Vukic Milena D, Vukovic Nenad L, Popovic Suzana Lj, Todorovic Danijela V, Djurdjevic Predrag M, Matic Sanja D, Mitrovic Marina M, Popovic Ana M, Kacaniova Miroslava M, Baskic Dejan D
University of Kragujevac, Faculty of Science, Department of Chemistry, R. Domanovica 12, 34000 Kragujevac, Serbia.
University of Kragujevac, Faculty of Medical Sciences, Centre for Molecular Medicine and Stem Cell Research, Svetozara Markovica 69, 34000 Kragujevac, Serbia.
Saudi Pharm J. 2020 Jan;28(1):136-146. doi: 10.1016/j.jsps.2019.11.015. Epub 2019 Dec 16.
Acetylshikonin (AcSh), as a red colored pigment found in roots of the plants from family , 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 H NMR spectroscopy, and determine cytotoxic activity. Phase solubility test indicated formation of A-type binary system (substrate/ligand ratio was 1:1 M/M), with stability constant Ks of 306.01 M. 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 (C-H and C-H), as well as protons from hydroxyl groups (-0.013 and -0.009, respectively) attached to C and C 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.
乙酰紫草素（AcSh）是一种存在于紫草科植物根部的红色色素，具有出色的细胞毒性活性。由于其疏水性，生物利用度较差，本研究旨在采用共沉淀法制备乙酰紫草素/β-环糊精（AcSh/β-CD）包合物，通过紫外可见光谱、红外光谱和核磁共振氢谱对所得体系进行表征，并测定细胞毒性活性。相溶解度试验表明形成了A型二元体系（底物/配体比为1:1 M/M），稳定常数Ks为306.01 M。采用光谱方法观察到β-CD孔内层与AcSh之间形成了非共价键。萘醌部分（C-H和C-H）的两个质子（-0.020 ppm）以及萘醌部分连接到C和C碳上的羟基质子（分别为-0.013和-0.009）的化学位移发生了显著变化，表明AcSh分子从芳香侧进入β-CD腔。通过MTT试验和克隆形成试验测定了对HCT-116和MDA-MB-231细胞系的细胞毒性活性。通过流式细胞术评估游离AcSh和包合物的作用机制。与游离AcSh相比，AcSh/β-CD对HCT-116细胞显示出更强的短期效应，对两种细胞系均显示出更优异的长期效应。包合物比游离AcSh更有效地诱导更明显的细胞周期停滞和自噬抑制，并诱导细胞内活性氧积累增加。总之，AcSh/β-CD二元体系在对测试肿瘤细胞系的细胞毒性活性方面表现出更好的性能。
