Recent Advances in Anthocyanin Analysis and Characterization

Abstract

Anthocyanins are a class of polyphenols responsible for the orange, red, purple and blue colors of many fruits, vegetables, grains, flowers and other plants. Consumption of anthocyanins has been linked as protective agents against many chronic diseases and possesses strong antioxidant properties leading to a variety of health benefits. In this review, we examine the advances in the chemical profiling of natural anthocyanins in plant and biological matrices using various chromatographic separations (HPLC and CE) coupled with different detection systems (UV, MS and NMR). An overview of anthocyanin chemistry, prevalence in plants, biosynthesis and metabolism, bioactivities and health properties, sample preparation and phytochemical investigations are discussed while the major focus examines the comparative advantages and disadvantages of each analytical technique.

Fig. (1)

Chemical structure of some common polyphenolic compounds: phenolic acids, gallic acid (1) and caffeic acid (2); a stilbene, resveratrol (3); a lignan, secoisolariciresinol (4); and a flavonoid, cyanidin (5).

Fig. (2)

The chemical structures of flavanol, (±)-catechin (6); flavanone, naringenin (7); flavone, apigenin (8); isoflavone, genistein (9); flavonol, quercetin (10); and anthocyanins, cyanidin (5).

Fig. (3)

Structural transformations of cyanidin (5) pigments with change in pH, modified from [16].

Fig. (4)

General representation of the biosynthetic pathway of anthocyanins, specifically pelargonidin (18), cyanidin (5), and delphinidin (19), modified from [101]. The intermediate compounds consist of naringenin chalcone (11), naringenin (7), dihydrokaempferol (12), dihydroquercetin (13), dihydromyricetin (14), leucopelargonidin (15), leucocyanidin (16), and leucodelphinidin (17). Catalysts are acetyl-CoA carboxylase (ACCase), phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), 4-coumarate:CoA ligase (4CL), chalcone synthase (CHS), calcone isornerase (CHE), flavanone 3-hydroxylase (F3H), flavonoid 3′-hydroxylase (F3′H), flavonoid 3′,5′-hydroxylase (F3′5′H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), flavonoid 3-O-glucosyltransferase (3-GT), anthocyanin acyltransferase (AAT), and anthocyanin rnalonyltranferase (MAT).

Fig. (5)

A side-by-side comparison of an LC analysis (upper) and CE analysis (lower) of a 2002 vintage Tannat red wine. The conditions are described in the original paper and the peak identification for each follows. The LC chromatogram (A): 1, delphinidin-3-glucoside (dp); 2, cyanidin-3-glucoside (cy); 3, petunidin-3-glucoside (pt); 4, pt-3-glucoside pyruvic acid dimer; 5, peonidin-3-glucoside (pn); 6, malvidin-3-glucoside (mv); 7, mv-3-glucoside pyruvic acid derivative; 8, dp-3-(6-acetyl)glucoside; 9, mv-3-glucoside catechin dimer; 10, pt-3-(6-acetyl) glucoside; 11, mv-3-glucoside catechin dimer; 12, pn-3-(6-acetyl)glucoside; 13, mv-3-(6-acetyl)glucoside; 14, mv-3-(6-coumaryl)glucoside; The CE electrophoregram (B): 1, rnv-3-(6-coumaryl)glucoside; 2, mv-3-(6-acetyl)glucoside; 3, pn-3-(6-acetyl)glucoside; 4, mv-3-glucoside; 5, pn-3-glucoside; 6, mv-3-glucoside catechin dimer; 7, mv-3-glucoside catechin dimer; 8, pt-3-(6-acetyl)glucoside; 9, mv-3-glucoside pyruvic acid derivative; 10, pt-3-glucoside pyruvic acid derivative; 11, pt-3-glucoside; 12, dp-3-glucoside; 13, cy-3-glucoside. Modified from Calvo et al. [120] and used with permission from Elsevier Publishers.

Fig. (6)

Separation of common glucosylated anthocyanins in acidic electrolyte at pH=2 (left) and basic electrolyte at pH=9 (right) with appropriate mass spectrometry data included for each anthocyanins. The two figures demonstrate the difference in migration order with acidic versus basic media. Modified from Bednar et al. [129] and reprinted with permission from Wiley VCH Publishers.

Fig. (7)

Base peak chromatogram from the HPLC analysis of a South African Pinotage red wine with detection at 520 nm. The separation of anthocyanins is demonstrated and peak identification can be found in the original paper. Modified from de Villiers et al [130] and used with permission from Elsevier Publishers.

Fig. (8)

LC-ESI/MS/MS of anthocyanins in black raspberries. (A) HPLC chromatogram of anthocyanins at 520 nm. Peak labels: (1) cyanidin 3-glucoside (cy 3-gluc); (2) cyanidin 3-sambubioside (cy 3-sam); (3) cyanidin 3-(2-xylosylrutinoside) (cy 3-xylosylrutin); (4) cyanidin 3-rutinoside (cy 3-rutin); and (5) pelargonidin 3-rutinoside (pg 3-rutin). Precursor-ion analysis of (B) m/z 287 (cyanidin) and (C) m/z 271 (pelargonidin). Product-ion analysis of (D) cy 3-xylosylrutin (m/z 727); (E) cy 3-rutin (m/z 595); and (F) pg 3-rutin (m/z 579). Modified from Tian et al. [135] and used with permission from Elsevier Publishers.