1. Onion decreases the ovariectomy-induced osteopenia in young adult rats
Tsang-Hai Huang a
, Roman C. Mühlbauer g
, Chih-Hsin Tang e
, Hsiun-Ing Chen b
,
Guan-Liang Chang c
, Yi-Wei Huang d
, Yu-Ting Lai d
, Hsin-Shi Lin a
,
Wei-Ting Yang c
, Rong-Sen Yang f,⁎
a
Institute of Physical Education, Health and Leisure Studies, National Cheng-kung University, No. 1, University Road, Tainan City 701, Taiwan
b
Department of Physiology, National Cheng-kung University, No. 1, University Road, Tainan City 701, Taiwan
c
Institute of Biomedical Engineering, National Cheng-kung University, No. 1, University Road, Tainan City 701, Taiwan
d
Department of Life Sciences, National Cheng-kung University, No. 1, University Road, Tainan City 701, Taiwan
e
Department of Pharmacology, School of Medicine, China Medical University, No. 91, Xue-shi Road, Taichung City 404, Taiwan
f
Department of Orthopaedics, National Taiwan University and Hospital, No. 7, Chung-Shan South Road, Taipei City 100, Taiwan
g
Bone Biology Group, Department of Clinical Research, University of Bern, Murtenstrasse 35, CH-3010 Bern, Switzerland
Received 21 July 2007; revised 15 November 2007; accepted 30 January 2008
Available online 29 February 2008
Edited by: Robert Recker
Abstract
It has been suggested that fruit and vegetable consumption are associated with good bone health. Onion, in particular, has been verified in its
efficacy in bone resorption activity. In this study, we further investigated the effects of an onion-containing diet on ovariectomy-induced bone
loss using methods of serum marker assay, histomorphometric analysis and biomechanical tests. Sixty-four female Wistar rats (14-week-old)
with sham operations or ovariectomy were assigned to 6 groups: CON, sham-operated control group; OVX, ovariectomized group; ALN,
ovariectomized rats treated with alendronate (1 mg/kg/day, p.o.); and 3% ON, 7% ON and 14% ON, ovariectomized rats fed with diets
containing 3%, 7% and 14% (wt/wt) onion powder, respectively. Animals were sacrificed after a six-week treatment course. In the serum marker
assay, alendronate and all three onion-enriched diets significantly decreased serum calcium level (pb0.05). Both 14% ON group and the ALN
group even showed similarly lower level of serum osteocalcin ( pb0.05), suggesting a down-regulation of bone turnover. The
histomorphometric analysis showed that ovariectomy markedly decrease bone trabeculae. The ALN and 14% ON rats were 80% and 46%
higher, respectively, in BV/TV than the OVX rats ( pb0.05), and the rats fed with onion-enriched food showed a lesser ovariectomy-induced
bone loss in a dose-dependent manner. Additionally, both ALN and 14% ON groups had significantly more trabecular number, less separated
trabeculae, and fewer osteoclasts ( pb0.05), but the protective efficacy from the 14% onion-enriched diet was slightly inferior to that of
alendronate. Ovariectomy also significantly decreased tissue weight and biomechanical strength in the OVX group ( pb0.05). The ALN and
14% ON groups equivalently showed a lesser decrease in tissue weight, though the difference was not significant. On the other hand, both the
ALN and 14% ON groups represented similar biomaterial properties of femurs, and both reduced the ovariectomy-induced decrease in bending
load and bending energy ( pb0.05). The present study further verified that an onion-enriched diet could counteract ovariectomy-induced bone
loss and deterioration of biomechanical properties.
© 2008 Elsevier Inc. All rights reserved.
Keywords: Osteoporosis; Phytotherapy; Alendronate; Osteoclast; Menopause
Introduction
The increasing incidence of osteoporosis and its related frac-
tures are important global health issues. Osteoporosis affects
about 75 million people in Europe, USA and Japan [1]. In
addition, one-third of women, as well as one-fifth of men over 50,
may develop osteoporotic fractures in their lifetimes [2–4]. It was
Bone 42 (2008) 1154–1163
www.elsevier.com/locate/bone
⁎ Corresponding author. Department of Orthopaedics, College of Medicine,
National Taiwan University and Hospital, No. 7, Chung-Shan South Road,
Taipei (100), Taiwan. Fax: +886 2 23936577.
E-mail address: rsyang@ntuh.gov.tw (R.-S. Yang).
8756-3282/$ - see front matter © 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.bone.2008.01.032

2. predicted that, by 2050, more than 50% of worldwide osteo-
porotic hip fractures would occur in Asia [5]. Among the
pharmacological treatments, hormone therapy (HT), selective
estrogen receptor modulators (SERMs) and bisphosphonate have
been shown to be effective either in increasing bone mineral
density (BMD) and/or reducing fracture rates [6]. However, the
side effects of these medications, including gastrointestinal
tolerance problems in bisphosphonate and the potential malig-
nancies in HT [7,8], may preclude their long-term use. Growing
evidence of the benefits of natural foods for bone health pro-
vide an alternative option for prevention and/or treatment of
osteoporosis.
Cross-sectional studies have shown that people, including
postmenopausal women, healthy women, and elderly men,
consuming more fruits and vegetables accumulated a higher
bone mineral density (BMD) [9–15]. In addition, increased fruit
and vegetable consumption showed benefits for bone health and
bone development in young people [15–17]. In animal studies,
it had been demonstrated that short-term treatment with diverse
fruits, vegetables and herbs could effectively reduce bone
resorption activity [18–20]. One recent study showed the effects
of 5%, 15%, and 25% prune-enriched diets in reversing bone
loss in young adult ovariectomized rats [21]. The 25% prune-
enriched diet even showed an equally protective effect on bone
volume ratio (BV/TV, %) as 17β-estradiol E2. Similarly, a
summary of animal studies strongly suggested the positive
effects of onion on good bone health. A short-term treatment
(10 days) with onion (0.03–1.5 g/rat/day) has been shown to
decrease the ovariectomy-induced bone resorption rate in a
dose-dependent manner [22]. A dose at 1 g/rat/day has even
served as a positive control treatment for down-regulating bone
resorption [19,20]. The aim of this study was to investigate
whether an onion-enriched diet could prevent estrogen
deficiency induced osteopenia. For this purpose, serum bone
markers and long bone biomechanical testing were measured
and tissue histomorphometry was performed to investigate the
dose-dependent bone protective effects of onion on 14-week-
old ovariectomized Wistar rats for a period of six weeks.
Materials and methods
Animals
Female Wistar rats (n=64) were housed under controlled conditions including
a room temperature of 22±1 °C with a 12:12 h light–dark cycle. All animals were
fed with standard Purina Rodent Chow 5001 (Labdiet®, Richmond, IN, U.S.A.)
containing 0.95% calcium and 1.07 % phosphate (wt/wt of dry food) and tap water
ad libitum. The procedures of the animal study, including the raising, feeding, and
the whole surgical process, followed the APS's “Guiding Principles in the Care and
Use of Animals” and were approved by the Committee of Animal Study at National
Cheng Kung University (Document Serial No. 940204).
Experimental design and treatments
The use of rats younger than 3 months is not suitable for ovariectomy due to the
concurrence of bone remodeling and bone growth [23]. Thus, we used adult virgin
female Wistar rats with an age of 14 weeks. One week before the ovariectomy or
sham operation, all animals (13 weeks old) were given demineralized water and a
diet of “semi-purified” diet powder (AIN-76A, TestDiet®, Richmond, IN, USA)
with animal protein (20% casein) containing 0.52% calcium and 1.12% phosphate.
Three days after surgery, sham-operated animals (n=8) were set as the control
group and ovariectomized rats (n=56) were randomly assigned to five groups
(Fig. 1): the OVX group (n=11), fed with the AIN-76A diet without any addition;
the ALN group (n=12), fed with the AIN-76A diet+alendronate (Calbiochem Co.,
Merck Taiwan Ltd, Taiwan) at a dose of 1 mg/kg b.w./day (p.o.), which has been
used by others in either growing or aged ovariectomized rats [24,25]; and three
onion treatment groups (n=11 for each), the 3% ON, 7% ON and 14% ON groups,
fed with an AIN-76A diet containing 3%, 7% and 14% (wt/wt) onion powder,
respectively. The onion powder (Chiseng®, Taipei, Taiwan), which was made from
fresh onion without any additives, is commercially available. The treatment course
for all groups was six weeks (Fig. 1).
The female Wistar rats ate, on average, 15–20 g/day of AIN-76A powder
according to the evaluation in the week before ovariectomy surgery. Since three or
four rats were housed per cage, we used ad libitum feeding. Thus, the diet for each
group was freshly prepared everyday and was given at a mean amount of 25 g per rat.
Serum bone marker assay
After the treatments were completed, the animals were sacrificed under deep
anesthesia with a high dose of sodium pentobarbital (intraperitoneally, 65 mg/kg)
and bloodletting via carotid arteries. Blood samples were collected and
centrifuged (4 °C) at 1500 ×g for 20 min. Serum was separated immediately
and was stored at −75 °C for future bone metabolic marker assay. Serum calcium,
and phosphorus were determined with an automatic chemistry analyzer
Fig. 1. Experimental design. Animals were fed semi-purified AIN-76A powder starting at 13 weeks old; sham or ovariectomy (OVX) operations were performed at
14 weeks old, treatments with onion diet or alendronate for each group were begun 3 days after surgery and maintained for 6 weeks. ALN, alendronate-treated group;
CON, control group; ON, onion-treated groups; OVX, ovariectomized group.
1155T.-H. Huang et al. / Bone 42 (2008) 1154–1163

3. (Beckman Synchron LX20, Diamond Diagnostics, Holliston, MA, USA).
ELISA kits, including RatTRAP™, Rat-MID™ and Rat/Mouse IGF-I (Nordic
Bioscience Diagnostics, Herlev, Demark), were used to analyze serum tartrate-
resistant acid phosphatase (TRAP) activity, osteocalcin, and insulin-like
growth factor-I (IGF-I), respectively. Assay procedures followed the instruc-
tions included in package of each kit. A microplate photometer (Multiskan
Ascent, Thermo Fisher Scientific, Inc., Waltham, MA, USA) was used to
measure the absorbance of each well after a series of sample preparations and
reactions.
Bone samples preparation
The left tibiae were removed, fixed with a 4% neutral paraformaldehyde in
PBS solution ( pH 7.4) for 48 h and then decalcified with a 10% ethylenediamine
tetraacetic acid (EDTA) solution (pH 7.4) at 4 °C for 4 weeks. After decal-
cification, each bone sample was cut along the coronal plane and embedded with
paraffin for further tissue section and histological staining. For geometric and
biomechanical measurement, the left femora were removed and the soft tissues
were cleaned. Then, the bone tissues were wrapped in 0.9% sodium chloride
soaked gauze and aluminum foil, and stored at −20 °C for future biomechanical
testing.
Bone histomorphometric analysis
Histological staining, including hemotaxylin and eosin (H & E) staining and
tartrate-resistant acid phosphates (TRAP) staining, was performed on two
nonconsecutive sections (5 μm) for each sample, which were the 1st and the 11th
sections in sequence. H & E staining was performed according to our previous
studies [26]. The histomorphometric study of the metaphysis of the proximal
tibiae was performed as previously described with image analysis software
(Image Pro Plus 6.1 for Windows; Media Cybernetics, Silver Spring, MD, USA)
[27]. Parameters measured included the bone volume ratio (BV/TV, %); mean
thickness of the trabeculae (Tb.Th, μm); trabecular number (Tb. N., 1/mm); and
trabecular separation (Tb. Sp., mm). TRAP staining with a standard kit (Sigma-
Aldrich Co., St. Louis, MO, USA) was used to determine the presence of
osteoclasts. The TRAP-positive multinucleated osteoclast along the metaphy-
seal trabecular bone surfaces were counted, and the number of osteoclasts per
unit trabecular tissue area (N. Oc/T. A, 1/mm2
) was measured as an index of
bone resorption [27].
Femur weight measurements
Before biomechanical testing, femora were thawed at room temperature and
weighed as wet weight. After the completion of biomechanical testing, the
fractured bone specimens were immersed in a solvent (2 vol of chloroform
combined with 1 vol of methanol) for 1 week and then dried at 80 °C for 24 h
[28]. The fat-free dry weight (FFDW) of each femur was then measured.
Biomechanical three-point bending testing and geometry measurements
Mechanical properties of long bone tissues were determined by a three-point
bending test [29]. The material testing system was designed by our lab with a
50 lb load cell (MDB-50, Transducer Techniques, Inc., Temecula, CA, USA),
which was connected to a data acquisition system (Model: iNstruNet, GW
Instruments, Inc. Somerville, MA, USA) in a personal computer. The span of the
two support points was 20 mm, the deformation rate was 0.05 mm/sec and
sampling frequency was 50 Hz. Load-displacement data were used for further
computing of the extrinsic material properties, including max load, ultimate load
to failure (fracture load), energy to maximal load, energy to fracture load, and
linear stiffness. The failure sites of all bone specimens were photographed under
a ×25 magnification by a high-resolution digital camera (COOLPIX 4500,
Nikon, Japan) [29]. Cross-sectional parameters, including cortical bone
thickness and cross-sectional area of cortical bone (CSA), were measured
from the photographs using the software Image Pro Plus 6.1 for Windows
(Media Cybernetics, Silver Spring, MD, USA). The cross-sectional moment of
inertia (CSMI) of the failure site was measured by the methods of Turner and
Burr for irregular cross-sections [30]. Based on the assumption that a bone cross-
section is made up of many rectangular elements (pixels), each with a height of h
and a width of w, the equation is shown as follows:
I ¼
Xn
i¼1
wh3
=12 þ whd2
i
À Á
where I is CMSI, n is the number of pixels, d is the distance from the center of
the element of the area to a given axis on the cross-section. All of these
parameters were obtained using the software Image Pro Plus 6.1 for Windows
(Media Cybernetics, Silver Spring, MD, USA).
Statistical analysis
The data are presented as means±SEM. For the data of serum markers,
histomorphometry and body weight gain, one-way analysis of variance
(ANOVA) was used to evaluate the differences among groups. For geometric
and biomechanical data, one-way analysis of covariance (ANCOVA) was used
to evaluate the differences among groups, and body weight data was set as a
covariate. When significant levels ( pb0.05) were revealed, pair-wise
comparisons between groups were made using the Fisher's least significant
difference (LSD) method. Power values regarding the size of groups were
calculated using α=0.05. Statistical analysis software, SPSS (11.5 version,
SPSS, Chicago, IL), was adopted for processing the data of the present study.
Results
Body weight gain (one-way ANOVA)
After surgery, body weight gain was typically and consistently
higher in the ovariectomized rats in each group when compared
with the sham-operated control rats (pb0.05) (Table 1). Except
for the slightly lower body weight gain of the ALN and 14% ON
group in the first week, the interventions we used, including
alendronate and three onion-enriched diets, did not significantly
affect body weight gain throughout the experimental period.
Serum bone markers (one-way ANOVA)
After the six-week treatment, the ALN and three onion-
enriched groups similarly showed significantly lower serum
calcium levels as compared to the CON and OVX groups
(pb0.05) (Table 2). In osteocalcin assay, the ALN group showed
Table 1
Body weight gain (gm) during the treatment period
CON OVX ALN 3% ON 7% ON 14% ON Power
1st week 13.9±
1.3c
35.0±
1.8a
28.8±
2.3b
32.1±
2.0ab
31.5±
1.5ab
28.0±
2.5b
1.000
2nd week 27.1±
2.9b
50.7±
3.3a
46.9±
3.6a
53.4±
2.9a
48.6±
3.0a
44.5±
4.5a
0.990
3rd week 32.1±
3.4b
60.5±
5.1a
58.3±
3.9a
65.5±
3.6a
65.0±
4.7a
58.7±
5.0a
0.994
4th week 36.8±
4.4b
67.9±
5.1a
63.8±
4.1a
71.2±
3.4a
61.7±
4.5a
61.6±
5.1a
0.991
5th week 36.1±
4.2b
76.8±
5.7a
73.2±
3.2a
79.5±
4.1a
67.5±
5.4a
66.9±
4.8a
1.000
6th week 44.9±
4.6b
81.5±
5.4a
76.3±
5.0a
79.5±
3.9a
68.2±
5.9a
75.1±
5.8a
0.984
Data were presented as mean±SEM. Power, power values calculated using
α=0.05; a–c
Mean±SEM values within a group not sharing a common super-
script are significantly different ( pb0.05).
1156 T.-H. Huang et al. / Bone 42 (2008) 1154–1163

4. the lowest value and was significantly lower than the OVX and
3% ON groups (pb0.05). The 14% ON groups also showed
significantly lower serum osteocalcin level when compared to the
3% ON group (pb0.05). The TRAP and IGF-I assays in both
ALN group and 14% ON group also showed a numeric lower
level as compared with CON group or OVX group, but not
reaching statistical significance.
Histomorphometry study (one-way ANOVA)
As shown in Figs. 2 and 3, the ovariectomy procedure caused
significantly less bone volume ratio (BV/TV, %), less trabecular
number (Tb. N., 1/mm), and increased trabecular separation
(Tb. Sp., mm) in the OVX group as compared with the CON
group ( pb0.05). In addition, the CON rats had a significantly
higher BV/TV than the rats in the ALN and three onion-
enriched diet groups ( pb0.05) (Figs. 2A and 3). Alendronate-
treated rats showed a significantly higher BV/TV as compared
to the rats in the OVX, 3% ON and 7% ON groups ( pb0.05),
but not the rats in the 14% ON group. The 14% ON group was
significantly higher in BV/TV than the OVX and 3% ON
groups ( pb0.05). Linear regression analysis showed that the
BV/TV data were significantly related to the ratio of onion-
contained in the diet (r=489, pb0.05) (Fig. 2B).
Further histomorphometric analysis of architecture para-
meters showed that trabecular sections were 14–17% thicker in
rats fed 7% and 14% onion-enriched diets than those in the ALN
group, but not at a significant level (Fig. 2C). Though the 14%
ON group was significantly less in trabecular number than the
CON and ALN groups, the rats in those three groups had
significantly higher trabecular number and less trabecular sepa-
ration as compared to the OVX, 3% ON and 7% ON groups
( pb0.05) (Fig. 2D and E). In osteoclast number per trabecular
area, the OVX group was significantly higher than the other five
groups ( pb0.05) (Fig. 2F).
Femur weight, geometry, and biomechanical testing
(one-way ANCOVA)
In order to eliminate the effects of body weight on size-
related parameters and extrinsic biomechanical properties, an
analysis of covariance was used to adjust and analyze the mean
values using body weight as a covariate [29]. The CON group
was significantly higher in femoral wet weight than the OVX,
3% ON and 7% ON groups ( pb0.05) (Table 3). The ALN and
the 14% ON groups were numerically higher in wet weight than
the three other ovariectomized groups, but did not show a
significant difference. The CON group was also significantly
higher in femoral fat-free dry weight (FFDW) than all
ovariectomized groups ( pb0.05). However, the ALN group
showed a smaller decrement of FFDW and was higher than the
OVX ( p=0.057) and 3% ON groups ( p=0.007). As compared
to the CON group, the five ovariectomized groups had lower
values for three cross-sectional geometric measurements,
ranging from −2.0% to −13.6% lower (Table 3), but showed
no statistical significance. Also, the ALN and 14% ON groups
showed less decrease in CSA, cortical thickness and CSMI as
compared with OVX group.
Regarding the femoral three-point bending test, the CON rats
showed the highest maximal load value and were significantly
different from rats in the OVX, 3% ON and 7% ON group
(Fig. 4A). In fracture load value, the CON group was also the
highest, but only significantly higher than the 3% ON group.
Treatments with alendronate and 14% onion-enriched diets could
prevent the deterioration of bending strength; the ALN and 14%
ON groups were significant higher in values of maximal load and
fracture load than the 3% ON group (pb0.05) (Fig. 4A and B).
Paralleling loading values, the CON group showed the highest
energy to maximal load and fracture load with significantly higher
data than those of the OVX group (pb0.05) (Fig. 4C and D).
Again, the alendronate and 14% onion-enriched diets respectively
preserved the properties of bone bending energy. The ALN group
showed significantly higher energy to maximal load than the
OVX and 3% ON groups, and significantly higher energy to
fracture load than the OVX group. On the other hand, the 14% ON
group also had a higher bending energy than the OVX group in
energy to maximal load (p=0.032) and fracture load (p=0.059),
though a statistically significant difference was not revealed in the
later measurement (Fig. 4D). In bending stiffness, the CON group
was significant higher than all ovariectomized groups (pb0.05)
except for the 14% ON group. The rats treated with 14% ON
showed a slightly higher stiffness and were significantly higher
than the 3% ON group (Fig. 4E).
Discussion
In this study we showed that onion-enriched diets could
counteract the ovariectomy-induced osteopenia in young adult
rats in a dose-dependent manner. The efficacies of the highest
dose of onion (14%) in preventing bone loss were similar to
those of alendronate treatment in many measurements.
Body weight gain
Previous work showed that onion is effective in intact male
rats and does not affect specific body weight [19,20]. A previous
study using rutin, an extract from onion, as a treatment showed
no effect on ovariectomy-induced weight gain [31]. Therefore,
there is no reason to assume that onion displays an estrogen-like
Table 2
Serum markers of bone metabolism
CON OVX ALN 3% ON 7% ON 14% ON Power
Calcium
(mg/dl)
9.68±
0.07a
9.43±
0.1a
8.96±
0.09b
8.90±
0.11b
8.83±
0.15b
8.83±
0.10b
1.000
Phosphorus
(mg/dl)
4.73±
0.25
5.17±
0.28
4.73±
0.42
5.68±
0.29
5.49±
0.35
4.85±
0.18
0.546
TRAP
(U/L)
1.84±
0.43
1.47±
0.12
1.55±
0.17
1.95±
0.12
1.68±
0.14
1.75±
0.19
0.314
Osteocalcin
(ng/ml)
123.4±
23.2bc
166.7±
17.0ab
109.2±
8.7c
199.3±
13.9a
150.6±
21.0abc
129.1±
19.8bc
0.927
IGF-I
(ng/ml)
931.9±
74.8
1020.2±
58.7
913.9±
46.9
1003.2±
78.4
976.2±
49.5
899.0±
65.6
0.221
Data were presented as mean±SEM. Power, power values calculated using
α=0.05;a–c
Mean±SEM values within a group not sharing a common superscript
are significantly different (pb0.05).
1157T.-H. Huang et al. / Bone 42 (2008) 1154–1163

5. Fig. 2. Histomorphometric analysis of proximal tibiae among different groups. A. bone volume ratio (BV/TV, %); B. linear regression between BV/TV and dietary onion consumption ratio; C. mean thickness of the
trabeculae (Tb.Th, μm); D. trabecular number (Tb. N., 1/mm); E. trabecular separation (Tb. Sp., mm). F. number of osteoclasts per unit tissue area (N. Oc/T. A, 1/mm2
). Ovariectomy caused much decrease of BV/TV
(%), and trabecular number, increased trabecular separation and number of osteoclasts. However, the ALN and 14% ON groups could counteract the ovariectomy-induced changes. Power, power values calculated using
α=0.05; a–d
Mean±SEM values within a group not sharing a common superscript are significantly different ( pb0.05).
1158T.-H.Huangetal./Bone42(2008)1154–1163

6. effect. In order to minimize the interference from body weight-
induced mechanical loading on bone, alendronate was chosen
as positive control treatment due to its nominal effect on
ovariectomy-induced body weight gain [32]. During the six-
week treatment period, all five ovariectomized groups showed
similar trends of body weight gain. Neither the alendronate, nor
the three different onion-enriched diets showed any obvious
modification in the increment of body weight gain induced by
ovariectomy and, thus, reduced the interference from body
weight on bone metabolism among different treated groups.
Serum bone markers
Except for serum TRACP 5b activity, the ALN group showed
lower levels in serum mineral level and markers of bone
formation. It has been previously verified that alendronate
prevents bone loss through down-regulating bone turnover [33–
35]. Since the high turnover may lead to loss of bone, such a
down-regulation of bone turnover may be beneficial to the bone
metabolism. The 14% ON group's equivalent results suggested
that a high onion-enriched diet could counteract bone loss through
a similar pathway. In contrast with previous studies investigating
the effects of dried plums [21,36], our serum markers seemed not
suggest enhanced bone formation activity from onion-enriched
diets. This might be due to the different status of subjects. Since
Arjmandi et al. and Deyhim et al. treated their subjects after a
period of estrogen deficiency [21,36], their studies demonstrated a
major effect of dried plums in bone restoration. However, our
subjects were treated immediately after ovariectomy. Moreover,
onion and dried plums might act on bone metabolism through
different pathways, which need to be further clarified.
Regarding bone resorption markers, TRACP 5b has been
proven as a sensitive marker for detecting bone resorption;
however, this sensitivity seemed to be limited within the initial
Table 3
Tissue weight, geometry measurements
CON OVX ALN 3% ON 7% ON 14%ON Power
Wet weight, mg 1165.2±40.2a
1047.5±33.5b
1109.0±32.6ab
1020.1±36.3b
1045.8±35.6b
1079.1±33.8ab
0.556
(−10.1%) (−4.8%) (−12.5%) (−10.2%) (−7.4%)
FFDW, mg 704.5±19.2a
594.3±16.0bc
638.6±15.6b
571.9±17.3c
593.5±17.0bc
603.7±16.1bc
0.992
(−15.6%) (−9.4%) (−18.8%) (−15.6%) (−14.3%)
CSA, mm2
6.40±0.28 5.81±0.23 6.09±0.22 5.77±0.25 5.86±0.25 6.20±0.23 0.290
(−9.2%) (−4.8%) (−9.8%) (−8.4%) (−3.1%)
Cortical bone thickness of the CS, mm 0.70±0.02 0.66±0.02 0.68±0.02 0.66±0.02 0.65±0.02 0.69±0.02 0.344
(−5.9%) (−2.1%) (−6.3%) (−7.8%) (−2.0%)
CSMI mm4
5.55±0.44 4.83±0.37 5.21±0.36 4.80±0.40 5.12±0.39 5.28±0.37 0.156
(−13.1%) (−6.2%) (−13.6%) (−7.8%) (−5.0%)
Data were presented as means±SEM. FFDW, fat-free dry weight; CSA, cross-sectional area of cortical bone in fracture site; CSMI, cross-sectional moment of inertia.
Data in parentheses were percentage differences relative to the CON group. Power, power values calculated using α=0.05; a–c
Mean±SEM values within a group not
sharing a common superscript are significantly different ( pb0.05).
Fig. 3. Histological sections of metaphysis of the proximal tibiae in rats of different groups. The ovariectomy caused serious loss of trabeculae in the proximal tibiae of
the OVX group, but alendronate and 14% onion-contained diet significantly protected bone from ovariectomy-induced osteopenia. An onion-containing diet preserved
more trabeculae close to the endo-cortical surface, especially in the 7% ON group. Trabeculae near by cortical bone were thicker (arrows) than those located in the
central of medullar canal (arrow heads); Bar, 1 mm.
1159T.-H. Huang et al. / Bone 42 (2008) 1154–1163

7. two weeks after ovariectomy or orchidectomy [37]. Since
TRACP 5b is released from osteoclasts, the serum activity of
TRACP 5b could be partially affected by the absolute number
of osteoclasts. Therefore, it was speculated that the serum
TRACP 5b activity turn back to sham level due to the decrease
in trabecular area and absolute osteoclast number as well [37].
This might be able to explain the inconsistency between the
serum TRACP activity and histological osteoclast number per
trabeculae area (N. Oc/T. A, Fig. 2F).
Histomorphometry analysis
Previous studies showed that onion at a dose of 1 g/rat/day
could inhibit bone resorption shown as about a 20% decrease of
Fig. 4. Evaluations of the midshaft femur biomechanical properties by using a three-point bending test. A. loading force to maximal load; B. loading force to tissue
fracture; C. energy to maximal load; D. energy to tissue fracture; E. bending stiffness of tissue. Ovariectomy significantly decreased the bending load, bending energy,
and stiffness as compared with controls. However, the ALN and 14% ON groups showed effects to counteract the deteriorations of these parameters. Power, power
values calculated using α=0.05; a–c
Mean±SEM values within a group not sharing a common superscript are significantly different ( pb0.05).
1160 T.-H. Huang et al. / Bone 42 (2008) 1154–1163

8. [H3
]-tetracycline urinary excretion [18–20]. Furthermore, a
higher dose of onion (1.5 g/rat/day) could blunt [H3
]-tetracycline
urinary excretion up to 26% in ovariectomized rats [22]. In the
present study, we further investigated the effects of onion on
ovariectomy-induced loss of trabecular bone tissue. As a positive
control treatment, alendronate significantly prevented trabecular
bone loss in bone volume ratio (BV/TV) and kept better archi-
tecture as compared to the OVX group (Figs. 2 and 3). However,
the dose of alendronate (1 mg/kg/day) used in the present study
could not fully counteract the effects of ovariectomy when
compared to control rats (Fig. 2).
On the other hand, the rats treated with onion-enriched diets
also showed a good preservation of BV/TV. A positive linear
relationship between BV/TV and onion-enrichment ratio sug-
gested the impact of onion is dose-dependent (Fig. 2A and B).
The highest onion-treated (14%) group showed similar effects to
the alendronate-treated group. Only the highest onion dose
prevented a decrease in the number of trabeculae (Fig. 2D).
Interestingly, the 7% ON and 14% ON groups both showed
numerically higher average trabecular thickness (Fig. 2C). Since
the trabeculae near the endo-cortical surface were thicker on
average than those located in the center of the medullar canal, the
preservation of more trabeculae in the central medullary canal in
the ALN rats made their thickness of trabeculae (Tb.Th) on
average slightly lower (Fig. 2C). A decreased number of TRAP-
positive osteoclasts in onion-treated groups and the ALN group
indicated that onion-enriched diets could act similarly to
alendronate through a pathway of bone resorption inhibition
(Fig. 2F). This decreased number of osteoclasts confirmed the
onion's anti-resorptive effects on bone, which has previously
been suggested [18–20]. The 3% ON group failed to show
preservation of BV/TV, which might be because the lower dose
of onion treatment was only able to inhibit osteoclastic activity
during the later phase of estrogen deficiency, when the bone
resorption rate was slower.
Tissue weight, geometry and strength
Typically, ovariectomy induced a body weight gain in the
present study. According to Frost's mechanostat theory [38], this
increment of body weight causes a higher loading on bone, thus
preserving bone strength to a certain extent. In addition, it has
been suggested that the ovariectomy-induced higher body weight
might partially compensate for the deterioration of bone quality
[39]. Several studies failed to reveal the ovariectomy-induced
loss of bone strength in the midshaft of the femur, which might be
due to the interference of this increased body weight [21,40,41].
Therefore, we compared the cross-sectional parameters and long
bone three-point bending data among groups using a one-way
analysis of covariance (ANCOVA) with body weight data as a
covariate. Again, alendronate, as well as a 14% onion-enriched
diet, better maintained bone quality in biomaterial properties
(Fig. 4A–D), and the 14% ON group even showed a slightly
higher stiffness than other ovariectomized groups (Fig. 4E).
On the other hand, the effect of ovariectomy on tissue weight
was relatively modest, and moreover, only numeric differences
were shown between the CON group and the OVX group in
cross-sectional cortex measurements (Table 3). Nevertheless,
the ALN and 14% ON groups still showed tendencies toward
minor decreases in tissue weight or cross-sectional cortex mea-
surements. Cross-sectional parameters, such as cortical thick-
ness, cortical area or moment of inertia, would contribute to long
bone mechanical properties [30]. However, the significant dif-
ferences shown in three-point bending test were not revealed in
cross-sectional measurements. This inconsistent data might
be explained by the following: (1) Geometric parameters in
midshaft femur were less affected by ovariectomy, which might
be due to the lack of a Haversian system and vessels in cortical
bone of young adult rats [42–44]; (2) According to the formula
of three-point bending, a certain amount of change in CSMI and
cross-sectional diameter would cause a fourfold change in
loading values of three-point bending when the tested sample
was an ideal cylinder with an equilibrium material property [30].
Therefore, a slight change in cross-sectional parameters would
cause significant variation in loading values; (3) Other than
geometric factors, it has been suggested that the strength of bone
tissue could also be related to compositional measures, such as
tissue density, collagen content or ash fraction, as well as the
microstructure parameters, including collagen cross-linking or
fiber orientation [45,46]. Further studies could be useful in
determining the preventive effects of an onion-enriched diet on
composition and microstructure of osteoporotic bone.
Possible mechanisms
Limited evidence was available to explain the actions of onions
and other vegetables and fruits. One possible mechanism of bone
restoration by those natural foods might be related to their base
excess [10,14], which is believed to retain calcium in bones.
However, Mühlbauer et al. showed that onion could enhance bone
mineral through a pharmacological inhibition on bone resorption
rather than its base excess [19]. The pharmacological inhibition
effects might be from various extracts of onion, including rutin,
and several phytoestrogens (coumestrol, isoflavones). These
extracts have also been proven to have individual benefits for
the bone of ovariectomy animals [31,47–49]. Recently, another
extract of onion, gamma glutamyl peptide, has been proven to
inhibit osteoclastic activity in vitro, and further animal study is
awaited [50]. Aside from the effects of a specific extract, the
synergistic effects of extracts from natural foods could be also
important. As noted by Mühlbauer [51], the working dose of rutin
used by Horcajada-Molteni et al. [31] is six times higher than its
contentin 1 g of onionand 700–800 times higher than its content in
1 g of leeks or wild garlic, which implies that those extracts might
be more effective when taken together, rather than separately.
Limitations and applicability in human diet habits
Animals in this study were kept in a controlled environment
and fed with a standard semi-purified diet, which is far from the
practice of real human life. Natural inhibitors contained in our
daily diets and various lifestyle factors (ex. alcohol consump-
tion, smoking etc.) would counteract the benefits from onion
and vegetables as well.
1161T.-H. Huang et al. / Bone 42 (2008) 1154–1163

9. Though we verified onion as another whole food that may
prevent bone loss in animal osteoporotic model, it would not
be applicable to suggest that people consume large amounts
of a specific fruit or vegetable as a strategy to prevent bone
loss. Notwithstanding those limitations mentioned above, there
are still ways and clues that encourage us to apply a potentially
anti-resorptive diet in our lives. Indirect evidence from human
studies has suggested that eating more fruits and vegetables
would benefit bone health [9–17]. Besides, plenty of vegetables
and herbs commonly consumed by humans have been men-
tioned about their similarly inhibitive effects on bone resorption
in animal studies [18–20,22]. Therefore, inclusion of an appro-
priate amount of these vegetables and herbs would keep variety
in the daily diet and could be an effective and more practical
way to decrease the incidence of osteoporosis in humans.
Conclusion
In summary, our study showed that high onion-enriched diets
are able to decrease ovariectomy-induced osteopenia and
deterioration of biomechanical strength. Further investigations
would be valuable in investigating the cellular mechanisms of
onion on bone homeostasis. Although it remains unclear how
those natural foods benefit bone health, increasing the intake of
fruits and vegetables might offer a potentially alternative way to
improve bone health.
Acknowledgments
This study was supported by a grant from the National
Science Council (NSC-94-2314-B-006-033, Taiwan). Deep
appreciation for English editing assistance from Miss Jae Cody.
References
[1] Who are candidates for prevention and treatment for osteoporosis?
Osteoporos Int 1997;7:1–6.
[2] Kanis JA, Johnell O, Oden A, Sembo I, Redlund-Johnell I, Dawson A. Long-
term risk of osteoporotic fracture in Malmo. Osteoporos Int 2000;11:669–74.
[3] Melton III LJ, Atkinson EJ, O'Connor MK, O'Fallon WM, Riggs BL. Bone
density and fracture risk in men. J Bone Miner Res 1998;13:1915–23.
[4] Melton III LJ, Chrischilles EA, Cooper C, Lane AW, Riggs BL. Perspective.
How many women have osteoporosis? J Bone Miner Res 1992;7:1005–10.
[5] Gullberg B, Johnell O, Kanis JA. World-wide projections for hip fracture.
Osteoporos Int 1997;7:407–13.
[6] Goltzman D. Discoveries, drugs and skeletal disorders. Nat Rev Drug
Discov 2002;1:784–96.
[7] Reid IR. Pharmacotherapy of osteoporosis in postmenopausal women:
focus on safety. Expert Opin Drug Saf 2002;1:93–107.
[8] Yeh IT. Postmenopausal hormone replacement therapy: endometrial and
breast effects. Adv Anat Pathol 2007;14:17–24.
[9] Chen YM, Ho SC, Woo JL. Greater fruit and vegetable intake is associated
with increased bone mass among postmenopausal Chinese women. Br J
Nutr 2006;96:745–51.
[10] New SA, Robins SP, Campbell MK, Martin JC, Garton MJ, Bolton-Smith
C. Dietary influences on bone mass and bone metabolism: further evidence
of a positive link between fruit and vegetable consumption and bone
health? Am J Clin Nutr 2000;71:142–51.
[11] Okubo H, Sasaki S, Horiguchi H, Oguma E, Miyamoto K, Hosoi Y.
Dietary patterns associated with bone mineral density in premenopausal
Japanese farmwomen. Am J Clin Nutr 2006;83:1185–92.
[12] Prynne CJ, Mishra GD, O'Connell MA, Muniz G, Laskey MA, Yan L.
Fruit and vegetable intakes and bone mineral status: a cross sectional study
in 5 age and sex cohorts. Am J Clin Nutr 2006;83:1420–8.
[13] Tucker KL, Chen H, Hannan MT, Cupples LA, Wilson PW, Felson D.
Bone mineral density and dietary patterns in older adults: the Framingham
Osteoporosis Study. Am J Clin Nutr 2002;76:245–52.
[14] Tucker KL, Hannan MT, Chen H, Cupples LA, Wilson PW, Kiel DP.
Potassium, magnesium, and fruit and vegetable intakes are associated with
greater bone mineral density in elderly men and women. Am J Clin Nutr
1999;69:727–36.
[15] Vatanparast H, Baxter-Jones A, Faulkner RA, Bailey DA, Whiting SJ.
Positive effects of vegetable and fruit consumption and calcium intake
on bone mineral accrual in boys during growth from childhood to ado-
lescence: the University of Saskatchewan Pediatric Bone Mineral Accrual
Study. Am J Clin Nutr 2005;82:700–6.
[16] McGartland CP, Robson PJ, Murray LJ, Cran GW, Savage MJ, Watkins
DC. Fruit and vegetable consumption and bone mineral density: the
Northern Ireland Young Hearts Project. Am J Clin Nutr 2004;80:1019–23.
[17] Tylavsky FA, Holliday K, Danish R, Womack C, Norwood J, Carbone L.
Fruit and vegetable intakes are an independent predictor of bone size in
early pubertal children. Am J Clin Nutr 2004;79:311–7.
[18] Muhlbauer RC, Li F. Effect of vegetables on bone metabolism. Nature
1999;401:343–4.
[19] Muhlbauer RC, Lozano A, Reinli A. Onion and a mixture of vegetables,
salads, and herbs affect bone resorption in the rat by a mechanism inde-
pendent of their base excess. J Bone Miner Res 2002;17:1230–6.
[20] Muhlbauer RC, Lozano A, Reinli A, Wetli H. Various selected vegetables,
fruits, mushrooms and red wine residue inhibit bone resorption in rats.
J Nutr 2003;133:3592–7.
[21] Deyhim F, Stoecker BJ, Brusewitz GH, Devareddy L, Arjmandi BH.
Dried plum reverses bone loss in an osteopenic rat model of osteoporosis.
Menopause 2005;12:755–62.
[22] Muhlbauer RC, Li F, Lozano A, Reinli A, Tschudi I. Some vegetables
(commonly consumed by humans) efficiently modulate bone metabolism.
J Musculoskelet Neuronal Interact 2000;1:137–40.
[23] Kalu DN, Liu CC, Hardin RR, Hollis BW. The aged rat model of ovarian
hormone deficiency bone loss. Endocrinology 1989;124:7–16.
[24] Azuma Y, Oue Y, Kanatani H, Ohta T, Kiyoki M, Komoriya K. Effects of
continuous alendronate treatment on bone mass and mechanical properties
in ovariectomized rats: comparison with pamidronate and etidronate in
growing rats. J Pharmacol Exp Ther 1998;286:128–35.
[25] Ito M, Azuma Y, Takagi H, Komoriya K, Ohta T, Kawaguchi H. Curative
effect of combined treatment with alendronate and 1 alpha-hydroxyvitamin
D3 on bone loss by ovariectomy in aged rats. Jpn J Pharmacol 2002;89:
255–66.
[26] Huang TH, Yang RS, Hsieh SS, Liu SH. Effects of caffeine and exercise on
the development of bone: a densitometric and histomorphometric study in
young Wistar rats. Bone 2002;30:293–9.
[27] Parfitt AM, Drezner MK, Glorieux FH, Kanis JA, Malluche H, Meunier PJ.
Bone histomorphometry: standardization of nomenclature, symbols, and
units. Report of the ASBMR Histomorphometry Nomenclature Committee.
J Bone Miner Res 1987;2:595–610.
[28] Honda A, Umemura Y, Nagasawa S. Effect of high-impact and low-
repetition training on bones in ovariectomized rats. J Bone Miner Res
2001;16:1688–93.
[29] Huang TH, Lin SC, Chang FL, Hsieh SS, Liu SH, Yang RS. Effects of
different exercise modes on mineralization, structure, and biomechanical
properties of growing bone. J Appl Physiol 2003;95:300–7.
[30] Turner CH, Burr DB. Basic biomechanical measurements of bone: a
tutorial. Bone 1993;14:595–608.
[31] Horcajada-Molteni MN, Crespy V, Coxam V, Davicco MJ, Remesy C,
Barlet JP. Rutin inhibits ovariectomy-induced osteopenia in rats. J Bone
Miner Res 2000;15:2251–8.
[32] Ogawa K, Hori M, Takao R, Sakurada T. Effects of combined elcatonin
and alendronate treatment on the architecture and strength of bone in
ovariectomized rats. J Bone Miner Metab 2005;23:351–8.
[33] da Paz LH, de Falco V, Teng NC, dos Reis LM, Pereira RM, Jorgetti V.
Effect of 17beta-estradiol or alendronate on the bone densitometry, bone
1162 T.-H. Huang et al. / Bone 42 (2008) 1154–1163

10. histomorphometry and bone metabolism of ovariectomized rats. Braz J
Med Biol Res 2001;34:1015–22.
[34] Frolik CA, Bryant HU, Black EC, Magee DE, Chandrasekhar S. Time-
dependent changes in biochemical bone markers and serum cholesterol in
ovariectomized rats: effects of raloxifene HCl, tamoxifen, estrogen, and
alendronate. Bone 1996;18:621–7.
[35] Iwamoto J, Seki A, Takeda T, Sato Y, Yamada H, Yeh JK. Comparative
effects of alendronate and alfacalcidol on cancellous and cortical bone
mass and bone mechanical properties in ovariectomized rats. Exp Anim
2006;55:357–67.
[36] Arjmandi BH, Khalil DA, Lucas EA, Georgis A, Stoecker BJ, Hardin C.
Dried plums improve indices of bone formation in postmenopausal
women. J Womens Health Gend Based Med 2002;11:61–8.
[37] Alatalo SL, Peng Z, Janckila AJ, Kaija H, Vihko P, Vaananen HK. A novel
immunoassay for the determination of tartrate-resistant acid phosphatase
5b from rat serum. J Bone Miner Res 2003;18:134–9.
[38] Frost HM. Bone “mass” and the “mechanostat”: a proposal. Anat Rec
1987;219:1–9.
[39] Peng ZQ, Vaananen HK, Zhang HX, Tuukkanen J. Long-term effects of
ovariectomy on the mechanical properties and chemical composition of rat
bone. Bone 1997;20:207–12.
[40] Cai DJ, Zhao Y, Glasier J, Cullen D, Barnes S, Turner CH. Comparative
effect of soy protein, soy isoflavones, and 17beta-estradiol on bone
metabolism in adult ovariectomized rats. J Bone Miner Res 2005;20:
828–39.
[41] Chachra D, Lee JM, Kasra M, Grynpas MD. Differential effects of
ovariectomy on the mechanical properties of cortical and cancellous bone
in rat femora and vertebrae. Biomed Sci Instrum 2000;36:123–8.
[42] Bagi CM, Ammann P, Rizzoli R, Miller SC. Effect of estrogen deficiency
on cancellous and cortical bone structure and strength of the femoral neck
in rats. Calcif Tissue Int 1997;61:336–44.
[43] Martiniakova M, Grosskopf B, Omelka R, Vondrakova M, Bauerova M.
Differences among species in compact bone tissue microstructure of
mammalian skeleton: use of a discriminant function analysis for species
identification. J Forensic Sci 2006;51:1235–9.
[44] Ruth EB. Bone studies. II. An experimental study of the Haversian-type
vascular channels. Am J Anat 1953;93:429–55.
[45] Friedman AW. Important determinants of bone strength: beyond bone
mineral density. J Clin Rheumatol 2006;12:70–7.
[46] van der Meulen MC, Jepsen KJ, Mikic B. Understanding bone strength:
size isn't everything. Bone 2001;29:101–4.
[47] Arjmandi BH, Alekel L, Hollis BW, Amin D, Stacewicz-Sapuntzakis M,
Guo P. Dietary soybean protein prevents bone loss in an ovariectomized rat
model of osteoporosis. J Nutr 1996;126:161–7.
[48] Draper CR, Edel MJ, Dick IM, Randall AG, Martin GB, Prince RL.
Phytoestrogens reduce bone loss and bone resorption in oophorectomized
rats. J Nutr 1997;127:1795–9.
[49] Ye SF, Saga I, Ichimura K, Nagai T, Shinoda M, Matsuzaki S. Coumestrol
as well as isoflavones in soybean extract prevent bone resorption in
ovariectomized rats. Endocr Regul 2003;37:145–52.
[50] Wetli HA, Brenneisen R, Tschudi I, Langos M, Bigler P, Sprang T. A
gamma-glutamyl peptide isolated from onion (Allium cepa L.) by bioassay-
guided fractionation inhibits resorption activity of osteoclasts. J Agric Food
Chem 2005;53:3408–14.
[51] Muhlbauer RC. Rutin cannot explain the effect of vegetables on bone
metabolism. J Bone Miner Res 2001;16:970–1.
1163T.-H. Huang et al. / Bone 42 (2008) 1154–1163