Betulinic Acid Mitigates Estrogen Deficiency–Related Femoral Bone Loss: Evidence from an Ovariectomized Rat Model

Onural Özhan; Hilal Sahin; Mehmet Fatih Korkmaz; Azibe Yıldız; Ersoy Kekilli; Nigar Vardı; Hakan Parlakpınar

doi:10.62425/rtpharma.1871984

Betulinic Acid Mitigates Estrogen Deficiency–Related Femoral Bone Loss: Evidence from an Ovariectomized Rat Model

Abstract

Objective: Postmenopausal osteoporosis (OP) is characterized by estrogen deficiency–induced bone loss and deterioration of trabecular microarchitecture. Betulinic acid (BA), a natural pentacyclic triterpenoid, has demonstrated antioxidant and osteoprotective properties. This study aimed to evaluate the histopathological effects of BA on femoral bone tissue in ovariectomized rats. Methods: Thirty-two female Wistar albino rats were randomly divided into four groups (n=8): Sham, OVX, BA+OVX (BA administered before OVX), and OVX+BA (BA administered after OVX). BA was given orally at 3 mg/kg/day. Pelvic bone mineral density (BMD) was assessed using dual-energy X-ray absorptiometry (DEXA). Serum calcium levels were measured, and femoral bone tissues were evaluated histopathologically for trabecular thinning and loss of connectivity. Results: OVX resulted in a significant reduction in pelvic BMD and marked deterioration of trabecular bone architecture compared with the Sham group. BA administration prior to OVX significantly attenuated OVX-induced bone loss, as evidenced by higher pelvic BMD and preserved trabecular structure compared with the OVX group (p<.05). In contrast, BA treatment initiated after OVX did not significantly reverse bone loss or histopathological damage. Serum calcium levels did not differ significantly among the groups. Conclusion: BA exerts a protective effect against OVX-induced bone loss when administered prior to estrogen deficiency, preserving both bone mineral density and trabecular microarchitecture. These findings suggest that BA may have a preventive, rather than therapeutic, role in estrogen deficiency–related (OP).

Keywords

Supporting Institution

TUBITAK

Project Number

TUBITAK Grant Agreement No : 1919B011700680

Ethical Statement

Ethics committee approval was received for this study from the ethics committee of İnönü University (Date: March 29, 2016, Number: 2016/A-47).

References

Aeimlapa, R., Wongdee, K., Tiyasatkulkovit, W., Kengkoom, K., Krishnamra, N., & Charoenphandhu, N. (2019). Anomalous bone changes in ovariectomized type-2 diabetic rats: inappropriately low bone turnover with bone loss in an estrogen-deficient condition. American Journal of Physiology. Endocrinology and Metabolism, 317(4), E646-E657. https://doi.org/10.1152/ajpendo.00093.2019
Anish, R., Soumya, N., Nair, A., & Rauf, A. (2023). Standardization of Sprague-Dawley rats as a postmenopausal osteoporotic model through biochemical marker evaluation and Dexa scan. Exploratory Animal and Medical Research, 13(1), 39-48. https://doi.org/10.52635/eamr/13.1.39-48
Arslan, A. K., Yaşar, Ş., Çolak, C., & Yoloğlu, S. (2018). R Shiny Paketi ile Kruskal Wallis H Testi için İnteraktif Bir Web Uygulaması [An Interactive Web Application for Kruskal Wallis H Test with R Shiny]. Annals of Health Sciences Research, 7(2), 49-55.
Atwa, A., Mostafa, M., Song, S.-J., & Sarhan, M. (2021). The Femoral Head Epiphysis of Ovariectomized Rats as A Site for Studies on Osteoporosis: Microstructural Changes Evaluations. Egyptian Academic Journal of Biological Sciences, 13, 1-12. https://doi.org/10.21608/eajbsd.2021.145983
Bilgiç, Y., Alkuş, H., Özhan, O., Üremiş, M. M., Vardı, N., Arslan, A. K.,…Türköz, Y. (2022). Protective and Therapeutic Effects of Betulinic Acid on Acetic Acid-Induced Experimental Colitis Model. Journal of Enterocolitis, 1, 6-11. https://doi.org/10.5152/Jenterocolitis.2022.220203
Boyd, S., Davison, P., Müller, R., & Gasser, J. (2006). Monitoring individual morphological changes over time in ovariectomized rats by in vivo micro-computed tomography. Bone, 39(4), 854-862. https://doi.org/10.1016/j.bone.2006.04.017
Campbell, G., Buie, H., & Boyd, S. (2008). Signs of irreversible architectural changes occur early in the development of experimental osteoporosis as assessed by in vivo micro-CT. Osteoporosis International, 19, 1409-1419. https://doi.org/10.1007/s00198-008-0581-7
Cheng, C., Chen, L.-R., & Chen, K.-H. (2022). Osteoporosis Due to Hormone Imbalance: An Overview of the Effects of Estrogen Deficiency and Glucocorticoid Overuse on Bone Turnover. International Journal of Molecular Sciences, 23(3), 1376. https://doi.org/10.3390/ijms23031376

Choi, H., Jeong, B.-C., Kook, M., & Koh, J. (2016). Betulinic acid synergically enhances BMP2-induced bone formation via stimulating Smad 1/5/8 and p38 pathways. Journal of Biomedical Science, 23(1), 45. https://doi.org/10.1186/s12929-016-0260-5
De Villiers, T. (2023). Bone health and menopause: Osteoporosis prevention and treatment. Best practice & research. Clinical Endocrinology & Metabolism, 38(1), 101782. https://doi.org/10.1016/j.beem.2023.101782
Dereka, X., Emfietzoglou, R., & Lelovas, P. (2025). Effects of Ovariectomy and Low-Calcium Diet on Six Different Sites of the Rat Skeleton. Biomimetics, 10(7), 474. https://doi.org/10.3390/biomimetics10070474
Eastell, R., O’Neill, T., Hofbauer, L., Langdahl, B., Reid, I., Gold, D., & Cummings, S. (2016). Postmenopausal osteoporosis. Nature Reviews Disease Primers, 2, 16069. https://doi.org/10.1038/nrdp.2016.69
Emmanuelle, N., Marie-Cécile, V., Florence, T., Jean-François, A., Françoise, L., Coralie, F., & Alexia, V. (2021). Critical Role of Estrogens on Bone Homeostasis in Both Male and Female: From Physiology to Medical Implications. International Journal of Molecular Sciences, 22(4), 1568. https://doi.org/10.3390/ijms22041568
Fischer, V., & Haffner-Luntzer, M. (2021). Interaction between bone and immune cells: Implications for postmenopausal osteoporosis. Seminars in Cell & Developmental Biology, 123, 14-21. https://doi.org/10.1016/j.semcdb.2021.05.014
Francisco, J. I., Yu, Y., Oliver, R. A., & Walsh, W. R. (2011). Relationship between age, skeletal site, and time post-ovariectomy on bone mineral and trabecular microarchitecture in rats. Journal of Orthopaedic Research, 29(2), 189-196. https://doi.org/10.1002/jor.21217
Franco, F., Natali, A., & Costa, N. (2024). Caffeine and Ovariectomy Debilitate Bone Health in Rats on Calcium-deficient Diet. Anais da Academia Brasileira de Ciencias, 96(suppl 1), e20230095. https://doi.org/10.1590/0001-3765202420230095
Giulia, M., Alessio, D., Gabriela, C., Massimo, L., Chiara, N., Marco, C., Nicola, B., Fabrizio, G., Sonia, F., & Chiara, V. B. (2019). Osteoporosis-related variations of trabecular bone properties of proximal human humeral heads at different scale lengths. Journal of the Mechanical Behavior of Biomedical Materials, 100, 103373. https://doi.org/10.1016/j.jmbbm.2019.103373
Guo, X., Yu, X., Yao, Q., & Qin, J. (2022). Early effects of ovariectomy on bone microstructure, bone turnover markers and mechanical properties in rats. BMC Musculoskeletal Disorders, 23(1), 316. https://doi.org/10.1186/s12891-022-05265-1
Hsu, S.-H., Chen, L.-R., & Chen, K.-H. (2024). Primary Osteoporosis Induced by Androgen and Estrogen Deficiency: The Molecular and Cellular Perspective on Pathophysiological Mechanisms and Treatments. International Journal of Molecular Sciences, 25(22), 12139. https://doi.org/10.3390/ijms252212139
Huang, L., Zhu, L., Ou, Z., Chaoyang, Kong, L., Huang, Y.,…Yi, J. (2021). Betulinic acid protects against renal damage by attenuation of oxidative stress and inflammation via Nrf2 signaling pathway in T-2 toxin-induced mice. International immunopharmacology, 101(Pt B), 108210. https://doi.org/10.1016/j.intimp.2021.108210
Hurwitz, S. (1996). Homeostatic control of plasma calcium concentration. Critical Reviews in Biochemistry and Molecular Biology, 31(1), 41-100. https://doi.org/10.3109/10409239609110575
Jeong, D. H., Kwak, S. C., Lee, M. S., Yoon, K. H., Kim, J. Y., & Lee, C. H. (2020). Betulinic Acid Inhibits RANKL-Induced Osteoclastogenesis via Attenuating Akt, NF-κB, and PLCγ2-Ca(2+) Signaling and Prevents Inflammatory Bone Loss. Journal of Natural Products, 83(4), 1174-1182. https://doi.org/10.1021/acs.jnatprod.9b01212
Kim, H.-N., Ponte, F., Nookaew, I., Ozgurel, S. U., Marques-Carvalho, A., Iyer, S.,…Almeida, M. (2020). Estrogens decrease osteoclast number by attenuating mitochondria oxidative phosphorylation and ATP production in early osteoclast precursors. Scientific Reports, 10(1), 11933. https://doi.org/10.1038/s41598-020-68890-7
Laib, A., Kumer, J., Majumdar, S., & Lane, N. (2001). The Temporal Changes of Trabecular Architecture in Ovariectomized Rats Assessed by MicroCT. Osteoporosis International, 12, 936-941. https://doi.org/10.1007/s001980170022
Lao, X. Y., Sun, Y., Zhao, Z., Liu, J., & Ruan, X. (2025). Pharmacological effects of betulinic acid and its protective mechanisms on the cardiovascular system. Fitoterapia, 183, 106561. https://doi.org/10.1016/j.fitote.2025.106561
Lean, J., Jagger, C., Kirstein, B., Fuller, K., & Chambers, T. (2005). Hydrogen peroxide is essential for estrogen-deficiency bone loss and osteoclast formation. Endocrinology, 146(2), 728-735. https://doi.org/10.1210/en.2004-1021
Lei, X., Cheng, T., Dong, H.-Y., Xia, J., Hong, Y., Cheng, G., Wang, Y. J., & Chen, T. (2025). Study on the alteration of gut microbiota in ovariectomized rats and the impact of estrogen intervention over a 3-month period. PLOS One, 20(9), e0330108. https://doi.org/10.1371/journal.pone.0330108
Li, Y., Tseng, W.-J., De Bakker, C., Zhao, H., Chung, R., & Liu, X. (2021). Peak Trabecular Bone Microstructure Predicts Rate of Estrogen-Deficiency-Induced Bone Loss in Rats. Bone, 145, 115862. https://doi.org/10.1016/j.bone.2021.115862
Liu, X. L., Li, C., Lu, W., Cai, W., & Zheng, L. (2015). Skeletal site-specific response to ovariectomy in a rat model: change in bone density and microarchitecture. Clinical Oral Implants Research, 26(4), 392-398. https://doi.org/10.1111/clr.12360
Lo, Y.-C., Chang, Y.-H., Wei, B.-L., Huang, Y.-L., & Chiou, W. (2010). Betulinic acid stimulates the differentiation and mineralization of osteoblastic MC3T3-E1 cells: involvement of BMP/Runx2 and beta-catenin signals. Journal of Agricultural and Food Chemistry, 58(11), 6643-6649. https://doi.org/10.1021/jf904158k
Lorentzon, M., Johansson, H., Harvey, N., Liu, E., Vandenput, L., McCloskey, E., & Kanis, J. (2021). Osteoporosis and fractures in women: the burden of disease. Climacteric, 25, 4-10. https://doi.org/10.1080/13697137.2021.1951206
Manolagas, S. (2010). From estrogen-centric to aging and oxidative stress: a revised perspective of the pathogenesis of osteoporosis. Endocrine Reviews, 31(3), 266-300. https://doi.org/10.1210/er.2009-0024
Mao, H., Wang, W., Shi, L., Chen, C., Han, C., Zhao, J.,…Huo, J. (2021). Metabolomics and physiological analysis of the effect of calcium supplements on reducing bone loss in ovariectomized rats by increasing estradiol levels. Nutrition & Metabolism, 18(1), 76. https://doi.org/10.1186/s12986-021-00602-y
Marcucci, G., Domazetovic, V., Nediani, C., Ruzzolini, J., Favre, C., & Brandi, M. (2023). Oxidative Stress and Natural Antioxidants in Osteoporosis: Novel Preventive and Therapeutic Approaches. Antioxidants, 12(2), 373. https://doi.org/10.3390/antiox12020373
Marques-Carvalho, A., Kim, H.-N., & Almeida, M. (2023). The role of reactive oxygen species in bone cell physiology and pathophysiology. Bone Reports, 19, 101664. https://doi.org/10.1016/j.bonr.2023.101664
Mashadiyeva-Bayramova, S. A., Aliyeva, D. M., Zulfugarova, P. A., Rustamzada, A. A., & Bayramov, A. A. (2025). Calcium Homeostasis: Physiological Regulation by Parathyroid Hormone and Vitamin D. International Journal of Medical Science and Health Research, 9(4), 48-53. https://doi.org/10.51505/ijmshr.2025.9404
O’Sullivan, L., Allison, H., Parle, E., Schiavi, J., & McNamara, L. (2019). Secondary alterations in bone mineralisation and trabecular thickening occur after long-term estrogen deficiency in ovariectomised rat tibiae, which do not coincide with initial rapid bone loss. Osteoporosis International, 31, 587-599. https://doi.org/10.1007/s00198-019-05239-5
Park, S. B., Lee, Y. J., & Chung, C. (2010). Bone mineral density changes after ovariectomy in rats as an osteopenic model: stepwise description of double dorso-lateral approach. Journal of Korean Neurosurgical Society, 48(4), 309-312. https://doi.org/10.3340/jkns.2010.48.4.309
Park, S. Y., Kim, H.-J., Kim, K. R., Lee, S., Lee, C., Park, K.-K., & Chung, W. (2014). Betulinic acid, a bioactive pentacyclic triterpenoid, inhibits skeletal-related events induced by breast cancer bone metastases and treatment. Toxicol Appl Pharmacol, 275(2), 152-162. https://doi.org/10.1016/j.taap.2014.01.009
Percie du Sert, N., Hurst, V., Ahluwalia, A., Alam, S., Avey, M. T., Baker, M.,…Würbel, H. (2020). The ARRIVE guidelines 2.0: Updated guidelines for reporting animal research. BMC Veterinary Research, 16(1), 242. https://doi.org/10.1186/s12917-020-02451-y
Ríos, J., & Máñez, S. (2017). New Pharmacological Opportunities for Betulinic Acid. Planta Medica, 84, 8-19. https://doi.org/10.1055/s-0043-123472
Roberts, B., Giorgi, M., Oliviero, S., Wang, N., Boudiffa, M., & Dall’Ara, E. (2019). The longitudinal effects of ovariectomy on the morphometric, densitometric and mechanical properties in the murine tibia: A comparison between two mouse strains. Bone, 127, 260-270. https://doi.org/10.1016/j.bone.2019.06.024
Rocabado, J. M. R., Kaku, M., Nozaki, K., Ida, T., Kitami, M., Aoyagi, Y., & Uoshima, K. (2018). A multi-factorial analysis of bone morphology and fracture strength of rat femur in response to ovariectomy. Journal of Orthopaedic Surgery and Research, 13(1), 318. https://doi.org/10.1186/s13018-018-1018-4
Sardar, A., Gautam, S., Sinha, S., Rai, D., Tripathi, A. K., Dhaniya, G.,…Trivedi, R. (2022). Nanoparticles of naturally occurring PPAR-γ inhibitor betulinic acid ameliorates bone marrow adiposity and pathological bone loss in ovariectomized rats via Wnt/β-catenin pathway. Life Science, 309, 121020. https://doi.org/10.1016/j.lfs.2022.121020
Shaheen, M., Basudan, A., Niazy, A., Van Den Beucken, J., Jansen, J., & Alghamdi, H. (2021). Histological and Histomorphometric Analyses of Bone Regeneration in Osteoporotic Rats Using a Xenograft Material. Materials, 14(1), 222. https://doi.org/10.3390/ma14010222
Spiardi, R., & Geara, A. (2022). Normal Regulation of Serum Calcium. Hypercalcemia, 1-17. https://doi.org/10.1007/978-3-030-93182-7_1
Wawrzyniak, A., & Balawender, K. (2022). Structural and Metabolic Changes in Bone. Animals, 12(15), 1946. https://doi.org/10.3390/ani12151946
Wei, J., Li, Y., Liu, Q., Lan, Y., Wei, C., Tian, K.,…Yang, Y. (2020). Betulinic Acid Protects From Bone Loss in Ovariectomized Mice and Suppresses RANKL-Associated Osteoclastogenesis by Inhibiting the MAPK and NFATc1 Pathways. Frontiers in Pharmacology, 11, 1025. https://doi.org/10.3389/fphar.2020.01025
Weitzmann, M., & Pacifici, R. (2006). Estrogen deficiency and bone loss: an inflammatory tale. The Journal of Clinical Investigation, 116(5), 1186-1194. https://doi.org/10.1172/jci28550
Yang, K., Cao, F., Xue, Y., Tao, L., & Zhu, Y. (2022). Three Classes of Antioxidant Defense Systems and the Development of Postmenopausal Osteoporosis. Frontiers in Physiology, 13, 840293. https://doi.org/10.3389/fphys.2022.840293
Yoon, K.-H., Cho, D.-C., Yu, S.-H., Kim, K., Jeon, Y., & Sung, J. (2012). The Change of Bone Metabolism in Ovariectomized Rats: Analyses of MicroCT Scan and Biochemical Markers of Bone Turnover. Journal of Korean Neurosurgical Society, 51, 323-327. https://doi.org/10.3340/jkns.2012.51.6.323
Yousefzadeh, N., Kashfi, K., Jeddi, S., & Ghasemi, A. (2020). Ovariectomized rat model of osteoporosis: a practical guide. EXCLI Journal, 19, 89-107. https://doi.org/10.17179/excli2019-1990
Zhang, C., Li, H., Li, J., Hu, J., Yang, K., & Tao, L. (2023). Oxidative stress: A common pathological state in a high-risk population for osteoporosis. Biomedicine & pharmacotherapy, 163, 114834. https://doi.org/10.1016/j.biopha.2023.114834

Details

Primary Language

English

Subjects

Medical Pharmacology

Journal Section

Research Article

Authors

Onural Özhan ^*
0000-0001-9018-7849
Türkiye

Hilal Sahin
0009-0007-5778-3332
Türkiye

Mehmet Fatih Korkmaz
0000-0001-7498-6763
Türkiye

Azibe Yıldız
0000-0001-5686-7867
Türkiye

Ersoy Kekilli
0000-0003-2127-269X
Türkiye

Nigar Vardı
0000-0003-0576-1696
Türkiye

Hakan Parlakpınar
0000-0001-9497-3468
Türkiye

Early Pub Date

April 27, 2026

Publication Date

-

Submission Date

January 26, 2026

Acceptance Date

April 20, 2026

Published in Issue

Year 2026 Number: Advanced Online Publication

DOI

https://doi.org/10.62425/rtpharma.1871984

IZ

https://izlik.org/JA22JA32XZ

Cite

RIS / Bibtex

APA

Özhan, O., Sahin, H., Korkmaz, M. F., Yıldız, A., Kekilli, E., Vardı, N., & Parlakpınar, H. (2026). Betulinic Acid Mitigates Estrogen Deficiency–Related Femoral Bone Loss: Evidence from an Ovariectomized Rat Model. Recent Trends in Pharmacology, Advanced Online Publication. https://doi.org/10.62425/rtpharma.1871984

AMA

1.Özhan O, Sahin H, Korkmaz MF, et al. Betulinic Acid Mitigates Estrogen Deficiency–Related Femoral Bone Loss: Evidence from an Ovariectomized Rat Model. Recent Trends in Pharmacology. 2026;(Advanced Online Publication). doi:10.62425/rtpharma.1871984

Chicago

Özhan, Onural, Hilal Sahin, Mehmet Fatih Korkmaz, et al. 2026. “Betulinic Acid Mitigates Estrogen Deficiency–Related Femoral Bone Loss: Evidence from an Ovariectomized Rat Model”. Recent Trends in Pharmacology, no. Advanced Online Publication. https://doi.org/10.62425/rtpharma.1871984.

EndNote

Özhan O, Sahin H, Korkmaz MF, Yıldız A, Kekilli E, Vardı N, Parlakpınar H (April 1, 2026) Betulinic Acid Mitigates Estrogen Deficiency–Related Femoral Bone Loss: Evidence from an Ovariectomized Rat Model. Recent Trends in Pharmacology Advanced Online Publication

IEEE

[1]O. Özhan et al., “Betulinic Acid Mitigates Estrogen Deficiency–Related Femoral Bone Loss: Evidence from an Ovariectomized Rat Model”, Recent Trends in Pharmacology, no. Advanced Online Publication, Apr. 2026, doi: 10.62425/rtpharma.1871984.

ISNAD

Özhan, Onural - Sahin, Hilal - Korkmaz, Mehmet Fatih - Yıldız, Azibe - Kekilli, Ersoy - Vardı, Nigar - Parlakpınar, Hakan. “Betulinic Acid Mitigates Estrogen Deficiency–Related Femoral Bone Loss: Evidence from an Ovariectomized Rat Model”. Recent Trends in Pharmacology. Advanced Online Publication (April 1, 2026). https://doi.org/10.62425/rtpharma.1871984.

JAMA

1.Özhan O, Sahin H, Korkmaz MF, Yıldız A, Kekilli E, Vardı N, Parlakpınar H. Betulinic Acid Mitigates Estrogen Deficiency–Related Femoral Bone Loss: Evidence from an Ovariectomized Rat Model. Recent Trends in Pharmacology. 2026. doi:10.62425/rtpharma.1871984.

MLA

Özhan, Onural, et al. “Betulinic Acid Mitigates Estrogen Deficiency–Related Femoral Bone Loss: Evidence from an Ovariectomized Rat Model”. Recent Trends in Pharmacology, no. Advanced Online Publication, Apr. 2026, doi:10.62425/rtpharma.1871984.

Vancouver

1.Onural Özhan, Hilal Sahin, Mehmet Fatih Korkmaz, Azibe Yıldız, Ersoy Kekilli, Nigar Vardı, Hakan Parlakpınar. Betulinic Acid Mitigates Estrogen Deficiency–Related Femoral Bone Loss: Evidence from an Ovariectomized Rat Model. Recent Trends in Pharmacology. 2026 Apr. 1;(Advanced Online Publication). doi:10.62425/rtpharma.1871984