Petschenka Georg, Züst Tobias, Hastings Amy P, Agrawal Anurag A, Jander Georg
Institute of Phytomedicine, University of Hohenheim, Stuttgart, Germany.
Department of Systematic and Evolutionary Botany, University of Zürich, Zürich, Switzerland.
Methods Enzymol. 2023;680:275-302. doi: 10.1016/bs.mie.2022.08.003. Epub 2022 Sep 7.
The biosynthesis of cardiac glycosides, broadly classified as cardenolides and bufadienolides, has evolved repeatedly among flowering plants. Individual species can produce dozens or even hundreds of structurally distinct cardiac glycosides. Although all cardiac glycosides exhibit biological activity by inhibiting the function of the essential Na/K-ATPase in animal cells, they differ in their level of inhibitory activity. For within- and between-species comparisons of cardiac glycosides to address ecological and evolutionary questions, it is necessary to not only quantify their relative abundance, but also their effectiveness in inhibiting the activity of different animal Na/K-ATPases. Here we describe protocols for characterizing the amount and toxicity of cardenolides from plant samples and the degree of insect Na/K-ATPase tolerance to inhibition: (1) an HPLC-based assay to quantify the abundance of individual cardenolides in plant extracts, (2) an assay to quantify inhibition of Na/K-ATPase activity by plant extracts, and (3) extraction of insect Na/K-ATPases for inhibition assays.
强心苷的生物合成大致可分为甲型强心苷和乙型强心苷，在开花植物中已多次进化。个别物种可产生数十种甚至数百种结构不同的强心苷。尽管所有强心苷都通过抑制动物细胞中必需的钠钾ATP酶的功能而表现出生物活性，但它们的抑制活性水平有所不同。为了在种内和种间比较强心苷以解决生态和进化问题，不仅有必要量化它们的相对丰度，而且要量化它们抑制不同动物钠钾ATP酶活性的有效性。在这里，我们描述了用于表征植物样品中甲型强心苷的含量和毒性以及昆虫钠钾ATP酶对抑制的耐受程度的实验方案：（1）基于高效液相色谱的分析方法，用于量化植物提取物中单个甲型强心苷的丰度；（2）一种用于量化植物提取物对钠钾ATP酶活性抑制作用的分析方法；（3）提取昆虫钠钾ATP酶用于抑制分析。
