Background and ObjectivesTraditional Chinese Medicine (TCM) Fufang or formula Xianlinggubao (XLGB) is a prescribed TCM drug in China registered for prevention and treatment of osteoporosis. Fufang in TCM is comprised of a group of herbal compounds contributing in group to the treatment efficacy. The present study aims to identify the bioactive fraction(s) in XLGB extract that account(s) dominantly for its osteogenic effects.MethodsThe extract of XLGB formula was separated into three fractions using chromatography, i.e., XLGB-A, XLGB-B and XLGB-C. They were administrated to 4-month old ovariectomized (OVX) mice for 6 weeks to determine which bioactive fraction(s) were more effective for preventing OVX-induced bone loss evaluated by microCT, biomechanical testing and biochemical markers. The main peaks of the key fraction were identified using reference compounds isolated from the fraction. In addition, the effects of the composite compounds in XLGB-B on osteoblasts’ proliferation and mineralization were evaluated in UMR 106 cells.ResultsXLGB-B with a yield of 13.0% from herbal Fufang XLGB was identified as the most potential one among the three fractions for prevention of OVX-induced bone loss confirmed with bone mass, bone microarchitecture, bone strength and bone turnover markers. Nine compounds in HPLC fingerprint were identified in the XLGB-B fraction, including phenylpropanoids from Herba Epimedii, terpenes from Radix Dipsaci and coumarins from Fructus Psoraleae. In addition, the identified compounds effectively promoted proliferation and/or mineralization of osteoblast-like UMR 106 cells in vitro.ConclusionXLGB-B with defined phytochemical structures was screened as the key fraction that demonstrated preventive effects on OVX-induced bone loss in mice. The present study laid down a foundation towards a new generation of herbal Fufang characterized with “less herbal materials for achieving equal treatment efficacy” in development strategy of TCM for prevention of OVX-induced osteoporosis.