葛根芩连汤成分间自组装纳米粒改善伊立替康所致肠毒性作用研究

Improvement of irinotecan-induced enterotoxicity by self-assembled nanoparticles between components of Gegen Qinlian Decoction

目的基于中药经典复方葛根芩连汤(Gegen Qinlian decoction,GQD)合煎过程中成分间相互作用,探讨其代表性成分葛根素、小檗碱、汉黄芩苷、黄芩苷、甘草酸、巴马汀体外结合形成自组装纳米粒的性能及改善伊立替康(CPT-11)所致肠毒性作用的药理活性,为来源于中药汤剂的活性成分间的自组装纳米粒提供参考。方法采用溶剂挥发法分别制备小檗碱-汉黄芩苷自组装纳米粒(berberine-wogonoside nanoparticles,Ber-Wog NPs)、小檗碱-葛根素自组装纳米粒(berberine-puerarin nanoparticles,Ber-Pue NPs)、黄芩苷-葛根素自组装纳米粒(baicalin-puerarin nanoparticles,Bai-Pue NPs)、黄芩苷-巴马汀自组装纳米粒(baicalin-palmatine nanoparticles,Bai-Pal NPs)、黄芩苷-甘草酸自组装纳米粒(baicalin-glycyrrhizic acid nanoparticles,Bai-GA NPs)5种组分自组装纳米粒,测定其粒径分布、多分散指数(polydispersity index,PDI)、包封率、载药量,采用透射电子显微镜(transmission electron microscope,TEM)观察其微观形貌,紫外光谱和傅里叶红外光谱考察成分间相互作用;考察5种组分自组装纳米粒缓解CPT-11致小鼠迟发性腹泻的药理作用。结果组分间按1∶1投药比,制备所得Ber-Wog NPs、Ber-Pue NPs、Bai-Pue NPs、Bai-Pal NPs、Bai-GA NPs的平均粒径分别为(198.09±4.64)、(216.29±5.31)、(173.53±5.46)、(185.75±3.38)、(242.10±12.30)nm;PDI分别为0.14±0.06、0.13±0.06、0.19±0.03、0.19±0.01、0.21±0.05,TEM观察均为球状纳米粒,除Ber-Wog NPs外,其余4种纳米粒包封率均较高。紫外可见吸收光谱和红外光谱提示,5种纳米粒的组装均是通过分子间非共价键作用形成;分子对接模型进一步提示,其形成机制与分子间静电相互作用或氢键相关。药效试验结果显示,制剂组可缓解小鼠体质量降低,腹泻指数降低,结肠组织炎症因子肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-1β(interleukin,IL-1β)含量降低,IL-10含量升高;苏木精-伊红染色(hematoxylin-eosin staining,HE染色)显示结肠黏膜组织病变显著缓解。结论GQD来源的几种代表性成分可高效自组装形成纳米粒,且具有类似GQD缓解CPT-11所致肠毒性作用,为研究传统中药汤剂药效物质基础形态与药效的相关性提供了新思路。

Objective Based on the interactions among the components in the combined decoction process of the classical Chinese medicine compound Gegen Qinlian Decoction(葛根芩连汤,GQD),we investigated the performance of its representative components,puerarin,berberine,baicalin,baicalin,glycyrrhizic acid,and pamadine,in combining with each other in vitro to form self-assembled nanoparticles,and their pharmacological activities in ameliorating the enterotoxicity effects induced by irinotecan(CPT-11),so as to provide references for self-assembled nanoparticles among active ingredients originating from the traditional Chinese medicine broths.Methods The five self-assembled berberine-wogonoside nanoparticles(Ber-Wog NPs),berberine-puerarin nanoparticles(Ber-Pue NPs),baicalin-puerarin nanoparticles(Bai-Pue NPs),baicalin-palmatine nanoparticles(Bai-Pal NPs),and baicalin-glycyrrhizic acid nanoparticles(Bai-GA NPs)were prepared by solvent evaporation method.Then,the particle size,polydispersity index(PDI),drug entrapment efficiency and drug loading efficiency of the nanoparticles were measured.The morphology was observed by transmission electron microscopy,and the interaction between the components was investigated by the UV-vis absorption spectroscopy and Fourier transform infrared spectroscopy.In addition,the pharmacological effects of five self-assembled nanoparticles on alleviating delayed diarrheam mice model induced by irinotecan(CPT-11)in mice were investigated.Results The average particle size of Ber-Wog NPs,Ber-Pue NPs,Bai-Pue NPs,Bai-Pal NPs,Bai-GA NPs were(198.09±4.64),(216.29±5.31),(173.53±5.46),(185.75±3.38),(242.10±12.30)nm,respectively.The average PDI was 0.14±0.06,0.13±0.06,0.19±0.03,0.19±0.01,0.21±0.05,respectively.Furthermore,the five self-assembled nanoparticles were spheroidal nanoparticles observed by transmission electron microscopy.The other four nanoparticles have higher encapsulation efficiency except Ber-Wog NPs.The UV-vis absorption spectroscopy and Fourier transform infrared spectroscopy suggested that the five assembly nanoparticles were formed by intermolecular hydrophobic forces and the molecular docking modeling further suggested that the formation mechanism of nanoparticles was related to intermolecular electrostatic interactions or hydrogen bonding.The pharmacodynamic results showed that the preparation group relieved weight reduction,diarrhea index was decreased,the content of inflammatory factors tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β)in the colonic tissues was decreased,and interleukin-10(IL-10)was increased;Hematoxylin-eosin staining(HE staining)showed that the lesions of the colonic mucosal tissues were significantly relieved.Conclusion Several representative components from GQD can efficiently self-assemble to form nanoparticles,and have similar effects to GQD in alleviating enterotoxicity caused by CPT-11,which provides a new way to study the correlation between the basic form of pharmacodynamic substances and pharmacodynamic of traditional Chinese medicine decoction.