目的 評價(jià)經(jīng)不同干燥與提取方式處理后的人源胎盤樣品對斑馬魚貧血模型的影響。方法 取健康產(chǎn)婦新鮮胎盤，經(jīng)-40℃冷卻后于真空冷凍干燥機(jī)中冷凍干燥，研磨成粉，制成胎盤凍干粉；取健康產(chǎn)婦的新鮮胎盤，于90℃熱風(fēng)循環(huán)烘箱中烘干，研磨成粉，制成胎盤烘干粉。胎盤凍干粉或烘干粉分別經(jīng)胰蛋白酶酶解提取、梯度溶劑（依次為水、50%乙醇水、無水乙醇、二氯甲烷）提取。發(fā)育至56 h（56 hpf）的健康野生型AB系斑馬魚分為對照組、模型組、給藥組，對照組給予斑馬魚養(yǎng)殖水，模型組給予0.1 μg·mL^-1苯肼溶液制備貧血模型，給藥組給予苯肼與胎盤凍干粉或烘干粉各提取樣品，樣品質(zhì)量濃度分別為10、20、50、100 μg·mL^-1，培養(yǎng)至3 dpf，采用O-dianisidine染色法對斑馬魚進(jìn)行紅細(xì)胞染色。結(jié)果 與對照組比較，模型組紅細(xì)胞染色積分吸光度顯著下降（P＜0.001）。與模型組比較，胎盤凍干粉酶解樣品100 μg·mL^-1組，水提樣品10、20、50、100 μg·mL^-1組，醇提樣品20、100 μg·mL^-1組，50%醇提樣品10、20 μg·mL^-1組，二氯甲烷提取樣品10、20、50、100 μg·mL^-1組紅細(xì)胞染色積分吸光度顯著增加（P＜0.05、0.001）；胎盤凍干粉酶解樣品20 μg·mL^-1組，水提樣品10、20 μg·mL^-1組，醇提樣品20、50、100 μg·mL^-1組，二氯甲烷提取樣品50、100 μg·mL^-1組紅細(xì)胞染色積分吸光度顯著增加（P＜0.05、0.01、0.001）。經(jīng)酶解、水提取及二氯甲烷提取的胎盤凍干粉樣品和經(jīng)水提取、醇提取及二氯甲烷提取的胎盤烘干粉樣品作用呈濃度相關(guān)性，其中凍干粉水提取樣品100 μg·mL^-1效果最佳，染色后的紅細(xì)胞積分吸光度達(dá)到對照組的94.77%。結(jié)論 人源胎盤樣品處理方式可優(yōu)先選擇凍干技術(shù)結(jié)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 0.1 μg·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 100 μg·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 100 μg·mL^-1 group, water extraction 10, 20, 50, 100 μg·mL^-1 group, alcohol extraction 20, 100 μg·mL^-1 group, 50% alcohol extraction 10, 20 μg ·mL^-1 group and dichloromethane extraction 10, 20, 50 and 100 μg·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 20 μg·mL^-1 group, water extraction 10, 20 μg·mL^-1 group, alcohol extraction 20, 50, 100 μg·mL^-1 group, and dichloromethane extraction 10, 20, 50 and 100 μg·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 100 μg·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.

R285.5

山東省自然科學(xué)基金面上項(xiàng)目（ZR2021MC055）；齊魯工業(yè)大學(xué)（山東省科學(xué)院）科教產(chǎn)融合試點(diǎn)工程項(xiàng)目（2022PX022）；山東省自然科學(xué)基金優(yōu)青項(xiàng)目（ZR2020YQ60）；國家自然科學(xué)基金青年項(xiàng)目（42006090）；齊魯工業(yè)大學(xué)（山東省科學(xué)院）科教產(chǎn)融合創(chuàng)新試點(diǎn)工程項(xiàng)目（2020KJC-ZD08）