目的 研究大孔吸附树脂法结合斑马鱼评价富集纯化补骨脂总黄酮的制备工艺。方法 以补骨脂的黄酮类代表成分（新补骨脂异黄酮、补骨脂甲素、补骨脂二氢黄酮甲醚和corylifol A）为指标，考察不同类型大孔吸附树脂的静态、动态吸附和解吸附能力，优选大孔吸附树脂类型；综合斑马鱼形态变化和存活率，评价大孔吸附树脂有机溶剂残留的安全性，优选不同厂家大孔吸附树脂；进一步考察上样药液质量浓度、体积流量、上样量、吸附时间、洗脱剂乙醇体积分数和用量等因素对黄酮纯化工艺的影响，并对优选纯化工艺进行验证，比较大孔吸附树脂纯化前后新补骨脂异黄酮、补骨脂甲素、补骨脂二氢黄酮甲醚、corylifol A、补骨脂宁、补骨脂定、补骨脂乙素、4'-O-甲基补骨脂查耳酮、补骨脂素和异补骨脂素（黄酮类和香豆素类），以及补骨脂酚的含量。结果 优选出D101大孔吸附树脂对补骨脂黄酮成分同时具有较好的吸附能力和解吸附能力；10个厂家D101大孔吸附树脂醇浸液对斑马鱼形态和致死率的影响差别较大，其中D2和D5的大孔吸附树脂安全性较好。优化的D101大孔吸附树脂纯化总黄酮制备工艺为补骨脂药液上样质量浓度为0.5 g/mL生药，上样量为1倍柱体积（BV），上样体积流量为1 mL/min，吸附时间为1.5 h，用1 BV 30%乙醇洗去未被吸附的成分，再用4 BV 90%乙醇洗脱黄酮成分，所得补骨脂总黄酮提取物中8种黄酮成分含量约是纯化前的1.3～5.1倍，黄酮成分总量约占上述所测11种成分总量的93.6%，提取物中总黄酮质量分数约为54%。结论 斑马鱼评价可快速辨别大孔吸附树脂的安全性差异，D101大孔吸附树脂可有效富集纯化补骨脂黄酮类成分，优选的工艺简单、稳定、重复性好。

Objective To optimize the enrichment and purification process of total flavonoids from Psoraleae Fructus by macroporous adsorptive resin. Methods Four representative flavonoids of Psoraleae Fructus (neobavaisoflavon, bavachin, bavachinin A and corylifol A) were selected as index components, the static and dynamic adsorption and desorption capacity of different types of macroporous adsorptive resins were investigated, and the optimal macroporous adsorptive resin type was screened out; morphological changes and survival rate of zebrafish were used to evaluate the safety of organic solvent residues in macroporous adsorptive resin from different manufacturers, and the preferred macroporous adsorptive resin manufacturer was selected; the effects of the mass concentration of sample solution, flow rate, volume of loading solution, adsorption time, ethanol volume fraction and volume of the eluent on the purification process of flavonoids were further investigated, the optimized purification process was verified, and the contents of neobavaisoflavone, bavachin, bavachinin A, corylifol A, corylin, psoralidin, isobavachalcone, 4'-O-methylbroussochalcone, psoralen, isopsoralen (flavonoids, coumarins) and bakuchiol before and after purification with macroporous adsorptive resins were compared. Results The selected D101 macroporous adsorptive resin had both excellent adsorption and desorption capacity for flavonoids from Psoraleae Fructus; The effects of D101 macroporous adsorptive resin ethanol extracts from 10 manufacturers on zebrafish morphology and mortality were significantly different, among which D2 and D5 macroporous adsorptive resins had better safety. The optimum purification process of total flavonoids purified by D101 macroporous adsorptive resin was as follows: the loading solution mass concentration of Psoraleae Fructus was 0.5 g/mL, the volume of loading solution was 1 BV, the sample flow rate was 1 mL/min, the adsorption time was 1.5 h, the unabsorbed components were washed with 1 BV of 30% ethanol, and then the flavonoid components were eluted with 4 BV of 90% ethanol. The content of eight flavonoids in the total flavonoids extract of Psoraleae Fructus was about 1.3—5.1 times of that before purification, the total content of eight flavonoids accounted for about 93.6% of the total measured 11 components, and the total flavonoids content in the extract was about 54%. Conclusion Zebrafish evaluation can quickly identify the safety differences of macroporous adsorptive resin; D101 macroporous adsorptive resin can effectively enrich and purify the flavonoids of Psoraleae Fructus; And the optimized process is simple, stable and reproducible.

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“重大新药创制”科技重大专项资助（2017ZX09301056）；国家中医药管理局2022年中央财政转移支付地方项目（2022GJJZDYJS-01）；江苏省中医药科技发展计划项目（YB2020022）；江苏省中医药科技发展计划项目（ZD202225）；江苏省中医药科技发展计划项目（QN202214）；江苏省医学创新团队项目（CXTDB2017003）