β-ecdysterone accumulation and regulation in Ajuga lobata suspension culture.
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摘要: 植物源蜕皮激素——β-蜕皮甾酮(β-EC)是一种细胞生长调节激素,在医疗和生物农药等方面有极高的应用价值。利用匍枝筋骨草细胞中含有较高β-EC这一特性,为从改善细胞悬浮培养、监测及加入添加物等途径找出一个可以高效、快速生产β-EC的生物学方法,通过紫外分光光度法和高效液相色谱等研究方法在前期已建立的匍枝筋骨草细胞悬浮培养体系的基础上,进一步研究了细胞培养过程中的营养消耗、次生代谢产物生成的动力学曲线;并探索了向细胞悬浮培养体系中添加L-苯丙氨酸(L-PAL)、甲羟戊酸(MVA)、α-蒎烯、松油醇、NO(硝普钠SNP)等来提高匍枝筋骨草细胞中β-EC含量。结果表明:匍枝筋骨草细胞生长符合逻辑斯蒂方程曲线“S”型,生长周期共19 d并在第11天进入细胞生长稳定期;电导率与细胞干质量、蜕皮甾酮的积累呈负相关,因而,可利用电导率的变化确定最佳细胞收获时间;多次继代会弱化细胞生产蜕皮甾酮的能力;通过将α-蒎烯、MVA、SNP联合添加于弱化的细胞中,可以显著提高细胞悬浮培养体系中β-EC的含量,联合添加的适宜组合和最优浓度为:α-蒎烯50 μL/L、MVA 10 mg/L、SNP 80 μmol/L。Abstract: Ecdysterone is a naturally occurring steroid hormone secreted by arthropods, including insects, to regulate the molting process of larvae. Ecdysterone also has many pharmacological functions such as promoting cell growth and inducing human epidermal cell differentiation.Suspension culture of Ajuga lobata D. Don cells provides a method of synthesis of the phytoecdysteroid β-ecdysterone (β-EC). In this study, we tried to characterize the culture conditions to optimize β-EC production. Growth of A. lobata cells fit the logistic equation curve, with a growth cycle of 19 days and the stationary phase of 11 to 17 days. Medium conductivity was negatively correlated with dry cell weight and β-EC accumulation, thus could be used to determine the optimal time for cell harvest. Continuous subculture reduced β-EC synthesis, but supplementing medium with β-EC precursors mevalonic (MVA), metabolic inhibitors α-pinene and elicitor NO could significantly promote cell growth and influence β-EC accumulation. Combination of α-pinene, MVA and SNP significantly elevated β-EC accumulation, thus may synergistically enhance β-EC synthesis in A. lobata. The optimal concentrations of α-pinene, MVA and NO donor SNP in the suspension culture were 50 μL/L, 10 mg/L, 80 μmol/L, respectively.
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Key words:
- Ajuga lobata /
- kinetics /
- precursors /
- elicitors /
- metabolic inhibitor /
- β-ecdysterone /
- metabolic regulation
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