beta-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.

Relative exposure to beta-carbolines norharman and harman from foods and tobacco smoke.

Norharman and harman are two heterocyclic beta-carboline (9H-pyrido[3,4-b]indole) alkaloids with biological and potential toxicological activity that appear in foodstuffs and environmental sources. To assess the occurrence and distribution of these compounds and to estimate the exposure levels based on the detected amounts, numerous samples of foodstuffs and cigarette smoke were analysed by solid-phase extraction and high-performance liquid chromatography-fluorescence. The levels found of beta-carbolines were highly variable. Low processed foodstuffs (i.e. milk, yoghurt, uncooked meats and fish) did not contain norharman and harman above the detection limit. Others, however, contained relatively high concentrations (at the tens of ng g(-1) or microg l(-1) level) depending on the processing conditions as, for example, 'well-done' cooked meat and fish. The highest amounts of norharman and harman were found in brewed coffee (29-207 microg l(-1)), sauces (soy sauce and Tabasco, among others; 4-252 microg l(-1)), 'well done' cooked meat and fish (57-160 ng g(-1)), toasted bread (42-160 ng g(-1)), and fermented alcoholic beverages (n.d.-41 mug l(-1)). beta-Carbolines also occurred in a high amount in the mainstream of cigarette smoke (207-2780 ng/cigarette), which is an important contributor to daily exposure to these compounds. Based on these results, it is concluded that the daily exposure to beta-carbolines in humans might be from tens to hundreds of micrograms, with cigarette smoke, coffee, certain seasonings, cooked foods and alcoholic beverages, in this order, being the major contributors. Many other foodstuffs might also contribute with minor amounts of norharman and harman. Foods and tobacco smoke might be potential contributors to the reported endogenous presence of beta-carbolines in humans.

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

Norharman and harman, two heterocyclic beta-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 beta-carboline alkaloid in brewed coffee at levels up to 9.34 microg g(-1) in instant ground coffee compared with harman, which had levels up to 1.67 microg g(-1). The two beta-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 beta-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 beta-carbolines are an important exogenous source of these alkaloids in humans.

Tetrahydro-beta-carboline-3-carboxylic acid compounds in fish and meat: possible precursors of co-mutagenic beta-carbolines norharman and harman in cooked foods.

The presence of tetrahydro-beta-carbolines and beta-carbolines was studied in raw, cooked and smoked fish and meat. 1,2,3,4-Tetrahydro-beta-carboline-3-carboxylic acid (THCA) usually was the major beta-carboline found, whereas 1-methyl-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid (MTCA) appeared in smoked and 'well done' cooked samples. THCA was detected in raw fish (nd-2.52 micrograms/g), cooked fish (nd-6.43 micrograms/g), cooked meats (nd-0.036 microgram/g), smoked fish (0.19-0.67 microgram/g) and smoked meats (0.02-1.1 micrograms/g). Smoked and cooked samples contained higher amounts of THCA and MTCA than raw products. Deep cooking of fish and meat increased both THCA and MTCA, and this was accompanied by the formation of more beta-carbolines, norharman and harman. The tetrahydro-beta-carbolines THCA and MTCA were chemical precursors of the co-mutagens norharman and harman during cooking. These and previous results confirm that foods are an important source of beta-carbolines in humans.

Tetrahydro-beta-carbolines, potential neuroactive alkaloids, in chocolate and cocoa.

Tetrahydro-beta-carbolines (THbetaCs), potential neuroactive alkaloids, were found in chocolate and cocoa. 6-Hydroxy-1-methyl-1,2, 3,4-tetrahydro-beta-carboline (6OHMTHbetaC), 1,2,3, 4-tetrahydro-beta-carboline-3-carboxylic acid (THCA), 1-methyl-1,2,3, 4-tetrahydro-beta-carboline-3-carboxylic acid (MTCA) in both diastereoisomers (1S,3S and 1R,3S), and 1-methyl-1,2,3, 4-tetrahydro-beta-carboline (MTHbetaC), besides serotonin and tryptamine biogenic amines, were identified and quantified in dark chocolate, milk chocolate, cocoa, and chocolate-containing cereals by RP-HPLC-fluorescence and HPLC-MS. For each THbetaC, the concentration ranges were determined: 6OHMTHbetaC (0.16-3.92 microg/g), THCA (0.01-0.85 microg/g), 1S,3S-MTCA (0.35-2 microg/g), 1R,3S-MTCA (0.14-0.88 microg/g), and MTHbetaC (nd-0.21 microg/g). The highest content was generally found in chocolates and cocoas, but cereals containing chocolate also showed an appreciable amount of THbetaCs. The possible biological implications of this novel group of alkaloids in chocolate are discussed.

Analysis of the bioactive alkaloids tetrahydro-beta-carboline and beta-carboline in food.

Simple tetrahydro-beta-carbolines (THbetaCs) and beta-carbolines (betaCs) are naturally occurring alkaloids in foods and food processing. This paper reviews the methods employed for their analysis. Procedures for THbetaC and betaC 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 THbetaCs with methyl chloroformate, or anhydrides are accomplished before GC-MS. Quantitative analysis of THbetaCs and betaCs 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 THbetaCs and betaCs. Fragmentation by collision induced dissociation or tandem mass spectrometry helps to complete trace identification. The occurrence of biologically relevant THbetaCs and betaCs in foods highlights the interest of accomplishing their analysis. Foods containing those compounds represent a source of possible THbetaCs and betaCs in humans.

Analysis of tetrahydro-beta-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.

1-methyl-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid and 1,2, 3,4-tetrahydro-beta-carboline-3-carboxylic acid in fruits.

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), as two diastereoisomers (1S,3S and 1R,3S), occurred in commercial fruits. Citrus fruits exhibited the highest content; other fruits contained very low levels or none at all. The content of MTCA was as follows: orange, 0.35-2.47 microg/g; lemon, 0.15-2.05 microg/g; grapefruit, 1.12-8.37 microg/g; mandarin, 0.57-2.5 microg/g; banana, nd-0.74 microg/g; pear, nd-0.017 microg/g; grape, 0.01-0.22 microg/g, tomato, 0.05-0.25 microg/g; and apple, nd-0.012 microg/g). THCA, if present, usually occurred at <0.05 microg/g. Fruit ripening and softening during storage were accompanied with a significant increase of MTCA, in both pears and bananas. Those and previous results confirm that foods are an important source of tetrahydro-beta-carbolines in humans.

Presence of tetrahydro-beta-carboline-3-carboxylic acids in foods by gas chromatography-mass spectrometry as their N-methoxycarbonyl methyl ester derivatives.

Various tetrahydro-beta-carboline-3-carboxylic acids (TH beta C-3-COOH) are identified in commercial foods and drinks by GC-MS. Positive identification of 1-methyl-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid (MTCA) is demonstrated in soy and tabasco sauces, wine, beer, wine vinegar, cider, orange juice, toasted bread, blue cheese and yoghurt. 1,2,3,4-Tetrahydro-beta-carboline-3-carboxylic acid (THCA) occurs in toasted bread, beer, cider, wine vinegar, soy and tabasco sauce, orange juice and blue cheese. MTCA and THCA are reported for the first time in several of these products. MTCA appears as a mixture of two diastereoisomers with the same mass spectra. MTCA is the major TH beta C-3-COOH in foodstuffs except for toasted bread that contains more THCA. GC-MS analysis of N-methoxycarbonyl methyl ester derivatives of TH beta C-3-COOHs was used for chemical identification. Those derivatives were synthesized in a qualitatively using methyl chloroformate or methyl chloroformate and diazomethane reagents. Electron impact mass spectra of N-methoxycarbonyl-TH beta C-3-COOH methyl esters are reported and fragmentation assigned and discussed. These results prove the presence of TH beta C-3-COOHs in commercial foodstuffs suggesting their uptake during the daily consumption of foods.

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.

Reversed-phase HPLC analysis of peptides in standard and dairy samples using on-line absorbance and post-column OPA-fluorescence detection.

Absorbance and post-column o-phthalaldehyde (OPA)-fluorescent detection were used to analyse standard and dairy peptides following reverse-phase HPLC. Using both detection systems on-line provides additional information on the presence of peptides in dairy products. The detection response depends on the amino acid composition of the peptide involved. Among the peptides used, glutathione, lysine-containing peptides and peptides with glycine as the N-terminal residue give the highest fluorescence after the OPA post-column reaction. Absorbance is more sensitive than fluorescence for peptides with aromatic amino acids. Different parameters, such as the flow rate of OPA, the amount of mercaptoethanol in the OPA reagent and the temperature of reaction, substantially influence the fluorescent response of peptides. The interest of using on-line absorbance and fluorescence detection is highlighted by analysing peptides from skim milk and from a tryptic hydrolysate.