Targeting inhibition of microtubule affinity regulating kinase 4 by Harmaline: Strategy to combat Alzheimer's disease.

Microtubule-affinity regulating kinase 4 (MARK4) is linked with the development of cancer, diabetes and neurodegenerative diseases. Due to its direct role in the hyperphosphorylation of tau protein, MARK4 is considered as an attractive target to fight Alzheimer's disease (AD) and neuroinflammation. In the present study, we have selected Harmaline (HAR), an alkaloid of Paganum harmala, to investigate its MARK4 inhibitory potential and its binding mechanism. Molecular docking and fluorescence binding studies were carried out to estimate the binding affinity of the HAR with the MARK4. We observed an excellent binding affinity of HAR to the MARK4 (K = 107 M-1), further complemented by isothermal titration calorimetric measurements. In addition, HAR significantly inhibits the kinase activity of MARK4 (IC50 value of 4.46 µM). Structural investigations suggested that HAR binds to the active site pocket and forms several non-covalent interactions with biologically important residues of MARK4. All-atom molecular dynamics simulation studies further advocated that the MARK4-HAR complex is stabilized throughout the trajectory of 200 ns and causes a little conformational change. All these findings suggest that HAR is a potential MARK4 inhibitor that can be implicated in managing MARK4-associated diseases, including AD.

Ultrasound-assisted solid phase microextraction-HPLC method based on Fe3O4@SiO2-NH2-molecularly imprinted polymer magnetic nano-sorbent for rapid and efficient extraction of harmaline from Peganum harmala extract.

In the present study, a magnetic molecularly imprinted polymer (MMIP) was synthesized for the extraction of harmaline from Peganum harmala by dispersive solid-phase microextraction (DSPME). The MMIP for selective and intelligent extraction of harmaline with excellent functionality and high selectivity was synthesized using the sol-gel method with functionalized superparamagnetic core-shell nanoparticles, ethylene glycol dimethacrylate (EDMA) as a cross-linker, methacrylic acid (MAA) as a functional monomer, and 2,2-azobisisobutyronitrile (AIBN) as a porogen. To study the properties and morphology of the coated polymer, FT-IR spectroscopy, FESEM, TEM images, and VSM were used. The DSPME-HPLC-UV equipment was used to quantify and analyze the data obtained from harmaline extraction. In this research, the efficiency of the synthesized polymer in harmaline extraction was modeled and optimized using the response surface methodology based on central composite design (RSM-CCD). In addition, for modeling the isotherm of harmaline sorption by the MMIP, Langmuir and Freundlich isotherm equations were used. The obtained results showed that the extraction of harmaline with the MMIP was well described with Freundlich isotherm. The results of the validation of the method showed that the measurement of harmaline in the concentration range of 1.0-4000 ng mL-1 followed a linear relationship (R2 = 9986.0). Moreover, the accuracy or repeatability index (% RSD) was determined to be < 10, and the LOQ and LOD values were 0.526 and 0.158 ng mL-1, respectively. The results of this study showed that the DSPME technique by using the synthesized MMIP as an effective sorbent with high efficiency and capacity could be utilized for pre-concentration and extraction of harmaline from real and complex samples.

A rapid analytical strategy for the determination of ayahuasca alkaloids in non-ritualistic approaches by UHPLC-MS/MS.

INTRODUCTION: Ayahuasca is a beverage composed by a mixture of herbs which contain the compound N,N-dimethyltriptamine (DMT) and the ß-carbolines. Although its use is legalized in Brazil only for religious and spiritual ceremonies, there is a growing black market specialized in the distribution of these compounds in form of herbal material through internet and mail. The purpose of this work was the development of an ultra-high-performance liquid chromatography-tandem mass spectrometry method for the determination of ayahuasca alkaloids and its application in seized ayahuasca products. METHODS: An aliquot of seized products was weighted and diluted with methanol. An aliquot of this solution was added with internal standard (DMT-d6), followed by injection in the analytical system. RESULTS: The limit of quantitation was 10ng/mL for DMT and 25ng/mL for harmine, harmaline and tetrahydroharmine. The concentration ranges used were 10-100ng/mL for DMT, harmine and harmaline and all analytes presented a coefficient of determination (r2)≥0,99. Analysis of four seized samples presented concentrations of DMT ranging between 31.5 and 46.5mg/g. Presence of ß-carbolines was not detected in the products. The variability of DMT concentrations can be correlated with the potential intoxications described in the literature. CONCLUSION: This work successfully established a determination method for ayahuasca alkaloids in herbal material. In addition, the workflow proved to be simple, rapid and useful to estimate the concentration of psychoactive compounds in seized materials, leading to further investigation of ayahuasca ritualistic or recreational exposure.

Validation of an analytical method for the determination of the main ayahuasca active compounds and application to real ayahuasca samples from Brazil.

Ayahuasca is a brew prepared from the water decoction of two Amazonian plants, which is legally used for religious, cultural or therapeutic activities. The potential use of ayahuasca as a natural or phytotherapeutic drug is directly linked to the action of its active compounds and their connection with the therapeutic efficacy of the beverage. In this context, the aim of the present study was to establish a selective, sensitive and reproducible analytical method for the quantification of the main active ayahuasca compounds. Thirty-eight samples from the state of São Paulo, Brazil, were analyzed and the simultaneous quantifications of N,N-dimethyltryptamine (DMT), tetrahydroharmine (THH), harmine (HME) and harmaline (HML) were performed. This study enabled the development of a fast validated analytical method with minimal matrix interference and high reproducibility for the tracing of active ayahuasca compound concentrations for the first time. This method is important as an auxiliary tool for the study of active compound effects in biological responses using different multi-omic platforms.

A rapid and simple method for the determination of psychoactive alkaloids by CE-UV: application to Peganum Harmala seed infusions.

The ß-carboline alkaloids of the harmala (HAlks) group are compounds widely spread in many natural sources, but found at relatively high levels in some specific plants like Peganum harmala (Syrian rue) or Banisteriopsis caapi. HAlks are a reversible Mono Amino Oxidase type A Inhibitor (MAOI) and, as a consequence, these plants or their extracts can be used to produce psychotropic effects when are combined with psychotropic drugs based on amino groups. Since the occurrence and the levels of the HAlks in natural sources are subject to significant variability, more widespread use is not clinical but recreational or ritual, for example B. caapi is a known part of the Ayahuasca ritual mixture. The lack of simple methods to control the variable levels of these compounds in natural sources restricts the possibilities to dose in strict quantities and, as a consequence, limits its use with pharmacological or clinical purposes. In this work, we present a fast, simple, and robust method of quantifying simultaneously the six HAlks more frequently found in plants, i.e., harmine, harmaline, harmol, harmalol, harmane, and norharmane, by capillary electrophoresis instruments equipped with the more common detector UV. The method is applied to analyze these HAlks in P. Harmala seeds infusion which is a frequent intake form for these HAlks. The method is validated in three different instruments in order to evaluate the transferability and to compare the performances between them. In this case, harmaline, harmine, and harmol were found in the infusion samples. Copyright © 2016 John Wiley & Sons, Ltd.

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

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.

Direct infusion ESI-IT-MSn alkaloid profile and isolation of tetrahydroharman and other alkaloids from Bocageopsis pleiosperma maas (Annonaceae).

INTRODUCTION: The Annonaceae family is known as a promising abundant source of secondary metabolites, especially annonaceous acetogenins, terpenoids and isoquinoline-derived alkaloids. Although widely investigated from the phytochemical viewpoint, this family still presents some largely unexplored genera, e.g. the Bocageopsis. OBJECTIVE: To investigate the alkaloid content of Bocageopsis pleiosperma Maas using direct infusion electrospray ionisation ion trap tandem mass spectrometry (ESI-IT-MS(n)) analysis. METHODOLOGY: Dichloromethane extracts of aerial parts were subjected to acid-base partitioning to yield the alkaloidal fractions. These fractions were analysed by direct infusion into a (+)ESI-IT-MS(n) system. The alkaloidal fraction from the leaves was also obtained on a large scale and subjected to chromatographic separation. RESULTS: The tentative MS(n) -based identification of alkaloids in leaves, twigs and trunk bark showed that aporphine alkaloids were restricted to the leaves and twigs, tetrahydroprotoberberine alkaloids were only found in the twigs and trunk bark while benzylisoquinoline alkaloids were found in the leaves, twigs and trunk bark. Chromatographic separation of the leaf alkaloidal fraction yielded the aporphine alkaloids nornuciferine, asimilobine and isoboldine, the ß-carboline alkaloid tetrahydroharman and some mixtures containing benzylisoquinoline and aporphine alkaloids, all described for the first time in the Bocageopsis genus. Furthermore, tetrahydroharman has not previously been reported in the Magnoliales order. CONCLUSION: Direct infusion ESI-IT-MS(n) analysis of alkaloids allowed fast recognition of alkaloidal classes previously reported in the Annonaceae family, aiding the chromatographic step and allowing a selective isolation of compounds previously not identified in the Bocageopsis genus.

Fast determination of harmala alkaloids in edible algae by capillary electrophoresis mass spectrometry.

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 ß-carboline 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 standard-addition 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.

Composition, standardization and chemical profiling of Banisteriopsis caapi, a plant for the treatment of neurodegenerative disorders relevant to Parkinson's disease.

ETHNOPHARMACOLOGICAL RELEVANCE: Banisteriopsis caapi, a woody vine from the Amazonian basin, is popularly known as an ingredient of a sacred drink ayahuasca, widely used throughout the Amazon as a medicinal tea for healing and spiritual exploration. The usefulness of Banisteriopsis caapi has been established for alleviating symptoms of neurological disorders including Parkinson's disease. AIM OF THE STUDY: Primary objective of this study was to develop the process for preparing standardized extracts of Banisteriopsis caapi to achieve high potency for inhibition of human monoamine oxidases (MAO) and antioxidant properties. The aqueous extracts prepared from different parts of the plant collected from different geographical locations and seasons were analyzed by HPLC for principal bioactive markers. The extracts were simultaneously tested in vitro for inhibition of human MAOs and antioxidant activity for analysis of correlation between phytochemical composition of the extracts and bioactivities. MATERIALS AND METHODS: Reversed-phase HPLC with photodiode array detection was employed to profile the alkaloidal and non-alkaloidal components of the aqueous extract of Banisteriopsis caapi. The Banisteriopsis caapi extracts and standardized compositions were tested in vitro for inhibition of recombinant preparations of human MAO-A and MAO-B. In vitro cell-based assays were employed for evaluation of antioxidant property and mammalian cell cytotoxicity of these preparations. RESULTS: Among the different aerial parts, leaves, stems/large branches and stem bark of Banisteriopsis caapi, HPLC analysis revealed that most of the dominant chemical and bioactive markers (1, 2, 5, 7-9) were present in high concentrations in dried bark of large branch. A library of HPLC chromatograms has also been generated as a tool for fingerprinting and authentication of the studied Banisteriopsis caapi species. The correlation between potency of MAO inhibition and antioxidant activity with the content of the main active constituents of the aqueous Banisteriopsis caapi extracts and standardized compositions was established. Phytochemical analysis of regular/commercial Banisteriopsis caapi dried stems, obtained from different sources, showed a similar qualitative HPLC profile, but relatively low content of dominant markers 1, 2, 7, and 9, which led to decreased MAO inhibitory and antioxidant potency compared to Banisteriopsis caapi Da Vine. CONCLUSION: The ethnopharmacological use of bark of matured stem/large branch of Banisteriopsis caapi as well as whole matured stem is supported by the results obtained in this investigation. Among various constituents of Banisteriopsis caapi, harmine (7), harmaline (6) and tetrahydroharmine (5) are responsible for MAO-A inhibition, while two major proanthocyanidines, epicatechin (8) and procyanidine B2 (9) produce antioxidant effects. The compounds 1-9 can serve as reliable markers for identification and standardization of Banisteriopsis caapi aerial parts, collected in different seasons and/or from different geographical regions.

High-performance thin-layer chromatography densitometric method for the quantification of harmine, harmaline, vasicine, and vasicinone in Peganum harmala.

Peganum harmala Linn. seed is a reputed drug of the Indian systems of medicine. We report a simple high-performance thin-layer chromatography (HPTLC) densitometric method for the quantification of 4 alkaloids, viz., harmine, harmaline, vasicine, and vasicinone from P. harmala seed. The 4 compounds were resolved in ethyl acetate-methanol-ammonia (7 + 1 + 0.3, v/v/v) mobile phase. The method was validated for precision, repeatability, and accuracy. Instrumental precision was 0.27, 1.53, 0.39, and 1.15% [relative standard deviation (RSD)] and repeatability of the method was 1.01, 0.79, 0.13, and 1.62% RSD for harmine, harmaline, vasicine, and vasicinone, respectively. Accuracy of the method was checked by a recovery study conducted at 3 different levels, and the average recovery was 97.88% for harmine, 97.69% for harmaline, 98.38% for vasicine, and 98.28% for vasicinone. The 4 compounds were quantified in P. harmala seed using the method, and it was found to contain 0.44, 0.096, 0.25, and 0.0007% (w/w) of each, respectively. The proposed HPTLC densitometric method for the quantification of the 4 compounds was found to be simple, precise, specific, sensitive, and accurate. It can be used for routine quality control of P. harmala seed and has the ability to quantify the 4 marker compounds in the formulations containing P. harmala. It also can be used to quantify any of these marker compounds in other herbal drugs.

Analysis of hallucinogenic constituents in Amanita mushrooms circulated in Japan.

The constituents of seven mushrooms sold as Amanita muscaria or Amanita pantherina (five A. muscaria and two A. pantherina) and four "extracts purported to contain A. muscaria" products that are currently circulated in Japan were determined. All mushroom samples were identified as A. muscaria or A. pantherina by macroscopic and microscopic observation. The dissociative constituents, ibotenic acid (IBO) and muscimol (MUS), were extracted with 70% methanol twice and determined by gas chromatography/mass spectrometry. The IBO (as the hydrate)/MUS contents were in the range of <10-2845ppm/46-1052ppm in the cap of A. muscaria and 188-269ppm/1554-1880ppm in the cap of A. pantherina. In the caps, these compounds had a tendency to be more concentrated in the flesh than in the cuticle. On the other hand, the IBO/MUS contents in the stem were far lower than in the caps. In the "extracts purported to contain A. muscaria" products, IBO/MUS were detected below the lower limit of calibration curve (<10ppm/<25ppm) or not detected. However, these samples contained other psychoactive compounds, such as psychoactive tryptamines (5-methoxy-N,N-diisopropyltryptamine and 5-methoxy-N,N-dimethyltryptamine), reversible monoamine oxidase inhibitors (harmine and harmaline) and tropane alkaloids (atropine and scopolamine), which were not quantified. This is the first report of the chemical analysis of Amanita mushrooms that are circulated in the drug market.

Various alkaloid profiles in decoctions of Banisteriopsis caapi.

Twenty nine decoctions of Banisteriopsis caapi from four different sources and one specimen of B. caapi paste were analyzed for N,N-dimethyltryptamine (DMT), tetrahydroharmine (THH), harmaline and harmine. Other plants were also used in the preparation of these products, typically Psychotria viridis, which provides DMT. There were considerable variations in alkaloid profiles, both within and between sample sources. DMT was not detected in all samples. Additional THH may be formed from both harmine and harmaline during the preparation of these products. The alkaloid composition of one decoction sample did not change significantly after standing at room temperature for 80 days, but the initial acidic pH was neutralized by natural fermentation after 50 days.

HPLC method for the analysis of harmol, harmalol, harmine and harmaline in the seeds of Peganum harmala L.

A simple and sensitive method for separation and determination of harmol, harmalol, harmine and harmaline has been developed and validated. Harmol, harmalol, harmine and harmaline were separated using a Metasil ODS column by isocratic elution with flow rate 1.5 ml/min. The mobile phase composition was Isopropyl alcohol-Acetonitrile-Water-Formic acid (100:100:300:0.3) (v/v/v/v) and pH adjusted 8.6 with triethylamine. Spectrophotometric detection was carried out at 330 nm. The linear range of detection for harmol, harmalol, harmine and harmaline were between 9.375-250, 30.750-246, 31.250-500 and 31.000-248 microg/ml, respectively. The method described was suitable for the determination of harmol, harmalol, harmine and harmaline in the seeds of Peganum harmala L.

Exudation of fluorescent beta-carbolines from Oxalis tuberosa L roots.

Root fluorescence is a phenomenon in which roots of seedlings fluoresce when irradiated with ultraviolet (UV) light. Soybean (Glycine max) and rye grass (Elymus glaucus) are the only plant species that have been reported to exhibit this occurrence in germinating seedling roots. The trait has been useful as a marker in genetic, tissue culture and diversity studies, and has facilitated selection of plants for breeding purposes. However, the biological significance of this occurrence in plants and other organisms is unknown. Here we report that the Andean tuber crop species Oxalis tuberosa, known as oca in the highlands of South America, secretes a fluorescent compound as part of its root exudates. The main fluorescent compounds were characterized as harmine (7-methoxy-1-methyl-beta-carboline) and harmaline (3, 4-dihydroharmine). We also detected endogenous root fluorescence in other plant species, including Arabidopsis thaliana and Phytolacca americana, a possible indication that this phenomenon is widespread within the plant kingdom.

Formation of tetrahydroharman (1-methyl-1,2,3,4-tetrahydro-beta-carboline) by Helicobacter pylori in the presence of ethanol and tryptamine.

Helicobacter pylori contains alcohol dehydrogenase which oxidizes ethanol to acetaldehyde. In the present study, H. pylori cytosol was incubated in a buffered media at pH 6.0 and 7.4 in the presence of ethanol and tryptamine. Under these conditions, tetrahydroharman (1-methyl-tetrahydro-beta-carboline) was produced as a condensation product of tryptamine and acetaldehyde. At pH 6.0, 20.60 +/- 5.00% of the added tryptamine was converted to tetrahydroharman, while 27.00 +/- 4.80% (mean +/-SD) was converted at pH 7.4. Similar reactions between acetaldehyde and other dietary amines seem likely. Such biogenic alkaloids, if formed in vivo, might contribute to the dysphoric effects of alcohol.

Fluorescence of harmol, harmalol and 2-hydroxycarbazole in concentrated hydroxide solutions.

Room temperature electronic absorption and fluorescence spectra of harmol, harmalol and 2-hydroxycarbazole have been obtained in concentrated aqueous potassium hydroxide solutions. The appearance of a new fluorescence band for all these compounds in media of H- greater than 16, has been ascribed to the emission of excited dianions formed by deprotonation. Acidity constants have been estimated from the Föster-Weller method.

Synthesis and characterization of novel biologically active platinum(II) and palladium(II) complexes of some beta-carboline alkaloids.

The preparation of novel biologically active platinum(II) and palladium(II) complexes of some beta-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 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.