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[beta]-Ecdysone has bone protective but no estrogenic effects in ovariectomized rats.


Estrogens exert beneficial effects in the bone. Their chronic use however bares several risks. Therefore intensive search for non-estrogenic, bone protective compounds is going on. We observed that an extract of Tinospora cordifolia has antiosteoporotic effects and identified 20-OH-Ecdysone ([beta]-Ecdysone = Ecd) as a possible candidate for this action.

Ovariectomized (ovx) rats were treated orally over 3 months with no Ecd (control) or 18, 57 or 121 mg Ecd/day/animal. Estradiol-17[beta] benzoate (E2) 159 [micro]g/day/animal) fed animals served as positive controls. Bone mineral density (BMD) of tibia was measured by quantitative computer tomography, serum Osteocalcin and CrossLaps were measured in a ligand binding assay. Utilizing an estrogen receptor (ER) containing cytosolic extract of porcine uteri the capability of Ecd to bind to ER was tested.

Ecd did not bind to ER. BMD was reduced by more than 50% in the control. In the Ecd animals BMD was dose dependently higher. Serum CrossLaps was lower in the Ecd and E2 group while serum Osteocalcin levels were decreased in the E2 but increased in the Ecd fed animals.

Ecd has an antiosteoporotic effect which does not involve activation of ER.

[c] 2010 Elsevier GmbH. All rights reserved.




Tinospora cordiofolia

Quantitative computer tomography




Ever since the publication of increased mammary cancer risk and cardiovascular incidences following hormone replacement therapy (Rossouw et al. 2002) an intensive search for alternatives has begun. Phytoestrogens, such as isoflavones derived from soy, red clover or hops were promoted but they have similar though milder effects than estradiol-17[beta] benzoate (E2) (Nelson et al. 2006; Rimoldi et al. 2007; Seidlova-Wuttke et al. 2008). We have recently published that extracts of Tinospora cordifolia (in Ayurveda medicine also known as Guduchi) has antiosteoporotic effects (Kapur et al. 2008) and in searching for active compounds in this plant extract we identified an ecdysteroid namely 20-OH-Ecdysone ([beta]-Ecdysone = Ecd). Plants produce ecdysteroids to cause metamorphosis of larvae into pupae to protect themselves against herbivory by caterpillars (Bathori et al. 2008).

Some estrogenic effects of compounds supposed to replace HRT are undesired. Cytosolic extracts of porcine uteri contain both estrogen-receptor (ER) subtypes namely ER-[alpha] and ER-[beta] and can be used to verify or deny binding properties of test compounds to these two ERs (Jarry et al. 2003). Similarly, the so-called uterotrophy assay is recommended by the OECD to test for estrogenicity (Yamasaki et al. 2003) and utilizes the capability of estrogenic compounds to stimulate uterine weights in ovariectomized (ovx) rats. Another sensitive parameter to test for estrogenicity is the serum LH level which is increased in ovx animals due to the lacking negative feedback of E2 and consequently decreased by estrogenic compounds (McNeilly et al. 2003).

Beneficial effects of estrogens are bone protective actions. Particularly the metaphysis of the tibia of mice and rats was shown to react excessively sensitive to withdrawal or replacement of estrogens and by a variety of methods it was shown that demineralisation of the metaphysis of the tibia amounts to 50% or more within 3 months following ovx (Garner et al. 1991; Wronski 1985; Seidlova-Wuttke et al. 2003a,b, 2008; Perry et al. 2005; Klinck and Boyd 2008). Quantitative computer tomography (qCT) proved to be a means to measure a variety of bone parameters in living small animals (Helterbrand et al. 1997) and was therefore used by others and ourselves to assess effects of a variety of estrogenic compounds on several bone parameters including bone mineral density (BMD) (Seidlova-Wuttke et al. 2003a,b, 2008; Sheng et al. 2007; Wei et al. 2007; Coelingh Bennink et al. 2008).

There are surrogate parameters of bone metabolism available: following ovx the osteoblast products Osteocalcin (OC) increases in the serum (Seidlova-Wuttke et al. 2003a; French et al. 2008) and markers of osteoclast activity are the CrossLaps, which are metabolic breakdown products of bone specific collagen I are also high in ovx rats (Seidlova-Wuttke et al. 2003a,b; Coelingh Bennink et al. 2008; French et al. 2008). This indicates that both osteoblast and osteoclast activities are increased in the absence of E2 and that in ovx animals the activity of osteoclasts are more stimulated than of osteoblasts which results in more bone resorption than formation (Wronski 1985; Seidlova-Wuttke et al. 2003b; Seidlova-Wuttke et al. 2003a). These markers are therefore widely used to study putative antiosteoporotic effects of test substances.

Due to the possible antiosteoporotic effects of Ecd we tested its estrogenicity utilizing ligand binding assays, the uterotrophy assay and the effects on serum LH levels. Furthermore we determined bone mineral densities at different doses of oral Ecd administration and compared them with the known antiosteoporotic effect of E2 after 3 months of treatment.

Materials and methods

Female Sprague-Dawley rats were used for the present experiments. Allowance to perform the experiments was obtained from the Bezirksregierung Braunschweig (permission No. Az. 33,42502-082/06). The test substance was 20-OH-Ecdysone (=[beta]-Ecdysone = Ecd) commercially available through Changzhou Dahua Imp. and Export (Group) Corp. Ltd. Changzhou, Jiangsu, China, which had a 97.2% purity. For control purposes estradiol-17[beta] (E2) benzoate (Sigma-Aidrich Chemie GmbH Munich, Germany) was tested.

3 months old rats (Winkelmann, Borken, Germany) weighing 250[+ or -]10g were adjusted to our animal facilities (5 animals/cage; light on 06:00 a.m. to 06:00 p.m., relative humidity 55%) and were kept on soy-protein, isoflavone-poor pelleted food (ssniff, V 1355, R-Z, poor in phytoestrogens) in which isocaloric protein supplementation was secured by added potato proteins. After 3 weeks of adjustment animals were anaesthetized with Isoflurane (Florene, Abbott Laboratories Ltd., Maidenhead, UK) weighed and subjected to quantitative computer tomography with the XCT Research SA, Stratec Medizintechnik, Pforzheim, Germany, for determination of bone densities (BMD) of the cancellous and cortical parts of the metaphysis of the tibia. The scanner was positioned at the epiphysis of tibia and a coronal computed radiograph (scout view) was carried out. The scout view was used to position the scanner at the site of measurement. Two tomographic slices at a distance of 3.75 or 4.25 mm distal of the reference line were used for determination of cancellous bone parameters. A third slice was taken 15 mm distal of the reference line for determination of cortical bone parameters.

Image acquisition, processing and calculation of the results were performed using the software package XCT5.40 (STRATEC Inc.). Cancellous density was calculated in the cross sections with a lower and upper density threshold of 280 and 710 mg/[cm.sup.3], respectively. All values above 710 mg/[cm.sup.3] were considered to be cortical bone. One week later rats were ovariectomized (ovx) again under Isoflurane anaesthesia. After ovx control animals were maintained on soy-free potato protein enriched food, the other animals were divided into 3 groups (n = 10/group) and after ovx treated orally for 3 months with E2 and Ecd containing food on soy-free basis.

Body weights of animals were determined once per week. The estimation of daily intake of E2 and Ecd per animal was calculated from weekly measured food consumption per cage divided by 7 and the number of animals per cage (Table 1). After a treatment period of 3 months animals were again subjected to qCT measurement of the metaphysis of the tibia, then decapitated and blood samples were collected from the trunk. The uteri were removed, cleaned of adherent tissue and weighed.
Table 1

Average daily food and test substance intake and the final body
weights (BW) of the animals are detailed in the table (sf = soy free).

              Average daily food  Average daily     Mean BW [+ or -]
              intake              substance intake  SEM (g)
              (g/day/animal)      (mg/day/animal)

ovx, sf       17.32 [+ or -] 1.3  -                 324.7 [+ or -]

ovx + Ecd 18  18.0 [+ or -] 1.1   18.02             324.4 [+ or -]

Ovx + Ecd 57  18.9 [+ or -] 1.3   56.5              333.2 [+ or -]

Ovx+Ecd 121   19.3 [+ or -] 1.7   120.8             344.5 [+ or -]

Ovx + E2      15.9 [+ or -] 3.9   0.159             274.5 [+ or -]

The following bone parameters were calculated utilizing the XCT 5.40 software package:

- Density of cancellous bone, the endosteal structures with a density between 280 and 710 mg/[cm.sup.3].

- Cortical density, the cortical area where mineral density was above 710mg/[cm.sup.3].

- On the basis of these data a strain strength index (SSI) can be calculated which, after adjustment to the body weight of the animals gives an index of stability of the bone.

To test the estrogenicity of Ecd in a cytosolic estrogen-receptor preparation the method of Jarry et al. (2003) was employed to prepare a cytosolic fraction from porcine uteri. These receptor preparations contain both, ER-[alpha] and ER-[beta]. Instead of a tritium-labelled tracer, 16[[alpha]-[.sup.125]I-labelled estradiol (2200Ci/mmoI) was used. Samples were measured in triplicates.

Serum analyses

The blood samples were centrifuged (3000 x g, 20 min) and the serum stored at -20 [degrees]C for further analysis. Luteinizing hormone (LH) was measured by specific RIA supplied by the National Hormone and Pituitary Program of the NIH (Dr A.F. Parlow, Harbor General Hospital, Torrance, CA, USA), as described previously (Roth et at. 2001). Serum E2 was assayed with a commercially available kit (DSL, Sinsheim, Germany). Serum OC was determined using a human system (Elecsys, Roche, Mannheim, Germany) and the C-terminal telopeptides of type I Collagen were measured with an ELISA (RatLaps ELISA: Nordic Biosciences, Herlev, Denmark).

To recover Ecd from serum enzymatic hydrolysis of potential metabolites was performed before serum extraction. A volume of 500 u.1 serum was extended with 500 [micro]1 NH acetate buffer (pH 5.0) containing 1 mg [beta]-glucuronidase (Helix Pomatia [beta]-Glucuronidase Type H1: Sigma, Taufkirchen) and incubated overnight at 37 [degrees]C The Strata X solid-phase extraction method (8B-S100-UBJ, Phe-nomenex, Aschaffen burg) with a polymeric sorbent was used according to the instructions of the manufacturer. The eluted volume was evaporated to dryness and reconstituted with 100 [micro]1 EtOH. For HPLC a volume of 20 u.1 was chromatographed over a NC 2504.6 mm Hypersil-ODS 5.0 [micro]m column (Bischoff, Leonberg, Germany). Ecd was detected at 254 nm.

Statistical evaluation

Data were expressed as means [+ or -] standard errors of the means (SEM). Significant differences between the control- and treatment groups were analysed by one-way ANOVA followed by Dunnett's post hoc test for multiple comparisons (Prism [TM], Graph Pad, San Diego, USA), p values < 0.05 were considered statistically significant.


Food intake of ovx controls and Ecd treated animals did not vary significantly (~ 18-20 g/day/animal) while the E2 fed animals ate significantly less food (15.3 g/day/animal). In the animals receiving the food with the highest Ecd amounts the serum concentrations were 1.2 x [10.sup.-6] M. In sera of animals receiving lower amounts of Ecd the assay sensitivity was at its limit. Serum E2 levels in the E2 fed animals were 73 [+ or -] 24.4 pg/ml. In the other animals E2 concentrations were below the detection limit of the assay, i.e. < 5 pg/ml (Table 1).

Fig. 1 shows the ligand binding activity of E2 and Ecd on the cytosolic ER preparation. While E2 readily displaces the radioactively labelled estradiol; Ecd was totally ineffective even at concentrations of [10.sup.-3] M.


Fig. 2a details a marked increase of uterine weights in the E2 treated controls. This effect was not shared by any of the 3 doses of Ecd.


Serum LH levels were high in ovx animals under control food and significantly suppressed in the animals fed with the E2 containing pellets. The animals under Ecd had also reduced serum LH levels and this effect was most marked in the animals treated with the intermediate dose of Ecd (Fig. 2b).

The effects of E2 and Ecd on cancellous and cortical densities and on the trabecular surface in the metaphysis of the tibia are detailed in Fig. 3a-c. Cancellous densities (Fig. 3a) were reduced by more than 50% in the ovx controls and were only slightly reduced in the E2 treated in comparison to the pre-treatment values. Also the 3 doses of Ecd partially prevented the reduction of cancellous density, i.e. the development of osteoporosis. Interestingly the highest dose of Ecd was less effective in this respect than the intermediate dose. This higher density of the cancellous structure was due to an increase of the trabecular surface as demonstrated in Fig. 3b. Cortical BMDs (Fig. 3c) increased in all ovx animals in comparison to the pre-ovx values statistically significant. The weight related strain strength index was lowest in the ovx, highest in the E2 and at intermediate values in the Ecd treated animals (Fig. 4).



Serum OC levels were high in ovx animals and remained at the high values in the Ecd treated animals while the E2 application resulted in a significant reduction of OC (Fig. 5a). The metabolic breakdown products of bone specific collagen 1, the CrossLaps, were also high in ovx animals and reduced by Ecd and E2 (Fig. 5b).



In the present experiment we demonstrate a marked effect of Ecd to prevent osteoporosis and to reduce serum LH levels in ovx animals. As demonstrated by the E2-ligand binding assay and by the lacking effect of any of the 3 Ecd doses to stimulate uterine weights, it is safe to conclude that the observed effects in the bone and in the hypothalamo-pituitary axis are not due to an estrogenic effect of the ecdysteroids. Also androgenic effects of Ecd were not found previously (Semeikin et al. 1991; Gorelick-Feldman et al. 2008). What then are the mechanisms of action of Ecd? Ecd is known to have protein anabolic effects in the mammalian organism. In the human Ecd was shown to increase lean body mass. Particularly muscular training in combination with an Ecd treatment causes an increase in muscular mass (Bathori et al. 2008) Such increase in muscular mass occurrs also in the ovx rats resulting in increased mechanical stimulation of the bone. Mechanical stimulation of the bone tissue is known to have bone protective effects (Flieger et al. 1998; Rubin et al. 2002; Ferretti et al. 2003; Luu et al 2008).

Attempts to demonstrate Ecd receptors in mammalian tissues failed. There is some evidence however, that Ecd can stimulate transactivation via stimulation of the retinoid X receptor (RXR) (Thomas et al. 1993; Yao et al. 1993). Among others RXR may dimer-ize with the peroxisome proliferator activated receptors (PPARs) (Michalik et al. 2006), the retinoic receptor (RAR) (Germain et al. 2006) or the Vitamin D (VD) receptor (Baudino et al. 1998; Farmer et al. 2000; Bathori et al. 2008). Vitamin D promotes intestinal calcium resorption and stimulates the production of osteoblastic osteobalcin (Nagpal et al. 2001) a protein necessary for mineralization of the protein bone matrix. Hence, Ecd stimulated transactivation of a RXR/VD dimer may be one of the factors by which Ecd exerts its beneficial effects in the bone. In favour of this explanation is our observation that osteocalcin, an osteoblast product, was significantly increased in the Ecd treated animals, an effect also exerted by VD (Shiraishi et al. 2000; Peleg et al. 2002).

The PPARs are known to be intimately involved in fat metabolism. PPAR [alpha] agonists are clinically used as lipolytic drugs (Guerre-Millo et al. 2000; Berger and Moller 2002; Michalik et al. 2006) while PPAR [gamma] agonists predispose the development of lipocytes at the cost of osteoblasts. This action would not explain the antiosteoporotic effects of Ecd. Possibly, the ecdysteroid has selective PPAR modulator effects (SPPAR effects) effects and recruits in the bone more co-repressors than co-activators to suppress adipocyte formation. Such organ selectivity is known for estrogen receptors where selective estrogen-receptor modulators (SERM) have estrogen antagonistic effects in the mammary gland but act as estrogen agonist in the bone. Also retinoids are known to have bone protective effects (Weston et al. 2003) and the transactivational effects of the RXR/RAR dimer may result in increased osteoblast activity. Taken together Ecd appears to have fundamentally different mechanism of action from that of E2 which--following activation of the ER-[alpha]--decreases bone metabolism by decreasing osteoclast activity more pronounced than osteoblast activity, thereby preventing the development of osteoporosis (Wronski 1985; Seidiova-Wuttke et al. 2003a,b).

The LH reducing effect of Ecd deserves also a comment. As mentioned above this is a typical estrogenic effect but the lack of binding activity to ERs in ligand binding assays and the lacking effects in the uteri of ovx animals deny an estrogenic effect. The high LH levels following ovx are due to an overactivity of the GnRH-pulse generator (Tataryn et al. 1980; Kronenbevg et al. 1984; Ushiroyama et al. 1999; Sturdee 2008) which is believed to be reason for the occurrence of hot flushes in the climacteric/postmenopausal women. Hence, reduction of the overactivity of the GnRH-pulse generator may effectively ameliorate climacteric complaints in women. The mechanism of action of Ecd on LH levels is explainable on that basis.

In an in vitro system of cortical neurons from newborn rats Ecd potentiated the effects on GABA induced inhibition (Tsujiyama et al. 1995). GABA was shown previously to inhibit the activity of the hypothalamic GnRH-pulse-generator thereby inhibiting pulsatile pituitary LH release (Mansky et al. 1982; Herbison et al. 1991; Jarry et al. 1992; Kimura et al. 2004) and a number of estrogen receptive neurons were shown to use GABA as neurotransmitter (Flugge 1986; Curran-Rauhut and Petersen 2002). Hence, the GABA potentiating effects of Ecd may explain the decreased serum LH levels.

In summary, our results demonstrate beneficial effects of Ecd in the bone. The lack of activity in the ER-ligand binding assay and the lack of uterotrophic activities of any of the 3 tested doses as well as the opposite effects of Ecd in comparison to E2 on serum osteocalcin levels make it unlikely the Ecd acts via estrogenic mechanism. Clearly more research is needed to unravel the mechanism of Ecd action.


The authors thank Mrs. Heidi Bruggemann-Meyer and Mrs. Maria Metten for the excellent serum analysis.


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Dana Seidlova-Wuttke, David Christel, Priya Kapur, Ba Tiep Nguyen, Hubertus Jarry, Wolfgang Wuttke *

Department of Endocrinology, University Medical Center Goettingen, Robert-Koch-Str. 40. D-37075 Goettingen, Germany

* Corresponding author. Tel.: +49 551 396714; fax: +49 551 396518.

E-mail address: (W. Wuttke).

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Author:Seidlova-Wuttke, Dana; Christel, David; Kapur, Priya; Nguyen, Ba Tiep; Jarry, Hubertus; Wuttke, Wolf
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:4EUGE
Date:Sep 1, 2010
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