A "novel" protocol for the analysis of hydroxycinnamic acids in leaf tissue of chicory (Cichorium intybus L., Asteraceae)

Abstract

A "novel" protocol is presented for easy and reliable estimation of soluble hydroxycinnamate levels in Cichorium intybus L. leaf tissue in large-scale experiments. Samples were standardized by punching 6 discs per leaf, and hydroxycinnamates were extracted by submerging the discs in 80% ethanol with 5% acetic acid for at least 48 h in the darkness at 4°C. Residual dry mass of the discs was used for a posteriori correction of compound levels. Chlorophyll was eliminated by chloroform, and the aqueous phases were transferred to microplates, dried, and dissolved in 50% methanol for HPLC analysis and storage. An HPLC program of 8 min was developed for the analysis of the extracts. Comparisons with extractions of liquid nitrogen powders indicated that the novel extraction method was reliable. No degradation of the major hydroxycinnamates-caftaric, chlorogenic, and chicoric acids-was observed, during maceration at ambient temperatures, or after storage for 1 year.

Figure 1

Chromatographic profiles of chicory leaves at 280 nm. Dotted line and continuous line represent HPLC profiles obtained for the same fresh leaf tissue area with a “conventional” liquid nitrogen grinding and the “novel” method, respectively.

Figure 2

Histogram representing the contents (arbitrary unity) in caftaric acid (grey), chlorogenic acid (black), and chicoric acid (white) for each of the 24 different leaves analyzed by four inexperienced testers. The differences obtained between the manipulators are showed by the standard deviations (no significant differences; see Table 2).

Figure 3

HPLC profile of chicory leaves with the “short program,” using the “novel” method. Peak identifications: (1) caftaric acid, (2), chlorogenic acid, (3) caffeic acid (insert), and (4) chicoric acid. Chromatogram was recorded at 320 nm.