蛹虫草活性成分的提取分离及体外抗氧化作用研究

目的 以抑制蛋白酪氨酸磷酸酶1B(PTP1B)活性为导向,筛选蛹虫草子实体有效部位,分离纯化单体化合物,并检测其体外抗氧化作用。方法 通过硅胶色谱柱,半制备高效液相等色谱技术对蛹虫草子实体进行分离纯化,检测各组分对PTP1B的抑制活性;应用核磁碳谱、氢谱数据分析鉴定单体化合物结构;利用MTT法检测单体化合物对PC12细胞的增殖作用,及其对过氧化氢(H₂O₂)损伤的PC12细胞存活率的影响,试剂盒检测单体化合物对细胞乳酸脱氢酶(LDH)、超氧化物歧化酶(SOD)活性及丙二醛(MDA)含量的影响。结果 发现5个对PTP1B具有较强抑制作用的活性成分,将其中活性最高的成分进一步分离纯化,获得单体化合物,经鉴定为β-D-吡喃葡萄糖基-9-甲基-4,8鞘氨醇(5),其对PTP1B具有较强的抑制活性,半抑制浓度(IC₅₀)为(3.42±0.59) μmol·L^-1。该单体化合物对H₂O₂诱导的PC12细胞氧化损伤具有明显的保护作用。结论 首次以抑制PTP1B活性为导向,在蛹虫草中分离得到脑苷脂类单体化合物,其具有抑制PTP1B作用和体外抗氧化活性,为蛹虫草用于糖尿病的预防和治疗奠定理论基础。

Extraction and Separation of Active Components from Cordyceps militaris and Antioxidant Effect In Vitro

OBJECTIVE To screen the active fraction of Cordyceps militaris fruiting body with the inhibition of PTP1B activity, isolate and purify the monomeric compounds, and detect their antioxidative activity in vitro. METHODS The traditional extraction and separation method was used to isolate and purify the fruit body of Cordyceps militaris through silica gel column chromatography and semi-preparative high performance liquid chromatography. The data were analyzed by nuclear magnetic carbon spectroscopy and hydrogen spectroscopy (by which spectrum analysis was written clearly). The inhibitory activity of each component on PTP1B were detected. The proliferation of PC12 cells and the effect on the survival rate of PC12 cells damaged by H₂O₂ was detected by MTT assay. The effects of monomer compounds on LDH, SOD and MDA contents of cells were detected. RESULTS Five active components were found to have strong inhibitory effect PTP1B, and the most active component was further isolated and purified to obtain the monomer compound, which identified as β-D-glycopyranosyl-9-methyl-4,8-sphingadienine (5). It has a strong inhibitory activity against PTP1B with an IC₅₀ of (3.42±0.59) μmol·L^-1. And the monomer compound has obvious protective effect on H₂O₂-induced oxidative damage of PC12 cells. CONCLUSION This monomeric compound is isolated from Cordyceps militaris for the first time. It has the inhibitory effect on PTP1B and antioxidative activity in vitro. It lays a theoretical foundation for the prevention and treatment of Cordyceps militaris in diabetes.