JOURNAL OF BONE AND MINERAL RESEARCH

Volume 20, Number 11, 2005

Published online on July 18, 2005; doi: 10.1359/JBMR.050716
© 2005 American Society for Bone and Mineral Research

Isopropanolic Extract of Black Cohosh Stimulates Osteoprotegerin

Production by Human Osteoblasts
Volker Viereck,1,2 Carsten Gründker,1,2 Stephanie C Friess,1 Karl-Heinz Frosch,3 Dirk Raddatz,4 Michael Schoppet,5
Thomas Nisslein,6 Günter Emons,1 and Lorenz C Hofbauer5

ABSTRACT: An isopropanolic extract (iCR) from the rhizomes of Cimicifuga racemosa (black cohosh) is used
an alternative in the treatment of menopausal symptoms, and animal studies suggest positive skeletal effects.
iCR stimulated osteoblastic OPG protein secretion by 3- to 5-fold as early as 12 h without affecting RANKL
expression. The iCR effect, abrogated by the pure estrogen receptor antagonist ICI 182,780, also enhanced
ALP activity (4-fold) and osteocalcin expression (3-fold), possibly contributing to the skeletal effects of black
cohosh.
Introduction: Despite its positive effects on the skeleton, estrogen replacement therapy is no longer recom-
mended as first-line therapy for the prevention and treatment of postmenopausal osteoporosis because it
increases cardiovascular, thromboembolic, and breast cancer risk. Recently, herbal therapeutics such as an
isopropanolic extract (iCR) from the rhizomes of Cimicifuga (=Actaea) racemosa (black cohosh) are gaining
interest as an alternative in the treatment of menopausal symptoms. Whereas animal studies in rats suggest
positive skeletal effects, the mechanism of its actions on bone cells remain unclear. RANKL is essential for
osteoclast formation and activation, while osteoprotegerin (OPG) neutralizes RANKL.
Materials and Methods: In this study, we assessed the effects of iCR on OPG and RANKL mRNA steady-
state levels by semiquantitative RT-PCR and on protein production by an ELISA system in human osteoblasts
(hOBs).
Results: Under serum-free conditions, treatment with iCR increased OPG mRNA levels and protein secretion
of hOBs by 2- to 3-fold in a dose-dependent manner, with a maximum effect at a 106-fold dilution of iCR
(p < 0.001) after 24–48 h. Time-course experiments indicated a stimulatory effect of iCR on osteoblastic OPG
protein secretion by 3- to 5-fold (p < 0.001) as early as 12 h, whereas RANKL expression was very low and
was not found to be modulated by iCR. Of note, the stimulatory effect of iCR on OPG production was
abrogated by the pure estrogen receptor antagonist ICI 182,780. Moreover, iCR enhanced two osteoblastic
differentiation markers, bone-specific alkaline phosphatase activity and osteocalcin expression, by up to 4- and
3-fold, respectively (p < 0.001).
Conclusions: Our data suggest that iCR enhances differentiation and increases the OPG-to-RANKL ratio of
normal human osteoblasts. These effects may contribute to the positive skeletal effects of black cohosh.
J Bone Miner Res 2005;20:2036–2043. Published online on July 18, 2005; doi: 10.1359/JBMR.050716
Key words: black cohosh, osteoprotegerin, osteoblasts, osteoblastic differentiation

INTRODUCTION erogeneous group of nonsteroidal plant-derived com-

pounds that include isoflavones, lignins, flavonoids, and
adverse effects,(1) estrogen replace-
B ECAUSE OF SERIOUS
ment therapy is no longer recommended as first-line
therapy for the prevention and treatment of postmeno-
coumestans that share structural similarities with naturally
occurring or synthetic estrogens. Phytoestrogens exhibit es-
trogen-like effects at various reproductive and nonrepro-
pausal osteoporosis.(2) Selective estrogen receptor modula-
ductive estrogen target tissues, including bone.(1) However,
tors (SERMs) such as raloxifene, bisphosphonates, and
safety concerns regarding their origin and processing as well
PTH are proposed as effective alternatives. However, these
as poor standardization with high variability of concentra-
compounds are unable to treat menopausal symptoms, and
tions may limit the use of phytoestrogen preparations.
some may even aggravate them. Phytoestrogens are a het-
In contrast, extracts of the rootstock of Cimicifuga
(⳱Actaea) racemosa (black cohosh) do not contain phy-
Dr Nisslein is an employee of Schaper & Brümmer Co., manu- toestrogenic substances, represent standardized and well-
facturer of iCR (Remifemin). All other authors have no conflict of characterized pharmaceuticals, and are increasingly consid-
interest. ered as an alternative to synthetic hormone therapy.(3) In

1
Department of Obstetrics and Gynecology, Georg-August-University of Göttingen, Göttingen, Germany; 2These authors contributed
equally; 3Department of Trauma Surgery, Georg-August-University of Göttingen, Göttingen, Germany; 4Department of Gastroenter-
ology and Endocrinology, Georg-August-University of Göttingen, Göttingen, Germany; 5Department of Medicine, Philipps-University of
Marburg, Marburg, Germany; 6Schaper & Brümmer Co., R & D-Department of Veterinary Medicine, Saltzgitter, Germany.

2036
BLACK COHOSH STIMULATES OSTEOBLASTIC OPG PRODUCTION
an ovariectomized rat model of osteoporosis, extracts of
black cohosh decreased urinary excretion of cross-links;
however, the positive effect on trabecular BMD and on
bone quality as assessed by mechanical testing was weaker
than that of raloxifene.(4) In a similar study on orchidecto-
mized rats, extracts of black cohosh mitigated bone loss at
the tibial metaphysis after 3 months.(5) However, compared
with estrogen and testosterone, extracts of black cohosh
had a weaker effect on suppression on bone formation
markers serum osteocalcin and collagen-1␣.(5) Of note, con-
current analysis of skeletal and uterine effects by black co-
hosh in an ovariectomized rat model revealed weak protec-
tive effects on bone loss and on reduction of serum levels of
osteocalcin and cross-laps, but no increase in uterine
weight.(6) In a small randomized controlled trial of 62
women, black cohosh alleviated menopause symptoms
without affecting endometrial thickness of the uterus.(7)
However, its effects on bone turnover markers were am-
biguous, and BMD or fracture reduction was not as-
sessed.(7) Despite these preliminary results from preclinical
studies and one small study in humans, the possible mecha-
nisms of black cohosh extracts on the skeleton remain un-
clear.
Estrogens regulate osteoblast and osteoclast functions
through activation of high affinity receptors (ER).(8,9) Re-
cent studies have suggested that the antiresorptive effects of
estrogens are largely mediated through direct effects on
osteoclasts and by osteoblast-derived paracrine factors that
modulate osteoclast formation and activation.(10) Osteo-
protegerin (OPG) is secreted by osteoblastic lineage
cells(11,12) and acts as a decoy receptor for RANKL, the
essential cytokine for osteoclast formation and activa-
tion.(13,14)
The expression of RANKL and OPG by osteoblastic
lineage cells is modulated by a variety of hormones and
cytokines.(14) We have shown that skeletal ER agonists,
including 17␤-estradiol,(15,16) raloxifene,(17) and the phy-
toestrogen genistein,(16) enhanced the expression of OPG
mRNA and protein by estrogen-responsive human osteo-
blasts. Here we report that an isopropanolic extract of black
cohosh stimulates OPG production by primary human tra-
becular osteoblasts that predominantly express ER-␣.
MATERIALS AND METHODS
Materials
Cell culture medium and supplements were purchased
from Gibco-BRL (Karlsruhe, Germany), and culture flasks
and dishes were from Nunc (Roskilde, Denmark). Unless
otherwise stated, all other chemicals were purchased from
Sigma (Munich, Germany). Isopropanolic extract of root-
stock of black cohosh (iCR) was kindly provided by
Schaper & Brümmer as a stock solution containing 75 mg/
ml in relation to the dry residue.
Cell culture
Bone specimens were obtained from the iliac crest of five
patients (four premenopausal women of 29, 37, 39, and 43
2037
years, respectively, and 1 man of 33 years) undergoing
trauma surgery after fractures. None of the patients had
overt bone or autoimmune diseases, and none were taking
medications known to affect bone and mineral metabolism
(diuretics, glucocorticoids, immunosuppressants, bisphos-
phonates, or anticonvulsants). All participants provided
written informed consent, and the study was approved by
the Institutional Review Board of the University of Göttin-
gen. The experiments were conducted using cells from in-
dividual patients without sample pooling.
First-passage human osteoblastic (hOB) cells from pri-
mary cultures of trabecular bone explants were used as
described elsewhere.(16,18) These hOB cells have been
shown to further differentiate under appropriate culture
conditions and represent an osteoblastic phenotype within
the matrix maturation phase.(16,18) The cells (plating den-
sity: 4000 cells/cm2) were grown in phenol red–free MEM
supplemented with 10% charcoal-stripped FCS (cs-FCS)
from Allgaeu BioTech Service (Goerisried, Germany) at
37°C. Cells were cultured in serum-free MEM supple-
mented with 0.125% (wt/vol) BSA for 4 days before RNA
isolation. Cultures were analyzed on day 16, 6 days after
they had reached confluence.
For dose–response experiments, the hOB cells were
treated with extract dilutions ranging from 10−9 to 10−5
compared with vehicle (ethanol) for a constant exposure
time of 48 h. For time-course experiments, the hOB cells
were treated with iCR at a 107-fold dilution for 0, 6, 12, 24,
48, and 72 h, respectively. To determine whether the stimu-
latory effects of iCR on OPG production are directly and
specifically mediated by the ER, we used a combination of
iCR (107-fold dilution for 72 h) and the pure ER antagonist
ICI 182,780 (for the last 48 h before RNA isolation). To
evaluate whether iCR may counteract the inhibitory effects
of glucocorticoids on OPG mRNA and protein produc-
tion,(19) hOB cells were treated with either vehicle, iCR (at
various concentrations for 72 h), or dexamethasone (10−8 M
for the last 24 h before RNA isolation).
RT-PCR analysis
Total RNA was isolated using the RNeasy total RNA
extraction kit from Qiagen (Hilden, Germany). The cDNA
was synthesized at 38°C for 1 h from 1 ␮g of total RNA in
a total volume of 40 ␮l containing 3 mM MgCl2, 75 mM
KCl, 50 mM Tris-HCl (pH 8.3), 10 mM DTT, 0.4 mM
dCTP, dGTP, dATP, and dTTP, respectively, 40 U RNase
inhibitor, 400 U Moloney murine leukemia virus (M-MLV)
reverse transcriptase, and 80 pM poly-dT15 primer from
Roche Molecular Biochemicals (Mannheim, Germany).
Aliquots of the total cDNA were amplified in each PCR
reaction in a 15-␮l reaction mixture containing 20 pmol of
5⬘ and 3⬘ primer each, 50 mM KCl (pH 8.3), 10 mM Tris-
HCl (pH 9.0), 1.5 nM MgCl2, and 0.02 mM dCTP, dGTP,
dATP, and dTTP, respectively, and 0.5 U Taq polymerase
(Roche Molecular Biochemicals). Each cDNA sample was
run in triplicate for each PCR reaction.(16)
Competitive RT-PCR was performed using exogenous
DNA competitors (mimics) as internal control that were
synthesized with the PCR mimic construction kit from
2038
TABLE 1. PRIMER SEQUENCES AND PCR CONDITIONS USED
Gene product Primer sequence
ER-␣-865 Sense
TGC TTC AGG CTA CCA TTA TGG A
ER-␣-988 Antisense
GTT TTT ATC AAT GGT GCA CTG GTT
ER-␤-724 Sense
TCA AAA GAG TCC CTG GTG TGA AG
ER-␤-849 Antisense
CTC TTT GAA CCT GGA CCA GTA ACA G
␤-actin-1031 Sense
CTG GCA CCC AGC ACA ATG
␤-actin-1098 Antisense
CCG ATC CAC ACG GAG TAC TTG
hPR-2024 (M15716) Sense
AAA AAC TGC CCA GCA TGT CG
hPR-2074 Antisense
GAC CAT GCC AGC CTG ACA G
hRANKL-530 (AF019047) Sense
CGT TGG ATC ACA GCA CAT CAG
hRANKL-580 Antisense
CAT GAG CCA TCC ACC ATC G
Clontech (Palo Alto, CA, USA). The ribosomal house-
keeping gene L7, OPG, bone-specific alkaline phosphatase
(BALP), and osteocalcin (OC) mRNA were analyzed as
reported elsewhere.(20) PCR products were analyzed by
agarose gel electrophoresis and visualized by ethidium bro-
mide staining under UV light. In all experiments, the ex-
pression of each gene was quantified as target to mimic
ratio and normalized to the ribosomal housekeeping gene,
L7. To ensure specificity of the PCR products, the ampli-
fication products were sequenced with the ABI Prism sys-
tem from Perkin Elmer (Weiterstadt, Germany).
Real-time quantitative RT-PCR for ER-␣, ER-␤
subtypes, progesterone receptor, and RANKL
To quantitate low expression of mRNA levels for steroid
hormone receptors (ER-␣, ER-␤ subtypes, and progester-
one receptor [PR]) and RANKL, real-time quantitative
RT-PCR was carried out using subtype-specific primers in
an ABI Prism 7000 sequence detection system (Applied
Biosystems, Foster City, CA, USA) as previously de-
scribed.(20) Primer sequences are summarized in Table 1.
PCR reaction was set up with Sybr Green PCR Mastermix
(Invitrogen, Karlsruhe, Germany) containing 0.3 ␮M prim-
ers each and 1 ␮l of RT product in a 25-␮L volume. A
two-step amplification protocol was chosen, consisting of
initial denaturation at 95°C for 2 minutes followed by 45
cycles with 15-s denaturation at 95°C and a 30-s annealing/
extension at 60°C. Finally, a dissociation protocol was per-
formed to control specificity of amplification products. For
time-course and dose experiments, expression of the cDNA
of interest was measured relative to the expression of the
housekeeping gene ␤-actin by the comparative threshold-
cycle (CT) method as described earlier.(20) Receptor/␤-actin
ratios were calculated for each time-point and dose, divided
VIERECK ET AL.
IN THIS STUDY FOR REAL-TIME PCR
PCR product size (bp) Annealing (°C) Reference
124 60 17
126 60 31
68 60 20
51 60
51 60
by receptor/␤-actin ratio of the respective control, and pre-
sented as percentage of control.
Protein assays
For the determination of total protein and total ALP
activity, the cells were lysed by repeated freeze-thawing
cycles and subsequent sonification. Lysates were processed
by centrifugation (10 minutes at 10,000g). The soluble pro-
tein fraction was quantified using the BioRad protein assay
with an albumin standard (Munich, Germany) by measur-
ing the release of p-nitrophenol from p-nitrophenol phos-
phate at 37°C and a pH of 10.5 as described by Boyan et
al.(21) Samples were measured in duplicates, and activity is
reported as nanomole per microgram DNA per minute in-
cubation time. Measurement of ALP activity had an intra-
assay CV of 3–5.4% using three identical samples in five
intra-assay experiments.
Measurement of OC secretion was performed in dupli-
cate with an immunoradiometric assay (Osteomedical,
Bünde, Germany). The CV for the OC assay was deter-
mined using three identical samples in 20 intra-assay and 20
interassay experiments. The detection range of the kit was
0.9–267 ng/ml with an intra-assay CV between 3.2% and
4.9% and an interassay CV of 4.0–8.9%.
For OPG protein measurement, conditioned medium
was harvested from cultured cells, centrifuged to remove
debris, and stored at −80°C until used. OPG protein secre-
tion was determined in triplicate measurements with an
immunoassay from Immundiagnostik (Bensheim, Ger-
many).(16) The OPG assay has a lower limit of detection of
0.5 pM. The intra-assay CV (measuring two samples 16
times) is between 8% and 10%, and the interassay CV
(measuring two samples 7 times) is between 12% and 15%.
BLACK COHOSH STIMULATES OSTEOBLASTIC OPG PRODUCTION 2039

Statistical analysis

Each of the experiments was reproduced at least three

times using first-passage cells from primary osteoblastic cul-
tures derived from individual donors. Values are expressed
as the mean ± SE of triplicate measurements of individual
hOB cultures, and data obtained from representative ex-
periments are shown. For analysis of time-courses and dose
responses, values were compared by multiple-measurement
ANOVA and corrected by Student-Newman-Keul’s test for
differences between groups. A p value of <0.05 was consid-
ered statistically significant. Standard software from Stat-
View 5.0 (SAS Institute, Cary, NC, USA) was used for
statistical analyses.

RESULTS

First, we analyzed the effects of isopropanolic extracts of

iCR on the expression of ER-␣, ER-␤, and PR mRNA. iCR
increased ER-␣ but not ER-␤ (Fig. 1A) mRNA expression.
PR mRNA increased dose- (Fig. 1B) and time-dependently
(Fig. 1C) after treatment with iCR.
To characterize the stimulatory effects of iCR on OPG
gene expression and protein secretion, dose responses and
time-courses were performed in primary first-passage hOB
cells along with negative controls (ethanol vehicle) and
positive controls (17␤-estradiol). iCR increased OPG
mRNA steady-state levels (as assessed by semiquantitative
RT-PCR) and protein secretion (as assessed by ELISA
measurement of conditioned medium; p < 0.0001 by
ANOVA for protein) in a dose-dependent manner (Figs.
2A and 2B). At the most effective dose of iCR (106-fold
dilution for 48 h), the induction of OPG mRNA levels and
protein concentrations was 2.5- and 2.9-fold, respectively.
These stimulatory effects of iCR were also similar to those
of 17␤-estradiol (Fig. 2B). In the hOB system, iCR in-
creased OPG mRNA levels as assessed by RT-PCR and
OPG protein secretion as assessed by an ELISA system in
a time-dependent fashion (Figs. 2C and 2D). The maximum
effect on OPG protein level occurred after 72 h and was
4.6-fold (and 2.5-fold at the gene expression level). Of note,
upregulation of OPG gene expression and protein secretion
by iCR was comparable with that induced by 17␤-estradiol
(Fig. 2D).
As shown previously, hOB cells display a characteristic
expression pattern of various osteoblastic lineage markers
(secretion of type 1 collagen and OC; expression of ALP)
under appropriate culture conditions, and therefore are
suitable for studying the effects of hormones and drugs on
osteoblastic differentiation.(16–18) Analysis of cellular mark-
ers of osteoblastic differentiation revealed that iCR en-
hanced ALP activity and OC secretion. Specifically, iCR
FIG. 1. Induction of ER-␣ and PR mRNA by extracts of iCR.
time- and dose-dependently stimulated BALP mRNA ex- (A) RT-PCR analysis of ER-␣ (left) and ER-␤ mRNA (right)
pression by up to 3-fold (Figs. 3A and 3C) and enzyme levels from hOB cultured with iCR or vehicle (V). The absolute
activity of total ALP by up to 4-fold (Figs. 3B and 3D), expression of ER-␣ vs. ER-␤ is >100:1 in the hOB model as as-
respectively (p < 0.0001 by ANOVA). iCR (106-fold dilu- sessed by PCR analysis.(17) (B) Dose response and (C) time-
course of iCR and 17␤-estradiol (10−8 M) exposure on PR mRNA.
tion) also led to an increase of OC mRNA by 2-fold (com- Numbers for iCR represent n-fold dilution (−log) of the iCR stock
pared with the 24-h control; p < 0.001 by ANOVA; Fig. solution. Numbers for 17␤-estradiol (E) represent the dose (in
4A). At the protein level, iCR time-dependently increased –log M). **p < 0.01, ***p < 0.001. E, estrogen.
2040
OC protein secretion by 2-fold (p < 0.0001 by ANOVA)
with a maximum effect after 48 h (Fig. 4B).
The iCR-induced enhancement of OPG mRNA levels
and protein secretion was dose-dependently abrogated by
co-treatment with the pure ER antagonist ICI 182,780 (by
59% for OPG protein at a dilution of 10−6 for 48 h; p < 0.01;
Fig. 5). Treatment with ICI 182,780 alone inhibited OPG
VIERECK ET AL.
FIG. 2. Dose- and time-dependent stimula-
tion of OPG production by iCR. (A) RT-
PCR analysis of OPG mRNA levels from
hOB cells cultured for 72 h with various
doses of iCR. Numbers for iCR represent n-
fold dilution (−log) of the iCR stock solution.
(B) OPG protein secretion (ELISA) from
hOB treated as described in A. (C) RT-PCR
analysis of OPG mRNA levels from hOB
cells cultured for the time indicated with iCR
(D) OPG protein secretion (ELISA) from
medium as described in A. *p < 0.05, **p <
0.01, ***p < 0.001. E, 17␤-estradiol.
FIG. 3. Stimulation of ALP activity by iCR.
(A) RT-PCR analysis of BALP mRNA lev-
els from hOB cells cultured for the time in-
dicated with iCR compared with vehicle. (B)
Total ALP activity of hOB cells cultured for
the time indicated with iCR. (C) RT-PCR
analysis of BALP mRNA levels from hOB
cells cultured for 72 h with various doses of
iCR. (D) Total ALP activity of hOB cells
cultured for 72 h with various doses of iCR.
Numbers for iCR represent n-fold dilution
(−log) of the iCR stock solution. *p < 0.05,
**p < 0.01, ***p < 0.001.
mRNA levels and protein production only weakly (by 11%
and 9%, respectively; p > 0.05 for protein). We also evalu-
ated whether iCR may counteract the inhibitory effects of
glucocorticoids on OPG mRNA and protein production.(19)
iCR dose-dependently abrogated the inhibitory effects of
dexamethasone on OPG mRNA and protein production
(Fig. 6).
BLACK COHOSH STIMULATES OSTEOBLASTIC OPG PRODUCTION
FIG. 4. Time-dependent stimulation of OC by iCR. (A) RT-
PCR analysis of OC mRNA levels from hOB cells cultured for
the time indicated with iCR compared with vehicle. (B) OC pro-
tein secretion measured by ELISA from medium as described in
A. *p < 0.05, **p < 0.01, ***p < 0.001.
DISCUSSION
In clinical practice, extracts from black cohosh are gain-
ing interest in the treatment of menopausal symptoms.(1,3)
However, whereas black cohosh extracts alleviated meno-
pause symptoms without affecting the endometrial thick-
ness of the uterus, its effects on biochemical markers of
bone metabolism were ambiguous, and the effects on BMD
or fracture reduction were not assessed.(7) Recent data
from animal studies on rats have suggested that extracts
from black cohosh partially protect against bone loss and
deterioration of biomechanical properties after ovariec-
tomy(4) and orchidectomy.(5)
In this study, we show that an isopropanolic extract (iCR)
is capable of enhancing the production of OPG by human
osteoblasts and increases osteoblastic differentiation mark-
ers OC and BALP. The increase of OPG production is
dose- and time-dependent, occurs at the gene expression
and protein levels, is substantial (up to 5-fold), and was
abrogated by the pure antiestrogen ICI 182,780. In addi-
tion, iCR prevented inhibition of osteoblastic OPG produc-
tion by glucocorticoids,(19) suggesting that iCR may coun-
teract the detrimental skeletal effects of glucocorticoids on
the endogenous antiresorptive cytokine OPG.
Both osteoblasts and osteoclasts express ER and are thus
2041
FIG. 5. The pure ER antagonist ICI 182,780 (ICI) prevented
stimulation of OPG by iCR. (A) RT-PCR analysis of OPG
mRNA levels from hOB cells cultured for 72 h with iCR, ICI, or
both. Numbers for iCR represent n-fold dilution (−log) of the iCR
stock solution. Numbers for ICI represent doses (−log M). (B)
OPG protein secretion measured by ELISA from hOB cells
treated as described in A. **p < 0.01, ***p < 0.001.
direct target cells of estrogen action.(8,9) The hOB cells used
in our experiments display a characteristic pattern of gene
expression and protein production of various osteoblastic
differentiation markers (type I collagen, BALP, and OC),
which are upregulated by phenotypic differentiation and
were stimulated by different agents such as dexamethasone
and vitamin D, indicating that they represent the mature
osteoblastic phenotype.(18) RT-PCR analysis of hOB cells
revealed marked ER-␣ and PR mRNA levels, whereas
ER-␤ mRNA levels were low and inconsistently present.
Therefore, these results indicate that iCR induces produc-
tion of the antiresorptive cytokine OPG by human osteo-
blasts that predominantly express ER-␣. This is consistent
with our previous studies that show enhancement of OPG
production under these cell culture conditions by other
2042
FIG. 6. ICR prevented glucocorticoid-induced inhibition of
OPG. (A) RT-PCR analysis of OPG mRNA levels from hOB
cells cultured for 72 h with iCR, dexamethasone (DEX), or both.
Numbers for iCR represent n-fold dilution (−log) of the iCR stock
solution. Numbers for dexamethasone (DEX) represent the dose
in –log M. (B) OPG protein secretion measured by ELISA from
hOB treated as described in A. **p < 0.01, ***p < 0.001.
skeletal ER agonists, including 17␤-estradiol,(15,16) raloxi-
fene,(17) and the phytoestrogen genistein.(16) Because OPG
production is a function of osteoblastic cell maturation, en-
hancement of OPG by iCR may, at least in part, be related
to the stimulatory effects on osteoblastic differentiation as
previously reported for other agents that stimulate OPG
production by hOB cells, including 17␤-estradiol,(15,16) ra-
loxifene,(17) genistein,(16) bisphosphonates,(22) and leptin.(23)
Of note, estrogen agonists have pleiotropic skeletal effects
and regulate a variety of osteoblast-derived paracrine fac-
tors and cytokines, including IL-1, IL-6, TNF-␣, macro-
phage colony-stimulating factor, prostaglandin E2, TGF-␤,
and OPG.(24) Thus, OPG regulation by iCR may represent
only one mechanism for its potential positive effects on the
skeleton.
VIERECK ET AL.
Estrogen replacement therapy has been shown to effec-
tively prevent bone loss and reduce fracture risk in post-
menopausal women.(25) However, because of the adverse
effects of exogenous estrogen administration, alternative
therapy options to conventional estrogen replacement
therapy are mandatory, especially for women who are at
increased risk for breast cancer or with a past history of
breast cancer.(1,26,27) Ideally, drugs used in these patients
should have a profile of a SERM with pronounced ER
agonistic effects on the skeleton, lack of ER agonistic ef-
fects on the uterus and vascular system, and ER antagonis-
tic effects on the breast.
Binding of ingredients of black cohosh to ER might be
one explanation for its bone protective activity.(28,29) The
observation of ER-binding, putative estrogen-agonistic ac-
tivity on bone tissue, but absence of stimulatory effects on
mammary and endometrial tissue, has led to the proposal to
classify black cohosh as a SERM. However, like many other
herbal extracts, iCR is a multicomponent drug with diverse
pharmacologically active substances. Recent publications
showed that growth of the ER+ breast cancer cell line
MCF-7 was inhibited through enhanced apoptosis by either
whole iCR extract or its main components (triterpene gly-
cosides and cinnamic acid esters) in vitro.(30,31) The absence
of proliferative effects on critical estrogen-responsive tis-
sues should stimulate more basic and clinical research(32)
into the question of whether iCR might qualify as a safe(r)
alternative to estrogen replacement therapy.(33) Interest-
ingly, preclinical findings suggest that dopaminergic or se-
rotoninergic activity may contribute to the capability of
black cohosh extracts to ameliorate menopausal symp-
toms.(29,34) Considerable efforts by the manufacturer to iso-
late the active ingredient(s) of black cohosh extracts are
ongoing (T Nisslein, unpublished data, 2005).
In summary, our findings indicate that isopropanolic ex-
tracts from black cohosh act on human osteoblastic cells to
increase the secretion of OPG, a potent inhibitor of bone
resorption. These in vitro data in conjunction with preclini-
cal animal data(4–6) and positive data from short-term ex-
posure in humans(7) provide a rationale to evaluate the
long-term skeletal and extraskeletal effects of black cohosh
extracts in postmenopausal women in randomized con-
trolled trials.
ACKNOWLEDGMENTS
We acknowledge the excellent technical assistance of H
Schulz, F Scheve, and U Niebergall. This work was sup-
ported by grants from Forschungsförderungsprogramm
2002 of the University of Göttingen to VV and from Deut-
sche Forschungsgemeinschaft Grants Ho 1875/3-1 to LCH
and Ho 1875/5-2 to LCH and MS.
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Address reprint requests to:
Volker Viereck, MD
Department of Obstetrics and Gynecology
University of Göttingen
Robert-Koch-Street 40
D-37075 Göttingen, Germany
E-mail: viereck@med.uni-goettingen.de
Received in original form April 26, 2005; revised form June 24,
2005; accepted July 13, 2005.