Isolation, Purification And Identification Of α - Glucosidase Inhibitors From Chlorella Growth Factor

In recent years, the incidence of diabetes is increasing, more and more attention has been payed to the anti-diabetes drug. α-glycosidase inhibitor is a new material to treat diabetes, it has been widely used to reduce postprandial hyperglycemia. There are three kinds of α-glycosidase inhibitors in clinical, namely acarbose, voglibose and miglitol, but they are limited to use because of its similar adverse reactions. So choosing an efficient and safe inhibitor is a hot field to treat type II diabetes and develop hypoglycemic health food.Single cell algae chlorella is a kind of unicellular algaehigh, containing protein, low fat, high sugar, a variety of vitamins and minerals, with a variety of nutrition and health care function, it widely distributed, quikly grew and reproducted. Chlorella growth factors(CGF) is different from the other micro algae, which contains a variety of components and has many physiological functions, such as blood glucose, blood pressure and so on. Predictably, CGF has broad application in food, beverage, health care products, medicine and other fields. Until now, only a few literatures that CGF is a new type of α-glycosidase inhibitor have been reported, and they are rare in further study on the isolation and purification. The extraction technology and its physiological activity research have been a hot pot all over the world, such as Europe, America, Japan and Taiwan.For CGF, this paper mainly focus on three aspects: the confirmination of CGF extraction method, the establishment of the best screening model and isolation and identification of active ingredients of CGF.Firstly,on the basis of previous work, with high-pressure hot water extraction preparation technique of CGF(best) on chlorella growth factors(CGF) is extracted, CGF is produced at a rate of 19.6%. High pressure of the water extraction yield CGF is higher, and the operation is simple and economic price.Then, the screening model of the best α-glycosidase inhibitor was established. the best enzymatic reaction conditions were: the substrate concentration for 10 m M, enzyme concentration of 0.28U/ml, the reaction time for 30 min. The model for screening was sensitive and high reliable. And the concentration was positively related to the inhibition rate.Furthermore, the active substances of CGF were identified. Inhibitory activity of macromolecules and small molecules were quantitatively detected by using the above screening model, and to a certain extent, the active region was locked. At the same time, the thermal stability and p H stability. Research shows that, the active substance mainly existed in the small molecule and it has good heat stability, but its alkali stability is not very good.Finally, further purification small molecule of CGF was conducted by chromatography methods orderly. The chromatography technologies contain ion exchange chromatography(732, strong acid type), gel filtration chromatography(Sephadex-G10), thin layer chromatography(TLC) and high performance liquid chromatography( HPLC). The results revaled that it was obtained and has obvious inhibitory activity of α-glucosidase enzyme inhibition activity. The experiment condition are: detection wavelength 210 nm, mobile phase: 95% acetonitrile, flow rate: 1.0ml/min, rentation time: 7.7min and 12.6min. This provides technical guidance for large-scale preparation.