In vitro assays for bioactivity-guided isolation of endocrine active compounds in Vitex agnus-castus

Introduction: Extracts of Vitex agnus-castus (VAC) are used for the treatment of premenstrual symptoms. The mechanism of action was proposed to be dopaminergic and estrogenic in nature. To isolate these endocrinologically active substances, receptor ligand binding assays and cell cultures were used as in vitro systems to monitor the bioactivity-guided chromatographic separation. Methods: Estrogen receptor (ER) and Dopamine D-2-receptor ligand binding assays were performed with human recombinant receptor protein. Both known ERs (ER alpha and ER beta) were used. Cultures of anterior pituitary cells were prepared from 3 months old female rats. The concentrations of prolactin (PRL) and of cAMP in the supernatants were measured by radio immunoassay (RIA). Starting material for the isolation of single constituents was the aqueous ethanol 70% (v/v) VAC extract BNO 1095. The fractionations were performed with high speed counter current or Sephadex-LH-20 chromatography. Structure elucidation was conducted with GC-MS, LC-MS, H-1 NMR and C-13 NMR. Results: The bio-guided fractionation of BNO 1095 resulted in the isolation of dopaminergic bicyclic diterpenes. The fraction with the highest dopaminergic activity was a mixture of diterpenes of the clerodane type, designated as "BNO-diterpenes". These newly isolated diterpenes inhibited cAMP formation and PRL-release in rat pituitary cell cultures. The estrogenic compounds in BNO 1095 were identified as the flavonoids penduletin and apigenin. Both substances are specific ligands for the ER beta. Conclusion: Using the strategy of bioactivity-guided fractionation we were able to isolate new dopaminergic diterpenes with a high specific activity which contribute significantly to the PRL-lowering activity of VAC. The therapeutical potential of the ER beta specific ligands penduletin and apigenin needs further investigation. (c) 2006 Elsevier Ireland Ltd. All rights reserved.