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Chinese Medicine
Open Access
Research
The effects of Rhizoma Curculiginis and Rhizoma Drynariae extracts
on bones
Ricky WK Wong*, Bakr Rabie, Margareta Bendeus and Urban Hägg
Address: Biomedical and Tissue Engineering Research Group, University of Hong Kong, Prince Philip Dental Hospital, 34 Hospital Road, Sai Ying
Pun, Hong Kong SAR, China
Email: Ricky WK Wong* - fyoung@hkucc.hku.hk; Bakr Rabie - rabie@hkusua.hku.hk; Margareta Bendeus - sambende@hkusua.hku.hk;
Urban Hägg - euohagg@hkusua.hku.hk
* Corresponding author
Abstract
Background: Rhizoma Curculiginis (Xianmao) and Rhizoma Drynariae (Gusuibu) are 'Yang-tonifying'
traditional Chinese herbal medicines used to strengthen bones. This investigation aims to assess
the systemic effect of extracts of Rhizoma Curculiginis and Rhizoma Drynariae on bone
histomorphology and formation, and their local effect on bone healing.
Methods: For the investigation of the systemic effect, thirty 8-week-old male BALB/c mice were
divided into three groups: (1) control group, ten mice fed daily with distilled water; (2) Rhizoma
Curculiginis group, ten mice fed daily with distilled water mixed with Rhizoma Curculiginis extract; (3)
Rhizoma Drynarie group, ten mice fed daily with distilled water mixed with Rhizoma Drynarie extract.
The mice were fed for five weeks before sacrifice. Twenty micro-tomographic slices with an
increment of 0.25 mm were prepared to cover the proximal end of the left tibia of each mouse.
Quantitative morphometry of the bone structure was performed. For the investigation of the local
effect on bone healing, two bone defects (5 × 10 mm) were created in the parietal bone of each of
the three New Zealand white rabbits. Two defects in the first animal were grafted with collagen
matrix with Rhizoma Curculiginis extract; two defects in the second animal were grafted with
collagen matrix with Rhizoma Drynarie extract; two defects in the third (control) animal were
grafted with collagen matrix alone. The animals were sacrificed on day 14 and the defects were
dissected and prepared for histological and ultrastructural assessment.
Results: Rhizoma Curculiginis and Rhizoma Drynariae extracts altered the bone histomorphology,
both increasing the trabecular number by 10% (P = 0.002). Rhizoma Curculiginis extract increased
bone density by 3.13% (P = 0.122) and Rhizoma Drynariae extract increased bone density by 6.45%
(P = 0.005). Both Rhizoma Curculiginis and Rhizoma Drynariae extracts induced new bone formation
on the margins of the defects.
Conclusion: Two 'Yang-tonifying' herbs, Rhizoma Curculiginis and Rhizoma Drynariae, were
demonstrated to have systemic effects on bone histomorphology and formation as well as local
bone healing.
Published: 19 December 2007
Chinese Medicine 2007, 2:13 doi:10.1186/1749-8546-2-13
Received: 2 May 2007
Accepted: 19 December 2007
This article is available from: http://www.cmjournal.org/content/2/1/13
© 2007 Wong et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0
),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Chinese Medicine 2007, 2:13 http://www.cmjournal.org/content/2/1/13
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Background
For thousands of years, traditional Chinese medicine has
been used to treat bone diseases and to promote bone
healing [1]. According to Chinese medicine theories,
herbs that are 'Yang-tonifying' and act on Shen (kidney)
strengthen bones. Shen, in traditional Chinese medicine,
is related to bones [1]. These effects had not been demon-
strated by modern scientific methods; thus, we intended
to investigate whether the crude extracts of these herbs can
indeed increase bone formation. New leads of therapeutic
chemicals may provide possibilities for treating oste-
oporosis, reduce bone resorption after bone grafting sur-
gery or promote bone healing after bone surgery or
fracture.
One of the 'Yang-tonifying' herbs for strengthening bones
is Rhizoma Curculiginis (Xianmao), the dried rhizome of
Curculigo orchioides Gaertn. (Amaryllidaceae) [2]. The
'Yang-tonifying' effect of Rhizoma Curculiginis is hormone-
like. Studies show that oral administration of 10 g/kg of
the decoction of Rhizoma Curculiginis significantly
increased the weight of the lobus anterior hypophysis,
ovary and uterus in rats. Rhizoma Curculiginis can potenti-
ate the luteotrophic activity of the hypothalamus-pitui-
tary-ovary system [3]. In traditional Chinese medicine,
Rhizoma Curculiginis is used to reinforce the Yang in Shen
to treat impotence, limpness of the limbs, arthritis of the
lumbar and knee joints, and to strengthen tendons and
bones [2]. Current research mainly focuses on identifying
the chemical components of this herb [4,5]. Thirteen
cycloartane triterpene saponins named curculigosaponins
A to M were isolated and identified in Rhizoma Curculiginis
[6,7]. Two other triterpenes, curculigol and 31-methyl-3-
oxo-20-ursen-28-oic acid, were also isolated from Rhi-
zoma Curculiginis [8,9]. Moreover, phenyl glycosides cur-
culigoside B, curculigines B and C, and an aliphatic
compound 25-hydroxy-33-methylpentatricontan-6-one
were identified in Rhizoma Curculiginis [7,9].
Rhizoma Drynariae (Gusuibu), the dried rhizome of peren-
nial pteridophyte Drynaria fortunei (Kunze) J. Sm. (Poly-
podiaceae), is another 'Yang-tonifying' herb for treating
bone diseases [2]. According to traditional Chinese medi-
cine theories, Rhizoma Drynariae acts on Shen and Gan
(liver) and is used for replenishing Shen, treating defi-
ciency syndrome of the kidney and promoting the healing
of fracture and relieving pain; it is also used in treating
traumatic injuries and bone fracture [2]. The extract of
Rhizoma Drynariae which contains flavonoid and triterpe-
noid compounds was shown to increase bone cell viabil-
ity, intracellular total proteins, alkaline phosphatase and
acid phosphatase [10]. Naringin, a major flavonoid com-
ponent, went up by 1%. The triterpenes isolated from Rhi-
zoma Drynariae include 24-ethyl-9, 19-cyclolanost-25-en-
3; 3-ol, hop-22(29)-ene and fern-9(11)-ene [2]. Our lab-
oratory recently demonstrated that naringin, a flavonoid
component increased new bone formation locally [11].
Micro-computed tomography (micro-CT) permits non-
invasive imaging and quantitative morphometry of the
bone structure in two and three dimensions [12,13]. For
visualizing trabecular architecture, micro-CT is better than
conventional methods of histological sectioning, e.g. pla-
nar radiography and medical computed tomography [14].
Micro-CT can produce images with resolution in tens of
microns to show micro-architecture of bone and bone
grafts [15,16]. Micro-CT was commonly used for studying
the effects of different agents on bone histomorphology
[17,18]. By using mice as an animal model [19], it is pos-
sible to compare the systemic effect of different agents on
bones with normal mice as control. However, for studying
the local effect of different agents on the healing of bone
defects, it is necessary to use other animal models (e.g.
rabbits) that allow histological examination of bones and
other tissues surrounding the agents and to utilize a carrier
that allows the release of these agents. The present study
aims to investigate the systemic effect of crude extracts of
Rhizoma Curculiginis and Rhizoma Drynariae on bone his-
tomorphology in normal mice using micro-CT and the
local effect of these extracts on a bone defect in rabbits.
Methods
Identification and preparation of Rhizoma Curculiginis
and Rhizoma Drynariae extracts
Rhizoma Curculiginis and Rhizoma Drynariae were pur-
chased in a local Chinese medicine store and were identi-
fied morphologically, histologically and chemically
according to standard Chinese herbal identification pro-
cedures [20,21]. Initially, the morphology and histology
of the herbs were compared with standard photographs
obtained from the School of Traditional Chinese Medi-
cine, the University of Hong Kong. The actual identifica-
tion procedures were performed by the authors in the
Hard Tissue Laboratory, the University of Hong Kong.
Thin layer chromatography was used to separate the com-
ponents of Rhizoma Curculiginis ethanolic extract. Potas-
sium ferrocyanide (2%) and ferric chloride were added to
the extract, which produced bluish spots (compared with
the standards), an indication of the authenticity of the
herb [21]. Methanolic extract of Rhizoma Drynariae was
used for thin layer chromatography. The extract was sepa-
rated by benzene-methanol-butanone (at a ratio of 3:1:1)
and tested with ferric chloride-ethanol solution (1%).
Brownish colored extract was compared with naringin
standard [20]. A sample of each herb was stored in the
Hard Tissue Laboratory. Rhizoma Curculiginis and Rhizoma
Drynariae extracts were prepared according to the protocol
for commercial production of injection preparation of tra-
ditional Chinese medicine in China [22]. For every 4 g of
Rhizoma Curculiginis or Rhizoma Drynariae powders, 40 ml
Chinese Medicine 2007, 2:13 http://www.cmjournal.org/content/2/1/13
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of distilled water was added and the mixtures were boiled
with stirring on a hot plate for 4 hours. Distilled water was
added occasionally to prevent the mixtures from drying.
The final volume of the mixtures was made up to 4 ml by
adding distilled water. The mixtures were cooled to room
temperature and then centrifuged. The supernatants were
collected and filtered with a 0.22 μm sterile syringe filter
into a sterile glass bottle. The extracts contained 1 g/ml of
Rhizoma Curculiginis or Rhizoma Drynariae. This method of
extraction is widely used for obtaining water soluble frac-
tions in Chinese medicinal herbs [23].
Investigation of systemic effect of Rhizoma Curculiginis
or Rhizoma Drynariae extracts
All animals were obtained from the Laboratory Animal
Unit at the University of Hong Kong where they were kept
under standard conditions. The temperature was kept
between 22°C and 24°C. The light cycle was from 8 am to
8 pm daily. Each animal was kept individually in a cage
and fed with standard diet in the Laboratory Animal Unit.
The animal handling and experimental protocol was
approved by the Committee for the Use of Living Animals
in Teaching and Research, the University of Hong Kong.
The mice were bred by the Laboratory Animal Unit, the
University of Hong Kong. The doses of both Rhizoma Cur-
culiginis and Rhizoma Drynariae for human were 0.2 g/kg/
day as suggested in the Chinese Pharmacopeia [2]. The
doses of the herbs for mice were estimated in a pilot study.
Thirty 8-week-old male BALB/c mice were divided into
three groups as follows:
1. Control group (C
mice
): Ten BALB/c mice fed with nor-
mal diet and distilled water.
2. Rhizoma Curculiginis group (XM
mice
): Ten BALB/c mice
fed with normal diet and distilled water mixed with Rhi-
zoma Curculiginis extract (0.5 g Rhizoma Curculiginis in 250
ml of water).
3. Rhizoma Drynariae group (G
mice
): Ten BALB/c mice fed
with normal diet and distilled water mixed with Rhizoma
Drynariae extract (0.5 g Rhizoma Drynariae in 250 ml of
water).
The mice were kept individually (one animal per cage) for
five weeks before sacrificed. The drinking solutions, which
did not contain any suspending herbal particulates, were
freshly prepared every day.
The bone samples (proximal tibia) were carefully dis-
sected after sacrifice of the mice. The sample was then
fixed with buffered saline and placed onto a sample
holder. The proximal tibia is a long bone which can be
easily inserted in the micro-CT chamber, allowing easy
standardization in specimen positioning. These proce-
dures were originated from other similar micro-CT studies
[18]. The investigator was blinded to the treatment of each
mouse. The morphometric parameters were determined
automatically by computer without human interference.
The bone samples were scanned through 360° by a com-
pact fan-beam-type tomography instrument μCT20
(Scanco Medical AG, Bassersdorf, Switzerland). Samples
were placed in an air-tight cylindrical sample holder filled
with formaldehyde to preserve the sample for the dura-
tion of the measurement. The sample holder was marked
with an axial alignment line on the outside of the tube to
allow consistent positioning of the specimens within the
holder. By connecting the alignment notch to the sample
holder with the μCT20 turnable, precise positioning of the
bone within seconds was achievable. A typical analysis
consisted of a scout view to ensure accurate and consistent
positioning of slides, selection of the examination vol-
ume, automatic positioning, measurement, offline recon-
struction and evaluation. Scout (enlarged) views were
taken to ensure the precision of finding the anatomic loca-
tion in each bone, a sample size estimated from similar
studies was selected to minimize the effect of the location
variation.
Modified protocols from Zhang et al. [24] and Ishimi et al.
[18] were followed: twenty micro-CT slices/sections (0.25
mm apart) were taken to cover the proximal end of the left
tibia. The most proximal slice was 1.5 mm away from the
proximal end to avoid possible morphological variation
in the proximal head (Figure 1). Quantitative morphom-
etry of the bone structure was performed with the μCT20
computer system. The fibula was excluded from measure-
ment. The micro-CT reconstruction parameters were as
follows: sigma: 1.2; support: 2; threshold: 140; increment/
scan thickness: 0.25 mm; resolution: high (1024 × 1024
pixels).
Data were analyzed using statistical analysis software
Graphpad Instat (v.2.04a). The arithmetic mean and
standard deviation (SD) were calculated for each group.
The means (XM
mice
and C
mice
; G
mice
and C
mice
) were com-
pared by the Welch's unpaired t test which does not
assume equal variances, with P < 0.05 chosen as the criti-
cal level of statistical significance.
Investigation of local effect of Rhizoma Curculiginis or
Rhizoma Drynariae extracts
The methodology and animal model used in the current
study have been described previously [11]. Six 10 × 5 mm
2
full-thickness bone defects were created in the parietal
bones of three inbred New Zealand white rabbits. The rab-
bits were five months old (adult stage) and weighed
between 3.5 kg and 4.0 kg. The animal handling and
experimental protocol was approved by the Committee
Chinese Medicine 2007, 2:13 http://www.cmjournal.org/content/2/1/13
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on the Use of Live Animals in Teaching and Research, the
University of Hong Kong. In the experiment, two defects
in the first animal were grafted with collagen matrix with
Rhizoma Curculiginis extract; two defects in the second ani-
mal were grafted with collagen matrix with Rhizoma Dry-
narie extract; two defects in the third (control) animal
were grafted with collagen matrix alone. The animals were
pre-medicated one hour before surgery with oxytetracy-
cline hydrochloride (200 mg/ml, 30 mg/kg body weight)
and buprenorphine hydrochloride (0.3 ml/kg body
weight), supplemented with diazepam (5 mg/ml, 1 mg/kg
body weight). In order to maintain the level of neurolep-
tanalgesia, increments of Hypnorm (0.1 ml/kg) were
given at 30-minute intervals during the operation.
The surgical procedure consisted of the creation of two 10
× 5 mm full-thickness (approximately 2 mm) cranial
defects, devoid of periosteum, using templates, in the
parietal bones. The defects were produced using round
stainless steel burs (1 mm in diameter) on a low speed
dental drill. Outlines of the defects were made initially by
making holes of full thickness the parietal bone using a
stainless steel wire template bent to the required size of
the defect. The holes were joined to complete the process.
During the cutting of the bones, copious amount of sterile
saline was used for irrigation and to minimize thermal
damage to the tissues. In the first experimental animal, the
defects were filled with purified absorbable fibrillar colla-
gen matrix (Collagen Matrix, NJ, USA) with 0.2 ml 1 g/ml
Rhizoma Curculiginis extract. In the second experimental
animal, the defects were filled with the same collagen
matrix with 0.2 ml 1 g/ml Rhizoma Drynarie extract. The
grafts were prepared 15 minutes before grafting. In the
control animal, the defects were grafted with 0.02 g of the
same collagen matrix mixed with 0.2 ml water for injec-
tion.
All wounds were closed with interrupted 3/0 black silk
sutures. No attempt was made to approximate the perios-
teum to prevent the barrier effect. Postoperatively, the rab-
bits were given oxytetracycline hydrochloride daily for ten
days and buprenorphine hydrochloride for two weeks.
The animals were monitored under a standardized proto-
col during postoperative period with the supervision of
the veterinary surgeon for any unhealthy signs or side
effects. Medication for the animals during this period was
as follows: 30 mg/kg of oxytetracycline hydrochloride
intramuscular injection every 30 minutes; 50 μg/kg of
Temgesic subcutaneous injection daily (for two weeks);
60 ml of saline and 10 ml Dextran 40 (10% dextrose solu-
tion in 0.9% saline solution) subcutaneous injection daily
until appetite recovered; 10 ml of Dextran 40 in 350 ml
drinking water daily until appetite recovered; vitamin B1,
B6 and B12.
Two weeks after surgery, the animals were sacrificed with
sodium pentobarbitone. Immediately after death, defects
and surrounding tissues were removed for histological
preparation.
Tissues were fixed in 10% buffered saline solution, dem-
ineralized with K's Decal Fluid (sodium formate/formic
acid) and double embedded in celloidin-paraffin wax.
Serial, 5-μm-thick sections of the whole defect were cut
perpendicular to the long axis. The slides were stained
with Periodic acid-Schiff stain which allowed easy identi-
fication of new bone formation.
For the investigation of the ultra-structure of new bone
formation, some tissues of the animal grafted with Rhi-
zoma Drynarie extract was fixed with Karnovsky solution,
decalcified in K's Decal Fluid and double embedded in
celloidin-paraffin wax. The specimens were cut into 3-μm-
thick serial sections, perpendicular to the long axis, and
stained with hematoxylin and eosin. Histological sections
were used to locate the area of the interface between the
graft and the host bone on the tissue blocks. The areas of
interest were then fixed, post-fixed in osmium tetroxide
(OsO
4
) and trimmed into 1 mm blocks and embedded in
resin. Semi-thin sections from resin block were sectioned
and stained with toluidine blue for further orientation
and reduction of the block face. Ultra-thin sections (90
nm thickness) were cut with a diamond knife to and
mounted on metal grids (200 meshes). Sections were then
stained with uranyl acetate and lead citrate, and examined
under a transmission electron microscope (EM208S, Phil-
lips Electron Optics BV, Netherlands).
Scout view (× 3) of tibia (left)Figure 1
Scout view (× 3) of tibia (left). The proximal end of the tibia
was covered by the 20 sections. The first, most proximal
slice was 1.5 mm away from the proximal end of the tibia.
Sample of micro-CT scan section (right). The fibula was
excluded during analysis (× 20).
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Results
Investigation of systemic effect of Rhizoma Curculiginis
or Rhizoma Drynariae extracts
All animals remained in excellent health throughout the
course of the experiment. There was no evidence of side
effects in any of the animals. The mean (SD) weight of the
mice was 28.46 (1.03) g and there was no significant dif-
ference in weight between the experimental and control
groups before and after the experiment. The mean (SD)
daily water consumption of each mouse was 6.6 ml. There
was no significant difference in daily water consumption
between the XM
mice
: 6.7 (1.1) ml, G
mice
: 6.5 (1.1) ml and
C
mice
groups: 6.6 (1.3) ml.
Micro-CT scanning was performed on the proximal tibia
of the XM
mice
, G
mice
and C
mice
groups. 2D-histomorphom-
etry was calculated from the micro-CT system. Compared
with the C
mice
control group, trabecular number increased
10.00%; trabecular thickness and trabecular separation
decreased 7.01% and 9.66% respectively in the XM
mice
group. The low trabecular separation is consistent with
the high trabecular number, i.e. finer trabecular pattern
with more and thinner trabeculae. In the XM
mice
group,
the low trabecular separation is also consistent with
increased bone surface to tissue volume and bone surface
to bone volume ratios which went up 10.03% and 6.68%
respectively. In the G
mice
group, compared with the C
mice
control group, trabecular number increased 10.00%;
trabecular thickness and trabecular separation decreased
3.78% and 9.66% respectively. Rhizoma Drynariae extract
caused a lower trabecular thickness, which was consistent
with the overall increase in the bone volume/tissue vol-
ume ratio (up 6.45%), an indicator of bone density (Table
1).
For method error analysis, readings of ten sections
recorded at two separate micro-CT scanning sessions by
the same examiner were randomly drawn and the read-
ings were then compared. The method error of the image
analysis ( ) is 0.003194 mm
2
, where d is the differ-
ence between the two readings of a pair and n is the
number of double readings. The error was insignificant
compared with the results. The duplicate intra-observer
registrations of the 10 randomly drawn sections were also
insignificant (P = 0.1679).
Investigation of local effect of Rhizoma Curculiginis or
Rhizoma Drynariae extracts
All animals remained in excellent health throughout the
course of the experiment and recovered rapidly after oper-
ation. There was no evidence of side effects or infection in
any of the animals.
In the rabbits grafted with Rhizoma Curculiginis or Rhizoma
Drynariae extracts in collagen matrix, new bone was
formed at the host bone – graft interface tended to grow
across the defect (Figures 2 and 3). Integration of the Rhi-
zoma Curculiginis or Rhizoma Drynariae extracts and colla-
gen with the recipient bed was characterized by the
appearance of new bone. No cartilage was found. At
higher magnification, new bone could be seen growing
around a collagen matrix fragment and tended to grow
towards and amalgamate with the collagen matrix. The
presence of bone cells indicated that new bone was
formed.
In the control rabbit, little new bone was formed at the
host bone/graft interface. Some collagen fibers were
present at the center of the defects (Figure 4). In all rab-
bits, the defects were healed, with fibrous tissue bridging
across the defects.
d
n
2
2
∑
Table 1: Micro-CT scan and 2D-morphometry
Parameter C
mice
XM
mice
G
mice
Mean (SD) Mean (SD) % diff (P-value) Mean (SD) % diff (P-value)
Bone volume/tissue volume (%) 17.748 (1.073) 18.304 (1.096) 3.13%↑ (0.122) 18.892 (1.207) 6.45%↑ (0.005*)
Trabecular number (1/mm) 0.781 (0.080) 0.860 (0.049) 10.00%↑ (0.002*) 0.859 (0.054) 10.00%↑ (0.002*)
Trabecular thickness (mm) 0.291 (0.022) 0.271 (0.010) 7.01%↓ (0.002*) 0.280 (0.013) 3.78%↓ (0.069)
Trabecular separation (mm) 0.321 (0.036) 0.290 (0.020) 9.66%↓ (0.002*) 0.290 (0.020) 9.66%↓ (0.002*)
Bone surface/bone tissue volume (1/mm) 2.454 (0.246) 2.700 (0.156) 10.03%↑ (0.002*) 2.699 (0.165) 10.00%↑ (0.002*)
Bone surface/bone volume (1/mm) 14.067 (1.122) 15.006 (0.644) 6.68%↑ (0.005*) 14.625 (0.653) 4.00%↑ (0.070)
C
mice
: control group
XM
mice
:Rhizoma Curculiginis group
G
mice
: Rhizoma Drynariae group
↑:increase
↓:decrease
SD: standard deviation
% diff: % difference
*: P < 0.05
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The area of interest for ultra-structural study was taken
near the newly formed bone and its surrounding connec-
tive tissue. At the mineralization front (F), osteoblasts
(OB) with abundant rough ER and newly entrapped oste-
ocytes (OC) could be seen (Figure 5).
Discussion
According to traditional Chinese medicine, the herbs that
affect Shen have effects on bones [2]. Two Shen-affecting
herbs, namely Rhizoma Curculiginis and Rhizoma Drynar-
iae, were used to test this hypothesis. The non-destructive
and precise micro-tomography system for measuring
static morphometrical and architectural parameters of
bones [14] showed systemic effects of Rhizoma Curculiginis
and Rhizoma Drynariae extracts on bones.
The administration of Rhizoma Curculiginis extract for five
weeks changed the trabecular pattern of the long bone in
mice. The increase in bone density, indicated by the bone
volume/tissue volume ratio, was not statistically signifi-
cant. Further research is needed to investigate whether the
bone remodeling rate is increased by the administration
of Rhizoma Curculiginis extract and what biological mech-
anisms are.
This study showed that both Rhizoma Curculiginis and Rhi-
zoma Drynariae altered bone histomorphological pattern
and that Rhizoma Drynariae increased bone density. In
addition, both of these herbal extracts induced new bone
formation on the margins of the bone defects, whereas lit-
tle new bone formation was seen in the control groups.
The ultra-structural study of the new bone formed in the
Transmission electron micrographs show the area near the newly formed bone of bone defect grafted with Rhizoma Dry-nariae extract in collagen matrixFigure 5
Transmission electron micrographs show the area near the
newly formed bone of bone defect grafted with Rhizoma Dry-
nariae extract in collagen matrix. (F: mineralization front; OB:
osteoblasts; OC: osteocytes; original magnification × 3500).
Photomicrograph of bone defect grafted with Rhizoma Dry-nariae extract in collagen matrix on day 14Figure 3
Photomicrograph of bone defect grafted with Rhizoma Dry-
nariae extract in collagen matrix on day 14. New bone can be
seen spanning the defect. Some collagen matrix (C) remained
at the center of the bone defect (Periodic acid-Schiff stain;
original magnification × 40).
Photomicrograph of bone defect grafted with Rhizoma Curcu-liginis extract in collagen matrix on day 14Figure 2
Photomicrograph of bone defect grafted with Rhizoma Curcu-
liginis extract in collagen matrix on day 14. New bone (N) can
be seen spanning the defect. H: Host bone. Some collagen
matrix (C) remained at the center of the bone defect (Peri-
odic acid-Schiff stain; original magnification × 40).
Photomicrograph of bone defect grafted with collagen matrix (positive control) on day 14Figure 4
Photomicrograph of bone defect grafted with collagen matrix
(positive control) on day 14. No bone could be seen across
the defect except a little new bone near the ends of the host
bone (H). Collagen matrix (C) remained across the bone
defect (Periodic acid-Schiff stain, original magnification × 40).
Chinese Medicine 2007, 2:13 http://www.cmjournal.org/content/2/1/13
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rabbit with Rhizoma Drynariae extract was to investigate
the structure of the new bone. The identification of oste-
oblasts and osteocytes in the newly formed bone showed
that the newly formed hard tissue was actually bone
instead of calcification of the collagen fibers.
While this study aims to identify the effect of crude
extracts of Rhizoma Curculiginis and Rhizoma Drynariae on
bones, we can speculate the action mechanism of the
effect. The bone anabolic ability of Rhizoma Drynariae
shown in this study is likely to be related to angiogenesis
and/or osteogenesis through the up-regulation of the
expression of osteogenic factors. The mechanism of the
osteogenic effect of Rhizoma Drynariae can be partially
explained by the activation of BMP-2 gene expression
through the HMG-CoA reductase inhibition effect of nar-
ingin. Further studies are required to investigate whether
other active components of Rhizoma Drynariae are also
osteogenic. In Rhizoma Curculiginis, a new orcinol gluco-
side that has potent antioxidative activities similar to that
of naringin was identified [5]. It is possible that orcinol
glucoside also affects the redox reaction of the HMG-CoA
transformation to mevalonate and triggers the BMP-2
gene expression. Ko and Leung [25] recently reported that
'Yang-tonifying' herbs enhance ATP generation capacity in
mitochondria and exhibit antioxidant effect. Whether the
APT generation and antioxidant effect are related to the
osteogenic effect is of interest in our future studies. It is
well known that the effect of any compound on bones is
dependent on the site of the bone. As femur neck and
lumbar are most susceptible to fracture, future studies of
the effects of Rhizoma Curculiginis and Rhizoma Drynariae
on femur neck and lumber are also warranted.
Conclusion
Two 'Yang-tonifying' herbs, Rhizoma Curculiginis and Rhi-
zoma Drynariae, were demonstrated to have systemic
effects on bone histomorphology and formation as well as
local bone healing.
Competing interests
The author(s) declare that they have no competing inter-
ests.
Authors' contributions
RW conceived the research design. RW, AR, MB and UH
contributed to the research work, supervision of the tech-
nicians, and drafting of the manuscript. All authors read
and approved the final manuscript.
Acknowledgements
We thank Y Y Chui and S Yeung for their technical assistance and the staff
of the Laboratory Animal Unit of the University of Hong Kong for their
assistance in the care of the animals. This work was supported by the RCG
grant 10205950/23438/08003/301/01 from the University of Hong Kong.
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