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 pharmacological experimental results, the effective extraction of EF-AS based on coupling of wet ultra-fine pulverization extraction and membrane clarification was verified, which can provide a green manufacturing extraction technology and new ideas for the future development of new Chinese medicines.

R285.5

国家自然科学基金资助项目(81861138042)