This chemical profiling study investigated the different formulations of ayahuasca and proposes that the identified compounds can serve as reliable markets for the potency/composition of ayahuasca brews.
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.
Papers cited by this study that are also in Blossom
Callaway, J. C., Brito, G. S., Neves, E. S. · Journal of Psychoactive Drugs (2011)
Samoylenko, V., Rahman, M. M., Tekwani, B. L. et al. · Journal of Ethnopharmacology (2010)
Schwarz, M. J., Houghton, P. J., Rose, S. et al. · Pharmacology Biochemistry and Behavior (2003)
Papers in Blossom that reference this study
Banisteriopsis caapi, a woody vine from the Amazon used traditionally as an ingredient of the psychoactive brew ayahuasca, contains β-carboline alkaloids that act as monoamine oxidase (MAO) inhibitors and has been reported to alleviate symptoms in neurological disorders including Parkinson’s disease. Earlier phytochemical studies identified harmine, harmaline and tetrahydroharmine (THH) among other β-carbolines, as well as various terpenoids. Recent work by the study team on a cultivated Hawaiian plant material named Da Vine additionally isolated two tetrahydro-β-carboline glycosides (banistenoside A and B), a new tetrahydronorharmine analogue, and antioxidant flavan-3-ols (epicatechin and procyanidin B2). Wang and colleagues set out to develop standardised aqueous extracts of B. caapi with high potency for inhibition of human MAOs and antioxidant activity, and to perform chemical profiling across plant parts, seasons and sources. The study aimed to quantify marker compounds (including β-carbolines and proanthocyanidins), formulate standardised pharmaceutical compositions from isolated markers, and test those preparations in vitro for MAO inhibition and antioxidant effects to explore correlations between phytochemistry and bioactivity.
The researchers analysed material from B. caapi cultivar Da Vine grown in Hawaii and from commercially sourced matured stems. Fresh leaves, stems and large branches were collected from Oahu and Hilo between 2007 and 2008; a voucher specimen was deposited at the Harold Lyon Arboretum. Plant extracts were prepared using either water maceration (coffee-maker style) or an accelerated solvent extractor (ASE) with water as solvent; extracts were freeze-dried for analysis. Phytochemical profiling used reversed-phase HPLC with photodiode array detection on a Gemini C18 column. Separate chromatographic methods were employed for alkaloids and catechins, with detection wavelengths chosen to optimise sensitivity for particular β-carbolines and proanthocyanidins. Calibration curves were prepared for markers 1–9 across defined concentration ranges; isolated markers and reference standards (harmol, harmine, harmaline, epicatechin) were used for peak assignment and quantification. Peak purity was evaluated from PDA data and samples were injected in triplicate to assess precision. Standardised pharmaceutical compositions were prepared by combining stock DMSO solutions of key active constituents: tetrahydroharmine (THH, 5), harmaline (6), harmine (7), epicatechin (8) and procyanidin B2 (9) in three different ratios (compositions 1–3) intended to simulate the range observed in natural extracts. B. caapi extract DMSO solutions (10 mg/mL) were used as references. Bioactivity assays comprised in vitro inhibition kinetics against recombinant human MAO-A and MAO-B and a cell-based assay of antioxidant activity. MAO-A IC50 values were determined from five concentrations ranging from 0.0001 to 1 μg/mL; MAO-B inhibitory effects of crude extracts were screened at 100, 10 and 1 μg/mL. Intracellular reactive oxygen species (ROS) generation was measured in HL-60 cells using the DCFH-DA method across multiple concentrations. Most experiments were performed in duplicate (bioassays) or triplicate (HPLC injections) as reported.
HPLC profiling consistently assigned nine marker compounds (banistenoside A and B [1,2], THNH [3], harmol [4], THH [5], harmaline [6], harmine [7], epicatechin [8] and procyanidin B2 [9]) across fresh and dried samples of leaves, stems, bark and large branches. Alkaloid chromatograms showed approximately 10 dominant signals and catechin analysis revealed two main peaks; three additional, not fully characterised peaks with UV spectra consistent with THH analogs were also observed. Peak purity checks showed no gross impurities for the majority of markers. Marker contents were higher in dried material than fresh. Using ASE, leaves and young stems yielded higher marker contents, whereas maceration was more efficient for mature plant material. The dried bark of matured stem/large branch contained the highest concentrations of dominant β-carboline markers and proanthocyanidins: for example, ranges reported included banistenoside A (1) 0.31–0.71%, banistenoside B (2) 0.81–1.93%, harmine (7) 0.28–0.67%, THH (5) 0.04–0.1%, epicatechin (8) 0.43–0.67% and procyanidin B2 (9) 0.16–0.49% in some samples. Whole dried matured stems and debarked stems showed lower but still notable amounts. Harmaline (6) was consistently present only in small amounts (≈0.02–0.05%), and THNH (4) and harmol (3) were minor markers. Commercial stem extracts (BCEx-1 to BCEx-4) displayed assignable peaks for 1–9 but generally contained lower amounts of major markers compared with Da Vine dried materials; epicatechin was an exception in one commercial sample (BCEx-4) which showed higher epicatechin content. In vitro MAO-A inhibition assays showed that pharmaceutical compositions 2 and 3 were the most potent, with IC50 values of 0.027 μg/mL and 0.024 μg/mL respectively. These potencies were comparable to two Da Vine standardised extracts (IC50 0.029 μg/mL and 0.032 μg/mL). Composition 1, containing lower amounts of harmaline and harmine, had weaker MAO-A inhibition (IC50 ≈0.047 μg/mL) similar to a commercial dried stem extract (BCEx-4, IC50 ≈0.059 μg/mL) that had comparable 6 and 7 contents. Antioxidant activity measured in the HL-60 cell assay tracked with amounts of epicatechin and procyanidin B2: compositions 2 and 3 had antioxidant potency similar to a commercial extract containing comparable 8 and 9 levels, whereas whole dried matured stem material and composition 1 exhibited weaker antioxidant effects. Notably, the large branch extract (BCdBS) displayed stronger antioxidant activity than compositions 2 and 3 and BCEx-4 despite having lower measured 8 and 9, suggesting additional antioxidant constituents in that extract.
Wang and colleagues prepared and chemically profiled multiple standardised aqueous extracts of B. caapi using maceration and ASE methods, identifying β-carboline alkaloids and proanthocyanidins as principal bioactive markers. Marker concentrations were highest in dried bark of matured stem/large branch, followed by whole dried stem and debarked stem; dried material generally contained more markers than fresh. The study found that variation in MAO-A inhibitory and antioxidant activities across extracts and prepared compositions correlated with differing phytochemical compositions, particularly levels of THH (5), harmaline (6), harmine (7), epicatechin (8) and procyanidin B2 (9). Pharmaceutical compositions assembled from these active constituents (compositions 2 and 3) produced in vitro MAO-A inhibitory and antioxidant activity comparable to that of Da Vine stem extracts. The authors propose an optimal marker composition for a standardised B. caapi phytopharmaceutical preparation (reported ranges: 1 0.81–3.93%, 2 0.50–10.72%, 5 0.29–3.48%, 6 0.12–0.59%, 7 1.26–3.71%, 8 0.6–5.4% and 9 0.9–7.2%). They note that the two glycosides 1 and 2, though major markers, appeared inactive and warrant further study of their aglycones. Finally, the authors link the observed MAO inhibition and antioxidant properties to traditional uses of B. caapi in ayahuasca preparations and suggest potential relevance for neurodegenerative conditions such as Parkinson’s disease, given the roles of MAO inhibition and oxidative stress in neurodegeneration.
Banisteriopsis (Family: Malpighiaceae) is a tropical South American genus with 92 species distributed mainly in Brazil, Bolivia, Colombia, Ecuador, and Peru. B. caapi (Spruce ex Griseb.) Morton is an ingredient of a popular sacred and psychoactive drink ayahuasca, also known as Caapi, Pinde, Natema or Yaje. It is widely employed for prophecy, divination, and as a sacrament in the northern part of South America. Traditionally, this drink is prepared by boiling the stems of B. caapi and adjuvant plant, either Psychotria viridis (chacruna) or Diplopterys cabrerana (oco yagé). It should be noted that the identities of different Banisteriopsis species are incompletely known due to the paucity of fertile collections and lack of detailed taxonomic study. There are at least thirty different varieties of B. caapi that natives of Amazon have knowledge of and have different uses. Earlier chemical investigation have reported the presence of β-carboline alkaloids harmine, harmaline and tetrahydroharmine (THH) as the principal monoamino oxidase (MAO) inhibitors, together with harmol, harmine N-oxide, harmic acid methyl ester, harmalinic acid, harmic amide, acetylnorharmine, and ketotetrahydronorharmine, from B. caapi. In addition, pyrrolidinesshihunine and (S)-(+)-dihydroshihunine, and terpenoidsstigmasterol, β-sitosterol, ursolic acid, oleanolic acid and nerolidol were also reported. The alkaloid content of B. caapi was determined previously by GC/MS, LC/MSand HPLC, suggesting the content of harmine is highest among βcarbolines, followed by THH and harmaline. Recent investigations at our laboratories on B. caapi Da Vine, a cultivar propagated by cuttingsand collected from Oahu, Hawaii, have yielded two new tetrahydro-β-carboline-derived alkaloidal glycosides namely, banistenoside A (1) and banistenoside B (2) as major markers, a new natural THH analog tetrahydronorharmine (THNH) (3), and four known MAO inhibitors β-carbolines harmol (4), tetrahydroharmine (THH) (5), harmaline (6) and harmine (7). In addition, two potent antioxidant flavan-3-ols (-)-epicatechin (8)and its dimer (-)-procyanidin B2 (9), were isolated for the first time from the genus Banisteriopsis. In this paper, we report the quantification of markers in B. caapi Da Vine, performed by RP-HPLC in different parts of the plant collected in different seasons and/ or different geographical regions, as well as regular/ commercial sample of B. caapi. Based on β-carboline alkaloids (5-7) and proanthocyanidines (8 and 9) compositions of the standardized aqueous extracts of Da Vine a few pharmaceutical compositions were prepared and evaluated for inhibition of MAO-A and antioxidant properties.
The HPLC system consisted of a Model 2695 Alliance Separations Module equipped with a 2996 photodiode array detector, and a computerized data station equipped with Waters Empower 2 software (Waters, Milford, MA). Separation was achieved on a Gemini C18 110Å column (Phenomenex, 150 × 4.6 mm I.D.; 5 μm particle size; Phenomenex Inc., Torrance, CA, USA) and operated at 30 °C. The column was equipped with a 2 cm LC-18 guard column (Phenomenex Inc., Torrance, CA, USA). Plant material was extracted by either Coffee Maker (Mr. Coffee®, ISX-43) or Accelerated Solvent Extractor (Dionex®, ASE-200) using H 2 O as a solvent. Water extracts were freeze-dried using Freeze Dry System (Labconco®, Freezone 4.5). Samples were dried using a Savant Speed Vac Plus SC210A Concentrator. The isolated compounds were visualized by observing the TLC plates under UV light at 254 or 365 nm, followed by spraying separately with Dragendorff's and/or 1% vanillin-H 2 SO 4 spray reagents.
Fresh leaves, stems, and large branches of B. caapi were collected from the island of Oahu, Hawaii, in August and November 2007, and June 2008, as well as from Hilo (Big island), Hawaii, USA, in October, 2007. This plant, a cultivar known as Da Vine, is produced from cuttings of South American mother plant transplanted in Hawaii. A reference specimen was collected from Oahu, and a voucher specimen (HLA # 7835) was deposited at the Herbarium of Harold Lyon Arboretum, University of Hawaii. The collector of the voucher specimen of the plant at Lyon Arboretum was Dr. Kenneth M. Nagata (Accession # L-81.0727; collector # 2789; dated 03/13/1984). The regular B. caapi (mature stems) samples analyzed in this study (BC-Ex-1 -BC-Ex-4) were procured online from different commercial sources and also through NCNPR sources during 2005-2007. All samples used in this work are preserved using the standard procedures for collection, drying, grinding and packaging at the NCNPR.
The detailed methods for the preparation of standardized extracts from stem and other plant parts of B. caapi cultivar Da Vine and regular/ commercial samples were described in our previous report.
The marker compounds 1-9 were isolated and identified from B. caapi cultivar Da Vine as previously described(Fig.). The reference standards of harmol, harmine, harmaline, and (-)-epicatechin were purchased from Sigma-Aldrich (St. Louis, MO), while others were available in our laboratories.
For catechin analysis, the mobile phase consisted of water (A) and acetonitrile (B), both containing 0.1% acetic acid which were applied in the following gradient elution: 92% A/ 8% B hold for 5 min; increased to 22% B in next 35 min. The total run time was 40 min, and flow rate was 0.7 mL/min. The peaks were assigned based on their retention times and UV spectra. Detection wavelength was at 279 nm. The injection volume was 10 μL. For alkaloid analysis, the mobile phase consisted of acetonitrile containing 0.1% acetic acid (X) and 50 mM ammonium acetate (pH = 4.2) (Y), and applied in the following gradient method: 0 -20 min, 5% X/ 95% Y to 13% X/ 87% Y; 20-25 min, 13% X/ 87% Y to 22% X/ 78% Y; 25-30 min, 22% X/ 78% Y to 25% X/ 75% Y. Each run was followed by a 5 min wash with 100% acetonitrile and an equilibration period of 15 min. The flow rate was adjusted to 0.85 mL/min. The injection volume was 10 μL. Detection wavelengths were used at 293 nmandandandAn individual stock solution of the standard compounds (1-9) was prepared at a concentration of 1.0 mg/mL in methanol or methanol/water (50:50, v/v). The calibration curve was prepared at five different concentration levels. The range of the calibration curve was 1.0-500 μg/mL for banistenoside A (1), banistenoside B (2), tetrahydronorharmine (3), tetrahydroharmine () and (-)-epicatechin (8), 5.0-500 μg/mL for (-)-procyanidine B2 (9), and 0.2-100 μg/mL for harmol (4), harmaline (6) and harmine (7), respectively, for HPLC-UV analysis.
Tetrahydroharmine (5) (0.3 mg), harmaline (6) (1.0 mg), harmine (7) (1.0 mg), epicatechin (8) (1.0 mg) and procyanidin B2 (9) (0.6 mg), each separately, were dissolved in appropriate amount of DMSO to make individual stock solutions with concentration 1.5 mg/mL for compounds 5 and 9, and 1.0 mg/mL for 6-8. These stock solutions were combined in appropriate calculated ratio and the total volume of each stock solution was normalized to 0.5 mL by DMSO. This gives the standardized compositions 1-3 with concentrations of 5-9 assigned in the Table. Calculations for composition preparation were based on simulation of natural B. caapi extract (5 mg) to be dissolved in 0.5 mL DMSO giving a stock solution with concentration equivalent to 10 mg/mL. B. caapi extract DMSO solutions (10 mg/mL, 0.5 mL) were used as the references. All prepared DMSO solutions were used as stocks for further MAO-A and antioxidant assays carried out in duplicates.
Recombinant human monoamine oxidase A (MAO-A) and monoamine oxidase B (MAO-B) were obtained from BD Biosciences (Bedford, MA), and the assay procedures used as described previously. The IC 50 values for extracts, pure compounds and reference standards were determined at five concentrations of 1, 0.1, 0.01, 0.001 and 0.0001 μg/mL for MAO-A, whereas for MAO-B, percent inhibitions of all crude extracts were evaluated at three concentrations, 100, 10 and 1 μg/mL. All experiments were carried out in duplicate.
The effect of samples on the generation of intracellular reactive oxygen species (ROS) in myelomonocytic HL-60 cells was determined by the DCFH-DA (2′,7′-dichlorofluorescin diacetate) method (i.e., six concentrations of.91 and 1.95 μg/mL were used), as described previously.
The qualitative and quantitative analyses of isolated markers 1-9 were carried out on a reversed phase C-18 column by HPLC according to published methods for β-carboline alkaloids(using harmine, harmaline, harmol and harmane as standards) and green tea catechins(using catechin and epicatechin as standards). These markers (1-9) were consistently assigned and quantified in fresh and dried samples of young and mature stem, bark, debarked stem, large branch and leaves (Table). B. caapi aqueous extracts demonstrated 10 dominating signals in the HPLC chromatograms for alkaloids, while 2 major peaks were identified in the chromatogram of catechins analysis. For the alkaloid analysis, compounds 1, 2, 4 and 5 (banistenoside A, banistenoside B, THNH, THH) were detectable at 293 nm, whereas a wavelength of 320 nm was more suitable for sensitive monitoring of 3 and 7 (harmol and harmine), and 367 nm for 6 (harmaline). Peaks 1, 2 and 4 (Rt 17.74, 18.91, 22.66 min) were isolated and identified for markers 1, 2 and 4, respectively, together with known biomarkers 3 and 5-7 as peaks 3, 5, 6 and 7.08 min) in the HPLC (Figure). Three additional peaks A, B and C at Rt 16.92, 20.84 and 19.50 were also detected and identified (not isolated) as THH analogs, according to UV spectra (Fig.). In the analysis of catechins, epicatechin (8) and procyanidin B2 (9), with Rt 22.53 and 20.25 min, were detectable at 279 nm (Figure). Two additional peaks at Rt ca 7.0 and 14.0 were also detected but not identified. Initial screening experiment showed that an acidic mobile phase containing acetonitrile (0.1% HOAc) and 50 mM ammonium acetate at a pH 4.2 enhances peak shape and separation of β-carbolines, with a flow rate of 0.85 mL/min, while the two catechins were conveniently eluted using acetonitrile (0.1% HOAc) and Water (0.1% HOAc) with a flow rate of 0.7 mL/min. In both the cases, reversed phase column (Gemini C18, 5 μm, 150 × 4.6 mm) was used and peak purity was confirmed by studying the photodiode array (PDA) data of all peaks of interest; no indication for gross impurities could be found for majority of the markers. All samples (extracts and standards) were injected in triplicate, indicated the precision of the method. The isolated markers (1-9) were employed for chemical profiling of various extracts of B. caapi Da Vine plant parts, collected in different seasons from two different Islands of Hawaii; thereby markers 1-9 were consistently assigned and quantified from aqueous extracts prepared either by maceration or ASE extractor (Table). Compounds 1, 2, 4, 8 and 9 were not reported previously as markers from the genus Banisteriopsis, but being main markers, especially 8 and 9 are indicators of potent antioxidant value of the material, were quantified. The content of all markers was significantly higher in the dried sample compared to the corresponding fresh material. Using ASE extraction method, the leaves and young stems afforded higher yields of extracts with higher markers content, while maceration appears to be more efficient for matured plants. The contents of dominant β-carboline derived markers were significantly higher in dried bark of matured stem/ large branch [banistenoside A (1; 0.31, 0.71%), banistenoside A (2; 0.81, 1.93%), harmine (7; 0.28, 0.67%), THH (5; 0.04, 0.1%), and epicatechin (8; 0.67, 0.43%) and procyanidin B2 (9; 0.49, 0.16%)], followed by dried matured stem/ large branch [1 (0.21, 0.27%), 2 (0.38, 0.57%), 5 (0.15, 0.21%), 7 (0.23, 0.35%), 8 (0.06, 0.17%) and 9 (0.12, 0.62%)]. The bioactive marker harmaline (6) was present in small amounts only (0.02-0.05%), compared to harmine (7), while THNH (4) and harmol (3) were present in these samples as minor markers. The content of the two major markers (1 and 2) was comparable to the total amount of all markers (3-9) in the large branch. The active alkaloid 7 was more predominant in most of the stem extracts, followed by 5 and 6. Incidentally, harmine (7) was reported as the major biomarker in various B. caapi, followed by 5 and 6 (, which was in agreement with our observation. The content of β-carbolines in fresh or dried leaves were much less than the other plant parts. Analysis of regular/ commercial B. caapi stem extracts revealed consistently assignable and quantifiable peaks for 1-9, however, the content of the major markers 1 (0.15, 0.17%), 2 (0.07, 0.15%), 7 (0.04, 0.13%) and 9 (0.03, 0.38%) were found to be relatively less in BCEx-1 and BCEx-4 (Table), compared to corresponding dried matured stem/ large branch of Da Vine; while epicatechin (8) was the only marker present in higher amounts in BCEx-4 (0.25% vs. 0.06-0.17%). No significant variations were found for the same plant parts collected in different season and/or different locations.
The in vitro MAO-A inhibitory and antioxidant activities of the standardized extracts and pharmaceutical compositions, prepared from the active constituents identified and isolated from B. caapi cultivar Da Vine, were evaluated. Three MAO-A inhibitory β-carboline alkaloids, THH (5), harmaline (6) and harmine (7), and two antioxidants, epicatechin (8) and procyanidin B2 (9), were used for the preparation of the standardized compositions. Three compositions were prepared based on optimum range of marker constituents in standardized B. caapi aqueous extracts consisting 5 (0.29-3.48%), 6 (0.12-0.59%) 7 (1.26-3.71%), 8 (0.6-5.4%) and 9 (0.9-7.2%); composition 1 with the lowest concentration of the compounds 5-9, composition 2 with intermediate amounts and composition 3 with the highest amounts of these compounds (Table). Three pharmaceutical compositions together with the standardized extracts of B. caapi (Da Vine) and B. caapi from commercial sources were subjected to in vitro evaluation of MAO-A inhibitory and antioxidant activities. Compositions 2 and 3 showed most potent MAO-A inhibitory activity (IC 50 0.027 μg/mL and 0.024 μg/mL, respectively), which is comparable to two different B. caapi (Da Vine) standardized extracts (IC 50 0.029 μg/mL and 0.032 μg/mL, respectively) (Table). The amount of the two most potent MAO-A inhibitor β-carboline alkaloids harmaline (6) and harmine (7) of the standardized extracts of B. caapi (Da Vine) dried bark of large branch (BCdBS: 0.16% and 3.71%) and whole dried matured stem (BCDVSBig-07-08: 0.48% and 3.66%) was found to be similar to those of compositions 2 (0.36% and 2.49%) and 3 (0.59% and 3.71%). On the other hand, composition 1 with the lowest amounts of 6 and 7 (0.12% and 1.26%, respectively) showed MAO-A inhibitory activities (IC 50 0.047 μg/mL vs. 0.059 μg/mL) similar to commercial B. caapi dried matured stem extract (BCEx-4) with comparable concentrations of 6 and 7 (0.24% and 1.36%). The content of 6 and 7 in the BCEx-4 was found to be lower than the two Da Vine standardized extracts used for comparison. The antioxidant activity of compositions 2 and 3 was found to be equally potent to those of B. caapi commercial extract due to the presence of similar amounts of active antioxidant constituents 8 and 9 (Table). However, B. caapi (Da Vine) whole dried matured stem showed weak antioxidant activity, like composition 1, due to lower amounts of 8 and 9. On the other hand, B. caapi large branch extract (BCdBS) was found to be more potent than compositions 2, 3 and BCEx-4, although they had lower amounts of 8 and 9, suggesting the presence of additional antioxidant constituent(s). Collectively, standardized/ pharmaceutical compositions containing principal bioactive constituents (5-9) demonstrated in vitro MAO-A inhibitory and antioxidant activity generally comparable to those of B. caapi aqueous extracts with corresponding composition of bioactive constituents.
Various standardized extracts of B caapi were prepared, using maceration and ASE extraction methods, to determine high potency of MAO-A inhibitory and antioxidant activities. The predominant chemical and bioactive markers were present in high concentrations in dried bark of matured stem/ large branch, followed by whole dried stem and debarked stem [i.e., with the range of concentration of markers: 1 (0.21-0.71%), 2 (0.38-1.93%), 5 (0.04-0.21%), 7 (0.23-0.67%), 8 (0.06-0.67%) and 9 (0.12-0.49%)], while harmaline (6) was present in small amounts only (0.02-0.05%). The variation of MAO-A inhibitory and antioxidant activities of various standardized extracts (Table) and compositions (Table) may be attributed to their different phytochemical compositions with respect to β-carboline alkaloids (5-7) and proanthocyanidins (8 and 9), respectively. Thus, the low content of key bioactive markers (5, 7 and 9) in regular sample of B. caapi dried stems BCEx-1 (0.1%, 0.04%, 0.03%), compared to those of dried matured stems/ large branch, stem bark and debarked stem of Da Vine, is likely to be responsible for relatively less potent MAO-A inhibitory and antioxidant activities. However, the presence of two inactive major marker glycosides, based on azepino[1,2-a] tetrahydro-β-carboline nucleus (1 and 2), warrants further biological investigation of their aglycones. Collectively, various dried stem extracts demonstrated potent MAO-A inhibitory and antioxidant activity, thereby suggesting an optimal marker composition consisting 1 (0.81-3.93%), 2 (0.50-10.72%), 5 (0.29-3.48%), 6 (0.12-0.59%) 7 (1.26-3.71%), 8 (0.6-5.4%) and 9 (0.9-7.2%) in a standardized B. caapi phytopharmaceutical preparation. Interestingly, pharmaceutical composition 2 and 3, prepared using only active constituents (compounds 5-9) of B. caapi demonstrated similar in vitro MAO-A inhibitory and antioxidant activity compared to those displayed by B. caapi stem extracts (Table). Traditionally, B. caapi is used as an adjuvant plant with Psychotria viridis (chacruna) or Diplopterys cabrerana (oco yage) for the preparation of the scared drink Ayahausca, which is widely used throughout the Amazon as a psychoactive tea for healing and spiritual exploration. It was established that monoamine oxidase (MAO) inhibitory activity of β-carboline alkaloids of B. caapi protects the psychoactive constituent dimethyltryptamine (DMT) of Psychotria viridis or Diplopterys cabrerana from oral degradation (i. e., caused by MAO). Therefore, the presence of different β-carboline alkaloids (3-7) as main chemical markers reaffirms the traditional use of B. caapi as an adjuvant plant for the preparation of Ayahausca. Inhibition of MAO by harmine and harmaline provide protection against neurodegeneration, and has a potential therapeutic value for the treatment of Parkinson's disease. In addition, oxidative stress induced by reactive oxygen species has been strongly associated with the pathogenesis of neurodegenerative disorders, including Alzheimer's diseases. Collectively, these results support traditional uses of B. caapi stem extract in the treatment of PD. Furthermore, this investigation suggested that the standardized extract of B. caapi with assigned composition of markers may be valuable for neurodegenerative diseases due to combined effect of proanthocyanidins (8 and 9) and β-carboline alkaloids (5-7); the former responsible for antioxidative, MAO-B inhibitory activities, and antineurodegenerative effects, while the latter increases the release of dopamine from brain cellsand inhibiting MAO's, thus preventing its breakdown. UV Spectra of standards and unidentified Alkaloids.
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