Extraction,Purification And Activity Study Of Roselle Anthocyanins

Roselle is a high quality medicinal and food plant.Roselle calyx is rich in anthocyanins,which is an excellent natural pigment with many biological activities.In this study,the dried roselle flower was used as the raw material to establish the process of separation and purification of anthocyanins,and the stability and chemical structure of the anthocyanins were analyzed.The antioxidation and anticancer activities of the Delphinidin-3-O-sambubioside were also explored.Using roselle calyx as raw material,the effects of ethanol concentration,solid-liquid ratio,and extraction time and extraction temperature on the anthocyanin extraction content were studied by single-factor experiments and orthogonal experiments.Extracted twice under the optimized conditions(50%ethanol concentration,solid-to-liquid ratio 1:15,extraction time 30 min,extraction temperature 55?),the anthocyanin extraction rate was 98.41%.The crude extract of roselle anthocyanin was purified by ethyl acetate extraction and membrane separation,which could remove some of the flavonoids and macromolecular impurities.After further purification by X-5 macroporous adsorption resin,the food-grade anthocyanin samples were prepared.The spectral analysis and liquid chromatography analysis showed that the anthocyanin sample after separation of the column contained two anthocyanins,and the final purified roselle anthocyanin sample had a color value of 15199.8 U/g.The effects of light,heat,pH,and metal ions on the food-grade anthocyanins were investigated.The results showed that the anthocyanins had poor stability to high temperature and strong light;anthocyanins would decompose rapidly when placed at temperatures above 60? and under direct sunlight for a long time.The pH value had a great influence on the color and structure of the anthocyanins solution,the solution or beverage of anthocyanins should be stored at acidic conditions.Common metal ions such as Na+,K+,Mg2+,Ba2+,and NH4+ had a color-enhancing effect on anthocyanins,which would delay their degradation,but Al3+,Fe3+,and Fe2+ could directly destroy the anthocyanins structure.The food grade anthocyanins were further purified by semi-preparative liquid chromatography.Two anthocyanin monomer components were collected.The structure of the anthocyanin component was analyzed by HPLC-MS,IR,NMR techniques.The two components were delphinidin-3-sambubioside and Cyanidin-3-sambubioside,respectively.The antioxidant capacity of delphinidin-3-O-sambodiglycoside was analyzed by removing DPPH radical,ABTS radical and Fe3+ reducing ability,indicating that it had good antioxidant activity in vitro.The anticancer activity of delphinidin-3-O-sambodiglycoside was analyzed by vitro cell experiments.MTT assay showed that delphinidin-3-O-sambodiside could inhibite the proliferation of Hela cells,and its IC50 value was 65?g/mL;flow cytometry Annexin V/PI double staining and PI single staining experiments showed that delphinidin-3-O-sambodiside could induce apoptosis and reflect its anti-cancer activity.