Preparation And Bioactive Activity Of Anthocyanin From Padus Racemosa

Padus racemosa is one of the Rosaceae padus deciduous trees. There are a lot of species in padus, which are widely distributed in our country, the fruit maturation is from May to October, its shape is ovoid and diameter is 8 to 10 mm, its flavor is bitter and sweet. Padus racemosa is rich in nutrition, contains a variety of ingredients. The skin is reddish brown to purple, contains a variety of anthocyanin. Anthocyanin is natural water-soluble pigment. It has become a hot spot in biology, food science and pharmacy because of its biological activity such as hypoglycemic, hypolipidemic, , antioxidation, antitumor and other functions. At present, the research of Padus racemosa focuses on the growth characteristics, cultivation and breeding, but the studies of the anthocyanin mainly focus on the anthocyanin preparation. So the extraction process and main bioactive activity of Padus racemosa anthocyanin were studied. The overall goal of the studies is to provide further information for research and reasonable application of Padus racemosa anthocyanin.In order to determine the optimum extraction conditions of anthocyanin from Padus racemosa, three methods were used, such as ethanol extraction, ultrasonic method and cellulase method. AB-8 macroporous resin was used to study the purification of anthocyanin from Padus racemosa under the condition of static and dynamic. Further, the stability of anthocyanin from Padus racemosa influenced by physical factors (light,temperature and pH),chemistry factors (H2O1,Vc and sucrose) and metal ions(Na+,Ca2+and Al3+) were studied. Its antitumor activity in chemistry was evaluated by DPPH method, salicylic acid method, NBT method. The protection of Padus racemosa anthocyanin on Ins-1 cell was studied with morphological observation, MTT assay, DCFH-DA method and flow cytometry method. At last, in the level of animal, the research established high glucose and high fat mouse model by inject STZ, blood glucose and serum total cholesterol (CHO), triglycerides (TG), and high-density lipoprotein (HDL-C) were measured.The optimum ethanol extraction of Padus racemosa anthocyanin is:material ratio of 1:20 g/mL, ethanol 75%, leaching temperature of 75 ℃, for 1.5 h, under this condition, anthocyanin content is 0.792±0.012 mg/g. The optimum ultrasonic method of Padus racemosa anthocyanin is:the material ratio of 1:30 g/mL, ethanol 60%, ultrasonic power 300 W, ultrasonic time 45 min, under the optimum technological conditions, Padus racemosa anthocyanin content is 0.897±0.023 mg/g. The optimum cellulase method of Padus racemosa anthocyanin is:temperature 60℃ , enzyme dosage 9 mg/g, material ratio of 1:35 g/mL, pH 3.5. Under the optimum conditions, anthocyanin content is 0.956±0.027 mg/g. Those three methods were compared, acidic cellulase method has the highest extraction rate of anthocyanin.The macroporous resin AB-8 was used to purify Padus racemosa anthocyanin. Eventually, the research of the static case were determined, the best condition: anthocyanin concentration of crude extract is 8 mg/mL, temperature is 20 ℃,50 mL anthocyanin 8 g resin add coarse extraction solution, desorption agent of 70% ethanol, the pH value is 3 and the temperature is 60 ℃, the determination of anthocyanin natural pigment is 31.4. On the dynamic case, the optimal condition:the concentration of the sample solution is 8 mg/mL, the adsorption velocity is 0.01 mL/s, column 2 times, desorption agent ethanol concentration is 70%, the flow rate is 0.01 mL/s, the pH value is 3, the determination of anthocyanin natural pigment is 57.1. Two methods were compared, dynamic purification has better effect than static purification. And the stability experimental results show the stability of Padus racemosa anthocyanin was easily influenced by the physical factors (light, temperature and pH), it should be stored in the dark, low temperature and acid medium. The chemistry factors (H2O2) have a hypochromic effect on the Padus racemosa anthocyanin, it should avoid contacting with oxidants. Therefore, reducing agent Vc, food additive sucrose and metal ions (Na+, Ca2+, Al3+) have no significant effects on Padus racemosa anthocyanin.Padus racemosa anthocyanin have strong antioxidant activity in vitro in a certain concentration range, it can scavenge DPPH radical, O radical and OH radical, but its scavenging ability is weaker than Vc. The Ins-1 cell- can be protected by Padus racemosa anthocyanin through morphological observation and MTT assay, the Padus racemosa anthocyanin have strongly scavenge reactive oxygen species in the method of DCFH-DA. Therefore, the result of Padus racemosa anthocyanin detected by flow cytometry also showed that it can inhibit apoptosis. At last, the experimental result in animal showed that anthocyanin of Padus racemosa can effectively reduce the blood glucose and have the function of reducing blood lipid, but the exact mechanism needs to be further studied.