Effects of beta-carboline alkaloids on the object recognition task in mice

beta-carboline alkaloids are found in several medicinal plants and display a variety of actions on the central nervous, muscular and cardiovascular systems. The aim of the present study was to evaluate the effects of systemic administration of beta-carboline alkaloids on object recognition in mice. Adult Swiss mice received an intra-peritoneal injection (i.p.) of alkaloids (1.0, 2.5 or 5.0 mg/kg) 30 min before training in an object recognition task. The fully aromatic beta-carbolines, harmine and harmol, induced an enhancement of short-term memory (STM) at all doses tested when compared to controls. Harmaline, a dihydro beta-carboline and inverse agonist of the MK-801 binding site on the N-methyl-d-aspartate (NMDA) receptor, also induced an enhancement of both short-term memory (STM) and long-term memory (LTM). These results demonstrate that systemic administration of beta-carboline alkaloids can improve object recognition memory in mice.     

Protective effect of harmalol and harmaline on MPTP neurotoxicity in the mouse and dopamine-induced damage of brain mitochondria and PC12 cells

The present study elucidated the protective effect of beta-carbolines (harmaline, harmalol, and harmine) on oxidative neuronal damage. MPTP treatment increased activities of total superoxide dismutase, catalase, and glutathione peroxidase and levels of malondialdehyde and carbonyls in the basal ganglia, diencephalon plus midbrain of brain compared with control mouse brain. Coadministration of harmalol (48 mg/kg) attenuated the MPTP effect on the enzyme activities and formation of tissue peroxidation products. Harmaline, harmalol, and harmine attenuated both the 500 microM MPP(+)-induced inhibition of electron flow and membrane potential formation and the 100 microM dopamine-induced thiol oxidation and carbonyl formation in mitochondria. The scavenging action of beta-carbolines on hydroxyl radicals was represented by inhibition of 2-deoxy-D-ribose degradation. Harmaline and harmalol (100 microM) attenuated 200 microM dopamine-induced viability loss in PC12 cells. The beta-carbolines (50 microM) attenuated 50 microM dopamine-induced apoptosis in PC12 cells. The compounds alone did not exhibit significant cytotoxic effects. The results indicate that beta-carbolines attenuate brain damage in mice treated with MPTP and MPP(+)-induced mitochondrial damage. The compounds may prevent dopamine-induced mitochondrial damage and PC12 cell death through a scavenging action on reactive oxygen species and inhibition of monoamine oxidase and thiol oxidation.     

Mitochondrial dysfunction and biotransformation of β-carboline alkaloids,harmine and harmaline,on isolated rat hepatocytes

The cytotoxic effects and biotransformation of harmine and harmaline, which are known β-carboline alkaloids and potent hallucinogens, were studied in freshly isolated rat hepatocytes. The exposure of hepatocytes to harmine caused not only concentration (0–0.50 mM)- and time (0–3 h)-dependent cell death accompanied by the formation of cell blebs and the loss of cellular ATP, reduced glutathione, and protein thiols but also the accumulation of glutathione disulfide. Of the other analogues examined, the cytotoxic effects of harmaline and harmol (a metabolite of harmine) at a concentration of 0.5 mM were less than those of harmine. The loss of mitochondrial membrane potential and generation of oxygen radical species in hepatocytes treated with harmine were greater than those with harmaline and harmol. In the oxygen consumption of mitochondria isolated from rat liver, the ratios of state-3/state-4 respiration of these β-carbolines were decreased in a concentration-dependent manner. In addition, harmine resulted in the induction of the mitochondrial permeability transition (MPT), and the effects of harmol and harmaline were less than those of harmine. At a weakly toxic level of harmine (0.25 mM), it was metabolized to harmol and its monoglucuronide and monosulfate conjugates, and the amounts of sulfate rather than glucuronide predominantly increased with time. In the presence of 2,5-dichloro-4-nitrophenol (50 μM; an inhibitor of sulfotransferase), harmine-induced cytotoxicity was enhanced, accompanied by decrease in the amount of harmol-sulfate conjugate, due to an increase in the amount of unconjugated harmol and the inhibition of harmine loss. Taken collectively, these results indicate that (a) mitochondria are target organelles for harmine, which elicits cytotoxicity through mitochondrial failure related to the induction of the MPT, mitochondrial depolarization, and inhibition of ATP synthesis; and (b) the toxic effects of harmine are greater than those of either its metabolite harmol or its analogue harmaline, suggesting that the onset of harmine-induced cytotoxicity may depend on the initial and/or residual concentrations of harmine rather than on those of its metabolites.     

Beta-carboline and quinoline alkaloids in root cultures and intact plants of Peganum harmala

Alkaloid profiles of root and shoot cultures, seedlings and mature plants were analysed by capillary GLC and GLC-MS. beta-Carboline alkaloids, such as harmine, harmaline dominate in normal and root cultures transformed by Agrobacterium rhizogenes, as well as in roots and fruits of the plant. In shoots, flowers and shoot cultures quinoline alkaloids such as peganine, deoxypeganine, vasicinone and deoxyvasicinone widely replace the beta-carboline alkaloids. In root cultures, the formation of beta-carboline alkaloids can be induced by methyljasmonate and several other elicitors indicating that these alkaloids are part of the reactive chemical defence system of Peganum harmala.     

Determination of harmane and harmine in human blood using reversed-phased high-performance liquid chromatography and fluorescence detection

A number of tremorogenic beta-carboline alkaloids have been found in common plant-derived foodstuffs, beverages, and inhaled substances. Because of their natural presence in the food chain, there is a growing concern regarding the potential risks of certain essential tremors associated with the long-term, low-level dietary exposure to these alkaloids. The purpose of this study was to develop an effective analytical method to determine blood levels of two major beta-carboline derivatives, harmane and harmine. Human blood was extracted with ethyl acetate and methyl-t-butyl ether (2:98) under an alkaline condition. After evaporation of organic solvent, the samples were reconstructed in methanol. The samples were fractionated on a 250 x 4.6-mm C18 reversed-phase column with an isocratic mobile system consisting of 17.5 mM potassium phosphate buffer (ph 6.5) and methanol (30:70), followed by an on-line fluorescence detection. The method had the detection limit to determine 206 and 81 pg/ml of harmane and harmine, respectively, in 10 ml of human blood. The intraday precision (C.V.) at 25 ng/ml was less than 6.7 and 3.4% for harmane and harmine, respectively. The interday precision was 7.3% for harmane and 5.4% for harmine. The method has proven sensitive, reproducible, and thus useful for both laboratory and clinical studies of beta-carboline toxicities.     

Acute Biphasic Effects of Ayahuasca

Ritual use of ayahuasca, an amazonian Amerindian medicine turned sacrament in syncretic religions in Brazil, is rapidly growing around the world. Because of this internationalization, a comprehensive understanding of the pharmacological mechanisms of action of the brew and the neural correlates of the modified states of consciousness it induces is important. Employing a combination of electroencephalogram (EEG) recordings and quantification of ayahuasca''s compounds and their metabolites in the systemic circulation we found ayahuasca to induce a biphasic effect in the brain. This effect was composed of reduced power in the alpha band (8–13 Hz) after 50 minutes from ingestion of the brew and increased slow- and fast-gamma power (30–50 and 50–100 Hz, respectively) between 75 and 125 minutes. Alpha power reductions were mostly located at left parieto-occipital cortex, slow-gamma power increase was observed at left centro-parieto-occipital, left fronto-temporal and right frontal cortices while fast-gamma increases were significant at left centro-parieto-occipital, left fronto-temporal, right frontal and right parieto-occipital cortices. These effects were significantly associated with circulating levels of ayahuasca’s chemical compounds, mostly N,N-dimethyltryptamine (DMT), harmine, harmaline and tetrahydroharmine and some of their metabolites. An interpretation based on a cognitive and emotional framework relevant to the ritual use of ayahuasca, as well as it''s potential therapeutic effects is offered.     

Protective Effect of 1-Methylated β-Carbolines Against 3-Morpholinosydnonimine-Induced Mitochondrial Damage and Cell Viability Loss in PC12 Cells

The present study investigated the effect of 1-methylated beta-carbolines (harmaline, harmalol and harmine) on change in the mitochondrial membrane permeability and cell death due to reactive nitrogen species in differentiated PC12 cells. beta-Carbolines, caspase inhibitors (z-LEHD.fmk and z-DQMD.fmk) and antioxidants (N-acetylcysteine, dithiothreitol, melatonin, carboxy-PTIO and uric acid) depressed cell viability loss due to 3-morpholinosydnonimine (SIN-1) in PC12 cells. beta-Carbolines inhibited the nuclear damage, the decrease in mitochondrial transmembrane potential, the cytochrome c release, the formation of reactive oxygen species and the depletion of GSH caused by SIN-1 in PC12 cells. beta-Carbolines decreased the SIN-1-induced formations of 3-nitrotyrosine, malondialdehyde and carbonyls in PC12 cells. The results show that 1-methylated beta-carbolines attenuate SIN-1-induced mitochondrial damage. This results in the inhibition of caspase-9 and -3 and apoptotic cell death in PC12 cells by suppressing the toxic actions of reactive oxygen and nitrogen species, including the GSH depletion.     

Personality, Psychopathology, Life Attitudes and Neuropsychological Performance among Ritual Users of Ayahuasca: A Longitudinal Study

Ayahuasca is an Amazonian psychoactive plant beverage containing the serotonergic 5-HT_2A agonist N,N-dimethyltryptamine (DMT) and monoamine oxidase-inhibiting alkaloids (harmine, harmaline and tetrahydroharmine) that render it orally active. Ayahuasca ingestion is a central feature in several Brazilian syncretic churches that have expanded their activities to urban Brazil, Europe and North America. Members of these groups typically ingest ayahuasca at least twice per month. Prior research has shown that acute ayahuasca increases blood flow in prefrontal and temporal brain regions and that it elicits intense modifications in thought processes, perception and emotion. However, regular ayahuasca use does not seem to induce the pattern of addiction-related problems that characterize drugs of abuse. To study the impact of repeated ayahuasca use on general psychological well-being, mental health and cognition, here we assessed personality, psychopathology, life attitudes and neuropsychological performance in regular ayahuasca users (n = 127) and controls (n = 115) at baseline and 1 year later. Controls were actively participating in non-ayahuasca religions. Users showed higher Reward Dependence and Self-Transcendence and lower Harm Avoidance and Self-Directedness. They scored significantly lower on all psychopathology measures, showed better performance on the Stroop test, the Wisconsin Card Sorting Test and the Letter-Number Sequencing task from the WAIS-III, and better scores on the Frontal Systems Behavior Scale. Analysis of life attitudes showed higher scores on the Spiritual Orientation Inventory, the Purpose in Life Test and the Psychosocial Well-Being test. Despite the lower number of participants available at follow-up, overall differences with controls were maintained one year later. In conclusion, we found no evidence of psychological maladjustment, mental health deterioration or cognitive impairment in the ayahuasca-using group.     

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.     

Structure-activity relationship study of beta-carboline derivatives as haspin kinase inhibitors

Haspin is a serine/threonine kinase that phosphorylates Thr-3 of histone H3 in mitosis that has emerged as a possible cancer therapeutic target. High throughput screening of approximately 140,000 compounds identified the beta-carbolines harmine and harmol as moderately potent haspin kinase inhibitors. Based on information obtained from a structure-activity relationship study previously conducted for an acridine series of haspin inhibitors in conjunction with in silico docking using a recently disclosed crystal structure of the kinase, harmine analogs were designed that resulted in significantly increased haspin kinase inhibitory potency. The harmine derivatives also demonstrated less activity towards DYRK2 compared to the acridine series. In vitro mouse liver microsome stability and kinase profiling of a representative member of the harmine series (42, LDN-211898) are also presented.     

Are tissue cultures of Peganum harmala a useful model system for studying how to manipulate the formation of secondary metabolites?

This article reviews our present knowledge on the formation of tryptophan derived secondary metabolites in tissue cultures of Peganum harmala. With the presence of -carboline alkaloids and serotonin, P. harmala contains two rather simple, interrelated biosynthetic pathways. The long term disadvantage of low and unstable productivity of P. harmala suspension culture has recently been overcome by establishing highly productive hairy root cultures. The first -carboline alkaloid biosynthetic enzymes, specific for the O-methylation of harmalol and harmol as well as for the oxidation of harmaline to harmine, have been detected in these cultures, and they should thus provide a suitable source for studying the yet unknown initial two enzymatic steps of -carboline alkaloid biosynthesis. Seedlings of P. harmala have also been successfully transformed with constructed strains of Agrobacterium, as demonstrated by the overexpression of a tryptophan decarboxylase gene from Catharanthus roseus in cultures of P. harmala. In such transgenic cultures a large overproduction of serotonin was observed. The relative simplicity of these pathways and the rather easy handling of the cultures could make P. harmala a useful and attractive model system for studying the interaction, regulation and manipulation of secondary pathways in cultured cells.Abbreviations TDC tryptophan decarboxylase - tdc gene of tryptophan decarboxylase     

beta-Carbolines, psychoactive compounds in the mammalian body. Part II: Effects

The biochemical and the pharmacological effects of beta-carbolines in animals and man are reviewed. Biochemical studies have revealed beta-carbolines' several actions, including inhibition of MAO-A, competitive inhibition of 5-HT uptake, general inhibition of Na+ dependent transports, binding to benzodiazepine and opiate receptors and probable action on dopamine receptors, which may all participate to a variable degree in the actions of different beta-carbolines. Many early in vivo studies, however, have concentrated on some harmala alkaloids, particularly harmaline or harmine. The effects of beta-carbolines in man are compared in this review with the symptoms of alcohol withdrawal. However, no human studies have been reported with those tetrahydro-beta-carbolines shown to occur in human body in normal conditions or after alcohol intake. To prove any connections of beta-carbolines with the withdrawal syndromes or other neurological and psychiatric diseases means that these compounds have to be shown to have abnormal central nervous system concentrations in these diseases. The physiological role of beta-carbolines has yet to be shown. They may act as neuromodulators and some, especially 6-methoxytetrahydro-beta-carboline, may have an endocrinological function. It has been suggested that some beta-carbolines act as the physiological ligands (agonists) of the benzodiazepine receptors, but the physiological beta-carbolines so far known seem to have other effects, such as the inhibition of MAO-A or 5-HT uptake in low concentrations.     

Simultaneous determination of nineteen hallucinogenic tryptamines/beta-calbolines and phenethylamines using gas chromatography-mass spectrometry and liquid chromatography-electrospray ionisation-mass spectrometry

To investigate the trend of non-controlled drugs of abuse, simultaneous analytical methods were developed using GC-MS and LC-ESI-MS for 8 tryptamines/beta-carbolines, 6 phenethylamines of typically non-controlled substances in Japan, and, additionally, five legally controlled tryptamines and phenethylamines originally found in fungi or plants. Moreover, the proposed methods were applied to analyses of these drugs in 99 kinds of products (a total number of 123 products purchased at adult shops or via the Internet over the past 2 years in Japan), which potentially advertised psychotropic/psychoactive effects. The samples were extracted with methanol under ultrasonication. After centrifugation, the extracts were filtered prior to injections. GC-MS analysis was performed using a DB-5MS capillary column. Regarding the LC-ESI-MS analysis; the separation of the target drugs was optimized on an ODS column in acetonitrile/MeOH (7:3)-10 mM ammonium formate buffer (pH 3.5)/acetonitrile (95:5) by a linear gradient program and a quantitative analysis was carried out by the monitoring of each [M+H]+ in the positive ion mode of ESI-MS. As a result of the analyses using GC-MS and LC-ESI-MS, 5-MeO-DIPT (the synthetic substance known by the street name "Foxy") was found in 8 out of the 99 kinds of products. Additionally, AMT (from brown powder), DMT (from dried plant), harmine and harmaline (from dried plant) were also found in some of the 99 products. These analytical methods could be useful for the investigation of the distribution of the non-controlled psychotropic tryptamines/beta-carbolines and phenethylamines in the market.     

Effects of ATP and magnesium ions on the fluorescence of harmala alkaloids. Restrictions for the use of harmala alkaloids as fluorescent probes for (Na+ + K+)-ATPase.

1. Harmine and harmaline were investigated as potentially useful fluorescent inhibitors of (Na+ + K+) activated ATPase. 29 From spectroscopic measurements both compounds were shown to form 1 : 1 complexes with ATP, the dissociation constants being 0.65 mM and 1.83 mM for harmine and harmaline respectively. Addition of Mg2+ and enzyme further affected these equilibria. 3. Although it was possible to demonstrate a competitive effect of harmine at the sodium-loading site of the enzyme, other inhibitory effects, including inhibitions of ouabain binding and the ouabain-insensitive ATPase were found. 4. It was concluded that the harmala alkaloids can inhibit (Na+ + K+)-activated ATPase in a complex way involving both Na- and ATP-binding sites. This severely limits their usefulness as spectroscopic probes.     

Glucuronidation of 3-O-methylnoradrenaline, harmalol and some related compounds

1. The following compounds were glucuronidated in the presence of UDP-glucuronic acid and a microsomal preparation made from guinea-pig liver: (14)C-labelled 3-O-methyladrenaline, 3-O-methylnoradrenaline, 3-methoxytyramine and 4-hydroxy-3-methoxyphenethanol, as well as unlabelled harmalol and harmol. 2. [(14)C]Homovanillic (4-hydroxy-3-methoxyphenylacetic) acid was not a substrate for the microsomal glucuronyltransferase. 3. The K(m) values for harmalol and harmol were 0.69x10(-4)m and 0.50x10(-4)m respectively. 4. The K(m) values for UDP-glucuronic acid, in the presence of (14)C-labelled 3-O-methylnoradrenaline, harmalol and harmol as aglycones, were 0.57x10(-4)m, 0.44x10(-4)m and 2.20x10(-4)m respectively. 5. Mg(2+) added at 2.5-10mm activated glucuronyltransferase, with harmalol as substrate. Concentrations above 10mm inhibited the enzymic activity. 6. The overall, or net, transglucuronidating activity of microsomal preparations of the liver, with harmalol as substrate, was greatest for guinea pig, and very much lower for rabbit, mouse and rat.     

Development and validation of column-switching high-performance liquid chromatographic methods for the determination of a potent AII receptor antagonist, TCV-116, and its metabolites in human serum and urine

Column-switching HPLC methods have been developed and validated for the determination of a new antihypertensive prodrug, TCV-116 (I), and its metabolites, CV-11974 (II) and CV-15959 (III), in human serum and urine. Initial sample cleanup was achieved by extracting the analytes into an organic solvent. After chromatographing on an ODS column with a mobile phase consisting of acetonitrile and an acidic phosphate buffer, the zone of the analyte's retention was heart-cut onto a second ODS column with a mobile phase of acetonitrile and a phosphate buffer at a higher pH. Complete separation of the analytes and the endogenous peaks was accomplished by the two-dimensional chromatography. Good precision and linearity of the calibration standards, as well as the inter-day and intra-day precision and accuracy of quality control samples, were achieved. The limit of quantitation (LOQ), using 0.5 ml of serum, was 2 ng/ml for I, 0.8 ng/ml for II, and 0.5 ng/ml for III. The LOQ for urine sample was 10 ng/ml for II and III. Stability of the analytes during storage, extraction, and chromatography processes was established. The results illustrate the versatile application of column switching to method development of multiple analytes in various biological matrices. The methods have been successfully used for the analyses of I and its metabolites in thousands of clinical samples to provide pharmacokinetics data.     

Human monoamine oxidase is inhibited by tobacco smoke: beta-carboline alkaloids act as potent and reversible inhibitors

Monoamine oxidase (MAO) is a mitochondrial outer-membrane flavoenzyme involved in brain and peripheral oxidative catabolism of neurotransmitters and xenobiotic amines, including neurotoxic amines, and a well-known target for antidepressant and neuroprotective drugs. Recently, positron emission tomography imaging has shown that smokers have a much lower activity of peripheral and brain MAO-A (30%) and -B (40%) isozymes compared to non-smokers. This MAO inhibition results from a pharmacological effect of smoke, but little is known about its mechanism. Working with mainstream smoke collected from commercial cigarettes we confirmed that cigarette smoke is a potent inhibitor of human MAO-A and -B isozymes. MAO inhibition was partly reversible, competitive for MAO-A, and a mixed-type inhibition for MAO-B. Two beta-carboline alkaloids, norharman (beta-carboline) and harman (1-methyl-beta-carboline), were identified by GC-MS, quantified, and isolated from the mainstream smoke by solid phase extraction and HPLC. Kinetics analysis revealed that beta-carbolines from cigarette smoke were competitive, reversible, and potent inhibitors of MAO enzymes. Norharman was an inhibitor of MAO-A (K(i)=1.2+/-0.18 microM) and MAO-B (K(i)=1.12+/-0.19 microM), and harman of MAO-A (K(i)=55.54+/-5.3nM). Beta-carboline alkaloids are psychopharmacologically active compounds that may occur endogenously in human tissues, including the brain. These results suggest that beta-carboline alkaloids from cigarette smoke acting as potent reversible inhibitors of MAO enzymes may contribute to the MAO-reduced activity produced by tobacco smoke in smokers. The presence of MAO inhibitors in smoke like beta-carbolines and others may help us to understand some of the purported neuropharmacological effects associated with smoking.     

Simultaneous determination of isosorbide dinitrate and its mononitrate metabolites in human plasma by capillary gas chromatography with electron-capture detection

A method for the simultaneous determination of isosorbide dinitrate (ISDN) and its mononitrate metabolites (2- and 5-ISMN) in human plasma by capillary gas chromatography with electron-capture detection was developed. Two internal standards were used: isomannide dinitrate (IMDN) for the determination of ISDN and isomannide mononitrate (IMMN) for the determinations of 2- and 5-ISMN. After addition of the internal standards, the compounds were isolated from plasma by solid-liquid extraction. They were determined by gas chromatography using an electron-capture detector. The reproducibility and accuracy of the method were found suitable in the range of concentrations 2.5–83 ng/ml for ISDN, 2.6–208 ng/ml for 2-ISMN and 2.3–1010 ng/ml for 5-ISMN. The limit of quantitation (LOQ) was about 2.5 ng/ml for each compound. The method was applied to clinical samples.     

Limitations of feeding experiments in studying alkaloid biosynthesis in Peganum harmala callus cultures

Feeding and trapping experiments to Peganum harmala callus cultures were limited by compartmentation; exogenous substrates were detoxified by precipitation, presumably as polymers or conjugates, or by conversion to water-soluble products, such as phenols and glucosides, easily stored in vacuoles. Alkaloid-producing and non-alkaloid-producing callus cultures were readily able to convert tryptamine to 5-hydroxytryptamine and harmaline to dihydroruine (8-hydroxyglucosylharmaline). Phenolic substrates, including 5- and 6-hydroxy-tryptophan, 5- and 6-hydroxytryptamine and harmalol, were not metabolized. In alkaloid-producing callus cultures, radioactivity from [methylene-¹⁴C]-l-tryptophan and [methyl-¹⁴C-]-harmaline was incorporated into harmine. The dilution of radioactivity was 30000- and 2-fold respectively.