Eurycoma longifolia as a potential alternative to testosterone for the treatment of osteoporosis: Exploring time-mannered proliferative, differentiative and morphogenic modulation in osteoblasts

J Ethnopharmacol. 2017 Jan 4:195:143-158. doi: 10.1016/j.jep.2016.10.085. Epub 2016 Nov 3.


Ethnopharmacological relevance: Eurycoma longifolia (EL) has been well-studied traditionally as a chief ingredient of many polyherbal formulations for the management of male osteoporosis. It has also been well-recognised to protect against bone calcium loss in orchidectomised rats.

Aim of the study: To evaluate the effects of EL on the time-mannered sequential proliferative, differentiative, and morphogenic modulation in osteoblasts compared with testosterone.

Materials and methods: Cell proliferation was analysed using MTS assay and phase contrast microscopy. Osteogenic differentiation of MC3T3-E1 cells was assessed through a series of characteristic assays which include crystal violet staining, alkaline phosphatase (ALP) activity and Van Gieson staining. Taken together, the bone mineralization of extra cellular matrix (ECM) was estimated using alizarin red s (ARS) staining, von kossa staining, scanning electron microscopic (SEM) and energy dispersive x-ray (EDX) analysis.

Results: The cell proliferation data clearly revealed the efficiency of EL particularly at a dose of 25µg/mL, in improving the growth of MC3T3-E1 cells compared with the untreated cells. Data also showed the prominence of EL in significantly promoting ALP activity throughout the entire duration of treatment compared with the testosterone-treated cells. The osteogenic differentiation potential of EL was further explored by analysing mineralization data which revealed that the calcified nodule formation (calcium deposition) and phosphate deposition was more pronounced in cells treated with 25µg/mL concentration of EL at various time points compared with the untreated and testosterone treated cells. The scanning electron microscopic (SEM) analysis also revealed highest globular masses of mineral deposits (identified as white colour crystals) in the ECM of cultured cells treated with 25µg/mL concentration of EL.

Conclusion: Compared to testosterone, greater potential of EL in promoting the proliferation and osteogenic differentiation of MC3T3-E1 cells provides an in vitro basis for the prevention of male osteoporosis. Thus, we anticipate that EL can be considered as an alternative approach to testosterone replacement therapy (TRT) for the treatment of male osteoporosis.

Keywords: 5α-Dihydrotestosterone (PubChem CID: 10635); Ascorbic acid (PubChem CID: 54670067); Calcium (PubChem CID: 5460341); Cell proliferation; Crystal violet (PubChem CID: 11057); Ethanol (PubChem CID: 702); Eurycoma longifolia; Eurycomanone (PubChem CID: 102004821); Male osteoporosis; Morphogenic characteristics; Osteogenic differentiation; Paraformaldehyde (PubChem CID: 71309502); Phosphate buffered saline (PubChem CID: 24978514); Silver nitrate (PubChem CID: 24470); Testosterone; β-glycerophosphate (PubChem CID: 2526).

Publication types

  • Comparative Study

MeSH terms

  • Alkaline Phosphatase / metabolism
  • Animals
  • Biomarkers / metabolism
  • Bone Density Conservation Agents / isolation & purification
  • Bone Density Conservation Agents / pharmacology*
  • Calcification, Physiologic / drug effects*
  • Cell Differentiation / drug effects*
  • Cell Line
  • Cell Proliferation / drug effects*
  • Collagen / metabolism
  • Dihydrotestosterone / pharmacology*
  • Dose-Response Relationship, Drug
  • Eurycoma / chemistry*
  • Extracellular Matrix / metabolism
  • Mice
  • Osteoblasts / drug effects*
  • Osteoblasts / metabolism
  • Osteoblasts / ultrastructure
  • Osteogenesis / drug effects*
  • Osteoporosis / drug therapy*
  • Osteoporosis / metabolism
  • Osteoporosis / pathology
  • Osteoporosis / physiopathology
  • Phytotherapy
  • Plant Extracts / isolation & purification
  • Plant Extracts / pharmacology*
  • Plants, Medicinal
  • Time Factors
  • Tumor Protein, Translationally-Controlled 1


  • Biomarkers
  • Bone Density Conservation Agents
  • Plant Extracts
  • Tpt1 protein, mouse
  • Tumor Protein, Translationally-Controlled 1
  • Dihydrotestosterone
  • Collagen
  • Alkaline Phosphatase