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Methods for the isolation and purification of ethanol-insoluble, phenolic esters in mint Majak, Walter


Previous kinetic, isotopic studies have suggested that "insoluble" phenolic esters are the direct precursors of lignin. Heretofore, the "insoluble" esters have been detected by the chromatographic examination of gross hydrolysis products of ethanol-insoluble residues and/or acetone powders. In this study, new, definitive methods were developed for the isolation and purification of the ethanol-insoluble, phenolic esters of Mentha arvensis. "Insoluble" conjugates of caffeic (compound 1), ferulic (compound 2) and para-coumaric (compound 3) acids were purified and were shown to be electrophoretically and chromatographically homogeneous. A multiplicity of "insoluble" pools of caffeic acid was detected in Mentha. Two pools of "insoluble" caffeic acid were distinguished on the basis of their anionic mobility at pH 1.9. An "insoluble" macromolecular caffeyl conjugate was partially purified. Structural investigations indicated that the caffeic acid moiety of compound 1 was covalently associated with amino acids. Radioactive sulfur was incorporated into compounds 1, 2 and 3 and the evidence suggested that the ³⁵S resided in an anionic, acidic group. Ultra-violet spectroscopy demonstrated that the phenolic moieties of compounds 1, 2 and 3 were linked via their respective carboxyl groups. Time-course tracer studies were performed, for the first time, on a purified, "insoluble" pool of caffeic, ferulxc and para-coumaric acid. When ¹⁴CO₂ and ¹⁴C-phenylalanine were administered, a decline in the specific activity of compounds 1,2 and 3 was indicated in two out of three experiments. The specific activity of the caffeoyl moiety of compound 1 followed a parallel course in each experiment. Compound 2, the ferulic acid-containing conjugate, consistently possessed the highest specific activity as compared to compounds 1 and 3. The specific activities of various pools of caffeic acid were compared at the six-hour metabolic period. The specific activity of "soluble" caffeic acid was 2-3 times greater than the specific activity of the caffeoyl moiety of compound 1. The caffeic acid moiety of the high-molecular-weight conjugate was not radioactive. The author suggests that compound 2 is an "active, insoluble ester" which may be directly involved in lignification.

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