| Diabetes is a serious disease, although the medicine can effectively control blood glucose, but there are a variety of side effects. A lot of natural compounds can also control the blood glucose with little side effect at lower cost, therefore, they become a research hotpot of hypoglycemic drugs. Moringa oleifera (Moringa oleifera.Lam), also named as drumstick tree (Drumstick tree), is a member of the Moringaceae family. It is a tropical plants of unique economic value. Moringa oleifera leaves contains a large number of bio-active substances, the total flavones was reported to have activity of lower blood glucose, increase the serum SOD and reduce the MDA in diabetes mice. There is no reports of hypoglycemic components and mechanism of Moringa oleifera leaves flavones. This research focused on the extraction and separation of flavones from Moringa oleiferaleaves, and systemic characterization of the mechanisms of its main components isoquercitrin in lowering the blood glucose level. The main contents of this research are as follows:1. The conditions for microwave-assisted extraction of total flavones from Moriga oleifera leaves were optimized by response surface analysis. On the basis of single-factor experiments, the relationship between main extraction conditions including ethanol concentration, microwave power, extraction time and liquid-solid ratio were modeled using a 4-factor,3-level Box-Behnken experimental design. The established model was analyzed by response surface methodology to obtain the optimal extraction conditions. The optimal extraction conditions were as follows: ethanol concentration is 58%, microwave power is 397w, extraction time is 8min, and liquid-solid ratio is 59:1 (mL/g). Under these conditions, the estimated and observed value of maximal extraction yield of total flavonoids were 3.49% and 3.45%, which is consistent with the expected results.2. The optimal separation condition of moringa oleifer folvonse was studied, and its main components were measured. The optimal separation condition by HPD-750 were as follows:the sample concentration is 2.5mg/mL, adsorption time is 2h, in pH6.0; 2BV water is used for washing away surface bound flavones, and then 3BV 70%(volume fraction) ethanol is used for completely eluting the flavones from the resin. The highest recovery rate of flavones was 85.31%, and the purity of flavones was improved to 56.7%, compared to 15.3% as before the purification. The purified flavones were then extracted by petroleum ether, ethyl acetate and butanol; almost all of them exist in ethyl acetate phase, with a purity of 71.8%. The concentration of isoquercitrin, chlorogenic acid, quercetin and kaempferol in crude flavones ethyl acetate extracts, n-butyl alcohol extracts were measured by RP-HPLC. The main component of crude flavones was extracted by ethyl acetate. In the ethyl acetate extracts, isoquercitrin was the highest component, with composition of 41.42%. Isoquercitrin is the main component of Moringa oleifera leaf flavones.3. In vitro study on moringa oleilera and isoquercitrin showed that both of them promoted the glucose consumptionin HepG2 cells within 24h. The Moringa oleifera leaf flavones (0.156mg/mL) promoted the glucose consumption, which resulted in a significant decrease of glucose levels in this cell type. Isoquercitrin(0.156mg/mL) could reach the same significant level, which means isoquercitrin possessed significant hypoglycemic activity. The final inference, Isoquercitrin is probably the main hypoglycemic activity component of Moringa oleifera leaf flavones.4. The activity of isoquercitrin in decreasing glucose level was studied in vivo as well. STZ induced type-2 diabetes rat model were used. Intragastric administration of isoquercitrin (10mg·kg-1·d-1, or 30 mg·kg-1·d-1), sitagliptin (10 mg·kg·-1·d-1) or saline were applied for 21 days. Results demonstrated that isoquercitrin treatments ameliorate clinical symptoms of these diabetic rats, including lower blood sugar level, reduced insulin resistance, less oxidative stress and inflammatory reaction, higher oral glucose tolerance, increased serum insulin and GLP-1 content. Also, isoquercitrin treatments strengthened the function of islet β cells, and its role of reducing the risk of cardiovascular disease in diabetes may be better than sitagliptin.5. The effects of isoquercitrin on pancreatic β cells were observed. Pancreases tissues from all four treatment groups were processed by HE, Gomori stainings. Results showed that high-dose isoquercitrin or sitagliptin treatment appeared to have islet protection effects. Morphologically, islet structure was intact,and islet tissue and acini boundaries were clear, the quantity and quality of healthy islet cells were higher, only a small amount of cells were underwent degeneration or necrosis. Therefore, isoquercitrin could protecet β cells in diabetic rats.6. The effect of isoquercitrin on liver was also evaluated. Liver tissues from all four treatment groups were morphologically examined by HE staining, biochemical indexes of liver were measured by using blood samples, and the cell-cycle distributions and apoptosis rate of liver cells were tested by flow cytometry. The results showed that isoquercitrin had similar effect as sitagliptin on liver tissues. There were less degenerated liver cells, and more healthy liver cells; hepatic cords were arranged as normal, the liver tissues looked normal morphologically; disrupted lipid metabolisms were corrected in these diabetic rats, and liver protein synthesis were maintained; the population of liver cells in G0/G1 phase was reduced, G2+M ratio was increased, therefore, the hepatic cell apoptosis was inhibited, the cell proliferation was promoted. Overall, liver function in isoquercitrin treated diabetic rats were improved and recovered.7. The mechanism of decreasing cellular glucose by isoquercitrin was studied.The expression of several genes in livers of diabetic rats with or without treatments were quantified by fluorescence quantitative PCR. The results showed that, isoquercitrin had similar effects as sitagliptin in modulating diabetic-related signaling pathway. The DPP-4 expression decreased after treatment in diabetic rats, which suggested isoquercitrin is a DPP-4 inhibitors. Isoquercitrin treatment also increased InsR expression, provided more binding sites for insulin, which made body had more hypoglycemic activity.Isoquercitrin treatment increased the expression of PI3K and Akt; activation of the PI3K/Akt pathway may play a role in inhibiting apoptosis and enhancing proliferation of islet and liver cells. Isoquercitrin treatment also increased PKAand PKCa expression, which are important signals in promoting glucose metabolisms by inhibited the glucose synthesis, and promoted glycogen metabolism. The blood glucose level decreased in diabetic rats after isoquercitrin treatment probably through a comprehensive regulation and signaling system.From the above results, isoqueritrin from moringa oleifera leaf flavones has the hypoglycemic activity, the mechanism is mainly reflected in the following ways:First, inhibit the activity of DPP-4, which allows the role of GLP-1 in lowering blood glucose; Second, activation of the PI3K/Akt pathway results in up-regulating InsR,PKA and PKCa expression, promoting the proliferation and inhibiting the apoptosis of islet P cell, increasing insulin secretion, and reducing the blood glucose levels; Third, promote hepatocyte proliferation and inhibit the apoptosis, enhance the liver function in adjusting blood fat, prevent the high fat damage the liver tissues. These effects are all associated with isoquercitrin dose. Under the influence of isoquercitrin, the blood glucose of diabetes rats decreased through a series of comprehensive regulation. |
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