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[摘要]
目的 建立高效液相色谱(HPLC)法同时检测蝉花培养物4种核苷类成分,分析不同斜面培养基及固体培养基对4种核苷类成分的影响。方法 建立4种核酸类成分——尿苷、鸟苷、腺苷、N6-(2-羟乙基)腺苷的HPLC检测方法,应用Waters Symmetry shield TMRP C18(250 mm×4.6 mm,5 μm)色谱柱;流动相为水-甲醇,梯度洗脱;体积流量1.0 mL/min;柱温30℃;进样量10 μL;检测波长260 nm;外标法测定4种核苷类成分水平。对包括假单胞菌选择培养基(PSA)、沙氏葡萄糖琼脂培养基(SDAY,1 000 mL中含葡萄糖量分别为20、30、40 g)在内的斜面培养基和包括江苏农垦(SCM)、烟农24(YN-24)、烟农15(YN-15)、济麦22(JM-22)在内的固体培养基培养的蝉花培养物中4种核酸类成分水平进行测定,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,4种核苷类成分分离度良好,精密度、稳定性、重复性、加样回收率、耐用性均符合要求;4种不同的斜面培养基对尿苷成分基本无影响(P>0.05),PSA对腺苷、鸟苷、N6-(2-羟乙基)腺苷的水平有明显促进作用(P<0.05);固体培养基SCM的4种核苷类成分的水平较高。结论 培养基种类对蝉花培养物核苷类成分变化有直接的影响,确定蝉花虫草的生产工艺时,应充分考虑培养基的选择,同时结合产品成分和生产成本等因素进行综合评价。
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[Abstract]
Objective The HPLC method was established to simultaneously determine the contents of four kinds of nucleosides in culture of Cordyceps cicadae, and to analyze the effect of different culture medium on nucleoside component content. Methods The HPLC method was applied to determining the contents of four kinds of nucleosides such as uridine, guanosine, adenosine, and N6-(2-hydroxyethyl) adenosine in C. cicadae. Chromatography was carried out on a Waters Symmetry shield TMRP C18 column (250 mm×4.6 mm, 5 μm) using gradient elution program at the volume flow rate of 1.0 mL/min. Mobile phase was water-methanol, column temperature was 30℃, the sample weight was 10 μL and detection wave length was 260 nm. External standard method was used to calculate the content of nucleoside component. The contents of four kinds of nucleosides were measured in culture of C. cicadae 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 (SCM), Yannong 24 (YN-24), Yannong 15 (YN-15), and Jimai 22 (JM-22). The SPSS 16.0 statistical software was applied to verifying and find out the reason causing the difference. Results The separation degree of four kinds of nucleosides was good, and the precision, stability, repetition, sample recovery rate, and durability of HPLC method have been verified to meet the requirements. There is no effect of four different slant medium on the content of uridine. The guanosine, adenosine, and N6-(2-hydroxyethyl) adenosine levels in slant culture medium of PSA were higher than those of other slant culture medium. The levels of four kinds of nucleosides in solid culture medium of SCM were higher than those of others. Conclusion The media types have a direct impact on nucleosides in culture of C. cicadae. Therefore, to determine C. cicadae production process, should give full consideration to the choice of the medium, while the combination of product composition and production costs and other factors comprehensive evaluation.
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[基金项目]
药用虫生真菌中试及产业化平台的建立