University of Victoria

Malonated flavonoids in maize --O. Ceska and E. D. Styles Two acylated anthocyanins in maize have been characterized recently by Harborne and Self (Phytochemistry 26:2417, 1987); they are: 1) cyanidin 3-(6"-malonylglucoside) and 2) cyanidin 3-dimalonylglucoside. Compounds acylated with aliphatic dicarboxylic acids are relatively unstable in solutions containing HCl. Standard extraction procedures using methanolic HCl result in intermediate methyl ester formation and the eventual loss of the acyl group. Substitution of a weaker acid such as acetic or formic acid allows for the extraction of the acylated compounds without change. In the past we have always extracted anthocyanin-containing tissues with methanolic HCl, and we have always obtained several anthocyanin spots on TLC chromatograms in addition to the simple cyanidin 3-glucoside. These extra spots were almost certainly the result of an interaction between HCl and the malonated anthocyanins. When we use acetic acid in aqueous methanol for the extraction we get only two acylated anthocyanins in addition to the spot of cyanidin 3-glucoside. In tissues that have predominantly pelargonidin compounds (pr pr genotypes) we find, as with the cyanidin compounds, the simple 3-glucoside of pelargonidin plus two acylated derivatives, and in tissues that have peonidin (cherry pericarp), we find the simple 3-glucoside peonidin, and again, two acylated derivatives.

Malonated anthocyanins can be easily separated by electrophoresis on Whatman No. 3 paper in acetate buffer of pH 4.4 at 40V/cm and 0.5mA/cm. The usual time quoted in the literature is 2 hours, but we have found that a much longer time is required for good separation (up to 18 hours).

When we analyze the methanolic extracts of tissues that normally have strong anthocyanin concentration but are brown because of a1, a2, or bz2 blocks, we consistently find two flavonol compounds in addition to the quercetin and isorhamnetin 3-glucosides. They behave similarly on TLC but their Rf values in 15% acetic acid are slightly higher than those of the other flavonols. Only the flavonol based on quercetin was isolated in sufficient quantity for partial characterization. Total acidic hydrolysis as well as enzymic hydrolysis gave quercetin and glucose. Diagnostic UV spectra were identical with quercetin 3-glucoside. On chromatography in water the Rf value of quercetin 3-glucoside was 0.8 whereas the Rf value of unknown flavonol was 80, suggesting the presence of acyl group. Paper electrophoresis under the same conditions as described for malonated anthocyanins was used to find out if malonic acid was present. As a control, crude methanol-acetic acid-water (8:1:1) extract of cherry Pl pericarp containing malonated anthocyanins was used. Anionic mobilities were 2.5 cm for flavonol, 2.5 cm for cyanidin 3-malonylglucoside and 5 cm for cyanidin 3-dimalonylglucoside. This confirmed the presence of malonic acid in quercetin 3-glucoside. However, on saponification no malonic acid could be detected, probably due to the small amount of compound available. FAB mass spectrum gave clearly [M]+ = 551 which is molecular weight of quercetin 3-monomalonylglucoside but fragmentation indicating loss of malonic acid and mass of the aglycone did not show well. Again, the amount of compound was probably inadequate. Work is now in progress to isolate more of this flavonol so saponification and FAB MS can be repeated.

Malonylation is considered an important step because it appears to stabilize anthocyanins in the acidic environment of the vacuole (Harborne, Phytochemistry 25:1887, 1986) and also it may facilitate the transport of flavonoid glucosides through the tonoplast into the vacuole (Mattern et al., Planta 167:183, 1986).

Malonated anthocyanins are fairly widespread (Harborne, Phytochemistry 25:1887, 1986), but only a few malonated flavonols have been reported. Quercetin 3-malonylglucoside, together with other malonated flavonoids, were produced by parsley cell cultures after irradiation with UV (Kreuzaler and Hahlbrock, Phytochemistry 12:1149, 1973). Quercetin 3-malonylglucoside was also reported by Woeldecke and Hermann (Z. Naturforsch. 29C:335, 1974) and more recently by Geslin and Verbist (J. Natur. Prod. 48:111, 1985) from Salicornia europaea.

It is not clear why malonated quercetin 3-glucoside should be produced in maize only when there is a block in anthocyanin synthesis. Perhaps malonyltransferase can accept other substrates, in this case quercetin 3-glucoside, if no anthocyanins are available. Or perhaps malonated quercetin 3-glucoside is formed as a reaction of the plant to obvious stress. In the case of the above-mentioned genotypes, brown pigments form in the place of anthocyanins, and frequently their accumulation is accompanied by tissue necrosis, the ultimate stress response.

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