Research On The Technology Of Preparation Of Blueberry Anthocyanins Aglycones And Its Inhibitive Effect On The Proliferation Of HepG2 Cells

Wild Blueberry is a kind of small pure blue berries,rich in anthocyanins,minerals,vitamins and other nutrients,which has high economic value.As a natural plant pigment,anthocyanins have broad application prospects in food and medicine industry,which is due that they have been found to possess many of biological activities,such as antioxidant,anti-tumor,hypoglycemic and so on.There is a great deal of research that have shown that anthocyanins are mostly in the form of glycosides that have the characteristics of higher molecular weight,poorer membrane permeability,direct-absorbed difficultly by the body,while anthocyanin aglycones that are the removal of glycosides are greatly enhanced in biological activity and utilization,hence,blueberry anthocyanin aglycone monomer substances were prepared and purified by using wild blueberry fruit as raw material,and the inhibitive effect of anthocyanin aglycones on the proliferation of HepG2 cells was preliminarily analyzed in this paper,this can provides a certain reference for further study of the functional activity and the potential value in food and medicine of anthocyanin aglycones.Firstly,the crude extract of blueberry anthocyanin glycosides was prepared by using extraction-partition-column chromatography-solid phase extraction combined technologies,and then,the method for preparing anthocyanin aglycones of blueberry was obtained via comparing and analyzing the effects of enzymatic hydrolysis(β-glucosidase and cellulase)and acid hydrolysis(acetic acid,citric acid and hydrochloric acid).The results were as follows:(1)The active ingredient of anthocyanins from blueberry was effectively extracted by the extraction and partition process,the dissolution rate of anthocyanins was improved by ultrasonic-assisted treatment,and finally the separation of anthocyanin and non-anthocyanin impurities was realized;(2)The purification process with column chromatography significantly improved the anthocyanin glycoside samples,and the crude and purified extracts of anthocyanins were obtained,respectively.(3)The method of hydrochloric acid hydrolysis is effective,which can obtain anthocyanin aglycones of blueberry.The crude extract of blueberry anthocyanin aglycones was prepared by the purification technologies of macro porous resin chromatography-solid phase extraction,whose products have a purity of 72.59%.By optimizing the semi-preparative high performance liquid chromatography method,the overall purity of the crude extract of blueberry anthocyanin aglycones was significantly improved,and the preparation of anthocyanin aglycone monomers was prepared effectively,which can provide fresh ideas for anthocyanin aglycone monomers,While laying a foundation for further study of its functional activity.Four semi-preparative components were prepared by semi-HPLC,these purities of which were above 90%,which provided a technical reference for the preparation of standardized substances.The content of anthocyanin aglycones in the four semi-preparative components was analyzed by HPLC.The four components’ differences of the retention time and the peak sequence in the chromatographic column were used to deduce in order: delphinidin,cyanidin,petunidin,a mixture of peonidin and malvidin.Based on the HPLC method,this chapter conducts in-depth analysis and identification by mean of HPLC-DAD-ESI-MS.These monomers structure of components obtined by semi-HPLC were identified via scanning the mass-to-charge ratio of fragment ions which make use of mass spectrometry.The results were consistent with those of the HPLC method.In this paper,the inhibitive effect of anthocyanin aglycones on the proliferation of HepG2 cells was preliminarily studied.The antioxidant activity of anthocyanin aglycones of wild blueberry was mainly evaluated by the methods of radicals scavenging of DPPH and ABTS.The results were that,blueberry anthocyanin aglycones have a strong antioxidant effect.Based on the results of antioxidant evaluation,by carrying out the cell level experiment,the effect of anthocyanin aglycones on the proliferation of HepG2 cells was significantly inhibited at the concentration of 200 ~ 1000μg / m L,and the IC50 value was between 50 ~ 200μg / m L.By comparing the anti-tumor effects of 24,48 and 72 h,it was found that the anti-cancer effect of anthocyanin aglycones of blueberry were more prominent at 72 h,which could provide a new idea for the treatment of liver cancer.