Quantification of plant cardenolides by HPLC, measurement of Na+/K+-ATPase inhibition activity, and characterization of target enzymes

Abstract

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.

Keywords: Cardenolide; Cardiac glycoside; Digitoxin; Enzyme assay; HPLC; Inhibition; Insect; Na(+)/K(+)-ATPase; Ouabain; Spectrophotometric; Target site insensitivity.