Effects of a herbal extract on the bone density, strength and markers of bone turnover of mature ovariectomized rats

Effects of a herbal extract on the bone density, strength and markers of bone turnover of mature ovariectomized rats

Min Xu

Abstract: For many decades, the Chinese have been using herbal medications to treat bone diseases. To examine effects of an extract of ten medicinal herbs on estrogen deficiency bone loss, ten-month-old female rats were randomly divided into three groups: ovariectomized (OVX), OVX treated with herbs (OVX-M) 4 ml/day by gavage, and OVX treated with estrogen (OVX-E) 10 mg subcutaneously (s.c.) twice per week. The bone mineral density (BMD) of the left femur (fBMD), spine (sBMD) and global body (gBMD) were measured at baseline and at 4, 8 and 12 weeks using a Hologic QDR 2000 dual-energy X-ray densitometer. Tibial strength was tested using the Instron Model 5566 electro-mechanical testing machine. The urinary pyridinoline creatinine ratio (Pyd/Cr), deoxypyridinoline creatinine ratio (Dpd/Cr), plasma alkaline phosphatase (ALP), calcium (CA), phosphorus (P) and albumin (ALB) were also determined. Uterine weight was determined at 12 weeks. The results showed that percent changes of fBMD in the OVX (n = 9), OVX-E (n = 8) and OVX-M (n = 8) rats at the 12-week time point were -11.8 [+ or -] [4.6.sup.c], 1.8 [+ or -] [3.1.sup.a], -7.6 [+ or -] [1.9.sup.abc] (p < 0.05-0.001, a: vs. OVX, b: vs. OVX-E, c: vs. baseline); sBMD were -10.7 [+ or -] [4.6.sup.c], -0.3 [+ or -] [5.5.sup.a], -5.9 [+ or -] [3.5.sup.abc]; and gBMD were -4.8 [+ or -] [2.3.sup.c], 0.1 [+ or -] [2.4.sup.a], -2.7 [+ or -] [2.6.sup.abc], respectively. Further, the tibia maximum breaking stress and flexural modulus of elasticity in OVX-M rats (295 [+ or -] [33.sup.a], 18194 [+ or -] [3264.sup.a]) were significantly higher (p < 0.005-0.001) than that in OVX rats (189 [+ or -] 83, 10309 [+ or -] 4930), and similar to OVX-E rats (298 [+ or -] [35.sup.a], 18766 [+ or -] [2620.sup.a]). Additionally, the herbal extract reduced the urinary Pyd/Cr, Dpd/Cr and plasma ALP increment followed OVX and was not associated with a rise in uterine weight. In conclusion, the herbal extract demonstrated a therapeutic effect to inhibit bone resorption and to reduce estrogen-dependent bone loss without uterine stimulation. It may have potential as a new approach in treating and preventing postmenopausal osteoporosis (PMOP).

Keywords: Herb Medicine; Bone Mineral Density; Bone Strength; Bone Marker; Ovariectomized Rat.

Introduction

Osteoporosis is a rapidly growing global health problem. It is characterized by low bone mass and microarchitectural deterioration of bone tissue leading to enhanced bone fragility. Bone mineral density (BMD) and bone mineral content (BMC) are significantly reduced, and the incidence of fracture increases (Garnero and Delmas, 1997; Hans et al., 1997). Quality of life in fracture patients can be seriously reduced (Hall et al., 1999) and five year mortality after hip or vertebral fracture is about 20% in excess of that expected (Center et al., 1999; Cooper, 1997). In China, the population of elderly people is more than one hundred million with a high incidence of osteoporosis in those aged 60 years and above. Thus, over 60 million Chinese may be affected, with every one in ten of them sustaining a subsequent fracture (Tong et al., 1996; Zhu et al., 1990).

The decline of gonadal function plays a key role in postmenopausal osteoporosis (PMOP), and there is a general agreement that hormone replacement therapy (HRT) is reliable in treating and preventing PMOP and reducing the risk of fracture (Notelovitz, 1997; Prince et al., 1991). Other potential benefits of HRT are cardioprotection, which may be related to the effects of estrogen on lipid profile and fibrinogen levels (Gibaldi, 1997). However, patients have to take HRT for a long time, and the balance between long-term benefits and risks remain unclear. Some epidemiological investigations have suggested that long-term HRT increase the risk of breast cancer, endometrial cancer, endometriosis and venous thrombo-embolism (Colditz, 1997; Gibaldi, 1997; Whitehead and Godfree, 1997). The identification of medicines for PMOP that are both effective and safe is still one of the most important aims in osteoporosis research.

Few Chinese women take hormone preparations regularly for health purposes. In postmenopausal women the main reason for this is concern over side effects such as postmenopausal bleeding (Christiansen and Riis, 1990; Prince et al., 1991) and the dangers of certain malignant tumors. Another important reason is cultural attachment to traditional herbal medicines. We have previously reported a preliminary study of the effects of a Chinese herbal preparation on bone density in the ovariectomized (OVX) rat model of PMOP. The results showed that this herbal preparation significantly reduced the bone loss (Xu et al., 1994). Based on this previous study, we have tested a new preparation of these herbs. We used OVX rats, which are an accepted model of PMOP (Kalu et al., 1991; Mitlak et al., 1994; Wronski et al., 1986, 1989 and 1991) and have undertaken observations on bone mass, strength and biochemical markers of bone turnover, to further study the potential role of the herbal medicine on postmenopausal bone loss.

Materials and Methods

Herbal Extract

The herbal extract consisted of Psoralea corylifolia L. (Leguminosae), Epimedium koreanum Nakai. (Berberidaceae), Cuscuta chinensis Lam. (Convolvulaceae), Cistanche deserticola Y.C.Ma. (Orobanchaceae), Eucommia ulmoides Oliv. (Eucommiaceae), Astragalus membranaceus (Fisch.) Bge. (Leguminosae), Pueraria thomsonii Benth. (Leguminosea), Ziziphus jujuba Mill. (Rhamnaceae), Salvia miltiorrhiza Bge. (Labiatae) and Angelica sinensis (Oliv.) Diels. (Umbelliferae) (Cathey Herbal Laboratories Pty. Ltd. Sydney, NSW, Australia). Specimens of these plant materials were authenticated by the Department of Pharmacognosy, Guangzhou University of Chinese Medicine. An aqueous extract was made by boiling equal weights of each herb three times to make a decoction (1 g/ml, w/v).

Animal and Treatment

Twenty-seven female six-month-old Sprague-Dawley strain rats (Animal Resources Center, Murdoch, Western Australia) were housed at 26[degrees]C on 12-hour light, 12-hour dark cycles until they were ten months old (average body weight 286 [+ or -] 18 g). They were randomly divided into three groups, an ovariectomized group (OVX), OVX group treated with medical herbal decoction (OVX-M), and an OVX group treated with estrogen (OVX-E). They were pair-fed 15 g of rat diet per day containing 0.4% calcium and 0.3% phosphorus (Glen Forrest Stockfeeders, Glen Forrest, Western Australia) from 8 weeks before OVX to 4 weeks after OVX, and then pair-fed 17 g of this diet until the end of the 12-week experiment. Tap water was freely available.

From the 2nd day to 12 weeks after operation, the rats in OVX-M group were given the herbal decoction 4 ml/day by gavage. The rats in other groups were given water by gavage. The rats in OVX-E group were given 10 [micro]g estradiol s.c. twice per week, and the rats in other groups were injected vehicle castor oil alone. The bone mass of the rats in all three groups was analyzed by DXA measurement at baseline and 4, 8 and 12 weeks after ovariectomy. The fasting blood samples were taken at the same time points for biochemical analyses. Urine samples at baseline, 1, 4, 8 and 12 weeks of postoperation were collected after a 24-hour fasting. The protocol was carried out according to the Australia National Health and Medical Research Council guidelines for animal research and approved by the Animal Experimentation Ethics Committee of the University of Western Australia.

DXA Analyses

The BMD (g/[cm.sup.2]) of the left femoral (fBMD), spinal (sBMD) and global (gBMD) sites at baseline, 4, 8 and 12 weeks postovariectomy were determined with a Hologic QDR 2000 dual-energy X-ray bone densitometer using the small animal software (Hologic, Waltham, MA, USA). Following anesthesia with a peritoneal injection of a 1:1 mixture of ketamil and xylazil-20 (Troy Laboratories Pty. Ltd., NSW, Australia), the rat was placed in the prone position on a lucite block. The left femur was scanned with the Hologic rat subregion Hi-res scan protocol (version 4.57), the whole body was scanned with the Hologic rat whole body scan protocol (version 5.57) in which the lumber spine was analyzed as a subregion of interest.

To evaluate the short-term precision of the DXA instrument and analytic technique used in this study, 10 rats were scanned twice with an interval of 16 days before ovariectomy. The coefficient of variations (CV) was calculated using the following formula: CV = [([summation of][d.sup.2]/mean).sup.-2]/2n x 100%. The short-term CV of femoral, spinal, global BMD were 1.39-3.52%, respectively. The long-term stability of the measurements was assured by scanning the manufacturers spine phantom using spine analysis software and a frozen rat using the small animal software (Mitlak et al., 1994). The results of these tests were evaluated using the Shewart Rules for long-term stability (Orwoll and Oviatt, 1991).

Biomechanical Analyses

An electro-mechanical (universal) testing machine (Model 5566, Instron, High Wycombe, UK) used in conjunction with Instron Merlin software (version 4.03) was employed for all tests. The left tibia from each rat of the three groups was cleaned of its surrounding soft tissue and maintained at -20[degrees]C until analysis. The tibiae were loaded to failure in a three-point bending apparatus. The outer two supporting points were fixed 20 mm apart with a single central point positioned at the midpoint of the specimen. All loading points were 3 mm in diameter. The diameter of the midpoint of each tibia was measured and recorded. The central loading point was displaced, and the load and displacement recorded until the specimen had been broken. The bending stiffness was then derived from the slope of the linear region of the resulting load versus displacement curve, and the bending stress and flexural modulus were calculated using the cross-section diameter.

Biochemical Analyses

Urinary pyridinoline (Pyd) and deoxypyridinoline (Dpd) were extracted from urine specimens by isolation on a cellulose column. They were then eluted from the column in 0.5% n-heptafluorobutyric acid ready for loading onto ion-paired reverse-phase high-pressure liquid chromatography using a Waters 700 WISP Autosampler, a Waters 600 pump (Milford, MA, USA), a Shimadzu RF551 fluorescence detector (Tokyo, Japan), and a Beckman Ultrasphere ODS 5 [micro]m, 4.6 mm x 15 cm column (CA, USA.). The fluorescence detector was programmed to measure Pyd and Dpd with [lambda]ex/[lambda]em 295/395 nm at 17 minutes and then to switch to [lambda]ex/[lambda]em 275/320 nm to detect isodesmosine as an internal standard (Randall et al., 1996). Urinary creatinine (Cr) was measured using a Technicon Axon analyzer (Bayer Diagnostics, Sydney, Australia). The results of pyridinoline and deoxypyridinoline were expressed as Pyd/Cr ratio and Dpd/Cr ratio. The intra- and interassay CV’s were 2% and 10%, respectively. Plasma alkaline phosphatase (ALP), calcium (Ca), phosphorus (P) and albumin (ALB) were determined using an automatic biochemistry analyses techniques (Biotecnic Analyzers BT-2245, ARCO and ARCO-PC, Biotecnica Instruments s.r.l. Roma, Italy). All of these indexes were measured with special kits purchased from the Unison Biotech Co. Ltd. (Taiwan). The intra- and interassay CV’s were 1.4-4.4% and 4.1-7.1%, respectively.

Long-term Toxicity Assessment

Samples of liver, kidney, heart, lung, spleen, brain and uterus of the three groups of rats were collected after 12 weeks of treatment and sent to the Department of Pathology, University of Western Australia and Guangzhou University of Chinese Medicine. The tissues were fixed by 10% formalin and embedded in paraffin. Tissue slices were stained with hematoxylin and eosin and examined under the optical microscope. Additionally, the uterine weights were recorded at the end of the experiment.

Statistical Analyses

The data were analyzed using the SPSS for Windows (version 9.0, SPSS Inc. Chicago, IL, USA), using both longitudinal and cross-sectional analyses. A Split-plot repeated measures design was used to test the treatment and time effects for the analysis of the longitudinal changes of bone mass and biochemical data. Pairwise comparisons of baseline data with successive measurements over time and pairwise comparison of treatment at a particular time were performed to evaluate treatment effects within and between the groups. Post-treatment measurements of bone strength and uterine weight were analyzed for group differences using One-way ANOVA model. The differences between the groups were evaluated using LSD post-hoc test. All tests were performed at 0.05 significance level.

Results

Effects on Bone Mass and Strength

At 4, 8 and 12 weeks after operation, OVX resulted in significant reductions in femoral, spinal and global BMD (p < 0.001), while estrogen treatment prevented the reduction in BMD values at all three sites and postoperative time points (Tables 1 and 2, and Figs. 1 and 2). Compared with OVX rats, the decrease in BMD values of OVX-M rats was reduced significantly at all three sites, and most markedly at 12 weeks. The reduction of bone loss varied from 30 to 50% at the various sites. The results of the three-point bending test on the tibiae collected at 12 weeks showed maximum stress, and flexural modulus values in the OVX-E group were significantly higher than that of the OVX group (p < 0.002). The results in the OVX-M group were not different to the OVX-E group and significantly higher than the OVX group (p < 0.002).

[FIGURES 1-2 OMITTED]

Effects on Bone Turnover and Plasma Ca, P and Alb

After operation, urinary Pyd/Cr and Dpd/Cr in OVX group rose significantly at all time points from 1 to 12 weeks (Tables 3 and 4, and Fig. 3). The increments were 69-114% for Pyd/Cr and 81-239% for Dpd/Cr compared with the baseline (p < 0.01-0.001). Both the herbal extract and estrogen treatment prevented the rise in Pyd/Cr and Dpd/Cr followed OVX. There was no statistical significance between OVX-M and OVX-E rats in Pyd/Cr but the Dpd/Cr levels were lower in the OVX-E rats.

[FIGURE 3 OMITTED]

Plasma ALP levels were significantly higher than baseline in the OVX rats at all time points (p < 0.001) and in the OVX-E group only at 12 weeks (p < 0.01). Although ALP value rose in the OVX-M group compared to baseline (p < 0.01), the values were significantly lower than the OVX group at all time points (p < 0.05-0.001) and not significantly higher than the OVX-E group except at 8 weeks. Overall the treatments had no long-term effects on plasma Ca, P or ALB levels (Table 4).

Long-Term Toxicity Assessment

No significant histological abnormality could be found in liver, kidney, heart, lung, spleen, brain or uterus after the 12-week treatment. The uterus weight of OVX-E rats (0.50 [+ or -] 0.11 g, mean [+ or -] SD) was significantly higher (p < 0.05) than the OVX rats (0.09 [+ or -] 0.02 g) and OVX-M rats (0.18 [+ or -] 0.09 g). There was no significant difference in uterus weight between the OVX and OVX-M groups.

Discussion

Chinese herbal medicine has been widely used in clinical practice to treat bone disease for thousands of years and will undoubtedly continue to be used as a cost-effective alternative to commercial pharmaceutical products by traditional users of these therapies. In addition, Chinese herbal medicines may be the source of compounds that are suitable for further development as potential therapeutic agents that are suitable for the treatment and prevention of postmenopausal osteoporosis. We have therefore scientifically examined a decoction of commonly used medicinal herbs in order to examine their efficacy in preventing bone loss.

Ovariectomy in the mature rat causes significant bone loss, which can be precisely and accurately measured by DEXA analysis (Mitlak et al., 1994, Rozenberg et al., 1995). Our data confirms the detection of significant bone loss in these mature OVX rats at femur, spine and whole body sites present at 4 weeks and increasing until 12 weeks although at a slower rate. The OVX rat is an excellent animal model for evaluating potential therapeutic regiments for the prevention or treatment of postmenopausal osteoporosis in preclinical studies (Jiang et al., 1997). This model has been widely used for studying the effect of estrogen (Sato et al., 1995), tamoxifen (Kalu, 1991), droloxifene (Ke et al., 1995), idoxifene (Nuttall et al., 1998) and raloxifene (Frolik et al., 1996; Li et al., 1998) on the skeleton. In the raloxifene, idoxifene and tamoxifen studies, these selective estrogen receptor modulators significantly reduced the fall in bone mass that followed OVX, but did not completely prevent the bone loss observed in OVX rats (Frolik et al., 1996; Li et al., 1998; Nuttall et al., 1998). In the current study, the herbal preparation was most effective for preventing bone loss at the femur site at 4-12 weeks, whilst by 12 weeks there was a significant effect at the spine and whole body site. These data are similar to the effects of well-characterized selective estrogen receptor modulators.

The BMD decrease in the OVX rat model is correlated with a reduction of Ca content and ash weight of the bone tissue (Keenan et al., 1997; Lu et al., 1994), and accompanied with an impairment of bone histology and biomechanics properties (Jiang et al., 1997; S hen et al., 1995). It is recognized that bone quality depends not only on the amount of bone mass, but also on the mechanical ability and the spatial distribution of bone material (Ott, 1993; Recker, 1989). Testing bone biomechanical parameters as end points is necessary in the preclinical research and development programs of new antiosteoporotic drugs (Bonjour et al:, 1999). In our study, a three-point bending test was performed with tibias of the three groups of rats for assessment of the efficacy of the herbal extract on bone mechanical properties. The results indicated that estrogen deficiency results in a reduction in maximum stress and flexural modulus of bone. By contrast, herbal therapy, similar to HRT, ameliorates the effect of estrogen deficiency to reduce stiffness and strength, and therefore makes an important contribution to bone quality and resistance to fracture in the OVX rat.

The biochemical data suggested that the herbal effects on bone were similar to estrogen in that bone turnover were suppressed compared to the OVX control rats. After OVX, urinary Pyd/Cr, Dpd/Cr and plasma ALP in OVX rats rose at all postoperative time points. These changes were significantly ameliorated both by herbal and estrogen treatments. No difference was observed between OVX-M and OVX-E rats for Pyd/Cr at any time point, for Dpd/Cr at 1 and 8 weeks, and for ALP at 4 and 12 weeks. The effects of the herbal preparation on bone turnover were similar to that of raloxifene, idoxifene and tamoxifen reported in other studies (Frolik et al., 1996; Nuttall et al., 1998; Xu et al., 1994). Biochemical testing of plasma Ca, P and ALB in this work showed no adverse effects of the herbal preparation. Similarly, the histological studies and the data on uterus weight showed no safety concerns. The absence of a stimulatory effect on the uterus does not exclude an estrogen-like mechanism of action in light of evidence that other estrogen-like partial agonists do not stimulate uterine growth.

The nature of the compounds having these beneficial effects on the skeleton is uncertain. However, preliminary chemical analysis of some of the herbs has shown the presence of various phytoestrogens. For example, Psoralea corylifolia contains coumestrol (Ji and Xu, 1995), Astragalus membranaceus contains formononetin (Toda and Shirataki, 1999), and Pueraria root contains genistein and daidzin (Ohshima et al., 1988; Wang et al., 1998). Additionally, it has also been suggested that tanshinone and isoporalen in these herbs have estrogen-like activity, and may be classed as phytoestrogens (Draper et al., 1997). In recent years, data from animal studies and clinical trials present some evidence for the potential of phytoestrogens in prevention and treatment of osteoporosis. Administration of the phytoestrogens coumestrol (Fanti et al., 1998), genistein (Ishida et al., 1998) and daidzin (Ishida et al., 1998) ameliorated the OVX-induced bone loss in the rat model.

In conclusion, these studies show significant beneficial effects of a Chinese herbal preparation on skeletal mass and strength without significant adverse effects. These data indicate that vigorous efforts to isolate the active agent or agents should be undertaken together with testing of this preparation in human studies.

Table 1. Femoral, Spinal and Global BMD in the three

Groups of Rats (Mean [+ or -] SD, g/[cm.sup.2])

Week

Group 0 4

Baseline Value

fBMD OVX 0.293 0.265

[+ or -] 0.012 [+ or -] 0.013

OVX-M 0.289 0.271

[+ or -] 0.020 [+ or -] 0.019

OVX-E 0.301 0.292

[+ or -] 0.016 [+ or -] 0.019

Group — —

diff.

sBMD OVX 0.197 0.187

[+ or -] 0.010 [+ or -] 0.012

OVX-M 0.199 0.189

[+ or -] 0.014 [+ or -] 0.012

OVX-E 0.204 0.203

[+ or -] 0.013 [+ or -] 0.015

Group — —

diff.

gBMD OVX 0.137 0.134

[+ or -] 0.005 [+ or -] 0.004

OVX-M 0.138 0.134

[+ or -] 0.006 [+ or -] 0.004

OVX-E 0.139 0.139

[+ or -] 0.005 [+ or -] 0.005

Group — —

diff.

Week

Group 4

% Change

fBMD OVX -9.7 ([double dagger])

[+ or -] 2.0

OVX-M -6.4 ([double dagger])

[+ or -] 2.7 (a)

OVX-E -3.1 ([dagger])

[+ or -] 2.2 (a b)

Group F = 21.435,

diff. p < 0.001

sBMD OVX -5.1 ([double dagger])

[+ or -] 3.9

OVX-M -5.1 ([double dagger])

[+ or -] 2.4

OVX-E -0.7

[+ or -] 2.9 (a b)

Group F = 7.014,

diff. p < 0.002

gBMD OVX -2.5 ([double dagger])

[+ or -] 1.4

OVX-M -3.3 ([dagger])

[+ or -] 1.9

OVX-E -0.6

[+ or -] 0.6 (a b)

Group F = 10.546,

diff. p = 0.001

Week

Group 8

Value % Change

fBMD OVX 0.263 -10.2 ([double dagger])

[+ or -] 0.012 [+ or -] 3.3

OVX-M 0.271 -6.4 ([double dagger])

[+ or -] 0.017 [+ or -] 2.2 (a)

OVX-E 0.303 0.7

[+ or -] 0.017 [+ or -] 2.4 (a b)

Group — F = 60.281,

diff. p < 0.001

sBMD OVX 0.178 -9.8 ([double dagger])

[+ or -] 0.013 [+ or -] 3.9

OVX-M 0.181 -9.3 ([double dagger])

[+ or -] 0.013 [+ or -] 3.7

OVX-E 0.201 -1.9

[+ or -] 0.017 [+ or -] 4.6 (a b)

Group — F = 21.387,

diff. p < 0.001

gBMD OVX 0.131 -4.6 ([double dagger])

[+ or -] 0.005 [+ or -] 1.5

OVX-M 0.132 -4.4 ([dagger])

[+ or -] 0.005 [+ or -] 1.3

OVX-E 0.138 -1.5 *

[+ or -] 0.005 [+ or -] 1.1 (a b)

Group — F = 16.324,

diff. p < 0.001

Week

Group 12

Value % Change

fBMD OVX 0.259 -11.8 ([double dagger])

[+ or -] 0.013 [+ or -] 4.6

OVX-M 0.268 -7.6 ([double dagger])

[+ or -] 0.021 [+ or -] 1.9 (a)

OVX-E 0.307 1.8

[+ or -] 0.020 [+ or -] 3.1 (a b)

Group — F = 95.250,

diff. p < 0.001

sBMD OVX 0.176 -10.7 ([double dagger])

[+ or -] 0.013 [+ or -] 4.6

OVX-M 0.188 -5.9 ([double dagger])

[+ or -] 0.017 [+ or -] 3.5 (a)

OVX-E 0.203 -0.3

[+ or -] 0.015 [+ or -] 5.5 (a b)

Group — F = 30.836,

diff. p < 0.001

gBMD OVX 0.131 -4.8 ([double dagger])

[+ or -] 0.004 [+ or -] 2.3

OVX-M 0.135 -2.7

[+ or -] 0.006 [+ or -] 2.6 (a)

OVX-E 0.139 0.1

[+ or -] 0.004 [+ or -] 2.4 (a b)

Group — F = 35.129,

diff. p = 0.001

Analysis of the group time-treatment effect using a split-plot repeated

measures design indicated significant interactions between groups and

time points for femoral BMD (F = 7.269, p < 0.001), spinal BMD

(F = 3.542, p = 0.014), and global BMD (F = 4.461, p = 0.004).

Univariate testing was undertaken to examine the significance of

inter-group differences at each time point. Pairwise comparisons for

analyzing pre- and post-treatment differences of intra-groups and

comparing the individual % change amongst three groups at different

time points were also undertaken (*: p < 0.05, ([dagger]): p < 0.01 and

([double dagger]): p < 0.001 vs. baseline; (a): p < 0.005-0.001 vs.

group OVX; (b): p < 0.005-0.001 vs. group OVX-M).

Table 2. Bone Strength of the Tibia in the Three Groups of Rats

(Mean [+ or -] SD)

Group n Maximum Stress (MPa) Flexural Modulus (MPa)

OVX 7 189.4 [+ or -] 82.8 10309 [+ or -] 4930

OVX-M 8 295.0 [+ or -] 32.9 * 18194 [+ or -] 3264 ([dagger])

OVX-E 8 297.7 [+ or -] 34.8 * 18766 [+ or -] 2620 ([dagger])

One-way F = 9.741, F = 12.157,

ANOVA p = 0.001 p < 0.001

The significance between groups was evaluated by One-way ANOVA and the

LSD post-hoc tests (*: p = 0.01, ([dagger]): p < 0.001 vs. group OVX).

Table 3. Urine PYD/CR, DPD/CR and Plasma ALP in the three Groups

of Rats (Mean [+ or -] SD)

Week

Group 0 1

Pyd/Cr OVX 52.33 88.47 ([double

(nmol/mmol) dagger])

[+ or -] 11.01 [+ or -] 23.01

OVX-M 42.55 66.54

[+ or -] 9.86 [+ or -] 14.14 (a)

OVX-E 35.25 50.59

[+ or -] 8.48 [+ or -] 7.07 (a)

Group diff. F = 1.325, F = 6.526,

p = 0.271 p = 0.002

Dpd/Cr OVX 22.54 40.85 ([dagger])

(nmol/mmol)

[+ or -] 10.80 [+ or -] 11.81

OVX-M 19.46 26.66

[+ or -] 2.39 [+ or -] 6.38 (a)

OVX-E 13.15 22.44

[+ or -] 4.29 [+ or -] 6.64 (a)

Group diff. F = 1.178, F = 4.932

p = 0.313 p = 0.009

ALP OVX 89.45

(U/L) [+ or -] 41.20

OVX-M 79.43

[+ or -] 9.76

OVX-E 80.47

[+ or -] 17.74

Group diff. F = 0.584,

p = 0.561

Week

Group 4

Pyd/Cr OVX 112.04 ([double dagger])

(nmol/mmol) [+ or -] 53.15

OVX-M 75.06

[+ or -] 16.43 (a)

OVX-E 79.96 ([double dagger])

[+ or -] 36.25 (a)

Group diff. F = 7.372,

p = 0.001

Dpd/Cr OVX 76.42 ([double dagger])

(nmol/mmol) [+ or -] 33.24

OVX-M 41.98 ([dagger])

[+ or -] 14.30 (a)

OVX-E 25.91

[+ or -] 12.82 (a b)

Group diff. F = 35.165,

p < 0.001

ALP OVX 139.76 ([double dagger])

(U/L) [+ or -] 30.78

OVX-M 110.91 ([dagger])

[+ or -] 16.19 (a)

OVX-E 91.99

[+ or -] 13.28 (a)

Group diff. F = 11.014,

p < 0.001

Week

Group 8

Pyd/Cr OVX 103.37 ([double dagger])

(nmol/mmol) [+ or -] 32.42

OVX-M 62.52

[+ or -] 19.12 (a)

OVX-E 55.73

[+ or -] 13.26 (a)

Group diff. F = 12.129,

p < 0.001

Dpd/Cr OVX 67.56 ([double dagger])

(nmol/mmol) [+ or -] 17.46

OVX-M 34.78 *

[+ or -] 11.95 (a)

OVX-E 22.93

[+ or -] 15.30 (a)

Group diff. F = 28.286,

p < 0.001

ALP OVX 136.34 ([double dagger])

(U/L) [+ or -] 27.92

OVX-M 113.07 ([dagger])

[+ or -] 12.44 (a)

OVX-E 72.62

[+ or -] 7.38 (a b)

Group diff. F = 19.431,

p < 0.001

Week

Group 12

Pyd/Cr OVX 100.58 ([double dagger])

(nmol/mmol) [+ or -] 35.13

OVX-M 59.06

[+ or -] 11.26 (a)

OVX-E 55.85

[+ or -] 15.48 (a)

Group diff. F = 11.380,

p < 0.001

Dpd/Cr OVX 67.27 ([double dagger])

(nmol/mmol) [+ or -] 21.94

OVX-M 34.75 *

[+ or -] 12.87 (a)

OVX-E 17.21

[+ or -] 8.12 (a b)

Group diff. F = 34.016,

p < 0.001

ALP OVX 152.22 ([double dagger])

(U/L) [+ or -] 26.71

OVX-M 110.41 ([dagger])

[+ or -] 29.97 (a)

OVX-E 112.91 ([dagger])

[+ or -] 28.31 (a)

Group diff. F = 10.583,

p < 0.001

Analysis of the group time-treatment effect using split-plot repeated

measures design indicated significant interactions between groups and

time points for Dpd/Cr (F = 5.325, p < 0.001) and ALP (F = 3.415,

p = 0.005), but not for Pyd/Cr (F = 1.191, p = 0.313). Univariate

testing was undertaken to examine the significance of inter-group

differences at each time point. Pairwise comparisons for analyzing

pre- and post-treatment differences of intra-groups and comparing the

individual values amongst three groups at different time points was

also undertaken (*: p < 0.05, ([dagger]): p < 0.01 and ([double

dagger]) p < 0.001 vs. baseline; (a): p < 0.05-0.001 vs. group OVX;

(b): p < 0.05-0.001 vs. group OVX-M).

Table 4. Plasma Calcium, Phosphorus and Albumin in the Three Groups

of Rats (Mean [+ or -] SD)

Week

Group 0 4

Ca OVX 2.97 [+ or -] 0.17 2.86 [+ or -] 0.09

(mmol/L) OVX-M 2.95 [+ or -] 0.17 2.78 [+ or -] 0.06

OVX-E 2.99 [+ or -] 0.49 2.88 [+ or -] 0.13

Group diff. F = 0.089, p = 0.915 F = 0.625, p = 0.538

P OVX 1.21 [+ or -] 0.19 1.34 [+ or -] 0.19

(mmol/L) OVX-M 1.29 [+ or -] 0.09 1.31 [+ or -] 0.13

OVX-E 1.52 [+ or -] 0.29 1.35 [+ or -] 0.12

(a b)

Group diff. F = 4.277, p = 0.018 F = 0.086, p = 0.918

ALB OVX 39.01 [+ or -] 3.67 42.24 [+ or -] 2.51 *

(mmol/L) OVX-M 40.51 [+ or -] 2.75 43.49 [+ or -] 3.94 *

OVX-E 41.51 [+ or -] 3.06 48.98 [+ or -] 4.67

([double dagger])

(a b)

Group diff. F = 1.757, p = 0.181 F = 13.931, p < 0.001

Week

Group 8 12

Ca OVX 2.96 [+ or -] 0.12 2.95 [+ or -] 0.19

(mmol/L) OVX-M 2.81 [+ or -] 0.18 2.98 [+ or -] 0.17

OVX-E 3.01 [+ or -] 0.10 2.87 [+ or -] 0.17

Group diff. F = 2.201, p = 0.119 F = 0.649, p = 0.526

P OVX 1.12 [+ or -] 0.15 1.51 [+ or -] 0.36

([dagger])

(mmol/L) OVX-M 1.38 [+ or -] 0.17 1.31 [+ or -] 0.18

OVX-E 1.15 [+ or -] 0.12 1.57 [+ or -] 0.48

([dagger])

Group diff. F = 3.142, p = 0.050 F = 2.775, p = 0.070

ALB OVX 35.14 [+ or -] 2.81 38.53 [+ or -] 3.37

([dagger])

(mmol/L) OVX-M 36.39 [+ or -] 3.49 39.37 [+ or -] 2.36

([dagger])

OVX-E 41.99 [+ or -] 2.29 41.24 [+ or -] 3.10

(a b)

Group diff. F = 14.399, p < 0.001 F = 2.107, p = 0.130

Analysis of the group time-treatment effect using split-plot repeated

measures design indicated significant interactions between groups and

time points for plasma P (F = 2.785, p = 0.018), but not for Ca

(F = 0.894, p = 0.504) and ALB (F = 2.091, p = 0.066). Univariate

testing was undertaken to examine the significance of inter-group

differences at each time point. Pairwise comparisons for analyzing

pre- and post-treatment differences of intra-groups and comparing the

individual value amongst three groups at different time points was also

performed (*: p < 0.05, ([dagger]): p < 0.01 and ([double dagger]):

p < 0.001 vs. baseline; (a): p < 0.05-0.001 vs. group OVX;

(b): p < 0.05-0.001 vs. group OVX-M).

Acknowledgments

This work was supported by an Ad-Hoc Medical Scholarship of the University of Western Australia and Fok Ying Tung Education Foundation. It is a partial research of the Australia-China joint project (1997-C 12). We are thankful for the help of Professor Lai Xiaoping and Du Biaoyan in identifying plant materials and examining tissue specimens. We also thank Dr. Warrren Kett and Sonia Dunn for their technical assistance in urinary pyridinoline and deoxypyridinoline assay, and the help of Dr. Dorota Doherty in the statistical analyses.

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Min Xu, *, ([paragraph]) Ian M. Dick, * Robert Day, ([double dagger]) Drew Randall ([section]) and Richard L. Prince *, ([dagger]), ([parallel])

* Departments of Medicine and ([dagger]) Endocrinology and Diabetes, Sir Charles Gairdner Hospital

([double dagger]) Department of Medical Physics, Royal Perth Hospital, University of Western Australia, Western Australia

([section]) Department of Clinical Biochemistry, Western Australian Centre for Pathology and Medical Research Western Australia

([paragraph]) Guangzhou University of Chinese Medicine, Guangzhou, P.R. China

([parallel]) Western Australia Institute of Medical Research, Western Australia

Correspondence to: Assoc. Prof. R.L. Prince, Department of Medicine, University of Western Australia, Sir Charles Gairdner Hospital, Nedlands, WA 6009, Australia. Tel: (+61) 8-9346-2847, Fax: (+61) 8-9346-3221, E-mail: rlprince@cyllene.uwa.edu.au

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