| Objective:Glabridin,an isoflavonoid originally isolated from the roots of Glycyrrhiza glabra L.,has numerous biological activities,including whitening,anti-inflammatory,antioxidant,and phytoestrogenic activities.Glabridin has been widely used in cosmetic applications as whitening agents.In recent years,the state has paid increasing attention to the safety and efficacy of cosmetic.Multiple regulations and restrictions were introduced to require that cosmetic should have sufficient safety and stability,as well as a certain efficacy.As a plant extract,the quality of glabridin products is affected by unknown residual impurities resulting from the extraction,separation,and storage processes.However,current quality control methods cannot comprehensively detect unknown or unidentified impurities in glabridin products.Therefore,these approaches are not sufficiently accurate to predict the safety of the product for organisms.Thus,key questions about the application of glabridin still remain: 1)lack of criteria on biosafety evaluation;2)key impurity which affects the quality of products is unclear;3)toxicological mechanism is not elucidated.The aims of this thesis were to compare toxicities of commercially available glabridin products using a zebrafish model and to identify unknown impurities.Meanwhile,the teratogenic mechanism of glabridin and impurities during zebrafish embryonic development was preliminarily explored.Methods:1.In total,10 glabridin samples from different sources were quantified using high-performance liquid chromatography.Sample toxicities were evaluated using a zebrafish model and cell model.2.To identify the impurity,the samples with the different toxicity were analyzed by ultra-high-performance liquid chromatography coupled with quadrupole-Orbitrap mass spectrometry.The toxicity of related impurities was verified in the zebrafish model.3.Zebrafish embryos were treated with glabridin,the phenotypes were observed and recorded under fluorescence microscope.Whole-mount in situ hybridization and q PCR were used to assess early development-related genes m RNA expression.Phalloidin stains and transmission electron microscopy analysis were used to evaluate the subtle changes in the myofibril alignment.To explore the molecular mechanism of how glabridin affects muscle early development of zebrafish embryos,RNA-seq was carried out for the transcriptome analysis.Validation of indicated RNA-seq differential gene expression using q PCR.Results:1.Although glabridin content in the samples was similar,there were significant differences in biosafety.This phenomenon could not be totally reflected by HPLC.2.Higher contents of glabrone and glabrol were identified in the sample with the highest toxicity.Glabrol exacerbates glabridin toxicity in zebrafish embryos,whereas glabrone did not have any toxic effect.3.Glabridin causes shorter body length and inflammatory reactions in zebrafish embryos.In further studies,we found that glabridin exerted myotoxicity.Glabridin incubation induced a significant elevation in gene expression for myog,myofibril misalignment and ultrastructural anomalies in myocyte sarcomeres.RNA-Seq analysis indicates that glabridin activate distinct pathways that are relevant for the ER stress（atf6,perk,ern2,hspa5）,apoptosis（chop,atf3,junba,fosab,fosl2）and autophagy（lc3,p62,becn1）.Glabrol impurity has similar toxicological mechanism to that of glabridin,and exacerbates the toxic effect of glabridin by above mechanism.Conclusions:1.There are significant differences in biosafety of commercially available glabridin products.Therefore,further refinement of quality control system of glabridin products will be required.Zebrafish embryos is high sensibility to the biosafety of glabridin.The tendency from zebrafish model is consistent with the results of the cells model validation.Thus,it is feasible to use zebrafish model to build the biosafety evaluation for glabridin products.2.Glabrol was identified as a key impurity that increased glabridin toxicity.Therefore,it is necessary to add the requirement of glabrol content in the quality control system of glabridin products.3.Glabridin exerted myotoxicity in zebrafish embryos.Glabridin increased the expression of ER stress,apoptosis and autophagy pathway related genes,which ultimately affects muscle normal development. |
