ETHNOPHARMACOLOGICAL RELEVANCE: Masiningan is a traditional medicine consisting of six crude drugs that have been used for treating constipation and diabetes mellitus in both Japan and China. Masiningan has been reported to have significant PTP1B inhibitory activity and to affect cells in the insulin-signaling pathway. The aim of the present study is to identify the PTP1B inhibitory compounds in Masiningan.MATERIALS AND METHODS: Bioactivity peaks were identified by analytical HPLC profiling and PTP1B inhibitory activity profiling of sub-fractions from Masiningan extract. The bioactive compounds were isolated by tracking two identified bioactive peaks, and the chemical structures were determined by spectroscopic analyses. The bioactive compounds were further investigated for their inhibitory effect against PTP1B by enzymatic kinetic analysis, molecular docking simulation, inhibitory selectivity against other PTPs, and cellular activity in the insulin signal transduction pathway.RESULTS: From Masiningan, magnolol (1) and chrysophanol (2) were isolated as compounds that exhibited significant dose-dependent inhibitory activities against PTP1B, with IC50 values of 24.6 and 12.3muM, respectively. Kinetic analysis revealed that 1 is a non-competitive and that 2 is a competitive PTP1B inhibitor. In the molecular docking simulation, compound 2 was stably positioned in the active pocket of PTP1B, and the CDOCKER energy was calculated to be 24.3411kcal/mol. Both compounds demonstrated remarkably high selectivity against four PTPs and revealed cellular activity against the insulin signal transduction pathway.CONCLUSIONS: Magnolol (1) and chrysophanol (2) were identified as the principle PTP1B inhibitory active compounds in Masiningan, and their actions were investigated in detail. These findings demonstrated the effectiveness of Masiningan on diabetes mellitus through the inhibition of PTP1B at a molecular level as well as the potential of magnolol (1) and chrysophanol (2) as lead compounds in future anti-diabetes drug development.