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The work highlighted interaction of harmalol, harmaline and harmine with human serum albumin by biophysical and biochemical assays. Presence of serum protein in the media negatively affects the cyt...

β-carboline alkaloids of the harmala group (HAlks)—a family of compounds with pharmacologic effects—can be found at trace levels ( −1 algae) in the edible invasive algae Undaria pinnatifida , known commonly as wakame. In this study, we present a simple and sensitive method to detect and quantify at low parts-per-trillion levels the six HAlks more frequently found in those plants. The method is based on on-line solid phase extraction capillary electrophoresis mass spectrometry using a C 18 sorbent. First, the methodology was optimized and validated with standard solutions through the use of ultraviolet (UV) and mass spectrometry (MS) detection. Second, the optimized method for MS detection was applied to an analysis of the HAlks in U. pinnatifida extracts. The method achieved limits of detection between 2 and 77 pg mL −1 for standards, producing an analyte preconcentration of about 1000-times in comparison to CE-MS. Some matrix effects were observed for the complex wakame extracts, especially for the most polar HAlks (harmol and harmalol), which bear aromatic hydroxyl groups. Harmine, harmaline, and norharmane were not detected in the algal extracts, whereas harmane was found at 70 pg mL −1 (70 ng kg −1 dry algae). The results underscored that C 18 -SPE-CE-MS may be considered as a powerful method to detect trace levels of alkaloids and other bioactive small molecules in complex plant extracts.

A simple method based on micellar liquid chromatography (MLC) was developed to enable the simultaneous determination of norharmane, harmane, harmol, harmalol, harmine, and harmaline in phytotherapic samples of Passiflora incarnata L., Passiflora alata Dryander and urine with virtually no sample preparation. Chromatographic separation was performed under 30 min using a C18 HPLC column and isocratic elution with an aqueous micellar mobile phase (phosphate buffer pH 8.0 containing 220 mmol L−1 of sodium dodecyl sulfate and 0.5% of triethylamine) containing only 3% (v/v) of acetonitrile. Fluorescence detection allowed limits of quantification below 4.5 ng g−1. Recoveries in controlled samples were close to 100% and the sample analysis enabled the quantification of harmol as the principal β-carboline found in the phytotherapic samples, whereas another three were detected. In urine samples, the determination of harmol was made after 8 hours of drug ingestion. Both repeatability and intermediary precision, for a...

Background Harmalol, a beta carboline alkaloid, shows remarkable importance in the contemporary biomedical research and drug discovery programs. With time, there is emerging interest in search for better anti-cancer drugs of plant origin with high activity and lower toxicity. Most of the chemotherapeutic agents due to their non-specific target and toxicity on active healthy cells, use is often restricted, necessitating search for newer drugs having greater potentiality. Objective The review highlighted the interaction of harmalol with nucleic acids of different motifs as sole target biomolecules and in vitro cytotoxicity of the alkaloid in human cancer cell lines with special emphasis on its apoptotic induction ability. Methods Binding study and in vitro cytotoxicity was performed using several biophysical techniques and biochemical assays, respectively. Results Data from competition dialysis, UV and fluorescence spectroscopic analysis, circular dichroism, viscometry and isothermal calorimetry shows binding and interaction of harmalol with several natural and synthetic nucleic acids, both DNA and RNA, of different motifs. Furthermore, apoptotic hallmarks like internucleosomal DNA fragmentation, membrane blebbing, cell shrinkage, chromatin condensation, change of mitochondrial membrane potential, comet tail formation and ROS (reactive oxygen species) dependent cytotoxicity being analyzed in the harmalol treated cancer cells. Conclusion These results stating the therapeutic role of harmalol, will lead to the interesting knowledge on the cytotoxicity, mode, mechanism, specificity of binding and correlation between structural aspects and energetics enabling a complete set of guidelines for design of new drugs.

Dioxins are known to cause several human cancers through activation of the aryl hydrocarbon receptor (AhR). Harmaline and harmalol are dihydro-β-carboline compounds present in several medicinal plants such as Peganum harmala. We have previously demonstrated the ability of P. harmala extract to inhibit TCDD-mediated induction of Cyp1a1 in murine hepatoma Hepa 1c1c7 cells. Therefore, the aim of this study is to examine the effect of harmaline and its main metabolite, harmalol, on dioxin-mediated induction of CYP1A1 in human hepatoma HepG2 cells. Our results showed that harmaline and harmalol at concentrations of (0.5-12.5μM) significantly inhibited the dioxin-induced CYP1A1 at mRNA, protein and activity levels in a concentration-dependent manner. The role of AhR was determined by the inhibition of the TCDD-mediated induction of AhR-dependent luciferase activity and the AhR/ARNT/XRE formation by both harmaline and harmalol. In addition, harmaline significantly displaced [(3)H]TCDD in the competitive ligand binding assay. At posttranslational level, both harmaline and harmalol decreased the protein stability of CYP1A1, suggesting that posttranslational modifications are involved. Moreover, the posttranslational modifications of harmaline and harmalol involve ubiquitin-proteasomal pathway and direct inhibitory effects of both compounds on CYP1A1 enzyme. These data suggest that harmaline and harmalol are promising agents for preventing dioxin-mediated effects.

β-Carboline alkaloids are the main chemical constituents of the plant Peganum harmala, while they also could be formed endogenously and found in coffee, alcoholic beverages and tobacco. Considering the fact that the possibility of herb–drug interactions has recently received great attention worldwide, the aim of the current study was to assess the potential for the metabolism-based drug–drug interactions arising from five β-carboline alkaloids (harmine, harmaline, harmalol, harmol and harmane) from P. harmala in vitro. With microsome incubation assays and UPLC/HPLC methods, the inhibitions on human liver CYP3A4 and CYP2D6 enzymes by those β-carboline alkaloids were studied kinetically. Harmine, harmol and harmane exhibited noncompetitive inhibition on the activity of CYP3A4 with Ki values of 16.76, 5.13 and 1.66 μm, respectively. These β-carboline alkaloids were also found to be both substrates and inhibitors for CYP2D6. Harmaline, harmine and harmol showed typical competitive inhibition on the activity of CYP2D6 with Ki values of 20.69, 36.48 and 47.11 μm, respectively. The inhibition of the two major CYP enzymes by those β-carboline alkaloids suggested that changes in the pharmacokinetics of co-administered drugs were likely to have occurred. Therefore, caution should be exercised for possible drug interactions of medicinal plants containing those β-carboline alkaloids and CYP substrates. Copyright © 2011 John Wiley & Sons, Ltd.

Melanin synthesis is regulated by melanocyte specific enzymes and related transcription factors. β-carboline alkaloids including harmaline and harmalol are widely distributed in the environment including several plant families and alcoholic beverages. Presently, melanin content and tyrosinase activity were increased in melanoma cells by harmaline and harmalol in concentration- and time-dependent manners. Increased protein levels of tyrosinase, tyrosinase-related protein-1 (TRP-1), and TRP-2 were also evident. In addition, immunofluorescence and Western blot analyses revealed harmaline and harmalol increased cAMP response element binding protein phosphorylation and microphthalmia-associated transcription factor expression. In addition to studying the signaling that leads to melanogenesis, roles of the p38 MAPK pathways by the harmaline and harmalol were investigated. Harmaline and harmalol induced time-dependent phosphorylation of p38 MAPK. Harmaline and harmalol stimulated melanin synthesis and tyrosinase activity, as well as expression of tyrosinase and TRP-1 and TRP-2 indicating that these harmaline and harmalol induce melanogenesis through p38 MAPK signaling.

β-Carboline alkaloids are present in medicinal plants such as Peganum harmala L., which have been used as folk medicine in anticancer therapy. Recently, they have drawn attention because of their antitumor activities. Despite considerable interest and investigations on alkaloid-DNA complexes, reports on alkaloid-RNA interaction are very limited. This study is the first attempt to investigate the binding of β-carboline alkaloids (harmine, harmane, harmaline, harmalol, and tryptoline) with yeast RNA. The effect of alkaloid complexation on RNA aggregation and condensation was investigated in aqueous solution at physiological conditions, using constant RNA concentration (6.25 mM) and various alkaloid:polynucleotide (phosphate) ratios of 1:240, 1:160, 1:80, 1:40, 1:20, 1:10, 1:5, 1:2, and 1:1. Fourier transform infrared and UV-visible spectroscopic methods were used to determine the ligand-binding modes, the binding constants, and the stability of alkaloid-RNA complexes in aqueous solution. Spectroscopic evidence showed major binding of alkaloids to RNA with overall binding constants of K(harmine)-RNA = 2.95 × 10⁷ M⁻¹, K(harmane)-RNA = 5.62 × 10⁵ M⁻¹, K(harmaline)-RNA = 7.47 × 10⁵ M⁻¹, K(harmalol)-RNA = 4.32 × 10⁵ M⁻¹, and K(tryptoline)-RNA = 3.21 × 10⁵ M⁻¹. The affinity of alkaloids-RNA binding is in the order of harmine harmaline harmane harmalol tryptoline. No biopolymer secondary structural changes were observed upon alkaloid interaction and RNA remains in the A-family structure in these complexes.

Preliminary work on Passiflora alata leaves failed to detect harmane alkaloids using LC. The aim of this work was to investigate the production of harmane alkaloids through the cell culture of P. alata, inducing its precursor (L-tryptophan). The leaf explants presented satisfactory results after disinfection, and the callus formation was initiated in MS media with adequate quantities of phytohormones. Sixty days after inoculation, calli were inoculated in the optimized semi-solid MS media, with and without the addition of L-tryptophan (50, 100, 200 mg/L) and kept in standard conditions for 90 days. Calli were collected on days 6, 16, 26, 36, and 90, followed by acid-base extraction, and analysed by LC. The results showed an absence of harmane, harmin, harmol, harmalol, and harmaline. With L-tryptophan feeding, two peaks were detected, collected and analysed through positive mode electrospray [ESI(+)-MS] and sequential analysis in tandem ESI(+)-MS/MS. The spectra obtained were very similar, with a repetition of the more intense ions, and consecutive loss of 68 Da units, attributed to the heterocycle pyrazole. It appeared that this transformation was not related to any enzymatic pathway previously described for the plant from L-tryptophan, and the biosynthesis of β-carboline alkaloids in callus culture of P. alata were not observed in this work.As folhas de varias espécies de Passiflora são utilizadas como ansioliticas e sedativas. Passiflora alata Curtis, Passifloraceae consta em três edições da farmacopéia brasileira, porem não há muitos estudos sobre sua composição química. No passado, enfatizava-se a ação conjunta de alcalóides e flavonóides. Em trabalho anterior, não foi detectada a presença de alcalóides harmanicos através de CLAE. Assim, decidiu-se investigar a produção dos mesmos através de cultivo celular, introduzindo seu precursor metabólico L-triptofano. Os explantes foliares apresentaram resultados satisfatorios para germinação apos assepsia, e a formação de calo foi iniciada em meio MS com quantidades adequadas de fitohormonios, previamente determinadas. Sessenta dias após a inoculação os calos foram repicados para meio semi-solido com e sem L-triptofano (50, 100, 200 mg/L), mantidos por 90 dias em condições padrão. Amostras foram coletadas com 6, 16, 26, 36, e 90 dias, realizada extração acido-base e o extrato analisado por CLAE. Os resultados mostraram a ausência de harmana, harmina, harmol, harmalol e harmalina. Dois picos presentes nas amostras com L-triptofano foram coletados e analisados através de espectrometria de massas, electrospray modo positiva [ESI(+)-MS] e analise em tandem ESI(+)-MS/MS. Os espectros correspondentes foram similares, mostrando a perda consecutiva de 68 Da, atribuídos ao pirazol. Este fato aponta para uma transformação não enzimática, não relacionada a uma biossintese previamente descrita para alcalóides β-carbolínicos.

Beta-carboline alkaloids including harmalol, harmaline, norharmane, harmol, harmine and harmane are important constituents of the medicinal plant, Perganum harmala L. (Zygophylaceae), which has been used in traditional medicine. In the present study, the antiplatelet activities of six beta-carboline alkaloid compounds were investigated in vitro. At a concentration of 200 microM, these compounds have no effect on arachidonic acid (AA)-, thrombin- and U46619 (a thromboxane A2 mimic)-stimulated platelet aggregation. On the contrary, it was revealed that collagen-induced platelet aggregation could be inhibited by these compounds with different potencies (harmane and harmine were most potent, harmol had medium potency, and harmol, norharmane, harmalol and harmaline had a weak, non significant effect), indicating a selective inhibition on collagen-mediated platelet activation. Consistently, further study revealed that collagen-mediated phospholipase (PL) Cgamma2 and protein tyrosine phosphorylation, cytosolic calcium mobilization and arachidonic acid liberation were completely inhibited by harmane and harmine in a concentration-dependent manner, while the other compounds were only partially or not effective at all. Taken together, these results indicate that three of these six beta-carboline alkaloids can selectively affect collagen-induced platelet aggregation with different potencies; in particular, harmane and harmine were most potent, and their antiplatelet activities may be mediated by inhibiting PLCgamma2 and protein tyrosine phosphorylation with sequential suppression of cytosolic calcium mobilization and arachidonic acid liberation, indicating that harmane and harmine have a potential to be developed as a novel agent for atherothrombotic diseases.

Chemical composition of the floral nectar of Peganum harmala, a herbaceous medicinal perennial of the family Zygophyllaceae, was analysed using high-performance liquid chromatography technique. The nectar sugar detection experiments resulted in 33.1, 39.8 and 27.4%, respectively, for fructose, glucose and sucrose, upon which the nectar was classified as hexose rich. In addition, 11 proteinaceous amino acids were recognised and quantified in the nectar. Concentration of the insects' favoured amino acid, prolin, was markedly high. Furthermore, among four detected alkaloids, harmalol and harmine as the two beta-carboline derivatives were identified. These findings may confer a better understanding upon outcrossing processes and favour the plant-pollinator relationships.

In capillary electrophoresis (CE), resolution (Rs) and selectivity (α) are criteria often used in practice to optimize separations. Nevertheless, when these and other proposed parameters are considered as an elementary criterion for optimization by mathematical maximization, certain issues and inconsistencies appear. In the present work we analyzed the pros and cons of using these parameters as elementary criteria for mathematical optimization of capillary electrophoretic separations. We characterized the requirements of an ideal criterion to qualify separations within the framework of mathematical optimizations and, accordingly, propose: -1- a new elementary criterion (t') and -2- a method to extend this elementary criterion to compose a global function that simultaneously qualifies many different aspects, also called multicriteria optimization function (MCOF). In order to demonstrate this new concept, we employed a group of six alkaloids with closely related structures (harmine, harmaline, harmol, harmalol, harmane and norharmane). On the basis of this system, we present a critical comparison between the new optimization criterion t' and the former elementary criteria. Finally, aimed at validating the proposed methods, we composed an MCOF in which the capillary-electrophoretic separation of the six model compounds is mathematically optimized as a function of pH as the unique variable. Experimental results subsequently confirmed the accuracy of the model., Laboratorio de Investigación y Desarrollo de Métodos Analíticos (LIDMA)

The beta-carboline alkaloids found in medical plants and in a variety of foods, beverages and cigarette smoke have a range of action in various biological systems. In vitro studies have demonstrated that these alkaloids can act as scavengers of reactive oxygen species. In this paper, we report the in vivo antioxidative properties of the aromatic (harmane, harmine, harmol) and dihydro-beta-carbolines (harmaline and harmalol) studied by using Saccharomyces cerevisiae strains proficient and deficient in antioxidant defenses. Their antimutagenic activity was also assayed in S. cerevisiae and the antigenotoxicity was tested by the comet assay in V79 cell line, when both eukaryotic systems were exposed to H(2)O(2). We show that the alkaloids have a significant protective effect against H(2)O(2) and paraquat oxidative agents in yeast cells, and that their ability to scavenge hydroxyl radicals contributes to their antimutagenic and antigenotoxic effects.

The use of algae as a foodstuff is rapidly expanding worldwide from the East Asian countries, where they are also used for medical care. Harmala alkaloids (HAlk) are a family of bioactive compounds found in the extracts of some plants, including wakame (Undaria pinnatifida), an edible marine invasive algae. HAlks are based on a characteristic βcarboline structure with at least one amino ionizable group. In this work, we report the successful separation of a mixture of six HAlks (harmine, harmaline, harmol, harmalol, harmane, and norharmane) by capillary electrophoresis ion-trap mass spectrometry (CE-IT-MS) in less than 8 min. Optimum separation in fused-silica capillaries and detection sensitivity in positive-ion mode were achieved using a background electrolyte (BGE) with 25 mmol L⁻¹ ammonium acetate (pH 7.8) and 10 % (v/v) methanol, and a sheath liquid with 60:40 (v/v) isopropanol–water and 0.05 % (v/v) formic acid. The separation method was validated in terms of linearity, limits of detection and quantification, repeatability, and reproducibility. Later, a sample pretreatment was carefully optimized to determine HAlks in commercial wakame samples with excellent recovery and repeatability. For the complex wakame extracts, the MS–MS fragmentation patterns of the different HAlks were useful to ensure a reliable identification. The complete procedure was validated using the standardaddition calibration method, determining matrix effects on the studied compounds. Harmalol, harmine, and harmaline were naturally present in the samples and were quantified at very low concentrations, ranging from 7 to 24 μg kg⁻¹ dry algae., Facultad de Ciencias Exactas, Laboratorio de Investigación y Desarrollo de Métodos Analíticos (LIDMA), Centro de Investigación y Desarrollo en Tecnología de Pinturas

The partial least squares regression method has been applied for simultaneous spectrophotometric determination of harmine, harmane, harmalol and harmaline in Peganum harmala L. (Zygophyllaceae) seeds. The effect of pH was optimized employing multivariate definition of selectivity and sensitivity and best results were obtained in basic media (pH>9). The calibration models were optimized for number of latent variables by the cross-validation procedure. Determinations were made over the concentration range of 0.15-10 microg mL(-1). The proposed method was validated by applying it to the analysis of the beta-carbolines in synthetic quaternary mixtures of media at pH 9 and 11. The relative standard errors of prediction were less than 4% in most cases. Analysis of P. harmala seeds by the proposed models for contents of the beta-carboline derivatives resulted in 1.84%, 0.16%, 0.25% and 3.90% for harmine, harmane, harmaline and harmalol, respectively. The results were validated against an existing HPLC method and it no significant differences were observed between the results of two methods.

Keeping in view the interesting chemistry and pharmacological importance of harmine series of bases – the β-carboline alkaloids, a number of new derivatives of tetrahydroharmine (1) and harmalol (2) have been prepared and characterized through spectral studies. Some of these derivatives showed spasmolytic activity. It was observed that all the N-acyl tetrahydroharmine derivatives are stable, not labile and no ring opening occurs in these compounds, as reported recently.

Ayahuasca is a South American psychotropic beverage prepared from plants native to the Amazon River Basin. It combines the hallucinogenic agent and 5-HT(2A/2C) agonist N,N-dimethyltryptamine (DMT) with beta-carboline alkaloids showing monoamine oxidase-inhibiting properties. In the present paper, an analytical methodology for the plasma quantification of the four main alkaloids present in ayahuasca plus two major metabolites is described. DMT was extracted by liquid-liquid extraction with n-pentane and quantified by gas chromatography with nitrogen-phosphorus detection. Recovery was 74%, and precision and accuracy were better than 9.9%. The limit of quantification (LOQ) was 1.6 ng/ml. Harmine, harmaline, and tetrahydroharmine (THH), the three main beta-carbolines present in ayahuasca, and harmol and harmalol (O-demethylation metabolites of harmine and harmaline, respectively) were measured in plasma by means of high-performance liquid chromatography (HPLC) with fluorescence detection. Sample preparation was accomplished by solid-phase extraction, which facilitated the automation of the process. All five beta-carbolines were measured using a single detector by switching wavelengths. Separation of harmol and harmalol required only slight changes in the chromatographic conditions. Method validation demonstrated good recoveries, above 87%, and accuracy and precision better than 13.4%. The LOQ was 0.5 ng/ml for harmine, 0.3 ng/ml for harmaline, 1.0 ng/ml for THH, and 0.3 ng/ml for harmol and harmalol. Good linearity was observed in the concentration ranges evaluated for DMT (2.5-50 ng/ml) and the beta-carbolines (0.3-100 ng/ml). The gas chromatography and HPLC methods described allowed adequate characterization of the pharmacokinetics of the four main alkaloids present in ayahuasca, and also of two major beta-carboline metabolites not previously described in the literature.

The present study elucidated the protective effect of beta-carbolines (harmaline, harmalol and harmine) against oxidative damage of brain mitochondria, synaptosomes and PC12 cells induced by either dopamine or 6-hydroxydopamine. Harmaline, harmalol and antioxidant enzymes (superoxide dismutase/SOD and catalase) decreased the alteration of mitochondrial swelling and membrane potential induced by 200 microM dopamine or 100 microM 6-hydroxydopamine. Deprenyl attenuated the dopamine-induced mitochondrial dysfunction but did not reduce the effect of 6-hydroxydopamine. While beta-carbolines inhibited the electron flow in mitochondria, they did not enhance the depressant effect of catecholamines. beta-Carbolines and antioxidant enzymes reversed the depression of synaptosomal Ca2+ uptake induced by 10 microM catecholamines. The compounds inhibited the catecholamine-induced thioredoxin reductase inhibition, thiol oxidation and carbonyl formation in mitochondria and synaptosomes. beta-Carbolines decreased the reactive species-induced deoxyribose degradation. Harmaline and harmalol reduced the catecholamine-induced loss of the transmembrane potential and of cell viability in PC12 cells. beta-Carbolines alone did not show a significant cytotoxic effect on PC12 cells. The results suggest that beta-carbolines may attenuate the dopamine- or 6-hydroxydopamine-induced alteration of brain mitochondrial and synaptosomal functions, and viability loss in PC12 cells, by a scavenging action on reactive oxygen species and inhibition of thiol oxidation.

A high performance liquid chromatographic method was developed to determine all types of β-carboline alkaloids in medicinal and edible plants. The alkaloids, including internal standards, were purified from plant homogenates by serial extractions after reacting with fluorescamine. The extracts were analysed twice using reversed-phase chromatography with fluorometric detection optimized for each individual analyte. β-Carbolines and tetrahydro-β-carbolines were analysed in the first run, while dihydro-β-carbolines were analysed in the second. The simultaneous separation of harmol, norharman, harman, harmine, harmalol, harmaline, tetrahydronorharman, tetrahydroharman and two internal standards was achieved within 17 min by an isocratic elution. β-Carbolines, dihydro-β-carbolines and tetrahydro-β-carbolines could be quantitatively determined in concentrations of 0.01–50.0 ng/mL. In replicate spiking experiments, the mean recovery was 71–110% and the relative standard deviation was 0.3–10.1% within and between assays. Application of the proposed method has revealed that medicinal plants and dried edible plants contain β-carboline alkaloids at ng/g to µg/g levels. Norharman and harman were distributed in all the tested plants, while harmol and harmalol were found only in some plant species. However, no significant amounts of tetrahydro-β-carbolines were detected in any plant materials tested, suggesting that the oxidation of tetrahydro-β-carbolines to β-carbolines occurs during the drying and/or the storing process. The β-carboline alkaloids may be responsible for the pharmacological effects of certain medicinal plants. Copyright © 1999 John Wiley & Sons, Ltd.

The β-carboline alkaloids of the harmane group (harmine, harmol, harmaline and harmol) are found at high levels in some plants and occur in many natural foodstuffs and beverages. Both harmane and norharman also occur naturally in mammalian neural tissues. The harmane alkaloids are often difficult to fully fractionate using conventional high-performance liquid chromatography. We report here the successful separation of a mixture of six harmane alkaloids (harmane, norharman, harmine, harmaline, harmol and harmalol) to baseline resolution using micellar electrokinetic chromatography. The alkaloids were detected by UV absorption using diode-array spectrophotometry, which allowed characterization of individual peaks. The fractionation was rapid and was highly reproducible, with complete resolution of all six compounds within 14 min. Harmalol could also be detected directly using laser-induced fluorescence.

Ethnopharmacological relevance Seeds and aerial parts of Peganum harmala L. are widely used in Algeria as anti-inflammatory remedies. Evaluation of Peganum harmala total alkaloids extracts and pure β-carboline compounds as an anti-inflammatory treatment by the inhibition of an enzyme key of inflammatory, myeloperoxidase (MPO) and HPLC quantification of the alkaloids from the different parts of plant. Materials and methods MPO inhibition was tested using taurine chloramine test. The inhibition of LDL oxidation induced by MPO was carried out. The molecular docking analysis of Peganum harmala alkaloids on MPO was performed using the Glide XP docking protocol and scoring function and the redox potential of alkaloids was determined using an Epsilon potentiostat. The concentration of harmala alkaloids was determined using HPLC analysis. Results The HPLC profiling of the active total alkaloids indicates that β-carboline e.g. harmine, harmaline, harmane, harmol and harmalol are major components. As β-carbolines resemble tryptamine, of which derivatives are efficient inhibitors of MPO, the harmala alkaloids were tested for their activity on this enzyme. Total alkaloids of the seeds and of the aerial parts strongly inhibited MPO at 20 µg/mL (97±5% and 43±4%, respectively) whereas, at the same concentration, those of the roots showed very low inhibition (15±6%). Harmine, harmaline and harmane demonstrated a significant inhibition of MPO at IC50 of 0.26, 0.08 and 0.72 µM respectively. These alkaloids exerted a similar inhibition effects on MPO-induced LDL oxidation. Molecular docking analysis of Peganum harmala alkaloids on MPO showed that all active Peganum harmala alkaloids have a high affinity on the active site of MPO (predicted free energies of binding up to −3.1 kcal/mol). Measurement of redox potentials versus the normal hydrogen electrode clearly differentiated (i) the high MPO inhibitory activity of harmine, harmaline and harmane (+1014, 1014 and 1003 mV, respectively); and (ii) the low activity of harmalol and harmol (+629/778 and 532/644 mV, respectively). A reverse phase HPLC method has been developed to determine simultaneously five alkaloids of Peganum harmala. Seeds contained all five β-carboline derivatives with the main active alkaloids, harmaline and harmine, being up to 3.8% and 2.9%, respectively. Up to 3.2% of harmine was determined in the roots. The four β-carboline derivatives, harmine, harmaline, harmane and harmalol were identified in the aerial parts. The highest inhibitory effect observed in seeds and the moderate effect of aerial parts could be explained by their harmine and harmaline content. In contrast, the very weak inhibition of the root extract, despite the presence of harmine, may tentatively be explained by the high concentration of harmol which can reduce Compound II of MPO to the native form. Conclusion The inhibition of MPO by Peganum harmala β-carboline alkaloids, herein reported for the first time, may explain the anti-inflammatory effect traditionally attributed to its herbal medicine.

An integrated derivatization-chemiluminescence detection system was used for the high-performance liquid chromatographic determination of β-carboline alkaloids such as harmol, harmalol, norharmane, harmane, harmine and harmaline by the chemiluminescence produced in their reaction with the bis(2,4-dinitrophenyl)oxalate (DNPO)-hydrogen peroxide system. The ability of the proposed detection system based on its zero-dead-volume to overcome the problems associated with the use of this oxalate ester in peroxyoxalate chemiluminescence detection in HPLC was exploited to develop a sensitive chromatographic method for the determination of the above-mentioned hallucinogens. The method excels over other alternatives based on different detection techniques, such as UV and fluorescence spectroscopy, in terms of limits of detection. The proposed method was validated by determining the alkaloids (the identification and quantification of some are reported for the first time) in Heliconiini butterfly specimens with good results.

The continuous addition of reagent technique was used for the determination of hallucinogenic alkaloids such as harmaline, harmalol, harmine, harmol and harmane by measuring the chemiluminescence (CL) produced in the reaction with the bis(2,4-dinitrophenyl) oxalate/hydrogen peroxide system; this was the first reported use of peroxyoxalate chemiluminescence for these hallucinogens. The ability of this technique to reduce background emission in peroxyoxalate CL determinations was exploited to develop a sensitive method for the determination of the above mentioned hallucinogens. The method features linear responses over 2 orders of magnitude, relative standard deviations of ca. 3% and detection limits in the picomole range, in addition to a high selectivity. Also it compares favourably in terms of sensitivity with that based on another well-known peroxyoxalate reagent, viz. bis(2,4,6-trichlorophenyl) oxalate. It also excels other alternatives based on different techniques in terms of sensitivity. The proposed method was validated for analysis of real samples by applying it to the determination of harmaline in plasma samples.

Summary Treatment of cell smears (chicken blood, Trypanosoma cruzi epimastigotes, Ehrlich ascites tumor cells and mouse spleen) with simple β -carboline alkaloids induced a strong bluish white fluorescence emission of condensed chromatin and basophilic cytoplasm under ultraviolet excitation. The compounds used were harmine, harmol, harman, norharman, harmalol and harmaline (25 – 50 μg/ml aqueous solutions for 2 – 3 min). Spectrofluorometric studies on harmine solutions in vitro (emission peak at 420 nm) showed fluorescence quenching at high concentrations as well as in the presence of DNA. Microscopic fluorescence features of these new fluorochromes and possible binding modes to nucleic acids are discussed.

Tryptophan decarboxylase (TDC) activity, and serotonin and harmane alkaloids levels of a Peganum harmala root culture were followed over a growth cycle. A close relationship between the peak of TDC activity and the highest specific content of serotonin was found, while such a correlation was not observed for the β-carboline alkaloids, the main constituents of the root cultures. The content of serotonin, but not of the alkaloids was greatly enhanced by feeding tryptamine. Trials to identify individual biosynthetic steps of alkaloid biosynthesis by feeding were only partially successful, because only the oxidation of dihydro-β-carbolines to aromatic harmane alkaloids was observed when the various alkaloids were added to the root cultures. Tracer experiments with tryptophan revealed the presence of 5− and 6-hydroxytetrahydronorharmane in the root cultures which, however, should not be biosynthetic intermediates of the major alkaloids, harmalol and harmine.

A recent study showed that N-acylserotonin derivatives have strong inhibitory activity against tyrosinase. To clarify the role of the 5-hydroxy group in the indole ring, 2-, 4-, 5-, 6-, and 7-hydroxyindole and 11 related compounds such as 5-hydroxyindan and 6-hydroxyquinoline were tested for their inhibition of catecholase activity of tyrosinase from human HMVII melanoma cells. 6-Hydroxyindole (5) and 7-hydroxyindole (6) were potent inhibitors, while 5-hydroxyindole (4) was a weaker inhibitor than the above-mentioned compounds (IC50 = 20, 79, 366, and 342 μM for 5, 6, 4, and kojic acid, respectively). 2-Hydroxycarbazole was also active (IC50 = 190 μM), 5-hydroxyindan, 4-aminophenol, and harmalol were slightly active, and other compounds were inactive as an inhibitor. A similar pattern of inhibition was found with these compounds against mouse B16 melanoma tyrosinase, but with some differences from that for HMV-II tyrosinase. Kinetic analysis with HMV-II tyrosinase showed that the inhibition by hydroxyindoles 4, 5, and 6 was competitive with respect to the substrate LDOPA. Melanin formation in HMV-II cells was suppressed by 14% with 10 μM 5 without cytotoxicity, but 30 or 100 μM 5 decreased the cell viability. The present results suggest that 6-hydroxyindole is a potential and useful pharmacophore of antimelanogenic agents and that the position of a phenolic hydroxy group in a specifi c heterocyclic ring such as in indole is possibly optimized to yield more active inhibitors for tyrosinase.

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 β-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 IC50 of 27 μg/l whereas root extracts strongly inhibited MAO-A with an IC50 of 159 μg/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 β-carbolines should contribute to the psychopharmacological and toxicological effects of this plant and could be the basis for its purported antidepressant actions. © 2010 Elsevier Ltd. All rights reserved.

Simple and sensitive methods for the separation and quantification of beta-carboline alkaloids in foods and beverages by HPLC with electrochemical detection at carbon nanotubes-modified glassy carbon electrodes (CNTs-GCE) are reported. Electrode modification with multi-wall CNTs produced an improved amperometric response to beta-carbolines, in spite of the working medium consisting of methanol:acetonitrile: 0.05 mol L(-1) Na(2)HPO(4) solution of pH 9.0 (20:20:60). On the contrary to that observed at a bare GCE, a good repeatability of the amperometric measurements carried out at +900 mV versus Ag/AgCl (R.S.D. of 3.2% for i(p), n=20) was achieved at the CNTs-GCE. Using an Ultrabase C(18) column and isocratic elution with the above mentioned mobile phase, a complete resolution of the chromatographic peaks for harmalol, harmaline, norharmane, harmane and harmine, was achieved. Calibration graphs over the 0.25-100 microM range with detection limits ranging between 4 and 19 ng mL(-1), were obtained. The HPLC-ED at CNTs-GCE method was applied to the analysis of beer, coffee and cheese samples, spiked with beta-carbolines at concentration levels corresponding to those may be found in the respective samples. The steps involved in sample treatment, such as extraction and clean-up, were optimized for each type of sample. Recoveries ranging between 92 and 102% for beer, 92 and 101% for coffee, and 88 and 100% for cheese, at sub-microg mL(-1) or g(-1) analytes concentration levels were achieved.

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.

β-Carboline alkaloids, found in medicinal plants, tobacco smoke and well-cooked foods, have shown a variety of actions in biological systems related to their interaction with DNA. Therefore, these alkaloids can be considered potentially mutagenic. In this work, the genotoxic, mutagenic, and cytotoxic activities of three aromatic β-carboline alkaloids (harman, harmine, and harmol) and two dihydro-β-carboline alkaloids (harmaline and harmalol) were evaluated by means of the Salmonella/microsome assay ( Salmonella typhimurium TA98, TA97, TA100, and TA102) and SOS chromotest ( Escherichia coli PQ37) with and without metabolic activation. Moreover, harman and harmine were analyzed by the micronucleus assay in vivo. It was shown that genotoxicity was inhibited by the addition of S9 mix for aromatic β-carbolines harman and harmol in TA97. However, harmine showed signs of mutagenicity only in the presence of S9 mix in TA98 and TA97 frameshift strains. In the SOS chromotest, only harman induced SOS functions in the absence of S9 mix. Dihydro-β-carbolines were not genotoxic in any of the microorganisms used. The negative responses obtained in the micronucleus assay indicated that harman and harmine were not able to induce chromosomal mutations.

This article reviews our present knowledge on the formation of tryptophan derived secondary metabolites in tissue cultures of Peganum harmala. With the presence of β-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 β-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 β-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.

The preparation of novel biologically active platinum(II) and palladium(II) complexes of some β-carboline alkaloids (harmaline, harmalol, harmine, and harmane) is described. These complexes, characterized on the basis of their CHN elemental analysis, infrared, Raman, and 1H and 13C nuclear magnetic resonance spectral data, were shown to have the empirical formula [M(alkaloid)Cl2], M = Pt, Pd. The antitumor and antiviral activities of some of these complexes have been demonstrated.

A study of some emission parameters (quantum yield and fluorescence lifetime) for six alkaloid derivatives of β-carboline is presented. Depending on pH value, various species can exist in solution. In this work we have studied only two of them: cationic and neutral species (see fig. 1). The variation of quantum yield and fluorescence lifetime has been analysed for different substituted β-carboline rings and for some derivatives which do not present the aromatic character on the pyrimidinic nucleus.

1. 1. Two fractions of aryl acylamidase (EC 3.5.1.13) were further separated from rat brain extracts at pH 7.5 by ammonium sulfate precipitation and Bio-Gel chromatography. 2. 2. 1,2,3,4-Tetrahydro-β-carboline competitively inhibited (67%) fraction-1 but slightly inhibited (13%) fraction-2. Tetrahydroharman, 6-hydroxy-tetrahydroharman and harminic acid slightly inhibited both fractions. Harmalol inhibited fraction-1 but enhanced fraction-2. 6-Methoxy-harman, 6-methoxy-harmalan and harmaline enhanced both fractions. 3. 3. Pargyline did not affect either fraction. Methiothepin, cyproheptadine and chlorimipramine inhibited fraction-1 but stimulated fraction-2. 4. 4. Neostigmine moderately (30%) inhibited AAA-2 but did not have any significant effect on AAA-1. 5. 5. These results indicate that the β-carboline compounds might play a role in regulating activity of AAA-1 and 2 in brain. 6. 6. Both fractions might be related to serotonergic neurons but only AAA-2 might be associated with acetylcholinesterase.

The actions of five harmala alkaloids on the sodium dependent high affinity choline uptake activity in rat striatal synaptosomes was investigated. All five compounds were found to be competitive inhibitors of the uptake system. Harmalol (Ki approximately 3.4 microM) and 2-methylharmine (Ki approximately 5.7 microM) were found to be relatively potent inhibitors in a series with an ascending order of inhibitory potency of harmaline less than 2-methylharmaline less than harmine less than 2-methylharmine less than harmalol.

Unusual behaviour of harmaline and harmalol by excited state proton transfer has been studied and the acidity constants for the different prototropic equilibria in the ground and excited singlet states have been reported.

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- 14 C]- l -tryptophan and [methyl- 14 C-]-harmaline was incorporated into harmine. The dilution of radioactivity was 30000- and 2-fold respectively.

Glucuronidation and sulfation of the phenolic — OH group of harmol and harmalol by the rat in vivo , and by rat liver subcellular fractions in vitro have been studied. In vivo harmol was extensively excreted (59% of the dose in 3 hr) in bile and urine, mostly as harmol-sulfate (70%) but also as harmol-glucuronide (30%). Harmalol was also excreted in bile and urine (50% of the dose in 3 hr), mostly as harmalol-glucuronide (70%), with only a trace of harmalol-sulfate present (less than 3%). In vitro kinetic parameters of conjugating activities towards both substrates were determined. Harmol and harmalol were glucuronidated by UDP glucuronyltransferase at comparable rates. Phenolsulfotransferase converted harmol readily to its sulfate-conjugate, whereas harmalol was a very poor substrate of this enzyme. Thus, the excretory pattern of harmol and harmalol can be explained by different rates of conjugation of these substrates by UDP glucuronyltransferase and phenolsulfotransferase, as found in vitro .

The formation of the glucuronides of harmol and harmalol has been demonstrated in an enzyme system with UDPGA as the glucuronyl donor and guinea pig liver microsomes as the source of UDP-glucuronyltransferase. The subsequent separation of the glucuronide from its aglycone is achieved by thin-layer chromatography and the identification of the conjugated products has been established (1) by hydrolysis with β-glucuronidase and Glusulase and the subsequent identification of the aglycones, (2) by the stoichiometric relation between the amounts of glucuronide formed and of aglycone disappearing, (3) by the linear relationship between the amount of glucuronide formed and the concentration of added UDPGA, and (4) by the lack of formation of glucuronide when UDPG or UDPAG is substituted for UDPGA, or when UDPGA is omitted from the reaction mixture. This glucuronidating reaction with harmol or harmalol provides a method for the measurement of UDPGA in tissues. Likewise, UDPG can be determined after its oxidation to UDPGA by NAD and UDPG dehydrogenase. In this way, both UDPG and UDPGA in various tissues of a number of species of animals have been measured.

The distribution and metabolic fate of [3H]harmaline-HCl were studied in rats. Thirty min after subcutaneous injection, high radioactivity was found in the small intestine, liver, adrenals, kidneys and lungs. A rapid turnover and elimination was evident after the first hour, as most of the tissues, except the liver, kidneys and intestines, had decreased nearly 50 per cent in levels of radioactivity. About 40 per cent of the harmaline was bound to human serum or rat serum proteins in vitro. The blood levels, however, were low at all times in vivo. The peak concentration in the brain occurred at 1 hr postinjection. The major route of excretion of harmaline and its metabolites was through the kidneys; a total of 62 per cent of the injected dose was excreted in the urine during 96 hr as compared to only 11·5 per cent in the feces over the same period. The major fate of harmaline in rats was demethylation to form harmalol, which was predominantly excreted as the glucuronide conjugate. Six to 10 per cent of the radioactivity was identified as the sulfate conjugate of harmol, which was formed by the dehydrogenation of harmalol. During the first 8 hr, unchanged harmaline in the urine amounted to about 25 per cent; however, this decreased to only 7 per cent during the 8–24 hr period.

—Two uridine nucleotides, UDPG and UDPGA, have been measured in brain by a procedure whereby they are extracted from tissue with subsequent utilization, either directly with (UDPGA) or after dehydrogenation (UDPG), in a glucuronidation reaction with harmol or harmalol as cosubstrate. The concentrations of UDPG and UDPGA in brains of a few species of animals examined are, respectively, 11.5–23.3 μmoles/100 g and 0.7–2.5 μmoles/100 g tissue. The ratios of UDPGA to UDPG range from 4% to 11%. The role and importance of these uridine nucleotides in brain are discussed.

The effect of imipramine. desipratnine, harmol, harmalol and some monoamine oxidase inhibitors of the hydrazine type on rat liver UDP glucuronyltransferase (EC 2.4.1.17) activity has been investigated. Substrates of the enzyme were p-nitrophenol and bilirubin; with p-nitrophenol only Triton X-100 activated microsomes were used as enzyme source, with bilirubin both not-activated and Triton X-100 activated microsomes were used. The degree of inhibition obtained with the various inhibitors was dependant on the substrate used and on the pretreatment of the microsomes. It is concluded that most of the effects were caused by the action of the compounds on microsomal membrane structure, which affects UDP glucuronyltransferase activity, and that the physiological relevance of inhibition of the enzyme in vitro is questionable.

A new oxamide, N , N ′-[(3-hydroxy-5-methyl)phenyl]-oxamide has been isolated from an aqueous extract of Peganum harmala seeds. The structure was established together with harmine (7-methoxy-1-methyl-9H-pyrido[3,4- b ]-indole), harmaline (4,9-dihydro-7-methoxy-1-methyl-3H-pyrido[3,4- b ]indole) and harmalol (4,9-dihydro-1-methyl-3H-pyrido[3,4- b ]indol-7-ol).