The effect of black cohosh extract and risedronate coadministration

The effect of black cohosh extract

and risedronate coadministration

on bone health in an

ovariectomized rat model

Amy L. Inselman

*, Elysia A. Masters

, Jalina N. Moore

Rajiv Agarwal

, Audrey Gassman

, Gemma Kuijpers

3‡

Richard D. Beger

, Kenneth B. Delclos

, Sybil Swift

5†

Luísa Camacho

, Michelle M. Vanlandingham

, Daniel Sloper

Noriko Nakamura

, Gonçalo Gamboa da Costa

Kellie Woodling

, Matthew Bryant

, Raul Trbojevich

Qiangen Wu

, Florence McLellen

and Donna Christner

Division of Systems Biology, National Center for Toxicological Research, U.S. Food and Drug

Administration, Jefferson, AR, United States,

Ofﬁce of New Drug Products, Ofﬁce of Pharmaceutical

Quality, Center for Drug Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD,

United States,

Division of Urology, Obstetrics and Gynecology, Center for Drug Evaluation and

Research, U.S. Food and Drug Administration, Silver Spring, MD, United States,

Division of Biochemical

Toxicology, National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson,

AR, United States,

Ofﬁce of Dieta ry Supplement Program, Center for Food Safety and Nutrition, U.S.

Food and Drug Administration, College Park, MD, United States,

Ofﬁce of the Center Director, National

Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, AR, United States,

Ofﬁce of Scien tiﬁc Coordination, National Center for Toxicological Research, U.S. Food and Drug

Administration, Jefferson, AR, United States

Preparations of black cohosh extract are sold as dietary supplements marketed to

relieve the vasomotor symptoms of menopause, and some studies suggest it may

protect against postmenopausal bone loss. Postmenopausal women are also

frequently prescribed bisphosphonates, such as risedronate, to prevent

osteoporotic bone loss. However, the pharmacodynamic interactions between

these compounds when taken together is not known. To investigate possible

interactions, 6-month-old, female Sprague-Dawley rats underwent bilateral

ovariectomy or sham surgery and were treated for 24 weeks with either

vehicle, ethinyl estradiol, risedronate, black cohosh extract or coadministration

of risedronate and black cohosh extract, at low or high doses. Bone mineral

density (BMD) of the femur, tibia, and lumbar vertebrae was then measured by

dual-energy X-ray absorptiometry (DEXA) at weeks 0, 8, 16, and 24. A high dose of

risedronate signiﬁcantly increased BMD of the femur and vertebrae, while black

cohosh extract had no signiﬁcant effect on BMD individually and minimal effects

upon coadministration with risedronate. Under these experimental conditions,

black cohosh extract alone had no effect on BMD, nor did it negatively impact the

BMD-enhancing properties of risedronate.

KEYWORDS

black cohosh, risedronate, bone mineral density, dietary supplements, bisphosphonates,

postmenopausal osteoporosis

OPEN ACCESS

EDITED BY

Patricia Rijo,

Lusofona University, Portugal

REVIEWED BY

Srinivasa Rao Sirasanagandla,

Sultan Qaboos University, Oman

Roberta Okamoto,

São Paulo State University, Brazil

*CORRESPONDENCE

Amy L. Inselman,

[email protected]

†

PRESENT ADDRESS

Sybil Swift, Scientiﬁc and Regulatory Affairs,

CBD Industries, LLC, Charlotte, NC,

United States

‡

Gemma Kuijpers, Retired, Laurel, MD,

United States

RECEIVED 03 January 2024

ACCEPTED 01 April 2024

PUBLISHED 16 April 2024

CITATION

Inselman AL, Masters EA, Moore JN, Agarwal R,

Gassman A, Kuijpers G, Beger RD, Delclos KB,

Swift S, Camacho LD, Vanlandingham MM,

Sloper D, Nakamura N, Gamboa da Costa G,

Woodling K, Bryant M, Trbojevich R, Wu Q,

McLellen F and Christner D (2024), The effect of

black cohosh extract and risedronate

coadministration on bone health in an

ovariectomized rat model.

Front. Pharmacol. 15:1365151.

doi: 10.3389/fphar.2024.1365151

Gassman, Kuijpers, Beger, Delclos, Swift,

Camacho, Vanlandingham, Sloper, Nakamura,

Gamboa da Costa, Woodling, Bryant,

Trbojevich, Wu, McLellen and Christner. This is

an open-access article distributed under the

terms of the Creative Commons Attribution

License (CC BY). The use, distribution or

reproduction in other forums is permitted,

provided the original author(s) and the

original publication in this journal is cited, in

accordance with accepted academic practice.

No use, distribution or reproduction is

permitted which does not comply with these

terms.

Frontiers in Pharmacology frontiersin.org01

TYPE Brief Research Report

PUBLISHED 16 April 2024

DOI 10.3389/fphar.2024.1365151

1 Introduction

Dietary supplements are often viewed as a safe alternative for the

prevention and treatment of disease, frequently leading to their

usage being under-reported to physicians. Combining dietary

supplements with prescription medications, however, may alter

the efﬁcacy, and even safety, of a medication. The present study

was designed to evaluate the potential pharmacodynamic

interactions of black cohosh extract, a dietary supplement

marketed to relieve the vasomotor symptoms of menopause, and

the FDA-approved osteoporosis drug risedronate, prescribed to

post-menopausal women to improve bone health.

Black cohosh extract is made from the roots and rhizomes of

Actaea racemosa L. (synonym Cimicifuga racemosa (L.) Nutt), a

perennial plant native to North America (Betz et al., 2009). Dietary

supplements containing black cohosh extract are available in a

variety of forms (dried extracts, liquid extracts, dried whole herb)

and vary widely in their chemical composition, with some

standardized on the ratio of herbal drug to native extract and

others to total triterpene glycoside content ( National Institutes of

Health, 2023). While there are no data available on the speciﬁc

number of individuals who take black cohosh extracts, it was the

20th ranked top-selling herbal supplement in 2021 (Smith et al.,

2022). Black cohosh has a long history of use for women’s

reproductive health (Foster, 1999), with numerous clinical studies

investigating whether it can alleviate the vasomotor symptoms of

menopause (Nappi et al., 2005; Osmers et al., 2005; Newton et al.,

2006; Wuttke et al., 2006; Bai et al., 2007; Geller et al., 2009; Leach

and Moore, 2012; Franco et al., 2016). While some studies indicated

a positive effect (lessening of symptoms), others indicated worsening

of symptoms or shown no beneﬁt over placebo. The inconsistent

reports on the efﬁcacy of black cohosh products may be due to the

variability of the test article used, as few include characterization or

standardization (Swanson, 2002). Adulteration of black cohosh

products with related species of Actaea has also been

documented (Foster, 2013).

In addition to providing relief from the vasomotor symptoms of

menopause, extracts of black cohosh have also been reported to protect

against postmenopausal bone loss. Qui and others demonstrated that

25-acetylcimigenol xylopyranoside (ACCX), a component isolated

from black cohosh, inhibited receptor activator of nuclear factor

kappa B ligand (RANKL)-induced osteoclast differentiation of

mouse bone marrow macrophages (Qiu et al., 2007). Black cohosh

extract has also been associated with decreased bone loss in the

ovariectomized (OVX) rat model, as well as increased bone mineral

density (BMD) and enhanced callus formation in a OVX rat tibia

fracture healing model (Nisslein and Freudenstein, 2003; Kolios et al.,

2010; Seidlová-Wuttke et al., 2012).

Risedronate sodium is one of several bisphosphonates approved

by the U.S. FDA for the treatment and prevention of osteoporosis in

postmenopausal women and in 2012 was the leading branded oral

bisphosphonate on the market in the U.S. (U.S. Securities and

Exchange Commission, 2012). Bisphosphonates prevent bone loss

by binding hydroxyapatite and inhibiting the bone resorbing action

of osteoclasts through inhibition of RANKL (Yasuda et al., 1998).

Nitrogen-containing bisphosphonates, such as risedronate, also

block osteoclast activity by inhibiting the enzyme farnesyl

pyrophosphate synthase (FPPS) (Kavanagh et al., 2006; Russell,

2011). It is possible that black cohosh extract and risedronate

both protect against bone loss; risedronate via binding

hydroxyapatite and inhibition of FPPS, and risedronate and black

cohosh extract via inhibition of RANKL-mediated osteoclast

differentiation. However, it is not known whether there are

pharmacodynamic interactions when taken together that could

impact the efﬁcacy of FDA-approved bisphosphonates.

Here we investigated the individual and combined effects of

black cohosh extract and risedronate at high and low doses, as well as

ethinyl estradiol as a positive control, on BMD in the OVX rat, an

established model of postmenopausal osteoporosis (Kalu, 1991).

Risedronate treatment increased BMD compared to the OVX-

vehicle controls, while black cohosh extract had no effect on

BMD when administered individually. When co-administered

with risedronate, black cohosh extract had some positive effects;

however, BMD increases were not signiﬁcantly different from

animals administered risedronate alone. Taken together, the

results suggest that black cohosh extract did not inhibit the

effectiveness of risedronate.

2 Materials and methods

2.1 Test compounds

Risedronate sodium (Cat. No. SML0650), ethinyl estradiol (Cat

No. E4876) and carboxymethylcellulose (CMC; Cat. No. C4888)

were purchased from Sigma-Aldrich (St. Louis, MO). A

standardized black cohosh dry extract (Cat. No. 398014; USA

sourced, water/ethanol extraction; total triterpene glycoside

content, 2.7%) was obtained from Euromed USA, Inc. (Presto,

PA) and was considered a representative market sample. Detailed

methods for test compound characterization/veriﬁcation and dose

certiﬁcation are provided in the Supplementary Data S1.

2.2 Animals and experimental design

Animal procedures were approved by the NCTR Institutional

Animal Care and Use Committee and followed the guidelines set

forth in the Care and Use of Laboratory Animals (National Research

Council, 2011). Animal rooms were maintained at 23

C±3

C with a

relative humidity of 50% ± 20% and a 12-h light/dark cycle. Animals

were housed in solid-bottom polysulfone cages with microisolator

tops. Millipore-ﬁltered tap water was provided in glass bottles with

silicone stoppers. Animals were maintained on 5K96 veriﬁed casein

diet 10 IF (LabDiet, St. Louis, MO), to minimize background

exposure to phytoestrogens; detailed analysis of isoﬂavone

measurements are provided in the Supplementary Data S1. Food

and water were provided ad libitum.

A total of 230 virgin female Sprague-Dawley rats were purchased

from Harlan Industries (Indianapolis, IN) and delivered at

approximately 3 months of age. At 6 months of age, animals were

assigned to treatment groups such that the mean initial weights were

comparable and underwent bilateral ovariectomy or a sham surgery.

In brief, surgery was conducted under isoﬂurane anesthesia. Bilateral

dorsal incisions were made to allow visualization of the viscera. The

ovaries were located, removed by cauterization, and incisions closed

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with wound clips. In the sham control group, surgery was conducted

as described, but ovaries were left intact. Animals recovered for

7 days before initial BMD assessment; dosing began the following

day and continued for 24 weeks (Figure 1A).

Animals were euthanized at 1 year of age by over-exposure to

carbon dioxide followed by exsanguination. Liver, kidney (paired),

and uterine weights were obtained. OVX animals were examined to

verify removal of ovarian tissue; three animals (n = 2 OVX-vehicle;

n = 1 low dose black cohosh extract/high dose risedronate) were

excluded from analysis, due to incomplete ovariectomy.

Four animals died during the study or were euthanized before

the scheduled euthanasia date due to health concerns. One of the

animals had a nephroblastoma, one had focal caseous

pleuropneumonia due to a gavage accident, and the cause of

morbidity in the other two animals could not be determined.

2.3 Dose selection and treatment groups

There was a total of 12 treatment groups with 18 animals

assigned to each group (Table 1).

Risedronate was solubilized in Millipore

-ﬁltered water and the

animals were dosed twice weekly (Monday and Thursday) by

subcutaneous injection at 1.5 or 5 μg/kg bw, modeling previous

bone pharmacology studies (Li et al., 1999; Yao et al., 2007; Cheng

et al., 2009; Uyar et al., 2009; Shahnazari et al., 2010).

Dry black cohosh extract was mixed with 0.5% aqueous CMC.

Animals were dosed daily by gavage at 10 or 100 mg/kg bw using an

automated Hamilton Microlab

pump (Hamilton Co., Reno, NV).

Black cohosh dose selection was based on literature reports

demonstrating a positive effect on bone with no reported toxicity

(Seidlová-Wuttke et al., 2003a; Kolios et al., 2010). Additionally, the

10 mg/kg bw dose is similar to the upper end of the suggested human

dose for treatment of menopausal symptoms (20–40 mg twice per

day) (Reagan-Shaw et al., 2008).

Ethinyl estradiol, was solubilized in 0.3% aqueous CMC and

administered daily by gavage at 2.5 or 15 μg/kg bw. Initially, doses of

10 and 100 μg/kg bw were selected, based on literature reports

demonstrating that oral concentrations of 30 μg/kg bw reversed

OVX-induced bone loss, with a dose as high as 100 μg/kg bw

showing no evidence of toxicity (Ke et al., 1997; Picherit et al.,

2000; Coelingh Bennink et al., 2008). However, after problems with

solubility and toxicity (i.e., weight loss) concentrations were adjusted

downward after three or four weeks of treatment.

2.4 Dual-energy X-ray absorptiometry

BMD of the femur, tibia, and L1-L4 lumbar vertebrae were

measured by DEXA at weeks 0 (one day prior to start of dosing), 8,

16, and 24 (one day prior to euthanasia). The DEXA instrument

(PIXImus, Lunar GE Medical Systems, Madison, WI) was calibrated

using the manufacturer’s phantom mouse. All animals were

anesthetized with isoﬂurane and placed on the DEXA tray in the

ventral position with the limbs held splayed. Each animal was

positioned to scan the right leg, then positioned to scan the spine

FIGURE 1

Study design to evaluate changes in bone quality in the ovariectomized rat, an established model of postmenopausal osteoporosis. (A) At 6 months

of age, female Sprague Dawley rats underwent sham or bilateral ovariectomy (OVX). Following a 7-day recovery period, animals were treated with vehicle

or test articles for 24 weeks. (B) Representative DEXA images show regions of interest in shaded red boxes used to quantify bone mineral density (BMD) for

the femur, lumbar vertebrae (L1-L4), and tibia. All images depicted are from the same rat. (C) Estradiol levels, measured by ELISA, were signiﬁcantly

greater in sham anima ls compared to OVX-vehicle animals (p < 0.0001, n = 12). The lower limit of detection in the assay was 3 pg/mL (dashed line) .

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area. Each region was scanned twice with repositioning of the animal

between scans. The software’s inclusion and exclusion areas were

used to outline the femur, tibia, and lumbar vertebrae regions of

interest (ROI) (Figure 1B). BMD data for each ROI were analyzed by

averaging the technical replicates. To limit repeated isoﬂurane

exposure, only a subset of animals were subjected to DEXA

scanning at weeks 8 and 16 (n = 10/group); all animals were

scanned at weeks 0 and 24.

2.5 ELISA assays

For measurement of estradiol levels and serum bone markers,

blood was collected at euthanasia by cardiac puncture into

Vacutainer

tubes (BD, Franklin Lakes, NJ). Samples were

centrifuged at 3,000 xgfor 10 min at room temperature; the

serum was aliquoted and stored at −80

C until use.

Serum estradiol was measured using a mouse/rat estradiol

ELISA kit (Cat. No. ES180S-100) from Calbiotech (Spring Valley,

CA) per the manufacturer’s instructions. Samples were read on a

Molecular Devices Spectramax M2 spectrophotometer (Sunnyvale,

CA) and analyzed with Softmax Pro 5 software. The lower limit of

detection (LOD) for estradiol was 3 pg/mL; samples below the LOD

were set to zero for analysis.

2.6 Statistical analysis

Differences between groups were analyzed using GraphPad

Prism Version 6.0 (GraphPad Software, Inc., LaJolla, CA).

Differences between multiple treatment groups at single

timepoints (body weight, organ weights, and BMD) were

evaluated using one-way ANOVA followed by Sidak’s post-hoc

test for multiple comparisons; comparisons included vehicle vs.

all other groups, low risedronate treatment vs. low/high black

cohosh extract + low risedronate treatment and high risedronate

treatment vs. low/high black cohosh extract + high risedronate

treatment. Difference in estradiol levels were evaluated using

unpaired t-tests. p values less than 0.05 were considered

signiﬁcant. Data are presented as means ± standard deviation

(SD), unless noted.

3 Results

3.1 Estradiol levels

Serum estradiol levels were quantiﬁed in a subset of animals

from the sham and OVX-vehicle groups. Three of the twelve sham

animals had estradiol levels below the LOD, while all twelve OVX-

vehicle animals had undetectable levels (Figure 1C). The average

estradiol concentration of the sham animals was signiﬁcantly greater

than the OVX-vehicle-treated group, which were 3.11 and 0 pg/mL,

respectively.

3.2 Body and organ weights

Body weights of animals treated with risedronate or black

cohosh extract, alone or with coadministration, did not

TABLE 1 Treatment groups and dosing.

Treatment group Compound(s) administered Concentration Route and Frequency of administration

1 Sham CMC 0.5% Oral Gavage; daily

2 Vehicle (OVX) CMC 0.5% Oral Gavage; daily

3 Lo EE2 Ethinyl Estradiol 2.5 μg/kg bw Oral Gavage; daily

4 Hi EE2 Ethinyl Estradiol 15.0 μg/kg bw Oral Gavage; daily

5 Lo Ris Risedronate 1.5 μg/kg bw Subcutaneous; biw

6 Hi Ris Risedronate 5.0 μg/kg bw Subcutaneous; biw

7 Lo BC Black Cohosh Extract 10 mg/kg bw Oral Gavage; daily

8 Hi BC Black Cohosh Extract 100 mg/kg bw Oral Gavage; daily

9 Lo BC + Lo Ris Black Cohosh Extract 10 mg/kg bw Oral Gavage; daily

Risedronate 1.5 μg/kg bw Subcutaneous; biw

10 Lo BC + Hi Ris Black Cohosh Extract 10 mg/kg bw Oral Gavage; daily

Risedronate 5.0 μg/kg bw Subcutaneous; biw

11 Hi BC + Lo Ris Black Cohosh Extract 100 mg/kg bw Oral Gavage; daily

Risedronate 1.5 μg/kg bw Subcutaneous; biw

12 Hi BC + Hi Ris Black Cohosh Extract 100 mg/kg bw Oral Gavage; daily

Risedronate 5.0 μg/kg bw Subcutaneous; biw

CMC, carboxymethylcellulose.

biw = twice a week.

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signiﬁcantly differ from OVX-vehicle controls. Estradiol and sham

surgery groups had lower mean body weights than OVX-vehicle

controls from week 0 through 24 (Figure 2A), with the difference

reaching statistical signiﬁcance at week 24 (Figure 2B).

Ovariectomy decreased uterine weights in all groups relative to

the sham controls (Figure 2C). However, a dose-related increase in

uterine weight was observed in the low and high ethinyl estradiol

treatment groups (1.7 and 2.8-fold increase vs. OVX-vehicle,

respectively). High ethinyl estradiol treatment and sham surgery

groups also showed increased liver and kidney weights, respectively,

at the time of sacriﬁce compared to OVX-vehicle controls

(Figures 2D, E).

3.3 BMD

Femur, vertebrae, and tibia BMD were measured at 0, 8, 16 and

24 weeks. Mean BMD for ethinyl estradiol, risedronate, black cohosh

extract and coadministration treatment groups were plotted

separately to visualize trends over time (Figure 3A). Longitudinal

BMD measurements revealed similar trends for femur and vertebrae

BMD measurements across treatment groups, while tibia BMD

trends were less apparent. In the femur and vertebrae, BMD of

ethinyl estradiol (red) and risedronate (blue) groups trend higher

than OVX-vehicle control but remain lower than sham surgery from

0 to 24 weeks. In contrast, treatment with black cohosh extract

(green) showed minimal changes in BMD compared to OVX-

vehicle control in the femur and vertebrae. Coadministration of

risedronate and black cohosh extract (purple) produced modest

changes in BMD of all ROIs compared to risedronate treatment

alone. Net changes in tibial BMD were minimal across all control

and treatment groups.

BMD at week 24 was normalized to baseline (week 0) for each

animal and plotted for each ROI (Figures 3B–D). Mean BMD

measurements of the femur, vertebrae, and tibia at weeks 0 and

24 are shown in Supplementary Tables S2-S4. Statistical analysis

conﬁrmed that sham surgery femurs had signiﬁcantly higher BMD

compared to OVX-vehicle (Figure 3B). In all high-dose risedronate

treatment groups, individually and when co-administered with

black cohosh extract, the BMD of the femur at week 24 was

signiﬁcantly greater compared to OVX-vehicle control. Black

cohosh extract alone had no statistically signiﬁcant effect on

femur BMD, as compared to OVX-vehicle control. Interestingly,

low risedronate when co-administered with high-dose black cohosh

extract showed a signiﬁcant increase in femur BMD as compared to

OVX-vehicle control, while low risedronate treatment alone had no

signiﬁcant effect. However, femur BMD in this high black cohosh

extract/low risedronate group was not signiﬁcantly different than

low-dose risedronate treatment alone (p = 0.6281).

Vertebral BMD followed similar trends as the femur (Figure 3C);

however, the magnitude of changes in BMD were the largest among the

three ROIs. The sham surgery group showed the highest mean vertebral

BMD at week 24 and was 1.3-fold greater than OVX-vehicle control.

Both ethinyl estradiol dose groups also had signiﬁcantly higher vertebral

FIGURE 2

Longitudinal body, terminal body, and absolute organ weight measurements. (A) Mean weekly body weight measurements are plotted for each

treatment group. Due to signiﬁcant reductions in body weight, doses of EE2 were adjusted downward to 2.5 and 15 μg/kg body weight per day after three

or four weeks of treatment for load one and load two animals. Animals in loads three and four only received the lowered doses. (B) Sham surgery and

EE2 treatment groups had signiﬁcantly lower body weight measurements at week 24, while all risedronate and black cohosh extract treatment

groups showed no signiﬁcant differences compared to OVX-vehicle control. (C–E) Uterine, liver and kidney weights at week 24 showed no signiﬁcant

differences associated with risedronate or black cohosh extract treatments compared to OVX-vehicle. Data plotted as mean ± SD. Signiﬁcance was

evaluated by one-way ANOVA with Sidak’s post-hoc for multiple comparisons. * Indicates difference vs. OVX-vehicle control; *p < 0.05, **p < 0.01, ***p <

0.001, ****p < 0.0001; n = 16–18. EE2 = ethinyl estradiol; BC = black cohosh extract; Ris = risedronate sodium; Veh = OVX-vehicle control.

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FIGURE 3

BMD of femur, vertebrae and tibia following treatment with black cohosh extract and/or risedronate. (A) Longitudinal measurement of BMD at 0, 8,

16, and 24 weeks of treatment is plotted to show the effects of EE2, risedronate, black cohosh extract and risedronate + black cohosh extract

combination treatments, with sham and vehicle data shown on each graph. Data plotted as mean ± SEM; n = 9–10. (B–D) BMD at week 24 was

normalized to week 0 for each animal. After normalization, signiﬁcant differences in BMD were associated with Hi EE2, Hi Ris and combined BC + Ris

treatments, as compared to vehicle control. No statistically signiﬁcant differences were observed between Lo Ris vs. Lo/Hi BC + Lo Ris or between Hi Ris

vs. Lo/Hi BC + Hi Ris groups. Data plotted as mean ± SD. Signiﬁcance was evaluated by one-way ANOVA with Sidak’s post-hoc for multiple comparisons. *

Indicates difference vs. OV X-vehicle control; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; n = 14–18. EE2 = ethinyl estradiol; BC = black cohosh

extract; Ris = risedronate sodium; Veh = OVX-vehicle control.

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BMDs at week 24 compared to the OVX-vehicle control. The vertebral

BMD of the high-dose risedronate group was signiﬁcantly larger (1.2-

fold greater) than the OVX-vehicle control, whereas the low-dose

risedronate treatment was statistically similar. There was no

signiﬁc ant effect on verteb ral BMD in the low or high-do se black

cohosh extract groups. Low risedronate co-administered with either low

or high doses of black cohosh extract led to statistically signiﬁcant

increases in vertebral BMD. Lastly, high-dose risedronate co-

administered with low or high doses of black cohosh extract also

showed signiﬁcant increases in vertebral BMD, as compared to

OVX-vehicle controls; however, the coadministration of black

cohosh extract and risedronate did not signiﬁcantly increase

vertebral BMD compared to corresponding doses of risedronate

alone (Lo BC + Lo Ris vs. Lo Ris, p = >0.9999; Hi BC + Lo Ris vs.

Lo Ris, p = 0.7238; Lo BC + Hi Ris vs. Hi Ris, p = 0.2805; Hi BC + Hi Ris

vs. Hi Ris, p = 0.9919).

Finally, normalized week 24 tibia BMD measurements showed very

few statistically signiﬁcant differences (Figure 3D). In the tibia, only low

black cohosh extract/high risedronate and high black cohosh extract/

low risedronate groups had statistically greater BMDs, as compared to

OVX-vehicle control. Again, tibial BMD values in the coadministration

groups were not statistically different from the corresponding doses of

risedronate alone (Lo BC + Hi Ris vs. Hi Ris, p = 0.9945; Hi BC + Lo Ris

vs. Lo Ris, p = 0.0588). Notably, the BMD in sham surgery tibias was not

signiﬁcantly different from OVX-vehicle controls (p = 0.2763), despite

being the highest of all groups at week 24.

Changes in serum bone markers, osteocalcin, bone-speciﬁc

alkaline phosphatase and C-terminal telopeptide, were also

measured as markers for bone metabolism at week 24. Overall,

there were no statistically signiﬁcant differences between control

and treatment groups (Supplementary Table S5, Figure S1).

4 Discussion

To ease the symptoms of menopause, many women turn to

dietary supplements with little consideration of the possible

unintended effects on the efﬁcacy of their prescription

medications. Here we investigated whether an extract of black

cohosh had any pharmacodynamic interactions with risedronate

effecting postmenopausal bone loss in the OVX rat model.

Ovariectomy decreased serum estradiol concentration, uterine

weight (−81.8%), and BMD of the right femur (−6.1%), lumbar

vertebrae (−19.5%) and right tibia (−1.4%) at 24 weeks, as expected.

Ethinyl estradiol was included as a positive reference control to

demonstrate suitability of the OVX rat model. Both low (2.5 μg/kg

bw) and high doses (15 μg/kg bw) were tested; the low dose was

expected to have minimal effects on bone loss and the higher dose

was expected to normalize bone parameters. While the high dose did

not completely normalize all biomarkers to the levels in sham

animals, ethinyl estradiol treatment raised BMD levels over that

of OVX-vehicle control animals in the femur, lumbar vertebrae, and

tibia. Increases in the lumbar vertebrae were statistically signiﬁcant.

Together the data conﬁrmed bone loss related to reductions in

estrogen and appropriateness and sensitivity of the model for

assessing interactions of black cohosh extract with risedronate.

Risedronate was selected for evaluation with black cohosh

extract due to its prevalent use to treat osteoporosis in

postmenopausal women and its demonstrated effectiveness in

increasing BMD in OVX rats (Li et al., 1999; Yao et al., 2007;

Cheng et al., 2009; Uyar et al., 2009; Shahnazari et al., 2010). Despite

variations in dosing, previous studies were consistent in ﬁnding

increased BMD, trabecular bone thickness, and volume of cortical

bone area with risedronate treatment. In this study, both the low and

high doses of risedronate had positive effects on BMD of the femur

and the lumbar vertebrae when compared to OVX-vehicle control.

As with ethinyl estradiol, BMD of the tibia was not signiﬁcantly

affected by risedronate treatment when compared to the OVX-

vehicle group. Neither dose of risedronate inﬂuenced body weight or

uterine weight, aside from the effects of ovariectomy, as expected

(Erben et al., 2002).

In contrast to the positive effectsobservedonBMDinresponseto

risedronate, black cohosh extract had no effect when given alone at high

or low doses. After 24 weeks of treatment, BMD of the femur, lumbar

vertebrae and tibia were statistically similar to OVX-vehicle control

animals. Additionally, there were no differences in serum bone marker

levels upon treatment with black cohosh extract. The lack of an impact

on bone health upon treatment with black cohosh extract contrasts with

others, which have previously reported positive associations with black

cohosh extract on BMD, urine markers of bone turnover and bone

morphometry (Seidlová-Wuttke et al., 2003b; Nisslein and

Freudenstein, 2003; Kolios et al., 2010).

Chronic black cohosh extract administration in OVX rats also

had no effect on body or uterine weights, which is consistent with

previous reports demonstrating the lack of estrogenic effects of black

cohosh extract. This ﬁnding was supportive of those in recent

reports by Seidlová-Wuttke et al. and the National Toxicology

Program, who also reported no effect of black cohosh extract on

uterine weights or pubertal development in rats and mice (Seidlová-

Wuttke et al., 2009; Mercado-Feliciano et al., 2012; Seidlová-Wuttke

et al., 2013).

Importantly, when co-administered with risedronate, black

cohosh extract did not counteract the BMD enhancing properties

of risedronate. Increases in BMD were observed with certain dosage

combinations but were dependent on the bone type measured. In the

vertebrae, coadministration of black cohosh extract and risedronate,

irrespective of the dose, had positive effects on BMD. Interestingly,

the addition of a high dose of black cohosh extract with the low dose

of risedronate produced a signiﬁcant increase in BMD compared to

vehicle in the femur and tibia, while BMD of animals administered a

low dose of risedronate alone was not signiﬁcantly different from

vehicle. This modest increase may suggest that black cohosh extract

enhances the bone-protective effects of low doses of risedronate.

However, this difference was not statistically signiﬁcant when

comparing the coadministration groups with the same dose of

risedronate treatment alone.

A consistent ﬁnding of this study was the varying patterns of

BMD response across speciﬁc bones measured. Speciﬁcally, we

found the greatest effect of OVX-induced bone loss and

treatment-related recovery in the vertebrae, then the femur, and

minimal changes in the tibia. It is known that postmenopausal bone

loss in humans and in the OVX rat model, is primarily attributed to

resorption of trabecular bone rather than cortical bone (Laib et al.,

2001; Shin et al., 2012). Therefore, bones with higher composition of

trabecular bone, such as the vertebrae, will exhibit increased rates of

bone remodeling compared to those composed of more cortical

Frontiers in Pharmacology frontiersin.org07

Inselman et al. 10.3389/fphar.2024.1365151

bone, such as the diaphysis of long bones (Thompson et al., 1995;

Shin et al., 2012). In addition to composition, bone size and

mechanical loading will inﬂuence rates of remodeling, which

explains the larger changes observed in the femur versus tibial BMD.

While changes in bone strength (i.e., mass, stiffness) or quality

cannot be ruled out, as they were not evaluated in this study, BMD is

generally regarded as a good predictor of fracture risk and bone

health (Uyar et al., 2009). Further, DEXA scanning and

measurement of serum biomarkers are common, non-invasive

and clinically relevant analyses for osteoporosis screening in

postmenopausal women. It is important to note that, although

the rat has been useful for predicting effects in humans,

differences in human and rat bone physiology exist, which may

limit the translation of the effects observed to postmenopausal

women. For example, unlike humans, rats do not develop

spontaneous bone fractures in response to declining estrogen

levels. Additionally, the bones of rats do not stop growing and

lack a well-developed Haversian-based remodeling system that

occurs in human cortical bones (Lelovas et al., 2008).

Importantly, due to the lack of characterization and/or

standardization for many preparations of black cohosh, the results

and conclusions drawn from this study are limited to the speciﬁc

water/ethanol extract of black cohosh used and the dose regimen

described. The extraction method has been shown to play a critical

role in the chemical proﬁle and biological activity of black cohosh extract

(Jiang et al., 2008). While our study utilized a water/ethanol extract,

many of the previous studies in the OVX rat model used an iso-

propanolic extract. Comparison of extraction methods have found that

the ethanolic and iso-propanolic extracts are quantitatively different in

their triterpene glycoside and polyphenolic composition (Jiang et al.,

2008) and thus, may explain some of the differences observed in BMD

between studies. The age of the animal at ovariectomy may also have

affected the observed BMD response upon treatment with black cohosh

extract. Ovariectomy in this study was performed when the animals were

6 months of age, opposed to 3 months of age in previous studies

(Seidlová-Wuttke et al., 2003b; Nisslein and Freudenstein, 2003;

Kolios et al., 2010). Yousefzadeh and others have suggested that for

osteoporosis research the preferred age for ovariectomy in rats is

6–9monthsold(Yousefzadeh et al., 2020). Animals in this age-range

have fewer confounding effects of age-related changes in bone growth.

The present study demonstrates that alone the water/ethanol

standardized extract of black cohosh tested did not affect BMD or

serum bone biomarker levels in an OVX rat model for osteoporosis.

When given in combination with risedronate, black cohosh extract

did not negatively impact the positive BMD-enhancing properties of

the oral bisphosphonate. While increases in BMD was observed in

select combinations and bone regions, the increases were not

signiﬁcantly different than those of risedronate alone. Taken

together, there does not appear to be any pharmacodynamic

synergistic effects of the FDA-approved drug risedronate and the

dietary supplement black cohosh.

Data availability statement

The original contributions presented in the study are included in

the article/Supplementary Material, further inquiries can be directed

to the corresponding author.

Ethics statement

The animal study was approved by the NCTR Institutional

Animal Care and Use Committee. The study was conducted in

accordance wit h the loc al legislation and institutional

requirements.

Author contributions

AI: Conceptualization, Data curation, Formal Analysis,

Supervision, Writing–original draft, Writing–review and editing.

EM: Data curation, Formal Analysis, Writing–original draft,

Writing–review and editing. JM: Formal Analysis,

Writing–review and editing. RA: Conceptualization,

Writing–review and editing. AG: Conceptualization,

Writing–review and editing. GK: Conceptualization,

Writing–review and editing. RB: Conceptualization,

Writing–review and editing. KD: Conceptualization,

Writing–review and editing. SS: Conceptualization,

Writing–review and editing. LC: Conceptualization, Investigation,

Writing–review and editing. MV: Investigation, Writing–review and

editing. DS: Investigation, Writing–review and editing. NN:

Investigation, Writing–review and editing. GG: Investigation,

Writing–review and editing. KW: Investigation, Writing–review

and editing. MB: Investigation, Writing–review and editing. RT:

Investigation, Writing–review and editing. QW: Investigation,

Writing–review and editing. FM: Investigation, Writing–review

and editing. DC: Conceptualization, Writing–review and editing.

Funding

The author(s) declare ﬁnancial support was received for the

research, authorship, and/or publication of this article. This project

was supported by the FDA’s Chief Scientist Challenge Grants (OCS-

challenge) under protocol NCTR E-0758301.

Acknowledgments

The authors would like to acknowledge Dr. Deborah K. Hansen,

a retired employee of the NCTR, for her contributions to the study.

Dr. Hansen was involved in study design, primary data analysis and

drafting of the manuscript. The authors would also like to

acknowledge Dr. Ikhlas Khan at the University of Mississippi for

his assistance in obtaining the black cohosh test article. In addition,

the authors recognize the tremendous support of the NCTR animal

care staff, especially Florene Lewis. The authors also acknowledge

Andy Matson with the NCTR Diet Preparation Group for his

assistance in preparing the dose formulations, Patricia Porter-Gill

in the Division of Biochemical Toxicology for her assistance in the

DEXA data analysis, and Ralph Patton and Kristie Voris with TPA

Inc. for their assistance with measuring estradiol levels. A special

thank you to Tom Schmitt and Lisa Pence for their efforts to develop

an LCMS assay to measure risedronate and to Robert Felton in the

Ofﬁce of Scientiﬁc Coordination for his advice on the

statistical analysis.

Frontiers in Pharmacology frontiersin.org08

Inselman et al. 10.3389/fphar.2024.1365151

Conﬂict of interest

The authors declare that the research was conducted in the

absence of any commercial or ﬁnancial relationships that could be

construed as a potential conﬂict of interest.

Publisher’s note

All claims expressed in this article are solely those of the authors

and do not necessarily represent those of their afﬁliated

organizations, or those of the publisher, the editors and the

reviewers. Any product that may be evaluated in this article, or

claim that may be made by its manufacturer, is not guaranteed or

endorsed by the publisher.

Supplementary material

The Supplementary Material for this article can be found online

at: https://www.frontiersin.org/articles/10.3389/fphar.2024.1365151/

full#supplementary-material

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