Figure 3. DPA is the active factor released from PIB.
(A and B) PIB culture supernatant was extracted with methanol/H2O/dichloromethane, and the substances in polar and nonpolar phases were subjected to contraction measurement. The contraction inhibition rate of the substances in the 2 phases was calculated (n = 3). (C) The substances in the dichloromethane phase were analyzed with HPLC with a C18 column. The arrow indicates the extra peak (red) of PIB medium in contrast to LB medium. The experiments were repeated 3 times. (D) The contraction-inhibiting activity for each eluted fraction of HPLC was measured. The fraction about 10.2 minutes after elution showed the highest activity. (E) The major resulting fragment ions are indicated in mass spectra of active fraction peaks and were extracted as DPA. The numbers indicate molecular weights of fragment ions. (F) cDPA had a substantial inhibitory effect on contraction. (G) Quantitation of the inhibitory effect of DPA (n = 4). (H) Colonic transit test with bead expulsion time in vehicle- and DPA-treated mice (n = 10). Data are presented as mean ± SD. **P < 0.01 (B, paired 2-tailed t test; H, unpaired 2-tailed t test).