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Cistanoside A (Cis A), an active phenylethanoid glycoside isolated from Cistanche deserticola Y. C. Ma, has received our attention because of its possible role in the treatment of osteoporosis. In the present study, we evaluated the effects of Cis A on an ovariectomized (OVX) mice model and investigated its underlying molecular mechanisms of action. After 12 weeks of orally-administrated intervention, Cis A (20, 40 and 80 mg/kg body weight/day) exhibited significant antiosteoporotic effects on OVX mice, evidenced by enhanced bone strength, bone mineral density and improved trabecular bone microarchitecture. Meanwhile, the activities of bone resorption markers, including tartrate-resistant acid phosphatase (TRAP), deoxypyridinoline (DPD) and cathepsin K, were decreased, and the bioactivity of bone formation marker alkaline phosphatase (ALP) was increased. Mechanistically, Cis A inhibited the expression of TNF-receptor associated factor 6 (TRAF6), an upstream molecule that is shared by both nuclear factor kappa-light chain enhancer of activated B cells (NF-κB) and phosphatidylinositol 3-kinase (PI3K)/Akt pathways and subsequently suppressed the levels of receptor activators of nuclear factor kappaB ligand (RANKL), downregulated the expression of NF-κB and upregulated osteoprotegerin (OPG), PI3K and Akt, which means Cis A possessed antiosteoporotic activity in ovariectomized mice via TRAF6-mediated NF-kappaB inactivation and PI3K/Akt activation. Put together, we present novel findings that Cis A, by downregulating TRAF6, coordinates the inhibition of NF-κB and stimulation of PI3K/Akt pathways to promote bone formation and prevent bone resorption. These data demonstrated the potential of Cis A as a promising agent for the treatment of osteoporosis disease.

The volatile components of the Hui formula "Ha Hei Lili" were extracted by steam distillation extraction (SD) and supercritical CO2 fluid extraction, and the structures were analyzed and identified by GC-MS.The GC-MS conditions were set as follows: Rxi-5Sil MS quartz capillary column (30 m x 0.25 mm, 0.25 μm), the initial temperature of 50 degrees C to keep 1 min, to 10 degrees C/min heating to 120 degrees C, maintained 3 min, then to 3 degrees C/min heating to 200 degrees C, maintained 3 min, and then to 5 degreesC/min heating to 290 degrees C, maintained until completion of analysis; helium as the carrier gas, column flow rate 1.0 ml/min, split ratio 25: 1, inlet temperature 250 degrees C, EI ionization source 70 eV, ion source temperature 230 degrees C, scan range of m/z 35 - 500.Yield of volatile oil were 0.21% and 5.44% extracted by SD and SFE methods, respectively; and for SD method, 36 kinds of compounds were identified, accounted for 87.02% of total mass of volatile oil; for SFE method, 38 kinds of constituents were identified, accounted for 97.47% of total mass of volatile oil.The type of constituents contained in the volatile oil extracted by SD and SFE methods are totally different; and GC-MS can be used to identify the structures and relative content of volatile components, the results of this study can provide an experimental basis for development and utilization of Hui formula "Ha Hei Lili".