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Analysis and Antioxidant Capacity of AnthocyaninPigments. Part IV: Extraction of AnthocyaninsMaría José Navas a , Ana María Jiménez-Moreno a , Julia Martín Bueno b , Purificación Sáez-Plaza a & Agustin G. Asuero aa Department of Analytical Chemistry, Faculty of Pharmacy, The University of Seville,Seville, Spainb Department of Analytical Chemistry, Higher Polytechnic School, The University of Seville,Seville, SpainVersion of record first published: 27 Aug 2012.
To cite this article: María José Navas , Ana María Jiménez-Moreno , Julia Martín Bueno , Purificación Sáez-Plaza & AgustinG. Asuero (2012): Analysis and Antioxidant Capacity of Anthocyanin Pigments. Part IV: Extraction of Anthocyanins, CriticalReviews in Analytical Chemistry, 42:4, 313-342
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Critical Reviews in Analytical Chemistry, 42:313–342, 2012Copyright c© Taylor and Francis Group, LLCISSN: 1040-8347 print / 1547-6510 onlineDOI: 10.1080/10408347.2012.680343
Analysis and Antioxidant Capacity of AnthocyaninPigments. Part IV: Extraction of Anthocyanins
Marıa Jose Navas,1 Ana Marıa Jimenez-Moreno,1 Julia Martın Bueno,2
Purificacion Saez-Plaza,1 and Agustin G. Asuero1
1Department of Analytical Chemistry, Faculty of Pharmacy, The University of Seville, Seville, Spain2Department of Analytical Chemistry, Higher Polytechnic School, The University of Seville, Seville, Spain
Anthocyanins are naturally occurring polyphenol compounds that impart orange, red, purple,and blue color to many fruits, vegetables, grains, flowers, and plants. In recent decades, in-terest in anthocyanin pigments has increased due to their possible utilization as natural foodcolorants and especially because their consumption has been linked to protection against manychronic diseases. It seems that anthocyanin posseses strong antioxidant properties leading toa variety of health benefits. Coupled to increasing consumer demand, food manufacturershave moved towards increased usage of approved natural colors. Despite the great potentialin applications that anthocyanins represent for food, pharmaceutical, and cosmetic industries,their use has been limited because of their relative instability and low extraction percentage.Growing demands have been made on sample pretreatment, and over time some novel extrac-tion techniques have been developed. Solid phase extraction, countercurrent chromatography,adsorption, pressurized liquid or fluid extraction, and microwave-assisted extraction are en-vironmentally friendlier in terms of using smaller amount of solvents (often nontoxic) andreducing working time. The past few years have been characterized by wide interest in thesetechniques, and many contributions describing these methods have been published. The aim ofthis article is to review the literature available on the most important procedures proposed forthe extraction of anthocyanins; the use of non-thermal technologies in the assisted extractionof anthocyanins will be covered in a separate report.
Keywords Anthocyanins, extraction
INTRODUCTIONIn the human diet, anthocyanins are found in red wine
(He et al., 2012a, 2012b), certain varieties of cereals (Abdel-Aal et al., 2006, 2011), and certain leafy and root vegetables(aubergines, cabbage, beans, onions, radishes), but they are mostabundant in fruits (Bueno et al., 2012a; Mercadante and Bobbio,2008; Kuskoski et al., 2002). A high intake of anthocyanin-richfood has been linked to health-protecting effects (Tsuda, 2012;He and Giusti, 2010). The dietary consumption of grapes andtheir products, for example, is associated with a lower incidenceof degenerative disease and certain types of cancer (Xia et al.,2010). Anthocyanins are glycosylated derivatives of the 3,5,7,3′-tetrahydroxyflavylium cation also known as anthocyanidin andthey comprise the largest group of water-soluble pigments in theplant kingdom (Harborne, 1967; Harborne and Grayer, 1988).Chemical structure, color, and intake of anthocyanins are the
Address correspondence to Agustin G. Asuero, Dept. of AnalyticalChemistry, Faculty of Pharmacy, The University of Seville, 41012Seville, Spain. E-mail: [email protected]
subject of a previous report (Bueno et al., 2012b). The sugarcompound increases the chemical stability of anthocyanidin,therefore anthocyanins are the chemical form most prevalent inberries, grapes, and wine. The number of hydroxyl groups, thenature and number of sugars, and the position of these attach-ments determine the difference between the individual antho-cyanins (Bueno et al., 2012b; Kong et al., 2003). Sugars pro-vide additional sites for structure modifications, as they may beacilated with aromatic or aliphatic acids, e.g., p-coumaric, caf-feic, ferulic, sinapic, acetic, malonic, or p-hydroxybenzoic acid.
Anthocyanins have an antioxidant activity (Kuskoski et al.,2005, 2003), i.e., they act as reducing agents, hydrogen donors,and single oxygen quenchers. This property depends to a largeextent upon the number and location of the OH groups present,the extent of strutural conjugation, and the presence of electron-donating and electron-withdrawing substituents in the ring struc-ture (Lapornik et al., 2005; Kuskoski et al., 2004). The rangeof color associated with anthocyanins is the result of variedsubstitution of the parent C6C3C6 aglycone nucleus, acylationspatterns, and various environmental influences (Bueno et al.,
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2012b; Rodriguez-Saona and Wrolstad, 2001). It is possibleto extract colors from various plant sources, but for economicreasons, grape skins, a byproduct of the wine industry, are thecommon source of anthocyanins (Scotter, 2010; Francis andMarkakis, 1989). Because of their highly reactive nature antho-cyanins readily degrade or react with other constituents to formcolorless or brown compounds (Cavalcanti et al., 2011). Loss ofanthocyanins also occurs in the presence of oxygen and variousenzymes and as a result of high-temperature processing (Bridleand Timberlake, 1997).
Overall, cyanidin is the most common anthocyanidin foundin foods (Manach et al., 2004), followed by pelargonidin, peoni-din, and delphinidin, then by petudinin and malvidin (Jackmanand Smith, 1992). The anthocyanin content in foods (fruits,berries, etc.) is generally proportional to color intensity andreaches values up to 2–4 g/kg fresh weight in blackcurrants andblackberries, increasing as the fruit ripens (Basu et al., 2010).Wine contains approximately 200–350 mg anthocyanins/L, andinvolved anthocyanins are transformed into various complexstructures as the wine ages (He et al., 2012a, 2012b; Jordheim,2007). There is also significant interest in the identification ofpyrranoanthocyanins, which are products formed from antho-cyanins during processing of fruit juices and the aging of wines(Jing and Giusti, 2011).
Documentation of the anthocyanin content of different botan-ical sources is important for determination of structural diversityas well as for food and health perspectives (Jordheim, 2007).The anthocyanin composition in berries has been used as abotanical tool for taxonomic classification of plants, as the an-thocyanin profile tends to be characteristic of a plant, similar toa fingerprint (Jing and Giusti, 2011). The pigmentation of plantsis rarely due to a single anthocyanin, and several food sourcesof anthocyanins contain significant levels (Scotter, 2010, 2011a,2011b). These include grapes, blackcurrants, elderberries, cran-berries, and cherries and the aqueous extracts of roselle (thecalyces of hibiscus) and of red cabbage.
Among the food plants available in nature, grapes arethe major source of anthocyanins. Pigment content in grapesranges between 30 and 75 mg/100 g and varies greatly accord-ing to cultivar, season, and environmental factors (Bridle andTimberlake, 1997). Depending upon the grape variety, the sepa-rate anthocyanin content usually goes from at least 4 to over 16anthocyanins (Rein, 2005). The Concord variety has 31 antho-cyanins, the greatest number in any simple cultivar (Mazza andFrancis, 1995). Some anthocyanin profiles are rather simple,such as the cases of strawberries, where 80–90% of the pigmentcomposition is just one pigment, pelargonidin-3-glucoside (Jingand Giusti, 2011). Blueberries have the most complex antho-cyanin profile among common berries, containing over 25 in-dividual anthocyanins, from five different aglycones (cyanidin,peonidin, malvidin, petunidin, and delphinidin) and three differ-ent glycosilating sugars and acetylated monoglycosides of theseanthocyanins (Howard et al., 2012; Jing and Giusti, 2011). Thus,the preparation of a full range of reference materials is difficult
and expensive (Scotter, 2010), due to the potential number ofanthocyanins present in a given fruit or vegetable extract. How-ever, if the main anthocyanin of the extract is known or can beidentified, the total anthocyanin content may then be expressedin terms of the major component (Prodanov et al., 2005; Giustiand Wrolstadt, 2001), and only one purified reference standardis then required for quantitation.
Metabolites are the intermediates and end products of cel-lular regulatory processes, and their levels can be regarded asthe ultimate response of biological systems to genetic or en-vironmental changes (Bueno et al., 2012a; Traka and Mithen,2011; Pereira et al., 2005). Differences due to volatility, polar-ity, solubility, and chromatographic behavior mean that multiplemethods will need to be deployed to analyze different subsetsof metabolites (Jones and Kinghorn, 2005; Ward et al., 2003).In this context (Seger and Sturm, 2007; Pereira et al., 2005)high-performance liquid chromatography (HPLC), mass spec-troscopy (MS), Fourier transform-infrared spectroscopy (FT-IR), spectroscopy methods (UV, fluorescence), and coupledgas chromatography-mass spectrometry (GC-MS) have alreadybeen successfully applied to plant metabolite profiling. Anotherpotentially powerful tool for plant metabolite analysis is high-resolution nuclear magnetic resonance (NMR) spectroscopy, inparticular, 1H NMR (Krishnan et al., 2005). The use of chemo-metric methods on data obtained by spectrometer analyses to-gether with recent advances in computer technology allows thedevelopment of multivariate data analysis as a powerful toolin the evaluation of food quality (Ferrari et al., 2011; Saurina,2010; Winterhalter, 2007; Wrolstad and Durst, 2007; Debordeet al., 2005).
Much of the literature on food colors is concerned not somuch with the role of natural pigments as food additives as withthe composition and analysis of those pigments (Schoefs, 2002,2003, 2005) that are inherently present in foodstuffs. This iscertainly the case with anthocyanins (Scotter, 2011a, 2011b).Extraction of anthocyanins from natural sources may be morefavorable than laboratory synthesis because of the labile natureof the compounds (Castaneda-Ovando et al., 2009). For natu-ral products, extraction of anthocyanins typically involves someform of solid-liquid extraction, followed by solid phase and/orliquid-liquid extraction to help remove unwanted species, suchas phenolic acids, sugars, proteins, and other flavonoids (Barnes,2010; Barnes et al., 2009). In recent years, various novel extrac-tion techniques have been developed (Wijngaard et al., 2012;Ignat et al., 2011; Kataoka, 2010) for the extraction of nutraceu-ticals from plants, including countercurrent chromatography,ultrasound-assisted extraction, microwave-assisted extraction,supercritical fluid extraction, and high hydrostatic pressure ex-traction (HHP).
Polyphenol analytical chemistry has been the subject of var-ious studies in this journal (Bueno et al., 2012a, 2012b; Navaset al., in press; Escudero, 2011; Pyrzynska and Pekal, 2011;Biesaga and Pyrzynska, 2009; Pappas and Schaich, 2009; Pohlet al., 2009; Zima et al., 2009; Marszall and Kaliszan, 2007;
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ANTHOCYANIN PIGMENTS. PART IV: EXTRACTION 315
Escarpa and Gonzalez, 2001). This article aims to overview theextraction approaches that have been either developed or appliedto anthocyanins. It brings together information from a scatteredliterature to give an accessible account of assisted extractionmethods for anthocyanins. However, the use of non-thermaltechnologies in the assisted extraction of anthocyanins will becovered in a separate report.
ANTHOCYANIN SOLVENT EXTRACTIONThe analysis of anthocyanins is complex as a result of their
ability to undergo structural transformations and complexationreactions (Bueno et al., 2012a, 2012b). Anthocyanins may bepart of complexes, may occur in complex matrices, and mayexist in a variety of protonated, deprotonated, hydrated, and iso-meric forms, and the relative proportion of these molecules isstrongly dependent on pH. Acid dissociation constants (Asueroand Michalowski, 2011; Asuero, 1989, 2007; Asuero et al.,1986a, 1986b; Herrador et al., 1987) are important physico-chemical parameters that describe the extent of ionization offunctional groups as a function of pH (Bueno et al., 2011a,2012b); they are of vital importance in the analysis of bioactivecompounds as well as in the interpretation of their mechanismof action. Their antiradical properties, i.e., the ability to reactquickly and efficiently with electron-deficient radicals, dependon the acidity of phenolic hydroxyl groups and on the stability ofthe formed radical (Leopoldini et al., 2011; Estevez et al., 2010;Musialik et al., 2009; Pina et al., 2012a; Pina et al., 2012b).
Typical procedures for isolation and characterization of pureanthocyanins consist of several steps: (i) extraction of the plantmaterial, followed by a preliminary purification step, (ii) frac-tionation of the mixture followed by isolation of pure pigments,and finally (iii), characterization and identification of pure an-thocyanins (Strack et al., 1989). Methods such as solid-phaseextraction (SPE), as shown later, use solid adsorbents to extractphytochemicals from liquid matrixes such as juices. It is easy,rapid, and economical compared to solvent extraction. However,SPE is perhaps more often used in sample cleanup, purificationor pre-concentration than in extraction because of the selectivityand saturation of the adsorbents (de la Rosa et al., 2009; Tsaoand Deng, 2004).
Extraction is a very important stage in the isolation, iden-tification, and use of anthocyanins (Andersen and Markham,2006). The recovery of anthocyanins is commonly performedthrough a solvent-extraction procedure and the solvent type, sol-vent concentration, liquid-to-solid ratio, temperature, and timeare important parameters to be optimized (Zou et al., 2011).The extracting solution should be slightly acidic to maintainthe flavylium cation form, which is red and stable in highlyacidic medium, but not so acidic to cause partial hydrolysis ofthe acyl moieties in acylated anthocyanins (Welch et al., 2008).The structural diversity, together with the susceptibility of an-thocyanins to heat, pH, metal complexing, and copigmentation,complicates the protocols of extraction and analysis from bothplant material and biological fluids (Mazza et al., 2004). Antho-
cyanins are heat-sensitive, so high temperatures must be avoidedduring extraction and concentration, i.e., <30◦C (Andersen andMarkham, 2006).
Anthocyanins, like flavonoids in general, have aromatic ringscontaining polar substituent groups (hydroxyl, carboxyl, andmethoxyl) and glucosil residues that altogether produce a polarmolecule (Bueno et al., 2012a, 2012b; Delgado-Vargas et al.,2000). While flavonoid glycosides are more polar, aglyconesare extracted with alcohols or alcohol-water mixtures. Antho-cyanins are extracted with cold acidified solvents (polar organicsolvents, water) under mild conditions (Giusti and Jing, 2008).The organic solvent is usually methanol, but many other sol-vents may be used such as acetone, ethanol, or acetonitrile. Thissolvent system denatures the cell membranes, simultaneouslydissolving the anthocyanins and stabilizing them (Naczk andShahidi, 2006). The acid employed is usually acetic acid (about7%) or trifluoroacetic acid (TFA; about 3%), whereas the or-ganic solvent content varies from 50% to 100% of the mixture(Nicoue et al., 2007; Robards, 2003). The use of mineral acidcan lead to the loss of attached acyl group (Dai and Mumper,2010; Barnes et al., 2009). Sulfured water has also been used asextraction solvent in seeking a reduction of the use of organicsolvents as well as the cost of extraction (Cacae and Mazza,2002).
Anthocyanins are normally extracted with methanol contain-ing 0.5% trifluoracetic acid (TFA) (v/v). Black beans (Phase-olus vulgaris) were also presoaked in water containing 0.5%TFA (Jordheim, 2007; Jordheim et al., 2006). In this case thiswas done to improve the extraction yields of anthocyanins be-cause direct methanolic extractions provide very poor yield.The extraction was performed in a refrigerator (5◦C) at lowtemperatures to avoid hydrolysis of potential acyl groups in theanthocyanin structure and degradation. After extraction the ex-tract was filtered, and the methanol was removed by evaporationunder reduced pressure at relatively low temperatures (<30◦C).
Anthocyanins are also extracted under cold conditions withmethanol, if it contains 2–10% formic acid. This process is verygood for grape extraction despite the fact that the final water-alcohol extract will include some non-polyphenolic substancessuch as sugars, amino acids, proteins, and pigments (Shi et al.,2005). Thus, the extract must be purified. If appreciable amountsof lipids, chlorophylls, or unwanted polyphenols are suspectedto be present in anthocyanin-containing extracts, they may beremoved by washing with ethyl acetate, petroleum ether, ethylether, or diethyl ether (Rodriguez-Saona and Wrolstadt, 2001).A procedure used acetone as the extracting solvent followedby separation of the aqueous phase by addition of chloroform,isolating and partially purifying the pigments in this way. Theaqueous portion contains the anthocyanin, phenolics, sugar, or-ganic acids, and other water-soluble compounds, whereas thechoroform phase contains the lipids, carotenoids, chorophyllpigments, and other nonpolar compounds. High recoveries ofanthocyanins are obtained requiring little concentration or fur-ther purification (Rodriguez-Saona and Wrolstad, 2001).
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316 M. J. NAVAS ET AL.
Column chromatography has been also employed for frac-tionation of phenolic extracts. This method, though often laborintensive and solvent consuming, ensures that greater amountsof fractions can be obtained for use in subsequent isolationand identification of pure substances (Dai, 2009; Andersen andMarkham, 2006).
The composition of anthocaynins and polyphenols is highlydependent on the extraction temperature (Holtung et al., 2011;Ruenroengklin et al, 2008), which reflects the conflicting actionsof solubilization and analyte degradation. Under cold extraction,color degradation was significantly lower and extraction timeswere 15-fold longer (Ramirez Rodrıgues et al., 2011). A 50%methanol extract shows maximum recovery for anthocyaninsbunga kantan (Etlingera elatior Jack.) inflorescence (Wijekoonet al., 2011). Eight different solvent mixtures containing acetoneor methanol, pure or combined with an acid (acetic, formic, hy-drochloric), were tested for their efficiency for extraction of phe-nolic compounds from strawberries belonging to five groups ofpolyphenols including anthocyanins (Kajdzanoska et al., 2011).
Solvent extraction offers good recovery of antioxidant phy-tochemicals from various samples. The primary drawback ofthe traditional extraction procedure is that the obtained finalextracts often require subsequent concentration and cleanupprior to analysis. Furthermore, when considering the extractionof bioactive compounds, which are unstable and thermolabileand are found in low concentrations, traditional extraction tech-niques would not be the most suitable option (Routray and Orsat,2012). However, the use of large amounts of organic solventsposes health and safety risks and is environmentally unfriendly.There are many alternative methods that either eliminate or re-duce significantly the use of organic solvents, e.g., microwave-assisted extraction (MAE), supercritical fluid extraction (SFE),and pressurized liquid extraction (PLC), whose use is increas-ingly popular in the extraction of antioxidant phytochemicals.Some of these methods offer identical, if no better, extrac-tion efficiency and cost effectiveness (Tsao and Deng, 2004).Table 1 shows the advantages of limitations of these extractionprocedures.
SOLID PHASE EXTRACTIONSolid phase extraction (SPE) was developed in the 1980s and
has emerged as a powerful tool for chemical isolation and pu-rification (Santana et al., 2009). The goals of SPE are usuallyretention and elution of an analyte from a sample, removal ofcontaminants and interfering substances, and sample concen-tration (Kole et al., 2011). It is based on the same principle ofaffinity-based separation as liquid chromatography and over-comes the limitations of liquid-liquid extraction (Zwir-Ferencand Bizink, 2006). It requires application of samples in a liquidstate, a proper extraction being the first preparation step of solidsamples. Simple filtration or centrifugation and then a filtrate orsupernatant is applied to the SPE cartridge.
SPE on C18 cartridges (because polyphenol molecules havehydrophobic groups, nonpolar absorbents should be used) or
Sephadex is commonly used for the initial purification of thecrude anthocyanin extracts (Giusti and Jing, 2008; Rodriguez-Saona and Wrolstad, 2001). Although they are available in nor-mal phase, reverse-phase, and ion-exchange modes, reverse-phase adsorbents are used as the main adsorption materialsin SPE (McDonald and Bouvier, 2001). The anthocyanins arebound strongly to these adsorbents through their unsubstitutedhydroxyl groups and are separated from unrelated compoundsby using a series of solvents or increasing polarity (Mazza et al.,2004; Kong et al., 2003).
Currently used methods for anthocyanin extraction are nons-elective and result in solutions with large amounts of undesirableproducts such as sugars, acids, amino acids, and proteins that re-quire removal. Crude extracts have been purified by removal ofsugars, acids, and water-soluble compounds with C18 cartridgespreviously activated with methanol, followed by water or 0.01%aqueous HCl or 3% formic acid (Mazza et al., 2004; Wang andSporns, 1999). Partitioning of extracts with ethylacetate hasbeen shown to remove interferences prior to LC-MS analysis(Giusti et al., 1999). Anthocyanins were recovered from dilutedfruit juice or wine (after removal of ethanol) by elution froma C18 cartridge with an aqueous eluent at a low pH (Corradiniet al., 2011). However, SPE was used to obtain anthocyanin-rich extracts from berry species (Denev et al., 2010) and toobtain a pigment-rich fraction in anthocyanins from grape skins(Kneknopoulos et al., 2011) as well as in the anthocyanin anal-ysis of cranberry fruit and cranberry fruit products (Brown andShipley, 2011), prior to the HPLC final determination. Shah andChapman (2009) used mixed-mode cation exchange SPE to pu-rify anthocyanins from tulip extracts in 50:50 methanol:waterwith 0.1% formic acid, and Ling et al. (2009) have usedhydrophilic-lipophilic balanced (HLB) SPE to extract antho-cyanins from human tissue homogenates. Automated off-lineSPE can be used in combination with LC-MS-MS to meetspecific needs (Dumont et al., 2010). Applications of SPE toanthocyanins are compiled in Table 2.
COUNTERCURRENT CHROMATOGRAPHYSeparation by countercurrent chromatography (CCC) is
based on the partition of a phytochemical (Sarker et al., 2005;Tsao and Deng, 2004). CCC is a technique that allows the frac-tionation and isolation of pure compounds to yield the largeamounts required for identification by MS and NMR method-ologies (Yao et al., 2012; Andersen and Markham, 2006), orfor further utilization as standards in analytical methods, or asbioactive compounds for biological studies (Hillebrand et al.,2009). In certain cases CCC can replace traditional techniqueslike low-pressure columns or semi-preparative and preparativeHPLC, since CCC can provide great resolution and greateryields at lower costs (Valls et al., 2009).
High-speed countercurrent chromatography (HSCCC), a rel-atively new technique, is the most advanced CCC form in termsof partition efficiency and separation time (Du, 2011). Un-like other chromatographic techniques, HSCCC does not use
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ANTHOCYANIN PIGMENTS. PART IV: EXTRACTION 317
TABLE 1Advantages and disadvantages of pretreatment techniques (Ignat et al., 2011; Kivilompolo, 2009; Tsao and Deng, 2004; Wang
and Weller, 2006; Jakubowska et al., 2005)
Technique Advantages Limitations
SFE (CO2) CO2 is chemically inactive, has low toxicity, and isenvironmentally friendly, nonflammable, andcheap
Reduces use of organic solventsLow operation temperature possibleRapid, automatable, selective
CO2 is nonpolar and addition of modifier is oftenneeded
Expensive instrumentationOptimizing the method is demanding and often
matrix dependentHigh concentrations of organic modifiers lead to
reduced selectivityPHWE (pressurized
hot waterextraction)
Water is environmentally friendly, nontoxic,nonflammable, and cheap
Reduces use of organic solventsAlso suited to wet samples
High temperature and pressure make demands onenergy and material
No commercial instrument availableNot suitable for labile compoundsDirty extracts (if high temperature)
MAE (microwave-assistedextraction)
Reduced extraction timeReduced solvent usageImproved extraction yieldProcess simplicity and low cost
It is necessary to remove the solid residue duringMAE
The efficiency of microwaves can be very poorwhen either the target compounds or the solventsare nonpolar, or when they are volatile
SPE (solid phaseextraction)
Reduced lab timeEasy manipulationHigher concentration factorNo problem with the miscibility of solventsEasy adaptable for very selective extractionEasy automation
ChannelingLimited flow ratesInsufficient equilibration time for quantitative
uptakeIncomplete elutionMemory effects from previous extraction (though
these disadvantages are more pronounced only inchelating resins)
a solid support as the stationary phase (Sethi et al., 2009) andtherefore has many advantages over conventional chromatogra-phy (Berthod et al., 2009). It has been used for separation ofanthocyanins in the pigment mixtures extracted from varioussamples. Anthocyanins were successfully fractionated based ontheir polarities into the biphasic mixture of tert-butyl methylether/n-butanol/acetonitrile/water acidified with trifluoroaceticacid (TFA) (Dai and Mumper, 2010). Wine is an importantsource of dietary antioxidants because of its phenolic compoundcontent (Noguer et al., 2008). CCC, because of its gentle sep-aration conditions, is ideally suited to the analysis of variousgroups of wine constituents (Winterhalter, 2009). Selected ap-plications of the CCC method applied to anthocyanins are shownin Table 3.
ADSORPTIONProcessing of solutions containing phenolics by adsorption-
desorption enables the recovery and purification of bioactivecompounds or fractions from plants (Soto et al., 2011). Althoughmost reported information deals with experiments at the labscale, studies conducted in pilot plants and industrial facilities
are also available (El-Shafey et al., 2005). Commercial resinsare widely used at the pilot scale, e.g. recovery of anthocyaninsfrom grape pomace (Kammerer et al., 2005). The versatility,simplicity, low cost, high efficiency, and ease in upscaling ofadsorption processes make them an attractive possibility for theselective removal/recovery of phenolic compounds (Soto et al.,2011). Adsorption shows a number advantages over separationscarried out by partition with organic solvents, such as selectivity,environmental impact, and toxicological effects.
Adsorption onto polymeric resins of anthocyanins from grapepomace, Ficus racemosa L. fruit, mulberries, eggplant peel, andred cabbage were reported as well as selective recovery fromcitrus byproducts containing anthocyanins. Cation-exchangeresins and clays have been used for anthocyanin recovery frompomegranate juice and red cabbage extracts, respectively. Ta-ble 4 contains a number of applications of adsorbent and elutingagents proposed for the selective recovery of anthocyanins.
PRESSURIZED FLUID EXTRACTIONSupercritical fluid extraction (SFE) represents an interest-
ing alternative technique to conventional solid-liquid extraction
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TAB
LE
2Se
lect
edap
plic
atio
nsof
solid
phas
eex
trac
tion
(SPE
)ap
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dto
anth
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Type
ofm
atri
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alyt
eE
xtra
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n/cl
eanu
pte
chni
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Sorb
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Fina
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sR
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ence
Arc
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logi
cal
sam
ples
/mal
vidi
nSo
nica
tion
and
cent
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gatio
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us/R
PSP
E
SPE
cart
ridg
e(A
gile
ntA
ccuB
ond
OD
S-C
18)
LC
-MS/
MS
Bar
nard
etal
.,20
11
Que
ensl
and
red
win
es/p
heno
licpr
ofile
sN
on-a
ntho
cyan
inph
enol
ics
prev
ious
lyex
trac
ted/
SPE
C18
reve
rsed
-pha
sepa
ckin
g(A
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ssoc
iate
s,D
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eld,
Ill.)
HPL
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AD
-MS
Gin
jom
etal
.,20
11
Gra
pesk
ins
ofpi
not
noir
/ant
hocy
anin
san
dre
late
dco
mpo
unds
Ext
ract
ion
with
MeO
H/S
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rom
atog
raph
y
Am
berl
iteX
AD
-7re
sin
Mul
tilay
erco
ilco
unte
rcur
rent
chro
mat
ogra
phy
(ML
CC
C)
and
ESI
-MS
Kne
knop
oulo
set
al.,
2011
Dif
fere
ntbi
olog
ical
tissu
es/p
rocy
anid
ins,
anth
ocya
nins
,and
alka
loid
s
Off
-lin
eliq
uid-
solid
extr
actio
n(L
SE)-
SPE
and
off-
line
LSE
-µSP
E
OA
SIS
HL
Bca
rtri
dges
(60
mg,
Wat
ers
Cor
p.,
Milf
ord,
Mas
s.)
UPL
C-E
I-M
S/M
SSe
rra
etal
.,20
11b
Five
berr
ysp
ecie
s/an
thoc
yani
nsE
xtra
ctio
nw
ithci
tric
acid
and
cent
rifu
gatio
n/SP
EA
mbe
rlite
XA
D-7
Spec
trop
hoto
met
ryD
enev
etal
.,20
10
Win
em
atri
ces
and
grap
eju
ice/
anth
ocya
nins
and
othe
rph
ytoc
hem
ical
s
Liq
uid-
liqui
dex
trac
tion/
auto
mat
edof
f-lin
eSP
E
LC
-MS-
MS
Dum
onte
tal.,
2010
Win
e/an
thoc
yani
nsSP
EPo
lym
erph
ase
Stra
taSD
B-L
,(P
heno
men
ex,T
orra
nce,
USA
)
LC
/MS
Har
tman
ova
etal
.,20
10
Red
kiw
ifru
it/an
thoc
yani
nco
mpo
nent
sE
xtra
ctio
nw
ithE
tOH
and
HC
OO
H/S
PEX
AD
-7H
PLC
-DA
D,L
C-M
S/M
S,an
dG
C-M
SC
omes
key
etal
.,20
09
Hum
ansa
liva,
plas
ma,
and
oral
tissu
e/cy
anid
in3-
gluc
osid
ean
dot
her
3-su
bstit
uted
cyan
idin
sfr
ombi
oadh
esiv
ebl
ack
rasp
berr
yge
l
Cen
trif
ugat
ion
and
supe
rnad
antd
ilutio
n(s
aliv
a).S
ampl
essp
iked
with
inte
rnal
stan
dard
(tis
sue,
plas
ma)
HL
Bca
rtri
dge
LC
-MS/
MS
Lin
get
al.,
2009
Hum
anpl
asm
a/an
thoc
yani
nsSP
Ew
itha
cent
rifu
gatio
npr
evio
usO
asis
HL
B1
cm3
(10
mg)
extr
actio
nca
rtri
dges
LC
/MS
Nak
amur
aet
al.,
2009
318
Dow
nloa
ded
by [
Lau
rent
ian
Uni
vers
ity]
at 1
3:15
24
Febr
uary
201
3
Red
tulip
bloo
ms
(Tul
ipa
darw
inhy
brid
‘Ape
ldoo
rn’)
/ant
hocy
anin
san
dfla
vono
lgly
cosi
des
50/5
0m
etha
nol/w
ater
with
0.1%
form
icac
id,
soni
catio
nan
dfil
trat
ion
DSC
-MC
AX
SPE
cart
ridg
e,10
0m
g/3
mL
(527
83-U
),H
PLC
/DA
D/E
SI-M
S/M
SSh
ahan
dC
hapm
an,
2009
Ace
rola
/pul
psan
dju
ices
/ant
ioxi
dant
com
poun
ds
SPE
Wat
ers
C18
cart
ridg
esH
PLC
-DA
DM
ezad
riet
al.,
2008
Vio
letc
aulifl
ower
and
red
cabb
age/
anth
ocya
nin
profi
les
SPE
C18
HPL
C-M
S/M
SL
oSc
alzo
etal
.,20
08
Red
win
es/p
heno
licco
mpo
unds
SPE
with
prio
rce
ntri
fuga
tion
HL
Bw
ithN
-vin
ylpy
rrol
idon
e-di
viny
lben
zene
copo
lym
er
HPL
CPe
rez-
Mag
arin
oet
al.,
2008
Hib
iscu
s/an
thoc
yani
nsSP
EA
mbe
rlite
XA
D-2
(por
esi
ze9
nm,p
artic
lesi
ze0.
3–1.
2m
m)
CE
-ESI
-MS
Segu
ra-C
arre
tero
etal
.,20
08
Gre
ekre
dw
ines
/phe
nolic
finge
rpri
ntSP
Eun
der
vacu
umC
18(B
aker
bond
J.T.
Bak
erSP
EC
18)
UV
-vis
and
mid
-IR
with
mul
tivar
iate
data
anal
ysis
Tara
ntili
set
al.,
2008
“Vin
hoV
erde
”gr
apes
/phe
nolic
com
poun
dsan
dor
gani
cac
ids
Solid
-liq
uid
extr
actio
n/SP
EC
hrom
abon
dC
18no
nen
d-ca
pped
and
end-
capp
edco
lum
ns
HPL
C/D
AD
Dop
ico-
Gar
cıa
etal
.,20
07
Wild
mul
berr
y(M
orus
nigr
aL
.)/a
ntho
cyan
inpi
gmen
tsE
xtra
ctio
nw
ithM
eOH
/wat
er/a
cetic
acid
/pol
yam
idSP
F
Poly
amid
e(C
C-6
,M
ache
rey-
Nag
el,
Ger
man
y)
HPL
C/D
AD
and
MS
Has
sim
otto
etal
.,20
07
Com
mer
cial
juic
es/f
ood
auth
entic
itySP
ESe
p-Pa
kV
ac(6
cm3 ,1
g)C
18ca
rtri
dge
(Wat
ers,
Milf
ord,
Mas
s.)
Tota
lrefl
ecta
nce
IRH
eet
al.,
2007
Def
atte
dbr
anof
dark
blue
grai
ned
whe
at/a
ntho
cyan
ins
Ext
ract
ion
with
EtO
H/S
PEA
ccuB
ond
IIO
DS-
C18
(500
mg,
Agi
lent
)H
PLC
and
ESI
-MS
Hu
etal
.,20
07
Red
cabb
age/
anth
ocya
nins
SPE
C18
LC
-MS
McD
ouga
llet
al.,
2007
(Con
tinu
edon
the
next
page
)
319
Dow
nloa
ded
by [
Lau
rent
ian
Uni
vers
ity]
at 1
3:15
24
Febr
uary
201
3
TAB
LE
2Se
lect
edap
plic
atio
nsof
solid
phas
eex
trac
tion
(SPE
)ap
plie
dto
anth
ocya
nins
(Con
tinu
ed)
Type
ofm
atri
x/an
alyt
eE
xtra
ctio
n/cl
eanu
pte
chni
que
Sorb
ent
Fina
lana
lysi
sR
efer
ence
Ber
ries
/ant
hocy
anin
sE
xtra
ctio
nus
ing
EtO
Hal
one
orE
tOH
acid
ified
/SPE
C-1
8so
lid-p
hase
extr
actio
nca
rtri
dge
HPL
C-E
SI-M
S/M
S)N
icou
eet
al.,
2007
Hum
anpl
asm
aan
dur
ine/
anth
ocya
nins
SPE
Oas
isH
LB
SPE
cart
ridg
es(1
mL
;Wat
ers,
Els
tree
,U
K)
HPL
Can
dM
SC
ooke
etal
.,20
06
Aqu
eous
extr
acto
fre
dra
dish
/ant
hocy
anin
sSP
EO
asis
HL
B-c
artr
idge
(1m
L,
30m
g;W
ater
s,M
ilfor
d,M
ass.
)
HPL
C/D
AD
/MS
and
GC
/MS
Fles
chhu
teta
l.,20
06
Red
win
e/no
npol
ymer
ican
dpo
lym
eric
phen
ols
SPE
C18
Sep-
Pak
cart
ridg
e(F
ishe
rSc
ient
ific,
1g)
HPL
C-U
V-v
issp
ectr
osco
pyPi
nelo
etal
.,20
06
Age
dre
dw
ine/
poly
phen
ols
SPE
LiC
hrop
rep
RP
18(p
artic
ular
size
25–4
0µ
m)
HPL
C-D
AD
,HPL
-ESI
-MS
and
mul
tista
geM
Sfr
agm
enta
naly
sis
Sun
etal
.,20
06
Uri
nesa
mpl
es/a
ntho
cyan
inm
etab
olite
sSP
ESe
p-Pa
kC
18Pl
usca
rtri
dge
(Wat
ers,
Milf
ord,
Mas
s.)
HPL
C-E
SI-M
S-M
San
dH
PLC
with
UV
-vis
Felg
ines
etal
.,20
05
Hum
anse
rum
and
urin
e/to
tal
anth
ocya
nins
SPE
C18
cart
ridg
es(S
upel
clea
nE
NV
I-18
6m
L20
00m
g;Si
gma;
lot#
SP24
19C
)
RP-
HPL
C-D
AD
Kay
etal
.,20
05
Hum
anur
ine
and
seru
m/a
ntho
cyan
inm
etab
olite
s
SPE
C18
cart
ridg
es(S
upel
clea
nE
NV
I-18
6m
L50
0m
g;Si
gma,
Oak
ville
,Ont
.,C
anad
a)
HPL
C-D
AD
,MS,
GC
,and
enzy
mic
tech
niqu
esK
ayet
al.,
2004
Ber
ries
ofSm
ilax
aspe
raL
/ant
hocy
anin
sM
eOH
extr
actio
n/SP
EC
-18
Sep-
Pak
cart
ridg
e(W
ater
sC
orpo
ratio
n,M
ilfor
d,M
ass.
)
HPL
C-D
AD
-MS
Lon
goan
dV
asap
ollo
,20
06
Oliv
efr
uits
/phe
nolic
com
poun
dsM
eOH
extr
actio
n/SP
EIS
OL
UT
EC
18no
n-en
d-ca
pped
(NE
C)
RP
HPL
C/D
AD
and/
orH
PLC
-DA
D/E
SI-
MS/
MS
Vin
haet
al.,
2004
320
Dow
nloa
ded
by [
Lau
rent
ian
Uni
vers
ity]
at 1
3:15
24
Febr
uary
201
3
Uri
nesa
mpl
es/s
traw
berr
yan
thoc
yani
nsSP
ESe
p-Pa
kC
18Pl
us(W
ater
s,M
ilfor
d,M
ass.
)H
PLC
-ESI
-MS-
MS
and
HPL
C-U
V-v
isib
leFe
lgin
eset
al.,
2003
Red
win
e/pi
gmen
tsSP
EC
18H
PTL
CL
ambr
ieta
l.,20
03Pl
asm
aan
dur
ine/
anth
ocya
nins
SPE
Oct
adec
ylsi
lane
cart
ridg
e(S
ep-P
akC
18)
HPL
C/D
AD
Cao
etal
.,20
01
Bot
anic
alsu
pple
men
traw
mat
eria
ls/a
ntho
cyan
ins
SPE
Sep-
pak
Plus
C-1
8ca
rtri
dge
(1m
L)
(Wat
ers,
Milf
ord,
Mas
s.).
HPL
Can
dL
C/E
S-M
SC
hand
raet
al.,
2001
Aus
tria
nre
dw
ines
/phe
nolic
com
poun
dsSP
EC
-18
colu
mns
Mid
-IR
and
UV
-vis
spec
tros
copy
Ede
lman
net
al.,
2001
Food
s/an
thoc
yani
nsE
xtra
ctio
nw
ithm
etha
nol
orac
eton
eC
18(S
PE)
cart
ridg
esor
Seph
adex
LC
-UV
,LC
-MS,
and
CE
(cap
illar
yel
ectr
opho
resi
s)
Da
Cos
taet
al.,
2000
Gra
pesk
ins/
anth
ocya
nins
Ext
ract
ion
with
EtO
H-w
ater
-HC
lso
lutio
n/SP
E
Sep-
Pak
C-1
8ca
rtri
dge
(Wat
ers)
ESI
-MS
Favr
etto
and
Flam
ini,
2000
Ber
ries
ofth
ehy
brid
grap
ecu
ltiva
rs/a
ntho
cyan
ins
Ext
ract
ion
with
EtO
H-w
ater
-HC
lso
lutio
n/SP
E
Sep-
Pak
C-1
8ca
rtri
dge
(Mill
ipor
e-W
ater
s)H
PLC
-UV
-vis
Flam
inia
ndTo
mas
i,20
00
Finn
ish
cran
berr
y/an
thoc
yani
nan
dor
gani
cac
ids
SPE
CH
sorb
ent1
00m
g,SA
Xso
rben
t,10
0m
gSp
ectr
opho
tom
etry
and
HPL
CH
uopa
laht
ieta
l.,20
00
Hun
gari
anre
dw
ines
/pig
men
tsSP
ESi
lica,
octa
decy
lsili
ca,
cyan
opro
pyl,
alum
ina
(aci
dic,
basi
c,ne
utra
l),d
iol,
amin
opro
pyl,
flori
sil,
acce
llpl
usQ
MA
,car
bon
blac
k
Mul
tiwav
elen
ghsp
ectr
opho
tom
etry
with
asp
ectr
alm
appi
ngte
chni
que
and
byR
PH
PLC
Kis
set
al.,
2000
Bla
ckbe
anan
dbl
ackb
erry
/ant
hocy
anid
ins
Ext
ract
ion
with
ethy
lac
etat
e/SP
EC
18SP
Eca
rtri
dge
HPL
C-U
V-v
issp
ectr
osco
pyD
aoet
al.,
1998
Bla
ckch
okeb
erry
(Aro
nia
mel
anoc
arpa
var
Ner
o)/a
ntho
cyan
ins
SPE
Serd
olit
PAD
IV,A
mbe
rlite
XA
D-7
HPL
C-U
V-v
isK
raem
er-S
chaf
halte
ret
al.,
1998
Bla
ckbe
ans
(Pha
seol
usvu
lgar
isL
.)/a
ntho
cyan
ins
Ext
ract
ion
with
HC
lin
MeO
H/S
PEE
xtre
lut2
0ca
rtri
dge
(Mer
ck,
Dar
mst
adt,
Ger
man
y)H
PLC
-DA
D,T
LC
,GC
,U
V-v
is,M
S,an
dN
MR
Take
oka
etal
.,19
97
321
Dow
nloa
ded
by [
Lau
rent
ian
Uni
vers
ity]
at 1
3:15
24
Febr
uary
201
3
TAB
LE
3Se
lect
edex
ampl
esof
coun
terc
urre
ntch
rom
atog
raph
icm
etho
dsap
plie
dto
anth
ocya
nins
Sam
ple/
anal
yte
Solv
ents
yste
mE
lutio
nm
ode/
com
men
tR
efer
ence
Gra
pesk
ins
from
Viti
svi
nife
racv
.pin
otno
ir/a
ntho
cyan
ins
Tert
-but
ylm
ethy
leth
er/n
buta
nol/a
ceto
nitr
ile/w
ater
acid
ified
with
0.01
%T
FA(2
:2:0
.1–1
.8:5
v/v/
v/v)
.ML
CC
C
Aqu
eous
laye
r(l
ower
)as
stat
iona
ryph
ase.
Step
grad
ient
elut
ion
tose
para
tean
thoc
yani
nol
igom
ers
from
grap
ean
thoc
yani
ns
Kne
knop
oulo
set
al.,
2011
Bla
ckca
rrot
/pig
men
tco
mpo
sitio
nTe
rt-b
utyl
met
hyle
ther
/n-b
utan
ol/
acet
onitr
ile/w
ater
(2/2
/1/5
v/v/
v/v)
,aci
difie
dw
ith0.
1%T
FA
Stat
iona
ryph
ase:
less
dens
ela
yer
Isol
atio
nof
pure
anth
ocya
nin
stan
dard
sby
HSC
CC
inhi
gham
ount
s
Mon
tilla
etal
.,20
11a
Dif
fere
ntB
oliv
ian
purp
leco
rn(Z
eam
ays
L.)
vari
etie
s/ph
enol
icco
mpo
unds
Tert
-but
ylm
ethy
leth
er/n
-but
anol
/ac
eton
itrile
/wat
er(2
:2:1
:5v/
v/v/
v),a
cidi
fied
with
0.1%
TFA
Stat
iona
ryph
ase:
less
dens
ela
yer.
HSC
CC
sepa
rate
dfiv
efr
actio
nsas
wel
las
the
coil
resi
due
Mon
tilla
etal
.,20
11b
Stra
wbe
rry/
antio
xida
ntac
tivity
ofan
thoc
yani
nco
mpo
unds
Tert
-but
ylm
ethy
leth
er/n
-but
anol
/ac
eton
itrile
/wat
er(2
.75:
1.25
:1:5
),ac
idifi
edw
ith0.
1%T
FAac
id
Stat
iona
ryph
ase:
the
light
er(o
rgan
ic)
phas
e.Tw
ofr
actio
ns:
pela
rgon
idin
-3-g
luco
side
and
pela
rgon
idin
-3-r
utin
osid
e
Cer
ezo
etal
.,20
10
Japa
nese
purp
lesw
eetp
otat
o(I
pom
oea
bata
tas
L.)
vari
etie
s/an
thoc
yani
ns
Tert
-but
ylm
ethy
leth
er/n
-but
anol
/ac
eton
itrile
/wat
er(1
:3:1
:5,
v/v/
v/v)
,aci
difie
dw
ith0.
1%T
FA
Stat
iona
ryph
ase:
less
dens
ela
yer.
HSC
CC
show
sad
vant
ages
with
resp
ectt
oH
PLC
Mon
tilla
etal
.,20
10
Cor
ozo
(Bac
tris
guin
eens
is)
frui
t/ant
hocy
anin
pigm
ents
Tert
-but
ylm
ethy
leth
er/n
buta
nol/
acet
onitr
ile/w
ater
(2:2
.1:5
,v/
v/v/
v),a
cidi
fied
with
0.1%
TFA
Stat
iona
ryph
ase:
less
dens
ela
yer.
HSC
CC
allo
ws
the
iden
tifica
tion
ofcy
anid
in-3
-O-(
mal
onyl
)gl
ucop
yran
osid
e
Oso
rio
etal
.,20
10
Ber
ries
(bla
ckbe
rrie
s,bl
ack
chok
eber
ries
,and
grap
es)/
anth
ocya
nins
,st
ilben
es,a
ndpr
oant
hocy
anin
s
Dif
fere
ntty
pes
ofth
eal
l-ch
rom
atog
raph
icte
chni
ques
ofco
unte
rcur
rent
chro
mat
ogra
phy
asw
ell
asse
mi-
synt
hetic
stra
tegi
es
Win
terh
alte
ret
al.,
2010
Frui
tsan
dve
geta
bles
Isol
atio
nof
diff
eren
tnon
-acy
late
dan
dac
ylat
edcy
anid
ing
deri
vativ
esH
illeb
rand
etal
.,20
09
Purp
lesw
eet
pota
to/a
ntho
cyan
ins
Tert
-but
ylm
ethy
leth
er/n
-but
anol
/ac
eton
itrile
/wat
er/T
FA(1
:4:1
:5:0
.01,
v/v)
.
Stat
iona
ryph
ase:
uppe
ror
gani
cph
ase.
Four
acyl
ated
cyan
idin
san
dpe
onid
ins
obta
ined
byH
SCC
C
Qiu
etal
.,20
09
322
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Febr
uary
201
3
Red
win
e(M
onas
trel
l)(a
ntio
xida
ntac
tivity
)/po
lyph
enol
icfr
actio
ns(i
nclu
ding
poly
mer
s)
Tert
-but
ylm
ethy
leth
er/n
-but
anol
/ac
eton
itrile
/wat
er(2
/2/1
/5)
acid
ified
with
0.1%
TFA
.HSC
CC
Org
anic
phas
eas
stat
iona
ry.
Four
frac
tions
:pol
ymer
icco
mpo
unds
,m
alvi
din-
3-gl
uco-
side
,pe
onid
in-3
-glu
cosi
de,a
ndvi
sitin
A
Nog
uer
etal
.,20
08
Pota
to(S
olan
umtu
bero
sum
L.)
Var
ietie
s/an
thoc
yani
nsTe
rt-b
utyl
met
hyle
ther
/n-b
utan
ol/
acet
onitr
ile/w
ater
(2:2
:1:5
v/v/
v/v)
,aci
difie
dw
ith0.
1%T
FAac
id
Mob
ileph
ase:
the
low
erla
yer.
Four
maj
orfr
actio
ns:a
ntho
cyan
ins
and
3-ch
loro
geni
cac
id(1
–3);
and
5-ch
loro
geni
cac
id(4
)
Eic
hhor
nan
dW
inte
rhal
ter,
2005
Red
win
e(c
aber
nets
auvi
gnon
(60%
)an
dta
nnat
(40%
)/pi
gmen
ts
Tert
-but
ylm
ethy
leth
er/n
-but
anol
/ac
eton
itrile
/wat
er(2
/2/1
/5),
acid
ified
with
0.1%
TFA
Stat
iona
ryph
ase:
the
orga
nic
phas
e.Se
para
tion
ofan
thoc
yani
n-de
rive
dpi
gmen
tsby
HSC
CC
Sala
set
al.,
2005
Cam
u-ca
mu
(Myr
ciar
iadu
bia)
/ant
hocy
anin
sTe
rt-b
utyl
met
hyle
ther
(MT
BE
)/n-
buta
nol/a
ceto
nitr
ile/w
ater
(2:2
:1:5
,v/v
/v/v
)ac
idifi
edw
ith0.
1%T
FA
Stat
iona
ryph
ase:
less
dens
ela
yer.
Ant
hocy
anin
sw
ere
isol
ated
byH
SCC
CZ
anat
taet
al.,
2005
Tea,
shal
lota
nd,
endi
ve/fl
avon
olgl
ycos
ides
;fr
uitj
uice
s,pu
rple
carr
ot/a
ntho
cyan
ins
HSC
CC
and
ML
CC
Cte
chni
ques
appl
ied
toth
eis
olat
ion
offla
vono
lgly
cosi
des
Deg
enha
rdte
tal.,
2004
Bilb
erry
frui
tcru
deex
trac
t(V
acci
nium
myr
till
us,
Eri
cace
ae)/
anth
ocya
nins
Met
hylt
ert-
buty
leth
er–n
-bu
tano
l–ac
eto-
nitr
ile–w
ater
–TFA
(1:4
:1:5
:0.0
1,v/
v).H
SCC
C
Upp
eror
gani
cph
ase
asst
atio
nary
.Pr
epar
atio
nof
pure
anth
ocya
nins
(del
phin
idin
and
cyan
idin
-3-O
-sam
bubi
osid
es)
Du
etal
.,20
04
Blo
odor
ange
(Cit
rus
sine
nsis
(L.)
Osb
eck)
juic
e/an
thoc
yani
nsan
dpy
rano
anth
ocya
nins
Met
hylt
ert-
buty
leth
er/n
-but
anol
/ac
eton
itrile
/wat
er(1
:3:1
:5,
v/v/
v/v)
,aci
difie
dw
ith0.
1%T
FA
Mob
ileph
ase:
dens
erla
yer
Eig
htfr
actio
nsob
tain
edH
illeb
rand
etal
.,20
04
Win
efr
omSo
uth
Afr
ican
grap
eva
riet
ypi
nota
ge(p
inot
noir
×)
Ads
orpt
ion
onA
mbe
rlite
XA
D-7
resi
nan
dpu
rific
atio
nby
HSC
CC
New
deri
vativ
esof
mal
vidi
n-3-
gluc
osid
e,m
alvi
din-
3-(6
′′ -ac
etyl
gluc
osid
e)an
dm
alvi
din-
3-(6
-cou
mar
oyl-
gluc
osid
e)
Schw
arz
and
Win
terh
alte
r,20
04
Gra
pesk
ins/
olig
omer
ican
thoc
yani
nsTe
rt-b
utyl
met
hyle
ther
/n-b
utan
ol/
acet
onitr
ile/w
ater
acid
ified
with
TFA
(2/2
/x/5
);M
LC
CC
with
grad
ient
elut
ion
Frac
tiona
tion
ofan
thoc
yani
nsas
gluc
osid
esan
dth
eco
rres
pond
ing
acet
ylat
ed,c
oum
aroy
late
d,an
dca
ffeo
ylat
edde
riva
tives
Vid
alet
al.,
2004
a
(Con
tinu
edon
the
next
page
)
323
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Febr
uary
201
3
TAB
LE
3Se
lect
edex
ampl
esof
coun
terc
urre
ntch
rom
atog
raph
icm
etho
dsap
plie
dto
anth
ocya
nins
(Con
tinu
ed)
Sam
ple/
anal
yte
Solv
ents
yste
mE
lutio
nm
ode/
com
men
tR
efer
ence
Gra
pesk
inex
trac
t/olig
omer
ican
thoc
yani
nsTe
rt-b
utyl
met
hyle
ther
/n-b
utan
ol/
acet
onitr
ile/w
ater
with
TFA
(2/2
/x/5
)
ML
CC
Cco
uple
dw
ithst
epgr
adie
ntel
utio
n.Id
entifi
catio
nby
mas
ssp
ectr
omet
ry
Vid
alet
al.,
2004
b
Phyt
oste
rol,
chlo
rina
ted
s-tr
iazi
nes,
flavo
noid
s,th
eafla
vins
,ant
hocy
anin
s,sp
orav
irid
ins,
etc.
Tert
-but
ylm
ethy
let
her/
n-bu
tano
l/ace
toni
trile
/wat
er(2
:2:1
:5),
acid
ified
with
0.01
%T
FA
Aqu
eous
phas
eas
mob
ile.
Five
anal
ogs
ofan
thoc
yani
nssu
cces
sful
lyse
para
ted.
Che
net
al.,
2003
Trad
esca
ntia
pall
ida
leav
es,
purp
leco
rn,e
lder
berr
yju
ice,
red
win
e,an
dbl
ackb
erri
es/a
ntho
cyan
ins
n-bu
tano
l/ter
t-bu
tylm
ethy
let
her/
acet
onitr
ile/w
ater
.I:2
:2:1
:5v/
v/v/
vpl
us0.
1%T
FA.I
I:T
hesa
me
plus
0.1%
TFA
.III
:3:1
:1:5
v/v/
v/v
with
0.1%
TFA
Seve
ralh
undr
edm
illig
ram
sof
pure
anth
ocya
nins
wer
eob
tain
edw
ithin
asi
ngle
CC
Cru
n.H
SCC
Cof
fers
seve
rala
dvan
tage
sov
erH
PLC
.
Schw
arz
etal
.,20
03
Red
win
e,re
dbe
et,G
arde
nia
jasm
inoi
des,
blac
kte
a/na
tura
lpig
men
ts(a
ntho
cyan
ins.
..)
HSC
CC
Isol
atio
nof
hydr
ophi
licth
earu
bigi
nsfr
ombl
ack
tea
Deg
enha
rdta
ndW
inte
rhal
ter,
2001
Red
onio
ns(A
lliu
mce
pa)
and
flow
ers
oftu
lipcu
ltiva
r“L
osA
ngel
os”/
anth
ocya
nins
Tert
-but
ylm
ethy
let
her/
n-bu
tano
l/ace
toni
trile
/wat
er(2
/2/1
/5),
plus
0.01
%T
FA
Frag
ilean
thoc
yani
nsw
ithal
ipha
ticac
ilatio
nin
sem
ipre
para
tive
scal
eTo
rska
nger
poll
etal
.,20
01
Red
win
e,gr
ape
skin
extr
acts
,an
dot
her
frui
tand
juic
eex
trac
ts/p
olym
eric
anth
ocya
nins
;ace
tyla
ted
anth
ocya
nins
Tert
-but
ylm
ethy
leth
er/n
-but
anol
/ac
eton
itrile
/wat
er(2
/2/1
/5),
plus
0.1%
TFA
.Eth
ylac
etat
e-n-
buta
nol-
wat
er(4
/1/5
)ac
idifi
edw
ith0.
1%T
FA
HSC
CC
prov
ides
ato
olfo
rth
ech
emis
tto
isol
ate
larg
equ
antit
ies
ofpu
rest
anda
rds
Deg
enha
rdte
tal.,
2000
b
Red
win
ean
dgr
ape
skin
extr
acts
/mal
vidi
n-3-
gluc
osid
e
Red
pigm
ents
first
clea
ned
upon
anA
mbe
rlite
XA
D-7
colu
mn.
HSC
CC
Puri
tyan
did
entit
yof
isol
ated
com
poun
dsch
ecke
dby
HPL
C-D
AD
,N
MR
,and
MS
Deg
enha
rdte
tal.,
2000
a
Cha
mpa
gne
vint
age
bypr
oduc
ts/m
alvi
din-
3an
dpe
onid
in-3
-glu
cosi
des
Eth
ylac
etat
e/n-
buta
nol/w
ater
acid
ified
with
TFA
Cen
trif
ugal
part
ition
chro
mat
ogra
phy
was
appl
ied
for
the
first
time
Ren
ault
etal
.,19
95
CC
C:c
ount
ercu
rren
tchr
omat
ogra
phy;
HSC
CC
:hig
h-sp
eed
coun
terc
urre
ntch
rom
atog
raph
y;M
LC
CC
:mul
tilay
erco
ilco
unte
rcur
rent
chro
mat
ogra
phy;
TFA
:tri
fluor
oace
ticac
id.
324
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ANTHOCYANIN PIGMENTS. PART IV: EXTRACTION 325
TABLE 4Examples of adsorbents and eluting agents proposed for the selective recovery of phenolic compounds
Solution (target phenoliccompounds) Adsorbent
Eluting and/or regeneratingagent References
Grape skins of pinot noir(anthocyanins)
XAD-7 resin 50% aqueous methanol Kneknopoulos et al.,2011
Raw juice (flavonoids andanthocyanins)
Macroporous adsorptive resinsD141
Anthocyanins eluted withethanol-H2O (30:70, v/v),and flavonoids withethanol
Li et al., 2011
Blood oranges (anthocyanins) NKA-9 macroporous resin 50% ethanol with citric acid(pH 2.5)
Cao et al., 2010
Muscadine grape pomace(polyphenolics)
Amberlite XAD-4 resins 100% methanol Cardona et al., 2009
Ficus racemosa fruit(anthocyanins)
Amberlite XAD-4 resin Acidified methanol (1%HCl)
Sarpate et al., 2009
Eggplant peel (anthocyanins) Methacrylic resin ReliteEXA-31
Acidified H2O ethanol 1:1;1% w/v of citric acid
Todaro et al., 2009
Red cabbage (anthocyanins) Tonsil Terrana 580 FF clay andAmberlite XAD-7 resin
70:30:5 v/v/w ethanol, waterand citric acid
Lopes et al., 2007
Orange juice (anthocyanins,hesperidin, hydroxycynnamates,nariturin, limonin)
Relite EXA 118 Ethanol, 75–100% Scordino et al., 2007
Cassis, commercial powderobtained from blackcurrant juice(anthocyanins)
Previously cleaned C18material or sea sand
MeOH/H2O (1:1, v/v) Manhita et al., 2006
Grape pomace extracts(anthocyanins)
Nonpolar SDVB resin Methanol, ethanol, propanol Kammerer et al.,2005
Red wine from Vitis vinifera L. cvGraciano grapes (anthocyanins)
Cell walls of different strainsof Saccharomyces
10 mL formic acid:methanol (10:90)
Morata et al., 2005
Orange juice(cyanidin-3-glucoside, hesperin)
Relite EXA 118 Methanol, 75–100% Scordino et al., 2005
Brassica oleraceae (anthocyanins) Amberlite XAD-7 70% Ethanol Coutinho et al., 2004Wines from the South African
grape variety pinotage (pinotnoir × cinsault) (acetylated andcoumaroylated anthocyanins)
Amberlite XAD-7 resin Purification by high-speedcountercurrentchromatography andsemi-preparativehigh-performance liquidchromatography
Schwarz andWitnterhalter,2004
Different cultivars of mulberry(anthocyanins)
D3520, D4020, X-5, NKA-9,D101A, and AB-8 resins
Acidified ethanol (0.5%(v/v) of hydrochloric acid)
Liu et al., 2004
Wastewater of orange juiceprocessing (hesperidin,anthocyanins, hydroxycinnamicacids)
Kastell S-112 96% Ethanol NaOH/10%ethanol
Di Mauro et al.,2000, 2002
Pomegranate juices (antioxidantactivity, anthocyanins)
Bio-Rad cation-exchange resinAG50W-X8
Water and methanol Gil et al., 2000
Hungarian red wines (pigmentsand anthocyanins)
Silica, octadecylsilica,cyanopropyl, alumina, diol,aminopropyl, florisil, accellplus QMA, carbon black.
Acetonitrile, methanol,tetrahydrofuran, anddioxane, each containing10% (v/v) concentratedformic acid.
Kiss et al., 2000
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326 M. J. NAVAS ET AL.
with lower solvent consumption and lower working temperature(Temelli and Seifried, 2011; Garcıa-Salas et al., 2010; Pereiraand Meireles, 2010). It is a form of liquid extraction where theusual liquid solvent phase has been replaced by a supercriticalfluid; i.e., a substance that is above its critical point. Amonga wide variety of supercritical fluids, carbon dioxide is essen-tially the only convenient supercritical extraction solvent usedbecause of its comparatively low critical temperature and pres-sure (Sticher, 2008; Nahar et al., 2005; Handa et al., 2008). Thismethod provides an alternative to the pretreatment of the plantmaterials, replacing toxic organic solvents (e.g., hexane).
The extraction of anthocyanins by using supercritical CO2
(SC-CO2) methods requires high pressures and the presenceof an organic co-solvent (methanol, ethanol, water) in highpercentage due to the polarity of anthocyanins (Ignat et al.,2011; Serra et al., 2010). High concentrations of polar organicmodifiers added to obtain the best extraction yield lead, however,to reduced selectivity. Bleve et al. (2008) described a method forthe purification of anthocyanins from grape skin extracts as liq-uid matrix, by using CO2 under liquid and subcritical conditions.The solid residues generated from blueberries, cranberries, andraspberries after pressing were extracted by conventional sol-vent extraction or by SC-CO2 extraction (Laroze et al., 2010).SFE of bioactive compounds from grape peel by using 6–7%ethanol as modifier has been carried out (Ghafoor et al., 2010).
Fractionated high-pressure extraction is performed in orderto obtain anthocyanin-rich extracts. A first step with SC-CO2
followed by a second step with mixtures of CO2 and ethanolwere applied to cherries (Serra et al., 2010, 2011a); the productderived from the second CO2-EtOH (90:10 V/V) step extractionexhibited activity against colon cancer (Serra et al., 2011a). SC-CO2 fluid extraction followed by enhanced solvent extractionwith CO2-EtOH-H2O mixtures allows obtaining anthocyanin-rich fractions in the second step (Seabra et al., 2010b), fromelderberry pomace. Supercritical fluid extraction, with 6–7%ethanol as modifier, was applied for the extraction of valuablecompounds, e.g., total anthocyanins, from grape peel (Ghafooret al., 2010).
Pressurized liquid extraction (PLE), also known under thetrade names of accelerated solvent extraction (ASE), pressurizedfluid extraction (PFE), pressurized solvent extraction (PSE), orenhanced solvent extraction (ESE), is a relatively new technol-ogy for extraction of phytochemicals under high temperatureand pressure and is partly derived from SFE (Sticher, 2008).In PLE, pressure is applied to allow the use as extraction sol-vents of liquids at temperatures greater than their normal boilingpoint. The method was first described in 1995 (Richter et al.,1995, 1996). The combined use of high pressures and tempera-tures provides faster extraction processes (generally completedwithin a few minutes, owing to increased solubility, better des-orption, and enhanced diffusion) that require small amounts ofsolvents (Dai and Mumper, 2010; Wibisono et al., 2009; Buldiniet al., 2002; Ramos et al., 2002). There are two ways to per-form PLE, either in the static or dynamic mode. In both cases,
under conditions of elevated pressure and temperature, the masstransfer rates are accelerated according to Fick’s law of dif-fusion. Both commercially available and laboratory-assembledPLE systems are used (Kivilompolo, 2009).
Therefore, extraction solvents, including water, that showlow efficiency in extracting phytochemicals at low tempera-tures may be much more efficient at elevated PLE temperatures(Dai and Mumper, 2010). The use of water as an extractionsolvent in PLE is the so-called pressurized hot water extrac-tion (PHWE) (Teo et al., 2010). PHWE is also referred to assubcritical water extraction, superheated water extraction, high-temperature water extraction, extraction using hot compressedwater, and extraction with water at elevated temperatures andpressures.
Subcritical water appears to be an excellent alternative to or-ganic solvents to extract anthocyanins and other phenolics fromdried grape skin and possibly other grape byproducts (Ju andHoward, 2003), and from industrially generated apple pomace(Wijngaard and Brunton, 2009). PHWE is usually performed indynamic mode with water flowing constantly through the sam-ple, but static extraction is also possible (Kivilompolo, 2009).Water is heated up to 200◦C and the change in dielectric con-stant of the water with the temperature leads water to behavelike an organic solvent. For example, the dielectric constant ofwater at 200◦C is equal to 36, which is close to that of methanol(Dai and Mumper, 2010; Dai, 2009).
A dynamic subcritical fluid extraction system (80% aque-ous ethanol) has been used for extraction of flavonoids (an-thocyanins) from food processing waste such as grape pomace(Srinivas et al., 2011). Anthocyanins have been extracted fromred grape pigments by PLE in a static model (Hohnova et al.,2008). A static bath reactor has also been used for the extractionof anthocyanins from red onion (Pettersson et al., 2010). Pres-surized hot water containing 5% ethanol was used to extractanthocyanins from red cabbage (Arapitsas and Turner, 2008;Arapitsas et al., 2008). Anthocyanins were extracted from redpomace in acidified aqueous methanolic and aqueous ethanolicsolvents and identified and quantified in the extracts by HPLC-MS and HPLC (Monrad et al., 2010).
Residence time of the extracted solute must be minimized inorder to prevent possible degradation of anthocyanin moietiesor their possible reaction with sugar and other products. Thereis some evidence that side reactions generating antioxidant moi-eties may occur in pressurized hot water (Bravi et al., 2012; Kingand Srinivas, 2009). At first, the extraction effect dominates, butdegradation effects soon take over (Pettersson et al., 2010), asoccurs with anthocyanins from red onion. Anthocyanins presentin black carrot were extracted with pressurized acidified water;anthocyanin degradation became significant above 100◦C (Giziret al., 2008).
At the present, no commercial instrument is available and,thus, the equipment has been self-built or modified from otherinstruments. The difference from commercial instruments forSFE and PLE or ASE is that PHWE equipment can tolerate
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ANTHOCYANIN PIGMENTS. PART IV: EXTRACTION 327
TABLE 5Factors influencing pressurized liquid extraction
Pressurized liquid extractionMatrix characteristics Nature of the analyte
Position and bonding degree ofthe analyte
Particle sizeWater content
Extraction kind Controlled by solubilityControlled by diffusion
Extraction operation DynamicStatic
Factors influencing theextraction
Solvent typeTemperatureTime
Extraction enhancers Drying agentDispersing agentsOther additives
temperatures over 300◦C (Kivilompolo, 2009; Hyotylainen,2009). The high temperature makes demands on the materialof the extraction vessels and sealing rings. In pressurized hotwater extraction the most important parameter affecting extrac-tion is the temperature.
The other significant parameters are pressure, flow rate ofthe water, and extraction time. In addition, the matrix needs tobe taken into consideration, and choosing the proper analytecollection system can increase selectivity of the extraction. Themost important parameters affecting the extraction in PLE areshown in Table 5 (Dai and Mumper, 2010; Mustafa and Turner,2011; Kivilompolo, 2009). Response surface methodology wasusually used to optimize the response values in the extractionof anthocyanins from different sources (Ghafoor et al., 2010;Wijngaard and Brunton, 2009; Arapitsas and Turner, 2008).Recent applications of PLE to anthocyanin extractions are listedin Table 6.
MICROWAVE-ASSISTED EXTRACTIONMicrowave-assisted extraction (MAE), or simply microwave
extraction, is a relatively new technology for extracting solu-ble products into a fluid from a wide range of materials usingmicrowave energy (Dai and Mumper, 2010; Garcıa-Salas et al.,2010; Jain et al., 2009). The use of MAE in natural productextraction started in the late 1980s (Delazar et al., 2012). Thistechnique allows one to extract compounds more selectivelyand more rapidly (usually in less than 30 minutes) with similaror better recovery than traditional extraction processes (Garcıa-Salas et al., 2010; Escribano-Bailon and Santos-Buelga, 2003).Microwaves directly heat the solvent or solvent mixture, thusaccelerating the speed of heating. Besides the advantage of highextraction speed, MAE also enables a significant reduction in
the consumption of organic solvent (Mandal et al., 2007; Wangand Weller, 2006).
The application of microwave energy to the samples may beperformed using two technologies: closed vessels under con-trolled pressure and temperature (PMAE with a multimodecavity) or open vessels at atmospheric pressure (FMAE usingthe waveguide as a single-mode cavity) (Kivilompolo, 2009;Sticher, 2008). The closed-vessel MAE system is quite similarto PLE technology, as the solvent is heated and pressurized inboth systems. The main difference is in the means of heating,either by microwave energy or by conventional oven heating.Consequently, as for PLE, the number of influential parametersis reduced, thus making the application of this technique quitesimple to use (Camel, 2001).
Microwaves are a non-ionizing electromagnetic radiationwith a frequency from 300 to 300000 MHz. In order to avoidinterference with radio communications, domestic and com-mercial systems generally operate at 2450 MHz. Even thoughmicrowave energy as a source of heat has been used in analyticallaboratories since the late 1970s, their application to enhanceextraction is very recent (Handa, 2008; Sticher, 2008). Exten-sive use began around 10 years ago, with the commercializationof several extraction instruments.
In MAE, it is important that the extraction solvent has goodpolarity because solvents with high dielectric constants (polar)can absorb more microwave energy and therefore result in betterextraction efficiency (Dai and Mumper, 2010; Jain et al., 2009;Rawa-Adkonis et al., 2003). Water or other polar solvent istherefore often added as a modifier in order to achieve an optimaldielectric constant of the extraction solvent. However, differingopinions also exist: when solvents of low dielectric constants areused, all the microwave energy may be directed to the samplematerial, and the moisture inside the cellular structure absorbsthe energy so quickly that it erupts and breaks the cell wall,releasing the phytochemicals to the surrounding solvent.
Due to the particular effects of microwaves on matter (namelydipole rotation and ionic conductance), heating with microwavesis instantaneous and occurs in the heart of the sample, leadingto very fast extractions (Sticher, 2008). The results obtained sofar have concluded that microwave radiation causes no degra-dation of the extracted compounds, unless the temperature inthe vessel rises too high. At the same time, a specific effect ofmicrowaves on plant material has been found (Sticher, 2008).They interact selectively with the free water molecules presentin the solid matrix, leading to rapid heating and temperatureincrease, resulting in rupture of the plant tissue and release ofsolutes into the solvent (Mandal et al., 2007; Wang and Weller,2006).
MAE (especially PMAE) is used in analytical protocols, veryoften to investigate extraction parameters including pressure andtemperature, extraction time, microwave power, solvent nature,and volume, or in comparative studies of this and other re-cent techniques (such as SFE or PLE) with classical extractionmethods for particular applications (de la Rosa et al., 2009).
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3
TAB
LE
6A
pplic
atio
nof
pres
suri
zed
fluid
extr
actio
nto
the
anal
ysis
ofan
thoc
yani
ns
Mat
rix/
com
poun
dsSo
lven
tT
Pres
sure
/cyc
les
Ext
ract
ion
time
Tech
niqu
eR
efer
ence
Che
rrie
s(P
runu
sav
ium
)/an
tican
cer
activ
itySu
perc
ritic
alC
O2,
CO
2:e
than
ol(9
0:10
,v/v
)50
◦ C25
±1
MPa
15,6
0,90
min
TL
C,H
PLC
-DA
DSe
rra
etal
.,20
11a
Free
ze-d
ried
sunb
elt(
Viti
sla
brus
caL
.)gr
ape
pom
ace/
flavo
noid
s
Hyd
roet
hano
licac
idifi
edw
ithor
gani
cac
ids
60◦ –
140◦ C
10.3
MPa
Srin
ivas
etal
.,20
11
Lyop
hiliz
edgr
ape
resi
dues
/pol
yphe
nols
and
anth
ocya
nins
CO
2,C
O2-m
etha
nol
40◦ C
110
bar
HPL
C-D
AD
,L
C-M
SFl
oris
etal
.,20
10
Gra
pepe
el/to
talp
heno
ls,
antio
xida
nts,
and
tota
lan
thoc
yani
ns
Supe
rcri
tical
CO
2/6
–7%
etha
nola
sm
odifi
er45
◦ –46
◦ C16
0–16
5kg
cm−2
30m
inU
V-v
isib
leG
hafo
oret
al.,
2010
Ber
ries
ofcr
owbe
rry/
flavo
nols
and
othe
rph
enol
icco
mpo
unds
Supe
rcri
tical
carb
ondi
oxid
eH
PLC
-DA
D,G
CL
aaks
onen
etal
.,20
10
Solid
resi
dues
gene
rate
dfr
ombl
uebe
rrie
s,cr
anbe
rrie
s,an
dra
spbe
rrie
s
Supe
rcri
tical
CO
2et
hano
las
co-s
olve
nt60
◦ C80
–300
bar
2h
UV
-vis
ible
,G
C-M
SL
aroz
eet
al.,
2010
Dri
edre
dgr
ape
pom
ace/
anth
ocya
nins
Met
hano
l/wat
er/f
orm
icac
id(6
0:37
:3v/
v/v)
;10,
30,
50,a
nd70
%et
hano
lin
wat
er,v
/v
40◦ ,
60◦ ,
80◦ ,
100◦ ,
120◦ ,
and
140◦ C
6.8
Mpa
/1H
PLC
-MS
Mon
rad
etal
.,20
10
Red
onio
n/an
thoc
yani
nsU
ltrap
ure
wat
er-e
than
ol-f
orm
icac
id11
0◦ C1–
3ba
r10
min
HPL
C-D
AD
Pette
rsso
net
al.,
2010
Gra
pesk
inex
trac
ts/p
olyp
heno
ls,
tota
lphe
nolic
Met
hano
land
etha
nol
40◦ –
120◦ C
15M
pa/3
5m
inH
PLC
,EPR
spec
tros
copy
Polo
vka
etal
.,20
10
Eld
erbe
rry
(Sam
bucu
sni
gra
L.)
pom
ace/
anth
ocya
nins
Supe
rcri
tical
CO
2w
ithet
hano
l-H
2O
mix
ture
s31
3K
20M
PaSt
atic
and
dyna
mic
TL
C,H
PLC
-UV
Seab
raet
al.,
2010
b
Eld
erbe
rry
pom
ace/
anth
ocya
nins
CO
2/e
than
ol/H
2O
313
K21
MPa
45m
inU
V-v
isan
dH
PLC
-DA
DSe
abra
etal
.,20
10a
Swee
tche
rry
vari
ety
from
Port
ugal
(“Sa
co”)
/ant
hocy
anin
phen
olic
cont
ent,
antio
xida
ntac
tivity
Supe
rcri
tical
CO
2,C
O2
and
etha
nol(
10–1
00%
,v/v
)50
◦ C25
MPa
1pl
us1.
5h
HPL
C-D
AD
,LC
-D
AD
-MS/
MS
Serr
aet
al.,
2010
328
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Febr
uary
201
3
Gra
pepo
mac
e/an
thoc
yani
ns,
resv
erat
rol,
cate
chin
,and
epic
atec
hin
CO
2w
ithw
ater
oret
hano
las
coso
lven
ts80
◦ C30
0ba
rC
asas
etal
.,20
09
Bla
ckcu
rran
t(R
ibes
nigr
um)
juic
e/ph
enol
icco
mpo
unds
,an
thoc
yani
ns
Supe
rcri
tical
CO
2H
PLC
-DA
DSa
ndel
leta
l.,20
09
Gra
pepo
mac
e/so
lubi
lity
ofan
thoc
yani
nsW
ater
and
etha
nol
25◦
and
75◦ C
/16
min
Han
sen
solu
bilit
ypa
ram
eter
Srin
ivas
etal
.,20
09
Red
cabb
age/
anth
ocya
nins
Dio
nex
acce
lera
ted
solv
ent
100◦ C
50ba
r/1
6m
inH
PLC
/DA
D-
ESI
/Qtr
apM
S
Ara
pits
aset
al.,
2008
Red
cabb
age/
anth
ocya
nins
Wat
er/e
than
ol/f
orm
icac
id(9
4/5/
1,v/
v/v)
99◦ C
50ba
r/1
7m
inH
PLC
/DA
DA
rapi
tsas
and
Tur
ner,
2008
Gra
pesk
inex
trac
ts/
anth
ocya
nins
CO
230
◦ –40
◦ C10
0–13
0ba
rH
PLC
-DA
D,M
SB
leve
etal
.,20
08
Bla
ckca
rrot
/ant
hocy
anin
sW
ater
with
sulf
uric
,citr
ic,
and
lact
icac
ids
50◦ –
150◦ C
50ba
r10
min
HPL
C/U
V-v
isG
izir
etal
.,20
08
Red
grap
esk
in/a
ntho
cyan
ins
Met
hano
land
etha
nol
40◦ –
120◦ C
15M
pa/3
5m
inH
PLC
-DA
DH
ohno
vaet
al.,
2008
Red
grap
em
arc
(var
iety
ofR
efos
k)/a
ntho
cyan
ins
CO
2A
mbi
ent
tem
pera
ture
10–1
8M
PaSp
ectr
opho
tom
etry
Vat
aiet
al.,
2008
App
lean
dpe
ach
pom
aces
/pol
yphe
nols
CO
2+
etha
nol(
20%
)55
.7◦ –
58.4
◦ C;
50.9
◦ –52
.3◦ C
54.–
57M
Pa;
50.6
–51
MPa
40m
inSp
ectr
opho
tom
etry
Has
bay
Adi
leta
l.,20
07
App
leju
ice/
anth
ocya
nins
Wat
er,e
than
ol(2
5–10
0%,
v/v)
,and
met
hano
l(2
5–10
0%,v
/v)
40◦ –
180◦ C
/32
min
Spec
trop
hoto
met
ry,
HPL
C-M
SK
amm
erer
etal
.,20
07
Mal
vidi
n3,
5-di
gluc
osid
e/de
term
inat
ion
ofdi
ffus
ion
coef
ficie
nt
Car
bon
diox
ide
and
met
hano
las
coso
lven
tV
alid
ityof
diff
eren
teq
uatio
ns
Dif
fusi
onco
effic
ient
Supe
rcri
tical
chro
-m
atog
raph
y/U
Vde
tect
or
Man
tell
etal
.,20
04
(Con
tinu
edon
the
next
page
)
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TAB
LE
6A
pplic
atio
nof
pres
suri
zed
fluid
extr
actio
nto
the
anal
ysis
ofan
thoc
yani
ns(C
onti
nued
)
Mat
rix/
com
poun
dsSo
lven
tT
Pres
sure
/cyc
les
Ext
ract
ion
time
Tech
niqu
eR
efer
ence
Frui
tber
rysu
bstr
ates
/ant
hocy
anin
sSu
bcri
tical
deio
nize
dan
dM
illi-
Q-p
urifi
edne
atw
ater
;aci
difie
dw
ater
120◦ –
160◦ C
4.0
MPa
40m
inSp
ectr
opho
to-m
etry
,H
PLC
Kin
get
al.,
2003
Red
grap
epo
mac
e/an
thoc
yani
nsC
O2
mod
ified
with
met
hano
l56
0ba
r15
min
Man
tell
etal
.,20
03a
Dif
fusi
onco
effic
ient
ofm
alvi
din
3,5-
digl
ucos
ide
CO
2-m
etha
noli
nne
arcr
itica
lreg
ion
40◦ ,
50◦ ,
and
60◦ C
100,
200,
300,
and
400
bar
Supe
rcri
tical
chro
-m
atog
raph
y/U
Vde
tect
or
Man
tell
etal
.,20
03b
Dri
edre
dgr
ape
skin
/an
thoc
yani
nsan
dto
tal
phen
olic
s
Aci
difie
dw
ater
Aci
difie
d60
%m
etha
nol
80◦ –
100◦ C
60◦ C
10.1
Mpa
/35
min
HPL
C-D
AD
Juan
dH
owar
d,20
03
Ber
ries
/ant
hocy
anin
sSu
bcri
tical
wat
erw
ithet
hano
l11
0◦ –16
0◦ C40
bar
40m
inH
PLC
Kin
g,20
02
Gol
den
Del
icio
usap
ple
peel
and
pulp
/pol
yphe
nols
Hyd
rom
etha
nolic
mix
ture
s20
◦ –14
0◦ C10
00p.
s.i/2
5m
inH
PLC
-DA
DA
lons
o-Sa
lces
etal
.,20
01
Ant
hocy
anin
san
dot
hers
Pres
suri
zed
carb
ondi
oxid
e70
◦ C40
0ba
r24
hT
LC
,UV
-vis
,NM
R,
MS
Jay
etal
.,19
91
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ANTHOCYANIN PIGMENTS. PART IV: EXTRACTION 331
TABLE 7Selected applications of microwave-assisted extraction (MAE) techniques for anthocyanins
Sample/analyte Comment Result Technique Reference
Wine lees/anthocyanin:malvidin-3-glucoside
Ethanol 75%, HCl 1% inwater 1:10 (w/v) ratio.MAE 200 W for 17 min
Better extraction efficiencyin a much shorter timethan the conventionalmethod
HPLC-DAD Perez-Serradillaand de Castro,2011
Grape skins (Tintilla deRota)/anthocyanins(acylderivatives)
40% methanol, T =100◦C, 500 W power,and 25 mL and 5 min asextraction volume andtime
Extraction solvent is themost important variablefor the recovery. Notablereduction in the extractiontime from 5 h to 5 min
HPLC-MS Liazid et al., 2011
Blackcurrant pulp/vitamin Cand anthocyanin content
MAFM: 560 W power,pulp load of 65 g,drying time of 8 min,and pulp thickness of4.46 mm
Positive effects up to certainlevel and then a negativeeffect on both vitamin Cand anthocyanin content
UV-vis Zheng at al., 2011
Two anthocyanin extractsfrom purple sweet potato
Microwave baking andacidified electrolyzedwater (MB-AEW)
Extraction yield byMB-AEW was 35.0%,much higher than that byMB-EtOH technology(14.2%)
UV-vis Lu et al., 2010
Purple corn (Zea mays L.)cob/anthocyanins
MAE: 19 min extractiontime; solid-liquid ratio1:20, 555 W
Cyanidin, pelargonidin, andpeonidin-3-glucosides,and malonatedcounterparts
HPLC-MS Yang and Zhai,2010
Dried cranberry/cyanidin-3-and peonidin-3galactosides andarabinosides
VMD VMD and freezing-dryingpreserved betterantioxidant activity aswell as anthocyanincontent than hot air-drying
HPLC Leusink et al., 2010
Fresh and driedraspberries/anthocyanincontent, total antioxidantcapacity, and otherproperties
MIVAC, HAD/MIVAC90 min at 3000 W and avacuum pressure of 20torr (2.6 KPa).
Anthocyanins shown to beless heat stable thanpolyphenols
RP-HPLC Mejia-Meza et al.,2010
Colorado potato breedingprogram/anthocyanins andother compounds;antioxidant properties
Microwave cooking:1.0 min/30 g FW at700 W full power
Major phenoliccompounds,anthocyanins,and glycoalkaloid contentwere investigated
LC/MS Stushnoff et al.,2008
Red grapeskins/anthocyanins
Sample weight-to-solventvolume ratio equal to0.06 and T = 50◦C withMeOH
Recoveries of MAE (faster),UAE, and macerationmethods were 90, 82, and98%, respectively
LC-MS/MS Ghassempour et al.,2008
Redraspberries/anthocyanins
Ratio of solvents tomaterials 4:1 (mL/g),extraction time 12 min,and power 366 W
43.42 mg of Acys/100 g offresh fruits expressed ascyanidin-3-glucoside.About 98.33% redpigments
HPLC-MS Sun et al., 2007
MAE: microwave-assisted extraction; MAFM: microwave-assisted foam mat drying; VMD: vacuum-microwave drying; MB: microwave baking;MIVAC: microwave-vacuum; HAD/MIVAC: combination of hot-air drying and microwave-vacuum.
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332 M. J. NAVAS ET AL.
Comparisons of extraction efficiency and susceptibility to ma-trix effects, selectivity, time and solvent reduction, level of au-tomation, and simplicity of the operating procedures are dis-cussed in the literature (Sticher, 2008). MAE may also be usedin isolation protocols.
Less polar solvents are used for extraction of flavonoidaglycones and more polar solvents are used for extraction offlavonoid glycosides and anthocyanins, as flavonoids vary inpolarity. The conventional extraction of flavanoids often in-volves the addition of an acid (Routray and Orsat, 2010). Usingmicrowave extraction can, in most cases, reduce the need foran acid (Kothari and Seshadri, 2010; Xiao et al., 2008; Gaoet al., 2006). However, in cases where acids are used, such asfor the extraction of anthocyanins (Sun et al., 2007; Yang andZhai, 2010), less concentrated acids can be used with microwaveas compared with conventional methods of extraction withoutaffecting the final yield (Routray and Orsat, 2010).
The optimal conditions for the extraction of anthocyanins inred raspberries (Sun et al., 2007), purple corn (Yang and Zhai,2010), red grape skin (Ghassempour et al., 2008), and grapes(Liazid et al., 2011) with MAE have been devised with theaid of statistical designs, e.g., central composite rotate design,Box-Behnken design, to factorial designs at three level, andfractional factorial experiment, respectively. The applications ofmicrowave-assisted extraction to the recovery of anthocyaninsare summarized in Table 7.
CONCLUDING REMARKSPhenolics can be extracted from fresh, frozen (Kuskoski,
2006a, 2006b), or dried plant samples. Usually, before extrac-tion plant samples are treated by milling, grinding, and homoge-nization, which may be preceded by air-drying or freeze-drying(Dai, 2009). Solubility of phenolics is governed by such param-eters as chemical nature of the plant sample and polarity of thesolvent used. There is no universal extraction procedure suit-able for extraction of all plant phenolics, as plant materials maycontain different quantities of phenolics with varying natures,associated, in addition, with other components such as carbo-hydrates and proteins. A mixture of phenolics, which may alsocontain non-phenolic substances such as sugar, organic acids,and fats, will be extracted from plant materials, depending onthe solvent extraction system used. This means that additionalsteps may be required to remove those unwanted components.
In preparing anthocyanin-rich phenolic extracts from plantmaterials, the most commonly used solvents are methanol,ethanol, and acetone, at a composition of 70–80% in water(Mazza et al., 2004). Acidic (pH of approximately 1.5) condi-tions are usually preferred for the extraction of anthocyaninsin order to minimize pH effects on the flavylium equilib-ria. The concentrated extract can be then extracted with hex-ane, petroleum ether, or diethyl ether to remove unwantedlipophilic compounds after vacuum evaporation of the sol-vent (Jackman and Smith, 1992). An advantage of the ace-tone/chloroform method over the methanol method is that the
aqueous anthocyanin is not contaminated with lipophilic com-ponents (Rodriguez-Saona and Wrolstad, 2001).
Conventional solvent extraction methods often have limitedapplications because of the long extraction times and precau-tions needed to protect the highly reactive phenol species fromdegradation processes (Robards, 2003; Tura and Robards, 2002;Antolovich et al., 2000). A feature of conventional extractionis that it influences the integrity of flavonoid glycosides duringprolonged extraction, thus affecting reproducibility (Routrayand Orsat, 2010; Stalikas, 2007). The growing demand for newextraction techniques amenable to automation with reduced sol-vent consumption and analysis times has seen increasing use inthe extraction of anthocyanins of SPE, CCC, adsorption, PLE(ASE), and MAE (Scotter, 2011a, 2011b; Dai and Mumper,2010).
The applications of many of these developments have beensummarized in tabular form (Tables 2–6); the authors intendto condense some of the applications of anthocyanin separa-tion. Optimization experiments were performed using surfaceresponse methodology. The significance of anthocyanin extrac-tion is not limited to the analytical context, for it has importantpractical applications in the food industry (Shi et al., 2005).Grape peel and seed are good sources of important bioactivecomponents such as phenolics, anthocyanins, and antioxidants.Recovery of these components and their proper utilization is im-portant in the development of functional foods (Ghafoor et al.,2011). It is not the purpose of this review to go into the theoryof the separation methods, which is adequately covered in otherreviews, but to describe the application of the methods.
The SPE process is usually carried out to remove polar non-phenolic compounds (sugar, organic acids) from plant crudeextracts. SPE is becoming popular since it is rapid, economical,and sensitive and because different cartridges with a great varietyof sorbents may be used (Tsao and Deng, 2004). Anthocyanisare usually isolated in a C-18 semi-preparative cartridge, whileother phenolics are eluted using ethyl acetate. Anthocyanins canthen be recovered for futher analysis using acidified methanol(Takeoka and Dao, 2008). The extracts can also be purifiedusing ion exchange resins. Processing of solutions containingphenolics by adsorption-desorption enables the recovery andpurification of bioactive compounds or fractions from plants(Soto et al., 2011).
Conventional methods such as low-pressure chromatogra-phy and preparative reversed phase chromatography are used tofractionate or isolate pure products from plants. However, bothare tedious and time and solvent consuming and require mul-tiple chromatographic steps (Di et al., 2011). Countercurrentchromatography (CCC) can be an excellent alternative. The bigadvantage of CCC is that it uses no solid matrix and the roleof the liquid phases, namely, liquid stationary phase and mobilephase, can be switched during a run. Thus, there is no irreversiblesample adsorption and recovery is 100% (Dai, 2009).
Dissolving power is dependent on the density of the fluid,and by changing pressure and temperature this property can be
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ANTHOCYANIN PIGMENTS. PART IV: EXTRACTION 333
controlled (Kivilompolo, 2009). The unique properties of super-critical fluids, liquid-like (high density and dissolving power)and gas-like (low viscosity, low surface tension, and high diffu-sion rate), make the extraction faster than conventional extrac-tion processes (Brunner, 2010; Sekhon, 2010). Carbon dioxideis nonpolar, but with small additions of a more polar modifier, itcan also be used as an extraction fluid for polar compounds, likeanthocyanins (Bravi et al., 2012). Supercritical carbon dioxide(SC-CO2) methods are ideal for the extraction of natural prod-ucts from plant materials; they are especially recommended forthe extraction of thermolabile compounds when low tempera-tures are required (Ignat et al., 2011; Marostica Junior et al.,2010; Wang and Weller, 2006). Industrial applications of SFEpreceded analytical use of this technique.
Pressurized liquid extraction (PLE) is a solid-liquid extrac-tion process using organic solvents at an elevated temperatureand applying higher pressure to maintain the solvent in its liq-uid state, even at temperatures above its boiling point (Wangand Weller, 2006). In addition, pressure helps to force the sol-vent into the matrix pores, improving in this way the efficiencyof the extraction process (Ramos et al., 2002). Extraction atelevated temperatures increases solubility, diffusion rate, andmass transfer, coupled with the ability of the solvent to disruptthe analyte-matrix interactions. PLE thus allows fast extractionowing to increased solubility, better desorption, and enhanceddiffusion, and the extraction is generally completed within afew minutes. The extraction of value-added products in foodand nutraceutical industries using pressurized liquids involvesa careful selection of solvent and optimal temperature condi-tions to achieve maximum yield (Srinivas et al., 2009). PLE isa green technique that can enhance extraction rates of bioactivecompounds (Wijngaard and Brunton, 2009).
Water, an inexpensive and environmentally friendly solvent,is an ideal solvent for industrial extraction of phenolics, butits use is limited due to poor extraction efficiency at low tem-peratures (Ju and Howard, 2003). At room temperature wa-ter has a high boiling point, dielectric constant, and polarity,but when the temperature is increased, the properties of wa-ter change significantly. At higher temperatures, the diffusionrate increases and the dielectric constant, viscosity, and surfacetension decrease. The permittivity of heated water resemblesthat of an organic solvent, e.g., the dielectric constants of waterand methanol are 30 (at 220◦C) and 33 (at ambient tempera-ture), respectively. This leads water to behave like an organicsolvent, dissolving a wide range of medium- and low-polaritycompounds (Dai and Mumper, 2010; Teo et al., 2010). Pres-surized hot water extraction above and below the boiling pointof water has been demonstrated to be an affective analyticaltechnique (King, 2006) to recovery anthocyanins from varioussources (Arapitsas and Turner, 2008).
Microwave-assisted extraction (MAE) is a relatively new ex-traction technique that combines microwave and the use of tra-ditional solvent (Delazar et al., 2012; Santana et al., 2009). Ex-traction using microwave energy is largely an unexplored area
(Ignat et al., 2011), although by using microwaves to mediatethe extraction, it is possible to maintain mild conditions andachieve superior extraction (Routray and Orsat, 2010). For ther-molabile compounds, reduced extraction times should minimizeanalyte degradation, although the effects of temperature are notalways intuitive.
Although many traditional sample-preparation methods forflavonoids are still in use (Stalikas, 2007; Andersen andMarkham, 2006), there are trends in recent years towards: (1)use of smaller initial sample sizes, small volumes, or no or-ganic solvents; (2) greater specificity or greater selectivity inextraction; (3) higher recoveries or better reproducibility; and(4) increased potential of automation (Liu et al., 2008), as shownin this review. The use of non-thermal technologies in the as-sisted extraction of anthocyanins will be covered in a separatereport.
ACKNOWLEDGMENTSThis work was supported by the Junta de Andalucia (Spain)
through Grant Excellence Research Project P06-FQM-02029,for which the authors are grateful.
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