基于纳米粒整体吸收的提高藏药波棱瓜子中木脂素生物利用度的作用机制研究

Herpetospermum caudigerum is widely used for the treatment of hepatitis as a well-known Tibetan medicine with obvious curative effect. Its main active ingredients are herpetrione (Her) and other bioactive lignans with poor water solubility and low oral bioavailability. Our research group adopted nanosuspensions (NS) technology to greatly increase its bioavailability. However, the key scientific issues whether Her-NS is absorbed as molecules or integral nanoparticles and how to improve bioavailability still remain unclear..The project aims to understand the in vivo biological behavior and elucidate the mechanisms of NS improving bioavailability using the water-quenching fluorescent dyes hybrid technology. Based on the quenching effect and fluorescence switching ability, we characterize the structural alterations of the particles to achieve its "visualization" in vivo. The cellular model and in situ perfusion model will be established to investigate the cell uptake and transport and intestinal uptake of particles with different sizes. Lymphatic transportation of particles is evaluated by sampling via lymphatic cannulation. The pharmacokinetics and tissue distribution of Her-HNS after oral administration and injection in rats are also evaluated and the pharmacokinetic parameters and AUC of liver are calculated based on fluorescence quantification and HPLC quantification to elucidating the role of particles in enhancing oral bioavailability, so as to confirm Her-NS is absorbed as integral particles into the blood as main contribution. This project firstly addresses the fact that the mechanisms of NS increasing the bioavailability of insoluble components is unclear, resolves the issues of commonness in the industry and promotes the development of traditional ethnomedicine industry.

藏药波棱瓜子是藏医治疗肝病的常用药物，疗效确切，其主要活性成分为波棱甲素(Her)等难溶性木脂素，生物利用度低。课题组采用纳米混悬(NS)技术大大提高了其生物利用度。但Her-NS是以分子还是纳米粒形式整体吸收、如何提高生物利用度等关键科学问题仍不清楚。.本项目运用水淬灭探针杂化技术，瞄准体内生物学行为，阐明NS提高其生物利用度的作用机制。基于淬灭效应和荧光转化能力，表征粒子结构变化，实现生物体内“可视化”；建立细胞模型和单向肠灌流法，考察Her-NS细胞摄取转运及肠道摄取，研究口服经淋巴途径吸收转运，研究Her-NS经大鼠口服和注射后药动学和组织分布，计算荧光定量与HPLC定量，阐明粒子在增强口服生物利用度作用机制；以期证实Her-NS以纳米粒整体形式吸收入血作为主要贡献而提高生物利用度。本项目首次正视NS提高难溶性成分生物利用度机制不清事实，解决行业共性问题，推动传统民族医药产业发展。

藏药波棱瓜子是藏医治疗肝病的常用药物，疗效确切，其主要活性成分为波棱甲素(Her) 等难溶性木脂素，生物利用度低。课题组采用纳米混悬(NS)技术大大提高了其生物利用度。但 Her-NS是以分子还是纳米粒形式整体吸收、如何提高生物利用度等关键科学问题仍不清楚。本项目运用水淬灭探针杂化技术，瞄准体内生物学行为，阐明NS提高其生物利用度的作用机制。.主要完成以下内容：一、应用水淬灭荧光探针进行杂化，通过单因素实验优化，成功制备了两种不同粒径的波棱甲素无定型纳米粒（Her-ANPs），即Her-ANPs-200、Her-ANPs-450。粒径分别为200 nm和450 nm；PDI小于0.2。体外验证实验表明，ACQ探针荧光信号的变化可用来指示ANPs的状态变化；可用于Her-ANPs体内命运的研究。二、Her-ANPs口服后体内药动学及组织分布：不同粒径的Her-ANPs能够在胃肠道内留存至少8 h。组织分布结果显示，在肝脏中观察到荧光信号可维持24-36 h。三、通过单向肠灌流实验观察发现，Her-ANPs可以整体形式被肠上皮细胞摄取，且大粒径的Her-ANPs-450更易被回肠整体摄取。四、粒子的细胞摄取及转运研究建立了Caco-2、Caco-2/HT29-MTX以及Caco-2/HT29-MTX/Raji 3种细胞模型，证明了不同粒径的Her-ANPs口服后均有部分以整体ANPs的形式吸收进入细胞，并受黏液层影响，小粒径的ANPs可通过提高粘液屏障穿透能力来改善口服药物的吸收。五、尾静脉注射两种粒径的Her-ANPs，大鼠全身均可观察到荧光信号，保持至少36 h；小粒径的Her-ANPs-200更快进入血液循环，而Her-ANPs-450在血液中有较长的驻留时间。组织分布动力学研究结果表明，Her-ANPs注射后均可被各脏器摄取并有较长的驻留时间。六、对口服和注射Her-ANPs粒子的组织分布动力学进行分析比较，以TRE计算的Her-ANPs-200/450口服后绝对生物利用度分别为2.41%、1.75%。本项目发表相关学术论文14篇，其中SCI论文8篇 ，申请国家发明专利1项。