祖师麻主要成分抗炎活性的体外研究

Study on anti-inflammatory activity of the main component of Girald Daphne Bark in vitro

目的 观察祖师麻主要成分的抗炎活性.方法 分离提取祖师麻化学成分川木香醇D、左旋松脂酚、瑞香新素、西瑞香素、黄瑞香苷A、瑞香素、黄瑞香苷B,采用CCK-8实验进行细胞毒性评价.将细胞按随机数字表法分为对照组、模型组和化合物组.对照组和模型组各加入50μl培养液,化合物组分别加入50.00、25.00、12.50、6.25、3.12μg/ml西瑞香素、黄瑞香苷A、黄瑞香苷B溶液,模型组和化合物组再加入4μg/ml脂多糖50μl,刺激24 h.采用Griess试剂法测定NO释放量,采用ELISA法检测TNF-α分泌量.结果 西瑞香素、黄瑞香苷A、黄瑞香苷B在50μg/ml下对小鼠巨噬细胞RAW264.7细胞具有较弱的细胞毒性,而其他化合物细胞毒性作用较明显.与模型组比较,12.50、25.00、50.00μg/ml黄瑞香苷B可抑制RAW264.7细胞NO[（271.86±20.92）%、（256.48±20.92）%、（199.31±15.16）%比（358.62±28.64）%]、TNF-α[（647.87±115.79）%、（618.42±87.52）%、（588.33±87.94）%比（1035.06±58.29）%]生成（P〈0.05或P〈0.01）;25.00、50.00μg/ml黄瑞香苷A可抑制RAW264.7细胞NO[（234.99±34.28）%、（167.36±25.76）%比（358.62±28.64）%]、TNF-α[（691.76±60.37）%、（534.01±41.60）%比（1035.06±58.29）%]生成（P〈0.05或P〈0.01）;12.50、25.00、50.00μg/ml西瑞香素可抑制RAW264.7细胞NO[（283.89±36.69）%、（243.08±48.19）%、（225.92±33.67）%比（358.62±28.64）%]、TNF-α[（713.77±121.96）%、（670.62±18.70）%、（599.62±68.62）%比（1035.06±58.29）%]生成（P〈0.05或P〈0.01）.结论 黄瑞香苷A、黄瑞香苷B和西瑞香素具有抗炎作用,可降低脂多糖诱导的RAW264.7细胞NO释放量和TNF-α分泌量,发挥抗炎作用.

Objective To investigate the anti-inflammatory activity of the main active ingredients in the dried stem bark of Daphne giraldii Nitsche.Methods Severialchemical compounds like vladinol D, pinoresinol, daphneticin, daphnoretin, daphnetin, giraloid A and giraldoid B were isolated from the stem barks. The CCK-8 experiemnts were analyzed for the cytotoxicity study. The cells were divided into the control group, the model group and the treatment group according to random number table method. The control group and the model group were added with 50μl culture medium. Moreover, treatment group was added with different concentrations （50.00, 25.00, 12.50, 6.25, 3.12μg/ml） of the solutions of giraloid A, giraldoid B and daphneticin. Then, RAW264.7 cells were treated with 50μl LPS （4μg/ml） for 24 h in the model group and treatment group. Griess reagent was used to determine the amount of NO release, and the secretion of TNF-α was detected by ELISA kit.Results Cytotoxicity test indicated that giraldoid A （50.00μg/ml）, giraldoid B （50.00μg/ml） and daphneticin （50.00μg/ml） showed noobvious cytotoxicity. Giraldoid B （12.50, 25.00, 50.00μg/ml） could inhibit the production of NO （271.86% ± 20.92%, 256.48% ± 20.92%, 199.31% ± 15.16%vs.358.62% ± 28.64%） and TNF-α （647.87% ±115.79%, 618.42% ± 87.52%, 588.33% ± 87.94%vs. 1035.06% ± 58.29%） in RAW264.7 induced by LPS compared with the model group. Giraldoid A （25, 50μg/ml） could inhibit the production of NO （234.99% ± 34.28%, 167.36% ± 25.76% vs.358.62%±28.64%） and TNF-α （691.76% ± 60.37%, 534.01% ± 41.60% vs. 1035.06% ± 58.29%） in RAW264.7 induced by LPS compared with the model group. Daphneticin （12.5, 25, 50μg/ml） could inhibit the production of NO （283.89% ± 36.69%, 243.08% ± 48.19%, 225.92% ± 33.67% vs.358.62% ± 28.64%） and TNF-α （713.77% ± 121.96%, 670.62% ± 18.70% vs. 1035.06% ± 58.29%） in RAW264.7 induced by LPS compared with the model group.Conclusions Giraldoid A, giraldoid B and daphneticin exhi bited anti-inflammatory effect through inhibiting the release of NO and the production of TNF-α in RAW264.7 induced by LPS.