NMR structural characterization and inhibition of colon cancer cells by components of Citrus aurantium L

Introduction: Epidemiological studies suggest that dietary limonoids and phytosterols offer protection from some types of cancers (1). In the present study, cancer preventive components from sour orange (Citrus aurantium L.) were isolated and identified for the first time. Isolated compounds were tested for the inhibition of human colon cancer cells (HT-29) proliferation, apoptosis. Methods: Sour oranges (Citrus aurantium L.) seed powder (4800 g) was extracted using a Soxhlet apparatus with hexane (15 L) for 24 h for the removal of fatty matter. The defatted powder was extracted for 8 h with ethyl acetate (15 L) at 60-70°C. Purification was carried out by column chromatography using dichloromethane and acetone in ratio 7:3 to obtain limonexic acid and sitosterol glucoside at 4:6. Both the compounds were subjected to HPLC analysis using gradient mobile phase (A) 3mM phosphoric acid and (B) acetonitrile. Cell viability, cell cycle analysis and apoptosis assessment was performed using MTT assay, flow cytometry measurements. and acridine orange and ethidium bromide staining under Olympus FV1000 confocal microscope. Two isolated compounds were characterized by extensive analysis of H and C NMR experiments on a Bruker Biospin Avance 400 NMR spectrometer (H frequency = 400.13 MHz, C frequency = 100.62 MHz) at 298 K using 5-mm broad band inverse probe head equipped with shielded z-gradient and XWIN-NMR software version 3.5 using TMS as an internal reference. One-dimensional H and C spectra were obtained using one pulse sequence. Two-dimensional NMR experiments such as H-H DQF-COSY, edited H-C HSQC and H-C HMBC were performed to solve the structure of isolated compounds. Results: Purity of the isolated compounds was analyzed by TLC and HPLC. Comprehensive analysis of H and C 2D NMR experiments of isolated compounds facilitated us to identify and assign the compounds as limonexic acid and sitosterol glucoside (Fig. 1). Typical H and C NMR spectra of limonexic acid with assignments is displayed in Fig. 2. Proton spectra of the compounds were complex with many overlapped peaks in the region 0.98 to 2 ppm (sitosterol glucoside) and 1.2 to 2.8 ppm (limonexic acid). In order to resolve the chemical shift values of CH and CH2 protons from the overlapped region, isolated compounds were subjected to edited