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花青素.ANTHOCYANIN

Introduction:

Anthocyanins represent a large group of water-soluble plant pigments of the 2-phenylbenzophyrylium (flavylium) structure (Kuhnau, 1976). The class, "Anthocyanins", consists of some 200 or more compounds (Parkinson & Brown, 1981) chemically combined to a sugar moiety (glucose < rhamnose < galactose < xylose < arabinose) of which the most common are:

What Is It?

Anthocyanins are naturally occurring compounds that impart color to fruit, vegetables, and plants. Derived from two Greek words meaning plant and blue, anthocyanins are the pigments that make blueberries blue, raspberries red, and are thought to play a major role in the high antioxidant activity levels observed in red and blue fruits and vegetables. Anthocyanins are also largely responsible for the red coloring of buds and young shoots and the purple and purple-red colors of autumn leaves. Close to 300 anthocyanins have been discovered.

Each fruit and vegetable has its own anthocyanin profile, providing a distinct "fingerprint." Red wine, for example, contains over 15 anthocyanin monomers (type of chemical compound), the varying proportions of which, depending on the type of grape, establish the various shades of the wine's color.  

Researchers are attempting to identify the specific bioactivity of each anthocyanin in relation to human health. Variation in pigment results from different degrees of acidity and alkalinity. Intense light and low temperatures favor the development of anthocyanin pigments.  

All plant materials contain various pigments, some of which change color as the pH of the plant tissue is changed (for example, by the addition of vinegar or other acids while cooking or processing). An average anthocyanin is red in acid, violet in neutral, and blue in alkaline solution. In fact, when cooking a food that is red, such as red cabbage, it may be helpful to add an acidic substance such as vinegar (or tomato juice or lemon juice) to prevent the food from turning purple.  

Many factors influence the stability of anthocyanins. Heat- and light-sensitive, anthocyanin pigments can easily be destroyed during the processing of fruits and vegetables. In particular, in the presence of a high sugar concentration, anthocyanins are rapidly destroyed, thus processed foods containing large amounts of sugar or syrup would not have the same amount of anthocyanins as their unprocessed counterparts.

Biology:  

     Anthocyanins are members of a class of nearly universal, water-soluble, terrestrial plant pigments that can be classified chemically as both flavonoid and phenolic. They are found in most land plants, with the exception of the cacti and the group containing the beet. They contribute colors to flowers and other plant parts ranging from shades of red through crimson and blue to purple, including yellow and colorless. (Every color but green has been recorded).

Anthocyanins apparently play a major role in two very different plant processes: for one, attracting insects for the purpose of pollination. Advantage is made of the fact that the pigments absorb strongly in the UV (ultraviolet), visually attracting insects but with light wavelengths that are invisible to humans. These pigments play a major role in plant pollination - and in predation in carnivorous plants, attracting insects into the trap apparatus. (Anthocyanins play a very versatile role in pollination, especially in the Bromeliaceae. Certain bromeliads turn a vivid red just before and during pollination but soon revert to the original green color characteristic of the photosynthesis pigment, chlorophyll. Anthocyanins are not a biochemical dead end but rather a dynamic signalling device that can be switched on when needed by the plant to assist in pollination. They are then degraded by plant enzymes when no longer needed to attract pollinators to flowers.)      

In their second major role, anthocyanin-related pigments serve as a UV screen and are produced in response to exposure of the plant to UV radiation, protecting the plant's DNA from damage by sunlight. (UV causes the paired strands of genetic material in the DNA double helix to become cross-linked, preventing cell division and other vital cellular processes like protein production).      

And in a third, and no less significant role, anthocyanins serve as anti-feedents, their disagreeable taste serving to deter predatory animals.

In a related defense mechanism, anthocyanin production can be induced by ionizing radiation, which can damage DNA as readily as UV can. Chemical messengers apparently signal the damage to DNA and induce anthocyanin production in these plants.     

The biosynthesis of this class of pigment is accomplished by a series of enzymes that are bound to cell membranes and that help convert two central biochemical building blocks derived from photosynthesis (acetic acid and the amino acid phenylalanine) found in the cell's cytoplasm through a series of discrete chemical steps into the final pigments, which are then excreted on the other side of the membrane into vacuoles in the epidermal cell layer. Significant genetic change in the DNA coding for the production of these enzymes results in loss of pigment production.     

Plants having no pigmentation themselves in cultivation were subsequently demonstrated to produce anthocyanin in tissue culture.      

Environmental factors affecting anthocyanin production included light (intensity and wavelength, with blue and UV being most effective), temperature, water and carbohydrate levels, and the concentrations of the elements nitrogen, phosphorous and boron in the growth medium. Anthocyanin production can be induced by light, blue being the most effective color. Low light levels also induce the formation of different flavonoid pigments, which is another interesting adaptive response on the part of plants. (Tillandsias, for example, develop a bright red coloration due to induced anthocyanin production if grown in strong light. For some additional observations on possible alternate roles for anthocyanin in Tillandsia,

Anthocyanins are poorly absorbed from the gastrointestinal:
Anthocyanins (notably delphinidin) extracted from concord grapes were administered to rats by either gavage  or by percutaneous injection  andtheurinetestedforunchanged anthocyanins by an HCl-acid red test (Horwitt, 1933). Anthocyanin was detected in the urine of rats administered anthocyanin by the percutaneous route but not by gavage. In studies in dogs (Horwitt, 1933) administered anthocyanin  by gastric fistula, no urinary coloration was demonstrated. However, in the rabbit, 1-2%ofanoraldoseofanthocyanin (500 mg) was present in the urine as the unchanged pigment. It should benotedthatthe HCl-acid red test used in this study would onlydetectunchangedanthocyaninsScheline, 1978). Iftheanthocyaninsweretransformedintocolourlesspseudobasesorpaleanhydrolasesprior to absorption and excretion, they would not be detected (Kuhnau, 1976).

Clinical studies have reported anthocyaninuria in patients witha beet allergy, following the ingestion of large amounts of beets (Zindler & Colovos, 1950).
　

Tissue disposition of anthocyanosides derived from Vaccinium myrtillus (approximately 25% anthocyanins) was examined in Charles River rats following intraperitoneal (i.p.) or intravenous (i.v.) injection. Following acute administration by either route, anthocyanins were found to distribute rapidly into the tissues.

Accumulation was primarily in the kidney, skin, liver, heart and lung (Lietti & Forni, 1976). There was also some indication of lymph node uptake of the anthocyanins. Elimination of the compound occurred primarily via the kidney (25-29%/24 hours) and bile (15-18%/24 hours). Because of the high urinary excretion rate in these studies, the anthocyanins are considered to be eliminated by both glomerular filtration and renal tubular excretion (Lietti &Forni, 1976).
Evolution:

Anthocyanin-type pigments are found only in terrestrial plants. They are not found in animals, marine plants or in microorganisms. It is theorized that anthocyanin production is an evolutionary response to plants first venturing onto the stark primordial landscape under intense UV radiation. (Significant screening of the earth's surface from the effects of UV radiation didn't occur until after the advent of terrestrial plants. Oxygen in large amounts first had to be generated by the photosynthesis of land plants before the UV-screening ozone layer was formed).

The evolution of insect vision to respond to the unique wavelengths of light presented by flowering plants is an interesting scenario, as is the evolution of these plants to take advantage of the insect's attraction to the sight of anthocyanins. Obviously, the plants came first and developed anthocyanins as a defense mechanism long before the first insect evolved. Flowering plants

subsequently found in anthocyanin a handy way to attract pollinators. Carnivorous plants took advantage of the pollination attraction mechanism to serve as an effective visual lure for their prey.

Chemistry:

Anthocyanin pigments are assembled from two different streams of chemical raw materials in the cell: both starting from the C2 unit acetate (or acetic acid) derived from photosynthesis, one stream involves the shikimic acid pathway to produce the amino acid phenylalanine. The other stream (the acetic acid pathway) produces 3 molecules of malonyl-Coenzyme A, a C3 unit. These streams meet and are coupled together by the enzyme chalcone synthase (CHS), which forms an intermediate chalcone via a polyketide folding mechanism that is commonly found in plants. The chalcone is subsequently isomerized by the enzyme chalcone isomerase (CHI) to the prototype pigment naringenin, which is subsequently oxidized by enzymes like flavonoid hydroxylase and coupled to sugar molecules by enzymes like UDP-O-glucosyl transferase to yield the final anthocyanins. More than five enzymes are thus required to synthesize these pigments, each working in concert. Any even minor disruption in any of the mechanism of these enzymes by either genetic or environmental factors would halt anthocyanin production.
　

Anthocyanins differ from other natural flavonoids in the range of colors that can be derived from them and by their ability to form resonance structures by changes in pH.1 The purported health benefits of red wine are thought to be derived from their phenolic content, principally flavonoids, which have demonstrated powerful antioxidant properties against low density lipoprotein oxidation.2,3 The general antioxidant effect of red wines correlate with their total phenolic content.4,5 Anthocyanins are the main class of flavonoids in red wines, which gives red wine its color and contributes to its powerful antioxidant properties. The 3-glucoside anthocyanins: delphinidin, cyanidin, petunidin and malvidin, are present in red wines, but malvidin 3-glucoside, malvidin 3-glucoside acetate and malvidin 3-glucoside coumarate are the most abundant.6 Anthocyanins exist in an aqueous phase in a mixture of four molecular species. Their relative color depends upon pH. At pH 1-3 the flavylium cation is red colored, at pH 5 the colorless carbinol pseudo base (pb) is generated, and at pH 7-8 the blue purple quinoidal base (qb) is formed. The high levels of anthocyanins in berries contribute to their powerful antioxidant activity.

The positively charged oxygen atom in the anthocyanin molecule makes it a more potent hydrogen- donating antioxidant compared to oligomeric proanthocyanidins (OPCs) and other flavonoids.

Anthocyanins are versatile and plentiful flavonoid pigments found in red/purplish fruits and vegetables, including purple cabbage, beets, blueberries, cherries, raspberries and purple grapes. Within the plant they serve as key antioxidants and pigments contributing to the coloration of flowers. Our online experiments archive includes instructions for using red cabbage juice as a pH indicator, and answers in our archives describe how to perform pigment chromatography.

Red cabbage contains pigments call anthocyanins. The pigments give it the red/purplish color. Anthocyanins belong to group of chemical compounds called flavonoids.

For most pH indicators, the compound acquires a proton at low pH (lots of H+) but looses it at higher pH. This seemingly minor alteration is sufficient to alter the wavelengths of light reflected by the compound, thus creating the color change with respect to pH. Anthocyanins behave somewhat inversely in that the pigments "gain" an -OH at basic pH, but loose it at acidic pHlink below describes the chemistry with structures if to see the details.

The chemistry behind pH, acids and bases.. An acidic solution contains an excess of protons or H+. pH is a measure of how 'acidic' a solution is. The lower the pH, the more acidic the solution. In chemical terms, pH means "the negative log of the concentration of protons" in solution. Chemistry should recognize this as pH = -log[H+]. If the concentration of H+ is .01M, the pH will be:

-log[.01] = -log[10^-2] = -(-2) = 2 (very acidic!).

"Neutral" solutions (water, e.g.) have a pH of 7. This number coicides with the amount of H+ naturally formed in water from the equilibrium reaction: H2O <--> H+ + OH- (H+ experimentally known to be ~10^-7M; OH- is also the same concentration). "Basic" solutions have a pH greater than 7 - meaning they have less free H+ than that of neutral water.

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. They are water soluble flavonoid derivatives, which can be glycosylated and acylated.

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.
　

Anthocyanin structure:

                             Carbon ring B substitution

         Compound               3'              5'

         pelargonidin           -H              -H

         cyanidin               -OH             -H

         delphinidin            -OH             -OH

         peonidin               -OCH3           -H

         petunidin              -OCH3           -OH

         malvidin               -OCH3           -OCH3

The blue to red colour imparted by the anthocyanins depends largely upon the pH of the medium (Francis, 1977). The anthocyanins normally exist as glycosides; the aglycone component alone is extremely unstable.

The anthocyanin pigments present in grape-skin extract consist of diglucosides, monoglucosides, acylated monoglucosides, and acylated diglucosides of peonidin, malvidin, cyanidin, petunidin and delphinidin. The amount of each compound varies depending upon the variety of grape and climatic conditions.












Effect of pH on Anthocyanins:
The colour and stability of an anthocyanin in solution is highly dependent on the pH. They are most stable and most highly coloured at low pH values and gradually lose colour as the pH is increased. At around pH 4 to 5, the anthocyanin is almost colourless. This colour loss is reversible, and the red hue will return upon acidification. This characteristic limits the application of anthocyanins as a food colourant to products that have a low pH.

(Note: The anthocyanins from cranberry fruit were extracted into water, and the pH of the solutions were adjusted from 1 to 13.)
The loss of colour, as pH is increased, can be monitored by measuring the absorption spectrum of the pigment using a spectrophotometer. There is a decrease in the peak at 515 nm as pH is increased, indicating that the red hue is being lost. The loss of red colour with pH implies that there is an equilibrium between two forms of the anthocyanin. These are the red flavylium cation and the colourless carbinol base. The flavylium cation, as the name implies, has a positive charge associated with it, while the carbinol base is a hydrated form of the anthocyanin.










The vast majority of the anthocyanin in solution is accounted for by these two species. But there are actually 2 additional species or forms of anthocyanin in solution - the blue quinoidal base, which is also in a pH dependent equilibrium with the flavylium cation, and the colorless chalcone.

There are very small amounts of the quinoidal base or the chalcone present at any pH. As well, at high pH values, there may be irreversible changes to the structure of the anthocyanin causing permanent loss of the red hue, even at acidic pH values.


Sulfur dioxide & Hydrogen peroxide:
Anything which interupts the conjugated double bond system of the anthocyanin causes a loss of colour. The presence of the positive oxonium ion next to the C-2 position makes anthocyanins particularly susceptible to nucleophilic attack by compound such as sulfur dioxide or hydrogen peroxide.
Anthocyanins form a colourless addition complex with sulfur dioxide by forming a flaven-4-sulfonic acid. This addition can be reversed under low pH conditions (pH 1) to yield the coloured anthocyanin. While this loss of colour is not desirable in many foods, sulfur dioxide is used to decolorize maraschino cherries during their processing.


Sulfur dioxide is commonly used as a food preservative. It is very effective in inhibiting the growth of some yeast and in preventing enzymatic browning reaction catalyzed by polyphenol oxidase. Although still widely used during food processing, the use of sulfur dioxide is restricted, as it may cause serious reactions in sensitve individuals, such as those suffering from asthma. It is illegal to use sulfur dioxide on fresh fruits and vegetables.

Oxidizing agents such as hydrogen peroxide can effectively decolourize anthocyanins, causing ring cleavage at the C-2 and C-3 positions to form a o-benzoyloxyphenyl acetic acid ester under acidic conditions. One possible source of hydrogen peroxide is from the oxidation of ascorbic acid.

Anthocyanins & Metals: Coordination complexes:
Some metals, such as Fe+3 and Al+3 form deeply coloured coordination complexes with anthocyanins that have ortho-dihydroxy groups on the B-ring. The effect of ferrous ammonium sulfate on the colour of anthocyanins extracted from different berries is shown here. Such metalo-anthocyanin complexes have been found to produce discolouration in some canned fruit products, including pears and peaches.


Chromatography:

The process involves equilibria between a molecule being dissolved in a solvent and being adsorbed on a surface. In general polar solvents will tend to dissolve polar molecules better and less polar molecules to a lesser extent. In the presence of a surface, such as alumina or silica gel a polar solvent will more readily dissolve polar molecules and the polar molecules are less likely to be adsorbed on the surface. Hence a moving polar solvent solvent would move a polar molecule more readily than a non polar one. If a non polar solvent were used, the non polar molecules would more readily dissolve and the more polar molecules would be adsorbed on the surface. This is a brief qualitative discription of the physical relationships that give chemists very powerful analytical tools to separate and identify organic molecules.

Molecular Structures of Anthocyanines:
Anthocyanin R R'
cyanidin OH H
delphinidin OH OH
malvidin O-CH3 O-CH3
pelargonidin H H
peonidin O-CH3 H
Petunidin O-CH3 OH

Fig. 1. Cellular uptake of CY, DP and RE on NHF, CaCo-2 and HeLa cells. Cells were incubated with 200 µM anthocyanins or 100 µM RE for 24 h and the cellular uptake was evaluated by HPLC. Three independent experiments were performed. Bars indicate standard deviation of the mean. In the right panel three representative chromatograms are reported (MEM, membrane; CYT, cytoplasm).

Anthocyanins are sensitive to thermal processes, yielding a loss in the desirable hue and an increase in a brown hue as the pigment degrades and polymerizes. Figure 1 shows the effect temperature on colour stability of anthocyanin. A known concentration of anthocyanin was prepared in distilled water and the content was heared for 4 hr at each temperature.

Figure 1. Effect of temperature on colour stability of anthocyanin (100 mg/100ml). o 20oC, • 75oC, p50oC, n 100oC

Metabolism:
Studies in rats have shown that some anthocyanins (notably pelargonidin, delphinidin, malvidin) were subject to degradation by intestinal bacteria (Griffiths & Smith, 1972a, b). p-hydroxyphenyl- lactic acid was detected in the urine of rats following the oral administration of pelargonidin (a 3',3-diglycoside of pelargonidin). Decoloration of "anthocyanin" by rat caecal cell extracts has been reported (Haveland-Smith, 1981). Anthocyanin extracts incubated with human faecal suspensions for 2-3 days remained unchanged (as measured by a reduction in suspension colour).

The presence of 2 unidentified metabolites in the urine of rats after gavage with 100 mg of delphinidin has also been reported (Scheline, 1978). Rats gavaged with malvidin (a 3',5'-diglycoside of malvidin) had 3 unidentified metabolites present in the urine.
These studies suggest that some of the metabolites of anthocyanin(aglycones) can be absorbed. Metabolism of anthocyanins may occur to a limited degree by ring fission and/or glycoside hydrolysis of the anthocyanins (Parkinson & Brown, 1981). Cyanidin, the most widespread anthocyanin, has not been shown to be attacked by intestinal bacteria (Scheline, 1968; Griffiths & Smith, 1972a).

Both pelargonidin and delphinidin have been shown to inhibit aldoreductase in the lens of rats (Varma & Kinoshita, 1976). In other studies, anthocyanin-3-monoglycosides (namely petunidin-, delphinidin- and malvidin-) extracted from grapes were found to increase the activity of alpha glucan phosphorylase and glutamic acid dicarboxylase but inhibit glycerol dehydrogenase, malate dehydrogenase and hexokinase (Carpenter et al., 1967).

Other studies have shown that anthocyanins are capable of chelating ions such as copper (Somaatmadja et al., 1964) and iodide (Moudgal et al., 1958). The iodide ion was observed in vitro to form a stable complex with the anthocyanins (Moudgal et al., 1958).

TOXICOLOGICAL STUDIES:
Special studies on mutagenicity. Cyanidin chloride was not mutagenic when examined in the Ames assay using Salmonella typhimurium strain TA-98 with and without metabolic activation (arochlor 1254 induced rat liver S-9 fraction)
(MacGregor & Jurd, 1978). Structure-activity testing of a large group of flavonols for mutagenic response in this assay system indicated that compounds of flavylium class were inactive.

Cyanidin and delphinidin were inactive in the Ames assay system using 5 different strains of Salmonella typhimurium (TA-1535, TA-100, TA-1537, TA-1538 and TA-98) with and without activation (Brown & Dietrich, 1979).

Anthocyanin was tested in both the Ames test using Salmonella typhimurium TA-1538 for mutagenicity and in another in vitro test employing E. coli Wf2 for induction of DNA damage. In both assay procedures with or without metabolic
activation (using either rat caecal extracts or rat liver microsomes) anthocyanins were not found to induce any response (Haveland-Smith, 1981). Negative findings were also reported for the anthocyanins in a gene conversion assay using S. cerevisiae D4 (Haveland-Smith, 1981).

Special studies on reproduction:

A 2-generation reproduction study was performed in rats
(Sprague-Dawley) ingesting a grape-skin extract preparation that was prepared by spray drying the liquid form of the extract after addition of a carrier material (malto-dextrose). The preparation contained approximately 3% anthocyanins. The test group received dietary levels of 7.5% or 15% of the grape-skin extract throughout the study. There were two concurrent control groups, one receiving the basal diet, the other receiving a diet containing 9% of the malto-dextrin used as a carrier to the grape-skin extract preparation. The F2a generation (10/litter culled at 4 days) were maintained for 21 days post-partum, then autopsied. No differences in reproduction performance or indices including pup viability were apparent between control and dosed groups. At the high-dose level, both the F1a and F2a rats exhibited lower body weights than the concurrent controls. Body weights of the F2 pups in the 7.5% group were marginally depressed. However, it should be noted that the decrease in body weights was accompanied by a concomitant decrease in food intake. At week 6 and at termination of the studies, haematological and blood serum chemistry and urinalyses were carried out in the F1a group. There were no compound-related effects. At week 18 of the study, rats in the F1a group were sacrificed and absolute and relative organ weights determined, and a complete histological study was carried out in the principal organs and tissues. Decrease in organ weights of the liver, adrenal and thyroid occurred in the 15% group. There were no compound- related histological effects (Cox & Babish,1978a).

Anthocyanins link with sugar molecules to form anthocyanins; besides chlorophyll, anthocyanins are probably the most important group of visible plant pigments. Anthocyanins, a flavonoid category, were found in one study to have the strongest antioxidizing power of 150 flavonoids. (Approximately 4,000 different flavonoids have been identified.)

The U.S. Department of Agriculture recently tested the abilities of berry varieties to protect against oxidative damage. In general, blackberries have the highest antioxidant capacity of any fruit. Different varieties of the same species have varying amounts of anthocyanins. The varietal cultivars with the highest antioxidative capacity against superoxide radicals, hydrogen peroxide, and other oxidants are hull, thornless, and jewel raspberries; early black cranberries; and Elliot blueberries.

Anthocyanidins and their derivatives, many found in common foods, protect against a variety of oxidants through a number of mechanisms. For example, red cabbage anthocyanins protect animals against oxidative stress from the toxin paraquat. Cyanidins, found in most fruit sources of anthocyanins, have been found to "function as a potent antioxidant in vivo" in recent Japanese animal studies. In other animal studies, cyanidins protected cell membrane lipids from oxidation by a variety of harmful substances. Additional animal studies confirm that cyanidin is four times more powerful an antioxidant than vitamin E. The anthocyanin pelargonidin protects the amino acid tyrosine from the highly reactive oxidant peroxynitrite. Eggplant contains a derivative of the anthocyanidin delphinidin called nasunin, which interferes with the dangerous hydroxyl radical-generating system—a major source of oxidants in the body.