Biochemical Systematics and Ecology 29 (2001) 819–825 Alkaloids of Erythroxylum (Erythroxylaceae) species from Southern Brazil José Angelo S. Zuanazzi*, Valéria Tremea, Renata P. Limberger, Marcos Sobral, Amélia T. Henriques Curso de Pós-Graduação em Ciências Farmaceˆuticas, Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul, Avenida Ipiranga, 2752. CEP 90.610-000. Porto Alegre, Brazil Received 10 February 2000; received in revised form 7 December 2000; accepted 18 December 2000 Abstract A new alkaloid identified as 3b,6b-ditigloyloxynortropane as 3b,7b-ditigloyloxynortropane, 4-hydroxyhygrinic acid, methylecgonidine and tropacocaine have been isolated from the leaves of Erythroxylum argentinum. The new structure was established by means of spectroscopic techniques. Four other species E. deciduum, E. microphyllum, E. pelleterianum and E. cuneifolium collected in the state of Rio Grande do Sul were screened for methylecgonidine and tropacocaine by CG/MS. Two of these plants contain tropacocaine and two contain methylecgonidine. GC/MS analysis of all 5 species for cocaine proved fruitless. The chemotaxonomic significance of these results is discussed. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Erythroxylum; Erythroxylaceae; Tropane alkaloids; Cocaine; Chemotaxonomy 1. Introduction Erythroxylaceae is a family of four genera, distributed especially in the tropical areas. Erythroxylum is the largest genus with about 250 species (Brachet et al., 1997). The Neotropical taxa, in particular E. coca, have been cultivated in South America for at least 5000 years and have been the object of many ethnobotanical investigations (Bohm et al., 1982). With the exception of the cocaine-producing species, this genus is surprisingly poorly investigated and the chemical composition *Corresponding author. Fax: +51-316-5437. E-mail address: Zuanazzi@farmacia.ufrgs.br (J.A.S. Zuanazzi). 0305-1978/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 3 0 5 - 1 9 7 8 ( 0 1 ) 0 0 0 2 2 - 9 820 J.A.S. Zuanazzi et al. / Biochemical Systematics and Ecology 29 (2001) 819–825 of several species used in traditional medicine remains unknown (Brachet et al., 1997). The genus is characterized by the presence of tropane alkaloids, tannins, terpenes and phenylpropanoids (Evans, 1981; Ansell et al., 1993). According to Holmsted and coworkers (1977), cocaine is produced in quantity only in the cultivated varieties of Erythroxylum. In other species, morphologically close to E. coca and E. novogranantense, cocaine is not produced. However, Aynilian and coworkers (1974) found traces of cocaine (0.00008–0.00882%) by GC/FID analysis of five herbarium specimens of Erythroxylum, including E. deciduum and E. pelleterianum, subjects of the present work. E. argentinum Schulz is a shrub or a small tree widespread in Argentina and Southern Brazil that has also been used in folk medicine as stomachic and in the treatment of sinusitis. In a pharmacological investigation, analgesic and antiinflammatory activities have been confirmed using in-vivo systems (Chaves et al., 1988). Previously, three alkaloids, tropacocaine, hygrine and cuscohygrine were isolated from this species (El-Iman et al., 1985) but the other four have received little attention. The five species are shrubs or small trees widespread in Southern Brazil and are commonly named: ‘‘cocão’’ or ‘‘fruto-de-pomba’’ (Sobral, 1987). In this work, we investigated the alkaloid composition in E. argentinum leaves and analysed E. cuneifolium, E. microphyllum, E. deciduum and E. pelleterianum for the presence of tropacocaine, methylecgonidine and cocaine by GC/MS. 2. Materials and methods 2.1. Plant materials Leaves were collected in 1998 from September to December in different localities in Rio Grande do Sul (Brazil). Voucher specimens were deposited at the ICN Herbarium (UFRGS- Brazil): E. argentinum Schulz (Guaı́ba; Sobral 1172), E. deciduum Saint-Hilaire (Erechim; Zuanazzi s.n., ICN 117396), E. microphyllum Saint-Hilaire (Porto Alegre; Sobral s.n., ICN 48860), E. pelleterianum Saint-Hilaire (Tenente Portela; Sobral 1352a) and E. cuneifolium (Martius) Schulz (São José dos Ausentes; Sobral 8898). 2.2. Extraction of alkaloids and TLC Dried leaves were soaked with 25% aqueous NH4OH and then exhaustively extracted with CH2Cl2 in a Soxhlet apparatus. The concentrated extract was partitioned into dilute HCl (0.1 N) and after separation the aqueous layer was made alkaline (pH 9) with a 25% NH4OH solution, and further partitioned against CH2Cl2. The organic layer was concentrated under reduced pressure to afford a crude alkaloid fraction. 4-Hydroxyhygrinic acid (10 mg) in crystalline form was directly obtained from this fraction by dilution of the extract with CH2Cl2. The J.A.S. Zuanazzi et al. / Biochemical Systematics and Ecology 29 (2001) 819–825 821 crystals were re-dissolved in MeOH and Et2O was added dropwise until the crystals reformed. The crude alkaloid fraction (5 g) of E. argentinum was partially resolved under vacuum liquid chromatography on Al2O3 60 (neutral) using a gradient hexane, CH2Cl2 and MeOH, successively to give 9
100 ml fractions. Fraction 3, eluted with CH2Cl2:MeOH (99:1), was submitted to preparative TLC on several Si gel plates with CHCl3:MeOH (90:10) affording tropacocaine (13 mg) as the major product and methylecgonidine (8 mg) (1). From fraction 4, eluted with CH2Cl2:MeOH (98:2), 3b,6b-ditigloyloxynortropane (4 mg) (2) was isolated by preparative TLC on Si gel plates with CHCl3–MeOH (90:10). 2.3. Analytical GC The alkaloid extracts of the five species and references (tropacocaine, methylecgonidine and cocaine) were dissolved in chloroform and analyzed by GC/MS using a Shimadzu QP5000, in a 30 m
0.25 mm i.d. fused-silica capillary DB-5 column (film thickness 0.25 mm). Helium was the carrier gas with a flow rate of 1.0 ml/min. GC/MS was performed in the EI mode at 70 eV. The oven temperature was programmed from 100 to 3208C at 208C/min (14 min). Injector and detector were set at 250 and 2808C, respectively. 2.4. NMR analysis Spectra in NMR 1H, 1H-1H COSY experiments and 13C were recorded on a Varian VXR-200, 200 MHz (1H) and 50 MHz (13C) in CD3OD. Chemical shifts are given in ppm relative to TMS. 3. Results 3.1. Alkaloids identification in E. argentinum From the leaves of E. argentinum four alkaloids were isolated of which three were already known. 4-Hydroxyhygrinic acid and tropacocaine have been isolated from the species growing in Argentina (Iñigo and Pomilho, 1984) and cultivated in The Botanic Garden of Sydney (Evans, 1981), respectively. Methylecgonidine has been isolated from the seeds of the both E. coca and E. novogranatense, but not from E. argentinum. 3b,6b-ditigloyloxynortropane is reported here as a new alkaloid. NMR data for methylecgonidine are presented since no previous reports were available in the literature, only physical data (melting point and refractive index) of a sample prepared from cocaine was found (Matchett and Levine, 1941). The mass spectrum of methylecgonidine (1) afforded [M]+ at m/z 181 suggesting a molecular formula of C10H15NO2. The 1H NMR spectrum showed signals at d3.90 and 3.32 attributed to the bridgehead protons in the tropane ring. A triplet at d6.82 was 822 J.A.S. Zuanazzi et al. / Biochemical Systematics and Ecology 29 (2001) 819–825 Table 1 1 H and 13C NMR assignments for methylecgonidine Chemical shifts 1 1 2 3 4a 4b 5 6a 6b 7a 7b N–CH3 O–CH3 CO H 3.90 (d, J ¼ 5:2 Hz) } 6.82 (t, J ¼ 3:3 Hz) 2.70 (d, J ¼ 19:8 Hz) 1.95 (m) 3.32 (m) 2.20 (m) 1.55 (m) 2.20 (m) 1.82 (m) 2.4 (s) 3.75 (s) } 13 C 58.8 141.6 135.8 34.3 57.3 31.8 29.9 36.1 51.8 180.9 assigned to the olefinic proton. The remaining protons were assigned by means of 1 H-1H COSY experiments. The 1H and 13C NMR data are shown in Table 1. Compound 2 presented an EIMS fragmentation pattern consistent with a disubstituted 3,6-diol nortropane ester. Fragments at m/z 80 (7%), 81 (6%) and J.A.S. Zuanazzi et al. / Biochemical Systematics and Ecology 29 (2001) 819–825 823 82 (8%) could be assigned to pyridinium and related ions (Evans and Woolley, 1978). The ions [M-99] at m/z 208 (84%) and 83 (100%) and 55 (56%) corresponded to the loss of a tigloyl moiety. Structure 2 was confirmed by 1H-NMR spectroscopy presenting a 3b-linkage (d5.16, m, W1/2=30 Hz) whereas the attachment of the second ester residue at C-6 was evident from its characteristic multiplet (dd) at d5.34 (J6a,7a and J6a,7b, 7.0 and 2.0 Hz, respectively) (Al-Said et al., 1986; Muñoz et al., 1996). In effect, in all natural C-6 or C-7 substituted tropane alkaloids, the groups appear to be exo (Evans, 1981; Christen et al., 1993; Doerk-Schmitz et al., 1994). The absence of an N–CH3 signal pointed toward a nortropane ring, in agreement with the MS data. The two bridgehead protons produced two singlets at d3.70 and 3.65. The signals at d6.82 and 6.87 (dq, J ¼ 7:0 and 1.5 Hz) were attributed to the olefinic protons of tiglic acid (E-isomer) (San Martin et al., 1987) and confirmed the structure as 3b,6b-ditigloyloxynortropane (2). However, our data obtained from mass and NMR spectra could not define an unequivocal structure for this compound, a second structure with the tigloyl residue at C-7 is equally possible. 3.2. CG analysis Tropacocaine and methylecgonidine were identified in E. pelleterianum and E. microphyllum, respectively. Cocaine was not detected in any species. E. argentinum, E. pelleterianum, E. cuneifolium and E. deciduum presented in the chromatogram a unknown compound (P1) with ions at m/z: 229 (26%), 228 (42%), 211 (26%), 185 (14%), 171 (5%), 143 (8%), 129 (30%), 115 (12%), 102 (93%), 97 (19%), 73 (51%), 60 (95%) and 41 (100%). This alkaloid was the major component in all four species. Furthermore, the chromatograms of E. deciduum and E. pelleterianum showed another unknown compound at the same retention time as cocaine (12.8 min). However, the fragmentation pattern was different from that observed for cocaine which presented characteristic fragments at m/z 82 (96%), 182 (base peak) and 303 (M+-24%). This unknown compound showed fragments at m/z 100 (82%), 85 (28%), 71 (53%), 57 (100%) and 43 (95%). 4. Discussion Many minor alkaloids in were detected E. argentinum, but due to their paucity it was not possible to obtain more revealing information by NMR spectroscopy. Schulz (1907) divided the genus Erythroxylum in 19 sections. Apparently the alkaloid distribution is not related to this division. Tropacocaine is present in only two species of different sections (E. pelleterianum, section Rhabdophyllum and E. argentinum, section Archerythroxylum) and as well as methylecgonidine identified in E. argentinum (Archerythroxylum) and E. microphyllum (section Microphyllum). Our results are in agreement with previous findings that no significant quantity of cocaine is detected in species other than E. coca and E. novogranatense, including 824 J.A.S. Zuanazzi et al. / Biochemical Systematics and Ecology 29 (2001) 819–825 those morphologically related (Holmstedt et al., 1977). The presence of cocaine in E. deciduum (already challenged by Plowman and Rivier, 1983) and E. pelleterianum reported by Aynilian and coworkers (1974) was not supported by our results with GC/MS where the peak at the same retention time as cocaine had a significantly different mass spectrum. Tiglic acid esterifying tropine was reported for Erythroxylum species (E. australe } Johns and Lamberton, 1967; E. australe } Griffin, 1978; and E. argentinum, the present paper) as well as for species of the genera Datura and Brugmansia of the Solanaceae. In spite of sharing this chemical feature, the two families are not closely related taxonomically. 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