Journal of Chemical Ecology

, Volume 45, Issue 1, pp 50–60 | Cite as

Toxicity of Milkweed Leaves and Latex: Chromatographic Quantification Versus Biological Activity of Cardenolides in 16 Asclepias Species

  • Tobias ZüstEmail author
  • Georg Petschenka
  • Amy P. Hastings
  • Anurag A. Agrawal


Cardenolides are classically studied steroidal defenses in chemical ecology and plant-herbivore coevolution. Although milkweed plants (Asclepias spp.) produce up to 200 structurally different cardenolides, all compounds seemingly share the same well-characterized mode of action, inhibition of the ubiquitous Na+/K+ ATPase in animal cells. Over their evolutionary radiation, milkweeds show a quantitative decline of cardenolide production and diversity. This reduction is contrary to coevolutionary predictions and could represent a cost-saving strategy, i.e. production of fewer but more toxic cardenolides. Here we test this hypothesis by tandem cardenolide quantification using HPLC (UV absorption of the unsaturated lactone) and a pharmacological assay (in vitro inhibition of a sensitive Na+/K+ ATPase) in a comparative study of 16 species of Asclepias. We contrast cardenolide concentrations in leaf tissue to the subset of cardenolides present in exuding latex. Results from the two quantification methods were strongly correlated, but the enzymatic assay revealed that milkweed cardenolide mixtures often cause stronger inhibition than equal amounts of a non-milkweed reference cardenolide, ouabain. Cardenolide concentrations in latex and leaves were positively correlated across species, yet latex caused 27% stronger enzyme inhibition than equimolar amounts of leaf cardenolides. Using a novel multiple regression approach, we found three highly potent cardenolides (identified as calactin, calotropin, and voruscharin) to be primarily responsible for the increased pharmacological activity of milkweed cardenolide mixtures. However, contrary to an expected trade-off between concentration and toxicity, later-diverging milkweeds had the lowest amounts of these potent cardenolides, perhaps indicating an evolutionary response to milkweed’s diverse community of specialist cardenolide-sequestering insect herbivores.


Cardiac glycoside Coevolution Macroevolutionary escalation Mode of action Monarch butterfly Na+/K+ ATPase Phylogenetic chemical ecology Plant-insect interactions Structure-activity relationships Target site insensitivity 



We thank Eamonn Patrick for technical support and performing parts of the experiment, Katalin Böröczky, Steve Broyles, Ron White, Hongxing Xu, Navid Movahed, and Georg Jander for help with the purification and HRMS analysis of labriformin, uscharin, and voruscharin, Ivan Keresztes for performing the NMR analysis of labriformin, and members of the Phytophagy Laboratory at Cornell University ( for discussion. This work was supported by German Research Foundation grant PE 2059/1-1 to GP, Swiss National Science Foundation grants P300P3-151191 and PZ00P3-161472 to TZ, and NSF-DEB-1118783 and a Templeton Foundation grant to AAA.

Supplementary material

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© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Institute of Plant SciencesUniversity of BernBernSwitzerland
  2. 2.Institut für InsektenbiotechnologieJustus-Liebig-Universität GiessenGiessenGermany
  3. 3.Department of Ecology and Evolutionary BiologyCornell UniversityIthacaUSA
  4. 4.Department of EntomologyCornell UniversityIthacaUSA

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