Fruit Anthocyanins Colorful Sensors of Molecular Milieu Robert D. Curtrlght, James A. Rynearson, and John ark well' Center for Biological Chemistry, University of Nebraska-Lincoln. Lincoln, NE 68583
One of the most obvious characteristics of the biosphere is the almost overwhelming concentration of color in all but the most arid regions. The cool greens produced when blue and red photons are absorbed by chlorophyll and carotenoid molecules dominate in temoerate and tro~icalregions. Against this verdant backdroi, nature has ielected contrastine colors to hiehlieht fruits and flowers. Some of these colors, the rich r 2 oFtomatoes or the pure yellow of the daffodil flower. are oroduced bv carotenoids. An additional group of natural pigments, the anthocyanins, are resoonsible for manv of the red and blue colors observed in flowers such as the peony or codower, in berries, and in the leaves, stems (rhubarb) and roots (radishes) of many plants. The anthocyanins are a subclass of the flavonoids (131,that also includes pigments responsible for the yellow (saillower)and white colors of some flowers. Except for the use of fruit and red cabbage extracts as pH indicators (4.51, the chemistr, of the anthowanins is not usually explored in secondaj or ~ n d e r ~ ~ a d uchemistry ate courses. This class of molecules has the potential to beof interest to students and teachers alike, to be useful in illustrating chemical principles such as acid-base equilibria, pK., light absorption, effects of changes in conjugated double bonds, etc., and we have prepared this article to provide background and references as a starting point for the further exploration of this class of compounds. Most naturally occurring anthocyanins (Greek: anthos, flower; kyanos, blue) occur as glycosides of one of several aglycone cores (Fig. 1).Substitution of the third ring of the aglycone with -OH or - 0 M e groups a t the 3'- or 5'-positions (Rand K, respectively in Figure 1)produces a range of changes in the electron distribution over the molecules that result in a shift from the oranee wlor of ela are on id in (strawberries) to the deep blue color of malvikn (biueberries). Anthocvanins usuallv contain a sinele elvcoside unit -" but many anthocyanins contain two, three, or more sugars attached at multiple positions, or occurring as oligosaccharide side chains. Adiscussion of the carbohydrate moieties of the anthocyanins is beyond the scope of this article. The anthocyanins are naturally occurring pH indicators, and this property has been the subject of previous articles (6,7)related to chemical education. It is through the work of Bmuillard ( 8 . 9 , that our current understanding of the pH-induced color changes of the anthocyanins is based. In Homewhat simplified terms, the anthocyanin exists in dilute acid solutions as a positively charged oxonium ion termed a flavylium cation (Fig. 1).This results in an extended conjugation of double bonds through the three rings of the anthocyanin, the absorption of photons in the visible region of the spectrum (wavelength of maximum absorbance between 480 and 550 nm) and a moderately strong molar absorptivity (in the range of 25,000 to 50,000 M-' em-'). The colored flavylium cation is in equilibrium with a colorless pseudo-base form, which begins to predominate as the pH of the solution is raised to values
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' Corresponding author. 682
Journal of Chemical Education
Agylwne cyanidin peanidin delphinidin petunidin maividin pelargonidin
R
R'
OH OCH3 OH OCH3 OCH3
OH OH OCH3
H
H
H H
Figure 1. Chemical structures of the two forms of anthocyanin aglywne wres responsible for the primary wlor change between pH 1 and 5 greater than three. The addition of the water molecule in the formation of the ~seudo-basefrom the cation form of the molecule disrupt; the conjugation of double bonds between the second and third rines ., and results in the absomtion of photons in the ultraviolet region rather than in the visible. This DH-based eauilibrium omvides a dramatic demonstratio; that increasing the number of wnjugated double bonds in a molecule lowers the energy level of the electronic transition between the ground state and the excited states, causing the absorption of photons of greater wavelength. In addition to serving as potential models for classroom and independent experiments on acid-base chemistry and the nature of conjugated double bonds, the dramatic color changes involvedin these systems appear to increase student interest. It is possible to obtain commercial sources of anthocyanins suitable for experiments (e.g., malvidin-3.5digluwside can be purchased from Sigma Chemical Co.), but we decided to explore whether similar results could be obtained usinglow-costmaterials purchased from the local market. The advantages of fruit as a starting material is that thev usuallv contain few other comooundsthat absorb in the same spectral regions as the anthocyanins. The major disadvantage is that whereas most fruits contain only a limited number of aglycone types, they usually contain a variety of glycoside derivatives, so that students actually will be studying a mixture of related compounds rather than a pure compound. Cranberry juice is a source of the monoglycosides of peonidin and cyanidin that gave good results in the types of experiments that could be carried out in high school or undergraduate laboratories. This product is available
readily on a national basis and generally is available from only one supplier (Ocean Spray), resulting in a uniform product. When the pH of cranberry juice is adjusted from 5 to 1, the increase in the absorbance of a peak at 515 nm (Fig. 1)results in a dramatic change from pale ta bright red. Atypical experiment with cranbeny juice is shown in Figure 2. The juice from a newly opened bottle typically has a pH of 2.6 to 2.8. A dilution of the juice with water to
Proportion At=
[Ail - 10(~K.-~M [AT + [AOHI- 1 + lo(PK.-pH)
Theor.Abs. = (hH1 - %H5) x
lo[~K.-~H.) l +10(@.-pH.)
(3) (4)
Apnl andA n5 are the absorbance values at pH 1and pH 5, respective&, and pHx is the experimentally determined pH. If the value for is kept in separate cell and &ked as an 2.0 absolute reference while the experimental and theoretical absorbance~are plotted versus pH, it becomes possible for students 1.5 ta fit a theoretical curve to the experimental data points by empirical manipulation. We routinely find a mid-point (roughly equal to the pKJ for the absor1.0 bance change of approximately 3.1. Quantitation of the amount of anthocyanins in the cranberry juice is possible using the extinc0.5 tion coefficients developed by Fuleki and Francis for aqueous solutions of cranberry juice at 515 n m (10).These values were 0.0 developed for use on solutions 350 400 450 500 550 600 adjusted to pH 1and pH 4.5, but we find that pH values of 1and 5 Wavelength Inn) give similar results. For the above experiment with juice d;Figure 2. Absorption spectra of a 50% solLtlon ofOcean Spray Cranberry Juice at pH values of 1.4.2.6. luted to 50% of the original con. 3.8. and 5.0. Increasing the pH decreases the aosorbance ar 51 5 nm centration the total amount of anthocyanins (gI100 mL of bottled juice) can be calculated as 50% of initial concentration and adjustment to pH 1pro(Apm -Apn~)/0.387.Students could then calculate the total duces a final absorbance at 515 nm of a ~ ~ m x i m a t e1.2. lv amount of anthocyanins per bottle. An additional experiExperiments have been carried out in w6Lh 100 mL of the ment would be to let students a t t e m ~ to t estimate ---- ~~~-the ~ - - diluted iuice is first adiusted to DH 1with concentrated amount of cranbeny juice in a sample of cranapple juice (a HCI and then the pH is"increasedhowly by the addition of mixture of cranbem and a ~ ~iuices). l e in which the antho--~--4 M NaOH dropwise or in which one aliquot of diluted juice cyanin contribution"from aiple:uice is negligible. is increased in pH with NaOH while an identical aliquot is Other ex~erimentsthat mav be a ~ ~ r o ~ r ionce a t e stuincrementally adiusted to DH 1 with HCI. At various DH dents are ikerested in the craiberry juice is to use chemiintervals, aliquois are removed from the solution and {he cal reactions to disrupt the conjugated double bond system absorbance at 515 nm determined with a suectro~hotomein the anthocyanins. Addition of solid sodium sulfite or ter. Similar results were obtained w h e n b ~was deterdrovwise addition of 3% H701will cause a bleaching of the mined using either a research-grade pH meter or a handanthocyanins due to, resiec%vely, chemical addicons or held pH sensor of the type commonly found in high schools. The response of the anthocyanins in solutions should obey the Henderson-Hasselbalch Equation (eq 1).This equation is transformed readily to give the ratio of flavylium cation (At)to oseudo-base (AOH)(en 2) or the ~ r o ~ o r t i oofn 3) as a the total an&ocyanin in the flavdium for& (iq function of pH. Because the absorption at 515 nm is due ta the flavylium cation form and the pseudo-base is colorless at that wavelength, the absorbance at any pH relative to the absorbance at pH 1is proportional to the fraction of anthocyanin molecule in the flavylium form. The raw pH and absorbance data were entered into a microcomputer spreadsheet, corrected for the residual absorbance at DH5 &d compa&d to a theoretical plot generated (eq 4) fo; the same pH values.
a
~~
~~~
~
~
~~
~
.. .
Figure 3. Response of absorbanceat 515 nm to pH for a 50%dilution of Ocean Spray Cranberry Juice. Volume 71 Number 8 August 1994
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oxidation (11).Students also may undertake a quantitative titration of cranberry juice, measuring pH versus eauivalents of acid or base added. rather than change in absorbance. Cranberry juice contains four predominant oraanic acids (12!-citric. malic, auinic. and benzoic-and they provide a strong buffering of-the juice as known equivalents of either acid or base are added and the effect on pH is observed. Another anthocvanin source that we initially studied t s the anthocyan& of the was the grape. ~ i ~ e r i m e non European grape (Vitis uinifera) found in red wine (Gallo Classic Burgundy) produced a near theoretical response of 515 nm absorbance versus pH, with a midpoint for the absorbance change a t a pH of approximately 2.9. These grapes are reported to contain primarily monoglycosides of neonidin. cvanidin. malvidin. ~etunidin.and del~hinidin ill). To prokde a source mor~&ceptableinseconiary and undermaduate education. we examined non-fermented juice gf concord grapes (k labrusca) in which the glycosidesof malvidin and delphinidin are dominant (11).The absorbance change with concord grape juice as the pH was adjusted between 1and 5 was not as well defined as for the wine sample and did not fit well to a computer-generated theoretical curve using- the Henderson-Hasselbalch eauatinn. . . . . . .
In addition to cranberry juice, we explored other sources of materials from the market, some of which have the advantage of containing anthocyanins with only one predominant aglycone core (Table 2). All of these sources were found to be easy to work with and provided good results. The samples of-strawberries and bheberry skins and red onion skins weregroundin a mortar and pestle with water prior to filtering. Filtration wascarried out with Whatman No. 1 paper. Raspbemy contains primarily cyanidin anthocyanins and twosou&s of juiceproduced similar absorption spectra and pH values for the midpoint of absorbance change. Mature blueberries contain primarily malvidin, but also significant amounts of delphinidin cyanidin, ~etunidin.and peonidin anthowanins (13).and the two sources had absorption peaks &th similar wavelengths and the pH values for the midpoint of absorbance change were the lowest of the examined sources. Strawberry contains primarily p e l a r m ~ d i nanthowanins and had an absorptibn peak with the lowest wavelength of the tested sources. The skin of the red onion contains primarily cyanadin anthocyanins. We hope that the above discussion and experiments will stimulate increased interest in the colorful anthocyanins as a source of research and experimentation in high school and undergraduate laboratories. We have used our experiments with anthocyanins to develop a number of specific learning experiences at both levels. For example, if a class grinds a leafin a small amount of methanol with a mortar and pestle, the chlorophyll and carotenoid pigments are released into the green solution. If the extract is diluted with an eaual volume of cranbem iuice and filtered throueh whatman No. 1paper, a solukon neither green nor red-is oroduced. Place 1mL of the mixture into each of four small test tubes. To each tube then add, in a chemical fume hood, an equal volume of water, methanol, isoamyl alcohol or chloroform. After mixing, the latter two tubes will partition into a green (organic) phase and a red (aaueous) phase, demo&trating&fferekial partitioning into;mmiscible solvents. The meen isoamyl alcohol phase will be above the aqueous fhase; where&, the green chloroform phase will be on the bottom, demonstrating the effect of density. Finally, if one drop of dilute acid or base is added
684
Journal of Chemical Education
Table 2. Characteristics of Anthocyanins from Various Sources
Source
MajoraAnthocyanins Present (3)
haXb (nm) Midpoint pH
Cranberryc Pn- and Cy-3-glywsides
515
3.1
~aspberty~~y-3-g~ywside
517
2.9
Red onion'
519
2.9
Cy-3-glymsides
aAbbreviations:Cy, cyanadin; Mv, malvidin; Pg, plargonidin; Pn, Peonidin. b p 1~in aqueous solution. 'Ocean Spray Cranberly Juice CocMail, diluted to 50% original concentration. %ole Pure 8 LioM Countw Ras~benvConcernrate. diluted to 25% oriainal concentration. wSrnucker'sRaspberry Syrup, diluted to 20% original concentration and TI-
.
,
.
-
tcmd
'~ilteredaqueous enran of blueberry skins. gSrnucker's Bluebeny Syrup, diluted to 20% original mncentrafion and filtered. "F tered aqdeOUS enran ol strawoernes. F ltered aqueoLs enran ol red on on skns.
to each of the tubes, different color changes will occur in the aqueous phase, but not in the organic phases. Thus, a number of different chemical principles are tied together in a simple and colorful experiment. We encourage anyone with an interest in obtaining our experiments, in sharing their own protocols, or in discussing the development of additional experiments, to contact us. Acknowledgment We are most g r a t e l l to F. J. Francis of the University of Massachusetts for his help and assistance. We gratefully acknowledge the help of Mark Bevins in obtaining spectra of the anthoevanins. This research was s u ~ w r t e dbv the University 2 Nebraska, Lincoln ~enter'for~iological Chemistrv. Center for Curriculum and Instruction Summer ~ a z Fellowship t ~ Program. This is manuscript No. 92-4 of the College of A~riculturalSciences and Katural Resources, university ofkebraska. Literature Cited
4. Summerlir., L C k m i s h y #Common S u b n c ~ s8ih.a-Burdett: : Monistown, NJ, 1979: oo 97-98 5. Bor&ford. C.; Summerlin. L in C k m b l Aeliuitiu; American Chmieel Society:
...
~~
Wsahingtrm, DC, 1988; pp 9244. 8Mebane. R. C.; RyblZ T R.J. Chem. Educ. I N , 62,285. 7. Fmster, M. J. C k m . Edue 1978.55.107-108. 8. Bmuillard, R. in A n t h a p n i m a.Fmd Colors; Markab, P Acedmic Press: New York, 1982, pp 1-40. 9. Bmuillerd, R.PhytoehPm 1885,2e, 1311-1323. 10. N e b , T.;Frwck.F.J. J. F a d Sei 1868.33.78-83. P in Chemistn of Wiwmokiw; Gould, R. F.; Am. Chem. Sac.: 11. Rib&reau4ay~n, Washington, DC,1974; pp 6O-87. 12. Ran&. E J. i n E d c m l i o n o f W i O o f h i t s a n d l & l a b l e d ; Pettes,H. E.:AM Publishing Co.:Westport, CT, 198s; pp 19%218. 13. Ballingtan, J.R.; Ballinger, W. E.; Manem, E. P J. Am. Soe Bar(. Sci. lsB7,112, 859-864.
