Determination of harmane and harmine in human blood using reversed-phased high-performance liquid chromatography and fluorescence detection.
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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. (+info)
Harmane produces hypotension following microinjection into the RVLM: possible role of I(1)-imidazoline receptors.
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The beta-carboline, harmane (0.1 - 1.0 nmol) produces dose dependent hypotension when microinjected unilaterally into the rostral ventrolateral medulla (RVLM) of the anaesthetized rat. The potency of harmane on blood pressure is similar to that of the imidazoline, clonidine. The hypotensive effects of both clonidine and harmane are reversed by microinjection of the relatively I(1)-receptor selective antagonist efaroxan (20 nmol). These results are consistent with harmane acting at an I(1)-receptor in the RVLM. This is the first report of an endogenous ligand for I(1)-receptors that has central effects on blood pressure. (+info)
Comparative study on the vasorelaxant effects of three harmala alkaloids in vitro.
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Three psychological active principles from the seeds of Peganum harmala L., harmine, harmaline and harmalol, showed vasorelaxant activities in isolated rat thoracic aorta preparations precontracted by phenylephrine or KCl with rank order of relaxation potency of harmine > harmaline > harmalol. The vasorelaxant effects of harmine and harmaline (but not harmalol) were attenuated by endothelium removal or pretreatment with a nitric oxide (NO) synthase Nomega-nitro-L-arginine methyl ester. In cultured rat aortic endothelial cells, harmine and harmaline (but not harmalol) increased NO release, which was dependent on the presence of external Ca2+. In endothelium-denuded preparations, pretreatment of harmine, harmaline or harmalol (3-30 microM) inhibited phenylephrine-induced contractions in a non-competitive manner. Receptor binding assays indicated that all 3 compounds interacted with cardiac alpha1-adrenoceptors with comparable affinities (Ki value around 31 - 36 microM), but only harmine weakly interacted with the cardiac 1,4-dihydropyridine binding site of L-type Ca2+ channels (Ki value of 408 microM). Therefore, the present results suggested that the vasorelaxant effects of harmine and harmaline are attributed to their actions on the endothelial cells to release NO and on the vascular smooth muscles to inhibit the contractions induced by the activation of receptor-linked and voltage-dependent Ca2+ channels. The vasorelaxant effect of harmalol was not endothelium-dependent. (+info)
Effects of reserpine on the adrenal medulla of the spontaneously hypertensive rat.
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1. Reserpine administration resulted in a larger initial decline in adrenal catecholamines in spontaneously hypertensive rats (SHR) than in normotensive Wistar rats (NWR); the difference was eliminated by pretreatment with cholisdondamine. 2. Reserpine also produced a larger increase in SHR catecholamines and dopamine beta-hydroxylase several days later; chlorisondamine pretreatment did not prevent the increases, although it did slightly slow the increases. 3. Vesicles from SHR, or NWR incubated with reserpine in vitro demonstrated equivalent inhibition of adenosine 5'-triphosphate (ATP)-Mg-2+-stimulated adrenaline uptake. 4. Recovery of uptake was more rapid in SHR than in NWR after reserpine inhibition, and this was associated with a burst of new vesicle synthesis in the SHR; chlorisondamine pretreatment reduced the number of new, immature vesicles in reserpine-treated SHR. 5. Both SHR and NWR secreted equal proportions of their adrenal catecholamine contents after nicotine administration. 6. These data suggest that the sympatho-adrenal system of the SHR exhibits an enhanced reflex response to reserpine but that reserpine is equally effective in SHR and NWR in producing blockade of vesicular catecholamine transport; these alterations can affect markedly the actions of autonomic drugs in the SHR. (+info)
Collision-induced dissociation actualized the H+-promoted reaction as observed in vitro; harman formation from beta-carboline-type monoterpenoid glucoindole alkaloids.
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The fragmentation from beta-carboline-type monoterpenoid glucoindole alkaloids to harman, which is a hypothetical pathway to generate simple beta-carbolines, was actualized in the collision-induced dissociation in MS. (+info)
Inhibitors alter the spectrum and redox properties of monoamine oxidase A.
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Monoamine oxidase A (MAO A) catalyses the oxidation of both neurotransmitter and ingested amines. The mechanism of catalysis involves the covalently bound FAD cofactor. Although substrates and inhibitors alter the thermodynamic and kinetic properties of the flavin, how the ligands interact with the flavin is unknown. This work characterises the spectral changes that occur on inhibitor binding to MAO A and examines how the binding influences the flavin. The inhibitors, D-amphetamine, harmine, tetrindole, and befloxatone all induce similar (but not identical) changes in the spectrum of MAO A, consistent with stacking of inhibitor with the flavin in the active site. D-Amphetamine, harmine, and tetrindole stabilise the semiquinone form of FAD during reduction of MAO A by dithionite and no further reduction of these inhibitor-MAO A complexes has been observed. In contrast, semiquinone is never observed during reduction of the befloxatone-MAO A complex. Instead, partial reduction directly to the FADH(2) form occurs extremely slowly. Thus, inhibitor binding has a strong, structure-dependent influence on the environment of the flavin that alters its electronic properties. (+info)
Contribution of individual cytochrome P450 isozymes to the O-demethylation of the psychotropic beta-carboline alkaloids harmaline and harmine.
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The psychotropic beta-carboline alkaloids, showing high affinity for 5-hydroxytryptamine, dopamine, benzodiazepine, and imidazoline receptors and the stimulation of locus coeruleus neurons, are formed endogenously from tryptophan-derived indolealkylamines through the Pictet-Spengler condensation with aldehydes in both plants and mammals. Cytochromes P450 1A1 (18.5), 1A2 (20), and 2D6 (100) catalyzed the O-demethylation of harmaline, and CYP1A1 (98.5), CYP1A2 (35), CYP2C9 (16), CYP2C19 (30), and CYP2D6 (115) catalyzed that of harmine (relative activities). The dehydrogenation/aromatization of harmaline to harmine was not carried out by aromatase (CYP19), CYP1A2, CYP2C9, CYP2D6, CYP3A4, pooled recombinant cytochromes P450, or human liver microsomes (HLMs). Kinetic parameters were calculated for the O-demethylations mediated by each isozyme and by pooled HLMs. K(cat) (min(-1)) and K(m) Awake M) values for harmaline were: CYP1A1, 10.8 and 11.8; CYP1A2, 12.3 and 13.3; CYP2C9, 5.3 and 175; CYP2C19, 10.3 and 160; and CYP2D6, 39.9 and 1.4. Values for harmine were: CYP1A1, 45.2 and 52.2; CYP1A2, 9.2 and 14.7; CYP2C9, 11.9 and 117; CYP2C19, 21.4 and 121; and CYP2D6, 29.7 and 7.4. Inhibition studies using monoclonal antibodies confirmed that CYP1A2 and CYP2D6 were the major isozymes contributing to both harmaline (20% and 50%, respectively) and harmine (20% and 30%) O-demethylations in pooled HLMs. The turnover numbers for CYP2D6 are among the highest ever reported for a CYP2D6 substrate. Finally, CYP2D6-transgenic mice were found to have increased harmaline and harmine O-demethylase activities as compared with wild-type mice. These findings suggest a role for polymorphic CYP2D6 in the pharmacology and toxicology of harmine and harmaline. (+info)
Microbial metabolites of harman alkaloids.
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Several microorganisms showed the ability to transform the harman alkaloids, harmaline (1), harmalol (2) and harman (5). Harmaline (1) and harmalol (2) were converted by Rhodotorula rubra ATCC 20129 into the tryptamines, 2-acetyl-3-(2-acetamidoethyl)-7-methoxyindole (3) and 2-acetyl-3-(2-acetamidoethyl)-7-hydroxyindole (4), respectively. Harman (5) was biotransformed by Cunninghamella echinulata NRRL 3655 into 6-hydroxyharman (6) and harman-2-oxide (7). (+info)