High-Purity Anthocyanins Isolation Using Solid Phase Extraction Tehniques
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View metadata, citation and similar papers at core.ac.uk brought to you by CORE REVIEW ARTICLES High-Purity Anthocyanins 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 anthocyanin 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 flavonoids 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 anthocyanidins (Markham et al., 2000). The concentrations of cyaninde, delphinidin, pelargonidin 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: (cyanidin) 50%, (delphinidin) 12%, (pelargonidin) 12%, (paeonidin) 12%, (petunidin) 7% and (malvidin) 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 apigeninidin, aurantinidin, capensinidin, anthocyanins, the sugars positions is often C3, europinidin, hirsutidin, 6-hydroxycyanidin, Cyanidin-3-O-galactoside Cyanidin-3-O- Delphinidin-3-galactoside glucoside Delphinidin-3-arabinoside Peonidin-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 luteolinidin, 5-methylcyanidin, pulchellidin, rosinidin, and tricetinidin 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 repre sen- 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