Studies on polyphenols and lignin of Astronium urundeuva wood

Journal of the Brazilian Chemical Society

Drumond, Mariza G.¹  Universidade Federal de Uberlândia, Uberlândia, Brasil¹  Morais, Sérgio A.L.¹  Piló-Veloso, Dorila¹  Nascimento, Evandro A.¹  Queiroz, Carla R.A.A.¹  Universidade Federal de Minas Gerais, Belo Horizonte, Brazil¹  Fundação Centro Tecnológico de Minas Gerais, Belo Horizonte, Brazil¹ 

关键词: lignin;    polyphenols;    Astronium urundeuva;    cerrado vegetation;   

DOI  :  10.1590/S0103-50531999000600005

学科分类：化学（综合）

来源: SciELO

【 摘 要 】

Astronium urundeuva wood is well known due to its very good mechanical, biological and chemical resistance and therefore it is widely used in Brazil1. In spite of this, we could not find any systematic study on the chemical composition of this wood in the literature. In order to investigate whether there is a relationship between resistance and chemical constitution, we have initiated studies to characterize the polyphenols and the lignin structure of this wood. To obtain a lignin mostly free of polyphenol contamination, we employed the Björkmann procedure adapted for Eucalyptus grandis wood cultivated in the central region of Brazil. This wood also contains a large amount of polyphenols2. Polyphenol studies were carried out using classical chemical techniques3-7 associated with modern high performance liquid chromatography (HPLC)8-13 and high resolution liquid and solid state 13C-NMR11,14,15 and FTIR spectroscopies. This work presents the results of our studies on the Astronium urundeuva wood composition.  Materials and Methods Astronium urundeuva woodA stem of a thirty year old tree from Uberlândia (Triângulo Mineiro) was disk cut and air dried for 12 months.ExtractsA ball-milled wood sample (100 g) was sequentially extracted in a soxhlet apparatus with benzene, benzene: ethanol (2:1), chloroform, and water. The isolated extracts were dried in an oven at 70 °C for 12 h and the residual air-dried wood powder (A) was used for MWL preparation. Another sample of ball-milled wood (50 g) was stirred with 300 mL of methanol in a beaker at room temperature for 24 h and, then, the mixture was filtered and the methanol evaporated under reduced pressure, at 40 °C, yielding the extract B. A third sample of ball-milled wood (50 g) was submitted to an extraction with methanol: water (80:20) in a beaker at room temperature with stirring for 24 h. Then the mixture was filtered and the methanol was evaporated under reduced pressure, at 40 °C. The remaining aqueous extract (C) was divided in four fractions and freeze-dried for its conservation. One fraction of C was used for total phenols analysis and another for proanthocyanidins determination; a third was extracted with ethyl ether, and the fourth fraction was used in the HPLC analysis as well as the ethereal extract.High performance liquid chromatographyHPLC analysis was carried out in a chromatograph equipped with diode array detector and Hypersil ODS (200 x 4 mm i. d.) column protected by a precolumn of the same material. Two solvent mixtures were employed for elution: X: MeOH:H3PO4 (999:1) and Y: H2O:H3PO4 (999:1) and the gradient profile was: 0 min - 40 min, 20% - 100% X; 40 min - 45 min, 100% X. The flow rate was 1 mL/min and column oven temperature 30 °C. Detection was carried out at 270 ± 15 nm, 325 ± 75 nm and 365 ± 2 nm. The compound identification was based on a private library of UV spectra of phenolics and flavanoids and on retention times.Total phenol contentsTotal phenol content was determined by the Folin-Ciocalteu method2. 2,5 mL of Folin-Ciocalteu reagent (Fluka, diluted 10 times by water) and 2 mL of aqueous solution of sodium carbonate (75 g L-1) were added to 0.5 mL of the aqueous extract (C), and the mixture was kept 5 min at 50 Â°C. After cooling, absorbance was measured at 760 nm. Aqueous solution of gallic acid (2 mg – 40 mg) was used as standard. The molar absorptivity obtained in our conditions was 22.3 x 103.Proanthocyanidin contentsProanthocyanidin determination was carried out by the vanillin method4. 1 mL of the aqueous extract was mixed with 2 mL of a freshly prepared vanillin solution (1 g/ 100 mL of 70% H2SO4) and maintained 15 min at 20 °C. The absorption was measured at 500 nm. Calibration was performed with catechin aqueous solutions (2 mg - 40 mg mL-1) and a molar absorbance of 34.3 x 103 was attained in our conditions.Anthocyanidin contents3-4 To a reaction vial were added 6 mL of concentrated HCl:n-BuOH (2:3, v v-1), 1 mL of aqueous extract and 0.9 mg of FeSO4.7H2O. The vial was closed and left in the oven for 40 min at 90 °C. After cooling, 0.1 mL of reaction mixture was dissolved in 1 mL of MeOH:H2O (1:1) and injected in the HPLC chromatograph.Milled wood ligninMWL was prepared from A according to Björkmann technique16 adapted by us to Eucalyptus grandis wood: the dried wood powder (100 g) was extracted in a soxhlet apparatus with ketone-water (9:1) and the organic solvent was evaporated under reduced pressure at 70 °C. After that, the aqueous mixture was acidified with diluted HCl until pH 2 and the precipitated lignin was filtered and washed with a small amount of water. The lignin was dried at 70 Â°C for 12 h.Easily and hardly hydrolysable polysaccharidesThe polysaccharides were determined according to the Kiesel-Semiganovski method17: 4.0 g of A were refluxed with 2% HCl for 3 h and filtered. The residue obtained was washed and dried at 105 °C. The weight difference corresponds to the easily hydrolysable polysaccharides (EHP). Then, the residue was treated with 80% H2SO4 for 2 h at room temperature and, thereafter, it was diluted up to 1 L and refluxed for 3 h. The mixture was filtered and the residue was washed and dried at 105 °C. The weight loss was due to the hardly hydrolysable polysaccharides (HHP).HolocelluloseHolocellulose content was determined applying Browning procedure18 to non-extracted ball-milled wood. The obtained holocellulose still showed a pale brown color.NMR measurementsSolid state 13C-NMR spectra were recorded on a BRUKER DRX spectrometer operating at a frequency of 100.6 MHz using the pulse sequence VACP, pulse delay 0.5 s, acquisition time 25 ms, rotor speed 4000 rpm, and 1520 scans were collected. Liquid 13C-NMR spectra were recorded at the same frequency using DMSO-d6 as solvent and pulse sequence ZGPG30, power gated and angle of 30° at the principal pulse; pulse delay was 1.0 s and 10240 scans were collected.FTIR spectraThey were recorded on a PERKIN-ELMER SPECTRUM 1000 spectrometer in KBr pellets.  Results and Discussion Table 1 presents the composition of Astronium urundeuva wood. The total of substances extracted by benzene, benzene: ethanol (2:1, v v-1), water and chloroform was very large 18.19%. However, when only methanol was used as extraction solvent, at room temperature, the extract yield was still greater: 23.85%. The sum of extracts, Klason lignin, EHP, plus HHP was 95.70%. Holocellulose plus Klason lignin and extracts reached, however, 105.03%. These discrepancies are considered normal18 and reflect the difficulties in analyzing woody materials.   In spite of Astronium urundeuva wood being very hard, its content of structural polymer cellulose is low as can be verified from the low content of hardly hydrolysable polysaccharides. However, the relatively high content of lignin and extractives is not sufficient to impart resistance to wood species against chemical and biological attacks. For instance, Eucalyptus grandis wood cultivated in the same region also contains approximately 40% of lignin plus extracts and, nevertheless, it is well known that this wood easily degrades under a tropical climate. The structure of lignin or more probably the composition of phenolics could account for the great resistance of Astronium urundeuva. Figure 1 presents the IR spectrum of the MWL in KBr pellet. The band profile in the finger print region is typical of lignins19-20.   Figure 2 shows the IR spectrum of the methanolic extract B. The band distribution is characteristic of tannins21. In addition, the great intensity of the band at 1513 cm-1 suggests that B is contaminated by lignin, as was the

【 授权许可】

Unknown