Carvacrol and HP-β-Cyclodextrin Complexes: Extensive Characterization and Potential Cytotoxic Effect in Human Colorectal Carcinoma Cells

Abstract

The aim of this study was to obtain solid carvacrol-cyclodextrin (CD) complexes for use in the pharmaceutical industry. To this end, the complexation of carvacrol at different pH values was studied in detail, to determine the type of CD and the reaction environment that supported the highest amount of encapsulated carvacrol. Evidence of the capability of hydroxypropyl-β-cyclodextrins (HP-β-CD) to form inclusion complexes with carvacrol (K_C = 5042 ± 176 L mol^-1) and more high complexation efficiency (2.824) was demonstrated for HP-β-CDs using two different energy sources, ultrasound (US) (K_C = 8129 ± 194 L mol^-1 24 h) and microwave irradiation (MWI) (K_C = 6909 ± 161 L mol^-1), followed by spraying the resulting solution in a spray dryer. To confirm complex formation, the complexes were characterized using various instrumental methods to corroborate the carvacrol incorporation into the hydrophobic cavity of HP-β-CD. The obtained carvacrol solid complexes were analyzed by 1H nuclear magnetic resonance (¹H-NMR) and 2D nuclear magnetic resonance (ROSEY), differential scanning calorimetry (DSC), thermogravimetric analysis (TG) and Fourier transform infrared spectroscopy (FTIR) characterization. The structures of the resulting complexes were also characterized by molecular modeling. Furthermore, 1 mM HP-β-CD-carvacrol complex has been shown to reduce cell proliferation in HCT-116 colorectal cancer cells by 43%, much more than in a healthy lung fibroblast MRC-5 cell line (11%).

Keywords: HP-β-cyclodextrins; carvacrol; cell viability; chemical characterization; microwave irradiation; solid complexes.

Figure 1

Phase diagrams of carvacrol with α-, β-, and HP-β-CDs at different pH values: pH 3.5 (●), pH 5.5 (○), pH 6.5 (■), pH 7.0 (□), and pH 8.5 (♦), at 25 °C. Values represent the means of triplicate determination. Inset pH 3.5 (●) at 25 °C.

Figure 2

(a) Structure of carvacrol with H-atoms. (b) Carvacrol dissociation equilibrium.

Figure 3

Carvacrol complexes with HP-β-CD at pH 7.0, performed at 24 h (●) and 48 h by microwave irradiation (MWI) (○) and ultrasound (US) (□). The experiments were performed in triplicate.

Figure 4

Micrographies of HP-β-CDs (a), MWI complexes (b), and US complexes (c).

Figure 5

(a) Drying process yield (g kg⁻¹) and (b) encapsulation efficiency (g kg⁻¹) of carvacrol-HP-β-CDs complexes, prepared via US (grey bars) and MWI (black bars). The experiments were performed in triplicate.

Figure 6

Stability of the solid complexes during storage. MWI at 25 °C (red line) and 8 °C (blue line), with US complexes at 25 °C (red dashed line) and 8 °C (blue dashed line).

Figure 7

ROESY spectrum of the carvacrol-HP-β-CD complex in methanol-d₄.

Figure 8

Structure of the carvacrol inclusion complex, with HP-β-CDs.

Figure 9

(a). Curves of the DSC carvacrol (pink line), MWI carvacrol-HP-β-CDs (red line), and HP-β-CDs (green line). (b). The TG curves of HP-β-CD (green line), MWI HP-β-CD/carvacrol (red line), and carvacrol (blue line).

Figure 10

The FTIR spectrum of HP-β-CDs (green line), carvacrol-HP-β-CDs complexes by US (red line), and carvacrol (blue line). The vertical lines indicate the maximum of the HP-β-CDs curve.

Figure 11

The dose-dependent effect of carvacrol and HP-β-CD-carvacrol complexes on (A) HCT-116 colorectal cancer cells and (B) MRC-5 lung fibroblast cells, with viability (72 h incubation). The data shown represent the averaged and SD values of the triplicates.

Figure 12

The release of carvacrol, both free and complexed, with HP-β-CD in the presence of HCT-116 cells, at 37 °C, after 7 days of treatment, where n = 3.