LC-MS/MS-Based Profiling of Tryptophan-Related Metabolites In
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molecules Article LC-MS/MS-Based Profiling of Tryptophan-Related Metabolites in Healthy Plant Foods 1, 2, 2 3 Sara Vitalini y , Michele Dei Cas y , Federico Maria Rubino , Ileana Vigentini , Roberto Foschino 3 , Marcello Iriti 1,* and Rita Paroni 2 1 Department of Agricultural and Environmental Sciences, Università degli Studi di Milano, 20133 Milan, Italy; [email protected] 2 Department of Health Sciences, Università degli Studi di Milano, 20142 Milan, Italy; [email protected] (M.D.C.); [email protected] (F.M.R.); [email protected] (R.P.) 3 Department of Food, Environmental and Nutritional Sciences, Università degli Studi di Milano, 20133 Milan, Italy; [email protected] (I.V.); [email protected] (R.F.) * Correspondence: [email protected] These authors contributed equally to this work. y Academic Editor: Paula B. Andrade Received: 1 December 2019; Accepted: 8 January 2020; Published: 13 January 2020 Abstract: Food plants contain hundreds of bioactive phytochemicals arising from different secondary metabolic pathways. Among these, the metabolic route of the amino acid Tryptophan yields a large number of plant natural products with chemically and pharmacologically diverse properties. We propose the identifier “indolome” to collect all metabolites in the Tryptophan pathway. In addition, Tryptophan-rich plant sources can be used as substrates for the fermentation by yeast strains to produce pharmacologically active metabolites, such as Melatonin. To pursue this technological development, we have developed a UHPLC-MS/MS method to monitor 14 Tryptophan, Tryptamine, amino-benzoic, and pyridine metabolites. In addition, different extraction procedures to improve the recovery of Tryptophan and its derivatives from the vegetal matrix were tested. We investigated soybeans, pumpkin seeds, sesame seeds, and spirulina because of their botanical diversity and documented healthy effects. Four different extractions with different solvents and temperatures were tested, and water extraction at room temperature was chosen as the most suitable procedure to extract the whole Tryptophan metabolites pattern (called by us “indolome”) in terms of ease, high efficiency, short time, low cost, and sustainability. In all plant matrices, Tryptophan was the most abundant indole compound, while the pattern of its metabolites was different in the diverse plants extracts. Overall, 5-OH Tryptamine and Kynurenine were the most abundant compounds, despite being 100–1000-fold lower than Tryptophan. Melatonin was undetected in all extracts, but sesame showed the presence of a Melatonin isomer. The results of this study highlight the variability in the occurrence of indole compounds among diverse food plants. The knowledge of Tryptophan metabolism in plants represents a relevant issue for human health and nutrition. Keywords: Tryptophan; Tryptamine; indole compounds; matrix extraction; soya; Pumpkin; Sesame; Spirulina; nutraceuticals; functional foods; mass spectrometry 1. Introduction For several decades the benefits of the dietary styles abundant in fruit, vegetables, and legumes have been emphasized in terms of longevity, healthy ageing, and low morbidity from degenerative diseases, such as type 2 diabetes, metabolic syndromes, cardiovascular disease, neurodegenerative and neurobehavioral disorders, and some types of cancers. As an example, the Mediterranean diet can be considered the archetype of a health-promoting lifestyle, including moderate physical activity, Molecules 2020, 25, 311; doi:10.3390/molecules25020311 www.mdpi.com/journal/molecules Molecules 2020, 25, 311 2 of 14 and neurobehavioral disorders, and some types of cancers. As an example, the Mediterranean diet can be considered the archetype of a health-promoting lifestyle, including moderate physical aactivity, high intake a high of low-fatintake of dairy low-fat products dairy and products healthy and lipids healthy (from lipids seafood), (from as wellseafood), as low as consumption well as low ofconsumption refined sugars, of refined red meat, sugars, and saturatedred meat, fatsand [saturated1,2]. fats [1,2]. TheThe hugehuge phytochemicalphytochemical diversitydiversity ofof plantplant foodsfoods isis oneone ofof thethe reasonsreasons whywhy well-balancedwell-balanced vegetable-basedvegetable-based dietsdiets consistentlyconsistently aaffordfford healthhealth benefits.benefits. Plants typically containcontain hundredshundreds ofof phytochemicalsphytochemicals arisingarising from from di ffdierentfferent secondary secondary metabolic metabolic pathways pathways and contribute and contribute to plant to defense plant fromdefense competitors from competitors and pests and [3]. pests Varieties [3]. thatVarieties humans that selected humans for selected food use for have food lower use contentshave lower of toxiccontents and of off toxic-tasting and phytochemicals off-tasting phytochemicals and a higher and content a higher of bulk content and micronutrients, of bulk and micronutrients, especially of vitamins,especially essential of vitamins, fatty acids,essential amino fatty acids, acids, and amino antioxidants. acids, Phenylpropanoids,and antioxidants. Phenylpropanoids, a class of widespread a phenylalanineclass of widespread derivatives phenylalanine including polyphenols derivatives (flavonoids,including polyphenols stilbenes, and (flavonoids, proanthocyanidins), stilbenes, have and beenproanthocyanidins), the most investigated have dietarybeen the phytochemicals most investigat ined the dietary last decades, phytochemicals thus representing in the thelast paradigm decades, ofthus the representing relationship the between paradigm food of and the health relationship [4,5]. between food and health [4,5]. TheThe aminoamino acidacid TryptophanTryptophan (TRP)(TRP) isis anan irreplaceableirreplaceable constituentconstituent ofof thethe humanhuman diet,diet, sincesince thethe humanhuman genomegenome doesdoes notnot includeinclude thethe enzymesenzymes thatthat areare necessarynecessary forfor itsits de-novode-novo biosynthesis,biosynthesis, butbut onlyonly thosethose that that perform perform some some biotransformations biotransformations and and those those responsible responsible for break-down for break-down of the indoleof the nucleusindole nucleus (Figure 1(Figure). An important 1). An important class of neuro-transmitters, class of neuro-transmitters, such as Serotonin, such as Serotonin, is derived fromis derived TRP, thefrom source TRP, ofthe which source is of only which in the is diet.only Ain furtherthe diet. biotransformed A further biotransformed derivative ofderivative Serotonin, of MelatoninSerotonin, (MEL),Melatonin is also (MEL), an important is also an neurohormone, important neurohormone with multiple, with biological multiple eff ectsbiological that include effects the that circadian include regulationthe circadian of sleep.regulation of sleep. FigureFigure 1.1. SchemeScheme ofof thethe proposedproposed pathwayspathways ofof Tryptophan.Tryptophan. TryptophanTryptophan cancan bebe metabolizedmetabolized alongalong thethe KynurenineKynurenine pathwaypathway alongalong threethree didifferentfferent branchesbranches toto AnthranilicAnthranilic acid,acid, 3-OH3-OH Kynurenine,Kynurenine, andand KynurenicKynurenic acid,acid, depending depending to theto enzymaticthe enzymatic repertoire repert ofoire the cells.of the Tryptophan cells. Tryptophan can also be metabolizedcan also be alongmetabolized the Melatonin along the pathway Melatonin through pathway the synthesis through ofthe 5-OH synthesis Tryptamine of 5-OH (Serotonin) Tryptamine and (Serotonin) by using preferentiallyand by using dipreferentiallyfferent enzymes different depending enzymes on thedepending cellular on repertoire. the cellular repertoire. Recently, we suggested that indole compounds, in particular MEL (a potent neurohormone Recently, we suggested that indole compounds, in particular MEL (a potent neurohormone produced in traces from TRP in the pineal gland of mammals), discovered in some typical Mediterranean produced in traces from TRP in the pineal gland of mammals), discovered in some typical foods, such as wine and nuts, may represent a new element in this scenario, further corroborating the Mediterranean foods, such as wine and nuts, may represent a new element in this scenario, further protective effects of diets rich in plant products [6–8]. corroborating the protective effects of diets rich in plant products [6–8]. In addition, the chemical feature of indole is widely represented in the phytochemicals’ structures, In addition, the chemical feature of indole is widely represented in the phytochemicals’ which range in complexity from simple derivatives of the amino acid TRP to the high chemical diversity structures, which range in complexity from simple derivatives of the amino acid TRP to the high of the huge family of indole alkaloids. chemical diversity of the huge family of indole alkaloids. 2 Molecules 2020, 25, 311 3 of 14 Of note in plants, phenylalanine and TRP share a common biosynthetic route together with tyrosine, the shikimic acid or aromatic amino acid pathway unique of plants [3]. According to the USDA Food Composition Database (https://ndb.nal.usda.gov/ndb/), the sources that contain very high levels of free TRP include soy seeds (0.68 g TRP/100 g), pumpkin seeds (0.58 g TRP/100 g), and spirulina (0.93 g TRP/100 g). Some studies have pointed out the ability of Saccharomyces cerevisiae and other high producer yeast strains to produce MEL by biotrasformation of TRP.