J. Braz. Chem. Soc., Vol. 18, No. 6, 1132-1135, 2007. Printed in Brazil - ©2007 Sociedade Brasileira de Química 0103 - 5053 $6.00+0.00 A rt ic le A rt ic le A rt ic le A rt ic le A rt ic le *e-mail: vilegasw@gmail.com New Flavone from the Leaves of Neea theifera (Nyctaginaceae) Daniel Rinaldo, Clenilson M. Rodrigues, Juliana Rodrigues, Miriam Sannomiya, Lourdes C. dos Santos and Wagner Vilegas* Instituto de Química, Universidade Estadual Paulista, CP 355, 14801-900 Araraquara-SP, Brazil Neea theifera Oerst. (Nyctaginaceae) é amplamente utilizada na medicina popular brasileira para tratamento de úlceras gástricas e inflamação. A investigação fitoquímica das folhas de Neea theifera permitiu o isolamento e identificação da nova flavona luteolina-7-O-[2''-O-(5'''- O-feruloil)-β-D-apiofuranosil]-β-D-glucopiranosídeo (1) além dos oito compostos conhecidos vitexina, isovitexina, isoorientina, orientina, vicenina-2, crisoeriol, apigenina e luteolina. A identificação química foi realizada por métodos espectroscópicos incluindo RMN-2D, bem como análises no UV e IES-EM. Neea theifera Oerst. (Nyctaginaceae) is widely used in Brazilian folk medicine for the treatment of gastric ulcers and inflammation. Phytochemical investigation of the leaves of Neea theifera afforded the isolation of the new flavone luteolin-7-O-[2''-O-(5'''-O-feruloyl)-β- D-apiofuranosyl]-β-D-glucopyranoside (1) besides the eight-known compounds vitexin, isovitexin, isoorientin, orientin, vicenin-2, chrysoeriol, apigenin and luteolin. Their chemical identification was established by NMR spectroscopic methods including 2D-NMR, as well as UV and ESI-MS analyses. Keywords: Neea theifera, Nyctaginaceae, flavone, luteolin-7-O-[2''-O-(5'''-O-feruloyl)-β- D-apiofuranosyl]- β-D-glucopyranoside Introduction The Nyctaginaceae family comprises around 300 species in 30 genera.1 Phytochemical investigation with plants from this family is still scarce. Previous phytochemical studies of Boerhavia coccinea and B. erecta led to the isolation of tannins and saponins,2 while B. diffusa yielded dihydroisofuranoxanthone,3 rotenoids4 and lignans.5 Betacianins and flavonoids were isolated from Bougainvillea glabra,6 already flavonoids and phenolic compounds were isolated from B. spectabillis.7,8 Saponins were isolated from Colignonia scandens Benth.9 and from Pisonia umbellifera.10 In our search for bioactive natural products from São Paulo State, Brazil, we have examined the leaves of Neea theifera Oerst. This species is popularly known as ‘Capa- rosa-do-campo’ that grow wild in cerrado lands of Brazil.1 They are used in folk medicine for the treatment of gastric ulcers and inflammation.11 To the best of our knowledge, we could not find any previous phytochemical investigation with plants belonging to this genus. The present paper describes the isolation, purification and structure elucidation of the nine compounds from the leaves of N. theifera. Experimental Plant material Neea theifera Oerst. was collected in March 2005, at Corumbataí, Itirapina city, São Paulo State, Brazil, and authenticated by Prof. Dr. Jorge Yoshio Tamashiro from the Instituto de Biologia, Unicamp, São Paulo. A voucher specimen (HUEC 1396) is on file of the Herbarium of the Universidade Estadual de Campinas, Brazil. Extraction and isolation The dried leaves of N. theifera (953.5 g) were powdered and extracted successively with CHCl3 and MeOH. The methanolic extract (3.0 g) was subjected to RP-C18 CC (40.0 cm × 2.5 i.d.) eluted with H2O:MeOH (9:1), H2O:MeOH (1:1) and MeOH giving 3 fractions: fr. 9:1 (1.8 g), fr. 1:1 (0.86 g) and fr. MeOH (0.34 g), 1133Rinaldo et al.Vol. 18, No. 6, 2007 respectively. The fr. 1:1 was chromatographed on Sephadex LH-20 with H2O:MeOH (1:1) as eluent. Fractions (4.0 mL) were collected and checked by TLC [Si gel plates, CHCl3:MeOH:n-PrOH:H2O (5:6:1:4), organic phase] giving 426 fractions. Fractions 53-58, 59- 61, 66-68, 162-170, 190-206, 233-254 and 284-301 were identified as vitexin (12 mg), isovitexin (26 mg), orientin (16 mg), vicenin-2 (8 mg), chrysoeriol (5 mg), apigenin (4 mg) and luteolin (4 mg), respectively. Fraction 74-82 (60 mg) was further purified by semi-preparative HPLC and afforded to obtain pure isoorientin (6 mg) and pure compound (1) (8 mg). General experimental procedures Melting point was measured on a digital MQ APF- 301 (Microquímica®, Brazil) apparatus. UV spectrum was recorded on a HACH UV-Vis DR/4000 spectrophotometer in MeOH. IR spectrum was obtained using Shimadzu FT- IR 8300 spectrophotometer in KBr disk. NMR analyses and 2D experiments were run on Varian® INOVA 500 operating at 500 MHz for 1H and 125 MHz for 13C (11.7 T), using TMS as internal standard. The ESI-MS spectra were obtained with a Fisons Platform spectrometer in negative mode (70 V). The samples were dissolved in MeOH and injected directly. HREIMS was performed by using an ultrOTOFQ-ESI-TOF Mass Spectrometer Bruker Daltonics® instrument. The compound (1) was dissolved in MeOH, mixed with the internal calibrant, and introduced directly into the ion source by direct infusion. TLC analyses were performed on silica gel 200 µm (Sorbent Technologies®) and visualized using UV light (254 and 365 nm). Semi-preparative HPLC analysis Semi-preparative HPLC analysis was obtained on a HPLC Varian® ProStar 210 system equipped with a Varian® 330 photodiode array detector with a Phenomenex® Luna(2) RP18 column (40 × 2.5 cm , 10 µg) and Reodyne injector of 100 µL. A binary gradient elution system with solvent A (0.05% TFA in H2O) and solvent B (0.05% TFA in CH3CN) was applied with linear gradient formation initially with 68:32 (A:B) at 35 min, and then it was changed to 55:45 (A:B) at 5 min, and finally 55:45 (A:B) at 30 min. The flow-rate was 4.7 mL min–1. Luteolin-7-O-[2''-O-(5'''-O-feruloyl)-β-D-apiofuranosyl]- β-D-glucopyranoside Yellow amorphous powder. mp 250-252 °C. UV (MeOH) λmax/nm: 206, 249 and 334. IR (KBr) νmax/cm-1: 3433, 1655 and 1605. ESI-MS m/z: 755 [M-H]–, 579 [M- E-feruloyl-H]–, 561 [M-E-feruloyl-H2O-H]–, 447 [M-E- feruloyl-apiose-H]–, 285 [M-E-feruloyl-apiose-glucose- H]–. HRESIMS [M-H]– m/z 755.1902 (calculated for C36H36O18 –H, 755.1823). For 1H and 13C NMR analyses see Table 1. Results and Discussion The methanolic extract of the dried powdered leaves of N. theifera was purified by column chromatography (CC) on a Sephadex LH-20 column followed by purification by HPLC-DAD, afforded the isolation of the new compound (1). In addition, eight-known compounds were identified by comparison of their spectroscopic properties with published data: vitexin, isovitexin, isoorientin, orientin, vicenin-2, chrysoeriol, apigenin and luteolin.12-14 Compound (1) was isolated as a yellow solid (mp 250-252 °C). The UV spectral data showed absorption bands at 249 nm and 334 nm. The IR spectrum presented bands at 3433 cm–1 (OH), at 1655 cm–1 (C=O) and 1605 cm–1 (C=C). The molecular formula of compound (1) was calculated as C36H36O18 from its HRESIMS, which showed a [M-H]– at m/z 755.1902 (calculated for C36H36O18-H, 755.1823). The negative ESI-MS (70 V) exhibited the pseudomolecular ion [M-H]– at m/z 755. Key fragmentation ions occurred at m/z 579 [M-E- feruloyl-H]–, m/z 561 [M-E-feruloyl-H2O-H]–, m/z 447 [M-E-feruloyl-apiose-H]– and m/z 285 [M-E-feruloyl- apiose-glucose-H]–. The 1H NMR spectrum (Table 1) showed signals at 7.34 (1H, d, J 8.0 Hz), 7.35 (1H, brs) and at 6.88 (1H, d, J 8.0 Hz) assigned to H-6', H-2' and H-5' respectively, two doublets at 6.68 (1H, d, J 2.0 Hz) and 6.36 (1H, d, J 2.0 Hz), attributed to H-8 and H-6 of the A-ring, and one singlet at 6.54 (1H, s) attributed to H-3 typical of a luteolin derivative. Signals at 6.17 (1H, d, J 16 Hz, H- α), 7.30 (1H, d, J 16 Hz, H-β), 7.07 (1H, d, J 1.5 Hz, H-2''''), 6.69 (1H, d, J 8.0 Hz, H-5'''') and 6.87 (1H, d, J 8.0 and 1.5 Hz, H-6'''') suggested the presence of an E- feruloyl unit.15 A signal at 3.74 (3H, s) indicates the presence of a methoxyl group.13 NOESY experiment showed correlation between signal at 3.74 (OMe) and at 7.07, thus establishing the methoxyl group at position 3'''' of the E-feruloyl unit. A doublet at 5.20 (1H, d, J 7.5 Hz, H-1'') and a singlet at 5.38 (1H, s) in the 1H NMR spectrum revealed the presence of two anomeric hydrogen from two sugar 1134 New Flavone from the Leaves of Neea theifera (Nyctaginaceae) J. Braz. Chem. Soc. units. The TOCSY experiment with irradiation at 5.20 displayed the spin system of the β-D-glucopyranoside unit, whereas irradiation at 5.38 resulted only in the singlet at 3.75 (1H, s) suggesting an apiofuranosyl unit. The coupling constant of the anomeric proton at 5.20 (1H, d, J 7.5 Hz) indicated that the present glucose unit has a β-configuration.13 The 13C NMR experiment presented 35 signals, from which 15 were attributed to the aglycone, 9 to the E-feruloyl unit, 6 to the β-D-glucopyranosyl unit, and with 5 was possible determined a apiofuranosyl unit.14 The apiose unit was characterized through 1H and 13C NMR experiments compared to the literature data. In the 1H NMR spectrum, apiose unit with OH linked to C-1''' and OH linked to C-2''' in trans configuration presents constant coupling J1,2 0-1 Hz, whereas cis configuration is characterized by J1,2 3-4 Hz.16,17 The chemical shift of C-1 in 13C NMR experiments in pyridine-d 5 to the α-D- apiofuranoside is 105 and 112 to β-D-apiofuranoside,18 whereas in DMSO-d 6 these isomers produce signals at 108 and 109, respectively.19,20,21 Thus, the apiose unit in (1) was identified as being a β-D-apiofuranoside. The structure and bonds of these units on compound (1) was established from gHMQC and gHMBC experiments. gHMQC experiment showed direct correlations between carbons and the respective hydrogens (Table 1). gHMBC experiments showed long-range correlations between the hydrogen signal at 5.20 (H-1'’ glucose) and the carbon signal at 162.4 (C-7 aglycone), and between the hydrogen signal at 3.52 (H-2'’ glucose) and the carbon signal at 108.2 (C-1''' apiose). Besides, the chemical shift of the C-2'' of the glucose ( 75.8) unit was clearly dishielded (+3) compared to the chemical shift of the analogous carbon resonance of a non-substituted glucose unit ( 72.9), supporting the glucose (1→2) apiose linkage.14 The gHMBC experiment also showed Table 1. NMR spectral data of compound 1 (11.7 T, DMSO-d 6 )a Position 1H NMR ( H) 13-C NMR ( C) gHMBC ( H) 2 164.4 H-3 3 6.54 s 103.0 4 181.7 5 12.91 161.2 H-6 6 6.36 d (2.0) 99.1 H-8 7 162.4 H-1''; H-6; H-8 8 6.68 d (2.0) 94.4 H-6 9 156.9 H-8 10 105.4 H-6; H-3; H-8 1' 121.6 2' 7.35 brs 113.4 H-6' 3' 149.3 H-5' 4' 145.0 H-2'; H-6' 5' 6.88 d (8.0) 115.9 6' 7.34 d (8.0) 119.1 Glucose 1'' 5.20 d (7.5) 97.8 H-2'' 2'' 3.52 t (7.5; 7.5) 75.8 3'' 3.45 t (7.5; 7.5) 76.7 H-2'' 4'' 3.20 t (7.5; 7.5) 69.8 H-2'' 5'' 3.48 m 77.0 H-4''; H-3'' 6'' a) 3.50 m b) 3.71 d (10.0) 60.5 Apiose 1''' 5.38 s 108.2 H-2'''; H-2'' 2''' 3.75 s 76.6 3''' 77.5 H-2'''; H-4''' 4''' a) 3.78 d (9.5) b) 4.04 d (9.5) 73.8 H-5'''; H-2''' 5''' 4.06 brs 66.6 H-2'''; H-4''' E-Feruloyl α 6.17 d (16.0) 113.7 H-β β 7.30 d (16.0) 144.9 H-6''''; H-2'''' 1'''' 125.4 H-α; H-5'''' 2'''' 7.07 d (1.5) 110.9 H-6''''; H-β 3'''' 147.8 -OCH3; H-5'''' 4'''' 149.3 H-6''''; H-2'''' 5'''' 6.69 d (8.0) 115.4 H-6'''' 6'''' 6.87 dd (8.0; 1.5) 122.9 H-2''''; H-β -OCH3 3.74 s 55.5 C=O 166.3 H-5'''; H-β aChemical shifts ( ) are in ppm, and coupling constants (J in Hz) are given in parentheses. Figure 1. Structure of compound (1). α β 1 4 2 3 5 6 7 8 9 10 1' 2' 3' 4' 5' 6'1’’2’’ 3’’ 4’’ 5'' 6'' 1’’’ 2’’’ 3’’’ 4’’’ 5’’’ 1’’’’ 2’’’’ 3’’’’ 4’’’’ 5’’’’ 6’’’’ O OOH OH O OH O O OH OH OHHa Hb Ha O OH OH O O O OH CH3 Hb α β 1 4 2 3 5 6 7 8 9 10 1' 2' 3' 4' 5' 6'1’’2’’ 3’’ 4’’ 5'' 6'' 1’’’ 2’’’ 3’’’ 4’’’ 5’’’ 1’’’’ 2’’’’ 3’’’’ 4’’’’ 5’’’’ 6’’’’ O OOH OH O OH O O OH OH OHHa Hb Ha O OH OH O O O OH CH3 Hb 1135Rinaldo et al.Vol. 18, No. 6, 2007 correlation between the hydrogen signal at 4.06 (H-5''' apiose) and the carbon signal at 166.3 (E-feruloyl, C=O) thus evidencing the esterification at this position. The foregoing evidences in combination with the downfield shift of the C-5''' apiofuranosyl ( 66.6) when compared to a non-acylated analogue ( 62.4) also supported this conclusion. Therefore, compound (1) was identified as luteolin-7-O-[2''-O-(5'''-O-feruloyl)-β-D-apiofuranosyl]-β- D-glucopyranoside (Figure 1). A few reports were observed describing the presence of flavonoids in the Nyctaginaceae family. Until now only flavonols were related, such as kaempferol and quercetin from the Bougainvillea glabra6 and B. spectabillis.7 Being thus, Neea theifera is the unique species of Nyctaginaceae, which produces flavones. These data are important because reveal to be able in the future to establish a taxonomic marker in the genus or in this species. Acknowledgments We thank Dr. Nivaldo Boralle from Instituto de Química UNESP de Araraquara-SP for recording the NMR spectra, to Dr. Norberto Peporine Lopes from Faculdade de Filosofia Ciências e Letras USP de Ribeirão Preto-SP for HRESIMS analysis, to Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) for funding and fellowships to D. Rinaldo and M. Sannomiya, to Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for grants to W. Vilegas. Supplementary Information Supplementary data are available free of charge at http://jbcs.sbq.org.br, as PDF file. References 1. Durigan, G.; Baitello, J. B.; Franco, G. A. D. C.; de Siqueira, M. F.; Plantas do Cerrado Paulista : Imagens de uma Paisagem Ameaçada, Páginas & Letras Editora e Gráfica: São Paulo, 2004. 2. Edeoga, H. O.; Ikem, C. I.; S. Afr. J. Bot. 2002, 68, 382. 3. Ahmed, B.; Yu, C. P.; Phytochemistry 1992, 31, 4382. 4. Lami, N.; Kadota, S.; Kikuchi, T.; Chem. Pharm. Bull. 1991, 39, 1863. 5. Lami, N.; Kadota, S.; Kikuchi, T.; Momose, Y.; Chem. Pharm. Bull. 1991, 39, 1551. 6. Heuer, S.; Richter, S.; Metzger, J. W.; Wray, V.; Nimtz, M.; Strack, D.; Phytochemistry 1994, 37, 761. 7. Chang, W. S.; Lee, Y. J.; Lu, F. J.; Chiang, H. C.; Anti-Cancer Res. 1993, 13, 2165. 8. Chang, W. S.; Chang, Y. H.; Lu, F. J.; Chiang, H.; C. Anti- Cancer Res. 1994, 14, 501. 9. De Feo, V.; Piacente, S.; Pizza, C.; Soria, R. U.; Biochem. Syst. Ecol. 1998, 26, 251. 10. Lavaud, C.; Beauviere, S.; Massiot, G.; Lemenolivier, L.; Bourdy, G.; Phytochemistry 1996, 43, 189. 11. Duke, J.; Vasquez, R.; Amazonian Ethnobotanical Dictionary, CRC Press: Boca Raton, 1994. 12. Mabry, T. J.; Markham, K. R.; Thomas, M. B.; The Systematic Identification of Flavonoids, Springer: New York, 1970. 13. Harborne, J. B.; The Flavonoids: Advances in Research Since 1986, Chapman and Hall: London, 1996. 14. Agrawal, P. K.; Carbon 13 of Flavonoids, Elsevier: New York, 1989. 15. dos Santos, L.C.; Piacente, S.; Pizza, C.; Toro, R.; Sano, P. T.; Vilegas, W.; Biochem. Syst. Ecol. 2002, 30, 451. 16. Angyal, S. J.; Bodkin, C. L.; Mills, J. A.; Pojer, P. M.; Aust. J. Chem. 1977, 30, 1259. 17. Tronchet, J. M. J.; Tronchet, J.; Carbohydr. Res. 1974, 34, 263. 18. Kitagawa, I.; Sakagami, M.; Hashiuchi, F.; Zhou, J. L.; Yoshikawa, M.; Ren, J.; Chem. Pharm. Bull. 1989, 37, 551. 19. Jung, M. J.; Kang, S. S.; Jung, Y. J.; Choi, J. S.; Chem. Pharm. Bull. 2004, 52, 1501. 20. Mathias, L.; Vieira, I. J. C.; Braz-Filho, R.; Rodrigues-Filho, E. A.; J. Nat. Prod. 1998, 61, 1158. 21. Bashir, A.; Hamburger, M.; Gupta, M. P.; Solis, P. N.; Hostettmann, K.; Phytochemistry 1991, 30, 3781. Received: December 20, 2006 Web Release Date: September 4, 2007 FAPESP helped in meeting the publication costs of this article. J. Braz. Chem. Soc., Vol. 18, No. 6, S1-S17, 2007. Printed in Brazil - ©2007 Sociedade Brasileira de Química 0103 - 5053 $6.00+0.00 Supplem entary Inform ation Supplem entary Inform ation Supplem entary Inform ation Supplem entary Inform ation Supplem entary Inform ation *e-mail: vilegasw@gmail.com New Flavone from the Leaves of Neea theifera (Nyctaginaceae) Daniel Rinaldo, Clenilson M. Rodrigues, Juliana Rodrigues, Miriam Sannomiya, Lourdes C. dos Santos and Wagner Vilegas* Instituto de Química, Universidade Estadual Paulista, CP 355, 14801-900 Araraquara-SP, Brazil Figure S1. UV spectrum of (1) (MeOH). 1.00 Wavelength / nm In te n si ty / a u 1.25 400 0.75 250 0.25 0.50 200 2 0 6 .2 0 2 4 9 .4 9 3 3 4 .1 9 300 350 Figure S2. IR spectrum of (1) (KBr disk). 29 25.84 343 3.0 9 4000 3000 2000 1000 594.04 819 .70 1031.86 10 74.29 1122 .50 1 176.51 12 59.44 1332 .73 1 380.95 145 2.3 1 1514.03 1 604.68 165 4.8 3 1 683.76 170 3.0 4 23 72.30 29 25.84 343 3.0 9 29 25.84 343 3.0 9 4000 3000 2000 1000 Wavenumber / cm-1 T ra n sm itt a n ce / % 0 10 20 30 40 50 60 70 80 90 100 594.04 819 .70 1031.86 10 74.29 1122 .50 1 176.51 12 59.44 1332 .73 1 380.95 145 2.3 1 1514.03 1 604.68 165 4.8 3 1 683.76 170 3.0 4 23 72.30 29 25.84 343 3.0 9 S2 New Flavone from the Leaves of Neea theifera (Nyctaginaceae) J. Braz. Chem. Soc. Figure S3. 1H NMR spectrum of (1) (DMSO-d 6 , 11.7 T, TMS, ppm). 5.5 5.0 4.5 4.0 3.5 6.176.101.65 1.60 3 .7 3 6 3 .7 5 1 3 .7 6 1 3 .7 8 0 4 .0 22 4 .0 4 1 4 .0 5 6 5 .1 9 0 5 .2 0 5 5 .3 7 6 7.4 7.3 7.2 7.1 7.0 6.9 6.8 6.7 6.6 6.5 6.4 6.3 6.2 6.1 4.013.653.65 1.831.791.771.70 1.71 6 .1 5 5 6 .1 8 7 6 .3 53 6 .3 5 8 6 .5 4 5 6 .6 7 2 6 .6 8 1 6 .6 85 6 .6 8 8 6 .8 4 8 6 .8 5 1 6 .8 6 1 6 .8 7 7 7 .0 6 4 7 .0 6 7 7 .2 8 2 7 .3 1 4 7 .3 3 4 7 .3 5 1 6 .8 6 8 5.5 5.0 4.5 4.0 3.5 6.176.101.65 1.60 3 .7 3 6 3 .7 5 1 3 .7 6 1 3 .7 8 0 4 .0 22 4 .0 4 1 4 .0 5 6 5 .1 9 0 5 .2 0 5 5 .3 7 6 5.5 5.0 4.5 4.0 3.5 6.176.101.65 1.60 3 .7 3 6 3 .7 5 1 3 .7 6 1 3 .7 8 0 4 .0 22 4 .0 4 1 4 .0 5 6 5 .1 9 0 5 .2 0 5 5 .3 7 6 7.4 7.3 7.2 7.1 7.0 6.9 6.8 6.7 6.6 6.5 6.4 6.3 6.2 6.1 4.013.653.65 1.831.791.771.70 1.71 6 .1 5 5 6 .1 8 7 6 .3 53 6 .3 5 8 6 .5 4 5 6 .6 7 2 6 .6 8 1 6 .6 85 6 .6 8 8 6 .8 4 8 6 .8 5 1 6 .8 6 1 6 .8 7 7 7 .0 6 4 7 .0 6 7 7 .2 8 2 7 .3 1 4 7 .3 3 4 7 .3 5 1 6 .8 6 8 7.4 7.3 7.2 7.1 7.0 6.9 6.8 6.7 6.6 6.5 6.4 6.3 6.2 6.1 4.013.653.65 1.831.791.771.70 1.71 6 .1 5 5 6 .1 8 7 6 .3 53 6 .3 5 8 6 .5 4 5 6 .6 7 2 6 .6 8 1 6 .6 85 6 .6 8 8 6 .8 4 8 6 .8 5 1 6 .8 6 1 6 .8 7 7 7 .0 6 4 7 .0 6 7 7 .2 8 2 7 .3 1 4 7 .3 3 4 7 .3 5 1 6 .8 6 8 δ / ppm δ / ppm 5.5 5.0 4.5 4.0 3.5 6.176.101.65 1.60 3 .7 3 6 3 .7 5 1 3 .7 6 1 3 .7 8 0 4 .0 22 4 .0 4 1 4 .0 5 6 5 .1 9 0 5 .2 0 5 5 .3 7 6 7.4 7.3 7.2 7.1 7.0 6.9 6.8 6.7 6.6 6.5 6.4 6.3 6.2 6.1 4.013.653.65 1.831.791.771.70 1.71 6 .1 5 5 6 .1 8 7 6 .3 53 6 .3 5 8 6 .5 4 5 6 .6 7 2 6 .6 8 1 6 .6 85 6 .6 8 8 6 .8 4 8 6 .8 5 1 6 .8 6 1 6 .8 7 7 7 .0 6 4 7 .0 6 7 7 .2 8 2 7 .3 1 4 7 .3 3 4 7 .3 5 1 6 .8 6 8 5.5 5.0 4.5 4.0 3.5 6.176.101.65 1.60 3 .7 3 6 3 .7 5 1 3 .7 6 1 3 .7 8 0 4 .0 22 4 .0 4 1 4 .0 5 6 5 .1 9 0 5 .2 0 5 5 .3 7 6 5.5 5.0 4.5 4.0 3.5 6.176.101.65 1.60 3 .7 3 6 3 .7 5 1 3 .7 6 1 3 .7 8 0 4 .0 22 4 .0 4 1 4 .0 5 6 5 .1 9 0 5 .2 0 5 5 .3 7 6 7.4 7.3 7.2 7.1 7.0 6.9 6.8 6.7 6.6 6.5 6.4 6.3 6.2 6.1 4.013.653.65 1.831.791.771.70 1.71 6 .1 5 5 6 .1 8 7 6 .3 53 6 .3 5 8 6 .5 4 5 6 .6 7 2 6 .6 8 1 6 .6 85 6 .6 8 8 6 .8 4 8 6 .8 5 1 6 .8 6 1 6 .8 7 7 7 .0 6 4 7 .0 6 7 7 .2 8 2 7 .3 1 4 7 .3 3 4 7 .3 5 1 6 .8 6 8 7.4 7.3 7.2 7.1 7.0 6.9 6.8 6.7 6.6 6.5 6.4 6.3 6.2 6.1 4.013.653.65 1.831.791.771.70 1.71 6 .1 5 5 6 .1 8 7 6 .3 53 6 .3 5 8 6 .5 4 5 6 .6 7 2 6 .6 8 1 6 .6 85 6 .6 8 8 6 .8 4 8 6 .8 5 1 6 .8 6 1 6 .8 7 7 7 .0 6 4 7 .0 6 7 7 .2 8 2 7 .3 1 4 7 .3 3 4 7 .3 5 1 6 .8 6 8 δ / ppm δ / ppm 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 6.174.013.65 1.651.711.70 DMSOÁgua 2 .4 99 3 .4 48 3 .7 36 TMS 0 .0 0 1 2 .9 1 3 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 6.174.013.65 1.651.711.70 DMSOÁgua 2 .4 99 3 .4 48 3 .7 36 TMS 0 .0 0 1 2 .9 1 3 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 6.174.013.65 1.651.711.70 DMSOÁgua 2 .4 99 3 .4 48 3 .7 36 TMS 0 .0 0 1 2 .9 1 3 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 6.174.013.65 1.651.711.70 DMSOÁgua 2 .4 99 3 .4 48 3 .7 36 TMS 0 .0 0 1 2 .9 1 3 δ / ppmδ / ppm S3Rinaldo et al.Vol. 18, No. 6, 2007 Figure S4. 1H NMR 1D-NOESY of (1) (DMSO-d 6 , 11.7 T, ppm). 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 7 .0 67 3. 7 36 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 7 .0 67 3. 7 36 δ / ppm 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 7 .0 67 3. 7 36 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 7 .0 67 3. 7 36 δ / ppm S4 New Flavone from the Leaves of Neea theifera (Nyctaginaceae) J. Braz. Chem. Soc. Figure S5. 13C NMR spectrum of (1) (DMSO-d 6 , 11.7 T, ppm). 200 150 100 50 0 DMSO-d6 3 9 .5 0 1 6 0 .2 9 0 7 2 .2 5 8 1 8 1 .7 2 9 200 150 100 50 0 DMSO-d6 3 9 .5 0 1 6 0 .2 9 0 7 2 .2 5 8 1 8 1 .7 2 9 200 150 100 50 0 DMSO-d6 3 9 .5 0 1 6 0 .2 9 0 7 2 .2 5 8 1 8 1 .7 2 9 200 150 100 50 0 DMSO-d6 3 9 .5 0 1 6 0 .2 9 0 7 2 .2 5 8 1 8 1 .7 2 9 δ / ppmδ / ppm 180 175 170 165 160 155 150 145 140 135 130 125 120 115 110 105 100 95 9 4 .3 7 3 9 7 .7 6 5 9 9 .1 3 4 1 0 3 .0 5 5 1 0 5 .3 7 5 1 0 8 .2 3 8 1 1 0 .9 3 2 1 1 3 .4 4 2 1 1 3 .7 3 2 1 1 5 .4 1 0 1 1 5 .9 3 9 1 1 9 .0 9 1 1 2 1 .6 3 2 1 2 2 .8 7 1 1 2 5 .3 9 6 1 4 4 .9 5 9 1 4 7 .8 0 1 1 4 9 .2 8 2 1 4 9 .9 9 11 5 6 .9 1 9 1 6 1 .2 2 1 1 6 2 .4 4 1 1 6 4 .3 7 41 6 6 .2 8 5 1 8 1 .7 2 9 1 1 1 .9 7 6 75 70 65 60 5 5 .5 4 4 6 0 .2 9 0 6 0 .5 3 7 6 6 .6 0 96 9 .8 1 7 7 2 .2 5 8 7 3 .7 6 7 7 5 .7 7 0 7 6 .6 3 0 7 6 .7 1 5 7 7 .0 2 5 7 7 .4 9 0 180 175 170 165 160 155 150 145 140 135 130 125 120 115 110 105 100 95 9 4 .3 7 3 9 7 .7 6 5 9 9 .1 3 4 1 0 3 .0 5 5 1 0 5 .3 7 5 1 0 8 .2 3 8 1 1 0 .9 3 2 1 1 3 .4 4 2 1 1 3 .7 3 2 1 1 5 .4 1 0 1 1 5 .9 3 9 1 1 9 .0 9 1 1 2 1 .6 3 2 1 2 2 .8 7 1 1 2 5 .3 9 6 1 4 4 .9 5 9 1 4 7 .8 0 1 1 4 9 .2 8 2 1 4 9 .9 9 11 5 6 .9 1 9 1 6 1 .2 2 1 1 6 2 .4 4 1 1 6 4 .3 7 41 6 6 .2 8 5 1 8 1 .7 2 9 1 1 1 .9 7 6 180 175 170 165 160 155 150 145 140 135 130 125 120 115 110 105 100 95180 175 170 165 160 155 150 145 140 135 130 125 120 115 110 105 100 95 9 4 .3 7 3 9 7 .7 6 5 9 9 .1 3 4 1 0 3 .0 5 5 1 0 5 .3 7 5 1 0 8 .2 3 8 1 1 0 .9 3 2 1 1 3 .4 4 2 1 1 3 .7 3 2 1 1 5 .4 1 0 1 1 5 .9 3 9 1 1 9 .0 9 1 1 2 1 .6 3 2 1 2 2 .8 7 1 1 2 5 .3 9 6 1 4 4 .9 5 9 1 4 7 .8 0 1 1 4 9 .2 8 2 1 4 9 .9 9 11 5 6 .9 1 9 1 6 1 .2 2 1 1 6 2 .4 4 1 1 6 4 .3 7 41 6 6 .2 8 5 1 8 1 .7 2 9 1 1 1 .9 7 6 75 70 65 60 5 5 .5 4 4 6 0 .2 9 0 6 0 .5 3 7 6 6 .6 0 96 9 .8 1 7 7 2 .2 5 8 7 3 .7 6 7 7 5 .7 7 0 7 6 .6 3 0 7 6 .7 1 5 7 7 .0 2 5 7 7 .4 9 0 75 70 65 60 5 5 .5 4 4 6 0 .2 9 0 6 0 .5 3 7 6 6 .6 0 96 9 .8 1 7 7 2 .2 5 8 7 3 .7 6 7 7 5 .7 7 0 7 6 .6 3 0 7 6 .7 1 5 7 7 .0 2 5 7 7 .4 9 0 180 175 170 165 160 155 150 145 140 135 130 125 120 115 110 105 100 95 9 4 .3 7 3 9 7 .7 6 5 9 9 .1 3 4 1 0 3 .0 5 5 1 0 5 .3 7 5 1 0 8 .2 3 8 1 1 0 .9 3 2 1 1 3 .4 4 2 1 1 3 .7 3 2 1 1 5 .4 1 0 1 1 5 .9 3 9 1 1 9 .0 9 1 1 2 1 .6 3 2 1 2 2 .8 7 1 1 2 5 .3 9 6 1 4 4 .9 5 9 1 4 7 .8 0 1 1 4 9 .2 8 2 1 4 9 .9 9 11 5 6 .9 1 9 1 6 1 .2 2 1 1 6 2 .4 4 1 1 6 4 .3 7 41 6 6 .2 8 5 1 8 1 .7 2 9 1 1 1 .9 7 6 75 70 65 60 5 5 .5 4 4 6 0 .2 9 0 6 0 .5 3 7 6 6 .6 0 96 9 .8 1 7 7 2 .2 5 8 7 3 .7 6 7 7 5 .7 7 0 7 6 .6 3 0 7 6 .7 1 5 7 7 .0 2 5 7 7 .4 9 0 180 175 170 165 160 155 150 145 140 135 130 125 120 115 110 105 100 95 9 4 .3 7 3 9 7 .7 6 5 9 9 .1 3 4 1 0 3 .0 5 5 1 0 5 .3 7 5 1 0 8 .2 3 8 1 1 0 .9 3 2 1 1 3 .4 4 2 1 1 3 .7 3 2 1 1 5 .4 1 0 1 1 5 .9 3 9 1 1 9 .0 9 1 1 2 1 .6 3 2 1 2 2 .8 7 1 1 2 5 .3 9 6 1 4 4 .9 5 9 1 4 7 .8 0 1 1 4 9 .2 8 2 1 4 9 .9 9 11 5 6 .9 1 9 1 6 1 .2 2 1 1 6 2 .4 4 1 1 6 4 .3 7 41 6 6 .2 8 5 1 8 1 .7 2 9 1 1 1 .9 7 6 180 175 170 165 160 155 150 145 140 135 130 125 120 115 110 105 100 95180 175 170 165 160 155 150 145 140 135 130 125 120 115 110 105 100 95 9 4 .3 7 3 9 7 .7 6 5 9 9 .1 3 4 1 0 3 .0 5 5 1 0 5 .3 7 5 1 0 8 .2 3 8 1 1 0 .9 3 2 1 1 3 .4 4 2 1 1 3 .7 3 2 1 1 5 .4 1 0 1 1 5 .9 3 9 1 1 9 .0 9 1 1 2 1 .6 3 2 1 2 2 .8 7 1 1 2 5 .3 9 6 1 4 4 .9 5 9 1 4 7 .8 0 1 1 4 9 .2 8 2 1 4 9 .9 9 11 5 6 .9 1 9 1 6 1 .2 2 1 1 6 2 .4 4 1 1 6 4 .3 7 41 6 6 .2 8 5 1 8 1 .7 2 9 1 1 1 .9 7 6 75 70 65 60 5 5 .5 4 4 6 0 .2 9 0 6 0 .5 3 7 6 6 .6 0 96 9 .8 1 7 7 2 .2 5 8 7 3 .7 6 7 7 5 .7 7 0 7 6 .6 3 0 7 6 .7 1 5 7 7 .0 2 5 7 7 .4 9 0 75 70 65 60 5 5 .5 4 4 6 0 .2 9 0 6 0 .5 3 7 6 6 .6 0 96 9 .8 1 7 7 2 .2 5 8 7 3 .7 6 7 7 5 .7 7 0 7 6 .6 3 0 7 6 .7 1 5 7 7 .0 2 5 7 7 .4 9 0 δ / ppmδ / ppm δ / ppm S5Rinaldo et al.Vol. 18, No. 6, 2007 Figure S6. TOCSY spectrum of (1) (DMSO-d 6 , 11.7 T, ppm): a) Apiose; b) Glucose. 5.0 4.5 4.0 3.5 3. 18 0 3. 19 8 3. 21 5 3. 43 9 3. 45 3 3. 47 7 3. 50 8 3. 52 5 3. 53 9 3. 55 8 3. 68 6 3. 70 6 5. 19 0 5. 20 5 3. 48 9 3. 46 8 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 3. 75 1 5. 37 6 b) a) 5.0 4.5 4.0 3.5 3. 18 0 3. 19 8 3. 21 5 3. 43 9 3. 45 3 3. 47 7 3. 50 8 3. 52 5 3. 53 9 3. 55 8 3. 68 6 3. 70 6 5. 19 0 5. 20 5 3. 48 9 3. 46 8 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 3. 75 1 5. 37 6 5.0 4.5 4.0 3.5 3. 18 0 3. 19 8 3. 21 5 3. 43 9 3. 45 3 3. 47 7 3. 50 8 3. 52 5 3. 53 9 3. 55 8 3. 68 6 3. 70 6 5. 19 0 5. 20 5 3. 48 9 3. 46 8 5.0 4.5 4.0 3.5 3. 18 0 3. 19 8 3. 21 5 3. 43 9 3. 45 3 3. 47 7 3. 50 8 3. 52 5 3. 53 9 3. 55 8 3. 68 6 3. 70 6 5. 19 0 5. 20 5 3. 48 9 3. 46 8 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 3. 75 1 5. 37 6 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 3. 75 1 5. 37 6 b) a) δ / ppm δ / ppm 5.0 4.5 4.0 3.5 3. 18 0 3. 19 8 3. 21 5 3. 43 9 3. 45 3 3. 47 7 3. 50 8 3. 52 5 3. 53 9 3. 55 8 3. 68 6 3. 70 6 5. 19 0 5. 20 5 3. 48 9 3. 46 8 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 3. 75 1 5. 37 6 b) a) 5.0 4.5 4.0 3.5 3. 18 0 3. 19 8 3. 21 5 3. 43 9 3. 45 3 3. 47 7 3. 50 8 3. 52 5 3. 53 9 3. 55 8 3. 68 6 3. 70 6 5. 19 0 5. 20 5 3. 48 9 3. 46 8 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 3. 75 1 5. 37 6 5.0 4.5 4.0 3.5 3. 18 0 3. 19 8 3. 21 5 3. 43 9 3. 45 3 3. 47 7 3. 50 8 3. 52 5 3. 53 9 3. 55 8 3. 68 6 3. 70 6 5. 19 0 5. 20 5 3. 48 9 3. 46 8 5.0 4.5 4.0 3.5 3. 18 0 3. 19 8 3. 21 5 3. 43 9 3. 45 3 3. 47 7 3. 50 8 3. 52 5 3. 53 9 3. 55 8 3. 68 6 3. 70 6 5. 19 0 5. 20 5 3. 48 9 3. 46 8 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 3. 75 1 5. 37 6 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 3. 75 1 5. 37 6 b) a) δ / ppm δ / ppm S6 New Flavone from the Leaves of Neea theifera (Nyctaginaceae) J. Braz. Chem. Soc. δ / ppm (F1) 180 160 140 120 100 80.0 60.0 40.0 20.0 δ / p pm (F 2 ) 13.0 12.0 11.0 10.0 9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0 -1.0 200 δ / ppm (F1) 180 160 140 120 100 80.0 60.0 40.0 20.0 δ / p pm (F 2 ) 13.0 12.0 11.0 10.0 9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0 -1.0 200 Figure S7. gHMQC spectrum of (1) (DMSO-d 6 , 11.7 T, TMS, ppm). Figure S8. gHMBC spectrum of (1) (DMSO-d 6 , 11.7 T, TMS, ppm). 200 180 160 140 120 100 80.0 60.0 40.0 20.0 13.0 12.0 11.0 10.0 9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0 -1.0 δ / ppm (F1) 200 180 160 140 120 100 80.0 60.0 40.0 20.0 δ / p pm (F 2 ) 13.0 12.0 11.0 10.0 9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0 -1.0 200 180 160 140 120 100 80.0 60.0 40.0 20.0 13.0 12.0 11.0 10.0 9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0 -1.0 δ / ppm (F1) 200 180 160 140 120 100 80.0 60.0 40.0 20.0 δ / p pm (F 2 ) 13.0 12.0 11.0 10.0 9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0 -1.0 S7Rinaldo et al.Vol. 18, No. 6, 2007 Figure S9. ESI-MS spectrum of (1) (negative mode, 70 V). 755 285 284 283 561 286 299 447 365 543448 579 562 563 580 581 595 753 756 836 757 835771 801 820 837 200 250 300 350 400 450 500 550 600 650 700 750 800 850 m/z 0 100 A b un d an ce / % 365 595 771 Figure S10. 1H NMR spectrum of vitexin (DMSO-d6, 4.7 T, TMS, ppm). 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 2.252.20 1.091.011.00 DMSO 4 .7 0 4 .7 5 6 .2 1 6 .7 1 6 .9 0 7 .9 6 8 .0 0 1 3 .1 3 6 .8 6 TMS 8.0 7.5 7.0 6.5 6.0 5.5 5.0 2.252.20 1.091.02 1.01 4. 70 4. 75 6. 21 6. 7 1 6. 86 6. 90 7. 96 8. 00 2 .4 9 0 .0 0 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 2.252.20 1.091.011.00 DMSO 4 .7 0 4 .7 5 6 .2 1 6 .7 1 6 .9 0 7 .9 6 8 .0 0 1 3 .1 3 6 .8 6 TMS 8.0 7.5 7.0 6.5 6.0 5.5 5.0 2.252.20 1.091.02 1.01 4. 70 4. 75 6. 21 6. 7 1 6. 86 6. 90 7. 96 8. 00 2 .4 9 0 .0 0 S8 New Flavone from the Leaves of Neea theifera (Nyctaginaceae) J. Braz. Chem. Soc. Figure S11. 13C NMR spectrum of vitexin (DMSO-d6, 4.7 T, ppm). 200 150 100 50 0 DMSO 3 9 .5 0 6 1 .3 0 7 0 .5 57 0 .9 1 7 3 .4 6 7 8 .7 1 8 1 .8 2 9 8 .3 51 0 2 .4 4 1 0 4 .6 4 1 1 5 .8 9 1 2 1 .6 2 1 2 8 .9 3 1 5 6 .0 1 1 6 0 .4 4 1 6 1 .2 3 1 6 3 .8 5 1 8 2 .0 1 180 170 160 150 140 130 120 110 100 90 80 70 60 61 .3 0 70 .5 57 0. 91 7 3. 46 7 8. 71 81 .8 2 98 .3 5 10 2 .4 4 10 4. 64 1 15 .8 9 12 1. 62 12 8. 93 15 6 .0 1 16 0 .4 4 16 1 .2 3 16 3 .8 5 18 2. 01 1 63 .2 9 200 150 100 50 0 DMSO 3 9 .5 0 6 1 .3 0 7 0 .5 57 0 .9 1 7 3 .4 6 7 8 .7 1 8 1 .8 2 9 8 .3 51 0 2 .4 4 1 0 4 .6 4 1 1 5 .8 9 1 2 1 .6 2 1 2 8 .9 3 1 5 6 .0 1 1 6 0 .4 4 1 6 1 .2 3 1 6 3 .8 5 1 8 2 .0 1 180 170 160 150 140 130 120 110 100 90 80 70 60 61 .3 0 70 .5 57 0. 91 7 3. 46 7 8. 71 81 .8 2 98 .3 5 10 2 .4 4 10 4. 64 1 15 .8 9 12 1. 62 12 8. 93 15 6 .0 1 16 0 .4 4 16 1 .2 3 16 3 .8 5 18 2. 01 1 63 .2 9 Figure S12. ESI-MS spectrum of vitexin (negative mode, 70 V). S9Rinaldo et al.Vol. 18, No. 6, 2007 Figure S13. 1H NMR spectrum of the mixture of isovitexin and vitexin (DMSO-d6, 4.7 T, TMS, ppm). 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 6.054.08 2.052.02 1.041.00 TMS DMSO 0 .0 0 2 .4 9 4 .5 7 4 .6 2 4 .7 0 4 .7 5 6 .2 1 6 .4 1 6 .7 1 6 .8 6 6 .9 0 7 .8 6 7 .9 0 7 .9 6 8 .0 0 1 3 .1 2 1 3 .5 6 2.08 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 6.054.08 2.053.04 1.012.04 1.04 4. 57 4 .6 2 4. 70 4 .7 5 6. 21 6. 41 6. 71 6. 86 6. 90 7 .8 6 7. 90 7. 96 8. 00 2.08 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 6.054.08 2.052.02 1.041.00 TMS DMSO 0 .0 0 2 .4 9 4 .5 7 4 .6 2 4 .7 0 4 .7 5 6 .2 1 6 .4 1 6 .7 1 6 .8 6 6 .9 0 7 .8 6 7 .9 0 7 .9 6 8 .0 0 1 3 .1 2 1 3 .5 6 2.08 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 6.054.08 2.052.02 1.041.00 TMS DMSO 0 .0 0 2 .4 9 4 .5 7 4 .6 2 4 .7 0 4 .7 5 6 .2 1 6 .4 1 6 .7 1 6 .8 6 6 .9 0 7 .8 6 7 .9 0 7 .9 6 8 .0 0 1 3 .1 2 1 3 .5 6 2.08 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 6.054.08 2.053.04 1.012.04 1.04 4. 57 4 .6 2 4. 70 4 .7 5 6. 21 6. 41 6. 71 6. 86 6. 90 7 .8 6 7. 90 7. 96 8. 00 2.08 Figure S14. ESI-MS spectrum of the mixture of isovitexin and vitexin (negative mode, 70 V). S10 New Flavone from the Leaves of Neea theifera (Nyctaginaceae) J. Braz. Chem. Soc. Figure S15. 1H NMR spectrum of isoorientin (DMSO-d6, 4.7 T, TMS, ppm). 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 2.10 1.341.251.00 DMSO Água 2 .4 9 3 .4 5 4 .5 4 4 .5 9 6 .4 7 6 .6 6 6. 8 9 7 .3 8 7 .4 2 1 3 .5 7 1.05 0 TMS 0 .0 0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 2.10 1.341.251.05 1.03 4. 54 4. 59 6. 47 6. 66 6. 85 6. 89 7. 38 7. 42 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 2.10 1.341.251.00 DMSO Água 2 .4 9 3 .4 5 4 .5 4 4 .5 9 6 .4 7 6 .6 6 6. 8 9 7 .3 8 7 .4 2 1 3 .5 7 1.05 0 TMS 0 .0 0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 2.10 1.341.251.05 1.03 4. 54 4. 59 6. 47 6. 66 6. 85 6. 89 7. 38 7. 42 Figure S16. 13C NMR spectrum of isoorientin (DMSO-d6, 4.7 T, ppm). 200 150 100 50 0 DMSO 3 9 .5 0 6 0 .4 8 7 0 .3 8 7 0 .8 1 7 3 .2 37 8 .4 8 8 1 .7 6 9 3 .7 9 1 0 3 .0 0 1 0 3 .5 9 1 0 9 .0 3 1 1 3 .4 3 1 1 9 .2 6 1 2 1 .6 2 1 4 6 .0 1 1 4 9 .9 8 1 5 6 .5 1 1 6 0 .9 0 1 6 3 .9 5 1 8 2 .1 8 180 170 160 150 140 130 120 110 100 90 80 70 60 6 0. 48 70 .3 8 7 0. 81 73 .2 378 .4 8 81 .7 6 93 .7 9 10 3. 00 1 03 .5 9 10 9 .0 3 11 3. 43 11 6 .3 1 1 19 .2 6 12 1. 62 14 6. 01 14 9. 98 15 6. 5 1 16 0 .9 0 1 63 .9 5 1 82 .1 8 200 150 100 50 0 DMSO 3 9 .5 0 6 0 .4 8 7 0 .3 8 7 0 .8 1 7 3 .2 37 8 .4 8 8 1 .7 6 9 3 .7 9 1 0 3 .0 0 1 0 3 .5 9 1 0 9 .0 3 1 1 3 .4 3 1 1 9 .2 6 1 2 1 .6 2 1 4 6 .0 1 1 4 9 .9 8 1 5 6 .5 1 1 6 0 .9 0 1 6 3 .9 5 1 8 2 .1 8 180 170 160 150 140 130 120 110 100 90 80 70 60 6 0. 48 70 .3 8 7 0. 81 73 .2 378 .4 8 81 .7 6 93 .7 9 10 3. 00 1 03 .5 9 10 9 .0 3 11 3. 43 11 6 .3 1 1 19 .2 6 12 1. 62 14 6. 01 14 9. 98 15 6. 5 1 16 0 .9 0 1 63 .9 5 1 82 .1 8 S11Rinaldo et al.Vol. 18, No. 6, 2007 Figure S17. ESI-MS spectrum of isoorientin (negative mode, 70 V). Figure S18. 1H NMR spectrum of orientin (DMSO-d6, 4.7 T, TMS, ppm). 4.7 4.6 1.24 4. 66 3 4. 68 2 13 12 11 10 9 8 7 6 5 4 3 2 1 0 1.241.051.00 0.94 0.80 TMS DMSOÁgua 0 .0 00 2 .4 87 3 .3 33 4 .6 63 4 .6 826 .2 54 6 .6 19 6 .8 61 7 .4 63 7 .5 07 7 .5 24 1 3 .1 4 3 7.5 7.4 7.3 7.2 7.1 7.0 6.9 6.8 6.7 6.6 6.5 6.4 6.3 6.2 6.1 1.050.94 0.82 0.800.79 6. 2 54 6. 61 9 6. 84 5 6. 86 1 7. 46 3 7. 50 7 7. 52 4 4.7 4.6 1.24 4. 66 3 4. 68 2 4.7 4.6 1.24 4. 66 3 4. 68 2 4.7 4.6 1.24 4. 66 3 4. 68 2 13 12 11 10 9 8 7 6 5 4 3 2 1 0 1.241.051.00 0.94 0.80 TMS DMSOÁgua 0 .0 00 2 .4 87 3 .3 33 4 .6 63 4 .6 826 .2 54 6 .6 19 6 .8 61 7 .4 63 7 .5 07 7 .5 24 1 3 .1 4 3 13 12 11 10 9 8 7 6 5 4 3 2 1 0 1.241.051.00 0.94 0.80 TMS DMSOÁgua 0 .0 00 2 .4 87 3 .3 33 4 .6 63 4 .6 826 .2 54 6 .6 19 6 .8 61 7 .4 63 7 .5 07 7 .5 24 1 3 .1 4 3 7.5 7.4 7.3 7.2 7.1 7.0 6.9 6.8 6.7 6.6 6.5 6.4 6.3 6.2 6.1 1.050.94 0.82 0.800.79 6. 2 54 6. 61 9 6. 84 5 6. 86 1 7. 46 3 7. 50 7 7. 52 4 7.5 7.4 7.3 7.2 7.1 7.0 6.9 6.8 6.7 6.6 6.5 6.4 6.3 6.2 6.1 1.050.94 0.82 0.800.79 6. 2 54 6. 61 9 6. 84 5 6. 86 1 7. 46 3 7. 50 7 7. 52 4 S12 New Flavone from the Leaves of Neea theifera (Nyctaginaceae) J. Braz. Chem. Soc. Figure S19. 13C NMR spectrum of orientin (DMSO-d6, 4.7 T, ppm). 4.7 4.6 1.24 4. 66 3 4. 68 2 13 12 11 10 9 8 7 6 5 4 3 2 1 0 1.241.051.00 0.94 0.80 TMS DMSOÁgua 0 .0 00 2 .4 87 3 .3 33 4 .6 63 4 .6 826 .2 54 6 .6 19 6 .8 61 7 .4 63 7 .5 07 7 .5 24 1 3 .1 4 3 7.5 7.4 7.3 7.2 7.1 7.0 6.9 6.8 6.7 6.6 6.5 6.4 6.3 6.2 6.1 1.050.94 0.82 0.800.79 6. 2 54 6. 61 9 6. 84 5 6. 86 1 7. 46 3 7. 50 7 7. 52 4 4.7 4.6 1.24 4. 66 3 4. 68 2 4.7 4.6 1.24 4. 66 3 4. 68 2 4.7 4.6 1.24 4. 66 3 4. 68 2 13 12 11 10 9 8 7 6 5 4 3 2 1 0 1.241.051.00 0.94 0.80 TMS DMSOÁgua 0 .0 00 2 .4 87 3 .3 33 4 .6 63 4 .6 826 .2 54 6 .6 19 6 .8 61 7 .4 63 7 .5 07 7 .5 24 1 3 .1 4 3 13 12 11 10 9 8 7 6 5 4 3 2 1 0 1.241.051.00 0.94 0.80 TMS DMSOÁgua 0 .0 00 2 .4 87 3 .3 33 4 .6 63 4 .6 826 .2 54 6 .6 19 6 .8 61 7 .4 63 7 .5 07 7 .5 24 1 3 .1 4 3 7.5 7.4 7.3 7.2 7.1 7.0 6.9 6.8 6.7 6.6 6.5 6.4 6.3 6.2 6.1 1.050.94 0.82 0.800.79 6. 2 54 6. 61 9 6. 84 5 6. 86 1 7. 46 3 7. 50 7 7. 52 4 7.5 7.4 7.3 7.2 7.1 7.0 6.9 6.8 6.7 6.6 6.5 6.4 6.3 6.2 6.1 1.050.94 0.82 0.800.79 6. 2 54 6. 61 9 6. 84 5 6. 86 1 7. 46 3 7. 50 7 7. 52 4 Figure S20. ESI-MS spectrum of orientin (negative mode, 70 V). S13Rinaldo et al.Vol. 18, No. 6, 2007 Figure S21. 1H NMR spectrum of vicenin-2 (DMSO-d6, 11.7 T, TMS, ppm). 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 2.082.272.251.00 TMS DMSO Água 0 .0 00 2 .5 03 3 .4 11 4 .7 55 4 .7 75 4 .7 99 4 .8 19 6 .7 93 6 .9 1 3 8 .0 14 8 .0 30 1 3 .7 3 5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 2.082.272.25 1.08 4. 75 5 4. 77 5 4 .7 9 9 4. 81 9 6. 79 3 6. 89 7 6. 91 3 8. 01 4 8. 03 0 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 2.082.272.251.00 TMS DMSO Água 0 .0 00 2 .5 03 3 .4 11 4 .7 55 4 .7 75 4 .7 99 4 .8 19 6 .7 93 6 .9 1 3 8 .0 14 8 .0 30 1 3 .7 3 5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 2.082.272.25 1.08 4. 75 5 4. 77 5 4 .7 9 9 4. 81 9 6. 79 3 6. 89 7 6. 91 3 8. 01 4 8. 03 0 Figure S22. 13C NMR spectrum of vicenin-2 (DMSO-d6, 11.7 T, TMS, ppm). 200 150 100 50 0 DMSO -d6 3 9 .5 0 5 9 .8 6 6 0 .2 8 6 1 .3 3 6 9 .0 4 7 0 .6 1 7 1 .9 67 2 .2 8 7 7 .8 9 7 8 .8 4 8 0 .9 1 8 1 .9 6 1 0 2 .6 1 1 0 5 .3 0 1 0 7 .5 9 1 1 5 .8 9 1 2 1 .5 3 1 2 9 .0 3 1 5 5 .1 3 1 5 8 .6 7 1 6 1 .2 6 1 6 4 .0 5 1 8 2 .3 1 180 170 160 150 140 130 120 110 100 90 80 70 60 5 9 .8 6 6 0 .2 8 6 9 .0 4 7 0 .6 17 2 .2 8 7 3 .4 3 7 7 .8 9 7 8 .8 4 8 0 .9 1 8 1 .9 6 1 0 2 .6 1 1 0 5 .3 0 1 0 7 .5 9 1 1 5 .8 9 1 2 1 .5 31 2 9 .0 3 1 5 5 .1 31 5 8 .6 7 1 6 1 .2 6 1 6 4 .0 5 1 8 2 .3 1 7 4 .1 5 1 0 3 .6 6 200 150 100 50 0 DMSO -d6 3 9 .5 0 5 9 .8 6 6 0 .2 8 6 1 .3 3 6 9 .0 4 7 0 .6 1 7 1 .9 67 2 .2 8 7 7 .8 9 7 8 .8 4 8 0 .9 1 8 1 .9 6 1 0 2 .6 1 1 0 5 .3 0 1 0 7 .5 9 1 1 5 .8 9 1 2 1 .5 3 1 2 9 .0 3 1 5 5 .1 3 1 5 8 .6 7 1 6 1 .2 6 1 6 4 .0 5 1 8 2 .3 1 200 150 100 50 0 DMSO -d6 3 9 .5 0 5 9 .8 6 6 0 .2 8 6 1 .3 3 6 9 .0 4 7 0 .6 1 7 1 .9 67 2 .2 8 7 7 .8 9 7 8 .8 4 8 0 .9 1 8 1 .9 6 1 0 2 .6 1 1 0 5 .3 0 1 0 7 .5 9 1 1 5 .8 9 1 2 1 .5 3 1 2 9 .0 3 1 5 5 .1 3 1 5 8 .6 7 1 6 1 .2 6 1 6 4 .0 5 1 8 2 .3 1 180 170 160 150 140 130 120 110 100 90 80 70 60 5 9 .8 6 6 0 .2 8 6 9 .0 4 7 0 .6 17 2 .2 8 7 3 .4 3 7 7 .8 9 7 8 .8 4 8 0 .9 1 8 1 .9 6 1 0 2 .6 1 1 0 5 .3 0 1 0 7 .5 9 1 1 5 .8 9 1 2 1 .5 31 2 9 .0 3 1 5 5 .1 31 5 8 .6 7 1 6 1 .2 6 1 6 4 .0 5 1 8 2 .3 1 7 4 .1 5 1 0 3 .6 6 S14 New Flavone from the Leaves of Neea theifera (Nyctaginaceae) J. Braz. Chem. Soc. Figure S23. TOCSY spectrum of vicenin-2 (DMSO-d6, 11.7 T, ppm, irradiation of 4.809). 5.0 4.5 4.0 3.5 3.0 3 .2 76 3 .2 95 3 .3 19 3 .3 39 3 .3 67 3 .3 86 3 .4 05 3 .4 22 3 .5 0 8 3 .5 15 3 .5 33 3 .6 46 3 .7 59 3 .7 773 .8 70 3 .8 88 3 .9 06 4 .7 55 4 .7 7 5 4 .7 9 9 4 .8 1 9 5.0 4.5 4.0 3.5 3.0 3 .2 76 3 .2 95 3 .3 19 3 .3 39 3 .3 67 3 .3 86 3 .4 05 3 .4 22 3 .5 0 8 3 .5 15 3 .5 33 3 .6 46 3 .7 59 3 .7 773 .8 70 3 .8 88 3 .9 06 4 .7 55 4 .7 7 5 4 .7 9 9 4 .8 1 9 Figure S24. ESI-MS spectrum of vicenin-2 (negative mode, 70 V). S15Rinaldo et al.Vol. 18, No. 6, 2007 Figure S25. 1H NMR spectrum of chrysoeriol (DMSO-d6, 11.7 T, ppm). 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 3.222.25 1.09 0.90 0.96 DMSOÁgua 2 .5 00 3 .3 58 3 .8 90 6 .1 89 6 .1 93 6 .5 016 .8 75 6 .9 28 6 .9 467 .5 49 7 .5 61 TMS 0 .0 00 6.95 6.80 6.65 6.50 6.35 6.15 1.09 1.00 0.90 0.96 6 .1 8 9 6. 19 3 6. 50 1 6. 50 5 6. 87 5 6. 92 8 6. 9 45 7.59 7.50 2.25 7. 54 8 7. 56 1 7 .5 65 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.08.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 3.222.25 1.09 0.90 0.96 DMSOÁgua 2 .5 00 3 .3 58 3 .8 90 6 .1 89 6 .1 93 6 .5 016 .8 75 6 .9 28 6 .9 467 .5 49 7 .5 61 TMS 0 .0 00 6.95 6.80 6.65 6.50 6.35 6.15 1.09 1.00 0.90 0.96 6 .1 8 9 6. 19 3 6. 50 1 6. 50 5 6. 87 5 6. 92 8 6. 9 45 7.59 7.50 2.25 7. 54 8 7. 56 1 7 .5 65 Figure S26. UV spectrum of chrysoeriol (MeOH). 50 100 150 200 250 300 350 nm 2 0 6 . 4 5 2 5 0 . 5 0 3 4 6 . 1 9 50 100 150 m A U 200 250 300 350 wavelenght / nm 2 0 6 . 4 5 2 5 0 . 5 0 3 4 6 . 1 9 S16 New Flavone from the Leaves of Neea theifera (Nyctaginaceae) J. Braz. Chem. Soc. Figure S27. 1H NMR 1D-NOESY of chrysoeriol (DMSO-d6, 11.7 T, ppm, irradiation of 3.890). 9 8 7 6 5 4 3 2 1 0 3 .8 90 7 .5 4 8 9 8 7 6 5 4 3 2 1 0 3 .8 90 7 .5 4 8 Figure S28. ESI-MS spectrum of chrysoeriol (negative mode, 70 V). S17Rinaldo et al.Vol. 18, No. 6, 2007 Figure S29. Cromatograms of the co-injections of a) apigenin isolated + apgenin Sigma® (1:1 m/m) and b) luteolin isolated + luteolin Sigma® (1:1 m/m) (C18, 250 × 4.60 mm i. d. × 5 µm). A binary gradient elution system with solvent A (0.05% TFA in H2O) and solvent B (0.05% TFA in CH3CN) was used, with linear gradient starting from 68:32 (A:B) at 20 min and then changed to 75:35 (A:B) for 40 min. The flow-rate was 1.0 mL min-1. λ 254 nm. 10 20 30 40 50 60 0.0 0.5 1.0 1.5 0 50 100 150 200 250 100 200 300 400 200 250 300 350 nm 2 0 9 . 1 3 2 6 6 . 4 6 3 3 5 . 8 9 100 200 300 400 500 600 700 200 250 300 350 nm 2 0 7 . 8 6 2 5 2 . 7 8 3 4 7 . 1 8 10 20 30 40 50 60 0.0 0.5 1.0 1.5 0 50 100 150 200 250 100 200 300 400 200 250 300 350 nm 2 0 9 . 1 3 2 6 6 . 4 6 3 3 5 . 8 9 100 200 300 400 500 600 700 200 250 300 350 nm 2 0 7 . 8 6 2 5 2 . 7 8 3 4 7 . 1 8 10 20 30 40 50 60 time / min 0.0 0.5 1.0 1.5 A U 0 50 100 150 200 250 m A U 100 200 300 400 200 250 300 350 nm 2 0 9 . 1 3 2 6 6 . 4 6 3 3 5 . 8 9 100 200 300 400 m A U 200 250 300 350 nm 2 0 9 . 1 3 2 6 6 . 4 6 3 3 5 . 8 9 100 200 300 400 500 600 700 200 250 300 350 nm 2 0 7 . 8 6 2 5 2 . 7 8 3 4 7 . 1 8 100 200 300 400 500 600 700 m A U 200 250 300 350 nm 2 0 7 . 8 6 2 5 2 . 7 8 3 4 7 . 1 8 a) b)