BIOCHEMICAL FEATURES OF ORGANIC EXTRACTIVES FROM Eucalyptus AND Corymbia WOODS USING ETHANOL AS A SOLVENT

This study aims to evaluate chemical characteristics, antioxidant and antibacterial activities of organic compounds extracted from three Eucalyptus wood and Corymbia maculate wood using ethanol as a solvent. To obtain the ethanolic extracts, 15 g of a powdered wood sample was mixed with 150 mL of ethyl alcohol 99 % PA by constant mechanical stirring, which was further magnetically mixed at 60 oC for 24 h. The extractives were analyzed utilizing percent yield, Fourier-transform infrared spectrum, inhibitory index (measured after antimicrobial tests), antioxidant activity, and condensed tannins content. The Eucalyptus dunnii extract showed the highest percent yield. The infrared spectra of all the extractives presented similar profiles, with remarkable bands ascribed to the presence of lipophilic extracts, sterols, fatty acids, and other hydroxylated substances, such as carbohydrates and phenolic compounds. In all cases, the higher the concentration of the 1Federal University of Pelotas, Postgraduate Program in Materials Science and Engineering, Pelotas, Rio Grande do Sul, Brazil. 2Federal University of Rio Grande do Sul, Postgraduate Program in Metallurgical and Materials Mining Engineering, Porto Alegre, Rio Grande do Sul, Brazil. 3Federal University of Pelotas, Postgraduate program in Biochemistry and Bioprospecting, Pelotas, Rio Grande do Sul, Brazil. ♠Corresponding author: gattodarci@gmail.com Received: 22.04.2020 Accepted: 30.05.2021 Maderas. Ciencia y tecnología 2021 (23): 58, 1-8 Universidad del Bío-Bío 2 extractive was, the higher the antioxidant activity was. The antioxidant activity of Eucalyptus saligna extract stood out since overcame that of the positive control (ascorbic acid). Regarding the condensed tannins content, that extract from Eucalyptus grandis excelled.


INTRODUCTION
Wood is composed of structural macromolecules of high molecular weight, namely cellulose, hemicellulose, and lignin. Aside of them, extractives are low molecular weight substances from wood. They are often hydrophobic or lipophilic secondary metabolites and do not influence in the tree growth (Saha Tchinda et al. 2018).
The amount and composition of the wood extractives may vary in function of both radial and axial position in the wooden trunk. Moreover, factors associated with the forest also play significant roles, such as conditions related to both tree growth and wood storage (Saha Tchinda et al. 2018). Some wood extractives are soluble in both water and neutral organic solvents and, besides of that, most of them are located in bark (Morais et al. 2005).
According to Valette et al. (2017), the wood extractives are biosynthetized in trees to avoid injuries attributed to biotic (like fungi and insects) and abiotic (like rain, sunlight, wind, among other) agents. Other authors also reported strong correlations between the content of some wood extractives and the wood durability (Kirker et al. 2015, Pometti et al. 2009).
Eucalyptus-based woods present several types of extractives, including essential oils, fatty acid esters, as well as small amounts of inorganic substances (Santos et al. 2016). The essential oils from Eucalyptus woods are widely applied in chemical, cosmetic, and pharmaceutic industries (Albuquerque et al. 2017). The high added value of these compounds also may encourage researches on their production and characterization.
Extractives and essential oils obtained from plants are important sources of natural antioxidants (Hayat et al. 2010, Luna et al. 2010. Some recent studies reported secondary metabolites in the composition of some wood extractives, such as phenols (Jiang et al. 2017), terpenoids (Andrew et al. 2013), and flavonoids (Takahashi et al. 2004). Yamakoshi et al. (1992) and Nakayama et al. (1990) affirmed that extractives obtained from Eucalyptus macrocarpa and Eucalyptus perriniana were effective against the proliferation of some Gram-positive bacteria (namely Staphylococcus aureus and Bacillus subtilis). Moreover, Takahashi et al. (2004) studied extractives obtained from Eucalyptus leaves and flavonoids from Corymbia maculate wood and reported great performances against the growth of microbes and fungi.
For Wu et al. (2019), a feasible use of a wood extractive must be done after an effective and cheap extraction process. Ethanol is a commonly used solvent for obtaining several compounds from wood and other vegetable sources, such as extractives and/or essential oils (Hofmann et al. 2015). The ethanol soluble compounds from wood include fatty acid esters, long-chain alcohols, steroids, phenolic compounds, and glycosides (Sjöström and Alén 1998, Gullichsen and Paulapuro 1999, Sun and Sun 2002, Morais et al. 2005. This solvent can be obtained from renewable sources (like sugar cane) using well-known and cheap routes. This is also commonly combined with other organic solvents, like methanol and benzene, which may allow an improved extraction yield or even a selective extraction of particular substances (Abdul Mudalip et al. 2013, Peng et al. 2017).
This study aimed to evaluate chemical features and both the antioxidant and antibacterial activities of ethanol extractives obtained from three Eucalyptus woods and a Corymbia maculate wood.

Raw materials and ethanol extractions
Fifteen 22-28-year-old exotic trees from Eucalyptus dunnii, Eucalyptus saligna, Eucalyptus grandis, and Corymbia maculate were felled in homogeneous forests located in Tapes/Brazil. Wood flakes cut from the sapwood of each specie were crushed until pass through a 40 mesh sieve and be retained in a 60 mesh sieve.
A high purity ethanol solution (99 %) was purchased from Sigma Aldrich. 15 g of powdered wood sample and 150 mL of ethanol were placed into 250 mL Erlenmeyer and kept under magnetic stirring at 60 ºC for 24 h. After that, the solvent was evaporated using a Heidolph Rotary Evaporator (Laborota 4002 equipment) and then the flask was hermetically closed and stored under -4 ºC for further analyses. Yield of the ethanol extraction was calculated according to TAPPI T 204 cm-97 (1997), as shown in Equation 1. Besides, chemical groups were evaluated by Attenuated Total Reflection-Fourier transform infrared spectroscopy (ATR-FTIR), in which 32 scans were performed at the 4000 cm -1 to 600 cm -1 range, 4 cm -1 resolution, and 2 mm·s -1 scanner velocity, Equation 1.
Where: Y is the percent yield, m e is the mass of the flask plus the evaporated extract, m f is the mass of the flask, and m w is the mass of the wood sample.

Test organisms
Antimicrobial activity was evaluated using gram-positive standard strains from Staphylococcus aureus (ATCC 25923) and Enterococccus faecalis (ATCC 51299), as well as gram-negative standard strains from Escherichia coli (ATCC 25922) and Salmonella typhimurium (ATCC 14028). These microorganisms were provided by the Oswaldo Cruz Foundation (FIOCRUZ). The evaluated strains were kept in Mueller-Hinton agar at 4 °C and reactivated prior to the antimicrobial evaluation.
The antimicrobial assays with these bacteria were carried out following the broth microdilution method, indicated by Clinical and Laboratory Standards Institute (CLSI 2018). Extractives were diluted in a 0,5 % dimethyl sulfoxide (DMSO) solution and then placed into 96-well microplates (Kasvi®), reaching concentrations that ranged from 0,0078 mg·mL -1 to 1 mg·mL -1 . 100 μL of the DMSO-based solution was used as emulsifier to control the overall sterility and a mixture of DMSO and extractive (ratio of 1:1) was used to control the microbial growth. The bacteria were suspended in a 0,9 % saline solution until reach 0,5 McFarland standard. For that, the optical density was accessed using a UV-VIS spectrophotometer adjusted at 630 nm wavelength until reach an absorbance of 0,08-0,1 range. Then, the bacterial solution was adjusted to a final concentration of 3E4 colony-forming unit mL -1 and the microplates were incubated at 37 ºC for 24 h. Afterwards, a 20 mL of 0,02 % Resazurin (acquired from Sigma Aldrich) was used to reveal the microbial growth in each well. The bacteria were indicated in pink colour and its minimal inhibitory concentration (MIC) was determined.

Antioxidant activity
The antioxidant activity was determined using 2,2-diphenyl-1-picrylhydrazyl (DPPH) as free radial, following that methodology described by Brand-Williams et al. (1995). Variable concentrations of each extractive were progressively incorporated in a 300 mL of a methanol-DPPH solution with 2,7 mL of methanol and incubated for 15 min in darkness. After that, a UV-VIS spectrum was obtained in a UV-M51 equipment (Bel Ptotonics brand) adjusted for a wavelength of 517 nm. Both a reference sample and two control samples were also prepared and measured. The latter one had 2,7 mL of methanol and 300 μL of DPPH, while both ascorbic acid and rutin (Vetec brand) were used as control samples at the following concentrations: 1,0 mg.mL-1, 0,5 mg.mL-1, 0,25 mg.mL-1 and 0,15 mg.mL-1. All analyses were performed in triplicate. Inhibition of the DPPH radical at different extract concentrations was calculated using Equation 2.
Where: A DPPH is the absorbance of the DPPH radical without samples, A Extract is the absorbance of extracts mixed with DPPH radical and A Blank is the absorbance of ethanol.

Condensed tannins content
The condensed tannins content was determined according to the vanillin method, described by Morrison et al. (1995). Firstly, a vanillin solution was prepared using equal volumes of 1 g of vanillin in 100 mL of methanol and 8 mL of concentrated HCl in 100 mL of methanol. This solution was incorporated with 0.1 mL of extractive sample (with a concentration of 50 mg·mL -1 ) and 0,9 mL of methanol. Then, it was left in water bath for 20 min and the absorbance was read at 500 nm. All analyses were performed in triplicate and the corrected spectra were converted to catechin equivalents from standard curves (Missio et al. 2017).

Statistical analyses
The data were arranged in a completely randomized design. Normality of the data and homogeneity of variances were verified by the Shapiro-Wilk and Levene tests, respectively. Whenever the null hypothesis was rejected, a Fisher test (at a confidence level of 95 %) was performed to compare the means.

RESULTS AND DISCUSSION
The studied wood species presented a wide range regarding the values of yields of ethanol extractions (Table 1), which are in agreement with a previous work by Gomide et al. (2010), that reported that the main chemical compounds from wood may vary among genders, species, parts from a same tree, as well as they are affected by microclimatic factors, soil conditions, tree age, and so on. The Eucalyptus dunnii presented the highest yield of extractives, which is ascribed to some of its intrinsic features, like anatomical, chemical and macroscopic properties. The obtained values for yield of extractives were higher than those reported by Silvério et al. (2006). These authors compared different solvents applied for extractions performed with a Eucalyptus grandis wood and reported the following yields: 2,17 %, 0,53 %, 0,55 %, and 2,48 %, which are respective to extractions with acetone, chloroform, dichloromethane, and an ethanol:toluene (1:2) mixture. Figure 1 presents infrared spectra for all extractives, in which prominent peaks were found at 1040 cm -1 , 1089 cm -1 , and 1386 cm -1 , which represent C-O bonds. A remarkable slope was visualized at 2978 cm -1 , which represents C-H bonds. The peak at 3326 cm -1 appears in all the spectra and can be ascribed to O-H bonds from some alcohols (like β-sitosterol) commonly found in wood extractives. This also indicates that probably other hydroxylated compounds may be present, like phenolic groups (Gullichsen andPaulapuro 1999, Morais et al. 2005). Similarly shaped spectra were reported by Abdul Mudalip et al. (2013), who carried out an elucidative study on hydrogen bonds from a pure ethanol solution. The above mentioned spectra show peaks at 2984 cm -1 , 2978 cm -1 , 2971 cm -1 , 2967 cm -1 , 2899 cm -1 , 2897 cm -1 , and 2879 cm -1 correspondent to C-H stretching, as well as CH, CH2, and CH3 groups commonly found in aliphatic compounds, like fatty acids, fatty esters, and long-chain alcohols. Silverstein et al. (2002) found the same organic compounds and associated them to peaks at 2954 cm -1 , 2919 cm -1 , and at 2850 cm -1 . Other minor peaks at 1748 cm -1 , 1741 cm -1 , and 1735 cm -1 are associated to C=O bonds from esters (Sjöström and Alén 1998). Figure 2 presents the different % inhibition values of the extracts from Eucalyptus and Corymbia maculate in function of their concentrations. The E. saligna extract showed the greatest inhibition of the DPPH radical, followed by the extract of Corymbia maculate, E. dunnii and E. grandis. Regarding the positive control, E. saligna extract showed greater inhibition than ascorbic acid (except at the concentration of 0,125 mg·mL -1 ), and in relation to the positive control rutin, all extracts showed greater inhibition in all concentrations. Also, the extractive from the Eucalyptus saligna presented a higher inhibition than the ascorbic acid, which may be attributed to its high amounts of both phenolic and hydroxyl groups.  Schumack et al. (2018) ascribed significant inhibitory actions to commercial essential oils extracted from Eucalyptus ssp. woods against two bacteria, namely E. coli and S. aureus. Regarding the same bacteria, Estanislau et al. (2001) reported similar results for essential oils extracted from both Eucalyptus grandis wood and Eucalyptus saligna wood. These essential oils were obtained by hydro-distillation and differ from those extracts studied here since wood extractives obtained by ethanolysis do not encompass essential oils (Silveira et al. 2012).
It is expected that those extractives with high contents of phenolic, fatty acids, and steroids may present high antioxidant activities. Chang (2000) and (Gomes and Canhoto 2003) reported phenolic compounds for similar wood species. Bio-based antioxidants can replace synthetic antioxidants, when equivalent or superior inhibitions of enzymatic lipid oxidation are reached (Bandoniene and Murkovic 2002). Natural antioxidants can act as inhibitors against free radicals, chelators, and oxygen scavengers and these compounds include flavonoids, phenolic acids, terpenes, tocopherols, phospholipids, and polyfunctional organic acids (Gómez 2003, Ribeiro 2007. Wood extractives with these substances are known due to their antioxidant, anti-inflammatory, anticarcinogenic and antimicrobial characters (Hras et al. 2000).
The extractives from Eucalyptus grandis wood presented the highest condensed tannin content, followed in a decreasing order by Eucalyptus saligna wood, Corymbia maculate wood, and Eucalyptus dunni wood (Table 2). That result for the Eucalyptus grandis wood indicates its high potential for particular applications, when certain features are needed, such as antibacterial, antiviral and protein-binding (Salminen 2018, Zeller 2019. The condensed tannin content is directly related to the amount of phenolic groups. In plants, condensed tannins are obtained by liquid-solid extractions and are composed of sugars, proteins, lipids and some minor phenolic compounds (Brown et al. 2017).

CONCLUSIONS
Among the wood species, the Eucalyptus dunnii wood presented the highest extractives content after the ethanol extractions. All the wood extractives yielded such infrared spectra, which were similar to lipophilic extractives, like sterols, fatty acids, and other hydroxylated substances, such as some carbohydrates and phenolic compounds. The antioxidant activities were directly related to the extractives content and the Eucalyptus saligna wood stood out, since its inhibition was higher than that of the ascorbic acid. The wood extractives showed a great potential related to the production of condensed tannins, especially the Eucalyptus grandis wood. On the other hand, there were no promise results regarding the reported antimicrobial activities and, in this sense, we recommend further studies dealing with higher concentrations of wood extractives.