Harmol

Human CYP2D6 in the Brain Is Protective Against Harmine-Induced Neurotoxicity: Evidence from Humanized CYP2D6 Transgenic Mice.[Pubmed:32761352]

Mol Neurobiol. 2020 Nov;57(11):4608-4621.

CYP2D6 metabolically inactivates several neurotoxins, including beta-carbolines, which are implicated in neurodegenerative diseases. Variation in CYP2D6 within the brain may alter local inactivation of neurotoxic beta-carbolines, thereby influencing neurotoxicity. The beta-carboline harmine, which induces hypothermia and tremor, is metabolized by CYP2D6 to the non-hypothermic/non-tremorgenic Harmol. Transgenic mice (TG), expressing human CYP2D6 in addition to their endogenous mouse CYP2D, experience less harmine-induced hypothermia and tremor compared with wild-type mice (WT). We first sought to elucidate the role of CYP2D in general within the brain in harmine-induced hypothermia and tremor severity. A 4-h intracerebroventricular (ICV) pretreatment with the CYP2D inhibitor propranolol increased harmine-induced hypothermia and tremor in TG and increased harmine-induced hypothermia in WT. We next sought to specifically demonstrate that human CYP2D6 expressed in TG brain altered harmine response severity. A 24-h ICV propranolol pretreatment, which selectively and irreversibly inhibits human CYP2D6 in TG brain, increased harmine-induced hypothermia. This 24-h pretreatment had no impact on harmine response in WT, as propranolol is not an irreversible inhibitor of mouse CYP2D in the brain, thus confirming no off-target effects of ICV propranolol pretreatment. Human CYP2D6 activity in TG brain was sufficient in vivo to mitigate harmine-induced neurotoxicity. These findings suggest that human CYP2D6 in the brain is protective against beta-carboline-induced neurotoxicity and that the extensive interindividual variability in CYP2D6 expression in human brain may contribute to variation in susceptibility to certain neurotoxin-associated neurodegenerative disorders.

High Content Screening Using New U2OS Reporter Cell Models Identifies Harmol Hydrochloride as a Selective and Competitive Antagonist of the Androgen Receptor.[Pubmed:32560058]

Cells. 2020 Jun 16;9(6). pii: cells9061469.

Prostate cancer is the most commonly diagnosed malignancy in men. Its growth mainly relies on the activity of the androgen receptor (AR), justifying the use of androgen deprivation therapy as a gold standard treatment for the metastatic disease. Inhibition of the androgen axis using second generation antagonists has improved patients' survival, but is systematically confronted to resistance mechanisms, leading to a median survival that does not exceed 5 years. Counteracting this resistance has been the object of a large number of investigations, with a particular emphasis towards the identification of new AR inhibitors, whether they antagonize the receptor by a competitive or a non-competitive binding. To this end, many high content screens have been performed, to identify new non-steroidal AR antagonists, using a variety of approaches, but reported somewhat controversial results, depending on the approach and on the cell model that was used for screening. In our study, we used the U2OS osteosarcoma cells stably transfected with AR or ARv7 and a luciferase reporter as a previously validated model to screen the Prestwick Phytochemical library. The results of our screen identified ellipticine, Harmol, and harmine hydrochloride as confirmed hits. Surprisingly, we could demonstrate that Harmol hydrochloride, previously identified as a non-competitive inhibitor of AR or a weak inhibitor of androgen signaling, was actually a competitive antagonist of AR, which inhibits the growth of VCaP prostate cancer line, at concentrations for which it did not affect the growth of the AR negative DU145 and PC3 cells. Interestingly, we also report for the first time that Harmol hydrochloride was selective for AR, as it could not alter the activity of other nuclear receptors, such as the glucocorticoid receptor (GR), the progesterone receptor (PR), or the mineralocorticoid receptor (MR). Additionally, we demonstrate that, conversely to enzalutamide, Harmol hydrochloride did not show any agonistic activity towards the pregnane X receptor (PXR), a master regulator of drug metabolism. Together, our results shed light on the importance of the cellular context for the screening of new AR antagonists. They further indicate that some of the potential hits that were previously identified may have been overlooked.

Automated piezoelectric spraying of biological and enzymatic assays for effect-directed analysis of planar chromatograms.[Pubmed:31153601]

J Chromatogr A. 2019 Sep 27;1602:458-466.

Bioanalytical questions are more and more solved by bioassays directly in situ the planar separation. If compared to chemical derivatization in situ, several reagent applications on the same chromatogram make the workflow for enzymatic and biological assays more complex. Hence, if compared to piezoelectric spraying of chemical derivatization reagents, an assay transfer to the piezoelectric spraying technique was much more challenging. Important aspects were investigated, i.e., plate pre-wetting, spraying nozzle type and applied volumes for microorganism suspension as well as enzyme and substrate-chromogenic solutions. Finally, with the newly developed piezoelectric spraying procedures for the application of biological (Aliivibrio fischeri) and enzymatic (acetyl- and butyrylcholinesterase) assays, several obstacles of the state-of-the-art automated immersion were avoided such as the (1) required high volumes of solutions, (2) tailing of highly water-soluble zones upon slow plate withdrawal, (3) zone distortion or shift observed after previous buffer salt applications or long/slow immersion times/speeds, (4) gradual inactivation of the enzyme solution along with its ongoing re-use, and (5) lack of covering the whole plate surface. The benchmarking of both techniques also showed that simplicity remains the key argument for immersion. As proof of concept, piezoelectrically sprayed autograms were compared with those of immersion, by taking the example of Peganum harmala (P. h.) seed extract. The plate background and thus homogeneity of the applied solutions were found to be almost comparable. Three bands among the pronounced fluorescent bands were responsible for the most antibacterial activity of P. h. seed extract in the A. fischeri bioassay and were also inhibiting the AChE. These AChE and three further BChE inhibitors were detected, whereby the AChE inhibition was twice as strong as the BChE inhibition. By their in situ HRMS spectra, the active zones in the P. h. seed extract were assigned to be the AChE-inhibiting beta-carboline alkaloids, harmine, harmaline and ruine, as well as the BChE-inhibiting quinazoline alkaloids, vasicine and deoxyvasicine, and the beta-carboline alkaloid Harmol. For the first time, the found inhibitors were calculated equivalently to the well-known ChE-inhibitor physostigmine, and thus, piezoelectric spraying was proven to be suited for quantifications.

[N-containing compounds from seeds of Paganum harmala].[Pubmed:31090324]

Zhongguo Zhong Yao Za Zhi. 2019 Apr;44(8):1601-1606.

To investigate the N-containing compounds in the seeds of Paganum harmala,fourteen compounds were finally isolated from the 95% Et OH extract of P. harmala seeds by using various chromatographic techniques including silica gel,MCI resin,and ODS column chromatography as well as the semi-preparative HPLC. Depending on spectroscopic techniques and comparison with the reported data in the literatures,the structures of all compounds were identified as N-[3-(2-amino-4-methoxyphenyl)-3-oxopropyl]acetamide(1),dehydroharmalacidine(2),h armalacidine(3),harmine N-oxide(4),harmine(5),tetrahydroharmine(6),demethylharmalacidine(7),Harmol(8),tet rahydroHarmol(9),harmindol beta-D-glucopyranoside(10),tryptophyl beta-D-glucopyranoside(11),pegaminebeta-D-glucopyranoside(12),vasicol(13) and vasicinone(14). Among them,1 was a new compound,and 2 and 10 were obtained as natural products for the first time.

Potential Pharmacokinetic Drug(-)Drug Interaction Between Harmine, a Cholinesterase Inhibitor, and Memantine, a Non-Competitive N-Methyl-d-Aspartate Receptor Antagonist.[Pubmed:30978991]

Molecules. 2019 Apr 11;24(7). pii: molecules24071430.

Harmine (HAR) is a beta-carboline alkaloid widely distributed in nature. It exhibits psychopharmacological effects of improving learning and memory. However, excessive dose of HAR can cause central tremor toxicity, which may be related to the glutamate system. Memantine (MEM) is a non-competitive N-methyl-d-aspartate receptor antagonist. It can be used for the treatment of Alzheimer's disease and also can block the neurotoxicity caused by glutamate. Therefore, combination of HAR and MEM would be meaningful and the pharmacokinetics investigation of HAR and MEM in combination is necessary. A ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was established and validated for the simultaneous quantitative determination of MEM, HAR and Harmol (HOL), a main metabolite of HAR, in rat plasma after oral administration of HAR and MEM in combination (5.0 mg/kg of MEM combined with 20.0, 40.0, 80.0 mg/kg of HAR). The contents of HAR and HOL were determined after oral administration of HAR (20.0, 40.0 and 80.0 mg/kg), and the content of MEM was determined after oral administration of MEM (5.0 mg/kg). Blood samples were collected from each rat at 0 (pre-dose), 0.08, 0.17, 0.25, 0.33, 0.50, 0.75, 1.0, 2.0, 4.0, 8.0, 12.0 and 24.0 h after administration. The maximum peak concentration (Cmax) of MEM was obviously decreased, and the area under the plasma concentration versus time curve from zero to time t (AUC(0-t)) and mean residence time (MRT) were significantly increased after combination with HAR. The Cmax and AUC(0-t) of HAR and its metabolite HOL were increased after combination with MEM. These findings suggested that co-administration of HAR and MEM could extend their residence time in rats, and then might increase the efficacy for treatment of Alzheimer's disease. Therefore, this study will provide a basis for the rational combined application of HAR and MEM.

Subchronic toxicity and concomitant toxicokinetics of long-term oral administration of total alkaloid extracts from seeds of Peganum harmala Linn: A 28-day study in rats.[Pubmed:30970283]

J Ethnopharmacol. 2019 Jun 28;238:111866.

ETHNOPHARMACOLOGICAL RELEVANCE: The seeds of Peganum harmala Linn, in which the most abundant active compounds are harmaline and harmine, have been widely used as a traditional medicine in various countries to treat a broad spectrum of diseases including asthma, cough, depression, Parkinson's and Alzheimer's diseases. However, few studies on long-term or subchronic toxicity of seeds of P. harmala were reported after overdose. AIM OF THE STUDY: To investigate the subchronic toxicity and concomitant toxicokinetics of total alkaloid extracts from seeds of P. harmala (TAEP) after oral administration for four weeks in rats. MATERIALS AND METHODS: The subchronic toxicity and concomitant toxicokinetics of TAEP were evaluated after 28-day oral administration in rats at daily dose levels of 15, 45, and 150mg/kg. The signs of toxicity and mortality were monitored and recorded daily. The body weight and average food consumption were measured weekly. The analyses of hematology, biochemistry, urine, relative organ weights and histopathology were conducted at the termination of treatment and recovery phase. For concomitant toxicokinetics study, the plasma toxicokinetic parameters, tissue distribution, and excretion of predominant ingredients harmaline and harmine in TAEP and metabolites harmalol and Harmol were tested. RESULTS: Following initial repeated exposure to high-dose (150mg/kg/day) of TAEP excitotoxic reaction, such as tremor, was observed, but tolerated on the fourth day after multiple dosing. The significant alterations in blood glucose and lipid metabolism in liver were observed, but recovered after four weeks of drug withdrawal. The no-observed-adverse-effect level (NOAEL) of TAEP was considered to be 45mg/kg/day under the present study conditions. There were no significant gender differences in most indexes of subchronic toxicity throughout the experimental period with the exception of food consumption and body weight. In concomitant toxicokinetics study, the alterations of dynamic characteristic for harmaline, harmine and metabolite Harmol after multiple oral administration at three doses had been observed. Harmaline, harmine and metabolites harmalol and Harmol were widely distributed in organs and there was no accumulation in the tissues examined. The reduction of harmaline and metabolite harmalol in brain after multiple dosing at dose of 150mg/kg might be closely related to the tremor tolerance. The main excretory pathway for metabolites harmalol and Harmol was urinary excretion via kidney. CONCLUSIONS: The results revealed that TAEP at doses of 15 and 45mg/kg/day in rats might be safe. Excitotoxic reaction such as tremor occurred initially at dose of 150mg/kg/day, however, the toxicity was tolerant and reversible. In addition, harmaline and harmine in TAEP had a quick absorption into blood and metabolized to harmalol and Harmol, and there was no drug accumulation in the detected tissues. Further studies should be investigated to clarify the mechanisms of tremor tolerance and neurotoxicity of TAEP.

Natural beta-carboline alkaloids regulate the PI3K/Akt/mTOR pathway and induce autophagy in insect Sf9 cells.[Pubmed:30765058]

Pestic Biochem Physiol. 2019 Feb;154:67-77.

The beta-carboline alkaloids are a large group of naturally occurring and synthetic indole alkaloids with remarkable pharmacological properties. Furthermore, these alkaloids have also been reported to be effective agents for controlling many pests and plant pathogenic nematodes. However, studies on these potential insecticidal components are scarce. The previous finding that these bioactive compounds can induce programmed cell death in cancer cell lines provided a new insight for exploration of their toxicological mechanisms on insects. In the present study, the cytotoxicity of five natural harmala alkaloids was measured, and the autophagy-inducing effect was confirmed in the Spodoptera frugiperda Sf9 cultured cell line. The results demonstrated that these alkaloids inhibited the proliferation of Sf9 cells in a dose- and time-dependent manner, and the unsaturated beta-carboline alkaloids, harmine and Harmol, exhibited stronger autophagy induction activity based on monodansylcadaverineand LysoTracker Red staining. Many autophagy-related genes were increased after beta-carbolines treatment at the RNA level, and the protein expression of Sf-Atg8 was also confirmed to increase after treatment. In addition, the primary autophagic signaling pathway, the PI3K/Akt/mTOR pathway, was altered during the procedure. Furthermore, experiments with special inhibitors and activators were performed to confirm the effect of beta-carbolines on this pathway. The results suggested that the PI3K/Akt/mTOR pathway primarily regulated harmine-induced autophagy in insect cells, and this finding may potentially benefit the application of these promising bioactivity components.

The alkaloids of Banisteriopsis caapi, the plant source of the Amazonian hallucinogen Ayahuasca, stimulate adult neurogenesis in vitro.[Pubmed:28706205]

Sci Rep. 2017 Jul 13;7(1):5309.

Banisteriopsis caapi is the basic ingredient of ayahuasca, a psychotropic plant tea used in the Amazon for ritual and medicinal purposes, and by interested individuals worldwide. Animal studies and recent clinical research suggests that B. caapi preparations show antidepressant activity, a therapeutic effect that has been linked to hippocampal neurogenesis. Here we report that harmine, tetrahydroharmine and harmaline, the three main alkaloids present in B. caapi, and the harmine metabolite Harmol, stimulate adult neurogenesis in vitro. In neurospheres prepared from progenitor cells obtained from the subventricular and the subgranular zones of adult mice brains, all compounds stimulated neural stem cell proliferation, migration, and differentiation into adult neurons. These findings suggest that modulation of brain plasticity could be a major contribution to the antidepressant effects of ayahuasca. They also expand the potential application of B. caapi alkaloids to other brain disorders that may benefit from stimulation of endogenous neural precursor niches.

Harmine is an inflammatory inhibitor through the suppression of NF-kappaB signaling.[Pubmed:28551404]

Biochem Biophys Res Commun. 2017 Jul 29;489(3):332-338.

Harmine is a major constituent in a hallucinogenic botanical mixture ayahuasca and medical plant Peganum harmala L. The plant is used for various illnesses and exhibits anti-inflammatory activity. However, the active constituents remain unclear. Here, we screened the seven alkaloids in P. harmala for their anti-inflammatory activity using an nuclear factor-kappaB (NF-kappaB) reporter assay. We found that harmine and Harmol could inhibit NF-kappaB transactivity. As the most abundant compound, harmine inhibited tumor necrosis factor-alpha (TNF-alpha)- and lipopolysaccharides (LPS)-induced NF-kappaB transactivity and nuclear translocation in mouse macrophage RAW 264.7 cells. The mRNA and protein levels of NF-kappaB downstream inflammatory cytokines also reduced. In an LPS-challenged mouse model, harmine markedly averted inflammatory damage of the lung, and decreased serum TNF-alpha, interleukin-1beta (IL-1beta) and IL-6 levels. Our data indicate that harmine may exert the anti-inflammatory effect by inhibition of the NF-kappaB signaling pathway and harmine is probably responsible for the anti-inflammatory effects of P. harmala.

UVA Photoactivation of Harmol Enhances Its Antifungal Activity against the Phytopathogens Penicillium digitatum and Botrytis cinerea.[Pubmed:28326067]

Front Microbiol. 2017 Mar 7;8:347.

Phytopathogenic fungi responsible for post-harvest diseases on fruit and vegetables cause important economic losses. We have previously reported that Harmol (1-methyl-9H-pyrido[3,4-b]indol-7-ol) is active against the causal agents of green and gray molds Penicillium digitatum and Botrytis cinerea, respectively. Here, antifungal activity of Harmol was characterized in terms of pH dependency and conidial targets; also photodynamic effects of UVA irradiation on the antimicrobial action were evaluated. Harmol was able to inhibit the growth of both post-harvest fungal disease agents only in acidic conditions (pH 5), when it was found in its protonated form. Conidia treated with Harmol exhibited membrane integrity loss, cell wall disruption, and cytoplasm disorganization. All these deleterious effects were more evident for B. cinerea in comparison to P. digitatum. When conidial suspensions were irradiated with UVA in the presence of Harmol, antimicrobial activity against both pathogens was enhanced, compared to non-irradiated conditions. B. cinerea exhibited a high intracellular production of reactive oxygen species (ROS) when was incubated with Harmol in irradiated and non-irradiated treatments. P. digitatum showed a significant increase in ROS accumulation only when treated with photoexcited Harmol. The present work contributes to unravel the antifungal activity of Harmol and its photoexcited counterpart against phytopathogenic conidia, focusing on ROS accumulation which could account for damage on different cellular targets.

Indole Alkaloids from Plants as Potential Leads for Antidepressant Drugs: A Mini Review.[Pubmed:28293192]

Front Pharmacol. 2017 Feb 28;8:96.

Depression is the most common illness observed in the elderly, adults, and children. Antidepressants prescribed are usually synthetic drugs and these can sometimes cause a wide range of unpleasant side effects. Current research is focussed on natural products from plants as they are a rich source of potent new drug leads. Besides Hypericum perforatum (St. John's wort), the plants studied include Passiflora incarnata L. (passion flower), Mitragyna speciosa (kratom), Piper methysticum G. Forst (kava) and Valeriana officinalis L. Harman, Harmol, harmine, harmalol and harmaline are indole alkaloids isolated from P. incarnata, while mitragynine is isolated from M. speciosa. The structure of isolated compounds from P. methysticum G. Forst and V. officinalis L. contains an indole moiety. The indole moiety is related to the neurotransmitter serotonin which is widely implicated for brain function and cognition as the endogenous receptor agonist. An imbalance in serotonin levels may influence mood in a way that leads to depression. The moiety is present in a number of antidepressants already on the market. Hence, the objective of this review is to discuss bioactive compounds containing the indole moiety from plants that can serve as potent antidepressants.

Parts-per-trillion detection of harmala alkaloids in Undaria pinnatifida algae by on-line solid phase extraction capillary electrophoresis mass spectrometry.[Pubmed:28081815]

Anal Chim Acta. 2017 Feb 15;954:60-67.

beta-carboline alkaloids of the harmala group (HAlks)-a family of compounds with pharmacologic effects-can be found at trace levels (<25 mug kg(-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 C18 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 C18-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.

Antifungal activity of beta-carbolines on Penicillium digitatum and Botrytis cinerea.[Pubmed:27889171]

Food Microbiol. 2017 Apr;62:9-14.

beta-carbolines (betaCs) are alkaloids widely distributed in nature that have demonstrated antimicrobial properties. Here, we tested in vitro six betaCs against Penicillium digitatum and Botrytis cinerea, causal agents of postharvest diseases on fruit and vegetables. Full aromatic betaCs (harmine, Harmol, norharmane and harmane) exhibited a marked inhibitory effect on conidia germination at concentrations between 0.5 and 1 mM, while dihydro-betaCs (harmalina and harmalol) only caused germination delay. Harmol showed the highest inhibitory effect on both fungal pathogens. After 24 h of exposure to 1 mM Harmol, conidia revealed a severe cellular damage, exhibiting disorganized cytoplasm and thickened cell wall. Harmol antimicrobial effect was fungicidal on B. cinerea, while it was fungistatic on P. digitatum. Conidia membrane permeabilization was detected in treatments with Harmol at sub-inhibitory and inhibitory concentrations, for both pathogens. In addition, residual infectivity of P. digitatum on lemons and B. cinerea on blueberries was significantly reduced after exposure to this alkaloid. It also inhibited mycelial growth, preventing sporulation at the highest concentration tested. These results indicate that Harmol might be a promising candidate as a new antifungal molecule to control causal agents of fruit diseases.

Antiviral activity of natural and synthetic beta-carbolines against dengue virus.[Pubmed:27568370]

Antiviral Res. 2016 Oct;134:26-33.

Dengue virus (DENV) is the most prevalent mosquito borne viral pathogen worldwide. In this work we first evaluated the antiviral activity of natural and synthetic beta-carbolines against DENV-2 multiplication in cell cultures. We determined that the natural beta-carboline Harmol and a synthetic harmine derivative, 9N-methylharmine, exhibit inhibitory effect on DENV-2 production without virucidal activity. The active compounds were inhibitory of all DENV serotypes, being DENV-2 the more susceptible to their antiviral action. The mode of action of 9N-methylharmine against DENV-2 was further explored. We determined that the derivative neither affects viral adsorption-internalization events nor viral RNA synthesis. The quantification of intracellular and extracellular viral genomes and infectious virus particles indicated that 9N-methylharmine would impair the maturation and release of virus particles to the extracellular medium affecting the spreading of the infection. Furthermore, we also determined that 9N-methylharmine antiviral activity is not related to the ability of the compound to downregulate p38 MAPK phosphorylation.

Quality criterion to optimize separations in capillary electrophoresis: Application to the analysis of harmala alkaloids.[Pubmed:27443250]

J Chromatogr A. 2016 Aug 19;1460:190-6.

In capillary electrophoresis (CE), resolution (Rs) and selectivity (alpha) 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.