In each case, the reaction was allowed to proceed at 37°C for 0, 7.5, 15, 22.5, and 30 min as described in a previous section. Statistical analysis Statistical and curve-fitting analyses were performed using Prism 4.0 (GraphPad Software Inc.). The data are expressed as means ± SEM. Differences between groups were assessed by one-way analysis of variance (ANOVA), followed by Student–Newman–Keul’s test. Values of percentage inhibition of EM selleck inhibitor degradation were calculated using following formula, which was described earlier (Tomboly et al., 2002): $$ \textInhibition \left( \% \right)

\, = \, \left( k_0 – k_\texti \right)/k_0 \times 100, $$where k 0 the rate constant of degradation without inhibitor, k i the rate constant of degradation with inhibitor. Results Effect of inhibitors on degradation of EMs by DPP IV We evaluated EMDB-2 and EMDB-3 for their inhibitory effect on degradation BLZ945 in vivo of EMs by DPP IV. Diprotin A was included in the study for comparison. Degradation of EMs was analyzed by reversed phase HPLC. Effects of 30 min incubation of EM-2

with DPP IV in the absence and presence of inhibitors are shown in Fig. 2. The chromatographic peak area of EM-2 was found to decrease greatly in the sample without inhibitors. Diprotin A almost completely suppressed enzymatic cleavage of EM-2, while EMDB-2 and EMDB-3 only partially protected EM-2 against hydrolysis. Degradation rates and half-lives of EMs alone and in the presence of inhibitors are collected in Table 1. Different rates of degradation of EM-1 and EM-2 by DPP IV were observed. EM-1 was PARP activity about 1.5 times more resistant to DPP IV than EM-2, which is in agreement with the data obtained by others (Tomboly et al., 2002; Grass et al., 2002; Fujita and Kumamoto, 2006; Keresztes et al., 2010). EMDB-2 and EMDB-3 increased EM-1 and EM-2 half-lives two- to threefold. The effects of inhibitors on degradation of EMs after 30 min incubation with DPP IV are summarized in Table 2. EMDB-3 appeared to be a better aminophylline DPP IV inhibitor than EMDB-2. The Lineweaver–Burk plots revealed that both tested compounds acted as competitive inhibitors of DPP IV (Fig. 3). Fig. 2 Effect

of inhibitors on the degradation of EM-2 by DPP IV. The reaction mixture was incubated at 37°C for 30 min in the absence (a) and presence of diprotin A (b), EMDB-2 (c), and EMDB-3 (d). Asterisk indicates the peak derived from the inhibitor added Table 1 Degradation rates (k) and half-lives (t 1/2) of EMs incubated with DPP IV alone and in the presence of inhibitors Inhibitor DPP IV EM-1 EM-2 100 × k (1/min) t 1/2 (min) 100 × k (1/min) t 1/2 (min) Without inhibitor 4.12 ± 0.2 16.7 ± 0.52 6.30 ± 0.31 10.9 ± 0.64 Diprotin A 0.13 ± 0.01 530 ± 14.5*** 0.18 ± 0.01 383 ± 20.2*** Tyr-Pro-Ala-NH2 (EMDB-2) 3.02 ± 0.09 22.9 ± 1.14* 3.48 ± 0.13 19.8 ± 0.75* Tyr-Pro-Ala-OH (EMDB-3) 2.51 ± 0.12 27.5 ± 1.21* 2.52 ± 0.13 27.4 ± 1.41* * P < 0.05, *** P < 0.