Preparation Of High-purity Blueberry Anthocyanin Extracts And Induced Apoptosis Of Tumor Cells

Nowadays, Blueberry industry is showing great potential in the international anddomestic market. Despite the late start of the blueberry industry in our country, it has beenone of the fastest growing new industries. In recent years, most of the researches onanthocyanins from blueberries limited to structural identification and preparation ofanthocyanin crude extracts, there are only a few studies on preparation of high purity ofanthocyanin extracts and monomers. On the aspect of anti-tumor activity of anthocyanins,abroad studies have showed that anthocyanins could inhibit tumor growth and blockcarcinogens, however, the effects of anthocyanins and acylcones on the inhibition ofmelanoma cell research have been rarely reported. This paper focuses on the followingresearch aspects:(1) optimized the content and structure detection methods of anthocyanins,(2) established a natural process for producing high-purity blueberry anthocyanin extract,(3)studied the purification of blueberry anthocyanins and monomers,(4) prepared anthocyaninacylcones using chemical degradation pathway, and then evaluated the stability and chemicalstructure,(5) explored the in vitro and in vivo anti-tumor effects of blueberry anthocyanins.Firstly, both pH differential method and HPLC method employed to determine the totalanthocyanins content (TAC) of blueberry fruits. Through the pH differential method, theTAC in Wild blueberries blueberry fruit reached up to144.72±4.49mg/100g fresh fruit, andthe TAC was216.50±6.26mg/100g in cultivated blueberry fruits. Through the HPLCmethod, the TAC in wild blueberry was173.15±8.16mg/100g fresh fruit, compared with245.52±11.54mg/100g fresh fruit in cultivated blueberry. By contrast, both methods areapplicable to the content analysis of anthocyanin, what is more, the former is more feasiblefor the determination of total content, and the latter is more suitable for the analysis ofmonomeric anthocyanins.Based on the understanding of total anthocyanin content, three techniques of UV-vis,HPLC and HPLC-ESI-MS methods were combined to analyze the anthocyanin profiles inwild and cultivated blueberries. There were13glycosylated anthocyanins in wild blueberry,and12glycosylated and two acylated anthocyanins in cultivated blueberry species.Malvidin-3-O-glucoside is the most abundant in the wild blueberry, accounting for28%of the total anthocyanin content, reaching up to48.44mg/100g fresh fruit, while malvidin-3-O-galactoside is the predorminent monomer, accounting for21%of the total anthocyanincontent, reaching to51.45mg/100fruit.The separation process of blueberry anthocyanins was carried out using extraction,partition, macroporous resin purification, gel chromatography purification. In the extractionprocess, it was found that70%ethanol acidified with hydrochloride was suitableanthocyanins extraction, which is not suitable for the extraction of acylated anthocyaninsfrom cultivated blueberries. In the partition process, ethyl acetate could effectively removemost of the non-anthocyanin flavonoids from the crude extracts of anthocyanin after threetimes of partition. In the process of Amberlite XAD-7HP macroporous resin purification,35%ethanol was used to elute the anthocyanins, and the purity of free-dried anthocyaninsapproached32%where the non-anthocyanin flavonoids were not detected. In the process ofSephadex LH-20gel column chromatography, it was found that the crude extracts ofanthocyanins could be successfully separated into three segments using25%ethanol as theeluent, their purities were59.53%,68.04%and65.79%, respectively. Through an analysis ofHPLC-PDA, segment one mainly contained malvidin-3-O-glucoside (86.8%), segment twocontained petunidin-3-O-glucoside (47.3%), segment three contained delphinidin-3-O-glucoside (51.6%).A small semi-preparative C18column (4.6×250,5μm) was tested to purify the crudeextracts of blueberry anthocyanins. And then, the medium-sized semi-preparative C18column (20.0mm×250mm,5μm) was used to isolate the components obtained fromSephadex LH-20. Three high-purity blueberry anthocyanin monomers were prepared aftertwice injection. The profiles and purities of three anthocyanin monomers were determinedusing combined techniques, such as UV-vis, HPLC and HPLC-ESI-MS. Three monomerswere malvidin-3-O-glucoside (purity:97.7%), petunidin-3-O-glucoside (purity:99.3%) anddelphinidin-3-O-glucoside (purity:95.4%), respectively. All of the three monomers can beused as references for anthocyanin analysis from other natural products, even the productionof high added blueberry products. Furthermore, the semi-preparative HPLC process in thispaper can lay a foundation for the scale production of anthocyanin standards.The UV-vis spectrums between the blueberry anthocyanin glycosides and aglycone forms were studied.The maximum absorption wavelength of anthocyanin aglycone was521nm, which blueshifted14nm compared with anthocyanins glycosides. When theconcentration of hydrochloric acid was2to4mol/L, hydrolysis time was40to60min, it cancompletely hydrolyzed anthocyanins glycosides. There were five basic anthocyaninaglycones in wild and cultivated blueberries, and delphinidin derivatives are the mostaffluent, followed by cyanidin derivatives.In order to evaluate the anti-tumor effects of blueberry anthocyanins, we first comparedthe antioxidant capacities of blueberry anthocyanin samples. We investigated the DPPH andABTS radical scavenging abilities, and found that the crude extracts of blueberryanthocyanins have stronger antioxidant ability than that of other materials, and the segmentone and Vitamin C ranked the last. Further, the crude extracts were used to evaluate theanti-tumor effect of blueberry anthocyanins. In vitro experiments, the concentration ofanthocyanins in the range of200~1000μg/mL could strongly inhibited the proliferation ofmurine melanoma cells. in vivo experiments, the murine melanoma model was firstestablished, and the results showed that2000μg/mL of anthocyanin extracts showed a higherinhibition capacity on murine melanoma cells than20μg/mL of DOX (doxorubicin) group,while200μg/mL of anthocyanin extracts were not better than DOX group. Through acomparison on the lung weight among different groups, the invasion of melanoma couldcause an increase in organs weight, but which could not affect the total weight of mice.This paper systematically analyzes the contents and profiles of anthocyanins inblueberries. We established a natural process for separation and purification of blueberryanthocyanins and then explored the anti-tumor effects of blueberry anthocyanins in vitro andin vivo. The present results will help to reveal the potential nutritional value of blueberries,and lay a foundation for the further study on the structure and functions of blueberryanthocyanins. It is expected to provide a new way for the development of high value-addedproducts of blueberry anthocyanins, to expand the application of anti-tumor foods or naturalmedicines.