Dietary bioactive components that play a role in improving skeletal health have received considerable attention in complementary and alternative medicine practices as a result of their increased efficacy to combat chronic diseases. The objectives of this study were to evaluate the additive or synergistic effects of dried plum and fructooligosaccharides (FOS) and to determine whether dried plum and FOS or their combination in a soy protein-based diet can restore bone mass in ovarian hormone deficient rats. For this purpose, 72 3-month-old female Sprague-Dawley rats were divided into six groups (
The postmenopausal period typically occupies one-third of a woman's life [
Conventional health care providers often administer several medications focusing on the pathogenic process of osteoporosis rather than seeking alternatives that maximize the inherent healing ability of a person's body. As a result, considerable interest has been generated recently by the desire of postmenopausal women to find more natural ways to combat osteoporosis. Scientists have turned to explore the health benefits of plant-based bioactive compounds because they contain vitamins and minerals that are essential to bone health and a number of synthetic and natural phytochemicals that have been shown to positively influence bone metabolism. For instance, soy protein and its isoflavones have been shown to improve bone mass in both animal models of osteoporosis [
Seventy-two female, 3-month-old Sprague–Dawley rats (Harlan Sprague-Dawley Inc., Indianapolis, IN) were individually housed in an environmentally controlled facility. Guidelines for the ethical care and treatment of animals from the Animal Care and Use Committee at Oklahoma State University were strictly followed. After 5 days of acclimation, the rats were divided into six groups (
One day prior to necropsy, rats were placed in metabolic cages and urine was collected. At the end of the 60 day treatment period, rats were anesthetized with a mixture of ketamine and xylazine (100 and 5 mg kg−1 body weight, resp.) and bled from the abdominal aorta. Blood samples were collected, and serum was separated by centrifugation at 1500 g for 20 min at 4°C. Aliquots of serum and urine were frozen and kept at −20°C for later analyses. The femurs, tibiae and fourth lumbar vertebrae were removed, cleaned and freed of surrounding soft tissues. The left tibiae were stored in 70% ethanol for histomorphometric analyses while the other bones were frozen at −20°C until analyses. Uteri were collected, blotted and weighed to confirm the success of ovariectomy.
A commercially available radioimmunoassay kit was used to measure serum 17
BMD and BMC of the whole body, right femurs and fourth lumbar vertebrae were assessed by DXA equipped with appropriate software for use with small laboratory animals and isolated bones. Whole-body BMD and BMC were assessed the day of the surgery, 45 days after surgery to assure that bone loss has occurred due to ovariectomy and at the end of 60 days of dietary treatment for evaluating treatment effects. To ash the bones, they were dried, weighed and placed in a muffle furnace at
Femoral strength was assessed by three-point bending performed on a material testing system (Instron 5543, Canton, MA). The femur was placed in a three-point bending fixture such that the posterior surface rests on the lower supports and the upper support touches the anterior surface. One lower support was placed touching the posterior surface at the distal end of the femur, at the point where the metaphysis begins to widen, 5–8 mm from the distal end. During this test, the anterior surface was under compression and the posterior surface was under tension. An initial load
(
After the mechanical test, the cross-sectional surface at the fracture site was prepared flat for tracing and subsequent inertial and area calculations. The ultimate load, yield load and the stiffness of the specimen were measured from the load-displacement curve. Ultimate stress, yield stress and modulus of elasticity of the specimen were calculated using beam-bending theory using values for second moment of area.
At 8 days and at 1 day prior to necropsy, rats were subcutaneously injected with calcein (8 mg kg−1 body weight; Sigma, St Louis, MO) for double-flurochrome labeling of bone to determine active mineralization sites and rate of bone formation using histomorphometry. Quantitative histomorphometry of the left tibia was used to examine the dynamics of the bone changes and bone cell activities due to ovariectomy and dietary treatments.
The tibiae were cut at 1 mm distal to the tibio-ibula junction (TFJ) and 19 mm proximal to the TFJ to obtain a proximal portion for cancellous bone histomorphometry. Both the central diaphysis and proximal tibia were placed in Villanueva stain for 72 h and then returned to 70% ethanol. During the next 14 days, the specimens were dehydrated in graded ethanol and acetone and then embedded individually in modified methyl methacrylate. Sections were analyzed with a light/epifluorescent microscope and a video camera interfaced with the BIOQUANT TCW software (R&M Biometrics, Nashville, TN).
Data analyses involved computation of means and SEM for each of the treatment groups using SAS (Version 8.2, SAS Institute, Cary, NC). Analysis of variance and least square means were calculated using the general linear model procedure and the means were compared using Fisher's least significant difference for comparing groups. Differences were considered significant at
The success of the surgical procedure was confirmed as the rats in Ovx groups experienced atrophy of uterine tissue (Table
Effects of ovariectomy (Ovx) and various dietary treatments on food intake and body and uterine weights.
Parameters | Sham-casein | Ovx + casein | Ovx + soy | Ovx + soy + plum | Ovx + soy+ FOS | Ovx + soy + plum + FOS |
---|---|---|---|---|---|---|
Food intake (g/d) | 14.1 ± 0.4 | 14.0 ± 0.3 | 13.9 ± 0.3 | 13.9 ± 0.3 | 14.1 ± 0.4 | 14.2 ± 0.3 |
Initial | 231 ± 1 | 231 ± 1 | 232 ± 1 | 231 ± 1 | 230 ± 1 | 232 ± 1 |
Final | 281 ± 4b | 317 ± 4a | 315 ± 4a | 319 ± 4a | 327 ± 4a | 316 ± 4a |
Uterine weight (mg) | 606 ± 33a | 185 ± 31b | 182 ± 30b | 169 ± 30b | 132 ± 31b | 141 ± 31b |
Values are mean ± SE;
Whole-body BMD and BMC prior to Ovx were not different among the groups (data not shown). However, 45 days after ovariectomy, all Ovx rats on average had 5% lower (
Effects of ovariectomy (Ovx) and various dietary treatments on whole body, right femur and fourth lumbar BMD and BMC.
Parameters | Sham-casein | Ovx + casein | Ovx + soy | Ovx + soy + plum | Ovx + soy + FOS | Ovx + soy + plum + FOS |
---|---|---|---|---|---|---|
Whole body | ||||||
BMC (g) | 10.268 ± 0.164 | 10.397 ± 0.164 | 10.297 ± 0.157 | 10.648 ± 0.157 | 10.779 ± 0.164 | 10.625 ± 0.164 |
BMD (mg/cm2) | 172.4 ± 1.4a | 159.8 ± 1.4c | 161.4 ± 1.4c | 165.2 ± 1.4b,c | 167.2 ± 1.4b | 167.5 ± 1.4b |
Right femur | ||||||
BMC (mg) | 473.9 ± 8.8a | 435.0 ± 8.8c | 447.5 ± 8.4b,c | 450.1 ± 8.4a,b,c | 464.6 ± 8.8a,b | 454.9 ± 6.4a,b,c |
BMD (mg/cm2) | 250.7 ± 2.5a | 226.2 ± 2.5c | 232.9 ± 2.4b,c | 235.5 ± 2.4b | 238.2 ± 2.5b | 237.5 ± 2.5b |
Ash (%) | 68.2 ± 0.5a | 66.6 ± 0.5b | 65.9 ± 0.5b | 66.8 ± 0.5a,b | 67.2 ± 0.5a,b | 66.3 ± 0.5b |
Fourth Lumbar | ||||||
BMC (mg) | 152.4 ± 3.7a | 128.4 ± 3.7d | 131.5 ± 3.6c,d | 138.5 ± 3.6b,c,d | 142.0 ± 3.7b | 140.7 ± 3.7b,c |
BMD (mg/cm2) | 246.2 ± 3.8a | 215.0 ± 3.8d | 219.7 ± 3.7c,d | 222.5 ± 3.7c,d | 228.1 ± 3.8b,c | 232.3 ± 3.8b |
Ash (%) | 63.3 ± 0.6a | 56.3 ± 0.6c | 57.0 ± 0.6b,c | 58.5 ± 0.6b | 58.5 ± 0.6b | 58.7 ± 0.6b |
Values are mean ± SE;
Effects of ovariectomy (Ovx)
and various dietary treatments on change in whole-body bone
mineral density (BMD). Bars represent mean + SE;
The right femoral BMC and BMD were significantly reduced in ovariectomized rats in comparison with intact rats (Table
Similar to the femur, ovariectomy significantly reduced BMC, BMD and % ash of the fourth lumbar, a site mostly composed of trabecular bone (Table
In Ovx rats, ovariectomy somewhat reduced ultimate load, an indicator of the material properties of bone, compared with sham rats (Figure
Effects of
ovariectomy (Ovx) and various dietary
treatments on ultimate load of the left
femur. Bars represent mean + SE;
Ovariectomy caused an increase in trabecular thickness (Tb Th), trabecular separation (Tb Sp) and mineral surface as percent of bone surface (MS/BS) (Table
Effects of ovariectomy (Ovx) and various dietary treatments on histomorphometric parameters of the left proximal tibia.
Parameters | Sham-casein | Ovx + casein | Ovx + soy | Ovx + soy + plum | Ovx + soy + FOS | Ovx + soy + plum + FOS |
---|---|---|---|---|---|---|
BV/TV (%) | 11.9 ± 1.9a | 8.1 ± 1.9a,b | 6.7 ± 1.9a,b | 5.2 ± 1.9b | 8.3 ± 1.9a,b | 9.9 ± 1.9a,b |
Tb Th ( | 46 ± 5c | 63 ± 5a,b | 58 ± 5a,b,c | 53 ± 5b,c | 61 ± 5a,b | 69 ± 5a |
Tb N ( | 2.46 ± 0.27a | 1.09 ± 0.27b | 1.19 ± 0.27b | 0.99 ± 0.27b | 1.26 ± 0.27b | 1.42 ± 0.27b |
Tb Sp ( | 441 ± 198b | 1359 ± 198a | 1032 ± 198a | 1125 ± 198a | 959 ± 198a,b | 942 ± 198a,b |
MS/BS (%) | 2.2 ± 2.0b | 10.2 ± 2.0a | 10.1 ± 2.0a | 8.9 ± 2.0a | 12.1 ± 2.0a | 6.5 ± 2.0ab |
Values are mean ± SE;
As expected, ovariectomy significantly reduced serum levels of E2, thus confirming the success of the surgery (Table
Effects of ovariectomy (Ovx) and various dietary treatments on serum and urinary parameters.
Parameters | Sham-casein | Ovx + casein | Ovx + soy | Ovx + soy + plum | Ovx + soy + FOS | Ovx + soy + plum + FOS |
---|---|---|---|---|---|---|
Serum | ||||||
17- | 21.9 ± 5.4a | 7.2 ± 1.9b | 6.4 ± 1.5b | 5.3 ± 1.0b | 3.8 ± 0.4b | 4.9 ± 0.9b |
ALP (U/L) | 41.7 ± 4.3b | 51.4 ± 4.3a,b | 49.8 ± 4.1a,b | 55.3 ± 4.1a | 60.9 ± 4.4a | 52.7 ± 4.3a,b |
Urine Dpd | 39.9 ± 8.9b | 98.9 ± 25.8a | 57.1 ± 3.7b | 66.9 ± 17.8a,b | 48.0 ± 3.2b | 54.2 ± 3. 1b |
(nmol/mmol creatinine) |
Values are mean ± SE;
Inhibiting further bone loss and reducing the risk of fracture are hallmarks for an effective antiosteoporotic agent. Although there are a number of conventional medicines for preventing and/or treating osteoporosis, these medicines are not devoid of side effects and are also costly. In addition, the long-term patient adherence rates are low. The purpose of our research is to find functional foods that can act as such agents by preventing bone loss or promoting bone restoration. The present study was designed to determine if combining soy protein with FOS, dried plum or both was more effective at restoring bone loss as a result of ovarian hormone deficiency.
The findings of this study show that while a soy protein-based diet alone was not sufficient to reverse the loss in whole-body, femoral and fourth lumbar BMD due to ovariectomy, the combination of soy protein with other bioactive components such as FOS and dried plum was able to improve BMD of osteopenic Ovx rats. Soy protein alone offers minimal beneficial effect on BMD while FOS and dried plum provide additive effects in terms of bone density in this animal model. In general, the effect of soy protein on bone is controversial and inconclusive. Soy protein has been shown to have no significant effect [
The higher whole-body BMD in diets supplemented with FOS (Ovx + soy + FOS, Ovx + soy + FOS + plum) suggests that FOS, a mixture of indigestible and fermentable sugars, has a bone reversal effect. FOS has been shown to increase absorption of minerals from the colon [
Combining FOS and dried plum significantly improved whole-body BMD, femoral BMC and BMD and fourth lumbar BMD. The bone protective effect of the combination of soy with dried plum and FOS may be due to its ability to decrease bone resorption, as shown by lower urinary Dpd excretion. The dietary bioactive components caused a reduction in bone resorption and maintained the increased rates of bone formation due to ovariectomy, thereby resulting in a net bone gain in bone density. It is interesting to note that whole-body BMD improvements were similar in the soy protein diets combined with FOS alone (Ovx + soy + FOS) and with both FOS and dried plum (Ovx + soy + plum + FOS); however, there seems to be distinct differences in the ability to improve bone health at different sites. Soy plus FOS seemed to improve BMD the greatest at the right femur while greatest improvement in fourth lumbar BMD was seen in the soy plus dried plum and FOS group.
In the present study, deterioration of Ovx-induced trabecular microarchitecture was restored by adding dried plum to a soy diet. Trabecular thickness increase due to Ovx has been suggested to be a compensation for lost trabecular connectivity [
Animals on a soy + FOS + plum diet showed an 8% increase in fourth lumbar BMD and a 5% increase in both whole-body and right femur BMD. These percent increases are considered clinically relevant when results are compared with studies using approved medications for treating osteoporosis. For instance, Ito et al. [
The results from this study are in agreement with those from our previous observations [
Relevant to the present study, it may be worth investigating the mechanisms underlying the additive effects of soy with the different bioactive compounds to provide an explanation for these findings. Adding dried plum and/or FOS to a soy-based diet can offer improvements beyond that of soy alone and results suggest that some of the combinations may affect different areas with varying degrees. Therefore, it is necessary to investigate if these bioactive compounds either together or independently can also restore bone mass in postmenopausal women. These positive effects of dried plum and FOS on biomarkers of bone metabolism have resulted in an improvement of BMD in this animal model. FOS and dried plum combined with soy in the diet provides a valuable alternative to conventional osteoporosis therapies due to their anti-resorptive and anabolic properties.