The effects of Cosmos caudatus (ulam raja) on dynamic and cellular bone histomorphometry in ovariectomized rats
Abstract
Background
Cosmos caudatus is a local plant which has antioxidant properties and contains high calcium. It is also reported to be able to strengthen the bone. This report is an extension to previously published article in Evidence Based Complementary and Alternative Medicine (doi:10.1155/2012/817814). In this study, we determined the effectiveness of C. caudatus as an alternative treatment for osteoporosis due to post-menopause by looking at the dynamic and cellular paramaters of bone histomorphometry.
Methods
Forty female Wistar rats were divided into four groups i.e. sham operated, ovariectomized, ovariectomized treated with calcium 1% ad libitum and ovariectomized force-fed with 500 mg/kg C. caudatus extract. Treatment was given six days a week for eight weeks.
Results
Dynamic and cellular histomorphometry parameters were measured. C. caudatus increased double-labeled surface (dLS/BS), mineral appositional rate (MAR), osteoid volume (OV/BV) and osteoblast surface (Ob.S/BS). C. caudatus also gave better results compared to calcium 1% in the osteoid volume (OV/BV) parameter.
Conclusions
C. caudatus at the 500 mg/kg dose may be an alternative treatment in restoring bone damage that may occur in post-menopausal women.
Background
This paper reported the findings which were an extension to an article recently published [1].
Low bone mass and deterioration of bone tissue microarchitectural which are observed in osteoporosis may eventually lead to increase bone fragility and tendency to fracture [2,3]. Osteoporosis has many negative impacts on public health by increasing death rates and economic costs due to the impact of the fracture [2]. Increasing age is a major risk for osteoporosis and fractures [4].
To date, there are several treatments available to treat osteoporosis such as bisphosphonates, calcitonin, estrogen and/or hormone therapy, selective estrogen receptor modulator, anabolic agents and others [5]. However, there are many studies reporting that these treatments have side effects such as estrogen treatment can pose risk of breast cancer [6], ovarian and endometrial cancer [7] to the osteoporosis patients even though it can reduce the fracture of hip, vertebrae and other bones [8].
Natural products have been proven to affect bone metabolism by involving a complex balance between deposition, mineralization and resorption of bone matrix [9]. Natural substances that were often studied and were observed to have osteoprotective effects were phytoestrogens such as ferutinin [10], genistein [11] and 8-prenylnaringenin [12].
Cosmos caudatus or better known among locals as ulam raja is a popular herb in Malaysia [13]. Methanol extract of C. caudatus was found to have antioxidant activity [14,15]. In addition, C. caudatus was also found to have antifungal activity [16] and can stimulate bone formation [17]. In analogy with other vegetal substances already demonstrated to mime osteoprotective functions as described above, C. caudatus was considered to be able to act on bone tissue. Thus, this study was done to determine the effects of C. caudatus on dynamic and cellular bone histomorphometry of ovariectomized rats which will serve as a model for post-menopausal women.
Methods
Animals and treatment
This study utilized thirty-two young adult (3 months) female Wistar rats, which were obtained from the Laboratory Animal Resource Unit, Faculty of Medicine, Universiti Kebangsaan Malaysia. The rats, weighing 190 g-260 g, were randomly assigned to four groups with eight rats in each group. Group 1 was sham operated (Sham) while the second group was ovariectomized control group (OVX). The third and fourth groups were ovariectomized and treated with calcium 1% (Ca) ad libitum and force-fed with 500 mg/kg C. caudatus extract (CC) respectively. The ovariectomy and sham operation protocol was carried out as previously described [18]. Rats were left recuperating for 1 week before commencing the treatment. Treatment was given six days a week for eight weeks. All rats were injected with two doses of calcein nine days and two days before sacrificed. The study was approved by the Universiti Kebangsaan Malaysia Animal Ethics Committee with the approval code of PP/FAR/2008/NORAZLINA/12-AUGUST/225-SEPT-2008-AUG-2009.
Diet, Cosmos caudatus and calcium 1%
All rats received normal rat chow obtained from Gold Coin, Malaysia. Water extraction method was used to obtain 500 g/300 ml concentration of C. caudatus aqueous extract. Extraction was carried out by School of Chemical Sciences & Food Technology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia as previously described [19]. The 500 mg/kg dose was prepared by mixing C. caudatus with deionized water in ratio 3:7. Calcium 1% solution was prepared by mixing 1 g of hemicalcium lactic acid (Sigma Chemical CO., USA) with 100 ml deionized water.
Bone histomorphometry
After 8 weeks of treatment, rats were sacrificed via euthanasia with diethyl ether. The left femora were removed and the distal parts were fixed in 70% ethanol. Cellular parameters measured were osteoid volume (OV/BS, osteoid volume over bone surface), osteoid surface (OS/BS, osteoid surface over bone surface), osteoclast surface (Oc.S/BS, osteoclast surface over bone surface), osteoblast surface (Ob.S/BS, osteoblast surface over bone surface) and eroded surface (ES/BS, eroded surface over bone surface). Bone samples were decalcified according to the method described by Hermizi et al. [20]. In addition, undecalcified bone samples were used to measure dynamic parameters i.e. single labeled surface (sLS/BS, single labeled surface over bone surface), double-labeled surface (dLS/BS, double-labeled surface over bone surface), mineralized surface (MS/BS, mineralized surface over bone surface), mineral appositional rate (MAR), and bone formation rate (BFR/BS, bone formation rate over bone surface). Preparation of bone samples and measurement techniques were carried out as previously described [20].
Statistical analysis
All data were subjected to normality test using the Kolmogorov test. For normally distributed data, the statistical test used was analysis of variance, followed by Tukey’s honestly significance difference test. While non-normally distributed data were analysed using Mann–Whitney and Kruskal Wallis tests. Data analysis was performed using Statistical Package for Social Sciences (18.0.1; SPSS, Inc., Chicago, IL) software. Results were expressed as mean ± standard error of the mean (SEM).
Results
Figure 1 shows the photomictorographs of the trabecular bone of the distal part of femurs analysed using fluorescence microscope. Calcein acts as a fluorescent marker which labels the bone surface thus the double-labeled surface (dLS) and single-labeled surface (sLS) parameters may be determined. From the photos, it was observed that the OVX group had more sLS than dLS while the Ca and CC groups had more dLS than sLS.
Photomicrographs of trabecular bone labelled with calcein. Arrows indicating dLS (double-labeled surface) and sLS (single-labeled surface).
Table 1 shows the dynamic bone histomorphometric results. The Ca and CC groups showed significant increase (p < 0.05) in dLS/BS compared to the sham group. For mineral appositional rate parameter (MAR), the Ca and CC groups showed a significant increase (p < 0.05) compared to the sham and OVX group.
The effects of Cosmos caudatus and calcium on dynamic histomorphometry parameters in ovariectomized rats
| Dynamic parameters | Sham | OVX | Ca | CC |
|---|---|---|---|---|
| Single labeled surface (%) | 75.44 ± 4.00 | 78.99 ± 3.35 | 73.8 ± 6.33 | 73.1 ±6 .33 |
| Double-labeled surface (%) | 18.5 ± 3.33 | 24.68 ± 5.08 | 28.38 ± 2.78* | 38.9 ± 4.10* |
| Mineralized surface (%) | 58.69 ± 1.10 | 63.78 ± 1.18 | 60.55 ± 2.86 | 63.55 ± 3.10 |
| Mineral appositional rate (μm/day) | 1.52 ± 0.12 | 1.52 ± 0.13 | 3.20 ± 0.38#* | 3.60 ± 0.09#* |
| Bone formation rate (μm3/μm2/day) | 1.00 ± 0.09 | 1.08 ± 0.02 | 1.58 ± 0.27 | 1.48 ± 0.29 |
Sham sham-operated rats.
OVX ovariectomized rats.
Ca ovariectomized rats supplemented with 1% calcium.
CC ovariectomized rats supplemented with 500 mg/kg Cosmos caudatus.
* significant vs sham group (p < 0.05).
# significant vs OVX group (p < 0.05).
Data is presented as mean ± S.E.M.
The results for the cellular parameters are shown in Table 2. The osteoblast surface (Ob.S/BS) of the Ca and CC groups were increased significantly (p < 0.05) compared to the OVX group. Osteoid volume (OV/BS) was significantly increased (p < 0.05) in the sham and CC groups compared to the OVX group. In addition, osteoid volume of CC group was also higher (p < 0.05) compared to the Ca group. No significant differences were seen in the other parameters.
The effects of Cosmos caudatus and calcium on cellular histomorphometry parameters in ovariectomized rats
| Cellular parameters | Sham | OVX | Ca | CC |
|---|---|---|---|---|
| Osteoblast surface (%) | 30.92 ± 8.78 | 8.75 ± 5.15* | 55.32 ± 10.2# | 70.4 ± 7.78# |
| Osteoclast surface (%) | 52.43 ± 12.59 | 65.56 ± 13.04 | 62.96 ± 6.68 | 36.12 ± 1.11 |
| Eroded surface (%) | 12.32 ± 6.00 | 38.89 ± 14.70 | 36.53 ± 9.15 | 22.62 ± 4.66 |
| Osteoid volume (%) | 13.13 ± 1.92 | 4.54 ± 2.46* | 8.66 ± 1.07 | 18.27 ± 0.53#@ |
| Osteoid surface (%) | 72.95 ± 2.66 | 51.29 ± 3.43 | 63.08 ± 5.62 | 71.85 ± 7.09 |
Sham sham-operated rats.
OVX ovariectomized rats.
Ca ovariectomized rats supplemented with 1% calcium.
CC ovariectomized rats supplemented with 500 mg/kg Cosmos caudatus.
* significant vs sham group (p < 0.05).
# significant vs OVX group (p < 0.05).
@ significant vs Ca 1% (p < 0.05).
Data is presented as mean ± S.E.M.
Discussion
Ovariectomy caused a state of estrogen deficiency which may lead to bone loss. The use of rats as a model for postmenopausal osteoporosis has been discussed and significant bone loss may be seen as early as 14 days in the proximal tibial metaphysis [21]. The ovariectomy-induced osteopenia observed in rats are similar to skeletal responses in post-menopausal women [22].
In this study, the ovariectomized group did not differ to sham-operated group in terms of dynamic histomorphometry parameters (Table 1). These findings were in agreement with our previous study except in one parameter. The previous study showed that the ovariectomized rats had lower mineral appositional rate compared to the sham-operated group [23]. In terms of cellular histomorphometry parameters, ovariectomy caused reduction in osteoid volume as compared to the sham group. Other parameters were not significantly different compared to sham even though declining values were observed. However, in another study it was observed that cellular parameters deteriorated in the ovariectomized group as compared to the sham group [24]. The discrepancies seen in the histomorphometry parameters between ovariectomized and sham-operated groups were surprising and it may be attributed to the small sample size used in our study. In addition, the technique used in measuring the histomorphometric parameters may not be sensitive since others have found that the use of high-throughput technique such as microcomputed tomography was able to yield more significant results [25].
The effects of C. caudatus on bone biochemical markers were previously investigated in which we used the doses of 100, 200 and 300 mg/kg. It was observed that all doses of C. caudatus were able to prevent the increase in interleukin-1 and pyridinoline in the ovariectomized group. However, no significant changes were seen in the bone histomorphometry parameters (unpublished data).
Following that, we proceeded with a higher dose i.e. 500 mg/kg and look at the bone histomorphometry paramaters. Parts of the results from this study were previously published in which we observed C. caudatus was able to improve some of the structural bone histomorphometry parameters as compared to ovariectomized group [1]. In this article, we reported the remaining half of the findings i.e. the dynamic and cellular bone histomorphometry parameters.
No significant differences were observed in single-labeled surface between the treatment groups. High single-labeled surface reflects high bone resorption rate [26]. Thus, the absence of significant differences in this parameter indicates that Ca 1% and C. caudatus did not affect the resorption process. However, this study showed that groups treated with Ca 1% and C. caudatus had higher double-labeled surface. The presence of high double-labeled surface indicates high bone formation and active mineralization process [27,28]. The results suggested that 500 mg/kg C. caudatus extract may play a role in stimulating bone formation in ovariectomized rats. Calcium, on the other hand, has been used as a treatment for osteoporosis [29]. Daily calcium supplementation has been shown to prevent bone loss in postmenopausal women [30].
Mineral appositional rate (MAR) is the distance between two fluorescence labels which were formed when calcein was injected on the ninth day and second day before rats were sacrificed [31]. MAR represents the osteoblastic activities in the bone [32]. High MAR value in the Ca and CC groups is suggestive of high osteoblastic activity. This is in agreement with osteoblast surface value which was higher in the Ca and CC groups compared to OVX.
Osteoid volume (OV/BV) was found to increase significantly in the sham and CC groups compared to the OVX group. The CC group also had a higher osteoid volume compared to the Ca group. Osteoid is bone matrix that is not mineralized yet. The increase in osteoid volume may occur due to the increase in osteoid formation or by the disruption that has occurred during the process of mineralization of bone [33]. The increase in osteoid volume in the CC group showed that C. caudatus may have a role in osteoid formation. This effect of C. caudatus is an advantage over Ca 1%. Cosmos caudatus was found to have high phenolic content of the flavonoids class (which include catechin, epicatechin, myricetin and luteolin) and was observed to possess free-radicals scavenging activity [34]. These properties of C. caudatus may also contribute to its bone-protective effects. It is also reported that C. caudatus contains 270 mg calcium per 100 g of the plant [35]. This composition, in addition to its antioxidative property, may contribute to the superiority of C. caudatus as opposed to calcium supplementation.
Calcium supplementation in this study was given in drinking water at the concentration of 1%. This method of administration was used in our previous study [36] and has also been used by others [37]. The advantage of using this route of administration is that it minimizes the stress from using injection or oral gavage. However we did not measure serum levels of calcium to ensure adequate calcium intake which can further verify the differences seen between the C. caudatus group and the Ca 1% group.
No significant differences in osteoid surface (OS/BS) were observed between all the groups. Although the percentage of osteoid surface is higher in the Ca and CC groups than in the OVX group, significant difference was not observed. This may be attributed to the small sample size used in this study.
There were no significant differences in osteoclast surface (Oc.S/BS) between all the groups. However, based on a previous study, there was a significant increase in Oc.S/BS for ovariectomized rats [38]. In addition, post-menopausal condition can stimulate the formation of osteoclast by the production of nuclear factor-kβ ligand (RANKL) and tumor necrosis factor (TNF) by monocytes and T cell, and this phenomenon tends to occur in women with osteoporosis [39]. C. caudatus did not seem to cause any effects on osteoclast surface which implies C. caudatus has no effects on resorption process.
Conclusions
C. caudatus has beneficial effects on dynamic and celluar bone histomorphometry parameters in ovariectomized rats. C. caudatus was also observed to be superior to Ca 1% in increasing the osteoid volume parameter. Thus, C. caudatus has the potential to become an alternative treatment in restoring bone damage in post-menopausal women.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
NM participated in the design of the study and drafted the manuscript. ZS carried out the analysis and performed the statistical analysis. ESMR participated in interpreting the bone histomorphometry analysis. NM helped in the design of the study and helped to draft the manuscript. All authors read and approved the final manuscript.
Acknowledgments
The authors are grateful to Prof Dr Abdul Salam Babji from Faculty of Science and Technology, Universiti Kebangsaan Malaysia for the preparation of the plant extract. The authors thank Ms. Azizah Osman, Ms. Sinar Suriya Muhamad, Mr. Arizi Aziz, Ms. Mazliadiyana Mazlan, Mr. OK Azuan Ghazali and Mr. Imran Mohd Ali for their technical assistance. The authors expressed their gratitude to UKM grant FF-193-2008 for funding this project.
References
- 1. The effects of Cosmos caudatus on structural bone histomorphometry in ovariectomized ratsEvid Based Complement Alternat Med20122012817814[PubMed][Google Scholar]
- 2. Current pharmacological approaches to prevent and treat post menopausal osteoporosisRecent Pat Endocr Metab Immune Drug Discov200934253[PubMed][Google Scholar]
- 3. Consensus Development ConferenceDiagnosis, prophylaxis and treatment of osteoporosisAm J Med199394646650[PubMed][Google Scholar]
- 4. Hip fractures in the elderly: a world-wide projectionOsteoporosis Int199226285289[PubMed][Google Scholar]
- 5. Nutrition: effect of vegetables on bone metabolismNature1999401343344[PubMed][Google Scholar]
- 6. Effect of hormone replacement therapy on breast cancer risk: Estrogen versus estrogen plus progestinJ Natl Cancer Inst200092328332[PubMed][Google Scholar]
- 7. Hormone replacement therapy: current controversiesClin Endocrinol200358249261[PubMed][Google Scholar]
- 8. Effects of conjugated equine estrogen in postmenopausal women with hysterectomy: the Women’s health initiative randomized controlled trialJ Am Med Assoc20042911417011712[Google Scholar]
- 9. Natural products as alternative treatments for metabolic bone disorders and for maintenance of bone healthPhytother Res20072199112[PubMed][Google Scholar]
- 10. Influence of ferutinin on bone metabolism in ovariectomized rats. II: role in recovering osteoporosisJ Anat20102174856[PubMed][Google Scholar]
- 11. Dietary phytoestrogens and their effect on bone: evidence from in vitro and in vivo, human observational, and dietary intervention studiesAm J Clin Nutr2003783 Suppl593S609S[PubMed][Google Scholar]
- 12. Tissue specificity of 8-prenylnaringenin: protection from ovariectomy induced bone loss with minimal trophic effects on the uterusJ Steroid Biochem Mol Biol200597299305[PubMed][Google Scholar]
- 13. Malay herbal medicine in Germencheh Negeri Sembilan, MalaysiaFitoterapia1999701014[PubMed][Google Scholar]
- 14. Flavonoid content and antioxidant activity of vegetables from IndonesiaFood Chem2010121412311235[PubMed][Google Scholar]
- 15. Antioxidant activity of plants methanolic extracts containing phenolic compoundsAfr J Biotechnol200983484489[Google Scholar]
- 16. Phenylpropane derivates from roots of Cosmos caudatusPhytochem199539409412[PubMed][Google Scholar]
- 17. Sayuran Tradisional Ulam dan Penyedap Rasa2000Bangi: Universiti Kebangsaan Malaysia
- 18. Bone oxidative changes during early fracture healing of postmenopausal osteoporosis rat modelAsian J Animal Vet Adv2011611931203[PubMed][Google Scholar]
- 19. Antioxidative activities of some Malaysian herbal water extractsASEAN Food J2007146168[Google Scholar]
- 20. Effects of nicotine administration and nicotine cessation on bone histomorphometry and bone biomarkers in Sprague–Dawley male ratsCalcif Tissue Int2011884147[PubMed][Google Scholar]
- 21. The laboratory rat as an animal model for osteoporosis researchComp Med2008585424430[PubMed][Google Scholar]
- 22. Assessing bone quality-animal models in preclinical osteoporosis researchBone199517343S352S[PubMed][Google Scholar]
- 23. Comparison of the effects of tocotrienol and estrogen on the bone markers and dynamic changes in postmenopausal osteoporosis rat modelAsian J Anim Vet Adv20127225234[PubMed][Google Scholar]
- 24. Labisia pumila protects the bone of estrogen-deficient rat model: a histomorphometric studyJ Ethnopharmacol20121421294299[PubMed][Google Scholar]
- 25. Analysis of change patterns of microcomputed tomography 3-dimensional bone parameters as a high-throughput tool to evaluate antiosteoporotic effects of agents at an early stage of ovariectomy-induced osteoporosis in miceInvest Radiol2006419704712[PubMed][Google Scholar]
- 26. Laboratory histopathology: a complete reference1994London: Churchill Livingstone Publisher
- 27. Beneficial effects of tocotrienol and tocopherol on bone histomorphometric parameters in sprague–dawley male rats after nicotine cessationCalcif Tissue Int2009846574[PubMed][Google Scholar]
- 28. Tocotrienol offers better protection than tocopherol from free radical–induced damage of rat boneClin Exp Pharmacol Physiol200532761770[PubMed][Google Scholar]
- 29. Preventing osteoporosis-related fractures: an overviewAm J Med2006119S3S11[PubMed][Google Scholar]
- 30. The effect of calcium supplementation on bone loss in 32 controlled trials in postmenopausal womenOsteoporosis Int2009201221352143[PubMed][Google Scholar]
- 31. Bone histomorphometry: standardization of nomenclature, symbols and unitsJ Bone Miner Res19876595610[PubMed][Google Scholar]
- 32. Bone histomorphometry: analysis of trabecular bone dynamicBone histomorphometry: technique and interpretations1983Boca Raton: CRC Press109131[Google Scholar]
- 33. High fluoride intake causes osteomalacia and diminished bone strength in rats with renal deficiencyBone199619595601[PubMed][Google Scholar]
- 34. Total phenolic compounds, flavonoids, and radical scavenging activity of 21 selected tropical plantsJ Food Sci2010751C28C35[PubMed][Google Scholar]
- 35. Nutriweb Malaysia: nutrition composition database – ulam rajahttp://www.nutriweb.org.my/cgi-bin/dbrecords.cgi?comp=Minerals&submit_do=View&food_no=105105&part1=Raw+and+Processed&fname=Ulam+raja&group=Vegetables&mname=Ulam+raja
- 36. Effect of dietary calcium on spinal bone fusion in an ovariectomized rat modelJ Korean Neurosurg Soc2012524281287[PubMed][Google Scholar]
- 37. Effects of calcium supplements on fracture healing in a rat osteoporotic modelJ Orthop Res2010281216511656[PubMed][Google Scholar]
- 38. Histologic evidence for osteopenia and increased bone turnover in ovariectomized ratsBone198672119123[PubMed][Google Scholar]
- 39. Estrogen deficiency increases osteoclastogenesis up-regulating T cells activity: a key mechanism in osteoporosisBone200843192100[PubMed][Google Scholar]