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β-cyclodextrin encapsulation of curcumin elicits an altered mode of angiogenin inhibition: In vitro and in vivo studies 1 1 Introduction 1 2 Materials and methods 2 2.1 Materials and sample preparation 2 2.2 Methods 3 2.2.1 Preparation of curcumin-β-cyclodextrin (Cur-βCD) inclusion complex 3 2.2.2 Quantification of curcumin loading in the inclusion complex 3 2.2.3 UV–Vis spectroscopy 3 2.2.4 Morphology and size determination 3 2.2.5 Fourier transform infrared spectroscopy (FTIR) 3 2.2.6 1H and 13C NMR spectroscopy 3 2.2.7 Agarose gel based assay 3 2.2.8 Inhibition kinetics 3 2.2.9 Docking studies 3 2.2.10 Accessible surface area (ASA) calculations 3 2.2.11 Fluorescence spectroscopy 3 2.2.12 Determination of protein:ligand binding stoichiometry 3 2.2.13 Isothermal titration calorimetry 3 2.2.14 Chorioallantoic membrane (CAM) assay 4 2.2.15 Statistical analyses 4 3 Results and discussion 4 3.1 Characterization of the formed Cur-βCD inclusion complex 5 3.1.1 Analysis of UV–Vis spectra, size and morphology of the inclusion complex 5 3.1.2 FTIR Study 5 3.1.3 1H and 13C NMR spectra analysis 5 3.2 Interactions of hAng with Cur and Cur-βCD 6 3.2.1 Anti-ribonucleolytic activity of Cur/Cur-β

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UND

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nonfiction