Anthocyanins are water soluble ("watercolor") phenolic pigments dissolved inside leaf cells. Anthocyanins are a group of different colored pigments where each base color changes as the cell environment changes. Anthocyanins generate red, pink, purple and blue colors. They are the pigments found in bronzed or darkleaved trees. Anthocycnins also color some tree flowers, fruits, and new tissues. The colored blush of new growth in many trees is from anthocyanin pigments. Anthocyanins make cherries, cranberries, apples and beets reddish while making grapes, blueberries and plums blue. The more common forms of anthocyanin yield orange-red, purple-red, bluish-purple, rosy-red, and a host of purple colors. Each pigment does not have a set color, but ranges widely in appearance based upon the conditions of the cell where it is dissolved. In the cellís water solution, anthocyanins are not stable for long periods.
Structure and Solubility of Anthocyanins
Anthocyanins are widely distributed in plants, and are responsible for the pink, red, purple and blue hues seen in many flowers, fruits and vegetables.
The aglycone is referred to as an anthocyanidin. There are 6 commonly occurring anthocyanidin structures. However, anthocyanidins are rarely found in plants - rather they are almost always found as the more stable glycosylated derivatives, referred to as anthocyanins. Sugars are present most commonly at the C-3 position, while a second site for glycosylation is the C-5 position and, more rarely, the C-7 position. The sugars that are present include glucose, galactose, rhamnose, and arabinose. The sugars provide additional sites for modification as they may be acylated with acids such as p-coumaric, caffeic, ferulic, sinapic, acetic, malonic or p-hydroxybenzoic acid. Because of the diversity of glycosylation and acylation, there are at least 300 naturally occurring anthocyanins.
Recently, there has been interest in anthocyanins, not only for their colour properties, but due to their activity as antioxidants.
Major anthocyanin forms occurring at wine pH
naturally occurring anthocyanins are in equilibrium between the color
flavylium cation and the colorless hydrated form. The equilibrium is driven
to the left as the pH of the wine is decreased and to the right as pH
is increased. At pH's above 4.5 other destabilizing reactions begin, such
as ring opening of the C ring.
Sources of Anthocyanins and Health Benefits
Widely distributed among flowers, fruits, and vegetables, anthocyanins belong to a group of plant compounds called flavonoids. Flavonoids are a subclass of plant polyphenols that may have antioxidant abilities and are being studied for their anticancer potential. Currently under investigation for their ability to inhibit LDL cholesterol, prevent blood clotting, and defend cells against dangerous carcinogens, anthocyanins may prove to be significant compounds in human health.
Resolution of Fruit Anthrocyanins
To prepare anthocyanin pigments for TLC they were first extracted in methanol/HCl (99/1, v/v) extraction also can be done using ethanol/HCl (99/1, v/v), but the release of the pigments was slower. Sources of anthocyanins that worked well for these studies included red apples (Malus pumila) blueberries (Vaccinium corymbosom), cranberries (Vaccinium macrocarpon), strawberries (Fragaria anannassa) and red cabbage (brassica oleracea). Extractions were carried out by grinding 5 to 10 grams of tissue in the extraction fluid or simply by covering apple peels, whole berries, or pieces of red cabbage with the extraction medium and allowing it to stand for 1 to 2 hours or overnight if covered. If the extracts be analysed following hydrolysis, the pigments can be concentrated with a rotary evaporator or by simply leaving the open beaker in a fume hood until concentrated.
Samples of concentrated apple, blueberry, strawberry, cranberry, and red cabbage extract were spotted on large cellulose TLC plates (20 cm x 5 cm) wht glass plate backing that required over two hours of development but gave excellent resolution. Somewhat smaller cellulose plates with plastic backing (7.5 cm x 4 cm) required only 20 minutes of development but produced somewhat less resolution. Development was carried out in a chamber or covered beaker equilibrated with a solvent mixture composed of concentrated HCl/ formic acid/water, in a ratio of 19.0/39.6/41.4 by volume.
There appear to be two types of anthocyanins in both cranberry and strawberry, and three types in blueberry. The migration of compounds during chromotographic separation is reported commonly as an Rf value. This is a ratio of distance migrated from the orgin by the compound of interest versus the distance migrated by the solvent front. Thus, a compound not migrating from the origin would have an Rf of 0, a compound migrating with the solvent front would have an Rf of 1.0, and a compound migrating half as far as the solvent front would have an Rf of 0.5. All the spots except the one found in red cabbage have Rf values ranging between 0.32 and 0.62; such values usually correspond to monoglycosylated anthocyanins, which migrate in the solvent more slowly than the diglysolated anthocyanins. Thus, the initial assumption is that the anthocyanin in red cabbage is a diglycoside; whereas, the other anthocyanins are monoglycosides. Broadly speaking the pelargonidins appear as pink, orange red, or scarlet. The cyanidins appear as crimson or magenta; whereas, the delphinidins appear as mauve or blue.
Hydrolysis of Anthocyanins
If the sugars are removed from the anthocyanin by acid hydrolysis, the remaining molecule is an aglycone. Aglycones, being less polar, will migrate more slowly on the TLC plate than either the mono or diglycosides using the solvent. The analysis of extracts prior to and following hydrolysis will reveal a shift in the pattern of resolved pigments.
Acid hydrolysis was accomplished by mixing 1.0 mL of the extracted pigment with 1.0 mL of 4 M HCl in a test tube and heating the contents in a water bath or heat block at approximately 80 C for varying time intervals. The test tube was covered with a glass marble during hydrolysis to prevent the escape of vapours. Acid hydrolysis should be carried out in a fume hood. We found that the addition of acid without concurrent heating of the anthocyanin extracts results in very little hydrolysis in a 24h period. Following hydrolysis for the desired times, the tubes can be stored in the cold for up to a week with little evidence of additional hydrolysis.
After hydrolysis for each time interval, a small sample was removed from the heated mixture and spotted onto the TLC plate. When the hydrolysis experiment was complete, the spots on the origin of the TLC plate represented the different lengths of time over which hydrolysis had occurred.
Identification of Anthocyanidin Types from Hydrolysis of Food Extracts
The apple affords a simple and direct example of hydrolysis. In the first lane there is only one spot. By its Rf, it is interpreted to be a monoglycoside. In the second lane there are two spots. This is interpreted to mean that at intermediate times, some of the monoglycoside remains and some of it has been hydrolysed to form the aglycone. In the last lane there is only one spot. The decrease in Rf indicates that it is an aglycone and that hydrolysis of the sugar moieties is complete. This interpretation is consistent with a published report concerning the anthocyanin found in apple peel. In each case, the number of unhydrolysed pigments is the same as the number of spots that migrate as completely hydrolysed pigments. The difference in migration is that the monoglycosides are more soluble in the developing solution than are aglycones; thus, the difference in their Rf values.
A single spot interpreted as a monoglycoside may represent more than one type of molecule. Cranberries, for example, have four types of anthocyanins, but they yield only two spots. The pigments are cyanidin 3-galactoside, cyanidin 3-arabinoside, peonidin 3-galactoside, and peonidin 3-arabinoside. The structures of galactose, an ahexose, and arabinose, a pentose can be found in any biochemistry or organic chemistry textbook. After TLC development, the cyanidin 3-galactoside and cyanidin 3-arabinoside are unresolved and constitute one spot; whereas, the peonidin 3-galactoside and peonidin 3-arabinoside are contained in the second spot. Following complete hydrolysis and subsequent TLC development for cranberries, it is found two anthocyanidins or aglycone spots.
The red cabbage anthocyanin is apparently a diglycoside. The unhydrolysed pigment migrates as one spot very near the solvent front. The completely hydrolysed pigment migrates as a single aglycone. In the case of the partial hydrolysis there are four spots; one dilycoside, two monoglycosides, and one aglycone. The table indicates the types of anthocyanins present in the various foods.