Antidiabetic activity of isoquercetin in diabetic KK -Ay mice
© Zhang et al; licensee BioMed Central Ltd. 2011
Received: 8 October 2011
Accepted: 2 December 2011
Published: 2 December 2011
Tartary buckwheat bran is an important natural source of quercetin and isoquercetin. Quercetin and isoquercetin are both powerful α-glucosidase inhibitors. Although the IC50 of isoquercetin as α-glucosidase inhibitor was much higher than that of quercetin, the bioavailability of isoquercetin was higher than that of quercetin. Hence, we are interested in the antidiabetic effect of isoquercetin in diabetic KK -Ay mice.
The hypoglycemic effect of isoquercetin in a type 2 diabetic animal model (KK-Ay mice) was studied. Isoquercetin was administrated at doses of 50, 100 and 200 mg/kg for 35 days.
It was found that fasting blood glucose concentration was decreased with the 200 mg/kg group (p < 0.01) the most efficient compared with the diabetic control group. In addition, there was significant decrease in plasma C-peptide, triglyceride, total cholesterol and blood urea nitrogen levels after 35 days. Meanwhile, glucose tolerance was improved, and the immunoreactive of pancreatic islets β-cells was promoted.
These results suggest that isoquercetin had a regulative role in blood glucose level and lipids, and improved the function of pancreatic islets. Isoquercetin may be useful in the treatment of type 2 diabetes mellitus.
Diabetes, especially type 2 diabetes, is rapidly becoming an enormous health burden by decreasing quality of life and causing death and disability in the whole world, all at a huge economic cost. Diabetes is, in part, related to the amount of carbohydrates in the diet. Acting as a key enzyme for carbohydrate digestion, intestinal α-glucosidase is one of the glucosidases located at the epithelium of the small intestine. α-Glucosidase has been recognized as a therapeutic target for modulation of postprandial hyperglycemia, which is the earliest metabolic abnormality to occur in type 2 diabetes mellitus [1, 2]. The inhibition on intestinal α-glucosidases would delay the digestion and absorption of carbohydrates and consequently suppress the postprandial hyperglycemia [3, 4].
Tartary buckwheat bran is an important natural source of quercetin and isoquercetin. Quercetin and isoquercetin are both powerful α-glucosidase inhibitors . The IC50 of quercetin was about five times lower than that of acarbose which has been used as effective α-glucosidase inhibitors to delay glucose absorption in clinical . Quercetin significantly decreased the plasma glucose level and improved the biochemical profiles in the streptozocin-induced diabetic rats in a dose-dependent manner .
Diabetic KK-Ay mice have been frequently used as an animal model for noninsulin-dependent diabetes [8, 9]. The symptoms of this animal model are similar to diabetic patients and the mice exhibit metabolism abnormalities such as an absolute or relative lack of insulin, hyperglycemia and glucose intolerance, higher lipid and so on. We are interested in the antidiabetic effect of isoquercetin in KK -Ay mice. Although the IC50 of isoquercetin as α-glucosidase inhibitor was much higher than that of quercetin , the bioavailability of isoquercetin was higher than that of quercetin . Morand et al.  also demonstrated that isoquercetin was better absorbed than quercetin by rats. Furthermore, isoquercetin has been shown to play protective roles against lipid peroxidation and oxidative stress . Accumulated evidence has suggested that diabetic patients are under oxidative stress with an imbalance between the free radical-generating and radical-scavenging capacity. The increased free radical production and reduced antioxidant defense may partially mediate the initiation and progression of diabetes-associated complications [13, 14]. Here we reported our investigations on the effect of isoquercetin on diabetic KK-Ay mice.
Materials and methods
Preparation of isoquercetin from Tartary Buckwheat
The isoquercetin was prepared from tartary buckwheat provided by the Chinese National Genebank (Beijing, China) using the method of Chang and Muir . Briefly, buckwheat was extracted with 50% methanol aqueous solution at 40°C for 3 hours. After vacuum filtration at 50°C, the supernatants were combined and concentrated to one-third of the volume under a reduced pressure using a rotary evaporator. The concentrate was dispersed in 500 ml of water (solid:liquid ratio = 1:100). Then the dispersion was heated to 80°C and the pH adjusted to 4, followed by the addition of naringin enzyme. After further purification by a preparative HPLC method, the isoquercetin purity was 96.7%, which was detected by an analytical HPLC procedure.
Design of animal experiment
Composition of the Experimental Diet
soybean meal (%)
soybean powder (%)
wheat flour (%)
wheat bran (%)
fish powder (%)
Yolk powder (%)
Fasting blood glucose levels and oral glucose tolerance test (OGTT)
Blood samples were taken from the tail vein weekly after overnight fasting. Glucose was determined according to the method of Yao et al.  using glucose analyzes (ACCU-CHEK Active, Roche, Shanghai, China). On the morning of OGTT, fasting animals were given glucose orally (2 g/kg). Blood glucose levels were measured at 0 (before oral glucose), 30, 60, and 120 min after glucose administration.
Plasma biomarker analyses
Blood was collected from abdominal artery into a heparin-coated tube and centrifuged at 1000 g for 15 min at 4°C, the plasma was colleted and stored at -20°C until analysis . Plasma insulin (DSL-1600 Insulin RIA kit, Diagnostic Systems Laboratories, USA) level was measured based on a radio-immunometric assay. C-peptide (ADL, San Diego, CA) and glucagon (RapidBio Laboratory, Calabasas, CA) were determined using commercial enzyme-linked immunosorbent assay kits. Plasma total cholesterol, triglycerides, and blood urea nitrogen (BUN) were measured using an autobiochemical analyzer (Hitachi 7600, Japan) [19–21].
Immunohistochemical evaluation on pancreas
The pancreas was removed immediately after sacrifice and rinsed in ice-cold saline. The tissue samples were fixed in paraformalclehyde, dehydrated in a graded series of ethanol, and embedded in paraffin wax before sectioning. Sections were immersed in a solution of 3% H2O2 for 10 min then preincubated with nonimmune serum for 15 min and subsequently replaced with the mouse anti-insulin antibody (1:200, SP-9000, ZYMED, CA) for incubation at 4°C for 16 h. Biotinylated goat anti-mouse immunoglobulin was used as a secondary antibody. They were labeled with streptavidin peroxidase followed by incubation with the secondary antibody at 37°C for 30 min. The localization of the antigen was indicated by a brown color obtained with 3-amino-9-ethyl-carbazole (AEC) as a chromogenic substrate for peroxidase activity. Slides were counterstained with hematoxylin for microscopic observation. The specificity of the immunohistochemical staining was checked by omission of the primary antibody or by using an inappropriate antibody (antigastrin). More than 10 islets in each mice group were randomly selected and transferred to a pathology image analyzing system. Staining signals of the islets selected on the captured image were converted to gray density which can be automatically calculated as a staining intensity per unit area.
All values were expressed as mean ± SD. Data were analyzed using one-way analysis of variance (ANOVA). Differences with p < 0.05 were considered to be significant.
Results and discussion
Food intake and body weight
Effect of Isoquercetin on Body Weight and Food Intake (week) in Diabetic KK-Ay
Initial body wt (g)
Final body wt (g)
Food intake (g)
control diabetic mice
36.49 ± 1.72
37.51 ± 1.42
11.56 ± 0.97
diabetic mice given 200 mg of isoquercetin/kg
35.43 ± 1.35
36.86 ± 1.87
12.32 ± 1.19
diabetic mice given 100 mg of isoquercetin/kg
37.21 ± 1.79
37.75 ± 1.68
10.08 ± 0.95
diabetic mice given 50 mg of isoquercetin/kg
37.09 ± 1.40
37.93 ± 1.53
11.53 ± 1.62
Fasting blood glucose levels and oral glucose tolerance
Plasma biomarker levels
Isoquercetin (200 mg/kg) significantly lowered the levels of plasma insulin and C-peptide (p < 0.01), which is a byproduct of insulin production. Newly C-peptide levels are measured instead of insulin levels because insulin concentration in the portal vein ranges from two to ten times higher than in the peripheral circulation. The liver extracts about half the insulin reaching it in the plasma, but this varies with the nutritional state. The pancreas of patients with type 1 diabetes is unable to produce insulin and therefore they will usually have a decreased level of C-peptide, whereas C-peptide levels in type 2 patients are normal or higher than normal. Measuring C-peptide in patients injecting insulin can help to determine how much of their own natural insulin these patients are still producing.
Plasma Parameters in Diabetic KK-Ay a
total cholesterol (mmol/L)
control diabetic mice
0.84 ± 0.09
107.73 ± 4.65
1.03 ± 0.12
4.98 ± 0.63
11.79 ± 2.40
diabetic mice given 200 mg of isoquercetin/kg
0.65 ± 0.21 a
67.29 ± 6.37c
0.83 ± 0.14 b
3.79 ± 0.67 a
9.35 ± 0.49 a
diabetic mice given 100 mg of isoquercetin/kg
0.76 ± 0.13
72.81 ± 7.58 b
0.86 ± 0.09 a
4.30 ± 0.36
11.26 ± 0.81
diabetic mice given 50 mg of isoquercetin/kg
0.81 ± 0.26
103.52 ± 8.29
0.90 ± 0.11 a
4.74 ± 0.52
10.94 ± 1.38
The most common lipid abnormalities in diabetes are hypertriglyceridemia and hypercholesterolemia . In the present study, plasma lipids including cholesterol and triglycerides in diabetic mice were elevated (Table 3). Isoquercetin (200 mg/kg) decreased the level of triglyceride and total cholesterol may be due to the increased insulin releasing capacity .
Diabetic hyperglycemia induces the elevation of plasma urea nitrogen, which is considered to be a marker of renal dysfunction . As shown in Table 3, plasma urea in isoquercetin group (200 mg/kg) reduced significantly plasma urea nitrogen by 12.8% (p < 0.05) compared with the value of the diabetic control group, indicating that isoquercetin may capable of ameliorating the impaired diabetic kidney function in addition to its hypoglycemic control.
Immunohistochemical level on pancreas
Isoquercetin had a regulative role in blood glucose level and lipids, and improved the function of pancreatic islets. Isoquercetin may be useful in the treatment of type 2 diabetes mellitus.
The present study was supported by the Talent Fund (for Guixing Ren) of the Chinese Academy of Agricultural Sciences and the Institute Fund (#2060302-2-09) from The Ministry of Sciences and Technology.
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