Abstract:
Abstract: As one kind of important hedge materials, Ligustrum quihoui Carr. has been widely used in landscaping. Currently, a number of researchers are engaged in the identification of the chemical structure for the leaves of Ligustrum quihoui Carr.. However, the chemical ingredients in the fruits have not been well studied up till now, especially the study on the anthocyanins is rare. In order to promote the application of Ligustrum quihoui Carr. on food and medicine fields, the present research focuses on the determination of anthocyanin content and monomer composition of Ligustrum quihoui Carr. fruits. The study adopted the combined techniques of ultraviolet-visible spectroscopy spectrum (UV-vis), reversed phase high-performance liquid chromatography (RP-HPLC), high-performance liquid chromatography - diode array detector - electrospray ionization - tandem mass spectrometry (HPLC-DAD-ESI-MS/MS) and acid hydrolysis, as well as the preparation process of anthocyanin extracts using extraction, concentration, partition, column chromatography and freeze-drying technologies. The UV-vis spectrum results verified the existence of anthocyanins in the fruit part, and the total anthocyanin content was 499±18.42 mg per 100 g fresh fruits, which was determined by the pH differential method. Two glycosylated anthocyanin monomers (cyanidin-3-O-glucoside and petunidin-3-O-glucoside) were successfully identified from the fruits of Ligustrum quihoui Carr. by means of RP-HPLC, HPLC-DAD-ESI-MS/MS technologies, and then testified through the identification of anthocyanin aglycones prepared by acid hydrolysis of anthocyanin glycosides. We also found that, petunidin-3-O-glucoside was the dominating form in the fruits of Ligustrum quihoui Carr., which could become a new focus of our further research. A natural and simple preparation process of anthocyanin extracts was obtained by the combined chromatographic separation techniques. A kind of safe and cheap extractant was employed to extract anthcoyanin glycosides instead of the use of harmful methanol, acetone, trifluoroacetic acid etc., which was composed of 70% ethanol (v/v) and 0.1 mol/L HCl with a mixing proportion of 9:1 (v/v). After the anthocyanin extracts were filtered and concentrated, ethyl acetate was used to remove fat-soluble impurities from the anthocyanin extracts in the partition process, and the extraction was carried out for 3 times. Because of the existence of lots of water-soluble impurities (reducing sugars, proteins and polar flavonoids) in anthocyanin extracts, the aqueous anthocyanin extracts were loaded onto a column (2.6 cm × 50 cm) of cation-exchange resin (Amberlite XAD-7HP, particle size: 20-60 meshes), washed by deionized water (containing 0.01% HCl, v/v) to remove water-soluble ingredients, and then eluted by 40% aqueous ethanol (containing 0.01% HCl, v/v) to collect anthocyanin eluents, concentrated and freeze-dried; finally the extract was obtained with a purity of 35% and a yield of 0.6%. In view of the current purity of anthocyanin extracts, it was estimated that there were still many impurities mixed with the anthocyanin extracts, which needed a further separation using gel chromatography, high-speed countercurrent chromatography, preparative-HPLC etc. Because of the simplicity of anthocyanin profiles and high content of petunidin-3-O-glucoside monomer in Ligustrum quihoui Carr. fruit, it was promising to separate high-purity petunidin-3-O-glucoside monomer from the anthocyanin extracts using the combined purification techniques in our next experiments. The present research can promote the comprehensive development of the Ligustrum quihoui Carr. fruit, and meanwhile lay a foundation for further research on the bio-activity of anthocyanins in Ligustrum quihoui Carr. fruit and its potential application in food and medicine fields.