蛹虫草及其培养基中主要核苷类成分的分析比较

Comparative Analysis of Nucleotides between Fruiting Body of Cordyceps militaris (L.ex Fr.) Link and Cultured Medium

采用高效液相色谱法测定虫草中的核苷类成分,优化后的色谱条件为YMC-Polyamine柱(250mm×4.6mm,5μm);采用梯度洗脱,流动相:乙腈-水(V/V):0～15min为90:10,15～20min为86.5:13.5,20～30min为75:25,30～35min为70:30;流速:1mL/min;柱温:30℃;检测波长:259nm;进样量:10μL。结果表明:胸腺嘧啶、虫草素、尿嘧啶、腺苷、腺嘌呤、尿苷、鸟嘌呤、次黄嘌呤均能得到较好分离,该方法稳定性好、精密度高、重现性好,适用于虫草中的核苷类成分的分析。经分析发现,蛹虫草子实体核苷类物质组成大致相似,但含量差异非常显著,同时发现蛹虫草培养基残基及固体发酵产物中虫草素含量较高,其他核苷类成分很少,因此认定蛹虫草培养基残基及固体发酵产物是非常优良的分离纯化虫草素的原料。

A high performance liquid chromatographic （HPLC） method was developed for the determination of nucleotides.Meanwhile,a comparative analysis of nucleotides between fruiting body of Cordyceps militaris （L.ex Fr.） Link and cultured medium was conducted by the developed method.YMC-Polyamine column （5 μm,250 mm × 4.6 mm） was used for the chromatographic separation.A mobile phase composed of acetonitrile and double distilled water at a flow rate of 1 mL/min was used for gradient elution and the elution program was as follows：acetonitrile/water （90：10,V/V） for 15 min → （86.5：13.5） for 20 min→（75：25） for 30 min → （70：30） for 35 min.Other chromatographic conditions were set as follows：column temperature 30 ℃,detection wavelength 259 nm,and injection volume 10 μL.Results showed that a good separation of thymine,cordycepin,uracil,adenosine,adenine,uridine,guanine and hypoxanthine was achieved.This method was stable,precise and reproducible and was successfully applied to the determination of nucleosides in commercial Cordyceps militaris （L.ex Fr.） Link samples.HPLC analysis revealed that a basic similarity of nucleotide kind was observed in various commercial samples of fruiting body of Cordyceps militaris （L.ex Fr.） Link.But the contents of nucleotides exhibited significant differences.A high content of cordycepin was found in cultured medium and solid fermentation products,but other nucleotides had a low content.These results demonstrate the excellence of cultured medium and solid fermentation products as a source of cordycepin.