N‑Methylated Derivatives of Tyramine in Citrus Genus Plants: Identification of N,N,N‑Trimethyltyramine (Candicine) Luigi Servillo,* ,† Alfonso Giovane, † Nunzia D’Onofrio, † Rosario Casale, † Domenico Cautela, ‡ Giovanna Ferrari, § Maria Luisa Balestrieri, † and Domenico Castaldo §,∥ † Dipartimento di Biochimica, Biofisica e Patologia Generale, Seconda Universita ̀ degli Studi di Napoli, Via L. De Crecchio 7, 80138 Napoli, Italy ‡ Stazione Sperimentale per le Industrie delle Essenze e dei derivati dagli Agrumi, Azienda Speciale della Camera di Commercio di Reggio Calabria, Via Tommaso Campanella 12, 89127 Reggio Calabria, Italy § Dipartimento di Ingegneria Industriale e ProdAL scarl, Universita ̀ degli Studi di Salerno, Via Ponte Don Melillo 1, 84084 Fisciano, Salerno, Italy ∥ Ministero dello Sviluppo Economico, Via Molise 2, Roma, Italy ABSTRACT: The distribution of tyramine and its methylated derivatives, N-methyltyramine and N,N-dimethyltyramine, was investigated in tissue parts (leaves and fruits) of several plants of Citrus genus by liquid chromatography−electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). In the course of our study we discovered the occurrence of N,N,N- trimethyltyramine in all citrus plants examined. This quaternary ammonium compound, known to act in animals as a neurotoxin, was recognized and characterized by mass spectrometric analysis. The substance, never described before in the Citrus genus, is also known as candicine or maltoxin. Results indicate that N,N,N-trimethyltyramine is consistently expressed in leaves of clementine, bitter orange, and lemon. Conversely, low levels were found in the leaves of orange, mandarin, chinotto (Citrus myrtifolia), bergamot, citron, and pomelo. In the edible part of the fruits, N,N,N-trimethyltyramine was found at trace levels. KEYWORDS: N-methylated tyramine derivatives, N,N,N-trimethyltyramine, candicine, Citrus plants, biotic stress ■ INTRODUCTION In the course of evolution, vegetal organisms have developed the ability to synthesize a multitude of chemical compounds, some of which are nonessential for their normal growth and development. These compounds are produced with the aim of contributing to the plant’s defense from herbivores and pathogens. 1−3 Decarboxylation of aromatic amino acids, catalyzed by specific decarboxylases, is aimed at the biosyn- thesis of biogenic amines such as tryptamine, tyramine, and phenylethylamine. These compounds are then used as starting substrates for the production of more complex phytochem- icals, 4,5 which are toxic for the plant aggressors. 1,6−8 Despite considerable progress in understanding defense mechanisms against biotic stress, only little research on plant tyrosine decarboxylase (TYDC) has been reported. 9−12 TYDC belongs to the family of pyridoxal 5′-phosphate-dependent enzymes that decarboxylate tyrosine or L-dopa to form tyramine or dopamine, respectively. 13,14 Except for a study on a TYDC involved in synephrine biosynthesis in Citrus genus, 15 further studies on specific TYDC in Citrus genus plants have not yet been reported. Tyramine represents the starting substrate of at least two different and fundamental metabolic routes leading to the formation of protoalkaloids. Specifically, in the first metabolic route, tyrosine decarboxylation catalyzed by TYDC produces tyramine, from which octopamine is then derived through the hydroxylation reaction catalyzed by tyramine-β-hydroxylase. Successively, N-methylation of octop- amine forms N-methyloctopamine, commonly known as synephrine. In the second metabolic route, N-methyltyramine and N,N-dimethyltyramine (hordenine) are formed from tyramine by two consecutive methylation steps. Moreover, synephrine can be also produced through hydroxylation of N- methyltyramine. Octopamine and synephrine, both present in Citrus genus plants, are of pharmacological relevance. 16−21 Another substance, N, N,N-trimethyltyramine (Figure 1), known as candicine or maltoxin, was first discovered in barley malt rootlets (Hordeum distichon L.) 22,23 and acts in animals as a neurotoxin. 24,25 In this investigation we studied the distribution of N- methylated tyramine derivatives in several plants of Citrus genus and quantitated their levels in leaf tissue. ■ MATERIALS AND METHODS Reagents. Tyramine, N-methyltyramine, N,N-dimethyltyramine, phenylalanine, methyl iodide, and 0.1% solution of formic acid in water were from Sigma−Aldrich (Milan, Italy). SPE-C18 columns for flash chromatography were obtained from Phenomenex (Anzola Emilia, Italy). All other solvents and reagents used were of analytical grade. Synthesis and Purification of N,N,N-Trimethyltyramine. For the conversion of tyramine into its quaternary ammonium compound N,N,N-trimethyltyramine, we used a modified heterogeneous-phase reaction employing methyl iodide as the methylation agent in the presence of KHCO 3 . 26,27 Tyramine (200 mg) was dissolved in 20 mL of methanol, and then 1 g of KHCO 3 and, subsequently, 10 mL of Received: January 10, 2014 Revised: March 5, 2014 Accepted: March 6, 2014 Published: March 17, 2014 Article pubs.acs.org/JAFC © 2014 American Chemical Society 2679 dx.doi.org/10.1021/jf5001698 | J. Agric. Food Chem. 2014, 62, 2679−2684