Components of Banisteriopsis caapi,a Plant Used in the Preparation of the Psychoactive Ayahuasca,Induce Anti-Inflammatory Effects in Microglial Cells

Banisteriopsis caapi is used to prepare the psychoactive beverage ayahuasca, and both have therapeutic potential for the treatment of many central nervous system (CNS) conditions. This study aimed to isolate new bioactive compounds from B. caapi extract and evaluate their biological activity, and that of the known β-carboline components of the plant (harmine, harmaline, and tetrahydroharmine), in BV-2 microglial cells, the in vivo activation of which is implicated in the physiopathology of CNS disorders. B. caapi extract was fractionated using semipreparative liquid chromatography (HPLC-DAD) and the exact masses ([M + H]⁺ m/z) of the compounds in the 5 isolated fractions were determined by high-resolution LC-MS/MS: F1 (174.0918 and 233.1289), F2 (353.1722), F3 (304.3001), F4 (188.1081), and F5 (205.0785). Harmine (75.5–302 µM) significantly decreased cell viability after 2 h of treatment and increased the number of necrotic cells and production of reactive oxygen species at equal or lower concentrations after 24 h. F4 did not impact viability but was also cytotoxic after 24 h. Most treatments reduced proinflammatory cytokine production (IL-2, IL-6, IL-17, and/or TNF), especially harmaline and F5 at 2.5 µM and higher concentrations, tetrahydroharmine (9.3 µM and higher), and F5 (10.7 µM and higher). The results suggest that the compounds found in B. caapi extract have anti-inflammatory potential that could be explored for the development of treatments for neurodegenerative diseases.     

Evaluation of the Cytotoxicity of Ayahuasca Beverages

Ayahuasca is a beverage consumed at shamanic ceremonies and currently has gained popularity on recreational scenarios. It contains beta-carboline alkaloids and N,N-dimethyltryptamine, which possesses hallucinogenic effects. Only a few studies have elicited the psychoactive effects and the dose of such compounds on neurological dopaminergic cells or animals. In this work, we aimed to study the cytotoxic effects of these compounds present in ayahuasca beverages and on five different teas (Banisteriopsis caapi, Psychotria viridis, Peganum harmala, Mimosa tenuiflora and Dc Ab (commercial name)) preparations on dopaminergic immortalized cell lines. Moreover, a characterization of the derivative alkaloids was also performed. All the extracts were characterized by chromatographic systems and the effect of those compounds in cell viability and total protein levels were analyzed in N27 dopaminergic neurons cell line. This is the first article where cytotoxicity of ayahuasca tea is studied on neurological dopaminergic cells. Overall, results showed that both cell viability and protein contents decreased when cells were exposed to the individual compounds, as well as to the teas and to the two mixtures based on the traditional ayahuasca beverages.     

Methodology for and the determination of the major constituents and metabolites of the Amazonian botanical medicine ayahuasca in human urine

Ayahuasca, also known as caapi or yage among various South American groups, holds a highly esteemed and millennia-old position in these cultures' medical and religious pharmacopeia. There is now an increasing interest in the potential for modern medical applications of ayahuasca, as well as concerns regarding its increasing potential for abuse. Toxicological and clinical research to address these issues will require information regarding its metabolism and clearance. Thus, a rapid, sensitive and specific method for characterization and quantitation of the major constituents and of the metabolites of ayahuasca in urine is needed. The present research provides a protocol for conducting such analyses. The characteristics of the method, conducted by sample dilution and using HPLC-electrospray ionization (ESI)-selected reaction monitoring (SRM)-tandem mass spectrometry, are presented. The application of the analytical protocol to urine samples collected from three individuals that were administered ayahuasca has also been demonstrated. The data show that the major metabolite of the hallucinogenic component of ayahuasca, N,N-dimethyltryptamine (DMT), is the corresponding N-oxide, the first time this metabolite has been described in in vivo studies in humans. Further, very little DMT was detected in urine, despite the inhibition of monoamine oxidase afforded by the presence of the harmala alkaloids in ayahuasca. The major harmala alkaloid excreted was tetrahydroharmine. Other excretion products and metabolites were also identified and quantified. The method described would be suitable for use in further toxicological and clinical research on ayahuasca.     

Simultaneous Production of Psilocybin and a Cocktail of β-Carboline Monoamine Oxidase Inhibitors in “Magic” Mushrooms

The psychotropic effects of Psilocybe “magic” mushrooms are caused by the l -tryptophan-derived alkaloid psilocybin. Despite their significance, the secondary metabolome of these fungi is poorly understood in general. Our analysis of four Psilocybe species identified harmane, harmine, and a range of other l -tryptophan-derived β-carbolines as their natural products, which was confirmed by 1D and 2D NMR spectroscopy. Stable-isotope labeling with ¹³C₁₁-l -tryptophan verified the β-carbolines as biosynthetic products of these fungi. In addition, MALDI-MS imaging showed that β-carbolines accumulate toward the hyphal apices. As potent inhibitors of monoamine oxidases, β-carbolines are neuroactive compounds and interfere with psilocybin degradation. Therefore, our findings represent an unprecedented scenario of natural product pathways that diverge from the same building block and produce dissimilar compounds, yet contribute directly or indirectly to the same pharmacological effects.     

In Vitro Study of the Bioavailability and Bioaccessibility of the Main Compounds Present in Ayahuasca Beverages

Ayahuasca is a psychoactive beverage that contains the psychoactive compound N,N-dimethyltryptamine and β-carboline alkaloids. This study aims at determining in vitro the bioavailability and bioaccessibility of the main compounds present in decoctions of four individual plants, in a commercial mixture and in four mixtures of two individual plants used in the preparation of Ayahuasca. The samples were subjected to an in vitro digestion process, and the Caco-2 cell line was used as an absorption model. The integrity and permeability of the cell monolayer were evaluated, as well as the cytotoxicity of the extracts. After digestion and cell incubation, the compounds absorbed by the cell monolayer were quantified by high-performance liquid chromatography coupled to a diode array detector. The results showed that compounds such as N,N-dimethyltryptamine, Harmine, Harmaline, Harmol, Harmalol and Tetrahydroharmine were released from the matrix during the in vitro digestion process, becoming bioaccessible. Similarly, some of these compounds, after being incubated with the cell monolayer, were absorbed, becoming bioavailable. The extracts did not show cytotoxicity after cell incubation, and the integrity and permeability of the cell monolayer were not compromised.     

Methodology for determining major constituents of ayahuasca and their metabolites in blood

There is an increasing interest in potential medical applications of ayahuasca, a South American psychotropic plant tea with a long cultural history of indigenous medical and religious use. Clinical research into ayahuasca will require specific, sensitive and comprehensive methods for the characterization and quantitation of these compounds and their metabolites in blood. A combination of two analytical techniques (high-performance liquid chromatography with ultraviolet and/or fluorescence detection and gas chromatography with nitrogen-phosphorus detection) has been used for the analysis of some of the constituents of ayahuasca in blood following its oral consumption. We report here a single methodology for the direct analysis of 14 of the major alkaloid components of ayahuasca, including several known and potential metabolites of N,N-dimethyltryptamine and the harmala alkaloids in blood. The method uses 96-well plate/protein precipitation/filtration for plasma samples, and analysis by HPLC-ion trap-ion trap-mass spectrometry using heated electrospray ionization to reduce matrix effects. The method expands the list of compounds capable of being monitored in blood following ayahuasca administration while providing a simplified approach to their analysis. The method has adequate sensitivity, specificity and reproducibility to make it useful for clinical research with ayahuasca.