View metadata, citation and similar papers at core.ac.uk brought to you by CORE

REVIEW ARTICLES

High-Purity Isolation Using Solid Phase Extraction Tehniques

1 2 1 Zoriţa DIACONEASA *, Dumitriţa RUGINĂ , Carmen SOCACIU 1 Faculty of Food Science and Technology, University of Agricultural Science and Veterinary Medicine Cluj-Napoca,2 Romania, Calea Mănăştur 3-5, 400372, Cluj-Napoca, Romania; Faculty of Veterinary Medicine,[email protected] University of Agricultural Science and Veterinary Medicine Cluj- Napoca, Romania, Calea Mănăştur 3-5, 400372, Cluj-Napoca, Romania; *Corresponding author, e-mail:

Bulletin UASVM Food Science and Technology 73(1) / 2016 ISSN-L 2344-2344; Print ISSN 2344-2344; Electronic ISSN 2344-5300 DOI: 10.15835/buasvmcn-fst:11986

ABSTRACT

Anthocyanins are water soluble pigments used as natural colorants which have been extendedly studied due to their beautiful colors, and more important, beneficial health potential. Published studies regarding anthocyanins health benefits have been performed using crude extracts from natural sources with no any data about the purity of the tested extract. The impurities from the crude extracts are mostly other phenolic compounds; which can also have biological proprieties. In this case the beneficial potential of crude extract is not attributed only to anthocyanin. Thus, data regarding anthocyanins purity are required for the biological research. Currently, most investigations on anthocyanins are focused on their stability, as well as their purification and identification. In this paper,Keywords the most: recent anthocyanins, techniques purification, in the chemical SPE investigation of the anthocyanins purification are summarized.

1. DEFINITION CHEMISTRY AND DISTRIBUTION found in many vegetal sources, such as roots, leaves, flowers and fruits (Brouillard, 1982). They Anthocyanins are plants pigments wich can be found mostly in plants, while small amounts belong to the class named being can founds in mosses and ferns (Delgado-Vargas F. responsible for the vibrant color in many plantset al., 2000; sare found Fossen to et have al., 2002;a more Welch compelx et al., chemical 2008). vegetables and fruits (Giusti and Wrolstad, 2003). The antocyanins found in flowers and ornamental Anthocyanins (of the Greek anthos = flower and kianos = blue) are the most important plants stucture than those found in fruits and vegetables. pigments. due to theyir chemical stucture, are For example, in blueberries the anthocyanins themmore anstable important and also role soluble as natural in aqueous food colorants media have a simple chemical structure, but comparing and acidic conditions, proprieties which give to number of those from ornamental plants and flowers they are in a higher number (Welcha et (Ahmadiani, 2012, Castanelo Osvaldo et al., 2009; al., 2008). They are usually found in the vacuole Socaciu 2007; Pazmino-Duran et al., 2001). Also of plants, covering fruits and vegetables this they have an important role in protecting plants layer being called outer epidermal peel or flower against ultraviolet-induced damage because of petal. For particular species, the anthocyanins are the colorful appearance of fruits, vegetables, localized in pelicular regions of the cell vacuole, and flowers, they attract animals helping to seed named anthocyanoplasts (Pecket and Small, dispersal and pollination. Anthocyanins can be 1980). More exactly if the vacuolar concentration 2 et al.

DIACONEASA of anthocyanins has high values, the coloring vegetables, cerealsred wine, also anthocyanins of the vegetable, or flower petal is more interse can be found in roots, bulbs, cole crops, grasses even darkens. Also the color hue can be affected and many other sources (Markham et al., 2000; by vacuolar concentration causing the difference Harborne et al., 1998). The glycoside derivatives between bright red and deep red pigmentation of the three non-methylated (Markham et al., 2000). The concentrations of cyaninde, , are the anthocyanins found in fruits and vegetables are most common in nature, being found in 80% of observed to be in edible parts in range from 0.1% pigmented leaves, 69% in fruits and 50% in flowers up to 1.0% dry weight (Delgado-Vargas et al., 2000; (Dey and Harborne, 1993). The distribution of Mazza and Miniati, 1993). The main sources of the six common anthocyanidins in fruits and anthocyanin are red fruits as berries and red grapes, vegetables is: () 50%, (delphinidin) 12%, (pelargonidin) 12%, (paeonidin) 12%, () 7% and () 7%. The glycoside derivatives more widespread in nature are 3-monosides, 3-biosides, 3, 5- and 3, 7-diglucosides being 2.5 more frequent than the 3, 5-diglucosides (Castaneda-Ovando et al., 2009; Kong et al., 2003). The most common sugars realted to anthocyanins chemistry are glucose, galactose, rhamnose, xylose and arabinose (Fig. 1). Moreover these sugars molecules can be linked Fig. 1 as acylated form with aliphatic acids and also with aromatic acids such as p-coumaric, caffeic, . Tipycaly glycosilation and acylation of ferulic, sinapic, gallic or p-hydroxybenzoic acids anthocyanins (Castaneda-Ovando et al., 2009) (Figure 1). Chromane ring

Visible max.(nm) Substitution Name Visible color in MeOH-HCl R1 R2 Magenta Cyanidine (Cy) OH H 535 3 Paeonidine (Pn) OCH H 532 Pelargonidine (Pg) H H 520 Malvidin (Mv) OCH3 OCH3 Purple 542 Delphinidin (Dp) OH OH Red 546 3 PetunidinFig. 2. (Pt) OCH OH 543

Structure of the most commonly anthocyanidins (adapted from Harborne, 1998) Bulletin UASVM Food Science and Technology 73(1) / 2016 3

High-Purity Anthocyanins Isolation Using Solid Phase Extraction Tehniques

However, anthocyanins are known for their C5, or C7 and possibly esterification of the sugars different and complex acylation patterns witch (Welch 2008) (Fig.2). are attached on different sugar (Andersen et The main differences between their structure al., 1995; Giusti et al., 1998). The large variety are mainly the number of hydroxylated groups, of anthocyanins sources found in nature makes the nature and the number of bonded sugars, them a very complicated and fascinating group the aliphatic or aromatic carboxylates bonded of natural plant pigments (García-Beneytez et al., to the sugar in the molecule and the position 2003). of these bonds (Castanelo Osvaldo 2009; Kong Anthocyanins are the most oxidized flavonoids et al., 2003). The published data reports more with the C ring fully unsaturated and a hydroxyl at than 500 different anthocyanins (He and Giusti position 3 (Castanelo Osvaldo 2009; Welch 2008; 2011; Andersen and Jordheim, 2006) and 23 Gehm, 1997). The anthocyanidins (or aglycons) anthocyanidins (Andersen and Jordheim, 2006; are mainly consist of an aromatic ring [A] bonded Kong et al., 2003; Rein, 2005) but only six of them to an heterocyclic ring [C] that contains oxygen, are comonly founds in edible plants (Clifford, which is also bonded by a carbon–carbon bond 2000). The most common anthocyanin found in to a third aromatic ring [B] (Castanelo Osvaldo plants and berries is cyanidine (Harborne, 1998) 2009; Konczak and Zhang, 2004). When the and cyaninide-3-glucoside (Kong et al., 2003). anthocyanidins are found in their glycoside form There are also other uncommon 11 anthocyanidins (linked to a sugar moiety) they are known as including , , , anthocyanins, the sugars positions is often C3, , , 6-hydroxycyanidin,

Cyanidin-3-O-galactoside Cyanidin-3-O- Delphinidin-3-galactoside glucoside

Delphinidin-3-arabinoside -3- Petunidin-3-galactoside galactoside

Malvidin-3-glucoside Malvidin-3- Malvidin-3-arabinoside galactoside

Fig. 3

. Chemical stuctures of common anthocyanins Bulletin UASVM Food Science and Technology 73(1) / 2016 4 et al.

DIACONEASA 3. ANTHOCYANINS ISOLATION , 5-methylcyanidin, , , and have also been detected Till now many kinds of chromatographic in plants (Table 1) (Hou, 2003). The UV-Vis spectral methods have been detailed for anthocyanins data showed that anthocyanins have a maximum isolation and purification. The most repre­sen­ absorption at 217 nm in UV region and at 542 nm tative are high-performance liquid chromatogra­ in the visible region. Figure 3 presents the major phy, paper chromatography, thin-layer chroma­ anthocyanins2. ANTHOCYANINS’ found in edible STABILITY berries. tography, column chromatography high speed countercurrent chromatography. one of the main techniques used for isolating the compounds from Anthocyanins stability is high infuenced by complex mixtures isPreparative chromatography their chemical structure. The pure compunds are is but this tehnique has an the only inconvenient, low stable and they can may undergo degradations is high-priced. Capillary electrophoresis is another (Giusti and Wrolstad, 2003). The anthocyanins method used in order to isolate anthocyanins color is based on the fully conjugated 10-electron (Giusti 2007; Segura-Carretero et al., 2008). The A-C ring π-system, with some contribution by the most convenient method is solid-phase extraction B ring as well. When this is disrupted, the color is (SPE) because is a very popular technique relatively lost as when anthocyanins are in high pH medium simple, allowing the elimination of polarand or bleached by bisulfite (He, 2004; Waterhouse, non-phenolics impurities in one step and do not 2002). imply any sophisticate equipment (Diaconeasa et The anthocyanins stability can be affected also al., 2015; Bunea et al., 2013; Rugină et al., 2012; by several factors such as pH, temperature, light, He and3.1. Giusti,Principles 2011; of solid-phaseXiaoke and Zhimin,extraction 2011; chemical structure, co-pigmentation, acylation, Takeoka and Dao, 2002). oxygen, solvents, enzymes, flavonoids, proteins and metallic ions. Thermal stability of anthocyanins SPE is usually used to clean up the sample can be easily influenced bytemperature and before using a chromatographic or other analytical pH. Also the the oxygen can interact with other method, in order to quantify the amount of analytes components, like sugars and ascorbic acid wich can or to evaluate the purity of the sample. Briefly, affect anthocyanins stability (Welch et al., 2008). this technique is used to remove the interfering In the last decades antocyanins stability became compounds of a mixture in order to obtain a pure a interesting subject due to their potential heath extract only with the compounds of interest. benefits and aslo their aplication in food industy The mechanism work of SPE is pretty similar as as alternative to artificial colorants (Ahmadiani, to that of the liquid chromatography, involves 2012; Rein, 2005). the existence of a solid (stationary) and a liquid (mobile) phase and the interest compounds are

Fig.5. Fig.4. The chemical structure of MCX sorbent and an SPE clean up procedure (from http:// anthocyanin molecule at varied pH conditions (He and Bulletin UASVMservices.leatherheadfood.com) Food Science and Technology 73(1) / 2016 Giusti, 2011) 5

High-Purity Anthocyanins Isolation Using Solid Phase Extraction Tehniques separed according to their physical and chemical mode resin. Also the compounds with no positive properties. The common procedure consists in charge can be removed (Fig. 5). loading the sample that needs to be purified in the Therefore an innovative mixed-mode antho­ SPE column, removing the unwanted compounds, cyanin isolation method utilizing the cation- usually impurities, and collecting the desired exchange and reversed-phase mechanisms of the compounds with another solvent (Fig. 4) (He Oasis MCX SPE cartridge was developed (He and and Giusti, 2011; Pavlovic et al., 2010; Kraemer- Giusti, 2011). The purity of the extracts purified Schafhalter et al., 1998). using this methods was found to be highest than In a recent sudy, a new sorbent Oasis MCX, other SPE methods. Recently, a new research was described and developed for anthocyanins project regarding sorbent capacity, anthocyanin purification. The SPE methods comparison was recovery and also anthocyanin purity was done using C-18 cartriges, Oasis MCX and LH20 reported. The results obtained using Oasis MCX sorbents reagarding the purity of the obtained SPE cartridges were found to be superior to the purified extracts by HPLC. The results showed a commonlyCONCLUSIONS used SPE methods (Simmons, 2012). higher purity for anthocyanin fraction purified on Oasis MCX than the other sorbents used LH20 and C-18 (He and Giusti, 2011). The SPE technique has This paper presents an brief overview more3.2 mechanisms Solid-phase for extraction purification, techniques briefly these regarding antocyanins chemistry and also the are presented Reversed-phase below: technique most used methods for antocyanins purification. The recent studies have demonstrated that anthocyanins play an important role in the The most powerful and used sorbent for biology of the plant, and also they can prevent 18 anthocyanin purification is C due to theyr degenerative diseases such as cardiovascular simplicity. The purification procedure consist diseases, diabetes and cancer. Being coloured in the existence of a non-polar stationary phase molecules, antocyanins can play an important and a polar mobile phase (Rodriguez-Saona role of attraction or repulsion of animals, birds or and Wrolstad, 2001). Therefore, the non-polar insects. In this way they are helping for pollination constituents such as anthocyanins and also other in flowers and seedin vitro dispersal and in vivoin fruits. phenolic are stay attached onto the non-polar A high purity for anthocyanin rich extracts is 18 C sorbent, while the polar compounds will be required for all studies, but also removed by the polar mobile phase. Ethyl acetate for pharmaceutical and food industry, where can can be used to remove a part of non-anthocyanin be used as natural colorants. Pure anthocyanins phenolicMixed-mode in order techniqueto obtain a high purity extracts extracts can be used in the food industry as food (Rodriguez-Saona and Wrolstad, 2001). colorants and also they have a high value in nutraceutical industry. Further study regarding Recently was developed a new mechanism anthocyanins potential health benefits are the mail of anthocyanins purification, based on a mixed objective of many researchers. Asin a vitro consequence, or in vivo mode between cation exchange and reversed- treatmentsthe antocyanin administration. chemistry, stability and also the phase interactions. This method provide a extracts purity is require step for better selectivity of the SPE sorbent for putified Acknowledgments. compounds (He and Giusti,2011). So far this under the frame method was used in order to detect drugs in water This paper was published and aslo urine samples and lately was applied of a national grant financed for antocyanins putification from edible sources by Romanian National Authority, “Anthocyaninsfor Scientific (Pavlovic et al., 2010; Bierman and Sanchez, 2010 liposomesResearch, UEFISCDI,systems andproject their number involvement PN-II-RU- on He and Giusti, 2011). molecularTE-2014-4-0944, signalling 16/01.10.2015 of skin diseases”. Based on anthocyanin properties to acquire a positive charge in acidic conditions, they h The work developed a hypothesis that positively charged of the second author was supported by UEFISCDI anthocyanin molecules (pK ~ 2.8) may interact National Project PN-II-RU-TE-2014-4-1135, 14/ with the negatively charged groups on the mixed- 01.10.2015. Bulletin UASVM Food Science and Technology 73(1) / 2016 6 et al.

DIACONEASA REFERENCES solid-phase extraction method using mixed mode cation- exchange chromatography.

Journal of Chromatography, A 1. Ahmadiani Neda (2012). AnthocyaninThe anthocyaninsBased Blue. Analysis Colorants. The Ohio State University and1218:7914-7922 biological activities of anthocyanins. 2. Andersen OM, Jordheim M (2006). 19. Kong J, Chia L, Goh N, Chia T, Brouillard R (2003). In: Andersen O.M., Markham K.R. (Eds): Flavonoids Phytochemistry, Chemistry, Biochemistry and Applications. CRC Press, Solid-phase64: 923-933. extraction (SPE) - a comparison of 16 Taylor and Francis, Boca Raton: 471–551. 20. Kraemer-Schafhaltermaterials for the purification A, Fuchs of H, anthocyanins Pfannhauser from W (1998). Aronia 3. Bunea A, Rugină D, Sconţa Z, Pop R, Pintea A, Socaciu C, melanocarpa var Nero Tăbăran F, Struijs K, Van Camp J (2013). Anthocyanins determination in various cultivars of highbush , Journal of the reactions Science ofand Food color and blueberries and their antiproliferation and apoptosis stabilityAgriculture, of berry78(3)435-440. anthocyanins properties in B16-F10 metastatic murine melanoma cells, 21. Rein M (2005). Phytochemistry, 95:436-44 . University of Helsinki, 4. Brouillard R (1982). Chemical Structure of Anthocyanins. Department of Applied Chemistry and Microbiology, Food In: Anthocyanins as Food Colors, Markakis, P. (Ed.). Chemistry Division Academic Press, New York, London, Paris, pp: 1-40. 22. Rugină D, Sconţa Z, Leopold L, Pintea A, Bunea A, Socaciu 5. Welch Cara R, Wu Q and James ES (2008). Recent Advances C (2012). Antioxidant Activities of Chokeberry Extracts in Anthocyanin Analysis and Characterization, Curr Anal and the Cytotoxic Action of Their Anthocyanin Fraction on Chem, 4(2): 75–101. HeLa Human Cervical Tumor Cells”, Journal of Medicinal Food, 15, TBD, 15(8): 700-706 6. Castaneda-Ovando A, Pacheco-Hernandez MDL, Paez- Anthocyanic vacuolar inclusions Hernandez ME, Rodriguez JA, Galan Vidal CA (2009). 23. Markham- their nature KR, Gould and KS, significance Winefield inCS, flower Mitchell colouration KA, Bloor. Chemical studies of anthocyanins: A review. Food Chem; SJ, Boase MR (2000). 113:859-871. Anthocyanins in fruits, Phytochemistry, 55: 327-336. 7. Clifford MN (2000). Anthocyanins – nature, occurrence vegetables and grains and dietary burden. Journal of the Science of Food and 24. Mazza G, Miniati E (1993). Development and Agriculture, 80(7)1063–1072. optimization of the SPE. CRC procedure Press, Ino; for Boca determination Raton, FL of 8. Diaconeasa Z, Leopold L, Rugină D, Ayvaz H, Socaciu C 25. Pavlovicpharmaceuticals DM, Babic in waterS, et samplesal (2010). by HPLC-diode array (2015). Antiproliferative and antioxidant properties detection. 33 of anthocyanin rich extracts from blueberry and blackcurrant juice. Int J Mol Sci, 16:2352-65. JournalAnthocyanins of Separation from Science, banana : bracts258-267. (Musa 9. Vargas DF, Jimenez AR, Paredes-Lopez O (2000). Natural 26. Pazmino-Duranparadisiaca) as EA,potential Giusti food MM, colorants. Wrolstad RE, Gloria pigments: carotenoids, anthocyanins, and betalains-- MBA (2001). Food Chem, characteristics, biosynthesis,Nutrigenomics processing, and Proteomicsand stability, in Occurrence, loca­tion 73:327-332. HealthCrit Rev and Food Disease: Sci Nutr Food 40:173. Factors and Gene Interactions, and development of anthocyanoplasts­ 10. Hou De-Xing (2009). 27. Pecket RC, Small CJ (1980). . Extraction,.Phytoche­ Chapter 16. Cancer-Preventive Effects and Molecular isolation,mistry, 19:2571–2576. and purification of anthocyanins. Actions of Anthocyanins, edited byPlantYoshinori phenolics. Mine, Methods Kazuo 28. Rodriguez-Saona LE, Wrolstad RE (2001) inMiyashita, plant biochemistryFereidoon Shahid(2nd printing) Current Protocols in Food Analytical Chemistry. Hoboken, NJ, John 11. Dey PM, Harborne JB, (Eds) 1993. Polyphenols Wiley & Sons, Inc.: F1.1.1-F1.1.11 Extraction from Foods.in , Academic, London 12. Bailon Escribano MT, Santos-Buelga C (2003). 29. Segura-Carretero A, Puertas-MejíaSelective MA, Cortacero- extraction, : Methods in Polyphenol Analysis Ramírezseparation, S, Beltrán and identificationR, Alonso-Villaverde of anthocyanins C, Joven J, Dinelli from (eds.C. Santos-Buelga, G. Williamson). Royal Society of G,Hibiscus Fernández-Gutiérrez sabdariffa L. using A solid (2008). phase extraction-capillary Chemistry, Cambridge, United Kingdom, pp. 1–16. electrophoresis-mass spectrometry (time-of-flight /ion trap) 13. Fossen T, Slimestad R, Ovstedal ChapterD, Andersen 5. Anthocyanins, OM (2002). Biochem Syst Ecol 30:855. Methods of Analysis for Functional Foods and Nutraceuticals . Electrophoresis, 29(13):2852-61. 14. Takeoka G And Lan D (2002). 30. Socaciu C (2007). Food colorants: chemical and functional , Commercial Applicability of an Innovative Anthocyaninproperties. Boca Purification Raton: Taylor Technique, & Francis. Utilizing 616-617 Mixed-Mode Edited by W . JeffreyAnalisys Hurst of CRC anthocyanins Press Print Food ISBN: Colorants 978-1- 31. SimmonsSolid-Phase ST Extraction (2011). Chemical56676-824-5 and eBookFunctional ISBN: Properties, 978-1-4200-1467-9. 15. Giusti MM, 2007. Wine Phenolics , The Ohio State University 429–547, ISBN: 978- 0-8493-9357-0 32. Waterhouse AL (2002). Chapter. Ann. 15. N.Y.Isolation Acad. Sci., 957, 21-36. 16. Giusti MM, Wrolstad RE (2003). Acylated14 anthocyanins Characterization of Bioactive Compounds in Fruits and 33. XiaokeCereals FruitH, Zhimin and CerealX (2011). Bioactives from edible sources and their applicationsPhytochemical in food Methods:systems. BiochemicalA Guide to Modern Engineering Techniques Journal of Plant: 217-225. Analysis. Edited by Özlem 17. Harborne JB and Harborne AJ (1998). Tokuşoğlu and Clifford Hall III CRC Press ,319–336 Print High-purity isolation of ISBN: 978-1-4398-0665-4 anthocyaninKluwer Academic mixtures Publishers, from fruits London, and vegetables UK – a novel 18. He J and Giusti MM (2011).

Bulletin UASVM Food Science and Technology 73(1) / 2016