Extraction of Phenolic Compounds from Raspberry Press Cake

SCIENTIFIC WORKS OF THE INSTITUTE OF HORTICULTURE,LITHUANIAN RESEARCH CENTRE FOR AGRICULTURE ANDFORESTRY AND LITHUANIAN UNIVERSITY OF AGRICULTURE.SODININKYSTĖ IR DARŽININKYSTĖ. 2010. 29(2).Extraction of Phenolic Compounds from RaspberryPress CakeRamunė Bobinaitė 1, 2 , Pranas Viškelis 2 , Loreta Buskienė 21Kaunas University of Technology, Radvilėnu pl. 19, LT-50254Kaunas, e-mail ramune.bobinaite@stud.ktu.lt2Institute of Horticulture, Lithuanian Research Centre forAgriculture and Forestry, Kauno 30, LT-54333 Babtai,Kaunas distr., Lithuania, e-mail biochem@lsdi.ltProducing berry-based juices and nectars the seeds are removed from the berry pulp asunwanted ingredient. This by-product contains valuable, bioactive constituents which mightbe extracted and used in the production of food functional ingredients or nutraceuticals.The aim of this preliminary study was to choose the most suitable conditions for theextraction of phenolic compounds, and ellagic acid in particular, from raspberry press cake.For this purpose three-step batch extractions of dried raspberry press cake were performedwith 80 % ethanol (at ambient temperature and 50 °C), and water (at 50 °C). Prior to freezedryingof the extracts, maltodextrin (MD) was added in order to enable the drying process.The highest dry extract yields with added MD were obtained with water at 50 °C and80 % ethanol at 50 °C, in total 23.77 and 23.69 % of the dry raspberry press cake. Though dryaqueous extract yield was high, it had the lowest DPPH radical scavenging activity and totalphenolics, anthocyanins and ellagic acid contents comparing with 80 % ethanol extracts at 25and 50 °C. The highest amounts of total phenolics, total anthocyanins and total ellagic acidfrom raspberry press cake were extracted at 50 °C with 80 % ethanol. Very strong correlationswere found between total phenolics content and radical scavenging activity (r = 0.98), andbetween total phenolics content and total ellagic acid content of the dry extracts (r = 0.97).Key words: phenolics, anthocyanins, ellagic acid, radical scavenging, raspberry presscake.Introduction. Oxidative stress of cellular lipids, proteins and nucleic acids hasbeen associated with the development of several diseases and conditions such ascardiovascular diseases, cancer, Parkinson’s and Alzheimer’s diseases, etc. (Ameset al., 1993; Halliwell, Gutteridge, 2007).Cellular oxidation is usually caused by free radical species produced by normalcellular metabolism or encountered in our environment. Dietary antioxidants such aspolyphenols that are present in foods of plant origin may help to protect cells against theoxidative damage caused by free radicals (Prior, 2003; Wang, Jiao, 2000). It confirms19

was applied. The intention of pre-extraction was to eliminate as much interferingsubstances (sugars, organic acids, etc.) as possible from the final extracts. Unfortunately,aqueous extract obtained during pre-extraction process was not dried andwas not further analysed in the recent study.Solvent to ground press cake ratios during three extraction steps were as follows;5 : 1 (I-st step), 3 : 1 (II-nd step), 3 : 1 (III-rd step). After the extraction ethanol wasrecovered in a rotary vacuum evaporator (Büchi, Flavil, Switzerland). In order toimprove the drying process and textural and/or physical properties of the powderedextract, liquid aqueous extracts were mixed with maltodextrin (final MD concentrationwas 10 % of the dry extract weight). After the addition of MD, extracts werefreeze-dried at – 70 °C and kept in the hermetically sealed containers until analysed.A n a l y s i s o f A n t h o c y a n i n s . For anthocyanins analysis dried extractwith MD were re-dissolved in methanol acidified with 0.1 N HCl. The absorptionof the extracts was measured using a Cintra 202 UV/Vis spectrophotometer (GBCScientific Equipment, Australia) at 535 nm. The concentration of anthocyanins wasdetermined from a calibration curve and expressed in mg of cyanidin-3-glucoside inone gram of dry extract.A n a l y s i s o f P h e n o l i c C o m p o u n d s . For total phenolics assay,dried extracts with MD were re-dissolved in pure methanol. The phenolic contentof the samples was determined using the Folin-Ciocalteu reagent according to themethod of Slinkard and Singleton (1977) using gallic acid as a standard. The reagentwas prepared by diluting a stock solution (Sigma-Aldrich Chemie GmgH, Steinheim,Germany) with distilled water (1/10, v/v). Samples (1.0 mL, three replicates)were introduced into test cuvettes followed by 5.0 mL of Folin-Ciocalteu’s phenolreagent and 4.0 mL of Na 2CO 3(7.5 %). The absorbance of all samples was measuredat 765 nm using a Genesys-10 UV/Vis (Thermo Spectronic, Rochester, USA) spectrophotometerafter incubation at ambient temperature for 1 h. Total concentrationof phenolic compounds was determined from calibration curve and expressed in mgof gallic acid equivalents in one gram of dry extract.A n t i o x i d a n t a c t i v i t y a s s a y . For antioxidant activity assay one gramof dried extracts with MD were re-dissolved in 125 ml of pure methanol. The radical-scavengingactivity (RSA) of the extracts against stable 2.2-diphenyl-1-picrylhydrazylhydrate DPPH• (Sigma-Aldrich, Germany) was determined by a slightlymodified spectrophotometric method of Brand-Williams et al. (1995). DPPH• methanolicsolution (2 mL, 6 × 10 -5 M) was mixed with 20 µL of prepared extract. The reactionwas carried out at ambient temperature. The decreasing absorbance at 515 nmdue to the scavenging of DPPH• was measured with a spectrometer Genesys-10 UV/Vis every 60 s for a period of 30 min to attain reaction equilibrium (Burda, Oleszek,2001). Simultaneously, the absorption of a blank sample containing the same amountof methanol and DPPH• solution was measured daily. The measurements were performedin triplicates.The radical scavenging activity of the berry extracts was expressed as the percentinhibition of the DPPH radical:RSA = (A B- A S)/A B× 100 %where AB is the absorption of blank sample (t = 0 min) and AS is the absorptionof tested sample (t = 30 min).21

Fig. 3. Sorbtion of the dry extracts obtained with water at 50 °C3 pav. Sausų ekstraktų, išekstrashuotų vandeniu 50 °C temperatūroje, sorbcijaThe lowest hyghroscopicity had dry ethanolic extracts obtained at 50 °C (Fig. 4).All the dry extracts obtained at third extraction step were the least hygroscopic ascompared to the respective extracts obtained at thirst and second extraction steps.Fig. 4. Sorbtion of the dry extracts obtained with 80 % EtOH at 50 °C4 pav. Sausų estraktų, išekstrahuotų 80 % EtOH 50 °C temperatūroje, sorbcija24

Fig. 5. Sorbtion of the dry extracts obtained with 80 % EtOH at 50 °Cwith hot water pre-extraction5 pav. Sausų ekstraktų, išekstrahuotų 80 % EtOH 50 °C temperatūroje,taikant priešekstrakciją karštu vandeniu, sorbcijaIt is well established that total phenolics content contribute greatly to overallantioxidant activity of the plant-derived extracts. The highest total phenolics content,as expressed per one gram basis of a dry extract with MD, was found in the extractsobtained with 80 % ethanol at 50 °C, where 20 min pre-extraction with hot water wasapplied (Fig. 6). The average total phenolics value of the dry extracts obtained duringall three extraction steps was 90.84 mg g -1 .Fig. 6. Total phenolics content in the extracts6 pav. Bendras fenolinių junginių kiekis ekstraktuose25

Although, the highest extract yields were received at 50 °C using water as a solvent,the dry aqueous extracts had the lowest contents of phenolic compounds; the averagevalue of the extracts obtained during three extraction steps was only 30.19 mg g -1 .The data indicates that pure water is not an efficient solvent for phenolic compoundsextraction from raspberry press cake, because it yields the extracts of a low purity.It should be noted that, although the extract yields reduced with each repeated stepof extraction (for all the solvent and extraction regimes used) the dry extract qualityregarding the total phenolics content per one gram basis, remained approximately thesame for all three extraction steps.Investigated dry ethanolic extracts of raspberry press cake had higher amountsof total phenolics in comparison with data reported by Peschel et al. (2006) for thedry methanolic extracts of apple, strawberry and pear wastes (52.2, 59.8 and 18.4 mgin g, respectively).Only small quantities of raspberry pulp (where all anthocyanin pigments areconcentrated) stays in raspberry press-cake after juice pressing, therefore anthocyaninfraction comprised a very small part of the total phenolics content found in dry raspberrypress cake extracts. Naturally, the highest amounts of anthocyanins were extractedduring the thirst extraction step (Fig. 7). The highest percentage of anthocyanins of thetotal amount of phenolic compounds was found in the dry aqueous extracts obtainedat 50 °C (I-st stage 9.44 %, II-nd stage 6.96 % and III-rd stage 5.88 %). Howeveras expressed per one gram basis of the dry extract, higher amounts of anthocyaninswere found in the ethanolic extracts extracted at 50 °C and at ambient temperature(see Fig. 7).Fig. 7. Total anthocyanins content in the extracts7 pav. Bendras antocianinų kiekis ekstraktuose26

Comparing to the total ellagic acid content (the sum of free ellagic acid and ellagicacid released after hydrolysis), relatively low levels of free ellagic acid were presentin the dry extracts of raspberry press cake (see Figs. 8 and 9). This indicates that ellagitanninswere the major source of ellagic acid following the hydrolysis.In the investigated dry extracts free ellagic acid comprised from 6.01 to 18.15 %of the ellagic acid content released after acid hydrolysis. The lowest concentrations offree ellagic acid were found in the dried aqueous extracts extracted at 50 °C (Fig. 8).This could be explained by low solubility of ellagic acid in water.Fig. 8. Free ellagic acid content in the extracts8 pav. Laisvos elago rūgšties kiekis ekstraktuoseSolubility of ellagic acid increases in organic solvents such as ethanol or methanol.Ellagitannins are more water-soluble as compared to ellagic acid however dry aqueousextracts also had the lowest total ellagic acid contents (Fig. 9). Regarding total ellagicacid content dried ethanolic extracts obtained during I-st, II-nd and III-rd extractionsteps remained of approximately the same quality. The total ellagic acid content in dryaqueous extract obtained during III-rd extraction step was more than two times higheras compared to the extract obtained during I-st extraction step (Fig. 9).The dry extracts obtained with 80 % ethanol at 50 °C, where 20 min pre-extractionwith hot water was applied, had the highest radical scavenging activity – on average55.5 % (Fig. 10). However using this type of extraction, the lowest extraction yieldswere received, because in this study the pre-extraction water with part of the extractivecompounds was not concentrated and further evaluated. Anyway evaluating per onegram basis of the dry extract pre-extraction with hot water yielded extracts of higherpurity in terms of phenolics content and as a consequence with higher antiradicalactivity (Figs. 6 and 10). The dry aqueous extracts (50 °C) were found to have thelowest radical scavenging activity – on average 16.8 % (Fig. 10).27

Fig. 9. Total ellagic acid content in the extracts9 pav. Bendras elago rūgšties kiekis ekstraktuoseFig. 10. Radical scavenging activity in the extracts10 pav. Ekstraktų radikalų sujungimo aktyvumasIt is well established that antioxidant activity strongly correlates to phenoliccompound concentration in berry extracts (Jakobek et al., 2007) and in berry presscake extracts (Vatai, Škerget, Knez., 2007; Spigno, Tramelli, de Faveri., 2007). Theresults of our investigation are in agreement with those reported by other scientists;very strong correlation (r = 0.98) was found between the total phenolics content and28

adical-scavenging activity of raspberry press cake extracts. Furthermore, very strongcorrelation (r = 0.97) was found between total phenolics content and total ellagic acidcontent of the investigated extracts.Conclusions. Extracts yields and the yields of phenolic compounds were affectedby the type of extraction solvent and extraction temperature. In this study,aqueous ethanol at 50 °C was recognised as the best solvent-temperature system fora high extract yield and high phenolic compounds yield. As expressed per one grambasis of dry extract pre extraction with hot water yielded the higher purity extractregarding the total penolics content, total ellagic acid content and radical scavengingactivity. The high content of antioxidant phenolics found in the extracts suggests thatraspberry press cake may be an economically interesting phytochemical source forthe nutraceutical and functional food market.However more research is required to establish bioavailability and real beneficialeffects of natural antioxidants from raspberry press cake in vivo.Gauta 2010 06 09Parengta spausdinti 2010 06 12References1. Ames B. N., Shigena M. K, Hagen T. M. 1993. Oxidants, antioxidants andthe degenerative diseases of aging. Proceedings of the National Academy ofScience, 90: 7 915–7 922.2. Beekwilder J., Jonker H., Meesters P., Hall R. D., Van Der Meer I. M., De VosC. H. R. 2005. Antioxidants in raspberry: on-line analysis links antioxidant activityto a diversity of individual metabolites. Journal of Agriculture and FoodChemistry, 53: 3 313–3 320.3. Brand-Williams W., Cuvelier M. E., Berset C. 1995. Use of a free radical methodto evaluate antioxidant activity. Lebensmittel-Wissenschaft und Technologie,28: 25–30.4. Burda S., Oleszek W. 2001 Antioxidant and antiradical activities of flavonoids.Journal of Agriculture and Food Chemistry, 49: 2 774–2 779.5. Corhout J., Peiters L. A., Claeys M., Vanden Berghe D. A., Vleitinck A. J. 1991.Antiviral ellagitannins from Spondia mombin. Phytochemistry, 30: 1 129–1 130.6. Daniel E. M., Krupnick A. S., Heur Y. H., Blinzler J. A., Nims, R. W., Stoner G. D.1989. Extraction stability and quantitation of ellagic acid in various fruits andnuts. Journal of Food Composition and Analysis, 3: 338–349.7. Duel C. L. 1996. Strawberries and Raspberries. In: L. P. Somogyj, D. M. Barrett,Y. Hui (eds.), Processing fruits Science and Technology: Major ProcessedProducts. Technolmic Publishing Company, Inc., Lancaster.8. Hakkinen S. H., Karenlampi S. O., Heinonen I. M., Mykkanen H. M., TorronenA. R. 1999. Screening of selected flavonoids and phenolic acids in 19 berries.Food Research International, 32: 345–353.9. Halliwell B., Gutteridge J. N. C. Free radical biology and medicine (4th ed.).Oxford University Press, USA.29

10. Jakobek L., Seruga M., Novok I., Medvidovic-Kosanovic M. 2007. Flavonols,phenolic acids and antioxidant activity of some red fruits. Deutsche Lebensmittel-Rundschau, 103: 369–378.11. Koponen J. M., Happonen A. M., Mattila P. H., Törrönen A. R. 2007. Contents ofanthocyanins and ellagitannins in selected foods consumed in Finland. Journalof Agriculture and Food Chemistry, 55: 1 612–1 619.12. Larrosa M., Tomas-Barberan F., Espin J. 2006. The dietary hydrolysable tanninpunicalagin releases ellagic acid that induces apoptosis in human colonadenocarcinoma Caco-2 Cells by using the mitochondrial pathway. Journal ofNutritional Biochemistry, 17: 611–625.13. Peschel W., Sanchez-Rabaneda F., Diekmann W., Plescher A., Garzis I.,Jimenez D., Lamuela-Raventos R., Buxaderas S., Codina C. 2006. An Industrialapproach in the search of natural antioxidants from vegetable and fruit wastes.Food Chemistry, 97: 137–150.14. Priyadarsini K. J., Khopde S. M., Kumar S. S., Mohan H. 2002. Free radicalstudies of ellagic acid, a natural phenolic antioxidant. Journal of Agriculture andFood Chemistry, 50: 2 200–2 206.15. Prior R. L. 2003. Fruits and vegetables in the prevention of cellular oxidativedamage. American Journal of Clinical Nutrition, 78 (Suppl.): 570–578.16. Siriwoharn T., Wrolstad R. E. 2004. Polyphenolic composition of Marion andEvergreen blackberries. Journal of Food Science, 69: 233–240.17. Slinkard K., Singleton V. L. 1977. Total phenol analysis: Automation and comparisonwith manual methods. American Journal of Enology and Viticulture, 28:49–55.18. Spigno G., Tramelli L., de Faveri M. 2007. Effects of extraction time, temperatureand solvent on concentration and antioxidant activity of grape marc phenolics.Journal of Food Engineering, 81: 200–208.19. Szajdek A., Borowska E. J. 2008. Bioactive compounds and health-promotingproperties of berry fruits: a review. Plant Foods for Human Nutrition, 63: 147–156.20. Šipailienė A., Venskutonis P. R., Pritykina N., Jasinauskienė D., Bobinaitė, R.2009. Antioxidant and antimicrobial properties of raspberry press-cake extracts.Cheminė technologija, 52: 62–68.21. Vatai T., Škerget M., Knez Ž. 2007. Extraction of phenolic compounds fromelder berry and different grape marc varieties using organic solvents and/or supercriticalcarbon dioxide. Journal of Food Engineering, 90: 246–254.22. Vattem D. A. Shetty K. 2005. Biological functionality of ellagic acid: a review.Journal of Food Biochemistry, 29: 234–266.23. Viškelis P., Bobinaitė R. 2007. Fenolinių junginių ir antocianinų kiekis nokstantavietėms. Maisto chemija ir technologija, 41: 115–120.24. Vrhovsek U., Palchetti A., Reniero F., Guillou C., Masuero D., Mattivi F. 2006.Concentration and mean degree of polymerization of Rubus ellagitannins valuatedby optimized acid methanolysis. Journal of Agriculture and Food Chemistry,54: 4 694–4 475.30

25. Wang S. Y. Jiao H. 2000. Scavenging capacity of berry crops on superoxideradicals, hydrogen peroxide, hydroxyl radicals, and singlet oxygen. Journal ofAgriculture and Food Chemistry, 48: 5 677–5 684.SODININKYSTĖ IR DARŽININKYSTĖ. MOKSLO DARBAI. 2010. 29(2).Fenolinių junginių ekstrakcija iš aviečių išspaudųR. Bobinaitė, P. Viškelis, L. BuskienėSantraukaGaminant uogų sultis ir nektarus, kaip šalutinis perdirbimo produktas gaunamos išspaudos.Uogų išspaudose lieka biologiškai vertingų medžiagų, kurias išgavus būtų galima panaudotigaminant ingredientus funkcionaliems maisto produktams arba farmacijos pramonėje.Šio preliminaraus tyrimo tikslas – nustatyti tinkamiausias fenolinių junginių, tarp jų –elago rūgšties, ekstrakcijos iš aviečių išspaudų sąlygas. Tuo tikslu buvo atliekamos trijų pakopųišspaudų ekstrakcijos. Ekstrakcijai buvo naudojami 80 proc. etanolis (kambario ir 50 °Ctemperatūroje) ir vanduo (50 °C temperatūroje). Siekiant pagerinti džiovinimo procesą, priešliofilizavimą į ekstraktus buvo įdėta maltodekstrino (MD).Didžiausia sauso ekstrakto išeiga su MD gauta ekstrahuojant vandeniu arba 80 proc.etanoliu 50 °C temperatūroje. Bendras gauto ekstrakto kiekis nuo išdžiovintų aviečių išspaudųkiekio buvo atitinkamai 23,77 ir 23,69 procento. Nors sauso vandeninio ekstrakto išeiga buvodidžiausia, jo DPPH radikalų sujungimo aktyvumas buvo mažiausias, taip pat vandeniu buvoišekstrahuoti mažiausi fenolinių junginiu, antocianinų ir elago rūgšties kiekiai. Didžiausiasbendras fenolinių junginių, antocianinų ir elago rūgšties kiekis buvo rastas etanoliniuoseekstraktuose, ekstrahuotuose 50 °C temperatūroje. Labai stiprios koreliacijos buvo nustatytostarp sausuose ekstraktuose esančio fenolinių junginių kiekio ir ekstraktų DPPH radikalų sujungimoaktyvumo (r = 0,98) bei tarp ekstraktuose esančio fenolinių junginių kiekio ir bendroelago rūgšties kiekio (r = 0,97).Reikšminiai žodžiai: fenoliniai junginiai, antocianinai, elago rūgštis, radikalų sujungimas,aviečių išspaudos.31