茶多酚-蛋白质聚集体增效纳米硒的途径及分子机制

Functional modification of selenium nanoparticles (SeNPs) with bioactive templets has been widely proved as an effective approach for the development of novel selenium supplements with high bioactivity but low toxicity and new food formulations with outstanding function. Though it has merited increasing attention, the efforts of investigators in this field have not been entirely successful because of the limited knowledge on the binding mechanisms between templets and SeNPs. Our previous research found that the self-assembled aggregates of tea polyphenols and proteins in tea infusions were excellent templates to strongly stabilize SeNPs and remarkably enhance their bioactivity. It successfully broke through the limitation in the stabilization of SeNPs via bioactive small molecules such as tea polyphenols. To further explore the underlining mechanisms, SeNPs will primarily be prepared using stimulating aggregates assembled with tea polyphenol and model protein (bovine serum albumin) at a given ratio as templets in the present proposal. In addition, the physicochemical properties of SeNPs will be characterized and their in vitro antioxidant activity will be evaluated on the hydrogen peroxide induced hepatic cell line (LO2 cells), in order to correlate the stability and bioactivity of SeNPs with the constitution of tea polyphenol-protein aggregates. Furthermore, the conformational changes of protein will be analyzed and the molecular interactions among proteins, tea polyphenols, and SeNPs will be subsequently explored via the novel Saturation-Transfer-Difference Nuclear Magnetic Resonance strategy and the Isothermal Titration Microcalorimetry, aiming at the construction of the molecular model for the stabilization of SeNPs by the aggregates. Moreover, the effects on the expressions of key cytokines in LO2 cells by SeNPs will be studied using Western blot and Quantitative Real-time PCR to discover their antioxidant targets. This research is expected to exploit the approach for the functional modification of SeNPs by tea spontaneously-assembled aggregates and to elucidate the underlining mechanism from the innovative perspectives in chemical molecules and the in vitro cell lines. It will provide a scientific theoretical basis for the improvement of the security and functions of SeNPs as well as the application of the combination between selenium and tea.

以活性模板增效纳米硒是发展高效安全硒制剂的有效途径，然而其理论原理至今仍进展缓慢。本团队前期研究发现，茶汤中茶多酚、蛋白质等自发结合形成的聚集体能够作为模板充分稳定纳米硒并增强其活性，突破了当前茶多酚等小分子活性模板难以有效稳定纳米硒的技术难题。为充分阐释这一新发现的深入机理，本项目拟以茶多酚单体和模型蛋白质的自组装聚集体为模板构建纳米硒，系统表征其物化特性，利用体外细胞系评价其抗氧化活性，探索纳米硒稳定性、活性与聚集体组成的相关性；进而分析蛋白质空间构象的变化，重点采用核磁共振转移饱和差谱、等温滴定微量热等探究蛋白质与茶多酚、纳米硒相互作用的动态过程，建立聚集体稳定纳米硒的分子模型；同时考察纳米硒对细胞抗氧化关键因子的调控，探讨聚集体增效纳米硒的作用靶点，以期在化学分子层面和细胞水平上揭示茶汤聚集体增效纳米硒的途径和机制，为新型高效纳米硒的安全利用和茶叶功能的广泛发挥提供科学的理论依据。

纳米硒是高效安全的新型硒制剂，但其稳定性差，极易聚集沉淀而失去活性。目前主要通过引入蛋白质、多酚等模板与纳米硒结合，使之稳定或增强其功效。然而，采用单一模板难以同时实现对纳米硒的稳定化和功能强化，限制了多功能纳米硒的开发利用。针对这一难题，本项目提出利用茶多酚和蛋白质的自发相互作用，探索能够一步稳定并增效纳米硒的新途径，并揭示其内在的相互作用机理和构效机制。取得了系列重要的研究成果：（1）建立了以茶多酚-蛋白质的自组装聚集体为模板合成高活性、高安全性、高稳定性纳米硒的方法，并获得其适宜的合成条件，即亚硒酸钠浓度为5mM、亚硒酸钠/维生素C配比为1:8、蛋白质浓度为3mg/mL、茶多酚浓度为3mM、反应温度为40℃；（2）成功合成了四种茶多酚-蛋白质-纳米硒，为椭圆形或圆形的纳米级无定型单质硒颗粒，直径约30~70nm，分散性良好，表面带正电荷，硒含量约4.6~4.7mM，茶多酚保留率约34~58%，且具有比亚硒酸钠更强的生物活性和更低的细胞毒性；（3）揭示了茶多酚、蛋白质、纳米硒主要通过氢键、疏水相互作用、范德华力结合，涉及蛋白质表面的疏水位点、-OH及-C=O-等基团，并引起蛋白质空间结构变得松散，从而更利于三者的相互作用；（4）证实了茶多酚的没食子酰基、顺式构象及B环的儿茶酚结构和蛋白质较大的分子量均会增强茶多酚与蛋白质、茶多酚与蛋白质-纳米硒的相互作用；（5）获得了热处理、pH、离子、储藏条件等因素影响茶多酚-蛋白质-纳米硒稳定性的规律；（6）已发表论文4篇、其中SCI农林科学1区TOP期刊论文2篇，获授权国家发明专利5件、已申请4件，培养毕业博士生1名、硕士生3名。上述研究成果为茶多酚-蛋白质-纳米硒的合成与应用奠定了理论研究基础，为新型纳米硒制剂的研发提供了新策略，为协同发挥茶多酚和纳米硒的健康功效提供了新思路，对发展复合型食品功能因子、拓展茶叶综合利用领域具有重要的借鉴和参考价值，对改善我国居民微量元素营养状态、助力健康中国建设具有积极意义。

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