Effects of storage and extraction procedures on yields of lipophilic metabolites from the brown seaweeds Dictyota ciliolata and D. menstrualis

Abstract

Investigations focused on the ecological roles of marine secondary metabolites have become common, but marine ecologists have rarely assessed how methodologies used in sample preparation affect the extractability and stability of secondary metabolites and, thus, measurements of intraspecific and interspecific compound variance. We assessed various procedures for storing, drying, and extracting samples of 2 chemically defended brown seaweeds Dictyota ciliolata and D. menstrualis. These plants contain the diterpenoid alcohols pachydictyol A, dictyol B acetate, and dictyol E that are relatively stable under all test conditions. In contrast, the related diterpenoid dialdehyde, dictyodial, decomposed when plant tissues or crude extracts were stored at -25°C for 13 to 27 wk or when tissues or extracts were freeze-dried or subjected to high vacuum (<0.01 torr), methods that are commonly used in studies of marine chemical ecology. The stability of dictyodial was species-specific, degrading more in D. ciliolata than in D. menstrualis. During a few extractions, dictyodial reacted with methanol (MeOH) to yield an artifact resulting from the addition of 2 molecules of MeOH per molecule of dictyodial. A mixture of 2:1 dichloromethane (DCM) and MeOH tended to extract the lipophilic secondary metabolites better than MeOH or DCM alone. Metabolites were also afforded some protection against degradation when fresh tissue was submerged in 2:1 DCM:MeOH during storage at -25°C. Results of this investigation indicate that storage, extraction, and quantification methods need to be optimized for analyses of individual compounds and that even identical compounds can behave differently when they occur in different species.