Purple pepper plants, an anthocyanin powerhouse: Extraction, separation and characterization

Most plants have multiple anthocyanins present that produce their color. In contrast, the foliage of the purple pepper plant (Capsicum annuum L.) contains high concentrations of a single anthocyanin delphinidin-3- p-coumaroylrutinoside-5-glucoside (Dp-3-p-coumrut-5-glc) in the foliage, making it very unique. This provides an excellent platform to extract the single anthocyanin at high concentrations.;A food-grade extraction method was developed using 1% hydrochloric acid and 200 proof ethanol (1% HCl/EtOH) in order to remove the intact anthocyanin. A separation method using High Performance Liquid Chromatography (HPLC) was developed to identify Dp-3-p-coumrut-5-glc. The retention time was compared with the Blue Ribbon Iris, a known source of Dp-3- p-coumrut-5-glc. The HPLC results confirmed the presence of Dp-3- p-coumrut-5-glc in the pepper extract, but the chromatograms also demonstrated the presence of additional highly colored compounds. The extract was injected onto the HPLC and the major anthocyanin peak (Dp-3-p -coumrut-5-glc) was collected over the course of multiple injections. The collected fractions were dried down and re-solubilized in 1% HCl/methanol for analysis by mass spectrometry.;A HPLC coupled to a photodiode array detector and an electrospray ionization tandem mass spectrometer (LC-PDA-ESI-MS/MS) was utilized to characterize Dp-3- p-coumrut-5-glc. The precursor compound was confirmed at m/z 919 with product ions at m/z 757, 465 and 303 by comparing against plant extracts of freeze-dried purple pepper foliage, Chinese eggplant and Chinese celery. The extract's structure was elucidated by Nuclear Magnetic Resonance (NMR) by analyzing both proton (1H) and carbon (13C) spectra. The 1H and 1 3C data matched very well with previous NMR data of Dp-3- p-coumrut-5-glc elucidated in eggplant peels. The major difference was that the trans isomer of Dp-3-p-coumrut-5-glc greatly dominated over the cis in the purple pepper extract.