New Biflavonoid and Other Flavonoids from the Leaves of Chimarrhis turbinata and their Antioxidant Activities

Um novo biflavonol denominado chimarrosídeo (1) e oito flavonóis glicosilados adicionais (2-9) foram isolados das folhas de Chimarrhis turbinata. Suas estruturas foram elucidadas com base nos dados dos experimentos de RMN 1D e 2D, como: 3-O-rutinosilquercetina (2), 3-Orutinosil-kaempferol (3), 3-O-galactopiranosil-(6→1)-ramnopiranosil kaempferol (4), 3-O-βgalactopiranosil-(6→1)-α-ramnopiranosil-quercetina (5), 6-hidroxirutina (6), 3-Ogalactopiranosil-kaempferol (7), 3-O-glucopiranosil-kaempferol (8) e 3-O-ramnopiranosil(6→1)-glucopiranosil-(4→1)-ramnopiranosil-kaempferol (9). Adicionalmente, a catequina (10) e a procianidina B-3 (catequina-(4α→8)-catequina) (11) também foram isoladas. O extrato bruto, frações e compostos isolados foram avaliados quanto às suas propriedades antioxidantes no teste em CCD aspergida com solução de β-caroteno, e teste espectrofotométrico utilizando o radical livre 1,1-difenil-2-picrilidrazila (DPPH). Os flavonóides 2, 5, 6, 10 e 11 apresentaram forte atividade antioxidante quando comparados com os padrões BHT e rutina.


Introduction
In recent years, flavonoids have been widely recognized as a major class of secondary metabolites with antioxidant properties due to their ability to scavenge free radicals. [1][2][3] Antioxidant is a broad classification for molecules that may act prior to, or during, a free radical chain reaction at initiation, propagation, termination, decomposition, or subsequent reaction of oxidation products as sensitive targets. 4 There are several diseases, whose causes and severity are linked to oxidation, mainly those associated with oxygen free radicals, which have been implicated as mediators of degenerative and chronic deteriorative, inflammatory, and autoimmune diseases, 5 such as rheumatoid arthritis, 6 diabetes, vascular disease and hypertension, cancer and hyperplasic diseases, 7 cataract formation and aging processes. 8 Radical-mediated pathologies such as ischemia reperfusion, asthma and many others involving an imbalance in pro-oxidantantioxidant processes. 9 In our search for antioxidant compounds from Amazonian plant species, we examined constituents of Chimarrhis turbinata DC Rodr.. (Rubiaceae) leaves, collected in the city of Belém, Pará State, Brazil. The chemical composition of C. turbinata has been studied previously, and bioactive indole alkaloids were identified. [10][11][12] In this paper, we describe the isolation

Results and Discussion
Chimarrhoside (1) was obtained as an amorphous pale yellow powder from the EtOH extract and exhibited in its mass spectrum a molecular ion peak at m/z 1171 [M+H]+ obtained from ESIMS. The IR and UV spectra of 1 revealed the presence of hydroxyl (3410 cm -1 ) and phenolic groups (270, 295, 344 nm, 1690 cm -1 ). Part of the 1 H and 13 C NMR signals of compound 1 were observed in duplicate, suggesting a dimer ( Table 1). Comparison of the 13 C NMR values of 1 with literature data 13 evidenced the kaempferol aglycone and additional similar spectral features to those of 3, also isolated in this work, except for the duplication of some signals for carbons arising from a dimer structure. In the 1 H NMR spectrum of 1, signals for two kaempferol units as two meta-coupled A-ring hydrogens (H-6/H-8) and two pairs of coupled B-ring hydrogens (H-3'/H-5' and H-2'/H-6') for each aglycone were observed, in addition to four anomeric hydrogens at δ 5.12 (1H, d, J 7.7 Hz), 5.02 (1H, d, J 7.7 Hz), 4.40 (1H, br s), 4.60 (1H, br s), 20 hydroxymethine and 2 hydroxymethylene hydrogens. The 13 C NMR spectrum of 1 revealed the presence of two kaempferol moieties and 24 hydroxymethine and hydroxymethylene signals which, analyzed together with 1 H NMR data (Table  1) were consistent with the presence of two diglycoside moieties identified as rutinosyl and α-L-rhamnopyranosyl-(1→6)-β-D-galactopyranoside. This assumption was supported by signals at δ 103.1 / 67.1 and δ 104.0 / 65.9, assigned to C-1'' C-6'' of the glucopyranosyl and of the galactopyranosyl moiety respectively. Those signals also showed HMQC correlations with H-1'' (anomeric) and H-6'' (Table 1); as well as signals at δ 100.9/16.4 and δ 100.4/16.6, assigned to C-1'''/C-6''' of the two rhamnopyranosil moieties, respectively, which also showed HMQC correlations with H-1''' (δ 4.4 0 and 4.60) and H-6''' (δ 1.17 and 1.11). On the basis of the above data, the chemical shifts assigned for each sugar: the glucosyl and galactosyl moieties were confirmed taking into account the values at δ 103.1, 76.6, 75.7, 74.3, 70.1 and 67.1 as well as δ 104.0, 73.9, 73.6, 71.5, 68.2 and 65.9 attributed to glucose and galactose, respectively. 15 According to literature data, using the same solvent, the chemical shifts of hydroxymethines carbons in glucose are quite deshielded when compared to those of galactose. The glucosyl/galactosyl groups are linked to the rhamnosyl residue at C-6", because the signal assigned to this carbon, in both sugar moieties, was markedly displaced downfield at δ 67.1/65.9 (∆δ +6), respectively when compared with the 13 C NMR spectrum of free hydroxymethylenes in glucose/galactose. This also was confirmed by the observation of a HMBC long-range correlation of the anomeric hydrogen signal of the rhamnosyl group at δ 4.40 / 4.60 with C-6" of the glucosyl moieties at 67.1 and 65.9, respectively.
Additionally, the site of glycosylation at C-3 of each kaempferol moiety was evidenced from downfield shifts of C-2 (∆δ +9) and C-4 (∆δ +2) and upfield shift of C-3 (∆δ -3) when compared to the aglycone, 15 as well as from HMBC correlations of H-1'' to C-3a/3b within each kaempferol diglycoside moiety ( Figure 1). Further HMBC correlations of H-6 and H-8 with C-7 (δ 164.6) confirmed the assignments for ring A and suggested the interflavonoidic linkage at C-7 due to the observed downfield shift of its signal when compared to the monomer. 15 Moreover, ESIMS analysis showed peaks of m/z 298 (70) and 133 (40) resulting from Retro Diels-Alder (RDA) fragmentations. From moiety A wherein bonds 1 and 3 undergo scission, lead to formation of ions I and II as well as scission of bonds 0 and 2 led to the formation of fragment m/z 433 (30). The fragment m/z 298 (70), was generated by both RDA suffered by moiety A and moiety B ( Figure 2). Additional fragments observed in the mass spectra are in accordance with those reported for kaempferol. 16 These data, combined with HPLC analysis, which showed a lower retention time for compound 1 (5.7 min) when compared to monomeric flavonol diglycosides 2 (9.8 min) and 3 (13.8 min.) ( Figure  3), confirmed the proposed structure for compound 1 as Further studies of the EtOAc extract from leaves of C. turbinata resulted in 10 known flavonoids already reported in the literature:   (Figure 4). 13-15, 18, 19-22, 26-28 The radical scavenging effects obtained for compounds 2-11 assayed with DPPH are shown in (Table 2) using as reference the antioxidant standard rutin (IC 50 21.34 μg mL -1 ) and BHT (IC 50 62.50 μg mL -1 ). These results indicate that the free-radical scavenging activity of these J. Braz. Chem. Soc. micromolecules is due to its hydrogen-donating ability, provided by the ease of stabilization of the phenoxyl radical after reduction of DPPH and is enhanced by the presence of catechol groups and the α,β-unsaturated carbonyl moiety, as evidenced by the IC 50 values for compounds 2, 5, 6, 10, and 11. 24 It is also evident from Table 2, that ortho-dihydroxy moiety plays more important role for this type of activity than the α,β-unsaturated carbonyl, as the lack of the latter, e.g. in compound 10, contributed in a lesser extent to the loss of activity, as observed for compounds 3, 4 and 7-9, due to the missing catechol group. On the other side, the   presence of an additional catechol group as in compound 11, rendered as the most effective in scavenging free radicals, evidenced by its IC 50 value, lower that that for rutin used as standard compound.

General experimental procedures
Commercial β-carotene (Aldrich) and DPPH (Aldrich) were used in the antioxidant assays. NMR spectra were recorded on a Varian Unity 500 MHz spectrometer at 25 °C and referenced to the residual proton solvent resonance (CD 3 OD at δ 3.33 and 49.0 for 1 H and 13 C NMR, respectively). IR spectra were recorded on an FT-IR-Nicolet, model EMACT-40 Perkin Elmer 1600 FT-IR spectrophotometer, in the range 500-4000 cm -1 . ESIMS spectra were acquired using an ESI capillary voltage of 3 kV and a cone voltage of 10-20 eV with argon. Silica gel 60H (230-400 μ), (60-230 μ) (Merck), Sephadex LH-20 (Pharmacia Biotech) and XAD-16 (Sigma) were used in column chromatography. TLC plates were illuminated under UV light at 254 and 366 nm. For preparative HPLC, Varian Star Dynamax model SD-1 pump, ODS Phenomenex,, Luna column (250 x 21.20 mm, 5 μm), and pre-Column (50 x 10.00 mm) were used in this study. For semi-preparative HPLC, a Supelco ODS column 8 (250 mm x 10 mm x 5 μm) was used. Peaks were detected using Varian model 320-chromatointegrator connected to a UV detector. All EIMS spectra were obtained by direct insertion of the samples, using an electric cone potential of +70 eV. Leaves of C. turbinata (powdered and air-dried material 1.2 kg) obtained from the second collection were extracted exhaustively with EtOH at room temperature. The EtOH solutions were evaporated under vacuum to give a residue (57.1 g). This residue was dissolved in EtOH:H 2 O (90:10), and then extracted with n-hexane to give a n-hexane fraction (28.9 g). The EtOH:H 2 O fraction was extracted with CH 2 Cl 2 , EtOAc and n-BuOH successively, to give a CH 2 Cl 2 fraction (4.0 g), EtOAc fraction (4.1 g), n-BuOH fraction (12.4 g) and H 2 O-soluble residues (7.6 g), respectively, which were screened by β-carotene test on TLC.

β-Carotene bleaching experiments
TLC plates, after elution and drying, were sprayed with a solution of β-carotene (Aldrich) (0.02%) in CH 2 Cl 2 . Plates were placed under natural light until discoloration of background was observed. The persisting yellow spots indicated the presence of antioxidant substances. 17 Determination of the radical scavenging activity 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical was used in methanol (100 μmol L -1 ). 2. 0 mL of the reagent was added to a 1.0 mL aliquot of the compounds, previously dissolved in methanol, with yield final concentrations of 100, 80, 40, 20, 10 and 5 μmol L -1 . Each mixture was shaken and maintained for 30 min at room temperature, in the dark. Rutin and BHT were used as standard compounds. DPPH solution (2.0 mL) in methanol (1.0 mL) served as control. Absorbances of the resulting solutions were measured using a Milton Roy 20 D spectrophotometer at 517 nm and the percent inhibition was determined by comparison with a MeOH treated control group.