Antidiabetic Activity and Potential Mechanism of Amentoflavone in Diabetic Mice

Aim: To investigate the anti-diabetic activity of amentoflavone (AME) in diabetic mice, and to explore the potential mechanisms. Methods: Diabetic mice induced by high fat diet and streptozotocin were administered with amentoflavone for 8 weeks. Biochemical indexes were tested to evaluate its anti-diabetic effect. Hepatic steatosis, the histopathology change of the pancreas was evaluated. The activity of glucose metabolic enzymes, the expression of Akt and pAkt, and the glucose transporter type 4 (GLUT4) immunoreactivity were detected. Results: AME decreased the level of glucose, total cholesterol (TC), triglyceride (TG), low density lipoprotein cholesterol (LDL-C) and glucagon, and increased the levels of high density lipoprotein cholesterol (HDL-C) and insulin. Additionally, AME increased the activity of glucokinase (GCK), phosphofructokinase-1 (PFK-1), and pyruvate kinase (PK), and inhibited the activity of glycogen synthase kinase-3 (GSK-3), phosphoenolpyruvate carboxykinase (PEPCK), and glucose-6-phosphatase (G-6-Pase). Mechanistically, AME increased superoxide dismutase (SOD), decreased malondialdehyde (MDA), activation of several key signaling molecules including pAkt (Ser473), and increased the translocation to the sedimenting membranes of GLUT4 in skeletal muscle tissue. Conclusions: AME exerted anti-diabetic effects by regulating glucose and lipid metabolism, perhaps via anti-oxidant effects and activating the PI3K/Akt pathway. Our study provided novel insight into the role and underlying mechanisms of AME in diabetes.


Introduction
Diabetes mellitus is a complex, heterogeneous, and polygenic disease which is characterized by increasing circulating glucose concentrations associated with abnormalities in carbohydrate, protein and fat metabolism caused by the complete or relative insufficiency of insulin secretion and/or insulin action. According to the International Diabetes Federation, there will be around 693 million people suffering from diabetes in 2045 [1].
Treatment of diabetes mellitus involves diet control, exercise and the use of insulin and/or oral hypoglycemic drugs. However, many oral hypoglycemic agents have a number of serious adverse effects [2]. The adverse reactions limit the use of antidiabetic drugs. Therefore, looking for new antidiabetic drugs which have little adverse reaction appears to be very urgent. Traditional and natural indigenous medicines on diabetes prevention and treatment have become a research focus, and a large number of natural plants/plant products have been evaluated for their anti-diabetic effects [3]. Table 1. Effects of amentoflavone (AME) on body weight and Fasting Blood Glucose (FBG) levels in diabetic mice ( _ x ± SD, n = 10).

Effects of AME on Oral Glucose Tolerance Test in Diabetic Mice
Results of the oral glucose tolerance test conducted on diabetic mice fed with AME are shown in Table 2 in diabetic control mice, blood glucose reached the highest level at 30 min after oral glucose ingestion, and this hyperglycemia was maintained until 60 min, and then began to decrease. AMEII significantly prevented (p < 0.05) the increase in blood glucose levels after glucose administration at 120 min in comparison with the diabetic control group. AMEI could also decrease blood glucose levels at different time, although these did not reach statistical significance. RG had an obvious hypoglycemic effect at 0 min and 120 min compared with the diabetic group (p < 0.05).  Table 3 showed the levels of insulin and glucagon in serum in each group, it indicated that the level of insulin decreased significantly in diabetic control mice (p < 0.01) while the level of glucagon increased significantly (p < 0.01), whereas the RG, AMEI, AME II increased the insulin levels, and the RG and AME II showed significant changes when compared with the diabetic control mice (p < 0.01), moreover, the RG, AME II decreased the glucagon levels significantly (p < 0.01). Table 3. Effects of AME on insulin and glucagon levels in diabetic mice ( _ x ± SD, n = 10).

Group
Insulin (

Effects of AME on Lipids and Lipoprotein in Diabetic Mice
As shown in Table 4, the serum levels of TG, TC, HDL-C and LDL-C were significantly raised (p < 0.05, p < 0.01) compared with normal control mice. After 8 weeks of treatment, the RG, AMEI, AME II could significantly lowered the levels of TG, LDL-C (p < 0.05, p < 0.01), while the RG, AMEI, AME II could significantly increase the HDL-C level in comparison to the diabetic controls (p < 0.01). Although the whole AME group could decrease the level of TC, only the AME II showed an obvious trend (p < 0.05), and RG could also decrease the level of TC significantly (p < 0.05). 2.5. Effects of AME on the Level of Alanine Aminotransferase (ALT), Aspartate Aminotransferase (AST), Blood Urea Nitrogen (BUN) and Creatinine (CREA) in Serum and Liver iNdex and Kidney Index in Diabetic Mice As shown in Table 5 that the levels of ALT, AST and liver index had significantly increased in diabetic mice compared with normal mice (p < 0.01), and the AMEI, AMEII and RG significantly decreased the level of ALT, AST and liver index (p < 0.01) in comparison to diabetic control mice. The levels of BUN, CREA and kidney index of all the groups have no obvious change. Table 5. Effects of AME on the level of ALT, AST, BUN and CREA in serum and liver index and kidney index in diabetic mice ( _ x ± SD, n = 10).

Effects of AME on Hepatic Steatosis
AS is shown in Figure 1, there is a high degree of steatosis with severe cytoplasmic vacuoles and swelling of hepatocytes of diabetic control mice. After being fed with AME, there is an obvious prevention of the fatty deposition in hepatocytes. Particularly, AMEII showed a greater improvement of the steatosis and had almost the same effect as RG.

Effects of AME on Hepatic Steatosis
AS is shown in Figure 1, there is a high degree of steatosis with severe cytoplasmic vacuoles and swelling of hepatocytes of diabetic control mice. After being fed with AME, there is an obvious prevention of the fatty deposition in hepatocytes. Particularly, AMEII showed a greater improvement of the steatosis and had almost the same effect as RG.  Figure 1. Effects of AME on hepatic steatosis in mice. The tissues were surgically excised and subjected to histological study by staining with hematoxylin and eosin. Magnification: 400×. The scale bar represents a length of 20 µm.

Effects of AME on the Histopathology Change of the Pancreas
As was shown in Figure 2, the pancreatic sections of the DC group mice stained with HE showed the severe necrotic changes of pancreatic islets, especially in the center of the islets. Nuclear changes, karyolysis, disappearing of nucleus and in some places residue of destructed cells were visible. The relative reduction of the size and number of islets, especially around the large vessel, and a severe reduction of the number of B cells was obvious. Administration of AME and RG led to the protection of the histomorphologic change of the pancreas, increasing the number of the B cells. In particular, AMEII almost repaired the histomorphologic change of the pancreas. As was shown in Figure 2, the pancreatic sections of the DC group mice stained with HE showed the severe necrotic changes of pancreatic islets, especially in the center of the islets. Nuclear changes, karyolysis, disappearing of nucleus and in some places residue of destructed cells were visible. The relative reduction of the size and number of islets, especially around the large vessel, and a severe reduction of the number of B cells was obvious. Administration of AME and RG led to the protection of the histomorphologic change of the pancreas, increasing the number of the B cells. In particular, AMEII almost repaired the histomorphologic change of the pancreas.  Table 6 indicated that the MDA level significantly increased (p < 0.01) whereas the SOD activity significantly decreased (p < 0.01) in diabetic mice compared with normal control mice. AME II and RG significantly increased (p < 0.01) the SOD activity, AME II and RG significantly decreased (p < Figure 2. Effects of AME on the histopathology of the pancreas histomorphologic change of pancreas. The tissues were surgically excised and subjected to histological study by staining with hematoxylin and eosin. Magnification: 400×. The scale bar represents a length of 20 µm. Table 6 indicated that the MDA level significantly increased (p < 0.01) whereas the SOD activity significantly decreased (p < 0.01) in diabetic mice compared with normal control mice. AME II and RG significantly increased (p < 0.01) the SOD activity, AME II and RG significantly decreased (p < 0.01) the MDA level, while the AMEI had no obvious change compared with the diabetic control group. Table 6. Effects of AME on malondialdehyde (MDA) level and superoxide dismutase (SOD) activity in diabetic mice ( _ x ± SD, n = 10).  Table 7 showed that the activity of GCK, PFK-1 and PK of the diabetic group decreased significantly (p < 0.05, p < 0.01), while the activity of GSK-3, PEPCK and G-6-Pase of the diabetic group increased obviously (p < 0.05, p < 0.01) compared with normal control mice. The AMEI, AME II and RG could significantly increase the activity of GCK, PFK-1 and PK (p < 0.05, p < 0.01), and decrease the activity of GSK-3, PEPCK and G-6-Pase (p < 0.05, p < 0.01) in comparison to diabetic control mice. Table 7. Effects of AME on the activity of glucose metabolic enzymes in liver of diabetic mice ( _ x ± SD, n = 10).

Effects of AME on Protein Expression of Akt Serine Phosphorylation (Ser-473)in Muscle of Diabetic Mice
As is shown in the Figures 3 and 4. The pAkt(Ser-473) expression of the diabetic control mice significantly decreased 61% in diabetic control mice than that in the normal control mice, and the AME II significantly increased pAkt(Ser-473) expression by 69.23% above that of diabetic control mice (p < 0.01), and the RG group could increase the expression of pAkt(Ser-473) significantly (p < 0.01).  Table 7 showed that the activity of GCK, PFK-1 and PK of the diabetic group decreased significantly (p < 0.05, p < 0.01), while the activity of GSK-3, PEPCK and G-6-Pase of the diabetic group increased obviously (p < 0.05, p < 0.01) compared with normal control mice. The AMEI, AME II and RG could significantly increase the activity of GCK, PFK-1 and PK (p < 0.05, p < 0.01), and decrease the activity of GSK-3, PEPCK and G-6-Pase (p < 0.05, p < 0.01) in comparison to diabetic control mice. Table 7. Effects of AME on the activity of glucose metabolic enzymes in liver of diabetic mice (⎯x ± SD, n = 10).

Effects of AME on Protein Expression of Akt Serine Phosphorylation (Ser-473)in Muscle of Diabetic Mice
As is shown in the Figures 3 and 4. The pAkt(Ser-473) expression of the diabetic control mice significantly decreased 61% in diabetic control mice than that in the normal control mice, and the AME II significantly increased pAkt(Ser-473) expression by 69.23% above that of diabetic control mice (p < 0.01), and the RG group could increase the expression of pAkt(Ser-473) significantly (p < 0.01).    . The effect of AME on pAkt, AKT proteins (n = 10), # p < 0.05, ## p < 0.01 compared with normal control group; * p < 0.05, ** p < 0.01, compared with diabetic control group.

Effects of AME on GLUT4 Immunoreactivity in the Skeletal Muscle Tissue of Diabetic Mice
As shown in Figures 5 and 6, the GLUT4 immunoreactivity in sections of skeletal muscle from all subjects expressed distinct granular reactions in association with the cell surface. Few grains were found in deeper parts of the cytoplasm. Due to its distinct granular appearance, the GLUT4 immunoreactivity could be quantified by counting. The GLUT4 integral optical density (IOD) of the diabetic control mice significantly decreased compared with that of normal control mice (p < 0.01), and the RG, AMEI, AME II could significantly increase the GLUT4 IOD (p < 0.01).  . The effect of AME on pAkt, AKT proteins (n = 10), # p < 0.05, ## p < 0.01 compared with normal control group; * p < 0.05, ** p < 0.01, compared with diabetic control group.

Effects of AME on GLUT4 Immunoreactivity in the Skeletal Muscle Tissue of Diabetic Mice
As shown in Figures 5 and 6, the GLUT4 immunoreactivity in sections of skeletal muscle from all subjects expressed distinct granular reactions in association with the cell surface. Few grains were found in deeper parts of the cytoplasm. Due to its distinct granular appearance, the GLUT4 immunoreactivity could be quantified by counting. The GLUT4 integral optical density (IOD) of the diabetic control mice significantly decreased compared with that of normal control mice (p < 0.01), and the RG, AMEI, AME II could significantly increase the GLUT4 IOD (p < 0.01).  . The effect of AME on GLUT4 immunoreactivity in the skeletal muscle tissue of diabetic mice. (n = 10), # p < 0.05, ## p < 0.01 compared with normal control group; * p < 0.05, ** p < 0.01, compared with the diabetic control group.

Discussion
Insulin resistance and insufficiency of insulin secretion are the basis of type 2 diabetes [6]. Insulin resistance is a condition where the body tissues become resistant to insulin, resulting in a marked decrease of glucose metabolism in response to insulin [7]; which is associated with dyslipidemia [8].  . The effect of AME on pAkt, AKT proteins (n = 10), # p < 0.05, ## p < 0.01 compared with normal control group; * p < 0.05, ** p < 0.01, compared with diabetic control group.

Effects of AME on GLUT4 Immunoreactivity in the Skeletal Muscle Tissue of Diabetic Mice
As shown in Figures 5 and 6, the GLUT4 immunoreactivity in sections of skeletal muscle from all subjects expressed distinct granular reactions in association with the cell surface. Few grains were found in deeper parts of the cytoplasm. Due to its distinct granular appearance, the GLUT4 immunoreactivity could be quantified by counting. The GLUT4 integral optical density (IOD) of the diabetic control mice significantly decreased compared with that of normal control mice (p < 0.01), and the RG, AMEI, AME II could significantly increase the GLUT4 IOD (p < 0.01).

Discussion
Insulin resistance and insufficiency of insulin secretion are the basis of type 2 diabetes [6]. Insulin resistance is a condition where the body tissues become resistant to insulin, resulting in a marked decrease of glucose metabolism in response to insulin [7]; which is associated with dyslipidemia [8]. Figure 6. The effect of AME on GLUT4 immunoreactivity in the skeletal muscle tissue of diabetic mice. (n = 10), # p < 0.05, ## p < 0.01 compared with normal control group; * p < 0.05, ** p < 0.01, compared with the diabetic control group.

Discussion
Insulin resistance and insufficiency of insulin secretion are the basis of type 2 diabetes [6]. Insulin resistance is a condition where the body tissues become resistant to insulin, resulting in a marked decrease of glucose metabolism in response to insulin [7]; which is associated with dyslipidemia [8]. Studies have shown that the high fat diet feeding rats develop insulin resistance [9]. Meanwhile, streptozotocin has been known to selectively target and destroy the pancreatic β-cell by necrosis [10]. Our previous studies indicated that the total flavonoids of Selaginella tamariscina (Beauv.) Spring had hypoglycemic and hypolipidemic effects on the diabetic rats induced by high fat diet and low dose streptozotocin [4]. Therefore, we selected the mice model by high-fat diet following low-dose streptozotocin in the study to evaluate the effect of AME on the treatment of type 2 diabetes. Normally, diabetes is detected by measuring glucose blood levels. In addition, diabetic mice have impaired glucose tolerance. Additional load of glucose is found to impair the tolerance further, and an index of insulin sensitivity obtained from the oral glucose tolerance test (OGTT) [11]. Therefore, our research investigated FBG and OGTT during AME treatment. From the results obtained, AME at a dose of 40 mg/kg produced a statistically significant decrease in FBG in comparison to the diabetic control group. From the data obtained in the OGTT, it was clear that AME at a dose of 40 mg/kg blocked an increase in blood glucose levels after 120 min of glucose administration clearly, these results imply that AME has the ability to increase peripheral utilization of glucose, it has the same hypoglycemic effect as rosiglitazone.
The glucose metabolism is complex. At the molecular level, a variety of hormones can affect blood glucose levels. Insulin and glucagon play a prominent role in the regulation of glucose metabolism. They vary with the changes in the blood glucose level, and in turn regulate the blood glucose level precisely. Insulin is the unique hormone that down-regulate blood glucose level. In contrast to the action of insulin, glucagon is a hormone promoting the catabolism. It can promote glycogenolysis and gluconeogenesis, and increase the blood glucose level significantly. In the diabetic group, the insulin level was significantly reduced, while the level of glucagon was significantly increased. However, after the administration of AME, the level of insulin was significantly increased and the level of glucagon was significantly decreased. It implies that the regulation of insulin and glucagon may be one of the possible factors responsible for the anti-diabetic activity of AME in diabetic mice.
Diabetes is associated with profound alterations in the plasma lipid and lipoprotein profile. In uncontrolled type 2 diabetes mellitus, there will be an increase in TC, LDL-C, VLDL-C, TG and a decrease in HDL-C which contributes to the coronary artery disease [12]. In addition, hypertriglyceridemia is also an important maker of insulin resistance [13]. In our previous study, a rise in blood glucose was accompanied with the lipid metabolism disorder. In the present study, the rise in blood glucose was accompanied with a marked increase in TC, LDL-C, and TG in diabetic mice. AME could decrease the TC, TG, and LDL-C value while elevate the HDL-C value. It is implying that the AME could alter the disorder of lipid metabolism.
When there is damage in the liver, the level of ALT and AST in serum can increase [14]. BUN is the terminal product of protein metabolism; when kidney was damaged, the level of BUN in the blood increased. Serum creatinine concentration is widely used as an index of renal function [15]. Therefore, we detected the level of ALT, AST, BUN and CREA in serum to show the influence of AME on the liver and kidney function. In the present study, the level of ALT, AST and liver index was significantly increased in diabetic mice, it suggested that the liver was necrotized in diabetic mice. However, there was no obvious change on the BUN, CREA level and kidney index. AME could significantly decrease the level of ALT, AST and liver index; what's more, AME didn't change the level of BUN, CREA and kidney index. It implies that AME could effectively ameliorate hepatic injury in diabetic mice, and AME didn't have renal injury.
Hepatic steatosis is an important marker of metabolic dysfunction, the type 2 diabetes mellitus is likely responsible for many cardiometabolic risk factors associated with hepatic steatosis [16]. In the study, AME ameliorated hepatic lipid accumulation of hepatic steatosis. The high dose of AME had an obvious effect.
In type 2 diabetes mellitus, defective insulin secretion is caused by beta cell dysfunction, and the number of the beta cell was reduced [17]. According to our study, the relative reduction of the size and number of islets, especially around the large vessel, and also severe reduction of the number of B cells was obvious in diabetic mice. AME could protect the histomorphologic change of the pancreas, adding the number of the B cells. In particular, the high dose of AME almost repaired the histomorphologic change of the pancreas.
The balance between the source and outlet of blood glucose keeps the blood glucose at the normal level. The blood glucose is from the digestive absorption of food, glycogenolysis and gluconeogenesis. The outlet of the blood is glycolysis, aerobic oxidation, glycogen synthesis, transforming into other sugars, lipid and amino acid et al. The whole process of the reaction is an enzymatic reaction. Some of enzymes in the reaction play an important role. In the process of glycolysis, hexokinase which is called glucokinase (GCK) in hepatocytes, phosphofructokinase-1(PFK-1) and pyruvate kinase (PK) are very important kinases. GCK can catalyze the phosphorylation of glucose to glucose 6-phosphate. The reduction of GCK activation may involve insulin resistance [18]. PFK-1 catalyzes the transfer of a phosphoryl group from ATP to fructose 6-phosphate to yield fructose 1,6-bisphosphate. PFK-1 reaction is essentially irreversible under cellular conditions, and it is the first "committed" step in the glycolytic pathway. Pyruvate kinase (PK) catalyzes the transfer of the phosphoryl group from phosphoenolpyruvate to ADP, the pyruvate kinase reaction is essentially irreversible under intracellular conditions and is an important site of regulation. The activity of PK plays a very important role in the level of blood sugar [19]. During the process of glycogen synthesis, glycogen synthase kinase-3(GSK-3) retains the activity of glycogen synthase by its phosphorylation, and then the glycogen synthesis is reduced. Restraining the activity of GSK-3 in this way can increase the activity of glycogen synthase, and increase the glycogen synthesis to decrease the level of blood sugar. GSK-3 can regulate the gene expression of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G-6-Pase) in the process of gluconeogenesis [20]. PEPCK is an important kinase in gluconeogenesis. G-6-Pase represents a second important gluconeogenic enzyme and also catalyzes the last step in glycogenolysis [21]. In addition, high fructose diet feeding increased expression of PEPCK and G-6-Pase [22]. In our study, in the diabetic group, the activity of GCK, PFK-1, PK was significantly reduced, while the activity of GSK-3, PEPCK, G-6-Pase was significantly increased compared with the normal group. However, after the administration of AME, we observed a significant increase in the activity of GCK, PFK-1, PK, and a significant decrease in the activity of GSK-3, PEPCK, and G-6-Pase. It is suggested that the pertinent anti-diabetic mechanism of AME may be related to promoting glycolysis, aerobic oxidation and glycogen synthesis, and to restraining the gluconeogenesis. Now, studies have found that oxidative stress has become an important factor in the pathogenesis of diabetes [23]. SOD is the first enzyme of the scavenger enzyme series to protect tissues against oxygen free radicals by catalyzing the removal of superoxide radicals, which damage the membrane and biological structures [24]. MDA reflected the degree of lipid peroxidation and increased malonaldehyde production, which played an important role in the progression of diabetic pancreas damage [25]. Our results corroborated these observations. In the present study, an increase level of MDA and a decreased activity of SOD were noticed in diabetic mice. After the administration of AME for 8 weeks, a significant increase of SOD activities associated with decreased MDA levels in liver, indicating that antioxidative stress could be one of the mechanisms by which AME alleviated the high blood glucose status in diabetic mice.
The disorder of insulin signal transduction plays an important role in the pathogenesis of diabetes, so it has important significance with the study of insulin signal transduction [26], and the metabolic function of insulin is mainly mediated by the PI3K/Akt pathway [27]. The PI3K/Akt pathway is briefly introduced as follows. After insulin binding with an insulin receptor, the PI3K is activated, and then a series of biological effects is produced. In addition, the Akt is activated. Akt is an extremely important signaling protein in the pathway. Studies have shown that the phosphorylation of Akt (Ser473) is the primary phosphorylated form after the activation of Akt [28]. Generally, the phosphorylation level of Akt(Ser473) was used to reflect the activity of the PI3K/Akt pathway. The activation of Akt could obviously improve the impaired ability of GLUT4 translocation from intracellular storage vesicles to the cell membrane of the mice fed with a high fat diet. GLUT4 over expression in db/db mice ameliorates diabetes [29]. In our present study, the Akt protein of diabetic mice had no obvious change. However, the phosphorylation level of Akt(Ser473) of diabetic mice was decreased significantly while the protein of GLUT4 in skeletal muscle was decreased significantly. This suggests that activation of Akt is in disorder, and the effects of insulin cannot respond, leading to the disorder of the glucose metabolism. AME could markedly increase the level of phosphorylation Akt (Ser473) and increase the expression of GLUT4 in skeletal muscle of diabetic mice. This implies that AME may activate the PI3K/Akt pathway and promote GLUT4 transporting glucose to have a hypoglycemic effect.
In summary, this research revealed that amentoflavone, an active compound of Selaginella tamariscina (Beauv) Spring, had the effect of ameliorating the glucose, lipid metabolism disorder, and the hepatic lipid accumulation of hepatic steatosis, and repairing the histomorphologic change of pancreas. Moreover, AME had the effect of regulating the enzyme activity of glucose metabolism, increasing the insulin secretion and improving the insulin signal transduction in target tissues. These benefits may be via anti-oxidant effects and activating the PI3K/Akt pathway. However, evaluation of the hypoglycemic of amentoflavone in the complex mechanism needs to be further explored.

Preparation of AME
Dried Selaginella tamariscina (Beauv) Spring was purchased from the Ben Cao Guo Yao Tang (Zhengzhou, China) and appraised by Chen Sui-qing of the Henan University of Traditional Chinese Medicine. AME was isolated from Selaginella. The purity of the AME prepared in our laboratory is more than 98% as determined by assays of high performance liquid chromatography (HPLC) (Figure 7). All doses were given AME powder dissolved in distilled water according to the weight of the mice. mice was decreased significantly while the protein of GLUT4 in skeletal muscle was decreased significantly. This suggests that activation of Akt is in disorder, and the effects of insulin cannot respond, leading to the disorder of the glucose metabolism. AME could markedly increase the level of phosphorylation Akt (Ser473) and increase the expression of GLUT4 in skeletal muscle of diabetic mice. This implies that AME may activate the PI3K/Akt pathway and promote GLUT4 transporting glucose to have a hypoglycemic effect. In summary, this research revealed that amentoflavone, an active compound of Selaginella tamariscina (Beauv) Spring, had the effect of ameliorating the glucose, lipid metabolism disorder, and the hepatic lipid accumulation of hepatic steatosis, and repairing the histomorphologic change of pancreas. Moreover, AME had the effect of regulating the enzyme activity of glucose metabolism, increasing the insulin secretion and improving the insulin signal transduction in target tissues. These benefits may be via anti-oxidant effects and activating the PI3K/Akt pathway. However, evaluation of the hypoglycemic of amentoflavone in the complex mechanism needs to be further explored.

Preparation of AME
Dried Selaginella tamariscina (Beauv) Spring was purchased from the Ben Cao Guo Yao Tang (Zhengzhou, China) and appraised by Chen Sui-qing of the Henan University of Traditional Chinese Medicine. AME was isolated from Selaginella. The purity of the AME prepared in our laboratory is more than 98% as determined by assays of high performance liquid chromatography (HPLC) ( Figure  7). All doses were given AME powder dissolved in distilled water according to the weight of the mice.

Experimental Model and Drug Treatment
Mice were maintained under standard laboratory conditions (temperature: 25 ± 2 • C, humidity: 60 ± 5%, 12 h dark/light cycle), and fed with a standard laboratory diet and fed a standard laboratory diet and water.
After a 1-week acclimation period, mice were randomly divided into two groups. The normal control group (10 mice) was fed with a basic diet, whereas the experimental group was fed with a high fat diet (consisting of 18% fat, 20% carbohydrate, 3% egg and 59% basic diet (w/w) which made by the Animal Experimental Center of Zhengzhou University) for a period of 8 weeks. After 8 weeks of dietary manipulation, the experimental mice were fasted overnight and were intraperitoneally injected with a freshly prepared solution of streptozotocin (100 mg/kg) in 0.1M citrate buffer (pH 4.21) to induce diabetic model, while the normal control mice were given the 0.1M citrate buffer in a dose volume of 1 mL/kg respectively. The mice with fasting plasma glucose level of above 11.1 mmol/L 72 h after injection of streptozotocin were considered diabetic and only uniformly diabetic mice were induced in the study.
The mice were divided into six groups: Group NC-normal control mice; Group DC-diabetic control mice; Group RG-diabetic mice treated with rosiglitazone (4 mg/kg, ig.); Group AMEI-diabetic mice treated with AME (20 mg/kg, ig.); Group AME II-diabetic mice treated with AME (40 mg/kg, ig.); the mice were treated for 8 weeks. Blood samples were collected from the mice fasted for 4 h previously every 4 weeks, and serum glucose levels were estimated. OGTT was performed the day before mice were sacrificed. At the end of the experiment, blood samples were collected from the eyes (venous pool) and centrifuged at 2900× g for 10 min to separate the plasma from the whole blood and stored at −80 • C until assayed. The hepatics and skeletal muscles were harvested, frozen in liquid N 2 , and subsequently stored at −80 • C until required.
All experimental animals were overseen and approved by the Institutional Animal Care and Use Committee of Henan University of Traditional Chinese Medicine before and during experiments.

Oral Glucose Tolerance Test (OGTT)
An oral glucose tolerance test (OGTT) was performed using an oral dose of glucose (2 g/kg) for 2 h after 8 consecutive weeks of AME or rosiglitazone treatment. Animals were food-restricted and were given only water to drink throughout the time prior to the OGTT procedure. The blood samples were collected from each group just before glucose administration (0 min) and at 30, 60 and 120 min after glucose administration. Plasma glucose concentrations were determined by the glucose oxidase method.

Biochemical Assays
Glucose levels were estimated by commercially available glucose kits based on the glucose oxidase method. TC, TG, HDL-C, LDL-C, ALT, AST, BUN and CREA levels were measured by using commercial assay kits according to the manufacture r 's directions. The liver index and kidney index were calculated as follows: liver index = the weight of the liver/the body weight, kidney index = the weight of the kidney/the body weight. The contents of MDA and the activity of SOD were determined by commercially available kits according to the manufacturer's instructions.

Morphological Evaluation
The liver and the pancreas were stored in 10% formalin, and then the tissue was embedded in paraffin, sliced at 5 µm thickness, and dyed with hematoxylin and eosin (HE) staining. The pathological changes of the liver and the pancreas were assessed and photographed under an Olympus BX-51 microscope.

Enzyme-Linked Immunosorbent Assay (ELISA)
The liver homogenate was prepared, and then the activity of GCK, PFK-1, PK, GSK-3, PEPCK, G-6-Pase was analyzed by the enzyme-linked immunosorbent assay (ELISA) method according to the respective manufacturer's instructions.

Western Blot Analysis
Protein was extracted from tissues following the commercial kits' instructions. Protein concentration was determined by using the BCA protein assay kit. Equal amounts of protein samples were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to the PVDF membrane. The membrane was blocked in 5% (v/v) non-fat milk in TBST (20 mM Tris-HCL, 137 mM NaCl, 0.1% (v/v) Tween-20) for 2 h at room temperature, and then blotted with a primary antibody (Akt at 1:500, pAkt at 1:2500) overnight at 4 • C. After being washed (6 × 5 min) in TBST buffer, the membrane was detected with the second anti-body (1:1000) for 1h at room temperature, followed by additional washes (6 × 5 min) in TBST. Bound antibody was visualized by enhanced chemiluminescence. The intensity of target proteins and reference protein were quantified using Gene tools. The mean value for samples from normal group mice on each immunoblot, expressed in densitometry units, was adjusted to a value of 1.0. All experimental sample values were then expressed relative to this adjusted mean value.

Immunohistochemical Assay
Skeletal muscle tissue samples were fixed for 48 h, embedded in paraffin, and then sliced. The thickness of the paraffin section was 5 µm. The section was dewaxed to water, then the 3% H 2 O 2 was used to inactive endogenous enzymes for 10 min at room temperature, then being washed (3 × 3 min) in PBS (135 mM NaCl, 2.7 mM KCl, 1.5 mM KH 2 PO 4 , 8 mM Na 2 HPO 4 , pH 7.2). The section was immersed in the citrate buffer (pH 6.0), and heated by microwave, and then cooled, and washed (3 × 3 min) in PBS. The blocking solution I was added in the section for 20 min at room temperature, and then the excess liquid was shaken out. The section was incubated for 1 h with rabbit anti-GLUT4 antibody (1:500), followed by additional washes (3 × 3 min) in PBS. Then the biotinylated goat anti-rabbit antibody was added in the section for 50 min at 37 • C, and then being washed (3 × 3 min) in PBS; then the peroxidase complex was added in the section for 50 min at room temperature, followed by additional washes (3 × 3 min) in PBS. Then the section was colored by the DAB kit; and then the section was observed and photographed by electric microscope (OLYMPUS BX61). Moreover, the photos were analyzed by Image pro plus6.0. (3 paraffin sections were chosen for each group, and 5 photos were taken for each section.)

Statistical Analysis
Results are presented as mean ± SD, and the comparison between groups were performed by one-way ANOVA. p values < 0.05 were considered statistically significant.

Conclusions
AME exerted anti-diabetic effects by regulating glucose and lipid metabolism, possible via anti-oxidant effects and activating the PI3K/Akt pathway. Our study provided novel insight into the role and underlying mechanisms of AME in diabetes.

Conflicts of Interest:
The authors declare no conflict of interest.