The Anti-Angiogenic Effect of Cynara cardunculus L. subsp. cardunculus Waste Product

Abstract

Cynara cardunculus L. subsp. cardunculus (Cynara cardunculus L. var. sylvestris (Lam.) Fiori), the wild cardoon, is known for its culinary applications and potential health benefits. Due to this, and given the growing interest in circular economies, deepening our under-standing of the effects of wild cardoon leaf waste on angiogenesis and collagenase activity represents a valuable opportunity to valorise agricultural byproducts as health-promoting ingredients. In this study, the waste product of wild cardoon leaves was extracted to examine its chemical composition and biological activities. Analytical techniques identified several bioactive compounds, including flavonoids, hydroxycinnamic acids such as dicaffeoyl-succinoylquinic acids, and luteolin-7-O-rutinoside. In vivo tests in zebrafish embryos and the chick chorioallantoic membrane demonstrated dose-dependent antiangiogenic effects, particularly enhanced by the complexation with hydroxypropyl-β-cyclodextrin (HP-β-CD). Considering the link between angiogenesis and collagenase, the potential effects of the extract on collagenase activity was investigated. The extract alone inhibited collagenase with an IC₅₀ value comparable to that of the standard inhibitor while its complexed form exhibited a 4.5-fold greater inhibitory activity. A molecular docking study examined the interaction between the main compounds and collagenase. In conclusion, wild cardoon leaves can represent a valuable source of bioactive compounds. This study demonstrated that the complexation of the extract with cyclodextrin determines an increase in its biological activity.

Keywords: angiogenesis; chick chorioallantoic membrane; collagenase; hydroxypropyl-β-cyclodextrin; wild cardoon; zebrafish.

Figure 1

¹H NMR spectrum portions of the extract; for simplicity, only the signals relating to caffeoylquinic acid have been indicated (the scale is in ppm).

Figure 2

HPLC-PDA chromatogram at 360 nm of wild cardoon leaf extract (SyEt80). Chromatographic conditions are described in the text. Peak identifications are given in Table 1 and text.

Figure 3

Binding mode of all compounds in collagenase: (a) representation of the binding mode of all compounds in human collagenase. In green: ligand 1CGL, in magenta: 1,5-dicaffeoyl-3-succinoylquinic acid, in purple: 1,5-dicaffeoyl-4-succinoylquinic acid, in yellow: epigallocatechin gallate, in cyan: luteolin 7-O-rutinoside, in orange: chlorogenic acid. (b) Representation of the binding mode of all compounds in collagenase from Clostridium histolyticum. In green: ligand 7ZOC, in magenta: 1,5-dicaffeoyl-3-succinoylquinic acid, in purple: 1,5-dicaffeoyl-4-succinoylquinic acid, in yellow: epigallocatechin gallate, in cyan: luteolin 7-O-rutinoside, in orange: chlorogenic acid.

Figure 4

TGA thermograms of the extract/HP-β-CD complex in comparison with the free components.

Figure 5

Antiangiogenic activity of SyEt80 and of SyEt80 complexed with HP-β-CD (0.5%, w/v) in the CAM assay. Results are expressed as % inhibition vs. negative control. Retinoic acid (1 µg/egg) = positive control. Mean ± SD (n = 10). * p < 0.05; ** p < 0.01 vs. control: Student’s t-test.

Figure 6

Representative images of the chick chorioallantoic membrane (CAM) assay showing the effects of SyEt80 extract and its complex with HP-β-CD on angiogenesis at different doses. (A) Control; (B) retinoic acid (positive control); (C–E) SyEt80 and (F–H) SyEt80 complexed with HP-β-CD at 25, 50, and 100 µg/egg, respectively. The images illustrate a dose-dependent antiangiogenic effect of the extract, which is enhanced by HP-β-CD complexation (scale bar = 1000 μm).

Figure 7

Antiangiogenic activity of SyEt80 (10, 20, and 40 μg/embryo) free and complexed with HP-β-CD (0.5%, w/v) in zebrafish embryos. Results are expressed as % inhibition vs. negative control. 2-methoxyestradiol (2 µM) = positive control. Mean ± SD (n = 10). ** p < 0.01 vs. negative control: Student’s t-test.