Effect Of Pomegranate Flower Polyphenol On Glucose Of Diabetes Rats And Mechanism Research

Objective:Pomegranate flower in traditional Uygur medicine is wildly used for the treatment of the disease with a little "as" san-duo symptom, modern medical research have found that pomegranate flower extract has effective hypoglycemic activity, In this study, effect of PFP on blood glucose, blood lipids and insulin resistance of diabetic rats were observed through extraction of pomegranate flower polyphenols （PFP） and establishment of diabetic rats, then hypoglycemic mechanism of PFP was further explored, which provide a theoretical data and basic information for the development and effective use of Uighur local herbs.Methods:PARTâ… Pomegranate flower polyphenol extractionD101 macroporous resin purification method was used to extract pomegranate polyphenols; with total phenol content as evaluation index, finally 68% of pomegranate flower polyphenols （PFP） were got from the effective part of pomegranate flower. And orthogonal experiment was used to study different factors on the leaching rate of pomegranate flower polyphenols in order to get optimum extraction process as follows: pomegranate flower was extracted for 2 times at 50℃, each extraction time covered 1h; the solution with 60% ethanol and solid-liquid was mixed as 1∶20 （g∶ml） .PARTâ…¡Pharmacodynamic studies of PFP on diabetic rats1.Establishment of diabetic rats modelsThe models is established by feeding with high fat diet plus intraperitoneal injection of streptozotocin （STZ） 45mg·kg^-1.The rats whose fasting plasma glucose （FPG） ≥11.1mmol·L^-1 are identified as diabetic model rats. Diabetic model rats tend to drink, eat and urinate more than normal and like lying down, whose hair become gloomy and FPG, insulin levels insulin resistance become higher. Characteristics of model rats close to the human type 2 diabetes.2.Administration and Measurement of indexesWith metformin (100mg·kg^-1) as a positive control, diabetes rats fed with pomegranate flower polyphenols (100mg·kg^-1, 300mg·kg^-1) dose respectively for four weeks, then Weight, FPG, GLR, Fins, IRI, lipid profiles were detected.Results: Compared with diabetic models rats, fasting plasma glucose of rats in two-dose group and metformin group effectively reduced, as well as plasma triglycerides, low-density lipoprotein cholesterol, total cholesterol. while ratio of high-density lipoprotein to total cholesterol （HDL-C/TC） was higher. （P<0.05, P<0.01）; Serum insulin levels as well as IRI were reduced significantly, while ISI was effectively elevated in two dose group. （P<0.05, P<0.01）. In addition, PFP showed a protective effect on liver tissue and islet B cells of diabetic rats by pathology examination under light microscopy.PARTâ…¢Hypoglycemic mechanism of PFP1.Effects of PFP on glycogen synthesis and mechanism research Effects of PFP on glycogen synthesis were evaluated by measuring the following items: MG and HG content, skeletal muscle HK activity ,GK activity; then Western Blot assay was applied for checking GSK-3βand AMPKαandP-AMPKαprotein expression in skeletal muscle .Results: Compared with diabetic models rats, MG level in both group （P<0.05, P<0.01） and HK activity in PFPâ…¡group （P<0.05） were effectively elevated; however HG and GK activity in liver showed no significant change （P>0.05）; GSK-3βprotein expression effectively reduced in PFPâ… group （P<0.01）; P-AMPK/AMPK significantly increased in PFPâ… group （P<0.05）.2.Effect of PFP on IR of diabetic ratsSerum FFA was measured by UV-visible spectrophotometry; serum IL-6 and IL-18 were checked by ELISA method; antioxidant activity of PFP was evaluated by clearance of DPPH·and·OH in Mn²⁺-H₂O₂-PAR system in Vitro; then SOD、CAT、GSH and MDA in liver and skeletal muscle were detected respectively; PPARα/γmRNA level by RT-PCR and PPARα/γprotein expression by Western blot were measured.Results: Compared with model rats, 1)Serum FFA （P<0.05） and IL-6 （P<0.01） were significantly higher in two-dose group; however IL-18 had no significant change （P>0.05）. 2) In vitro, PFP (0.08mg·ml^-1) in the clearance rate of·OH 46%is about three times of Vc (0.2mg·ml^-1) in Mn²⁺-H₂O₂-PAR system. PFP (0.02mg·ml^-1) with scavenging rate of DPPH·87%is about 5 times of Vc (0.2mg·ml^-1). Antioxidant activity of PFP in liver: Compared with model rats,CAT significantly increased in both PFP group （P<0.05）; SOD was significant higher （P<0.01） in PFPâ…¡group; MDA was significant lower in PFPâ… group （P<0.05）. In skeletal muscle : SOD were significantly elevated in PFPâ…¡group （P<0.01）; MDA was significantly reduced, GSH was significant increased in both PFP group. （P<0.05, P<0.01）. 3) Compared with model rats,PPARγmRNA （P<0.01） in both dose group and PPARαmRNA in PFPâ… group （P<0.01） significantly increased; PPARγprotein expression in PFPâ… group （P<0.05） and PPARαprotein expression in both PFP group （P<0.05）, were effectively upgrulated.3.Effect of PFP on vascular endothelium of diabtic ratsPlasma ET-1, ATâ…¡, TXB2 and 6-Keto-PGF1a were detected by ELISA. Results: compared with model rats, ATâ…¡and ET-1 concentrations were significantly reduced in two-dose group （P<0.05）, and significant reduction of TXB2 （P<0.01） and increment of 6-Keto-PGF1a （P<0.01; P<0.05） caused a significant reduction in TXB2 and 6-Keto-PGF1a ratio （P<0.01）.Conclusion:1) PFP perform hypoglycemic function in diabetic rats, possibly related to the following aspects: reduction of blood glucose in diabetic rats was probably induced by incremental glycogen content of skeletal muscle.and incremental skeletal muscle glycogen content was possibly caused by enhancement of hexokinase activity, which is the rate-limiting enzyme in glycogen synthesis process. In addition, another possible hypoglycemic mechanism was related to reduction of the protein expression of GSK-3βand significant enhancement of AMPK activity. 2) PFP are able to alleviate insulin resistance level in diabetic rats: the possible mechanism related to the following aspects: reducution of serum TG, FFA and serum inflammatory mediators IL-6 in diabetic rats and antioxidant activity; moreover, PFP are able to improve insulin sensitivity in liver tissue of diabetic rats by enhancing both protein expression of PPARγand PPARα, function as dual PPARα/γagonists and improve insulin sensitivity in skeletal muscle by reducing GSK-3βexpression and enhancing AMPK activity in skeletal muscle. 3) Protective effect of PFP on vascular endothelial of diabetic rats: the results of plasma ET-1, ATâ…¡, TXB2 and 6-Keto-PGF1a suggested that: PFP may have preventative and protective effect on vascular complications of type 2 diabetes.