Journal of Ethnopharmacology 141 (2012) 331–337 Contents lists available at SciVerse ScienceDirect Journal of Ethnopharmacology journa l h o me page: www.elsevier.com/locate/jethpharm Evaluation of Cameroonian plants towards experimental bone regeneration Florence Tsofack Ngueguim a,∗,1 , Mohd Parvez Khan b,1 , Jean Hubert Donfack c , Jawed Akhtar Siddiqui b , Deepshikha Tewari b , Geet K. Nagar b , Satish C. Tiwari d , Dimo Theophile a , Rakesh Maurya d , Naibedya Chattopadhyay b a Department of Animal Biology and Physiology, Faculty of Science, University of Yaounde 1, P.O. Box 812, Yaounde, Cameroon b Endocrinology Division, CSIR-Central Drug Research Institute, Lucknow 226001, India c Department of Biomedical Sciences, Faculty of Science, University of Dschang, P.O. Box, 67, Dschang, Cameroon d Medicinal and Process Chemistry Division, CSIR-Central Drug Research Institute, Lucknow 226001, India a r t i c l e i n f o Article history: Received 14 November 2011 Received in revised form 2 February 2012 Accepted 26 February 2012 Available online 5 March 2012 Keywords: Fracture Bone regeneration Elephantopus mollis Spilanthes africana a b s t r a c t Ethnopharmacological relevance: Elephantopus mollis, Spilanthes africana, Urena lobata, Momordica multi- flora, Asystasia gangetica and Brillantaisia ovariensis are used in Cameroonian traditional medicine for the treatment of bone diseases and fracture repair. The aim of this study was to evaluate the effect of ethanolic extracts of six Cameroonian medicinal plants on bone regeneration following bone and marrow injury. Materials and methods: Ethanol extract of Cameroonian medicinal plants were administered (each extract at 250, 500 and 750 mg/kg doses) orally to adult female Sprague–Dawley rats having a drill hole injury (0.8 mm) in the femur diaphysis. Vehicle (gum-acacia in distilled water) was given to the control group. After 12 days of treatment, animals were euthanized and femur bones collected. Confocal microscopy of fractured bone was performed to evaluate bone regeneration (calcein labeling). Only active plant extracts were used for further experiments. Thus, callus was analyzed by microcomputed tomography. Osteogenic effects of the extracts were evaluated by assessing mineralized nodules formation of bone marrow stromal cells and osteoblast recruitment at drill hole site by immunohistochemistry. Results: Ethanolic extract of the leaves and twigs of Elephantopus mollis (EM) and whole plant of Spilanthes africana (SA) dose-dependently stimulated bone regeneration at the drill hole site. EM at 250 and 750 mg/kg doses and SA at 750 mg/kg dose significantly increased mineral deposition compared to controls. Both extracts at 500 and 750 mg/kg doses improved microarchitecture of the regenerating bone evident from increased bone volume fraction, trabecular thickness, trabecular number, and decreased trabecular separation and structure model index. EM and SA extracts increased the formation of mineralized nodules from the bone marrow stromal cells. In addition, EM and SA extracts increased osteoblast recruitment at the drill hole site evident from increased Runx-2 positive cells following their treatments compared to control. Conclusion: Ethanolic extracts of EM and SA accelerate fracture repair in rats via stimulatory effects on osteoblast differentiation and mineralization, thereby justifying their traditional use. © 2012 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Bone remodeling cycle is a tightly regulated physiological process exhibited by normal adult skeleton and is essential for maintaining the bone quality. This cycle is regulated mainly by two types of cells, osteoblasts and osteoclasts. The former cells are responsible for bone formation and the latter for bone resorption (Rodan and Martin, 2000; Seeman and Delmas, 2006). Abnormali- ties in bone remodeling result in a variety of bone-related diseases including Paget’s disease and osteoporosis, leading to increased ∗ Corresponding author. E-mail address: tsngueguim@yahoo.fr (F.T. Ngueguim). 1 Contributed equally for this work. risk of fragility and fracture. Several pharmacological interventions are in clinical use to reduce the fracture risk including, bispho- sphonates, selective estrogen receptor modulators (SERMs) and calcitonin (Gerstenfeld and Einhorn, 2003; Delaney, 2006; Gass and Dawson-Hughes, 2006). In spite of the availability of these clini- cal agents, the research still continues due to the lack of adequate benefit-to-risk ratio. There is no orally active pharmacological agent available for rapid fracture repair. Traditional mode of medicine worldwide has abundant mention of herbal extracts having positive effect on fracture repair. However, validation of this effect through controlled studies is scarce in the literature. Regeneration of bone at the fracture/injury site requires activation of osteoblast func- tion. Metabolic bone diseases including primary and secondary osteoporosis is essentially caused by the deficiency in osteoblast 0378-8741/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2012.02.041