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Estrogenic properties of isoflavones derived from Millettia griffoniana.


In most developing countries, 70-80% of the population still resort to traditional medicine for their primary health care. This medicine utilises medicinal plants which are traditionally taken as concoction and infusion. The root and stem bark of Millettia griffoniana (Leguminosae), has been reported to contain isoflavonoids, alkaloids, and diterpenoids. The possible benefit of some bioactive isoflavones derived from M. griffoniana prompted us to screen them for estrogenic activity. Six isoflavones and coumarin derived from M. griffoniana (bail) namely, compound nos. 1-6 (Fig. 1) were tested for their potential estrogenic activities in three different estrogen receptor alpha (ER[alpha])-dependent assays. In a yeast-based ER[alpha] assay, all test substances and 17[beta]-estradiol as endogenous agonist, showed a significant induction of [beta]-galactosidase activity. The test compounds at the concentration of 5 X [10.sup.-6] M could achieve 59-121% of the [beta]-galactosidase induction obtained with [10.sup.-8] M 17[beta]-estradiol (100%). In the reporter gene assay based on stably transfected MCF-7 cells (MVLN cells), the estrogen responsive induction of luciferase was also stimulated by the M. griffoniana isoflavones. In Ishikawa cells, all substances exhibited estrogenic activity revealed by the induction of alkaline phosphatase (AlkP) activity. The estrogenic activities of isoflavones from M. griffoniana could be completely suppressed by the pure estrogen antagonist, ICI 182,780, suggesting that the compounds exert their activities through ER[alpha]. Although all substances showed estrogenic effects, 4'-methoxy-7-O-[(E)-3-methyl-7-hydroxymethyl-2,6-octadienyl]isoflavone (7-O-DHF), Griffonianone C (GRIF-C), and 3',4'-dihydroxy-7-O-[(E)-3,7-dimethyl-2,6-octadienyl]isoflavone (7-O-GISO) were found to be the most potent of tested substances. In summary, estrogenic activities of the isoflavones derived from M. griffoniana were described for the first time using reporter gene assays and the estrogen-inducible AlkP Ishikawa model.

[c] 2005 Elsevier GmbH. All rights reserved.

Keywords: Isoflavones; In vitro bioassays; [beta]-galactosidase; Luciferase; Alkaline phosphatase; Root bark; Leguminosae


Phytoestrogens are plant-derived compounds with estrogenic or antiestrogenic properties (Umland et al., 2000). The 2-phenylnaphthalene-type chemical structure of phytoestrogens is equivalent to that of steroid hormones and they have been found to directly interact with estrogen receptors (ERs), though their ability for binding ERs is only 1/500-1/1000 of that of estradiol (Tham et al., 1998; Umland et al., 2000). The scientific interest in phytoestrogens is enhanced by the hope for a potential medical use like in hormone replacement therapy (HRT). Phytoestrogens are then hoped to be beneficial to relieve a variety of symptoms of menopause and help prevent bone resorption. The aim of this study was to characterise compounds derived from Millettia griffoniana (Bail) (Leguminosae) (Yankep et al., 1998) towards potential estrogenic properties. Plants of the genus Millettia are reputed to yield isoflavonoids (Dewick, 1994; Yankep et al., 2001), and diterpenoids (Dagne et al., 1990) with insecticidal, piscicidal, and molluscicidal properties (Dewick, 1986). Crude extracts from root and stem bark of M. griffoniana are used in traditional medicine in some village communities of Cameroon to treat boils, insects bits, inflammatory affections like pneumonia and asthma, sterility, amenohrrea and menopausal disorders. Although some plants of the genus Millettia are commonly used for the treatment of various conditions, their estrogenic or antiestrogenic properties have not yet been studied in the literature.

Validated and reproducible in vitro bioassays are utilised to investigate and define the hormonal activity of chemicals (Sohoni and Sumpter, 1998). Many previous studies have shown that isoflavones exert estrogenic or antiestrogenic activities in several in vitro test systems (Shelby et al., 1996; Zava and Duwe, 1997; Breinholt and Larsen, 1998). In this study, one screening assay and two independent in vitro bioassays were used to assess and to validate estrogenic effects of some pure substances derived from M. griffoniana. The screening assay was based on the recombinant yeast strain, Saccharomyces cerevisiae expressing human estrogen receptor [alpha] (hER[alpha]) together with a reporter Lac-Z gene, coding for the [beta]-galactosidase (Sohoni and Sumpter, 1998). This assay is capable of detecting low estrogenic activities, e.g. levels of estradiol ranging between 0.1 and 1 nmol/l (Petit et al., 1995).


The first bioassay used was based on the estrogen induction of luciferase in MVLN cells, which are MCF-7 cell derivatives stably transfected with a vitellogenin-2 promotor/luciferase reporter gene (Pons et al., 1990). These cells express endogenous ER[alpha]. The second bioassay used is the endometrial Ishikawa model, which has been reported to produce a specific, natural and dose-dependent increase in alkaline phosphatase (AlkP) in response to estrogens (Holinka et al., 1986). Estradiol is shown to be capable of inducing AlkP at very low concentrations, for example, 1 pmol/l (Littlefield et al., 1990) (Fig. 1).

Materials and methods


17[beta]-estradiol was obtained from Sigma (Taufkirchen, Germany). Chlorophenol red [beta]-D-galactopyranoside (CPRG) and p-nitrophenylphosphate (PNPP) were purchased from Roche (Mannheim, Germany). Dulbecco's Modified Eagle Medium/Ham's F-12 (DMEM/F-12) without phenol red, foetal calf serum (FCS), and trypsin-EDTA were purchased from Biochrom (Berlin, Germany). ICI 182,780 was purchased from Tocris (Bristol, UK) or was supplied from Schering AG (Berlin, Germany), while Insulin was obtained from Invitrogen (Karlsruhe, Germany). The collection of the plant materials, the extraction, the isolation, and the purification of test substances (Fig. 1) 4'-O-geranylisoquiritigenin (4-O-GIQ), 7-O-geranylformononetin (7-O-GF), Griffonianone E (GRIF-E), Griffonianone C, 3',4'-dihydroxy-7-O-[(E)-3,7-dimethyl-2,6-octadienyl] isoflavone and 4'-methoxy-7-O-[(E)-3-methyl-7-hydroxymethyl-2,6-octadienyl]isoflavone were done as previously described by Yankep et al. (1998, 2001). The analysis of the NMR-proton spectrum and the mass spectrum of the six compounds studied in this work clearly show that all them have a purity of over 90%.

Yeast recombinant screen

The estrogen-inducible screening assay in the yeast strain S. cerevisiae was used as previously described (Routledge and Sumpter, 1997). Cells are stably transfected with the DNA sequence of the hER[alpha]. The system also contains expression plasmids composed of two estrogen-responsive elements (ERE) regulating the expression of the reporter Lac-Z gene that encodes the enzyme [beta]-galactosidase (Sohoni and Sumpter, 1998; Routledge and Sumpter, 1997).

Yeasts were handled as previously described (Tham et al., 1998). Stock solutions and the test compounds were prepared in DMSO and added to clear 96-well polystyrene plates (TPP, Switzerland), to a maximum concentration of 1% DMSO. Plates were seeded with 200 [micro]l per well of the assay medium; plates were then sealed and incubated at 32[degrees]C for 2-3 days.

Substrate conversion (colour development) was measured at 565 and 690 nm using a plate reader. The readings at 690 nm were used to correct for the increase in turbidity due to growth of the yeast. Samples were tested in quadruplicates, and a standard curve for E2 ([10.sup.-12]-[10.sup.-8]M) was included in each assay. The concentration of the test substances generally ranged from [10.sup.-8] to 5 x [10.sup.-6]M.

Luciferase-reporter-gene assay

MVLN cells were obtained from M. Pons, Institut National de la Sante et de la Recherche (INSERM, Montpellier, France). This cell line is based on human MCF-7 cells containing an estrogen regulated luciferase reporter gene driven by an ERE of the vitellogenin A2 gene fused to the thymidine-kinase-promoter (Gagne et al., 1992; Pons et al., 1992). Therefore, the specific transcription activity of a test chemical is directly related to the activity of luciferase measured. MVLN cells were maintained as previously described (Pons et al., 1992). On the day of induction, the medium was changed against fresh 1% DCC-FCS medium and cells were treated with the test compounds ([10.sup.-10]-[10.sup.-5]M), or 17[beta]-estradiol (positive control), and vehicle (DMSO). After 24 h, cells were harvested and treated for luciferase assay. The luciferase was extracted as described (Pons et al., 1992). To compare data, the protein content of each extract was measured using the bicinchoninic acid (BCA) protein assay, with bovine serum albumin (BSA) as standard protein. Luciferase activity was calculated in relative light units (RLU) per mg of protein.

This experiment was performed at least three times, with duplicates for each treatment.

Alkaline phosphatase assay with Ishikawa cells

The AlkP activity was measured according to the method of Wober et al. (2002). Briefly, Ishikawa cells (human endometrial adenocarcinoma cells) were maintained in DMEM/F12 medium containing 5% DCC-FCS and additional insulin-transferrin-selenium (Gibco/BRL). Cells were harvested, resuspended in culture medium and plated in 12-well plates (125 000 cells/well). Test compounds (dissolved in DMSO) were then added. A dose-dependent analysis was performed with concentrations ranging from [10.sup.-10] to [10.sup.-5] M. After 72h treatment, cells were harvested and resuspended in reaction buffer (274 mM mannitol, 100 mM CAPS, 4 mM MgCl[.sub.2]. pH 10.4). Cell lysates were prepared by ultrasonic disintegration on ice. one hundred microlitres of cell lysate was added to 325 [micro]l of PNPP substrate. The substrate (PNNP) is hydrolysed to p-nitrophenol and the kinetics of the product formation were read at 405 nm. The data are shown as mean [+ or -] standard error of mean (SEM) of three independent experiments performed in duplicate for each dose.

Statistical analysis

The results are expressed as mean [+ or -] SEM. ANOVA followed by a post hoc multiple comparison was performed. Dunnett's test (XLSTAT-Pro version 7.1, Addinsoft,) was used to compare increasing doses of the test compounds with the respective control. P-values <0.05, <0.005, and <0.001 were considered significant. In Fig. 5 data were compared using t-test. A P-value less than 0.005 was considered significant (Figs. 2-5).




We have screened for the potential estrogenic activity of six compounds extracted from M. griffoniana and validated results in two different bioassays. At the concentration of 5 x [10.sup.-6]M 4'-O-geranylisoquiritigenin, 7-O-geranylformononetin, Griffonianone E, Griffonianone C, 3',4'-dihydroxy-7-O-[(E)-3,7-dimethyl-2, 6-octadienyl]isoflavone, and 4'-methoxy-7-O-[(E)-3-methyl-7-hydroxymethyl-2,6-octadienyl]isoflavone could achieve 59-121% of the gene induction obtained with [10.sup.-8] M 17[beta]-estradiol (100%) in the yeast system (Figs. 2a and b). Once the compounds from M. griffoniana have been shown to possess some estrogenic activities in estrogen-inducible yeast receptor assay, we tested them in a luciferase-reporter-gene assay; where the isoflavones induced the luciferase activity in a dose-dependent manner (Figs. 3a and b). However, this induction was still moderate in comparison to estradiol (E2, [10.sup.-8] M). At the concentration [10.sup.-6] M, all six isoflavones displayed 37-122% of estrogenic activity. Only griffonianone-E and 3',4'-dihydroxy-7-O-[(E)-3,7-dimethyl-2,6-octadienyl]isoflavone M exhibited estrogenic activity at a concentration of [10.sup.-5] M. As observed in the recombinant yeast screen, different potentials of phytoestrogens to stimulate the luciferase induction were measured in MVLN cells. The ranking was as follows: 7-O-DHF>7-O-GF>GRIF-C>GRIF-E>7-O-GISO>4-O-GIQ, as delineated from their activities at a concentration of [10.sup.-6] M.



When MVLN cells were simultaneously treated with estradiol or test substances and with the pure ER antagonist, ICI 182,780 at a concentration of 5 x [10.sup.-7] M (Fig. 5a) expression of luciferase activity was suppressed. For all test compounds, the ICI 182,780 treatment resulted in expression levels which were under the level of the negative control (DMSO), whereas this concentration of ICI 182,780 did not completely reduce the stimulation due to E2 ([10.sup.-8]M) to basal level.

In the endometrial Ishikawa system, all test compounds (Figs. 4a and b) displayed a lower estrogenic activity than estradiol ([10.sup.-7]M). For griffonianone E, and 3',4'-dihydroxy-7-O-[(E)-3,7-dimethyl-2,6-octadienyl]isoflavone, the maximum AlkP activity was found at the concentration of [10.sup.-5]M. The most effective concentration for 4'-O-geranylisoquiritigenin, 7-O-geranylformononetin, griffonianone C, and 4'-methoxy-7-O-[(E)-3-methyl-7-hydroxymethyl-2,6-octadienyl] isoflavone was [10.sup.-6]M. Compared to the negative control (cells treated with vehicle only), the isoflavones induced 2-4.5-fold increases in AlkP activity. As for estradiol [10.sup.-7]M, it exhibited a 5.9-fold increase.

Simultaneous treatment of Ishikawa cells with ICI 182,780 (5 x [10.sup.-7]M), and estradiol ([10.sup.-7]M) or test substances showed (Fig. 5b) a significant suppression of the AlkP induction.


For all six isoflavones tested, a positive screening result could be verified by an estrogenic response induced in the two bioassay systems. The use of the recombinant yeast system for screening purposes presents several advantages including the lack of known endogenous receptors, use of media that is devoid of steroids (Zacharewski, 1998), and its weak biotransformation activity (Holinka et al., 1986; Le Guevel and Pakdel, 2001). In this system, the estrogenic activity of a substance results from its direct interaction with the ER (Zacharewski, 1998). The ER previously has shown an ability to bind to an array of compounds with a degree of structural diversity (Kuiper et al., 1998). However, the potency of each substance may be due to its affinity to the ERs. Although the yeasts present several advantages, many factors can affect the estrogenic activity of a substance when tested in this yeast-based system (Zacharewski, 1998). Disadvantages of the system may arise by the inability to distinguish antiestrogens such as ICI 182,780 from (4-hydroxy)tamoxifen, which exert partial agonist activity in yeast (Legler et al., 2002), as well as differences in permeability for compounds through the yeast cell wall (Lyttle et al., 1992). For these reasons and for verification of the effects of isoflavones from M. griffoniana, it was necessary to confirm the primary results of the yeast system in additional bioassays. We tested the substances in two other systems based on human cells, namely the MVLN cells and Ishikawa cells. The induction of luciferase in MVLN cells in response to the treatment with test compounds confirmed their estrogenic potency. This corroborates the results of Demirpence et al. (1993) who found this cell line a very effective tool to screen natural or synthetic molecules classified as full or partial (anti)estrogens.

E2 as well as the isoflavones showed moderate estrogenic potency in Ishikawa cells, this could be explained by the fact that Ishikawa cells, like normal uterine cells, possess specific metabolism pathways for steroid conversion that modulate the sensitivity of the uterus to estrogens (Falany and Falany, 1996).

The simultaneous treatment of both cell lines each with isoflavones and the pure estrogen antagonist ICI 182,780 suppressed estrogenic effects induced by the test substances. This result supports our hypothesis that transactivation activity of 4'-O-geranylisoquiritigenin, 7-O-geranylformononetin, Griffonianone E, Griffonianone C, 3',4'-dihydroxy-7-O-[(E)-3,7-dimethyl-2,6-octadienyl]isoflavone, and 4'-methoxy-7-O-[(E)-3-methyl-7-hydroxymethyl-2,6-octadienyl]isoflavone can be mainly attributed to ER binding thereby mediating a transcriptional response. Howell et al. (2000) found that the aptitude of estrogenic agonists to activate or inhibit the transcription in a ligand-dependent or -independent manner is completely attenuated by ICI 182,780. This antagonist blocks not only the functional activity of the ERs, but it considerably reduces cellular levels of ERs (Howell et al., 2000).

The estrogenic effects of isoflavones derived from M. griffoniana observed in our study could reinforce the use of this plant in the traditional medicine to treat menopause-related symptoms and amenorrhoea. In fact, the rising level of oestrogen causes the endometrium to become thicker and more richly supplied with blood vessels and glands. Amenorrhoea is usually related to insufficient production of estradiol, so substances with estrogenic properties may be used to promote menstruation. In the case of menopausal women, such substances may also be beneficial for the relieve of some unpleasant menopausal symptoms, but still the risk of promotion of endometrial and breast cancer has to be included as most estrogenic compounds also induce cell proliferation, thereby increasing the risk of cancer. To judge the common use of M. griffoniana in the treatment of boils, insect bites and asthma will require establishment of additional test systems addressing additional endpoints of functions of substances derived from M. griffoniana.

Our study was focused on assays which address mainly the ER[alpha] because it appears that the binding affinities of compounds for ERs correlate well with phytoestrogen-induced increased in binding of ER[alpha] to ERE (Kostelac et al., 2003). However, it is essential to assess tested compounds in further assays which address estrogen receptor [beta] (ER[beta]) subtype only, since epidemiological studies suggest that phytoestrogens like genistein and daizein reduce the risk of breast and prostate cancers. The preferential binding of isoflavones to ER[beta] may explain why these compounds reduce the risk of cancers in these organs. The much higher concentrations required for stimulating cell growth than for binding may also explain the antiproliferative properties of some phytoestrogens (Morito et al., 2001).


We thank S. Kolba and A. Beyer for technical assistance. We are grateful to Babila J. Tachu, Institutfur Biologie, Humboldt Universitat zu Berlin, for critical reading and editing of the manuscript. The author of this paper is sponsored by the German Academic Exchange Service (DAAD).


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G.J.M. Ketcha Wanda (a), D. Njamen (b), E. Yankep (c), M. Tagatsing Fotsing (c), Z. Tanee Fomum (c), J. Wober (a), S. Starcke (a), O. Zierau (a), G. Vollmer (a,*)

(a) Molecular Cell Physiology und Endocrinology, Technical University Dresden, Mommsen Street 13, Dresden, Germany

(b) Laboratory of Animal Physiology, Department of Animal Biology and Physiology, Faculty of Science, University of Yaounde 1, P.O. Box 812, Yaounde, Cameroon

(c) Department of Organic Chemistry, Faculty of Science, University of Yaounde 1, P.O. Box 812, Yaounde, Cameroon

Received 26 July 2004; accepted 30 June 2005

*Corresponding author. Tel.: +351 463 31922; fax: + 351 463 31923.

E-mail address: (G. Vollmer).
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Author:Wanda, G.J.M. Ketcha; Njamen, D.; Yankep, E.; Fotsing, M. Tagatsing; Fomum, Z. Tanee; Wober, J.; Sta
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Geographic Code:4EUGE
Date:Feb 1, 2006
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