山核桃叶总黄酮抑制TBBPA诱导斑马鱼胚胎发育毒性的研究

摘要

四溴双酚A (tetrabromobisphenol A,TBBPA)是新型高关注度污染物之一,对鱼类具有强毒性.山核桃叶中的黄酮类化合物具有抗氧化和抗细胞凋亡等作用.通过硅胶柱洗脱、薄层色谱(TLC)检测对山核桃叶进行提取和纯化,得到黄酮类化合物产物B;设置4个实验组(空白对照,0.5 mg/L TBBPA,1.0 mg/L产物B,0.5 mg/LTBBPA和1.0 mg/L产物B),记录斑马鱼胚胎发育过程中的形态学指标;同时,检测不同处理组中活性氧(reac-tive oxygen species,ROS)水平和凋亡相关基因的表达情况.结果发现,山核桃叶提取的总黄酮产物B中包含50％的查尔酮、27％的黄酮或者黄酮醇、18％的二氢黄酮或者二氢黄酮醇、5％的异黄酮等22种黄酮类化合物;富含黄酮类化合物的产物B对TBBPA诱导斑马鱼胚胎的卵凝结率、畸形率及32h血流障碍等指标具有显著降低作用;可显著抑制TBBPA诱导的ROS水平升高;对TBBPA诱导的凋亡基因p53上调和Bcl-2下调均有显著改善作用.实验结果表明山核桃叶总黄酮通过降低ROS水平,抑制凋亡基因的异常表达,发挥抗氧化和抑制凋亡作用.实验结果有助于了解山核桃叶有效成分总黄酮对TBBPA诱导的斑马鱼胚胎毒性的保护作用,为水生态系统和人体健康危害的防治提供新思路.%Tetrabromobisphenol A (TBBPA) is one of the highly concerned emerging contaminants.It has fierce toxicity to fish.Leaves of Carya cathayensis Sarg.have showed obvious antioxidant and anti-apoptosis effects as they contain large quantity of flavonoid compounds.Herein,total flavonoid compounds were separated and extracted from leaves of Carya cathayensis Sarg.through silica gel column elution and thin layer chromatography (TLC) detection.Four experimental groups (blank control,0.5 mg/L of TBBPA,1.0 mg/L of total flavonoid compounds,0.5 mg/L of TBBPA and 1.0 mg/L of total flavonoid compounds) were set up.During the development of zebrafish embryo under TBBPA exposure,indices of morphological toxicity were recorded,reactive oxygen species (ROS) production level and apoptotic gene expression were analyzed.Results indicated that 22 types of total flavonoid compounds were found,mainly including 50％ chalcone,27％flavonol,18％ flavanone and 5％ isoflavone.Morphological toxicity indices induced by TBBPA,such as spawn coagulation rate,malformation rate and blood flow disorders,were improved significantly after treatment with total flavonoid compounds.Meanwhile,the rising ROS level induced by TBBPA was significantly inhibited,and the up-regulated expression of p53 and down-regulated expression of Bcl-2 induced by TBBPA were also inhibited.Total flavonoid in leaves of Carya cathayensis Sarg.were found to have antioxidant and anti-apoptosis effects by decreasing the level of ROS and inhibiting abnormal expressions of apoptosis genes.The results contributed to the acquaintance of protective effects of total flavonoid,which are the active ingredients in leaves of Carya cathayensis Sarg.,against TBBPA-induced developmental toxicity in zebrafish embryo and larvae,providing a new idea for protecting aquatic ecosystems and human health.