Archives of Pharmacal Research

, Volume 31, Issue 2, pp 216–224

Effects of ginkgo biloba on in vitro osteoblast cells and ovariectomized rat osteoclast cells

Articles Drug Efficacy and Safety


Ginkgo biloba extract (GBE) has a selective estrogen receptor modulator (SERM)-like biphasic effect on estrogen, and could be a potential alternative to hormone replacement therapy (HRT). Here, we investigated whether GBE can ameliorate estrogen-depleted osteoporosis in in vitro osteoblast cells and in estrogen-deprived ovariectomized (OVX) rats, a classical animal model for postmenopausal osteoporosis. GBE (50–150 μg/mL) significantly increased ALP (Alkaline phosphatase) activity of osteoblast cells, indicating that GBE promotes osteoblast mineralization. OVX rats exposed to GBE (100 and 200 mg/kg/day, oral treatment), raloxifene (3 mg/kg/day, oral treatment) or estradiol (E2, 10 μg/kg/day, subcutaneous injection) decreased osteoclast resorptive activity compared with OVX rats. GBE and raloxifene did not increase uterine weight compared with OVX rats, while E2 and Sham control did, suggesting that GBE has no uterotrophic activity, which is a disadvantage of estrogen therapy. In OVX rats, GBE did not restore severe bone density loss induced by OVX, indicating that GBE may be insufficient as therapeutic material for severe osteoporosis. However, despite its no effects on bone density loss in OVX rats, GBE did stimulate osteoblast differentiation and antiosteoclastic activity in vitro. Therefore, GBE may have preventive potential on osteoporosis as do other phytoestrogens.

Key words

Ginkgo biloba extracts Osteoporosis Ovariectomized rat Osteoclast Osteoblast 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Adlercreutz, H. and Mazur, W., Phytoestrogens and western diseases. Ann. Med., 29, 95–120 (1997).PubMedGoogle Scholar
  2. Arjmandi, B. H., Birnbaum, R. S., Juma, S., Barengolts, E., and Kukreja, S. C., The synthetic phytoestrogen, ipriflavone, and estrogen prevent none loss by different mechanisms. Calcif. Tissue Int., 66, 61–65 (2000).PubMedCrossRefGoogle Scholar
  3. Beck, V., Rohr, U., and Jungbauer, A., Phytoestrogens derived from red clover: an alternative to estrogen replacement therapy? J. Steroid Biochem. Mol. Biol., 94, 499–518 (2005).PubMedCrossRefGoogle Scholar
  4. Bellows, C. G., Aubin, J. E., and Heersche, J. N. M., Initiation and progression of mineralization of bone nodules formed in vitro-the role of alkaline-phosphatase and organic phosphate. Bone Miner., 14, 27–40 (1991).PubMedCrossRefGoogle Scholar
  5. Bellows, C. G., Heersche, J. N., and Aubin, J. E., Inorganic phosphate added exogenously or released from β-glycerophosphate initiates mineralization of osteoid nodules in vitro. Bone Miner., 17, 15–29 (1992).PubMedCrossRefGoogle Scholar
  6. Boyle, W. J., Simonet, W. S., and Lacey, D. L., Osteoclast differentiation and activation. Nature, 423, 337–342 (2003).PubMedCrossRefGoogle Scholar
  7. Brayboy, J. R., Chen, X. W., Lee, Y. S., and Anderson, J. J. B., The protective effects of ginkgo biloba extract (EGb 761) against free radical damage to osteoblast-like bone cells (MC3T3-E1) and the proliferative effects of EGb 761 on these cells. Nutr. Res., 21, 1275–1285 (2001).CrossRefGoogle Scholar
  8. Brzexinski, A. and Debi, A., Phytoestrogens: the “Natural” selective estrogen receptor modulators? Eur. J. Obstet. Gynecol. Reprod. Biol., 85, 47–51 (1999).CrossRefGoogle Scholar
  9. Dempster, D. W. and Lindsay, R., Pathogenesis of osteoporosis. Lancet, 341, 797–805 (1993).PubMedCrossRefGoogle Scholar
  10. Dew, J., Eden, J., Beller, E., Magarcy, C., Schwartz, P., Crca, P., and Wren, B., A cohort study of hormone replacement therapy given to women previously treated for breast cancer. Climacteric, 1, 137–142 (1998).PubMedCrossRefGoogle Scholar
  11. Faber, A., Bouvy, M. L., Loskamp, L., van de Berg, P. B., Egberts, T. C., and de Jong-van den Berg, L. T., Dramatic change in prescribing of hormone replacement therapy in The Netherlands after publication of the Million Women Study: a follow-up study. Br. J. Clin. Pharmacol., 60, 641–647 (2005).PubMedCrossRefGoogle Scholar
  12. Fanti, P., Monier-Faugere, M. C., Geng, Z., Schmidt, J., Morris, P. E., Cohen, D., and Malluche, H. H., The phytoestrogens genistein reduces bone loss in short-term ovariectomized rats. Osteoporos. Int., 8, 274–281 (1998).PubMedCrossRefGoogle Scholar
  13. Felsen, D. T., Zhang, Y., Hannan, M. T., Kiel, D. P., Wilson, P. W., and Anderson, J. J., The effect of postmenopausal estrogen therapy on bone density in elderly woman. N. Engl. J. Med., 329, 1141–1146 (1993).CrossRefGoogle Scholar
  14. Genge, B. R., Sauer, G. R., Wu, L. N. Y., McLean, F. M., and Wuthier, R. E., Correlation between loss of alkaline-phosphatase activity and accumulation of calcium during matrix vesicle-mediated mineralization. J. Biol. Chem., 263, 18513–18519 (1988).PubMedGoogle Scholar
  15. Greendale, G. A., Reboussin, B. A., Hogan, P., Barnabei, V. M., Shumaker, S., Johnson, S., and Barrett-Connor, E., Symptom relief and side effects of postmenopausal hormone: results from the postmenopausal estrogen/progestin interventions trial. Obstet. Gynecol., 92, 982–988 (1998).PubMedCrossRefGoogle Scholar
  16. Hallworth, R. B., Prevention and treatment of postmenopausal osteoporosis. Pharm. World Sci., 20, 198–205 (1998).PubMedCrossRefGoogle Scholar
  17. Hedblad, B., Merio, J., Manjer, J., Engstrom, G., Berglund, G., and Janzon, L., Incidence of cardiovascular disease, cancer and death in postmenopausal women affirming use of hormone replacement therapy. Scand. J. Public Health, 30, 12–19 (2002).PubMedCrossRefGoogle Scholar
  18. Hiroi, H., Inoue, S., Watanabe, T., Goto, W., Orimo, A., Momoeda, M., Tsutsumi, O., Taketani, Y., and Muramatsu, M., Differential immunolocalization of estrogen receptor a and b in rat ovary and uterus. J. Mol. Endocrinol., 22, 37–44 (1999).PubMedCrossRefGoogle Scholar
  19. Hsu, H., Lacey, D. L., Dunstan, C. R., Solovyev, I., Colombero, A., Timms, E., Tan, H. L., Elliott, G., Kelley, M. J., Sarosi, I., Wang, L., Xia, X. Z., Elliott, R., Chiu, L., Black, T., Scully, S., Capparelli, C., Morony, S., Shimamoto, G., Bass, M. B., and Boyle, W. J., Tumor necrosis factor receptor family member RANK mediates osteoclast differentiation and activation induced by osteoprotegerin ligand. Proc. Natl. Acad. Sci. USA, 96, 3540–3545 (1999).PubMedCrossRefGoogle Scholar
  20. Ibrahim, N. K. and Hortobagyi, G. N., The evolving role of specific estrogen receptor modulators (SERMs). Surg. Oncol., 8, 103–123 (1999).PubMedCrossRefGoogle Scholar
  21. Ikeda, S., Tsurukami, H., Ito, M., Sakai, A., Sakata, T., Nishida, S., Takeda, S., Shiraishi, A., and Nakamura, T., Effect of trabecular bone contour on ultimate strength of lumbar vertebra after bilateral ovariectomy in rats. Bone, 28, 625–633 (2001).PubMedCrossRefGoogle Scholar
  22. Ishida, H., Uesugi, T., Hirai, K., Toda, T., Nukaya, H., Yokotsuka, K., and Tsuji, K., Preventive effects of the plant isoflavones, daidzin and genistin, on bone loss in ovariectomized rats fed a calcium-deficient diet. Biol. Pharm. Bull., 21, 62–66 (1998).PubMedGoogle Scholar
  23. Kalu, D. N., The ovariectomized rat model of postmenopausal bone loss. Bone Miner., 15, 175–191 (1991).PubMedCrossRefGoogle Scholar
  24. Kanno, S., Anuradha, C. D., and Hirano, S., Localization of zinc after in vitro mineralization in osteoblastic cells. Biol. Trace Elem. Res., 83, 39–47 (2001).PubMedCrossRefGoogle Scholar
  25. Kanno, S., Hirano, S., and Kayama, F., Effects of phytoestrogens and environmental estrogens on osteoblastic differentiation in MC3T3-E1 cells. Toxicology, 196, 137–145 (2004).PubMedCrossRefGoogle Scholar
  26. Kim, H. J., Bae, Y. C., Park, R. W., Choi, S. W., Cho, S. H., Choi, Y. S., and Lee, W. J., Bone-protecting effect of safflower seeds in ovariectomized rats. Calcif. Tissue Int., 71, 88–94 (2002).PubMedCrossRefGoogle Scholar
  27. Kwan, T. S., Padrines, M., Theoleyre, S., Heymann, D., and Fortun, Y., IL-6, RANKL, TNF-alpha/IL-1: interrelations in bone resorption pathophysiology. Cytokine Growth Factor Rev., 15, 49–60 (2004).CrossRefGoogle Scholar
  28. Lieberman, S., A review of the effectiveness of Cimicifuga racemosa (black cohosh) for the symptoms of menopause. J. Women’s Health, 7, 525–529 (1998).CrossRefGoogle Scholar
  29. Mackie, E. J., Osteoblasts: Novel roles in orchestration of skeletal architecture. Int. J. Biochem. Cell Biol., 35, 1301–1305 (2003).PubMedCrossRefGoogle Scholar
  30. MacLennan, A. H., Lawton, B., and Baber, R. J., Hormone replacement therapy and the breast. Studies must determine the evidence. BMJ, 324, 915 (2002).PubMedCrossRefGoogle Scholar
  31. Manolagas, S. C., Birth and death of bone cells: Basic regulatory mechanisms and implications for the pathogenesis and treatment of osteoporosis. Endocr. Rev., 21, 115–137 (2000).PubMedCrossRefGoogle Scholar
  32. Manolagas, S. C. and Jilka, R. L., Bone marrow, cytokines, and bone remodeling. Emerging insights into the pathophysiology of osteoporosis. N. Engl. J. Med., 332, 305–311 (1995).PubMedCrossRefGoogle Scholar
  33. Messina, M. and Hughes, C., Efficacy of soyfoods and soybean isoflavones supplements for alleviating menopausal symptoms is positively related to initial hot flush frequency. J. Med. Food, 6, 1–11 (2003).PubMedCrossRefGoogle Scholar
  34. Miyake, M., Arai, N., Ushio, S., Iwaki, K., Ikeda, M., and Kurimoto, M., Promoting effect of kaempferol on the differentiation and mineralization of murine pre-osteoblastic cell line MC3T3-E1. Biosci. Biotechnol. Biochem., 67, 1199–1205 (2003).PubMedCrossRefGoogle Scholar
  35. MWS, Million Women Study Collaborators, Breast cancer and hormonereplacement therapy in the Million Women Study. Lancet 362, 419–427 (2003).Google Scholar
  36. Novack D. V., Estrogen and bone: Osteoclasts take center stage. Cell Metabolism, 6, 254–256 (2007).PubMedCrossRefGoogle Scholar
  37. O’Connell, D., Robertson, J., Henry, D., and Gillespie, W., A systemic review of the skeletal effects of estrogen therapy in postmenopausal women II. An assessment of treatment effects. Climacteric, 1, 112–123 (1998).PubMedCrossRefGoogle Scholar
  38. Oh, S. M. and Chung, K. H., Estrogenic activities of Ginkgo biloba extracts. Life Sciences, 74, 1325–1335 (2004).PubMedCrossRefGoogle Scholar
  39. Oh, S. M. and Chung, K. H., Antiestrogenic activities of Ginkgo biloba extracts. J. Steroid Biochem. Mol. Biol., 100, 167–176 (2006).PubMedCrossRefGoogle Scholar
  40. Pang, J. L., Ricupero, D. A., Huang, S., Fatma, N., Singh, D. P., Romero, J. R., and Chattopadhyay, N., Differential activity of kaempferol and quercetin in attenuating tumor necrosis factor receptor family signaling in bone cells. Biochem. Pharmacol., 71, 818–826 (2006).PubMedCrossRefGoogle Scholar
  41. Pepping, J., Black cohosh: Cimicifuga racemosa. Am. J. Health Syst. Pharm., 56, 1400–1402 (1999).PubMedGoogle Scholar
  42. Picherit, C., Coxam, V., Bennetau-Pelissero, C., Kati-Coulibaly, S., Davicco, M. J., Lebecque, P., and Barlet, J. P., Daidzein is more efficient than genistein in preventing ovariectomy induced bone loss in rats. J. Nutr., 130, 1675–1681 (2000).PubMedGoogle Scholar
  43. Prouillet, C., Mazière, J. C., Mazière, C., Wattel, A., Brazier, M., and Kamel, S., Stimulatory effect of naturally occurring flavonols quercetin and kaempferol on alkaline phosphatase activity in MG-63 human osteoblasts through ERK and estrogen receptor pathway. Biochem. Pharmacol., 67, 1307–1313 (2004).PubMedCrossRefGoogle Scholar
  44. Riggs, B. L. and Melton, L. J., Involutional osteoporosis. N. Engl. J. Med., 26, 1676–1684 (1986).CrossRefGoogle Scholar
  45. Sagraves, R., Estrogen therapy for postmenopausal symptoms and prevention of osteoporosis. J. Clin. Pharmacol., 35, 2S–10S (1995).PubMedGoogle Scholar
  46. Setchell, K. D. and Lydeking-Olsen, E., Dietary phytoestrogens and their effect on bone: evidence from in vitro and in vivo, human observational, and dietary intervention studies. Am. J. Clin. Nutr., 78(suppl), 593S–609S (2003).PubMedGoogle Scholar
  47. Shumaker, S. A., Legault, C., Rapp, S. R., Thal, L., Wallace, R. B., Ockene, J. K., Hendrix, S. L., Jones, B. N., Assaf, A. R., Jackson, R. D., Kotchen, J. M., Wassertheil-Smoller, S., and Wactawski-Wende, J., WHIMS Investigators, Estrogen plus progestin and the incidence of dementia and mild cognitive impairment in postmenopausal women. The Women’s Health Initiative Memory study: a randomized controlled trial. JAMA, 289, 2654–2662 (2003).CrossRefGoogle Scholar
  48. Stanford, C. M., Jacobson, P. A., Eanes, E. D., Lembke, L. A., and Midura, R. J., Rapidly forming apatite mineral in an osteoblastic cell-line (UMR 106-01 BSP). J. Biol. Chem., 270, 9420–9428 (1995).PubMedCrossRefGoogle Scholar
  49. Suda, T., Takahashi, N., Udagawa, N., Jimi, E., Gillespie, M. T., and Martin, T. J., Modulation of osteoclast differentiation and function by the new members of the tumor necrosis factor receptor and ligand families. Endocrinol. Rev., 20, 345–347 (1999).CrossRefGoogle Scholar
  50. Ukeda, H., Shimamura, T., Tsubouchi, M., Harada, Y., Nakai, Y., and Sawamura, M., Spectrophotometric assay of superoxide anion formed in maillard reaction based on highly water-soluble tetrazolium salt. Anal. Sci., 18, 1151–1154 (2002).PubMedCrossRefGoogle Scholar
  51. Wattel, A., Kamel, S., Mentaverri, R., Lorget, F., Prouillet, C., Petit, J. P., Fardelonne, P., and Brazier M., Potent inhibitory effect of naturally occurring flavonids quercetin and kaempferol on in vitro osteoclastic bone resorption. Biochem. Pharmcol., 65, 35–42 (2003).CrossRefGoogle Scholar
  52. Weistein, R. S. and Manolagas, S. C., Apoptosis and osteoporosis. Am. J. Med., 108, 153–164 (2000).CrossRefGoogle Scholar
  53. Weitzmann, M. N. and Pacifici, R., Estrogen deficiency and bone loss: an inflammatory tale. J. Clin. Invest., 116, 1186–1194 (2006).PubMedCrossRefGoogle Scholar
  54. WHI, Writing Group for the Women’s Health Initiative Investigators, Risks and benefits of estrogen plus progestin in healthy postmenopausal women. Principal results from the Women’s Health Initiative randomized controlled trial. JAMA 288, 321–333 (2002).CrossRefGoogle Scholar
  55. WHI, Writing Group for the Women’s Health Initiative Investigators, Effects of conjugated equine estrogen in postmenopausal women with hysterectomy. JAMA 291, 1701–1712 (2004).CrossRefGoogle Scholar
  56. Wuttke, W., Jarry, H., Westphalen, S., Christoffel, V., and Seidlová-Wuttke, D., Phytoestrogens for hormone replacement therapy? J. Steroid Biochem. Mol. Biol., 83, 133–147 (2003).CrossRefGoogle Scholar
  57. Xie, F., Wu, C. F., Lai, W. P., Yang, X. J., Cheung, P. Y., Yao, X. S., Leung, P. C., and Wong, M. S., The osteoprotective effect of Herba epimedii (HEP) extract in vivo and in vitro. Evid. Based Complement Alternat. Med., 2, 353–361 (2005).PubMedCrossRefGoogle Scholar
  58. Yamazaki, I. and Yamaguchi, H., Characteristics of an ovariectomized osteopenic rat model. J. Bone Miner. Res., 4, 13–22 (1989).PubMedCrossRefGoogle Scholar
  59. Yasuda, H., Shima, N., and Nakagawa, N., Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis inhibitory factor and is identical to TRANCE/RANKL. Proc. Natl. Acad. Sci. U.S.A., 95, 3597–3602 (1998).PubMedCrossRefGoogle Scholar
  60. Yudoh, K., Matsuno, H., Nakazawa F., Katayama, R., and Kimura, T., Reconstituting telomerase activity using the telomerase catalytic subunit prevents the telomere shorting and replicative senescence in human osteoblasts. J. Bone Miner. Res., 16, 1453–1464 (2001).PubMedCrossRefGoogle Scholar

Copyright information

© The Pharmaceutical Society of Korea 2008

Authors and Affiliations

  • Seung Min Oh
    • 1
  • Ha Ryong Kim
    • 1
  • Kyu Hyuck Chung
    • 1
  1. 1.College of PharmacySungkyunkwan UniversitySuwonKorea

Personalised recommendations