Journal of Chromatography A, 1319 (2013) 166–171
Contents lists available at ScienceDirect
Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Short communication
Application of pH-zone-refining countercurrent chromatography for the separation of indole alkaloids from Aspidosperma rigidum Rusby Mariana N. Vieira a,b,∗ , Suzana G. Leitão a , Paula C.C. Porto a , Danilo R. Oliveira a , Shaft Corrêa Pinto a,c , Raimundo Braz-Filho d , Gilda G. Leitão e a
Universidade Federal do Rio de Janeiro, Faculdade de Farmácia, CCS, Bl. A 2o andar, Ilha do Fundão 21941-590, RJ, Brazil Institute of Food Chemistry, Technische Universität Braunschweig, Schleinitzstrasse 20, 38106 Braunschweig, Germany c Curso de Farmácia/Campus UFRJ-Macaé, Rua Aluisio da Silva Gomes, 50, Granja dos Cavaleiros, Macaé 27930-560, RJ, Brazil d Laboratório de Ciências Químicas, Universidade Estadual do Norte Fluminense Darcy Ribeiro, Av. Alberto Lamego, 2000, Campos dos Goytacazes 28013-602, RJ, Brazil e Universidade Federal do Rio de Janeiro, Núcleo de Pesquisas de Produtos Naturais, CCS, Bl. H, Ilha do Fundão 21941-590, RJ, Brazil b
a r t i c l e
i n f o
Article history: Received 22 May 2013 Received in revised form 10 October 2013 Accepted 12 October 2013 Available online 22 October 2013 Keywords: Indole alkaloids Aspidosperma pH-zone-refining countercurrent chromatography
a b s t r a c t Species of Aspidosperma (Apocynaceae) are characterized by the occurrence of indole alkaloids, but few recent reports on Aspidosperma rigidum Rusby chemical constituents were found. The present work shows the application of pH-zone refining countercurrent chromatography on the separation of alkaloids from the barks of A. rigidum. In this study, the dichloromethane extract was fractionated with the solvent system composed of methyl-tert-butyl ether and water with different concentrations of the retainer triethylamine in the organic stationary phase and formic or hydrochloric acids as eluters in the aqueous mobile phase, in order to evaluate the most suitable condition. In each experiment, from circa 200 mg of the dichloromethane extract of A. rigidum, three major alkaloids were isolated and identified as 3␣aricine (circa 17 mg), isoreserpiline (ca. 22 mg) and 3-reserpiline (ca. 40 mg), with relative purity of 79%, 89% and 82% respectively, in a one-step separation of 2 h. Two of them – 3␣-aricine and isoreserpiline – were isolated and identified for the first time in this species. © 2013 Elsevier B.V. All rights reserved.
1. Introduction High-speed countercurrent chromatography (HSCCC) is an hydrodynamic preparative technique based on the distribution coefficient (K) of substances between the two phases of a biphasic solvent system, where one of them is the stationary phase and the other acts as mobile phase [1,2]. Specially for the case of ionizable molecules like organic acids and bases, a method proposed by Ito [3,4] allows the separation process also according to pKa values and hydrophobicity of the substances. For the separation of alkaline organic compounds, this method consists on the addition of a basic retainer to the stationary phase and an acidic eluter to the mobile phase [4]. pH-Zone refining CCC shows some advantages when compared to conventional CCC, for example the increase of the sample loading capacity, the high concentration of the fractions and the possibility of monitoring the analysis by measuring the pH value of each
∗ Corresponding author. Tel.: +49 17656523379; fax: +49 55 21 2562 6413. E-mail addresses: [email protected], [email protected] (M.N. Vieira). 0021-9673/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.chroma.2013.10.044
fraction collected. On the other hand, one obvious disadvantage is that the analyte must be ionic (or ionizable) [4]. Furthermore, such approach in does not allow continuous operation, due to the fact that the system has to be re-established again after each run. Nevertheless, this method was successfully applied to the separation of several types of alkaloids [5–8], including those of the indole type, which are also the major components of Aspidosperma rigidum Rusby [9,10]. Species of Aspidosperma genus (Apocynaceae) are generally trees found in Central and South America, commonly known in the North of Brazil as “Carapanaúba”, which means mosquito’s tree [11]. In this region, the teas made from its barks are popularly used to treat several diseases [12–14]. To date, only nine alkaloids were described for A. rigidum. extracts: 3-reserpiline, burnamine, picraline, caboxine A, caboxine B, isocaboxine, (-) carapanaubine, isocarapanaubine and haplocidine [10,15]. Based on the ethnopharmacological background and on the lack of recent studies about this plant, the aim of the present work was to apply the modern technique of CCC in the pHzone refining mode for the separation of the components present in the dichloromethane extract of Carapanaúba (A. rigidum Rusby).
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2. Experimental
167
Table 1 pH-Zone refining experiments A to D, solvent system MtBE-H2 O.
2.1. Plant material
Experiment
Retainer (TEA) concentration (mM)
Eluter concentration
Samples of A. rigidum Rusby were collected in August 2008, in the Brazilian Amazon region of Oriximiná (Pará state), at Jauari community (S 01◦ 15.326 , W 056◦ .02437 ). Plants were collected as part of a bioprospecting project in quilombola communities from Oriximiná that received authorization by the Directing Council of Genetic Heritage (Conselho de Gestão do Patrimônio Genético), through the Resolution no. 213 (6.12.2007), published in the Federal Official Gazette of Brazil on December 27, 2007. Samples were identified by Dr. Washington Marcondes-Ferreira, from Universidade Estadual de Campinas, Campinas, Brazil. A voucher specimen is deposited at the Instituto Nacional de Pesquisas da Amazônia, INPA, herbarium (Manaus, AM), under the registration INPA 233366. Dried and ground barks (506 g) of A. rigidum were submitted to extraction by maceration in percolator first with hexane, than dichloromethane, followed by ethyl acetate and methanol, in this order.
A B C D
15 10 10 5
10 mM formic acid 10 mM formic acid 15 mM formic acid 5 mM hydrochloric acid
2.2. Choice of the solvent systems The solvent systems tested by test tubes assay were composed of MtBE-CH3 CN–water in different proportions, as proposed by Ito [4]. First, two test tubes containing the solvent system to be tested were prepared. Then, the acid to be used as eluter was added to one of them and the base (retainer) was added to the other. Different acids were tested, such as acetic, formic, sulfuric, trifluoroacetic and hydrochloric acids; the base used was triethylamine. Later, small amounts of the extract were dissolved in both tubes and equal amounts of each phase from the two tubes were spotted separately in a silica gel 60 F254 TLC plate (Merck, Darmstadt, Germany, Art. 5554) developed with ethyl acetate:acetone:water (25:8:2) and a drop of a concentrated ammonium hydroxide solution. The result was visualized under UV light (264 and 366 nm) in a Spectronline Model CC-80 (Spectronics Corporation, USA) spectrometer and then the TLC plate was stained with Dragendorff’s [16] reagent to detect the alkaloids. The K values were estimated using the equation formulated by Conway [1]. 2.3. CCC apparatus Separations were performed on a P.C. Inc. (Potomac, MD, USA) countercurrent chromatograph equipped with a triple polytetrafluoroethylene multi-layer coil (15 ml + 80 ml +280 ml, 1.6 mm i.d.) equilibrated by a counterweight. The rotation speed is adjustable from 0 to 1000 rpm. The 80 ml coil was used in all experiments. The solvents were pumped with a HPLC Solvent Delivery System Model M-45 Waters (USA) and the fractions were collected in a Super Fraction Collector SF-2120 Advantec (Japan). 2.4. pH-Zone refining experiments A to D Separations were performed by pH-zone refining CCC in the reverse-displacement mode. The two-phase solvent system utilized was composed of MtBE and water and prepared as described by Ito and Ma [5]. Different concentrations of the retainer (TEA) in the organic stationary phase and of the eluter formic acid in the aqueous mobile phase were tested, as follows: experiment (A)—15 mM TEA and 10 mM formic acid, experiment (B)—10 mM TEA and 10 mM formic acid, and experiment (C)—10 mM TEA and 15 mM formic acid. Also, an experiment using 5 mM TEA and 5 mM hydrochloric acid was performed (experiment D). To prepare the sample solution approximately 200 mg of the dichloromethane
extract of the barks of A. rigidum were dissolved in a mixture of 3 ml of the organic stationary phase with the retainer (TEA) and 3 ml of the aqueous mobile phase free of the acidic eluter. 2.5. Separation procedures First, the column was filled with the organic stationary phase, saturated with the mobile phase and containing the retainer (TEA), in a flow rate of 5 ml/min. Then, the rotation was turned on at the speed of 850 rpm and the sample solution was injected through the sample port, using a 5 ml loop. After this, the aqueous mobile phase containing the acidic eluter was pumped at a flow rate of 2 ml/min. About 60 fractions of 4 ml were collected (rotation was turned off at tube 40). Fractions were analyzed by TLC using the same conditions as specified in Section 2.3 and pooled together by means of chromatographic similarity. Retention of the stationary phase was determined by measuring the volume of stationary phase displaced until the solvent front; i.e. the first tube that presented two phases, then subtracting it from the total coil volume and calculating the percentage of stationary phase that remained inside the coil [17]. The pH values of each fraction were manually measured with a portable pH meter model Q400BC, from Quimis (Brazil). 2.6. Identification of the isolated substances The fractions containing the isolated alkaloids were concentrated in rotary evaporator (under reduced pressure, at 40 ◦ C) and then identified by the combination of 1D-/2D-NMR and MS spectral data. NMR experiments were performed on a Varian 500 MHz spectrometer. All samples were dissolved in deuterated chloroform, and tetramethylsilane (TMS) was used as internal standard. Chemical shifts (ı) are reported in ppm. The MS experiments were made by direct injection on a GC–MS QP 5000 Shimadzu equipment, using electron impact ionization at 70 eV. UV data were obtained from HPLC-DAD injections. 2.7. HPLC analyses The relative purity of the fractions was obtained by HPLC in a Merk-HITACHI LaChrom (Germany) apparatus L-7000, with UV/DAD detector L-7450, using a reversed phase ODS reversible analytical column (25 cm × 4.6 mm, 5 m) from Rexchrom Regis (USA) at room temperature of 25 ◦ C. The mobile phase solvent system consisted of a mixture of H2 O/TFA (pH 3; 0.025%) and CH3 CN/TFA (0.025%) in a stepwise gradient as follows: from 65:35 to 55:45 (0 to 10 min); from 55:45 to 40:60 (10.1 to 15 min); from 40:60 to 25:75 (15.1 to 28 min), and 0:100 from 28.1 to 30 min. The injection volume was 20 l, the flow rate 1.0 ml/min and detection was performed at 250 nm. The dichloromethane extract, as well as the fractions containing the separated alkaloids, were dissolved in a mixture (1:1) of the mobile phase components. LC–MS experiments were performed, in order to determinate the molecular weight of the isolated alkaloids. Samples ran in a HCT-Ultra ETD II (Bruker Daltonics) equipment, with a Prontosil ˚ and pre-column C18-Aq column of 250 × 2.0 mm, 5 micron (100 A)
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Fig. 1. TLC plates with the results of the pH-zone refining CCC. Experimental conditions: TLC—silica-gel plates developed with: ethyl-acetate:acetone:water (25:8:2) and a drop of a concentrated NH4 OH solution, detection: Dragendorff’s reagent; CCC—coil volume: 80 ml, flow-rate: 2 ml/min, rotation speed: 850 rpm, sample loading: 200 mg, fraction size: 4 ml, solvent system: MtBE–water; (A) 15 mM TEA in the OSP and 10 mM formic acid in the AMP, SPR: 67%; (B) 10 mM TEA in the OSP and 10 mM formic acid in the AMP, SPR: 40%; (C) 10 mM TEA in the OSP and 15 mM formic acid in the AMP, SPR: 67%; (D) 5 mM TEA in the OSP and 5 mM hydrochloric acid in the AMP, SPR: 55%. OSP—organic stationary phase, AMP—aqueous mobile phase, SPR—retention of stationary phase.
Fig. 2. Chemical structures of the alkaloids isolated from A. rigidum Rusby (1, 2 and 4).
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Fig. 3. HPLC analyses of the dichloromethane extract from the barks of A. rigidum (A) and the isolated alkaloids (B, C, and D). Experimental: Rexchrom Regis reversed phase ODS reversible analytical column (25 cm × 4.6 mm, 5 m), temperature of 25 ◦ C, mobile phase: H2 O/TFA (pH 3; 0.025%) and CH3 CN/TFA (0.025%) stepwise gradient from 65:35 to 55:45 (0 to 10 min); from 55:45 to 40:60 (10.1 to 15 min); from 40:60 to 25:75 (15.1 to 28 min) and 0:100 from 28.1 to 30 min, flow rate 1.0 ml/min, detection: 250 nm, injection volume: 20 l.
of the same material, at room temperature of 20 ◦ C. The mobile phase solvent system consisted of a mixture of H2 O/TFA (pH 3; 0.025%) and CH3 CN/TFA (0.025%) in a linear gradient from 80:20 to 10:90 (0 to 50 min). The injection volume was 10 l, the flow
rate 0.25 ml/min and detection was performed by DAD ( between 200 and 700 nm) and MS. The MS measurements were made in the range of m/z 100 to 2000 with electrospray ionization in the positive mode. The fragments obtained represented m/z [M+ + 1].
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3. Results and discussion Preliminary thin layer chromatography (TLC) analysis of the extracts obtained from the bark of A. rigidum showed the presence of four major alkaloids in the dichloromethane extract when stained with the Dragendorff’s reagent and, therefore, this was chosen for the separation experiments. As alkaloids can be regarded as weak organic bases, the reverse mode displacement pH-zone CCC technique was applied for the purification of these compounds. According to Ito [4] the suitable two-phase solvent system for pH-zone refining CCC is the one in which Kacid 1 in the experiments from the acidic condition, what was observed when adding formic acid to the test tubes, and Kbase 1 in the experiments from the basic condition. The solvent systems tested were composed of MtBE-CH3 CN–water in different ratios, as follows: 1:0:1 (S1), 4:1:5 (S2), 6:3:8 (S3) and 2:2:3 (S4) (v/v/v). Three of these (S2 to S4) showed an ideal Kacid value, but Kbase ≈ 1. The best condition was achieved when no CH3 CN was added to the system, making the system MtBE and water the most suitable choice (data not shown). 3.1. Fractionation of the A. rigidum dichloromethane extract Four experiments were performed in order to optimize the concentrations of the retainer base (TEA) and eluter acid (Table 1). Each of the three first experiments (A to C) provided, from circa 200 mg of the dichloromethane extract of A. rigidum, around 17 mg of 3␣-aricine, 1, 22 mg of isoreserpiline, 2, and 40 mg of 3reserpiline, 4, in a one-step separation of 2 h. Fig. 1 shows the results of the four pH-zone refining experiments (A to D) as analyzed by both TLC and pH measurements of each fraction tube. In experiment A (Fig. 1A) the elution of the alkaloids begins after around 54 min, coinciding with the decrease of the mobile phase’s pH value. First, 3-reserpiline, 4, elutes as a mixture with a small amount of 3 (non identified alkaloid) until isoreserpiline, 2, starts to elute. After another pH decrease, 2 elutes, followed by 3␣-aricine, 1. The stationary phase retention was of 67% and the pH flat zones are observed at pH 6.5, 5.0, 3.7 and 2.7. In experiment B (Fig. 1B), elution of the alkaloids starts around 10 min earlier than in A. First, a small decrease in the mobile phase pH was observed, representing the beginning of the elution of the mixture of 3-reserpiline, 4, and 3. This lasts for 6 min, then the pH decreases again and elution of isoreserpiline, 2, starts. In this experiment, there is an increase in resolution between 2 and 1, in comparison with A. The stationary phase retention was of 40% and the pH flat zones are observed at pH 9.0, 5.5, 3.5 and 2.4. Fig. 1C shows the result of experiment C. Here, elution begins even faster than in B, with the decrease of the pH values observed at around 38 min after the run started. In this experiment, although 3-reserpiline, 4, eluted in a mixture with 3, the proportion of the latter was significantly smaller. It is worth noting that also cleaner fractions of 2 were obtained, as well as those containing isoreserpiline, as the increased resolution was maintained. This fact was corroborated by the observation of an additional pH flat zone, compared to experiments A and B. These were at pH 7.3, 5.9, 4.7, 3.6 and 2.7. The stationary phase retention was of 67%. All the experiments showed a reasonable separation of the components from the alkaloidic dichloromethane extract of A. rigidum and allowed the isolation of three alkaloids – 1, 2 and 4 – with relative purity of 79%, 89% and 82% respectively. In all experiments, alkaloid 3 eluted in mixture either with 2 or 4. Comparing the experiments, it was possible to observe that increasing the eluter acid concentration in the aqueous mobile phase resulted in a shorter retention time of the analytes. Reducing the concentration of the retainer base in the upper organic phase changed the
stationary phase properties, increasing the resolution between the alkaloids. The pH-zone refining CCC method is characterized by the production of pH-zones according to pKa and hydrophobicity of the analytes [17], but in experiments A to C the major components were eluted with an abrupt variation from basic to acid in a few minutes in all experiments. In terms of chromatogram, it could represent a peak sharpening, that occurs when the K of the retainer (Kr ) falls between the analyte’s K under acidic (Kacid ) and basic (Kbase ) conditions [4]. This fact can be due to the utilization of an organic acid, which is relatively weak when compared to a mineral acid, in the aqueous mobile phase. Although it is not usual to add an organic modifier to the inorganic (aqueous) phase, this work showed that in some cases it can provide the most suitable condition for the separation of the compounds. On the other hand, 3␣-aricine, 1, did not seem to be affected by the change in the retainer’s and/or eluter’s concentrations. It eluted at the latest plateau during circa 18 min, after the retainer be entirely removed from the system, which means that its Kacid and Kbase values are greater than Kr [4]. In order to analyze the influence of the chemical nature of the acid in the separation, an experiment using the inorganic hydrochloric acid, instead of the organic formic acid was also made. As shown in Fig. 1D the pH flat zones were better defined. Elution of the alkaloids started after around 40 min, yielding fractions containing only 3-reserpiline, 4, followed, however, by mixtures of 3 + 2 and 3 + 1. The stationary phase retention was of 55% and the pH flat zones are observed at pH 5.6, 5.0 and 2.6. The four pH-zone refining experiments (A to D) afforded the separation of indole alkaloids from the dichloromethane extract of the barks of A. rigidum to different extents. By using hydrochloric acid it was possible to separate 3-reserpiline, 4, from the other alkaloids, but not to resolve 2 from 1. With the organic formic acid; however, this separation was possible in experiment C, although a major overlapping of the other components was observed.
3.2. Identification of the alkaloids Identification of the alkaloids 1, 2 and 4 was carried out by the combination of data from HPLC-UV, LC–ESI–MS and 1D-/2DNMR analyses. Therefore, 1 was identified as 3␣-aricine (MW 382 u) [18–21], 2 as isoreserpiline (MW 412) and 4 as 3-reserpiline (MW 412) [18,19,21], as shown in Fig. 2. Compound 3 remains unidentified. 3˛-aricine (1): UV and 1 H-NMR data [18–21]; 13 C NMR (125 MHz, CD3 OD) ␦ 168.16 (C-22), 155.48 (CH-17), 153.60 (C10), 134.95 (C-2), 131.85 (C 13), 127.17 (C-8), 111.17 (CH-12), 110.25 (CH-11), 109.49 (C-16), 106.42 (C-7), 99.49 (CH-9), 72.15 (CH-19), 60.43 (CH-3), 55.66 (CH2 -21, 54.83 (MeO-10), 53.48 (CH2 5), 50.21 (MeO-22), 38.44 (CH-20), 33.32 (CH2 -14, 31.24 (CH-15), 21.04 (CH2 -6), 17.43 (CH3 -18). MS-EI (70 eV): m/z 281 (29%), 253 (50%), 199 (37%), 186 (100%).isoreserpiline (2): UV, 1 H and 13 C-NMR data [18,19,21]. MS-EI (70 eV): m/z 311 (40%), 283 (74%), 229 (46%), 216 (100%). 3ˇ-reserpiline (4): UV, 1 H and 13 C-NMR data [18,19,21]. MS-EI (70 eV): m/z 311 (59%), 283 (100%), 216 (50%).
3.3. Analysis of the fractions by HPLC The extracts, as well as the separated fractions, were analyzed by HPLC and three intense peaks could be detected (Fig. 3A), which correspond, respectively, to compounds 2 and 4 (Rt 16.27 min, coeluted), 1 (Rt 17.84 min) and 3 (Rt 21.28 min). Although alkaloids 2 and 4 showed the same retention time in this HPLC conditions,
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they were well separated in the pH zone refining CCC experiments, as shown by their NMR data.
offering the resources to run the LC–MS analysis. This work was partially supported by CNPq (scholarship and grant).
4. Conclusions
References
The overall results of our work demonstrate that the pH-zone refining counter-current chromatography was successfully applied to the fractionation of indole alkaloids from the dichloromethane extract of Aspidosperma rigidum Rusby. Two of them – 3␣-aricine, 1, and isoreserpiline, 2 – were isolated and identified for the first time in this species. We also demonstrated that both organic and inorganic acids can be used as eluters in the aqueous mobile phase of the pH-zone refining countercurrent fractionation of these indole alkaloids. Although the profile of the experiments was different, all of them produced efficient separations. The present method may be applied to various other indole alkaloids from natural products.
[1] Y. Ito, W. Conway, Crit. Rev. Anal. Chem 17 (1986) 65. [2] W. Conway, Countercurrent Chromatography: Apparatus, Theory and Applications, VCH Publishers Inc., New York, 1989. [3] A. Weisz, A.L. Scher, K. Shinomiya, H.M. Fales, Y. Ito, J. Am. Chem. Soc 116 (1994) 704. [4] Y. Ito, Y. Ma, J. Chromatogr. A 753 (1996) 1. [5] Y. Ma, Y. Ito, E. Sokolosk, H.M. Fales, J. Chromatogr. A 685 (1994) 259. [6] J.H. Renault, J.M. Nuzillard, G. Le Crouerour, P. Thepenier, M. Zeches-Hanrot, L. Le Men-Olivier, J. Chromatogr. A 849 (1999) 421. [7] F. Yang, J. Quan, T. Zhang, Y. Ito, J. Chromatogr. A 822 (1998) 316. [8] F. Yang, T. Zhang, R. Zhang, Y. Ito, J. Chromatogr. A 829 (1998) 137. [9] R.R. Arndt, S.H. Brown, N.C. Ling, P. Roller, C. Djerassi, J.M. Ferreira, B. Gilbert, E.C. Miranda, S.E. Flores, Phytochemistry 6 (1967) 1653. [10] M.M. Pereira, R.L.R.P. Jácome, A.F.C. Alcântara, R.B. Alves, D.S. Raslan, Quim. Nova 30 (2007) 970. [11] A.G. Cunha, Dicionário Histórico das Palavras Portuguesas de Origem Tupi, 5th ed., Universidade de Brasília, São Paulo, Companhia Melhoramentos; Brasília, 1999. [12] M.K. Rai, G.A. Cordell, J.L. Martinez, M. Marinoff, L. Rastrelli, Medicinal Plants: Diversity and Drugs, Science Publishers, Enfield, 2011, pp. 160. ˜ [13] J. Sanz-Biset, S. Canigueral, J. Ethnopharmacol. 145 (2013) 67. [14] V.B. Oliveira, M.S.M. Freitas, L. Mathias, R. Braz-Filho, I.J.C. Vieira, Rev. Bras. Pl. Med. 11 (2009) 92. [15] M. Reina, W. Ruiz-Mesia, L. Ruiz-Mesia, R. Martínez-Díaz, A. González-Coloma, Z. Naturforsch. 66 c (2011) 225. [16] H. Wagner, S. Bladt, E.M. Zgainski, Plant Drug Analysis, Springer-Verlag, Berlin/Heidelberg, 1984. [17] Y. ITO, J. Chromatogr. A 1065 (2005) 145. [18] P. Gabetta, G. Mustich, Spectral Data of Indole Alkaloids, Inverni Della Beffa, Italy, 1975. [19] A. Bruyn, W. Zhang, M. Budesinsky, Magn. Reson. Chem 27 (1989) 935. [20] R. Verpoorte, E. Kos-Kuyck, T.A. Tsoi, C.L.M. Ruigrok, G. Jong, A.B. Svendsen, Planta Med. 48 (1983) 283. [21] M. Lounasmaa, A. Tolvaner, Heterocycles 24 (1986) 3229.
Acknowledgments The authors are thankful for Dr. Ma’s help with the pH-zone refining practical application and Dr. Ito’s explanations about some theoretical doubts. In addition, we would like to thank Prof. Dr. R. Braz-Filho and Prof. Dr. B. Gilbert, for the help with the identification of the alkaloids. We are also deeply indebted to the Centro Nacional de Ressonância Magnética Nuclear Jiri Jonas and LAMAR NPPN-UFRJ, Rio de Janeiro, for the NMR experiments and to ARQMO, Associac¸ão de Comunidades Remanescentes de Quilombos do Município de Oriximiná, Oriximiná-PA, Brazil, for supervising the plant collection. Furthermore, we wish to acknowledge the help provided by Prof. Dr. Peter Winterhalter and Dr. Gerold Jerz (Technische Universität Braunschweig, Germany) for
Contents lists available at ScienceDirect
Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Short communication
Application of pH-zone-refining countercurrent chromatography for the separation of indole alkaloids from Aspidosperma rigidum Rusby Mariana N. Vieira a,b,∗ , Suzana G. Leitão a , Paula C.C. Porto a , Danilo R. Oliveira a , Shaft Corrêa Pinto a,c , Raimundo Braz-Filho d , Gilda G. Leitão e a
Universidade Federal do Rio de Janeiro, Faculdade de Farmácia, CCS, Bl. A 2o andar, Ilha do Fundão 21941-590, RJ, Brazil Institute of Food Chemistry, Technische Universität Braunschweig, Schleinitzstrasse 20, 38106 Braunschweig, Germany c Curso de Farmácia/Campus UFRJ-Macaé, Rua Aluisio da Silva Gomes, 50, Granja dos Cavaleiros, Macaé 27930-560, RJ, Brazil d Laboratório de Ciências Químicas, Universidade Estadual do Norte Fluminense Darcy Ribeiro, Av. Alberto Lamego, 2000, Campos dos Goytacazes 28013-602, RJ, Brazil e Universidade Federal do Rio de Janeiro, Núcleo de Pesquisas de Produtos Naturais, CCS, Bl. H, Ilha do Fundão 21941-590, RJ, Brazil b
a r t i c l e
i n f o
Article history: Received 22 May 2013 Received in revised form 10 October 2013 Accepted 12 October 2013 Available online 22 October 2013 Keywords: Indole alkaloids Aspidosperma pH-zone-refining countercurrent chromatography
a b s t r a c t Species of Aspidosperma (Apocynaceae) are characterized by the occurrence of indole alkaloids, but few recent reports on Aspidosperma rigidum Rusby chemical constituents were found. The present work shows the application of pH-zone refining countercurrent chromatography on the separation of alkaloids from the barks of A. rigidum. In this study, the dichloromethane extract was fractionated with the solvent system composed of methyl-tert-butyl ether and water with different concentrations of the retainer triethylamine in the organic stationary phase and formic or hydrochloric acids as eluters in the aqueous mobile phase, in order to evaluate the most suitable condition. In each experiment, from circa 200 mg of the dichloromethane extract of A. rigidum, three major alkaloids were isolated and identified as 3␣aricine (circa 17 mg), isoreserpiline (ca. 22 mg) and 3-reserpiline (ca. 40 mg), with relative purity of 79%, 89% and 82% respectively, in a one-step separation of 2 h. Two of them – 3␣-aricine and isoreserpiline – were isolated and identified for the first time in this species. © 2013 Elsevier B.V. All rights reserved.
1. Introduction High-speed countercurrent chromatography (HSCCC) is an hydrodynamic preparative technique based on the distribution coefficient (K) of substances between the two phases of a biphasic solvent system, where one of them is the stationary phase and the other acts as mobile phase [1,2]. Specially for the case of ionizable molecules like organic acids and bases, a method proposed by Ito [3,4] allows the separation process also according to pKa values and hydrophobicity of the substances. For the separation of alkaline organic compounds, this method consists on the addition of a basic retainer to the stationary phase and an acidic eluter to the mobile phase [4]. pH-Zone refining CCC shows some advantages when compared to conventional CCC, for example the increase of the sample loading capacity, the high concentration of the fractions and the possibility of monitoring the analysis by measuring the pH value of each
∗ Corresponding author. Tel.: +49 17656523379; fax: +49 55 21 2562 6413. E-mail addresses: [email protected], [email protected] (M.N. Vieira). 0021-9673/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.chroma.2013.10.044
fraction collected. On the other hand, one obvious disadvantage is that the analyte must be ionic (or ionizable) [4]. Furthermore, such approach in does not allow continuous operation, due to the fact that the system has to be re-established again after each run. Nevertheless, this method was successfully applied to the separation of several types of alkaloids [5–8], including those of the indole type, which are also the major components of Aspidosperma rigidum Rusby [9,10]. Species of Aspidosperma genus (Apocynaceae) are generally trees found in Central and South America, commonly known in the North of Brazil as “Carapanaúba”, which means mosquito’s tree [11]. In this region, the teas made from its barks are popularly used to treat several diseases [12–14]. To date, only nine alkaloids were described for A. rigidum. extracts: 3-reserpiline, burnamine, picraline, caboxine A, caboxine B, isocaboxine, (-) carapanaubine, isocarapanaubine and haplocidine [10,15]. Based on the ethnopharmacological background and on the lack of recent studies about this plant, the aim of the present work was to apply the modern technique of CCC in the pHzone refining mode for the separation of the components present in the dichloromethane extract of Carapanaúba (A. rigidum Rusby).
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2. Experimental
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Table 1 pH-Zone refining experiments A to D, solvent system MtBE-H2 O.
2.1. Plant material
Experiment
Retainer (TEA) concentration (mM)
Eluter concentration
Samples of A. rigidum Rusby were collected in August 2008, in the Brazilian Amazon region of Oriximiná (Pará state), at Jauari community (S 01◦ 15.326 , W 056◦ .02437 ). Plants were collected as part of a bioprospecting project in quilombola communities from Oriximiná that received authorization by the Directing Council of Genetic Heritage (Conselho de Gestão do Patrimônio Genético), through the Resolution no. 213 (6.12.2007), published in the Federal Official Gazette of Brazil on December 27, 2007. Samples were identified by Dr. Washington Marcondes-Ferreira, from Universidade Estadual de Campinas, Campinas, Brazil. A voucher specimen is deposited at the Instituto Nacional de Pesquisas da Amazônia, INPA, herbarium (Manaus, AM), under the registration INPA 233366. Dried and ground barks (506 g) of A. rigidum were submitted to extraction by maceration in percolator first with hexane, than dichloromethane, followed by ethyl acetate and methanol, in this order.
A B C D
15 10 10 5
10 mM formic acid 10 mM formic acid 15 mM formic acid 5 mM hydrochloric acid
2.2. Choice of the solvent systems The solvent systems tested by test tubes assay were composed of MtBE-CH3 CN–water in different proportions, as proposed by Ito [4]. First, two test tubes containing the solvent system to be tested were prepared. Then, the acid to be used as eluter was added to one of them and the base (retainer) was added to the other. Different acids were tested, such as acetic, formic, sulfuric, trifluoroacetic and hydrochloric acids; the base used was triethylamine. Later, small amounts of the extract were dissolved in both tubes and equal amounts of each phase from the two tubes were spotted separately in a silica gel 60 F254 TLC plate (Merck, Darmstadt, Germany, Art. 5554) developed with ethyl acetate:acetone:water (25:8:2) and a drop of a concentrated ammonium hydroxide solution. The result was visualized under UV light (264 and 366 nm) in a Spectronline Model CC-80 (Spectronics Corporation, USA) spectrometer and then the TLC plate was stained with Dragendorff’s [16] reagent to detect the alkaloids. The K values were estimated using the equation formulated by Conway [1]. 2.3. CCC apparatus Separations were performed on a P.C. Inc. (Potomac, MD, USA) countercurrent chromatograph equipped with a triple polytetrafluoroethylene multi-layer coil (15 ml + 80 ml +280 ml, 1.6 mm i.d.) equilibrated by a counterweight. The rotation speed is adjustable from 0 to 1000 rpm. The 80 ml coil was used in all experiments. The solvents were pumped with a HPLC Solvent Delivery System Model M-45 Waters (USA) and the fractions were collected in a Super Fraction Collector SF-2120 Advantec (Japan). 2.4. pH-Zone refining experiments A to D Separations were performed by pH-zone refining CCC in the reverse-displacement mode. The two-phase solvent system utilized was composed of MtBE and water and prepared as described by Ito and Ma [5]. Different concentrations of the retainer (TEA) in the organic stationary phase and of the eluter formic acid in the aqueous mobile phase were tested, as follows: experiment (A)—15 mM TEA and 10 mM formic acid, experiment (B)—10 mM TEA and 10 mM formic acid, and experiment (C)—10 mM TEA and 15 mM formic acid. Also, an experiment using 5 mM TEA and 5 mM hydrochloric acid was performed (experiment D). To prepare the sample solution approximately 200 mg of the dichloromethane
extract of the barks of A. rigidum were dissolved in a mixture of 3 ml of the organic stationary phase with the retainer (TEA) and 3 ml of the aqueous mobile phase free of the acidic eluter. 2.5. Separation procedures First, the column was filled with the organic stationary phase, saturated with the mobile phase and containing the retainer (TEA), in a flow rate of 5 ml/min. Then, the rotation was turned on at the speed of 850 rpm and the sample solution was injected through the sample port, using a 5 ml loop. After this, the aqueous mobile phase containing the acidic eluter was pumped at a flow rate of 2 ml/min. About 60 fractions of 4 ml were collected (rotation was turned off at tube 40). Fractions were analyzed by TLC using the same conditions as specified in Section 2.3 and pooled together by means of chromatographic similarity. Retention of the stationary phase was determined by measuring the volume of stationary phase displaced until the solvent front; i.e. the first tube that presented two phases, then subtracting it from the total coil volume and calculating the percentage of stationary phase that remained inside the coil [17]. The pH values of each fraction were manually measured with a portable pH meter model Q400BC, from Quimis (Brazil). 2.6. Identification of the isolated substances The fractions containing the isolated alkaloids were concentrated in rotary evaporator (under reduced pressure, at 40 ◦ C) and then identified by the combination of 1D-/2D-NMR and MS spectral data. NMR experiments were performed on a Varian 500 MHz spectrometer. All samples were dissolved in deuterated chloroform, and tetramethylsilane (TMS) was used as internal standard. Chemical shifts (ı) are reported in ppm. The MS experiments were made by direct injection on a GC–MS QP 5000 Shimadzu equipment, using electron impact ionization at 70 eV. UV data were obtained from HPLC-DAD injections. 2.7. HPLC analyses The relative purity of the fractions was obtained by HPLC in a Merk-HITACHI LaChrom (Germany) apparatus L-7000, with UV/DAD detector L-7450, using a reversed phase ODS reversible analytical column (25 cm × 4.6 mm, 5 m) from Rexchrom Regis (USA) at room temperature of 25 ◦ C. The mobile phase solvent system consisted of a mixture of H2 O/TFA (pH 3; 0.025%) and CH3 CN/TFA (0.025%) in a stepwise gradient as follows: from 65:35 to 55:45 (0 to 10 min); from 55:45 to 40:60 (10.1 to 15 min); from 40:60 to 25:75 (15.1 to 28 min), and 0:100 from 28.1 to 30 min. The injection volume was 20 l, the flow rate 1.0 ml/min and detection was performed at 250 nm. The dichloromethane extract, as well as the fractions containing the separated alkaloids, were dissolved in a mixture (1:1) of the mobile phase components. LC–MS experiments were performed, in order to determinate the molecular weight of the isolated alkaloids. Samples ran in a HCT-Ultra ETD II (Bruker Daltonics) equipment, with a Prontosil ˚ and pre-column C18-Aq column of 250 × 2.0 mm, 5 micron (100 A)
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Fig. 1. TLC plates with the results of the pH-zone refining CCC. Experimental conditions: TLC—silica-gel plates developed with: ethyl-acetate:acetone:water (25:8:2) and a drop of a concentrated NH4 OH solution, detection: Dragendorff’s reagent; CCC—coil volume: 80 ml, flow-rate: 2 ml/min, rotation speed: 850 rpm, sample loading: 200 mg, fraction size: 4 ml, solvent system: MtBE–water; (A) 15 mM TEA in the OSP and 10 mM formic acid in the AMP, SPR: 67%; (B) 10 mM TEA in the OSP and 10 mM formic acid in the AMP, SPR: 40%; (C) 10 mM TEA in the OSP and 15 mM formic acid in the AMP, SPR: 67%; (D) 5 mM TEA in the OSP and 5 mM hydrochloric acid in the AMP, SPR: 55%. OSP—organic stationary phase, AMP—aqueous mobile phase, SPR—retention of stationary phase.
Fig. 2. Chemical structures of the alkaloids isolated from A. rigidum Rusby (1, 2 and 4).
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Fig. 3. HPLC analyses of the dichloromethane extract from the barks of A. rigidum (A) and the isolated alkaloids (B, C, and D). Experimental: Rexchrom Regis reversed phase ODS reversible analytical column (25 cm × 4.6 mm, 5 m), temperature of 25 ◦ C, mobile phase: H2 O/TFA (pH 3; 0.025%) and CH3 CN/TFA (0.025%) stepwise gradient from 65:35 to 55:45 (0 to 10 min); from 55:45 to 40:60 (10.1 to 15 min); from 40:60 to 25:75 (15.1 to 28 min) and 0:100 from 28.1 to 30 min, flow rate 1.0 ml/min, detection: 250 nm, injection volume: 20 l.
of the same material, at room temperature of 20 ◦ C. The mobile phase solvent system consisted of a mixture of H2 O/TFA (pH 3; 0.025%) and CH3 CN/TFA (0.025%) in a linear gradient from 80:20 to 10:90 (0 to 50 min). The injection volume was 10 l, the flow
rate 0.25 ml/min and detection was performed by DAD ( between 200 and 700 nm) and MS. The MS measurements were made in the range of m/z 100 to 2000 with electrospray ionization in the positive mode. The fragments obtained represented m/z [M+ + 1].
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3. Results and discussion Preliminary thin layer chromatography (TLC) analysis of the extracts obtained from the bark of A. rigidum showed the presence of four major alkaloids in the dichloromethane extract when stained with the Dragendorff’s reagent and, therefore, this was chosen for the separation experiments. As alkaloids can be regarded as weak organic bases, the reverse mode displacement pH-zone CCC technique was applied for the purification of these compounds. According to Ito [4] the suitable two-phase solvent system for pH-zone refining CCC is the one in which Kacid 1 in the experiments from the acidic condition, what was observed when adding formic acid to the test tubes, and Kbase 1 in the experiments from the basic condition. The solvent systems tested were composed of MtBE-CH3 CN–water in different ratios, as follows: 1:0:1 (S1), 4:1:5 (S2), 6:3:8 (S3) and 2:2:3 (S4) (v/v/v). Three of these (S2 to S4) showed an ideal Kacid value, but Kbase ≈ 1. The best condition was achieved when no CH3 CN was added to the system, making the system MtBE and water the most suitable choice (data not shown). 3.1. Fractionation of the A. rigidum dichloromethane extract Four experiments were performed in order to optimize the concentrations of the retainer base (TEA) and eluter acid (Table 1). Each of the three first experiments (A to C) provided, from circa 200 mg of the dichloromethane extract of A. rigidum, around 17 mg of 3␣-aricine, 1, 22 mg of isoreserpiline, 2, and 40 mg of 3reserpiline, 4, in a one-step separation of 2 h. Fig. 1 shows the results of the four pH-zone refining experiments (A to D) as analyzed by both TLC and pH measurements of each fraction tube. In experiment A (Fig. 1A) the elution of the alkaloids begins after around 54 min, coinciding with the decrease of the mobile phase’s pH value. First, 3-reserpiline, 4, elutes as a mixture with a small amount of 3 (non identified alkaloid) until isoreserpiline, 2, starts to elute. After another pH decrease, 2 elutes, followed by 3␣-aricine, 1. The stationary phase retention was of 67% and the pH flat zones are observed at pH 6.5, 5.0, 3.7 and 2.7. In experiment B (Fig. 1B), elution of the alkaloids starts around 10 min earlier than in A. First, a small decrease in the mobile phase pH was observed, representing the beginning of the elution of the mixture of 3-reserpiline, 4, and 3. This lasts for 6 min, then the pH decreases again and elution of isoreserpiline, 2, starts. In this experiment, there is an increase in resolution between 2 and 1, in comparison with A. The stationary phase retention was of 40% and the pH flat zones are observed at pH 9.0, 5.5, 3.5 and 2.4. Fig. 1C shows the result of experiment C. Here, elution begins even faster than in B, with the decrease of the pH values observed at around 38 min after the run started. In this experiment, although 3-reserpiline, 4, eluted in a mixture with 3, the proportion of the latter was significantly smaller. It is worth noting that also cleaner fractions of 2 were obtained, as well as those containing isoreserpiline, as the increased resolution was maintained. This fact was corroborated by the observation of an additional pH flat zone, compared to experiments A and B. These were at pH 7.3, 5.9, 4.7, 3.6 and 2.7. The stationary phase retention was of 67%. All the experiments showed a reasonable separation of the components from the alkaloidic dichloromethane extract of A. rigidum and allowed the isolation of three alkaloids – 1, 2 and 4 – with relative purity of 79%, 89% and 82% respectively. In all experiments, alkaloid 3 eluted in mixture either with 2 or 4. Comparing the experiments, it was possible to observe that increasing the eluter acid concentration in the aqueous mobile phase resulted in a shorter retention time of the analytes. Reducing the concentration of the retainer base in the upper organic phase changed the
stationary phase properties, increasing the resolution between the alkaloids. The pH-zone refining CCC method is characterized by the production of pH-zones according to pKa and hydrophobicity of the analytes [17], but in experiments A to C the major components were eluted with an abrupt variation from basic to acid in a few minutes in all experiments. In terms of chromatogram, it could represent a peak sharpening, that occurs when the K of the retainer (Kr ) falls between the analyte’s K under acidic (Kacid ) and basic (Kbase ) conditions [4]. This fact can be due to the utilization of an organic acid, which is relatively weak when compared to a mineral acid, in the aqueous mobile phase. Although it is not usual to add an organic modifier to the inorganic (aqueous) phase, this work showed that in some cases it can provide the most suitable condition for the separation of the compounds. On the other hand, 3␣-aricine, 1, did not seem to be affected by the change in the retainer’s and/or eluter’s concentrations. It eluted at the latest plateau during circa 18 min, after the retainer be entirely removed from the system, which means that its Kacid and Kbase values are greater than Kr [4]. In order to analyze the influence of the chemical nature of the acid in the separation, an experiment using the inorganic hydrochloric acid, instead of the organic formic acid was also made. As shown in Fig. 1D the pH flat zones were better defined. Elution of the alkaloids started after around 40 min, yielding fractions containing only 3-reserpiline, 4, followed, however, by mixtures of 3 + 2 and 3 + 1. The stationary phase retention was of 55% and the pH flat zones are observed at pH 5.6, 5.0 and 2.6. The four pH-zone refining experiments (A to D) afforded the separation of indole alkaloids from the dichloromethane extract of the barks of A. rigidum to different extents. By using hydrochloric acid it was possible to separate 3-reserpiline, 4, from the other alkaloids, but not to resolve 2 from 1. With the organic formic acid; however, this separation was possible in experiment C, although a major overlapping of the other components was observed.
3.2. Identification of the alkaloids Identification of the alkaloids 1, 2 and 4 was carried out by the combination of data from HPLC-UV, LC–ESI–MS and 1D-/2DNMR analyses. Therefore, 1 was identified as 3␣-aricine (MW 382 u) [18–21], 2 as isoreserpiline (MW 412) and 4 as 3-reserpiline (MW 412) [18,19,21], as shown in Fig. 2. Compound 3 remains unidentified. 3˛-aricine (1): UV and 1 H-NMR data [18–21]; 13 C NMR (125 MHz, CD3 OD) ␦ 168.16 (C-22), 155.48 (CH-17), 153.60 (C10), 134.95 (C-2), 131.85 (C 13), 127.17 (C-8), 111.17 (CH-12), 110.25 (CH-11), 109.49 (C-16), 106.42 (C-7), 99.49 (CH-9), 72.15 (CH-19), 60.43 (CH-3), 55.66 (CH2 -21, 54.83 (MeO-10), 53.48 (CH2 5), 50.21 (MeO-22), 38.44 (CH-20), 33.32 (CH2 -14, 31.24 (CH-15), 21.04 (CH2 -6), 17.43 (CH3 -18). MS-EI (70 eV): m/z 281 (29%), 253 (50%), 199 (37%), 186 (100%).isoreserpiline (2): UV, 1 H and 13 C-NMR data [18,19,21]. MS-EI (70 eV): m/z 311 (40%), 283 (74%), 229 (46%), 216 (100%). 3ˇ-reserpiline (4): UV, 1 H and 13 C-NMR data [18,19,21]. MS-EI (70 eV): m/z 311 (59%), 283 (100%), 216 (50%).
3.3. Analysis of the fractions by HPLC The extracts, as well as the separated fractions, were analyzed by HPLC and three intense peaks could be detected (Fig. 3A), which correspond, respectively, to compounds 2 and 4 (Rt 16.27 min, coeluted), 1 (Rt 17.84 min) and 3 (Rt 21.28 min). Although alkaloids 2 and 4 showed the same retention time in this HPLC conditions,
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they were well separated in the pH zone refining CCC experiments, as shown by their NMR data.
offering the resources to run the LC–MS analysis. This work was partially supported by CNPq (scholarship and grant).
4. Conclusions
References
The overall results of our work demonstrate that the pH-zone refining counter-current chromatography was successfully applied to the fractionation of indole alkaloids from the dichloromethane extract of Aspidosperma rigidum Rusby. Two of them – 3␣-aricine, 1, and isoreserpiline, 2 – were isolated and identified for the first time in this species. We also demonstrated that both organic and inorganic acids can be used as eluters in the aqueous mobile phase of the pH-zone refining countercurrent fractionation of these indole alkaloids. Although the profile of the experiments was different, all of them produced efficient separations. The present method may be applied to various other indole alkaloids from natural products.
[1] Y. Ito, W. Conway, Crit. Rev. Anal. Chem 17 (1986) 65. [2] W. Conway, Countercurrent Chromatography: Apparatus, Theory and Applications, VCH Publishers Inc., New York, 1989. [3] A. Weisz, A.L. Scher, K. Shinomiya, H.M. Fales, Y. Ito, J. Am. Chem. Soc 116 (1994) 704. [4] Y. Ito, Y. Ma, J. Chromatogr. A 753 (1996) 1. [5] Y. Ma, Y. Ito, E. Sokolosk, H.M. Fales, J. Chromatogr. A 685 (1994) 259. [6] J.H. Renault, J.M. Nuzillard, G. Le Crouerour, P. Thepenier, M. Zeches-Hanrot, L. Le Men-Olivier, J. Chromatogr. A 849 (1999) 421. [7] F. Yang, J. Quan, T. Zhang, Y. Ito, J. Chromatogr. A 822 (1998) 316. [8] F. Yang, T. Zhang, R. Zhang, Y. Ito, J. Chromatogr. A 829 (1998) 137. [9] R.R. Arndt, S.H. Brown, N.C. Ling, P. Roller, C. Djerassi, J.M. Ferreira, B. Gilbert, E.C. Miranda, S.E. Flores, Phytochemistry 6 (1967) 1653. [10] M.M. Pereira, R.L.R.P. Jácome, A.F.C. Alcântara, R.B. Alves, D.S. Raslan, Quim. Nova 30 (2007) 970. [11] A.G. Cunha, Dicionário Histórico das Palavras Portuguesas de Origem Tupi, 5th ed., Universidade de Brasília, São Paulo, Companhia Melhoramentos; Brasília, 1999. [12] M.K. Rai, G.A. Cordell, J.L. Martinez, M. Marinoff, L. Rastrelli, Medicinal Plants: Diversity and Drugs, Science Publishers, Enfield, 2011, pp. 160. ˜ [13] J. Sanz-Biset, S. Canigueral, J. Ethnopharmacol. 145 (2013) 67. [14] V.B. Oliveira, M.S.M. Freitas, L. Mathias, R. Braz-Filho, I.J.C. Vieira, Rev. Bras. Pl. Med. 11 (2009) 92. [15] M. Reina, W. Ruiz-Mesia, L. Ruiz-Mesia, R. Martínez-Díaz, A. González-Coloma, Z. Naturforsch. 66 c (2011) 225. [16] H. Wagner, S. Bladt, E.M. Zgainski, Plant Drug Analysis, Springer-Verlag, Berlin/Heidelberg, 1984. [17] Y. ITO, J. Chromatogr. A 1065 (2005) 145. [18] P. Gabetta, G. Mustich, Spectral Data of Indole Alkaloids, Inverni Della Beffa, Italy, 1975. [19] A. Bruyn, W. Zhang, M. Budesinsky, Magn. Reson. Chem 27 (1989) 935. [20] R. Verpoorte, E. Kos-Kuyck, T.A. Tsoi, C.L.M. Ruigrok, G. Jong, A.B. Svendsen, Planta Med. 48 (1983) 283. [21] M. Lounasmaa, A. Tolvaner, Heterocycles 24 (1986) 3229.
Acknowledgments The authors are thankful for Dr. Ma’s help with the pH-zone refining practical application and Dr. Ito’s explanations about some theoretical doubts. In addition, we would like to thank Prof. Dr. R. Braz-Filho and Prof. Dr. B. Gilbert, for the help with the identification of the alkaloids. We are also deeply indebted to the Centro Nacional de Ressonância Magnética Nuclear Jiri Jonas and LAMAR NPPN-UFRJ, Rio de Janeiro, for the NMR experiments and to ARQMO, Associac¸ão de Comunidades Remanescentes de Quilombos do Município de Oriximiná, Oriximiná-PA, Brazil, for supervising the plant collection. Furthermore, we wish to acknowledge the help provided by Prof. Dr. Peter Winterhalter and Dr. Gerold Jerz (Technische Universität Braunschweig, Germany) for
