Projection Experiment: Anthocyanins as pH-Indicators and Complexing Agents

Projection Experiment - Chemistry en miniature

Anthocyanins as pH-Indicators and Complexing Agents

Peter Keusch

German version

Chemicals:

96 % ethanol

1 N HCl

aluminium chloride

0.5 % aqueous solution of ammonia

Hazards and safety precautions:

Ethanol is highly flammable.

Safety glasses, gloves and good ventilation required.

Experimental procedure:

10 mL of ethanol are used to extract the dye from the petals of two red roses. 1 mL of the dye extract is pipetted into each of four test tubes. In addition, one drop of HCl is added to the solutions in T2, T3 und T4. 5 drops of NH₃ solution are added to the solution in T3. A Pasteur pipette contains some crystals of aluminum chloride in its tip. The pipette is placed in T4.

Results:

Test tube 1	dye extract	pale-red
Test tube 2	dye extract, HCl	red
Test tube 3	dye extract, HCl, NH₃	blue
Test tube 4	dye extract, HCl, AlCl₃	violet

Photo 1

Discussion:

· Anthocyanin is the common name of a flavonoid pigment that possesses a hydroxylated 2-phenylbenzopyrilium chromophore. Anthocyanins are localized in the cell vacuole and produce blue, purple or red colors of many flower blooms and fruits. They have a sugar moiety, most often attached to the 3 position or both the 3 and 5 positions together (1).

· The anthocyanins are broken down by acids into sugars and the corresponding dye components (anthocyanidins). By acid hydrolysis flavylium salts (oxonium salts) are formed, whose cations are resonance-stabilized (2).

· The three anthocyanidins found in roses are cyanidin, peonidin and pelargonidin.

· The color of anthocyanidins is pH dependent. The anthocyanidin system undergoes a variety of molecular transformations as the pH changes. In aqueous solutions, anthocyanidins exist as essentially five molecular species in chemical equilibrium: red flavylium cation, colorless carbinol pseudo base, purple quinoidal base, blue quinoidal base anion, and yellowish chalcone.

Structures of cyanidin in aqueous solution under varying pH

Degradation of cyanidin in strong alkaline medium

The color of anthocyanins depends on the acidity of the medium. At acidic pH = 1-3, anthocyanidins exist predominantly in the form of the red flavylium cation (2-phenylchromenylium cation). Increasing the pH leads to a decrease in the color intensity and the concentration of the flavylium cation which undergoes hydration to produce the colorless carbinol pseudobase (hemiacetal or chromenol). The conjugated 2-benzopyrilium system is disrupted due to a nucleophilic attack of water at the 2-position of the anthocyanidin skeleton. A rapid proton loss of the flavylium cation takes place as the pH shifts higher. Now the equilibrium is shifted toward a purple quinoidal anhydrobase at pH < 7 and a deep blue ionized anhydrobase at pH < 8 (4). When pH increases further the carbinol form yields, through opening of the central pyran ring, the light yellow chalcone. The color of the alkaline solutions can be reverted by changing the pH back to acidic. The anthocyanidin equilibrium forms shift back to the equilibrium where the red colored flavylium cation predominates. However, if the pH value is too high and unstable ionic chalcones have already formed, recovery of the flavylium form can not be achieved by simple re-acidification. Chalcone is converted to an a-diketone via keto-enol tautomerism. Subsequent breakdown of the chalcone leads to carboxylic acid (i.e. substituted benzoic acid) originating from the B-ring and hxdroxyaldehyde (i.e. 2,4,6-trihydroxybenzaldehyde) from the A-ring of the parent anthocyanin (5).

· Al ³⁺ forms a violet complex with the red flavylium cation.

Why is the cornflower blue and rose red when both flowers contain the same anthocyanin?"
In 1913 Richard Willstätter, a german chemist who had won the Nobel Prize in 1915, made the striking observation that the same pigment can give rise to different colors. Thus, the same pigment, cyanin, is found in the blue cornflower and in the red rose. Willstätter attributed the variety of flower colors to different pH values in cell fluid. Indeed, anthocyanin changes its color with pH: it appears red in acidic, violet in neutral, and blue in basic aqueous solution (4), (5). Shibata, a japanese botanist, has examined pH of a variety of flowers and found that the flower cell fluid is usually weakly acidic or neutral. Studies have shown, that the vacular pH of rose is generally pH 4.5 - 5.5. The vacuolar pH in the cells of the cornflower bloom is 4.6. Evidently, the blue color of the cornflower can not be explained by Willstätter's pH theory.

The plant cell vacuole is maintained at pH range of 4 to 6, at which the predominant portion of the total anthocyanin content would be expected to exist in the colourless carbinol pseudobase form and a smaller portion in the quinoidal form. However, anthocyanins normally exhibit red colouration in situ and the occurence of natural colourless pseudobase is extremely rare. The reason for this is that the color variation and stabilization of anthocyanins in aqueous solution is in the first place a result of self-association, co-pigmentation with polyphenols such as flavones and flavonols, respectively, and 'intramolecular sandwich-type stacking' (Goto amd Kondo, 1991; Brouillard and Dangles, 1994). In addition, metal complexation seems to be chiefly responsible for blue flower coloration. Phacelianin from the blue petals of Phacelia campanularia may take both an inter- and intramolecular stacking form and shows the blue petal color by molecular association and the co-existence of a small amount of trivalent metal ions (Al³⁺ or Fe³⁺). Nevertheless Willstätter's pH-theory for explaining flower color variation is still to be found in major text books of organic chemistry.

Meanwhile, the 'mystery' about the blue cornflower seems to be solved. Kosaku Takeda and colleagues has found that the molecular structure of the cornflower pigment (protocyanin complex) comes from the arrangement of four metal ions (one ferric iron, one magnesium and two calcium ions) which bind a complex of six molecules each of a succinyl anthcyanin and a malonylflavone (copigment). "This tetrametal complex may represent a previously undiscovered type of supermolecular pigment," says Takeda (Picture 3).

Picture 2: Cornflower
(Centaurea cyanus)

Picture 3: Stereo image of the cornflower protocyanin complex (seen from above)
Ca (black) is complexed by flavone glycosides, Fe (pink) and Mg (green) are complexed by anthocyanines (blue)

"Heavenly Blue" - an exceptional case
The change in color of Ipomoea tricolor is an exceptional case supporting the pH theory. The 'Morning Glory' or 'Heavenly Blue' changes its color from purplish red in the bud to blue in the fully open flower (Picture 4). One and the same pigment, a triacylated anthocyanin, is responsible for both colors. By direct measurement of the vacuolar pH using a proton sensitive microelectrode, was demonstrated that the change of petal color is accompanied by an unusual increase of the vacuolar pH in the petal epithelium from 6.6 to 7.7.

Picture 4: 'Heavenly Blue' - petals in the bud (A) and in the fully open flower (B)

The increase of vacuolar pH in the petals during flower-opening is due to an active transport of Na⁺ and / or K⁺ from the cytosol into the epidermal vacuoles through a sodium- or potassium-driven Na⁺(K⁺) / H⁺ exchanger. This systematic ion transport maintains the weakly alkaline vacuolar pH, producing the sky-blue petals. Over-alkalization is prevented by enzymes. The blue anhydrobase anion of HBA (Heavenly Blue Anthocyanin) must be stabilized by 'stacking'.

In general, stabilization of anthocyanins is caused namely by self-association, copigmentation and intramolecular 'stacking'. There are two types of copigmentations which both stabilize anthocyanins by a similar mechanism. Intermolecular copigmentation happens when anthocyanin weakly binds to compounds such as flavonoids, phenolic acids and alkaloids. Intramolecular copigmentation happens when the aromatic acyl group in acylated anthocyanin interacts with the anthocyanidin of the anthocyanin, linked by the sugar component. The two types of copigmentation allow formation of 'stacked complexes' by hydrophobic anf charge-transfer interaction and the occurence of hydrogen bonds and Van der Waals forces. Copigmentation enhance color intensity (hyperchromic effect) and the shifting to longer visible maximum wavelength (bathochromic effect, 'blueing' effect).

The 'heavenly blue anthocyanin'(HBA) is composed of peonidin with six molecules of glucose and three molecules of caffeic acid. Each caffeate is esterified with one glucose residue and forms a glycosidic bond with another glucose residue (6). The folded structure of HBA yields a so-called 'sandwich stacking'. Peonidin and acyl residue are arranged parallel to each other. This means that the planar aromatic residue of the acyl group can fold over the planar anthocyanin chromophore, interacting with its p-system. The glucosyl residues are considered as spacers, which enable the folding of acyl group on the aglycone to take place, thus protecting it against the nucleophilic attack of water and subsequent formation of a carbinol anthocyanidin nucleus. HBA is stabilized by an intramolecular 'stacking' of the three caffeoyl residues to the chromophore.
Polyacylated anthocyanins such as HBA have shown unusual stability during pH changes. Seven of twelve structurally related anthocyanins, being investigated at the Université Louis Pasteur (Strasbourg, France), possess chemical structures that allow a folding of the 'side-arms' constituted by the acyl-glycosyl chains attached to positions 3’ and / or 7 of the aglycone. The stacking of one or even two of the aromatic residues of these 'arms' on the central anthocyanidin nucleus (Picture 5), very effectively precludes hydration.

"But suddenly some new blue seemingly is seen ...."
· Some metals, such as Fe ³⁺ and Al ³⁺ form stable deeply colored coordination complexes with anthocyanins that bear ortho-dihydroxyphenyl structure on the B-ring (7) . The experiment above shows that Al ³⁺ is complexed by anthocyanidins of the rose to form a purple color. So far in the petals of roses no metal complexes were found.

Aluminium complex of cyanidin-3-glucoside

Rainer Maria Rilke
Blue Hydrangea
Just like the last green in a colour pot
So are these leaves, withered and wrecked
Behind the flower umbels, which reflect
A hue of blue only, more they do not.
Reflections are tear-stained, inaccurate,
As if they were about to cease,
And like old blue notepaper sheets
They wear some yellow, grey and violet,
Washed-out like on a children’s apron,
Outworn and now no more in use:
We contemplate a small life’s short duration.
But suddenly some new blue seemingly is seen In just one umbel, and we muse Over a moving blue delighting in the green.

translated by Guntram Deichsel

The blue colored pigment-copigment-metal complex of Hortensia sepals (Hydrangea macrophylla) is well known. However, Hydrangea flower color is varied from red, through purple to blue. All colors are caused from the same pigment, delphinidin 3-glucoside (1) and the same co-pigments, 5-O-caffeoylquinic acid, 5-O-p-coumaroylquinic acid, and 3-O-caffeoylquinic acid, with Al³⁺ ion. The vacuolar pH in the cells of blue sepals is 4.1 and that in the cells of red sepals is 3.3.

Picture 5: Hortensia (Hydrangea macrophylla)

It is commonly known to most gardeners that two factors affect the color in Hydrangea macrophylla cultivars: soil acidity and the presence of aluminium in the soil. Acidic soil increases the availabiliy of Al³⁺ in the soil and leads to a change of flower colour from pink to blue. In alkaline soil (lime) aluminium salt is tied up. The plants are unable to absorb the existing aluminium and the flowers will not bloom blue. Also, when aluminium in the soil is used up the flower color will be red or pink again. If the soil is watered with aluminium salts aluminium will be accumulated in the petals and the color turns blue. Only red and pink colored Hortensia are suitable for the "breeding" of blue flowers. They contain the dye component delphinidin.

References:
Les anthocyanes
Copigmentation reactions and color stability of berry anthocyanins
Anthocyanin-aluminium and -gallium complexes in aqeous solutions
Blue flower color development by anthocyanins: from chemical structure to cell physiology
Study on synthesis of 5-O-acylquinic acid analogs involved in blue color development of hydrangea

General experimental instructions and index of experiments