北虫草中6种核苷类成分的含量测定与质量控制

Determination of six nucleosides in Cordyceps militaris and its quality control

目的建立同时测定北虫草中尿苷、肌苷、鸟苷、腺苷、虫草素和N6-(2-羟乙基)腺苷含量的定量分析方法,建立北虫草中尿苷、腺苷、虫草素的定性分析方法,对北虫草进行质量控制研究。方法采用高效液相色谱法对北虫草中6种核苷类成分进行定量分析,Shiseido CAPCELL PAK MG C18色谱柱(4.6 mm×250 mm,5μm),以甲醇-水为流动相,梯度洗脱,检测波长260 nm,流速1.0 m L·min^(-1),柱温25℃。采用薄层色谱法对北虫草中3种核苷类成分进行定性分析,硅胶SGF_(254)薄层板,乙酸乙酯–丙酮–氨水体积分数为2.5∶6∶1.5作为展开剂,置于波长254 nm的紫外光灯下检视。结果尿苷、肌苷、鸟苷、腺苷、虫草素和N6-(2-羟乙基)腺苷的定量测定分别为1.223~156.66、3.914~125.25、1.959~125.38、1.223~156.66、1.225~156.75和3.906~125.00μg·m L^(-1),呈良好的线性关系,r≥0.999 6;平均加样回收率分别为98.31%、101.12%、100.72%、99.96%、100.29%和100.40%,RSD均小于3.0%,在20批北虫草样品的平均含量分别为0.043%、0.097%、0.022%、0.082%、0.986%和0.175%,不同来源北虫草样品中核苷类的含量差别较大。北虫草薄层色谱中尿苷、腺苷、虫草素的分离度良好,特征性强,可以用于北虫草的定性鉴别。结论以核苷类成分作为指标性成分所建立的定性定量分析方法简便,专属性强,重复性好,可实现对北虫草进行质量控制的目的。

AIM To establish a qualitative analyses method for determining nucleosides （uridine, inosine, guanosine, adenosine, cordycepin and N6- （2-hydroxyethyl）- adenosine） simultaneously, and establish a quantitative analyses method for determining uridine, adenosine and cordycepin in Cordyceps militaris to control its quality. METHODS A high performance liquid chromatography （HPLC） method was developed for simultaneous determination of six nucleosides on a Shiseido CAPCELL PAK C18 MG column （4.6 mm × 250 mm, 5 μm） with gradient elution of methanol and water as mobile phase at a flow rate of 1.0 mL· min-1. The column temperature was maintained at 25 % and the wave length was detected at 260 nm. A thinlayer chromatography （TLC） method was established by using ethyl acetate-acetone-ammonia volume fraction of （2.5 ： 6 ： 1.5） as a developing solvent on high performance silica gel precoated plate （SGF254）. Three nucleosides were taken as control and were identified under UV 254 nm. RESULTS Uridine, inosine, guanosine, adenosine, cordycepin and N6- （2-hydroxyethyl）-adenosine showed good linearity in ranges of 1. 223-156.66, 3.914-125.25, 1.959-125.38, 1.223-156.66, 1.225-156.75 and 3.906-125.00 μg·mL-1 with r ≥ 0.999 6, and the average recoveries were 98.31%, 101.12%, 100.72%, 99.96%, 100.29% and 100.40% with RSD 〈 3.0%, respectively. The contents of six nucleosides in 20 batches of Cordyceps militaris from different sources differed significantly with the average contents were 0.043%, 0.097%, 0.022%, 0.082%, 0.986% and 0.175%, respectlively. The uridine, adenosine and cordycepin had obviously characteristic with good separating in TLC which could be used to identify Cordyceps militaris. CONCLUSION The qualitative and quantitative analyses established by taking six characteristic nucleosides as the markers are simple, selective and reproducible for quality control of Cordyceps militaris.