Printer Friendly

Subtype-specific activation of estrogen receptors by a special extract of Rheum rhaponticum (ERr 731[R]), its aglycones and structurally related compounds in U2OS human osteosarcoma cells.


The special extract ERr 731[R] from the roots of Rheum rhaponticum is the major constituent of Phytoestrol[R] N which is used for the alleviation of menopausal symptoms. Recently, we demonstrated that ERr 731[R] and its aglycones trans-rhapontigenin and desoxyrhapontigenin as single test substances do not activate the estrogen receptor-[alpha] (ER[alpha]) in human endometrial adenoarcinoma cells. However, these substances together with the structurally related hydroxystilbenes cis-rhapontigenin, resveratrol and piceatannol activated the ER[beta]-dependent reporter gene activity. To investigate if these substances are tissue selective ER activators, ERr 731[R] and the single test substances were examined in bone-derived U2OS cells stably expressing ER[alpha] or transiently expressing ER[beta]. In the ER[alpha] expressing U2OS cells, a weak, but statistically significant ER[alpha]-coupled luciferase activity was detected with ERr 731[R] and desoxyrhapontigenin which was 10-times lower than with [10.sup.-8]M 17[beta]-estradiol. In the ER[beta] test system, all test substances significantly induced the luciferase activity in a magnitude comparable to 17[beta]-estradiol. All effects were abolished with the pure ER antagonist ICI 182 780, indicating an ER-specific effect. Intracellular actions were also examined with the glycosylated ERr 731[R] constituents rhaponticin and desoxyrhaponticin. Treatment of U2OS cells with defined mixtures of both glycosides resulted in a reporter gene activity comparable to that of ERr 731[R], thereby providing evidence for the existence of cellular uptake mechanisms for glycosylated hydroxystilbenes. This report confirms the strong ER[beta]-dependent activity of ERr 731[R] and provides evidence for a tissue selective ER agonistic activity by ERr 731[R] and its aglycones, as demonstrated by the activation of ER[alpha] in bone cells but not in endometrial cells.

[c] 2007 Elsevier GmbH. All rights reserved.

Keywords: Estrogen receptor; Estrogen responsive element; Hydroxystilbenes; Phytoestrol N; Rhapontigenin; Desoxyrhapontigenin; Resveratrol; Piceatannol; Osteosarcoma cells; Menopause; Climacteric


Hormone therapy (HT) is very effective in relieving climacteric complaints during perimenopause and post-menopause as well as preventing the long-term consequences of estrogen deficiency such as osteoporosis (Lindsay and Tohme, 1990; Skafar et al., 1997). HT works by replacing one or both of the two female sex steroids estrogen and progestogen. Due to the association of HT with an increased risk of breast and endometrial cancer and venous thromboembolism (Rosendaal et al., 2002; Ross et al., 2000), many women refuse to take HT. These patients however still seek relief from their climateric symptoms and thus, the demand for alternative treatments, in particular herbal remedies, which are claimed to not display the potential risks associated with HT, are high.

Additionally, untreated postmenopausal cessation of ovarian estrogen production leads to the loss of bone mass and density, a condition commonly referred to as osteoporosis. During the reproductive period of females, the mass and density of the bone is maintained by a subtle balance of the activity of the bone forming osteoblastic and bone degrading osteoclastic cells, which are regulated by estradiol. Two basic mechanisms can explain the bone sparing activity of estradiol, (a) the stimulation of osteoblasts or (b) the inhibition of osteoclasts. The main bone protective activity of estradiol appears to result from the suppression of osteoclasts rather than from the stimulation of osteoblasts. However, there are also indications that estradiol physiologically stimulates osteoblasts, since it has been shown to induce gene expression in these cells e.g. osteocalcin or collagen type-1. These results make it desirable for any HT replacement therapy to have, in addition to the relief of menopausal symptoms, osteoporosis protection properties.

The special extract from the roots of Rheum rhaponticum (L.), referred to as ERr 731[R] (trade name Phytoestrol[R] N), has been regularly prescribed for climacteric complaints since 1993, without the occurrence of any safety related side effects such as endometrial hyperplasia, spotting or breakthrough bleeding. Recently, a 12 week double-blind, placebo-controlled clinical trial in 109 perimenopausal women has been completed demonstrating the clinical efficacy (proof of principle of the biological activity) of ERr 731[R] (Heger et al., 2006; Kaszkin-Bettag et al., 2007). However, in contrast to the clinical effectiveness of ERr 731 [R], little to date has been known about the molecular mechanism of either the extract or its potential metabolites, that can explain the clinical observations.

The plant Rheum rhaponticum, commonly known as Sibiric Rhubarb, originates from Central Asia. It was introduced into Europe in the 17th century, and has been cultivated since then in Western Europe, East Asia and the United States. The standardised extract ERr 731[R] (drug:extract ratio 16-26:1, extraction solvent calciumoxide:water 1:38 (mass/mass)), consists mainly of rhaponticin and desoxyrhaponticin and small amounts of the aglycones trans-rhapontigenin and desoxyrhapontigenin (both together about 5%). When ingested, however, it is proposed that large amounts of the aglycones may appear due to the deglycosylation of rhaponticin and desoxyrhaponticin by intestinal bacteria (Park et al., 2002; Kobashi and Akao, 1997). So far, it is not clear which molecule(s) are implicated as the primary mediators of the clinical effects of ERr 731[R] The structures of trans-rhapontigenin and desoxyrhapontigenin are shown in Fig. 1. Both molecules have a hydroxystilbene backbone and are structurally related to resveratrol (Aggarwal et al., 2004).

From the clinical observations with ERr 731[R], the question arises whether ERr 731[R] exerts its biological effects via binding to, and activation of, estrogen receptors. In a first in vitro study we investigated ERr 731[R], the aglycones of its constituents, trans-rhapontigenin and desoxyrhapontigenin, as well as the structurally related compounds cis-rhapontigenin, piceatannol and resveratrol for potential activity on the estrogen receptors-a (ER[alpha]) and -[beta] (ER[beta]) (Wober et al., 2007). ER[alpha]-mediated activities were investigated using two independent assays, (1) the yeast ER[alpha] reporter assay, and (2) stimulation of alkaline phosphatase in the human endometrial adenocarcinoma Ishikawa cell line expressing ER[alpha]. ER[alpha] and ER[beta] dependent activities were investigated in the human endometrial HEC-1B adenocarcinoma cells transiently transfected with the human ER[alpha] or ER[beta]. In this study we failed to detect any ER[alpha] dependent activity in either endometrial cells lines, or any antiestrogenic activity in the yeast system. In contrast, the total extract ERr 731[R], its aglycones trans-rhapontigenin and desoxyrhapontigenin as well as cis-rhapontigenin, resveratrol and piceatannol significantly stimulated ER[beta] dependent reporter gene activities similar to that seen with 10nM 17[beta]-estradiol (E2).

Many plant derived estrogenic compounds apparently exhibit selective estrogen receptor modulating- (SERM)-like properties (Cos et al., 2003) and have therefore been referred to phyto-SERMS (Vollmer and Zierau, 2004). SERM activity arises from a combination of the ER selectivity and an organ selectivity which may be mediated through other mechanisms. As mentioned above, bone is one of the major target organs for HT and therefore, it is important to know whether bone protection is mediated by ER-[alpha], ER-[beta] or via both receptors. The availability of selective agonists for the respective ER subtypes have demonstrated that bone protection is largely mediated through ER-[alpha] (Hillisch et al., 2004; Harris, 2007), balanced in a Ying-Yang-like way by ER-[beta] as shown from the studies with ER knockout animals (Lindberg et al., 2003).

U2OS human osteosarcoma cells (Ponten and Saksela, 1967) have therefore been used to elucidate the action of ERr 731[R] and the single test substances on the ER activation in bone-derived cells. ER[alpha] activity was investigated using U2OS cells stably transfected with the human ER[alpha] (U2OS-ER[alpha]). ER[beta] activity was investigated using U2OS cells transiently transfected with an expression plasmid of the human ER[beta] and a reporter construct consisting of a synthetic double estrogen response element (ERE) and a luciferase reporter ((ERE)2-tk-Luc; U2OS-ER[beta]).

These studies have shown that in U2OS-ER[alpha] cells a weak but significant activation of the ER[alpha] with ERr 731[R], desoxyrhapontigenin and resveratrol was detectable. These studies confirmed and extended our earlier work in human endometrial adenocarcinoma cells, and showed that the total extract ERr 731[R], its aglycones trans-rhapontigenin and desoxyrhapontigenin as well as cis-rhapontigenin, resveratrol and piceatannol markedly stimulated the ER[beta] dependent reporter gene activity in U2OS-ER[beta] cells. It is therefore concluded that the total extract ERr 731[R] and its aglycones are potential tissue selective ER agonists having beneficial effects on the bone when used clinically as HT replacement therapy.

Material and methods


17[beta]-estradiol (Estr-1,3,5(10)-trien-3,16[alpha], 17[beta]-triol) was obtained from Sigma-Aldrich (Deisenhofen, Germany), Diarylproprionitril (DPN), Propylpyrazoltriol (PPT) and ICI 182 780 (Faslodex[R]) were from Tocris Biosciences (Bristol, UK). The extract ERr 731[R], trans- and cis-rhapontigenin and desoxyrhapontigenin, as well as the 90:10 and 10:90 mixtures of rhaponticin and desoxyrhaponticin were provided by Chemisch-Pharmazeutische Fabrik, Carl Muller Apotheker GmbH & Co. KG (Goppingen, Germany). Resveratrol was from Sigma Aldrich (Deisenhofen, Germany) and piceatannol from Fisher Scientific (Schwerte, Germany). The chemical structures of the hydroxystilbenes are shown in Fig. 1.

Cell culture systems and plasmids

U2OS cells were from ATCC/Promochem (Wesel, Germany). U2OS cells stably transfected with the ER[alpha] (U2OS-ER[alpha] cells), the ER[beta] expression plasmid and the (ERE)[.sub.2]-tk-Luc reporter plasmid were kindly provided by Dr. Luisella Toschi (Schering AG, Berlin).

Reporter gene assays in U2OS cells stably transfected with ER[alpha] (U2OS-ER[alpha] cells)

U2OS-ER[alpha] cells were cultured routinely in DMEM-F12 medium containing 10% fetal calf serum (FCS) and 0.5mg/ml G418 (Calbiochem/VWR, Darmstadt, Germany). Experiments were performed in DMEM-F12 Medium containing 5% dextran coated charcoal (DCC) stripped FCS and 0.5mg/ml G418. For transfection, U2OS-ERa cells were transferred to a 24 well plate (30,000 cells/well) and transfected with l00ng of the (ERE)[.sub.2]-tk-Luc reporter plasmid using a liposome protocol (DOTAP; Roth, Karlsruhe, Germany) and a DOTAP:DNA ratio of 3:1. Luciferase activity was measured with a commercial kit (Promega, Mannheim, Germany) according to the manufacturer's instructions.

Reporter gene assays in U2OS cells transiently transfected with ER[alpha] (U2OS-ER[alpha] cells)

U2OS cells, which do not express any ERs, were cultured routinely in DMEM-F12 Medium containing 10% FCS. Experiments were performed in DMEM-F12 Medium containing 5% DCC. For transfection, U2OS cells were transferred to a 24 well plate (45,000 cells/well) and co-transfected with 100ng of the ER[beta] expression plasmid and 600 ng (ERE)[.sub.2]-tk-Luc reporter plasmid using a liposome protocol (DOTAP; Roth, Karlsruhe, Germany) and a DOTAP:DNA ratio of 3:1. Luciferase activity was measured with a commercial kit (Promega, Mannheim, Germany) according to the manufacturer's instructions.

Treatment of cell cultures with the test substances

All test substances were dissolved initially in DMSO and were used at a final DMSO concentration of 0.1%. All assays were performed with different concentrations of the test substances specifically adapted to the respective assay in order to obtain dose-response relationships (see legends to Figures). To investigate whether the effects are exclusively mediated by ER activation, cells were incubated with the effective doses of the test substances in the absence or presence of a 500 nM concentration of the pure antiestrogen ICI 182 780. This dose is 50 x the [IC.sub.50]. This dose represents a 50-fold molar excess to the effective dose of [10.sup.-8]M E2 which corresponds to a 10 x effective excess of antagonist, since the relative binding affinity of ICI 182 780 is approximately 20% of the binding affinity of E2 (Wakeling and Bowler, 1987, 1992). In order to test whether the glycosides of ERr 731[R] are biologically active in inducing ER reporter gene activity, 1:10 and 10:1 mixtures of rhaponticin and desoxyrhaponticin were tested at the effective dose of 10 ng/ml.


Data presentation and statistics

To compare the effects of the total extract ERr 731[R] with those obtained with the single test substances, the concentrations of all single substances in Figs. 5-7 are presented as molarity as well as ng/ml or [micro]g/ml. The concentration of [10.sup.-8] M for 17[beta]-estradiol (E2) corresponds to 0.0027 [micro]g/ml.

The data represent the means of at least 3 independent determinations[+ or -]standard deviation. Data shown in the figures are representative of at least 3 individual experiments with similar results. Statistical analysis was performed by the Student's t-test. A probability of <0.05 was defined as significant. *p<0.05, **p<0.01, ***p<0.001, compared to the solvent control.


Fig. 2 shows the results of the control experiments testing the specificity of the U2OS cells transfected with ER[alpha] or ER[beta]. In each case the cells were incubated with different concentrations of the pure ER[alpha] agonist PPT, or the pure ER[beta] agonist DPN. The ER[beta] cell lines responded to DPN but not to PPT, whilst the ER[alpha] cell lines responded to PPT and not DPN (Fig. 2).

Effect of ERr 731[R] on ER[alpha] activation

The effect on ER[alpha] dependent activity was tested in stably transfected U2OS-ER[alpha] cells. In contrast to our previous findings in human endometrial adenocarcinoma cells, a weak but statistically significant induction of luciferase reporter gene activity was detectable in U2OS-ER[alpha] cells even at he lowest ERr 731[R] concentrations (0.001 [micro]g/ml; Fig. 3a). The signal was not further enhanced by increasing the concentration, instead it decreased at highest concentrations used. Although statistically significant, this weak ERr 731[R] mediated ER[alpha] activation of the U2OS cells is less then 10% of the effect detectable in the same cells treated with [10.sup.-8] M E2 (equivalent to 0.0027 [micro]g/ml E2).


Effect of ERr 731[R] on ER[beta] activation

After stimulation of the U2OS-ER[beta] cells with ERr 731[R] a statistically significant ER[beta]-mediated induction of reporter gene activity was detected (Fig. 3b). The maximal activation was already present at ERr 731[R] concentrations of 0.001 [micro]g/ml and was not further enhanced by increasing the concentration, but instead it decreased towards higher concentrations. The activity level at the lowest dose was comparable to that obtained with [10.sup.-8] M E2 (Fig. 3b).

In order to investigate whether the induction of the reporter gene activation by ERr 731[R] is exclusively mediated by the ER[beta] cells were co-incubated with ERr 731[R] and the pure antiestrogen ICI 182 780. ICI 182 780 completely abolished the ERr 731[R] induced ER[R] activation (Fig. 4) indicating that the observed effect of ERr 731[R] is ER[beta]-specific.

Effect of the aglycones of ERr 731[R] and the structurally related hydroxystilbenes piceatannol and resveratrol on ER[beta] activation

The action of the ERr 731[R] aglycones trans-rhapontigenin and desoxyrhapontigenin on the estrogen receptors has not been reported, whilst ER[alpha] and ER[beta] activation by the structurally related hydroxystilbenes resveratrol and piceatannol has already been described (Gehm et al., 2004; Maggiolini et al., 2005). No investigations on the activation of estrogen receptors in bone cells by these compounds were done so far.

The effects of trans-rhapontigenin and desoxyrhapontigenin on ER[alpha] and ER[beta] activation in the respective U2OS cell systems were compared with piceatannol and resveratrol. In addition, cis-rhapontigenin (which is not present in the total extract of ERr 731[R], but which may potentially appear in the organism after isomerisation of trans-rhapontigenin) was also tested in these cell systems.

Desoxyrhapontigenin displayed a weak, but statistically significant ER[alpha] activation in U2OS-ER[alpha] cells in a dose-dependent manner (Fig. 5a). A statistically significant stimulation of the test system was also observed with the lowest concentration of resveratrol (Fig. 5b) but no subsequent dose-dependency was detectable. The ER[alpha] activation of the cells with desoxyrhapontigenin was similar to that obtained with ERr 731[R] (Fig. 3a) being approximately 10% of the effect of [10.sup.-8]M E2.

All single test compounds induced an ER[beta] dependent reporter gene activity in U2OS-ER[beta] cells (Fig. 6). Trans- and cis-rhapontigenin showed similar potency. The U2OS-ER[beta] cells responded with high activity even at the lowest concentrations of the test substances (0.01 [micro]M). A comparable ER[beta] activity level was achieved with the same concentration of E2. As expected, resveratrol and piceatannol also activated the reporter gene activity (Fig. 6).


To investigate if the effects of the single substances were exclusively mediated via ER[beta] activation, U2OS cells were co-incubated with effective doses of the test substances and 500 nM of the pure antiestrogen ICI 182 780. As a positive control, the selective ER[beta] activator DPN was also tested. ICI 182 780 completely abolished the stimulation of the ER[beta] reporter gene activity in U2OS cells by all test compounds (Fig. 7).

Effects of mixtures of rhaponticin and desoxyrhaponticin on ER activation

The observation that ERr 731[R] induces estrogenic responses through induction of reporter gene activity was not necessarily expectable, because the extract constituents are predominately present as glycosides (as rhaponticin and desoxyrhaponticin) which would not be expected to enter the cells by passive diffusion. However, it has been demonstrated that glycosylated herbal substances can cross cell membranes of the isolated rat small intestine (Andlauer et al., 2000) and oral cancer cells (Browning et al., 2005) and are then intracellularly active. We therefore investigated whether defined mixtures of the glycosides rhaponticin and desoxyrhaponticin (10:1 and 1:10) induce ER dependent reporter gene activity in the present cell lines.

Fig. 8 shows that the responses on ER-[beta] are mimicked by defined mixtures of the glycosides in proportions similar to those found in the extract (Fig. 8). Both ERr 731[R] and the rhaponticin/desoxyrhaponticin mixture induced ER-dependent reporter gene activity in a similar manner. These experiments confirmed our previous observations of ER activation by the total extract ERr 731[R].


The investigations presented here were conducted to investigate and identify the effect of ERr 731[R] on the ER activation in the bone. To date, no experimental studies on an estrogenic activity of this special extract and its metabolites in bone cells have been performed.

In the U2OS-ER[alpha] test system, ERr 731[R] produced a weak but significant stimulation of the ER[alpha] mediated reporter gene activity which was 10-times lower than that obtained with [10.sup.-8] M E2. In contrast, ERr 731[R] and all five single test substances activated the ER[beta]-coupled reporter gene activity in the bone-derived cell culture model to a degree comparable to that of [10.sup.-8]M E2.

It is remarkable that already the lowest concentration of the extract induced this ER[beta]-dependent reporter gene activity. In the same dosage range ERr 731[R] also induced ER[beta]-dependent reporter gene activity in endometrial cells, which was detectable for 1 ng/mL and statistically significant for 10 ng/mL (Wober et al., 2007). However, high potency of stilbene derivatives on estrogen receptors is not surprising. For example, the synthetic diethylstilbestrol has to be regarded as one of the most potent estrogens known to date (Kuiper et al., 1998). Also resveratrol, a natural hydroxystilbene, had been demonstrated to activate ER[beta]-dependent reporter gene activity (Wober et al., 2007).


The extract ERr 731[R] contains several natural hydroxystilbenes which may act additive or even synergistic. This assumption is hypothetic in nature and certainly needs further experimental proof particularly in in vivo experiments which are currently under way.

It has been postulated that the relative activity of phytoestrogens on estrogen receptors are dependent on the polar basic structure of the molecule and on the distance and relative position of two polar groups (Cos et al., 2003; Harris et al., 2005). In the case of rhaponticin and desoxyrhaponticin the positions of the hydroxyl group relative to the stilbene background differ (Fig. 1), which might explain the differences in their activities on the ERs.

The strong effect of all substances on the ER[beta] activation was completely abolished by the antiestrogen ICI 182 780 indicating that this activation was exclusively mediated via ER[beta]. The latter results confirm our previous finding on ER[beta] specificity of ERr 731[R] in endometrial cell lines and provide another important piece of evidence that the hydroxystilbenes and their derivatives are the principal candidate molecules for the bioactivity of the total extract ERr 731[R]. The precise contribution of each component of the extract still remains to be elucidated in more detail.

Since ERr 731[R] consists almost exclusively of the glycosides rhaponticin and desoxyrhaponticin, the question was whether a fixed combination of these two glycosylated compounds has a similar effect on the ER activation. The results with the mixtures of rhaponticin/desoxyrhaponticin were comparable to that of ERr 731[R] demonstrating that in principle, this defined mixture of these glycosides was intracellularly active.


The question arises how the extract exerts its biological activity in cell cultures. The glycosides rhaponticin and desoxyrhaponticin are not a subject to passive transmembrane diffusion in contrast to the lipophilic aglycones rhapontigenin and desoxyrhapontigenin. However, there are two possible explanations for the intracellular effects of ERr 731[R]: (a) the substances actively cross the membrane by transporters or pumps and act intracellularly, or (b) they modulate transcription via membrane bound estrogen receptors.

The active transportation of ERr 731[R] seems more likely since the transport of glycosylated phytoestrogens has previously been shown (Andlauer et al., 2000; Browning et al., 2005). Genistin, the glycosylated derivative of genistein, has also been shown to cross cell membranes of the isolated rat small intestine (Andlauer et al., 2000), and of oral cancer cells (Browning et al., 2005). The uptake mechanisms, however, are unknown at present. Our results with 10:1 and 1:10 mixtures of rhaponticin and desoxyrhaponticin may represent another example for uptake of glycosylated phytoestrogens, since these mixtures clearly induce ER-[beta] dependent reporter gene activity. Organic anion transporting polypeptides (OATP) transporters represent candidate molecules for the glycosylated hydroxystilbenes to be actively transported across the plasma membrane. These transporters exhibit a wide substrate spectrum including molecules such as cardiac glycosides, steroid conjugates and hormones in general (Konig et al., 2006). Although conclusive evidence is lacking at present, these data represent a plausible explanation for the ER activation with ERr 731[R] in transfected cells.



The assumption that active transporter systems are responsible for the uptake of glycosylated phytoestrogens might potentially also offer an explanation why a dose-dependent increase in reporter gene activity could not be observed in the current experiments, instead an almost "inverse" dose response relationship became apparent. It might be assumed that at the higher doses of the extracts tested, the uptake capacity of the transporters became saturated, and the observed results may be due to inability of the transporter to deliver more substance internally, rather than being related to the external concentrations of the extract. This hypothesis needs to be verified by further investigations.

Rapid actions originating by membrane bound estrogen receptors e.g., association with G-protein receptor coupled pathways (Boulware and Mermelstein, 2005), recruitment of intracellular tyrosine kinases to membrane bound estrogen receptors and concomitant activation of either MAP-kinase or AKT-dependent pathways (Song et al., 2005) and finally, through orphan membrane bound G-protein coupled receptors (Filardo and Thomas, 2005) appeared unlikely for ERr 731[R] We predominantly observed ER[beta] dependent activation of the reporter gene activities while most of the known membrane initiated actions of estrogens were described to be triggered primarily by ER[alpha] (Song et al., 2005). Nevertheless, the activation of membrane bound estrogen receptors might be discussed as an alternative pathway to explain the ER[alpha] agonistic activity of ERr 731[R] in the U2OS cells.

The question arises whether the biological responses triggered by ERr 731[R] have to be regarded as SERM (selective estrogen-receptor modulating) properties. SERMs are a class of ER ligands that exert cell and tissue specific responses and may functionally operate as agonists or antagonists (McDonnell, 1999). Although there is no unifying definition for a SERM or SERM-properties some crucial observations are helpful to explain the pharmacology of SERMs (for review see (McDonnell, 2000): (1) the discovery of the ER[beta]; (2) the observation that the conformation of the two receptor subtypes is influenced by the nature of the bound ligand; and (3) the identification of receptor-associated comodulators, proteins that can enhance (co-activators) or repress (co-repressors) receptor transcriptional activity. All of these activities have been shown to be important in SERM pharmacology and at least two are met by ERr 731[R] receptor subtype and tissue selectivity. ERr 731[R] and its constituents predominantly activate ER-[beta]. In addition, a tissue-specific ER-[alpha] activity is identified, however, it has to be clearly stated that the latter was weakly although significantly detectable in bone cells and most importantly, undetectable in endometrial cells (Wober et al., 2007). This is indicative of SERM-properties which need to be characterized in depth in future experiments. In addition, ER[alpha] activity is a prerequisite for bone protection. Whether or not the detected ER[alpha]-dependent activity of ERr 731[R] is sufficient for bone protection is subject of in vivo experiments.

In conclusion, in bone-derived U2OS cells the special extract ERr 731[R] acts primarily through the ER[beta] with an activity that is comparable to that of the single test substances. In addition, we demonstrated for the first time a weak but significant activation of the ER[alpha] in bone cells and it is tempting to speculate that a long-term treatment of patients with ERr 73[R] may have a beneficial effect on the bone. Further supporting experimental and clinical studies are in progress.


We thank Dr. Andrew Richardson, Health Research Services Ltd. Hungerford, UK, for helpful scientific discussions and proof-reading of the manuscript. We also thank Dr. Marianne Heger for her scientific contribution. We thank Antje Beyer and Susanne Kolba for their expert technical support.

The work was supported by the Chemisch-Pharmazeutische Fabrik Goeppingen, Carl Muller, Apotheker, GmbH u. Co. KG, Goeppingen, Germany. The authors declare that there is no conflict of interest that would prejudice its impartiality.


Aggarwal, B.B., Bhardwaj, A., Aggarwal, R.S., Seeram, N.P., Shishodia, S., Takada, Y., 2004. Role of resveratrol in prevention and therapy of cancer: preclinical and clinical studies. Anticancer Res. 24, 2783-2840.

Andlauer, W., Kolb, J., Furst, P., 2000. Absorption and metabolism of genistin in the isolated rat small intestine. FEBS Lett. 475, 127-130.

Boulware, M.I., Mermelstein, P.G., 2005. The influence of estradiol on nervous system function. Drug News Perspect. 18, 631-637.

Browning, A.M., Walle, U.K., Walle, T., 2005. Flavonoid glycosides inhibit oral cancer cell proliferation--role of cellular uptake and hydrolysis to the aglycones. J. Pharm. Pharmacol. 57, 1037-1042.

Cos, P., De Bruyne, T., Apers, S., Vanden Berghe, D., Pieters, L., Vlietinck, A.J., 2003. Phytoestrogens: recent developments. Planta Med. 69, 589-699.

Filardo, E.J., Thomas, P., 2005. GPR30: a seven-transmembrane-spanning estrogen receptor that triggers EGF release. Trends Endocrinol. Metab. 16, 362-367.

Gehm, B.D., Levenson, A.S., Liu, H., Lee, E.J., Amundsen, B.M., Cushman, M., Jordan, V.C., Jameson, J.L., 2004. Estrogenic effects of resveratrol in breast cancer cells expressing mutant and wild-type estrogen receptors: role of AF-1 and AF-2. J. Steroid Biochem. Mol. Biol. 88, 223-234.

Harris, D.M., Besselink, E., Henning, S.M., Go, V.L., Heber, D., 2005. Phytoestrogens induce differential estrogen receptor alpha- or beta-mediated responses in transfected breast cancer cells. Exp. Biol. Med. (Maywood) 230, 558-568.

Harris, H.A., 2007. Estrogen receptor-beta: recent lessons from in vivo studies. Mol. Endocrinol. 21, 1-13.

Heger, M., Ventskovskiy, B.M., Borzenko, I., Kneis, K.C., Rettenberger, R., Kaszkin-Bettag, M., Heger, P.W., 2006. Efficacy and safety of a special extract of Rheum rhaponticum (ERr 731) in perimenopausal women with climacteric complaints: a 12-week randomized, double-blind, placebo-controlled trial. Menopause 13, 744-759.

Hillisch, A., Peters, O., Kosemund, D., Muller, G., Walter, A., Schneider, B., Reddersen, G., Elger, W., Fritzemeier, K.H., 2004. Dissecting physiological roles of estrogen receptor alpha and beta with potent selective ligands from structure-based design. Mol. Endocrinol. 18, 1599-1609.

Kaszkin-Bettag, M., Ventskovskiy, B.M., Kravchenko, A., Rettenberger, R., Richardson, A., Heger, P.W., Heger, M., 2007. The special extract ERr 731 of the roots of Rheum rhaponticum decreases anxiety and improves health state and general well-being in perimenopausal women. Menopause.

Kobashi, K., Akao, T., 1997. Relation of intestinal bacteria to pharmacological effects of glycosides. Biosci. Microflora 16, 1-7.

Konig, J., Seithel, A., Gradhand, U., Fromm, M.F., 2006. Pharmacogenomics of human OATP transporters. Naunyn. Schmiedebergs Arch. Pharmacol. 372, 432-443.

Kuiper, G.G., Lemmen, J.G., Carlsson, B., Corton, J.C., Safe, S.H., van der Saag, P.T., van der Burg, B., Gustafsson, J.A., 1998. Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor beta. Endocrinology 139, 4252-4263.

Lindberg, M.K., Moverare, S., Skrtic, S., Gao, H., Dahlman-Wright, K., Gustafsson, J.A., Ohlsson, C., 2003. Estrogen receptor (ER)-beta reduces ERalpha-regulated gene transcription, supporting a "ying yang" relationship between ERalpha and ERbeta in mice. Mol. Endocrinol. 17, 203-208.

Lindsay, R., Tohme, J.F., 1990. Estrogen treatment of patients with established postmenopausal osteoporosis. Obstet. Gynecol. 76, 290-295.

Maggiolini, M., Recchia, A.G., Bonofiglio, D., Catalano, S., Vivacqua, A., Carpino, A., Rago, V., Rossi, R., Ando, S., 2005. The red wine phenolics piceatannol and myricetin act as agonists for estrogen receptor alpha in human breast cancer cells. J. Mol. Endocrinol. 35, 269-281.

McDonnell, D.P., 1999. The molecular pharmacology of SERMs. Trends Endocrinol. Metab. 10, 301-311.

McDonnell, D.P., 2000. Selective estrogen receptor modulators (SERMs): a first step in the development of perfect hormone replacement therapy regimen. J. Soc. Gynecol. Investig. 7, S10-S15.

Park, E.K., Choo, M.K., Yoon, H.K., Kim, D.H., 2002. Antithrombotic and antiallergic activities of rhaponticin from Rhei Rhizoma are activated by human intestinal bacteria. Arch. Pharm. Res. 25, 528-533.

Ponten, J., Saksela, E., 1967. Two established in vitro cell lines from human mesenchymal tumours. Int. J. Cancer 2, 434-447.

Rosendaal, F.R., Helmerhorst, F.M., Vandenbroucke, J.P., 2002. Female hormones and thrombosis. Arterioscler Thromb. Vase. Biol. 22, 201-210.

Ross, R.K., Paganini-Hill, A., Wan, P.C., Pike, M.C., 2000. Effect of hormone replacement therapy on breast cancer risk: estrogen versus estrogen plus progestin. J. Natl. Cancer Inst. 92, 328-332.

Skafar, D.F., Xu, R., Morales, J., Ram, J., Sowers, J.R., 1997. Clinical review 91: female sex hormones and cardiovascular disease in women. J. Clin. Endocrinol. Metab. 82, 3913-3918.

Song, R.X., Zhang, Z., Santen, R.J., 2005. Estrogen rapid action via protein complex formation involving ERalpha and Src. Trends Endocrinol. Metab. 16, 347-353.

Vollmer, G., Zierau, O., 2004. What are phytoestrogens and phyto-SERMS. Pharm. Unserer Zeit 33, 378-383.

Wakeling, A.E., Bowler, J., 1987. Steroidal pure antioestro-gens. J. Endocrinol. 112, R7-R10.

Wakeling, A.E., Bowler, J., 1992. ICI 182,780, a new antioestrogen with clinical potential. J. Steroid. Biochem. Mol. Biol. 43, 173-177.

Wober, J., Moller, F., Richter, T., Unger, C., Weigt, C., Jandausch, A., Zierau, O., Rettenberg, R., Kaszkin-Bettag, M., Vollmer, G., 2007. Activation of estrogen receptor-beta by a special extract of Rheum rhaponticum (ERr 731[R]), its aglycones and structurally related compounds. J. Steroid Biochem. Mol. Biol. 107, 191-201.

Frank Moller (a), Oliver Zierau (a), Anett Jandausch (a), Reinhard Rettenberger (b), Marietta Kaszkin-Bettag (c), Gunter Vollmer (a,*)

(a) Molekulare Zellphysiologie & Endokrinologie, Institut fur Zoologie, Technische Universitat Dresden, 01062 Dresden, Germany

(b) Chemisch-Pharmazeutische Fabrik Goppingen, Carl Muller, Apotheker, GmbH u. Co. KG, 73033 Goppingen, Germany

(c) Health Research Services Ltd., 68789 St. Leon-Rot, Germany

Received 5 March 2007; accepted 2 September 2007

*Corresponding author. Tel.: + 49 351 46331922; fax: +49 351 46331923.

E-mail address: (G. Vollmer).
 R1 R2 R3

Rhaponticin OH OC[H.sub.3] O-Glc
Desoxyrhaponticin H OC[H.sub.3] O-Glc
Rhapontigenin OH OC[H.sub.3] OH
Desoxyrhapontigenin H OC[H.sub.3] OH
Resveratrol H OH OH
Piceatannol OH OH OH

Fig. 1. Structure of hydroxystilbene derivatives.
COPYRIGHT 2007 Urban & Fischer Verlag
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2007 Gale, Cengage Learning. All rights reserved.

Article Details
Printer friendly Cite/link Email Feedback
Author:Moller, Frank; Zierau, Oliver; Jandausch, Anett; Rettenberger, Reinhard; Kaszkin-Bettag, Marietta; V
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:4EUGE
Date:Nov 1, 2007
Previous Article:Randomised, placebo-controlled, double-blind, split-face study on the clinical efficacy of Tricutan[R] on skin firmness.
Next Article:Antibacterial effect of some essential oils administered alone or in combination with Norfloxacin.

Terms of use | Privacy policy | Copyright © 2021 Farlex, Inc. | Feedback | For webmasters