Print version ISSN 0103-5053
J. Braz. Chem. Soc. vol.15 no.4 São Paulo July/Aug. 2004
Mariana H. ChavesI; Nidia F. RoqueII, *; Mariane C. Costa Ayres
IDepartamento de Química, Universidade Federal do Piauí, Campus Ininga, 64049-550 Teresina - PI, Brazil
IIInstituto de Química, Universidade de São Paulo, CP 26077, 05513-970 São Paulo - SP, Brazil and Instituto de Química, Universidade Federal da Bahia, 40170-290 Salvador - BA, Brazil
Three mixtures of steroids (1a+1b, 2a+2b and 3a+3b) and two flavonoid glycosides (4 and 5) were isolated from the ethanol extract of branches of Porcelia macrocarpa (Warm.) R. E. Fries (Annonaceae). The steroids Stigmasta-4,25-dien-3-one (2a) and (22E)-Stigmasta-4,22,25-trien-3-one (2b) are news. The structures were elucidated by NMR and MS data and comparison with literature data of model compounds.
Keywords: Porcelia macrocarpa, Annonaceae, steroids, flavonoids
Três misturas de esteróides (1a+1b, 2a+2b e 3a+3b) e dois flavonóides glicosilados (4 e 5) foram isolados do extrato etanólico dos galhos da Porcelia macrocarpa (Warm.) R. E. Fries (Annonaceae). Os esteróides Estigmasta-4,25-dien-3-ona (2a) e (22E)-Estigmasta-4,22,25-trien-3-ona (2b) são substâncias novas. As estruturas foram determinadas com base na análise de dados espectrais de RMN e de massas e por comparação com dados de substâncias da literatura usadas como modelos.
Porcelia macrocarpa is a botanical species from Annonaceae family and the only one of the genus occurring in Brazil.1 This species has a very diversified chemical composition, revealed by isolation of acetogenins, lignanamides, hydroxycinnamoyltyramines, sesquiterpenes and alkaloids, including azapolycyclics, from different parts, previously investigated, of the same specimen.2-6
This paper describes the isolation and identification of three steroid mixtures and two flavonoid glycosides from the ethanol extract of the branches of Porcelia macrocarpa.
Results and Discussion
The ether soluble part from the ethanol extract of the branches of Porcelia macrocarpa was submitted to a partition between hexane and aqueous methanol. Chromatographic separation of the hexane phase afforded two steroid mixtures (M1 and M2) and the hydroalcoholic phase gave, after the same procedure, one mixture of steroid glycosides (M3). An insoluble material precipitated in the interface between diethyl ether and water, during the partition from the ethanol extract, afforded two flavonoid glycosides 4 and 5.
A GCMS analysis of the M1 mixture showed two compounds 1a and 1b in a 5:2 relationship, where the major compound presents a molecular peak [M]+ at m/z 412 and the minor one at m/z 410. The 1H NMR spectrum (Table 1) of the mixture M1 showed signals at d 3.51 (m), 5.34 (m) and between d 0.6 and 1.6 attributed to H-3, H-6 and methyl hydrogens, respectively, from a D5-3b-hydroxy sterol with an allylic methyl group.7 Signals at d 5.23-5.17 and 4.72-4.62 suggested additional double bonds in the steroidal nucleus. The 1H and 13C NMR data agree with the structure of D5 sterols [dC 71.8 (oxygenated CH-3), 140.7 (C-5) and 121.7 (CH-6) ] containing additionally one (1a, [M]+ at m/z 412) and two double bonds (1b, [M]+ at m/z 410) located at the side chain. The side chain of both steroids sustain an ethyl group at C-24, as in sitosterol. The presence in the mixture of two methylidene groups (C=CH2) was deduced by 13C signals corresponding to sp2 non-hydrogenated and methylene carbon atoms [dC 147.5 and 111.4 (1a); 148.6 and 109.5 (1b)]. Thus, the second double bond of 1a was placed at C-25 to justify the 13C signals and the allylic methyl group in the 1H NMR (Table 1 and 2). The peak at m/z 84 (19%) attributed to fragment 1a-F originated by a McLafferty rearrangement (Table 3 and Figure 1) is in accordance with this suggestion. The compound 1a has been isolated before from the marine green alga Codium iyengarii.8 The low relative intensity of the peak at m/z 84 (3%) observed in the mass spectrum of 1b is justified by the presence of another peak at m/z 138 (43%) attributed to the fragment 1b-D formed also by a McLafferty rearrangement involving the additional double bond localized between the carbon atoms C-22 and C-23 (Table 3 and Figure 1). This third double bond is present only in 1b, as indicated by the 1H NMR multiplet integration at d 5.23-5.17 and by the molecular ion. The CH signals at d 137.2 and 130.0 observed in the 13C NMR spectrum (Table 2) are in accordance with the proposed structure. Compound 1b is also described in the literature.
The GC-MS of M2 showed two compounds, in the same proportion of M1, with molecular peaks at m/z 410 and 408 (Table 3). The 1H NMR spectrum (Table 1) was very similar with that of M1, but presented a signal at d 5.72 instead of the signals at d 3.51 and 5.34, suggesting a D4 conjugated double bond as in sitostenone.9 The 13C NMR spectra (Table 2) showed signals that confirmed this suggestion, when compared with literature data of model compounds.10 The MS fragmentation followed the same pattern as M1 (Figure1 and Table 3). Hydrogenation of M2 mixture, at room pressure, gave the steroid sitostenone, as proved by GCMS and 1H NMR spectra.9 M2 is then composed by a mixture of 2a and 2b which are described here for the first time.
The 1H NMR spectrum (Table 1) of M3 differs from that of M1 only in the carbinolic hydrogen region. It showed signals between, d 4.05 and 5.07, from a sugar unit. The H-3 multiplet is deshielded by 0.4 ppm suggesting that the mixture is composed by, glycopyranosyl steroids, with b configuration on the anomeric carbon, evidenced by the doublet at d 5.02 (7.6 Hz).11 The 13C NMR (Table 2) data suggested that the compounds are steroid glucosides.11,12 All the other signals are compatible with the structure of 3a and 3b. The steroid 3a has been isolated from Clerodendron colebrookianum (Verbenaceae).11
The 1H NMR spectrum of 4 (Table 4) showed five signals in the aromatic region with the pattern of the flavonoid quercetin.13 Besides of those a singlet at d 3.84 (6H) and signals between d 3.05 and d 5.35 were assigned to methoxyl groups and sugar hydrogens respectively. A doublet at d 0.95 suggested that rhamnose should be present in the molecule and the singlet at d 12.5 indicated the presence of one hydroxyl at C-5 of the aglycone. The 13C NMR spectrum (Table 5) showed 29 signals indicating that two sugar units and two methoxyl groups are present in the flavonoid. The chemical shifts of the sugar moiety are consistent with those of rutinosyl (rhamnopyranosyl-(1®6) glucopyranosyl). Comparison between the 13C NMR data of 4 with those of 7,4'-dimethoxyquercetin (6)13,14 showed that C-3 is shielded and C-2, C-4 and C-10 are deshielded in agreement with the glycosylation at C-3 of 4.14-16
The 1H NMR spectrum of 5 (Table 4) had the same pattern as that of 4 but the signal of C-5 OH was absent. The 13C NMR spectrum showed 33 signals indicating the presence of 3 sugar units in the dimethoxyflavonoid, one of them should be linked to C-5. Signals of rutinosyl and glucosyl groups were present. Hydrolysis of 5 led to the isolation of 7,4'-dimethoxyquercetin (6).14 To determine which group, glicosyl or rutinosyl is bounded to C-5, a COLOC 1H-13C was obtained and a long range correlation between d 4.84, assigned to the anomeric hydrogen of glucose and d 158.6, assigned to C-5 of the flavonoid was observed. To confirm the structure of 5 as quercetin-5-O-glucoside-3-O-rutinoside-7,4'-dimethyl ether a comparison between the 13C NMR data of 4 and 5 and those of 5-glucopyranosyl luteolin and luteolin17 was made. The difference found in the chemical shifts of rings A and C of those flavonoids confirmed the proposed structure, as showed in Table 6.
The low resolution mass spectra were obtained in a INCOS 50 Finnigan-Mat instrument operating at 70 eV coupled to a GLC 3400 Varian, capillary column (DB-5, 30 m x 0.25 mm), det. 280°. Temperature programming from 120 °C to 180 °C, at 10°/min, then 40°/min to 300 °C. The 1H and 13C NMR spectra, using CDCl3, DMSO-d6 as solvent and TMS as internal reference, were run in a Bruker AC 200. For the column separations Merck silica gel, 63-200 mm was used.
The branches of P. macrocarpa (Warm.) R.E. Fries were collected at Instituto of Botânica de São Paulo in June, 1991. A voucher specimen is deposited in the herbarium of the Instituto de Botânica, São Paulo, Brazil under reference SP76791.
Extraction and isolation of the compounds
Dried and powdered branches (800 g) of P. macrocarpa were extracted with EtOH. The EtOH extract, after concentration in vacuum, was partitioned between Et2O/H2O (1:2) giving a water-soluble fraction (31 g), an ether soluble (20 g) and an insoluble interface (2.0 g). The ether soluble part was then partitioned between MeOH-H2O (9:1) and hexane.
The hexane phase (10 g) was chromatographed on silica gel column eluted with hexane with increasing amounts of EtOAc. Two fractions were eluted with hexane-EtOAc (9:1). These fractions were, separately, submitted to further purification on silica gel column, eluted with hexane-EtOAc (95:5). The less polar fraction afforded a mixture of steroids (8 mg, 2a+2b) and the more polar fraction afforded another mixture of steroids (64 mg, 1a+1b).
The MeOH-H2O layer (10 g) was chromatographed on silica gel column eluted with CHCl3 with increasing amounts of MeOH. A mixture of steroid glycosydes (27 mg, 3a+3b) precipitated from the CHCl3-MeOH (9:1) eluate. The insoluble interface (2.0 g) was chroma Steroids and Flavonoids of tographed on silica gel column eluted with CHCl3 and increasing amounts of MeOH. The fractions CHCl3-MeOH (7:3) and (1:1) treated with MeOH, gave insoluble materials which were, respectively, the flavonoid glicosydes 4 (12 mg) and 5 (168 mg).
Hydrogenation of M1 (2a+2b). The mixture of 2a+2b (3 mg) dissolved in CHCl3 was maintained under hydrogen atmosphere, for one hour, using Pd/C as catalyst to give, after filtration and evaporation of the solvent, 2 mg of sitostenone.
EIMS m/z 412 [M]+(15), 370 (10), 327(5), 289(15), 229(50), 124(100). 1H NMR (CDCl3, 500 MHz, d): 5.75 (s, 1H), 2.35-2.45 (m, 2H), 1.15 (s, 3H), 0.93 (d, 3H), 0.83-0.88 (2d,6H), 0.73(s, 3H).
Flavonoid 5 (50 mg) were hidrolyzed with 20 mL of 2 mol L-1 HCl at 80 ºC for 1 h. The precipitate was filtrated, washed with water and after drying afforded 19 mg of 6.
1H NMR: See Table 1; 13C NMR: See Table 2; EIMS m/z 410 [M]+ (5), 381 (5), 300 (17), 273 (9), 272 (23), 271 (52). 255 (27), 213 (18), 187 (12), 185 (15), 173 (18), 171 (13), 163 (19), 161 (23), 159 (41), 147 (34), 145 (48), 143 (21), 138 (43), 137 (42), 133 (47), 131 (29), 129 (17), 123 (16), 121 (32), 191 (38), 109 (95), 105 (58), 95 (100), 84 (3), 79 (65), 69 (31), 67 (64), 57(21), 55(87).
IR (KBr) nmax /cm-1: 1677; 1H NMR: See Table 1; 13C NMR: See Table 2; Stigmasta-4,25-dien-3-ona. 410 (18), 381 (1), 327 (37), 312 (8), 297 (20), 281 (20), 271 (10), 270 (15), 269 (30), 245 (19), 229 (14), 207 (54), 149 (17), 148 (12) 147 (26), 138 (5), 135 (18), 134 (11), 133 (25), 124 (26), 123 (20), 121 (21), 119 (20), 109 (22), 107 (25), 105 (26), 95 (32), 93 (27), 91 (32), 84 (52), 83 (20), 81 (33), 79 (32), 69 (40), 67 (32), 55 (100).
IR (KBr) nmax/cm-1: 1677; 1H NMR: See Table 1; 13C NMR: See Table 2; EIMS m/z 408 [M]+(8), 379 (14), 299 (28), 298 (26), 283 (17), 281 (24), 271 (36), 270 (36), 269 (100), 253 (27), 229 (15), 207 (54), 177 (19), 175 (21), 161 (25), 159 (19), 149 (32), 147 (44), 145 (25), 138 (25), 137 (37), 135 (29), 133 (24), 131 (22), 124 (22), 123 (26), 121 (36), 119 (29), 110 (24), 109 (86), 107 (44), 105 (43), 95 (86), 93 (63), 91 (63), 84 (6), 81 (92), 79 (66), 77 (35), 69 (32), 67 (68), 55 (99)53 (23).
This work was supported by CAPES, CNPq and FAPESP. The authors are grateful to CAPES-PICDT (M.H.C.) and CNPq (N.F.R.) for awards of scholarships. They are also grateful to Dr. Claudia M. Young, Instituto de Botânica, SEMA, São Paulo for the plant material.
1. Murray, N.A.; Sistematic Botany Monographs 1993, 40, 89. [ Links ]
2. Chaves, M. H.; PhD Thesis, Universidade de São Paulo, Brazil, 1996. [ Links ]
3. Chaves, M. H.; Roque, N. F.; Phytochemistry 1997, 44, 523. [ Links ]
4. Chaves, M. H.; Roque, N. F.; Phytochemistry 1997, 46, 879. [ Links ]
5. Brochini, C. B.; Núñez, C. V.; Moreira, I. C.; Roque, N. F.; Chaves, M. H.; Quim. Nova 1999, 22, 37. [ Links ]
6. Chaves, M. H.; Santos, L.A.; Lago, J. H. G.; Roque, N. F.; J. Nat. Prod. 2001, 64, 240. [ Links ]
7. González, A.G.; Bermejo, J.; Mediavilla, M.J.; Toledo, F.J.; Rev. Latinoamer. Quim. 1984, 15, 107. [ Links ]
8. Ahmad, V.U.; Aliya, R.; Perveen, S.; Shameel, M.; Phytochemistry 1992, 31, 1429. [ Links ]
9. Tandon, S.; Rastogi, R.P.; Planta Med. 1976, 29, 190. [ Links ]
10. Oger, J.-M.; Richomme, P.; Bruneton, J.; Guinaudeau, H.; J. Nat. Prod. 1991, 54, 273. [ Links ]
11. Goswami, P.; Kotoky, J.; Chen, Z.-N.; Lu, Y.; Phytochemistry 1996, 41, 279. [ Links ]
12. Rauwald, H.-W.; Sauter, M.; Schilcher, H.; Phytochemistry 1985, 24, 2746. [ Links ]
13. Agrawal, P.K.; Carbon-13 NMR of Flavonoids, Elsevier Science: Amsterdan, 1989. [ Links ]
14. Agrawal, P.K.; Rastogi, P.R.; Heterocycles 1981, 16, 2181. [ Links ]
15. Markham K.R.; Ternai,; B. Tetrahedron 1976, 32, 2607. [ Links ]
16. Markham K.R.; Ternai, B.; Stanley R.; Geiger, H.; Mabry, T.J.; Tetrahedron 1978, 34, 1389. [ Links ]
17. Harbone, J.B.; Mabry, T.J.; The Flavonoids: Advances in Reserarch, Chapman and Hall LTD: Londres, 1982. [ Links ]
18. Nasr, C.; Haag-Berrurier, M.; Lobstein-Guth, A.; Anton, R.; Phytochemistry 1986, 25, 770. [ Links ]
19. Inigo,R. P. A.; Iglesias, D. I. A. N.; Phytochemistry 1988, 27, 1230. [ Links ]
Received: August 19, 2002
Published on the web: May 17, 2004
FAPESP helped in meeting the publication costs of this article.