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Journal of the Brazilian Chemical Society

versión impresa ISSN 0103-5053

J. Braz. Chem. Soc. vol.23 no.2 São Paulo feb. 2012 



Antiprotozoal alkaloids from Psychotria prunifolia (Kunth) Steyerm



Lucilia KatoI,*; Cecília M. A. de OliveiraI; Emiret O. FariaI; Laryssa C. RibeiroI; Brenda G. CarvalhoI; Cleuza C. da SilvaII; Ivânia T. A. SchuquelII; Silvana M. O. SantinII; Celso V. NakamuraIII; Elisandra A. BrittaIII; Nathielle MirandaIII; Amadeu H. IglesiasIV; Piero G. DelpreteV

IInstituto de Química, Universidade Federal de Goiás, Campus II, Samambaia, 74001-970 Goiânia-GO, Brazil
IIDepartamento de Química, Universidade Estadual de Maringá, Av. Colombo, 5790, 87020-900 Maringá-PR, Brazil
IIIDepartamento de Farmácia e Farmacologia, Universidade Estadual de Maringá, Av. Colombo, 5790, 87020-900 Maringá-PR, Brazil
IVWaters Technologies do Brasil LTDA, 125 / 27, Alphaville, 06455-020 Barueri-SP, Brazil
VHerbier de Guyane, Institut de Recherche pour le Développement (IRD), UMR AMAP, B.P. 165, 97323 Cayenne Cedex, French Guiana, France




The continuity of the phytochemical study of crude extracts of P. prunifolia's roots and branches led to the isolation of five indole-β-carboline alkaloids. Among them, the 10-hydroxy-iso-deppeaninol and N-oxide-10-hydroxy-antirhine derivatives are described here for the first time. The structures were achieved through 1D and 2D NMR, IR and HRMS analyses. The branches and roots crude extracts and the alkaloids 14-oxoprunifoleine and strictosamide showed selective activity against L. amazonensis, with IC50 values of 16.0 and 40.7 µg per mL, respectively.

Keywords: β-carboline alkaloids, antiprotozoal, Trypanosoma, Leishmania, Psychotria prunifolia, Rubiaceae


A continuidade do estudo fitoquímico de P. prunifolia com a análise dos extratos etanólicos obtidos a partir de suas raízes e galhos levou ao isolamento de cinco alcaloides indol-β-carbolínicos dos quais, dois derivados, o 10-hidróxi-iso-deppeaninol e o N-óxido-10-hidróxi-antirhina são descritos pela primeira vez. As estruturas foram determinadas por análise de técnicas espectroscópicas de IV, EMAR e RMN (1H e 13C, 1D e 2D). A avaliação da atividade frente à Leishmania amazonensis e Trypanosoma cruzi, mostrou que os extratos brutos e os alcaloides 14-oxoprunifoleína e estrictosamida inibiram as formas promastigotas de L. amazonensis, com valores de CI50 de 16,0 e 40,7 µg per mL, respectivamente.




The genus Psychotria, one of the largest genera of the Rubiaceae, has a long history of indigenous use as a component of the hallucinogenic beverage ayahuasca and also in traditional medicines used to treat microbial infections, inflammatory disease and complications of pregnancy. Pharmacological studies of Psychotria species, such as P. umbelata Tonn, P. leiocarpa Cham. & Schltdl, and P. insularum A. Gray revealed that their crude extracts possessed analgesic and allelophatic properties, as well as depressed the central nervous system and decreased the inflammatory action of cyclooxygenase.1-5

P. prunifolia is an understory shrub ranging from Venezuela, throughout the Amazon basin, to Bolivia and southern Brazil, with its southern limit in the state of São Paulo. Previous phytochemical studies carried out on several species of Psychotria have resulted in the identification of polypyrrolidine indole, monoterpenoid indole, and isoquinoline alkaloids.6-8 In our laboratory, phytochemical study of the leaves of P. prunifolia collected in Brazilian Cerrado have led to the isolation of β-carboline alkaloids and triterpenes.9

Numerous reports in the current literature provide evidence that alkaloids possessing an indole moiety could display important antiprotozoal activities. For example, alkaloids like harmane have exerted antiproliferative effects toward parasites of the genus Trypanosoma.10,11 The bis-indole alkaloids ramiflorines A and B isolated from Aspidosperma ramiflorum exhibited remarkable activity in in vitro assays against promastigote forms of L. amazonensis.12 In addition, indole alkaloids isolated from the bark of Corynanthe pachyceras (Rubiaceae) and Kopsia griffithii (Apocynaceae) showed activity against promastigotes of L. major and L. donavani.13,14

In this study, further phytochemical work was performed on P. prunifolia with particular attention to their alkaloid constituents, resulting in the isolation of two new beta carboline derivatives (1 and 3), besides the three known compounds 2, 4, and 5. Here we report the structural determination of the new alkaloids and the antiprotozoal activity of the crude extracts of P. prunifolia and the major alkaloids 4 and 5.


Results and Discussion

Structural elucidation of new alkaloids

Alkaloid 1 [α]25 = -21.5º (MeOH, c = 0.085) was obtained as a yellowish oil and was positive with Dragendorff's reagent. Analysis of HRMS data indicated that compound 1 had the molecular formula C19H22N2O3 (m/z 326.1630), [(M+H)+ observed at m/z 327.1693, calculated 327.1709, C19H23N2O3 ].

The 1H (1D and 2D COSY), 13C ({1H}, DEPT 90º and DEPT 135º), HMQC and HMBC NMR spectra revealed the presence of four sp3 methylene units (two of which were oxygenated), two olefinic carbons in a vinyl group, five aromatic and two aliphatic methines, and six sp2 quaternary carbons. A 1,2,4-trisubstituted aromatic and 1,2,3 trisubstituted-pyridine ring were present in the structure, as evidenced by signals at δH 7.42 (d, J 2.4 Hz, H-9), 7.36 (d, J 9.0 Hz, H-12), 7.05 (dd, J 9.0, 2.4 Hz, H-11), and at δH 8.06 (d, J 5.4 Hz, H-5) and 7.84 (d, J 5.4 Hz, H-6) correlated to δC 106.6, 113.7, 120.4, 135.7 and 114.6 in the HMQC spectra, respectively. HMBC correlations among H-6/C-8 (δC 123.1) and C-2 (δC 136.9), together with the correlation observed for H-5/C-3 (δC 145.5) and C-7 (δC 130.6), helped us to assign the presence of an indole β-carboline moiety substituted in position 10 or 11 by a hydroxyl group. The 3J HMBC correlation H-11/C-13 (δC 137.5), in addition to the NOE correlation between H-6/H-9 and the coupling pattern from aromatic protons with JH9-H11 2.4 and JH11-H12 9.0 Hz, corroborated the hydroxyl at C-10 (δC 152.6).

Apart from the indole β-carboline ring, the spectral data (HMQC and 13C) showed two methine groups at δC 36.4 and 51.0, two oxygenated methylenes (δC 61.4 and 64.4), and one vinyl portion at δC 118.7 (methylene) and 138.1 (methine). Information from COSY and HMQC data showed spin systems ranging for H-14/H-15/H-16/H-17, H-15/H-20/H-21, and H-18/H-19/H-20. The 2J HMBC correlation observed between H-14/C-3 and 3J observed for H-14/C-16 and H-14/C-20 permitted us to suggest the connection between the aliphatic unit and the indolic β-carboline unit (Figure 1). Further HMBC correlations are described in Table 1. All these data were consistent with a new alkaloid similar to 10-hydroxy derivative of deppeaninol, which was assigned as alkaloid 1 identified as 10-hydroxy-iso-deppeaninol. The relative configuration proposed is that observed of corynantheine-heteroyohimbine alkaloids. Deppeaninol was isolated from Deppea blumenaviensis (Rubiaceae) and described by Kan-Fan et al.15



Alkaloid 2 was obtained as a brownish oil and was positive to Dragendorff's reagent. Its molecular formula was determined by HRMS, which exhibited a molecular ion [M+H]+ observed at m/z 313.1920 (calculated m/z 313.1916, C19H25N2O2) consistent with a molecular formula of C19H24N2O2 (m/z 312.1838). The 1H (1D and 2D COSY), 13C ({1H}, DEPT 90º and DEPT 135º), HMQC and HMBC NMR spectra data showed the presence of six sp3 methylene units (one of which was oxygenated), two olefinic carbons (one of which was a methylene unit), three aromatic and three aliphatic methines, and five sp2 quaternary carbons. The resonances and the 1H NMR J values at δH 6.80 (d, J 2.4 Hz), 6.68 (dd, J 8.7 and 2.4 Hz) and 7.17 (d, J 8.7 Hz) were attributed to a trisubstituted aromatic system with a 10 or 11-substitution pattern.

In addition to these assignments, the HMBC correlations from H-5a (δH 3.57, dd, J 12.6 and 6.0 Hz) to C-7 (δC 106.0) and from H-6a (δH 2.82, dd, J 16.2 and 4.8 Hz) and H-6b (δH 3.01, m) to C-7 (δC 106.0) and C-2 (δC 130.5) indicated the presence of a tetrahydro-β-carboline moiety in the structure of alkaloid 2.

Analysis of COSY data indicated proton spin systems corresponding to H-16/H-17, H-3/H-14/H-15 and H-18/H-19/H-20/H-21, suggesting a cyclic terpene scaffold. HMBC correlations from H-18 to C-20 and H-21 to C-20/C-15 further revealed the connectivities in the terpene unit. The 3J HMBC correlations among H-6 to C-2, H-5 to C-17 and H-14 to C-2 led to the direct connection of the indole and terpene moieties. All of these assignments, combined with the literature data, permitted us to identify alkaloid 2 as 10-hydroxy-antirhine, which was previously isolated from Ochrosia alyxiodis.16

Alkaloid 3 (6.2 mg) [α]25 = +108.2º (MeOH, c = 0.135) was isolated as a brown oil and was also positive with Dragendorff's reagent. The HR ESI-MS exhibited a quasi-molecular [M-H]- ion peak at m/z 327.1712, (calculated 327.1709, C19H23N2O3) consistent with the molecular formula C19H24N2O3. (m/z 328.1787). This ion was 16 amu higher than that of compound 2, which is typical for N-oxide derivatives.

The signals at δH 6.71 (d, J 2.1 Hz, H-9), 6.60 (dd, J 8.7 and 2.1 Hz, H-11) and 7.08 (d, J 8.7 Hz) suggest the same pattern of aromatic moiety. In the COSY spectra could be identified spin systems of H-9/H-11/H-12, H-5/H-6, H-3/H-14, H-16/H-17 and H-18/H-19. The HMBC experiment showed correlation among H-5/C-17, H-6/C-2, H-14/C-2/C-3/C-15, H-16/C-15, H-18/C-20 and H-21/C-15/C-20, establishing a connection between the terpene and indole units. The analysis of all the spectral data suggested that 3 possessed the same skeleton of 2, but with differences in the chemical shifts at the carbons C-3, C-5 and C-17 of Δδ +14.6, +16.6 and +11.1, respectively. This deshielding effect observed in the 13C could be attributed to the influence of an N-oxide at the N-4 position and permitted us to assign alkaloid 3 as a 10-hydroxy-antirhine N-oxide derivative. The alkaloids 4 and 5 were isolated previously9 and identified as 14-oxoprunifoleine and strictosamide, respectively.

Antiprotozoal assay

In this study, the crude extracts of roots and branches and the major alkaloids 4 and 5 were evaluated for in vitro antiprotozoal activity against promastigotesof Leishmania amazonensis and epimastigotes of Trypanosoma cruzi strains. Table 3 summarises the antiprotozoal activity data for the ethanolic crude extracts and the known alkaloids (4 and 5). In both cases, L. amazonensis was the most sensitive protozoan. The ethanolic root extract was the most active among the extracts assayed. For comparison, the two alkaloids 4 and 5 showed activity against promastigote forms of L. amazonensis with IC50 values of 16.0 and 40.7 µg per mL, respectively.


Table 2





Plant material

Fresh material of P. prunifolia was collected in September 2007 in the municipality of Goiânia and identified by Piero Delprete of the Federal University of Goiás, at Bosque A. Saint-Hilaire. The plants were found in understory vegetation of seasonal semi-deciduous forest, at 16º36'12"S, 49º15'41"W and 850 m altitude. The voucher specimen, Delprete 10323, was deposited at the Herbarium (UFG) of the Federal University of Goiás, Goiânia.9

Extraction and isolation

The air-dried and powdered branches (149 g) were successively extracted with EtOH. The resulting extract was filtered and concentrated under reduced pressure to yield 17 g. An amount of 12 g of the resulting ethanolic extract was added to 10% aq. HOAc (100 mL). The resulting suspension was incubated at 5 ºC overnight. The suspension was then filtered, and the acidic aqueous phase was extracted with CHCl3 (3 × 150 mL). The combined organic layers were treated with Na2SO4 and filtered, affording a CHCl3 acidic fraction (0.8 g - fraction A).

The aqueous layer was adjusted to pH 8-9 with a saturated aq. NaHCO3 solution, and then again extracted with CHCl3 (3 × 150 mL). The combined organic layers were treated with Na2SO4 and filtered, affording CHCl3 basic fractions (0.2 g - fraction B). This process was repeated using EtOAc as solvent to afford an EtOAc basic fraction (1.35 g - fraction C).

Compounds 1 (4 mg), 2 (18 mg) and 5 (12 mg) were obtained from fraction C (1.0 g) by repeated CC on silica gel 60 (EtOAc-MeOH-NH4OH eluent system in gradient form) and successive purification by preparative TLC on silica gel with EtOAc-MeOH-NH4OH (77:20:3). The fraction A after successive treatment by CC on silica gel 60 (CHCl3-MeOH-NH4OH eluent system in gradient form) and preparative TLC (CHCl3-MeOH 75-25) yielded 6 mg of compound 4. Compound 3 (6.3 mg) was isolated by successive fractionation by CC on silica gel 60 (CHCl3-MeOH eluent system in gradient form) from ethanolic extract from branches (3.88 g).

The crude extract of roots (9 g) was obtained by extraction with ethanol from 150 g of powdered roots. Then 5 g of this ethanolic extract was submitted to the same acid-base treatment described above and yielded CHCl3 acid fraction (0.06 g - fraction D), CHCl3 basic (0.04g - fraction E) and AcOEt basic fraction (0.1g - fraction F). Preparative TLC from fraction F (61 mg) using CH2Cl2-MeOH (20-1) as eluent system furnished 5 (8.5 mg).

General procedures

IR spectra were recorded with a FTIR Bomem MB100 using KBr pellets. Optical rotations were obtained with a Bellingham+Stanley Ltd ADP 440. NMR spectra were recorded with a Varian Mercury spectrometer operating at 300.1 MHz for 1H and at 75.5 MHz for 13C. CD3OD was used as the solvent, with Me4Si (TMS) used as the internal standard. HRMS was performed with a Synapt HDMS spectrometer in positive (or negative) ionisation modes of electrospray ionisation (ESI) (Waters Corporation). TLC was conducted using precoated Kiesegel 60 F254 plates (Merck and M. Nagel). The spray developing reagents used for TLC were 50% H2SO4 in CH3OH and Dragendorff's reagent.

Antiprotozoal assay17

The effects of the extract and alkaloids were evaluated in promastigotes of L. amazonensis and epimastigotes of T. cruzi (Y strain). For the assay, epimastigote forms of T. cruzi (Y strain) were harvested during the exponential phase of growth, resuspended in liver infusion tryptose broth supplemented with 10% inactivated foetal bovine serum (Gibco Invitrogen Corporation, New York, NY, USA) and plated on 24-well plates at a concentration of 1 × 106 cells per mL. One millilitre of diluted compound was included in each well and incubated for 96 h at 28 ºC. Cell density was determined by counting the parasites in a hemocytometer chamber (Improved Double Neubauer) under a light microscope. All assays were performed in duplicate on separate occasions. The results were expressed as percentage of inhibition in relation to the control. The 50% inhibitory concentration (IC50) was determined by logarithm regression analysis of the obtained data.

Parasites were treated with different concentrations (10.0, 50.0, 100.0, 500.0 and 1000.0 µg mL-1) of crude extract and 1.0, 5.0, 10.0, 50.0 or 100.0 µg mL-1 of isolated alkaloids (4 and 5).


Supplementary Information

Supplementary data (NMR, HRMS and IR spectra and spectroscopic data) is available free of charge at as PDF file.



We acknowledge the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Nível Superior (Capes) and Fundação de Apoio à Pesquisa (Funape)-UFG for financial support.



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Submitted: May 19, 2011
Published online: December 6, 2011



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