基于固定化酶体内反应器的宝藿苷Ⅰ定向转化系统研究

BaohuosideⅠ has a good anti-hepatocarcinoma activity, but it is very difficult to obtain and its poor biopharmaceutical characteristic also limits the clinical application. We have carried out a lot of research on the intestinal hydrolysis process for icariin and immobilized enzyme technology in the previous study. Considering the metabolic pathway for converting icariin to baohuosideⅠby intestinal hydrolase and the reduced metabolic rate of icariin in the pathological condition, an oral biotransformation system based on immobilized enzyme was constructed to promot the oriented biotransformation of icariin to baohuosideⅠwith stronger antitumor activity in vivo. In this system, β-glucosidase was selected as the hydrolase for icariin and immobilized by thiolated chitosan modified mesoporous silica nanoparticles. By improving its enzymatic properties, conversion efficiency and intestinal adhesion capability, icariin was fully hydrolyzed to baohuosideⅠand absorbed into the blood to achieve the anti-hepatocarcinoma effect through the P-gp inhibition of ginsenoside Rh2. This biotransformation system not only solved the source problem of baohuosideⅠ, but also enhanced its oral absorption and anti-hepatocarcinoma effect. In this study, formulation optimization, intestinal absorption, tissue distribution, pharmacokinetics, pharmacodynamics and safety evaluation for the immobilized enzyme system will be investigated, which will provide new ideas and new methods for research on other similar flavonoid glycosides.

宝藿苷Ⅰ具有很好的抗肝癌活性，但存在获取困难、生物药剂学特性差、难以临床应用等问题。本项目基于淫羊藿苷在体内可被肠道酶、肠道菌转化为宝藿苷Ⅰ的代谢途径和病理状态下代谢速率减慢的事实，在对固定化酶生物转化技术深入研究的基础上，设计构建了一种能够促进淫羊藿苷在体内定向转化为抗肿瘤活性更强的宝藿苷Ⅰ的口服固定化酶生物转化系统。该系统以β-葡萄糖苷酶为淫羊藿苷水解酶，采用巯基化壳聚糖修饰的介孔二氧化硅纳米粒对其进行固定化，通过提高其酶学性能、转化效率和肠道黏附性，促进淫羊藿苷定向、高效地转化为宝藿苷Ⅰ，并在人参皂苷Rh2的P-gp抑制作用下，促进宝藿苷Ⅰ的肠道吸收转运，从而提高其体内生物利用度和抗肝癌药效。本项目将围绕固定化酶生物转化系统的构建与评价、肠吸收、药效药动学、组织分布、安全性评价等方面进行研究，为其它类似黄酮类成分的转化吸收和充分利用提供新思路和新方法。

宝藿苷Ⅰ具有很好的抗肝癌活性，但存在获取困难、生物药剂学特性差、难以临床应用等问题。淫羊藿苷是中药淫羊藿中含量较高的一种黄酮成分，其口服到肠道后会被肠道酶（菌）水解为活性更强的宝藿苷Ⅰ吸收，但在病理状态下由于肠道酶（菌）活力下降、数量减少会导致淫羊藿苷不能被有效水解为宝藿苷Ⅰ吸收，继而影响其药效发挥。针对上述问题，本研究提出以下两个解决方案：（1）采用固定化酶体外生物转化淫羊藿苷的方法高效获取吸收更好、活性更强的宝藿苷Ⅰ；（2）探索将固定化酶口服作为淫羊藿苷体内生物转化器的给药方式，促进淫羊藿苷在肠道实时酶解为宝藿苷Ⅰ吸收，提高抗肝癌药效。. 本研究分别制备了交联纳米SiO2固定化蜗牛酶和交联介孔硅SBA-15固定化β-葡萄糖苷酶，并对固定化酶的制备工艺、理化表征、最适酶解条件和酶学性能进行研究，结果发现制备的固定化酶载酶量高、稳定性好，可在体外高效水解淫羊藿苷为宝藿苷Ⅰ，重复利用5次后的剩余酶活可保持在80%以上，为宝藿苷Ⅰ的绿色工业化生产奠定实验基础；此外还采用吸附-交联法制备了交联介孔硅SBA-15固定化纤维素酶，围绕其制备工艺、理化表征、体外仿生酶解、小鼠体内荧光示踪及抗肝癌药效进行研究，结果发现固定化纤维素酶经灌胃给药后，表现出较强的肠道滞留能力，与淫羊藿苷联合给药，可显著提高其抗肝癌药效，抑瘤率为63.1%,且对心、肝、脾、肺、肾等脏器组织无明显毒副作用。. 综上，本项目基于淫羊藿苷的肠道水解代谢规律和固定化酶生物转化的关键技术，将固定化酶口服作为淫羊藿苷的体内生物转化器，促进淫羊藿苷在肠道定向、高效地水解为抗肝癌活性更强的宝藿苷Ⅰ，不但解决了宝藿苷Ⅰ获取困难的问题，还促进了其在肠道的吸收转运，提高了抗肝癌药效，该研究突破了传统先获取原料药再制剂成型研究思路的局限，为其它类似黄酮苷类成分的转化吸收和充分利用提供了新的研究思路和研究方法。