The changes of extractive contents of young Swietenia mahagoni (L.) Jacq trees during heartwood formation

Sodium carbonate, sodium hydroxide, aluminum chloride, and Folin-Ciocalteu’s phenol reagent were purchased from Merck (Darmstadt, Germany), while standard components of gallic acid and D-glucose were purchased from Sigma-Aldrich (Chemie GmbH, USA).

The powder (0.8 to 1.5 g) was successively extracted by n-hexane and methanol solutions for 6 hours in a Soxhlet apparatus, while hot water refluxing in separating the extraction for 3 hours. The number of percolations during the extraction was 8 cycles per hour. Furthermore, the solvent was evaporated by a rotary evaporator, then the extract was dried in an oven (103±2°C) and the extractive content was quantified.

The total phenolic content (TPC) was determined by the Folin-Ciocalteu method (Singleton et al., 1999). 2.5 ml of diluted Folin Ciocalteu phenol reagent and distilled water (1:9, v/v) were mixed with 0.5 ml of the methanolic extract (0.25 mg/ml) in a 9 ml glass. After an interval of 2 min, 7.5% aqueous Na₂CO₃ (2 ml) was added, and the mixture was allowed to stand for 30 min at ambient temperature. Then, the absorbance for testing of standard solutions was performed against the blank at 765 nm with a Visible spectrophotometer (model VIS-WPA S800⁺). In addition, TPC was performed as the mean ± standard deviation of five tree replication measurements and was expressed as milligrams gallic acid equivalents (mg GAE/g dried extract).

Total soluble polysaccharides (TSP) were measured according to DuBois et al. (1956). Meanwhile, the hot water extract was dissolved in distilled water (0.25 mg/ml). 1 ml of the solution was mixed with 1 ml phenol reagent solution 5% (5 g in 100 ml in distilled water) before adding 5 ml of sulfuric acid (98%). Furthermore, the mixture was allowed to stand for 20 min at room temperature, and the absorbance for testing of standard solutions was performed against the blank with the same procedure (without extract) at 490 nm, using a Visible spectrophotometer (model VIS-WPA S800⁺). The TSP was performed as the mean ± standard deviation of five tree replication measurements and was expressed as milligrams glucose equivalents (mg GE/g dried extract).

The data were statistically handled using the SPSS program (version 16 IBM, New York, USA). Analysis of variance (ANOVA) was conducted and the significant differences were set at a 95% confidence level. Also, two-way ANOVA was applied to determine the effect of tree age and axial position (bottom and top) on the extractive content, TPC, and TSP. All the obtained data were normally distributed after analysis, and Duncan’s test was performed to evaluate groups that differ.

The average value of n-hexane, methanol, and hot water extractive content from bottom to top parts were 2.39 to 0.71%, 7.81 to 1.61%, and 5.21 to 1.01%, respectively (Table 3). In addition, Figure 2a showed that the total extractive content ranged from 3.93 to 13.7% of dried wood. Methanol solution has the highest contribution of total extractives, it ranged from 25 to 70.2% based on extractive weight (Figure 2b). Meanwhile, the value for hot water and n-hexane ranged from 13.6 to 50.3% and 10 to 29%, respectively.

Figure 2

Figure 3 showed that the TPC in the methanol extracts ranged from 107.3 to 480.2 mg GAE/g dried extract. Furthermore, the amount of TSP ranged from 160 to 688 mg GE/g dried extract with the mean values of 440.3 mg GE/g dried extract (Figure 4).

Figure 3

Figure 4

The ANOVA of extractive contents showed significant tree age and axial position interactions (Table 2). Prior to heartwood formation (1 to 3 years) (Table 3) generally the extractive content increased with the increasing tree age at the bottom of the tree, where the content of hexane and hot water extract increased significantly from the age of 1 to 2 and 1 to 3 years. Meanwhile, the methanol extract slightly decreased from the age of 2 to 3 years, after slightly increasing from the age of 1 to 2 years. Furthermore, the total extractive content increased significantly from the age of 1 to 3 years. At the top of the tree, hexane and hot water extract decreased significantly (hot water extract) at the age of 3 years, although slightly increased previously at the age of 1 to 2 years. Meanwhile, a significant decrease was found in the methanol extract (from tree age of 1 to 2 and 1 to 3 years) and in the total extractive content (from tree age of 1 to 3 years and 2 to 3 years).

From the age of 3 to 4 years in sapwood, the levels of hexane and methanol increased with tree age at the base of the tree, while the reverse pattern was found in the amount of hot water extract and total extractives, where the hot water content decreased significantly. At the top part, a slight increase in the extractive content was found in the amount of hexane and hot water extract, while the content of methanol and total extractives were found to decrease. After the formation of heartwood (4 to 5 years), the content of methanol and hot water extract increased (except for hexane) with tree age in the bottom sapwood. The same pattern was shown in heartwood, except for the hot water extract which slightly increased. Further at the top of the tree, both in sapwood and heartwood, the extractive content of methanol and hot water increased significantly from the age of 4 to 5 years. A similar trend was shown by the total extractive content, where its level drastically increased with increasing tree age.

In the axial position, the extractive content generally descends from the bottom to the top of the tree before heartwood formation (Table 3, Figure 2). A significant reduction was found in the amount of hexane extract at the age of 3 years and hot water at the age of 2 and 3 years. The reverse trend was found at the age of 1 year, where the extractive content increased significantly in hexane and methanol extracts. Furthermore, a similar pattern was also shown in the total extractive content, where the levels decreased significantly at the age of 3 years and increased significantly at the age of 1 year.

In addition, from the age of 3 to 4 years, the content of extractive patterns was similar to before heartwood formation (except for methanol extract at the age of 3 years and hexane extract at the age of 4 years, which slightly decreased). After the formation of heartwood, at the age of 4 years both in sapwood and heartwood, the extractive content decreased significantly from the bottom to the top of the tree. The same trend was also shown at the age of 5 years, although the decrease was not significant (except for methanol extract). Furthermore, in the radial profiles, extractive content in the heartwood both at the base and at the upper part of the tree was higher than in the sapwood at the age of 4 years. The same pattern was also shown at the age of 5 years, where the content of hexane extract in heartwood was significantly larger compared to sapwood at the bottom and a similar trend was shown by the methanol extract at the top of the tree.

There was a significant interaction between age and axial position in TPC in the ANOVA test (Table 2). Before heartwood formation (Figure 3), TPC increased with tree age. It increased significantly from the age of 1 to 2 years at the bottom of the tree. A similar trend was also found in the TPC at the age of 3 to 4 years at the bottom sap-wood part, although TPC decreased at the top of the tree. However, a reverse pattern was found from the age of 4 to 5 years after heartwood formation, where the TPC increased significantly at the top sapwood part.

Axially, before heartwood formation (1 to 3 years), the TPC content decreased from the bottom to top part except for 1-year-old trees, which slightly increased from the base to the upper part of the trees. The same pattern was shown during heartwood formation. A significant decrease was found at the age of 4 years in sapwood. In addition, a similar trend was also shown after the formation of heartwood, where both in sapwood and heartwood (4 and 5 years old), the TPC content significantly decreased from the bottom to the top of the tree. Furthermore, in the radial variation, both at the age of 4 and 5 years, the difference in TPC between sap-wood and heartwood was more visible at the bottom than at the top, where the TPC of heartwood was higher than that in the sapwood samples.

The ANOVA of TSP showed significant tree age and axial position interactions (Table 2). Generally, the TSP level increased with the increasing tree age before the formation of heartwood (Figure 4), where both at the bottom and top of the tree, a significant increase was found from the age of 1 to 2 years. Meanwhile, at the top part, the amount of TSP from the age of 2 to 3 years decreased significantly. Furthermore, the TSP content slightly increased again from the age of 3 to 4 years both at the bottom and top of the tree in sapwood. Then, TSP at the bottom both in sapwood and heartwood decreased significantly from the age of 4 to 5 years after the formation of heartwood. In contrast, the amount of TSP increased significantly at the top heartwood of the tree.

In axial position, the TSP increased from the bottom to the top of the tree (except for the age of 3 years) before heartwood formation. TSP from the age of 3 to 4 years, with the axial pattern being the same as that of TPC, decreased drastically from the base sapwood to the upper sapwood. Furthermore, the TSP trend at the age of 4 and 5 years was the same as that of TCP at the same age where the TSP level dropped from the bottom to the top of the tree. Meanwhile in the radial variation, TSP in sapwood was higher than in heartwood. A significant difference was found at the age of 4 years at the top, and at 5 years at the base of the tree.

Theoretically, the non-polar solution (n-hexane) removed fats, waxes, resins, and sterols (Lukmandaru, 2011). Meanwhile, cold and hot water-soluble extractives dissolve mineral salts, sugars, tannins, phenolic compounds, dyes, pectins, free acids, and others (Fengel & Wegener, 1989; Han & Rowell, 1997). The extractive contents of this result were slightly lower compared to that of Batubara et al. (2009, 2010, 2012) and da Silva et al. (2013) who observed the values of methanol and hot water extractive in other Swietenia. The value obtained for S. macrophylla in Moto Grosso, Brazil was 5.93% to 7.97%. Meanwhile, 4.01% to 14.83% was obtained for Swietenia sp. wood in Samarinda, Indonesia. Taylor et al. (2008) obtained that the yield of toluene/ethanol (2:1), 95% ethanol, and hot water extract ranged from 11.8% to 28.1% of mahogany wood from Central and South America. Additionally, Jankowsky & Galvão (1979) reported that the amounts of alcohol-benzene and water-soluble extract in S. macrophylla wood were 7.03% and 8.64%, respectively. The observed yield of extractive of S. mahagoni was lower compared to the reported values because the samples were used from a young age.

In contrast, extractive levels of this study were higher compared to the content of petrol ether (0.8%), acetone (3.2%), methanol (3.4%), hot water (1.1%), and cold water (2.1%) (Rutiaga Quiñones et al., 1998a). Gala et al. (2020) found that the extractive content of ethanol and hot water solution of S. mahagoni wood from Malang, Indonesia was 4.85% and 4.95%, respectively. In addition, the contribution of the three solvents in the total of extractive contents was similar to the results of Morais & Pereira (2012), where methanol contributed greatly to dissolve the extract. The proportions of particular components in wood depend on the species, the climate, the time of year, the site conditions, the part of the tree, and the health condition (Bikovens et al., 2013; Miranda et al., 2017; Szczepkowski et al., 2007; Zobel & Sprague, 1998). In addition, the composition and amount of extractives depend on the solvent used (e.g., ethanol-benzene mixture, ethanol, acetone, dichloromethane, and ethyl ether) (Lachowicz et al., 2019).

TPC of this study was lower compared to previous research. Batubara et al. (2012) reported that TPC in Swietenia sp. was 673.8 mg GAE/g. In Swietenia species, such as S. macrophylla, it was 6.81% (da Silva et al., 2013). Furthermore, the same pattern was also shown by TSP, the levels of which were lower compared to the value reported by Rutiaga Quiñones et al. (1998b). They reported the content in S. macrophylla as 63.3%, dominated by the glucose component.

In general, the extractive content increased with increasing tree age. The same pattern has been reported in previous studies in Eucalyptus globulus Labill., Eucalyptus grandis W. Hill, Eucalyptus urophylla S.T. Blake, and Tectona grandis L. f. (Miranda & Pereira, 2002; Lukmandaru, 2009; Santana et al., 2012; Lestari et al., 2016). The vascular cambium of older trees is more likely to divide and multiply into parenchyma cells, so that the formation of parenchyma cells increases. Therefore, the extractive content tends to rise (Hamidah et al., 2009). In addition, a significant increase in the extractive content was also found after the formation of heartwood (4 to 5 years) at the top of the tree. This content increased due to the largest contribution from the extractive content of methanol and hot water which increased significantly after heartwood formation. This may be related to non-structural carbohydrate (NSC) and phenolic compounds, considering that methanol and hot water solutions are the best solvents to dissolve sugar and phenolic components (Fengel & Wegener, 1989; van Putten et al., 2014). This argument was supported by the significantly increased TSP and TPC content at the top of the tree. Piispanen & Saranpää (2001) reported that some TSP (sucrose and glucose content) are more abundant in the top parts of the crown, such as the stems, which are closer to photosynthesizing leaves, where the assimilation of CO₂ takes place. In addition, Magel et al. (1994) reported that the high amount of the main monosaccharide components in sapwood could be a high demand of NSC which were degraded for energy and the carbon skeleton needed for metabolic activities, such as cell respiration, photosynthesis and the formation of secondary metabolites, such as phenolics. In addition, the significant decrease in the TSP content after the formation of heartwood (4 to 5 years) at the base of the tree may be related to the formation of heartwood, where sugar is converted into phenolic components. This is indicated by the increased TPC content at the base both in sapwood and heartwood after the formation of heartwood.

However, in this study it was also found that the extractive content decreased from a certain tree age, namely in the hot water extract at the bottom and in the methanol extract at the top part from the age of 3 to 4 years. In addition, especially at the top of the tree from 1 to 3 years, the extractive content decreased significantly. This may be influenced by the certain proportion of sugar components which decreases with age at the top of the tree. Berrocal et al. (2004) reported that the sugar component, such as xylose, galactose, and arabinose in Pinus radiata D. Don decreased with tree age (1 to 15 years old). In addition, Rencoret et al. (2011) reported that the hot water extractive of E. globulus clone wood from Pontevedra, Spain, decreased with tree ages of 1 month, 18 months, and 9 years. Stolarski et al. (2011) observed in willow shoots that the hot water extract decreased from the age of 1, 2, and 3-year-old trees in the species obtained from North of Poland. Miranda & Pereira (2002) reported that the total extractive content of E. globulus from Portugal decreased from 3 to 6 years old. In addition, a drastic decrease in methanol and TSP levels at the top of 3-year-old trees suggests that a lot of TSP was transferred from the top to bottom part in the process of heartwood formation in 4-year-old trees at the base. This finding was supported by the high extractive content of hot water at the bottom part of 3-year-old trees, where the hot water solvent is known as a good solvent of sugar components (van Putten et al., 2014). Prayitno (1992) reported that the axial position in the bottom part has the highest extractive content.

In axial variation, the extractive content at the bottom part was greater compared to the top of the tree (except for 1-year-old trees). A similar trend was reported in previous work in other species, such as superior teakwood, Styrax sumatrana J.J. Sm., and Eucalyptus globulus (Iswanto et al., 2019; Gominho et al., 2015; Zulkahfi et al., 2020). Gominho et al. (2001) and Caron et al. (2013) also found the high concentrations of extractives at the base of hardwood. The high extractive content at the base part may be due to the intense polymerization reaction during the aging process of the tree. Meanwhile, the high content at the top part is caused by the vicinity to the canopy as a place of photosynthesis (Lukmandaru et al., 2021). Yeh et al. (2006) found that benzene alcohol extractives in the top part were higher compared to the bottom. This is because the living moiety on the top of a tree was higher. Meanwhile, TPC and TSP generally decreased from the bottom to the top of the tree (after 2 years of age). A similar pattern was also found by Neverova et al. (2013) in Larix sibirica Ledeb. However, a reverse trend was found in previous studies that TPC increased from the base to the top of the tree in other tropical species, such as superior teakwood aged 11 years and Eucalyptus pellita F. Muell. from natural forest (Arisandi et al., 2019a; Zulkahfi et al., 2020). This might be one of the characteristics of the species S. mahagoni compared to other tropical wood species in the period until the age of 5 years. Other reasons might be due to differences in tree age, species, and growth location (Hillis, 1987; Freire et al., 2005).

In the radial profiles, the extractive content in heartwood was higher than in the sapwood part. A similar trend has been reported in other species, such as Tectona grandis, Acacia melanoxylon R. Br., Eucalyptus pellita, and Eucalyptus globulus (Miranda et al., 2006; Lourenço et al., 2008; Lukmandaru, 2011; Morais & Pereira, 2012; Arisandi et al., 2019a, 2020). Studies on the pigments of the heartwood parts were almost never carried out in mahogany (Yazaki, 2015). Rastogi & Mehrota (1993) reported that the heartwood of S. mahagoni contained cycloswietenol, lupleol, hedergenin benzoate, cycloartenol, and -sitosterol. In addition, the hot water extractive from mahogany bark exhibits a strong red color, and is usually used as a tannin for leather tanning. Bark extracts found in hot water solution were (+)-catechin, (–)-epicatechin and a new amorphous compound, switemacrophyllanin (Falah et al., 2008).

The high levels of extractives in heartwood are related to the heartwood formation process, where the accumulation of extractives is a key feature in the formation of heartwood (Hillis, 1971). Umezawa (2000) reported that heartwood is richer in polyphenols and resin acids (diterpenes). In heartwood (with a lower pH condition), the most abundant of the soluble sugars (xylose, mannose, and arabinose) were derived from hydrolyses. This is supported by the decreasing TSP content from sap-wood to heartwood as well as a reverse pattern found in TPC. Thus, the decline in TSP from sapwood to heartwood indicates that the content of certain TSP compounds is metabolized at the sapwood-heartwood boundary (Niamké et al., 2011). Hillis & Hasegawa (1963) postulated that NSC becomes the main source of carbon framework for synthesizing extractives of heartwood (phenolic compounds).

In the transition zone, the amount of starch can be hydrolyzed to glucose by hydrolyzing enzymes (amyloglycosidases) (Magel et al., 1997, 2001). In other species, like teak, key NSC catabolism enzymes, such as succinate dehydrogenase and glucose-6-phosphate are involved in reducing NSC and accumulate in the inner sap-wood and in the transition zone (Data & Kumar 1987). Niamké et al. (2010) reported that the depletion of NSC from sapwood to heartwood suggests that they may be precursors for the synthesis of wood extractives (phenolic compounds) during heartwood formation. Burtin et al. (1998) reported that hydrojuglone glucoside was the precursor of juglone, and their polymers in heartwood Juglans nigra L., whereas dihydrorobinetin was the precursor of robinetin synthesis in Robinia pseudoacacia L. (Magel et al., 1994). However, this study was limited in the general composition of TSP and TPC. Therefore, further research is needed to investigate the changes in sugar and phenolic compositions in the component level, in order to investigate what compounds play an important role in the heartwood formation of S. mahagoni.