The chemistry of anthocyanins

This class of flavonoids that creates the brilliant red, orange and blue colours of fruits and flowers also packs a potent antioxidant punch. Fabio Galvano and a team of Italian researchers look at the chemistry, analytical methods and health benefits for this fruitful category

Anthocyanins (antos is Greek for flower, and kyanos means blue,) are one of the six subgroups of the large and widespread group of plant constituents known as flavonoids. They are of nutritional interest because of their marked daily intake (180-215mg/day in the US), which is much higher than the daily intake (23mg/day) estimated for other flavonoids,1 and their reported positive effects in the treatment of various diseases.2

Due to their particular chemical structure, anthocyanins are characterised by an electron deficiency, which makes them very reactive toward reactive oxygen species (ROS), also known as free radicals; they are consequently considered to be powerful natural antioxidants.

In the last few years, much attention has been given to the possible protection exerted by natural antioxidants present in plant-based foods toward tissue injury mediated by ROS. Since cyanidin and its glycosides represent one of the major groups of naturally occurring anthocyanins, their antioxidant and biological properties have generated considerable interest among numerous researchers.

Chemical structure
Anthocyanins are water-soluble glycosides of polyhydroxyl and polymethoxyl derivatives of 2-phenylbenzopyrylium or flavylium salts. Individual anthocyanins differ in the number of hydroxyl groups present in the molecule; the degree of methylation of these hydroxyl groups; the nature, number and location of sugars attached to the molecule; and the number and the nature of aliphatic or aromatic acids attached to the sugars in the molecule.1,2,3 Hundreds of anthocyanins have been isolated and chemically characterised by spectrometric tools; cyanidins and their derivatives are the most common anthocyanins present in vegetables, fruits and flowers.4

Anthocyanins share a basic carbon skeleton in which hydrogen, hydroxyl or methoxyl groups can be found in six different positions. In fruits and vegetables, six basic anthocyanin compounds predominate, differing both in the number of hydroxyl groups present on the carbon ring and in the degree of methylation of these hydroxyl groups. The identity, number and position of the sugars attached to the carbon skeleton are also variable; the most common sugars that can be linked to carbon-3, carbon-5 and, sometimes, carbon-7, are glucose, arabinose, rhamnose or galactose. On this basis, it is possible to distinguish monosides, biosides and triosides.5

Another important variable that contributes to the chemical structure of anthocyanins is the acylating acid that may be present on the carbohydrate moiety. The most frequent acylating agents are caffeic, ferulic, sinapic and p-coumaric acids, although aliphatic acids such as acetic, malic, malonic, oxalic and succinic acids may also occur. Up to three acylating acids may be present simultaneously.

The natural shielding offered by the three-dimensional structure of anthocyanins protects them from aqueous attack, thus preventing hyperchromic and batho-chromic shifts on the phenolic hydroxyl ion present in the carbon skeleton. Such an effect is lost at high temperatures such as those that occur during industrial processing of anthocyanin-rich vegetables. Therefore, anthocyanins are often partially degraded in many foods stored for long periods of time.6

This variability in the chemical structure of anthocyanins accounts for the large number of compounds belonging to the anthocyanin family, and allows researchers to assign chemical/molecular fingerprints to many different plant species on the basis of their anthocyanin composition.

Analytical methods
HPLC separation combined with diode array detection (DAD) is the most common method for qualitative and quantitative analysis of anthocyanins.5 Recently, electrospray ionization mass spectrometry (ESI-MS) has been introduced and is a powerful tool for anthocyanin characterisation in complex food matrices. ESI-MS is also a promising technique for detection of anthocyanin metabolites in human plasma.

Spectrophotometric measurements: Anthocyanin absorption spectra depend strictly on pH. At neutral pH, freshly prepared anthocyanin solutions show characteristic maxima of absorption, one in the ultraviolet region (approximately 240nm) and two in the visible region (approximately 415 and 520nm). The wavelengths of these absorbance peaks can differ slightly by a few nanometers among various anthocyanins, depending on the precise structure of each anthocyanin. However, it is not possible to discriminate spectrophotometrically among the various anthocyanins simply on the basis of their absorption spectra. The maximum of absorption at 520nm in the visible region is the most common wavelength used in the spectrophotometric measurement of total anthocyanins.

Since such anthocyanin determination is rapid and cost-effective and can also be carried out in raw materials with no sample hydrolysis, it is often adopted by food industries to perform a rapid analysis of the total anthocyanin content in foods and beverages.7 This method can therefore be used as a tool for rapid screening of total anthocyanin content in fruits and vegetables for nutritional food supplements companies.8 Spectral similarities in the majority of anthocyanins make this determination relatively feasible, with the anthocyanins being reported as external standard equivalents; in most of cases, cyanidin-3-glucoside (C3G) is the reference anthocyanin used.

This procedure, however, is unsatisfactory for precise characterisation of the anthocyanins present in raw food mixtures. Variability in the sample extraction media (water, acidic water, acidic organic solvent, etc.), chemical properties of anthocyanins, and co-pigmentation of anthocyanins with other secondary metabolites present in the sample solution can cause an inaccurate correlation between absorbance and actual anthocyanin concentration.9 Moreover, the spectrophotometric method does not provide any specificity as far as the molecular identity of the anthocyanin present in the material is concerned.

Extraction, hydrolysis and HPLC: The difficulties related to anthocyanin determination are often associated with sample preparation before analysis. Treatment of anthocyanin-containing material before analysis is highly variable according to the plant or food matrix being analysed, as well as the aim of the research. Extraction from dry matrices is usually carried out using water/methanol mixtures acidified by hydrochloric, trifluoroacetic or formic acids, although, for some anthocyanins, excessive acidic conditions may result in partial hydrolysis of the glycosidic moiety. To obtain satisfactory results, specific solvent mixture extraction has been developed. For example, a hexane-based solvent mixture has been used to extract anthocyanins from lipid-containing sources such as Citrus sinensis.10

For wines or fruit juices naturally rich in anthocyanins, it is possible to directly analyse the raw materials using HPLC. This procedure is rapid and avoids problems related to extraction recovery. However, in many of the studies reported, a fast clean-up of the raw extracts using a C-18 Sep-Pak cartridge was performed before analysis to eliminate carbohydrates and other polar substances. A further separation of anthocyanins from other flavonoids can be achieved by washing the C-18 cartridge with ethyl acetate before elution with methanol.11

Direct HPLC analysis of anthocyanin-rich extracts could result in a chromatogram with a large number of peaks, making it difficult to identify individual anthocyanins. In fact, due to the huge variability of the sugars and organic acids forming the glycosidic moiety, the number of anthocyanins present in a sample can be 15-20 times greater than the number of aglycone forms.

In order to establish the definitive anthocyanins composition and concentration in a sample, the most reliable method is to release the aglycone portion of an anthocyanin molecule using acid hydrolysis, which reduces the number of peaks on the chromatogram to six. Optimal anthocyanin hydrolysis is obtained by incubating samples in methanol containing 1-3M HCl, in a boiling water bath for 20-60 minutes.

HPLC separation of anthocyanins has been performed using a variety of different chromatographic conditions, using mostly reversed-phase columns. The mobile phase is always acidic (12

Single wavelength detection is most commonly performed at 520nm, while data acquisition between 200 and 800nm is advisable when DAD detection is available. Although UV/visible spectra of anthocyanins are fairly similar, tentative identification of compounds can be achieved by comparison with the spectra of reference compounds.

A major challenge in quantification of anthocyanins using HPLC is the poor availability of commercial standards, particularly for those compounds that exist in the glycoside form. It is possible to overcome this problem by choosing a reference glycoside that has a known molar extinction coefficient for calibration.8 In this case, it is important to select a reference compound with the same aglycone moiety of the predominant anthocyanin in the sample.

ESI-MS: This uses low voltage as well as atmospheric pressure, and is versatile as an ionisation technique. The positive charge of anthocyanins at low pH values permits their easy detection using low voltages since other potentially interfering compounds are not usually ionised. The application of this mass spectrometry technique to anthocyanin analysis was reported by researchers in 1999, who applied electrospray and tandem mass spectroscopy (ES-MS and MS-MS) for anthocyanin characterisation.11

Several studies took advantage of this powerful technique to provide a fingerprint of anthocyanin composition in rose hips,13 red onions,14 various berries12 and dry ingredients for the food industry.8 MALDI-TOF mass spectrometry has been proposed as a valid tool for rapid screening of anthocyanins present in foods.15

Antioxidant properties
There is some evidence that chronic diseases such as cancer and cardiovascular disease may occur as a result of oxidative stress. An overall feature of anthocyanins is their natural electron deficiency, which makes them particularly reactive toward ROS. Thus, their antioxidant potency is modulated by their chemical structure. In fact, by varying the position and type of chemical groups present on the aromatic rings of anthocyanin molecules, their capacity to accept unpaired electrons from free radicals, and thus their antioxidant activity, increases or decreases.

Among the potential properties exhibited by cyanidins in particular, antioxidant activity is certainly the most studied, as confirmed by numerous in vitro and in vivo reports. In vitro cyanidin antioxidant activity has been widely demonstrated using several types of measurements, such as automated assays to measure oxygen radical absorbance capacity (ORAC),17 Trolox (vitamin E analogue) equivalent antioxidant activity (TEAC),18 total oxyradical scavenging capacity (TOSC),19 ferric reducing ability of plasma (FRAP) assays20 and inhibition of lipid peroxidation.21

Tests have been conducted on pure compounds as well as on fruit and vegetable extracts rich in cyanidins. Although the conditions used in such studies differed considerably, leading to difficulties in comparison of data, it was found that overall antioxidant activity of cyanidins was higher than that of vitamin E and Trolox and was comparable to that of synthetic antioxidants such as tert-butylhydroquinone (TBHQ), butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA).

Studies of the mechanisms controlling the antioxidative properties of cyanidins seem to be far from complete, although several hypotheses have been proposed, including the ability of cyanidins to act as free radical scavengers, to chelate metal ions and to inhibit lipoprotein oxidation.16

Some studies suggest that the aglycone form (cyanidin) has higher efficacy than its glycosides in inhibiting lipid peroxidation. Such studies hypothesize that antioxidant activity is due to the aglycone moiety being present in cyanidins, and that the number of sugar residues at the C3 position is crucial for this activity, such that an increase in the number of sugar units at C3 would lead to a decrease in antioxidant activity. The lower antioxidant activity of glycosidic forms of cyanidins when compared with aglycone forms might be explained by the bulkiness of the glycoside structure, which may hinder its cellular uptake.22

However, describing aglycones as having a higher antioxidant activity than glycosides may oversimplify the situation, and a systematic study of both the aglycone and glycosidic forms is necessary. The C3G reaction with an alkylperoxyl radical leads to the formation of the oxidation product protocatechuic acid, which can also scavenge free radicals, suggesting that C3G would produce another radical by reacting with biological radicals in vivo.23 Interestingly, some studies have demonstrated the ability of C3G to form a stable copigmentation complex with ascorbic acid and DNA,24,25 which is protected from oxidative damage, since when these compounds are exposed individually, they showed severe oxidative damage.

The model of Cu2+-mediated human low-density lipoprotein cholesterol oxidation was used to demonstrate that the antioxidant capacity of C3G was higher than resveratrol and ascorbic acid, and was independent of pH variations in the range of 4 to 7.4.16 Moreover, the authors showed direct interactions between C3G and ROS and hypothesized that C3G protection of plasma LDL cholesterol oxidation is due to its ROS scavenging activity rather than to any metal-chelating properties.

Antioxidant properties also have been verified by various in vitro biological animal models, such as bovine cells, rabbit erythrocytes and rat liver microsomes, as well as human cells (endothelial cells, LPS/IFN-activated RAW 264.7 macrophage cells, red blood cells).22,26,27,28,29

Lowering LDL
High plasma LDL cholesterol is a significant risk factor in occurrence of coronary heart disease; regular consumption of foods containing high levels of antioxidants is thought to account for the lower incidence of coronary artery disease in countries around the Mediterranean.30 For these reasons, LDL cholesterol has also been widely used as a model to clarify if foods rich in cyanidins such as grapes, red wines, black rice and others may act as in vivo inhibitors of LDL cholesterol oxidation.31,32

Indeed, it may be possible to imagine an important role for C3G as a dietary antioxidant since significant in vivo studies confirm that C3G acts as a potent antioxidant during acute oxidative stress in both rats and humans.33,34

Cyanidins may also demonstrate antimutagenic activity. It has been shown that cyanidins in purple sweet potato (Ipomoea batatas) are able to reduce the mutagenicity and/or carcinogenicity of compounds such as tryptophan pyrolysates, which occur in broiled beef, 2-amino3-methylimidazo(4,5-f) quinoline isolated from baked fish, or benzo[a]pyrene-4-nitroquinoline-1-oxide and dimethyl sulfoxide extracted from grilled beef.35

A natural food colourant, extracted from a pigmented variety of Zea mays and composed mainly of C3G, was shown to have dietary efficacy in suppressing the incidence and number of colorectal adenomas and carcinomas induced in rats by 1,2-dimethylhydrazine and 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine, the latter of which is a heterocyclic amine that occurs abundantly in cooked meat and fish and is able to produce colon tumours in rats.36 It has also been implicated in the aetiology of human colon cancer.37 Grapefruit juice, a well-known source of cyanidins, was able to inhibit colon DNA damage caused by 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine when administered to rats.

Raspberry extract also demonstrates an ability to inhibit proliferation of HepG2 human liver cancer cells.19 The fruits of four Vaccinium species containing cyanidins (bilberry, cranberry, lowbush blueberry and lingonberry) have demonstrated anticarcinogenic properties.38 Moreover, cyanidins have been shown to inhibit the epidermal growth factor receptor of the human vulva carcinoma cell line A431,39 as well as induce production of the tumour necrosis factor.28

Although structure-activity relationships need further investigation, there is evidence cyanidins may even have antioxidant properties higher than other naturally occurring antioxidants. Experimental indications from animal studies have shown that consumption of cyanidins can reduce ROS-mediated cell and tissue damage caused by increases in oxidative stress.

Since some cyanidin-rich foods and beverages, such as red wines, are important in diets such as those common in the Mediterranean, other cyanidin-rich foods, such as pigmented oranges, might also be important components in such diets.

Fabio Galvano is at the University of Reggio Calabria, Italy. Luca La Fauci, Giuseppe Lazzarino and Giacomo Galvano are at the University of Catania, Italy. Vincenzo Fogliano and Alberto Ritieni are at the University Federico II in Naples, Italy. Respond: [email protected]

This article originally appeared on the website of IFIS: International Food Information Service,

Cyanidin riches from the plant kingdom
Cyanidins are considered the most widespread anthocyanins in the plant kingdom. They occur in crops such as beans, fruits and vegetables, and also in red wines. This means daily human intake of these compounds from plant-based diets is significant. One of the most important dietary sources of cyanidin-3-glucoside (C3G) is pigmented oranges, namely Moro, Sanguinello and Tarocco cultivars, which typically grow in Sicily, Italy,1 as well as in Florida, US,2 although such pigmented oranges are rarely cited as food sources of cyanidins.

Other sources of cyanidins are red grapes, fruits of Vaccinium (blueberry, bilberry), Rubus (blackberry, raspberry) and Ribes (redcurrant and blackcurrant) species, and some pigmented varieties of rice, corn, potato and onion.

Various authors, using different analytical methods, have reported the presence of cyanidins in many fruits and vegetables. However, most of the studies do not report quantitative estimation of cyanidin and its derivatives.

—FG, et al

1. Amorini AM, et al. Activity and mechanism of the antioxidant properties of cyanidin-3-O-?-glucopyranoside. Free Rad Res 2001; 35:953-66.
2. Lee HS. Characterization of major anthocyanins and the color of red-fleshed Budd Blood orange (Citrus sinensis). J Agric Food Chem 2002; 50:1243-6.

A range of research still beckons
The amount of experimental evidence demonstrating the important properties of cyanidins continues to increase.

Antioxidant activity is certainly the most studied of the proposed cyanidin properties, with the cyanidin antioxidative mechanism being predominantly related to their redox activities, which play an important role in neutralising free radicals.

Other protective mechanisms are their ability to bind metal ions, their capacity to suppress radical-generating enzymes (eg, cytochrome P450 and xanthine oxidase), and their protective effect on antioxidant vitamins (C and E) and detoxifying enzymes (eg, glutathione S-transferase). Moreover, it has yet to be established whether the aglycone or the glycoside form of these compounds has the higher antioxidant activity, and indeed whether either form demonstrates any pro-oxidant characteristics.

The experimental data available on cyanidins/cyanins shows considerable variability, making direct comparison of data difficult. This is due to several factors including:

  • the diversity of experimental models used
  • use of cyanidins in both isolated chemical and food extract forms
  • insufficient information provided regarding analytical techniques.
Apart from the antioxidant properties of cyanidins, which have been demonstrated by numerous studies, other biological activities of cyanidins have not been as extensively studied. Indeed, their antimutagenic and anticarcinogenic activities have been studied only in bacterial assays and with anthocyanin-rich extracts, respectively. Further analysis of these properties in other experimental models is necessary.

In conclusion, in order to establish whether cyanidins really are capable of positively influencing the incidence and progression of many chronic diseases, much work is still necessary.

—FG, et al

Select Antho Suppliers

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