Drynaria fortunei J. Sm. improves the bone mass of ovariectomized rats through osteocalcin-involved endochondral ossification Yong-Eng Lee a,b , Hwa-Chang Liu c , Yi-Ling Lin b , Shing-Hwa Liu d , Rong-Sen Yang c , Ruei-Ming Chen b,e,f,n a Department of Orthopedic Surgery, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan b Cell Physiology and Molecular Image Research Center, Taipei Medical University's Wan-Fang Hospital, Taipei, Taiwan c Department of Orthopedic Surgery, National Taiwan University Hospital, Taipei, Taiwan d Institute of Toxicology, College of Medicine, National Taiwan University, Taipei, Taiwan e Graduate Institute of Medical Sciences, Taipei Medical University, Taipei, Taiwan f Anesthetics Toxicology Research Center, Taipei Medical University Hospital, Taipei, Taiwan article info Article history: Received 1 May 2014 Received in revised form 1 September 2014 Accepted 13 October 2014 Available online 23 October 2014 Keywords: Drynaria fortunei J. Sm. Bone mass Ovariectomy Osteocalcin Endochondral ossification abstract Aim of this study: Our previous study showed that Drynaria fortunei J. Sm. (Kunze), a traditional Chinese medical herb, can promote osteoblast differentiation and maturation. This study was further aimed to confirm the traditional effects of Kunze on the bone mass of ovariectomized rats. Materials and methods: Female Wistar rats were given an ovariectomy and then administered the water extract of Kunze (WEK). Systemic and tissue toxicities of WEK were assessed. A biomechanical test, bone mineral contents, and bone histomorphometry were analyzed to determine the effects of the WEK on the bone mass. Levels of osteocalcin (OCN) in bone tissues were determined by immunohistochemistry and immunoblotting. The effects of naringin, one of the bioactive compounds of the WEK, on the bone mass were evaluated. Results: A bilateral ovariectomy in rats caused a time-dependent decrease in levels of serum 17β- estradiol. Exposure of ovariectomized rats to the WEK at 0.5 and 1 g/kg body weight/day for 1, 2, 3, and 6 months did not induce systemic or tissue toxicities. Biomechanical testing and a bone mineral content analysis showed that the ovariectomy decreased the bone torsion force and bone ash in time-dependent manners. In comparison, after exposure to the WEK, the ovariectomy-induced reductions in the bone torsion force and bone ash were significantly alleviated. In parallel, results of a bone histomorphometric assay further revealed that the ovariectomy caused significant diminution in the production of prehypertrophic chondrocytes and trabecular bone but enhanced hypertrophic chondrocyte numbers in the growth plate. However, exposure to the WEK lowered ovariectomy-induced changes in these cellular events. As to the mechanism, the WEK increased OCN biosynthesis in bone tissues of ovariectomized rats. Administration of naringin to ovariectomized rats caused significant amelioration of the bone strength, bone mineral contents, and trabecular bone amounts. Conclusions: This study shows that the WEK can translationally promote the bone mass in ovariecto- mized rats through stimulating OCN-involved endochondral ossification. & 2014 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Osteoporosis, a progressive bone disease, is characterized by a decrease in the bone mass and density which can induce bones to weaken and be more likely to break (Adams, 2012). In the clinic, osteoporosis is also called as a silent disease because it proceeds without pain or symptoms until bone fracture occurs (Bradbury et al., 2012). Bone fractures typically linked to osteoporosis are very dangerous and can cause patients to suffer permanent disabilities or even death (Edwards et al., 2012). A variety of risk factors contribute to the causes of osteoporosis, including low levels of sex hormones, inadequate calcium intake, heredity, chronic diseases, lifestyle habits, medication use, and aging (Rubin et al., 2013). Among these, sex hormones are reported to play the most crucial roles in regulating bone remodeling and maintaining bone mass. However, post-menopausal hormone Contents lists available at ScienceDirect journal homepage: www.elsevier.com/locate/jep Journal of Ethnopharmacology http://dx.doi.org/10.1016/j.jep.2014.10.016 0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved. Abbreviations: ALT, aminotransferase; AST, aspartate aminotransferase; BUN, bilirubin; CRE, creatinine; LDH, lactate dehydrogenase; OCN, osteocalcin; OPN, osteopontin; UA, uric acid; WEK, water extracts of Kunze n Corresponding author at: Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, 250 Wu-Xing St., Taipei, 110, Taipei, Taiwan. Tel.: þ886 2 27361661x3222; fax: þ886 2 86621119. E-mail address: rmchen@tmu.edu.tw (R.-M. Chen). Journal of Ethnopharmacology 158 (2014) 94–101