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Journal of the Brazilian Chemical Society

Print version ISSN 0103-5053

J. Braz. Chem. Soc. vol.22 no.11 São Paulo Nov. 2011 



Phenylpropanoid substituted flavan-3-ols from Trichilia catigua and their in vitro antioxidative activity



Flávia O. ResendeI; Edson Rodrigues-FilhoII; Heinrich LuftmannIII; Frank PetereitIV; João C. Palazzo de MelloI, *

IPrograma de Pós-Graduação em Ciências Farmacêuticas, Universidade Estadual de Maringá, Av. Colombo No. 5790, 87020-900 Maringá-PR, Brazil
IIDepartamento de Química, Universidade Federal de São Carlos, Rod. Washington Luiz, km 235, 13905-905 São Carlos-SP, Brazil
IIIOrganisch-Chemisches Institut, Universität Münster, Corrensstrasse 40, D-48149, Münster, Germany
IVInstitut für Pharmazeutische Biologie und Phytochemie, Universität Münster, Hittorfstrasse 56, D-48149, Münster, Germany




The new phenylpropanoid substituted flavan-3-ol apocynin E, together with eight known compounds, epicatechin, procyanidin B2, procyanidin B4, procyanidin C1, cinchonain Ia, cinchonain Ib, cinchonain IIb, and cinchonain IIa were isolated from an acetone-H2O extract of the air-dried stem bark of Trichilia catigua. The cinchonain Ia e Ib were reevaluated to its estereochemistry. All the compounds were characterized by spectroscopic analysis including 1D and 2D nuclear magnetic resonance (NMR) and mass spectrometry (MS) of their peracetate derivatives. The absolute configuration of the phenylpropanoid moiety was determined by circular dichroism (CD) spectra and by analyzing the anisotropic effects in the Dreiding model and nuclear Overhauser effect (NOESY NMR) experiments. The nine isolated compounds showed higher radical scavenging activity and reducing power than ascorbic acid and Trolox in the free-radical 2,2-diphenyl-1-picrylhydrazyl and Fe3+-Fe2+ reduction assay systems.

Keywords: Trichilia catigua, Meliaceae, cinchonains, proanthocyanidins, antioxidative activity


O novo flavan-3-ol contendo um substituinte fenilpropanoide apocinina E, juntamente com oito substâncias conhecidas, epicatequina, procianidina B2, procianidina B4, procianidina C1, cinchonaína Ia, cinchonaína Ib, cinchonaína IIb e cinchonaína IIa foram isoladas a partir do extrato acetona-H2O das cascas secas de Trichilia catigua. As substâncias cinchonaína Ib e cinchonaína IIb foram reavaliadas em relação à sua estereoquímica. Todas as substâncias foram caracterizadas por análises espectroscópicas incluindo ressonância magnética nuclear (NMR) 1D e 2D e espectrometria de massa (MS) de seus derivados acetilados. A configuração absoluta do substituinte fenilpropanoide foi determinada por espectropolarimetria de dicroísmo circular (CD) e pela análise do efeito anisotrópico empregando modelo de Dreiding, e por experimento de espectroscopia de efeito nuclear Overhauser (NOESY NMR). As nove substâncias mostraram maior atividade sobre o sequestro de radical livre e poder de redução do que o ácido ascórbico e Trolox frente ao radical livre 2,2-difenil-1-picrilhidrazila e no sistema do teste de redução do ferro (Fe3+-Fe2+).




Trichilia catigua A. Juss. (Meliaceae), commonly known as "catuaba", is a plant native to South and Central America and widely distributed in Brazil.1 However, the identity of the plant source is often uncertain, and stem barks from several different species of Erythroxylum or Anemopaegma, all regionally known as "catuaba", are sold commercially. Marques2 described the differences among the species known as "catuaba" and determined that T. catigua is the main species that is sold as "catuaba" in Brazil. The same conclusion was reached by Kletter et al.3 and Daolio et al.4 The bark of T. catigua has been traditionally used as a health and mental tonic, and especially as a sexual stimulant.3,5 Some pharmacological studies of this bark have reported antimicrobial, trypanocidal, antinociceptive, vasorelaxant or dopamine-mediated antidepressant-like effects.5-10 The bark contains a high concentration of polyphenols (6.96 ± 0.11%; RSD 1.57%) including flavan-3-ols and phenylpropanoid derivatives,5,11 as well as tropane alkaloids.3 Other species, T. claussenii and T. lepidota, were evaluated and yielded sesquiterpenoids.12 We further investigated the constituents of T. catigua bark and isolated nine polyphenolic constituents, including one new compound. The isolated polyphenols showed high radical scavenging and antioxidative activity in DPPH (2,2-diphenyl-1-picryl-hydrazyl) and Fe3+-Fe2+ assays.


Results and Discussion

Isolation of polyphenols from bark of Trichilia catigua

Multiple chromatography on Sigma-Sephadex® LH-20 and high-speed countercurrent chromatography (HSCCC) of the ethyl acetate fraction originating from an acetone/H2O (7:3) extract of the air-dried stem bark of T. catigua yielded eight known compounds together with a new compound. The known compounds were identified as epicatechin (1), procyanidin B2 [epicatechin-(4β 8)-epicatechin] (2), procyanidin B4 [catechin-(4α 8)-epicatechin] (3), procyanidin C1 [epicatechin-(4β 8)-epicatechin-(4β 8)-epicatechin] (4), cinchonain Ia (5), cinchonain Ib (6), cinchonain IIb (8), and cinchonain IIa (9) by comparison of the physical and spectral data [1H nuclear magnetic resonance (NMR), 1H-1H correlation spectroscopy (COSY NMR), [α]D20, mass spectrometry (MS), circular dichroism (CD)] of their peracetates with published data.5,13-18 The new compound was isolated as its peracetate 7a and identified as apocynin E (7).

Structure of phenylpropanoid substituted flavan-3-ols

Cinchonain Ia (5) and Ib (6) were first described by Nonaka and Nishioka13 and their structures were revised by Chen et al.,15 based on nuclear Overhauser spectroscopy (NOESY NMR), nuclear Overhauser effect (NOE) and CD data. Several reports have been published with ambiguous stereochemistry of both compounds.5,11,16,19,20 The now generally accepted structures for cinchonanin Ia and Ib are outlined in Figure 1.



Compounds 5a and 6a showed the same [M − H] signal at m/z 451.4 in negative ESI-MS (electrospray ionization mass spectrometry). The presence of a basic flavan-3-ol skeleton in each molecule was readily confirmed by the observation of AMX2-type resonances at δ 5.20 (brs) and 5.43 (m), 5.46 (brs) and 5.46 (m), and between 2.72 and 3.08 (m, 2H), respectively ascribable to H-2, H-3 and H-4, for peracetates 5a and 6a. The appearance of an aromatic proton singlet at δ 6.50 for 5a and δ 6.63 for 6a indicated the occurrence of a C8 or C6 attached linkage in the flavan A-ring, while the aromatic ABX-type resonances at δ 7.11-7.33, and the chemical shifts and coupling constants were in good agreement with those of the epicatechin peracetate (1a) (Table 1). 13C NMR spectra revealed the presence of a carbonyl (δ 167.3 and 167.1), a β-methine carbon (δ 35.1 and 34.9), a α-methylene (δ 36.8 and 36.5) and an additional 1,3,4-trisubstituted aromatic ring system for 5a and 6a, respectively. This suggests the presence of a phenylpropanoid linked to the A-ring of the epicatechin unit through a carbon-carbon linkage. According to Nonaka and Nishioka13 and Tang et al.,21 the C-8 resonance (δ 111.5 for 5a and δ 111.1 for 6a) relative to the C-6 (δ 104.8 for 5a and δ 104.5 for 6a) demonstrates the occurrence of a carbon-carbon linkage at the C-8 position for both compounds. The HMBC (heteronuclear multiple-bond correlation spectroscopy) spectra of 5a and 6a clearly indicated a strong correlation between H-7" and C-8 and C-9, H-8" and C-8, C-9" and C-1", and H-6 and C-5, C-7, C-8, and C-10, which would be impossible if the substituent were attached at the C-6 position, and therefore confirmed the position of the phenylpropanoid moiety at C-8.

For compounds 5a and 6a, the S- or R-absolute configuration of the β-methine carbon in the phenylpropanoid substituent was determined based on 1H NMR, 1D- or 2D-NOESY NMR experiments and CD data. The NMR spectrum of 5a revealed resonances due to the H-2 and the ABX-type B-ring in accordance with those of epicatechin (see Experimental part). On the other hand, in acetone-d6, 6a displays the H-2 resonance at δ 5.46 and the B-ring proton resonances at δ 7.03 (d, J 1.8 Hz, H-2'), δ 7.14 (d, J 5.4 Hz, H-5') and δ 7.17 (m, H-6'). The H-2 resonance is shifted downfield, while H-2' and H-6' are shifted upfield compared with those of epicatechin (Table 1). This implies that these protons are anisotropically affected by the aromatic ring in the phenylpropanoid moiety.13 Examination of the Dreiding model indicated that in the case of the β-configuration of the methine proton (H-7"), the direction of the B-ring to the aromatic ring of the phenylpropanoid moiety leads to the concentration of the electronic cloud, thus causing a shielding effect at the B-ring protons and an opposite effect at H-2. These effects explain the upfield shift of the B-ring proton resonances and the downfield shift of the H-2 resonance in compound 6a. CD data for 5a show strong negative Cotton effects at 234 and 280 nm and a positive effect at 255 nm, whereas 6a exhibited Cotton effects of opposite signs, indicating that these Cotton effects are mainly affected by the configuration at the β-methine carbon, and are largely unaffected by the asymmetrical flavan C-2 and C-3 atoms; and hence that the absolute configuration at the β-methine carbon in 5a is S and in 6a is R. From the 2D-NOESY NMR experiments, a small effect between the H-7" and the H-2' and H-6' of the B-ring was observed for compound 5a. Another important observation is the cross peak between H-2(C) and H-2" and H-6". This could only be the case if the proton 7" in 5a is in α-orientation. The compound 6a with a β-orientation shows in the NOESY NMR spectrum a strong cross peak between H-7" and the H-2' and H-6' of the B-ring, and H-2" and H-6" of the phenylpropanoid moiety. No effect between H-2(C) and H-2" or H-6" was observed. This situation was confirmed with a Dreiding model, and established compound 5 as cinchonain Ia and compound 6 as cinchonain Ib.

Compound 7a showed the [M + Na]+ signal at m/z 785.4 in the positive ESI-MS. The HR-ESI-MS (positive-ion mode) exhibited a pseudo-molecular ion peak at m/z 785.1688 [M + Na]+ (calcd. for 785.6561). The 1H and 13C NMR data are almost similar to those of cinchonain Ib (6a), with the exception of a two-proton singlet corresponding to the 1,3,4,5-substituted B-ring, and proved that compound 7a contained an epigallocatechin unit. This was also confirmed by the 1H-1H COSY and HMBC spectra. The R absolute configuration of the β-methine carbon of 7a was determined by comparing the 1H NMR, NOESY NMR and CD data with those of 5a and 6a. The H-2 and H-3 resonances of 7a suggested that the β-methine carbon configuration is the same as 6a. The Cotton effect in the CD spectrum of 7a is in perfect agreement with those found for 6a. Furthermore, the NOESY NMR spectrum shows no effect between the aromatic ring systems, and H-2' and H-7", confirming the absolute configuration as R. Thus, it was concluded that the structure of compound 7 corresponds to apocynin E. To the best of our knowledge, 7 has been isolated for the first time from a natural source.

Structure of phenylpropanoid substituted dimeric flavan-3-ols

Compounds 8 and 9 were shown to be proanthocyanidin dimers substituted by a phenylpropanoid moiety at the A-ring. Both substances showed the same [M − H] peak at m/z 739.4 in negative ESI-MS. Compound 9a exhibited a pseudo-molecular ion peak at m/z 619.1785 [(calcd. for C61H54O26 + 2 NH4)2+ 619.1789] in the HR-ESI-MS (positive-ion mode), ascribable to molecular formula C61H54O26, and showed a peak at m/z 1220.4 [M + NH4]+ at ESI-MS. The occurrence of two constituent flavan-3-ol units with 2,3-cis configuration in 8a and 9a and a phenylpropanoid moiety was deduced from the small coupling constants (J2,3 < 2 Hz) of the heterocyclic H-2 and H-3 protons and additional methylene (δ 2.53 and 3.26) and methine (δ 4.16 and 4.92) proton resonances, respectively. The HMBC spectrum of 8a and 9a strongly favored the 4 8 interflavonoid bond and the existence of a phenylpropanoid substituent linked to the C-8(A) of the "upper" flavan units in both compounds. Furthermore, 2D correlations between H-6(A), H-7", and H-8" and the carbon C-8(A) at δ 110.2 for 8a and 111.6 for 9a strongly favored the existence of a phenylpropanoid substituent linked to the C-8 (A) of the "upper" flavan units in 8a and 9a.

The relative configuration of 8a was determined based on anisotropic effects by comparison of 1H NMR data with those of the peracetate of procyanidin B2. The downfield shift of the H-2(C) resonance at δ 5.86 and the upfield shift of the B-ring proton resonances H-2' and H-6' at δ 6.96 (d, J 2.1 Hz) and δ 7.05 (dd, J 2.1 and 8.4 Hz) for 8a, respectively, in comparison to the equivalent protons in procyanidin B2 supported the S-configuration at C-7". In addition, the 4β 8-linkage and thus the R-configuration at C-4(C) was confirmed by the strong positive Cotton effect observed between 220 and 230 nm, and 280 nm in the CD spectrum.22-27 The missing NOE effect between H-2(C) and H-4(C) strongly supported the proposed 4β orientation of the interflavanoid bond. The high-amplitude positive Cotton effect was caused by the phenylpropanoid substituent with an S-configuration at C-7". No NOE effect between H-7" and H-2(C) was observed, confirming the information from the CD data. Thus, it was concluded that the structure of compound 8 corresponds to cinchonain IIb.

The absolute configuration of the β-methine carbon in 9a was determined based on CD data and NOESY NMR experiments. In general, the 4S-linkage between two flavan units produces negative Cotton effects at 220-240 and 275 nm in the CD spectra.22-27 Such negative Cotton effects can be observed in the spectrum of compound 9a, in addition to a positive Cotton effect at 255 nm. The latter effect is (probably) due to the S-configuration of the phenylpropanoid substituent. The 4β 8 interflavanoid-linkage was determined by the NOESY NMR experiment, where no effect between H-2(C) and H-4(C) was observed. Therefore it was concluded that the structure of compound 9 is cinchonain IIa.

Physical data from the well documented 8 and 9 are in good agreement with the literature values.14

Antioxidative activity of Trichilia catigua polyphenols

The antioxidant effects of the polyphenols are mainly due to their redox properties, which allow them to act as reducing agents or hydrogen-atom donors. Thus, natural antioxidants function as free-radical scavengers and chain-breakers, complexers of pro-oxidant metal ions and quenchers of singlet-oxygen formation.28 The radical-scavenging properties of polyphenols can be shown by the effects on DPPH radical as a model compound for lipid radicals or lipid peroxide radicals.29,30 The effects of the polyphenols isolated from T. catigua bark on the DPPH radical scavenging and their reducing power were therefore examined.

The DPPH radical scavenging activities of all the tested polyphenols were stronger than the effect of ascorbic acid and Trolox (a water-soluble analogue of α-tocopherol), as shown in Table 2. The differences between the monomers cinchonain Ia and Ib and also between the dimers cinchonain IIb and IIa were not statistically significant. This implies that the stereochemistry does not influence the DPPH radical scavenging activity. According to the literature, the radical scavenging activity of polyphenols is mainly due to the number of hydroxyl groups in the molecule.18,31 This could be confirmed, in this study, for compounds containing only flavan-3-ol units in their structures. However, for the phenylpropanoid substituted flavan-3-ols, the capacity of scavenging DPPH radicals of cinchonain monomers does not differ from procyanidin B2 and the activity of the cinchonain dimers does not differ significantly from procyanidin C1. Obviously the number of catechol type B-rings to form quinone intermediates is more responsible for the radical scavenging activity than the number of hydroxyl groups, especially A-Ring hydroxyl groups.



The reducing power of a compound may serve as a significant indicator of its potential antioxidative activity, and therefore can reduce the oxidized intermediates of lipid peroxidation processes, so that they can act as primary and secondary antioxidants.32,33 The presence of reducing substances causes the reduction of the Fe3+/ferricyanide complex to the ferrous form. Therefore, Fe2+ can be monitored by measuring the formation of Perl's Prussian blue at 700 nm.34 Figure 2 shows the reducing power of the compounds isolated from T. catigua compared to Trolox and ascorbic acid. Like the radical scavenging activity, the reducing capacity of these compounds depended on the concentration.



The absence of a significant difference (P < 0.05) between the reducing capacity of the cinchonain monomers and the cinchonain dimers, in contrast to the series of flavan-3-ol analogues, suggests that the reducing ability of substituted phenylpropanoids is largely unaffected by the number of hydroxyl groups or catechol-type B-rings.



General experimental procedures

NMR spcetra of the peracetates (Varian Mercury 300BB) were recorded in acetone-d6 or CDCl3 at ambient temperature with TMS as internal standard. Some compounds were recorded in CDCl3 to confirm the positions of some protons, or to better define them. ESI-MS was carried out on a Quattro LCZ (Waters-Micromass, Manchester, UK) and HR-ESI-TOF-MS (high resolution-electrospray ionization-time-of-flight-mass spectroscopy) on a microTof (Bruker-Daltonics, Bremen, Germany). Optical rotations were measured in acetone and CD data were obtained in MeOH (Jasco 815 spectrometer). Compounds were revealed by spraying with 1% ethanolic FeCl3 solution. Analytical TLC (thin-layer chromatography) was carried out on precoated aluminum sheets (Kieselgel 60 F254, Merck) with EtOAc/formic acid/H2O (18:1:1; system Sl). Preparative TLC was performed on silica gel plates (Kieselgel 60 F254, 1.0 mm) using toluene/Me2CO (7:3; system S2). Acetylations were performed in pyridine/Ac2O (1:1) at ambient temperature.

Plant material

Stem bark of T. catigua A. Juss. was collected in January 1997 in Caitité City (Bahia State, Brazil) and identified as described elsewhere.35 A voucher specimen is deposited in the Herbarium of the Municipal Botanical Museum of Curitiba City, Paraná State, Brazil (65 901).

Extraction, isolation and identification of compounds

The data of extraction, isolation and identification of all compounds are presented as supplementary information.

Radical scavenging effects of Trichilia catigua polyphenols on the DPPH radical

To a DPPH radical solution in MeOH (1 mmol L-1, 0.3 mL), a solution of the test polyphenol in MeOH (3.0 mL) was added, and the reaction mixture was left to stand for 30 min at room temperature. The scavenging activity of each polyphenol at 1, 2, 4, 6, 8 and 10 µmol L-1 was estimated by measuring the absorption of the mixture at 517 nm, which reflects the amount of DPPH radical remaining in the solution. The scavenging activity was expressed as the EC50 (µmol L-1), the concentration of polyphenol required for scavenging 50% of DPPH radical in the solution.28

Reducing power of the polyphenols by Fe3+ - Fe2+ transformation

Different concentrations of the polyphenols (20, 40, 60, 80 and 100 µmol L-1) in 1 mL of distilled water were mixed with phosphate buffer (2.5 mL, 0.2 mol L-1, pH 6.6) and potassium ferricyanide [K3Fe(CN)6] (2.5 mL, 1%). The mixture was incubated at 50 oC for 20 min. Trichloroacetic acid (2.5 mL, 10%) was added to the mixture, which was then centrifuged for 10 min at 1000× g. The upper layer of solution (2.5 mL) was mixed with distilled water (2.5 mL) and FeCl3 (0.5 mL, 0.1%), and the absorbance was measured at 700 nm. Higher absorbance of the reaction mixture indicated greater reducing power.36

Statistical analysis

Results were assessed by one-way analysis of variance (ANOVA) and represent the mean ± standard deviation of three different measurements. The significant differences were determined by the Tukey test with P < 0.05.


Supplementary Information

The data of extraction, isolation and identification of all compounds are presented as Supplementary Information together with 29 spectra referring to the isolated compounds. Supplementary data are available free of charge at as pdf file.



We thank Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação Araucária, Instituto Nacional de Ciência e Tecnologia para Inovação Farmacêutica (INCT_if), Financiadora de Estudos e Projetos (FINEP) for financial support and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for a fellowship. Our gratitude to Prof. Dr. W. Villegas (Instituto de Química, Universidade Estadual Paulista, Brazil) for technical support on MLCCC. The technical assistance provided by A. Arantes and C. R. Novello is gratefully acknowledged. Our gratitude is to Dr. J. W. Reid for the English text revising and to Dr. I. T. A. Schuquel for NMR spectra. The valuable observations of anonymous reviewers and Prof. Dr. Daneel Ferreira contributed significantly to the improvements in the manuscript.



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Submitted: July 16, 2010
Published online: September 6, 2011
FAPESP has sponsored the publication of this article.



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Supplementary Information

Extraction, isolation and identification of compounds

Air-dried stem bark (500 g) was extracted with Me2CO/H2O (7:3, 5.0 L). The combined extracts were filtered and evaporated under reduced pressure to 0.5 L and lyophilized (71 g). This fraction was redissolved in 710 mL H2O and extracted with EtOAc (8.5 L). After evaporation of solvents, the EtOAc extract and the remaining H2O phase gave dark-brown solids of 24 and 47 g, respectively. A portion (13 g) of the EtOAc extract was subjected to column chromatography (CC) on Sigma-Sephadex® LH-20 (500 × 60 mm; eluents: 30% EtOH (6.7 L), 40% EtOH (3.2 L), 50% EtOH (4 L), 70% EtOH (1.3 L), EtOH (1.5 L), 50% MeOH (1 L), MeOH (1 L) and 70% Me2CO (3 L); 10 mL fractions) to yield 32 main fractions (indicated below with roman numerals). All main fractions were further separated by high-speed countercurrent chromatography (HSCCC), which was carried out with the solvent system EtOAc/n-PrOH/H2O (140:8:80) on a P. C. Inc. high-speed countercurrent separator-extractor, flow rate of 1 mL min-1 (10 mL), using the upper phase as mobile phase (these fractions are indicated below with asterisks). Unresolved subfractions were subjected to TLC (thin-layer chromatography) preparation using toluene/Me2CO (7:3; system S2).

Epicatechin (1)

Fraction XII (test tubes 671-765; 265 mg) was subjected to a new CC on Sephadex® LH 20 (25 × 270 mm; eluents 5% EtOH (1 L), 10% EtOH (0.5 L), 15% EtOH (0.5 L), and 70% acetone (0.5 L); 3 ml fractions) to yield 4 subfractions. Subfraction 3 (test tubes 320-485; 113 mg) was identified as epicatechin, a light-yellow amorphous powder; [α]D20° –8 (c 0.1, MeOH); ESI-MS at m/z 313 ([M + Na]+). These data are for the free phenol; the 1H NMR data are identical to those of an authentic sample as peracetate in acetone-d6 (1a), see Table 1.

Procyanidin B2 (2)

Fraction XVI (test tubes 920-999; 514 mg) was subjected to HSCCC to yield five subfractions. Subfraction 4 (test tubes 75-108; 256 mg) was identified as procyanidin B2, a light-brown amorphous powder; [α]D20° + 3 (c 0.1, MeOH); ESI-MS at m/z 577.4 ([M − H]), 601.5 ([M + Na]+). These data are for the free phenol. CD (c 0.02, MeOH, peracetate): [θ]240 + 11380, [θ]280 + 7910; the 1H NMR data are identical to those of an authentic sample as peracetate;17,23 13C NMR (CDCl3, 75 MHz) δ 155.3 (C, C-5(A)), 154.2 (C, C-9(D)), 149.1 (C, C-7(A)), 149.0 (C, C-5(D)), 147.9 (C, C-9(A)), 147.8 (C, C-7(D)), 141.7ª and 142.0ª (C, C-3' and C, C-4'(E)), 141.6b and 141.9b (C, C-3' and C, C-4'(B)), 136.5 (C, C-1'(B)), 134.4 (C, C-1'(E)), 125.0 (CH, C-6'(E)), 124.4 (CH, C-6'(B)), 123.1 (CH, C-5'(B)), 122.8 (CH, C-5'(E)), 122.4 (CH, C-2'(E)), 122.2 (CH, C-2'(B)), 116.7 (C, C-8(D)), 111.7 (C, C-10(D)), 111.6 (C, C-10(A)), 110.3 (CH, C-6(D)), 108.6 (CH, C-8(A)), 107.2 (CH, C-6(A)), 77.1 (CH, C-2(F)), 73.6 (CH, C-2(C)), 71.0 (CH, C-3(C)), 66.8 (CH, H-3(F)), 33.9 (CH, C-4(C)), 26.6 (CH2, C-4(F)); a and b = interchangeable.

Procyanidin B4 (3)

Fraction XVII (test tubes 1000-1080; 767 mg) was subjected to a new CC on Sephadex® LH 20 (34 × 340 mm; eluents 1% EtOH (1.3 L), 3% EtOH (0.6 L), 5% EtOH (0.2 L), 8% EtOH (0.5 L), 12% EtOH (1.2 L), 14% EtOH (0.5 L), 16% EtOH (1.2 L), 18% EtOH (3.0 L), 20% EtOH (1.0 L), 25% EtOH (0.7 L), 40% EtOH (1.0 L), and Me2CO (0.7 L; 4 mL fractions) to yield 15 subfractions. Subfraction 11 was peracetylated and purified by preparative TLC (system S2, Rf 0.33) to give 16 mg of compound 3a, a light-brown amorphous powder. 3a: [α]D20° –14.6 (c 0.13, Me2CO). ESI-MS at m/z 997.5 ([M − H]), 1021.5 ([M + Na]+); CD (c 0.1; MeOH): [θ]240 – 4280, [θ]280 – 7140; the 1H NMR data are identical to those published by Thompson et al.23 and Yamaguti-Sasaki et al;17 13C NMR (acetone-d6, 75 MHz) δ 156.7 (C, C-9(C)), 154.6 (C, C-9(D)), 150.7 (C, C-5(A)), 150.5 (C, C-7(A)), 149.5 (C, C-5(D)), 148.9 (C, C-7(D)), 143.4ª, 143.2ª, 143.0ª and 142.8ª (2 × C, C-3'(B/E) and 2 × C, C-4'(B/E)), 136.9 (C, C-1'(E)), 136.8 (C, C-1'(B)), 126.0 (CH, C-6'(E)), 125.7 (CH, C-6'(B)), 124.4 (CH, C-5'(B)), 123.9 (CH, C-5'(E)), 123.7 (CH, C-2'(B)), 123.1 (CH, C-2'(E)), 118.4 (C, C-8(D)), 116.6 (C, C-10(A)), 111.9 (C, C-10(D)), 111.3 (CH, C-6(A)), 110.7 (CH, C-6(D)), 109.0 (CH, C-8(A)), 79.5 (CH, C-4(C)), 78.2 (CH, C-2(F)), 70.9 (CH, C-3(C)), 67.6 (CH, C-3(F)), 37.4 (CH, C-2(C)), 27.3 (CH2, C-4(F)); a and b = interchangeable.

Procyanidin C1 (4)

Fraction XXI (test tubes 1371-1440; 347 mg) was subjected to HSCCC to yield six subfractions. Subfraction 5 (test tubes 96-102; 19 mg) was identified as procyanidin C1 by comparison of physical data of its peracetate. Compound 4 is a light-brown amorphous powder; [α]D20° + 7 (c 0.1, MeOH); ESI-MS at m/z 865.5 ([M − H]), 889.4 ([M + Na]+); CD (c 0.02, MeOH): [θ]240 + 2527, [θ]280 +1863. 4a: 1H NMR data are identical to those published by Yamaguti-Sasaki et al;17 13C NMR (CDCl3, 75 MHz) δ 111.2 (CH, C-6(G)), 110.9 (CH, C-6(D)), 107.7 (CH, C-8(A)), 107.6 (CH, C-6(A)), 77.2# (CH, C-2(I)), 77.2# (CH, C-3(C)), 75.2 (CH, C-3(F)), 70.7 (CH, C-2(C)), 66.8 (CH, C-3(I)), 35.8 (CH, C-4(C)), 35.3 (CH, C-4(F)), 34.6 (CH, C-2(F)), 26.6 (CH2, C-4(I)); # = overlapping.

Cinchonain Ia (5)

Fraction XVIII (test tubes 1081-1112; 353 mg) was subjected to HSCCC to yield eight subfractions. Subfraction 2 (test tubes 19-22; 136 mg) was identified as cinchonain Ia, a white amorphous powder; [α]D20° – 35 (c 0.1, MeOH); ESI-MS at m/z 451.4 ([M − H]), 475.3 ([M + Na]+); CD (c 0.002, MeOH): [θ]234 – 22741, [θ]255 + 5053, [θ]280 – 20214. 5a: 1H NMR (acetone-d6, 300 MHz): see Table 1; 13C NMR (acetone-d6, 75 MHz) δ 167.3 (CO, C-9"), 152.6 (C, C-9), 151.7 (COC, C-7), 150.7 (C, C-5), 143.5 (C, C-3"), 143.4 (C, C-3'), 143.1 (C, C-4'), 142.5 (C, C-4"), 141.3 (C, C-1"), 137.1 (C, C-1'), 125.4 (CH, C-6'), 125.3 (CH, C-2"), 124.2 (CH, C-5'), 124.9 (CH, C-6"), 123.1 (CH, C-5"), 122.8 (CH, C-2'), 111.5 (C, C-8), 109.9 (C, C-10), 104.8 (CH, C-6), 77.6 (CH, C-2), 67.1 (CH, C-3), 36.7 (CH2, C-8"), 35.1 (CH, C-7"), 26.6 (CH2, C-4).

Cinchonain Ib (6)

Fraction XIX (test tubes 1113-1295; 707 mg) was subjected to HSCCC to yield six subfractions. Subfraction 2 (test tubes 23-25; 292 mg) was identified as cinchonain Ib, a light-brown amorphous powder; [α]D20° + 3 (c 0.1, MeOH); ESI-MSat m/z 451.4 ([M − H]), 475.4 ([M + Na]+); CD (c 0.002, MeOH): [θ]234 + 3369, [θ]255 – 1212, [θ]280 + 1347. 6a: 1H NMR (acetone-d6, 300 MHz): see Table 1; 13C NMR (acetone-d6, 75 MHz): δ 167.1 (CO, C-9"), 152.6 (C, C-9), 152.0 (COC, C-7), 150.8 (C, C-5), 143.7 (C, C-3"), 143.2 (C, C-3'), 143.1 (C, C-4'), 142.6 (C, C-4"), 141.2 (C, C-1"), 137.2 (C, C-1'), 125.8 (CH, C-6'), 125.4 (CH, C-2"), 124.8 (CH, C-6"), 124.2 (CH, C-5'), 122.9 (CH, C-2'), 123.2 (CH, C-5"), 111.1 (C, C-8), 109.7 (C, C-10), 104.5 (CH, C-6), 78.1 (CH, C-2), 67.3 (CH, C-3), 36.5 (CH2, C-8"), 34.9 (CH, C-7"), 26.5 (CH2, C-4).

Cinchonain IIb (8)

Fraction XIX (test tubes 1113-1295; 707 mg) was subjected to HSCCC to yield six subfractions. Subfraction 4 (test tubes 42-58; 60.4 mg) was identified as cinchonain IIb, a light-brown amorphous powder; [α]D20° + 22 (c 0.1, MeOH); ESI-MS at m/z 739.4 ([M − H]), 763.4 ([M + Na]+); CD (c 0.002, MeOH): [θ]234 –49617, [θ]255 + 2756, [θ]280 + 11026. 8a: 1H NMR (acetone-d6, 300 MHz) δ 7.28 (d, 1H, J 8.7 Hz, H-5"), 7.28 (dd, 1H, J 2.7, 6.3 Hz, H-6'(E)), 7.23 (d, 1H, J 1.8 Hz, H-2'(E)), 7.22 (d, 1H, J 8.4 Hz, H-5'(E)), 7.17 (d, 1H, J 8.4 Hz, H-5'(B)), 7.10 (dd, 1H, J 2.4, 8.7 Hz, H-6"), 7.05 (dd, 1H, J 2.1, 8.4 Hz, H-6'(B)), 6.96 (d, 1H, J 2.1 Hz, H-2'(B)), 6.88 (d, 1H, J 2.4 Hz, H-2"), 6.76 (s, 1H, H-6(D)), 6.40 (s, 1H, H-6(A)), 5.86 (brs, 1H, H-2(C)), 5.24 (m, 1H, H-3(F)), 5.17 (m, 1H, H-3(C)), 4.89 (brs, 1H, H-2(F)), 4.54 (d, 1H, J 1.8 Hz, H-4(C)), 4.16 (1H, brd, ΣJ 6.6 Hz, H-7"), 2.98 (1H, dd, J 4.5, 17.7 Hz, H-4eq(F)), 2.85 (1H, m, H-4ax(F)), 2.78 (dd, 1H, J 1.5, 16.5 Hz, H-8"eq), 2.28 (dd, 1H, J 0.9, 16.5 Hz, H-8"ax); 13C NMR (acetone-d6, 75 MHz) δ 167.1 (CO, C-9"), 154.7 (C, C-9(D)), 152.8ª (C, C-9(A)), 150.6b (C, C-7(D)), 150.4 (COC, C-7(A)), 149.6ª (C, C-5(A)), 149.2b (C, C-5(D)), 143.6d (C, C-3"), 143.4c (C, C-4'(B)), 143.3c (C, C-3'(B)), 143.2e (C, C-3'(E)), 142.9d (C, C-4"), 142.4e (C, C-4'(E)), 141.6 (C, C-1"), 136.9 (C, C-1'(E)), 136.8 (C, C-1'(B)), 126.1 (CH, C-6"), 125.6 (CH, C-6'(B)), 125.3 (CH, C-6'(E)), 124.6 (CH, C-2'(E)), 124.4 (CH, C-5'(B)), 124.2 (CH, C-5"), 123.3 (CH, C-5'(E)), 123.2 (CH, C-2'(B)), 122.6 (CH, C-2"), 117.2 (C, C-8(D)), 112.5 (C, C-10(D)), 111.7 (C, C-10(A)), 111.5 (CH, C-6(D)), 110.2 (C, C-8(A)), 104.8 (CH, C-6(A)), 77.9 (CH, C-2(F)), 75.8 (CH, C-2(C)), 71.3 (CH, C-3(C)), 67.3 (CH, C-3(F)), 36.9 (CH2, C-8"), 35.0 (CH, C-4(C)), 34.6 (CH, C-7"), 26.9 (CH2, C-4(F)); a, b, c, d, e = interchangeable.

Cinchonain IIa (9)

Fraction XXI (test tubes 1371-1440; 347 mg) was subjected to HSCCC to yield six subfractions. Subfraction 3 (test tubes 51-55; 31 mg) was identified as compound 9. A light-brown amorphous powder; [α]D20° – 5 (c 0.1, acetone); ESI-MS at m/z 739.4 ([M − H]), 763.4 ([M + Na]+) and at m/z 1220.4 [M + NH4]+; HR-ESI-MS positive-ion mode at m/z 619.1785 [(calcd for C61H54O26 + 2 NH4)2+ 619.1789]; CD (c 0.002, MeOH): [θ]234 – 15160, [θ]255 + 9096, [θ]280 – 13645. 9a: 1H NMR (acetone-d6, 300 MHz) δ 7.45 (d, 1H, J 1.8 Hz, H-2'(E)), 7.36 (m, 3H, H-5'(B,E), H-2'(B)), 7.30 (dd, 1H, J 1.8, 8.7 Hz, H-6'(B)), 7.22 (dd, 1H, J 1.8, 8.4 Hz, H-6'(E)), 7.22 (dd, 1H, J 1.8, 8.4 Hz, H-6'(E)), 7.12 (m, 3H, H-2", H-5", H-6"), 6.64 (s, 1H, H-6(A)), 6.61 (s, 1H, H-6(D)), 5.67 (m, 1H, H-3(F)), 5.41 (brs, 1H, H-2(C)), 5.40 (brs, 1H, H-2(F)), 5.34 (m, 1H, H-3(C)), 4.92 (brd, 1H, ΣJ 7.2 Hz, H-7"), 4.67 (brs, 1H, H-4(C)), 3.52 (dd, 1H, J 7.8 and 16.5 Hz, H-8"ax(F)), 3.20 (dd, 1H, J 1.8, 4.5 Hz, H-4eq(F)), 3.01 (m, 1H, H-8"eq), 2.88 (m, 1H, H-4ax(F)); 13C NMR (acetone-d6, 75 MHz) δ 167.8 (CO, C-9"), 152.7 (C, C-9(D)), 152.4 (COC, C-7(A)), 151.1 (C, C-9(A)), 150.7 (C, C-5(A)), 149.7ª (C, C-7(D)), 148.4ª (C, C-5(D)), 143.5b, 143.3b, 143.3b, 143.1b, 142.8b and 142.7b (4 × C, C-3',4'(B/E) and 2 × C, C-3",4"), 141.9 (C, C-1"), 137.4c and 136.3c (2 × C, C-1'B/E), 125.1 (CH, C-6'(B)), 125.4d, 125.1d and 124.1d (3 × CH, C-2",5",6"), 124.2 (CH, C-6'(E)), 122.8 (CH, C-2'(E)), 122.5# (CH, C-2'(B)), 122.5# (CH, C-5'(B)), 122.5# (CH, C-5'(E)), 118.4 (C, C-8(A)), 112.1 (C, C-10(A)), 111.9 (C, C-10(D)), 111.6 (CH, C-6(D)), 111.2 (C, C-8(D)), 105.3 (CH, C-6(A)), 77.6 (CH, C-2(F)), 75.8 (CH, C-2(C)), 72.1 (CH, C-3(C)), 67.3 (CH, C-3 (F)), 35.6# (CH2, C-8"), 35.6# (CH, C-4(C)), 35.5 (CH, C-7"), 26.8 (CH2, C-4(F)); a, b, c, d = interchangeable and # = overlapping.

Apocynin E (7)

Fraction XVIII (test tubes 1081-1112; 353 mg) was subjected to HSCCC to yield eight subfractions. Subfraction 3 (test tubes 23-36; 45 mg) was peracetylated and purified by preparative TLC (system S2, Rf 0.31) to give 15 mg of compound 7a. 7a: A light-brown amorphous powder; [α]D20° + 6.2 (c 0.08, acetone); ESI-MS at m/z 785.4 ([M + Na]+); HR-ESI-MS (positive-ion mode) at m/z 785.1688 [M + Na]+ (calcd for 785.6561); CD (c 0.002, MeOH): [θ]234 + 2527, [θ]255 – 1263, [θ]280 + 4422; 1H NMR (acetone-d6, 300 MHz): see Table 1; 13C NMR of the peracetate 7a (acetone-d6, 75 MHz) δ 167.1 (CO, C-9"), 152.4 (C, C-9), 151.9 (COC, C-7), 150.7 (C, C-5), 144.3 (C, C-3'), 143.6 (C, C-3"), 142.5 (C, C-4"), 142.1 (C, C-4'), 141.0 (C, C-1"), 136.8 (C, C-1'), 135.6 (C, C-5'), 125.8 (CH, C-6"), 124.7 (CH, C-5"), 122.6ª (CH, C-6'), 119.9a,# (CH, C-2'), 119.9 (CH, C-2")#,111.1 (C, C-8), 109.7 (C, C-10), 104.6 (CH, C-6), 77.8 (CH, C-2), 67.1 (CH, C-3), 36.4 (CH2, C-8"), 34.7 (CH, C-7"), 26.5 (CH2, C-4); a = interchangeable and # = overlapping.



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