基于生物转化和代谢分析构建非营养高倍甜味剂斯梯夫苷系的代谢产物谱

Stevioside family (as called steviosides) includes major sweet components from stevia rebaudiana (Bertoni) and enzymatic transglucosyled steviosides, their metabolism can affect the safety and bioactivities. In this proposal, some rare steviosides will be prepared by extraction from the plant and enzymatic synthesis. Metabolism kinetics analysis using rats microPET will be applied to identify the specific organs or parts in rats that will be chosen for investigating the metabolites and detailed kinetics. The phase I metabolites will be then enzymatically up-scaled to enhance subsequent analysis of the phase II metabolism. Therefore the metabolites profile of the steviosides will be established based on the metabolism analysis and the supplementary enzymatic synthesis. In addition, gastric juice with controlled artificial factors will be used to stimulate the production of unpredictable metabolites for analyzing phase I metabolism in some extreme conditions. Metabolites produced in those extreme conditions will be supplementary help for elucidating the metabolism. Meanwhile, pharmacokinetics and enzymology studies will be conducted using the kinetics constants of UDP-glucuronosyltransferases (UGTs ) catalyzed phase II metabolism reaction. The secondary conformation variation of the selected UGT will be determined by circular dichroism, and interaction energy between the steviosides and the UGT protein will be calculated. Therefore, the affiliation and recognization between single UGT isomer and steviosides' member could be analyzed. Toxicity of steviosides and their metabolites on human hepatic cells will be investigated. The completion of current project will lead to the elucidating of the structure-function relationships of the steviosides; their toxicity on hepatic cells and their effect on the metabolism enzymes' activities will be revealed. The proposed work will benefit researches on steviosides' safety and bioactivity; provide suggestion for better use of the non-nutritive sweetener.

斯梯夫苷系化合物包括甜菊糖中的主要甜味成分和酶促转苷的斯梯夫苷，它们的代谢会直接影响其安全性和生物活性。本课题将以仿生酶促合成和植提纯化来制备部分稀少品种，以大鼠MicroPET的经时代谢分析，引导分析特定代谢点的代谢产物和动力学，以酶促合成的重要I相代谢产物来补给II相体外代谢分析，从而构建斯梯夫苷系代谢产物谱；并以人胃液结合环境模拟应激来解析特异环境的I相非酶水解代谢，发掘应激环境下的可能代谢物。同时将结合药代动力学和酶学，拟合计算II相酶促糖醛酸酯化的动力学参数，以圆二色谱解析酶蛋白二级结构变化和底物-蛋白结合能计算，协同分析尿苷二磷酸葡萄糖醛酸转移酶中的相关同工酶对不同斯梯夫苷系化合物的识别和亲和行为和酶活构效关系。进而研究斯梯夫苷系及其代谢产物的人肝细胞毒性，发掘其构效关系与代谢酶活影响构效关系间的关联，将有助于甜菊糖安全性和生物活性的研究，并给其合理使用提供理论基础。

本课题以仿生酶促合成、水解以及植提、仿生制备部分斯梯夫苷系化合物的稀少品种，以酶促合成斯梯夫苷系化合物的重要I相代谢产物来补给II相代谢体外代谢分析；并以人胃液结合环境模拟应激来解析特异环境的I相代谢非酶水解代谢，发掘应激环境下的应激代谢产物，构建了斯梯夫苷系化合物的代谢产物谱。没有发现除甜菊醇和甜菊双糖苷以外的I相代谢产物。所有JECFA准用的斯梯夫苷系化合物包括斯梯夫苷 (St)和莱鲍迪苷A (RA)以及I相代谢产物甜菊双糖苷的水解产物都是甜菊醇。而甜菊醇的II相代谢以尿甘二磷酸葡糖醛酸苷转移酶 (UDP-glucuronosyltransferases, UGTs)催化甜菊醇的羧基和5ˊ-二磷酸尿苷葡糖醛酸 (三钠) (UDPGA)的羟基的反应产物为主，其中在众多UGT酶中UGT2B7和UGT1A3是催化甜菊醇II相代谢主要的酶，甜菊醇在HLM和UGT2B7下的Km分别为8.68±1.66和6.26±0.84 μM，Vmax分别为0.343±0.017和0.210±0.014 μmol/min/g protein。用Auto dock 模拟甜菊醇与UGT2B7的分子对接发现，甜菊醇与UGT2B7的结合自由能为-8.35 kcal/mol，UGT2B7的活性口袋由Arg 352，Leu 347，Lys 343，Phe 339，Tyr 354，Lys 355和Leu 353氨基酸组成，其中Lys 343与甜菊醇形成氢键。.进一步地，对斯梯夫苷系化合物及其主要代谢产物甜菊醇对消化道细胞的细胞毒性进行了广泛研究。构建了消化道正常细胞和癌细胞的受体群，研究发现甜菊醇对于多种肝、肠、胃癌细胞的抑癌活性接近常用化疗药物5-FU (5-Fluorouracil)和阿霉素，且呈浓度和时间依赖关系，并利用流式、western blot、miRNA芯片以及RT-PCR等分析了甜菊醇抑制消化道癌细胞的可能作用机制。