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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 To establish an HPLC method for the determination of ergosterol in cicadas culture cultured in different culture stages and in different media. Methods The ergosterol HPLC method was established. The elution was carried out on an Agilent Eclipse XDB-C18 column (4.6 mm × 250 mm, 5 μm) with a mobile phase of 100% methanol and a volume flow rate of 1.0 m L / min. The column temperature was 35 ℃ ; Injection volume 10μL; detection wavelength 281 nm; external standard method for the determination of ergosterol levels. Pseudomonas selective medium (PSA), Sabouraud dextrose agar medium (SDAY, glucose content of 20, 30, 40 g in 1 000 mL, respectively) were used in different culture stages of liquid and solid medium, Jiangsu Academy of Agricultural Sciences, Jiangsu Nongken, tobacco farmers 24, tobacco farmers 15 and Jimai 22 cultivated cicadae culture cicadaera culture were measured by SPSS16.0 statistical software to determine the results of multiple statistical analysis to verify and identify The reason for the difference. Results The HPLC method was established. The separation of ergosterol was good. The RSD of precision, stability and repeatability were 0.30%, 0.07% and 1.37%, respectively. The recovery rate was 104.3%. The reproducibility of experiment and durability were good, Which could be used for the detection of ergosterol in the cicadas culture. The growth curves of ergosterol showed different characteristics in different stages of liquid-solid culture. There were significant differences in the ergosterol levels in the cicada flowers cultures cultured in different media, , The ergosterol level increased with the increase of the amount of glucose. The levels of ergosterol in solid medium Jimai 22 and Yannong 15 were significantly higher than those of Yannong 24 and Jiangsu Nongken. Conclusion The growth curve of ergosterol in liquid-solid incubation has different characteristics and has certain selectivity for the medium, which provides the basis for the determination and optimization of the culture process of Artificial cicadas. It also lays the foundation for targeted drug development.