Chicory (Cichorium intybus) accumulates caffeic acid esters with important significance for human health. In this study, we aim at a better understanding of the biochemical pathway of these bioactive compounds. Detailed metabolic analysis reveals that C. intybus predominantly accumulates caftaric and chicoric acids in leaves, whereas isochlorogenic acid (3,5-diCQA) was almost exclusively accumulated in roots. Chlorogenic acid (3-CQA) was equally distributed in all organs. Interestingly, distribution of the four compounds was related to leaf age. Induction with methyljasmonate (MeJA) of root cell suspension cultures results in an increase of 3-CQA and 3,5-diCQA contents. Expressed sequence tag libraries were screened using members of the BAHD family identified in Arabidopsis and tobacco as baits. The full-length cDNAs of five genes were isolated. Predicted amino acid sequence analyses revealed typical features of BAHD family members. Biochemical characterization of the recombinant proteins expressed in Escherichia coli showed that two genes encode HCTs (hydroxycinnamoyl-CoA:shikimate/quinate hydroxycinnamoyltransferases, HCT1 and HCT2) whereas, three genes encode HQTs (hydroxycinnamoyl-CoA:quinate hydroxycinnamoyltransferases, HQT1, HQT2, and HQT3). These results totally agreed with the phylogenetic analysis done with the predicted amino acid sequences. Quantitative real-time polymerase chain reaction analysis of gene expression indicated that HQT3, HCT1, and HCT2 might be more directly associated with CQA accumulation in cell culture in response to MeJA elicitation. Transient expression of HCT1 and HQT1 in tobacco resulted in a higher production of 3-CQA. All together these data confirm the involvement of functionally redundant genes in 3-CQA and related compound synthesis in the Asteraceae family.

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A method for the simultaneous determination of free chlorogenic acids (CGA) and sesquiterpene lactones (STL) in chicory root and its dried (flour) and roasted (grain) forms is described. The method uses one extraction and one analysis for all chicory root products. Various solvents with low to high polarity, such as methanol, chloroform, or n-hexane, were tested alone, in combination in different proportions or with acidified or neutral aqueous solvent. The water/chloroform/methanol (30/30/40, v/v/v) mixture generated the best extraction yield, 21% higher than alcohol mixtures. The profiling of CGA and STL content was performed through a conventional HPLC-DAD method using a PFP core shell column in a fast single run. Good retention time and area repeatability (RDD mean % 0.46 and 5.6, resp.) and linearity (R 2 ≥ 0.96) were obtained. The STL and chlorogenic acids levels determined were 254.7 and 100.2 μg/g of dry matter in the root, 792.5 and 1,547 μg/g in flour, and 160.4 and 822.5 μg/g in the roasted grains, respectively. With an average recovery of 106% and precision of 90%, this method is rapid, reproducible, and straightforward way to quantify the chlorogenic acids and STL in chicory raw material and end products.

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.