太子参抗T2DM活性多糖及其口服递药轨迹探究

Pseudostellaria heterophylla (Miq.) Pax (Taizishen, TZS) strengthens Qi and generates Fluids. TZS has been used as a key drug for the treatment of diabetes mellitus on many prescriptions of traditional Chinese medicine. In our series studies on the pharmacodynamics anti type 1 and type 2 diabetes mellitus （T2DM） of TZS named as No.2 Zhe-Shen, produced in Zherong County, Fujian Province; the polysaccharides in specific range of molecular weight of 50～210kDa lower blood sugar who type 2 diabetic rat induced by streptozocin (STZ) and improve the insulin resistance. Polysaccharide oral absorption mechanism of hypoglycemic effect is unknown. In this project, the effective fractions will be separated & purified by different kinds of sephadex column chromatography and ultrafiltration system to get homogeneous polysaccharide. The composition and structures will be identified with chemical and spectral methods (HPLC、IR、1D／2D-NMR). To establis HPLC-FLD method and use fluorescence/ radioactive nuclide tracing aPHPn, the active / passive and transcytosis mode will be investigated with Caco-2 cell model in vitro. The absorption of paracellular pathway with gastrointestinal epithelial cells tight regulation will be investigated with CLSM in vivo. The distribution of aPHPn will be investigated by MRI&PET/CT. The expressions of of the brush border of the small intestine epithelial cell glucose transporter SGLTl、GLUT2 mRNA and P-gp protein of biologic markers are tested by RT-PCR and western blotting methods. To explore the polysaccharide oral absorption of drug delivery trail.

太子参益气生津，是中药降糖方中常用核心药味之一。课题前期发现50～210kDa分子量范围太子参多糖显著降低2型糖尿病（T2DM）大鼠血糖、改善胰岛素抵抗。然而多糖所致生物活性与口服吸收，一直是争论的焦点和研究的难点。本项目分离纯化多糖有效部位获得均一多糖，集多种波谱分析技术，表征化学结构。以葡萄糖转运、消耗、促胰岛素分泌离体细胞模型，获知活性均一多糖（aPHPn）。建立HPLC-FLD或HPLC-GCP检测方法，荧光/放射性核素标记示踪，Caco-2体外模拟研究aPHPn主动/被动和胞吞转运模式；多模式小动物活体成像技术（MRI&PET/CT）研究大鼠口服多糖体内以完整分子全身分布的比率；探寻多糖口服吸收的递药轨迹。

通过本项目研究发现，太子参多糖包含两类，一类葡聚糖，另一类是果胶多糖暨杂多糖；50~200 kDa分子量范围的粗多糖具有很好的降血糖作用，是抗二型糖尿病（T2DM）的活性部位。通过本项目的实施，从中分离获得了5个均一多糖，命名为H-1-1-1、H-1-2、H-1-3、0.2M-1-3-2、0.5MSC-F，其分子量分别为1.5×105、1.4×104、5.3×104、2.1×104、4.7×104 Da，并表征出化学结构。经体外细胞模型筛选，其中2个活性均一多糖（H-1-2和0.5MSC-F），具有增加细胞对葡萄糖的消耗，增加细胞对葡萄糖的摄取量，以及促进胰岛细胞分泌胰岛素的作用；通过Caco-2 体外模型研究，H-1-2和0.5MSC-F活性均一多糖，在模拟人胃、肠液中稳定，能够整体吸收；H-1-2和0.5MSC-F的转运吸收方式均为被动吸收，H-1-2葡聚糖主要利用网格蛋白介导内吞途径进入Caco-2细胞，而0.5MSC-F多糖并不是以胞吞的形式进入细胞，可能是由于细胞内外的压力差进入细胞。核素99mTc标记，SPECT/CT示踪；多糖口服进入大鼠体内，随着时间的变化，经食道、胃、大肠、小肠至膀胱，放射性物质在小肠分布量大；99mTc–H-1-2大鼠口服，4 h后摄入的大约50%的放射性物质滞留在小肠，99mTc–0.5MSC-F，2 h后摄入的大约40%的放射性物质滞留在小肠，通过肠粘膜吸收进入体循环。.本项目研究结果，为开发太子参多糖抗糖尿病药物或保健食品，奠定了药效物质基础。