高效液相色谱定量分析比较4种虫草药材的核苷类成分
[摘要] 为比较4种常见虫草类药材(冬虫夏草、蛹虫草、虫草菌丝体和蝉花)中核苷类成分的差异,该研究以0.5%磷酸溶液提取样本,色谱柱为Zorbax SB-Aq(4.6 mm×150 mm,5 μm),流动相为0.04 mol·L-1磷酸二氢钾溶液(A)和乙腈(B),梯度洗脱,流速为0.8 mL·min-1,检测波长260 nm,柱温30 ℃,对4类药材产品进行分析。已知核苷以外标一点法进行计算,未知成分以腺苷为对照进行计算,并对测定结果进行聚类分析,同时利用指纹图谱相似度软件对不同样本进行了比较。结果显示,冬虫夏草、蛹虫草、虫草菌丝体、蝉花4种药材中的核苷类成分差异较大,聚类分析和指纹图谱分析均可有效区分冬虫夏草和其他3种虫草药材。该研究建立的核苷测定方法准确可靠,可用于虫草类产品的品质评价。
[关键词] 虫草;高效液相;核苷;聚类分析;指纹图谱
[Abstract] To compare the main nucleosides in Cordyceps genus herbs (C. sinensis, C. millitaris, Hirsutella sinensis and C. sobolifera), an HPLC method for simultaneous determination of uridine, inosine, guanosine, adenosine and cordycepine in Cordyceps genus herbs was developed. The sample was extracted with 0.5% phosphoric acid solution to prepare test solution. The separation was performed on a Zorbax SB-Aq (4.6 mm×150 mm, 5 μm) column with gradient elution by 0.04 mol·L-1 potassium dihydrogen phosphate solution and acetonitrile, column temperature 30 ℃,flow rate 0.8 mL·min-1,and detection wavelength 260 nm. The content of nucleosides in four Cordyceps genus herbs was evaluated by fingerprint analysis and hierarchical cluster analysis (HCA). The calibration curves of five nucleosides showed good linear regression (r>0.99) and the average recoveries were between 95.0% and 105.0%. The contents of the five nucleosides in the four Cordyceps genus herbs were different and could be obviously distinguished by HCA. The fingerprint analysis result showed that the similarity between C. sinensis and the others was less than 0.9. The method was accurate and reliable, which can be used for quality control of Cordyceps genus herbs.
[Key words] Cordyceps;HPLC;nucleoside;hierarchical cluster analysis;fingerprint
doi:10.4268/cjcmm20161320
冬虫夏草Cordyceps sinensis为传统名贵中药材,具有免疫调节、抗肿瘤、抗衰老等功效[1-3]。由于其资源稀缺,无法满足广大消费者的需求,市场上出现了多种类似药材,其中蛹虫草C. militaris、虫草菌丝体Hirsutella sinensis和蝉花C. sobolifera为3种最为常见的虫草类药材[4-7]。冬虫夏草为冬虫夏草真菌寄生在蝙蝠蛾科昆虫幼虫上形成的子座和幼虫尸体的干燥复合体[8];蛹虫草是蛹草真菌以虫蛹为寄主或在人工培养基上培养形成的子实体[9];虫草菌丝体为中华被毛孢真菌通过液体深层培养技术生产的菌丝体[10];蝉花为蝉花真菌寄生于蝉类若虫形成的菌虫复合体[11]。为了比较虫草类产品的品质,本研究室前期发表了高效液相色谱质谱联用方法对冬虫夏草、蛹虫草和虫草菌丝体的定性分析研究,结果显示尿苷、肌苷、鸟苷、腺苷和虫草素为虫草类药材的主要核苷类成分[12-13]。本研究在前期研究基础上采用高效液相色谱方法定量分析了4种虫草类药材的核苷类成分,分别对15批冬虫夏草药材、7批蛹虫草药材、5批虫草菌丝体药材和4批蝉花药材中的主要核苷类成分进行了含量测定,并用聚类分析对这些药材的核苷含量数据进行了分类,运用相似度比较软件对核苷类指纹图谱进行了比较。
1 材料
Agilent1260高效液相色谱仪(在线脱气机,四元泵,自动进样器,柱温箱,DAD检测器);Agilent6530四级杆飞行时间质谱检测器(QTOF);XS205DR型天平(梅特勒-托利多仪器有限公司);KQ-500DB超声清洗仪(昆山市超声仪器有限公司)。