大豆功能糖提高异黄酮生物利用度的组-效关系及互作调控机制

Low bioavailability of soybean isoflavones has emerged as a key restrictive factor to exhibit their nutritional benefits. The applicant already screened and found that soybean isoflavones induced the expression of intestinal phase II enzymes and efflux transporters (proteins), leading to their very low bioavailability, while soybean functional carbohydrates could significantly promote the intestinal absorption of these isoflavones. However, the composition-activity relationship of the tested oligosaccharide or polysaccharide fractions, and their mechanisms underlying regulation and control of specific interaction are not clear, and it is the urgent need for the deep investigation from the perspective of metabolism, genes and molecules. In this study, we will explore the composition, primary structure, and molecular weight of different soybean functional carbohydrates and their inhibitory effects on the expression of phase II enzymes (SULT and UGT) and efflux transporters (P-gp and MRP1), as well as the time- and dose-effect relationship of the isoflavone bioavailability enhancement to reveal the active carbohydrate fractions with the interaction effect. Furthermore, under oral ingestion of the functional carbohydrate fraction with high activity, isoflavones or their combination in rats, this investigation will test whether microRNA (miRNA) derived from rat small intestine directly regulates the mRNA and protein expressions of the metabolic enzymes or transporters, and also investigates whether it indirectly control the expression of the metabolic enzymes and transporters through miRNA-mediated Keap1-Nrf2/ARE signaling pathway. This study will elucidate the new target and interaction mechanism of soybean functional carbohydrates in improving the intestinal absorption of soybean isoflavones, and to provide a scientific basis for the interaction theory of food ingredients and development of novel foods.

低生物利用度已突显为大豆异黄酮发挥营养作用的主要制约因素。申请人已筛选发现大豆异黄酮诱导肠II相代谢酶和外排转运蛋白表达而生物利用度极低，且大豆功能糖可显著促进其肠吸收，但该寡糖或多糖组分的组-效关系及其具体互作调控机制不清楚，急需从代谢、基因与分子水平深入探究。本项目研究大豆不同功能糖的组成、一级结构和分子量等组分结构特征与大鼠小肠II相代谢酶（SULT、UGT）和外排转运蛋白（P-gp、MRP1）的表达抑制及异黄酮生物利用度提升的时效和量效关系，揭示高效互作调控的功能糖组分；进而探究高活性功能糖组分、异黄酮及其联合摄入中大鼠小肠miRNA是否直接靶向代谢酶或转运蛋白的mRNA及蛋白表达，并考察miRNA是否介导Keap1-Nrf2/ARE信号通路，间接调控代谢酶和转运蛋白的表达。阐明大豆功能糖调控异黄酮肠吸收的新靶标及两者的互作机制，为食品成分互作增效理论及新型食品创制提供科学依据。

目前，国内外对大豆主要成分功能糖与染料木黄酮等异黄酮研究多局限于单一组分的分离、纯化及功能评价，其结果难以准确反映食品多组分共存并相互作用的营养特征。本项目研究发现，大豆水苏糖寡糖、多糖等功能糖抑制小肠II相代谢酶和外排转运蛋白表达的营养新功能，揭示其通过该新功能拮抗类黄酮小肠首过代谢进而增强其肠吸收和生物学活性的互作效应机制。.率先发现大豆染料木黄酮等异黄酮摄入显著诱导小肠II相代谢酶（SULT和UGT）和外排转运蛋白（P-gp、MRP1）表达而导致小肠首过代谢机制的发生（葡萄糖醛酸化或硫酸化的类黄酮被肠上皮细胞的转运蛋白外排至肠腔），从而极大地降低其生物利用度，其中异黄酮酯化度与抑制效应成正相关的构效关系。进一步的机制研究发现，共摄入的大豆功能糖可有效抑制小肠II相代谢酶和外排转运蛋白的表达，据此对抗异黄酮首过代谢，增强其生物利用度及营养功效；并发现水苏糖等功能糖与小肠上皮细胞膜上的HSP90β蛋白结合改变其外泌体miRNA谱，突破了功能糖益生元是小肠“过客”的传统认知，揭示了大豆功能糖调控小肠上皮细胞外泌体miRNA谱的新功能，也阐明了功能糖-小肠miRNA-结肠菌群的轴向调控关系，证实了功能糖下调的小肠miR-30a-5p显著抑制结肠（粪便）Lactobacillus reuteri的增殖，而miRNA被证实不能介导Keap1-Nrf2/ARE信号通路的调控，因而小肠miRNA与代谢酶或转运蛋白的调控关系需进一步的探究。这些研究发现为功能糖和类黄酮功能因子的互作增效理论发展及复合功能食品研发提供科学依据。.相关成果已在Mol. Nutr. Food Res.、J. Agric. Food Chem.、Food Funct.、J. Funct. Foods等SCI期刊上共发表论文33篇，被SCI期刊大量引用，已完成合同的全部任务。此外，部分研究成果正在整理中，拟发表。