Italic 6 - Science & Technology of Biomass: Advances and Challenges

September, 5-8. 2011

EXTRACTION POLYPHENOLICS OPTIMIZATION OF EXTRACTION OF POLYPHENOLICS FROM EUCALYPTUS

GLOBULUS BARK USING RESPONSE SURFACE METHODOLOGY

Ins Mota1, Paula Pinto1, Gabriel Sousa2, Catarina Novo1, Eduardo Silva1 and Alrio Rodrigues1
1) Laboratory of Separation and Reaction Engineering LSRE, Associate Laboratory LSRE/LCM, Faculty of Engineering, University of Porto, Portugal 2) RAIZ, Research Institute of Forestry and Paper, Quinta de S. Francisco, Apart. 15, 3801-501 Eixo, Portugal

Abstract
The bark of Eucalyptus globulus wood is one of the main by-products of pulp and paper industry in Portugal. In comparison with wood, this biomass contains a higher percentage of polyphenolic compounds that could be extracted by environmental friendly processes employing polar solvents such as ethanol and water. The overall aim is to increase the bark chain-value by the preferential extraction of this family of compounds, well known by its biological activity and valuable chemical properties for a wide range of applications. In this work the bark was characterized concerning its chemical constituents. Extraction experiments were planned according to a Box-Behnken design in order to analyse, and to optimize, the effect of the temperature and time of extraction and also the percentage of ethanol on the solvent focusing the highest total phenolic content (as gallic acid equivalent, GAE), antioxidant activity (as mmol of acid ascorbic equivalent, AAE) and proanthocyanidins (as mimosa extract equivalent, MEE). A maximum of selectivity for total phenolic compounds of 32.2% wGAE/wextract was achieved for 264 min, 82.5oC and 52% of ethanol corresponding to about 2% of bark weight. These conditions are close to those ones leading to maximum values of compounds with antioxidant activity (208 mmol AAE/100 g extract) and proanthocyanidins (14% wMEE/wextract).

Introduction
Eucalyptus globulus wood is the main raw material for the production of pulp for high quality paper production in Portugal. Around 2 million ton of pulp are annually produced in Portugal generating about 190 thousand ton of bark as by-product.[1] The bark of E. globulus has a chemical composition near to that of wood with respect to macromolecular components. The most significant difference is the higher content of soluble compounds in water and alkaline solution of the bark, mainly due to the higher content of polyphenolic compounds, which is one of the reasons that prevent its mill integration to pulp production; hence, bark is used for energy co-generation. However, the fraction of polyphenolics includes high value-added compounds that could be extracted by polar solvents. This group of compounds is simultaneously diverse and complex and there are only a few studies concerning its fine characterization. [2-5] Due to their properties, polyphenolics can find several applications as anti-oxidant, as anti-microbiologic additive, as precursors for chemical industry or as source of phenol group, for instance, for adhesive production [6]. Recently, application-driven research of polar extracts of E. globulus bark has been published, showing the emergent interest on this field and potential herein. [7-9] The integration of low-cost processes and technologies to increment the chain-value of bark is a key step for implementing the biorefinery concept in pulp and paper industry. In this work experimental design was employed to find the best extraction conditions (in particular, time, temperature and ethanol percentage) in order to maximize the total phenolic content and

Session n Ins Mota

Italic 6 - Science & Technology of Biomass: Advances and Challenges

September, 5-8. 2011

antioxidant activity in extracts of E. globulus bark. This work was performed in the framework of a National project BIIPP-Integrated Biorefinery in Pulp and Paper Industry.

Experimental
1. Characterization of bark Bark was collected at Portucel Soporcel industrial unit of Cacia, Portugal. Tappi standard methods [10] were used for bark characterization. For monosaccharide quantification, acid hydrolysis and acid methanolysis were applied. The products of hydrolysis were quantified by HPAEC-PAD. The products of acid methanolysis (24 h, 100oC, HCl 2M in anhydrous methanol) were trimethylsylilated and analysed by GC-MS, allowing the quantification of the neutral and acid monosaccharides. 2. Design of experiments To analyse the influence of the independent variables, time (30-360 min) X1, temperature (25140oC) X2, and % of ethanol (0-80%) X3 on several dependent variables, bark extraction experiments were planned according to Box-Behnken model with three central point replicates, maintaining the liquid:solid ratio of 8. The extractions were performed in M/K digesters with time control and liquid recirculation using 500 g of bark for 4 L of extractive medium. Response surfaces were built using the Unscrambler software, with adjustment of the experimental results to a linear model with the incorporation of significant quadratic and interaction effects. Statistic significance was determined at 95% confidence interval. 3. Extract characterization and dependent variables Extraction yield was given by the total non-volatile solids (weight of dry extract at 105oC) - variable dependent Y1. Total phenolic compounds (TFC) were determined by Folin-Ciocalteu method as described in literature [10] and expressed as g of gallic acid equivalent (GAE) per 100 g of bark (Y2) or per 100 g of extract (Y5) (on dried basis). The antioxidant activity was determined by means of ferric reducing antioxidant power (FRAP) assay as described elsewhere [10] and results were expressed as mmol of ascorbic acid equivalents (AAE) per 100 g of bark (Y3) or per 100 g of extract (Y6). Proanthocyanidins were accessed by the method of butanol-acid as described by Hagerman [10]. For calibration, solutions of commercial extract of mimosa (considered as containing only proanthocyanidins) were used and the results are expressed as g of mimosa extract equivalents (MEE) per 100 g of bark (Y4) or 100 g of extract (Y7). The carbohydrates in extracts were accessed by acid methanolysis as describe for bark characterization.

Results and discussion

The chemical composition of bark is summarized in table 1. Bark composition is similar to wood of E. globulus for the main components, although with a slight low value for lignin and high inorganic content. The high values of solubility are probably due to the high content of phenolics extracted. Table 1 Composition of E. globulus bark
Entry 1 2 3 4 5 5.1 5.2 5.3 5.4
o

Parameter Ashes (550 C, 12 hours) Ethanol/Toluene extractives (soxhlet, 6 h) Lignin (Klason+soluble) Total polysaccharides + acetic acid (from acetyl groups hydrolysis in xylans) Solubility in, Cold water (48 h, 2.5g bark, 300mL, room temperature) Hot water (3h, 2.5g bark, 300 mL, reflux) NaOH 1% (1 h, 1 g, 200 mL, reflux) NaOH 0,1% (1 h, 1 g, 200 mL, reflux)

% wt dry basis 2.3 0.06 2.2 0.15 19.3 0.98 76.7 2.1 2.4 0,41 8.1 0,13 24.8 0,59 16.0 0,16

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Italic 6 - Science & Technology of Biomass: Advances and Challenges

September, 5-8. 2011

It is possible that a fraction of tannins could account for total lignin content (19%), otherwise, the sum of the entries 1-4 would not close the bark weight. On the other side, the solubility values could be influenced with other compounds such as soluble polysaccharides. Table 2 shows the coefficients of the obtained models adjusted for the following expression:

Y = 0+ i =1 i i + i =1 j =1+1 ij i j + i =1 ij i2 + . Some of the insignificant terms were

3 2 3 3

neglected in order to improve models significance. It is also mentioned the significance, lack of fit and the correlation parameter for each model. In this study all models are considered significant and the obtained R2 values supports the adequacy of the models and, therefore, can be quite useful as predicting tools. Table 2 Response model coefficients and statistical parameters from ANOVA analysis
Response 0 1 2 3 12 13 23 1 2 3
p-value
2 2 2

Y1

Y2

Y3
-1

Y4
-1

Y5
-3

Y6

Y7

Model Lack of fit

2

-3,05 1,05x10-2 -2 9,19 x10 -2 -3,97 x10 2,67 -2,50 2,95 0,0002 0,0016 0,941

-2,47x10 2,43 x10-3 -2 1,82 x10 -3 1,55 x10 -1,39x10-1 -1 1,74x10 -1 -7,39x10 0,0010 0,0676 0,905

-5,85x10 1,38 x10-2 -1 1,05 x10 -3 9,00 x10 -4,28 <0,0001 0,1623 0,918

-3,33x10 6,96 x10-4 -3 5,22 x10 -3 3,81 x10 -1 -4,55x10 0,0004 0,1281 0,849

20,86 1,33 x10-2 -2 3,96 x10 -1 1,11x10 -5,46 -2,63 -6,62 -7,47 0,0353 0,0983 0,814

145,38 7,69x10-2 -1 1,34x10 -1 7,37x10 -35,76 -13,60 -49,04 -47,47 0,0302 0,1291 0,823

8,03 2,81 x10-3 -2 1,52 x10 -2 8,60 x10 -4,55 -5,88 0,0021 0,1794 0,842

Examples of response surface plots for extraction yield and TFC concerning its selectivity are shown in figure 1.

Figure 1 Response surface plots showing the effects of independent variables for extraction yield (Y1, %w/w) and for total phenolic content (Y5, gGAE/100g extract) The responses surfaces and statistical parameters have shown a significant linear effect between total extraction yield and the independent variable temperature. The maximum extraction yield was reached for the boundaries of the tested conditions (360 min, 140C and 0% of ethanol): 21.7% wt., but with a rather low content of total phenolic compounds, 9% wGAE/wextract. The interaction between time and temperature is more significant concerning selectivity of phenolics and antioxidant activity than the isolated variables: for a fixed % of ethanol, the responses surfaces showed opposite effects on selectivity of the variables time and temperature. A minimum of 20% of ethanol is required to achieve a remarkable increase of yield on phenolics and anti-oxidant activity compounds and proanthocyanidins. TFC and antioxidant activity show the same behaviour in the studied experiment domain and one can also observe a significant linear correlation (0,9866) between both these variables. The analysis of the response surfaces allowed selecting the extraction conditions for obtaining a high quality extract concerning total phenolics and antioxidant activity. Table 3 summarizes the predicted values under the optimum selectivity conditions for total phenolic compounds.
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Italic 6 - Science & Technology of Biomass: Advances and Challenges

September, 5-8. 2011

Table 3 Predicted values of the responses under the optimum conditions for selectivity of total phenolic content (X1=264 min; X2=82,5C; X3=52%)
Response Predicted value

Y1 %(w/w) 5.24

Y2 gGAE/
100gbark

Y3 mmolAAE/
100gbark

Y4 gMEE/
100gbark

Y5 gGAE/ 100gextract 32.2

Y6 mmolAAE/
100gextract

Y7 gMEE/ 100gextract 14.0

1.9

11.8

0.77

208.4

The acid methanolysis of the extracts revealed that the main monosaccharide residue was glucose followed by arabinose and galactose. It was shown that temperature and % ethanol significantly affect the total carbohydrate content in the extracts.

Conclusions
The Box-Behnken design use was effective for estimating the effect of time, temperature and ethanol concentration for the extraction of polyphenolic compounds with antioxidant activity. The effect on total yield of extraction time is enhanced with the increase of temperature and with the decrease of % of ethanol in the extractive medium. Focusing on the extraction selectivity, the maximum value obtained for total phenolics was 32.2% wGAE/wextract for the extraction conditions of 264min, 82.5C and 52% ethanol. Under these conditions it is possible to obtain 1.9% w/wbark of total phenolics, 11.8% mmolAAE/wbark of antioxidant compounds and 0.77% wMEE/wbark of proanthocyanidins. This work demonstrated the bark potential as a source of phenolic compounds with antioxidant activity that can be extracted in moderate conditions. Considering a 45 000 tons of bark produced annually, about 900 tons of antioxidant phenolic material can be obtained.

References
1. CELPA - Portuguese Paper Industry Association, Statistical report 2010, CELPA: Lisbon, http://www.celpa.pt/, accessed May 2011. 2. Cadahia, E.; Conde,E.; Simn, F.; Garca-Vallejo, M. Holzforchung 1997, 51(2): p. 125-129. 3. Charrier, B.; Marques,M.; Haluk, J. Holzforchung 1992, 46(1): p. 87-89. 4. Kim, J.; Lee, I.; Yun, B.; Chung, S.; Shim, G.; Koshino, H.; Yoo, I. Phytochemistry 2001, 57(4): p. 587-591. 5. Conde, E.; Cadaha, E.; Garca-vallejo, M.; Tomas-Barderan, F. Wood and Fiber Science, 1995, 27: p. 379-383. 6. Pizzi, A.; Tannins: Major Sources, Properties and Applications, in Monomers, Polymers and Composites from Renewable Resources, M.N. Belgacem and A. Gandini, Editors, 2008, Elsevier: Oxford. p. 179-199. 7. Pinto, P.; Sousa, G.; Amaral, L.; Silvestre, A.; Neto, C.; Crispim, F. Water-Based Technology for Recovery of a Polyphenolic Fraction from Eucalyptus globulus Bark. in 2nd Nordic Wood Biorefinery Conference (NWBC 2009), 2009, Helsinki, Finland. 8. Vzquez, G.; Fontela, E.; Santos, J.; Freire, M.; Gonzlez-lvarez, J.; Antorrena, G. Industrial Crops and Products, 2008, 28(3): p. 279-285. 9. Vzquez, G.; Gonzlez-lvarez, J.; Santos, J.; Freire, M.; Antorrena, G.; Industrial Crops and Products, 2009, 29(2-3): p. 364-370. 10. Hagerman, A., The Tannin Handbook, http://www.users.muohio.edu/hagermae/, acessed 30 June 2011. 2011.

Acknowledgements
Mr. Nigel Dunning and Mimosa Extract Company (Pty) Ltd (South Africa) for gently providing the mimosa extract. This work was carried out under the Project BIIPP No. 11551 - Integrated Biorefinery Industry Pulp and Paper - funded by the European Regional Development Fund (ERDF) through the Operational Programme for Competitiveness Factors (POFC) of the National Strategic Reference Framework (NSRF).
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