Flavonoids from Chiococca braquiata (Rubiaceae)

Article J. Braz. Chem. Soc., Vol. 15, No. 4, 468-471, 2004. Printed in Brazil - ©2004 Sociedade Brasileira de Química 0103 - 5053 $6.00+0.00 Flavonoids from Chiococca braquiata (Rubiaceae) a a b ,a Marcia N. Lopes , André C. de Oliveira , Maria Cláudia M. Young and Vanderlan da S. Bolzani* a Instituto de Química, Universidade Estadual Paulista, CP 355, 14801-970 Araraquara - SP, Brazil b Seção de Fisiologia e Bioquímica de Plantas, Instituto de Botânica, CP 4005, 10051 São Paulo - SP, Brazil Um flavonol inédito, 4’-metoxikaempferol-7-(acetiloxi)-3,5-O-D-L-ramnosídeo (1), três flavonóides conhecidos, apigenina, 7-O-metoxiquercetrina e quercetrina e quatro triterpenos, Damirina, E-amirina, ácido oleanólico e ácido ursólico foram isolados das folhas de Chiococca braquiata. As estruturas dessas substâncias foram elucidadas com base em seus dados espectroscópicos. A new flavonol 4’-methoxykaempferol-7-(acetyloxy)-3,5-O-D-L-rhamnoside (1), was isolated from the leaves of Chiococca braquiata, along with three known flavonoids apigenin, 7-Omethoxyquercetrin and quercetrin and four triterpenes D-amirin, E-amirin, ursolic and oleanolic acids . Their structures were established on the basis of spectroscopic methods. Keywords: Chiococca braquiata, Rubiaceae, flavonoids Introduction The genus Chiococca (Rubiaceae) with 22 species is endemic of the American Continent, and occurs from North America to Brazil. Several Chiococca species have been traditionally used in these regions for the treatment of numerous human ailments including inflammation, antivirus, anti-edema and as aphrodisiac. 1 Other plants belonging to the Rubiaceae family have yielded a number of interesting biologically active compounds,2,3 including seco-iridoids with mild DNA-activity isolated from Chiococca alba.4 C. braquiata, however, has not been subjected to phytochemical analysis or assayed for any biological activity. In an ongoing quest to identify biologically active compounds from the Brazilian Rubiaceae plant species, antifungal evaluation using Cladosporium sphaerospermum and C. cladosporioides was performed on CH2Cl2-MeOH (2:1, v/v) extract from C. braquiata leaves, which exhibited strong antifungal activity. Bioassay-guided fractionation of the bioactive extract led to the isolation of the inactive flavonoids 4’methoxykaempferol-7-(acetyloxy)-3,5-O-D-L-rhamnoside (1), apigenin, 7-O-methoxyquercetrin and quercetrin. The triterpenes D-amirin, E-amirin, ursolic and oleanolic acids * e-mail: bolzaniv@iq.unesp.br were also isolated. The structure of the new derivative 1 and the known compounds were elucidated by spectroscopic methods, mainly 2D NMR and MS. Experimental General procedures For column chromatography silica gel 60 (Merck 230400 mesh) and Sephadex LH-20 were used. TLC analysis carried out on silica gel 60 F254. IR spectrum was recorded on a Nicolet Spectrometer. UV spectra were recorded on a Perkin-Elmer UV/Vis Spectrometer Lambda 14P. The ESMS spectra were obtained on a VG Platform II Spectrometer. NMR spectra were recorded in DMSO-d6 or CDCl3 on a Vol. 15, No. 4, 2004 Flavonoids from Chioccoca braquiata (Rubiaceae) Varian Unit 500 instrument at 500 MHz for 1H and 125 MHz for 13C, using TMS as internal standard. The DEPT experiments were performed using polarization transfer pulses of 90 and 135o. Plant material Leaves of Chiococca braquiata Ruiz & Pav. (now Chiococca alba (L.) Hitchc.) were collected around Lagoa do Abaeté, BA, Brazil and identified in the Botanical Institute, SMA, SP. A voucher no. 1934 specimen has been deposited in the herbarium Maria Eneida P. K. Fidalgo, SP, Brazil. Bioassay The antifungal activity against C. cladosporioides was performed using direct bioautography with 10 PL of the solutions of crude extracts and pure compounds, which were prepared in different concentrations ranging from 300 to 10 Pg respectively, as described elsewhere.5 Extraction and isolation of compounds Air-dried powdered leaves (2.0 kg) were extracted with CH2Cl2-MeOH (2:1) at room temperature. The CH2Cl2MeOH extract was evaporated in vacuum to give a crude extract (120 g), which was partitioned into equal volumes of MeO-H2O (80%) and hexane (3x). The hydromethanolic extract was concentrated to 60% and was subsequently extracted using CHCl3 (3x) and EtOAc (3x). These were evaporated to give a hexane phase (5 g), a chloroform phase (12 g), an ethyl acetate phase (4.7 g) and a hydromethanolic phase (42 g). The ethyl acetate phase was applied to a column of Sephadex LH-20 (30 g) eluted with MeOH-H2O with increasing polarity to give 17 fractions. Fraction 8 (123 mg) was applied again to a column of Sephadex LH-20 (5 g) eluted with MeOH to give 6 fractions. The sub-fractions 8.2 and 8.3 yielded mixture of triterpenes D-amirin and Eamirin (39 mg) and ursolic and oleanolic acids (64 mg), respectively. Fractions 11 and 12 were combined (176 mg) and further purified by reverse phase preparative thin-layer chromatography in MeOH-H2O (8.5:1.5) leading to the isolation of 4’-methoxykaempferol-7-(acetyloxy)-3,5-OD-L-rhamnoside (1; 21 mg). Fraction 13 (460 mg) was applied to a silica gel column (230-400 mesh) eluted with ethyl acetate containing increasing concentration of methanol (up to 100%) to give apigenin (2; 174 mg). Fraction 14/15 (111 mg) was applied to a column of Sephadex LH-20 (2 g) eluted with MeOH to give 7-O- 469 methoxyquercetrin (3; 60 mg). Fractions 16/17 (175 mg), after recristalization in acetone, gave quercetrin (4; 165 mg). 4’-Methoxykaempferol-7-(acetyloxy)-3,5-O-a-Lrhamnoside (1). Colorless powder; IR (film) Qmax /cm-1: 1695, 1688, 1600, 1443, 1213, 1060; ES-MS m/z (rel. int.) [M+Na]+ 657 (6.5), 634 (4 ), 363 (98), 317 (100), 170 (56), 147 (25); UV MeOH Omax /nm: 341, 259; 1H and 13C NMR data see Table 1. Apigenin (2). Colorless gum, identified by comparison (UV, 1H and 13C NMR) with literature data.6 7-O-Methoxyquercetrin (3). Colorless gum, identified by comparison (UV, 1H and 13C NMR) with literature data.7 Quercetrin (4). Colorless needles, mp 188-190 0C (MeOH); identified by comparison (UV, 1H and 13C NMR) with literature data.8 Table 1. NMR spectral data of flavonol 1 Position GH a 2 3 4 5 6 7 8 9 10 1’ 2’-6’ 3’-5’ 4’ 1”, 1”’ 2”, 2”’ 3”, 3”’ 4”, 4”’ 5”, 5”’ 6”’6”’ OMe C=O CH3-CO 6.63 (d, J 1.25) 6.32 (d, J 1.25) 7.75 (d, J 8.0) 6.89 (d, J 8.0) 5.30 ( br s) 3.92 (br dd, J 9.0, 11.0) 3.48 (dd, J 9.0, 10.8) 3.08 m 3.28 (m) 0.87 (d, J 6.5) 3.79 (s) 1.87 (s) GCb HMBCa (H) 156.5 s 2‘,6‘ 134.2 s 1“ 177.6 s 161.7 s 6, 1“‘ 98.0 d 6, 8 165.1 s 6, 8 92.0 d 6 157.6 s 8 105.0 s 6 119.3 s 3’/ 5’ 130.5 d 3’/5’ 115.8 d 5’/6’ 161.7 s 2’/6’, OCH3 101.8 d 2”/2”’3”/3”’ 72.5 d 72.6 d 74.4 d 71.1 d 17.5; 18.5 q 56.0 q 174.7 s COCH3 24.3 q - a,b, Spectra in DMSO-d6. Assignments were made with the aid of the DEPT and 2D-shift-correlated HMQC and 1H- 1H COSY spectral data. Chemical shift in G, multiplicities and coupling constants (J) are in parentheses. Spectra were recorded at 500 MHz for 1H and 125 MHz for 13C. Results and Discussion The bioactive soluble EtOAc part of a CH2Cl2:MeOH (2:1, v/v) extract, prepared from the leaves of C. braquiata, was chromatographed over Sephadex LH-20 column and preparative TLC to afford a new flavonoid 1, along with the know compounds apigenin (2), 6 7-O-methoxyquercetrin (3),7 quercetrin (4),8 D-amirin, E-amirin, ursolic and oleanolic acids.9 470 Lopes et al. Compound 1 was isolated as a white amorphous powder, with molecular formula C30H34O15 deduced from the [M+Na]+ peak at m/z 657 in the ES-MS and supported by 13C and 1H NMR data. Its UV spectrum exhibited characteristic absorbance bands of flavonols10 at 259 and 341 nm. The 1H and 13C NMR spectra (Table 1) revealed two set signals, which features indicated a flavonoid with glycosidic and acetyl groups. The 1H NMR signals attributed to the aglycone at d 7.75 (2H, d, J 8.0 Hz), 6.89 (2H, d, J 8.0 Hz), identified as a 2H AA’ and a 2H XX’system, 6.63 (1H, d, J 1.25 Hz), 6.32 (1H, d, J 1.25 Hz) showed characteristic pattern of kaempferol.5 The 1H NMR spectra also showed signals at G 5.30 (2H, br s), 3.92 (2H, br, dd, J 9.0, 11.0 Hz), 3.48 (2H, dd, J 9.0, 10.8 Hz), 3.28 (2H, m), 3.08 (2H, m) and 0.87 (6H, d, J 6.5 Hz), which revealed the presence of at least two glycosyl moieties, clearly evidenced by integration area of the peaks corresponding to these signal values. In addition, a methoxyl and an acetyl groups were also confirmed by two singlets at G 3.79 (56.0 q) and G 1.87 (24.3 q) and 174.7 (s), respectively. The identification of the sugar moieties as two D-rhamnopyranosides was determined from the chemical shifts, multiplicity of the signals, and absolute values of the coupling constants in the 1H NMR and 1H-1H COSY spectra as well as 13C NMR data. The signals observed in the 13C NMR spectrum (Table 1) at G 101.8 (d), 74.4 (d), 72.6 (d), 72.5 (d), 71.1 (d), 18.5 and 17.5 (q) clearly indicated that the sugar moiety was rhamnose. In addition, the evidence of two distinct signals for characteristic methyl groups of rhamnose deeply aided the proposal of two rhamnose moieties in compound 1. The ES-MS ions peaks at m/z 365 [M-(2x rham) + Na]+, m/z 363 [M-(294) + Na]+ and m/z 186 [C6H11O4 + Na]+ also emphasized the presence of two rhamnopyranoside units in compound 1. The first indication of the positions C-3 and C-5 as substitution sites in 1 was evidenced from the 1H NMR spectrum, which do not showed the typical signals assigned to H-3 and the HO-C-5 quelate. The HMBC spectrum (Table 1) confirmed these positions as glycosylation sites due to the connectivities that were observed between G 5.30 (1H, br s, H-1”) and 134.2 (C-3) and the correlation between G 5.30 (1H, br s, H-1”’) and 161.7 (C-5). The correlation between the signal corresponding to -OCH3 at G 3.79 with C-4’ at d 161.7 also was important to attribute all substitutions in 1, if we consider that the only remain place linkage for the acetyl group should be at hydroxyl group in C-7. In fact, by the NOESY experiments (Figure 1) was observed a weak spatial correlation between the signal at G 6.89 (H-3’ and/or H-5’ with the resonance at G 3.79 corresponding to -OMe. The absence of cross peaks correlation between H-6 and H-8 J. Braz. Chem. Soc. with the CO of the acetyl group could suggested that this group was not attached to the hydroxyl at C-7. However, a correlation observed in the HMBC spectrum (Table 1) between the anomeric hydrogens at G 5.30 and C-5 (161.7) and C-3 (134.2) indicated that the second sugar unit was located at C-3, and thus corroborated the acetylation site at C-7. These findings confirmed the substitution pattern for compound 1. The absence of a bathochromic shift in the UV (methanol) spectrum after the addition of AlCl3 was other important evidence for the substitution of the 5OH, as well as the substitution at C-7 being indicated by the absence of a bathochromic shift in band II (ring A) upon addition of NaOAc. To our knowledge, 1 is a new flavonol derivative assigned as 4’-methoxykaempferol-7(acetyloxy)-3,5-O-D-L-rhamnoside (1). The strong antifungal activity detected in the crude extract (minimum amount required for the inhibition of fungal growth on TLC plates = 10 Pg for C. cladosporioides, standard nystatin = 1.0 Pg) was decreased proportionally during fractionation procedures. The detection limit values of the CHCl3, and EtOAc bioactive solubles (50 and 30 Pg, respectively) suggested that the antifungal activity was substantially lost during guidedfractionating of the EtOAc extract with fungus C. cladosporioides. The very weak activity detected for pure flavonols 1-4 (detection limit values higher than 300 Pg) indicated that their individual activities are not potent enough to be considered for practical use or to justify the strong activity detected in the crude extract. The antifungal activity against C. cladosporioides was significantly enhanced when a combination of the flavonols 1, 2, 3 and 4 where tested. The detection limit of compound 1 against C. cladosporioides was reduced from 350 to 100 Pg when it was combined with compounds 2 (300), 3 (400), and 4 (250). Based on these observations, the synergistic activity of these compounds, and probably of others not isolated in this study, against this fungus, could be inferred. Acknowledgments This work was funded by grants from Biota-FAPESP and CNPq. A. C. de Oliveira thanks CAPES for providing a scholarship. V. da S. Bolzani and M. C. M. Young also are grateful to CNPq for the fellowships of scientific productivity. We thank Dr. J. M. David (UFBA) for facilities in the provision of plant material. References 1. El-Hafiz, M.A.; Weniger, B.; Quirion, J.C.; Anton, R.; Phytochemistry 1991, 30, 2029. Vol. 15, No. 4, 2004 Flavonoids from Chioccoca braquiata (Rubiaceae) 2. Bolzani, V.S.; Young, M.C.M.; Izumisawa, C.M.; Trevisan, L.M.V.; J. Braz. Chem. Soc. 1996, 7, 157. 3. Bolzani, V.S.; Young, M.C.M.; Furlan, M.; Cavalheiro, A.J.; Araújo, A.R.; Silva, D.H.S., Lopes, M.N.; Recent Res. Devel. Phytochem. 2001, 5, 19. 4. Carbonezi, C.A.; Martins, D.; Young, M.C.M.; Lopes, M.N.; 471 7. Shen, Z.; Theander, O.; Phytochemistry 1985, 24, 155. 8. Markham, K.R.; Ternai, B.; Stanley, R.; Geiger, H.; Mabry, T.J.; Tetrahedron 1978, 34, 1389. 9. Mahato, S.B.; Kundu, A.P.; Phytochemistry 1994, 37, 1517. 10. Markham, K.R.; Techniques of Flavonoid Identification, Academic Press: Tokyo, 1982. Furlan, M.; Rodrigues Filho, E.; Bolzani, V.S.; Phytochemistry 1999, 51, 781. 5. Homans, A.L.; Fuchs A.; J. Chromatogr. 1970, 51, 327. Received: October 28, 2003 Published on the web: May 17, 2004 6. Wagner, H.; Chari, V.M.; Sonnenbichler, J.; Tetrahedron Lett. 1976, 21, 1799. FAPESP helped in meeting the publication costs of this article.