| Diabetes is a metabolic disease that blood glucose higher than normal, with cardiovascular disease and tumor make up human "three health killer". China has become the country that have the largest number of people with diabetes, the incidence of typeⅡdiabetes accounts of about 95%, the trend become the younger age and more mortality. In nature, flavonoids mainly comes from the secondary metabolites of plant, usually with basic mother nucleus of benzchromones. Now, there have found 4000 many kinds of flavonoids, the structure is complicated and it has many pharmacological activities, such as antioxidation, antitumor, hpyerglycemic, hypotensive, bacteriostasis, treat of respiratory system diseases and so on. Flavonoids is one of the important active ingredients of mulberry root bark, it have hpyerglycemic and the effect is remarkable, but the mechanism is unclear. There are great theory significance that explore the hpyerglycemic mechanism of mulberry root bark flavonoids and develop a good hyperglycemic medicine. This study based on the extraction technology of total flavonoids from the mulberry root bark, investigate the extraction time, concentration of ethanol and mass concentration raw materials that have greater influence on the extraction yield of total flavonoids of mulberry root bark. Then, the mulberry root bark extraction through the silicagel column chromatography, separation and purification, and use the HPLC analyze the flavones compounds, establish HPLC fingerprint. On this basis, screening the enzymes activity in vitro of the sample from mulberry root bark. At last, we use the 3T3-L1 preadipocyte cells set up insulin resistance model that investigate the hypoglycemic mechanism of mulberry root bark, for the development of hypoglycemic drugs of mulberry root bark to provide theory and technology support. The main research results as follows:(1) Process optimization of total flavonoids extract from mulberry root bark: select mulberry root bark of tang 10 breed as raw materials, and extraction time, concentration of ethanol and mass concentration raw materials as conditions of single factor, ultrasonic assisted extraction, Box-Benhnken central composite design combined with response surface analysis methodogy(RSM) was further used to optimize the extraction technological condition, the yield of total flavonoids from mulberry root barks as response value, establish regression model, using the Design- Expert software regression analysis of experimental results, decide the ultrasonic assisted extraction technology of total flavonoids from mulberry root bark conditions as follows: extraction time 1.66 h, mass concentration raw material 0.04 g/m L, volume fraction of ethanol 79.70 %. Actual operation revise extracting time 1.6 h, mass concentration raw material 0.04 g/m L, volume fraction of ethanol 80%. Using the response surface analysis optimize extraction technology of total flavonoids from mulberry root bark conditions has good practical application value, for mulberry root bark functional ingredients research laid the raw materials foundation.(2) Extraction, separation and HPLC analysis: It used silicagel column chromatography, prep-HPLC methods for the total flavonoids from mulberry root bark extract and purification, get four compounds, identified as ursolic acid, β-sitosterol, morusin and betulinic acid. At the same time, the flavonoids from mulberry root bark extract with petroleum ether, ethyl acetate and n-butyl alcohol, get the sample of petroleum ether phase, ethyl acetate phase and n-butyl alcohol phase and water phase, using HPLC analyze the fingerprint of flavonoids from mulberry root bark, comparative the flavonoids content of mulberry root bark, the results showed that the most abundant is kaempferide, followed by mulberrin, the least amount is isoquercitrin and Mulberrin E. Through this part of the experiment, for the flavonoids from mulberry root bark HPLC fingerprint setted up laid the theoretical basis, and supply the sample for the enzyme activity tests.(3) Evaluation hypoglycemic activity of sample in vitro: The four monomer compounds and different polar extraction parts from the mulberry root bark as the test samples, comprehensive survey the α-glucosidase, sucrose, PTP1 B enzyme inhibition activity in vitro, and DPPH radical scavenging capacity and FRAP antioxidant capacity in vitro. The results show that the obtained samples from mulberry root bark have the influence of three types enzymes of hpyerglycemic, but the inhibition differences between strong and weak. Crude extraction from mulberry root bark, ethyl acetate extraction phase and morusin have the remarkable inhibiting effection, and at the same time show significant inhibition for the two enzymes(α-glucosidase and PTP1B). In the evaluation of antioxidant activity, n-butanol extraction phase showed strong DPPH radical scavenging ability and total antioxidant capacity(FRAP) at the same time, Crude extraction and ethyl acetate extraction show a strong total antioxidant capacity(FRAP), and morusin is weak of two kinds of antioxidant capacity.(4) Study of hypoglycemic mechanism: using 3T3-L1 preadipocytes set up insulin resistance model, detect the total flavonoid from mulberry root bark influence on proliferation and differentiation, as well as the key factors PPARγ, IR, PI3 K, Akt, GLUT-4, PTP1 B gene expression and protein expression in the PI3K-Akt signaling pathways. The result show as follows, compared with model group, the total flavonoids from mulberry root bark can improve 3T3-L1 adipocytes insulin resistance, up-regulated PPARγ, IR, PI3 K, Akt and GLUT-4 gene expression level, down-regulated PTP1 B gene expression level, but the protein expression not present the same as genes expression, it consider may be related to gene introns, exons and transcriptional regulation or other factors. In conclusion, the total flavonoid from mulberry root bark hypoglycemic effect may be associated with activate PI3K- Akt signaling pathways. |
