Screening for food-grade anti-amyloidgenic compounds in food stuffs by using human cystatin C and improvement of their functionalities

半胱氨酸蛋白酶抑制剂CystatinC是一种半胱氨酸蛋白酶抑制剂，广泛存在于哺乳动物的组织和体液中，在内源性半胱氨酸蛋白酶的胞外调节中起重要作用。人半胱氨酸蛋白酶抑制剂C已经显示出容易在患有阿尔茨海默病或遗传性半胱氨酸蛋白酶抑制剂C淀粉样血管病的患者的脑中引起淀粉样蛋白沉积。由于在发达国家，不可治愈的疾病的发病率一直在稳步上升，人们对食品中食品级抗淀粉样蛋白化合物的筛选越来越感兴趣。构建编码人胱抑素C的合成基因，并将其不仅插入巴斯德毕赤酵母载体pPCIZa-C（Invitrogen，卡尔斯巴德，CA）中，而且插入大肠杆菌载体pQE-30和pQE-32中。将重组人胱抑素C用于筛选食品中具有淀粉样蛋白活性的化合物。结果表明，植物类黄酮和酚酸类化合物能够在体外抑制半胱氨酸蛋白酶抑制剂的淀粉样纤维形成。人参皂苷Rg 1与人参的单糖表现出强烈的抗淀粉样蛋白对半胱氨酸蛋白酶抑制剂，而β-Re与二糖链没有。绿原酸及其衍生物也表现出抗淀粉样变性，虽然它可能有一个趋势，在肠道消化一段时间内被削弱。这些观察结果可用于设计新型抑制剂作为淀粉样蛋白相关疾病的治疗药物。

Cystatin C, a cysteine proteinase inhibitor, is ubiquitous in mammalian tissues and body fluids, and plays an important role in extracellular regulation of endogenous cysteine protainases. A human cystatin C has been shown to easily cause amyloid deposits in the brains of patients suffering from Alzheimer's disease or hereditary cystatin C amyloid angiopathy. Since the incidence of the incurable diseases has been steadily on the rise in developed nations, there has been an increased interest in screening for food-grade anti-amyloidgenic compounds in food stuffs. A synthetic gene encoding human cystatin C was constructed and inserted not only into a Pichia pastoris vector, pPCIZa-C (Invitrogen, Carlsbad, CA) but also into a Escherichia colt vectors pQE-30 and pQE-32. The resulting recombinant human cystatin C was used for screening of bioactive compounds with amyloidgenicity in food stuffs. As the result, it was demonstrated that flavonoids and phenolic acids from plants are capable of in vitro inhibition of amyloid-fibril formation of cystatin. Ginsenoside Rg1 with a monosaccharide from gingseng showed strong anti-amyloidgenicity towards cystatin, whereas ginsenoside Re with a disaccharide chain did not. Chlorogenic acid and its derivatives also exhibited anti-amyloidgenicity, although it may have a tendency to be attenuated over a period of digestion in the intestinal tract. These observations can be useful for novel inhibitors' design as therapeutic anents for amyloid-associated diseases.