Investigation Of The Chemical Structure Of Anti-amyloidogenic Constituents Extracted From Resina Draconis

Amyloid diseases are a group of amyloidosis disorders characterized by the alternative folding of proteins,which can lead to the formation,deposition,accumulation of toxic and misfolded aggregates.Protein aggregation has been connected to more than 30 proteinopathies,ranging from neurodegeneration to type 2Diabetes and systemic amyloidosis.In recent years,some progress has been made in inhibiting or treating amyloid diseases by inhibiting the formation of amyloid fibrils and oligomers.Many plant-derived small molecules with the ability to inhibit the formation of amyloid fibrils have gradually attracted great attention.Due to the characteristics of stable structure,functions and multiple targets,natural products can not only intervene in the occurrence of diseases at different stages of amyloid fibril formation,but also reduce the by-effects caused by chemotherapy.In our previous screening of natural herbal medicines for anti-amyloidgenic diseases in Yunnan,we found that the natural medicine Resina Draconis can be used as a good candidate.Therefore,in-depth excavation and exploration of the bio-active components against amyloid fibrillogenesis in Resina Draconis has become the core of this study.In this study,Hen Egg White Lysozyme（HEWL）was firstly utilized as a typical amyloid model protein to investigate the potential effects of Resina Draconis on protein fibrillogenesis.Solvent extraction and Thin-Layer Chromatography（TLC）were used to isolate and prepare the effective components of the traditional Chinese medicine Resina Draconis.The inhibitory effect of the extract on amyloid fibrillation in vitro was investigated using thioflavin T and 8-anilinonapthalene-1-sulfonic acid spectrofluorometric analyses.Finally,the effect of the bio-active components on the structure of HEWL in the early stages of fibrillogenesis was determined by molecular docking simulation.The results are as follows:（1）In this study,a screening system in vitro for inhibitors of HEWL fibrosis was constructed.The induction conditions were as follows:HEWL（1 mg/m L）was dissolved in glycine-hydrochloride（50 m M,p H=2.0）solution and incubated at 57℃and 140 rpm for 7 days.（2）The ethanol extract of Resina Draconis（RDEE）was obtained by solvent extraction method,and it was proved that RDEE had a significant dose-dependent inhibitory effect on amyloid aggregation,and the half-inhibitory concentration(IC₅₀value)was 209 ng/m L.（3）To further refine the bio-active ingredients,TLC was utilized and obtained 14kinds of isolates with different polarities,especially Sample 11 had a significantly inhibitory effect(IC₅₀=4.23 ng/m L).In addition,MTT assay was used to detected that that sample 11 had the ability to improve the viability of PC12 cells treated by HEWL fibrils,and the fibrils concentration should be less than or equal to 5μM.（4）To explore the structural information of the bio-active ingredients,LC-Q-TOF/MS was used to identify the functional ingredients of Sample 11 against amyloid fibrillogenesis,and 10 kinds of compounds were analyzed.Finally,molecular docking analyzed the binding conformations of typical bio-active ingredients（pterostilbene and 7,4-dihydroxy-8-methyl flavonoid）and HEWL,which laid a foundation for the study of the mechanism of amyloid aggregation inhibition.