Natural alkaloid from seeds of PEGANUM
Harmaline targets multiple molecular targets including monoamine oxidase A (MAO-A), NMDA receptors, and DYRK1A kinase. As a reversible MAO-A inhibitor, it increases the availability of monoamines such as serotonin, norepinephrine, and dopamine. By binding to the NMDA receptor as an inverse agonist, it modulates glutamatergic signaling. The compound also acts as a DYRK1A kinase inhibitor and exhibits antioxidant effects. These diverse targets contribute to its psychoactive and neuropharmacological effects.

Harmaline and its derivatives were identified as anti-MDR agents against various highly resistant and Pakistani MDR clinical isolates of E. coli. These compounds may serve as the leads for further studies towards the development of treatment against the infections caused by MDR E. coli.[1]

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Experiments were conducted on Control (Salin) and Experiment (Harmaline) groups, generating a dataset for developing predictive models. Because the dataset has a limited number of samples, we utilized models that are effective with small datasets. Among different groups of regression models (linear, ensemble, and tree models), the ensemble models, specifically the LGB method, can achieve better performance. The results demonstrate accurate prediction of first spike latency, with an average mean squared error of 0.0002 and mean absolute error of 0.01 in 10-fold cross-validation. The research suggests the potential of machine learning in forecasting the first spike latency, allowing reliable estimation without the need for extensive animal testing. This intelligent predictive system facilitates efficient analysis of first spike latency changes in both healthy and unhealthy brain cells, streamlining experimentation and providing more detailed insights into the captured signals.[2]

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In vitro, Harmaline hydrochloride dihydrate inhibits MAO-A activity, increasing monoamine availability. It binds to the NMDA receptor as an inverse agonist. The compound acts as a DYRK1A kinase inhibitor and exhibits antioxidant effects. Harmaline and its derivatives have been identified as anti-MDR agents against various highly resistant clinical isolates of E. coli. The compound can interact with DNA and other intracellular targets. These diverse in vitro activities confirm its multi-target pharmacological profile.

Depression is a mental disorder characterised by persistent low mood, anhedonia and cognitive impairment that affects an estimated 3.8% of the world's population, including 5% of adults. Peganum harmala L. (P. harmala) is a medicinal plant and has been reported to be effective against Alzheimer's disease, Parkinson's disease and depression. The present study was aimed to evaluate the behavioral and pharmacological effects of P. harmala seed extract in rats exposed to chronic unpredictable mild stress (CUMS) in vivo and to investigate the mechanism of action. CUMS-exposed rats were treated with P. harmala extract (75 and 150 mg/kg, i.p.) for 2 weeks. HPLC analysis was used to determine the concentration of Harmaline and harmine alkaloids in the extract. Heavy metal analysis in seeds was performed by ICP-MS. Our results showed that P. harmala at the dose of 150 mg/kg significantly reduced the depressive-like behaviors in CUMS-exposed rats, as evidenced by increased sucrose consumption in the sucrose preference test (SPT), decreased immobility time in the forced swim test (FST) and plasma corticosterone levels, increased the time spent in open arms in the elevated plus maze (EPM), and improved memory and learning in the passive avoidance test (PAT). In addition, P. harmala decreased monoamine oxidase-A (MAO-A) levels, and increased serotonin (5-HT), dopamine (DA), and noradrenaline (NA) levels in the brains of rats exposed to CUMS. P. harmala decreased the expression of the pro-inflammatory transcription factor nuclear factor-κB (NF-κB), and increased the antioxidant nuclear factor erythroid 2-related factor 2 (Nrf2) in rat brain. Furthermore, P. harmala improved brain-derived neurotrophic factor (BDNF) and tropomyosin receptor kinase B (TrkB) protein expression in rat brain. In conclusion, P. harmala at a dose of 150 mg/kg is more effective in preventing depressive-like behavior in CUMS-exposed rats by improving neurotransmitter levels, reducing oxidative stress, suppressing neuroinflammation and activating the BDNF/TrkB pathway, all of which are important in the pathogenesis of depression.[2]

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In vivo, Harmaline causes mice to tremble by binding to the NMDA receptor, an effect used as a pharmacological model for studying essential tremor and other movement disorders. The compound's MAO-A inhibitory activity suggests potential for in vivo studies in depression and anxiety models. Its effects on pregnancy in rats, including abortifacient effects, have been reported, indicating significant biological activity. However, specific pharmacokinetic and efficacy data are limited in the available literature.

The MAO-A inhibitory activity is assessed using fluorometric or radiometric enzyme assays. Recombinant human MAO-A or MAO-B enzyme is incubated with various concentrations of Harmaline and a suitable substrate such as kynuramine. The reaction product is quantified by fluorescence spectroscopy, and IC50 values are calculated from dose-response curves. NMDA receptor binding is assessed using radioligand binding assays or electrophysiological recordings. DYRK1A kinase inhibitory activity is assessed using kinase assays with appropriate substrates.

Multidrug resistance (MDR) is a major challenge in the treatment of infectious diseases. The MDR in urinary tract infection causing bacteria, such as Escherichia coli, has made treatment of UTI very difficult.Objective: The aims of the current study were to synthesize a library of harmaline derivatives, and to evaluate their activity against various strains of multi-drug resistance (MDR) E. coli.Method: Harmaline derivatives were synthesized by the reaction of harmaline (1) with various acid halides and anhydrides. These compounds were subjected to susceptibility determination by in vitro MTT assay. The changes in morphology of the bacterial cells after the treatment with harmaline (1) and its new derivatives 2 and 3 were studied through scanning electron, atomic force and fluorescence microscopy. Effect of harmaline and its derivatives on the production of Reactive Oxygen Species (ROS) in MDR E. coli was assessed through lucigenin chemiluminescence assays.Results: The selected compounds assisted the fluorescently labeled dye DiBAC4(3) to bind to the lipid rich intra-cellular entities, and thus produced a sharp green fluorescence by easily penetrating into the compound-induced depolarized membrane of MDR E. coli. These compounds have also triggered a significant generation of ROS from bacterial cells as compared to the conventional antibiotics. The current study demonstrated that harmaline (1), and its derivatives 2 and 3 were identified as anti-MDR agents against MDR strains of E. coli. Antibacterial effect of compounds 1-3 on MDR E. coli is possibly due to membrane depolarization due to ROS-induced damage to the bacterial cell membrane.[1]

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For cellular studies, various cell lines are cultured in appropriate media. Cells are treated with Harmaline hydrochloride dihydrate at various concentrations for 24-72 hours. Cell viability is assessed using MTT or similar assays. MAO-A activity in cell lysates may be measured. For anti-MDR studies, bacterial cultures are treated with the compound and growth inhibition is assessed. DNA interaction studies may be performed using spectroscopic or electrophoretic methods.

In vivo studies for Harmaline are conducted in rodent models. For tremor studies, Harmaline is administered via intraperitoneal injection, and tremor intensity is measured using electromyography or behavioral scoring. For MAO-A inhibition studies, behavioral tests such as the forced swim test or tail suspension test may be used. For reproductive toxicity studies, pregnant rats are treated with Harmaline, and effects on pregnancy outcomes are assessed. However, specific published protocols for Harmaline hydrochloride dihydrate are limited.

Specific pharmacokinetic data for Harmaline hydrochloride dihydrate are not extensively reported. Harmaline is known to be rapidly absorbed and to cross the blood-brain barrier. The compound undergoes metabolism via hepatic enzymes. Pharmacokinetic studies would be required to determine parameters such as half-life, Cmax, and bioavailability for the hydrochloride dihydrate salt form. The compound's water solubility facilitates formulation for in vivo administration.

Harmaline hydrochloride dihydrate has significant biological activity and potential toxicity. MAO inhibitors can cause hypertensive crisis when combined with tyramine-rich foods (cheese effect). The compound's abortifacient effects in rats indicate potential reproductive toxicity. Tremor induction demonstrates significant CNS effects. Comprehensive toxicology studies would be required before any therapeutic development. The compound should be handled with extreme caution.

[1]. Harmaline and its Derivatives Against the Infectious Multi-Drug Resistant Escherichia coli. Med Chem. 2017;13(5):465-476.
[2]. Peganum harmala L. seed extract attenuates anxiety and depression in rats by reducing neuroinflammation and restoring the BDNF/TrkB signaling pathway and monoamines after exposure to chronic unpredictable mild stress. Metab Brain Dis . 2024 Aug 22. doi: 10.1007/s11011-024-01416-6.
[3]. Comparison of Regression Methods to Predict the First Spike Latency in Response to an External Stimulus in Intracellular Recordings for Cerebellar Cells. Stud Health Technol Inform . 2024 Aug 22:316:796-800.

Background: This study aimed to elucidate the potential anticancer activities and mechanisms of alkaloid extracts from P. harmala, namely, halamine (HAR) and halamine (HAL), in HCT-116 colorectal cancer cells. Methods and Results: Alkaloids from P. harmala seeds were extracted. HCT-116 cells were treated with P. harmala alkaloid extracts, HAR, and HAL. Cytotoxicity was assessed using the MTT assay. Apoptosis activity was detected by flow cytometry and acridine orange (AO)/ethidium bromide (EB) double staining, and cell cycle distribution was analyzed by flow cytometry. The mRNA expression levels of Bcl-2-associated X protein (Bax) and glycogen synthase kinase-3β (GSK3β) were detected by real-time quantitative PCR. Furthermore, the expression levels of Bax, Bcl-2, GSK3β, and p53 proteins were detected by Western blotting. The results showed that P. harmala alkaloid extracts HAR and HAL exhibited significant cytotoxicity in HCT-116 cells after 24 and 48 hours of treatment. We found that camel thorn alkaloid extract can induce apoptosis in HCT116 cells and arrest their cell cycle in the G2 phase. We also observed downregulation of GSK3β and Bcl-2 expression and upregulation of Bax and p53 expression. Conclusion: The results of this study indicate that camel thorn alkaloid extract has anticancer activity and can be further studied and developed into future anticancer chemotherapy drugs. Mol Biol Rep. 2024 Jun 13;51(1):732. doi: 10.1007/s11033-024-09655-7. https://pubmed.ncbi.nlm.nih.gov/38872006/

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Halmin is a Halman alkaloid in which the Halman skeleton is substituted with a methoxy group at the C-7 position and the 3,4 bond is reduced. It has a nocturnal effect. It is derived from the hydride of Halman.

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Harmin is reportedly found in passion fruit (Passiflora phoenicia), water flea (Daphnia pulex), and other organisms with relevant data. LOTUS - Natural Products Database. A β-carboline alkaloid isolated from the seeds of PEGANUM.

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Therapeutic Uses
/EXPL THER/ Oxidative modification of low-density lipoprotein (LDL) particles is associated with the development of atherosclerosis. Antioxidants that prevent LDL oxidation may help alleviate atherosclerosis. We investigated the protective effects of P. harmala extract (P extract) and two major alkaloids (harmin and harmine) from its seeds against copper sulfate-induced low-density lipoprotein (LDL) oxidation. The extract (P extract) and its compounds exhibited inhibitory effects by determining the production of malondialdehyde (MDA) and conjugated dienes, as well as the hysteresis period. Furthermore, both harmine and harmin reduced the rate of vitamin E depletion and demonstrated significant free radical scavenging capacity (DPPH). However, harmine showed significantly higher antioxidant capacity than harmin in scavenging or preventing free radicals and inhibiting oxidation-induced LDL protein (apolipoprotein B) aggregation. These results suggest that P. harmala compounds may be a major source of compounds for inhibiting copper-induced LDL oxidation.

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Absorption, Distribution, and Excretion
Harmin is known to be an inhibitor of monoamine oxidase type A (MAO) in the adult brains of various animals. Studies have found that fetuses born to mother rats injected with harmin 2-4 hours before cesarean section have elevated levels of dopamine and serotonin (5-HT). Similar stimulating effects have been observed with the norepinephrine metabolite 3-methoxy-4-hydroxyphenylethylene glycol (MHPG), but without a significant effect on norepinephrine itself. The dopamine metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) and the 5-HT metabolite 5-hydroxyindoleacetic acid (5-HIAA) are both decreased under the same treatment. These results suggest that halamine or one of its metabolites may cross the placental barrier and affect the fetal brain system, not only as an inhibitor of type A MAO (i.e., relatively 5-HT specific), but also as an stimulator of aldosterone reductase or catechol-O-methyltransferase (COMT), or as a drug that inhibits the binding, efflux, or turnover of biogenic amine metabolites (such as MHPG). PMID:2465555 Okonmah AD et al.; Pharmacology 37 (3): 203-8 (1988)

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Metabolism/Metabolites
Psychoactive β-carboline alkaloids have high affinity for serotonin, dopamine, benzodiazepines, and imidazoline receptors and can stimulate locus coeruleus neurons, which are endogenously formed in plants and mammals by tryptophan-derived indolylamines via Pictet-Spengler condensation with aldehydes. Cytochrome P450 1A1 (18.5), 1A2 (20), and 2D6 (100) catalyzed the O-demethylation of halamine, while CYP1A1 (98.5), CYP1A2 (35), CYP2C9 (16), CYP2C19 (30), and CYP2D6 (115) catalyzed the O-demethylation of halamine (relative activity). Aromatases (CYP19), CYP1A2, CYP2C9, CYP2D6, CYP3A4, a mixture of recombinant cytochrome P450, or human liver microsomes (HLM) could not catalyze the dehydrogenation/aromatization of halamine to halamine. Kinetic parameters of the O-demethylation reactions mediated by each isoenzyme and recombinant HLM were calculated. Halmin's K(cat) (min(-1)) and Ku(uM) values 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. Halmin's K(cat) and Ku(uM) values 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 are the major isoenzymes for halamine (20% and 50%, respectively) and halamine (20% and 30%, respectively) O-demethylation in mixed human liver microsomes (HLM). The turnover rate of CYP2D6 is among the highest reported for CYP2D6 substrates to date. Finally, CYP2D6 transgenic mice exhibited increased halamine and halamine O-demethylase activities compared to wild-type mice. These findings suggest that polymorphic CYP2D6 plays a role in the pharmacology and toxicology of halamine and halamine. PMID:12649384 Yu AM et al.; Journal of Pharmacology and Experimental Therapy 305 (1): 315-22 (2003)

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Mechanism of Action
Three psychoactive components extracted from the seeds of Peganum harmala L.—harmin, harine, and harol—all exhibited vasodilatory activity in isolated rat thoracic aortic specimens pre-constricted with phenylephrine or potassium chloride, with the vasodilatory efficacy in the order: harmin > harine > harol. Endothelial removal or pretreatment with nitric oxide synthase Nω-nitro-L-arginine methyl ester attenuated the vasodilatory effects of harmin and harine (but not harol). In cultured rat aortic endothelial cells, harmin and harine (but not harol) increased NO release, and this process depended on the presence of extracellular Ca2+. In endothelial-desorbed vascular preparations, pretreatment with halamine, halmalin, or halmol (3–30 μM) non-competitively inhibited phenylephrine-induced contraction. Receptor binding assays showed that all three compounds interacted with cardiac α1-adrenergic receptors with similar affinity (Ki values approximately 31–36 μM), but only halamine showed a weaker interaction with the 1,4-dihydropyridine binding site of cardiac L-type Ca2+ channels (Ki value 408 μM). Therefore, these results suggest that the vasodilatory effects of halamine and halmalin are attributed to their action on endothelial cell NO release and their action on vascular smooth muscle to inhibit contraction induced by receptor coupling and voltage-dependent Ca2+ channel activation. The vasodilatory effect of halmol is endothelial-independent. PMID:11325023 Shi CC et al; Jpn J Pharmacol 85 (3): 299-305 (2001)

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Harmaline Hydrochloride Dihydrate is a β-carboline alkaloid that inhibits MAO-A, binds to NMDA receptors as an inverse agonist, and inhibits DYRK1A kinase. It induces tremor in mice and has abortifacient effects. No clinical trials or approvals exist. For research use only.

C13H19CLN2O3

286.7546

286.108

6027-98-1

2723643

Typically exists as solid at room temperature

426.4ºC at 760 mmHg

232-240 °C

211.7ºC

PEGANUM

2.65

4

4

1

19

302

0

Cl[H].O(C([H])([H])[H])C1C([H])=C([H])C2=C(C=1[H])N([H])C1C(C([H])([H])[H])=NC([H])([H])C([H])([H])C=12.O([H])[H].O([H])[H]

LCEKUHFBUFUSSY-UHFFFAOYSA-N

InChI=1S/C13H14N2O.ClH.2H2O/c1-8-13-11(5-6-14-8)10-4-3-9(16-2)7-12(10)15-13;;;/h3-4,7,15H,5-6H2,1-2H3;1H;2*1H2

7-methoxy-1-methyl-4,9-dihydro-3H-pyrido[3,4-b]indole;dihydrate;hydrochloride

Harmaline hydrochloride dihydrate; UNII-5B4DGH2M9R; 6027-98-1; 63885-08-5; 5B4DGH2M9R; 7-methoxy-1-methyl-4,9-dihydro-3H-pyrido[3,4-b]indole;dihydrate;hydrochloride; MFCD00150052; 7-Methoxy-1-methyl-4,9-dihydro-3H-pyrido[3,4-b]indole hydrochloride dihydrate;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)