沙冬青种子总黄酮活性成分分析及对小鼠免疫功能的影响

本研究旨在通过高效液相色谱-质谱联用（LC-MS）和高效液相色谱（HPLC）技术对沙冬青种子总黄酮主要活性成分进行分析及含量测定，并探索沙冬青种子总黄酮对小鼠免疫功能的影响。采用LC-MS分析沙冬青种子总黄酮主要活性成分，在HPLC优化条件下用标准品对主要活性成分的含量进行测定；采用MTT法和ELISA试剂盒检测沙冬青种子总黄酮对小鼠脾淋巴细胞增殖和IL-2、IL-4生成的影响，以及总黄酮对肝脏KCs细胞的增殖和NO、NOS含量的影响；运用定量溶血分光光度法和血清溶血素HC₅₀测定法检测不同浓度沙冬青种子总黄酮对小鼠抗体生成细胞和血清溶血素的影响。结果显示，沙冬青种子总黄酮中主要活性成分为芒柄花素、7，3'-二羟基-4'-甲氧基黄酮、黄豆黄素和穗花杉双黄酮，其中，芒柄花素含量高达52.48%，其次为7，3'-二羟基-4'-甲氧基黄酮，含量为11.20%。在2.5~100 μg/mL总黄酮作用下，沙冬青种子总黄酮对脾淋巴细胞的增殖和细胞因子的产生具有明显的促进作用，尤其在20 μg/mL时脾淋巴细胞增殖率和IL-2的分泌量分别高达217.62%和159.661 pg/mL，与空白对照组相比差异极显著（P<0.01）；在40 μg/mL时，脾淋巴细胞IL-4的分泌量高达149.274 pg/mL，极显著高于空白对照组（P<0.01）。沙冬青种子总黄酮对KCs细胞的增殖及NO和NOS分泌也具有明显促进作用，在60 μg/mL时，与空白对照组相比，KCs细胞增殖率达67.77%，NO和NOS的分泌量分别达180.106和29.942 μmol/L（P<0.01）。此外，沙冬青种子总黄酮能明显促进小鼠体内抗体生成细胞和血清溶血素含量的增加。综上表明，沙冬青种子总黄酮主要活性成分为芒柄花素和7，3'-二羟基-4'-甲氧基黄酮。沙冬青种子总黄酮对小鼠免疫活性细胞的增殖、相关免疫细胞因子和抗体的产生与释放等都具有显著的促进作用。

Analysis of Active Components of Total Flavonoids from the Seeds of Ammopiptanthus mongolicus and Its Effects on Immune Function in Mice

This study was aimed to analyze and determine the main active components of total flavonoids in the seeds of Ammopiptanthus mongolicus by high performance liquid chromatography-mass spectrometry (LC-MS) and high performance liquid chromatography (HPLC),and explore the effects of total flavonoids in the seeds of Ammopiptanthus mongolicus on immune function in mice.The main active components in total flavonoids of the seeds of Ammopiptanthus mongolicus was analyzed by LC-MS,and the content of the main active components was determined by standard substance under the optimized conditions of HPLC.The effects of proliferation of splenic lymphocytes and KCs were detected by MTT method,the contents of IL-2,IL-4,NO and NOS were measured by ELISA Kit,the antibody-producing cells and hemolysin in mice serum were explored by quantitative hemolytic spectrophotometry and HC₅₀ determination.The results showed that the main compounds of total flavonoids from the seeds of Ammopiptanthus mongolicus were manshank,7,3'-dihydroxy-4'-methoxyflavone,glycitein and amentoflavone,among them,the content of manshank was 52.48%,and the content of 7,3'-dihydroxy-4'-methoxyflavone was 11.20%.The total flavonoids from the seeds of Ammopiptanthus mongolicus had obvious promoting effect on the proliferation of splenic lymphocytes and the production of cytokines,especially at 20 μg/mL total flavonoids,the proliferation rate of splenic lymphocytes and the secretion of IL-2 reached 217.62% and 159.661 pg/mL,respectively,which were extremely significantly higher than that of blank control group (P<0.01);At 40 μg/mL total flavonoids,the secretion of IL-4 in splenic lymphocytes reached 149.274 pg/mL,which was extremely significantly higher than that of blank control group (P<0.01).The total flavonoids from the seeds of Ammopiptanthus mongolicus also significantly promoted the proliferation and NO and NOS secretion of KCs cells.At 60 μg/mL total flavonoids,compared with blank control group,the proliferation rate of KCs cells was 67.77%,the secretion of NO and NOS reached 180.106 and 29.942 μmol/L (P<0.01),respectively.In addition,the total flavonoids could increase the contents of antibody-producing cells and hemolysin in mice serum.The results suggested that the main compounds of total flavonoids from the seeds of Ammopiptanthus mongolicus were manshank and 7,3'-dihydroxy-4'-methoxyflavone.The total flavonoids from the seeds of Ammopiptanthus mongolicus could obviously promote the proliferation of immunocompetent cells,the production and release of related immune cytokines and antibody in mice.