【作者】 孙敏；

【导师】 李娇；

【作者基本信息】 山西大学 ， 食品科学与工程， 2023， 硕士

【摘要】 杨梅素（MYR）是一种多酚类黄酮物质,属于黄酮类化合物中的黄酮醇类,广泛存在于天然植物中,具有抗氧化、抗菌、抗蛋白质淀粉样纤维化等生物活性,在体内能够发挥抗衰老、逆转神经退行性病变等多种药理功效,但因其结构的特殊性,导致其水溶性、稳定性较差,限制了其在食品、药品中的广泛应用。本研究利用辣根过氧化物酶（HRP）催化杨梅素得到杨梅素氧化产物（MYRox）,利用萃取、层析等方法对其进行纯化,得到不同组分的杨梅素衍生物,并运用波谱、质谱等手段对该衍生物进行表征。通过体外活性检测、对模式生物秀丽隐杆线虫的作用,探究了杨梅素氧化前后体内外生物活性的变化,筛选得到了生物活性更强且比较稳定的杨梅素衍生物,为杨梅素安全、合理、广泛的应用,尤其是在抗菌、预防蛋白质构象病等方面的应用提供了理论依据。1.MYRox的制备、纯化和表征。利用HRP催化氧化MYR得到MYRox,通过紫外可见光谱、高效液相色谱和高分辨液质联用仪对MYRox进行监测、表征,结果显示MYRox在300 nm处有特殊吸收峰,是由多个化合物组成的混合物。通过液液萃取和层析法对MYRox进行分离、纯化,得到水溶性杨梅素衍生物（MYRow）和有机相杨梅素衍生物（MYRoo）。分别利用高分辨液质联用仪和基质辅助激光解吸飞行时间质谱对上述得到的MYRow和MYRoo进行分析,推测其可能为杨梅素二聚体和醌类物质。2.MYR及MYRox体外生物活性确定。体外抗氧化实验结果显示,MYR及MYRox均具有良好的自由基清除能力和Fe³⁺还原能力。与MYR相比,MYRox保留了MYR大部分的体外抗氧化活性,对DPPH、ABTS、羟自由基清除率的IC₅₀依次为:30.62μM、97.11μM、3.795 m M,对铁离子的还原能力保留度达90%左右。金黄色葡萄球菌增殖抑制实验结果显示,MYRox最小抑菌浓度为256μg/m L,低于MYR的512μg/m L,最小杀菌浓度为512μg/m L,低于MYR的1024μg/m L,表明与MYR相比,MYRox对金黄色葡萄球菌具有更好抑制作用。以溶菌酶（Lys）为蛋白模型的抗蛋白质淀粉样纤维化结果显示,MYR及MYRox均能够有效抑制Lys的淀粉样纤维化,且MYRox作用效果要强于MYR。3.MYR及MYRox抑菌机制探究。金黄色葡萄球菌增殖抑制实验结果显示,MYRox中发挥抑菌作用的主要成分是MYRoo。进一步对MYR及MYRoo抑菌机制进行探究,发现MYR及MYRoo能够增加金黄色葡萄球菌的细胞膜通透性、改变其细胞表面疏水性和胞内活性氧水平,并改变胞内蛋白及DNA状态,且MYRoo作用效果更明显。4.MYR及MYRow体内外抗淀粉样纤维化作用探究。体外抗淀粉样纤维化结果表明,MYRox中起抗淀粉样纤维化作用的主要成分为MYRow,即杨梅素二聚体。与MYR相比,MYRow对Lys和牛胰岛素淀粉样纤维化抑制作用更强。利用阿尔茨海默症模型线虫验证MYRow在体内是否有同样的抗淀粉样纤维化作用,结果显示MYR及MYRow均能有效预防Aβ_1-42聚集、积累对GMC101引起的神经毒性作用,且MYRow作用更强。综上所述,杨梅素经HRP改造氧化后体内外生物活性发生了变化,体外抗氧化能力略有降低,但抗菌作用及体内外抗淀粉样纤维化活性均有提高。据此,从杨梅素氧化产物中筛选得到了两种不同活性的杨梅素衍生物,一种水溶性好、抗蛋白质淀粉样纤维化活性强（MYRow）,另一种溶于有机相抗菌活性更强（MYRoo）。该研究为杨梅素的合理、安全应用,尤其是抗氧化、抗蛋白质淀粉样纤维化和抗菌等方面的应用提供了实验基础。同时,也为拓宽杨梅素的应用范围提供了理论依据。更多还原

【Abstract】 Myricetin（MYR）is a polyphenolic flavonoid,belonging to the flavonol group of flavonoids,which is widely found in natural plants and has many biological activities such as antioxidant,antibacterial and anti-amyloid fibrillation,and can exert various pharmacological effects in vivo such as anti-aging and reversal of neurodegenerative lesions.However,the water solubility and stability of myricetin are poor due to its structural peculiarities,which limit its wide application in food and pharmaceuticals.In this study,horseradish peroxidase（HRP）was used to catalyze MYR to obtain myricetin oxidation product（MYRox）.And MYRox was purified by extraction and chromatography to obtain myricetin derivatives of different components,and the derivatives were characterized by using wave and mass spectrometry.The in vitro activity assay and the effect on the model organism Caenorhabditis elegans were investigate to determine the changes of biological activities of myricetin after oxidation,and the stronger biologically active and more stable myricetin derivatives were screened.This study provided a theoretical basis for the safe,rational and wide application of myricetin,especially in antibacterial and protein conformational diseas prevention.1.Preparation,purification and characterization of MYRox.MYRox was prepared by catalytic oxidation of MYR with HRP.MYRox was monitored and characterized by UV-visible spectroscopy,high performance liquid chromatography and high-resolution liquid mass coupling instrument,and the results showed that MYRox has a strong absorption at 300 nm and was a mixture of several compounds.The MYRox was isolated and purified by liquid-liquid extraction and chromatography to obtain myricetin water-soluble oxidation products（MYRow）and myricetin organic phase oxidation products（MYRoo）.The MYRow and MYRoo were analyzed by high-resolution liquid mass coupling instrument and Matrix assisted laser desorption time of flight mass spectrometry,respectively,and were presumed to be possible myricetin dimer and quinone.2.Changes in biological activities of MYR and MYRox in vitro.The results of in vitro antioxidant experiments showed that both MYR and MYRox had good free radical scavenging ability and Fe³⁺reducing ability.Compared with MYR,MYRox retained most of the in vitro antioxidant activity of MYR,with IC₅₀values of 30.62μM,97.11μM,and3.795 m M for DPPH,ABTS,and hydroxyl radical scavenging respectively,and about90%retention of Fe³⁺reduction ability.The results of proliferation inhibition assay for S.aureus showed that the minimum inhibitory concentration of MYRox was 256μg/m L,which was lower than 512μg/m L of MYR.The minimum bactericidal concentration was512μg/m L,which was lower than 1024μg/m L of MYR.These data indicated that MYRox had better inhibitory effect on S.aureus compared with MYR.The results of anti-amyloid fibrillation using lysozyme（Lys）as a protein model showed that both MYR and MYRox could effectively inhibit amyloid fibrillation of Lys,and the effect of MYRox was stronger than that of MYR.3.Inhibition mechanism of MYR and MYRox on S.aureus were investigated.The results of the proliferation inhibition assay of S.aureus showed that MYRoo was the main functional component of MYRox.Further,the inhibition mechanism of MYR and MYRoo were investigated.The results showed that MYR and MYRoo could increase the cell membrane permeability,change the cell surface hydrophobicity（CSH）and intracellular reactive oxygen species（ROS）level,and affect the intracellular protein and DNA of S.aureus.And MYRoo had a more obvious effect on them overall.4.Exploration of the anti-amyloid fibrillation effect of MYR and MYRow in vitro and in vivo.The results of anti-amyloid fibrillation in vitro showed that the main component of MYRox that plays an anti-amyloid fibrillation role is MYRow,a myricetin dimer.Compared with MYR,MYRow had a stronger inhibitory effect on Lys and bovine insulin（BI）amyloid fibrillation.Using the Alzheimer’s disease model nematode to verify whether MYRow had the same anti-amyloid fibrillation effect in vivo,the results showed that both MYR and MYRow were effective in preventing the neurotoxic effects caused by Aβ_1-42aggregation and accumulation on GMC101,but MYRow was more potent.In conclusion,the in vitro and in vivo bioactivities of myricetin were changed after oxidation,and the in vitro antioxidant capacity was slightly reduced,but the antibacterial effect and anti-amyloid fibrillation activity were increased.Accordingly,two different active myricetin derivatives were screened from myricetin oxidation products,one with good water solubility and anti-amyloid activity（MYRow）,and the other with stronger antibacterial activity（MYRoo）.This study provides an experimental basis for the rational and safe application of MYR,especially in antioxidant,anti-amyloid fibrillation and antibacterial applications.At the same time,it broadened the application scope of MYR.更多还原