Recently as I was mowing my lawn, I noticed that some of the florets on white clover have a slight pink or purple color (Figure 1). Because anthocyanins often impart red, blue, and purple color to plants, fruits, and flowers,^1,2 I wondered if these tiny pink florets in white clover contained anthocyanins. Of course, anthocyanins are routinely used as acid-base indicators in science classes, and red cabbage is a widely used source of anthocyanins. I reasoned that if white clover contained anthocyanins, these common flowers could provide yet another easily obtained source of an anthocyanin-based acid-base indicator.  

Figure 1: White clover. Slight pink and purple color can be observed in the florets.

I set out to extract anthocyanins from white clover by first collecting about 30 white clover samples in a bowl. Next, I added a quarter cup of tap water, and heated the flowers in a microwave for one minute. When I removed the bowl from the microwave and poured off the water, I saw no indication of pink or purple color – no anthocyanins. Instead, I was quite surprised to see that the water had taken on a deep yellow color (Figure 2, Video 1).

Figure 2: Boiling white clover in water yields a yellow-colored mixture.

On a whim I decided to add some vinegar to the yellow-colored mixture. Upon doing so the yellow color disappeared. Adding baking soda to the colorless mixture caused the yellow color to reappear (Video 1). I found that I could cause the yellow color to appear upon addition of a variety of household bases and disappear upon addition of several household acids. While I was unsuccessful in extracting anthocyanins from white clover, I had nevertheless discovered that the white clover contained an acid-base indicator, after all! But where did this colorless-to-yellow acid-base indicator come from?

A bit of literature searching turned up a likely candidate: anthoxanthins (Figure 3). Anthoxanthins are a class of chemical compounds that are similar in structure to anthocyanins, and they often impart white or yellow color to plants and flowers.^3-8 Anthoxanthins, are known to appear yellow colored in the presence of base, but colorless in the presence of acid.^3-4

Figure 3: Chemical structure of apigenin, an anthoxanthin

You can see some of my explorations I did with white clover below (Video 1).

Video 1: White Clover Color Chemistry, Tommy Technetium YouTube Channel, July 2, 2021.

In the future I’m hoping to carry out some other investigations with other white-colored plants that might contain anthoxanthins, such as daisies or cauliflower. I’m also hoping to use white clover as a source of an acid-base indicator in my classes from time to time. If you try some similar experiments with white colored plants and flowers and learn anything new, please be sure to let me know in the comments.

Happy experimenting!  

References

2. Alkema, J.; Seager, S. L. The Chemical Pigments of Plants, J. Chem. Educ. 1982, 59, 183-186.

3. Asenstorfer, R. E.; Wang, Y.; Mares, D. J. Chemical Structure of flavonoid compounds in wheat (Triticum aestivum L.) flour that contribute to the yellow color of Asian alkaline noodles. Journal of Cereal Science 2006, 43, 108-119.

4. Pate, K. M.; Rogers, M.; Reed, J. W.; van der Munnik, N.; Vance, S. Z.; Moss, M. A. Anthoxanthin polyphenols attenuate Ab oligomer-induced neuronal responses associated with Alzheimer’s disease, CNS Neurosci. Ther. 2017, 23, 135-144.

5. Blank, F. The anthocyanin pigments of plants. The Botanical Review 1947, 13, 241-317.

6. Geissman, T. A. The chemistry of flower color variation. J. Chem. Educ. 1949, 26, 657-655.

7. Lawrence, W. J. C.; Price, J. R. The genetics and chemistry of flower color variation. Biol. Rev. 1940, 15, 35-57.

8. Dutta, S.; Halder, S. A colourful food palette: health benefits and beyond. International Journal of Current Research 2021, 13, 16596-16600.