Exploration Of The In Vitro And In Vivo Biological Activities Of Myricetin Modified By Horseradish Peroxidase

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.