Estradiol-antagonistic activity of phenolic compounds from leguminous plants

PHYTOTHERAPY RESEARCH Phytother. Res. (2007) Published online in Wiley InterScience ANTAGONISTIC ACTIVITY OF PHYTOCHEMICALS (www.interscience.wiley.com) DOI: 10.1002/ptr.2327 1 Estradiol-antagonistic Activity of Phenolic Compounds from Leguminous Plants† B. Pinto1*, A. Bertoli2, C. Noccioli2, S. Garritano3, D. Reali1 and L. Pistelli2 1 Dipartimento di Patologia Sperimentale, Biotecnologie Mediche, Infettivologia ed Epidemiologia, University of Pisa, Pisa, Italy Dipartimento di Chimica Biorganica e Biofarmacia, University of Pisa, Pisa, Italy IARC, 150 Cours Albert-Thomas, 69372 Lyon Cedex 08, France 2 3 Natural flavonoids are currently receiving much attention because of their estrogenic and antiestrogenic properties. Six isoflavones (isoprunetin, isoprunetin 7-O-β-D-glucopyranoside, isoprunetin 4′′,7-di-O-β -D-glucopyranoside, genistein, genistein 7-O-β-D-glucopyranoside, daidzein), four flavones (luteolin, luteolin 7-O-β -D-glucopyranoside, luteolin 4′′-O-β-D-glucopyranoside, licoflavone C), isolated from Genista morisii and G. ephedroides (two Leguminosae plants of the Mediterranean area) together with two structurally related pterocarpans, bitucarpin A and erybraedyn C, isolated from Bituminaria bituminosa (Leguminosae), were tested for the antagonist activity by a yeast based estrogen receptor assay (Saccharomyces cerevisiae RMY326 ER-ERE). Most compounds inhibited the estradiol-induced transcriptional activity in a concentration dependent manner. In particular, for the flavone luteolin 77% inhibition of the induced β-galactosidase activity was observed. Interestingly, licoflavone C exhibited a dose-dependent antagonistic activity at concentrations up to 10−4 M, but stimulated β-galactosidase expression at higher concentrations resulting in a U-shaped-like dose-response curve. Copyright © 2007 John Wiley & Sons, Ltd. Keywords: phytoestrogens; antagonistic activity; yeast assay; Leguminosae. INTRODUCTION Flavonoids are widespread natural compounds found in different plant tissues such as flowers, roots, leaves and stems. They constitute the largest group of secondary metabolites involved in several functions in plants. Additionally, they are important in the human and animal diet and have a biomedical interest since they show numerous beneficial health effects. They inhibit cell proliferation, act as antioxidant agents and display antiallergic, antiinflammatory, apoptotic, antitumor, antithrombic and antihypertensive activities (Havsteen, 2002; Osoki and Kennelly, 2003). Pterocarpans are employed as antioxidants, but also display antifungal, antiviral and antibacterial properties as well. Recently, bitucarpin A and erybraedyn C were demonstrated to exhibit anticlastogenic activity against mytomicin C and bleomycin C (Pistelli et al., 2003; Maurich et al., 2004). Several flavonoid derivatives, including isoflavonoids and flavones, are being studied for their estrogenic and antiestrogenic properties. As estrogen agonists, phytoestrogens mimic endogenous estrogens and cause estrogenic effects. As estrogen antagonists, they may block or alter estrogen receptors (ERs) and modify the estrogenic response (Brzezinski and Debi, 1999). As a part of a project addressed to characterize selected Leguminosae plants from the Mediterranean area for their phytochemicals and to evaluate the biological * Correspondence to: Dr Barbara Pinto, Dipartimento di Patologia Sperimentale, Biotecnologie Mediche, Infettivologia ed Epidemiologia, University of Pisa, via San Zeno, 37 56127 Pisa, Italy. E-mail: b.pinto@med.unipi.it † Dedicated to the memory of Professor Ivano Morelli (1940 –2005). Copyright © 2007 John Wiley & Sons, Ltd. Copyright © 2007 John Wiley & Sons, Ltd. properties, the antiestrogenic activities of six isoflavones (isoprunetin, isoprunetin 7-O-β-D-glucopyranoside, isoprunetin 4′,7-di-O-β-D-glucopyranoside, genistein, genistein 7-O-β-D-glucopyranoside, and daidzein), four flavones (luteolin, luteolin 7-O-β-D-glucopyranoside, luteolin 4′-O-β-D-glucopyranoside, licoflavone C) in an estrogen-inducible yeast assay were tested. The estrogenic/antiestrogenic activities of two structurally related pterocarpans, bitucarpin A and erybraedyn C, were also analysed. MATERIALS AND METHODS Phytochemicals. The pure compounds, isoprunetin (4′7-dihydroxy, 5-methoxyisoflavone), isoprunetin 7-Oβ-D-glucopyranoside, isoprunetin 4′,7-di-O-β-D-glucopyranoside, luteolin (3′,4′,5′,7′-tetrahydroxyflavone), luteolin 7-O-β-D-glucopyranoside, luteolin 4′-O-β-Dglucopyranoside, genistein, genistein 7-O-β-D-glucopyranoside, daidzein, were obtained from Genista morisii Colla aerial parts, a leguminous shrub, endemic to southwest Sardinia (Italy), according to the separation procedure previously described by Giachi et al. (2002). Licoflavone C was isolated from Genista ephedroides aerial parts, as reported in Pistelli et al. (1998). The pterocarpans, erybraedin C and bitucarpin A, were purified from Bituminaria bituminuosa, aerial parts, growing in the Elba Isle (Italy), according to data published elsewhere (Pistelli et al., 2003). The structural elucidation of the pure isolated compounds was performed by comparison of 1H and 13C NMR data with those reported in the literature or by direct comparison with authentic samples available in our laboratories. Received 22 December 2006 Phytother. Res. (2007) Revised July 2007 DOI: 310.1002/ptr Accepted 20 July 2007 2 B. PINTO ET AL. Yeast assay. The estrogenic/antiestrogenic activities of the compounds were tested on a recombinant S. cerevisiae yeast strain (RMY326 ER-ERE) lacking in uracil and tryptophan containing the human estrogen receptor α (hERα) and a Xenopus laevis vitellogenin estrogenresponsive element (ERE) linked to a reporter gene lacZ encoding the enzyme β-galactosidase. Plasmid [pG/ ER(G)] was used as the yeast expression vector for ERα and [pUCΔSS-ERE] as its β-galactosidase reporter plasmid (Liu and Picard, 1998). The induction of transcription of the reporter gene by the complex receptorligand is detected by spectophotometry. 17β-Estradiol (E2), 4-hydroxytamoxifen (OHT) and chromogenic substrate (O-nitrophenyl β-D-galactopyranoside, ONPG) were purchased from Sigma. Chlorophenol red-β-D-galactopyranoside (CPRG) was purchased from Roche Diagnostics. Test compounds, E2 and OHT, were dissolved in dimethyl sulphate (DMSO). Genistein was dissolved in methanol. Serial dilutions of the compounds were added to the yeast culture so that the concentration of solvent did not exceed 1.1% (v/v). Compounds were tested at concentrations at which they showed estrogenic activity (Garritano et al., 2005) namely, 10−8–10−3 M final concentration for licoflavone C and genistein, 10−6–10−3 M for all others. Licoflavone C and genistein were tested at lower concentrations because in a previous study they were estrogenic at concentrations lower than the other compounds. OHT was tested at 10−8–10−5 M final concentration. At higher concentrations it strongly inhibited the yeast cell growth. 17β-Estradiol was used as a positive control, vehicle as the negative control. All experiments were performed in triplicate. Assay for agonistic activity. The method utilized has previously been described (Pinto et al., 2004, 2005). The β-galactosidase activity was normalized to the number of cells assayed and expressed as Miller units using the following formula (Miller, 1972): β-gal units = (1000 × OD420/578)/(t × V × OD600) where t is the length of incubation (min) and V is the volume of culture used in the assay (mL). For each experiment, β-gal activity induction of compounds was presented as the mean ± SD of three replicates for each concentration and expressed as a percentage of the activity obtained with 10−8 M E2 (positive control) (Gong et al., 2003). Assay for antagonistic activity. In order to measure the antagonistic activity of the compounds, yeast cultures were grown overnight with 10−9 M of estradiol in the presence of increasing concentrations of the single plant phytochemical (concentration range 10−6–10−3 M; 10−8– 10−3 M for licoflavone C and genistein) and the β-gal activity was determined. For reference 10−9 M 17βestradiol (E2) and vehicle were used as positive and negative controls, respectively. The ability of the test compounds to inhibit the estradiol-dependent transcriptional activity was expressed as inhibition (%) of the enzymatic activity elicited by E2 (Ahn et al., 2004). Since luteolin and luteolin 7-glucoside confer on the yeast culture a yellow colour which persists during the assay, the experimental protocol was modified to test antagonistic activity and the chromogenic substrate o-nitrophenyl β-D-galactopyranoside (ONPG) was subCopyright © 2007 John Wiley & Sons, Ltd. stituted with chlorophenol red-β-D-galactopyranoside (CPRG) (Clontech, 1999; Garritano et al., 2005). RESULTS AND DISCUSSION The chemical structures of phytochemicals used in this study are shown in Fig. 1. The standard OHT, a typical ER antagonist in yeast, exhibited strong inhibitory effects on β-gal expression induced by 10−9 M E2. At a concentration of 10−5 M (3.87 µg/mL), OHT showed 60% inhibition. The inhibitory effects of OHT could not be determined at concentrations higher than 10−5 M because of its strong cytotoxic effect on yeast cells. The flavonoids tested showed different degrees of antagonistic activity (Figs 2 and 3). Luteolin and isoprunetin were the most effective inhibitors tested, but the inhibitory effects were lower than that of OHT (Fig. 2A and 2B). In particular, luteolin which did not display any significant estrogenic activity in a previous work (β-gal activity = 4.20% ± 0.75, Garritano et al., 2005), inhibited (77%) estradiol-induced enzymatic activity at the higher concentration tested, and this result is in agreement both with the literature data reporting an inhibitory action of this compound on in vitro proliferation of MCF-7 cells and in vivo uterine hypertrophy in rats (Markaverich et al., 1988) and with the observation that compounds that possess weaker estrogenic activities may exert more appreciable inhibitory action especially at high concentrations (Hwang et al., 2006). In general, the flavones (luteolin, luteolin 4′-glc and luteolin 7-glc) inhibited the estradiol-induced reporter gene activity in a concentration dependent manner (Fig. 2A), with the exception of licoflavone C. Since prenylated isoflavones are reported to be highly antiestrogenic in yeast systems (Ahn et al., 2004; Ito et al., 2006; Okamoto et al., 2006), licoflavone C, which contains a prenyl group in the 8-position on the A ring, was hypothesized to exhibit high antagonistic activity. This compound showed appreciable dose-dependent antagonistic activity at concentrations up to 10−4 M (37.7% inhibition), while at concentrations of 10−3 M no inhibitory activity was observed (8.0% inhibition of β-gal activity of E2), resulting in U-shaped dose-response curves (Fig. 3). U-shaped dose-response curves have been observed only for phytoestrogens (Collins et al., 1997; Almstrup et al., 2002). This behaviour could be explained by the high estrogenic activity of this flavone (Table 1) (Garritano et al., 2005) and seems to be in good agreement with the observation that none of the prenylated isoflavones with strong inhibitory effects showed induction of β-galactosidase activity (Ahn et al., 2004; Okamoto et al., 2006). As shown in Table 1, compounds that elicited high estrogenic activity in our previous work, exhibited low or no antagonistic activity in this study. The isoflavones isoprunetin and isoprunetin 7-glc inhibited the induction of β-galactosidase by 52.6% and 38.5%, respectively (Fig. 2B). In previous work, these compounds showed a relatively weak estrogenic action and β-gal activities were 38.09% ± 3.51and 34.79% ± 4.39, respectively (Table 1) (Garritano et al., 2005). This study demonstrated that they possess mixed agonistic/antagonist activity in an in vitro yeast assay. Phytother. Res. (2007) DOI: 10.1002/ptr ANTAGONISTIC ACTIVITY OF PHYTOCHEMICALS 3 Figure 1. Chemical structures of the tested compounds. Figure 2. Inhibitory effects of flavonoids and 4-hydroxytamoxifen (OHT) on β-gal activity induced by E2 in yeast. The yeast strain was incubated with 1 nM 17β-estradiol in the presence or absence of isolated compounds. The activity of compounds is expressed as a percentage of the β-galactosidase activity induced by 1 nM E2 alone (100%). Values are the mean ± SD. Flavones and pterocarpans (A), isoflavones (B). Copyright © 2007 John Wiley & Sons, Ltd. Phytother. Res. (2007) DOI: 10.1002/ptr 4 B. PINTO ET AL. Table 1. Comparison of estradiol agonistic and antagonistic activity of flavonoids Concentration (10−3 M) Compound Genistein Genistein 7-glc Daidzein Licoflavone C Luteolin 4′-glc Luteolin 7-glc Isoprunetin Isoprunetin 7-glc Luteolin Bitucarpin A Isoprunetin 4′,7-di glc a b Agonistic activity (β-gal %)a 82.88 75.55 63.42 82.38 65.99 46.55 38.09 34.79 4.20 2.33 1.44 ± ± ± ± ± ± ± ± ± ± ± Antagonistic activity (% inhibition of E2) 8.88 4.75 11.4 10.45 5.3 6.44 3.51 4.39 0.75 0.21 0.7 (*)b 14.7% 16.4% 8.0% 20.0% 29.1% 52.6% 38.7% 77.0% 37.6% 23.2% Values are expressed as mean ± SD. (*) Genistein was cytotoxic at 10−3 M; at 10−4 M it showed 27.4% inhibition. Figure 3. Inhibitory effects of licoflavone C on β-gal activity induced by E2 in yeast. The activity of compound is expressed as a percentage of the β-galactosidase activity induced by 1 nM E2 alone (100%). Values are the mean ± SD. Figure 4. Effects of addition of 1 nM E2 and increasing concentration of compounds on yeast cell growth. Yeast growth was measured as optical density at 600 nm (OD600). The reduction of the growth is expressed as a percentage of the OD600 measured for cells treated with 1 nM E2 alone. Values are the mean ± SD. The inhibitory effect of isoprunetin 4′,7-diglc was low at a concentration of 10−3 M, as it slightly decreased the induction of β-galactosidase (23.2% inhibition), while at the lower concentration tested it showed 33.0% inhibition. This compound also showed an anomalous behaviour for agonistic activity, as the induction of βgal activity increased to the lower doses progressively (Garritano et al., 2005). Genistein 7-glc and daidzein, poorly inhibited the β-gal expression. Genistein was cytotoxic at a concentration of 10−3 M and at 10−4 M showed 27.4% inhibition. The estrogenic/antiestrogenic activities of the two pterocarpans were also evaluated. Bitucarpin A did not show estrogenicity (β-gal activity, 2.33% ± 0.21) and it weakly inhibited the β-galactosidase activity of estradiol (Fig. 2A). Erybraedin C was tested at a final concentration 10−6 M only, because it strongly inhibited the yeast cell growth at higher concentrations. At this concentration, neither estrogenic activity nor inhibitory effect on estradiol activity was observed for this compound. The observed reduction of β-gal activity induced by E2 of luteolin, isoprunetin, luteolin 7-glc and bitucarpin A was not due to the inhibition of yeast cells proliferation since in co-treated cells, these flavonoids did not affect yeast cell growth at the higher concentration tested in the assay but it rather stimulated the yeast cell growth (Fig. 4). Copyright © 2007 John Wiley & Sons, Ltd. CONCLUSION The flavonoids tested in this study showed different degrees of antagonist activity. Luteolin and isoprunetin showed higher inhibitory effects by inhibiting the βgalactosidase activity mediated by 17 β-estradiol by more than 50%. Compounds with no estrogenic activity (such as luteolin and bitucarpin A) exhibited high to weak inhibitory activity. Relatively weak estrogenic compounds (such as isoprunetin or isoprunetin 7 glc) had partial antagonistic activity. As the observed reduction of β-gal activity induced by these phytochemicals would not be due to the inhibition of yeast cell proliferation, other mechanisms for the decrease in estrogen-induced β-gal expression by E2 should be examined. Most of the phytoestrogens exhibited antiestrogenic activity in a dose-dependent fashion. In contrast, licoflavone C acted as an estradiol inhibitor within a concentration range and was estrogenic Phytother. Res. (2007) DOI: 10.1002/ptr ANTAGONISTIC ACTIVITY OF PHYTOCHEMICALS at higher concentrations. These data suggest that the presence of a prenyl group at the C8-position of the skeleton is not predictive of the antagonistic activity of this flavone, as observed for some isoflavones (Okamoto et al., 2006). In these experiments, compounds with strong estrogenic activity generally showed a low inhibitory action, and this is true both for flavones and isoflavones and independently of the presence of the prenyl group at the 8-position on the A-ring. These results indicate that it is possible to predict the degree of antagonistic activity only for compounds that display strong estrogenic activity. These compounds are supposed not to function as antiestrogens. Isoprunetin 4′,7 diglc had an anomalous behaviour in this assay, since it showed a progressive enhancement of inhibitory activity at a lower concentration. Literature data report that phytoestrogens have strong agonistic and antagonistic effects both on estrogen receptors α and β. Some isoflavones such as genistein and daidzein induce receptor-dependent transcription relatively better with the ERβ than with ERα, while 5 equol, a daidzein metabolite, and 8-prenylnaringenin are more active on the ERα (Kuiper et al., 1997; Morito et al., 2001; Bovee et al., 2004). However, studies of the tissue distributions and expression patterns of these receptors indicate that the ERα isoform has a broad expression pattern and it seems the more important receptor type in the mammary gland, uterus, testis, adrenal and the pituitary, the lung and hypothalamus are positive for both ERα and ERβ, whereas ERβ has a more focused pattern, with high levels in the ovary, prostate and epididymis and the developing brain, with differences in humans compared with other species (Enmark and Gustafsson, 1999; Carpino et al., 2004; Fried et al., 2004). Moreover, some studies suggest that the biological functions of the ERβ protein may be dependent on the presence of ERα in certain cell types and tissues (Couse et al., 1997). The use of an in vitro test on a S. cerevisiae yeast strain modified with the estrogen receptor alpha (ERα) is then reliable for measuring the biological activity of natural compounds from plants. REFERENCES Ahn EM, Nakamura N, Akao T, Nishihara T, Hattori M. 2004. Estrogenic and antiestrogenic activities of the roots of Moghania philippinensis and their constituents. Biol Pharm Bull 27: 548–553. Almstrup K, Fernandez MF, Petersen JH, Olea N, Skakkebaek NE, Leffers H. 2002. Dual effects of phytoestrogens result in U-shaped dose-response curves. Environ Health Perspect 110: 743–748. Bovee TF, Helsdingen RJ, Rietjens IM, Keijer J, Hoogenboom RL. 2004. Rapid yeast estrogen bioassays stably expressing human estrogen receptors alpha and beta, and green fluorescent protein: a comparison of different compounds with both receptor types. J Steroid Biochem Mol Biol 91: 99–109. Brzezinski A, Debi A. 1999. Phytoestrogens: the ‘natural’ selective estrogen receptor modulators? Eur J Obstet Gynecol Reprod Biol 85: 47–51. Carpino A, Bilinska B, Siciliano L, Maggiolini M, Rago V. 2004. Immunolocalization of estrogen receptor beta in the epididymis of mature and immature pigs. Folia Histochem Cytobiol 42: 13–17. Clontech Yeast Protocol Handbook. 1999. CLONTECH Laboratories, Inc. Collins BM, McLachlan JA, Arnold SF. 1997. The estrogenic and antiestrogenic activities of phytochemicals with the human estrogen receptor expressed in yeast. Steroids 62: 365– 372. Couse JF, Lindzey J, Grandien K, Gustafsson JÅ, Korach KS. 1997. Tissue distribution and quantitative analysis of estrogen receptor-α (ERα) and estrogen receptor–β (ERβ) messenger ribonucleic acid in the wild-type and ERα-knockout mouse. Endocrinology 138: 4613–4621. Enmark E, Gustafsson JÅ. 1999. Oestrogen receptors – an overview. J Intern Med 246: 133–138. Fried G, Andersson E, Csoregh L et al. 2004. Estrogen receptor beta is expressed in human embryonic brain cells and is regulated by 17beta-estradiol. Eur J Neurosci 20: 2345–2354. Garritano S, Pinto B, Giachi I, Pistelli L, Reali D. 2005. Assessment of estrogenic activity of flavonoids from Mediterranean plants using an in vitro short-term test. Phytomedicine 12: 143–147. Giachi I, Manunta A, Morelli I, Pistelli L. 2002. Flavonoids and isoflavonoids from Genista morisii. Biochem System Ecol 30: 801–803. Gong Y, Chin HS, Lim LS, Loy CJ, Obbard JP, Yong EL. 2003. Clustering of sex hormone disruption in Singapore’s marine environment. Environ Health Perspect 111: 1448 – 1453. Copyright © 2007 John Wiley & Sons, Ltd. Havsteen BT. 2002. The biochemistry and medical significance of the flavonoids. Pharmacol Ther 96: 67–202. Hwang CS, Kwak HS, Lim HJ et al. 2006. Isoflavone metabolites and their in vitro dual functions: they can act as an estrogen agonist or antagonist depending on the estrogen concentration. J Steroid Biochem Mol Biol 101: 246 –253. Ito C, Itoigawa M, Kumagaya M et al. 2006. Isoflavonoids with antiestrogenic activity from Millettia pachycarpa. J Nat Prod 69: 138 –141. Kuiper GG, Carlsson B, Grandien K et al. 1997. Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors α and β. Endocrinology 138: 863–870. Liu JW, Picard D. 1998. Bioactive steroids as contaminants of the common carbon source galactose. FEMS Microbiol Lett 159: 167–171. Markaverich BM, Gregory RR, Alejandro MA, Clark JH, Johnson GA, Middleditch BS. 1988. Methyl p-hydroxyphenyllactate: an inhibitor of cell proliferation and an endogenous ligand for nuclear type II binding sites. J Biol Chem 263: 7203– 7210. Maurich T, Pistelli L, Turchi G. 2004. Anti-clastogenic activity of two structurally related pterocarpans purified from Bituminaria bituminosa in cultured human lymphocytes. Mutat Res 561: 75–81. Miller JH. 1972. Assay for beta-galactosidase. In Experiments in Molecular Genetics, Miller JH (ed.). Cold Spring Harbour Laboratory Press: New York. Morito K, Hirose T, Kinjo J et al. 2001. Interaction of phytoestrogens with estrogen receptors α and β. Biol Pharm Bull 24: 351–356. Okamoto Y, Suzuki A, Ueda K et al. 2006. Anti-estrogenic activity of prenylated isoflavones from Millettia pachycarpa: implications for pharmacophores and unique mechanisms. J Health Sci 52: 186–191. Osoki AL, Kennelly EJ. 2003. Phytoestrogens: a review of the present state of research. Phytother Res 17: 845–869. Pinto B, Garritano S, Reali D. 2005. Occurrence of estrogen-like substances in the marine environment of the Northern Mediterranean sea. Mar Poll Bull 50: 1681–1685. Pinto B, Picard D, Reali D. 2004. A recombinant yeast strain as a short term bioassay to assess estrogen-like activity of xenobiotics. Ann Ig 16: 579–585. Pistelli L, Bertoli A, Giachi I, Manunta A. 1998. Flavonoids from Genista ephedroides. J Nat Prod 61: 1404 –1406. Pistelli L, Noccioli C, Appendino G, Bianchi F, Sterner O, Ballero M. 2003. Pterocarpans from Bituminaria morisiana and Bituminaria bituminosa. Phytochemistry 64: 595–598. Phytother. Res. (2007) DOI: 10.1002/ptr