{Accelerated solvent extraction of carrot - response surface methodology optimization} J. Serb. Chem. Soc. 83 (11) 1223–1228 (2018) UDC 633.43+66.061.3:547.565: JSCS–5145 544.032.732:615.279 Short communication 1223 SHORT COMMUNICATION Accelerated solvent extraction of bioactive compounds from carrot – Optimization of response surface methodology VANJA ŠEREGELJ1#, VESNA TUMBAS ŠAPONJAC1*#, ANAMARIJA MANDIĆ2, GORDANA ĆETKOVIĆ1#, JASNA ČANADANOVIĆ-BRUNET1#, JELENA VULIĆ1# and SLAĐANA STAJČIĆ1# 1Faculty of Technology, University of Novi Sad, Bulevar cara Lazara 1, 21000 Novi Sad, Serbia and 2Institute of Food Technology, University of Novi Sad, Bulevar cara Lazara 1, 21000 Novi Sad, Serbia (Received 19 March, revised 9 August, accepted 10 August 2018) Abstract: Carrot is considered to be rich in bioactive antioxidants, both lipophilic (carotenoids) and hydrophilic (phenolic compounds). In the present study, the conditions for accelerated solvent extraction (ASE) of bioactive compounds from carrots (Daucus carota L.) were optimized using response surface methodology (RSM). Box–Behnken design was employed for the experimental design to obtain the optimized combination of extraction temperature, time, and number of extraction cycles. Total carotenoid content (TCar), total polyphenol content (TPh), free radical scavenging activity (SA) and reducing power (RP) of the obtained extracts were used as responses for the optimization. Considering the four quality indicators, the ideal extraction conditions were found to be: 120 °C, 60 min and three extraction cycles. Under these conditions, predicted values of 28.84 mg β-carotene/100 g for TCar; 530.81 mg GAE/100 g for TPh; 2572.29 μmol TE/100 g for SA and 1336.26 μmol TE/100 g for RP were obtained with high desirability (0.975) and no significant difference (p < 0.05) with the experimental values. Keywords: carotenoids; polyphenols; scavenging activity; reducing power; ext- raction. INTRODUCTION Bioactive compounds in plants have gained popularity for their beneficial effects on human health.1 Storage root of carrot (Daucus carota L.) provides an important source of carotenoids and polyphenols in human diets. The extraction process is the first step towards recovery of natural antioxidants from plants. * Corresponding author. E-mail: vesnat@uns.ac.rs # Serbian Chemical Society member. https://doi.org/10.2298/JSC180531068S ________________________________________________________________________________________________________________________Available on line at www.shd.org.rs/JSCS/ (CC) 2018 SCS. 1224 ŠEREGELJ et al. Optimum extraction methods depend on the characteristics of the bioactive com- pounds and the diversity of the tissue plant structures.2 Therefore, optimization of the extraction conditions is a starting point in obtaining the highest yields of target compounds. Accelerated solvent extraction (ASE) is an automated extract- ion technique that uses elevated pressure and temperature to achieve efficient extraction in a very short time, using lower solvent volume and resulting in higher extraction yields.3 Mustafa et al. reported that both time and temperature and the interaction between these two factors significantly affect the extraction yield of carotenes.4 The aim of this study was to optimize the extraction con- ditions, i.e., time, temperature and number of extraction cycles, for isolation of bioactive compounds from carrots using the ASE technique, taking into account the total carotenoid and polyphenol contents, free radical scavenging activity and reducing power. EXPERIMENTAL Plant material Fresh carrots (Daucus carota L.) were purchased from a local supermarket. After wash- ing, fresh carrots were chopped in a kitchen blender (B 800 E, Gorenje, Slovenia), freeze- -dried at –40 °C (Martin Crist Alpha 2-4, Osterode, Germany), ground and stored at –20 °C until use. Experimental design The optimization of the extraction conditions was established by response surface methodology (RSM). The experimental plan was based on three variables at three levels, referred to as Box–Behnken design. The design consisted of 15 experimental runs, including three replicates at the central point.5 The independent variables were extraction time (X1: 20–60 min), temperature (X2: 40–120 °C) and number of extraction cycles (X3: 1–3). The coded values of the independent variables were –1, 0 and 1. The actual values were chosen from the preliminary studies, and the corresponding coded values of three independent variables are given in Table I. Accelerated solvent extraction procedure A Dionex ASE 350 (Thermo Scientific, Waltham, MA, USA) system was used for the extraction of the carrots using 100 % ethanol. For this purpose, a stainless-steel Dionex cell was filled with a diatomaceous earth (to reduce the volume of the extraction solvent) and the carrot sample (0.5 g) in the ratio 4:1. To prevent the collection of suspended particles in the extract, a cellulose filter was placed at the bottom of the cell. Finally, the cell was placed in the cell tray and used for extraction under the conditions obtained from the RSM guided experimental design. Glass vials were used to collect the extracts, which were stored at −20 °C until further use. Total carotenoid content (TCar) The contents of carotenoids in the carrot extracts were analyzed spectrophotometrically by the method of Nagata and Yamashita6 using the extraction solvent as blank. The content of total carotenoids was calculated using the equation: TCar (mg β-carotene per100 ml) = 0.216A663–1.22A645–0.304A505+0.452A453 (1) ________________________________________________________________________________________________________________________Available on line at www.shd.org.rs/JSCS/ (CC) 2018 SCS. ACCELERATED SOLVENT EXTRACTION OF BIOACTIVE COMPOUNDS FROM CARROT 1225 where A663, A645, A505 and A452 represent the absorbance measured at 663, 645, 505 and 453 nm, respectively. The total carotenoid content is expressed as mg of β-carotene equivalents per 100 g sample. Total polyphenol content (TPh) The total polyphenol content in carrot extracts was determined spectrophotometrically by Folin–Ciocalteau method adapted to microscale.7 The results are expressed as gallic acid equi- valents (GAE) per 100 g sample. Radical scavenging activity by DPPH assay (SA) The levels of free radical scavenging activity (SA) of the carrot extracts on the 2,2- -diphenyl-1-picrylhydrazyl (DPPH•) radical were measured spectrophotometrically in a 96- -well microplate, according to Girones-Vilaplana et al.8 The SA values were calculated using the following equation: SA = 100(AC − AS)/AC (2) where AC is the absorbance of the control and AS is the absorbance in the presence of extracts. The results are expressed as µmol trolox equivalents (TE) per 100 g of sample. Reducing power (RP) The reducing power of the extracts was determined by the method of Oyaizu9 adapted for a 96-well microplate. A calibration curve was made with trolox and the results are expres- sed as µmol TE per 100 g of sample. RESULTS AND DISCUSSION In order to understand the effect of the extraction parameters on the effici- ency of carotenoids and polyphenols extraction, and antioxidant activity of carrot extracts, experimental design was prepared and evaluated by RSM (Table I and Figs. S-1 and S-2 and Table S-I of the Supplementary material to this paper). The medium time (40 min), lowest temperature (40 °C) and one extraction cycle (experiment 9) were the least suitable for the isolation of carotenoids, while the longest extraction (60 min), medium temperature (80 °C) and 3 cycles (expe- riment 8) yielded the highest amount of TCar. Mustafa et al. obtained the highest yield of α- and β-carotene in extracts of carrot by-products using the following ASE extraction conditions: 60 °C and 10 min extraction time (5 cycles of 2 min each).4 However, the carrot samples were fresh and the pressure was constant (50 bar), while in the present study, the carrot samples were freeze-dried and the pressure varied in dependence on the temperature. The amount of TCar in the carrot extracts ranged from 8.42–29.01 mg 100 g–1, which corresponds with the reports of Mustafa et al.4 for the content of α- and β-carotene in carrot by-pro- ducts (10.3–27 mg 100 g–1). The highest TPh amount was obtained using the highest temperature (120 °C), with 3 cycles and a moderate duration of the pro- cess (40 min, experiment 12), whereas the lowest TPh was obtained when the extraction was performed longer (60 min), in 2 cycles and at a relatively low temperature (40 °C, experiment 2). Liu et al. reported that elevated temperature improves diffusion rates and solubility in extraction solvents.10 According to ________________________________________________________________________________________________________________________Available on line at www.shd.org.rs/JSCS/ (CC) 2018 SCS. 1226 ŠEREGELJ et al. Herrero et al. ASE with high temperature is an effective way to increase the recovery of bioactive compounds.11 Longer extraction times, higher extraction temperature, and more extraction cycles also resulted in higher SA and RP of the extracts. Both SA and RP reached the highest values (2615.99 and 1324.80 μmol TE 100 g–1, respectively) when the extraction was performed using the para- meters: 120 °C, 60 min and 2 cycles (experiment 4). TABLE I. Experimental design, total carotenoid (TCar) and polyphenol (TPh) contents, radical scavenging activity (SA) and reducing power (RP) of carrot extracts; the results are presented as mean values of three replicates ± SD Run t / min (X1) t / °C (X2) na (X3) TCarb TPhc SAd RPe 1 20 (–1) 40 (–1) 2 (0) 8.98±0.01 94.21±0.98 404.30±1.59 278.05±0.48 2 60 (+1) 40 (–1) 2 (0) 11.07±0.02 78.51±0.13 473.96±10.38 274.82±0.30 3 20 (–1) 120 (+1) 2 (0) 24.55± 0.01 322.71±0.69 1135.87±3.01 997.85±6.53 4 60 (+1) 120 (+1) 2 (0) 24.20±0.01 470.01±3.03 2615.99±23.41 1324.80±5.27 5 20 (–1) 80 (0) 1 (–1) 22.01±0.01 127.79±0.37 619.30±6.90 569.97±1.91 6 60 (+1) 80 (0) 1 (–1) 23.72±0.01 157.11±0.43 532.43±1.29 730.67±8.40 7 20 (–1) 80 (0) 3 (+1) 24.24±0.01 154.88±0.07 658.47±1.12 483.99±2.28 8 60 (+1) 80 (0) 3 (+1) 30.57±0.01 232.15±0.37 949.60±0.02 649.73±0.85 9 40 (0) 40 (–1) 1 (–1) 8.42±0.01 87.26±0.50 733.54±1.07 192.66±1.40 10 40 (0) 120 (+1) 1 (–1) 24.14±0.03 454.65±0.60 2356.49±1.41 921.57±0.81 11 40 (0) 40 (–1) 3 (+1) 12.57±0.002 107.79±0.82 657.94±3.26 129.56±1.00 12 40 (0) 120 (+1) 3 (+1) 29.01±0.02 472.74±0.84 2071.03±10.79 1040.04±6.07 13 40 (0) 80 (0) 2 (0) 26.02±0.02 144.14±0.19 824.66±2.28 469.38±1.86 14 40 (0) 80 (0) 2 (0) 26.95±0.01 143.26±0.01 997.41±2.11 429.32±0.73 15 40 (0) 80 (0) 2 (0) 27.50±0.01 208.80±0.21 837.78±1.72 514.45±2.13 aNumber of cycles; bmg β-carotene/100 g; cmg GAE/100 g; dμmol TE/100 g; eμmol TE/100 g The experimental values of all quality indicators obtained in the optimization experiments (Table I) were analyzed by single and multi-response optimization and the results are reported in Table II and Figs. S-1 and S-2. TABLE II. Single and multi response optimization of the extraction parameters Optimization Variable code Variable value Optimal response X1 X2 X3 X1 X2 X3 TCara TPhb SAc RPd Single response (TCar) 0 0.21 0.95 40 88.4 3 30.80 – – – Single response (TPh) 1 1 –1 60 120 1 – 472.78 – – Single response (SA) 1 1 1 60 120 3 – – 2572.28 – Single response (RP) 1 1 0.59 60 120 3 – – – 1325.67 Multi response 1 1 1 60 120 3 28.84 530.81 2572.29 1336.26 amg β-carotene/100 g; bmg GAE/100 g; cμmol TE/100 g; dμmol TE/100 g It was found that single-cycle extraction applying a high temperature (120 °C) for a longer time (60 min) ensures the maximum extraction of carotenoids. ________________________________________________________________________________________________________________________Available on line at www.shd.org.rs/JSCS/ (CC) 2018 SCS. ACCELERATED SOLVENT EXTRACTION OF BIOACTIVE COMPOUNDS FROM CARROT 1227 The optimal conditions maximizing TPh were lower temperature and duration, in several cycles. Considering sample 4 that showed the highest SA and RP, it could be concluded that the higher temperature (120 °C) and longer extraction time (60 min) are the main contributors to these responses. The simultaneous optimization of multiple responses is the main concern for industrial applications, especially in view of energy cost reduction. The optimal extraction conditions for all observed responses were 120 °C for 60 min with three extraction cycles. CONCLUSIONS Response surface methodology (RSM) and Box–Behnken design were dev- eloped to determine the optimum process parameters of carrot ASE extraction. The optimal conditions to obtain the highest extraction yield of carotenoids and polyphenols in carrot extracts, as well as maximum antioxidant activity were: 120 °C, 60 min in 3 extraction cycles. Under the optimal conditions, the experi- mental values were in agreement with the predicted values. SUPPLEMENTARY MATERIAL Analysis of variance (ANOVA) of the modelled responses, as well as single- and multi- -response optimization of the influence of extraction parameters on the total carotenoid contents (TCar), total polyphenol contents (TPh), scavenging activity (SA) and reducing power (RP) of carrot extracts are available electronically at the pages of journal website: http:// //www.shd.org.rs/JSCS/, or from the corresponding author on request. Acknowledgements. This work was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia under Grant No. TR 31044 and COST Action 15136. И З В О Д ОПТИМИЗАЦИЈА ЕКСТРАКЦИЈЕ БИОАКТИВНИХ ЈЕДИЊЕЊА ИЗ ШАРГАРЕПЕ РАСТВАРАЧИМА ПОД ПРИТИСКОМ МЕТОДОМ ОДЗИВНИХ ПОВРШИНА ВАЊА ШЕРЕГЕЉ1, ВЕСНА ТУМБАС ШАПОЊАЦ1, АНАМАРИЈА МАНДИЋ2, ГОРДАНА ЋЕТКОВИЋ1, ЈАСНА ЧАНАДАНОВИЋ-БРУНЕТ1, ЈЕЛЕНА ВУЛИЋ1 и СЛАЂАНА СТАЈЧИЋ1 1Технолошки факултет Нови Сад, Универзитет у Новом Саду, Булевар цара Лазара 1, 21000 Нови Сад и 2Научни институт за прехрамбене технологије, Универзитет у Новом Саду, Булевар цара Лазара 1, 21000 Нови Сад Шаргарепа се сматра богатим извором биоактивних антиоксидативних једињења, и липофилних (каротеноиди) и хидрофилних (полифеноли). У овом раду извршена је оптимизација екстракције биоактивних компонената из шаргарепе (Daucus carota L.) растварачима под притиском, употребом методе одзивних површина (RSM). Експери- менти су планирани употребом Box–Behnken дизајна у циљу одређивања оптималне комбинације екстракционе температуре, времена и броја циклуса екстракције. Садржај укупних каротеноида (TCar), укупних полифенола (TPh), способност хватања радикала (SA) и редукциона способност (RP) добијених екстраката коришћени су као одзиви за оптимизацију. Узимајући у обзир ове одзиве добијени су следећи оптимални услови екс- тракције: 120 °C, 60 min и три екстракциона циклуса. Под овим експерименталним условима предвиђене вредности TCar (28,84 mg β-каротена на 100 g), TPh (530,81 mg ________________________________________________________________________________________________________________________Available on line at www.shd.org.rs/JSCS/ (CC) 2018 SCS. 1228 ŠEREGELJ et al. GAE на 100 g), SA (2572,29 μmol TE на 100 g) и RP (1336,26 μmol TE на 100 g) су добијене са високим фактором пожељности (0,975) и без статистички значајне разлике (p < 0,05) у поређењу са експериментално добијеним вредностима. (Примљено 19. марта, ревидирано 9. августа, прихваћено 10. августа 2018) REFERENCES 1. C. O. Perera, G. M. Yen, Int. J. Food Prop. 10 (2007) 201 2. V. T. Nguyen, Recovering Bioactive Compounds from Agricultural Wastes, Wiley, New York, 2017 3. J.-H. Kang, S. Kim, B. K. Moon, Food Chem. 205 (2016) 140 4. A. Mustafa, L. M. Trevino, C. Turner, Molecules 17 (2012) 1809 5. V. Tumbas Šaponjac, J. Čanadanović-Brunet, G. Ćetković, M. Jakišić, S. Djilas, J. Vulić, S. Stajčić, Molecules 21 (2016) 584 6. M. Nagata, I. Yamashita, J. Jpn. Soc. Food Sci. Technol. 39 (1992) 925 7. E. González-Molina, D. A. Moreno, C. García-Viguera, J. Agric. Food Chem. 56 (2008) 8. А. Girones-Vilaplana, P. Mena, D. A. Moreno, C. Garcia-Viguera, J. Sci. Food Agric. 94 (2014) 1090 9. M. Oyaizu, Jpn. J. Nutr. 44 (1986) 307 10. H. Liu, Y. Zhang, Q. Li, Y. Zou, J. Shao, S. Lan, J. Liq. Chromatogr. Relat. Technol. 34 (2011) 2653 11. M. Herrero, M. Castro-Puyana, J. A. Mendiola, E. Ibañez, TrAC, Trends Anal. Chem. 43 (2013) 67. ________________________________________________________________________________________________________________________Available on line at www.shd.org.rs/JSCS/ (CC) 2018 SCS. << /ASCII85EncodePages false /AllowTransparency false /AutoPositionEPSFiles true /AutoRotatePages /None /Binding /Left /CalGrayProfile (Dot Gain 20%) /CalRGBProfile (sRGB IEC61966-2.1) /CalCMYKProfile (U.S. Web Coated \050SWOP\051 v2) /sRGBProfile (sRGB IEC61966-2.1) /CannotEmbedFontPolicy /Error /CompatibilityLevel 1.4 /CompressObjects /Tags /CompressPages true /ConvertImagesToIndexed true /PassThroughJPEGImages true /CreateJobTicket false /DefaultRenderingIntent /Default /DetectBlends true /DetectCurves 0.0000 /ColorConversionStrategy /CMYK /DoThumbnails false /EmbedAllFonts true /EmbedOpenType false /ParseICCProfilesInComments true /EmbedJobOptions true /DSCReportingLevel 0 /EmitDSCWarnings false /EndPage -1 /ImageMemory 1048576 /LockDistillerParams false /MaxSubsetPct 100 /Optimize true /OPM 1 /ParseDSCComments true /ParseDSCCommentsForDocInfo true /PreserveCopyPage true /PreserveDICMYKValues true /PreserveEPSInfo true /PreserveFlatness true /PreserveHalftoneInfo false /PreserveOPIComments true /PreserveOverprintSettings true /StartPage 1 /SubsetFonts true /TransferFunctionInfo /Apply /UCRandBGInfo /Preserve /UsePrologue false /ColorSettingsFile () /AlwaysEmbed [ true ] /NeverEmbed [ true ] /AntiAliasColorImages false /CropColorImages true /ColorImageMinResolution 300 /ColorImageMinResolutionPolicy /OK /DownsampleColorImages true /ColorImageDownsampleType /Bicubic /ColorImageResolution 300 /ColorImageDepth -1 /ColorImageMinDownsampleDepth 1 /ColorImageDownsampleThreshold 1.50000 /EncodeColorImages true /ColorImageFilter /DCTEncode /AutoFilterColorImages true /ColorImageAutoFilterStrategy /JPEG /ColorACSImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /ColorImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /JPEG2000ColorACSImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /JPEG2000ColorImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /AntiAliasGrayImages false /CropGrayImages true /GrayImageMinResolution 300 /GrayImageMinResolutionPolicy /OK /DownsampleGrayImages true /GrayImageDownsampleType /Bicubic /GrayImageResolution 300 /GrayImageDepth -1 /GrayImageMinDownsampleDepth 2 /GrayImageDownsampleThreshold 1.50000 /EncodeGrayImages true /GrayImageFilter /DCTEncode /AutoFilterGrayImages true /GrayImageAutoFilterStrategy /JPEG /GrayACSImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /GrayImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /JPEG2000GrayACSImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /JPEG2000GrayImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /AntiAliasMonoImages false /CropMonoImages true /MonoImageMinResolution 1200 /MonoImageMinResolutionPolicy /OK /DownsampleMonoImages true /MonoImageDownsampleType /Bicubic /MonoImageResolution 1200 /MonoImageDepth -1 /MonoImageDownsampleThreshold 1.50000 /EncodeMonoImages true /MonoImageFilter /CCITTFaxEncode /MonoImageDict << /K -1 >> /AllowPSXObjects false /CheckCompliance [ /None ] /PDFX1aCheck false /PDFX3Check false /PDFXCompliantPDFOnly false /PDFXNoTrimBoxError true /PDFXTrimBoxToMediaBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXSetBleedBoxToMediaBox true /PDFXBleedBoxToTrimBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXOutputIntentProfile () /PDFXOutputConditionIdentifier () /PDFXOutputCondition () /PDFXRegistryName () /PDFXTrapped /False /CreateJDFFile false /Description << /ARA /BGR /CHS /CHT /CZE /DAN /DEU /ESP /ETI /FRA /GRE /HEB /HRV (Za stvaranje Adobe PDF dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. 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