目的 制备难溶性药物反式西红花酸（trans-crocetin，TC）与甲基-β-环糊精（methyl-β-cyclodextrin，MβCD）的包合物（TC-MβCD），并评价包合物对TC溶解度、安全性和有效性的影响。方法 采用单因素实验和Box-Behnken响应面法筛选环糊精种类等工艺参数，采用冷冻干燥法制备TC-MβCD。采用扫描电子显微镜（SEM）、UV法、体外释放度测定法等对TC-MβCD包合物进行表征，采用HPLC法测定TC含量。采用Caco-2细胞模型研究TC与TC-MβCD的体外转运机制。采用斑马鱼鱼卵/胚胎发育保护模型评价TC-MβCD包合物的安全性，采用体外乳腺癌4T1细胞和裸鼠体内乳腺癌MCF-7肿瘤模型对比研究TC及TC-MβCD的体内外抗肿瘤活性。结果 在5种环糊精中，MβCD对TC的增溶效果最好，优化包合工艺参数为料液比33%，搅拌温度60℃，搅拌时间120 min。TC-MβCD冻干粉中TC含量2.2%，增溶效果显著，复溶性良好。SEM和UV法表明TC被MβCD的空穴结构包合，TC-MβCD冻干粉可在0.1%聚山梨酯80-pH 6.8介质中释放，其拟合曲线符合一级释药模型。Caco-2细胞模型表明TC转运机制为被动扩散，包合促进吸收。斑马鱼鱼卵/胚胎发育试验发现，TC-MβCD包合物对斑马鱼的孵化率、斑马鱼胚胎的存活率和心率几乎没有影响，表明包合物安全性好。乳腺癌4T1细胞体外抗肿瘤表明TC-MβCD的IC₅₀明显低于TC，说明包合物对乳腺癌细胞有更强的抑制作用。裸鼠体内MCF-7肿瘤模型表明，相同药物剂量下，TC-MβCD的抑制率为33.71%，远高于TC的16.86%。结论 采用相溶解度法筛选出合适的环糊精，通过冷冻干燥制备TC-MβCD冻干粉，并对其进行评价。TC被MβCD包合后，显著增加了TC的溶解度，提高了TC的抗肿瘤活性和安全性，为TC包合物制剂工业化生产提出理论依据。

Objective To prepare insoluble drugs trans-crocetin (TC) and methyl-β-cyclodextrin (MβCD) inclusion complex (TC-MβCD), and evaluate the effect of inclusion complex on the solubility, safety and effectiveness of TC. Methods The kinds of cyclodextrins and other process parameters were selected by single factor test and Box-Behnken response surface method, and TC-MβCD was prepared by freeze-drying method. The inclusion complex of TC-MβCD was characterized by scanning electron microscope, UV method and in vitro release assay, and the content of TC was determined by HPLC. Caco-2 cell model was used to study the transport mechanism of TC and TC-MβCD in vitro. The safety of TC-MβCD inclusion complex was evaluated by using zebrafish egg/embryo development protection model. The anti-tumor activity of TC and TC-MβCD in vivo and in vitro was compared by using 4T1 breast cancer cell anti-tumor model in vitro and MCF-7 tumor model in nude mice.Results Among the five cyclodextrins, MβCD had the best solubilization effect on TC. The optimized inclusion process parameters were as follows:solid-liquid ratio 33%, stirring temperature 60℃, stirring time 120 min. The content of TC in TC-MβCD lyophilized powder was 2.2%, which had remarkable solubilization effect and good resolubility. Scanning electron microscopy and UV method showed that TC was encapsulated by the hole structure of MβCD, and TC-MβCD lyophilized powder could be released in the range of 0.1% Tween80-pH 6.8, The fitting curve accorded with the first-order drug release model. Caco-2 cell model showed that the transport mechanism of TC was passive diffusion, and inclusion promoted absorption. Zebrafish egg/embryo development test found that TC-MβCD inclusion complex had little effect on zebrafish hatching rate, zebrafish embryo survival rate and heart rate, indicating that the inclusion complex was safe. The anti-tumor activity of 4T1 cells in vitro showed that the IC₅₀ of TC-MβCD was significantly lower than that of TC, indicating that the inclusion compound had a stronger inhibitory effect on breast cancer cells. The MCF-7 tumor model in nude mice showed that under the same drug dose, the inhibition rate of TC MβCD was 33.71%, much higher than 16.86% of TC. Conclusion Appropriate cyclodextrins were screened out by phase solubility method, and TC-MβCD lyophilized powder was prepared by freeze-drying and evaluated. The inclusion of TC by MβCD significantly increases the solubility of TC, improves the antitumor activity and safety of TC, and provides a theoretical basis for the industrial production of TC inclusion complex preparations.

R283.6

国家自然科学基金青年基金项目(81403114)