Occurrence, formation from D‑fructose and 3‑deoxyglucosone, and activity of the carbohydrate-derived β‑carbolines in foods

Occurrence, Formation from D‑Fructose and 3‑Deoxyglucosone, and Activity of the Carbohydrate-Derived β‑Carbolines in Foods Tomás Herraiz*and Fernando Vera Cite This: J. Agric. Food Chem. 2021, 69, 6650−6664 Read Online ACCESS Metrics & More Article Recommendations ABSTRACT: β-Carbolines are naturally occurring bioactive alkaloids. In this work, carbohydrate-derived β-carbolines (βCs), 1- (1,3,4,5-tetrahydroxypent-1-yl)-β-carboline isomers (1a/b), 1-(1,4,5-trihydroxypent-1-yl)-β-carboline (2), 1-(1,5-dihydroxypent-3en-1-yl)-β-carboline (3), and 1-(1,2,3,4,5-pentahydroxypent-1-yl)-β-carboline (4) were identified and analyzed in commercial foods. The concentrations of βCs 1−4in foods ranged from undetectable to 11.4 μg/g levels, suggesting their intake in the diet. Processed foods contained higher amounts than fresh or unprocessed foods, and the highest content was found in processed tomato and fruit products, sauces, and baked foods. βCs 1−3were formed in foods during heating, and 1a/b were the main compounds. The formation of carbohydrate-derived βCs was studied in model reactions of tryptophan and carbohydrates. They formed in reactions of tryptophan with glucose under acidic conditions at temperatures higher than 80 °C. The formation of 1a/b was favored, but 2−3 increased at high temperatures. Noticeably, the βCs 1−3formed in the reactions of tryptophan with fructose or sucrose, and the formation from fructose was much higher than from glucose. Thus, fructose was the main carbohydrate involved in the formation of 1−3, whereas sucrose gave these βCs after acid hydrolysis. It is shown for the first time that the mechanism of formation of βCs 1−3 occurs from the sugar intermediate 3-deoxyglucosone that reacts with tryptophan affording these carbohydrate-derived βCs. A mechanism of reaction to give βCs 1−3is proposed that relies on the tautomerism (keto−enediol or enamine−imine) of intermediates involved in the reaction. Carbohydrate βCs 1−4were assessed as inhibitors of monoamine oxidase (MAO), as antioxidants, and for their interaction with DNA. They were not good inhibitors of MAO-A or -B, were poor antioxidants, and did not appreciably interact with DNA. KEYWORDS: carbohydrate-derived β-carbolines, alkaloids, tryptophan, 3-deoxyglucosone, Maillard reaction, advanced glycation ■INTRODUCTION β-Carbolines (9H-pyrido[3,4-b]indole) (βCs) are indole alkaloids occurring in foods, plants, and biological systems. 1,2 These alkaloids are bioactive compounds that exhibit an array of biological, pharmacological, and toxicological activities. 1−3 They act on the central nervous system (CNS) through interaction with serotonin uptake, benzodiazepine receptor and imidazoline binding sites, and inhibit monoamine oxidase (MAO) and kinase enzymes. 3 The βCs norharman and harman occurring in foods and cigarette smoke are potent inhibitors of MAO. 4,5 These compounds exert antidepressant and behavioral effects owing to their effects on neurotransmitters and inhibition of MAO, and have been involved in drug and alcohol addictions. 6−8 Some βCs are neuroprotectants or are involved in neurogenesis, 9 while others can be bioactivated by N-methylation affording endogenous neurotoxins (β-carbolinium cations) analogues to 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxin. 3 In addition, some βCs are co-mutagenic, bind to DNA, and react with hydroxyl radical (OH•). 10,11 Depending on the pyridoindole ring oxidation, there are tetrahydro-β-carbolines (THβCs) and aromatic β-carbolines (βCs). THβCs are produced through a Pictet−Spengler reaction from indole-ethylamines or indole-ethylamino acids and aldehydes or α-keto acids. Tetrahydro-β-carboline-3carboxylic acids (THβC-3-COOHs) arise from a reaction between L-tryptophan and aldehydes. These reactions occur in foods, and a number of THβCs including carbohydrate-derived THβCs have been identified and quantified in foods. 1,12 Aromatic βCs arise from the oxidation of THβCs. 13 Among them, the βCs norharman and harman are two main compounds that occur in foods and which are generated in meats and fish during cooking. 13,14 These βCs arise from the corresponding THβC-3-COOHs that are the most abundant THβCs in foods. 15 In addition, other aromatic βCs such as those derived from carbohydrates may appear in foods (Figure 1). 1,16−19 Thus, glucose-derived aromatic βCs have been found in a number of foods and human urine. 1,17,20,21 Some of these βCs have been also recently found in fruits of Nitraria tangutorum. 22 However, a few reports exist so far on the occurrence and activity of these compounds, whereas their mechanism of formation remains unknown. In this regard, carbohydrate-derived THβCs appear in reactions of tryptoReceived: April 19, 2021 Revised: May 20, 2021 Accepted: May 21, 2021 Published: June 3, 2021 Articlepubs.acs.org/JAFC © 2021 American Chemical Society 6650 https://doi.org/10.1021/acs.jafc.1c02281 J. Agric. Food Chem. 2021, 69, 6650−6664 Downloaded via CSIC on April 19, 2022 at 09:07:02 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. phan with carbohydrates. 12,23,24 It has been proposed that the carbohydrate-derived aromatic βCs might arise from the corresponding pentahydroxypentyl-THβC-3-COOH (PHPTHβC-3-COOH) formed from glucose and tryptophan (Figure 1). 1,12,16,23 In fact, the compound PHP-THβC-3COOH has been previously reported in foods and relatively high amounts found in tomato products, fruit juices, and jams, 12 and also found in human urine. 25,26 As the βCs exhibit bioactive and/or toxic actions and they may appear in tissues and biological fluids, the exposure to these compounds in the diet is a matter of interest. The purpose of this work was to study the occurrence of carbohydrate-derived βCs in a wide range of commercial foods and, subsequently, investigate their mechanisms of formation highlighting the precursors and intermediates involved. Finally, the study assesses the activity of carbohydrate-derived βCs identified in foods as MAO inhibitors, antioxidants, and their interaction/intercalation with DNA. ■MATERIALS AND METHODS Chemical Compounds and Foods. Commercial samples of foods were purchased from local supermarkets to cover a wide range of foods and used to analyze carbohydrate-derived β-carbolines. D- (+)-Glucose monohydrate, lactose, and maltose were obtained from Merck, D-(-)-fructose from Sigma, D-(+)-sucrose from Scharlau, and 3-deoxy-D-glucosone from Biosynth-Carbosynth. L-Tryptophan, tryptamine, L-tryptophan methyl ester, harman, and norharman were purchased from Sigma. 1-(1,2,3,4,5-Pentahydroxypent-1-yl)-1,2,3,4tetrahydro-β-carboline-3-carboxylic acid (PHP-THβC-3-COOH) was obtained from L-tryptophan and D-glucose as previously. 12 The carbohydrate-derived β-carbolines, 1-(1,3,4,5-tetrahydroxypent-1-yl)- β-carboline diastereoisomers (1a/b), 1-(1,4,5-trihydroxypent-1-yl)-βcarboline (2), and 1-(1,5-dihydroxypent-3-en-1-yl)-β-carboline (3) were obtained from a reaction of glucose with tryptophan heated in pH 1 and isolated with C18 column chromatography, as previously, 17 and 1-(1,2,3,4,5-pentahydroxypent-1-yl)-β-carboline (4)obtained following oxidation of PHP-THβC-3-COOH 23 (Figure 1). The spectral data of these βCs have been reported. 12,17,20,22,23 The purity of βCs was higher than 90% by high-performance liquid chromatography (HPLC). 1-Ethyl-β-carboline (EβC) was obtained from the chemical oxidation of 1-ethyl-1,2,3,4-tetrahydro-β-carboline3-carboxylic acid (ETCA) with sodium dichromate and solvent extraction. 13 Monoamine oxidase (MAO) enzymes were obtained from Corning (Gentest), horseradish peroxidase (HRP), calf thymus DNA (type I highly polymerized), ethidium bromide, ascorbic acid, catechin, quercetin, and 3,3′,5,5′-tetramethylbenzidine (TMB) from Sigma-Aldrich, and H2O2from Scharlau. Isolation of Carbohydrate-Derived β-Carbolines by SolidPhase Extraction (SPE). Samples of solid foods (4−7 g) were added with 0.6 M HClO4(15−20 mL), homogenized using an ultraturrax homogenizer, and subsequently centrifuged at 10000 rpm for 15 min at 0−5°C. Liquid samples of foods (juices, beverages, vinegars, teas) or diluted samples (e.g., soy sauce) were centrifuged and acidified with HCl 0.1 M. The isolation of carbohydrate βC was carried out by solid-phase extraction (SPE) using propylsulfonic acid-derivatized silica PRS columns (Bond Elut, 500 mg, 3 mL size, Varian, Harbor City, CA) as previously with minor modifications. 14 The procedure of isolation was optimized for the recovery of carbohydrate βCs. The conditioning of PRS columns was made with methanol and 0.1 M HCl. Aliquots (5 mL) were spiked with 125 μL of 1-ethyl-β-carboline solution (EβC) (0.2 mg/L) as internal standard (IS) and subsequently loaded onto PRS columns using a vacuum manifold. After washing with deionized water (6 mL), the carbohydrate βCs were eluted with 3 mL of 0.4 M K2HPO4(pH 9.1) followed with 3 mL of 0.4 M K2HPO4(pH 9.1)/methanol (1:1). The eluates were joined and injected into HPLC and HPLC mass spectrometry (MS) Figure 1. Chemical structures of carbohydrate-derived βCs 1-4 and the reaction of tryptophan with glucose to give 1-(1,2,3,4,5-pentahydroxypent1-yl)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (PHP-THβC-3-COOH). The compounds are: 1-(1,3,4,5-tetrahydroxypent-1-yl)-β-carboline isomers (1a/b), 1-(1,4,5-trihydroxypent-1-yl)-β-carboline (2), 1-(1,5-dihydroxypent-3-en-1-yl)-β-carboline (3), and 1-(1,2,3,4,5-pentahydroxypent-1-yl)-β-carboline (4). Journal of Agricultural and Food Chemistry pubs.acs.org/JAFC Article https://doi.org/10.1021/acs.jafc.1c02281 J. Agric. Food Chem. 2021, 69, 6650−6664 6651 columns. The evaluation of the performance and quantitative analysis were carried out as mentioned below. Formation of Carbohydrate-Derived βCs in Model Reactions and Foods. Studies using model reactions containing Ltryptophan and carbohydrates, both in low and high concentrations, were carried out to evaluate the effects of pH, temperature, and carbohydrate on the formation of the carbohydrate-derived βCs as follows: (a) two concentrations of tryptophan (0.5 or 2 g/L) and equimolar concentrations of glucose (5 or 40 g/L), fructose (4.54 or 36.6 g/L), or sucrose (8.5 or 69.1 g/L) in phosphate solutions (100 mM) adjusted to different pHs (1.3, 3.1, 5, 7.4, and 9) were placed in glass tubes with ground-glass stoppers and reacted in an oven for 20 h (80 or 90 °C). In addition, controls with only tryptophan or carbohydrates were also carried out. (b) Solutions of tryptophan (2 g/ L) and glucose (40 g/L), fructose (36.6 g/L), or sucrose (69.1 g/L) in phosphate buffer pH 3.1 were reacted at different temperatures to cover a wide range (from 37 to 130 °C). (c) Reactions were also carried out with tryptophan (2 g/L) and maltose (72.7 g/L) or lactose (72.7 g/L) at pH 1.3 and 3.1 (90 °C, 20 h). On the other hand, tryptamine (2 g/L) or tryptophan methyl ester (2 g/L) instead of tryptophan were reacted with glucose (40 g/L) (pH 3.1, 3 h, 110 °C). An aliquot of the reactions was injected into the reversed-phase (RP)-HPLC, analyzed by a diode array detector (DAD) and fluorescence as mentioned below, and subjected to HPLC-MS for compound identification. On the other hand, to study the formation of carbohydrate βCs in processed foods, white grapes and tomatoes (cherry) were subjected to drying in an oven at 80 °C and the carbohydrate βCs were isolated by SPE as reported above and analyzed by RP-HPLC. The same analysis was done with the raw samples. To study the conversion of possible precursors into products, compounds PHP-THβC-3-COOH, 1, and 4(100-500 μM, pH 1−7) were heated in an oven (140 °C, 1.4−4 h, or 110 °C, 3 h) or microwave, reconstituted to the original volume, and injected into HPLC and HPLC-MS. On the other hand, PHP-THβC-3-COOH was also oxidized (2 mM H2O2, 2 h, 37 °C or 0.025 mg/mL HRP, 500 μMH 2O2, 40 min, 37 °C) and injected into HPLC and HPLCMS columns. Finally, to highlight the mechanism of formation, model reactions of 3-deoxyglucosone (0.1 mg/mL) and tryptophan (0.5 mg/ mL) in phosphate solutions adjusted to pH 1.3 or 3.1 were carried out at 90 and 110 °C for 2−4 h, and the products formed were analyzed by RP-HPLC and identified by HPLC-MS. All of the reactions were carried out in duplicate. Chromatographic and Quantitative Analyses of Carbohydrate-Derived βCs in Foods and Model Reactions, and Identification by HPLC-MS. Chromatographic analysis of carbohydrate βCs was performed using a 1050 high-performance liquid chromatograph with a 1100 series DAD (Agilent) and a 1046A fluorescence detector controlled by a Chemstation (Agilent Technologies). A 150 mm ×3.9 mm, 5 μm, Novapak C18 column (Waters) was used for HPLC separation. Eluents were 50 mM ammonium phosphate buffer adjusted to pH 3 with phosphoric acid (eluent A) and 20% of eluent A in acetonitrile (eluent B). The gradient was 0% B to 32% B in 8 min, then 90% B at 18 min, and 100% B at 20 min. The flow rate was 1 mL/min, the oven temperature was 40 °C, and the injection volume was 20 μL. The carbohydrate βCs isolated from foods by SPE were analyzed by HPLC with fluorescence detection at 300 nm of excitation and 433 nm of emission. Quantitative analysis was obtained using standard solutions of known concentration of 1against EβC used as an internal standard (IS) that were carried out through the entire SPE isolation procedure. The use of IS allowed us to correct for possible matrix effects occurring during SPE. The standard solutions were made in an acidic media similar to those used in SPE of foods and covered a range of concentrations found in foods (25−1000 μg/L). A linear calibration curve (r2= 0.99) of peak area of the βC1divided by IS area vs concentration of the compound was obtained and used for quantitation. The performance and validation of the method gave good repeatability (2.5% RSD, n= 4), accuracy (3.4% mean error, n= 4), and recovery (95.7%, n= 4) (compound 1, 100 μg/L). The recoveries in the SPE after spiking the food samples with 1(1000 μg/ L) were 95.8, 97.2, 92.3, 85.2, and 97% (n= 2) for tomato puree, jam, bread, chocolate, and pineapple juice, respectively. The analysis of carbohydrate βCs in model reactions was carried out by HPLC with DAD and fluorescence detection. Quantitation was carried out with calibration curves of βC1response at 254 nm and fluorescence (300 nm excitation and 433 nm emission) against concentration (0.01−1 mM range). The concentration of PHP-THβC-3-COOH in model reactions was determined by HPLC with absorbance detection at 280 nm and calculated from a calibration curve of response against concentration of this compound (0.01−1 mM range). For identification purposes, the chromatographic peaks of carbohydrate βCs 1-4 in foods and model reactions were co-injected with authentic standards, and the DAD and fluorescence spectra were obtained. Identification was confirmed by HPLC-MS. For that, SPE extracts from foods and aliquots of model reactions were analyzed by HPLC-MS. SPE fractions were concentrated using a vacuum concentrator and analyzed by HPLC-MS (electrospray ionization (ESI) mode). The HPLC-MS analysis was carried out with an apparatus HPLC-MS Agilent equipped with 1200 series quaternary pump and DAD coupled to a 6110 single-quadrupole MSD detector working in electrospray ionization mode (API-ESI). Chromatographic separation was accomplished with a 3.9 mm ×150 mm Novapak C18 column (Waters) with eluents 0.5% formic acid (A) and 0.5% formic acid in acetonitrile (B), and using a linear gradient from 0 to 100% B in 15 min, a flow rate of 0.6 mL/min, and a temperature of 40 °C. Mass spectra were acquired in ESI positive-ion ionization mode at various fragmentor voltages (90 and 150 V) with an acquisition mass range of 50−800 u. The conditions in the mass spectrometer were: gas temperature, 350 °C; drying gas flow, 12 L/min; nebulizing gas pressure, 35 psig; and capillary voltage, 3000 V. Activity of Carbohydrate-Derived βCs as Inhibitors of MAO, Antioxidants, and DNA-Interaction Agents. The activity of carbohydrate βCs 1−4and norharman and harman as inhibitors of MAO enzymes was studied as previously. 27 Briefly, protein fractions containing MAO-A or -B (Corning-Gentest) were diluted to the desired concentrations in 100 mM potassium phosphate buffer (pH 7.4). A 0.2 mL reaction mixture containing 0.01 mg/mL protein, carbohydrate βCs (1-4), norharman or harman (from 0 to 50 μM range), and 0.25 mM kynuramine in 100 mM potassium phosphate (pH 7.4) was incubated at 37 °C for 40 min. After incubation, the reaction was stopped by the addition of 2 N NaOH (75 μL), followed by the addition of 70% HClO4(25 μL), and the sample was centrifuged (10 000g) for 10 min. The supernatant (20 μL) was injected into the HPLC, and the kynuramine deamination product 4hydroxyquinoline was determined by RP-HPLC-diode array detector at 320 nm. The inhibitors clorgyline (MAO-A) and R-deprenyl (MAO-B) were used as positive control for inhibition. Incubations were carried out in duplicate, and IC50 values calculated using GraphPad Prism software. The antioxidant activity of carbohydrate βC1−4was assessed by scavenging of the radical TMB•+(blue color) that is eliminated in the presence of antioxidants. 28 For that, the radical cation TMB (TMB•+) was obtained from TMB (250 μM), HRP (25 μg/mL), and H2O2 (100 μM) that reacted at room temperature in 100 mM buffer phosphate (pH 7.4) for 1 min. Subsequently, compounds 1−4(30 μM) and the antioxidants ascorbic acid (30 μM), catechin (30 μM), or quercetin (30 μM) were added to the mixture containing TMB cation radical, and the elimination of the radical (decoloration) followed at 650 nm over time (10 min). The activity of compounds 1−4was compared with that of antioxidants. The interaction of compounds 1−4with calf thymus DNA was studied by UV−vis spectroscopy and compared with ethidium bromide, a known intercalating agent. 29,30 The interaction between DNA and a ligand molecule can be examined by studying the shifting of the position of the maximum and the intensity of the absorption bands from when the ligand is free in solution or when is bound with DNA. 31 For that, solutions (final volume 500 μL) of carbohydrate βC 1−4(from 25 to 50 μM), or ethidium bromide (25 μM) in 10 mM trisaminomethane (TRIS) buffer (pH 7.4) placed in a quartz cuvette were added with successive volumes of calf thymus DNA (0, 2.5, 5, Journal of Agricultural and Food Chemistry pubs.acs.org/JAFC Article https://doi.org/10.1021/acs.jafc.1c02281 J. Agric. Food Chem. 2021, 69, 6650−6664 6652 7.5, 10, and 10 μL corresponding to 0, 6, 18, 36.4, 61.7, and 85 μM DNA calculated with εat 260 nm), mixed, and UV−vis spectra were acquired from 220 to 800 nm over time (after 25 min of each addition of DNA) in a spectrophotometer (T70+ UV−vis spectrophotometer PG Instruments). ■RESULTS Identification and Occurrence of Carbohydrate βCs in Foods and Model Reactions. The carbohydrate-derived βCs 1−3(Figure 1) occurring in foods and model reactions of tryptophan and carbohydrates were analyzed by HPLC and identified by UV−vis and fluorescence spectra as well as by coelution with authentic standards. Identification was confirmed by HPLC-MS (Figure 2). They were identified as 1- (1,3,4,5-tetrahydroxypent-1-yl)-β-carboline isomers (1a/b),1- (1,4,5-trihydroxypent-1-yl)-β-carboline (2), and 1-(1,5-dihydroxypent-3-en-1-yl)-β-carboline (3)inagreementwith previous results. 17,20 The compounds appeared in model reactions of tryptophan and glucose but also in the reactions of tryptophan and fructose (Figure 2). The mass spectra afforded the protonated molecular ion [M + H]+and a fragmentation pattern dominated by successive loss of water (m/z18) and fragmentation losses of C3H6O3in 1,C 2H4O2in 2, and CH2O and C4H8Oin3. 17 The compounds have different C-1′ configurations that were separated by HPLC in the case of 1a/ bdiastereoisomers. Besides 1−3, the compounds 1-(1,2,3,4,5pentahydroxypent-1-yl)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (PHP-THβC-3-COOH) and 1-(1,2,3,4,5-pentahydroxypent-1-yl)-β-carboline (4) were identified in foods and model reactions of tryptophan and glucose (Figures 1 and 3). 12 Moreover, on the basis of the mass spectra and fragmentation pattern, two additional β-carbolines were identified as 1-(1,4,5trihydroxypent-2-en-1-yl)-β-carboline (5) and 1-(1-keto-3,4,5Figure 2. (A) HPLC-MS extracted ion chromatograms (EIC) and ESI-mass spectra (ionization voltage, 150 V) of the carbohydrate-derived βCs 1−3in model reaction of tryptophan (2 g/L) and fructose (36.6 g/L) (90 °C, pH 3.1, 20 h). (B) HPLC-MS extracted ion chromatograms (ionization voltage, 150 V) of the carbohydrate-derived βCs 1−3in concentrated tomato paste. The m/zions are: 303 [M + H]+, 285 [303-18]+ and 195 [285-C3H6O3]+in 1a/b; 287 [M + H]+, 269 [287-18]+, 251 [269-18]+, and 209 [269-C2H4O2]+in 2; and 269 [M + H]+, 251 [269-18]+, and 197 [269-C4H8O]+in 3. Compounds are as in Figure 1. Journal of Agricultural and Food Chemistry pubs.acs.org/JAFC Article https://doi.org/10.1021/acs.jafc.1c02281 J. Agric. Food Chem. 2021, 69, 6650−6664 6653 trihydroxypent-1-yl)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (6)(Figure 3) in model reactions of tryptophan and glucose or fructose, and in reactions of tryptophan and fructose, respectively. These compounds were collected from the HPLC column and afforded high-resolution mass spectra (ESI-Q-TOF-MS, Agilent) of m/z285.1223 [M + H]+ (calculated for [C16H16N203+H]+, 285.1234) (5) and m/z 349.1370 [M + H]+(calculated for [C17H20N2O6+H]+, 349.1394) (6). The carbohydrate βCs 1−3were analyzed in commercial foods by HPLC-fluorescence after SPE (examples are shown in Figure 4). Their content ranged from undetected to several μg/g or mg/L levels, with 1a/b being the major compounds (Table 1). These β-carbolines appeared in the highest content in processed tomato products, including tomato juice, fried tomato sauce, ketchup, tomato concentrate, and dried tomato. Low processed samples such as natural tomato puree (from crushed tomatoes) did not contain or contained a very low Figure 3. (A) HPLC-MS extracted ion chromatograms (EIC) and ESI-mass spectra (ionization voltage, 150 V) of the carbohydrate-derived βC4 and PHP-THβC-3-COOH in model reactions of tryptophan (0.5 g/L) and glucose (5 g/L) (90 °C, pH 1.3, 20 h) (compounds are as in Figure 1). (B) HPLC-MS extracted ion chromatograms (EIC) and ESI-mass spectra (ionization voltage, 150 V) of compounds identified as (1,4,5trihydroxypent-2-en-1-yl)-β-carboline (5) and 1-(1-keto-3,4,5-trihydroxypent-1-yl)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (6) in reactions of tryptophan (2 g/L) and fructose (36.6 g/L) (90 °C, pH 3.1, 20 h). The m/zions are: 319 [M + H]+, 301 [319-18]+, and 211 [301-C3H6O3]+in 4; 367 [M + H]+, 350 [367-17]+, 332 [350-18]+, and 294 [367-73]+in PHP-THβC-3-COOH; 285 [M + H]+, 267 [285-18]+, 207 [267-C2H4O2]+, 249 [267-18]+, and 237 [267-CH2O]+in 5; and 349 [M + H]+, 332 [349-17]+, 314 [332-18]+, and 276 [349-73]+in 6. Journal of Agricultural and Food Chemistry pubs.acs.org/JAFC Article https://doi.org/10.1021/acs.jafc.1c02281 J. Agric. Food Chem. 2021, 69, 6650−6664 6654 level of carbohydrate βCs, and tomato juice elaborated from concentrate juice contained higher amounts than juices made not from concentrate. They appeared in relatively high amounts in processed fruit products such as dried fruits, jams and juices with those from pineapple, pineapple and grape or tropical juice containing the highest levels, whereas other juices such as orange or apple juices contained low amounts. The carbohydrate βCs were present in sauces including soy sauce. They were also present in baked foods such as bread and breakfast cereals and bars, biscuits, and cookies. In contrast, flours from wheat, corn, and rice did not have carbohydrate βCs. These compounds were present in chocolate, whereas very low levels were found in coffee or tea. Most nuts contained very low amounts of 1−3. In contrast, carbohydrate βCs 1−3appeared in dehydrated fruits, particularly in raisins and tomatoes. A number of foods did not contain or contained very low amounts or traces of carbohydrate βCs 1−3. Those include fresh, smoked or cooked meat or fish, or dairy products such as milk, cheese, or yogurt. They were not found in sausages with exception of some cured sausages. Neither, they occurred in alcoholic beverages such as wines, liquors, or distilled beverages and trace levels appeared in beer. The compounds were not found in vinegars made from wine or cider, but balsamic vinegar that is made from heated must was an exception. The proportion of 1−3in foods varied greatly, but usually 1a/b were the main carbohydrate βCs. Nevertheless, relatively high levels of 2and 3appeared in some samples of baked foods and breads. The presence of βCs 4and 5was studied in foods, but they only appeared in a few samples, and the content of 4ranged from undetectable in most samples to less than 0.2 μg/g in samples of tomato products (juices, sauces, fried, and dried tomato) and dried grapes. Insights into the Mechanism of Formation of Carbohydrate βCs. The content of carbohydrate βCs 1−3 (Table 1) was high in processed foods and low in fresh or little processed foods, suggesting that these βCs formed during food processing (e.g., tomato puree vs fried tomato sauce or tomato juices made not from concentrate vs those made from concentrate). This fact was confirmed further in the laboratory as the carbohydrate βCs were formed during the heating and drying process of grapes and tomatoes (Figure 5). As shown in model reactions, compounds 1−3can occur from a reaction of tryptophan with glucose (Figure 5). This formation highly increased under acidic conditions and with increasing temperature. It needed high temperatures as no formation of carbohydrate βCs 1−3occurred at room temperature or under physiological conditions (37 °C), whereas low or trace amounts appeared at 60 °C (e.g., in the reaction of 2 g/L tryptophan and 40 g/L glucose, pH 3.1, 20 h). However, the formation of these compounds occurred when heating at 80 °C or higher temperatures. Remarkably, the relative proportions of compounds 1−3changed with temperature, and 2and 3were favored over compounds 1a/b at high temperatures (100 °C and above) (Figure 5). L-Tryptophan reacts with glucose affording PHP-THβC-3COOH (Figure 1). 12 This THβC-3-COOH occurred in foods such as processed tomato products, fruit juices, and jams, and increased under acidic conditions and with increasing temperature. 12 Here, PHP-THβC-3-COOH appeared in model reactions of tryptophan and glucose in higher amounts than the carbohydrate βCs 1−3(Figure 5). PHP-THβC-3-COOH might be a possible precursor of the aromatic carbohydrate βCs 1−3by oxidative decarboxylation and dehydration. 16 However, no appreciable formation of the carbohydrate βCs 1−3occurred when PHP-THβC-3-COOH was heated at a high temperature (e.g., 110−140 °C) or under oxidative conditions (H2O2and peroxidase). Instead, it afforded the corresponding oxidation and decarboxylation product 1- (1,2,3,4,5-pentahydroxypent-1-yl)-β-carboline (4) and norharman (Figure 6). Indeed, βC4appeared in model reactions of tryptophan and glucose at acidic pH along with PHP-THβC-3COOH (Figure 5B). No formation of βCs 1-3 resulted from 4 during heating at high temperatures (110−140 °C), and Figure 4. HPLC chromatograms of carbohydrate βCs isolated in tomato juice (a), raisins (b), and chocolate (c) obtained after SPE cleanup. Compounds are as in Figure 1. Journal of Agricultural and Food Chemistry pubs.acs.org/JAFC Article https://doi.org/10.1021/acs.jafc.1c02281 J. Agric. Food Chem. 2021, 69, 6650−6664 6655 Table 1. Concentration of Carbohydrate βCs (μg/L1or μg/g2) Determined in Foods a 1a 1b 2 3 food samples mean SD range mean SD range mean SD range mean SD range tomato juice1(n= 11) 1494 1019 15−3575 1365 914.5 12.2−3097 274.3 230.4 5.1−797.8 54.3 53.6 nd−123.1 tomato juice (concentrate)1(n= 8) 1918 841.4 971−3575 1762 715.5 1020−3097 352.0 222.9 150.6−798 72.4 52.0 nd−123.1 tomato juice (not concentrate)1(n= 3) 365.9 304.3 15−575 305.1 269.7 12.2−543.2 67.18 54.31 5.1−105.9 6.1 10.6 nd−18.4 pineapple juice1(n= 14) 111.3 113.7 nd−308.2 102.1 111.3 nd−319 16.9 23.4 nd−85.1 2.1 8.3 nd−32.3 tropical juice1(n= 4) 142.1 113.9 49.3−304 154.0 122.3 46.4−317.3 26.04 9.846 15.05−34.35 43.13 86.25 nd−172.5 pineapple + grape juice1(n=8) 158.9 135.0 9.6−363.5 154.3 100.8 9.5−294.4 34.83 38.43 nd−93.2 3.9 4.8 nd−12.3 orange juice1(n= 6) 28.5 23.2 nd−78 32.6 34.7 nd−110.9 13.7 21.64 nd−62.8 18.15 16.19 nd−49.8 juice (apple, peach, grape)1(n= 15) 29.8 37.6 nd−103 36.4 45.7 nd−132.9 18.98 18.9 nd−50.7 nd fresh vegetable beverage/cream2(n =7) 0.027 0.024 nd−0.056 0.027 0.022 nd−0.051 0.003 0.007 nd−0.02 0.002 0.004 nd−0.01 jam2(n= 11) 0.044 0.077 0.002−0.27 0.037 0.055 0.002−0.19 0.013 0.012 nd−0.034 0.005 0.010 nd−0.030 fried tomato sauce2(n =9) 1.78 1.44 0.44−4.97 1.57 1.34 0.42−4.6 0.28 0.17 0.054−0.61 0.054 0.059 nd−0.15 natural tomato puree2 (n=6) 0.003 0.007 nd−0.018 0.002 0.005 nd−0.013 0.002 0.005 nd−0.011 0.002 0.002 nd−0.009 tomato puree conc.2 (n=1) 7.6 6.4 1.6 0.49 ketchup2(n= 8) 1.43 1.38 0.24−4.3 1.28 1.19 0.24−3.8 0.26 0.22 0.024−0.71 0.070 0.074 nd−0.21 sauce2(n= 8) 0.63 0.47 0.2−1.55 0.65 0.48 0.2−1.6 0.22 0.24 nd−0.7 0.038 0.06 nd−0.16 soy sauce1(n= 3) 954.6 236.1 713.2− 1185 1050 118.1 965.6−1185 968.8 104.2 803−1088 wine vinegar1(n=4) nd nd nd nd cider vinegar1(n=2) nd nd nd nd vinegar balsamic1(n= 1) 319.5 385.0 94.3 nd fried/caramelized onion2(n=3) 0.19 0.18 0.044−0.39 0.18 0.19 0.031−0.40 0.12 0.205 nd−0.36 nd dried tomato2(n= 2) 2.35 3.0 0.22−4.48 2.26 2.97 0.15−4.4 0.97 1.33 0.029−1.91 0.34 0.37 0.08−0.60 dried fruit2(n= 8) 0.11 0.10 nd−0.26 0.10 0.11 nd−0.26 0.033 0.05 nd−0.14 0.004 0.012 nd−0.035 raisin (dried grape)2 (n= 12) 0.78 0.78 0.07−2.85 0.79 0.63 0.10−1.93 0.54 0.44 nd−1.46 0.026 0.040 nd−0.12 nut2(n= 6) 0.02 0.017 nd−0.05 0.025 0.019 nd−0.05 0.06 0.04 0.025−0.13 nd french fries2(n=2) nd nd nd nd chocolate2(n= 7) 0.24 0.15 0.13−0.58 0.265 0.19 0.008−0.68 0.26 0.18 0.09−0.59 0.03 0.053 nd−0.135 breakfast cereal/bar2 (n=9) 0.23 0.25 0.01−0.64 0.25 0.273 0.008−0.71 0.17 0.23 nd−0.79 0.028 0.055 nd−0.16 bread2(n= 3) 0.04 0.03 0.01−0.07 0.05 0.03 0.012−0.077 0.31 0.086 0.21−0.38 0.017 0.003 nd−0.03 bread toasted2(n= 3) 0.30 0.12 0.19−0.43 0.31 0.15 0.18−0.47 1.15 0.35 0.76−1.46 0.17 0.025 0.14−0.19 cookies/baked good2 (n=8) 0.05 0.065 nd−0.178 0.06 0.063 nd−0.167 0.06 0.084 nd−0.26 0.007 0.006 nd−0.013 flour (wheat, corn, rice)2(n=4) nd nd nd nd tea1(n= 4) 1.7 1.2 nd−2.7 1.8 1.2 nd−2.7 1.03 1.28 nd−2.6 nd coffee1(n= 12) 8.7 10.7 nd−25.4 6.6 9.3 nd−22 nd nd milk1(n=4) nd nd nd nd yogurt2(n=3) nd nd nd nd cheese2(n=5) nd nd nd nd cooked meat (pork/ beef)2(n=4) nd nd nd nd cooked fish2(n=3) nd nd nd nd smoked fish2(n=4) nd nd nd nd sausage2(n=4) nd nd nd nd cured sausage2(n= 2) 0.04 0.008 0.03−0.05 0.06 0.014 0.054−0.074 0.02 0.01 0.017−0.031 nd wine1(n=6) nd nd nd nd beer1(n= 5) 29.2 40 nd−98 31.4 45 nd−108 5.3 9 nd−20.5 nd alcoholic beverages/ liqueur1(n=6) nd nd nd nd Journal of Agricultural and Food Chemistry pubs.acs.org/JAFC Article https://doi.org/10.1021/acs.jafc.1c02281 J. Agric. Food Chem. 2021, 69, 6650−6664 6656 neither compound 2or 3resulted from 1under the same conditions. Then, compounds 1−3formed in a distinct way to PHP-THβC-3-COOH and 4. A free carboxylic group was required because the reaction to give 1−3occurred with tryptophan, but tryptamine did not afford the compounds when reacted with glucose at high temperatures and under acidic conditions, whereas tryptophan methyl ester afforded only trace amounts likely due to ester hydrolysis. Interestingly, the carbohydrate βCs 1−3were formed when tryptophan reacted with fructose or sucrose under acidic conditions and heating. Under the same conditions, the formation of βCs 1−3from fructose or sucrose was much higher than from glucose (Figure 7A and also Figures 7B and 5B). The reaction was also highly favored in acidic pH although the formation of 1−3from fructose occurred even in up to pH 5 to a low extent (Figure 7). It increased with temperature, and high temperatures favored 2and 3over 1 (Figure 7C). Under acidic conditions (pH 1−3) and heating, the formation of 1−3from sucrose was close to fructose; however, it was much lower at pH 5 (Figure 7). Compared with sucrose, other disaccharides such as maltose or lactose gave very low levels or trace amounts of 1−3under the same conditions (pH 3.1, 90 °C, 20 h). In model reactions of tryptophan and fructose, compound 6(1-(1-keto-3,4,5trihydroxypent-1-yl)-THβC-3-COOH) was detected (Figure 3). This compound was isolated by collecting the corresponding peak at the exit of the HPLC column, and after heating (110 °C, 3 h) or oxidation with 0.025 mg/mL HRP, 500 μM H2O2(40 min, 37 °C) gave the corresponding aromatic βC (m/zat 301 (MH+) and 211 MH+-C3H6O3) but not 1−3. The presence of this THβC could indicate a possible involvement of 3-deoxyglucosone in the reaction. Indeed, when authentic 3deoxyglucosone (0.1 mg/mL) was reacted with tryptophan (0.5 mg/mL) under acidic conditions (pH 1.3 and 3.1) at 90 Table 1. continued a nd: not detected. Figure 5. (A) Formation of carbohydrate βCs in grapes (a) and tomatoes (b) samples dried in an oven at 80 °C compared with raw samples. (B) Formation of carbohydrate βCs 1−4(a) and PHP-THβC-3-COOH (b) in model reactions of tryptophan (0.5 g/L) and glucose (5 g/L) at various pHs (90 °C, 20 h). (C) Formation of carbohydrate βCs 1−3with temperature and time (h) in model reactions of tryptophan (2 g/L) and glucose (40 g/L) at pH 3.1. Results are average of duplicates. Journal of Agricultural and Food Chemistry pubs.acs.org/JAFC Article https://doi.org/10.1021/acs.jafc.1c02281 J. Agric. Food Chem. 2021, 69, 6650−6664 6657 °C and 110 °C, the carbohydrate βCs 1−3were formed (Figure 8). The concentrations produced (110 °C and pH 3.1, 2 h) were 27.2 ±0.42 μM(1a), 22.8 ±1.8 μM(1b), 24.5 ± 0.5 μM(2), and 14.0 ±0.7 μM(3). Activity of the Carbohydrate-Derived βCs 1−4as MAO Inhibitors, Antioxidants, and DNA-Interaction Agents. Inhibition of MAO, antioxidant effects, and interaction with DNA could be expected as biological activities of the carbohydrate-derived βCs. The carbohydrate βCs 1−4 were studied as inhibitors of human MAO-A and -B and none of the compounds significantly inhibited MAO compared to the βCs harman and norharman, which were good inhibitors (Figure 9A). 5,27 The antioxidant activity of 1−4was studied by scavenging of TMB+•cation radical. The carbohydrate βCs had almost no antioxidant activity compared with ascorbic acid, catechin, or quercetin (Figure 9B). The βCs have been previously reported to interact with DNA. 32 The interaction of βCs 1−4with DNA was studied by spectrophotometry UV− vis following standard procedures. 31 None of the compounds showed a relevant interaction or intercalation with calf thymus DNA evidenced by changes in UV−vis spectra compared with DNA and compounds alone (Figure 9C). In the same experiment, ethidium bromide, a well-known DNA intercalator, interacted with DNA as shown by hypochromicity and the change in the maximum of the absorption band (bathochromicity) from 480 to 510−520 nm (Figure 9C). 29 ■DISCUSSION The carbohydrate-derived βCs were identified and quantified in foods. The βCs 1−3occurred in many commercial foods, and the compounds 4and 5were identified as minor compounds. The βCs 1−3occurred in higher amounts in processed foods, particularly those subjected to heating processes. They formed in a reaction of L-tryptophan with glucose at a high temperature and acidic pH. Noticeably, the βCs 1−3also formed from other carbohydrates such as fructose and sucrose (Figures 2 and 7), and under the same conditions (e.g., molar concentration, reaction time, pH, and temperature), the formation of the βCs 1−3from fructose occurred at a much higher rate than from glucose (Figures 5 and 7). The formation of 1−3from fructose and sucrose also increased with the temperature and acidic pH. Fructose reacted with tryptophan to give 1−3,whereassucrose hydrolyzed under acidic pH and heating releasing fructose that reacted with tryptophan to give the compounds. No significant reaction was observed from tryptophan and carbohydrates under physiological conditions (37 °C and pH 7.4). The results show that foods containing tryptophan and carbohydrates subjected to processing conditions including heating or cooking will afford the carbohydrate βCs 1−3. The results obtained indicate that fructose and sucrose (when hydrolyzed to fructose) should be the main carbohydrate precursors of the carbohydrate βCs 1−3in foods. Fructose and sucrose are naturally present in foods and/or are added during food production or cooking (e.g., high fructose corn syrup, HFCS). Many foods are naturally rich in fructose such as fruits, dried fruits, and fruit juices. Some foods are also added with fructose and processed by heating such as fried tomato sauce, salad dressings, snack foods, baked foods, fast foods, breakfast cereals, cereal bars, and jams. Some of these foods contained relatively high levels of 1−3. Sucrose is naturally present in foods, and it is also added during processing (heating) in foods such as fried tomato sauce and ketchup. Then, fructose or sucrose addition to processed and cooked foods could increase the carbohydrate-derived βC1−3in foods containing tryptophan. The βC1has been isolated from the juice of ripe fruits of N. tangutorum (desert cherry), and called tangutorids E (1a) and F (1b). 22 Other βCs arising from highly processing (dehydrating) conditions such as flazin and perlolidin (furfural derivatives of βCs) were also identified in those fruits. 22 However, as shown here, the βCs 1−3occurred in many foods including dried fruits (Table 1). Therefore, the classical nomenclature initially used for carbohydrate βCs 17 was preferred here because these compounds are mainly formed during processing/heating and usually do not appear in fresh fruits. Without more data on the biogenesis and quantitative levels of 1a/b in Nitraria fruits, it can be expected Figure 6. RP-HPLC chromatograms (A 254 nm) of PHP-THβC-3-COOH (200 μM, pH 5) before (A) and after heating at 140 °C for 4 h (B). The compound was redissolved when dried during heating. Journal of Agricultural and Food Chemistry pubs.acs.org/JAFC Article https://doi.org/10.1021/acs.jafc.1c02281 J. Agric. Food Chem. 2021, 69, 6650−6664 6658