不同培养因素对蝉花培养物中麦角甾醇的影响

Effect of different culture factors on ergosterol content in culture of Cordyceps cicadae

目的建立HPLC法对不同培养阶段及不同培养基培养的蝉花培养物中麦角甾醇水平进行测定。方法建立麦角甾醇HPLC检测方法,应用Agilent Eclipse XDB-C18色谱柱(4.6 mm×250 mm,5μm);流动相为100%甲醇,等度洗脱;体积流量1.0 m L/min;柱温35℃;进样量10μL;检测波长281 nm;外标法测定麦角甾醇水平。对液体、固体培养基不同培养阶段,假单胞菌选择培养基(PSA)、沙氏葡萄糖琼脂培养基(SDAY,1 000 m L中含葡萄糖量分别为20、30、40 g),以及包括江苏农垦、烟农24、烟农15和济麦22在内的固体培养基培养的蝉花培养物中麦角甾醇的水平进行测定,利用SPSS16.0统计软件对测定结果进行多重统计分析,验证并找出引起差异的原因。结果建立的HPLC法,麦角甾醇分离度良好,精密度、稳定性、重复性的RSD分别为0.30%、0.07%、1.37%,加样回收率为104.3%,实验重现性、耐用性良好,均符合要求,可用于蝉花培养物中麦角甾醇的检测;液-固不同培养阶段麦角甾醇生长曲线的表现特征不同;不同培养基培养的蝉花培养物中麦角甾醇水平差异显著,斜面培养基中,随着葡萄糖量的增加,麦角甾醇水平增加;固体培养基济麦22和烟农15的麦角甾醇的水平显著高于烟农24和江苏农垦。结论液-固培育阶段麦角甾醇的生长曲线特征不同,且对于培养基具有一定的选择性,为人工蝉花培养工艺的确定及优化提供了依据,也为针对性的药物开发奠定基础。

Objective The measurement method was established to determine the content of ergosterol in culture of Cordyceps cicadae, and the content of ergosterol was measured in cicada fruiting bodies for different culture medium and different stages. Methods The HPLC method was applied to determine the content of ergosterol in C. cicadae. Chromatography was carried out on an Agilent Eclipse XDB-C18 column （4.6 mm ×μ250 mm, 5 μm） using isocratic elution program at the volume flow rate of 1.0 mL/min. Mobile phase was 100% methanol, column temperature was 35 ℃, the sample weight was 10 μL and detection wave length was 281 nm. External standard method was used to calculate the content of ergosterol. The content of ergosterol was measured in C. cicadae fruiting bodies for different culture medium including different slope medium such as Pseudomonas Selection Medium （PSA） and Sabouraud＇s Glucose Agar Medium （SDAY） containing 20, 30, or 40 g glucose sugar in 1000 mL medium; The different solid medium were Jiangsu state farms, Yannong 24, Yarmong 15, and Jimai 22 and different stages in liquid and solid medium. The SPSS 16.0 statistical software was applied to verify and find out the reason causing the difference. Results The separation degree of ergosterol was good, RSD of precision, stability, and repetition was 0.30%, 0.07%, and 1.37% respectly, sample recovery rate （104.3%）, reproducibility, and durability of HPLC method have been verified to meet the requirements. It could be used for the detection of ergosterol in C. cicadae. The growth curves of ergosterol in different stages of liquid-solid cultures had different Characteristics. Theergosterol level was significantly different in different cultures. The ergosterol levels increased with the increase of amount of glucose in slant culture medium. The ergosterol levels in solid culture medium of Jimai 22 and Yarmong 15 were significantly higher than those of Yarmong 24 and Jiangsu Agribusiness. Conclusion It has been confirmed that ergosterol has selectivity for the medium. The growth curves of ergosterol in different stages of liquid-solid cultures have different characteristics. This experiment is conducive to optimization of the process of C. cicadae.