PharmaNutrition 2 (2014) e149–e154
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b-Cryptoxanthin rich paprika extract prevents ovariectomy induced bone loss in Wistar rats Rahul S. Chaudhari a , Vikas B. Mankumare a , J. Shankaranarayanan b,1, Jayant V. Deshpande b,1, T.K. Sunilkumar b,1, Sadhana Sathaye a, * a b
Department of Pharmaceutical Sciences & Technology, Institute of Chemical Technology, N.P. Marg, Matunga (E), Mumbai, M.S. 400019, India OmniActive Health Technologies Ltd., Mumbai,India
A R T I C L E I N F O
A B S T R A C T
Article history: Received 18 May 2014 Received in revised form 14 August 2014 Accepted 18 August 2014
Recent studies show that high intake of carotenoids is helpful in preventing bone loss in post-menopausal women. Carotenoids like b-cryptoxanthin increases calcium and phosphorus deposition in cultured osteoblasts. b-Cryptoxanthin is also reported to have anabolic property in cultured osteoblast cells. It can also inhibit bone resorption process. Paprika and its dietary products are rich in carotenoids like b-cryptoxanthin. Carotenoids from the paprika oleoresin are also reported to have very good bioavailability in humans. The present study was carried out to investigate the effect of b-cryptoxanthin rich paprika extract on animal model of ovariectomy induced bone loss. Treatment with the test drug was given for 6 weeks. The b-cryptoxanthin rich paprika extract significantly reduced urinary excretion of pyridinium crosslinks, indicating its probable anti-resorptive property. The mechanical strength and density of cancellous bones are significantly improved. Thus the paprika extract may have potential role as a sustainable nutritional approach to improving bone health in post-menopausal condition. ã 2014 Elsevier B.V. All rights reserved.
Keywords:
b-Cryptoxanthin Ovariectomy Postmenopausal osteoporosis Paprika
1. Introduction Postmenopausal osteoporosis is a disease characterized by asymptomatic bone loss which leads to disabling skeletal fractures. The National Institute of Health consensus conference has defined osteoporosis as a skeletal disorder characterized by compromised bone strength and increased skeletal fragility accompanied by low bone mineral density (BMD) and micro-architectural deterioration [1]. According to the WHO criteria, osteoporosis is defined as a BMD that lies 2.5 standard deviations or more below the average value for young healthy women (a T-score of < 2.5 S.D.) [2]. Worldwide, osteoporosis causes more than 8.9 million fractures
Abbreviations: ALP, alkaline phosphatase; BPE, b-Cryptoxanthin rich paprika extract; BMD, bone mineral density; BMU, basic multicellular units; OB, osteoblasts; OC, osteoclasts; OVX, ovariectomized; TRAP, tartrate resistant acid phosphatase; TPBT, three point bending test. * Corresponding author. Tel.: +91 22 33612218; fax: +91 22 33611020. E-mail addresses:
[email protected] (R.S. Chaudhari),
[email protected] (V.B. Mankumare),
[email protected] (J. Shankaranarayanan),
[email protected] (J.V. Deshpande),
[email protected] (T.K. Sunilkumar),
[email protected] (S. Sathaye). 1 Address: New Technology Centre, A-10 Road No. 1, Wagle Industrial Estate, Thane (W) Pin: 400 064, India. http://dx.doi.org/10.1016/j.phanu.2014.08.001 2213-4344/ ã 2014 Elsevier B.V. All rights reserved.
annually, resulting in an osteoporotic fracture every 3 s [3]. It is estimated to affect 200 million women worldwide [3]. The old bone tissue is continuously removed and replaced by new tissue through the process called bone remodeling. Bone remodeling occurs via the coordinated action of osteoblasts OBs and osteoclasts OCs. The activities of OCs and OBs are combined into temporary anatomical spaces called basic multicellular units (BMUs) [4]. Estrogen deficiency leads to significant increase in the number of BMUs because of increased activation frequency, which is the number of new remodeling units activated per unit time [5]. This increased activation frequency expands the remodeling space, increases cortical bone tissue porosity, and enlarges the tissue resorption area on trabecular bone [6]. Carotenoid such as retinal is reported to have deleterious effect on bone at large doses. High intake of vitamin A leads to reduced bone density because of accelerated bone resorption and develops osteoporotic lesions in laboratory animals [7–9]. However, the effects of carotenoids on bone health have not been completely understood. Recent clinical studies show that high intake of carotenoids is helpful in preventing bone loss in post-menopausal women [9]. Epidemiological studies suggest that carotenoids have bone sparing effect in osteoporotic women [9]. Carotenoids are present in vegetables and fruits. Uchiyama and Yamaguchi [10] showed that carotenoid b-cryptoxanthin increases calcium and phosphorous deposition in cultured osteoblasts. It also increased
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the protein synthesis in bone tissue [10]. b-Cryptoxanthin directly stimulates bone formation in cultured bone tissues and also have inhibitory effect on bone resorption [11]. Paprika (Capsicum annuum L.) and its dietary products contain a variety of carotenoids [12]. b-Cryptoxanthin is the major carotenoid present in the paprika oleoresin [12]. Carotenoids from the paprika oleoresin are also reported to have very good bioavailability in humans [13]. The unique bone sparing property of b-cryptoxanthin led us to hypothesize that b-cryptoxanthin rich paprika extract (BPE) may prevent bone loss in ovariectomized rat model of osteoporosis. Thus the objective of the present study was to investigate the effect of b-cryptoxanthin rich paprika extract (BPE) on ovariectomy induced bone loss in experimental animals. 2. Materials and methods
collected from abdominal aorta and serum was separated and stored at 70 C until analysis. The left femur and tibia was carefully dissected and cleaned from surrounding muscle tissue. The bone samples were stored in 50% ethanolic saline at 70 C until analysis. 2.4. Biochemical analysis Alkaline phosphatase (ALP) and tartrate resistant acid phosphatase (TRAP) levels were analyzed in freshly separated serum samples. Activity of ALP was determined by spectrophotometric kinetic assay using p-nitrophenyl phosphate (p-NPP) as a substrate [16]. Activity of TRAP was determined by spectrophotometric kinetic assay using a-napthylphosphate as a substrate [17]. Urine calcium was measured by using commercially available calcium estimation kit (Accurex, India), based on o-cresolphthalein complexion method [18].
2.1. Paprika extract The Paprika extract rich in b-cryptoxanthin was obtained as a gift sample from OmniActive Health Technologies Ltd. The carotenoid content of paprika extract was estimated using gradient HPLC. Separation of carotenoids was carried out using reverse phase gradient elution. The compounds were identified by comparing the respective retention time with analytical standards. Chromatographic analysis showed the presence of b-cryptoxanthin as a major constituent (80% of total carotenoids) and traces of a-cryptoxanthin. 2.2. Animals and surgical procedure Virgin female Wistar rats were used for the study. Rats were 3 months old and weighed between 250 and 300 g. Rats were bilaterally ovariectomized (n = 16) to produce estrogen deficiency induced bone loss. Sham surgery was carried out (n = 8) to minimize the surgery stress. Rats were anesthetized with an intraperitoneal injection of ketamine hydrochloride (75 mg/kg) and xylazine (10 mg/kg). Procedure was performed by making a small 1 cm incision in flank region. The ovary surrounded by fat was exposed and the vessels supplying to the ovary were ligated and ovary was excised. Sham surgery was performed in the same manner without excising the ovary. The wound was then closed by using absorbable suture. Tramadol (5 mg/kg i.m.) was administered to each animal to minimize the post surgery pain. Animals were housed individually and allowed to recover for 1 week. The OVX rats were randomly divided into two groups, ovariectomized control and treatment group. 2.3. Study design Sham control and OVX control animals were administered orally with corn oil (pharmaceutical grade) (Kamani Oils, India) which was used as vehicle. The other OVX group was administered BPE equivalent to 20 mg b-cryptoxanthin/100 g of rat. The dose of BPE was selected as per doses mentioned in the earlier reports of b-cryptoxanthin for the studies carried out on bone physiology [14]. Drug treatments were started a week after ovariectomy with 2 ml/kg dosing volume and continued for 6 weeks. All rats were pair-fed and the quantity of food consumed matched with the sham group. Water was provided ad libitum. The experimental procedure was approved by Institutional Animal Ethics Committee (ICT/IAEC/2011/P08). On the last day of treatment urine was collected by micturation induced by manual pressure [15] from overnight fasted animals and preserved at 20 C till further analysis. At the end of the experiment, rats were euthanized by overdose of CO2. Blood was
2.5. Urinary excretion of pyridinium crosslinks Urinary excretion of pyridinium crosslinks (pyridinoline and deoxypyridinoline) was determined using commercially available ELISA assay kit (Quidel Corp., USA). The values obtained by ELISA assay were corrected for creatinine concentration to minimize variation. 2.6. Bone density Left femur bones were carefully isolated and cleaned from adhering tissues. Bone volume and density were measured using the method based on Archimedes’ principle [19,20]. 2.7. Bone mechanical strength Mechanical strength of bones was measured by three point bending test (TPBT) [21] for tibia using Brookfield CT3 texture analyzer. Tibia was placed on 2 supports 13 mm apart. Load was applied to midshaft, with applicator-head speed of 10 mm/min. The load applicator head compressed the middle of the tibial shaft with linearly increasing force, until fracture occurred. The load at which bone fractures was recorded. 2.8. Qualitative scanning electron microscopy The frozen right femurs were placed in 5% sodium hypochlorite solution for 4 h. The bones were then dehydrated in ethanol and dried, mounted on stubs and coated with gold using a sputter coater. The bones were examined on a JEOL JSM-6380LA scanning electron microscope. Qualitative observations of bone resorption at the epiphyseal edges were carried out. 2.9. Statistical methods The significance of difference between groups was estimated using student’s t-test. p-values of less than 0.05 were considered to indicate statistically significant differences. Statistical analysis was performed using GraphPad Prism 5.00 software (GraphPad, San Diego, CA, USA). 3. Results 3.1. Urinary calcium excretion The OVX control group showed significantly high urinary calcium levels as compared to sham control group. The calcium excretion in the BPE treatment remained high.
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3.2. Serum alkaline phosphatase (ALP) Alkaline phosphatase activity was significantly increased in OVX rats as compared with sham operated animals. However, BPE treatment group showed alkaline phosphatase activity comparable with sham control. 3.3. Serum tartrate-resistant acid phosphatase (TRAP) Increased activity of tartrate resistant acid phosphatase (TRAP) was observed in OVX rats as compared with sham operated animals. However, BPE treatment group showed no change in tartrate resistant acid phosphatase activity when compared to OVX control. 3.4. Urinary excretion pyridinium crosslinks Significantly (p > 0.05) high urinary excretion of pyridinium crosslinks were observed on OVX control group. Pyridinium crosslinks levels were significantly reduced in BPE treatment group when compared to OVX control and are comparable to sham operated group. 3.5. Bone density Significant reduction in bone density was observed in OVX control group. The BPE treatment significantly (p < 0.01) improved the bone density which is comparable to sham control animals. 3.6. Bone mechanical strength (three point bending test) The load bearing capacity was significantly reduced in OVX control animals as observed in TPBT. BPE treatment animals show significant (p < 0.01) improvement in mechanical strength as compared to OVX control animals. 3.7. Qualitative scanning electron microscopy The scanning electron photomicrographs (Fig. 7) clearly indicate an extensive resorption in OVX control animals when compared with sham operated animals. Observation of the number of resorption pits and depth on the surface clearly demonstrates the beneficial effects of BPE in inhibiting bone resorption process. 4. Discussion In the present study, we investigated the anti-osteoporotic potential of commercially available paprika extract. Paprika fruit is rich in carotenoid content. b-Cryptoxanthin is the major carotenoid reported in paprika [12]. Our chromatographic analysis showed the presence of b-cryptoxanthin as a major constituent (80% of total carotenoids) and traces of a-cryptoxanthin. It has been shown that b-cryptoxanthin has a stimulatory effect on osteoblastic bone formation and an inhibitory effect on osteoclastic bone resorption in vitro [10]. b-Cryptoxanthin also has anabolic effect on in-vitro cultured osteoblasts [14]. Carotenoids from paprika oleoresin are reported to have good bioavailability in humans [12]. Owing to the stimulatory and anabolic effect of b-cryptoxanthin on bone tissue and inhibitory effect on osteoclastic bone resorption, it was decided to evaluate its anti-osteoporotic potential in estrogendeficiency induced osteoporosis. Ovariectomized rat model is a well established model to mimic human postmenopausal bone loss and has been reported by many researchers previously [22–24]. Young adult female Wistar rats of 12 weeks of age [25] have been used in the study. Rats of this age
Fig. 1. Effect of BPE (0.20 mg/100 g p.o., 6 weeks) on urinary calcium excretion (n = 8/group). Each value represents the mean SEM. *p < 0.05 compared with the OVX control value.
group are skeletally mature and do not have features like rapidly growing bone or age-related bone loss. Prolonged of oral administration of BPE was found to have a preventive effect on loss of bone mineral density and mechanical strength of cancellous bone. OVX-induced changes in bone density and bone mechanical strength were clearly restored by oral administration of b-cryptoxanthin (Figs. 5 and 6). These observations demonstrate that oral administration of BPE can prevent bone loss in OVX rats. Bone loss after ovariectomy is because of high bone turnover where the rate of bone resorption exceeds the rate of bone formation. In the present study, bone turnover was assessed by serum ALP and TRAP, commonly used bone remodeling markers. ALP denotes bone formation activity whereas TRAP signifies bone resorption. The process of resorption and formation are always coupled and this is termed as bone remodeling or turnover. In the young adults the amount of bone formed is balanced by the amount resorbed resulting in maintenance of bone mass. Estrogen deficiency greatly increases the resorption process relative to the formation resulting in the negative bone balance. This may lead to reduced bone mass, deterioration of bone microarchitecture, and increased susceptibility to fractures [21]. OVX control animals showed increased bone turnover, as demonstrated by increased ALP and TRAP activity, than SH control rats. BPE did not affect the bone turnover as evidenced by unchanged serum levels of ALP and TRAP (Figs. 2 and 3). Pyridinium crosslinks (pyridinoline and deoxypyridinoline) are the bone collagen metabolites that appear in the urine when bone resorption process is accelerated hence considered as the early important marker of osteoporosis [26]. OVX control animals
Fig. 2. Effect of BPE (0.20 mg/100 g p.o., 6 weeks) on serum alkaline phosphatase activity (n = 8/group). Each value represents the mean SEM.
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Fig. 3. Effect of BPE (0.20 mg/100 g p.o., 6 weeks) on serum tartrate resistant acid phosphatase (n = 8/group). Each value represents the mean SEM.
Fig. 5. Effect of BPE (0.20 mg/100 g p.o., 6 weeks) on bone density (n = 8/group). Each value represents the mean SEM. **p < 0.01when compared with the OVX control value.
showed significant increase in pyridinoline crosslinks in urine which indicated accelerated bone resorption process caused due to estrogen deficiency. BPE significantly reduced urinary excretion of pyridinium crosslinks, suggestive of probable antiresorptive property of BPE (Fig. 4). Six weeks after ovariectomy, urinary excretion of calcium in OVX rats was significantly higher than the sham operated controls (Fig. 1). To confirm the calcium resorption from bones, we determined pyridinium crosslinks in urine as discussed earlier. Pyridinium crosslinks that appear in the urine are more specific markers of bone resorption process [26]. Increased urinary excretion of calcium along with increased pyridinium crosslinks in urine confirmed the process of bone resorption due to estrogen deficiency. These results suggest that the rapid bone loss occurs immediately after ovariectomy which is in consistence with the earlier findings reported by Goulding et al. [27]. BPE treatment showed reduction in urinary calcium excretion indicating its ability to correct the negative calcium balance by increasing calcium incorporation by osteoblasts to the bone tissue (Fig. 1). This effect may be correlated to the anabolic and anti-resorptive property of b-cryptoxanthin on cultured bone cells [11]. Bone density was measured using The Archimedes’ principle; it is the standard method for determination of density (g/cm3) of bones of small animals. It yields results statistically comparable with the non invasive dual-energy X-ray absorptiometry (DEXA) [28]. Cessation of the ovarian function in humans leads to increase in bone turnover, a negative bone balance, and a net decrease in bone density; these changes are also evident in surgically ovariectomized rats. A significant OVX induced decrease in bone
density was observed in OVX control group. BPE treatment significantly increased the bone density (Fig. 5). This may be attributed to the anabolic property of BPE reported earlier by Uchimaya and Yamaguchi [10]. Bone fragility can be defined broadly as the susceptibility to fracture. One function of bones is to carry loads. Fractures occur when loads exceed the bone strength, so weakened bones should be considered fragile. For example, osteoporotic vertebral bodies might fracture during normal daily activities such as opening a window or rising from a chair. These non traumatic or fragility fractures result from substantially weakened bones. On the other hand, long bone fracture results mainly from trauma associated with falls or impact. During a traumatic loading, such as a fall to the ground, fracture will occur if the energy from the fall exceeds the mechanical energy that the bone can absorb. Osteoporotic bones absorb very little energy before breaking (failure load) and are therefore more susceptible to fracture resulting from trauma. As per the WHO guidelines for preclinical evaluation of osteoporosis, measurement of failure load of cancellous bones (tibiae) is recommended as the ultimate determining factor in evaluation of osteoporosis in laboratory animals (WHO 1998) [28]. In this study, the bone mechanical strength was measured using three point bending test (ISO 149) the force at which fracture occurs was measured [29]. In ovariectomized animals there was significant decrease in maximal bearable load values for tibial mid-shaft indicating a significant loss of cancellous bone. BPE treatment significantly increased the maximal bearable load values of cancellous bone indicating increased bone mechanical strength and protection from bone fragility (Fig. 6).
Fig. 4. Effect of BPE (0.20 mg/100 g p.o., 6 weeks) on urinary excretion of pyridinium crosslinks (n = 8/group). Each value represents the mean SEM. *p < 0.05 when compared with the OVX control value.
Fig. 6. Effect of BPE (0.20 mg/100 g p.o., 6 weeks) on bone mechanical strength (n = 8/group). Each value represents the mean SEM. **p < 0.01 when compared with the OVX control value.
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The distal metaphyseal region of femur that contains both cortical and trabecular bone is very sensitive to estrogen deprivation and results in rapid and profound osteopenia [30]. We utilized the scanning electron microscopy technique to determine the pattern of bone resorption at the epiphyseal region of distal femur, which predominantly contains the areas on bone resorption. Porous and erosive appearance of femur at the epiphyseal edges was more pronounced and prevalent in OVX animals when compared with sham operated. Treatment with BPE decreased the number of resorption pits and maintained the intactness and integrity of the surface indicating its usefulness in the prevention of bone loss (Fig. 7).
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5. Conclusion In conclusion, it has been demonstrated that the prolonged oral administration of b-cryptoxanthin rich paprika has a preventive effect on bone loss as indicated by restored bone density and mechanical strength of cancellous bone in OVX rats. Our results suggest utility of b-cryptoxanthin rich paprika a sustainable nutritional approach to improving bone health in postmenopausal women. Conflicts of interest The authors declare no conflict of interest. Acknowledgements Authors are thankful to Mr. Jaynt Sancheti, Mr. Gauresh Somani, Mr. Pankaj Jain, Mr. Sachin Patil, Mr. Sagar Dhanawde, Ms. Manali Taskar, and Ms. Aditi Patil for their kind help in animal dissections and bone isolation. Layperson’s summary Fruit and vegetable intake is positively associated with bone density. The exact components of fruits and vegetables which confer benefit to bone are still to be clarified. Carotenoids are brightly colored compounds produced by plants and are present in fruits and vegetables. The intake of carotenoid rich food is reported to have positive impact on bone health. Thus our objective of this investigation is to study the effect of carotenoid rich food on overall bone health including bone density and mechanical property (ability to bear loads) in animal model of postmenopausal osteoporosis. b-Cryptoxanthin is a brightly red colored carotenoid present in paprika. b-Cryptoxanthin have antioxidant property and help prevent aging process. We induced the postmenopausal osteoporotic condition in laboratory animals by removing both ovaries of adult female Wistar rats. The removal of ovaries creates estrogen hormone deficiency which leads to weakened bones. We treated the ovariectomized rats with b-cryptoxanthin rich paprika extract. The treatment with b-cryptoxanthin rich paprika extract was continued till 6 weeks after ovariectomy procedure. We found improved bone density and mechanical strength in major load bearing bones of rats. The experimental procedure stated in the manuscript was approved by Institutional Animal Ethics Committee (ICT/IAEC/2011/P08). Our findings suggest that the paprika extract rich in b-cryptoxanthin may have potential nutritional value in preventing the progress and improving the overall bone health in elderly osteoporotic patients.
References
Fig. 7. Scanning electron photomicrographs (500) demonstrating resorptive pits and surface in remodeling sites at the epiphyseal edges of femur from rats of sham operated (Fig. 7a), ovariectomized (Fig. 7b) and BPE treated (Fig. 7c). Note the increased number of resorption pits (red circle) in OVX group compared with sham and treated group. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
[1] NIH. “Osteoporosis Prevention, Diagnosis, and Therapy”. NIH Consensus Statement Online March 27-29 2000;17(1):1–36. [2] WHO. “Assessment of Fracture Risk and its Application to Screening for Postmenopausal Osteoporosis,” Report of a WHO Study Group. Geneva: World Health Organization; 1994 (WHO Technical Report Series, No. 843). [3] Johnell O, Kanis JA. An estimate of the worldwide prevalence and disability associated with osteoporotic fractures. Osteoporos Int 2006;17:1726–33, doi: http://dx.doi.org/10.1007/s00198-006-0172-4. 16983459. [4] Jilka RL. Biology of the basic multicellular unit and the pathophysiology of osteoporosis. Med Pediatr Oncol 2003;41:182–5, doi:http://dx.doi.org/ 10.1002/mpo.10334. 12868116. [5] Compston JE. Sex steroids and bone. Physiol Rev 2001;81(1):419–47. 11152762.
154
R.S. Chaudhari et al. / PharmaNutrition 2 (2014) 149–154
[6] Jilka RL, Takahashi K, Munshi M. Loss of estrogen upregulates osteoblastogenesis in the murine bone marrow. Evidence for autonomy from factors released during bone resorption. J Clin Invest 1998;101:1942–50, doi:http:// dx.doi.org/10.1172/JCI1039. 9576759. [7] Forsyth KS, Watson RR, Gensler HL. Osteotoxicity after chronic dietary administration of 13-cis-retinoic acid, retinyl palmitate or selenium in mice exposed to tumor initiation and promotion. Life Sci 1989;45:2149–56, doi: http://dx.doi.org/10.1016/0024-3205(89)90081-7. 2601568. [8] Rohde CM, Manatt M, Clagett-Dame M, Deluca HF. Vitamin A antagonizes the action of vitamin D in rats. J Nutr 1999;129:2246–50. 10573558. [9] Promislow JH, Goodman-Gruen D, Slymen DJ, Barrett-Connor E. Retinol intake and bone mineral density in the elderly: the rancho bernardo study. J Bone Miner Res 2002;17:1349–58, doi:http://dx.doi.org/10.1359/ jbmr.2002.17.8.1349. 12162487. [10] Uchiyama S, Yamaguchi M. Oral administration of b-cryptoxanthin prevents bone loss in ovariectomized rats. Int J Mol Med 2007;17:15–20. [11] Yamaguchi M, Uchiyama S. b-cryptoxanthin stimulates bone formation and inhibits bone resorption in tissue culture in vitro. Mol Cell Biochem 2004;258:137–44, doi:http://dx.doi.org/10.1023/B: MCBI.0000012848.50541.19. 15030178. [12] Pérez-Gálvez A, Martin HD, Sies H, Stahl W. Incorporation of carotenoids from paprika oleoresin into human chylomicrons. Br J Nutr 2003;89:787–93, doi: http://dx.doi.org/10.1079/BJN2003842. 12828795. [13] Parker RS. Bioavailability of carotenoids in human subjects. Eur J Clin Nutr 1997;51(Suppl. 1):S86–90. 9023490. [14] Uchiyama S, Sumida T, Yamaguchi M. Oral administration of b-cryptoxanthin induces anabolic effects on bone components in the femoral tissues of rats in vivo. Biol Pharm Bull 2004;27(2):232–5. 14758041. [15] Weiss J, Taylor GR, Zimmermann F, Nebendahl K. Collection of body fluids. Laboratory Rat. New York: Academic Press; 2000. p. 485–510. [16] Neumann H, Van Vreedendaal M. An improved alkaline phosphatase determination with p-nitrophenyl phosphate. Clin Chim Acta 1967; 17(2):183–7, doi:http://dx.doi.org/10.1016/0009-8981(67)90117-9. [17] Cooper JD, Turnell DC, Price CP. The estimation of serum acid phosphatase using alpha-naphthyl phosphate as substrate: observations on the use of fast red TR salt in the assay. Clin Chim Acta 1982;126(3):297–306, doi:http://dx. doi.org/10.1016/0009-8981(82)90304-7. 7151283. [18] Connerty HV, Briggs AR. Determination of serum calcium by means of orthocresolphthalein complexone. Am J Clin Pathol 1966;45:290–6. 5932858.
[19] Kalu DN. Evaluation of the pathogenesis of skeletal changes in ovariectomized rats. Endocrinology 1984;115:507–12, doi:http://dx.doi.org/10.1210/endo115-2-507. 6745166. [20] Lee YB, Lee HJ, Kim KS, Lee JY, Nam SY, Cheon SH, et al. Evaluation of the preventive effect of isoflavone extract on bone loss in ovariectomized rats. Biosci Biotechnol Biochem 2004;68:1040–5, doi:http://dx.doi.org/10.1271/ bbb.68.1040. [21] Peng Z, Tuukkanen J, Zhang H. The mechanical strength of bone in different rat models of experimental osteoporosis. Bone 1994;15:523–32, doi:http://dx. doi.org/10.1016/8756-3282(94)90276-3. 7980963. [22] Kalu DN, Liu CC, Hardin RR, Hollis BW. The aged rat model of ovarian hormone deficiency bone loss. Endocrinology 1989;124:7–16, doi:http://dx.doi.org/ 10.1210/endo-124-1-7. 2909382. [23] Cai DJ, Zhao Y, Glasier J, Cullen D, Barnes S, Turner CH, et al. Comparative effect of soy protein, soy isoflavones and 17-b estradiol on bone metabolism in adult ovariectomized rats. J Bone Miner Res 2005;20:828–39, doi:http://dx.doi.org/ 10.1359/JBMR.041236. 15824856. [24] Horcajada-Molteni MN, Crespy V, Coxam V, Davicco MJ, Rémésy C, Barlet JP, et al. Rutin inhibits ovariectomy-induced osteopenia in rats. J Bone Miner Res 2000;15:2251–8, doi:http://dx.doi.org/10.1359/ jbmr.2000.15.11.2251. 11092407. [25] Zhang Y, Lai WP, Leung PC, Wu CF, Wong MS. Short- to mid-term effects of ovariectomy on bone turnover, bone mass and bone strength in rats. Biol Pharm Bull 2007;30:898–903. 17473432. [26] Seibel MJ, Robins SP, Bilezikian JP. Urinary pyridinium crosslinks of collagen: specific markers of bone resorption in metabolic bone disease. Trends Endocrinol Metab 1992;3(7):263–70, doi:http://dx.doi.org/10.1016/10432760(92)90129-O. 18407110. [27] Goulding A, Gold E, Lewis-Barned NJ. Effects of hysterectomy on bone in intact rats, ovariectomized rats, and ovariectomized rats treated with estrogen. J Bone Miner Res 1996;11(7):977–83, doi:http://dx.doi.org/10.1002/ jbmr.5650110715. 8797119. [28] WHO. Guidelines for Preclinical Evaluation and Clinical Trials in Osteoporosis. Geneva: World Health Organization; 1994. [29] ISO 178:2010(E): Determination of flexural properties, 2010-12-15, International Organization for Standardization, Geneva, Switzerland. [30] Westerlind KC, Wronski TJ, Ritman EL, Luo ZP, An KN, Bell NH, et al. Estrogen regulates the rate of bone turnover but bone balance in ovariectomized rats is modulated by prevailing mechanical strain. Proc Natl Acad Sci U S A 1997;94:4199–204, doi:http://dx.doi.org/10.1073/pnas.94.8.4199. 9108129.
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