Journal of Ethnopharmacology 118 (2008) 271–279

Contents lists available at ScienceDirect

Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jethpharm

Antiosteoporotic chemical constituents from Er-Xian Decoction,

a traditional Chinese herbal formula
Luping Qin a , Ting Han a , Qiaoyan Zhang a,∗ , Dapeng Cao b , Hua Nian a ,
Khalid Rahman c , Hanchen Zheng a
a
School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433, PR China
b
College of Chinese Medicinal Materials, Jilin Agricultural University, Changchun 130118, PR China
c
School of Biomolecular Science, Faculty of Science, Liverpool John Moores University, Byrom Street, Liverpool L3 3AF, UK

a r t i c l e i n f o a b s t r a c t

Article history: Aim of the study: Er Xian Decoction (EXD), a traditional Chinese medicine formula, has long been used
Received 15 September 2007 for the treatment of osteoporosis and menopausal syndrome in China. The present study was designed
Received in revised form 29 March 2008 to investigate the antiosteoporotic constituents of EXD, and evaluate their antiosteoporotic effects in
Accepted 9 April 2008
ovariectomized rats.
Available online 15 April 2008
Materials and methods: Osteoblasts in neonatal calvaria cultures and osteoclasts derived from rat marrow
cells were used to bioactivity-guided screen the active constituents. The proliferation of osteoblast was
Keywords:
assayed by MTT methods. The activity of ALP and TRAP was measured by p- nitrophenyl sodium phosphate
Er-Xian Decoction
Osteoporosis
assay. The antiosteoporotic effects of icariin (1), anemarsaponin B II (8) and berberine (6) were verified
Active constituents by using OVX rats model. The bone mineral density (BMD) was measured by dual-energy X-ray absorp-
tiometry using the small animal scan mode. The undecalcified longitudinal proximal tibial metaphysical
(PTM) sections were cut and stained for the bone histomorphometric analysis.
Results: Bioactivity-guided fractionation has led to the successful isolation of antiosteoporotic con-
stituents, i.e., icariin (1), icariside I (2), baohuoside I (3), mangiferin (4), neomangiferin (5), berberine (6),
anemarsaponin B (7), anemarsaponin BII (8), anemarsaponin C (9), anemarrhenasaponin I (10), rubiadin-
1-methyl ether (11) and obaculactone (12) from EXD. Further study showed that icariin (1), anemarsaponin
BII (8) and berberine (6) increased the BMD in ovariectomized rats, and icariin (1) not only increased
the bone formation, but also inhibited bone resorption; anemarsaponin BII (8) mainly increased bone
formation and berberine (6) only inhibited the bone resorption in ovariectomized rats.
Conclusion: Our findings demonstrate that multiple ingredients are responsible for antiosteoporotic activ-
ity in traditional Chinese medicine formula Er-Xian decoction.
© 2008 Elsevier Ireland Ltd. All rights reserved.

1. Introduction have been shown to possess antiosteoporotic activities (Cassidy,

Osteoporosis is the most frequent bone remodeling disease,
is defined by a low bone mass and a high risk of fractures,
and is a major health problem for elderly women. For several
years, estrogen replacement therapy (ERT) has been used to pre-
vent osteoporosis in postmenopausal women (Tuner et al., 1994).
However, studies have also reported that estrogen can lead to
a higher incidence of breast carcinoma, endometrial cancer, and
cardiovascular disease (Davison and Davis, 2003). Plants derived
medicines display less adverse effects and have been a part of tra-
ditional healthcare in China for thousands of years. Many of these
∗ Corresponding author. Tel.: +86 21 25074579; fax: +86 21 25074574.
E-mail address: zqy1965@163.com (Q. Zhang).
2003).
Er-Xian Decoction (EXD), a traditional Chinese medicine for-
mula, has been used for the treatment of osteoporosis disorders
(Wang et al., 1998), menopausal syndrome (Liu et al., 2005), and
aging diseases for several decades (Shen et al., 1995). The daily
dose of EXD is composed of six Chinese herbs, including 9–15 g
Epimedium sagittatum (Siebold & Zucc.) Maxim. (Berberidaceae,
whole herb), 6–15 g Curculigo orchioides Gaertn. (Amaryllidaceae,
rhizome), 4.5–9 g Anemarrhena asphodeloides Bge (Liliaceae, rhi-
zome), 4.5–9 g Phellodendron chinense C.K. Schneid. (Rutaceae,
bark), 9 g Morinda officinalis F.C. How. (Rubiaceae, root), and 9 g
Angelica sinensis (Oliv.) Diels (Apiaceae, root). Sometimes the for-
mula is composed of 10 g above-mentioned herbs, respectively.
Usually daily dose is decocted two to three times and decoction
is combined to be taken by oral administration. These six herbs

0378-8741/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.jep.2008.04.009
272 L. Qin et al. / Journal of Ethnopharmacology 118 (2008) 271–279
are succinctly and excellently combined in the formula, with the
properties of being warm but not dry, cold but not stagnant, sup-
plementary but not hot, and enjoying the therapeutic merits of
warming Shen-yang, invigorating Shen-essence, nourishing Shen-
yin, purging Xiang-fire, harmonizing Chong-Ren meridian, and
balancing yin-yang (Li et al., 2007). In the clinical application, the
formula composition often is modified by adding or subtracting
adjuvant drug to treat the related disease with perimenopausal syn-
drome and to meet the need of treatment of present accompanying
symptoms (Chen and Fang, 1998; Li, 2004).
Many studies have demonstrated that EXD stimulated the secre-
tion of testosterone of the leydig cell, progesterone of luteal cell and
E2 of granulose cell (Liu et al., 2005), markedly increased adreno-
corticotropic hormone (ATCH) level in kidney-yang deficiency
rats through up-regulating the expression ATCH mRNA (Zheng et
al., 2003), improved the amount of gonadotrop in-releasing hor-
mone (GnRH), luteinizing hormone (LH) and follicle-stimulating
hormone (FSH) in the anterior pituitary and concentration of
testosterone (T) in blood, and decreased the concentration of LH
in blood (Fang et al., 1997). Our previous studies have shown
that EXD contributes significantly to the prevention or treat-
ment of the development of bone loss induced by ovariectomy
in rats, and has positive effects on bone while only displaying
minor effects on the uterus, an important factor in the treat-
ment of osteoporosis (Nian et al., 2006). These findings give some
insight into the antiosteoporotic mechanism of EXD in relation to
endocrinology.
In EXD formula, there are various kinds of chemical con-
stituents. The isoflavones from Epimedium sagittatum, phenols
and phenolic glycosides from Curculigo orchioides are phytoe-
strogen, and possess the estrogen-like activity (Wang and Lou,
2004; Vijayanarayana et al., 2007). These kinds of compounds,
which have aroused general concern, have the capacity to bind
to the estrogen receptors and maybe decrease the bone loss like
estrogen (Messina et al., 2000). The alkaloids from Phelloden-
dron chinense such as berberine have been reported to prevent a
decrease in BMD in vivo by inhibiting osteoclastic bone resorption
(Li et al., 1999). The steroidal saponins from Anemarrhena asphode-
loides, are somewhat similar to mammalian estrogens in chemical
structure, perhaps is correlated with activity of estrogens, which
associated with the estrogen receptor dependent pathway and
antiosteoporotic properties. Besides above-mentioned compounds,
there are anthraquinones from Morinda officinalis and polysac-
charide from Angelica sinensis and Morinda officinalis. However,
the antiosteoporotic chemical constituents of EXD have not been
identified.
In osteoporosis, the osteoblast formation and function decreases
whilst the osteoclast formation and recruitment increases, this
causes a relative increase of osteoclastic bone resorption over
osteoblastic bone formation. The bone formation is related to
osteoblastic proliferation, alkaline phosphatase activity, osteocal-
cin and collage synthesis; and the bone resorption is associated
with osteoclast formation and differentiation, and tartrate-
resistant acid phosphatase activity. In this study, the active
constituents of EXD were identified and investigated for their
antiosteoporotic effects in ovariectomized rats.
2. Materials and methods
2.1. Apparatus
NMR spectra were obtained with a Bruker DRX-500 spectrome-
ter at 500 MHz for 1 H NMR and 125 MHz for 13 C NMR expressed in
ı values with reference to TMS as internal standard, and coupling
constants J (Hz). EI-MS was recorded on a Varian MAT-212 mass
spectrometer; melting points were determined on a RY-2 melting
point apparatus and are uncorrected. IR spectra were recorded on a
Bruker Vector 22 spectrometer with KBr pellet. Column chromatog-
raphy was executed on silica gel (200–300 mesh, Yantai, PR China),
and Sephadex LH-20 (Pharmacia). TLC analysis was run on HSGF254
precoated silica gel plates (10–40 mm, Yantai, PR China). The sol-
vents used for isolation and purification were purchased from
Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). RPMI-
1640 medium, ␣-MEM medium and fetal calf serums (FCSs) were
purchased from Gibco (USA). 1,25-Dihydroxyvitamin D3 , dexam-
ethasone, naphthol AS-BI phosphate, pararosaniline, coomassie
brilliant blue G-250 and 2-mercaptoethanol were purchased from
Sigma (USA). The elutes on macroporous resins were dissolved in
culture medium. The test compounds were dissolved in ethanol at
a concentration of 10 mmol/L and diluted to an appropriate con-
centration using culture medium, and the final concentration of
ethanol was adjusted to 0.01%. Ethylene glycol methyl ether, potas-
sium sodium tartrate, disodium 4-nitrophenylphosphate, Triton
X-100, and 4-nitrophenol were of domestic AR grade.
2.2. Animals
The SD rats were purchased from the Experimental Animal Cen-
ter of the Second Military Medical University.
2.3. Plant material
The six plant materials Epimedium sagittatum (Berberidaceae,
whole herb, 2004021801), Curculigo orchioides (Amaryllidaceae,
rhizome, 2004021802), Anemarrhena asphodeloides (Liliaceae,
rhizome, 2004021803), Phellodendron chinense (Rutaceae, bark,
2004021804), Morinda officinalis (Rubiaceae, root, 2004021805),
Angelica sinensis (Apiaceae, root, 2004021806) were obtained from
Shanghai Hua Yu Chinese Herbs Co. Ltd. and identified by Prof. H.C.
Zheng of the Department of Pharmacognosy, School of Pharmacy of
the Second Military Medical University, Shanghai, PR China. Their
voucher specimens are available in the herbarium of this Depart-
ment. All crude drugs were air-dried and powdered mechanically.
2.4. Extraction and fractionation procedures
Each crude drug (5 kg) was powdered and mixed, then extracted
3 times with boiling water (150 L, 3× 2 h). The total filtrate was
evaporated to 30 L under reduced pressure to obtain the water
extracts of EXD and these were extracted with CH3 Cl to give CH3 Cl
extracts (35 g). The residue was dissolved in water and passed
through ZTC-1 macroporous resin, and eluted with water, 10%, 30%,
50%, 70% and 95% (v/v) ethanol successively. The various elutes
were evaporated to dryness in under vacuum to give 6 fractions
(water: 5600 g, 10% ethanol: 320 g, 30% ethanol: 400 g, 50% ethanol:
170 g, 70% ethanol: 5.8 g, and 95% ethanol: 3.2 g), respectively.
CH3 Cl extracts (35 g) were rechromatographed on silica gel with
CH2 Cl2 –CH3 OH (100:1–1:1) to give berberine (230 mg), rubiadin-
1-methyl ether (60 mg), obaculactone (210 mg). The elutes of 10%
ethanol on macroporous resin was chromatographed on silica gel
with CHCl3 –CH3 OH (7:1 to 1:2) and on sephadex LH-20 with
CH3 OH and CH3 OH–H2 O to give the anemarsaponin B (70 mg),
anemarsaponin B-II (10 g), anemarsaponin C (200 mg), anemar-
rhenasaponin I (70 mg). The elutes (400 g) of 30% (v/v) ethanol
on macroporous resin were chromatographed on silica gel with
CHCl3 –CH3 OH and on sephadex LH-20 with CH3 OH and H2 O to give
icariin (10 g), neomangiferin (38 mg). The elutes (170 g) of 50% (v/v)
ethanol on macroporous resin were chromatographed on silica gel
with CHCl3 –CH3 OH (10:1 to 1:1) and on sephadex LH-20 with
 L. Qin et al. / Journal of Ethnopharmacology 118 (2008) 271–279
CH3 OH and H2 O to give icariside I (50 mg), baohuoside I (70 mg),
mangiferin (30 mg).
2.5. Cell cultures
Wistar rats, which were 3–4 days old, were purchased from
the Experimental Animal Center of the Second Military Medical
University, Shanghai, China. Primary osteoblastic cells were pre-
pared from the calvarias of newborn rats according to literature
(Liu et al., 1995). These cells had typical properties of osteoblast
s such as alkaline phosphates activity. Rat marrow cells
were collected as described by Zambonin et al. (1982). Pri-
mary osteoblastic cells (1 × 108 L−1 ) and bone marrow cells
(1 × 109 L−1 ) were co-cultured in ␣-MEM medium containing 10%
FCS, 1,25-dihydroxyvitamine D3 (10 nmol/L) and dexamethasone
(100 nmol/L) at 37 ◦ C in a humidified atmosphere of 5% CO2 for 10
days in six-well culture dishes (1.5 ml per well). Prior to plating the
cells, a cover glass (5 mm × 5 mm) or bone slices (40 ␮m thick) were
placed into culture dishes. The formation of osteoclast-like MNCs
(multinucleated osteoclasts) was confirmed by the staining of TRAP
and resorption pit formed on bone slices.
2.6. Assay for osteoblast proliferation and alkaline phosphatase
(ALP) activity
The primary osteoblasts in neonatal rat calvarias cultures were
treated with test substances or control (ethanol, final concentration
0.01 % (v/v)) for 48 h, and prior to the end of culture, MTT was added
to each well and incubated for 4 h after which the medium was
discarded, and 100 ␮L of DMSO was added to each well. The cells
were incubated for 20 min; the UV absorbance was measured at
540 nm at ELx 800 universal microplate reader (Bio-Tek) and used
as an indicator of osteoblast proliferation.
Primary osteoblasts were plated at 1 × 105 cells per well in 24-
well dishes. The ALP activity was measured according to literature
(Owen et al., 1990). Total protein was assayed by the method of
Bradford (1976). The activity of alkaline phosphatase was expressed
as nanomoles of p-nitrophenol liberated per minute per milligram
protein.
2.7. Assay for osteoclastic TRAP activity
After cell cultures, the cells were washed twice with PBS buffer
and the TRAP activity of osteoclasts was measured as described
previously (Qin et al., 2003). Briefly, the cells were washed twice
with PBS and 10 ␮L 0.1% Triton X-100 was added for 10 min to lyse
the cells. Then 100 ␮L substrate solution (preparation of reactive
solution: 0.4 g p-nitro-disodium phenyl phosphate was dissolved
in deionized water, 2.0 g of potassium tartrate was added followed
by 150 ml of deionized water, pH was adjusted to 3.5 with 1 M HCl
and the final volume adjusted to 200 ml) was added and incubated
for 30 min at 37 ◦ C. To stop the reaction, 100 ␮L NaOH (1 mol/L)
was added to each well. The samples and standards were diluted in
NaOH 20 mmol/L, and the absorbance was measured at 405 nm. The
nanomolar number of p-nitrophenol in each well was calculated
and at the same time, the positive cells for TRAP were counted.
The activity of TRAP was expressed as nanomoles of p-nitrophenol
liberated per minute per 100 osteoclasts.
2.8. Animal experimental protocol
Ninety female Sprague–Dawley rats, 12 weeks of age, were
purchased (SLACOM Experimental Animal Company of Shanghai,
China) and acclimated to conditions for 1 week before the experi-
ment. The experimental animals were housed in an air-conditioned
273
room with 12 h/12 h light-dark illumination cycles at constant tem-
perature 24 ± 0.5 ◦ C and humidity (45–50%). Food and drinking
water were supplied ad libitum. The rats were weighed weekly
during the experimental period.
Ten rats were sham-operated and treated with vehicle (deion-
ized water) as aging control (sham + Veh). The remaining rats were
bilaterally ovariectomized and randomly divided into nine groups
with 10 rats per group. They were treated with vehicle (water),
nylestriol (1 mg kg−1 , ig, weekly) or test substances for 12 weeks.
Rats received treatments po starting from 1 day after surgeries.
For in vivo fluorochrome labels, tetracycline (20 mg/kg) and calcein
(10 mg/kg) were injected to the rats on days −14, −13 and days −4,
−3 before being sacrificed. Success of ovariectomy was confirmed
at necropsy by failure to detect ovarian tissue and by observation
of marked atrophy of uterine horns. At the end of the treatment,
the femurs were cleaned off from adhering soft tissues, and then
enclosed with gauze saturated by PBS and stored in a freezer at
−80 ◦ C. The BMD (bone mineral density) was determined by dual-
energy X-ray absorptiometry (LUNAR Co. Ltd., USA) using the small
animal scan mode.
In addition, the left proximal tibia metaphysis were opened to
expose the marrow cavity using an isomet low speed saw (Buech-
ler Ltd., USA) and fixed in 10% phosphate buffer formalin for 24 h.
They were then dehydrated in ethanol, defatted in xylene and
embedded undecalcified in methyl methacrylate. The frontal sec-
tions were cut at 4- and 10-␮m thickness with microtome (Leica
RM 2155, Germany). The 4-␮m section was stained with Goldner’s
Trichrome staining for static histomorphometric measurements,
the unstained 10-␮m sections were used for dynamic histomor-
phometric analyses.
This experiment was approved by the Bioethic Committee of
the Second Military Medical University, and the procedures of the
experiment were strictly according to generally accepted interna-
tional rules and regulations.
2.9. Statistics
The results of each experiment were expressed as
mean ± standard deviation. Significance of results was evaluated
using Student’s test.
3. Results and discussion
The water extracts of Er-Xian Decoction was fractionated by
liquid extracts and macroporous resin, silica gel and Sephadex
chromatography. All fractions were tested for their osteoblast pro-
liferation, alkaline phosphatase and osteoclastic TRAP activity. The
elutes on macroporous resin by water and 70% (v/v) ethanol did not
show any activity on osteoblast and osteoclast. The CH3 Cl extracts
only decreased the osteoclastic TRAP activity at the concentration
of 10 and 20 ␮g/ml; however, did not increase osteoblast prolif-
eration and ALP activity at the same concentration. The elutes on
macroporous resin by 10%, 30% and 50% (v/v) ethanol respectively
showed significant activities on osteoblastic proliferation, alkaline
phosphatase activity and osteoclastic TRAP activity at the concen-
tration of 10 and 20 ␮g/ml (Table 1). These fractions displayed
stronger activities were thus chosen for further isolation. Finally,
20 compounds were obtained, and 12 of them showed significant
activity on osteoblast and osteoclast. These active compounds were
identified as icariin (1), icariside I (2), baohuoside I (3), mangiferin
(4), neomangiferin (5), berberine (6), anemarsaponin B (7), ane-
marsaponin BII (8), anemarsaponin C (9), anemarrhenasaponin I
(10), rubiadin-1-methyl ether (11) and obaculactone (12). Their
chemical structure was shown in Fig. 1.
274 L. Qin et al. / Journal of Ethnopharmacology 118 (2008) 271–279

Table 1
Effects of various fractions of water extracts of EXD on osteoblast and osteoclast (n = 8, x ± S.D.)

Fractions Concentration Osteoblast proliferation Osteoblastic ALP activity Osteoclastic TRAP activity
(␮g/ml) absorbance (550 nm) (nmol/(mg min)) (nmol/(min 100 osteoclastic cell))

Control 0.71 ± 0.01 11.3 ± 0.6 10.0 ± 1.2

Extracts of EXD 200 1.00 ± 0.07** 21.6 ± 1.2** 6.2 ± 0.7**

400 1.16 ± 0.10** 22.4 ± 2.1** 5.8 ± 0.7**

CH3 Cl extracts 10 0.71 ± 0.04 13.1 ± 1.2 5.8 ± 0.6**

20 0.72 ± 0.06 12.8 ± 0.9 5.6 ± 0.8**

Water elutes 10 0.74 ± 0.03 12.2 ± 1.2 9.0 ± 1.1

20 0.71 ± 0.01 12.0 ± 1.0 10.2 ± 1.3

10% Ethanol elutes 10 0.99 ± 0.06** 18.3 ± 1.5** 6.0 ± 0.7**

20 1.17 ± 0.11** 18.6 ± 1.5** 5.8 ± 0.9**

30% Ethanol elutes 10 0.91 ± 0.02** 21.4 ± 1.8** 6.4 ± 0.8**

20 0.94 ± 0.02** 22.8 ± 1.0** 5.8 ± 1.0**

50% Ethanol elutes 10 0.95 ± 0.03** 22.8 ± 1.1** 6.1 ± 1.1**

20 1.19 ± 0.13** 22.1 ± 2.0** 5.5 ± 1.0**

70% Ethanol elutes 10 0.70 ± 0.02 11.5 ± 1.2 8.1 ± 1.5

20 0.74 ± 0.02 11.4 ± 1.4 8.4 ± 1.5

*P < 0.05, **P < 0.01 compared with control.

Icariin (1): yellow needle; ESIMS: m/z 675 [M−H]− ; molecular
formula: C33 H40 O15 ; 1 H NMR (DMSO-d6 , ı, ppm): 12.57 (1H, s, 5-
OH), 7.89 (2H, d, J = 9.0 Hz, 2 ,6 -H), 7.12 (2H, d, J = 9.0 Hz, 3 ,5 -H),
6.63 (1H, s, 6-H), 5.30 (1H, s, Rha-1-H), 5.16 (1H, t, J = 7.0 Hz, 12-H),
4.99 (1H, d, J = 5.0 Hz, Glu-1-H), 3.84 (3H, s, 4 -OCH3 ), 1.67 (3H, s,
14-CH3 ), 1.59 (3H, s, 15-CH3 ), 0.79 (3H, d, J = 6.0 Hz, Rha-6-CH3 ); 13 C
NMR (DMSO-d6 , ı, ppm): 157.21 (C-2), 134.60 (C-3), 178.22 (C-4),
160.45 (C-5), 98.08 (C-6), 161.34 (C-7), 108.27 (C-8), 152.95 (C-9),
105.54 (C-10), 21.34 (C-11), 122.06 (C-12), 130.99 (C-13), 17.76 (C-
14), 25.35 (C-15), 122.20 (C-1 ), 130.46 (C-2 ), 114.01 (C-3 ), 159.01
(C-4 ), 114.01 (C-5 ), 130.46 (C-6 ), 55.43 (4 -OCH3 ), 100.51 (Glu-
C1 ), 73.30 (Glu-C2 ), 76.55 (Glu-C3 ), 69.62 (Glu-C4 ), 77.13 (Glu-C5 ),
60.60 (Glu-C6 ), 101.94 (Rha-C1 ), 70.60 (Rha-C2 ), 70.27 (Rha-C3 ),
71.06 (Rha-C4 ), 70.00 (Rha-C5 ), 17.36 (R-C6 ). 1 H and 13 C NMR were
identical with literature values (Wang et al., 2005).
Icariside I (2): yellow powder; ESIMS: m/z 529 [M−H]− ; molec-
ular formula: C27 H30 O11 ; 1 H NMR (DMSO-d6 , ı, ppm): 12.44 (1H, s,
5-OH), 9.65 (1H, s, 3-OH), 8.15 (2H, d, J = 9.0 Hz, 2 ,6 -H), 7.15 (2H, d,
J = 9.0 Hz, 3 ,5 -H), 6.61 (1H, s, H-6), 5.21 (1H, t, J = 6.5 Hz, 12-H), 5.01
(1H, d, J = 7.0 Hz, Glu-1-H), 3.84 (3H, s, 4 -OCH3 ), 1.77 (3H, s, 14-CH3 ),
1.63 (3H, s, 15-CH3 ); 13 C NMR (DMSO-d6 , ı, ppm): 146.90 (C-2),
136.03 (C-3), 176.37 (C-4), 160.15 (C-5), 97.48 (C-6), 160.69 (C-7),
108.24 (C-8), 152.79 (C-9), 104.49 (C-10), 21.44 (C-11), 122.34 (C-
12), 131.13 (C-13), 17.89 (C-14), 25.44 (C-15), 123.38 (C-1 ), 129.36
(C-2 ), 114.15 (C-3 ), 158.31 (C-4 ), 114.15 (C-5 ), 139.36 (C-6 ), 55.42
(4 -OCH3 ), 100.53 (Glu-C1 ), 73.28 (Glu-C2 ), 76.46 (Glu-C3 ), 69.60
(Glu-C4 ), 77.14 (Glu-C5 ), 60.59 (Glu-C6 ). The 1 H and 13 C NMR were
in agreement with literature values (Wang et al., 2005).
Baohuoside I (3): yellow powder; ESIMS: m/z 513 [M−H]− ;
molecular formula: C27 H30 O10 ; 1 H NMR (DMSO-d6 , ı, ppm): 12.53
(1H, s, 5-OH), 10.85 (1H, s, 7-OH), 7.85 (2H, d, J = 9.0 Hz, 2 ,6 -H),
7.11 (2H, d, J = 9.0 Hz, 3 , 5 -H), 6.30 (1H, s, H-6), 5.26 (1H, s, Rha-
1 -H), 5.14 (1H, t, 12-H), 3.84 (3H, s, 4 -OCH3 ), 1.67 (3H, s, 14-CH3 ),
1.61 (3H, s, 15-CH3 ), 0.78 (3H, d, J = 6.0 Hz, Rha-6 -CH3 ); 13 C NMR
(DMSO-d6 , ı, ppm): 153.75 (C-2), 134.41 (C-3), 177.95 (C-4), 161.24
(C-5), 98.30 (C-6), 161.60 (C-7), 105.90 (C-8), 156.69 (C-9), 104.15
(C-10), 21.12 (C-11), 122.24 (C-12), 130.97 (C-13), 17.73 (C-14), 25.37
(C-15), 122.37 (C-1 ), 130.36 (C-2 ), 114.01 (C-3 ), 158.82 (C-4 ),
114.01 (C-5 ), 130.36 (C-6 ), 55.44 (4 -OCH3 ), 101.93 (Rha-C1 ), 70.59
(Rha-C2 ), 70.28 (Rha-C3 ), 71.10 (Rha-C4 ), 70.03 (Rha-C5 ), 17.41 (Rha-
C6 ). The 1 H and 13 C NMR were in agreement with literature values
(Wang et al., 2005).
Mangiferin (4): yellowish needle; ESIMS: m/z 404 [M−H2 O]+ ;
molecular formula: C19 H18 O11 ; 1 H NMR (DMSO-d6 , ı, ppm):
3.42–4.75 (m), 6.37 (1H, s, 4-H), 6.86 (1H, s, 5-H), 7.39 (1H, s, 8-H),
10.37 (3H, br, C3 , C6 , C7 -OH), 13.72 (1H, s, C1 -OH); 1 C NMR (DMSO-
d6 , ı, ppm): 161.4 (C-1), 107.5 (C-2), 163.6 (C-3), 93.4 (C-4), 156.3
(C-4a), 102.6 (C-5), 153.8 (C-6), 143.6 (C-7), 108.1 (C-8), 111.8 (C-8a),
179.1 (C-9), 101.3 (C-9a), 150.8 (C-10a), 73.1 (Glu-C1 ), 70.5 (Glu-C2 ),
78.8 (Glu-C3 ), 70.2 (Glu-C4 ), 81.5 (Glu-C5 ), 61.4 (Glu-C6 ). The 1 H
and 13 C NMR were in agreement with literature values (Hong et al.,
1997).
Neomangiferin (5): yellowish needle; ESIMS: m/z 583 [M−H]− ;
molecular formula: C25 H28 O16 ; 1 H NMR (DMSO-d6 , ı, ppm):
3.40–4.75 (m), 4.52 (1H, d, J = 7 Hz, glu-1 -H), 4.63 (1H, d, J = 9.8 Hz,
glu-1 -H), 6.64 (1H, s, 4-H), 6.95 (1H, s, 5-H), 7.50 (1H, s, 8-H),
14.35 (3H, br, C1 , C3 , C6 -OH); 1 C NMR (DMSO-d6 , ı, ppm): 161.8
(C-1), 107.3 (C-2), 163.8 (C-3), 93.3 (C-4), 156.3 (C-4a), 103.6 (C-5),
164.4 (C-6), 145.9 (C-7), 112.1 (C-8), 106.0 (C-8a), 178.1 (C-9), 100.7
(C-9a), 154.8 (C-10a), 73.2 (Glu-C1 ), 70.6 (Glu-C2 ), 79.1 (Glu-C3 ),
70.2 (Glu-C4 ), 81.4 (Glu-C5 ), 61.4 (Glu-C6 ), 103.1 (Glu-C1 ), 73.4
(Glu-C2 ), 76.5 (Glu-C3 ), 69.5 (Glu-C4 ), 77.2 (Glu-C5 ), 60.7 (Glu-
C6 ). The 1 H and 13 C NMR were in agreement with literature values
(Hong et al., 1997).
Berberine (6): yellow needle; ESIMS: m/z 335 [M−H]− ; molec-
ular formula: C20 H18 NO4 . 1 H NMR (DMSO-d6 , ı, ppm): 3.2 (2H, t,
J = 6.0 Hz, H-4), 4.07, 4.10 (3H each, s, 2× OCH3 ), 4.95 (2H, t, J = 6.0 Hz,
H-3), 6.18 (2H, s, O-CH2 -O), 7.09 (1H, s, H-5), 7.80 (1H, s, H-8), 8.0
(1H, d, J = 9.0 Hz, H-13), 8.2 (1H, d, J = 9.0 Hz, H-14), 8.98 (1H, s, H-
15), 9.92 (1H, s, H-16). 13 C NMR (DMSO-d6 , ı, ppm): 105.6 (C-1),
130.8 (C-2), 133.2 (C-3), 108.6 (C-4), 121.5 (C-5), 26.5 (C-6), 55.3 (C-
7), 145.6 (C-8), 120.6 (C-9), 149.9 (C-10), 150.5 (C-11), 126.9 (C-12),
123.7 (C-13), 143.8 (C-14), 120.6 (C-15), 147.8 (C-16), 137.6 (C-17),
102.2 (O-CH2 -O), 62.1 (OCH3 ), 57.2 (OCH3 ). The 1 H and 13 C NMR
were in agreement with literature values (Hu et al., 2007).
Anemarsaponin B (7): white powder; ESIMS: m/z 903 [M+H]+ ;
molecular formula: C45 H74 O18 . 1 H NMR (DMSO-d6 , ı, ppm): 0.61
(3H, s, 18-CH3 ), 0.87 (3H, s, 19-CH3 ), 0.89 (3H, d, J = 6.5 Hz, 27-CH3 ),
1.55 (3H, s, 21-CH3 ), 4.08 (1H, d, J = 8 Hz, glu-1 -H), 4.27 (1H, d,
J = 7 Hz, gal-1 -H), 4.41 (1H, d, J = 8 Hz, glu-1 -H). 13 C NMR (DMSO-
d6 , ı, ppm): 30.1 (C-1), 26.4 (C-2), 73.7 (C-3), 29.8 (C-4), 35.9 (C-5),
26.4 (C-6), 25.9 (C-7), 34.6 (C-8), 39.7 (C-9), 34.0 (C-10), 34.5 (C-
11), 39.4 (C-12), 43.2 (C-13), 54.1 (C-14), 30.5 (C-15), 83.5 (C-16),
63.7 (C-17), 14.0 (C-18), 23.5 (C-19), 102.8 (C-20), 11.4 (C-21), 151.5
L. Qin et al. / Journal of Ethnopharmacology 118 (2008) 271–279 275

Fig. 1. Chemical structures of isolated compounds from EXD.

276 L. Qin et al. / Journal of Ethnopharmacology 118 (2008) 271–279

Table 2
Effects of isolated compounds on osteoblast and osteoclast (n = 8, x ± S.D.)

Compound Osteoblast proliferation absorbance Osteoblastic ALP activity Osteoclastic TRAP activity
(550 nm) (nmol/(mg min)) (nmol/(min 100 osteoclaste))
10−6 mol/L 10−5 mol/L 10−6 mol/L 10−5 mol/L 10−6 mol/L 10−5 mol/L

Control 0.76 ± 0.02 0.76 ± 0.02 11.0 ± 1.0 11.0 ± 1.0 9.5 ± 1.1 9.5 ± 1.1
Icariin 1.06 ± 0.07** 1.13 ± 0.09** 14.0 ± 0.7** 14.2 ± 0.8** 6.4 ± 0.5* 6.2 ± 0.3**
Icariside I 1.01 ± 0.07** 1.15 ± 0.10** 11.5 ± 0.8 12.1 ± 0.8 8.4 ± 0.8 6.6 ± 0.4**
Baohuoside I 1.07 ± 0.11** 1.17 ± 0.08** 14.5 ± 1.0** 15.2 ± 1.0** 5.9 ± 0.4** 5.6 ± 0.5**
Mangiferin 0.79 ± 0.03 0.78 ± 0.07 11.5 ± 1.2 12.3 ± 1.4 6.0 ± 0.6** 5.8 ± 0.2**
Neomangiferin 0.77 ± 0.05 0.78 ± 0.09 11.2 ± 1.0 11.2 ± 0.8 5.8 ± 0.4** 5.7 ± 0.3**
Berberine 0.80 ± 0.04 0.78 ± 0.08 12.3 ± 0.8 12.4 ± 1.0* 5.9 ± 0.4** 5.8 ± 0.4**
Anemarsaponin B 0.83 ± 0.04** 1.01 ± 0.08** 14.6 ± 1.1** 14.8 ± 1.1** 8.3 ± 0.8 8.6 ± 0.6
Anemarsaponin BII 1.02 ± 0.09** 1.09 ± 0.06** 13.4 ± 0.9** 13.6 ± 1.1** 8.0 ± 1.5 7.9 ± 0.9
Anemarsaponin C 1.00 ± 0.10** 1.06 ± 0.05** 14.7 ± 1.0** 15.0 ± 1.3** 8.6 ± 0.8 8.4 ± 0.6
Anemarrhenasaponin I 1.02 ± 0.09** 1.06 ± 0.09** 14.6 ± 0.9** 15.1 ± 1.3** 7.4 ± 0.8* 7.6 ± 0.7*
Rubiadin-1-methyl ether 0.79 ± 0.05 0.81 ± 0.07 11.6 ± 1.2 11.9 ± 1.3 8.4 ± 1.0 7.3 ± 0.8*
Obaculactone 0.78 ± 0.07 0.80 ± 0.04 12.3 ± 1.5 12.2 ± 1.0 6.0 ± 0.3** 6.3 ± 0.4**

*P < 0.05, **P < 0.01 compared with control.

(C-22), 32.5 (C-23), 22.7 (C-24), 33.7 (C-25), 73.8 (C-26), 16.7 (C- 14), 79.1 (C-15), 91.4 (C-16), 61.5 (C-17), 18.1 (C-18), 24.2 (C-19),
27), 103.2 (gal-C1 ), 78.9 (gal-C2 ), 73.3 (gal-C3 ), 67.9 (gal-C4 ), 73.0 40.8 (C-20), 16.6 (C-21), 110.3 (C-22), 37.7 (C-23), 33.7 (C-24), 28.7
(gal-C5 ), 61.0 (gal-C6 ), 103.6 (glu-C1 ), 74.6 (glu-C2 ), 76.6 (glu- (C-25), 22.9 (C-26), 22.8 (C-27), 102.5 (Gal-C1 ), 81.6 (Gal-Gal-C2 ),
C3 ), 70.0 (glu-C4 ), 76.8 (glu-C5 ), 61.0 (glu-C6 ), 100.6 (glu-C1  ), 75.2 (Gal-Gal-C3 ), 69.9 (Gal-Gal-C4 ), 76.6 (Gal-Gal-C5 ), 62.2 (Gal-
74.9 (glu-C2  ), 75.9 (glu-C3 ), 70.0 (glu-C4 ), 76.9 (glu-C5 ), 60.0 C6 ) 105.9 (Glu-C1 ), 76.7 (Glu-C2 ), 77.8 (Glu-C3 ), 71.5 (Glu-C4 ), 78.2
(glu-C6  ). The 1 H and 13 C NMR were in agreement with literature (Glu-C5 ), 62.6 (Glu-C6 ). The 1 H and 13 C NMR were in agreement
values (Dong and Han, 1992). with literature values (Meng and Xu, 1998).
Anemarsaponin B-II (8): white powder, ESIMS: m/z 921 [M+H]+ ; Rubiadin-1-methyl ether (11): yellow needle, ESIMS: m/z 267
molecular formula: C45 H76 O19 . 1 H NMR: 0.88 (3H, s, 18-CH3 ), 0.98 [M−H]− ; molecular formula: C16 H12 O4 ; 1 H NMR (DMSO-d6 , ı,
(3H, s, 19-CH3 ), 1.03 (3H, d, J = 6.5 Hz, 27-CH3 ), 1.32 (3H, s, 21-CH3 ), ppm): 2.16 (3H, s, 2-CH3 ), 3.79 (3H, s, 1-OCH3 ), 7.51 (1H, s, 4-H),
4.80 (1H, d, J = 8 Hz, glu-1 -H), 4.91 (1H, d, J = 8 Hz, gal-1 -H), 5.28 7.82–7.91 (2H, m, 6-H, 7-H), 8.10–8.16 (2H, m, 5-H, 8-H), 11.1 (1H,
(1H, d, J = 7 Hz, glu-1 -H). 13 C NMR (DMSO-d6 , ı, ppm): 30.6 (C-1), br s, 3-OH). 13 C NMR (DMSO-d6 , ı, ppm): 160.1 (C-1), 134.4 (C-2),
26.7 (C-2), 74.8 (C-3), 30.6 (C-4), 36.6 (C-5), 26.5 (C-6), 26.5 (C-7), 161.5 (C-3), 108.9 (C-4), 133.2 (C-4a), 126.0 (C-5), 133.7 (C-6), 134.5
35.2 (C-8), 40.3 (C-9), 34.9 (C-10), 20.9 (C-11), 40.1 (C-12), 40.9 (C- (C-7), 126.6 (C-8), 135.0 (C-8a), 182.5 (C-9), 108.9 (C-9a), 180.1 (C-
13), 56.1 (C-14), 32.1 (C-15), 80.9 (C-16), 63.7 (C-17), 16.4 (C-18), 10), 133.0 (C-10a), 9.5 (CH3 ), 60.5 (OCH3 ). The 1 H and 13 C NMR were
23.7 (C-19), 40.1 (C-20), 16.1 (C-21), 110.4 (C-22), 36.8 (C-23), 28.0 in agreement with literature values (Yang et al., 1992).
(C-24), 34.1 (C-25), 75.1 (C-26), 17.2 (C-27), 102.2 (gal-C1 ), 81.4 (gal- Obaculactone (12): white needle, ESIMS: m/z 470 [M+ ]; molec-
C2 ), 76.2 (gal-C3 ), 69.5 (gal-C4 ), 76.5 (gal-C5 ), 62.5 (gal-C6 ), 105.7 ular formula: C26 H30 O8 ; 1 H NMR (DMSO-d6 , ı, ppm): 7.71 (1H, s,
(glu-C1 ), 75.2 (glu-C2 ), 78.0 (glu-C3 ), 71.3 (glu-C4 ), 78.3 (glu- H-21), 7.65 (1H, t, J = 1.4 Hz, H-23), 6.51 (1H, d, J = 1.2 Hz, H-22), 5.48
C5 ), 62.5 (glu-C6 ), 104.8 (glu-C1 ), 74.9 (glu-C2 ), 78.1 (glu-C3  ), (1H, s, H-l7), 4.92 (1H, d, J = 13.0 Hz, H-l9), 4.48 (1H, d, J = 13.0 Hz,
71.4 (glu-C4  ), 77.7 (glu-C5 ), 61.9 (glu-C6 ). The 1 H and 13 C NMR H-l9), 4.12 (2H, s, H-1, 15), 3.13 (1H, t, J = 15.0 Hz), 2.77 (1H, d,
were in agreement with literature values (Dong and Han, 1992). J = 16.2 Hz), 2.63 (1H, dd, J = 3.6, 16.5 Hz), 2.56 (1H, br s), 2.46 (1H,
Anemarsaponin C (9): white powder, ESIMS: m/z 903 [M+H]+ ; dd, J = 2.7, 16.2 Hz), 2.28 (1H, dd, J = 2.9, 14.7 Hz), 1.77 (3H, m), 1.35
molecular formula: C45 H74 O18 . 1 H NMR (DMSO-d6 , ı, ppm): 0.71 (1H, m), 1.19 (3H, s), 1.12 (3H, s), 1.03 (3H, s), 1.01 (3H, s). 13 C NMR
(3H, s, 18-CH3 ), 1.01 (3H, s, 19-CH3 ), 1.08 (3H, d, J = .8 Hz, 27-CH3 ), (DMSO-d6 , ı, ppm): 208.1 (C O, C-7), 170.3 (C O, C-3), 167.4 (C O,
1.68 (3H, s, 21-CH3 ), 2.54 (1H, d, J = 10.3 Hz, 17-H), 4.86 (1H, d, C-l6), 143.5 ( CH, C-23), 141.9 ( CH, C-21), 120.4 (C-14), 65.0 (C-19),
J = 7.8 Hz, Glu-1 -H), 4.99 (1H, d, J = 7.3 Hz, Glu-1 -H), 5.49 (1H, d, 58.2 (C-5), 54.0 (C-l5), 46.7 (C-9), 45.5 (C-l0), 37.8 (C-13), 36.3 (C-6),
J = 7.3 Hz, Glu-1-H). 13 C NMR (DMSO-d6 , ı, ppm): 31.0 (C-1), 26.8 (C- 35.8 (8 (C-2), 9.9 (9 ( CH3 ), 29.4 (C-l2), 21.6 ( CH3 ), 19.8 ( CH3 ),
2), 75.2 (C-3), 30.9 (C-4), 36.8 (C-5), 26.8 (C-6), 26.8 (C-7), 35.1 (C-8), 17.7 (C-11), 17.2 ( CH3 ). The 1 H and 13 C NMR were in agreement
40.1 (C-9), 35.1 (C-10), 21.3 (C-11), 40.0 (C-12), 43.8 (C-13), 54.7 (C- with literature values (Liang and Li, 1995).
14), 31.3 (C-15), 84.5 (C-16), 64.6 (C-17), 14.3 (C-18), 24.0 (C-19),
103.5 (C-20), 11.8 (C-21), 152.3 (C-22), 34.3 (C-23), 23.6 (C-24), 33.6 Table 3
(C-25), 75.2 (C-26), 17.1 (C-27), 101.9 (glu-C1 ), 83.1 (glu-C2 ), 78.5 Effects of icariin, berberine and anemarsaponin BII from EXD on the BMD of ovaric-
(glu-C3 ), 71.7 (glu-C4 ), 78.2 (glu-C5 ), 62.8 (glu-C6 ), 105.9 (glu- tomized rat (n = 10)
C1 ), 77.0 (glu-C2 ), 77.9 (glu-C3 ), 71.5 (glu-C4 ), 78.5 (glu-C5 ), 62.6 Groups BMD (g/cm2 )
(glu-C6 ), 105.1 (glu-C1  ), 75.2 (glu-C2 ), 78.5 (glu-C3 ), 71.6 (glu-
Sham 0.271 ± 0.011**
C4  ), 78.2 (glu-C5  ), 62.8 (glu-C6 ). The 1 H and 13 C NMR were in OVX control 0.247 ± 0.009
agreement with literature values (Ma et al., 1996). OVX + nylestriol 0.261 ± 0.011**
Anemarrhenasaponin-I (10): white powder, ESIMS: m/z 757 OVX + icariin (25 mg/kg) 0.254 ± 0.008
[M−H]− ; molecular formula: C39 H66 O14 . 1 H NMR (DMSO-d6 , ı, OVX + icariin (125 mg/kg) 0.258 ± 0.008**
OVX + berberine (30 mg/kg) 0.248 ± 0.007
ppm): 0.88 (3H, d, 18-CH3 ), 0.98 (3H, s, 19-CH3 ), 1.03 (3H, s, 27-CH3 ),
OVX + berberine (90 mg/kg) 0.259 ± 0.007**
1.34 (3H, d, 21-CH3 ), 4.90 (1H, d, Glu-1-H), 5.27 (1H, d, Gal-1-H), OVX + anemarsaponin BII (50 mg/kg) 0.259 ± 0.007**
5.10 (1H, dd). 13 C NMR (DMSO-d6 , ı, ppm): 31.0 (C-1), 27.2 (C-2), OVX + anemarsaponin BII (100 mg/kg) 0.257 ± 0.008*
75.5 (C-3), 31.1 (C-4), 37.1 (C-5), 27.0 (C-6), 26.8 (C-7), 36.4 (C-8), 
P < 0.01 compared with sham group; *P < 0.05, **P < 0.01 compared with OVX
40.4 (C-9), 35.4 (C-10), 21.2 (C-11), 41.2 (C-12), 41.3 (C-13), 60.9 (C- control group.
 L. Qin et al. / Journal of Ethnopharmacology 118 (2008) 271–279 277

Table 4
Effects of icariin, beriberine and anemarsaponin BII on static parameters of proximal tibia cancellous bone histomorphometry in rats induced by ovariectomy (n = 10)

Groups Percent trabecular area (%) Trabecular thickness (␮m) Trabecular number (mm−1 ) Trabecular separation (␮m)

Sham 24.0 ± 2.9 70.1 ± 10.8 3.4 ± 0.3 222.3 ± 22.8

OVX control 5.3 ± 1.1 43.6 ± 1.4 1.3 ± 0.2 1267.4 ± 127.6
OVX + nylestriol 13.6 ± 2.3** 57.4 ± 4.8* 2.4 ± 0.3* 372.5 ± 52.1**
OVX + icariin (25 mg/kg) 8.2 ± 3.4 64.0 ± 8.9* 1.3 ± 0.6 1203.0 ± 368.2
OVX + icariin (125 mg/kg) 6.9 ± 3.3 60.1 ± 6.8* 1.1 ± 0.5 732.9 ± 383.8*
OVX + berberine (30 mg/kg) 5.2 ± 3.0 58.1 ± 5.0* 0.9 ± 0.5 1298.7 ± 604.6
OVX + berberine (90 mg/kg) 6.2 ± 1.4 64.4 ± 5.8* 1.0 ± 0.3 1036.8 ± 368.5
OVX + anemarsaponin BII (50 mg/kg) 7.1 ± 3.3 58.8 ± 5.0 1.2 ± 0.5 792.5 ± 141.4**
OVX + anemarsaponin BII (100 mg/kg) 12.2 ± 1.6** 69.3 ± 9.9* 1.2 ± 0.6 768.5 ± 145.1**

P < 0.01 compared with sham group; *P < 0.05, **P < 0.01 compared with OVX control group.

Among active compounds, icariin, icariside I, baohuoside I
increased significantly osteoblast proliferation and ALP activity, and
inhibited the osteoclast TRAP activity at a concentration of 10−5 M
and 10−6 M (Table 2). Icariin also caused an increase of bone min-
eral densities in ovariectomized rats at dose of 125 mg/kg (Table 3).
Furthermore, the histomorphometric analysis results showed that
in OVX rats treated with icariin, the trabecular thickness increased,
and trabecular separation decreased; % label perimeters increased
and osteoclast number decreased (Tables 4 and 5). This indicated
that the reduction of bone loss in OVX rats by icariin was due to
the increase on the bone formation and the decrease on the bone
resorption.
Anemarsaponin B, anemarsaponin BII, anemarsaponin C and
anemarrhenasaponin I, which are steroidal saponin compounds,
significantly stimulated osteoblastic proliferation and ALP activity,
but did not show any activity on osteoclast (Table 2). In ovariec-
tomized rat, anemarsaponin BII (100 mg/kg) caused an increase of
bone mineral densities (Table 3), an increase in the trabecular thick-
ness and % trabecular area, and reduction in trabecular separation in
OVX rats. The parameters of bone formation such as % label perime-
ters, bone formation rate/bone volume (BFR/BV), increased at the
dose of 100 mg/kg (Tables 4 and 5). These findings indicated that
Anemarsaponin BII caused the reduction of bone loss through the
promotion of bone formation but not the inhibition of bone resorp-
tion, and this is different from estrogen. In addition, flavonoids
constituent mangiferin, neomangiferin from Anemarrhena aspho-
deloides did not show any activity on osteoblast at a concentration
of 10−6 M and 10−5 M, respectively (Table 2), but showed inhibitory
activity on the TRAP activity of osteoclast.
Berberine and obaculactone, which comes from the bark of Phel-
lodendron chinense, inhibited TRAP activity of osteoclast and did not
have any effects on the proliferation and ALP activity of osteoblast
(Table 2). In ovariectomized rats, berberine increased bone den-
sity and trabecular thickness, and decreased the osteoclast number
at the dose of 30 mg/kg and 90 mg/kg (Tables 3–5). These results
showed that the inhibitory effects on bone loss of berberine and
obaculactone were produced through the inhibition of bone resorp-
tion but not the promotion of bone formation.
Rubiadin-1-methyl ether, which is anthraquinones from
Morinda officinalis, did not show any activity on osteoblast, but
showed inhibitory activity on the TRAP activity of osteoclast at
a concentration of 10−6 M and 10−5 M (Table 2). And in Morinda
officinalis, there are various anthraquinones constituents including
rubiadin, rubiadin-1-methyl, 1-hydroxyanthraquinone and so on
(Yang et al., 1992), and it have been reported that aqueous extract
of Morinda officinalis could evidently suppress the bone loss in
osteoporotic mice induced by sciatic neurectomy surgery (Seo et
al., 2005). Therefore, rubiadin-1-methyl ether and its derivatives
may be the antiosteoporotic constituents in Morinda officinalis.
In addition, there are polysaccharide substances from Morinda
officinalis and Angelica sinensis in EXD. In the bioactivity guided
fraction process, they existed in the water elutes on macroporous
resins, and did not show any activity on osteoblast and osteo-
clast. It is not clear whether these substances really did not have
antiosteoporotic activity or their effects were counteracted by other
substances.
Phenols and phenolic glycosides from Curculigo orchioides pos-
sess the estrogen-like activity (Vijayanarayana et al., 2007). But in
our study, these compounds were not obtained. Whether these
compounds possess the antiosteoporotic activity, this is worth of
being further studied.
The theory of traditional Chinese medicine thinks that the action
of a single drug is usually limited, and some of them may pro-
duce certain side effects or even toxicity, but when several drugs
are applied together, they will display their superiority over a sin-
gle drug in the treatment of disease. Our findings showed that
both isoflavonoids and steroidal saponins constituents in EXD could
stimulate osteoblast proliferation, alkaline phosphatase activity,
and thus increase the bone formation, and isoflavonoids, berberine,
obaculactone and rubiadin-1-methyl ether could inhibit the TRAP
activity of osteoclast and bone resorption, and thus reduce the bone
loss. We also found that EXD increase the bone density of ovariec-
tomized rat more than any single compounds in it (results was
not shown in this paper). These results indicated that these com-
pounds used simultaneously can enhance the therapeutic effects,
and further explained the traditional Chinese medicine theory that

Table 5
Effects of icariin, berberine and anemarsaponin BII on dynamic parameters of proximal tibia cancellous bone histomorphometry in rats induced by ovariectomy (n = 10)

Groups % Label Mineral rate (␮m day−1 ) BFR/BV (%/year) Osteoclast number

sham 22.9 ± 5.1 0.78 ± 0.08 141.7 ± 54.1 3.5 ± 1.8

OVX control 33.8 ± 2.9 1.21 ± 0.08 402.6 ± 44.9 11.9 ± 1.5
nylestriol 24.1 ± 3.7* 0.76 ± 0.13* 194.2 ± 48.9* 10.2 ± 2.7
icariin (25 mg/kg) 34.0 ± 9.3 1.09 ± 0.17 363.2 ± 138.2 8.0 ± 1.9*
icariin (125 mg/kg) 38.5 ± 3.7* 1.02 ± 0.21 314.8 ± 178.7 3.0 ± 2.2**
berberine (30 mg/kg) 30.4 ± 14.8 1.24 ± 0.12 510.8 ± 139.0 9.5 ± 3.0
berberine (90 mg/kg) 39.6 ± 4.5 1.15 ± 0.19 426.2 ± 40.9 8.4 ± 0.8*
anemarsaponin BII (50 mg/kg) 27.8 ± 4.9 1.16 ± 0.19 334.1 ± 59.9 14.2 ± 1.3
anemarsaponin BII (100 mg/kg) 45.5 ± 2.9** 1.28 ± 0.11 517.5 ± 104.0* 11.5 ± 1.8

P < 0.01 compared with sham group; *P < 0.05, **P < 0.01 compared with OVX control group.
278 L. Qin et al. / Journal of Ethnopharmacology 118 (2008) 271–279
drugs of similar action, if used simultaneously, strengthen the ther-
apeutic effect for serious disease, or two or more ingredients with
similar properties and effects are used in combination to reinforce
each other’s action. Secondly, these compounds in combination in
EXD not only can increase bone density, reduce bone loss, but also
improve other syndrome of osteoporosis, and thus broaden the
therapeutic scope in the treatment of complex conditions.
According to the theory of traditional Chinese medicine, in EXD,
Epimedium sagittatum and Phellodendron chinense is an essential
ingredient, plays a leading curative role aiming at the cause or the
main syndrome of menopausal syndrome and osteoporosis. Cur-
culigo orchioides, Morinda officinalis helps strengthen the curative
effect of the Epimedium sagittatum, and Anemarrhena asphode-
loides helps Phellodendron chinense to play a full role. Angelica
sinensis are the ingredient to cooperate with above five drugs
to strengthen the therapeutic effects and treat the accompany-
ing disease or syndromes. Our findings showed that essential
antiosteoporotic compounds are isoflavonoids from Epimedium
sagittatum and berberine from Phellodendron chinense, steroidal
saponins from Anemarrhena asphodeloides and rubiadin-1-methyl
ether from Morinda officinalis helps further improve therapeutic
effects. Active constituents from Angelica sinensis did not show any
activity on the bone. Perhaps the major activities of Angelica sinen-
sis are mainly to nourish the blood, promote blood circulation, and
relieve pain and other syndromes. This will be further studied in
the future work. These results further supported the viewpoint of
traditional Chinese medicine.
The theory of traditional Chinese medicine thinks that the kid-
ney governs bones. It means that the development of the bones
depend on the functions of the kidney. In EXD formula, Epimedium
sagittatum, Curculigo orchioides and Morinda officinalis have the
effects of invigorating kidney and strengthening bones. In our study,
isoflavonoids from Epimedium sagittatum and Rubiadin-1-methyl
ether from Morinda officinalis also showed the effects of reducing
bone loss. This further verified the thoughts of traditional Chinese
medicine that the bone could be strengthened by nourishing kid-
ney.
4. Conclusion
In the present study, we examined the extracts and fractions
of Er-Xian Decoction together with isolated compounds for their
antiosteoporotic activities. The isolation of active constituents
was achieved by monitoring the proliferation and ALP activity
of osteoblastic cells and TRAP activity of osteoclastic cells. The
icariin (1), icariside I (2), baohuoside I (3) and anemarrhenas-
aponin I (10) not only increased the osteoblast proliferation and
ALP activity, but also decreased the TRAP activity of osteoclast.
The anemarsaponin B (7), anemarsaponin BII (8), anemarsaponin
C (9) only stimulated osteoblast activity, whilst mangiferin (4),
neomangiferin (5), berberine (6), rubiadin-1-methyl ether (11)
and obaculactone (12) only reduced the osteoclastic TRAP activity.
Furthermore, the antiosteoporotic effects of major active chem-
ical constituents icariin, berberine and anemarsaponin BII were
investigated in ovarictomized rats, and they all increased the
bone mineral density, restored the change of bone histologi-
cal morphology, and displayed strong antiosteoporotic activity.
These twelve compounds isolated from EXD are responsible for
antiosteoporotic activity. These findings also confirm that multi-
ple ingredients are responsible for therapeutic effects in traditional
Chinese medicine formula. Further studies on the action of syn-
ergistic, competition, equilibrium and feedback among active
compounds, and their antiosteoporotic pathway and integrative
mechanisms are in progress and will be the subject of another
paper.
Acknowledgment
The authors are indebted to the National Natural Science Foun-
dation of China (Project No. 90209043).
References
Bradford, M.M., 1976. A rapid and sensitive method for the quantization of micro-
gram quantities of protein utilizing the principle of protein–dye binding.
Analytical Biochemistry 72, 248–254.
Cassidy, A., 2003. Dietary phytoestrogen and bone health. Journal of the British
Menopause Society 9, 17–21.
Chen, S.G., Fang, F., 1998. The Zhi An Shen’ s experience of Ex Xian Tang treat-
ment for menopausal Syndrome. Jiangxi Journal of Traditional Chinese Medicine
29, 5.
Davison, S., Davis, S.R., 2003. Hormone replacement therapy: current controversies.
Clinical Endocrinology 58, 249–261.
Dong, J.X., Han, G.Y., 1992. Studies of the active constituents of Anemarrhena aspho-
deloides Bunge. Acta Pharmaceutica Sinica 27, 26–32.
Fang, Z.Q., Si, F.C., Song, J.M., Liao, H., Shi, J.L., 1997. Effect of Erxian decoction and its
partial constituents on the hypothalamic–pituitary–gonadal axis in aged rats.
Geriatrics and Health Care 3, 12–14.
Hong, Y.F., Han, G.Y., Guo, X.M., 1997. Isolation and structure determination of xan-
tone glycosides of Anemarrehena asphodeloides. Acta Pharmaceutica Sinica 32,
473–475.
Hu, W.Y., Du, J.A., Qian, D.W., Wang, D.W., 2007. Studies on chemical constituents
in fresh fleshy scaleleaf of Lifium lancifolium. China Journal of Chinese Materia
Medica 32, 1656–1659.
Li, H.Y., Miyahara, T., Tezuka, Y., Namba, T., Suzuki, T., Dowaki, R., Watanabe, M.,
Nemoto, N., Tonami, S., Seto, H., 1999. The effect of kampo formulae on bone
resorption in vitro and in vivo. II. Detail study of berberine. Biological and Phar-
maceutical Bulletin 22, 391–396.
Li, J.J., Li, J.T., Fu, J.P., 2007. Erxian Tang introduction of a Chinese herbal formula, clin-
ical practice, and experimental studies. Chinese Journal of Integrated Medicine
13, 67–73.
Li, Y., 2004. Er Xian Tang combined with Gan Mei Da Zao Tang was treated in 118
female case of menopausal syndrome. Shanghai Journal of Traditional Chinese
Medicine 38, 43–44.
Liang, L., Li, G.Y., 1995. Studies on the chemical constituents of Phellodendron chinense
var. glabriusculum. Zhong Yao Cai 18, 85–86.
Liu, Q., Shi, J.R., Yang, Y., Fang, Z.Q., Liang, S.H., Guo, R.X., 2005. Effects on secretory
function of rat gonad by Er-Xian Decoction and its disassembled prescriptions.
China Journal of Chinese Materia Medica 30, 1023–1026.
Liu, Z.D., Zang, H.S., Ouyang, Y.P., 1995. Biological study of growth pattern of
newborn rat calvaria osteoblastic cell in vitro. Acta Anatomic Sinica 26,
157–159.
Ma, W.P., Dong, J.X., Wang, B.J., Yan, X.Z., 1996. Studies on the furostanol
saponins from Anemarrhena asphodeloides Bunge. Acta Pharmaceutica Sinica
31, 271–277.
Meng, Z.Y., Xu, S.X., 1998. Saponins from Anemarrhena asphodeloides Bge. Chinese
Journal of Medicinal Chemistry 8, 135–136.
Messina, M., Messina, V., Soyfoods, 2000. Soybean isoflavones and bone health: a
brief overview. Journal of Renal Nutrition 10, 63–68.
Nian, H., Qin, L.P., Zhang, Q.Y., Zheng, H.C., Yu, Y., Huang, B.K., 2006. Antiosteo-
porotic activity of Er-Xian Decoction, a traditional Chinese herbal formula, in
ovariectomized rats. Journal of Ethnopharmacology 108, 96–102.
Owen, T.A., Aronow, M., Shalhoub, V., Barone, L.M., Wilming, L., Tassinari, M.S.,
Kennedy, M.B., Pockwinse, S., Lian, J.B., Stein, G.S., 1990. Progressive development
of the rat osteoblast phenotype in vitro: reciprocal relationship in expression of
genes associated with osteoblast proliferation and differentiation during for-
mation of the bone extracellular matrix. Journal of Cellular Physiology 143,
420–430.
Qin, L.P., Zhang, Q.Y., Tian, Y.P., Zheng, H.C., Huang, M., Huang, B.K., 2003. Total
coumarins from fruits of Cnidium monnieri (TCFC) inhibit the formation and dif-
ferentiation of multinucleated osteoclasts derived from rat marrow cells. Acta
Pharmacologica Sinica 24, 181–186.
Seo, B., Ku, S.K., Cha, E.M., Park, J.H., Kim, J.D., Choi, H.Y., Lee, H.S., 2005. Effect of
Mornidae radix extracts on experimental osteoporosis in sciatic neurectomized
mice. Phytotherapy Research 19, 231–238.
Shen, X.H., Fang, Z.Q., Wu, D.X., 1995. Effect of Er-Xian Decoction and its disassem-
bled prescription on enzyme activity and their gene expression of antioxidant
enzymes in aging rat. Chinese Journal of Integrated Traditional and Western
Medicine 15, 672–674.
Tuner, R.T., Riggs, B.L., Spelsberg, T.C., 1994. Skeletal effects of estrogen. Endocrine
Reviews 15, 275–300.
Vijayanarayana, K., Rashmi, S.R., Chandrashekhar, K.S., Subrahmanyam, E.V.S., 2007.
Evaluation of estrogenic activity of alcoholic extract of rhizomes of Curculigo
orchioides. Journal of Ethnopharmacology 114, 241–245.
Wang, C.H., Zhang, Z.H., Ma, J., 1998. Treatment of 50 cases of osteoporosis with
Er-Xian Decoction. Shan Xi Zhong Yi 19, 205.
Wang, M.Q., Peng, X., Gan, Q.F., 2005. Studies on chemical constituents of
Epimedium brevicornum Maxim. Research and Practice of Chinese Medicine 19,
39–42.
 L. Qin et al. / Journal of Ethnopharmacology 118 (2008) 271–279 279

Wang, Z.Q., Lou, Y.J., 2004. Proliferation-stimulating effects of icaritin and
desmethylicaritin in MCF-7 cells. European Journal of Pharmacology 504,
147–153.
Yang, Y.J., Shu, H.Y., Min, Z.D., 1992. Anthraquinones isolated from Morinda
officinalis and Damnacanthusindicus. Acta Pharmaceutical Sinica 27,
358–364.
Zambonin, Z.A., Teti, A., Primavera, M.V., 1982. Isolated osteoclasts in primary cul-
ture: first observation on structure and survival in culture media. Anatomy and
Embryology 165, 405–413.
Zheng, X.W., Bao, S.Z., Li, R.Q., Song, H., Liu, M.Z., 2003. Effects of Er-Xian Decoction
on gene expression of ACTH in pituitary tissue in kidney-yang deficiency rats.
Zhongguo Yi Yao Xue Bao 18, 716–717.