Effect of β-cyclodextrin encapsulation on cytotoxic activity of acetylshikonin against HCT-116 and MDA-MB-231 cancer cell lines

Milena D Vukic; Nenad L Vukovic; Suzana Lj Popovic; Danijela V Todorovic; Predrag M Djurdjevic; Sanja D Matic; Marina M Mitrovic; Ana M Popovic; Miroslava M Kacaniova; Dejan D Baskic

doi:10.1016/j.jsps.2019.11.015

Effect of β-cyclodextrin encapsulation on cytotoxic activity of acetylshikonin against HCT-116 and MDA-MB-231 cancer cell lines

Saudi Pharm J. 2020 Jan;28(1):136-146. doi: 10.1016/j.jsps.2019.11.015. Epub 2019 Dec 16.

Authors

Affiliations

¹ University of Kragujevac, Faculty of Science, Department of Chemistry, R. Domanovica 12, 34000 Kragujevac, Serbia.
² University of Kragujevac, Faculty of Medical Sciences, Centre for Molecular Medicine and Stem Cell Research, Svetozara Markovica 69, 34000 Kragujevac, Serbia.
³ University of Kragujevac, Faculty of Medical Sciences, Department of Genetics, Svetozara Markovica 69, 34000 Kragujevac, Serbia.
⁴ University of Kragujevac, Faculty of Medical Sciences, Department of Internal Medicine, Svetozara Markovica 69, 34000 Kragujevac, Serbia.
⁵ University of Kragujevac, Faculty of Medical Sciences, Doctoral Academic Studies, Svetozara Markovica 69, 34000 Kragujevac, Serbia.
⁶ University of Kragujevac, Faculty of Medical Sciences, Department of Biochemistry, Svetozara Markovica 69, 34000 Kragujevac, Serbia.
⁷ Master Academic Studies, Department of Biology and Ecology, Faculty of Sciences, University of Novi Sad, Trg Dositeja Obradovića 2, 21000 Novi Sad, Serbia.
⁸ Department of Fruit Sciences, Viticulture and Enology, Faculty of Horticulture and Landscape Engineering, Slovak University of Agriculture, Tr. A. Hlinku 2, 94976 Nitra, Slovakia.
⁹ Department of Bioenergy and Food Technology, Faculty of Biology and Agriculture, University of Rzeszow, Zelwerowicza St. 4, PL-35601 Rzeszow, Poland.
¹⁰ Public Health Institute, Nikole Pašića 1, 34000 Kragujevac, Serbia.

Abstract

Acetylshikonin (AcSh), as a red colored pigment found in roots of the plants from family Boraginaceae, showed excellent cytotoxic activity. Due to its hydrophobic nature, and thus poor bioavailability, the aim of this study was to prepare acetylshikonin/β-cyclodextrin (AcSh/β-CD) inclusion complex by using coprecipitation method, characterize obtained system by using UV/VIS, IR and ¹H NMR spectroscopy, and determine cytotoxic activity. Phase solubility test indicated formation of A_L-type binary system (substrate/ligand ratio was 1:1 M/M), with stability constant Ks of 306.01 M^-1. Formation of noncovalent bonds between inner layer of the hole of β-CD and AcSh was observed using spectroscopic methods. Notable changes in chemical shifts of two protons (-0.020 ppm) from naphthoquinone moiety (C₆-H and C₇-H), as well as protons from hydroxyl groups (-0.013 and -0.009, respectively) attached to C₅ and C₈ carbons from naphthoquinone part indicate that the molecule of AcSh enters the β-CD cavity from the aromatic side. Cytotoxic activity against HCT-116 and MDA-MB-231 cell lines was measured by MTT test and clonogenic assay. Mechanisms of action of free AcSh and inclusion complex were assessed by flow cytometry. In comparison to free AcSh, AcSh/β-CD showed stronger short-term effect on HCT-116 cells and superior long-term effect on both cell lines. Inclusion complex induced more pronounced cell cycle arrest and autophagy inhibition, and induced increase in accumulation of intracellular ROS more effectively than free AcSh. In conclusion, AcSh/β-CD binary system showed better performances regarding cytotoxic activity against tested tumor cell lines.

Keywords: Acetylshikonin; Cytotoxicity; Inclusion complex; β-Cyclodextrin.