薄层色谱-反相高效液相色谱法分离纯化蟾酥中的内源性钠泵抑制因子

 采用薄层色谱法分离中药蟾酥中内源性哇巴因样物质 ,以三氯甲烷 甲醇 水 (体积比为 75∶2 0∶5 )溶液为展开剂 ,将所获得的活性物用反相高效液相色谱法纯化并经 2 2 0nm及 30 0nm检测后 ,以人红细胞86Rb摄取率试验测定其生物活性 ,得到了匀质的、紫外吸收波长为 2 5 0nm、可明显抑制钠泵活性的一种新的哇巴因样物质。实验结果为薄层色谱 高效液相色谱法用于内源性、低相对分子质量的生物样品的分离及纯化提供了依据。

Separation and Purification of an Endogenous Inhibitor ofSodium Pump from Chansu by Thin-Layer Chromatography andReversed-Phase High Performance Liquid Chromatography

An endogenous inhibitor of the sodium pump from the Chinese medication Chansu was purified. The dry substance Chansu was extracted with methanol. The dry residue dissolved in water and filtered subsequently through membrane filters with the exclusion size of 1000 Da, 3000 Da and 10000 Da in a Filtron Pro Vario-3-System and applied to thin-layer chromatographic plate made of Silica gel 60 F254 + 366 developed with a mixture of CHCl3-MeOH-H2O(75:20:5, volume ratio). The fractions with Rf 0.55 inhibiting the sodium pump were purified on an HPLC C18-RP column using a linear H2O-methanol gradient with 220 nm and 300 nm DAD detection. The bioactivity was measured by 86Rb-uptake into human red blood cells. The results showed that a low molecular weight, water soluble compound, which inhibited the sodium pump activity in the red blood cells and had a maximum absorbance at 250 nm was isolated from the Chinese medication Chansu. Several mg of the compound in pure state could be obtained from 1 kg Chansu. It was different from ouabain and proscillaridin A in chemical structure, because ouabain and proscillaridin A show a UV maximum absorption at 220 nm and 300 nm, while the new inhibitor at 250 nm.