Isolation,Purification And Antitumor Activity Of Anthocyanins From Lonicera Caerulea 'Beilei' Fruit

Lonicera caerulea 'Beilei' is the first approved cultivar of Lonicera caerulea in China.It is also the main cultivar of cultivated area in northeastern China.The cultivation area is increasing year by year.However,the basic processing and utilization of fruit processing,physiological functions and other basic theoretical and technical aspects of the study is lagging behind,restricting the development of its industry speed and level.Lonicera caerulea 'Beilei'fruit rich in anthocyanins,such substances have been confirmed with anti-cell proliferation,induction of tumor cell apoptosis and other functions.In recent years,the extraction of anthocyanins from plant sources,the study of their physiological activities and to be used,has become a hot spot in the field of research and development of plant resources.In this paper,the separation and extraction of anthocyanins from Lonicera caerulea 'Beilei' and the study of antitumor activity were carried out.The method of response surface methodology was used to optimize the process parameters of anthocyanin from Lonicera caerulea 'Beilei'.The purification method of anthocyanin from Lonicera caerulea 'Beilei' was studied by media screening.And the anti-tumor effect and related mechanism of anthocyanin from Lonicera caerulea 'Beilei' were studied systematically from in vitro and in vivo animal models.The results of the study can provide theoretical basis and technical reference for the further development and utilization of the resources.The main results are as follows:(1)The method of ultrasonic countercurrent extraction of anthocyanin from Lonicera caerulea 'Beilei' was established.The optimum technological parameters were as follows:three-step countercurrent extraction,70%(v/v)ethanol aqueous solution,solid-liquid ratio 1:10(g/ml),ultrasonic time 28min,ultrasonic temperature 30 ?,ultrasonic power 120W The yield of buds was 5.22%.Under the optimum conditions.Compared with the ultrasonic method(89.86%),the extraction rate of the process increased by 10%.(2)The separation and purification of different separation media were compared from three aspects:static adsorption,static analysis and dynamic flow elution.A novel resin XDA-6 suitable for preparation of anthocyanin from Lonicera caerulea 'Beilei' was screened out.The pure anthocyanins were separated by this resin,and four anthocyanin groups,B1(25.35±0.23%)?B2(42.91±0.32%)?B3(31.12±0.32%)?B4(24.87±0.26%),with purity of more than 20%were obtained.The purity of the B2 component(purity:42.91 ±0.32%)was 8.06%higher than that of the previous product(34.85%purity)prepared with X-5 resin.(3)Four Lonicera caerulea 'Beilei' anthocyanin(B1-B4,purity higher than 20%)had the best proliferative effect on SMMC-7721 cells,and had no cytotoxicity to normal cells 293T,but their anti-MCF-7 cell proliferation is not obvious.Among the four isolates,B2 group had the best proliferative effect on SMMC-7221 cells(IC50 value was 0.123mg/mL).According to the results of flow cytometry,fluorescence staining and electron microscopy,the B2 component not only had anti-SMMC-7721 cell proliferation activity but also induced apoptosisof SMMC-7721 cells(cell apoptosis rate was 66.32 ± 1.05%).The mechanism of action is:B2 component can block the cell cycle in G2/M phase,DNA damage,reduce mitosis,leading to apoptosis.(4)Each dose group of the B2 anthocyanin component had anti-tumor effect on H22 tumor-bearing mice.The anti-tumor effect of the high dose group was the best,and the tumor inhibition rate reached 46.30 ± 2.20%.And it can significantly improve the survival status of H22 tumor-bearing mice and immune organ function.The anti-tumor activity mechanism is:by increasing the activity of SOD and GSH-Px in mice,the level of lipid peroxidation and the antioxidant capacity were increased.By regulating the levels of immune cytokines IL-2,IFN-?and TNF-a,especially in the liver levels of IL-2 and TNF-?,improve immune activity,direct killing tumor cells.(5)The main anthocyanin type of anthocyanin B2 in Lonicera caerulea 'Beilei' with the highest antitumor activity was.caravir-3-glucoside,and its content was 42.91± 0.32%.