Inhibitory activity of Euonymus alatus against alpha-glucosidase in vitro and in vivo.

The major goal in the treatment of diabetes mellitus is to achieve near-normal glycemic control. To optimize both fasting blood glucose and postprandial glucose levels is important in keeping blood glucose levels as close to normal as possible. alpha-Glucosidase is the enzyme that digests dietary carbohydrate, and inhibition of this enzyme could suppress postprandial hyperglycemia. The purpose of this study was to test the inhibitory activity of methanol extract of Euonymus alatus on alpha-glucosidase in vitro and in vivo to evaluate its possible use as an anti-diabetic agent. Yeast alpha-glucosidase inhibitory activities of methanol extract of E. alatus were measured at concentrations of 0.50, 0.25, 0.10, and 0.05 mg/ml. The ability of E. alatus to lower postprandial glucose was studied in streptozotocin-induced diabetic rats. A starch solution (1 g/kg) with and without E. alatus extract (500 mg/kg) was administered to diabetic rats by gastric intubation after an overnight fast. Plasma glucose levels were measured at 30, 60, 90, 120, 180, and 240 min. Plasma glucose levels were expressed in increments from baseline, and incremental areas under the response curve were calculated. Extract of E. alatus,which had an IC(50) value of 0.272 mg/ml, inhibited yeast alpha-glucosidase activity in a concentration-dependent manner. A single oral dose of E. alatus extract significantly inhibited increases in blood glucose levels at 60 and 90 min (p<0.05) and significantly decreased incremental response areas under the glycemic response curve (p<0.05). These results suggest that E. alatus has an antihyperglycemic effect by inhibiting alpha-glucosidase activity in this animal model of diabetes mellitus.

Introduction

Diabetes is the fifth leading cause of death among Koreans (Korea National Statistical Office, 2006). The prevalence of diabetes mellitus is increasing markedly because of an aging population, increased urbanization, and more sedentary lifestyles (King et al.,1998). Keeping blood glucose levels close to normal and preventing diabetic complications are the major goals in the treatment of diabetes mellitus (DCCT Research Group, 1993; UKPDS Group, 1998). Cardiovascular disease (CVD), a major complication of diabetes, is the major cause of morbidity and mortality in patients with diabetes (Centers for Disease Control and Prevention, 1999). Achieving near-normal glycemic control in patients with diabetes mellitus is associated with sustained and decreased rates of diabetes-related cardiovascular complications (DCCT Research Group, 1993; UKPDS Group, 1998). Optimizing both fasting blood glucose and postprandial glucose levels is important in achieving near-normal glucose levels (Abrahamson, 2004). Avignon et al. (1997) reported that postprandial glucose levels could be a better marker of glycemic control than fasting blood glucose levels in patients with type 2 diabetes. Microvascular and macrovascular complications are strongly associated with postprandial hyperglycemia (Baron, 1998; Haller, 1998; Jenkins et al.,1988; Mooradian & Thurman, 1998).

At present, α-glucosidase inhibitors are the most common oral agents used to decrease postprandial hyperglycemia, since they can delay the digestion of dietary carbohydrates, resulting in retardation of glucose absorption (Saito et al.,1998; Sels et al., 1999; Stand et al., 1999). In addition, numerous studies have been carried out to isolate effective and safe α-glucosidase inhibitors from natural products, including plant materials, as alternative hypoglycemic agents for diabetes that can be used in addition to conventional treatments (Joo et al., 2006; Li et al., 2005; Shim et al., 2003; Youn et al.,2004).

Euonymus alatus, known as 'gui-jun woo' in Korea, has been used in Asian folk medicine to treat tumors, regulate blood circulation, and relieve pain in countries including Korea and China (Kim et al., 2006; Park et al., 2005). However, there is not enough scientific evidence to support the medical use of E. alatus. Euonymus alatus has anticancer (Lee et al., 1993) and antioxidative (Oh et al.,2005; Seo et al.,2003) properties and is effective in preventing hyperglycemia and hyperlipidemia in mice fed high-fat diets (Park et al.,2005). Activation of mRNA expression of PPARγ by E. alatus extract could improve insulin resistance and hyperlipidemia and thus help to prevent obesity-related type 2 diabetes.

Controlling postprandial glucose levels is an also important strategy in the prevention of type 2 diabetes (Jermendy, 2005). Clinical studies have documented that α-glucosidase inhibitor is effective in controlling both fasting and postprandial hyperglycemia in patients with diabetes (Balflour & McTavish, 1993; Coniff et al.,1995; Holman et al.,1999), and the relative risk of type 2 diabetes could be decreased by α-glucosidase inhibitors in subjects with impaired glucose tolerance and obesity (Jermendy, 2005). Thus, in this study we measured the α-glucosidase inhibitory activity of E. alatus in vitro and in vivo to evaluate its possible use as an anti-diabetic agent.

Materials and Methods

Reagents

Yeast α-glucosidase, p-nitropheny-α-D-glucopyranoside, soluble starch, and streptozotocin (STZ) were purchased from Sigma Chemical Co (St. Louis, MO, USA). A glucose assay kit was obtained from Yeongdong Co (Seoul, South Korea).

Preparation of the methanol extract

Euonymus alatus was obtained from a local market in Busan, Korea. Leaves of Euonymus alatus was powdered and extracted with ten volumes of methanol for 12 h three times at room temperature. The solvent was removed by rotary evaporation at 40℃. The extraction yield was 9.5%, and the extract was dissolved in dimethylsulfoxide (DMSO) at a concentration of 5 mg/ml to be used as a test sample.

Measurement of yeast α-glucosidase inhibitory activity in vitro

Yeast α-glucosidase inhibitory activity was determined using the chromogenic method developed by Watanabe et al. (1997). Yeast α-glucosidase (0.7 U) dissolved in 100 mM phosphate buffer (pH 7.0) containing 2 g/l bovine serum albumin, and 0.2 g/l NaN3, and 5 mM p-nitrophenyl-α-D-glucopyranoside in the same buffer (pH 7.0) were used as an enzyme and a substrate solution, respectively. The enzyme solution (50 µl) and 10 µl of the test sample at various concentrations were mixed, and absorbance at 405 nm was measured using a microplate reader (model 550, BioRad, Hercules, CA, USA). After incubation for 5 min, 50 µl of the substrate solution were added and incubated for an additional 5 min. The increase in absorbance from time zero was measured, and inhibitory activity was calculated as a percentage of the blank control. The inhibitory activities of the E. alatus extract and acarbose, a positive control, against α-glucosidase were measured at concentrations of 0.50, 0.25, 0.10, and 0.05 mg/ml. The measurements were performed in triplicate, and the IC50 value, i.e., the concentration of the extract that results in 50% inhibition of maximal activity, was determined.

Animals

Male Sprague-Dawley rats weighing between 250 and 280 g were purchased from Bio Genomics, Inc. (Seoul, Korea). The rats were housed individually in stainless steel wire-bottomed cages and located in a room where temperature (23-27℃), humidity (50-60%), and light (0600-1800 hr light and 1800-0600 hr dark cycle) were controlled. The animals were fed a commercial chow (Samyang Co., Seoul, Korea) ad libitum for 14 d after arrival. They were rendered diabetic by intravenous injection of STZ (60 mg/kg) in citrate buffer, pH 4.5. Blood samples were taken from the tail tip after 7 d, and blood glucose concentration was measured using a glucometer (Glucotrend Roche Diagnostics, United Kingdom). Animals showing fasting blood glucose levels higher than 200 mg/dl were considered diabetic and used for further study. All animals continued to be fed commercial chow.

Measurement of postprandial blood glucose

The effect of E. alatus extract on postprandial glucose was measured in STZ-induced diabetic rats (n=16). The rats were randomly divided into two groups. After an overnight fast, fasting blood samples were collected from the tail tip. The rats were given soluble starch (1 g/kg) alone or starch with methanol extract of E. alatus (500 mg/kg) by gastric intubation. Blood samples were collected from the tail tip at 30, 60, 90, 120, 180, and 240 min. Food was withheld during the test. Blood samples were centrifuged at 3,000 rpm for 15 min. Plasma glucose was measured using a commercial glucose oxidase kit (Yeongdong Co., Seoul, Korea). The plasma glucose level was expressed in increments from the baseline. Incremental areas under the response curve (AUC) were calculated using the trapezoidal rule with fasting levels as the baseline. All experiments were performed according to the guidelines of animal experimentation approved by the Animal Resource Center at Inje University.

Statistical analysis

Increment plasma glucose level and AUC of the glucose response curve were expressed as mean ± standard error (SE). All statistical analyses were performed using SAS (version 8.02). Differences in incremental plasma glucose levels and AUC between the control group and the Euonymus alatus group were assessed using Student's t-test. Significance was defined as p<0.05.

Results

Inhibition of α-glucosidase activity in vitro

The inhibitory activity of the methanol extract of E. alatus against yeast α-glucosidase is shown in Fig. 1. The methanol extract of E. alatus inhibited yeast α-glucosidase activity by 69.4, 48.1, 24.5, 11.3, and 4.2% at concentrations of 0.50, 0.25, 0.10, 0.05, and 0.025 mg/ml in vitro, respectively. Acarbose, an α-glucosidase inhibitor used as an oral hypoglycemic agent, inhibited enzyme activity by 26.5, 20.1, 11.4, and 4.5% at concentrations of 0.50, 0.25, 0.10, and 0.05 mg/ml, respectively. The E. alatus extract had an IC50 value of 0.272 mg/ml.

Fig. 1

Dose-dependent inhibition of yeast α-glucosidase activity of The inhibitory activities of the methanol extract of Euonymus alatus or acarbose were measured at concentrations of 0.5, 0.25, 0.1, and 0.05 mg/ml. □, Euonymus alatus extract; ♦, acarbose. Values represent means of triplicate measurements.

Inhibition of α-glucosidase activity in vivo

The plasma glucose response to a single oral dose of starch (1 g/kg) alone or starch with E. alatus extract (500 mg/kg) is shown in Fig. 2. The incremental plasma glucose levels of the rats that consumed starch were 53.8 ± 8.5, 91.5 ± 5.5, 115.3 ± 10.3, 80.3 ± 8.6, 36.1 ± 5.2, and -0.8 ± 3.2 mg/dl at 30, 60, 90, 120, 180, and 240 min, respectively. The incremental plasma glucose levels of the rats that consumed E. alatus extract with starch were 39.4 ± 10.0, 70.8 ± 5.6, 87.8 ± 7.4, 64.8 ± 5.6, 23.1± 4.2, -4.6 ± 3.8 mg/dl at 30, 60, 90, 120, 180, and 240 min, respectively. Consumption of E. alatus extract significantly decreased incremental plasma glucose levels at 60 and 90 min (p<0.05). The AUC for the glucose response of the E. alatus group (9,350 ± 668 mg·min/dl) was significantly lower than that of the control group (12,541 ± 951 mg·min/dl, p<0.05, Table 1).

Fig. 2

Postprandial glucose response to In the control group (□), starch (1 g/kg) was administered orally to rats after an overnight fast. In the Euonymus alatus group (♦), starch (1 g/kg) plus Euonymus alatus methanol extract (500 mg/kg) was administered orally to rats after an overnight fast. Values represent mean ± SE (n = 8). *Significantly different at p<0.05.

Table 1

Area under the curve (AUC) of postprandial glucose responses of STZ-induced diabetic rats

Control group: Starch (1 g/kg) was administered orally to a rat after an overnightfast. Euonymus alatus group: starch (1 g/kg) with the methanol extract of Euonymus alatus (500 mg/kg) was administered orally to a rat after an overnight-fast.

Values represent mean ± SE (n=8).

*Significantly different at p<0.05.

Discussion

α-Glucosidase is a key enzyme in carbohydrate digestion in the small intestine (Li et al., 2005). Therefore, α-glucosidase inhibitors could delay digestion of dietary carbohydrates to reduce postprandial glucose. In fact, α-glucosidase inhibitors have become the most common oral agents used to improve postprandial hyperglycemia since their introduction in the early 1990s (Mooradian & Thurman, 1999). However, because the chronic use of synthetic α-glucosidase inhibitors can have undesirable side effects, such as flatulence, diarrhea, and abdominal cramping, their use may be limited (Mooradian & Thurman, 1999). Therefore, attention has focused on natural substances that show potent inhibitory activity against α-glucosidase and have fewer side effects (Joo et al., 2006; Li et al., 2005; Shim et al., 2003; Youn et al., 2004). Galls of Rhus chinensis (Shim et al., 2003), Commelina communis L. (Youn et al.,2004), flowers of Punica granatum (Li et al., 2005), and Saururus chinensis Baill (Joo et al., 2006) have shown potent inhibitory activity against α-glucosidase.

In this study, we investigated the inhibitory effect of E. alatus against α-glucosidase to elucidate the possible use of E. alatus as an antihyperglycemic agent. Inhibition activity of the methanol extract of E. alatus against yeast α-glucosidase was about 2.6 times that of acarbose in vitro at a concentration of 0.5 mg/ml (Fig. 1). Euonymus alatus extract showed inhibitory activity against yeast α-glucosidase in a dose-dependent manner and had an IC50 value of 0.272 mg/ml.

We determined the effect of E. alatus on postprandial hyperglycemia in STZ-induced diabetic rats after consumption of starch. Postprandial plasma glucose peaked 90 min after consumption of starch in the control group. The E. alatus extract significantly suppressed incremental plasma glucose at 60 and 90 min (Fig. 2) and significantly decreased the AUC for the glucose response curve. These data demonstrate that E. alatus exerts α-glucosidase inhibitory activity in vivo to decrease postprandial glucose levels. Inoue et al. (1997) reported that an α-glucosidase inhibitor that flattens the peak postprandial blood glucose levels reduces the AUC of the blood glucose response curve. In our study, E. alatus extract decreased both incremental blood glucose at the peak time point and the AUC.

Postprandial hyperglycemia is one of the earliest observable abnormalities in diabetes mellitus. Postprandial hyperglycemia is highly correlated with glycated hemoglobin levels and is a better predictor of glycated hemoglobin levels than fasting glucose (Soonthornpun et al.,1999). Glycated hemoglobin is highly associated with a higher risk of cardiovascular disease and coronary heart disease mortality (Campbell et al.,2001). Postprandial hyperglycemia is strongly correlated with risks for micro- and macrovascular complications of diabetes (Campbell et al.,2001). Bastyr et al. (2000) demonstrated that diabetes therapy focused on lowering postprandial glucose rather than fasting glucose could be a better treatment.

In conclusion, E. alatus showed strong inhibitory activity against α-glucosidase in vitro and in vivo. Thus, chronic consumption of E. alatus could be helpful in improving hyperglycemia and preventing diabetic complications. Further study to identify the active component responsible for the inhibition of α-glucosidase is strongly recommended.