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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 the immunosuppressive activity and nephrotoxic related chromatographic peaks in the HPLC chromatogram of Tripterygium wilfordii by spectral analysis, and provide the basis for determining the immunosuppressive activity and toxicity of Tripterygium wilfordii. Methods: Eight medicinal materials of Tripterygium wilfordii were extracted and purified respectively, and the fingerprint of Tripterygium wilfordii extract was established by high performance liquid chromatography (HPLC). Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation and stimulated by PHA and lipopolysaccharide (LPS). They were divided into blank group, model group (PHA + LPS), tripterygium wilfordii group. The contents of IFN-γ and IL-2 in the supernatant were detected 48 hours after treatment with Tripterygium wilfordii, and the IC50 values were calculated. Nephrotoxicity of Tripterygium wilfordii was evaluated by CCK8 assay using mouse glomerular mesangial cells (GNM-SV40) as a model, and IC50 values were calculated. The fingerprint of Tripterygium wilfordii was identified and the immunosuppressive efficacy and cytotoxicity data were normalized and analyzed by partial least square method. Results: In the immunosuppressive efficacy test, Tripterygium wilfordii extract could significantly inhibit the secretion of IFN-γ and IL-2 of PBMC induced by PHA and LPS (P <0.05), and the immunosuppressive efficacy of Tripterygium wilfordii was significantly different Among them, Tripterygium wilfordii was the strongest in Fujian, Hunan and Yunnan. In vitro nephrotoxicity results showed that Tripterygium wilfordii was more virulent in Fujian, Hunan and Yunnan, but its IC50 was much higher than that of immunosuppressive IC50 (90 folds). According to spectrum analysis based on partial least squares, the peaks of 1,8,5,3,2 were the major contributors (the absolute value of regression coefficient> 0.3) when the IFN-γIC50 value was taken as toxicity index. When IL -2 was the main contribution peak (the absolute value of regression coefficient was> 0.3) when the IC50 value was the toxicity index, and the peak with the peak value of 16-29 was the secondary contribution peak (the absolute value of regression coefficient was <0.3). When the IC50 value of in vitro nephrotoxic GNM-SV40 cells was used as a toxic index, peaks 1,8 and 3 were the major contributors (absolute value of regression coefficient> 0.3), and peaks 15 and 29 were secondary contributors (absolute regression coefficient was absolute Value <0.3). Conclusion: Peaks 1, 3, and 8 are the main pharmacodynamic peaks and the main toxicity peaks. Peaks 2 and 5 are the main drug-free non-toxic peaks. Peaks 15-29 have a certain contribution to pharmacodynamics and toxicity , But smaller.