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New electrochemical method for the determination of β-carboline alkaloids, harmalol and harmine, in human urine samples and in Banisteriopsis caapi
Microchemical Journal 118 (2015) 95–100
Contents lists available at ScienceDirect
Microchemical Journal journal homepage: www.elsevier.com/locate/microc
New electrochemical method for the determination of β-carboline alkaloids, harmalol and harmine, in human urine samples and in Banisteriopsis caapi Dalibor Stanković a,⁎, Eda Mehmeti b, Lubomir Svorc c, Kurt Kalcher b a b c
Innovation Centre Faculty of Chemistry, University of Belgrade, Studentski trg 12-16, Belgrade, Serbia Institute of Chemistry — Analytical Chemistry, Karl-Franzens University Graz, A-8010 Graz, Austria Institute of Analytical Chemistry, Faculty of Chemical and Food Technology, Slovak University of Technology in Bratislava, SK-812 37, Bratislava, Slovak Republic
a r t i c l e
i n f o
Article history: Received 12 June 2014 Received in revised form 20 August 2014 Accepted 20 August 2014 Available online 27 August 2014 Keywords: Harmalol Harmine Banisteriopsis caapi Differential pulse voltammetry
a b s t r a c t In this paper a new electrochemical method for determination of the β-carboline alkaloids, harmalol and harmine, using differential pulse voltammetry is presented. Experimental parameters such as pH, scan rate, pulse amplitude and pulse time were optimized to characterize their electrochemical behavior and to find best analytical conditions. Thus, in the presence of ascorbic acid, uric acid, dopamine, the developed method exhibits excellent performance for the determination of harmalol and harmine for a linear concentration range between 1–50 μM and 1–75 μM with a detection limit of 0.6 μM and 0.2 μM, respectively. The proposed method was successfully applied for the determination of these alkaloids in a sample of an Ayahuasca liana (Banisteriopsis caapi) and in model human urine samples with good recoveries. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The β-carboline alkaloids, harmalol and harmine (Fig. 1.) are present in a great variety of medicinal plants and they are also endogenously produced in human and animal tissues as a product of secondary metabolism [1]. They possess diverse biological properties due to their capability to bind to benzodiazepine or imidazoline receptors, such as cardiovascular actions, hypotensive, hallucinogenic or antimicrobial actions and tremorogenesis [2,3]. The β-carboline alkaloids have been of interest of South-American ethnic tribes due to their psychotropic properties. These compounds affect the content of neurotransmitters by strong reversible inhibition of monoamine oxidase and the inhibition of acetylcholinesterase [4–6]. The biological significance of β-carbolines seems to be manifested also in cytotoxic as well as neuroprotective properties. Since they are structurally similar to 1-methyl-4-phenyl-11,2,3,6-tetrahydropyridine (MTPT) [7], which induces a Parkinsonianlike syndrome in animals, they have been postulated to act as endogenous neurotoxins. However, β-carbolines protect neurons against the exocytotoxic effects of dopamine and glutamate [8], and display a protective effect on oxidative neuronal damage through a scavenging action on reactive oxygen species [9–12]. They show effective antioxidant properties. Moreover, they have a significant protective effect against H2O2 and paraquat-induced oxidative agents in yeast cells, ⁎ Corresponding author. E-mail address: [email protected] (D. Stanković).
http://dx.doi.org/10.1016/j.microc.2014.08.007 0026-265X/© 2014 Elsevier B.V. All rights reserved.
and their ability to scavenge hydroxyl radicals contributes to their antimutagenic and antigenotoxic effects. β-carbolines exhibit cytotoxicity with regards to HL60 and K562 leukemia cell lines [13–17]. Natural sources of these alkaloids are different organisms, most of which are plants, most notably the Middle Eastern plant harmal or Syrian rue (Peganum harmala) and the South American vine Banisteriopsis caapi, also known as yage or ayahuasca. Recently, there are various techniques for the analysis of carboline alkaloids in different biological samples such as HPLC with electrochemical [18], photodiode array (DAD) [19] or fluorescence [20,21] detection as well as GC–MS [20–22], HPLC–MS and CE–MS [23]. Most of these methods require several time-consuming manipulation steps, sophisticated instruments and special training of the operating personnel. Some recent electrochemical studies on alkaloids showed that boron-doped diamond can be a useful electrode material for their voltammetric determination [24], nevertheless glassy carbon represents an inexpensive alternative, supposed that the oxidation potentials are not too high, which has also the advantage of allowing simple and effective cleaning of its surface, i.e., polishing. Differential pulse voltammetry (DPV) is one of the most sensitive electrochemical techniques for the determination of traces of numerous compounds because of its remarkably low detection limit. Other advantageous features of DPV include relatively low cost instrumentation and the capability for simultaneous determination. The aim of this work was to develop a new, simple, inexpensive and sensitive method for determination of harmalol and harmine. Another aspect of the new method was its successful application to the
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D. Stanković et al. / Microchemical Journal 118 (2015) 95–100
Fig. 1. Structure of harmalol (A) and harmine (B).
determination of harmalol and harmine in urine samples and B. caapi with ascorbic acid (AA), dopamine (DA), and uric acid (UA) as most common interferences.
The liana B. caapi was treated according the procedure described in [25], which can be summarized shortly as: 2 g of dried and powdered seeds of P. harmala was macerated four times with 50 ml of methanol at 50 °C for 1 h. The extracts were combined and evaporated to dryness. The residue was dissolved in 50 ml HCl (2%) then filtered. The filtrate was extracted two times with 20 ml petroleum ether. The aqueous acid layer was basified and extracted four times with 50 ml chloroform. The chloroform layer was combined and evaporated to dryness, then residues were dissolved in 25 ml methanol. An aliquot (100 μL) of the prepared extract was added to 9.9 mL of supporting electrolyte. 3. Results and discussion
2. Experimental 2.1. Apparatus, solvents and reagents Water was purified with a cartridge purification system (Milli-Q); it had a resistivity of at least 18 MΩ cm and was used for preparing all solutions in use. Harmalol, harmine, ascorbic acid, uric acid, dopamine, boric acid, sodium hydroxide, acetic acid and phosphoric acid were purchased from Sigma Aldrich (analytical grade) and used as received without any further purification. Stock solutions (10− 3 M) of the analytes were prepared in water. Calibration standard solutions were prepared from the stock solutions by appropriate dilution with supporting electrolyte. Britton–Robinson buffer solutions (BRBS) were prepared by mixing of phosphoric acid, acetic acid and boric acid (all at 40 mM) and adjusting the pH with sodium hydroxide (0.2 M) to the desired value. All pH values were measured with a pH meter (model 1234, Orion). Cyclic voltammetric (CV) and differential pulse voltammetric measurements were performed using an electrochemical system PalmSens (Netherlands). The cell (10 mL) contained a three-electrode system, a glassy carbon working electrode, an Ag/AgCl (saturated KCl) reference electrode and a Pt counter electrode. All potentials in this paper are reported as values against the Ag/AgCl reference electrode at an ambient temperature. Prior to each experiment, the working electrode was polished with alumina (grain size 0.05 μm) followed by rinsing with water and sonication. The potential was swept over the range from 0 to 1.5 V (vs. Ag/AgCl) at different scan rate for CV, and from 0 to 1.3 V vs. (Ag/AgCl) in DPV mode with the optimized parameters (scan rate 25 mV/s, pulse amplitude 30 mV, step potential 5 mV and pulse width 40 ms). 2.2. Sample preparation The urine samples were collected from two different volunteers; to avoid in vivo drug administration they were spiked with stock solution of the alkaloids. Each 0.1 mL of fresh urine sample was taken and diluted to 10 mL of Britton–Robinson buffer at pH 6 and then directly analyzed.
3.1. Characterization Since the pH is one of the most important factors in the electrochemical behavior of organic compounds, its influence on the peak potential and peak current of investigated alkaloids was examined with cyclic voltammetry. Both, harmalol and harmine, give electrochemical responses in CV which is strongly pH dependent. Oxidation of harmalol occurs at around 0.5 V with a much smaller signal at 0.8 V, whereas the oxidation of harmine proceeds at 0.9 V. With increasing of pH of the electrolyte solution, the peak potential of the two alkaloids shifted linearly to less positive value, with a slope of − 0.365 and − 0.882 V/pH for harmalol and harmine, respectively (Fig. 2A and B). The shape of the peak potential dependence on pH were in good agreement with previously found pKa values for harmine (pKa1 8.0 and pKa2 14.4) and for harmalol (pKa1 8.6 and pKa2 11.3) respectively [25]. These results and the slopes of the peak potential dependence on pH indicate that protons participate in the oxidation reaction, but it is difficult to conclude from these data the ratio between protons and transferred electrons, because they deviate strongly from ideal Nerstian values. The proposed overall mechanism of the electrode reaction of both investigated alkaloids is given in Scheme 1. Oxidation is irreversible for both analytes. From Fig. 2 can be concluded that pH 6 is the most suitable pH for the determination of harmalol and harmine. At pH 6 both alkaloids show good quasi-linearity of the peak current on the square root of the scan rate (R2 = 0.984 for harmine and R2 = 0.973 for harmalol) (Fig. 3) which indicates that the mass transport is mainly controlled by diffusion. Nevertheless, the trend of data in both cases is not ideally linear but exhibits some slight curvature which, in combination with non-zero intercepts on the current axis, might suggest some other effects to contribute to the generation of the current. The signals provide good potential separation with a peak-to-peak value of 0.45 V which is sufficient for their possible simultaneous determination. 3.2. Optimization of DPV parameters The DPV method was applied as sensitive voltammetric method to investigate the relation between the peak current and concentration
Fig. 2. Cyclic voltammograms of harmalol (A) and harmine (B) at pH 6 in BRBS on a glassy carbon electrode with a scan rate of 100 mV/s; inserts: effect of pH on the peak current (▲) and the peak potential (■).
D. Stanković et al. / Microchemical Journal 118 (2015) 95–100
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Scheme 1. Proposed electrode reactions for A) harmine and B) harmalol under optimized electrochemical parameters.
of harmalol and harmine (calibration curves). The corresponding voltammetric parameters such as pulse amplitude, pulse time and scan rate were optimized by changing one parameter while the others were kept constant. Both alkaloids behaved almost identical,
therefore, the optimization process can be described independent on the analyte. When varying the pulse time the peak currents increased from 10 to 40 ms and decreased again with higher values, so the most suitable peak
Fig. 3. Dependence of the peak current on the square root of the scan rate for harmalol (A) and harmine (B).
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Fig. 4. DP voltammograms for different concentrations of harmine from 1 to 75 μM (A) and harmalol from 1 to 50 μM (B); supporting electrolyte: BRBS pH 6; scan rate 25 mV/s; pulse time 40 ms; pulse amplitude 30 mV; glassy carbon electrode.
Fig. 5. A) Dependence of the DPV peak current on the concentration of harmine (A) and of harmalol (B); experimental parameters as in Fig. 4.
current was chosen as 40 ms. When increasing the pulse amplitude from 10 to 75 mV the peak current increased accompanied with a widening of the peak width at the same time. The best peaks with respect to shape and height were obtained for a pulse amplitude of 30 mV. A scan rate of 25 mV/s was chosen as the best value with respect to peak height and analysis time. 3.3. Calibration curves and analytical applications Calibration plots for the simultaneous determination of harmalol and harmine were established in DPV mode applying the optimized experimental parameters. The DP voltammograms for different concentrations of harmalol and harmine and calibration curves are presented
in Fig. 4. As can be seen, evident partly from the CVs already, the current in differential pulse mode is not made by a simple single oxidation, but seems to consist of two overlapping peaks for each alkaloid. The evaluation of the peak current was done by a tangent fit and enumeration of the highest current in the tangent range. As a consequence of the more complex oxidation behavior of the alkaloids, both showed two linear ranges for the calibration curves (Fig. 5). Both, harmine and harmalol yielded a primary linear relation between current and concentration from 1 to 5 μM the corresponding equations and correlation coefficients of I = 0.004 + 0.076C, R2 = 0.9972 for harmine and I = − 0.021 + 0.084C, R2 = 0.9865 for harmalol, where C is concentration in μM and I the current in μA. The second range for higher concentrations extends to 75 μM (harmine) and 50 μM (harmalol) and can be described by I = 0.296 + 0.015C (R2 = 0.9927, with the same meaning of variables as above) and I = 0.094 + 0.054 C (R2 = 0.9969) for harmine and harmalol, resp. At concentrations higher than listed the currents level off. The detection limits were 0.6 μM for harmalol and 0.2 μM for harmine based on a ratio three times signal to background noise. The repeatability was evaluated as 2.3% and 1.9% RSD from 5 repetitive measurements of 25 μM of each, harmine and harmalol. From the results it can be concluded that the proposed method provides good sensitivity and repeatability for the determination of harmalol and harmine. Table 1 Determination of harmalol and harmine in Banisteriopsis caapi.
Fig. 6. DP voltammograms for determination of harmalol and harmine in urine sample, experimental parameters as in Fig. 4.
Sample
Harmalol
Harmaline
Harmine
Banisteriopsis caapi
Not expected [27] not found
0.03–0.83% expected [27]
0.31–8.6% expected [27] 0.6% found
D. Stanković et al. / Microchemical Journal 118 (2015) 95–100 Table 2 Determination of alkaloids in urine samples. Sample
Harmine added (μM)
Harmalol added (μM)
Harmine found (μM)
Harmalol found (μM)
Recovery of harmine (%)
Recovery of harmalol (%)
1 1 2
5.00 20.00 5.00
5.00 20.00 5.00
4.99 19.70 5.18
5.38 22.14 5.26
99.8 98.5 103.6
107.6 110.7 105.3
99
was evaluated with recovery experiments. As can be seen from the figure the proposed method enables the determination of both alkaloids in the presence of some interfering compounds with satisfactory recovery values. The signal of harmalol is superimposed on a matrix signal of the urine, but still can be evaluated with sufficient precision by the tangent fit method. These results confirm that the developed method is also suitable for determination of harmalol and harmine in urine samples. (See Table 1.)
3.4. Interferences
Fig. 7. DP voltammograms of different concentrations of harmalol and harmine in the presence of 50 μM of each, ascorbic acid, dopamine and uric acid; experimental parameters as in Fig. 4.
The application of the new method in biological samples was one of the primary requirements for its validation. The concentrations were estimated by DPV from the calibration curves for the proposed method. B. caapi sample was prepared as previously described [26]. The concentration of harmalol and harmine was determined after addition of 100 μL of the sample to 10 mL of supporting electrolyte. It was found that the concentration of harmine was in good agreement with literature data [27]. The proposed method was successfully applied to urine analysis of harmalol and harmine without any previous preparation (Fig. 6, Table 2). Generally, after drug intake some unmetabolized amounts usually secrete in the patient's urine. Standard additions of 5 and 10 μM of harmalol and harmine each caused current increments at the expected potentials allowing quantitative estimation. The accuracy
Perhaps the main characteristics of every method for the determination of significant analytes in biological samples lie in its inherent selectivity. Losing of neurons from the substantia nigra of the brain causes Parkinson's disease and when damaged, these neurons stop producing dopamine and compromise the brain's ability to control movement. The reason for this can be that free radicals and toxic particles normally deactivated in the body are responsible, which can be controlled by antioxidants. B. caapi was established for alleviating these symptoms, which contains β-carboline alkaloid inhibitors as active constituent used for treatment of Parkinson's disease [28,29]. As ascorbic acid is known as a major antioxidant in the human body, influence on the determination of harmalol and harmine of ascorbic acid, dopamine and uric acid, which often may coexist in the body fluids, was investigated. Fig. 7. shows the DP voltammograms of different concentrations of harmalol and harmine in the absence and in the presence of ascorbic acid, uric acid and dopamine in a concentration of 50 μM. As can be seen the peak current for the oxidation of both alkaloids increases with increasing concentration whereas the response of the interferents remains almost constant. Fig. 8A and B shows the interferences between harmalol and harmine themselves. The concentration of one of the alkaloids was kept constant while the concentration of the other was varied. The peak current of harmalol increased with increasing its concentration while the peak current of harmine remained constant when its concentration was constant; the resultant calibration curve of harmalol had the same characteristics as in the absence of harmine. In the case where the concentration of harmalol was constant the peak current of harmine increased with increasing concentration and had same linear ranges as without harmalol. This study finally proved that both alkaloids can be determined simultaneously with this new method even in the presence of higher concentrations of major interferents present in human samples, namely ascorbic acid, uric acid and dopamine.
Fig. 8. DP voltammograms of different concentrations of harmine (in the presence of 25 μM of harmalol, A) and harmalol (in the presence of 25 μM of harmine, B); experimental parameters as in Fig. 4.
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4. Conclusions In this study a new analytical method for the determination of harmalol and harmine was developed using a glassy carbon electrode. Cyclic voltammetry was used for the characterization of the electrochemical behavior of both alkaloids, and DPV for their quantification. The proposed method is simple, inexpensive and rapid for the determination of the target analytes compared to other analytical techniques previously described in the literature. The practical utility was successfully demonstrated in the analysis of real samples of the ayahuasca liana B. caapi and of model human urine samples. There was no influence of compounds such as ascorbic acid, uric acid or dopamine which might be present in body fluids. Based on this fact, the proposed method offers fast, low cost, simple, selective and sensitive possibilities for analysis of these alkaloids in biological samples without any complex pretreatment of samples. Acknowledgments This work was supported by the Ministry of Education and Science of the Republic of Serbia (project no. OI 172030) and the Grant Agency of the Slovak Republic (grant no. 1/0051/13). E.M. acknowledges financial support from the Austrian government. References [1] T.G. Bidder, D.W. Schomaker, H.S. Boetger, H.E. Evans, J.T. Cummins, Harmane in human platelets, Life Sci. 25 (1979) 157–164. [2] J. Lutes, J.F. Lorden, M. Beales, G.A. Lotmans, Tolerance to the tremorogenic effects of harmaline: evidence for altered olivo-cerebellar function, Neuropharmacology 27 (1988) 849–855. [3] Y.B. Guan, E.D. Louis, W. Zheng, Toxicokinetics of tremorogenic natural products, harmane and harmine, in male Spraqe–Dawley rats, J. Toxicol. Environ. Health A 64 (2001) 645–660. [4] X.Y. Zheng, Z.J. Zhang, G.X. Chou, T. Wu, X.M. Cheng, C.H. Wang, et al., Acetylcholinesterase inhibitive activity-guided isolation of two new alkaloids from seeds of Peganum nigellastrum buNge by an in vitro TLC-bioautographic assay, Arch. Pharm. Res. 32 (2009) 1245–1251. [5] X.Y. Zheng, L. Zhang, X.M. Cheng, Z.J. Zhang, C.H. Wang, Z.T. Wang, Identification of acetylcholinesterase inhibitors from seeds of plants of genus Peganum by thin-layer chromatography–bioautography, J. Planar Chromatogr. Mod. TLC 24 (2011) 470–474. [6] H. Kim, S.O. Sablin, R.R. Ramsay, Inhibition of monoamine oxidase A by betacarboline derivatives, Arch. Biochem. Biophys. 337 (1997) 137–142. [7] M.J. Schwarz, P.J. Houghton, S. Rose, P. Jenner, A.D. Lees, Activities of extract and constituents of Banisteriopsis caapi relevant to parkinsonism, Pharmacol. Biochem. Behav. 75 (2003) 627–633. [8] R. Albores, E.J. Neafsey, G. Drucker, J.Z. Fields, M.A. Collins, Mitochondrial respiratory inhibition by N-methylated beta-carboline derivatives structurally resembling N-methyl-4-phenylpyridine, Proc. Natl. Acad. Sci. U. S. A. 23 (1990) 9368–9372. [9] P. Maher, J.B. Davis, The role of monoamine metabolism in oxidative glutamate toxicity, J. Neurosci. 20 (1996) 6394–6401.
[10] S.L. Lee, E.S. Han, Y.Y. Jang, J.H. Han, H.W. Ha, D.E. Kim, Protective effect of harmalol and harmaline on MPTP neurotoxicity in the mouse and dopamineinduced damage of brain mitochondria and PC12 cells, J. Neurochem. 2 (2000) 521–531. [11] D.H. Kim, Y.Y. Jang, E.S. Han, C.S. Lee, Protective effect of harmaline and harmalol against dopamine and 6-hydroxydopamine- induced oxidative damage of brain mitochondria and synaptosomes, and viability loss of PC12 cells, Eur. J. NeuroSci. 13 (2001) 1861–1872. [12] T.H. Park, O.S. Kwon, S.Y. Park, E.S. Han, C.S. Lee, N-methylated β- carbolines protect PC12 cells from cytotoxic effects of MPP+ by attenuation of mitochondrial membrane permeability change, Neurosci. Res. 46 (2003) 349–358. [13] D.J. Moura, M.F. Richter, J.M. Boeira, J.A. PêgasHenriques, J. Saffi, Antioxidant properties of b-carboline alkaloids are related to their antimutagenic and antigenotoxic activities, Mutagenesis 22 (2007) 293–298. [14] D.L. Bemis, J.L. Capodice, P. Gorroochurn, A.E. Katz, R. Buttyan, Anti-prostate cancer activity of a beta-carboline alkaloid enriched extract from Rauwolfia vomitoria, Int. J. Oncol. 29 (2006) 1065–1073. [15] Q. Chen, R. Chao, H. Chen, X. Hou, H. Yan, Sh. Zhou, W. Peng, A. Xu, Antitumor and neurotoxic effects of novel harmine derivatives and structure-activity relationship analysis, Int. J. Cancer 114 (2004) 675–681. [16] J. Jiméne, L. Riverón-Negrete, F. Abdullaev, J. Espinosa-Aguirre, R. Rodríguez-Arnaiz, Cytotoxicity of the b-carboline alkaloids harmine and harmaline in human cell assays in vitro, Exp. Toxicol. Pathol. 60 (2008) 38–44. [17] F. Jahaniani, S.A. Ebrahimi, N. Rahbar-Roshandel, M. Mahmoudian, Xanthomicrol is the main cytotoxic component of Dracocephalum kotschyii and a potential anticancer agent, Phytochemistry 66 (2005) 1581–1586. [18] L. Agüí, C. Peña-Farfal, P. Yáñez-Sedeño, J.M. Pingarrón, Determination of betacarboline alkaloids in foods and beverages by high-performance liquid chromatography with electrochemical detection at a glassy carbon electrode modified with carbon nanotubes, Anal. Chim. Acta. 585 (2007) 323–330. [19] T. Herraiz, C. Chaparro, Analysis of monoamine oxidase enzymatic activity by reversed-phase high performance liquid chromatography and inhibition by betacarboline alkaloids occurring in foods and plants, J. Chromatogr. A 1120 (2006) 237–243. [20] T. Herraiz, C. Chaparro, Human monoamine oxidase is inhibited by tobacco smoke: beta-carboline alkaloids act as potent and reversible inhibitors, Biochem. Biophys. Res. Commun. 326 (2005) 378–386. [21] H. Tsuchiya, High-performance liquid chromatographic analysis of beta-carbolines in human scalp hair, J. Chromatogr. A 1031 (2004) 325–330. [22] S. Casal, E. Mendes, J. Fernandes, M.B.P.P. Oliveira, M.A. Ferreira, Analysis of heterocyclic aromatic amines in foods by gas chromatography–mass spectrometry as their tert-butyldimethylsilyl derivatives, J. Chromatogr. A 1040 (2004) 105–113. [23] J. Stockigt, Y. Sheludko, M. Unger, I. Gerasimenko, H. Warzecha, D. Stockigt, HPLC–, CE– and CE/MS analysis of selected alkaloid groups, J. Chromatogr. A 967 (2002) 85–113. [24] L. Švorc, D. Stanković, K. Kalcher, Boron-doped diamond electrochemical sensor for sensitive determination of nicotine in tobacco products and anti-smoking pharmaceuticals, Diam. Relat. Mater. 42 (2014) 1–7. [25] A.M.C. Pereira, T.R. Rodrigues, J.H. Yariwake, Quantification of harman alkaloids in sour passion fruit pulp and seeds by a novel dual SBSE–LC/Flu (stir bar sorptive extraction–liquid chromatography with fluorescence detector) method, J. Braz. Chem. Soc. 00 (2014) 1–12. [26] M. Kartal, M.L. Altun, S. Kurucu, HPLC method for the analysis of harmol, harmalol, harmine and harmaline in the seeds of Peganum harmala L, J. Pharm. Biomed. Anal. 31 (2003) 263–269. [27] J.C. Callaway, G.S. Brito, E.S. Neves, Phytochemical analyses of Banisteriopsis caapi and Psychotriy viridis, J. Psychoactive Drugs 37 (2005) 145–150. [28] J.R. Sanchez-Ramos, Banisterine and Parkinson's Disease, 14 (1991) 391–402. [29] M. Serrano-Duenas, F. Cardozo-Pelaez, J.R. Sanchez-Ramos, Effect of Banisteriopsis caapi extract on Parkinson's disease, Sci. Rev. Alternat. Med. 5 (2001) 127–132.
Contents lists available at ScienceDirect
Microchemical Journal journal homepage: www.elsevier.com/locate/microc
New electrochemical method for the determination of β-carboline alkaloids, harmalol and harmine, in human urine samples and in Banisteriopsis caapi Dalibor Stanković a,⁎, Eda Mehmeti b, Lubomir Svorc c, Kurt Kalcher b a b c
Innovation Centre Faculty of Chemistry, University of Belgrade, Studentski trg 12-16, Belgrade, Serbia Institute of Chemistry — Analytical Chemistry, Karl-Franzens University Graz, A-8010 Graz, Austria Institute of Analytical Chemistry, Faculty of Chemical and Food Technology, Slovak University of Technology in Bratislava, SK-812 37, Bratislava, Slovak Republic
a r t i c l e
i n f o
Article history: Received 12 June 2014 Received in revised form 20 August 2014 Accepted 20 August 2014 Available online 27 August 2014 Keywords: Harmalol Harmine Banisteriopsis caapi Differential pulse voltammetry
a b s t r a c t In this paper a new electrochemical method for determination of the β-carboline alkaloids, harmalol and harmine, using differential pulse voltammetry is presented. Experimental parameters such as pH, scan rate, pulse amplitude and pulse time were optimized to characterize their electrochemical behavior and to find best analytical conditions. Thus, in the presence of ascorbic acid, uric acid, dopamine, the developed method exhibits excellent performance for the determination of harmalol and harmine for a linear concentration range between 1–50 μM and 1–75 μM with a detection limit of 0.6 μM and 0.2 μM, respectively. The proposed method was successfully applied for the determination of these alkaloids in a sample of an Ayahuasca liana (Banisteriopsis caapi) and in model human urine samples with good recoveries. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The β-carboline alkaloids, harmalol and harmine (Fig. 1.) are present in a great variety of medicinal plants and they are also endogenously produced in human and animal tissues as a product of secondary metabolism [1]. They possess diverse biological properties due to their capability to bind to benzodiazepine or imidazoline receptors, such as cardiovascular actions, hypotensive, hallucinogenic or antimicrobial actions and tremorogenesis [2,3]. The β-carboline alkaloids have been of interest of South-American ethnic tribes due to their psychotropic properties. These compounds affect the content of neurotransmitters by strong reversible inhibition of monoamine oxidase and the inhibition of acetylcholinesterase [4–6]. The biological significance of β-carbolines seems to be manifested also in cytotoxic as well as neuroprotective properties. Since they are structurally similar to 1-methyl-4-phenyl-11,2,3,6-tetrahydropyridine (MTPT) [7], which induces a Parkinsonianlike syndrome in animals, they have been postulated to act as endogenous neurotoxins. However, β-carbolines protect neurons against the exocytotoxic effects of dopamine and glutamate [8], and display a protective effect on oxidative neuronal damage through a scavenging action on reactive oxygen species [9–12]. They show effective antioxidant properties. Moreover, they have a significant protective effect against H2O2 and paraquat-induced oxidative agents in yeast cells, ⁎ Corresponding author. E-mail address: [email protected] (D. Stanković).
http://dx.doi.org/10.1016/j.microc.2014.08.007 0026-265X/© 2014 Elsevier B.V. All rights reserved.
and their ability to scavenge hydroxyl radicals contributes to their antimutagenic and antigenotoxic effects. β-carbolines exhibit cytotoxicity with regards to HL60 and K562 leukemia cell lines [13–17]. Natural sources of these alkaloids are different organisms, most of which are plants, most notably the Middle Eastern plant harmal or Syrian rue (Peganum harmala) and the South American vine Banisteriopsis caapi, also known as yage or ayahuasca. Recently, there are various techniques for the analysis of carboline alkaloids in different biological samples such as HPLC with electrochemical [18], photodiode array (DAD) [19] or fluorescence [20,21] detection as well as GC–MS [20–22], HPLC–MS and CE–MS [23]. Most of these methods require several time-consuming manipulation steps, sophisticated instruments and special training of the operating personnel. Some recent electrochemical studies on alkaloids showed that boron-doped diamond can be a useful electrode material for their voltammetric determination [24], nevertheless glassy carbon represents an inexpensive alternative, supposed that the oxidation potentials are not too high, which has also the advantage of allowing simple and effective cleaning of its surface, i.e., polishing. Differential pulse voltammetry (DPV) is one of the most sensitive electrochemical techniques for the determination of traces of numerous compounds because of its remarkably low detection limit. Other advantageous features of DPV include relatively low cost instrumentation and the capability for simultaneous determination. The aim of this work was to develop a new, simple, inexpensive and sensitive method for determination of harmalol and harmine. Another aspect of the new method was its successful application to the
96
D. Stanković et al. / Microchemical Journal 118 (2015) 95–100
Fig. 1. Structure of harmalol (A) and harmine (B).
determination of harmalol and harmine in urine samples and B. caapi with ascorbic acid (AA), dopamine (DA), and uric acid (UA) as most common interferences.
The liana B. caapi was treated according the procedure described in [25], which can be summarized shortly as: 2 g of dried and powdered seeds of P. harmala was macerated four times with 50 ml of methanol at 50 °C for 1 h. The extracts were combined and evaporated to dryness. The residue was dissolved in 50 ml HCl (2%) then filtered. The filtrate was extracted two times with 20 ml petroleum ether. The aqueous acid layer was basified and extracted four times with 50 ml chloroform. The chloroform layer was combined and evaporated to dryness, then residues were dissolved in 25 ml methanol. An aliquot (100 μL) of the prepared extract was added to 9.9 mL of supporting electrolyte. 3. Results and discussion
2. Experimental 2.1. Apparatus, solvents and reagents Water was purified with a cartridge purification system (Milli-Q); it had a resistivity of at least 18 MΩ cm and was used for preparing all solutions in use. Harmalol, harmine, ascorbic acid, uric acid, dopamine, boric acid, sodium hydroxide, acetic acid and phosphoric acid were purchased from Sigma Aldrich (analytical grade) and used as received without any further purification. Stock solutions (10− 3 M) of the analytes were prepared in water. Calibration standard solutions were prepared from the stock solutions by appropriate dilution with supporting electrolyte. Britton–Robinson buffer solutions (BRBS) were prepared by mixing of phosphoric acid, acetic acid and boric acid (all at 40 mM) and adjusting the pH with sodium hydroxide (0.2 M) to the desired value. All pH values were measured with a pH meter (model 1234, Orion). Cyclic voltammetric (CV) and differential pulse voltammetric measurements were performed using an electrochemical system PalmSens (Netherlands). The cell (10 mL) contained a three-electrode system, a glassy carbon working electrode, an Ag/AgCl (saturated KCl) reference electrode and a Pt counter electrode. All potentials in this paper are reported as values against the Ag/AgCl reference electrode at an ambient temperature. Prior to each experiment, the working electrode was polished with alumina (grain size 0.05 μm) followed by rinsing with water and sonication. The potential was swept over the range from 0 to 1.5 V (vs. Ag/AgCl) at different scan rate for CV, and from 0 to 1.3 V vs. (Ag/AgCl) in DPV mode with the optimized parameters (scan rate 25 mV/s, pulse amplitude 30 mV, step potential 5 mV and pulse width 40 ms). 2.2. Sample preparation The urine samples were collected from two different volunteers; to avoid in vivo drug administration they were spiked with stock solution of the alkaloids. Each 0.1 mL of fresh urine sample was taken and diluted to 10 mL of Britton–Robinson buffer at pH 6 and then directly analyzed.
3.1. Characterization Since the pH is one of the most important factors in the electrochemical behavior of organic compounds, its influence on the peak potential and peak current of investigated alkaloids was examined with cyclic voltammetry. Both, harmalol and harmine, give electrochemical responses in CV which is strongly pH dependent. Oxidation of harmalol occurs at around 0.5 V with a much smaller signal at 0.8 V, whereas the oxidation of harmine proceeds at 0.9 V. With increasing of pH of the electrolyte solution, the peak potential of the two alkaloids shifted linearly to less positive value, with a slope of − 0.365 and − 0.882 V/pH for harmalol and harmine, respectively (Fig. 2A and B). The shape of the peak potential dependence on pH were in good agreement with previously found pKa values for harmine (pKa1 8.0 and pKa2 14.4) and for harmalol (pKa1 8.6 and pKa2 11.3) respectively [25]. These results and the slopes of the peak potential dependence on pH indicate that protons participate in the oxidation reaction, but it is difficult to conclude from these data the ratio between protons and transferred electrons, because they deviate strongly from ideal Nerstian values. The proposed overall mechanism of the electrode reaction of both investigated alkaloids is given in Scheme 1. Oxidation is irreversible for both analytes. From Fig. 2 can be concluded that pH 6 is the most suitable pH for the determination of harmalol and harmine. At pH 6 both alkaloids show good quasi-linearity of the peak current on the square root of the scan rate (R2 = 0.984 for harmine and R2 = 0.973 for harmalol) (Fig. 3) which indicates that the mass transport is mainly controlled by diffusion. Nevertheless, the trend of data in both cases is not ideally linear but exhibits some slight curvature which, in combination with non-zero intercepts on the current axis, might suggest some other effects to contribute to the generation of the current. The signals provide good potential separation with a peak-to-peak value of 0.45 V which is sufficient for their possible simultaneous determination. 3.2. Optimization of DPV parameters The DPV method was applied as sensitive voltammetric method to investigate the relation between the peak current and concentration
Fig. 2. Cyclic voltammograms of harmalol (A) and harmine (B) at pH 6 in BRBS on a glassy carbon electrode with a scan rate of 100 mV/s; inserts: effect of pH on the peak current (▲) and the peak potential (■).
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Scheme 1. Proposed electrode reactions for A) harmine and B) harmalol under optimized electrochemical parameters.
of harmalol and harmine (calibration curves). The corresponding voltammetric parameters such as pulse amplitude, pulse time and scan rate were optimized by changing one parameter while the others were kept constant. Both alkaloids behaved almost identical,
therefore, the optimization process can be described independent on the analyte. When varying the pulse time the peak currents increased from 10 to 40 ms and decreased again with higher values, so the most suitable peak
Fig. 3. Dependence of the peak current on the square root of the scan rate for harmalol (A) and harmine (B).
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Fig. 4. DP voltammograms for different concentrations of harmine from 1 to 75 μM (A) and harmalol from 1 to 50 μM (B); supporting electrolyte: BRBS pH 6; scan rate 25 mV/s; pulse time 40 ms; pulse amplitude 30 mV; glassy carbon electrode.
Fig. 5. A) Dependence of the DPV peak current on the concentration of harmine (A) and of harmalol (B); experimental parameters as in Fig. 4.
current was chosen as 40 ms. When increasing the pulse amplitude from 10 to 75 mV the peak current increased accompanied with a widening of the peak width at the same time. The best peaks with respect to shape and height were obtained for a pulse amplitude of 30 mV. A scan rate of 25 mV/s was chosen as the best value with respect to peak height and analysis time. 3.3. Calibration curves and analytical applications Calibration plots for the simultaneous determination of harmalol and harmine were established in DPV mode applying the optimized experimental parameters. The DP voltammograms for different concentrations of harmalol and harmine and calibration curves are presented
in Fig. 4. As can be seen, evident partly from the CVs already, the current in differential pulse mode is not made by a simple single oxidation, but seems to consist of two overlapping peaks for each alkaloid. The evaluation of the peak current was done by a tangent fit and enumeration of the highest current in the tangent range. As a consequence of the more complex oxidation behavior of the alkaloids, both showed two linear ranges for the calibration curves (Fig. 5). Both, harmine and harmalol yielded a primary linear relation between current and concentration from 1 to 5 μM the corresponding equations and correlation coefficients of I = 0.004 + 0.076C, R2 = 0.9972 for harmine and I = − 0.021 + 0.084C, R2 = 0.9865 for harmalol, where C is concentration in μM and I the current in μA. The second range for higher concentrations extends to 75 μM (harmine) and 50 μM (harmalol) and can be described by I = 0.296 + 0.015C (R2 = 0.9927, with the same meaning of variables as above) and I = 0.094 + 0.054 C (R2 = 0.9969) for harmine and harmalol, resp. At concentrations higher than listed the currents level off. The detection limits were 0.6 μM for harmalol and 0.2 μM for harmine based on a ratio three times signal to background noise. The repeatability was evaluated as 2.3% and 1.9% RSD from 5 repetitive measurements of 25 μM of each, harmine and harmalol. From the results it can be concluded that the proposed method provides good sensitivity and repeatability for the determination of harmalol and harmine. Table 1 Determination of harmalol and harmine in Banisteriopsis caapi.
Fig. 6. DP voltammograms for determination of harmalol and harmine in urine sample, experimental parameters as in Fig. 4.
Sample
Harmalol
Harmaline
Harmine
Banisteriopsis caapi
Not expected [27] not found
0.03–0.83% expected [27]
0.31–8.6% expected [27] 0.6% found
D. Stanković et al. / Microchemical Journal 118 (2015) 95–100 Table 2 Determination of alkaloids in urine samples. Sample
Harmine added (μM)
Harmalol added (μM)
Harmine found (μM)
Harmalol found (μM)
Recovery of harmine (%)
Recovery of harmalol (%)
1 1 2
5.00 20.00 5.00
5.00 20.00 5.00
4.99 19.70 5.18
5.38 22.14 5.26
99.8 98.5 103.6
107.6 110.7 105.3
99
was evaluated with recovery experiments. As can be seen from the figure the proposed method enables the determination of both alkaloids in the presence of some interfering compounds with satisfactory recovery values. The signal of harmalol is superimposed on a matrix signal of the urine, but still can be evaluated with sufficient precision by the tangent fit method. These results confirm that the developed method is also suitable for determination of harmalol and harmine in urine samples. (See Table 1.)
3.4. Interferences
Fig. 7. DP voltammograms of different concentrations of harmalol and harmine in the presence of 50 μM of each, ascorbic acid, dopamine and uric acid; experimental parameters as in Fig. 4.
The application of the new method in biological samples was one of the primary requirements for its validation. The concentrations were estimated by DPV from the calibration curves for the proposed method. B. caapi sample was prepared as previously described [26]. The concentration of harmalol and harmine was determined after addition of 100 μL of the sample to 10 mL of supporting electrolyte. It was found that the concentration of harmine was in good agreement with literature data [27]. The proposed method was successfully applied to urine analysis of harmalol and harmine without any previous preparation (Fig. 6, Table 2). Generally, after drug intake some unmetabolized amounts usually secrete in the patient's urine. Standard additions of 5 and 10 μM of harmalol and harmine each caused current increments at the expected potentials allowing quantitative estimation. The accuracy
Perhaps the main characteristics of every method for the determination of significant analytes in biological samples lie in its inherent selectivity. Losing of neurons from the substantia nigra of the brain causes Parkinson's disease and when damaged, these neurons stop producing dopamine and compromise the brain's ability to control movement. The reason for this can be that free radicals and toxic particles normally deactivated in the body are responsible, which can be controlled by antioxidants. B. caapi was established for alleviating these symptoms, which contains β-carboline alkaloid inhibitors as active constituent used for treatment of Parkinson's disease [28,29]. As ascorbic acid is known as a major antioxidant in the human body, influence on the determination of harmalol and harmine of ascorbic acid, dopamine and uric acid, which often may coexist in the body fluids, was investigated. Fig. 7. shows the DP voltammograms of different concentrations of harmalol and harmine in the absence and in the presence of ascorbic acid, uric acid and dopamine in a concentration of 50 μM. As can be seen the peak current for the oxidation of both alkaloids increases with increasing concentration whereas the response of the interferents remains almost constant. Fig. 8A and B shows the interferences between harmalol and harmine themselves. The concentration of one of the alkaloids was kept constant while the concentration of the other was varied. The peak current of harmalol increased with increasing its concentration while the peak current of harmine remained constant when its concentration was constant; the resultant calibration curve of harmalol had the same characteristics as in the absence of harmine. In the case where the concentration of harmalol was constant the peak current of harmine increased with increasing concentration and had same linear ranges as without harmalol. This study finally proved that both alkaloids can be determined simultaneously with this new method even in the presence of higher concentrations of major interferents present in human samples, namely ascorbic acid, uric acid and dopamine.
Fig. 8. DP voltammograms of different concentrations of harmine (in the presence of 25 μM of harmalol, A) and harmalol (in the presence of 25 μM of harmine, B); experimental parameters as in Fig. 4.
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4. Conclusions In this study a new analytical method for the determination of harmalol and harmine was developed using a glassy carbon electrode. Cyclic voltammetry was used for the characterization of the electrochemical behavior of both alkaloids, and DPV for their quantification. The proposed method is simple, inexpensive and rapid for the determination of the target analytes compared to other analytical techniques previously described in the literature. The practical utility was successfully demonstrated in the analysis of real samples of the ayahuasca liana B. caapi and of model human urine samples. There was no influence of compounds such as ascorbic acid, uric acid or dopamine which might be present in body fluids. Based on this fact, the proposed method offers fast, low cost, simple, selective and sensitive possibilities for analysis of these alkaloids in biological samples without any complex pretreatment of samples. Acknowledgments This work was supported by the Ministry of Education and Science of the Republic of Serbia (project no. OI 172030) and the Grant Agency of the Slovak Republic (grant no. 1/0051/13). E.M. acknowledges financial support from the Austrian government. References [1] T.G. Bidder, D.W. Schomaker, H.S. Boetger, H.E. Evans, J.T. Cummins, Harmane in human platelets, Life Sci. 25 (1979) 157–164. [2] J. Lutes, J.F. Lorden, M. Beales, G.A. Lotmans, Tolerance to the tremorogenic effects of harmaline: evidence for altered olivo-cerebellar function, Neuropharmacology 27 (1988) 849–855. [3] Y.B. Guan, E.D. Louis, W. Zheng, Toxicokinetics of tremorogenic natural products, harmane and harmine, in male Spraqe–Dawley rats, J. Toxicol. Environ. Health A 64 (2001) 645–660. [4] X.Y. Zheng, Z.J. Zhang, G.X. Chou, T. Wu, X.M. Cheng, C.H. Wang, et al., Acetylcholinesterase inhibitive activity-guided isolation of two new alkaloids from seeds of Peganum nigellastrum buNge by an in vitro TLC-bioautographic assay, Arch. Pharm. Res. 32 (2009) 1245–1251. [5] X.Y. Zheng, L. Zhang, X.M. Cheng, Z.J. Zhang, C.H. Wang, Z.T. Wang, Identification of acetylcholinesterase inhibitors from seeds of plants of genus Peganum by thin-layer chromatography–bioautography, J. Planar Chromatogr. Mod. TLC 24 (2011) 470–474. [6] H. Kim, S.O. Sablin, R.R. Ramsay, Inhibition of monoamine oxidase A by betacarboline derivatives, Arch. Biochem. Biophys. 337 (1997) 137–142. [7] M.J. Schwarz, P.J. Houghton, S. Rose, P. Jenner, A.D. Lees, Activities of extract and constituents of Banisteriopsis caapi relevant to parkinsonism, Pharmacol. Biochem. Behav. 75 (2003) 627–633. [8] R. Albores, E.J. Neafsey, G. Drucker, J.Z. Fields, M.A. Collins, Mitochondrial respiratory inhibition by N-methylated beta-carboline derivatives structurally resembling N-methyl-4-phenylpyridine, Proc. Natl. Acad. Sci. U. S. A. 23 (1990) 9368–9372. [9] P. Maher, J.B. Davis, The role of monoamine metabolism in oxidative glutamate toxicity, J. Neurosci. 20 (1996) 6394–6401.
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