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Title: Authors: Source: Document Type: Subject Terms: Chemotaxonomy: Simple tests for distinguishing between anthocyanins and betacyanins. Nielson, Lynette R. Harley, Suzanne M. Journal of Biological Education; Summer96, Vol. 30 Issue 2, p88, 3p, 3 charts Article *ANTHOCYANINS *CHEMOTAXONOMY *BIOLOGY -- Study & teaching Presents four tests to teach biology students about chemotaxonomy by distinguishing between anthocyanins and betacyanins. Families of the caryophyllales that contain betalains; Sources of anthocyanins and betacyanins; Procedures for conducting the test. 0021-9266 9607105117 Academic Search Premier

Abstract:

Full Text Word Count: 1600 ISSN: Accession Number: Database:

Section: Practical Biology

CHEMOTAXONOMY: SIMPLE TESTS FOR DISTINGUISHING BETWEEN ANTHOCYANINS AND BETACYANINS

Four easily performed tests can be done to differentiate plants on the basis of the presence of anthocyanin or betacyanin pigments

Introduction
The red, purple, and blue colours found in many plants are due to two classes of water soluble pigments, anthocyanins and betacyanins. The anthocyanins are flavonoids, a class of phenolic molecules, and are synthesized via the shikimic acid pathway. They are widespread in the plant kingdom, even occasionally being found in mosses and gymnosperms, as well as the angiosperms (Salisbury and Ross, 1992). Betalains, a group of pigments that includes the betacyanins, are restricted to 10 families of flowering plants in the order Caryophyllales (table 1). Betalains are indole-derived alkaloids and, as such, contain nitrogen (Daniel and Johns, 1982). Traditionally, these pigments have been used in various class exercises and demonstrations to illustrate phenomena such as plasmolysis (Kamrin and LaVan, 1984), membrane integrity (Moore, 1974), and pH (Forster, 1978; Kamrin and LaVan, 1984; Sae, 1990). For us, these pigments have additional interest, as they can be used to introduce the concept of chemotaxonomy. A method to teach students about chemotaxonomy by examining betalains has been published (Daniel and Johns, 1982), but the procedures require equipment that is not always available for student use. Also, this procedure emphasizes differences among the betalains for illustrating taxonomy. We wanted a more introductory level of analysis to acquaint students with the idea that taxonomy need not be based solelyon morphological features. This led us to develop a laboratory exercise, in which students use easily performed tests to determine whether a plant's colour is due to anthocyanin or betacyanin. As betacyanins and anthocyanins do not occur in the same plant (Cronquist, 1981; Salisbury and Ross, 1992) and are so different chemically (Harborne, 1973; Salisbury and Ross, 1992), students should be able to determine

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which of the two is present fairly unambiguously. So that students can draw conclusions on the basis of a variety of data, we selected four tests. These tests are: observing the colour of the pigment under acidic conditions, observing pigment colour under alkaline conditions, measuring pigment migration in thin layer or paper chromatography, and comparing absorption spectra (Harborne, 1973). The number and type of tests to be done can be adjusted to match the available equipment and student sophistication.

Procedures
Plant material Suggested sources of anthocyanins and betacyanins are listed in table 2. These plants are readily available from grocers, florists, or gardens; some, such as lettuce and amaranth, are easily started from seed. Fruits and flowers can be collected when in season and stored in a freezer until needed. Preparation of pigment extracts The plant material is homogenized, using 20 g of plant tissue and 200 cm3 methanolic HCl (1 per cent concentrated [37-38per cent] HCl in methanol), in a blender. The homogenate is then clarified by filtering through a Buchner funnel lined with filter paper. If the filtrate is not deep red in colour, the pigment concentration can be increased by using the filtrate to extract a second 20 g of tissue. Extracts can be stored for a, few days in a refrigerator; a cryoprecipitate sometimes forms which can be removed by filtering. Reactions of the pigments to acidic conditions First, 3 cm3 of extract and 3 cm3 of 4 tool dm-3 HCl are mixed in a test-tube with a capacity of at least 12 cm3. After the colour is noted, the test-tube is placed in a boiling water bath for 5 min. The colour of the anthocyanins will be stable, but the betacyanins will lose colour on boiling. Reactions of the pigments to alkaline conditions To observe the effect of alkaline conditions on the pigments, 2 cm3 of extract and 10 cm3 of water are placed in a 125 cm3 flask. Then, while swirling the flask, 1-2 cm3 of 2 moi dm-3 NaOH is added to the flask drop-wise. Anthocyanins should change to a blue-green colour that eventually fades, while betacyanins should change to a stable yellow colour. Migration in thin layer or paper chromatography For chromatography of the pigments, either silica-coated thin layer plates (on glass or plastic backing) or chromatography paper can be used. Extracts are spotted approximately 2 cm up from the bottom edge using capillary pipettes. We generally cut 20 x 20 cm silica-coated sheets into four 10 x 10 cm plates. Students have enough room on the smaller plates to spot five samples. Pigment concentration can be built up, if necessary, by applying a spot, waiting for it to dry, and then applying more extract at the same mark. A pencil can be used to mark an application line, identify spots, and mark the solvent front. The solvent mixture for the chromatography, BAW, is 1-butanol: glacial acetic acid:water, 4:1:5 (v/v/v). The three liquids are mixed, and the phases are allowed to separate. The upper phase is poured into a chromatography tank or other suitable closed container to a depth of about 1.5 cm. The chromatography plate or paper with the dry sample spots is then placed into the tank, being careful not to immerse the sample spots in the BAW. The chromatograph is developed until the solvent has moved approximately 7 cm beyond the sample application line. The solvent front is marked, and the chromatograms are allowed to dry before determining the Rf of each pigment. Anthocyanins migrate in the lower third of the chromatograph, but betacyanins barely, if at all, leave the point of application.

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Absorption spectra This portion of the analysis requires the use of a visible light spectrophotometer, such as a Spectronic 20. To keep the absorbance readings on scale, each extract should have an absorbance reading at 530 nm that is 0.7 or less. Extracts can be diluted with methanolic HCl, if necessary. Absorbance readings are taken at 10 nm intervals from 480-580 nm, to determine the range where an extract shows 95-100 per cent maximal absorbance. Anthocyanins show maximal absorbance between 505-535 nm, and betacyanins have maximal absorbance between 530-555 nm.

Discussion
Students are provided with pigment standards (we generally use cranberry extract for anthocyanin and beet root extract for betacyanin), so that they can see 'typical' results for each of the tests. A summary of test results is given in table 3. Differences between anthocyanins and betacyanins should be fairly clear, although the presence of chlorophyll in leaf extracts can make colour changes with pH difficult to see. Since anthocyanins and betacyanins give such distinct results in the various tests, students should be able to decide which of the two pigment classes is present in a particular plant. In order to enhance the investigative nature of laboratory work, we do not tell students which type of pigment is present until conclusions have been reported. The number and type of tests performed can be adjusted to the level of the students. We have had college students, working in pairs, prepare extracts and perform all four tests within a 3-h laboratory period. The first three tests were completed by 12-14-year-olds within a 75-min extended class period, when extracts were prepared in advance. The first two tests require minimal equipment. The volumes of reagents are approximate, and we have found that disposable '1 cm3, droppers and 10 cm3 graduated cylinders are suitable for most of the measurements. The third test provides a practical application of thin layer or paper chromatography, while the fourth test does the same with spectrophotometry.

Table I Families of the Caryophyllales that contain betalains. Two additional families, the Caryophyllaceae and the Molluginaceae, contain anthocyanins instead of betalains (Cronquist, 1981)
Family Achatocarpaceae Aizoaceae Amaranthaceae Bascellaceae Cactaceae Chenopodiaceae Didiereaceae Nyctaginaceae Phytolaccaceae Portulacaceae Representative genera Achatocarpus, Paulothamnus Lampranthus, Lithops (stone plant), Mesembryanthemum (ice plant) Amaranthus, Celosia, Gomphrena Anredera, Basella Opuntia, Schlumbergera Beta, Chenopodium (goosefoot, pigweed), Spinacia Didierea Mirabills (four o'clock), Bougainvillea Phytolacca (pokeweed), Rivina Calandrinia, Portulaca

Table 2 Sources of anthocyanins and betacyanins

Anthocyanins African violet--flowers Coleus--leaves Cranberry--fruits Betacyanins Amaranth (love-lies-bleeding)--leaves Beet--root Bougainvillea--flowers

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Geranium--flowers Plum--fruits Raspberry--fruits Begonia--red leaves Red cabbage--leaves Red lettuce--leaves Strawberry--fruits

Christmas cactus (Schlurnbergera)--flowers Cockscomb (Celosia)--flowers Purslane (Portulaca)--flowers

Table 3 Responses to tests for distinguishing anthocyanin and betacyanin pigments (adapted from Harborne, 1973)
Legend for Chart: A B C D E A Pigment Test, Heat with HCl Test, Add NaOH Test, Chromatography in BAW Test, Absorption maximum B D Anthocyanin colour stable low Rf (0.1-0.4) Betacyanin colour disappears very low Rf (0.0-0.1) C E colour changes to blue-green; fades 505-535 nm colour changes to yellow; stable 530-555 nm

References
Cronquist, A. (1981) An integrated system of classification of flowering plants. New York, USA: Columbia University Press. Daniel, T.F. and Johns, T. (1982) A laboratory on chemotaxonomy: the systematic distribution of betalains. The American Biology Teacher, 44(5), 308-310. Forster, M. (1978) Plant pigments as acid-base indicators--an exercise for junior high school. Journal of Chemical Education, 55(2), 107-108. Harborne, J.B. (1973) Phytochemical methods. London: Chapman and Hall. Kamrin, A.A. and LaVan, J.S. (1984) Demonstrating osmosis and anthocyanins using purple onion. The American Biology Teacher, 46(2), 116-117. Moore, T.C. (1974) Research experiences in plant physiology, a laboratory manual. New York, USA: Springer-Verlag. Sae, A. (1990) Of cabbages and anthocyanins. The Science Teacher, 55(7), 16-18.

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Salisbury, F.B. and Ross, C.W. (1992) Plant physiology. 4th edn. Belmont, CA, USA: Wadsworth Publishing Company. ~~~~~~~~ By Lynette R. Nielson and Suzanne M. Harley At the time of writing, Mrs Lynette R. Nielson was an undergraduate (majoring in biology teaching) at Weber State University. She now teaches at North Davis Junior High School, Clearfield, UT. Suzanne M. Harley is Associate Professor of Botany, Weber State University, Ogden, UT 84408-2504, USA. E-mail: sharley@weber.edu. (Please address any correspondence to Professor Harley.) Copyright of Journal of Biological Education is the property of Institute of Biology and its content may not be copied or e-mailed to multiple sites or posted to a listserv without the copyright holder`s express written permission. However, users may print, download, or e-mail articles for individual use. Source: Journal of Biological Education, Summer96, Vol. 30 Issue 2, p88, 3p Item: 9607105117

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