Monoamine oxidase inhibitors in South American hallucinogenic plants: tryptamine and beta-carboline constituents of ayahuasca

This study (1984) investigated the alkaloid composition of field samples, consisting of the bark and leaf specimens used for brewing ayahuasca, which were also compared directly with other ayahuasca mixtures. Their compositions were generally similar, with little variation in the constituents of brews made by different ayahuasqueros.

Abstract

“Introduction: Ayahuasca is a hallucinogenic beverage derived by boiling the bark of the Malpighiaceous liana Banisteriopsis caapi together with the leaves of various admixture plants, viz. Psychotria viridis, Psychotria carthagenensis , or Diplopterys cabrerana . B. caapi contains harmine, harmaline, and tetrahydroharmine while the admixtures contain N,N-dimethyltryptamine (DMT). DMT, a potent hallucinogen, is inactive orally due to degradation by visceral monoamine oxidase (MAO). The beta-carbolines, however, are highly active reversible inhibitors of MAO and may protect the DMT from deamination by MAO and render it orally active. This mechanism has been proposed to underlie the oral activity of ayahuasca but has not been experimentally confirmed.

Methods: In the present study the constituents of the admixture plants and the alkaloids of eight ayahuasca samples from Peru were qualitatively and quantitatively analyzed using two-dimensional thin-layer chromatography (TLC), high pressure liquid chromatography (HPLC) and gas chromatography/mass spectrometry (GC/MS). Several B. caapi cultivars were quantitatively compared for variations in alkaloid content. Three admixture plants used rarely in the manufacture of ayahuasca were also screened for alkaloids. A selected sample of beta-carbolines were screened for activity as MAO inhibitors using an in vitro assay system, and structure/activity relationships were compared. Inhibition observed with single compounds was compared with the activity of selected samples of ayahuasca which were screened in the system and also with the activity of mixtures of beta-carbolines.

Results: The levels of DMT and beta-carbolines found in the ayahuasca samples examined in the present study were an order of magnitude greater than the levels reported in a previous study. Ayahuasca was found to be an extremely effective inhibitor of MAO in vitro and the degree of inhibition was directly correlated with the concentration of MAO-inhibiting beta-carbolines. Inhibition experiments using mixtures of beta-carbolines indicated that their effects in combination are additive, rather than synergistic or antagonistic.

Discussion: Implications of the results in understanding the pharmacology of ayahuasca are discussed.“

Authors: Dennis J. McKenna, G.H.N. Towers & F. Abbott

Summary

Ayahuasca is a hallucinogenic beverage derived from boiling the bark of Bunisteriopsis caapi together with the leaves of various admixture plants. The admixture plants contain NJ-dimethyltryptamine (DMT), which is inactive orally due to degradation by visceral monoamine oxidase (MAO).

Several B. caapi cultivars were quantitatively compared for variations in alkaloid content, and p-carbolines were screened for activity as MAO inhibitors. Ayahuasca was found to be an extremely effective inhibitor of MAO in vitro, and its effects were additive, rather than synergistic or antagonistic.

Introduction

The hallucinogenic beverage ayahuuscu is widely used for medicinal, ritual and recreational purposes by the aboriginal and mestizo populations inhabiting the Amazon Basin. It is prepared by boiling the bark of the Malpighiaceous jungle liana B. cuupi together with the leaves of various admixture plants.

Rivier and Lindgren (1972) found that the p-carboline alkaloids harmine, harmaline, and tetrahydroharmine (THH), and DMT were the major active constituents of uyuhuuscu, and that the concentrations of these compounds in the plant make it unlikely that they contribute significantly to the pharmacological activity of uyuhuuscu.

The active alkaloids of uyuhuuscu are now known, but the pharmacology of the drug remains to be clarified. In particular, the role of intestinal and hepatic MAO in deamination of indole is still unclear.

Ayahuasca samples

Ayahuasca samples were identified by the name of the ayahuusquero from whom they were obtained, and also by a number. Some samples were diluted with methanol, but no obvious difference could be detected.

Two-dimensional TLC was carried out using Polygram Silica Gel G UV254 precoated plates (Began Instrument). Five microlitre aliquots of the material to be analyzed were applied to the origin using a Microcap applicator. Solvent 1 was prepared in a separatory funnel, the upper phase was collected for TLC, and the plates were developed in Solvent 1 and Solvent 2 at ambient temperature for 2-3 days. The plates were examined under short- and long-wave UV light.

Stock solutions of harmine, harmaline, tetrahydroharmine and DMT were made up to a precise concentration of 1 mg/ml. A SP4100 integrator was calibrated by making single 20-111 injections of the calibration mixture at each concentration level.

Ayahuasca samples were diluted with methanol and injected into a HPLC. Two replicates of each ayahuasca sample were prepared, and each replicate sample was injected twice during separate runs following calibration of the integrator using the standard mixtures.

Ayahuasca samples that had been diluted with methanol in the field were quantified for alkaloids in terms of mg/g dry wt of the lyophilized sample. Two replicates of each sample were prepared, and each replicate was injected twice onto the HPLC.

B. caapi stems were dried, ground to powder, and extracted in methanol for 24 h. The extracts were concentrated, filtered, and the volume adjusted to 25 ml with methanol.

Alkaloid precipitation tests and TLC of uncommon admixture plants

The uncommon admixture plants were screened for alkaloids using the method of Farnsworth and Euler (1962). The alkaloids were detected using Meyer’s, Valser’s, or Dragendorff’s reagents, and the samples were dried, examined under UV light, and sprayed with Dragendorff’s modified reagent.

Preparation of rat-liver cy tosol

Rat liver was derived from mature female Wistar rats, perfused with cold 0.1 M sodium phosphate buffer (pH 7.2), removed from the abdominal cavity, and homogenized in cold buffer to a smooth paste. The diluted cytosol fraction was sonicated for 10 s and kept on ice for the experiment.

The assay was conducted at 37.5°C in 13 X 100 mm disposable test tubes and the percent inhibition of tubes containing varied concentrations of inhibitor was calculated relative to these controls.

Ayahuasca samples were diluted with chilled methanol, the white non-alkaloidal precipitate removed by filtration, and the reconstituted solution was quantified using the HPLC method described above. The inhibitor assays were conducted using harmine, harmaline and tetrahydroharmine.

Results and discussion

The alkaloids in uyahuuscu, B. cuupi and admixture plants were analyzed using two-dimensional TLC and HPLC, and in vitro MAO inhibition was studied using se&ted p-carboline standards, mixtures of P-carboline standards, and ayuhuascu preparations.

Ayuhuuscu brews

Eight samples of uyuhuascu preparations were obtained from uyuhuusqueros mg alkaloid, of which 30 mg is harmine, 10 mg is THH and 25 mg DMT. This dosage is well within the range at which the /3-carbolines are effective as MAO inhibitors.

The alkaloid levels found in the five samples from Pucallpa exceed the levels reported by Rivier and Lindgren (1972) in samples collected on the upper Rio PurGs by at least an order of magnitude.

Alkaloid content of B. caapi cultivars

All of the ayahuasqueros that we interviewed recognized several different kinds of ayahuasca, which were distinguished by the types of admixture plants which were added, and the type of Bani, steriopsis vine which was utilized. There were no outstanding morphological differences between the three or four kinds of B. caapi which we collected.

There is no consistent correlation between alkaloid content and recognized cultivars of B. caupi, probably because of the age of the plant and the soil, light, water, and other environmental conditions affecting the growth of the particular specimen.

DMT-con taining admixtures

In Peru, Psychotria viridis R. JZ P. is the admixture plant most frequently used in the preparation of ayahuasca. DMT is the single major base in all Psychotria viridis collections. The Psychotria viridis samples analyzed contained substantial amounts of DMT, between 1 and 1.6 mg/g dry wt in the leaves. No other alkaloids were detected in any of the Psychotria samples with the exception of DMCK no. 139, which contained a trace constituent with a mass spectrum corresponding to that reported for 2-methyl-tetrahydro-/3-carboline.

Psychotria species, including Psychotria uiridis, have tiny spine-like extensions of the mid-rib on the abaxial surface of the leaf. These structures may be inhabited by mites, and are the key feature used to identify the “proper” Psychotria species for use as admixtures.

All the Psychotria uiridis collections which we analyzed possessed espinas (spines), and all contained DMT; the single specimen which lacked these structures also contained no tryptamines or other alkaloids.

Alkaloids in uncommon admixture plants

Ayahuasca is usually prepared using Diplopterys cabrerana or Psychotria sp., but other admixtures have been identified, although nothing is known of their chemical or biodynamic properties. During fieldwork in Peru, three collections of plants were made. The material from one of the collections gave an unambiguously positive test for palmatine, a typical quatemary base of the benzylisoquinoline family which characterizes the Menispermaceae. Although palmatine is one of the commonest alkaloids in nature, investigations of its pharmacology are surprisingly sparse. It has been found to inhibit the effects of epinephrine, 5HT and ACTH, and to have anticholinesterase activity.

The enzyme MAO is widely distributed throughout various tissues in vertebrates and invertebrates and functions as a detoxification mechanism to protect the nervous and cardiovascular systems from toxic biogenic amines. MAO consists of two species, MAO-A and MAO-B, which are activated by substituted p-phenylethylamines, /3-phenylethanolamine derivatives having a free p-hydroxyl group, and 5HT. The specific metabolism of DMT has not been investigated, but it is likely that it is a substrate for both MAO-A and MAO-B.

Most MAO inhibitors belong to one of four classes: hydrazine derivatives, phenylcyclopropylarnine derivatives, N-benzyl-N-methyl propargylamine, or 2methyl-3-piperidinopyrazine. The major exception is the /I-carboline derivatives, which are widespread in nature.

The MAO inhibitory activity of several compounds was assessed in vitro using rat liver cytosol fraction and 14C-labelled Shydroxytryptamine creatinine sulphate as substrate. The ISo values correspond to the molar concentration of inhibitor at which enzyme activity is 50% inhibited with respect to controls lacking inhibitor.

The conclusions suggested by the present study regarding the structure/activity relationships of p-carbolines as MAO inhibitors are in agreement with the previous studies. For example, the fully aromatic and dihydro-fl-carbolines are significantly more potent inhibitors than analogues in which the piperidine ring is fully saturated. Buckholtz and Boggan (1977) found that harman, lacking substituents on the aromatic ring, was less active than the 7-methoxylated analogue but approximately equipotent to the 6-methoxylated fully aromatic compounds. 2-Me-6-methoxy-tetrahydro-carboline (2Me-GMeO-THBC), a compound reported from Virola sp., exhibited the greatest inhibitory activity of any of the 6-methoxylated p-carbolines tested, and was approximately an order of magnitude more active than THH.

MAO inhibition by ayahuasca samples and by combinations of /3-carbolines were compared. Ayahuasca showed nearly identical inhibitory activity to harmine, harmaline and THH, indicating that harmine and THH alone can account for most of the MAO inhibition exhibited by ayahuasca.

Dr. C.-K. Wat carried out the GC/MS analysis of the admixtures and Dr. G. Bellward developed the MAO methodology. Financial support from the Natural Sciences and Engineering Research Council of Canada is gratefully acknowledged.