Cell-based assays and molecular simulation reveal that the anti-cancer harmine is a specific matrix metalloproteinase-3 (MMP-3) inhibitor.

The biological activities of harmine have been a much clearer picture in recent years, which include anti-tumor, anti-inflammation and cytotoxic properties. Numerous in vitro and in vivo animal models have confirmed its activities, but its mode of action remains a relative unsolved issue. We therefore investigated harmine for its effects on MMP-3 and the molecular interaction was also simulated. The human glioma cancer cell line, U-87 MG cells, was subjected to different concentrations (1-10 µM) of harmine for 24 h. Methylthiazol tetrazolium (MTT) test, half maximal inhibitory concentration (IC50), western blot analysis, enzyme-linked immunosorbent assay and molecular docking through BIOVIA DiscoveryStudio™ were performed. These results showed that although harmine stimulation in vitro has very little or no effects on MMP-3 expression by U-87 MG cells, the treatment of harmine decreases MMP-3 activity in a dose dependent manner. It was further calculated that 7.9 µM is the IC50 towards MMP-3. Using a molecular dynamic simulation approach, we identified the N2, methyl of C1 and benzene ring of harmine interact with Zn2+ (2.4 Å), His205 (2.4 Å) and His211 (2.4 Å) as well as Val163 (2.7 Å) at the active site of MMP-3, respectively, and thus conferred a striking specific binding advantage. Taken altogether, the present study evidences that harmine acts as an MMP-3 inhibitor specially targeting the enzymatic active site and possibly efficiently ameliorates MMP-3-driven malignant and inflammatory diseases.

Pharmacological effects of harmine and its derivatives: a review.

Harmine is isolated from the seeds of the medicinal plant, Peganum harmala L., and has been used for thousands of years in the Middle East and China. Harmine has many pharmacological activities including anti-inflammatory, neuroprotective, antidiabetic, and antitumor activities. Moreover, harmine exhibits insecticidal, antiviral, and antibacterial effects. Harmine derivatives exhibit pharmacological effects similar to those of harmine, but with better antitumor activity and low neurotoxicity. Many studies have been conducted on the pharmacological activities of harmine and harmine derivatives. This article reviews the pharmacological effects and associated mechanisms of harmine. In addition, the structure-activity relationship of harmine derivatives has been summarized.

Harman and norharman, metabolites of entomopathogenic fungus Conidiobolus coronatus (Entomopthorales), disorganize development of Galleria mellonella (Lepidoptera) and affect serotonin-regulating enzymes.

Naturally occurring entomopathogenic fungi such as Conidiobolus coronatus are important regulatory factors of insect populations. GC-MS analysis of fungal cell-free filtrates showed that C. coronatus synthesizes two ß- carboline alkaloids: harman and norharman. Significantly higher levels of both alkaloids are produced by C. coronatus in minimal postincubation medium than in rich medium. The beta-carboline alkaloids may have an effect on the nervous system of insects and their behavior. Harman and norharman were applied to Galleria mellonella larvae (a parasite of honeybees) either topically or mixed with food. Larvae received alkaloids in three concentrations: 750, 1000 or 1250 ppm. The effect on the survival and further development of larvae was examined. Both harman and norharman delayed pupation and adult eclosion, and inhibit total monoamine oxidase activity. In addition, they increased the serotonin concentration and decreased the monoamine oxidase A level in the heads of the moths. It is likely that the alkaloids were metabolized by the insects, as their effect wore off 24 hours after topical application. This is the first study to show that C. coronatus produces alkaloids. Its aim was to identify the actions of ß-carboline alkaloids on insect development and serotonin-regulating enzymes. Knowledge of the potential role of harman and norharman in the process of fungal infection might lead to the development of more effective and environmentally-friendly means of controlling insect pests.

Anti HSV-2 activity of Peganum harmala (L.) and isolation of the active compound.

Genital herpes is a sexually transmitted disease caused by herpes simplex virus type 2 (HSV-2). Nucleoside analogues such as acyclovir (ACV) are the usual therapy for treating HSV infection. However, the overuse of this drug has led to the emergence of resistant strains. Therefore, the search for new alternative or complementary molecules to overcome this obstacle is needed. In this objective, Peganum harmala was investigated for its HSV-2 activity. The organic extracts of the different plant organs were evaluated for their cytotoxicity on Vero cells by the MTT test and anti HSV-2 activity by plaque reduction assay. Only the methanol seeds extract was active with a 50% inhibitory concentration (IC50) and a selectivity index (SI) of 161 and 13.2 µg/mL, respectively. In addition, the study of the antiviral mode of action revealed that this extract exerts a virucidal action both during the entry of viruses and the release of the newly formed virions, whereas no cell protection effect was observed. The active compound was isolated by bio-guided purification using thin layer chromatography (TLC) and identified by GC-MS and HPLC-DAD-ESI-MSn as harmine. The combination of harmine standard compound with ACV showed a combination index (CI) of 0.5 indicating that these two compounds have a synergic effect. This data suggests that harmine could be associated to ACV to improve the treatment of genital herpes essentially for the immunocompromised patients.

Effects of the Natural ß-Carboline Alkaloid Harmine, a Main Constituent of Ayahuasca, in Memory and in the Hippocampus: A Systematic Literature Review of Preclinical Studies.

Harmine is a natural ß-carboline alkaloid found in several botanical species, such as the Banisteriopsis caapi vine used in the preparation of the hallucinogenic beverage ayahuasca and the seeds of Syrian rue (Peganum harmala). Preclinical studies suggest that harmine may have neuroprotective and cognitive-enhancing effects, and retrospective/observational investigations of the mental health of long-term ayahuasca users suggest that prolonged use of this harmine-rich hallucinogen is associated with better neuropsychological functioning. Thus, in order to better investigate these possibilities, we performed a systematic literature review of preclinical studies analyzing the effects of harmine on hippocampal neurons and in memory-related behavioral tasks in animal models. We found two studies involving hippocampal cell cultures and nine studies using animal models. Harmine administration was associated with neuroprotective effects such as reduced excitotoxicity, inflammation, and oxidative stress, and increased brain-derived neurotrophic factor (BDNF) levels. Harmine also improved memory/learning in several animal models. These effects seem be mediated by monoamine oxidase or acetylcholinesterase inhibition, upregulation of glutamate transporters, decreases in reactive oxygen species, increases in neurotrophic factors, and anti-inflammatory effects. The neuroprotective and cognitive-enhancing effects of harmine should be further investigated in both preclinical and human studies.

Microwave-assisted extraction of three bioactive alkaloids from Peganum harmala L. and their acaricidal activity against Psoroptes cuniculi in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Peganum harmala L. is a perennial herbaceous, glabrous plant that grows in semi-arid conditions, steppe areas and sandy soils. It is used to treat fever, diarrhoea, subcutaneous tumours, arthralgia, rheumatism, cough, amnesia and parasitic diseases in folk medicines. In this paper, we aimed to develop a simpler and faster method for the extraction of three alkaloids from Peganum harmala L. than other conventional methods by optimizing the parameters of a microwave-assisted extraction (MAE) method, and to investigate the acaricidal activities of three compounds against Psoroptes cuniculi. MATERIALS AND METHODS: After optimizing the operating parameters with the single factor experiment and a Box-Behnken design combined with a response-surface methodology, a MAE method was developed for extracting the alkaloids from the seeds, and a high-performance liquid chromatography was used to quantify these compounds. An in vitro experiments were used to study the acaricidal activities. RESULTS: The optimal conditions of MAE method were as follows: liquid-to-solid ratio 31.3:1mL/g, ethanol concentration 75.5%, extraction time 10.1min, temperature 80.7°C, and microwave power 600W. Compared to the heat reflux extraction (HRE, 60min) and the ultrasonic-assisted extraction (UAE, 30min) methods, MAE method require the shortest time (10min) and obtain the highest yield of three compounds (61.9mg/g). Meanwhile, the LT50 values for the vasicine (1.25 and 2.5mg/mL), harmaline (1.25 and 2.5mg/mL), harmine (1.25 and 2.5mg/mL) and MAE extract (100mg/mL) against Psoroptes cuniculi were 12.188h, 9.791h, 11.994h, 10.095h, 11.293h, 9.273h and 17.322h, respectively. CONCLUSIONS: The MAE method developed exhibited the highest extraction yield within the shortest time and thus could be used to extract the active compounds from Peganum harmala L. on an industrial basis. As the active compounds of Peganum harmala L., vasicine, harmalin and harmine presented the marked acaricidal activities against Psoroptes cuniculi, and could be widely applied for the treatments of acariasis in animals.

Harmaline and hispidin from Peganum harmala and Inonotus hispidus with binding affinity to Candida rugosa lipase: In silico and in vitro studies.

The inhibitory effect of phenolic compounds and alkaloids of Inonotus hispidus and Peganum harmala on Candida rugosa lipase was investigated, also, their antioxidant activities using DPPH, ABTS and phosphomolybdenum were studied in this paper. The phenolic extracts have shown a stronger antiradical activity than the alkaloids extracts. The enzymatic inhibition produced by these extracts is described here for the first time. The results have shown that the phenolic and the alkaloid extracts are good inhibitors of C. rugosa lipase. Thus, the inhibitor molecules (harmaline and hispidin) have been isolated from P. harmala and I. hispidus. Their structures were elucidated by (1)H NMR analysis. Molecular docking has been achieved using AutoDock Vina program to discuss the nature of interactions and the mechanism of inhibition. Therefore, these isolated molecules could be used in the treatment of candidiasis.

Main alkaloids of Peganum harmala L. and their different effects on dicot and monocot crops.

Alkaloids with allelopathic activity are not as well-known as other allelochemicals. Our study revealed that total alkaloids from seeds of the medicinal plant Peganum harmala L. possessed significant growth inhibitory effect on four treated plants, with dicot plants (lettuce and amaranth) being more sensitive than the tested monocot plants (wheat and ryegrass). Further investigation led to the isolation of harmaline and harmine as the main active ingredients in the total alkaloids of P. harmala seeds. Harmaline exerted potent inhibitory effects on seedling growth of treated plants, especially dicots, inhibiting root elongation of lettuce and amaranth by 31% and 47% at a very low concentration (5 µg/mL), whereas harmine exhibited much weaker non-selective inhibitory effect on the plants. Considering the high yield and poor utilization of P. harmala in China, we anticipate that this plant could be exploited as an alternative weed management tool in the future.

Cerebroprotective effect of isolated harmine alkaloids extracts of seeds of Peganum harmala L. on sodium nitrite-induced hypoxia and ethanol-induced neurodegeneration in young mice.

The aim of the study was to isolate the harmine alkaloids from the seeds of Peganum harmala (TAPH) and its cerebroprotective effect on cognitive deficit mice. The tested doses of TAPH were screened for Sodium nitrite induced hypoxia and Ethanol induced neurodegeneration using behavioral models. The TAPH was found to be non-neurotoxic and Psychoactive by preventing the motor impairment and increasing the locomotion activity of animals in Rota rod and Actophotometer respectively. TAPH (5, 2.5 and 1.25 mg kg(-1) p.o.) significantly (p < 0.001) protected the Sodium nitrite induced memory impairment by decreasing the time require to find the water bottle in special water bottle case model. In Elevated Plus Maze (EPM) and Passive Shock Avoidance paradigm (PSA) the TAPH shown improved acquisition and retention memory significantly (p < 0.001) by decreasing the Transverse Latency Time (TLT) and increasing the Step Down Latency (SDL), respectively in dose dependent manner. The results were well supported by biochemical parameters, by inhibiting the Acetylcholinestrase (p < 0.01) activity, increasing the GSH (p < 0.001) level and decreasing the TBARS (p < 0.001) level of whole brain. Moreover TAPH has shown the significant Monoamine oxidase-A (MAO-A) inhibition action (p < 0.001), hence it reduces the metabolism of epinephrine, 5-HT and other monoamines and enhances the action of these neurotransmitters indirectly; this adrenergic system plays an important role in learning and memory. Further, TAPH (5 mg kg(-1)) protect the DNA fragmentation of frontotemporal cortex of the brain from hypoxic effect induced by Sodium nitrite in Gel Electrophoresis studies. The results were comparable to their respective standards. Hence, harmine alkaloids are potential enough to utilize in the management of Neurodegenerative disorders of the type Alzheimer's diseases.

Library-based discovery of DYRK1A/CLK1 inhibitors from natural product extracts.

The dual specificity tyrosine-phosphorylation-regulated kinase DYRK1A possesses diverse roles in neuronal development and adult brain physiology, and increased activity has been linked to neurodegenerative diseases. Very few inhibitors of this kinase have been reported up to now. Screening of a library of > 900 plant and fungal extracts afforded 25 extracts with IC50s < 10 µg/mL against DYRK1A. To identify the active constituents, the extracts were submitted to a process integrating physicochemical data with biological information, referred to as HPLC-based activity profiling. Follow-up investigation of four extracts led to the targeted isolation of harmine (1, IC50 0.022 µM) from Peganum harmala, emodin (3, IC50 4.2 µM) from Cassia nigricans, kaempferol (4, IC50 0.91 µM) from Cuscuta chinensis, and 3,8-di-O-methylherbacetin (11, IC50 8.6 µM), 3,3',4'-tri-O-methylmyricetin (12, IC50 7.1 µM) and ombuin (15, IC50 1.7 µM) from Larrea tridentata as the active constituents. Active extracts and compounds were also tested on the closely related cdc2-like kinase CLK1. Finally, the selectivity profile of compounds was evaluated by including other members of the DYRKs and CLKs families. While the flavonoids and emodin did not show significant differences in the potency of their activities, harmine (1) was most active against DYRK1A, CLK1, and CLK4, and less potent against the other kinases, with selectivity ranging from 2- to 20-fold.

Simiranes A and B: erythroxylanes diterpenes and other compounds from Simira eliezeriana (Rubiaceae).

The first phytochemical study of Simira eliezeriana Peixoto (Rubiaceae) allowed the isolation and structural determination of two new diterpenes named simirane A [(5R,6R,8R,9R,10S,11S,13S)-6ß,11ß-dihydroxy-2,4(18),15-erythroxylatrien-1-one] (1) and simirane B [(5S,8R,9R,10S,11S,13S)-11ß-hydroxy-2,4(18),15-erythroxylatrien-1-one] (2), together with seven known compounds: sitosterol (3), stigmasterol (4), campesterol (5), coniferaldehyde (6), vanillin (7), pinoresinol (8) and harman (9) from the bark of the plant. The structures of the compounds were established on the basis of spectroscopic methods, including 1-D and 2-D NMR, HRESI-MS and CD analysis and comparisons with available literature data of known compounds.

A new compound, jolynamine, from marine brown alga Jolyna laminarioides.

A new compound, jolynamine (1), was isolated from the marine brown alga Jolyna laminarioides collected from the coast of Karachi, Pakistan. In addition, four known compounds, namely saringosterol (2), loliolide (3), methyl-4-hydroxybenzoate (4) and propyl-4-hydroxybenzoate (5), were isolated for the first time from the marine brown alga Iyengaria stellata, and two known compounds, namely 3,4,5-trimethylaniline (6) and harmine (7), were isolated for the first time from the marine brown alga Melanothamnus afaqhusainii. Compound 6 is synthetically known but was isolated for the first time from a natural source. The structures of these compounds were elucidated with the help of powerful spectroscopic techniques. Furthermore, the methanolic extracts of both algae showed anti-microbial activities against various bacteria and fungi.

[Study on chemical constituents of Peganum multisectum].

OBJECTIVE: To study the chemical consituents of Peganum multisectum. METHODS: The compounds were isolated by chromatographic methods and their structures were elucidated by physicochemical properties and spectral analysis. RESULTS: Six alkaloids were isolated and identifiedas deoxyvasicinone (1), l-vasicinone (2), harmine (3), vasicine (4), evodiamin (5), and fagomine (6). CONCLUSION: Compounds 5 and 6 are isolated from this genus for the first time.

Antibacterial and antifungal activities of (beta)-carboline alkaloids of Peganum harmala (L) seeds and their combination effects.

The ß-carboline alkaloids of Peganum harmala L were extracted through a bioassay-guided fractionation and their antimicrobial activities were investigated. Results revealed significant differences (P>0.05) between compounds depending on the microorganism tested and the application method. When examined individually, harmine was the most effective against Proteus vulgaris, Bacillus subtilis and Candida albicans where inhibition zones ranged between 21.2 and 24.7 mm. Potentiality of the alkaloids was increased when applied as binary mixtures suggesting a kind of synergistic interaction with inhibition zones reaching 31.5mm with the total alkaloidal extract. We recommended the use of such compounds as new antimicrobial biorationals.

Separation and purification of harmine and harmaline from Peganum harmala using pH-zone-refining counter-current chromatography.

pH-zone-refining counter-current chromatography was successfully applied to the separation of alkaloids from a crude extract of Peganum harmala L. using a multilayer coil planet centrifuge. The experiment was performed with a two-phase solvent system composed of methyl tert-butyl ether/THF/water (2:2:3 by volume) where triethylamine (10 mM) was added to the upper organic stationary phase as a retainer and hydrochloric acid (5 mM) to the aqueous mobile phase as an eluter. From 1.2 g of the crude extract, 554 mg harmine and 325 mg harmaline were obtained each with a purity of over 96% as determined by HPLC. The structures of the isolated compounds were identified by electron ionization MS (EI-MS), (1)H NMR, and (13)C NMR.

Antifungal activity of the termite alkaloid norharmane against the mycelial growth of Metarhizium anisopliae and Aspergillus nomius.

Antifungal activity of norharmane, a beta-carboline alkaloid found in termites (Isoptera, Rhinotermitidae) was tested against two entomopathogenic fungi, Metarhizium anisopliae and Aspergillus nomius. It was determined that, at physiological concentration (10 microg ml(-1)), norharmane had no significant effect on A. nomius mycelial growth rate but reduced M. anisopliae growth rate by 11.9%. Contrary to previous findings, we suggest that norharmane has a limited role in disease resistance against fungal pathogens in individual subterranean termites, and we discuss the potential role of this chemical at a colony level.

Alkaloids from the seeds of Peganum harmala showing antiplasmodial and vasorelaxant activities.

Bioassay-guided purification from the seeds of Peganum harmala led to the isolation of harmine (1), harmaline (2), vasicinone (3), and deoxyvasicinone (4). Harmine (1) and harmaline (2) showed a moderate in vitro antiplasmodial activity against Plasmodium falciparum. Quinazoline alkaloid, vasicinone (3), showed a vasorelaxant activity against phenylephrine-induced contraction of isolated rat aorta.

Effect of norharmane in vitro on juvenile hormone epoxide hydrolase activity in the lower termite, Reticulitermes speratus.

The aromatic beta-carboline norharmane was determined in workers, nymphs, and ergatoids of Reticulitermes speratus (Kolbe) (Isoptera: Rhinotermitidae) by gas chromatography/mass spectrometry. Norharmane levels in workers, nymphs, and ergatoids collected in May (approximately 4.3 ng/termite) were higher than those in November (approximately 0.2 ng/termite). Fluorescence of norharmane was observed in histological sections in whole animals and in the fat body. Norharmane, at a final concentration of 1 mM, stimulated juvenile hormone epoxide hydrolase (JHEH) activity of enzyme extract from ergatoids, but inhibited JHEH activity at higher concentrations. The elevated JHEH activity stimulated by norharmane should accelerate juvenile hormone metabolism in R. speratus.

A study on the antitumoral and differentiation effects of peganum harmala derivatives in combination with ATRA on leukaemic cells.

Plant derived agents may exert a new approach to the treatment of leukaemia. The present study was an evaluation of proliferation, cytotoxicity and differentiation of harmine and harmaline on HL60 cells, alone or in combination with ATRA and G-CSF. Counting of cells, viability, MTT assay, morphology, NBT reduction and flow cytometry analysis were performed using CD11b and CD 14 monoclonal antibodies. The data showed that harmine and harmaline reduced proliferation in dose and time dependent manner. Optimal antiproliferative concentration of these agents was chosen. However, both agents in higher doses were cytotoxic. Combination of ATRA, G-CSF and each agent alone, particularly harmaline in optimal dose, resulted in partially additive decrease in cell proliferation. Cells treated with both harmaline and ATRA demonstrated some morphological changes and NBT positivity, but the extent of changes observed following treatment with harmaline was less than ATRA. Flow cytometric analysis showed that ATRA induced a neutrophilic differentiation, while harmaline led to a predominantly monocytic differentiation. Combination of harmine and harmaline with ATRA and G-CSF did not change the extent of differentiation, and the cells differentiated into the neutrophilic lineage. This shows that the direction of differentiation is dominantly determined by ATRA. These preliminary data implies a new approach in treatment of leukemia.

Efficacy of planar chromatography coupled to (tandem) mass spectrometry for employment in trace analysis.

HPTLC/MS by a plunger-based extraction device was shown to be an appropriate technique for quantitative planar chromatography, even in trace analysis. Reproducible extraction from silica gel phases in the lower-pg range distinguishes this technique from other approaches. Repeatability of the MS signal showed a mean RSD of 12.5% for the example of Harman, a heterocyclic aromatic amine. Analytical response within a plate and over various plates/days showed determination coefficients of 0.9915 and 0.9488, respectively. Limit of detection (LOD) by a single quadrupole was better than 40 pg and limit of quantitation (LOQ) by a tandem mass spectrometer better than 20 pg. LOQ/LOD obtained were of similar magnitude as reported for HPLC/MS methods, however, this order of sensitivity was shown for the first time in the field of HPTLC/electrospray ionization MS.