Zanthoxylamide protoalkaloids based on N-phenylethyl cinnamamides: a series of natural and synthetic safe umami taste enhancing components assessed in silico and in vivo toxicity assays in zebrafish embryos

Among 546 plant members of family Rutaceae, the genus Zanthoxylum is widely known. It is commonly used in cooking, as a spice, and also in alternative medicine for the treatment of carminative, dyspepsia, anthelmintic, stomachic disorders and fever. On the basis of these uses, scientists have selected these plants for phytochemical studies, isolating, characterized and evaluated a hundred secondary metabolites and alkaloids such as coumarins, flavonoids, limonoids, and volatile oils, identifying a series of leading compounds with antidiarrheal, antimicrobial, antileishmanial, anticholinesterase, antiprotozoal, larvicidal and antioxidant activities.

Indeed, genus Zanthoxylum contains a wide variety of amides, and those derived from L-phenylalanine and cinnamic acids are known as zanthoxylamide protoalkaloides. Armatamide, rubecenamide, lemairamin, rubemamine and zanthosine are the representative molecules of this family and recently the scientific community have become interested in their biological activities. Structure-taste relationship studies of cinnamamides against the umami taste (one of the five basic tastes together with sweetness, sourness, bitterness, and saltiness) have revealed that the 3,4-dimethoxycinnamic moiety is the responsible for modulating receptors associated with the umami taste in both in vitro and in vivo studies. Due to their outstanding umami properties, these protoalkaloids will be used in preparation of human food very soon, but before that, their toxicological profile needs to be studied and validated in vertebrate models related with humans like the zebrafish embryo model.

Recently, Dr. Carlos E. Puerto Galvis and Prof. Vladimir V. Kouznetsov (Industrial University of Santander, Colombia) developed a mild and efficient method for the synthesis, at gram-scale, of diverse zanthoxylamide protoalkaloids through the direct amidation between commercially available phenylethylamines and cinnamic acids, obtaining a series of 29 substituted N-phenylethyl cinnamamides using the tris-(2,2,2-trifluoroethyl) borate (B(OCH₂CF₃)₃) as a catalyst in excellent yields.

Then, performing in silico computational methods against eight toxicity risks (mutagenic, hepatotoxicity, carcinogenicity, immunotoxicity, tumorigenic, cytotoxicity, reproductive effects and irritant), the authors found that the tested molecules exhibited moderate immunotoxicity and possible reproductive effects, while among different biological targets, these protoalkaloids showed a high affinity to the amine oxidase A (AOFA) and prostaglandin G/H synthase 1 (PGH1 receptors.

Finally, the acute toxicity toward zebrafish embryos (96 hpf-LC₅₀, 50% lethal concentration) was also determined in the present study for the synthesized zanthoxylamide protoalkaloids, and with the toxicity data of the selected reference compounds, an in vivo toxicity scale was established in which the average LC₅₀ for the zanthoxylamide protoalkaloids fit in the safe region, indicating their low toxicity (LC₅₀ values around 217 μM) on living organisms and their possible environmental impact.