Phenylpropanoid Substituted Flavan-3-ols from Trichilia catigua and their In Vitro 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 (Fe-Fe).


Introduction
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.Marques 2 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][7][8][9][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. 3Other species, T. claussenii and T. lepidota, were evaluated and yielded sesquiterpenoids. 12e 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 Fe 3+ -Fe 2+ assays.

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/H 2 O (7:3) extract of the air-dried stem bark of T. catigua yielded eight known compounds together with a new compound.4][15][16][17][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 Nishioka 13 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. 5,11,16,19,20The now generally accepted structures for cinchonanin Ia and Ib are outlined in Figure 1.5.46 (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 d 6.50 for 5a and d 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 d 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 (d 167.3 and 167.1), a b-methine carbon (d 35.1 and 34.9), a a-methylene (d 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 Nishioka 13 and Tang et al., 21 the C-8 resonance (d 111.5 for 5a and d 111.1 for 6a) relative to the C-6 (d 104.8 for 5a and d 104.5 for 6a) demonstrates the occurrence of a carbon-carbon linkage at the C-8 position for both compounds.The HMBC (heteronuclear multiplebond 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 b-methine carbon in the phenylpropanoid ).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. 13Examination of the Dreiding model indicated that in the case of the b-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 b-methine carbon, and are largely unaffected by the asymmetrical flavan C-2 and C-3 atoms; and hence that the absolute configuration at the b-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 a-orientation.The compound 6a with a b-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 1 H and 13 C 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 1 H-1 H COSY and HMBC spectra.The R absolute configuration of the b-methine carbon of 7a was determined by comparing the 1 H NMR, NOESY NMR and CD data with those of 5a and 6a.The H-2 and H-3 resonances of 7a suggested that the b-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.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 (J 2,3 < 2 Hz) of the heterocyclic H-2 and H-3 protons and additional methylene (d 2.53 and 3.26) and methine (d 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 d 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 1 H NMR data with those of the peracetate of procyanidin B2.The downfield shift of the H-2(C) resonance at d 5.86 and the upfield shift of the B-ring proton resonances H-2' and H-6' at d 6.96 (d, J 2.1 Hz) and d 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".3][24][25][26][27] The missing NOE effect between H-2(C) and H-4(C) strongly supported the proposed 4b 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 b-methine carbon in 9a was determined based on CD data and NOESY NMR experiments.3][24][25][26][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 4b → 8 interflavanoidlinkage 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. 28The 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. 29,30The 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 a-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. 18,31his 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. 32,33The presence of reducing substances causes the reduction of the Fe 3+ /ferricyanide complex to the ferrous form.Therefore, Fe 2+ can be monitored by measuring the formation of Perl's Prussian blue at 700 nm. 34Figure 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.

Experimental
General experimental procedures NMR spcetra of the peracetates (Varian Mercury 300BB) were recorded in acetone-d 6 or CDCl 3 at ambient temperature with TMS as internal standard.Some compounds were recorded in CDCl 3 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 resolutionelectrospray 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 FeCl 3 solution.Analytical TLC (thin-layer chromatography) was carried out on precoated aluminum sheets (Kieselgel 60 F 254 , Merck) with EtOAc/formic acid/H 2 O (18:1:1; system Sl).Preparative TLC was performed on silica gel plates (Kieselgel 60 F 254 , 1.0 mm) using toluene/Me 2 CO (7:3; system S2).Acetylations were performed in pyridine/Ac 2 O (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. 35A 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 mmol 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 EC 50 (mmol L -1 ), the concentration of polyphenol required for scavenging 50% of DPPH radical in the solution. 28

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.*e-mail: mello@uem.br

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 AMX 2 -type resonances at d 5.20 (brs) and 5.43 (m), 5.46 (brs) and

Figure 1 .
Figure 1.Structures of the compounds isolated from T. catigua.

Figure 2 .
Figure 2. Reducing power of compounds isolated from T. catigua.

Table 1 .
1H NMR spectral data of the peracetate compounds

1a, 5a, 6a and 7a (
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 739.4in negative ESI-MS.Compound 9a exhibited a pseudo-molecular ion peak at m/z 619.1785 [(calcd.for C 61 H 54 O 26 + 2 NH 4 ) 2+ 619.1789] in the HR-ESI-MS (positive-ion mode), ascribable to molecular formula C 61 H 54 O 26 , and showed a peak at m/z 1220.4 [M + NH 4 ] +

Table 2 .
Radical scavenging activity of compounds isolated from T. catigua on the DPPH radical Equal letters indicate absence of significant difference (P < 0.05); different letters indicate significant difference (P < 0.05); RSD = relative standard deviation.