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Earthworms are known as a source of a traditional medicine, and bioactive components have been reported. We have reported that a fraction (U3EE) with molecular mass under 3 kDa from the water extract of Eisenia fetida inhibits porcine pancreatic α-amylase (PPA) activity with a half-maximal inhibitory concentration (IC5 of 73.7 ± 4.0 mg/mL. Here we purified PPA-inhibitory components from U3EE by sequential procedures of 85 %-ethanol (EtOH) extraction, solid-phase extraction (SPE), and RP-HPLC. The water eluate from SPE of the 85 %-EtOH

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