Effects of D-psicose on Glycemic Response 203 Table 1. Inhibitory effect of rare sugars on glucose release by α-amylase and α-glucosidase Values are the means ± SD of four experiments. Relative rates are shown as 100% without inhibitor. Means with different superscripts within a column are significantly different (p<0.05, one-way ANOVA and Fisher’s PLSD tests). Enzymes α-Amylase (rat small intestine) α-Amylase (rat pancrease) α-Amylase (human saliva) α-Amylase (bacillus subtilis) α-Amylase (aspergillus oryzae) Sucrase (rat small intestine) Maltase (rat small intestine) Substrate/ inhibitor 1 10 1 10 1 10 1 10 1 10 1 10 1 10 glucose concentration induced by oral carbohydrate (sucrose, maltose or starch) tolerance tests [19]. In addition, we demonstrated that plasma glucose and insulin concentrations were lower in all days the rats were fed a D-psicose diet than in the rats fed a sucrose diet for 8 weeks [20]. These findings suggest that D-psicose may inhibit the activities of α- amylase and α-glucosidase. However, inhibitory effects of D-psicose on enzymes in the digestive tract were not clarified. In this study, we investigated the effects of D- psicose on the activities of α-amylases and α-glucosidases in vitro, and evaluated the effects of D-psicose on the in vivo postprandial glycemic response using rats. Materials and Methods All procedures involving animals were approved by the Animal Care Committee of Kagawa University. Inhibitory effect of D-psicose on activities of α-amylases and α-glucosidases in vitro D-Psicose and D-allose were donated from Rare Sugar Research Center, Kagawa University. α-Amylases from bacillus subtilis and aspergillus oryzae were purchased from Wako Pure Chemical Industries (Osaka, Japan). Three male Wistar rats (3-week-old) obtained from Japan SLC (Shizuoka, Japan) were killed by decapitation. The small intestines and pancreases of the rats were obtained immediately after death, rinsed with ice-cold saline, and stored at –40°C until use. All of the collected mucosa from the three rats was homogenized together with a 5 mmol/l EDTA-phosphate buffer (pH 7.0) and centrifuged at 4°C for Vol. 45, No. 2, 2009 D-Fructose 100.8 ± 1.0a 101.2 ± 2.0 100.7 ± 0.4 98.5 ± 1.7 101.3 ± 1.5 101.5 ± 0.9 102.4 ± 3.1 99.1 ± 0.2 95.0 ± 1.7a 98.8 ± 0.5 99.6 ± 2.6a 98.1 ± 1.5a 99.5 ± 3.4a 100.9 ± 0.5 D-Psicose Relative rate (%) 83.0 ± 1.9c 100.2 ± 1.4 100.4 ± 1.1 98.0 ± 1.2 96.2 ± 3.3 99.9 ± 2.3 102.3 ± 5.3 99.1 ± 0.2 89.3 ± 0.6b 97.5 ± 0.3 74.5 ± 3.9b 92.2 ± 2.3b 85.4 ± 2.6b 101.0 ± 0.3 D-Allose 89.5 ± 1.9b 100.4 ± 0.6 101.6 ± 1.1 101.3 ± 2.9 103.7 ± 3.3 99.3 ± 2.3 101.7 ± 2.9 98.3 ± 0.8 95.3 ± 1.1a 98.0 ± 1.0 77.9 ± 1.3b 96.4 ± 1.3ab 90.2 ± 5.6ab 99.7 ± 0.2 10 min at 15000 × g. The extract was used to assay of the activities of α-amylase, sucrase, and maltase by methods of Caspary and Graf [12] and Dahlqvist [21]. For the pancreatic α-amylase assay, the tissues were homogenized together with a 10 mmol/l phosphate buffer (pH 7.0), and the homo- genate was used for amylase assay using the method of Whelan [22]. The standard assay mixture contained a 40 μl substrate solution (20 mg/ml soluble starch, sucrose, and maltose, for assay of α-amylase, sucrase, and maltase, respectively), and 80 μl of a test carbohydrate solution (final concentration, 0.4–4.0 mg/ml D-fructose, D-psicose, and D- allose). The reaction was initiated by the addition of 80 μl of appropriate dilutions of dried enzyme powders (from bacillus subtilis and aspergillus oryzae) or enzyme solutions (from the small intestine and pancreas). The reaction mixture was incubated for 60 min at 37°C, and then the glucose concentration of the mixture was determined by the kits (Glucose CII-Test, Wako Pure Chemical Industries). The relative enzyme activities were calculated from glucose release. Relative rates were shown as 100% without inhibitors. Effect of D-psicose on plasma glucose concentrations after carbohydrate loading in rats To evaluate the potency of D-psicose in vivo, the effects of D-psicose on plasma glucose concentrations after glucose, sucrose, maltose, and soluble starch loading, were examined. One hundred forty-four male Wistar rats (6 months old) were purchased from Japan SLC (Shizuoka, Japan) and acclimatized for a week under standard laboratory condi- tions (22 ± 2°C, 60% humidity). The light/dark cycle was 12 h with lights on from 8:00 h to 20:00 h. Rats were