Stability-increasing effects of anthocyanin glycosyl acylation

Accepted Manuscript Stability-increasing effects of anthocyanin glycosyl acylation Chang-Ling Zhao, Yu-Qi Yu, Zhong-Jian Chen, Guo-Song Wen, Fu-Gang Wei, Quan Zheng, Chong-De Wang, Xing-Lei Xiao PII: DOI: Reference:

S0308-8146(16)31095-0 http://dx.doi.org/10.1016/j.foodchem.2016.07.073 FOCH 19534

To appear in:

Food Chemistry

Received Date: Revised Date: Accepted Date:

10 March 2016 10 July 2016 10 July 2016

Please cite this article as: Zhao, C-L., Yu, Y-Q., Chen, Z-J., Wen, G-S., Wei, F-G., Zheng, Q., Wang, C-D., Xiao, X-L., Stability-increasing effects of anthocyanin glycosyl acylation, Food Chemistry (2016), doi: http://dx.doi.org/ 10.1016/j.foodchem.2016.07.073

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

REVIEW

Stability-increasing effects of anthocyanin glycosyl acylation

Chang-Ling Zhaoa*, Yu-Qi Yub, Zhong-Jian Chenc, Guo-Song Wena, Fu-Gang Weib, Quan Zhengd, Chong-De Wange, Xing-Lei Xiaob

a

College of Agronomy and Biotechnology, Yunnan Agricultural University, Kunming 650201,

China b c d e

Miaoxiang Sanqi Industrial Corporation Ltd. of Wenshan City, Wenshan 663000, China Sanqi Research Institute, Wenshan University, Wenshan 663000, China Training Department, Zhejiang College of Construction, Hangzhou 311231, China College of Plant Protection, Yunnan Agricultural University, Kunming 650201, China

* Corresponding author at: College of Agronomy and Biotechnology, Yunnan Agricultural University, 452 Fengyuan Road, Kunming 650201, Yunnan Province, P. R. China E-mail address: [email protected] (C.-L. Zhao).

1

Abbreviations: C=C, carbon-carbon double bond; –COOH, carboxyl; Cy, cyanidin; Dp, delphinidin; E, trans; GC, p-coumaryl-glucose unit; Mv, malvidin; –OH, hydroxyl; Pg, pelargonidin; pKH, hydration constant; Pn, peonidin; Pt, petunidin; SAR, structure-activity relationship; UV, ultraviolet; Z, cis

Keywords: Anthocyanin glycosyl acylation Stability Acylated anthocyanins Effects Mechanism

2

ABSTRACT This review comprehensively summarizes the existing knowledge regarding the chemical implications of anthocyanin glycosyl acylation, the effects of acylation on the stability of acylated anthocyanins and the corresponding mechanisms. Anthocyanin glycosyl acylation commonly refers to the phenomenon in which the hydroxyl groups of anthocyanin glycosyls are esterified by aliphatic or aromatic acids, which is synthetically represented by the acylation sites as well as the types and numbers of acyl groups. Generally, glycosyl acylation increases the in vitro and in vivo chemical stability of acylated anthocyanins, and the mechanisms primarily involve physicochemical, stereochemical, photochemical, biochemical or environmental aspects under specific conditions. Additionally, the acylation sites as well as the types and numbers of acyl groups influence the stability of acylated anthocyanins to different degrees. This review could provide insight into the optimization of the stability of anthocyanins as well as the application of suitable anthocyanins in food, pharmaceutical and cosmetic industries.

3

1 Introduction

Anthocyanins, which are considered flavonoids, are the glycosylated products of anthocyanidins, which are oxygenated derivatives of a 2-phenylbenzopyrylium (flavylium) cation containing two benzoyl rings (A and B) separated by an oxygen-containing six-membered heterocyclic ring (C) (Fig. 1) (Brouillard, 1982; Castañeda-Ovando, Pacheco-Hernández, de Lourdes, Elena, Rodríguez, & Galán-Vidal, 2009). In recent years, more than 30 naturally occurring anthocyanidins have been identified (Ananga, Georgiev, Ochieng, Phills, & Tsolova, 2013). However, only six of these anthocyanidins are common (i.e., cyanidin (Cy), delphinidin (Dp), malvidin (Mv), pelargonidin (Pg), peonidin (Pn) and petunidin (Pt)) (Francis & Markakis, 1989) (Fig. 1). In nature, the hydroxyl groups (–OHs) of the substituted glycosyls (i.e., the sugar moieties) of anthocyanins are typically acylated by organic acids via ester bonds, which is referred to as anthocyanin glycosyl acylation, to yield acylated anthocyanins (Osawa, 1982). In fact, the majority of all known anthocyanins are acylated ones (Andersen & Jordheim, 2006), and, in many plant species, anthocyanins are located in vacuoles as their acylated forms (Nakayama, Suzuki, Nishino, 2003). Glycosyl acylation is one of the key factors for creating diversity within anthocyanin molecules (Mazza & Miniati, 1993), and the types, numbers, and linkage positions of the acyl groups substantially increase the types of anthocyanins (Andersen & Jordheim, 2006).

Fig. 1.

4

In addition to chlorophyll, anthocyanins are most likely the most significant group of visible, water-soluble and vacuolar plant pigments (Kong, Chia, Goh, Chia, & Brouillard, 2003), and these compounds can exist in nearly all land plant parts including the roots, stems, leaves, flowers and fruits (Winkel-Shirley, 2001). Recently, numerous studies have demonstrated that anthocyanins possess a wide range of pharmacological activities (Nabavi et al., 2015) (e.g., antioxidant, anti-inflammatory, antimicrobial (Hribar & Ulrih, 2014), antimutagenic (Yoshimoto, Okuno, Yamaguchi, & Yamakawa, 2001), anti-aging, and anti-carcinogenic properties, microcirculation improvement (He & Giusti, 2010)). Therefore, anthocyanins possess great potential for application in various food, pharmaceutical and cosmetic industries (Giusti & Wrolstad, 2003; Castañeda-Ovando et al., 2009). However, the relatively poor in vitro and in vivo (in plant cells or digestive tracts of animals) chemical instability of anthocyanins has been a critical drawback that limits their wide and effective applications (Harborne, 1964; Giusti & Wrolstad, 2003; Castañeda-Ovando et al., 2009). Numerous studies have confirmed that anthocyanin glycosyl acylation affects the in vitro and in vivo chemical stability of acylated anthocyanins (Brouillard, 1982; Andersen & Jordheim, 2006; Sasaki, Nishizaki, Ozeki, & Miyahara, 2014). Nonetheless, the chemical connotation of anthocyanin glycosyl acylation and the effects of acylation on the stability of acylated anthocyanins have not been systematically reviewed. Therefore, for the first time, this review comprehensively summarizes the existing knowledge regarding the chemical implications and typical physiochemical effects of anthocyanin glycosyl acylation. In addition, we highlight the detailed effects of acylation on the in vitro and in vivo chemical stability of acylated anthocyanins and the corresponding latent stabilization mechanisms to provide valuable insight for optimizing the stability of anthocyanins as well as selecting and applying suitable anthocyanins in various food, pharmaceutical and cosmetic industries. 5

2 Chemical implications and general physicochemical effects of anthocyanin glycosyl acylation

2.1 Chemical implications of anthocyanin glycosyl acylation

Chemically, all of the –OHs of anthocyanins including those of the glycosyl groups and anthocyanidins can be esterified by acids under specific conditions. However, the glycosyl –OHs are commonly acylated with organic acids (Fig. 2) (Brouillard, 1982; Francis & Markakis, 1989; Giusti & Wrolstad, 2003; Escribano-Bailón, Santos-Buelga, & Rivas-Gonzalo, 2004; BąkowskaBarczak, 2005; Andersen & Jordheim, 2006) and rarely acylated with inorganic ones (e.g., sulfuric acid) (Toki, Saito, Ueda, Chibana, Shigihara, & Honda, 1994). As a result, in general, anthocyanin glycosyl acylation, which is sometimes referred to as anthocyanin acylation, primarily refers to the phenomenon where the –OHs of anthocyanin glycosyls are partially or totally esterified by various organic acids (Brouillard, 1982; Francis & Markakis, 1989; Bąkowska-Barczak, 2005; Sasaki et al., 2014). As a post-biosynthetic modification of anthocyanins, anthocyanin glycosyl acylation is believed to be catalyzed by acyl transferases and primarily occur in the cytosol (Sasaki et al., 2014) and vacuoles (Sasaki et al., 2013, 2014), which are synthetically represented by various acylation sites as well as numerous types and various numbers of acyl groups.

Fig. 2.

6

2.1.1 Acylation site involved in anthocyanin glycosyl acylation Due to the hierarchy of the chemical structures, i.e., anthocyanidin glycosylation and the following glycosyl acylation, of the acylated anthocyanins and the difference in the acylation degrees of anthocyanin glycosyls, the acylation site of anthocyanin glycosyls is not only related to the two levels, i.e., the glycosylation and glycosyl acylation, of the molecular structures but also to the acyl numbers of the acylated anthocyanins. On one hand, the acylation site is fundamentally defined by the two structural levels of the acylated anthocyanins. First, for the glycosyls acylated with organic acids, how do these glycosyls attach to specific positions on the aglycons (i.e., anthocyanidins)? Many previous studies have indicated that the glycosyl groups typically attached to the C3 or C5 position (Harborne, 1964; Markham, 1982), C7 position (Saito, Tatsuzawa, Yazaki, Shigihara, & Honda, 2007), and C3′ or C5′ position (i.e., on the B-ring) (Fig. 1) (Andersen & Jordheim, 2006). Second, for the glycosyls acylated with organic acids, which – OHs on the glycosyl groups are acylated? Numerous previous studies have demonstrated that, for monoacylated anthocyanins, the acyl groups are frequently linked at the C6′′–OH of the C3monosaccharide (Harborne, 1964; Honda & Saito, 2002; Giusti & Wrolstad, 2003), but less frequently at the C2′′–OH (Reiersen, Kiremine, Byamukama, & Andersen, 2003), C3′′–OH (Andersen & Fossen, 1995), C4′′–OH (Fossen, Østedal, Slimestad, & Andersen, 2003), and C4′′′– OH of the C3-disaccharide or the C6′′′- or C4′′′′- OH of the C3- disaccharide and C5- monosaccharides (Fossen, Rayyan, Holmberg, Nateland, & Andersen, 2005). Infrequently, one acyl may simultaneously link the two glycosyls which are located at different positions on the anthocyanidins. For example, for the anthocyanidin 3, 5-diglucoside in Dianthus caryophyllus petals, a maloyl concurrently linked the glucosyls at the C3 and C5 positions to form a macrocyclic ring (Sasaki et al., 2013), and this type of acylated anthocyanin is believed to be 7

limited to Caryophyllaceae (Nakayama et al, 2000). On the other hand, the diversity of acylation sites is further positively associated with the number of acyl groups. When more acyl groups are present, the sites are more uncommon. For example, for the tetraacylated cyanin of the redpurple flowers of x Laeliocattleya cv Mini Purple, the three glucosyl groups possessing four acyl groups attach to the C3, C7 and C3′ position of Cy (Tatsuzawa, Saito, Yokoi, Shigihara, & Honda, 1994).

2.1.2 Acyl type involved in anthocyanin glycosyl acylation The organic acids acylating the sugar moieties of anthocyanins include aliphatic and aromatic (phenolic) acids (Fig. 2). The aliphatic acids do not have aromatic rings in their molecular structures but contain straight or branched carbon chains or nonaromatic rings, which are referred to as alicyclic rings. The aliphatic acids include malonic acid, which is the most frequent aliphatic acyl group in acylated anthocyanins (Harborne, 1964; Takeda, Harborne, & Self, 1986) and acetic, malic, succinic, tartaric, oxalic and erucic acids (Francis & Markakis, 1989; Andersen & Jordheim, 2006) as well as glutaric acid (Tatsuzawa et al., 2009) (Fig. 2). Additionally, in 2006, the anthocyanins acylated by lactic acid were identified in trace amounts in some red wines (Alcalde-Eon, Escribano-Bailón, Santos-Buelga, & Rivas-Gonzalo, 2006). The phenolic acids are distinguished by one functional group consisting of a carboxylic acid and two constitutive carbon frameworks (i.e., the hydroxycinnamic and hydroxybenzoic structures) and commonly substituted with one or more –OHs (Herrmann, 1995). The most common phenolic acids are derivatives of hydroxycinnamic acids (e.g., p-coumaric, caffeic, ferulic, sinapic, and 3, 5-dihydroxycinnamic acids) and hydroxybenzoic acids (e.g., p-hydroxybenzoic and gallic acids) (Osawa, 1982; Francis & Markakis, 1989; Andersen & Jordheim, 2006) (Fig. 2). In nature, some

8

glucosylated hydroxycinnamic acids (e.g., β-D-glucopyranosyl- p-coumaric or caffeic acids) can also acylate anthocyanin glycosyls (Saito, Toki, Kuwano, Moriyama, Shigihara, & Honda, 1995; Saito et al., 1996). In addition, some anthocyanins are even acylated by both aromatic and aliphatic acids (Andersen & Jordheim, 2006), and one anthocyanin may simultaneously contain two or more different types of acyl groups (e.g., the Lobelinin A, which was isolated from the bluish violet petals of Lobelia erinus flowers, possesses one coumaryl, one malonyl and two caffeyls, and Lobelinin B has four different types of acyls including coumaryl, malonyl, caffeyl and ferulyl) (Kondo, Yamashiki, Kawahori, & Goto, 1989).

2.1.3 Acyl number involved in anthocyanin glycosyl acylation Different acylated anthocyanins may contain different quantities of acyl groups. In general, an acylated anthocyanin possesses one acyl, which is referred to as monoacylated (e.g., the acetylated one, Mv 3-(6′′-acetylglucoside)-5-glucoside from Geranium sylvaticum flowers) (Andersen, Viksund, & Pedersen, 1995). Occasionally, two or more organic acids can acylate different –OHs of the glycosyls, respectively, and the acids may also consecutively join to the same –OH of one glycosyl, yielding diacylated or polyacylated anthocyanins (BąkowskaBarczak, 2005) (e.g., the diacylated pelargonin from Verbena hybrid flowers (Toki, Saito, Kuwano, Terahara, & Honda, 1995), triacylated cyanins from the flowers of Ipomoea asarifolia (Pale, Kouda-Bonafos, Nacro, Vanhaelen, & Vanhaelen-Fastré, 2003), and tetraacylated delphinin cinerarin from the blue garden cineraria, Senecio cruentus (Goto, Kondo, Kawai, & Tamura, 1984)). Naturally, one plant organ may contain several anthocyanins possessing different numbers of acyl groups (e.g., Ajuga reptans flowers have diacylated and triacylated

9

cyanins, and triacylated delphinins) (Terahara, Callebaut, Ohba, Nagata, Ohnishi-Kameyama, & Suzuki, 2001).

2.2 General physicochemical effects of anthocyanin glycosyl acylation on acylated anthocyanins

2.2.1 General physical effects Physically, anthocyanin glycosyl acylation typically decreases the polarity, increases the molecular size, and changes the spatial structures of the acylated anthocyanins. In general, the organic acids that acylate the –OHs of anthocyanin glycosyls have comparatively low polarity (Fig. 2). Therefore, anthocyanin glycosyl acylation decreases the polarity of the acylated anthocyanins (da Costa, Horton, & Margolis, 2000; Escribano-Bailón et al., 2004). Different acyl groups bear the carbon chains with different lengths or volumes (Fig. 2), and as the carbon chain length of the acyl group increases, the acylation on the anthocyanins results in a decrease in the water solubility (Brouillard, 1982; Osawa, 1982; Giusti & Wrolstad, 2003; Escribano-Bailón et al., 2004). Due to their specific sizes, shapes, and molecular structures, the acyl groups, especially the aromatic ones, can increase the molecular sizes of the anthocyanins and, concurrently, confer steric hindrance effects on the anthocyanins (Mazza & Brouillard, 1987), which may simultaneously alter the ring orientation of the anthocyanin molecules (Kay, Mazza, Holub, & Wang, 2004).

2.2.2 General chemical effects In theory, all of the previously physical effects of anthocyanin glycosyl acylation can influence the chemical reactivity of anthocyanins. For example, the decrease in the polarity

10

induced by the acyl groups can strengthen the capacity of anthocyanins to partition into nonpolar or low-polar domains of in vitro or in vivo media (Jang, Zhou, Nakanishi, & Sparrow, 2005), and enhance the access of the acylated anthocyanins to lipid phases or peroxyls and alkoxyl radicals (Heim, Tagliaferro, & Bobilya, 2002). The acylation-induced changes in the ring orientation of the anthocyanin molecules can influence the ease by which the hydrogen atoms from the –OHs of the anthocyanins are donated to free radicals as well as the capacity of the anthocyanins to support unpaired electrons (Kay et al., 2004). In addition, the special anthocyanin glycosyl acylations induced by some aliphatic dicarboxylic acids (e.g., malic, malonic, oxalic and succinic acids) or by some aromatic acids, such as p-coumaric, caffeic, ferulic and sinapic acids (Fig. 2), can convert anthocyanin flavylium cations to zwitterions, which affects the reactivity of the anthocyanins in subsequent reactions (Harborne & Grayer, 1988).

3 Effects of anthocyanin glycosyl acylation on the in vitro and in vivo chemical stability of acylated anthocyanins

3.1 Effects of acylation on the stability of acylated anthocyanins

Anthocyanin glycosyl acylation can substantially enhance the resistance of acylated anthocyanins to a variety of physicochemical and biochemical (digestive enzymes) factors (e.g., heat, light, pH changes, hydrogen peroxide (H2O2), and gastrointestinal and pancreatic digestion) in nature, during processing and storage or after being taken up by animals (Honda & Saito, 2002; Giusti & Wrolstad, 2003). In nature, for plant organs (e.g., flowers and fruits) colored by anthocyanins, acylated anthocyanins primarily contribute to the stable colorations (Baublis, 11

Spomer, & Berber-Jimenez, 1994). During processing and storage, acylated anthocyanins are more stable than their nonacylated analogs. For example, for heat resistance, the acylated anthocyanins from red cabbage were more stable to heating at 80℃ than the unacylated anthocyanins from red grapes, black currants and elderberries (Dyrby, Westergaard, & Stapelfeldt, 2001). For light resistance, acylated anthocyanins were more stable to light than the unacylated ones in fruit juices (Inami, Tamura, Kikuzaki, & Nakatani, 1996), and the acylated anthocyanins synthesized by lipase-catalyzed transesterification were also more stable against illumination with white fluorescent light than their non-acylated glucosides (Nakajima, Sugimoto, Yokoi, Tsuji, & Ishihara, 2003). With regards to responses to increases in pH, acylated anthocyanins are more resistant to increased pH values than unacylated ones (Delgado-Vargas, Jiménez, & Paredes-López, 2000; Giusti & Wrolstad, 2003), and unacylated anthocyanins were stable only at the pH values at which their flavylium cations dominated (Heredia, Francia-Aricha, Rivas-Gonzalo, Vicario, & Santos-Buelga, 1998). For responses to H2O2, the diacylated anthocyanins in red radish extract were more stable than the monoacylated ones (Matsufuji et al., 2007). In addition, after being taken up by animals, the acylated anthocyanins are substantially more stable against pancreatic digestion than the nonacylated forms (Kurilich, Clevidence, Britz, Simon, & Novotny, 2005; McDougall, Fyffe, Dobson, & Stewart, 2007).

3.2 Main mechanisms that acylation enhances the stability of acylated anthocyanins

3.2.1 General physicochemical mechanism - low polarity and steric hindrance of acyl groups As stated above, anthocyanin glycosyl acylation results in a decrease in the polarity of anthocyanins (da Costa et al., 2000; Escribano-Bailón et al., 2004) and steric hindrance effect for

12

probable ruinous ion attack of the anthocyanidin framework, especially in aqueous environments (Mazza & Brouillard, 1987). Therefore, acylation can lower the sensitivity of the anthocyanins to the nucleophilic attack by the water and sulfite in the media, preventing the anthocyanin molecules from changing into the colorless pseudobase or chalcone structures with pH changes (i.e., contributing to the high stability of the anthocyanins) (Bąkowska-Barczak, 2005; Zhao et al., 2014).

3.2.2 Stereochemical mechanism - intramolecular copigmentation resulting from acyl groups and isomerization from hydroxycinnamic acyl groups (i) Intramolecular copigmentation resulting from acyl groups Based on the spatial structures of acylated anthocyanins, the efficient enhancement of the stability of the anthocyanins resulting from anthocyanin glycosyl acylation in a wide range of slightly acidic to neutral aqueous media may be due to the stacking effect of the acyl groups, especially the aromatic ones, with the pyrylium ring of the anthocyanidin flavylium cations (Fig. 1) (Brouillard, 1982; Nakayama et al., 2003), and the most fundamental and crucial stacking is intramolecular copigmentation (Dangles, Saito, & Brouillard, 1993; Figueiredo, George, Tatsuzawa, Toki, Saito, & Brouillarda, 1999). The intramolecular copigmentation of acylated anthocyanins occurs within specific anthocyanin molecules where the anthocyanidins function as chromophores and the acyl groups as electron-donor copigments (Brouillard, 1982; Saito et al., 1995; Figueiredo et al., 1999; Castañeda-Ovando et al., 2009). Subsequently, when the acyl groups come into contact with the residuary part of the acylated anthocyanins aside from the acyl groups, they form covalent ground-state charge-transfer complexes that possess efficient ultrafast energy dissipation pathways. In particular, in these complexes, fast energy transfers occur from

13

the excited acyl groups to the anthocyanins followed by adiabatic proton transfers (da Silva, Paulo, Barbafina, Eisei, Quina, & Maçanita, 2012). As a prerequisite for intramolecular copigmentation, the anthocyanin glycosyls that are acylated by organic acids are typically attached to the C3 or C5 site of the anthocyanidins (Fig. 1) (Harborne, 1964; Markham, 1982). These glycosyl groups, especially those attached to the C3 site, are very flexible and can rotate freely (Dangles et al., 1993). In aqueous environments, due to hydrophobic forces, the glycosyl flexibility and the rotation possibility of the B-ring with respect to the polarizable and planar benzopyrylium ring (i.e., the A- and C- ring) jointly result in folding of the planar aromatic acyl groups over the benzopyrylium ring, forming π-π interactions (π-complex) where both the double bonds and aromatic rings of the acyl groups are involved (Fig. 1 and 3) (Dangles et al., 1993; Figueiredo, Elhabiri, Toki, Saito, Dangles, & Brouillard, 1996; Figueiredo et al., 1999). A typical case in point is that, a diacylated cyanin (i.e., Cy 3-O-(3′′-O-trans (E)-caffeoyl-6′′-O-E-caffeoylβ-D-glucopyranoside)) was reported to easily form a "sandwich" structure in which the flexible and collapsible sugar chain acts like a ribbon, putting the two caffeoyl groups on both sides of the plane of the 2-phenylbenzopyran framework (Fig. 3) (Figueiredo et al., 1999). Therefore, the stability-enhancing effect of the intramolecular copigmentation of acylated anthocyanins in slightly acidic or neutral aqueous environments may be primarily due to the stereospecific blockage of the aromatic acyl groups. In particular, the intramolecular hydrophobic interactions between the benzopyrylium ring and the aromatic acyl groups can cause an effective physical hindrance to the nucleophilic attack of the OH- in media at the C2 and/or C4 positions of anthocyanidins at acidic to neutral pH ranges to produce a colorless pseudobase or chalcone (Fig. 1) (Zhao et al., 2014), which results in a decrease in the hydration constant (pKH) of the acylated anthocyanins (Figueiredo et al., 1996). For diacylated anthocyanins, the direct and significant

14

effect of the “sandwich” structure favors better overlap and stronger interaction between the two acyl groups and the π-system of the benzopyrylium ring (Fig. 3 B), improving the stability of the anthocyanins to a great extent (Figueiredo et al., 1996, 1999; Nakayama et al., 2003).

Fig. 3.

For different acylated anthocyanins, the possibility and effectiveness of intramolecular copigmentation vary greatly. As one of the characteristics of anthocyanidin glycosylation (Zhao et al., 2014), the length of the anthocyanin glycosyl groups is a distinct factor that influences the occurrence of intramolecular copigmentation prior to acylation (Andersen & Jordheim, 2006). During the process of establishing an intramolecular copigmented complex, the acylated glycosyl groups act as spacers to enable folding of the acyl group(s) on the benzopyrylium ring. Therefore, the compositions and sizes of the glycosyl groups must guarantee that the groups are sufficiently long to form the complex. For Pg 3-(caffeyl) sophorosyl-5-glucoside, the sophorosyl spacer is too short to allow a sufficiently close contact between the caffeyl and the benzopyrylium ring, so the caffeyl can not adequately fold on the ring. In addition, two sugar residues are necessary to function as the spacers to allow folding of the acyl moiety, resulting in higher chromophore integrity (Dangles et al., 1993). (ii) Isomerization resulting from hydroxycinnamic acyl groups The hydroxycinnamic acids acylating the –OHs of the anthocyanin glycosyl groups, such as pcoumaric, caffeic, 3, 5-dihydroxycinnamic, ferulic and sinapic acids (Fig. 2), have E- and cis (Z)isomers due to the existence of the nonrotable double bonds in their side chains. Therefore, the hydroxycinnamic acid residues of acylated anthocyanins may occur in both E and Z configurations (Andersen & Jordheim, 2006). In nature, many anthocyanins with aromatic 15

acylation (e.g., the Cy and Dp 3-(6''-p-coumarylglucoside)-5-(6'''-malonylglucoside) in Labiatae) occur as a mixture of E and Z isomers (Saito & Harborne, 1992). However, the Z-acyl groups of the anthocyanins may be nearly parallel to the anthocyanidin moiety, but the E-acyl groups are present in quasi-perpendicular conformations. In addition, the E form is more stable than the Z one (George, Figueiredo, Toki, Tatsuzawa, Saito, & Brouillard, 2001). In vivo, the hydroxycinnamic acyl groups exist predominately in the E configuration (George et al., 2001; Yoshida, Okuno, Kameda, Mori, & Kondo, 2003; Andersen & Jordheim, 2006), or infrequently in the Z one (Jordheim, Måge, & Andersen, 2007). Additionally, the in vivo conversion between the two isomers is believed to be rare (George et al., 2001; Andersen & Jordheim, 2006). Nevertheless, mutual conversion between the two isomers has been demonstrated in vitro both with artificial (366 nm and UV) and natural (sun) irradiation, and this conversion is referred to as E-Z photoisomerization (Yoshida, Okuno, Kameda, & Kondo, 2002; Yoshida et al., 2003). The E→Z isomerization can significantly increase the stability of the acylated anthocyanins by inducing resistance in the anthocyanins to pyrylium ring hydration (George et al., 2001; Yoshida et al., 2002; Zhao et al., 2014). For example, the Mv and Dp pairs of 3-[6''-(E/Z-p-coumaryl) glucoside]-5-[6'''-(malonyl) glucosides] are structurally compared, and the Z forms may achieve a more coplanar arrangement of the anthocyanin molecules (George et al., 2001). Therefore, the molecules may be less prone to undergo hydration reactions to yield colorless anthocyanin forms (Zhao et al., 2014).

3.2.3 Photochemical mechanism – light energy- absorbing and transferring effects of acyl groups with conjugated double bonds

16

Some aromatic acids that acylate anthocyanin glycosyls, such as p-coumaric, caffeic, 3, 5dihydroxycinnamic, ferulic and sinapic acids, contain carbon-carbon double bonds (C=Cs) in their side chains (Fig. 2). Spectroscopically, conjugated systems in the acid molecules are constructed between the double bonds in the side chains and the three double bonds of the benzene rings, conferring the light energy- absorbing and potential electron- donating abilities on the corresponding aromatic acyls of the anthocyanins (Markham, 1982). In fact, previous studies have reported that the potential ability of acyl groups to donate electrons to anthocyanins (Dangles et al., 1993; Stintzing, Stintzing, Carle, Frei, & Wrolstad, 2002), resulting in enhanced stability of the acylated anthocyanins under light irradiation. For example, in 1996, acylated anthocyanins were proved to be more stable to light than the unacylated ones in fruit juices (Inami et al., 1996). Furthermore, in 2003, the light energy absorbed by cinnamoyl groups of acylated anthocyanins was postulated to be transferred to anthocyanidins and released without any isomerization reaction or degradation of the anthocyanins. Therefore, the anthocyanins might be stable for a long period of time under strong solar irrradiation, which was confirmed by the relatively unchanging coloration (Yoshida et al., 2003).

3.2.4 Biochemical mechanism – membrane carrier-combining hindrance induced by acyl groups and inhibitory effects of acylated anthocyanins on digestive enzymes of animals For the anthocyanins taken up by human beings or animals, the higher stability (i.e., lower bioavailability) of the acylated anthocyanins in the digestive systems compared to that of nonacylated ones may be primarily related to two biochemical reasons. (i) Membrane carriercombining hindrance induced by the acyl groups. In the tissues, organs or systems of animals, the interaction between anthocyanins and their membrane carriers (e.g., the bilitranslocase

17

located in the sinusoidal domain of liver plasma membrane and in the epithelial cells of gastric mucosa) may be initiated via the hydrophilic moieties of the anthocyanins (Passamonti, Vrhovsek, & Mattivi, 2002). As stated above, the acyl groups typically decrease the polarity (i.e., water solubility) of the acylated anthocyanins and exert steric hindrance effects on the anthocyanins. Therefore, in comparison with nonacylated anthocyanins, the interaction between the acylated anthocyanins and the membrane carriers may be more difficult (Passamonti et al., 2002). (ii) Inhibitory effects of acylated anthocyanins on digestive enzymes. Kinetic experiments have confirmed that anthocyanins can inhibit the activities of some enzymes that catalyze anthocyanin

degradation

(e.g.,

α-glucosidase)

(Adisakwattana,

Ngamrojanavanich,

Kalampakorn, Tiravanit, Roengsumran, & Yibchok-Anun, 2004; Homoki et al., 2016), acylated anthocyanins exhibit stronger inhibitory effects on immobilized α-glucosidase (e.g., maltase) that mimics the membrane-bound one at the small intestine than nonacylated anthocyanins, and anthocyanin deacylation results in a marked reduction of the inhibitory activity on the immobilized maltase (Matsui, Ueda, Oki, Sugita, Terahara, & Matsumoto, 2001), which may contribute to the higher stability of the acylated anthocyanins in the digestive systems (Kurilich et al., 2005; McDougall et al., 2007).

3.2.5 Environmental mechanism - acidifying capacity of the acyl groups possessing a free carboxyl Dicarboxylic acids, such as oxalic, malonic, succinic, malic and tartaric acids, can acylate the –OHs of anthocyanin glycosyl groups (Fig. 2). Therefore, the corresponding acyl groups (i.e., oxalyl, malonyl, succinyl, maloyl and tartaroyl) of the acylated anthocyanins still possess free carboxyl groups (–COOHs). In aqueous solutions, the dissociation of the protons of the free acyl

18

–COOHs converts –COOHs to –COO-s, which results in the formation of anthocyanin zwitterions (Takeda et al., 1986; Harborne & Grayer, 1988). In addition, the dissociated protons decrease the pH values of the environmental solutions containing anthocyanins (e.g., the vacuolar sap of petals), which can be defined as the environment-acidifying capacity of the dicarboxylic acyl groups of acylated anthocyanins. As a result, more acidic environments enhance the stability of anthocyanins by facilitating the production of the protonated forms (flavylium cations) of the anthocyanins (Zhao et al., 2014). For example, the pKa of malonic acid is 2.83, and for anthocyanidin 3-(6″-malonylglucosides), the deprotonation of the malonyl group increases the acidity of the medium and protects the anthocyanin from degradation induced by pH increases (Figueiredo et al., 1999).

3.3 Acylation features that influence the stability of acylated anthocyanins

The three aspects including the acylation sites as well as the types and numbers of acyl groups of anthocyanin glycosyl acylation influence the stability of acylated anthocyanins to different degrees. However, in nature, the influence is integrative and interactive (Matsufuji et al., 2007).

3.3.1 Influence of the acylation site on the stability of acylated anthocyanins The effects of the acylation site of acylated anthocyanins on the stability of anthocyanins are primarily represented by two factors. (i) The attachment sites of the acylated glycosyl groups on the anthocyanidins influence the likelihood of intramolecular copigmentation and the stability of the copigmented complex. First, the contribution of the acylated glycosyl groups that attach to the A- and C- or B- ring to the occurrence of the copigmentation and the stability of the complex

19

is different. In recent years, numerous studies have demonstrated that, due to the rotation of the C2-C1′ single bond in anthocyanidins (Fig. 1) and the attachment of the acylated glycosyl groups to the B-ring but not to the A- or C- ring, the intramolecular copigmentation of the acylated anthocyanins occurs more effortlessly, resulting in a more stable copigmented complex. For example, in 1992, the caffeic acid that esterified the glycosyl at the C3′ position of Dp produced a copigmentation effect. However, when the caffeic acid esterified the glycosyl at the C5 position of Dp, no significant copigmentation effect was observed (Yoshida, Kondo, & Goto, 1992). In 2001, the triacylated Dp 3, 7, 3′, 5′-tetraglucosides from the berries of two Dianella species effectively formed intramolecular copigmented complexes due to the p-coumaryl-glucose units (GC) at the C7, C3′ and C5′ positions, and the order of effectiveness of the intramolecular copigmentation of the Dp tetraglucosides bearing the GCs was determined to be: 3′, 5′-GC>7GC>3-GC (Bloor, 2001). In addition, a 2002 study reported that more stable complexes were formed when aromatic acids acylated the glycosyls on the B-ring rather than those on the A-ring (Fig. 1) (Yoshida et al., 2002). Secondly, the contribution of the acylated glycosyl groups attached to different sites on the A- and C- ring to the occurrence of the copigmentation and the stability of the complex is also different. For example, for the diacylation of Cy 3-sophoroside-5glucoside with coumaric and sinapic acids, the 5-glycosylation of the Cy 3-sophoroside-5glucoside might hinder the free rotation of the aromatic acyl of the C5 glycosyl, reducing the shielding effect of the acyl on the other face of the flavylium nucleus (Stintzing et al., 2002). Similarly, the Dp 3-E-coumaroylrutinoside-5-glucoside from violet pepper (Capsicum annuum) peels was less stable than the Dp 3-rutinoside from eggplant (Solanum melongena) peels just due to the hindrance from the additional C5-glycosyl of the former delphinin to the coumaroyl rotation folding over the flavylium nucleus (Sadilova, Stintzing, & Carle, 2006). Finally, for the

20

diacylated anthocyanins that have two glycosyl groups attaching to different sites of the anthocyanidins are acylated by the same or different organic acids, the attachment sites of the acylated glycosyl groups on the anthocyanidins fundamentally determine the remoteness between the two glycosyl groups and the possibility of folding of the two acyl groups with respect to the benzopyrylium ring (Fig. 3 B). For example, the two glycosyl-acyl groups attaching to the C3′ and C7 sites respectively resulted in the production of a minimum energy “sandwich”-typed intramolecular copigmented complex much more easily than the two glycosylacyl groups attaching to the C3 and C5 sites respectively (Fig. 1 and 3 B) (Figueiredo et al., 1999; Stintzing et al., 2002). When the two glycosyl-acyl “side chains” of the diacylated anthocyanins whose two glycosyls were both acylated by phenolic acids attached to the C3 and C5 sites respectively, the folding of the acyl groups did not appear to accommodate the same coplanarity of the different π-electron moieties, and when the “side chains” attached to C3′ and C7 sites respectively, the C3′-chain folded “over” the central chromophore (benzopyrylium ring) and the C7-chain folded “under” the chromophore, resulting in a sandwich-typed conformation with minimum energy and the superposition of two aromatic acyl groups over the chromophore (Figueiredo et al., 1999). The low-energy “sandwich” could more effectively protect the anthocyanin from hydration reactions at different pH values compared to the anthocyanins whose “side chains” attached to C3 and C5 respectively (Figueiredo et al., 1999). A similar acylation pattern of glycosyl groups at the C3′ and C7 positions has been reported for the anthocyanins in Commelinaceae (Baublis & Berber-Jimenez, 1995), Orchidaceae and Senecio (Harborne & Williams, 2001), which may universally indicate that suitable distances between acylated glycosyl groups may facilitate effective intramolecular copigmentation of polyacylated anthocyanins. (ii) The attachment sites of the acyl groups on the glycosyl groups are related to

21

the stability of the anthocyanins. For example, for the acylated anthocyanins from Allium victorialis stem, the malonylation at the 3′′-position in the sugar moiety resulted in better stability than that at the 6′′ position (Andersen & Fossen, 1995). Therefore, the acylation sites including the attachment sites of the acylated glycosyl groups on the anthocyanidins and the attachment sites of the acyl groups on the glycosyl groups jointly influence the stability of the acylated anthocyanins due to their effects on the efficiency of the intramolecular copigmentation and the stability of the intramolecular copigmented complexes of the anthocyanins.

3.3.2 Influence of the acyl type on the stability of acylated anthocyanins Many studies have confirmed that the contribution from the two types of acyl groups (i.e., aliphatic and aromatic ones) to the stability of acylated anthocyanins is different. (i) Aliphatic acyl groups. The effect of aliphatic acyl groups on the stability of anthocyanins is fundamentally related to the fact that the acyl groups effectively decrease the water solubility of the anthocyanins in aqueous media (da Costa et al., 2000; Escribano-Bailón et al., 2004) and generate steric hindrance effects for the possible ion attack of the anthocyanidins (Mazza & Brouillard, 1987). Therefore, aliphatic acyl groups primarily protect and stabilize the anthocyanin molecules due to comparatively high hydrophobic and steric hindrance effects (Bąkowska-Barczak, 2005). Due to their specific molecular shapes and sizes (Fig. 2), different aliphatic acyl groups exhibit different potential to lower the polarity of the anthocyanins (e.g., maloyl>acetyl>malonyl>succinyl) (Andersen et al., 1995; Escribano-Bailón et al., 2004), which results in differential steric hindrance effects (Mazza & Brouillard, 1987). Therefore, these groups possess

different

abilities to

stabilize

anthocyanins.

For

example,

Cy 3-

glucuronylglucoside is more stable than Cy 3-malonylglucoside, indicating that the glucuronyl

22

may possess a stronger ability to stabilize the Cy 3-glucoside than the malonyl (Saito et al., 1988). (ii) Aromatic acyl groups. In comparison with aliphatic acyls, aromatic ones possess a much stronger ability to stabilize acylated anthocyanins because they facilitate the formation of intramolecular copigmented complexes of the anthocyanins (Brouillard, 1982; Dangles et al., 1993; Figueiredo et al., 1999). Undoubtedly, due to their particular molecular structures (i.e., the length and configuration of their side chains) and the presence or absence of double bonds in the chains (Fig. 2), different aromatic acyl groups exhibit different effects on the occurrence of intramolecular copigmentation and contribution to enhancing the stability of the acylated anthocyanins. For example, as reported in 1989, the glycosyl acylation of anthocyanins with caffeic acid induced more stable properties than p-coumaric acid for acylated anthocyanins (Francis & Markakis, 1989). In 2001, the acylation of anthocyanin with caffeic or ferulic acid was determined to be important for the inhibitory effects of anthocyanins on immobilized αglucosidase (e.g., maltase) (Matsui et al., 2001). In 2002, the cyanin acylated by cinnamic acids was reported to substantially increase the pKH of the anthocyanins, and the ferulic acid moiety of Cy 3-xylosyl-glucosylgalactoside increased the pKH more than the sinapoyl residue (Stintzing et al., 2002). In 2007, the pelargonins acylated by p-coumaric or ferulic acids from red radish extract were determined to be more stable at a pH of 7 than those acylated by caffeic acids (Matsufuji et al., 2007). In addition, under acidic gastric digestion conditions, anthocyanins with sinapic acid exhibited lower stability compared with those with other hydroxycinnamic acids (McDougall et al., 2007). In general, based on recent studies, the acyl type, especially the acyl groups that induce intramolecular copigmentation, in anthocyanin glycosyl acylation primarily affects the stability of the acylated anthocyanins at higher pH values (Matsufuji et al., 2007), and

23

the acyl groups that are larger in sizes or have a higher hydrophobicity or more free –OHs can confer much higher stability on the anthocyanins (Figueiredo et al., 1996).

3.3.3 Influence of the acyl number on the stability of acylated anthocyanins Numerous studies have indicated that, as the number of acyl groups in acylated anthocyanins increases, the anthocyanins become more stable, and di-, tri- and poly- acylated anthocyanins typically have increased stability over the monoacylated analogs (Brouillard, 1982). For example, as reported in 1999, the red radish extracts with diacylated anthocyanins were more stable than the red-fleshed potato (Solanum tuberosum) extracts with monoacylated anthocyanins (Rodriguez-Saona, Giusti, & Wrolstad, 1999). In 2001, the stabilization of flower colors was remarkably reported to be substantially dependent on the number of aromatic acids present in the polyacylated anthocyanins (Honda & Saito, 2002), and in 2007, the diacylated pelargonins in the red radish extract were confirmed to be more stable to fluorescence light (5000 lx) at a pH of 3 than monoacylated anthocyanins (Matsufuji et al., 2007). In theory, the effects of the acyl number on the stability of the acylated anthocyanins result from two aspects. (i) The acyl numbers are closely related to some basic physicochemical properties of the acylated anthocyanins. The numbers are negatively related to the water solubility (Brouillard, 1982; Kondo et al., 1989; Giusti & Wrolstad, 2003; Escribano-Bailón et al., 2004) and positively related to the previously discussed steric hindrance effects and electrondonating potential of the acyl groups in acylated anthocyanins (Mazza & Brouillard, 1987; Figueiredo et al., 1999; Stintzing et al., 2002). Therefore, more acyl groups provide higher stability to anthocyanins. (ii) Acyl numbers, especially those of aromatic acyls, are positively associated with the efficiency of the intramolecular copigmentation effect for acylated

24

anthocyanins. In general, the copigmentation effects of monoacylated anthocyanins are not as efficient as those of di- and poly- acylated ones (Figueiredo et al., 1999; Bąkowska-Barczak, 2005). In addition, for acylated anthocyanins, intramolecular copigmentation primarily occurs in those containing two or more aromatic acyl groups (Nakayama et al., 2003). In the intramolecular copigmented complexes of monoacylated anthocyanins, only one side of the benzopyrylium ring of the anthocyanidins can be protected by one plane of the acyl group (i.e., primarily the aromatic one), but the other side of the ring can not be protected and is easily attacked by water molecules. Therefore, for monoacylated anthocyanins, only a partial stabilization can be established via intramolecular copigmentation (Dangles et al., 1993; Figueiredo et al., 1996, 1999). In contrast, for di- or poly- acylated anthocyanins, the 2phenylbenzopyran scaffold is tightly clamped between the planes of the two or more aromatic acyl groups in the intramolecular copigmented complexes (Figueiredo et al., 1996). Therefore, the two faces of the benzopyrylium ring are simultaneously sheltered from nucleophilic attack (Fig. 3 B), resulting in anthocyanins with much higher stability. In general, the number of acyl groups, especially that of intramolecular acyl groups, primarily contribute to the stability of the acylated anthocyanins with regards to light and heat at lower pH values (Matsufuji et al., 2007).

4 Conclusions and future research suggestions

Anthocyanin glycosyl acylation, which is typically the final step in anthocyanin biosynthesis (Brouillard, 1982; Osawa, 1982; Nakayama et al., 2003), refers to the –OHs of glycosyl groups being esterified by aliphatic or aromatic acids (Fig. 2), which is synthetically influenced by the acylation sites as well as the types and numbers of acyl groups. These factors influence the 25

reactivity of anthocyanin by primarily increasing the polarity and molecular size as well as changing the spatial structure of the anthocyanins. Numerous studies have confirmed that anthocyanin glycosyl acylation typically increases the in vitro and in vivo chemical stability of acylated anthocyanins. In particular, the acylation can substantially enhance the resistance of acylated anthocyanins to heat, light, pH changes, H2O2, and gastrointestinal and pancreatic digestion in nature and during processing and storage or after being taken up by animals (Honda & Saito, 2002; Giusti & Wrolstad, 2003). Due to the diversity of the physicochemical and biochemical factors that affect the chemical stability of acylated anthocyanins, the primary mechanisms by which the acylation enhances the stability of acylated anthocyanins involve general physicochemical, stereochemical, photochemical, biochemical and environmental aspects. Physicochemically, the acylation decreases the polarity of anthocyanins and creates steric hindrance effects to lower the sensitivity of the anthocyanins to nucleophilic attack. Stereochemically, the intramolecular copigmentation that is primarily caused by aromatic acyl groups produces an effective physical hindrance to nucleophilic attack of media OH- at the C2 and/or C4 positions of anthocyanidins (Fig. 1 and 3) (Dangles et al., 1993; Figueiredo et al., 1999). The anthocyanins bearing E-hydroxycinnamic acyl groups are more stable than those bearing Z-hydroxycinnamic acyl groups (George et al., 2001), and the E→Z isomerization of the hydroxycinnamic acyl groups can significantly increase the stability of anthocyanins by inducing resistance to pyrylium ring hydration (George et al., 2001; Yoshida et al., 2002; Zhao et al., 2014). Photochemically, the aromatic acyl groups containing C=Cs in their side chains are conjugated systems. Therefore, these groups possess light energy-absorbing and potential electron-donating abilities, which can contribute to the stability of anthocyanins under light irradiation (Inami et al., 1996, Yoshida et al., 2003). Biochemically, the acyl-induced low

26

polarity and steric hindrance effects of anthocyanins hinder hydrophilic initiation of the interaction between anthocyanins and their membrane carriers (Passamonti et al., 2002), and acylated anthocyanins exhibit stronger inhibitory effects on digestive enzymes, which contributes to the higher stability of anthocyanins in digestive systems (Matsui et al., 2001; Kurilich et al., 2005; McDougall et al., 2007). Environmentally, some acyl groups possess free carboxyl groups, and the dissociation of the carboxyl protons decreases the pH values of the environmental solutions, enhancing the stability of anthocyanins by facilitating the production of the protonated forms (i.e., flavylium cations) (Figueiredo et al., 1999; Zhao et al., 2014). Based on the main mechanisms, the acylation sites as well as the types and numbers of acyl groups influence the stability of acylated anthocyanins to different degrees. First, the acylation sites of the acylated glycosyl groups on anthocyanidins and the sites of the acyl groups on the glycosyl groups jointly influence the stability of the acylated anthocyanins. The acylated glycosyl groups attached to the B-ring favor intramolecular copigmentation and the stability of the copigmented complex more than those attached to the A- or C-ring (Yoshida et al., 2002). However, a C5glycosyl group may hinder intramolecular copigmentation (Stintzing et al., 2002; Sadilova et al., 2006), and the attachment sites of two or more than two acylated glycosyl groups on the anthocyanidins determine the distances between the glycosyl groups and the possibility of folding of the acyl groups (Figueiredo et al., 1999; Honda & Saito, 2002; Stintzing et al., 2002). Secondly, different aliphatic and aromatic acyl groups contribute differently to enhancing the stability of acylated anthocyanins (Saito et al., 1988; Francis & Markakis, 1989; Matsufuji et al., 2007), and aromatic acyl groups exhibit a much stronger capacity to stabilize anthocyanins than aliphatic ones (Brouillard, 1982; Dangles et al., 1993; Figueiredo et al., 1999; Giusti & Wrolstad, 2003). The type of acyl group, especially the acyl groups that induce intramolecular

27

copigmentation, primarily affects the stability of the anthocyanins at higher pH values (Matsufuji et al., 2007), and the acyl groups that are larger or have a higher hydrophobicity or more free hydroxyl groups can confer much higher stability to the anthocyanins (Figueiredo et al., 1996). Finally, as the number of acyl groups on the acylated anthocyanins increases, the stability of the anthocyanins increases, and di-, tri- and poly- acylated anthocyanins typically exhibit increased stability over the monoacylated analogs (Brouillard, 1982). In practice, due to the poor chemical stability of nonacylated anthocyanins, their applications in food, pharmaceutical and cosmetic industries are severely limited (Harborne, 1964; Osawa, 1982; Castañeda-Ovando et al., 2009). In contrast, acylated anthocyanins may be more suitable for various applications due to their higher stability (Francis & Markakis, 1989; Giusti & Wrolstad, 2003; Bąkowska-Barczak, 2005). Theoretically, all of the techniques that result in acylation, reacylation or deacylation of glycosyl groups in anthocyanins may significantly modify the stability of the anthocyanins (Nakayama et al., 2003), which can optimize the industrial applications of the anthocyanins. Despite the substantial progress that has been made in studying the effects of anthocyanin glycosyl acylation on the stability of acylated anthocyanins over the past several decades, a detailed structure-activity relationship (SAR) between glycosyl acylation and the stability of anthocyanins is still needed, and three fundamental aspects must be thoroughly elucidated. First, the chemical diversity of the acyl groups is very complicated (i.e., the length and ring-forming features of the carbon chains, the numbers of free –COOHs, and the length, configuration and isomerization of the side chains of the aromatic acyl groups) (Fig. 2). The effects of the chemical and spatial structures of the acyl groups on the stability of acylated anthocyanins may be independent or synergetic. Therefore, the chemical attributes and their respective stability-related targets of the acyl groups should be carefully investigated. Second, the in vitro and in vivo

28

factors that affect the chemical stability of acylated anthocyanins are physical, chemical and biological in nature and contribute to the diversity of mechanisms by which the acylation improves the in vitro and in vivo chemical stability of anthocyanins. The functional details of the acylation under specific factors should be systematically established. Finally, the probable relationships between acylation and anthocyanin stability in animal cells, tissues and organs have been rarely investigated. Therefore, much attention must be focused on establishing scientific in vivo and/or in vitro experimental systems along with suitable determination methods. In the future, with the rapid deepening of the understanding of the SAR of anthocyanin glycosyl acylation and the stability of acylated anthocyanin, industrial applications of anthocyanins will become possible.

Conflict of interest The authors have declared no conflict of interest.

Acknowledgement Financially, this work was supported by the National Natural Science Foundation of China: (i) “Ecophysiological effects of Panax notogensing anthocyanins on Panax notogensing (No. 31260091)”. (ii) “Golden section-typed purpling of the aerial stems of one-year-old Panax notoginseng plants and its relationship with the saponin accumulation (No. 31460065)”.

References Adisakwattana, S., Ngamrojanavanich, N., Kalampakorn, K., Tiravanit, W., Roengsumran, S., & YibchokAnun, S. (2004). Inhibitory activity of cyanidin-3-rutinoside on alpha-glucosidase. Journal of Enzyme Inhibition and Medicinal Chemistry, 19(4), 313–316. 29

Alcalde-Eon, C., Escribano-Bailón, M. T., Santos-Buelga, C., & Rivas-Gonzalo, J. C. (2006). Changes in the detailed pigment composition of red wine during maturity and ageing: A comprehensive study. Analytica Chimica Acta, 563(1–2), 238–254. Ananga, A., Georgiev, V., Ochieng, J., Phills, B., & Tsolova, V. (2013). Production of anthocyanins in grape cell cultures: a potential source of raw material for pharmaceutical, food, and cosmetic industries. In D. Poljuha, & B. Sladonja (Eds.). The Mediterranean Genetic Code - Grapevine and Olive (pp. 247–287). Rijeka Croatia: InTech. Andersen, Ø. M., & Fossen, T. (1995). Anthocyanins with an unusual acylation pattern from stem of Allium victorialis. Phytochemistry, 40(6), 1809–1812. Andersen, Ø. M., & Jordheim, M. (2006). The Anthocyanins. In Ø. M. Andersen, & K. R. Markham (Eds.). Flavonoids: Chemistry, Biochemistry and Applications (pp. 471–553). Boca Raton: CRC Press. Andersen, Ø. M., Viksund, R. I., & Pedersen, A. T. (1995). Malvidin 3-(6-acetylglucoside)-5-glucoside and other anthocyanins from flowers of Geranium sylvaticum. Phytochemistry, 38(6), 1513–1517. Bąkowska-Barczak, A. (2005). Acylated anthocyanins as stable, natural food colorants-a review. Polish Journal of Food Nutrition Sciences, 14/55(2), 107–116. Baublis, A. J., & Berber-Jimenez, M. D. (1995). Structural and conformational characterization of a stable anthocyanin from Tradescantia pallida. Journal of Agricultural and Food Chemistry, 43(3), 640–646. Baublis, A., Spomer, A., & Berber-Jimenez, M. D. (1994). Anthocyanin pigments: comparison of extract stability. Journal of Food Science, 59(6), 1219–1221, 1233. Bloor, S. J. (2001). Deep blue anthocyanins from blue Dianella berries. Phytochemistry, 58(6), 923–927. Brouillard, R. (1982). Chemical structure of anthocyanins. In P. Markakis (Ed). Anthocyanins as Food Colors (pp. 1–40). New York: Academic Press. Castañeda-Ovando, A., Pacheco-Hernández, M., de Lourdes, P. -H., Elena, M., Rodríguez, J. A., & GalánVidal, C. A. (2009). Chemical studies of anthocyanins: A review. Food Chemistry, 113(4), 859–871. da Costa, C. T., Horton, D., & Margolis, S. A. (2000). Analysis of anthocyanins in foods by liquid chromatography, liquid chromatography-mass spectrometry and capillary electrophoresis. Journal of Chromatography A, 881(1–2), 403–410. 30

da Silva, P. F., Paulo, L., Barbafina, A., Eisei, F., Quina, F. H., & Maçanita, A. L. (2012). Photoprotection and the photophysics of acylated anthocyanins. Chemistry, 18(12), 3736–3744. Dangles, O., Saito, N., & Brouillard, R. (1993). Anthocyanin intramolecular copigment effect. Phytochemistry, 34(1), 119–124. Delgado-Vargas, F., Jiménez, A. R., & Paredes-López, O. (2000). Natural pigments: carotenoids, anthocyanins, and betalains-characteristics, biosynthesis, processing, and stability. Critical Reviews in Food Science and Nutrition, 40(3), 173–289. Dyrby, M., Westergaard, N., & Stapelfeldt, H. (2001). Light and heat sensitivity of red cabbage extract in soft drink model systems. Food Chemistry, 72, 431–437. Escribano-Bailón, M. T., Santos-Buelga, C., & Rivas-Gonzalo, J. C. (2004). Anthocyanins in cereals. Journal of Chromatography A, 1054(1–2), 129–141. Figueiredo, P., Elhabiri, M., Toki, K., Saito, N., Dangles, O., & Brouillard, R. (1996). New aspects of anthocyanin complexation. Intramolecular copigmentation as a means for colour loss? Phytochemistry, 41(1), 301–308. Figueiredo, P., George, F., Tatsuzawa, F., Toki, K., Saito, N., & Brouillarda R. (1999). New features of intramolecular copigmentation by acylated anthocyanins. Phytochemistry 51(1), 125–132. Fossen, T., Østedal, D. O., Slimestad, R., & Andersen, Ø. M. (2003). Anthocyanins from a Norwegian potato cultivar. Food Chemistry, 81, 433–437. Fossen, T., Rayyan, S., Holmberg, M. H., Nateland, H. S., & Andersen, Ã. M. (2005). Acylated anthocyanins from leaves of Oxalis triangularis. Phytochemistry, 66(10), 1133–1140. Francis, F. J., & Markakis, P. C. (1989). Food colorants: Anthocyanins. Critical Reviews in Food Science and Nutrition, 28(4), 273–314. George, F., Figueiredo, P., Toki, K., Tatsuzawa, F., Saito, N., & Brouillard, R. (2001). Influence of trans-cis isomerisation of coumaric acid substituents on colour variance and stabilisation in anthocyanins. Phytochemistry, 57(5), 791–795. Giusti, M. M., & Wrolstad, R. E. (2003). Acylated anthocyanins from edible sources and their applications in food systems. Biochemical Engineering Journal, 14(3), 217–225. 31

Goto, T., Kondo, T., Kawai, T., & Tamura, H. (1984). Structure of cinerarin, a tetra-acylated anthocyanin isolated from the blue garden cineraria, Senecio cruentus. Tetrahedron Letters, 25(52), 6021–6024. Harborne, J. B. (1964). Plant polyphenols. XI. Structure of acylated anthocyanins. Phytochemistry, 3(2), 151– 160. Harborne, J. B., & Grayer, R. -J. (1988). The Anthocyanins. In J. B. Harborne (Ed). The Flavonoids: Advances in Research Since 1980 (pp. 1–20). London: Chapman and Hall. Harborne, J. B., & Williams, C. A. (2001). Anthocyanins and other flavonoids. Natural Product Reports, 18(3), 310–333. He, J., & Giusti, M. M. (2010). Anthocyanins: natural colorants with health-promoting properties. Annual Review of Food Science and Technology, 1(3): 163–187. Heim, K. E., Tagliaferro, A. R., & Bobilya, D. J. (2002). Flavonoid antioxidants: chemistry, metabolism and structure-activity relationships. Journal of Nutritional Biochemistry, 13(10), 572–584. Heredia, F. J., Francia-Aricha, E. M., Rivas-Gonzalo, J. C., Vicario, I. M., & Santos-Buelga, C. (1998). Chromatic characterization of anthocyanins from red grapes – I. pH effect. Food Chemistry, 63, 491–498. Herrmann, K. (1995). The shikimate pathway: early steps in the biosynthesis of aromatic compounds. Plant Cell, 7(7), 907–919. Homoki, J. R., Nemes, A., Fazekas, E., Gyémánt, G., Balogh, P., Gál, F., et al. (2016). Anthocyanin composition, antioxidant efficiency, and α-amylase inhibitor activity of different Hungarian sour cherry varieties (Prunus cerasus L.). Food Chemistry, 194, 222–229. Honda, T., & Saito, N. (2002). Recent progress in the chemistry of polyacylated anthocyanins as flower color pigments. Heterocycles, 56(1–2), 633–692. Hribar, U., & Ulrih, N. P. (2014). The metabolism of anthocyanins. Current Drug Metabolism, 15(1), 3–13. Inami, O., Tamura, I., Kikuzaki, H., & Nakatani, N. (1996). Stability of anthocyanins of Sambuskus canadensis and Sambuskus nigra. Journal of Agricultural and Food Chemistry, 44(1), 3090–3096. Jang, Y. P., Zhou, J., Nakanishi, K., & Sparrow, J. R. (2005). Anthocyanins protect against A2E photooxidation and membrane permeabilization in retinal pigment epithelial cells. Photochemistry and Photobiology, 81(3), 529–536. 32

Jordheim, M., Måge, F., & Andersen, Ø. M. (2007). Anthocyanins in berries of ribes including gooseberry cultivars with a high content of acylated pigments. Journal of Agricultural and Food Chemistry, 55(14), 5529–5535. Kay, C. D., Mazza, G., Holub, B. J., & Wang, J. (2004). Anthocyanin metabolites in human urine and serum. British Journal of Nutrition, 91(6), 933–942. Kondo, T., Yamashiki, J., Kawahori, K., & Goto, T. (1989). Structure of lobelinin A and B, novel anthocyanins acylated with three and four different organic acids, respectively. Tetrahedron Letters, 30(44), 6055–6058. Kong, J. -M., Chia, L. -S., Goh, N. -K., Chia, T. F., & Brouillard, R. (2003). Analysis and biological activities of anthocyanins. Phytochemistry, 64(5), 923–933. Kurilich, A. C., Clevidence, B. A., Britz, S. J., Simon, P. W., & Novotny, J. A. (2005). Plasma and urine responses are lower for acylated vs nonacylated anthocyanins from raw and cooked purple carrots. Journal of Agricultural and Food Chemistry, 53(16), 6537–6542. Markham, K. R. (1982). Techniques of flavonoid identification (p. 113). London, New York, Toronto, Sydney, San Francisco: Academic Press. Matsufuji, H., Kido, H., Misawa, H., Yaguchi, J., Otsuki, T., Chino, M., et al. (2007). Stability to light, heat, and hydrogen peroxide at different pH values and DPPH radical scavenging activity of acylated anthocyanins from red radish extract. Journal of Agricultural and Food Chemistry, 55(9): 3692–3701. Matsui, T., Ueda, T., Oki, T., Sugita, K., Terahara, N., & Matsumoto, K. (2001). alpha-Glucosidase inhibitory action of natural acylated anthocyanins. 2. alpha-Glucosidase inhibition by isolated acylated anthocyanins. Journal of Agricultural and Food Chemistry, 49(4), 1952–1956. Mazza, G., & Brouillard, R. (1987). Recent developments in the stabilization of anthocyanins in food products. Food Chemistry, 25, 207–225. Mazza, G., & Miniati, E. (1993). Types of anthocyanins. In G. Mazza, & E. Miniati (Eds). Anthocyanins in Fruits, Vegetables, and Grains (pp. 1–28). Boca Raton: CRC Press. McDougall, G. J., Fyffe, S., Dobson, P., & Stewart, D. (2007). Anthocyanins from red cabbage-stability to stimulated gastrointestinal digestion. Phytochemistry, 68(9), 1285–1294. 33

Nabavi, S. F., Habtemariam, S., Daglia, M., Shafighi, N., Barber, A.J., & Nabavi, S. M. (2015). Anthocyanins as a potential therapy for diabetic retinopathy. Current Medicinal Chemistry, 22(1), 51–58. Nakajima, N., Sugimoto, M., Yokoi, H., Tsuji, H., & Ishihara, K. (2003). Comparison of acylated plant pigments: light-resistance and radical-scavenging ability. Bioscience, Biotechnology, and Biochemistry, 67(8), 1828–1831. Nakayama, M., Koshioka, M., Yoshida, H., Kan, Y., Fukui, Y., Koike, A., et al. (2000). Cyclic malyl anthocyanins in Dianthus caryophyllus. Phytochemistry, 55(8), 937–939. Nakayama, T., Suzuki, H., & Nishino, T. (2003). Anthocyanin acyltransferases: specificities, mechanism, phylogenetics, and applications. Journal of Molecular Catalysis B-Enzymatic, 23(2–6), 117–132. Osawa, Y. (1982). Copigmentation of anthocyanins. In P. Markakis (Ed). Anthocyanins as Food Colors (pp. 41–69). New York: Academic Press. Pale, E., Kouda-Bonafos, M., Nacro, M., Vanhaelen, M., & Vanhaelen-Fastré, R. (2003). Two triacylated and tetraglucosylated anthocyanins from Ipomoea asarifolia flowers. Phytochemistry, 64(8), 1395–1399. Passamonti, S., Vrhovsek, U., & Mattivi, F. (2002). The interaction of anthocyanins with bilitranslocase. Biochemical and Biophysical Research Communications, 296(3), 631–636. Reiersen, B., Kiremine, B. T., Byamukama, R., & Andersen, Ø. M. (2003). Anthocyanins acylated with gallic acid from chenille plant, Acalypha hipsida. Phytochemistry, 64(4), 867–871. Rodriguez-Saona, L. E., Giusti, M. M., & Wrolstad, R. E. (1999). Color and pigment stability of red radish and red-fleshed potato anthocyanins in juice model systems. Journal of Food Science, 64(3), 451–456. Sadilova, E., Stintzing, F. C., & Carle, R. (2006). Anthocyanins, colour and antioxidant properties of eggplant (Solanum melongena L.) and violet pepper (Capsicum annuum L.) peel extracts. Zeitschrift Fur Naturforschung C-A Journal of Biosciences, 61(7–8), 527–535. Saito, N., & Harborne, J. B. (1992). Correlations between anthocyanin type, pollinator and flower colour in the labiatae. Phytochemistry, 31(9), 3009–3015. Saito, N., Tatsuzawa, F., Yazaki, Y., Shigihara, A., & Honda, T. (2007). 7-polyacylated delphinidin 3, 7diglucosides from the blue flowers of Leschenaultia cv. Violet Lena. Phytochemistry, 68(5), 673–679.

34

Saito, N., Tatsuzawa, F., Yokoi, M., Kasahara, K., Iida, S., Shigihara, A., et al. (1996). Acylated pelargonidin glycosides in red-purple flowers of Ipomoea purpurea. Phytochemistry, 43(6), 1365–1370. Saito, N., Toki, K., Kuwano, H., Moriyama, H., Shigihara, A., & Honda, T. (1995). Acylated cyanidin 3rutinoside-5, 3′-diglucoside from the purple-red flower of Lobelia erinus. Phytochemistry, 39(2), 423– 426. Sasaki, N., Matsuba, Y., Abe, Y., Okamura, M., Momose, M., Umemoto, N., et al. (2013). Recent advances in understanding the anthocyanin modification steps in carnation flowers. Scientia Horticulturae, 163, 37– 45. Sasaki, N., Nishizaki, Y., Ozeki, Y., & Miyahara, T. (2014). The role of acyl-glucose in anthocyanin modifications. Molecules, 19(11), 18747–18766. Stintzing, F. C., Stintzing, A. S., Carle, R., Frei, B., & Wrolstad, R. E. (2002). Colour and antioxidant properties of cyanidin-based anthocyanin pigments. Journal of Agricultural and Food Chemistry, 50(21), 6172–6181. Tatsuzawa, F., Saito, N., Mikanagi, Y., Shinoda, K., Toki, K., Shigihara, A., et al. (2009). An unusual acylated malvidin 3-glucoside from flowers of Impatiens textori Miq. (Balsaminaceae). Phytochemistry, 70(5), 672–674. Tatsuzawa, F., Saito, N., Yokoi, M., Shigihara, A., & Honda, T. (1994). An acylated cyanidin glycoside in the red-purple flowers of x Laeliocattleya cv mini purple. Phytochemistry, 37(4), 1179–1183. Terahara, N., Callebaut, A., Ohba, R., Nagata, T., Ohnishi-Kameyama, M., & Suzuki, M. (2001). Acylated anthocyanidin 3-sophoroside-5-glucosides from Ajuga reptans flowers and the corresponding cell cultures. Phytochemistry, 58(3), 493–500. Toki, K., Saito, N., Kuwano, H., Terahara, N., & Honda, T. (1995). Acylated anthocyanins in verbena flowers. Phytochemistry, 38(2), 515–518. Toki, K., Saito, N., Ueda, T., Chibana, T., Shigihara, A., & Honda, T. (1994). Malvidin 3-glucoside-5glucoside sulphates from Babiana stricta. Phytochemistry, 37(3), 885–887. Winkel-Shirley, B. (2001). Flavonoid biosynthesis: A colorful model for genetics, biochemistry, cell biology, and biotechnology. Plant Physiology, 126(2), 485–493. 35

Yoshida, K., Kondo, T., & Goto, T. (1992). Intramolecular stacking conformation of gentiodelphin, a diacylated anthocyanin from Gentiana makinoi. Tetrahedron, 48(21), 4313–4326. Yoshida, K., Okuno, R., Kameda, K., & Kondo, T. (2002). Prevention of UV-light induced E, Z-isomerization of caffeoyl residues in the diacylated anthocyanin, gentiodelphin, by intramolecular stacking. Tetrahedron Letters, 43(35), 6181–6184. Yoshida, K., Okuno, R., Kameda, K., Mori, M., & Kondo T. (2003). Influence of E, Z-isomerization and stability of acylated anthocyanins under the UV irradiation. Biochemical Engineering Journal, 14(3), 163–169. Yoshimoto, M., Okuno, S., Yamaguchi, M., & Yamakawa, O. (2001). Antimutagenicity of deacylated anthocyanins in purple-fleshed sweetpotato. Bioscience Biotechnology and Biochemistry, 65(7), 1652– 1655. Zhao, C. L., Chen, Z. J., Bai, X. S., Ding, C., Long, T. J., Wei, F. G., et al. (2014). Structure-activity relationships of anthocyanidin glycosylation. Molecular Diversity, 18(3): 687–700.

36

Figure captions:

Fig. 1. Chemical structures of the six common anthocyanidins in nature. Atom numbering and ring nomenclature of the 2-phenylbenzopyrylium (flavylium) core are marked.

Fig. 2. Organic acids that acylate the hydroxyl groups of anthocyanin glycosyl groups. A: Acetic acid; B: Oxalic acid; C: L-lactic acid; D: Malonic acid; E: Succinic acid; F: Malic acid; G: Tartaric acid; H: Glutaric acid; I: Erucic acid; J: p-hydroxybenzoic acid; K: Gallic acid; L: (E)-p-coumaric acid; M: (E)-caffeic acid; N: (E)-3, 5-dihydroxycinnamic acid; O: (E)-ferulic acid; P: (E)-sinapic acid. A~I are aliphatic acids. J~P are phenolic acids.

Fig. 3. Chemical structure and suggested intramolecular copigmentation complex of cyanidin 3-O-(3′′-O-E– caffeoyl-6′′-O-E-caffeoyl-β-D-glucopyranoside). A: Chemical structure; B: Suggested intramolecular copigmentation complex. Atom numbering and ring nomenclature are included. The middle parallelogram in B indicates the plane formed by the benzopyrylium ring, and the upper and under ones indicate the planes formed by the two caffeoyl groups.

37

R1 3' HO

8 7

A

C

5

4

6

2'

+ O 1

1'

5' 6'

2

OH 4'

B R2

3 OH

OH

R1=H, R2=H: pelargonidin (Pg) R1=OH, R2=H: cyanidin (Cy) R1=OH, R2=OH: delphinidin (Dp) R1=OCH3, R2=H: peonidin (Pn) R1=OCH3, R2=OH: petunidin (Pt) R1= OCH3, R2=OCH3: malvidin (Mv)

Fig. 1. Chemical structures of the six common anthocyanidins in nature. Atom numbering and ring nomenclature of the 2-phenylbenzopyrylium (flavylium) core are marked.

O

O

O

OH

O

O

OH H C 3

HO

HO

O

CH3

O

A

OH HO

HO

OH

B

O

O

OH

OH

C

OH HO O

O

D

E

F

OH

O

OH

O

O

O

HO OH

HO

O

OH

G

HO

CH3 HO

(CH2)11

H

(CH2)7

OH

I

HO

J HO

HO

O

O

HO

HO

O

O

HO

OH OH HO

OH

HO

K

L

H3CO

M

OH

N

H3CO

O

HO

O

HO

OH H3CO

O

OH

P

Fig. 2. Organic acids that acylate the hydroxyl groups of anthocyanin glycosyl groups. A: Acetic acid; B: Oxalic acid; C: L-lactic acid; D: Malonic acid; E: Succinic acid; F: Malic acid; G: Tartaric acid; H: Glutaric acid; I: Erucic acid; J: p-hydroxybenzoic acid; K: Gallic acid; L: (E)-p-coumaric acid; M: (E)-caffeic acid; N: (E)-3, 5-dihydroxycinnamic acid; O: (E)-ferulic acid; P: (E)-sinapic acid. A~I are aliphatic acids. J~P are phenolic acids.

OH

HO

8 7 6A 5 OH

+ O 1 C 23 4

OH

3' 2' B 4' 1' 5' 6'

O OH OH

H O 1'' HO

OH 2''

O

H

O

H 3'' O

6'' O H 4''OH

HO

B

OH

H 5'' HO

O

O+ O

C

H H

A

O HO H

OH

HO

O

HO

A

O H

OH

OH OH

O

H HO

B

Fig. 3. Chemical structure and suggested intramolecular copigmentation complex of cyanidin 3-O-(3′′-O-E–caffeoyl-6′′-O-E-caffeoyl-β-D-glucopyranoside). A: Chemical structure; B: Suggested intramolecular copigmentation complex. Atom numbering and ring nomenclature are included. The middle parallelogram in B indicates the plane formed by the benzopyrylium ring, and the upper and under ones indicate the planes formed by the two caffeoyl groups.

Highlights •

Anthocyanin glycosyl acylation commonly refers to the phenomenon in which the hydroxyl groups of anthocyanin glycosyls are esterified by aliphatic or aromatic acids.

•

Anthocyanin glycosyl acylation is synthetically represented by the acylation sites as well as the types and numbers of acyl groups.

•

Anthocyanin glycosyl acylation generally increases the in vitro and in vivo chemical stability of acylated anthocyanins.

•

The mechanisms of the stability-increasing effect mainly involve typical physicochemical, stereochemical, photochemical, biochemical or environmental aspects under specific conditions.

•

The acylation sites as well as the types and numbers of acyl groups influence the stability of acylated anthocyanins to different degrees.

38