Introduction

Eucalyptus has been widely used for industrial purposes, especially in the paper industry. In the paper industry, only the eucalyptus wood tissue is utilized, with the leftovers, mainly barks in large quantity. In Thailand, the amount of bark produced in the pulp and paper industry is more than 4 million tons per year. The suitable eucalyptus variety for pulp and paper production in Thailand has been studied by Senjuntichai’s work (2014). Eucalyptus bark is usually fed into a gasifier and converted to CO for further use as clean energy in a biomass power plant (Gonzalez et al. 2011; Guerra et al. 2016), or used as a fertilizer (Yadav et al. 2002). Direct use of the bark in the boiler generally reduces the boiler efficiency, resulting from tar deposit on the heating surface of the boiler. Laaksometsä et al. (2009) have reported an increase in boiler efficiency by lignin extraction, thus reducing environmental pollution. It is a well-known fact that Eucalyptus bark contains large quantity of bioactive compounds (Piwowarska and González-Alvarez 2012) such as triterpenic (TT) acid and their acetyl derivatives (betulinic acid, betulonic, oleanolic and ursolic) (de Melo et al. 2012; Domingues et al. 2010, 2011a, b), ellagic acid rhamnosides (Kim et al. 2001) and tannin (Pinto et al. 2013). Some of these compounds have antioxidant properties which can replace synthetic additives such as butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), propylgallate and tert-butylhydroquinone (TBHQ). Even though these synthetic compounds possess highly effective antioxidant properties, they also carry carcinogenic effect (Baydar et al. 2007; Iyer et al. 2015). For example, BHA and BHT have been suspected of causing some health problems, such as liver swelling and influence on liver enzyme activities (Siddhuraju and Becker 2003). These concerns lead to the growing interest in the study of secondary plant metabolites. Most of the previous studies (Almeida et al. 2016; Hossain et al. 2010) have focused on antioxidants from natural sources, especially from medicinal plants, in order to obtain environmentally friendly and low-cost antioxidants that can be used as antioxidants in food and pharmaceutical products (de Melo et al. 2012; Mirabella et al. 2014). Furthermore, they could be used as a component in cosmetics for anti-aging, anti-wrinkle and whitening activities.

In order to obtain the antioxidants, a proper extraction process is required. Among the available extraction processes, the solvent extraction is mostly used to extract bioactive compounds. The important factors in this process are the type of solvents, extraction time and temperature (Dai and Mumper 2010). The efficiency of the extraction process mostly depends on the extraction technique. It has been reported that traditional techniques such as a one-stage, maceration or Soxhlet, extraction provide a high extraction yield. However, these techniques usually require long extraction time and a large quantity of solvent (Aspé and Fernández 2011). Many researchers have developed alternative techniques to improve the efficiency and reduce the environmental impact of the extraction process, for example, combination of mechanic partitioning (Maroušek, 2014a), steam explosion (Maroušek 2013a, b), enzymatic hydrolysis (Maroušek et al. 2015), pressure shockwaves (Maroušek et al. 2013; Maroušek 2014b), microwave-assisted extraction (MAE) or ultrasound-assisted extraction (UAE) (Liazid et al. 2010; Oniszczuk and Podgórski 2015). The MAE technique is based on the absorption of microwave energy from water contained in the plant matrix. During the process, the internal heating will promote the cell disruption to facilitate the liberation of compounds into the solvent (Wang and Weller 2006). Among the extraction techniques, one-stage extraction is one of the most commonly used techniques, because of its high efficiency, relatively inexpensive equipment and simple operational process (Li et al. 2006).

Therefore, in this work, the one-stage technique was selected for antioxidant extraction due to its simplicity and high production yield. The aim of this research is to find the optimal condition for extraction of the total phenolic compounds from Eucalyptus globulus bark using ethanol, methanol and dichloromethane as extractants. The extraction system was improved by continuous mixing during the extraction period. The Folin–Ciocalteu colorimetric method was used to measure the total phenolic compounds. DPPH radical scavenging capacity assay and ferric reducing antioxidant power (FRAP) method were used to determine the antioxidant activity of the extract. Furthermore, thermal degradation and heating value of the bark before and after extraction were carried out to compare the fuel properties. GC-MS analyses were performed to identify the volatile compounds. The rationale of this work lies in the fact that a solid–liquid extraction process may be used in order to realize more benefits than the simple gasification of the bark. Through the extraction process, useful chemicals may be obtained for further use with little sacrifice of the heating value of the bark. The proposed conceptual process of this technology will be later discussed in detail.

Materials and methods

Chemical and reagent

Methanol (MeOH), ethanol (EtOH), dichloromethane (DCM), Folin–Ciocalteu reagent, sodium carbonate anhydrous (Na2CO3), gallic acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH) and butylated hydroxytoluene (BHT) were purchased from Sigma (Singapore) in analytical reagents grade.

Extraction and concentration

Preparation of the Eucalyptus globulus bark extract

Eucalyptus globulus bark was obtained from Phoenix Pulp & Paper Public Company Limited (PPPC), Thailand. The bark was cleaned and air-dried at 50 °C for 12 h. After that, the bark was crushed in a ball mill (in house building with 3 m3 (0.15 m of diameters and 0.17 m of height) of mill size and 10 mm balls) before passing through a 1–3 mm sieve. The proximate analysis of bark is given in table S.1 (Supplementary materials).

Extraction and concentration

The conventional solid–liquid extraction was performed by soaking the ground eucalyptus bark with various solvents including methanol (MeOH), ethanol (EtOH) and dichloromethane (DCM). The total volume of extraction was 100 ml with various ratios of solid and liquid at 1:10, 1:15 and 1:20 g (solid)/ml (solvent). The extraction was performed under a shaking speed of 200 rpm at 30 °C for 6 h. After the extraction was completed, all solid phase was removed. The liquid phase was retained and evaporated by using a rotary vacuum R-205 (Buchi, Switzerland) at 40 °C, vacuum pressure of 300 mbar with the rotating speed of 90 rpm. The extract was weighed and expressed in a unit of milligram per gram dry bark (mg/g DW). The extract was reconstituted in methanol for further analysis. All experiments were conducted in 3 replicates.

Determination of antioxidant activity

DPPH radical scavenging activity

The antioxidant activity of each extract was analyzed using free radicals from 2,2-diphenyl-1-picrylhydrazyl (DPPH) method (Blois 1958) with some modification. The principle of the assay is based on the color change of the DPPH solution from purple to yellow, as the radical is quenched by the antioxidants. When a solution of DPPH is mixed with a substance that can donate hydrogen, the reduced form of DPPH is obtained, and the solution which is initially violet turns yellow. This change in color was monitored by visible spectroscopy at 517 nm (UV-1201 spectrometer, Shimadzu, Japan). Briefly, 1 ml of a sample solution with different concentrations (10–100 mg/l for methanol and ethanol and 500–5000 mg/l for dichloromethane) of each extraction solvent and solid–liquid ratio was mixed with 4 ml of a DPPH solution (0.051 mmol l−1) in methanol (ethanol or dichloromethane depending on each extraction solvent). The reaction mixtures were kept in the dark for 30 min at room temperature. The absorbance of the DPPH solution in the absence of the bark extract under analysis was also measured as a control. Butylated hydroxytoluene (BHT) was used as reference compound (Brand-Williams et al. 1995). All experiments were conducted in 3 replicates. The DPPH radical scavenging activity was calculated by the following equation:

$$\% \;{\text{DPPH}}\;{\text{radical}}\;{\text{scavenging}}\;{\text{activity}} = \left( {{\text{Abs}}_{\text{control}} - {\text{Abs}}_{\text{sample}} } \right)/{\text{Abs}}_{\text{control}} \times 100$$

where Abscontrol is the absorbance of the control, and Abssample is the absorbance of the DPPH in each sample at 517 nm. DPPH scavenger activity is defined by the IC50 values. The IC50 (mg/l) values are determined from the equation reported in Supplementary information (Fig. S2–S4).

Ferric reducing antioxidant power (FRAP) assay

This method was modified from Benzie and Strain (1996). The FRAP reagent was prepared from the mixture of 10 mM 2,4,6-tripyridyl-s-triazine (TPTZ) solution in 40 mM HCl, 20 mM FeCl3·6H2O solution and 0.3 M acetate buffer (pH 3.6) in a proportion of 1:1:10 (v/v). The FRAP reagent was freshly prepared and heated to a temperature of 37 °C. The absorbance of each sample was measured at 600 nm using an UV–Vis spectrophotometer (UV-1201 spectrometer, Shimadzu, Japan). The FRAP values of each sample were determined using the standard curve prepared from 50 to 1000 μM FeSO4·7H2O.

Determination of total phenolic compounds

The total phenolic compounds of extract were analyzed by Folin–Ciocalteu colorimetric method (Slinkard and Singleton 1977) with gallic acid as positive control; 125 µl of extract in methanol was mixed with 500 µl of distilled water before addition of 125 µl of Folin–Ciocalteu reagent. The reaction was continued for 6 min, after addition of 1.25 ml of 7 % w/v sodium carbonate. Finally, the sample volume was adjusted to 3 ml with distilled water and placed in the dark room for 90 min. The absorbance of each sample was observed at 760 nm by UV–Vis spectrophotometer (UV-1201 spectrometer, Shimadzu, Japan) and expressed as milligrams of gallic acid equivalent per grams dry bark (mg GAE/g DW).

Thermogravimetric analyses (TGA)

Thermogravimetric analyzer (TGA 50) was used for investigating thermal degradation of both raw bark and extracted bark. All samples were milled and ground to less than 1 mm before testing; 10 mg of each sample was loaded into an alumina crucible and heated at a programmed temperature. The sample was held at 25 °C for 30 min. The temperature was then increased at a heating rate of 10 °C min−1 up to 700 °C and held for 10 min. The weight losses were recorded and analyzed.

The heating value of Eucalyptus globulus bark

The calorific value of raw bark and extracted bark was determined by Gallenkamp Auto Bomb™ adiabatic calorimeter (Gallenkamp, UK), following the method outline in ASTM E711 (ASTM International, 1987).

Gas chromatography (GC) and mass spectroscopy (MS) analysis

The extract identification and quantification of low molecular weight compounds were carried out by GC-MS analysis using an Agilent 6890 N (Agilent, Singapore) model connected to MS Agilent 5973 inert mass selective detector (Agilent, Singapore). The sample preparation was as follows: 20 mg of extract was dissolved in 250 μl of ethanol and passed through 0.45 μm filter membrane to clean the sample. DB-5 ms capillary column (Agilent 122-5532, Singapore) of 30 m long, with a 0.25 mm internal diameter and 0.25 µm film thicknesses, was used. Helium at a flow rate of 1 ml/min was used as a carrier gas. One microliter of this sample was injected into the column. The injection temperature was at 250 °C with a split rate of 10.0 ml/min. The column temperature was programmed at 80 °C for 2 min, then 10 °C/min increment up to 200 °C. Finally, a heating rate of 5 °C/min was performed up to 280 °C and held for 15 min. The identification of any low molecular weight aromatic compounds as TMS derivatives produced from the experiments was completed by comparing their mass spectra with those in the Agilent ChemStation Database.

Results and discussion

Influence of type of solvents on E. globulus bark extraction

Three types of solvents were used to compare the effect of solvents on extraction. The extraction process follows that of the conventional extraction technique with shaking at 50 °C for 6 h, while high temperature was used to shorten the extraction time. As shown in Fig. 1, the weight of the extract obtained from methanol was approximately two times higher than those from other solvents in similar condition. This suggests that methanol provided the highest extraction efficiency in the test condition.

Fig. 1
figure 1

Amount of extract from extraction of 10 g of eucalyptus bark in dichloromethane (DCM), ethanol (EtOH) and methanol (MeOH) under a shaking speed of 200 rpm at 30 °C for 6 h. t test of two sample assuming unequal variance is performed; asterisk statistical analysis of different solvents with the same ratio

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The effect of solvent quantity on extraction yield was examined by varying the bark and solvents at the ratios of 1:10, 1:15 and 1:20 (gram bark/ml solvent). For comparison among the same solvent but different ratios, dichloromethane and methanol at a ratio of 1:20 were found to be the most effective for extraction, while no difference was observed for ethanol. This may be due to the unsaturation of the extract dissolved in those organic solvents. The result suggested that methanol is the most effective solvent for this extraction condition, especially at the ratio of 1:20 which is due to higher polarity of methanol compared with that of other solvents. The statistical analysis confirmed that the methanol provides significantly higher extraction yield (at p value <0.05) compared to ethanol and dichloromethane. In addition, the extract yields from ethanol and dichloromethane showed no difference. Moreover, the extraction yields were similar to the previous studies of eucalyptus bark extraction (2.48 % extraction yield for ethanol and 2.70 % extraction yield for methanol) (Vázquez et al. 2008).

Total phenolic content of extracts

In the present work, the total phenolic content in each sample was investigated. As shown in Fig. 2, the sample extracts using ethanol and methanol at a ratio of 1:20 provide the best condition for the total phenolic content. This result correlates with the extraction yield in the previous experiment. The maximum total phenolic contents obtained at the ratio of 1:20 were 3.18 ± 0.03, 3.13 ± 0.03 and 0.64 ± 0.06 mg GAE/g DW for ethanol, methanol and dichloromethane extracts, respectively. This suggests that methanol and ethanol provide a better yield of phenolic compound than dichloromethane in the experimental condition. The statistical analysis confirms the significant difference of the phenolic content of DCM extract when compared with other solvents at the similar ratio (with p value <0.05). High polarity of methanol and ethanol is one of the important parameters to enhance the extraction efficiency, and in this case, to obtain polar phenolic compounds. The polarity of methanol, ethanol and DCM are 0.762, 0.654 and 0.269, respectively (Reichardt and Welton 2010). The highest total phenolic content obtained in this work is lower than that from other plant sources, including eucalyptus leaves (62.10 ± 2.49 mg GAE/g DW), black tea (24.9 ± 0.2 mg GAE/g DW) and purple potato peels (4.74 ± 0.07 mg GAE/g DW), as given in Table 1. The methanol extraction used in this study gave a higher yield (3.13 ± 0.03 mg GAE/g DW, with methanol) than that of Santos et al. (0.819 mg GAE/g DW, with methanol/water) (Santos et al. 2011). This possibly suggests that the continuous mixing and polarity of solvent could enhance the extraction yield. The polarity of solvent affects the total phenolic contents of the extract, for example, flavonoid glycosides and more polar aglycones can be extracted by alcohols or alcohol–water mixtures, while less polar flavonoids (isoflavones, flavanones, methylated flavones and flavonols) usually can be extracted by chloroform, dichloromethane, diethyl ether or ethyl acetate (Stankovic et al. 2011). Therefore, apart from the high amount of total phenolic content acquired, choice of solvents suitable for specific groups of phenolic compounds has to be considered. It is also need to be considered that even though ethanol does not show the best extraction yield (based on previous section), ethanol gives the comparable phenolic content to the methanol extracts. Ethanol could be used as a better alternative for downstream processing, especially in the medical industry due to its lower cytotoxicity compared with methanol.

Fig. 2
figure 2

Total phenolic content of 10 g of eucalyptus bark extract in dichloromethane (DCM), ethanol (EtOH) and methanol (MeOH). t test of two sample assuming unequal variance is performed. Asterisk statistical analysis of different solvents with the same ratio

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Table 1 Total phenolic contents of various materials
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Antioxidant activity test

Antioxidant activity of sample extracted using DPPH test

The antioxidant activity was determined by the ability of molecules to provide hydrogen atoms to reduce the free radical DPPH. This causes the formation of the DPPH-H, non-radical form that can be detected under the spectrophotometer at 517 nm. The antioxidant activity obtained in this work was expressed in terms of the amount of extract required for decreasing 50 % of DPPH concentration (IC50). Thus, butylated hydroxytoluene (BHT), a synthetic phenolic molecule, was used as a reference compound to evaluate the antioxidant activity of tested samples. Table 2 shows IC50 of the samples extracted with different solvents at various ratios with BHT as standard anti-oxidant. The antioxidant activities show a similar trend to the total phenolic content as mentioned in Sect. 3.2. The antioxidant activities were the lowest (5113.98 ± 347.16 mg/l) in the sample extracted by DCM at 1:10 ratio, while the sample extracted by ethanol at 1:20 ratio provided the highest activity (30.53 ± 1.76 mg/l). It is clear that the extracts by ethanol and methanol have similar antioxidant properties. It has been reported by Ham et al. (2015) that an aqueous ethanol is effective in extracting phenolic compounds and provides the highest DPPH radical scavenging activity. All extracts in the present work show lower antioxidant activities than those of BHT compound (IC50 = 15.1 mg/l). However, the antioxidant activity of E. globulus bark extracts in this study is comparable to Syrian and Egyptian coriander, a natural source of polyphenol compound, with their IC50 values being 36 ± 3.22, and 32 ± 2.8 mg/l, respectively (Msaada et al. 2013).

Table 2 IC50 of the samples extracted by methanol, ethanol and dichloromethane, at the ratio of 1:10, 1:15 and 1:20 (g sample/ml solvent)
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Antioxidant activity of E. globulus bark extract using ferric reducing antioxidant power (FRAP) assay

Total antioxidant of E. globules bark extract was analyzed by FRAP, with Trolox as a reference compound. As shown in Fig. 3, the result correlated with the DPPH experiment, the antioxidant activity of the sample extracted by methanol is statistically higher than the sample extracted by ethanol (p value <0.05), and 100 times higher than that by DCM (data not shown). The highest antioxidant power was found in the sample extracted from 1:10 methanol (630 ± 15.76 of µM Fe2SO4). It is comparable to the standard reagent, Trolox at a concentration of 50 mg/ml (678.5 of µM Fe2SO4). It should be mentioned here that the work of Henderson et al. (2015) with manuka honey reported the FRAP value of 677 ± 78 of µM Fe2SO4 with UMF of 15 which is in a similar range to our work.

Fig. 3
figure 3

Ferric reducing antioxidant power (FRAP) value of E. globulus bark extracted by ethanol and methanol (t test of two sample assuming unequal variance is performed. Asterisk statistical analysis among different solvents, number sign statistical analysis of solvent with the same ratio)

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Thermogravimetric analysis (TGA) value of E. globules barks before and after extraction

Thermogravimetric curves of E. globulus bark before and after extraction are presented in Fig. 4. The decomposition of both untreated and treated bark is quite similar and can be divided into three stages. The first stage (region 1 in Fig. 4) is related to moisture loss, which was around 15 % weight loss at 0–140 °C (da Silva et al. 2009). The second stage is the degradation of tars and volatile organics which occurred at 140 and 520 °C, and the decomposition was observed at 70 % weight loss of untreated bark and 75 % weight loss of bark after extraction. The last stage, the degradation of ash contents in both untreated and treated bark occurred at a temperature higher than 520 °C. The percentage weight loss of both untreated and treated bark show some difference at high temperatures (at 400–700 °C). In addition, the residual weight of the untreated bark is slightly higher than that of the treated bark. This might correlate to the amount of phenolic contents and other compounds which were eliminated in the extraction process.

Fig. 4
figure 4

Percentage weight loss of Eucalyptus globulus bark before and after extraction with ethanol extracted at the ratio of 1:15 (g of sample/ml of solvent)

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Heating value of E. globulus bark after extraction

The quality of fuel can be determined by the amount of heat generated from a unit mass of fuel (in kJ/kg). The calorific value is considered as an important parameter for comparing one fuel with another. As listed in Table 3, the mean calorific values of untreated and treated bark were 7754 and 7449.74 kJ/kg, respectively. A smaller calorific value in the treated bark might result from the loss of some compounds during the extraction process. This similarity in the calorific values suggests that the treated bark can still be used as a fuel with little loss of heating value.

Table 3 Heating value of treated and untreated E. globulus bark
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Identification of the extracts by GC-MS

Gas chromatography–mass spectroscopy (GC-MS) was used to identify the volatile compounds in the extracts by ethanol at the ratio of 1:20, which gave the highest yield. The result is given in Table 4 with nine identified compounds including 2,4-bis-(1,1-dimethylethyl)-phenol, 2,2′-[(1-methyl-1,2-ethanediyl)bis(nitrile-methylidyne)]bis-phenol, (S)-2-iodo-1-methoxy-(1-methoxyethyl)benzene, 1-methyl-3,3-ethylenedioxy-1,2,3-trihydro-2-oxo-1-benzazole, lidocaine, methyl n-nitro cinnamate, silicic acid, γ-sitosterol and β-amyrin. In a study by Domingues et al. (2011a, b), the dichloromethane extracted samples from other eucalyptus species contained highly valued compound of triterpenoids such as β-sitosterol and β-amyrin. β-Sitosterol has been studied for its anti-tumor and thermal-induced protein denaturation inhibitory activities (Rauf et al. 2015). γ-Sitosterol can be considered for developing into a potent anti-diabetic drug (Balamurugan et al. 2012), and β-amyrin (C30H50O), the derivative of triterpenes, showed anti-anxiety and anti-depressive actions through α1 receptor blockade (Jeon et al. 2015). Lidocaine, a class Ib anti-arrhythmic agent and local anesthetic agent, has emerged within the pediatric literature as an anti-epileptic drug (AED) in neonatal status epilepticus (SE) (Zeiler et al. 2015). It is worth mentioning here that the present study successfully extracted three compounds, namely γ-sitosterol, β-amyrin and lidocaine, which have medicinal applications.

Table 4 Chemical composition (%) of methanol extracts of E. globulus bark analyzed by gas chromatography–mass spectroscopy (GC-MS)
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Proposed conceptual process for clean technology

Figure 5 shows the conventional process and a proposed process of eucalyptus bark. As discussed in Sect. 3.5, the heating value of the bark after the solid–liquid extraction process hardly changes. The conventional process in using eucalyptus bark (Fig. 5a) to produce syngas for electricity generation is shown in Fig. 5a. The syngas was not the only product from this process. CO2 and tar were obtained as by-products (Baloch et al. 2016). The presence of tar in the gas results in tar deposit on the heating surface of the boiler which reduces its efficiency. Thus, the extracted bark may be decreased the tar deposit effect because some hydrocarbon in bark might be eliminated. In the proposed conceptual process as illustrated in Fig. 5b, phenolic and fine chemical extraction of eucalyptus bark is obtained. The obtained chemical fines could be further used to produce high-value fine chemicals (as mentioned in Sect. 3.6). The products from extraction process will be separated by vacuum evaporation. The solvent can also be extracted and recycled in the extraction process. It can be seen that the proposed conceptual process not only produce extracted bark but also products which can be upgraded to products with high commercial values. Furthermore, the proposed conceptual process could be considered as an alternative clean technology for increasing the value of eucalyptus bark and reducing environmental pollution.

Fig. 5
figure 5

a Conventional process using eucalyptus bark. b Proposed concept process using eucalyptus bark

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Conclusions

Ethanol and methanol, with the ratio of bark to solvent of 1:20, was found to give the best condition for extraction of E. globulus bark, with slight difference in total phenolic content and antioxidant activity. The methanol extraction provides the greatest amount of extracts among the three tested solvents. Ethanol, despite yielding comparatively lower amounts of extract, provides better antioxidant activity than that of methanol. TGA of untreated bark and treated bark had some difference at high temperatures (at 400–700 °C). The heating value results showed no significant difference between the untreated E. globulus bark (ca. 7803 kJ/kg) and the extracted barks (ca. 7570 kJ/kg). The GC-MS results revealed that useful compounds with potential medicinal application can be derived from the bark before it is ultimately used as a source of energy. More importantly, it has been demonstrated that the proposed conceptual process is feasible with greater commercial and environmental benefits. However, further study is needed before the actual benefits can be realized.