目的 对传统中药煎煮自组装现象进行改良，应用微沉淀法制备甘草新型自组装纳米粒(glycyrrhiza novel self-assembled nanoparticles，GN-SAN)，将其与传统水煎煮形成的甘草 SAN(glycyrrhiza decoction self-assembled nanoparticles，GD-SAN)进行系统比较，进一步探究GN-SAN对2，4-二硝基氯苯(2，4-dinitrochlorobenzene，DNCB)诱导的小鼠特应性皮炎的治疗作用。方法 采用煎煮结合微沉淀法制备GN-SAN，以粒径、PDI和ζ电位为评价指标，通过单因素试验联合Box-Behnken设计-响应面法对煎煮时间、磁力搅拌转速、磁力搅拌时间、磁力搅拌温度、旋转蒸发温度以及生药质量浓度进行优化，筛选最优处方工艺。将优化后的GN-SAN与GD-SAN进行比较，扫描电子显微镜(scanning electron microscope，SEM)和透射电子显微镜(transmission electron microscope，TEM)观察形态;高效液相色谱仪(HPLC)、紫外分光光度计(UV)以及bicinchoninic acid(BCA)试剂盒检测各SAN中小分子活性成分(芹糖甘草苷、甘草苷、芹糖异甘草苷、异甘草苷、甘草酸、甘草查耳酮A)、多糖以及蛋白的含量。采用DNCB对小鼠背部皮肤刺激，建立特应性皮炎模型。将小鼠分为空白组，模型组，阳性药组，GN-SAN凝胶低、中、高剂量组，观察各小鼠背部皮肤皮损变化，对皮损组织病理变化、炎症因子表达、脏器指数等指标进行检测。结果 GN-SAN的最佳处方工艺:甘草经8倍量水煎煮1h，得GD-SAN。再经6倍70％乙醇煎煮1h后得醇提液，合并2次提取液，600r/min磁力搅拌20min，60 ℃旋转蒸发除醇并浓缩至0。2g/mL，即得GN-SAN。形成的SAN性质稳定，为形态均一的圆球形纳米粒，粒径为(189。5±0。3)nm，多分散指数为0。138±0。130，ζ电位为(-31。4+0。8)mV。其粒径大小、均匀性、稳定性及有效成分转移率相对于传统SAN均提高，且GN-SAN对皮炎有良好的治疗效果。结论 采用煎煮法结合微沉淀法制备GN-SAN，其工艺简单、主要成分含量高，稳定性好;制成的GN-SAN性能优良、抗炎作用显著，为甘草纳米制剂进一步开发应用奠定基础。
Formation of novel self-assembled nanoparticles of Glycyrrhiza uralensis and anti-inflammatory evaluation
Objective To refine the traditional decoction self-assembly phenomenon,the Glycyrrhiza novel self-assembled nanoparticles(GN-SAN)were constructed by microprecipitation method,and compared with Glycyrrhiza decoction self-assembled nanoparticles(GD-SAN)in detail,and to further explore the therapeutic effect of GN-SAN on 2,4-dinitrochlorobenzene(DNCB)-induced atopic dermatitis in mice.Methods GN-SAN was prepared by decoction combined with microprecipitation,and the particle size,PDI and ζ potential were used as evaluation indexes,and the decoction time,magnetic stirring speed,magnetic stirring time,magnetic stirring temperature,rotary steaming temperature and mass concentration of raw materials were optimized by single factor test combined with Box-Behnken design-response surface method,and the optimal prescription process was screened.Compare the optimized GN-S AN with GD-S AN,the morphology of SAN was observed by scanning electron microscopy(SEM)and transmission electron microscopy(TEM),and the contents of small molecule active components(liquiritin apioside,liquiritin,isoliquiritin apioside,isoliquiritin,glycyrrhetinic acid,licochalcone A),polysaccharides and proteins were detected by high-performance liquid chromatography(HPLC),ultraviolet spectrophotometer(UV)and bicinchoninic acid(BCA)kit.DNCB was used to stimulate the dorsal skin of mice,and an atopic dermatitis model was established.The mice were divided into blank group,model group,positive drug group,GN-SAN gel low,medium and high dose groups,and the changes of skin lesions on the back of the mice were observed,and the histopathological changes,inflammatory factor expression,organ index and other indicators of skin lesions were detected.Results The optimal prescription process of GN-SAN was as follows:Glycyrrhiza uralensis was decocted with 8 times the amount of water for 1 h to obtain the GD-SAN.After decoction of 6 times of 70％ethanol for 1 h,the alcohol extract was obtained.Combining the two extracts,stirring magnetically at 600 r/min for 20 min,removing alcohol by rotary evaporation at 60 ℃ and concentrating to 0.2 g/mL to obtain GN-SAN.The formed SAN is a stable spherical nanoparticle with a morphological homogeneous particle size of(189.5±0.3)nm,a polydispersity index of 0.138±0.130,and a ζ potential of(-31.4±0.8)mV.Compared with traditional SAN,the particle size,uniformity,stability and effective component transfer rate were improved,and GN-SAN had a good therapeutic effect on dermatitis.Conclusion The preparation of GN-SAN by decoction method combined with microprecipitation method is simple and the content of main components is high and stable,and the prepared GN-SAN has excellent performance and significant anti-inflammatory effect,which lays a foundation for the further development and application of G.uralensis nano-preparations.
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