基于斑马鱼毒/代同步进程法分析巴戟天配伍淫羊藿减毒作用  

Analysis of Folium Epimedium toxicity in combination with Radix Morindae Officinalis based on zebrafish toxicity/metabolism synchronization

作　　者：凌洁 王茉 陈颖[1,2] 宋捷[1,2] 孙娥[1,2] 石子琪 封亮[1,2] 贾晓斌[1,2] 韦英杰[1,2] 

机构地区：[1]南京中医药大学第三临床医学院,江苏南京210028 [2]江苏省中医药研究院,国家中医药管理局中药释药系统重点研究室,江苏南京210028 [3]解放军93318部队医院,辽宁开原112300

出　　处：《药学学报》2018年第1期74-83,共10页Acta Pharmaceutica Sinica

基　　金：国家自然科学基金资助项目(81573833,81603500,81503253); “重大新药创制”科技重大专项(2017ZX 09301-056,2017ZX 09301-051); 2015年度中医药行业科研专项(201507004-10); 江苏省自然科学基金资助项目(BK20141507); 江苏省六大人才高峰资助项目(2013-YY006); 2014年度江苏省第四期“333工程”科研资助项目(BRA2014348)

摘　　要：基于体内转化的毒性研究至关重要,但难以实时动态观察,本文提出斑马鱼毒/代(毒性/代谢)同步进程法,并用于揭示淫羊藿中毒及其配伍巴戟天后的减毒作用。将受精后1天(1 dpf)的健康斑马鱼胚胎置于不同浓度淫羊藿水煎液(YYH)、淫羊藿黄酮组分(YTF)、代表黄酮成分(朝藿定C和淫羊藿苷)及分别配伍巴戟天水煎液(BJT)的供试液中,同时设置空白溶媒(0.4%DMSO)对照组,观察记录给药后1~5天(2~6 dpf)的鱼中毒和死亡情况,显微镜观察3 dpf斑马幼鱼形态,用SPSS16.0计算不同供试液在2~6 dpf的鱼半数致死浓度(LC50);同时,分别选取各样品的一个相对安全浓度,每天同步取药液分析淫羊藿代表黄酮成分动态代谢变化。结果表明:淫羊藿中主要黄酮成分朝藿定A/B/C和淫羊藿苷经斑马鱼作用后动态转化为以箭藿苷C和宝藿苷I为主的代谢物,其中宝藿苷I主要来自朝藿定A/B和淫羊藿苷,原形和代谢物均不会致鱼中毒;而箭藿苷C主要来自朝藿定C,箭藿苷C的动态增加与YYH、YTF及朝藿定C的毒性产生密切相关;配伍BJT后,淫羊藿主要黄酮成分(如朝藿定C)代谢速度减慢,伴随产生的毒性也减缓。斑马鱼毒/代同步进程法揭示淫羊藿中朝藿定C代谢转化为箭藿苷C后增加毒性,是淫羊藿潜在致毒关键黄酮成分,巴戟天通过减缓朝藿定C代谢速度而减毒。研究为中药毒性物质发现及配伍减毒提供新思路与方法。Metabolic transformation in vivo is a critical approach in the study of toxicity, but real-time dynamic observation of the transformation is difficult. We proposed that zebrafish toxicity/metabolism synchronization may be used in the analysis of toxicity of Folium Epimedium（Yinyanghuo for Chinese, YYH） and the toxicity may be reduced by Radix Morindae Officinalis（Bajitian for Chinese, BJT）. Healthy zebrafish embryos 1 day post fertilization（1 dpf） were exposed to different concentrations of YYH, total flavonoids of YYH（YTF）, representative flavonoids（epimedin C and icariin） and their respective in combination with BJT. Death numbers of the embryos or larvals were counted during 1-5 days after dosing（2-6 dpf）; embryonic micro-morphology of zebrafish（3 dpf） was observed and pictures were taken. The blank vehicle（0.4% DMSO） was used in the control group, and LC50 value of 2 to 6 dpf was calculated by SPSS16.0. A relative safe concentration was sampled every day to analyze the dynamic metabolites changes of major flavonoids of YYH. The results showed that epimedin A/B/C（EA/EB/EC） and icariin, the major flavonoids of YYH, were dynamically transformed into major metabolites of sagittatoside C（SC） and baohuoside I（BI） by zebrafish. BI was mainly derived from EA, EB and icariin. Neither original form nor their metabolite BI can cause zebrafish poisoning. SC was mainly derived from EC, and its accumulation was closely related to the toxicity of YYH, YTF and EC. After combination with BJT, the metabolism of EC was slowed down and the toxicity was alleviated. Zebrafish toxicity/metabolism synchronization revealed that the toxicity of EC of YYH was increased after metabolism into SC, which maybe the key potential poisonous factor of YYH, and BJT can reduce the toxicity by slowing down the metabolism rate of EC. The data provides new ideas and methods in the study of toxic substances in Chinese medicine and mechanism of detoxicity by combination.

关 键 词：斑马鱼 淫羊藿 巴戟天 毒性 代谢 

分 类 号：R965.2[医药卫生—药理学]