Cabbage, colours and cleaning products: A citizen science inspired review of anthocyanin extractions that can be attempted at home

Iain A. Smellie*, Iain L. J. Patterson*, Isobel Everest**

*University of St Andrews School of Chemistry, North Haugh, St Andrews, United Kingdom, **Bedford Girls’ School, Bedford, United Kingdom

Background

The travel restrictions and stay at home orders associated with the COVID-19 pandemic in the spring/summer of 2020, led to many educators focusing on practical activities that could be attempted at home. The use of plant-derived pH indicators proved to be a very popular theme¹ since the necessary flowers, fruit and vegetables were readily accessible and a selection of acids and bases could easily be obtained from grocery stores or from online retailers. Many people shared their results on Twitter using #Gardenindicators and the large number of results obtained were compiled and curated online as an open access spreadsheet by Isobel Everest.² This resource has been popular and should continue to prove useful to educators who wish to extract anthocyanins from plants and examine their pH dependent colour changes (or interactions with transition metal ions³). The online spreadsheet is still available free of charge and represents an extension to a lead resource that was originally published in 1985.⁴

Focus on extraction methods

A wide range of plant material has been examined by online contributors on Twitter, the key to a successful outcome has been finding suitable methods for isolating samples containing anthocyanins. The best-known example is arguably extraction from red cabbage,⁵ this simply requires soaking the vegetable tissue in hot water for a short period of time. However, other plant tissues have proved to be harder to work with and other methods were employed by contributors. Here we attempt to summarise representative examples of methods that have been employed in the isolation of anthocyanins from a range of plant sources as part of #Gardenindicators investigations. We hope that this resource will prove to be a useful tool for educators at all levels who wish to conduct pH change (or metal ion detection experiments) using anthocyanins from plants. We don’t aim to provide an exhaustive list of all of the available methods, rather, the objective is to provide readers with a range of different options based on our experiences and to highlight the relevant literature. Example methods and pictures have been provided in an accompanying supporting information document. In most cases, the materials required are available from domestic retailers (grocery stores, hardware stores, online vendors), however some laboratory specific examples are noted too.

anthocyanin_extracts_from_black_beans_.png

Figure 1 – Anthocyanin extracts from black beans using cold water (effects of varied pH shown).

1 – Extractions using cold water

In certain cases, extractions can be very simple, since these do not require the use of hot water or any additional chemicals. A good example is the use of black beans, these must be soaked overnight prior to cooking, and the soak solution is a good source of anthocyanins.⁶ This approach has the advantage that there is no associated food waste and a large quantity of extract is obtained (a 500 g packet of beans is typically soaked in 2-2.5 litres of cold water). A black bean extract where the pH has been varied is shown in figure 1, this example is a good illustration of the equilibrium between the flavylium and carbinol forms of the anthocyanins in solution (equation 1).  The samples at low pH (A and B) show the main species in solution are red flavylium ions. In contrast, the samples shown at pH 5 and pH 6 (D and E) are faded, this observation is due to hydrolysis of flavylium ions to form the corresponding colourless hemiketal.

flavylium_ionhemiketal_adduct_equilibrium_of_malvidin-3-o-glucoside_.png

Equation 1 – Flavylium ion/hemiketal adduct equilibrium of malvidin-3-O-glucoside (black beans also contain delphinidin-3-O-glucoside and petunidin-3-O-glucoside).

Frozen fruit can also be a good source of anthocyanins, the freezing process aids the extraction by damaging cell walls. Blueberries in particular have been found to be very easy to work with,⁷ adding a small volume of water (10-20 mL) to a handful of frozen berries will release some colour into solution, gentle mashing with a spoon gives a rich extract within a minute or two. Although not readily available, if access is possible, liquid nitrogen can also be very effective. We have successfully extracted anthocyanins from poinsettia by immersing the red leaves in liquid nitrogen for 20-30 seconds and then carefully breaking the brittle frozen leaves with a spatula. The resulting powdery material was stirred with 10 mL of water for a few minutes to afford a useable anthocyanin sample. It is also possible to freeze flower petals in a domestic freezer, crush the brittle petals and extract anthocyanins using cold water. An example of this process is outlined in the supporting information document (log into your ChemEd X account to access the Supporting Information).

2 – Extractions using hot water

As discussed above, extraction of red cabbage is probably the best-known method that uses hot water to extract anthocyanins.⁵ Most procedures suggest adding pre-boiled water from a kettle, although heating water in a domestic microwave oven can work well too. Red cabbage has the advantage that it does not usually need much mechanical action on the plant tissue to release anthocyanins from the cells. In addition to red cabbage, we have found that poinsettia, petunias and lobelia all gave useful extracts when the flower petals and water were placed in a suitable container and heated for a minute or 2 in a microwave oven. Once heating was complete, best results were obtained after the petals were mashed with a wooden stick, metal spoon or fork. We have also found that red begonia petals provide good sample extracts if soaked and mashed in hot water (see figure 2). Overall, this option is the best to start with if a plant is being extracted for the first time, however this method is not always successful and other options are described below. Very recently, Tom Kuntzleman⁸ has expanded the range of flower-derived pH indicators by conducting experiments with clover flowers. In this case, a hot aqueous extract from clover was found to change from yellow to colourless on addition of acid. This behaviour of the extract is different to most other flowers investigated, in this instance, the indicator chromophore is believed to be an anthoxanthin species, rather than an anthocyanin.

anthocyanin_extracts_from_begonia_flowers_.png

Figure 2 – Anthocyanin extracts from begonia flowers using hot water (effects of varied pH shown).

3 – Extractions using water and detergent

Rather than using boiling or near boiling water, it is also possible to use tepid water as an alternative. This option may be preferable if younger children are involved in a practical activity to extract anthocyanins. Detergents, such as household dish soap, are used in practical activities that require breaching cell walls of various tissues to extract DNA samples.⁹ We have successfully applied this principle to extractions of plant tissue. For example, we have found that soaking red cabbage, rose petals and campanula petals in a mixture of detergent in warm water (40-50 °C) can be an effective way to obtain useful extracts containing anthocyanins. Very good results were obtained by soaking the plant tissue in cold water/detergent mixture overnight, however satisfactory results were frequently obtained within 2-3 hours. An example extraction result is shown in figure 3, campanula flowers were soaked in warm water/detergent for 30 minutes (the petals in this case were almost completely decolourised).

anthocyanin_extracts_from_campanula_flowers_.png

Figure 3 – Anthocyanin extracts from campanula flowers using cold water and detergent (effects of varied pH shown).

4 – Extractions in aqueous acetic acid

Extractions 1-3 use methods where the pH is ca. 7, it is also possible to extract anthocyanins at lower pH. This approach can be advantageous since the flavylium ion forms that exist at pH <4 are easily identified by eye due to their intense red, pink, or mauve colours. The effect of weak acids on red cabbage is well documented in the context of making “sauerkraut” from red cabbage¹⁰ (in this process, fermentation produces lactic acid which makes the cabbage tissue turn from purple to red). We have found that soaking red cabbage in a 4% aqueous solution of acetic acid (either prepared from glacial acetic acid or using household distilled vinegar)¹¹ provides a strongly coloured extract containing anthocyanins (see figure 4). The pH of the solution can be adjusted by the addition of bases afterward if desired.

effect_of_4_aqueous_acid_on_red_cabbage_tissue_after_a_prolonged_soaking_time.png

Figure 4 – Effect of 4% aqueous acid on red cabbage tissue after a prolonged soaking time.

Using acidic solutions has an advantage in certain cases, since colourless hemiketal forms of anthocyanins can be the dominant species in solution between pH 5 and pH 7 (see equation 1). As a result, a weakly coloured extract may be obtained that does not look like a promising indicator, however, if anthocyanins are present, the strongly coloured flavylium forms will usually be clearly visible when the extract pH is lowered. An example of this situation is shown in figure 5, where the extraction of anthocyanins from azalea flowers is shown in hot water and cold water/detergent mix. In both cases, the pH of the initial extract is ~5, under those conditions the colourless hemiketal forms are dominant. Adjusting the extract pH to <4 results in the solutions becoming red/pink in colour as flavylium ions are formed. A similar outcome is observed when dilute acetic acid is used to extract the flower tissue, the low pH extract is immediately observed to be red in colour.

anthocyanin_extracts_from_azalea_petals_.png

Figure 5 – Anthocyanin extracts from azalea petals using dilute acetic acid and water/detergent (effects of varied pH shown).

Following on from the method of extraction 4, it is possible to use other acids for extractions too (HCl being relatively easy to source). This approach has been highlighted before, although in that instance HCl/alcohol mixtures were employed.¹² We found it was possible to modify the earlier method by using domestic cleaning materials designed to remove limescale. Some of the formulations available commercially¹³ were very useful for extracting anthocyanins from flowers that had proved to be resistant to other methods. In this case the formulation contained HCl (5-10%), sulfamic acid (5-10%) mixed with surfactants, this mixture proved to be particularly effective, especially after soaking flower petals overnight (see figure 6).

anthocyanin_extracts_from_delphinium_petals_.png

Figure 6 – Anthocyanin extracts from delphinium petals using aqueous acid/detergent (effects of varied pH and metal ions shown).

6 – Extractions using organic solvents

A small number of investigations have also included the use of organic solvents. However, these are not favoured due to the fire hazards and disposal issues associated with these materials when they are used outside a laboratory environment. A method that involves soaking chopped red cabbage in propan-2-ol^14,15 (available commercially as “rubbing alcohol”) has been reported and we have found this to work well and give very concentrated extracts. We have also found that mixtures of ethanol and water can be effective,¹⁶ extracts from blue hydrangea flowers were obtained using a 40% aqueous solution of ethanol (see figure 7). A recent publication has also reported that acetone can be used to extract anthocyanins from petunia, rose and lisianthus petals.¹⁷

anthocyanin_extracts_from_hydrangea_sepals_.png

7 – Adjusting the pH of extracts containing anthocyanins

The examples outlined in sections 1-6 include the use of a variety of acids and bases to alter the colour of the anthocyanin mixtures. Some suggestions for solutions to give a range of pHs are shown in Table 1. The solutions listed will give approximately the desired pH when diluted with an equal volume of neutral plant extract. In areas with a domestic water supply containing significant quantities of dissolved calcium and magnesium bicarbonates, it is advisable to use distilled or deionised water. Alternatively, mains water can be used if boiled prior to use. if any carbonates are present after boiling, they can be removed by allowing them to settle out on cooling. The experiments illustrated in the earlier sections have been conducted in Scotland, where the mains water is generally considered “soft” or moderately soft.¹⁸ The supporting information contains information about where to source the materials outlined in table 1 and how to prepare a buffer solution made from potassium bitartrate (“cream of tartar”).

Table 1 - Selected solutions for adjusting the pH of anthocyanin extracts.

selected_solutions_for_adjusting_the_ph_of_anthocyanin_extracts.png

Summary

Since the summer of 2020 we have conducted anthocyanin extractions from a variety of plant sources at home using domestic equipment and chemicals available from retail outlets. From these initial experiments, we have repeatedly tested a selection of different techniques and have summarised these approaches in this article. We hope that these findings will be useful to educators in schools and Universities, for example those who currently use red cabbage to make pH indicator and wish to expand the repertoire. These activities are also well suited to open-ended/discovery practical tasks since the variety of anthocyanin-containing plants is very large and offer a wide range of results to consider. To help guide practical enquiries, the online #GardenIndicators spreadsheet is freely available and contains a large amount of useful information about the expected colours of extracts when pH is varied, or certain metal ions are added.  

References

1. Schultz, M.; Callahan, D. L.; Miltiadous, A. COVID-19 Development and use of kitchen chemistry home practical activities during unanticipated campus closures. J. Chem. Educ., 2020, 97 (9), 2678.
2. “#Gardenindicators Spreadsheet” https://docs.google.com/spreadsheets/d/e/2PACX-1vQWeVCZ0I52kL48z7uzm7fkFDqDpCDFdS4P3ck5pEDXrHlt62dSKDZPed6rb62kHoP8bp496JCjcOLO/pubhtml (Accessed 11th November 2021)
3. Kuntzleman, T. “Aqueous red cabbage extracts: More than just a pH indicator” Chemical Education Xchange (chemedx.org). (Accessed 11th November 2021)
4. Mebane, R. C.; Rybolt, T. R., Edible Acid-Base indicators. J. Chem. Educ., 1985, 62 (4), 285.
5. Fortman, J. J.; Stubbs, K. M. Demonstrations with red cabbage indicator. J. Chem. Educ., 1992, 69 (1), 66.
6. Takeoka, G. R.; Dao, L. T.; Full, G. H.; Wong, R. Y.; Harden, L. A.; Edwards, R. H.; Berrios, J. J.; Characterization of Black Bean (Phaseolus vulgaris L.) Anthocyanins. J. Agric. Food Chem., 1997, 45 (9), 3395.
7. Kuntzleman, T. “Berries are red, berries are blue…I’ve got a berry surprise for you!” Chemical Education Xchange (chemedx.org). (Accessed 21st July 2021).
8. Kuntzleman, T. “Clover Color Chemistry” Chemical Education Xchange (chemedx.org). (Accessed 11th November 2021).
9. Nordell K. J.; Jackelen, A. L.; Condren, S. M.; Lisensky, G. C.; Ellis, A. B. Liver and Onions: DNA Extraction from Animal and Plant Tissues". J. Chem. Educ., 1999, 76 (3), 400A.
10. Linder, J. L.; Aljic, S.; Shroof, H. M.; Di Giusto, Z. B.; Franklin, J. M.; Keaney, S.; Le, C. P.; George, O. K.; Castaneda, A. M.; Fisher, L. S.; Young, V. A.; Kiefer, A. M. Exploring Acid−Base Chemistry by Making and Monitoring Red-Cabbage Sauerkraut: A Fresh Twist on the Classic Cabbage-Indicator Experiment. J. Chem. Educ., 2019, 96 (2), 304.
11. “non-brewed condiment” is available in grocery stores, this is an inexpensive cheap vinegar substitute (suitable for those with wheat allergies) that is a 4.5% solution of food grade acetic acid in water).
12. Markwell, J.; Curtright, R.; Rynearson, J. A. Anthocyanins: Model Compounds for Learning about more than pH. J. Chem. Educ., 1996, 73 (4), 306.
13. “Unilever safety data sheet, Domestos Zero Limescale” https://www.beaucare.com/media/catalog/product/b/r/brado007_domestos_zero_limescale.pdf (Accessed 11th November 2021).
14. Suzuki, C., Making Colourful Patterns on Paper Dyed with Red Cabbage Juice. J. Chem. Educ., 1991, 68 (7), 588.
15. Kajiya, D. Demonstrating purple color development to students by showing the highly visual effects of aluminum ions and pH on aqueous anthocyanin solutions. J. Chem. Educ., 2020, 97 (11), 4084-4090.
16. Garber, K. C. A.; Odendaal, A. Y.; Carlson, E. E. Plant Pigment Identification: A Classroom and Outreach Activity. J. Chem. Educ., 2013, 90 (6), 755.
17. Sampaio, C. I.; Sousa, L. F.; Dias, A. M., Separation of Anthocyaninic and Nonanthocyaninic Flavonoids by Liquid–Liquid Extraction Based on Their Acid–Base Properties: A Green Chemistry Approach. J. Chem. Educ., 2020, 97 (12), 4533.
18. Scottish Water: Water Quality Data, https://www.scottishwater.co.uk/Help-and-Resources/Document-Hub/Key-Publications/Water-Quality (accessed November 11, 2021)  

Acknowledgements – Firstly, we would like to offer our thanks to all the contributors to the #Gardenindicators spreadsheet. The results from this example of citizen science have inspired many of the experiments described in this article. We would also like to thank Mrs G. C. Smellie and Mr B. F. Patterson for providing many of the flowers and gardening materials for this study since July 2020.

Supporting Information - (log into your ChemEd X account to find the supporting information below.) Experimental procedures (and safety information) are provided in addition to photographs from trials conducted with various flowers, fruit, vegetables and beans.