基于药效权重评价的杜仲叶-刺五加复方“湿法超微提取-膜澄清耦合”工艺筛选研究  

Study on process screening for Eucommiae Folium-Acanthopanacis Senticosi Radix et Rhizoma seu Caulis via evaluation of medicinal efficacy weighting using wet ultra-micro pulverization extraction and membrane clarification

作　　者：龙利娜 钟文蔚 周丹 郭立玮[3,4,5] 聂红 LONG Li-na;ZHONG Wen-wei;ZHOU Dan;GUO Li-wei;NIE Hong(Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research,College of Pharmacy,Jinan University,Guangzhou 510632,China;International Cooperative Laboratory of Traditional Chinese Medicine Modernization and Innovative Drug Development of Chinese Ministry of Education(MOE),College of Pharmacy,Jinan University,Guangzhou 510632,China;Guangzhou Bio-Green Biotechnology Co.,Ltd.,Guangzhou 511455,China;National Engineering Research Center of Pharmaceutical Processing Technology of Traditional Chinese Medicine and Drug Innovation,Guangzhou 510240,China;Guangzhou Dayuan Studio of Membrane Science and Technology for Traditional Chinese Medicine,Guangzhou 510091,China)

机构地区：[1]暨南大学药学院中药药效成分及新药研究广东省重点实验室,广东广州510632 [2]中国教育部中药现代化与创新药物开发国际合作实验室,广东广州510632 [3]广州百奥格林生物科技有限公司,广东广州511455 [4]中药制药过程技术与新药创制国家工程研究中心,广东广州510240 [5]广州达原中药膜科研技术工作室,广东广州510091

出　　处：《中草药》2022年第7期2053-2063,共11页Chinese Traditional and Herbal Drugs

基　　金：国家自然科学基金资助项目(81861138042)。

摘　　要：目的基于对湿法超微粉碎提取与膜澄清耦合工艺的研究,用细胞存活率及细胞上清液中炎症因子水平筛选提取工艺,并以杜仲叶-刺五加提取液的免疫、抗炎效应对所筛选的工艺进行验证。方法根据杜仲叶-刺五加提取液的体外抗炎实验结果,筛选杜仲叶与刺五加的比例、提取方法(传统热回流提取工艺和湿法超微粉碎提取)、湿法超微粉碎提取时间和膜澄清耦合的影响等提取条件,用MTT法测定细胞存活率,并分析杜仲叶与刺五加提取物对脂多糖(lipopolysaccharide,LPS)诱导小鼠单核巨噬细胞RAW264.7增殖和抗炎活性的影响。结果以免疫调节和抗炎药理效应筛选的杜仲叶-刺五加提取的2种工艺产物表明,湿法超微粉碎提取工艺优于传统热回流方法提取,该工艺下的杜仲叶-刺五加提取液对LPS诱导RAW264.7细胞炎症具有良好的抑制作用,可降低炎症细胞上清液中一氧化氮(nitric oxide,NO)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-1β(interleukin-1β,IL-1β)、IL-6水平,能降低M1型巨噬细胞标志物诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)表达,增加M2型巨噬细胞标志物Arginase表达,增加M1型和M2型巨噬细胞的吞噬作用。结论通过湿法超微粉碎提取与膜澄清耦合制备的杜仲叶-刺五加提取液可以调节炎症过程中的相关细胞因子,从而产生积极的免疫调节和抗炎作用。在体外抗炎实验的药效权重评价基础上,验证了湿法超微粉碎提取与膜澄清耦合可以对杜仲叶-刺五加进行有效提取,为中药新药研发中绿色提取技术应用方面提供了新思路。Objective To screen the extraction process by cell viability and expression of inflammatory substances in cell culture fluid based on the research of wet ultra-micro pulverization extraction(WUPE)coupled with membrane clarification,and the extracts of Duzhongye(Eucommiae Folium,EF)and Ciwujia(Acanthopanacis Senticosi Radix et Rhizoma seu Caulis,AS)under the best process were investigated for immune and anti-inflammatory effects.Methods Based on the results of cellular pharmacological experiments of extracts,the extraction conditions concerning the drug ratio of EF and AS,the choice of extraction method(comparing the extracts obtained by traditional hot reflux extraction process with those obtained by wet ultra-fine micronization extraction process),the effect of extraction time of wet ultra-fine micronization and clarification coupling by microfiltration were screened;Cell viability was determined by MTT method;Effects of EF and AS extracts on proliferation and anti-inflammatory activity of RAW264.7 cells induced by lipopolysaccharide(LPS)were investigated.Results The products of two processes of EF-AS extract screened for immunomodulatory and anti-inflammatory pharmacological effects showed that wet ultra-micronized extraction process was superior to the traditional hot reflux method of extraction,EF-AS extract had a good inhibitory effect on inflammation of RAW264.7 cells induced by LPS,decreased the levels of nitric oxide(NO),tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β)and IL-6 in the supernatant of inflammatory cells,decreased M1-like cells marker inducible nitric oxide synthase(iNOS)expression,increased M2-like cells marker Arginase expression,increased the phagocytosis of M1-and M2-like cells.Conclusion EF-AS extract prepared by wet ultra-fine pulverization extraction and membrane clarification could modulate cytokines in the inflammatory process,resulting in positive immunomodulatory and anti-inflammatory effects.Based on the evaluation of the pharmacological efficacy weights of the in vitro ph

关 键 词：杜仲叶 刺五加 湿法超微粉碎提取 微滤膜澄清 抗炎 药效权重评价 

分 类 号：R285.5[医药卫生—中药学]