Green approaches for the extraction of antioxidants from eucalyptus leaves

Industrial Crops & Products 138 (2019) 111473

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Green approaches for the extraction of antioxidants from eucalyptus leaves

T

Beatriz Gullón, Abel Muñiz-Mouro, Thelmo A. Lú-Chau, María Teresa Moreira, Juan M. Lema, ⁎ Gemma Eibes Dept. of Chemical Engineering, Institute of Technology, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain

A R T I C LE I N FO

A B S T R A C T

Keywords: Microwave Ultrasounds Deep eutectic solvents Phenolic composition Eucalyptus leaves Antioxidants

Leaf extracts of Eucalyptus globulus present a wide range of phenolic compounds with interest in the pharmaceutical, health, agricultural, cosmetic and food industries because of their medicinal properties. Although conventional solvent extraction is the most extensively used methodology to extract and isolate these active compounds, the excessive consumption of time and energy makes its application relatively inefficient. This study investigates other more cost-effective and environmentally friendly techniques for the isolation of antioxidant phenolic compounds, such as enzyme assisted extraction (EAE), microwave assisted extraction (MAE), ultrasound assisted extraction (UAE), and deep eutectic solvent (DES) extraction. First, the conventional extraction was used to evaluate the effect of the particle size and the liquid-to-solid ratio. Next, relevant parameters of the green extraction techniques were optimized, such as time of extraction, enzyme and dose used, microwave power, and type of DES. The optimized extracts from MAE, UAE, DES and conventional extraction were characterized by UHPLC-ESI-MS, showing a similar phenolic profile with 26 tentatively identified compounds. Finally, the conditions leading to the extracts with the highest content in phenolics were evaluated in terms of specific energy consumption. The lowest specific energy consumption was obtained with MAE, with a value more than 13 times lower than conventional extraction.

1. Introduction In recent years there has been a considerable growing interest in secondary plant metabolites associated with their potential use in the food and pharmaceutical industries as natural antioxidants to replace synthetic additives. In parallel, awareness on food safety has created concern on synthetic antioxidants, which have been the subject of controversy due to their long-term toxicological potential associated with their regular intake as an industrial additive (Shahidi and Ambigaipalan, 2015). In this context, it is justifiable that the demand for natural antioxidants has increased dramatically, so that the market for these products is 50% higher than for their synthetic counterparts (Berdahl et al., 2010). Eucalyptus globulus, a member of the Myrtaceae family, is a tall, perennial tree originating in Australia, which has been cultivated extensively in subtropical and Mediterranean regions. Due to its fast growth and the density of its wood, E. globulus is widely used in papermaking. In the European Union, Spain and Portugal are major producers of eucalyptus, with a total combined cultivated area of 1.4 million ha. These eucalyptus plantations generate a significant amount of biomass residues in the form of branches and leaves, estimated at 2.8

⁎

million tons per year for Spain and Portugal (Silva-Fernandes et al., 2015). Beyond its use in the production of paper pulp, there are other opportunities for the valorization of eucalyptus waste. Based on the biorefinery concept, alternatives have been proposed for the synthesis or separation of minority components present in this type of wastes, ranging from chemical commodities to byproducts with high added value (Mota et al., 2012). E. globulus leaves present a wide range of compounds, including monoterpenes, such as 1,8-cineole, sesquiterpene-phloroglucinol derivatives, flavonoids, tannins and related polyphenols (Amakura et al., 2009). Eucalyptus extracts and essential oils have been used in the pharmaceutical, health, agricultural, cosmetic and food industries because of their medicinal properties (Vecchio et al., 2016). Indeed, looking back on the traditional use of eucalyptus leaves, traditional remedies for the treatment of various diseases such as pulmonary tuberculosis, influenza, fungal infections and diabetes have been formulated from its active ingredients (Boulekbache-Makhlouf et al., 2013a,b). Furthermore, the ethanolic extract of eucalyptus leaves presents a strong antioxidant capacity, confirming its usefulness as a natural antioxidant food additive (Amakura et al., 2009).

Corresponding author. E-mail address: [email protected] (G. Eibes).

https://doi.org/10.1016/j.indcrop.2019.111473 Received 21 December 2018; Received in revised form 7 June 2019; Accepted 10 June 2019 0926-6690/ © 2019 Elsevier B.V. All rights reserved.

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2.2. Conventional extraction

Investigating between the different possibilities for the extraction and isolation of active compounds such as polyphenols, conventional solvent extraction emerges as the most extensively used methodology used for more than a century. However, the excessive consumption of time and energy makes its application relatively inefficient, not to mention the need to completely recover the solvent to avoid contamination of the final product (Ameer et al., 2017). These disadvantages have triggered researchers to explore more cost-effective and environmentally friendly techniques for extracting phenolics from a wide range of plant matrices. The application of enzymes for the extraction of bioactive ingredients from plants appears as an attractive alternative. Enzyme-assisted extraction (EAE) can save processing time and energy, and potentially provide a more reproducible extraction process on a commercial scale (Puri et al., 2012). On the other hand, microwave-assisted extraction (MAE) is gaining merits due to higher extraction yields, lower processing costs and higher quality of the final product (Nayak et al., 2015; Pongmalai et al., 2015). Another green alternative is the ultrasound-assisted extraction process (UAE), in which the use of ultrasound can enhance the extraction process by increasing mass transfer between the solvent and the plant material (Vilkhu et al., 2008). Recently, deep eutectic solvents (DES), which consist of a mixture of organic compounds with a significantly lower melting point than either of the two individual components, have been proposed as versatile alternatives to common organic solvents (Paiva et al., 2014). The main advantages of DES are their availability, low cost, biodegradability and environmental friendliness of the components (Duan et al., 2016). In particular, for the extraction of bioactive compounds present in eucalyptus leaves, extraction with conventional solvents is the most common method used (Gullón et al., 2017; Boulekbache-Makhlouf et al., 2013a,b). In fact, there are few bibliographical references that address extraction processes under the perspective of green technologies. Microwave (Bhuyan et al., 2015) and ultrasound (Bhuyan et al., 2017) assisted extraction of bioactive compounds from E. robusta leaves has been previously reported. In the particular case of E. globulus leaves, only recently has supercritical fluid extraction been reported (Rodrigues et al., 2018). The main aims of the present study were to compare the suitability of several green extraction methods such as EAE, MAE, UAE and the use of DES for the extraction of phenolic compounds from eucalyptus leaves. In a first series of research, conventional extraction was used to evaluate the effect of the particle size and the liquid-to-solid ratio. The relevant parameters of these green extraction techniques were then evaluated and the extracts were characterized in terms of their polyphenolic profile. Finally, the conditions leading to the extracts with the highest content in phenolics, were evaluated in terms of specific energy consumption.

The effect of three particle sizes was evaluated: i) small (S): leaves were grounded in a coffee mill and sieved to obtain particles with d < 0.5 mm; ii) medium (M): leaves were crushed in a domestic grinder to obtain particles in the range 0.5 < d < 2 mm; ii) large (L): leaves were manually cut with scissors with dimensions of 10 × 10 mm. Eucalyptus leaves (2 g) were extracted in an orbital shaker (Adolf Kühner AG, Birsfelden, Switzerland) at 50 °C, 120 rpm, using aqueous ethanol as solvent (56%, v:v) and for 225 min, based on the conditions described in Gullón et al. (2017). The liquid-to-solid ratio (LSR) was varied in the range of 3–20 mL/g. The extracts were filtered through filter paper (Whatman Ashless, Grade 42) and the filtrate was stored at -20 °C until later use. 2.3. Enzyme assisted extraction Enzyme-assisted extraction experiments were performed with three enzymatic cocktails: commercial cellulase preparation (Celluclast 1.5 L), a multi-enzyme complex containing a wide range of carbohydrases, including arabanase, cellulase, β-glucanase, hemicellulase and xylanase (Viscozyme L) and another multi-enzyme complex containing mainly β-glucanase and xylanase (Ultraflo L). The selection of the enzymatic cocktails was based on previously published works (Pinelo et al., 2008; Huynh et al., 2014) using cellulase, β-glucanase, xylanase and hemicellulase for the extraction of phenolics from apple skin and cauliflower leaves. The experimental protocol was similar to the one described in the conventional extraction experiments (Section 2.2), with an LSR of 10 mL/g, 56% ethanol (v:v), 50 °C and 225 min. An enzyme/substrate ratio (ESR) of 5% (v:w) was considered according to Huynh et al. (2014). Based on the results obtained, ESR of 5 and 10% (v:w) were considered for mixtures of Celluclast 1.5 L and Viscozyme in water at 50 °C and 30 min. The experiments were carried out in duplicate. 2.4. Microwave assisted extraction A domestic microwave oven (Team International) was used for the extraction of eucalyptus leaves at a LSR of 10 mL/g, using water as solvent. The conditions for the ultrasound-assisted extraction were: 56% ethanol (v:v) and LSR of 10 mL/g. Two Erlenmeyer flasks (500 mL), with a final volume of 100 mL, were used as duplicates. Different power (low, medium-low and medium) and irradiation periods (2, 3 and 7 min) were evaluated. To prevent boiling of solvent mixtures, the maximum irradiation times were reduced to 3 and 2 min for low-medium and medium power, respectively. Evaporation losses (< 20%) were accounted for the characterization of the extracts. The experiments were performed in duplicate.

2. Materials and methods 2.5. Ultrasound assisted extraction 2.1. Materials The conditions for the ultrasound assisted extraction were: 56% ethanol (v:v), LSR of 10 mL/g and 50 °C. Erlenmeyer flasks (100 mL) with a final volume of 20 mL were used. The ultrasound extraction was carried out in an ultrasonic bath (Branson CPX 3800 H), with temperature and time control, working at a frequency of 40 kHz. The dimensions of the equipment were 290 × 150 × 150 mm, which allowed the positioning of six flasks at the same time. Different extraction times (15, 30, 45, 90 and 120 min) were evaluated. The experiments were carried out in duplicate

E. globulus leaves were harvested in March 2016 from cultivated plants located in Ourense, Spain. Samples were cleaned, air dried for 4 days to 9% average moisture, ground, sieved to separate the 0.5 mm fraction, packaged in sealed plastic bags and stored at −20 °C until use, to prevent polyphenols degradation. All the reagents were of analytical grade. Ethanol, methanol, gallic acid, rutin, trolox (6-hydroxy-2,5,7,8tetramethylchroman-2-carboxylic acid), Folin-Ciocalteu reagent, ABTS (2,2′-azino-di(3-ethylbenzothiazoline-6-suslfonic acid), TPTZ (2,4,6-tri (2-pyridyl)-S-triazine), DPPH (2,2-diphenyl-1-picrylhydrazyl), sodium carbonate, sodium acetate 3-hydrate, potassium persulfate, acetic acid, hydrochloric acid, iron(III) chloride hexahydrate and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich (Barcelona, Spain). Celluclast 1.5 L, Viscozyme L and Ultraflo L enzymes were provided by Novozymes.

2.6. Eutectic liquid extraction Four different deep eutectic solvents were investigated for the extraction of eucalyptus leaves. ChE was synthesized according to Qi et al. (2015): choline chloride and ethylene glycol (molar ratio 1:2) were 2

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spectrometry and TOF high resolution mass spectrometry (LC-TOF MS) was used for the tentative identification of the phenolic compounds present in the extracts. Extracts were diluted 50 times in ethanol:water mixtures (56%). Sample injection (5 μL) and LC separation was performed on an Elute UHPLC (Bruker Daltonics Inc, Billerica, MA). An Intensity Solo C18 column (2.1 mm × 100.0 mm, 2 μm) (Bruker Inc, Billerica, MA) was used. A binary gradient of 0.1% aqueous formic acid for mobile phase A and 0.1% formic acid in methanol for mobile phase B at a flow rate of 0.25 ml/min was applied. The linear gradient was from 5 to 100% B for the time range from 0.4 to 7 min and maintained for 5 min. Samples were ionized using an ESI source in negative ion mode. Typical operating conditions were 4000 V capillary voltage, 500 V end plate offset, 2.5 bar nebulizer pressure, 6.0 L/min dry gas, 200 °C dry heater.

heated to 80 °C for 2 h under magnetic agitation. Extractions were conducted in ChE:water mixtures (80:20, v:v). ChX, ChGlu and CaGlu were synthetized using a freeze-drying method as described by Nam et al. (2015). Briefly, each component was added to a 50 mL conical tube: choline chloride and xylitol (molar ratio 5:1), choline chloride and D-(+)-glucose (molar ratio 1:1) and citric acid and D-(+)-glucose (molar ratio 1:1), for ChX, ChGlu and CaGlu synthesis, respectively. After dissolving the mixed components in the smallest amount of distilled water, the mixture was centrifuged at 4000 rpm for 10 min, followed by cooling at −32 °C. The added water was removed by freezedrying and the resulting DESs were stored until further use. The extraction solvent consisted of the eutectic liquid dissolved in distilled water at a ratio of 7:3 (w:w). 2.7. Determination of total phenolic (TPC) and flavonoid content (TFC)

2.10. Statistical analysis Total phenols content (TPC) was determined by the Folin-Ciocalteu method (Singleton and Rossi, 1965) with slight modifications. Briefly, 0.5 mL of the diluted extracts was mixed with 2.5 mL of Folin Ciocalteau reagent previously diluted with water (1:10, v/v). After stirring in a vortex, 2.0 mL of Na2CO3 (75 g/L) was added and the mixture was shaken vigorously. The samples were incubated at room temperature for 1 h and the absorbance of each sample was measured at 760 nm. Gallic acid was the reference standard and the results were expressed as mg of gallic acid equivalents (GAE)/g dried leaf. Each assay was carried out in triplicate, and the mean value was calculated. Total flavonoid content (TFC) was measured using the method described by Blasa et al. (2005). An aliquot of diluted extracts (1 mL) was mixed with 0.3 ml 5% NaNO2. After 5 min, 0.3 mL AlCl3 (10%) was added. At 6 min, 2 mL NaOH (1 M) was added to the mixture. The samples were vigorously shaken; after 5 min of incubation, the absorbance was determined at 510 nm, considering rutin as standard. The results were expressed in mg of rutin equivalents (RE)/g dried leaf as an average of three replicates.

A statistical analysis was conducted for the correct comparison of the results using the software R v.2.12.0 (The R Foundation for Statistical Computing). First, a one-way analysis of variance (ANOVA) was carried out to determine whether the results obtained under different conditions were considerably different. Then, if ANOVA analysis confirms the existence of a significant difference (p < 0.05), a post-hoc analysis (Tukey’s HSD) was performed for a level of significance (α) of 0.05. 2.11. Determination of the specific energy consumption The specific energy consumption (SEC, kWh/g) was defined as the amount of energy consumed per unit mass of total phenols in the extract (Eq. (1)):

SEC =

EC·1000 TPC·dw·Nflasks

(1)

The energy consumption (EC, kWh) was experimentally determined using an energy meter (Brennenstuhl, EM 230). The mass of total phenols was calculated knowing the TPC (mg GAE/g dw) and the biomass used (dw, g). Nflasks represents the number of flasks handled at the same time in each extraction: 29 flasks were arranged in the orbital shaker, 2 flasks in the microwave oven and 6 flasks in the ultrasound bath.

2.8. Determination of the antioxidant capacity The DPPH (α,α-Diphenyl-β-picrylhydrazyl) radical scavenging assay was conducted according to the method described in Gullón et al. (2017). Briefly, 2 mL of a methanolic solution of DPPH (6·10−5 M) was added to 0.2 mL of an ethanolic solution of the extract. The decrease in absorbance at 515 nm was recorded after 16 min. The trolox equivalent antioxidant capacity (TEAC) was measured using the ABTS assay as described by Gullón et al. (2017). The ABTS radical cation (ABTS•+) was obtained by the reaction of 7 mM ABTS stock solution with 2.45 mM potassium persulfate (final concentration). The mixture was placed at room temperature and protected from light for 12–16 h before use. The next step consisted on diluting the ABTS•+ solution with phosphate buffer saline (PBS, pH 7.4) to an absorbance of ≈0.70 at 734 nm. Six minutes after the addition of 2 mL of diluted ABTS•+ solution to 20 μL of diluted extracts, the decrease in the absorbance was recorded. The ferric reducing antioxidant power (FRAP) assay was performed according to the procedure described by Gullón et al. (2017). In short, the reagent was prepared by mixing 25 mL of 300 mM acetate buffer (pH 3.6) and 2.5 mL of a 10 mM 2,4,6-tripyridyl-s-triazine (TPTZ) solution in 40 mM HCl solution and 2.5 mL of a 20 mM FeCl3·6H2O solution. The diluted extracts (0.1 mL) were mixed with 3 mL of the FRAP reagent. The absorbance was recorded after 6 min at 593 nm. Trolox was used as standard and results were expressed as mg of trolox equivalents (TE)/g dried leaf. Each assay was carried out in triplicate.

3. Results and discussion 3.1. Conventional extraction 3.1.1. Selection of the particle size One of the determining variables in the efficiency of any extraction system corresponds to the selection of particle size, an aspect that has not been previously addressed for the extraction of bioactive compounds from E. globulus. It is generally recognised that a smaller particle size favours the extraction of the target compounds. In a related paper, García et al. (2017) evaluated the size of E. camaldulensis leaf in the extraction of essential oils by steam distillation, and unexpectedly, they concluded that the entire leaf resulted in the highest extraction yield compared to the cut leaf. The question therefore arises as to how much the particle size must be reduced in order to obtain a high quality extract. Table 1 shows the characterization of the extracts obtained from the three particle sizes evaluated. As expected, the smaller the particle size, the higher the TPC, TFC and the antioxidant activities. This is due to the larger surface area in contact with the solvent, thus facilitating the migration of polyphenols to the medium. However, considering the practical application of this technology, grinding eucalyptus leaves into very small particles cannot only have a

2.9. UHPLC-TOF MS analysis of extracts Liquid chromatography coupled with trapped ion mobility 3

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Table 1 Characterization of phenolic content, flavonoid content and antioxidant activity of extracts obtained from eucalyptus leaves of different particle sizes. S: d < 0.5 mm; M: 0.5 < d < 2 mm; L: 10 mm × 10 mm. Size

TPC (mg GAE/g db)

S M L

94.1 ± 1.1 87.6 ± 2.0 63.8 ± 2.7

a b c

TFC (mg RE/g db) 45.5 ± 1.4 41.2 ± 2.5 32.3 ± 1.7

a b c

FRAP (mg TE/g db)

DPPH (mg TE/g db)

112.4 ± 2.1 a 101.2 ± 0.8 b 85.3 ± 1.2 c

180.1 ± 0.8 185.4 ± 2.1 165.3 ± 1.1

a b c

ABTS (mg TE/g db) 165.3 ± 1.3 144.4 ± 2.1 115.5 ± 3.2

a b c

Different letters in each column indicate significant differences (p ≤ 0.05).

from E. camaldulensis leaves (Wong-Paz et al., 2015). In this work, the effect of LSR on ultrasound-assisted extraction of polyphenols was studied, and the highest LSR evaluated: 12 mL/g, was necessary to reach maximum TPC levels. In conventional solvent extraction, increasing the liquid/solid ratio (both at constant temperature and time) improves solvent recovery by maximizing the driving force, i.e. the concentration of the gradient. However, it should be borne in mind that higher liquid-to-solid ratios lead to increased solvent consumption and waste production, resulting in higher extraction and waste management costs. Therefore, the LSR selected for the subsequent experiments was 10 mL/g.

significant economic impact, but can also lead to a difficult separation of plant waste extract once the extraction process is complete (Ameer et al., 2017). In this sense, an additional clean-up step may be necessary after the extraction of the phenolic compounds from the plant matrices. With these considerations in mind, the medium particle size was selected for further optimization studies. 3.1.2. Liquid-to-solid ratio Several authors have demonstrated the importance of the LSR in the extraction efficiency of phenolic compounds from different lignocellulosic biomass (Wong-Paz et al., 2015). However, to the best of our knowledge, the effect of LSR on the extraction of E. globulus leaves has not been examined. Fig. 1 shows the characterization of the extracts obtained at different LSR, in the range of 3 to 20 mL/g. As expected, phenolic and flavonoid content as well as antioxidant activities increased with LSR. However, the results achieved using the highest LSR were not statistically different (p ≤ 0.05) from those achieved at 10 mL/g. These results correlate well with the results of polyphenol extraction

3.2. Enzyme-assisted extraction Enzyme-assisted extraction (EAE) has been applied for the extraction of bioactive components in plant materials such as sweet potato, citrus peel, carrot, grape skin and soybean (Marathe et al., 2017), however, the extraction of eucalyptus leaves by enzymes has not yet been addressed. EAE is a safe, green and novel approach for the extraction of bioactive compounds. This method has been used for the extraction of lipids, polysaccharides, oils, natural pigments and phenolic compounds from various sources, as reviewed by Puri et al. (2012). Enzyme-aided extraction depends mainly on the ability of enzymes (mainly cellulase, pectinase and hemicellulase) to disintegrate and hydrolyze cell wall materials, thus allowing the easy release of the intracellular compounds of interest in the bulk solution. Operational conditions such as temperature, pH, enzyme activity, particle size of the target raw material and time are important in the enzyme-assisted extraction (Marathe et al., 2017). In this work, three different commercial carbohydrases cocktails were evaluated and compared with the extraction in the absence of enzymes (Table S1, Supplementary material). Enzyme supplementation did not promote phenolic extraction or antioxidant activity after 225 min of extraction in ethanol-water mixtures. It has been reported that enzyme extraction may be a potential alternative to conventional solvent based extraction methods. To assess whether enzymes could replace the use of solvents, enzyme-assisted extraction was performed in an aqueous medium and compared with the control in the absence of enzyme (Table S1, Supplementary material). Again, the combination of enzymes did not enhance antioxidant phenolic extraction, even when the dosage of enzymes was increased to 10% (v:w). Thus, the use of these particular carbohydrases did not play a relevant role in this application. Previously, carbohydrases (cellulases, xylanases, hemicellulases and pectinases) had been successfully used for the extraction of phenols from bay leaves (Boulila et al., 2015) or ginkgo biloba leaves (Chen et al., 2011). Probably, the nature of eucalyptus leaves requires a different type or combination of enzymes for cell wall hydrolysis (Marathe et al., 2017). 3.3. Microwave assisted extraction

Fig. 1. Conventional solvent extraction of eucalyptus leaves. Effect of the liquid:solid ratio (LSR) on the A) total phenolic content (TPC, white bar) and total flavonoid content (TFC, grey bar) and B) on the antioxidant activities of the extracts, measured by FRAP (dotted bar), DPPH (striped bar) and ABTS (black bar). Different letters in each series indicate significant differences (p ≤ 0.05).

Microwave-assisted extraction (MAE) is currently gaining attention due to the higher extraction rate and superior quality of products at lower cost (Belwal et al., 2018). Solvent polarity (dielectric constant), extraction time, irradiation power, temperature and contact surface 4

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Table 2 Properties of the extracts obtained under different conditions of microwave-assisted extraction. Power level Low

Medium-low Medium

Time 2 4 7 2 3 2

TPC (mg GAE/g dw) 55.9 70.0 79.4 65.4 69.2 65.5

± ± ± ± ± ±

1.3 2.0 2.1 3.1 2.1 4.5

a b c b b b

TFC (mg RE/g dw) 27.3 33.6 39.6 31.2 33.8 32.4

± ± ± ± ± ±

0.7 0.7 2.5 0.7 1.7 1.2

FRAP (mg TE/g dw)

a

a

73.2 ± 2.5 90.0 ± 3.4 b 105.8 ± 0.3 c 93.4 ± 0.9 b 102.6 ± 1.4 c,d 100.1 ± 0.8 d

b c b b b

DPPH (mg TE/g dw) 122.5 130.7 141.2 138.3 124.2 135.3

± ± ± ± ± ±

5.1 4.3 4.7 7.5 7.0 5.4

a a,b b,c b,d a,d a,c,d

ABTS (mg TE/g dw) 143.6 161.9 187.4 176.4 172.1 164.1

± ± ± ± ± ±

2.7 0.5 8.1 1.8 7.9 6.6

a b c b,c b b

Different letters in each column indicate significant differences (p ≤ 0.05).

area have been recognized as the main MAE parameters affecting polyphenol extraction (Routray and Orsat, 2014). In this work, we focus our attention on irradiation power and extraction time. Table 2 presents the characteristics of the extracts obtained after MAE of eucalyptus leaves. The highest TPC, TFC and antioxidant activity was obtained at lower power for an extraction time of 7 min. When the power increased, the extraction period was shortened to 3 and 2 min, for low-medium and medium power levels, respectively. At extraction times longer than those evaluated, the mixtures reached their boiling points. In order to avoid the degradation of thermolabile compounds, only temperatures below the boiling point were evaluated (Belwal et al., 2018). MAE has been previously used for the extraction of phenolics from eucalyptus leaves. In this sense, Bhuyan et al. (2015) studied MAE of Eucalyptus robusta leaf and, at optimal conditions (3 min, 600 W and LSR 50 mL/g) obtained a TPC of 58.40 mg GAE/g, and a TFC of 19.1 RE/g, values lower than those obtained here. 3.4. Ultrasound assisted extraction UAE has been also proposed as a novel green method for phenolic extraction. It is considered as one of the most efficient, inexpensive and simplest existing extraction systems that could be suitable for largescale operation (Tomšik et al., 2016). Acoustic effects accompanied by ultrasound cavitation phenomena enhance mass transfer during UAE, allowing greater solvent penetration into the sample matrix and hence, facilitating the release of extractable compounds (Tiwari, 2015). The most important parameters affecting UAE are ultrasonic power and frequency, temperature, extraction time, matrix and solvent properties (Tiwari, 2015). In the present study, different extraction times were evaluated maintaining the rest of the parameters. The highest values of TPC, TFC and antioxidant activities were obtained after 90 min of UAE (Fig. 2). In a previous research, the highest phenolic content observed for the UAE of E. robusta leaf was obtained under the highest tested conditions of ultrasonic power (250 W), extraction time (90 min) and temperature (60 °C) (Bhuyan et al., 2017). However, in the present work, maintenance of ultrasonic cavitation for a longer period resulted in a reduction in all performance indicators evaluated. In this case, the phenolic content of the extracts obtained after 120 min of UAE was 15% lower than that of the 90-min extraction, and the antioxidant activity was even lower, showing a 37% TE reduction for the ABTS assay. It has been reported that high ultrasonic potency may lead to low phenolic recovery, probably caused by the degradation of certain sensitive bioactive compounds (Xie et al., 2014; Tomšik et al., 2016). In this case, maintaining the ultrasound cavitation for a period longer than 90 min may have caused the loss of antioxidant activity by degradation of certain phenolics, such as flavonoids (Paniwnyk et al., 2001; Figueiras Abdala et al., 2017).

Fig. 2. Ultrasounds assisted extraction of eucalyptus leaves. Effect of the extraction time on the A) total phenolic content (TPC, white bar) and total flavonoid content (TFC, grey bar) and B) on the antioxidant activities of the extracts, measured by FRAP (dotted bar), DPPH (striped bar) and ABTS (black bar). Different letters in each series indicate significant differences (p ≤ 0.05).

ammonium salts (e.g., choline chloride) with naturally derived uncharged hydrogen-bond donors (e.g., vitamins, amines, sugars, alcohols, carboxylic acids). DES have unique physicochemical properties and, thanks to the ability to design their properties for a given purpose, their low vapor pressure and non-flammability, their low ecological footprint and attractive price, have become a topic of growing interest to both research and industry (Paiva et al., 2014). The structure of DES determines their physicochemical properties and consequently greatly influences the efficiency of the extraction of biologically active compounds. In the present work, we evaluated the capability of four different DES for the extraction of antioxidant phenolics from eucalyptus leaves (Table 3). Three of them were based on choline chloride, which contained ethylene glycol (ChE), xylitol (ChX) or glucose (ChGlu) as hydrogen bond donors. The fourth solvent tested was citric acid and glucose (CaGlu). These DES have previously been used for the extraction of phenolic compounds in plant materials (Espino et al., 2016; Wei et al., 2015). The phenolic content of the extracts using DES was substantially lower compared to the extract obtained with ethanol/water

3.5. Deep eutectic solvent extraction In recent years, the use of deep eutectic solvents (DES) as a green alternative to conventional solvents has expanded considerably (Espino et al., 2016). DES are generally based on mixtures of relatively cheap and readily available components, such as non-toxic quaternary 5

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Table 3 Characterization of the extracts obtained using natural deep eutectic solvents for 60 min at 50 °C. Solvent

TPC (mg GAE/g dw)

Ethanol-water 56% (v/v) Water ChE ChX ChGlu CaGlu

73.3 56.7 69.9 31.7 39.5 36.4

± ± ± ± ± ±

4.8 0.8 1.0 2.5 0.3 0.7

a

TFC (mg RE/g dw) 45.3 26.5 45.4 16.9 18.3 19.1

b a c d c,d

± ± ± ± ± ±

1.7 0.7 0.8 1.2 2.2 1.4

FRAP (mg TE/g dw)

a

92.4 50.3 66.3 48.9 63.6 49.4

b a c c c

± ± ± ± ± ±

5.2 1.9 2.7 2.5 3.4 1.1

a

DPPH (mg TE/g dw)

ABTS (mg TE/g dw)

a

98.6 ± 3.4 a 180.4 ± 3.4 b 89.9 ± 4.0 c 87.2 ± 1.5 c 82.3 ± 2.6 c 82.5 ± 2.2 c

179.8 ± 2.8 121.8 ± 5.0 b 68.0 ± 2.1 c 39.5 ± 3.4 d 48.9 ± 2.6 e 36.5 ± 1.0 d

b c b c b

Different letters in each column indicate significant differences (p ≤ 0.05). Table 4 Tentative identification of phenolic compounds in eucalyptus leaves extracts obtained under conventional, MAE, UAE and DES technologies. [M−H]−

RT (min)

Compound

m/z

Conv

MAE

UAE

DES

Conv

MAE

UAE

DES

3-O-methylellagic acid 3’-a-rhamnoside Cypellocarpin C Cypellocarpin C Cypellogin A or B Cypellogin A or B Digalloylglucose Ellagic acid Ellagic acid hexose Eucaglobulin or Globulusin B Gallic acid Glucoside of dimethylellagic acid HHDP galloylglucose HHDP galloylglucose Methylellagic acid Methylellagic acid hexoside Methylellagic acid hexoside Methylellagic acid hexoside Methylellagic acid-acetylrhamnoside Methylellagic acid pentoside Pedunculagin Pedunculagin Pentagalloylglucose Quercetin 3-O-a-rhamnoside Quercetin 3-O-b-D-glucuronide Quercetin 3-O-b-galactoside-6”-O-gallate Quercetin 3-O-glucoside Rutin Sideroxylonal A or B Sideroxylonal A or B Tellimagrandin II Tetragalloylglucose Trisgalloyl HHDP glucose Valoneic acid dilactone

461.0725 519.1872 519.1872 629.1876 629.1876 483.078 300.999 463.0518 497.1664 169.0142 491.1559 633.0733 633.0733 315.0146 477.1402 477.1402 477.1402 503.0831 447.0569 783.0686 783.0686 939.1109 447.0933 477.0675 615.0992 463.0882 609.1461 499.161 499.161 937.0953 787.0999 951.0745 469.0049

4.16 5.27

4.16 5.29

4.16 5.27

0.34 0.52

0.15 0.27

−0.15 −0.18

5.91 4.88 2.5 4.06 3.22 3.7 2.33 5.63 2.28 1.77 4.58 3.22 6.57 7.49 4.71 4.23

5.91 4.88 2.51 4.06 3.23 3.7 2.36 5.63 2.26 1.54 4.59

5.91 4.88 2.49 4.05 3.21 3.7 2.37 5.62 2.28 1.42 4.58

0.4 −0.05 −0.21 −0.21 0.24 −0.32 0.05 −0.73 −0.06 −0.28 0.08

0.13 0.5 −0.05 0.25

1.87 2.97 4.14 3.75 3.51 3.81 3.79 9.52 11.31 2.6 2.79 2.36 2.88

1.74 2.96 4.14 3.74 3.5 3.79 3.78 9.52 10.37 2.61 2.77 2.36 2.88

0.22 0.77 −0.23 0.18 0.41 0.32 −0.23 0.27 0.31 0.03 0.44 0.89 0.22

0.33 0.66 0.13 0.14 0.29 0.18 0.08 0 −0.07 −0.22 −0.11 0.57 0.07

0.11 −0.2 −0.06 −0.05 −0.6 −0.03 0.44 −0.56 −0.32 −0.26 −0.15 0.58 0.03 −0.22 −0.66 −0.3 0.26 −0.2

−0.05 0.15 0.17 0.43 0.28 0.06 −0.06 0.23 −0.07 0.01 0.23 0.36 0.25 0.37 0.1 −0.04 0.13 0.39 −0.07

1.76 2.97 4.14 3.74 3.5 3.79 3.78 9.52 11.35 2.61 2.78 2.35 2.88

6.57 7.48 4.7 4.23 1.47 1.84 2.96 4.14 3.74 3.5 3.8 3.78 9.52 11.28 2.58 2.77 2.36 2.88

0.44 0.42 0.19 0.1 0.16 0.19 0.04 0.46 0.29 0.5 0.24 0.5 0.54 0.38 0.46 0.21

0.11 0.62 0.14 0.08 0.41 0.28 0.02 −0.04 0.32 0.31 0.05

6.58 7.51 4.71 4.24

4.16 5.25 4.92 5.91 4.88 2.5 4.05 3.22 3.7 2.38 5.62 2.28 1.75 4.58 3.22 6.57 7.51 4.7 4.23

theor

Delta (mDa)

−0.05 0.62 −0.47 −0.04 −0.06 0.05 0.07 0 0.73 −0.19 0.18 1.02 0.11

Table 5 Characterization of the extracts and specific energy consumption at the optimal conditions for the different technologies used for the extraction of antioxidant phenolics. Extraction

Solvent (%)

Time (min)

TPC (mg GAE/g dw)

conventional MAE UAE DES

Ethanol (56) Ethanol (56) Ethanol (56) ChE* (80)

225 7 90 60

87.9 79.4 84.0 69.9

± ± ± ±

0.38 a 2.1 b 0.23 c 1.0 d

TFC (mg RE/g dw) 40.7 39.6 47.2 45.4

± ± ± ±

0.78 a 2.5 a 0.2 b 0.8 b

FRAP (mg TE/ g dw)

DPPH (mg TE/g dw)

ABTS (mg TE/g dw)

SEC† (kWh/g GAE)

101.6 ± 0.65 a 105.8 ± 0.3 a 84.7 ± 1.64 b 66.3 ± 2.7 c

188.0 ± 3.68 a 141.2 ± 4.7 b 156.6 ± 4.17 c 68.0 ± 2.1 d

144.9 ± 0.8 a 187.4 ± 8.1 b 241.1 ± 6.13 c 89.9 ± 4.0 d

0.177 0.013 0.082 0.059

* Choline chloride/Ethylene glycol (molar ratio 1:2). † Specific energy consumption, calculated from Eq. (1).

compounds (Radoševic et al., 2016; Nam et al., 2015). In the present research, the DES assayed did not show an additive antioxidant activity and led to a lower recovery of antioxidant phenolics compared to aqueous ethanolic extraction. This study should be complemented by evaluating other different DES families and considering the possibility to tailor new DESs with enhanced efficiency by combining the more effective components (Nam et al., 2015).

blends, and even compared to water extraction. Only ChE extraction led to a TPC and TFC similar to those of ethanol extraction; however, the antioxidant activity of this extract was markedly lower, particularly in the DPPH assay. Only a few studies have reported the antioxidant activity of the extracts obtained by DES. In some cases, DES extracts have been reported to show substantially higher antioxidant activity, which was explained by the activity derived from any of the DES-forming

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since MAE required 1.0 Wh/g dm (energy expressed per gram of dried eucalyptus leaf). Our results also agree with those from Pongmalai et al. (2015), who observed that MAE exhibited the highest energy efficiency compared with UAE, UAE + MAE and Soxhlet extraction. On the other hand, Rosa et al. (2017) obtained much higher values of specific energy consumption, in the range 0.37–38 kW h/g of recovered phenolic compounds, when using MAE to extract phenolic compounds from Juglans regia L. Considering the total phenolic and flavonoid content as well as the antioxidant capacity the best results were obtained with the conventional extraction method followed by MAE. However, taking also into account the extraction time and specific energy consumption, MAE was selected as the best extraction method.

3.6. Comparative of the different extraction technologies 3.6.1. Qualitative assessment of eucalyptus extracts by LC–MS The genus Eucalyptus is a unique source of a wide spectrum of secondary metabolites and these metabolites have been tested for their antitumor, anti-inflammatory, and antioxidative activities, and they have also shown antibacterial, antifungal, antileishmanial, and antiviral effects (Brezáni and Šmejkal, 2013). Extracts obtained using conventional, MAE, UAE and DES technologies were analyzed by UHPLC-ESIMS. Compounds previously described in the literature (BoulekbacheMakhlouf et al., 2013a,b; Gomes et al., 2018; Santos et al., 2012) were tentatively identified based both on the accurate mass and the comparison of the theoretical with the measured isotopic pattern. In all cases, the deviation of the theoretical with the measured mass was lower than 1.5 ppm and the mSigma value was lower than 50. The four extracts presented a similar composition, with 26 different compounds tentatively identified (Table 4). The main differences in the extracts corresponded to the presence of certain isomers and the abundance of the phenolic compounds (data not shown). As for instance, the compound with the highest peak area corresponded to sideroxylonal A or B (m/z 499.161) in the four extracts. However, the peak area of sideroxylonal on the DES extract was 2.6 times higher than that of the conventional extract. Other compounds with high signal intensity in the four extracts were quercetin 3-O-b-D-glucuronide (m/z 477.0675), eucaglobulin or globulusin (m/z 497.1664), ellagic acid (m/ z 300.999) and methylellagic acid pentoside (m/z 447.0569). Sideroxylonal, a formylated phloroglucinol compound, is an important chemical defense in eucalypts. Soliman et al. (2014) reported that sideroxylonal B could kill cancer cells (MCF7, HEP2 and CaCo) but did little damage to normal cells and hence, it is selectively active. Globulusin B is a glucose ester of oleuropeic acid and gallic acid, which is a potent scavenger of free radicals, more potent than that of ascorbic acid (Brezáni and Šmejkal, 2013). Globulusin A suppresses in vitro inflammatory cytokine production (TNF-α, IL-1β) in THP-1 cells and exerts antimelanogenic activity as tested on B16F1 mice melanoma cells. Eucaglobulin is a regioisomer of globulusin B which possesses potent antioxidant and anti-inflammatory activities and inhibits melanogenesis (Brezáni and Šmejkal, 2013). Quercetin 3-O-β-glucuronide (Q3G), a glucuronide conjugate of quercetin, is present in various medicinal herbs like Polygonum perfoliatum, Hypericum hirsutum, Nelumbo nucifera, Hedychium coronarium and green beans (Yang et al., 2016; Park et al., 2016). This compound has been reported to exert antioxidant, anti-inflammatory, antiviral and anticancer activities and showed beneficial effects in the reduction and prevention of various diseases, including neurodegenerative diseases (Li et al., 2017; Park et al., 2016). Ellagic acid and its glycosides are commonly present in plants such as raspberry, pomegranate, oak, birch leaves and many herbs (Lee et al., 2005). Ellagic acid has been reported to show antioxidant, anti-adipogenic and cancer chemopreventive effects (Ferreres et al., 2013).

4. Conclusions Small particle size and high liquid to solid ratio favors the extraction and isolation of bioactive compounds. However, grinding eucalyptus leaves into very small particles cannot only have a significant economic impact, but can also lead to a difficult separation of plant waste extract once the extraction process is complete. Furthermore, high liquid-tosolid ratios lead to increased solvent consumption and waste production, resulting in higher extraction and waste management costs. Hence, a compromise must be made to select the most adequate particle size and the liquid to solid ratio. In this research, we have investigated different green extraction processes aiming to reduce energy consumption and to explore alternative solvents such as DES. Enzyme supplementation did not promote phenolic extraction or antioxidant activity, even with high enzymatic doses. The extract obtained with choline chloride/ethylenglycol showed lower antioxidant activities, compared to the other extracts. On the other hand, intensification of extraction using ultrasounds and microwaves allowed the reduction of the extraction time to 90 and 7 min, respectively, while maintaining the quality of the extract. This resulted in a reduction of the specific energy consumption compared to that of the conventional extraction, which was 2 and 13 times lower for UAE and MAE, respectively. Extracts from eucalyptus leaves have been demonstrated a rich source of biologically active polyphenolic compounds, such as sideroxylonal, quercetin 3-O-b-D-glucuronide, eucaglobulin or globulusin, ellagic acid and methylellagic acid pentoside, among others. Acknowledgements This work was funded by the Spanish Ministry of Economy and Competitiveness (CTQ2014-58879-JIN). The authors belong to the Galician Competitive Research Group GRC 2013–032 and to the strategic group CRETUS (AGRUP2015/02). These programs are co-funded by FEDER (EU). B. Gullón would like to express their gratitude to the Spanish Ministry of Economy and Competitiveness for financial support (Grant reference IJCI-2015-25305). Authors would like to thank the use of RIAIDT-USC analytical facilities.

3.6.2. Specific energy consumption According to Table 5, there are substantial differences between the specific energy consumption in conventional and non-conventional extraction technologies. As expected, conventional extraction yielded the highest value (0.177 kW h/g GAE), followed by UAE which required half of the energy consumed under conventional conditions. The reduction of time in DES extraction resulted in a lower value of specific consumption. The lowest specific energy consumption was obtained with MAE, with a value more than 13 times lower than conventional extraction. Menéndez et al. (2014) evaluated energy consumption in the extraction of fatty acids from microalgae using MAE and UAE technologies. The lowest energy consumption was obtained by MAE, with the lowest value of 0.9 Wh/g dm, and energy consumption with UAE was 2–7 times higher. Similar results were obtained in the present work,

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