金茵利胆胶囊的体外抗炎活性评价及其谱效关系_曹凡.pdf

海军军医大学学报2023 年 4 月第 44 卷第 4 期http:/Academic Journal of Naval Medical University,Apr.2023,Vol.44,No.4 418 论 著 收稿日期 2022-01-24 接受日期 2022-04-29基金项目 中药大品种品牌价值提升示范研究项目（202190025），国家现代农业产业技术体系资助项目（CARS-21），咸阳市 2020 年度重大科技专项（2020K01-20），国家自然科学基金青年科学基金（81904047），陕西省千人计划区域青年人才项目（2018）Supported by Demonstration Research Project on Brand Value Enhancement of Large Chinese Medicine Varieties(202190025),Project of National Modern Agricultural Industrial Technology System(CARS-21),2020 Major Science and Technology Project of Xianyang(2020K01-20),National Natural Science Foundation of China for Young Scholars(81904047),and Regional Youth Talent Project of“Thousand Talents”Program of Shaanxi Province(2018).作者简介 曹 凡，硕士生 E-mail:*通信作者（Corresponding authors）.Tel:029-38182205,E-mail:;Tel:029-38185060,E-mail:金茵利胆胶囊的体外抗炎活性评价及其谱效关系曹 凡1，宋忠兴2，陈 琳2*，唐志书2,3*，倪 健4，张德柱51.陕西中医药大学药学院，西安 7120462.陕西省中药资源产业化协同创新中心，秦药特色资源研究开发国家重点实验室（培育），陕西省创新药物研究中心，陕西中医药大学，咸阳 7120833.中国中医科学院中药资源中心，北京 1007004.北京中医药大学北京中医药研究院，北京 1000295.陕西盘龙药业集团股份有限公司，西安 710025 摘要 目的 评价金茵利胆胶囊的体外抗炎活性，并研究其抗炎谱效关系。方法 建立脂多糖（LPS）诱导的小鼠单核巨噬细胞（RAW264.7 细胞）炎症模型，检测细胞培养上清液中一氧化氮（NO）、TNF-和IL-6 等的含量以评价金茵利胆胶囊的抗炎活性。采用均匀设计法制备金茵利胆胶囊组成饮片的不同配伍样本，采用HPLC建立其指纹图谱，并进行抗炎活性测定；以细胞培养上清液中NO、TNF-和IL-6 的抑制率为药效指标，与共有峰峰面积经灰色关联分析构建抗炎谱效关系。结果 金茵利胆胶囊提取物（0.251.00 mg/mL）能抑制LPS诱导的RAW264.7 细胞分泌NO、TNF-和IL-6。经对照品比对指认出其中 7 个色谱峰，分别为新绿原酸、绿原酸、隐绿原酸、1,3-二咖啡酰奎宁酸、对羟基苯乙酮、柚皮苷、新橙皮苷。抗炎谱效关系分析结果表明，23 个共有峰均具有一定的抗炎贡献度，其中峰 21（柚皮苷）、峰 23（新橙皮苷）与 3 个细胞炎症指标（NO、TNF-、IL-6）的关联度均0.74。结论 金茵利胆胶囊发挥抗炎作用是多种成分共同作用的结果。本研究结果可为金茵利胆胶囊抗炎药效物质基础及其质量控制提升提供参考。关键词 金茵利胆胶囊；单核巨噬细胞；抗炎活性；均匀设计；谱效关系中图分类号 R 286.5文献标志码 A文章编号 2097-1338(2023)04-0418-09Evaluation for in vitro anti-inflammatory activity of Jinyin Lidan capsule and its spectrum-effect relation CAO Fan1,SONG Zhong-xing2,CHEN Lin2*,TANG Zhi-shu2,3*,NI Jian4,ZHANG De-zhu51.College of Pharmacy,Shaanxi University of Chinese Medicine,Xian 712046,Shaanxi,China2.Shaanxi Collaborative Innovation Center of Chinese Medicine Resources Industrialization,State Key Laboratory of Research and Development of Characteristic Qin Medicine Resources(Cultivation),Shaanxi Innovative Drug Research Center,Shaanxi University of Chinese Medicine,Xianyang 712083,Shaanxi,China3.Chinese Medicine Resource Center,China Academy of Chinese Medical Sciences,Beijing 100700,China4.Beijing Academy of Chinese Medicine,Beijing University of Chinese Medicine,Beijing 100029,China5.Shaanxi Panlong Pharmaceutical Group Co.Ltd.,Xian 710025,Shaanxi,China Abstract Objective To evaluate the in vitro anti-inflammatory activity of Jinyin Lidan capsule and study its anti-inflammatory spectrum-effect relation.Methods The lipopolysaccharide(LPS)-induced inflammation model of mouse mononuclear macrophages(RAW264.7 cells)was established,and the contents of nitric oxide(NO),tumor necrosis factor (TNF-)and interleukin 6(IL-6)in the supernatant of RAW264.7 cells were determined to evaluate the anti-inflammatory activity of Jinyin Lidan capsule.Different compatible samples of Jinyin Lidan capsules were prepared by uniform distribution design method,their fingerprints were established by high-performance liquid chromatography(HPLC),and their anti-inflammatory activity was determined.The inhibitory rates of NO,TNF-and IL-6 in the supernatant were used as pharmacodynamic indexes,and the correlation between the anti-inflammatory spectrum and the common peak area was established by grey correlation DOI：10.16781/j.CN31-2187/R.20220091 419 金茵利胆复方制剂金茵利胆胶囊由茵陈、金钱草、郁金和枳壳 4 种中药饮片加工制成，适用于肝郁气滞及肝胆湿热引起的胁痛、胃痛、食少纳呆等。金茵利胆复方制剂在临床上对非酒精性脂肪 肝1、新生儿高胆红素血症2、慢性胆囊炎3、急性梗阻性化脓性胆管炎4等疾病有一定的探索和用药经验。文献中也有对金茵利胆复方制剂的质量控制方法5-8，但并不能全面反映金茵利胆胶囊的质量，其抗炎物质基础的研究更是鲜有报道。谱效关系的研究能够较好地表征中药化学成分与药效的关联，是快速筛选活性化合物的一种研究方法。本实验通过建立脂多糖（lipopolysaccharide，LPS）诱导的RAW264.7 细胞炎症模型，以金茵利胆胶囊为基础筛选出适宜的工作浓度，并建立以金茵利胆胶囊组成药材饮片为基础的均匀设计样品HPLC指纹图谱，考察其体外抗炎活性，采用灰色关联分析方法研究其抗炎谱效关系，探讨金茵利胆胶囊发挥抗炎作用的物质基础，为金茵利胆胶囊全面的质量控制提供科学依据。1 材料和方法1.1 药品、细胞与试剂 金茵利胆胶囊（批号 20201003，陕西盘龙药业集团股份有限公司）；小鼠单核巨噬细胞 RAW264.7（中国科学院上海细胞库）；FBS（货号2120140）、RPMI 1640培养基（货号 2049224）、PBS（货号UB150259）均购于以色列 Biological Industries公司；LPS（货号 L6529,美国 Sigma 公司）；MTT（货号 922N053，上海碧云天生物技术有限公司）；DMSO（货号 HG/T5345-2018,天津市科密欧化学试剂有限公司）；无水对氨基苯磺酸（sulfanilic acid anhydrous，SUL；货号 DC5BA0731）、N-（1-萘）乙二胺盐酸盐analysis.Results The Jinyin Lidan capsule extract(0.25-1.00 mg/mL)reduced the secretion of NO,TNF-and IL-6 in LPS-induced inflammatory RAW264.7 cells.Seven chromatographic peaks were identified by reference substance comparison,and they were neochlorogenic acid,chlorogenic acid,cryptochlorogenic acid,1,3-dicaffeoylquinic acid,p-hydroxyacetophenone,naringin,and neohesperidin.The analysis results of anti-inflammatory spectrum-effect relation showed that 23 common peaks had a certain degree of anti-inflammatory contribution,and the correlation degree of peak 21(naringin)and peak 23(neohesperidin)with the 3 cellular inflammatory indicators(NO,TNF-,and IL-6)was greater than 0.74.Conclusion The anti-inflammatory effect of Jinyin Lidan capsule is the result of the joint action of various components,and the res