Estrogenic activity of two standardized red clover extracts (Menoflavon) intended for large scale use in hormone replacement therapy.

Isoflavones are the most potent estrogenic compounds in red clover extracts. Standardized extracts have been discussed as an alternative for hormone replacement therapy. Variation due to extraction procedure and natural seasonal variation and variations originating from agricultural conditions have prevented the large scale use of such phytochemicals. An improved extraction procedure and careful analysis of the raw material yielded in a highly standardized preparation (Menoflavon) with an average isoflavone content of approximately 9% (dry weight) determined by HPLC. The estrogenic activity has been further evaluated by a yeast two plasmid system using estrogen receptor alpha (ER alpha) and estrogen receptor beta (ER beta). An estrogenic activity corresponding to a transactivational capacity of ca. 18 microg 17 beta-estradiol per g red clover extract for ER alpha and ca. 78 microg 17 beta-estradiol per g red clover for ER beta was obtained. The difference is explained by the higher affinity of ER beta to isoflavones than that observed for ER alpha. Calculation of potency from isoflavone content measured by HPLC yielded a comparable potency to that experimentally determined by the bioassay. The high content of isoflavones as well as the higher transactivational potency for ER beta than ER alpha make these extracts interesting candidates for HRT.

Transcriptional activities of estrogen receptor alpha and beta in yeast properties of raloxifene.

Raloxifene represents a potent compound for the prevention and treatment of osteoporosis and cardiovascular disease in postmenopausal women. Raloxifene exhibits targeted antiestrogenicity in breast and uterus, but acts as an agonist in bone and liver. This synthetic selective estrogen receptor modulator binds both estrogen receptors alpha and beta. The molecular mechanisms by which raloxifene exerts agonistic or antagonistic activity are still not resolved. Therefore, the binding behavior of raloxifene to estrogen receptors and its effects on DNA binding and transactivation were studied. The equilibrium binding affinity of raloxifene by displacing radiolabeled 17beta-estradiol exhibited a similar affinity behavior to that of its natural ligand. Using BIACORE technology with an immobilized estrogen response element, we showed that 17beta-estradiol and raloxifene increased the binding of estrogen receptor alpha to the DNA, suggesting a ligand-dependent dimerization. The influence of the ligands to the binding of estrogen receptor beta was lower. We may conclude that unliganded estrogen receptor alpha binds as a monomer whereas in the presence of 10(-8) M 17beta-estradiol or higher, homodimers are formed that interact with the estrogen response element. Transactivation studies in a yeast reporter system in a ligand-dependent manner resulted in a similar potency of raloxifene to estrogen receptor beta compared to the control testosterone. Subeffective doses of raloxifene combined with 17beta-estradiol did not shift the efficiency, whereas saturating concentrations of 17beta-estradiol combined with increasing concentrations of raloxifene altered the response induced by 17beta-estradiol. In this pure system, the antagonistic activity of raloxifene could not be detected as was expected by the results from ligand competition analysis.

Analysis of mitogenic activity of proteins after separation by gel electrophoresis.

We have used a combination of gel electrophoresis and a cell culture assay in microplates to analyse mitogenic activity in tissue extracts. The procedure is a modification of the method described by Kuo et al. The proteins were separated by native gel electrophoresis or isoelectric focusing. The gel was sliced and defined pieces were transferred into tissue culture inserts fitting in 96 well microplates, which contained the test cells. The proteins diffused from the gel slices directly into the culture supernatant and the mitogenic effects were evaluated by a colorimetric assay (MTT or phosphatase activity). Human interleukin 2 was used to demonstrate the feasibility of the method by evaluating the mitogenic effect on the cell line CTLL-2. Extracts of bovine pituitary glands were separated by native gel electrophoresis and isoelectric focusing and several protein bands could be identified which showed a distinct mitogenic effect on human endothelial cells. The method is very sensitive and allows rapid screening of protein mixtures for bioactive fractions.