30 Nova Biotechnologica et Chimica 12-1 (2013) DOI 10.2478/nbec-2013-0003 © University of SS. Cyril and Methodius in Trnava OPTIMIZATION OF THE ANTIOXIDANT EXTRACTION FROM ELEUTHEROCOCCUS SENTICOSUS ROOTS BY RESPONSE SURFACE METHODOLOGY MIROSLAV ONDREJOVIČ1,2, DANIELA CHMELOVÁ3, TIBOR MALIAR1 1Department of Biotechnology, Faculty of Natural Sciences, University of SS. Cyril and Methodius, J. Herdu 2, Trnava, SK-917 01, Slovak Republic (miroslav.ondrejovic@ucm.sk) 2Food Research Institute, Department Biocentrum, Kostolna 5, Modra, SK-900 01, Slovak Republic 3Department of Biochemistry and Biotechnology, Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture, Tr. A. Hlinku 2, Nitra, SK-949 76, Slovak Republic Abstract: Eleutherococcus senticosus is known as adaptogen with benefits in general health promotion. The aim of this study was to investigate the effect of major extraction parameters on extraction yield of antioxidants measured by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging activity. Secondly, content of total polyphenols was evaluated. Optimal conditions of the extraction were processed by response surface methodology. The independent variables of extraction were proposed as temperature, solid–liquid ratio and solvent composition. For the optimal antioxidant extraction, E. senticosus is suitable to extract by 23 % (v/v) aqueous ethanol at 70 °C in ratio 53 mL of extraction solvent per g of plant material. The optimal conditions calculated for the extraction of total polyphenols were very similar (70 °C, 22 % (v/v) aqueous ethanol) expect solid-liquid ratio which indicates need of increasing of solid-liquid ratio to 91 mL of extraction solvent per g of plant material. Key words: extraction, Eleutherococcus senticosus, antioxidant activity, polyphenols, response surface methodology 1. Introduction Eleutherococcus senticosus, known as Siberian ginseng or Acanthopanax senticosus, belong to the Araliaceae family, is distributed in China, Korea and Russia. E. senticosus has been known in traditional Chinese medicine due to putative benefits in general health promotion, vitality, stamina, restoration of homeostasis, chemoprevention, wound healing, longevity and other indications (KITTS and HU, 2000). By detailed research, it was found that the active compounds of E. senticosus include ginsenosides, polysaccharides, peptides, polyacetylenes, phenols and enzymes (XIANG et al., 2008). The major active compounds are acanthoside D, eleutherosides I, K, L, M (triterpenic saponins), eleutherosides B, B1, D, E (phenylpropane derivates) (KITTS and HU, 2000), senticoside, β-sitosterol, sesamin (DAVYDOV and KRIKORIAN, 2000; LEE et al., 2004; LI et al., 2006). Today, the extracts from different parts of E. senticosus are studied for their biological and pharmacological Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 16.01.20 14:25 UTC Nova Biotechnologica et Chimica 12-1 (2013) 31 effects include antioxidant, antidiabetes, anticancer, anti-inflammatory, imunoregulatory and immunomodulating, antimicrobial and antiviral activities (FUJIKAWA et al., 1996; HIBASAMI et al., 2000; YI et al., 2002; JUNG et al., 2003). Several works are focused on the medical efficacy of E. senticosus mediated via its antioxidant actions, e.g. inhibiting lipid peroxidation and oxidative injury to DNA (LEE et al., 1998; NAVAL et al., 2007; LEE et al., 2008; CHEN et al., 2010; LEE et al., 2011). Except for protection against oxidative reaction, the antioxidant activity of E. senticosus is associated with their traditional using as adaptogen by the prevention of human body against faster organ aging. Antioxidants from E. senticosus belonging to polar and less polar components can be extracted by various extraction solvent at different extraction conditions. Determination of optimal values of extraction parameter is significant point in design of extraction process for obtaining of biological active compounds from plant material. In this regard, the response surface methodology (RSM) is a powerful tool that can provide a complete optimal condition to improve a various technology processes (BOX and WILSON, 1951). It is a collection of statistical and mathematical techniques and it has important applications in the design, development and formulation of new products as well as in the improvement of existing products (MYERS and MONTGOMERY, 1995). The aim of this study was to evaluate the effects of the extraction temperature, solvent composition and solid-liquid ratio on the extraction yield of antioxidants. Secondary, total polyphenol content was determined to find possible relations between this parameter and antioxidant activity. This work provides preliminary data for making products from E. senticosus with high antioxidant activity which would be used as food additives or nutraceutical supplement with specific effects to human health. 2. Material and methods 2.1 Chemicals Ethanol 96 %, methanol, Folin-Ciocalteu reagent and sodium carbonate were obtained from Mikrochem (SK). Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2- carboxylic acid), 2,2-diphenyl-1-picrylhydrazyl (DPPH) and gallic acid were obtained from Sigma-Aldrich (D). 2.2 Plant material The roots of E. senticosus (F-Dental Hodonin, CZ) were an aliquot amount of the tested material cut at particle size < 0.1 cm. 2.3 Extraction procedure For determination of optimization ranges, 1 g of dried roots of E. senticosus was extracted by extraction solvent (aqueous ethanol with concentration 0, 10, 30, 50, 70, Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 16.01.20 14:25 UTC 32 Ondrejovič, M. et al. 90 and 96 % (v/v) of ethanol) in solid-liquid ratio (10, 25, 50, 75 and 100 mL per gram of plant material) at various temperatures (20, 30, 50 and 70 °C) during 60 minutes. After 60 minutes, the solution was centrifuged for 2 minutes at 4,000 RPM and the supernatant was used for analysis. 2.4 Experimental design Three factors, five level experiment was carried out with tested, independent variables- temperature (22, 34, 52, 70 and 82 °C), solvent composition (3.3, 10, 20, 30 and 36.7 % (v/v) ethanol) and solid-liquid ratio (8.2, 25, 50, 75 and 91.8 mL/g). Real variables values were transformed into non-dimensional coded form (Table 1). Table 1. Independent variables in original and coded form and experimental results for response variables TEAC [mg/g plant material] and total polyphenols [mg/g plant material]. Stand. order Temperature [°C] Solvent composition [% EtOH] Solid-liquid ratio [mL/g] TEAC [mg/g plant material] Total polyphenols [mg/g plant material] 1 34 (-1) 10 (-1) 25 (-1) 15.0 8.8 2 52 (0) 20 (0) 50 (0) 30.9 18.5 3 34 (-1) 30 (1) 75 (1) 20.5 15.9 4 70 (1) 30 (1) 25 (-1) 31.5 17.5 5 70 (1) 10 (-1) 75 (1) 26.1 21.8 6 70 (1) 10 (-1) 25 (-1) 18.9 13.0 7 34 (-1) 30 (1) 25 (-1) 14.7 9.5 8 70 (1) 30 (1) 75 (1) 27.4 21.5 9 52 (0) 20 (0) 50 (0) 30.9 18.0 10 34 (-1) 10 (-1) 75 (1) 24.4 10.7 11 52 (0) 3.3 (-1.682) 50 (0) 18.2 9.8 12 52 (0) 20 (0) 91.8 (1.682) 23.3 20.2 13 82 (1.682) 20 (0) 50 (0) 26.4 12.8 14 52 (0) 20 (0) 8.2 (-1.682) 10.1 6.2 15 22 (-1.682) 20 (0) 50 (0) 17.9 9.1 16 52 (0) 36.7 (1.682) 50 (0) 14.3 12.3 17 52 (0) 20 (0) 50 (0) 28.9 17.5 Measured dependent variables were Trolox equivalent antioxidant capacity (TEAC) in mg/g of plant material and total polyphenols in mg/g of plant material. Experimental data were fit by the polynomial regression of the second order (Eq. 1), and regression coefficients (bi) were calculated. (1) where Xi are independent variables responsible for response Y and bi are regression coefficients, describing relations of the measured properties to coded levels of the ∑∑∑∑ − < = === +++= 1 1 2 2 11 0 k ji i j jiiji k i ii k i ii XXbXbXbbY Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 16.01.20 14:25 UTC Nova Biotechnologica et Chimica 12-1 (2013) 33 selected parameters (Table 1). For computer and statistical processing, the Statgraphics Plus 5.1 (Statpoint Technologies, USA) was applied. 2.5 Analysis of the response variables 2.5.1 Trolox equivalent antioxidant capacity TEAC was determined using the method proposed by YEN and CHEN (1995) by with modification to microplate form. DPPH (0.012 %; w/v) was dissolved in pure methanol. The radical stock solution was prepared fresh daily. The DPPH solution (100 μL) was added to 25 μL extract. The mixture was shaken and allowed to stand at room temperature for 10 minutes. The decrease in absorbance was monitored at 540 nm. The results were corrected for dilution and expressed in mg Trolox per gram of plant material. 2.5.2 Total polyphenols Total polyphenol content was measured using Folin–Ciocalteu colorimetric method (SINGLETON et al., 1999). Plant extracts (20 μL) were mixed with 20 μL Folin– Ciocalteu reagent and incubated at room temperature for 5 minutes. Following the addition of 200 μL 20 % Na2CO3 (w/v) to the mixture, total polyphenols were measured at 690 nm. The results were expressed as gallic acid equivalents, mg per gram of plant material. 2.6 Statistical analyses All experiments were realized in triplicate and statistical analysis was calculated by Statgraphics Plus 5.1. 3. Results and discussion 3.1 Selection of optimization ranges The extraction efficiency of natural compounds from the plant material is dependent on some parameters such as temperature, time, solid-liquid ratio, solvent composition and others. All these parameters were put in optimization aimed to maximal antioxidant activity. The polyphenol content in plant extracts is often employed to content of antioxidant constituents (WU et al., 2004). This dependence was evaluated also in our work. The influence of some above-mentioned parameters was partly reported in literature (CHEN et al., 2010; LEE et al., 2011; PARK et al., 2006; WANG, 2012), but optimization for the extraction of antioxidants and polyphenols from E. senticosus using RSM have not been reported. In the literature, for antioxidant extraction, various extraction solvents such as water, methanol, acetone or their water solutions was used (PARK et al., 2006; WANG, 2012). For this purpose, methanol appeared to be an effective extraction solvent. Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 16.01.20 14:25 UTC 34 Ondrejovič, M. et al. The temperature plays an important role in the extraction of active substances from plant materials. In this work, we studied antioxidant extraction from E. senticosus at four different temperatures: 20, 30, 50 and 70 °C by measuring kinetics of extraction within 1 hour because it was necessary to determine the extraction time as a fixed parameter in the optimization. After one hour of extraction, the antioxidant activity of prepared extracts no longer increases (Fig. 1). Therefore, in all experiments, we used extraction parameter – time as fixed parameter with value - one hour. According to Fig. 1, the most suitable temperature for extraction of antioxidants from E. senticosus was 70 °C. In the literature, the extraction of antioxidants was carried out at 70 °C by distillated water during 3 hours (PARK et al., 2006). On the other hand, KIM et al. (2002) and LEE et al. (2011) have done extraction antioxidant activity at laboratory temperature. On the basis of this information, we have been selected the temperature range of 34 – 70 °C. Transport of active compounds from plant material to extraction solvent is diffusion process affected by various factors, but primary attribute of extraction is the solubilization of extracted compounds. Solubilization of target compounds is affected also by solubilization of ballast compounds. Therefore, selection of extraction solvent volume needed for extraction target compounds is very important. The highest antioxidant activity was measured at up to 50 mL/g of plant material. In the literature, authors used various solid-liquid ratios as 13.3 (LEE et al., 2011), 35 (WANG, 2012) and 100 mL/g of plant material (PARK et al., 2006). Therefore, it can be considered that optimal value of parameter solid-liquid ratio will be located between values 25 – 75 mL/g of plant material. Fig. 1. The kinetic of antioxidant extraction by various temperature (20, 30, 50 and 70 °C) during 1 hour by 50 mL to g of plant material expressed as mg of TEAC per g of plant material. In the context with solubilization of target compounds, solvent polarity is also important. For extraction of active compounds from E. senticosus, authors of other studies recommend using of polar extraction solvent such as ethanol or methanol and Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 16.01.20 14:25 UTC Nova Biotechnologica et Chimica 12-1 (2013) 35 their water mixtures. Because methanol is toxic and unauthorized for food industry, aqueous solution of ethanol with concentrations of ethanol varied between 0 and 96 % (v/v) was used for antioxidant extraction from E. senticosus. The highest antioxidant activity was determined in extracts prepared by 10 and 20 % (v/v) aqueous ethanol. Opposed to the work of LEE et al. (2011) was the highest antioxidant activity in extracts prepared from E. senticosus by 40 – 80 % (v/v) aqueous ethanol. Based on the measured results, the optimization ranges of solvent composition were selected 10 – 30 % (v/v) aqueous solution of ethanol. 3.2 Extraction optimization by response surface methodology (RSM) Optimal conditions of the extraction were calculated by the software Statgraphics Plus 5.1., processed by RSM approach. In Table 1, TEAC and polyphenols are presented. Based upon the regression analysis results, we can state, that compared dependent variable expressed self-independent relation. 3.2.1 Multiple linear regression For the purpose of the fitting the presented results in Table 1, polynomial regression of the second order (Eq. 1) had regression coefficient R2 = 0.95 for TEAC as parameter Y1, R2 = 0.91 for total polyphenols as parameter Y2. 3.2.2 Regression coefficient analysis Regression coefficients of the model for TEAC and total polyphenols obtained by multiple polynomial regression are presented in Table 2. Dependent variable in coded form (Table 1) allow direct interpretation of the effect (linear, quadratic and interaction) of the independent variables to dependent variables and visualization by 3D surface plots (Fig. 2) assisted visualization of the statistically important factors (marked as bold in the Table 2) obtained from statistical analysis. Table 2. Regression coefficients of the model polynomial regression of the second order for dependent variables - TEAC [mg/g plant material] and total polyphenols [mg/g plant material]. Model parameters TEAC Total polyphenols Constant effect -34.11 -20.8457 Temperature [°C] (A) 0.673912 0.653699 Solvent composition [% EtOH] (B) 1.30007 0.83962 Linear Effect Solid-liquid ratio [mL/g] (C) 1.03881 0.213897 A × A -0.00558012 -0.0052778 B × B -0.0390238 -0.0166475 Quadratic effect C × C -0.00598639 -0.00143291 A × B 0.0125748 -0.00116015 A × C -0.00335388 0.00123142 Interaction effect B × C -0.00736462 -0.000160227 Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 16.01.20 14:25 UTC 36 Ondrejovič, M. et al. 3.3 Determination and experimental validation of the optimal conditions Optimal values of the parameters for extraction TEAC and total polyphenols from E. senticosus are presented in Table 3. Predicted value for extraction TEAC (70 °C, 53 mL per g of plant material, 23 % (v/v) aqueous ethanol) and total polyphenols (70 °C, 91 mL per g of plant material, 22 % (v/v) aqueous ethanol) from E. senticosus as dependent parameters were comparable with experimentally measured value at the level of the statistical significance at p < 0.05. Achieved results confirm the possibility to predict the course of the extraction of active substances from E. senticosus by the model under particular experimental conditions. Fig. 2. The relation of the dependent variables from the temperature and solvent composition at constant solid-liquid ratio 50 mL per g of plant material; A – TEAC [mg of TEAC per g of plant material]; B – total polyphenols [mg of gallic acid per g of plant material]. Table 3. Optimal extraction parameters for maximizing yield of TEAC and total polyphenols and comparison of the predicted values of the dependent variables and experimentally measured values at these optimal conditions. Optimal extraction parameters Temperature [°C] 70 70 Solvent composition [% EtOH] 23 22 Solid-liquid ratio [mL/g] 53 91 TEAC [mg/g plant material] Total polyphenols [mg/g plant material] Predicted values 32.1 22.8 Experimental values 31.4 21.3 A B Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 16.01.20 14:25 UTC Nova Biotechnologica et Chimica 12-1 (2013) 37 In the work Lee et al. (2011) not found the optimal conditions for antioxidant extraction from E. senticosus. They describe that both concentration ethanol in extraction solvent and extraction time weren´t affected antioxidant extraction. In comparison with our experiment, they used low value of solid-liquid ratio, which cannot allow achieving sufficient antioxidant extraction. 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