Comparison of the in vitro estrogenic activities of compounds from hops (Humulus lupulus) and red clover (Trifolium pratense)

Abstract

Because the prevailing form of hormone replacement therapy is associated with the development of cancer in breast and endometrial tissues, alternatives are needed for the management of menopausal symptoms. Formulations of Trifolium pratense L. (red clover) are being used to alleviate menopause-associated hot flashes but have shown mixed results in clinical trials. The strobiles of Humulus lupulusL. (hops) have been reported to contain the prenylflavanone, 8-prenylnaringenin (8-PN), as the most estrogenic constituent, and this was confirmed using an estrogen receptor ligand screening assay utilizing ultrafiltration mass spectrometry. Extracts of hops and red clover and their individual constituents including 8-PN, 6-prenylnaringenin (6-PN), isoxanthohumol (IX), and xanthohumol (XN) from hops and daidzein, formononetin, biochanin A, and genistein from red clover were compared using a variety of in vitro estrogenic assays. The IC50 values for the estrogen receptor alpha and beta binding assays were 15 and 27 microg/mL, respectively, for hops and 18.0 and 2.0 microg/mL, respectively, for the red clover extract. Both of the extracts, genistein, and 8-PN activated the estrogen response element (ERE) in Ishikawa cells while the extracts, biochanin A, genistein, and 8-PN, significantly induced ERE-luciferase expression in MCF-7 cells. Hop and red clover extracts as well as 8-PN up-regulated progesterone receptor (PR) mRNA in the Ishikawa cell line. In the MCF-7 cell line, PR mRNA was significantly up-regulated by the extracts, biochanin A, genistein, 8-PN, and IX. The two extracts had EC50 values of 1.1 and 1.9 microg/mL, respectively, in the alkaline phosphatase induction assay. On the basis of these data, hops and red clover could be attractive for the development as herbal dietary supplements to alleviate menopause-associated symptoms.

Figure 1

Formation of A) 8-PN from precursors from hops and B) genistein and daidzein from precursors from red clover.

Figure 2

Negative ion electrospray LC-MS chromatograms showing the ultrafiltration mass spectrometric screening of a hop extract for ligands to ERα and ERβ. Denatured ER was used as a control for non-specific binding and specific binding is indicated by increases in the chromatographic peak areas. IX and 8-PN were detected as the highest affinity ligands to both ERα and ERβ.

Figure 3

ERE-Luciferase induction in Ishikawa and MCF-7 cells by hop and red clover extracts, and their respective compounds. Cells were treated with either extracts (2 μg/mL) or pure compounds (100 nM) for 24 h, and then analyzed for chemiluminescence. Results were normalized for tranfection efficiency and they are shown as a fold induction relative to the level observed in cells treated with solvent only. Results are the mean of two determinations ± SD. Significant different from the control value was determined by one-way ANOVA with follow-up Dunnett test * 0.1 > p > 0.05. ** p < 0.05.

Figure 4

PR mRNA expression levels in the Ishikawa cell line by hops, red clover, and their respective compounds. Ishikawa cells were treated with either extracts (2 μg/mL) or pure compounds (100 nM) for 96 h, and then analyzed for PR mRNA. Results are shown as a fold induction relative to the level observed in cells treated with solvent only. Results are the mean of nine determinations ± SD with the exception of the hop extract, daidzein, and biochanin A where n = 6. * 0.1 > p > 0.05. ** p < 0.05.

Figure 5

PR mRNA expression levels in the MCF-7 cell line by hop and red clover extracts, and their respective compounds. MCF-7 cells were treated with either extracts (20 μg/mL) or pure compounds (100 nM) for 24 h, and then analyzed for PR mRNA. Results are shown as a fold induction relative to the level observed in cells treated with solvent only. Results are the mean of nine determinations ± SD with the exception of xanthohumol where n=6. * 0.1 > p > 0.05. ** p < 0.05.