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  • The use of recombinant ER and ER binding


    The use of recombinant ERα and ERβ binding assays offers a relatively inexpensive, rapid technique for screening compounds for potential direct novobiocin receptor modulatory activity and understanding the mechanisms of existing herbal medicines. For background on the protein production system used to generate the ER used in this ERBA and the functional characterization, including, coactivator and DNA-binding, please see (Bhavnani et al., 2008; Bolger et al., 1998; Bramlett et al., 2001; Chaudhri et al., 2014; Kim et al., 2005; Lin et al., 2013; Margeat et al., 2003; Tamrazi et al., 2002; Weber and Fussenegger, 2009). We will include novobiocin references to various functional assays that can be used in conjunction with the ERBA further in the discussion. A method was modified and validated to accommodate the binding assay for crude plant extracts (Obourn et al., 1993). Validation experiments determined appropriate radioligand and receptor concentrations required to construct a standard curve by which estrogen binding equivalents could be calculated. The possibility of matrix interference was rigorously tested. Linearity, parallelism and quench studies all made it evident that there was little matrix interference. One of the samples failed to demonstrate linearity in the volume tests; this sample, a hops extract, was a commercial preparation of hops concentrate and the only tested extract having higher binding affinity for ERα than ERβ. The cause of this remains unknown but may be related to commercial processing. ERα showed significantly less affinity for the phytoestrogens than ERβ. Based on EBE concentrations, the most potent ERα binding extracts were that from alfalfa which approached that of kudzu (Pueraria montana var. lobata (Willd.) Maesen & S.M.Almeida ex Sanjappa & Predeep) (Gray et al., 2015). Additionally, some samples had no detectable binding to ERα; however, all extracts showed binding to ERβ. Compared to ERα, ERβ had a higher affinity for all but one (hops concentrate) of the plant extracts, similar to that of a reporter gene assay for the determination of ER activities (Andres et al., 2015). The EBE concentrations in the ERβ binding test were 3-fold higher for alfalfa, 7-fold higher for licorice, and 33-fold higher for red clover than for ERα. Dietary isoflavones have differing activity depending on the cell and tissue, stimulating an estrogen receptor-dependent transcriptional response and promoted growth of estrogen-dependent MCF-7 cells in culture (Verma and Goldin, 1998; Zava et al., 1997). Glycitein displayed greater affinity with ERβ than ERα in a study using two different cells lines (Tchoumtchoua et al., 2016). Biochanin A is reported to affect steroidogenesis and ERβ expression (Nynca et al., 2013). Formononetin, found in red clover, was reported to decrease proliferation of MCF-7 cells through ERβ and insulin-like growth factor-1 receptor (IGF-1R) pathways (Chen et al., 2013, 2011). Estrogenic effects of red clover and alfalfa in animals and binding at ERα and ERβ have been reported (Chandsawangbhuwana and Baker, 2014; Ferreira-Dias et al., 2013; Hong et al., 2011; Liu et al., 2001a, b). Licorice root extract and two of its novel estrogenic compounds, glabrine and isoliquiritigenin, have estrogenic activity in cell proliferation and transactivation assays (Chansakaow et al., 2000; Maggiolini et al., 2002; Tamir et al., 2001, 2000). The preferential binding to one ER type compared to the other is referred to as a selective estrogen receptor modulator (SERM). The SERM activity of licorice has been examined in functional assays of ovarian cancer cells, beta-cells and breast cancer cells that can be utilized in conjunction with the ERBA (Bae et al., 2018; Boonmuen et al., 2016; Liu et al., 2001a, b; Mersereau et al., 2008). An example of ERβ crosstalk with protein kinase B / Akt was reported (Bae et al., 2018). A novel mechanism of cellular protection by these phytoestrogens may involve ERβ-mediated induction of manganese superoxide dismutase (Robb and Stuart, 2014).