HPLC法检测蛇足石杉内生真菌胶胞炭疽发酵液中石杉碱甲和石杉碱乙的含量

Detection of Huperzine A and Huperzine B in fermentation broth of endophytic fungus Colletotrichum gloesporioides from Huperzia serrate by HPLC

摘要:

建立了高效液相色谱 (HPLC) 测定蛇足石杉 (Huperzia serrate) 内生真菌胶胞炭疽发酵液中石杉碱甲 (Huperzina A) 和石杉碱乙 (Huperzine B) 含量的方法，并以此方法检测胶胞炭疽发酵液中石杉碱甲和石杉碱乙含量的积累。内生真菌发酵液经氯仿萃取、甲醇溶解、过滤后进行高效液相检测分析，选用Agilent Eclipse plus-C18 色谱柱 (250 mm×4.6 mm, 5 μm)，以0.015 mol/L乙酸铵 (pH 6.8) 和甲醇溶液 (70∶30) 为流动相进行等度洗脱，流速1 mL/min，检测波长为308 nm，连续检测内生真菌胶胞炭疽发酵液中第6–15天石杉碱甲和石杉碱乙的含量积累。结果表明，发酵提取液中的石杉碱甲和石杉碱乙可在25 min内进行很好的分离和分析，石杉碱甲在1.50?48.00 μg/mL范围内线性关系良好 (相关系数r为0.999 5)，石杉碱乙在0.25?7.50 μg/mL范围内线性关系良好 (相关系数r为0.999 7)，石杉碱甲和石杉碱乙的平均加标回收率分别为106.83%、108.06%，相对标准偏差 (RSD) 分别为3.34%、3.60%。该方法简便、快速、精密度高、结果准确，适用于内生真菌发酵液中石杉碱甲和石杉碱乙含量检测。在发酵过程中，内生真菌发酵液中石杉碱甲和石杉碱乙的含量呈现先增后减，随后有所增加继而又减少的趋势。石杉碱甲和石杉碱乙的含量分别在内生真菌发酵第14天、第8天达到最高，分别为12.417 0 μg/mL、4.660 3 μg/mL。该方法学的建立为内生真菌胶胞炭疽合成石杉碱甲与石杉碱乙的机制研究提供了检测手段，从而有利于药物新资源的开发。

关键词:高效液相色谱，次生代谢产物，千层塔，内生真菌，石杉碱甲，石杉碱乙

Abstract:

In this study, we established a rapid and efficient HPLC method to determine the accumulation of Huperzine A and Huperzine B in the fermentation broth of endophytic fungus Colletotrichum gloesporioides from Huperzia serrate. The chloroform extracts of fermentation broth were dissolved in methanol and filtered before injection for HPLC analysis. The analysis was performed on an Agilent Eclipse plus-C18 column (250 mm×4.6 mm, 5 μm) by isocratic elution. The mobile phase was 0.015?mol/L ammonium acetate-methanol (70:30, V/V), the flow rate was 1 mL/min and the detection wavelength was set at 308 nm. Huperzine A and Huperzine B could be well separated within 25 min. Good linearity of Huperzine A was found in the range of 1.50?48.00 μg/mL (r=0.999 5), and that of huperzine B was in 0.25?7.50 μg/mL (r=0.999 7). The average recoveries of Huperzine A and Huperzine B were 106.83% and 108.06%, respectively (RSD=3.34%, 3.60%). The results demonstrate that this method can detect the content of huperzine A and huperzine B in fermentation broth simply, rapidly, accurately and in good reproducibility. Under the optimized conditions, the accumulated content of huperzine A and huperzine B were measured from the sixth to the fifteenth day. Huperzine A and Huperzine B reached the highest (12.417 0 μg/mL and 4.660 3 μg/mL, respectively) at the fourteenth and eighth days. The analysis methodology could contribute to the future study of huperzine A and huperzine B biosynthesis in C. gloeosporioides, consequently facilitate the development of new drug resources.

Key words:HPLC, secondary metabolites, Huperzia serrate, endophytic fungi, Huperzine A, Huperzine B