In the present study, icaritin (ICT) nanosuspensions were prepared by antisolvent-precipitation method to enhance its dissolution rate and oral bioavailability. Firstly, different stabilizers were evaluated on their feasibility to form stable ICT nanosuspension. Hydroxypropyl methylcellulose (HPMC) was proved its superiority both in terms of particle size reduction and short-term physical stability. Three experimental parameters, viz., types of HPMCs, concentration of ICT in acetone and time length of ultrasonication were investigated on their impacts on particle size. Subsequently, central composite design was applied to evaluate the impacts of concentration of stabilizer solution and volume of ICT acetone solution injected on the particle size distribution of nanosuspension. The optimized formulation was determined as following: 0.16 mL of ICT acetone solution (10 mg/mL) injected into 2 mL of HPMC E3 solution (0.02% w/v) under ultrasonication for 10 seconds. The particle size, polydispersity index and zeta potential of nanosuspension were 216.6±12.4 nm, 0.124±0.013 and -13.8±1.4 mV, respectively. Nanosuspension was observed by transmission electron microscope (TEM) morphology. Raw ICT and freez-dried nanosuspension were also characterized by scanning electron microscope (SEM) morphology. Both of nanosuspension particles in the solution and in the freeze-dried form were rod shape. The X-ray diffraction (XRD) and differential scanning calorimetry (DSC) analysis showed that the characteristic peaks of ICT was maintained but with reduced peak intensity, indicating that the crystallinity of drug in nanosuspension was much lower than the raw material. Drug in nanosuspension completely dissolved within 15 min with much higher dissolution rate. After oral administration of ICT nanosuspensions and raw suspensions to rats, nanosuspension exhibited significant increased AUC0–36h and Cmax by 2.0 and 4.7 folds respectively than those of raw suspension. Meanwhile, the decreased Tmax indicated that drug in nanosuspension could reach the peak concentration much faster than the raw suspension. The effect of ICT on osteoblast proliferation and differentiation was examined in the culture system of MC3T3-E1 cells. ICT (10-9 -10-6 M) has no promoting proliferation effect. ICT showed statistically significant promoting differentiation effect (1.75 fold of the control group at 10-6 M). The serum pharmacological method was employed to investigate the effects of ICT nanosuspension and raw suspension on proliferation and differentiation on MC3T3-E1 cell line. Two formulations, ICT nanosuspensions and raw suspensions, were orally administered to rats and blood samples were withdrawn at 10 min, 1.5 h and 24 h. The total concentrations of ICT in serum were determined after enzyme hydrolysis with β-glucuronidase/sulphatase. The serum was added into the culture system of MC3T3-E1 cells. When MC3T3-E1 cells were exposed to culture medium containing serum of 10 min and 1.5 h after administration with ICT nanosuspension, cell viability was significantly increased to (120.1±9.0) % and (116.6±9.9) % compared to the control group, respectively. The serum of 24 h after administration with ICT nanosuspension has no effect on proliferation. When MC3T3-E1 cells were exposed to culture medium containing serum of 10 min, 1.5 h and 24 h after administration with raw suspension, no effect on cell viability was observed. The culture medium containing serum of 10 min and 1.5 h after administration with ICT nanosuspension both induced increase of ALP activity (1.41-fold) compared to control group (p0.05). These results suggested that ICT nanosuspension was superior to raw suspension on enhancing proliferation and differentiation of osteoblast. In conclusion, the present study suggested that ICT nanosuspension increased the dissolution rate as well as the oral bioavailability of ICT. ICT has differentiation enhancing effect on osteoblasts. However, based on the result of experiment employed serum pharmacological method, the metabolites of ICT showed significant proliferation and differentiation promoting effect in vitro. ICT nanosuspension was very promising to be developed into a new formulation with anti-osteoprosis activity. Keywords: Icaritin, Nanosuspension, Antisolvent-precipitation, Ultrasonication, Dissolution rate, Oral bioavailability, Osteoporosis

本研究利用反溶劑沉澱法製備淫羊藿苷元（Icaritin，ICT）納米混懸液以提 高其溶出度及口服生物利用度。首先選擇不同種類的穩定劑製備了 ICT 納米混 懸液，利用羥丙基甲基纖維素（HPMC）製備出的納米混懸液粒徑小且分佈均勻、 短期穩定性好。通過單因素實驗考察不同型號的 HPMC、ICT 丙酮溶液濃度以及 超聲時間對粒徑的影響。在以上影響因素為最優的情況下，利用星點設計-效應 面優化法考察穩定劑濃度和注入 ICT 丙酮溶液的體積對粒徑的影響。獲得最優 處方及工藝如下：將 0.16 mL 的 ICT 丙酮溶液（10 mg/mL）在超聲條件下注入 到 0.02%（w/v）HPMC E3 溶液中並繼續超聲 10 sec。最優處方的粒徑、多分散 係數及 zeta 電位分別為：（216.6±12.4）nm，0.124±0.013，（-13.8±1.4）mV。 利用透射電鏡表徵納米混懸液的形態，並利用掃描電鏡表徵原藥及納米混懸液凍 幹後的形態，發現納米混懸液與納米混懸液凍幹後的顆粒形態為棒狀。通過 X 光衍射掃描和差示掃描量熱分析發現，製備成納米混懸液後，ICT 原藥的晶體峰 位置不變但強度下降，提示結晶度比原晶體下降。溶出實驗顯示，ICT 在納米混 懸液中 15 min 內完全溶出，比原晶體的溶出速度更快也更完全。大鼠口服 ICT 納米混懸液和原藥粗混懸液後，納米混懸液的 AUC0–36h 和 Cmax 比原藥粗混懸液 顯著提高，分別約為原藥粗混懸液的 2.0 倍和 4.7 倍。而 Tmax 較原藥粗混懸液降 低，說明納米混懸液可以在體內血漿中更快速達峰。 研究 ICT 對體外培養 MC3T3-E1 細胞系增殖和分化的影響。ICT 在 10-9 -10-6 M 摩爾濃度下對 MC3T3-E1 增殖無影響。ICT 表現出明顯的促進分化的作用（10-6 M 摩爾濃度下的鹼性磷酸酶活性是空白對照組的 1.75 倍）。利用血清藥理學方 法評價 ICT 納米混懸液和原藥粗混懸液對成骨細胞增殖和分化的作用。ICT 納米 混懸液和原藥粗混懸液分別口服給予大鼠後，於 10 min、1.5 h 和 24 h 時采血。 血清中 ICT 總濃度經 β-葡萄糖醛酸酯酶/硫酸酯酶酶解後進行檢測，並將血清加 入到 MC3T3-E1 細胞培養體系中。口服 ICT 納米混懸液後 10 min 和 1.5 h 含藥血 清和空白對照組相比，細胞存活率分別提高至（120.1±9.0）%和（116.6±9.9） %，24 h 含藥血清無作用。而口服原藥粗混懸液 10 min、1.5 h 和 24 h 含藥血清 對細胞存活率沒有顯著提高。口服納米混懸液後，10 min 和 1.5 h 含藥血清均可 提高鹼性磷酸酶活性為空白對照組的 1.41 倍，表現出顯著的促進鹼性磷酸酶活性的作用（p0.05）。上述結果提示，納米混懸液對成骨細胞的增殖和分化的作 用均有促進作用，其效果優於原藥粗混懸液。 本研究證明，ICT 納米混懸液可以提高體外溶出度和口服生物利用度，同時 提高藥物的達峰速度。ICT 具有促進成骨細胞分化的作用，而血清藥理學結果顯 示，ICT 的代謝產物可以有效促進成骨細胞增殖與分化。ICT 納米混懸液是一種 有潛力被開發成抗骨質疏鬆的口服納米製劑。 關鍵字：淫羊藿苷元，納米混懸液，反溶劑沉澱法，超聲，溶出速度，口服生物 利用度，骨質疏鬆