目的 基于斑马鱼代谢-效/毒（M-Act/Tox）联合评价箭叶淫羊藿代谢、抗骨质疏松效应及毒性。方法 用受精后1~6 d（1~6 dpf）斑马鱼评价箭叶淫羊藿（生药质量浓度200、500、1 000、1 500、2 000 μg/mL）的安全性，观察鱼脏器形态、计数死亡数并计算斑马鱼半数死亡浓度（LC₅₀）；用5 dpf斑马鱼暴露于箭叶淫羊藿（生药250 μg/mL）24 h，分析6个代表性黄酮成分朝藿定A（EA）、朝藿定B（EB）、朝藿定C（EC）、淫羊藿苷、箭藿苷C（SC）与宝藿苷I（BI）变化；用25 μmol/L泼尼松龙诱导斑马鱼骨质疏松模型，采用茜素红对培养至8 dpf的各组斑马鱼幼鱼骨骼染色，进行显微检测、数码成像，并用图像软件定量分析骨骼染色区域来评价箭叶淫羊藿抗骨质疏松活性。结果 箭叶淫羊藿在500μg/mL及以上质量浓度时致斑马鱼中毒（脏器变形或死亡），毒性与给药质量浓度和时间相关；经斑马鱼作用后，EA、EB、EC及淫羊藿苷几近全部转化，代谢物以SC与BI为主；箭叶淫羊藿在一定质量浓度（6.25、12.5、25 μg/mL）具显著的抗泼尼松龙诱导斑马鱼骨丢失作用。结论 用斑马鱼有效评价了箭叶淫羊藿的代谢、抗骨质疏松效应与毒性。斑马鱼M-Act/Tox联合法有机整合了斑马鱼代谢方法、骨质疏松模型及毒性评价法的优势，实现基于体内过程的效应/毒性评价，为抗骨质疏松中药筛选提供新思路与方法。

Objective To evaluate the metabolism, anti-osteoporosis efficacy and toxicity of Epimedium sagittatum based on zebrafish M-Act/Tox integration method. Methods The safety of E. sagittatum (crude drug 200, 500, 1 000, 1 500, and 2 000 μg/mL) was evaluated with 1-6 dpf zebrafish, the morphology of fish organs was observed and the number of deaths was counted and the half death concentration of zebrafish (LC₅₀) was calculated; The 5 dpf zebrafish were exposed to E. sagittatum (crude drug 250 μg/mL) for 24 h, six representative flavonoids, including epimedin A, epimedin B, epimedin C, icariin, sagittatoside C, and baohuoside I, were analyzed; The zebrafish osteoporosis model was induced with 25 μmol/L prednisolone, microscopic detection and digital imaging of zebrafish larvae of each group cultured to 8 dpf were performed using alizarin red, and the bone staining area was quantitatively analyzed by image software to evaluate the anti-osteoporosis activity of E. sagittatum. Results E. sagittatum caused zebrafish poisoning at crude drug 500 μg/mL and above concentration (organs deformation or death), and toxicity was related to drug concentration and exposing time; After the action of zebrafish, epimedin A, epimedin B, epimedin C, and icariin were almost completely transformed, and sagittatoside C and baohuoside I were the main metabolites; E. sagittatum had significant anti-prednisolone-induced bone loss in zebrafish at a certain concentration (crude drug 6.25, 12.5, and 25 μg/mL). Conclusion The metabolism, anti-osteoporosis efficacy and toxicity of E. sagittatum are evaluated using zebrafish. The zebrafish M-Act/Tox integration method integrates the advantages of zebrafish metabolism, osteoporosis model and toxicity evaluation method, and realizes the effect/toxicity evaluation based on in vivo process, providing new ideas and method for screening anti-osteoporosis Chinese medicine.

R285.5

国家自然科学基金资助项目（81573833）；"重大新药创制"科技重大专项资助（2017ZX09301-056）；中医药行业科研专项资助（201507004-10）