HPLC identification of anthocyanins in maize endosperm

--Elizabeth E. O. Caldwell and Peter A. Peterson

High performance liquid chromatography was used to analyze the anthocyanin content of pooled aleurone and starchy endosperm. Tissue was collected from mature, fully-colored, red, C-(lineC) sh Bz wx kernels.

Individual kernels were prepared for analysis as follows:

1. pericarp and embryo tissues removed

2. 1% HCl in water and the remaining tissue (endosperm including aleurone) ground to a slurry

3. supernatant lyophilized

4. pigments redissolved in 1% HCl in methanol

Anthocyanins were separated using a C18 reverse phase column (Lichrosorb 1ORP18, 25 x 0.5cm) and detected at 540nm. The first eluent was 15% acetic acid, 1.5% phosphoric acid, 83.5% water. The second eluent was acetonitrile. The following gradient was a modification of one published by Asen and Griesbach (J. Amer. Soc. Hort. Sci. 108:845-850, 1983):
Time Eluent 1 Eluent 2
0 to 10 min 9% 1%
10 to 50 min 99% to 80% 1% to 20%

Individual anthocyanins were identified by comparing the unknown retention times with results from extracts of lyophilized USDA-83 petunia petals. The USDA-83 standard was provided by Dr. Robert Griesbach along with identification of its anthocyanin content.

By comparing the maize and USDA-83 extracts, three acylated anthocyanins were identified in the C-(lineC) sh Bz wx mature endosperm tissue. These include: peonidin-3-(caffeylrutinoside)-5-glucoside, peonidin-3-(p-coumarylrutinoside)-5-glucoside, and malvidin-3(caffeylrutinoside)-5-glucoside. Peonidin has been reported in maize aleurone but not these derivatives.

Please Note: Notes submitted to the Maize Genetics Cooperation Newsletter may be cited only with consent of the authors

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