Biological evaluation of isoflavonoids from Genista halacsyi using estrogen-target cells: Activities of glucosides compared to aglycones

Abstract

The purpose of this study was to evaluate the response of estrogen target cells to a series of isoflavone glucosides and aglycones from Genista halacsyi Heldr. The methanolic extract of aerial parts of this plant was processed using fast centrifugal partition chromatography, resulting in isolation of four archetypal isoflavones (genistein, daidzein, isoprunetin, 8-C-β-D-glucopyranosyl-genistein) and ten derivatives thereof. 7-O-β-D-glucopyranosyl-genistein and 7,4΄-di-O-β-D-glucopyranosyl-genistein were among the most abundant constituents of the isolate. All fourteen, except genistein, displayed low binding affinity for estrogen receptors (ER). Models of binding to ERα could account for the low binding affinity of monoglucosides. Genistein and its glucosides displayed full efficacy in inducing alkaline phosphatase (AlkP) in Ishikawa cells, proliferation of MCF-7 cells and ER-dependent gene expression in reporter cells at low concentrations (around 0.3 μM). ICI182,780 fully antagonized these effects. The AlkP-inducing efficacy of the fourteen isoflavonoids was more strongly correlated with their transcriptional efficacy through ERα. O-monoglucosides displayed higher area under the dose-response curve (AUC) of AlkP response relative to the AUC of ERα-transcriptional response compared to the respective aglycones. In addition, 7-O-β-D-glucopyranosyl-genistein and 7,4΄-di-O-β-D-glucopyranosyl-genistein displayed estradiol-like efficacy in promoting differentiation of MC3T3-E1 cells to osteoblasts, while genistein was not convincingly effective in this respect. Moreover, 7,4΄-di-O-β-D-glucopyranosyl-genistein suppressed lipopolysaccharide-induced tumor necrosis factor mRNA expression in RAW 264.7 cells, while 7-O-β-D-glucopyranosyl-genistein was not convincingly effective and genistein was ineffective. However, genistein and its O-glucosides were ineffective in inhibiting differentiation of RAW 264.7 cells to osteoclasts and in protecting glutamate-challenged HT22 hippocampal neurons from oxidative stress-induced cell death. These findings suggest that 7-O-β-D-glucopyranosyl-genistein and 7,4΄-di-O-β-D-glucopyranosyl-genistein display higher estrogen-like and/or anti-inflammatory activity compared to the aglycone. The possibility of using preparations rich in O-β-D-glucopyranosides of genistein to substitute for low-dose estrogen in formulations for menopausal symptoms is discussed.

Isoflavonoid Profiling and Estrogen-Like Activity of Four Genista Species from the Greek Flora

“…Having as reference standards the compounds isolated in our previous study of G. halacsyi [ 10 ], we confirmed the presence of two diglucosides of genistein [8- C ,4′- O -di-glucopyranosyl genistein ( 1 ) and 7,4′-di- O -glucopyranosyl genistein ( 2 )], five isoflavone monoglucosides [8- C -glucopyranosyl orobol ( 3 ), 7- O -glucopyranosyl isoprunetin ( 4 ), 8- C -glucopyranosyl 3′- O -methylorobol ( 5 ), 7- O -β- d -glucopyranosyl genistein ( 6 ), and 8- C -glucopyranosyl genistein ( 7 )], as well as seven isoflavonoid aglycones [daidzein ( 8 ), genistein ( 9 ), isoprunetin ( 10 ), 5- O -methylorobol ( 11 ), 8-methoxyformononetin ( 12 ), 3′-methoxyisoprunetin ( 13 ), and biochanin A ( 14 )] in some or all of the eight extracts ( Table 1 and Supplementary Material, Table S1 ). Of note, the isoflavonoids of Table 1 were previously isolated from a conventional MeOH extract of the aerial parts of G. halacsyi , using fast centrifugal partition chromatography followed by prep-TLC and/or silica and/or sephadex column chromatography of the fractions to deliver isolated compounds (>95% purity), as assessed by several spectrometric methods [ 10 ]. The data of Table 1 show that isoflavonoids 4 , 5 , 8 , 9 , 11 , 13 , and 14 were detected in all four Genista species, while 12 was not found in G. acanthoclada and G. hassertiana , 2 and 10 were not detected in G. depressa and G. millii, and 1 , 3 , 6 , and 7 were not observed in G. depressa .…”

“…The data of Table 1 show that isoflavonoids 4 , 5 , 8 , 9 , 11 , 13 , and 14 were detected in all four Genista species, while 12 was not found in G. acanthoclada and G. hassertiana , 2 and 10 were not detected in G. depressa and G. millii, and 1 , 3 , 6 , and 7 were not observed in G. depressa . We have reported that 2 , 6 , and 7 , are the main constituents of the aerial parts of G. halacsyi [ 10 ]. The limited phytochemical compositions of the aerial parts of G. millii and, in particular, of G. depressa , with 2 missing from the first and 6 missing from both, compared to the respective compositions of G. acanthoclada and G. hassertiana are possibly related to the comparatively lower yields of the EtOAc extract of G. millii and the MeOH extract of G. depressa .…”

“…Regarding the phytochemical composition of the MeOH extracts, in particular, only six isoflavonoids are featuring in the MeOH extract of G. depressa as compared to ten in that of G. millii and to thirteen in those of G. acanthoclada and G. hassertiana . We have previously reported that at the concentration of 1 μM, 2 and 6 displayed similar and higher, respectively, estrogen-like activity in Ishikawa cells compared to genistein, compatible with full conversion to the aglycone by cell membrane glucosidases, while 7 exhibited weak activity at this concentration, reflecting its ability to enter the cell using glucose transporters in the cell membrane as well as its metabolic stability [ 10 ]. Given that 2 is absent from G. millii extracts and 2 and 6 from G. depressa extracts, one may expect substantial differences in their estrogen-like activities compared to those of G. acanthoclada and G. hassertiana .…”

“…The estrogen-like activities of isoflavones render them potentially therapeutic agents for the prevention and/or treatment of health issues due to estrogen deficiency, including increased risk for cardiovascular disease, menopausal symptoms, and post-menopausal osteoporosis [ 5 , 8 , 9 ]. Based on our ongoing effort in highlighting the biodiversity of Greek flora as a unique source of bioactive agents [ 10 , 11 , 12 ] and especially on our previous research in identifying isoflavonoids with estrogen-like activity from G. halacsyi [ 10 ], we selected four Genista species of Greek flora, namely G. acanthoclada , G. depressa , G. hassertiana, and G. millii , for phytochemical and biological evaluation. We report herein for the first time a comparative phytochemical characterization of the four Genista species, utilizing a uHPLC-HRMS identification approach, and an evaluation of their estrogen-like activity, as assessed using human endometrial and breast adenocarcinoma cells.…”

Application of Various Molecular Modelling Methods in the Study of Estrogens and Xenoestrogens

“…Docking plant-derived substances into ER and comparing their binding energies and interactions (above all hydrogen bonding) with those of estradiol is a common practice. Such studies deliver information on conformational flexibility [ 81 ], the ability of the investigated molecules to selectively modulate ERα/β ability [ 82 , 83 ], their possible reductive influence on breast cancer risk [ 84 ], or their applicability to reduce menopausal symptoms [ 85 ]. Most importantly, data derived in this way very often show good consistency with experiments [ 86 ].…”