The bark of Eucalyptus sideroxylon as a source of phenolic extracts with anti-oxidant properties

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The bark of Eucalyptus sideroxylon as a source of phenolic extracts with anti-oxidant properties Isabel Miranda ∗ , Leandro Lima, Teresa Quilhó, Sofia Knapic, Helena Pereira Centro de Estudos Florestais, Instituto Superior de Agronomia, Universidade de Lisboa, Tapada da Ajuda, 1349-017 Lisboa, Portugal

a r t i c l e

i n f o

Article history: Received 8 September 2015 Received in revised form 18 November 2015 Accepted 5 December 2015 Available online xxx Keywords: Bark Eucalyptus sideroxylon Anatomy Chemical composition Phenolic extracts Anti-oxidants Tannins

a b s t r a c t Barks are today viewed as a potential resource for biorefineries given their chemical richness and diversity. This paper describes for the first time the chemical composition of Eucalyptus sideroxylon bark and the antioxidant properties of its polar extractives. The bark is thick, deeply furrowed and dark colored. Large pockets of kino were observed formed by the breaking down of tissues of the outer phloem. The mean chemical composition of E. sideroxylon bark was: ash 1.3%; total extractives 55.7%, mainly corresponding to polar compounds that were soluble in ethanol and water, lignin 13.1% and suberin 1.9%. The polysaccharides composition showed predominance of glucose and xylose (80.0% and 11.0% of total neutral monosaccharides respectively). The ethanol–water bark extract had a high content of phenolics: total phenolics 440.7 mg gallic acid equivalent/g extract, flavonoids 204.4 mg catechin equivalent/g extract and tannins 395.0 mg catechin equivalent/g extract. The antioxidant activity corresponded to 648.8 mg Trolox/g of extract, and FRAP values to 5247 mM Fe2+ /g of extract. E. sideroxylon bark can therefore be extracted with green solvents to yield polar extractives with a potential valorization based on their chemical functionalities and bioactivity. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Tree barks are complex biomass components with a large structural and chemical diversity among species. Barks have been used traditionally e.g., as a source of drugs, materials and energy, and are today viewed as a potential resource for biorefineries (e.g., Le Normand et al., 2014). Knowledge on bark characteristics is however limited to a small number of species, mostly those with high wood commercial exploitation. Eucalyptus sideroxylon A. Cunn. ex Woolls (Myrtaceae family) (also known as Red Ironbark, Mugga Ironbark or Mugga) is a species native to Australia where it exists in open forests in New South Wales, extending to Queensland and Victoria. It is a small to medium sized, occasionally tall, tree that can be easily recognized by its hard, deeply furrowed bark of dark grey to black color, and impregnated with kino, and by its white, pink or red flowers (Bean, 2010). The species has been introduced in various regions where it grows well withstanding dry climates, poor soils and frost, sometimes used in arid zones (Jayawickrama et al., 1993)

∗ Corresponding author. E-mail address: [email protected] (I. Miranda).

E. sideroxylon has a hard and dense wood (sideroxylon means iron wood) with high durability that is used in construction and outdoor uses e.g., sleepers, posts, piers, boatbuilding. A few phytochemical and toxicological studies were carried out on extracts of E. sideroxylon heartwood and leaves in relation to chemical composition and biological activities (Hillis and Hasegawa, 1962; Hillis and Isoi, 1965; Hart and Hillis, 1974; Hillis et al., 1974), as well as on its essential oil (Ahmadouch et al., 1985; Dellacassa et al., 1990; Satoh et al., 1992; Ashour, 2008; Vuong et al., 2015). Little information was found on the bark of this species. The mature bark of E. sideroxylon is persistent to the small branches, hard and deeply furrowed, dark brown to black (Boland et al., 1992). This species belongs to the “Ironbark” group of eucalypts with others e.g., Eucalyptus paniculata, Eucalyptus crebra, or Eucalyptus siderophloia as described by Chattaway (1955a) as a furrowed bark, with cracks extending through the rhytidome to the outermost phloem. Information on the bark structure of E. sideroxylon is however scarce in spite of the numerous anatomical studies of barks in the genus Eucalyptus (Chattaway, 1955b,c,d; Alfonso, 1987; Quilhó et al., 1999, 2000). The presence of kino in E. sideroxylon bark is well recognized (Boland et al., 1992). Kino is a wood exudate found in many Myrtaceae species including the eucalypts that is characterized by a deep rich coloring, with high polyphenols and tannin content, and

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astringency. Tippett (1986) studied the pathological anatomy of 28 Eucalyptus species that exhibited kino veins in the phloem, including E. sideroxylon. The mechanism of kino formation was recently reviewed (Locher and Currie, 2010) and still a matter of discussion. A number of kinos have been used for centuries as astringents for control of diarrhea and the Australian aborigines used eucalypt kinos to tan skins into leather (Hillis 1986, 1989; Von Martius et al., 2012; Locher et al., 2013). The tannin content of E. sideroxylon bark has also been considered namely for production of adhesives (Fechtal and Riedl, 1993). In recent years, the composition of bark extractives has been studied for several Eucalyptus species, focusing on the extraction of phenolic compounds as natural antioxidants in Eucalyptus globulus (Vázquez et al., 2012; Santos et al., 2012 and Mota et al., 2012; Conde et al., 1995), Eucalyptus camaldulensis and Eucalyptus rudis (Cadahía et al., 1997; Conde et al., 1996), Eucalyptus exserta (Li and Xu, 2012), Eucalyptus astringens, Eucalyptus cladocalyx, Eucalyptus occidentalis and E. sideroxylon (Fechtal and Reidl, 1991) as well as on the lipophilic composition of bark extracts in E. globulus (Domingues et al., 2010; Freire et al., 2002) E. grandis, Eucalyptus urograndis and Eucalyptus maidenii (Domingues et al., 2011). Studies on bark as a feedstock for fermentable sugars of Eucalyptus urophylla × Eucalyptus grandis and E. grandis clones were also made (Bargatto, 2010; Lima et al., 2013). Bark as potential fiber supply to the pulp industry was studied for E. globulus (Miranda et al., 2013). In this paper we describe the anatomy and chemical composition of E. sideroxylon bark with the objective to analyze its potential within a biorefinery route of bark use, namely related with the presence of extractives, and their high phenolic content and antioxidant properties. 2. Material and methods The bark of E. sideroxylon was taken from three trees harvested with 6 years of age, with an average breast height diameter of 16.2 cm, from an eucalypt arboretum located in the fields of the School of Agriculture, University of Lisbon (ULisboa), at Tapada da Ajuda, Lisboa, Portugal (38◦ 42 N; 09◦ 10 W). The bark was separated manually from a disc taken at breast height. 2.1. Microscopic observations The bark samples were impregnated with DP 1500 polyethylene glycol. Transversal, tangential and radial sections of approximately 17 ␮m thickness were prepared with a Leica SM 2400 microtome using Tesafilm 106/4106 adhesive for sample retrieval (Quilhó et al., 1999). The sections were stained with a triple staining of chrysodine/acridine red and astra blue and mounted on Eukitt. Light microscopic observations were made using Leica DM LA and photomicrographs were taken with a Nikon Microphot-FXA. The terminology follows Richter et al. (1996). 2.2. Chemical characterization The bark samples were fractionated using a cutting mill (Retsch SM, 2000) with an output sieve of 1 × 1 mm2 . The ground material was sieved in a vibratory apparatus, and the 40–60 mesh fraction was used for chemical analysis. Mineral content was calculated gravimetrically according to TAPPI Standard Method (T 211 om-93). 2 g of the bark material was incinerated in a muffle furnace at 525 ◦ C overnight and the combustion residue was weighed and reported as ash content of the original sample. Extractives were determined by successive Soxhlet extraction of approximately 2 g of the sample with dichloromethane, ethanol and water during 6 h, 16 h and 16 h respectively. The extractives

solubilized by each solvent were determined using the mass difference from the mass of the solid residue after drying at 105 ◦ C, and reported as percent of the original sample (TAPPI T204 om-88). Suberin content was determined on 1.5 g of the extractivefree sample by refluxing with 100 mL of a 3% NaOCH3 solution in CH3 OH during 3 h (Pereira, 1988). The sample was filtrated, washed with methanol, again refluxed with 100 mL CH3 OH for 15 min and filtrated. The combined filtrates were acidified to pH 6 with 2 M H2 SO4 and evaporated to dryness. The residue was suspended in 50 mL water and the alcoholysis products recovered with dichloromethane in three successive extractions, each with 50 mL dichloromethane. The combined extracts were dried over anhydrous Na2 SO4 and evaporated to dryness. The suberin extracts, that include the fatty acid and fatty alcohol monomers of suberin, were quantified gravimetrically, and the results expressed in percent of the initial dry mass. The determination of lignin content was made by acid hydrolysis as Klason lignin (TAPPI T222 om 98) and acid soluble lignin (TAPPI UM 250). Klason lignin was determined on 0.35 g of the material after suberin removal that were treated with a 72% sulphuric acid solution in a 30 ◦ C water-bath for 1 h, after which the acid concentration was reduced to 3% with water and the hydrolysis was completed in an autoclave at 120 ◦ C (1.2 bar) for 3 h. The reaction mixture was filtered in a G3-porosity glass filter, the residue, dried in an oven at 105 ◦ C and weighed as the Klason lignin. Aliquot from the aqueous acidic filtrate was used for soluble-lignin determination by spectroscopy at 250 nm wavelength (absorptivity coefficient of 110 g L−1 cm−1 ). The remainder of the acidic solution was kept for sugar analysis. Klason lignin and acid-soluble lignin were reported as percent of the original sample and combined to give the total lignin content. The polysaccharides were estimated by determining the neutral monosaccharides monomers released by the total acid hydrolysis used for lignin determination in the extractive-free and suberinfree samples. The neutral sugar monomers were determined by high performance anion exchange chromatography (HPAEC) using a Dionex ICS-3000 system equipped with an electrochemical detector. The separation was performed with Aminotrap plus CarboPac SA10 anion-exchange columns. The mobile phase was an aqueous 2 nMNaOH solution at a flow rate of 1.0 mL/min at 25 ◦ C. The polysaccharide content was determined by the sum of the individual sugar masses. 2.3. Phenolic content of the bark extract Approximately 1 g of the ground bark was extracted with ethanol/water (50/50, v/v) with a solid–liquid ratio 1:10 (m/v) for 60 min at 50 ◦ C using an ultrasonic bath. The insoluble materials were removed by filtration and the supernatant extract was stored at 4 ◦ C. The solid residue was dried and the extraction yield was calculated as the percent mass loss of the starting material. Total phenolic content was determined by the Folin–Ciocalteu method using gallic acid as standard (Singleton and Rossi, 1965). An aliquot (100 ␮L) of the extract was mixed with 4 mL of the Folin–Ciocalteu reagent and after 6 min, 4 mL of a 7% Na2 CO3 solution was added. After 15 min of incubation in a bath at 45 ◦ C, absorbance at 760 nm was read versus a prepared blank. A calibration curve was built using gallic acid as a standard (0–150 ␮g/mL). The total phenolic content was expressed as milligrams of gallic acid equivalents (GAE)/g of the dry bark extract. Total flavonoids content was determined by an aluminium chloride colorimetric assay (Zhishen et al., 1999). An aliquot (1.0 mL) of the extract was mixed with 4.0 mL of deionized water followed by 0.3 mL of a 5% NaNO2 solution. After 5 min, 0.3 mL of a 10% AlCl3 ·6H2 O solution was added to the mixture. After 5 min, 2.0 mL of 1 M NaOH solution was added, and the total volume was adjusted

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to 10.0 mL with deionized water. The absorbance was measured at 510 nm, corrected using a blank, and the results were expressed as mg of (+)-catechin equivalents (CE)/g of the dry bark extract. Tannins content was determined by the vanillin-H2 SO4 method (Sun et al., 1998). An aliquot (1.0 mL) of the extract was mixed with 2.5 ml of 1.0% (m/v) vanillin in absolute methanol and then with 2.5 mL of 25% (v/v) sulphuric acid in absolute methanol for vanillin reaction with the polyphenols in the extract. The blank solution was prepared in the same procedure without vanillin. Absorbances were recorded at 500 nm after 15 min, and the results were expressed as mg of (+)-catechin equivalents (CE)/g of the dry bark extract. 2.4. Antioxidant activity of the bark extract The antioxidant activity of the bark ethanol/water extract was determined using 2,2-diphenyl-1-picrylhydrazyl hydrate(DPPH) and expressed in terms of the amount of extract required to reduce by 50% the DPPH concentration (IC50 ) and in terms of Trolox equivalents (TEAC) on a dry extract or bark base (mg Trolox/g dry mass) First, different dilutions of the initial extract and of Trolox (0.2 mg/mL) in methanol were prepared. An aliquot of 100 ␮L of each methanolic solution of the extract and Trolox was added to 3.9 mL of a DPPH methanolic solution (24 ␮g/mL). The blank sample consisted of 100 ␮L of methanol added to 3.9 mL of DPPH solution. After 30 min incubation at room temperature in the dark, the absorbance was measured at 515 nm. The radical scavenging activity of each sample was calculated by the DPPH inhibition percentage as follows: I% = [(Abs0 − Abs1 )/Abs0 ] × 100, where Abs0 was the absorbance of the blank and Abs1 was the absorbance in the presence of the extract at different concentrations. The IC50 inhibiting concentration, which represents the concentration of a sample necessary to sequester 50% of the DPPH radicals, was obtained by plotting the inhibition percentage against the extract concentration. The scavenging effect on the DPPH radical of the extract was also expressed as the Trolox equivalent antioxidant capacity (TEAC) calculated from the calibration curve with the Trolox solution concentrations and the percentage of scavenging effect on the DPPH radical as: TEAC(mgTroloxequivalent/mgextractorbark)

Fig. 1. Bark of Eucalyptus sideroxilon. (For interpretation of the references to color in the text, the reader is referred to the web version of this article.)

HCl; 2.5 mL 20 mmol/L FeCl·6H2O) were added. The absorbance was measured in comparison with a blank at 593 nm. Aqueous solutions of known Fe(II) concentrations in the range of 0–1500 ␮mol/L (FeSO4 ·7H2 O) were used for the calibration curve, and the results were expressed as ␮mol Fe(II)/g dry mass. 3. Results and discussion

= [(%inhibitionsample + n)/s × m], where n and s represent the intercept and the slope of the Trolox calibration curve for DPPH and m is the sample amount in mg dry basis. The FRAP (ferric reducing antioxidant power) assay was followed as described by Benzie and Strain (1996), with modifications. 100 ␮L bark extract solutions and 300 ␮L of distilled water were added to 3.0 mL of the FRAP reagent (25 mL acetate buffer, 300 mmol/L, pH 3.6 (acetic acid and sodium acetate); 2.5 mL 10 mmol/L 2,4,6-tripyridyl-s-triazine (TPTZ) in 40 mmol/L Table 1 Summative chemical composition (% of total dry mass) of the bark of Eucalyptus sideroxylon. Mean Ash Extractives total Dichloromethane Ethanol Water Suberin Lignin total Klason lignin Soluble lignin

1.26 55.74 1.72 20.10 33.93 1.92 13.11 12.11 1.01

± ± ± ± ± ± ± ± ±

0.48 3.51 0.08 0.84 3.02 0.11 1.26 0.19 1.15

3.1. Structure and anatomy The bark of E. sideroxylon is thick with an average 4.6 cm, and a conspicuous appearance showing considerable furrows that reveal the underlying reddish brown phloem; at the outside, a dark colored periderm is fractured as scales (Fig. 1). This is consistent with the descriptions of a persistent E. sideroxylon bark, hard and deeply furrowed, dark brown to black (Boland et al., 1992). This species belongs to the “Ironbark” group of eucalypts described by Chattaway (1955a) as having a furrowed bark, due to cracks which extend through the rhytidome to the outermost phloem. The main anatomical features are summarized in Fig. 2. The periderm includes a thin phellem with two types of cells (suberized cells that were stained with sudan and lignified cells) and a poorly developed phelloderm (Fig. 2A). The non-collapsed phloem is thin and shows tangential layers of fibers that alternate with thin layers of axial parenchyma cells and sieve tube elements (Fig. 2B); the collapsed phloem includes some dilatation tissue as a result of tree growth i.e., expanded axial parenchyma cells (Fig. 2C). The rays are mainly uniseriate (Fig. 2D) and homogenous (Fig. 2E) and clusters of expanded parenchyma cells and sclereids are present (Fig. 2E). Large pockets of kino were observed formed by the breaking down of tissues of the outer phloem (Fig. 3). This has been pointed

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Fig. 2. Microscopic structure of Eucalyptus sideroxylon bark. (A)—periderm including the lignified (lig) and suberized (sub) cells of the phellem and 1 cell of the phelloderm (Fm); deposits of suberin (arrow). (transverse section) (B)—phloem with non-collapsed phloem and collapsed phloem (transverse section); (C)–(E)—collapsed phloem (C), transverse section (D), tangential section (CEX, expanded parenchyma cells) (E), radial section (SC, sclereids). Scale bar = 50 ␮m.

out by Chattaway (1955a) for this species. Strong impregnations of kino in the bark related with its hardness were also mentioned by Cunningham et al. (2011). In spite of its obvious singular external appearance, the bark of E. sideroxylon at this age has a similar structure to barks of other young eucalypt species, in accordance with observations of Chattaway (1955b,c,d), Quilhó et al. (1999) and Alfonso (1987). 3.2. Chemical composition

Fig. 3. Large pockets in the outer phloem of the Eucalyptus sideroxylon. Scale bar = 100 ␮m.

The chemical composition of E. sideroxylon bark is summarized in Table 1. A comparative table of the present results with other published data for other Eucalyptus species is included (Table 2). E. sideroxylon bark shows a remarkable high content of extractives, amounting to 55.7% of the initial dry bark. The polar compounds extracted by ethanol and water represent about 97% of the total extractives. The lipophilic compounds soluble in dichloromethane represented only 3% of the total extractives. This content in extractives is well above the values found in the bark of other eucalypt species: e.g., 28.1% and 28.3% for E. grandis × urophylla and E. grandis, respectively (Lima et al., 2013), 6.5% and 6.0% for E. globulus (Miranda et al., 2013; Neiva et al., 2014) or between 3.5% and 9.5% for Eucalyptus salignaa, E. grandis, E. urophylla, E. camaldulensis, Eucalyptus citriodora, Eucalyptus paniculata and Eucalyptus pellita (Andrade et al., 2010). It is worth pointing out that the wood of these same trees had also a high content of extractives, corresponding to 13.5% of the wood, where the hydrophilic extractives were also dominant (Neiva et al., 2015). The high content of polar extractives is clearly related with the anatomical features of the bark and with the presence of the kino pockets (Fig. 3), and in line with the very dark colored bark (Fig. 1).

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Table 2 Results of total extractives suberin and total lignin for Eucalyptus sideroxylon bark and results given in literature for barks of other Eucalyptus species (% of total dry mass). Species

Extractives

Suberin

Lignin

Authors

E. sideroxylon E. camaldulensis E. citriodora E. globulus

55.7 23.5 13.0 6.5 6.0 6.3–8.5 – – 28.3 26.6 – 18.0 28.1 25.8 20–24 13.5 16.0 24.0 17.0

1.9 – – 0.9 – – – – – – – – – – – – – – –

13.1 23.5 24.0 29.2 18.5 18.6 19.2 28.0 21.6 14.7 26.2 22.0 22.2 19.7 21–24 37.0 32.0 27.0 23.0

Present work Andrade et al. (2010) Andrade et al. (2010) Miranda et al. (2013), Neiva et al. (2004), Sakai (2001), Vázquez et al. (2008) and Yadav et al. (2002)

E. grandis

E. grandis × urophylla

E. paniculata E. pellita E. saligna E. urophylla

Table 3 Monosaccharide composition of the bark of Eucalyptus sideroxylon, in % of total neutral monosaccharides detected by HPLC. Monosaccharide

% Of neutral sugars

Glucose Xylose Galactose Arabinose Mannose Rhamnose

80.01 11.06 4.25 2.42 1.96 0.20

± ± ± ± ± ±

1.32 0.83 0.16 0.36 0.28 0.06

E. sideroxylon bark contained 1.9% suberin (4.6% reported to extractive-free bark). This value is similar to what was reported for E. globulus bark (0.98%, Miranda et al., 2013). The low content in suberin is a consequence of the anatomical structure of the bark that showed a small amount of suberized phellem tissue in the periderm (Fig. 2) Total lignin represented 13.1% of the bark, corresponding to 29.6% of the extractive-free bark. The extent of lignification was not substantially different from barks of other eucalypt species, e.g., between 18.5% and 25.8% in E. globulus (Sakai, 2001; Vázquez et al., 2008; Miranda et al., 2013; Neiva et al., 2014), 21.6% and 22.2% respectively for E. grandis × urophylla and E. grandis (Lima et al., 2013) and 27% in E. saligna, E. grandis (22%), E. urophylla (23%), E. camaldulensis (23.5%), E. citriodora (24%), E. paniculata (37%) and E. pellita (32%) (Andrade et al., 2010). The composition of neutral sugars released by total hydrolysis is presented in Table 3. Glucose content dominates corresponding to 80.1% of total monosaccharides with xylose as the second most important sugar (11.1% of total monosaccharides). The monomeric composition of polysaccharides is similar to the composition of other eucalypt barks in relation to the predominance of glucose and of a substantial xylose content, although the individual compositions vary somewhat with species, as shown by e.g., Miranda et al. (2013) and Vázquez et al. (2008) for E. globulus, Bargatto (2010) for E. grandis × E. urophylla and E. grandis barks. The ash content of bark was 1.26%, which is low in relation to some previously reported values for eucalypt barks, e.g., in E. globulus 4.7% (Vázquez et al., 2008), 2.9% (Miranda et al., 2013) and 2.3% (Mota et al., 2012). Bargatto (2010) reported 4.1% and 7.1% ash content for E. grandis × urophylla and E. grandis barks, respectively.

Lima et al. (2013), Bragatto (2010), Yu et al. (2010) and Andrade et al. (2010)

Lima et al. (2013), Bragatto (2010) and Andrade et al. (2010) Andrade et al. (2010) Andrade et al. (2010) Andrade et al. (2010) Andrade et al. (2010)

Table 4 Extraction yield, total phenolic content, tannins and flavonoids content and antioxidant activity of Eucalyptus sideroxylon bark extract by DPPH radical scavenging, expressed as IC50 value, in ␮g extract/mL and as mg of Trolox equivalents/g of dry extract and bark, and the for FRAP antioxidant activity in mM Fe2+ /g of bark. Extraction yield (%) Total phenolic content (mg GAE/g of extract) Tannins (mg catechin/g of extract) Flavonoids (mg catechin/g of extract) Antioxidant capacity TEAC (mg Trolox/g of extract) Antioxidant capacity TEAC (mg Trolox/g of bark) IC50 values (␮g extract/mL) IC50 Trolox in ethanol–water (␮gTrolox/mL) FRAP (mM Fe2+ /g of extract) FRAP (mM Fe2+ /g of bark)

50.04 ± 8.46 440.70 ± 35.15 395.01 ± 51.96 204.36 ± 26.38 648.79 ± 4.75 317.92 ± 1.74 2.25 ± 0.77 2.90 5247.01 ± 446.05 87692.31 ± 10734.52

3.3. Phenolic content in the bark extracts The yield of ethanol–water extraction and the quantification of polyphenols are given in Table 4. The extraction yield of 50.0% was very similar to the content of the polar extractives determined by sequential solvent extraction (54.0%, Table 1), thereby confirming the very high content of soluble material in this bark and the possibility of their almost complete removal. The phenolic and polyphenolic nature of the extract is shown by the high contents of total phenolics, flavonoids and tannins (Table 4). Total phenolics correspond to 440.7 mg GAE/g extract (219.9 mg GAE/g of bark), tannins to 395.0 mg CE/g extract (194.4 mg CE/g of bark) and flavonoids to 204.3 mg CE/g extract (100.6 mg CE/g of bark). These values are significantly higher when compared to those found in the literature for the total phenolics content in extracts of Eucalyptus barks. Luis et al. (2014) referred for ethanol extract of E. globulus stump bark 253.1 mg GAE/g of extract, and Vázquez et al. (2008) reported for E. globulus bark extracted with ethanol:water and methanol:water 223 mg GAE/g of extract and 201 mg GAE/g of extract respectively. Santos et al. (2012) found for methanol:water bark extracts of E. grandis, E. urograndis and E. maidenii values in the range of 203.9 and 385.6 mg GAE/g of extract. Puttaswamy et al. (2014) reported for Eucalyptus tereticornis bark 198 mg GAE/g aqueous methanolic extract. If reported in relation to bark mass, the phenolics content of E. sideroxylon bark extract is well above those reported in the literature: Cadahía et al. (1997) referred for E. camaldulensis, E. globulus and E. rudis barks, respectively, 93.3, 22.8 and 3.4 mg GAE/g of bark and Conde et al. (1996) for E. camaldulensis,

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E. globulus and E. rudis barks referred values in the range of 2.5 and 91.6 mg/g of bark. Total flavonoids concentration was higher than the values found in the literature: Luis et al. (2014) reported for E. globulus stump bark 8.5 mg quercetin equivalent/g extract in ethanol:water extract and Puttaswamy et al. (2014) for E. tereticornis bark 160 ␮g rutin/mg of extract. The tannins content in the extract of E. sideroxylon bark was also very high when compared to the values found in the extracts of other eucalypt barks: 29.0 mg GAE/g extract in ethanol:water E. globulus stump extracts (Luis et al., 2014), 39.2, 7.4 and 0.5 mg CE/g of bark in the methanolic extract of E. camaldulensis, E. globulus and E. rudis barks (Cadahía et al., 1997), and 103 ␮g tannic acid/mg extract in the methanolic extract of E. tereticornis bark (Puttaswamy et al., 2014) These results clearly confirm the potential of E. sideroxylon bark as a source of tannins and other phenolics, as shown by its use based on empirical knowledge of the Australian aborigines. 3.4. Anti-oxidant activity of bark extracts Two methods were applied to determine antioxidant activity of the bark extract: the DPPH assay to determine the free radical scavenging activity and the FRAP assay to determine the ferric ion reducing power. The free radical-scavenging activity of E. siderolylon bark extract was expressed in terms of the amount of extract required to reduce by 50% the DPPH concentration (IC50 ) and also in terms of Trolox equivalents (TEAC) on a dry extract base (mg Trolox/mg extract). The results show that the extract of E. sideroxylon bark has a strong free radical scavenging activity with an IC50 value of 2.25 ␮g/mL, as compared to Trolox (IC50 of 2.90 ␮g/mL) used as standard. This antioxidant activity is significantly higher than that reported for E. grandis. E. urograndis and E. maidenii bark MeOH·H2 O extracts for which the IC50 values were, respectively, 6.26 ␮g/mL, 6.14 ␮g/mL and 8.24 ␮g/mL compared with 2.17 ␮g/mL for ascorbic acid and 18.22 ␮g/mL for BHT (3,5-di-tert-4-butylhydroxytoluene) (Santos et al., 2012). Luis et al. (2014) determined the scanvenging activity of the hydroalcoholic extract of E. globulus stump bark that showed comparatively less antioxidant activity with an IC50 value of 11.32 ␮g/mL (compared with 2.23 ␮g/mL for gallic acid and 4.32 ␮g/mL for quercetin). The antioxidant activity expressed as mg of Trolox/g of bark (317.92 mg Trolox/g of bark, corresponding to 648.79 mg Trolox/g extract) show also values significantly higher than those reported for E. grandis. E. urograndis and E. maidenii bark extracts expressed in terms of ascorbic acid equivalents (AAE) on a bark basis,respectively, 36.73, 53.18 and 34.45 mg AAE/g bark (Santos et al., 2012) or for the barks of other species as Quercus suber cork extracts between 9.15 and 15.59 mg AAE/g cork (Santos et al., 2010). The reducing ability of the extract was 5247.0 mM Fe2+ /g extract (corresponding to 1384.49 mM Fe2+ /g bark). Significantly, lower FRAP antioxidant activity, expressed in ascorbic acid equivalentes, were obtained for E. globulus bark extracts using organic solvents of different polarity, water and an alkaline aqueous solution, in the range of 4.70–11.96 mmol ascorbic acid equivalent (AAE)/100 g bark (Vázquez et al., 2012). Boni et al. (2014) evaluated the antioxidant activity of aqueous and methanolic extract of bark of Spondias mombin (Anacardiaceae) reporting values for ferric reducing antioxidant properties (FRAP) of 266 and 456.8 ␮mol Fe2+ /g of extract using ascorbic acid as a reference 2056.10 ␮mol Fe2+ /g. For aqueous extracts from barks of Cinnamomum zeylanicum and Pinus maritima, respectively values of 309.23 and 360.76 mg gallic acid equivalent (GAE)/g extract for total phenols and 6.48 and 6.45 mmol Fe2+ /g of extract for the FRAP antioxidant activity (Dudonné et al., 2009).

These results allow considering E. sideroxylon bark as an interesting source of phenolics and of antioxidant extracts 4. Conclusions E. sideroxylon bark was characterized for the first time as regards the anatomical structure and chemical composition. The striking chemical feature of E. sideroxylon bark is the high content of extractives, especially of polar extractives that are rich in phenolics, namely flavonoids and tannins. Extracts may be obtained in very high yields and the crude solution show high anti-oxidant activities. This bark can therefore be considered as a source of polar extractives with a potential valorization based on their chemical functionalities and bioactivity. Acknowledgments This work is part of the research activities of Centro de Estudos Florestais (CEF), a research unit supported by FCT—Fundac¸ão para a Ciência e a Tecnologia (UID/AGR/00239/2013) and of the research project EucPlus—New processes and uses for eucalypt woods (PTDC/AGR-CFL/119752/2010). We thank Dr. Paula Soares for providing the samples and the information about the trial, which was sponsored by CELPA – Associac¸ão da Indústria Papeleira (Portuguese Pulp and Paper Industry Association) as well as the assistance of Marília Pirralho for the anatomical observations. References Ahmadouch, A., Bellakdar, J., Berrada, M., Denier, C., Pinel, R., 1985. Chemical analysis of essential oils from five species of Eucalyptus acclimatized in Morocco. Fitoterapia 56, 209–220. Alfonso, V., 1987. Anatomical characterization of wood and bark of the main species of Eucalyptus L’Herit, cultivated in Brazil. In: Ph.D. Thesis. Instituto de Biociências da Universidade de Sao PauIo, Brazil. Andrade, M.C.N., Minhoni, M.T.A., Sansígolo, C.A., Zied, D.C., 2010. Chemical analysis of the wood and bark of different Eucalyptus types before and during the shiitake cultivation. Rev. Árvore 34, 165–175. Ashour, H.M., 2008. Antibacterial, antifungal, and anticancer activities of volatile oils and extracts from stems, leaves, and flowers of Eucalyptus sideroxylon and Eucalyptus Torquata. Cancer Biol. Ther. 7, 399–403. Bargatto, J., 2010. Evaluation of the potential use of Eucalyptus spp. bark for bioethanol production. In: Ph.D. Thesis. Universidade de S. Paulo, Escola Superior de Agricultura Luiz de Queirós, Piracicaba, Brazil. Bean, A.R., 2010. A new subspecies of Eucalyptus sideroxylon A. Cunn. ex Woolls (Myrtaceae) from Queensland. Austrobaileya 8, 139–141. Benzie, I.F.F., Strain, J.J., 1996. The ferric reducing ability of plasma (FRAP) as a measure of antioxidant power: the FRAP assay. Anal. Biochem. 239, 70–76. Boland, D.J., Brooker, M.I.H., Chippendale, G.M., Hall, N., Hyland, B.P.M., Johnston, R.D., Kleinig, D.A., Turner, J.D., 1992. Forest Trees of Australia. CSIRO Publishnig, Collingwood, Victoria, Australia. Boni, A.N.R., Ahua, K.M., Kouassi, K., Yapi, H., Djaman, A.J., Nguessan, J.D., 2014. Comparison of in-vitro antioxidant activities and total phenolic contents in water and methanol extracts of stems sark of Spondias mombin. Res. J. Pharm. Biol. Chem. Sci. 5, 1457–1468. Cadahía, E., Conde, E., Simón, B.F., García-Vallejo, M.C., 1997. Tannin composition of Eucalyptus camaldulensis, E. globulus and E. rudis. Part II. Bark. Holzforchung 51, 125–129. Chattaway, M.M., 1955a. The anatomy of bark. VI. Peppermints, boxes, iron barks, and other eucalypts with cracked and furrowed barks. Aust. J. Bot. 3, 170–176. Chattaway, M.M., 1955b. The anatomy of bark II. Oil gland in Eucalyptus species. Aust. J. Bot. 3, 23–27. Chattaway, M.M., 1955c. The anatomy of bark. III. Enlarged fibres in bloodwoods (Eucalyptus spp.). Aust. J. Bot. 3, 28–38. Chattaway, M.M., 1955d. he anatomy of bark.IV. Radially elongated cells in the phelloderm of species of Eucalyptus. Aust. J. Bot. 3, 39–47. Conde, E., Cadahia, E., García-Vallejo, M.C., Tomas-Barberan, F., 1995. Low molecular weight polyphenols in wood and bark of Eucalyptus globulus. Wood Fiber Sci. 27 (4), 379–383. Conde, E., Cadahia, E., Díez, R., García-Vallejo, M.C., 1996. Polyphenolic composition of bark extracts from Eucalyptus camaldulensis, E. globulus and E. rudis. Holz Roh Werkst. 54, 175–181 http://www.researchgate.net/journal/ 0018-3768 Holz als Roh-und Werkstoff. Cunningham, G.M., Mulhan, W.E., Milthorpe, P.L., Leigh, J.H., 2011. Plants of Western New South Wales. CSIRO Publishnig, Collingwood, Victoria, Australia. Dellacassa, E., Menéndez, P., Moyna, P., Soler, E., 1990. Chemical composition of Eucalyptus essential oils grown in Uruguay. Flavour Fragance J. 5, 91–95.

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Please cite this article in press as: Miranda, I., et al., The bark of Eucalyptus sideroxylon as a source of phenolic extracts with anti-oxidant properties. Ind. Crops Prod. (2015), http://dx.doi.org/10.1016/j.indcrop.2015.12.003