基于斑马鱼多模态评价杜仲配...豆素成分的代谢及毒_效影响_高一波.pdf

中草药 中草药 2023 年年 3 月月 第第 54 卷卷 第第 5 期期 Chinese Traditional and Herbal Drugs 2023 March Vol.54 No.5 1449 药理与临床 药理与临床 基于斑马鱼多模态评价杜仲配伍补骨脂对其香豆素成分的代谢及毒/效影响 高一波1,2,3，李佳妍1,3,4，石子琪1,2,3，宁 青1,2,3*，韦英杰1,2,3*1.南京中医药大学附属中西医结合医院，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.南京中医药大学第三临床医学院，江苏 南京 210028 4.烟台毓璜顶医院 药学部，山东 烟台 264000 摘 要：目的 用斑马鱼多模态评价杜仲配伍补骨脂对其香豆素糖苷的代谢及毒/效影响。方法 用受精后 16 d（day after fertilization，dpf）的斑马鱼评价杜仲提取物（lignan extract of Eucommiae Cortex，LEEC）配伍补骨脂香豆素提取物（coumarin extract of Psoraleae Fructus，CEPF）以及代表成分松脂醇二葡萄糖苷（pinoresinol diglucoside，PDG）配伍补骨脂苷（psoralenoside，PSS）和异补骨脂苷（isopsoralenoside，IPSS）的安全性，观察斑马鱼胚胎发育情况，包括胚胎致畸、致死情况，并计算斑马鱼半数死亡浓度（half lethal concentration，LC50）；用 16 dpf 斑马鱼暴露于上述配伍组合物，分析 PSS 和/或 IPSS 及其代谢物补骨脂素（psoralen，PS）和/或异补骨脂素（isopsoralen，IPS）的动态变化；用 25 mol/L 泼尼松龙诱导斑马鱼骨质疏松模型，采用茜素红对培养至 8 dpf 的各给药组斑马鱼幼鱼骨骼染色，进行显微观察、数码成像，并用图像软件定量分析骨骼染色区域来评价上述配伍组合抗骨质疏松活性。结果 LEEC 配伍 CEPF 及 PDG 分别配伍 PSS 和 IPSS，可改善斑马鱼心包、卵黄囊等形态畸变，减少鱼死亡率，提高鱼致畸或致死浓度；能明显减慢 PSS 和 IPSS 脱糖基转化为潜在肝损伤代谢产物 PS 和 IPS 的速率，减慢毒性代谢物的产生是杜仲配伍补骨脂减毒的重要因素；LEEC 配伍 CEPF 以及 PDG分别配伍 PSS 和 IPSS 可显著增加斑马鱼头骨累积光密度值，提示具一定增效作用。结论 采用斑马鱼多模态高效评价了杜仲配伍对 CEPF 及补骨脂香豆素代表成分的代谢与毒/效作用的影响，为杜仲配伍补骨脂的减毒、增效作用研究提供高效的方法与思路。关键词：斑马鱼；补骨脂；杜仲；毒性；代谢；骨质疏松；补骨脂素；异补骨脂素；补骨脂苷；异补骨脂苷；松脂醇二葡萄糖苷 中图分类号：R285.5 文献标志码：A 文章编号：0253-2670(2023)05-1449-12 DOI:10.7501/j.issn.0253-2670.2023.05.011 Evaluation of effects of Eucommiae Cortex combined with Psoraleae Fructus on metabolism and toxicity/efficacy of coumarin components based on zebrafish multimodal analysis GAO Yi-bo1,2,3,LI Jia-yan1,3,4,SHI Zi-qi1,2,3,NING Qing1,2,3,WEI Ying-jie1,2,3 1.Affiliated Hospital of Integrated Traditional Chinese and Western Medicine,Nanjing University of Chinese Medicine,Nanjing 210028,China 2.Key Laboratory of New Drug Delivery Systems of Chinese Materia Medica,Jiangsu Province Academy of Traditional Chinese Medicine,Nanjing 210028,China 3.The Third Clinical Medical College,Nanjing University of Chinese Medicine,Nanjing 210028,China 4.Department of Pharmacy,Yantai Yuhuangding Hospital,Yantai 264000,China 收稿日期：2022-11-03 基金项目：国家“重大新药创制”科技重大专项（2017ZX09301056）；国家中医药管理局 2022 年中央财政转移支付地方项目（2022GJJZDYJS-01）；江苏省中医药科技发展计划项目（ZD202225）；江苏省中医药科技发展计划项目（YB2020022）；江苏省中医药科技发展计划项目（QN202214）；江苏省医学创新团队项目（CXTDB2017003）作者简介：高一波，男，硕士研究生，研究方向为中药学。Tel:13482439556 E-mail:*通信作者：宁 青，女，助理研究员，研究方向为中药物质基础及中药药理学。E-mail: 韦英杰，女，研究员，博士生导师，研究方向为中药物质基础与质量控制。Tel:(025)52362109 E-mail: 1450 中草药 中草药 2023 年年 3 月月 第第 54 卷卷 第第 5 期期 Chinese Traditional and Herbal Drugs 2023 March Vol.54 No.5 Abstract:Objective To evaluate the effects of Duzhong(Eucommiae Cortex)combined with Buguzhi(Psoraleae Fructus)on its metabolism and toxicity/efficacy of coumarin components with zebrafish multimodal analysis.Methods Zebrafish 16 d after fertilization(16 dpf)was used to evaluate the safety of lignan extract of Eucommiae Cortex(LEEC)combined with coumarin extract of Psoraleae Fructus(CEPF)and the representative components of pinoresinol diglucoside(PDG)combined with psoralenoside(PSS)and isopsoralenoside(IPSS),zebrafish embryonic development including embryonic teratogenicity and lethality were observed,and half death concentration(LC50)of zebrafish was calculated;Zebrafish were exposed to the above compatible compositions at 16 dpf to analyze the dynamic changes of PSS and/or IPSS and their metabolites psoralen(PS)and/or isopsoralen(IPS);The zebrafish osteoporosis model was induced with 25 mol/L prednisolone,microscopic observation and digital imaging of zebrafish larvae of each treatment group cultured to 8 dpf were performed using alizarin red,and the bone staining area was quantitatively analyzed by image software to evaluate the anti-osteoporosis activity of the above compatibility combinations.Results LEEC combined with CEPF and PDG combined with PSS and IPSS could alleviate zebrafish morphology distortion of heart,yolk sac,etc.and reduce fish mortality,and increase teratogenic or lethal concentrations of fish,significantly slowed down the conversion rate of PSS and IPSS to potential liver injury metabolites PS and IPS,and slowed down the production of toxic metabolites was an important factor for the attenuating effect of Eucommiae Cortex combined with Psoraleae Fructus;LEEC combined with CEPF and PDG combined with PSS and IPSS could increase the cumulative optical density of zebrafish skull,suggesting a certain synergistic effect.Conclusion The effects of Eucommiae Cortex compatibility on metabolism and toxicity/efficacy of CEPF and its representative components were high efficiently evaluated by zebrafish multimodal analysis,so as to provide efficient methods and ideas for indicating the attenuating and synergistic effects of Eucommiae Cortex compatibility with Psoraleae Fructus.Key words:zebrafish;Psoraleae Fructus;Eucommiae Cortex;toxicity;metabolism;osteoporosis;psoralen;isopsoralen;psoralenoside;isopsoralenoside;pinoresinol diglucoside 补骨脂为豆科植物补骨脂 Psoralea corylifolia L.的干燥成熟果实，温肾助阳、纳气平喘、温脾止泻，用于治疗肾阳不足、阳痿遗精、遗尿尿频、腰膝冷痛、肾虚作喘、五更泄泻；外用消风祛斑，治疗白癜风、斑秃1。补骨脂始载于雷公炮炙论，临床应用