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.
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.
Papers in Blossom that reference this study
Bouso, J. C., Andión, Ó., Cantillo, J. et al. · Psychedelics (2026)
Calnan, M., Blest-Hopley, G., Busch, C. et al. · Brain and Behavior (2025)
Suay, D., Aicher, H. D., Kometer, M. et al. · NeuroImage (2025)
Egger, K., Redondo, J. J., Müller, J. et al. · Biomedicine & Pharmacotherapy (2025)
Ayahuasca is a traditional Amazonian hallucinogenic brew made by boiling the vine Banisteriopsis caapi with leaves of various admixture plants, commonly Psychotria viridis or Diplopterys cabrerana. Earlier chemical surveys identified three beta-carboline alkaloids in B. caapi—harmine, harmaline and tetrahydroharmine (THH)—and N,N-dimethyltryptamine (DMT) in the admixture plants. Because DMT is inactive orally due to degradation by visceral monoamine oxidase (MAO), it has been hypothesised that the beta-carbolines act as reversible MAO inhibitors to protect DMT from deamination and thereby enable the oral activity of the brew; however, this mechanism had not been experimentally demonstrated in ayahuasca preparations themselves. McKenna and colleagues set out to characterise the alkaloid constituents of multiple ayahuasca brews and their source plants and to test directly whether ayahuasca and its beta-carboline constituents inhibit MAO in vitro. The study combined ethnobotanical sampling of brews and plants from Peru with chromatographic identification and quantitation (two‑dimensional thin‑layer chromatography (TLC), high‑performance liquid chromatography (HPLC), and gas chromatography/mass spectrometry (GC/MS)), and assessed MAO inhibition using a rat‑liver cytosol assay to explore structure–activity relationships among selected beta‑carbolines and mixtures approximating ayahuasca composition.
Field sampling comprised eight ayahuasca brews obtained from ayahuasqueros around Iquitos, Pucallpa and Tarapoto in Peru, preserved either undiluted or diluted 1:1 with methanol to arrest fermentation. Stem samples of several Banisteriopsis caapi cultivars and leaves of admixture plants (primarily Psychotria viridis and a Diplopterys cabrerana clone) were also collected and vouchered when possible. Phytochemical analysis used two‑dimensional TLC to profile crude brews and purified alkaloid fractions, with visualisation under short‑ and long‑wave UV and Ehrlich's reagent for tryptamines. Quantitative determinations were performed on a Varian HPLC system with reverse‑phase column and UV detection at 260 nm; calibration used external standards of harmine, harmaline, THH and DMT at known concentrations. Sample preparation procedures differed by material type (crude brews, methanol‑preserved brews, dried B. caapi stems, and methanol extracts of admixture leaves) but generally involved methanolic extraction, filtration, solvent removal, acid/base partitioning for plant extracts, and injection of aliquots in replicate runs. GC/MS (Finnigan model 1020) was used to confirm the identity of DMT in admixture leaf extracts by matching retention time and mass spectra to authentic DMT standard. Uncommon admixture plants were screened for alkaloids using precipitation reagents (Meyer's, Valser's, Dragendorff's) and TLC when only limited material was available. MAO inhibition assays employed whole rat‑liver cytosol prepared from perfused Wistar rat livers. The enzyme assay used 14C‑labelled 5‑hydroxytryptamine (5‑HT) as substrate, incubated at 37.5°C; reaction products were extracted into ethyl acetate and quantified by liquid scintillation counting. Stock solutions of selected beta‑carbolines were prepared in 0.1 N HCl and assayed over serial 1:10 dilutions to generate concentration–response data; boiled blanks and active‑enzyme, no‑inhibitor controls were run in parallel. Two ayahuasca brews (Don Fidel no. 1 and Don Juan no. 2) were reconstituted from methanol‑preserved samples, quantified by HPLC (total alkaloid 3.5 mg/ml and 4.8 mg/ml respectively), diluted serially, and tested for MAO inhibition. Assays also compared activity of (a) equimolar mixtures of harmine, harmaline and THH and (b) an "ayahuasca analogue" mixture with the same total alkaloid concentration but with relative proportions matching measured brews (approximately 69% harmine, 26% THH, 4.6% harmaline); DMT was not included in the analogue mixtures.
Chemical profiling showed that harmine, harmaline and THH were the major beta‑carbolines present in all analysed ayahuasca brews; harmalol was detected in only one sample. DMT was detected in the admixture plants and in most brews, but was absent from the single Tarapoto brew examined, which employed Psychotria carthagenensis rather than the more common Psychotria viridis. Two‑dimensional TLC, HPLC and GC/MS confirmed the identities: Psychotria viridis leaf samples consistently contained DMT (reported between 1 and 1.6 mg/g dry weight), and the Diplopterys cabrerana sample (Plowman 6040) contained DMT at about 1.74 mg/g dry weight with a trace of 5‑hydroxy‑DMT. Quantitative HPLC revealed substantial variation in alkaloid content across brews and plant material. The Pucallpa lyophilised samples showed notably higher total alkaloid content than samples from Iquitos and Tarapoto; one Pucallpa sample was reported as 75.7 mg alkaloid/g dry weight, comprised approximately of 76% harmine, 10.6% THH and 7.6% DMT. Using the average of five Pucallpa samples, the authors calculated that a 100‑ml dose could contain about 728 mg total alkaloid (467 mg harmine, 160 mg THH, 41 mg harmaline and 60 mg DMT), substantially exceeding levels reported in an earlier study by at least an order of magnitude. Analysis of dried B. caapi stems from different cultivars showed wide variation in total alkaloid content, from 1.8 to 13.6 mg/g, with no simple correlation to vernacular cultivar designations. Screening of less common admixture plants found that Abuta grandifolia yielded a positive alkaloid test and is reported to contain palmatine (a benzylisoquinoline), whereas Justicia pectoralis showed no alkaloids in the tested collection. In vitro MAO assays produced three principal findings. First, ayahuasca preparations were potent inhibitors of MAO in the rat‑liver cytosol system: Don Fidel no. 1 and Don Juan no. 2, quantified at 3.5 mg/ml and 4.8 mg/ml total alkaloid respectively, inhibited MAO strongly even when serially diluted across many orders of magnitude. The extracted text reports that Don Fidel still produced greater than 40% inhibition at a highly diluted concentration reported as 10^-5 of full strength; Don Juan's sample exceeded 50% inhibition even at a high dilution, although the exact dilution for that result is ambiguously reported in the extraction. Second, assays of single beta‑carboline standards showed structure–activity relationships: fully aromatic and dihydro‑beta‑carbolines were substantially more potent MAO inhibitors than fully saturated analogues; methoxy substitution tended to increase potency relative to hydroxyl substitution at comparable positions; certain methylations (for example N‑methylation) enhanced activity in some analogues. Third, mixtures approximating ayahuasca composition were assayed: an equimolar mixture of harmine, harmaline and THH and an "ayahuasca analogue" (same total alkaloid but proportions matching measured brews) had similar inhibitory potencies (reported IS0 values are presented in the paper but the extracted text does not clearly show the full numeric exponents). The authors conclude that the combined activity of harmine and THH can account for most of the MAO inhibition observed with the brews, and that the overall activity of mixtures is not greater than that of the most active component(s) present.
McKenna and colleagues interpret these findings as the first direct empirical evidence that ayahuasca preparations inhibit monoamine oxidase in vitro and that the degree of inhibition correlates with the concentration of MAO‑inhibiting beta‑carbolines. They note that the high levels of beta‑carbolines and DMT measured in their Pucallpa samples—considerably greater than levels reported in an earlier study—support the ethnopharmacological hypothesis that beta‑carbolines render DMT orally active by preventing its deamination, thereby providing a plausible pharmacological mechanism for the brew's psychoactivity. The authors caution that the present results derive from an in vitro rat‑liver cytosol assay and represent only an initial step toward understanding ayahuasca pharmacology. They state explicitly that in vivo studies in animals and humans are required before firm conclusions about the brew's effects in living organisms can be drawn. Variation in alkaloid composition between regions and between B. caapi cultivars is emphasised, with preparation methods (length and repetition of boiling, concentration) and environmental factors proposed as contributors to observed differences in alkaloid load; the extracted text indicates that Pucallpa preparations are simmered and concentrated for many hours, which may account for higher alkaloid concentrations relative to other regional methods. Additional implications discussed by the authors include the possibility that ayahuasca made from B. caapi without DMT‑containing admixtures would have a different pharmacology and that at substantially higher concentrations the beta‑carbolines alone could produce psychotropic effects. They also highlight that non‑tryptamine admixtures such as Abuta grandifolia (containing palmatine) could modulate the brew's effects in ways that are not yet characterised and call for further pharmacological work on these admixture plants. Finally, the report acknowledges limitations in botanical sampling (the need for broader systematic sampling of B. caapi cultivars and consideration of environmental variables) and the ambiguity of some numerical assay details in the extracted text; they reiterate the necessity of follow‑up in vivo and clinical investigations to clarify the pharmacodynamics and safety of ayahuasca.
The hallucinogenic beverage ayahuuscu (Quechua for "vine of the souls") is widely used for medicinal, ritual and recreational purposes by the aboriginal and mestizo populations inhabiting the Amazon Basin. While uyahuascu is the most common term for the drug in the Peruvian Amazon, in different regions it is known by various vernacular names, including yuge, cuupi, nutemu and pinde. The bitter, coffee-colored beverage is prepared by boiling the bark of the Malpighiaceous jungle liana B. cuupi (Spruce ex Griseb.) Morton together with the leaves of various admixture plants, the addition of which is believed to intensify or modify the effect. Many of the admixture plants used remain uncharacterized either botanically or chemically, however those used most commonly are Psychotriu viridis Ruiz and Pavon, Psychotriu curthagenensis Jacq. and Diplop terys cubrerunuGates; the latter is also a Malpighiaceous liana formerly known as Bunisteriopsis rusbyunu. The most complete study to date of the chemistry of the uyuhuuscu beverage, and of the source-plants and admixture plants used in its manufacture, is that of; these investigators studied uyuhuuscu samples and vouchered botanical material collected among the Sharanahua and Culina tribes of the upper Rio Purtis in Peru. Using GC/MS as the primary analytical tool, Rivier and Lindgren found that the P-carboline alkaloids harmine, harmaline, and tetrahydroharmine (THH), and DMT were the major active constituents of uyuhuusca (cf. Fig.). The p-carbolines are constituents of B. cuupiwhile DMT has been reported in the two Psychotria spp.as well as in D. cubrerunu. Hashimoto andhave reported the isolation of six p-carboline bases from the leaves of B. cuupi in addition to the three main constituents (viz. harmic amide, acetyl norharmine, ketotetrahydronorharmine, harmine N-oxide, harmic acid methyl ester and harmalinic acid). However, the extremely low concentrations of these compounds in the plant (0.007-0.0001%) make it unlikely that they contribute significantly to the pharmacological activity of uyuhuuscu. Although the active alkaloids of uyuhuuscu are now known, certain aspects of the pharmacology of the drug remain to be clarified. DMT is known to be a potent hallucinogen but is also known to be inactive when ingested orally, probably due to deamination by intestinal and hepatic MAO. Several studieshave shown that indole-3-acetic acid (IAA) is a major metabolite of DMT both in vitro and in vivo which suggests that it is a substrate for MAO. Further support for the importance of MAO in the metabolism of DMT is found in the fact that the behavioural effects and tissue levels of DMT in rats are potentiated by pretreatment with the MAO inhibitor iproniazid, a specific inhibitor of MAO-A, However,point out that incubation of DMT in rat whole brain homogenates resulted in the formation of N-methylt~ptamine ~NMT), DMT-~-oxide, and Z-me~yl-~tr~ydro-~-c~boline as well as IAA. These workers suggest that NMT may be the primary metabolite of DMT, and that IAA is formed secondarily as a result of the oxidative deamination of NMT. NMT is oxidized nine times more rapidly than DMT by MAO, while DMT-N-oxide is essentially resistant to metabolism by MAO under aerobic conditions. The &carbolines present in B. caupi are known to be highly active reversible inhibitors ofand are probably also hallucinogenic. The psychotomimetic activity of the p-carbolines is not well understood because there are relatively few clinical investigations of the effects of p-carbolines on human subjects.reported harmaline to be hallucinogenic at oral doses of 4 mg/kg but could not report similarly unequivocal results for harmine or tetrahydroharmine.reported harmine was orally inactive at levels approaching 12 mg/kg. It has been suggestedthat the hallucinogenic properties of the crude aya-huasc~ brew result from a synergistic interaction among the various constituents; specifically, that it results from an oral activation of the DMT through the inhibition of MAO by the /I-carbolines. This mechanism would render the DMT orally active by blocking its degradation by visceral MAO. Although this mechanism is reasonable and has long been accepted in the ethnopharmacological literature, the effect of ayahuasca on monoamine oxidase has not been expe~en~ly detested. This paper presents the results of our phytochemical and pharmacological investigations of ayahuasca, including an evaluation of its effect on MAO in vitro.
Collection numbers cited throughout this paper refer to the personal collection numbers of D. A yahuasca samples Ayahu~ca samples used for analysis are identified in this paper by the name of the ayahuusquero from whom they were obtained, and also by a number. In some cases more than one sample was obtained from the same person. Some of the ayahuasca samples were diluted approximately 1: 1 with methanol at the time of collection in order to arrest fermentation and prevent biological transformation of the products. Other samples were not diluted. No obvious difference between the alkaloid content of methsol-d~uted samples and und~u~d ayahuascu samples could be detected by HPLC or TLC.
Two-dimensional TLC was carried out using 10 X 10 cm Polygram Silica Gel G UV254 precoated plates (Began Instrument). The origin was marked with pencil in the lower left hand corner of the plate, 1.0 cm from the bottom and left-hand edge. Five microlitre aliquots of the material to be analyzed (consisting either of the crude ayahuasca samples or in the case of the admixture plants of a methanol solution of the purified alkaloid fraction) were applied to the origin using a Microcap applicator. The applied sample was dried under a gentle stream of air, and then developed in the first direction using ether/Z-butanone/conc. NH,OH, 5: 4 : 1 (Solvent 1). Solvent 1 was freshly prepared in a separatory funnel and the upper phase was collected for TLC. Following development in Solvent 1, the plate(s) were removed and allowed to air dry in a fume hood. Development in the second dimension was commenced when the plates were completely free of Solvent 1, indicated by the absence of any solvent odour. Plates were then rotated 90' to the left with respect to their position in Solvent 1, and developed in Solvent 2, consisting of n-propanol/l.5% NH40H, 9: 2; this solvent was stable for 2-3 days at room temperature if kept sealed in a ground-glass stoppered flask. Development in both Solvent 1 and 2 was carried out at ambient temperature in an unlined 10 X 30 X 26 cm glass chromatographic tank containing approx. 50 ml of solvent. Following development in Solvent 2, plates were removed and air-dried in a fume hood for 30 min to 1 h or overnight on the laboratory bench. Plates were examined under short-and long-wave UV light to visualize the alkaloids. DMT and tetrahydro-fl-carbolines are visible as dark spots under short-wave UV while the aromatic and dihydro-@-carbolines give characteristic strong fluorescent colours under long-wave UV. Duplicate plates were sprayed with Ehrlich's reagentwhich gives blue to violet colours with DMT and other tryptamine derivatives following exposure to HCl' vapours. Aromatic and d~ydro-~-c~bol~es do not react with Ehrlich's reagent, however THH gives a characteristic robin's egg blue colour which develops over 24 h. This slow reaction can be used to conveniently distinguish THH from its more aromatic analogues and also from the tryptamines which give darker blue reactions that appear within 30 mm of exposure to WC1 vapours. A TLC plate containing aliquots of known &carboline and tryptamine standards was developed simultaneously with the sample plates; constituent in the samples co~e~ond~g to known standards could thus be readily identified by comparison of the sample plates with the "standard" plate (cf. "ayahuasca analogue", Fig.).
A Varian model 5000 HPLC interfaced with a Spectra-physics model SP4100 computing integrator was used for the qu~ti~tive analysis of the uyahuasca samples, the B. caupi cultivars, and the DMT-containing admixture plants. Constituents were detected by UV absorption at 260 nm with a Varian model 634 variable wavelength UV/visible spectrophotometer. Column consisted of a Varian Micropak MCH-10 reverse phase column, 30 cm X 4 mm i.d. Solvents were methanol/water containing 0.05% triethylamine. A gradient elution program was used for the analysis, from 60-90% methanol at a rate of l%/min. Solvent flow rate was 2 ml/mm. Samples were applied to the column via a Rheodyne model 7125 syringe loading sample injector fitted with a 20-~1 sample loop. Samples were loaded onto the sample injector using a 25~1 Hamilton no. 702 microlitre syringe fitted with a no. 22 gauge 90" bevelled needle.
Quantitative analyses were carried out using the external standard program supplied with the SP4100 compu~ng integrator. In this method, the column is calibrated with a mixture containing known concentrations of standard compounds. The integrator program calculates the peak area of each standard in the calibration mixture and normalizes the response factor. Calibration at two or more concentrations enables the integrator to generate the coefficients of a linear equation relating sample concentration to peak area. This calibration data is stored in the memory circuits of the integrator and used to caiculate the concentration of components of interest in subsequent sample runs by comparison of the peak areas of sample component with the peak areas of standards in the calibration mix. Stock solutions of harmine, harmaline, tetrahydroharmine and DMT standards were made up to a precise concentration of 1 mg/ml. Equal aliquots from the stock solutions were combined, giving a calibration mixture of 0.25 mg/ml of each component; 1: 1 dilution of a portion of this mixture with methanol gave a second calibration mixture in which the concentration of each component was 0.125 mg/ml. The SP4100 integrator was calibrated by making single 20-111 injections of the calibration mixture at each concentration level. The integrator was recalibrated following every five sample injections.
Ayahuasca samples. The ayahuasca samples which had not been diluted with methanol on collection were diluted for analysis so that the alkaloids present were within the concentration range of the calibration standards. A 1.5-ml aliquot of the crude preparation was diluted to 15 ml with chilled methanol and a white, flocculent, proteinaceous precipitate which separated from solution was removed by filtration. This diluted preparation, after filtration through a Pasteur pipette plugged with glass wool, was injected directly into the 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. Values reported in Tableare the means of these four replicate injections. Values given are 10X the actual value measured since the samples measured were l/10 the concentration of the undiluted brew. The ayahuasca samples which had been diluted with methanol in the field were quantified for alkaloids in terms of mg/g dry wt of the lyophilized sample. Fifteen millilitres of the methanol-diluted sample was evaporated on a steam bath, frozen, then lyophilized. A portion of the freeze-dried residue was ground to a fine powder and 100 mg was transferred to a 100 ml round-bottom flask. Ten millilitres 100% methanol was added and the solution was extracted over a steam bath for 5 min. The methanol was removed with a Pasteur pipette and filtered through glass wool. The extraction was repeated using a second lo-ml aliquot of methanol. The filtered extracts were combined, and the final volume was adjusted to 10 ml. Twenty microlitre aliquots of this solution were injected onto the HPLC. As with the previous samples, two replicates of each sample were prepared, and each replicate was injected twice. Figures given in Tableare the average of these four replicate injections. B. caapi cultilars. Qua&it&ion of the alkaloid content of the B. caapi cultivars was carried out on stem samples which had been dried under low heat (<6O"C) in a plant dryer. The air-dried stems were ground to powder in a Wiley mill and 5 g was extracted 2X in 100 ml methanol for 24 h on a rotary shaker. The combined extracts were concentrated under vacuum, filtered through glass wool and the volume adjusted to 25 ml with methanol. Ten microlitre aliquots were injected directly onto the HPLC. Four replicate injections of each sample were made, and figures given in Tablereflect the mean of these four replicate injections. ~~T-~o~tui~i~g adm~ture plants. All of the ~s~c~otri~ uiridis samples were analyzed using methanol-preserved leaf material collected in the field; the Diplopterys cabrerana sample consisted of freeze-dried leaves derived from a greenhouse propagated clone of Plowman 6040. The methanol was decanted from the methanol-preserved material and the remaining solid matter was frozen, then lyophilized. The freeze-dried leaf material was powdered and extracted overnight on a rotary shaker with methanol (10-20 ml/g dry wt). The L). eabreruna leaves were extracted directly with methanol and otherwise treated in the same manner as the Psychotria samples. The methanol extracts were filtered, combined with the original methanol used to preserve the samples, and concentrated in a rotary evaporator to a known volume. The crude rne~~ol extracts were sealed and stored at 4°C. For purposes of alkaloid quantitation, an aliquot of the methanolic extract equivalent to 2.0 g dry wt of the freeze-dried leaf material was transferred to a 50 ml round-bottom flask and evaporated to dryness on a rotary evaporator. The residue was shaken with 5 ml of 1 N HCl and filtered. The acidic filtrate was washed with 1 X 5 ml CH&&, and the organic layer discarded. The aqueous layer was basified to pH 8-9 with saturated NaHCO, and 2 N NaOH. The basic aqueous fraction was extracted with 3 X 5 ml CH2C12. The organic layer was dried over anhydrous Na$O,, evaporated to dryness, redissolved in methanol, and filtered through glass wool. Twenty microlitre aliquots of this purified alkaloid fraction were injected into the HPLC. Concentration of the DMT present was determined by comparison with a standard curve constructed by injecting known amounts of DMT standard. Figures given in Tableare means f S.E. of 2-5 replicate injections.
DMT-containing admixtures were screened by GC/MS to confirm that the major indole base detected was DMT. The instrument used was a Finnigan model 1020 automated GC/MS interfaced with a Perkin-Elmer Sigma 3B gas chromatograph. The chromatograph was equipped with a 30 m X 0.25 mm SE-54 fused silica capillary column (J & W Scientific). The chromatograph was ~mperatur~pro~med from an initial ~m~rature of 180°C to a final temperature of 250°C. The ramp rate was 3*C/min initiated 3 min after injection of the sample. Injector block temperature was 250°C, detector temperature 260°C. The carrier gas was helium. One microlitre aliquots of DMT standard, or of the purified alkaloid extracts of the leaf samples were injected. Eluted compounds were detected as peaks in the reconst~c~d ion c~omatogr~ (RIG) generated by the mass spectrometer data system. Under these conditions, DMT had a retention time of 9.5-10 min, base peak: m/z 58, M' : 188. The indole base present in the Diplopterys cabreruna and Psychotria leaf extracts had a mass spectrum and retention time identical with the standard.
The uncommon admixture plants were screened for alkaloids following the method of. Material used in the analysis was preserved in methanol, and this was worked up in a manner identical to that described above for the Psychotria samples. Aliquots of the acidic filtrate were tested with l--2 drops of either Meyer's, Valser's, or Dragendorff's reagent. Appearance of either a marked turbidity or a heavy precipitate on addition of the reagent was interpreted as a positive alkaloid reaction; slight turbidity indicated possible traces of alkaloid. Composition of the reagents used is given in. After testing with the precipitation reagents, the acidic aqueous solutions were basified to pH 8-9 with saturated NaHC03 and 2 N NaOH and extracted with 3 X 5 ml CHzCll. The organic layer was collected, evaporated to dryness under vacuum, and the residue taken up in 2.0 ml methanol. Five microlitre aliquots of this final methanol fraction were applied to precoated Polygram silica gel plates UVzs4, and the plate was developed in one direction in butanol/acetic acid/water, 4 : 1: 1. Following development, the plates were air-dried, examined under short-and long-wave UV light, and sprayed with Dragendorff's modified reagent,.
Rat-liver, derived from mature female Wistar rats, was used as the source of MAO for the assays. The rats were killed by stunning followed by cervical dislocation. The abdominal cavity was opened by removal of the belly skin and peritoneum and the liver perfused with cold 0.1 M sodium phosphate buffer (pH 7.2)by injection into the hepatic vein with a bevelled no. 22 stainless steel syringe. The perfused liver was removed from the abdominal cavity and immediately placed in cold buffer. Portions of liver were transferred to a glass homogenizer and homogenized in cold buffer to a smooth paste (approx. 1 g liver/3 ml buffer). The homogenate was centrifuged at 3000 X g for 20 min to remove nuclear fragments. The supernatant was collected and diluted 1: 1 with buffer. The diluted cytosol fraction was sonicated for 10 s. This diluted whole cytosol fraction was kept on ice for the duration of the experiment, and was used without further centrifugation.
The assay was conducted at 37.5'C in 13 X 100 mm disposable test tubes. Volume of the reaction mixture was'0.5 ml. Composition of the reaction mixture in controls lacking added inhibitor consisted of 375 ~1 sodium phosphate buffer, 100 ~1 whole cytosol and 25 ~1 labelled substrate. The substrate consisted of 5-hydroxy [ side-chain-2-14C] tryptamine creatinine suiphate (Amersham). The labelled substrate was diluted with cold carrier ~hydroxyt~p~~e (5HT) to a specific activity of 0.835 mCi/mmol and a concen~ation of 2 X 10m3 M. At this dilution, addition of a 25-~1 aliquot to the reaction mixture results in a final volume of 500 ~1 and a 5HT concentration of 0.1 mM. containing a total of 0.04 PCi of labelled 5HT. All components of the reaction mixture except the labelled substrate were added to the reaction tubes which were maintained at 0°C in an ice bath. Inactivated control blanks, identical in composition to the active controls, were prepared by immersing the tubes in boiling water for 5 min. Prior to initiation of the assay, the reaction tubes were immersed in a 37.5% water bath and equilibrated for 5 min. The assay was initiated by adding a 25-~1 aliquot of the diluted substrate to the tubes, mixing for 1-2 s on a vortex mixer, and replacing in the water bath. Following addition of the substrate the assay tubes were incubated for 30 min at 37.5%. After 30 min the reaction was terminated by adding 1.0 ml of 1.0 M citrate. This was followed by the addition of 0.5 ml saturated NaCl, and 2.0 ml ethyl acetate to each tube. Tubes were then vortexed for 5 s each and centrifuged at 2000 rev./ min for 10 min to separate the organic and aqueous layers. In this method, (cf. Tipton and Youdim, 1976) the labelled reaction products are soluble in the organic (upper) phase while the protonated substrate remains in the lower aqueous phase. Following centrifugation, a l.O-ml aliquot of the upper phase was transferred to a scintillation vial containing 9.0 ml Aquasol-2 (New England Nuclear). Scintillation vials were counted for 10 min in a Searle Isocap 300 Liquid Scintillation Counter.
Stock solutions of various @-carbolines, dissolved in 0.1 N HCl, were prepared so that addition of a 50-~1 aliquot to a reaction mixture would result in an inhibitor concentration of 10q3 M and a total volume of 500 ~1. Stock solutions for lower concentrations were prepared by making serial 1: 10 dilutions, over a concentration range from 10m3 to lo-" M. Assay mixtures containing inhibitor consisted of 325 yl buffer, 100 ~1 whole cytosol, 50 ~1 i~ibitor solution of the approp~a~ concen~ation and 25 ~1 substrate. The inhibitor solution was added just before the assay tubes were transferred to the warm water bath, so that the incubation time of the cytosol plus inhibitor at 37.5"C was 5 min. Boiled blanks containing denatured enzyme were included in each run. In addition controls containing active enzyme but no inhibitor were also assayed simultaneously (inhibitor was replaced either with sodium phosphate buffer or with 50 ~1 0.1 N HCl). There was no appreciable difference between controls in which buffer replaced the inhibitor and those in which 0.1 N HCl replaced the inhibitor. The controls lacking inhibitor represented 0% MAO inhibition; and the percent inhibition of tubes containing varied concentrations of inhibitor was calculated relative to these controls, by dividing the amount of labelled reaction products recovered (measured as cpm) by the amount recovered from the control tubes. The background count represented by the activity detected in the boiled blanks (usually 200-300 cpm) was subtracted from both control counts and inhibitor counts. The ayahuasca samples used in the assay were Don Fidel no. 1 and Don Juan no. 2. Five milliliter aliquots of each were diluted to 50 ml with chilled methanol and the white non-alkaloidal precipitate removed by filtration. The filtered solution was then evaporated to dryness under reduced pressure and resolubilized in 5 ml 0.01 N HCl to reconstitute the original volume. A 1: 1 dilution of this reconstituted solution was used to quantify the alkaloids present in the mixture using the HPLC method described above. This quantification showed the reconstituted samples to contain 3.5 mg/ml and 4.8 mg/ml total alkaloid, respectively. Serial 1: 10 dilutions of the reconstituted samples, ranging from 0 to lo-', were made using 0.01 N HCl. Fifty microlitre aliquots of the appropriately diluted uyahuuscu solutions were added to the reaction mixture in the same manner as the inhibitors, and percent inhibition was calculated relative to the controls (cf. Fig.). Inhibitor assays were also conducted using an equimolar mixture of harmine, harmaline and tetrahydroharmine. A mixture of these compounds in which each constituent was present in the same proportion as found in the uyuhuusca samples but in which the overall concentration was approximately equivalent to the concentration of the equimolar solution was also assayed (cf. Fig.). Points plotted in Figs.andare means + S.E. of four replicate determinations. Standard errors less than 1% are not shown. See the Results and Discussion for an evaluation and comparison of these various inhibitor assays.
The alkaloidal constituents of a number of uyahuuscu brews, cultivars of B. cuupi and a variety of admixture plants were qualitatively and quantitatively investigated using two-dimensional TLC and HPLC as the primary analytical tools. Admixture samples were also analyzed using gas chromatography/mass spectrometry (GC/MS). Some admixture plants were screened for alkaloids using precipitation tests and TLC. In vitro MAO inhibition was studied using se&ted p-carboline standards, mixtures of P-carboline standards, and ayuhuascu preparations. Details of the analytical and experimental methods are presented in the Experimental section.
During the course of ethnobotanical fieldwork in the spring of 1981, eight samples of uyuhuascu preparations were obtained from uyuhuusqueros living on the outskirts of the Peruvian towns of Iquitos, Pucallpa and Tarapoto. These samples were qualitatively analyzed using two-dimensional TLC and quantified using HPLC. Identification of alkaloids was based on comparison with authentic standards. Comparison of the TLC profiles of the eight samples (Fig.) shows that the major constituents vary little from sample to sample; different batches made by the same u~u~~~s~~e~o (cf. Don Fidel nos. 1 and 2, and Don Juan, nos. 1 and 2) are generally similar, and there is also httie variation in the constituents of brews made by different ~~~~~usq~e~os, Harmine, harmol, harmaline and THH were found to be the major P-carbolines present in all of the samples, while harmalol was not detected in any samples save one (Don Milton no. 1). DMT was found in all samples except that from Tarapoto. No other si~~i~~t Ehr~ch-positive spots were detected. Known constituents wereidentified by comparison with a mixture of authentic standards (aya~~~ca "analogue", upper left in Fig.). Traces of fluorescent compounds not corresponding to any of the P-carboline standards were also detected in most samples; it is assumed that these represent P-carbolines of undetermined structure. Absence of DMT in the sample from Tarapoto is significant, since this is the only sample in which Psychotria carthagenensis was employed as the admixture rather than the more commonly used P. vi~~is, The quantitative HPLC analysis of five undiluted samples prepared by two ayahuasgueros in Pucallpa shows little variation from batch to batch, either in total alkaloid content or in the proportions of ~onsti~en~ (Table). The rne~~ol-Druid samples were Iyophihzed and their alkaloid contents compared on a dry weight basis (Table). These samples showed considerable differences in alkaloid content (expressed as mg alkaloid/g dry wt). The sample from Pucallpa had the highest total alkaloid content (75.7 mg/g dry wt) of which 76% was harmine, 10.6% was THH, and 7.6% was DMT. The samples from Iquitos and Tarapoto generally had lower total alkaloid levels, and also differed in the proportions of different constituents. The reason for the difference in relative proportion of harmine and THH in the Pucallpa samples and those from other regions of Peru could be related to the type of B. caapi cultivar employed to prepare the drink, or to the method of preparation, Environmental variables, such as the type of soil in which the cultivar is grown, or different conditions of exposure to sunlight, may also be involved, Tableshows, however, that all of the aya~~asca samples from Pucallpa ~onsis~n~y show approximately the same relative proportions of harmine to THH to harmaline. This may indicate that all of these samples were prepared either from the same B. caapi cultivar or clones of the same cultivar. This would not be surprising since Don Juan is the uncle of Don Fidel and they often collaborate in the preparation of their brews, Summ~ing the results of their qu~titative studies, Rivier and Lindgren (1972) state that a typical 200 ml dose of ayahuasca contains a total of 65 mg alkaloid, of which 30 mg is harmine, 10 mg is THH and 25 mg DMT. This is some 10-30 times less than the dosage at which the /3-carbolines are hallucinogenically active in the pure form (cf. Introduction), although it is well within the range at which they are effective as MAO inhibitors. Twenty five milligrams is just below the threshold dose for DMT when this compound is injected intramuscularly (Szara, 1957) but it is possible that the threshold may be lowered under conditions of MAO inhibition. Commenting on their findings, Rivier and Lindgren (1972, p. 127) conclude: "In view of these results, new pharmacological experiments for a better understanding of the hallucinogenic action of ayahuasca seem necessary ". The alkaloid levels found in the five samples from Pucallpa (Table) exceed the levels reported byin samples collected on the upper Rio PurGs by at least an order of magnitude. Thus, (based on the average of the five samples) a lOO-ml dose of the Pucallpa ayahuasca (cf. Table) contains 728 mg total alkaloid, of which 467 mg is harmine, 160 mg is THH, 41 mg is harmaline and 60 mg is DMT. This is well above the threshold dose for DMT but is still considerably below the hallucinogenic dose level for the p-carbolines. In practice the typical dose ingested in the Pucallpa ceremonies rarely exceeds 75 ml and is usually closer to 55-60 ml. The relatively large differences in the alkaloid content of the upper Purls ayahuasca analyzed by Rivier and Lindgren and the Pucallpa ayahuasca analyzed in the present study may be readily explained by the differences in the method of preparation in the two regions. In the upper Purls method, stems of B. caupi totalling about 900 cm in length and l-4 cm diameter are cut into short sections, crushed, and packed in a 15-Q metal vessel together with alternating layers of leaves of Psychotria sp. Ten litres of water are added and the mixture is boiled for 1 h, strained and cooled. The mixture is then consumed without further processing. The method employed in Pucallpa starts out similarly but the mixture is boiled for a much longer time, approximately lo-15 h. The water may be drained off and replaced with fresh water several times during this boiling process. The separate batches are combined, allowed to cool, and filtered through a strainer or cheesecloth. The plant material is removed from the cooking pot and discarded, and then the strained ayahuasca is poured back into the pot and simmered over a low fire until it has been concentrated to about half its original volume. The 5 or 6 L of ayahuasca obtained from this process are transferred to wine or beer bottles and stoppered with corks. In this form the brew may be kept for up to 6 months without refrigeration.
All of the ayahuasqueros that we interviewed during our field studies in Peru recognized several different "kinds" of ayahuasca which were claimed to vary in their psychological effect. The differentiation of these varieties of ayahuasca was based in part on the types of admixture plants which were added, and in part on the type of B. caapi which was utilized. Several types of B. caapi were generally recognized by these practitioners and were distinguished by different adjectives e.g. "cielo" ayahuasca, "lucero" ayahuasca, "rumi" ayahuasca. Some claimed to distinguish aqmany as 10 kinds of Bani, steriopsis vine (the term ayahuasca is indiscriminately applied either to the B. caapi vine or to the beverage made from it) but most were familiar with only two or three kinds. Presumably these "kinds" of B. caapi are referable to different cultivars, races, or chemical or morphological varieties of B. caapi. There were no outstanding morphological differences between the three or four kinds of B. caapi which we collected, and the relevant voucher specimens have all been determined as B. caapi by taxonomic specialists in the Malpighiaceae (W.R. Anderson and B. Gates, University of Michigan). HPLC analysis of the alkaloid levels in the dried stems of three of the recognized varieties plus one specimen (DMCK no. 125) for which the vernacular name is unknown has shown that there is considerable variation between samples (Table). The lowest level was found in DMCK no. 126 which contained 1.8 mg/g total alkaloid, while DMCK no. 125 contained the highest level, 13.6 mg/g. There appears to be no consistent correlation of alkaloid content with particular recognized cultivars, however. The variation observed probably has more to do with the age of the plant, and the soil, light, water, and other environmental conditions affecting the growth of the particular specimen. The amounts of alkaloids are in the same general range as those detected in the B. caupi samples analysed by. Further clarification of this question of possible chemical or morphological differences between recognized types of B. caupi cultivars would require a systematic sampling of as many different individuals of each type as possible; climatic, edaphic and other environmental factors should also be considered.
In Peru, the admixture plant employed most frequently in the preparation of ayahuasca appears to be Psychotria viridis R. JZ P. We encountered only one ayahuasquero during our fieldwork who preferred to use another species of Psycho tria, tentatively identified as Psycho tria carthagenensis Jacq. Interestingly, no alkaloids of any kind were detected in this collection (DMCK no. 109, Tarapoto) however, all of the Psychotria viridis collections contained DMT as the single major base. Identity of the compound was confirmed by GC/MS and comparison of its HPLC retention time, TLC Rf and Ehrlich's colour reaction with that of an authentic standard (Table). The Psychotria viridis samples analyzed contained fairly substantial amounts of DMT, between 1 and 1.6 mg/g dry wt in the leaves. No alkaloid was detected in fruits or stems of P. viridis. No other alkaloids were detected in any of the Psychotria samples with the exception of DMCK no. 139, in which a trace constituent with a mass spectrum corresponding to that reported (Rivier and Lindgren, 1972) for 2methyl-tetrahydro-/3-carboline was detected. A single sample of Diplopterys cabrerana (Plowman 6040), the Malpighiaceous admixture, was available for analysis and this also contained DMT together with an extremely trace amount of 5-hydroxy-DMT. The alkaloid extract of dried leaves of the original (1976) collection of Plowman 6040 had an ion chromatogram that was essentially identical to leaf extracts of greenhouse propagated clones of this specimen. Plowman 6040 contained slightly higher levels of DMT (1.74 mg/g dry wt) than the Psychotria viridis samples, but otherwise was indistinguishable in terms of alkaloid content. Although Plowman 6040 was collected in Tarapoto where it was being utilized as an ayahuasca admixture, this use of Diplopterys cabreruna in Peru is uncommon; this species is the usual admixture in Southern Colombia and Ecuadorand in fact Plowman 6040 was originally brought to Tarapoto as a live cutting from the Rio Pastaza in Ecuador (Plowman, pers. commun.). Psychotria viridis, or less frequently, Psychotria carthagenensis are the ad- mixtures of choice in Peru and few of our informants in Peru were familiar with D. cabrerana under its common names, chagro-panga or ocoyage. An ethnobotanically interesting aspect of folk taxonomy related to the identification of the "proper" Psychotria spp. for use as admixtures came to light during the course of fieldwork. Some Psychotria species, including Psychotria uiridis, possess tiny spine-like extensions of the mid-rib on the abaxial surface of the leaf. These appear to be slightly swollen glandular structures which may be equivalent to the "dolmatia" found in some Psycho tria spp. (Gentry, pers. commun.). A dolmatium is a part of a leaf, petiole, stem or other plant part that is inhabited by ants or other insects. The dolmatia-like structures of the Psychotria uiridis specimens we examined appeared to be much too small to accomodate ants of any visible size, but may be inhabited by mites (Prance, pers. commun.). These tiny dolmatialike structures were pointed out by the ayahuasqueros we interviewed as the key feature used to identify the chacrunas suitable for use as admixtures. "Chacruna" is the vernacular term for Psychotria. All of the ayahuasqueros except one insisted that these structures -termed by them espinas (spines) -had to be present; plants lacking espinas were regarded as false chacrunas and were considered to have no value as admixtures. Indeed, all of the Psychotria uiridis collections which we analyzed (cf. Table) possessed these structures, and all contained DMT; the single specimen which lacked these structures also contained no tryptamines or other alkaloids. This specimen (DMCK no. 109) may correspond to
Jacq., although the collection is sterile and the identification therefore tentative; further doubt is cast on the identification by the fact that Rivier and Lindgren (1972) reported DMT in Psychotria carthagenensis, while none was detected in DMCK no. 109.
Ayahuasca is usually prepared using one of the DMT-containing admixture plants mentioned above, either Diplopterys cabrerana or a Psychotria sp., less commonly, however, other admixtures are utilized, either in conjunction with the tryptamine-containing admixtures, or in place of them. Many of these admixtures have been identified in the ethnobotanical literature) although virtually nothing is known of their chemical or biodynamic properties. This would appear to be a promising area for further research. During our fieldwork in Peru three collections were made of plants which were stated by informants to be used as admixtures to ayahuasca (Table). One of these, Z'eliostachya lanceolata, has been discussed byas an admixture, but the other two, Abuta grandifolia (Menispermaceae) and Cornutia odoratahave not previously been reported as admixtures. Plant material from these collections, preserved in methanol, were screened for alkaloids using alkaloid precipitation tests and TLC (Table). Insufficient material was available to permit further chemical characterization. The only collection giving an unambiguously positive test was Abuta grandifozia). This species has recently been reportedto contain 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. One studyfound that palmatine inhibited the effect of epinephrine on blood pressure of rabbits, on the isolated rat seminal vesicle and on the toad hind-leg; its derivative dl-tetrahydropalmatine inhibited the effect of 5HT on isolated rat uteri, colon and stomach. Palmatine also exhibited anticholinesterase activity. Both alkaloids had ACTH and bactericidal activity. Some of these properties may be antagonistic to the effects of the fl-carbolines while others may be synergistic. For instance, harmaline causes an increase in 5HT concentration in the whole brain, while harmine causes a significant decrease in acetylcholine in brain; on the other hand, harmine strongly inhibits the ATP-Mg'+ dependent uptake of norepinephrine into isolated adrenal medullary vesicles. Whether the -- overall effect of palmitine is agonistic or antagonistic to the action of the p-carbolines, there seems little doubt that addition of the bark of Abuta grandifolia to ayahuasca could modify its effect. Further investigations of the pharmacology of this and many other admixture plants are needed in order to clarify their cont~bution to the effects of ~~a~u~ca. Justiciu pectoralis (Acanthaceae) is also included in Table. Justicia pectoralis is not used as an uyahuasca admixture but has been reported as an admixture to the Virola snuffs, and these authors have suggested that it may be used by itself as an hallucinogenic snuff. No ~p~ines or alkaloids of any other type were detected in our collection of this species. W/MS analysis of extracts of Justicia pectomfis indicate that it contains the coumarin derivative umbelliferone and the quaternary nitrogen derivative betaine (MacRae, unpublished data).
The mitochondrially-localized enzyme, MAO, plays an important role in mammalian metabolism. MAO catalyzes the oxidative deamination of biogenic monoamines including tyramine, tryptamine, serotonin, norepinephrine, dopamine and other monoamines, according to the general reaction equation: RCH,NH, + O2 + H,O = RCHO + NH3 + H20, The enzyme is widely distributed throughout various tissues in vertebrates and invertebrates, including the liver, brain, small intestine, blood plasma and platelets, heart and lungs. The most significant metabolic function of MAO is related to its ability to inactivate endogenously produced monoamines such as serotonin or dopamine, which function as CNS neurotransmitter substances). Considerable experimental evidence has been accumulatedwhich indicates that the visceral MAO system functions as a detoxification mechanism serving to protect the nervous and cardiovascular systems from toxic biogenic amines ingested in the diet and formed as a result of aromatic amino acid decarboxylation. Evidence based on substrate specificity and selective sensitivity to certain MAO inhibitors indicates that MAO consists of two species, designated.carried out a kinetic evaluation of the two species using mixed substrate assays and concluded that the substrates for species A activity were substituted p-phenylethylamines and /3-phenylethanolamine derivatives having a free p-hydroxyl group; and 5HT. Substrates for species B activity included substituted benzylamine and /3-phenylethylamine derivatives, but not substituted p-phenylethanolamines. Tryptamine, 5-methoxytryptamine, dopamine and the m-O-methyl derivatives of doparnine are substrates for both MAO-A and MAO-B.point out that substances which interact with MAO-A such as serotonin and norepinephrine have more polar aromatic rings than substances which interact with MAO-B (e.g. /3-phenylethylamine, benzylamine). In general adding a polar hydroxyl group to /I-phenylethylamines (e.g. tyramine) or removing one from serotonin to form tryptamine results in a common substrate. The specific metabolism of DMT has not been investigated, but if it conforms to the above rule it would be a substrate for both MAO-A and MAO-B since it lacks aromatic substituents. Further evidence for the existence of two species of MAO comes from experiments using selective MAO inhibitors. Most of the MAO inhibitors which have been the focus of biochemical and clinical investigations belong to one of four classes, viz. hydrazine derivatives, phenylcyclopropylarnine derivatives, N-benzyl-N-methyl propargylamine, or 2methyl-3-piperidinopyrazine. These are synthetic compounds which are not known to have corresponding analogues in nature. The major exception is the /I-carboline derivatives, a group of tricyclic indole alkaloids which are widespread in nature (Allen and Holmstedt, 1980) (cf. Fig.). These compounds are biosynthetically closely related to tryptamine and its derivatives and can be readily synthesized from tryptamine derivatives.were the first to demonstrate that harmaline and related compounds
In order to assess the influence of structural variation of the @-carboline skeleton (cf. Fig.) on the MAO inhibitory activity of these compounds, a selected group of standards (Table) was assayed in vitro using a ratliver cytosol fraction as the source of enzyme and 14C-labelled Shydroxytryptamine creatinine sulphate as substrate. Tablelists ISo values for the present experiment and also includes the values reported byandby the particular substrate used, the IS,, values reported in Tablefor the present study are not directly comparable to those reported byor: In generd, however, 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. Thus, for example, the fully aromatic and dihydro-fl-carbolines are significantly more potent inhibitors than analogues in which the piperidine ring is fully saturated (cf. harmine, harmaline, and THH). Inhibitory potency is roughly equivalent between the 7-methoxylated fully aromatic and dihydro derivatives harmine and harmaline, but of the 6-methoxylated analogues the fully aromatic compound is more potent. The 7-substituted (3-carbolines were generally more potent inhibitors than the corresponding 6-substituted analogues in our study and that ofbutfound 6-methoxy-harman was equipotent with harmine while 6-methoxyharmalan was a more active inhibitor than harmaline. Hydroxyl substitution on the aromatic ring results in a less active compound than the corresponding methoxy-substituted compound (cf. harmine and harmol). Buckholtz andand the present study 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. McIsaac and Estevez (1966) reported harman to be somewhat more active than either harmine or 6-methoxy-harman. Lack of a methyl group at Cl (cf. harman and norharman) resulted in reduced activity in our assays and in those of, butreported somewhat greater activity for norharmane than for harmane. We found 2-methyl-6-methoxy-tetrahydro-~carboline (2Me-GMeO-THBC), a compound reported from Virola sp. ( Agurellto be in the same general range of potency as harman and harmol. Thus 2-Me-6-MeO-THBC 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. The greater activity of this compound with respect to THH and the other 6-methoxylated analogues may be due primarily to the methylation of the piperidine nitrogen. This compound was not investigated by eitheror; the former authors, however, reported that acetylation of the piperidine nitrogen resulted in a compound lacking inhibitory activity.
Once the relative inhibitory activity of a representative sample of flcarbolines had been evaluated in our assay system, the next step was to evaluate the activity of appropriately diluted ayahuasca samples. The activity exhibited by the ayahuasca samples was compared to that shown by a mixture of harmine, harmaline and THH, which are known to be the major B-carbolines found in cryahuasca. Two types of standard mixtures were used for comparison. The first mixture was an equimolar mixture of harmine, harmaline and THH; the second contained approximately the same molar concentration of total alkaloid as the first mixture, but in this case the proportion of each constituent reflected the proportions present in the ayahuasca brews; these proportions and concentrations had previously been determined by quantitative HPLC. Thus, this "ayahuasca analogue" (as the second mixture was termed) contained 69% harmine, 26% THH and 4.6% harmaline; DMT, which comprised 6.1% of the total alkaloids, was not included in the "analogue" mixture. Two of the u~a~~~cu samples from Pucallpa, Don Juan sample no. 2 and Don Fidel sample no. 1, were analyzed using qu~ti~tive HPLC in order to determine the concentration of alkaloids in the undiluted samples. Total alkaloid concentration was determined to be 4.8 mg/ml and 3.5 mg/ml, respectively. The samples were then subjected to serial 1: 10 dilutions so that the most dilute solution was 1 X lo-' as concentrated as the undiluted sample. Fifty microlitre aliquots of the appropriate dilutions from each sample were assayed for MAO inhibitory activity using the in vitro rat-liver cytosol system (Fig., Table). Figureshows clearly that both uyahuasca samples are extremely effective MAO inhibitors; Don Fidel's no. 1 still showed >40% inhibition of the enzyme at 10M5 full strength, while Don Juan's no. 2 exceeded 50% ~hibition even at one in-rn~ion~ (lo-') the concent~tion of the un~u~d brew. These in vitro results indicate that uyuhuascu is active as an MAO inhibitor even when diluted by many orders of magnitude. These observations constitute the first empirical demonstration of the effect of uyahuascu on MAO and provide evidence for the hypothesis that the hallucinogenic properties of ayahuusca are due to its inactivation of visceral MAO and consequent oral potentiation of the DMT in the preparation. It should be emphasized, however, that the present in vitro study represents only the first step toward understanding the pharmacology of uyuhuusca. Further investigations are required, particularly in vivo studies of the action of uyahuuscu in both animals and humans, before the pharmacology of this Amazonian drug is fully elucidated. Another point worth mentio~ng is the fact that on occasion uy~~~~ca is prepared from B. ceupi alone without the addition of any admixture plants; in these instances, DMT would be absent from the preparation. This would alter its pharmacology and presumably the hallucinogenic effects, if present, would be due to the p-carbolines alone. In this case concentrations of p-carboiines considerably greater than those measured in our samples would be required if a non-synergistic mechanism is assumed. It is informative to compare the 7% MAO inhibition produced by an equimolar mixture of harmine, harmaline, and THH with the inhibition elicited by single components of this mixture (Fig., Table). The equimolar solution inhibited 50% of the enzyme at a concentration of 3.16 X lo-' M (cf. Table) (in this case, moiarity of the solution is based on the pounds collectively is not greater than the activity of the most active compound of the group. This supposition is further supported by the assay of the "ayahuasca analogue" (Fig., Table) in which the total alkaloid concentration is equal to the ~oncen~tion of the equimolar mixture, but the proportions of harmine, THH, and harmaline approximate those found in Don Juan sample no. 2, i.e. 69% harmine, 26% THH and 4.6% harmaline. ISo values for the ayahuasca "analogue" are nearly identical with those of the equimolar mixture (3.98 X lo-' and 3.16 X lo-', respectively) indicating that the combination of harmine and THH alone can account for most of the MAO inhibition exhibited by ayahuasca. While harmaline is equivalent to or slightly stronger than harmine, it is essentially a trace component in ayahuasca and probably does not contribute significantly to the MAO inhibition which this drug elicits.
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