Exploration On Identification Rules Of Anthocyanin Compounds In Blueberry Fruits

As the nutritional value of blueberries is accepted increasingly by mankind, blueberries have become more and more popular in the world. With the growing demand for blueberries, the output of the world’s blueberry is increasing day by day. At present, many regions in China are working on the cultivation of blueberries and researches on the functional components in blueberries. Because of the high content of anthocyanins in blueberry fruit and many benefits of anthocyanins on the human body, lots of researchers at home and abroad are engaged in the studies of blueberry fruit anthocyanins. Although there are a number of advanced identification methods of anthocyanins, it is difficult to identify the structure of anthocyanins by a single method. The combined identification technologies for anthocyanins have become a research hotspot. In this paper, based on the existing research foundation of our research group, the anthocyanin profiles in blueberry fruits was studied by different methods such as aglycone preparation by acid hydrolysis, UV spectrophotometry, HPLC and LC-MS. A series of identification rules was established for the composition of anthocyanins in blueberry species, which could provide a method for the identification of anthocyanins in other blueberries, even a theoretical reference for anthocyanin identification in more plants.Five anthocyanin aglycones were prepared by acid hydrolysis from the blueberry fruit, and identified by high performance liquid chromatography(HPLC). The results showed that the order of retention time in HPLC was as follows: delphinidin < cyanidin < petunidin < peonidin < malvidin. Through a investigation on the maximum absorption wavelengths of different anthocyanin aglycones in UV-vis spectrum, we found that the five anthocyanin aglycones have a comparable UV-vis spectrum.The UV-Vis spectrum of anthocyanins from three kinds of blueberry anthocyanins and some other fruits were investigated. It was concluded that the proportional distribution of anthocyanins and non-anthocyanin flavonoids were distinctive in each kind of fruits. In addition to the large amount of anthocyanins, the contents of non-anthocyanins in wild blueberries were higher, in contrast, the content of flavonoids in blueberries was lower. The UV-vis spectrum of anthocyanin monomer in blueberry were analyzed by high pressure liquid chromatography, it was concluded that three anthocyanin monomers in cultivated blueberries were distinctive with most of the other anthocyanin monomers, which need a further experiment for the confirmation of chemical structures.The structure of anthocyanins in blueberry fruit was identified by high performance liquid chromatography-electrospray ionization mass spectrometry(HPLC-ESI-MS). Twelve glycosylated anthocyanins were identified from the wild blueberry fruit, and 14 anthocyanin monomers identified from cultivated blueberry fruit, including 12 glycosylated anthocyanin forms, 2 acylated anthocyanin forms. The characteristics of blueberry anthocyanins on HPLC were studied. The fragmentation characteristics of different anthocyanins on the mass spectra were analyzed. The identification rules of anthocyanin monomer in blueberry fruit were summarized. The present research provides a theoretical and experimental basis for the further identification of more complex anthocyanins from new blueberry cultivars or other plants.