Isolation of Flavonoids from Dipteryx Odorata by High Performance Liquid Chromatography Da Cunha, C

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Artigo Isolation of Flavonoids from Dipteryx odorata by High Performance Liquid Chromatography da Cunha, C. P.; Godoy, R. L. O.;* Braz Filho, R. Rev. Virtual Quim., 2016, 8 (1), 43-56. Data de publicação na Web: 3 de janeiro de 2016 http://rvq.sbq.org.br

Isolamento de Flavonoides de Dipteryx odorata por Cromatografia Líquida de Alta Eficiência Resumo: Dipteryx odorata (Aubl) Willd, família Fabaceae, é uma espécie nativa da floresta Amazônica, Brasil. O objetivo do trabalho foi isolar e identificar os flavonoides do endocarpo de D. odorata minimizando custos com solventes, tempo e resíduos. Seis flavonoides, 3',4',7-triidroxiflavona, 3',4',7-triidroxiflavanona, 3',4',6-triidroxiaurona, 3',4',5,7-tetraidroxiflavona, 2',3,4,4'-tetraidroxichalcona e 2',4,4'-triidroxichalcona foram isolados. Os flavonoides 3',4',7-triidroxiflavona e 2',3,4,4'-tetraidroxichalcona foram identificados pela primeira vez nesta espécie. Palavras-chave: Cumaru; Fabaceae; CLAE.

Abstract Dipteryx odorata (Aubl.) Willd, Fabaceae family, is a native species from the Amazon forest, Brazil. The goal of the work was to isolate and identify the flavonoids in the endocarp from D. odorata in order to minimize costs with solvents, time and residues. Six flavonoids, 3’,4’,7-trihydroxyflavone, 3’,4’,7-trihydroxyflavanone, 3’,4’,6- trihydroxyaurone, 3’,4’,5,7-tetrahydroxyflavone, 2’,3,4,4’-tetrahydroxychalcone and 2’,4,4’-trihydroxychalcone were isolated. The flavonoids 3’,4’,7-trihydroxyflavoneand 2’,3,4,4’-tetrahydroxychalcone were identified for the first time in this species. Keywords: Tonka bean; Fabaceae; HPLC.

* Embrapa Agroindústria de Alimentos, Laboratório de Cromatografia Líquida, Avenida das Américas, 29501, Guaratiba, CEP 23020-470, Rio de Janeiro-RJ, Brasil. [email protected] DOI: 10.5935/1984-6835.20160004 Rev. Virtual Quim. |Vol 8| |No. 1| |43-56| 43

Volume 8, Número 1 Janeiro-Fevereiro 2016

Revista Virtual de Química ISSN 1984-6835

Isolation of Flavonoids from Dipteryx odorata by High Performance Liquid Chromatography Carolina P. da Cunha,a Ronoel Luiz de O. Godoy,b,* Raimundo Braz-Filhoc‡ a Universidade Federal Rural do Rio de Janeiro, Departamento de Química, Rodovia BR 465, Km 7, Campus Universitário, CEP 23897-000, Seropédica-RJ, Brasil. b Embrapa Agroindústria de Alimentos, Laboratório de Cromatografia Líquida, Avenida das Américas, 29501, Guaratiba, CEP 23020-470, Rio de Janeiro-RJ, Brasil. c Universidade Estadual do Norte Fluminense Darcy Ribeiro, Laboratório de Ciências Químicas, CCT, Avenida Alberto Lamego 2000, Horto – 28013-602, Campos dos Goytacazes-RJ, Brasil. ‡Pesquisador Visitante Emérito – FAPERJ/UENF/UFRRJ * [email protected]

Recebido em 3 de janeiro de 2016. Aceito para publicação em 3 de janeiro de 2016

1. Introduction 2. Material and Methods 2.1. Plant Material and Sample Preparation 2.2. Chromatographic conditions for flavonoid separation 2.3. Isolation and structural characterization of the substances 2.4. Nuclear Magnetic Resonance Spectrometry Conditions 2.5. UPLC-Mass Spectrometry Conditions 2.6. Derivatization of isolated flavonoids 3. Results 3.1. Fractionation of the extract 3.2. Optimization of separation and collection of compounds present in the fraction of ethyl acetate 3.3. Identification of the separated peaks 3.4. Preparation of derivatives 4. Conclusion

1. Introduction locally as “cumaru”, is a high arboreal species native to the Amazon area.1 Due to its high content of coumarin, the plant’s seeds have significant commercial value and are widely Dipteryx odorata (Aubl.) Willd. (syn. used in the perfumery, cigarettes and Coumarouna odorata Aubl); Fabacea family, cosmetics industries.1,2 commonly known as “tonka bean” tree or

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Popularly, it is used to aid in the An alternative involves the optimization of treatment of ulcers, ear infections, chromatographic methods to reduce these respiratory and cardiac disorders.1 The seeds consequences. Thus, the objective of this are characterized by acaricidal activity,3 work was the isolation and the identification which revealed anti-carcinogenic effects, of flavonoids from the endocarp minimizing especially against breast cancer, potential costs, time and solvent’s residues. phytotoxicity4 and cancer chemopreventive.5

Previous phytochemical investigations of this plant resulted in the isolation of 2. Material and Methods 2,6 5,7 coumarins, cassanediterpenoids, isoflavonoids,8-10 isoflavolignans,5fatty acids4 and lupane triterpenoids.11 Isoflavones, 2.1. Plant Material and Sample chalcones and aurones represents some Preparation flavonoids that have already been isolated from different parts of this species. From the endocarp, only diterpenoids were isolated Dipteryx odorata fruits were collected at but the presence of flavonoids has not been Ducke Reserve – National Institute for investigated in this plant structure. Amazonian Research, Manaus, AM, Brazil. The voucher specimen (INPA00143012) is The flavonoids are secondary metabolites, deposited in the Herbário INPA. having a C6-C3-C6 carbon framework. Variations in the basic carbon skeleton and The fruits were dried at room the oxidation state lead to main classes of temperature and broken up, separating the flavonoids: chalcones, aurones, flavanones, endocarp material from the seed. The dihydroflavonols, flavones, flavonols, endocarp (3000 g) was grinded in a ball mill isoflavones, flavan-3-ols and and extracted with petroleum ether (5 L) by anthocyanidins.12 Flavonoids receive maceration for 6 days. The solid residue of considerable attention in the literature, the maceration was dried at room especially for being awarded several temperature and extracted by maceration for biological activities to this class, such as 6 days, this time with ethyl ether (5 L). antioxidant activity, anti-inflammatory, Dried ethyl ether extract (15 g) was 13-15 antitumor and antiviral. fractionated by ultraturrax agitation and The high performance liquid filtration under vacuum on sintered glass chromatography (HPLC) is the most wide funnel plate, with n-hexane, spread technique and it is used for analytical dichloromethane, ethyl acetate and scale separation of flavonoids. The major methanol in order of increasing polarity. advantage over others chromatographic From the dried fractions 1 mg was separated methods is that it provides high resolution and re-suspended with 1 mL of and sensitive quantitative analysis in a single methanol:water (1:1), and centrifuged. The operation.16 When the goal is to obtain the supernatant was analyzed by HPLC-PDA. compounds individually isolated with sufficient purity and quantity for identification purposes, the preparative scale 2.2. Chromatographic conditions for separation of flavonoids by classic liquid flavonoid separation chromatography is most commonly used. However, it is a time consuming and The HPLC analysis was carried out on a laborious process17 and uses large volume of Waters Alliance® 2695 system (Waters, MA, solvents, causing an expressive USA) equipped with an auto sampler, PDA environmental impact and economic detector (2996, Waters, MA, USA). The demand. chromatographic separation was optimized

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using reverse phase column (C18, YMC 250 x were reported in Hz. Heteronuclear spectra 1 13 1 1 4.6 mm, with a particle size of 5 μm) at a 2D HSQC ( H- C-COSY- JCH) and HMBC ( H- 13 n temperature of 45 °C. The mobile phase C-COSY- JCH, n=2 and n=3) were acquired consisted of water:acetic acid 98:2 (A) and with 8 transitions/128 increments and 4 acetonitrile:acetic acid 98:2 (B). The flow rate transitions/128 increments respectively. For was 1.3 mL.min-1 and the gradient program homonuclear spectra 2D 1H-1H-COSY spectral was 0–6 min, 20–40 % B; 6–12 min, 40–50 % widths of 5000 Hz in both dimensions and the B; 12–14 min, 20–20 % B. The PDA detection normal number of transitions were used. wavelengths were set at 200 nm and 600 nm.

2.5. UPLC-Mass Spectrometry Conditions 2.3. Isolation and strutural characterization of the substances MS spectra was carried out on a Waters

Acquity UPLC system (Waters, MA, USA) The flavonoids separation was performed equipped with an auto sampler and by HPLC analytical scale using a reverse phase connected to the mass spectrometer Q-TOF column (C18, YMC 250 x 4.6 mm, with 5 μm Synapt detector (Waters, MA, USA) by particle) and the isolation was performed electron spray ionization (ESI) or with direct through the automated collection at the infusion on mass spectrometer. The isolation detector output, using a selector valve was done on a reverse phase column (C18, channels (RV500-104/550-104, Rheodyne®) ACQUITY UPLC BEH 150 x 2.1 mm and 1.7 as a fraction collector. The valve was mM) at a temperature of 45 ºC. The mobile programmed to collect one flavonoid through phase consisted of acetonitrile (A) and 0.1% each channel according to its retention time. formic acid in water (B). The flow rate was In order to increase the amount of each 0.35 mL.min-1 and the gradient program was: isolated flavonoid, successive injections and 0-5 min, 90-85 % B; 5-10.5 min, 85-81 % B; collections were performed automatically. 10.5-11 min, 81-80 % B; 11-19 min, 80-30 % B; 19-25 min, 30-40 % B; 25-26 min, 40-90 % Isolated flavonoids were dried by rotary B; 26-30 min, 90-90 % B. evaporator and their structures were determined by interpretation of spectral Mass spectrometer was operated in data, mainly the one furnished by 13C NMR positive mode, analyzer mode V, with and 1H NMR (1D and 2D) and ESI-MS, capillary voltage, cone voltage and extraction including methyl derivatives prepared by cone voltage set to 3000, 25 and 4000 V reaction with diazomethane to confirm the respectively. The desolvatation gas flow rate structures. The purity of the isolated was set to 750 L/h at a temperature of 500 ºC compounds was evaluated by HPLC-PDA. and the source temperature was set to 120 °C.

2.4. Nuclear Magnetic Resonance Spectrometry Conditions 2.6. Derivatization of isolated flavonoids

The spectral data was carried out on a Flavonoids isolated from the D. odorata Bruker AVANCE (operating in 499.80 MHz were subjected to reaction with and 125.69 MHz to 1H and 13C, respectively). diazomethane. The diazomethane was

The chemical shifts (δH e δC in ppm) were synthesized from the reaction of potassium referenced based on the residual values of hydroxide with nitrosomethylurea.18 the corresponding solvent MeOD used (δH 3.31 andδC 49.15 ppm). Coupling constants (J)

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3. Results solvents of different polarities: n-hexane, dichloromethane, ethyl acetate and methanol. The four extracts were analyzed by high performance liquid chromatography in 3.1. Fractionation of the extract reversed phase column with photodiode array detector. The chromatogram was The crude extract of the D. odorata reported at λ 253 nm (Figure 1). endocarp was fractionated with organic

Figure 1. Chromatogram of the D. odorata endocarp crude extract, n-hexane (n-C6H12), dichloromethane (CH2Cl2), ethyl acetate (EtOAc) and methanol (MeOH) fractions. Injection volume: 50 µL; concentration of the crude extract and fractions: 1 mg.mL-1

The crude, n-hexane and dichloromethane resolution, minimizing the time and the extracts showed one major peak at 7.8 min, solvent spent in the isolation without losing characterized as coumarin (1) by analysis of the quality and purity of the final product. accurated mass spectrum. The ethyl acetate The isolation of the flavonoids from D. extract did not reveal the presence of odorata involved the use of a HPLC analytical coumarin (1) and showed a higher scale system connected to the selector valve concentration of flavonoids than the other channels Rheodyne® in the output of the extracts. For that reason it was submitted to detector (Figure 2). Successive injections and HPLC separation and purification/collection collections were performed to obtain the of these components. flavonoids isolated in quantity and purity appropriated to achieve good quality spectra and for the preparation of derivatives when 3.2. Optimization of separation and necessary. The separation of flavonoids was collection of compounds present in the monitored by a photodiode array detector fraction of ethyl acetate (PDA) at λ 253 nm.

Each of the valve channels was The chromatographic separation was programmed to open and close according to optimized in order to minimize the running the retention time of each substance of time and get a good chromatographic interest. The valve has six channels, five

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channels were used for flavonoid collection the output to channel 2, where is the and one channel for waste, thus five reservoir flavonoid 2. In 5.5 minutes when flavonoids were collected at each injection. the flavonoid finished eluting, the valve For instance, at the beginning of the injection returns to the channel 1, and so on until the the valve output is selected on channel 1, end of the process. Each flavonoid was where is the waste. In 5.3 minutes the elution collected by a different channel reducing the of flavonoid 2 starts, then the valve change contamination.

Figure 2. Flavonoids collection schema. Channel 1: waste reservoir; channel 2-5: flavonoid 2-5 reservoir; channel 6: flavonoid 6 or 7 (Source: own file)

From the ethyl acetate extract (40,6 mg) and environmental viability, therefore of the D. odorata were isolated the spending only 10 days using high flavonoids 2 (1,2 mg, purity 93 %), 3 (1,3 mg, performance liquid chromatograph and only purity 90 %), 4 (1,5 mg, purity 96 %), 5 (1,0 6.9 L of acetonitrile, 370 mL of acetic acid and mg, purity 94 %), 6 (0,9 mg, purity 96 %) and 11 L of water. It is important to notice that 7 (0,8 mg, purity 98 %) (Figure 3) with automatic injection and collection were used, sufficient purity and quantity for structural eliminating the full time work of the analyst elucidation. The flavonoids 2 and 6 were and enabling the application of this identified for the first time in this species. methodology overnight, and for that not The methodology has proven effective in the changing the laboratory routine. isolation of flavonoids along with economic

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OH OH 3' 4' OH 3' 4' OH 2' 2' 1 8 8 1 HO 9 O 2 5' HO 9 O 2 5' 8 1 1' 1' 9 O O 7 6' 7 6' 7 2 6 4 3 6 4 3 10 3 5 5 10 6 10 5 4 O R O 1 3 2 R=H 5 R=OH

R OH 3 3' 4 OH 4' OH 2 1 2' 3' 1 7 HO 2' OH 5 HO 6 8 O 1' 1 5' 4' b 6 6' 1' 5 3 2 10 5' a 9 4 6' O O 4 6 R=OH 7 R=H Figure 3. Structures of seven substances isolated from the D. odorata

3.3. Identification of the separated peaks 112.75 (C-2’), 145.63 (C-3’), 149.40 (C-4’), 114.89 (C-5’), 118.82 (C-6’). Comparison of

these NMR spectral data with values The structures of the isolated flavonoids described in the literature19 allowed the were determined by interpretation of identification of the compound 2 as 3’,4’,7- spectral data, specially the one furnished by trihydroxyflavone. ESI-HRMS (positive mode) 13C NMR and 1H NMR (1D and 2D), ESI-MS revealed peaks at m/z 271.0662 253.0787, and UV, including of methyl derivatives 225.0811, 197.0809, 161.0441, 137.0380, prepared by reaction with diazomethane to 135.0627 (Figure 4). confirm the structures. Compound 3. UV max: 277, 311 nm.1H- Compound 1 was identified by UV NMR (MeOH-d4, 500 MHz) δH ppm: 5.34 (1H, (max=270 nm) and ESI-HRMS spectral data. dd, J = 12.9, 2.8 Hz, H-2 - axial position), 2.71 The ESI-HRMS (positive mode) showed peaks (1H, dd, J = 17.0, 2.8 Hz, H-3a – equatorial at m/z 147.0834 and 103.0997, attributed to position), 3.03 (1H, dd, J = 17.0, 12.9 Hz, H-3b protonated molecular ion ([M+H]+, calc m/z – axial position), 7.75 (1H, d, J = 8.8 Hz, H-5), 147.0446) and elimination of CO2 by 6.52 (1H, d, J = 8.8 Hz, H-6), 6.38 (1H, s, H-8), + protonated molecular ion ([M+H-CO2] , m/z 6.95 (1H, s, H-2’), 6.81 (1H, d, J = 8.2 Hz, H- 103.0548). These spectral data allowed the 5’), 6.82 (1H, d, J = 8.2 Hz, H-6’). 13C-NMR identification of the compound 1 as (MeOH-d4, 125 MHz) δC ppm: 79.68 (C-2), coumarin. 43.63 (C-3), 192.14 (C-4), 128.45 (C-5), 110.33 (C-6), 165.41 (C-7), 102.40 (C-8), 164.14 (C-9), Compound 2. UV max: 253, 342 nm.1H- 113.56 (C-10), 130.62 (C-1’), 113.27 (C-2’), NMR (MeOH-d4, 500 MHz) δH ppm: 6.67 (1H, s, H-3), 8.00 (1H, d, J = 8.8 Hz, H-5), 6.96 (1H, 145.09 (C-3’), 145.43 (C-4’), 114.82 (C-5’), d, J = 8.8 Hz, H-6), 7.00 (1H,br s, H-8), 7.43 117.82 (C-6’). Comparison of these NMR spectral data with values described in the (1H, brs, H-2’), 6.94 (1H, d, J = 8.1 Hz, H-5’), 20 7.44 (1H, d, J = 8.1 Hz, H-6’). 13C-NMR literature allowed the identification of the compound 3 as 3’,4’,7-trihydroxyflavanone (MeOH-d4, 125 MHz) δC ppm: 164.67 (C-2), 103.76 (C-3), 178.89 (C-4), 126.35 (C-5), (butin). ESI-HRMS (positive mode) revealed 115.40 (C-6), 163.46 (C-7), 102.08 (C-8), peaks at m/z 273.0901, 255.0872, 227.0918, 158.24 (C-9), 115.81 (C-10), 122.56 (C-1’), 163.0550, 137.0423 (Figure 5).

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Compound 4. UV max: 395 nm.1H-NMR compound 6 as 2’,3,4,4’- (MeOH-d4, 400 MHz) δH ppm: 7.63 (1H, d, J = tetrahydroxychalcone (butein). ESI-HRMS 8.00 Hz, H-4), 6.73 (1H, d, J = 8.00 Hz, H-5), (positive mode) revealed peaks at m/z 6.73 (1H, H-7), 6.73 (1H, d, H-10), 7.55 (1H, s, 273.1023, 255.0872, 227.0973, 163.0550, H-2’), 6.87 (1H, d, J = 8.00 Hz, H-5’), 7.26 (1H, 137.0423 (Figure 7). d, J = 8.00 Hz, H-6’). 13C-NMR (MeOH-d , 100 4 Compound 7. UV  : 229, 370 nm.1H- MHz) δ ppm: 146.29 (C-2), 183.07 (C-3), max C NMR (MeOH-d , 400 MHz) δ ppm: 7.65 (1H, 125.45 (C-4), 113.29 (C-5), 168.40 (C-6), 97.97 4 H d, J = 15.0 Hz, H-α), 7.81 (1H, d, J = 15.0 Hz, (C-7), 166.98 (C-8), 113.44 (C-9), 112.71 (C- H-β), 7.66 (2H, d, J = 8.2 Hz, H-2/H-6), 6.87 10), 124.11 (C-1’), 117.52 (C-2’), 145.34 (C- (2H, d, J = 8.2 Hz, H-3/H-5), 6.31 (1H, s, H-3’), 3’), 148.01 (C-4’), 115.27 (C-5’), 125.01 (CH- 6.44 (1H, d, J = 8.2 Hz, H-5’), 8.01 (1H, d, J = 6’). Comparison of these NMR spectraldata 8.2 Hz, H-6’). 13C-NMR (MeOH-d4, 100 MHz) with values described in the literature21 δ ppm: 192.14 (C=O), 116.92 (C-α), 144.30 allowed the identification of the compound 4 C (C-β), 130.47 (C-2/C-6), 115.54 (C-3/C-5), as 3’,4’,7-trihydroxyaurone (sulfuretin). ESI- 160.19 (C-4), 154.41 (C-2’), 102.41 (C-3’), HRMS (positive mode) revealed peaks at m/z 165.01 (C-4’), 107.78 (C-5’), 132.00 (C-6’). 271.0783, 253.0670, 225.0701, 215.0865, Comparison of these NMR spectraldata with 197.0757, 137.0380 (Figure 6). 23 values described in the literature allowed

Compound 5. UV max: 252, 348 nm.1H- the identification of compound 7 as 2’,3,4’- NMR (MeOH-d4, 500 MHz) δH ppm: 6.57 (1H, tetrahydroxychalcone (isoliquiritigenin). ESI- s, H-3), 6.23 (1H, s, H-6), 6.47 (1H, s, H-8), HRMS (positive mode) revealed peaks at m/z 7.41 (2H, sl, H-2’/H-6’), 6.93 (1H, sl, H-5’). 257.0919, 239.0935, 211.0967, 147.0587,

13C-NMR (MeOH-d4, 125 MHz) δC ppm: 137.0380 (Figure 7). 164.80 (C-2), 102.46 (CH-3), 182.47 (C-4), The proposed fragmentation mechanisms 161.80 (C-5), 98.81 (C-6), 93.66 (C-8), 158.03 to justify the principals peaks observed in the (C-9), 103.86 (C-10), 122.27 (C-1’), 112.78 (C- ESI-HRMS of all flavonoids were summarized 2’), 145.67 (C-3’), 149.64 (C-4’), 115.41 (C-5’), in the Figures 4 (compounds 2 and 5), 5 118.90 (C-6’). Comparison of these NMR (compound 3), 6 (compound 4) and 7 spectraldata with values described in the 22 (compounds 6 and 7). The similarity revealed literature allowed the identification of by the fragmentations reaction of these compound 5 as 3’,4’,5,7-trihydroxyflavone flavonoids, such as retro-Diels-Alder reaction, (luteolin). ESI-HRMS (positive mode) revealed was also used to define the number of peaks at m/z 269.0679, 241.0630, 161.0394, hydroxyl groups present on rings A and B 135.0584 (Figure 4). (Figures 4 to 7). Through the 1D and 2D NMR

Compound 6. UV max: 229, 379 nm.1H- spectral data involving comparison with the NMR (MeOH-d4, 500 MHz) δH ppm: 7.59 (1H, literature was possible to postulate the d, J = 14.8 Hz, H-α), 7.76 (1H, d, J = 14.8 Hz, substitution pattern of each ring. Ultraviolet H-β), 7.22 (1H, s, H-2), 6.86 (1H, d, J = 7.9 Hz, spectra were also useful in the structural H-5), 7.15 (1H, d, J = 7.9 Hz, H-6), 6.32 (1H, s, classification of the compounds, contributing H-3’), 6.45 (1H, d, J = 8.8 Hz, H-5’), 7.99 (1H, to the identification of the flavonoids class. d, J = 8.8 Hz, H-6’). 13C-NMR (MeOH-d4, 125 The compounds 3 and 6 showed very MHz) δC ppm: 192.05 (C=O), 116.86 (C-α), similar fragmentation and the same 144.70 (C-β), 126.98 (C-1), 114.37 (C-2), protonated molecular ion [M+H]+. As 145.48 (C-3), 148.61 (C-4), 115.24 (C-5), expected, the UV absorption of compound 6 122.29 (C-6), 113.24 (C-1’), 165.17 (C-2’), revealed expressive difference when 102.46 (C-3’), 166.12 (C-4’), 107.86 (C-5’), compared with the compound 3, this 131.92 (C-6’). Comparison of these NMR information suggested that these compounds spectraldata with values described in the 20 are the isomers chalcone and flavanone, literature allowed the identification of respectively. The ionization energy used in

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+ + the mass spectra can lead to interconversion H2O-CO] and [M+H-H2O-2CO] . Compounds 6 between these isomers explaining the and 7 (Figure 7) revealed the fragments + + similarity between the mass spectra. [M+H-H2O] and [M+H-H2O-CO] and compound 3 (Figure 5) revealed the Compounds 2, 5 (Figure 4) and 4 (Figure 6) + + + fragments [M+H-H2O] , [M+H-CO] . revealed the fragments [M+H-H2O] , [M+H-

TOF MSMS 271.00ES+ 271.0662 100 a 137.0380

161.0441 % 135.0627 272.0894

109.0475 141.0885 197.0809 273.1023 225.0811 253.0787 0 m/z 100 120 140 160 180 200 220 240 260 280 300 320

TOF MSMS 287.00ES+ 100 153.0298 b

% 135.0584

137.0423 241.0630 161.0394 179.0502 269.0679 0 m/z 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280

OH OH c OH2 O OH OH O H2O HO O HO O HO O HO O

R O R O H+ R OH + R+ O + 5+H R=OH C15H11O6 5d R=OH C15H9O5 5 R=OH C15H10O6 m/z calc.: 287.0550 m/z calc.: 269.0444 m/z calc.: 286.0477 m/z exp.: non detected m/z exp.:269.0679 2 R=H C H O + + + 15 10 5 2+H R=H C15H11O5 2d R=H C15H9O4 m/z calc.: 270.0528 m/z calc.: 271.0601 m/z calc.: 253.0495 m/z exp.:271.0662 m/z exp.:253.0787 CO

OH OH2 HO O O HO OH O HO O C C OH C R C HO R H CO R O 5b R=OH C H O + 5c/2c C H O + 5a/2a C H O + 7 5 4 9 5 3 2f R=H C H O + 8 7 2 m/z calc.: 153.0182 m/z calc.: 161.0233 13 9 2 5e R=OH C H O + m/z calc.: 135.0441 m/z exp.: 153.0298 m/z exp. (5c): 161.0394 m/z calc.: 197.0597 14 9 4 m/z exp. (5):135.0584 + m/z exp.: 197.0809 m/z calc.: 241.0495 2b R=H C7H5O3 m/z exp. (2c): 161.0441 m/z exp.: 241.0630 m/z exp. (2): 135.0627 m/z calc.:137.0233 + 2e R=H C14H9O3 m/z exp.: 137.0380 m/z calc.: 225.0546 m/z exp.: 225.0811 Figure 4. Mass spectra in positive mode of compound 2 (a) and 5 (b) and proposed MS fragmentation pathway(c)

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TOF MSMS 273.00ES+ 273.0901 100 a 163.0550

137.0423 % 255.0872 274.1110 164.0604 256.0916 145.0431 227.0918 0 m/z 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300

OH b OH OH OH OH O O H HO O HO O HO O H2O HO O

O OH OH OH + 3 C H O H 15 12 5 3+H+ C H O + + + m/z calc.: 272.0685 15 13 5 3+H C15H13O5 3c C15H11O4 m/z calc.: 273.0757 m/z calc.: 273.0757 m/z calc.: 255.0652 m/z exp.:273.0901 m/z exp.:273.0901 m/z exp.: 255.0872 HO O OH CO OH C OH HO OH HO O + 3a C7H5O3 m/z calc.:137.0233 m/z exp.:137.0423 O H OH 3+H+ C H O + 15 13 5 + m/z calc.: 273.0757 3d C14H11O3 m/z exp.:273.0901 m/z calc.: 227.0703 m/z exp.:227.0918

OH OH OH OH

C O O + + 3b C9H7O3 3b C9H7O3 m/z calc.:163.0395 m/z calc.:163.0395 m/z exp.:163.0550 m/z exp.:163.0550 Figure 5. Mass spectra in positive mode of compound 3 (a) and proposed MS fragmentation pathway (b)

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TOF MSMS 271.00ES+ 100 137.0380 a

225.0701 % 215.0865 197.0757 271.0783 141.0885 105.0529 169.0813 201.0720 226.0798 253.0670 187.0922 272.0833 149.0388 m/z 0 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300

OH b OH OH2 OH OH O O HO O HO O HO O HO O

O O OH O H2O H+ 4d C H O + 4 C15H10O5 + + 4+H+ C H O + 15 9 4 m/z calc.: 270.0528 4+H C15H11O5 15 11 5 m/z calc.: 253.0495 m/z calc.: 271.0601 m/z calc.: 271.0601 m/z exp.: 253.0670 m/z exp.: 271.0783 m/z exp.: 271.0783 CO OH OH OH OH HO O O CO HO O O O O OH C OH + OH 4e C14H9O3 + 4b C14H11O4 + m/z calc.: 225.0552 + + 4a C7H5O3 m/z calc.: 243.0652 4+H C15H11O5 m/z calc.: 137.0233 m/zexp.: 225.0701 m/z exp.: non detected m/z calc.: 271.0601 m/z exp.: 137.0380 m/z exp.: 271.0783 OH OH O CO O O CO O O O

O OH OH + O 4f C13H9O2 + + 4e C H O + 4b C14H11O4 4c C13H11O3 m/z calc.: 197.0603 14 9 3 m/z calc.: 243.0652 m/z calc.: 215.0704 m/z exp.: 197.0757 m/z calc.: 225.0552 m/zexp.: 225.0701 m/z exp.: non detected m/z exp.: 215.0865 Figure 6. Mass spectra in positive mode of compound 4 (a) and proposed MS fragmentation pathway (b)

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da Cunha, C. P. et al.

TOF MSMS 273.00ES+ 273.1023 100 a 163.0550

% 137.0423

255.0872 274.1049 164.0698 256.0974 145.0475 227.0973 0 m/z 100 120 140 160 180 200 220 240 260 280 300 320

TOF MSMS 257.00ES+ 100 137.0380 b

257.0919 % 147.0587

258.1120 119.0654 148.0672 211.0967 239.0935 0 m/z 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 R R c R OH OH OH

HO OH HO OH HO O HO O

C OH O + OH + + OH H 6+H R=OH C15H13O5 m/z calc.: 273.0757 6d/7d C H O + 6 R=OH C15H12O5 7 5 3 m/z calc.: 272.0685 m/z exp.:273.1023 m/z calc.:137.0233 7+H+ R=H C H O + m/z exp.(6d):137.0423 7 R=H C15H12O4 15 13 4 m/z calc.: 256.0736 m/z calc.: 257.0808 m/z exp. (7d): 137.0390 m/z exp.: 257.0919 H2O

R OH R R O O R

HO HO HO C HO + OH 6c R=OH C9H7O3 OH OH m/z calc.:163.0395 + + 6b R=H C14H11O3 m/z exp.:163.0550 6a R=OH C15H11O4 + + m/z calc.: 255.0652 6a R=OH C15H11O4 m/z calc.: 227.0703 7c R=H C9H7O2 m/z calc.: 255.0652 m/z exp.: 227,0973 m/z calc.: 147.0441 m/z exp.: 255.0872 + + m/z exp.: 255.0872 7b R=H C14H11O2 m/z exp.: 147.0587 7a R=H C15H11O3 + m/z calc.: 239,0703 7a R=H C15H11O3 m/z calc.: 211.0754 m/z exp.: 239,0935 m/z calc.: 239,0703 m/z exp.: 211,0967 m/z exp.: 239,0935 Figure 7. Mass spectra in positive mode of compound 6 (a) and 7 (b) and proposed MS fragmentation pathway (c)

3.4. Preparation of derivatives trimethoxyflavanone (3M), 3',4',6- trimethoxyaurone (4M) was observed the

addition of 42 mass units confirming the The methylated flavonoids were analyzed existence of three hydroxylated groups in the by mass spectrometry. In the spectra of original flavonoid. In the spectrum of 3',4',7- 3',4',7-trimethoxyflavone (2M), 3',4',7- 54 Rev. Virtual Quim. |Vol 8| |No. 1| |43-56| da Cunha, C. P. et al.

trimethoxy-5-hidroxyflavone (5M) and 3,4,4'- 2 Sullivan, G. Occurrence of Umbelliferone in trimethoxy-2'-hidroxychalcone (6M) the the Seeds of Dipteryx odorata (Aubl.) Willd. addition of 42 mass units confirmed the Journal of Agricultural and Food Chemistry existence of three hydroxyl groups located at 1982, 30, 609. [CrossRef] [PubMed] positions compatibles with conditions to 3 methylation and one involved in Gleye, C.; Lewin, G.; Laurens, A.; Jullian, J-C.; intramolecular hydrogen bond that do not Loiseau, P.; Bories, C.; Hocquemiller, R. react with diazomethane. Analogous result Acaricidal activity of tonka bean extracts. was observed in the spectrum of 4,4'- Synthesis and structure-activity relationships of bioactive derivatives. Journal of Natural trimethoxy-2'-hidroxychalcone (7M) the Products 2003, 66, 690. [CrossRef] [PubMed] addition of 28 mass units confirmed the 4 existence of two hydroxyl groups at positions Oliveros-Bastidas, A. J.; Demuner, A. J.; to methylation and one involved in Barbosa, L. C. A. Chemical characterization by intramolecular hydrogen bond. Spectra of GC-MS and phytotoxic potential of non-polar derivatives confirmed unequivocally the and polar fractions of seeds of Dipteryx structures of the isolated flavonoids. odorata (Aubl.) Willd. from venezuelan regions. Química Nova 2013, 36, 502. [CrossRef] 5 Jang, D. S.; Park, E. J.; Hawthorne, M. E.;

4. Conclusion Vigo, J. S.; Graham, J. G.; Cabieses, F.; Santarsiero, B. D.; Mesecar, A. D.; Fong, H. H. S.; Mehta, R. G.; Pezzuto, J. M.; Kinghor, A. D. Flavonoids 2, 3, 4, 5, 6 and 7 were isolated Potential cancer chemopreventive from D. odorata with sufficient purity and constituents of the seeds of Dipteryx odorata quantity for structural elucidation through (tonka bean). Journal of Natural Products interpretation of UV, NMR and MS spectra 2003, 66, 583. [CrossRef] [PubMed] data. The flavonoids 2 and 6 were identified 6 for the first time in this species. The Griffiths, L. A. On the co-occurence of methodology is effective in the isolation of coumarin, o-coumaric and melilotic acid in flavonoids and viable both economically and Gliricidia sepium and Dipteryx odorata. environmentally. Journal of Experimental Botany 1962, 15, 169. [CrossRef]

7 Godoy, R. L.; Lima, P. D.; Pinto, A. C.; De Acknowledgments Aquino, F. R. Diterpenoids from Dipteryx odorata. Phytochemistry 1989, 28, 642. [CrossRef] The authors are grateful to the Empresa 8 Brasileira de Pesquisa Agropecuária Hayashi, T.; Thomson, R. H. Isoflavones (Embrapa), Fundação de Amparo à Pesquisa from Dipteryx odorata. Phytochemistry 1974, do Estado do Rio de Janeiro (FAPERJ), 13, 1943. [CrossRef] Conselho Nacional de Desenvolvimento 9 Nakano, T.; Alonso, J.; Grillet, R.; Martin, A. Científico e Tecnológico (CNPq) and Isoflavonoids of the Bark of Dipteryx odorata Coordenação de Aperfeiçoamento de Pessoal Willd. (Aubl.). Journal of the Chemical Society, de Nível Superior (CAPES). Perkin Transactions 1 1979, 9, 2107. [CrossRef] References 10 Socorro, M. P.; Pinto, A. C.; Kaiser, C. R. New isoflavonoid from Dipteryx odorata. Zeitschriftfuer Naturforschung, B: ChemSci 1 Carvalho, P. E. R. Cumaru-ferro Dipteryx 2003, 58, 1206. [Link] odorata. Comunicado Técnico Embrapa CNPF 2010, 225, 1. [Link] Rev. Virtual Quim. |Vol 8| |No. 1| |43-56| 55

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11 Nakano, T., Suarez, M. Studies on the C.; Cardoso, M. A. R. Determinação estrutural neutral constituints of the bark of Dipteryx de flavonoides com análise de espectros de odorata. Planta Medica 1969, 18, 79. RMN de 1H 1D e reação com diazometano. [CrossRef] [PubMed] Revista Universidade Rural. Série Ciências

12 Exatas e da Terra 2006, 25, 46. [Link] 19 Grotewold, E.; The Science of Flavonoids, Carvalho, M. G.; Gomes, M. S. R; Oliveira, Springer: Ohio, 2006. [CrossRef] M. C. C; Silva, C. J.; Carvalho, A. G. Chemical 13 Coutinho, M. A. S.; Muzitano, M. F.; Costa, constituents from Piptadenia rígida Benth., S. S. Flavonoides: Potenciais Agentes Fabaceae, “angico”. Brazilian Journal of Terapêuticos para o Processo Inflamatório. Pharmacognosia, 2010, 21, 397. [CrossRef] Revista Virtual de Química 2009, 1, 241. 20 Tian, G.; Zhanga, U.; Zhanga, T.; Yang, F.; [CrossRef] Ito Y. Separation of flavonoids from the seeds 14 Cazarolli, L. H.; Zanatta, L.; Alberton, E. H.; of Vernonia anthelmintica Willd by high- Figueiredo, M. S. R. B.; Folador, P.; Damazio, speed counter-current chromatography. R. G.; Pizzolatti, M. G.; Silva, F. R. M. B. Journal of Chromatography A 2004, 1049, Flavonoids: Prospective Drug Candidates. 219. [CrossRef] Mini-Reviews in Medicinal Chemistry 2008, 8, 1429. [CrossRef] [PubMed] 21 Wang, J.; Wang, N.; Yao, X.; Kitanaka, S. 15 Ravishankar, D.; Rajora, A. K.; Greco, F.; Structures and Anti-histamine Activity of Osborn, H. M. I. Flavonoids as prospective Chalcones & Aurones Compounds from compounds for anti-cancer therapy. The Bidens parviflora Willd. Asian Journal of International Journal of Biochemistry & Cell Traditional Medicines 2007, 1, 23. [Link]

Biology 2013, 45, 2821. [CrossRef] [PubMed] 22 16 Harbone, J. B.; Williams, C. A. Advances in Park, Y.; Moon, B-H.; Lee, E.; Lee, Y.; Yoon, flavonoid research since 1992. Y.; Ahn, J-H.; Lim, Y. Spectral Assignments and Reference Data. 1H and 13C-NMR data of Phytochemistry 2000, 55, 481. [CrossRef] 17 hydroxyflavone Derivatives. Magnetic Molnár-Perl, I.; Füzfai, Z. Chromatographic, capillary eletrophoretic and capillary Resonance in Chemistry 2007, 45, 674. eletrochromatographic techniques in the [CrossRef] [PubMed] 23 analysis of flavonoids. Journal of Zhao, X.; Mei, W.; Gong, M.; Zuo, W.; Bai, Chromatography A 2005, 1073, 201. H.; Daí, H. Antibacterial Activity of the [CrossRef] [PubMed] Flavonoids from Dalbergia odorifera on 18 Carvalho, M. G.; Junior, J. G. R.; Cavatti, L. Ralstonia solanacearum. Molecules 2011, 16, C.; Suzart, L .R.; Cornelius, M. T. F.; Silva, V. 9775. [CrossRef] [PubMed]

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