This study (2010) shows evidence of the potential efficacy of ayahuasca in the treatment of Parkinson’s disease. Several extracts are examined and scored according to the different (expected) effects.
Abstract
“Aim of the study: Parkinson’s disease is a neurological disorder mostly effecting the elder population of the world. Currently there is no definitive treatment or cure for this disease. Therefore, in this study the composition and constituents of the aqueous extract of B. caapi for monoamine oxidases (MAO) inhibitory and antioxidant activities were assessed, which are relevant to the prevention of neurological disorders, including Parkinsonism.
Materials and methods: The aqueous extract of B. caapi stems was standardized and then fractionated using reversed-phase (RP) chromatography. Pure compounds were isolated either by reversed-phase (RP) chromatography or centrifugal preparative TLC, using a Chromatotron®. Structure elucidation was carried out by 1D and 2D NMR, Mass, IR and Circular Dichroism spectroscopy and chemical derivatization. Chemical profiling of the extract was carried out with RP-HPLC. The inhibitory activity of MAO-A, MAO-B, acetylcholinesterase, butyrylcholinesterase and catechol-O-methyl transferase enzymes, as well as antioxidant and cytotoxic activities of both B. caapi extract and isolated compounds were evaluated.
Results: An examination of the aqueous extracts of B. caapi cultivar Da Vine yielded two new alkaloidal glycosides, named banistenoside A (1) and banistenoside B (2), containing “azepino[1,2-a]tetrahydro-β-carboline” unique carbon framework. One additional new natural tetrahydronorharmine (4), four known β-carbolines harmol (3), tetrahydroharmine (5), harmaline (6) and harmine (7), two known proanthocyanidines (−)-epicatechin (8) and (−)-procyanidin B2 (9), and a new disaccharide β-D-fructofuranosyl-(2→5)-fructopyranose (14) together with known sacharose (15) and β-D-glucose (16) were also isolated. In addition, the acetates of 1, 2, 8, 9, 14 and 15 (compounds 10–13, 17, 18) were also prepared. Harmaline (6) and harmine (7) showed potent in vitro inhibitory activity against recombinant human brain monoamine oxidase (MAO) -A and -B enzymes (IC50 2.5 and 2.0 nM, and 25 and 20 µM, respectively), and (−)-epicatechin (8) and (−)-procyanidin B2 (9) showed potent antioxidant and moderate MAO-B inhibitory activities (IC50 <0.13 and 0.57 µg/mL, and 65 and 35 µM). 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. Analysis of regular/commercial B. caapi dried stems showed a similar qualitative HPLC pattern, but relatively low content of dominant markers 1, 2, 7, and 9, which led to decreased MAO inhibitory and antioxidant potency.
Conclusion: Collectively, these results give additional basis to the existing claim of B. caapi stem extract for the treatment of Parkinsonism, including other neurodegenerative disorders.”
Authors: Volodymyr Samoylenko, Md. Mostafizur Rahman, Babu L. Tekwani, Lalit M. Tripathi, Yan-Hong Wang, Shabana I. Khan, Ikhlas A. Khan, Loren S. Miller, Vaishali C. Joshi & Ilias Muhammad
Summary
The aim of this study was to assess the antioxidant and MAO inhibitory activities of B. caapi for the prevention of neurological disorders, including Parkinsonism.
The aqueous extract of B. caapi stems was standardized and then fractionated using reversed-phase (RP) chromatography. The isolated compounds were characterized by 1D and 2D NMR, Mass, IR and Circular Dichroism spectroscopy and chemical derivatization.
Results – Aqueous extracts of B. caapi cultivar Da Vine were used to make two new alkaloidal glycosides, which contained azepino[1,2-a]tetrahydro–carboline unique carbon framework. These compounds showed potent inhibitory and antioxidant activity against recombinant human brain monoamine oxidase (MAO) -A and -B enzymes.
- Introduction
Banisteriopsis caapi is a tropical South American genus with 92 species distributed mainly in Brazil, Bolivia, Colombia, Ecuador, and Peru. It is an ingredient of the popular sacred and psychoactive drink Ayahuasca, which is used for prophecy, divination, and as a sacrament in the northern part of South America.
Parkinson’s disease is caused by a loss of neurons from substantia nigra of the brain, and can be treated with antioxidants as adjuvant with dopamine agonist or MAO inhibitors. B. caapi stem extract contains MAO inhibitor harmine, which can improve motor function in PD patients.
Banisteriopsis caapi has at least thirty different uses in Amazon, and several compounds have been isolated. These include two new -carboline alkaloidal glycosides, a new tetrahydronorharmine, four known -carbolines, two proanthocyanidines, and a new disaccharide.
2.1. General experimental procedures
Optical rotations, IR spectra, and circular dichroism spectra were measured on an AUTOPOL IV® instrument, a Bruker Tensor 27 FTIR instrument, and a Olis DCM 20 CD spectrometer, respectively. A HPLC system was used. The NMR spectra were acquired on a Bruker Avance DRX-400 instrument at 400 MHz (1H), 100 MHz (13C) in CDCl3 or CD3OD, and the multiplicity determinations and 2D NMR spectra were obtained using standard Bruker pulse programs. Plant material was extracted by Coffee Maker or Accelerated Solvent Extractor, freeze-dried, and analyzed by TLC, CPTLC, and flash CC. The isolated compounds were visualized by observing under UV light at 254 or 365 nm, followed by spraying separately with Dragendorff’s and/or 1% vanillin-H2SO4 spray reagents.
2.2. Plant material
Fresh leaves, stems, and large branches of B. caapi cultivar Da Vine were collected from the island of Oahu, Hawaii, and Hilo (Big island), Hawaii, USA, and preserved using the standard procedures for collection, drying, grinding, and packaging at the NCNPR.
Different plant parts of B. caapi were extracted using hot water by two different methods, and the dried material was ground into small pieces before processing.
Water extracts were prepared by ASE-200 using distilled H2O for four times at 100 °C, 3.45 MPa of N2, 30 min. The optimal method was developed and used, giving strong yellow-brown colored water extract.
Based on the MAO-A and antioxidant activity of initial extracts, the most active extracts were identified and bulk extracted using a coffee maker to yield 60 g of dried pale-yellow powder.
2.4. Bioassay-guided fractionation and characterization of isolated compounds
The freeze-dried powdered aqueous extract was subjected to RP-CC over C-18 silica gel, eluted with MeCN, and then evaporated under reduced pressure to obtain fractions A-N. Fraction A was purified by CPTLC, fraction B was purified by CPTLC, fraction D was purified by CPTLC, fraction I was purified by CPTLC, fraction L was purified by RP-flash chromatography, and fraction L-2 was the mixture of fractions L-1 and L-2.
Using the above procedures, 9 compounds were isolated from aqeuous stem extracts of samples collected in 2007 and 2008. They were identified as THH, harmaline, harmine, epicatechin and procyanidin B2, respectively.
The CD data of 8 was in agreement with those recorded for an authentic sample of ()-2R,3R-epicatechin.
()-2R,3R-epicatechin-4,8-()-2R,3R-epicatechin ()-procyanidin B2, whose physical and spectral data were identical to those reported in the literature, was synthesized.
13C NMR spectra are superimposed on each other for C-2, 97.8, 81.3, 76.6, 75.4, 70.4, 69.8, 68.3, 64.5, 62.9, 61.2 and C-1.
Fraction L-2 was acetylated with acetic anhydride and then dried under vacuum to give the solid residue, which was subjected to CPTLC and yielded 10 and 18, together with fraction L-2-1.
Banistenoside A heptaacetate (10), a colorless solid, is a derivative of banistenoside A, a cyanogenic glycoside.
Banistenoside B heptaacetate (11) is a colorless solid with a molecular weight of 805.2693 (MH) and 827.2518 (MNa).
Acetylation of ()-epicetechin (8) was performed in pyridine and acetic anhydride was added dropwise to the solution while stirring. The solid residue was dried under vacuum and purified by flash chromatography over silica gel.
Fraction H-1 was treated with acetic anhydride and dried under vacuum to give acetates 13 and 17.
()-Procyanidin B2 acetate (13) is a colorless solid with properties similar to those reported in the literature. It has a molecular weight of 1016.2859 (MNH4), 1021.2346 (MNa), and 1037.2160 (MK).
2.5. 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 obtained from BD Biosciences and used in the kynuramine deamination assay to determine the effect of extracts on MAO-A and MAO-B. The IC50 values for extracts, pure compounds and reference standards were determined at five concentrations. The reactions were carried out in 0.1 M potassium phosphate buffer at pH 7.4, with an inhibitor dissolved in DMSO or in buffer (if not dissolved in DMSO). The formation of 4-hydroxyquinoline was determined fluorometrically by SpectraMax M5 fluorescence plate reader.
2.6. In vitro cytotoxicity assay
The in vitro cytotoxic activity of SK-MEL was determined against four human cancer cell lines, monkey kidney fibroblasts and pig kidney epithelial cells. The IC50 values were determined from dose response curves of percent growth inhibition against test concentrations.
2.7. Cell Based Assay for Antioxidant Activity (Rosenkranz et al., 1992; Scudiero et al., 1988)
The effect of samples on the generation of ROS in Myelomonocytic HL-60 cells was determined by the DCFH-DA (2′,7′-dichlorofluorescin diacetate) method. Vitamin C and trolox were used as the positive controls in each assay. Non-fluorescent DCFH-DA diffuses into cells, where cytoplasmic esterases hydrolyse the DCFH-DA to DCFH, which is then oxidized by ROS in HL-60 cells to 2′,7′-dichlorofluorescein. The cytotoxicity of the test materials was also determined by XTT method.
- Results and discussion
A bioassay-guided fractionation of crude aqueous extract of B. caapi revealed potent antioxidant and moderate MAO-B inhibitory activities, and two new alkaloidal glycosides, as major markers, and four known MAO inhibitors, CA’s 3 and 5 – 7.
3.1. Structure elucidation
Compound 1 was analyzed by ESIHRMS and showed absorption bands at 226, 269 and 290 nm, typical of THH chromophore, and IR bands at 3550 and 1740 cm1, attributed to hydroxyl and amide groups, respectively.
The 13C NMR spectrum of compound 2 showed the presence of a glucose moiety, a methoxyl group, a THH base skeleton, four oxymethine carbons and a quaternary amide carbonyl. The presence of amide carbonyl and hydroxyl groups was suggested from the IR spectrum.
Acetylation of a fraction containing mixture of 1 and 2 afforded heptaacetates 10 and 11, which were subsequently separated and purified by CPTLC. Compound 10 was found to be levorotatory, and its 1H and 13C NMR spectra displayed signals for seven acetyl groups, a methoxyl and a glucose moiety. The 13C and 1H NMR spectra suggested the presence of tetra-O-acetyl–D-glucose moiety, and a 2D NMR COSY spectrum showed correlations between four acetoxy protons at h-10, h-11, h-12 and h-135.72, suggesting the structure of compound 10 was confirmed by a 2D NMR HMBC experiment.
Compound 11 was also dextrorotatory, had seven acetyl groups, and was fused with a seven-member ring via five oxygenated carbons C-10 – C-14. Its 1H and 13C NMR spectra were similar to those of acetate 10, except for significant shielding of protons H-1, H-10 and H-12. The structure of ring A-C was established by HMBC experiments, which revealed 2J and 3J-correlation of protons and carbons, and a free indole N-H proton and C-7-OMe group.
The stereochemistry of C-1 stereocenter of compound 10 and 11 was established by Circular Dichroism spectra and NOESY correlations. The CD spectra displayed a high amplitude strong positive Cotton effect in the 310 – 320 nm regions.
The 1H NMR spectrum of 10 showed small coupling constant (J = 4.3 Hz) compared to 11 (J = 10 Hz), suggesting cis- and trans-relationships between H-1 and H-10 protons in these two compounds, respectively. The spatial orientation of the relevant protons was confirmed by NOESY spectrum.
NOESY spectra of 11 showed correlations between H-1, H-11 and H-13, as well as H-11 and H-12, suggesting these protons are cis and placed at the -face of the molecule, and the structures of banistenoside A (1) and banistenoside B (2) were confirmed.
Compound 4 was isolated as gum and analyzed for C12H14N2O by ESIHRMS. The 1H and 13C NMR spectra of 4 were generally similar to those observed for THH (5), except for the differences associated with the presence of a methylene group at C-1.
Compound 14 was isolated as gum and homogenous on TLC. Its 13C NMR spectrum was generally similar to those observed for fructan-type disaccharide with two fructofuranosyl units, but had a nonsymmetrical dimmer, instead of a symmetrical dimmer. Compound 17 was found to be dextrorotatory and had 8 acetyl groups, 12 carbon signals, 4 triplets, 6 doublets and 2 singlets. Its structure was fully substantiated by 2D NMR COSY, HMQC, HMBC and NOESY experiments on 14-Ac (17).
Physical and spectroscopic data were used to determine the structures of harmol (3), THH (5), harmaline (6), harmine (7), ()-2R,3R – epicatechin (8) and ()-2R,3R – epicatechin-48-()-2R,3R – epicatechin (procyanidin B2) (9) as well as their corresponding peracetate 12 and 13.
3.2. Biological activity
The hot aqueous extracts of fresh and dried large branches of B. caapi and isolated compounds 1 – 9 were evaluated for MAO inhibitory and antioxidant activities, as well as cytotoxicity against selected human cancer and mammalian (VERO) cells.
Fluorescence interference was used to measure MAO-B and MAO-A inhibitory activities. The MAO-A activity of extracts of fresh/ dried stem/ bark/ debarked large branch was 2500-fold more potent than MAO-B, and the activity of most potent MAO-A inhibitors harmine (7) or harmaline (6) was >10,000-fold more potent than MAO-B.
Large branches of B. caapi showed strong antioxidant activity, and were also found to be able to inhibit the formation of Reactive Oxygen Species (ROS) and show cytotoxic activity against human breast, melanoma, skin and prostate cancer, and mammalian VERO and kidney cell lines.
3.3. HPLC analyses and quantification
The isolated markers 1 – 9 were employed for chemical profiling of B. caapi Da Vine, and the results showed that most of the dominant chemical and bioactive markers were present in high concentrations in dried bark of large branch.
- Conclusion
This appears to be the first report of banistenoside A and banistenoside B, containing an unique “azepino[1,2-a]tetrahydro–carboline” carbon framework, either from natural or synthetic sources. These compounds are biogenetically analogous to mitragynine-type alkaloid dihydrocadambine, and may have potential hallucinogenic or behavioral properties.
The presence of two potent antioxidants, ()-epicatechin (8) and ()-procyanidin B2 (9), in B. caapi stem extract provides significant added value for the protection of neuronal cells damage by oxidative free radicals, and has a potential therapeutic value for the treatment of Parkinson’s diseases.