Effects of rice protein hydrolysate on bone fracture properties in ovariectomized rats
Article information
Abstract
[Purpose]
Osteoporosis is a global health concern, particularly in developed countries with aging populations, where the incidence of postmenopausal osteoporosis is increasing. Various nutrients have been studied as potential interventions, and components derived from regionally familiar foods may enhance acceptability and adherence. In this study, we focused on rice, a staple food in Asia and Africa. Recent studies have suggested that rice-derived peptides may help prevent bone fragility. Therefore, we examined the effects of rice protein hydrolysate (RPH) on bone health in ovariectomized rats.
[Methods]
In Study 1, which was designed to prevent osteoporosis, 5-week-old female Sprague-Dawley rats were acclimated for one week and then underwent ovariectomy (OVX). They were divided into three groups: a control group fed a normal diet and two experimental groups fed diets containing 0.01% or 0.1% RPH for eight weeks. In Study 2, which aimed at promoting recovery from osteoporosis, the setup was similar; however, after OVX, rats were fed a low-calcium diet for four weeks to induce osteoporosis, followed by the same diet groups as in Study 1 for an additional four weeks.
[Results]
Study 1 showed that RPH significantly increased bone-breaking force (0.01% RPH, p < 0.01; 0.1% RPH, p < 0.05). In Study 2, RPH intake reduced serum tartrate-resistant acid phosphatase (TRAP) levels, a marker of bone resorption (both p < 0.05). However, a significant effect on bone mineral density was observed only in the 0.01% RPH group in Study 1 (p < 0.05).
[Conclusion]
These findings suggested that RPH may help treat postmenopausal osteoporosis.
INTRODUCTION
Osteoporosis is a global issue and a systemic skeletal disease characterized by bone mass loss and weakened bone structure, leading to increased fragility and fracture risk [1]. The number of postmenopausal osteoporosis cases is increasing in developed countries with aging populations. In the European Union, approximately 21% of women aged 50–84 years, representing over 22 million women, have osteoporosis [2]. The USA reports 1.5 million osteoporosis-induced fractures annually [3], and Japan has an estimated 12.8 million patients with osteoporosis [4]. However, fewer than 20% of high-risk cases, such as those involving proximal femur fractures, receive drug therapy [5]. This low treatment rate, combined with high medical costs and untreated patients in Japan and other countries, highlights the urgent need for better management [6].
Nutritional therapy plays an important role in preventing and treating osteoporosis, with previous studies highlighting the impact of nutrients on bone health. A meta-analysis of Japanese women aged 40–69 years showed that those with low calcium (Ca) intake (approximately < 350 mg/day) had twice the risk of vertebral fractures around menopause compared to those with high Ca intake (approximately > 700 mg/day) [7]. In addition, a randomized controlled trial demonstrated that daily consumption of soy-based foods attenuated lumbar spine bone loss in perimenopausal women [8]. These findings suggest that nutritional interventions, as well as pharmacotherapy, can effectively prevent osteoporosis.
Nutritional therapy insights may be found in everyday foods. For example, people in Europe and North America consume more calcium through dairy products than those in the Asia-Pacific and South America, which impacts bone health and vertebral fracture risk [9,10]. This suggests that culturally familiar foods may play a significant role in shaping nutritional strategies for bone health. For example, rice proteins have been shown to exhibit lipid-lowering and antidiabetic effects, and in diabetic rat models, they have also been reported to influence bone quality and strength [11]. Moreover, rice protein hydrolysate-derived peptides have demonstrated additional bioactivities, including antidepressant effects and the promotion of ghrelin secretion [12,13]. Based on these findings, we hypothesized that rice-derived peptides could also benefit bone health in postmenopausal osteoporosis, possibly through their effects on bone resorption and remodeling.
Furthermore, postmenopausal osteoporosis is a widely accepted model for investigating bone loss due to estrogen deficiency, which accelerates bone resorption and disrupts the bone remodeling balance. Therefore, in the present study, we used ovariectomized rats to evaluate the effects of rice protein hydrolysate (RPH) on bone fracture properties, with the aim of exploring its potential as a culturally relevant nutritional intervention and a potential new source for promoting bone health. To investigate the potential of RPH in the prevention and treatment of postmenopausal osteoporosis, we conducted two separate experiments. Study 1 was designed to evaluate the preventive effects of RPH by initiating treatment immediately after ovariectomy, whereas Study 2 aimed to assess its therapeutic effects using a recovery model after osteoporosis had been induced through a low-calcium diet. By using these complementary approaches, we aimed to clarify the potential role of RPH in maintaining or restoring bone strength under estrogen-deficient conditions.
METHODS
Rice protein hydrolysate
Rice protein hydrolysate (RPH) was prepared from rice endosperm protein (REP) purified using a previously described alkaline extraction method [14]. REP was digested with subtilisin (Amano Enzyme, Aichi, Japan). The digests were subsequently boiled for 10 min to inactivate the enzyme, and then freeze-dried and stored at -20°C until further use. The RPH used in this study was confirmed to be stable based on the quantitative analysis of a specific sequence using LC-MS/MS as a quality control. Previous studies have also reported that RPH has physiological effects that stimulate ghrelin secretion [12].
Experimental design
This study included two investigations, each of which is described in Figure 1. In Study 1, female Sprague-Dawley rats (n = 24, 5 weeks old) underwent ovariectomy (OVX) after a 1-week acclimation period. The animals were then randomly categorized into three experimental groups, including a group consuming a normal diet (Ca, P = 0.3%) (CON, n = 8), a group consuming a diet containing 0.01% RPH (n = 8), and a group consuming a diet containing 0.1% RPH (n = 8). The duration of the experiment was eight weeks. Study 1 was conducted based on a method previously described [15,16], which evaluated the effects of RPH on bone strength in OVX rats. In Study 2, female Sprague-Dawley rats (n = 24, 5 weeks old) were acclimated for 1 week, subjected to OVX, and fed a low-Ca diet (Ca = 0.01%, P = 0.3%) for four weeks to create an osteoporosis rat model. The feed was then changed and the animals were randomly divided into three experimental groups. The groups were the same as those in Study 1, and the duration of the experiment was four weeks. The design of Study 2 was adapted from the model reported by [17], which combined OVX and dietary calcium deficiency to induce bone loss in OVX rats. The duration of the experiment was eight weeks. Table 1 lists the composition of the experimental diets. In this study, we employed a combined OVX and Ca-deficient approach in young rats. This model has been previously reported as an osteoporosis model and has been shown to exhibit similar increases and decreases in bone mineral density (BMD) and bone strength to those observed in models using older rats [17]. Although young rats were used in this study, the target human population was postmenopausal women, as the model effectively reproduced estrogen deficiency-induced bone loss when combined with a low-calcium diet. The selected RPH doses (0.01% and 0.1%) were determined based on preliminary internal experiments, which suggested that 0.1% RPH had potential effects on bone parameters. Considering the practical importance of minimizing the intake volume and cost for future applications in humans, we included a 1/10 lower dose (0.01%) to evaluate whether similar effects could be observed at a reduced level. Based on the estimated feed intake under ad libitum conditions, the corresponding human equivalent doses were approximately 400 and 40 mg/d for the 0.1% and 0.01% groups, respectively. Rats were housed individually in regular cages (13 × 13 × 22 cm), with the room maintained at 22℃ ± 2℃ and a constant 12:12 h light/dark cycle (light, 7:00 am–7:00 pm) in all experiments. All animal care and experimental procedures were approved by the Animal Experimental Ethics Committee of the University of Tsukuba (approval number: 20-452) and conducted according to the Guidelines for the Care and Use of Laboratory Animals.
Schematic diagram of the experimental protocol.
Compositions of the experimental diets
Daily Data Collection and Specimen Harvesting
Body weight and food intake were measured every two days. All rats were fasted for 2 h before dissection. Under isoflurane anesthesia, whole blood was drawn from the abdominal aorta using a syringe, and the rats were euthanized. Serum samples were obtained by centrifugation at 4°C at 3,000 rpm for 15 min. The serum was frozen at −80°C to measure bone metabolism markers and hormones. The femur, tibia, and lumbar spine were harvested after the rats died. The femurs were removed from the adherent connective tissue, and bone strength was promptly measured. The tibia and lumbar spine were preserved in 70% ethanol after removing the soft tissue for bone area, bone mineral content (BMC), and BMD measurements.
Bone Strength Measurement Using Three-Point Bending Test
A three-point bending test (DYN-1255, IIO DENKI, Tokyo, Japan) was used to assess the strength of the femoral mid-shaft as previously described (distance between the fulcrums, 1 cm; plunger speed, 100 mm/min; full scale, 50 kg; chart speed, 120 cm/min) [18]. Two femurs were used for this test, one on each side. Breaking force is the loading weight (gravitational acceleration) required for bone breaking. Breaking energy is the workload caused by bone breakage.
BMC, Bone Area, and BMD Measurements Using Dual-Energy X-ray Absorptiometry
Dual-energy X-ray absorptiometry (DXA; QDR-4500A, Hologic Inc., Tokyo, Japan) was used to measure the BMC, bone area, and BMD of the tibia and L4-L5 lumbar spine. The analyzed regions were selected based on a previous study [19]. For DXA measurements, two tibiae were used, one from each side. All scans were performed in small-animal mode with high regional resolution.
Bone Metabolic Marker and Serum Hormone Measurement
Serum bone-specific alkaline phosphatase (Bone-ALP) activity, a bone formation marker, and serum tartrate-resistant acid phosphatase (TRAP) activity, a bone resorption marker, were measured as previously described [20]. Serum IGF-1 levels were determined using an enzyme-linked immunosorbent assay (Mouse/Rat IGF-1 sandwich ELISA kit; Proteintech Group, Inc., IL, USA), following the manufacturer’s protocol.
Statistical Analysis
All data were expressed as mean ± standard error (SE). Statistical analysis was conducted using a one-way analysis of variance in Studies 1 and 2. When significant differences were detected, Tukey’s post hoc comparison test was used for multiple comparisons. Additionally, we examined the dose-dependent effects of RPH using the Jonckheere-Terpstra test. The significance level was set at p < 0.05. Statistical Package for the Social Sciences (ver. 29.0; SPSS Inc., Chicago, IL, USA) was used for all statistical analyses.
RESULTS
Body weight, Food intake, and Food efficiency
Table 2 presents data on body weight, food intake, and food efficiency. None of these parameters demonstrated significant differences with RPH treatment.
Body weight, food intake and food efficiency
Bone strength Measurement
Figure 2 shows the breaking force and breaking energy of the femur, assessed using a three-point bending test. In Study 1, both 0.01% and 0.1% RPH groups demonstrated significantly higher breaking force compared to the control group (0.01% RPH, p < 0.01; 0.1% RPH, p < 0.05). A trend test also revealed a significant dose-dependent increase in this parameter (p < 0.05). Similarly, a trend test revealed a significant dose-dependent increase in breaking energy (p < 0.05). In Study 2, the 0.1% RPH group exhibited significantly higher breaking force compared to the control group (p < 0.05), and a trend test revealed again a significant dose-dependent effect (p < 0.05).
Breaking force and breaking energy of femur.
(a) Breaking force in Study 1. (b) Breaking force in Study 2. (c) Breaking energy in Study 1. (d) Breaking energy in Study 2. CON: Control group. RPH 0.01%: Rice Protein Hydrolysate 0.01% group. RPH 0.1%: Rice Protein Hydrolysate 0.1%. Bars are mean ± SE (n = 8 per group). (a), (b), (c), and (d) were analyzed using one-way ANOVA with Tukey’s posterior comparison test and we examined the dose-dependent effects of Rice Protein Hydrolysate using the Jonckheere-Terpstra test. *p < 0.05, **p < 0.01 versus CON group. “Trend ” indicates a significant trend by the Jonckheere-Terpstra test.
BMD, BMC, and Bone Area
Figure 3 shows the BMD and Table 3 shows the BMC and bone area of the tibia and lumbar spine. In Study 1, the 0.01% RPH group showed a significantly higher BMD of the lumbar spine (p < 0.05) compared to the control group. Additionally, the tibial bone area, lumbar spine bone area, and lumbar spine BMC demonstrated dose-dependent increases according to the trend test (p < 0.05). Conversely, Study 2 showed no statistically significant differences in any of the parameters.
Bone mineral density (BMD) of lumbar spine and tibia.
(a) BMD of tibia in Study 1. (b) BMD of tibia in Study 2. (c) BMD of lumbar in Study 1. (d) BMD of lumbar in Study 2. CON: Control group. RPH 0.01%: Rice Protein Hydrolysate 0.01% group. RPH 0.1%: Rice Protein Hydrolysate 0.1%. Bars are mean ± SE (n = 8 per group). (a), (b), (c), and (d) were analyzed using one-way ANOVA with Tukey's posterior comparison test. *p < 0.05 versus CON group.
Bone area (Area) and bone mineral content (BMC) of lumbar spine and tibia
Bone Metabolic Markers and Hormone
Table 4 presents data on bone metabolic markers and hormones. In Study 1, no statistically significant differences were observed in any of the parameters. In Study 2, the 0.01% and 0.1% RPH groups showed significantly lower TRAP levels compared to the control group (p < 0.05), and a trend test revealed a significant dose-dependent decrease (p < 0.05).
Bone metabolism marker and serum hormone
DISCUSSION
This study investigated the potential effects of rice protein hydrolysate (RPH) intake on bone parameters in ovariectomized rats. Study 1 focused on the prevention of bone loss, whereas Study 2 examined the potential for improvement. In Study 1, bone fracture properties, particularly femur-breaking force, were higher in the RPH group than in the control group. In Study 2, although no significant increase in bone strength was observed, RPH intake was associated with a reduction in serum TRAP levels, a marker of bone resorption. These findings highlight the potential of RPH as a supportive nutritional approach in the treatment of postmenopausal osteoporosis.
As mentioned above, RPH intake significantly increased femur-breaking force in ovariectomized rats. Although no significant differences were observed in breaking energy, a trend toward higher values was observed in the RPH groups. Bone strength is generally determined by approximately 70% BMD and 30% bone quality [21]. While BMD changes were not statistically significant in the present study, with some exceptions as shown in Fig. 3, the observed improvement in bone strength may be partly attributed to changes in bone quality. Bone substances are broadly categorized according to structural properties, such as bone microstructure and geometry, and material properties, such as hydroxyapatite and collagen composition, crystal orientation, bone turnover, and microdamage accumulation [22]. The present study did not evaluate these structural or material characteristics in detail, which limited our ability to identify specific contributors to the increased bone strength. Previous studies revealed that ovariectomized osteoporotic rats demonstrated a decrease in BV/TV and Tb. N and an increase in SMI (shift from plate to rod structure) compared to sham [23,24]. Therefore, the control groups in studies 1 and 2 may have had similar bone structures. RPH may suppress or ameliorate bone structure deterioration; however, explaining this using the data from the present study alone is difficult. In summary, RPH had limited effects on BMD-related parameters, but significantly improved bone strength.
The present study examined bone metabolism markers and serum IGF-1 levels to explore potential contributors to bone quality and material mechanical properties. IGF-1 was measured due to its established role in promoting bone formation by stimulating osteoblast activity [25]. However, no significant differences in serum IGF-1 levels were observed between groups. One possible explanation for this is that the animals used in this study were young and had naturally high baseline IGF-1 levels, which made it difficult to detect treatment-related changes. Additionally, the duration and dose of RPH administration may have been insufficient to elicit systemic hormonal responses. In contrast, in Study 2, which employed a recovery model following osteoporosis induction, serum TRAP, a bone resorption marker, was significantly reduced in the RPH groups. This finding suggests that RPH suppresses osteoclast activity, thereby reducing bone resorption during the recovery phase of bone loss. One possible explanation is that bone resorption is more actively upregulated in the low-calcium, post-ovariectomy phase, which makes the suppressive effect of RPH more detectable. These findings suggest that RPH may help prevent bone loss primarily by suppressing bone resorption rather than enhancing bone formation through IGF-1. Although the underlying mechanism is not yet clear, it is possible that RPH affects signaling pathways involved in osteoclastogenesis. Future studies are needed to clarify this mechanism.
Research on the effects of rice-derived components on bone health is limited, making the findings of this study valuable. Previous studies with ovariectomized rats have shown that Superhongmi rice lowered PTH, NTx-1, and CTx-1 levels, improving bone metabolism [26]. Similarly, Keunnunjami rice reduced these markers and increased 17-β-estradiol [27], demonstrating effectiveness in addressing bone metabolism deterioration. However, these rice varieties, which have shown beneficial effects in previous studies, are colored-rind types rich in polyphenols and flavonoids with high antioxidant capacity [28]. In contrast, the rice used in the present study was a non-pigmented, polished variety lacking significant amounts of polyphenols. Therefore, the observed effects may be attributed to bioactive peptides derived from rice proteins rather than antioxidant compounds. This suggests that RP may act via different biological pathways, such as the modulation of osteoclast activity or bone remodeling, independent of polyphenol-mediated mechanisms. Although no consistent dose-dependent effect was observed in the current study, it is noteworthy that a minimal dose of 0.01% RPH exerted significant effects on bone-breaking force. This suggests that RPH may be effective even at low intake levels. Further investigation is warranted to determine the optimal intake range and elucidate the underlying mechanisms. Taken together, while previous studies have reported the beneficial effects of rice-derived compounds in various contexts, our findings suggest that rice peptides may improve bone strength through a mechanism distinct from that involving polyphenols or other micronutrients. These findings highlight the potential of RPH as a novel dietary component to support bone health.
The present study has several limitations. First, we did not include a sham-operated control group, which limited our ability to distinguish the specific effects of ovariectomy from those of natural growth or other confounding factors. Second, although young rats were used in this study, previous reports have shown that this model exhibits bone changes comparable to those observed in aged ovariectomized rats. Therefore, we believe that this limitation does not significantly affect the overall conclusions. However, these findings may not fully capture the bone-loss patterns typically observed in the elderly human population. Third, specific bone quality parameters, such as hydroxyapatite crystallinity and collagen composition, were not evaluated, which could have provided further insight into the mechanisms underlying the observed changes in bone strength. Future studies should address these limitations to more comprehensively evaluate the effects of RPH on bone health.
In conclusion, bone fracture properties, particularly the femur-breaking force, were higher in the RPH group than in the control group during the prevention phase of the study using ovariectomized rats. In the improvement phase, RPH intake was associated with reduced serum TRAP levels, suggesting possible suppression of high bone turnover. Although no significant increase in bone mineral density was observed, these findings suggested that RPH may influence bone quality and resorption dynamics. Further research is needed to confirm these observations and clarify the underlying mechanisms.
Acknowledgements
We thank MARUZEN-YUSHODO Co., Ltd. and Editage (www.editage.co.kr) for the English language editing.
This research was supported by a research grant in collaboration with KAMEDA SEIKA Co., Ltd.
KK, HF, KO, and NO declare no conflicts of interest. YH, TM, KU, and HT are employees of KAMEDA SEIKA Co. Ltd..