Amburanins A and B from Amburana cearensis: Daphnodorin-Type Biflavonoids that Modulate Human Neutrophil Degranulation

Dois novos biflavonoides 3,5,7,4’-tetraidroxiflavanona-(2→O→4’:3→3’)-2’,4’,6’,4tetraidroxidiidrochalcona (1) e 3,5,7,4’-tetraidroxiflavanona-(2→O→7:3→8)-3,4’,5,7tetraidroxiflavona (2), denominados amburanina A e amburanina B, respectivamente, foram isolados da casca do caule de Amburana cearensis, e as estruturas deles foram elucidadas com base em análises espectroscópicas e por comparação com dados da literatura. Os efeitos de 1 e 2 sobre a resposta pro-inflamatória de neutrófilos humanos foram investigados (0,1; 1; 25; 50 e 100 μg mL). Ambos compostos inibiram em torno de 92% a degranulação de neutrófilos a partir da concentração de 25 μg mL, e reduziram em até 53% a atividade da mieloperoxidase humana, indicando o potencial deles como substâncias anti-inflamatórias.


General experimental procedure
Melting points (uncorr.)were determined with a Mettler Toledo FP82HT apparatus (Columbus, OH, USA), with a heating rate of 2 °C min -1 .Optical rotations were measured on a Perkin-Elmer 341 polarimeter (at 589 nm) in MeOH at 20 °C.Electronic circular dichroism (ECD) spectra were recorded in high purity MeOH on a JASCO J-815 CD spectrometer (Easton, MD, USA) at 25 °C using a 1 cm path length quartz cuvette.ECD spectra for both compounds were obtained at concentrations of 0.10, 0.08, 0.06, 0.04, and 0.02 mmol mL -1 in order to determine the concentration which produced the highest quality data.UV spectra were obtained on a Varian Cary 50 Conc UV-Visible spectrophotometer (Mulgrave, Australia).Infrared (IR) spectra were recorded with a Perkin-Elmer FT-IR 1000 spectrometer (Waltham, MA, USA), using KBr pellets.Nuclear magnetic resonance (NMR) experiments were performed on a Bruker DRX-500 spectrometer (Bruker Biospin, Rheinstetten, Germany) at room temperature, using dry DMSO-d 6 as solvent (Cambridge Isotope Laboratories), and were internally referenced to residual undeuterated solvent signals (d H 2.50 and d C 39.51 ppm).High resolution mass spectra (HR-MS) were recorded on a Q-TOF Xevo mass spectrometer (Waters, Milford, USA), utilizing an electrospray ionization (ESI) source as ionization method.Column chromatography was run on silica gel 60 (70-230 mesh, VETEC) and Sephadex LH-20 (Amersham Pharmacia Biotech).Thin layer chromatography (TLC) was performed on precoated silica gel polyester sheets (Merck) and monitored by UV detection and vanillin-perchloric acid reagent detection.High performance liquid chromatography (HPLC) purification was performed on a Waters 1525 (Milford, MA, USA) chromatograph, equipped with a binary pump, Rheodyne injector (200 µL loop), and photodiode-array detector (Waters-2996 PDA), using a Waters X-Terra RP-18 column (250 × 4.6 mm, 5 µm) at 35 ºC in a thermostatic oven.HPLC MeOH was purchased from Tedia Co, and HPLC grade water (18 mΩ) obtained by a Milli-Q purification system (Millipore, Bedford, MA, USA).

Plant material
Trunk bark of A. cearensis was collected in the Quixeramobim region, Ceará State, Northeastern Brazil, in September 2002.Voucher specimens (# 837 and 847) were deposited at the Prisco Bezerra Herbarium, and identified by Dr. Afrânio G. Fernandes, a botanist at the Departamento de Biologia, Universidade Federal do Ceará.

Myeloperoxidase activity assay
The aforementioned method 16 was modified to evaluate the effect of the bioflavonoids on MPO activity.Human neutrophils were stimulated with PMA, and the MPO-rich supernatant was separated by centrifugation.Aliquots of the supernatant were incubated at 37 °C with compounds 1 and 2 (25, 50, and 100 µg mL -1 ), DMSO (1% v/v, vehicle) or HBSS (untreated cells) for 15 min, before determining MPO activity.
After 3 h under a 5% CO 2 atmosphere, the cells were washed with PBS, and the formazan product was dissolved in 100 µL of DMSO.The absorbance was recorded at 550 nm; 17 its values decreased as a function of cell death.

Results and Discussion
The ethanol extract of A. cearensis trunk bark was subjected to liquid-liquid partitioning (EtOAc/H 2 O) to yield an EtOAc phase that was further separated by either silica gel or Sephadex LH-20 column chromatography.Subsequent HPLC and spectroscopic analysis of purified compounds led to the isolation and structural characterization of biflavonoids 1 and 2, along with known compounds vanillic acid, protocatechuic acid, quercetin and amburoside A. 9 Compound  O 11 , 557.1084) in its HRESIMS spectrum.A broad and intense IR absorption band centered at 3335 cm -1 confirmed the presence of hydroxy groups, while an intense band with a shoulder at 1641 cm -1 suggested the presence of conjugated carbonyl functionalities.Additional IR absorption bands at 1252 and 1171 cm -1 were attributed to phenolic and tertiary alcohol C-O stretching frequencies, respectively.
The 1 H NMR spectrum for compound 1 in dry DMSO-d 6 displayed two methylene proton triplets at d H 2.87 and 3.29 (t, 2H, J 7.7 Hz, H-IIβ and H-IIa), and two m-coupled doublets at d H 5.86 and 5.95 (d, 1H, J 2.0 Hz, H-I8 and H-I6), the latter characteristic of the I-A-ring aromatic protons.Five sharp singlets at d Η 6.51, 9.17, 9.77, 11.70 and 13.17 (s, 1H), in addition to a broad singlet at d H 11.02 (s, 2H) characterized seven hydroxy groups.Four pairs of two-proton doublets at d H 6.62, 7.02 (d, 2H, J 8.4 Hz, H-II3, 5 and H-II2, 6), 6.72 and 7.25 (d, 2H, J 8.7 Hz, H-I3', 5' and H-I2', 6') were characteristic of two para-disubstituted aromatic rings, while a singlet at d H 5.96 (s, 1H, H-II5') was associated with the single hydrogen on the pentasubstituted benzene ring.A gradient-selective correlation spectroscopy (gs-COSY) spectrum showed, as expected, scalar coupling between the two aliphatic methylenes and hydrogen at d The 13 C NMR data (Table 1) showed the presence of two carbonyl carbons; one at d A further conclusion from the chemical shifts of these carbons is that there were not oxygenated ortho-carbons, as such species would lead to more shielded chemical shifts (144 ≤ d C ≤ 148). 18his suggested that the A-ring oxygenation pattern followed the common phloroglucinol A-ring theme for flavonoids. 18A saturated quaternary carbon bearing an oxygen atom at d C 80.5 (C-I3) was readily characterized, while the dioxygenated carbon at d 118.6 (C-I2) was identified by comparison with the chemical shifts of dihydroflavonol moieties from the daphnodorins isolated from Daphne odora Thunb. 19he heteronuclear single quantum coherence (HMQC) spectrum permitted the correlation of all protonated carbons (Table 1).The heteronuclear multiple-bond correlation (HMBC) data were of crucial importance for confirming structural assignments and correctly positioning the hydroxy groups and all non-protonated carbons.From Table 1 it is evident that the assignments of C-6 and C-8 could mistakenly be interchanged, as C-6 is slightly more deshielded than C-8. 18Unambiguous assignment was, however, possible due to several key HMBC correlations.The hydrogen at d  Thus, the structure of 1 was assigned as 3,5,7,4'-tetrahydroxyflavanone-(2→O→4':3→3')-2',4',6',4-tetrahydroxydihydrochalcone, an adduct of the flavonol kaempferol and the dihydrochalcone phloretin, and named amburanin A. Comparison of the 13 C NMR chemical shifts reported for these two monomers with those of compound 1, shows agreement, taking into account the empirical influence of the substitution effect. 18This effect is particularly noticeable for C-II3' as it is deshielded by 12.7 ppm due to the "ipso" effect of C-I3, and for C-I3 as it is deshielded by 6.8 ppm due to the a-effect of C-II3'. 18lthough compound 1 possessed a negative specific rotation, ([a] D 20 −15.9, c 0.24, MeOH), the experimental ECD spectrum (not shown) lacked distinct Cotton effects (CEs) at all concentrations examined, indicating a lack of enantiomeric purity.
Compound 2 was obtained as a yellow powder, with a molecular formula of C 30 H 18 O 12 as revealed by a HREIMS quasi-molecular ions at m/z 593.0679 [M+Na] + (calcd.for C 30 H 18 O 12 Na, 593.0696); m/z 571.0853 [M+H] + (calcd.for C 30 H 19 O 12 , 571.0877).Its FT-IR spectrum exhibited a broad absorption band centered at 3433 cm -1 , characteristic of hydroxy groups, and an absorption band with a shoulder at 1640 cm -1 characteristic of C=O stretching.Absorption bands at 1258 cm -1 and 1168 cm -1 were characteristic of phenolic and tertiary alcohol C-O stretching frequencies, similar to those observed for 1.
The 13 C NMR (CPD) spectrum of 2 was similar to that of compound 1 and showed 26 resonances between d C 80 and 192, two with prominent intensities.From Table 1, it is evident that the dihydroflavonol moiety is the same for both compounds.The chemical shift of the second carbonyl absorption (d C 177.1, C-II4), however, indicated that the second C 6 -C 3 -C 6 unit was a flavonol moiety instead of a dihydrochalcone.This was supported by the lack of the two saturated methylene carbons seen in compound 1.Two sp 2 carbons at d 136.9 (C-II3) and 148.3 (C-II2) were instead present, further supporting the assignment of a flavonol moiety.
Although compound 2 possessed a low magnitude negative specific rotation of [a] D 20 −12.4 (c 0.21, MeOH), the experimental ECD spectrum (Figure S17 in the Supplementary Information section) contained distinct CEs which appeared to correspond to the multiple UV maxima for the compound.Attempts to interpret these CEs based on the precedent of the daphnodorins, 19,20 however, revealed a series of significant differences between ECD data for the daphnodorins with their flavan and flavan-3-ol constituents units, and compound 2.However, the negative CE at ca. 320 nm presumably indicates (2S, 3S) absolute configuration for this compound based on the ECD data of daphnodorins F and H with similar configuration at C-2 and C-3.The low magnitude of the specific rotation of compound 2 presumably reflects a low degree of enantiomeric purity when compared to the reported values for daphnodorins F and H (-120.0 and -170.4,respectively). 16,19Although the two new amburanins A and B (1 and 2) are here described for the first time, analogous dihydrofurantype biflavonoids, the daphnodorins, have been reported from Glycyrrhiza glabra L. (Fabaceae) 21 and several species of the genus Daphne (Thymelaeaceae) including D. odora, 18,20 D. acutiloba Rehd., 22 D. genkwa Sieb.et Zucc., 23 D. giraldii Nitsche 24 and D. tangutica Maxim. 25][29][30] In previous studies, 2,4,7,13,31 it has been shown that hydroalcohol extract, coumarin, isokaempferide, amburoside A, vanillic acid and afrormosin from A. cearensis display various pharmacological properties.These molecules showed anti-inflammatory activity in rodents by inhibiting paw edema and the accumulation of inflammatory cells into the peritoneal cavity of mice.In addition, isokaempferide and amburoside A were able to inhibit human neutrophil degranulation, myeloperoxidase activity and the secretion of TNF-a by human neutrophils, showing no cytotoxicity at the concentrations investigated.2][33] Additional investigations into the possible anti-inflammatory activities of amburanins A and B were therefore deemed worthwhile, as these compounds could contribute to the biological activities of A. cearensis.
The recruitment and activation of polymorphonuclear leukocytes (PMNs) is considered one of the main defense mechanisms of innate immunity.5][36] As part of the first line of host defense, PMNs possess bactericidal activities and are involved in pathogen adherence, chemotaxis towards infected cells, and phagocytosis of pathogens and damaged cells. 37Activation of various PMNs during inflammatory processes results in the release of complement components, reactive oxygen species and lysosomal enzymes, including MPO.9][40] In addition, MPO has direct effects on endothelial cells since its internalization by these cells is followed by the intracellular production of oxidants. 41he possible effects of biflavonoids 1 and 2 on the neutrophil degranulation were assessed by measuring MPO release by PMA-stimulated cells.Exposure of neutrophils to compounds 1 and 2 (0.1-100 µg mL -1 ) caused an inhibition of MPO release (Figure 4) that was comparable to the effects of indomethacin (36 µg mL -1 ), a non-selective cyclooxygenase inhibitor used as reference compound.At concentrations higher than 10 µg mL -1 , biflavonoids 1 and 2 inhibited MPO activity by 45.5 and 52.7%, respectively; at 36 µg mL -1 , indomethacin inhibited MPO activity by 45% (Figure 5).Compared with the control group (115.2 ± 3.8%), 1 and 2 did not interfere significantly in the cell viability at concentrations ranging from 10 to 100 µg mL -1 ; the reference compound Triton X-100 reduced the cell viability to 12.1% (Figure 6).These results provide evidence that the biflavonoids amburanins A and B have anti-inflammatory potential through the modulation of human neutrophil degranulation, but this effect seems is related at least in part to a direct effect of the biflavonoids on the MPO activity.Furthermore, the anti-inflammatory activity of these biflavonoids seems not to be related to a cytotoxic effect, since these compounds did not affect the neutrophil viability, as assayed by the MTT assay.In this context, because neutrophils play important roles in the pathophysiology of several inflammatory diseases and the MPO activity correlates well with leukocytes infiltration in inflamed regions, 42 it is likely that pharmacological activities of amburanins A and B reported here have beneficial effects in the treatment of inflammatory diseases.

Conclusions
The chemical investigation of Amburana cearensis trunk bark permitted the isolation of two unusual biflavonoids, amburanins A and B (compounds 1 and 2).These compounds displayed inhibition of human neutrophil pro-inflammatory responses, such as degranulation and MPO activity, suggesting potential anti-inflammatory effects similar to those of co-occurring amburoside A, isokaempferide and afrormosin. 7,31These findings corroborate the folk usage of this plant, justifying its pharmacological potential for the treatment of inflammatory diseases such as asthma.

Figure 2
shows additional important C-H correlations observed in the HMBC spectrum.

Figure 4 .
Figure 4. Effects of biflavonoids 1 and 2 on the human neutrophil degranulation assayed using release MPO as marker.Freshly isolated cells (5 × 10 6 ) were preincubated with the indicated concentration of 1 or 2 prior to the addition of PMA (0.1 mg mL -1 ).Data are expressed as percentage of inhibition of MPO release by the tested compounds.Numbers represent mean ± standard error of the mean (SEM).

Figure 5 .
Figure 5. Effects of biflavonoids 1 and 2 on the activity of human neutrophil myeloperoxidase (MPO).The MPO-rich supernatant obtained from PMA-stimulated neutrophils was incubated with compound 1 or 2 at the concentrations indicated in the graph.Data are expressed as percentages of inhibition of MPO activity by the tested compounds.Numbers represent mean ± SEM.

Figure 6 .
Figure 6.Evaluation of biflavonoids 1 and 2 toxicity towards human neutrophils.Data from two to eight samples.*p < 0.05 (ANOVA and Tukey's post hoc test).