Article pubs.acs.org/IECR
Extraction of Polyphenolic Compounds from Eucalyptus globulus Bark: Process Optimization and Screening for Biological Activity Inês Mota,† Paula C. Rodrigues Pinto,*,† Catarina Novo,†,‡ Gabriel Sousa,‡ Olinda Guerreiro,§,∥ Â ngela R. Guerra,§,∥ Maria F. Duarte,§,∥ and Alírio E. Rodrigues† †
LSRE-Laboratory of Separation and Reaction Engineering, Associate Laboratory LSRE/LCM (Laboratory of Catalysis and Materials), Faculty of Engineering, University of Porto, Rua Dr. Roberto Frias, 4200-465 Porto, Portugal ‡ RAIZ, Research Institute of Forestry and Paper, Quinta de S. Francisco, Apart. 15, 3801-501 Eixo, Portugal § Centro de Biotecnologia Agrícola e Agro-Alimentar do Baixo Alentejo e Litoral (CEBAL)/Instituto Politécnico de Beja (IPBeja), 7801-908 Beja, Portugal ∥ Centre for Research in Ceramics & Composite Materials (CICECO), University of Aveiro, 3810-193 Aveiro, Portugal S Supporting Information *
ABSTRACT: The bark of Eucalyptus globulus is a promising source of polyphenolic compounds that could be extracted employing polar solvents. Extraction experiments were planned according to the Box−Behnken design to evaluate the effect of temperature and time of extraction and the ethanol/water ratio on the dependent variables total phenolic content (TPC; as gallic acid equivalents, GAE), antioxidant activity (AA; as millimoles of ascorbic acid equivalents, AAE), proanthocyanidins (Pac; as mimosa extract equivalents, MEE), Stiasny number (SN), and coextracted total carbohydrates (TC). Response surface models were developed, and statistical analysis of variance was performed. A maximum of TPC of 32% (wGAE/wextract) was achieved for 264 min, 82.5 °C, and 52% ethanol corresponding to about 2% of the bark weight. These conditions are close to those leading to maximum values of compounds with AA (2.1 mmolAAE/gextract) and Pac 14% (wMEE/wextract). TC in the extract and the SN predicted were 22.7% (wTC/wextract) and 37, respectively. Preliminary studies concerning the biological activity of some extracts have demonstrated their differential capacity to reduce human breast cancer cell proliferation. antioxidants, such as butylated hydroxytoluene (BHT).14 Other end-uses are well described in the literature.15 Regarding the extraction of polyphenols from plant material, the operating conditions play an important role in extract quality and extraction efficiency. The temperature, time of extraction, type of solvent used and solid/liquid ratio are some of the factors to consider in optimizing the extraction efficiency in terms of quality and yield.16−18 The existing literature discloses that an alcohol/water mixture is a better extractive medium for total phenolic content19 and antioxidant activity20,21 when compared to single solvents or aqueous solutions of Na2SO3 or NaOH, confirming that the extractive medium is a key variable in the extractive process of the bark components. Table S1 (Supporting Information) summarizes some of the literature data concerning the yield and the content of a polar fraction of E. globulus bark. Detailed analysis of polar extracts has been attempted,22−24 and new advances were recently achieved through the identification and quantification of 29 compounds, 16 of them referenced for the first time in E. globulus bark.19 Besides the extraction medium, other process variables such as time and temperature have been reported as factors with influence on the extract characteristics.6,25,26 These are certainly
1. INTRODUCTION Eucalyptus globulus is the main fiber source for the pulp and paper industry in Southern Europe (Portugal and Spain) and Australia. Portugal produces about 2 million tons/year of E. globulus pulp, and this sector is responsible for the management of 1.5 million m3 of E. globulus wood (with bark) from national forests in intensive short rotation plantations.1 Log debarking is the first operation in the industrial plant, generating large amounts of bark, which represents about 11% of the stem dry weight.2 This biomass residue is used as fuel for heating and electricity to mill operation, and it is not incorporated into the pulping process mainly due to its high content of extractives, in particular those of polyphenolic nature. This drawback of bark as raw material for pulp production can be turned into an advantage considering its potential as a source of high-added-value compounds. The interest in developing processes for the recovery of polyphenolic compounds from bark and other sources3−6 has increased remarkably in the past decade to find alternatives to the nonrenewable sources of chemical commodities and simultaneously to valorize industrial byproducts. Polyphenolic compounds from higher plants could be regarded as highadded-value compounds due to their recognized antimicrobial and antitumor activities and radical scavenger action (which is the basis of antioxidant proprieties).7−13 Currently, plant polyphenols have been incorporated into nutraceuticals and food supplements and constitute an alternative to the synthetic © 2012 American Chemical Society
Received: Revised: Accepted: Published: 6991
January 11, 2012 April 3, 2012 April 7, 2012 April 8, 2012 dx.doi.org/10.1021/ie300103z | Ind. Eng. Chem. Res. 2012, 51, 6991−7000
Industrial & Engineering Chemistry Research
Article
important contributions to the knowledge of E. globulus bark that would lead to the recognition of this material as a source of valuable compounds; however, the most promising extractive medium, ethanol (or methanol)/water, has never been explored considering a systematic variation of the alcohol/water ratio, time and temperature of extraction focusing on maximum polyphenolic material. The Box−Behnken design (BBD)27 has been used for process optimization of extractions of polyphenolic compounds from different sources.25,28,29 Clear trends and conclusions about the influence of process conditions (independent variables) on selected parameters (dependent variables) could be achieved from a reduced number of experiments. Besides this, the statistical analysis reveals which variable effects and their interactions are relevant to select a desirable set of process conditions. The aim of this study was to evaluate the effect of temperature, time, and ethanol/water ratio on E. globulus bark extraction using the BBD. The following dependent variables were studied: total phenolic content, antioxidant activity, proanthocyanidins, Stiasny number and total carbohydrates. Response surface models were developed and statistical analysis of variance (ANOVA) was performed. Finally, an extract in the optimal operating conditions (for maximum extraction of phenolic compounds) was obtained and the experimental values were compared with the predicted ones. Some of the extracts were selected to proceed for a preliminary evaluation of the antiproliferative capacity against an estrogen receptor-negative human breast cancer cell line (MDA-MB-231).
The dependent variables (Yn) studied were the total extraction yield, total phenolic compounds (TPC), FRAP (ferric reducing/antioxidant power) antioxidant activity (AA), proanthocyanidins (Pac), total carbohydrate content (TC), and the Stiasny number (SN) of the extracts. Experimental results were modeled according to a seconddegree polynomial described by the following equation: 3
Yn = β0 +
2
3
∑ βi Xi + ∑ ∑ i=1
i=1 j=1+1
3
βijXiXj+ ∑ βiiXi 2 + ε i=1
Yn is the dependent variable, β0 is the model constant, βi, βij, and βii represent the model coefficients for the linear interaction and quadratic effects of the independent variables, respectively. Xi and Xj are the independent variables coded between −1 (lower limit) and +1 (upper limit), and ε is the experimental error. The statistical analysis was performed using ANOVA, which established the model significance, the significance for each polynomial coefficient, and the determination coefficient R2. The statistical significance of each model was improved through a “backward elimination” process, deleting some insignificant dependent terms (p > 0.05). Unscramber v9.2 software was used to estimate the optimal extraction conditions for TPC by means of regression analysis and to obtain three-dimensional response surface plots. The models were validated performing the experiments in the optimal extraction conditions and comparing the values predicted by each model with the experimental data. 2.4. Characterization of the Extracts. 2.4.1. Total Nonvolatile Solids. Nonvolatile solids in the extracts were quantified following standard method TAPPI T652m-89. Briefly, 25 mL of extract was added to dried crucibles containing calcined sand. The crucibles with sample were dried at 105 °C until constant weight. Total nonvolatile solids of the extracts is a measure of the extraction yield, calculated as the percentage weight of nonvolatile solids per 100 g of oven-dry bark (Y1, %, w/ wbark). All samples were analyzed in duplicate. 2.4.2. Total Phenolic Compounds. TPC was quantified by the Folin−Ciocalteu method32 as described by Cadahı ́a and coworkers.33 Aliquots of the diluted extracts (0.5 mL) were transferred into the test tubes and 2.5 mL of Folin−Ciocalteu reagent (Merck) (diluted with water, 1:10, v/v), and 2 mL of NaCO3 (≥99.9%, Merck) 75 g/L was added. The mixture was kept for 5 min at 50 °C. After 10 min of cooling, the absorbance was measured at 760 nm. A blank was prepared at the same time as samples using 0.5 mL of extraction solvent. Gallic acid (98%, Acros Organics) was the standard used for the calibration curve (concentration range 10−100 μg/mL), and the results were expressed as total phenolic compounds in gallic acid equivalents (GAE) per 100 g of bark (Y2, TPCbark, % wGAE/wbark) or per 100 g of extract (Y5, TPCextract, % wGAE/ wextract). The analyses were done in triplicate, and the mean value was calculated. 2.4.3. Antioxidant Activity. AA was determined by the FRAP assay, a simple and reliable method that depends upon the reduction of ferric 2,4,6-tripyridyl-s-triazine [Fe(III)−TPTZ] to the ferrous 2,4,6-tripyridyl-s-triazine [Fe(II)−TPTZ] complex by a reducing agent at low pH.34 This complex has an intense blue color that can be monitored at 593 nm. An aliquot (0.1 mL) of each diluted extract was transferred into test tubes and 3.0 mL of freshly prepared FRAP reagent was added. FRAP reagent was prepared with 25 mL of acetate buffer 0.3 M, pH 3.6 (sodium
2. MATERIALS AND METHODS 2.1. Material Sampling and Preparation. E. globulus bark was collected at the end of the debarking process at a pulp mill (Portucel Soporcel Group, Cacia, Portugal) and air-dried in the dark until a constant moisture content (near 15 wt %). The composition of the E. globulus bark (expressed as weight percent, oven-dry basis) is, briefly, total polysaccharides 72% (quantified after acid hydrolysis30 and methanolysis31), total lignin 19%, ash 2.3% and ethanol/toluene extractives 2.2%, parameters quantified as described by Pinto and coworkers.30 2.2. Extraction of Bark: General Equipment and Conditions. Batch extractions were performed in a M/K System batch digester (5 L capacity) with temperature and time control and liquid recirculation over the bark using mixtures of ethanol/water at different ratios as described in section 2.3. The liquid/solid ratio was maintained constant at 8 L/kg (4 L of extractive medium for 500 g oven-dry bark). Before each batch extraction, the moisture of the bark was quantified and taken into consideration for the volume of water in the extractive medium. The heating rate of the extractions was 2 °C/min; after the selected temperature was reached, the predefined contact time began. At the end of each run, the liquid was quickly cooled to room temperature, filtered, bubbled with nitrogen, and stored in the freezer. 2.3. Experimental Design. Time, temperature, and ethanol concentration are the independent variables selected. The influence of time (X1, 30−195−360 min), temperature (X2, 25−82.5−140 °C), and ethanol concentration (X3, 0−40− 80%) on several dependent variables was studied according to the BBD with three experiments in the central point to estimate the experimental error variance (Table S2, Supporting Information). 6992
dx.doi.org/10.1021/ie300103z | Ind. Eng. Chem. Res. 2012, 51, 6991−7000
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Article
converted to homopolysaccharide (Y8, TC, %, wTC/wextract). The analyses were done in duplicate and the mean value was calculated. 2.4.6. Stiasny Number. The Stiasny number, which is a measure of the reactivity of the polyphenols toward formaldehyde, allows evaluation of the potential of the extract components to produce adhesives.41 It was determined according to the procedure proposed by Hillis and Urbach.42 For each assay, 2.5 mL of HCl 10 M, and 5.00 mL of formaldehyde (37%, Sigma) were added to a 100 mL aqueous solution containing 45−100 mg of the dried extract. The mixture was heated under reflux for 30 min and filtered through a 0.2 μm filter. The precipitate was washed with warm water (100 mL) and then dried at 105 °C. SN is the yield of formaldehyde-condensable tannins (precipitate) expressed as percentage of the starting material weight (Y9, SN). The analyses were done in duplicate and the mean value was calculated. 2.4.7. Biological Activity. 2.4.7.1. Cell Culture. The MDAMB-231 human breast cancer cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA) and maintained in Dulbecco's modified Eagle's medium (4.5 g/L glucose and L-glutamine) (Lonza, Verviers, Belgium), supplemented with 10% heat-inactivated fetal bovine serum (Lonza, Verviers, Belgium) and a 1% penicillin−streptomycin mixture solution (Lonza, Verviers, Belgium). The cells were incubated in an atmosphere of 95% air and 5% CO2 at 37 °C (C150, Binder GmbH, Tuttlingen, Germany). MDA-MB-231 cells were subcultured every other day. Extracts 1, 9, and 10 were dissolved in cell culture medium and extracts 4, 8, 11, and 12 were dissolved in dimethyl sulfoxide (DMSO, cell culture grade, Sigma-Aldrich) to a final concentration below 1%; controls received DMSO only. 2.4.7.2. Analysis of Cell Viability. Cells were seeded in 96well plates at 2 × 105 cells/mL. After 24 h, the cells were incubated with the extracts at various concentrations. Cell viability was estimated by 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay (Calbiochem) as previously described.43 This assay relies on the ability of living cells to reduce metabolically the MTT salt in an insoluble purple formazan salt which can be quantified by spectrophotometry. After 45 h of incubation with the extracts, 20 μL of MTT stock solution was added to each well (final concentration 0.5 mg/mL), followed by an incubation period of 3 h. A DMSO/ethanol (1:1) solution was then added to dissolve the formazan crystals, and the absorbance was read with a spectrophotometer (Helios Alpha, Thermo Scientific, Bremen, Germany) at 570 nm. The results were expressed as the percentage of cell viability relative to that of the control (cells without any test compound). IC50, defined as the extract concentration necessary to cause 50% inhibition of cell viability, was calculated by plotting the percentage of cell viability compared to that of a control (no added extract) against the different extract concentrations tested. All experimental results were performed at least in triplicate (n = 3) and the data were expressed as means ± standard deviation. ANOVA (SigmaPlot Software Inc., San Jose, CA) was used to test for differences between extract activities. Where differences did exist, the source of the differences at a p < 0.05 significance level was identified by a pairwise multiplecomparison procedure (Student−Newman−Keuls test). The final graphs were plotted using GraphPad Prism 5.04 (GraphPad Software Inc., La Jolla, CA).
acetate trihydrate 99.5%, Merck), 2.5 mL of TPTZ (≥99%, Fluka) 0.01 M in 0.04 M HCl (prepared with HCl 37%, Panreac) and 2.5 mL of FeCl3·6H2O (>98%, Sigma-Aldrich) 0.02 M. The absorbance was recorded after 5 min at 593 nm against a blank containing 0.1 mL of solvent. The relative activities of the samples were calculated from the calibration curve of L-ascorbic acid (Sigma-Aldrich) standard solutions (50−1000 μmol/L) under the same experimental conditions and expressed as millimoles of ascorbic acid equivalents (AAE) per 100 g of bark (Y3, AAbark, mmolAAE/100 gbark) and per gram of extract (Y6, AAextract, mmolAAE/gextract). The analyses were done in triplicate, and the mean value was calculated. 2.4.4. Proanthocyanidins. Pac was quantified by the Bate− Smith reaction (butanol−acid method) as recommended by several authors.35−37 This method is based on the oxidative depolymerization of proanthocyanidins with 1-butanol−HCl reagent, leading to the production of anthocyanidins that absorb at 550 nm (red color). Thus, each extract (1.00 mL) was transferred into a test tube, and 6.0 mL of butanol−HCl reagent (1-butanol:HCl = 95:5, v/v) (prepared with 1-butanol, 99.8%, Sigma-Aldrich) and 0.2 mL of 2% ferric ammonium sulfate (99%, Sigma-Aldrich) in 2 M HCl were added. The reaction was carried out at 100 °C for 50 min. After cooling, the absorbance was recorded at 550 nm against a blank prepared with 1 mL of the solvent instead of the extract. Pac was calculated according to a calibration plot for a solution of commercial extract of mimosa (sample kindly provided by Mimosa Extract Co. (Pty) Ltd., South Africa) between 0 and 0.5 mg/mL. The results were expressed as mimosa extract equivalents (MEE) per 100 g of bark (Y4, Pacbark, %, wMME/ wbark) or per 100 g of extract (Y7, Pacextract, %, wMME/wextract). The analyses were done in duplicate, and the mean value was calculated. 2.4.5. Carbohydrate Analysis: Acid Methanolysis. The dried extracts (10−12 mg) were subjected to acid methanolysis with 2 mL of HCl solution in anhydrous methanol (2 M HCl/ MeOH) (prepared with 3 M HCl/anhydrous methanol, from Supelco and anhydrous methanol, 99.8%, from Sigma-Aldrich) at 100 °C as described in the literature.38 Initially, the methanolysis was performed at different reaction times (4, 17, and 24 h) for extract 3, aiming to select the reaction time that leads to the maximum of quantified monosaccharides. A reaction time of 17 h was selected for the methanolysis of all the extracts. Afterward, methanolysates were neutralized with 120 μL of pyridine (99%, Aldrich) containing sorbitol (≥98%, Sigma), used as an internal standard and evaporated to dryness in a vacuum evaporator. Derivatization was performed before GC analysis with 80 μL of pyridine, 150 μL of bis(trimethylsilyl)trifluoroacetamide (≥99%, Fluka), and 50 μL of chlorotrimethylsilane (≥99.0%, Fluka). The reaction occurred at 80 °C for 30 min. The identification of each trimethylsilyl (TMS) methyl glycoside derivative was performed by GC−MS using spectral data reported in the literature39,40 and obtained from the analysis of standards. GC− MS and GC−FID analyses were performed with the same equipment and conditions as described in the literature.31 The calibration was performed with reference monosaccharides rhamnose (>99%) and xylose (>99%), both purchased from Merck, glucuronic acid (>98%) from Alfa Aesar, glucose (99.5%) from Sigma-Aldrich, galactose (>99%), arabinose (>99%), and mannose (>99%) from Acros Organics, and galacturonic acid (≥97%) from Fluka. TC was calculated for each extract as the sum of all monosaccharide quantified and 6993
dx.doi.org/10.1021/ie300103z | Ind. Eng. Chem. Res. 2012, 51, 6991−7000
Industrial & Engineering Chemistry Research
