基于酪蛋白磷酸肽/壳聚糖自组装构建月见草素纳米粒及其吸收机制研究

Oenothein B (OeB) abundant in eucalyptus leaf is a kind of antioxidant phytochemicals with a brilliant commercial prospect. But its poor oral-bioavailability is often the main reason for limiting their application. To solve this problem, a novel polyelectrolyte nanoparticle (PNP) delivery carrier fabricated by self-assembly behavior of casein phosphopeptides and chitosan, was developed to deliver OeB in this project. And the two materials had unique encapsulation and targeting delivery effects on OeB. The model groups including build conditions model and interaction forces model between casein phosphopeptides and chitosan were built based on the analysis of specific nanoparticle characteristics on the biological fate of OeB encapsulated in PNP, which was helpful to further understand self-assembling theory, predict stability of PNP, and optimize fabrication process. In addition, the physical and chemical properties and the biological fate of PNP were systematically studied by using in vitro and in vivo models to determine OeB PNPs oral digestion, absorption, distribution, metabolism and potential toxicity. The overall objective of this project is to address the lack of knowledge why the bioavailability of OeB can be substantially increased by encapsulation in PNP. At the completion of this project, we will have gained detailed fundamental knowledge about the impact of specific characteristics of the OeB PNP on the mechanisms of digestion within the GI tract, and provided theoretical basis for developing high-efficiency antioxidant health products.

桉叶中富含的月见草素(OeB)是一种极具开发前景的抗氧化功能活性物，但较低的口服生物利用度限制其应用。项目基于乳源生物活性肽-酪蛋白磷酸肽与天然活性多糖-壳聚糖自组装，合理设计新型聚电解质纳米粒来输送OeB，以改善其吸收缺陷，两种原料针对OeB具有独特的承载和靶向传递效果。项目通过分析构建过程中影响因素与纳米粒生物表征指标之间的相关性，优化构建条件，建立构建模型组，包括构建条件模型和作用力模型两部分。模型组的构建有利于更好的理解自组装理论、预测输送系统的稳定性和优化纳米粒构建过程。在模型构建的基础上，项目借助体内外实验模型，重点研究OeB纳米粒口服释放、吸收、代谢机制及潜在毒性，系统讨论纳米尺度粒子在通过胃肠道时的生物学过程基本问题。最终阐释纳米技术如何提高OeB生物利用度这一科学问题。该研究结果为纳米尺度物质吸收机制的理论体系提供基础数据，为研究高效的抗氧化保健新产品提供理论依据。

桉叶中富含的月见草素B是一种极具开发前景的抗氧化功能活性物，但较低的口服生物利用度限制其应用。本论文以广林9号桉叶为原料进行OEB的单体大量制备及体内外稳定性研究，通过基于食品级乳源生物活性肽-酪蛋白磷酸肽与天然活性多糖-壳聚糖（自组装，构建可控的CPPs/CS聚电解质纳米输送体系实现OEB的包埋。同时，结合纳米粒的表征指标，研究京尼平交联对OEB纳米粒在胃酸性环境下的保护作用，并对其体内外释放进行研究。从研究三者相互作用出发，进一步指导OeB纳米载体小球的构建条件、方式，从而实现OeB的有效包埋。同时，优化了适合大规模生产的制备方法，对制备纳米小球的稳定性、缓释作用进行评价。最后研究了纳米小球在Caco-2细胞模型中吸收机制。.研究结果如下：1）溶剂极性、热处理、pH、生化酶的种类及浓度均对OEB的稳定性有不同程度的影响， OEB在小鼠的体内代谢过程研究结果表明其在体内不稳定、易分解，不能直接进入血液。由此可见，OEB在体内外均不稳定；2）以相图作为指导，从CPP/OeB/CS共同作用所形成聚合物的粒径、数量、体系稳定性等方面综合考虑，选择体系终浓度为1.4 mg/mL，CPP/CS/OeB三者比例为3:3:1的条件制备聚电解质纳米小球，此时纳米小球粒径为156.1±2.2 nm，带电量22.02±1.3 mV，光强652.1 kcps、多分散性系数0.214；3）OeB-GNP的最佳工艺为：京尼平体系浓度为0.4 mg/mL，交联时间12h；离心条件为13400g，30min；超声条件为100W，45min。工艺优化后所得OeB-GNP参数为：粒径范围170~290 nm，CountRate范围为500~700 kcps，带电量18.06±1.8 mV，多扩散系数0.200±0.05，OeB包封率为99%，载药量为15%；4）成功地建立Caco-2细胞模型，并考察OeB-GNP在Caco-2单层细胞的摄取和转运特征，利用倒置荧光显微镜观Caco-2细胞摄取效果，发现OeB-GNP能很好地附着在Caco-2细胞表面，利用采用高效液相色谱法（HPLC）和动态光散射法进行检测，发现OeB-GNP能以整体形式透过细胞模型；5）CCGB的小鼠体内释放结果表明：GP交联的OEB纳米粒在体内、外可实现缓释，纳米化后OEB的体内稳定性显著提高。

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