雷公藤的免疫抑制活性及毒性的谱效关系研究

Spectrum-effect relationship of immunosuppressive effects and toxicity of Tripterygium wilfordii HooK F.

目的：通过谱效分析探讨雷公藤药材提取物高效液相色谱图中免疫抑制活性及肾毒性相关色谱峰,为确定雷公藤免疫抑制活性及毒性物质基础提供依据。方法：对8个产地雷公藤药材分别进行提取纯化,采用高效液相色谱法（HPLC）建立各产地雷公藤提取物的指纹图谱。利用密度梯度离心法分离新鲜人外周血单个核细胞（PBMC）,给予凝集素（PHA）和脂多糖（LPS）共刺激造模,分为空白组、模型组（PHA＋LPS）、雷公藤处理组。给予雷公藤处理48 h后检测上清中IFN-γ和IL-2的含量,并计算IC50值。以小鼠肾小球系膜细胞（GNM-SV40）为模型利用CCK8法考察雷公藤的肾毒性,计算IC50值。将各产地雷公藤的指纹图谱信息及免疫抑制药效、细胞毒性数据进行标准化处理后利用偏最小二乘法进行谱效关系分析。结果：免疫抑制药效试验中,雷公藤提取物可显著抑制PHA和LPS诱导PBMC免疫细胞因子IFN-γ和IL-2的分泌（P〈0.05）,不同产地雷公藤免疫抑制药效差异较大,其中以福建、湖南和云南产雷公藤药效最强。体外肾毒性研究结果显示,福建、湖南和云南产雷公藤毒性较大,但其IC50值要远远高于免疫抑制IC50值（90倍以上）。根据基于偏最小二乘法的谱效分析结果显示,以IFN-γIC50值为毒性指标时,1,8,5,3,2号峰为主要贡献峰（回归系数绝对值〉0.3）;当以IL-2 IC50值为毒性指标时,1,8,3,5号峰为主要贡献峰（回归系数绝对值〉0.3）,16~29号峰为次要贡献峰（回归系数绝对值〈0.3）;当以体外肾毒性GNM-SV40细胞IC50值为毒性指标时,1,8,3号峰为主要贡献峰（回归系数绝对值〉0.3）,15~29号峰为次要贡献峰（回归系数绝对值〈0.3）。结论：1,3,8号峰既是主要的药效峰又是主要的毒性峰,2和5号峰为主要药效无毒峰,15~29号峰对药效和毒性都有一定的贡献,但都较小。

OBJECTIVE To investigate relationship between marker peaks in HPLC and immunosuppressive effect and relationship between marker peaks in HPLC and toxicity of extracts of Tripterygium wilfordii HooK F.（TWHF）,then provide evidences for confirming immunosuppressive and toxic components in TWHF.METHODS Eight batches of TWHF were extracted and purified,and then HPLC was used to construct fingerprint chromatography.Human peripheral blood mononuclear cells（PBMC）were separated from fresh whole blood by density gradient centrifugation method,and stimulated by phytoagglutinins（PHA）and lipopolysaccharide（LPS）,and then divided into three groups：control group,model group and test group.Contents of IFN-γand IL-2 were measured by ELISA after treatment for 48 h then IC50 values were calculated.CCK8 was used to evaluate toxicity of TWHF on GNM-SV40 cells then IC50 values were calculated.Fingerprint chromatography and cell experiment data were normalized,spectrum-effect relationship was investigated by partial least squares（PLS）.RESULTS According to result of immunosuppressive experiment,levels of IFN-γand IL-2 were significantly lowered by extract of TWHF（P〈0.05）.Potency of TWHF from different origins differed greatly,and TWHF from Fujian,Hunan and Yunnan were the best.Toxicities of TWHF from Fujian,Hunan and Yunnan were also stronger.Based on results of PLS,among 29 marker peaks,peak 1,8,5,3 and 2 contributed greatly to decrease of IFN-γ（absolute value of coefficient〉0.3）;peak 1,8,5 and3 contributed greatly to decrease of IL-2（absolute value of coefficient〉0.3）;peak1,8 and3 contributed greatly to toxicity on GNM-SV40 cells（absolute value of coefficient〉0.3）.CONCLUSION Immunosuppressive activity and toxicity of TWHF have the closest correlation with peak1,3 and 8in HPLC fingerprints.Peak2 and 5 are main effective peaks without toxicity.Peak 15-29 have certain effects on immunosuppressive activity and toxicity,but not significant.