Tetrahydro-β-carboline Alkaloids that Occur in Foods and Biological Systems Act as Radical Scavengers and Antioxidants in the ABTS Assay

Tetrahydro- &#103 -carboline alkaloids that occur in foods such as wine, seasonings, vinegar and fruit products (juices, jams) acted as good radical scavengers (hydrogen- or electron donating) in the ABTS (2,2'-Azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)) assay, and therefore, they could contribute to the beneficial antioxidant capacity attributed to foods. In contrast, the fully aromatic &#103 -carbolines norharman and harman did not show any radical scavenger activity in the same assay. During the reaction with ABTS^.++ radical cation, tetrahydro- &#103 -carboline-3-carboxylic acid such as 1-methyl-1,2,3,4-tetrahydro- &#103 -carboline-3-carboxylic acid (MTCA) and 1-methyl-1,2,3,4-tetrahydro- &#103 -carboline-1,3-dicarboxylic acid (MTCA-COOH) were converted to harman, whereas 1,2,3,4-tetrahydro- &#103 -carboline-3-carboxylic acid (THCA) and 1,2,3,4-tetrahydro- &#103 -carboline-1,3-dicarboxylic acid (THCA-COOH) afforded norharman. These results suggest that food and naturally-occurring tetrahydro- &#103 -carboline alkaloids if accumulated in tissues, as reported elsewhere, might exhibit antioxidant activity.     

Naturally-occurring tetrahydro-β-carboline alkaloids derived from tryptophan are oxidized to bioactive β-carboline alkaloids by heme peroxidases

β-Carbolines are indole alkaloids that occur in plants, foods, and endogenously in mammals and humans, and which exhibit potent biological, psychopharmacological and toxicological activities. They form from naturally-occurring tetrahydro-β-carboline alkaloids arising from tryptophan by still unknown way and mechanism. Results in this research show that heme peroxidases catalyzed the oxidation of tetrahydro-β-carbolines (i.e. 1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid and 1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid) into aromatic β-carbolines (i.e. norharman and harman, respectively). This oxidation followed a typical catalytic cycle of peroxidases through redox intermediates I, II, and ferric enzyme. Both, plant peroxidases (horseradish peroxidase, HRP) and mammalian peroxidases (myeloperoxidase, MPO and lactoperoxidase, LPO) catalyzed the oxidation in an efficient manner as determined by kinetic parameters (V_MAX and K_M). Oxidation of tetrahydro-β-carbolines was inhibited by peroxidase inhibitors such as sodium azide, ascorbic acid, hydroxylamine and excess of H₂O₂. The formation of aromatic β-carbolines by heme peroxidases can help to explain the presence and activity of these compounds in biological systems.     

Mutagenicities of nitrosated carboline derivatives

Food-borne amines have been considered as the potential precursors of endogenous carcinogenic N-nitroso compounds in humans. A compound which yields a direct mutagen after nitrite treatment was isolated from soy sauce and was identified as 1-methyl-1,2,3,4-tetrahydro-2-carboline-3-carboxylic acid (MTCA) (Wakabayashi, et al., 1983). The mutagenicities of other carboline derivatives such as harman, norharman, harmaline, harmalol, harmine, and harmol were studied. Like MTCA, the nitrosated carboline derivatives showed higher mutagenic activity as compared to their corresponding parent compounds. The demethylated analogue of MTCA, 1,2,3,4-tetrahydro-2-carboline-3-carboxylic acid was synthesized and its nitrosated products were shown to be mutagenic to Salmonella typhimurium TA 100 and TA 98. The potent mutagen Trp-P-2 is a typical 3-carboline derivative. The mutagenicity of Trp-P-2 was suppressed remarkably after nitrosation. Several 3-carboline derivatives also showed the similar property. Nitrosation of MTCA gave several derivatives which were isolated and showed direct mutagenicity to Salmonella typhimurium TA 98. Further characterization of these new carboline derivatives is in progress.     

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.     

The occurrence of 2-methyl-1,2,3,4-tetrahydro-β-carboline and variation in alkaloids in Phalaris arundinacea

2-Methyl-1,2,3,4-tetrahydro-β-carboline was isolated from reed canarygrass (Phalaris arundinacea L.) and the occurrence of 2-methyl-6-methoxy-1,2,3,4-tetrahydro-β-carboline confirmed. Clones of reed canarygrass that contained N,N-dimethyltryptamine or 2-methyl-1,2,3,4-tetrahydro-β-carboline did not contain their respective methoxy or hydroxy derivatives. Five of the 12 clones tested contained either or both of 5-methoxy-N,N-dimethyltryptamine and 2-methyl-6-methoxy-1,2,3,4-tetrahydro-β-carboline. The data suggest that clones that contain gramine are not likely to contain N,N-dimethyltryptamine and/or β-carbolines. Thus, an inverse biosynthetic relationship between gramine and the tryptamine and β-carboline alkaloids seems to exist. However, further work is needed to firmly establish any such relationship between these alkaloids.     

Phenylethanolamine N-methyltransferase has beta-carboline 2N-methyltransferase activity: hypothetical relevance to Parkinson's disease

Mammalian brain has a β-carboline 2N-methyltransferase activity that converts β-carbolines, such as norharman and harman, into 2N-methylated β-carbolinium cations, which are structural and functional analogs of the Parkinsonian-inducing toxin 1-methyl-4-phenylpyridinium cation (MPP⁺). The identity and physiological function of this β-carboline 2N-methylation activity was previously unknown. We report pharmacological and biochemical evidence that phenylethanolamine N-methyltransferase (EC 2.1.1.28) has β-carboline 2N-methyltransferase activity. Specifically, purified phenylethanolamine N-methyltransferase (PNMT) catalyzes the 2N-methylation (21.1 pmol/h per unit PNMT) of 9-methylnorharman, but not the 9N-methylation of 2-methylnorharmanium cation. LY134046, a selective inhibitor of phenylethanolamine N-methyltransferase, inhibits (IC₅₀ 1.9 μM) the 2N-methylation of 9-methylnorharman, a substrate for β-carboline 2N-methyltransferase. Substrates of phenylethanolamine N-methyltransferase also inhibit β-carboline 2N-methyltransferase activity in a concentration-dependent manner. β-Carboline 2N-methyltransferase activity (43.7 pmol/h/mg protein) is present in human adrenal medulla, a tissue with high phenylethanolamine N-methyltransferase activity.

We are investigating the potential role of N-methylated β-carbolinium cations in the pathogenesis of idiopathic Parkinson’s disease. Presuming that phenylethanolamine N-methyltransferase activity forms toxic 2N-methylated β-carbolinium cations, we propose a novel hypothesis regarding Parkinson’s disease—a hypothesis that includes a role for phenylethanolamine N-methyltransferase-catalyzed formation of MPP⁺-like 2N-methylated β-carbolinium cations.     

柴达木盆地唐古特白刺种子的化学成分研究

从唐古特白刺种子75%乙醇提取物中分离得到6种化合物,应用波谱方法及与文献对照的手段鉴定为:胡萝卜苷(daucosterol,1),4-hydroxypipecolic acid(2),槲皮素(quercetin,3),尿囊素(allantoin,4),1,2,3,4-tet-rahydro-1-methyl-β-carboline-3-carboxylic acid(5),L-tyrosine(6)。除槲皮素外的其他五种化合物均为首次从该植物中分离得到。     

Synthesis and receptor binding of oxytocin analogs containing conformationally restricted amino acids

Summary We report the solid-phase synthesis and receptor-binding properties of eleven oxytocin analogs (Mpa-Xxx-Ile-Gln-Asn-Cys-Sar-Arg-Gly-NH₂) containing non-coded amino acids in position 2: D-α- and L-α-(2-indanyl)glycine, R,S-6-methoxy-2-aminotetralin-2-carboxylic acid, D- and L-pentafluorophenylalanine, D,L-2,4-dimethylphenylalanine, D,L-2,4,6-trimethylphenylalanine, R,R- and S,S-1,2,3,4-tetrahydro-1-methyl-β-carboline-3-carboxylic acid and R- and S-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid. Some of these amino acid analogs (2-indanylglycine and D-pentafluorophenylalanine) were earlier successfully applied for the synthesis of potent bradykinin antagonists [1, 2]. Their receptor bindings were tested on isolated guinea-pig uterus, rat liver and rat kidney inner medulla plasma membranes. The extent of binding of the peptides to the oxytocin receptor was in several cases was even higher than that of the parent hormone (oxytocin). However, the real pharmacological value of these analogs can be evaluated only afterin vivo measurements of their inhibition of uterine motor activity.     

Antioxidant effects of tetrahydro-beta-carboline derivatives identified in aged garlic extract

1,2,3,4-Tetrahydro-beta-carboline derivatives (THbetaCs) are formed through Pictet-Spengler chemical condensation between tryptophan and aldehydes during food production, storage and processing. In the present study, in order to identify the antioxidants in aged garlic extract (AGE), we fractionated it and identified four THbetaCs; 1-methyl-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acids (MTCC) and 1-methyl-1,2,3,4-tetrahydro-beta-carboline-1,3-dicarboxylic acid (MTCdiC) in both diastereoisomers using liquid chromatography mass spectrometry (LC-MS). Interestingly, these compounds were not detected in raw garlic, but the contents increased during the natural aging process of garlic. In in vitro assay systems, all of these compounds have shown strong hydrogen peroxide scavenging activities. (1S, 3S)-MTCdiC was found to be stronger than the common antioxidant, ascorbic acid. MTCC and MTCdiC inhibited AAPH-induced lipid peroxidation. Both MTCdiCs also inhibited LPS-induced nitrite production from murine macrophages at 10-100 microM. Our data suggest that these compounds are potent antioxidants in AGE, and thus may be useful for prevention of disorders associated with oxidative stress.     

Evaluation of the oxidation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to toxic pyridinium cations by monoamine oxidase (MAO) enzymes and its use to search for new MAO inhibitors and protective agents

Monoamine oxidase (MAO) enzymes catalyze the oxidative deamination of amines and neurotransmitters and inhibitors of MAO are useful as neuroprotectants. This work evaluates the human MAO-catalyzed oxidation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a dopaminergic neurotoxin, to the directly-acting neurotoxic metabolites, 1-methyl-4-phenyl-2,3-dihydropyridinium (MPDP⁺) and 1-methyl-4-phenylpyridinium (MPP⁺) measured by High-Performance Liquid Chromatography (HPLC), and this approach is subsequently used as a new method for screening of MAO inhibitors and protective agents. Oxidation of MPTP by human MAO-B was more efficient than by MAO-A. R-Deprenyl, a known neuroprotectant, norharman (β-carboline), 5-nitroindazole and menadione (vitamin K3) inhibited MAO-B and reduced the formation of toxic pyridinium cations. Clorgyline and the β-carbolines, harman and norharman, inhibited the oxidation of MPTP by MAO-A. Cigarette smoke, as well as the naturally occurring β-carbolines (norharman and harman) isolated from smoke and coffee inhibited the oxidation of MPTP by MAO-B and/or MAO-A, suggesting protective effects against MPTP. The results show the suitability of the approach used to search for new MAO inhibitors with eventual neuroprotective activity.     

Side reactions in the preparation of 1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid

Summary During a study on the preparation of the conformationally restricted analogue of tryptophan into 1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid1 by a Pictet-Spengler condensation with formaldehyde, two site products were detected:N-hydroxymethyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid2 and a dimer3 of two 1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid units linked by a methylene group. Their structures were determined by HPLC-MS and 2D NMR spectroscopy. By changing the isolation procedure, theN-hydroxymethyl compound was removed. Treatment of the mixture with TFA in water converted the dimer into 1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid1.     

Effects of harman and norharman on the mutagenicity and binding to DNA of benzo[a]pyrene metabolites in vitro and on aryl hydrocarbon hydroxylase induction in cell culture

Harman and norharman, two β-carboline derivatives known to exist in certain foods and to be formed during pyrolysis of tobacco and meat, were tested for mutagenic activity in the presence of benzo[a]pyrene, mouse liver enzymes, and Salmonella typhimurium TA98 in vitro. Both harman and norharman inhibit benzo[a]pyrene mutagenicity, benzo[a]pyrene metabolism (as measured by aryl hydrocarbon hydroxylase activity), and the binding of all benzo[a]pyrene metabolites to DNA in vitro. Moreover, harman and norharman are quite toxic to cultures of hepatoma-derived H-4-II-E and Hepa-1 established cell lines and therefore were found to be very weak inducers of aryl hydrocarbon hydroxylase activity.     

New alkaloids from Banisteriopsis caapi

Three new alkaloids isolated from Banisteriopsis caapi, were identified as harmic amide (1-carbamoyl-7-methoxy β-carboline), acetyl norharmine (1-acetyl-7-methoxy β-carboline) and ketotetrahydronorharmine (7-methoxy-1,2,3,4-tetrahydro-1-oxo-β-carboline)     

Pharmacokinetic and pharmacodynamic factors contributing to the convulsant action of β-carboline-3-carboxylic acid esters

Both the methyl ester of β-carboline-3-carboxylic acid and the 6, 7-dimethoxy-4-ethyl derivative of this compound are potent convulsants in rodents, while the ethyl ester of β-carboline-3-carboxylic acid does not cause convulsions, even when administered at very high doses. The rate of degradation of these compounds by rat plasma ($\text{in vitro}$) parallels their potencies as convulsants. In contrast, 3-carboethoxy-β-carboline was found to potently elicit tonic and clonic convulsions in the squirrel monkey ($\text{Saimiri sciureus}$). Furthermore, the rate of degradation of 3-carboethoxy-β-carboline in monkey plasma ($\text{in vitro}$) is negligible compared with rats. No significant differences were observed in either the potency or efficacy of GABA to inhibit [³H] β-carboethoxy-β-carboline binding in rat and monkey brain. These data strongly suggest that pharmacokinetic, as well as pharmacodynamic, factors may determine the pharmacologic profile of these β-carboline-3-carboxylic acid esters.     

Synthesis and receptor binding of oxytocin analogs containing conformationally restricted amino acids

We report the solid-phase synthesis and receptor-binding properties of eleven oxytocin analogs (Mpa-Xxx-Ile-Gln-Asn-Cys-Sar-Arg-Gly-NH₂) containing non-coded amino acids in position 2: D-- and L--(2-indanyl)glycine, R,S-6-methoxy-2-aminotetralin-2-carboxylic acid, D- and L-pentafluorophenylalanine, D,L-2,4-dimethylphenylalanine, D,L-2,4,6-trimethylphenylalanine, R,R- and S,S-1,2,3,4-tetrahydro-1-methyl--carboline-3-carboxylic acid and R- and S-1,2,3,4-tetrahydro--carboline-3-carboxylic acid. Some of these amino acid analogs (2-indanylglycine and D-pentafluorophenylalanine) were earlier successfully applied for the synthesis of potent bradykinin antagonists [1,2]. Their receptor bindings were tested on isolated guinea-pig uterus, rat liver and rat kidney inner medulla plasma membranes. The extent of binding of the peptides to the oxytocin receptor was in several cases was even higher than that of the parent hormone (oxytocin). However, the real pharmacological value of these analogs can be evaluated only after in vivo measurements of their inhibition of uterine motor activity.     

Kinetics of Parallel Electron Transfer from β-Carotene to Phenoxyl Radical and Adduct Formation Between Phenoxyl Radical and β-Carotene

Phenoxyl radicals generated by laser flash photolysis were found to react with β-carotene with concomitant β-carotene bleaching in two parallel reactions with similar rates: (i) formation of a β-carotene adduct with a (pseudo) first order rate constant of 1-1.5 ± 10⁴ s^-1 with absorption maximum around 800 nm, and (ii) formation of a β-carotene radical cation with a (pseudo) first order rate constant of 2-3 ± 10⁴ s^-1 with absorption maximum around 920 nm. Both β-carotene radicals decay on a similar time scale and have virtually disappeared after 100 ms, the β-carotene adduct by a second order process. Oxygen had no effect on β-carotene bleaching or radical formation and decay. The reduction of phenoxyl radicals by β-carotene may prove important for an understanding of how β-carotene acts as an antioxidant.     

Asymmetric bioreduction of racemic 5,6,7,8-tetrahydro-8-methyl-1,3-dimethoxynaphthalen-6-one to the corresponding chiral β-tetralols

The synthesis of racemic 5,6,7,8-tetrahydro-8-methyl-1,3-dimethoxynaphthalen-6-one 1 was performed and the bioreduction to the corresponding β-tetralols was studied with respect to the stereochemical course and optical purity of the products; in particular the 6S,8S enantiomer corresponding to the dimethyl derivative of the natural compound feroxidin was isolated. The biomass of: Aspergillus niger, Cladosporium cladosporioides, Candida lypolitica, Bacillus megatherium, Rhodotorula minuta, R. flava, R. rubra, Beauveria bassiana and Baker's yeast were used as biocatalysts. Relative and absolute configurations of the obtained β-tetralols were established by comparison with those of the natural feroxidin.     

Simultaneous determination of l-tryptophan impurities in meat products

L-tryptophan has been used as a feed additive for swine and poultry and as a nutrient supplement for humans. However, some impurities in l-tryptophan have been reported as causative components of eosinophilia-myalgia syndrome. Therefore, from a safety perspective, it is important to analyze meat samples for these impurities. This study aims to develop an analytical method for the simultaneous detection of l-tryptophan impurities in meat products using LC–MS/MS. Among the various impurities, detection methods for (S)-2-amino-3-(5-hydroxy-1H-indol-3-yl)propanoic acid (5-hydroxytryptophan) (HTP), 1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (MTCA), 3a-hydroxy-1,2,3,3a,8,8a-hexahydropyrrolo-[2,3-b]-indole-2-carboxylic acid (PIC), and 1,1′-ethylidenebistryptophan (EBT) and 2-(3-indoylmethyl)-l-tryptophan (IMT) were developed. The developed method allowed simultaneous determination of these four impurities in 5 min. No interferences from the matrix were observed, and the method showed good sensitivity to each analyte. The method detection limit and limit of quantification in meat matrices were below 11.2 and 35.7 μg/kg, respectively.

     

Occurrence of Stereoisomers of 1-(2′-Pyrrolidinethione-3′-yl)-1,2,3,4- tetrahydro-β-carboline-3-carboxylic Acid in Fermented Radish Roots and Their Different Mutagenic Properties

Stereoisomers of the tetrahydro-β-carboline derivative, 1-(2′-pyrrolidinethione-3′-yl)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (PTCC), were formed from L-tryptophan with 4-methylthio-3-butenyl isothiocyanate, and their mutagenic properties and contents in different types of the radish products were studied. The isomers were identified as (1S ^*, 3S ^*, 3′R ^*)- and (1R ^*, 3S ^*, 3′R ^*)-PTCCs; the former was found as the major compound but had no mutagenic activity, while the latter was mutagenic toward Salmonella typhimurium TA 98 in the presence of a rat microsomal fraction. Both (1S ^*, 3S ^*, 3′R ^*)- and (1R ^*, 3S ^*, 3′R ^*)-PTCC were detected in a ratio of about 4:1 in a product fermented for 8 months, but only a trace was apparent in products manufactured within a few weeks.