不同干燥与提取方式处理的人源胎盘防治贫血活性差异研究

Anti-anemia activity of human placenta from different drying and extraction methods

目的评价经不同干燥与提取方式处理后的人源胎盘样品对斑马鱼贫血模型的影响。方法取健康产妇新鲜胎盘,经−40℃冷却后于真空冷冻干燥机中冷冻干燥,研磨成粉,制成胎盘冻干粉;取健康产妇的新鲜胎盘,于90℃热风循环烘箱中烘干,研磨成粉,制成胎盘烘干粉。胎盘冻干粉或烘干粉分别经胰蛋白酶酶解提取、梯度溶剂(依次为水、50%乙醇水、无水乙醇、二氯甲烷)提取。发育至56 h(56 hpf)的健康野生型AB系斑马鱼分为对照组、模型组、给药组,对照组给予斑马鱼养殖水,模型组给予0.1μg·mL^(−1)苯肼溶液制备贫血模型,给药组给予苯肼与胎盘冻干粉或烘干粉各提取样品,样品质量浓度分别为10、20、50、100μg·mL^(−1),培养至3 dpf,采用O-dianisidine染色法对斑马鱼进行红细胞染色。结果与对照组比较,模型组红细胞染色积分吸光度显著下降(P<0.001)。与模型组比较,胎盘冻干粉酶解样品100μg·mL^(−1)组,水提样品10、20、50、100μg·mL^(−1)组,醇提样品20、100μg·mL^(−1)组,50%醇提样品10、20μg·mL^(−1)组,二氯甲烷提取样品10、20、50、100μg·mL^(−1)组红细胞染色积分吸光度显著增加(P<0.05、0.001);胎盘冻干粉酶解样品20μg·mL^(−1)组,水提样品10、20μg·mL^(−1)组,醇提样品20、50、100μg·mL^(−1)组,二氯甲烷提取样品50、100μg·mL^(−1)组红细胞染色积分吸光度显著增加(P<0.05、0.01、0.001)。经酶解、水提取及二氯甲烷提取的胎盘冻干粉样品和经水提取、醇提取及二氯甲烷提取的胎盘烘干粉样品作用呈浓度相关性,其中冻干粉水提取样品100μg·mL^(−1)效果最佳,染色后的红细胞积分吸光度达到对照组的94.77%。结论人源胎盘样品处理方式可优先选择冻干技术结合水提取方式。

Objective To explore the anti-anemia activity of human placenta from different drying and extraction methods.Methods Fresh placentas from healthy mothers were taken,cooled at−40°C,freeze-dried in a vacuum freeze-dryer,ground into powder,and made into placenta freeze-dried powder.The fresh placenta of healthy puerpera was dried in a hot air circulation oven at 90℃and ground into powder to make placenta baking powder.Placenta freeze-dried powder or baking powder were extracted by trypsin enzymolysis and gradient solvent(water,50%ethanol water,anhydrous ethanol,dichloromethane).The healthy wild type AB zebrafish of 56 h post fertilization(56 hpf)were divided into control group,model group and administration group.The control group was given zebrafish culture water,the model group was given 0.1μg·mL^(−1)phenylhydrazine solution to prepare anemia model,and the administration group was given phenylhydrazine and placenta freeze-dried powder or baking powder extract samples.The sample concentrations were 10,20,50 and 100μg·mL^(−1)respectively,and cultured to 3 dpf.O-dianisidine staining was used to stain zebrafish red blood cells.Results Compared with control group,the optical density of erythrocyte staining integral in model group decreased significantly(P<0.001).Compared with model group,the optical density of erythrocyte staining integral of placenta freeze-dried powder enzymolysis sample 100μg·mL^(−1)group,water extraction 10,20,50,100μg·mL^(−1)group,alcohol extraction 20,100μg·mL^(−1)group,50%alcohol extraction 10,20μg·mL^(−1)group and dichloromethane extraction 10,20,50 and 100μg·mL^(−1)group were significantly increased(P<0.05,0.001).Compared with model group,the optical density of erythrocyte staining integral of placenta baking powder enzymolysis sample 20μg·mL^(−1)group,water extraction 10,20μg·mL^(−1)group,alcohol extraction 20,50,100μg·mL^(−1)group,and dichloromethane extraction 10,20,50 and 100μg·mL^(−1)group were significantly increased(P<0.05,0.01,0.001).The effect of enzymolysis,water extraction and dichloromethane extraction of placenta freezedried powder,and water extraction,alcohol extraction and dichloromethane extraction of baking powder was correlated with dose.The effect of water extraction of placenta freeze-dried powder 100μg·mL^(−1)group was the best,and the integrated optical density of red blood cells after staining reached 94.77%of control group.Conclusion Freeze-drying followed by water extraction process was the preferred recommended production process to obtain better anti-anemia activity.