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Taxonomic significance of alkaloids and iridoid glucosides in the tribe Psychotrieae (Rubiaceae)
Biochemical Systematics and Ecology 32 (2004) 1187–1195 www.elsevier.com/locate/biochemsyseco
Taxonomic significance of alkaloids and iridoid glucosides in the tribe Psychotrieae (Rubiaceae) Sı´lvia Lopes, Gilsane L. von Poser, Vitor A. Kerber, Fabiane M. Farias, Eduardo L. Konrath, Paulo Moreno, Marcos E. Sobral, Jose´ A.S. Zuanazzi, Ame´lia T. Henriques Programa de Po´s-Graduac¸a˜o em Cieˆncias Farmaceˆuticas, Universidade Federal do Rio Grande do Sul (UFRGS), Av. Ipiranga, CEP 90.610-000, 2752 Porto Alegre, RS, Brazil Received 30 August 2002; accepted 30 April 2004
Abstract Leaves of 15 Brazilian species of Psychotria, three of Rudgea and Palicourea rigida, were analyzed for their alkaloid and iridoid content. Alkaloids were found in three of Rudgea and 14 species of Psychotria, and iridoids were found in Psychotria leiocarpa, which produces asperuloside and deacetylasperuloside. Palicourea rigida yielded no alkaloids but loganin was isolated. The results illustrate the significance of the alkaloids in the chemotaxonomy of some taxa of Psychotrieae. The phytochemical data indicate that the American species of Psychotria with Palicourea could be joined to form the genus Heteropsychotria. # 2004 Elsevier Ltd. All rights reserved. Keywords: Psychotria; Palicourea; Rudgea; Rubioideae; Rubiaceae; Alkaloids; Iridoid glucosides; Taxonomy
1. Introduction The tribe Psychotrieae (subfamily Rubioideae) comprises about 50 genera; some of them with unclear generic limits due to the lack of adequate morphological characters to define their boundaries. The generic delimitation of this tribe has been the object of investigations by several authors (Taylor, 1989, 1996; Nepokroeff et al., 1999) initially using purely morphological characters, but more recent
Corresponding author. Tel./fax: +55-51-3316-5243. E-mail address: [email protected] (A.T. Henriques).
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molecular data suggest that there should be a revision of generic limits, mainly in the Psychotria alliance. Psychotria L., distributed in tropical regions is one of the largest genera of flowering plants with 1000–1650 species worldwide and is taxonomically complex (Nepokroeff et al., 1999). Petit (1964, 1966) and Steyermark (1972) recognized three subgenera: Psychotria (pantropical), Tetramerae (includes some species from Africa and Madagascar) and Heteropsychotria (includes the remainder of the species of Psychotria in the neotropics). The division into subgenera is based on morphological characters and geographical distribution. Species of Palicourea Aubl. and Rudgea Salisb. are most closely related to members of subg. Heteropsychotria based on their shared persistent and continuously connate stipules, versus the deciduous stipules found in other Psychotria (Taylor, 1989, 1996). Molecular phylogenetic analysis proposed the fusion of Palicourea and the subgenus Heteropsychotria in a genus of its own (Nepokroeff et al., 1999). If this viewpoint is accepted, this new inclusive genus would be named Psychotrophum P. Browne, based on Taylor’s (1996) arguments of priority rules. The present paper is part of a search for alkaloids and iridoid glucosides in native Rubiaceae species of Brazil. It deals with the chemical screening of 15 species of Psychotria, (subg. Heteropsychotria), Palicourea rigida H.B.K., three species of Rudgea and contributes to the systematic analysis of these taxa.
2. Materials and methods 2.1. Plant material The species of Psychotria surveyed are shrubs to treelets of tropical and subtropical forest formations of southern and southeastern Brazil; those of Rudgea are treelets from southern Brazilian coastal forests and Palicourea rigida is a shrub from rocky areas of central Brazil. Voucher specimens were deposited in the herbarium of the Universidade Federal do Rio Grande do Sul (ICN). Collected species, voucher number and sites of collection are presented in Table 1. 2.2. Chemical methods 2.2.1. Iridoid detection The dried leaves were powdered, extracted with EtOH and the concentrated extracts were partitioned in Et2O–H2O. The aqueous phases were concentrated giving the crude extracts. These crude extracts were analyzed by TLC eluting with CH2Cl2/MeOH (4:1) and submitted to 1H NMR analysis which can detect concentration in iridoids lower than 0.01% (Jensen et al., 1988). A larger batch of Psychotria leiocarpa (180 g) and Palicourea rigida (50 g) (which showed the characteristic 1H NMR signals of iridoid glycosides) was extracted as described above. The concentrated aqueous extracts (8.2 and 3.4 g, respectively) were submitted to column chromatography on silica gel using a CHCl3/MeOH gradient system followed by preparative TLC eluting with CH2Cl2/MeOH (4:1, V/V)
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Table 1 Species of Psychotrieae investigated for alkaloids and iridoid glucosides contents Species
Site of collection
Voucher number
Palicourea rigida H.B.K. Psychotria alba Ruiz et Pav. P. barbiflora DC. P. brachyceras Mu¨ll.Arg.
Bom Jardim de Minas, MG Blumenau, SC Blumenau, SC Porto Alegre, RS
P. carthagenensis Jacq. P. deflexa DC. P. hancorniifolia Benth. P. kleinii L.B.Sm. et Downs P. leiocarpa Cham. Et Schltdl. P. longipes Mu¨ll.Arg. P. myriantha Mu¨ll.Arg. P. nuda (Cham. Et Schltdl.) Wawra P. pleiocephala Mu¨ll.Arg. P. pubigera Schltdl. P. suterella Mu¨ll.Arg. P. umbellata Vell. Rudgea heurckii Mu¨ll.Arg. R. jasminoides (Cham.) Mu¨ll.Arg. R. recurva Mu¨ll.Arg.
Porto Alegre, RS Blumenau, SC Juiz de Fora, MG Blumenau, SC Porto Alegre, RS Blumenau, SC Tenente Portela, RS Blumenau, SC Juiz de Fora, MG Blumenau, SC Santo Antonio da Patrulha, RS Morretes, PR Peruı´be, SP Torres, RS Blumenau, SC
Sobral 8180 Sobral 9054 Sobral 9056 Sobral e Kerber 7899— UFRGS—08/1995 Sobral 7901 Sobral 9055 Sobral 8216 Sobral 8469 Sobral 7898 Sobral 8912 Sobral 9044 Sobral 8913 Sobral 8213 Sobral 8911 Sobral 8336 Hatschbach (MBM 48571) Sobral 7320 Sobral 9052 Sobral 9053
ES, Espı´rito Santo; MG, Minas Gerais; PR, Parana´; RS, Rio Grande do Sul; SC, Santa Catarina; SP, Sa˜o Paulo.
providing (1) asperuloside (13 mg) and (2) deacetylasperuloside (9 mg) from Psychotria leiocarpa and (3) loganin (37 mg) from Palicourea rigida. The products were identified by 1H NMR and 13C NMR (El Naggar and Beal, 1980; Boros and Stermitz, 1990; Gonza´lez and Dieck, 1996; Peng et al., 1999). No iridoid glucosides were detected in the other species analyzed.
,
,
2.2.2. Asperuloside (1) 1 H NMR (400 MHz, D2O) d: 5.75 (d, J ¼ 2:2 Hz, H-1), 7.37 (d, J ¼ 1:8 Hz, H-3), 3.60–3.10 (m, H-5), 5.68 (br d, J ¼ 7 Hz, H-6), 5.90 (br s, H-7), 3.60–3.10 (m, H-9), 4.85 (br s, H-10), 2.10 (s, AcO), 4.70 (d, 9.0 Hz, H-10 ), 4.20–3.15 (m, H-20 , H-30 , H-40 and 50 ), 3.90–3.68 (m, H-60 ).
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13
C NMR (100 MHz, D2O) d: 99.1 (C-1), 150.1 (C-3), 105.2 (C-4), 36.4 (C-5), 86.5 (C-6), 128.5 (C-7), 142.9 (C-8), 44.1 (C-9), 61.5 (C-10), 173.5 (C-11), 93.3 (C-10 ), 73.2 (C-20 ), 76.9 (C-30 ), 70.2 (C-40 ), 76.3 (C-50 ), 61.5 (C-60 ), 173.7 (CO–AcO). 2.2.3. Deacetylasperuloside (2) 1 H NMR (400 MHz, D2O) d: 5.70 (d, J ¼ 2 Hz, H-1), 7.33 (d, J ¼ 2 Hz, H-3), 3.55–3.15 (m, H-5), 5.66 (br d, J ¼ 7 Hz, H-6), 5.70 (br s, H-7), 3.55–3.15 (m, H-9), 4.15 (br s, H-10), 4.80 (d, 8.0 Hz, H-10 ), 4.45–3.10 (m, H-20 , H-30 , H-40 and 50 ). 13 C NMR (100 MHz, D2O) d: 99.3 (C-1), 152.4 (C-3), 106.2 (C-4), 37.2 (C-5), 87.1 (C-6), 129.2 (C-7), 147.7 (C-8), 44.9 (C-9), 61.9 (C-10), 174.3 (C-11), 93.1 (C-10 ), 73.6 (C-20 ), 77.0 (C-30 ), 70.6 (C-40 ), 76.1 (C-50 ), 61.5 (C-60 ). 2.2.4. Loganin (3) 1 H NMR (400 MHz, D2O) d: 5.35 (d, J ¼ 4:0 Hz, H-1), 7.34 (s, H-3), 3.05 (m, H-5), 2.10–1.70 (m, H-6), 4.08 (m, H-7), 2.10 (m, H-9), 1.05 (d, J ¼ 7:5 Hz, H-10), 4.60 (d, 8.0 Hz, H-10 ), 4.45–3.15 (m, H-20 , H-30 , H-40 and 50 ), 3.90–3.68 (m, H-60 ), 3.65 (s, OMe). 13 C NMR (100 MHz, D2O) d: 97.4 (C-1), 151.7 (C-3), 114.1 (C-4), 31.0 (C-5), 41.3 (C-6), 75.8 (C-7), 40.8 (C-8), 46.0 (C-9), 13.2 (C-10), 171.2 (C-11), 99.8 (C-10 ), 74.0 (C-20 ), 77.0 (C-30 ), 70.2 (C-40 ), 77.5 (C-50 ), 61.5 (C-60 ), 53.0 (OMe). 2.2.5. Alkaloid detection The crude alkaloid extracts of dried leaves, obtained by classical acid/base partition, were directly evaluated by HPLC/PDA detector. Aliquots of these extracts were loaded onto a Waters 2690 HPLC system (Millipore Corp.) fitted with a 3:9 150 mm Nova-Pak C18-4 lm column (Waters), preceded by a guard-column, eluted at 1 ml min1 from 0 to 15 min with a linear gradient solvent system from 50:50 (V/V) methanol/water to methanol. The analysis continued isocratically for other 20 min. Eluting compounds were monitored with a Waters Millenium (version 2.15.01) and a Waters 996 photodiode array detector, which measured absorbance (200–400 nm) every 1.8 s with 4.8 nm resolution. The presence of indole UV spectra (instead of the indoline chromophore from the polyindolinic alkaloids) was observed and taken as indicative for the presence of monoterpene indole alkaloids. From some species, the alkaloids were isolated and identified: Psychotria umbellata yielded umbellatine (4) and the derivatives 3,4-dehydro-18,19-b-epoxyumbellatine, N4-[1-(2a-hydroxypropyl)]-umbellatine and N4-[1-(2a-hydroxypropyl)]-umbellatine (Kerber, 1999); from Psychotria brachyceras, brachycerine (5) was isolated (Kerber et al., 2001); lyaloside (6) and strictosamide (7) were found in Psychotria suterella (De Santos et al., 2001); myrianthosines A (8) and B (9) were isolated from Psychotria myriantha (Farias, 2004) while Psychotria leiocarpa afforded N,b-d-glucopyranosil vincosamide (10) (Lopes, 1998).
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3. Results and discussion The main metabolites found in pantropical Psychotria (subgenus Psychotria) are alkaloids of polyindoline type and one of the most frequently reported is quadrigemine A (11). This group of compounds is derived from condensation of several N-methyltryptamine moieties, and they seem to be characteristic of the species from subgenus Psychotria (De Santos et al., 2001). With the exception of P. colorata (Elisabetsky et al., 1997; Verotta et al., 1998) and P. glomerulata (Solı´s et al., 1995), these alkaloids have not been detected in neotropical Psychotria (subgenus Heteropsychotria) which have, instead, yielded monoterpene indole alkaloids (Solı´s et al., 1993; Achenbach et al., 1995; Lopes, 1998; Kerber, 1999; De Santos et al., 2001; Kerber et al., 2001). The two species cited above were formerly included in the genus Cephaelis, which is considered by some authors (Steyermark, 1972; Taylor, 1996; Solı´s et al., 1997) as a synonym of Psychotria (subgenus Hetero-
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psychotria). However, the main type of alkaloids of Cephaelis, such as emetine and cephaeline (Southon and Buckinghan, 1989), derive from tyrosine, instead of tryptophan. Thus, from the chemosystematic viewpoint, the merging of these two genera seems to be unlikely.
Only a few species of Palicourea, an exclusively neotropical genus, have been analyzed for alkaloid content. P. dominguensis (Ripperberger, 1982), P. fendlerii (Nakano and Martin, 1976), P. alpina (Woo Ming and Stuart, 1975) and P. ovalis (Garcia et al., 1997) present polyindolines. The compounds described are dimers in contrast with the ones found in Psychotria, which contain several degrees of polymerization. Both genera also produce monoterpene indole alkaloids, derived from tryptamine (Morita et al., 1989; Valverde et al., 1999), in addition to the polyindoline alkaloids. The total alkaloids fraction of the leaves of 15 Brazilian Psychotria species, three Rudgea species and Palicourea rigida were analyzed by HPLC/PDA and peaks presenting UV spectra characteristic for the indole chromophore (223 and 280 nm) were detected in 14 Psychotria and three Rudgea species. This chromophore was not detected in Psychotria carthagenensis and Palicourea rigida. In P. suterella, it was found substances with the extended indol chromophore (237, 289 and 334 nm) characterizing b-carbolines (De Santos et al., 2001). In P. umbellata together with the indole group it was also observed compounds with UV spectra for 5-6-dihydrob-carboline (236 and 320 nm) (Kerber, 1999). No alkaloids were detected in P. carthagenensis (Lopes et al., 2000) and Palicourea rigida, and only traces of alkaloids were found in Psychotria hancorniifolia. Glucoside monoterpene indole alkaloids were isolated from P. brachyceras (Kerber et al., 2001), P. leiocarpa (Lopes, 1998), P. suterella (De Santos et al., 2001) and P. umbellata (Kerber, 1999). This type of alkaloid was firstly found in the neotropical Heteropsychotria subgenus in the species P. dichroa (Cephaelis dichroa) (Solı´s et al., 1993) and Psychotria correae (Cephaelis correae) (Achenbach et al., 1995). Palicourea alpina (Stuart and Woo-
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Ming, 1974), P. markgravii (Morita et al., 1989) and P. adusta (Valverde et al., 1999) also contain monoterpene indole alkaloids and such data could corroborate with the inclusion of part of Palicourea in the subgenus Heteropsychotria. Therefore, alkaloids can be a useful tool to distinguish groupings within a complex genus such as Psychotria, as previously proposed (Solı´s et al., 1995). No chemical investigations of Rudgea species were previously reported. In addition to the alkaloids, iridoid glucosides such as asperuloside (1) and deacetylasperuloside (2) were located in some species of Psychotria. Inouye et al. (1988) reported the occurrence of these compounds in P. rubra (Lour) Poir., P. serpens L. and P. manillensis Bartl. Ex D.C. In addition, Gonza´lez and Dieck (1996) isolated asperuloside from aerial parts of a plant named by them as ‘‘Psichotria mariniana’’. In the ethanolic extract of the leaves of Psychotria leiocarpa, the mentioned iridoids asperuloside and deacetylasperuloside were found only in small amounts. On the other hand, Palicourea rigida presents high concentrations of loganin (3), never found in species of Psychotria, although it is one of the intermediates in the biosynthetic pathway of monoterpene indole alkaloids. This product presents the same configuration (8b) of the iridoids frequently isolated from species of subfamily Rubioideae. This is the first report of iridoids in Palicourea. The results are summarized in Table 2. In conclusion, our findings reinforce the proposition from Solı´s et al. (1995) that alkaloids are important taxonomic markers and Taylor’s (1996) hypothesis that the Table 2 Alkaloids and iridoids in Psychotria, Palicourea and Rudgea species Species
Iridoid glucosides
Alkaloids
Palicourea rigida H.B.K. Psychotria alba Ruiz et Pav. P. barbiflora DC. P. brachyceras Mu¨ll.Arg. P. carthagenensis Jacq. P. deflexa DC. P. hancorniifolia Benth. P. kleinii L.B.Sm. et Downs P. leiocarpa Cham. Et Schltdl.
Loganin (3) n.d. n.d. n.d. n.d. n.d. n.d. n.d. Asperuloside (1); deacetylasperuloside (2) n.d. n.d. n.d. n.d. n.d. n.d. n.d.
n.d. i.c. i.c. Brachycerine (5) n.d. i.c. i.c. i.c. N,b-d-Glucopyranosil vincosamide (10) i.c. Myrianthosines A (8) and B (9) i.c. i.c. i.c. Lyaloside (6); strictosamide (7) Umbellatina (4) and derivatives i.c. i.c. i.c.
P. P. P. P. P. P. P.
longipes Mu¨ll.Arg. myriantha Mu¨ll.Arg. nuda (Cham. Et Schltdl.) Wawra pleiocephala Mu¨ll.Arg. pubigera Schltdl. suterella Mu¨ll.Arg. umbellata Vell.
Rudgea heurckii Mu¨ll.Arg. R. jasminoides (Cham.) Mu¨ll.Arg. R. recurva Mu¨ll.Arg.
n.d. n.d. n.d.
n.d., not detected; i.c., indole chromophore (detected by HPLC/PDA).
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American Psychotria, together with Palicourea, could be joined to form the genus Heteropsychotria. Additionally, the data achieved are in agreement with the statements of Nepokroeff et al. (1999) that some groups previously assigned to Psychotria (i.e. subg. Heteropsychotria plus Palicourea) are more closely related to other genera in the Psychotrieae than they are to other species of Psychotria.
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Taxonomic significance of alkaloids and iridoid glucosides in the tribe Psychotrieae (Rubiaceae) Sı´lvia Lopes, Gilsane L. von Poser, Vitor A. Kerber, Fabiane M. Farias, Eduardo L. Konrath, Paulo Moreno, Marcos E. Sobral, Jose´ A.S. Zuanazzi, Ame´lia T. Henriques Programa de Po´s-Graduac¸a˜o em Cieˆncias Farmaceˆuticas, Universidade Federal do Rio Grande do Sul (UFRGS), Av. Ipiranga, CEP 90.610-000, 2752 Porto Alegre, RS, Brazil Received 30 August 2002; accepted 30 April 2004
Abstract Leaves of 15 Brazilian species of Psychotria, three of Rudgea and Palicourea rigida, were analyzed for their alkaloid and iridoid content. Alkaloids were found in three of Rudgea and 14 species of Psychotria, and iridoids were found in Psychotria leiocarpa, which produces asperuloside and deacetylasperuloside. Palicourea rigida yielded no alkaloids but loganin was isolated. The results illustrate the significance of the alkaloids in the chemotaxonomy of some taxa of Psychotrieae. The phytochemical data indicate that the American species of Psychotria with Palicourea could be joined to form the genus Heteropsychotria. # 2004 Elsevier Ltd. All rights reserved. Keywords: Psychotria; Palicourea; Rudgea; Rubioideae; Rubiaceae; Alkaloids; Iridoid glucosides; Taxonomy
1. Introduction The tribe Psychotrieae (subfamily Rubioideae) comprises about 50 genera; some of them with unclear generic limits due to the lack of adequate morphological characters to define their boundaries. The generic delimitation of this tribe has been the object of investigations by several authors (Taylor, 1989, 1996; Nepokroeff et al., 1999) initially using purely morphological characters, but more recent
Corresponding author. Tel./fax: +55-51-3316-5243. E-mail address: [email protected] (A.T. Henriques).
0305-1978/$ - see front matter # 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.bse.2004.04.015
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molecular data suggest that there should be a revision of generic limits, mainly in the Psychotria alliance. Psychotria L., distributed in tropical regions is one of the largest genera of flowering plants with 1000–1650 species worldwide and is taxonomically complex (Nepokroeff et al., 1999). Petit (1964, 1966) and Steyermark (1972) recognized three subgenera: Psychotria (pantropical), Tetramerae (includes some species from Africa and Madagascar) and Heteropsychotria (includes the remainder of the species of Psychotria in the neotropics). The division into subgenera is based on morphological characters and geographical distribution. Species of Palicourea Aubl. and Rudgea Salisb. are most closely related to members of subg. Heteropsychotria based on their shared persistent and continuously connate stipules, versus the deciduous stipules found in other Psychotria (Taylor, 1989, 1996). Molecular phylogenetic analysis proposed the fusion of Palicourea and the subgenus Heteropsychotria in a genus of its own (Nepokroeff et al., 1999). If this viewpoint is accepted, this new inclusive genus would be named Psychotrophum P. Browne, based on Taylor’s (1996) arguments of priority rules. The present paper is part of a search for alkaloids and iridoid glucosides in native Rubiaceae species of Brazil. It deals with the chemical screening of 15 species of Psychotria, (subg. Heteropsychotria), Palicourea rigida H.B.K., three species of Rudgea and contributes to the systematic analysis of these taxa.
2. Materials and methods 2.1. Plant material The species of Psychotria surveyed are shrubs to treelets of tropical and subtropical forest formations of southern and southeastern Brazil; those of Rudgea are treelets from southern Brazilian coastal forests and Palicourea rigida is a shrub from rocky areas of central Brazil. Voucher specimens were deposited in the herbarium of the Universidade Federal do Rio Grande do Sul (ICN). Collected species, voucher number and sites of collection are presented in Table 1. 2.2. Chemical methods 2.2.1. Iridoid detection The dried leaves were powdered, extracted with EtOH and the concentrated extracts were partitioned in Et2O–H2O. The aqueous phases were concentrated giving the crude extracts. These crude extracts were analyzed by TLC eluting with CH2Cl2/MeOH (4:1) and submitted to 1H NMR analysis which can detect concentration in iridoids lower than 0.01% (Jensen et al., 1988). A larger batch of Psychotria leiocarpa (180 g) and Palicourea rigida (50 g) (which showed the characteristic 1H NMR signals of iridoid glycosides) was extracted as described above. The concentrated aqueous extracts (8.2 and 3.4 g, respectively) were submitted to column chromatography on silica gel using a CHCl3/MeOH gradient system followed by preparative TLC eluting with CH2Cl2/MeOH (4:1, V/V)
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Table 1 Species of Psychotrieae investigated for alkaloids and iridoid glucosides contents Species
Site of collection
Voucher number
Palicourea rigida H.B.K. Psychotria alba Ruiz et Pav. P. barbiflora DC. P. brachyceras Mu¨ll.Arg.
Bom Jardim de Minas, MG Blumenau, SC Blumenau, SC Porto Alegre, RS
P. carthagenensis Jacq. P. deflexa DC. P. hancorniifolia Benth. P. kleinii L.B.Sm. et Downs P. leiocarpa Cham. Et Schltdl. P. longipes Mu¨ll.Arg. P. myriantha Mu¨ll.Arg. P. nuda (Cham. Et Schltdl.) Wawra P. pleiocephala Mu¨ll.Arg. P. pubigera Schltdl. P. suterella Mu¨ll.Arg. P. umbellata Vell. Rudgea heurckii Mu¨ll.Arg. R. jasminoides (Cham.) Mu¨ll.Arg. R. recurva Mu¨ll.Arg.
Porto Alegre, RS Blumenau, SC Juiz de Fora, MG Blumenau, SC Porto Alegre, RS Blumenau, SC Tenente Portela, RS Blumenau, SC Juiz de Fora, MG Blumenau, SC Santo Antonio da Patrulha, RS Morretes, PR Peruı´be, SP Torres, RS Blumenau, SC
Sobral 8180 Sobral 9054 Sobral 9056 Sobral e Kerber 7899— UFRGS—08/1995 Sobral 7901 Sobral 9055 Sobral 8216 Sobral 8469 Sobral 7898 Sobral 8912 Sobral 9044 Sobral 8913 Sobral 8213 Sobral 8911 Sobral 8336 Hatschbach (MBM 48571) Sobral 7320 Sobral 9052 Sobral 9053
ES, Espı´rito Santo; MG, Minas Gerais; PR, Parana´; RS, Rio Grande do Sul; SC, Santa Catarina; SP, Sa˜o Paulo.
providing (1) asperuloside (13 mg) and (2) deacetylasperuloside (9 mg) from Psychotria leiocarpa and (3) loganin (37 mg) from Palicourea rigida. The products were identified by 1H NMR and 13C NMR (El Naggar and Beal, 1980; Boros and Stermitz, 1990; Gonza´lez and Dieck, 1996; Peng et al., 1999). No iridoid glucosides were detected in the other species analyzed.
,
,
2.2.2. Asperuloside (1) 1 H NMR (400 MHz, D2O) d: 5.75 (d, J ¼ 2:2 Hz, H-1), 7.37 (d, J ¼ 1:8 Hz, H-3), 3.60–3.10 (m, H-5), 5.68 (br d, J ¼ 7 Hz, H-6), 5.90 (br s, H-7), 3.60–3.10 (m, H-9), 4.85 (br s, H-10), 2.10 (s, AcO), 4.70 (d, 9.0 Hz, H-10 ), 4.20–3.15 (m, H-20 , H-30 , H-40 and 50 ), 3.90–3.68 (m, H-60 ).
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13
C NMR (100 MHz, D2O) d: 99.1 (C-1), 150.1 (C-3), 105.2 (C-4), 36.4 (C-5), 86.5 (C-6), 128.5 (C-7), 142.9 (C-8), 44.1 (C-9), 61.5 (C-10), 173.5 (C-11), 93.3 (C-10 ), 73.2 (C-20 ), 76.9 (C-30 ), 70.2 (C-40 ), 76.3 (C-50 ), 61.5 (C-60 ), 173.7 (CO–AcO). 2.2.3. Deacetylasperuloside (2) 1 H NMR (400 MHz, D2O) d: 5.70 (d, J ¼ 2 Hz, H-1), 7.33 (d, J ¼ 2 Hz, H-3), 3.55–3.15 (m, H-5), 5.66 (br d, J ¼ 7 Hz, H-6), 5.70 (br s, H-7), 3.55–3.15 (m, H-9), 4.15 (br s, H-10), 4.80 (d, 8.0 Hz, H-10 ), 4.45–3.10 (m, H-20 , H-30 , H-40 and 50 ). 13 C NMR (100 MHz, D2O) d: 99.3 (C-1), 152.4 (C-3), 106.2 (C-4), 37.2 (C-5), 87.1 (C-6), 129.2 (C-7), 147.7 (C-8), 44.9 (C-9), 61.9 (C-10), 174.3 (C-11), 93.1 (C-10 ), 73.6 (C-20 ), 77.0 (C-30 ), 70.6 (C-40 ), 76.1 (C-50 ), 61.5 (C-60 ). 2.2.4. Loganin (3) 1 H NMR (400 MHz, D2O) d: 5.35 (d, J ¼ 4:0 Hz, H-1), 7.34 (s, H-3), 3.05 (m, H-5), 2.10–1.70 (m, H-6), 4.08 (m, H-7), 2.10 (m, H-9), 1.05 (d, J ¼ 7:5 Hz, H-10), 4.60 (d, 8.0 Hz, H-10 ), 4.45–3.15 (m, H-20 , H-30 , H-40 and 50 ), 3.90–3.68 (m, H-60 ), 3.65 (s, OMe). 13 C NMR (100 MHz, D2O) d: 97.4 (C-1), 151.7 (C-3), 114.1 (C-4), 31.0 (C-5), 41.3 (C-6), 75.8 (C-7), 40.8 (C-8), 46.0 (C-9), 13.2 (C-10), 171.2 (C-11), 99.8 (C-10 ), 74.0 (C-20 ), 77.0 (C-30 ), 70.2 (C-40 ), 77.5 (C-50 ), 61.5 (C-60 ), 53.0 (OMe). 2.2.5. Alkaloid detection The crude alkaloid extracts of dried leaves, obtained by classical acid/base partition, were directly evaluated by HPLC/PDA detector. Aliquots of these extracts were loaded onto a Waters 2690 HPLC system (Millipore Corp.) fitted with a 3:9 150 mm Nova-Pak C18-4 lm column (Waters), preceded by a guard-column, eluted at 1 ml min1 from 0 to 15 min with a linear gradient solvent system from 50:50 (V/V) methanol/water to methanol. The analysis continued isocratically for other 20 min. Eluting compounds were monitored with a Waters Millenium (version 2.15.01) and a Waters 996 photodiode array detector, which measured absorbance (200–400 nm) every 1.8 s with 4.8 nm resolution. The presence of indole UV spectra (instead of the indoline chromophore from the polyindolinic alkaloids) was observed and taken as indicative for the presence of monoterpene indole alkaloids. From some species, the alkaloids were isolated and identified: Psychotria umbellata yielded umbellatine (4) and the derivatives 3,4-dehydro-18,19-b-epoxyumbellatine, N4-[1-(2a-hydroxypropyl)]-umbellatine and N4-[1-(2a-hydroxypropyl)]-umbellatine (Kerber, 1999); from Psychotria brachyceras, brachycerine (5) was isolated (Kerber et al., 2001); lyaloside (6) and strictosamide (7) were found in Psychotria suterella (De Santos et al., 2001); myrianthosines A (8) and B (9) were isolated from Psychotria myriantha (Farias, 2004) while Psychotria leiocarpa afforded N,b-d-glucopyranosil vincosamide (10) (Lopes, 1998).
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,
,
,
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,
,
,
3. Results and discussion The main metabolites found in pantropical Psychotria (subgenus Psychotria) are alkaloids of polyindoline type and one of the most frequently reported is quadrigemine A (11). This group of compounds is derived from condensation of several N-methyltryptamine moieties, and they seem to be characteristic of the species from subgenus Psychotria (De Santos et al., 2001). With the exception of P. colorata (Elisabetsky et al., 1997; Verotta et al., 1998) and P. glomerulata (Solı´s et al., 1995), these alkaloids have not been detected in neotropical Psychotria (subgenus Heteropsychotria) which have, instead, yielded monoterpene indole alkaloids (Solı´s et al., 1993; Achenbach et al., 1995; Lopes, 1998; Kerber, 1999; De Santos et al., 2001; Kerber et al., 2001). The two species cited above were formerly included in the genus Cephaelis, which is considered by some authors (Steyermark, 1972; Taylor, 1996; Solı´s et al., 1997) as a synonym of Psychotria (subgenus Hetero-
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psychotria). However, the main type of alkaloids of Cephaelis, such as emetine and cephaeline (Southon and Buckinghan, 1989), derive from tyrosine, instead of tryptophan. Thus, from the chemosystematic viewpoint, the merging of these two genera seems to be unlikely.
Only a few species of Palicourea, an exclusively neotropical genus, have been analyzed for alkaloid content. P. dominguensis (Ripperberger, 1982), P. fendlerii (Nakano and Martin, 1976), P. alpina (Woo Ming and Stuart, 1975) and P. ovalis (Garcia et al., 1997) present polyindolines. The compounds described are dimers in contrast with the ones found in Psychotria, which contain several degrees of polymerization. Both genera also produce monoterpene indole alkaloids, derived from tryptamine (Morita et al., 1989; Valverde et al., 1999), in addition to the polyindoline alkaloids. The total alkaloids fraction of the leaves of 15 Brazilian Psychotria species, three Rudgea species and Palicourea rigida were analyzed by HPLC/PDA and peaks presenting UV spectra characteristic for the indole chromophore (223 and 280 nm) were detected in 14 Psychotria and three Rudgea species. This chromophore was not detected in Psychotria carthagenensis and Palicourea rigida. In P. suterella, it was found substances with the extended indol chromophore (237, 289 and 334 nm) characterizing b-carbolines (De Santos et al., 2001). In P. umbellata together with the indole group it was also observed compounds with UV spectra for 5-6-dihydrob-carboline (236 and 320 nm) (Kerber, 1999). No alkaloids were detected in P. carthagenensis (Lopes et al., 2000) and Palicourea rigida, and only traces of alkaloids were found in Psychotria hancorniifolia. Glucoside monoterpene indole alkaloids were isolated from P. brachyceras (Kerber et al., 2001), P. leiocarpa (Lopes, 1998), P. suterella (De Santos et al., 2001) and P. umbellata (Kerber, 1999). This type of alkaloid was firstly found in the neotropical Heteropsychotria subgenus in the species P. dichroa (Cephaelis dichroa) (Solı´s et al., 1993) and Psychotria correae (Cephaelis correae) (Achenbach et al., 1995). Palicourea alpina (Stuart and Woo-
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Ming, 1974), P. markgravii (Morita et al., 1989) and P. adusta (Valverde et al., 1999) also contain monoterpene indole alkaloids and such data could corroborate with the inclusion of part of Palicourea in the subgenus Heteropsychotria. Therefore, alkaloids can be a useful tool to distinguish groupings within a complex genus such as Psychotria, as previously proposed (Solı´s et al., 1995). No chemical investigations of Rudgea species were previously reported. In addition to the alkaloids, iridoid glucosides such as asperuloside (1) and deacetylasperuloside (2) were located in some species of Psychotria. Inouye et al. (1988) reported the occurrence of these compounds in P. rubra (Lour) Poir., P. serpens L. and P. manillensis Bartl. Ex D.C. In addition, Gonza´lez and Dieck (1996) isolated asperuloside from aerial parts of a plant named by them as ‘‘Psichotria mariniana’’. In the ethanolic extract of the leaves of Psychotria leiocarpa, the mentioned iridoids asperuloside and deacetylasperuloside were found only in small amounts. On the other hand, Palicourea rigida presents high concentrations of loganin (3), never found in species of Psychotria, although it is one of the intermediates in the biosynthetic pathway of monoterpene indole alkaloids. This product presents the same configuration (8b) of the iridoids frequently isolated from species of subfamily Rubioideae. This is the first report of iridoids in Palicourea. The results are summarized in Table 2. In conclusion, our findings reinforce the proposition from Solı´s et al. (1995) that alkaloids are important taxonomic markers and Taylor’s (1996) hypothesis that the Table 2 Alkaloids and iridoids in Psychotria, Palicourea and Rudgea species Species
Iridoid glucosides
Alkaloids
Palicourea rigida H.B.K. Psychotria alba Ruiz et Pav. P. barbiflora DC. P. brachyceras Mu¨ll.Arg. P. carthagenensis Jacq. P. deflexa DC. P. hancorniifolia Benth. P. kleinii L.B.Sm. et Downs P. leiocarpa Cham. Et Schltdl.
Loganin (3) n.d. n.d. n.d. n.d. n.d. n.d. n.d. Asperuloside (1); deacetylasperuloside (2) n.d. n.d. n.d. n.d. n.d. n.d. n.d.
n.d. i.c. i.c. Brachycerine (5) n.d. i.c. i.c. i.c. N,b-d-Glucopyranosil vincosamide (10) i.c. Myrianthosines A (8) and B (9) i.c. i.c. i.c. Lyaloside (6); strictosamide (7) Umbellatina (4) and derivatives i.c. i.c. i.c.
P. P. P. P. P. P. P.
longipes Mu¨ll.Arg. myriantha Mu¨ll.Arg. nuda (Cham. Et Schltdl.) Wawra pleiocephala Mu¨ll.Arg. pubigera Schltdl. suterella Mu¨ll.Arg. umbellata Vell.
Rudgea heurckii Mu¨ll.Arg. R. jasminoides (Cham.) Mu¨ll.Arg. R. recurva Mu¨ll.Arg.
n.d. n.d. n.d.
n.d., not detected; i.c., indole chromophore (detected by HPLC/PDA).
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American Psychotria, together with Palicourea, could be joined to form the genus Heteropsychotria. Additionally, the data achieved are in agreement with the statements of Nepokroeff et al. (1999) that some groups previously assigned to Psychotria (i.e. subg. Heteropsychotria plus Palicourea) are more closely related to other genera in the Psychotrieae than they are to other species of Psychotria.
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