Experimental designs characterizing seasonal variations and solvent effects on the quantities of coumarin and related metabolites from Mikania laevigata.

Statistical design mixtures of acetone, chloroform, dichloromethane and ethanol were used to study the effects of different solvents and their mixtures on the quantities of coumarin and related metabolites extracted from Mikania laevigata samples harvested in each of the four seasons. RP-HPLC-DAD and both positive and negative modes of UPLC-MS analyses were used to determine relative quantities of coumarin, o-coumaric acid and melilotic acids in each season for all the mixture design extracts. The existence and measurement of the relative abundances of melilotic acid in Mikania laevigata have not been reported previously. Highest coumarin concentrations were encountered in the summer whereas its o-coumaric acid precursor and melilotic acid were most abundant in the spring. O-coumaric and melilotic acids concentrations were strongly correlated during the year. Also solvent effects were seen to be significant. Ethanol and 1:1 binary mixtures of ethanol and acetone extracted the largest quantities of coumarin whereas ethanolic binary and ternary mixtures with chloroform and dichloromethane provided the best yields of o-coumaric and melilotic acids. Statistical mixture models indicated that synergic binary interactions, especially those involving ethanol with acetone or chloroform, are important in the Mikania extraction process.