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.
Abstract
“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.”
Authors: Yan-Hong Wang, Volodymyr Samoylenko, Babu L. Tekwani, Ikhlas A. Khan, Loren S. Miller, Narayan D. Chaurasiy, Mostafizur Rahman, Lalit M. Tripathi, Shabana I.
Khan, Vaishali C. Joshi, Frank T. Wigger & Ilias Muhammad
Summary
Banisteriopsis caapi, a woody vine from the Amazonian basin, is used for alleviating symptoms of neurological disorders including Parkinson’s disease.
The aim of this study was to develop a process for preparing standardized extracts of B. caapi to achieve high potency for inhibition of human monoamine oxidases (MAO) and antioxidant properties.
Reversed-phase HPLC with photodiode array detection was used to profile the alkaloidal and non-alkaloidal components of B. caapi.
HPLC analysis of B. caapi dried stems showed that the main active constituents were present in high concentrations in the dried bark of large branches. This led to increased MAO inhibitory and antioxidant potency compared to B. caapi Da Vine.
The ethnopharmacological use of bark and stem of B. caapi is supported by the results obtained in this investigation. Several compounds, including harmine, harmaline and tetrahydroharmine, have been identified as having antioxidant effects.
- Introduction
Banisteriopsis caapi is an ingredient of ayahuasca, a popular sacred and psychoactive drink in the northern part of South America. There are at least thirty different varieties of B. caapi that natives of Amazon have knowledge of and have different uses. Earlier chemical investigation has reported the presence of harmine, harmaline and tetrahydroharmine (THH) as monoamino oxidase (MAO) inhibitors, along with harmol, harmine N-oxide, harmic acid methyl ester, harmalinic acid, acetylnorharmine, and ketotetrahydronorharmine, from B. caapi.
Recent investigations on B. caapi Da Vine, a cultivar propagated by cuttings and collected from Oahu, Hawaii, have yielded two new tetrahydro-carboline-derived alkaloidal glycosides, a new natural THH analog, and four known MAO inhibitors. In addition, two potent antioxidant flavan-3-ols and its dimer were isolated.
2.1. General experimental procedures
A HPLC system was used to separate compounds from plant material using a Gemini C18 110 column and a Waters Empower 2 software. The compounds were visualized by observing the TLC plates under UV light at 254 or 365 nm.
2.2. Plant material
Fresh leaves, stems, and large branches of B. caapi were collected from Oahu, Hawaii, as well as Hilo (Big island), Hawaii, USA, in August and November 2007, and June 2008, respectively. A reference specimen was collected from Oahu, and a voucher specimen was deposited at Lyon Arboretum.
2.4. Compounds and reference samples
The elution gradient was 92% A/8% B hold for 5 min, increased to 22% B in next 35 min, and flow rate was 0.7 mL/min.
Alkaloid analysis was performed using a gradient method with acetonitrile containing 0.1% acetic acid and 50 mM ammonium acetate (pH = 4.2) and a flow rate of 0.85 mL/min.
The calibration curve was prepared at five different concentration levels for banistenoside A, banistenoside B, tetrahydronorharmine (3), tetrahydroharmine (5) and ()-epicatechin (8), and ()-procyanidine B2 (9), respectively, for HPLC-UV analysis.
2.6. Preparation of standardized compositions
Tetrahydroharmine, harmaline, harmine, epicatechin, and procyanidin B2 were dissolved in DMSO to make individual stock solutions. These stock solutions were combined in appropriate calculated ratio to make standardized compositions 1-3 with concentrations of 5-9 assigned in the Table 2.
2.7. Inhibition kinetics assay using recombinant human MAO-A and MAO-B
Recombinant human monoamine oxidase A (MAO-A) and monoamine oxidase B (MAO-B) were used in the assay. The IC50 values for extracts, pure compounds and reference standards were determined at five concentrations.
2.8. Cell Based Assay for Antioxidant Activity
The effect of samples on the generation of intracellular reactive oxygen species (ROS) was determined by the DCFH-DA method.
3.1. HPLC Analyses and Quantification
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 -carboline alkaloids and green tea catechins. The aqueous extract of B. caapi demonstrated 10 dominating signals in the HPLC chromatograms for alkaloids, while 2 major peaks were identified in the chromatogram of catechins. Several additional peaks were detected but not identified. Initial screening experiment showed that -carbolines were best separated using an acidic mobile phase containing acetonitrile and ammonium acetate at pH 4.2, and two catechins were best separated using acetonitrile and water at pH 4.2.
Markers 1-9 were used to profile various extracts of B. caapi Da Vine plant parts, and were consistently assigned and quantified from aqueous extracts prepared either by maceration or ASE extractor. The content of all markers was significantly higher in the dried sample compared to the fresh material. The content of the major bioactive markers was higher in the matured stem/ large branch, followed by the leaves and young stems.
Analysis of regular/ commercial B. caapi stem extracts revealed that the content of the major markers 1-9 was consistently lower in BCEx-1 and BCEx-4 compared to corresponding dried matured stem/ large branch of Da Vine.
3.2. In vitro MAO-A Inhibitory Activity of Standardized Extracts and Pharmaceutical Compositions of β-Carboline Alkaloids and Proanthocyanidines
The in vitro MAO-A inhibitory and antioxidant activities of B. caapi standardized extracts and pharmaceutical compositions were evaluated. Three compositions were prepared based on optimum range of marker constituents in standardized B. caapi aqueous extracts.
Three pharmaceutical compositions were evaluated for MAO-A inhibitory and antioxidant activities. Compositions 2 and 3 showed most potent MAO-A inhibitory activity, and the amounts of the two most potent MAO-A inhibitor -carboline alkaloids harmaline (6) and harmine (7) in the standardized extracts of B. caapi (Da Vine) were similar to those of compositions 2 and 3. Compositions 2 and 3 were equally potent to B. caapi commercial extract, but B. caapi whole dried matured stem showed weak antioxidant activity, while B. caapi large branch extract was more potent than compositions 2, 3 and BCEx-4.
- Conclusion
Various standardized extracts of B caapi were prepared to determine high potency of MAO-A inhibitory and antioxidant activities.
The MAO-A inhibitory and antioxidant activities of various standardized extracts and compositions of B. caapi may be attributed to their different phytochemical compositions with respect to -carboline alkaloids and proanthocyanidins, respectively. However, the presence of two inactive major marker glycosides warrants further biological investigation.
B. caapi is used as an adjuvant plant for the preparation of Ayahausca, a psychoactive tea used throughout the Amazon as a healing and spiritual exploration tea.
The antidegenerative effects of B. caapi stem extract may be due to its antioxidative, MAO-B inhibitory, and antineurodegenerative effects, and its -carboline alkaloids, which increase the release of dopamine from brain cells and inhibit MAO’s, thus preventing its breakdown.