A Three-Step Process to Isolate Large Quantities of Bioactive Sesquiterpene Lactones from Cichorium intybus L. Roots and Semisynthesis of Chicory STLs Standards

Abstract

Sesquiterpene lactones (STLs) are a large group of terpenoids most commonly found in plants of the Asteraceae family, e.g., in chicory plants, displaying a wide range of interesting biological activities. However, further studies on the biological potential of chicory-derived STLs and analogues are challenging as only four of these molecules are commercially available (as analytical standards), and to date, there are no published or patented simple extraction–purification processes capable of large-scale STLs isolation. In this work, we describe a novel three-step large-scale extraction and purification method for the simultaneous purification of 11,13-dihydrolactucin (DHLc) and lactucin (Lc) starting from a chicory genotype rich in these STLs and the corresponding glucosyl and oxalyl conjugated forms. After a small-scale screening on 100 mg of freeze-dried chicory root powder, the best results were achieved with a 17 h water maceration at 30 °C. With these conditions, we managed to increase the content of DHLc and Lc, at the same time favoring the hydrolysis of their conjugated forms. On a larger scale, the extraction of 750 g of freeze-dried chicory root powder, followed by a liquid–liquid extraction step and a reversed-phase chromatography, allowed the recovery of 642.3 ± 76.3 mg of DHLc and 175.3 ± 32.9 mg of Lc. The two pure STLs were subsequently used in the context of semisynthesis to generate analogues for biological evaluation as antibacterial agents. In addition, other described chicory STLs that are not commercially available were also synthesized or extracted to serve as analytical standards for the study. In particular, lactucin-oxalate and 11,13-dihydrolactucin-oxalate were synthesized in two steps starting from Lc and DHLc, respectively. On the other hand, 11β,13-dihydrolactucin-glucoside was obtained after a MeOH/H2O (70/30) extraction, followed by a liquid–liquid extraction step and a reversed-phase chromatography. Together, this work will help facilitate the evaluation of the biological potential of chicory-derived STLs and their semisynthetic analogues.

Phenolics and Sesquiterpene Lactones Profile of Red and Green Lettuce: Combined Effect of Cultivar, Microbiological Fertiliser, and Season

“…To quantify all STL (hydroxyl, oxalyl, and glycosyl groups), extraction from the freeze-dried and ground leaves included maceration over a sufficient time period to promote hydrolysis on carbon 15 of the cyclopentenone ring [ 74 ]. In total, 300 mg of the freeze-dried lettuce leaf powder was mixed with 9 mL of methanol/water/acetic acid (75:23:2, v / v / v ) [ 75 ], vortexed for a few seconds, and shaken at 1000× g (Thermomixer, Eppendorf, Montesson, France) for 17 h at 25 °C.…”

“…To quantify all STL (hydroxyl, oxalyl, and glycosyl groups), extraction from the freezedried and ground leaves included maceration over a sufficient time period to promote hydrolysis on carbon 15 of the cyclopentenone ring [74]. In total, 300 mg of the freezedried lettuce leaf powder was mixed with 9 mL of methanol/water/acetic acid (75:23:2, v/v/v) [75], vortexed for a few seconds, and shaken at 1000× g (Thermomixer, Eppendorf, Montesson, France) for 17 h at 25 • C. The extract was centrifuged at 15,000× g (Sigma Centrifuge, Sigma Laboratory Centrifuges, Osterode, Germany) at 4 • C for 10 min and a supernatant (7 mL) was evaporated under reduced pressure with a vacuum concentrator.…”

Biocatalytic Regioselective O‐acylation of Sesquiterpene Lactones from Chicory: A Pathway to Novel Ester Derivatives

“…Given that we managed to extract a relatively larger amount of DHLc from chicory root, we selected this STL as the main substrate for this section of the study. [16] The lipase-catalyzed acylation with vinyl acetate as the acyl donor proceeded under similar reaction conditions as before. The reaction was conducted in 1 mL of 3/1:MTBE/ACN, with 20 mg of the immobilized lipase (Novozym 435), 10 mM of DHLc at 37 °C, along with the respective negative controls.…”

“…11β,13-dihydrolactucin used in this study was extracted in-house from lyophilized chicory root and purified via flash-column chromatography. The complete extraction protocol is described by Ruggieri et al [16] Novozym 435 (Immobilized lipase B from Candida antarctica), with a specific activity of 10,000 propyl laurate units per gram, was obtained from Novozymes.…”

“…Their structural characteristics have been described in detail in the literature and are reported in Figure 1. [14][15][16] Chicory-derived STLs have demonstrated promising antimicrobial properties. In a recent study, both 11β,13-dihydrolactucin (DHLc) and lactucopicrin (Lp) inhibited the growth of Pseudomonas aeruginosa, DHLp was also effective against Staphylococcus aureus, while DHLc showed promising results against different strains of Candida.…”