Addressing the phytochemical prospection of thermally treated Eucalyptus grandis wood

This work aimed to investigate phytochemical prospection in treated and untreated wood of Eucalyptus grandis to understand the dynamics of extractives in relation to heat treatment. Samples of E. grandis wood were collected and grouped into external and internal regions. Half of the samples from each region were submitted to heat treatment at 190 oC. From the treated and untreated samples, phytochemical tests were performed to detect classes of metabolites present in the E. grandis raw wood, hydrophilic extract and lipophilic extract. Phytochemical analysis detected the presence of alkaloids, phenolic compounds and triterpenoids in all hydrophilic extracts of the studied species. Presence of flavanonols, flavanones and saponins was detected only in the internal region of the wood. Tannins, leucoanthocyanidins, triterpenoids and saponins were influenced by heat treatment. The classes of flavonoids, xanthones and alkaloids are the most resistant to heat treatment. The phytochemical analysis made it possible to identify a new class of extractives that emerged after heat treatment, flavones.


Introduction
Wood is a basic, versatile and renewable resource widely used in various applications, and remains indispensable to everyday life and human culture due to its aesthetic aspect and characteristic properties. Despite its great benefits, this lignocellulosic material has undesirable properties, such as dimensional instability, hygroscopicity and susceptibility to biological degradation, thus limiting its use in several applications (Hoseinzadeh et al., 2019;Chien et al., 2018;Borges & Quirino, 2004). To solve these problems, chemical approaches have been used to increase dimensional and thermal stability, and biological resistance in recent years (Yang et al., 2014;Hung et al., 2010;Evans, 2009;Wu et al., 2004). However, Development, v. 9, n. 11, e94591110537, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i11.10537 4 chemical modification is costly and time consuming, requiring cumbersome processing. Therefore, heat treatment, a physical modification, has attracted attention in the academic and industrial spheres due to its economic and sustainable aspect.
Heat treatment is said to be an effective process to improve physical and aesthetic properties, besides being considered a method of wood preservation. It consists of applying high temperatures ranging from 160 to 250 °C, with no use of toxic chemical agent, thus being an environmentally acceptable and technically more attractive treatment (Sandberg et al., 2017;Zhu et al., 2014;Poncsak, 2011). Some studies have shown that high temperature treatment reduces the moisture and dimensional shrinkage of wood (Poncsak et al., 2009;Esteves et al., 2008). On the other hand, thermally treated wood suffers changes in chemical compositions, i.e., there are changes in structural components such as cellulose, hemicellulose, lignin and, the non-structural ones, such as natural extractives from wood cell wall (Zanuncio et al., 2014).
The extractives are non-structural components of wood, specifically concentrated in the core, and commonly produced by the tree as defensive compounds against environmental stresses, besides having great influence on wood properties, being quite affected by the action of heat (Klock et al., 2005;Taylor et al., 2012;Singh et al., 2012;Kirker et al., 2013). Most of the original wood extractives disappear with the heat treatment, especially the more volatile compounds, while new extractives appear as structural polymer degradation products. In this perspective, a better understanding of the differences between extracts during heat treatment is necessary to enable their use in different fields, such as pharmaceutical or cosmetic (Esteves et al., 2008).
Phytochemical studies are important mainly when not all chemical studies of certain plant species are available, aiming to know the chemical classes and evaluate their presence in them, identifying groups of relevant secondary metabolites (Carvalho, 2009;Simões et al., 2001). In addition, phytochemical analysis in plants confirms the presence of alkaloids, steroids, flavonoids, coumarin, saponins, glycosides and phenols (Gröcer et al., 1998). The studies about wood extractives have been stimulated in the discovery and characterization of new chemical structures, taxonomic classification of species, tree growth processes, obtaining of new products and by-products of commercial value and the determination of problems related to some uses of wood (Santos et al., 2017;Barbosa et al., 2005;Klock et al., 2005).
Therefore, the detailed analysis of the chemical composition, and particularly of the less abundant and studied components present in non-modified and thermally modified wood, is a relevant issue to be studied. Thus, the present work was aimed at investigating the Research, Society and Development, v. 9, n. 11, e94591110537, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i11.10537 5 phytochemical prospection of the thermally modified Eucalyptus grandis wood, in order to understand the dynamics of the extractives in relation to the heat action.

Sampling and preparation of material
The wood in this study came from six trees of Eucalyptus grandis Hil Ex Maiden with 23 years of age. These trees were provided by the company Quinvale, located in Barra do Piraí, Rio de Janeiro State, Brazil, whose geographical coordinates are 22°43'23" latitude (S) and 44°08'08" longitude (W) and at an average altitude of 446 meters. The woods were processed, generating boards of 2.5 x 12.5 x 50.0 cm, and only those considered free of defects were selected, totaling 104 samples ( Figure 1). In order to evaluate the action of heat on the wood and its influence on the chemical composition, the wood samples were divided into external and internal regions, the former closer to the bark and the latter closer to the pith. For the thermal treatment, a program of 6 hours and 30 minutes program with four stages was adopted: (1) temperature increase up to

Preparation of organic extracts
The woods of E. grandis were processed in a Willey-type knife mill. To obtain a greater homogenization of the particles, the fraction sieved through the 40 mesh and 60 mesh was used, according to the procedures pointed out by the standard TAPPI T204 cm-97 (TAPPI, 1997). To obtain the organic extracts of the material, it was conditioned to a paper filter cartridge of 15.0g of the wood samples treated and not thermally treated. Then, the material was submitted to the extraction cycle using cyclohexane, ethyl acetate and methanol solvents for 12 hours in a Soxhlet apparatus, using 400 mL of each solvent (Abreu et al., 2006). To obtain the organic extract, the content from the extraction was submitted to separation of each solvent through the application of rotavapor, responsible for the evaporation of the solvent, to obtain a concentrated solution of wood extractives. The concentrates were transferred to a container until reaching the complete evaporation of the solvent at room temperature.

Phytochemical evaluation
Phytochemical tests for the identification of non-structural components present in woods in this study were performed based on methodologies proposed by Costa (1995), Matos (1997) and Rodrigues et al., (2010). The analyses were carried out from raw wood, hydrophilic extract (methanol) and lipophilic extract (cyclohexane). All tests were performed in duplicate.

Phytochemical prospection of thermally modified E. grandis wood
When performing the phytochemical analysis with the thermally modified wood, the presence of alkaloids was observed in the heartwood and sapwood regions ( Figure 5). The result is obtained after the appearance of flocculent precipitates after the addition of Dragendorff's reagent. With the thermal treatment, in both extracts (lipophilic and hydrophilic), most of the extractives disappeared from the wood ( Figure 5). These findings are in accordance with those of Nuopponen et al. (2003), who reported that fats and waxes are the first compounds to disappear from wood after thermal treatment, followed by fatty acids and resins. Some authors, such as Esteves; Graça and Pereira (2008); Pierre et al. (2011), andSilva (2012), Zanuncio et al. (2014), show that with the increased rigor of the heat treatment, extractives decrease at temperatures above 170 °C, mainly at 240 °C. Hakkou et al. (2006), when conducting a study with thermally treated Fagus sylvatica (beech) wood, observed that the alterations in the contents of extractives began to be significant in the thermally treated wood above 200 °C, this behavior is due to the beginning of the decomposition of hemicellulose (Alén et al., 2002;Hakkou et al., 2006), that is, the quantity of extractives reaches a peak around 240 °C and decreases with the increase in temperature, which leads to their volatilization.
After the heat treatment, the presence of specific classes of extractives (flavones, flavonols and xanthones) in the internal region of E. grandis wood can be verified ( Figure 5).
These chemical compounds can come from the degradation of other classes of extractives, allowing their appearance after the wood is subjected to the action of heat. Among these components, flavonols and xanthones are more resistant to heat treatment. Sung et al. (2019), in a recent study, provided information about thermally treated citrus fruit peel, and their results showed that heat treatment contributed to the significant release of phenolic and flavonoid compounds present in the peel. According to Kabera et al. (2014), flavonoids are widely distributed in plants and various functions are associated with these secondary metabolites, for example, the coloring of flowers, producing the yellow, red or blue pigmentation of petals, and serving as an attraction for pollinating animals. Negi et al. (2013) highlights the importance of the pharmacological properties of xanthones and flavonoids, emphasizing the similarity of their structures and chromatographic behavior in plants. In addition, this class may have a certain connection with the coloring of the wood because, after the heat treatment, it undergoes a change in its color pattern, becoming dark. region; E = external region; D = detected; ND = not detected; NP = not performed. Research, Society and Development, v. 9, n. 11, e94591110537, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i11.10537 13

Conclusions
The phytochemical investigation detected the presence of alkaloids, phenolic and triterpenoid compounds in the hydrophilic extract of the untreated E. grandis wood. Saponins were detected only in the hydrophilic extract of the internal region.
The extractives of E. grandis wood were influenced by the heat treatment. Almost all the extractives were volatilized, with alkaloids and flavonoids being more resistant classes and, due to the chemical reactions, a new class of extractives (flavones) was found in the thermally treated wood. Further studies are recommended to be carried out to carry out a more detailed characterization of the metabolites that resist heat treatment and understanding the action of heat on these compounds.