semi-quantitative RT-PCR analysis indicated that ICS at low concentrations induced ERα-mediated luciferase activity in MCF-7 cells and promoted the ER downstream target gene pS2 and PR trans-activation. These effects were inhibited by ICI 182,780, a special antagonist of ER, indicating that an ER-mediating pathway was involved. Alkaline phosphatase (AP) expression in Ishikawa cells showed that ICS at low concentrations stimulated AP expression. Our current study is the first to demonstrate that ICS has significant estrogenic activity in vitro. ICS may be useful as a supplement to hormone replacement therapy and in dietary supplements.

Establishment and application of co-transfection screening method for phytoestrogen active constituents / 中国中药杂志

<p><b>OBJECTIVE</b>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.</p><p><b>METHOD</b>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.</p><p><b>RESULT</b>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.</p><p><b>CONCLUSION</b>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.</p>