桦树皮中桦木酸重结晶纯化工艺优化及其对斑马鱼毒性效应研究

Optimization of purification process for recrystallization of betulinic acid from birch bark and its toxic effects on Danio rerio

中文关键词:  桦树皮  桦木酸  超声辅助提取  重结晶  斑马鱼  急性毒性

英文关键词:birch bark  betulinic acid  ultrasonic-assisted extraction  recrystallization  Danio rerio  acute toxicity

中文摘要:

      目的 优化桦树皮中桦木酸的提取纯化工艺, 并研究其对斑马鱼(Danio rerio)的毒性效应。方法 采用超声辅助乙醇提取桦木酸, 通过单因素实验及响应面分析优化工艺参数, 再经重结晶法进行纯化, 探究其对模式生物斑马鱼的毒性效应, 观察并计算斑马鱼的半数致死浓度(half lethal concentration, LC50)、生存率、畸形率和主要毒性靶器官。结果 经优化验证后, 最佳工艺条件为乙醇浓度75%、料液比1:25 (g/mL)、超声温度45℃、超声时间30 min, 桦木酸得率达(9.15±0.01) mg/g。以异丙醇为溶剂进行桦木酸重结晶, 纯化的最佳工艺参数为料液比1:25 (mg/mL)、重结晶温度0℃, 重结晶时间24 h, 此时桦木酸纯度达到96.49%。桦木酸对斑马鱼的LC50为0.905 mg/mL, 致畸的主要表现为心包水肿、卵黄囊水肿、肾性水肿和胃肠道发育延迟, 质量浓度为0.2~0.6 mg/mL的桦木酸没有显示出急性毒性作用。结论 优化后的纯化工艺可有效提取桦树皮中的桦木酸, 且桦木酸在受试剂量下对斑马鱼胚胎及幼鱼无明显的急性毒性及致畸作用。

英文摘要:

      Objective To optimize the extraction and purification process of betulinic acid from birch bark, and investigate its toxic effects on zebrafish (Danio rerio). Methods Betulinic acid was extracted by ultrasonic-assisted ethanol, the process parameters were optimized by single-factor experiments and response surface analysis, and then purified by recrystallization to investigate the toxic effects on the model organism zebrafish, and the half lethal concentration (LC50), survival rate, deformation rate and main toxic target organs were observed and calculated. Results After optimization and verification, the optimum process conditions were 75% ethanol concentration, 1:25 (g/mL) material-to-liquid ratio, 45℃ sonication temperature and 30 min sonication time, and the extraction rate of betulinic acid reached (9.15±0.01) mg/g. Betulinic acid recrystallization was performed with isopropyl alcohol as solvent, the optimal process parameters for the purification were the material-liquid ratio of 1:25 (mg/mL), recrystallization temperature of 0℃ and recrystallization time of 24 h. The purity of betulinic acid was 96.49%. The LC50 of betulinic acid for zebrafish was 0.905 mg/mL, the main teratogenic manifestations were pericardial edema, yolk sac edema, nephrogenic edema and delayed gastrointestinal development, and birchic acid at concentrations of 0.2?0.6 mg/mL did not show acute toxic effects. Conclusion The optimized purification process is effective in extracting betulinic acid from birch bark, which has no significant acute toxicity or teratogenic effects on zebrafish embryos and larvae at reagent levels.