Tomás Herraiz

β-Carboline alkaloids in Peganum harmala and inhibition of human monoamine oxidase (MAO)

Peganum harmala L. is a multipurpose medicinal plant increasingly used for psychoactive recreational purposes (Ayahuasca analog). Harmaline, harmine, harmalol, harmol and tetrahydroharmine were identified and quantified as the main beta-carboline alkaloids in P. harmala extracts. Seeds and roots contained the highest levels of alkaloids with low levels in stems and leaves, and absence in flowers. Harmine and harmaline accumulated in dry seeds at 4.3% and 5.6% (w/w), respectively, harmalol at 0.6%, and tetrahydroharmine at 0.1% (w/w). Roots contained harmine and harmol with 2.0% and 1.4% (w/w), respectively. Seed extracts were potent reversible and competitive inhibitors of human monoamine oxidase (MAO-A) with an IC(50) of 27 microg/l whereas root extracts strongly inhibited MAO-A with an IC(50) of 159 microg/l. In contrast, they were poor inhibitors of MAO-B. Inhibition of MAO-A by seed extracts was quantitatively attributed to harmaline and harmine whereas inhibition by root extracts came from harmine with no additional interferences. Stems and leaves extracts were poor inhibitors of MAO. The potent inhibition of MAO-A by seed and root extracts of P. harmala containing beta-carbolines should contribute to the psychopharmacological and toxicological effects of this plant and could be the basis for its purported antidepressant actions.

Endogenous and dietary indoles: a class of antioxidants and radical scavengers in the ABTS assay.

Indoles are very common in the body and diet and participate in many biochemical processes. A total of twenty-nine indoles and analogs were examined for their properties as antioxidants and radical scavengers against 2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) ABTS•+ radical cation. With only a few exceptions, indoles reacted nonspecifically and quenched this radical at physiological pH affording ABTS. Indoleamines like tryptamine, serotonin and methoxytryptamine, neurohormones (melatonin), phytohormones (indoleacetic acid and indolepropionic acid), indoleamino acids like l-tryptophan and derivatives (N-acetyltryptophan, l-abrine, tryptophan ethyl ester), indolealcohols (tryptophol and indole-3-carbinol), short peptides containing tryptophan, and tetrahydro-β-carboline (pyridoindole) alkaloids like the pineal gland compound pinoline, acted as radical scavengers and antioxidants in an ABTS assay-measuring total antioxidant activity. Their trolox equivalent antioxidant capacity (TEAC) values ranged from 0.66 to 3.9 mM, usually higher than that for Trolox and ascorbic acid (1 mM). The highest antioxidant values were determined for melatonin, 5-hydroxytryptophan, trp-trp and 5-methoxytryptamine. Active indole compounds were consumed during the reaction with ABTS•+ and some tetrahydropyrido indoles (e.g. harmaline and 1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid ethyl ester) afforded the corresponding fully aromatic β-carbolines (pyridoindoles), that did not scavenge ABTS•+. Radical scavenger activity of indoles against ABTS•+ was higher at physiological pH than at low pH. These results point out to structural compounds with an indole moiety as a class of radical scavengers and antioxidants. This activity could be of biological significance given the physiological concentrations and body distribution of some indoles.

Analysis of the bioactive alkaloids tetrahydro-β-carboline and β-carboline in food

Abstract Simple tetrahydro-β-carbolines (THβCs) and β-carbolines (βCs) are naturally occurring alkaloids in foods and food processing. This paper reviews the methods employed for their analysis. Procedures for THβC and βC isolation and clean-up to remove interfering compounds are carried out by liquid–liquid extraction, and/or better solid-phase extraction under both reversed-phase (C18) and cation-exchange mechanisms. Chemical derivatizations of THβCs with methyl chloroformate, or anhydrides are accomplished before GC–MS. Quantitative analysis of THβCs and βCs is made by RP-HPLC (C18) with fluorescence detection providing good selectivity and sensitivity. For the same reasons, HPLC–MS is increasingly applied to these compounds. Electrospray and atmospheric pressure chemical ionization easily produce protonated molecules (M+H)+ of both THβCs and βCs. Fragmentation by collision induced dissociation or tandem mass spectrometry helps to complete trace identification. The occurrence of biologically relevant THβCs and βCs in foods highlights the interest of accomplishing their analysis. Foods containing those compounds represent a source of possible THβCs and βCs in humans.

Identification and occurrence of the bioactive β-carbolines norharman and harman in coffee brews

Norharman and harman, two heterocyclic β-carboline alkaloids with biological activity, were found in brewed coffee. Identification and analysis were carried out by HPLC-MS and RP-HPLC-fluorescence, respectively. All tested samples of brewed coffee including ground coffee, decaffeinated coffee, instant coffee and espresso contained both norharman and harman in variable amounts. Norharman was the major β-carboline alkaloid in brewed coffee at levels up to 9.34 μg g-1 in instant ground coffee compared with harman, which had levels up to 1.67 μg g-1. The two β-carbolines appeared to be formed during roasting of the coffee beans. It is concluded that drinking coffee is a major exogenous dietary source of these bioactive β -carboline alkaloids previously reported as mild psychoactive compounds in animal studies and in vitro co-mutagens. These results support our previous conclusion that foods containing β -carbolines are an important exogenous source of these alkaloids in humans.

Oxidative Metabolism of the Bioactive and Naturally Occurring β-Carboline Alkaloids, Norharman and Harman, by Human Cytochrome P450 Enzymes

Norharman and harman are naturally occurring beta-carboline alkaloids exhibiting a wide range of biological, psychopharmacological, and toxicological actions. They occur in foods and tobacco smoke and also appear endogenously in humans. In this research, metabolic and kinetic studies with cytochrome P450 enzymes and human liver microsomes showed that beta-carbolines were efficiently oxidized to several ring-hydroxylated and N-oxidation products that were subsequently identified and quantified. 6-Hydroxy- beta-carboline (6-hydroxynorharman and 6-hydroxyharman) was a major metabolite efficiently produced (high kcat and low Km) by P450 1A2 and 1A1 and to a minor extent by P450 2D6, 2C19 and 2E1. 3-Hydroxy-beta-carboline (3-hydroxynorharman and 3-hydroxyharman), another major metabolite, was specifically produced by P450 1A2 and 1A1, whereas beta-carboline-N(2)-oxide (harman-2-oxide and norharman-2-oxide) was produced by P450 2E1. The same pattern of metabolism was confirmed for human liver microsomes. Oxidative metabolism for harman was slightly higher than norharman, but norharman showed lower Km values. The oxidation of beta-carbolines is a detoxication route performed mainly by P450 1A2 and 1A1, with the participation of P450 2D6, 2C19, and 2E1, as additional contributors. Then, individual variations in the levels and activity of these P450s may influence biotransformation of beta-carboline alkaloids and their ultimate biological effects. beta-Carbolines were previously reported as comutagens and/or inhibitors of mutagens activated by P450 1A enzymes such as heterocyclic amines and polycyclic hydrocarbons. Results in this work show that beta-carbolines are good ligands and substrates for P450 1A2/1A1, contributing to the explanation of some of their toxicological effects.

Evaluation of solid-phase extraction procedures in peptide analysis

Solid-phase extraction (SPE) procedures for peptide isolation and fractionation, based on non-polar and ionic interactions, were evaluated using small synthetic peptides and casein enzymatic hydrolysates. SPE based on hydrophobic phases is a useful, efficient and rapid procedure for peptide extraction and concentration. It allows a successful peptide fractionation using eluents that contain an increasing content of acetonitrile in the presence of trifluoroacetic acid. Differences regarding selectivity are observed between sorbents. Non-polar interaction with C18 sorbents is adequate for the isolation of very polar and hydrophobic peptides. CN sorbents are only adequate for very hydrophobic peptides. PH, CH, C8 and C2 sorbents are useful for isolating and fractionating hydrophobic and very non-polar peptides, but generally not for very polar peptides. Ionic solid-phase extraction using Accell Plus cartridges of QMA (quaternary methylammonium) and CM (carboxymethyl) are very useful for the fractionation of peptide mixtures into basic, acidic and neutral pools of peptides. It can be concluded that SPE using these procedures is a useful tool for the isolation and fractionation of peptides from biological and food samples.

Comparative prediction of the retention behaviour of small peptides in several reversed-phase high-performance liquid chromatography columns by using partial least squares and multiple linear regression

The retention behaviour of small standard peptides in reversed-phase high-performance liquid chromatography (RP-HPLC) is evaluated. Chromatographic retention of small peptides highly correlates with the summing of the hydrophobic contributions of their respective amino acid residues. Prediction of peptide retention time is accomplished in four different RP columns (Ultrasphere Octyl, Ultrasphere ODS, Polymeric reversed phase PLRP-S and Nova-pak C-18) under the same chromatographic conditions. The elution profiles of a mixture of 25 small standard peptides are highly correlated among the four RP columns. In addition, RP columns exhibit similar behaviour as far as the pattern of amino acid contribution to the retention of small peptides is concerned. Comparative prediction of peptide retention times is carried out using Multiple Linear Regression (MLR) and Partial Least Squares (PLS) regression. PLS regression provides better prediction ability than MLR. Following the PLS procedure, a reasonable estimation of peptide retention times, after calculating the corresponding amino acid retention coefficients, is possible.

Sample preparation and reversed phase-high performance liquid chromatography analysis of food-derived peptides

Abstract Reversed phase-high performance liquid chromatography (RP-HPLC) is an indispensable technique in food peptide research. This paper briefly reviews the methods for isolation and RP-HPLC analysis of food-derived peptides. A critical aspect is the sample preparation and clean-up. For this, selective precipitation, ultrafiltration, size exclusion chromatography, ion-exchange chromatography and solid phase extraction (SPE) are used. SPE with non-polar or ion-exchange cartridges, is an effective and rapid method for peptide isolation, fractionation and concentration. RP-HPLC is accomplished on C 18 silica columns using shallow gradients of acetonitrile containing 0.1% TFA. Detection is by far-UV absorbance although selective detection by fluorometry on-line provides complementary and selective information. HPLC coupling to mass spectrometry (MS) and tandem MS/MS is increasingly applied to food-derived peptides. The research topics and the analytical strategies dealing with food-derived peptides are summarized.

Inhibition of the bioactivation of the neurotoxin MPTP by antioxidants, redox agents and monoamine oxidase inhibitors

Monoamine oxidase (MAO) enzymes located in human mitochondria oxidize neurotransmitters and bioactivate the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) by oxidation to directly-acting neurotoxic pyridinium cations (MPDP⁺/MPP⁺) that produce Parkinsonism. Antioxidants and MAO inhibitors are useful as neuroprotectants. Naturally-occurring substances, antioxidants and redox agents were assessed as inhibitors of the oxidation (bioactivation) of MPTP by human mitochondria and MAO enzymes. Methylene blue, 5-nitroindazole, norharman (β-carboline), 9-methylnorharman (9-methyl-β-carboline) and menadione (vitamin-K analogue) highly inhibited the oxidation of MPTP to the neurotoxic species, MPDP⁺/MPP⁺, in human mitochondria (IC₅₀ of 0.18, 3.1, 9.9, 7.3, and 12.6 μM, respectively). Inhibition by methylene blue was similar to R-deprenyl (IC₅₀ of 0.15 μM), a known neuroprotectant. The naturally-occurring β-carbolines, harmine, harmaline and tetrahydro-β-carboline, and the antioxidants, melatonin, resveratrol, quercetin and catechin showed little or no inhibition. Oxidation of MPTP in mitochondria was performed by human MAO-B and the above active compounds were also inhibitors of this isozyme. Norharman and 5-nitroindazole were competitive inhibitors of MAO-B whereas methylene blue inhibited MPTP oxidation (IC₅₀ of 50 nM) under a mixed type and predominantly uncompetitive mechanism. Methylene blue, 5-nitroindazole, norharman, 9-methylnorharman and menadione inhibit MAO-B in mitochondria and afford protective effects, as suggested by a reduced conversion of MPTP to neurotoxic species.

Analysis of tetrahydro-β-carboline-3-carboxylic acids in foods by solid-phase extraction and reversed-phase high-performance liquid chromatography combined with fluorescence detection.

The presence and analysis of two tetrahydro-beta-carboline-3-carboxylic acids in foods are studied. Sample preparation with benzenesulfonic acid strong cation-exchange columns followed by RP-HPLC-fluorescence allowed a reliable analysis and spectral characterization of 1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid (THCA) and 1-methyl-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid (MTCA). Experimental data showed that upon oxidation tetrahydro-beta-carboline-3-carboxylic acids gave rise to beta-carbolines (norharman and harman) that were also chromatographically separated and their fluorescent profile monitored. This approach was useful to confirm identification of tetrahydro-beta-carboline-3-carboxylic acids in foods. Several foods and beverages contained THCA and MTCA in varying proportions. Their occurrence in foods implies that diet is a source of these compounds in humans.